Sec e o y esponses o in ac glomus cells in hin
slices o a ca o id body o hypoxia
and e ae hylammonium
Rica do Pa dal, Uwe Ludewig, Julia Ga ci
´a-Hi sch eld, and Jose
´Lo
´pez-Ba neo*
Depa amen o de Fisiologı´a, Facul ad de Medicina y Hospi al Uni e si a io Vi gen del Rocı´o, Uni e sidad de Se illa, E-41009, Se illa, Spain
Communica ed by Clay M. A ms ong, Uni e si y o Pennsyl ania School o Medicine, Philadelphia, PA, Decembe 1, 1999 ( ecei ed o e iew
Augus 12, 1999)
We ha e de eloped a hin-slice p epa a ion o whole a ca o id
body ha allows us o pe o m pa ch-clamp eco ding o mem-
b ane ionic cu en s and o moni o ca echolamine sec e ion by
ampe ome y in single glomus cells unde di ec isual con ol. In
no moxic condi ions (P
O
2⬇140 mmHg; 1 mmHg ⴝ133 Pa), mos
glomus cells did no ha e measu able sec e o y ac i i y, bu ex-
posu e o hypoxia (P
O
2⬇20 mmHg) elici ed he appea ance o a
la ge numbe o spike-like exocy o ic e en s. This neu osec e o y
esponse o hypoxia was ully e e sible and equi ed ex acellula
Ca
2ⴙ
in lux. The a e age cha ge o single quan al e en s was 46 ⴞ
25 C (nⴝ218), which yields an es ima e o ⬇140,000 ca echol-
amine molecules pe esicle. Addi ion o e ae hylammonium
(TEA; 2–5 mM) o he ex acellula solu ion induced in mos (>95%)
cells es ed (nⴝ32) a sec e o y esponse simila o ha elici ed by
low P
O
2. Cells non esponsi e o hypoxia bu ac i a ed by exposu e
o high ex e nal K
ⴙ
we e also s imula ed by TEA. A sec e o y
esponse simila o he esponses o hypoxia and TEA was also
obse ed a e ea men o he cells wi h ibe io oxin o block
selec i ely Ca
2ⴙ
- and ol age-ac i a ed maxi-K
ⴙ
channels. Ou da a
u he show ha memb ane ion channels a e c i ically in ol ed in
senso y ansduc ion in he ca o id body. We also show ha in
in ac glomus cells inhibi ion o ol age-dependen K
ⴙ
channels
can con ibu e o ini ia ion o he sec e o y esponse o low P
O
2.
ca o id-body slice 兩O2sensing 兩glomus cell sec e ion
Adecade has passed since pa ch-clamp expe imen s on
dispe sed glomus cells om abbi and a ca o id bodies
showed ha hey we e elec ically exci able and con ained
O
2
-sensi i e ol age-dependen K
⫹
channels (1–6). Al hough
some o he elec ophysiological p ope ies and he kind o
O
2
-sensi i e K
⫹
channel o glomus cells a ied among animal
species, hese obse a ions ga e s ong suppo o a uni ied
memb ane model o chemo ansduc ion based on he capabili y
o glomus cells o sense educ ions o O
2
ension (P
O
2) h ough
he closu e o K
⫹
channels, which in u n lead o Ca
2⫹
in lux,
ansmi e elease, and ac i a ion o he a e en ibe s o he
sinus ne e (see e s. 7 and 8 o e iew). Di ec e idence ha
indi idual glomus cells wo ked as O
2
-sensi i e neu osec e o y
elemen s was ob ained by moni o ing cy osolic [Ca
2⫹
] and
quan al ca echolamine sec e ion in single Fu a 2-loaded cells (7,
9–11). Nume ous in es iga o s ha e ound O
2
-sensi i e ol age-
dependen channels in o he issues (in addi ion o he ca o id
body), whe e hese channels pa icipa e in a a ie y o cellula
elec ical, mechanical, o sec e o y esponses o acu e hypoxia
(see e s. 12 and 13 o e iew).
The p ecise ole o ol age-dependen K
⫹
channels in he
chemo ecep i e p ope ies o he ca o id body emains, howe e ,
con o e sial, because he e a e esea che s who belie e ha he
O
2
-sensi i e memb ane elec ical e en s in glomus cells a e no
di ec ly in ol ed in senso y ansduc ion. A majo a gumen
suppo ing his no ion is ha pha macological agen s, such as
e ae hylammonium (TEA), 4-aminopy idine (4-AP), o
cha ybdo oxin (CTX), which can inhibi he O
2
-sensi i e K
⫹
cu en s in pa ch-clamped glomus cells, do no elici neu al
discha ges (o sec e ion) in whole ca o id-body p epa a ions
(14–16). Al hough i has been epo ed ha applica ion o CTX
can depola ize glomus cells (17), i has also been shown ha TEA
and 4-AP ail o depola ize o o inc ease cy osolic [Ca
2⫹
]in
dispe sed a glomus cells (18). This las wo k (18) desc ibed an
O
2
-sensi i e, K
⫹
-selec i e leak conduc ance, esis an o TEA
and 4-AP, which seemed o con ibu e o he depola iza ion o
he cells in esponse o low P
O
2. In consequence, i was p oposed
ha closu e o he leak K
⫹
channels is an obliga o y ini ial e en
in low P
O
2-induced exci a ion o a glomus cells.
Despi e he conside able amoun o in o ma ion on ca o id-
body unc ion al eady a ailable, he unde s anding o he O
2
-
sensing mechanisms is p og essing a a ela i ely slow pace.
Resea ch on he ca o id body is hampe ed by i s small size,
abundance o connec i e issue, and p o use ascula iza ion, all
o which make i di icul o ob ain a iable p epa a ion o
enzyma ically dispe sed cells wi h consis en physiological p op-
e ies. These limi a ions a e pa icula ly impo an when s udy-
ing he cellula esponses o hypoxia, because sensi i i y o O
2
is
a labile p ocess ha seems o be easily des oyed by uncon olled
expe imen al a iables (13). In sea ch o an op imal app oach o
in es iga e ca o id-body physiology, we ha e de eloped a hin-
slice p epa a ion o he whole o gan ha does no equi e
enzyma ic ea men o mechanical dis up ion o he issue.
He ein, we show ha he ca o id-body slice p epa a ion used in
combina ion wi h single-cell ampe ome y allows he s udy o
he elec ophysiology and sec e o y ac i i y o in ac cells in
almos op imal condi ions. Ou s udy, which uses his echnique,
u he suppo s he iew ha ol age-dependen memb ane ion
channels pa icipa e c i ically in chemosenso y ansduc ion.
Me hods
Ca o id-Body Slicing. Expe imen s we e pe o med on ca o id
bodies isola ed om Wis a a s o ages be ween pos na al day
5 and 20. Animals we e anes he ized by injec ion o 0.2 mg
兾
kg
sodium pen oba bi al (Eu a-Lende , Mad id). Comple e ca o id
bi u ca ions we e quickly emo ed and placed on ice-cooled and
O
2
-sa u a ed modi ied Ty ode’s solu ion (in mM: 148 NaCl
兾
2
KCl
兾
3 MgCl
2
兾
10 Hepes
兾
10 glucose, pH 7.4). The ca o id body
was dissec ed om he adjacen a e y and hen included in 3%
(w
兾
ol) low-mel ing-poin aga ose (FMC). A e quick cooling
on ice, he aga ose block was glued wi h cyanoac yla e o he
s age o a ib a ome chambe and co e ed by he same cold,
O
2
-sa u a ed Ty ode’s solu ion. Slices 100- o 150-
␮
m- hick we e
Abb e ia ions: TEA, e ae hylammonium; 4-AP, 4-aminopy idine; CTX, cha ybdo oxin;
IbTX, ibe io oxin; TH, y osine hyd oxylase.
*To whom ep in eques s should be add essed. E-mail: [email p o ec ed].
The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This
a icle mus he e o e be he eby ma ked “ad e isemen ” in acco dance wi h 18 U.S.C.
§1734 solely o indica e his ac .
A icle published online be o e p in : P oc. Na l. Acad. Sci. USA, 10.1073兾pnas.030522297.
A icle and publica ion da e a e a www.pnas.o g兾cgi兾doi兾10.1073兾pnas.030522297
PNAS
兩
Feb ua y 29, 2000
兩
ol. 97
兩
no. 5
兩
2361–2366
PHYSIOLOGY
cu wi h s anda d azo blades. The esul ing slices (usually ou )
we e washed wice wi h cold s e ile PBS and placed on 35-mm
Pe i dishes wi h DMEM (GIBCO
兾
BRL) supplemen ed wi h
1% ( ol/ ol) penicillin
兾
s ep omycin (GIBCO
兾
BRL) and 10%
( ol
兾
ol) FBS (BioWhi ake ). The slices we e main ained a
37°C in a 5% CO
2
incuba o 48–96 h be o e he expe imen s. A
his ime, ma u a ion o he O
2
-sensi i e mechanisms, which
occu s a ound pos na al day 10 (19, 20) we e almos comple e.
Sensi i i y o he glomus cells o changes in P
O
2did no seem o
depend on he age o he animals used.
Expe imen al Se up, Pa ch-Clamp Reco ding, and Ampe ome ic Mea-
su emen o Sec e ion. In mos aspec s, ca o id-body slices we e
managed ollowing he p ocedu es desc ibed o hin b ain slices
(21, 22). Fo he expe imen s, a slice was ans e ed o a
eco ding chambe (⬇200-
␮
l olume) placed on he s age o an
up igh Zeiss mic oscope (Axioskop) equipped wi h long dis-
ance wa e imme sion objec i es. Once in he chambe , he
ca o id-body slice was con inuously pe used by g a i y ( low 1–2
ml
兾
minu e) wi h a solu ion con aining (in mM) 147 NaCl, 2.7 o
4.5 KCl, 23 NaHCO
3
, 1 MgCl
2
, 2.5 CaCl
2
, and 10 glucose. A ly a
made o sil e wi e wi h glued ine Nylon h eads was used o
hold he slice o he bo om o he chambe . The s anda d
(no moxic) solu ion was bubbled wi h a gas mix u e o 5% CO
2
,
20% O
2
, and 75% N
2
(P
O
2⬇140 mmHg). Hypoxia was ob ained
by con inuously bubbling he solu ion in one o he ese oi s
wi h 5% CO
2
and 95% N
2
. The pH o he no moxic and hypoxic
solu ions was 7.4. The ime cou se o P
O
2changes in he chambe
was de e mined wi h a ca bon- ibe elec ode ha was nega i ely
pola ized and wo ked as an O
2
elec ode. A e swi ching om
no moxia o hypoxia, comple e equilib a ion o he new solu ion
in he chambe equi ed be ween 1 and 2 minu es. P
O
2in he
chambe du ing exposu e o hypoxia eached a alue o ⬇20
mmHg. All pha macological agen s used (ion channel blocke s
and ion chela o s) we e ob ained om Sigma and we e added o
he ba h solu ion. The osmolali y o he solu ion (⬇300 millios-
mol
兾
kg) was main ained by educing he concen a ion o NaCl.
In he high K
⫹
solu ions, KCl eplaced NaCl equimola ly. In all
he expe imen s, he empe a u e o he solu ions and he
chambe was be ween 34 and 37°C. Memb ane cu en s we e
eco ded by using he whole-cell con igu a ion o he pa ch-
clamp echnique as adap ed in ou labo a o y (23, 24). We used
low- esis ance pipe es (1–3 M⍀), capaci y compensa ion, and
sub ac ion o linea leakage and capaci i e cu en s. Se ies
esis ance compensa ion was be ween 40% and 50%. Pipe e
solu ion con ained (in mM) 125 KCl, 4 MgCl
2
,4MgATP,10
Hepes, and 10 EGTA (pH 7.2 and osmolali y o 285–290
milliosmol
兾
kg). Ionic cu en s we e eco ded wi h an EPC-8
pa ch-clamp ampli ie (HEKA Elec onics, Lamb ech
兾
P alz,
Ge many), il e ed a 3 kHz, digi ized, and s o ed on a compu e .
Sec e o y e en s we e eco ded wi h a 10-
␮
m ca bon- ibe
elec ode connec ed o he inpu o a high-gain cu en o ol age
con e e (10, 25). The elec ode was pola ized o ⫹750 mV, a
alue mo e posi i e han he oxida ion po en ial o dopamine
and he o he ca echolamines in glomus cells (10), and posi-
ioned nea a cell unde isual con ol by using a piezoelec ic
manipula o . Ampe ome ic cu en s we e also eco ded wi h
he EPC-8 pa ch-clamp ampli ie . Cu en s we e il e ed a 100
Hz and digi ized a 250 Hz be o e s o age on compu e . Da a
acquisi ion and analysis o ionic and ampe ome ic cu en s we e
done wi h an ITC-16 in e ace (Ins u ech, G ea Neck, NY) and
PULSE兾PULSEFIT so wa e ( e sion 8.11, HEKA Elec onics).
Ty osine Hyd oxylase (TH) Immunocy ochemis y. Ca o id-body
slices simila o hose used o he expe imen s desc ibed abo e
we e ixed o e nigh a 4°C wi h PBS
兾
4% ( ol/ ol) pa a o mal-
dehyde. A e washing in PBS
兾
0.1% T i on X-100 (PBTx),
sec ions we e incuba ed wi h PBS
兾
0.3% H
2
O
2
o 2 h and hen
Fig. 1. Mo phological appea ance o cells in hin slices o a ca o id body. (A) Low-magni ica ion iew o a slice a ew hou s a e cu ing. (B) Typical cell
agg ega e (glome ulus) in a ca o id-body slice main ained o 48 h in a CO2incuba o . Indi idual cells, like he one indica ed by he a ow, can be clea ly
di e en ia ed. (C) Ca o id-body slice s ained wi h an ibodies an i- y osine hyd oxylase. No e he ypical appea ance o glomus cells wi h la ge nuclei and he
o ganiza ion o he ca o id body in glome uli.
2362
兩
www.pnas.o g Pa dal e al.
wi h PBTx
兾
10% ( ol/ ol) FCS
兾
1 mg/ml BSA o ano he 2h o
block nonspeci ic si es. A e incuba ion, a polyclonal an i-TH
an ibody (1:1,000; Chemicon) was added o e nigh . A e wash-
ing in PBTx, sec ions we e incuba ed again wi h bio inyla ed
an i- abbi an ibody (1:200; Pie ce) o 12 h. The ea e , sec ions
we e incuba ed wi h he ABC ki (Pie ce) o 2 h, and speci ically
bound an ibody was e ealed by using 3,3⬘-diaminobenzidine
(Sigma) as ch omogen. A e washing in PBS, sec ions we e
moun ed on slides and co e slipped.
Resul s
Fig. 1Aillus a es he ypical appea ance o a a ca o id-body
slice o ound shape (⬇400
␮
m in diame e ) su ounded by he
connec i e capsule and a ached issue. Du ing he ew hou s
a e slicing, he his ological appea ance o he sec ions was
a he uni o m, wi hou clea sepa a ion be ween he glandula ,
connec i e, and ascula issues. As incuba ion ime p og essed,
he slice la ened, and he ex u e became less uni o m, wi h he
appea ance o cell agg ega es clea ly sepa a ed om he su -
ounding issue and wi h nume ous sphe ical o o oid cells 8–12
␮
m in diame e (Fig. 1B). These cell agg ega es, simila o he
glome uli desc ibed in his ological p epa a ions o he ca o id
body (26), we e nicely obse ed in slices immunos ained wi h
an i-TH an ibodies showing ypical clus e s o TH-posi i e
glomus cells (Fig. 1C). We did mos o ou expe imen s on slices
incuba ed o a leas 48 h, because hese slices had be e
de ined glome uli ha seemed o con ain a la ge numbe o
cells esponding o changes in P
O
2(see below). Fo he mea-
su emen o whole-cell cu en s o sec e o y ac i i y, he pa ch-
clamp and ca bon- ibe elec odes, espec i ely, we e placed
adjacen o a well iden i ied cell wi hin a clus e (e.g., he cell
nea he a ow in Fig. 1B). We we e able o main ain ca o id-
body slices in good condi ion o up o 5 days. The longe i y o
he slices was no s udied sys ema ically, al hough i seems ha ,
wi h special ca e, long-las ing o gano ypic cul u es migh easily
be achie ed.
A e se ing up he slice p epa a ion, ou main goal was o
s udy he sec e o y esponse o hypoxia o in ac cells in he slice
(see below); howe e , we also es ed whe he cells in he
glome uli could be pa ch-clamped and he e o e we e amenable
o elec ophysiological expe imen s. S able whole-cell eco d-
ings we e easily ob ained om glomus cells ha had inwa d and
ou wa d ol age-dependen cu en s quali a i ely simila o he
cu en s seen in enzyma ically dispe sed ca o id-body cells (Fig.
2A Middle and Bo om). Ne peak inwa d cu en a ⫹20 mV was,
in mos cells, be ween 50 and 200 pA; howe e , in some cases,
Fig. 2. Elec ophysiological and ampe ome ic eco dings om indi idual glomus cells in he slice. (A Top) Mac oscopic cu en s eco ded om a glomus cell
by using he whole-cell con igu a ion o he pa ch-clamp echnique. (A Middle) Reco dings o inwa d and ou wa d cu en s om a cell ha had pa icula ly la ge
as inwa d cu en . Reco dings elici ed by depola iza ions o ⫹20 and ⫹40 mV om he holding po en ial o ⫺80 mV a e supe imposed. (A Bo om)
Supe imposed eco dings om a di e en glomus cell elici ed by depola izing pulses om ⫺80 mV o ⫹20 mV illus a ing he e e sible educ ion o he cu en
by TEA. c and a e he con ol and eco e y aces, espec i ely. (B Top) Measu emen o sec e o y ac i i y om glomus cells by ampe ome y. A la ge spike-like
exocy o ic e en is shown a an expanded ime base. (B Middle) Sec e o y esponse o a glomus cell o hypoxia (PO2⬇20 mmHg) and o high ex acellula
po assium. (B Bo om) Example o a cell ha was insensi i e o changes o PO2(non esponsi e cell) bu ha main ained he ypical sec e o y esponse o high
ex acellula po assium.
Pa dal e al. PNAS
兩
Feb ua y 29, 2000
兩
ol. 97
兩
no. 5
兩
2363
PHYSIOLOGY
as in he example shown in Fig. 2A Middle, we eco ded
unusually la ge inwa d cu en s. De ailed elec ophysiological
cha ac e iza ion o glomus cells in he slices was no a emp ed
in his wo k, bu he la ge inwa d cu en obse ed in his s udy
is simila o he Na
⫹
cu en desc ibed in a small popula ion o
dispe sed cells (27). In ou expe imen al condi ions, inpu
esis ance was be ween 1 and 3 G⍀, wi hin he ange o hose
epo ed in isola ed a and abbi glomus cells (18, 24). This
inding sugges s ha , unless whole-cell eco ding induced he
pa ch-clamped cell o uncouple, he deg ee o elec ical coupling
be ween neighbo ing glomus cells is no high. As shown be o e
in dispe sed cells (4, 27), he mac oscopic K
⫹
cu en was
e e sibly educed by exposu e o ela i ely low concen a ions
o TEA (Fig. 2A Bo om).
The sec e o y ac i i y o indi idual cells was s udied by
measu ing he cu en esul ing om he oxida ion o eleased
ca echolamine molecules (Fig. 2B Top). Single exocy o ic e en s
appea ed as spike-like signals ep esen ing he usion o indi-
idual ca echolamine gic esicles. An example o a la ge sec e-
o y spike is shown in Fig. 2B Top Inse . Unde no moxic
condi ions, mos cells did no show any measu able sec e o y
ac i i y o had some occasional exocy o ic e en s a a equency
o less han one pe minu e. Some cells we e, howe e , mo e
ac i e wi h a spon aneous sec e o y e en equency o 10 pe
minu e o highe . A e swi ching o he hypoxic solu ion, cells
esponded wi h a p og essi e inc ease in he equency and
ampli ude o he spikes ha pa ially used in o a b oad con-
cen a ion en elope (Fig. 2B Middle). Rep oducible esponses o
hypoxia we e obse ed h ee o ou imes in he cells i se e al
minu es o es we e allowed o eco e y be ween successi e
s imuli. Howe e , we ha e no s udied in de ail o he ac o s
egula ing he deple ion o eplenishmen o he eleasable
esicle pool in ou p epa a ion. Spike equency a he peak o
he esponse o hypoxia (measu ed in he minu e a e he ini ial
90 s o exposu e o hypoxia) was 51.8 ⫾22 spikes pe minu e (n⫽
6; mean ⫾SD). Gi en he p oximi y o he ampe ome ic
elec odes o he cell unde s udy, we hink ha mos o he
spikes eco ded we e due o exocy o ic e en s om he same cell;
howe e , we canno exclude he possibili y ha some small spikes
appea ing in he eco dings we e due o sec e o y e en s occu -
ing in neighbo ing cells. The p og essi e inc ease in spike
ampli ude du ing exposu e o hypoxia was a phenomenon ob-
se ed epe i i ely in di e en expe imen s, which p obably
esul ed om he in acy oplasmic usion o esicles occu ing
be o e elease (compound exocy osis; e . 11). A e swi ching
back o he no moxic solu ion, eco e y was as , and cells
no mally e u ned o con ol condi ions in less han 30 s.
As expec ed om elec ically exci able cells ha ing ol age-
dependen Ca
2⫹
channels, all glomus cells made a igo ous
sec e o y esponse when exposed o high ex acellula K
⫹
(see
Fig. 2B Middle). A e age quan al cha ge o sec e o y e en s
elici ed du ing exposu e o hypoxia was 46 ⫾25 C (mean ⫾SD;
n⫽218 e en s in six cells; see also Fig. 4A). This alue was
ob ained om he ime in eg al o selec ed spikes wi h he as
ising phase and slowe decay (Fig. 2B Top Inse ) ypical o
Fig. 3. The sec e o y esponse o hypoxia o glomus cells depends on
ex acellula Ca2⫹in lux. (Uppe ) Re e sible abolishmen o he esponse o
hypoxia du ing he blockade o Ca2⫹channels by addi ion o 0.2 mM CdCl2 o
he ex acellula solu ion. (Lowe ) Re e sible abolishmen o he esponse o
hypoxia by he emo al and chela ion o Ca2⫹ om he ex acellula solu ion.
Fig. 4. Sec e o y esponse o in ac glomus cells o TEA. (A Uppe ) In a cell
ha esponded o hypoxia, applica ion o TEA o he ba h elici ed a simila
e e sible sec e o y esponse. (A Lowe ) F equency dis ibu ion o he cha ge
o sec e o y e en s elici ed by hypoxia and 5 mM TEA in six di e en cells. No e
ha he pa ame e s o he dis ibu ion (means ⫾SD) a e he same in he wo
expe imen al condi ions. (B) Po en ia ion o he e ec o hypoxia by TEA. (C)
Sec e o y esponse o TEA in a cell ha was no esponsi e o hypoxia.
2364
兩
www.pnas.o g Pa dal e al.
sec e o y e en s occu ing in he memb ane acing he ampe o-
me ic elec ode (10, 25). Assuming ha wo elec onic cha ges
a e ans e ed in he oxida ion o a ca echolamine molecule, he
a e age numbe o molecules pe esicle is ⬇140,000 ⫾75,000
(mean ⫾SD), a alue simila o ou es ima e in dispe sed abbi
glomus cells (10). Besides he cells wi h cha ac e is ic sec e o y
esponse o hypoxia desc ibed abo e, we also obse ed non e-
sponsi e cells. Some o hese eco dings could ha e been om
ype II cells, which a e nonexci able (24) and appea in e min-
gled wi hin he glome ulus wi h glomus ( ype I) cells. Howe e ,
mos o he non esponsi e cells had he ypical appea ance o
glomus cells and showed a powe ul sec e o y esponse o high
ex acellula K
⫹
(Fig. 2B Bo om). In he O
2
-sensi i e glomus
cells, he neu osec e o y esponse o hypoxia was almos com-
ple ely and e e sibly abolished by he addi ion o Cd
2⫹
o he
ex acellula solu ion (Fig. 3 Uppe ) o a e emo al o ex a-
cellula Ca
2⫹
(Fig. 3 Lowe ). These obse a ions a e simila o
hose epo ed in abbi glomus cells (10, 11) and u he show
ha ansmi e elease elici ed by exposu e o physiological
le els o hypoxia (⬇20 mmHg o highe ) depends on he in lux
o ex acellula Ca
2⫹
.
In neona al and adul cells, he majo con ibu o s o he
O
2
-sensi i e mac oscopic K
⫹
cu en a e ol age- and Ca
2⫹
-
dependen K
⫹
channels, which a e blocked by ex e nal TEA (4,
17, 20, 27). Fo his eason, we es ed o see whe he applica ion
o TEA elici ed a sec e o y esponse simila o ha igge ed by
hypoxia. In mos cells s udied (31 o 32), TEA a concen a ions
o 2 o 5 mM, bo h o which p oduce blockade o a la ge
p opo ion o K
⫹
channels ( e s. 4, 17, and 27; Fig. 2A), induced
a clea sec e o y esponse (Fig. 4A Uppe ). This e ec o TEA
was obse ed e en in quiescen cells (wi hou measu able spon-
aneous quan al elease) and ega dless o whe he ex acellula
K
⫹
was 2.7 o 4.5 mM. Spike equency a he peak o he
esponse o 5 mM TEA (measu ed in he minu e a e he ini ial
90 s o exposu e o he blocke ) was 42 ⫾17 spikes pe minu e
(mean ⫾SD; n⫽6), and a e age quan al cha ge was 43 ⫾30
C (mean ⫾SD; n⫽275 e en s in six cells). These alues, as well
as he dis ibu ion o quan al e en s (Fig. 4A Lowe ), a e simila
o hose es ima ed wi h e en s elici ed by hypoxia, sugges ing
ha bo h TEA and exposu e o low P
O
2can igge he elease
o he same esicle pool. As shown in Fig. 4B, TEA also
po en ia ed he sec e o y esponse induced by hypoxia. We ha e
no es ed whe he his addi i e e ec is s ill p esen when high
(sa u a ing) concen a ions o TEA a e used; howe e , we ha e
eco ded one cell ha esponded o hypoxia bu was insensi i e
o TEA alone. As men ioned be o e, TEA could elici ca echol-
amine elease e en om cells ha we e no esponsi e o
changes in P
O
2(Fig. 4C). Thus, hese da a indica e ha di ec
blockade o he O
2
-sensi i e K
⫹
channels wi h TEA can elici a
igo ous sec e o y esponse om in ac glomus cells. Fu he
suppo o his no ion came om expe imen s wi h ibe io oxin
(IbTX), a selec i e blocke o he Ca
2⫹
- and ol age-ac i a ed
maxi K
⫹
channels ha can induce ca echolamine sec e ion in
clus e s o ca o id-body cells (28, 29). Fig. 5 illus a es ha IbTX
elici ed a sec e o y esponse om indi idual glomus cells simila
o ha e oked by hypoxia o TEA; washou o IbTX was,
howe e , slowe han eco e y om he o he s imuli.
Discussion
In his epo , we desc ibe he ca o id-body slice p epa a ion
and show ha his echnique allows us o s udy he elec o-
physiology and cellula esponses o single glomus cells o
hypoxia wi hou he need o enzyma ic ea men o mechan-
ical dis up ion o he issue. We ha e consis en ly eco ded
sec e o y ac i i y elici ed by hypoxia in single a glomus cells
by measu ing ca echolamine elease wi h a ca bon- ibe am-
pe ome ic elec ode. The neu osec e o y esponse o hypoxia
in single a glomus cells is quali a i ely simila o he esponse
ha we desc ibed be o e in dispe sed abbi glomus cells (10,
11). In bo h cases, ca echolamine elease e oked by physio-
logic low P
O
2le els (⬇20 mmHg o highe ) depends absolu ely
on ex acellula Ca
2⫹
in lux. Thus, ou da a gi e u he
suppo o he iew ha glomus cells beha e as O
2
-sensi i e
p esynap ic-like elemen s, in which Ca
2⫹
in lux igge ed by
hypoxia-induced depola iza ion is he undamen al e en lead-
ing o ansmi e elease and ac i a ion o he a e en senso y
ibe s wi h which he glomus cells o m synapses (7–11).
In e es ingly, we obse ed ha some cells ha had an appea -
ance ypical o glomus cells and ha esponded wi h exocy o ic
ca echolamine sec e ion o high ex acellula K
⫹
we e insensi i e
o changes in P
O
2. The insensi i i y o hese cells o hypoxia was
no ela ed o animal age and could be explained ei he by he
need o lowe P
O
2le els o become depola ized o by he ac
ha , in his subse o glomus cells, he O
2
-sensi i e machine y is
inac i a ed o uncoupled om he K
⫹
channels. We belie e ha
he las possibili y is mo e likely o wo easons. Fi s , like he
hypoxia- esponsi e cells, he non esponsi e ones we e s imu-
la ed by TEA. Second and mo e impo an ly, when we obse ed
a la ge numbe o non esponsi e cells in a pa icula expe imen ,
he lack o O
2
-sensi i i y ended o be main ained in all he slices
o he p epa a ion. The e o e, i seems ha insensi i i y o
hypoxia depends on uncon olled a iables, some o hem in-
oduced by he expe imen al p o ocol, which may modi y he
chemical s a us o he O
2
-sensing molecule o al e i s associa-
ion wi h he ion channels. In ac , we ealized long ago ha he
O
2
-sensi i i y o ion channels is a labile phenomenon ha can be
easily al e ed by cell dissocia ion p ocedu es (13).
Ano he impo an obse a ion in his s udy is ha ex e nal
applica ion o TEA and IbTX consis en ly elici ed a sec e o y
esponse ha in mos aspec s was simila o he esponse e oked
by hypoxia. This simila i y con as s wi h he lack o TEA e ec
on dispe sed a glomus cells (18) and indica es ha di ec
blockade o O
2
-sensi i e ol age-dependen K
⫹
channels, which
in hese cells a e hose blocked by TEA o IbTX (4, 17, 27, 28),
can lead o sec e ion. A co olla y o hese da a is ha inhibi ion
Fig. 5. Sec e o y esponse o in ac glomus cells o IbTX. In a cell ha
esponded o hypoxia (Uppe ), applica ion o IbTX (200 nM) o he ba h
elici ed a simila e e sible sec e o y esponse (Lowe ). No e ha eco e y
om IbTX is ela i ely slow p obably because o he high a ini y o oxin
binding o he channel.
Pa dal e al. PNAS
兩
Feb ua y 29, 2000
兩
ol. 97
兩
no. 5
兩
2365
PHYSIOLOGY

o ol age-dependen K
⫹
channels by hypoxia can con ibu e o
ini ia ion o he sec e o y esponse elici ed by low P
O
2.A
TEA- esis an leak K
⫹
cu en inhibi ed p e e en ially by anoxia
o ex eme low P
O
2le els has been desc ibed in dispe sed a
glomus cells (18). Whe he he leak and he TEA-sensi i e
channels a e p esen in he same a glomus cell ype and
whe he hei unc ions o e lap he same ange o P
O
2 alues a e
ques ions ha should be add essed in u u e expe imen al wo k.
Based on ou p esen da a, i is di icul o unde s and he
insensi i i y o TEA (o o CTX and 4-AP) o whole ca o id-
body p epa a ions epo ed by some au ho s (14–16). Al hough
he molecula iden i y and pha macology o he K
⫹
channels in
he glomus cell-a e en ibe synapse a e unknown (see, how-
e e , e s. 4, 5, 17, 27, and 30), i is logical o pos ula e ha ,
ega dless o he mechanisms in ol ed in he ansduc ion o he
hypoxic s imulus, he e mus be, a he synapse i sel and along
he a e en ne e ibe , se e al ypes o ol age-dependen K
⫹
channels whose blockade should al e ansmi e elease as well
as he equency and shape o he p opaga ed ac ion po en ials.
Because TEA as well as CTX and 4-AP a e ela i ely la ge,
poo ly lipid-soluble molecules, a possible explana ion could be
ha he blocke s do no di use a he app op ia e concen a ion
in o he ex acellula space o ca o id bodies ei he supe used
(wi h he su ounding connec i e capsule in ac ) o pe used
h ough he ca o id a e y.
We wish o hank D . Juanjo Toledo-A al o help in he TH
immunos aining o ca o id-body slices. R.P. is a p edoc o al ellow o
he Spanish Fo macio´n del Pe sonal In es iga o p og am. U.L. was
ecipien o a pos doc o al ellowship o he Eu opean Molecula
Biology O ganiza ion. Resea ch was suppo ed by he Spanish Di ec-
cio´n Gene al de Ensen˜auza Supe io , Fundacio´n La Caixa, and he
Andalusian go e nmen .
1. Duchen, M. R., Caddy, K. W. T., Ki by, G. C., Pa e son, D. L., Pon e, J. &
Biscoe, T. J. (1988) Neu oscience 26, 291–311.
2. Lo´pez-Ba neo, J., Lo´pez-Lo´pez, J. R., U en˜a, J. & Gonza´lez, C. (1988) Science
242, 580–582.
3. Delpiano, M. A. & Heschele , J. (1989) FEBS Le . 249, 195–198.
4. Pee s, C. (1990) Neu osci. Le . 119, 253–256.
5. Gan o nina, M. D. & Lo´pez-Ba neo, J. (1991) P oc. Na l. Acad. Sci. USA 88,
2927–2930.
6. S ea, A. & Nu se, C. A. (1991) P lu¨ge s A ch. 418, 93–101.
7. Lo´pez-Ba neo, J., Beno , A. R. & U en˜a, J. (1993) News Physiol. Sci. 8,
191–195.
8. Gonza´lez, C., Alma az, L. & Obeso, A. (1994) Physiol. Re . 74, 829–898.
9. Buckle , K. J. & Vaughan-Jones, R. D. (1994) J. Physiol. (London) 476,
423–428.
10. U en˜a, J., Fe na´ndez-Chaco´n, R., Beno , A. R., Al a ez de Toledo, G. &
Lo´pez-Ba neo, J. (1994) P oc. Na l. Acad. Sci. USA 91, 10208–10211.
11. Mon o o, R. J., U en˜a, J., Fe na´ndez-Chaco´n, R., Al a ez de Toledo, G. &
Lo´pez-Ba neo, J. (1996) J. Gen. Physiol. 107, 133–143.
12. Lo´pez-Ba neo, J. (1996) T ends Neu osci. 19, 435–440.
13. Lo´pez-Ba neo, J., Mon o o, R. J., O ega-Sa´enz, P. & U en˜a, J. (1998) in
Oxygen Regula ion o Ion Channels and Gene Exp ession, eds. Lo´pez-Ba neo, J.
& Wei , K. S. (Fu u a, New Yo k), pp. 127–144.
14. Doyle, T. P. & Donnelly, D. F. (1994) J. Appl. Physiol. 77, 2606–2611.
15. Osanai, S., Bue k, D. G., Mokashi, A., Chugh, D. K. & Lahi i, S. (1997) B ain
Res. 747, 324–327.
16. Lahi i, S., Roy, A., Rozano , C. & Mokashi, A. (1998) B ain Res. 794, 162–165.
17. Wya , C. N. & Pee s, C. (1995) J. Physiol. (London) 483, 559–565.
18. Buckle , K. J. (1997) J. Physiol. (London) 498, 649–662.
19. Donnelly, D. F. & Doyle, T. P. (1994) J. Physiol. (London) 475, 267–275.
20. Ha on, C. J., Ca pen e , E., Peppe , D. R., Kuma , P. & Pee s, C. (1997)
J. Physiol. (London) 501, 49–58.
21. Edwa ds, F. A., Konne h, A., Sakmann, B. & Takahashi, T. (1989) P lu¨ge s
A ch. 414, 600–612.
22. Schneggenbu ge , R., Lo´pez-Ba neo, J. & Konne h, A. (1992) J. Physiol.
(London) 445, 261–276.
23. Hamill, O. P., Ma y, A., Nehe , E., Sakmann, B. & Sigwo h, F. (1981) P lu¨ge s
A ch. 391, 85–100.
24. U en˜a, J., Lo´pez-Lo´pez, J. R., Gonza´lez, C. & Lo´pez-Ba neo, J. (1989) J. Gen.
Physiol. 93, 979–999.
25. Chow, R. H. & on Ruden, L. (1995) in Single Channel Reco ding, eds.
Sakmann, B. & Nehe , E. (Plenum, New Yo k), pp. 245–275.
26. Fidone, S. J. & Gonza´lez, C. (1986) in The Respi a o y Sys em II: Handbook o
Physiology, ed. Fishman, A. P. (Am. Physiol. Soc., Be hesda), pp. 557–621.
27. Lo´pez-Lo´pez, J. R., Gonza´lez, C. & Pe´ ez-Ga cı´a, M. T. (1997) J. Physiol.
(London) 499, 429–441.
28. Gal ez, A., Gimenez-Gallego, G., Reuben, J. P., Roy-Con ancin, L., Feigen-
baum, P., Kaczo o ski, G. J. & Ga cia, M. L. (1990) J. Biol. Chem. 264,
11083–11090.
29. Jackson, A. & Nu se, C. (1997) J. Neu ochem. 69, 645–654.
30. Gan o nina, M. D. & Lo´pez-Ba neo, J. (1992) J. Gen. Physiol. 100, 401–426.
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www.pnas.o g Pa dal e al.