Suppression of calcium release by calcium or procaine in

Transcription

1 3624 Journal of Physiology (1995), 485.2, pp Suppression of calcium release by calcium or procaine in voltage clamped rat skeletal muscle fibres J. Garcia and M. F. Schneider * Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, MD 21201, USA 1. Calcium transients were measured in fast-twitch rat skeletal muscle fibres stretched to #sm per sarcomere, and voltage clamped at a holding potential of -80 mv using the double-seal Vaseline gap technique. Resting calcium was monitored with fura-2 and the calcium transients were measured with antipyrylazo III. The rate of release of calcium from the sarcoplasmic reticulum was calculated from the calcium transient records. The temperature was 'C. 2. The steady-state calcium dependence of inactivation of release was studied with a two-pulse protocol in which 200 ms prepulses of different amplitudes elevated the internal calcium concentration to various levels. The inactivation of release was then measured in the test pulse that followed the prepulses. The calcium concentration at which the inactivation of release was half-maximal was um, the average number of bound calcium ions needed to cause inactivation was about three per release channel and the amount of release that could be inactivated was, on average, 2-48 times the steady level of release during the test pulses. 3. Procaine (03 mm) reversibly decreased the amplitude and the rate of rise of the calcium transient. Both the peak and the steady level of release were decreased by about 50%. The shape of the release waveform was not modified. Activation of the sarcoplasmic reticulum (SR) calcium-release channel or ryanodine receptor (Lai, Erickson, Rousseau, Liu & Meissner, 1988) in skeletal muscle appears to be controlled by voltage sensors localized in the transverse tubular membrane (Schneider & Chandler, 1973; Horowicz & Schneider, 1981; Rios & Brum, 1987). SR channel activation allows calcium efflux from the lumen of the SR to the myoplasm, leading to elevated myoplasmic free [Ca2+] and, consequently, calcium binding to the contractile proteins and other calcium-binding proteins. Local changes in calcium concentration at the site of release are also important, in that local calcium can influence the behaviour of the ryanodine receptor. Calcium-dependent modulation of the skeletal muscle SR calcium-release channel has been described in a variety of studies using skinned fibres (Endo, Tanaka & Ogawa, 1970; Donaldson, 1985; Stephenson, 1985), voltage clamped fibres (Baylor, Chandler & Marshall, 1983; Schneider & Simon, 1988; Simon, Klein & Schneider, 1991; Gy6rke & Palade, 1992), SR vesicles (Kim, Ohnishi & Ikemoto, 1983; Meissner, Darling & Eveleth, 1986), and purified SR release channels incorporated in bilayers (Smith, Imagawa, Ma, Fill, Campbell & Coronado, 1988). It has been suggested that in voltage clamped frog skeletal fibres, the elevation of myoplasmic calcium can cause a negative feedback in the form of calcium-dependent inactivation, of the release channel (Baylor et al. 1983; Schneider & Simon, 1988; Simon et al. 1991), and some reports of the effects of strong buffering of myoplasmic calcium with fura-2 are consistent with the removal of calcium-dependent inactivation by the added buffer (Baylor & Hollingworth, 1988; Jong, Pape, Chandler & Baylor, 1993). Moreover, strong buffering of myoplasmic calcium with fura-2 has also been reported to result in the loss of the peak (inactivating) component of release, which has been attributed to the prevention of a calcium-induced component of calcium release (Jacquemond, Csernoch, Klein & Schneider, 1991). Thus, modulation of the release channel by calcium may be important in regulating the calcium flux from the SR to the myoplasm after the initial activation of the channel by the voltage sensors. In the present experiments we have examined modulation of the release of calcium from the SR in rat isolated voltage clamped fast-twitch skeletal muscle fibres. We first explored the effects of increasing myoplasmic calcium concentration on the inactivation of SR calcium release. We found that * To whom correspondence should be addressed.

2 438 J Garcia and M. F Schneider J Physiol the peak rate of calcium release during a set test pulse decreased as the calcium concentration was elevated during a prepulse, indicating that a calcium-dependent inactivation process may be present in rat muscle as in frog muscle. We then examined the effects of the local anaesthetic procaine on calcium release. In skinned mammalian fibres procaine has been used as a 'specific' blocker of calcium-induced calcium release which was assumed to have little effect on depolarization-induced release (Donaldson, 1985, 1986). However, in our experiments, procaine was found to block both the peak and the steady level of the rate of calcium release during voltage clamp depolarizations, consistent with a non-specific action, as previously found for procaine in voltage clamped frog skeletal muscle fibres (Klein, Simon & Schneider, 1992). METHODS The experimental procedures and materials used in this study, briefly described here, were the same as those in Garcia & Schneider (1993). Fibre preparation Experiments were performed on cut segments of single fibres isolated from the extensor digitorum longus muscle of Sprague-Dawley rats. The rats, weighing g, were killed by inhalation of CO2 under National Institutes of Health (USA) guidelines. The whole muscle was placed in a beaker with normal Krebs-Ringer solution. A fibre segment was dissected in a depolarizing solution containing 0 4 mm Ca2+ and then transferred to a double Vaseline gap chamber containing a solution with 1 mm EGTA and no added Ca2. Fibres were stretched to 3'7-4 0 um per sarcomere and small notches were made beyond the Vaseline threads in both end-pools. The optical path length through the stretched fibres varied between 15 and 45 jum, and the length of the middle pool was m. After the Vaseline seals were formed, the solutions in the middle and end-pools were exchanged for those used throughout the experiment. Experiments were started between 40 and 90 min after the addition of the indicators to the end-pools and fibres were polarized to -80 mv at least 5 min before starting the recordings. Membrane current, membrane voltage and optical signals for calculating calcium transients were monitored simultaneously during voltage clamp depolarizations applied to the fibre. The actual recording time varied from fibre to fibre, ranging from 15 min to more than 60 min. Recordings were stopped when the leak current increased by more than about 10%. Leak current tended to remain relatively stable throughout the experiment but increased suddenly when the fibre deteriorated, at which point the experiment was terminated. Measurement of calcium transients Calcium transients in response to depolarizing voltage steps were measured as previously described (Klein, Simon, Sziics & Schneider, 1988; Garcia & Schneider, 1993). The internal solution contained two calcium-sensitive dyes: the absorbance dye antipyrylazo III (AP3; Scarpa, Brinley & Dubyak, 1978; ICN K & K Laboratories, Plainview, NY, USA) and the fluorescent dye fura-2 (Grynkiewicz, Poenie & Tsien, 1985; Molecular Probes, Eugene, OR, USA). We followed the procedures and methods for calculating the change in [Ca2+] (A [Ca2+]) and [Ca2+] described in KovaLcs, Rios & Schneider (1983) and Klein et al. (1988), as in our previous paper on rat skeletal muscle fibres (Garcia & Schneider, 1993). The calcium-independent absorbance or fluorescence due to each dye in the resting fibre was determined periodically. The respective values of AP3 and fura-2 concentrations were extrapolated linearly to the time of each stimulating pulse. The fluorescence emission F380 at 510 nm for excitation at the calciumsensitive wavelength of 380 nm before each pulse, was divided by the corresponding fluorescence F358 for excitation at the isobestic wavelength of 358 nm, to obtain the fura-2 ratio signal for calculating resting [Ca2+] for that pulse. Calcium transients were calculated from the AP3 signals. Calcium removal analysis and calculation of calcium release The calcium binding and transport properties of each fibre were characterized by fitting a specific binding and transport model (Brum, Rios & Schneider, 1988) to the decay of A[Ca2+] starting 15 ms after pulses of several amplitudes and/or durations. The fits were carried out using the general procedure for simultaneous fitting of the decays of multiple [Ca2+] transients developed by Melzer, Rios & Schneider (1986). The values of one or more parameters in the binding and transport system model were adjusted to produce a best fit of the decay of the A [Ca2+] predicted by the model to the measured decay of A[Ca2+] from 15 ms after the pulse, when release was assumed to be zero, to the end of the records. Records of different amplitudes and durations were fitted simultaneously for each fibre. Details regarding most of the specific myoplasmic calcium binding sites (both rapidly and slowly equilibrating) used in the previous (Garcia & Schneider, 1993) and present analyses (see figure legends) of rat fibres were similar to those used by Klein, Simon & Schneider (1990) for frog fibres. The rate of release of Ca2P from the SR was calculated for each [Ca2+] transient obtained with AP3 following the method developed by Melzer et al. (1984, 1987). Ca2P release during depolarization was calculated assuming that the Ca2+ removal system had the same characteristics during the pulse as determined from the fits to the decay of [Ca2+] after the pulses. Data acquisition and processing Optical signals (light intensities at 700, 850 and 510 nm) and fibre membrane electrical signals (current and voltage) were monitored as described (Garcia & Schneider, 1993) using custom computercontrolled hardware and software. The signals were successively sampled at 40,us intervals during 200,us. For sampling frequencies of 1-2 ms per point, each point in the stored records consisted of the average of five to ten determinations, respectively. Data were stored digitally and analysed with custom programs written in Fortran 77. Results are presented as means + S.E.M. Solutions The compositions of the solutions used were as follows. Krebs-Ringer solution (mm): NaCl, 145; KCl, 5; CaCl2, 2-5; MgSO4, 1; Na-Hepes, 10; and glucose, 10. Dissecting solution (mm): K2SQ4, 95; MgCl2, 10; CaCl2, 0 4; and Na-Hepes, 10. Mounting solution (mm): potassium glutamate, 150; MgCl2, 2; K2-EGTA, 1; and K-Hepes, 10. External solution (mm): TEAmethanesulphonate, 150; CaCl2, 2; MgCl2, 2; TEA-Hepes, 5; and TTX, Internal solution (mm): sodium glutamate, 130; Na- EGTA, 0-1; CaCl2, ; MgCl2, 5-5; Na-Hepes, 5; Na2-ATP, 5; sodium-phosphocreatine, 5; AP3, 1; fura-2, ; and glucose, 5. All the solutions were adjusted to ph 7'2 and 300 mosmol F- at C. The experiments were carried out at a holding potential of -80 mv and at 'C.

3 J Physiol Suppression of Ca2" release in rat muscle 439 RESULTS Inactivation of peak of calcium release Inactivation of calcium release was studied with a prepulse protocol similar to the one used by Simon et al. (1991) and is shown at the bottom of Fig. 1. An 80 ms test pulse to 0 mv was delivered in isolation or preceded by a 200 ms prepulse to different membrane potentials, indicated below the voltage records. In all the cases, a 10 ms step to -120 mv was applied immediately before the test pulse in order to reset the voltage sensors to the resting conditions. The calcium transients elicited with these command pulses are illustrated in the top row of the figure. The leftmost calcium transient was the first in the series and was obtained without a prepulse. The next four transients were obtained in response to test pulses following prepulses of increasing depolarization, from -40 to -10 mv in 10 mv increments. The rate of rise of [Ca2+] during the prepulses increased as the prepulse depolarization was increased. A small decrease in [Ca2+] was observed between the prepulse and the test pulse, corresponding to the 10 ms step to -120 mv. The decrease is better seen for the prepulses to -20 mv and -10 mv, where the rise in myoplasmic [Ca2+] is substantial by the end of the prepulse. Although the amplitude of the calcium transient at the end of the test pulses was bigger for stronger prepulses, the rate of rise of [Ca2+] during the test pulse was slower after the stronger prepulses. The dotted line indicates the amplitude of the first calcium transient obtained without a prepulse. The trace on the right of the row was obtained at the end of the series in response to a test pulse without prepulse. Its amplitude is somewhat smaller than the first calcium transient, probably due to some run-down of the fibre. The amplitude of the calcium transient for a given pulse generally tended to decline gradually during the course of most experiments. A [Ca2+] FM Rate of release 200 ms 2uM ms-' VI mv Figure 1. Calcium-dependent inactivation of the peak release The top row shows the calcium transients elicited with the command pulses displayed in the bottom row. The middle row corresponds to the rate of release calculated from each of the transients using a model for myoplasmic calcium binding and transport with a parvalbumin concentration of 585,m; the off-rate constants for calcium from parvalbumin and troponin are 1-7 s-i and 1.9 x 103 s-' respectively; the offrate constant for magnesium from parvalbumin is 3 8 s-'; the Vmax for the SR calcium pump is 1 4 /SM ms-' and all other removal model parameters are set to values given by Garcia & Schneider (1993). A test pulse to 0 mv lasting 80 ms was delivered in isolation or preceded by a 200 ms prepulse to several potentials. Test pulse and prepulse were separated by a 10 ms step to -120 mv. The dotted line in the top row indicates the maximum amplitude of the first calcium transient during the test pulse in the absence of a prepulse. As the prepulse calcium concentration was increased with stronger prepulses, the amplitude of the calcium transient at the end of the test pulses was bigger, but the rate of rise was slower. The slower rate of rise of the calcium transient is translated as an increasing inhibition of the peak of the release waveform (middle row). Fibre 33, stretched to 3-8 /sm per sarcomere, path length 35/um, temperature 16 C.

4 440 T. X 1 - x J Garcia and M F Schneider J Physiol The records in the middle row of Fig. 1 show the rate of release of calcium from the SR calculated from the calcium transients in the top row. The rate of release waveforms were not corrected for depletion in this or any of the other figures. As we have previously shown, in the absence of a prepulse, the rate of release in rat muscle consists of a fast initial peak that rapidly declines to a level of smaller amplitude (Garcia & Schneider, 1993). As in skeletal muscle from the frog (Simon et al. 1991), the initial peak of release during the test pulses was decreased as calcium levels increased during prepulses of increasing depolarization. Figure 2 shows superimposed the first, fourth and fifth calcium transients (left) and the corresponding rate of release records (right) from Fig. 1, obtained with a test pulse alone (thick line) and with prepulses to -20 mv (thin line) and -10 mv (dashed line), as shown in the pulse diagram. The superimposition of the records makes the effect of the prepulse on the calcium transient and rate of release more evident. The calcium level at the end of the prepulse to -10 mv was greater than for the larger test pulse to 0 mv without prepulse, because the duration of the prepulse was considerably longer (200 ms) than that of the test pulse (80 ms). The rate-of-release records show an increasing suppression of the peak during the test pulse with increasing [Ca2+] at the end of the prepulses. It is also noticeable from these records that the steady level of release was slightly reduced for the test pulses with the prepulse, to -10 mv. This prepulse produced the largest release of calcium, which may have caused significant A [Ca2+] calcium depletion from the SR and thus resulted in a smaller release in the test pulse after the prepulse to -10 mv. The fibre in Figs 1 and 2 showed the highest degree of suppression of the steady level during the test pulse. In a total of nine fibres, prepulses to -15 or -10 mv decreased the steady level of release during the test pulse to % compared with test pulses without prepulses. Quantification of inactivation of release We followed the same procedure as used by Simon et al. (1991) to quantify the degree of inactivation of the release during the test pulses as a function of calcium levels at the end of the prepulse. The expression P/S - 1, where P is the peak and S is the steady level of the rate of release during the test pulses, was used to provide a relative measure of the test pulse release that can be, but was not, inactivated. When the peak of release has the same amplitude as the steady level P/S - 1 would be zero, corresponding to maximal inactivation. Figure 3 shows a plot of the P/S - 1 as a function of the calcium concentration at the end of the prepulses, which was obtained by adding the change in [Ca2+] measured with AP3 at the end of each prepulse, to the resting [Ca2+] measured with fura-2 before the same prepulse. The four filled circles in the graph correspond to test pulses without prepulses, and were obtained before, after and during (two points) the series of pulses with prepulses. The data in Fig. 3 show that the release was suppressed rather steeply as the concentration of calcium increased, reaching a complete inactivation with -2 #m calcium. Rate of release 0-2 /FM 2 mm ms Vm (mv) -80 Vm (mv) 80 ms 80 ms Figure 2. Effect of prepulse stimulation on the calcium transient and the rate of release Calcium transients (left) and rate of release (right) obtained with an 80 ms test pulse to 0 mv without a prepulse (thick line) and with 200 ms prepulses to -20 mv (thin line) or -10 mv (dashed line). The traces were superimposed to show more clearly that the amplitude of the calcium transient increased and the rate of rise was slower during the test pulse when the fibre was stimulated with the prepulse protocol. The peak rate of release during the test pulse was decreased with increasing prepulse A [Ca2+] with a complete inhibition of the peak during the test pulse after the largest prepulse. Although in this fibre the steady level was decreased, it was much less affected than the peak of release. The steady level was not modified in the other fibres studied. The pulse protocol is shown at the bottom of the figure. Same fibre as in Fig. 1.

5 J Physiol Suppression of Ca2" release in rat muscle 441 Table 1. Parameters for the inactivation of the rate of release by calcium Fibre n [Ca2+]50 (M) P P79 fplk,s P963 1P P Mean + S.E.M [Ca2+]50, the concentration of calcium to attain half-maximal inactivation of release; n, the number of bound calcium ions needed to cause inactivation; fp/ks, the fraction of the peak release that can be inactivated and the ratio of peak to steady level during the test pulses. The P/S - 1 data were interpreted using the same model and analysis used previously to describe calcium-dependent partial inactivation of SR calcium release in frog skeletal fibres (Simon et al. 1991). In brief, the model for inactivation consists of two steps: a rapidly equilibrating simultaneous binding of n calcium ions to a receptor (R) to give the calcium-receptor complex (Ca.R), followed by a slower transition of CanR to the inactivated state CanR* (Schneider & Simon, 1988). The equilibrium constants for the first (fast) and second (slow) steps, KF and Ks, are given by R[Cpa ]n/canr and CanR/CanR*, respectively, where R, CanR and CanR* represent the fractions of SR calciumrelease channels in each of the respective states. With this model, P/S - 1 is given by the formula: P/S - 1 = (fp/ks) x K'/(K' + [Ca2+]), (1) from Simon et al. (1991), where fp is a correction factor to account for the increase in inactivation that develops between the end of the prepulse and the time of peak release during the test pulse; K' is equal to KFKs /(1 + Ks) O ~. 0 I [Ca2J (M) Figure 3. Dependence of inactivation of the peak rate of release on calcium concentration The symbols in the graph correspond to the fraction of peak rate of release, P/S - 1, that could be, but was not, inactivated during the test pulses as a function of calcium concentration prior to the test pulse, which was calculated as the sum of the resting calcium before stimulation and the amplitude of the calcium transient at the end of the 200 ms prepulse. *, values obtained when no prepulse was applied; 0, values when a prepulse was used. The smooth curve corresponds to the fitting of the data to eqn (1) in the text. The best-fit parameters for this fibre were: [Ca2+]50 = FM, n = 2-14, fp/ks = Fibre 28, stretched to 3-8jum per sarcomere, path length 32,um, temperature 150C.

6 442 J Garcia and M. F Schneider J Physiol Assuming fp to be the same for the test pulses after all prepulses, fp becomes a constant scale factor that does not influence the relative calcium dependence of P/S - 1. The maximum value of P/S - 1, which is reached as [Ca2+] approaches zero, is fp/ks and the [Ca2+] needed for halfmaximal inactivation, [Ca2+]5o, is the nth root of K'. The smooth curve through the points in Fig. 3 represents the best fit of eqn (1) to the P/S - 1 data. The values for K', fp/ks and n provided by fitting equation (1) to the data for the fibre in Fig. 1 and eight other fibres are presented in Table 1. From the different fibres, we found that on average, [Ca2+]50 was um (n = 9). The number, n, of bound calcium ions needed to inactivate release was -3, with a mean of '6. The mean value of fp/ks was These values are very similar to those found by Simon et al. (1991) for frog skeletal muscle. In resting conditions, when [Ca2+] is closer to zero, eqn (1) approximates fp/ks. The fraction of the peak release that can be inactivated and the ratio of peak to steady level during the test pulses is denoted by fp/k5, and is represented in the left part of the graph. The value of fp/ K. for the fibre in Fig. 3 was 1X96. Effect of procaine on the calcium transient and the rate of release Since procaine is purported to be a specific inhibitor of calcium-induced calcium release which does not suppress depolarization-induced calcium release, even at 10 mm concentration in mammalian peeled skeletal muscle fibres (Donaldson, 1985, 1986), we investigated the effects of procaine on SR calcium release in our voltage clamped mammalian fibres. Figure 4 shows the effect of 0 3 mm procaine on the calcium transient (upper row) and the SR rate of release (middle row). The fibre was depolarized using 60 ms pulses to 0 and 10 mv (bottom row) in control conditions (left column), after the addition of 03 mm procaine to the external solution (middle column), and upon returning to procaine-free solution (right column). Procaine reduced the amplitude and decreased the rate of rise of the calcium transient at both membrane potentials. The effect of procaine was present as soon as we were able to stimulate the fibre after a 1-2 min period, required to allow the temperature to equilibrate after addition of the drug. In different fibres, the maximal inhibition was reached in 3-5 min. The records in the middle row of Fig. 4 show that AControl Procaine Wash (0.3 mm) A[Ca2+] 1 /SM Rate of release 10 0n ms n n A5SM ms' ~~~~~~~~~~Vm (mv) Figure 4. Effect of procaine on the calcium transient and the rate of release of calcium from the SR The top row illustrates calcium transients obtained in response to 60 ms test pulses to 0 or 10 mv under control conditions (left), after the addition of 0 3 mm procaine (middle), and after washout (right). Procaine caused a partially reversible decrease in the calcium transient amplitude. The middle row illustrates the rate-of-release records calculated from the above calcium transients using a model for myoplasmic calcium binding and transport with a parvalbumin concentration of 387 Am; the off-rate constant for calcium from parvalbumin is 2f0 s-'; the off-rate constant for magnesium from parvalbumin is 11 6 s-'; the Vmax for the SR calcium pump is 2X3 fm ms-' and all other removal model parameters are set to values given by Garcia & Schneider (1993). Procaine reduced the amplitude of the peak of release and the steady level. The bottom row shows the voltage steps applied from the holding potential of -80 mv. Fibre 40, stretched to 3-8,um per sarcomere, path length 45 hum, temperature 16 C.

7 J Physiol Suppression of Ca2" release in rat muscle 443 procaine caused a decrease in the rate of release of about 50%, with little effect on the shape of the release waveform. The effect of 0 3 mm procaine was tested in six fibres, and in four of them we could maintain the fibre during washout of the drug from the external solution. In the four fibres with washout, the effect of procaine was reversible to different degrees, attaining 100% reversibility in one of the fibres. The right column in Fig. 4 shows the records obtained after procaine was eliminated from the bath in this experiment. The calcium transient and the rate of release showed a recovery of about 25%. In the four fibres with washout, 0 3 mm procaine produced a mean percentage inhibition of the peak and steady release of and %, respectively, at 0 mv. At 10 mv the inhibition was % for the peak release and % for the steady level. The inhibition of the steady level was calculated as the percentage change in the last 5-10 ms of release records not corrected for depletion of the SR. The amplitude of the peak of the release after washout was % (n= 4) compared with the peak release before procaine. The amplitude of the steady level recovered more after washout and was, on average, % (n = 4) compared with that before procaine. In an attempt to investigate whether the entire release or one of its components could be blocked completely, we tested higher concentrations of procaine (0 5 mm in two fibres and 1 mm in one) as used in frog muscle experiments (Klein et al. 1992), but the fibres showed an immediate increase of leak current which was not reversible, indicating that rat fibres may be more sensitive to procaine than are frog fibres. DISCUSSION Calcium-dependent inactivation of calcium release The experiments presented in this paper indicate that the rate of release of calcium from the SR in isolated rat skeletal muscle can be modulated by calcium. Elevations of myoplasmic calcium concentration produced by 200 ms prepulses to different membrane potentials caused a marked decrease of the peak rate of release during a subsequent test pulse. Inactivation of release showed a strong calcium dependence, indicating that on average, binding of about three calcium ions was necessary to inactivate each release channel. Experiments with frog muscle have led to the suggestion that the peak or inactivating component of release may be caused by a calcium-induced mechanism (Jacquemond et al. 1991; Csernoch, Jacquemond & Schneider, 1993). At the same time, the peak of release can be inactivated by increasing calcium concentrations (Simon et al. 1991). A similar behaviour has been observed for calcium release from heavy SR vesicles; calcium release is activated by micromolar concentrations of calcium and inhibited by higher concentrations (Kim et al. 1983; Meissner et al. 1986). Therefore, it seems that, in order to produce calcium-induced release in muscle fibres, the affinity of calcium binding sites for activation would be higher than the sites for inactivation, or that the activation process develops rapidly, whereas the inactivation occurs with a delay. Analysis of the calcium dependence of inactivation showed that about 0-22 /M free calcium caused half-maximal inactivation of release. This value, however, should be taken as a lower limit (Morgan, 1993), since we are measuring the transients with AP3 and using a calibration based on the assumption that all of the dye is available to react with calcium as in free solution, which may not apply to muscle fibres (see Maylie, Irving, Sizto & Chandler, 1987; Baylor & Hollingworth, 1988; Klein et al. 1988; and Konishi, Olson, Hollingworth & Baylor, 1988). Furthermore, the measurements of calcium transients with our apparatus in this preparation are an average over the total fibre volume within the optical path and, therefore, do not correspond to the local concentration of calcium in the vicinity of the release channel, which may be considerably higher. In any event, the value of [Ca2+]50 found in this paper, using rat fibres, is fairly close to the value of 0 34 /M reported for frog fibres using similar recording procedures (Simon et al. 1991). Inhibition of calcium release by procaine The local anaesthetic procaine has been widely used as a blocker of calcium-induced calcium release. Its direct effect has been studied recently, at the single channel level, on ryanodine receptor-ca2+release channels incorporated in lipid bilayers. Procaine decreased the channel open probability of the purified rabbit skeletal muscle channel twofold (Xu, Jones & Meissner, 1993), while it increased the longest closed time of the canine cardiac muscle channel (Zahradnikova & Palade, 1993). Both mechanisms would tend to decrease the total current through the channel. In the present paper we investigated whether procaine specifically blocks a calcium-induced component of release in voltage clamped rat fibres as previously claimed for mammalian peeled fibres (Donaldson, 1985, 1986). Procaine, at a concentration of 0 3 mm in the external solution, reversibly decreased the amplitude of the calcium transients. Both the peak and the steady level of the rateof-release were inhibited by about 50%. Therefore, we are inclined to think that, as in frog fibres (Klein et al. 1992), the effect of procaine might be non-specific even at 0 3 mm in voltage clamped rat fibres. In view of our findings it seems unlikely that any calcium release that is activated by solution change depolarization of peeled mammalian fibres in the presence of 10 mm procaine (Donaldson, 1985, 1986) could be related to the faster calcium release activated by voltage clamp (or action potential) depolarization of mammalian fibres. Our results also indicate that rat fibres

8 444 J Garcia and M. F Schneider J Physiol were more sensitive than frog fibres to procaine, since the maximum concentration that we could use was 0 3 mm, whereas in similar experiments, frog fibres could tolerate concentrations of 1 mm or more (Klein et al. 1992). Nevertheless, we believe that the effect of procaine we observed here was not due to toxicity of the anaesthetic because the transient was at least partially recovered after washout of the drug in three fibres and fully recovered in the fourth fibre. BAYLOR, S. M., CHANDLER, W. K. & MARSHALL, M. W. (1983). Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from arsenazo III calcium transients. Journal of Physiology 344, BAYLOR, S. M. & HOLLINGWORTH, S. (1988). Fura-2 calcium transients in frog skeletal muscle fibres. Journal of Physiology 403, BRUM, G., RIos, E. & SCHNEIDER, M. F. (1988). A quantitative model of calcium removal from the myoplasmic solution. Appendix to BRUM, G., Rios, E. & STEFANI, E. (1988). Effects of extracellular calcium on the calcium movements of excitation-contraction coupling in skeletal muscle fibres. Journal of Physiology 398, CSERNOCH, L., JACQUEMOND, V. & SCHNEIDER, M. F. (1993). Microinjection of strong calcium buffers suppresses the peak of calcium release during depolarization in frog skeletal muscle fibers. Journal of General Physiology 101, DONALDSON, S. K. (1986). Mammalian muscle fiber types: Comparison of excitation-contraction coupling mechanisms. Acta Physiologica Scandinavica 28, DONALDSON, S. K. B. (1985). Peeled mammalian skeletal muscle fibers. Possible stimulation of Ca2' release via a transverse tubule-sarcoplasmic reticulum mechanism. Journal of General Physiology 86, ENDO, M., TANAKA, M. & OGAWA, Y. (1970). Calcium-induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibers. Nature 228, FORD, L. E. & PODOLSKY, R. J. (1970). Regenerative calcium release within muscle cells. Science 167, GARCIA, J. & SCHNEIDER, M. F. (1993). Calcium transients and calcium release in rat fast-twitch skeletal muscle fibres. Journal of Physiology 463, GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca2' indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, GYORKE, S. & PALADE, P (1992). Calcium-induced calcium release in crayfish skeletal muscle. Journal of Physiology 457, HoRowIcz, P. & SCHNEIDER, M. F. (1981). Membrane charge moved at contraction thresholds in skeletal muscle fibres. Journal of Physiology 314, JACQUEMOND, V., CSERNOCH, L., KLEIN, M. G. & SCHNEIDER, M. F. (1991). Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibres. Biophysical Journal 60, JONG, D. S., PAPE, P., CHANDLER, W. K. & BAYLOR, S. M. (1993). Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle. Journal of General Physiology 120, KIM, D. H., OHNISHI, S. T. & IKEMOTO, N. (1983). Kinetic studies of calcium release from sarcoplasmic reticulum in vitro. Journal of Biological Chemistry 258, KLEIN, M. G., SIMoN, B. J. & SCHNEIDER, M. F. (1990). Effects of caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle fibres. Journal of Physiology 425, KLEIN, M. G., SIMON, B. J. & SCHNEIDER, M. F. (1992). Effects of procaine and caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle. Journal of Physiology 453, KLEIN, M. G., SIMON, B. J., SztCs, G. & SCHNEIDER, M. F. (1988). Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators. Biophysical Journal 53, KoNIsHI, M., OLSON, A., HOLLINGWORTH, S. & BAYLOR, S. M. (1988). Myoplasmic binding of Fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophysical Journal 54, KovACS, L., RIos, E. & SCHNEIDER, M. F. (1983). Measurement and modification of free calcium transients in frog skeletal muscle fibres by a metallochromic indicator dye. Journal of Physiology 343, LAI, F. A., ERICKSON, H. P., ROSSEAU, E., LIu, Q.-Y. & MEISSNER, G. (1988). Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 331, MAYLIE, J., IRVING, M., SIZTO, N. L. & CHANDLER, W. K. (1987). Calcium signals recorded from cut frog twitch fibers containing Antipyrylazo III. Journal of General Physiology 89, MEISSNER, G., DARLING, E. & EVELETH, J. (1986). Kinetics of rapid Ca2' release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides. Biochemistry 25, MELZER, W., RIos, E. & SCHNEIDER, M. F. (1984). The course of calcium release and removal in skeletal muscle fibers. Biophysical Journal 45, MELZER, W., Rios, E. & SCHNEIDER, M. F. (1986). The removal of myoplasmic free calcium following calcium release in frog skeletal muscle. Journal of Physiology 372, MELZER, W., Rios, E. & SCHNEIDER, M. F. (1987). A general procedure for determining calcium release from the sarcoplasmic reticulum in skeletal muscle fiber. Biophysical Journal 51, MORGAN, K. G. (1993). Cai versus [Ca2+]i. Biophysical Journal 65, Rios, E. & BRUM, G. (1987). Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle. Nature 325, SCARPA, A., BRINLEY, F. J. & DUBYAK, G. (1978). Antipyrylazo III, a 'middle range' Ca2+ metallochromic indicator. Biochemistry 17, SCHNEIDER, M. F. & CHANDLER, W. K. (1973). Voltage dependent charge movement in skeletal muscle. Nature 242, SCHNEIDER, M. F. & SIMON, B. J. (1988). Inactivation of calcium release from the sarcoplasmic reticulum in frog skeletal muscle. Journal of Physiology 405,

9 J Physiol Suppression of Ca2" release in rat muscle 445 SIMON, B. J., KLEIN, M. G. & SCHNEIDER, M. F. (1991). Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers. Journal of General Physiology 97, SMITH, J. S., IMAGAWA, T., MA, J., FILL, M., CAMPBELL, K. P. & CORONADO, R. (1988). Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum. Journal of General Physiology 92, STEPHENSON, E. W. (1985). Excitation of skinned muscle fibers by imposed ion gradients. Journal of General Physiology 86, TAKESHIMA, H., NISHIMURA, S., MATSUMOTO, T., ISHIDA, H., KANGAWA, K., MINAMINO, N., MATsuo, H., UEDA, M., HANAOKA, M., HIROSE, T. & NUMA, S. (1989). Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339, Xu, L., JONES, R. & MEISSNER, G. (1993). Effects of local anaesthetics on single channel behaviour of skeletal muscle calcium release channel. Journal of General Physiology 101, ZAHRADNiKOVA', A. & PALADE, P (1993). Procaine effects on single sarcoplasmic reticulum Ca2+ release channels. Biophysical Journal 64, Acknowledgements We thank Gerard Vaio and Alex Bustamante for technical assistance, Gabe Sinclair and Walt Knapick for constructing mechanical and optical apparatus and Jeff Michael and Chuck Leffingwell for electronics support. This work was supported by research grants from the NIH (RO1-NS23346, ROI-NS33578 and PO1-HL27867). Author's present address J. Garcia: Department of Physiology, Colorado State University, Fort Collins, CO 80523, USA. Received 18 July 1994; accepted 16 November 1994.