341
Journal of Physiology (1992), 453, pp. 341-366 With 13 figures Printed in Great Britain
EFFECTS OF PROCAINE AND CAFFEINE ON CALCIUM RELEASE FROM THE SARCOPLASMIC RETICULUM IN FROG SKELETAL MUSCLE
BY MICHAEL G. KLEIN, BRUCE J. SIMON* AND MARTIN F. SCHNEIDER From the Department of Biological Chemistry, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201, USA
(Received 25 April 1991) SUMMARY
1. Resting myoplasmic free [Ca2+] and [Ca21] transients (A[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 (Rrei) 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 A[Ca2+] and the corresponding Rrei 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 Rrei 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 A[Ca2+]. 4. Although 1 mM-procaine depressed Rrel it had no effect on intramembrane charge movements (IQ) calculated from membrane currents recorded simultaneously with A[Ca2+]. 5. Procaine (1 mM) reversibly inhibited the potentiating effect of 0 5 mM-caffeine on A[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 A[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 A[Ca2+] after 10-30 ms duration voltage steps compared to the bracketing control and wash runs without procaine. Rrei 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 * Present address: Electro-Biology, Inc., 6 Upper Pond Road, Parsippany, NJ 07054, USA. MS 9337
342
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
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. 8. The absence of a significant effect of procaine on the Rrel waveform and voltage dependence for long pulses suggests that the major component of suppression of release by procaine may be a non-specific block of SR calcium-release channels. INTRODUCTION
Contraction of skeletal muscle is initiated by the release of calcium from the sarcoplasmic reticulum (SR). The stimulus for SR calcium release is depolarization of the transverse tubules (TT) but the mechanism which couples this electrical signal to opening of calcium channels in the SR is unknown. It has been proposed that this coupling may involve a direct molecular link between the TT voltage sensors and the SR channels (Chandler, Rakowski & Schneider, 1976), or be mediated by a second messenger, possibly inositol 1,4,5-trisphosphate (1P3) (Vergara, Tsien & Delay, 1985) or calcium (Frank, 1980). The ability of calcium to open SR release channels from skeletal muscle via 'calcium-induced calcium release' (CICR) has been demonstrated in skinned fibres (Ford & Podolsky, 1970; Endo, Tanaka & Ogawa, 1970), isolated SR vesicles (Miyamoto & Racker, 1982) and in SR calcium-release channels incorporated into bilayers (Smith, Coronado & Meissner, 1986). However, the involvement of CICR in physiological activation of skeletal muscle has not been established and has been questioned (Endo, 1984, 1985). To investigate the role of CICR in skeletal muscle activation we have previously shown (Simon, Klein & Schneider, 1989; Klein, Simon & Schneider, 1990) that caffeine, a potentiator of CICR (Endo, 1975a, b, 1985), can cause skeletal muscle to exhibit [Ca2"] transients with properties suggestive of a regenerative CICR mechanism. In the presence of 0 5 mM-caffeine, the rate of calcium release (Rrei) is increased, the voltage dependence of release is shifted to the left, and the time course of decay of A[Ca2+] after repolarization is slowed. Most significantly, however, release can no longer be turned off rapidly by repolarization after relatively short (10-20 ms) pulses but continues for tens of milliseconds after the end of the pulse. This latter observation, uncoupling of TT voltage control of SR calcium release in the presence of caffeine, is qualitatively consistent with a positive feedback of [Ca2+] on calcium release that could be produced by CICR. Procaine has been shown to be a potent inhibitor of the potentiating effects of caffeine on calcium release in intact skeletal muscle (Feinstein, 1963), SR vesicles (Morii & Tonomura, 1983) and skinned skeletal muscle fibres (Endo, 1985), presumably by inhibiting CICR. Its lack of an effect on intact muscle at low concentrations in the absence of caffeine has been taken as an indication that CICR does not occur during physiological excitation-contraction (E-C) coupling (Endo, 1984, 1985), but this conclusion has been debated. The aim of the present investigation was to study the effects of procaine on SR calcium release in the presence and absence of caffeine to determine whether CICR played a significant role in E-C coupling under physiological conditions. If procaine could inhibit the effects of CICR observed in the presence of caffeine, then any effects of procaine on release in the absence of caffeine might be interpretable in terms of a 'negative caffeine'
EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE
343
effect due to procaine blocking CICR in the absence of caffeine. An abstract describing some of these results has appeared (Klein, Schneider & Simon, 1989). METHODS
Frogs (Rana pipiens, northern variety) were killed by decapitation and the spinal cord destroyed. Experiments were carried out on single skeletal muscle fibres dissected from ileofibularis or semitendinosus muscles and mounted in a double Vaseline-gap chamber as described previously (Klein et al. 1990). The fibres were stretched to sarcomere lengths of 3 8-42 ,sm to eliminate movement and voltage clamped to a holding potential of -100 mV. Two calcium indicators were used simultaneously: antipyrylazo III (Ap III), an absorbance dye with relatively low affinity for [Ca2+], and Fura-2, a fluorescent dye with high affinity for [Ca2+]. Ap III was used to measure calcium transients (A[Ca2+]) and Fura-2 was used to monitor resting [Ca2+] and small changes in
[Ca2+]. Details of optical procedures and methods for calculating A[Ca2+] from the Ap III absorbance changes and [Ca2+] from Fura-2 fluorescence changes were as described previously (Klein, Simon, Szucs & Schneider, 1988). Current records were analysed (Melzer, Schneider, Simon & Szucs, 1986) to obtain the non-linear component of current (IQ) due to intramembrane charge movement. All experiments were carried out at 6-10 'C. The internal solution applied to the cut ends contained (mM): 102-5 caesium glutamate, 5.5 MgCl2, 5 ATP (sodium salt), 4-5 sodium Tris-maleate buffer, 13-2 caesium Tris-maleate buffer, 0 1 EGTA, 5 creatine phosphate (sodium salt), 1 Ap III, 0 05 Fura-2, and 1 g 1-1 glucose. The external solution applied to the intact portion of the fibre in the middle pool contained (mM): 75 (TEA)2504, 5 C82SO2, 7-5 total CaSO4, 5 sodium Tris-maleate buffer and 10-7 g ml-' tetrodotoxin. The pH was adjusted to 7 0 at room temperature for both solutions. Procaine (0 32-1 mM), caffeine (0 5 mM) or a combination of the two drugs were dissolved in external solution. Drug effects were determined after changing the solution bathing the intact portion of the fibre in the middle pool to external solution containing the desired concentrations) of the drug(s). Solution changes were carried out by simultaneously pipetting solution to one side of the chamber while aspirating from the other. Each new solution (drug-containing or drug-free wash solution) applied to the middle pool was precooled to 4 'C so as to minimize any change in temperature of the fibre. The procedure for calculating the rate of calcium release (Rrei) from the SR from the A[Ca2+] records follows the method developed by Melzer, Rios & Schneider (1984, 1987), as described in detail in Klein et al. (1990). Briefly, the calcium removal properties of the fibre were characterized by fitting a calcium removal model to the decay of A[Ca2+] after several pulses elicited at various times during each experiment. In all cases in which the effect of a drug or drugs were investigated the [Ca2+] records used for characterizing the calcium removal properties of the fibre included records obtained both before applying the drug(s) and after washing the drug(s) from the chamber (Klein et al. 1990). The values of the removal model parameters that were obtained from the fit to the decay of the control and wash [Ca2+] records were then used to calculate the Rrel for each [Ca2+] transient, both before applying the drug(s), in the presence of drug(s) and after washing the drug(s) from the chamber. This procedure assumes that neither procaine, caffeine nor the combination of the two drugs altered the calcium removal properties of the fibre. The removal model used for the present calculations includes the calcium binding properties of troponin C, parvalbumin and the SR calcium pump (Klein et al. 1990). The troponin C sites were assumed to be present at a concentration of 250 #M with 'on' and 'off' rate constants of 1-3 x 108 M-1 s-1 and 1000 s-1. The 'on' rate constants for binding of calcium and magnesium to parvalbumin were assumed to be 1P6 x 108 M-1 s-1 and 4 x 104 M-1 s-1 respectively. The concentration of parvalbumin and the 'off' rate constants of calcium and magnesium binding were determined by least-squares fitting of the removal model to the decay of [Ca2+] transients after several pulses (above). The SR calcium pump was assumed to be in instantaneous equilibrium with [Ca2+] at a concentration of 200 /M with a dissociation constant between 1-5 and 3 ,UM. Throughout the text average values are given as the mean+ S.E.M. Statistical significance was determined using a two-tailed t test, with significance for P < 0 05.
12
PH Y 453
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
344
RESULTS
Effects of procaine on calcium transients and Rrei Figure 1 shows the effects of procaine on calcium transients for 60 or 100 ms pulses (bottom) to the indicated potentials from a holding potential of -100 mV. Panel A A
Control
B
Procaine
C
Wash A
[Ca2+]
1
JiM
V(MV) +20
200 ms
-20
-30 -40
-100 mV Fig. 1. Effect of 1 mM-procaine on [Ca2+] transients measured with Ap III. A, [Ca2+] transients before exposure to procaine for 60 or 100 ms pulses to the potentials (V) indicated on the left from a holding potential of -100 mV; B, [Ca2+] transients for the same pulses after the external solution was exchanged with one containing 1 mM-procaine (12 min exposure); C, after washing procaine from the fibre. Bottom traces, superimposed voltage records for all pulses in panel. Fibre 488; stretched to a sarcomere spacing of 3-9 ,um per sarcomere; temperature 9 'C; Ap III concentration 850-1169 /M.
of Fig. 1 presents A[Ca2+] measured in control external solution before applying procaine, panel B shows A[Ca2+] for the same potential steps as in the control but after changing the external solution to one containing 1 mM-procaine and panel C shows A[Ca2+] obtained within 2 min of returning to the procaine-free external solution. At each potential 1 mM-procaine depressed the A[Ca2+] signal to roughly half its control amplitude. This effect was reversible as shown by the traces in Fig. 1 C. The [Ca21] transients recorded after removing procaine from the external solution were actually slightly larger in this run than in the control, although this result was not consistently observed. For the fibre in Fig. 1 the resting [Ca2+] was 28 nm in control solution before applying procaine, 32 nm in the presence of 1 mm- procaine, and 30 nm after washing procaine from the chamber. In six fibres the mean resting [Ca2+] was 19-7 + 3-2 nm before procaine, 20-7 + 3-5 nm in 1 mM-procaine and 27-3 + 7-0 nm after washing procaine from the chamber. Thus, on average procaine reduced the resting [Ca2+] by 14 % as compared to the bracketing controls.
345 EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE Figure 2 presents Rrei records calculated from the A[Ca2+] records shown in Fig. 1. Panel A shows Rrel before application of procaine to the fibre. These records are similar to Rrei records reported previously (Melzer et al. 1984; Klein et al. 1990), showing a rapid early release of calcium from the SR followed by a partial A
Control
B
Procaine
C
Wash Rate of release 5 yMms1
V(mV)
+20 100 Ms
-20
-30 -40
---..~
-100 mV J--
Fig. 2. Effect of 1 mM-procaine on the rate of release of calcium (Rre,) from the SR. Rrei was calculated from the calcium transients in Fig. 1 (see Methods). Same format and conditions as in that figure, except that the time base is expanded twofold. The parameters which were varied in the removal model to give the least-squares fit to the decay of A[Ca2+] after the several pulses were the 'off' rate constant of Mg2+ from parvalbumin (5 3 s-1), and the maximum pump rate of the SR calcium pump (805 IM s-1). The total concentration of the Ca2+/Mg2+ sites on parvalbumin was set to 300 /M, and the 'off' rate constant of [Ca2+] binding to parvalbumin was set to 1-5 s-l. The values of the other parameters in the removal model used to calculate Rre, are given in the Methods section.
inactivation of release. Panel B shows Rrei records in the presence of 1 mM-procaine. Procaine decreased the amplitude of Rrei by about 50 % at each membrane potential but had little effect on the shape of the release waveform. Panel C shows Rrei after washing procaine from the chamber. The release amplitude largely recovered after the wash. The slight difference in the waveform of the Rrei records between the control and the wash (e.g. at -20 and + 20 mV), with the wash records exhibiting a somewhat smaller peak and a slower decline from the peak, is typical of changes of Rrel records over time in the absence of procaine or any other drug (Klein et al. 1990). The slowing of inactivation during the course of an experiment may be related to the loss with time from the myoplasm of a diffusible substance involved in inactivation of release (Schneider & Simon, 1988). In order to evaluate the effects of procaine on the peak and steady components of Rrei it was first necessary to correct the Rre, records for effects due to depletion of 12-2
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
346
calcium from the SR (Schneider, Simon & Szucs, 1987; Schneider, Simon & Klein, 1989; Klein et al. 1990). Figure 3 shows [Ca2l] transients (top row) and the corresponding Rrei (second row) recorded from a fibre for a 120 ms pulse to 0 mV in control conditions (A), during exposure to 1 mM-procaine (B) and after washing A Control
B Procaine
C
Wash 0.5 pim
A [Ca2+]
100 ms
Rrel
5
Rrei corrected for depletion
m s1
0
F 1F
-100 mV
Fig. 3. Correction for depletion of calcium from the SR during release in the presence and absence of procaine. Upper row, [Ca2+] transients elicited by a 120 ms depolarization to 0 mV (bottom row) in control (A), during a 9 min exposure to 1 mM-procaine (B) and after washing procaine from the fibre (C). Second row, Rrei calculated from the [Ca2+] transients above. Third row, Rre, after correction for depletion of [Ca2+], assuming the SR [Ca2+] content to be 900 /IM before the pulse. The removal parameters used to calculate rrel were koffMg-Parv = 7 2 sol, and Vm.. = 2145 #M s-1. The parvalbumin concentration was set to 600 #M. Fibre 486; [Ap III] 729-985 #m; temperature 9 'C; sarcomere length
4-1
Itm.
procaine from the chamber (C). As described above, procaine reversibly depressed both A[Ca2+] and Rrei without changing the Rrei waveform. The third row in Fig. 3 shows the Rrei records after correcting for the effects due to depletion of calcium from the SR, assuming that the SR calcium content was 900 AEM (equivalent concentration if all SR calcium were free in myoplasmic water) before the pulses in all cases. The main effect of the correction for depletion was to increase the amplitude and remove the slight downward slope of the 'steady' component of release. This is especially evident in the records measured under procaine-free conditions since the releases were larger and the SR was presumably depleted to a greater extent. In the fibre of Fig. 3, after correction for depletion procaine suppressed the peak and steady components of release by 54 and 58%, respectively, compared to the bracketing controls, indicating about the same degree of suppression of both the peak and steady components of release by procaine. Figures 2 and 3 indicate that 1 mm-procaine decreased the amplitude of the release
EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE 347 without major modification of the Rrei waveform. The ratio of the peak to the steady level of Rrel provides an estimate of the relative contribution of the peak component compared to the steady component of release (Simon et at. 1991). After correction for depletion the ratio of peak to steady level of Rrei was 7-1, 5-6 and 3-7 in control, procaine and wash, for pulses to -20 mV for the fibre in Figs 1 and 2 and was 3 9, 3 0, 2-7 for the fibre in Fig. 3 (pulses to 0 mV). Similar results were observed in other fibres. In five fibres exposed to 1 mM-procaine the average ratio of peak to steady level for pulses to -20 or 0 mV was 4-9 + 0 9 in control, 3-7 + 0'8 in procaine and 3 0 + 0 3 in the wash. The mean values of peak and steady levels of Rrel in control and wash reflect the slight run-down of the peak component of release with time which we observe routinely (above). The ratio of the peak to steady level in the presence of procaine decreased by 8-7 + 8-0 % compared to the average of bracketing controls (P < 0'4), while in the same fibres the peak amplitude of the release decreased by a mean of 41-6 + 88% (P < 0-001, data not shown). Thus the release waveform was relatively unchanged by procaine whereas the amplitude of the Rrel was suppressed significantly. Although the estimation of the initial SR calcium content from the correction for depletion is somewhat subjective, the records of Fig. 3 indicate that procaine had little effect on the SR calcium content as compared to the controls. The same observation was made in four other fibres in which depolarizations of sufficient duration were applied to estimate the SR content in control, 1 mM-procaine and subsequent procaine-free wash.
Voltage dependence of Rrei in procaine One possible mechanism whereby procaine might inhibit calcium release at a given potential is by shifting the voltage dependence of release to more positive potentials. We therefore examined the voltage dependence of release with and without procaine. Figure 4A is a plot of the peak Rrei vs. membrane potential, for the same fibre as in Figs 1 and 2, in controls (0), in 1 mM-procaine (@) and after washing the fibre with procaine-free external solution (El). Although in this fibre there was some run-down in the peak Rrei between the control and wash, procaine depressed peak Rrei by about the same percentage at all potentials. The continuous lines are non-linear leastsquares fits to the data of the cube of the Boltzmann equation R =Rmax[1+exp((V-V)/k)]-3, (1) where R is the peak rate of release at voltage V, Rmax is the maximum peak rate of release, V is the midpoint voltage and k is the steepness factor of the uncubed Boltzmann equation. This expression was used as an empirical means of comparing the voltage dependence of peak Rrei since it has been previously shown to provide an adequate fit for the voltage dependence of the peak Rrei (Melzer et al. 1986). Figure 4B shows the same data and curves as in A but normalized to the maximum peak Rrel determined from each fit of the cube of the Boltzmann equation in Fig. 4A. Figure 4B shows that 1 mM-procaine had little effect on the steepness or midpoint voltage of release. The mean+s.E.M. value of the parameters of the least-squares fit of eqn (1) are given in Table 1 for the fibre in Fig. 4 and for two others in which the complete
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
348
voltage dependence of the peak rate of release was determined in control, procaine and wash runs (rows 1 to 3, respectively). Table 1 also gives the corresponding parameters for the steady component of release for the same fibres. The records were first corrected for depletion of calcium from the SR, as described in connection with 20 A
15 10 Is 5
(a, 0
L-
0-0
1-0 - B Normalized 0)
0.8 0
°N 0.6 o 0.4 -
0.2
0-0 -80 I
-60
-40 -20 Membrane potential (mV)
0
20
Fig. 4. The voltage dependence of the peak Rrei before, during and after exposure to 1 mmprocaine. A, voltage dependence of the peak Rrei from the experiment in Fig. 2. The continuous lines are least-squares fits of the cube of Boltzmann equation (eqn (1)) to the data with the following parameter values: control (0), Rmax = 16-1 gM ms-', V = -37-3mV, k= 10OmV; procaine (*), Rmax=89 zuMms-1, V=-36-2mV, k= 11 1 mV; wash (a), Rmax = 13-8, M ms-1, V= -37-6 mY, k = 10-2 mV. B, the data and theoretical curves from A normalized to the value of Rmax determined from the leastsquares fit to each data set.
Fig. 3 before carrying out the fits to obtain the values in Table 1. The last two rows of Table 1 give the mean value of the ratio and difference, respectively, of the value in procaine to the mean of the values in the bracketing control and wash runs. The ratios show that in these fibres 1 mm-procaine suppressed the peak and steady release to 66 + 5 and 63 + 10 % of the bracketing controls, respectively. These changes were statistically highly significant (P < 0-01 orP < 0'05). The midpoint voltage, V, was not significantly different in procaine for either the peak or steady component of release. The steepness factor, k, was increased slightly in the presence of procaine compared to the bracketing control and wash, by 28 + 11 % for the peak component (P < 0-05)
:F^ECTS OF PROCAINE AND CAINE ON MUSCLE
349
and by 17 + 20% for the steady component (P < 04) of release. Thus, the main results of Fig. 4 and Table 1 are that Rrei was reduced by about the same percentage at all potentials in procaine but that the voltage dependence of release was unaffected by the procaine. Does procaine interact with Ap III? The apparent depression of calcium release in the presence of procaine might not reflect a true inhibition of release but might instead be an artifact due to an TABLE 1. Parameter values from the least-squares fit of the voltage dependence of the rate of release to the cube of the Boltzmann equation in the presence and absence of 1 mM-procaine k (mV) V (mV) RmaX (JCM ms-1)
Control Procaine Wash Procaine/ no procaine
P 19-9+93 139+7-1 204+8-3 0-66 + 005
8 6-4+22 4-6+ 19t 7-7+10 0-63 + 0-10
P S P -400+4-3 -34-9+40 93+0(4 -407 +42 -383 +5-7 13-5+ 16t -404+43 -382+59 118+1 1 1-28 + 0-11
S 15-1+1-6 17-8+2-6 15-4+19 1-17 + 020
-0 4+ 1-4 -2 1 + 1.1 Procaineno procaine Rmax is the maximum rate of release, V is the midpoint voltage and k the steepness factor from a least-squares fit of eqn (1) to the data from three fibres. Entries are given as the mean+ S.E.M. of the value. P is the peak value, S is the steady component of the rate of release after correction for depletion of calcium from the SR (see text). The estimates of the SR calcium content ranged from 700 to 1000 ,aM in the three fibres. The two lowermost rows are the ratio and difference, respectively, of the value in procaine and the average of the control and wash runs in the absence of procaine (no procaine). t indicates statistical significance at P < 005, 1 significance at P < 001.
interaction of procaine with Ap 111. To explore this possibility we compared [Ca2+] transients calculated from Ap III absorbance changes with those calculated from the Fura-2 fluorescence changes measured simultaneously. The top row in Fig. 5 shows A[Ca2+] determined from Ap III absorbance changes for a 100 ms depolarization to -20 mV before, during and after wash of 1 mM-procaine in the external solution. In this fibre 1 mM-procaine reversibly decreased the amplitude of A[Ca2+] by more than 80 %. The middle row shows the Fura-2 percentage saturation records calculated from the Fura-2 fluorescence signals at 380 nm measured simultaneously with the Ap III absorbance signal (Klein et al. 1988). Although it is clear the procaine also decreased the amplitude of the Fura-2 signal, it is difficult to make quantitative comparisons with Ap III using the Fura-2 saturation signals because Fura-2 is a nonlinear calcium indicator at these A[Ca2+] levels. In order to determine [Ca2+] from the Fura-2 percentage saturation signals we followed the procedure of Klein et al. (1988). The bottom row in Fig. 5 shows [Ca2+] transients determined from the Fura-2 signals (see legend to Fig. 5 for Fura-2 kinetic constants used to calculate [Ca2+]). Although much noisier than the corresponding Ap III [Ca2+] transients, the amplitudes and wave forms of the Fura-2 [Ca2+] transients are very similar to those recorded with Ap III. In particular, in the
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
350
presence of procaine (Fig. 5B) the [Ca2"] transient calculated using the Fura-2 fluorescence signal with the kinetic parameters determined in controls is nearly identical to the A[Ca2+] determined from the Ap III signal. We also measured the absorbance spectrum of Ap III in cuvettes (0-2 cm pathlength) containing internal solution plus 400 yaM-Ap III with zero added [Ca2+] Control
A
B
Procaine
C
Wash
A[Ca2+J
Fura-2
ration ~~~~~~~~~~~~~~~~satu
.J ................
...........................................
....
200 ms I.
j/ E ' [Ca2 ] -20
-100 mV Fig. 5. Comparison of [Ca2+] transients determined from Ap III absorbance and Fura-2 fluorescence signals in the presence of procaine. Top row, [Ca2+] transients for a 100 ms pulse from a holding potential of -100 to -20 mV in control (A), 1 mM-procaine (B, 10 min exposure) and wash (C). Second row, corresponding Fura-2 percentage saturation signals determined from the ratio -F380/F358 of fluorescence signals measured at 380 and 358 nm simultaneously with the Ap III absorbance signals (Klein et al. 1988). The dotted line below each Fura-2 record indicates the theoretical zero saturation level. Third row, [Ca2+] records calculated from the Fura-2 percentage saturation records. The values for kon (2-72 x 108 M-1 s-1) and koff (14-3 s-1) used to calculate the [Ca2+] records in the third row were obtained by fitting the Fura-2 fluorescence ratio signal to the A[Ca2+] from Ap III in the control and wash runs. Fibre 485; sarcomere spacing 4 0 ,um; temperature 10 °C; Ap III concentration 1388-1803 /.M.
and with 600 /IM [Ca2+], both in the presence and absence of 1 mM-procaine. A procaine difference spectrum was obtained (procaine-containing minus procaine-free solution) in nominally Ca2+-free and Ca2+-containing solutions. The difference spectra were essentially zero at wavelengths greater than 650 nm (data not shown). The greatest change was in the procaine difference spectrum in Ca2+-containing solution, where the presence of procaine resulted in a 4 % increase in absorbance at 700 nm as compared to the procaine-free [Ca2+]- difference spectrum. Thus procaine appears to have little effect on the ability of Ap III or Fura-2 to monitor calcium changes.
I_~ ~ ~ -10mV . '. I~ ~ ~ ~/Q
EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE
351
Does procaine affect the voltage sensor for E-C coupling? Although procaine did not inhibit release by shifting the midpoint (V) of the voltage dependence of release, its site of action may still be at the level of the T-tubule voltage sensor. Procaine could conceivably act by reducing the effective number of A
Control
V(mV)
B
Procaine
C
Wash
D Procaine - no procaine
** s~ ~ ~ ~ ~ ~ ~ *
+20 -2J
100 ms
-20 --' -
-20 -4
o -^'
-
-
0.
Fig. 6. Effect of procaine on intramembrane charge movement currents. Charge movement currents (IQ) were determined from total membrane currents measured simultaneously with the A[Ca2+] records shown in Fig. 1. Panels A, B and C show IQ measured before, during and after exposure to 1 mM-procaine, respectively. D, the difference between IQ measured in the presence and absence (average of control and wash) of procaine. Same fibre and conditions as in Fig. 1.
voltage sensors, or by altering their kinetics. To explore this possibility we measured intramembrane charge movement in the presence and absence of 1 mM-procaine. Figure 6 shows charge movement current (IQ) records obtained for the same pulses in which the [Ca2+] and Rre, records in Figs 1 and 2 were determined. Panels A and C show IQ calculated before and after the application of procaine. Panel B shows IQ records in the presence of 1 mM-procaine. Procaine appears to have very little effect on either the amplitude or the time course of the charge movement records, despite the fact that the [Ca2+] transients and Rrei were significantly depressed by procaine for the same pulses in the same fibre. In Fig. 6 the amount of charge moved at any voltage actually increased in the presence of procaine compared to control. However, the subsequent wash records (Fig. 6 C) exhibited a further increase in the charge moved at any voltage, indicating a systematic increase during the experiment in this fibre. A more direct comparison of the effects of procaine on IQ can be obtained by point by point subtraction of IQ in controls and procaine. Panel D of Fig. 6 shows the
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
352
difference between the records in the presence of procaine and the average of the bracketing control and wash IQ records at each voltage. The slight difference in charge is less than 4-2 % of the maximum charge moved at any voltage and may be due to drift in the linear properties of the fibre during the course of the experiment. A
Control
B Procaine + caffeine C
Wash
A
D
Caffeine
[Ca2N] 2 gLm
V(mV) +20
200 ms
-20
-40
Journal of Physiology (1992), 453, pp. 341-366 With 13 figures Printed in Great Britain
EFFECTS OF PROCAINE AND CAFFEINE ON CALCIUM RELEASE FROM THE SARCOPLASMIC RETICULUM IN FROG SKELETAL MUSCLE
BY MICHAEL G. KLEIN, BRUCE J. SIMON* AND MARTIN F. SCHNEIDER From the Department of Biological Chemistry, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201, USA
(Received 25 April 1991) SUMMARY
1. Resting myoplasmic free [Ca2+] and [Ca21] transients (A[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 (Rrei) 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 A[Ca2+] and the corresponding Rrei 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 Rrei 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 A[Ca2+]. 4. Although 1 mM-procaine depressed Rrel it had no effect on intramembrane charge movements (IQ) calculated from membrane currents recorded simultaneously with A[Ca2+]. 5. Procaine (1 mM) reversibly inhibited the potentiating effect of 0 5 mM-caffeine on A[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 A[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 A[Ca2+] after 10-30 ms duration voltage steps compared to the bracketing control and wash runs without procaine. Rrei 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 * Present address: Electro-Biology, Inc., 6 Upper Pond Road, Parsippany, NJ 07054, USA. MS 9337
342
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
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. 8. The absence of a significant effect of procaine on the Rrel waveform and voltage dependence for long pulses suggests that the major component of suppression of release by procaine may be a non-specific block of SR calcium-release channels. INTRODUCTION
Contraction of skeletal muscle is initiated by the release of calcium from the sarcoplasmic reticulum (SR). The stimulus for SR calcium release is depolarization of the transverse tubules (TT) but the mechanism which couples this electrical signal to opening of calcium channels in the SR is unknown. It has been proposed that this coupling may involve a direct molecular link between the TT voltage sensors and the SR channels (Chandler, Rakowski & Schneider, 1976), or be mediated by a second messenger, possibly inositol 1,4,5-trisphosphate (1P3) (Vergara, Tsien & Delay, 1985) or calcium (Frank, 1980). The ability of calcium to open SR release channels from skeletal muscle via 'calcium-induced calcium release' (CICR) has been demonstrated in skinned fibres (Ford & Podolsky, 1970; Endo, Tanaka & Ogawa, 1970), isolated SR vesicles (Miyamoto & Racker, 1982) and in SR calcium-release channels incorporated into bilayers (Smith, Coronado & Meissner, 1986). However, the involvement of CICR in physiological activation of skeletal muscle has not been established and has been questioned (Endo, 1984, 1985). To investigate the role of CICR in skeletal muscle activation we have previously shown (Simon, Klein & Schneider, 1989; Klein, Simon & Schneider, 1990) that caffeine, a potentiator of CICR (Endo, 1975a, b, 1985), can cause skeletal muscle to exhibit [Ca2"] transients with properties suggestive of a regenerative CICR mechanism. In the presence of 0 5 mM-caffeine, the rate of calcium release (Rrei) is increased, the voltage dependence of release is shifted to the left, and the time course of decay of A[Ca2+] after repolarization is slowed. Most significantly, however, release can no longer be turned off rapidly by repolarization after relatively short (10-20 ms) pulses but continues for tens of milliseconds after the end of the pulse. This latter observation, uncoupling of TT voltage control of SR calcium release in the presence of caffeine, is qualitatively consistent with a positive feedback of [Ca2+] on calcium release that could be produced by CICR. Procaine has been shown to be a potent inhibitor of the potentiating effects of caffeine on calcium release in intact skeletal muscle (Feinstein, 1963), SR vesicles (Morii & Tonomura, 1983) and skinned skeletal muscle fibres (Endo, 1985), presumably by inhibiting CICR. Its lack of an effect on intact muscle at low concentrations in the absence of caffeine has been taken as an indication that CICR does not occur during physiological excitation-contraction (E-C) coupling (Endo, 1984, 1985), but this conclusion has been debated. The aim of the present investigation was to study the effects of procaine on SR calcium release in the presence and absence of caffeine to determine whether CICR played a significant role in E-C coupling under physiological conditions. If procaine could inhibit the effects of CICR observed in the presence of caffeine, then any effects of procaine on release in the absence of caffeine might be interpretable in terms of a 'negative caffeine'
EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE
343
effect due to procaine blocking CICR in the absence of caffeine. An abstract describing some of these results has appeared (Klein, Schneider & Simon, 1989). METHODS
Frogs (Rana pipiens, northern variety) were killed by decapitation and the spinal cord destroyed. Experiments were carried out on single skeletal muscle fibres dissected from ileofibularis or semitendinosus muscles and mounted in a double Vaseline-gap chamber as described previously (Klein et al. 1990). The fibres were stretched to sarcomere lengths of 3 8-42 ,sm to eliminate movement and voltage clamped to a holding potential of -100 mV. Two calcium indicators were used simultaneously: antipyrylazo III (Ap III), an absorbance dye with relatively low affinity for [Ca2+], and Fura-2, a fluorescent dye with high affinity for [Ca2+]. Ap III was used to measure calcium transients (A[Ca2+]) and Fura-2 was used to monitor resting [Ca2+] and small changes in
[Ca2+]. Details of optical procedures and methods for calculating A[Ca2+] from the Ap III absorbance changes and [Ca2+] from Fura-2 fluorescence changes were as described previously (Klein, Simon, Szucs & Schneider, 1988). Current records were analysed (Melzer, Schneider, Simon & Szucs, 1986) to obtain the non-linear component of current (IQ) due to intramembrane charge movement. All experiments were carried out at 6-10 'C. The internal solution applied to the cut ends contained (mM): 102-5 caesium glutamate, 5.5 MgCl2, 5 ATP (sodium salt), 4-5 sodium Tris-maleate buffer, 13-2 caesium Tris-maleate buffer, 0 1 EGTA, 5 creatine phosphate (sodium salt), 1 Ap III, 0 05 Fura-2, and 1 g 1-1 glucose. The external solution applied to the intact portion of the fibre in the middle pool contained (mM): 75 (TEA)2504, 5 C82SO2, 7-5 total CaSO4, 5 sodium Tris-maleate buffer and 10-7 g ml-' tetrodotoxin. The pH was adjusted to 7 0 at room temperature for both solutions. Procaine (0 32-1 mM), caffeine (0 5 mM) or a combination of the two drugs were dissolved in external solution. Drug effects were determined after changing the solution bathing the intact portion of the fibre in the middle pool to external solution containing the desired concentrations) of the drug(s). Solution changes were carried out by simultaneously pipetting solution to one side of the chamber while aspirating from the other. Each new solution (drug-containing or drug-free wash solution) applied to the middle pool was precooled to 4 'C so as to minimize any change in temperature of the fibre. The procedure for calculating the rate of calcium release (Rrei) from the SR from the A[Ca2+] records follows the method developed by Melzer, Rios & Schneider (1984, 1987), as described in detail in Klein et al. (1990). Briefly, the calcium removal properties of the fibre were characterized by fitting a calcium removal model to the decay of A[Ca2+] after several pulses elicited at various times during each experiment. In all cases in which the effect of a drug or drugs were investigated the [Ca2+] records used for characterizing the calcium removal properties of the fibre included records obtained both before applying the drug(s) and after washing the drug(s) from the chamber (Klein et al. 1990). The values of the removal model parameters that were obtained from the fit to the decay of the control and wash [Ca2+] records were then used to calculate the Rrel for each [Ca2+] transient, both before applying the drug(s), in the presence of drug(s) and after washing the drug(s) from the chamber. This procedure assumes that neither procaine, caffeine nor the combination of the two drugs altered the calcium removal properties of the fibre. The removal model used for the present calculations includes the calcium binding properties of troponin C, parvalbumin and the SR calcium pump (Klein et al. 1990). The troponin C sites were assumed to be present at a concentration of 250 #M with 'on' and 'off' rate constants of 1-3 x 108 M-1 s-1 and 1000 s-1. The 'on' rate constants for binding of calcium and magnesium to parvalbumin were assumed to be 1P6 x 108 M-1 s-1 and 4 x 104 M-1 s-1 respectively. The concentration of parvalbumin and the 'off' rate constants of calcium and magnesium binding were determined by least-squares fitting of the removal model to the decay of [Ca2+] transients after several pulses (above). The SR calcium pump was assumed to be in instantaneous equilibrium with [Ca2+] at a concentration of 200 /M with a dissociation constant between 1-5 and 3 ,UM. Throughout the text average values are given as the mean+ S.E.M. Statistical significance was determined using a two-tailed t test, with significance for P < 0 05.
12
PH Y 453
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
344
RESULTS
Effects of procaine on calcium transients and Rrei Figure 1 shows the effects of procaine on calcium transients for 60 or 100 ms pulses (bottom) to the indicated potentials from a holding potential of -100 mV. Panel A A
Control
B
Procaine
C
Wash A
[Ca2+]
1
JiM
V(MV) +20
200 ms
-20
-30 -40
-100 mV Fig. 1. Effect of 1 mM-procaine on [Ca2+] transients measured with Ap III. A, [Ca2+] transients before exposure to procaine for 60 or 100 ms pulses to the potentials (V) indicated on the left from a holding potential of -100 mV; B, [Ca2+] transients for the same pulses after the external solution was exchanged with one containing 1 mM-procaine (12 min exposure); C, after washing procaine from the fibre. Bottom traces, superimposed voltage records for all pulses in panel. Fibre 488; stretched to a sarcomere spacing of 3-9 ,um per sarcomere; temperature 9 'C; Ap III concentration 850-1169 /M.
of Fig. 1 presents A[Ca2+] measured in control external solution before applying procaine, panel B shows A[Ca2+] for the same potential steps as in the control but after changing the external solution to one containing 1 mM-procaine and panel C shows A[Ca2+] obtained within 2 min of returning to the procaine-free external solution. At each potential 1 mM-procaine depressed the A[Ca2+] signal to roughly half its control amplitude. This effect was reversible as shown by the traces in Fig. 1 C. The [Ca21] transients recorded after removing procaine from the external solution were actually slightly larger in this run than in the control, although this result was not consistently observed. For the fibre in Fig. 1 the resting [Ca2+] was 28 nm in control solution before applying procaine, 32 nm in the presence of 1 mm- procaine, and 30 nm after washing procaine from the chamber. In six fibres the mean resting [Ca2+] was 19-7 + 3-2 nm before procaine, 20-7 + 3-5 nm in 1 mM-procaine and 27-3 + 7-0 nm after washing procaine from the chamber. Thus, on average procaine reduced the resting [Ca2+] by 14 % as compared to the bracketing controls.
345 EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE Figure 2 presents Rrei records calculated from the A[Ca2+] records shown in Fig. 1. Panel A shows Rrel before application of procaine to the fibre. These records are similar to Rrei records reported previously (Melzer et al. 1984; Klein et al. 1990), showing a rapid early release of calcium from the SR followed by a partial A
Control
B
Procaine
C
Wash Rate of release 5 yMms1
V(mV)
+20 100 Ms
-20
-30 -40
---..~
-100 mV J--
Fig. 2. Effect of 1 mM-procaine on the rate of release of calcium (Rre,) from the SR. Rrei was calculated from the calcium transients in Fig. 1 (see Methods). Same format and conditions as in that figure, except that the time base is expanded twofold. The parameters which were varied in the removal model to give the least-squares fit to the decay of A[Ca2+] after the several pulses were the 'off' rate constant of Mg2+ from parvalbumin (5 3 s-1), and the maximum pump rate of the SR calcium pump (805 IM s-1). The total concentration of the Ca2+/Mg2+ sites on parvalbumin was set to 300 /M, and the 'off' rate constant of [Ca2+] binding to parvalbumin was set to 1-5 s-l. The values of the other parameters in the removal model used to calculate Rre, are given in the Methods section.
inactivation of release. Panel B shows Rrei records in the presence of 1 mM-procaine. Procaine decreased the amplitude of Rrei by about 50 % at each membrane potential but had little effect on the shape of the release waveform. Panel C shows Rrei after washing procaine from the chamber. The release amplitude largely recovered after the wash. The slight difference in the waveform of the Rrei records between the control and the wash (e.g. at -20 and + 20 mV), with the wash records exhibiting a somewhat smaller peak and a slower decline from the peak, is typical of changes of Rrel records over time in the absence of procaine or any other drug (Klein et al. 1990). The slowing of inactivation during the course of an experiment may be related to the loss with time from the myoplasm of a diffusible substance involved in inactivation of release (Schneider & Simon, 1988). In order to evaluate the effects of procaine on the peak and steady components of Rrei it was first necessary to correct the Rre, records for effects due to depletion of 12-2
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
346
calcium from the SR (Schneider, Simon & Szucs, 1987; Schneider, Simon & Klein, 1989; Klein et al. 1990). Figure 3 shows [Ca2l] transients (top row) and the corresponding Rrei (second row) recorded from a fibre for a 120 ms pulse to 0 mV in control conditions (A), during exposure to 1 mM-procaine (B) and after washing A Control
B Procaine
C
Wash 0.5 pim
A [Ca2+]
100 ms
Rrel
5
Rrei corrected for depletion
m s1
0
F 1F
-100 mV
Fig. 3. Correction for depletion of calcium from the SR during release in the presence and absence of procaine. Upper row, [Ca2+] transients elicited by a 120 ms depolarization to 0 mV (bottom row) in control (A), during a 9 min exposure to 1 mM-procaine (B) and after washing procaine from the fibre (C). Second row, Rrei calculated from the [Ca2+] transients above. Third row, Rre, after correction for depletion of [Ca2+], assuming the SR [Ca2+] content to be 900 /IM before the pulse. The removal parameters used to calculate rrel were koffMg-Parv = 7 2 sol, and Vm.. = 2145 #M s-1. The parvalbumin concentration was set to 600 #M. Fibre 486; [Ap III] 729-985 #m; temperature 9 'C; sarcomere length
4-1
Itm.
procaine from the chamber (C). As described above, procaine reversibly depressed both A[Ca2+] and Rrei without changing the Rrei waveform. The third row in Fig. 3 shows the Rrei records after correcting for the effects due to depletion of calcium from the SR, assuming that the SR calcium content was 900 AEM (equivalent concentration if all SR calcium were free in myoplasmic water) before the pulses in all cases. The main effect of the correction for depletion was to increase the amplitude and remove the slight downward slope of the 'steady' component of release. This is especially evident in the records measured under procaine-free conditions since the releases were larger and the SR was presumably depleted to a greater extent. In the fibre of Fig. 3, after correction for depletion procaine suppressed the peak and steady components of release by 54 and 58%, respectively, compared to the bracketing controls, indicating about the same degree of suppression of both the peak and steady components of release by procaine. Figures 2 and 3 indicate that 1 mm-procaine decreased the amplitude of the release
EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE 347 without major modification of the Rrei waveform. The ratio of the peak to the steady level of Rrel provides an estimate of the relative contribution of the peak component compared to the steady component of release (Simon et at. 1991). After correction for depletion the ratio of peak to steady level of Rrei was 7-1, 5-6 and 3-7 in control, procaine and wash, for pulses to -20 mV for the fibre in Figs 1 and 2 and was 3 9, 3 0, 2-7 for the fibre in Fig. 3 (pulses to 0 mV). Similar results were observed in other fibres. In five fibres exposed to 1 mM-procaine the average ratio of peak to steady level for pulses to -20 or 0 mV was 4-9 + 0 9 in control, 3-7 + 0'8 in procaine and 3 0 + 0 3 in the wash. The mean values of peak and steady levels of Rrel in control and wash reflect the slight run-down of the peak component of release with time which we observe routinely (above). The ratio of the peak to steady level in the presence of procaine decreased by 8-7 + 8-0 % compared to the average of bracketing controls (P < 0'4), while in the same fibres the peak amplitude of the release decreased by a mean of 41-6 + 88% (P < 0-001, data not shown). Thus the release waveform was relatively unchanged by procaine whereas the amplitude of the Rrel was suppressed significantly. Although the estimation of the initial SR calcium content from the correction for depletion is somewhat subjective, the records of Fig. 3 indicate that procaine had little effect on the SR calcium content as compared to the controls. The same observation was made in four other fibres in which depolarizations of sufficient duration were applied to estimate the SR content in control, 1 mM-procaine and subsequent procaine-free wash.
Voltage dependence of Rrei in procaine One possible mechanism whereby procaine might inhibit calcium release at a given potential is by shifting the voltage dependence of release to more positive potentials. We therefore examined the voltage dependence of release with and without procaine. Figure 4A is a plot of the peak Rrei vs. membrane potential, for the same fibre as in Figs 1 and 2, in controls (0), in 1 mM-procaine (@) and after washing the fibre with procaine-free external solution (El). Although in this fibre there was some run-down in the peak Rrei between the control and wash, procaine depressed peak Rrei by about the same percentage at all potentials. The continuous lines are non-linear leastsquares fits to the data of the cube of the Boltzmann equation R =Rmax[1+exp((V-V)/k)]-3, (1) where R is the peak rate of release at voltage V, Rmax is the maximum peak rate of release, V is the midpoint voltage and k is the steepness factor of the uncubed Boltzmann equation. This expression was used as an empirical means of comparing the voltage dependence of peak Rrei since it has been previously shown to provide an adequate fit for the voltage dependence of the peak Rrei (Melzer et al. 1986). Figure 4B shows the same data and curves as in A but normalized to the maximum peak Rrel determined from each fit of the cube of the Boltzmann equation in Fig. 4A. Figure 4B shows that 1 mM-procaine had little effect on the steepness or midpoint voltage of release. The mean+s.E.M. value of the parameters of the least-squares fit of eqn (1) are given in Table 1 for the fibre in Fig. 4 and for two others in which the complete
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
348
voltage dependence of the peak rate of release was determined in control, procaine and wash runs (rows 1 to 3, respectively). Table 1 also gives the corresponding parameters for the steady component of release for the same fibres. The records were first corrected for depletion of calcium from the SR, as described in connection with 20 A
15 10 Is 5
(a, 0
L-
0-0
1-0 - B Normalized 0)
0.8 0
°N 0.6 o 0.4 -
0.2
0-0 -80 I
-60
-40 -20 Membrane potential (mV)
0
20
Fig. 4. The voltage dependence of the peak Rrei before, during and after exposure to 1 mmprocaine. A, voltage dependence of the peak Rrei from the experiment in Fig. 2. The continuous lines are least-squares fits of the cube of Boltzmann equation (eqn (1)) to the data with the following parameter values: control (0), Rmax = 16-1 gM ms-', V = -37-3mV, k= 10OmV; procaine (*), Rmax=89 zuMms-1, V=-36-2mV, k= 11 1 mV; wash (a), Rmax = 13-8, M ms-1, V= -37-6 mY, k = 10-2 mV. B, the data and theoretical curves from A normalized to the value of Rmax determined from the leastsquares fit to each data set.
Fig. 3 before carrying out the fits to obtain the values in Table 1. The last two rows of Table 1 give the mean value of the ratio and difference, respectively, of the value in procaine to the mean of the values in the bracketing control and wash runs. The ratios show that in these fibres 1 mm-procaine suppressed the peak and steady release to 66 + 5 and 63 + 10 % of the bracketing controls, respectively. These changes were statistically highly significant (P < 0-01 orP < 0'05). The midpoint voltage, V, was not significantly different in procaine for either the peak or steady component of release. The steepness factor, k, was increased slightly in the presence of procaine compared to the bracketing control and wash, by 28 + 11 % for the peak component (P < 0-05)
:F^ECTS OF PROCAINE AND CAINE ON MUSCLE
349
and by 17 + 20% for the steady component (P < 04) of release. Thus, the main results of Fig. 4 and Table 1 are that Rrei was reduced by about the same percentage at all potentials in procaine but that the voltage dependence of release was unaffected by the procaine. Does procaine interact with Ap III? The apparent depression of calcium release in the presence of procaine might not reflect a true inhibition of release but might instead be an artifact due to an TABLE 1. Parameter values from the least-squares fit of the voltage dependence of the rate of release to the cube of the Boltzmann equation in the presence and absence of 1 mM-procaine k (mV) V (mV) RmaX (JCM ms-1)
Control Procaine Wash Procaine/ no procaine
P 19-9+93 139+7-1 204+8-3 0-66 + 005
8 6-4+22 4-6+ 19t 7-7+10 0-63 + 0-10
P S P -400+4-3 -34-9+40 93+0(4 -407 +42 -383 +5-7 13-5+ 16t -404+43 -382+59 118+1 1 1-28 + 0-11
S 15-1+1-6 17-8+2-6 15-4+19 1-17 + 020
-0 4+ 1-4 -2 1 + 1.1 Procaineno procaine Rmax is the maximum rate of release, V is the midpoint voltage and k the steepness factor from a least-squares fit of eqn (1) to the data from three fibres. Entries are given as the mean+ S.E.M. of the value. P is the peak value, S is the steady component of the rate of release after correction for depletion of calcium from the SR (see text). The estimates of the SR calcium content ranged from 700 to 1000 ,aM in the three fibres. The two lowermost rows are the ratio and difference, respectively, of the value in procaine and the average of the control and wash runs in the absence of procaine (no procaine). t indicates statistical significance at P < 005, 1 significance at P < 001.
interaction of procaine with Ap 111. To explore this possibility we compared [Ca2+] transients calculated from Ap III absorbance changes with those calculated from the Fura-2 fluorescence changes measured simultaneously. The top row in Fig. 5 shows A[Ca2+] determined from Ap III absorbance changes for a 100 ms depolarization to -20 mV before, during and after wash of 1 mM-procaine in the external solution. In this fibre 1 mM-procaine reversibly decreased the amplitude of A[Ca2+] by more than 80 %. The middle row shows the Fura-2 percentage saturation records calculated from the Fura-2 fluorescence signals at 380 nm measured simultaneously with the Ap III absorbance signal (Klein et al. 1988). Although it is clear the procaine also decreased the amplitude of the Fura-2 signal, it is difficult to make quantitative comparisons with Ap III using the Fura-2 saturation signals because Fura-2 is a nonlinear calcium indicator at these A[Ca2+] levels. In order to determine [Ca2+] from the Fura-2 percentage saturation signals we followed the procedure of Klein et al. (1988). The bottom row in Fig. 5 shows [Ca2+] transients determined from the Fura-2 signals (see legend to Fig. 5 for Fura-2 kinetic constants used to calculate [Ca2+]). Although much noisier than the corresponding Ap III [Ca2+] transients, the amplitudes and wave forms of the Fura-2 [Ca2+] transients are very similar to those recorded with Ap III. In particular, in the
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
350
presence of procaine (Fig. 5B) the [Ca2"] transient calculated using the Fura-2 fluorescence signal with the kinetic parameters determined in controls is nearly identical to the A[Ca2+] determined from the Ap III signal. We also measured the absorbance spectrum of Ap III in cuvettes (0-2 cm pathlength) containing internal solution plus 400 yaM-Ap III with zero added [Ca2+] Control
A
B
Procaine
C
Wash
A[Ca2+J
Fura-2
ration ~~~~~~~~~~~~~~~~satu
.J ................
...........................................
....
200 ms I.
j/ E ' [Ca2 ] -20
-100 mV Fig. 5. Comparison of [Ca2+] transients determined from Ap III absorbance and Fura-2 fluorescence signals in the presence of procaine. Top row, [Ca2+] transients for a 100 ms pulse from a holding potential of -100 to -20 mV in control (A), 1 mM-procaine (B, 10 min exposure) and wash (C). Second row, corresponding Fura-2 percentage saturation signals determined from the ratio -F380/F358 of fluorescence signals measured at 380 and 358 nm simultaneously with the Ap III absorbance signals (Klein et al. 1988). The dotted line below each Fura-2 record indicates the theoretical zero saturation level. Third row, [Ca2+] records calculated from the Fura-2 percentage saturation records. The values for kon (2-72 x 108 M-1 s-1) and koff (14-3 s-1) used to calculate the [Ca2+] records in the third row were obtained by fitting the Fura-2 fluorescence ratio signal to the A[Ca2+] from Ap III in the control and wash runs. Fibre 485; sarcomere spacing 4 0 ,um; temperature 10 °C; Ap III concentration 1388-1803 /.M.
and with 600 /IM [Ca2+], both in the presence and absence of 1 mM-procaine. A procaine difference spectrum was obtained (procaine-containing minus procaine-free solution) in nominally Ca2+-free and Ca2+-containing solutions. The difference spectra were essentially zero at wavelengths greater than 650 nm (data not shown). The greatest change was in the procaine difference spectrum in Ca2+-containing solution, where the presence of procaine resulted in a 4 % increase in absorbance at 700 nm as compared to the procaine-free [Ca2+]- difference spectrum. Thus procaine appears to have little effect on the ability of Ap III or Fura-2 to monitor calcium changes.
I_~ ~ ~ -10mV . '. I~ ~ ~ ~/Q
EFFECTS OF PROCAINE AND CAFFEINE ON MUSCLE
351
Does procaine affect the voltage sensor for E-C coupling? Although procaine did not inhibit release by shifting the midpoint (V) of the voltage dependence of release, its site of action may still be at the level of the T-tubule voltage sensor. Procaine could conceivably act by reducing the effective number of A
Control
V(mV)
B
Procaine
C
Wash
D Procaine - no procaine
** s~ ~ ~ ~ ~ ~ ~ *
+20 -2J
100 ms
-20 --' -
-20 -4
o -^'
-
-
0.
Fig. 6. Effect of procaine on intramembrane charge movement currents. Charge movement currents (IQ) were determined from total membrane currents measured simultaneously with the A[Ca2+] records shown in Fig. 1. Panels A, B and C show IQ measured before, during and after exposure to 1 mM-procaine, respectively. D, the difference between IQ measured in the presence and absence (average of control and wash) of procaine. Same fibre and conditions as in Fig. 1.
voltage sensors, or by altering their kinetics. To explore this possibility we measured intramembrane charge movement in the presence and absence of 1 mM-procaine. Figure 6 shows charge movement current (IQ) records obtained for the same pulses in which the [Ca2+] and Rre, records in Figs 1 and 2 were determined. Panels A and C show IQ calculated before and after the application of procaine. Panel B shows IQ records in the presence of 1 mM-procaine. Procaine appears to have very little effect on either the amplitude or the time course of the charge movement records, despite the fact that the [Ca2+] transients and Rrei were significantly depressed by procaine for the same pulses in the same fibre. In Fig. 6 the amount of charge moved at any voltage actually increased in the presence of procaine compared to control. However, the subsequent wash records (Fig. 6 C) exhibited a further increase in the charge moved at any voltage, indicating a systematic increase during the experiment in this fibre. A more direct comparison of the effects of procaine on IQ can be obtained by point by point subtraction of IQ in controls and procaine. Panel D of Fig. 6 shows the
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
352
difference between the records in the presence of procaine and the average of the bracketing control and wash IQ records at each voltage. The slight difference in charge is less than 4-2 % of the maximum charge moved at any voltage and may be due to drift in the linear properties of the fibre during the course of the experiment. A
Control
B Procaine + caffeine C
Wash
A
D
Caffeine
[Ca2N] 2 gLm
V(mV) +20
200 ms
-20
-40
