P oc.
Na l.
Acad.
Sci.
USA
Vol.
92,
pp.
4715-4719,
May
1995
Physiology
Oxygen-sensi i e
calcium
channels
in
ascula
smoo h
muscle
and
hei
possible
ole
in
hypoxic
a e ial
elaxa ion
(02
sensing/hypoxia/cy osolic
Ca2+
oscilla ions)
A.
FRANCO-OBREGON,
J.
URENA,
AND
J.
LOPEZ-BARNEO*
Depa amen o
de
Fisiologia
Medica
y
Bio isica,
Uni e sidad
de
Se illa,
Facul ad
de
Medicina,
A enida
Sanchez
Pizjuan,
4,
E-41009,
Se ille,
Spain
Communica ed
by
Clay
M.
A ms ong,
Uni e si y
o
Pennsyl ania
Medical
Cen e ,
Philadelphia,
PA,
Feb ua y
15,
1995
( ecei ed
o
e iew
Decembe
10,
1994)
ABSTRACT
We
ha e
in es iga ed
he
modi ica ions
o
cy osolic
[Ca2
+]
and
he
ac i i y
o
Ca2+
channels
in
eshly
dispe sed
a e ial
myocy es
o
es
whe he
lowe ing
02
ension
(P02)
di ec ly
in luences
Ca2+
homeos asis
in
hese
cells.
Unclamped
cells
loaded
wi h
u a-2
AM
exhibi
oscilla-
ions
o
cy osolic
Ca22+
whose
equency
depends
on
ex acel-
lula
Ca2+
in lux.
Swi ching
om
a
P02
o
150
o
20
mmHg
leads
o
a
e e sible
a enua ion
o
he
Ca2'
oscilla ions.
In
ol age-clamped
cells,
hypoxia
e e sibly
educes
he
in lux
o
Ca2+
h ough
ol age-dependen
channels,
which
can
accoun
o
he
inhibi ion
o
he
Ca2+
oscilla ions.
Low
P02
selec i ely
inhibi s
L- ype
Ca2'
channel
ac i i y,
whe eas
he
cu en
media ed
by
T- ype
channels
is
unal e ed
by
hypoxia.
The
e ec
o
low
P02
on
he
L- ype
channels
is
ma kedly
ol age
dependen ,
being
mo e
appa en
wi h
mode a e
depola iza-
ions.
These
indings
demons a e
he
exis ence
o
02-
sensi i e,
ol age-dependen ,
Ca2'
channels
in
ascula
smoo h
muscle
ha
may
c i ically
con ibu e
o
he
local
egula ion
o
ci cula ion.
Oxygen
ension
(P02)
has
been
ecognized
o
decades
as
an
impo an
ac o
in
he
local
egula ion
o
ascula
one
in
i o
(1),
and
i
is
known
ha
hypoxia
causes
elaxa ion
o
sys emic
a e ies
in
i o
(2-4).
Ne e heless,
he
mechanisms
unde ly-
ing
his
physiological
esponse
o
low
P02
emain
la gely
unknown. Based
on
s udies
done
in
o gan
ba h
p epa a ions,
i
is
belie ed
ha
oxygen
in luences
ascula
esis ance
by
di ec ly
in e e ing
wi h
he
ise
o
cy osolic
Ca2+
equi ed
o
con ac ion
o
smoo h
muscle
cells
(4-7);
ne e heless,
e y
li le
is
unde s ood
abou
oxygen- egula ed
p ocesses
in
a e-
ial
myocy es.
I
has
been
sugges ed
ha
dila a ion
o
he
co ona y
a e y
(8,
9),
and
pe haps
o he
a e ies,
in
esponse
o
ex eme
hypoxia
migh
be
media ed
by
myocy e
hype po-
la iza ion
in
esponse
o
he
opening
o
ATP- egula ed
K+
channels.
Howe e ,
i
seems
unlikely
ha
his
is
he
only
unde lying
mechanism
because
he
sensi i i y
o
hypoxia
in
mos
a e ies
occu s
o e
a
physiologic
ange
o
P02
wi hou
a
comp omise
o
ene gy
me abolism
(3).
Since
in
he
pas
ew
yea s
02- egula ed
ion
channels
ha e
been
demons a ed
o
pa icipa e
in
a
numbe
o
cellula
unc ions
(10),
we
hypo h-
esized
ha
ascula
one
could
be
also
egula ed
by
di ec
modula ion
o
ol age-ga ed
Ca2+
channels
by
P02.
He e,
we
show
he
e e sible
inhibi ion
o
he
mac oscopic
Ca2+
cu en
o
a e ial
myocy es
in
esponse
o
hypoxia,
which
can
explain
he
supp ession
o
he
cy osolic
[Ca2+]
oscilla ions
in
he
same
cells
by
low
P02.
These
da a
demons a e
he
exis ence
o
02- egula ed
Ca2+
channels
in
ascula
smoo h
muscle
and
sugges
ha
hey
migh
pa icipa e
in
hypoxic
a e ial
elax-
a ion.
METHODS
Expe imen s
we e
pe o med
on
enzyma ically
dispe sed
smoo h
muscle
cells
om
he
celiac
and
emo al
a e ies
o
adul
abbi s.
In
b ie ,
a e ies
we e
emo ed,
placed
in
cold
(4°C)
Hanks'
balanced
sal
solu ion,
and
opened
longi udi-
nally.
Thei
ou e
and
inne
su aces
we e
cleaned
o
he
ad en i ia
and
endo helium,
espec i ely.
Pieces
o
a e y
(-
1
mm2)
we e
placed
in o
5
ml
o
Hanks'
solu ion
o
which
7
mg
o
papain,
5
mg
o
collagenase
(Sigma,
ype
IA),
and
3.5
mg
o
bo ine
se um
albumin
(Sigma,
ac ion
V)
had
been
added.
The
issue
was
hen
s o ed
in
his
enzyme
solu ion
o
1-6
h
a
6°C.
In
p epa a ion
o
he
dissocia ion
o
smoo h
muscle
cells,
he
issue
and
enzyme
solu ion
we e
placed
a
37°C
o
15-25
min
wi h
low
s i ing.
Upon
de ec ing
he
i s
ee
cells,
he
issue
was
hen
ans e ed
o
esh
Hanks'
solu ion
con aining
bo ine
se um
albumin
(10
mg/50
ml)
and
mechan-
ically
dissocia ed
h ough
he
i e-polished
ip
o
a
glass
pipe e.
Cells
we e
pla ed
on
pieces
o
poly(L-lysine)-coa ed
glass
co e slips.
Fo
expe imen s,
a
co e slip
was
placed
in
a
eco ding
chambe
o
-0.2
ml
wi h
con inuous
low
o
solu-
ion.
Ex e nal
solu ions
we e
equilib a ed
wi h
ei he
ai
(Po2
150
mmHg)
o
mix u es
o
N2
and
ai
o
ob ain
he
desi ed
02
concen a ions.
P02
in
he
chambe
was
moni o ed
wi h
an
02-sensing
elec ode
(11).
Cy osolic
[Ca2+]
was
es ima ed
in
unclamped
myocy es
loaded
wi h
u a-2
ace oxyme hyl
es e .
Expe imen s
we e
pe o med
on
an
in e ed
mic oscope
wi h
s anda d
op ical
componen s
and
equipped
o
epi luo es-
cence
and
dual-wa eleng h
pho ome y
(12,
13).
Calib a ion
o
he
luo escence
signals
in
e ms
o
[Ca2+]
was
pe o med
in
i o
as
desc ibed
(14).
Mac oscopic
calcium
cu en s
we e
eco ded
in
isola ion
using
he
whole-cell
con igu a ion
o
he
pa ch-clamp
echnique
(15,
16)
a e
blockade
o
he
ol age-
dependen
K+
channels.
Al hough
Na+
channels
a e
p ac i-
cally
absen
in
a e ial
myocy es,
some
expe imen s
we e
also
pe o med
wi h
e odo oxin
(0.2
A M)
added
o
he
ex e nal
solu ion.
The
holding
po en ial
was
ei he
-80
o
-70
mV.
Ba2+
was
used
as
cha ge
ca ie
ins ead
o
Ca2+
o
a o
he
low
o
cu en
h ough
he
Ca2+
channels,
and
ATP
was
added
o
he
in e nal
solu ion
o
p e en
he
wash-ou
o
he
channels.
The
composi ion
o
he
eco ding
solu ions
is
gi en
in
he
igu e
legends.
Capaci y
cu en
ansien s
we e
well
i ed
by
single
exponen ial
unc ions
wi h
a e age
ime
con-
s an s
o
120
±
45
,ls
(n
=
24)
and
113
±
30
As
(n
=
10)
(mean
±
SD)
o
celiac
and
emo al
myocy es,
espec i ely.
This
indica es
ha
despi e
he
la ge
size
o
he
cells
(-100
, m
in
leng h
and
6-8
, m
in
diame e ),
we
had
a
easonably
as
ol age-clamp
and,
hus,
we
could
di ec ly
moni o
ail
cu -
en s.
Vol age-clamp
speed
was
a o ed
by
ballis ic
cha ge
o
memb ane
capaci ance
and
he
use
o
low- esis ance
elec-
odes
(be ween
1
and
3
MQI)
(16).
Due
o
he
ela i ely
small
size
o
he
cu en s,
se ies
esis ance
was
no
sys ema ically
compensa ed.
Analog
cu en
signals
we e
low-pass
il e ed
*To
whom
ep in
eques s
should
be
add essed.
4715
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 .
P oc.
Na l.
Acad
ScL
USA
92
(1995)
(cu o
equency
be ween
3
and
10
kHz),
digi ized
a
a
sample
in e al
o
20
o
50
, s,
and
s o ed
on
compu e
o
analysis.
The
expe imen s
we e
conduc ed
a
oom
empe a u e
(22-
25°C).
RESULTS
The
e ec
o
hypoxia
on
single
a e ial
smoo h
muscle
cells
was
i s
s udied
by
moni o ing
he
modi ica ions
o
cy osolic
[Ca2+]
in
esponse
o
changes
in
P02
wi h
double-wa eleng h
mic o luo ime y.
Fu a-2-loaded
myocy es
s imula ed
wi h
a
pu ine gic
agonis
(ATP)
gene a ed
hy hmical
oscilla ions
o
cy osolic
[Ca2+].
In
all
myocy es
exhibi ing
egula
Ca2+
oscilla ions
ha
we e
hen
subjec ed
o
he
comple e
expe i-
men al
p o ocol
(n
=
8),
exposu e
o
hypoxia
(swi ching
om
a
ba h
solu ion
equilib a ed
wi h
a
P02
o
150
o
one
o
20
mmHg)
elici ed
a
ma ked
e e sible
educ ion
in
he
ampli-
ude
and
equency
o
he
Ca2+
spikes.
Hypoxic
ea men
e en ually
esul ed
in
he
comple e
supp ession
o
he
Ca2+
oscilla ions.
A
ep esen a i e
example
o
his
cellula
esponse
o
low
Po2
is
shown
in
Fig.
1A,
which
also
includes
he
signal
om
an
02-sensing
elec ode
placed
in
he
icini y
o
he
cell.
In
hose
myocy es
ha
had
a
ela i ely
high
es ing
[Ca2+]
(abo e
50
nM),
he
inhibi ion
o
he
oscilla ions
by
hypoxia
was
accompanied
by
a
dec ease
in
es ing
cy osolic
Ca2+
le els.
These
obse a ions
indica e
ha
low
P02
may
exe
i s
elaxing
ac ion
by
dec easing
in acellula
[Ca2+],
which
is
he
a iable
ha
de e mines
con ac ion
in
ascula
smoo h
muscle
(17-
20).
Al hough
la gely
due
o
Ca2+
elease
om
in acellula
s o es
(21,
22),
Ca2+
oscilla ions
in
exci able
(23)
and
nonex-
ci able
(24)
cells
equi e,
and
a e
acili a ed
by,
ansmem-
b ane
Ca;+
in lux.
In
acco d
wi h
his
idea,
depola iza ion
o
myocy es
wi h
60
mM
ex e nal
K+
elici s
an
inc ease
in
he
equency
o
he
Ca2+
oscilla ions
p eceding
a
main ained
ele a ion
o
cy osolic
[Ca2+]
(Fig.
1B).
An
opposi e
e ec
(abolishmen
o
he
Ca2+
spikes
and
dec ease
o
es ing
cy osolic
Ca2+)
was
obse ed
a e
b ie ly
emo ing
ex e nal
Ca2+
(Fig.
1
C)
o
blockade
o
ol age-ga ed
Ca2+
channels
wi h
ni edipine
(Fig.
1D).
The
hypoxic
supp ession
o Ca2+
oscilla ions
could
be
caused
by
a ious
02-dependen
cellula
p ocesses.
Re illing
o
s o es
p e iously
deple ed
wi h
ca eine
was
no
p e en ed
by
hypoxia.
Fu he mo e,
hypoxia
did
no
a ec
he
elease
o
Ca2+
om
in e nal
s o es
e oked
by
ei he
ca eine
(10
mM)
o
no epineph ine
(3
ApM).
The e o e,
one
o
he
ac ions
o
low
P02
migh
be
o
inhibi
Ca2+
in lux
h ough
ol age-dependen
channels.
This
was
di ec ly
es ed
by
eco ding
he
cu en
h ough
Ca2+
channels
in
whole-cell
pa ch-clamped
myocy es.
Cu en
sweeps
gene a ed
in
esponse
o
s ep
depola iza ions
o
+
10
mV
in
a
celiac
myocy e
exposed
o
no moxic
(con ol
and
eco e y)
and
hypoxic
ex e nal
solu ions
a e
shown
in
Fig.
2A.
The
eco dings
demons a e
an
-40%
e e sible
educ-
ion
in
cu en
ampli ude
upon
exposu e
o
low
P02.
The
ela ionship
be ween
cu en
ampli ude
and
P02
is
illus a ed
in
Fig.
2B,
whe e
he
alues
o
peak
cu en
(do s)
elici ed
by
depola izing
pulses
deli e ed
a
di e en
P02
le els
a e
plo -
ed.
The
g aphs
indica e
ha
he
e e sible
hypoxic
inhibi ion
o
cu en
ampli ude
occu s
oughly
wi h
he
ime
cou se
o
ba h
exchange
and
ha
i
is
pa icula ly
appa en
a
P02
le els
below
70
o
80
mmHg.
The
ime
cou se
o
he
inhibi ion
o
he
calcium
cu en
by
low
P02
was
simila
o
ha
o
ni edipine
block
(no
shown).
Fo
compa ison,
e e sible
hypoxic
inhi-
bi ion
o
Ca2+
channel
ac i i y
in
a
emo al
myocy e
is
illus a ed
in
Fig.
2C.
Simila
quali a i e
esul s
ha e
been
ob ained
in
all
cells
s udied
so
a
(n
=
33).
I
has
been
shown
ha
ascula
smoo h
muscle
cells
con ain
wo
majo
kine ically
A
150
E
/oxygen
ensiol
E
50
(P2)
+"
~~~60K+
200
200
-
"i"
+
100
0
K
n
s
C
.350
u
150
0
cn
2
50
5
o
D
+
N
.
u
0
>%
1
min
0
Ca2+,
0.5
mM
EGTA
0.5
FIM
ni edipine
1min
FIG.
1.
Oscilla ions
o
cy osolic
[Ca2+]
in
a e ial
myocy es
and
e ec
o
changes
in
ambien
oxygen
ension
(Po2).
(A)
Pa allel
eco dings
o
Po2
in
he
eco ding
chambe
and
in acellula
[Ca2+]
in
a
u a-2-loaded
myocy e
illus a ing
he
e e sible
inhibi ion
o
Ca2+
oscilla ions
in
esponse
o
hypoxia.
No e
ha
he
spikes
a e
supp essed
upon
eaching
ex eme
low
Po2
le els
(=20
mmHg).
A e age
es ing
cy osolic
[Ca2+]
was
61
±
28
nM
(mean
+
SD,
n
=
22)
and
oscilla ions
we e
igge ed
wi h
a
ansien
(=30
s)
exposu e
o
1
mM
ATP
(26).
The
a e age
equency
and
ampli ude
o
he
oscilla ions
we e
1.6
±
0.6
spikes
pe
min
and
271
±
172
nM
(n
=
8),
espec i ely.
(B)
Re e sible
inc ease
o
he
equency
o
Ca2+
oscilla ions
in
esponse
o
memb ane
depola iza ion
by
60
mM
ex e nal
K+.
Reini ia ion
o
he
oscilla ions
a e
eco e y
o
he
basal
Ca2+
le els
was
ob ained
by
a
b ie
pulse
o
1
mM
ATP.
(C
and
D)
Supp ession
o
Ca2+
oscilla ions
a e
b ie
emo al
and
chela ion
o
ex e nal
Ca2+
(C)
o
blockade
o
ol age-ga ed
Ca2+
channels
wi h
ni edipine
(D).
The
applica ion
o
he
di e en
es
solu ions
is
indica ed
by
he
ho izon al
ba s.
The
s anda d
ex e nal
solu ion
con ained
(in
mM)
140
NaCl,
2.7
KCI,
2.5
CaCl2,
1
MgCl2,
and
10
Hepes
(pH
7.35-7.40).
The
60
K+
solu ion
con ained
82.7
mM
NaCl
and
60
mM
KCl.
Ni edipine
(0.5
iM)
was
added
o
he
ex e nal
solu ion.
The
0
Ca2+,
EGTA
solu ion
con ained
4
mM
MgCI2
and
0.5
mM
EGTA.
4716
Pyilg:Fac
a
P oc.
Na l.
Acad.
Sci.
USA
92
(1995)
4717
A
hypoxia
con ol
2
3
B
o
150
C
CD
-
l!0
1
.
200-
0
0
a 0
0
50
100
150
200
ime
(s)
FIG.
2.
Inhibi ion
o
ol age-ga ed
Ca2+
channels
by
low
P02.
(A)
Mac oscopic
calcium
cu en s
eco ded
om
a
myocy e
dispe sed
om
he
celiac
a e y
du ing
15-ms
s ep
depola iza ions
o
+
10
mV
om
a
holding
po en ial
o
-80
mV.
Exposu e
o
hypoxia
(swi ching
om
an
ex e nal
solu ion
equilib a ed
wi h
P02
-
150
mmHg
o
ano he
wi h
P02
-
20
mmHg;
eco ds
2
and
3)
induces
an
inhibi ion
in
cu en
ampli ude.
Re e sibili y
is
illus a ed
by
he
eco e y
ace.
(B)
Pa allel
ime
cou ses
o
he
changes
o
P02
in
he
chambe
and
he
educ ion
in
cu en
ampli udc.
The
eco ds
shown
in
A
a e
indica ed
by
he
co esponding
numbe .
Cu en
ampli udes
we e
measu ed
immedia ely
be o e
he
end
o
he
depola izing
pulses.
(C)
Re e sible
educ ion
o
calcium
cu en
ampli ude
by
low
P02
(20
mmHg)
in
a
myocy e
dispe sed
om
he
emo al
a e y.
Depola izing
pulses
o
+10
mV
(15
ms)
we e
applied
om
a
po en ial
o
-80
mV.
No e
ha
a
slow
componen
o
he
ail
cu en
was
una ec ed
by
low
P02.
The
eco ding
solu ions
con ained
(in
mM)
he
ollowing:
Ex e nal
[140
NaCl,
2.7
KCI,
10
BaCl2,
and
10
Hepes
(pH
7.4)].
In e nal
(solu ion
in
he
pa ch
pipe e
and
inside
he
cell)
[100
CsCl,
25
CsF,
2
MgCl2,
10
Hepes,
10
EGTA,
5
bis(2-aminophenoxy)e hane-
N,N,N',N'- e aace a e,
and
4
MgATP
(pH
7.3)].
dis inc
popula ions
o
Ca2+
channels
(L
and
T
ype)
(25,
26)
ha ,
as
in
o he
cell
ypes
(16,
27),
can
be
iden i ied
by
hei
deac i a ion
ime
cou ses
as
slow-
(T)
and
as -
(L)
deac i a -
ing
channels.
In
many
emo al
and
celiac
myocy es
we
de ec
wo
componen s
o
he
ails.
The
slowly
deac i a ing
compo-
nen
o
he
ail
cu en ,
which
is
esis an
o
ni edipine
(Fig.
3A)
and
inac i a ed
by
a
small
depola izing
p epulse
(Fig.
3
B),
was
una ec ed
by
hypoxia
(Fig.
3
C)
in
all
cells
es ed
(see
also
Figs.
2C
and
4A).
These
obse a ions
indica e
ha
he
e ec
o
low
P02
is
selec i e
o
he
dihyd opy idine-sensi i e,
as -
deac i a ing
(L- ype),
channels,
whe eas
he
slow-deac i a ing
(T- ype)
Ca2+
channel
popula ion
is
una ec ed
by
changes
in
P02.
A
ema kable
cha ac e is ic
o
he
egula o y
ac ion
o
oxygen
on
Ca2+
channel
ac i i y
is
i s
s ong
ol age
depen-
dence.
Fig.
4A
shows
a
amily
o
calcium
cu en s
gene a ed
by
depola iza ions
om
-80
mV
o
he
indica ed
memb ane
po en ials.
Cu en
aces
eco ded
in
a
low
P02
solu ion
(H)
a e
compa ed
wi h
hose
ob ained
a
no mal
P02
(C).
Reco -
e y
om
hypoxia
was
almos
pe ec
and
an
example
is
shown
a
+10
mV
( ace
R).
Low
P02
p oduced
an
inhibi ion
o
cu en
ampli ude
ha
was
la ge
wi h
mode a e
depola iza-
ion.
Wi h
s onge
depola iza ion
he
e ec
o
hypoxia
was
almos
negligible.
In
celiac
myocy es,
he
a e age
inhibi ion
o
cu en
ampli ude
by
hypoxia
(Po2
-
20
mmHg)
was
43.17%
±
13.8%
(mean
±
SD,
n
=
12)
o
he
con ol
alue
a
0
mV,
bu
only
2.08%
+
7%
(n
=
8)
a
+20
mV.
In
emo al
myocy es,
hese
alues
we e
40%
+
27%
(n
=
7)
a
0
mV
and
10%
±
13%
(n
=
6)
a
+20
mV.
The
ol age
dependence
o
he
hypoxic
inhibi ion
o
Ca2+
channel
ac i i y
is
also
clea ly
e iden
in
Fig.
4B,
whe e
we
ha e
plo ed
he
a e age
cu en - ol age
ela-
ionship
unde
no moxic
( illed
symbols)
and
hypoxic
(open
symbols)
condi ions
ob ained
wi h
measu emen s
om
ou
cells.
DISCUSSION
Ou
esul s
indica e
ha
exposu e
o
low
P02
leads
o
inhibi-
ion
o
Ca2+
in lux
h ough
L- ype
Ca2+
channels
in
a e ial
myocy es.
This
is
selec i e
since
T- ype
Ca2+
channels
appea
o
be
una ec ed.
The
e ec
o
02
ension
on
he
Ca2+
channels
is
as ,
is
comple ely
e e sible,
and
occu s
wi h
P02
le els
wi hin
he
physiological
ange.
This
phenomenon
explains
he
dec ease
o
cy osolic
Ca2+
upon
exposu e
o
low
P02
and,
hus,
i
is
concei able
ha
i
con ibu es
o
he
hypoxic
elaxa ion
o
sys emic
a e ies.
In
ag eemen
wi h
p e ious
wo k
by
o he
au ho s
(28,
29),
we
ha e
no
de ec ed
so
a
any
modula o y
e ec
o
P02
on
he
mac oscopic
K+
cu en s
o
sys emic
myocy es.
Pha macological
s udies
done
in
isola ed
pe used
hea s
ha e
sugges ed
ha
hypoxic
asodila a ion
o
he
co -
ona y
a e ies
could
be
a
consequence
o
he
dec ease
o
in acellula
ATP
and
he
subsequen
hype pola iza ion
caused
by
he
opening
o
ATP-sensi i e
(KATP)
K+
channels
(8,
9).
Ac i a ion
o
KATP
channels
in
esponse
o
hypoxia
mos
likely
equi es
main ained
exposu e
o
ex eme
low
P02
(8,
30).
Thus,
al hough
possibly
impo an ,
his
p ocess
may
ac
on
a
slowe ,
mo e
p o ac ed,
ime
scale
han
he
acu e
esponse
o
Ca2+
channels
desc ibed
he e,
occu ing
o e
a
ull
ange
o
P02
alues
and
su ely
c i ical
o
an
immedia e
a e ial
elax-
a ion
in
esponse
o
hypoxia.
The
Ca2+
channels
selec i ely
egula ed
by
P02
a e
o
he
dihid opy idyne-sensi i e,
L- ype,
which
a e
b oadly
dis ib-
u ed
in
ascula
smoo h
muscle
and
a e
known
o
be
ac i a ed
by
no epineph ine
and
o he
asoac i e
agen s
(20,
26,
31).
In
mesen e ic
a e y
myocy es,
he
memb ane
po en ial- o ce
4
eco e y
00
msA
5
ms
C
hypoxia
5
ms
Physiology:
F anco
e
al.
1
P oc.
Na l.
Acad.
Sci.
USA
92
(1995)
I
| <
HP=-50
HP=-80
150
pA
5
ms
I
HP=-80
mV
HP=-50
mV
5
ms
FIG.
3.
Sepa a ion
o
as -
and
slowly
deac i a ing
componen s
in
he
Ca2+
ail
cu en s
and
selec i e
inhibi ion
o
he
as
componen
by
low
P02.
(A)
Supe posi ion
o
cu en
aces
eco ded
in
a
celiac
myocy e
du ing
15-ms
depola iza ions
o
+
10
mV
om
-80
mV
in
he
con ol
solu ion
and
a e
addi ion
o
0.2
u M
ni edipine
(uppe
panel).
The
ail
cu en s
in
he
wo
expe imen al
condi ions
a e
shown
a
a
expanded
ime
scale
in
he
middle
and
bo om
panels.
As
in
o he
cell
ypes
(16,
27),
he
ail
cu en s
ha e
as
and
slow
componen s
ha
mos
likely
ep esen
he
L- ype
( as -deac i a ing)
and
T- ype
(slow-deac i a ing)
Ca2+
channels
desc ibed
in
a e ial
smoo h
muscle
cells
(25,
26).
Ni edipine
almos
comple ely
abolished
he
as
componen
o
he
ail
bu
le
unal e ed
he
slow
componen .
This
is
shown
by
he
simila
ampli ude
o
single
exponen ial
unc ions
ha
in
he
wo
expe imen al
condi ions
we e
i ed
o
he
slow
componen
o
he
ail.
The
exponen ial
unc ions
we e
ex apola ed
o
he
onse
o
epola iza ion
(indica ed
by
downwa d
a ows).
(B)
Supe posi ion
o
cu en
aces
ob ained
om
a
emo al
myocy e
du ing
10-ms
depola iza ions
o
+
10
mV
om
he
indica ed
holding
po en ials
(HP)
(uppe
aces).
The
middle
and
bo om
panels
show
he
exponen ial
unc ions
i ed
o
he
slow..componen
o
he
ail
a
he
wo
holding
po en ials.
No e
ha
a
small
main ained
depola iza ion
( om
-80
o
-50
mV)
leads
o
>80%
educ ion
o
he
slow
componen
in
he
ail,
indica ing
inac i a ion
o
he
T- ype
Ca2+
channels.
The
as
componen
o
he
ail
was
almos
unal e ed.
(C)
Supe posi ion
o
cu en
aces
in
a
celiac
myocy e
gene a ed
du ing
15-ms
s ep
depola iza ions
o
+
10
mV
om
-80
mV
in
a
myocy e
exposed
o
no moxic
(C,
con ol,
P02
-
150
mmHg)
and
hypoxic
(H,
Po2
-
20
mmHg)
solu ions
(uppe
panel).
As
in
A,
he
eco dings
in
he
middle
and
bo om
panels
show
ha
he
slow
componen
o
he
ail
cu en s
was
una ec ed
by
low
P02
and
ha
he
educ ion
o
cu en
ampli ude
is
due
o
selec i e
inhibi ion
o
he
as
componen
o
he
ail
cu en .
ela ion
almos
pe ec ly
ma ches
wi h
he
ol age
dependence
o
Ca2+
channel
open
p obabili y
(Popen)
(20).
Thus,
is
has
A
VM
(mV)
H
C
0
-"
-
:;
~
~
-_-------"
+1
0
/
H
been
sugges ed
ha
his
channel
ype
is
a
majo
egula o
o
smoo h
muscle
ension
and
hence
o
a e ial
one
(20,
28,
31).
memb ane
po en ial
(mV)
B
-40
-20
'
0
'
0
20
40
60
.
.
..
*
-100-
-200-
3
CD
c
'D
0
a
C>
I
FIG.
4.
Vol age
dependence
o
he
inhibi o y
e ec
o
low
P02
on
he
Ca2+
channels.
(A)
Cu en
aces
eco ded
du ing
15-ms
s ep
depola iza ions
om
-80
mV
o
he
indica ed
memb ane
po en ials.
Sweeps
eco ded
om
he
same
celiac
myocy e
in
no moxic
(C,
P02
-
150
mmHg)
and
hypoxic
(H,
P02
-
20
mmHg)
solu ions
a e
supe imposed.
No e
ha
he
e ec
o
low
P02
is
la ge
wi h
mode a e
depola iza ions.
(B)
A e age
cu en - ol age
ela ion
in
no moxic
( illed
symbols)
and
hypoxic
(open
symbols)
solu ions.
Cu en
ampli udes
we e
measu ed
in
ou
celiac
myocy es
be o e
he
end
o
15-ms
depola izing
pulses.
Ve ical
ba s
a e
he
s anda d
e o
o
he
mean.
A
B
C
Icon ol
-slow
ail
i
con ol
I
ni edipine
-
B-
b
ms
)-
5
ms
4718
Pyilg:Fac
a
P oc.
Na l.
Acad
Sci.
USA
92
(1995)
4719
In e es ingly,
he
inhibi ion
o
he
calcium
cu en
by
low
Po2
is
mo e
p onounced
a
po en ials
be ween
-30
and
0
mV
(see
Fig.
4),
which
is
he
ange
a
which
he
popen- ol age
ela ion-
ship
o
he
channels
is
e y
s eep
(20,
31).
The e o e,
in
pa ially
depola ized
myocy es
low
P02
would
be
expec ed
o
ha e
a
majo
in luence
on
channel
Popen.
In
good
ag eemen
wi h
his
idea,
ea ly
s udies
had
al eady
shown
ha
he
sensi i i y
o
sys emic
a e ies
o
oxygen
is
mo e
ob ious
when
es ing
is
ca ied
ou
on
p econ ac ed
samples
using
low
o
mode a e
concen a ions
o
agonis
(5).
In
conclusion,
we
ha e
ound
in
a e ial
smoo h
muscle
a
ype
o
Ca2+
channel
modula ion
ha
may
pa icipa e
in
hypoxic
a e ial
dila a ion.
The
p ope ies
o
he
oxygen-
sensi i e
Ca2+
channels
make
hem
well
sui ed
o
ha ing
a
majo
physiological
ole
in
he
as
adap a ion
o
egional
a e ial
esis ance
o
he
deg ee
o
blood
oxygena ion.
The
exis ence
o
oxygen-sensi i e
K+
channels
in
a ious
issues
has
been
p e iously
desc ibed
(10)
bu
oxygen-sensi i e
Ca2+
channels
a e
wi hou
p eceden
in
he
li e a u e.
Besides
celiac
and
emo al
myocy es
( ep esen a i e
examples
o
isce al
and
skele al
muscle
a e ies),
we
ha e
obse ed
a
simila
egula-
o y
ac ion
o
P02
in
muscle
cells
dispe sed
om
a
and
abbi
mesen e ic
a e ies
as
well
as
om
he
main
unk
o
he
abbi
pulmona y
a e y.
This
sugges s
ha
02- egula ed
Ca2+
chan-
nels
a e
pe haps
dis ibu ed
h oughou
he
ci cula o y
sys em
and
in
di e en
species.
This
channel
ype
migh
be
in ol ed
in
some
ca dioci cula o y
dis u bances
such
as
hype ension.
This
wo k
was
suppo ed
by
g an s
om
he
Di ecci6n
Gene al
de
In es igaci6n
Cien ica
y
T6cnica
(DGICYT)
o
he
Spanish
Minis y
o
Science
and
Educa ion
and
he
Eu opean
Communi y
(DGXII).
A.F.-O.
is
an
In e na ional
Human
F on ie s
Science
P og am
Pos -
doc o al
Fellow.
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Spa ks,
H.
V.
(1980)
in
Vascula
Smoo h
Muscle,
Handbook
o
Physiology,
The
Ca dio ascula
Sys em,
eds.
Boh ,
D.
F.,
Somlyo,
A.
P.
&
Spa ks,
H.
V.
(Am.
Physiol.
Soc.,
Be hesda),
Vol.
3,
pp.
475-513.
2.
Hells and,
P.,
Johansson,
B.
&
No be g,
K.
(1977)
Ac a
Physiol.
Scand.
100,
69-83.
3.
De a ,
R.
(1980)
Am.
J.
Physiol.
238,
H761-H769.
4.
Ma io ,
J.
F.
&
Ma shall,
J.
(1990)
J.
Physiol.
(London)
422,
1-13.
5.
Chang,
A.
E.
&
De a ,
R.
(1980)Am.
J.
Physiol.
238,
H716-H728.
6.
an
B eemen,
C.
&
Saida,
K.
(1989)
Annu.
Re .
Physiol.
51,
315-329.
7.
Ebeigbe,
A.
B.,
Picka d,
J.
D.
&
Jenne ,
S.
(1980)
Q.
J.
Exp.
Physiol.
65,
273-292.
8.
Dau ,
J.,
Maie -Rudol ,
W.,
on
Becke a h,
N.,
Meh ke,
G.,
Gun he ,
K.
&
Goedel-Meinen,
L.
(1990)
Science
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on
Becke a h,
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A.
&
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J.
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(London)
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297-319.
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L6pez-Ba neo,
J.
(1994)
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133-135.
11.
Gan o nina,
M.
D.
&
L6pez-Ba neo,
J.
(1992)
J.
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Physiol.
100,
401-426.
12.
Toledo-A al,
J.,
U e ia,
J.,
Cas ellano,
A.
&
Lopez-Ba neo,
J.
(1993)
J.
Physiol.
(London)
472,
327-340.
13.
Alme s,
W.
&
Nehe ,
E.
(1985)
FEBS
Le .
192,
13-18.
14.
G ynkiewicz,
G.,
Poenie,
M.
&
Tsien,
R.
Y.
(1985)J.
Biol.
Chem.
260,
3440-3450.
15.
Hamill,
0.
P.,
Ma y,
A.,
Nehe ,
E.,
Sakmann,
B.
&
Sigwo h,
F.
(1981)
P liige s
A ch.
391,
85-100.
16.
Cas ellano,
A.
&
L6pez-Ba neo,
J.
(1991)
J.
Gen.
Physiol.
97,
303-320.
17.
Bla e ,
L.
A.
&
Wie ,
W.
G.
(1992)
Am.
J.
Physiol.
263,
H576-
H586.
18.
Weissbe g,
P.
L.,
Li le,
P.
J.
&
Bobik,
A.
(1989)
Am.
J.
Physiol.
256,
C951-C957.
19.
Gus a sson,
H.
&
Nilsson,
H.
(1993)
Ac a
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149,
283-291.
20.
Nelson,
M.
T.,
S anden,
N.
B.,
B ayden,
J.
E.
&
Wo ley,
J.
F.
(1988)
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