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. Neu obiol.,
6 (1975) 521--527.
4 Black, A,M.S., Com oe, J.H. and Jacob, L., Species di e ence in ca o id body esponse
o ca and dog o dopamine and se o onin, Ame . J. Physiol., 223 (1972) 1097--1102.
5 Bliimcke, S., Rode, J. and Niedo , H.R., The ca o id body a e oxygen de iciency,
Z. Zell o sch,, 80 (1967) 52--77.
6
Chen, I-Li, Ya es, R.O. and Duncan, D., Elec on mic oscopic localiza ion o b ogenic
amines in he ca o id body, J. Cell. Biol., 35 (1967) 40--51.
7
Chen, I-Li and Ya es, R.D,, Elec on mic oscopic adioau og apnic s udies o he
ca o id body ollowing injec ions o labeled biogenic amine p ecu som, J. Cell. Biol.,
42 (1969)794--803.
8 Chen, I-Li, Ya es, R.D. and Duncan, D., The e ec s o ese pine and hypoxia on he
amine-s o ing g anules o he hams e ca o id body, J. Cell Biol., 42 (1969) 804--814.
9 Chiocchio, S.R., Bisca di, A.M. and T amezzani, J.H., 5-Hyd oxy yp omine in he
ca o id body o he ca , Science, 158 (1967) 790--791.
10 Daly, M,i'de B,, ~mbe son, C~J. and Sch~e ze , A,, Obse a ions on he olume o
blood low and oxygen u ilisa ion o he ca o id body in he ca , J. Physiol. (Lond.),
125(1954) 67--89,
11 Da is, J,N,, B ain y osine hyd oxyla ion: al e a ion o oxygen a ini y in i o by
immobiliza ion o elec oshock in he a , J, Neu ochem., 27 (1976) 211--215.
12 Dea naley, D.P,, Fillenz, M, and Woods, R.I., The iden i ica ion o dopamine in he
abbi 's ca o id body, P oc, Roy. Soc. (Lond,), Se . B, 170 (1968) 195--203.
13 Debijadi, R., Pe o ic, L., Va agic, V. and S osic, N., E ec o hypoxic hypoxia on he
ca echolamine con en and some cy ochemical changes in he hypo halamus o he
a , Ae ospace Med,, 40 (1969) ~95--499.
.4 Fillenz, M,, The unc ion o Type I cell o he ca o id body, In M.J. Pu es (Ed.), Pe i-
phe ~ A e ial Chemo ecep o s, Camb idge Uni e si y P ess, Camb idge, 1975,
pp. 133--134.
15 Hells om, S. and Koslow, S.H., ii3iogenic amines in ca o id body o adul and in an
a s, a gas ch oma og aphic-mass spec ome ic assay, Ac a physiol. ~ and., 93 (1975)
540--547,
16 Hells om, S., Hanbaue , I, and Cos a, E,, Selec i e dec ease o dopamine con en in a
ca o id body du ing exposu e o hypoxic condi ions, B ian Res., 118 (1976) 352--355.
17 He onen, A., Kane a, L,, Ko kala, O. and Pa enen, S., E ec s o hypoxia and ghico-
99
co icoids on he his ochemically demons able ca echolamines o he newbo n a
ca o id body, Ac a physiol, scand., 86 (1972) 109--114.
18 Ho man, H. and Bi ell, J.H.W., The ca o id body in no mal and anoxic ~ ~ es: an
elec on mic oscope s udy, Ac a Ana . (Basel), 32 (1958) 297--311.
19 Hu wi z, D.A., Robinson, S.M. and Ba o sky, L, Beha io al dec emen s and b ain ca e-
cholamines in a s exposed o hypoba ic hypoxia, Psychopha macol., J9 (1971) 26--33.
20 Ji, S., discussion o pape by S. Heils om, en i led E ec s o hypoxia on ca o id body
Type IccUs and hei ca echolamines. In H. Acke , S. Fidone and D. Pallo (Eds.),
Func ion and Func ional Signi icance o he Ca o id Body, Sp inge~-Ve lag, in p ess.
21 Lei ne , L.-M. and Liaube , M.-J., Ca o id body oxygen consump ion o he ca in i o,
P l ige s A ch. ges. Physiol., 323 (1971) 315--322.
22 Mills, E. and Slo kin, T., Ca echolamine con en o he ca o id body in ca s en ila ed
wi h 8--40% oxygen, Li e Sci., 16 (1975) 1555--1562.
23 Sampson, S.R., Mechanism o e e en inhibi ion o ca o id body chemo ecep o s in
he ca , B ain Res., 45 (1972) 266--270.
24 Sampson, S.R., Amino , M.J., Ja e, R.A. and Vid uk, E.H., Analysis o inhibi o y
e ec o dopamine on ca o id body chemo ecep o s in ca s, Ame . J. Physiol., 230
(1976) 1494--1498.
25 S up el, M. and Ro l, J., Ac ion de l'anoxie e he di e en s ax de gaz ca bonique su
le con enu en no ad enaline e ad enaliI~e du ce eau de a , C.R. Soc. Biol. (Pa is),
155 (1961) 237--240.
26 Ve na, A., Con ibu ion a l'E ude de Glomus Ca o idien du Lapin, Rech~ ches Cy o-
Iogiques, Cy ochimiques e Expe imen ales, Doc o al Thesis (Bo deaux)~(1975 ).
27 Vog , M.,~The concen a ion o sympa hin in di e en pa s o he cen al ne ous
sys em unde no mal condi ions and a e he adminis a ion o d ugs, J. Physiol.
(Lond.), 123 (1954) 451--481.
28 Zapa a, P., E ec s o dopanline on ca o id chemo- and ba o ecep o s in i o, J. Physiol.
(Lond.), 244 (1975) 235--251.
29 Zapa a, P., Hess, A., Bliss, E.L. and Eyzagui e, C., Chemical, elec on mic oscopic and
physiological obse a ions on he ole o ca echolamines in he ca o id body, B ain Res.,
14 (1969) 473--496.