Ionic Cu en s in Dispe sed
Chemo ecep o Cells o he Mammalian
Ca o id Body
j. UREI~/A, J. LOPEZ-L6PEZ, C. GONZALEZ, and
j.
LOPEZ-BARNEO
F om he Depa amen o de Fisiologla y Bio isica, Facul ad de Medicina, Uni e sidad de
Se illa, 41009 Se illa, Spain
ABSTRACT Ionic cu en s o enzyma ically dispe sed ype I and ype II cells o
he ca o id body ha e been s udied using he whole cell a ian o he pa ch-clamp
echnique. Type II cells only ha e a iny, slowly ac i a ing ou wa d po assium cu -
en . By con as , in e e y ype I chemo ecep o cell s udied we ound (a) sodium,
(b) calcium, and (c) po assium cu en s. (a) The sodium cu en has a as ac i a ion
ime cou se and an ac i a ion h eshold a ~-40 mY. A all ol ages inac i a ion
ollows a single exponen ial ime cou se. The ime cons an o inac i a ion is 0.67
ms a 0 mV. Hal s eady s a e inac i a ion occu s a a memb ane po en ial o
~- 50 mV. (b) The calcium cu en is almos o ally abolished when mos o he
ex e nal calcium is eplaced by magnesium. The ac i a ion h eshold o his cu -
en is a ~-40 mV and a 0 mV i eaches a peak ampli ude in 6-8 ms. The
calcium cu en inac i a es e y slowly and only dec eases o 27% o he maximal
alue a he end o 300-ms pulses o 40 mV. The calcium cu en was abou wo
imes la ge when ba ium ions we e used as cha ge ca ie s ins ead o calcium
ions. Ba ium ions also shi ed 15-20 mV owa d nega i e ol ages he conduc ance
s. ol age cu e. Deac i a ion kine ics o he calcium cu en ollows a biphasic
ime cou se well i ed by he sum o wo exponen ials. A -80 mV he slow com-
ponen has a ime cons an o 1.3 _+ 0.4 ms whe eas he as componen , wi h an
ampli ude abou 20 imes la ge han he slow componen , has a ime cons an o
0.16 _+ 0.03 ms. These esul s sugges ha ype I cells ha e p edominan ly as
deac i a ing calcium channels. The slow componen o he ails may ep esen he
ac i i y o a small popula ion o slowly deac i a ing calcium channels, al hough
o he possibili ies a e conside ed. (c) Po assium cu en seems o be mainly due o
he ac i i y o ol age-dependen po assium channels, bu a small pe cen age o
calcium-ac i a ed channels may also exis . This cu en ac i a es slowly, eaches a
peak ampli ude in 5-10 ms, and he ea e slowly inac i a es. Inac i a ion is almos
comple e in 250-300 ms. The po assium cu en is e e sibly blocked by e ae h-
ylammonium. Unde cu en -clamp condi ions ype I cells can spon aneously i e
la ge ac ion po en ials. These esul s indica e ha ype I cells a e exci able and
Add ess ep in eques s o D . J. L6pez-Bameo, Depa amen o de Fisioiogla y Bio isica, Facul ad
de Medicina, A enida de S/mchez Pizju~.n, 4, 41009 Se illa, Spain. D . J L6pez-L6pez and D . C.
Gonz~lez' pe manen add ess is Depa amen o de Bioquimica y Biologia Molecula y Fisiologla,
Facui ad de Medicina, Uni e sidad de Valladolid, C/Ram6n y Cajal 5, 47005 Valladolid, Spain.
J. GgN. PHYSIOL. ~ The Rocke elle Uni e si y P ess 9 0022-1295/89/05/0979/21 $2.00
Volume 93 May 1989 979-999 979
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980 THE JOURNAL OF GENERAL PHYSIOLOGY" 9 VOLUME 93 9 1989
ha e a a ie y o ionic conduc ances. We sugges a possible pa icipa ion o hese
conduc ances in chemo ecep ion.
INTRODUCTION
The mammalian ca o id bodies a e pai ed o gans ha sense he le el o oxygen en-
sion (pO~) in a e ial blood. This senso y in o ma ion is sen o he cen al ne ous
sys em whe e i induces an adequa e en ila o y esponse (De Cas o, 1928; Hey-
mans e al., 1930; Fi zge ald and Lahi i, 1986). Among he di e en s uc u es o
he ecep o complex (glomus o ype I cells, sub en acula o ype II cells, and
ne e endings) ype I cells, he mos nume ous in he ca o id body, a e p esumably
he p ima y chemo ecep o s. Type I cells a e ich in sec e o y g anules con aining
dopamine, no epineph ine, and o he neu o ansmi e s and hey es ablish chemi-
cal synapses wi h ne e endings. A dec ease in pO~ ul ima ely esul s in ansmi e
elease by ype I cells and exci a ion o he a e en ibe s o he ca o id sinus ne e
(see o e iews Eyzagui e and Zapa a, 1968; Fidone and Gonz~lez, 1986).
The mechanism in ol ed in he ansduc ion o he hypoxic s imulus is unknown.
I has been ecen ly shown ha ei he low pO~ o high ex e nal po assium can
induce he elease o dopamine om ype I cells, which is dependen on ex e nal
calcium and is inhibi ed by calcium channel blocke s (Fidone e al., 1982; Obeso e
al., 1987). These da a sugges ha , in conco dance wi h he mechanism o s imulus-
sec e ion coupling in many sec e o y sys ems, memb ane depola iza ion may play a
pa in he esponse o ype I cells o hypoxia.
The p esen esea ch wo k was unde aken o cha ac e ize he elec ical p ope -
ies o he cellula elemen s o he ca o id body and o es he hypo hesis ha
memb ane ionic conduc ances could be al e ed by changes in pO~. F om an elec o-
physiological iewpoin ype I cells a e p ac ically unknown. Some p e ious elec o-
physiological s udies ha e been pe o med using in aceUula eco ding mic oelec-
odes, bu hey a e somewha con using and inconclusi e because hey we e p oba-
bly done on cells damaged by he mic oelec ode impalemen . I has been epo ed
ha ype I cells a e nonexci able and hei elec ical pa ame e s a e unal e ed by
hypoxia (Eyzagui e e al., 1983; Acke and Pie uschka, 1977). These expe imen s
we e, howe e , done on cells wi h an a e age es ing po en ial (-20 mV) ha is low
enough o p oduce comple e inac i a ion o he ol age-dependen memb ane ionic
conduc ances.
We ha e pe o med expe imen s in acu ely dispe sed cells o he ca o id body
subjec ed o whole-cell pa ch clamp, which is a echnique ha pe mi s accu a e elec-
ical measu emen s in small cells (Hamill e al., 1981). Ou da a demons a e ha
ype I cells ha e a a ie y o ol age-dependen ionic channels and ha hey a e able
o gene a e la ge sodium- and calcium-dependen ac ion po en ials. Type II cells a e
unexci able and on depola iza ion only gene a e a small ou wa d cu en . This
a icle concen a es on he ull desc ip ion o he sodium, calcium, and po assium
cu en s o ype I cells. The ollowing a icle illus a es ha in hese cells po assium
channel ac i i y is e e sibly dec eased by hypoxia, which may be he memb ane
mechanism esponsible o chemo ansduc ion. Some o his wo k has al eady
appea ed in a sho epo (L6pez-Ba neo e al., 1988).
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981
METHODS
Cell Dissocia ion and Cul u e
Expe imen s we e pe o med on cells dissocia ed om abbi ca o id bodies. The p o ocol
used o cell dissocia ion was wo ked ou a he Uni e si y o Valladolid in collabo a ion wi h
D . Beni o He e os.
Animals we e anes he ized and he whole egion o he ca o id a e y bi u ca ion was
emo ed. Fou ca o id bodies we e dissec ed unde a mic oscope, cleaned, and hen each one
o hem was cu in o wo o h ee small pieces. The issue was placed in a ial wi h 2 ml o a
Ca ~+- and Mg~+- ee Ty ode solu ion o he ollowing composi ion, in millimola : 140 NaCI,
4.7 KCi, 5 Na py u a e, 3 suc ose, 5 glucose, and 10 HEPES. This solu ion also con ained
ypsin (2 mg/ml), collagenase (2 mg/ml), and DNase (0.5 mg/ml). E e y 10 min he issue
was i u a ed. A e 20 min a 37~ he ial was cen i uged a 800 g o 5 ain. The pelle
was esuspended in ano he 2 ml o he same solu ion wi h collagenase (4 mg/ml), DNase (0.5
mg/ml), and albumin (5 mg/ml), and kep a 37~ o 20 min. The issue was i u a ed e e y
10 min. A he end o he incuba ion pe iod he p epa a ion was washed wice wi h he Ca ~+
and Mg2+- ee solu ion o emo e he enzymes, and he inal pelle was esuspended in 5 ml
o minimum essen ial medium supplemen ed wi h giu amine (1%), penicillin-s ep omicin
(2%), and e al cal se um (5%). Cells we e pla ed on sli e s o glass co e slips ea ed wi h
poly-/-lysine and kep in a CO~ incuba o a 37~ un il use (4-48 h a e pla ing). A e he
cells we e dissocia ed, mos o hem had a ound shape and wo dis inc popula ions could be
easily dis inguished. Type I cells had a diame e be ween 9 and 13 #m and a ypical bi e in-
gence appea ance unde he ligh mic oscope. A second popula ion o cells, classi ied as
pu a i e ype II cells, had a smalle and mo e uni o m diame e (be ween 5 and 7 um). These
wo cell ypes also had clea di e ences in hei elec ical p ope ies (see Resul s). A e 48 h
in cul u e many cells began o show g owing p ocesses and acqui ed a bipola o an i egula
shape.
Solu ions
Du ing he expe imen s a co e slip was ans e ed o a small chambe ha had a con inuous
low o solu ion ha could be changed in ~15-20 s. The composi ion o he eco ding solu-
ions is shown in Table I. In he ex and igu e legends solu ions a e indica ed as ex e nal/
in e nal. TI'X (500-1,000 nM) was added o he ex e nal solu ion o block Na channels and
in mos expe imen s 3 mM Mg-ATP we e added o he in e nal solu ion o e a d wash-ou o
Ca channels (Kos yuk, 1984; Fo scbe and Ox o d, 1985; Co a 1986). In some expe imen s
designed o eco d K cu en s, he in e nal solu ion con ained a known concen a ion o ee
Ca 2+, which was ob ained using Ca-EGTA bu e s. The concen a ions o Ca and EGTA used
in hese expe imen s a e indica ed in he igu e legends. Unless o he wise no ed, he pH o
he ex e nal and in e nal solu ions we e adjus ed o 7.4 and 7.2-7.3, espec i ely. Expe i-
men s we e pe o med a oom empe a u e (20-25~
Reco ding Techniques
Ionic cu en s o ype I cells we e eco ded using he whole-cell a ian o he pa ch-clamp
echnique (Hamill e al., 1981). Pa ch elec odes we e usually ab ica ed om so hema oc i
capilla ies (Hi schmann, Fede al Republic o Ge many) bu in se e al expe imen s bo osili-
ca e glass (Kimax 51) was also used. In ou expe imen al condi ions, cu en eco ded wi h
bo h ypes o elec odes we e indis inguishable. The elec ode ip was i e polished on a
mic o o ge and elec ode esis ance a ied be ween 1 and 2 M L The pa ch-clamp ampli ie
used in mos expe imen s was buil by us ollowing he s anda d design (Sigwo h, 1983; Ma -
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982 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 93 9 1989
eson and A ms ong, 1984; A ms ong and L6pez-Ba neo, 1987). The cu en - o- ol age
con e e was a Bu -B own OPA 111 (Tucson, AZ). F equency esponse was imp o ed by
using low esis ance elec odes, elec onic compensa ion o se ies esis ance, and a ela i ely
low eedback esis ance (100 M l). Cu en -clamp eco dings we e done in some cells using a
Lis pa ch-clamp ampli ie (model LPC-7; Adams and Lis Associa es, L d., G ea Neck,
NY).
Da a Acquisi ion and Analysis
An IBM-PC/AT compu e in e aced o he analog elec onics was used o pulse gene a ion
and o acquisi ion, display, s o age, and analysis o he da a. The in e ace, ab ica ed in ou
labo a o y, is buil on wo IBM p o o ype ca ds di ec ly connec ed o he expansion bus o
he compu e (U e ia e ai., 1989). The pulse gene a o has an 8 bi digi al- o-analog con-
e e (DCA 0808; Analog De ices, Inc., No wood, MA) and a p og ammable pe iphe al
in e ace (PPI 8255A-5; NEC Mic ocompu e s, Inc.) wi h he po s p og ammed in ou pu
TABLE I
Composi ion o Solu ions
Ex e nal
NaC1 KCI CaCI~ MgCI2 BaCI~ CdCI~ TEACI HEPES
140 Na 140 2.7 2.5-10 .... 10
140 Na, 10 Ba 140 2.7
-- -- 10 -- --
10
140 Na, 9 Mg 140 2.7 1 9 -- -- -- 10
140 Na, 0.5 Cd 140 2.7 1.5 -- -- 0.5 -- 10
100 Na, TEA 100 2.7 5 -- -- -- 40 10
In e nal
NaCI KCI K-glu ama e KF CsCi CsFI MgCI~ HEPES EGTA
130
K -- 30
80 20
-- -- 2 10
5-10
130 Cs .... 110 20
2 10
5-10
90 Cs, 40 Na 40 -- -- -- 70 20 2 10 5-10
All alues a e gi en in millimola .
mode (Pea man, 1977; Liu and Gibson, 1986). Cu en signals a e digi ized in a 12 bi ana-
log- o-digi al con e e (ADC, AD578; Analog De ices, Inc.). The 12 lines o pa allel ou pu
o he ADC a e ans e ed o he compu e RAM memo y by means o a PPI chip wi h he
po s p og ammed in inpu mode (Pea man, 1977). The acquisi ion sampling in e al can be
changed by he p og am o alues be ween 20 and 500/zs. Digi ized cu en signals a e dis-
played in an oscilloscope a e being con e ed in o analog o m. In his sys em each cu en
ace is de ined by 500 samples. In mos expe imen s linea ionic and capaci y cu en s we e
sub ac ed using a P/4 p ocedu e (Bezanilla and A ms ong, 1977). Du ing analysis ail cu -
en s we e i ed wi h one o he sum o wo exponen ials using a leas -squa es p ocedu e.
RESULTS
The da a p esen ed in his pape we e ob ained om o e 150 ype I chemo ecep-
o cells subjec ed o ol age clamp. These cells had an a e age capaci ance o 7.27
+_ 2.26 pF (mean + SD, n = 50). The elec ical p ope ies o ype II cells, which
gene a e only a iny ou wa d cu en , a e p esen ed a he end o he pape .
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983
Ionic Cu en Componen s
The majo componen s o ionic cu en eco ded in ype I cells a e shown in Fig. 1.
Wi h he s anda d high K in e nal solu ion, depola iza ion o 30 mV om a holding
po en ial o -70 mV elici ed a as inwa d cu en ollowed by a slowe ou wa d
cu en (Fig. 1 A). A he epola iza ion o he 8-ms pulse he cu en changes sud-
denly in di ec ion and a la ge inwa d ail cu en was eco ded. This esponse pa -
e n was obse ed in e e y cell s udied al hough he ampli ude o he di e en cu -
en componen s a ied om cell o cell.
The ou wa d cu en was mainly ca ied by K ions since i disappea ed when all
he K in he pipe e solu ion was eplaced by Cs. In his condi ion (Fig. 1 B) a as
and sus ained inwa d cu en , ollowed by an inwa d ail, we e eco ded by a depo-
A 140 Na.2.5Ca//130 K
J
/
/
.[
"i
140 Na,10Ca//130 Cs
!
140 Na,10 Ca,TTX//130 Cs
;
,i 1
2 ms -i
nA
0.5 nA
nA
FIGURE 1. Majo cu en compo-
nen s in ype I cells. (A) Cu en
eco ded du ing a ol age s ep o 30
mV wi h e u n o he holding po en-
ial (HP) o -70 mV a e 8 ms. (B)
Blockade o he ou wa d po assium
cu en by in e nal cesium. Pulse o
20 mV and HP o -80 inV. Calcium
cu en is eco ded in isola ion by
applica ion o he same ol age s ep
in he p esence o TTX (C). Solu ion
composi ion was as indica ed nex o
each ace. Expe imen s EN1988J
(A) and EN2188J (B and C).
la iza ion o 20 mV om a holding po en ial o -80 inV. The addi ion o TTX o
he ex e nal solu ion (Fig. 1 C) abolished he as componen o he inwa d cu en ,
which indica es ha i was due o he ac i i y o Na channels. Howe e , he slowly
ac i a ing inwa d cu en and he ail emained unal e ed. These wo las cu en
componen s we e he esul o he ac i i y o Ca channels since, as will be shown
below, hey disappea ed a e eplacemen o ex e nal Ca by Mg, o when 0.5 mM
Cd was added o he ex e nal solu ion. These esul s indica e he exis ence in ype I
chemo ecep o cells o ol age-dependen Na, Ca, and K channels.
P ope ies o he Sodium Cu en
Cu en - ol age ela ions.
Na cu en s we e eco ded in isola ion in cells whe e
in e nal K ions we e eplaced by Cs (o a mix u e o Cs and Na), and Ca channels
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THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME
93
9 1989
we e blocked by ex e nal Mg o Cd. A amily o Na cu en eco ds ob ained by
depola iza ions o he indica ed memb ane po en ials is shown in Fig. 2 A. The hold-
ing po en ial was -80 mV and he in e nal solu ion con ained 40 mM Na. Na cu -
en had a ol age-dependen as ac i a ion ime cou se, a 0 mV i eached a peak
in ~0.6-0.7 ms, and hen i inac i a ed comple ely. Cu en was inwa d a mem-
b ane po en ials mo e nega i e ha l ENa and ou wa d wi h mo e posi i e memb ane
po en ials.
The cu en - ol age ela ion o his expe imen is shown in Fig. 2 B, whe e he
peak cu en ampli ude is plo ed as a unc ion o he pulse memb ane po en ial
(VM). Ac i a ion h eshold was ~- 40 mV, he peak o he
I-V
cu e occu ed a a VM
A
:
.
-40 o 60 nV
9
'-. / Io.s ~A
1ms
-6O
1
-40 -20
FIGURE 2. Family o sodium cu -
IM'nA ,/
en s eco ded du ing depola iza-
-0.s / ions o -40, -20, 0, 20, 40, and 60
/Sp
mV om a HP o -80 mV (A). Peak
cu en ampli ude as a unc ion o
-0.4 he memb ane po en ial is plo ed in
B. The con inuous line was i ed by
-0.2 eye. Solu ions (in millimola ): 140
20 Na, 0.5 Cd//90 Cs, and 40 Na.
Expe imen MZ0288M.
--0.2 o/ VM' nV
5/
o 0 o + 10 mV, and he e e sal po en ial was a +32 mV, which was only 1 mV
apa om he ENa alue p edic ed by he Ne ns equa ion. Al hough inwa d Na
cu en s we e obse ed in e e y cell s udied he ampli ude a ied om cell o cell.
The maximal Na cu en ampli ude eco ded in cells wi hou in e nal Na was 0.46 _+
0.2 nA (mean -+ SD, n = 6).
Inac i a ion.
In ype I cells inac i a ion o he Na cu en was, as in o he p ep-
a a ions, a ol age-dependen p ocess. Fig. 3 A illus a es he ol age-dependence o
s eady-s a e inac i a ion by measu ing he peak Na cu en elici ed by a depola iza-
ion o 10 mV as a unc ion o V~ du ing a 50-ms condi ioning p epulse. In he
o dina e, cu en ampli ude is no malized wi h espec o i s alue in he absence o
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URENA ET AL.
IOniC Cu aUs o Glomus Cells
985
p epulse. Hal s eady-s a e inac i a ion was a ~-50 mV, and a -25 mV he non-
inac i a ed ac ion o IN, was < 10%. The inse in Fig. 2 B illus a es ha a 0 mV
inac i a ion was comple e in <6 ms and ha i s ime cou se could be well i ed by a
single exponen ial wi h a ime cons an o 0.67 ms. The plo in he same igu e
shows ha he ime cons an o inac i a ion dec eased wi h memb ane depola iza-
ion.
Closing kine ics. The
closing o Na channels was s udied in a ew cells by he
applica ion o sho depola izing pulses (0.5-1 ms), which p e en ed he de elop-
men o inac i a ion, and he eco ding o ail cu en s a he ins an o epola iza-
ion. Na ails we e well i ed by single exponen ials and a -80 mV he ime con-
A B
LK
"O
O
Z
0.5
VM,mV
' InA
; 2'0
VM,mV
FIGURE 3. (A) S eady-s a e inac i a ion o sodium cu en s. The plo ep esen s no malized
peak cu en ampli ude ob ained du ing a es s ep depola iza ion o 10 mV as a unc ion o
he memb ane po en ial du ing a 50-ms condi ioning p epulse. (B) Time cou se o inac i a-
ion. The inse is a sodium cu en eco ded by a ol age s ep o 0 inV. Inac i a ion is well
i ed by an exponen ial wi h a ime cons an o 0.67 ms. The plo ep esen s he ime con-
s an o inac i a ion (o dina e) measu ed in cu en s eco ded a a ious memb ane po en-
ials (abscissa). Lines we e i ed by eye. HP, -80 mV. Solu ions (in millimola ): 140 Na and
0.5 Cd//130 Cs. Expe imen s EN2888J (A) and FE0388J (B).
s an was be ween 60 and 70 #s. Thus, hese esul s indica e ha ype I chemo ecep-
o cells ha e a p ominen Na cu en wi h p ope ies simila o hose ound in
o he elec ically exci able cells.
P ope ies o he Calcium Cu en
Iden i ica ion o he cu en .
Ca cu en s we e eco ded in cells dialyzed wi h he
130 Cs solu ion. Fig. 4 A shows a ace o inwa d cu en in his expe imen al con-
di ion and wi h 10 mM Ca in he ex e nal solu ion. The pulse cu en was ollowed
by a la ge inwa d all cu en . Exposu e o a di e en ex e nal solu ion wi h less Ca
(9 mM Mg, 1 mM Ca) p oduced a ma ked educ ion o he s eady-s a e pulse cu -
en and he ail, al hough a small componen o inac i a ing inwa d Na cu en
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THE JOURNAL OF GENERAL
PHYSIOLOGY. VOLUME 93 9 1989
FIGURE 4. Iden i ica mn o
B
he calcium cu en . (A)
140 Na//130 Cs 140 Na,TTX//130 Cs
Inwa d cu en eco ded wi h
~,~ /~ ~~,.~a,z,~,,,~'~ ~ ~.9 ~,lCa he 140 mM Na, 10 mM Ca
9
Mg.1 Ca
=
;;( ,, --~:-:: ex e nal solu ion du ing a ol -
" :: ~~i" age s ep o 0 mV. On eplacing
/ . / calcium wi h magnesium (9
I 10 Ca
:
10 Ca ~ 0.SnA 9 mM Mg, 1 mM Ca) he s eady
; inwa d cu en and he ail
2ms
almos disappea bu a small
componen o ansien sodium cu en emains. In he p esence o TFX (B) he isola ed
calcium cu en eco ded in 10 mM Ca is almos abolished a e he in oduc ion o he 9
mM Mg, 1 mM Ca solu ion in he ba h. HP, -80 mV. Solu ions we e as indica ed in he
igu e. Expe imen s EN2788J (A) and EN2588J (B).
emained. A e Na channels we e blocked wi h TIX in ano he cell (Fig. 4 B), Ca
cu en s could be eco ded in o al isola ion. This cu en ac i a es slowly and a 0
mV i eaches a maximum in ~8 ms. Fig. 4 B also illus a es he almos comple e
disappea ance o he pulse cu en and he ail a e eplacemen o ex e nal Ca by
Mg. In o he expe imen s wi h low ex e nal Ca (1.5 mM) he addi ion o 0.5 mM Cd
o he ex e nal solu ion comple ely abolished he Ca cu en (see Fig. 2).
Cu en - ol age ela ions.
Fig. 5 (]e column, 10 Ca) shows calcium cu en s
eco ded a a ious memb ane po en ials. Cu en gene a ed du ing he pulse, indi-
ca ed be ween he a ows, ac i a es mo e apidly a mo e depola ized le els and is
ollowed by a as ail whose ampli ude inc eases wi h he ampli ude o he depola -
iza ion. These ails a e due o he low o Ca ions h ough he channels ha we e
open du ing each pulse, and hei ime cou se e lec s ha o he closing o he Ca
channels. Cu en ampli ude measu ed a he end o he pulse as a unc ion o pulse
VM'mV 10 Ca
-20 .... - .....
~ F
"!
":.:
13 nA
3ms
lO
Ba
i/
!
i
./
2
/
i
?
. . -. ~
- /
.j
FIGURE 5. Cu en h ough
calcium channels ca ied by cal-
cium and ba ium ions. Cu en s
a e he esul o depola iza ions
o he memb ane po en ials indi-
ca ed nex o each ace wi h 10
mM Ca o 10 mM Ba in he
ex e nal solu ion. The a ows
indica e he onse and he end o
he ol age s eps. All aces a e
om he same cell. HP, -80
inV. Solu ions (in millimola ):
140 Na, 10 Ca (10 Ba), and
T X//130 Cs. Expe imen
MZ0288K.
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URENA ET AL.
Ionic Cu en s o Glomus Cells
-60 -40 -20
0.4
0.6-
0.8-
VM,mV
20 40 60
1
I , I
IM,nA
987
FmURE 6. Calcium cu en - ol age
ela ion. The cu en measu ed a
he end o 8-ms pulses is plo ed as a
unc ion o he pulse memb ane
po en ial. The line was i ed by eye.
HP, -80 mV. Solu ions (in millimo-
la ): 140 Na, l0 Ca, and T X//130
Cs. Expe imen MZ0288K.
memb ane po en ial is plo ed in Fig. 6. This
I-V
plo shows ha he ac i a ion
h eshold was a --40 mV and ha he maximal inwa d cu en was ob ained a
+ ] 0 o + 20 mV. A hese I'M alues he a e age ampli ude o he Ca cu en mea-
su ed wi h 10 mM ex e nal Ca was 0.4 .+ 0.2 nA (mean _+ SD, n = 8).
The igh column o Fig. 5 shows eco dings ob ained in he same cell a e ex e -
nal Ca was eplaced by Ba. The aces illus a e ha Ba ions lowed h ough Ca
channels be e han Ca ions, as indica ed by he la ge size o he ails. In he ange
be ween 0 and + 20 mV, o al Ba conduc ance was abou wo imes la ge han Ca
conduc ance. Ba ions also a ec ed he conduc ance- ol age ela ion o Ca channels.
Fig. 7 plo s he no malized ampli ude o ail cu en s eco ded wi h 10 mM Ca
(do s) and 10 mMBa (squa es). A compa ison o bo h cu es indica es ha ex e nal
Ba shi ed he ac i a ion cu e 15-20 mV in he nega i e di ec ion.
Closing kine ics.
I has been shown in p e ious igu es ha a he ins an o
epola iza ion Ca cu en s we e ollowed by la ge ail cu en s. Examples o ails
eco ded on epola iza ion o -80 mV a e a pulse o + 40 mV a e shown in Fig. 8
using 10 mM ex e nal Ca (A) o Ba (C) as cha ge ca ie s. The decay o he ail
cu en had a clea biphasic ime cou se wi h a la ge as componen and a small
slow componen . The slow componen was i ed by an exponen ial ex apola ed o
@
N
m
E
Z
c
g
"0
c
0
0.5-
-~o -go -2~
FIGURE 7. Conduc ance- ol age e-
la ion o calcium channels wi h cal-
cium and ba ium as cha ge ca ie s.
Conduc ance, ep esen ed in he
o dina e, was measu ed om he
ampli ude o ail cu en s eco ded
a he ins an o epola iza ion o 10-
ms pulses. The pulse memb ane
po en ial is ep esen ed on he
abscissa. Reco dings a e om he
same cell ba hed in 10 mM Ca (do s)
and 10 mM Ba (squa es). Lines we e
d awn by eye. HP, -80 mV. Solu-
2~0 4J0 6~ ions (in millimola ): 140 Na, l0 Ca
VM,m (10 Ba), and TI'X//130 Cs. Expe i-
men MZ0288K.
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Published May 1, 1989
994
THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 98 9
1989
a a ions ( o example Sah e al., 1988). The cu en has a as ac i a ion and inac i-
a ion, is comple ely blocked by nanomola concen a ions o TTX, and is selec i ely
ca ied by Na ions. Ac i a ion o 1Na in ype I cells is simila o he Na cu en
eco ded in GHs cells (Ma eson and A ms ong, 1984) bu is somewha slowe han
he cu en om he squid axon (Hodgkin and Huxley, 1952; Bezanilla and A m-
s ong, 1977). The peak o he cu en - ol age cu e occu ed a 10 mV, which is
simila o Na cu en s eco ded in GHs (Dubinsky and Ox o d, 1984; Ma eson and
A ms ong, 1984) and ch oma in (Fenwick e al., 1982) cells, bu i is displaced
owa d posi i e ol ages i compa ed wi h Na cu en s eco ded in neu ons (Sah e
al., 1988) and neu oblas oma cells (Moolenaa and Spec o , 1978). These quan i a-
i e di e ences we e also seen in he ol age-dependence o s eady-s a e inac i a-
ion. In ou expe imen s hal o he Na channels we e inac i a ed a ~ -50 mV, a
alue simila o ha measu ed in GH3 cells (Dubinsky and Ox o d, 1984; Ma eson
and A ms ong, 1984), bu less nega i e han he one obse ed in cen al neu ons
(-75 mV, Sahe al., 1988). Inac i a ion o Na cu en s om ype I cells ollows a
single exponen ial ime cou se and is ela i ely as compa ed wi h ac i a ion. A sin-
gle exponen ial decay has also been obse ed in GH3 cells (Vandenbe g and Ho n,
1984) al hough a biexponen ial ime cou se was equi ed o i Na inac i a ion in a
and og ne e (Chiu, 1977; Neumcke and S amp li, 1982). Na cu en densi y in
ype I cells, assuming ha he e is no memb ane in olding, was 0.15 mA/cm ~. This
alue is wi hin he ange o he es ima ions done in GHs cells (Dubinsky and
Ox o d, 1984; Ma eson and A ms ong, 1984) bu i is abou 10 imes smalle han
in he squid axon. Thus, on quan i a i e g ounds he Na cu en o ype I cells
esembles he INa o GH s and ch oma in cells, bu di e s in some aspec s om he
cu en exis ing in neu ons and ne e cells.
Despi e he exis ence o a ela i ely low densi y o Na channels, ype I cells, like
o he sec e o y cells, can gene a e la ge ac ion po en ials. The ac ha in glomus
cells s eady-s a e inac i a ion o IN, is displaced in he posi i e di ec ion may con ib-
u e o a mo e e icien use o he Na channels a ailable (see also Ma eson and A m-
s ong, 1984). Res ing po en ials measu ed in p e ious in acellula eco dings pe -
o med in ype I cells we e in he neighbo hood o - 20 mV (Acke and Pie uschka,
1977; Eyzagui e e al., 1983). Ou esul s show ha >90% o gs, is inac i a ed a
-25 mV, which explains he unexci abili y o he in acellula ly eco ded cells,
which we e p obably damaged by he mic oelec odes.
Calcium cu en .
Blockade o Na channels by TTX e ealed in all ype I cells he
exis ence o a Ca cu en . This cu en esembles/ca om o he e eb a e p epa a-
ions in i s ime cou se, i s sensi i i y o ex e nal di alen ca ion block, and he abili y
o Ba o subs i u e o Ca as cha ge ca ie (Fenwick e al., 1982; Hagiwa a and
Ohmo i, 1982; Dubinsky and Ox o d, 1984; Ma eson and A ms ong, 1984). The
peak o he cu en - ol age cu e was a 10-20 mV, which is close o he alues
obse ed in o he cells. In equi alen expe imen al condi ions, he ampli ude o he
Ca cu en is somewha la ge in ype I cells han in GHs (Hagiwa a and Ohmo i,
1982; Dubinsky and Ox o d, 1984; Ma eson and A ms ong, 1984), ch oma in
(Fenwick e al., 1982), adenohypophysial (Co a, 1986), and panc ea ic be a (Hi ia
and Ma eson, 1988) cells. Wi h 10 mM ex e nal Ca, he peak Ca cu en ampli ude
was almos he same as he maximal Na cu en .
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URENA ET AL.
[Ol' ic Cu en s
o Glomus
Cells
995
Indica ions o he exis ence o mo e han one Ca channel ype ha e been
obse ed in a numbe o sec e o y cells (Co a, 1986; Ma eson and A ms ong,
1986; Hi ia and Ma eson, 1988; Taba es e al., 1989), neu ons (Ca bone and
Lux, 1984; Nowycky e al., 1985), and muscle (Bean, 1985). In hypophysial pa s
in e media, panc ea ic be a cells, and ad enoco ical cells, FD and SD Ca channels
a e dis inguished acco ding o hei di e en closing kine ics. FD channels also ha e
a highe h eshold and inac i a e mo e slowly han SD channels. Ca ails in mos
ype I cells ollow a clea biphasic ime cou se and a e well i ed by he sum o a
small slow exponen ial and a as la ge exponen ial. The ime cons an s o he wo
componen s a e compa able o hose measu ed in o he p epa a ions (Co a, 1986;
Ma eson and A ms ong, 1986).
Al hough he analysis o he deac i a ion kine ics o Ca cu en s sugges s he exis-
ence in ype I cells o wo Ca channel ypes, a numbe o expe imen al obse a ions
do no suppo his in e p e a ion. (a) Ca cu en o ype I cells inac i a e e y
slowly wi h a ime cou se ha esembles ha o FD channels om o he p epa a-
ions. (b) The ime cou se o ail cu en s eco ded a e sho and la ge depola iza-
ions is simila and is no a ec ed by a 500-ms condi ioning p epulse. In o he cells
he slow componen o he ails, which ep esen s he closing o SD channels, is
ma kedly educed a e long (>50 ms) depola iza ions o by a condi ioning depola -
izing p epulse. (c) The conduc ance- ol age ela ion wi h Ba ions as cha ge ca ie s
is displaced 15-20 mV owa d nega i e ol ages. Ba also slows down he closing o
he channels. These e ec s o Ba a e known o be media ed by hei in e ac ion wi h
FD channels. Finally, (d) in he absence o exogenous in e nal Mg-ATP he Ca cu -
en o giomus cells is e y labile and disappea s almos comple ely in 5-8 ain. This
p ope y is ypical o FD channels whe eas SD channels seem o be esis an o dilu-
ion o cy osolic componen s (Co a, 1986; Ma eson and A ms ong, 1986).
All oge he his e idence indica es ha Ca cu en in ype I cells is mainly
media ed by FD channels. A small popula ion o SD channels, esponsible o he
slow componen o he ails, may exis bu unequi ocal p oo s o hei exis ence
we e no ound. The possibili y s ill emains ha he slow componen o he ails is
ei he a esul o he ac i i y o Ca channels ha a e di e en om SD channels, o
ha i ep esen s a pa icula kine ic p ope y o FD channels in ype I cells.
Po assium cu en .
An ou wa d K cu en was eco ded in all ype I cells s ud-
ied. This cu en was eco ded wi h a la ge ampli ude in cells wi h in e nal Ca con-
cen a ions anging be ween 0.5 #M and <10 -1~ M, bu in a gi en cell i dec eased
in ampli ude a e wash-ou o Ca channels. Thus, a pe cen age o his cu en may
be due o he ac i i y o Ca-dependen K channels ac i a ed by he local ise in cy o-
solic Ca ha ollows Ca in lux h ough unc ional Ca channels (Ma y and Nehe ,
1985). Because o he apid disappea ance o Ca channels in ou p epa a ion, e en
in he p esence o 3 mM in e nal Mg-ATP, we cen e ed on he s udy o he ol age-
dependen K cu en a e wash-ou o Ca channels.
The ol age-dependen K cu en o ype I cells has a ypical sigmoid ac i a ion
ime cou se, a +40 mV i eaches hal maximal ampli ude in 3.5 ms, and he ea e
i slowly inac i a es. The cu en is quali a i ely simila o delayed K cu en s om
o he p epa a ions (Ad ian e al., 1970; Dubinsky and Ox o d, 1984; Ro sman and
T ube, 1986; Ma eson and Ca melie , 1988). I is, howe e , slowe han K cu en s
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996 TH~ JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 93 9 1989
o he squid axon o neu ons dispe sed om he squid gian ibe lobe (Hodgkin
and Huxley, 1952; A ms ong and L6pez-Ba neo, 1987), bu i s ac i a ion ime
cou se is simila o cu en om e eb a e sec e o y cells (Dubinsky and Ox o d,
1984; Ro sman and T ube, 1986). The K cu en in ype I cells inac i a ed almos
comple ely in 250 ms. This same beha io is ound in K cu en s om a numbe o
p epa a ions including squid neu ons (Llano and Bookman, 1986), GHs cells (Ma -
eson and Ca melie , 1988), and skele al muscle ibe s (Ad ian e al., 1970). The
ol age-dependen K cu en o ype I cells was, as we e o he K cu en s, almos
abolished by a la ge concen a ion o ex e nal TEA. In ou expe imen s he e e si-
bili y o he TEA blockade was almos pe ec .
Possible Pa icipa ion o Ionic Channels in he Physiology o Type I Cells
Al hough ype I cells ha e been conside ed he bes candida es o being he p i-
ma y chemo ecep o s since he ea ly wo k on he ca o id body, he basic mecha-
nisms in ol ed in his p ocess ha e emained la gely unknown ( o a e iew see Bel-
mon e and Gonz~lez, 1983). Recen in es iga ions ha e shown ha bo h hypoxia
and high ex e nal po assium, which p esumably causes memb ane depola iza ion,
p oduce sec e ion o dopamine in he ca o id body and ha his e ec is abolished
by Ca channel an agonis s (Fidone e al., 1982; Alma az e al., 1986; Obeso e al.,
1987). Thus, ype I cells may unc ion in a way simila o o he sec e o y sys ems in
which Ca in lux h ough ol age-ga ed memb ane Ca channels is a c i ical e en
leading o sec e ion. This idea could no be econciled wi h he ac ha ype I cells
impaled wi h mic oelec odes we e ound o be unexci able (Eyzagui e e al.,
1983), howe e , he esul s shown in his a icle demons a e ha hey ha e an
app eciable densi y o ol age-dependen sodium and calcium channels and ha
hey can epe i i ely gene a e ac ion po en ials. Na channels ha e, he e o e, an
impo an ole in spike gene a ion and by p oducing a as depola iza ion con ib-
u es o he opening o Ca channels, which a e well sui ed o as injec ion o Ca
in o he cy osol. The Ca-ac i a ed componen o he K cu en may pa icipa e in
spike epola iza ion, whe eas he slowly inac i a ing K channels a e mos p obably
in ol ed in pacemaking.
The indings epo ed he e led us o hypo hesize ha ionic channels in ype I cells
migh be egula ed by en i onmen al 02 ension and ha hey migh he e o e be
di ec ly implica ed in chemo ansduc ion. This hypo hesis was expe imen ally con-
i med as illus a ed in he ollowing a icle.
The au ho s wish o hank M s. Lola Gan o nina, Uni e si y o Se ille, o he aluable pa icipa-
ion in some expe imen s.
This esea ch was pa ially suppo ed by g an s PB86-250 and PB86-325 om Di eccion Gene al
de In esdgaci6n Cien i ica y T6cnica. J. R. L6pez-L6pez is a ellow o Fondo de In es igaciones
Sani a ias.
O iginal e sion ecei ed 25 July 1988 and accep ed e sion ecei ed 2 Decembe 1988.
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