Muscarinic receptor localization and function in rabbit carotid body

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. 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