Correlation Between Adenosine Triphosphate Levels, Dopamine Release and Electrical Activity in the Carotid Body: Support for the Metabolic Hypothesis of Chemoreception

64 B ain Resea ch, 348 (1985) 64-68 Else ie BRE 11141 Co ela ion Be ween Adenosine T iphospha e Le els, Dopamine Release and Elec ical Ac i i y in he Ca o id Body: Suppo o he Me abolic Hypo hesis o Chemo ecep ion ANA OBESO, LAURA ALMARAZ and CONSTANCIO GONZALEZ Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Un e sidad de Valladolid, 47005 Valladolid (Spain) (Accep ed Feb ua y 5 h, 1985) Key wo ds: ca o id body -- chemo ecep o s -- adenosine iphospha e (ATP) con en -- dopamine elease -- hypox ~a -- 2-deoxyglucose -- sodium cyanide An unsol ed issue o he a e ial chemo ecep o s is he mechanism by which hypoxia and o he na u al s imuli lead o an inc ease o ac i i y in he ca o id sinus ne e. Acco ding o he 'me abolic hypo hesis', he hypoxic ac i a ion o he ca o id body (CB) is media ed by a dec ease o he ATP le els in he ype I cells, which hen elease a neu o ansmi e capable o exci ing he senso y ne e endings. Using an in i o p epa a ion o ca CB, we epo ha ATP le els in he CB do in ac dec ease when he o gans a e exposed o mode - a e, sho las ing hypoxia (5 min 20% 02). Addi ionally, we ound ha dec eases in ATP le els induced by 2-deoxyglucose (2 mM) o sodium cyanide (0.1 aM) a e closely co ela ed wi h dopamine elease om ype I cells and elec ical ac i i y in he ca o id sinus ne e elici ed by hese agen s. The possible cause-e ec ela ionship o hese e en s is discussed. INTRODUCTION The ca o id bodies (CBs) a e a pai o chemo e- cep o o gans loca ed in he a ea o he ca o id bi u - ca ion, which a e ac i a ed by en i onmen al low 0 2 p essu e, low pH and high CO 2 p essu e 17. Senso y ne e ibe s o he ca o id sinus ne e (CSN) pen- e a e he o gan and o m synap ic-like con ac s wi h he ype I cells. As discussed in ecen e iews3,15 wo key p ob- lems in he unde s anding o he chemo ecep ion p ocess ha e been: (1) he loca ion o he chemosen- so wi hin he CB; and (2) he de ini ion o he ansduc ion mechanism(s) in biophysical and/o bio- chemical e ms. Dealing wi h he i s issue, he accu- mula ed e idence in ecen yea s a o s he idea ha he CB is a seconda y ecep o in G und es e mino- logy 16, he ype I cells being he chemosenso s. A ansmi e eleased a he synapse be ween he ype I cells and he senso y ne e endings would ul ima e- ly ac i a e he senso y ne e endings; howe e , he chemical iden i y o his ansmi e is no p esen ly es ablished. The ansduc ion mechanism o he CB chemo ecep o s emains la gely unknown, and among he hypo heses ad anced o chemo ansduc- ion, he 'me abolic hypo hesis' has ecei ed g ea a - en ion1,6,1s,26, 32. This hypo hesis es s on he obse - a ion ha all me abolic poisons a e powe ul che- mos imulan s, and pos ula es ha hypoxia, like he me abolic poisons, leads o an inc ease in CSN ac i - i y by p oducing a dec ease in adenosine iphospa e (ATP) le els, which in u n igge s he elease o ansmi e om he ype I cells. In a o o he me abolic hypo hesis a e he ecen obse a ions ha ype I cells elease dopamine (DA), a pu a i e neu o ansmi e , in a dose-de- penden ashion when CBs a e exposed o hypox- iam, n. On he o he hand, i is well documen ed ha : (1) ATP le els in b ain slices a e main ained a no - mal le els when he issue is supe used wi h 7-10% O2-equilib a ed solu ions 2°, which a e known o s ongly ac i a e he CB chemo ecep o sT, 2; and (2) in no o he s uc u e do me abolic poisons ac i a e he elease o neu o ansmi e s 19,29,3°,33,34. The e- o e, a alida ion o he me abolic hypo hesis will e- qui e ha he mode a e le els o hypoxia which a e Co espondence: C.G. Ma inez, Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Uni e sidad de Valladolid, 47005 Valladolid, Spain. 0006-8993/85/$03.30 © 1985 Else ie Science Publishe s B.V. (Biomedical Di ision) de ec ed by CB chemo ecep o s p oduce a dec ease in he ATP le els in his o gan, and ha me abolic poisons a e able o induce he elease o pu a i e neu o ansmi e s om he ype I cells. in he expe imen s o be desc ibed, i is shown ha mode a e and sho -las ing hypoxia educed he ATP le els in he CB. I is also shown ha 2-deoxyglucose (2-DG) and CN- educed he ATP con en o he CB, inc eased CSN ac i i y and induced elease o DA om he ype I cells. These indings p o ide ex- pe imen al suppo o he me abolic hypo hesis o chemo ansduc ion in his o gan. MATERIAL AND METHODS The ca o id bi u ca ions o adul ca s (2-3.5 kg), anes he ized wi h sodium pen oba bi al (30-40 mg/kg i.p., Sigma), we e emo ed and placed in a lu- ci e chambe illed wi h ice-cold 100% 0 2 equili- b a ed Ty ode 2. The CBs wi h hei ne es a ached we e p epa ed unde a dissec ing mic oscope (Lei z) o elec ophysiological eco ding a's p e iously de- sc ibed 12 To s udy he elease o DA, he p epa a ions we e i s incuba ed o 3 h a 37 °C in small ials placed in a me abolic shake wi h 500 ~ l o 100% O2-equili- b a ed Ty ode con aining 20 ~M [3H] y osine (3,5- [3H] y osine, 20 Ci/mmol; Ame sham); a he end o he incuba ion pe iod, he p epa a ions we e moun ed in a supe usion chambe 12 which allowed simul aneous eco ding o elec ical ac i i y in he CSN and collec ion o he supe usa es o analysis. The eleased [3H]DA and [3H]ca echol me aboli es we e adso bed on alumina a pH 8.6 and, a e ho - ough washing wi h dis illed wa e we e elu ed wi h 1 N hyd ocholo ic acid35. Thin-laye ch oma og aphic analysis o he elua es 13 e ealed ha [3H]DA and [3H]DOPAC accoun ed o mo e han 85% o he a- dioac i i y p esen in hem, he emainde being mos ly [3H] y osine. To al [3H]DA eleased is aken as he sum o [3H]DA and [3H]DOPAC. When ATP was o be measu ed, CBs we e dis- sec ed ee o he CSN o allow mo e comple e emo- al o su ounding connec i e issue. In hese expe i- men s, one CB om each animal was p ocessed as con ol while he con ala e al o gan was used o es he e ec s o s imula ion (hypoxia, 2-DG o CN-). All issues we e p eincuba ed o 25 min in scin illa- 65 ion ials a 37 °C wi h 5 ml o 100% O2-equilib a ed Ty ode, and inally incuba ed o 5 min in o he ials wi h 2 ml o he same p eincuba ion media (con ols) o ei he 20% O2-equilib a ed Ty ode (hypoxic- is- sues) o 100% O2-equilib a ed media con aining 2 mM 2-DG (2-DG- ea ed CBs) o 10 .4 M CN- (CN- ea ed CBs). All media con ained 5 mM glucose ex- cep in he case o he 2-DG expe imen s, in which 5 mM sodium-py u a e was subs i u ed o glucose. A he end o he incuba ion pe iod, he issues we e placed on p ecooled (-20 °C) homogenize pes les and immedia ely homogenized in 200 k l o ice-cold 0.6 N pe chlo ic acid; he samples we e cen i uged a 4 °C and he supe na an s neu alized wi h 2.4 N po assium bica bona e a 0-4 °C and cen i uged again. ATP was de e mined in he inal supe na an s ei he adioenzyma ically 14 o by a pho olumines- cence-based me hod 4. RESULTS The ATP le el ound in 9 CBs incuba ed wi h 100% O2-equilib a ed media was 4.3 + 0.40 x 10 -10 mo /CB. In he con ala e al o gans, incuba ed in he same media equilib a ed wi h ai , he le el was 3.4 + 0.33 x 10-10 mol/CB (P < 0.02). In con as , no di - e ence was ound in 6 pai s o mice supe io ce ical ganglia which we e ea ed iden ically (Fig. 1). This issue was chosen o compa ison because i s size and shape is simila o he ca CB, minimizing he di e - ences in O 2 di usion in bo h o gans. Since he same hypoxic s imulus applied o he same lengh o ime e oked a 7- old inc ease in he elease o [3H]DA and a simul aneous 6- old inc ease in CSN ac i i y10,12, he da a p esen ed in Fig. 1 sugges a possible link be- ween he dec ease in he ATP le els and he ac i a- ion o he CSN ia he elease o a neu o ansmi e om he ype I cells, as he me abolic hypo hesis pos- ula es. Fo he hypo hesis o be co ec , a simila co ela- ion be ween ATP le els, CSN ac i i y and [3H]DA elease mus be p esen in any si ua ion in which he ATP con en o he CB is lowe ed. The e ec s o 10 .4 M NaCN in he medium a e shown in Fig. 2. Fig. 2A shows he ypical p o ile o [3H]DA elease induced by CN--con aining medium; no e he as onse o e- lease, eaching a maximum wi hin 5 ain om he s a o CN- supe usion. On e u ning o CN-- ee 66 o , - X E D. I.- < 5- c.b. I 1- m~ iiiiiiiiiii!iiiiiii iiiii~iiiiiiiiiiiii iiiiiiiiiiiiiiiiiii ::::::::::::::::::: iiiiiiiiiiiiiiiiiii :i~iiiiiiiiiii!!!i! ~!:!!iii!i!i!i!i!i ~ [] Con ol D Hypoxic m* mE 3 ~ 180 -~ o 120 ~ ~2 i .~ ~ 601 0 3'0 6"0 9"0 3 ' 9 ' 1"5 min. min. S.c.g. ~ b 3 a. Fig. 1. E ec o 5 ain o hypoxic incuba ion (20% 02 equili- b a ed media) on he ATP con en o he ca CB and mice supe- io ce ical ganglia. * P < 0.02. solu ion, he elease slowly dec eased o he con ol le els. Fig. 2B shows he elec ical esponse induced by CN- in he same p epa a ion. Bo h esponses we e compa able o hose ob ained when CBs a e su- pe used wi h low 0 2 (20% 0 2 in N2)-con aining solu- ions ]2. The a e age esponse ob ained in 8 s imula- ion cycles om 4 expe imen s, exp essed as mul- iples o con ol, was 3.8 + 0.77 o [3H]DA elease and 6.8 + 0.59 o CSN ac i i y (Fig. 2C). Finally, when CBs we e incuba ed o 5 min in he p esence o 10 .4 M CN-, ATP le els dec eased o 55% o ha in con ala e al con ol o gans (P < 0.02; Fig. 2D). Since bo h hypoxia and CN- should dec ease ATP con en by impai ing i s p oduc ion, i was o g ea in- e es o see i a educ ion in he ATP le els p oduced by inc easing ATP expendi u es also inc eased bo h elec ical ac i i y in he CSN and [3H]DA elease om he ype I cells. As shown in Fig. 3A and B, he addi ion o 2-DG o he supe usion media (2 mmol/1) inc eased bo h pa ame e s in a simila way as CN- and hypoxia. Howe e , he ime cou se o hese in- c eases is slowed, p obably e lec ing di e en mech- anisms o ac ion. In Fig. 3C a e shown he a e aged esponses o [3H]DA elease and maximum CSN ac- i i y ob ained om 6 expe imen s. Fig. 3D shows ha he ATP con en in 4 CBs incuba ed o 5 min in 2 mM 2-DG dec eased by 38% compa ed o con a- la e al con ol o gans (P < 0.02). This concen a ion Fig. 2. A: ime cou se o CN- (10 4 M) induced elease o [3H]DA in a single expe imen in which CN was applied wice du ing 5 min (ho izon al ba s) de ining wo s imula ion cycles. The dashed a ea ep esen s ac ual elease induced by he i s applica ion o CN-, he s ippled ba ep esen s mean in e po- la ed basal elease du ing he i s s imula ion cycle o a 5 min pe iod (iden ical o he s imula ion pe iod). Dashed a ea di id- ed by s ippled ba equals induced elease in imes o e he con- ol. B: ca o id sinus ne e ac i i y in esponse o 10 .4 M CN- applied o 5 min (be ween a ows, same expe imen as A). One cu e (solid ci cles) co esponds o i s applica ion o CN- and he o he cu e (solid squa es) o he second, a ep esen s maximum ac i i y induced by CN- in he i s applica ion, b ep esen s basal ac i i y p io o he CN- applica ion; a/b equals induced ac i i y in imes o e he con ol. C: a e aged esponses o 10 4 M CN exp essed as imes o e he con ol. S ippled ba ep esen s elease o pH]DA; open ba ep esen s elec ical ac i i y. D: ATP con en in 6 con ol CBs (mean _+ S.E.M., open ba ) and in hei con ala e als incuba ed o 5 min wi h 10 -4 M CN- (s ippled ba ) * P < 0.02. ~. 120' J + 0 3'0 6'0 9"0 ain. a_ B £ g 1'2 1'8 ain. Fig. 3. Same as in Fig. 2 whe e he CBs a e supe used (A-C) o 5 min wi h a solu ion con aining 2 mM 2-DG. D shows he ATP con en ound in 4 con ol CBs and in hei con ala e als incuba ed o 5 min in he p esence o 2 mM 2-DG. * P < 0.02. 67 o 2-DG was chosen because i ep esen s a s imulus o abou he same s eng h as 10 -4 M CN-. DISCUSSION The da a p esen ed he e gene ally alida e he me abolic hypo hesis o chemo ecep ion and poin owa ds a link be ween dec ease in ATP le els, se- c e ion o pu a i e neu o ansmi e s and ac i a ion o he CSN. The ATP con en o he ca CB is ap- p oxima ely 4 x 10-10 ool/o gan. Wi h a mean weigh o 500 ¢ g 9, and wi h in acellula wa e close o 40% o weigh (pe sonal obse a ion), he ATP concen a ion in he CB is calcula ed o be 2.5 x 10-3 M. This concen a ion is simila o ha epo ed o a b ain 20, and li e 31 and mouse supe io ce ical ganglia ( his pape ). As shown in Fig. 1, he ATP con en o he CB de- c eased 21% wi h mode a e hypoxic s imula ion, while he e was no change in ATP in he supe io ce - ical ganglion. The ATP con en o b ain issue is also mo e esis an o hypoxia, and hus a b ain slices and synap osomes main ain no mal ATP le els e en a e 30 ain o incuba ion wi h a 7% O 2 equili- b a ed media 20. The exquisi e sensi i i y o CB ATP le els o hypoxia could be ela ed o he p esence in he ype I cells o a cy och ome oxidase wi h low a - ini y o oxygen 24. This would sugges ha he de- c ease in CB ATP le els should a ise om he speci - ic chemosensi i e issue (i.e. he ype I cells). Taking in o accoun ha his speci ic issue ep esen s abou 50% o CB olume 22, a 21% dec ease in ATP con en in he whole o gan should be an unde es ima ion o he eal ATP deple ion in he ype I cells. In con as , he 38-45% dec ease in ATP con en obse ed wi h 2-DG and CN- should esul om a homogeneous e- duc ion in ATP in all CB s uc u es. These conside a- ions sugges ha he ATP educ ion in ype I cells migh he qui e simila in all 3 expe imen al si ua- ions. This would be consis en wi h he obse a ion ha CN- and 2-DG p oduce abou he same inc ease in CSN ac i i y and [3H]DA elease as he hypoxic s imulus used in hese expe imen s n. I emains o be shown whe he hese 3 pa ame e s (ATP le els, [3H]DA elease and CSN ac i i y) a e linked in a causal ela ionship, as he me abolic hy- po hesis would sugges . I his indeed we e he case, hen he dec ease in ATP le els in he ype I cells should lead o he ac i a ion o a Ca 2+ conduc ance, because he elease o [3H]DA induced by hypoxia 12 and 2-DG and CN- is dependen on he p esence o ex acellula Ca 2+ (unpublished obse a ions). Wha e e he mechanism o his linkage, i seems o be speci ic o his chemosensi i e o gan because in o he s uc u es he sec e ion p ocess is no ac i a ed by hypoxia o me abolic poisonsS,19,33,34 despi e d as- ic educ ions in ATP con en 30. Al e na i ely, hese e ec s may be due o wo pa allel bu independen p ocesses, bu his would equi e ha 2-DG, applied o only 5 min, has ano he ac ion besides he educ- ion in ATP con en . The ela ionship be ween elease o [3H]DA and ac i i y in he CSN is con o e sial; exogenously ap- plied DA has been ound o be inhibi o y, exci a o y o bo h, depending on he dose, he animal species and he p epa a ion21,25,36 (see McQueen 23 o a e- iew). O he s ha e s essed ha he eal p oblem wi h DA ac ions in he CB is whe he exogenously applied amine has he same ac ion as he endoge- nously eleased subs ance. In ac , i has been ecen - ly sugges ed 5,27 ha endogenous DA may well be ex- ci a o y in he ca CB. This sugges ion is suppo ed by he indings epo ed in his pape and by he ob- se a ions p e iously published ha hypoxia11,12 and low pH 28 induce p opo ional inc eases in bo h syn- hesis and elease o [3H]DA and elec ical ac i i y in he CSN. None heless, i mus be ecalled ha ype I cells con ain many o he pu a i e neu o ansmi - e s 9. ACKNOWLEDGEMENTS We hank P o esso B. He e os o his aluable commen a ies in he p epa a ion o he manusc ip . This wo k was suppo ed by a g an om he C.A.I.C.T. o Spain. REFERENCES 1 Anichko , S.V. and Belenkii, M.L., Pha macology o he Ca o id Body Chemo ecep o s, Macmillan, New Yo k, 1963. 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 (1977) (Cell Physiol. 2) C35-C46. 3 Belmon e, C. and Gonzalez, C., Mechanisms o chemo e- cep ion in he ca o id body: possible models. In H. Acke 68 and R.G. 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