190
BRES 17074
B ain Resea ch, 5(~2 (199 ) 190-198
© 1991 Else ie Science Publishe s B.V. All igh s ese ed. 0006-8993/911503.50
A DONIS ~9939117074B
Musca inic ecep o localiza ion and unc ion in abbi ca o id body
B.G. Dinge , L. Alma az, T. Hi ano, K. Yoshizaki, C. Gonzalez. A. Gomez-Ni io and
S.J.
Fidone
Depa men o Physiology, School o Medicine. Uni e si y o U ah. Sal Lake Ci y, UT 84108 ~ U.S.A.
Accep ed 28 May 19911
Key wo ds: Ca o id body; Chemo ecep o : Musca inic ecep o : Choline gic ecep o : Au o adiog aphy; Recep o binding
Ace ylcholine and musca inic agonis s inhibi chemosenso y ac i i y in he abbi ca o id sinus ne e (CSN). Because he mechanism o
his inhibi ion is poo ly unde s ood, we ha e in es iga ed he kine ics and dis ibu ion o musca inic ecep o s in he abbi ca o id body wi h
he speci ic musca inic an agonis [SH]quinuclidinylbenzi a e ([3H]QNB). Equilib ium binding expe imen s iden i ied displaceable binding si es
(1/~M a opine) wi h a K d = 71.46 pM and a Bm~ x = 9.23 pmol/g issue. These binding pa ame e s and he pha macology o he displaceable
[SH]QNB binding si es a e simila o speci ic muscannic ecep o s iden i ied in nume ous o he ne ous, muscula and glandula issues.
Compa isons o speci ic binding in no mal and ch onic CSN-dene a ed ca o id bodies sugges ha musea inic ecep o s a e absen on a -
e en e minals in he ca o id body; howe e , nea ly 50% o he speci ic [3H]QNB binding is los ollowing ch onic sympa hec omy, sug-
ges ing he p esence o p esynap ic musca inic ecep o s on he sympa he ic inne a ion supplying he ca o id body ascula u e. Au o adio-
g aphic s udies ha e localized he emainde o [3H]QNB binding si es o Iobules o ype I and ype II pa enchymal cells. In sepa a e
expe imen s, he musca inic agonis s, oxo emo ine (100/~M) and be hanechol (100 ~ M) we e shown o inhibi bo h he elease o ca e-
cholamines and he inc eased CSN ac i i y e oked by nico ine (50/~M) s imula ion o he in i o ca o id body, Ou da a sugges ha mus-
ca inic inhibi ion in he abbi ca o id body is media ed by ecep o s loca ed on ype I cells which a e able o modula e he exci a o y ac ions
o ace ylcholine a nico inic si es.
INTRODUCTION
The ca o id body is an a e ial chemosenso y o gan
whose pa enchyma consis s o wo dis inc cell ypes in
close associa ion wi h a dense capilla y ne wo k. Type I
(glomus) cells possess abundan dense and clea co ed
synap ic esicles, mi ochond ia and a highly conspicuous
ough endoplasmic e iculum. Specialized e minals
o med by a e en ibe s om he ca o id sinus ne e
(CSN) synap ically appose he ype I cells while slende
cy oplasmic p ocesses o ype II cells, whose cy ology
esembles ha o Schwann cells, en elop he a e en
e minal- ype I cell complex. P esumably, esponses in
he CSN p oduced by exposu e o he ca o id body o
hypoxia, hype capnia o low pH esul om he elease
o a ansmi e om ype I cells which exci es he ad-
jacen a e en ne e e minals.
Ea ly pha macological s udies by Eyzagui e and his
colleagues (see e . 14 o e iew) o he ac ions o ace-
ylcholine (ACh) in he mammalian ca o id body we e
pe o med p ima ily in he ca , and e ealed ha nico-
inic agonis s exci e he chemo ecep o s and po en ia e
he esponse o he na u al s imuli, hypoxia and hype -
capnia (see e . 14 o e iew). La e in es iga ions wi h
he ca ca o id body demons a ed he p esence o ACh ~'
~7. choline ace yl ans e ase 1'2's7 and ace ylcholines e -
ase 25'26 in he specialized lobules o ca o id body pa en-
chymal ( ype l) cells. The a e en ibe s inne a ing he
o gan, on he o he hand. we e shown o be nea ly de-
oid o choline gic ac i i y ~6 9. Concu en wi h many o
hese obse a ions, he Loewi- ype expe imen s o Eyz-
agui e and his colleagues H'I2 led hem o sugges ha
an 'ACh-like" subs ance was eleased as a senso y ans-
mi e om he ype I cells o he ca ca o id body o
ac on he synap ically apposed a e en ne e e minals.
Howe e , since he o mula ion o his choline gic hy-
po hesis o chemo ansmission, nume ous s udies (see
e . 14) ha e demons a ed he p esence and elease o
mul iple neu oac i e subs ances om he ype I cells o
ca . abbi and a ca o id bodies, including ca echola-
mines (dopamine and no epineph ine) and neu opep-
ides (subs ance P and Me -enkepha in). The p esen -
day concep ha has hus e ol ed en isions some o all
o hese neu oac i e agen s ac ing in conce o de e -
mine he ne senso y ou pu o he o gan. Amongs such
possible in e ac ions, e idence has accumula ed ha ACh
Co espondence: S.J. Fidone, Depa men o Physiology, Uni e si y o
Lake Ci y, UT 84108, U.S.A.
U ah School o Medicine. 410 Chipe a Way. Resea ch Pa k, Sail
elease migh be coupled o he elease o ca echol-
amines 24. Indeed, ecen biochemical and au o adio-
g aphic s udies ha e demons a ed ha nico inic (a-bun-
ga o oxin) binding si es a e p esen on he ca e-
cholamine-con aining ype I cells in he ca ca o id body 6,
and u he mo e, ha he nico ine- and hypoxia-e oked
elease o dopamine, and he accompanying inc ease in
senso y discha ge, a e subs an ially educed in he p es-
ence o 50 nM a-bunga o oxin ~'.
A uni ied in e p e a ion o hese da a has un o u-
na ely been con ounded by he mo e ecen indings ha
ACh is inhibi o y o chemo ecep o ac i i y in he
abbi
ca o id
body 8'29, in con as o i s well-known exci a o y
ac ions in he ca . Pha macological s udies demons a ed
ha he chemosenso y inhibi ion in he abbi is medi-
a ed by musca inic ecep o s ~'2~. Howe e , he loca ion
o hese ecep o s in he ca o id body has no been ex-
amined, and consequen ly he mechanism o musca inic
inhibi ion is unknown.
In he p esen s udy, we ha e used he musca inic an-
agonis , [3H]quinuclidinylbenzila e ([3H]QNB), o quan-
i y and localize musca inic ecep o s in he abbi ca-
o id body. Biochemical and au o adiog aphic da a
indica e ha speci ic binding o [3H]QNB in abbi ca-
o id body, like ha o ~-bunga o oxin in he ca ca-
o id body, is localized o lobules o ype I cells and is
absen om he a e en e minals o he ca o id sinus
ne e (CSN). Companion s udies u ilizing choline gic ag-
onis s and an agonis s demons a e ha musca inic e-
cep o s loca ed on ype I cells a e able o inhibi bo h
CSN discha ge and he elease o ca echolamines om
hese cells e oked by nico ine.
A p elimina y epo o some o he au o adiog aphic
obse a ions was published ea lie in his jou nal 7.
MATERIALS AND METHODS
Adul New Zealand whi e abbi s o bo h sexes we e used in
hese expe imen s. In addi ion o no mal, unope a ed (con ol) an-
imals, wo g oups o abbi s we e ch onically dene a ed 12-15
days p io o he expe imen s. Unde pen oba bi al anes hesia (40
mg/kg, i. .) and wi h ascp ic su ge y, he CSN we e esec ed bi-
la e ally in one g oup o 7 abbi s, while in a second g oup o 6,
he supe io ce ical ganglia (SCG) we e emo ed bila e ally along
wi h hei ganglioglome ula ne es o he ca o id body.
Binding assay
Me hodological de ails o he binding expe imen s ha e been
published p e iously 7. B ie ly, ca o id bodies we e apidly emo ed
om no mal o ch onically denc a ed animals and dissec ed ee
o encapsula ing connec i e issue in ice-cold 100% O2-equilib a ed,
modi ied Ty ode's solu ion (in mM: NaC1, 112; KCI, 4.7; CaCI 2,
2.2; MgCI 2, 1.1; sodium glu ama e, 42; HEPES, 5; pH 7.43 a
37 °C wi h glucose 1 mg/ml). We weigh s we e de e mined on a
Cahn elec obalance equipped wi h a humidi ied chambe o p e-
en d ying o he issues. The issue samples we e ans e ed o
glass scin illa ion ials con aining 1 ml o modi ied Ty ode's solu-
ion, placed in a wa e ba h-shakc (100 Hz) and p eincuba ed o
191
20 min a 37 °C in he p esence o absence o compe ing d ugs
(10 -1° o 10 3 M a opine, QNB, be hanechol o nico ine). A sub-
sequen 60-min incuba ion pe iod began wi h he addi ion o 100
/A o media con aining [3H]QNB (33.1 Ci/mmol; New England Nu-
clea ). Following incuba ion, he issue samples we e washed in he
shake ba h o 90 min in 2 ml o esh bu e a oom empe a-
u e. Fo de e mina ion o adioac i i y, issue samples we e co n-
bus ed in a Packa d Model 306 sample oxidize , solubilized in 15
ml o Oxi luo and coun ed in a Pa ka d Model 3385 scin illa ion
spec ome e .
A u o adiog aphy
The ca o id bi u ca ion wi h he ca o id body was quickly e-
mo ed om he animal, and he ascula u c lushed wi h 3 ml o
ice-cold oxygena ed bu e ia he common ca o id a e y. P ein-
cuba ion and incuba ion imes we e educed o l0 and 20 min, e-
spec i ely, o enhance his ological p ese a ion, and he no mal
wash media was eplaced wi h phospha e bu e ed 1% pa a o mal-
dehyde and 1% glu a aldehyde. Tissue samples we e pos - ixed o
1 h in 1% OsO 4, dehyd a ed in a g aded se ies o e hanols and em-
bedded in A aldi e. Semi hin sec ions (0.5-1.0/~m) we e moun ed
on gela in-subbed glass slides, dipped in a 1:1 aqueous dilu ion o
Kodak NTB-2 emulsion and s o ed wi h desiccan a 4 °C o 8
mon hs. Au o adiog aphs we e de eloped in Dek ol (2.5 min a
18 °C), s ained wi h me hylene blue and pho og aphed h ough a
Zeiss Uni e sal Mic oscope. Au o adiog aphic g ain densi y o e
pa enchymal cell lobules was de e mined in 2000x colo pho o-
g aphs in which he episcopically illumina ed sil e g ains appea ed
as unambiguous ambe do s o e lying he me hylene blue-s ained
issue. The pho og aphs we e numbe coded and e alua ed in a
'double-blind' pa adigm. The a eas o pa enchymal cell lobules
we e subsequen ly es ima ed by imming and weighing he pho o-
g aphs, each o which included one o wo cell lobules.
Release s udies
The e ec s o musca inic agen s on he elease o [3H]ca e-
cholamines and CNS discha ge we e de e mined acco ding o p e-
iously published me hods ~5. B ie ly, pai s o ca o id bodies we e
incuba ed o 2 h in 25/~M [3H] y osine (spec. ac . 40-50 Ci/mmol),
a e which he o gans we e moun ed in a iny pla inum wi e bas-
ke suspended wi hin a closed, empe a u e- and humidi y-con-
olled chambe . The CSN om one ca o id body was d awn up
in o an adjacen suc ion- ype elec ode. Wi h he p epa a ion in
place, he ca o id bodies we e di ec ly benea h an a ay o 22-gauge
hypode mic needles which we e connec ed h ough a pe is al ic
pump o ese oi s con aining unlabeled modi ied Ty ode's solu-
ion. The solu ions we e p ehea ed and gas-equilib a ed in he es-
e oi s; inal hea ing o he solu ions o 37 °C was accomplished
wi h a se ocon olled hea ing sys em. The empe a u e o he d op
su ounding he ca o id body was di ec ly moni o ed h ough a sec-
ond he mis o which was posi ioned o abu agains he baske .
The ela i e humidi y in he chambe was main ained a 100% by a
cons an low o gas, sa u a ed wi h wa e apo . Unde con ol and
s imulus condi ions, he supe usa es we e equilib a ed wi h 100%
0 2. The s imulus solu ions con ained nico ine (50/~M) ei he alone
o in he p esence o a musca inic agonis (be hanechol o oxo em-
o ine, 1(1-4 M), which was in oduced 10 min be o e s imula ion.
The olumes o supe usa e pe collec ion pe iod a ied less han
5%. The supe usion a e in all expe imen s was 0.4 ml/min and
was es ablished in p elimina y expe imen s which assessed he i-
abili y o he p epa a ion, judged on he basis o a s able chemo e-
cep o esponse o e a 5 h supe usion pe iod.
The ac ion po en ials om he CSN ne e we e led h ough an
AC-coupled p eampli ie o an oscilloscope and a magne ic ape
eco de . The ampli ied signals we e also led h ough a window
disc imina o o: (1) a equency- o- ol age con e e o inal dis-
play o he spon aneous neu al ac i i y on a cha eco de ; and
(2) a digi al coun e -p in e which ead he o al numbe o ne e
impulses eco ded du ing he collec ion pe iod.
The supe usa es we e collec ed in ials con aining a ca ie so-
192
lu ion consis ing o 0.3 M ace ic acid, 1 mM asco bic acid, a a inal
pH o 3.6. The solu ions we e p ocessed wi h he alumina adso p-
ion me hod, and [3H]ca eeholamines ([3I--I]CA) we e ba ch elu ed
om columns wi h 1 N pe chlo ic acid 38. Radioac i i y was de e -
mined in a Packa d Model 1500 liquid scin illa ion coun e .
In sepa a e expe imen s, [3H]CA elease was e alua ed in ca-
o id bodies incuba ed in supe usion media con ained in glass
ials, as desc ibed p e iously 21.
RESULTS
Time cou se o [3H]QNB associa ion and washou om
he issue
P elimina y expe imen s es ablished condi ions o
washou o unbound [3H]QNB om in ac ca o id bod-
ies. A e 45 min o incuba ion in 400 pM [3H]QNB, ca-
o id bodies we e washed o up o 2 h wi h con inuous
agi a ion in 2 ml o bu e a oom empe a u e. Speci ic
A
• -, 8.
E
.~.s.
m
?
B
• Non-Speci ic Binding
• o~ '
" ~
°'• • F u i n
100 200 300 400
[~H-QNB] pM
O75'
@
U.
.~50"
C
O
m
100- e~ I~ =71.46pM
• Bmsx=9.23pmol/g
25- •e •~
-- i i ~
i
2 4 6 8
3H-QNB Bound [pmol/g]
Fig. 1.A: equilib ium binding da a om abbi ca o id bodies in-
cuba ed o 45 ain in [3H]QNB a a ious concen a ions. Each
poin ep esen s ~'-+ S.E.M. o 3-7 ca o id bodies. To al binding
no shown. B: Sca cha d analysis o a single: popula ion o binding
si es es ima es a K d = 71.46 pM and a Bma x = 9.23 pmol/g o issue.
binding (displaceable by a opine, ! 0-6 M) emained s a-
ble du ing his inse pe iod, as migh be expec ed om
he epo ed high a ini y o [3H]QNB o musca inic e-
cep o si es 4'41'42. In subsequen expe imen s, ca o id
bodies we e washed o 90 min because e en ion o non-
speci ic binding was no educed wi h longe inse pe i-
ods.
Sepa a e expe imen s es ablished he associa ion ime
cou se o 150 pM [3H]QNB, whe e non-speci ic binding
in he p esence o
10 -6
M a opine equilib a ed apidly
and emained a a s able alue, accoun ing o less han
10% o o al binding o up o 60 min. Speci ic binding
( o al binding minus non-speci ic binding) ose sha ply
du ing he ini ial 30 min and hen pla eaued. Subsequen
equilib ium s udies u ilized 45 min incuba ion pe iods in
o de o achie e s eady-s a e condi ions wi h lowe con-
cen a ions (<150 pM) o [3H]QNB.
Equilib ium kine ics and pha macology o [k~H]QNB
binding
Speci ic [3H]QNB binding in abbi ca o id body is
cha ac e ized by high a ini y and sa u abili Y. Fig. A
shows speci ic and non-speci ic binding a [3H]QNB con-
cen a ions be ween 20 and 390 pM. Speci ic binding
ose sha ply a low concen a ions o [3H]QNB and pla'
eaued a concen a ions abo e 200 pM. Non-speci ic
binding in he p esence o 10 -~' M a opine ne e ex-
ceeded 10% o o al binding, and inc eased linea ly o e
he concen a ion ange o [3H]QNB. In Fig. 1B, a Sca -
cha d analysis o he binding da a sugges s he exis ence
o a homogeneous popula ion o binding si es wi h a K d
o 71.46 pM and a B
.....
o 9.23 pmOl/g o ca o id body
issue. These K d alues a e simila o hose o [~H]QNB
binding o musca inic ecep o s in mammalian b ain (Kd
M 4
= 60 pM41), sympa he ic ganglion (K d = 830 p ) and
ileum (K, = 30 pM42).
120
T • A opine
100 • Bo hlnochol
O
Nico ine
G-
0
10 9 8 7 6 5 4 3
.Log [D ug], M
Fig. 2. Inhibi ion o [3HJQNB binding. Compelling d ugs we e in-
oduced in o he supe usion media 20 min p io O :inCuba ion in
[3H]QNB (150 pM). Incuba ion con inued wi h each compe ing
d ug o 45 min ollowed by a 90 min wash in esh media. Each
poin ep esen s da a (X -+ S.E.M.) om 4-6 ca o id bodies.
Compe i i e inhibi ion s udies o [3H]QNB binding
u he con i med he musca inic na u e o he binding
si e. Fig. 2 illus a es he ela i e po ency o 4 compe -
ing d ugs. The musca inic an agonis s, QNB and a o-
pine, we e po en inhibi o s o [3H]QNB binding in he
ca o id body, while he agonis , be hanechol, signi i-
can ly educed binding only a concen a ions abo e 10 -5
M. Nico ine also signi ican ly dec eased binding a e y
high concen a ions (10 -3 M), bu was ine ec i e a 10 -4
and 10 -5 . The o de o po ency o hese compe ing d ugs
o he [3H]QNB binding si e is simila o ha demon-
s a ed in o he s uc u es whe e [3H]QNB binds wi h
high a ini y o speci ic
musca inic
ecep o s 4'23'4~'42.
Localiza ion o speci ic [~H]QNB binding si es
As men ioned ea lie , he pu a i e chemosenso y is-
sue o he mammalian ca o id body consis s o lobules
o ype I (glomus) and ype II (sus en acula ) cells, o-
ge he wi h he associa ed senso y ne e e minals. In
addi ion, he o gan also ecei es a signi ican sympa-
he ic inne a ion om he nea by SCG. In o de o dis-
inguish musca inic binding si es on pa enchymal cells
e sus a e en o au onomic ne e e minals, we exam-
ined [3H]QNB binding in ca o id bodies ollowing
ch onic sec ion o he CSN o emo al o he SCG.
Fig. 3 shows ha 12-15 days ollowing esec ion o he
CSN, he numbe o [3H]QNB binding si es is unchanged
om no mal unope a ed issue. In con as , a e e-
mo al o he SCG and degene a ion o he sympa he ic
e minals in he ca o id body, [3H]QNB binding is e-
duced by 46%. These da a sugges ha musca inic e-
cep o s a e absen om CSN a e en ibe s and e mi-
nals, bu ha sympa he ic axons con ain nea ly one-hal
o ca o id body musca inic si es. An impo an ques ion
is whe he he emaining [3H]QNB binding ollowing
sympa hec omy is associa ed wi h he lobules o ype I
and ype I1 pa enchymal cells. The au o adiog aphic lo-
caliza ion o musca inic binding si es was examined in
ca o id bodies a e incuba ion in 400 pM [3H]QNB. Fig.
4A,B p esen s ypical au o adiog aphs showing sil e
g ain dis ibu ions o e pa enchymal cells in wo no mal
ca o id body Iobules. In hese p epa a ions, sil e g ains
a e clea ly associa ed wi h he lobules o ype I and ype
11 cells. In he p esence o 10 6 M a opine (Fig. 4C,
no mal), g ains a e spa se o e he issue.
Quan i ica ion o he g ain densi y o e lying lobules
o ype I and ype II cells om no mal, CSN-dene a ed
and sympa hec omized issue samples con i med he da a
om ou binding expe imen s (Fig. 5). Unde all 3 con-
di ions, issues incuba ed wi h 10 -~' M a opine (non-spe-
ci ic binding) displayed uni o mly low g ain coun s. Sim-
ila ly low 'backg ound' coun s we e measu ed o e he
lumen o blood essels (3.81 x 10 -3 -+ 0.89 × 10 3
193
g ains//~m2; no shown in he igu e). Non-speci ic
[3H]QNB binding in he au o adiog aphs was he e o e
minimal, which ag ees wi h ou binding da a. In he ab-
sence o a opine (i.e. o al binding), g ain coun s we e
signi ican ly highe in all p epa a ions (P < 0.005), e-
lec ing speci ic [3H]QNB binding. These quan i a i e
au o adiog aphic da a sugges ha speci ic binding wi hin
pa enchymal cell lobules was unchanged ollowing ei he
ch onic CSN dene a ion o ch onic emo al o he SCG
(i.e. o al binding was no signi ican ly di e en o he
3 condi ions, P > 0.10). Thus, degene a ion o he a -
e en o sympa he ic inne a ion o he ca o id body
does no al e he densi y o [3H]QNB binding si es
wi hin he pa enchymal cell lobules.
The e ec s o musca inic agen s on ca echolamine elease
and CSN discha ge
The e ec s o oxo emo ine on ca echolamine elease
and chemo ecep o discha ge o he CSN we e es ed
using ca o id bodies p eloaded wi h [3H]CA syn hesized
om [3H] y osine (see Ma e ials and Me hods). The o -
gans wi h hei a ached ne es we e moun ed in he in
i o eco ding chambe , and supe used wi h 100% 0 2-
equilib a ed media o 30-40 min p io o s imula ion.
[3H]CA elease and CSN discha ge we e hen moni o ed
du ing sequen ial 5 min p es imulus, s imulus and pos -
s imulus collec ion pe iods, which oge he comp ised a
single 's imulus cycle'. Following a 'con ol' nico ine-
100
m
z
?
e'-
°m
e-
Ill 50
m
E
0
z
T
No mal CSN Sympa h.
Dene .
Fig. 3. Speci ic [3H]QNB binding (X ± S.E.M.) in no mal ca o id
bodies and ollowing ch onic (12-15 days) esec ion o he ca o id
sinus ne e (CSN Dene .) o emo al o he supe io ce ical
ganglion (Sympa h.). Binding o each g oup was de e mined un-
de equilib ium condi ions (45 min incuba ion) in he p esence o
200 pM [3H]QNB. Each ba ep esen s da a om a leas 4 pai s
o ca o id bodies incuba ed in he p esence o absence o 1 k M a -
opine. *P < 0.0005 s no mal g oup.
194
Fig. 4. Au o adiog aphic dis ibu ion o [3H]QNB binding in Iobules o ype I and ype 1I cells in abbi ca o id bodies; (A and B) no mal.
(C) no mal ca o id body incuba ed in he p esence o 1 gM a opine. Au o adiog aphic g ains appea as black punc a e do s. Scale ba = 20
um.

1° 1
E
P
13
T
[~] To al Binding
Non-Speci ic Binding
E~ Speci ic Binding
No mal CSN-Dene . Sympa h.
Fig. 5. Quan i a i e au o adiog aphic da a (X _+ S.E.M.) o
[-~HJQNB binding in Iobules o ype I and ype II cells in no mal,
CSN-dene a ed (12-15 days) and sympa hec omized (12-15 days)
ca o id bodies. Sil e g ain densi y was de e mined in ca o id bod-
ies incuba ed in he p esence (non-speci ic binding) o absence ( o-
al binding) o 1 ,uM a opine. *P < 0.0005 s o al binding. See
ex o me hodological de ails.
s imulus cycle, he p epa a ion was supe used o 5 min
wi h 100% 0 2 media con aining
10 -4
M oxo emo ine,
and hen e-exposed o nico ine (50 #M) in he p esence
o he musca inic d ug. The da a in Fig. 6 show [3H]CA
elease and CSN discha ge exp essed as esponse a ios,
i.e. (s imulus minus con ol)/con ol, whe e a alue o 1
would indica e a 100% inc ease in elease (see e . 15).
Nico ine, in he absence o musca inic d ugs, inc eased
[3H]CA elease by 125%. Reco dings o CSN ac i i y
likewise showed ha nico ine e oked la ge inc eases in
2.0
°!o 1.5
n- 1.0
O
Q.
0.5
n-
0.0
T
(7)
3H-Release
[~ 50 IM Nico ine
50~ M Nico ine+
100HM Oxo emo lne
I
I
(6i
Peak A e age
CSN Discha ge
Fig. 6. E ec s o oxo emo ine (I(X)/~M) on he nico ine (50 ~uM)
e oked elease o [3HJCA and CSN discha ge in he abbi ca o id
body. The da a a e exp essed as esponse a ios
[(S-C)/C],
whe e
S ep esen s he [3H]CA eleased, o he ne e ac i i y, measu ed
du ing he s imulus pe iod; C is he con ol alue de e mined du -
ing an equi alen ime pe iod immedia ely p io o he s imulus.
Sec e . 15 o u he explana ion. CSN discha ge was e alua ed
bo h o i s peak esponse, as well as he a e aged ac i i y o e
he 5- ain s imulus pe iod. Values in pa en heses equal numbe o
measu emen s. *P < 0.01 compa ed o he da a ob ained wi h nic-
o ine alone.
195
2.0!
~
1.5
.o
c¢ 1.o
¢D
0
a.
I~ 0.5
Nico ine (50pM)
(4) [7"/'/] Nlco lna (50pM)+
~__ Be hanechol (IOOpM)
(4)
(4)
Peak A e age
Discha ge
Fig. 7. E ec o be hanechol (10 4 M) on CSN ac i i y e oked by
nico ine (50/~M). *P < 0.05 compa ed o esponse e oked by nic-
o ine alone. O he de ails as in Fig. 6.
bo h peak and a e age CSN discha ge du ing he 5 min
s imulus pe iod. In con as o hese nico inic e ec s,
oxo emo ine did no al e basal [3H]CA elease and
CSN discha ge. Howe e , in he p esence o oxo emo-
ine, [3H]CA elease and CSN discha ge e oked by nic-
o ine (50/~M) we e signi ican ly educed; he esponse
o nico ine e u ned o nea con ol alues a e a 1 h
wash wi h Ty ode's solu ion (100% O2-equilib a ed ). In
o he expe imen s which e alua ed d ug e ec s only on
ne e ac i i y (Fig. 7), he musca inic agonis be hane-
chol was shown o inhibi he nico ine-e oked inc ease
in CSN discha ge (P < 0.05).
The e ec s o be hanechol on he nico ine-e oked e-
0
O
o,9,
0.60
0.50 -
0.40 -
0.30 -
0.20-
0.10-
0.00
I---7 Nico ine (50~M)
(4) ~ Nico ine (50~M)+
ae hanechol (100pM)
(6)
---- 3H-CA Release --
Fig. 8. E ec o be hanechol (10 -4 M) on [3H]CA elease e oked
by nico ine (50/~M) in abbi ca o id bodies supe used in glass i-
als. Values in pa en heses equal numbe o obse a ions. *P <
0.025 compa ed o [3H]CA elease e oked by nico ine alone. Re-
sponse a io explained in legend o Fig. 6.
196
lease o [3H]CA was examined in a sligh ly di e en ex-
pe imen al p o ocol, in which ca o id bodies we e supe -
used in glass ials. The o gans we e s imula ed wi h
nico ine (50/~M) in he p esence o absence o his mus-
ca inic agonis . Fig. 8 shows ha
be hanechol (10 -4
M)
inhibi ed he nico ine-e oked elease o [3H]CA (ex-
p essed as a esponse a io) by mo e han 60%, hus
pa alleling ou esul s o he nico ine-e oked CSN ac-
i i y ob ained wi h he supe usion- eco ding chambe .
Collec i ely, hese da a clea ly demons a e ha musca-
inic agonis s can nega i ely modula e nico inic ac ions
in he abbi ca o id body.
DISCUSSION
In he p esen s udy, we ha e a emp ed o localize
musca inic ecep o s in he abbi ca o id body, and o
examine hei unc ional ela ionship o he physiologi-
cal esponse o his a e ial chemo ecep o o gan. Ou
p incipal indings sugges ha musca inic ecep o s a e
loca ed on he specialized ype I pa enchymal cells, and
ha hey modula e he nico ine-e oked elease o ca e-
cholamines om hese cells.
The equilib ium binding da a indica e ha [3H]QNB
associa es wi h a single popula ion o binding si es in he
ca o id body wi h a
K a
o 71.46 pM, which is simila o
he high a ini y binding o [3H]QNB o musca inic e-
cep o s in b ain 4x, sympa he ic ganglion 4, ileum 42,
hea 23, co ona y a e y 39, and ad enal medulla 31. Mo e-
o e , he o de o po ency o compe ing d ugs o he
[3H]QNB binding si es is consis en wi h he pha macol-
ogy o musca inic ecep o s in hese o he issues, whe e
speci ic an agonis s (QNB and a opine) possess a high
a ini y o he ecep o , in con as o he low a ini y
displayed by agonis s such as be hanecho123"4°-42. Al-
hough we ha e no pe o med addi ional binding expe -
imen s using pi enzepine o de ine he musca inic ecep-
o sub ype 3"9"z2"27, we ha e ound in p elimina y
expe imen s ha be hanechol educes he inc ease o
cAMP p oduced by o skolin in he ca o id body, sug-
ges ing a nega i e coupling o musca inic ecep o s o
adenyla e cyclase; in o he s uc u es nega i e coupling
occu s ia M e o M 3 musca inic ecep o s 2°'3°.
Because chemosenso y ansdue ion is hough o oc-
cu wi hin he lobules o he ype I and ype II pa en-
chymal cells (see e . 14), ou expe imen s we e de-
signed o elucida e he loca ion o pu a i e musca inic
ecep o s associa ed wi h hese lobules. Senso y ibe s
om he CSN con ibu e an impo an unc ional ele-
men o he chemo ecep o appa a us, namely he spe-
cialized e minals which o m synap ic apposi ions wi h
he ype I cells 18"33. In ou expe imen s, CSN-dene a-
ion did no educe he amoun o speci ic [3H]QNB
binding in abbi ca o id bodies, and a quan i a i e au-
o adiog aphic analysis o binding wi hin he pa enChy-
mal cell lobules likewise ailed o e eal changes in bind-
mg a e CSN degene a ion. These indings imply ha
musca inic ecep o s a e absen om he senso y ibe s
and hei e minals. Howe e . because hese ne e e -
minals ep esen less han 5% o he o al olume o he
ca o id body 32. a small educ ion in [3H]QNB binding
associa ed wi h CSN-dene a ion migh no ha e been
de ec ed in ou expe imen s. While his emains a pos-
sibili y, i is pe haps no consis en wi h he obse ed
46% educ ion in [3H]QNB binding ollowing degene a-
ion o sympa he ic ibe s o he ca o id body, which
likely also ep esen a simila ly small olume o issue.
The 46% educ ion o [3H]QNB binding in abbi ca-
o id body a e emo al o he SCG can be a ibu ed
o he loss o p esynap ic musca inic ecep o s associa ed
wi h he axon e minals o pos ganglionic sympa he ic
neu ons. In o he sympa he ically inne a ed issues, he
p esence o p esynap ic musca inic ecep o s was shown
in pha macological expe imen s which demons a ed he
abili y o musca inic agen s o inhibi [3H]no epineph-
ine elease 3~. While ou biochemical expe imen s show
a p o ound educ ion in o al o gan binding ollowing
sympa hec omy, ou au o adiog aphic da a om sym-
pa hec omized ca o id bodies sugges ha binding ~s un-
changed wi hin lobules o ype I and ype II cells. This
obse a ion is consis en wi h mo phological s udies
which demons a e ha sympa he ic axons a ely e mi-
na e wi hin he pa enchymal cell obules o he ca o id
body 3(~. bu ins ead o m abundan ne e endings on he
smoo h muscle o he ca o id body ascula u eL°'2s 3~,.
The unc ion o musca mic ecep o s in he ca o id
body can be in e ed om ou da a showing ha mus-
ca inic agen s inhibi he ele lsc o [3H]CA and CSN
discha ge e oked by nico ine Ea lie s udies by Mon l-
Bloch and Eyzagui e 2~ and by Doche y and McQueen s
showed ha musca mic d ugs q ACh and piloca pine) in-
hibi chemosenso y uni s o he CSN. Likewise. ou neu-
ochemical and elec ophysiological da a clea ly demon-
s a e ha musca inic ecep o s a e nega i ely coupled o
nico inic exci a ion o he abbi ca o id bod3.
The mechanism o in e ac ion o nico inic and musca-
inic ecep o s in he ca o id body is unknown. In many
o he species, bo h nico inic and musca inic agen s inde-
penden ly e oke he elease o CA om he ad enal me-
dulla. Howe e . in bo ine ad enal ch oma in cells, i
has been epo ed ha musca inic agen s inhibi CA e-
lease e oked by nico ine ~'3~. In his species, musca inic
agen s ac i a e guanyla e cyclase, hus inc easing he le -
els o cyclic GMP in medulla y cel s 43. Fu he mo e,
cGMP analogs and phosphodies e ase inhibi o s mimick
he musca inic e ec by dec easing CA elease in he
197
nico ine. These indings sugges ha mus-
p esence o " . s
ca inic ac ions which a e nega i ely coupled o nico inic
e ec s may be media ed by classical second messenge
sys ems and may in ol e p o ein phospho yla ion. In-
deed, i has been es ablished ha phospho yla ion o
nico inic ecep o s by a a ie y o p o ein kinases accel-
e a es ecep o desensi iza ion (see e . 34). P elimina y
expe imen s in ou labo a o y ha e in ac shown ha
sodium ni op usside (1.0 aM), an ac i a o o soluble
guanyla e cyclase, dep esses he CA elease e oked om
he abbi ca o id body by nico ine (50 k M).
The physiological ole o ACh and nico inic/musca -
inic ecep o s in he mechanisms o chemo ecep ion e-
main ill unde s ood, howe e . Ea ly expe imen s by
Eyzagui e and his collabo a o s demons a ed ha an
ACh-like subs ance is eleased om he ca o id body
du ing na u al s imula ion, mos p obably om he ype
I cells o he chemosenso y lobules ~'~2. The indings in
he p esen s udy sugges ha ACh eleased by hese
cells may ac a nico inic au o ecep o s o enhance CA
elease, while musca inic au o ecep o ac i a ion modu-
la es hese e ec s. Consis en wi h his schema a e p e-
limina y obse a ions in ou labo a o y which show ha
be hanechol (10 4 M) can inhibi he inc ease in [3H]CA
elease and CSN discha ge e oked by hypoxic s imula-
ion ~-~.
In summa y, a s udy o [3H]QNB binding in abbi
ca o id body has e ealed ha he ype I cells possess
speci ic musca inic ecep o s. Musca inic ecep o s a e
also associa ed wi h he sympa he ic inne a ion o he
o gan, while in con as , hey appea o be absen om
he chemosenso y CSN e minals. While he p ecise ole
o musca inic ecep o s in he chemo ecep o esponse
o na u al s imuli is no ully unde s ood, s udies o hei
e ec s on nico ine e oked CA eleased and CSN dis-
cha ge sugges ha musca inic ecep o s a e able o
modula e he ac ions o ACh a nico inic si es in he
abbi ca o id body.
Acknowledgemen s.
Suppo ed by USPHS G an s NS12636 and
NS07938, and DGICYT G an PB89/0358 (Spain).
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