Activation of Ca2+-dependent
K+ channels by cyanide in
guinea pig adrenal chromaffin cells
M.
Inoue and
I.
Imanaga
Department of Physiology, School of Medicine, Fukuoka University,
Fukuoka 814-01, Japan
 |
ABSTRACT |
The effects of
cyanide (CN) on whole cell current measured with the perforated-patch
method were studied in adrenal medullary cells. Application of CN
produced initially inward and then outward currents at
52 mV or
more negative. As the membrane potential was hyperpolarized, amplitude
and latency of the outward current (Io) by CN
became small and long, respectively. A decrease in the external
Na+ concentration did not affect
the latency for CN-induced
Io but enhanced
the amplitude markedly. The CN
Io reversed
polarity at
85 mV, close to the Nernst potential for
K+, and was suppressed by the
K+ channel blockers curare and
apamin but not by glibenclamide, suggesting that
Io is due to the
activation of Ca2+-dependent
K+ channels. Consistent with this
notion, the Ca2+-mobilizing
agents, muscarine and caffeine, also produced
Io. Exposure to
CN in a Ca2+-deficient medium for
4 min abolished caffeine- or muscarine-induced Io without
development of
Io, and addition
of Ca2+ to the CN-containing
solution induced
Io. We conclude
that exposure to CN produces
Ca2+-dependent
K+ currents in an external
Ca2+-dependent manner, probably
via facilitation of Ca2+ influx.
ATP; calcium pump; store sites; small-conductance calcium-dependent
potassium channel; mitochondria
 |
INTRODUCTION |
HYPOXIA OR METABOLIC INHIBITION induces cell death
through increases in intracellular concentrations of
Ca2+
([Ca2+]i)
and subsequent activation of
Ca2+-dependent enzymes, such as
protease (25). Thus much attention has been directed to mechanisms
related to Ca2+ increase.
Application of cyanide (CN) to cultured human skin epidermoid A-431
cells induced an increase in Ca2+
concentration, and the underlying mechanism was
Ca2+ influx via the
Na+/Ca2+
exchange system (18). By contrast, in glomus cells of the rabbit carotid body, hypoxia- and CN-induced
Ca2+ elevation was due to
Ca2+ release from mitochondria (2)
or to Ca2+ influx across
voltage-dependent Ca2+ channels
resulting from suppression of voltage-dependent
K+ channels and the subsequent
depolarization (22). It is also known that hypoxia in hippocampal
neurons (1) and cerebellar Purkinje cells (17) appears to mobilize
Ca2+ from inositol trisphosphate
(IP3)-sensitive store sites, the underlying mechanism being a reduction in NADH (17). To elucidate the
mechanism for Ca2+ elevation
evoked by hypoxia or mitochondrial inhibition, we evaluated effects of
CN on whole cell currents in dissociated adrenal chromaffin cells. Such
mechanisms may be involved in anoxia-induced secretion of
catecholamines from the adrenal medulla (4), a release that is vital
for survival (6). Furthermore, chromaffin cells originate from the
neural crest, as do peripheral neurons, and transform to neurons in the
presence of nerve growth factor (28). Thus findings obtained with
chromaffin cells would pave the way for treatment of neuronal disorders
and lead to a better understanding of neuron death and the consequent
brain damage (5).
 |
METHODS |
Whole cell recordings.
Adrenal medullae from female guinea pigs were treated with collagenase
to obtain dissociated chromaffin cells, as described elsewhere (14).
The whole cell current was recorded using the perforated-patch method
to diminish washout of cellular components (10, 12). Dissociated
chromaffin cells were left for a few minutes to facilitate attachment
to the bottom of the bath before being constantly perfused with
standard saline at a rate of ~1 ml/min. The standard saline contained
(in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.53 NaH2PO4,
5 D-glucose, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 2.6 NaOH. To study the effects of CN, the standard solution was exchanged with CN solution in which 5 mM NaCl was replaced
with 5 mM NaCN and the glucose was removed unless otherwise noted. In
Ca2+-free solutions, 3.6 mM
Mg2+ was substituted for 1.8 mM
Ca2+. The pH of all external
solutions was adjusted to 7.4. The current was recorded using an
Axopatch 200A amplifier and then fed into a brush recorder after
low-pass filtering at 5 Hz and into a videocassette recorder after
digitizing by an analog-to-digital converter. The pipette solution
contained (in mM) 120 potassium isethionate, 20 KCl, 10 NaCl, 10 HEPES,
and 2.6 KOH. The pH of the pipette solution was adjusted to 7.2. When
current-voltage (I-V) relationships were investigated, the pulse protocol consisting of a 50-ms negative pulse, 300-ms interval, and a 50-ms positive pulse was applied 10 times
in 10-mV steps from a holding potential. At the same time, currents
were stored on a diskette of a computer at a sampling interval of 0.1 ms after filtering at 3 kHz. The series resistance (20-30 M
)
was not compensated, and resulting voltage errors did not generally
exceed 5 mV in the I-V curves. On the
day of experiment, nystatin was added to the pipette solution at a
final concentration of 100 µg/ml. The membrane potential was
corrected for a liquid junction potential of
12 mV between the
nystatin solution and the standard saline. All experiments were carried
out at 23-25°C. Data were expressed as means ± SD unless
otherwise noted, and the Student's
t-test was used to determine
statistical significance.
Chemicals.
Sources of chemicals are as follows: (±)-muscarine chloride,
curare, and nystatin (Sigma); apamin and charybdotoxin (Peptide Institute, Japan); glibenclamide (Research Biochemicals International); collagenase (Yakult, Japan); NaCN (Hayashi Pure Chemical Industries, Japan).
 |
RESULTS |
Activation of
Ca2+-dependent
K+ channels by
CN.
Because chromaffin cells have
Ca2+-dependent
K+ channels (13, 20, 23), an
increase in
[Ca2+]i
could be detected as an increase in outward current
(Io) at membrane potentials positive to the equilibrium potential for K+ (
84 mV, under present
conditions). Figure
1A shows
that exposure to CN produced a gradual increase in
Io at
52
mV. The current began to develop with a latency of ~1 min and reached
a maximum of 37.0 ± 29.7 pA
(n = 16) at 3.7 ± 1.1 min (n = 16). The size of
Io evoked by CN
was not noticeably affected by addition of 10 mM glucose
(n = 3). To elucidate ionic mechanisms
without substantial contamination by the CN-induced inhibition of an
inwardly rectifying K+ current
(16), I-V relationships were examined
4 and 6 min after the application of CN. The
I-V relationship examined at the
maximum (b in Fig. 1,
A and
B) crossed a control
(a) at
82 mV (
85.3 ± 5.1 mV, n = 6), close to the
Nernst potential for K+. To
clarify voltage dependence of CN-induced
Io, CN-sensitive currents (Fig. 1D) were obtained by
subtracting currents in response to pulses before
(a) from those during exposure to CN
(b). The CN-sensitive current (Fig.
1C) changed polarity at
82
mV, and the conductance decreased with hyperpolarization. This
CN-induced Io was
enhanced markedly by a decrease in
Na+ concentration (135 mM
N-methyl-D-glucamine
in place of Na+). The
I-V relationship examined in the
Na+-deficient CN solution crossed
those in standard saline and CN solution at the same membrane potential
of
82 mV (Fig. 1B; reversal potential for CN-sensitive current enhanced by decrease in
[Na+],
84.3 ± 1.0 mV, n = 4, Fig. 1,
C and
Db). Restoration of physiological Na+ in the presence of CN induced
a transient Io
increase.

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Fig. 1.
Ionic mechanism for cyanide (CN)-induced outward current
(Io).
A: chart record of a whole cell
current at 52 mV. Standard saline was replaced with standard CN
solution (CN; 5 mM) or
Na+-deficient CN solution
[CN Na( )] during indicated periods (see
METHODS). In CN Na( ), 135 mM
Na+ was replaced equimolarly with
N-methyl-D-glucamine.
Upward and downward deflections were truncated currents in response to
100-mV pulse of 50 ms or to pulse protocol. Pulse protocol
consisting of 50-ms negative pulse, 300-ms interval, and 50-ms positive
pulse was applied in 10-mV steps 10 times.
B: current-voltage
(I-V) relationships examined in
saline (a in
A, ), CN solution
(b, ), and
Na+-deficient CN solution
(c, ).
C: CN-induced
I-V relationships in presence of 140 mV ( ) and 5 mM
Na+ ( ). CN-induced currents
were obtained by subtracting currents in response to pulses in saline
from those in standard CN solution and
Na+-deficient CN solution.
D: families of CN-sensitive currents
in 140 (a) and 5 mM
Na+
(b). Amplitudes of current were
measured at end of pulse. Arrows in this and following figures
represent 0-current level.
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To determine the K+ channel type
involved in CN-induced
Io, the effects
of K+ channel blockers were
studied. In Fig.
2A, bath
application of CN induced an inward current without a noticeable delay
and 4.5 min later began to induce
Io at
67
mV. This Io was
reversibly blocked by bath application of 0.1 mM curare, an agent known
to block a small-conductance
Ca2+-dependent
K+ channel (SK channel; Ref. 3,
23, 24). The I-V relationships before
and during application of curare crossed at
82 mV (Fig. 2B;
83.0 ± 3.6 mV,
n = 3), and the extent of suppression
was 71.4% (73.8 ± 25.1%, n = 3).
Apamin at 0.1 µM, another SK channel blocker (3), also inhibited the
Io by 33.3%
(Fig. 2C; 39.5 ± 22.2%,
n = 4), and the reversal potential for
apamin-sensitive currents was
87.0 mV (
86.3 ± 3.3 mV,
n = 4). In contrast, the current was
not suppressed either by 0.1 µM charybdotoxin
(n = 4), a blocker of a
large-conductance Ca2+-dependent
K+ channel (BK channel, Ref. 20;
21) or 10 µM glibenclamide (n = 3),
a blocker of an ATP-sensitive K+
channel (26). Figure 2D demonstrates
that
82 mV was the reversal potential for
K+ channels in this cell: addition
of 1 mM Ba2+ to the perfusate
abolished an inwardly rectifying
K+ channel (11).

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Fig. 2.
Suppression of CN-induced
Io by
Ca2+-dependent
K+ channel blockers.
A: chart record of a whole cell
current at 67 mV. CN, 0.1 mM curare (Cur), 0.1 µM apamin
(Apa), or 1 mM Ba2+ (Ba) was
applied during indicated period. B:
I-V relationships during ( ) and
after ( ) application of curare in presence of CN
(b in
A).
C:
I-V relationship before ( ) and
during ( ) apamin (c in
A).
D:
I-V relationship during ( ) and
after ( ) Ba2+.
I-V relationships were examined, as
explained for Fig. 1.
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Source of
Ca2+ involved in
channel activation.
The foregoing results suggest that the CN-induced
Io is due to
activation of Ca2+-dependent
K+ channels but not of
ATP-sensitive channels. We reported that guinea pig chromaffin cells
have intracellular store sites sensitive to caffeine and putatively to
IP3 (15). Thus
Ca2+ in these store sites is
possibly involved in the generation of Io by CN, as was
noted on a hyperpolarizing response to anoxia in hippocampal neurons
(1). Application of 10 mM caffeine later than 2 min of CN exposure
failed to induce
Io (Fig.
3A).
This event may not be due to involvement of stored
Ca2+ in the CN-induced
Io. Caffeine
applied at ~1 min of the exposure, a time when
Io had already
developed in response to CN, evoked Io for a longer
period. Similar results were observed in two other cells. One
possibility to account for the failure of caffeine to induce
Io at 2 min or
later would be depletion of Ca2+
from store sites, and this depletion probably results from a decrease
in Ca2+ pump activity due to
diminution in cellular ATP contents. This notion was supported by the
finding that restoration of the caffeine response after termination of
CN exposure required longer intervals than that in the absence of CN.
In four of five cells, application of caffeine 2-4 min after
termination of CN exposure, which lasted for >4 min, failed to evoke
Io (Fig.
3B), whereas an interval of 2-3
min was sufficient for reproduction of caffeine
Io in all seven
cells (13, 15). In addition, as CN exposure was lengthened, longer
intervals after washout of CN were required for reproduction of
Io. In Fig.
3B (open squares), caffeine applied
4.4 min after the washout induced
Io in the case of
3.5-min treatment but not in that of 10.6 min. The results suggest that
CN exposure for >4 min apparently depletes
Ca2+ from store sites and that
depleted Ca2+ are probably not
involved in the generation of
Io by CN.

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Fig. 3.
Effects of CN on
Io response to
caffeine. A: chart records of a whole
cell current. Holding potential, 52 mV. CN and/or 10 mM
caffeine (Caf) were applied during indicated periods (single and double
bars, respectively). Top and
bottom, continuous record.
B: times of caffeine application after
washout of CN are plotted against duration of CN treatment. Open and
filled symbols indicate success and failure of 10 mM caffeine to induce
an Io,
respectively. Same symbols mean same cells. Straight line shows rough
discrimination between failure and success.
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We then investigated effects of
Ca2+ removal on CN-induced
Io at
52
mV (Fig. 4). Administration of CN in the
absence of external Ca2+ did not
produce Io and
abolished Io in
response to 6 µM muscarine (n = 3, Fig. 4A) and to 10 mM caffeine
applied at 4 min (n = 8, Fig.
4B), indicating that CN depleted
Ca2+ in store sites without
producing Io. The
subsequent addition of 1.8 mM Ca2+
to the Ca2+-free CN solution,
however, resulted in a gradual development of
Io without a
latency (n = 6), and the evoked
current diminished after washout of
Ca2+ slowly with a half time
(T1/2) of 27 ± 8 s (n = 6). This slow decline
may not be due to sequestration of
Ca2+ into intracellular store
sites because the uptake was suppressed during exposure to CN (Fig.3).

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Fig. 4.
Dependence of CN-induced
Io on external,
but not internal, Ca2+.
A and
B: chart records of a whole cell
current at 52 mV. Different cells. Removal of
Ca2+ [Ca( )] and
application of CN were indicated by 1st and 2nd lines below each chart
record, respectively. Application of 10 mM caffeine (single bar) or 6 µM muscarine (M, double bar) was shown by 3rd lines. First 2 Ios evoked by
caffeine in B were out of scale.
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Mechanism for CN-induced increase in
[Ca2+]i.
There are several possibilities to account for the CN-induced increase
in
[Ca2+]i.
One is the reversed mode of
Na+/Ca2+
exchange due to accumulation of
Na+ at the inner surface of the
membrane. To explore this possibility, effects of ouabain, a
Na+-K+-adenosinetriphosphatase
(ATPase) inhibitor, were examined. Application of 20 µM
ouabain for up to 4 min did not induce
Io at
67
mV, whereas in the same cells CN consistently produced it
(n = 3). Moreover, a decrease in
Na+ concentration from 140 to 5 mM
enhanced Io in
response to CN. Figure
5A shows
that, at
52 mV, bath application of CN induced an inward current
without a noticeable delay and then apparently began to produce
Io at 72 s. When
a Na+-deficient CN solution was
perfused, the inward current did not develop
(n = 5) and the
Io began to
develop with a latency of 30 s (70 ± 34 s,
n = 5), which did not differ from that
(56 ± 35 s, n = 6) in the presence
of standard Na+. After a switch to
saline, the Io
was transiently enhanced and then diminished. On the other hand, after
Io had developed
in the CN solution, the decrease in
[Na+] of the CN
solution led to a time-dependent enhancement of the Io without a
noticeable delay (n = 3). The
restoration of standard Na+
concentration in the CN solution resulted in a decrease in
Io with
T1/2 of 23 ± 9 s (n = 6). These results indicate
that Na+ loading is not a
prerequisite for the generation of
Io by CN.

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Fig. 5.
Effects of a decrease in
Na+ concentration on
CN-induced Io.
A-C: chart records of a whole
cell current at 52 mV. Same cell. Standard saline was replaced
with standard CN solution (CN) or
Na+-deficient CN solution
[CN Na( )] during indicated periods. In CN
Na( ), 135 mM Na+ was
equimolarly replaced with
N-methyl-D-glucamine.
Downward deflections in C were
truncated currents in response to 100-mV pulse of 50 ms.
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In some experiments, application of CN initially induced an inward,
then an outward current (e.g., Figs. 2 and 5), and this inward current
was abolished by replacement of
Na+ with
N-methyl-D-glucamine.
Thus another possibility is that
Ca2+ flowing through a cation
channel mediates production of the
Io. If this is
indeed the case, then the cation channel involved in the inward current
should be activated for the generation of
Io. We thus
examined effects of CN at hyperpolarized membrane potentials, since the
amplitude of Io
is expected to decrease at such potentials with a decrease in the
driving force for K+ and the
voltage dependence of
Ca2+-dependent
K+ channels. Figure
6A shows
that as the membrane potential was hyperpolarized, production of an
inward current was dominant. At
77 mV, CN induced the inward
current alone over a period of 4 min. Similar results were observed in
all 3 cells held at
77 mV and in 12 of 16 cells at
67 mV
but not in 3 cells at
57 mV nor in 6 cells at
52 mV. In
Fig. 6B, the incidence and latency of
the development of
Io during
4 min
of exposure to CN were plotted against various membrane potentials. The
results suggest that the apparent absence of an inward current at
depolarized membrane potentials is due to masking by the concomitant
generation of Io.
Thus cation channels responsible for the inward current are probably
being activated for the generation of
Io.

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Fig. 6.
Voltage dependence of inward current and
Io in response to
CN. A: chart records of a whole cell
current at 57, 67, and 77 mV. CN was applied
during indicated periods. Same cell.
B: latency ( )
and incidence ( ) of development of
Io are plotted
against membrane potentials. Latencies of <4 min were calculated.
Number of cells examined was 3 at 77, 16 (4, latencies <4 min)
at 67, 3 at 57, and 6 at 52 mV.
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 |
DISCUSSION |
Involvement of
Ca2+-dependent
K+ channels.
In various types of cells, exposure to hypoxia or mitochondrial
inhibitors led to an increase in
[Ca2+]i.
In rabbit glomus cells (2) and mouse sensory neurons (7, 8), such an
increase was thought to be due to release of
Ca2+ from mitochondria, whereas in
hippocampal neurons (1) Ca2+ was
mobilized from IP3-sensitive store
sites. In chromaffin cells, application of CN reversibly induced an
increase in Io
with a latency that depended on a membrane potential. This generation of Io was
apparently not affected by the addition of glucose, but the suppression
of glycolysis with 2-deoxyglucose and iodoacetate resulted in a gradual
development of Io
(16). These results indicate that the production of
Io by CN could be
ascribed to depletion of cellular ATP due to chemical hypoxia. The
CN-induced Io
reversed polarity at membrane potentials close to the equilibrium potential for K+ and was
suppressed by the SK channel blockers apamin and curare but not
by the ATP-sensitive K+
channel blocker glibenclamide. In addition, CN
Io at
55
mV diminished rapidly within 10 s of intracellular access with a 5 mM
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA)-containing pipette solution, regardless of whether the
pipette solution contained ATP (16). These results indicate that
exposure to CN induced a
Ca2+-dependent
K+ current but not an
ATP-sensitive current. Application of 0.1 mM curare and 0.1 µM apamin
suppressed the current at
67 mV by 74 and 40%, respectively,
whereas 0.1 µM charybdotoxin, a BK channel blocker, had no
suppressing action. Thus SK channels may be involved in the current at
67 mV or more negative, but latency for the production of
Io was markedly
shortened at more positive membrane potentials. This finding suggests
that BK channels may also be responsible for the current at such
membrane potentials, since activation of BK channels, but not SK
channels, was enhanced by depolarizations (19), and the presence of the
channel was noted in rat (23) and bovine chromaffin cells (20).
The SK channel was suggested to contribute to resting membrane
potential in rat chromaffin cells (23). Although the present experiment
demonstrated the presence, it would not be a major one in the formation
of resting membrane potential in the guinea pig. As shown in Figs. 1
and 2, membrane conductance examined using the perforated-patch method
exhibited an inward-going rectification between
50 and
130 mV, and at more negative potentials, the conductance
diminished. These properties of membrane conductance were similar to
those noted under conditions in which intracellular Ca2+ was strongly chelated with 5 mM EGTA, and the inward rectification under these conditions was
ascribed to openings of inwardly rectifying K+ channels (11). Consistent with
this notion, addition of 1 mM Ba2+
to saline abolished the rectification (Fig. 2), whereas it suppressed about half the CN-induced
Io at
67
mV.
Putative mechanism for CN-induced outward current.
The present result indicates that
4-min exposure to CN depletes
Ca2+ from caffeine- and
muscarine-sensitive store sites. However, Ca2+ originating from the store
sites was not involved in activation of
Ca2+-dependent
K+ channels at
52 mV or
more negative. First, exposure to
Ca2+-free CN solution did not
induce Io,
whereas it abolished the response to caffeine or muscarine. Second,
Io developed in
response to CN even before the subsequent application of caffeine
evoked the current for a longer duration. Finally and most importantly, generation of the
Io entirely
depended on external Ca2+.
Addition of Ca2+ to
Ca2+-free CN solution produced the
Io without a
delay after exposure to CN for 3-6 min, whereas application of CN
in the presence of Ca2+ began to
produce Io with a
delay of ~1 min at
52 mV. This difference in latency could be
explained by impairment of Ca2+
sequestration and/or extrusion during CN treatment. When CN was applied in the presence of external
Ca2+,
Ca2+ flowing into the cell may be
extruded or sequestered into store sites during the latent period. In
contrast, when Ca2+-handling
mechanisms were blocked by CN,
Ca2+ flowing into the cell would
quickly accumulate above a concentration sufficient for openings of
Ca2+-dependent
K+ channels. In fact, the
Ca2+ pump in store sites was
suppressed during exposure to CN (Fig. 3). This suppression is probably
due to a decrease of cellular ATP content, since addition of 5 mM
glucose to CN solution restored in part the response to caffeine
(unpublished observations). This impairment of ATP-driven extrusion and
sequestration mechanisms alone could not account for CN-induced
Io. First, larger
amounts of Io
were evoked by application of caffeine within 2 min of CN exposure, at
which time Io had
already developed substantially. This result suggests that, during the
early period, at least Ca2+ pump
activity in store sites is not impaired. The larger generation of
Io may be due to
block of mitochondrial uptake of
Ca2+, as was noted in rat
chromaffin cells (9) and sensory neurons (29). Second, application of
cyclopiazonic acid, a specific inhibitor of the
Ca2+ pump in store sites (27), did
not induce Io at
52 mV (15). Thus inhibition of at least the
Ca2+ sequestration mechanism may
not produce an increase in Ca2+
concentratio sufficient for openings of
Ca2+-dependent
K+ channels. On the basis of these
results, Ca2+ influx seems to be
enhanced during CN exposure.
There are at least two possibilities to account for the CN-induced
Ca2+ influx. One possibility is
the reversed mode of
Na+/Ca2+
exchange. A decrease in ATP contents would be expected to result in
suppression of
Na+-K+-ATPase
and the subsequent accumulation of
Na+ beneath the plasma membrane.
This accumulation might be sufficient for
Na+/Ca2+
exchange to function in the reverse mode. However, this possibility is
questionable. First, application of 20 µM ouabain for 4 min did not
produce Io,
whereas that of CN consistently did so in the same cells. Second,
Io did not
develop when extracellular Na+
concentration decreased from 140 to 5 mM (unpublished observations). Furthermore, application of CN under
Na+-deficient conditions produced
Io with a latency
comparable to that for the current evoked under standard conditions.
This result suggests that Na+
loading is not a prerequisite for the CN-induced
Io and that Ca2+-ATPase in the plasma membrane
is mainly responsible for Ca2+
extrusion and
Na+/Ca2+
exchange contributes little in intact cells and during the latent period (cf. Ref. 29). In contrast, under metabolically suppressed conditions, Ca2+ may be extruded
mainly by
Na+/Ca2+
exchange. Thus the suppression or reversed operation of
Na+/Ca2+
exchange by replacement of Na+
with
N-methyl-D-glucamine
enhanced the CN-induced
Io with no substantial delay.
The second possibility regarding an increase in
Ca2+ concentration is a
Ca2+ influx through
Ca2+-permeable channels.
Application of CN initially produced inward and then outward currents
at membrane potentials more positive than
67 mV, whereas at a
more negative potential it generated an inward current alone during the
entire period. These results suggest that, at depolarized membrane
potentials, the inward current may be masked by concomitant generation
of Io. The inward
current elicited at
52 mV was apparently abolished by
substitution of N-methyl-D-glucamine
for 135 mM Na+ in CN solution,
suggesting that the current is due to activation of
Na+-permeable channels. This
CN-activated cation channel might have a substantial permeability to
Ca2+. To determine whether the
second possibility is tenable,
[Ca2+]i
has to be measured under voltage clamp conditions and compared with
amplitudes of CN-induced inward currents.
 |
ACKNOWLEDGEMENTS |
This study was supported by a Grant-in-Aid from the Ministry of
Education, Science, Sports, and Culture of Japan.
 |
FOOTNOTES |
Address reprint requests to M. Inoue.
Received 20 February 1997; accepted in final form 3 September
1997.
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REFERENCES |
1.
Belousov, A. B.,
J.-M. Godfraind,
and
K. Krnjevi
.
Internal Ca2+ stores involved in anoxic responses of rat hippocampal neurons.
J. Physiol. Lond.
486:
547-556,
1995[Abstract].
2.
Biscoe, T. J.,
and
M. R. Duchen.
Cellular basis of transduction in carotid chemoreceptors.
Am. J. Physiol.
258 (Lung Cell. Mol. Physiol. 2):
L271-L278,
1990[Abstract/Free Full Text].
3.
Blatz, A. L.,
and
K. L. Magleby.
Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle.
Nature
323:
718-720,
1986[Medline].
4.
Bülbring, E.,
J. H. Burn,
and
F. J. de Elio.
The secretion of adrenaline from the perfused suprarenal gland.
J. Physiol. Lond.
107:
222-232,
1948.
5.
Choi, D. W.
Cerebral hypoxia: some new approaches and unanswered questions.
J. Neurosci.
10:
2493-2501,
1990[Medline].
6.
Cryer, P. E.
Physiology and pathophysiology of the human sympathoadrenal neuroendocrine system.
N. Engl. J. Med.
303:
436-444,
1980[Medline].
7.
Duchen, M. R.
Effects of metabolic inhibition on the membrane properties of isolated mouse primary sensory neurones.
J. Physiol. Lond.
424:
387-409,
1990[Abstract].
8.
Duchen, M. R.,
M. Valdeolmillos,
S. C. O'Neill,
and
D. A. Eisner.
Effects of metabolic blockade on the regulation of intracellular calcium in dissociated mouse sensory neurones.
J. Physiol. Lond.
424:
411-426,
1990[Abstract].
9.
Herrington, J.,
Y. B. Park,
D. F. Babcock,
and
B. Hille.
Dominant role of mitochondria in clearance of large Ca2+ loads from rat adrenal chromaffin cells.
Neuron
16:
219-228,
1996[Medline].
10.
Horn, R.,
and
A. Marty.
Muscarinic activation of ionic currents measured by a new whole-cell recording method.
J. Gen. Physiol.
92:
145-159,
1988[Abstract].
11.
Inoue, M.,
and
I. Imanaga.
G protein-mediated inhibition of inwardly rectifying K+ channels in guinea pig chromaffin cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C946-C956,
1993[Abstract/Free Full Text].
12.
Inoue, M.,
and
I. Imanaga.
Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells.
Br. J. Pharmacol.
114:
419-427,
1995[Abstract].
13.
Inoue, M.,
and
H. Kuriyama.
Muscarine induces two distinct current responses in adrenal chromaffin cells of the guinea-pig.
Jpn. J. Physiol.
40:
679-691,
1990[Medline].
14.
Inoue, M.,
K. Ogawa,
N. Fujishiro,
A. Yano,
and
I. Imanaga.
Role and source of ATP for activation of nonselective cation channels by AlF complex in guinea pig chromaffin cells.
J. Membr. Biol.
154:
183-195,
1996[Medline].
15.
Inoue, M.,
Y. Sakamoto,
and
I. Imanaga.
Phosphatidylinositol hydrolysis is involved in production of Ca2+-dependent currents, but not non-selective cation currents, by muscarine in chromaffin cells.
Eur. J. Pharmacol.
276:
123-129,
1995[Medline].
16.
Inoue, M.,
Y. Sakamoto,
A. Yano,
and
I. Imanaga.
Cyanide suppression of inwardly rectifying K+ channels in guinea pig chromaffin cells involves dephosphorylation.
Am. J. Physiol.
273 (Cell Physiol. 42):
C137-C147,
1997[Abstract/Free Full Text].
17.
Kaplin, A. I.,
S. H. Snyder,
and
D. J. Linden.
Reduced nicotinamide adenine dinucleotide-selective stimulation of inositol 1,4,5-trisphosphate receptors mediates hypoxic mobilization of calcium.
J. Neurosci.
16:
2002-2011,
1996[Abstract].
18.
Kiang, J. G.,
and
R. C. Smallridge.
Sodium cyanide increases cytosolic free calcium: evidence for activation of the reversed mode of the Na+/Ca2+ exchanger and Ca2+ mobilization from inositol trisphosphate-insensitive pools.
Toxicol. Appl. Pharmacol.
127:
173-181,
1994[Medline].
19.
Lang, D. G.,
and
A. K. Ritchie.
Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line.
Pflügers Arch.
410:
614-622,
1987[Medline].
20.
Marty, A.
Ca-dependent K channels with large unitary conductance in chromaffin cell membrane.
Nature
291:
497-500,
1981[Medline].
21.
Miller, C.,
E. Moczydlowski,
R. Latorre,
and
M. Phillips.
Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle.
Nature
313:
316-318,
1985[Medline].
22.
Montoro, R. J.,
J. Ureña,
R. Fernández-Chacón,
G. A. de Toledo,
and
J. López-Barneo.
Oxygen sensing by ion channels and chemotransduction in single glomus cells.
J. Gen. Physiol.
107:
133-143,
1996[Abstract].
23.
Neely, A.,
and
C. J. Lingle.
Two components of calcium-activated potassium current in rat adrenal chromaffin cells.
J. Physiol. Lond.
453:
97-131,
1992[Abstract].
24.
Nomi, M.,
and
K. Kuba.
(+)-Tubocurarine blocks the Ca2+-dependent K+ channel of the bullfrog sympathetic ganglion cell.
Brain Res.
301:
146-148,
1984[Medline].
25.
Orrenius, S.,
D. J. McConkey,
G. Bellomo,
and
P. Nicotera.
Role of Ca2+ in toxic cell killing.
Trends Pharmacol.
10:
281-285,
1989.[Medline]
26.
Robertson, D. W.,
and
M. I. Steinberg.
Potassium channel modulators: scientific applications and therapeutic promise.
J. Med. Chem.
33:
1529-1541,
1990[Medline].
27.
Seidler, N. W.,
I. Jona,
M. Vegh,
and
A. Martonosi.
Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum.
J. Biol. Chem.
264:
17816-17823,
1989[Abstract/Free Full Text].
28.
Unsicker, K.
The chromaffin cell: paradigm in cell, developmental and growth factor biology.
J. Anat.
183:
207-221,
1993[Medline].
29.
Werth, J. L.,
and
S. A. Thayer.
Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons.
J. Neurosci.
14:
348-356,
1994[Abstract].
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