Localization and Function of Cat Carotid Body Nicotinic Receptors

B ain Resea ch, 339 (1985) 295- 304 295 Else ie BRE 10874 Localiza ion and Func ion o Ca Ca o id Body Nico inic Recep o s B. DINGER, C. GONZALEZ*, K. YOSHIZAKI** and S. FIDONE Depa men o Physiology, Uni e si y o U ah School o Medicine, Sal Lake Ci y, UT84108 (U.S.A. (Accep ed Oc obe 23 d, 1984) Key wo ds: ace ylcholine -- a-bunga o oxin -- au o adiog aphy -- ca o id body -- dopamine elease -- nico inic ecep o s Ace ylcholine and nico inic agen s exci e ca ca o id body chemo ecep o s and modi y hei esponse o na u al s imuli. The p esen expe imen s u ilized [125I]a-bunga o oxin ([125I]a-BGT) o localize wi hin he chemosenso y issue he possible si es o ac ion o exog- enous and endogenous nico inic choline gic subs ances. In i o equilib ium binding s udies o in ac ca o id bodies de e mined a K d o 5.57 nM and a Bma x o 9.21 pmol/g o issue. Ch onic sec ion (12-15 days) o he ca o id sinus ne e (CSN) did no change he amoun o displaceable oxin binding. In con as , he speci ic binding was educed by 46% ollowing emo al o he supe io ce ical gangli- on. Ligh mic oscope au o adiog aphy o no mal, CSN-dene a ed and sympa hec omized ca o id bodies e ealed displaceable bind- ing si es concen a ed in lobules o ype I and ype II cells. T ea men o ca o id bodies wi h 50 nM a-BGT in i o educed by 50% he elease o [3H]dopamine (syn hesized om [3H] y osine) caused by hypoxia o nico ine, and also signi ican ly educed he s imulus-. e oked discha ges eco ded om he CSN. The da a sugges (1) an absence o c -BGT binding si es on he a e en e minals o he CSN and (2) ha nico inic ecep o s loca ed wi hin pa enchymal cell lobules may modula e he elease o ca echolamines om hese cells. 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 Schwann cells, en elop he a e en e minal-glomus cell com- plex. In 1936, Heymans e al. 34 we e he i s o obse e ha small in aca o id doses o ace ylcholine (ACh) s imula ed espi a ion in a manne simila o na u al s imula ion o he ca o id body. In he ensuing dec- ades, nume ous s udies in es iga ed he ole o ACh in chemosensa ion and demons a ed ha small quan i ies o choline gic agonis s and an agonis s p o oundly al e ed chemo ecep o discha ge 20. Neu- ochemical in es iga ions indica ed ha he me abol- ic machine y necessa y o ACh syn hesis, s o age and inac i a ion appea o be associa ed wi h ype I cells in he ca o id body 2,3,23,24,31,35,36 and ha a e - en ibe s o he CSN a e nea ly de oid o choline gic ac i i y23, 28. In addi ion, i has been demons a ed ha an ACh-like subs ance is eleased upon s imula- ion o he o gan in i ol7, Is. While hese da a suppo he no ion ha ACh may play a ole in ca o id body chemo ecep ion, he p e- cise ac ions o his subs ance in he senso y p ocess has been elusi e, pa icula ly wi h espec o i s pu a- i e ac ion as a media o o ansmi e o chemosen- so y in o ma ion om he ype I cells o he a e en ibe s o he CSN. Disag eemen abou he unc ion o ACh s ems om he obse a ions ha (1) an icho- * P esen add ess: Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Valladolid, Spain. ** P esen add ess: Aki a Uni e si y School o Medicine, 1-1-1, Hondo, Aki a 010, Japan. Co espondence: S. J. Fidone, Depa men o Physiology, Uni e si y o U ah School o Medicine, 410 Chipe a Way, Resea ch Pa k, Sal Lake Ci y, U ah 84108, U.S.A. 96 line gic agen s comple ely block he e ec s o exoge- nously applied ACh, bu no he esponse o na u al s imuli 43.45,5I (bu see also e . 49); and (2) exoge- nous ACh enhances chemosenso y discha ge in he ca 17,e2 while simila doses in he abbi p oduce inhi- bi ion o discha ge 13.a¢'. A undamen al p oblem ega ding he ac ions o ACh in he ca o id body has been he unce ain y abou he locus o i s speci ic e ec s on ype 1 cells s a e en ne e e minals. P e ious a emp s o dem- ons a e pha macologically he chemosenso y in- ol emen o ACh ha e no been con incing, since nei he he si es o ac ion o he applied d ugs, no he nico inic s musca inic na u e o he choline gic ecep o s we e clea ly di e en ia ed. In he p esen s udy, we ha e se ou o cha ac e ize and localize nico inic ecep o s in he ca ca o id body using he labeled nico inic an agonis , [~251]a-bunga o oxin @2-q]a-BGT). We desc ibe he e he equilib ium ki- ne ics o oxin binding in no mal ca ca o id body, and he use o au o adiog aphic and biochemical echniques o assay oxin binding ollowing ch onic sec ion o he CSN o emo al o he supe io ce i- cal ganglion. In addi ion, he esul s o physiological and pha macological expe imen s indica e a unc- ional ole o nico inic ecep o s in modula ing he elease o ca echolamines om ype I cells. A p elim- ina y epo o hese indings has al eady been pub- lished 12. MATERIALS AND METHODS Th ee g oups o adul domes ic ca s o bo h sexes weighing 2.5-4.0 kg we e used in his s udy, In one g oup, he CSNs we e bila e ally esec ed 12-15 days p io o he binding assay. In a second g oup, he supe io ce ical ganglia, which p o ide he sym- pa he ic inne a ion o he ca o id body ascula u e, we e su gically emo ed also a 12-15 days p io o he binding assay. All su gical esec ion p ocedu es we e pe o med asep ically unde pen oba bi al an- es hesia. A hi d g oup o ca s consis ed o no mal unope a ed animals. Binding assay Ca o id bodies we e emo ed om anes he ized animals and cleaned o su ounding connec i e issue in a luci e chambe illed wi h O2-equilib a ed, ood- i ied Ty ode's solu ion (in mM: NaCI ! 2, KCI 4.7, CaC1 e 2.2, MgC1 e 1.1, Na glu ama e 42~ lEPES 5; pH 7.43 a 37 °C). We weigh was de e mined on a Cahn elec obalance i ed wi h a humidi ied chain. be o p e en d ying o ile issues. Tissue samples we e placed in ials con aining 1.5 m o~ ()2-ly ode's solu ion plus 1% bo ine se um albumin, and we e p eincuba ed in a wa e ba h shake a 37 °C m he p esence o absence o ei he D- ubocu a ine (D-T(': 10 -6 o 10 -3 M; Sigma), o ACh (1() -~' o I!) .~ M: Sig- ma) plus ese ine (10 -5 M; Sigma). Following 20 min o p eincuba ion, [ 25I]a-BGT (spec. ac . 138 Ci/m- mol, New England Nuclea ) was added o he ials o each inal concen a ions o om 1.0 o 45.0 nM. In- cuba ion con inued a 37 °C, a e which he issues we e washed in 10 ml o ice-cold Ty ode's solu ion. Fo biochemical analyses, issue samples we e di- ges ed o 4 h a 60 °C in a mix u e o 200/~ NCS (Ame sham) and 50/zl wa e . P io o coun ing in a liquid scin illa ion spec ome e , he diges ion mix- u e was neu alized wi h 750/A o ace ic acid (1.3%), and 15 ml o coun ing cock ail (PCSII, Ame sham) we e added o each ial. Speci ic binding was de ined as he binding in he absence o ei he 13- TC o ACh, less he binding in he p esence o hese subs ances. The da a a e exp essed as pmol adiola- beled-ligand bound/g o issue (_+ S.E.M. }, and e al- ua ed using S uden 's - es . A u o adiog aphy [125I]a-BGT issue samples o au o adiog aphy we e emo ed om he wash media, imme sed in a 0.1 M phospha e-bu e ed ixa i e (pH 7,6) con- aining 1% glu a aldehyde, 1% pa a o maldehyde and 0.01 M CaC12, and hen pos ixed in bu e ed 2% osmic acid solu ion, dehyd a ed in e hanol, and em- bedded in Epon. Semi hin sec ions we e cu , moun ed on glass slides and coa ed wi h Kodak NTB- 2 emulsion using a cons an a e wi hd awal appa a us (48 mm/min). Au o adiog aphs we e exposed o 6-7 weeks, de eloped in Dek ol, and s ained wi h me h- ylene blue. Release s udies The e ec s o a-BGT on he elease o [3H]dopa- mine and CSN discha ge we e de e mined acco ding o p e iously published me hods 21. B ie ly summa- ized, he ca o id body was incuba ed o 2-4 h in 40/~M [3H] y osine (spec. ac . 20-40 Ci/mmol), a e which he o gan was moun ed in a iny pla inum wi e baske suspended wi hin a closed, empe a u e- and humidi y-con olled chambe . The CSN 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 body was di ec ly benea h an a ay o 22-gauge hypode mic needles which we e connec ed h ough olle - ype pumps (Technicon) o ese oi s con aining unlabelled modi ied Ty ode solu ion. The solu ions we e p e- hea ed and gas-equilib a ed in he ese oi s; inal hea ing o he solu ions o 37 °C was accomplished wi h a se o-con 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 second 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 . This appa a us pe mi ed a choice o 4 solu- ions, and he same gas was used o humidi y he chambe and o equilib a e he chosen solu ion. Un- de con ol condi ions he supe usa e was equili- b a ed wi h 100% O 2, while s imulus solu ions in- cluded 10 -5 M nico ine (equilib a ed wi h 100% O2), o supe usa e equilib a ed wi h 30% 02. The d op a e om he needles was con olled by he speed o he pump, and he p ecise numbe o d ops ha ell om he ca o id body was moni o ed by means o pho ocells moun ed in he opening a he bo om o he chambe . 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 ab- lished in p elimina y expe imen s which assessed he iabili y o he p epa a ion, judged on he basis o a s able chemo ecep o esponse o e a 5 h supe u- sion 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 oscillo- scope 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 e- co 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 solu ion consis ing o 0.3 M ace ic acid, 1 mM asco bic acid and 100 ~M unlabeled DA, a a 297 inal pH o 3.6. The solu ions we e p ocessed wi h he alumina adso p ion me hod o eco e y o [3H]ca echolamines, as desc ibed by C ou 11. A e elu ion o [3H]DA wi h 0.3 M ace ic acid ( eco e y 65 _+ 5%), u he elu ion wi h 1 M HCI pe mi ed he eco e y o dihyd oxyphenyl ace ic acid (DO- PAC)25, 55, he p incipal DA ca aboli e in he ca o id body 29. The elua e o 0.3 M ace ic acid was u he analyzed using high- ol age pape elec opho esis; he elua e con aining [3H]DOPAC was di ec ly coun ed in a liquid scin illa ion spec ome e . [3H]DA elease is exp essed as he sum o [3H]DA and [3H]DOPAC. RESULTS Washou and ime cou se Ou ini ial expe imen s we e designed o de e - mine whe he p olonged washing would dissocia e signi ican amoun s o bound [ 125I] a-B GT om in ac (non-homogenized) samples o ca o id body issue. Ca o id bodies we e incuba ed o 3 h in 100 nM [125I]a-BGT in he absence o D-TC o ACh. The is- sue was hen ans e ed o wash ials con aining 10 ml o ice-cold Ty ode's solu ion. A e 20 h o washing, he le el o oxin binding was indis inguish- able om ha p esen a e 1, 2 o 6 h o washing. All subsequen expe imen s u ilized a 1 h wash pe i- od. Unde hese cohdi ions, non-speci ic binding ne e exceeded 35% o o al binding in he no mal ca o id body. The ime cou se o [lZSI]a-BGT binding is shown in 8 (n o c~ ~6 ..Q 4 0 E i j ~o do 1~o 2~o minu es Fig. 1. Associa ion ime cou se o [125I]a-BGT wi h he ca o id body. 0, o al binding; II, non-speci ic binding in he p esence o 10 -3 M ACh + 10 -5 M ese ine; A, speci ic binding. 298 • To al Binding A / • Nonspe¢i ic Binding ,-, 1.o .o • ~,_ 0.5 | lb 2'5 4'5 [1251"ec'lSl] (nM) B 2 4 6 8 1251-o,-86T Bound Fig. 2. A: concen a ion dependence o o al, non-speci ic and speci ic [125I]a-BGT binding in ca ca o id body. B: Sca cha d analysis o speci ic binding da a; K d = 5.57 nM, Bma x = 9.21 pmol/g o issue, = 0.951, P < 0.01. Fig. 1. A a concen a ion o 11.25 nM, displaceable binding (i.e. binding in he p esence o 10 -3 M ACh) pla eaued a app oxima ely 30 min and emained cons an o incuba ion imes up o 240 min. A lowe concen a ions o ACh o D-TC (10 -4 M), non-spe- ci ic binding con inued o inc ease wi h incuba ion imes longe han 1 h. This inding p obably e lec s a g adual inc ease in speci ic oxin binding, as has been shown o occu when he binding o a nea ly i e e si- ble ligand is blocked wi h d ugs ha bind e e sib- ly ]°. A 10 -3 M ACh o D-TC, non-speci ic binding was always signi ican ly less han o al binding (P < 0.005). In subsequen equilib ium s udies, issue samples we e incuba ed o 30 min, a e he ini ial 20 min p eincuba ion pe iod wi h o wi hou a blocking d ug. A [125I]a-BGT concen a ions below 11.25 nM, howe e , equilib ium may no ha e been a ained du ing he 30 min incuba ions, hus esul ing in small o e es ima es o he Ka. TABLE I Inhibi ion o [12s]a-BG T binding D ug Amoun bound (pmol/g) ++_ S. E. M. concen a ion ............................ A Ch l)- Tubocu a ine Con ol 7.64 + 0.52 7.64 + 0.52 10 -6 M 7.79 + 1.26 7.63 + 1.09 10 -5 M 5.50 _+ 0.88 4.61 _+ 0.42 10-4M 2.13 + 0.49 2.15 + 0.11 10-3M 1.20_+ 0.15 1.23 + 0.11 Concen a ion dependence and inhibi ion o binding Equilib ium kine ic s udies o [x25I]a-BGT binding in no mal ca ca o id bodies demons a ed a sa u- able componen which was displaceable by 10 -3 M ACh (Fig. 2A) o D-TC. Speci ic binding pla eaued a concen a ions abo e 11.25 nM oxin. The speci ic binding da a in he igu e a e i o a ec angula hy- pe bola by a 'bes - i ' compu e me hod. Non-speci - ic binding appea ed o be linea h oughou he con- cen a ion ange and ne e exceeded 27% o o al binding. The linea Sca cha d analysis p esen ed in Fig. 2B indica es a single popula ion o binding si es wi h a K~ o 5.57 nM and a Bma x o 9.21 pmol/g o issue ( = 0.951, P < 0.01). The Hill coe icien o he da a is 0.876, sugges ing non-coope a i e in e ac ion be- ween labeled oxin and he speci ic binding si es. Inhibi ion s udies showed ha pzsI]a-BGT binding was p og essi ely educed by inc easing concen a- ions o ei he D-TC o ACh (Table I). A concen a- ions be ween 10 -6 M and 10 -3 M, he wo d ugs we e simila in hei abili y o p e en oxin binding. In he p esence o 11.25 nM [125I]a-BGT, millimola concen a ions o D-TC o ACh educed he amoun o bound adioac i i y by 90%. E ec s o ca o id sinus ne e (CSN) dene a ion and sympa hec omy on [125I[a-BGT binding CSN-dene a ed and sympa hec omized ca o id 15] ~'~ TOTAL BINDING ~ NON-SPECIFIC BINDING ! IS E No mal nene a ed Sympa hec omized Fig. 3. [125I]c -BGT binding in no mal, CSN-dene a ed and sympa hec omized ( emo al o supe io ce ical ganglion) ca ca o id bodies. No e unal e ed speci ic binding wi h inc eased non-speci ic binding 12-15 days ollowing CSN-dene a ion. Sympa hec omy educed he speci ic binding by 46%. bodies we e s udied o u he localize c -BGT bind- ing. I has been shown ha degene a ion o ne e e - minals is comple e wi hin 12-15 days ollowing ei he CSN ansec ion o emo al o he supe io ce ical ganglion 33. Type I and ype II cells in he ca ca o id body appea la gely una ec ed by ei he p oce- du e 4,33. A e CSN dene a ion, speci ic [125I]a- BGT binding in ca ca o id bodies was unchanged (Fig. 3), sugges ing ha speci ic oxin binding si es a e no p esen on CSN a e en ibe s o hei e mi- nals. Non-speci ic binding, howe e , was inc eased in CSN-dene a ed ca o id bodies. The easons o his inding a e no appa en , bu may be ela ed o he Schwann cell p oli e a ion which has been shown o accompany ne e degene a ion 1. The ca o id body is inne a ed by sympa he ic ax- ons a ising om he supe io ce ical ganglion 16, and mos o hese ibe s a e pos -ganglionic axons which e mina e on ascula smoo h muscle wi hin he o - gan al. Because p esynap ic nico inic ecep o s ha e been demons a ed on he e minals o ad ene gic pos ganglionic neu ons 37,4s, educ ions in speci ic binding we e an icipa ed in sympa hec omized p ep- a a ions. Indeed, ou da a showed ha emo al o he supe io ce ical ganglion esul ed in a 46% de- c ease in speci ic binding, u he sugges ing ha nic- o inic ecep o s a e likely associa ed wi h pos gang- lionic sympa he ic axons in he ca o id body (Fig. 3). Non-speci ic binding in sympa hec omized p epa a- ions was simila o ha in CSN-dene a ed animals; 299 i.e. signi ican ly ele a ed abo e he le els o non- speci ic binding ound o no mally inne a ed ca o - id bodies. A u o adiog aphic localiza ion To localize speci ic [125I]a-BGT binding si es in he ca o id body, au o adiog aphic echniques we e em- ployed o de e mine whe he signi ican oxin bind- ing occu ed wi hin he lobules o ype I and ype II cells. As shown in Fig. 4, ollowing incuba ion in 11.25 nM [125I]a-BGT, he au o adiog aphic g ain dis ibu ion in no mal (A), CSN-dene a ed (B) and sympa hec omized (C) issues sugges ed ha he iodina ed oxin was concen a ed amongs he pa en- chymal cell lobules. This echnique did no pe mi isualiza ion o small ne e endings (<1.0 k m); no is he au o adiog aphic esolu ion su icien o di e - en ia e binding on ype I s ype II cells. The speci ic na u e o his binding wi hin he pa enchymal cell Io- bules was demons a ed by he absence o sil e g ains o e lobules ollowing incuba ion in he p es- ence o 10 -3 M ACh (Fig. 4D). Physiological and pha macological e ec s o a-BG T The e ec s o a-BGT on chemo ecep o ne e dis- cha ge and on endogenous ca echolamine elease we e es ed in ca o id bodies p eloaded wi h [3H]do- pamine ([3H]DA) syn hesized om [3H] y osine (see Me hods). P e ious s udies in ou labo a o y ha e es ablished a p opo ional ela ionship be ween he le el o hypoxia and he elease o [3H]DA om he ca o id body in i o 21. In hose s udies, [3H]DA e- lease was also co ela ed wi h he chemo ecep o dis- cha ge eco ded om he CSN. In he p esen expe - imen s, bo h hypoxia (media equilib a ed wi h 30% O2), as well as 10 -5 M nico ine we e used as s imuli o assessing he e ec s o a-BGT on ansmi e e- lease and ne e discha ge. The ca o id bodies wi h hei a ached ne es we e moun ed in he in i o eco ding chambe (see Me hods), and supe used wi h 100% O2-equili- b a ed media o 30-40 min p io o s imula ion. [3H]DA elease and CSN discha ge we e hen mon- i o ed du ing sequen ial 5 min p e-s imulus, s imulus and pos -s imulus collec ion pe iods, which oge he comp ised a single 's imulus cycle'. Twen y minu e es pe iods (100% 0 2 media) sepa a ed successi e 's imulus cycles'. A e one hypoxic and one nico ine- 31)0 Fig. 4. Ligh mic oscope au o adiog aphy o ca o id bodies incuba ed o 30 min in l 1.25 nM [12sI]a-BG'I. Da k punc mc sil e g ains a e localized o e lobules o ype I and ype II cells in no mal (A), CSN-dene a ed (B), and sympa hec omized (C! lissac. Sil e g ains a e spa se ollowing incuba ion in he p esence o 10 3 M ACh (D). Scale = 10/xm. =_ O 100" 50- a-BT (50 nM) DA Rel. 30~ 02 7 7-n I I I Oischa ge 10 -5 M Nico ine i , I! [jL lii DA To al Peak Rel. Discha ge Fig. 5. E ec s o a-BGT on [3H]DA elease, and o al and peak CSN discha ge e oked by hypoxia (30% 0 2 equilib a ed me- dia) o 10 5 M nico ine. Da a a e exp essed as pe cen ages o con ol esponses de e mined p io o he in oduc ion o 50 nM a-BGT. * and ** indica e P < 0.01 and P < 0.005 le els o signi icance, espec i ely. s imulus cycle, he p epa a ion was supe used o 1 h wi h 100% 02 media con aining 50 nM a-BGT, and hen e-exposed o each s imulus. The da a in Fig. 5 compa es [3H]DA elease and CSN discha ge du ing con ol-s imula ed s oxin-s imula ed collec- ion pe iods (exp essed as pe cen con ol esponse). T ea men wi h a-BGT inhibi ed app oxima ely 50% o he [3H]DA elease e oked by hypoxia o nico ine. Signi ican educ ions we e also obse ed in bo h o- al and peak CSN discha ge. These changes pe sis ed a e 2 h o washing, and hus e lec ed he i e e si- ble binding o a-BGT. These educ ions in he e- sponse o he s imuli could no ha e been asc ibed o de e io a ion o he in i o p epa a ion since p e- ious wo k in his labo a o y21 has shown ha s imu- lus epe i ions o e pe iods o mo e han 3 h esul in sligh inc eases, a he han dec eases, in he e- sponse a ios. DISCUSSION Alpha-bunga o oxin binds nea ly i e e sibly o pos synap ic nico inic ecep o s a mammalian neu- omuscula junc ions, whe e i causes p olonged blockage o synap ic ansmission 7. High-a ini y componen s o a-BGT binding ha e also been de- sc ibed o mammalian cen al ne ous sys em3S,42, 4 , and sympa he ic ganglia26. The oxin also blocks syn- 301 ap ic ansmission in og pa a e eb al ganglia 39 and in he pigeon cilia y ganglion 9. Bu in some issues (e.g. he supe io ce ical ganglion), a-BGT concen- a ions which no mally sa u a e he high-a ini y si es do no block he e ec s o endogenous ACh6,14,15, 44. Thus, he signi icance o high a ini y a- BGT binding is equi ocal in he absence o demon- s a ed physiological, biochemical o beha io al e - ec s. Ou expe imen s indica e ha [1251]a-BGT binding in ca ca o id body is no unlike i s in e ac ion wi h ecep o si es in nume ous o he issues. A 5.57 nM K a is consis en wi h alues es ablished in b ain (1.7 nM and 8.0 nM) 38.47 au onomic ganglia (-2.0-3.0 nM) 26 and mammalian skele al muscle (0.37 nM) 5. Mo eo e , a high-a ini y componen o oxin binding in he ca o id body is inc emen ally blocked in he p esence o inc easing concen a ions o ACh and D-TC, sugges ing ha he oxin binds o nico inic choline gic si es in his o gan. The kine ic da a a e consis en wi h he no ion ha ca ca o id bodies con ain high a ini y a-BGT binding si es simi- la o hose ound in skele al muscle whe e a-BGT selec i ely blocks pos synap ic nico inic ecep o s 7. Ou au o adiog aphic and biochemical s udies show ha no mal and CSN-dene a ed ca o id bod- ies bind compa able amoun s o [125I]a-BGT wi hin he lobules o ype I and ype II cells. The simples explana ion o his inding is ha a e en ne e e - minals o he CSN do no possess speci ic nico inic e- cep o s. Howe e , i mus be no ed ha ne e e mi- nals in he ca o id body ep esen less han 5% o he o al issue olume 52, and hus educ ions in [I251]a- BGT binding ollowing dene a ion may be beyond ou assay's limi s o de ec ion. An addi ional ac o which migh mi iga e agains de ec ion o ne e e - minal ecep o s is he abili y o Schwann cells o bind [125I]a-BGT 54. Since Schwann cells apidly p oli e - a e du ing ne e degene a ion, and hei inc eased numbe s pe sis o many mon hs ollowing ne e ansec ion 1, any educ ions in oxin binding which co espond o he loss o axons and ne e e minals may be masked by inc eases in Schwann cell oxin binding. On he o he hand, we did obse e signi i- can educ ions in oxin binding in sympa hec omized p epa a ions, whe e he loss o pos ganglionic ne e e minals p obably ep esen s an equally small ol- ume o ca o id body issue, and whe e degene a ion is also accompanied by Schwann cell p oli e a ion. Thus, ou da a sugges ha [I~'5I](,-BGT binding on a e en e minals may be absen , o quanli a i ely small, and ha mos o he choline gic nico inic e- cep o s wi hin he pa enchymal cell lobules a e local- ized o he ype I and/o ype II cells. Fu he mo e, he p esence o such ecep o s in he ca ca o id body is suppo ed by he indings o Hayashida and Eyza- gui e ~, who showed wi h in acellula eco ding ha small doses o ACh inc ease he ol age noise and depola ize he ype I cells. Addi ional e idence o a-BGT binding on ype 1 cells has been p o ided by Che and Ya esS. Thei s udies wi h ho se adish pe oxidase conjuga ed a- BGT e ealed binding o a selec subpopula ion o glomus cells in he a ca o id body. On he basis o dense-co ed esicle measu emen s, hese au ho s concluded ha he cells which bind he oxin co e- spond o he ype B glomus cells iden i ied ea lie by MacDonald and Mi chelP °. Howe e . m o he spe- cies, dense-co ed esicle s udies ha e no demon- s a ed subg oups o glomus cells 53. In o ma ion on his poin is lacking in he ca , and om ou da a i is no possible o de e mine whe he a-BGT b ads o a limi ed glomus cell popula ion. The e ec s o a-BGT on DA elease and chemo e- cep o discha ge e oked by hypoxia o nico ine con- i m he exis ence o speci ic nico inic si es in he ca ca o id body and, u he mo e, sugges ha a-BGT blocks ecep o s which a e unc ionally impo an R) no mal chemo ecep ion. In he ca o id body, DA is syn hesized and eleased by ype I cells 2~,2~ and hus, he abili y o a-BGT o a ec DA ou low, e oked ei he by hypoxia o nico ine, sugges s ~m choimc~ gic ecep o s loca ed on ype I cells m ca ca o id body modula e he eleasc , ca echolammes. (>,m-- sis en wi h his in e p e a ion, Hells om c al. ~-: ound ha deple ion o a ca o id body DA by hypo- xia was mimicked by me hacholine and an agonized by a opine. S udies o ansmi e ou low om he co pus s ia um e7 and hypo halamus s~j ha e likewise e ealed nico inic and musca inic ecep o s which, when ac i a ed, enhance he elease ~i [-~HIDA. Hence, he ca o id body may u ilize a ciloline gic- link mechanism o modula e DA elease, as has bee~ desc ibed o o he ca echolamine gic sys ems5% In summa y, a s udy o he localiza ion and unc- ion o [~25I]a-BGT binding in no mal and dene - a ed ca ca o id bodies indica es ha nico inic cho- line gic ecep o s a e loca ed on ype 1 cells (and pe - haps ype II cells), as well as on pos -ganglionic sym- pa he ic ne e e minals. The binding si es on ype I/ ype I1 cells a e cha ac e is ically nico inic and ap- pea o modula e bo h he elease o DA as well as he chemo ecep o discha ge. The appa en absence o [~25I]a-BGT binding si es on he CSN sugges s ha ca o id body chemosensa ion in he ca does no de- pend upon he ac i a ion o choline gic ecep o s lo- ca ed on a e en e minals apposed o ype I cells. ACKNOWLEDGEMENTS The au ho s wish o hank Ms. Vicki Skel on o skill ully p epa ing he manusc ip s. Resea ch sup- po ed by USPHS G an s NS 12636 and NS 07938. REFERENCES 1 Abe c ombie, M. and Johnson, M. 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