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1. Resting myoplasmic free [Ca2+] and [Ca2+] transients (delta [Ca2+]) were measured in single voltage-clamped frog skeletal muscle fibres in the presence and absence of procaine, caffeine or procaine plus caffeine using Fura-2 fluorescence and antipyrylazo III (Ap III) absorbance signals. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum (SR) was calculated from the calcium transients and corrected for the relatively small decline due to depletion of calcium from the SR. 2. Procaine (1 mM) reversibly suppressed delta [Ca2+] and the corresponding Rrel by about 40% for 60-100 ms depolarizing steps to -40 to +20 mV. Procaine had little effect on either the waveform or voltage dependence of the Rrel records. 3. [Ca2+] transients calculated from Fura-2 fluorescence changes in the presence or absence of procaine had similar time courses and amplitudes as those calculated from the Ap III absorbance changes suggesting that 1 mM-procaine did not interfere with the ability of Ap III or Fura-2 to monitor delta [Ca2+]. 4. Although 1 mM-procaine depressed Rrel it had no effect on intramembrane charge movements (IQ) calculated from membrane currents recorded simultaneously with delta [Ca2+]. 5. Procaine (1 mM) reversibly inhibited the potentiating effect of 0.5 mM-caffeine on delta [Ca2+]. The amplitude and waveform of the Rrel records were similar in control fibres and in the presence of 1 mM-procaine plus 0.5 mM-caffeine. 6. In the presence of 0.5 mM-caffeine delta [Ca2+] after 10-20 ms voltage steps exhibited an increase in the time to peak and a slower decay time course compared with caffeine-free controls, suggestive of significant calcium-induced calcium release in the presence of caffeine. These effects of caffeine were completely and reversibly blocked by 1 mM-procaine. 7. In the absence of caffeine, 1 mM-procaine caused a small decrease in time to peak of delta [Ca2+] after 10-30 ms duration voltage steps compared to the bracketing control and wash runs without procaine. Rrel turned off faster after 10 ms pulses in procaine than in the absence of procaine, but the turn-off of release was about equally fast with or without procaine after pulses of 20 ms or longer. The effect of procaine after 10 ms pulses in the absence of caffeine may indicate suppression of a component of calcium-induced calcium release in control that inactivates during the pulse.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The two calcium indicators Antipyrylazo III (AP III) and Fura-2 were used simultaneously to monitor free myoplasmic [Ca2+] in voltage-clamped cut segments of frog skeletal muscle fibres (8-10 degrees C). Antipyrylazo III was used for the relatively large [Ca2+] transients during 100-200 ms depolarizing pulses to -20 to 0 mV and for the rapid decline of [Ca2+] during the 200 ms after the pulses. Fura-2 was used to follow the slow decline of the small remaining elevation of [Ca2+] during the following 16 s (slow recovery period) and to monitor resting [Ca2+]. 2. From 1 to 16 s of the slow recovery period [Ca2+] declined with two exponential components, having time constants of 1.9 +/- 0.3 and 13.5 +/- 1.5 s (these and all other values are means +/- S.E.M. of eleven runs from seven fibres). At 1.2 s after the end of the pulses the amplitudes of the fast and slow exponential components of decline of [Ca2+] were 34 +/- 7 and 31 +/- 4 nM, respectively. The resting [Ca2+] in these runs was 40 +/- 4 nM. 3. The time course of calcium bound to parvalbumin [( Ca-Parv]) was calculated from the [Ca2+] records using literature values for the parvalbumin kinetic constants. From 1 to 16 s of the slow recovery period the total calcium [Ca]T outside the sarcoplasmic reticulum (SR) was assumed to equal [Ca-Parv] + [Ca-Fura]. During this period [Ca]T declined with two exponential components having time constants of 1.7 +/- 0.2 and 14.2 +/- 1.4 s, the same as those for [Ca2+]. Assuming the total concentration of parvalbumin cation binding sites to be 1000 microM, the fast and slow components of [Ca]T had amplitudes of 117 +/- 21 and 147 +/- 16 microM, respectively, at 1.2 s after the pulses. 4. The rate of decline of [Ca]T, -d[Ca]T/dt, was used as a measure of the net rate of removal of calcium from the myoplasm by the SR. From 3 to 16 s of the slow recovery period and in the resting fibre -d[Ca]T/dt varied with [Ca2+] according to A[Ca2+]n-L. The term A[Ca2+]n represents the pump rate and L represents a constant rate of calcium leak from the SR. 5. For 40 nM less than or equal to [Ca2+] less than or equal to 80 nM, the power n for the [Ca2+] dependence of pump rate was 3.9 +/- 0.6.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Resting myoplasmic [Ca2+] and [Ca2+] transients (delta [Ca2+]) were monitored using Fura-2 fluorescence and Antipyrylazo III absorbance signals from voltage-clamped segments of cut frog skeletal muscle fibres in the presence and absence of 0.5 mM-caffeine. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from delta [Ca2+]. 2. delta [Ca2+] and Rrel were increased in caffeine for all pulses. The decline of delta [Ca2+] was slower after a given pulse in caffeine than without caffeine. Resting [Ca2+] was slightly elevated in caffeine. 3. The voltage dependence of the peak value of Rrel and of the steady level of Rrel at the end of a 60-120 ms pulse were both shifted towards more negative voltages in caffeine. For relatively small pulses the voltage at which a given release waveform was observed was also shifted to more negative voltages. 4. Intramembrane charge movements measured in the same fibres in which the above changes in Rrel were observed showed no significant changes in caffeine. 5. In caffeine calcium release continued for many milliseconds after the end of a short (10 ms) pulse. Continued release after a pulse was not observed without caffeine and was probably due to positive feedback of elevated [Ca2+] on calcium release resulting from calcium-induced calcium release in caffeine. 6. Intramembrane charge movements after short pulses showed no change in caffeine that could account for the continued calcium release after the pulse. 7. Continued release after short pulses in caffeine decreased as the pulse duration was increased and was absent for pulses of 60 ms or longer. Rrel also inactivated during such pulses. 8. Relatively large and long conditioning pulses in caffeine suppressed both the peak Rrel and the continued release after short pulses. Peak release and continued release after short pulses recovered in parallel with increasing recovery time following suppression by a conditioning pulse in caffeine. 9. These results indicate that in the presence of caffeine, charge movement and calcium-induced calcium release both contribute significantly to the activation of sarcoplasmic reticulum calcium release during fibre depolarization. Release activated by either mechanism appears to be inactivated by calcium-dependent inactivation. A significant contribution of calcium-induced calcium release during depolarization in the absence of caffeine is not ruled out by present observations.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.

Intramembrane charge movement and myoplasmic free calcium transients (delta[Ca2+]) were monitored in voltage-clamped segments of isolated frog muscle fibres cut at both ends and mounted in a double Vaseline-gap chamber. The fibres were stretched to sarcomere lengths of 3.5-4.6 micron to minimize mechanical movement and the related optical artifacts. The over-all calcium removal capability of each fibre was characterized by analysing the decay of delta[Ca2+] following pulses of several different amplitudes and durations. The rate of sarcoplasmic reticulum (s.r.) calcium release was then calculated for each delta[Ca2+] using the calcium removal properties determined for that fibre. The calculated calcium release wave form reached a relatively early peak and then declined appreciably during a 100-150 ms depolarizing pulse. The voltage dependence of the peak rate of calcium release was steeper and was centred at more positive membrane potentials than the steady-state voltage dependence of charge movement in the same fibres. A considerable fraction of the total intramembrane charge was moved at potentials at which delta[Ca2+] and calcium release were only a few per cent of maximum. This 'subthreshold' charge may correspond to charge moved in preliminary transitions that precede a final charge transition that activates release. A 'stepped on' pulse protocol was used to experimentally separate the subthreshold charge movement from the charge movement of the final transitions that may control calcium release. The stepped on pulse consisted of a set 50 ms pre-pulse to a potential just at or below the potential for detectable delta[Ca2+] followed immediately by a test pulse of varying amplitude and duration. For a wide range of test pulse amplitudes and durations in the stepped on protocol the peak rate of calcium release was linearly related to the charge movement during the test pulse. This result points to a tight control of activation of s.r. calcium release by intramembrane charge movement. The voltage dependence of both charge movement and of the rate of calcium release could be fitted simultaneously with a three-state, two-transition sequential model in which charge moves in both transitions but only the final transition activates s.r. calcium release. A model with three identical and independent charged gating particles per channel gave an equally good fit to the data. Both models closely fit the charge movement and release data except within about 10 mV of the voltage at which release became detectable, where release varied more steeply with membrane potential than predicted by either model.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) in voltage clamped segments of frog skeletal muscle fibres was calculated from myoplasmic free calcium transients (delta[Ca2+]) measured with the calcium indicator dye Antipyrylazo III. 2. During a 100-200 ms depolarizing pulse Rrel reached an early peak and then declined markedly. The time course and extent of decline of Rrel were independent of membrane potential over a range of potentials where release activation varied severalfold. 3. For test pulses applied shortly after relatively large or long conditioning pulses Rrel completely lacked the early peak. The peak gradually recovered as the interval between the conditioning and test pulses was increased to 1 s. 4. A latency was often observed before the start of recovery of the peak in Rrel. The latency appeared to be correlated with the time for delta[Ca2+] to fall below a certain level, indicating that recovery of the peak might represent reversal of a calcium-dependent process. It was therefore proposed that the rapid decline in Rrel during a pulse was due to calcium-dependent inactivation of the SR calcium release channels. 5. Inactivation continued to develop during the interval between a relatively large 20 ms conditioning pulse and a test pulse applied 20 ms later. This was as expected for calcium-dependent inactivation of SR calcium release because of the elevated [Ca2+] between the conditioning and test pulses. It was not as expected for external membrane potential-dependent inactivation. 6. Small steady elevations in [Ca2+] due to relatively small 200 ms conditioning pulses produced marked inactivation of Rrel, indicating an apparent dissociation constant for calcium-dependent inactivation only slightly above resting [Ca2+]. 7. All observations could be well simulated by a two-step model for inactivation in which myoplasmic free calcium equilibrates rapidly with a high-affinity calcium receptor on the release channel and then the calcium-receptor complex undergoes a slower conformational change to the inactivated state of the channel. 8. An alternative model in which calcium binds to a soluble receptor (e.g. free calmodulin) and then the calcium-receptor complex binds to and directly inactivates the channel was shown to be formally identical to the preceding model. Either model could closely simulate all observations.

Myoplasmic free calcium transients delta [Ca2+] were monitored with the calcium indicators antipyrylazo III and fura-2 in voltage clamped cut frog skeletal muscle fibers, in the presence and absence of 0.5 mM caffeine. Without caffeine delta [Ca2+] began to decline within a few milliseconds of fiber repolarization for pulses of all durations. In caffeine delta [Ca2+] continued to rise for 10-60 ms after 10 or 20 ms depolarizing pulses, indicating that the release of calcium from the sarcoplasmic reticulum (SR) continued well after repolarization of transverse tubular (TT) membranes in the presence of caffeine. Caffeine also increased the peak amplitude of delta [Ca2+] for all pulses and slowed the decline of delta [Ca2+] after pulses of all durations. The rate of calcium release from the SR calculated from delta [Ca2+] showed that for 10 ms pulses in caffeine release did not turn off abruptly on repolarization but instead declined to zero with a time constant essentially the same as the time constant for inactivation of SR calcium release during depolarizing pulses in the presence or absence of caffeine. The observed loss of TT membrane potential control of SR calcium release in the presence of caffeine suggests the appearance of a significant component of cytosolic Ca2+-induced calcium release in caffeine.

1. Free intracellular calcium transients (delta[Ca2+] were monitored in cut segments of frog skeletal muscle fibres voltage clamped in a double Vaseline-gap chamber and stretched to sarcomere lengths that eliminated fibre movement. The measured calcium transients were used to calculate the rate of calcium release from the sarcoplasmic reticulum (s.r.) as previously described (Melzer, Rios & Schneider, 1984, 1987). 2. Conditioning pulses were found to suppress the rate of calcium release in test pulses applied after the conditioning pulse. Various combinations of conditioning and test pulses were used to investigate the basis of the suppression of calcium release by the conditioning pulse. 3. Using a constant test pulse applied at varying intervals after a constant conditioning pulse, recovery from suppression of release was found to occur in two phases. During the fast phase of recovery, which was completed within about 1 s, the rate of calcium release was smaller and had a different wave form than the unconditioned control release. The early peak in release that is characteristic of the control release wave form was absent or depressed. During the slow phase of recovery, which required about 1 min for completion, the release wave form was the same as control but was simply scaled down compared to the control. 4. Conditioning pulses also slowed the rate of decay of delta[Ca2+] after a constant test pulse, probably due to an increased occupancy by calcium of slowly equilibrating myoplasmic sites that bind some of the calcium released by the conditioning pulse. Since calcium binding to these sites contributes to the decay of delta[Ca2+], their increased occupancy would slow the decay of delta[Ca2+] following the test pulse. This effect was used to estimate the calcium occupancy of the slowly equilibrating sites. 5. Comparison of the time course of the slow recovery from suppression of release following a constant conditioning pulse with the time course of the loss of calcium from the slowly equilibrating myoplasmic calcium binding sites indicated that the two processes occurred in parallel. 6. Using a set 1 s recovery period and a constant test pulse but varying the amplitude and/or duration of the conditioning pulse, the degree of slowly recovering suppression of release was found to be directly related to the amount of calcium remaining outside of the s.r. at the start of the test pulse. 7. Points 3, 5 and 6 above indicate that the slow recovery from suppression of release may be due to slow recovery from depletion of calcium from the s.r.(ABSTRACT TRUNCATED AT 400 WORDS)

Myoplasmic free calcium transients were measured with antipyrylazo III in voltage clamped segments of frog skeletal muscle fibers and were used to calculate the rate of release (Rrel) of calcium from the sarcoplasmic reticulum. Intramembrane charge movement was measured for the same pulses in the same fibers. During a depolarizing pulse Rrel rose to an early peak and then decayed relatively rapidly but incompletely due to calcium-dependent inactivation (Schneider M.F., and B.J. Simon. 1988. J. Physiol. (Lond.). 405:727–745). Two approaches were used to determine release activation independent of the effects of inactivation: (a) a mathematical correction based on the assumption that inactivation was a process occurring in parallel with and independently of activation; (b) an experimental procedure in which release was maximally inactivated by a large short prepulse and then the remaining noninactivatable component of release was monitored during a subsequent test pulse. Both procedures gave the same time course of activation of release. Release activation paralleled the time course of intramembrane charge movement but was delayed by a few milliseconds.