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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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(beta -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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)
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB> × <IT>b</IT> × (R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)
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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

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).

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.

The expected increase of [Ca2+]i due solely to ICa could be calculated from the following equation
Expected &Dgr;[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <LIM><OP>∫</OP></LIM> −<IT>I</IT><SUB>Ca</SUB>d<IT>t</IT>/2<IT>F</IT> V
where <LIM><OP>∫</OP></LIM>-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.

                              
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Table 1.   B values obtained from five cells at various pulse durations

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.

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).

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).

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Discussion
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