Increased release of [3H]dopamine during low O2 stimulation of rabbit carotid body in vitro

Neu oscience Le e s, 6 (1977) 95--99 © Else ie /No h-Holland Scien i ic Publishe s L d. 95 INCP~ASED RELEASE OF [SH]DOPAMINE DURING LOW O2 STIMULATION OF RABBIT CAROTID BODY IN VITRO C. GONZALEZ and S. FIDONE* Depa men o Physiology, Uni e si y o U ah College o Medicine, Sal Lake Ci y, U ah 84108 (U.S.A.) (Recei ed July 30 h, 1977) (Accel~ ed Augus 3 d, 1977) SUMMARY Rabbi ca o id bodies syn hesized [3H]dopamine (DA) du ing a 3-h incuba- ion pe iod in modi ied Ty ode's solu ion con aining 40 uM [3H] y osine. Following bis loading pe iod, he ca o id bodies we e exposed o one addi- ional hou o unlabelled Ty ode's solu ion equilib a ed wi h ei he 10% oxygen in ni ogen o wi h 100% oxygen. The ca o id bodies exposed o low O2 eleased 81% mo e [3H]dopamine du ing his one-hou pe iod han he ca o id bodies exposed only o pu e oxygen. These da a sugges ha hypoxia induces elease o DA om he ca o id body. I is now well documen ed ha he mammalian ca o id body con ains he biogenic amines dopamine, no epineph ine and 5-hyd oxy yp amine [9,12,29], and ha o hese, dopamine (DA) is p esen in he issue in he highes concen a ion [12,15]. ~e e is also con incing e idence ha hese subs ances a e con ained p ima ily wi hin he glomus, o Type I, cells o he ca o id body [6,7]. Elec ophysiological and pha macological s udies in he ca [23,24,28], dog [4] and abbi (Mon i-Bloch and Eyzagui e, pe s. comm.) ha e shown ha exogenous DA al e s chemo ecep o ac i i y eco ded om he ca o id ne e, and i has been sugges ed ha du ing na u al s imula ion o he ca o id body elease o endogenous DA om he glomus cells may unc ion o modula e he ac i i y o chemo ecep o ne e ibe s which e mina e upon hese cells [2,14,28]. Howe e , p e ious s udies o he e ec s o na u al s imula ion on he DA s o es o he ca o id body ha e p oduced con lic ing esul s, and he e a e no ~ udies which ha e a emp ed o di ec ly demons a e ei he inc eased elease o unlo e o ca o id body DA du ing na al s imu]a- *Add ess co espondence o: S. Fidone, Ph.D., Associa e P o esso o Physiology, Uni e si y o U ah College o Medicine, 410 Chipe a Way, Rm. 156, Resea ch Pa k, Sal Lake Ci y, U ah 84108 (U.S.A.) 96 ion. On he basis o ul as uc u al and luo escence s udies, Ho man and Bi ~ell [18], Blumcke e al. [5] and He onen e al. [17] sugges ed ha hypoxic s imula ion o he ca o id body deple es he glomus cells o hei ca echolamines, whe eas wi h simila echniques A1-Lami and Mu ay [1], Chen e al. [8] and Vema [26] could ind no e idence o ca echolamine deple ion, e en unde condi ions o se e e anoxia. Using biochemical de e - mina ions o endogenous ca echolamines, Zapa a e al. [29] ound no change in ca ca o id body a e se e e hypoxia,'- while Mills and Slo kin [ 22] obse ed a signi ican dec ease in ca o id body DA wi h less se e e hypoxia in he same species. Mo e ecen ly, Hells om e al. [16] also obse ed a dec ease in a ca o id body DA le els wi h hypoxia. Howe e , hese s udies ha e been c i icized [20] as inconclusi e because ca echolamine syn hesis is known o be dep essed in o he issues wi h se e e hypoxia [11,13,19,25,27]. This ac , coupled wi h he acknowledged high oxygen dependence o he ca o id body [ 10,21], means ha a educ ion in issue DA le els wi h hypoxia need no e lec inc eased elease o DA, bu migh a :se om educed syn hesis as well. Thus, a mo e di ec app oach o he ques ion o DA elease du ing chemo- ecep o s imula ion is equi ed. In he p esen s udy, we ha e employed [~H] y osine as p ecu so o DA and ha e measu ed di ec ly [3H]DA eleased om he ca o id body in i o, and we epo he e ha low 02 (10% 02 in N2) esul s in an inc eased elease o DA. Ca o id bodies we e emo ed om adul New Zealand abbi s and quickly cleaned o su ounding connec i e issue in a chambe illed wi h an oxygena ed modi ied Ty ode's solu ion [3] : in aM, NaCI, 111.23; Na-glu ama e, 41.99; KC1, 4.69; CaC12, 2.16; MgCI2, 0.49; Hepes, 4.99; HC1, 2--4; glucose, 5.5. The issue was hen ans e ed o p eincuba ion o oxygena ed Ty ode's a 37°C con aining 40 ~m [3H] y osine (2 Ci/mM; Ame sham), 1 mM asco bic acid and 100 ~M 6-me hyl e ahyd obiop e in (Calbiochem). Following a p e- incuba ion pe iod o 3 h o allow o syn hesis o adequa e s o es o labelled ca echolamines, he issue samples we e di ided in o expe imen al and con ol g oups. The expe imen al g oup was ans e ed o incuba ion o a ial con- aining unlabelled Ty ode's solu ion a 37°C equilib a ed wi h 10% 02 in N2. The con ol issue samples we e ans e ed o a simila ial wi h Ty ode's solu ion equilib a ed wi h 100% O2. Bo h ials also con ained enzyma ic inhibi o s o DA ca abolism: he COMT inhibi o p opylgalla e (100 #M; Sigma) and he MAO inhibi o ha maline (5.7/~M; Sigma). A e one hou , he labelled dopamine in he supe usa es was analyzed as desc ibed below. In all, 8 animals, o 16 ca o id bodies we e used in he p esen se ies o expe imen s. The labelled dopamine was sepa a ed by high ol age pape elec opho esis on Wha man 54 a 32 V/cm o 104 V-h using a high ionic s eng h bu e (ace ic acid, 0.23 M; o mic acid, 0.25 M; NH4OH, 0.25 M; pH = 4.5). P io o elec opho esis, he supe usa e samples we e acidi ied o pH i 3.6 wi h ace ic acid, and asco bic acid was added o each a inal concen a ion o~ 10 aM; he samples we e d ied in a acuum cen i uge a 0°C and esuspended in 200 ~1 o a ca ie mix u e (pH = 3.6). The elec opho e ic channels we e ed h ough 97 200 |50 0 4- .4 0 ~: I00 I-.- ;¢ O 50 I00% 02 + IO% 0 2 SUPERFUSATE Fig. 1. Inc eased elease o [3H]DA om abbi ca o id bodies exposed o low 02. Ca o id bodies we e p eloaded wi h [3H]DA by incuba ion o 3 h in an oxygena ed modi ied Ty ode's solu ion con aining 40 uM [SH] y osine. The issue samples we e hen di ided in o expe imen al and con ol g oups and ans e ed o ials con aining unlabelled Ty ode's which had been equilib a ed wi h 10% O 2 in ni ogen (expe imen al) and wi h 100% O 2 (con ol). A e one hou , he supe usa es om hese ials we e analyzed o hei con en o [SH]DA. The ca o id body samples exposed o low O 2 (black ba ) eleased 81% (~ < 0.02) mo e [3H]DA han he con ol ca o id bodies (clea ba ) when compa ed on a i~sue weigh basis. Values gi en a e ± S.E. a adioch oma og am scanne (Packa d Model 7201), and he DA peaks we e combus ed in a sample oxidize (Packa d Model 306) a d coun ed ill a scin illa- ion coun e (Packa d Model 3385). When [SH]DA le els in he supe usa es we e compa ed be ween con ol (100% 02) and expe imen al (10% 02 in N2) ca o id bodies~ i was ound ha on a issue weigh basis he ials wi h issue samples exposed o low O2 con- ained 81% mo e [SH]DA (Fig. 1). These da a would sugges , he e o e, ha hypoxia b ings abou an inc eased elease o [SH]DA om he ca o id body. This obse a ion is consis en wi h he iew ha educ ion in ca o id body ca echolamines wi h hypoxia, obse ed by o he wo ke s in p e ious s udies, esul s a leas in pa om he elease o endogenous DA s o es. Whe he he e is, in addi ion o elease, a dep ession o DA syn hesis du ing hypoxia canno be de e mined om he p esen da a, bu expe imen s a e now in p og ess o esol e his poin . The elease o DA om he ca o id body, p esumably om . he glomus cells, may unc ion o modula e he ac i i y o a e en ibe s which e mina e upon hese cells. Howe e , he p ecise mode o ac ion o DA in ca o id body chemo ecep ion emains o be elucida ed, and he e is now 98 e idence ha he e e~ ; o exogenous DA on he chemosenso y discha ges o he ca o id ne e may be species dependen . Thus, in he ca , DA inhibi s he chemosenso y discha ges [23,24,28], bu appea s o be s imul~ o y in he dog [4] and abbi (Mon i-Bloch and Eyzagui e, pe s. comm.). A compa a i e s udy o he e ec s o hypoxia on he el~se o DA in hese di e en species is now in p og ess in ou labo a o y. ACKNOWLEDGEMENTS This wo k was supp0 ed by USPHS g an s NS-12636 and NS-07938, and by a g an om he U ah Hea Associa ion. REFERENCES 1 AI-Lami, F. and Mu ay, R.G., Fine s uc u e o he ca o id body o no mal and ~moxic ca s, Ana . Rec., 160 (1968) 697--718. 2 Biscoe, T.J., Ca o id body: s uc u e and unc ion, Physiol. Re ., 51 (1971) 437--495. 3 Ba on, M, and Eyzagui e, C., The mal esponses o ca o id body cells, J. 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