[3H]Spiroperidol binding in normal and denervated carotid bodies

Neu oscience Le e s, 21 (1981) 51-55 51 © Else ie /No h-Holland Scien i ic Publishe s L d. [3H]SPIROPERIDOL BINDING IN NORMAL AND DENERVATED CAROTID BODIES B. DINGER, C. GONZALEZ, K. YOSHIZAKI and S. FIDONE* Depa men o Physiology, Uni e si y o U ah College o Medicine, Sal Lake Ci y, UT 84108 (U.S.A.) (Recei ed Augus 29 h, 1980; Accep ed Sep embe 4 h, 1980) Speci ic dopamine ecep o s we e s udied in eshly dissec ed, unhomogenized abbi ca o id bodies incuba ed in [3H]spi ope idol. To al binding and non-speci ic binding we e de e mined in he absence and p esence o 0.2 #M (+)-bu aclamol, espec i ely. Speci ic binding in no mal ca o id bodies incuba ed a nea sa u a ing concen a ions (0.38 nM) was 1.63 _+ 0.58 pmol/g o issue. Ch onic sec ion o he ca o id sinus ne e (14 days) esul ed in a 64070 educ ion (P < 0.05) in speci ic binding. We conclude ha he majo i y o speci ic dopamine gic ecep o s a e loca ed on ca o id sinus ne e a e en e minals. The ca o id body is a chemosenso y o gan which e lexly in luences hea a e, aso esis ance and pulmona y en ila ion. Changes in blood pOE, pCO2 and pH a e ansla ed by his o gan in o app op ia e pa e ns o chemo ecep o discha ge on he ca o id sinus ne e (see e . 3). Howe e , he p ecise cellula and biochemical mechanisms in ol ed in his chemosenso y ansduc ion p ocess emain o be ully elucida ed. The pa enchyma o he ca o id body is composed o lobules, o glome uli, o ca echolamine-con aining ( ype I) cells which ecei e synap ic e mina ions om ibe s o he ca o id sinus ne e [4, 5, 15, 17]. The ype I cell/ne e e minal complex is en eloped by p ocesses o sus en acula , o ype II, cells. Blood low h ough he ca o id body u ilizes an ex ensi e ne wo k o enes a ed capilla ies and sinusoids, which pene a e he connec i e issue s oma su ounding he glome uli [23]. The ole o ca echolamines in he ca o id body has been in es iga ed in nume ous pha macological, physiological and biochemical s udies (see e s. 8 and 9). The ac ions o exogenously applied dopamine and dopamine gic agonis s on chemosenso y ac i i y has ecei ed conside able a en ion in ecen yea s. These s udies ha e shown ha dopamine ini ia es dose-dependen changes in ecep o po en ial and equency o chemo ecep o discha ge [6, 16, 18-20]. I emains unclea , howe e , whe he he pha macological e ec s o dopamine a e media ed ia i s di ec ac ion on he a e en e minals, o indi ec ly ia he ype I o o he ceils. In addi ion, because ca o id chemo ecep o discha ge is sensi i e o blood * To whom all co espondence should be add essed. 52 low [7, 14], in e p e a ions om he in i o e ec s o dopamine a e complica ed by possible accompanying and unce ain ascula changes [10]. Thus, while he exis ing e idence poin s o an impo an ole o dopamine in chemosenso y ansduc ion, an unde s anding o he unc ional ole o his subs ance would be conside ably enhanced by a demons a ion o he loca ion and na u e o dopamine gic ecep o s in his issue. We epo he e he esul s o ou s udy using he dopamine gic adioligand, [3H]spi ope idol [24]. Tissues om wo g oups o abbi s we e used in his s udy. In one g oup, he ca o id sinus ne es we e bila e ally esec ed unde pen oba bi al anes hesia 14 days p io o he binding assay; he o he g oup included no mal, unope a ed animals. Ca o id bodies we e emo ed o assay om pen oba bi al-anes he ized animals and cleaned o connec i e issue in ice-cold modi ied Ty ode's solu ion [1]. Each ca o id body was placed in a ial con aining 1.5 ml o Ty ode's wi h 0.1-1.45 nM [3H]spi ope idol (speci ic ac i i y - 25.1 Ci/mmol, New England Nuclea ) and incuba ed o 20 ain in a wa e ba h-shake a 37°C. The issues we e hen washed o 6 ain in 10 ml o ice-cold Ty ode's solu ion. Each ca o id body was weighed on a Cahn elec obalance i ed wi h a humidi ied chambe o p e en d ying o he issues. Ca o id bodies we e combus ed in a sample oxidize (Packa d Model 306, i ium eco e y 99+ %) be o e coun ing in a liquid scin illa ion spec ome e (Packa d Model 3385, E2/B app ox. 140 wi h Oxi luo -H20, New England Nuclea ). [3H]Hexadecane s anda ds we e used o cons uc quench co ec ion cu es h ough sample combus ion and coun ing. To al binding and non-speci ic binding we e de e mined in he absence and p esence, espec i ely, o 0.2/,M ( + )~ bu aclamol (Aye s Resea ch Labo a o ies) [22], he di e ence be ween o al and non-speci ic binding being de ined as he speci ic (displaceable) binding. The da a a e exp essed as pmol [3H]spi ope idol bound/g o issue (_+ S.E.) and he da a we e e alua ed using S uden 's - es . Ou esul s indica e he p esence o high a ini y dopamine gic ecep o s in he abbi ca o id body. Time cou se s udies showed ha speci ic binding o [3H]spi ope idol eached a pla eau a e 20 min o incuba ion and Sca cha d analysis o speci ic binding yielded a KD = 0.16 nM and a Bma~ = 2.15 pmol/g o issue. Fig. 1 shows he o al, non-speci ic and speci ic [3H]spi ope idol binding o no mal and dene a ed abbi ca o id bodies. Speci ic binding in no mal ca o id bodies was 1.63 + 0.58 pmol/g o issue, and in dene a ed issue was 0.58 + 0.28 pmol/g (s a is ically di e en a P < 0.05). Thus, ch onic ansec ion o he ca o id sinus ne e educed speci ic binding by 64%. These da a sugges ha he majo i y o speci ic dopamine gic ecep o s in abbi ca o id body a e associa ed wi h he e minals and/o ibe s o he ca o id sinus ne e. When hese indings a e conside ed in ligh o he epo ed pha macological e ec s o dopamine on he ecep o po en ial and chemo ecep o discha ge om abbi ca o id body [6, 18, 20], he esul s s ongly sugges he p esence o high a ini y dopamine gic ecep o s on he a e en e mina ions o his ne e. Fig. 1 also shows ha he non-speci ic binding o [3H]spi ope idol in bo h no mal and dene a ed ca o id bodies is la ge in compa ison o speci ic binding. This esul I _N I m. [] To al Binding [] Non- speci ic Binding • Sneci ic Bindinn 53 No mal Dene a ed Fig. 1. To al, non-speci ic and speci ic [3H]spi ope idol binding in no mal and ch onically dene a ed (ca o id sinus ne e ansec ion) abbi ca o id bodies. [3H]Spi ope idol concen a ion was 0.38 nM (nea sa u a ing). To al and non-speci ic binding we e de e mined in he absence and p esence, espec i ely, o 0.2 ~M (+)-bu aclamol, he di e ence be ween he wo being de ined as speci ic binding. Values a e exp essed in pmol [3H]spi ope idol bound/g issue ± S.E. To al binding exceeded non-speci ic binding in bo h no mal (n = 14, P < 0.025) and dene a ed (n = 40, P < 0.05) ca o id bodies, and speci ic binding in dene a ed o gans was educed by 64°7o (P < 0.05). can be a ibu ed in pa o he high deg ee o lipid solubili y o spi ope idol [22], combined wi h he una oidable p esence o adipocy es in in ac ca o id body p epa a ions. Wi h espec o speci ic binding in in ac issue, i is concei able ha in acellula accumula ion ia ca echolamine up ake mechanisms migh ha e in luenced ou kine ic da a. Howe e , se e al indings would end o a gue agains his possibili y: (1) neu olep ic d ugs simila o spi ope idol (e.g. halope idol) do no in luence dopamine up ake a he concen a ions used in ou s udy [22]; (2) he localiza ion o speci ic spi ope idol binding in in ac a s ia al issue is compa able o ha ound o homogena es [12, 13]; and (3) inhibi o s o dopamine up ake display a low a ini y o [3H]dopamine binding si es in s ia al memb anes [2]. The p ecise loca ion o he dopamine gic ecep o s emaining in dene a ed ca o id bodies canno be de e mined om ou p esen da a. These ecep o s could possibly be associa ed wi h ascula elemen s, sympa he ic ne e endings o o he non-glome ula s uc u es. Howe e , one in e es ing possibili y is ha dopamine gic ecep o s migh be loca ed on he ype I cells hemsel es. Such ecep o s would hen be analogous o he au o ecep o s which in luence dopamine me abolism in s ia al neu ons [11, 21, 25]. We a e cu en ly in es iga ing his possibili y by examining he e ec s o apomo phine and o he dopamine gic agonis s on he syn hesis and elease o ca echolamines om no mal and dene a ed abbi ca o id bodies. In conclusion, nume ous s udies in ecen yea s ha e sugges ed ha dopamine is a 54 likely candida e o neu o ansmi e o neu omodula o be ween ype I cells and hei a e en ne e e minals m he ca o id body. The esul s o he p esen s udy a e consonan wi h his hypo hesis and, in addi ion, suggesl he possible in ol emen o dopamine gic au o ecep o s in local eedback and egula ion o chemo ecep o mechanisms. This wo k was suppo ed by USPHS Resea ch G an s NS 12636 and NS 07938. We a e g a e ul o D . D.J. Ma shall o Aye s Resea ch Labo a o ies, Sain -Lau en , Quebec, Canada, o his kind assis ance in p o iding he bu aclamol o his s udy. 1 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, Ame . J. Physiol., ('ell Physiol., 2 (1977) C35 C46. 2 Bu , D.R., Enna, S.J., C eese, 1. and Snyde , S.A., Dopamine ecep o binding in he co pus s ia um o mammalian b ain, P oc. na . Acad. Sci. (Wash.), 72 (1975) 4655-4659. 3 Biscoe,I.J., Ca o id body: s uc u e and unc ion, Physiol. Re ., 51 (1971)437 495. 4 Dea naley, D.P., Fillenz, M. and Woods, R.I., The iden i ica ion o dopamine in he abbil's ca o id body, P oc. oy. Soc, B, 170 (1968) 195 * 203. 5 de Cas o, F., Su la s uc u e e I'inne a ion du sinus ca o idien de l'homme e des mammi e es. Nou eaux ai s su l'inne a iou el la onc ion du glomus ca o icum+ E udes ana omiques e physiologiques, T ab. Lab. In es , Biol. Uni . Mad ., 25 (1928) 331-380. 6 Doche y, R.J. and McQueen, D.S., ]'he e ec s o ace ylcboline and dopamine on ca o id chemosenso y ac i i y in he abbi , J. Physiol. (Lond.), 288 (1979) 411-423+ 7 Eyzagui e, C. and l.ewin, J., Chemo ecep o ac i i y o he ca o id body o he ca , J. Physiol. (Lond.), 159 (1961) 222-237. 8 Eyzagui e, C. and Fidone, S., T ansduc ion mechanisms in he ca o id body: glomus cells, pu a i e neu o ansmi e s and ne e endings, Ame . J. Physiol., Cell Physiol., in p ess. 9 Fidone, S., Gonzalez, C. and Yoshizaki, K., Pu a i e neu o ansmi e s in he ca o id body: he case o dopamine, Fed. P oc., 39 (1980) 2636-2640. 10 Goldbe g, L.I., Ca dio ascula and enal ac ions o dopamine: po en ial clinical applica ions, Pba macol. Re ., 24 (1972) 1-29. 11 Keh , W,, Ca lsson, A., Lindquis , M., Magnusson, T. and A ack, C., E idence o a ecep o + media ed eedback con ol o s ia al y osine hyd oxylase ac i i y, J. Pha m. Pha macol., 24 (1972) 744 747. 12 Ladu on, P. and Leysen, J., Speci ic in i o binding o neu olep ic d ugs in a b ain, Biochem. Pba macol., 26 (1977) 1003-1007. 13 Ladu on, P.M., Janssen, P.F.M. and Leysen, J.E., Spipe one: a ligand o choice o neu olep ic ecep o s, 3. Subcellula dis ibu ion o neu olep ic d ugs and hei ecep o s in a ious a b ain a eas, Biochem. Pha macol., 27 (1978) 327 328. 14 Landg en, S. and Neil, E., Chemo ecep o impulse ac i i y ollowing hemo hage, Ac a physiol. scand., 23 (1951) 158-167. 15 Le e , J.D., Lewis, P.R. and Boyd, J.D., Obse a ions on he ine s uc u e and his ochemis y o he ca o id body in he ca and abbi , J. Ana . (Lond.), 93 (1959) 478-490. 16 Llados, F. and Zapa a, P., E ec s o dopamine analogues and an agonis s on ca o id body chemosenso s in si u, J. Physiol. (Lond.), 274 (1978) 487-499. 17 McDonald, D.M. and Mi chell, R.A., The inne a ion o glomus cells, ganglion cells and blood essels in a ca o id body: a quan i a i e ul as uc u al analysis, J. Neu ocy ol., 4 (1975) 177-230. 18 Mon i-Bloch, L. and Eyzagui e, C., A compa a i e physiological and pha macological s udy o ca and abbi ca o id body chemo ecep o s, B ain Res., in p ess. 55 19 Sampson, S.R., Amino , M.J., Ja e, R.A. and Vid uk, E.H., Analysis o inhibi o y e ec o dopamine on ca o id body chemo ecep o s in ca s, Ame . J. Physiol., 230 (1976) 1494-1498. 20 Sampson, S.R. and Vid uk, E.H., Hype pola izing e ec s o dopamine on chemo ecep o ne e endings om ca and abbi ca o id bodies in i o, J. Physiol. (Lond.), 268 (1977) 211-221. 21 Sed all, G., Recep o eedback and dopamine u no e in CNS. In L.L. l e sen, S. l e sen and S. Snyde (Eds.), Handbook o Psychopha macology, Vol. 6, Plenum P ess, 1975, pp. 127-177. 22 Seeman, P., An i-schizoph enic d ugs - memb ane ecep o si es o ac ion, Biochem. Pha macol., 26 (1977) 1741-1748. 23 Seidl, E., On he mo phology o he ascula sys em o he ca o id body o ca and abbi and i s ela ion o he glomus Type I cells. In M.D. 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. 293-300. 24 Snyde , S.H., Bu , D.R. and C eese, 1., The dopamine ecep o o mammalian b ain: di ec demons a ion o binding o agonis and an agonis s a es, Neu osci. Abs ., 2 (1976) 28-49. 25 Wes all, T.C., Besson, M.-J., Gio guie , M.-F. and Glowinski, J., The ole o p esynap ic ecep o s in he elease and syn hesis o 3H-dopamine by slices o a s ia um, Naunyn- Schmiedebe g's A ch. Pha macol., 292 (1976) 279-287.