Effects of 2-Deoxy-D-Glucose on In Vitro Cat Carotid Body

B ain Resea ch, 371 (1986) 25-36 25 Else ie BRE 11605 E ec s o 2-Deoxy-D-Glucose on In Vi o Ca Ca o id Body A. OBESO, L. ALMARAZ and C. GONZALEZ Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Uni e sidad de Valladolid, 47005 Valladolid (Spain) (Accep ed Augus 20 h, 1985) Key wo ds: ca o id body -- a e ial chemo ecep o -- 2-deoxy-D-glucose -- adenosine iphospha e con en -- dopamine elease -- chemoa e en ac i i y -- me abolic hypo hesis The p ocess o chemosenso y ansduc ion in he a e ial chemo ecep o s is no well unde s ood. Acco ding o he me abolic hy- po hesis o chemo ecep ion, a dec ease in a e ial pO 2 will p oduce a dec ease in he adenosine iphospha e (ATP) con en in he chc- mosenso y ype I cells, leading o elease o a neu o ansmi e and inc eased senso y neu al ac i i y. The e is a pauci y o di ec ex- pe imen al suppo o his hypo hesis, and in he p esen wo k, we ha e es ed he pos ula es o he me abolic hypo hesis in an in i o p epa a ion o ca ca o id body using 2-deoxy-D-glucose as an ATP-deple ing agen . This p epa a ion, when supe used wi h Ty ode con aining 5 mM Na+-py u a e ins ead o glucose, esponds no mally o hypoxia, low pH and ace ylcholinc, and main ains no mal ATP le els. Unde hese condi ions, 2-deoxy-D-glucose is a chemos imulan , i.e. elec ical ac i i y in he ca o id sinus ne e is in- c eased, wi h a h eshold concen a ion o 0.25 mM and a maximum esponse a abou 2-4 aM. These concen a ions o 2-deoxyglu- cose e oke a dose-dependen elease o [3H]dopamine (syn hesized om pH] y osine) om he ca o id bodies which pa allels he elec ical ac i i y. The 2-deoxy-D-glucose-e oked elease and elec ical ac i i y is dependen on he p esence o ex acellula Ca 2+ . These same concen a ions o 2-deoxy-D-glucose signi ican ly educe he ATP con en o he ca o id bodies. The e en s pos ula ed b he me abolic hypo hesis, i.e. dec ease in ATP con en , elease o a pu a i e neu o ansmine and ac i a ion o he senso y ne e end- ings a e ound o occu simul aneously. A possible cause-e ec ela ionship be ween hese h ee e en s is discussed. INTRODUCTION The a e ial chemo ecep o s a e exci ed by de- c eases in paO2 and pH and by inc eases in paCO 2 and con ibu e o he espi a o y adjus men s seen in all physiological and pa hological si ua ions in which he e a e de ia ions om no mali y in blood gas en- sions and pH. The mechanisms by which hese ecep- o s de ec he changes in hose blood pa ame e s a e unknown. Acco ding o one hypo hesis, known as he me abolic hypo hesis 4,15,3~,39,55 (see e . 7 o a e- iew), he o e all ansduc ion p ocess in hese e- cep o s o he hypoxic s imuli will co espond o his sequence: hypoxia will dec ease adenosine iphos- pha e (ATP) le els in he ype i cells, which in u n will elease a neu o ansmi e capable o exci ing he senso y ne e endings. This hypo hesis is based on he assump ion ha because he me abolic poi- sons (inhibi o s o he espi a o y chain, uncouple s o oxida i e phospho yla ion and mi ochond ial ATPase inhibi o s) a e po en chemos imulan s hey will dec ease he ATP con en in he chemo ecep o s, as hey do in o he issues. The me abolic hypo hesis does no conside he mechanisms in ol ed in he de- ec ion o he o he wo physiological s imuli (p~,CO2, pn). In he p esen s udy, we ha e es ed he pos ula es o he me abolic hypo hesis by using 2-deoxy-D-glu- cose (2-DG) in an in i o p epa a ion o he ca ca- o id body (CB). 2-DG is an analogue o glucose which en e s he cells by he same ca ie as glucose. In he p esence o ATP, i is phospho yla ed by hexo- kinase o 2-deoxy-glucose-6-phospha e (2-DG-6P) and i is no u he me abolized 51,52. We ha e chosen 2-DG o es he me abolic hypo hesis because in ou expe imen al condi ions (p epa a ions main ained in py u a e-Ty ode ee o glucose), i is an e ec i e ATP-deple ing agen , i is ee o known side-ac ions Co espondence: C. Gonzalez, Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Uni e sidad de Valladolid, 47005 Valladolid, Spain. 0006-8993/86/$03.50 © 1986 Else ie Science Publishe s B.V. (Biomedical Di ision) and i s mechanism o deple ing ATP (inc eased ATP expendi u e) is di e en om ha o he mi ochon- d ial poisons which deple e ATP by blocking syn he- sis. The e o e, i 2-DG is a chemos imulan , his should allow a b oade o mula ion o he me abolic hypo hesis in he sense ha a educ ion in ATP le els, wha e e he mechanism, is accompanied by an in- c ease in chemo ecep o ac i i y. We ound ha CBs supe used wi h balanced saline con aining py u a e as a nu ien ins ead o glucose, esponded no mally o di e en s imuli (low O 2, low pH and ace ylcholine (ACh)). We ound also ha he p esence o 2-DG in he supe usion media (Ty ode-py u a e) caused a dose-dependen inc ease in he elec ical ac i i y o he ca o id sinus ne e (CSN) and in he elease o pH]dopamine ([3H]DA) om he ype 1 cells. This 2- DG-induced elease was dependen on he p esence o ex acellula Ca z+. Finally, we ound di ec ela- ionships amongs he chemo esponses, he amoun o 2-DG phospho yla ed and he dec ease in he ATP con en in he CB. Taken oge he , hese indings gi e expe imen al suppo o he me abolic hypo he- sis. Some o he esul s ha e been published m ab- s ac o m 42. MATERIALS AND METHODS Su ge y, Adul ca s (2-3.5 kg) we e anes he ized wi h sodi- um pen oba bi al (30-40 mg/kg, i.p.), acheos o- mized and placed on a espi a o . Subsequen su - ge y a ied sligh ly in he di e en ypes o expe i- men s: (1) When he elec ical ac i i y in he CSN was o be eco ded, a double liga u e was placed on he ex e nal ca o id a e y. A sec ion be ween he liga u es allowed be e iden i ica ion o he CSN which was dissec ed ou and emo ed oge he wi h he ca o id bi u ca ion. The block o issue was placed in a luci e chambe illed wi h ice-cold 100% O2-equilib a ed Ty ode con aining (in mM): NaC1, 140; KCI, 4.7: CaCI2, 2.2; MgC12, 1.1; HEPES, 10; glucose, 5.5; adjus ed o pH 7.4 wi h 1 N NaOH. (2) In he expe imen s in which he measu emen o elec- ical ac i i y was no equi ed (expe imen s o meas- u e ATP le els), a clamp was placed cephalad o he ca o id sinus and ano he on he common ca o id a - e y, and wi hin 40 s (25-40 s) he bi u ca ion was placed in he luci e chambe as be o e, in bo h cases, he ope a i e ield was kep oo s1 wi h w<i ~i saline. Elec ophysiological eco ding and elease ~/ [~ ]dc, pamine Unde a dissec ing scope (Lei z), he CBs wi h he CSN a ached we e p epa ed o eco ding as p e- iously desc ibed 19. In he expe imen s in which he elease o [3H]dopamine ([3H]DA) was o be meas- u ed, he p epa a ions we e incuba ed o 3 h in 500 d o 100% O~-equilib a ed Ty ode con aining he ca echolamine p ecu so [3H]- y osine (2,!~-[3HI y o - sine: 20 Ci/mmol, Ame sham) a a concen a ion o 20~M and con aining also l[10,uM o he y osine hy- d oxylase co ac o , 6-me hyl- e ahyd op e ine (Sig- ma) and 1 mM asco bic acid. Unde hese incuba ing condi ions, he CB syn he ized abou 20 pmol o [3H]DA (= 4 x 105 cpm) (unpublished). Whe he o no he elease o [3H]DA was moni o ed, he CBs we e moun ed in a d op supe usion chambe which allowed con inuous low- a e supe usion (0.4 ml/min) wi h di e en solu ions a 37 'XL eco ding o elec ical ac i i y in he whole CSN wi h a suc ion elec ode and collec ion o he supe usa es o la e analysis when needed ~. The supe usa e,~ we e col- lec ed in ials con aining a ca ie mix u e (glacial ace ic acid-20 mM asco bic acid-100 uM cold DA) in he app op ia e olume o yield a inal pH in he supe usa es o 3.2-3.6. The collec ion o he supe - usa es was made acco ding o he ollowing se- quence: 5 min p io o he applica ion o he s imulus (con ol), 5 min du ing he s imulus applica ion and 4 mo e ac ions o 5 and 10 min pos -s imulus, o aling 45 min (du a ion o a single s imulus c)clc: see Fig. 8). No mally, each expe imen consis ed o 4-6 s im- ulus cycles. The supe usa es we e analyzed o 31- - labelled ca echolamines eleased by adso p ion on acid-washed alumina a pH 8.6 and elu ion wi h I N HCI o eco e all 3H-labelled ca echol compounds 5s. Posi i e iden i ica ion o he elu ed 3H-labelled com- pounds was achie ed by hin-laye ch oma og aphy (TLC) analysis acco ding o Fleming and Cla k e~. Since he sum o [3H]DA and [-~Hldihyd oxyphenyl- ace ic acid ([3H]DOPAC) accoun ed o 80-85% o he adioac i i y p esen in he elua es ( he es being mos ly [3H] y osine), we e e o he eleased ma e i- al p esen in he elua es as [3H]DA. The s imulus-in- duced elease was e alua ed as ollows: he mean e- lease du ing wo con ol pe iods o wo consecu i e cycles was aken as basal elease/5 min du ing ha cycle, and he sum o any elease exceeding his basal elease in all cycle ac ions was conside ed s imulus- induced elease. The ac ion po en ials om he CSN we e led h ough an AC-coupled p eampli ie and displayed on an oscilloscope. The ampli ied signals we e also led h ough a window disc imina o o a digi al coun - e -p in e which ead he o al numbe o ac ion po- en ials du ing ixed pe iods o ime (1 o 10 s). The s imulus-induced elec ical ac i i y was e alua ed simila ly o he elease o [3H]DA; he maximum ac- i i y elici ed by a s imulus (Hz) was di ided by he mean basal ac i i y in he 5 ain p io o he applica- ion o he s imulus (Hz), hus gi ing induced ac i i y ela i e o con ol. AT[° measu emen s Taking in o conside a ion he labili y o ATP le - els 35, special p ecau ions we e aken: (1) As men- ioned be o e, he ca o id bi u ca ion was emo ed wi hin 40 s a e clamping he common ca o id a - e y. (2) The Ty ode in he luci e chambe was kep a 0-2 °C and enewed e e y 2 min o a oid wa ming and signi ican d ops in 0 2 ension. (3) The dissec ion and ho ough cleaning o he CB was comple ed wi hin 4-6 ain, bu in all ins ances he CBs emained in he chambe o 6 ain. ATP le els in bo h CBs o he same animal measu ed a his s age showed g ea a iabili y, p obably due o di e en deg ees o ma- nipula ion. (4) Incuba ion o he CBs in 100% 0 2- equilib a ed Ty ode a 37 °C o 15-60 ain esul ed in highe ATP le els and a d as ic educ ion in he a iabili y o he alues ob ained o bo h CBs o he same animal, and he e o e, he CBs we e sys ema i- cally p eincuba ed o 25 min a 37 °C. (5) The e ec o any gi en s imulus on he ATP le els was assessed on one CB om one animal, he con ala e al CB se ing as con ol. Bo h CBs we e p eincuba ed o 25 min; he con ol CB was incuba ed o 5 addi ional min in he con ol solu ion and he es CB was simi- la ly incuba ed o 5 min in he s imulus-con aining medium. (6) The CBs we e placed on p e-cooled (-20 °C) homogenize pes les and homogenized (glass o glass) in ice-cold 0.6 N PCA (200/zl). The samples we e cen i uged a 4 °C and he supe na- an s neu alized in he cold wi h 2.4 N po assium hi- 27 (0 "5 ~ E Z 20- 15- 10- 5- A 1 5 7 9 X 10 -'° moles ATP/CB 25- 20- 15- 10- 5 300 B 6DO 900 1200 pg Fig. 1. A: dis ibu ion o he ATP le els ound in he ca CB. ATP was assayed ei he adioenzyma ically o by a pho olumi- nescence-based assay; bo h me hods ga e he same le els, n = 70; X = 3.7 x 10 10 mol ATP/CB; S 2 ATP = 2.95. B: dis ibu- ion o CB weigh s in he ca . Shown o compa a i e pu poses is he dis ibu ion o CB weigh s om a p e ious s udy :5. n = 104; X = 559.0,ug/CB; S 2 = 47,961. The a iances o bo h dis- ibu ions a e no s a is ically di e en (P < 0.01 ). ca bona e, and a e 20 min cen i uged again. ATP was measu ed in he la es supe na an s ei he adio- enzyma ically 26 o by a pho oluminescence-based me hod 28. The sensi i i ies o bo h me hods we e be e han 3 x 10 -12 mol/assay and yielded he same ATP alues o he CB. Fig. 1A shows he dis ibu- ion o ATP le els measu ed in 70 con ol CBs and he weigh dis ibu ion o 104 CBs obse ed in a p e- ious wo k 25. The a iances o bo h dis ibu ions a e no s a is ically di e en (P < 0.01). RESULTS Supe usion condi ions In p elimina y expe imen s, i was ound ha in p epa a ions supe used wi h 100% O2-equilib a ed Ty ode con aining 5 mM glucose, only wi h high 2S 2-DG concen a ions (> 4 mM) in he supe usion media i was possible o ob ain an inc ease in he CSN ac i i y, and e en his inc ease had a e y slow onse . Supe usion wi h 100% O2-equilib a ed glucose- ee Ty ode, on he o he hand, p oduced a much la ge and as e exci a ion o he CB chemo ecep o s a 2-DG concen a ions well below I aM. The use o glucose-con aining Ty ode posed p oblems because in o de o ge he maximum (> 80%) exci a o y e - ec , i was calcula ed on he basis o compe i i e in- hibi ion kine ics ha 2-D(i should be aised o 20 mM. Since he CB chemo ecep o s a e e y sensi- i e o osmo ic changes 23, he indings could be mis- leading. On he o he hand, supe usion o he CB Hz 6OO- 2(X)-- Hypoxia //--q . ,/ I L pH•6.8• ..... _.. ,. _ I /?~,L...~_... i - ,:""..... ACh. 5 xlO-4M __..J ...... ~ ........ Fig. 2. Elec ical ac i i y o he CSN o one p epa a ion supe - used wi h Ty ode-glucose (con inuous acings) and Ty ode- py u a e (discon inuous acings). The expe imen s a ed wi h pe usion o he CB wi h 100% Oz-Ty ode-glucose , and e e y 45 min he CB was s imula ed i s wi h low O, (20% 02 bal- anced N2)-con aining solu ion, hen wi h a 100% O2-Ty o- de-glucose a pH 6.8 and inally wi h a 100% O,-Ty ode-glu- cose con aining 5 x 10 4 M ACh. The ea e , he supe usion was made wi h 100% O2-Ty ode-py u a e. A e allowing 1 h o wash ou he glucose, he same pa e n o s imula ion was ollowed. In di e en expe imen s, he o de o pe usion was al e na ed, bu in all ins ances he s imuli las ed 5 min (be ween a ows). I mus be s a ed ha he CB chemo ecep o s s a o inc ease hei elec ical ac i i y when he pe usion solu ion is equilib a ed wi h 60% O 2 in N2 a he supe usion a e used in he p esen expe imen s. Unde hese condi ions, a 20% 02- equilib a ed solu ion ep esen s a hypoxic s imulus o mode - a e o high in ensi y I9. wi h glucose- ee Ty ode was excluded a,~ an expe i- men al p epa a ion because a e abou~ i h ( ange. 20 o 80 min) in glucose- ee media, he e was a spon- aneous inc ease in he CSN ac i i y ollowed by un- esponsi eness o he p epa a ion o hypoxia-L As an al e na i e ene ge ic subs a c o glucose, py u a e (5 aM) was es ed. Wi h py u a e as sub- s a e, he esponse o hypoxia, low pH and ACh was indis inguishable om ha ob ained wi h glucose as subs a e (Fig. 2). Table 1 shows mean alues o he elec ical beha io o he CB when pedused wi h ei he glucose o py u a e. Nei he he basal ac i i y no he s imuli-induced esponses we e di e en om hose ob ained wi h glucose when he subs a e was py u a e. In addi ion, il was also ound ha he ATP le els measu ed a e 30 ain o incuba ion wi h glucose o py u a e we e no s a is ically di e en (Fig. 3). These indings, coupled o he ac ha un- de Ty ode-py u a e 2-D(', was a powe ul chemo- s imulan (see below), p omp ed us o use as pe u- sion media Ty ode-py u a e equilib a ed wi h 100% 02. E ec s o 2-DG on he chemo ecep o ac i y In Fig. 4 an expe imen is p esen ed showing he cha ac e is ics o he elec ical ac i i y ob ained on 4- N = NI ¢..) :~:~:~:5:~£ i~iii!i~iiiii):.x < iiiiii!i~ll " !I1 O E 2 .-.-.-...-.-.-i££:1 0 ii~N~i~! ...... .>>>:->z. × iiiiiiiii!i:i:;; !/@!il~i :;2::::::2:1 Fig. 3. ATP le els in ca CBs incuba ed o 30 min in Ty ode- py u a e (s iped ba ) o in Ty ode-glucose (clea ba ). Py u- a e: n = 14; X = 3.76; S.E.M. = 0.37. Glucose: n = 19: X = 3.48; S.E.M. = 0.37. All CBs o his igu e we e assayed by he luci e in-luci e ase me hod. Compa isons: - es , P > O,O5. 29 TABLE I Cha ac e is ics o he CB esponse o di e en s imuli du ing supe usion wi h glucose (G) and wi h py u a e (P) In all cases, he window disc imina o was adjus ed a he onse o he expe imen s o p o ide a basal ac i i y (supe usion wi h 100% O2-equilib a ed solu ion) in he ange o 25-50 ac ion po en ials. The coun ing windows emained unchanged o he es o he expe - imen . Da a ep esen means + S.E.M. No s a is ical di e ences ( - es ) we e obse ed in any o he pa ame e s e alua ed. Hypoxia pH6.8 ACh (5 × 10 4) G(n-9) P(n= 7) G(n= 16) P(n=14) G(n- 11) P(n=9) Basal ac i i y (BA) (Hz) 40 _+ 5 48 _+ 6 38 + 5 35 + 5 39 _+ 6 38 _+ 4 Peak ac i i y (PA) (Hz) 296 + 66 365 _+ 54 98 _+ 13 108 _+ 16 109 _+ 16 117 _+ 19 A e age ac i i y (AA) (Hz) 171 + 30 235 + 34 71 _+ 10 80 _+ 12 83 + 13 85 _+ 14 Time- o-peak (s) 253 + 20 233 + 35 210 + 17 235 _+ 13 77 _+ 16 56 _+ 13 PA/BA 7.4 7.6 2.6 3.1 2.8 3.1 AA/BA 4.3 4.9 1.9 2.3 2.1 2.2 supe using he CBs o 5 min wi h di e en concen- a ions o 2-DG. No e (Fig. 4 inse ) he dose-de- penden esponse. No e also ha he onse and he ime o peak o he esponse dec eased wi h inc eas- ing concen a ions o 2-DG. And inally i can be seen ha he eco e y ime ( ime o he elec ical ac- i i y o e u n o he p e-s imulus le els) inc eased wi h inc easing 2-DG (Fig. 4). Fig. 5 shows dose- esponse cu es o he e ec o 2-DG on he elec ical ac i i y o he CSN. The uppe cu e was ob ained om 10 CBs which immedia ely upon dissec ion we e moun ed in he supe usion- e- co ding chambe . The lowe cu e was ob ained om 8 CBs which had been incuba ed in Ty ode- 420 ........ , ...... ~PA.~ R,~ / ~ 91 2~ 8 300 l ........ ,..o, ............ | 180 g ILl 1 b 2'0 3'0 4'0 min, Fig. 4. Elec ical ac i i y elici ed in he ca o id sinus ne e by di e en concen a ions o 2-deoxyglucose in he supe using medium (Ty ode-py u a e equilib a ed wi h 100% 02). Inse : cha ac e is ics o he elec ical esponse. BA, basal ac i i y; PA, peak ac i i y; o PA, ime- o-peak ac i i y (s); eco e y ime, ime o he elec ical ac i i y o e u n o he basal ac i - i y (min) (single expe imen ). glucose o he 3 h pe iod p io o elec ophysiologic- al eco ding (Fig. 5). Since his di e ence in he e- sponse upon incuba ion is no obse ed wi h o he s imuli (hypoxia, low pH, CN ), i appea ed ha i mus ha e been ela ed o some speci ic ac o con- ce ned wi h he mechanism o ac ion o 2-DG. In his ega d i mus be men ioned ha in wo s uc u es ( he supe io ce ical ganglion and he ad enal me- dulla) emb yologically simila o he ype I cells 30,33, he e is an inc ease in he s o es o glycogen upon in- cuba ion o he s uc u es in i o wi h 5 mM glu- 12- T TO /,,, o 8 ~l .J T /// (~X ** ~ _ __-- _ n:8 .X//~, * ~, ~, mM 2-DG Fig. 5. Dose- esponse cu es o 2-deoxyglucose on he elec- ical ac i i y o he ca o id sinus ne e exp essed in ela ion o he con ol ( imes con ol ac i i y). In all ins ances, he CBs we e moun ed in he eco ding chambe and supe used o 1 h wi h 100% O2-equilib a ed Ty ode-py u a e be o e he ini ia- ion o he s imula ion wi h 2-deoxyglucose. (Q) Ob ained om 10 ca o id bodies (X + S.E.M.) immedia ely upon emo al om he animals. (m) Ob ained om 8 ca o id bodies p eincu- ba ed o 3 h in Ty ode-glucose. Compa isons: - es , ** P < 0.(]1 ; * P < (].05. cose 12,13,~7. I may be ha he b eakdown ol lhe gly- cogen du ing he eco ding pe iod gene a es glucose, which compe es wi h 2-DG a he phospho yla ion s ep (see Discussion; see also Fig. 9) E ec s q 2-DG on he elease o /-~H/DA . ' OI ll he ype 1 cells: co ela ion wi h he elec ical ac i i O, and ATP le els I is well-documen ed ha he exocy o ic elease o neu o ansmi e s and ho mones is a p ocess which equi es ATP 49. I is also well-documen ed ha in a g ea a ie y o sec e o y s uc u es (b ain issue, ad enal gland, sympa he ic endings, pla ele s), a de- c ease in ATP le els educes a he han inc eases 9 4 d ¸ 2 0 3 E 2 o "i 2" 1 A 1 5- 4- 3- 2- 1- T B T ' S " .--~E ec ical Ac . • .-.-.~H-DA Release i~ 3 - {' am 2-DG Fig. 6. A: e ec o di e en concen a ions o 2-deoxyglucose applied o 5 min (ho izon al ba s) on he elease o [3H]DA om he ca o id body (single expe imen ). B: dose- esponse cu es o 2-deoxyglucose on he elec ical ac i i y (O----Q) and [3H]DA elease (*-.-*) ob ained simul aneously in he same p epa a ions. Poin s ep esen means _+ S.E.M. o 8 expe imen s. he sec e o y p ocess 1~,,27-'9.~:~5~:. Since: he nbo c ac s a e opposi e o wha he me abolic ilypo hesis p edic s o he chemo ecep o s, i was oi glea in e - es o es ablish i s i 2-DG induced he elease o [~H]DA om ype 1 cells, and second i 2-I)G e- duced he ATP le els in he (B. Fig. 6A shows a single expe imen m which i is e i- den ha 2-DG applied o 5 ain induced a elease o [3H]DA om a p eloaded ('B which was dependen on he concen a ion o he glucose analogue. Fig. 6B shows he dose- esponse cu es o bo h he elease o [SH]DA and he elec ical ac i i y ob ained om 8 expe imen s. Fig. 7 shows he e ec s o 2-DG (1, 2 and 4 aM) on he ATP le els in CBs, compa ed o con ala e al o gans simila ly incuba ed in he absence o 2-DG. Fig. 7 also shows he ela i e a es o 2@H]DG phos- pho yla ion in he ange o concen a ions om 0.25 o 4 mM. The da a p esen ed in hese las wo igu es (Figs. 6 and 7) ce ainly sugges ha in he CB che- mo ecep o s he e is a close ela ionship be ween he dec ease in he ATP le els, he elease o a pu a i e neu o ansmi e and he genesis o ac i i y in he CSN, as he me abolic hypo hesis p oposes 5 % E 4. a_ 3 © C~ 2- "p<0 02 mM 2-DG 40 c~ -a0 o 20 n + ~.. ,< 10 L 4 Fig. 7. Phospho yla ion a es o 2-[~H]deoxy-D-g!ucose a di - e en concen a ions. The cu e was ob ained by adding ace amoun s o he labelled glucose analogue o he ials con aining he speci ied (abscissa) concen a ions o he unlabelled ana- logue: he incuba ion ime was 5 min. 2-[3H]Deoxy-D-glucose - 6-phospha e was isola ed om he unphospho yla ed suga by anion-exchange ch oma og aphy, as desc ibed by Gonzalez and Ga cia-Sancho 26. The ba s ep esen pe cen dec ease in ATP le els in ca o id bodies incuba ed o 5 ain in he p esence o 2-deoxyglucose wi h espec o hei pai ed con ols incu- ba ed in 2-deoxyglucose- ee media; da a a e means o 4-8 ca- o id bodies. S a is ical compa isons: pai ed - es . :' P < 0.02. Cha ac e is ics o he 2-DG-induced elease o /.~H/DA Time-cou se o [JH]DA elease. As shown in Fig. 4, he elec ical ac i i y elici ed by 2-DG ou las s i s applica ion, p obably because he phospho yla ion o he accumula ed 2-DG con inued wi hin he cells. The e o e, i was o in e es o co ela e he ime- cou se o 2-DO-elici ed elec ical ac i i y in he CSN wi h he ime-cou se o he 2-DG-induced elease o [3HiDA. In Fig. 8 is shown he ime-cou se o he 2-DO-induced elease o [3H]DA. No e ha he ac- ion wi h highes [SH]DA is he pos -s imulus pe iod, despi e he ac ha wi h 2 and 4 mM (see inse in Fig. 4), he peak o elec ical ac i i y occu ed du ing he s imulus pe iod. This appa en delay in [3H]DA elease should in ac be conside ed he esul o washou o eleased [3H]DA. No e also in Fig. 8, ha he ime equi ed o [SH]DA elease o e u n o p e-s imulus le els is abou he same as ha equi ed o he elec ical ac i i y o eco e o he basal le el (compa e Figs. 4 and 8). All he pa allelisms be ween 2-DG-elici ed elec ical ac i i y and [3H]DA elease sugges ha DA plays a p ominen ole in he genesis o he chemoa e en ac i i y, as al eady poin ed ou by o he in es iga o s ,.ls. 19.34.41 31 Ca e+ dependence o he 2-DG-e oked elease oJ [3H]DA. The Ca 2+ dependence o he 2-DG-e oked elease o [3H]DA was s udied in 4 expe imen s, and he esul s a e p esen ed in Fig. 9. In hese expe i- men s, he i s and ou h cycles we e made while pe using he p epa a ion wi h no mal Ca:+-Ty - ode, and he second and hi d cycles wi h 0 Ca> Ty- ode. No e ha he elease o [SH]DA in he wo con- ol cycles (Fig. 9A; Nos. 1 and 4) is qui e di e en , being much mo e p onounced in he las cycle. As al- eady men ioned (see Fig. 5), his di e ence p oba- bly is ela ed o he slow deple ion o he s o e o gly- cogen ha was buil up du ing he pe iod o incuba- ion p io o he eco ding pe iod. No e also ha , on he a e age, he 2-DG-e oked elease o [3H]DA in he wo cycles wi h 0 Ca -'+ is 141q- o he basal e- lease in hese same cycles, while in he wo con ol cycles he e oked elease is 320q- o he basal elease (P < 0.01). When 1 mM EGTA is added o he () Ca 2+ media, he e is no e oked elease (no shown). The a e age elec ical ac i i y induced by 2-DG was also educed in he wo cycles wi h ( Ca -'+ (Fig. 9B). Thus, i ep esen ed 175% o he basal ac i i y in 0 Ca 2+ and 273%, in he con ol cycles (P < ().02). In he p esence o EGTA, he elec ical ac i i y is un- o~ c¢ ,< E3 ± 300- 200- 100- n Con ol ,-- S imulus m ml m m 0.25 0.5 1 xlO-3M 2DG _I. ± .J.. m 4 Fig. 8. Time-cou se o he 2-deoxy-D-glucose-e oked elease o pH]DA; mean alues ob ained in 8 s imula ion cycles. In each case. he basal elease, elease in he 5 ain ac ion p io o he supe usion wi h 2-deoxy-D-glucose, was aken as 100qJ, ; in he o he ac ions, he elease is exp essed as a pe cen age o he basal elease. 500- 50O- a~ < 2O0 C3 ± 100 A OCJ* 1.6~.Mg'* 1 2 3 3 2 B "~ 400- :,,300 2. 0 200 0 19 7, 100 i ~.. ÷÷ OCa , 1.6mM.M~ 1 2 4 3 4 Cycle numbe Fig. 9. Calcium dependence o he 2-deoxy-D-glucose-e oked elease o [3H]DA and elec ical ac i i y in he ca o id sinus ne e. In each expe imen , a e he i s cycle o s imula ion (5 min supe usion wi h Ty ode-py u a e con aining 1 mM 2-deoxy-D-glucose), he p epa a ion was pe used o 30 min wi h Ty ode-py u a e wi h 0 Ca 2+ + 1.6 mM Mg 2+ p io o he ini ia ion o cycle 2; du ing cy- cles 2 and 3, he supe using medium was also Ca2+- ee. Be o e s a ing cycle 4, he p epa a ion was supe used o 30 min wi h no - mal Ca2+-Ty ode, and cycle 4 i sel was made unde no mal Ca 2~ -Ty ode. A: elease o [3H]DA. Emp y ba s: basal elease (100% I in each cycle; lined ba s: e oked elease (as pe cen o he basal) in each cycle. B: elec ical ac i i y in he ca o id sinus ne e. Emp y ba s: basal ac i i y p io o he supe usion wi h 2-deoxy-D-glucose in each cycle (100%); lined ba s: peak ac i i y e oked by 2-deoxy- glucose in each cycle as pe cen age o he basal ac i i y. The da a a e mean alues ob ained in 4 expe imen s. The s a is ical compa i- sons desc ibed in he ex we e made using a pai ed - esL s able and almos impossible o e alua e. Again, he pa allelism obse ed be ween elease o [3H]DA and he elec ical ac i i y in hese 0 Ca 2+ expe imen s sugges s a pa icipa ion o DA in he genesis o he elec ical ac i i y. DISCUSSION The p esen wo k has in es iga ed he me abolic hypo hesis o chemo ecep ion by co ela ing he e - ec s o 2-DG on he ATP le els in he CB wi h he e- lease o a pu a i e neu o ansmi e (DA) by he ype I cells and wi h he elec ical ac i i y in he CSN. As he me abolic hypo hesis pos ula es, he e is a di- ec and close ela ionship be ween he dec ease in ATP and he inc ease in DA elease and CSN ac i - i y. ATP le els in he CB The mean ATP con en ound in ou sample (n = 70) o CBs is 3.7 x 10 -10 mol/CB, wi h a dis ibu ion which was no s a is ically di e en om ha o he CB weigh s. The es ima ed in acellula concen a- ion o ATP in he CB is abou 2-3 aM, a concen a- ion qui e simila o ha ound in b ain issue 32 and li e 53. Ou indings con as wi h he da a ecen ly epo ed by Acke and S a linge I. These au ho s e- po ed ATP le els o 0.087 + 0.056 (S.D.) nmol/CB (n = 31), ha is, alues only abou 24% o ou s. In he same a icle, i was also epo ed ha se e e hy- poxia (p,O z = 25 o du ing 1 l min) did no modi y he ATP con en in he CB. Again, his obse a ion con as s wi h ou p e ious indings 47, showing ha a mode a e o in ense hypoxic episode (incuba ion in ai -equilib a ed Ty ode o 5 ain) signi ican ly e- duced he ATP con en in he CB. I is di icul o ec- oncile bo h se s o da a since hey we e ob ained om he same animal species; howe e , as poin ed ou in Ma e ials and Me hods (see also e . 35), he ATP s o es a e e y labile, and any mechanical and/o by- poxic auma o he CB issues can lead o apid de- ple ion o me abolically ac i e ATP deposi s. Since Acke and S a linge I assayed ATP in he CB di ec ly upon emo al om he animals, and conside ing he small size o he o gan, i may be ha hei dissec ion maneu e s we e esponsible o he lowe and g ea - ly dispe sed alues which hey epo ed. I his we e he case. i is concei able ha hey would no ha e obse ed a u he ATP deple ion ollowing a hypox- ic episode, The dec ease o he ATP le els obse ed in CBs in- cuba ed wi h py u a e in he p esence o 2-DG was expec ed conside ing ha 2°DG is a good subs a e o hexokinase and ha 2-DG-6P is no a subs a e o glucose phospha e isome ase 5,2.,31,51,5a. The de- c ease in ATP obse ed in he whole CB unde 2-DG should esul om a educ ion in ATP in all glomic s uc u es p obably in di ec p opo ion o hei me - abolic a e 51. Because he ype I cells a e conside ed he CB s uc u es wi h he highe me abolic a e 56, hey p obably con ibu e signi ican ly o he ob- se ed o e all ATP educ ion. None heless, enough ATP mus emain wi hin hese cells o suppo he exocy o ic Ca2+-dependen elease. 2-DG as a chemos imulan Any agen (me abolic poison) which in e e es wi h he gene a ion o phospha e-bond ene gy is a s imulan o he CB chemo ecep o s 4. Al hough he me abolic poisons ha e many side e ec s, seldom conside ed in he con ex o he chemo ecep ion p ocess, he ac ha all o hem a e known o de- c ease he ATP/ADP quo ien in o he s uc u es and a e powe ul s imulan s o he CB chemo ecep o s has been he basis o he p oposal 4 o he me abolic hypo hesis >,4<55 (see e s. 7 and 17 o e iews). As was s a ed in he in oduc ion, 2-DG was chosen o his s udy because o he appa en lack o side-e ec s and because i will lead o ATP deple ion by inc eas- ing ATP expendi u es a he han by blocking ATP syn hesis, especially unde ou condi ions o supe u- sion wi h Ty ode-py u a e. The di e ences in he dose- esponse cu es o 2-DG on he CSN ac i i y in non-incuba ed o incuba ed CBs a e, as al eady men- ioned, in all p obabili y due o he accumula ion o glycogen du ing he incuba ion pe iod. In addi ion o he obse a ions made in he supe io ce ical gan- glion 12,13 and ad enomedulla y cells 37, which show 33 ha on incuba ion in i o wi h glucose he e is an in- c ease in glycogen deposi s, we ha e shown 2 ha in CBs supe used wi h glucose- ee Ty ode, he ime equi ed o abolish he hypoxic esponse is much longe i he o gans a e p e iously incuba ed o 3 h in Ty ode con aining 5 mM glucose. This delay can only be in e p e ed as he esul o a buildup o en- e ge ic s o es du ing he incuba ion pe iod. In keep- ing wi h his no ion is also he ac ha he 2-DG phospho yla ion cu e, which has a hype bolic shape ( he co ela ion coe icien o I/V ( a e o phospho- yla ion) s 1/S (2-DG concen a ion) is 0.998). has an appa en K,, o 2.2 mM, which is much highe han ha o hexokinase o 2-DG5.~2,54, which sug- ges s a compe i i e inhibi ion o glucose wi h 2-DG e en in hese CBs ha had no been p e iously incu- ba ed. Co ela ion be ween ATP le els, elease o[" [-~H]DA and CSN ac i i y The da a p esen ed in Fig. 6 and in subsequen ig- u es show a close co ela ion be ween ATP le els, e- lease o [3H]DA and CSN ac i i y. The me abolic hy- po hesis pos ula es such a co ela ion, and e en mo e, pos ula es a cause-e ec ela ionship be- ween he dec ease in he ATP le els, he elease o he neu o ansmi e and he genesis o ac i i y in he CSN. A cause-e ec ela ionship be ween he de- c ease in he ATP le els and he elease o he pu a- i e neu o ansmi e DA seems o exis because hy- poxia and CN , bo h o which dec ease he ATP con- en in he CB, also induce a elease o DA which is CaZ+-dependen 43. How a dec ease in he ATP le els igge s he en y o Ca > in o he ype I cells is no known, bu depola iza ion o he ype I cells seems o be in ol ed because o ganic and ino ganic blocke s o he ol age-dependen Ca-'- channels inhibi he hypoxic as well as he high K+o-induced elease o DA 4a. These obse a ions aise ano he ques ion: how does he dec ease in he ATP le els induce depo- la iza ion o he ype cells. In his ega d, i is nec- essa y o p opose ha an ATP-dependen elec ogen- ic mechanism con ibu es o he main enance o he memb ane po en ial, as is also he case in o he s uc- u es (see e s. 9 and 20). Consis en wi h his sugges- ion a e he ollowing obse a ions: (1) Ouabain e- leases [3H]DA om he ype I cells 2, an e ec which is as in onse and occu s a lowe ouabain concen-