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-