Cons i u i e ac i i y and d ug
unc ional selec i i y o 5-HT2A
ecep o s in pos -mo em b ain
o subjec s wi h schizoph enia
I zia Mune a A a e
(cc) 2022 I zia Mune a A a e (cc by 4.0)
This doc o al hesis has been de eloped hanks o he inancial
suppo o a p edoc o al ellowship om he Basque Go e nmen (2018-
2022).
This wo k was unded by he Spanish Minis y o Science and
Inno a ion (SAF2017-88126R), he Basque Go e nmen (IT-616-13) and he
Cen e o Biomedical Resea ch in Men al Heal h, CIBERSAM.
ABREVIATION LIST
AA A aquidonic acid
AC Adenylyl cyclase
BB Basal binding
Ca2+ Calcium ion
CaMKII Calcium/calmodulin-dependen kinase II
CNS Cen al ne ous sys em
BRET Bioluminiscence esonance ene gy ans e
cAMP Cyclic adenosine monophospha e
DA Dopamine
DAG Diacylglyce ol
DLPFC Do sola e al p e on al co ex
(±)DOI 2,5-dime oxy-4-iodoamphe amine
DSM Diagnos ic and S a is ical Manual o Men al Diso de s
D2R Dopamine 2 ecep o
EC50 Concen a ion ha p omo es hal -maximal s imula o y e ec
Emax Maximal s imula o y e ec
ERK Ex acellula signal- egula ed kinases
FDA Food and d ug adminis a ion
GABA γ-aminobu i ic acid
GPCR G-p o ein coupled ecep o
GDP Guanosine diphospha e
GRK G-p o ein coupled kinase
GTP Guanosine iphospha e
5-HT 5-Hyd oxy yp amine (se o onin)
5-HT2AR Se o onin 2A ecep o
5-HT2AR(-/-) Knock-ou
5-HT2AR(+/+) Wild-Type
GWAS Genome-wide associa ion s udies
IC50 Concen a ion ha p omo es hal -maximal inhibi o y e ec
Imax Maximal inhibi o y e ec
IP3 Inosi ol 1,4,5- iphospha e
LSD D-lyse gic acid die hylamide
MAPK Mi ogen-ac i a ed-p o ein kinase
NBS Non-speci ic binding
NMDA N-me hyl-D-aspa a e
PCP Phencyclidine
PET Posi on emission omog aphy
PFC P e on al co ex
PIP2 Phospha idylinosi ol 4,5-biphospha e
PKC P o ein kinase C
PLA2 Phospholipase A2
PLC Phospholipase C
PMD Pos -mo em delay
PSD-95 Pos synap ic p o ein densi y 95
PTX Bo de ella pe ussis oxin
RSK-2 Ribosomal S6 kinase
GTPγS 5'-O-[gamma- hio] iphospha e
[35S]GTPγS Sulphu 35-labelled guanosine-5´-O-(γ- hio)- iphospha e
SNP Single nucleo ide polimo phism
SPA Scin illa ion p oximi y assay
7TM Se en ansmemb ane
Volinanse in MDL100907
INDEX
In oduc ion _________________________________________________ 1
1.1 Schizoph enia _____________________________________________________ 3
1.1.1 De ini ion _______________________________________________________ 3
1.1.2 Symp oma ology _________________________________________________ 3
1.1.3 Ae iology o schizoph enia __________________________________________ 4
1.1.3.1 Gene ics ______________________________________________________ 5
1.1.3.2 En i onmen al ac o s ____________________________________________ 5
1.1.3.3 Neu o ansmission sys ems al e a ions in schizoph enia _________________ 7
1.1.4 Mo phological b ain al e a ions _____________________________________ 11
1.2 G-p o ein coupled ecep o s (GPCRs) _________________________________ 12
1.2.1 Gene al aspec s ________________________________________________ 12
1.2.2 The basic mechanism o GPCR signalling ____________________________ 12
1.2.3 Theo y o d ug ecep o in e ac ion o GPCR __________________________ 17
1.2.4 GPCRs and biased signalling ______________________________________ 22
1.2.5 E alua ion o cons i u i e ac i i y, in e se agonism and unc ional selec i i y __ 25
1.3 Se o onin 2A ecep o (5-HT2AR) _____________________________________ 28
1.3.1 Gene al aspec s ________________________________________________ 28
1.3.2 Localiza ion and unc ion o 5-HT2AR in CNS __________________________ 29
1.3.3 5-HT2AR S uc u al biology ________________________________________ 33
1.3.4 5-HT2AR Signalling pa hways ______________________________________ 35
1.3.5 5-HT2AR ligands _________________________________________________ 37
1.3.5.1 Agonis s _____________________________________________________ 38
1.3.5.2 An agonis s ___________________________________________________ 40
1.3.6 5-HT2AR unc ional selec i i y ______________________________________ 41
1.4 5-HT2AR and schizoph enia _________________________________________ 46
1.4.1 Gene ic s udies _________________________________________________ 46
1.4.2 E alua ion o 5-HT2AR densi y, exp ession and unc ionali y in schizoph enia
subjec s. ___________________________________________________________ 47
1.5 An ipsycho ics____________________________________________________ 52
1.5.1 Fi s -gene a ion o an ipsycho ics (Typical an ipsycho ics) ________________ 52
1.5.2 Second-gene a ion o an ipsycho ics (A ypical an ipsycho ics) _____________ 53
1.5.3 Thi d-gene a ion o an ipsycho ics ___________________________________ 56
1.5.4 New gene a ion o an ipsycho ics ___________________________________ 56
Aims _______________________________________________________ 59
Subjec s, Ma e ials and Me hods _______________________________ 63
3.1 Human b ain samples ______________________________________________ 65
3.1.1 Subjec s selec ion, demog aphic cha ac e is ics, psychia ic diagnosis and
oxicological analysis _________________________________________________ 65
3.1.2 Demog aphic cha ac e is ics and diagnosis o subjec s included in pools used o
An ibody-cap u e [35S]GTPγS Scin illa ion p oximi y Assays (SPA) and Wes e n Blo
cha ac e iza ion assays _______________________________________________ 73
3.2 Animals: T ansgenic mice ___________________________________________ 74
3.3 D ugs __________________________________________________________ 75
3.4 Ma e ials ________________________________________________________ 77
3.5 Me hods ________________________________________________________ 79
3.5.1 An ibody-cap u e [35S]GTPγS Scincilla ion P oximi y Assay (SPA) __________ 79
3.5.1.1 P epa a ion o memb ane-en iched ac ion (P2 ac ion) ________________ 80
3.5.1.2 An ibody-cap u e [35S]GTPγS Scincilla ion P oximi y Assay (SPA) ________ 81
3.5.1.3 Ma hema ical and s a is ical analysis o he esul s ____________________ 85
3.5.2 Wes e n Blo expe imen s _________________________________________ 90
3.5.2.1 P epa a ion o memb ane en iched ac ion (P2 ac ion) ________________ 91
3.5.2.2 Gel elec opho esis, ans e ence and immunode ec ion ________________ 92
3.5.2.3 Ma hema ical and s a is ical analysis o esul s _______________________ 98
Resul s ___________________________________________________ 101
4.1 Func ional selec i i y o di e en se o onin 5-HT2AR an agonis s in pos -mo em
human b ain _______________________________________________________ 103
4.1.1 Concen a ion-e ec o di e en d ugs on Gαi1- and Gαq/11-p o ein coupling o 5-
HT2AR in pos -mo em human PFC _____________________________________ 103
4.1.2 E alua ion o maximal e ec o di e en d ugs on Gαi1-, Gαi2-, Gαi3-, Gαo-, and
Gαq/11-p o ein coupling o 5-HT2AR in pos -mo em human PFC _______________ 107
4.1.3 In ol emen o 5-HT2AR in he e ec induced by di e en d ugs on [35S]GTPγS
binding o Gαi1- and Gαq/11-p o eins in pos -mo em human PFC _______________ 109
4.1.4 E alua ion o he maximal e ec o di e en d ugs on Gαi1- and Gαq/11-p o ein
coupling o 5-HT2AR in knock-ou 5-HT2AR(-/-) and wild- ype 5-HT2AR(+/+) mice _____ 116
4.1.5 Summa y _____________________________________________________ 121
4.2 E alua ion o he unc ional coupling o 5-HT2AR o Gα-p o ein sub ypes in PFC o
schizoph enia subjec s, non-schizoph enia suicide subjec s and ma ched con ols by
an ibody-cap u e [35S]GTPγS scin illa ion p oximi y assay (SPA) _______________ 122
4.2.1 Basal [35S]GTPγS binding o Gαi1- and Gαq/11- p o eins in pos -mo em PFC o
schizoph enia subjec s, non-schizoph enia suicide subjec s and ma ched con ols 122
4.2.2 Modula ion o [35S]GTPγS binding o Gαi1- and Gαq/11-p o eins by he 5-HT2AR
in e se agonis pima anse in in pos -mo em PFC o schizoph enia subjec s, non-
schizoph enia suicide subjec s and ma ched con ols _______________________ 125
4.2.3 Modula ion o he [35S]GTPγS binding o Gαi1- and Gαq/11-p o eins by he 5-HT2AR
in e se agonis olinanse in in pos -mo em PFC o schizoph enia subjec s, non-
schizoph enia suicide subjec s and ma ched con ols _______________________ 132
4.2.4 Modula ion o [35S]GTPγS binding o Gαi1- and Gαq/11-p o eins by 5-HT2AR agonis
(±)DOI in pos -mo em PFC o schizoph enia subjec s, non-schizoph enia suicide
subjec s and ma ched con ols _________________________________________ 139
4.2.5 Rela ionship be ween e ec s o pima anse in, olinanse in and (±)DOI on
[35S]GTPγS binding o Gαi1- and Gαq/11-p o eins in pos -mo em human PFC _____ 145
4.2.6 Summa y _____________________________________________________ 149
4.3 E alua ion o immuno eac i i y o Gα-p o ein sub ypes in memb ane en iched
ac ions om PFC o pos -mo em b ain _________________________________ 150
4.3.1 Sui abili y o an ibodies o an ibody-cap u e [35S]GTPγS Scin illa ion P oximi y
Assay (SPA) and Wes e n Blo _________________________________________ 150
4.3.2 E alua ion o Gαi1- and Gαq/11-p o ein immuno eac i i y in pos -mo em PFC o
schizoph enia subjec s, non-schizoph enia suicide subjec s, and ma ched con ols 153
4.3.3 Summa y _____________________________________________________ 156
4.4 Func ional selec i i y o di e en an ipsycho ics in pos -mo em human b ain PFC.
Di e en ial G-p o ein biased ligand p ope ies a he 5-HT2AR _________________ 157
4.4.1 E alua ion o maximal e ec o di e en an ipsycho ic d ugs on Gαi1-, Gαi2-, Gαo-
and Gαq/11-p o ein coupling o 5-HT2ARs in pos -mo em human PFC ___________ 157
4.4.2 Pha macological cha ac e iza ion o he e ec s induced by clozapine and
ispe idone on [35S]GTPγS binding o Gαi1- and Gαq/11-p o eins in pos -mo em human
PFC _____________________________________________________________ 167
4.4.2.1 Clozapine ___________________________________________________ 168
4.4.2.2 Rispe idone _________________________________________________ 171
4.4.3 Summa y _____________________________________________________ 174
Discussion ________________________________________________ 177
5.1 E alua ion o 5-HT2AR unc ional coupling in human pos -mo em b ain wi h
di e en 5-HT2AR agonis / an agonis / in e se agonis d ugs _________________ 181
5.2 Cons i u i ely ac i e 5-HT2AR in pos -mo em human PFC ________________ 187
In oduc ion
4
cogni i e symp oms. Posi i e symp oms a e he co e ea u es o
schizoph enia, and include hallucina ions (mos equen ly audi o y),
disillusions and psycho ic beha iou , in which con ac wi h eali y is los .
Posi i e symp oms end o appea in episodes, and usually spaced in ime,
al hough some pa ien s ha e esidual long- e m psycho ic symp oms.
Nega i e symp oms a e cha ac e ized by impai ed mo i a ion, educ ion in
spon aneous speech and social wi hd awal (Liddle, 1987). Finally, cogni i e
symp oms include di icul ies in a en ion and concen a ion, lea ning, memo y
and execu i e unc ions (Joyce & Roise , 2007).
1.1.3 Ae iology o schizoph enia
Schizoph enia is a complex disease p obably caused by mul iple ae iological
ac o s. Thus, gene ic and en i onmen al ac o s a e known o ake pa in he
onse and de elopmen o schizoph enia (Sulli an e al., 2012; McCu cheon e
al., 2020). Mo eo e , a neu ode elopmen al ae iological hypo hesis has been
p oposed and associa ed wi h s uc u al, unc ional and neu ochemical b ain
changes. Those changes a ec se e al neu o ansmission sys ems and
ci cui s ha seem o be a ec ed in pa ien s wi h schizoph enia. E idence
poin s o abno mali ies in dopamine (DA), se o onin (5-HT) and glu ama e
neu o ansmi e sys ems in he pa hology o schizoph enia. E en so, none o
he hypo hesis seems o be su icien o explain he ull spec um o he
disease.
In oduc ion
5
1.1.3.1 Gene ics
Many epidemiological s udies ha e consis en ly shown a gene ic componen
in he de elopmen o schizoph enia, wi h an es ima ed he i abili y nea ly 80%
(Sulli an e al., 2003). In ecen yea s, se e al la ge-scale genomic s udies
ha e allow o s udy he con ibu ion o speci ic deoxy ibonucleic acid a ian s
and di e en ype o isk alleles. In consequence, schizoph enia is cu en ly
conside ed a highly polygenic diso de .
Genome-wide associa ion s udies (GWAS) ha e shown ha mul iple common
a ian s a e associa ed wi h schizoph enia (Schizoph enia Wo king G oup o
he Psychia ic Genomic Conso ium, 2014). Some o he mos eplica ed gene
associa ion in ol es pos synap ic densi y (PSD) p o eins, ac i i y- egula ed
cy oskele on-associa ed p o ein, N-me hyl-D-aspa a e (NMDA) ecep o ,
agile X men al e a da ion p o ein a ge s as well as o he neu ode elopmen
diso de s, ol age-ga ed calcium (Ca2+) channels, neu al cell adhesion
molecule and dopamine 2 ecep o (D2R) (Pa diñas e al., 2018; T ube skoy e
al., 2022). A widely accep ed associa ion o genes wi h schizoph enia a ises
in pa om many s uc u ally di e se alleles om complemen componen 4
genes ha is ela ed wi h he his ocompa ibili y complex (Seka e al., 2016).
1.1.3.2 En i onmen al ac o s
En i onmen al ac o s a e also in ol ed in he ae iology o schizoph enia.
Al hough s udies indica ed a s ong gene ic in luence and high he i abili y, i
has been p o en ha di e en en i onmen al ac o s can igge he disease in
people who al eady ha e a gene ic p edisposi ion, a e m known as “s ess-
ulne abili y” model (Os e al., 2010).
The en i onmen al ac o s ha e been ela ed wi h he neu ode elopmen al
hypo hesis o schizoph enia (Fa emi & Folsom, 2009). Nume ous s udies ha e
consis en ly epo ed inc eased incidence o schizoph enia associa ed o
In oduc ion
6
se e al ac o s ha a ec ea ly neu ode elopmen du ing p egnancy, including
ma e nal s ess, ma e nal in ec ions, nu i ional de ici s as well as bi h
complica ions (Jones e al., 1998; McG a h e al., 2010; B own, 2012) (Figu e
1.1). Se e al s udies ha e e ealed inc eased isk o de eloping psychosis
associa ed o childhood ad e si ies (Va ese e al., 2012). Fu he mo e, well
s ablished e idences show ha socioeconomic ac o s (Alla dyce & Boydell,
2006) and immig a ion (bo h i s and second gene a ions) show ela ionship
wi h a es o schizoph enia (Can o -G aae & Sel en, 2005). Toxic condi ions
also play a ole in schizoph enia. Thus, accumula ing e idence poin ed o
associa ion be ween cannabis use and psychosis (Figu e 1.1). These s udies
sugges ed ha ea ly ch onic exposu e o cannabis is associa ed wi h a highe
ulne abili y o psycho ic ou comes, including la e schizoph enia
de elopmen (Moo e e al., 2007; Iba a-Lecue e al., 2018) (Figu e 1.1).
The e o e, many candida e genes and en i onmen al isk ac o s seem o be
obus ly associa ed wi h schizoph enia.
In oduc ion
7
Figu e 1.1: Schema ic ep esen a ion o how en i onmen al ac o s can lead o psychia ic
diso de s, such as schizoph enia in o sp ing. In ec ion du ing p egnancy induces p o-
in lamma o y cy okines elease and immune sys em ac i a ion. Gene ic backg ound,
au oimmune s a us, and second hi s du ing childhood and adolescence (including s ess and
d ug abuse) combined wi h consequences o ma e nal in ec ion inc ease he likelihood o
o sp ing o de elop psychia ic diso de s in adul hood. Illus a ion o iginally c ea ed o his
hesis by N Co de o and adap ed om Es es & McAllis e , 2016.
1.1.3.3 Neu o ansmission sys ems al e a ions in
schizoph enia
Dopamine gic hypo hesis o schizoph enia
The mos widely known heo y o explain clinical symp oms and d ug esponse
o schizoph enia is he dopamine gic hypo hesis, which is based on di e en
indings. Fi s , clinical e ec s o ypical o i s -gene a ion an ipsycho ic a e
ela ed wi h hei abili y o block D2R (Seeman & Lee, 1975). Second, d ugs
ha inc ease DA ac i i y, such as amphe amine, p oduce psycho ic episodes
in heal hy indi iduals, and wo sened psychosis in schizoph enic pa ien s
(Liebe man e al., 1987).
Ma e nal immune
ac i a ion due o
in ec ion
S ess
D ug abuse
In oduc ion
8
Dopamine gic hypo hesis a gues hype ac i i y o dopamine gic ansmission
in mesolimbic and s ia al egions ha would be esponsible o posi i e
symp oms (hallucina ions, delusions). Howe e , nega i e and cogni i e
symp oms shown o be esis an o an ipsycho ics. Thus, dopamine gic
hypo hesis was e o mula ed pos ula ing ha a p e on al hypodopamine gia,
mo e han hype dopamine gia, would con ibu e o nega i e and cogni i e
symp oms (Da is e al., 1991; Howes & Kapu , 2009; McCu cheon e al.,
2020).
Dopamine ecep o s a e G-p o ein coupled ecep o s (GPCR) ha can be
di ided in o wo main ypes: dopamine D1 ecep o amily, including D1 and D5
dopamine ecep o s (D1R, D5R), and D2 ecep o amily, including D2, D3 and
D4 dopamine ecep o s (D2R, D3R, D4R). The D2R is a main a ge o ypical
and a ypical an ipsycho ic medica ion, which sugges ha his ecep o plays
a key ole in schizoph enia.
In i o neu oimaging, based on posi on emission omog aphy (PET) and
single pho on emission compu e ized omog aphy echniques (SPECT), ha e
been used o e alua e he s a us o D2R and D1R in schizoph enia pa ien s.
Ini ial s udies in basal ganglia we e inconsis en , wi h some o hem epo ing
inc eased D2R densi y and o he s no di e ences om con ols (Howes e al.,
2012; Cumming e al., 2021). Ele a ed D2R densi y was sugges ed o be a
consequence o ecep o up- egula ion a e long- e m an ipsycho ic
medica ion, since d ug-naï e pa ien s did no show such PET al e a ions
(Seeman, 2013). In i o pos -mo em binding s udies co obo a ed he inding
o enhanced s ia al D2R-binding densi y in schizoph enia (Seeman e al.,
1984; Zakzanis & Hansen, 1998). Mo eo e , a ecen s udy has e ealed an
absence o s ia al D2R hype ac i i y in pos -mo em b ain o schizoph enia
subjec s (Egusquiza e al., 2021). As o D2R, in i o obse a ions on D1R
densi y in p e on al co ex (PFC) o d ug-naï e schizoph enia pa ien s ha e
shown disc epancies (Cumming e al., 2021). In con as o his ea lie ocus
In oduc ion
9
on pos synap ic D1R and D2R dys egula ion, mo e ecen indings poin
owa ds a c i ical ole o p esynap ic dopamine gic neu o ansmission sys em
in schizoph enia. Thus, ele a ed p esynap ic DA syn hesis capaci y (Howes e
al., 2012; Fusa -Poli & Meye -Lindenbe g, 2013), highe synap ic DA
concen a ion (Abi-Da gham e al., 2000; Ca a aggio e al., 2015), and
amphe amine-induced DA elease (Howes e al., 2012; La uelle, 1998) ha e
been demons a ed in s ia um o schizoph enia subjec s. Con e sely,
e idence sugges a educ ion o amphe amine-induce DA elease in on al
co ex o schizoph enia pa ien s (Sli s ein e al., 2015), indica ing a p esynap ic
hypodopamine gia in his b ain a ea (Sli s ein e al., 2015).
Glu ama e gic hypo hesis o schizoph enia
Glu ama e is he majo exci a o y neu o ansmi e in he b ain. Glu ama e
in e ac s wi h selec i e iono opic and me abo opic ecep o s. Iono opic
ecep o g oup includes NMDA, kaina e and α-amino-3-hyd oxy-5-me hyl-
isoxazole-4-p opiona e (AMPA) ecep o sub ypes. Me abo opic glu ama e
ecep o s (mGluRs), which ac i a e G-p o ein signal ansduc ion, a e di ided
in g oups I (mGlu1, mGlu5), II (mGlu2, mGlu3) and III (mGlu6, mGlu4, mGlu7,
mGlu8) (Nakanishi, 1992; Niswende & Conn, 2010; Mugu uza e al., 2016).
I is well s ablished ha adminis a ion o NMDA ecep o an agonis s, such as
phencyclidine (PCP) o ke amine, can induce psychosis-like s a es and
cogni i e de ici s in heal hy humans. Ke amine and PCP a e non-compe i i e
glu ama e NMDA an agonis (Thomson e al., 1985). This ac ga e suppo o
he hypo hesis o a unc ional impai men o NMDA ecep o s in schizoph enia
(Ja i & Zukin, 1991; S one e al., 2008). In e es ingly, addi ion o
amphe amine o o he dopamine gic agonis as well as NMDA ecep o
an agonis also eassembles psycho ic symp oms, and e okes cogni i e and
nega i e symp oms, be e mimicking he pa hophysiolology o schizoph enia
In oduc ion
10
(K ys al e al., 2005). Fo his eason, non-compe i i e NMDA ecep o
an agonis adminis a ion has been chosen as a usual animal model o
schizoph enia.
Mul iple epo s ha e e alua ed he s a us o glu ama e in b ain o
schizoph enia pa ien s and i s ela ionship wi h di e en spec um o
symp oms. Thus, e idence ha impai ed glu ama e gic sys em migh be
implica ed in he cogni i e dys unc ion in schizoph enia is cu en ly
acknowledged (Moghaddam & Ja i , 2012).
Glu ama e hypo hesis is indeed closely ela ed o hype - and hypoac i i y o
dopamine gic pa hways in limbic and co ical a eas, espec i ely. Fi s , i has
been desc ibed an hypoac i i y o inhibi ion o NMDA ecep o s loca ed in γ-
aminobu i ic acid (GABA)e gic in e neu ons in mesolimbic dopamine gic
a eas. This would impai he inhibi o y onic s a e, esul ing in highe syn hesis
and elease o dopamine in hose a eas, which could p edispose o psycho ic
symp oms. In con as , cogni i e and nega i e symp oms could be ela ed wi h
a hypo unc ionali y o NMDA ecep o s in he co ex-b ains em p ojec ion,
which could make hypoac i e mesoco ical dopamine gic pa hways (Ja ii ,
2010).
Se o one gic hypo hesis o schizoph enia
The 5-HT hypo hesis o schizoph enia a ose om ea ly s udies on in e ac ions
be ween he hallucinogenic d ug D-lyse gic acid die hylamide (LSD) and 5-HT
(F eedman 1961). LSD and ela ed compounds p oduce men al dis u bances,
esembling hose occu ing a he onse o schizoph enia, and media e hei
cen al e ec s h ough 5-HT ecep o s. Mo eo e , psychedelic d ugs ha e
some chemical simila i ies wi h 5-HT s uc u e.
Among all he 5-HT ecep o sub ypes, abundan e idence indica e ha he
e ec s o hallucinogens a e media ed h ough he se o onin 2A ecep o (5-
In oduc ion
11
HT2AR) sub ype (González-Maeso e al., 2007; Geye & Vollenweide , 2008;
González-Maeso & Seal on, 2009; Madsen e al., 2019). In ela ion wi h hese
ac s, a ypical an ipsycho ic d ugs show high a ini y o 5-HT2AR, making his
a ge one o he mos widely s udied in schizoph enia (Mel ze e al., 1989). A
mo e de ailed e iew o 5-HT2AR physiology and in ol emen in schizoph enia
can be ound in sec ion 1.4.
1.1.4 Mo phological b ain al e a ions
PFC has long been implica ed in he pa hophysiology o schizoph enia,
especially in nega i e and cogni i e mani es a ions (Kolk & Rakic, 2022). In
ac , lesions o his b ain egion in animals and human migh p oduce la e
impai men s, as dis up ion in wo king memo y, educed impulsi e choice,
enhanced en i onmen al s imuli and beha iou al es ains. Thus,
abno mali ies in PFC ha e been la gely associa ed wi h schizoph enia and
o he psychia ic diseases (Xu e al., 2019).
I is well desc ibed ha schizoph enia pa ien s exhibi la e al en icula
enla gemen by 25% o olume, which is accompanied by a whole b ain
olume educ ion a ound 2% (Johns one e al., 1976; Haijma e al., 2013). The
en icles size inc eases p og essi ely a e he onse o he illness, as whole
b ain g ey ma e is educed o e ime ( an E p e al., 2016).
Mo phological s udies in pos -mo em b ains o schizoph enia pa ien s ha e
e ealed di e ences in cellula dis ibu ion, likely a ibu ed o al e ed neu onal
mig a ion ea ly in b ain de elopmen , loss o py amidal cells, mal o med cell
s uc u e, and dec eased numbe o GABA in e neu ons (Schmid & Mi nics,
2015).
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1.2 G-p o ein coupled ecep o s (GPCRs)
1.2.1 Gene al aspec s
GPCRs, also known as se en ansmemb ane (7TM) ecep o s, o m one o
he la ges memb ane ecep o sub amily o plasma memb ane p o ein in he
body. These ecep o s bind o di e se ype o ligands, such as ions, small
molecules and pep ides, linking hem o downs eam signalling (Alexande e
al., 2019).
GPCRs ha e an eno mous biomedical ele ance because hey a e in ol ed in
di e se physiological ac i i ies, and play a c ucial ole in pa hogenesis o
diso de s, being impo an as d ug- a ge s. Mo eo e , i is es ima ed ha
app oxima ely 35% o app o ed d ugs a ge GPCRs (Hause e al., 2017).
The 7TM ecep o supe amily is also e med as GPCRs because exe s
e ec s in esponse o di e en ligands h ough he associa ion wi h G-p o eins.
G-p o eins a e he e o ime ic guanine nucleo ide binding p o eins, cons i u ed
by h ee subuni s: α, β and γ. Howe e , GPCRs can also bind o o he cy osolic
adap o s, including β-a es ins, which elici G-p o ein independen ac i a ion
(Sy o a kina e al., 2016). G-p o eins media e he ea lies s ep in cell esponse
o ex e nal e en s, by linking cell su ace ecep o s o in acellula signalling
(S i am & Insel, 2018). G-p o eins se e as ansduce s o ampli ie s o signals
om GPCRs o in acellula e ec o s.
1.2.2 The basic mechanism o GPCR signalling
He e o ime ic G-p o eins ha e a c ucial ole in de ining he speci ici y and
empo al cha ac e is ics o cellula esponses. Upon ligand ac i a ion, he e is
a con o ma ional change o GPCRs ha inc ease hei a ini y o G-p o eins,
leading o G-p o ein ec ui men . The in e ac ion o a G-p o ein wi h an ac i e
ecep o s imula es he exchange o guanosine diphospha e (GDP) nucleo ide
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13
(inac i e con o ma ion) o guanosine iphospha e (GTP) (ac i e
con o ma ion) (Figu e 1.2). In he inac i e con o ma ion, Gα subuni is bound
o GDP as well as o Gβ and Gγ subuni s o ming a he e odime . A e ecep o
ac i a ion, Gα subuni bounds o GTP and dissocia es om he Gβγ dime
(Gilman, 1987; Ma inissen & Gu kind, 2001). A e wa ds, Gβγ subuni s
modula e downs eam cellula signalling pa hways, such as adenylyl cyclase
(AC), phospholipase and ion channels (Hamm, 1998). The signal e mina es
when he GTP is hyd olysed by he GTPase ac i i y o Gα subuni s, o GDP
and phospha e, and he Gβγ complex binds o Gα, o ming he inac i e G-
p o ein (Milligan & Kos enis, 2006; Hilge e al., 2018) (Figu e 1.2).
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20
Agonis s p esen a ini y o a a ge ecep o , as well as e icacy. Thus, hese
ligands a e able o bind he ecep o and subsequen ly p oduce a esponse.
Agonis s can di e in hei magni ude o he ecep o -p oduced s imulus, which
leads o hei cha ac e iza ion as ull o pa ial agonis . A ull agonis p oduces
maximal e ec , while a pa ial agonis exe s a submaximal esponse,
depending on hei in insic e icacy. In con as , an agonis s display a ini y bu
no in insic e icacy, since hey do no induce con o ma ional changes in he
ecep o o p omo e esponse. I s impac depends on hei compe ence o
educe he p obabili y o an agonis , such as endogenous ligand, o bind he
ecep o and dec easing endogenous cellula esponse.
On he o he side, Samama e al. p oposed ha he p esence o a ligand is no
necessa y o gene a e a cellula esponse o signalling (Samama e al., 1993),
which is de ined as cons i u i e ac i i y. Thus, he cons i u i e ac i i y consis s
on spon aneous au o-ac i a ion o he ecep o , adop ing a con o ma ion able
o igge in acellula signalling despi e ligand absence (Le kowi z e al., 1993).
In his sense, Cos a and He z ound ha ligands wi h nega i e in insic e icacy
we e able o dec ease he cons i u i e ac i i y (Cos a & He z, 1989; Chidiac e
al., 1994; Cos a & Co ecchia, 2005). These, ligands we e named as in e se
agonis s, and could be classi ied as ull o pa ial in e se agonis s, acco ding
o hei in insic e icacy.
Te na y complex model is he mos widely accep ed GPCR signalling model
(Lean e al., 1980). Acco ding o his model, ecep o is in a dynamic
equilib ium be ween inac i e (R) and ac i e (R*) con o ma ional s a es. Based
upon his model, neu al an agonis s ha e iden ical a ini ies o inac i e and
ac i e con o ma ional s a es, whe eas agonis s exhibi highe a ini y o he
ac i e s a e. Because agonis s ha e highe a ini y o he ac i e con o ma ion
o he ecep o , agonis binding s abilizes GPCR in i s ac i e s a e, shi ing he
dynamic equilib ium om R o R*.
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21
The maximal e ec o an agonis (e icacy) is di ec ly dependen upon he
di e en ial a ini y o ligand o inac i e ecep o con o ma ion (R) e sus ac i e
ecep o con o ma ion (R*). Con e sely, in e se agonis s exhibi highe a ini y
o inac i e ecep o s a e (R). The e o e, in e se agonis binding esul s in he
s abiliza ion o he inac i e s a e (R), shi ing he dynamic equilib ium om R*
o R. Howe e , he in e se agonis e icacy is also dependen upon he
magni ude o cons i u i e ac i i y (Figu e 1.5).
Figu e 1.5: Schema ic ep esen a ion o agonis , an agonis and in e se agonis binding o
di e en unc ional s a es o he GPCR. Agonis binds wi h high a ini y o ac i e con o ma ion
o ecep o (R*), whe eas in e se agonis p e e en ially binds and s abilizes ecep o in he
inac i e s a e (R). Neu al an agonis binds wi h he same a ini y bo h ac i e and inac i e
s a es o GPCR. Illus a ion om Mugu uza e al., 2013.
Acco ding o he ex ended e na y complex model, a ecep o is able o swi ch
om an ac i e o inac i e s a e in absence o a ligand (Sammama e al., 1993).
In his sense, in e se agonism is conside ed he pha macological p ope y o
a d ug o an agonize he ac ion o agonis s, and simul aneously dec ease
basal cons i u i e ac i i y o ecep o signalling. Consequen ly, demons a ion
o in e se agonis p ope ies equi es he exis ence o cons i u i e basal
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22
ac i i y ha will be educed by he in e se agonis . The dec ease o cons i u i e
ac i i y mus be sensi i e o blockade wi h an an agonis , and mus be absen
in knock-ou animals o he in ol ed ecep o (Aloyo e al., 2009). A e in e se
agonism scien i ic desc ip ion, many an agonis o iginally hough o be
“neu al” u ned ou o be in e se agonis ligands (S ange, 2002; Kenakin,
2004).
Al hough hese indings indica e a po en ial use ulness o in e se agonis s,
he e is li le in o ma ion abou he use o pha macological p ope ies o hese
d ugs in he apeu ics. Fo example, soma ic ecep o mu a ions leading o
cons i u i e ac i e ecep o s a e a causal ac o in ce ain diseases, such as
male p ecocious pube y (Kosugi & Mo i, 1995). Thus, in e se agonis s migh
be bene icial he e, since hey would dec ease ecep o s basal ac i i y induced
by he mu a ion. In his case, neu al an agonis s would p esumably be o li le
use, in absence o inc eased endogenous ligand le els (Lig e al., 2000).
O e all, he concep o in e se agonis migh p o ide new oppo uni ies in such
sc eening s a egies. Fo ha pu pose, unc ional assay desc ibing cons i u i e
ac i i y a e need. These assays could con ibu e o he iden i ica ion o agonis
as well as in e se agonis ligands.
1.2.4 GPCRs and biased signalling
As men ioned abo e, GPCRs can signal simul aneously h ough pa allel
pa hways such as he e o ime ic G-p o eins, β-a es ins and GRKs. In his
con ex , se e al ligands ha e been desc ibed o di e in hei abili y o engage
he di e en signalling pa hways coupled o he espec i e GPCR, a e m
called biased signalling o unc ional selec i i y (Azzi e al., 2003; Galand in e
al., 2007; Pe ez & Ka nik, 2005; Kenakin, 2011; Kenakin, 2012; Smi h e al.,
2018). Biased agonis s a e heo e ically able o s abilize di e en ecep o
con o ma ions, exhibi ing di e en a ini ies o he mul iple signal ansduce s
o he GPCR. They p oduce, as consequence, di e en cellula esponses. In
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23
con as , o he ligands can equally ac i a e all signalling pa hways. These
d ugs a e e med balanced agonis s o un-biased ligands.
In e es ingly, ac i a ing o inhibi ing speci ic signalling cascades ep esen s a
new app oach o e ec selec i i y, which could yield o imp o ed
he apeu ically e ec i e d ugs, wi h lowe side e ec s. In his way, he sea ch
o ligands o a pa icula signalling pa hway, a he han selec i e o speci ic
ecep o s, is one o he main challenges o d ug de elopmen nowadays
(Koma su e al., 2019).
The bes well-known example o biased signalling is p esen in opioids
pha macology (Che e al., 2021). Biased signalling in ol es a di e en ial
ac i a ion be ween Gαi-p o eins and β-a es ins. The e is e idence indica ing
ha he apeu ic e ec o μ-opioid ecep o agonis s, including analgesia, is
Gαi-p o ein media ed, while ad e se e ec s, such as espi a o y dep ession
and cons ipa ion, a e mo e ela ed o β-a es in ec ui men (Bohn e al., 1999).
In his sense, se e al opioid ligands we e de eloped in o de o ind G-p o ein
biased ligands wi h imp o ed he apeu ic e ec s. Recen ly, TRV130
(olice idine) demons a ed in i o unc ional selec i i y o Gα-p o eins, and
showed o be sa e o managemen o pain acco ding o a phase III clinical ial
(Singla e al., 2019). Howe e , o he s udies poin ed ou ha biased opioid
ligands de elopmen is con o e sial and ha u he esea ch is needed
s udying each d ug’s di e en signalling pa hways (Gillis e al., 2020; Kliewe
e al., 2020).
Ano he aspec o be conside ed o d ug de elopmen is he small change in
ligand s uc u e ha can esul in la ge changes in unc ional selec i i y p o iles
(Shonbe g e al., 2014). Fo example, LSD and lisu ide e oke dis inc in i o
cellula signalling and in i o esponses (González-Maeso e al., 2003,
González-Maeso e al., 2007), despi e he s uc u al ea u es and sha ed
a ini y o he 5-HT2AR. Mo eo e , ispe idone and i s ac i e me aboli e
palipe idone a e a ypical an ipsycho ics, which only di e in a single hyd oxyl
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24
g oup. They show di e en pha macological p o iles ha in luence in hei
unc ional selec i i y p o ile (Cla ke e al., 2013). These ac s highligh suppo
he impo ance o deep s uc u e- unc ional selec i i y ela ion s udies (Be g &
Cla ke, 2018).
Al hough many s udies ha e shed ligh on ligand bias p o iles o di e en
compounds, mos o he s udies a e limi ed o ew signalling pa hways, such
as G-p o eins e sus β-a es ins. Howe e , many o he possibili ies a e
easible, and all signalling pa hways should be conside ed when cha ac e izing
he indi idual ligand e icacy (Figu e 1.6).
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Figu e 1.6: Schema ic ep esen a ion o biased agonism on GPCR. Hypo he ical and
simpli ied example o μ-opioid ecep o biased agonism. G-p o ein signalling pa hway
unde goes he apeu ical e ec while β-a es ins cause side e ec s. Di ec in e ac ion o un-
biased ligands would esul s in ac i a ion o bo h signalling pa hways, wi h he apeu ic and
side e ec s. Howe e , unc ional o biased agonis s a ou p e e en ially one o he signal
cascades, like G-p o eins, p omo ing he apeu ic e ec s o e side e ec s.
1.2.5 E alua ion o cons i u i e ac i i y, in e se agonism and
unc ional selec i i y
GPCR implica ion in di e en diseases has inc eased he numbe o s udies
analysing di e en ligands p o ile o a ge hese ecep o s. Because o hei
high ele ance, se e al assays ha e been de eloped wi h a iew o
cha ac e ize he unc ional p o ile o di e en d ugs.
Classical unc ional assays measu ed downs eam messenge s, such as Ca2+
elease, IP3 accumula ion o cAMP p oduc ion, in o de o de e mine he
unc ional p o ile o a d ug a e binding a GPCR. Ad an ages o measu ing
second messenge s a e ha unmodi ied ecep o s, in na i e issues, can be
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26
s udied. These assays ha e been b oadly used o be e compa ison be ween
di e en publica ions.
E en i hese echniques a e widely used in assays wi h subs an ial
ampli ica ion, in se e al assays bo h ull and pa ial agonis could each he
same maximal esponse (Smi h e al., 2018). Mo eo e , i is no clea he
second messenge a ibu ion o speci ic G-p o ein sub ypes. The e o e, a
nea es unc ional quan i ica ion o d ug- ecep o in e ac ion was seen as
necessa y.
A di ec e alua ion o GPCR ac i a ion can be made by measu ing he
s imula ion o inhibi ion o guanine nucleo ide exchange on G-p o ein ecep o s
using adiolabelled GTP analogues. This app oach is called sulphu 35-
labelled guanosine-5´-O-(γ- hio)- iphospha e ([35S]GTPγS) binding assay.
Measu emen o G-p o ein ac i a ion is he unc ional consequence o ecep o
occupancy as ea lies e en , and i is no subjec ed o signal ampli ica ion
(González-Maeso e al., 2000; Ha ison & T ayno , 2003). Con en ional
[35S]GTPγS binding assays a e only limi ed o he measu emen o GPCR
coupling o Gαi/o-p o eins; p obably due o hei highe a es o nucleo ide
exchange and cons i u i e ac i i y (Sei e & Wenzel-Sei e , 2002).
Ne e heless, di e en assays ha e been de eloped in o de o measu e
di e en Gα-p o ein esponses such as [35S]GTPγS binding
immunop ecipi a ion assay using magne ic beads o scin illa ion p oximi y
assays (SPA) (Diez-Ala cia e al., 2021b). This assay combines classical
[35S]GTPγS binding assay wi h immunop ecipi a ion. This echnique can be
applied o s udying ecep o o in e es in cell cul u es and in na i e issues.
Mo eo e , his assay allows he e alua ion o cons i u i e ac i i y using
pha macological ools, as in e se agonis s, in na i e issue (Diez-Ala cia e al.,
2021b). Ne e heless, his [35S]GTPγS binding assay combined wi h
immunop ecipi a ion is cu en ly limi ed o Gα-p o eins, while β-a es in-
media ed esponse is no possible o be s udied as ye .
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In addi ion o p e ious assays, luo escence and bioluminescence esonance
ene gy ans e (FRET and BRET) assays, which a e echnologies o de ec
p o ein-p o ein in e ac ion and dynamic con o ma ional changes ha e been
de eloped o di ec ly moni o ing con o ma ional changes in GPCRs, G-
p o eins, and β-a es ins (Ange s e al., 2000; Galés e al., 2006; Zhou e al.,
2021; W igh & Bou ie , 2021). These assays a e sui able o moni o ing li e
cell e en s in eal ime, and display adap abili y o high- h oughpu sc eening.
Despi e hese ad an ages, he in oduc ion o luo escen dono and accep o
molecules needs o be conside ed as a ac o ha could in luence he obse ed
ou comes (Po ie & S o e, 2022). The use o BRET is only sui able o in i o
cell cul u e sys ems, whe eas in li e animals, he applica ion is limi ed o
s udies in supe icial loca ions. This assay is no sui able o he s udy o pos -
mo em issue igh now (D ino ec e al., 2012).
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1.3 Se o onin 2A ecep o (5-HT2AR)
1.3.1 Gene al aspec s
5-HT egula es a wide ange o physiological p ocesses in he cen al ne ous
sys em (CNS), including memo y, pe cep ion, cogni ion, emo ion, mood and
consciousness (Be ge e al., 2009). Dys unc ion o 5-HT sys em has been
implica ed in nume ous psychia ic diso de s (Hoye , 2020). The e o e,
pha macological manipula ion o 5-HT sys em has he apeu ic po en ial.
Mammalian 5-HT ecep o s a e now classi ied in o 14 s uc u ally and
pha macologically dis inc sub ypes (Hannon & Hoye , 2008), which a e
di ided in o se en main amilies (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5-
HT7 ecep o s), based on ope a ional (d ug ela ed cha ac e is ics),
ansduc ional and s uc u al cha ac e is ics (Figu e 1.7). A a s uc u al le el,
only 5-HT3 ecep o is a ligand-ga ed ion channel (Ma icq e al., 1991), whils
he es o 5-HT ecep o s a e membe s o GPCR supe amily (K oeze & Ro h,
1998).
The 5-HT2 ecep o s a e among he mos widely s udied 5-HT ecep o s. The
5-HT2 ecep o amily is di ided in h ee di e en ecep o s, e med as 5-HT2AR
5-HT2BR and 5-HT2CR, wi h 46-50% sequence homology (Hoye e al., 2002).
Mo eo e , ansmemb ane domains o 5-HT2AR and 5-HT2CR sha e 80%
sequence homology and e y close pha macological p o iles (Boess & Ma in,
1994). Thus, de elopmen o selec i e d ugs o each ecep o is a
undamen al challenge due o he simila binding pocke s. A p esen , i is no
easy ma e o disc imina e pha macologically 5-HT2 ecep o amily membe s,
due o he lack o uly selec i e 5-HT2AR o 5-HT2CR ligands.
Radioligand binding echniques we e ini ially used o cha ac e iza ion o 5-HT
ecep o s in mammalian b ain homogena es. Du ing he cou se o ea ly
in es iga ions using adioligands, wo classes o 5-HT binding si es we e
desc ibed. High a ini y si es o [3H]5-HT co esponded o he 5-HT1 ecep o
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sub ype, while low a ini y si es we e designa ed as 5-HT2 ecep o sub ype.
The disco e y o [3H]ke anse in as a easonably selec i e ligand o 5-HT2AR
(Leysen e al., 1982) u ned o be a huge ad ance, and g ea ly po en ia ed he
in es iga ion o 5-HT2AR (Leysen e al., 1982). Howe e , ke anse in also
shows mode a e a ini y o 5-HT2CR, among o he ecep o s (Choudha y e
al., 1992). Du ing he las yea s, new selec i e 5-HT2AR ligands ha e been
subsequen ly de eloped o he e alua ion o 5-HT2AR, such as [18F]al anse in,
[3H]MDL100907 (L´Es ade e al., 2018) and [11C]Cimbi-36 (E up e al.,
2014).
Sequence analysis o 5-HT2AR coding egions disclosed a high o e all gene ic
conse a ion ac oss species. The excep ion is a non-conse ed change, mos
no ably a esidue 242, co esponding o a se ine in humans and an alanine in
oden s. I has been desc ibed ha his change migh in luence in a ini y and
e icacy o a a ie y o 5-HT2AR agonis (López-Giménez & González-Maeso,
2018; Kim e al., 2020; Slocum e al., 2021).
1.3.2 Localiza ion and unc ion o 5-HT2AR in CNS
5-HT2AR and 5-HT2CR a e bo h exp essed in CNS. Pe iphe ally, 5-HT2AR is
ound in pla ele s, ascula smoo h muscle cells and ocula issue (Leysen,
2004). Ra he , 5-HT2BR is p ima ily ound in pe iphe y and, speci ically, in
human hea ca diac al es (Bona en u e e al., 2005) (Figu e 1.7).
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36
2006). The AA elease seems o be media ed by a complex mechanism ha
in ol es RhoA signalling, MAPK and ex acellula signal- egula ed kinases
(ERK). All o hem a e depending on Gα-p o eins, bu di e en om Gαq/11-
p o eins, such as Gα12/13- and Gαi/o-p o ein sub ypes (Ku asch-O baugh e al.,
2003a; Ku asch-O baugh e al., 2003b). Se e al s udies e ealed ha he 5-
HT2AR has abili y o igge he ac i a ion o Gαi/o-p o eins, a coupling
phenomenon ha unde lies hallucinogenic ac ions caused by psychedelic
d ugs (González-Maeso e al., 2007).
Addi ionally, se e al phospho yla ed downs eam p o eins ha e been
iden i ied by phosphop o eomic s udies a e 5HT2AR ac i a ion (Ka aki e al.,
2014). This includes ERKs, ibosomal S6 kinase (RSK-2) (S achan e al.,
2008) and a es ins (Schmid e al., 2008; Schmid & Bohn, 2010). Fu he mo e,
5-HT2AR in e ac ion wi h a la ge numbe o sca olding p o eins has been
desc ibed, including pos synap ic densi y p o ein 95 (PSD-95) and o he PSD-
95/discs la ge/zonula-occludens 1 (PDZ)-domain-con aining p o eins (Xia
G ay e al., 2003; Bécamel e al., 2004; Abbas e al., 2009), ca eolin-1
(Bha naga e al., 2004; Somme e al., 2009), and mic o ubule associa ed
p o ein A1 (MAP1A) (She le e al., 2006). Speci ically, 5-HT2AR in e ac ion
wi h PSD-95, ca eolin-1, RSK-2 and β-a es in2 ha e been desc ibed o be
essen ial o modula ion o he unc ional signalling.
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37
Figu e 1.10: Illus a ion o he main in acellula signal ansduc ion pa hways associa ed wi h
5-HT2AR. 5-HT2AR signalling in ol es he s imula ion o Gαq/11-p o eins, which p omo es PLC-
media ed ca alysis o PIP2 o IP3 and DAG hyd olysis, hus ac i a ing PKC and ele a ing
cy osolic Ca2+. Howe e , 5-HT2AR also ac i a es o he signalling pa hways. Fo example, i
media es AA elease, p esumably h ough he ac i a ion o PLA2, media ed by a complex
mechanism in ol ing Rho and p38. All hose mechanisms a e depending on Gα-p o eins,
di e en om Gαq/11, such as Gα12/13. 5-HT is sugges ed o ac i a e 5-HT2AR and assemble
p o eins, such as β-a es in2, S c and Ak . In con as , hallucinogenic 5-HT2AR agonis s do no
equi e his complex ec ui men , and elici di e en ial gene egula ion ia Gαi/o-p o ein
sub ypes. Illus a ion om Iba a-Lecue e al., 2021.
1.3.5 5-HT2AR ligands
5-HT2AR binding d ugs belong o s uc u ally di e se chemical classes:
indolealkylamines, phenylalkylamines, a ylpipe azines, alkylpipe idines,
alkylpipe azines and polycyclic/ icyclic agen s, among o he s (Wes kae mpe
& Glennon, 2002). 5-HT2AR d ugs (ei he agonis s, an agonis s o in e se
agonis s) ep esen some o he mos impo an d ugs in neu opsychia y,
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including a ypical o second-gene a ion an ipsycho ics, psychedelics and
an idep essan d ugs (Ba nes e al., 2021). The e o e, many e o s ha e
ocused on de elopmen o 5-HT2AR selec i e ligands. Howe e , many o he
ligands ha bind 5HT2AR also bind 5-HT2CR, wi h simila a ini ies, gi en he
sequence homology ound be ween he ansmemb ane po ions o bo h
ecep o s.
1.3.5.1 Agonis s
5-HT2AR agonis s and pa ial agonis s ha e adi ionally been di ided in o h ee
s uc u al g oups: e golines (LSD, lisu ide and pe golide), indolealkylamines
(psilocin) (bo h included in indoleamines g oup) and phenylalkylamines
(mescaline, 2,5-dime oxy-4-iodoamphe amine ((±)DOI)) (Halbe s and &
Geye , 2011; Nichols, 2016). The physiological ligand 5-HT is a non-selec i e
agonis ha binds o all 5-HT ecep o s. In 5-HT2 ecep o amily he a ini y
o de o his molecule is 5-HT2BR > 5-HT2AR > 5-HT2CR (Bax e e al., 1995).
Some o he 5-HT2AR agonis ligands display hallucinogenic e ec s in humans
(LSD, psilocybin, psilocin and mescaline). In his sense, classic hallucinogens
o se o one gic psychedelics a e desc ibed as subs ances capable o al e ing
hough s, pe cep ion, and mood, ia ac i a ion o 5-HT2AR (Glennon e al.,
1984; Vollenweide e al.,1998; González-Maeso e al., 2007; Halbe s ad ,
2015; Madsen e al., 2019). Despi e his e ec s has been epo ed o be
media ed by 5-HT2AR, classic hallucinogens a e no selec i e and bind o he
ecep o s.
In his sense, phenylalkylamine hallucinogens a e selec i e o 5-HT2
ecep o s, including 5HT2AR, 5HT2BR and 5-HT2CR. Among he di e en
molecules, (±)DOI has been desc ibed as a po en bu non-speci ic ligand o
5-HT2AR and 5-HT2CR. (±)DOI has o en been he agonis o choice o ex
i o/in i o s udies p obing 5-HT2AR-media ed unc ions (Nelson e al., 1999;
In oduc ion
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Pigo e al., 2012; Canal e al., 2013). [35S]GTPγS expe imen s in human pos -
mo em b ain and 5-HT2AR knock-ou animals con i med ha (±)DOI beha es
as 5-HT2AR and 5-HT2CR pa ial agonis (Diez-Ala cia e al., 2019; Ga cia-Bea
e al., 2019; Mune a-A a e e al., 2020).
Du ing he las yea s, a new class o ligands ha e been de eloped wi h highe
a ini y on 5HT2AR, based on N-benzylphene hylamine (NBOMe) sca old such
as n-(2-me hoxybenzyl)-2,5-dime hoxy-4-b omophenyle ylamine (25B-
NBOMe, Cimbi-36) and 25-CN-NBOH (Jensen e al., 2020). These molecules
a e sui able ools o PET imaging and pha macological s udies. 25-CN-NBOH
and Cimbi-36 show pa ial agonis ac i i y, exhibi ing highe a ini y o 5-HT2AR
s 5-HT2CR and 5-HT2BR (Hansen e al., 2014; Jensen e al., 2017).
On he o he hand, indolealkylamines like psilocin ( he ac i e me aboli e o
psilocybine) and e golines, like LSD, a e ela i ely non-selec i e 5-HT
ecep o s ligands, displaying mode a e o high a ini y o 5-HT1 and 5-HT2
ecep o s (Ro h, 2007; Nichols, 2016; Wacke e al., 2017). Mo eo e , LSD
binds wi h high a ini y o o he 5-HT ecep o s, bu also o dopamine ecep o s
(Halbe ad & Geye , 2011; Bo o o-Escuela e al., 2014).
Head- wi ch esponse (a apid side- o-side mo emen o head) ep esen he
mos widely used oden esponse o e alua e hallucinogenic e ec s induced
by psychedelics h ough 5-HT2AR. This is conside ed a c i ical es o in i o
e alua ion o unc ional esponse o psychedelics. Se e al s udies using
selec i e an agonis and 5HT2AR knock-ou animals ha e demons a ed ha
head- wi ch esponse is e y selec i e o 5-HT2AR, and is limi ed o
psychedelics (González-Maeso e al., 2007; Canal & Mo gan, 2012). Howe e ,
chemically closely ela ed 5-HT2AR agonis like lisu ide, e go amine and
pe golide (González-Maeso e al., 2003; González-Maeso e al., 2007) does
no induce his beha iou . Lisu ide and pe golide a e e med non-
hallucinogenic 5-HT2AR agonis s. Those compounds, as o he e golines,
p esen a e y complex polypha macological p o ile, showing a ini y o
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se e al amine gic ecep o s, including se o one gic and dopamine gic
ecep o s (Halbe s ad & Geye , 2011). Mo eo e , lisu ide and pe golide a e
conside ed an ipa kinsonian d ugs, due o hei high a ini y o dopamine
ecep o s and 5-HT1AR (Lang y & Clissold, 1990; Ma ona-Lewicka e al.,
2002).
1.3.5.2 An agonis s
One o he la ges and selec i e classes o 5-HT2AR an agonis a e he N-
alkylpipe idones, being ke anse in he mos widely used o yea s. Ke anse in
is selec i e o 5-HT2AR s 5-HT2CR (15-80 old) and 5-HT2BR (500-1000 old)
(Je man e al., 2001; Knigh e al., 2004; Diez-Ala cia e al 2019). Chemically,
i anse in is close o ke anse in, is highly po en , ela i ely selec i e and long
ac ing 5-HT2AR an agonis , bu is also desc ibed as in e se agonis (Bonhaus
e al., 1995). Ano he 5-HT2AR an agonis , chemically ela ed o ke anse in, is
he benzoylpe idine al anse in, ha has been desc ibed as po en and
selec i e 5-HT2AR an agonis wi h a 20- old g ea e a ini y o human 5-HT2AR
e sus human 5-HT2CR (Tan e al., 1999). Howe e , in e se agonis p ope ies
on 5-HT2AR ha e been p e iously epo ed o al anse in (Aloyo e al., 2009;
Diez-Ala cia e al., 2019).
O he 5-HT2AR-selec i e (o p e e ing) ligands ha e been de eloped such as
MDL100907, also known as olinanse in, and MDL-11,939. Volinanse in is a
po en 5-HT2AR an agonis and shows 300- old selec i i y o 5-HT2AR ecep o
e sus 5-HT2CR and o he GPCRs (So ensen e al., 1993; López-Giménez e
al., 1998). Volinanse in and i anse in we e e alua ed as an ipsycho ics o
schizoph enia; howe e , hey u n no o be success ul (Jones e al., 2020).
Nowadays, olinanse in is used as e e ence 5-HT2AR an agonis due o i s
high selec i i y.
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Recen ly, new d ugs as nelo anse in and epli anse in ha e been de eloped
o insomnia ea men . These compounds show high a ini y o 5-HT2AR wi h
20- old selec i i y e sus 5-HT2CR (Rinaldi-Ca mona e al., 1992; Al-Shamma
e al., 2010).
Pima anse in, also known as ACP-103, is desc ibed as a highly selec i e d ug
o 5-HT2AR, lacking a ini y o o he ecep o s excep 5-HT2CR (30- old lowe
selec i i y), and no signi ican ac i i y on any o he GPCR (Vano e e al.,
2006; Abbas & Ro h, 2008). The US Food and D ug Adminis a ion (FDA) has
app o ed his d ug o hallucina ions and delusions ea men associa ed wi h
Pa kinson´s disease psychosis (Cummings e al., 2014).
Some o he 5-HT2AR an agonis , al hough selec i e o 5-HT2A/2C ecep o s
bind wi h modes o high a ini y o dopamine gic, his amine gic, and/o
ad ene gic ecep o s. Thus, a ypical an ipsycho ics (e.g. ispe idone, clozapine
and olanzapine) and icyclic an idep essan s (e.g. ami ip yline, clomip amine,
and imip amine) also bind o 5HT2AR as an agonis (Ro h e al., 2004; Mel ze
& Massey, 2011; Mel ze , 2012).
1.3.6 5-HT2AR unc ional selec i i y
5-HT ecep o s, speci ically 5-HT2AR, we e among he i s GPCR o which
occu ence o unc ional selec i i y was sugges ed (Be g e al., 1998). Se e al
s udies suppo ha d ugs wi h high a ini y o 5-HT2AR (bo h agonis and
an agonis ) s abilize dis inc ecep o con o ma ions. This ac lead o biased
in e ac ions o unc ional selec i i y wi h a ious downs eam e ec o s, which
include he canonical Gαq/11-p o ein and non-canonical signalling (López-
Giménez & González-Maeso, 2018). In his sense, biased agonism has been
p oposed o explain he ac ha hallucinogenic and non-hallucinogenic d ugs
ac i a e he same popula ion o co ical 5-HT2AR, bu hey di e in Gα-p o ein
egula ion, ansc ip ome inge p in s, elec ophysiological esponses, as well
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as, beha iou al s a es (González-Maeso e al., 2003; González-Maeso e al.,
2007; Ka aki e al., 2014; Bane jee & Vaidya, 2020).
One o he i s e idence o unc ional selec i i y a ose om he inding o Be g
and co-wo ke s, ega ding abili y o his ecep o o signal no only ia Gαq-
dependen PLC ac i a ion, bu also ia PLA2 (Be g e al., 1998). Speci ically,
PLC-dependen IP inc ease and AA elease, ia PLA2 ac i a ion, we e
measu ed o demons a e he di e ence o e icacies depending on which
signal ansduc ion pa hway was ac i a ed. Acco ding o hose esul s, 5-HT
p e e en ially ac i a ed PLC-IP pa hway, whe eas LSD a ou ed PLA2-AA
pa hway (Be g e al., 1998; Ma í-Solano e al., 2015).
Hallucinogenic and non-hallucinogenic 5-HT2AR agonis s also di e en ially
in luence gene exp ession pa e ns (González-Maeso e al., 2003; González-
Maeso e al., 2007). This app oach we e es ed in cells and mouse
soma osenso y co ex, showing di e en ansc ip ome inge p in be ween
hallucinogenic and non-hallucinogenic 5-HT2AR agonis s. Thus, bo h
hallucinogenic and non-hallucinogenic d ugs induced c- os exp ession.
Howe e , ansc ip s eg -1 and e g-2 we e ac i a ed by hallucinogens-like
(±)DOI and LSD, bu exp ession o hese wo genes was una ec ed by non-
hallucinogenic agonis s (lisu ide and e go amine) (Figu e 1.11). These esul s
also indica ed ha all 5-HT2AR agonis s ac i a ed 5-HT2AR coupled o PLC,
whe eas hallucinogenic-dependen esponse in ol ed PTX-sensi i e
he e o ime ic Gαi/o p o eins. No ably his hypo hesis was alida ed by
quan i a i e phosphop o eomic app oach (Ka aki e al., 2014). This hypo hesis
was s ongly suppo ed by compa ison be ween unc ional selec i i y p o ile o
he hallucinogenic d ug (±)DOI wi h he non-hallucinogenic d ug pe golide in
pos -mo em human b ain co ex. Bo h 5-HT2AR agonis s induced Gαq/11-
p o ein ac i a ion, while Gαi1-p o ein s imula ion was only limi ed o he
hallucinogenic d ug (±)DOI (Mune a-A a e e al., 2020). In conclusion,
hallucinogenic agonis s, such as LSD and psilocybin, p omo e ac i a ion o
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canonical Gαq/11-p o ein cascade as well as a Gαi/o p o ein-media ed signalling
pa hway. In con as , chemically analogous 5-HT2AR agonis s lacking o
hallucinogenic p ope ies, like lisu ide, e go amine and pe golide, only
s imula e Gαq/11-p o ein-dependen pa hway.
O he s udy e ealed a dis inc signalling signa u e be ween hallucinogenic
and non-hallucinogenic 5-HT2AR agonis s. The au ho s no ed highe le els o
phospho-PLC, pERK, pCaMII, pCREB as well as highe le els o IP and DAG
p oduc ion a e ecep o s imula ion wi h (±)DOI han hose obse ed wi h
lisu ide (Bane jee & Vaidya, 2020).
Addi ionally, 5-HT2AR ac i a es o he signal ansduc ion cascades, such as β-
a es in, besides G-p o ein media ed PLC-β pa hway, in a ligand-dependen
manne . 5-HT and (±)DOI can di e en ially ac i a e 5-HT2AR in cellula models
and in i o head- wi ch esponse. By using mice lacking β-a es in-2, he
absence o head- wi ch esponse as demons a ed in p esence o 5-HT. Ak
phospho yla ion also seems o be p esen a e ac i a ion o β-a es in-2,
whe eas is absen in p esence o (±)DOI. Howe e , he hallucinogenic d ug
(±)DOI seems o media e head- wi ch esponse independen o β-a es in-2. In
conclusion, hese s uc u ally dis inc agonis s elici di e en signal
ansduc ion and a icking pa e ns upon 5-HT2AR ac i a ion (Schmid e al.,
2008; Schmid & Bohn, 2010).
Recen ly, i was sugges ed ha head- wi ch esponse o he hallucinogenic
d ug LSD was β-a es in-2-dependen and β-a es in-1-independen
(Rod iguiz e al., 2021). Howe e , non-hallucinogenic d ugs ha bind o 5-
HT2AR s imula ed β-a es in-2 ec ui men , in con as o p e ious indings
(Cao e al., 2022). The e o e, implica ion o β-a es in-2 in he hallucinogenic
esponse o 5-HT2AR agonis s needs u he esea ch.
On he o he hand, he a ypical an ipsycho ic clozapine inhibi s 5-HT2AR
signalling h ough a G-p o ein-dependen mechanism. I induces ecep o
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in e naliza ion and Ak phospho yla ion, ega dless o ecep o in e ac ion wi h
β-a es in-2. Thus, 5-HT and clozapine use dis inc molecula mechanisms o
achie e he same 5-HT2AR media ed downs eam e en s: Ak phospho yla ion
and ecep o in e naliza ion. This way, Ak phospho yla ion is equi ed o
clozapine-media ed e ec s supp ession, when his is s udied on
schizoph enic-like beha iou s induced by MK-801 and PCP adminis a ion in
mice (Schmid e al., 2014)
The complexi y o biased signalling o elici selec i e unc ional esponses has
p o ided an al e na i e a enue o de elop no el he apeu ics wi h inc eased
clinical e ec and less side e ec s. Howe e , he use o biased agonism and
o he pha macological p ope ies, such as in e se agonism o an agonism,
emains ques ionable o de elopmen o new he apies o schizoph enia and
dep ession.
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Figu e 1.11: Schema ic in acellula signalling pa hways o 5-HT2AR coupling o hei
downs eam e ec o s.
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1.5 An ipsycho ics
An ipsycho ics a e used as i s line medica ion o ea schizoph enia, and a e
bes classi ied in o wo ca ego ies: Fi s -gene a ion an ipsycho ics o ypical
an ipsycho ics (e.g., halope idol, chlo p omazine) and second-gene a ion
an ipsycho ics o a ypical an ipsycho ics (e.g., clozapine, ispe idone).
Exis ing medica ion o ea schizoph enia is e ec i e o ea ing posi i e
symp oms bu ha e li le impac on nega i e o cogni i e symp oms (Conn e
al., 2008; Leuch e al., 2009). This ac usually con ibu es o poo unc ional
ou come. Consequen ly, he e is an u gen need o iden i y new molecula
a ge s and o de elop mechanis ically no el compounds o mo e e ec i e
and be e - ole a ed an ipsycho ic agen s ha could imp o e he he apeu ic
e ec s and he sa e y p o ile.
1.5.1 Fi s -gene a ion o an ipsycho ics (Typical
an ipsycho ics)
The mechanism o ac ion o ypical an ipsycho ics is he an agonism o
dopamine D2R, D3R and/o D4Rs. This inding led o he hypo hesis ha he e
is a dys egula ion o dopamine gic sys em ha co esponds o a hype ac i i y
o mesolimbic pa hway, and a hypo unc ion o mesoco ical pa hway. Thus,
blockade o D2R is associa ed wi h a educ ion o dopamine and psycho ic
symp oms (Seeman, 1992; Ma de e al., 1993). The blockade o D2R is also
ela ed o ex apy amidal side e ec s and hype p olac inemia (Miyamo o e al.,
2008).
Halope idol is he p o o ypical ypical an ipsycho ic (Figu e 1.13). Typical
an ipsycho ics ha e he abili y o educe posi i e symp oms and isk o
elapse, imp o ing clinical ou comes o many pa ien s wi h schizoph enia.
Howe e , nea 30% o pa ien s ha e li le o no esponse o ypical
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an ipsycho ics and also null bene i o nega i e symp oms o cogni i e
impai men (Conley & Kelly, 2001; Legge e al., 2020).
Figu e 1.13: Chemical s uc u e o halope idol.
1.5.2 Second-gene a ion o an ipsycho ics (A ypical
an ipsycho ics)
Second-gene a ion o a ypical an ipsycho ics we e de eloped looking o
educe ex apy amidal side e ec s a he apeu ically e ec i e doses (Figu e
1.14). A ypical an ipsycho ics display highe a ini y o 5-HT2AR compa ing o
D2R amily, which explains lowe ex apy amidal e ec s (Mel ze e al., 1989;
Fa de e al., 1992). Hence, 5-HT2AR-D2R ac i i y a io has been conside ed o
be mo e ele an o p edic a educe side e ec liabili y han imp o ed e icacy
(Ebd up e al., 2011). Un o una ely, second-gene a ion an ipsycho ics ha e
an inc eased isk o weigh gain, and dis u bances in glucose and lipid
me abolism (Muench & Hame , 2010; Wes on-G een e al., 2013; G ajales e
al., 2019).
The disco e y o clozapine con ibu ed o he in oduc ion o new d ugs wi h
mo e bene icial pha macological p o ile han i s -gene a ion an ipsycho ics
(Mel ze e al., 1989). The complex pha macological p o ile o clozapine has
made he ask o de e mining i s mechanism o ac ion ex emely di icul .
Clozapine has easonable a ini y o a la ge numbe o ecep o s, including
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se e al his amine gic, se o one gic, ad ene gic, dopamine gic and choline gic
sub ype (Cowa d, 1992; Nuci o a e al., 2017). In addi ion o ac i i y on
dopamine and 5-HT2AR, clozapine is also 5-HT1AR pa ial agonis , which is
hough o be bene icial in e ms o educing cogni i e and nega i e symp oms.
Musca inic ecep o s a e also a ec ed by clozapine, by blocking M1, M2, M3
and M5 ecep o s (M1R, M2R, M3R, M5R), while s imula ed M4 ecep o (M4R).
Mo eo e , clozapine an agonizes his amine ecep o s, which is ela ed o
seda ion e ec s. Clozapine also blocks ad ene gic ecep o s, which causes
hypo ension and achyca dia (Cowa d e al., 1992; Nuci o a e al., 2017).
Clozapine is also associa ed wi h an ele a ed isk o po en ially le hal
hema o oxici y (ag anulocy osis and neu openia), an ad e se e ec ha
es ic s i s clinical use (Alphs e al., 1991). Consequen ly, new second-
gene a ion an ipsycho ics we e in oduced, such as ispe idone, olanzapine,
que iapine, among o he s, wi h an e o o educe side e ec s ela ed o blood
dysc asias.
Olanzapine is a chemical analogue o clozapine wi h simila pha macological
p ope ies. Howe e , i is no associa ed wi h a isk o ag anulocy osis. As
expec ed, olanzapine p esen s highe a ini y o 5-HT2AR han o DA ecep o s.
I also blocks his amine, musca inic and ad ene gic ecep o s bu is weake
compa ing wi h clozapine. Weigh gain and seda ion a e he mos equen side
e ec o olanzapine (Ful on & Goa, 1997; Leuch e al., 2013).
Que iapine ac s as D1R, D2R and 5-HT2AR an agonis as well as 5-HT1AR
pa ial agonis . Side e ec s induced by que iapine a e associa ed o α1-
ad ene gic and his amine gic an agonism (Miodownik & Le ne , 2006).
Rispe idone is ano he a ypical an ipsycho ic d ug. The apeu ic e ec o
ispe idone esul s om bo h D2R and 5-HT2AR an agonism, showing s onge
a ini y o 5-HT2AR han o D2R (Cohen, 1994). Mo eo e , his d ug also
causes α1-ad ene gic and his amine ecep o blockade. Rispe idone is no only
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e icien in ea ing posi i e symp oms, bu also nega i e and cogni i e
dis u bances. This ac makes i one o he mos commonly p esc ibed
an ipsycho ics (Mölle , 2005; Chopko & Lindsley, 2018).
Palipe idone is an ac i e me aboli e o ispe idone, which ac s a he same
ecep o s ange.
Figu e 1.14: Chemical s uc u e o clozapine, olanzapine, que iapine, ispe idone and
palipe idone.
In he las yea s, new second-gene a ion an ipsycho ics ha e been de eloped
like asenapine and lu asidone (Miyamo o e al., 2012). Howe e , he imp o ed
e icacy wi h espec o i s -gene a ion an ipsycho ics has ye o be
de e mined. Righ now, clozapine is he only d ug app o ed o esis an
schizoph enia ea men (Conley & Kelly, 2001).
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1.5.3 Thi d-gene a ion o an ipsycho ics
Ano he gene a ion o an ipsycho ics, such as a ipip azole, ha e also been
de eloped. This g oup o d ugs is known as hi d-gene a ion o an ipsycho ics
(Figu e 1.15). Unlike o he neu olep ics, hi d-gene a ion an ipsycho ics a e
no D2R an agonis bu D2R pa ial agonis (Da ies e al., 2004). In high
concen a ions o DA, hose an ipsycho ics compe e wi h DA, and esul s in
pa ial an agonism, leading o clinical bene i s. Con a y, when DA le els a e
low, a ipip azole can bind o D2R and ac as pa ial agonis . Mo eo e ,
a ipip azole also shows pa ial agonis p ope ies o 5-HT1AR. Con a y o
second-gene a ion an ipsycho ics, a ipip azole shows highe a ini y o D2R
han o 5-HT2AR (Chen e al., 2022). Thi d-gene a ion an ipsycho ics a e
e ec i e in alle ia ing psycho ic symp oms wi hou inducing ex apy amidal
side e ec s and hype p olac inemia, as well as lowe weigh gain and
me abolic liabili ies (Liebe man, 2004).
Figu e 1.15: Chemical s uc u e o a ipip azole.
1.5.4 New gene a ion o an ipsycho ics
Di e en e o s o ind new he apies o schizoph enia based on he use o 5-
HT2AR an agonis s, like i anse in and olinanse in, ha e ailed o be
he apeu ically use ul. Due o he lack o e icacy o mono he apy wi h selec i e
5-HT2AR an agonis , di e en esea ches indica e ha 5-HT2AR an agonism
alone is no enough o explain he e icacy o a ypical an ipsycho ics (Miyamo o
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e al., 2012). Thus, D2R blockade seems o be necessa y. Howe e , a po en
and selec i e 5-HT2AR d ug, pima anse in, was de eloped as a new
al e na i e o he ea men o psychosis (Figu e 1.16) (Mel ze & Ro h, 2013).
Pima anse in is he i s app o ed an ipsycho ic ha lacks o dopamine gic
a ini y (Hacksell e al., 2014). Un il now, pima anse in has been es ed as
adjunc i e he apy o schizoph enia in combina ion wi h halope idol and
ispe idone (Mel ze e al., 2012), and has gained FDA app o al o educe
delusions and hallucina ions in Pa kinson’s disease (Cummings e al., 2014).
Mo eo e , pima anse in has shown abili y o educe nega i e symp oms in
schizoph enia pa ien s (Buga ski-Ki ola e al., 2022). Pima anse in has been
desc ibed as 5-HT2AR in e se agonis . Howe e , e idence o his aspec needs
o be demons a ed (Vano e e al., 2006; Nu e al., 2017).
Figu e 1.16: Chemical s uc u e o pima anse in.
I has been ecen ly ecognized ha many second-gene a ion an ipsycho ics
a e 5-HT2AR in e se agonis s a he han neu al an agonis s (Weine e al.,
2001). In con as o an agonis s, in e se agonis s possess nega i e in insic
e icacy, and can a enua e basal cons i u i e signalling ac i i y.
O e all, a be e unde s anding o he mechanism o ac ion o an ipsycho ics
could lead o he design and de elopmen o mo e e ec i e and ole able
d ugs. His o ically, p omiscuous d ugs wi h polypha macological p o ile ha e
been hough o be mo e e ec i e o ea ing CNS disease, al hough his
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ep esen ed p esence o many se e e and po en ially li e- h ea ing side
e ec s. The e o e, design o selec i e d ugs ha would in e ac wi h de ined
molecula a ge s could p obably esul in mo e e ec i e and ole able d ugs.
Hence, biased agonism alone o in combina ion wi h in e se agonism a ise as
al e na i e app oaches o ecep o selec i i y in o de o imp o e he expec ed
unc ional esponses in schizoph enia.
Aims
Aims
61
Despi e much esea ch has been unde aken o de elop new an ipsycho ic
d ugs o ea ing e ec i ely he in insic impai men s, none o he medica ions
cu en ly a ailable has s ood ou o long-las ing e icacy wi hou de imen al
side e ec s. This is pa ially due o poo ly unde s ood neu obiology o
schizoph enia. The e o e, a deepe knowledge o schizoph enia a a
neu obiological le el would enable he iden i ica ion o molecula , cellula
and/o pa hway al e a ions, which could become he apeu ic a ge s o new
d ugs.
Se e al indings sugges ha he 5-HT2AR is in ol ed in he molecula
mechanism esponsible o psycho ic symp oms and hei ea men . On he
one side, he 5-HT2AR is esponsible o he hallucinogenic na u e o
psychedelic d ugs like LSD, psilocybin, mescaline and (±)DOI, which ac i a e
he 5-HT2AR. On he o he hand, a ypical an ipsycho ics commonly used in
schizoph enia would ac as an agonis o e en in e se agonis o he 5-HT2AR.
Di e en s udies by in i o PET neu oimaging and in i o pos -mo em s udies
ha e shown con lic ing esul s abou 5-HT2AR b ain densi y in schizoph enia
subjec s. These con lic ing epo s seem o be ela ed o he use o di e en
adio ace s binding o di e en con o ma ions o he ecep o . The e o e, 5-
HT2AR al e a ions in schizoph enia seem o be mo e ela ed o a ia ions in
molecula s a e o he ecep o a he han al e a ions in exp ession le els. In
his con ex , a ull cha ac e iza ion o 5-HT2AR in e se agonis s in human b ain
becomes an unme need o cla i y he unc ional s a us o his ecep o in
schizoph enia.
The 5-HT2AR is able o ac i a e bo h Gαq/11- and Gαi/o-p o eins depending on
he d ug binding p ope ies. In his con ex , ac i a ion o Gαi/o-p o eins by 5-
HT2AR agonis has been p oposed as a molecula inge p in o hallucinogenic
p ope ies. Mo eo e , highe s imula ion o Gαi1- bu no Gαq/11-p o eins in
esponse o he 5-HT2AR agonis (±)DOI has been desc ibed in pos -mo em
PFC o subjec s wi h schizoph enia. This inding could be in e p e ed as
Subjec s, Ma e ial and Me hods
68
Table 3.1:
Demog aphic cha ac e is ics, pos -mo em condi ions, cause o dea h and oxicological analysis o indi idual cases o schizoph enia
subjec s (
S), non-schzioph enia suicide (NSS) and hei espec i e con ols (C).
Case
Diagnos ic
Gende
(M/F)
Age
(yea s)
PMD(h
ou s)
S o age
(mon hs)
Cause o
dea h
Mechanism o dea h
B ain pH
D ug Blod Le els (mg/L)
B ain Toxicology (ng/g)
S 1
Schzioph enia
F
67
22
17
Na u al
Ca dio espi a o y ailu e
5.8
Nega i e
Nega i e
C 1
Con ol
F
66
17
83
Acciden
Road acciden
6.06
Nega i e
No pe o med
S 2
Schizoph enia
M
34
23
81
Suicide
Jump om a heigh
6.32
Nega i e
Co inine 23.1
C 2
Con ol
M
34
17
69
Acciden
Road Acciden
6.7
Nega i e
Nega i e
NSS 1
Pe sona
li y
diso de
M
34
7
199
Suicide
Hanging
No
pe o med
E hanol 2.7 g/L
Ci alop am 0.1
Oxaca bazepina 6.5
Ci alop am 1579.9
Co inine 3352.8
No ci alop am 368.2
S 3
Schizoph enia
F
53
18
98
Na u al
Haemo age 6.58
Alp azolam 0.05
Palipe idone 54.2
Co inine 503.2
Alp azolam 43.9
C 3
Con ol
F
51
10
70
Na u al
Ca dio espi a o y ailu e
6.3
Nega i e
Nega i e
S 4
Schizoph enia
M
32
21
87
Suicide
Jump om a heigh
6.65
Nega i e
Co inine 380.78
C 4
Con ol
M
33
23
95
Acciden
Road acciden
6.55
Nega i e
No pe o med
NSS 2
Pe sonali y
diso de
M
33
14
192
Suicide
Jump om a heigh
No
Pe o med
Lo azepam 0.03
Venla axine 0.16
Desme hyl enla axine 475.0
Lo azepam 1421.2
Midazolam 3.7
Olanzapine 6
S 5
Schizoph enia
M
45
36
15
Acciden
Choking
No
pe o med
No pe o med
Midazolam 1801.8
No diazepam 1247.1
Oxazepam 50.1
C 5
Con ol
M
44
23
98
Acciden
Road acciden
6.45
Nega i e
Nega i e
NSS 3
Pe sonali y
diso de
M
44
9
192
Suicide
Jump om a heihg
No
pe o med
E hanol 0.22 g/L
Alp azolam 0.01
Amisulp ide 1.4
Clomip amine 0.2
Reboxe ine 0.09
Alp azolam 141.4
Clomip amine 3040.7
Co inine 322.5
Reboxe ine 224.2
S 6
Schizoph enia
M
49
23
107
Acciden
Fall om a heigh
6.40
Nega i e
Co inine 391
Lo azepam 16.3
C 6
Con ol
M
49
19
93
Acciden
Road acciden
6.7
Nega i e
Nega i e
S 7
Schizoph enia
M
70
20
115
Suicide
Hanging
No
pe o med
No pe o med Nega i e
C 7
Con ol
M
71
22
115
Acciden
Fall om a heigh
5.92
Nega i e
Nega i e
Subjec s, Ma e ial and Me hods
69
S 8
Schizoph enia
F
74
9
118
Na u al
Ca dio espi a o y ailu e
No
pe o med
Phenoba bi al 9 Nega i e
C
8
Con ol
F
74
30
167
Acciden
Road acciden
No
pe o med
Nega i e Nega i e
S 9
Schizoph enia
M
46
20
129
Suicide
Jump om a heigh
6.41
Nega i e
Zuclopen hixol 110.8
Co inine 622.4
Lo azepam 25.5
C 9
Con ol
M
46
22
115
Na u al
Fall
6.48
Nega i e
Nega i e
NSS 4
Pe sonali y
diso de
M
47
4
195
Suicide
Jump om a heigh
No
pe o med
Pheny oine Nega i e
S 10
Schizoph enia
M
26
24
140
Suicide
Jump om a heigh
No
pe o med
Diazepam 0.27
Diazepam 356.5
No -diazepam 856.9
Oxazepam 15.1
C 10
Con ol
M
25
21
71
Acciden
Fi e
6.48
Nega i e
Nega i e
NSS 5
Pe sonali y
diso de
M
27
42
253
Suicide
Jump om a heigh
No
pe o med
Nega i e
Co inine 337.4
Diazepam 204.9
No diazepam 612
Oxazepam 65.8
S 11
Schizoph enia
F
75
18
140
Na u al
Ca dio espi a o y ailu e
No
pe o med
Nega i e Co inine 36.76
C 11
Con ol
F
79
24
213
Acciden
Road acciden
No
pe o med
Nega i e No pe o med
S 12
Schizoph enia
M
28
28
143
Suicide
Jump om a heihg
No
pe o med
Nega i e Co inine 93.79
C12
Con ol
M
29
13
116
Acciden
Fall om a heigh
6.44
Nega i e
Nega i e
NSS 6
Pe sonali y
diso de
M
28
5
259
Suicide
Jump om a heigh
No
pe o med
Nega i e Co inine 638.2
S 13
Schizoph enia
M
25
17
145
Suicide
Jump om a heigh
No
pe o med
Nega i e Co inine 153.1
C 13
Con ol
M
23
16
141
Acciden
Fall om a heigh
No
pe o med
Nega i e Nega i e
NSS 7
Obsessi e-
compulsi e
diso de
M
26
19
143
Suicide
Jump om a heigh
6.66
Clomip amine 0.5
Fluoxe ine 0.7
Flu oxamine 0.2
Que iapine 0.5
Fluoxe ine 15441
Flu oxamine 4094
No luoxe ine 4433.5
No que iapine 386.7
Que iapine 86
S 14
Schizoph enia
M
23
13
195
Suicide
Jump om a heigh
No
pe o med
No pe o med
Halope idol 136.5
Co inine 458.5
Que iapine 392.5
No que iapine 1309.4
C 14
Con ol
M
22
20
188
Acciden
Road acciden
No
pe o med
No diazepam 0.38
Co inine 371.6
Oxazepam 80.9
No diazepam 909
Subjec s, Ma e ial and Me hods
70
NSS 8
Pe sonali y
diso de
M
19
8
131
Suicide
Fall om a heigh
6.7 Nega i e Co inine 236.4
S 15
Schizoph enia
F
80
32
178
Na u al
Shock
No
pe o med
Nega i e No pe o med
C 15
Con ol
F
78
12
185
Na u al
Ca dio espi a o y ailu e
No
pe o med
Nega i e No pe o med
S 16
Schizoph enia
F
38
23
216
Suicide
Jump
om a heigh
No
pe o med
Nega i e Nega i e
C 16
Con ol
F
36
19
110
Acciden
Fall in on o a ain
6.51
Nega i e
Nega i e
NSS 9
Anxie y
diso de
F
39
19
214
Suicide
D ug o e dose
No
pe o med
No diazepam 0.13
E hanol 5.0 g/L
Diazepam 22.4
No diazepam 280.7
S 17
Schizoph enia
F
51
15
236
Na u al
Ca dio espi a o y ailu e
No
pe o med
Bu lomedil 66
Me amizol 4
Co inine 521.5
C 17
Con ol
F
51
38
230
Accid
en
Road acciden
No
pe o med
Nega i e No pe o med
S 18
Schizoph enia
M
62
28
240
Suicide
Jump om a heigh
No
pe o med
No pe o med
Ami ip yline 170.3
No yp iline 453
C 18
Con ol
M
62
23
243
Acciden
Road acciden
No
pe o med
Nega i e No Pe o med
NSS 10
Adap a i e
anxie y
diso de
M
62
19
271
Suicide
C ushing
No
pe o med
No pe o med Map o iline 257.9
No diazepam 1584.9
S 19
Schizoph enia
M
35
22
299
Suicide
Jump om a heigh
No
pe o med
Nega i e Nega i e
C 19
Con ol
M
36
22
286
Acciden
Road acciden
No
pe o med
E hanol 1.0 g/L No pe o med
NSS 11
Anxie y
diso de
M
36
15
181
Suicide
Gun sho
No
pe o med
Mi azapine 0.08 No Pe o med
S 20
Schizoph enia
M
49
41
292
Suicide
Hanging
No
pe o med
E hanol 0.49 g/L/
No pe o med
Co inine 127
Chlo p omazine 140.7
Lo azepam 388.6
Thio idazine 6079.5
C 20
Con ol
M
45
30
275
Acciden
Road acciden
6.82
E hanol 3.09 g/L
No pe o med
NSS 12
Anxie y
diso de
M
46
26
319
Suicide
Hanging
No
pe o med
No pe o med
Diazepam 91.3
No diazepam 175.2
S 21
Schizoph enia
F
56
24
125
Na u al
Ca dio espi a o y ailu e
No
pe o med
Alp azolam 0.03
Co inine 110
Alp azolam 38
C 21
Con ol
F
54
24
10
Acciden
Fall om a heigh
6.87
Nega i e
Nega i e
S 22
Schizoph enia
M
50
3
173
Suicide
D owning 7.09
No diazepam 0.4
Amisulp ide 75.9
Co inine 89
No diazepam 662.8
Oxazepam 47.6
T azodone 241.6
Subjec s, Ma e ial and Me hods
71
C22
Con ol
M
50
2
15
Na u al
Ca dio espi a o y ailu e
6.1
Nega i e
Nega i e
NSS 13
Pe sonali y
diso de
M
49
22
141
Suicide
Fall om a heigh
6.35
No diazepam 2.8
Tiap ide 5.4
Venla axine 1.2
Co inine 254.1
Desme hyl enla axine 265.6
Oxazepam 94.7
Tiap ide 973.5
Venla axine 2146.5
S 23
Schizoph enia
M
43
17
172
Na u al
Ca dio espi a o y ailu e
No
pe o med
Nega i e
Co inine 1003.9
Lo azepam 66
C 23
Con ol
M
41
15
7
Na u al
Ca dio espi a o y ailu e
No
pe o med
Nega i e Nega i e
Subjec s, Ma e ial and Me hods
72
All demog aphic cha ac e is ics, age, PMD, s o age ime and pH a e
summa ized in Table 3.2. As expec ed, no di e ences we e ound when
compa ed age be ween he h ee g oups (F[2,56]=2.08, p=0.1341). In he
same way, no s a is ical di e ences be ween g oups we e ound in ei he PMD
(F[2,56)]=1.99, p=0.1456) o pH alues (F[2,21]=0.17, p=0.8433). Howe e , a
longe s o age ime was obse ed in non-schizoph enia suicide subjec s
compa ed o con ols (F[2,56]=4.66, p=0.0135) (Table 3.2).
Table 3.2: Demog aphic and pos -mo em cha ac e is ics o schizoph enia, non-schizoph enia
suicide and con ol subjec s included in he s udy. G oup alues a e means±SEM.
*p<0.05 s con ol g oup (Bon e oni’s mul iple compa ison es ).
G oup
Gende
Age
(yea s)
PMD
pH
S o age ime
(mon hs)
Schizoph enia
7 F/16 M
48±4
22±2
6.5±0.1
150±15
Non-schizoph enia suicide
1 F/12 M
38±3
16±3
6.6±0.1
207±15*
Con ol
7 F/16 M
48±3
21±2
6.5±0.1
139±15
Subjec s, Ma e ial and Me hods
73
3.1.2 Demog aphic cha ac e is ics and diagnosis o subjec s
included in pools used o An ibody-cap u e [35S]GTPγS
Scin illa ion p oximi y Assays (SPA) and Wes e n Blo
cha ac e iza ion assays
The ini ial pha macological cha ac e iza ion o d ugs was pe o med wi h a
pool o DLPFC om di e en con ol subjec s no included in he es o he
s udy. In each memb ane p epa a ion, homogena es om six p e on al human
b ain samples we e used. Fi een samples collec ed be ween yea s 2016 and
2019 we e used o he whole s udy (30% men and 70% women) wi h a mean
age o 59±6 yea s, PMD o 11±2 hou s and s o age ime o 27±3 mon hs.
Absence o oxicological posi i e es o psycho opic d ugs in blood was
con i med in subjec s con ibu ing o hese pools.
Subjec s, Ma e ial and Me hods
74
3.2 Animals: T ansgenic mice
The 5-HT2AR knock-ou (5-HT2AR(-/-)) and wild- ype (5-HT2AR(+/+)) mice we e
gene ously dona ed by P o . R. Maldonado (Ba celona, Spain). Animals had
been o iginally gene a ed on a 129S6/S E backg ound u he back c ossed
in o he inb ed C57BL/6J line, ollowing s anda d p ocedu es (González-
Maeso e al., 2003; Weiss aub e al., 2006; González-Maeso e al., 2007;
O eja ena e al., 2011). A e dona ion, supplemen a y backc osses we e
pe o med. Animals we e geno yped by con en ional polyme ase chain
eac ion (PCR) and subsequen elec opho esis in ou labo a o y (da a no
shown), as desc ibed by Fio ica-Howells e al., (Fio ica-Hollowells e al., 2002).
Absence o 5-HT2AR exp ession in 5-HT2AR(-/-) mice was con i med by he lack
o [3H]ke anse in binding (Mugu uza e al., 2013) and Gαi1/Gαq/11-p o ein
ac i a ion by (±)DOI (Ga cia-Bea e al., 2019).
Expe imen s we e pe o med on adul (15-20 weeks old) C57BL/J6 mice.
Animals we e housed up o i e indi iduals unde s anda d labo a o y
condi ions (22±1°C, 55±5% ela i e humidi y, 12 h ligh /da k cycle and ee
access o s anda d oden chow and wa e ). E o s we e made o minimize he
numbe o animal used. The expe imen al p o ocols we e e iewed and
app o ed by he Local E hical Commi ee o Animal Resea ch o he Uni e si y
o he Basque Coun y (UPV/EHU, CEEA, Re . M20-2019-321). All
expe imen s we e ca ied ou in acco dance wi h he Eu opean Communi y
Council Di ec i e on “The P o ec ion o Animals Used o Scien i ic Pu pose”
(Eu opean Union Di ec i e 2010/63/UE) and Spanish Law (Royal dec ee
53/2013) o he ca e o labo a o y animals. Adul mice we e sac i iced by
ce ical disloca ion, b ains emo ed, co ex dissec ed and samples s o ed a -
80°C un il assay as p e iously desc ibed (Diez-Ala cia e al., 2016).
Subjec s, Ma e ial and Me hods
75
3.3 D ugs
-(±)-DOI: (±)-2,5-Dime hoxy-4-iodoamphe amine hyd ochlo ide (Sigma-
Ald ich; Sain Louis, Missou i, USA).
-Ke anse in: 3-[2-[4-(4-Fluo obenzoyl)-1-pipe idinyl]e hyl]-2,4[1H,3H]-
quinazolinedione a a e (Toc is; B is ol UK).
-MDL100907 ( olinanse in): (R)-(+)-α-(2,3-dime hoxyphenyl)-1-[2-(4-
luo ophenyl)e hyl]-4-pipidineme hanol (Sigma-Ald ich; Sain Louis, Missou i,
USA).
-Al anse in: 3-[2-[4-(4-Fluo obenzoyl)-1-pipe idinyl]e hyl]-2,3-dihyd o-2-
hioxo-4(1H)-quinazolinone hyd ochlo ide hyd a e (Sigma-Ald ich; Sain Louis,
Missou i, USA).
-Pima anse in (ACP-103): 1-(4-Fluo obenzyl)-3-(4-isobu oxybenzyl)-1-(1-
me hylpipe idin-4-yl)u ea (Axon Medchem; G oningen, The Ne he lands).
-Nelo anse in: 1-(3-(4-b omo-2-me hyl-2H-py azol-3-yl)-4-me hoxyphenyl)-3-
(2,4-di luo ophenyl)u ea (Axon Medchem; G oningen, The Ne he lands).
-Ri anse in: 6-[2-[4-[bis(4- luo ophenyl)me hylidene]pipe idin-1-yl]e hyl]-7-
me hyl-[1,3] hiazolo[3,2-a]py imidin-5-one (Sigma-Ald ich; Sain Louis,
Missou i, USA).
-Epli anse in: 4-[(E,3Z)-3-[2-(dime hylamino)e hoxyimino]-3-(2-
luo ophenyl)p op-1-enyl]phenol (Axon Medchem; G oningen, The
Ne he lands).
-MDL-11,939: α-phenyl-1-(2-phenyle hyl)-4-pipe idineme hanol (Toc is;
B is ol, UK).
-SB 242084: 6-Chlo o-2,3-dihyd o-5-me hyl-N-[6-[(2-me hyl-3-py idinyl)oxy]-
3-py idinyl]-1H-indole-1-ca boxyamide dihyd ochlo ide (Toc is; B is ol, UK).
-Clozapine: 8-Chlo o-11-(4-me hyl-1-pipe azinyl)-5H-
dibenzo[b,e][1,4]diazepine (Toc is; B is ol, UK).
-Rispe idone: 3-[2-[4-(6- luo o-1,2- benzisoxazol-3-yl)-1-pipe idinyl]e hyl]-
6,7,8,9- e ahyd o-2-me hyl-4H-py ido[1,2- a]py imidin-4-one (Sigma-Ald ich;
Sain Louis, Missou i, USA).
Subjec s, Ma e ial and Me hods
76
-A ipip azole: 7-{4-[4-(2,3-Dichlo ophenyl)-1-pipe azinyl]bu oxy}-3,4-dihyd o-
2(1H)-quinolinone one (Sigma-Ald ich; Sain Louis, Missou i, USA).
-Olanzapine: 2-Me hyl-4-(4-me hyl-1-pipe azinyl)-10H- hieno[2,3-
b][1,5]benzodiazepine (Sigma-Ald ich; Sain Louis, Missou i, USA).
-Palipe idone: 3-[2-[4-(6-Fluo o-1,2-benzisoxazol-3-yl)-1-pipe idinyl]e hyl]-
6,7,8,9- e ahyd o-9-hyd oxy-2-me hyl-4H-py ido[1,2-a]py imidin-4-one
(Toc is; B is ol, UK).
-Que iapine: 2-[2-(4-Dibenzo[b, ][1,4] hiazepin-11-yl-1-
pipe azinyl)e hoxy]e hanol hemi uma a e (Toc is; B is ol, UK).
-A opine: Endo-(±)-α-(Hyd oxyme hyl)benzeneace ic acid 8-me hyl-8-
azabicyclo[3.2.1]oc -3-yl es e (Sigma-Ald ich; Sain Louis, Missou i, USA).
-Phen olamine: 2-[N-(3-Hyd oxyphenyl)-p- oluidinome hyl]-2-imidazolidine
hyd ochlo ide (Sigma-Ald ich; Sain Louis, Missou i, USA).
-Ce i izine: [2-[4-[(4-Chlo ophenyl)phenylme hyl]-1-pipe azinyl]e hoxy]ace ic
acid dihyd ochlo ide (Sigma-Ald ich; Sain Louis, Missou i, USA).
-Raclop ide: 3,5-Dichlo o-N-[[(2S)-1-e hyl-2-py olidinyl]me hyl]-2-hyd oxy-6-
me hoxybenzamide (Toc is; B is ol UK).
-Halope idol: 4-[4-(4-Chlo ophenyl)-4-hyd oxypipe idino]-4′-
luo obu y ophenone (Sigma-Ald ich; Sain Louis, Missou i, USA).
Subjec s, Ma e ial and Me hods
77
3.4 Ma e ials
An ibodies
P ima y monoclonal an ibodies used o An ibody-cap u e [35S]GTPγS SPA
and Wes e n Blo expe imen s we e pu chased om San a C uz
Bio echnology, Inc (Cali o nia USA). They a e de ailed in sec ion 3.5.2, whe e
Wes e n Blo expe imen p o ocol is explained.
Fo Wes e n Blo assays, di e en luo escen seconda y an ibodies we e
used, such as Alexa Fluo ® 680 conjuga ed wi h goa an i-mouse, p o ided by
In i ogen (O egon, USA), and IRDyeTM conjuga ed wi h an i- abbi , p o ided
by Rockland Immunochemical (Pennsyl ania, USA).
Radioac i e compounds
Sul u 35 labelled guanosine-5’-O-(gamma- hio)- iphospha e ([35S]GTPγS)
wi h speci ic ac i i y o 1250 Ci/mmol was om Pe kinElme (Wal man, MA,
USA).
O he d ugs and chemicals sou ces
-Bio-Rad Labo a o ies (Cali o nia, USA): Ammonium pe sul a e (APS),
B ad o d P o ein Assay, 2x concen a ed Laemmli sample bu e , N-N-N-N´-
e ame hyle hylenediamine (TEMED), p e-s ained SDS-PAGE molecula
weigh s anda ds.
-Ca lo E ba Reagen s (Ba celona, Spain): Me hanol.
-GE Heal hca e (Buckinghamsi e, UK): Ni ocellulose memb anes (po e size:
0.45 μm) and Wha manTM cellulose 3 mm.
-In i ogen (Ba celona, Spain): DL-Di hio h ei ol (DTT), e hylenediamine
e ace ic acid (EDTA).
Subjec s, Ma e ial and Me hods
84
Table 3.4: Gene al p o ocol o [35S]GTPγS binding combined wi h SPA. BB: Basal binding
(binding alues in absence o any d ug), NBS: Non-speci ic binding (de ined as he emaining
[35S]GTPγS binding in p esence o 100 μM unlabelled GTPγS).
In gene al, in o de o ind a selec i e in e se agonis , ini ially, di e en d ugs
p e iously desc ibed as 5-HT2AR an agonis we e chosen and es ed o
in e se agonism p ope ies. A e ha , selec ed in e se agonis s compounds
we e used o in es iga e 5-HT2AR al e a ions coupling o Gα-p o eins in
schizoph enia subjec s. In an e o o e alua e he speci ici y and selec i i y o
hese d ugs, di e en selec i e an agonis as well as 5-HT2AR(+/+) and 5-
HT2AR(-/-) mice memb ane homogena es we e used o block he [35S]GTPγS
binding signal obse ed o each d ug.
BB
Agonis
Agonis +
An agonis
NBS
SPA incuba ion
bu e /DMSO
24 μl
12 μl
/
12 μl
GTPγS
/
/
/
12 μL
GDP
12 μl
12 μl
12 μl
12 μl
An agonis
/
/
12 μl
/
Memb anes
160 μl
160 μl
160 μl
160 μl
15 minu es o incuba ion a 30°C wi h gen le agi a ion (400 pm)
Agonis
/
12 μl
12 μl
/
30 minu es o incuba ion a 30°C wi h gen le agi a ion (400 pm)
[
35
S]GTPγS
5 μl
5 μl
5 μl
5 μl
90 minu es o incuba ion a 30°C wi h gen le agi a ion (400 pm)
De e gen
20 μl
20 μl
20 μl
20 μl
30 minu es o incuba ion a 22°C wi h gen le agi a ion (350 pm)
An ibodies
10 μl
10 μl
10 μl
10 μl
90 minu es o incuba ion a 22°C wi h gen le agi a ion (350 pm)
PVT P o ein A SPA
BEADS
50 μl
50 μl
50 μl
50 μl
180 minu es o incuba ion a 22°C wi h gen le agi a ion (350 pm)
Subjec s, Ma e ial and Me hods
85
3.5.1.3 Ma hema ical and s a is ical analysis o he esul s
Ma hema ical analysis
Resul s ob ained om he Mic obe a T ilux Scin illa ion coun e a e exp essed
as CCPM (co ec ed coun s pe minu e), which assumes he equi alency
be ween CCPM and DPM (disin eg a ions coun s pe minu e). In o de o
in e p e he esul s, da a om CCPM mus be ans o med in o em omole
[35S]GTPγS bound pe millig am p o ein ( mol/mg p o ein). The con e sion is
made conside ing he p o ein concen a ion used in he assay and by ollowing
he Equa ion 3.1. The p o ein concen a ion can in luence he [35S]GTPγS
binding as shown in Diez-Ala cia e al., 2021b. The e o e, p o ein con en o
he assay is measu ed a e each expe imen .
mol/ mg p o ein: CCPM coun s/(2,22 x 1250 x [assay p o ein concen a ion mg])
Equa ion 3.1: Resul s exp essed in mol/mg p o ein pe o med in [35S]GTPγS binding
combined wi h SPA. 1250 (Ci/mmol) co esponds o speci ic ac i i y o he adioligand 35S.
2,22 is a cons an o he ans o ma ion om Cu ie (Ci) o DMP (1 Ci=2,22x1012 DPM).
Basal binding (BB) o [35S]GTPγS, is de ined as [35S]GTPγS binding in
absence o any exogenous d ug. When an agonis o in e se agonis d ugs is
added o he assay he basal [35S]GTPγS binding is modula ed. When he d ug
is an agonis , an inc ease o [35S]GTPγS binding on [35S]GTPγS basal binding
will be obse ed. In con as , in e se agonis s will dec ease he [35S]GTPγS
basal binding.
The non-speci ic binding (NBS) in CCPM was sub ac ed om all CCPM
ob ained o each di e en condi ion (basal binding, s imula ion, inhibi ion,..).
Thus, speci ic [35S]GTPγS binding alues we e ob ained and used o u he
calcula ion and s a is ical analysis.
Subjec s, Ma e ial and Me hods
86
Concen a ion- esponse cu es o di e en d ugs we e pe o med in o de o
de e mine agonis , an agonis o in e se agonis p ope ies o each d ug. The
BB, in absence o he d ug, was conside ed as 100%, and speci ic binding
we e ans o med o pe cen age o basal binding. Thus, s imula ion o
inhibi ion e ec on he [35S]GTPγS binding in p esence o he d ug was de ined
in ela ion o espec i e basal binding. Each poin o he cu e was calcula e
wi h Equa ion 3.2 and 3.3.
% S imula ion: (S imula ion-NBS)/(BB-NBS)x100
Equa ion 3.2: Calcula ion o pe cen age o e basal binding pe o med in [35S]GTPγS binding
combined wi h SPA expe imen s in o de o de e mine agonis , an agonis o compe i ion
e ec s. NBS: Non-speci ic binding, BB: Basal binding.
% Inhibi ion: 100-[(Inhibi ion-NBS)/(BB-NBS)x100]
Equa ion 3.3: Calcula ion o pe cen age o basal binding pe o med in [35S]GTPγS binding
combined wi h SPA expe imen s in o de o de e mine in e se agonis , an agonis o
compe i ion e ec s. NBS: Non-speci ic binding. BB: Basal binding.
In some cases, in o de o es he e ec o [35S]GTPγS binding o di e en Gα-
p o ein sub ypes, ins ead o concen a ion- esponse cu es, a single
concen a ion (10 μM) o he d ug was used. This concen a ion was selec ed
because i gi es binding alues a ound he maximal e ec s (Emax/Imax) in he
concen a ion- esponse cu es. This concen a ion was selec ed o one-poin
concen a ion expe imen s. In his case, basal binding was de ined as 0% and
e ec s we e exp essed as posi i e o nega i e pe cen age changes espec o
basal condi ions
Pha macological pa ame e s o s imula ion o inhibi ion cu es o [35S]GTPγS
binding, maximal s imula o y o inhibi o y e ec s (Emax/Imax) and concen a ion
Subjec s, Ma e ial and Me hods
87
o he d ug ha de e mines hal maximal s imula ion o inhibi ion (EC50/IC50)
we e calcula ed. These pa ame e s we e ob ained by non-linea analysis using
G aphPad P imTM. Poin s we e i ed o he concen a ion- esponse cu e
shown in Equa ion 3.4 and 3.5. This model assumes ha concen a ion-
esponse cu e displays a s anda d slope, equal o a Hill slope (o slope ac o )
o 1.0. This is he expec ed slope when a ligand binds o a single ecep o
acco ding o he law o mass ac ion.
E= BB+(Emax-BB)(1+10(LogEC50-Log[X]))
Equa ion 3.4: Monophasic s imula o y concen a ion- esponse cu e (s anda d slope). E
co esponds o he e ec (% S imula ion o Equa ion 3.2) a he X concen a ion, BB
co esponds o he speci ic [35S]GTPγS binding in absence o agonis o basal binding
(assumed as 100%), Emax o he maximal e ec (%) and LogEC50 o he concen a ion o he
d ug ha de e mines hal maximal e ec .
E= BB+(Imax-BB)(1+10(LogIC50-Log[X]))
Equa ion 3.5: Monophasic inhibi o y concen a ion- esponse cu e (s anda d slope). E
co esponds o he e ec (% Inhibi ion o Equa ion 3.3) a X concen a ion, BB co esponds o
he speci ic [35S]GTPγS binding in absence o in e se agonis o basal binding (assumed as
100%), Imax o he maximal e ec (%) and LogIC50 o he concen a ion o he d ug ha
de e mines hal maximal e ec .
Pha macological pa ame e s Emax/Imax a e exp essed as mean±SEM (s anda d
e o o he mean). –LogEC50/IC50 a e exp essed as mean±SEM. These alues
a e used o u he s a is ical analysis. Hal -maximal s imula ion o inhibi ion
a e also exp essed as EC50/IC50 ha co esponds o he an iloga i hm o
LogEC50/IC50 mean alues.
Concen a ion- esponse cu es we e simul aneously co-analysed (all he
expe imen s o each expe imen al g oup oge he ) by non-linea eg ession o
Subjec s, Ma e ial and Me hods
88
he bes i assuming an one-si e model o [35S]GTPγS binding. This way,
global alues o each g oup we e ob ained. Resul s a e exp essed as he bes
i alue±95% con idence in e al.
S a is ical analysis
Di e en s a is ical analyses we e used o he sui able in e p e a ion o he
esul s.
Ini ially, all da a we e subjec ed o G ubb´s es , in o de o de ec and ejec
possible ou lie alues among expe imen al g oups. Whe he ob ained da a
displayed a Gaussian dis ibu ion was also es ed.
Resul s om one single-concen a ion expe imen s we e analyzed by one-
sample S uden ’s - es s basal alues, in o de o e alua e p esence o
absence o he e ec induced by d ugs on basal binding.
Two- ailed unpai ed S uden ´s - es was used o compa e wo di e en
condi ions. Fo example, o compa e he e ec o an agonis alone s agonis
co-incuba ed wi h an an agonis . When h ee di e en condi ions compa ison
was needed, one-way ANOVA analysis was pe o med ollowed by
Bon e oni´s pos -hoc analysis. This es was used, o example, o compa e
he modula ion o [35S]GTPγS binding be ween schizoph enia, non-
schizoph enia suicide and con ol g oups.
In o de o calcula e he con ibu ion e ec o wo di e en a iables, wo-way
ANOVA was used ollowed by Bon e oni´s pos -hoc analysis. This analysis
was app op ia e o s udy he e ec o di e en d ugs in a ious mice
geno ypes.
Pea son´s co ela ion coe icien was calcula ed o es possible associa ions
be ween independen co a iables (age, PMD and s o age ime) and
dependen unc ional esponses. When co ela ion was signi ican , analysis o
Subjec s, Ma e ial and Me hods
89
co a iance (ANCOVA) was pe o med be ween g oups (schizoph enia, non-
schizoph enia suicide and con ol subjec s) con olling o he independen
co a iable. ANCOVA analyses we e ca ied ou by using InVi oS a s a is ical
so wa e.
In all s a is ical e alua ions, di e ences we e conside ed signi ican a p<0.05.
A complemen a y analysis o po en ial di e ences be ween g oups was also
pe o med. Thus, global esul s ob ained om coanalysis o schizoph enia,
non-schizoph enia suicide and con ol subjec s da a we e subjec ed o u he
e alua ion (DeLean e al., 1978; Mo ulsky & Ransnas, 1987). This ex a
analysis compa ed he goodness o i o a model wi h and wi hou a se o
cons ains by means o an F es (based on he p inciple o ex a sum o
squa es). Fi s , se s o da a we e analysed sepa a ely (no cons ains) as
desc ibed abo e. O e all alue o he sum o squa es was sum o indi idual
alues om each i and, simila ly, he numbe s o deg ees o eedom. Nex ,
se s we e pooled, analysed simul aneously, and cons ained o sha e one o
mo e common pa ame e s (basal, Emax/Imax and LogEC50/IC50), which ga e
di e en alues o he sum o squa es and deg ees o eedom. Analysis ha
pe mi ed one o mo e pa ame e s sha ing, wi hou a signi ican inc ease in he
esidual a iance, was aken as he bes i . S a is ical signi icance o he
imp o emen was de e mined wi h a F es a p<0.05, and was exp essed as
F[DFn, DFd] whe e F is dis ibu ion alue, DFn a e deg ees o eedom in
nume a o and DFd a e deg ees o eedom in denomina o .
Subjec s, Ma e ial and Me hods
90
3.5.2 Wes e n Blo expe imen s
Wes e n Blo is a b oadly used echnique o de ec ion and iden i ica ion o
p o eins by means o an ibodies. The p ocess is di ided in o a ious s eps:
elec opho esis sepa a ion by molecula weigh o p o eins p esen in he
sample; p o ein ans e om he gel o ni ocellulose memb anes, which
immobilizes p o eins and make hem accessible o an ibodies; and exposi ion
o ni ocellulose memb anes o solu ion con aining he an ibodies ha
ecognize and bind speci ically a a ge p o ein.
In he p esen s udy, i s ly, Wes e n Blo expe imen s we e pe o med in o de
o cha ac e ize and e alua e he speci ici y o an ibodies used in an ibody-
cap u e [35S]GTPγS Scin illa ion P oximi y Assays (SPA) o each Gα-p o ein
sub ype. Fu he , immuno eac i e densi ies o Gαi1- and Gαq/11-p o eins we e
quan i ied in pos -mo em PFC o schizoph enia, non-schizoph enia suicide
subjec s and hei ma ched con ol subjec s. Simul aneously, densi y o
cy oskele al p o ein β-ac in was also measu ed, as a loading con ol.
Subjec s, Ma e ial and Me hods
91
Figu e 3.2: Schema ic ep esen a ion o Wes e n Blo assays. 1. Hea ing o samples a 95°C
o i e minu es; 2. Loading o an op imized quan i y o sample on o gel; 3. A e wa ds, unning
o gel elec opho esis a 60 V o 30 minu es and change o 140 V o 90 minu es. P o eins
sepa a e acco ding o size, wi h smalle p o eins mig a ing h ough he gel; 4. Assembling o
ans e sandwich wi h he gel nea he ca hode (-) and he memb ane nea he anode (+); 5.
Nega i ely cha ged p o eins will mig a e ou o he gel on o he memb ane by pe o ming
ans e a 0.3 A pe ay, o 90 minu es; 6. Blocking he memb ane in an app op ia e blocking
bu e (milk). Incuba ion o he memb ane wi h p ima y an ibody and, a e washing, incuba ion
wi h seconda y an ibody o 1 hou a oom empe a u e. Finally, de ec ion o image
luo escence. Illus a ion o iginally c ea ed o his hesis by J. DelaCues a-Ba u ia.
3.5.2.1 P epa a ion o memb ane en iched ac ion (P2 ac ion)
Memb ane P2 ac ions o pe o m Wes e n-blo assays we e p epa ed as
p e iously desc ibed o SPA assays (Sec ion 3.5.1.1). The day o expe imen ,
0.5 mg o P2 ac ion pelle s we e de os ed and esuspended in 125 μl o T is-
HCl 0.5 nM. These samples we e combined wi h 119 μl 2X Laemmli sample
bu e and 6 μl o β-me cap oe hanol, ob aining 2 mg/ml as a inal p o ein
con en . Samples om schizoph enia, non-schizoph enia suicide and con ol
subjec s we e p ocessed in pa allel on he same day.
Subjec s, Ma e ial and Me hods
92
3.5.2.2 Gel elec opho esis, ans e ence and
immunode ec ion
Elec opho esis in polyac ylamide gels
The elec opho esis in polyac ylamide gels in dena u alizing condi ions o
SDS-PAGE (Sodium Dodecyl Sulpha e Polyac ilamide Gel Elec opho esis) is
he mos used analy ical me hod o sepa a e di e en p o eins o a sample
acco ding o hei molecula weigh .
Du ing he elec opho esis, p o eins mig a e in i s place hough “s acking gel”.
S acking gel was p epa ed wi h 5% o polyac ylamide, 125 mM T is HCl, 0.1%
SDS, 0.07% p os a ic speci ic an igen (PSA) and 0.14 TEMED pH 6.8.
Laemmli-p epa ed samples we e hea ed a 95°C o i e minu es in a
The moblock du ing he p epa a ion o “s acking gel” (Figu e 3.2). P o ein
sepa a ion acco ding o hei molecula weigh s a ed in he “ unning gel”. This
gel was p epa ed in a 10% ac ylamide-bisac ylamide concen a ion in a
solu ion o 0.37 M T is HCl, 0.1% SDS, 0.07% PSA and 0.07% TEMED, pH
8.8. A comb om Te lonTM was inse ed in each s acking gel in o de o o m
15 lanes, whe e he samples un.
Samples we e loaded on hose 15-lane gels, sized 6 x 8 cm each (Figu e 3.3).
The loading p o ein and olume o samples in each line depended on he Gα-
p o ein o in e es (Table 3.5). The i s line o each gel was loaded wi h a
comme cial molecula weigh ma ke suspension (10-250 kDa, P ecision Plus
P o einTM, Dual Colo S anda ds, Bio-Rad). The es o lines we e loaded wi h
co esponding olume o he sample. Gel loading ske ch is shown in Figu e
3.3.
Subjec s, Ma e ial and Me hods
93
Figu e 3.3: Gel loading ske ch o a wes e n blo expe imen o an ibody cha ac e iza ion and
schizoph enia, non-schizoph enia suicide and con ol subjec s expe imen s. MW: Molecula
weigh ma ke , P2: memb ane-en iched ac ion, C: Con ol subjec s, SCH: Schizoph enia
subjec s, NSCHS: Non-schizoph enia suicide subjec s.
Table 3.5: Expe imen al condi ions o Wes e n Blo expe imen s wi h human, a and mouse
b ain samples. MW (Molecula weigh ).
P o ein
Loaded
p o ein/ olume
MW
(kDa)
Blocking
solu ion
Incuba ion
solu ion
P ima y
an ibody
dilu ion
Seconda y
an ibody
dilu ion
Gαi1
30 μg/
15 μl
40
5% non a
d y milk
Blocking solu ion
+ 0.1% Tween 20
1:200
1:10000
Gαi2
15 μg/
7.5 μl
42
5% non a
d y milk
Blocking solu ion
+ 0.1% Tween 20
1:300
1:8000
Gαi3
15 μg/
7.5 μl
40
5% non a
d y milk
Blocking solu ion
+ 0.1% Tween 20
1:300
1:8000
Gα
o
15 μg/
7.5 μl
45
5% non a
d y milk
Blocking solu ion
+ 0.1% Tween 20
1:500
1:8000
Gαq/11
24 μg/
12 μl
40
5% non a
d y milk
Blocking solu ion
+ 0.1% Tween 20
1:300
1:8000
Gα
s
15 μg/
7.5 μl
41
5% non a
d y milk
Blocking solu ion
+ 0.1% Tween 20
1:500
1:8000
Resul s
Resul s
103
4.1 Func ional selec i i y o di e en se o onin 5-HT2AR
an agonis s in pos -mo em human b ain
4.1.1 Concen a ion-e ec o di e en d ugs on Gαi1- and
Gαq/11-p o ein coupling o 5-HT2AR in pos -mo em human PFC
Concen a ion- esponse cu es in p esence o inc easing concen a ion o
di e en d ugs we e used o he unc ional coupling cha ac e iza ion o 5-
HT2AR o di e en Gα-p o eins. Unde his expe imen al condi ion, esponses
o speci ic [35S]GTPγS binding o Gαi1-p o eins (Figu e 4.1.A) o inc easing
concen a ions o MDL-11,939 (10-10–10-4 M) o ke anse in (10-10–10-4 M) we e
unal e ed, sugges ing ha hese d ugs ac as neu al an agonis s o he Gαi1-
p o ein-media ed pa hway. In con as , o he d ugs such as al anse in,
pima anse in, nelo anse in, i anse in, olinanse in and epli anse in displayed
a concen a ion-dependen inhibi o y esponse, which poin s ou ha hese
d ugs we e able o dec ease basal cons i u i e ac i i y.
When he canonical Gαq/11-p o ein pa hway o 5-HT2AR was s udied (Figu e
4.1.B), ke anse in inc eased [35S]GTPγS binding o Gαq/11-p o ein, displaying
agonis p ope ies. Con e sely, al anse in, pima anse in, nelo anse in and
MDL-11,939 p esen ed null e ec on Gαq/11-p o ein pa hway. Finally,
i anse in, olinanse in and epli anse in displayed concen a ion- esponse
inhibi ions o [35S]GTPγS binding o he canonical pa hway.
The e o e, i migh be concluded ha only MDL-11,939 ac s as neu al
an agonis o Gαi1- and Gαq/11-p o ein-coupling o 5-HT2AR in human b ain
co ex. In con as , he es o d ugs displayed unc ional selec i i y be ween 5-
HT2AR coupling o Gαi1- and Gαq/11-p o eins. The co esponding Emax/Imax and
EC50/IC50 alues o concen a ion- esponse cu es o each d ug a e shown in
Table 4.1.
Resul s
104
Figu e 4.1: Concen a ion- esponse cu es o speci ic [35S]GTPγS binding o Gαi1- and Gαq/11-
p o eins in esponse o addi ion o di e en 5-HT2AR d ugs. Concen a ion- esponse
s imula ion o inhibi ion o [35S]GTPγS binding o Gαi1- (Figu e 4.1.A) and Gαq/11- (Figu e 4.1.B)
p o eins by al anse in, pima anse in, nelo anse in, i anse in, olinanse in, ke anse in,
epli anse in and MDL-11,939 was pe o med in human PFC memb anes. The 100% dashed
line deno es he speci ic basal [35S]GTPγS binding o each GD-p o ein (BB). Each poin
ep esen s he mean±SEM alue om independen expe imen s ca ied ou in duplica e and
iplica e.
Resul s
105
The accu a e de e mina ion o e icacy is essen ial o ligand bias de ec ion.
Analysis o concen a ion- esponse cu es measu es d ug e icacy and
po ency, which a e ep esen ed by maximal inhibi o y o s imula o y esponses
(Emax/Imax), and he concen a ion ha p omo es hal -maximal e ec
(EC50/IC50), espec i ely. Pima anse in and nelo anse in showed he highes
e icacy o dec ease in almos a 30% [35S]GTPγS basal binding o Gαi1-p o ein
(Imax=-27±2% and -25±2%) as shown in Table 4.1. In he same way,
nelo anse in and pima anse in also showed highes po ency (IC50=8.4±0.3 nM
and 8.2±0.3 nM) (Table 4.1). None o he d ugs showed in insic posi i e
e icacy on he [35S]GTPγS basal binding o Gαi1-p o ein. Howe e , ke anse in
was able o inc ease he [35S]GTPγS binding o Gαq/11-p o ein by a 18%, wi h
a po ency o 994 nM (Table 4.1). Epli anse in dec eased he [35S]GTPγS
binding o Gαq/11-p o ein by a 16%, which co esponded o he highes
inhibi o y e icacy. In con as , epli anse in had he lowes po ency (IC50=3268
nM). In e ms o po ency, i anse in showed he lowes concen a ion equi ed
o each he hal -maximal e ec o [35S]GTPγS binding o Gαq/11-p o eins (IC50=
15.2 nM) (Table 4.1).
Resul s
106
Table 4.1: Pha macological pa ame e s o he concen a ion- esponse cu es o he speci ic
[35S]GTPγS binding o Gαi1- and Gαq/11-p o eins in esponse o addi ion o di e en 5-HT2AR
d ugs in human PFC memb anes. Da a a e shown as mean±SEM o n independen
expe imen s ca ied ou in duplica e o iplica e. Emax and Imax ep esen , in % o basal alues,
he maximal s imula ion and inhibi ion, espec i ely, es ima ed om non-linea i ing o he
concen a ion- esponse cu es. (-)logEC50/IC50 indica e he log o concen a ion (in nM) ha
p omo es hal -maximal s imula o y o inhibi o y e ec , and IC50/EC50 a e he espec i e an ilog
alues.
Gαi1-p o ein Gαq/11-p o ein
E
max
/
Imax (%)
(-)logEC
50
/IC5
0
IC
50
/EC50
(nM)
n
Emax
/
Imax (%)
(
-
)logEC50
/IC
50
IC
50
/EC50
(nM)
n
Al anse in
-19±3
6.7±0.4
205.3
6
/
/ /
6
Pima anse in
-25±2
8.2±0.3
6.1
5
/
/ /
8
Nelo anse in
-27±2
8.4±0.3
4.3
5
/
/ /
6
Ri anse in
-17±2
7.0±0.3
108.1
5
-
7±2
7.8±0.7
15.2
5
Volinanse in
-22±2
6.9±0.2
125.5
6
-10±2
6.3±0.7
530.9
6
Ke anse in
/
/ /
5
18±2
6.0±0.3
994.1
7
Epli anse in
-24±2
7.8±0.2
17.8
7
-16±4
5.5±0.5
3268
6
MDL
-
11,939
/
/ /
5
/
/ /
5
Resul s
107
4.1.2 E alua ion o maximal e ec o di e en d ugs on Gαi1-,
Gαi2-, Gαi3-, Gαo-, and Gαq/11-p o ein coupling o 5-HT2AR in
pos -mo em human PFC
In o de o elucida e he e ec s o p e iously e alua ed d ugs on [35S]GTPγS
binding, no only o Gαi1- and Gαq/11-p o eins bu also o o he PTX-sensi i e
Gα-p o eins (inhibi o y Gαi/o-p o eins), a new se o expe imen s was
pe o med. Fo hese expe imen s, [35S]GTPγS scin illa ion p oximi y assays
(SPA) coupled o immunop ecipi a ion wi h di e en selec i e an ibodies
agains Gαi1-, Gαi2-, Gαi3-, Gαo- and Gαq/11-p o ein sub ypes we e pe o med
a one single concen a ion (10 μM). This concen a ion induces e ec s e y
close o maximal e icacy. Al anse in, pima anse in and epli anse in induced
a s a is ically ele an inhibi ion o [35S]GTPγS binding o Gαi1-p o eins, while
hey did no show any e ec on o he inhibi o y Gα-p o eins (Table 4.2). Unde
hese expe imen al condi ions, nelo anse in and i anse in dec eased
[35S]GTPγS basal binding o Gαi1-, Gαi2-, Gαi3-, and Gαo-p o eins (Table 4.2).
Volinanse in p omo ed inhibi ion o [35S]GTPγS binding o Gαi1- and Gαo-
p o eins (Table 4.2). Ke anse in and MDL-11,939 we e unable o modi y
[35S]GTPγS binding o inhibi o y Gαi/o p o eins (Table 4.2). In gene al, he
maximal inhibi o y e ec s we e obse ed in Gαi1-p o eins (Table 4.2).
The esponses o [35S]GTPγS binding o Gαq/11-p o eins, a e addi ion o
di e en d ugs, we e simila o hose p e iously obse ed (see 4.1.1). Thus
i anse in, olinanse in and epli anse in induced an inhibi o y e ec on
[35S]GTPγS binding, whe eas ke anse in p omo ed s imula ion (Table 4.2).
Resul s
108
Table 4.2: S imula o y o inhibi o y e ec induced by one single concen a ion (10 μM) o
di e en d ugs (al anse in, pima anse in, nelo anse in, i anse in, olinanse in, ke anse in,
epli anse in and MDL-11,939) on he speci ic [35S]GTPγS binding o inhibi o y Gαi/o- and
Gαq/11-p o eins in human PFC memb anes. Da a a e mean±SEM o n independen
expe imen s ca ied ou in duplica e o iplica e, and exp ess he pe cen age alues ela ed
o basal [35S]GTPγS binding (0%). Da a we e analyzed using one sample - es s basal
binding (*p<0.05, **p<0.01, ***p<0.001).
D ug
Gα
i1
Gα
i2
Gα
i3
Gα
o
Gαq/11
Al anse in
-12±2***
(n=12)
-1±3
(n=10)
2±1
(n=11)
-4±5
(n=6)
4±2
(n=12)
Pima anse in
-20±3***
(n=10)
-2±2
(n=10)
3±2
(n=11)
-1±3
(n=5)
3±2
(n=13)
Nelo anse in
-22±3***
(n=9)
-
17±4**
(n=10)
-11±4*
(n=8)
-6±2*
(n=5)
3±3
(n=11)
Ri anse in
-18±2***
(n=9)
-15±4*
(n=6)
-10±3*
(n=18)
-15±5*
(n=7)
-11±3**
(n=11)
Volinan
s
e in
-16±2***
(n=9)
-2±3
(n=9)
-3±3
(n=9)
-4±1*
(n=7)
-7±3*
(n=10)
Ke anse in
-1±1
(n=5)
2±4
(n=6)
-2±3
(n=7)
-1±2
(n=5)
20±3**
(n=7)
Epli anse in
-19±5*
(n=5)
-2±4
(n=6)
0±5
(n=7)
5±2
(n=5)
-17±3***
(n=9)
MDL
-
11,939
3±3
(n=7)
0±2
(n=5)
-1±4
(n=5)
0±1
(n=5)
0±2
(n=7)
Resul s
109
4.1.3 In ol emen o 5-HT2AR in he e ec induced by di e en
d ugs on [35S]GTPγS binding o Gαi1- and Gαq/11-p o eins in
pos -mo em human PFC
The desc ibed indings in p e ious pa ag aphs sugges he in ol emen o 5-
HT2AR in he modula ion o [35S]GTPγS binding o Gαi1- and Gαq/11-p o eins
because e alua ed d ugs sha e ac i i y on 5-HT2AR. In o de o con i m he
in ol emen o 5-HT2AR, an agonism assays wi h selec i e 5-HT2AR d ugs
we e pe o med. Fo his pu pose, al anse in, pima anse in, nelo anse in,
i anse in, olinanse in, ke anse in, epli anse in and MDL-11,939 we e added
o SPA assays a 10 μM concen a ion in p esence o ke anse in (10 μM),
olinanse in (1 μM) o MDL-11,939 (1 μM). Ke anse in (pKi=8.5), olinanse in
(pKi=8.73) and MDL-11,939 (pKi=7.58) we e chosen as selec i e an agonis
agains 5HT2AR s 5HT2CR, acco ding o p e ious epo s (Knigh e al., 2004;
Bonhaus e al., 1995).
The selec i i y o di e en d ugs o 5-HT2AR was analysed by epea ed
measu es one-way ANOVA, whe e he d ug alone was compa ed wi h he d ug
co-incuba ed wi h he an agonis . A u he Bon e oni´s pos -hoc es would
delinea e he speci ic inhibi ion o di e en an agonis s.
Ke anse in, olinanse in and MDL-11,939 blocked he inhibi o y e ec s
exe ed by al anse in (-13±1%) (F[3,18]=13.28, p<0.0001) and pima anse in
(-15±3%) (F[3,19]=11.80, p=0.0001), on [35S]GTPγS binding o Gαi1-p o eins
(Tables 4.3 and 4.4) (Figu e 4.2.A). Mo eo e , since olinanse in had
unexpec edly demons a ed inhibi o y e ec on his ac i i y (-12±3%),
ke anse in and MDL-11,939 we e also es ed as po en ial an agonis s on his
e ec . A ull blockage o olinanse in-induced inhibi ion was obse ed
(F[2,15]=9.81, p=0.0019) (Tables 4.3 and 4.4) (Figu e 4.2.A), sugges ing
pa ial in e se agonism o his compound on he [35S]GTPγS binding o Gαi1-
p o eins.
Resul s
116
4.1.4 E alua ion o he maximal e ec o di e en d ugs on
Gαi1- and Gαq/11-p o ein coupling o 5-HT2AR in knock-ou 5-
HT2AR(-/-) and wild- ype 5-HT2AR(+/+) mice
To u he con i m he ole o 5-HT2AR in he s imula o y o inhibi o y e ec
exe ed on [35S]GTPγS binding o Gαi1- and Gαq/11-p o eins, SPA expe imen s
we e pe o med in b ain issue o knock-ou 5-HT2AR(-/-) and wild- ype 5-
HT2AR(+/+) mice. B ain memb anes we e incuba ed wi h a single concen a ion
(10 μM) o al anse in, pima anse in, nelo anse in, i anse in, olinanse in,
ke anse in, epli anse in and MDL-11,939 as well as speci ic an ibodies agains
Gαi1- (Figu e 4.3.A) and Gαq/11-p o eins (Figu e 4.3.B).
The e we e no di e ences in basal alues on [35S]GTPγS binding o Gαi1- and
Gαq/11-p o eins be ween 5-HT2AR(+/+) and 5-HT2AR(-/-) mice. Thus, he basal
binding alues o Gαi1-p o eins in wild- ype mice we e 3824±428 CCPM, and
3971±428 CCPM in knock-ou mice (p=0.8115, =0.24, d =14). Simila ly, he
basal binding alues o Gαq/11-p o ein we e 3160±329 CCPM in wild- ype
mice, and 3224±224 CCPM in knock-ou mice (p=0.8779, =0.16, d =16).
The compa ison be ween bo h geno ypes in esponse o di e en d ugs was
pe o med by a epea ed-measu es wo-way ANOVA, whe e condi ions we e
d ug and geno ype. This analysis was pe o med wice, co esponding o
e alua ion o [35S]GTPγS binding esponses o each Gα-p o ein. Since he
e ec s induced by d ugs a e a iable, i was expec ed o ind a signi ican
in e ac ion d ug x geno ype. A u he Bon e oni’s pos -hoc es would
delinea e he speci ic d ugs displaying di e en ial e ec s be ween wild- ype
and knock-ou animals.
In ag eemen wi h he esul s in human b ain samples, when wild- ype mice
b ain co ex memb anes whe e incuba ed wi h al anse in (-10±3%),
pima anse in (-13±2%), nelo anse in (-12±2%), i anse in (-12±3%),
olinanse in (-11±2%) and epli anse in (-12±3%), all o hem exe ed an
Resul s
117
inhibi o y e ec on [35S]GTPγS binding o Gαi1-p o eins (Table 4.5) (Figu e
4.3.A). Fu he mo e, ke anse in and MDL-11,939 did no change basal
[35S]GTPγS binding (Table 4.5) (Figu e 4.3.A).The signi ican inhibi ion
exe ed on [35S]GTPγS binding o Gαi1-p o eins by al anse in, pima anse in
and olinanse in was absen in knock-ou mice memb anes (Table 4.5)
(Figu e 4.3.A) These esul s con i m in ol emen o 5-HT2AR in he inhibi ion
o [35S]GTPγS binding o Gαi1-p o eins. On he o he hand, he inhibi ion
exe ed by nelo anse in, i anse in and epli anse in was also obse ed in
knock-ou mice, i.e. absence o 5-HT2AR (Table 4.5) (Figu e 4.3.A). This
esul s u he suppo ha inhibi o y e ec s on [35S]GTPγS binding o Gαi1-
p o eins exe ed by hese h ee d ugs a e no media ed by 5-HT2AR.
Nex , [35S]GTPγS binding o Gαq/11-p o eins was s udied in wild- ype 5-
HT2AR(+/+) mice. In a simila way o esul s in human on al co ex, i anse in (-
8±1%), olinanse in (-14±2%) and epli anse in (-10±1%) dec eased
[35S]GTPγS binding o Gαq/11-p o eins (Table 4.5) (Figu e 4.3.B).
Fu he mo e, al anse in, pima anse in, nelo anse in and MDL-11,939 did no
modi y he basal [35S]GTPγS binding o his canonical pa hway (Table 4.5)
(Figu e 4.3.B). Mo eo e , ke anse in induced a s imula o y (18±4%) esponse
in wild- ype animals, as desc ibed in human b ain (Table 4.5) (Figu e 4.3.B).
When hese d ugs we e s udied in knock-ou mice, hei e ec was null o
olinanse in and ke anse in, indica ing ha hei e ec s we e media ed by 5-
HT2AR. In con as , i anse in and epli anse in dec eased [35S]GTPγS binding
o Gαq/11-p o eins in knock-ou mice as in wild- ype mice, sugges ing he lack
o in ol emen o 5-HT2AR on hese e ec s (Table 4.5) (Figu e 4.3.B).
Resul s
118
Figu e 4.3: S imula o y o inhibi o y e ec induced by one single concen a ion (10 μM) o
di e en d ugs (al anse in, pima anse in, nelo anse in, i anse in, olinanse in, ke anse in,
epli anse in and MDL-11,939) on he speci ic [35S]GTPγS binding o Gαi1- (Figu e 4.3.A) and
Gαq/11- (Figu e 4.3.B) p o eins in b ain co ex memb anes o wild- ype 5-HT2AR(+/+) and knock-
ou 5-HT2AR(-/-) mice. Ba s a e mean±SEM o independen expe imen s ca ied ou in duplica e
o iplica e, and exp ess he pe cen age alues ela ed o basal [35S]GTPγS binding (0%).
Da a we e analysed using one sample - es s basal binding (*p<0.05, **p<0.01, ***p<0.001)
and wo-way ANOVA ollowed by Bon e oni´s pos -hoc es o d ug x geno ype e ec s
(#p<0.05, ###p<0.001).
Resul s
119
Table 4.5: Nume ical alues o esul s exp essed in Figu e 4.3. S imula o y o inhibi o y e ec
induced by one single concen a ion (10 μM) o di e en d ugs (al anse in, pima anse in,
nelo anse in, i anse in, olinanse in, ke anse in, epli anse in and MDL-11,939) on he
speci ic [35S]GTPγS binding o Gαi/o- and Gαq/11-p o eins in b ain co ex memb anes o wild-
ype 5-HT2AR(+/+) and knock-ou 5-HT2AR(-/-) mice. Values a e mean±SEM o n independen
expe imen s ca ied ou in duplica e o iplica e and exp ess he pe cen age alues ela ed o
basal [35S]GTPγS binding (0%). Da a we e analysed using one sample - es s basal binding
(*p<0.05, **p<0.01, ***p<0.001).
Gα
i1
-p o ein
Gα
q/11
-p o ein
5-HT
2A
R(+/+)
5-HT
2A
R(-/-)
5-HT
2A
R(+/+)
5-HT
2A
R(-/-)
Al anse in
-10±3* (n=5)
0±2 (n=6)
-1±3 (n=6)
-1±2 (n=7)
Pima anse in
-13±2** (n=5)
-2±2 (n=6)
2±2 (n=6)
-4±2 (n=7)
Nelo anse in
-12±2** (n=5)
-12±3* (n=6)
-1±3 (n=5)
-2±2 (n=8)
Ri anse in
-12±3* (n=5)
-11±3** (n=6)
-8±1*** (n=8)
-14±3** (n=7)
Volinanse in
-11±2** (n=5)
-1±2 (n=5)
-14±2*** (n=8)
-1±5 (n=8)
Ke anse in
-2±1 (n=5)
-2±3 (n=5)
18±4* (n=5)
2±1 (n=7)
Epli anse in
-11±3* (n=5)
-10±2** (n=6)
-10±2** (n=4)
-12±3** (n=5)
MDL-11,939
2±2 (n=5)
0±4 (n=6)
-2±5 (n=6)
0±4 (n=6)
Resul s
120
Table 4.6: Summa y o he epea ed measu es wo-way ANOVA analysis o he e ec s o
di e en d ugs on wild- ype 5-HT2AR(+/+) and knock-ou 5-HT2AR(-/-) mice. The esul s o a d ug
x geno ype in e ac ion sugges ha he esponse o he d ugs is a iable be ween bo h mice
geno ypes. A u he pos -hoc analysis was pe o med o disc imina e di e ences be ween
wild- ype and knock-ou animals o each d ug. Resul s a e shown in Figu es 4.3.A and 4.3.B.
Signi ican p alues a e shown in bold.
Gα
i1
-p o ein
Gα
q/11
-p o ein
DFn
DFd
F
P
DFn
DFd
F
p
In e ac ion
7
70
2.84
0.0116
7
105
7.02
<0.0001
D ug
7
70
8.25
<0.0001
7
105
20.24
<0.0001
Geno ype
1
70
10.44
0.0090
1
105
2.23
0.1558
Resul s
121
4.1.5 Summa y
5-HT2AR exp essed in human PFC displays unc ional coupling o he
canonical Gαq/11-p o eins bu also o o he cell pa hways h ough inhibi o y
Gαi/o-p o eins. Among hem, Gαi1-p o ein seems o be he p e e en ial sub ype
in ol ed in his al e na i e signaling pa hway.
The unc ional coupling o 5-HT2AR in human PFC shows cons i u i e ac i i y
because [35S]GTPγS binding o di e en Gα-p o ein subuni s can be inhibi ed
by di e en 5-HT2AR d ugs, p e iously assumed o be neu al an agonis .
The e o e, hese compounds should be conside ed po en ial in e se agonis s.
The blockage o in e se agonism by selec i e 5-HT2AR neu al an agonis s,
and he absence o his pha macological ea u e in 5-HT2AR(-/-) mice allow us
o iden i y hem as selec i e 5-HT2AR in e se agonis s.
Func ional ac i i y o he di e en d ugs as in e se agonis s o 5-HT2AR may
be di e en be ween Gαq/11- and Gαi1-p o ein pa hways. The e o e, a biased
in e se agonism is easible o his ecep o in human b ain.
Among he di e en compounds es ed, pima anse in displays highe and
mo e selec i e in e se e icacy in 5-HT2AR coupling o Gαi1-p o eins, lacking o
ac i i y on Gαq/11-p o ein ac i a ion. On he o he hand, olinanse in could be
conside ed a selec i e 5-HT2AR in e se agonis wi h in e se e icacy on he
coupling o bo h Gαq/11- and Gαi1-p o eins. The e o e, hese wo d ugs we e
selec ed as sui able ools o u he s udies o 5-HT2AR cons i u i e ac i i y in
pa hological condi ions.
Resul s
122
4.2 E alua ion o he unc ional coupling o 5-HT2AR o Gα-
p o ein sub ypes in PFC o schizoph enia subjec s, non-
schizoph enia suicide subjec s and ma ched con ols by
an ibody-cap u e [35S]GTPγS scin illa ion p oximi y assay
(SPA)
4.2.1 Basal [35S]GTPγS binding o Gαi1- and Gαq/11- p o eins in
pos -mo em PFC o schizoph enia subjec s, non-
schizoph enia suicide subjec s and ma ched con ols
Basal [35S]GTPγS binding o Gαi1-p o eins in co ical memb anes was no
s a is ically di e en be ween schizoph enia (162±7 mol/mg p o ), non-
schizoph enia suicide (175±9 mol/mg p o ) and con ol (159±5 mol/mg p o )
g oups (F[2,55]=1.13, p=0.35) (Figu e 4.4.A). Simila ly, basal [35S]GTPγS
binding o Gαq/11-p o eins was no s a is ically di e en be ween schizoph enia
(191±9 mol/mg p o ), non-schizoph enia suicide (198±9 mol/mg p o ) and
con ol subjec s (210±11 mol/mg p o ) (F[2,56]=0.95, p=0.39) (Figu e 4.4.B).
Resul s
123
Figu e 4.4: Indi idual alues o basal [35S]GTPγS binding o Gαi1-(A) and Gαq/11-(B) p o eins
in PFC memb anes o schizoph enia (n=23) ( ed squa e), non-schizoph enia suicide (n=13)
(blue iangle) and con ol subjec s (n=23) (black ci cle). The lines ep esen s mean±SEM
alues o each g oup.
In o de o e alua e he in luence o a iables such as age a dea h, sex, PMD
and s o age ime on basal [35S]GTPγS binding o bo h Gα-p o eins, linea
co ela ion analyses we e pe o med. Any o hese po en ial a iables showed
co ela ion wi h [35S]GTPγS basal binding alues. The absence o ela ionship
was also obse ed when conside ed sepa a ely hose h ee popula ion g oups
(Table 4.7).
Resul s
124
Table 4.7: E ec o age (yea s), PMD (hou s) and s o age ime (mon hs) on basal [35S]GTPγS
binding o Gαi1- and Gαq/11-p o eins.
Gαi1-p o ein basal binding ( mol/mg p o ein)
Schizoph enia Non-
schizoph enia
suicide
Con ol
p
n
p
n
p
n
Age
(yea s)
-0.2811
0.1939
23
-0.1690
0.5995
13
0.0150
0.9458
23
PMD
(hou s)
0.1012
0.6459
23
0.2500
0.4332
13
-0.0716
0.7452
23
S o age
ime
(mon hs)
-0.0175
0.9368
23
-0.0797
0.8054
13
0.0262
0.9056
23
Gαq/11-p o ein basal binding ( mol/mg p o ein)
Schizoph enia Non-
schizoph enia
suicide
Con ol
p
n
p
n
p
n
Age
(yea s)
-0.2783
0.1986
23
-0.3972
0.1790
13
0.1534
0.4847
23
PMD
(hou s)
0.1816
0.4070
23
-0.1876
0.5403
13
-0.0565
0.7978
23
S o age
ime
(mon hs)
-0.0823
0.7071
23
0.1802
0.5558
13
0.0778
0.7245
23
Resul s
125
4.2.2 Modula ion o [35S]GTPγS binding o Gαi1- and Gαq/11-
p o eins by he 5-HT2AR in e se agonis pima anse in in pos -
mo em PFC o schizoph enia subjec s, non-schizoph enia
suicide subjec s and ma ched con ols
Pima anse in (10-10-10-6 M) displayed concen a ion-dependen inhibi ion
cu es in schizoph enia, non-schizoph enia suicide and con ol g oups (Figu e
4.5). No di e ences in po ency o pima anse in we e obse ed be ween
g oups (schizoph enia: -logIC50=7.94±0.1; non-schizoph enia suicide: -
logIC50=8.15±0.0; con ol: -logIC50=8.01±0.1) (F[2,56]=2.26, p=0.0871)
(Table 4.8). The inhibi o y e ec o pima anse in was highe in schizoph enia
(Imax=-20±1%) han in con ol (Imax=-14±1%) and non-schizoph enia suicide
(Imax=-14±1%) g oups (F[2,56]=9.19, p=0.0004) (Table 4.8).
Figu e 4.5: Concen a ion- esponse cu es o speci ic [35S]GTPγS binding o Gαi1-p o eins in
esponse o pima anse in in human PFC memb anes. The 100% dashed line deno es speci ic
basal [35S]GTPγS binding o Gαi1-p o eins (BB). Each poin ep esen s he mean±SEM alue
om independen expe imen s. See coanalysis esul s in he ex .
gu e 4 5
: Concen a ion
esponse cu es o speci ic [
35
S]GTPγS binding o Gα
i1
p o
ei
ns
i
n
Resul s
132
4.2.3 Modula ion o he [35S]GTPγS binding o Gαi1- and Gαq/11-
p o eins by he 5-HT2AR in e se agonis olinanse in in pos -
mo em PFC o schizoph enia subjec s, non-schizoph enia
suicide subjec s and ma ched con ols
Gi en he absence o [35S]GTPγS binding modula ion by pima anse in, he
s a us o 5-HT2AR coupling o Gαq/11-p o eins could no be quan i ied by using
his d ug. In o de o e alua e whe he he enhanced cons i u i e ac i i y o 5-
HT2AR coupling o Gαi1-p o eins was selec i e o his pa hway, o Gαq/11-
p o ein canonical pa hway was also al e ed, ano he 5-HT2AR in e se agonis
should be es ed. Fo his pu pose, and acco ding o p e ious indings,
olinanse in was chosen as he mos sui able pha macological ool.
Volinanse in (10-9-10-5 M) displayed a concen a ion-dependen inhibi ion
cu es o [35S]GTPγS binding o Gαi1-p o eins in schizoph enia, non-
schizoph enia suicide and con ol g oups (Figu e 4.9). No di e ences in
po ency o olinanse in we e obse ed be ween g oups (schizoph enia: -
logIC50=6.45±0.1; non-schizoph enia suicide: -logIC50=6.53±0.1; con ol: -
logIC50=6.54±0.1) (F[2,56]=0.5830, p=0.5616) (Table 4.10). The inhibi o y
e ec o olinanse in was highe in schizoph enia subjec s (Imax=-18±1%) han
in con ol (Imax=-13±1%), as well as in non-schizoph enia suicide subjec s
(Imax=-14±1%) (F[2,56]=9.76, p=0.0002) (Table 4.10).
Resul s
133
Figu e 4.9: Concen a ion- esponse cu es o speci ic [35S]GTPγS binding o Gαi1-p o eins in
esponse o olinanse in in human PFC memb anes. The 100% dashed line deno es speci ic
basal [35S]GTPγS binding o Gαi1-p o eins (BB). Each poin ep esen s mean±SEM alue om
independen expe imen s.
Table 4.10: Pha macological pa ame e s o olinanse in concen a ion- esponse cu es o
[35S]GTPγS binding o Gαi1-p o eins in PFC memb anes o schizoph enia, non-schizoph enia
suicide and ma ched con ol subjec s. IC50 alues we e ob ained as an ilog o logIC50. Imax
ep esen s he expe imen al maximal inhibi o y e ec induced by olinanse in, and is
exp essed as pe cen age o espec i e basal [35S]GTPγS binding alue. Values a e
mean±SEM o n independen subjec s. Da a we e analysed using one-way ANOVA ollowed
by Bon e oni’s pos -hoc es (**p<0.01 s con ol and non-schizoph enia g oup).
G oup
Basal
( mol/ mg p o ein)
IC50
(nM)
Imax (%)
n
Schizoph enia
162±7
456±83
-18±1**
23
Non-schizoph enia suicide
175±9
359±67
-14±1
13
Con ol
159±5
341±42
-13±1
23
Resul s
134
An analysis o co a iance (ANCOVA) was u he pe o med o e alua e he
po en ial in luence o age a dea h, PMD and s o age ime. Sex di e ences
we e also e alua ed. Any o hese a iables modi ied he esul s and,
consis en wi h p e ious indings, di e ences be ween g oups in Imax alues
we e main ained (F[2,55]=8.13, p=0.0008).
A complemen a y s a is ical compa ison be ween g oups was conduc ed by
non-linea cu e i ing coanalysis o all indi idual expe imen s. The coanalysis
o concen a ion- esponse cu es o olinanse in demons a ed di e en
pa e ns o schizoph enia espec o con ol (F[3,178]=14.14, p<0.0001) and
non-schizoph enia suicide g oups (F[3,138]=8.09, p<0.0001) (Figu e 4.9).
Once s a is ical di e ences be ween cu es we e ob ained, u he con as s
we e pe o med o de ec whe he di e ences we e a ibu able o changes in
Imax and/o IC50 alues be ween g oups. Thus, he cu e i ing coanalysis
con i med ha schizoph enia subjec s showed a highe inhibi o y e ec o
olinanse in (Imax=-18%, 95%Cl 19% o 17%) han con ol (Imax=-13%, 95%Cl
14% o 12%) (F[1,178]=27.77, p<0.0001) and non-schizoph enia suicide
subjec s (Imax=-14%, 95%Cl 16% o 13%) (F[1,138]=11.85, p<0.0001). No
di e ences we e ob ained o IC50 alues be ween schizoph enia (IC50=355.
nM, 95%Cl 250.7 nM o 502.6 nM), non-schizoph enia suicide (IC50=302.2 nM,
95%Cl 186.3 nM o 477.4 nM) and con ol g oups (IC50=313.9 nM, 95%Cl
208.8 nM o 464.6 nM).
The selec i i y o inhibi o y e ec exe ed by olinanse in on [35S]GTPγS
binding o Gαi1-p o eins was es ed by using 5-HT2AR neu al an agonis MDL-
11,939 a 10 μM. In schizoph enia subjec s, olinanse in maximal inhibi o y
e ec on [35S]GTPγS binding (-17±1%) was blocked by he an agonis
(p<0.0001, =12.67, d =44) (Figu e 4.10). Simila ly, in non-schizoph enia
suicide g oup, he maximal inhibi ion p omo ed by olinanse in on [35S]GTPγS
binding o Gαi1-p o eins (-14±1%) was comple ely blocked by MDL-11,939
(p<0.0001, =8.50, d =24) (Figu e 4.10). As p e iously desc ibed (See 4.1.3),
Resul s
135
in con ol g oup, he maximal inhibi ion o [35S]GTPγS binding o Gαi1-p o eins
induced by olinanse in (-13±1%) was blocked by MDL-11,939 (p<0.0001,
=12.23, d =44) (Figu e 4.10).
Figu e 4.10: An agonism o he inhibi o y e ec induced by a maximal concen a ion o
olinanse in (10 μM) on speci ic [35S]GTPγS binding o Gαi1-p o eins in PFC memb anes o
schizoph enia, non-schizoph enia suicide and con ol subjec s. Volinanse in was co-incuba ed
wi h he selec i e 5-HT2AR an agonis MDL-11,939 (10 μM). Basal alues o speci ic
[35S]GTPγS binding o Gαi1-p o eins a e exp essed as 0%, and inhibi o y e ec s on espec i e
basal a e shown as pe cen age o basal alues. Each poin indica es indi idual alues. Ba s
ep esen mean±SEM o pe cen age alues o independen expe imen s. Da a we e analysed
using one sample - es s basal binding (****p<0.0001) and wo sample - es o compa e
olinanse in e ec s in absence o p esence o MDL-11,939. (####p<0.0001).
The modula ion o [35S]GTPγS binding o Gαq/11-p o eins by he 5-HT2AR
selec i e d ug olinanse in (10-9-10-5 M) was also es ed in schizoph enia, non-
schizoph enia suicide and con ol g oups. As expec ed, olinanse in displayed
concen a ion-dependen inhibi ion cu es in schizoph enia, non-
schizoph enia suicide and con ol g oups (Figu e 4.11). No di e ences we e
obse ed in po ency be ween g oups (schizoph enia: -logIC50=6.53±0.1; non-
Resul s
136
schizoph enia suicide: -logIC50=6.57±0.1; con ol: -logIC50=6.52±0.1
(F[2,56]=1.53, p=0.6617) (Table 4.11). Likewise, he inhibi o y e ec o
olinanse in was no di e en be ween g oups (schizoph enia: Imax=-16±1%;
non-schizoph enia suicide: Imax=-16±1%; con ol: Imax=-15±1%)
(F[2,56]=0.6861, p=0.5046).
Figu e 4.11: Concen a ion- esponse cu es o speci ic [35S]GTPγS binding o Gαq/11-p o eins
in esponse o olinanse in in human PFC memb anes. The 100% dashed line deno es
speci ic basal [35S]GTPγS binding o Gαq/11-p o eins (BB). Each poin ep esen s mean±SEM
alue om independen expe imen s.
Resul s
137
Table 4.11: Pha macological pa ame e s o olinanse in concen a ion- esponse cu es o
[35S]GTPγS binding o Gαq/11-p o eins in PFC memb anes o schizoph enia, non-
schizoph enia suicide and ma ched con ol subjec s. IC50 alues we e ob ained as an ilog o
logIC50. Imax ep esen s he expe imen al maximal s imula o y o inhibi o y e ec induced by
olinanse in, and is exp essed as pe cen age o espec i e basal [35S]GTPγS binding alue.
Values a e mean±SEM o n independen subjec s. Da a we e analysed using one-way
ANOVA.
A complemen a y s a is ical compa ison be ween g oups was conduc ed by
non-linea cu e i ing coanalysis o all indi idual expe imen s. The coanalysis
o concen a ion- esponse cu es o olinanse in demons a ed no di e en
pa e ns be ween schizoph enia and con ol g oups (F[3,178]=0.32, p=0.8138)
(Figu e 4.11). In he same way, he e was no di e ence be ween
schizoph enia and non-schizoph enia suicide g oups (F[3,138]=0.77,
p=0.5103) (Figu e 4.11). Finally, no changes we e ound be ween con ol and
non-schizoph enia suicide g oup (F[3,138]=2.17, p=0.0948) (Figu e 4.11).
G oup
Basal
( mol/ mg p o ein)
IC50
(nM)
Imax
(%)
n
Schizoph enia
191±9
341±54
-15±1
23
Non-schizoph enia suicide
198±9
310±47
-16±1
13
Con ol
210±11
368±52
-15±1
23
Resul s
138
The selec i i y o he inhibi o y e ec exe ed by olinanse in on [35S]GTPγS
binding o Gαq/11-p o eins was es ed by using he 5-HT2AR neu al an agonis
MDL-11,939 a 10 μM. In schizoph enia subjec s, olinanse in maximal
inhibi o y e ec on [35S]GTPγS binding (-15±1%) was also blocked by
an agonis (p<0.0001, =12.49, d =44) (Figu e 4.12). In non-schizoph enia
suicide g oup, he maximal inhibi ion p omo ed by olinanse in on [35S]GTPγS
binding o Gαq/11-p o eins (-15±1%), was also comple ely blocked by MDL-
11,939 (p<0.0001, =10.53, d =24) (Figu e 4.12). Addi ionally, in con ol g oup,
he maximal inhibi ion o [35S]GTPγS binding induced by olinanse in (-14±1%)
was also blocked by MDL-11,939 (p<0.0001, =11.73, d =44) (Figu e 4.12).
Figu e 4.12: An agonism o he inhibi o y e ec induced by a maximal concen a ion o
olinanse in (10 μM) on speci ic [35S]GTPγS binding o Gαq/11-p o eins in PFC memb anes o
schizoph enia, non-schizoph enia suicide and con ol subjec s. Volinanse in was co-incuba ed
wi h he selec i e 5-HT2AR an agonis MDL-11,939 (10 μM). Basal alues o speci ic
[35S]GTPγS binding o [35S]GTPγS a e exp essed as 0%, and inhibi o y e ec s on he
espec i e basal a e shown as pe cen age o basal alues. Each poin indica es indi idual
alues. Ba s ep esen mean±SEM o pe cen age alues o independen expe imen s. Da a
we e analysed using one sample - es s basal binding (****p<0.0001) and wo sample - es
o compa e olinanse in e ec s in absence o p esence o MDL-11,939 (####p<0.0001).
Resul s
139
4.2.4 Modula ion o [35S]GTPγS binding o Gαi1- and Gαq/11-
p o eins by 5-HT2AR agonis (±)DOI in pos -mo em PFC o
schizoph enia subjec s, non-schizoph enia suicide subjec s
and ma ched con ols
In o de o con i m p e ious esul s (Ga cia-Bea e al., 2019), he e ec o 5-
HT2AR selec i e agonis (±)DOI was e alua ed in schizoph enia, non-
schizoph enia suicide and con ol g oups a one single concen a ion (10 μM),
which co esponds o a submaximal concen a ion (Figu e 4.13). As expec ed,
(±)DOI inc eased [35S]GTPγS binding o Gαi1-p o eins in schizoph enia, non-
schizoph enia suicide and con ol g oups (Figu e 4.13). The s imula o y e ec
o (±)DOI was highe in schizoph enia han in con ol and non-schizoph enia
suicide g oups (F[1,56]=7.45, p=0.0014) (Table 4.12) (Figu e 4.13).
Resul s
140
Figu e 4.13: (±)DOI-induced e ec on he speci ic [35S]GTPγS binding o Gαi1-p o eins in
human PFC memb anes. (±)DOI was added a 10 μM concen a ion. Basal alues o speci ic
[35S]GTPγS binding o Gαi1-p o eins a e exp essed as 0%, and s imula o y e ec s on he
espec i e basal a e shown as pe cen age o basal alues. Each poin indica es indi idual
alues. Ba s ep esen mean±SEM o pe cen age alues o independen expe imen s. Da a
we e analysed using one sample - es s basal binding (****p<0.0001) and one-way ANOVA
ollowed by Bon e oni’s pos -hoc es (##p<0.01 s con ol and non-schizoph enia g oup).
Table 4.12: Pha macological pa ame e s o (±)DOI induced-e ec a one single concen a ion
(10 μM) on [35S]GTPγS binding o Gαi1-p o eins in PFC memb anes o schizoph enia, non-
schizoph enia suicide and ma ched con ol subjec s. Emax ep esen s he maximal s imula o y
e ec induced by (±)DOI, and is exp essed as pe cen age o espec i e basal [35S]GTPγS
binding alue. Values a e mean±SEM o n independen subjec s. Da a we e analyzed using
one-way ANOVA ollowed by Bon e oni’s pos -hoc es (**p<0.01 s con ol and non-
schizoph enia g oup).
G oup
Basal
( mol/ mg p o ein)
Emax
(%)
n
Schizoph enia
162±7
15±1**
23
Non-schizoph enia suicide
175±9
9±1
13
Con ol
159±5
10±1
23
Resul s
141
An analysis o co a iance (ANCOVA) was u he pe o med o e alua e he
po en ial in luence o age a dea h, PMD and s o age ime. Sex di e ences
we e also e alua ed. Any o hese a iables modi ied he esul s and,
consis en wi h p e ious indings, he di e ences be ween g oups in Imax alues
we e main ained (F[2,52]=3.13, p<0.05).
The selec i i y o s imula o y e ec exe ed by (±)DOI on [35S]GTPγS binding
o Gαi1-p o eins was es ed by using 5-HT2AR neu al an agonis MDL-11,939
a 10 μM. In schizoph enia subjec s, (±)DOI-induced maximal s imula o y
e ec on [35S]GTPγS binding o Gαi1-p o eins (15±1%) was blocked by he
an agonis (p<0.0001, =7.93, d =44) (Figu e 4.14). In non-schizoph enia
suicide g oup, he maximal s imula ion p omo ed by (±) DOI on [35S]GTPγS
binding o Gαi1-p o eins (9±1%) was also comple ely blocked by MDL-11,939
(p<0.0003, =4.17, d =24) (Figu e 4.14). In con ol g oup, he maximal
s imula ion o [35S]GTPγS binding induced by (±)DOI (10±1%) was also
an agonized by MDL-11,939 (p<0.0001, =5.86, d =44) (Figu e 4.14).
Re e ences
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5-HT2A ha zailea en ak ibi a e
kons i u iboa e a a makoen
hau ako asun un zionala:
eskizo enikoen pos -mo em
ga unean egindako lana
I zia Mune a A a e
LABURDUREN ZERRENDA
[35S]GTPγS 35 su ea ekin ma ka u ik dagen guanosina-5´-O-(γ- io)-
i os a oa
5-HT 5-Hid oxi ip amina (se o onina)
5-HT2AR Se o onina 2A ha zailea
5-HT2AR(-/-) Knock-ou
5-HT2AR(+/+) Wild-Type
7TM 7 domeinuko min z zeha eko ha zailea
AA Azido a akidonikoa
AC Adenila o ziklasa
BB Finkapen basala
Bolinanse in MDL100907
BRET Biolumineszen zia-e esonan zia bidezko ene gia- ans e en zia
Ca2+ Kal zio ioia
CaMKII Kal zio/kalmodulina en mendeko kinasa II
cAMP Adenosina mono os a o ziklikoa
D2R Dopamina 2 ha zailea
DA Dopamina
DAG Diazilglize ola
DLPFC Au e-ga unazal do sola e ala
(±)DOI 2,5-dime oxi-4-iodoam e amina
DSM Gaixo asun men alen diagnosia e a es a is ika eskulibu ua
EC50 Es imulazio e ek u maximoa lo zeko % 50 kon zen azioa
Emax Es imulazio e ek u maximoa
ERK Zelulaz kanpoko seinaleek e egula u iko kinasak
FDA D ogen e a elikagaien adminis azioak
GABA Azido-aminobu i ikoa
GPCR G-p o einei lo u iko ha zaileak
GDP Guanosina di os a oa
GRK G-p o einei lo u ako kinasa
GTP Guanosine i os a oa
GTPγS 5'-O-[gamma- io] i os a oa
GWAS Genoma-osoa en asoziazio-az e ke a
IC50 Inhibizio e ek u maximoa lo zeko % 50 kon zen azioa
Imax Inhibizio e ek u maximoa
IP3 Inosi ol 1,4,5- i os a oa
LSD Azido lise gikoa en D-die ilamida
MAPK Ak iba u ako mi ogenoen p o eina kinasa
NBS Finkapen ez espezi ikoa
NMDA N-Me il-D-aspa a oa
NSZ Ne bio sis ema zen ala
PCP Fen ziklidina
PET Posi oien igo pen bidezko omog a ia
PIP2 Fos a idilinosi ol 4,5-bi os a oa
PKC P o eina kinasa C
PLA2 A2 os olipasa
PLC C os olipasa
PMD Pos -mo em a ze apena
PSD-95 95 den si a e p o eina pos sinap ikoa
PTX Bo de ella pe ussis oxina
RSK-2 S6 kinasa e ibosomala
SNP nukleo ido baka eko polimo ismoa
SPA Immunop ezipi azioa ekin akopla u ako [35S]GTPγS
inkapen eknika
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Hipo esi dopamine gikoa en a abe a, sin oma posi iboak (haluzinazioak,
elda nioak) ga uneko e emu mesolimbiko-es ia alean ansmisio
dopamine gikoa en hipe ak ibi a ea en ondo io izango li a eke. Hala e e,
sin oma nega iboak e a kogni iboak an ipsiko ikoekiko e esis en eak di a. Ho i
dela e a, hipo esi dopamine gikoa bi o mula u egin zen, hipodopamine gia
ko ikala p oposa uz, e a ho i sin oma nega ibo e a kogni iboekin e laziona uz
(Da is e al., 1991; Howes e a Kapu 2009; McCu cheon e al., 2020).
Dopamina ha zaileak G-p o einei lo u ako ha zaileak (GPCR) di a. Bi amilia
nagusi an sailka u dai ezke: D1 dopamina-ha zaileen amilia, D1 e a D5
dopamina-ha zaileak ba ne ha zen di uena (D1R, D5R), e a D2 ha zaileen-
amilia, D2, D3 e a D4 dopamina ha zaileak ba ne ha zen di uena (D2R, D3R,
D3R). D2Ren blokeoa an ipsiko ikoen ekin za mekanismo nagusia da,
gehienba an ipsiko iko ipikoena. Ho i ho ela, ha zaile dopamine gikoak
un sezko ze egina du ela p oposa u da eskizo enian.
In i o eginiko neu oi udi az e ke ak, posi oien igo pen bidezko omog a ia
(PET) e a o oi baka a en igo pen bidezko omog a ia konpu a iza ua
(SPECT) e abiliaz, bes eak bes e, eskizo enia du en pazien een D2R e a D1R
ha zaileen egoe a ebalua zeko e abili di a. Hasie ako az e ke ek, emai za
kon aja iak au ki u zi uz en; ba zuek D2Ren igoe a adie aziz, e a bes e
ba zuek, be iz, kon olekin alde ik ez zu ela (Howes e al., 2012, Cumming e
al., 2021). D2Ren den si a e al ua, e egulazio mekanismoak di ela e a,
a amendu an ipsiko iko k onikoa en ondo io zela i adoki zen. Aldiz,
a mako ik jaso ez zu en pazien e eskizo enikoek ez zi uz en D2Ren igoe ak
au kez u, ha zaileen den si a ea en igoe a e a a amendua e laziona zen
zi uena (Seeman, 2013). Au eko emai zak inda u egin zi en in i o pos -
mo em giza ga unean D2Ren handipena azaldu ondo en (Seeman e al.,
1984, Zakzanis e a Hansen, 1998). Gaine a, duela gu xi a gi a a u ako
az e lan ba en a abe a, pos -mo em ga un eskizo enikoe an ez dago D2Ren
ak ibi a ea en handipena (Eguskiza e al., 2021). D2R- ekin bezala, in i o
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az e ke ek eskizo enikoen au e-ga unazalean D1R en den si a ea i bu uzko
emai za kon aja iak au ki u zi uz en, au e ik a amendu ik gabeko
pazien ee an (Cumming e al., 2021). Azkenengo ike ke ak, be iz, D1R e a
D2R pos sinap ikoen egoe an baino neu o ansmisio dopamine giko
pa asinpa ikoa az e ze a zuzendu di a. Hala, DA sin esi p esinap ikoa (Howes
e al., 2012; Fusa -Poli e a Meye -Lindenbe g, 2013), DA kon zen azio
sinap ikoa (Abi-Da gham e al., 2000; Ca a aggio e al., 2015) e a an e aminak
e agindako DA askapena (Howes e al. 2012; Lau elle, 1998) handiagoa
oga u da pazien e eskizo enikoen es ia umean. Bes alde, eskizo enia
du en pazien een au e-ga unazalean an e aminak e agindako DA en
askapena en mu izke a e e oga u da, e a ho ek hipodomapine gia ko ikal
p esinap ikoa dagoela i adoki zen du au e-ga unazalean (Sli s ein e al.,
2015).
Eskizo enia en hipo esi glu ama e gikoa
Glu ama oa ga uneko neu o ansmiso e ki zika zaile nagusia da, e a ha zaile
iono opiko e a me abo opiko ha zaileekin elka e agi en du. Ba e ik,
ha zaile iono opikoen aldeak NMDA, kaina oa e a α-amino-3-hid oxi-5-me il-
isoxazol-4-p opiona o (AMPA) ha zaile azpimo ak bil zen di u. Bes e ik,
glu ama oa en ha zaile me abo opikoak (mGluR) G-p o eina en bidezko
seinalea en ansdukzioa ak iba zen du e, e a I (mGlu1, mGlu5), II (mGlu2,
mGlu3) e a III (mGlu6, mGlu4, mGlu7, mGlu8) aldee an bana zen di a (Naka,
1992; Niswende e a Conlun, 2010; Mugu uza e al., 2016).
Ondo eza i a dago NMDA ha zaileen an agonis en adminis azioek, hala nola
en ziklidina (PCP) edo ke amina, psikosia en an zeko egoe ak e a de izi
kogni iboak e agin di zake ela gizaki osasun suengan. Ke amina e a PCPa
NMDA ha zaileen an agonis a ez-lehiako ak di a (Thomson e al., 1985).
Au kikun za ho ie an oina i u a, NMDA ha zailee an iza en den deso ekak
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eskizo enia ekin e lazioa izan dezakeela us e da (Ja i e a Zukin, 1991;
S one e al., 2008). Bi xia bada e e, an e amina edo bes e agonis a
dopamine giko ba , NMDA ha zaileen an agonis ekin ba e a adminis a zean
sin oma kogni ibo e a nega iboak so zen di u, eskizo enia en isiopa ologia
imi a uz (K ys al e al., 2005). Ho i dela e a, NMDA ha zaileen an agonis a ez
lehiako en adminis azioa animalia e edu gisa e abil zen da eskizo enia
az e zea helbu u du en ike lane an.
Ho ekin lo u a, hainba ike ke a an pazien e eskizo enikoen glu ama o mailak
neu u di uz e, sin oma mo a desbe dinekin e laziona zeko helbu ua ekin.
Ho ela, sis ema glu ama e gikoa en deso ekak eskizo enia en dis un zio
kogni iboekin e laziona u di a (Moghaddam e a Ja i , 2012). Izan e e, au e ik
azaldu ako hipe dopamine gia es ia ala e a hipodopamine gia ko ikala
hipo esi glu ama e gikoa ekin e laziona u di a. Lehenengo, NMDA ha zaileen
hipoak ibi a ea edo inhibizioa desk iba u da e emu dopamine giko
mesolinbikoe an, azido-aminobu i ikoa (GABA) aska zen du en
in e neu one an. Ho ek, egoe a oniko inhibi zailea i kal e egingo lioke, e a,
ondo ioz, dopamina en sin esi e a askapen handiagoa eka iko luke, sin oma
psiko ikoak e aginez. E a alde an ziz, sin oma nega iboak NDMA ha zaileen
hipo un zioak e agindako hipoak ibi a e dopamine giko ko ikala ekin
e laziona u di a (Ja ii , 2010).
Eskizo enia en hipo esi se o one gikoa
Eskizo enia en hipo esi se o one gikoa azido lise gikoa en D-die ilamida
(LSD) d oga haluzinogenoa en e a 5-HT en (F eedman 1961) a eko
in e akzioei bu uzko lehen ike ke e a ik so u zen. LSDak e a bes elako d oga
haluzinogenoek eskizo enia en sin oma posi iboen an ze akoak di en bu u
nahasmenak e agi en di uz e, se o onina ha zaileak ak iba uz. Gaine a, d oga
psikodelikoen egi u a kimikoa 5-HT neu o ansmiso ea en an ze akoa da. 5-
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HT ha zaile azpimo a guz ien a ean, ebiden zia uga ik adie az en du e
haluzinogenoek se o onina 2A ha zaile azpimo a ekin (5-HT2AR) lo zen di ela,
be en e aginak so zeko (González-Maeso e al., 2007; Geye e a
Vollenweide 2008; González-Maeso e a Seal on, 2009; Madsen e al., 2019).
Bes alde, a mako an ipsiko iko a ipikoek 5-HT2ARekiko a ini a e handia
e akus en du e, eskizo enian gehien az e u den i u a makologikoa bilaka uz
(Mel ze e al., 1989). 1.4 a alean 5-HT2ARen isiologia e a eskizo enian du en
inplikazioa en be ikuspen zeha zagoa au ki dai eke.
1.1.4 Ga uneko al e azio mo ologikoak
Au e-ga unazala eskizo enia en sin oma posi ibo e a kogni iboekin
e laziona u da (Kolk e a Rakic, 2022). Izan e e, ga uneko eskualde ho e an
eman dai ezkeen lesioak, animalie an e a gizakie an, epe luze ako ondo ioak
e agin di zake e; hala nola lan-memo ia en al e azioa, hau apen inpul so
mu iz ua, ingu umen-es imulu handiagoak suma zea e a jokabide-
mu izke ak. Be az, neu i handi ba ean au e-ga unazaleko anomaliak
eskizo enia ekin e a bes e gaixo asun psikia iko ba zuekin lo u di a, sin oma
amankomunak di uz en heinean (Xu e al., 2019).
Ondo desk iba u a dago eskizo enia pai a zen du en pazien een alboko
ben ikulua en %25eko zabal zea ema en dela, ga una en bolumen osoa en
%2ko mu izke a ekin (Johns one e al., 1976; Haijma e al., 2013).
Ben ikuluen amaina gu xika handi zen da gaixo asuna en hasie a ik,
ga una en ma e ia g isa mu iz u egi en den heinean (E p e al., 2016).
Eskizo enia du en pazien een ga unen pos -mo em az e ke a mo ologikoek
desbe din asunak e aku si di uz e banake a zelula ean; ziu enik ga una en
ga apen goiz ia ean emandako mig azio neu onala en al e azioekin, zelula
pi amidalen gale a ekin, egi u a zelula de o ma ua ekin e a GABA
in e neu onen kopu ua en mu izke a ekin lo uak (Schmid e a Mi nics, 2015).
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1.2 G-p o einei lo u iko ha zaileak (GPCR)
1.2.1 Ezauga i o oko ak
GPCR edo 7 domeinuko min z zeha eko ha zailea (7TM), go pu zeko min z
ha zaile amilia azpi alde nagusia osa zen du e. Ha zaileak ligando mo a
desbe dinak, hala nola ioiak, molekula xikiak e a pep idoak inka zeko edo
lo zeko ahalmena du e, zelulaz kanpoko seinaleak zelula en ba ne a
ansmi i zeko (Alexande e al., 2019).
GPCRek ga an zi biomediko handia du e, hainba ja due a isiologiko zein
pa ologia an pa e ha zen bai u e. Gaine a, a makoen ekin za mekanismoa
e agi eko i u nagusiak di a, me ka u a u ako a makoen %35a ho elakoa
izanik (Hause e al., 2017).
7TM ha zaileen supe amilia i GPCR e e dei zen zaie, be en ekin za G-
p o einen bi a ez be e zen du elako. G-p o einak guanina nukleo idoak lo zen
di uz en hi u azpi uni a ez osa u ik daude: α, β e a γ. Hala e e, GPCRak G-
p o einekin lo zeaz gain bes e p o eina zi oplasma ikoe a a lo u dai ezke, β-
a es ine a a bes eak bes e (Sy o a kina e al., 2016). G-p o einen ak ibazioa,
ho az, zelulaz kanpoko seinalea zelula en ba neko e an zun isiologikoan
bilaka zeko lehen u a sa izango da (S i am e a Insel, 2018). Modu ho e an,
G-p o einak ansduk o e edo GPCRek so u iko anpli ikado e gisa joka zen
du e, zelula ba uko e an zuna so zeko.
1.2.2 GPCRen seinalez apena en oina izko mekanismoa
G-p o eina he e o ime ikoek un sezko ze egina du e e an zun zelula en
espezi iko asuna e a ezauga iak de ini zeko. Ligandoen bidezko
ak ibazioa en ondo ioz, GPCRen kon o mazio aldake a ge a zen da, G-
p o einekiko a ini a ea a eago zen duena, e a G-p o einen e eklu amendua
e agi en duena. Ligando, ha zaile e a G-p o einen a eko elka ekin zak G-
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p o eina he e o ime ikoa en α-azpiuni a ea i lo u ik dagoen guanosina
di os a o (GDP) nukleo idoa aska zea daka , e a guanosina i os a o (GTP)
nukleo idoa ekin uka zea sus a zen du. GDP-GTP ukeak p o eina
he e o ime ikoa osa zen duen azpiuni a een e a ha zaileen a eko
disoziazioa e agi en du. Egoe a ho e an, Gα e a Gβγ azpiuni a eak hainba
e ek o eekin elka ekin za iza en du e, ligandoa en e an zuna zelula ba ne a
ansduzi zeko; hala nola, adenila o ziklasa (AC) os olipasa e a e e en
ionikoak (Gilman, 1987; Hamm, 1998; Ma inissen e a Gu kind, 2001).
Azpiuni a e ho ien seinalez apena amai zeko, e a p ozesu hao i e engabe
ge a zeko, G-p o einak be e o ma he e o ime iko a buel a u beha a du.
Ho e a ako, Gα-azpiuni a ea en GTPasa ak ibi a eak GTP nukeo idoa GDP a
hid oliza zen du, Gα-azpiuni a ea Gβγ dime oa en elka ke a ahalbide uz, e a
G-p o eina inak iboa osa uz be i o (Milligan e a Kos enis, 2006; Hilge e al.,
2018) (1.2 I udia).
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I udia 1.2: GPCR/G-p o eina ak ibazio e edua en i udikapen eskema ikoa. A seden-egoe an
edo egoe a inak iboan, G-p o eina he e o ime oak di a, e a osa u ik daude Gα azpiuni a eaz
(GDP ekin elka u a) e a Gβγ azpiuni a eaz (1a). Ligando ba en ak ibazioa en ondo en
(neu o ansmiso e gisa), GPCRak aldake a kon o mazionala su i zen du, G-p o eine a a
lo zea ahalbide zen duena (1b) e a GDP GTPaz uka zea sus a zen du Gα azpiuni a ea Gβγ
azpiuni a ea ekin bana uz (2). Momen u ho e an, G-p o eina egoe a ak iboan dago α e a βγ
azpiuni a eek zelula ba neko e ek o eekin elka e agingo du e, hainba seinalez apen bide
modula zeko ahalmena ekin (3). Seinalez apena amai zen da GTP molekula GDP a
hid oliza zean, G-p o eina e egula zaileen bi a ez (RGS) (4). Azkenik, konplexu
he e o ime ikoa osa zen da, G-p o einak egoe a inak ibo a buel a uz (5). Oli e ia e al.,
2019 en ilus azio egoki ua.
Agonis ak GPCRa ekin elka zeak, G-p o einak es imula zen di uz en
kon o mazio-aldake ak abia az eaz gain, G-p o einei lo u ako kinasen (GRK)
bidezko ha zailea en os o ilazioa e az en du e. Fos o ilazioak a es inen
e eklu amendua sus a zen du, GPCRen e egulazioa mu iz eko e a G-
p o einen mendeko seinalez apena a in zeko. Ho ez gain, β-a es inen e a G-
GPCR/G p o ein ac i a ion
GPCR
Ligandoa
α-azpiuni a ea
βγ-azpiuni a ea
RGS
P o eina e ek o eak (AC, GRIK, PLC)
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p o einekiko independien eak di en seinalez apenak ak iba u ahal di a,
ak iba u ako-mi ogenoen-p o ein-kinasen (MAPK) bidea ak iba uz (Wang e
al., 2018).
G-p o einekin e a β-a es inekin ba egi eaz gain, GPCRak bes e GPCRekin
elka e agin dezake e dime oak e a zeko; bai a goi-mailako oligome oak e e,
sa i an un sezkoak di enak GPCRen un zioa modula zeko (Milligan e al.,
2019).
G-p o einak
G-p o eina he e o ime ikoak o oko ean lau alde nagusi an sailka zen di a,
Gα azpiuni a ea en a abe a: Gαs/ol , Gαi/o, Gαq/11 e a Gα12/13 (Simon e al.,
1991). Ha zailea en ak ibazioa en ondo en, Gα-p o eina amilia bakoi zak
seinalez apen-bide desbe dinak ak iba zen di u, hainba e an zun isiologiko
e aginez (1.3 i udia).
Gαs amilia bi azpiuni a ez osa u a dago: Gαs-p o eina, zelula gehiene an
esp esa zen dena, e a Gαol -p o eina usaimen-neu ona sen so iale an
esp esa zen dena, ba ez e e (Weins ein e al., 2007). Gαs amiliako p o einek,
adenila o ziklasa (AC) es imula zen du e, be e ja due a ka ali ikoa es imula uz,
e a, ondo ioz, adenosina mono os a o ziklikoa en (cAMP) ekoizpena sus a zen
du (Milligan e a Kos enis, 2006). Ho ez gain, Gαi/o amiliak AC inhibi zen du
cAMP maila zelula ak mu iz uz (Busnelli e al., 2013). Gαi/o amilia Gαi1-,
Gαi2-, Gαi3-, Gαo- e a Gαz-p o eina azpimo a desbe dine an bana zen da. Gαi
azpiuni a eak zelula gehiene an esp esa zen di a, ga una ba ne, e a %85-95
homologia pa eka zen du e (Plumme e al., 2012). Gαo neu one an
esp esa zen da, ba ez e e, e a ga uneko G-p o eina uga iena da (S e nweis
e a Robishaw, 1984). Gαz-p o eina en esp esioa ehun neu onalean e a
plake e an muga zen da (Hul man e al., 2014). Buka zeko, Gαi/o amiliako kide
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guz iek (Gαz izan ezik) ka boxilo e minalean ondo kon se ba u ako zis eina
hondakin ba du e. E emu ho i adenosina di os a oa en (ADP) e ibosilazio-
gunea i dagokio, G-p o einen ak ibazioa en inhibizioa e agi en duena,
Bo de ella pe ussis oxina (PTX) ka aliza u os ean, (Mo is e a Malbon, 1999).
Ondo ioz, p o eina amilia ho iek PTX-sen iko gisa sailka u ohi di a.
Gαq/11 amiliako kideek os olipasa β (PLC-β) ak iba zeko ahalmena du e,
inosi ol 1,4,5- i os a oa en (IP3) e a diazilglize ola en (DAG) o makun za
bul za uz, min z plasma ikoan os a idilinosi ol 4,5-bi os a o ik (PIP2) abia u a.
IP3ak e e ikulu endoplasma ikoan kal zioa mugia az en du, DAG p o eina
kinasa C (PKC) ak iba zen duen bi a ean (We schu eck e al., 2005; Wilkie e
al., 2021). Gαq/11 amilia Gαq-, Gα11-, Gα14- e a Gα15/16 azpi-p o einez osa u ik
dago. Gα11- e a Gαq-p o einak edonon adie az en di a, e a aminoazidoen
%88an sekuen zia pa eka zen du e (Wilkie e al., 1991). Aldiz, Gα14- e a
Gα15/16-p o einen esp esioa ehun espezi ikoe a a muga ua dago; hala nola
gil zu une a a (Tanaka e al., 2000).
Azkenik, Gα12/13-p o eina amiliak Rho guanina nukleo idoen aldake a ak o ea
(RhoGEFs) es imula zen du, e a bi azpi- amilia an sailka zen da; Gα12- e a
Gα13-p o einak, zelula mo a gehiene an esp esa zen di enak (Siehle , 2009).
Nahiz e a hasie an GPCR bakoi za mo a baka eko G-p o einek es imula zeko
ahalmena zuela us e us e izan, gau egun oga u da GPCRek G-p o eina
desbe dine a a lo zeko ahalmena du ela, seinalez apen bide desbe dinak
ak iba uz.
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1.3 i udia: G-p o eina desbe dinen seinalez apena en i udikapen eskema ikoa. Ilus azioa:
Diez-Ala cia e al., 2016.
1.2.3 Fa mako e a GPCR ha zaileen a eko elka e aginen
eo ia
Fa makoek GPCRengan du en e agina un sezko bi ge ae en a abe ako da:
Lehenik e a behin, ligandoa ha zailea ekin lo u beha da, e a ho i a mako
bakoi za en a ini a ea en a abe akoa da. Biga enik, GPCR e a a makoa en
a eko lo u ak ha zailea en kon o mazio aldake a e agin dezake,
seinalez apen-sis ema ba ekin e laziona u ik dagoena, e a ho i a makoa en
e aginko asuna de i zo (Kenakin, 2002).
A ini a e a makologikoa a mako bakoi za ha zaile espezi iko ba ekin
elka zeko edo inka zeko duen gai asuna da. Gaine a, a ini a ea o eka-
disoziazioa en kons an ea en alde an zizkoa i dagokio (1/KD); hau da,
ha zaile kopu u osoa en %50a okupa zeko beha den a makoa en
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Fa makoak ga a ze ako o duan, kon uan ha zeko bes e alde di ba , egi u a
kimikoak hau ako asun un zionalean izan dezakeen e agina da (Shonbe g e
al., 2014). Adibidez, LDSak e a lisu ide a makoak in i o e a in i o e an zun
desbe dinak di uz e (González-Maeso e al., 2003, González-Maeso e al.,
2007), nahiz e a egi u a e a 5-HT2AR- ekiko a ini a e al ua pa eka u. Bes alde,
ispe idona e a be e me aboli o ak iboa, palipe idona, alde hid oxilo baka
ba ean be eiz en di a, e a biak a mako an ipsiko iko a ipikoak di a. Fa mako
ho ien ezauga i a makologikoak desbe dinak di a, e a a makoen
hau ako asun un zionala en ezauga ie an du ja o ia desbe din asunak
(Cla ke e al., 2013). Au kikun za guz i ho iek, egi u a-hau ako asun
un zionala en a eko e lazioa en az e ke a sakona en ga an zia azala az en
du e (Be g e a Cla ke, 2018).
Nahiz e a ike ke a askok ligandoen hau ako asun un zionala i bu uzko
in o mazioa a gi u du en, ike lan gehienak seinalez apen bide jakin ba zue a a
muga u a daude; hala nola G-p o einak edo β-a es inen bideak elka ekin
alde a uz. Hala e e, bes e seinalez apen bide desbe dinak e e ak iba u
dai ezke, e a ho iek e e kon uan ha u beha ko li a eke e aginko asun
ezauga iak desk iba ze ako o duan (1.6 i udia).
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1.6 i udia: GPCRen hau ako asun un zionala en i udikapen g a ikoa. μ-ha zaile opiodeen
adibide hipo e iko e a sinpli ika ua. G-p o einen ak ibazio edo seinalez apenak e agin
e apeu ikoa du; β-a es inen ak ibazioak, aldiz, e agin desi agai zak so zen di u. Agonismo
albo a u ik gabeko ligandoek bi seinalez apen bideak ak iba uko di uz e, e agin e apeu iko
nahiz e agin desi agai zak so uz. Aldiz, agonismo albo a ua au kez en du en agonis ek
seinalez apen bide ba ak iba zeko ahalmena du e, modu hau ako ean, e agin e apeu ikoak
sus a uz e a e agin desi agai zik so u gabe.
1.2.5 Ak ibi a e kons i u iboa en, alde an zizko agonismoa en
e a hau ako asun un zionala en ebaluazioa
GPCRen inplikazioak hainba gaixo asune an handi u egin du ha zailei
inka zeko ahalmena du en ligandoak ike zeko me odoen kopu ua. Ga an zi
handia du enez, hainba saiakun za egin di a a mako desbe dinen p o il
un zionala zehaz eko.
Saiakun za un zional klasikoak biga en mezula ien mailen neu ke an
oina i zen di a; hala nola Ca2+, IP3 en me a zea edo cAMP ekoizpenean,
Ligando albo a ua
Ligando ez-albo a ua
β-a es inak
β-a es inak
G-p o einak
G-p o einak
E agin
desi agai zak
E agin
desi agai zak
E agin
e apeu ikoak
E agin
e apeu ikoak
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bes eak bes e. Saiakun za ho iek a mako ba GPCRa ekin inka u os ean
duen p o il un zionala zehaz eko baliaga iak di a. Biga en mezula iak
neu zean, alda u gabeko ha zaileak az e dai ezke ja o izko ehune an.
Biga en mezula ien neu ke an oina i zen di en saiake ak ike kun za
desbe dine an e abili di a, e a ho ek aban aila suposa zen du, a gi alpenen
a ean emai zak alde a zea ahalbide zen duelako.
Biga en mezula ien neu ke an oina i zen di en eknikak asko e abil zen di en
a en, anpli ikazio maila al ua du en eknikak di a, e a, ondo ioz, agonis a
pa zialek e a agonis a osoek desbe din zea zaila da; e an zun maximo be a
izan dezake elako (Smi h e al., 2018). Gaine a, ez da a gi ge a zen biga en
mezula ien e an zuna zein G-p o eina azpimo a en ak ibazioa en menpekoa
den. Ho az, beha ezkoa da a makoen e a G-p o einen a eko
elka e agina en kuan i ikazio un zional hu bilagoa bu u zea.
GPCR ak ibazioa en ebaluazio zuzena egin dai eke, G-p o eine an ema en
den guanina nukleo idoen ukea en es imulazioa edo inhibizioa neu uz,
e adioak iboa e a ez-hid olizaga ia den GTP molekula e abiliz. Teknika honi
35 su ea ekin ma ka u ik dagen guanosina-5´-O-(γ- io)- i os a oa en
([35S]GTPγS) inkapen eknika esa en zaio. GPCR e a a makoa en a ean
ema en den lo u a en ondo ioz ge a zen den G-p o einen e an zun un zional
goiz ia a neu zen da, e a ez dago seinale-anpli ikazio en menpe (González-
Maeso e al., 2000; Ha ison e a T ayno , 2003). [35S]GTPγS inkapen eknika
konben zionala Gαi/o-p o eina e a GPCRen a ean ema en den akoplamendua
neu ze a muga zen di a; nukleo idoen aldake a asa al uena e a ak ibi a e
kons i u ibo al uena duen G-p o einen amilia delako Gαi/o (Sei e e a Wenzel-
Sei e , 2002). Hala e e, Gα-p o eina espezi ikoak neu zeko saiakun za
desbe dinak ga a u di a; hala nola, immunop ezipi azioa ekin akopla u ako
[35S]GTPγS inkapen eknika (SPA) (Diez-Ala cia e al., 2021b). Teknika
ho ek [35S]GTPγS inkapen eknika klasikoa G-p o eina bakoi za en kon ako
an igo pu zekin konbina zen di u. Me odologia kul ibo zelula e a a zein
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ja o izko ehune a a aplika u dai eke, ha zailee an aldake a ik e agin gabe.
Gaine a, eknika ho ek ak ibi a e kons i u iboa neu zea ahalbide zen du,
ja o izko ehunean, esna a makologikoak (alde an zizko agonis ak,
adibidez) e abiliz (Diez-Ala cia e al., 2021b). Hala e e,
immunop ezipi azioa ekin akopla u ako [35S]GTPγS inkapen eknika (SPA),
Gα-p o eine a a muga u ik dago. Ho i ho ela, β-a es inen e an zuna ezin da
o aindik neu u.
Au eko saiakun zez gain, luo eszen zia e a biolumineszen zia e esonan zia
bidezko ene gia- ans e en zian oina i zen di en saiakun zak ga a u di a
(FRET e a BRET). P o einen a eko elka ekin za e a kon o mazio-aldake a
dinamikoak de ek a zeko eknologiak di a, GPCR, G-p o einak, e a β-
a es inen kon o mazio aldake ak zuzenean neu zea baimen zen du enak
(Zhou e al., 2021; W igh e a Bou ie , 2021). Saiakun za hauek, zelula bizien
ge ae ak denbo a e elean moni o iza zea ahalbide zen du e, e a
e endimendu handiko de ekzio a egoki zeko auke a ema en du e. Teknika
ho iek aban ailak izan a en, de ekzio ako e abil zen di en molekula
luo eszen eek lo zen di en emai ze an izan dezake en e agina oga u
beha ko li za eke (Po ie e a S o e, 2022). BRET eknika en e abile a
muga u ik dago in i o zelula hazkun za sis eme a a, bizi ik dauden
animalie an ez bai a e abilga ia. Gaine a, o aingoz, e esonan zia bidezko
ene gia- ans e en zian oina i zen di en eknikak ez di a e abilga iak pos -
mo em ehuna az e zeko (D ino ec e al., 2012).
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1.3 Se o onina 2A ha zaileak (5-HT2AR)
1.3.1 O oko asunak
Ne bio sis ema zen alean (NSZ) 5-HTk hainba p ozesu isiologiko an pa e
ha ze du, o oimena, pe zepzioa, kognizioa, emozioak, gogo alda ea e a
kon zien zia, bes eak bes e (Be ge e al., 2009). Sis ema se o one gikoa en
dis un zioa gaixo asun psikia iko asko an inplika u ik dago (Hoye , 2020).
Ha zaile se o one gikoak egi u a e a ezauga i a makologikoen a abe a, 14
azpimo a desbe dine an sailka zen di a (Hannon e a Hoye , 2008), e a zazpi
amilia nagusi osa zen di uz e (5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5-
HT7 ha zaileak) (1.7 i udia). Egi u a i dagokionez, 5-HT3 ha zailea e e en
ionikoa den ha zaile baka a da (Ma icq e al., 1991). Gaine ako ha zaile
se o one ikoak, aldiz, G-p o eine a a lo u iko ha zaile me abo opikoak di a
(K oeze e a Ro h, 1998).
5-HT2 ha zaileak gehien ike u di en ha zaile se o one gikoen a ean daude.
5-HT2 ha zaileen amilia 3 ha zaile azpimo a desbe dine an bana zen da, 5-
HT2AR, 5-HT2BR e a 5-HT2CR, non %40-50 sekuen zia-homologia du en
(Hoye e al., 2002). Gaine a, 5-HT2ARen e a 5-HT2CRen min z a eko e emuek
%80eko sekuen zia-homologia du e, e a an zekoak di en p o il
a makologikoak pa eka zen di uz e (Boess e a Ma in, 1994). Ho i dela e a,
ha zaile bakoi za en za a mako hau ako ak ga a zea un sezko e onka da.
Gau egun, 5-HT2 ha zaileen azpimo a desbe dinak a makologikoki
sailka zea nahiko zaila da, ligando hau ako ik ez dagoelako.
Hasie an ba ean, 5-HT azpimo a desbe dinak sailka zeko e adioligandoen
inkapen eknikak e abili zi en. E adioligandoekin egindako lehenengo
ike ke an oina i u a, 5-HT molekula en bi inkapen e emu desbe din zeudela
desk iba u zi en. [3H]5-HT e adioligandoa en a ini a e al uko gunea 5-HT1
ha zailea en azpimo a i dagokio. A ini a e baxuko guneak, be iz, 5-HT2
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ha zaile gisa sailka u zi en. [3H]ke anse ina e adioligando hau ako a en
au kikun za au e apen zien i iko izuga ia izan zen, 5-HT2ARak ike zeko
(Leysen e al., 1982). Hala e e, ke anse inak 5-HT2CRekiko a ini a ea
au kez en du, bes e ha zaile ba zuen a ean (Choudha y e al., 1992). Azken
u e an, 5-HT2AR en az e ke a egi eko ligando hau ako be iak ga a u di a,
[18F]al anse ina, [3H]MDL100907 (L´Es ade e al., 2018) e a [11C]Cimbi-36,
bes eak bes e (E up e al., 2014).
5-HT2AR- en e emu kodi ika zaileen sekuen zia analisiek espezien a ean
kon se bazio gene iko handia dagoela baiez a u zu en. Hala e e, gizakion 242
honda ean se ina ba au ki zen bada e e, ka aska ie an alanina ba au ki zen
da. Aldake a ho ek 5-HT2AR agonis en a ini a e e a e aginko asunean
e agina izan dezakeela p oposa u da gaine a (López-Giménez e a González-
Maeso, 2018; Kim e al., 2020; Slocum e al., 2021).
1.3.2 5-HT2AR kokapena e a un zioa NSZean
5-HT2AR e a 5-HT2CR NSZean esp esa zen di a biak, e a pe i e ian, be iz, 5-
HT2AR plake e an; muskulu-zelule an e a begi ehune an au ki zen da (Leysen,
2004). Ai zi ik, 5-HT2BR ba ez e e pe i e ian esp esa zen da; zehazki, giza
biho z balbule an (Bona en u e e al., 2005) (1.7 i udia).
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1.7 i udia: Ha zaile se o one giko azpimo en i udikapen eskema ikoa (5-HT1-5-HT
7). 5-HT2
amilia 3 ha zaile mo a desbe dine an bana zen da (5-HT2AR, 5-HT2BR e a 5-HT2CR). 5-HT2
amiliako ha zaile mo a bakoi zak ga unean e a bes elako ehune an banake a desbe dina du.
5-HT2AR gehien ba esp esa zen da ga un azaleko V ge uzako neu ona pi amidale an. 5-
HT2CR ga unean e e esp esa zen da, baina hipokanpoan. Azkenik 5-HT2BR pe i e ian,
biho zeko balbulen zelule an dago. Ilus azioa: Mel ze e a Ro h, 2013.
5-HT2AR- en esp esioa ike zea posible izan da e adioligandoen inkapen
eknika, immunohis okimika, mik oskopia elek onikoa e a in si u hib idazio
eknikei eske . Bai pos -mo em e adiog a iak, bai in i o neu oi udia en
ike ke ek baiez a u ahal izan du e 5-HT2ARa en esp esioa giza ga unean
(Pazos e al., 1987; Fo u an e al., 2002). Giza ga unean gehien suena
eskualde ko ikale an (ba ez e e, au e-ga unazalean, pa ie alean e a
soma osen so ialean) esp esa zen da. E emu subko ikale an e a
hipokanpoan, be iz, den si a ea baxuagoan au ki zen da (Hoye e al., 1986;
Pazos e al., 1987; López-Giménez e al., 1997; Va näs e al., 2004; Beli eau
Ga unazala en 5. ge uza
Hipo alamoa
Biho z balbulen
ehun in e s iziala
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e al., 2017). Zehazkiago, pos -mo em ga une an egindako ike ke a ba ek
oga u zuen 5-HT2AR III e a IV ge uza ko ikale an, hipo alamoan, e a neu i
xikiagoan, hipokanpoan e a egi u a es ia ale an esp esa zen dela (Pazos e
al., 1987). Ho ez gain, ga unean au ki zen den 5-HT2ARen esp esioa
adina ekin mu iz u egi en da (G oss-Isse o e al., 1990; González-Maeso e
al., 2008; Moses-Kolko e al., 2011; Uchida e al., 2011; Mugu uza e al., 2013;
Diez-Ala cia e al., 2021a).
Gaine a, 5-HT2AR neu ona glu ama e giko ko ikale an au ki zen da, zehazki
dend i a apikale an (Jakab e a Goldman-Rackic, 1998; Mine e al., 2003;
San ana e al., 2004). In e neu ona GABAe gikoe an e e esp esa zen da
(Bu ne e al., 1995). O oko ean, 5-HT2AR sinapsi ondoko e emue an
koka zen da, ha zaile pos -sinap iko moduan (sinapsi glu ama e gikoei
dagokionez) (Jakab e a Goldman-Rakic, 1998). Hala e e, 5-HT2AR hein ba ean
e emu p esinap ikoan e e koka zen dela us e da, neu ona monoamine gikoen
axoie an, hain zuzen (Mine e al., 2003; Bécamel e al., 2017). Gaine a, 5-
HT2AR as ozi oe an e a mik oglian e e iden i ika u da (K abbe e al., 2012;
Ma in e a Nichols, 2016). Ugaz unen ga unean, 5-HT2AR esp esioa
handiagoa da akzio zi osolikoe an min z plasma ikoan baino (Co nea-Hebe
e al., 1999; Eas wood e al., 2001). Min z zi oplasma ikoan esp esa zen den
ha zailea G-p o eine a a lo u ik dagoela us e da, min ze ik ba ne a u iko
ha zailea ez bezala.
5-HT2AR Gαq/11-p o eine a a lo zen da gehienba , e a behin ak iba u a
dagoela,PLC iso o ma ak iba zen du, IP3k ekoiz eak Ca2+ en askapena
e agi en du, e a azkenik, PKC ak iba zen da (ikusi 1.3.4 a ala in o mazio
gehiago ako) (1.8 i udia). In i o espe imen uei eske oga u da 5-HT2ARen
ak ibazioa en ondo ioz au e-ga unazaleko neu onen ki zikaga i asuna
handi zen dela (A aneda e a And ade, 1991). Gaine a, agonis en
adminis azio ko ikalak ki zikaga i asun neu onala e a neu o ansmiso een
askapena handi zen di u (Ashby e al., 1990; A ano e al., 1999).
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Bes elako e an zun ba zuk, 5-HT2ARen bi a ez ema en di a; hipe e mia,
hipe lokomozioa e a e an zun endok inoak ba ne; hala nola ko isol, enina e a
p olak ina en ja ioa a eago zea (Gudelsky e al., 1986; Ba nes e a Sha p,
1999; Py liak e al., 2011). Bes e ike ke a ba zuek begien kliska zea 5-
HT2ARen bi a ez ge a zen dela oga u du e (Welsh e al., 1998a; Welsh e
al., 1998b; Romano e al., 2000; Ha ey, 2003).
Au e ga unazalean esp esa zen di en 5-HT2ARek haluzinogeno psikodelikoen
e an zun psiko omime ikoekin e laziona u di a. No malean haluzinogenoak,
LSD e a psilozibina bes eak bes e, e abil zen di a eskizo enia en sin oma
posi iboen an ze akoak di en sin omak e agi eko animalia e edue an
(Vollenweide e al., 1998; González-Maeso e al., 2007; González-Maeso e a
Seal on, 2009; Nichols, 2016). Egungo klinikan e abil zen di en an ipsiko iko
a ipikoen i u nagusia 5-HT2ARak di a. Klozapina, ispe idona e a olanzapina,
adibidez, 5-HT2ARen an agonis a edo alde an zizko agonis a gisa joka zen
du e (Mel ze , 1999).
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1.8 i udia: 5-HT2AR neu ona pi amidalen dend i e an koka zen da. D oga psikodelikoek 5-
HT2AR ak iba zen du e, e a zelula ba neko seinalez apen bide desbe dinak ak iba zen di a.
Ondo ioz, ak ibi a e neu onala a eago zen da, e a ho ek e e zelula ba neko bes elako
seinalez apen bideak ak iba zen di u. Ilus azioa: McClu e-Begley e a Ro h, 2022.
1.3.3 5-HT2ARen egi u a biologikoa
Azken hama kadan, GPCR mo a desbe dinen egi u a biologikoak zehaz u
di a, a makologia p o il desbe dineko ligandoekin konplexuak so zen
di uz enak. Ho ela, ligando-ha zaileen a eko e edu a mako o oak ga a u
di a, e a ondo ioz, min z zeha eko ha zaileen kon o mazio aldake ak
desk iba u di a, egi u a ak iboa e a uz e a seinalez apen bide desbe dine an
e aginez (Seyedabadi e al., 2022). Homologia-e eduek e a k is alezko
egi u ek, agonis a e a an agonis a desbe dinen akoplamendu-pun uak
zehaz eko auke a eman du e (Mozumde e al., 2020). Ha zaileen aminoazido
baka eko mu agenesia en bidez e a ondo engo espe imen u un zionalei
eske , G-p o einen e a GPCRen a ean ema en den lo u an pa e ha zen
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i o/ex i o ike ke e a ako agonis a e edu gisa e abili da (Nelson e al., 1999;
Pigo e al., 2012; Canal e al., 2013). [35S]GTPγS inkapen eknika en
bi a ez, giza ga un pos -mo em e a 5-HT2AR kock-ou animalie an egindako
espe imen ue an egiaz a u zen (±)DOI a makoak 5-HT2AR e a 5-HT2CRen
agonis a pa zial gisa joka zen duela (Diez-Ala cia e al., 2019; Ga cia-Bea e
al., 2019; Mune a-A a e e al., 2020).
Azken u ee an zeha , 5-HT2ARekiko a ini a e al uagoa du en a mako mo a
be ia ga a u da, N-ben zil ene ilamina (NBOMe) en egi u an oina i u a; hala
nola n-(2-me oxiben zil-2,5-dime oxi-4-b omo enile ilamina (25B-NBOMe,
Cimbi-36) e a N-(2-hid oxiben xil)-2,5-dime oxi-4-ziano enile ilamina (25-CN-
NBOH) (Jensen e al., 2020). Molekula be i ho iek esna egokiak di a PET
i udi bidezko eknikan e abili ako zein az e ke a a makologikoak egi eko. 25-
CN-NBOH e a Cimbi-36ak agonis a pa zial gisa ja du en du e, 5-HT2ARekiko
a ini a e handiagoa e aku siz, 5-HT2CR e a 5-HT2BRen aldean (Hansen e al.,
2014; Jensen e al., 2017).
Bes alde, psilozina (psilozibina en me aboli o ak iboa) e a e golinak (LSD,
adibidez) ha zaile se o one gikoen agonis a ez-hau ako ak di a, 5-HT1 e a 5-
HT2 ha zaileekiko a ini a ea du enak (Ro h, 2007; Nichols, 2016; Wacke e
al., 2017). Gaine a, LSD a makoa bes elako ha zaile se o one giko zein
ha zaile dopamine gikoe a a lo zen da a ini a e al ua ekin (Halbe ad e a
Geye , 2011; Bo o o-Escuela e al., 2014).
Head- wi ch e an zuna (alde ba e ik bes e a ema en den bu ua en mugimendu
azka a) psikodelikoen e an zun haluzinogenoa neu zeko gehien e abil zen
den ebaluazio oga da ka aska ie an. Ho ega ik, head- wich e an zuna
psikodelikoen ekin za in i o ebalua zeko oga un zional gisa sailka zen da.
An agonis a hau ako ak e a 5-HT2AR knock-ou animaliak e abiliz oga u da
head- wich e an zuna 5-HT2ARen bi a ez ema en dela, e a psikodelikoe a a
muga u ik dagoela (González-Maeso e al., 2007, Canal e a Mo gan, 2012).
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Hala e e, kimikoki oso an ze akoak di en molekulek, (lisu ide, e go amina e a
pe golide, bes eak bes e), ez du e head- wi ch e an zuna e agi en, nahiz e a
5-HT2ARekiko a ini a e al ua izan (González-Maeso e al., 2003; González-
Maeso e al., 2007). Ho i dela e a, lisu ide e a pe golide 5-HT2ARen agonis a
ez-aluzinogeno gisa sailka zen di a. Molekula ho iek, bes elako e golinak
bezala, p o il a makologiko oso konplexua du e, e a hainba ha zaile
amine gikoekiko kide asuna au kez en du e, ha zaile se o one gikoak e a
dopamine gikoak ba ne (Halbe s ad e a Geye , 2011). Gaine a, lisu ide e a
pe golide, pa kinson gaixo asuna en au kako a mako gisa e abil zen di a,
dopamina e a 5-HT1A ha zaileekiko du en a ini a e al ua dela-e a (Lang y e a
Clissold, 1990; Ma ona-Lewicka e al., 2002).
1.3.5.2 An agonis ak
N-alkilpipe idonak 5-HT2ARen aldeko an agonis a klase handiena e a
hau ako enen a ean dago. Ho ie a ik, ke anse ina izan da u ee an zeha
an agonis a gisa e abili izan den a mako nagusia. Ke anse inak
hau ako asun al uagoa dauka 5-HT2ARekiko 5-HT2CRekiko baino (15-80
aldiz), e a bai a 5-HT2BRekin alde a uz (500-1000 aldiz) (Je man e al., 2001;
Knigh e al., 2004; Diez-Ala cia e al 2019). Kimikoki, i anse ina e a
ke anse ina oso an zekoak di a, 5-HT2ARen an agonis a po en e e a hau ako
zein alde an zizko agonis a gisa desk iba u di enak biak (Bonhaus e al.,
1995). Bes e 5-HT2AR an agonis a, kimikoki ke anse ina ekin e laziona ua e e,
al anse ina da; 5-HT2ARen an agonis a po en e e a hau ako gisa desk iba u
dena, 5-HT2ARekin 20 aldiz a ini a e al uago ekin lo zen dena bes elako
ha zaileekin baino (Tan e al., 1999). Hala e e, alde an zizko agonis a
ezauga iak e e desk iba u zaizkio al anse ina i (Aloyo e al., 2009; Diez-
Ala cia e al., 2019).
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5-HT2ARen bes e ligando hau ako ba zuk e e ga a u di a; hala nola
MDL100907, bolinanse ina e e dei u ikoa, e a MDL-11,939. Bolinanse ina 5-
HT2ARen an agonis a po en ea da, e a 300 aldiz a ini a e al uagoa du 5-
HT2ARekiko, 5-HT2cRekiko baino, e a bai a bes e ha zaileekin konpa a uz
(So ensen e al., 1993; López-Giménez e al., 1998). Bolinanse ina e a
i anse ina an ipsiko iko gisa ebalua u zi en eskizo enia en a amendu ako,
hala e e, ez zu en a akas a ik izan, e a ondo ioz be en e abile a muga u egin
zen (Jones e al., 2020). Gau egun, bolinanse ina 5-HT2ARen an agonis a
e edu gisa e abil zen da, hau ako asun handia duelako.
Duela gu xi, a mako be iak ga a u di a loezina a a zeko, nelo anse ina e a
eplibanse ina, bes eak bes e. Konposa u be i ho iek 5-HT2ARekiko a ini a e
al ua au kez en du e, 20 aldiz hau ako asun gehiago 5-HT2ARekiko baino
(Rinaldi-Ca mona e al., 1992; Al-Shamma e al., 2010).
Pimabanse ina, be iz, ACP-103 bezala e e ezagu zen dena, 5-HT2ARen
a mako oso hau ako a da, bes e ha zaileekiko a ini a e ik ez duena, e a 5-
HT2ARekiko 30 aldiz a ini a e al uagoa au kez en duena 5-HT2CRekiko
baino(Vano e e al., 2006; Abbas e a Ro h, 2008). Pimabanse ina
Ame ike a ako Es a u Ba ue ako D ogen e a Elikagaien Adminis azioak (FDA)
a mako gisa ona u du Pa kinson gaixo asunean ge a zen den psikosia ekin
lo u ako haluzinazioak e a elda nioak a a zeko (Cummings e al., 2014).
5-HT2ARen bes e an agonis a ba zuk, nahiz e a 5-HT2A/2CRekiko hau ako ak
izan, ha zaile dopamine giko, his amine giko edo/e a ad ene gikoekiko
a ini a e e ain e a al ua ekin lo zen di a. Ho ie az gain, an ipsiko iko a ipikoak
(adibidez, ispe idona, klozapina e a olanzapina) e a an idep esibo iziklikoak
(adibidez, ami ip ilina, klomip amina e a imip amina) e e 5-HT2ARe a a lo zen
di a an agonis a gisa (Ro h e al., 2004; Mel ze e a Massey, 2011; Mel ze ,
2012).
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1.3.6 5-HT2ARen hau ako asun un zionala
Ha zaile se o one gikoak, 5-HT2AR be eziki, hau ako asun un zionala zuela
i adoki zen lehen GPCRe akoa izan zen (Be g e al., 1998). Zenbai ike ke en
a abe a, 5-HT2ARen agonis a zein an agonis ek, ha zailea en kon o mazio
desbe dinak egonko zen di uz e. Ho en ondo ioz, hau ako asun un zionala
so zen da seinalez apen bide desbe dinen bidez; Gαq/11-p o einen
seinalez apen bideaz gain bes elako seinalez apen bide al e na iboak
ak iba uz (López-Giménez e a González-Maeso, 2018). Ildo ho e an,
hau ako asun un zionala p oposa u da e an zun zelula desbe dina
e agi ea en enomenoa azal zeko, 5-HT2AR ko ikalen populazio be a
ak iba zen bai u e d oga haluzinogenoek, e a ez-haluzinogenoak di en
a makoek. Baina e an zunen a ean desbe din asunak az e u di a Gα-
p o eine an, geneen esp esioan, e an zun elek o isiologikoan e a po ae a
p obe an (González-Maeso e al., 2003; González-Maeso e al., 2007; Ka aki
e al., 2014; Bane jee e a Vaidya, 2020).
Hau ako asun un zionala en lehen ebiden zie akoa Be g e a
kolabo a zaileen au kikun za ik so u zen; ha zaileek Gαq/11-p o einen
mendeko PLCa en ak ibazioaz gain, PLA2 en ak ibazioa e e ge a zen zela
oga u zu enean (Be g e al., 1998). Zehazki, PLC mendeko IP mailak e a
PLA2 en ak ibazioa en ondo iozko AA en askapena neu u zu en,
seinalez apen bide bakoi za ak iba zeko e aginko asun ahalmena neu zeko.
Emai za ho ien a abe a, 5-HTak PLC-IP seinalez apen bidea ak iba zen zuen
be eziki, e a LSDak, be iz, PLA2-AA bidea (Be g e al., 1998; Ma í-Solano e
al., 2015).
Gaine a, 5-HT2AR haluzinogeno e a ez-haluzionogenoek e agin di e en ziala
du e gene-adie azpenean (González-Maeso e al., 2003; González-Maeso e
al., 2007). 5-HT2AR agonis a haluzinogeno e a ez-haluzinogenoen
p esen zian, gene e an zun desbe dina ema en dela oga u zu en zelula e a
saguen ga unazal soma osen so ialean. Ho i ho ela, d oga haluzinogenoak
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zein ez-haluzinogenoak c- os genea en adie azpena e agi en du e. Aldiz, e g-
1 e a e g-2 geneen adie azpena soilik ge a zen da LSD e a (±)DOI agonis a
haluzinogenoen p esen zian, baina azken bi geneen adie azpena ez da
alda zen lisu ide e a e go amina agonis a ez-haluzinogenoen p esen zian
(1.11 i udia). Emai za ho ien a abe a, 5-HT2ARen agonis a guz iek PLC ekin
akopla u iko 5-HT2AR ak iba zen di uz e. Haluzinogenoen mendeko
e an zunak, aldiz, PTX sen iko di en Gαi/o-p o einek bide a zen di uz e.
Ikuspegi be i ho i, os op o eomika kuan i a iboan oina i a u iko
espe imen uen bi a ez balioz a u zen (Ka aki e al., 2014). Gaine a, (±)DOI
agonis a haluzinogenoak e a pe golide agonis a ez-haluzinogenoak
hau ako asun un zional desbe dina au kez u zu en pos -mo em giza au e-
ga unazalean (Mune a-A a e e al., 2020). Bi agonis ek Gαq/11-p o einen
ak ibazioa e agi en du e; Gαi1-p o eina en ak ibazioa, be iz, (±)DOI d oga
haluzinogenoak soilik e agi en du (Mune a-A a e e al., 2020).
Ondo ioz, agonis a haluzinogenoek, LSD e a psilozibina kasu, Gαq/11-
p o einak zein Gαi/o-p o einak ak iba zen di uz e. Ai zi ik, lisu ide, e go amina
e a pe golide, 5-HT2ARen agonis ak izanik, nahiz e a egi u a kimiko an zekoa
izan, ez di uz e ezauga i haluzinogenoak, Gαq/11-p o einen mendeko bidea
baino ez du elako es imula zen.
Bes e ike ke a ba ek, 5-HT2ARen agonis a haluzinogenoak e a ez-
haluzinogenoak seinalez apen sinadu a desbe dina zu ela oga u zuen.
Egileek, os o ila u iko-PLC, pERK, pCREBII zein IP e a DAG ekoizpen maila
al uagoak neu u zi uz en (±)DOI en p esen zian lisu ide a makoa ekin
konpa a uz (Bane jee e a Vaidya, 2020).
Gaine a, 5-HT2ARek G-p o einekiko independen eak di en bes e
seinalez apen bideak ak iba zen di uz e, β-a es inak bes eak bes e. 5-HTak
e a (±)DOIk modu be eizga ian ak iba u di zake e 5-HT2ARak e edu
zelula e an e a head- wich e an zuna neu zean. β-a es ina-2 p o eina ik
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gabeko animalie an, 5-HTak head- wi ch e an zuna so zeko ahalmena
gal zen duela oga u zu en. Badi udi, Ak - en os o ilazioa ge a zen dela β-
a es ina-2 en ak ibazioa en ondo en, baina animalia ho ie an (±)DOI
a makoa en ak ibazioa en ondo en ez da Ak - en ak ibazio ik ge a zen. Hala
e a guz iz e e, (±)DOI a makoak e agindako head- wich e an zuna β-
a es ina-2a ekiko independen ea dela di udi. Egi u a kimiko desbe dina du en
agonis ek 5-HT2AR ak iba u ondo en seinalez apen bide desbe dinak
ak iba zeko ahalmena du ela ondo ioz a u zen (Schmid e al., 2008; Schmid
e a Bohn, 2010).
Be iki i adoki zen LSD d oga haluzinogenoak e agi en zuen head- wich
e an zuna e e β-a es ina-2-mendekoa zela, baina β-a es ina-1 ekiko
independen ea (Rod iguiz e al., 2021). Hala e e, bes e ike ke a ba ean oga u
zu en agonis a ez haluzinogenoek e e β-a es ina-2 e aka zeko ahalmena
zu ela, au eko au kikun za en emai zen kon a (Cao e al., 2022). Be az, β-
a es ina-2 e a haluzinogenoen ekin za mekanismoak ike ke a sakonagoa
beha du.
Bes alde, klozapina an ipsiko iko a ipikoak 5-HT2ARen seinalez apena
blokea zeko ahalmena du, G-p o einen bi a ez. Ha zailea zelulan
ba ne a zea (in e nalizazioa) e agi en du, e a bai a Ak - en os o ilazioa, nahiz
e a β-a es ina-2 ekin ez elka e agin. Be az, 5-HT e a klozapinak mekanismo
desbe dina e abil zen du e, 5-HT2ARen bi a ez, seinalez apen bide be dinak
induzi zeko: Ak - en os o ilazioa e a ha zailea en in e nalizazioa. Ho i
ho ela, klozapinak Ak os o ilazioa en bi a ez blokea zen du 5-HT2AR, e a
PCP edo MK-801 a makoek e agindako sin oma psiko ikoak e e blokea zeko
gai da.
Hau ako asun un zionala en konplexu asuna bide al e na iboa izan dai eke
a mako hau ako en ga apenean, e agin kliniko a eago uak e a e agin
desi agai z gu xi uak di uz en a makoak ga a zeko o duan. Hala e e,
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hau ako asun un zionala e a bes elako ezauga i a makologikoak, hala nola
alde an zizko agonismoa edo an agonismoa, a mako be ien mekanismo
a makologiko gisa zalan zan ja iak di a o aindik, dep esioa e a eskizo enia
gaixo asunen e apia gisa.
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1.11 I udia: 5-HT2ARen seinalez apen in azelula a en i udikapen eskema ikoa
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1.4 5-HT2ARak e a eskizo enia
Hainba ike lane an az e u du e 5-HT2ARen e a eskizo enia en a eko
ha emana. Eskizo enian 5-HT neu o ansmiso ea en inplikazioa zehaz ea
zaila izan bada e e (Halbe s ad & Geye , 2013), ike ke a gehienek 5-HT2AR
az e u du e an ipsiko iko a ipikoek 5-HT2AR blokea zeko ahalmenean
oina i u ik (Mel ze e al., 1989; Miyamo o e al., 2005). Gaine a, lehen
adie azi akoa i ja ai uz, hainba d ogek, sin oma psiko ikoak e agi en di uz e
5-HT2AR-en ak ibazioa en ondo ioz, bai gizakie an bai ka aska ie an;
psilozibinak, LSDak e a (±)DOIk, adibidez.
1.4.1 Ike ke a gene ikoak
HTR2A genea en aldae a e a eskizo enia en a eko e lazioa hainba ike lanek
p oposa u du e. Hala, HTR2A genea en edo p omo o ea en mu azioak
eskizo enia ekin e laziona u izan di a, nahiz e a, ho ie ako ba be a e e ez
den populazioan modu sendoan e eplika u. Izan e e, eskizo enian egindako
GWAS az e ke ek ez zu en inolako aldake a esangu a su ik au ki u HTR2A
gene aldae en a ean (Fa ell e al., 2015).
Gaine a, eskizo enian az e u di en HTR2A esp esioa en aldake ak
nukleo ido baka eko polimo ismoekin (SNPs) e e e laziona u izan di a: A-
1438G eskualde p omo o ean (Oha a e 1998), His452Ty eskualde
kodi ika zailean (Ozaki e al., 1996), e a T102C lehenengo exonean (A anz e
al., 1996), bes eak bes e. Az e lan ba zuk SPN ho iek eskizo enian pa e
ha zea bul za zen du en a en, bes e ike ke a ba zuek kon akoa oga u du e.
Ho ien a ean, zenbai ek i adoki du e A-1438G polimo ismoa eskizo enia
iza eko p obabili a ea ekin e laziona u ik dagoela (Pa sons e al., 2004;
Peñas-Lledo e al., 2007; Sáiz e al., 2007; Smi h e al., 2013). E a be ean,
oga u da A-1438G polimo ismoak e agina duela a amendu
an ipsiko ikoa en e an zunean e e (Yan e al., 2021).
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Azkenik, His452Ty ( s6314) eskizo enia ekin ze ikusia duela oga u zen,
baina a e gehiago an ipsiko ikoen e an zuna ekin lo u du e. Polimo ismoa
klozapina e a olanzapina en e an zun kliniko aldako a ekin e laziona u da
(Bi ke e al., 2000; Olajossy-Hilkesbe ge e al., 2011). Gaine a, HTR2A
s6314 5HT2A en adie azpen e a un zioan e agi en du, e a ho ek
eskizo enia en endo eno ipoa modula ze a lagun zen du, hala nola po ae a
kogni iboak e a ho i lo u ako ak ibi a e p e on ala (Blasi e al., 2013).
Bes alde, T102C ( s6313) e laziona u da populazio desbe dineko eskizo enia
gaixo asuna ekin, bai a ispe idona e a olanzapina en an ipsiko ikoen
e an zuna ekin (Pe onis e al., 2000; Ma iole i e al., 2020).
Labu bilduz, goiko da uen sendo asun ezak, 5-HT2ARen un zio, esp esio e a
an ispiko ikoen e an zunean e agina du en SPNei bu uzko e o kizunean egin
dai ezkeen ike ke a p ekliniko nahiz kliniko gehiga iak egi ea gehiago
jus i ika zen di u.
1.4.2 5-HT2ARen den si a ea, esp esioa e a un zionali a ea
subjek u eskizo enikoe an
Eskizo enia du en subjek uen pos -mo em ga unean 5-HT2ARen RNAm
ebalua zean emai za desbe dinak a gi a a u di a. Eskizo enia du en
subjek uen au e-ga unazalean, 5-HT2ARen RNAma en adie azpenean
mu izke ak nahiz aldake a ezak zi uz en emai zak a gi a a u di a azken
u eo an (Bu ne e al., 1996; He nandez & Sokolo , 1997; He nandez &
Sokolo , 2000; Lopez-Figue oa e al., 2004).
Duela gu xi, a amendu an ipsiko ikoa jaso zu en e a a amendu ik gabeko
subjek uen pos -mo em au e-ga unazalean 5-HT2ARen RNAm adie azpena
az e u zen. Emai zen a abe a, an ipsiko iko ik gabeko subjek u
eskizo enikoe an, 5-HT2ARen RNAm en adie azpena, kon olekin konpa a uz,
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sin oma ologia posi iboa mu iz eko gai asuna du e, eskizo enia pai a zen
du en pazien een kuad o klinikoa hobe uz. Hala e e, pazien een %30ak, gu xi
go a behe a, e an zun mu iz ua e akus en du e, edo inolako e an zunik ez
an ipsiko iko ipikoak ema ean. Gaine a, ez du e inolako onu a ik e agi en
sin oma nega ibo edo asaldu a kogni iboe an (Conley e a Kelly, 2001; Legge
e al., 2020).
1.13 i udia: Halope idola en egi u a kimikoa.
1.5.2 Biga en belaunaldiko an ipsiko ikoak (an ipsiko iko
a ipikoak)
Biga en belaunaldiko an ipsiko ikoak, edo an ipsiko iko a ipikoak, albo-
ondo io es api amidalak mu iz eko helbu ua ekin ga a u zi en (1.14 i udia).
An ipsiko iko a ipikoek a ini a e al uagoa au kez en du e 5-HT2ARekiko, D2R
amilia ekin alde a u a. ezauga i ho i sín oma ex api amidalen
mu izke a ekin e lazioana ua dago,e a ,be az, 5-HT2A/D2 ha zaileen a ini a e
e lazioa e agin desigai zen p o ila au eikus eko e albilga ia da (Ebd up e al.,
2011). Zo i za ez, biga en belaunaldiko an ipsiko ikoek pisu handi zea, e a
glukosa zein lipidoen me abolismoan aldake ak iza eko a iskua handi zen
du e (Muench e a Hame , 2010; Wes on-G een e al., 2013; G ajales e al.,
2019).
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Klozapina en ga apenak, lehen belaunaldiko a makoekin alde a u a, pe il
a makologiko hobe ua zu en a mako be ien ga apenean lagundu zuen
(Mel ze e al., 1989). Klozapina en p o il a makologiko konplexua dela e a,
oso zaila da a makoa en e agin klinikoen ekin za mekanismoa zehaz ea.
Klozapinak ha zaile desbe dinekiko a ini a e esangu a sua au kez en du;
ha zaile his amine giko, ad ene giko, dopamine giko e a koline gikoekiko,
bes eak bes e (Cowa d, 1992; Nuci o a e al., 2017). D2R e a 5-HT2ARe an
e agi eaz gain, klozapina 5-HT1R agonis a pa ziala e e bada, sin oma
kogni ibo e a nega iboen mu izke an inplika u ik daudenak. Ha zaile
muska inikoe an e e e agi en du; M1R, M2R, M3R e a M5R blokea uz, M4R
es imula zen duen bi a ean. Gaine a, ha zaile his amine gikoak blokea uz
sedazio e ek ua so zen du. Azkenik, klozapina ha zaile ad ene gikoak
blokea zen di u, hipo en sioa e a akika dia e aginez (Cowa d e al., 1992;
Nuci o a e al., 2017).
Klozapina a makoa hilga ia izan dai eke, hemo o oxiko asun-a iskua en
(ag anulozi osia e a neu openia) ondo ioz. Ho i dela e a, be e e abile a
klinikoa muga u ik au ki zen da (Alphs e al., 1991). Ho i ho ela, biga en
belaunaldiko an ipsiko iko be iak ga a u zi en; hala nola ispe idona,
olanzapina e a ke iapina, odol-disk asiekin lo u iko albo-ondo ioak mu iz eko
helbu ua ekin.
Olanzapina klozapina en analogo kimikoa da, an zeko ezauga i
a makologikoak di uena, baina ag anulozi osi a isku ik gabe. Espe o zen
moduan, olanzapinak 5-HT2ARekiko a ini a e handiagoa du ha zaile
dopamine gikoekiko baino. Ha zaile his amine gikoak, muska inikoak e a
ad ene gikoak blokea zen di u, baina po en zia gu xiago ekin, klozapin en
aldean. Hala e e, pisu handi zea e a sedazioa bezalako e agin desi agai zak
so zen di u olanzapinak (Ful on e a Goa, 1997; Leuch e al., 2013).
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Ke iapinak, be iz, D1R, D2R e a 5-HT2ARen an agonis a zein 5-HT1ARen
agonis a pa zial gisa ja du en du. Ke apinak e agindako albo-ondo iok
an agonismo α1-ad ene giko e a his amine gikoa ekin e laziona u ik daude
(Miodownik e a Le ne , 2006).
Rispe idona e e an ipsiko iko a ipikoa da. Rispe idona en e agin e apeu ikoa
5-HT2AR e a D2Ren blokeoan oina i zen da, baina 5-HT2ARekiko a ini a e
handiago ekin (Cohen, 1994). Ho ez gain, ispe idona α1-ad enoha zaileak
e a ha zaile his amine gikoak blokea zen di u. Rispe idona sin oma posi iboen
a amendu ako e aginko a iza eaz gain, nahasmen kogni ibo e a sin oma
nega iboen a amendu ako e e e aginko a da. Ondo ioz, gehien
p esk iba zen den an ipsiko ikoa da (Mölle , 2005; Chopko e a Lindsley, 2018).
Palipe idona ispe idona en me aboli o ak iboa da, e a p o il a makologiko
be a duela oga u da.
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1.14 I udia: Klozapina, olanzapina, que iapina, ispe idona e a palipe idona en egi u a
kimikoa.
Azken u eo an, biga en belaunaldiko an ipsiko iko be iak ga a u di a; hala
nola asenapina e a lu asidona (Miyamo o e al., 2012). Hala e e, lehen
belaunaldiko an ipsiko ikoen e aginko asuna o aindik ez da hobe u. Gau
egun, eskizo enia e esis en ea a a zeko ona u ik dagoen bo ika baka a
klozapina da (Conley e a Kelly, 2001).
1.5.3 Hi uga en belaunaldiko an ipsiko ikoak
Honezke o, hi uga en belaunaldiko an ipsiko ikoak e e ga a u di a; hala nola
a ipip azola (1.15 i udia). Bes e neu olep iko ba zuk ez bezala, hi uga en
belaunaldiko an ipsiko ikoak ez di a D2Ren an agonis ak, D2Ren agonis a
pa zialak baizik (Da ies e al., 2004). DA kon zen azio handie an, DA ekin
lehia zen di a, e agin klinikoa lo uz. Ai zi ik, DA en mailak xikiak di enean,
a ipip azola D2R- ekin elka u dai eke, e a agonis a pa zial gisa ja dun.
Klozapina
Olanzapina
Ke iapina
Rispe idona
Palipe idona
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Gaine a, a ipip azola 5-HT1Ren agonis a pa zial moduan a i zen da. Biga en
belaunaldiko an ipsiko ikoak ez bezala, a ipip azolak a ini a e handiagoa du
D2Rekiko 5-HT2ARekin alde a uz (Chen e al., 2022). Hi uga en belaunaldiko
an ipsiko ikoak sin oma psiko ikoak a in zeko e aginko ak di a, albo-ondo io
es api amidalik e a hipe p olak inemia ik e agin gabe. Gaine a, pisu
handi zea e a albo ondo io me aboliko gu xiago so zen di uz e (Liebe man,
2004).
1.15 i udia: A ip ip azola en egi u a kimikoa
1.5.4 An ipsiko ikoen belaunaldi be ia
5-HT2ARen blokeoan oina i u ako eskizo enia a a zeko a mako be iak
au ki zeko ahaleginak, i anse ina e a blonanse ina kasu, ez di a e apeu ikoki
e abilga iak izan. 5-HT2ARen an agonis a selek iboen e abile ak
mono e apian ez zuenez e aginko asun klinikoa au kez u, zenbai ike ke en
a abe a, 5-HT2ARen an agonismoa soilik ez da nahikoa an ipsiko iko a ipikoen
e aginko asun klinikoa azal zeko (Miyamo o e al., 2012). Hala, badi udi
D2Ren blokeoa beha ezkoa dela e agin klinikoa lo zeko. Hala e e,
pimabanse ina dei u iko 5-HT2ARen ligando po en e e a selek iboa ga a u zen
psikosia en a amendu ako al e na iba gisa (1.16 i udia) (Mel ze e a Ro h,
2013). Pimabanse ina a ini a e dopamine giko ik ez duen ona u ako lehen
an ipsiko ikoa da (Hacksell e al., 2014). O ain a e, pimabanse ina
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eskizo enia ako e apian lagunga i gisa p oba u da, halope idola e a
ispe idona a makoekin ba e a (Mel ze e al., 2012). Pimabanse inak FDA en
ona pena lo u du pa kinsonen gaixo asunean ge a zen di en elda nioak e a
haluzinazioak mu iz eko, (Cummings e al., 2014). Gaine a, pimabanse inak
eskizo enia du en pazien een sin oma nega iboak mu iz eko gai asuna duela
e e oga u da (Buga ski-Ki ola e al., 2022). Pimabanse ina 5-HT2ARen
alde an zizko agonis a gisa desk iba u den a en, beha ezkoa da
alde an zizko agonismoa oga zeko espe imen u gehiago egi ea (Vano e e
al., 2006; Nu e al., 2017).
1.16 i udia: Pimabanse ina en egi u a kimikoa.
Be iki ona u da biga en belaunaldiko an ipsiko iko gehienak 5-HT2ARen
alde an zizko agonis ak di ela, an agonis a neu alak izan beha ean (Weine
e al., 2001). An agonis ek ez bezala, alde an zizko agonis ek be ezko
e aginko asun nega iboa du e, e a oina izko seinalez apen-ja due a xiki u
dezake e.
O o ha , an ipsiko ikoen ekin za-mekanismoa hobe o ule zeak sendagai
e aginko ak e a jasanga iagoak diseina u e a ga a zea eka lezake.
His o ikoki, p o il poli a makologikoa du en a mako p omiskuoak e aginko ak
izan di a NSZ en gaixo asunak a a zeko, nahiz e a albo ondo io la i asko
Sa e a
330
e agin di zake en. Be az, helbu u molekula zeha za ekin elka e agi en du en
a mako selek iboen diseinuak sendagai e aginko en e a ona ga iagoen
ga apenean lagundu dezake. Hala bali z, agonismo albo a ua edo selek iboa
baka ka, zein alde an zizko agonismoa ekin ba e a, au kez en du en
a makoak, ha zaileen hau ako asuna en al e na iba gisa so u ahalko
li a eke, eskizo enian espe o di en e an zun un zionalak lo zeko.
Helbu uak
Helbu uakl
333
Nahiz e a ike ke a asko egin di en sendagai an ipsiko iko be iak ga a zeko,
gau egun esku a dauden sendagaien a ean ez da naba mendu epe luze a
e aginko asuna duenik, albo-ondo io kal ega i ik gabe. Ho i, eskizo enia en
neu obiologia i bu uzko jaki u ia eza i zo zaio. Be az, eskizo enia maila
neu obiologikoan sakonago ezagu uz ge o, aldake a molekula ak, zelula ak
e a/edo seinalez apen bideak iden i ika u ahal izango di a, bo ika be ie a ako
i u e apeu iko be iak bilaka u dai ezkenak.
Zenbai au kikun zek adi ze a ema en du enez, 5-HT2AR sin oma psiko ikoen
e a ho ien a amenduan e agi en du en mekanismo molekula e an
inplika u ik dago. Alde ba e ik, psikodelikoen (LSD, psilozina, meskalina e a
(±)DOI) izae a haluzinogenogenoa, 5-HT2ARen ak ibazioan oina i zen da.
Bes e ik, eskizo enian e abili ohi di en an ipsiko iko a ipikoek 5-HT2ARen
an agonis a edo alde an zizko agonis a gisa joka zen du e.
Neu oi udi az e ke ek, in i o PET e a in i o pos -mo em p oben bidez,
eskizo enia du en subjek ue an 5-HT2AR den si a ea i bu uzko emai za
kon aja iak e aku si di uz e. Txos en kon aja i ho iek ze ikusia dauka e,
an za, e abil zen di en e adio azado e desbe dinak ha zailea en
kon o mazio desbe dine a a inka zea ekin. Be az, badi udi eskizo eniako 5-
HT2ARen aldake ek ze ikusi gehiago du ela ha zailea en egoe a
molekula a en aldake ekin ha zailea en adie azpen maila en aldake ekin
baino.
5-HT2AR Gαq/11- zein Gαi/o-p o einak ak iba zeko gai da, ha zaile a inka zen
den a makoa en a abe a. Tes uingu u ho e an, ezauga i haluzinogenoen
az a na molekula a 5-HT2ARen agonis a bidezko Gαi1-p o einen ak ibazioan
oina i zen da. Gaine a, Gαi1-p o einen es imulazio handiagoa desk iba u da,
baina ez ho ela Gαq/11-p o einena; eskizo enia du en subjek uen au e-
ga unazala en pos -mo em ehunean e a 5-HT2ARen (±)DOI agonis a en
p esen zian egin zenean. Au kikun za ho i, 5-HT2ARen agonismo albo a u gisa
in e p e a dai eke, eskizo enikoe an Gαi1-p o eina bide haluzinogenoa en
Subjek uak, Ma e ialak and Me odoak
340
3.1 aula:
Eskizo enia aldea en (S), eskizo enia ez zu en suiziden aldea en (NSS) e a kon ol aldea en (C) ezauga i demog a ikoak, pos -mo em
egoe a, he io za
-kausa e a analisi oxikologikoa en emai zak.
Kasua
Diagnosia
Gene oa
(G/E)
Adina
(u e)
PMI
(o du)
Me a zea
(hilabe e)
He io za-
kausa
He io za-
mekanismoa
Ga un pH
Fa mako maila odolean
(mg/L) Ga un-
oxikologia (ng/g)
S 1
Eskizo enia
E
67
22
17
Na u ala
Po o ka diobaskula a
5,8
Nega iboa
Nega iboa
C 1
Kon ola
E
66
17
83
Is ipua
T a iko-is ipua
6,06
Nega iboa
Ez egina
S 2
Eskizo enia
G
34
23
81
Suizidioa
Al u a ik sal o
6,32
Nega iboa
Ko inina 23,1
C 2
Kon ola
G
34
17
69
Is ipua
T a iko-is ipua
6,7
Nega iboa
Nega iboa
NSS 1
Pe sonali a e
asaldu a
G
34
7
199
Suizidioa
U ka ua
Ez egina
E anola 2700
Zi alop am 0,1
Oxaka bazepina 6,5
Zi alop am 1579,9
Ko inina 3352,8
No zi alop am 368,2
S 3
Eskizo enia
E
53
18
98
Na u ala
Odulus ea 6,58
Alp azolam 0,05
Palipe idona 54,2
Ko inina 503,2
Alp azolam 43,9
C 3
Kon ola
E
51
10
70
Na u ala
Po o ka diobaskula a
6,3
Nega iboa
Nega iboa
S 4
Eskizo enia
G
32
21
87
Suizidioa
Al ue a ik sal o
6,65
Nega iboa
Ko inina 380,78
C 4
Kon ola
G
33
23
95
Is ipua
T a iko-is ipua
6,55
Nega iboa
Ez egina
NSS 2
Pe sonali a e
asaldu a
G
33
14
192
Suizidioa
Al ue a ik sal o Ez
egina
Lo azepam 0,03
Benla axina 0,16
Desme ilbenla axine 475,0
Lo azepam 1421,2
Midazolam 3,7
Olanzapine 6
S 5
Eskizo enia
G
45
36
15
Is ipua
I o a
Ez egina
Ez egina
Midazolam 1801,8
No diazepam 1247,1
Oxazepam 50,1
C 5
Kon ola
G
44
23
98
Is ipua
T a iko-is ipua
6,45
Nega iboa
Nega iboa
NSS 3
Pe sonali a e
asaldu a
G
44
9
192
Suizidioa
Al ue a ik sal o
Ez egina
E anola 220
Alp azolam 0,01
Amisulp ida 1,4
Klomip amina 0,2
Reboxe ina 0,09
Alp azolam 141,4
Klomip amina 3040,7
Ko inina 322,5
Reboxe ina 224,2
S 6
Eskizo enia
G
49
23
107
Is ipua
Al ue a ik e o ia 6,40
Nega iboa
Ko inina 391
Lo azepam 16,3
C 6
Kon ola
G
49
19
93
Is ipua
T a iko-is ipua
6,7
Nega iboa
Nega iboa
S 7
Eskizo enia
G
70
20
115
Suizidioa
U ka u
Ez egina
Ez egina
Nega iboa
C 7
Kon ola
G
71
22
115
Is ipua
Al ue a ik e o ia
5,92
Nega iboa
Nega iboa
S 8
Eskizo enia
E
74
9
118
Na u ala
Po o ka diobaskula a
Ez egina
Fenoba bi ala 9
Nega iboa
Subjek uak, Ma e ialak and Me odoak
341
C 8
Kon ola
E
74
30
167
Is ipua
Al ue a ik e o ia
Ez egina
Nega iboa
Nega iboa
S 9
Eskizo enia
G
46
20
129
Suizidioa
Al ue a ik sal o 6,41
Nega iboa
Zuklopen ixol 110,8
Ko inina 622,4
Lo azepam 25,5
C 9
Kon ola
G
46
22
115
Na u ala
Po o ka diobaskula a
6,48
Nega iboa
Nega iboa
NSS 4
Pe sonali a e
asaldu a
G
47
4
195
Suizidioa
Al ue a ik sal o
Ez egina
Feni oina Nega iboa
S 10
Eskizo enia
G
26
24
140
Suizidioa
Al ue a ik sal o
Ez egina
Diazepam 0,27
Diazepam 356,5
No -diazepam 856,9
Oxazepam 15,1
C 10
Kon ola
G
25
21
71
Is ipua
Su-is ipua
6,48
Nega iboa
Nega iboa
NSS 5
Pe sonali a e
asaldu a
G
27
42
253
Suizidioa
Al ue a ik sal o
Ez egina
Nega iboa
Ko inina 337,4
Diazepam 204,9
No diazepam 612
Oxazepam 65,8
S 11
Eskizo enia
E
75
18
140
Na u ala
Po o ka diobaskula a
Ez egina
Nega iboa
Ko inina 36,76
C 11
Kon ola
E
79
24
213
Is ipua
T a iko-is ipua
Ez egina
Nega iboa
Ez egina
S 12
Eskizo enia
G
28
28
143
Suizidioa
Al ue a ik sal o
Ez egina
Nega iboa
Ko inina 93,79
C12
Kon ola
G
29
13
116
Is ipua
Al ue a ik e o ia
6,44
Nega iboa
Nega iboa
NSS 6
Pe sonali a e
asaldu a
G
28
5
259
Suizidioa
Al ue a ik sal o
Ez egina
Nega iboa Ko inina 638,2
S 13
Eskizo enia
G
25
17
145
Suizidioa
Al ue a ik sal o
Ez egina
Nega iboa
Ko inina 153,1
C 13
Kon ola
G
23
16
141
Is ipua
Al ue a ik e o ia
Ez egina
Nega iboa
Nega iboa
NSS 7
Alsaldu a
obsesibo
-
konpul siboa
G
26
19
143
Suizidioa
Al ue a ik sal o 6,66
Klomip amina 0,5
Fluoxe ina 0,7
Fluboxamina 0,2
Ke iapina 0,5
Fluoxe ina 15441
Fluboxamina 4094
No luoxe ina 4433,5
No ke iapina 386,7
Ke iapina 86
S 14
Eskizo enia
G
23
13
195
Suizidioa
Al ue a ik sal o
Ez egina
Ez egina
Halope idol 136,5
Ko inina 458,5
Ke iapina 392,5
No ke iapina 1309,4
C 14
Kon ola
G
22
20
188
Is ipua
T a iko-is ipua
Ez egina
No diazepam 0,38
Ko inina 371,6
Oxazepam 80,9
No diazepam 909
NSS 8
Pe sonali a e
asaldu a
G
19
8
131
Suizidioa
Al ue a ik sal o 6,7 Nega iboa Ko inina 236,4
S 15
Eskizo enia
E
80
32
178
Na u ala
Shock
Ez egina
Nega iboa
Ez egina
C 15
Kon ola
E
78
12
185
Na u ala
Po o ka diobaskula a
Ez egina
Nega iboa
Ez egina
S 16
Eskizo enia
E
38
23
216
Suizidioa
Al ue a ik sal o
Ez egina
Nega iboa
Nega iboa
C 16
Kon ola
E
36
19
110
Is ipua
T enbide a e o ia
6,51
Nega iboa
Nega iboa
Subjek uak, Ma e ialak and Me odoak
342
NSS 9
An sie a e
asaldu a
E
39
19
214
Suizidioa
Fa mako gaindosia
Ez egina
No diazepam 0,13
E anola 5000
Diazepam 22,4
No diazepam 280,7
S 17
Eskizo enia
E
51
15
236
Na u ala
Po o ka diobaskula a
Ez egina
Bu lomedil 66
Me amizol 4
Ko inina 521,5
C 17
Kon ola
E
51
38
230
Is ipua
T a iko-is ipua
Ez egina
Nega iboa
Ez egina
S 18
Eskizo enia
G
62
28
240
Suizidioa
Al ue a ik sal o
Ez egina
Ez egina
Ami ip ilina 170,3
No ip ilina 453
C 18
Kon ola
G
62
23
243
Is ipua
T a iko-is ipua
Ez egina
Nega iboa
Ez egina
NSS 10
Moldae a
an sie a e
asaldu a
G
62
19
271
Suizidioa
C ushing
Ez egina
Ez egina Map o ilina 257,9
No diazepam 1584,9
S 19
Eskizo enia
G
35
22
299
Suizidioa
Al ue a ik sal o
Ez egina
Nega iboa
Nega iboa
C 19
Kon ola
G
36
22
286
Is ipua
T a iko-is ipua
Ez egina
E anol 1000
Ez egina
NSS 11
An sie a e
asaldu a
G
36
15
181
Suizidioa
A ma i oa
Ez egina
Mi azapina 0,08 Ez egina
S 20
Eskizo enia
G
49
41
292
Suizidioa
U ka ua
Ez egina
E anola 490
Ez egina
Ko inina 127
Klo p omazina 140,7
Lo azepam 388,6
Tio idazina 6079,5
C 20
Kon ola
G
45
30
275
Is ipua
T a iko-is ipua
6,82
E anola 3090
Ez egina
NSS 12
An sie a e
asaldu a
G
46
26
319
Suizidioa
U ka ua
Ez egina
Ez egina
Diazepam 91,3
No diazepam 175,2
S 21
Eskizo enia
E
56
24
125
Na u ala
Po o ka diobaskula a
Ez egina
Alp azolam 0,03
Ko inina 110
Alp azolam 38
C 21
Kon ola
E
54
24
10
Is ipua
Al ue a ik e o ia
6,87
Nega iboa
Nega iboa
S 22
Eskizo enia
G
50
3
173
Suizidioa
U e an i o a 7,09
No diazepam 0,4
Amisulp ida 75,9
Ko inina 89
No diazepam 662,8
Oxazepam 47,6
T azodona 241,6
C22
Kon ola
G
50
2
15
Na u ala
Po o ka diobaskula a
6,1
Nega iboa
Nega iboa
NSS 13
Pe sonali a e
asaldu a
G
49
22
141
Suizidioa
Al ue a ik e o ia 6,35
No diazepam 2,8
Tiap ida 5,4
Benla axina 1,2
Ko inina 254,1
Desme
illbenla axina
265,6
Oxazepam 94,7
Tiap ida 973,5
Benla axina 2146,5
S 23
Eskizo enia
G
43
17
172
Na u ala
Po o ka dioaskula a
Ez egina
Nega iboa
Ko inina 1003,9
Lo azepam 66
C 23
Kon ola
G
41
15
7
Na u ala
Po o ka diobaskula a
Ez egina
Nega iboa
Nega iboa
Subjek uak, Ma e ialak and Me odoak
343
Ezauga i demog a ikoak, adina, PMD, me a ze denbo a e a pH-a, 3.2 aulan
labu uak daude. Konpa ake a es a is ikoak egin zi enean ez zen di e en zia ik
au ki u, az e u ako hi u aldeen a ean, adinak alde a zean (F[2,56]=2,08,
p=0.1341). E a be ean, ez zen di e en zia esangu a su ik au ki u hi u aldeen
a ean PMD az e u zenean (F[2,56)]=1,99, p=0,1456), ez a pH-a az e u
zenean e e (F[2,21]=0,17, p=0,8433). Hala e e, eskizo enia aldea en laginen
me a ze denbo a luzeagoa izan zen kon ol aldea ekin e a eskizo enia ez
zu en suiziden aldea ekin konpa a uz (F[2,56]=4,66, p=0,0135) (3.2 aula).
3.2 aula: Ike lanean az e u ako eskizo enia aldea en, eskizo enia ez zu en suiziden
aldea en ezauga i demog a ikoak, nahiz pos -mo em ezauga iak. Talde bakoi za en
ba ezbes ekoa±SEM (ba ezbes ekoa en e o e es anda a):
*p<0,05 s kon ol aldea (Bon e oni en konpa azio anizkoi za en az e ke a)
Taldea
Gene oa
Adina(u eak)
PMD
pH
Me a ze
denbo a
(hilak)
Eskizo enia
7 E/16 G
48±4
22±2
6,5±0,1
150±15
Eskizo enia ez zu en
suizidak
1 E/12 G
38±3
16±3
6,6±0,1
207±15*
Kon ol
7 E/16 G
48±3
21±2
6,5±0,1
139±15
Subjek uak, Ma e ialak and Me odoak
344
3.1.2 Immunop ezipi azioa ekin akopla u ako [35S]GTPγS
inkapen eknika (SPA) e a Wes e n Blo ogen
ka ak e izazio ako e abili zen lagin mul zoa osa zen zu en
pa aideen ezauga i demog a ikoak.
Fa makoen hasie ako ka ak e izazio a makologikoa DLPFC lagin mul zo
ba ekin egin zen, gainon zeko oge an e abili ez zi ene a ik abia u a. Lagin
mul zoen min zak p es a zeko sei subjek uen au e-ga un-azal za iak
homogeneiza u zi en. Guz i a, 2016 e a 2019 bi a ean jaso ako hamabos
lagin e abili zi en ike lan oso ako (%30 gizonezkoak e a %70 emakumezkoak),
non ba ezbes eko adina 59±6 u ekoa zen, PMD 11±2 o dukoa e a me a ze
denbo a 27±3 hilekoa zen. Pa eha zaileen odolean egindako p oba
oxikologikoe an lo u ako emai zen a abe a, ez zeuka en a mako
psiko opiko ik hil zi enean.
Subjek uak, Ma e ialak and Me odoak
345
3.2 Animaliak: Sagu ansgenikoak
Lan hone an e abili ako 5-HT2AR knock-ou saguak (5-HT2AR(-/-)) e a ja o izko
edo Wild Type saguak (5-HT2AR(+/+)) R. Maldonado ka ed adunak (Ba zelona,
Espainia) eskuzabal asunez emanak izan zi en. Sagu ansgenikoak
lehenengo 129S6/S E anduian so u zi en, e a ondo en C57BL/6J
anduia ekin gu u za u zi en, labo a egiko p ozedu a es anda ak ja ai uz
(González-Maeso e al., 2003; Weiss aub e al., 2006; González-Maeso e al.,
2007; O eja ena e al., 2011). Animaliak jaso os ean, gu u za ze gehiga iak
egin zi en kolonia eska zeko. Gu e labo a egian ohiko polime izazio-ka ea en
e eakzioak (PCR) e a elek o o esiak egin zi en gu e saguak geno ipa zeko
(ez di a emai zak sa u), au e ik a gi a a u ako p ozedu ak ja ai uz (Fio ica-
Hollowells e al., 2002). 5-HT2AR(-/-) saguek 5-HT2AR esp esa zen ez zu ela
egiaz a zeko, [3H]ke anse ina ekin inkapen az e ke ak (Mugu uza e al., 2013)
e a (±)DOIk e agindako Gαi1/Gαq/11-p o einen ak ibazio az e ke ak (Ga cia-
Bea e al., 2019) egin zi en.
Ho e a ako, C57BL/J6 sagu heldu a ak e abili zi en (15-20 as eko
adina ekin). Animaliok bosnaka aldeka u zi en kaiole an, ohikoak di en
labo a egiko baldin zapean (22±1°C, %55±5 heze asun e la iboa, 12 h
a gi/ilun zikloa e a aska asun osoa ohiko ma aska ien jana ia e a u a
lo zeko). E abili ako animalien zenba ekoa mu iz eko ahalegina egin zen.
Gaine a, ja ai u ako espe imen uen p o okoloak Euskal he iko
Unibe si a eko (UPV-EHU) Animaliekin egi en den Espe imen azio ako E ika
Ba zo de ik (AEEB) ona uak izan di a (E e e en zia: M20-2019-321). E a
be ean, espe imen u guz ie an ja ai u ako p ozedu ek Eu opa Ba asuneko
(Eu opean Union Di ec i e 2010/63/UE) e a Espainiako (53/2013 e ege
dek e ua) legediek eza zen di uz en a auak be e zen di uz e, animalien
ongiza ea i dagokionean. Sagu helduak lepo dislokazio bidez hil zi en,
ondo en ga una a e a zi zaien e a ga un-azala disekziona u (Diez-Ala cia e
al., 2016). Laginak -80°C an go de zi en e abiliak izan a e.
Subjek uak, Ma e ialak and Me odoak
346
3.3 Fa makoak
-(±)-DOI: (±)-2,5-Dime oxi-4-iodoam e amina hid oklo u oa (Sigma-Ald ich;
Sain Louis, Missou i, EEBB).
-Ke anse ina: 3-[2-[4-(4-Fluo oben zoil)-1-pipe idinil]e il]-2,4[1H,3H]-
quinazolinadiona a a oa (Toc is; B is ol, E esuma Ba ua).
-MDL100907 (bolinanse ina): (R)-(+)-α-(2,3-dime oxi enil)-1-[2-(4-
luo o enil)e il]-4-pipidin me anola (Sigma-Ald ich; Sain Louis, Missou i, USA).
-Al anse ina: 3-[2-[4-(4-Fluo oben zoil)-1-pipe idinil]e il]-2,3-dihid o-2- ioxo-
4(1H)-kinazolinona hid oklo u o hid a oa (Sigma-Ald ich; Sain Louis,
Missou i, EEBB).
-Pimabanse ina (ACP-103): 1-(4-Fluo oben zil)-3-(4-isobu oxiben zil)-1-(1-
me ilpipe idin-4-il)u ea (Axon Medchem; G oningen, He behe eak).
-Nelo anse ina: 1-(3-(4-b omo-2-me il-2H-pi azol-3-il)-4-me oxi enil)-3-(2,4-
di luo o enil)u ea (Axon Medchem; G oningen, He behe eak).
-Ri anse ina: 6-[2-[4-[bis(4- luo o enil)me ilideno]pipe idin-1-il]e il]-7-me il-
[1,3] iazolo[3,2-a]pi imidin-5-ona (Sigma-Ald ich; Sain Louis, Missou i,
EEBB).
-Eplibanse ina: 4-[(E,3Z)-3-[2-(dime ilamino)e oximino]-3-(2- luo o enil)p op-
1-enil] enola (Axon Medchem; G oningen, He behe eak).
-MDL-11,939: α- enil-1-(2- enile il)-4-pipe idin me anola (Toc is; B is ol,
E esuma Ba ua).
-SB 242084: 6-Klo o-2,3-dihid o-5-me il-N-[6-[(2-me il-3-pi idinil)oxi]-3-
pi idinil]-1H-indol-1-ca boxiamida dihid oklo u oa (Toc is; B is ol, E esuma
Ba ua).
-Klozapina: 8-Klo o-11-(4-me il-1-pipe azinil)-5H-diben zo[b,e][1,4]diazepina
(Toc is; B is ol, E esuma Ba ua).
-Rispe idona: 3-[2-[4-(6- luo o-1,2- ben zisoxazol-3-il)-1-pipe idinil]e il]-
6,7,8,9- e ahid o-2-me il-4H-pi ido[1,2- a]pi imidin-4-ona (Sigma-Ald ich;
Sain Louis, Missou i, EEBB).
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-A ipip azola: 7-{4-[4-(2,3-Diklo o enil)-1-pipe azinil]bu oxi}-3,4-dihid o-2(1H)-
quinolinona (Sigma-Ald ich; Sain Louis, Missou i, EEBB).
-Olanzapina: 2-Me il-4-(4-me il-1-pipe azinil)-10H- ieno[2,3-
b][1,5]ben zodiazepina (Sigma-Ald ich; Sain Louis, Missou i, EEBB).
-Palipe idona: 3-[2-[4-(6-Fluo o-1,2-ben zisoxazol-3-il)-1-pipe idinil]e il]-
6,7,8,9- e ahid o-9-hid oxi-2-me il-4H-pi ido[1,2-a]pi imidin-4-ona (Toc is;
B is ol, E esuma Ba ua).
-Ke iapina: 2-[2-(4-Diben zo[b, ][1,4] iazepin-11-il-1-pipe azinil)e oxi]e anol
hemi uma a oa (Toc is; B is ol, E esuma Ba ua).
-A opina: Azido endo-(±)-α-(hid oxime il)ben zeno aze ikoa en 8-me il-8-
azabiziklo[3.2.1]ok -3-il es e a (Sigma-Ald ich; Sain Louis, Missou i, EEBB).
-Fen olamina: 2-[N-(3-Hid oxi enil)-p- oluidinome il]-2-imidazolidina
hid oklo u oa (Sigma-Ald ich; Sain Louis, Missou i, EEBB).
-Ze i izina: Azido [2-[4-[(4-klo o enyl) enilme il]-1-pipe azinil]e oxi]aze ikoa en
hid oklo u oa (Sigma-Ald ich; Sain Louis, Missou i, EEBB).
-Raclop idea: 3,5-Diklo o-N-[[(2S)-1-e il-2-pi olidinil]me il]-2-hid oxi-6-
me oxiben zamida (Toc is; B is ol, E esuma Ba ua).
-Halope idola: 4-[4-(4-klo o enil)-4-hid oxipipe idino]-4′- luo obu i o enona
(Sigma-Ald ich; Sain Louis, Missou i, EEBB).
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3.4 Ma e ialak
An igo pu zak:
Immunop ezipi azioa ekin akopla u ako [35S]GTPγS inkapen eknika (SPA)
e a Wes e n Blo ogak egi eko e abili zi en an igo pu z monoklonalak San a
C uz Bio echnology, Inc (Kali o nia EEBB) enp esa i e osi zi zaizkion.
An igo pu zen ezauga iak 3.5.2 a alean zehaz uak daude, non Wes e n Blo
espe imen uen p o okoloa e e azal zen den.
Wes e n Blo oge an bi an igo pu z sekunda io luo eszen e ezbe din e abili
zi en: ba a go ia, an i-sagu an igo pu za ekin elka u ako Alexa Fluo ® 680
(In i ogen, O egon, EEBB), e a bes ea be dea, an i-un xi an igo pu za ekin
elka u ako IRDyeTM 800 (Rockland Immunochemical, Pennsyl ania, EEBB).
Konposa u e adiak iboak:
Azu e 35az ma ka u ako guanosina-5’-O-(gamma- io)- i os a oa
([35S]GTPγS), ak ibi a e espezi ikoa 1250 Ci/mmol zuena e a Pe kinElme
labo a egia i e osia (Wal man, MA, EEBB).
Bes elako a mako e a konposa u kimikoak:
-Bio-Rad Labo a o ies (Cali o nia, EEBB): Amonio pe sul a oa (APS), B ad o d
P o ein Assay, Laemmli laginen anpoia 2 aldiz kon zen a ua, N-N-N-N´-
e ame ile ilenediamina (TEMED), au ez ma ka u ako SDS-PAGE pisu
molekula a en es anda ak.
-Ca lo E ba E eak iboak (Ba zelona, Espainia): Me anola.
-GE Heal hca e (Buckinghamsi e, E esuma Ba ua): Ni ozelulosa min zak
(po o amaina: 0.45 μm) e a Wha manTM zelulosa 3MM.
-In i ogen (Ba zelona, Espainia): DL-Di io ei ola (DTT), azido e ilenodiamino
e aze ikoa (EDTA).
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-Na ional diagnos ics (A lan a, GA, EEBB): %30 ak ilamida 30-%0,8
bisac ilamida.
-Pan eac S.A.U (Ba zelona, Espainia): Azido aze iko glaziala, azuk ea e a
HCl (%37).
-Sigma-Ald ich® (Sain Louis, Missou i, EEBB): Behia en se umeko albumina
(BSA), 2-bu anola, dime ilsul oxidoa (DMSO), azido e ileno glikol-bis(2-
aminoe ile he )-N,N,N′,N′- e aaze ikoa (EGTA), glizina, guanosina di os a oa
(GDP), guanosina 5'-O-[gamma- io] i os a oa (GTPγS), Igepal® CO-520, β-
me kap oe anola, MgCl2, NaCl, NaF, Na3VO4, P o easa Inhibi zaile Cock ail,
polioxie ilenoa (20) so bi an monolau a oa (TweenTM 20), sodio deoxikola oa
(SDC), sodio dodezil sul a oa (SDS), T is (hid oxime il)aminome ano
hid oklo u oa (T is HCl).
-Pe kin Elme (Wal ham, MA, EEBB): 96 pu zudun isoplaka e a A p o eina
i sa si ik du en polibinil oluenozko (PVT) SPA bola xoak.
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356
a mako bakoi zak [35S]GTPγS inkapenean neu u ako seinalea blokea zea
lo u zen.
3.5.1.3 Emai zen analisi ma ema ikoa e a es a is ikoa
Analisi ma ema iko
Mic obe a T ilux Scin illa ion coun e de ek agailuan lo u ako emai zak CCPM
(minu uko zuzendu ako kon u) gisa esp esa uak daude, non CCPMak e a
DPMak (minu uko desin eg azioak) baliokideak di ela ona zen den. Emai zak
in e p e a zeko, CCPM an dauden da uak inka u ako [35S]GTPγS en omol
p o eina milig amoko uni a e an bilaka u beha di a ( mol/mg p o eina). Da uen
bilaka zea egi eko, az e ke an e abili ako p o eina kon zen azioa kon uan
ha u beha da, e a 3.1 ekuazioa be e zen da. [35S]GTPγS inkapenean
p o eina kon zen azioak e agina izan dezake Diez-Ala cia e al., 2021
a ikuluan oga u zen moduan. Ho ega ik oga ho ie an p o eina edukia
neu zen da espe imen u bakoi zeko.
mol/ mg p o eina: CCPM kon uak/(2,22 x 1250 x [ ogako p o eina kon zen azioa mg])
3.1 ekuazioa: [35S]GTPγS inkapena e a SPA di uz en espe imen ue an lo u ako emai zak
mol/mg p o einako. 1250 (Ci/mmol) 35S e adioligandoak be ezkoa duen ak ibi a e
espezi ikoa i dagokio, 2,22 balioa kons an ea da Cu ie (Ci) uni a eak DPM an bilaka zeko. (1
Ci=2,22x1012 DPM).
[35S]GTPγS en inkapen basala (BB), a mako exogeno ik ezean ge a zen
den [35S]GTPγS en inkapena i dagokio. Agonis a edo alde an zizko agonis a
gehi zean [35S]GTPγSa en inkapen basala modula ua izango da. Izan e e,
agonis a gehi zean [35S]GTPγSa en inkapena handi zen da, ja o izko
[35S]GTPγS inkapen basala ekiko. Aldiz, alde an zizko agonis a gehi zean
[35S]GTPγSa en inkapen basala xiki zen da.
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357
Bes alde, inkapen ez espezi ikoa (NBS) CCPM an lo zeko, egoe a
espe imen al bakoi ze ik kenke a eginez a e a zen; inkapen basala,
es imulazioa, inhibizioa, e ab. Ho az, balio bakoi za i inkapen ez espezi ikoa
ken zean, [35S]GTPγSa en inkapen espezi ikoa lo u zen; kalkuluak e a analisi
es a is ikoa egi eko e abiliko zena.
Fa mako agonis a, an agonis a edo alde an zizko agonis en ezauga iak
ezagu zeko, kon zen azio-e an zun ku ba ba lo u zen a mako
bakoi za en za . Ho e a ako, a mako gabe lo u ako inkapen basala en
balioa inkapena en %100 gisa ona u zen. Ondo en, a makoa en za
lo u ako inkapen espezi ikoa po zen ai gisa bilaka u zen. Ho ela, lo u ako
[35S]GTPγS inkapena en es imulazioa edo inhibizioa inkapen basala en
a abe a adie azi zen. Ku ba osa zen du en pun u bakoi za en kalkulu ako 3.2
e a 3.3 ekuazioak e abili zi en:
%Es imulazioa en: (Es imulazioa-NBS)/(BB-NBS)x100
3.2 ekuazioa: [35S]GTPγS inkapena SPA ekin konbina zean lo u ako emai zen kalkulua
inkapen basala ekiko po zen ai izae a agonis a, an agonis a edo lehiako a desk iba zeko.
NBS: Finkapen ez espezi ikoa, BB: Finkapen basala.
%Inhibizioa en: 100-[(Inhibizioa-NBS)/(BB-NBS)x100]
3.3 ekuazioa: [35S]GTPγS inkapena SPA ekin konbina zean lo u ako emai zen kalkulua
inkapen basala ekiko po zen ai izae a agonis a, an agonis a edo lehiako a desk iba zeko.
NBS: Finkapen ez espezi ikoa, BB: Finkapen basala.
Kasu ba zue an, [35S]GTPγSak Gα-p o eina sub ipo desbe dine a a inka zeko
duen ahalmena az e zeko, kon zen azio-e an zuna en ku ba osoa egin
beha ean, kon zen azio baka ba en za (10 μM) azaldu ako inkapena soilik
az e u zen. Kon zen azio ho i auke a ze ako o duan, inkapen maximoa
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(Emax/Imax) e agi eko ahalmena zuen a makoa en kon zen azioa ha u zen,
kon zen azio-e an zuna en ku ba ik. Kasu ho ie an, inkapen basala %0 gisa
adie azi zen, e a a makoek e agindako e ek ua posi ibo edo nega ibo gisa
adie azi zen egoe a basala ekiko.
Es imulazioa en edo inhibizioa en pa ame o a makologikoen kalkulu ako
G aphPad P ismTM p og ama in o ma ikoa en bidez lo u ako analisi ez-lineala
e abili zen. Ho iekin, [35S]GTPγS en inkapena ekiko e ek u es imula zaile e a
inhibi zaile maximoak lo u zi en (Emax/Imax), bai a ho ien e diak lo zeko
beha ezkoa den a makoa en kon zen azioak e e (EC50/IC50). Az e ke e an
lo u ako pun u bakoi za 3.4 e a 3.5 ekuazioe an sa u zi en kon zen azio-
e an zun ku ba lo zeko. E edu ma ema iko ho ek ona u akoa en a abe a,
kon zen azio-e an zuna en ku ba en malda es anda a da, Hillen malda en
balioa (edo malda ak o ea) 1,0 izanik. Ho i ho ela, balio ho i izan beha ko
li za eke malda, baldin e a masen ekin za en legea en a abe a, a makoa
ha zaile ba e a soilik inka zen bada.
E= BB+(Emax-BB)(1+10(LogEC50-Log[X]))
3.4 ekuazioa: Es imulazioa en kon zen azio-e an zuna en ku ba mono asikoa (malda
es anda a duena). E e an zuna i dagokio (3.2 ekuazioa es imulazio %), X kon zen azioak
e agindakoa, BB [
35S]GTPγS en inkapen basala i dagokio, agonis a ezean lo u ako
inkapena (100% gisa adie azi a), Emax e ek u es imula zaile maximoa (%) e a LogEC50 e ek u
es imula zaile maximoa en e dia lo zeko beha ezkoa den a makoa en kon zen azioa.
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359
E= BB+(Imax-BB)(1+10(LogIC50-Log[X]))
3.5 ekuazioa: Es imulazioa en kon zen azio-e an zuna en ku ba mono asikoa (malda
es anda a duena). E e an zuna i dagokio (3.3 ekuazioa es imulazio %), X kon zen azioak
e agindakoa, BB [
35S]GTPγS en inkapen basala i dagokio, agonis a ezean lo u ako
inkapena (100% gisa adie azi a), Imax e ek u es imula zaile maximoa (%) e a LogIC50 e ek u
es imula zaile maximoa en e dia lo zeko beha ezkoa den a makoa en kon zen azioa.
Emax/Imax pa ame oak ba ezbes ekoa±SEM (ba ezbes ekoa en e o e
es anda a) gisa adie az en di a, e a –LogEC50/IC50 pa ame oak e e
ba ezbes ekoa±SEM gisa adie az en di a. Balio ho iek izango di a analisi
es a is ikoa egi eko e abiliko di enak. E ek u es imula zailea en edo
inhibi zailea en maximoa en e dia lo zeko gai den a makoa en
kon zen azioa EC50/IC50 gisa adie az en da. Pa ame o ho i, LogEC50/IC50
balioen ba ezbes ekoa en an iloga i moa egi ean lo zen da.
Ho i guz iaz gain, kon zen azio-e an zuna en ku bak ko-analisi bidez e e
az e u zi en ( alde be e ako egindako espe imen u guz ien analisia aldi
be ean), alde bakoi za en balio o oko ak lo zeko. Ho e a ako,
[35S]GTPγSa en inkapena leku baka ba e a hobekien doi zen zuen
e eg esio ez-lineala en e edua e abili zen. Emai zok, doikun za onena en
balioa ± %95eko kon idan za- a ea gisa adie azi zi en.
Analisi es a is ikoa
Lo u ako emai zen in e p e azio ako hainba analisi es a is iko egin zi en.
Has eko, lo u ako balio guz iak G ubb’s es analisia en bidez az e u zi en,
alde espe imen al bakoi zean egon zi ezkeen ou lie balioak de ek a u e a
baz e zeko.
Kon zen azio pun u baka a ekin egindako espe imen ue an lo u ako
emai zak lagin baka e ako S uden ’s es analisia en bidez az e u zi en;
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360
a makoak inkapen basala engan e agindako e ek ua ala e ek u-eza
az e zeko asmoz.
Bi buz ane ako S uden ´s - es pa eka ugabea e abili zen, bi egoe a
espe imen al az e u beha zi en bakoi zean; adibidez, agonis ak e agindako
e ek ua e a agonis ak an agonis a ekin ba e a e agindako e ek uak
alde a zeko. Hi u egoe a desbe din alde a zeko, aldiz, bide baka eko ANOVA
analisia e abili zen, Bon e oni en pos -hoc analisia ekin ba e a. Ho i izan zen
e abili ako analisia eskizo enia aldea, eskizo enia ez zu en suiziden aldea
e a kon ol aldea alde a zeko, [35S]GTPγSa en inkapena en modulazioa
az e ze ako o duan.
Bes alde, bi aldagaiek izan zezake en e agina az e zeko, bi bide ako ANOVA
e abili zen, Bon e oni en pos -hoc analisia ekin ba e a. Analisi ho i izan zen
egokiena geno ipo desbe dina zu en saguen aldeak alde a zeko, adibidez.
Aldagai independen een (adina, PMD e a me a ze denbo a) e a menpekoak
di en e agin un zionalen a eko e lazioa az e zeko Pea sonen ko elazio
koe izien ea kalkula u zen. Ko elazio ho i esangu a sua zen kasue an,
koba ian za analisia (ANCOVA) egin zen alde espe imen alen a ean
(eskizo enia, eskizo enia ez zu en suizidak e a kon olak). ANCOVA analisiak
egi eko InVi oS a so wa e es a is ikoa e abili zen.
Lan honen emai zak az e zean, lo u ako desbe din asunak es a is ikoki
esangu a su za jo zi en p<0,05 zela egiaz a u zenean.
Ho i guz iaz gain, analisi osaga iak e e egin zi en aldeen a eko
desbe din asun po en zialak az e zeko. Ho ela, eskizo enia, eskizo enia ez
zu en suizidak e a kon ol aldeko balioen koanalisian lo u ako emai za
globalei, bes e az e ke a gehiga i ba egin zi zaien. (DeLean e al., 1978;
Mo ulsky e a Ransnas, 1987) Analisi osaga i ho e an, e abili ako analisi
e eduen egoki asuna alde a u zen, beha ze mul zo ba aplika uz F analisia en
bidez (Balioen ka a ua en gehike a p in zipioan oina i u a).
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361
Has eko, balio mul zo bakoi za banaka az e u zen (beha u gabe). Ondo en,
balioen ka a ua en emai za lo zeko, doikun za bakoi zean lo u ako
baka kako balioen gehike a egi en da, aska asun g adu kopu ua ekin e e
egi en den moduan. Ondo en, balio mul zoak nahas u a e e az e zen di a, aldi
be ean guz iak, e a amankomunak di uz en pa ame o ba , edo gehiago,
pa eka ze a beha zen zaio doi u ako ku ba i (balio basalak, Emax/Imax e a
LogEC50/IC50). Ho ela, balio ezbe dinak lo zen di a balioen ka a uekin e a
aska asun g aduekin. Pa ame o ba edo gehiago pa eka zea baimen zen
zuen analisia jo zen doikun za egoki gisa, baldin e a ba ian zen onda a ez
bazuen esangu a suki handi zen. Egoki asuna en esagu a su asun
es a is ikoa F es bidez neu u zen, p<0,05 izanik, e a F[DFn, DFd] gisa
adie azi zen; non F balioen banake a i dagokio, DFn zenbaki zaileko
aska asun g aduei dagokio e a DFd izenda zaileko aska asun g aduei
dagokio.
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3.5.2 Wes e n Blo ogak
Wes e n Blo ogak luze e a zabal e abiliak izan di a p o einen de ekzio e a
iden i ikazio ako, an igo pu z espezi ikoak e abiliaz. P ozedu a hainba
pauso an egi en da: lehenengo, elek o o esia en bidez lagineko p o einak
bana u egi en di a, pisu molekula a en a abe a: ondo en, p o einak mugiezin
bilaka u e a ans e i u egi en di a, gele ik ni ozelulosa min ze a, p o einak
esku aga i izanik an igo pu zekin lo zeko. Azkenik, ni ozelulosa min zak
an igo pu z espezi ikoen disoluzioan sa zen di a, i u di en p o einak ezagu u
e a ho ie a a lo zeko.
Ike lan hone an, lehenengo helbu ua, immunop ezipi azioa ekin akopla u iko
[35S]GTPγS inkapen eknikan (SPA) e abili ako an igo pu zen ka ak e izazioa
e a espezi iko asuna oga zea izan zen, Gα-p o eina azpimo a
bakoi za en za . Behin an igo pu zak az e u a, Gαi1- e a Gαq/11-p o einen
den si a e immunoe eak iboa neu u genuen pos mo em au e-ga unazal
lagine an eskizo enia aldean, eskizo enia ez zu en suizida aldean e a
be aiekin pa eka u ako kon ol aldean. E a be ean, zi oeskele oa en za i den
β-ak ina p o eina e e neu u zen, ja i ako p o eina kopu ua kon olpean
iza eko asmoz.
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363
3.2 i udia: Wes e n Blo ogen adie azpen g a ikoa. 1. Laginak 95ºC an be o zen di a 5
minu uz; 2. Lagin kan i a e op imiza ua ja zen da gelean; 3. Gelen elek o o esia ja zen da
abian 30 minu uz e a 60 V pean, lehenengo, e a ondo en 90 minu uz 140 V pean. Ho ela,
p o einen banake a ema en da pisu molekula a en a abe a, e a xikienek mig azio handiena
jasango du e; 4. T ans e en zia ako sandwichak p es a zen di a; gela ka odo an z ja i ik (-)
e a min za anodo an z (+); 5. Nega iboki ka ga u ako p o einen mig azioa ge a zen da gele ik
min ze a, ans e en zia 0,3 A pean e a 90 minu uz luza zen da; 6. Min zen inkapen ez-
espezi ikoa e agoz en da esnea da aman Blocking anpoia e abili a. Ondo en, min za
an igo pu z p ima ioa ekin inkuba zen da, e a, ga bi u os ean, an igo pu z sekunda ioa ekin
inkuba zen da, o dube ez e a gi o enpe a u an. Buka zeko, i udien luo eszen zia en neu ke a
egi en da. I udia J. DelaCues a-Ba u ia en esi ako so u ako ilus azioa.
3.5.2.1 Min ze an abe as u ako laginen p es ake a (P2 za ikia)
Wes e n Blo ogak egi eko e abili zi en min zen laginen p es ake a, SPA
eknika egi eko e abili zi enen an ze a egin zen (3.5.1.1 a ala). Espe imen ua
egin zen egunean, 0,5 mg zi uz en P2 za ikia zu en pelle ak desizoz u os ean,
125 μl T is-HCl 0,5 nM anpoian be eseki zi en. Lagin ho iei, 119 μl 2X
Laemmli lagin anpoia e a β-me cap oe anola en 6 μl gehi u zi zaien, laginen
p o eina edukia 2 mg/ml izan zedin. Eskizo enia zu en subjek uen,
Lagina be o u 95 °C a a
Laginak elek o o esiko
gelean ka ga u
P o einen banake a
elek o o esia en bi a ez
Immunode ekzioa
P o einen ans e en zia min ze a
T ans e en zia ako gelen
p es akun za
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364
eskizo enia ez zu en suiziden e a kon olen laginak aldi be ean p ozesa u
zi en, egun be ean.
3.5.2.2 Gel elek o o esia, ans e en zia e a immunode ekzioa
Poliak ilamida gele an egindako elek o o esia
Poliak ilamida gele an egi en den elek o o esia edo SDS-PAGE (Sodium
Dodecyl Sulpha e Polyac ilamide Gel Elec opho esis), desna u alizazio
egoe an egi en da, e a lagin ba eko p o einen banake a egi eko (pisu
molekula a en a abe a) gehien e abil zen den eknika da.
Elek o o esian zeha , lehenengo, s acking gelean mig a zen du e p o einek.
Gela en za i ho en osake a %5 poliak ilamida zen, 125 mM T is HCl, %0,1
SDS, 0,07% an igeno p os a iko espezi ikoa (PSA) e a %0,14 TEMED zuen
disoluzioan (pH 6,8). Laemmli-e eak iboa ekin p es a u ako laginak 5 minu uz
be o u zi en 95ºC an The moblock gailuan, s acking gela p es a zen zen (3.2
i udia). Laginean dauden p o einen banake a, pisu molekula a en a abe a,
unnig gele a hel zen di enean has en da. Gela en za i ho en osake a %10
ak ilamida-bisak ilamida zen 0,37 M T is HCl, %0,1 SDS, %0,07 PSA e a
%0,07 TEMED zuen disoluzioan (pH 8,8). Te lonTM o azi ba sa u zi zaien
gelei 15 hila a so zeko, laginak ja iak zi en gela en za ian.
Ho ela lo u ako, 15 hila a an bana u ako gelen neu iak 6 x 8 cm izan zi en
(3. i udia). Hila a bakoi zean ja i ako bolumena e a p o eina kan i a ea az e u
beha eko Gα-p o eina en a abe akoa izan zen (3.5 aula). Gel bakoi za en
lehenengo hila an pisu molekula a en ma ka zaile kome ziala ja i zen (10-
250 kDa, P ecision Plus P o einTM, Dual Colo S anda ds, Bio-Rad). Gel
bakoi zean ja ai u zen laginen e a an igo pu zen ka ak e izazioan banake a
3.3 i udian adie azi a dago.
Subjek uak, Ma e ialak and Me odoak
365
3.3 i udia: Gel bakoi zean ja i ako laginen banake a, an igo pu zen ka ak e izazio
espe imen ue a ako goikoa e a aldeen a eko az e ke a egi eko behekoa (eskizo enia,
eskizo enia ez du en suizidak e a kon olak). MW: Pisu molekula en ma ka zailea, P2:
min ze an abe as u ako za ikien lagina, C: Kon ol subjek ua, SCH: Eskizo enia subjek ua,
NSCHS: Eskizo enia ez du en subjek u suizida.
P o eina e ekonbinan eak
Giza P2
Sagu P2
A a oi
P2
Subjek uak, Ma e ialak and Me odoak
372
Analisi es a is ikoa
An igo pu zen ka ak e izazioa egin zenean, lo u ako den si a een balioekin ez
zen inongo analisi ik egin, helbu ua Gα-p o eina azpimo a bakoi za ekiko
espezi ikoak zi en ala ez az e zea zelako. Ho i ho ela, pisu molekula a en
a abe a beha zen lekuan az a nak ikusgai zeudenean soilik neu u zi en.
Emai za guz iak G ubb´s es analisia en bidez az e uak izan zi en, ou lie
balio ik zegoen az e zeko, e a baiezko kasue an analisian baz e zeko.
Emai zek banake a gaussia a en a abe a bana uak zeuden e e az e u zen
kasu guz ie an.
Gaine a, kon ol aldea One-sample S uden ’s - es analisia en bidez az e u
zen pool lagina ekiko, be aien a eko desbe din asun esangu a su ik ez
zegoela oga zeko, %100 balioa lo zeko o duan.
Eskizo enia aldea, eskizo enia ez zu en suiziden aldea e a kon ol aldea
alde a zeko bide baka eko ANOVA analisia e abili zen, Bon e oni en pos -
hoc analisia ekin ba e a. Taldeen a eko emai zak desbe dinak zi ela adie azi
zen p<0,05 zen kasue an.
Aldagai independen een (adina, PMD e a me a ze denbo a) e a, GNAI1 e a
GNAQ p o einen esp esio mailen a eko e lazioa az e zeko Pea sonen
ko elazio koe izien ea kalkula u zen. Ko elazio ho i esangu a sua zen
kasue an, koba ian za analisia (ANCOVA) egin zen aldeen espe imen alen
a ean (eskizo enia, eskizo enia ez zu en suizidak e a kon olak). ANCOVA
analisiak egi eko InVi oS a so wa e es a is ikoa e abili zen (Mocke Media
20
