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Journal of Physiology (1992), 457, pp. 1-25 With 13 figures Printed in Great Britain

SOME PROPERTIES OF Ca2+-INDUCED Ca2+ RELEASE MECHANISM IN SINGLE VISCERAL SMOOTH MUSCLE CELL OF THE GUINEA-PIG BY A. V. ZHOLOS*, L. V. BAIDAN AND M. F. SHUBA From the Department of Nerve-Muscle Physiology, A.A.Bogomoletz Institute of Physiology, Academy of Sciences of the Ukraine, 252 601 GSP, Kiev 24, Ukraine

(Received 4 February 1991) SUMMARY

1. Late transient outward Ca2+-dependent K+ current (ILTO) correlated with Ca2+_ induced Ca2+ release mechanism was studied in relation to the calcium inward current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum using the whole-cell patch-clamp technique. 2. The voltage dependencies of peak ICa and ILTO were both bell shaped. However, the I- V curve of the outward current was shifted toward more positive potentials by about 60 mV in comparison to that for ICa* 3. Reduction in the external Ca2+ concentration resulted in a decrease of peak amplitude of both Icaand ILTO. However, caffeine-induced outward current was also decreased abruptly suggesting a rapid loss of stored Ca2+ upon lowering the external Ca2+ concentration. 4. Investigation of the relation Of ILTO to partially inactivated ICa showed that inactivation ofICa by approximately 65, 80 or 84 % of control (produced by prepulse to -20 mV for 2 s, shifting the holding potential to -20 mV for 30 s or by the ramp voltage command from -50 to + 10 mV, respectively) was without detectable effect on the ILTO generation. 5. Bath application of the Ca2+ antagonist nifedipine (300 nM) inhibited ICa by 81 % without affecting ILTO peak amplitude (92-0 + 5-6 % of control in six cells). The mean concentration-response curve for ICa inhibition was sigmoidal with the apparent dissociation constant of 86-9 nm, whereas that for the ILTO had a characteristic sharp transition indicating a definite threshold of Ca2+ influx for ILTO generation. 6. Application of Ca2+-free external solution during 500 ms of the time when Ica peaked inhibited the current by about 76 % whereas the ILTO during such an intervention remained virtually unchanged. 7. In double-pulse experiments, with conditioning and test pulses to + 10 mV from -50 mV and an interpulse interval of 600 ms, most of the cells (about 80%) showed larger outward current at the test pulse suggesting continued Ca2+ release triggered by Ca2+ influx during a short (50-200 ms) depolarizing prepulse. The * Present address: St George's Hospital Medical School, Department of Pharmacology and Clinical Pharmacology, Cranmer Terrace, London SW17 ORE.

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A. V. ZHOLOS, L. V. BAIDAN AND M. F. SHUBA

outward current could also be evoked at large positive potentials (presumably near the calcium equilibrium potential) where it did not occur normally by a prepulse to + 10 mV for 50 ms. The charge transferred by Ca2` current necessary to activate Ca2+ release in most of the cells was estimated to be from 6 to 20 pC. 8. The data are interpreted to suggest that the Ca2`-induced Ca2+ release mechanism operates in single ileal cells in a regenerative manner. Both fast initial and subsequent slow components of ICa could activate ILTO and consequently Ca2+ release implying physiological importance of Ca2+ release for amplification of Ca2+ influx and prolongation of the 'external Ca2+ signal' in excitation-contraction coupling in smooth muscle. INTRODUCTION

The role of Ca2+ in the excitation-contraction coupling of smooth muscle is well established. It is generally accepted that there are two main sources of Ca2+ for the cell contractile response, namely extracellular and intracellular. During the last few years advances in the patch-clamp method made it possible to quantify the Ca2+ influx through voltage- and receptor-operated Ca2+ channels in a variety of smooth muscle cells (Benham, Bolton & Lang, 1985; Mitra & Morad, 1985; Bean, Sturek, Puga & Hermsmeyer, 1986; Benham, Hess & Tsien, 1987; Yatani, Seidel, Allen & Brown, 1987; Benham & Tsien, 1988; Loirand, Mironneau, Mironneau & Pacaud, 1989; Hisada, Kurachi & Sugimoto, 1990; Lang, 1990; Matsuda, Volk & Shibata, 1990; Vivaudou, Singer & Walsh, 1991). According to several authors (Ganitkevich, Shuba & Smirnov, 1986; Yamamoto, Hu & Kao, 1989 a), the amount of Ca2+ entering the cell via voltage-dependent Ca2+ channels is more than sufficient to activate maximal contraction. This is consistent with the previous calculations of Ca2+ entry during the upstroke of the action potential (Bolton, 1979). However, the exact rise in intracellular Ca2+ concentration ([Ca2+]i) is difficult to calculate as Ca2' binding and Ca2+ extrusion properties are unknown. An amplification of this 'external Ca2+ signal' by the mechanism of Ca2+-induced Ca2+ release (CICR) from calcium stores has also been suggested (for review see Karaki & Weiss, 1988). However, the mechanisms) of this release is still not clear especially as to the role of voltagedependent Ca21 current (ICa) of an intact cell in regulating this process. In skeletal muscle, CICR occurs in a regenerative manner (Endo, Tanaka & Ogawa, 1970; Ford & Podolsky, 1970) whereas in cardiac muscle it is graded (Fabiato, 1985a, b; Callewaert, Cleemann & Morad, 1988). Although the exact mechanism underlying CICR is unclear, the sensitivity of the CICR mechanism to the rise in calcium produced by ICa is important. ICa can be functionally separated into two components, namely a fast initial transient current of large amplitude and a smaller subsequent slow component. Since the work of Fabiato (1985 b) on skinned cardiac cells it is generally believed that the first component triggers Ca2+ release whereas the second component loads the sarcoplasmic reticulum (SR). This is also believed to be the case for smooth muscle (for review see Karaki & Weiss, 1988), although direct supporting evidence for such a hypothesis has not yet been obtained. To address this issue, we have investigated the interactions between ICa and Ca2+_ activated K+ current ('K(Ca))* In a recent paper (Zholos, Baidan & Shuba, 1991) we described a slow late transient 'K(ca)(ILTO) in single isolated smooth muscle cells of

Ca2+_INDUCED Ca2+ RELEASE IN SMOOTH MUSCLE 3 the guinea-pig ileum that arises from an increase in Ca21 (which is the result of the CICR mechanism). The main finding of the present study is that significant reduction of ICa amplitude or elimination of either fast initial or subsequent slow voltageactivated Ca2+ entry did not prevent generation of the ILTO Thus, as far as the amplitude of the ILTO can be considered to be a measure of CICR, this mechanism in visceral smooth muscle cell seems to require rather small Ca2+ influx to be triggered and is maintained by self-regeneration. METHODS

The methods of cell isolation, electrical recordings and changes of the solutions were generally the same as previously described (Zholos et al. 1991). Briefly, adult guinea-pigs of either sex weighing 200-300 g were killed by decapitation following a sudden blow to the head. Cells were isolated after incubation of small pieces of the longitudinal layer of the guinea-pig ileum in Ca2+free physiological salt solution (PSS) containing 0-1 % collagenase, 0-1 % soybean trypsin inhibitor and 0 1% bovine serum albumin at 36 TC for 30-50 min. The PSS had the following composition (mM): NaCl, 120; KCl, 6; CaCl2, 2-5; MgCl2, 1-2; glucose, 11 5; HEPES, 10; pH adjusted to 7-3 with NaOH. External solutions with different concentrations of Ca2+ were prepared by substituting CaCl2 with equimolar amount of MgCl2. HighK+ pipette solution had the following composition (mM): KCl, 85; KH2P04, 30; MgSO4, 1; Na2ATP, 1; creatine, 5; glucose 20; EGTA, 0 3; HEPES, 10; pH adjusted to 7-3 either with NaOH (most of the experiments) or with KOH (when currents were measured at large positive potentials). K+-free PSS (KCl substituted by 10 mm TEA-Cl) and high-Cs' pipette solution (KCl and KH2PO4 substituted by 115 mm CsCl, 20 mm TEA-Cl added) were used for calcium current separation. Drugs used were: collagenase (Type I), caffeine, nifedipine, Ruthenium Red (all from Sigma); trypsin inhibitor, bovine serum albumin, adenosine 5'-triphosphate (ATP, sodium salt) (Reanal or Sigma); creatine (Merck); tetraethylammonium chloride (TEA; BDH or Sigma); N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES; Ferac); ethyleneglycol-bis-/Jaminoethylether-N,N'-tetraacetic acid (EGTA; Calbiochem). Temperature was measured by a microthermistor placed near the cell. Experiments were usually performed at room temperature (22-25 °C) and when needed the solution in the chamber could be heated to 36 °C within approximately 30 s. The voltage protocol used is indicated either in the text or in the figures. Series resistance compensation was applied to just below the point of ringing. Leakage current was not compensated except when indicated. RESULTS

Using the activity of Ca2+-dependent K+ channels as an indicator of [Ca2+]i increase during calcium release: voltage dependence of caffeine-induced outward current In this study we have attempted to analyse the Ca2+ release process in ileal smooth muscle cells by measuring Ca2+-activated K+ current (ILTO) resulting from activation of the CICR mechanism (Zholos et al. 1991). The rationale for using this approach was as follows: the high density, conductance and sensitivity to [Ca21]i of Ca2+-

dependent K+ channels, along with their fast activation by membrane depolarization or increases in [Ca2+]i allow the activity of these channels to be used as an intrinsic ' calcium indicator'. Recently, several studies on intestinal smooth muscle cells have been performed using these channels as a 'calcium indicator' (Komori & Bolton, 1990, 1991; Mayer, Loo, Snape & Sachs, 1990). We have discussed why we believe that the activation and inactivation of the ILTO (at + 10 mV) represents the time course of the [Ca2+]i transient elsewhere (Zholos et al. 1991). However, some precautions must be taken with this method. Among them, the determination of an

A. V. ZHOLOS, L. V. BAIDAN AND M. F. SHtUBA

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appropriate membrane test potential seems to be of prime importance. Considering the complex dependence of channel open probability (PO) on both [Ca2+]i and membrane potential (Inoue, Kitamura & Kuriyama, 1985; Benham, Bolton, Lang & Takewaki, 1986; Hu, Yamamoto & Kao, 1989a; Mayer et al. 1990) it is likely that B

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Fig. 1. The voltage dependence of caffeine-induced outward current. A, 10 mm caffeine was applied for 2 s at the end of each 10 s step pulse to different test potentials (as indicated at the bottom) evoked every 1 min and in the sequence as shown. The dashed line represents the baseline of the holding current at the holding potential of -50 mV. The results are typical of six observations. B, K+ conductance was calculated from current traces shown in A assuming an EK value of -76 mV and plotted against the membrane potential. At each test potential current amplitude was measured from a steady-state level before caffeine application.

large positive membrane potentials are preferable to resolve changes in [Ca2+]i at lower levels (and vice versa) as at these large potentials P. is saturated above some [Ca2+]i level (approximately 10' to 5 x 10'6 M at +40 mnV; Benham et al. 1986; Hu et al. 1989a; Mayer et al. 1990). In the experiments described below we were particularly interested in answering the question as to whether there is a reduction in calcium release associated with the reduction of calcium entry through the Ca2+ channels. Thus, possible saturation of the 'LTO at the test membrane potential chosen and at a given [Ca2+]i (which has yet to be established using direct measurements) represents a serious problem because if this were so, then significant reduction in calcium release would not be detected. Here we can given only an approximate estimation of maximum PO during the ILTO development based on indirect evidence. Single-channel conductance in these cells was found to be 245 +21 pS (n = 10) at 0 mV in cell-attached mode (115 mm K+ in the pipette solution; Zholos, Baidan & Shuba, 1990). Under a comparable K+ gradient (cells were exposed to high-K+ (115 mM) external solution) the mean membrane conductance was 2031 nS (from data shown in Fig. 2 of Zholos et al. 1991). This corresponds to about 830 channels simultaneously open. From the mean I-V relationship for the ILTO (see Fig. 2) the mean membrane conductance at 0 mV was about 55 nS, or about 550 open channels assuming single-channel conductance of 100 pS at a physiological K+ gradient. Finally, dividing the mean macroscopic

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outward current (2571 pA at + 10 mV, data from Zholos et al. 1991) by the appropriate unitary current (6-8 pA at + 10 mV and comparable K+ gradient; Inoue et al. 1985; Benham et al. 1986; Hu et al. 1989a) will give us a number in the range of 320-430. According to several authors (Singer & Walsh, 1987; Hu et al. 1989a) the channel density is 075-1 per ,tm2, or 3300-5000 Ca2+-activated K+ channels per cell. Taken together, this suggests a P. 01-0 25 at the peak of the ILTO0 These calculations suggest the 'LTO being far from saturation at + 10 mV and in the range of [Ca2+]i in these experiments. The possibility to evoke additional large outward current by 10 mm caffeine application at the moment when the ILTO peaked (Figs 5 and 6, for example) was also consistent with this conclusion. However, it was impossible to test further the range for the 'LTO possible saturation. Saturation of the current at about + 50 to + 70 mV (see explanations of data of Fig. 2 for further details) was most probably not due to P. saturation as it was followed by a decrease in current amplitude at more positive potentials suggesting a reduction in Ca2+ release. A more direct test of ILTO saturation was provided by examining the voltage dependence of outward current evoked by caffeine. Application of 10 mm caffeine always evoked larger outward current (Icaf) than the ILTO suggesting larger [Ca2+]i transients. Figure 1A illustrates an experiment which was based on the idea that as soon as the range of membrane potentials for Jaf saturation was determined this would give the lowest limit for the IETO investigation avoiding saturation. The cell was depolarized every 1 min to increasingly positive potentials by 10 s test pulses (20 mV increment was used to accelerate the protocol) and 10 mm caffeine was applied for 2 s at the end of each pulse. At -30 mV the response could hardly be seen, at least on this current scale, whereas at - 10 mV rapid decay to the baseline was characteristic for Icaf. At potentials positive to + 10 mV the second phase of [Ca2+]i elevation could also be resolved. The amplitude of Icaf was used to calculate maximal K+ conductance at each potential (assuming a reversal potential for K+ (EK) of -76 mV) which is directly related to PO of the individual channels. The P. started to saturate in this case at potentials positive to + 10 mV (Fig. 1B). In another five cells studied the saturation was always observed at potentials exceeding + 10 mV, or even + 50 mV (three of five). Thus, most of the experiments described below were performed using a test potential of + 10 mV which provided with a certain degree of confidence nonsaturation of the ILTO Additional advantages of this potential were also considered to be: (i) it provides a large driving force for K+ without significant contamination oftheILTO from other currents (about 10%); and (ii) it makes the modifications of 'Ca easier to study during any intervention as it is the potential for maximum 'Ca-

Voltage dependence of 'Ca and ILTO Cannell, Berlin & Lederer (1987), Callewaert et al. (1988) and Cleemann & Morad (1991) have shown similar bell-shaped relationships for'Ca and [Ca2+]i transients due to a graded CICR mechanism in heart muscle. Thus, if a similar mechanism operates in smooth muscle one could also expect a saturation of the current-voltage (I-V) relationship for the ILTO For these reasons I-V relationships for both 'Ca and ILTO were studied.

A. V. ZHOLOS, L. V. BAIDAN AND M. F. SHUBA

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Voltage steps applied from a holding potential of -50 mV were increased in steps of 20 mV to accelerate the protocol of the experiment and each sequence of pulses was followed by a pulse to + 10 mV to test the amount of run-down. Only those cells where run-down was less than 20 % for both ICa and ILTO during an experiment (mean values were of about 3 and 12%, respectively) were used. C 1.0

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10-7 10-5(M) [Nifedipine] Fig. 10. Effects of bath application of nifedipine on ICa and ILTO* A and B, current traces from two representative cells before (left), after cumulative application of nifedipine at concentrations indicated at the top and after wash-out of the drug (right). Complete recovery took about 3 min for 'ca (A) and 1 min for ILTO (B). In both cases depolarizing steps were applied from a holding potential of -50 to + 10 mV for 5 s duration every one minute. C, concentration-effect curves for ICa and ILTO (O and A, respectively). Abscissa: concentration of nifedipine on logarithmic scale; ordinate: peak current with nifedipine normalized to that without nifedipine (Inf/lc) Smooth curve for ICa was fitted to the Michaelis-Menten equation with the apparent dissociation constant of 86-9 nm. Points and vertical bars are averages and S.E.M., respectively, with the numbers of observations given in parentheses.

ILTO and fast initial component of ICa The ILTO was usually evoked by long-lasting depolarizing pulses of several seconds in duration. It was important to test, however, whether the CICR mechanism could be triggered by Ca2+ influx during a short depolarizing pulse comparable to the duration of the action potential in these cells. As the results above were mostly in favour of a positive feedback between [Ca2+]i elevation and Ca2+ release it was

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reasonable to suggest that initial ICa of large amplitude would be sufficient to trigger Ca2" release. This possibility was verified in experiments using three different experimental protocols. The protocol shown in Fig. I1 C was used to interrupt Ca2+ influx by deactivation of Ca21 channels. Figure llB compares Ca2+ traces obtained with PI and P2 pulses

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Some properties of Ca(2+)-induced Ca2+ release mechanism in single visceral smooth muscle cell of the guinea-pig.

1. Late transient outward Ca(2+)-dependent K+ current (ILTO) correlated with Ca(2+)-induced Ca2+ release mechanism was studied in relation to the calc...
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