Changes in intracellular Ca2+
concentration induced by L-type
Ca2+ channel current in guinea pig
gastric myocytes
Sung Joon
Kim1,
Seung Cheol
Ahn1,
Jin Kyung
Kim2,
Young Chul
Kim1,
Insuk
So1, and
Ki Whan
Kim1
1 Department of Physiology and
Biophysics, Seoul National University College of Medicine, and
2 Department of Anesthesiology,
Seoul National University Hospital, Seoul 110-799, Korea
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ABSTRACT |
We investigated
the relationship between voltage-operated
Ca2+ channel current and the
corresponding intracellular Ca2+
concentration
([Ca2+]i)
change (Ca2+ transient) in guinea
pig gastric myocytes. Fluorescence microspectroscopy was combined with
conventional whole cell patch-clamp technique, and fura 2 (80 µM) was
added to CsCl-rich pipette solution. Step depolarization to 0 mV
induced inward Ca2+ current
(ICa) and
concomitantly raised
[Ca2+]i.
Both responses were suppressed by nicardipine, an L-type
Ca2+ channel blocker, and the
voltage dependence of Ca2+
transient was similar to the current-voltage relation of
ICa. When pulse
duration was increased by up to 900 ms, peak
Ca2+ transient increased and
reached a steady state when stimulation was for longer. The calculated
fast Ca2+ buffering capacity
(B value), determined as the ratio of
the time integral of
ICa divided by
the amplitude of Ca2+ transient,
was not significantly increased after depletion of Ca2+ stores by the cyclic
application of caffeine (10 mM) in the presence of ryanodine (4 µM).
The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmic
reticulum Ca2+-ATPase inhibitor,
decreased B value by ~20% in a
reversible manner. When KCl pipette solution was used,
Ca2+-activated
K+ current
[IK(Ca)]
was also recorded during step depolarization. CPA sensitively
suppressed the initial peak and oscillations of IK(Ca) with
irregular effects on Ca2+
transients. The above results suggest that, in guinea pig gastric myocyte, Ca2+ transient is tightly
coupled to ICa
during depolarization, and global
[Ca2+]i
is not significantly affected by
Ca2+-induced
Ca2+ release from sarcoplasmic
reticulum during depolarization.
smooth muscle; sarcoplasmic reticulum; calcium buffer
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INTRODUCTION |
AS IN SKELETAL or cardiac muscle, intracellular
Ca2+ regulates smooth muscle
contraction. The level of intracellular
Ca2+ concentration
([Ca2+]i)
is determined by Ca2+ influx,
Ca2+ release from sarcoplasmic
reticulum (SR), and cytosolic Ca2+
buffering and removal mechanisms (16, 20, 29). In smooth muscle, the
role of Ca2+ release from SR is
thought to be largely due to agonist-induced stimulation especially
through the phospholipase C-inositol 1,4,5-trisphosphate cascade (16,
29). In addition, the presence of another
Ca2+ release mechanism,
Ca2+-induced
Ca2+ release (CICR), has also been
demonstrated in experiments performed to investigate the effects of
agonists and pharmacological intervention using caffeine or ryanodine
on
[Ca2+]i
(10, 13, 16, 29, 34).
Gastric smooth muscle shows rhythmic variations in membrane potential
in the form of slow waves, the configuration of which involves three
phases: 1) upstroke depolarization,
2) plateau phase, and
3) repolarization. The
excitation-contraction (E-C) coupling mostly occurs during the upstroke
depolarization and plateau phase of depolarization (31, 33). Whole cell
patch-clamp study involving smooth muscle cells shows
depolarization-activated Ca2+
inward current
(ICa) and
dihydropyridine-sensitive suppression of
ICa, which
suggests the importance of L-type
Ca2+ channels as a
Ca2+ influx pathway during
depolarization (20, 33).
It is well known that in cardiac myocyte, the
Ca2+ entry due to depolarization
triggers CICR from SR (4, 7, 17). Although Ca2+ release from cardiac SR is
both graded and regenerative, depending on experimental conditions
(e.g., the state of Ca2+ load in
SR; Ref. 18), it has been shown that the physiological macroscopic
increase of
[Ca2+]i
during depolarization (Ca2+
transients) is graded by the recruitment of active release clusters (17) or Ca2+ sparks, a
nonpropagating event in which Ca2+
is released from functional SR (4). In smooth muscle, the significance
of CICR in depolarization-evoked
Ca2+ transient is controversial,
depending on the tissue preparations (8, 10, 11, 13, 16, 21). Although
Ca2+ sparks are also observed in
smooth muscle cells using confocal microscopy (24, 26), it appears that
Ca2+ spark-induced transient
activations of Ca2+-activated
K+ current
[IK(Ca);
spontaneous transient outward currents (STOCs); Refs. 2, 24]
exert a tonic hyperpolarizing and inhibitory influence in vascular
smooth muscle (26). STOCs are also observed in guinea pig antral
myocytes under the whole cell patch clamp at a relatively low
Ca2+-buffered condition
[e.g., 0.1 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA) in the pipette solution]; in addition, the
application of caffeine or acetylcholine induces a large transient
increase of
IK(Ca), with
subsequent inhibition of STOCs (22), as in another report (2).
Electron microscopy has revealed the development of SR in various kinds
of smooth muscle cells (6, 9). They occupy 2-3% of cell volume in
phasic smooth muscle (vas deferens, portal vein, taenia coli) cells and
are distributed around the cell periphery as well as in deep cytoplasm
(6, 29). We also observed the presence of peripheral SR in guinea pig
gastric myocyte, but the exact volume was not estimated (unpublished
observations). The sarcolemma of smooth muscle shows well-developed
surface vesicles (caveolae), which in the case of guinea pig antral
myocytes increase the cell perimeter by a factor of 2.6 (unpublished
observations). It is known that the longitudinal tubules of peripheral
SR run between groups of caveolae, from which they are separated by a narrow gap (~10 nm; Ref. 6); the presumed accumulation of
Ca2+ in this narrow space has
evoked various hypotheses concerning the physiological roles of SR in
smooth muscle. These include 1) the
activation of CICR from SR by depolarization-induced
Ca2+ influx (E-C coupling; Ref. 6)
and 2) the sequestration of influx
Ca2+ by sarco(endo)plasmic
reticulum Ca2+-ATPase (SERCA),
followed by the vectorial release of
Ca2+ into the sarcolemma and
subsequent facilitation of the extrusion of
Ca2+ by
Na+/Ca2+
exchange or plasma membrane
Ca2+-ATPase (PMCA) (superficial
buffer barrier; Ref. 32). In relation to the latter hypothesis, it has
also been suggested that SR spontaneously and preferentially releases
stored Ca2+ toward the sarcolemma
for subsequent extrusion from the cell without increasing global
[Ca2+]i
(30).
This study was undertaken to determine the role of SR during
depolarization-induced Ca2+
transient in guinea pig gastric myocytes. Is it a source of activator Ca2+ and/or a sink? The
nature of the relationship between
ICa and [Ca2+]i
in canine gastric myocytes has been reported (33), but little information is available concerning the role of SR in
Ca2+ transient of gastrointestinal
myocytes. In amphibian gastric myocytes, tight regulation of
[Ca2+]i
increase by ICa
was reported (1), and the insignificant effect of ryanodine on
ICa-related
[Ca2+]i
increase has been briefly described; the authors deny the participation of CICR during depolarization-induced
Ca2+ increase (15). It cannot be
assumed, however, that in an experiment involving mammalian myocytes
the result would have been the same, so we attempted to characterize
the depolarization-induced Ca2+
transient in guinea pig antral circular myocytes and the role of SR in
the Ca2+ transient.
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MATERIALS AND METHODS |
Cell dissociation.
Guinea pigs of either sex weighing 300-350 g were exsanguinated
after stunning. The antral part of the stomach was cut, and the mucosal
layer was separated from the muscle layers in
Ca2+-free physiological salt
solution (PSS). The circular muscle layer was dissected from the
longitudinal layer using fine scissors and was cut into small segments
(2 × 3 mm). These were incubated in modified Kraft-Brühe
(K-B) medium (19) for 30 min at 4°C. They were then incubated for
15-25 min at 35°C in digestion medium [Ca2+-free PSS containing
0.15% collagenase (Wako or Sigma type IA), 0.05% dithioerythritol,
0.1% trypsin inhibitor, and 0.2% bovine serum albumin]. After
digestion, the supernatant was discarded, and the softened muscle
segments were returned to the modified K-B medium. Single cells were
dispersed by gentle agitation with a wide-bore glass pipette. Isolated
gastric myocytes were kept in this medium at 4°C until use. All
experiments were carried out within 12 h of harvesting cells, and the
bath solution was warmed by a circulating water jacket just before
perfusing the experimental bath. The temperature of the bath solution
was often monitored by a digital thermometer (model Gamma CS, Noronix
Electronics) and was maintained between 33 and 35°C.
Electrophysiological recordings.
Membrane currents were measured by employing the patch-clamp technique
in whole cell configuration, using an Axopatch 1D patch-clamp amplifier
(Axon Instruments). pCLAMP software v.5.6.2 and Digidata-1200 (both
from Axon Instruments) were used for the acquisition of data and
application of command pulses. The data were filtered at 5 kHz,
displayed on a computer monitor, and analyzed using pCLAMP and Origin
(Microcal Software).
Fluorescence measurements.
[Ca2+]i
was measured with a microfluorometer consisting of an inverted
fluorescence microscope (Diaphot 300, Nikon) with a dry-type fluorescence objective lens, a photomultiplier tube (type R 1527, Hamamatsu), and a PTI deltascan illuminator (Photon Technology International). Light was provided by a 75-W xenon lamp (Ushino), and,
to control excitation frequency, a chopper wheel alternated the light
path to monochromators (340 and 380 nm) with a frequency of 10 or 50 Hz. A 425-nm short-pass barrier filter was placed in the excitation
path to reduce background fluorescence. A short-pass dichroic mirror
passed emission light of <570 nm onto the photomultiplier tube, and
intensity at 510 nm was measured. A mechanical image mask was placed in
the emission path, thus limiting measurement to a single cell.
Fura 2 loading and estimation of
[Ca2+]i.
K5-fura 2 was dissolved in
distilled water to make a 10 mM stock solution and was added to the
pipette solution to make a final concentration of 80 µM. After a
whole cell configuration was achieved by rupture of the membrane patch,
fura 2 was loaded for 4-5 min before the start of the experiment.
[Ca2+]i
was calculated using the following equation (14)
where
Kd is the
effective dissociation constant of fura 2 and
b is the ratio of fluorescence signals
at 380 nm
(Sf 2/Sb2) without Ca2+
(Sf 2) and with saturating
Ca2+
(Sb2).
Rmin represents a ratio of 340/380
in the absence of Ca2+, and
Rmax represents this ratio when
Ca2+ concentration is at
saturation point. Rmin was
obtained by perfusing the cells with 10 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-containing solution (0.4 ± 0.01, n = 4), and
Rmax was obtained by stepping the
membrane potential to
200 to
250 mV, which caused cell
membranes to leak. This procedure induced a large increase in the
fluorescence ratio (R), which stabilized within 1-2 min. The R
value at 2 min after the hyperpolarizing step was taken as
Rmax, which was typically located
between 8 and 9 (8.6 ± 0.26, n = 15). The value of
Kd × b was estimated by perfusing the cells
with a pipette solution containing 10 mM BAPTA-buffered solution, where
Ca2+ concentration was clamped at
100 nM according to a computer program (28). Background fluorescence
was determined in cell-attached configuration and was omitted from the
respective wavelength.
An average cell volume of 4.2 pl was estimated from a typical cell
length and width of 250 and 8 µm, respectively, assuming that the
cell consisted of two cones connected base to base. The above geometric
assumption might be an overestimate, since each end of a myocyte is
usually very fine, and cytosolic space occupied by organelles should be
excluded. The reported volume of a visceral myocyte is usually between
2.5 and 3.5 pl (9), so the cell volume calculated was arbitrarily
reduced by 30%, and the remaining 70% (2.9 pl) was considered to be
the effective diffusible volume for fura 2.
Solutions and drugs.
Ca2+-free PSS used in the
isolation of cells contained (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 5 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and pH was adjusted to 7.4 with NaOH. For the experiment, 4 mM CaCl2 was added to the above
solution. Ca2+ at a concentration
higher than the usual physiological environment was used to enhance the
loading of SR with Ca2+ during the
experiment. Modified K-B medium (19) contained (in mM) 50 L-glutamate, 50 KCl, 20 taurine,
20 KH2PO4,
3 MgCl2, 10 glucose, 10 HEPES, and
0.5 EGTA, and pH was adjusted to 7.3 with KOH. The pipette solution
consisted of (in mM) 130 CsCl, 2.5 Na2ATP, 0.2 tris-GTP, 4 MgCl2, 10 HEPES, and 0.08 K5-fura 2, and pH was adjusted to
7.2 with CsOH. To record
IK(Ca),
K+-rich pipette solution was made
by replacing CsCl with KCl, and pH was adjusted with KOH. To apply
drugs, the experimental chamber (~0.1 ml) was superfused by gravity
at a rate of 2 ml/min. Significant differences were detected using
Student's unpaired t-test
(P < 0.05).
K5-fura 2 was purchased from
Molecular Probes (Eugene, OR). BAY K 8644 was purchased from Research
Biochemical International (Natick, MA). All other drugs were purchased
from Sigma (St. Louis, MO).
 |
RESULTS |
Ca2+
transient induced by the activation of L-type
Ca2+ channel
current.
In guinea pig gastric myocytes, held under voltage clamp at
80
mV, the resting
[Ca2+]i
was 102 ± 6.3 nM (n = 42). In Fig.
1, a single step pulse from
80 to 0 mV for 400 ms (a) or 150 ms
(c) evoked inward
ICa and simultaneously increased
[Ca2+]i
(bottom trace of Fig. 1,
Aa and
B).
ICa peaked within
10 ms and then decayed while
[Ca2+]i
continued to rise during the pulse period (see also Fig.
4A). Upon repolarization to
80 mV,
[Ca2+]i
slowly recovered to its resting level. Recovery was usually completed
within 15-20 s, although in some cells, almost 30 s were needed.
As a result of this slow recovery, repetitive stimulation with a train
of step pulses (1 or 1.6 Hz, in Fig. 1B,
b and
d, respectively) induced superimposed
increase of
[Ca2+]i.
Individual increase induced by repetitive stimulation became progressively smaller, however, and finally reached a ceiling level
after four to five pulses (Fig. 1B,
b and
d). Inward current was also
decreased by repetitive stimulation (Fig.
1Ab). This might have been due to
the short interpulse period, which was insufficient for complete
recovery from inactivation and/or
Ca2+-induced inactivation of
ICa. The
depolarizing pulse of 400 ms (Fig.
1Ba) induced larger
Ca2+ transient than 150 ms (Fig.
1Bc), and the peak concentration was
similar to those attained by trains of 150-ms pulses at 1 Hz (Fig.
1Bb) or at 1.6 Hz (Fig.
1Bd). A faster rate of
Ca2+ removal at a higher
[Ca2+]i
(1) as well as decreased
ICa seemed to
determine the highest ceiling level of
[Ca2+]i
induced by repetitive stimulation.

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Fig. 1.
Depolarization-evoked Ca2+ current
(ICa) and
Ca2+ transient. Depolarizing
single pulse (a and
c, 400 and 150 ms, respectively) or 7 repetitive pulses (b, 1 Hz) from
80 to 0 mV were applied to a gastric myocyte under whole cell
voltage clamp.
ICa and transient
changes of intracellular Ca2+
concentration
([Ca2+]i)
in response to step depolarizations
(Ca2+ transient) are shown in
A and
B with different time scales. Fourth
Ca2+ transient in
B
(d) is a response to repetitive
pulses with higher frequency (150 ms, 1.3 Hz, 16 pulses). In
A, a part of first
Ca2+ transient is plotted below
corresponding ICa
with expanded time scale (a).
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Depolarization-induced
ICa and
Ca2+ transients were concomitantly
enhanced by BAY K 8644 (0.5 µM, n = 3) and almost completely suppressed by nicardipine (2 µM,
n = 4), a dihydropyridine
Ca2+ channel agonist and
antagonist, respectively. Figure
2A shows a
typical response to the above agents. To observe the voltage dependence
of Ca2+ transient, various levels
of depolarization were applied (Fig. 2B). Measurable changes in
[Ca2+]i
required a depolarization of above
40 mV; on further
depolarization, up to 0 mV, the amplitude of
Ca2+ transient increased. The
voltage dependence of Ca2+
transient was bell-shaped, and this was similar to the well-known current-to-voltage relation of L-type
Ca2+ channel current (Fig.
2B). Under conditions of high
depolarization (above +20 mV), however, the voltage dependence of
Ca2+ transient did not exactly
overlap with the voltage dependence of
ICa. The results
shown in Figs. 1 and 2 suggest that depolarization-induced Ca2+ transient is closely related
to Ca2+ influx through
dihydropyridine-sensitive (L-type) voltage-operated Ca2+ channels
(VOCCs).

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Fig. 2.
Effects of Ca2+ channel agonist
and antagonist on
ICa and
Ca2+ transients.
A: depolarizing pulses (0 mV, 800 ms,
solid circles) were applied repetitively (0.05 Hz). Bath-applied BAY K
8644 (0.5 µM) increased size of inward current and peak amplitude of
Ca2+ transient (open bar and open
squares). Same cell was successively perfused with
nicardipine-containing solution (solid bar and solid squares), which
almost completely suppressed both inward current and
Ca2+ transient.
B: depolarization level was varied
(from 50 to +50 mV) to obtain current-voltage relationship
(solid square) and
[Ca2+]i-voltage
relationship (open circle) in gastric myocyte. In each cell, peak
values of inward current and peak amplitudes of
Ca2+ transient were normalized to
values obtained at 0 mV, and averaged values were plotted
(n = 11).
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Relation between the amount of
Ca2+ influx and
measured
[Ca2+]i.
To elucidate the relation between the amount of
Ca2+ influx and the amplitude of
Ca2+ transient, we changed the
duration of the depolarizing pulse (0 mV) from 5 to 1,300 ms (Fig.
3). A very short depolarization (5 ms) did
not induce a discernible change in the fluorescence ratio, whereas the
rapidly activating portion of inward current was noticeable (data not
shown). A pulse duration of 15 ms induced a noticeable change in
[Ca2+]i,
and as pulse duration increased, so did the amplitude of
Ca2+ transient. When the duration
of the depolarizing pulse was longer than 500 ms,
[Ca2+]i
reached its peak before this ended. In Fig.
3Ba, responses in each cell were
normalized to the value obtained by depolarization lasting 900 ms, and
averaged results from five cells were drawn against pulse duration.

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Fig. 3.
Pulse duration dependence of Ca2+
transients. A: pulse duration was
successively increased from 15 to 1,300 ms, and corresponding
Ca2+ transients from 1 representitive myocyte are shown. B: 5 cells were tested as shown in A, and
in each myocyte, amplitudes of
Ca2+ transient were normalized to
one obtained by depolarization of 900 ms
(a). From results shown in
a, calculated increase of
[Ca2+]i
by time-accumulated
ICa was divided
by amplitude of Ca2+ transient
(B value). In
b, calculated
B values were plotted against pulse
duration on a semi-logarithmic scale.
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The expected increase of
[Ca2+]i
due solely to ICa
could be calculated from the following equation
where

ICadt
is a time integral of the area under the curve of
Ca2+ current with reference to the
level of the holding current, F is the
Faraday constant, and V is the expected diffusible space of a single
myocyte (2.9 pl); the result was divided by the charge of
Ca2+. The calculated increase in
[Ca2+]i
induced by Ca2+ current was always
~200 (range 100-300) times greater than the measured change of
[Ca2+]i
during the pulse period. These ratios
(B values) were considered to
represent the Ca2+ buffering
capacity of single cells (8, 11, 21) and were plotted against pulse
duration (Fig. 3Bb; see also Table
1). Although
B values tended to decrease slightly
when pulse duration was increased from 15 to 100 ms, the difference was
not statistically significant.
By comparing the time course of the measured
[Ca2+]i
change with the time integral of
Ca2+ influx expected to accumulate
in a constant cell volume, we also examined the relationship between
the Ca2+ influx and the
Ca2+ transient. Figure
4 shows typical results where the time
courses of
[Ca2+]i
rise and the time integrals of
ICa with a pulse
duration of 400 ms were compared on the same time scale. Figure
4A shows three sequential responses
obtained from one cell with a pulse interval of 30 s, and traces in
Fig. 4B were from three different
cells. In each trace, the enveloping line of time-integrated
ICa and measured
[Ca2+]i
were plotted together. The general configurations appear that measured
and calculated
[Ca2+]i
increase were proportional and, in some cells, closely coincided (Fig.
4Bc). However, we could not be sure
that during the pulse period both time courses were always very similar
to each other. Even in the same cell, time course relations were
slightly variable during an experiment (Fig.
4A).

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Fig. 4.
Comparison between time course of
[Ca2+]i
increase and time-accumulated
ICa.
A: in same cell, repetitive
depolarizing pulses (0 mV, 400 ms) were applied with 30-s interval to
evoke ICa
(top traces), and corresponding
Ca2+ transients are plotted
together with same time scale (bottom
traces). Time-accumulated
ICa is
overplotted (smooth curves) on
Ca2+ transient trace with
different concentration scale (right vertical axis in lower traces).
B: traces for measured and calculated
(smooth curves)
[Ca2+]i
were obtained from 3 different cells to be compared.
Ca2+ currents are not shown
here.
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Effects of depleting CICR pool on
Ca2+ transient.
The results shown in Figs. 3 and 4 indicate that regenerative CICR was
not induced by Ca2+ influx during
depolarization. It was still possible, however, that localized CICR by
accumulated Ca2+ influx
contributed to Ca2+ transient. To
investigate this possibility, we examined the effects of caffeine and
ryanodine on the relationship between
Ca2+ transient and
ICa
(B value). Both drugs are known to
suppress the CICR process, although by different mechanisms; caffeine
releases Ca2+ from intracellular
stores by increasing the sensitivity of ryanodine receptor (RyR)
channels to Ca2+ while ryanodine
locks RyRs in a subconductance state at a micromolar range of
concentration and blocks RyRs at higher concentrations (10-500
µM; Refs. 27, 34).
Because it is known that ryanodine binds with RyR only when the gates
are open and locks them in an open state (27), we repetitively treated
the cell with caffeine in the presence of 4 µM ryanodine. We expected
that, by this procedure, the Ca2+
store for CICR would be depleted, and the further application of
caffeine would not be effective (16). As shown in Fig.
5A, short
application of 10 mM caffeine induced a transient
[Ca2+]i
increase, although this was <1 µM, which was different from other
reports (10-13). The relatively small amplitude of
caffeine-induced Ca2+ transient
might be due to the mode of drug application; instead of using the
rapid solution exchange system or puff ejection, the bath solution
containing caffeine was continuously superfused by gravity (12). After
confirming that the second or third application of caffeine was
ineffective, step depolarization was applied, and the resultant
Ca2+ transient was compared with
the control. B values were obtained by
dividing the calculated increase by measured increase of
[Ca2+]i
at 350-400 ms after the start of step depolarization. After this
treatment, both
ICa and
Ca2+ transient were smaller than
the initial control, and the B values were often increased (Fig. 5A). The
overall change in B value was,
however, statistically insignificant (113 ± 5.3%,
P > 0.05, n = 9).

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Fig. 5.
Effects of sarcoplasmic reticulum depletion on
ICa and
Ca2+ transient.
A: depolarizing pulses (0 mV, 400 ms)
were applied (arrows in bottom
trace) before and after cyclic application of
caffeine (10 mM) in presence of ryanodine (4 µM). Initial application
of caffeine induced transient increase of
[Ca2+]i,
whereas second application had no effect. Both amplitudes of
ICa
(top traces) and correspoding
Ca2+ transient were decreased
after such pharmacological treatment
(b).
B: B
values were obtained from 2 different groups (13 cells for each) to
which a high concentration (20 µM) of ryanodine was added (solid bar)
or not added (open bar) into pipette solution. Difference of
B values from 2 groups was not
significant (P > 0.05).
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In other cells, to directly block RyRs, a higher concentration (20 µM) of ryanodine was added to the pipette solution (27), and
B values obtained in this condition
were compared with those from control cells of the same aliquot (Fig.
5B). The averaged B value of the ryanodine-treated group
(176 ± 15.9, n = 13) appeared higher than that of the control (151 ± 13.3, n = 13), but the difference was not
significant (P > 0.05).
Effects of cyclopiazonic acid on ICa and
Ca2+ transient.
The results shown in Fig. 5 suggest that, during depolarization, the
contribution of CICR to the total increase of
[Ca2+]i
was insignificant. In the following experiment, we therefore examined
the role of SR as a buffer barrier for
Ca2+ influx, using cyclopiazonic
acid (CPA), a SERCA inhibitor (35). ICa,
Ca2+ transient, and the
B values were measured and compared
before and after treatment with 10 µM CPA. Although the amplitude of ICa decreased
slightly during this treatment (86 ± 2.7 and 88 ± 2.1% of control
ICa in the
presence and after washout of CPA, respectively,
n = 10), we were able to record the
effect of CPA in three cells without significant change of
ICa as shown in
Fig. 6. In Fig.
6B, the amplitude of
Ca2+ transient was higher in the
presence of CPA, whereas the amplitude of
ICa was not
changed (Fig. 6A). In nine cells
examined, 1 min of treatment with CPA decreased the
B values (80 ± 3.3% of control, P < 0.05), and this change was
reversed by washout of CPA (93 ± 2.5% of control).

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Fig. 6.
Effects of cyclopiazonic acid (CPA) on
ICa and
Ca2+ transient. Depolarizing step
pulses were repetitively applied before
(a), during
(b), and after
(c) treatment with CPA (10 µM).
Amplitudes of Ca2+ transients were
increased in presence of CPA (B),
whereas amplitudes of
ICa were not
enhanced (A). Basal
[Ca2+]i
was slightly increased by CPA and reversed by washout. Bar graph in
C summarizes effect of CPA on
B values in 9 different cells.
* B value was significantly
(P < 0.05) decreased (80 ± 3.3%
of control) by CPA and reversed by washout (93 ± 2.5% of
control).
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Effects of CPA on IK(Ca) and
Ca2+ transient.
The inhibitory effects of internal
Cs+ on depolarization-induced
Ca2+ transient and
depolarization-induced phasic contractions were reported in guinea pig
and rabbit cardiac myocytes, respectively (18, 23). Authors of both
reports suggested that inhibition of SR
Ca2+ release by dialyzed
Cs+ can explain the above
inhibitory effects. Cs+ dialysis
is a commonly used technique to record
ICa in
patch-clamp study, and so it was used in the present study. Possible
inhibitory effect of internal Cs+
on Ca2+ release might have
hindered the contribution from CICR to the Ca2+ transient in our results
(Figs. 4 and 5) as was shown in another smooth muscle preparation
(guinea pig coronary artery; Ref. 11). In ensuing experiments, we
therefore replaced the CsCl pipette solution with one that was
K+ rich and recorded
IK(Ca); we
assumed that this very sensitively reflects the subsarcolemmal
[Ca2+]i
([Ca2+]i,sl)
(12, 24, 26, 30).
Figure 7 shows the responses of two cells
to 3.6-s depolarization from
80 to 0 mV. This caused an abrupt
rise of outward current
[IK(Ca)]
that peaked to a nanoampere range within 100 ms and rapidly decayed to
a sustained level during depolarization, at which time, a number of
transient oscillations of outward currents often appeared. Although not
shown here, we confirmed that such large initial or oscillatory
transient outward currents are sensitively blocked by low concentration
(<1 mM) of tetraethylammonium, an inhibitor of maxi
K+ channel at this concentration
(3), in the bath solution, or by high concentration of
Ca2+ buffer (10 mM EGTA or BAPTA)
in the pipette solution.

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Fig. 7.
Relations between Ca2+-activated
K+ current
[IK(Ca)]
and Ca2+ transient.
A: with
K+-rich solution in pipette, step
depolarization (3.6 s) to 0 mV induced outward current with initial
transient peak and sustained current. Irregular small oscillations were
superimposed on sustained outward current level. Four current traces
were obtained by repetitive depolarization with 30-s interval in same
cell. B: in this myocyte, initial peak
current decayed very rapidly to a plateaulike sustained current with
higher amplitude and decreased again during depolarization period.
|
|
The amplitude of initial outward peak current, the rate of decay, and
the appearance of transient oscillations varied within individual
cells, and even in a single myocyte these properties varied with time.
In some cells, the amplitude of
IK(Ca) increased during the initial train of step depolarizations at 30-s intervals which started 3 or 4 min after rupture of the patch membrane and were
then sustained for a while or slowly decreased (Fig.
7A). In other cells, large
IK(Ca) was
obtained at the start of the pulse train, where the peak outward
current decayed very rapidly to a plateau level before decreasing
rather slowly during depolarization (Fig.
7B).
IK(Ca) recorded
in this myocyte did not confidently correlate with the peak amplitude
of Ca2+ transient, which was
simultaneously measured by fura 2 fluorescence ratio. In addition, as
the configuration of
IK(Ca) from each
cell could not be definitely categorized and varied with time, we were unable to perform the experimental protocol involving the cyclic application of caffeine in the presence of ryanodine, which took at
least 5-10 min. Moreover, a possible rundown or decrease of ICa during the
above treatment cast doubt on the interpretation of the change in
IK(Ca) and
Ca2+ transient. We therefore
observed the effect of short application (2-3 min) of CPA after
attaining sustained responses of
IK(Ca) and
Ca2+ transient. Figure
8 shows typical effects of CPA on
IK(Ca) and Ca2+ transient in three different
cells. Step depolarizations the same as in Fig. 7 were repetitively
applied every 30 s, and, after more than two similar responses of
IK(Ca) and
Ca2+ transient had been obtained,
10 µM CPA was applied. Although the control configurations of
IK(Ca) are quite
different between Fig. 8,
A and
B, common inhibitory effects were
observed after the application of CPA; the degree of
IK(Ca) inhibition
was greater than the decrease of
Ca2+ transient. In another cell,
Ca2+ transient was slightly
increased, whereas the
IK(Ca) were
sharply inhibited by CPA (Fig. 8C).
In five cells tested, peak
IK(Ca) amplitudes
had decreased to 49 ± 6.0% of control, whereas
Ca2+ transient had slightly
decreased to 93 ± 6.4% of control after ~90 s of CPA
application. In addition, transient oscillatory outward current
generation was completely eliminated by CPA.

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Fig. 8.
Effects of CPA on
IK(Ca) and
Ca2+ transient. Step
depolarizations as in Fig. 7 were applied repetitively every 30 s.
Responses from 3 different cells are shown in
A, B,
and C. In
A and
B, bath application of CPA (10 µM)
potently suppressed initial peak current and oscillatory outward
currents and slightly decreased amplitude of
Ca2+ transient as well. In
C, amplitude of
Ca2+ transient was slightly
increased, whereas transient and oscillatory outward currents were
almost completely suppressed by CPA.
|
|
 |
DISCUSSION |
In this study, we examined the relationship between voltage-dependent
Ca2+ influx and associated
Ca2+ transient in guinea pig
gastric myocytes. The results showed that the depolarization-induced
rise of
[Ca2+]i
strongly correlates with the amount of
Ca2+ influx through VOCCs, and in
the global change of
[Ca2+]i,
the contribution from SR Ca2+
release via the CICR process is insignificant. However, the large transient IK(Ca)
and the extent to which it changed seem to suggest the presence of an
inhomogeneous Ca2+ transient that
might be due to localized CICR. CPA inhibited the transient
and/or oscillatory outward current more than the Ca2+ transient. In addition, the
effect of CPA on Ca2+ buffering
capacity, demonstrated experimentally, implies that SR of guinea pig
gastric smooth muscle acts as a
Ca2+ buffering system.
Recent reports using various smooth muscle preparations and the whole
cell clamp method reached controversial conclusions regarding the role
of the CICR process during depolarization-induced Ca2+ transient. In an experiment
using rat portal vein myocytes, Grégoire et al. (13) found
evidence to show that CICR occurs during step depolarization. In
experiments using the same tissue, however, Kamishima and McCarron (21)
concluded that there was little CICR during depolarization and
suggested that the slow continued rise in
[Ca2+]i
after the termination of step depolarization was not due to CICR but to
sustained Ca2+ influx through
Ca2+-permeable cation channels
activated by elevated
[Ca2+]i.
In guinea pig urinary bladder smooth muscle, the earlier "phasic"
and later "tonic" component of
[Ca2+]i
rise was distinguished during the pulse period; the authors suggested
that since the phasic component was selectively abolished by caffeine
or ryanodine, a CICR mechanism operated during depolarization (10).
However, when the influx of Ca2+
was slowed by ramp depolarization, a significant
Ca2+ buffer function of SR was
also found in the same myocyte (35). In guinea pig coronary artery
smooth muscle, only a slow "creeping" [Ca2+]i
increase was observed during long depolarization, and the authors concluded that
ICa can trigger
CICR only locally and at a low amplitude (11).
In our results, although the averaged
B values seemed to be increased by
Ca2+ pool depletion, the
contribution of CICR was concluded to be statistically insignificant.
However, the presence of transient IK(Ca) and STOCs
cannot be explained without assuming cyclic
Ca2+ releases from SR to
subsarcolemmal space (2) or Ca2+
spark (24). Moreover, sensitive inhibition of
IK(Ca) by CPA (Fig. 8) appeared to demonstrate the presence of
Ca2+ release from SR, although the
same CPA decreased the B value in
another experiment (Fig. 6). Such contrasting effects of CPA might have
been due to two reasons: 1)
different ionic conditions (Cs+-
vs. K+-rich pipette solution in
Figs. 6 and 8, respectively), which modulate the probability of
Ca2+ release from SR (11, 18, 23),
and 2) vectorial
Ca2+ release during
Ca2+ transient toward
subsarcolemmal space without affecting global [Ca2+]i
(30, 32).
In guinea pig coronary arterial myocyte, creeping increase of
[Ca2+]i
during sustained depolarization was not observed in cells dialyzed with
Cs+ to block the
K+ current (11), and in guinea pig
cardiac myocyte, it was commented that
Cs+ dialysis had depressed the
amplitude of Ca2+ transient by
40% (18). The precise nature of the mechanism involved is not known,
but it may well be that the positive charge in SR is replaced with
K+ during each
Ca2+ release, and
Cs+ is not suitable for such
replacement, as the SR K+ channels
have been shown to have a very low conductance for
Cs+ (5). Although it is not
certain, such a negative effect of Cs+ might have affected our
experimental results in Figs. 5 and 6, and
Ca2+ release from SR is likely to
occur when KCl pipette solution is used.
Dissociation of the time courses between
IK(Ca) and
Ca2+ transient (Fig. 7) seems to
imply a dissociation between
[Ca2+]i,sl
and deep cytoplasmic
[Ca2+] during abrupt
stimulation (12, 30). It is also likely that the contribution of SR
Ca2+ release preferentially
affects
[Ca2+]i,sl,
since the IK(Ca)
was more sensitive than Ca2+
transient to inhibition by CPA (Fig. 8). In addition, the whole amplitude of outward current would include the current via
voltage-operated, Ca2+-independent
K+ channels, so the presented
inhibitory effects of CPA on
IK(Ca) (49 ± 6.0% of control amplitude) might as well have been underestimated. However, care should be taken when interpreting the change in IK(Ca), since the
Ca2+ dependence of the maxi
K+ channel is not linear
(3).
We suppose that the missing effect of CICR on global
Ca2+ transient of this gastric
myocyte might be because of the above two reasons (ionic conditions of
the pipette solution and spatially restricted influence from SR). This
restricted influence would be more plausible if distribution of the
functional RyR is spatially inhomogeneous, i.e., in the sarcolemmal
side of SR membrane. In cerebral arterial myocyte, the majority of
Ca2+ sparks (59%) arise close
(within 1 µm) to the sarcolemmal surface (26). Another possibility is
that depolarization produced a radial gradient of
[Ca2+]i
within the cell (25), recruiting RyRs in the vicinity of cell
membrane, and further release or diffusion is restricted by
Ca2+ buffers.
Once an increase in
[Ca2+]i
is initiated by the activation of
ICa, several
mechanisms will participate in the control of
[Ca2+]i.
The fast Ca2+ buffering system
(Ca2+-binding proteins and fura 2 included in the pipette) determines the level of initial
[Ca2+]i
during the step depolarization (11, 25), and the
Ca2+-removal processes (SERCA,
PMCA, and
Na+/Ca2+
exchange) are the principal mechanisms that finally bring
[Ca2+]i
to its resting level (32). The decrease of
B value by a SERCA inhibitor, CPA
(Fig. 6), seems to suggest the contribution of SR uptake to the
Ca2+ buffering capacity within 400 ms after step depolarization. However, we cannot completely exclude the
possibility that the fast Ca2+
buffering capacity had been consumed by
Ca2+ leaked from SR after the
application of CPA and decreased the B
values as a result.
To determine the Ca2+ buffering
capacity (B value) in this study, the
increase of total cytoplasmic Ca2+
concentration was approximated by dividing time integral of
Ca2+ influx by a number of numeric
constants including cytoplasmic diffusible water volume. In our
experiment, the B value was usually located between 150 and 250. Such values indicate that, if we assume
CICR does not occur, one of the 150-250
Ca2+ entering the cell appears as
a free Ca2+. A rather wide
variance of Ca2+ buffering
capacity might be inherent in our calculation process; averaged cell
volume was applied to all cells analyzed, and the even distribution of
Ca2+ and fura 2 was assumed. The
size of each cell would not, however, be the same, and because gastric
smooth muscle cells are long and spindle-shaped, it is still possible
that equilibrated diffusion of fura 2 was not attained within the
experimental time.
In summary, our results suggest that
Ca2+ influx through VOCCs plays a
key role in depolarization-induced
[Ca2+]i
increase in guinea pig gastric smooth muscle. During depolarization, the SR of this myocyte seems to serve as a kind of buffer barrier against Ca2+ influx rather than as
a source of global increase of
[Ca2+]i
via CICR. In gastric smooth muscle, the graded phasic contractions will
be attained by the regulation of
Ca2+ influx, which is determined
by the degree and duration of depolarization in each slow wave (31,
33). The localized Ca2+ release
and subsequent activation of
IK(Ca) might be
more important for the cessation of depolarization and repolarizing
mechanisms (26).
 |
ACKNOWLEDGEMENTS |
The [Ca2+]i measuring system for this
study was kindly donated by many supporters, including Dr. Gil-Ya Lee,
Director of Gil Medical Center.
 |
FOOTNOTES |
Address for reprint requests: K. W. Kim, Dept. of Physiology and
Biophysics, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea.
Received 5 March 1997; accepted in final form 6 August 1997.
 |
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