Alpha-bungarotoxin binding in cat carotid body

B ain Resea ch, 205 (1981) 187-193 187 © Else ie /No h-Holland Biomedical P ess Alpha-bunga o oxin binding in ca ca o id body B. DINGER, C. GONZALEZ, K. YOSHIZAKI and S. FIDONE* Depa men o Physiology, Uni e si y o U ah College o Medicine, 410 Chipe a Way, Sal Lake Ci y, U ah 84108 (U.S.A.) (Accep ed Augus 21s , 1980) Key wo ds: alpha-bunga o oxin -- ca o id body -- chemosensa ion The ca o id body is an a e ial chemosenso y o gan which de ec s changes in blood gas ensions and pH, and e lexly con ibu es o he ca dio espi a o y adjus - men s which occu du ing hypoxia, hype capnia and acidosis. Howe e , he senso y mechanisms in ol ed in ca o id chemo ecep ion emain o be elucida ed. Mo phologically, he ca o id body consis s o an associa ion o elemen al uni s, o glome uli, wi hin a connec i e issue s oma pene a ed by a dense capilla y ne 5. The glome uli a e comp ised o ca echolamine- ich ype I, o chie cells, which a e en- eloped by glial-like p ocesses o ype II, o sus en acula , cellsa,4,19. Senso y ibe s om he ca o id sinus ne e pene a e he glome uli o e mina e in synap ic-like apposi ion on ype I cells ,18, 21. Schwei ze and W igh 25 i s no ed he s imula o y e ec s o ace ylcholine (ACh) on ca o id chemo e lexes in he ca , and sugges ed ha his subs ance migh be in ol ed in he gene a ion o chemosenso y ac i i y. La e expe imen s cha ac e ized in de ail he exci a o y po ency o ACh and nico inic agonis s on he chemo ecep o discha ge om he ca ca o id body 7,9,10,24. They showed ha choline gic an agonis s abolish he sensi i i y o ACh and educe he esponse o na u al s imula ion. Mo e e- cen ly, i has been demons a ed ha chemically iden i iable ACh is p esen in he pa- enchymal issue o he ca ca o id body, a he han in he ibe s o e minals o he ca o id sinus ne e11,l~, 15. Al hough he si e(s) o ACh s o age in his issue has no been i mly es ablished, a high a ini y componen o choline up ake has been au o a- diog aphically localized o he ype I cells 12. Finally, he e is e idence ha ACh is e- leased om he ca o id body du ing na u al s imula ionS,L One in e p e a ion o hese indings is ha ACh is a senso y ansmi e in he ca ca o id body, and ha as such, his subs ance is eleased om he ype I cells by na u al s imula ion o ac i a e nico- inic ecep o s on neighbo ing senso y ne e e minals, he eby leading o he ini ia ion o chemosenso y impulses in he ca o id sinus ne e 1°. O he ecen s udies ha e shown, howe e , ha ACh di ec ly depola izes he ype I cells in bo h no mal and de- * To whom co espondence should be add essed. 188 ne a ed ca o id bodies 14, and ha choline gic an agonis s dep ess he elease o dopa- mine om hese cells 16. These da a aise he ques ion o whe he ACh ac s on he ne - e e minals di ec ly, and/o h ough a mechanism which in ol es ACh-induced e- lease o ca echolamines o o he subs ances om he ype I cells. In an e o o esol e his issue, we ha e a emped in he p esen s udy o localize nico inic ecep o s in he ca ca o id body using labelled a-bunga o oxin ([125I]a-BGT). The esul s will show ha mos , i no all, speci ic a-BGT binding si es in his o gan a e loca ed on non-neu al glome ula elemen s, p esumably on he ype I cells. Ca o id bodies we e emo ed om pen oba bi al-anes he ized ca s, placed in a luci e chambe illed wi h O2-equilib a ed, modi ied-Ty ode's solu ion (in mM: NaC1, 112; KC1, 4.7; CaC12, 2.2; MgClz, 1.1; sodium glu ama e, 42; HEPES, 5; pH 7.43 a 37 °C) and cleaned o su ounding connec i e issue wi h he aid o a dissec ing s e eo- mic oscope. To dis inguish be ween o al and non-speci ic a-BGT binding, ca o id bo- dies we e di ided in o wo g oups p io o incuba ion wi h he adiolabelled ligand. The issues we e i s p eincuba ed o 20 min in a wa e ba h shake a 37 °C in he p esence o absence o D- ubocu a ine (10-a-10 -6 M; Sigma), o ACh (10-a-10 -6 M; Sig- ma) plus ese ine (10 -5 M; Sigma). The p eincuba ion ials con ained 1.5 ml o Oz-Ty - ode's medium plus 1 ~ bo ine se um albumin. [125I]a-BGT (138 Ci/mM, New Eng- land Nuclea ) was hen added o each ial o each a inal concen a ion o 1-40 nM, and he incuba ion pe iod wi h he ligand was con inued o 30 min a 37 °C. The is- sues we e hen emo ed om he ials and washed o 1 h in 10 ml o ice-cold O2-Ty - ode's solu ion. Tissue samples o biochemical analysis we e placed in glass scin illa- ion ials and diges ed o 4 h a 60 °C in a mix u e o 200/~1 NCS (Ame sham) and 50 #1 wa e . P io o coun ing in a liquid scin illa ion spec ome e (Packa d 3385), he di- ges ion mix u e was neu alized wi h 750 #1 o ace ic acid (1.3 ~), and hen 15 ml o coun ing cock ail (PCS II, Ame sham) we e added o each ial. Tissue 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 a bu e ed 2 ~o osmic acid solu ion, dehyd a ed in e hanol, and embedded 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 hylene blue. In some animals, ca o id bodies we e dene a ed by an- sec ion o he ca o id sinus ne e 12-15 days p io o emo al o he o gans o expe i- men a ion. The binding o [125I]a-BGT in he ca ca o id body was concen a ion-depen- den , and he amoun o speci ic oxin binding, de ined as he binding displaceable by ACh o D- ubocu a ine, was maximal a a oxin concen a ion o 11 nM (6.44 :~ 0.28 mol/mg issue, n = 22 ca o id bodies). A his concen a ion, speci ic binding o [ 25I]a-BGT was linea o app oxima ely 20 ain, and hen apidly pla eaued so ha a e 30 min li le o no addi ional speci ic binding ook place. Nea ly all o he oxin binding could be p e en ed in he p esence o 10 -3 M ACh (91 ~o) o 10 -3 M D- ubocu- a ine (90 ~), and consequen ly ou da a sugges ha mos a-BGT binding si es in ca ca o id body a e nico inic ecep o si es. 189 Fig. 1. Binding o [125I]a-BGT in no mally inne a ed ca ca o id body. Concen a ion o [125I]a- BGT, 11 nM; incuba ion ime, 30 min. A-C: o al binding. D : non-speci ic binding in he p esence o ACh (10 -3 M) plus ese ine (10 -5 M). No e loss o speci ic binding om glome ula s uc u es. Scale: 5 #m. 190 Ligh mic oscope au o adiog aphy o ca ca o id bodies incuba ed wi h [125I]a- BGT demons a ed ha he speci ic oxin binding si es a e localized p ima ily wi hin he glome ula appa a us. This is shown in Fig. l, which compa es o al oxin binding (Fig. 1A-C) wi h he binding which emains (Fig. 1D) in he p esence o ACh (10 -3 M) plus ese ine (10 -5 M). Sil e g ains a e e iden in associa ion wi h ype I, and pe haps also ype II, cells. Biochemical and au o adiog aphic analysis o [125I]a-BGT bindingin dene a ed ca ca o id bodies indica ed ha he localiza ion and amoun o speci ic binding (6.33 ± 0.56 mol/mg issue, n = 17 ca o id bodies) was no signi ican ly di e en om con ol specimens (P ~ 0.1). Howe e , o al oxin binding was inc eased by 56 ~ (P < 0.01) in he dene a ed o gans. The eason o his inc ease in non-speci ic binding is unknown, bu may be ela ed o he hype ophy o ype lI cell p ocesses17,~0, 21 and/o o he p oli e a ion o Schwann cell elemen s 1 consequen o he degene a ion o he ca o id sinus ne e. Fig. 2A-C shows [125I]a-BGT binding in a dene a ed ca ca o id body, while Fig. 2D shows he oxin binding which emains in he p esence o ACh (10 -a M) plus ese ine (10 -5 M). The same pa e n o localiza ion and displacemen o binding si es seen in he no mally-inne a ed ca o id body is e iden in he dene a ed o gan; he sil e g ains appea concen a ed o e clus e s o glome ula cells. In summa y, ou esul s show ha [125I]a-BGT binding si es, displaceable by ACh o D- ubocu a ine, a e loca ed wi hin he glome ula appa a us o he ca ca o id body. Fu he mo e, ollowing degene a ion o he ca o id sinus ne e he amoun o his displaceable o speci ic binding is unchanged, al hough o al binding in he dene a ed o gan is signi ican ly inc eased. These da a demons a e, he e o e, ha speci ic a-BGT binding si es a e con ined p ima ily o non-neu al glome ula elemen s in he ca ca o id body, and hence a e absen om he senso y e minals o he ca o id sinus ne e. Ge mane o ou indings, howe e , a e he ecen obse a ions ha blockade o cu a e-displaceable a-BGT binding si es on chick sympa he ic neu ons does no a ec he choline gic-synap ic po en ial o he esponse o exogenous ACh eco ded om hese cells z. These obse a ions ha e been in e p e ed o mean ha a- BGT binding si es in his issue a e no equi alen o neu onal ACh ecep o s. P elimina y expe imen s in ou labo a o y, howe e , ha e demons a ed ha in ca ca o id body a-BGT blocks he inc ease in chemosenso y discha ge and he elease o dopamine elici ed by nico ine (10 -5 M). Since he ac ions o ACh in ca ca o id body seem o be la gely nico inic22, 23, he appa en absence o a-BGT binding si es on he senso y ne e endings sugges s ha ano he glome ula elemen , he ype I o ype II cell, may be in ol ed in media ing he exci a o y e ec s o ACh on chemosenso y ac i i y. The a ailable e idence would implica e he ype I cells in his capaci y, because choline gic agonis s and an agonis s al e dopamine elease om bo h no mal and dene a ed ca (unpublished obse a ions) and a 16 ca o id bodies, and he e o e poin o he likely p esence o ACh ecep o s on hese ca echolamine-con aining cells. Howe e , he mechanisms linking hese ACh ecep o s wi h exci a ion o chemo- senso y ne e ibe s emain o be elucida ed. 191 Fig. 2. Binding o [125IJa-BGT in ch onically dene a ed ( ansec ion o ca o id sinus ne e) ca ca o- id body. Concen a ion o [lZSI]a-BGT, 11 nM; incuba ion ime, 30 min. A-C: o al binding. D: non- speci ic binding in he p esence o ACh (10 a M) plus ese ine (10 -~ M). No e simila dis ibu ion o speci ic binding as in no mally inne a ed ca ca o id body (Fig. 1). Scale: 5 #m. 192 This wo k was suppo ed by Public Heal h Se ice G an s NS 12636 and NS 07938. 1 Aguayo, J., Epps, J., Cha on, L. and B ay, G. 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