Resea ch
Cell Calcium (1999) 24(Z), 87-96
8 Ha cou l B ace &Co. L d 1998
Func ional measu emen s o CCa2+1 in
-~
he endoplasmic e iculum u&g d
he pes i us o deli e a ge ed
aequo in
Ma ia Te esa Alonso, Ma ia Jose Ba e o, Es ela Ca nice o,
May e Mon e o, Ja ie Ga cia-Sancho, Ja ie Al a ez
Depa amen o de Bioquimica y Biologia Molecula y Fisiologia, lns i u o de Biologia y Gene ica Molecula (IBGM),
Facul ad de Medicina, Uni e sidad de Valladolid y CSIC, Valladolid, Spain
Summa y Changes in he ee calcium concen a ion o he endoplasmic e iculum ([Ca’+],,) play a cen al ole
con olling cellula unc ions like con ac ion, sec e ion o neu onal signaling. We ecen ly epo ed ha ecombinan
aequo in a ge ed o he endoplasmic e iculum (ER) [Mon e o M., B ini M., Ma saul R. e al. Moni o ing dynamic
changes in ee Ca2+ concen a ion in he endoplasmic e iculum o in ac cells. EMBO J 1995; 14: 5467-5475,
Mon e o M., Ba e o M.J., Al a ez J. [Ca2+] mic odomains con ol agonis -induced Ca2+ elease in in ac cells. FASEB J
1997; 11: 881-8861 can be used o moni o selec i ely [Ca2+le, in in ac HeLa cells. He e we ha e used a he pes
simplex i us ype 1 (HSV-1) based sys em o deli e a ge ed aequo in in o a numbe o di e en cell ypes including
bo h pos mi o ic p ima y cells (an e io pi ui a y cells, ch oma in cells and ce ebella neu ons) and cell lines (HeLa,
NIH3T3, GH, and PC12 cells). Func ional s udies showed ha he s eady s a e lumenal [Ca*+],, anged om a ound
300 pM in g anule cells o 800 ). M in GH,cells. InsP,-coupled ecep o s imula ion wi h agonis s like his amine (in HeLa,
NIH3T3 and ch oma in cells), UTP and b adykinin (in PC12 cells) o hy o opin- eleasing ho mone (TRH, in GH,cells)
p oduced a e y apid dec ease in lumenal [Ca’+],,. Ca eine caused a apid Ca2+ deple ion o he ER in ch oma in cells,
bu no in he o he cell ypes. Depola iza ion by high K+ p oduced an immedia e and e e sible inc ease o [Ca2+le in all
he exci able cells (an e io pi ui a y, GH,, ch oma in cells and g anule neu ons). We conclude ha deli e y o
ecombinan aequo in o he ER using HSV amplicon p o ides he i s di ec quan i a i e and dynamic measu emen s
o [Ca2+le, in se e al p ima y non-di iding cells.
INTRODUCTION
Changes in he concen a ion o cy osolic Caz+ ([Ca”],)
a e known o egula e many physiological esponses o
di e en ex acellula s imuli, bo h in exci able and non-
Recei ed 5 Ma ch 1998
Re ised 12 June 1998
Accep ed 16 June 1998
Co espondence o: D Ma ia Te esa Alonso, Depa amen o de Bioquimica y
Biologia Molecula y Fisiologia. Facul ad de Medicina, Uni e sidad de
Valladolid, 47005 Valladolid, Spain
Tel: +34 983 423085; Fax: +34 983 423588
E-mail: [email p o ec ed]
exci able cells. Recep o s imula ion can lead o a a ie y
o complex spa io empo al pa e ns o inc eases in [Ca’+],
like Ca2+ wa es and Ca2+ oscilla ions. The mechanisms
o Ca2+ elease om he ER, o Ca2+ en y ac oss he
plasma memb ane, and o seques e ing eleased Ca2+
unde lie hese complex esponses 13-51. The ER, o some
o i s subcompa men s, is belie ed o be he majo
cellula Ca2+ s o e, eleasing Ca2+ upon s imula ion o
InsP,-coupled plasma memb ane ecep o s o ia Ca2+-
induced Ca2+- elease (CICR), o cause ises in [Ca’+], [6].
Howe e , in spi e o e o s ocused on s udying he
di e en componen s o calcium signaling, di ec and
speci ic measu emen o [Ca2+] inside he ER o in ac
cells has been a di icul ask. Recen ly, he cloning o he
gene encoding o he pho op o ein aequo in has allowed
a7
88 M T Alonso, M J Ba e o, E Ca nice o, M Mon e o, J Ga cia-Sancho, J Al a ez
i s exp ession in mammalian cell cul u es. Fu he mo e,
he aequo in gene has been modi ied by including de ined
a ge ing signals. Thus,
a
numbe o aequo in chime as
a ge ed o di e en subcellula compa men s such as
mi ochond ia [7l, he nucleoplasm [8], cy osol [9], ER
[l,lO,ll]
and sa coplasmic e iculum [12] ha e been
gene a ed. The a ge ing s a egy was ully success ul in
e e y case, bu measu emen o [Ca*+], was pa icula ly
di hcul because o he high [Ca2+] in ha compa men ,
which p oduced a apid consump ion o aequo in [1,13].
We ecen ly sol ed his p oblem by using coelen e azine n,
a modi ied o m o he p os he ic g oup o aequo in which
educes he a e o luminescence and allows he pe o -
mance o long-las ing measu emen s o [caZ+],, in in ac
cells [2,14]. Howe e , al hough he use o hese chime as
has ep esen ed a landma k in he ield o subcellula
[Ca2+] in es iga ion, mos s udies ha e been pe o med in
one pa icula cell line (HeLa). This es ic ion is due o he
di hcul y in in oducing plasmid DNA in o p ima y
pos mi o ic cells by non- i al me hods such as Ca2+
phospha e, elec opo a ion o liposomes. To o e come his
limi a ion, we decided o de elop a deli e y sys em capable
o di ec ing he exp ession o ER- a ge ed aequo in in
di e en cell ypes and pa icula ly in p ima y cells, he
mos in e es ing ones om a physiological poin o iew.
He pes simplex i us ype 1 (HSV-1) is a neu o opic
i us wi h many unique ea u es ha make i sui able as
a gene ans e ec o o mammalian cells, bo h in i o
and in i o [ 151. I s cha ac e is ics include wide hos cell
ange, e icien in ec ion, abili y o deli e genes o bo h
di iding and non-di iding cells, long- e m pe sis ence
and he capaci y o accommoda e la ge molecules o
o eign DNA. In pa icula , he na u al opism o he pes
i us o in ec pos mi o ic neu ons is an ad an age ha
o e comes he di icul y o ans ec hese cells. In his
epo , we desc ibe an amplicon ha e icien ly exp esses
aequo in a ge ed o he ER (PHSVe AEQ) in a a ie y o
cell lines and non di iding p ima y cells, in pa icula
an e io pi ui a y cells (AP), ch oma in cells and g anule
neu ons. The co ec subcellula aequo in loca ion was
es ablished by immunocy ochemis y and by di ec
displaying o Ca-sensi i e luminescence in each in ec ed
cell ype. Ou esul s show ha deli e y o ecombinan
aequo in o he ER using HSV amplicon is a simple and
e icien me hod o moni o di ec ly [Ca2+],, in adul
p ima y cells such as AP cells, ch oma in and neu ons,
as well as in se e al cell lines in cul u e.
MATERIALS AND METHODS
Cell cul u e
NIH3T3,
wild ype HeLa cells and he e AEQmu -HeLa
cell clone EM26 [l] we e g own in DMEM supplemen ed
Cell Calcium (1998)24(2), 67-96
wi h 10% FCS, 2 mM glu amine, 100 U/ml penicillin and
100 mg/ml s ep omycin. The 2-2 cell line which
con ains he HSV- 1 IE2 gene [ 161 was g own in he same
medium. PC12 and GH, cells we e main ained in DMEM
wi h 10% e al cal se um and 5% ho se se um. An e io
pi ui a y (AP) cells we e isola ed om adul a s as
p e iously desc ibed [17]. Bo ine ad enal ch oma in
cells we e kindly p o ided by P o . An onio Ga cia,
Uni e sidad Au dnoma de Mad id, Spain [ 181. Ce ebella
g anule cells we e p epa ed om 4-7 day-old a s as
p e iously epo ed [ 191.
Exp ession o aequo in in cell cul u es using de ec i e
HSV ec o s
Plasmid cons uc ion
The chime& low-a ini y mu an o aequo in e AEQmu
has been desc ibed p e iously [l]. The e AEQmu cDNA
was isola ed om he o iginal ec o by EcoRI diges ion
and cloned in o he ecipien ec o pHSVpuc [20] o
gene a e he pHSVe AEQ. The pHSVlac amplicon ec o
which exp esses he P-galac osidase gene o
Esche idzia
coli
has been desc ibed p e iously [2 11.
Packaging o HSV- 1 ec o s
pHSVe AEQ DNA was packaged in o HSV-1 pa icles
using a dele ion mu an packaging sys em [22]. In b ie , 3
x lo5 2-2 cells we e seeded on 60 mm dishes and
ans ec ed wi h 6 pg o pHSVe AEQ o pHSVlac using
he lipo ec amine p ocedu e acco ding o he manu-
ac u e ’s p o ocol (Gibco, BRL, Mad id, Spain). One day
la e he medium was eplaced wi h 3 ml DMEM, 5% FBS
and cells we e in ec ed wi h -2 x lo6 plaque- o ming
uni s o 5dll.2 helpe i us which con ains a dele ion in
he IE2 gene o HSV-1 s ain KOS [23]. On he ollowing
day, i us was ha es ed and subsequen ly passaged on
esh 2-2 cells 3 imes o inc ease bo h he a io o ec o
o helpe and he o al amoun o i us.
Vec o i e ing
Ti e s we e measu ed in PC12 as desc ibed p e iously
[22]. Cells we e seeded in o poly-L-lysine coa ed 24-well
pla es a 5 x lo5 cells/well. The nex day, cells we e
in ec ed wi h a ious dilu ions o each i us and 24 h
la e he cells we e ixed in 4% pa a o maldehyde o 30
min. pHSVlac in ec ed cells we e de ec ed as p e iously
desc ibed [24]. pHSVe AEQ in ec ed cells we e isualized
by using a mouse an i-HA1 p ima y an ibody 1:200
(Boeh inge , Ge many) ollowed by an AP-conjuga ed
an i-mouse seconda y an ibody (Sigma, Spain). Alkaline
phospha ase was isualized using ni oblue e azolium
and 5-b omo-4-chlo o-3-indolyl phospha e acco ding o
he manu ac u e ’s ins uc ions (Sigma, Spain). S ained
0 Ha cou B ace & Co. L d 1998
Vi al ans e o ER- a ge ed aequo in 89
cells we e coun ed by mic oscopy and i e s o in ec ious
pa icles we e calcula ed. Ti e s o ec o we e exp essed
as in ec ious ec o pa icles (i p) pe ml.
In ec ion o cell cul u es
To assay o he capabili y o he HSV i uses o in ec
he di e en cell ypes, cul u es we e i s in ec ed wi h
pHSVlac. Cells we e seeded a semicon luency on o a 13
mm co e slip and in ec ed 24 h p io o expe imen s.
HeLa cells and NIH3T3 mouse ib oblas s we e in ec ed
wi h 1.2 x IO4 i phnl. PC12, GH, and AP cells we e
in ec ed wi h 4.8 x lo4 i phnl, ch oma lim cells wi h 3.6 x
lo4 i p/ml and g anule cells wi h 72 x 104 i phnl. Using
an an ibody agains HA1 we ou inely obse ed a ange
be ween 5-30% o cells exp essing aequo in. Con ol
expe imen s we e pe o med o es o cy o oxici y by
compa ing he e ec o ex acellula agonis s on [Caz+],
in bo h in ec ed and non in ec ed PC 12 cells (see below,
Fig. 4B). Mo eo e , measu emen s o ca echolamine
sec e ion om in ec ed bo ine ch oma in cells
s imula ed wi h ca eine o 70 mM K+ we e i ually
iden ical o he ones ob ained in unin ec ed cells (P.
Michelena and A. Ga cia, unpublished da a). Judging
om hese wo unc ional analyses, i al in ec ion did
no p oduce signi ican cy o oxici y in he es ed cell
ypes.
lmmuno luo escence
Cells we e ixed 24 h a e in ec ion wi h 4% pa a o m-
aldehyde in PBS o 20 min, washed wice wi h PBS and
once wi h TBS (100 mM T is-HCI pH 75, 150 mM NaCl).
Cells we e hen blocked o 5 min in TST (0.1% T i on X-
100, 1% FCS in TBS) and incuba ed wi h he an i-HA1
monoclonal an ibody 12CA5 (Boeh inge , dilu ion 1:200)
dilu ed in TST o e nigh a 4°C. A e wo washes in TBS,
samples we e incuba ed wi h an i-mouse IgG an ibody
FITC conjuga ed (dilu ion 1:200) o l-4 h a oom
empe a u e. Fluo escence was analyzed ou inely wi h a
Nikon Diapho mic oscope. Con ocal mic oscopy
analysis was pe o med in a Zeiss ISCM 3 10 appa a us
wi h a 488 nm band. In he [Caz+], expe imen s pe o -
med in he in ec ed PC12 cells (Fig. 4B), an abb e ia ed
immuno luo escence p o ocol was ca ied ou a he end
o he [Ca2+], measu emen s [25].
[Ca*+&, measu emen s
Cells we e seeded on o 13 mm poly-L-lysine- ea ed glass
co e slips and in ec ed 24 h p io o he expe imen s.
Be o e econs i u ing aequo in, [Ca2+],, was educed by
incuba ing he cells o 10 min a 37°C wi h he
sa coplasmic and endoplasmic e iculum Ca2+-ATPase
(SERCA) inhibi o 2,5-d& e -bu yl-benzohyd oquinone
A
lg hea y chain gene AEQ cDNA
*
I I
L VDJ CHl I;IBABI AEQ
e AEQ
, lkb ,
B
EcoR I
Fig. 1 (A) Schema ic map o he chime ic e AEQ cDNA. In he lg
moie y, coding and in onic egions a e ep esen ed by shaded
boxes and hick lines, espec i ely; in he aequo in moie y, coding
and non-coding sequences a e indica ed by a whi e box and a hin
line, espec i ely. The sho sequence encoding he HA1 ag and
he poin mu a ion (Asp11 9 + Ala, as e isk) a e indica ed. (B)
Schema ic diag am o he amplicon pHSVe AEQ plasmid. The
ansc ip ional uni con ains he IE 4/5 p omo e , he e AEQ
chime ic gene and a polyadenyla ion signal. Two gene ic elemen s
om HSV-1, he o i, and he HSV packaging sequences, allow
eplica ion and packaging o he amplicon. The p oka yo ic
sequences con ain a bac e ial o igin o eplica ion and an ampicillin
selec ion ma ke ha allow p opaga ion and ampli ica ion in E. co/i.
A simila ec o , ca ying he E. co/i /acZ gene in place o e AEQ
was used as a con ol.
(BHQ) 10 PM in KRB (K ebs-Ringe modi ied bu e : 125
mM NaCl, 5 mM KCl, 1 mM Na,PO,, 1 mM MgCl,, 5.5 mM
glucose, 20 mM HEPES, pH 74) supplemen ed wi h 3 mM
EGTA. Cells we e hen incuba ed o 1 h a oom
empe a u e in KRB con aining 0.5 mM EGTA, 10 j&l BHQ
and 0.5 mM coelen e azine n. The co e slip was hen
washed o 5 min in KRB con aining 0.5 mM EGTA, 5%
bo ine se um albumin and 10 j. M BHQ and inally placed
in he pe usion chambe o a pu pose-buil he mos a ed
luminome e . Expe imen s we e pe o med ei he a 37°C
0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 67-96
90 M T Alonso, M J Ba e o, E Camice o, M Mon e o, J Ga cia-Sancho, J Al a ez
Fig. 2 lmmunolocaliza ion o he e AEQ in he in ec ed cells. Con ocal images o HeLa cells (A) and GH, pi ui a y cells (B) in ec ed wi h he
pHSVe AEQ i us 24 h p io o he s aining wi h he an i-HA1 an ibody e ealed wi h an FITC-labeled an i-mouse seconda y an ibody.
o a 22°C. [Ca2+],, alues we e calcula ed om he
luminescence eco ds using an algo i hm which ollows
he calib a ion cu es p e iously epo ed [2,9,14].
[Ca*+], measu emen s
The cells we e seeded on o 12 mm poly-L-lysine ea ed
co e slips and in ec ed 24 h p io o he expe imen s.
Cells we e loaded wi h 4 @VI Fu a- and single-cell
[Ca”+], measu emen s and ime- esol ed digi al image
analysis we e pe o med as p e iously desc ibed [25].
RESULTS
P oduc ion o pHSVe AEQ i us and exp ession o
a ge ed aequo in
The s uc u e o he chime ic cDNA coding o aequo in
a ge ed o he ER has been p e iously desc ibed [l] and
is depic ed in Figu e 1A. This chime a esul s om he
usion o aequo in wi h he C- e minus o se e al domains
o he Ig@b hea y chain esponsible o a ge ing he gene
o he ER. The cons uc also includes an HA1 epi ope ag
o allow de ec ion by immunocy ochemis y and aequo in
cDNA ca ies a mu a ion (Asp1 19 + Ala) in one o he
h ee Caz+ binding si es o educe he a ini y o Ca2+ o
he pho op o ein. The pHSVe AEQ plasmid ec o is
shown schema ically in Figu e 1B. The e AEQ chime ic
cons uc is cloned
in
he
EcoRI
si e in o he pHSVpuc
amplicon [20] unde he con ol o he HSV-1 IE
(immedia e ea ly gene) 4/5 p omo e and hen packaged
in o i us pa icles as ou lined in Ma e ials and me hods.
Cell Calcium (1998) 24(2), 87-96
In o de o e i y he co ec exp ession and
localiza ion o he a ge ed p o ein deli e ed by he i al
ec o , cell cul u es we e in ec ed wi h he pH5Ve AEQ
i us and he ansduced p o ein was de ec ed by
immunochemis y using a monoclonal an ibody o he
HA1 epi ope o he aequo in moie y. Figu e 2 shows he
con ocal luo escence images o aequo in dis ibu ion in
wo ep esen a i e cell ypes: HeLa (Fig. 2A) and pi ui a y
GH, cells (Fig. 2B). The images show a clea pe inuclea
e icula e s aining ex ending o he pe iphe y o he cell
which is cha ac e is ic o a p o ein localized in he ER.
Simila pa e ns o localiza ion we e obse ed in he es
o he s udied cell ypes (da a no shown). De ailed
e idence showing ha he exp essed e AEQ usion
p o ein localizes speci ically in he lumen o he ER has
been epo ed p e iously in HeLa cells, i s om s udies
o colocaliza ion wi h he ER ma ke ERp72 [l] and mo e
ecen ly om elec on mic oscopy s udies [ 131.
Func ional measu emen s in cell lines
To in es iga e whe he he exp essed aequo in was ully
unc ional, we measu ed changes in he [Ca2+],,in in ec ed
HeLa cells and compa ed hem wi h hose ob ained using
a s able ans ec ed clone o HeLa cells (clone EM26, [l])
exp essing he same chime& aequo in cloned in he
mammalian exp ession ec o pcDNA unde he con ol o
he cy omegalo i us p omo e . Figu e 3 shows he ime
cou se o Ca2+ eloading and elease o he ER in
pHSVe AEQ in ec ed HeLa cells (Fig. 3A) and in he HeLa
EM26 clone (Fig. 3B). Bo h cell ypes we e deple ed o Caz+
and econs i u ed wi h coelen e azine n as desc ibed in
0 Ha cou B ace & Co. L d 1998
Vi al ans e o M- a ge ed aequo in
91
B
01 -
Fig. 3 Compa ison be ween he e ec o his amine on [Ca2+le in
pHSVe AEQ-in ec ed HeLa ceils (A) and in he HeLa EM26 clone
(B). Wild ype HeLa cells we e in ec ed wi h 1.2 x lo4 i plml24 h
p io o measu emen s. Bo h cells we e deple ed o CaZ+ and
econs i u ed wi h coelen e azine n o 1 h p io o measu emen s.
Whe e indica ed, medium con aining ei he only 1 mM CaCI, (Ca*+)
o wi h 100 FM his amine (His) was pe used. Measu emen s we e
pe o med a 37°C. The o al luminescence coun s pe co e slip
we e 0.36 0.13 x 10” coun s/s (cps, n = 3) in (A) and 12.8 2 1 .l x
1 O6 cps (n = 17) in (6). These expe imen s a e ep esen a i e o a
leas 3 simila ones.
‘Ma e ials and me hods! The addi ion o 1 mM ex a-
cellula Ca2+ p oduced a smoo h inc ease in [Ca2+le
leading o a s eady-s a e o 540 * 40 PM (mean SD, n =
3) wi hin 2 min in he pHSVe AEQ in ec ed HeLa cells
(Fig. 3A). This alue o [Ca2+],, was e y simila o 550
70 @VI (n = 45, [Z]), ob ained in he EM26 clone (Fig. 3B).
A subsequen challenge wi h 100 @I his amine e oked
an immedia e dec ease o [Ca2+],, in bo h cell ypes wi h
simila kine ics. His amine is known o mobilize
in acellula Ca2+ ia gene a ion o InsP, in hese cells
1261. Al hough he o al aequo in luminescence was
much lowe in he in ec ed HeLa cells han in he s able
clone (see igu e cap ion), he calib a ed signal showed
no signi ican di e ences be ween he dynamics o
[Ca2+],, in bo h ypes o cells. A simila pa e n o [Ca2+],,
esponse was ob ained when he ela ed ib oblas cell
line NIH3T3 was in ec ed (da a no shown).
Ra pheoch omocy oma PC12 cells a e a neu o-
sec e o y cell line e y equen ly used as a ne e cell
model because hey sha e a a ie y o p ope ies wi h
neu ons. Cell cul u es we e exposed o UTP and
b adykinin, agonis s coupled o polyphosphoinosi ide
A
350 ca=+
UTP BK
300 - -
5 250 -
z 200 -
5 150 -
100 -
50 -
o-
- Con ol
---- HSV in ec ed
600 i
:: h
1 min
-
Fig. 4 E ec s o UTP, b adykinin (BK) and high K+ on [Ca*+],,(A)
and ICa2+1- Bj in oHSVe AEQ-in ec ed PC12 cells. Cells we e
in ec ed & 4.8 ;( lo4 i p/ml24 h p io o measu emen s, deple ed
o CaZ+ and ei he econs i u ed wi h coelen e azine n ( o
measu ing [Ca*+],,, A) o loaded wi h Fu a- ( o measu ing
Ca*+l~. B). 100 uM UTP. 1 uM b ad kinin and 70 mM K+ we e
addez’when indica ed. in (B), an immuno luo escence assay using
he an i-HA1 an ibody was ca ied ou ‘on line’ a he end o he
[Ca2+], measu emen s o iden i y he in ec ed cells. The solid line is
he a e age o 57 con ol (non-in ec ed) cells and he dashed line
he a e age o 10 in ec ed cells in he same mic oscope ield. The
[Ca*+], inc emen s (measu ed a he [Cap+lc peak) we e (mean
SE): 435 24 nM (con ol) compa ed o 531 73 nM (in ec ed) o
UTP; 452 * 17 nM (con ol) and 575 46 nM (in ec ed) o BK; and
519 * 20 nM (con ol) compa ed o 430 * 62 nM (in ec ed) o high
K+. The o al luminescence coun s pe co e slip we e 1.86 0.63 x
lo6 cps (n = 5). These expe imen s a e ep esen a i e o a leas 3
simila ones.
hyd olysis h ough he P2x pu icep o [27] and he B2
ecep o [28], espec i ely. Figu e 4 shows he esul s
ob ained in PC12 cells in ec ed wi h pHSVe AEQ 24 h
p io o he measu emen s. The s eady-s a e le el o
[Ca2+le was 370 * 70 PM (n = 3) and addi ion o 100 PM
UTP igge ed a e y apid Caz+ elease (Fig. 4A). [Ca”],,
e u ned o he le el p io o s imula ion wi hin 2 min
and subsequen s imula ion wi h 1 l&i b adykinin (BK)
p oduced a u he e e sible dec ease in [Ca*+],,. These
indings a e consis en wi h he esul s in Figu e 4B
0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 87-96

92 M T Alonso, M J Ba e o, E Ca nice o, M Mon e o, J Ga cia-Sancho, J Al a ez
Ca
B
1600-
1400 -
Ca
* K
=
600.
C 1000
I
800
-
OL
E
600
,u
+o
*
200
0-
Ca
T=
/’
Ca Ca
TX T=
800.
2
&
600.
3
4*-
200
-
D
700.
600 -
2 500-
l 400
2 300
3 2oo.
IOO-
O-
F
800 -
5600.
Ca
TX
Fig. 5 E ec s o high K+ and TRH on [Ca2+le,and [Ca’+], in
pHSVe AEQ-in ec ed GH, cells (A,C,E) and AP cells (B,D,F). Cells
we e in ec ed wi h 4.8 x lo4 i p/ml 24 h p io o measu emen s,
deple ed o Ca2+and ei he econs i u ed wi h coelen e azine n ( o
measu ing [Ca*+]-., A-D) o loaded wi h Fu a- ( o measu ing
[Ca*+],, Eand F)yThe EP was e illed by incuba ion wi h 1 mM-
CaCI. (Ca) and ei he 50 mM K+ (A and B) o 100 nM TRH [C-F)
we e’ac ded when indica ed. The- o al lum nescence coun s’pe ’
co e slip we e 0.98 * 0.09 x 10” cps (n = 18) o GH,cells and 0.68
k 0.10 x lo6 cps (n = 19) o AP cells. T aces (E) and (F) ep esen
he a e age o 67 GH,and 57 AP ceils, espec i ely, p esen in he
same mic oscope ield. All he measu emen s we e pe o med a
22°C. Each expe imen is ep esen a i e o a leas 3 simila ones.
showing he cy osolic Ca2+ inc ease by he same
concen a ions o UTP and b adykinin in Fu a- loaded
PC12 cells. Depola iza ion wi h high K+ (70 mM) also
elici ed a [Ca2+], ise. In his igu e, he [Ca2+lc esponses in
con ol cells (a e age o 57 cells, solid line) and in in ec ed
cells (a e age o 10 cells, dashed line) a e also di ec ly
compa ed. The in ec ed cells we e iden i ied by immuno-
luo escence a he end o he expe imen in he same
mic oscope ield used o he Ca2+ imaging expe imen (see
‘Ma e ials and me hods’). Bo h he maximal alue and he
Cell Calcium (1998) 24(2), 87-96
in eg a ed a ea unde he peak o each agonis we e no
signi kan ly di e en in con ol and in ec ed cells. These
esul s indica e ha he ansduced aequo in was ully
unc ional and ha i al in ec ion did no modi y
unc ional pa ame e s such as agonis -induced Caz+
elease, sugges ing ha Ca2+ egula ion was essen ially
unal e ed in pHSVe AEQ in ec ed cells.
Func ional measu emen s in p ima y cells
To exploi he capaci y o he pes i us o in ec a wide
ange o cells, we nex exp essed a ge ed aequo in and
measu ed [Ca2+le in di e en p ima y cul u e cells. Mos
o he expe imen s we e ca ied ou a 22°C because he
a e o aequo in consump ion a his empe a u e is one
o de o magni ude lowe han a 37”C, and his allows
moni o ing [Ca2+],,du ing longe imes [14].
Pi ui a y cells a e a model o s udying he s imulus-
sec e ion coupling o pi ui a y ho mones. They espond
o hy o opin- eleasing ho mone (TRH) wi h a apid
inc ease in [Ca2+lc due bo h o elease o Ca2+ om
in acellula s o es and o s imula ed Ca2+ in lux om
he ex acellula medium [29]. They possess ol age-
dependen Caz+-channels ha ac i a e on depola iza ion
o he plasma memb ane, p oducing a as peak in
cy osolic Ca2+ [17,25]. In Figu e 5 we compa ed he
e ec s o bo h TRH and depola iza ion wi h 50 mM K+
on [Caz+],, and [Ca’+], in he GH, a pi ui a y cell line
(Fig. 5A,C,E) and in p ima y AP cells (Fig. 5B,D,F), om
which he cell line GH, de i es. Bo h cell ypes we e
in ec ed wi h pH.SVe AEQ i us du ing 24 h. Cells we e
deple ed o Ca2+ as desc ibed in Ma e ials and me hods
and hen eloaded by addi ion o 1 mM ex acellula Ca2+
as indica ed in he igu e. [Ca2+],, inc eased smoo hly
du ing 4-5 min and eached a s eady-s a e a 780 + 70
@VI and 600 + 150 @I in GH, and AP cells (mean SD, n
= 7) espec i ely. Depola iza ion wi h 70 mM KC1
induced a simila esponse in bo h pi ui a y cul u es,
esul ing in a apid o e shoo o [Ca2+],, om 780 @I up
o abou 1.3 mM in GH, cells (Fig. 5A) and nea 1.5 mM
in p ima y AP cul u es (Fig. 5B). In con as , s imula ion
wi h TRH esul ed in a apid and sus ained dec ease o
[Ca2+le , (by 470 150 )&l, n = 3) in GH, cells (Fig. 5C), bu
only in a mino dec ease in [Ca2+],, o a ound 70 )&I in AP
cells (Fig. 5D). The small CaZ+ esponse o TRH obse ed in
AP cells canno be a ibu ed o inhibi ion o Ca2+ elease
by cy osolic Ca2+, as ound o HeLa cells [2,14], because
loading he cells wi h BAPTA did no modi y he e ec o
TRH on [Ca2+],, (da a no shown). In con as o his
s iking di e ence in he [Ca2+],, esponses, measu emen s
o [Caz+], in GH, (Fig. 5E) and AP cells (Fig. 5F) loaded wi h
Fu a- and s imula ed wi h TRH showed e y simila
esponses. In bo h cases, mos o he indi idual cells (50-
750/0) esponded o TRH wi h a as and ansien [Ca2+],
0 Ha cou B ace & Co. L d 1998
Vi al ans e o ER- a ge ed aequo in 93
A
700
600
500
400
300
200
100
0
His Ca
B
700
600
3 500
b 400
+” 300
( I 0 200
2 100
0
Ca2+
Ca Ca
2
Fig. 6 E ec s o his amine and ca eine on [Ca*+],,in pHSVe AEQ-
in ec ed bo ine ch oma in cells. Cells we e in ec ed wi h 3.6 x lo4
i plml24h p io o measu emen s. (A) 10 FM his amine (His) o 10
mM ca eine (Ca ) was added when indica ed. (B) Two pulses o 10
mM ca eine we e added as indica ed. The o al luminescence
coun s pe co e slip we e 1.05 2 0.16 x lo8 cps (n = 14). O he
de ails as in Figu e 3. These expe imen s a e ep esen a i e o a
leas 3 simila ones.
inc ease. The a e age inc ease o [Ca2+], a he peak was
abou 600-800 no in bo h cell ypes.
One o he main ad an ages o he HSV amplicon
sys em is he abili y o in ec non-di iding cells such as
neu ons. This allows using he pHSVe AEQ i us o s udy
he dynamics o neu onal Caz+ s o es. In his epo , we
ha e employed wo di e en cell ypes: ch oma m and
ce ebella g anule cells. Ad enal medulla ch oma in cells
can be ega ded as a highly specialized o m o pos -
ganglionic sympa he ic neu ons. They de i e emb yo-
logically om he neu al c es , a e elec ically exci able
and elease esicle-con ained subs ances in esponse o
speci ic s imuli. They a e equen ly u ilized in s udies on
CICR mechanisms and he ela ionship be ween InsP,-
and yanodine-sensi i e s o es. He e, we ha e used
his amine o ac i a e he InsP,-sensi i e Ca*+ elease
mechanism and ca eine o ac i a e he CICR mechanism
p esen in hese cells 1301. Figu e 6 shows he esul s
ob ained in pHSVe AEQ in ec ed bo ine ch oma in cells.
The addi ion o 1 mM Ca2+ o Ca2+-deple ed ch oma in
cells induced an ele a ion o he [Ca2+le up o a s eady-
A
Ca2+
600
GllJ
B
600
I 500
Y 400
jg 300
+
c l 200
z 100
0
Ca2+
2
min
Fig. 7 E ec s o glu ama e (Glu) and high K+ on [Ca2+le in
pHSVe AEQ-in ec ed ce ebella a anule cells. Cells we e in ec ed
wi h 7.2 x 1 O4 i plml 24 h p io o-measu emen s. 100 PM
glu ama e (A) and 50 mM K+ (B) we e added as indica ed. The
o al luminescence coun s pe co e slip we e 0.19 0.07 x 1 O6 cps
(n = 4). O he de ails as in Figu e 3. These expe imen s a e
ep esen a i e o a leas 3 simila ones.
s a e o 650 100 mM (n = 13) wi hin 4-5 min (Fig. 6A).
Subsequen exposu e o 10 @VI his amine igge ed a
apid dec ease o [Ca2+le, o abou 60% o he ini ial le el.
A e washing ou he agonis , he ER eloaded wi h Ca*+
up o he same s eady-s a e, and he cells esponded o a
la e applica ion o 10 mM ca eine wi h a second d op
in [Ca2+],,. A simila pa e n was ob ained, bo h in ime
cou se and ampli ude, when he cells we e challenged
wi h wo consecu i e pulses o 10 mM ca eine (Fig. 6B).
These da a indica e ha wo di e en s imuli, ac ing
ei he ia InsP, ecep o s o ia yanodine ecep o s, a e
able o elease compa able amoun s o Ca*+ om he ER
in ch oma m cells.
Finally, a p ima y pos mi o ic neu onal cul u e was
used. We deli e ed he a ge ed aequo in in o a
ce ebella g anule cells by in ec ing hem wi h he
pHSVe AEQ i us. Figu e 7 illus a es a ep esen a i e
example in which g anule cells we e s imula ed ei he
wi h glu ama e o wi h 50 mM K+. The s eady-s a e le el
o [Caz+], was 300 + 60 I.~M (n = 3), somewha lowe han
in he o he cell ypes s udied. Addi ion o 100 PM
0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 87-96
94 M T Alonso, M J Ba e o, E Ca nice o, M Mon e o, J Ga cia-Sancho, J Al a ez
glu ama e (Fig. 7A) elici ed an inc ease in [Ca2+], ollowed
by a slow decline accele a ed by emo al o he agonis .
Me abo opic glu ama e ecep o agonis s like quisquala e
( 1 ClM) and ia% 1 aminocyclopen ane- 1 S,3Rdica boxylic
acid, (E dns-ACPD, 100 @VI) did no induce any signi ican
change in he [Ca2+], le el (da a no shown). This esul is
consis en wi h he lack o e ec o hese agonis s on
[Ca2+], in single cell imaging expe imen s in ou g anule
cell cul u es (da a no shown). Finally, a 2 min
depola iza ion pulse wi h 50 mM K+ (Fig. 7B) also induced
an inc ease in he [Caz+],, signal e y simila o ha e oked
by glu ama e.
DISCUSSION
Conside able in e es is p esen ly ocused on he
dynamics o [Ca2+],, due o i s ole as he main ese oi o
Ca2+, able o be apidly eleased in o he cy osol upon cell
s imula ion. Howe e , he es ima es o he s eady-s a e
[Ca2+],, epo ed in he li e a u e ha e been con lic ing,
p obably because o he di e en me hodologies used
[31]. Many o he measu emen s we e unde aken wi h
low-a ini y luo escen p obes, which ha e impo an
limi a ions such as non-speci ic localiza ion h oughou
he cell and in e e ence wi h Mg2+. In ac , due o he
need o elimina e he s ong dye signal a ising om he
cy osol, mos o hese s udies ha e been pe o med in
pe meabilized, quenched o pa ched cells 132,331. Se e al
o hese p oblems can be o e come using ER- a ge ed
pho op o eins. They a e speci ically localized in he ER,
hus allowing selec i e moni o ing o lumenal [Ca2+],,
changes in in ac cells. Fu he mo e, aequo in does no
display Mg2+-sensi i e luminescence and i has a g ea e
dynamic ange han luo escen dyes. Howe e , he i s
a emp s o measu e [Ca2+],, using ER- a ge ed aequo in
u ned ou o be pa icula ly di icul because o he apid
aequo in consump ion esul ing om he high [Caz+]
p esen in his compa men . Thus, no only he wild ype
[ 10,l 11, bu also he low-a ini y e sion o aequo in a e
endowed wi h a Ca2+ a&-&y oo high o eliably measu e
[Ca2+],, [ 1,131. We ha e ecen ly sol ed his p oblem by
using coelen e azine n, a chemically modi ied o m o he
p os he ic g oup ha educes he a e o luminescence,
hus allowing measu emen s o s eady-s a e [Caz+],,
a ound 500 @VI in HeLa cells [2,14]. These esul s we e in
close ag eemen wi h da a ecen ly epo ed, also in HeLa
cells, using an ER- a ge ed chime a dubbed ‘cameleon’
(34). The au ho s ound ha 90% o he cell popula ion
had [Ca”‘], a s eady s a e a ound 400 @VI [35]. The
s eady-s a e [Caz+],, es ima ed he e in he di e en cell
ypes s udied we e also in he same ange, om 300 l. ~ in
g anule cells o 800 l&I in GH, cells.
Up o now, s udies o he dynamics o [Ca2+],, using ER-
a ge ed cons uc s (aequo in- o cameleon-based) ha e
Cell Calcium (1998) 24(2), 87-96
been mos ly pe o med in adhe en cell lines (pa icula ly
HeLa cells) and e y limi ed in o ma ion is a ailable o
p ima y adul cells. The eason is ha non-di iding cells
a e e ac o y o he mo e common me hods o
in oduc ion o DNA like Ca2+ phospha e. He pes i al
ec o s a e a powe ul ool o gene ans e in o
pos mi o ic cells and, mo e speci ically, in o adul
neu ons. Mo eo e , HSV-1 can e icien ly in ec a wide
a ie y o es ablished lines o di e se o igin, hus
o e coming many o he limi a ions concomi an o DNA
ans ec ion o he selec ion o a s able ans ec ed clone.
Gi en he physiological ele ance o he Ca2+ s o es in
p ima y exci able cells, we decided o exploi he wide
hos ange o he HSV-1 sys em o deli e he ER-
a ge ed aequo in o a numbe o di e en cell ypes.
Thus, we ha e gene a ed a eplica ion de icien he pes
simplex ype 1 i us con aining he ER- a ge ed low
a ini y aequo in gene @HSVe AEQ. Vi al in ec ion o
bo h p ima y pos mi o ic cells and es ablished cell lines
led o he co ec exp ession o he chime ic aequo in
selec i ely in o he lumen o he ER as de e mined by
immunos aining and by unc ional [Ca2+],, s udies. The
ansduced chime ic ER- a ge ed aequo in displayed
Ca2+-sensi i e luminescence and could be easily used o
moni o [Ca2+],, changes elici ed by InsP,-gene a ing
agonis s, ca eine and ac i a ion o ol age-ga ed o
ecep o -ope a ed Ca2+ channels in a a ie y o cells. The
da a p esen ed he e a e he i s epo o di ec
measu emen s o he dynamics o [Ca2+],, in pi ui a y
cells (bo h GH, and AP cells), PC12, ch oma in and
ce ebella g anule cells. The use o an adeno i us ec o
o a ge wild ype aequo in o he ER has been ecen ly
epo ed 1361. Howe e , al hough he au ho s showed
exp ession o he apop o ein in he ER o he in ec ed
cells, no measu emen s o [Ca2+],, we e p o ided. As we
ha e al eady discussed, wild ype aequo in econs i u ed
wi h na i e coelen e azine canno p o ide eliable
measu emen s o [Ca2+],, due o he apid consump ion o
he p obe [ 131.
Rega ding he physiological da a epo ed in his s udy,
i was su p ising o ind ha TRH p oduced such a small
e ec in AP cells compa ed wi h ha ob ained in GH,
cells (compa e Fig. 5D and C), e en hough he agonis is
able o induce a compa able [Ca2+], inc emen in bo h
cell ypes (compa e Fig. 5E and F and see [ 17,251).
Appa en ly, a ela i ely modes mean dec ease o abou
70 @I in [Ca2+le is enough o p oduce a [Ca2+lc peak o
700 nM in 50-75~0 o he AP cells analyzed by single cell
imaging. This dissocia ion be ween he dec ease in
[Ca2+],, and he [Ca2+], peak is in ag eemen wi h ou
p e ious esul s in his amine-s imula ed HeLa cells,
whe e he changes in [Ca2+],, and [Ca”], we e di ec ly
compa ed [2,14]. Those esul s we e in e p e ed in e ms
o a s ong and apid inhibi ion o he InsP,-ga ed
0 Ha cou i B ace & Co. L d 1998
Vi al ans e o E/?- a ge ed aequo in 95
channels by mic odomains o high [Ca’+], in he icini y o
he channels. Howe e , expe imen s designed o es his
hypo hesis by loading he AP cells wi h BAPTA p io o
TRH s imula ion showed no accele a ion o he [Caz+],,
d op (da a no shown), sugges ing ha he mechanism
may be mo e complex in his case. Zn ac , he magni ude
o he esponse o [Ca*+],, o an InsP,-p oducing agonis
appea s o be qui e a iable depending on ei he he cell o
he agonis used. Thus, his amine in ch oma im cells o
o he InsP,-p oducing s imuli like UTP o b adykinin in
PC12 cells did induce a apid, la ge Ca*+ elease om he
ER (see Figs 6A & 4A). In any case, one impo an
conclusion om hese esul s is ha he e is no always a
di ec co ela ion be ween he heigh o he [Ca’+], peak
measu ed wi h luo escence indica o s and he amoun o
Ca eleased om he ER. Fo his eason, he con en o he
s o es canno be es ima ed accu a ely om he heigh o
he [Ca2+], peak induced by a Ca2+- eleasing agonis and
di ec measu emen o [Ca2+], becomes essen ial.
Gi en he na u al p opensi y o he pes i us o in ec
pos mi o ic neu ons, an immedia e applica ion o his
echnique is o s udy neu onal Ca2+ s o es. I has become
clea in ecen yea s ha in acellula Ca2+-s o age si es can
play a c ucial ole in egula ing complex Ca2+ signals and
neu onal esponses such as exci abili y, synap ic plas ici y,
elease o neu o ansmi e s and pe haps neu onal dea h
[371. I is well known ha s imula ion o glu ama e
ecep o s in neu onal cells elici s an inc ease o he [Ca2+lc
due o Ca2+ en y h ough bo h he NMDA ‘channel
complex and o he ol age-ga ed Ca2+ channels [38]. Ou
p esen esul s in ce ebella g anule cells (see Fig. 7A) show
ha s imula ion wi h glu ama e induces a apid inc ease in
[Ca2+le , which can be explained om he s imula ion o he
sa co- and endoplasmic e iculum Ca2+-ATPases (SERCA) by
he inc ease in [Ca2+], due o ac i a ed Ca2+ en y Al hough
ecen esul s sugges ha he iono opic ecep o -
media ed [Ca2+], signals migh also in ol e elease o Ca2+
om in acellula s o es by CICR 1391, ou da a do no
suppo his hypo hesis in ce ebella g anule cells. On he
o he hand, addi ion o quisquala e, o he mo e selec i e
me abo opic glu ama e ecep o agonis uns-ACPD, o
he neu onal cul u e did no p oduce any esponse ei he in
[Ca’+],, o in [Ca2+],, sugges ing ha ou cul u es o g anule
cells lack me abo opic glu ama e ecep o s.
S udies in a numbe o neu onal p epa a ions ha e
indica ed ha depola izing s imuli can ac i a e a calcium-
induced-calcium- eleased (CICR) mechanism [39].
Howe e , he in ol emen o CICR in he gene a ion o
[Caz+], peaks has been di licul o es ima e due o he
pa allel ac i a ion o plasma memb ane Ca2+ channels and
he subsequen massi e Ca2+ en y. Specula ions on he
ole o CICR ha e been qui e a iable depending on he
me hodology used o p oduce depola iza ion, leading o
con lic ing esul s in he li e a u e. In ou expe imen al
condi ions we did no obse e any sign o ac i a ion o
CICR upon a 2 min pulse wi h high K+ medium in GH,, AP
cells o g anule neu ons (see Figs 5A,B & 7B). Ins ead, high
K+ depola iza ion always elici ed a signi ican inc ease in
[Ca2+],, in all cells es ed. Again, he mos p obable
explana ion o his e ec is he s imula ion o he SERCA
by he inc ease in [Ca2+],. Ano he ool o en used o
examine he ole o CICR is he abili y o ca eine o elici
elease o in acellula Ca2+. Ou measu emen s o [Ca2+],,
in ad enal ch oma m cells (Fig. 6) con i m ha ca eine
mobilizes Ca2+ om he in acellula s o es in hese cells, a
esul consis en wi h ea lie epo s using luo ime ic
echniques o measu e [Ca”‘], [30,40].
In conclusion, he da a p esen ed he e demons a e ha
he HSV based echnology is an e icien me hod o
deli e he ER- a ge ed aequo in o a numbe o mam-
malian cells, bo h es ablished lines and pos mi o ic
p ima y cells. Fu u e de elopmen s may include p o-
ducing new HSV ec o s ca ying aequo in chime as
a ge ed o o he compa men s (e.g. nucleus, mi o-
chond ia) o exp essing o he a ge ed indica o genes
such as he ecen ly epo ed cameleons [34] ha allow
pe o ming single-cell imaging expe imen s. This
app oach will acili a e he s udy o key physiological
issues such as he ole o neu onal calcium s o es o he
beha io o unc ionally dis inc Ca2+ pools wi hin he ER.
ACKNOWLEDGEMENTS
We a e indeb ed o D F. Lim o helping us o es ablish he
HSV media ed gene ans e echnique in ou labo a o y
and o his e e p esen ad ise on he amplicon sys em. We
hank P. Ala &n o help wi h he imaging expe imen s and
bo h P. Ala con and D A. Sanchez o gene ously p o iding
AP cells. We acknowledge D P. Michelena and D A. Ga cia
o ch oma Bm cells. We also hank J. Fe nandez o
echnical assis ance. We a e g a e ul o S. Calleja o
assis ance wi h con ocal mic oscopy We specially hank D
T. Schimmang o his help ul commen s on he manusc ip .
This esea ch was suppo ed by g an s om Spanish
Go e nmen Agencies o Heal h Resea ch FIS 96/1443
(MTA), FIS 96/0456 (JA) and DGICYT PB92/0268 (JGS) and
ellowships o MJB and EC.
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2. Mon e o M., Ba e o MJ., Al a ez J. [Ca*+] mic odomains con ol
agonis -induced Ca2+ elease in in ac cells. FASEB / 1997; 11:
881-886.
3. Boo man M.D., Be idge MJ. The elemen al p inciples o
calcium signaling. Cell 1995; 83: 675-678.
0 Ha cou B ace & Co. L d 1998 Cell Calcium (1998) 24(2), 87-96