Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells

Ž. B ain Resea ch 754 1997 298–302 Sho communica ion E idence o wo ypes o nico inic ecep o s in he ca ca o id body chemo ecep o cells a´aab b Ana Obeso , Ma ia Angeles Gomez-Nino , Lau a Alma az , B uce Dinge , Sal a o e Fidone , ´˜ Cons ancio Gonzalez a,) ´ aDepa amen o de Bioquımica y Biologıa Molecula y Fisiologıa, Facul ad de Medicina, UniÕe sidad de Valladolid, 47005 Valladolid, Spain ´´ ´ b Depa men o Physiology, UniÕe si y o U ah School o Medicine, Sal Lake Ci y, UT, USA Accep ed 4 Feb ua y 1997 Abs ac Cu en concep s on he loca ion and unc ional signi icance o nico inic ecep o s in he ca o id body es on a -bunga o oxin binding and au o adiog aphic s udies. Using an in i o p epa a ion o he ca ca o id body whose ca echolamine deposi s ha e been labeled by w 3 xw 3 x p io incuba ion wi h he i ia ed na u al p ecu so H y osine, we ha e ound ha nico ine induces elease o H ca echolamines in a Ž. Ž. dose-dependen manne IC s9.81 m M . We also ound ha mecamylamine 50 m M comple ely abolished he nico ine-induced 50 Ž. elease, while a -bunga o oxin 100 nM; 20 imes i s binding Konly educed he elease by 56%. These indings indica e ha d chemo ecep o cells, and pe haps o he ca o id body s uc u es, con ain nico inic ecep o s ha a e no sensi i e o a -bunga o oxin and o ce a e ision o he cu en concep s on choline gic mechanisms in he ca o id body chemo ecep ion. Keywo ds: Ca o id body; Nico inic ecep o ; Nico ine; a -Bunga o oxin; Ca echolamine Ž. The signi icance o ace ylcholine ACh in he ca o id Ž. body CB a e ial chemo ecep o s has been deba ed h oughou he his o y o a e ial chemo ecep o s. Hey- wx mans e al. 17,18 disco e ed ha nico ine and exogenous ACh, ac ing a he le el o he CB, can e oke espi a o y e lexes compa able o hose p oduced by hypoxia and wx acidosis, and Schwei ze and W igh 24 obse ed ha Ž. p os igmine an inhibi o o ACh-es e ase mimicked he ac ion o ACh. These indings lead o he p oposal ha ACh would be he neu o ansmi e media ing he ac i a- ion o he senso y ne e endings o he ca o id sinus ne e Ž. CSN . This p oposal, known as he choline gic hypo hesis o CB chemo ecep ion, was highly deba ed, and in he six ies Eyzagui e’s labo a o y accumula ed sound expe i- men al e idence o suppo a nico inic ecep o -media ed ole o ACh in he genesis o he ac i i y in he CSN, in wx esponse o a a ie y o s imuli 9 . Howe e , he con o- e sy con inued because classical nico inic blocke s sup- p essed he exci a o y ac ions o nico inic agonis s, bu only educed in a a iable pe cen age he ac i a ion p o- wx duced by na u al s imuli 20,21 . )Ž. Co esponding au ho . Fax: q34 83 423588. Biochemically, i was shown ha he CB con ains ACh wx 12,16 , exp esses an adequa e ac i i y o choline ace yl wx ans e ase 16 , accumula es choline by a high a ini y wx sys em, and exhibi s a high u no e a e o ACh 10 . In spi e o all hese da a, he ole o ACh in he chemo ecep- ion p ocess emained elusi e. Radioligand binding and w125 xŽ au o adiog aphic s udies, using I a -bunga o oxin a - . BT as ligand in no mal, CSN-dene a ed and sympa hec- omized CBs, showed ha he high a ini y speci ic bind- ing was loca ed in chemo ecep o cells and sympa he ic wx endings, bu no in senso y ne e endings 4,5 . Elec o- physiological s udies ha e also shown he p esence o wx nico inic ecep o s in chemo ecep o cells 15,25 , and neu ochemically i has been shown ha chemo ecep o Ž. cells elease ca echolamines CA in esponse o nico inic wx agonis s 5,14 . These indings ha e been conside ed o indica e ha ACh ac s as a seconda y neu o ansmi e exclusi ely on he p esynap ic side o he chemo ecep o wx cell-senso y ne e ending synapse 11,13 . The e is, howe e , a inding ha was no sa is ac o ily explained: while classical nico inic blocke s ully elimi- wx na ed he nico ine-media ed exci a ion o he CSN 9,20 , a -BT a a concen a ion nea 10 imes he K, inhibi ed by d only 50% he elease o CA and CSN discha ges elici ed 0006-8993 97 $17.00 Copy igh q1997 Else ie Science B.V. All igh s ese ed. Ž. PII S0006-8993 97 00185-6 () A. Obeso e al. B ain Resea ch 754 1997 298–302 299 wx by nico ine 5 . In he ligh o ecen ad ances in he wx biology o he nico inic ecep o 3,19,23 we ha e es ed he possibili y ha chemo ecep o cells exp ess wo unc- ional sub ypes o nico inic ecep o s, one sensi i e and ano he insensi i e o a -BT. Using an in i o p epa a ion Ž. o ca CB whose ca echolamine CA deposi s ha e been w3x labeled by incuba ion wi h he na u al p ecu so H y o- sine, we ha e ound ha a -BT, a concen a ions 20 imes abo e i s binding K, inhibi s he nico ine e oked elease d w3x o H CA by only 56%, while he classical nico ine blocke mecamylamine a 50 m M inhibi ed he nico ine- e oked elease by 100%. These indings imply ha chemo ecep o cells exp ess nico inic ecep o s con aining wx a - a subuni s and he eby sensi i e o a -BT 19,23 , and 78 o he sub ypes o nico inic ecep o s wi h di e en a subuni s insensi i e o he oxin. Expe imen s we e pe o med wi h CBs o adul ca s Ž. 2.5–3.5 kg . The animals we e anes he ized wi h sodium Ž. pen oba bi al 40 mg kg; i.p. , and a e acheo omy, he ca o id bi u ca ions we e iden i ied, emo ed and placed in Ž a luci e chambe con aining ice-cold modi ied Ty ode in . mM : NaCl, 112; KCl, 4.7; CaCl , 2.2; MgCl , 1.1; 22 .wx sodium glu ama e, 42; HEPES, 5; glucose, 5.5 2 a pH 7.40 and equilib a ed wi h 100% O . 2 Unde a dissec ing mic oscope he CBs we e cleaned o su ounding issue and he ea e incuba ed in glass ials con aining 2 ml o Ty ode placed in a me abolic shake a a cons an empe a u e o 378C. The incuba ing solu ion Ž con ained 100 m M 6-me hyl- e ahyd op e ine a y osine .Ž hyd oxylase co ac o , 1 mM asco bic acid as a co ac o .w3xŽ o dopamine- b -hyd oxylase and 40 m M H y osine 20 . Ci mM; Ame sham , he na u al p ecu so o ca echol- Ž. amines CA . A e 2 h o incuba ion CA deposi s we e labeled so ha each CB has syn hesized 12 pmol o w3xŽ. 5 H dopamine DA , equi alen o 3=10 d.p.m., and w3xŽ. 1 pmol o H no epineph ine NE , equi alen o 3 =104d.p.m. A e he labeling pe iod he o gans we e ans e ed o new ials con aining 4 ml o p ecu so - ee Ty ode con inuously bubbled wi h 100% O sa u a ed wi h 2 wa e apo . The solu ion was enewed e e y 30 min du ing 2 h and disca ded; in his washing pe iod mos o he p ecu so as well as he labile pool o labeled CA we e w3x los , and a e wa ds he basal elease o H CA was s able wx o se e al hou s 1 . The ea e he incuba ing solu ions we e enewed e e y 10 min and collec ed o he ul e io w3x analysis in hei H CA con en . The collec ion o incuba ing solu ions was g ouped in s imula ion cycles, each cycle consis ing in a con ol sam- ple o de e mine he basal elease, a s imulus sample co esponding o he incuba ing pe iod in he p esence o Ž. he agen s es ed, and se e al pos -s imulus samples co - esponding o pe iods o incuba ion wi h con ol solu ions w3xŽ un il he elease o H CA e u ned o he basal le el see .Ž Fig. 1A . Nico inic blocke s i.e., mecamylamine and a - .Ž. BT we e included in he con ol pe iod s p io o nico ine applica ion o assu e an adequa e di usion o he d ug and block o nico inic ecep o s. A gi en p epa a ion could be subjec ed o one o mo e s imula ion cycles depending on he e e sibili y o he e ec o he agen unde s udy. The w3x analysis o he eleased H CA included: acidi ica ion o he collec ed incuba ing solu ions o pH 3.2 wi h a mix u e o glacial ace ic and asco bic acid o a oid deg ada ion o CA, bulk adso p ion o all ca echols eleased in o alumina a a pH o 8.6, in ense washing o alumina columns wi h dis illed wa e and bulk elu ion o all ca echols wi h 1 N HCl. Pa o he elua e was used o de e mine he o al w3x amoun o H CA eleased in each pe iod by liquid scin illa ion spec ome y, and he es was pooled wi h co esponden samples o a o al o ou expe imen s, d ied unde acuum, esuspended in he mobile phase con aining unlabeled ca echols as ca ie s o he labeled subs ances, and high pe o mance liquid ch oma og aphied o iden i y w3x Fig. 1. A: a s imula ion cycle o show he ime cou se o he elease o H ca echolamines induced by 100 m M nico ine in he ca ca o id body. The line Ž. Ž. c ossing he his og am sepa a es g aphically he basal elease below om he e oked elease abo e . B: log dose- esponse cu e o nico ine on he w3x elease o H ca echolamine by he ca ca o id body. The elease esponse is exp essed as imes basal elease. Da a we e i ed o he ollowing unc ion: wŽ. p x ysAyA 1qx x , whe e A is he maximum e ec , x is he concen a ion o nico ine, x is he IC and pis he Hill coe icien . The IC 22 0 2 050 50 was 9.81 m M. Da a a e means"S.E.M.; ns6. () A. Obeso e al. B ain Resea ch 754 1997 298–302300 w3xŽ he ac ual H ca echol eleased o ch oma og aphic de- wx. ails see e . 14 . The e ec o a gi en agen on he w3x elease o H CA was calcula ed in wo di e en ways: Ž. i s , he e oked elease abo e he dashed line in Fig. 1A was e e ed o he basal elease p io he applica ion o he s imulus and exp essed as imes basal elease, and second, he e oked elease by a gi en s imulus was e- e ed o he issue con en and exp essed as pe cen o he issue con en . The signi icance o he e ec s obse ed was assessed wi h a wo- ailed S uden - es o pai ed o unpai ed da a acco ding o expe imen al design. [3] E ec s o nico ine on he elease o H CA. Fig. 1A shows a s imula ion cycle wi h 100 m M nico ine. Nico- w3x ine-induced elease o H CA is ep esen ed by he d.p.m. abo e he dashed line c ossing he his og am. No e ha nea ly 80% o he e oked elease is collec ed du ing he 10 min pe iod co esponding o he nico ine applica ion, and ha du ing he pos -s imulus pe iods he e is a slow de- cline o he elease o each basal p e-s imula ion le els. This slow phase o he e oked elease is ep esen ed by he w3xw 3 x washing-ou o H CA ca aboli es; a pa he H CA eleased du ing he s imulus pe iod is aken ou by he w3x issues and deg aded, and he H ca aboli es a e slowly disposed by he cells. Fig. 1B shows a dose-e ec cu e w3x o nico ine on he elease o H CA. The e oked elease is exp essed as imes basal elease. A he lowes concen- Ž. a ion es ed 0.1 m M , nico ine inc eased he elease o w3x H CA by a ac o o 0.65 abo e basal elease; maximal esponse, co esponding o a elease o 14.4 imes abo e basal was ob ained wi h 50 m M nico ine, and he nico ine Ž. concen a ion p oducing hal maximal e ec IC was 50 9.81 m M. The same IC was ob ained i he e oked 50 elease was exp essed as pe cen age o issue con en , he w3x Fig. 2. Analy ical p o ile o he H ca echol eleased. In basal condi ions w3xŽ. H dihyd oxyphenyl ace ic acid DOPAC , he main ca aboli e o w3xw 3 x H dopamine, ep esen ed 60% o he o al H ca echols eleased. Du - w3x ing hypoxic s imula ion H DOPAC amoun ed o 20% o he eleased ma e ial and du ing nico inic s imula ion i ep esen ed 17%. The ca abo- w3x li es o H no epineph ine we e below de ec ion. Fig. 3. E ec s o mecamylamine and a -bunga o oxin on he elease o w3x H ca echolamines elici ed by nico ine. The le g oup o columns w3x ep esen s he elease o H CA elici ed by 50 m M nico ine du ing he Ž. Ž . s imulus S and he pos -s imulus PS pe iods. The middle g oup o Ž. columns shows he comple e inhibi ion o he nico ine 50 m M elease esponse p oduced by 50 m M mecamylamine, and he igh g oup o w3xŽ columns co esponds o he elease o H CA p oduced by nico ine 50 . m M in he p esence o 100 nM a -bunga o oxin. Da a a e means"S.E.M.; ns6 o nico ine and nico ine plus mecamylamine and ns8 o nico ine plus a -bunga o oxin. )P-0.001 in all he cases. basal elease in a 10 min pe iod ep esen ing 0.5% o he issue con en . w3x Fig. 2 shows he analy ical p o ile o he H ca echols ound in basal samples, in incuba ing samples co espond- Ž. ing o he s imula ion wi h nico ine 50 m M , and o compa a i e pu poses in incuba ing samples co esponding Ž o s imula ion wi h hypoxia incuba ion wi h 10% O -equi- 2 .w3xw 3 x lib a ed solu ions . The a io o H DA H NE ound in basal samples was 10.5, indica ing ha 91% o he o al w3xw 3 x H CA eleased was H DA and he emaining 9% was w3x H NE. These pe cen ages a e compa able o hose ound wx in he issues 22 , indica ing ha in basal condi ions bo h w3x H CA a e eleased a a a e p opo ional o hei issue le els. Du ing hypoxic s imula ion he elease o bo h w3xw 3 xw 3 x H CA inc eased, and he H DA H NE a io in he collec ed samples inc eased o 23, indica ing a p e e en ial w3x elease o H DA. On he con a y, du ing nico inic s imu- w3xw 3 x la ion he a io o H DA H NE in he collec ed sam- ples d opped o a alue o 4, indica ing a p e e en ial w3x elease o H NE. E en a mo e ma ked p e e en ial e- w3x lease o H NE du ing nico inic s imula ion has been wx p e iously epo ed in he abbi CB 14 . Fig. 3 shows he e ec o a -BT and mecamylamine on w3x he elease o H CA elici ed by nico ine. Since in p e i- ous expe imen s i was ound ha maximal a -BT binding Žwx. was a ained in 30 min see e . 5 , he oxin was included in he incuba ing solu ion o he h ee 10 min con ol pe iods p io o and du ing nico ine applica ion; () A. Obeso e al. B ain Resea ch 754 1997 298–302 301 mecamylamine was included in he incuba ing solu ion o a single 10 min con ol pe iod p io o and du ing nico ine Ž. applica ion. No e ha mecamylamine 50 m M comple ely blocked he elease induced by an iden ical concen a ion o nico ine. Howe e , a -BT a a concen a ion o 100 nM, wx which is nea ly 20 imes i s Kin binding expe imen s 5 , d only inhibi ed he nico ine-induced elease by 56%. This le el o inhibi ion is iden ical o ha ob ained wi h 50 nM wx a -BT 5 , and he e o e i was no necessa y o es highe concen a ions. Nei he nico inic blocke a ec ed he con- w3x ol uns imula ed o basal elease o H CA. The main inding o his wo k is ha mecamylamine w3x comple ely inhibi s he elease o H CA while a -BT only inhibi s 56%, indica ing ha he e is a se o nico inic ecep o s in he CB ha is insensi i e o a -BT. These esul s also imply ha he cu en concep s conce ning he iden i ica ion and loca ion o nico inic ecep o s in he CB, which a e based on a -BT binding and au o adiog aphic expe imen s, need o be e ised. w3x The dose- esponse o nico ine on he elease o H CA ob ained in he p esen s udy is displaced o he igh when wx compa ed o ha ob ained by Eyzagui e and Zapa a 7 o ACh on he ca CSN discha ges. They epo ed a h eshold Žy8. o he esponse o 68 nM 10 g ml , an IC o 50 0.68 m M and a maximal esponse a 6.8 m M; hese alues indica e a displacemen o he igh by mo e han an o de o magni ude o he dose- esponse cu e epo ed he e. A eady explana ion o his di e ence could be ha ACh inc eases CSN discha ges ac ing on senso y ne e endings and no on chemo ecep o cells; al e na i ely, he CA eleased om chemo ecep o cells could ep esen he d i e s o he CSN discha ges, in such a way ha small inc eases in he elease abo e basal p oduce signi ican inc eases in he CSN discha ges, and submaximal elease Ž p oduces maximal CSN ac ion po en ial equency see . below . I is impossible o compa e ou da a wi h hose ob ained in o he s udies, because in mos o hem ACh o nico ine we e adminis e ed in a-a e ially as bolus injec- ions o in he in lowing ubing o he eco ding chambe . w3x The p e e en ial elease o H NE obse ed du ing nico- inic s imula ion co ela es wi h he obse a ion ha nea ly 40% o he a -BT binding si es disappea a e ch onic CB wx sympa hec omy 5 and indica e ha a signi ican pa o w3x he H NE eleased comes om in aglomic sympa he ic endings. In ac , and con a y o he si ua ion in he abbi wx w 3x 14 , he p e e en ial elease o H NE obse ed unde nico inic s imula ion in he ca CB disappea s a e sympa- Ž. hec omy Gomez-Nino, unpublished . ´˜ Ou indings wi h mecamylamine s. a -BT clea ly demons a e ha pa o he nico inic ecep o s in he ca CB a e no sensi i e o he snake oxin. Al hough he p esen pha macological s udy does no allow o iden i y he sub ypes o nico inic ecep o s, i is clea ha chemo e- cep o cells should exp ess nico inic ecep o s wi h a o 7 a subuni s, because only hese subuni s bind a -BT; i is 8 also clea ha hey mus exp ess nico inic ecep o s wi h o he a subuni s, p obably o he sub ypes a o a 35 which a e he mos commonly ound in sympa he ic neu- wx ons, and PC12 cells 19 which a e emb yologically e- la ed o he CB. Ou indings in u n indica e ha p e ious iden i ica ion o nico inic ecep o s on he basis o a -BT binding and au o adiog aphic s udies ha e le ou pa o he nico inic ecep o s in he CB, and he possibili y exis s ha a pa o hese missed ecep o s a e loca ed in he senso y ne e endings. The unambiguous answe o his possibili y would equi e he demons a ion o he ecep- o s in he senso y ne e endings and o he demons a- ion o he message o hem in he chemo ecep o neu ons o he pe osal ganglion by immunohis ochemical and o in si u hyb idiza ion echniques. Howe e , he ac ha nico inic e ec s on CSN discha ges a e g ea ly diminished 2q2qwx o abolished in Ca - ee o Mg - ich solu ions 8,9 sugges s ha nico inic ecep o s a e p edominan ly o ex- clusi ely loca ed in chemo ecep o cells, and he eby ha nico inic e ec s on CSN discha ges a e media ed ia he Ca2q-dependen elease o o he neu o ansmi e s. In con- clusion, he p esen da a demons a ing ha a pa o he nico inic ecep o s a e no sensi i e o a -BT obliga es a e ision o he concep s o he choline gic mechanisms in wx he CB chemo ecep o s 6 . Acknowledgemen s Suppo ed by Spanish DGICYT G an PB92 0267 and by NIH G an s NS07938 and NS12636. Re e ences wx 1 Alma az, L., Gonzalez, C. and Obeso, A., E ec s o high po assium ´ w3x on he elease o H dopamine om he ca ca o id body in i o, J. ()Ž. Physiol. Lond. , 379 1986 293–307. wx 2 Ba on, M. and Eyzagui e, C., E ec s o empe a u e on some memb ane cha ac e is ics o ca o id body cells, Am. J. Physiol., 233 Ž. 1977 C35–C46. wx 3 Cla ke, P.B.S., The all and ise o neu onal alpha-bunga o oxin Ž. binding p o eins, T ends Pha macol. Sci., 13 1992 407–413. wx 4 Dinge , B., Gonzalez, C., Yoshizaki, K. and Fidone, S., Alpha- ´ Ž. bunga o oxin binding in ca ca o id body, B ain Res., 205 1981 187–193. wx 5 Dinge , B., Gonzalez, C., Yoshizaki, K. and Fidone, S., Localiza ion ´ and unc ion o ca ca o id body nico inic ecep o s, B ain Res., 339 Ž. 1985 295–304. wx 6 Eyzagui e, C., Fidone, S. and Nishi, K., Recen s udies on he Ž. gene a ion o chemo ecep o impulses. In: M.J. Pu es Ed. , The Pe iphe al A e ial Chemo ecep o s, Camb idge Uni e si y P ess, London, 1975, pp. 175–194. wx 7 Eyzagui e, C. and Zapa a, P., The elease o ace ylcholine om ca o id body issues. Fu he s udy on he e ec s o ace ylcholine and choline gic blocking agen s on he chemosenso y discha ge, J. ()Ž. Physiol. Lond. , 195 1968 589–607. wx 8 Eyzagui e, C. and Zapa a, P., Pha macology o pH e ec s on () ca o id body chemo ecep o s in i o, J. Physiol. Lond. , 195 Ž. 1968 557–588. wx 9 Eyzagui e, C. and Zapa a, P., A discussion o possible ansmi e () A. Obeso e al. B ain Resea ch 754 1997 298–302302 o gene a o subs ances in ca o id body chemo ecep o s. In: R.W. Ž. To ance Ed. , A e ial Chemo ecep o s, Blackwell Scien i ic Pub- lica ions, Ox o d, 1968, pp. 213–251. wx 10 Fidone, S., Wein aub, S., S a inoha, W., S i ling, C. and Jones, L., Endogenous ace ylcholine le els in ca ca o id body and he au o a- diog aphic localiza ion o a high a ini y componen o choline up ake. In: H. Acke , S. Fidone, D. Pallo , C. Eyzagui e, D.W. Ž. Lubbe s and R.W. To ance Eds. , Chemo ecep ion in he Ca o id ¨ Body, Sp inge -Ve lag, Be lin, 1977, pp. 106–113. wx 11 Fidone, S.J. and Gonzalez, C., Ini ia ion and con ol o chemo ecep- ´ Ž. o ac i i y in he ca o id body. In: A.P. Fishman Ed. , Handbook o Physiology. The Respi a o y Sys em, Am. Physiol. 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