42
INTRACELLULAR Ca2
+DEPOSITS AND
CATECHOLAMINE SECRETION BY
CHEMORECEPTOR CELLS OF THE RABBIT
CAROTID BODY
Ana Obeso, Asuncion Roche , Jose Ramon Lopez-Lopez, and
Cons ancio Gonzalez
Depa amen o
de
Bioquimica y Biologia Molecula yFisiologia
Facul ad
de
Medicina, Uni e sidad
de
Valladolid
47005 Valladolid, Spain
1.
INTRODUCTION
The pi o al ole
o
in acellula ee [Ca2
+]
luc ua ions in he con ol
o
cellula
unc ions such as con ac ion and sec e ion, including he elease
o
neu o ansmi e s,
was ecognized many decades ago (see Rubin, 1982). Mo e ecen ly, he lis
o
cellula
unc ions igge ed o modula ed by he le els
o
Ca2+
jhas g own eno mously. Addi ional
unc ions egula ed by [Ca2+l include neu onal exci abili y, synap ic plas ici y, gene ex-
p ession, cellula me abolism, cell di ision and di e en ia ion, and p og ammed cell dead
(Mille , 1991; Clapham, 1995). Pa alleling he g ow h in his lis
o
Ca2+
-con olled unc-
ions, amul iplici y
o
cellula mechanisms aimed a main aining es ing ee [Ca2+l in he
ange
o
100
nM
o mos cells has been desc ibed, allowing inc eases in Ca2+
jle els ha
a e speci ic in hei magni ude, ime cou se and spa ial dis ibu ion, acco ding o he cell
unc ion ac i a ed (Toescu, 1995).
Since Ca2+canno be me abolized, cells egula e hei cy oplasmic le els
o
ee Ca2+
h ough nume ous binding p o eins and in lux and e lux mechanisms (Fig 1). Ca2+in lux
o cell cy oplasm om he ex acellula milieu occu s ia ol age o ecep o ope a ed
channels o ia ye ill-de ined capaci a i e pa hways; he Na+/Ca2+exchange can also
p oduce in some ci cums ances ne in lux
o
Ca2+(Mille , 1991; Clapham, 1995). Ca2+e -
lux o he ex acellula space occu s agains elec ochemical g adien s, and he eby he
pumping ou
o
Ca2+is di ec ly (Ca2+pump) o indi ec ly (Na+/Ca2
+)
coupled o he hy-
d olysis
o
ATP.
In addi ion o he ex acellula space, he e a e in acellula s o es, ep esen ed p i-
ma ily by he smoo h endoplasmic e iculum, capable
o
accumula ing
Ca
2+a e y high
concen a ions (mM ange) ia speci ic
Ca2+
ATPases (Fig 1). The smoo h endoplasmic
F on ie s in A e ial Chemo ecep ion, edi ed by Zapa a e al.
Plenum P ess, New Yo k, 1996 279
280
A.
Obeso e al.
Na
Figu e
1.
Model o he egula ion
o
cellula calcium homeos asis. See ex o an explana ion.
e iculum
o
many cells may possess wo speci ic mechanisms capable
o
mobilizing he
accumula ed Ca2+in esponse o adequa e signals, he inosi ol 1,4,5- iphospha e ecep-
o s (IP3R) ha on IP] binding a e ensambled o p oduce aCa2+channel, and he yano-
dine ecep o -channel complex (RyR) equally pe mean o Ca2+(Pozzan e ai, 1994;
Simpson e aI, 1995). Many ex acellula signals, ho mones and neu o ansmi e s, pos-
sess ecep o s in he plasma cell memb ane ha ac i a e phospholipase Cleading
o
he
genesis
o
IP] in he cell in e io and o he elease
o
Ca2+ om he endoplasmic e icu-
lum ia he IP]R (Clapham, 1995); he RyR a e ac i a ed by local inc eases in ee Ca2+;
p oduced by Ca2+en e ing ia plasma cell memb ane (Lopez-Lopez e ai, 1995), and ep-
esen he subs a e o he long ime known p ocess
o
Ca2+
-induced Ca2
+ elease (Simp-
son e aI, 1995). The e o e, he endoplasmic e iculum may unc ion as asink o as a
sou ce o ee cy oplasmic Ca2+in di e en unc ional s a es
o
he cells, and addi ion-
ally he sink o sou ce unc ion
o
he endoplasmic e iculum migh a y om cell o
cell, depending on he deg ee
o
exp ession
o
he epe oi e
o
he molecules in ol ed in
he endoplasmic e iculum handling
o
Ca2+(Simpson e ai, 1995). A cy oplasmic le els
o
ee Ca2+highe han 500 nM (Clapham, 1995), i.e., du ing cell ac i a ion, mi ochon-
d ia may accumula e Ca2
+,
he eby unc ioning as e ec i e sinks o educe cy oplasmic
ee
Ca2+
concen a ion. Finally, many cy osolic cons i uen s including p o eins, and
small o ganic molecules can bind Ca2+ions con ibu ing o modula e local le els
o
cy o-
plasmic ee Ca2+(Clapham, 1995; Toescu, 1995). The p o eins wi h he capaci y o bind
Ca2+may in
um
acqui e ca aly ic ac i i ies when hei binding si es a e occupied by he
egula ing ion.
In he ca o id body (CB) chemo ecep o cells, ou knowledge on he ole
o
Ca2+in
cell signalling is a he es ic ed (see Gonzalez e ai, 1994).
I
was only in 1968 ha
Eyzagui e and Zapa a showed he Ca2+dependency
o
ca o id sinus ne e (CSN) dis-
cha ges elici ed by hypoxia, acidi y o low in e up ion.
In
1975, again Eyzagui e's
g oup (Eyzagui e e ai, 1975) epo ed ha ace ylcholine was incapable
o
gene a ing ac-
In acellula
Ca
l
+Deposi s
and
Ca echolamine Sec e ion
by
Chemo ecep o Cells 281
ion po en ials in he CSN in Caz+- ee solu ions. Some yea s la e , a ew ul as uc u al
s udies showed he appea ance
o
omega exocy o ic p o iles and/o coa ed pi s and es-
icles in chemo ecep o cells
o
a CBs incuba ed in Caz+-con aining (bu no in Caz+- ee)
ich
K
solu ions o in he p esence
o
he Caz+ionopho e A23187, concluding ha exocy-
osis was Caz+e-dependen (G onblad e ai, 1980). Consis en wi h ha , i was la e shown
ha he elease
o
dopamine (DA) elici ed by hypoxia and high
K
was
Ca
Z -dependen
in >95% (Fidone e ai, 1982; Alma az e ai, 1986; Obeso e ai, 1992). Mo e ecen ly, h ee
di e en labo a o ies (Gonzalez e ai, 1993; Lopez-Ba neo e ai, 1993; Buckle &
Vaughan-Jones, 1994) ha e used luo escen dyes o measu e [Caz+l in isola ed chemo e-
cep o cells, and ha e consis en ly ound ha mo e han 95%
o
he [Caz+l ise p oduced
by hypoxia is due o Caz+en e ing om he ex acellula space. In he case
o
acidic
s imula ion he
Ca
Z
dependence
o
he elease
o
DA was app oxima ely 80% (Obeso e
ai, 1992) and he
Ca2+;
signal was educed by asimila pe cen age in Caz+- ee media
(Buckle &Vaughan-Jones, 1993). A a iance wi h hose indings, Biscoe and Duchen
(1990) epo ed ha up o 40%
o
he hypoxic Caz
+
j ise was due o
Ca2+
en e ing he cy o-
plasm om in acellula s o es, and Biscoe e al (1989) could no de ec a ise in ee
Ca
z
+
j
du ing acidic s imula ion. In addi ion, Lahi i e al (1995) ha e ecen ly communica ed ha
he anoxic CSN discha ge was be e p ese ed in Caz+- ee media con aining hapsiga gin
han in i s absence, concluding ha Caz
+ om in acellula deposi s seems o con ibu e o
he anoxic chemo ecep ion.
2. METHODS
Using an in i o p epa a ion
o
he abbi CB whose ca echolamine deposi s ha e
been labeled by p io incuba ion
o
he o gans wi h hei na u al p ecu so 3H- y osine (Fi-
done &Gonzalez, 1982), we ha e s udied he signi icance
o
in acellula Caz+s o es as
possible sou ces
o
Caz+ o he sec e o y esponse, and as possible bu e s (sinks) o he
Ca
z+en e ing he cell cy oplasm du ing s imula ion. The ools used in he expe imen s in-
clude: ATP and b adykinin as possible gene a o s
o
IP3,and he eby as possible ac i a-
o s
o
he IP3
R; yanodine a submic omola concen a ion and ca eine as ac i a o s
o
he RyR; hapsiga gin as ablocke
o
he e iculum ATPase; u henium ed as ablocke
o
he mi ochond ial
Ca2+
unipo e ; and, ionomycin as a
Ca
z+ionopho e ha allows apid
equilib a ion
o
Ca
z+ h oughou cellula memb anes. The expe imen s ha e been pe -
o med in Caz+-con aining and in Ca2+- ee solu ions. The e ec s
o
he d ugs ha e been
es ed on hei abili y o igge asec e o y esponse in basal no moxic condi ions, o es
o a ole
o
in acellula Caz+s o es as sou ces
o
Caz+ o he sec e o y esponse, and on
hei abili y o modi y he in ensi y and ime cou se
o
he sec e o y esponse elici ed by
hypoxia and high
K .
In his way, we ha e es ed o he capaci y
o
he in acellula
s o es o ac as sou ces
o
Ca2+,
o as sinks
o
he Caz+en e ing om he ex acellula
space.
I
should be no ed ha mos
o
he expe imen s ha e been pe o med sampling he
incuba ing solu ions o hei analyses in 3H-ca echolamine
eH-CA)
con en e e y 2min,
he eby p o iding ahigh sensi i i y
o
he o e all p ocedu es capable
o
de ec ing <6 mol
o
labelled CA
«200
cpm), Which a e co ec ion o he speci ic ac i i y
o
3
H-CA in he
cells, ep esen s app oxima ely 120 mol
o
o al CA elease,
o
an amoun equi alen o
he basal elease/2 min in no moxic condi ions. Du ing s imula ion (hypoxia, high
K+
e
) he
elease inc eases ma kedly, and consequen ly he sensi i i y
o
he adioiso opic me hod
o de ec he e ec
o
ad ug on he s imulus induced elease dec eases.
A.
Obeso
e
al.
Figu e
2.
Time cou se
o
he disappea ance
o
high ex e -
nal
K+
induced elease
o
3H-ca echolamines upon emo al
o
calcium om he ex acellula space. Emp y ba s: e-
lease esponse
in
Ca2+-con aining solu ions a he begin-
ning and he end
o
he expe imen s. Slashed ba s: elease
esponse a e incuba ion in Ca2+- ee solu ions o he in-
dica ed pe iods. Release esponse (o dina e)
is
exp essed
as imes basal elease in no mal
K+
solu ions.
3. RESULTS AND DISCUSSION
Figu e 2shows he ime cou se
o
ex acellula
Ca
2
+washing ou . In designing hese
expe imen s, he ad an age ha he elease induced by high ex e nal
K+
is almos o ally
dependen on Ca2
+en e ing om he ex acellula space was aken. The e o e, he mini-
mum ime
o
incuba ion in
Ca
2
+ ee medium equi ed o abolish high
K
induced elease
would be equi alen o he ime equi ed o wash ou Ca2+ions om he incuba ing ial
and he CB ex acellula space. As shown in Figu e 2, one min
o
incuba ion in 0Ca2
+ e-
duced ma kedly he elease esponse, a e 4min in 0Ca2+ he elease was p ac ically
abolished, and on ein oduc ion
o
Ca
2+ he elease esponse eco e ed comple ely. In a
simila expe imen using hypoxic s imula ion (2% 02-equilib a ed solu ions, 2min), he
ime cou se
o
inhibi ion was compa able; in ac , a e 4min in 0Ca2
+ he elease induced
by hypoxia was comple ely abolished. These expe imen s con i m p e iously epo ed
indings wi h longe imes
o
incuba ion in
Ca
2+ ee solu ions (e.g. Obeso e ai, 1992),
and sugges ha in acellula Ca2+s o es do no
playa
signi ican ole in media ing he se-
c e o y esponse o hypoxia, unless chemo ecep o cells ha e an unusually as -emp ying
in acellula
Ca
2
+deposi s.
On
he o he hand, he p esen indings a e consis en wi h ob-
se a ions
o
Fidone (see Fidone e ai, 1977) and To ance (1977) showing ha he hal -
ime o bica bona e and suc ose wash ou , espec i ely, was close o
hal
aminu e.
Nex we pe o med ase ies
o
expe imen s aimed a mobilizing in acellula
Ca
2
+
s o es. In a i s g oup
o
expe imen s we es ed o he abili y
o
ATP o mobilize
Ca
2
+
s o es. In a ecen s udy, Spe gel &Lahi i (1993) showed ha ATP and analogs, bu no
adenosine, we e able o inc ease
CSN
discha ges in adose-dependen manne , concluding
ha he CB exp esses su ace P2 ype ATP ecep o s. Since P2 ecep o s a e coupled o
13
ype phospholipase C, i should be expec ed ha hei ac i a ion will lead o he o ma ion
o
IP3and ac i a ion
o IP
3R.
A
TP
a 100 and 1500
IlM
did no a ec he basal no moxic
elease
o
DA
ei he in
Ca
2+con aining
o
in Ca2+ ee solu ions sugges ing ha he ATP
ecep o s esponsible o he ac i a ion
o
CSN discha ges a e loca ed in as uc u e di e -
en om chemo ecep o cells. B adykinin, apo en mobilize
o
Ca2
+ om in acellula

In acellula
Ca
2+Deposi s
and
Ca echolamine Sec e ion by
Chemo ecep o
Cells
283
deposi s and po en sec e agogue in ch oma in cells (Augus ine &Nehe , 1992), was also
wi hou e ec on he basal elease
o
3H-CA om he CB, sugges ing ha chemo ecep o
cells do no possess b adykinin ecep o s o ha he endoplasmic e iculum Ca2+s o es a e
eally small. The same was ue o he es
o
he agen s es ed including ionomycin in
Ca2+- ee solu ions.
In conclusion, he pa icipa ion
o
endoplasmic e iculum and mi ochond ia
as
sou ces
o
Ca2+capable
o
al e ing he sec e o y esponse
o
chemo ecep o cells appea
o
be
o
sec-
onda y impo ance, implying ha chemo ecep o cells mus ely almos exclusi ely on
plasma memb ane mechanisms
o
main ain hei Ca2
+homeos asis. Al hough he p ecise iden-
i y
o
he Ca2
+s o es si es
is
no ully clea ,i appea s ha he smoo h endoplasmic e iculum
(SER) may be he p incipal place whe e he in acellula Ca2+ATPase
is
loca ed and he eby
he SER could be he main place whe e Ca2+is s o ed (Pozzan e
aI,
1994).
I
his
is
he case
in
chemo ecep o cells ou indings would co ela e wi h he appa en ly ea SER p o iles p e-
sen in he cells ha appea
o
be loca ed mainly in he p ocesses
o
chemo ecep o cells
(Ve na, 1975; McDonald, 1981). I should be men ioned ha pape s consis en wi h ou con-
clusion ha e been p esen ed in his Symposium. Thus, He e al (1996) communica ed ha en-
do helin Iinc eases up o i e imes he no moxic basal le els
o IP
3
,and he e o e
i
should
be expec ed ha
IP
3
R
o
he endoplasmic e iculum ep esen s he a ge
o
he IP3 o med. Al-
hough he cellula elemen in which he inc ease
o
IP3had occu ed was no iden i ied,
i
was shown ha endo helin Idid no a ec he basal elease
o
CA o he Ca2
+jle els in
chemo ecep o cells, bu s ill endo helin Iwas e ec i e
in
po en ia ing he elease
o DA
and
he Ca2+j ise p oduced by hypoxia. Those indings imply ha chemo ecep o cells possess en-
do helin ecep o s, and he e o e i could be expec ed ha
IP
3has inc eased in chemo ecep o
cells. The e o e, he lack
o
e ec on he basal elease
o
CA and Ca2+jwould imply ha he
cellula deposi s
o Ca
2+sensi i e o IP3a e eally small. The e ec s obse ed du ing s imula-
ion would be p oduced ia ol age-dependen Ca2+channels,
as
i
is
he case in o he sys-
ems. These pieces
o
in o ma ion u ge o explo e he dynamics
o
in acellula Ca2+s o es in
amo e di ec manne and o co ela e he da a wi h o he unc ions
o
he chemo ecep o
cells, besides he elease
o
neu o ansmi e s.
4.
ACKNOWLEDGMENTS
Suppo ed by Spanish DGICYT G an PB92/0267.
5.
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