Synthesis and Release of Catecholamines by the Cat Carotid Body in Vitro: Effects of Hypoxic Stimulation

B ain Resea ch, 374 (1986) 101-109 101 Else ie BRE 11718 Syn hesis and Release o Ca echolamines by he Ca Ca o id Body in Vi o: E ec s o Hypoxic S imula ion R. RIGUAL, E. GONZALEZ, C. GONZALEZ and S. FIDONE Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Valladolid (Spain) and Depa men o Physiology. School o Medicine, Uni e si y o U ah, Sal Lake Ci _ , UT ( U. S. A. ) (Accep ed Oc obe 9 b, 1985) Key wo ds: ca o id body -- chemo ecep o -- hypoxia -- ca echolamine -- dopamine elease -- ca o id sinus ne e ac i i y The ole o ca echolamines (CAs) in ca ca o id body chemo ecep ion has been con o e sial. On he basis o pha macological ex- pe imen s, i would appea ha endogenous dopamine (DA) may ac ei he as an inhibi o y o exci a o y ansmi e . Neu ochemical s udies on he e ec s o na u al s imula ion on he elease o ca o id body CAs in he ca ha e also been inconclusi e. In he p esen s udy, we ha e cha ac e ized he syn hesis and elease o CAs in he in i o ca ca o id body p epa a ion in esponse o di e en le els o hypoxic s imula ion and ha e co ela ed hese measu es wi h he chemosenso y ac i i y o he ca o id sinus ne e. The syn hesis o [3H]DA and [3H]no epineph ine was linea o a leas 4 h in ca o id bodies incuba ed wi h hei na u al p ecu so [~H] y osine. Syn- hesis o bo h [3H]CAs pla eaued when he [3H] y osine concen a ion in he media eached 40 uM, which is a concen a ion simila o ha ound in ca plasma. Exposu e o he animals o an a mosphe e o 10% 02 in N~ o 3 h p io o emo al and incuba ion o he ca- o id bodies wi h [3H] y osine esul ed in an app oxima ely 100% inc ease in he a e o [3H]DA syn hesis bu no change in [3H]no epi- neph ine syn hesis. This selec i e inc ease in [3H]DA syn hesis was no de ec ed when [3H]dihyd oxyphenylalanine was used as p e- cu so . Ca o id bodies i s incuba ed wi h [3H] y osine and la e supe used wi h solu ions equilib a ed wi h di e en gas mix u es (0 100% 0 2 in N2) exhibi ed an inc ease in [3H]DA elease and ca o id sinus ne e discha ge which we e in e sely ela ed o he oxygen concen a ion. This ela ionship was s onges o he weake s imuli (be ween 50% and 20% O 2 in N2), whe e bo h ne e ac i i y and [3H]DA elease inc eased almos in pa allel. Wi h lowe oxygen concen a ions (10% O 2 and 0% 02 equilib a ed solu ions), he in- c ease in he elease o [3H]DA was p opo ionally g ea e han he inc ease in ca o id sinus ne e discha ge. Ou esul s demons a e ha hypoxic s imula ion inc eases bo h he a e o syn hesis and elease o DA in he ca ca o id body. Al hough he p ecise ole o DA in his chemo ecep o o gan is p esen ly unknown, ou indings sugges ha his biogenic amine plays a di ec ole in gene a ing o con olling he elec ical ac i i y in he ca o id sinus ne e. INTRODUCTION The mammalian ca o id body is an a e ial chemo- ecep o o gan ac i a ed by low paO2, low pH and high p~CO2 ( e . 21). S uc u ally, he ecep o com- plex is o med by clus e s o wo ypes o cells, he ype I and ype II cells; he clus e s lie wi hin a sup- po ing connec i e issue ma ix con aining a dense capilla y ne 24, 41. Senso y ibe s o he ca o id sinus ne e (CSN) pene a e hese cell clus e s o end in synap ic apposi ion wi h ype I cells, which a e con- side ed o be p eneu al ( ecep o ) elemen s. Two pe ennial issues pe aining o a e ial chemo ecep- ion, conce n i s , whe he he ype 1 cells a e in ac he sole ansduce elemen s in his senso y ecep o , and, second, wha a e he unc ions o he se e al pu- a i e neu o ansmi e s con ained in hese cells (do- pamine (DA), no epineph ine (NE), ace ylcholine (ACh), Leu- and Me -enkephalin, subs ance P (SP) and se o onin (5-HT); o discussion, see e s. 2, 10, 12-14 and 22). In ecen yea s, pa icula a en ion has ocused on he ole o he ca echolamines (CAs), mos no ably DA, in he chemo ecep ion p ocess ( o e iew o hese s udies, see e . 12). The wo gene al expe i- men al app oaches which ha e been used in hese Co espondence: R. Rigual, Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Valladolid, Spain. 0006-8993/86/$03.50 © 1986 Else ie Science Publishe s B.V. (Biomedical Di ision) Ill2 s udies a e: (1) he co ela ion o neu ochemical changes in he me abolism o ca o id body (iAs (e.g. syn hesis, elease, ecep o localiza ion, e c. ) wi h changes in chemo ecep o ac i i y, and (2) he pha - macological cha ac e iza ion o he e ec s o ca e- cholamine gic agonis s and an agonis s on CSN dis- cha ge. In he ca , hese neu ochemical s udies disag ee in ega d o he e ec s o chemo ecep o s imula ion on CA con en o he ca o id body. Thus, Zapa a e al. 4a showed ha p olonged se e e hypoxic s imula- ion in i o (3 h; 5% O 2 in N 2, anes he ized ca ) o in i o (2 h in anoxia) ailed o modi y ca o id body CAs. On he o he hand, Mills and Slo kin 2s epo ed a ma ked dec ease in CA con en o he o gan a e only 40 ain o mild hypoxia (14% O 2 in N2). Fi z- ge ald e al. 15 also ound a dec ease in DA con en in c -me hyl-p- y osine ea ed ca s a e 30 ain o se- e e hypoxia (paO2 = 25 o ). Finally, S a linge e al. 37 obse ed ha 1 h o hypoxia (16% O2) p oduced only a ma ginal dec ease in NE con en o he ca o id body. S udies aimed a cha ac e izing he pha macolog- ical ac ions o exogenously applied DA and i s agon- is s and an agonis s ha e also p oduced some con- ounding esul s. A common inding om hese in- es iga ions has been ha DA may beha e ei he as an exci a o y o inhibi o y agen in he ca o id body, depending upon he dose o he adminis e ed agen 43, he animal species s udied 29, and in pa ic- ula whe he he o gan is s udied in i o o in i o (e.g. compa e e . 6 s 29 and e . 7 s 33). Howe e , as poin ed ou by McQueen 27, such con lic ing pha - macological s udies need no e lec eal di e ences in he ac ions o DA in hese di e en p epa a ions o species bu may simply esul om uncon olled expe imen al a iables such as ascula e ec s in i o o pe haps ecep o desensi iza ion in i o, e c. This explica ion no wi hs anding, i is e iden ha he unc ion o CAs in ca ca o id body chemo ecep- ion is no clea . I is well ecognized ha he ac ions o a gi en pu- a i e neu o ansmi e a e e lec ed in i s me abolic esponse o na u al s imula ion ,26,3~. Consequen ly, he p esen s udy was unde aken o cha ac e ize he e ec s o hypoxic s imula ion on he syn hesis o CAs by he ca ca o id body and o moni o di ec ly he e- lease o CAs om his o gan as a unc ion o he in- ensi y o hypoxic s imula ion and he elec ical ac i - i y o he ca o id sinus ne e MATERIAL,S AND METI OI)S Su gical p ocedu es. All expe imen s we e pe - o med wi h adul ca s (2-3.5 kg) anes he ized wi h sodium pen oba bi al (30-40 mg/kg, i.p.: Sigma). The animals we e acheos omized and a i icially en ila ed wi h a Palme espi a o (30 ml: 25 s o- kes/min). A e su gically exposing he a ea o he ca o id bi u ca ion, he ca o id body (in some expe i- men s, wi h he CSN a ached) was quickly emo ed and placed in a clea Luci e chambe illed wi h ice- cold 100% Ox-equilib a ed Ty ode solu ion (in aM): NACI, 112; KCI, 4.7: CaCI2, 2.2; MgCI> 1.1; Na-glu ama e. 44; N-2-hyd oxye hylpipe azine-N'- 2-e hanesul onic acid (HEPES), 5; glucose, 5.5: he pH was adjus ed o 7.40 wi h N NaOH. Using a dissec ing mic oscope equipped wi h cold illumina- ion, he ca o id body was cleaned o su ounding loose connec i e issue, capsule and pe iglome ula ascula plexus. Fo ch onic dene a ion expe i- men s, he CSN o he supe io ce ical ganglion was emo ed unila e ally unde asep ic condi ions 12--15 days p io o emo al o he ca o id body; he con a- la e al o gan hen se ed as con ol. Finally, in ano he g oup o expe imen s, ca s we e exposed o a 3-h pe iod in a chambe i~ con inu- ously lushed wi h 10% O, in N_, jus p io o emo al o he ca o id bodies; con ol animals o his "hypox- ic' g oup we e placed in he chambe o b ea he oom ai . The animals we e en ila ed wi h he co e- sponding gas mix u e du ing su ge y. Ca echolamine syn hesis. Ca o id bodies we e placedin mini ials con aining 0.5 ml o 100% O,- equilib a ed Ty ode solu ion, which also con ained 100/,M o he y osine hyd oxylase co ac o , 6-me h- yl e ahyd op e ine (6-MPHa; Sigma), 1 mM asco - bic acid and 10-80/,M [3H] y osine ([2,6 -~H] y o- sine, 1 Ci/mmol; Ame sham). Incuba ion p oceeded a 37 °C in a me abolic shake (New B unswick) a 75 pm o 0.5-4 h. In some expe imen s, [3H]dihy- d oxyphenylalanine, ([3H]DOPA; 10 uM; 1 Ci/ mmol, Ame sham) was used as p ecu so o [3H]CA syn hesis, and he y osine hyd oxylase co ac o was omi ed. A he end o he incuba ion pe iod, he is- sues we e washed in 10 ml o ice-cold 100% O2-equi- lib a ed Ty ode solu ion o 5 min o emo e excess labelled p ecu so , a e which he samples we e p ocessed o analysis. Ca o id bodies we e weighed on a Cahn elec obalance p o ided wi h a humidi ied chambe , homogenized and he [3H]CAs sepa a ed by high- ol age pape elec opho esis. As p e iously desc ibed ll, he pape s ips we e scanned o adio- ac i i y agains s anda d s ips con aining [3H] y o- sine, [3H]DA and [3H]NE. The a eas con aining hese subs ances we e combus ed in a sample oxidiz- e and coun ed in a scin illa ion coun e (40% coun - ing e iciency). Resul s he e a e exp essed as nmol/g issue (mean ca ca o id b.w ., 559 + 219 (S.D.) ~g; n = 70). In o he expe imen s pe o med a he De- pa men o Physiology in Valladolid, he CA le els we e de e mined by hin-laye ch oma og aphy, acco ding o he me hod o Fleming and Cla k 16, ex- cep ha he pla es we e s ained wi h a e icyanide- based eagen (0.05% po assium e icyanide + 1% e ychlo ide) o minimize quenching. The colo ed spo s we e sc aped o , elu ed by igo ous shaking (1 ain) in 1 ml o 1 N HC1 and coun ed wi h 30% e i- ciency. Resul s a e exp essed as pmol CA/ca o id body. Ca echolamine elease. These expe imen s we e ca ied ou as desc ibed p e iously TM. B ie ly, ca o id bodies we e incuba ed o 3 h wi h [3H] y osine o high speci ic ac i i y (20-40 Ci/mmol; Ame sham) o label issue CA s o es. A he end o he incuba ion pe iod, he o gans we e moun ed in a supe usion chambe which allowed simul aneous eco ding o CSN ac i i y and collec ion o he supe usa es con- aining he eleased labelled CAs. Collec ion pe iods (see Fig. 1) we e g ouped in o 's imulus cycles', wi h each cycle consis ing o a 5- ain con ol pe iod (su- pe usion wi h 100% Oz-equilib a ed Ty ode), ol- lowed by a 5- ain s imulus pe iod (supe usion wi h Ty ode equilib a ed wi h di e en low Oz-gas mix- u es; 's im' in Fig. 1), which was ollowed in u n by 4 pos -s imulus pe iods o 5 o 10 min du a ion (su- pe usion again wi h 100% Oz-equilib a ed Ty- ode). The o al du a ion o a single s imulus cycle was 45 min, and each expe imen consis ed o 3-5 consecu i e cycles. CSN ac ion po en ials we e am- pli ied o display on an oscilloscope and led h ough a window disc imina o o a coun e -p in e . The su- pe usa es om each pe iod we e collec ed in ials con aining a ca ie mix u e consis ing o 0.3 M ace- E O (D E "[3 C n S im 103 d E l 0 )< E "o ~OC Fig. 1. A: elease o [3H]CAs om he ca ca o id body du ing a single 's imulus cycle'; and B: hin-laye ch oma og aphic (TLC) iden i ica ion o he eleased ma e ial. In bo h A and B, he le -mos ba ep esen s he con ol pe iod (5 min), he ba labelled S im co esponds o he s imulus pe iod (5 min), and he emaining ba s a e o pos s imulus pe iods o 5, 10, 10 and 10 min du a ion. A shows he adioac i i y p esen in he alumi- na elua es; he solid po ion o each ba ep esen s he adioac- i i y iden i ied by TLC as [3H]DA + [3H]DOPAC, while he s ippled po ion ep esen s uniden i ied adiolabelled com- pounds. B shows [3H]DA (uppe ba s) and [3H]DOPAC (lowe ba s) p esen in he alumina elua es om he co esponding ac ions o he s imulus cycle. Owing o he small a e o [3H]NE syn hesis by he ca o id bodies, [3H]NE o i s ca abo- li es we e no measu ed in hese expe imen s. ic acid, 1.0 mM asco bic acid and 100 iM unlabelled DA, inal pH 3.6. The samples we e hen adso bed wi h alumina a pH 8.6 and elu ed wi h 1 N HC1 o e- co e [3H]CAs and [3H]ca echol ca aboli es. In some expe imen s, he 1 N HC1 elua es we e d ied in a acuum concen a o (Sa an ) and hin-laye ch o- ma og aphed a6. Since mos o he adioac i i y p es- en in he elua es could be iden i ied as [3H]DA + di- hyd oxyphenylace ic acid ([3H]DOPAC) (Fig. 1), he alumina elua es in mos expe imen s we e di ec - Ii14 13n E c- 60 l 4O 20 0 3H-Ty A zx 3H_DA _ _ 1 2 3 4 Time(h) o 3H-Ty B T 90 3H- DA ~:~ 60 Ol O E c 30-- 0 :- -~ ~ : - 20 40 60 80 Ty (/aM ) Fig. 2. A: ime-cou se o [3H]CA syn hesis om [3H] y osine. Ca o id bodies om ca s b ea hing oom ai we e incuba ed wi h 40/ M [3H] y osine in he p esence o 10 -4 M MPH 4 and 10 -3 M asco bic acid. A he end o he incuba ion pe iod, pH]DA, pH]NE and [3H] y osine we e sepa a ed by high- ol- age pape elec opho esis. B: syn hesis o [3H]CAs as a unc- ion o [3H] y osine concen a ion in he incuba ion media; in- cuba ion condi ions and measu emen o pH]CAs as in A. Da a a e means + S.E.M. o 5-8 ca o id bodies. ly coun ed and he esul an adioac i i y aken as a di ec measu e o [3H]DA elease. The elease o labelled CAs is exp essed in e ms o basal, o con ol, elease; he mean alue o e- lease om he con ol pe iods o wo consecu i e s imulus cycles was aken as basal elease/5 min du - ing ha in e ening cycle. Any elease abo e his basal le el du ing he s imulus pe iod was conside ed o be s imulus- ela ed elease. Basal e sus s imulus- ela ed discha ge om he CSN was e alua ed in a simila manne . RESULTS Syn hesis' o " [3H]ca echolamines by he ca ca o id body The i s g oup o expe imen s es ablished he ime-cou se o [3H]CA syn hesis om 40 uM [3Hl y- osine (Fig. 2A), a concen a ion simila o ha p es- en in ca plasma 39. Unde hese condi ions, syn hesis o bo h [3H]DA and [3H]NE was linea up o 4 h, in- dica ing adequa e a ailabili y o bo h p ecu so and co ac o . Nea maximal accumula ion o ee [3HI y- osine by he ca ca o id body was achie ed a e 3() ain o incuba ion, which was simila o ha ound o he abbi 1~. The in acellula concen a ion o [3H] y osine a his poin was es ima ed (assuming ha 40% o he ca o id body we weigh ep esen s in acellula wa e ) o be app oxima ely 100 uM, which is sa u a ing o y osine hyd oxylase 2<3-: The ela ionship be ween pH] y osine concen a- ion and [3H]CA syn hesis was s udied m ca o id bod- ies incuba ed o 2 h wi h he y osine hyd oxylase co ac o , 6-MPH 4, a a concen a ion o I(I ~ M. As shown in Fig. 2B, he syn hesis o bo h [-~H]DA and [3H]NE pla eaued a app oxima ely 40/¢M [3H] y o- sine, as p edic ed om he da a shown in Fig. 2A. The accumula ion o ee [3H] y osine by he issues inc eased linea ly wi h he media concen a ion. E ec s o sympa hec omy and CSN dene a ion on p H]ca echolamine syn hesis Sympa he ic ne e endings a e known o be p es- en in he ca o id body 24, and Table I (A) shows ha ollowing ch onic sympa hec omy ( emo al o he su- pe io ce ical ganglion 12-15 days p io o expe i- men a ion), he e was a 40% educ ion (P < 0.02 in [3H]NE syn hesis bu no change in [3H]DA syn hesis. F om hese da a and hose p esen ed in Fig. 2, he a- io o [3H]DA s [3H]NE syn hesis is abou 20:1. Conside ing ha he endogenous le els o DA and NE in he ca ca o id body a e simila 15, ~7 (also un- published da a), ou indings sugges a much slowe u no e a e o NE han o DA in he pa enchymal issue o he ca o id body. Addi ionally, since he en- dogenous con en o NE is unchanged a e sympa- hec omy (unpublished obse a ions), hese da a would also indica e ha he u no e a e o NE is highe o he sympa he ic endings han o he glo- mus cells. In ou p e ious s udies wi h he abbi , an TABLE I E ec s o sympa hec omy and CSN dene a ion on he a e o [3H]CA syn hesis in ca ca o id body Supe io ce ical ganglia and CSNs we e su gically emo ed 12-15 days p io o expe imen a ion. Dene a ed ca o id bod- ies and hei co esponding con ala e al con ols we e incu- ba ed o 3 h in 40/~M pH] y osine, 10 -4 6-MPH 4 and 10 3 M asco bic acid. Da a a e means + S.E.M. o 6-8 ca o id bodies and a e exp essed as pmol/ca o id body/3 h. [3H]Ty pH]DA FH]NE (A) Con ol 23.8 + 3.9 19.2 + 3.26 1.1 _+ 0.40 Sympa hec omy 27.37 + 5.17 18.0 _+ 5.62 0.66 _+ 0.26* (B) Con ol 17.2 + 2.82 20.7 + 3.8 1.0 + 0.18 Dea e en a ion 26.6 + 5.94 20.6 + 4.6 1.5 + 0.15" * P < 0.02 (pai ed - es ). 80% educ ion in [3H]NE syn hesis was ound o sympa hec omized ca o id bodies, wi h no change in [3H]DA syn hesis 11. These esul s migh mean ha he e is a mo e p o use sympa he ic inne a ion o he ca o id body in he abbi han in he ca . The e ec s o CSN dene a ion on [3H]CA syn he- sis was also in es iga ed because his ne e has been shown o exe ophic in luences on he ca o id body, including modi ica ions o y osine hyd oxyl- ase ac i i y and elec ical p ope ies o ype I cells 9,17. As shown in Table I (B), a 50% inc ease in he a e o ca o id body [3H]NE syn hesis (P < 0.02) was ound 12-15 days ollowing CSN ansec ion. This obse a ion is consis en wi h ou p e ious ind- ings o he abbi ca o id body, whe e a 17% in- c ease in [3H]NE syn hesis was obse ed 11. The s ud- ies o Vazquez-Nin e al. 40, which show ha sympa- he ic ne e endings in he ca ca o id body unde go sp ou ing a e CSN ansec ion, sugges a plausible explana ion o he obse ed inc ease in [3H]NE syn- hesis. E ec s o hypoxia on [3H]ca echolamine syn hesis om [~H] y osine s [3H]DOPA Unanes he ized animals we e exposed o 3 h in a chambe , ei he o 10% O2 in N2 (hypoxia) o o oom ai (no moxia). The ca o id bodies we e quick- ly emo ed and incuba ed ei he o 3 h in 40 pM [3H] y osine o o 2 h in 10 pM [3H]DOPA (see e . 11). The esul s o hese expe imen s a e p esen ed 105 in Fig. 3A ([3H] y osine) and Fig. 3B ([3H]DOPA). The hypoxic ca o id bodies incuba ed wi h [3H] y o- sine exhibi ed app oxima ely a 100% inc ease in he a e o [3H]DA syn hesis (P < 0.01) bu no change in [3H]NE syn hesis. Thus, he e ec o hypoxia in in- c easing only he a e o [3H]DA syn hesis in he ca ca o id body ag ees wi h ou p e ious indings o he abbi 13. We also obse ed a signi ican (P < 0.01) in- c ease in he accumula ion o ee [3H] y osine in hy- poxic ca o id bodies compa ed o con ol o gans. This inc ease in ee [3H] y osine migh ha e con ib- u ed o he obse ed inc ease in [3H]DA syn hesis, bu his con ibu ion should ha e been minimal be- cause (1) he e was no inc ease in [3H]NE syn hesis, A 80 60 -~ 4c 0 E c 2() No moxia Hypoxia Ty NE DA Ty NE DA 15 10 IJ 0 E - 5 No moxia Hypoxia o NF DA NF IDA Fig. 3. E ec s o hypoxic exposu e (10% 02 in N2; 3 h) o he animals on he syn hesis o [3H]CAs by he ca ca o id body. A: a e he hypoxic episode, ca o id bodies we e emo ed om he animals and incuba ed in he p esence o 40 ~M [3H] y o- sine, 10 4 M 6MPH 4 and 10 -3 M asco bic acid. The le els o [3H] y osine, [3H]DA and [3H]NE we e de e mined as in Fig. 2. B: a e iden ical hypoxic episodes, ca o id bodies we e incu- ba ed o 2 h wi h 10/~M [3H]DOPA. This incuba ion ime and concen a ion we e chosen because syn hesis was linea o up o 4 h wi h as much as 60pM [3H]DOPA. In bo h A and B, no - moxic animals we e simila ly exposed o oom ai in he cham- be . Da a a e means + S.E.M. o a leas 6 ca o id bodies. In A, y osine and DA in no moxia s hypoxia a e s a is ically ( - es ) di e en a he P < 0.01 le el. 106 (2) as shown in Fig. 2B, when ca o id bodies we e in- cuba ed wi h 80 ~ M [3H] y osine, he a e o [3H]CA syn hesis was no signi ican ly inc eased despi e a 100% inc ease in issue le els o ee [3H] y osine, and (3) a simila inc ease in [3H]DA syn hesis was obse ed in hypoxic abbi ca o id bodies wi hou any inc ease in ee [3H] y osine le els in he issues. Thus, he speci ic inc ease in [3H]DA syn hesis a e he hypoxic episode p obably esul ed om an in- c eased elease o endogenous DA du ing he hypox- ic episode and subsequen emo al o eedback inhi- bi ion exe ed by DA a he le el o y osine hyd ox- ylase, he a e-limi ing s ep in CA syn hesis 13.3°,35.42. Consis en wi h his in e p e a ion, ou esul s showed ha when he y osine hyd oxylase s ep is ob ia ed by incuba ion o he issue wi h pH]DOPA ins ead o [3H] y osine (Fig. 3B), he hypoxic epi- sode ailed o p oduce any change in he a e o [3H]DA syn hesis; he same lack o e ec o hypoxia was seen in abbi ca o id bodies incuba ed wi h [3H]DOPA 13, as well as in o he issues a e physiol- ogic s imula ion 36,42. E ec s o low oxygen on [3H]ca echolamine elease and CSN discha ge In p elimina y expe imen s, i was ound ha he basal e iux o [3H]DA (syn hesized om [3H] y o- sine) om ca ca o id bodies exhibi ed wo phases: one, las ing app oxima ely 2 h, in which he e iux dec eased e y apidly, and ano he , which we ol- lowed o up o 8 h, in which he basal e iux o [3H]DA dec eased e y slowly wi h ime. In s udying he s imulus-induced elease o [3H]CAs, he collec- ion pe iods o 's imulus cycles' (see Ma e ials and Me hods) we e begun a e he ca o id bodies had i s been pe used o 2 h wi h 100% Oz-equili- b a ed Ty ode. This p ecau ion was aken in o de o minimize possible e o s a ising om ime-depend- en p ocesses (e.g. emp ying o di e en [3H]CA pools), a he han s imulus- ela ed phenomena. Ad- di ionally, he o de o p esen a ion o he s imuli was andomized in he di e en expe imens o a oid sys ema ic e o s a ising om epea ed placemen o a gi en s imulus in he same egion o he [3H]DA de- cay cu el4. Fig. 4 p esen s a ypical expe imen con- sis ing o h ee s imulus cycles: he s imuli we e 40%, 20% and 10% O2 in N2 o he i s h ough hi d cy- cles. espec i ely, and he du a ion o s imula ion c l % E " l 0 < 121 I 4 / ,.., -~- ..... .~o*,. o~ A ~' ......... I(~*/o 02 :~ ' 250 ~ :l ! 5o ~ 40%02 20"/. 02 10"/. 02 I I 1 2 3 4 Time (In) Fig. 4. The ela ionship be ween CSN discha ge (inse )and [3H]DA elease om a ca o id body p e iously incuba ed o 3 h in he p esence o [3H] y osine (40 ~M; speclac . 20 Ci/ mmol), 10 -4 M 6 MPH 4 and asco bic acid (10 -3 M). P epa a- ion was supe used wi h solu ions equilib a ed wi h low-O 2- gas mix u es (40, 20 and 10% 02 in N2) a he imes indica ed in he igu e. The s ippled a eas ep esen s imulus-induced e- lease o [3H]DA. was 5 min in each cycle, As e iden om Fig, 4, he elease o [3H]DA was ela ed o he s eng h o he iow-O2 s imulus. CSN ac i i y eco ded du ing he s imulus pe iod in each cycle is shown a he op o Fig. 4. Al hough he peak CSN discha ge in esponse o 20% and 10% 0 2 in N2 we e simila , he ime- o- peak ac i i y was longe wi h 20% 0 2 , and he e- sponse ailed o adap . Fig. 5 summa izes he cha ac- e is ics o CSN esponse o low-O 2 s imuli, and he ela ionship o [3H]DA elease (da a a e exp essed as mean + S.E.M, o 8-15 esponses). Fig, 5A shows he ime- o-peak CSN discha ge om he onse o he s imulus, g aphed as a unc ion o he s eng h o he low-O2 s imulus. I can be seen ha his ime a ies om as li le as 1 min wi h he s onges s imulus, o mo e han 4 min o 50% 0 2 in N 2. This is simila o he esul s ob ained by Eyzagui e and Lewin8 wi h he in i o ca ca o id body p epa a ion, whe e he a e o change o CSN discha ge in esponse o hy- poxia was dependen on he s eng h o he hypoxic s imulus; hei imes- o-peak esponses we e simila o hose epo ed he e. Fig. 5B shows he ela ionship be ween CSN ac i - i y and elease o [3H]DA o di e en low-O2 s imu- li. Be ween 50% and 20% O2 in N 2 he e was a close ela ionship be ween elease and discha ge, bu wi h s onge s imuli (10% 02 and 0% 02 in N2), he e- lease o [3H]DA inc eased ma kedly wi hou any sig- ni ican u he inc ease in CSN ac i i y. This ela- ionship is quali a i ely simila o ha obse ed wi h he abbi ca o id body, al hough in he abbi he in- c ease in [3H]DA elease and CSN ac i i y was mo e closely ela ed a he highe s imulus s eng hs ~4. 3001 A 2o0 u'l , , 5O 40 3O 2O 10 0 °/o 02 in N2 o 0 u ;,,( .> o ~5 u L Ld 10 0 E~ o3H-DA eleased 13 A e age disc ha ge .~- I I I I I .50 40 30 20 10 0 :'/o 02 in N2 4O 2 O L_ O U 20 l O II/ O " < C3 ± O Fig. 5. Cha ac e is ics o CSN discha ge and [3H]DA elease in esponse o hypoxic s imula ion o he ca o id body (da a a e means + S.E.M. o 8-15 esponses o di e en low-O 2 s imu- li). A: ime- o-peak CSN discha ge in esponse o di e en low-O 2 s imuli. B: ela ionship be ween [3H]DA elease and a e age s peak CSN discha ge in ela ion o he in ensi y o he low-O~ s imulus. 107 DISCUSSION In he p esen s udy, we ha e desc ibed some cha - ac e is ics o [3H]CA syn hesis and elease in he in i o ca ca o id body p epa a ion. We ha e ound ha syn hesis o [3H]DA and [3H]NE om [3H] y o- sine was linea o up o 4 h and ha he a e o syn- hesis pla eaued when he media concen a ion o [3H] y osine eached 40 ~M, a le el simila o ha ound in ca plasma 39. These esul s indica e ha y- osine concen a ion is p obably no a limi ing ac o o CA syn hesis in i o in he ca ca o id body. Unde es ing condi ions, he a es o [3H]DA and [3H]NE syn hesis we e app oxima ely 10 and 0.5 nmol/g issue/h (6.5 and 0.35 pmol/ca o id body/h), espec i ely, yielding a [3H]DA:[3H]NE a io o ap- p oxima ely 20. The DA:NE a io o endogenous CAs in he ca ca o id body a ies conside ably om s udy o s udy (be ween 0.3 and 4.0; see e . 12 o e- iew), bu whiche e alues a e aken, ou esul s clea ly indica e a slowe u no e o NE han DA in he ca ca o id body. These da a a e in ag eemen wi h he obse a ions o Belmon e e al 3, who ound low le els o dopamine- l-hyd oxylase in he ca ca- o id body, and wi h he ecen s udies o S a linge e al.3V, who desc ibed o his animal a e y low a e o he in i o con e sion o y amine o oc opamine a e inhibi ion o monoamine oxidase. Since his con e sion is an index o dopamine- l-hyd oxylase ac i i y, he au ho s concluded ha he high NE con- en mus esul om ei he high e en ion and/o low deg ada ion o he amine, a he han om a high a e o syn hesis. Mo eo e , i has been demon- s a ed 15 ha exposu e o ca s o hypoxia esul ed in dec eased ca o id body DA wi hou any change in NE con en , which also sugges s a slow a e o NE u iliza ion du ing hypoxia. O cou se, he possibili y does exis ha he ca ca o id body in i o migh no syn hesize NE a he no mal in i o a e. Boadle- Bibe and Ro h 4 desc ibed dec eased NE syn hesis in i o o he a as de e ens, i.e. a highe DA:NE a- io o syn hesis in i o compa ed o in i o. In he guinea pig, on he o he hand, hese au ho s showed ha he DA:NE a ios we e he same o bo h he in i o and he in i o p epa a ions. I is no ewo hy, howe e , ha in hei expe imen s asco bic acid was absen om he in i o incuba ion media. This sub- s ance is an impo an co- ac o o he enzyme dopa- I~Jg mine- l-hyd oxylase, which con e s DA o NE 3~, and hus, he disc epan a ios which hey obse ed o he a migh ha e esul ed om a lack o asco bie acid. In ou expe imen s, he incuba ion media al- ways con ained 1 mM asco bic acid, and hus he like- lihood is small ha he low NE syn hesis which we obse ed wi h he in i o ca ca o id body p epa a- ion was due o a comp omised syn he ic machine y. The e ec s o CSN dene a ion and sympa hec o- nay on he a e o syn hesis o [3H]CAs in he ca ca- o id body a e simila o ou p e ious indings wi h he abbi L~, and wi h hose o Hanbaue and Hell- s om o he a TM, i.e. no change in DA syn hesis o con en ollowing ei he kind o dene a ion bu a signi ican dec ease in bo h NE syn hesis and con en a e sympa hec omy and a endency owa ds hei inc ease a e CSN dene a ion. The e ec s o hypoxia on [3H]CA syn hesis in he ca ca o id body a e also he same as hose p e iously epo ed o he abbi ~3. The speci ic inc ease in [3H]DA syn hesis sugges s ha NE may be con ained in di e en glomus cells han DA. In his ega d, i is in e es ing o no e ha wo subpopula ions o ype I cells ha e been dis inguished on he basis o ul a- s uc u al 2°,25, cy ochemicallg, 38 and immunohis o- chemical s udies 5, sugges ing ha NE may be p esen in only one o hese cell ypes. Howe e , he exis - ence o wo dis inc subpopula ions o ype I cells in he ca o id body has been a con o e sial issue, and his p oblem emains la gely un esol ed a he p es- en ime (see e . 12). The ela ionship be ween [3H]DA elease and CSN ac i i y a weak o mode a e le els o low-O e s imula ion sugges s ha endogenous DA may pa - icipa e in he genesis o he chemo ecep o e- sponse. Al hough s onge s imuli esul ed in disp o- po iona ely small inc eases in CNS discha ge m compa ison o he la ge inc eases in [3H]DA elease, his obse a ion migh be due in pa o he ollowing: (1) sho e du a ion o ac ion o he weake s imuli due o less s eep g adien s o exci a ion o he che- mo ecep o s (see ime- o-peak CSN esponse s s eng h o low-O 2 s imulus, Fig. 5A), (2) occlusion o he eco ded ne e spikes wi h s onge s inmli in ou whole ne e eco dings, and (3) sa u a ion o lhe neu al componen o he ecep o esponse and adap a ion o he chemo ecep o s a high s imulus s eng hs. In conclusion, ou s udy o he syn hesis and e- lease o [3H]CAs in he ca ca o id body has shown ha he basic neu ochemical aspec s o [~H]DA me- abolism in his o gan a e simila o hose desc ibed ea lie o he abbi and a ca o id body. These indings help esol e he con o e sy ega ding he e ec s o hypoxia on he elease o DA in he ca (see In oduc ion) and sugges ha he appa en species di e ences obse ed in pha macological expe i- men s do no accu a ely e lec he physiological unc ion o his amine. I is likely, he e o e, ha in all hese species his pu a i e neu o ansmi e may play analogously impo an oles in he chemo ecep- ion p ocess. ACKNOWLEDGEMENTS Suppo ed by a g an om he C.A.I.C.T~ o Spain and USPHS G an s NS-12636 and NS-07938. We g a e ully acknowledge he echnical assis ance o M . La y Jones and he e o s o Ms. Vicki Skel on in he p epa a ion o he manusc ip . 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