599
Journal of Physiology (1990), 425, pp. 599-626 With 11 figures Printed in Great Britain
EFFECTS OF CAFFEINE ON CALCIUM RELEASE FROM THE SARCOPLASMIC RETICULUM IN FROG SKELETAL MUSCLE FIBRES
13Y MICHAEL G. KLEIN, BRUCE J. SIMON* AND MARTIN F. SCHNEIDERt From the Department ofBiological Chemistry, University ofMaryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201, USA
(Received 4 October 1989) SUMMARY
1. Resting myoplasmic [Ca2+] and [Ca21] transients (A[Ca2+]) were monitored using Fura-2 fluorescence and Antipyrylazo III absorbance signals from voltageclamped segments of cut frog skeletal muscle fibres in the presence and absence of 0 5 mM-caffeine. The rate of release (Rrei) of calcium from the sarcoplasmic reticulum was calculated from A[Ca2+]. 2. A[Ca2+] and Rtei were increased in caffeine for all pulses. The decline of A[Ca2+] was slower after a given pulse in caffeine than without caffeine. Resting [Ca2+] was slightly elevated in caffeine. 3. The voltage dependence of the peak value of Rrei and of the steady level of Rrei at the end of a 60-120 ms pulse were both shifted towards more negative voltages in caffeine. For relatively small pulses the voltage at which a given release waveform was observed was also shifted to more negative voltages. 4. Intramembrane charge movements measured in the same fibres in which the above changes in Rrei were observed showed no significant changes in caffeine. 5. In caffeine calcium release continued for many milliseconds after the end of a short (10 ms) pulse. Continued release after a pulse was not observed without caffeine and was probably due to positive feedback of elevated [Ca2+] on calcium release resulting from calcium-induced calcium release in caffeine. 6. Intramembrane charge movements after short pulses showed no change in caffeine that could account for the continued calcium release after the pulse. 7. Continued release after short pulses in caffeine decreased as the pulse duration was increased and was absent for pulses of 60 ms or longer. Rre, also inactivated during such pulses. 8. Relatively large and long conditioning pulses in caffeine suppressed both the peak Rrei and the continued release after short pulses. Peak release and continued release after short pulses recovered in parallel with increasing recovery time following suppression by a conditioning pulse in caffeine. 9. These results indicate that in the presence of caffeine, charge movement and calcium-induced calcium release both contribute significantly to the activation of * Present address: Department of Physiology and Biophysics, F41, University of Texas Medical Branch, Galveston, TX 77550, USA. t Author for correspondence and reprint requests.
MS 7986
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M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
sarcoplasmic reticulum calcium release during fibre depolarization. Release activated by either mechanism appears to be inactivated by calcium-dependent inactivation. A significant contribution of calcium-induced calcium release during depolarization in the absence of caffeine is not ruled out by present observations. INTRODUCTION
Physiological activation of a skeletal muscle fibre is initiated by electrical depolarization of its transverse tubules (TT) which leads to calcium release from the neighbouring sarcoplasmic reticulum (SR). One suggested mechanism for signal transmission from the TT voltage sensors to the SR calcium release channels is the diffusion of 'trigger' calcium ions from the TT to the SR followed by activation of the SR channel by calcium-induced calcium release (cf. Frank, 1980). Alternatively, signal transmission could occur via a direct macromolecular coupling between the TT voltage sensors and the SR channels (Chandler, Rakowski & Schneider, 1976). In this case calcium-induced calcium release would not be involved in the initial release but still might provide a secondary component of release triggered by the elevated [Ca2+] resulting from the direct primary activation. However, the involvement of calciuminduced calcium release in physiological activation has not been established and has been questioned (Endo, 1984, 1985). The drug caffeine potentiates calcium-induced calcium release in skinned skeletal fibres (Endo, 1975, 1985), isolated skeletal SR vesicles (Miyamoto & Racker, 1982; Kim, Ohnishi & Ikemoto, 1983; Palade, 1987; Rousseau, LaDine, Liu & Meissner, 1988) and skeletal SR calcium release channels (Rousseau et al. 1988). We have therefore examined the effects of caffeine on calcium release in voltage-clamped segments of skeletal muscle fibres in order to determine possible characteristic manifestations of calcium-induced calcium release in a skeletal preparation with preserved TT control of SR calcium release. Submillimolar concentrations of caffeine have been shown previously to increase calcium transients resulting from action potential (Delay, Ribalet & Vergara, 1986; Konishi & Kurihara, 1987) and voltage clamp (Kovacs & SzUcs, 1983; Delay et al. 1986) depolarization of skeletal fibres. In our experiments 0 5 mM-caffeine increased calcium transients for all voltage clamp pulses (Simon, Klein & Schneider, 1989; present results). In order to further characterize the effects of caffeine on calcium release we used the measured calcium transients to calculate the rate of calcium release from the SR. In the presence of caffeine (0-5 mM) the rate of calcium release was increased for all pulses, the voltage dependence of release was shifted towards smaller depolarizations and the ability to turn off release rapidly by fibre repolarization after short pulses was decreased. The intramembrane charge movement currents generated by the TT voltage sensor were not detectably modified by caffeine, indicating that caffeine was acting on a step beyond the voltage sensor. Some aspects of the present results have been reported in abstract form (Klein, Simon & Schneider, 1989).
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
601
METHODS
General procedures Experiments were carried out on cut segments of single twitch fibres isolated from the semitendinosus or ileofibularis muscles of frogs (Rana pipiens, northern variety) maintained at room temperature, fed on crickets. The frogs were killed by stunning followed by decapitation. The cut segments were dissected and mounted in a double-Vaseline-gap chamber in a relaxing solution (Kovacs, Rios & Schneider, 1983). Fibres were stretched to 3-8-4-2 sm per sarcomere to eliminate movement (and the associated optical artifacts) and were notched just beyond the gaps in both end pools (Kovacs & Schneider, 1978; Kovacs et al. 1983) to minimize both the electrical impedance and the diffusion distance from the end-pool solution to the fibre interior. The pools of the chamber were connected for electrical recording of fibre membrane current and voltage and for voltage clamping (Kovacs et al. 1983; Melzer, Schneider, Simon & Szics, 1986a). Two calcium indicators were used simultaneously in these experiments: the absorbance dye Antipyrylazo III (AP III; ICN K and K Labs, Plainview, NY, USA) and the fluorescent dye Fura2 (Grynkiewicz, Poenie & Tsien, 1985; Molecular Probes, Eugene, OR, USA). A solution containing the two dyes was applied to both ends of the fibre and both dyes entered the fibre by diffusion (Klein, Simon, Szics & Schneider, 1988). The relatively lower-affinity calcium indicator AP III was used to monitor cytosolic free-calcium transients A[Ca2+] whereas the higher-affinity indicator Fura-2 was used for monitoring resting [Ca2+] and relatively small changes in [Ca2+] (Klein et al. 1988). The chamber was positioned for optical recording of resting fibre light absorbance and fluorescence and of absorbance and fluorescence changes resulting from depolarizing pulses. Details of all optical procedures and the methods for calculating A[Ca2+] and [Ca2+] from the optical measurements were as described by Klein et al. (1988) and the references cited therein. The 'internal' solution applied to the cut ends of the fibres contained (in mM): 1025 Cs+ glutamate, 5-5 MgCl2, 5 ATP (Na+ salt), 4-5 Na+ Tris-maleate buffer, 13-2 Cs' Tris-maleate buffer, 01 EGTA, 5 creatine phosphate (Na+ 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 (in mM):75 (TEA)2SO4,5 Cs2SO4, 7-5 total CaSO4,5 Na+ Tris-maleate buffer and 10-7 g ml-' tetrodotoxin, with or without 0 5 caffeine. Both solutions were adjusted to pH 7 0 at room temperature. Experiments were carried out at a holding potential of -100 mV and at 6-10 'C. Data were collected and stored as previously described (Klein et al. 1988) using a Z158 personal computer (Zenith Data Systems Corp., St Joseph, MI, USA) equipped with a DT2801A A-D converter (Data Translation, Marlboro, MA, USA) and interfaced to a custom computerprogrammable pulse generator (University of Rochester Department of Physiology Electronics Shop, Rochester, NY, USA) that controlled pulse sequences and data acquisition. For each pulse five different signals were monitored: fibre current, fibre membrane potential, changes in transmitted light intensities at 700 and 850 nm and fluorescent light emission at 510 nm (Klein et al. 1988). Each signal was filtered at 500 Hz using a 6-pole Bessel filter (746LT-3, Frequency Devices, Haverhill, MA, USA). The five signals were sampled successively at 40,us intervals. Each point in each data record was the average value of that signal over a 1 or 2 ms interval calculated as the average of five or ten values, respectively, sampled at 0-2 ms intervals. Data records were stored on floppy disc and were subsequently analysed using various custom programs written in FORTRAN 77. Curve-fitting programs were compiled and executed using a 32-bit processor board (DSI-32, Definicon Systems, Westlake Village, CA, USA) running in the personal computer. Records of fibre current were analysed as previously described (Melzer et al. 1986a) to obtain the current component IQ due to intramembrane charge movement. Before and after each sequence of depolarizing pulses a 20 mV hyperpolarizing pulse was applied to the fibre thirty-two times and the signal averaged. The average of the off-transients from the two resulting records was used to obtain the linear capacitative current transient (Melzer et al. 1986a). Currents for individual applications of each depolarizing pulse were corrected for capacitative current and for constant and slowly varying ionic current (Melzer et al. 1986a) to give the single sweep IQ records presented here.
Calcium release calculations The procedure used for calculating the rate of release (Rrei) of calcium from the SR from the measured calcium transients followed the general approach developed by Melzer, Rios & Schneider (1984, 1987; Schneider, Rios & Melzer, 1985). In the present case we first assigned properties to the
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M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
intrinsic rapidly equilibrating calcium binding sites in all fibres and then characterized the calcium removal capability of each fibre by fitting a calcium-removal model to the decay of A[Ca2+] after several pulses (method 1 of Melzer et al. 1987). The properties determined for the calcium removal system in each fibre were then used to calculate records of Rrei in that fibre. The specific intrinsic fibre components included in the present removal and release calculations were troponin C, parvalbumin and the SR calcium pump, with the implementation of these components in our calculations being similar to that previously described (Brum, Rios & Schneider, 1988a). The 'calcium-specific' sites on troponin C constitute a major group of intrinsic rapidly equilibrating sites. Troponin C sites were assumed to be present at 250 /M (cf. Baylor, Chandler & Marshall, 1983) and to have 'on' and 'off' rate constants of 1-3 x 108 M-1 s-1 and 103 s-1 (Melzer et al. 1987; Brum, Rios & Stefani, 1988b) in all fibres. The parvalbumin calcium/magnesium sites constitute the major group of slowly equilibrating sites. The 'on' rate constants for binding of calcium and of magnesium to the parvalbumin sites were assumed to be 1-6 x 108 M-1 s-1 and 4 x 104 M-1 s-1, respectively (Baylor et al. 1983; Brum et al. 1988b). The concentration of parvalbumin calcium/magnesium sites and the off rate constants for dissociation of calcium and of magnesium from parvalbumin were determined by a least-squares fit of the removal model prediction to the observed decay of A[Ca2+] after various pulses. The least-squares fit was carried out using a modified version of the computer fitting routine described first by Melzer, Rios & Schneider (1986b). The calcium binding sites on the SR calcium pump were assumed to be present at 200 fM and to equilibrate instantaneously with [Ca2+]. For simplicity, occlusion of pump sites during the pump cycle was arbitrarily ignored. The dissociation constant for calcium binding to the pump sites was set at a value between 1-5 and 3/M. The rate of calcium translocation by the pump was assumed to be proportional to the fraction of pump sites occupied by calcium. The maximum pump rate at full occupancy of pump sites was another parameter determined by the least-squares fit, giving a total of four removal model parameters that were determined by the fit. In some cases the decay of A[Ca2+] after the group of pulses used for the fit did not contain sufficient information to specify all four of the parameters normally determined by the least-squares fit. In such cases the value of the 'off' rate constant for calcium from parvalbumin was set at 1-5 s-1 and the values of the other three adjustable parameters were determined by the least-squares fit. The actual value used for the pump dissociation constant for each fibre was not determined by the fit but was chosen so as to allow the removal model to reproduce non-exponential components in the decay of A[Ca2+] that were observed occasionally after relatively large pulses. For the present experiments involving caffeine the calcium-removal properties of each fibre were determined by fitting the removal model simultaneously to the decays of calcium transients after pulses of several durations recorded both before applying caffeine and after washing caffeine from the chamber. Release records were calculated from the calcium transients recorded in both the control and wash conditions using the resulting values of the removal parameters. Release in the presence of caffeine was calculated assuming caffeine to have no effect on the calcium-removal system, so the same removal parameter values that were obtained by fitting the decay of A[Ca2+] records in the absence of caffeine were also used to calculate release in the presence of caffeine. However, changes in calcium removal due to changes in resting calcium were taken into account by the removal model in terms of changes in resting calcium binding to parvalbumin and the calcium pump. Any such predicted changes in removal in caffeine were used in calculating release in caffeine. Comparison of calcium transients recorded simultaneously with AP III and Fura-2 indicated the 0 5 mM-caffeine had no significant effect on calcium binding to either dye (Simon et al. 1989). The concentration of free magnesium was assumed to be constant at 1 mm during all measurements with or without caffeine. The calcium-binding properties assumed for troponin C were also assumed to be the same with or without caffeine. Changes in resting calcium occupancy of troponin C due to changes in resting [Ca2+] in caffeine were again taken into account in the release calculation. No attempt was made to quantify experimentally the extent of intrinsic rapid calcium binding in each fibre. Rather, the above values were simply assumed for the concentrations and rate constants or dissociation constants for the troponin C calcium-specific sites and the pump sites. For the range of [Ca2+] and A[Ca2+] encountered in our experiments the parameters assumed for the troponin C sites are close to those of a linear, fast calcium buffer whereas those assumed for the pump sites are those of a saturable instantaneous buffer. Since the size assumed for a fast buffer
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
603
influences the release amplitude (Melzer et al. 1987; Schneider, Rios & Melzer, 1987) the amplitudes of the release records presented here must be considered somewhat arbitrary. In general, the calculated rate of calcium release is independent of the nature of the model used to characterize removal of calcium from the myoplasm provided the removal model accurately describes the decay of A[Ca2+] after all depolarizing pulses (Melzer et al. 1987). However, the removal model considered by Melzer et al. (1987) contained only relatively slowly equilibrating calcium binding sites, representing a simplified view of calcium binding by parvalbumin. In the present usage (cf. Brum et al. 1988a), a more realistic model considers competition between Ca2+ and Mg2+ for sites on parvalbumin. In a resting fibre most of the sites are occupied by magnesium but a small fraction are metal free. These free parvalbumin sites represent a relatively rapid, saturable group of calcium binding sites which influence the early part of the release waveform. For typical values of parvalbumin concentration and resting [Ca2+] and [Mg2+], the free parvalbumin sites accounted for about 10-20 % of the calculated peak rate of release. Thus the properties assumed or obtained for parvalbumin did somewhat affect the calculated rapid calcium binding in the present analyses and influenced the amplitude of Rrei, especially at early times during a depolarization.
RESULTS
Effects of caffeine on calcium transients Figure 1 shows the effect of caffeine on calcium transients for 60 or 100 ms pulses (bottom) to the indicated membrane potentials. The top four rows present A[Ca2+] recorded with AP III. The records on the left were obtained before application of caffeine, those in the middle were for the same pulses but with 0.5 mM-caffeine in the external solution and those on the right were obtained after washing with caffeinefree external solution. Caffeine reversibly increased A[Ca2+] for all pulses. The slightly smaller calcium transients in the wash compared to control indicate a slight run-down of the fibre over the course of the experiment. The pulses to -50 mV caused a relatively large A[Ca2+] in the presence of caffeine, but gave little detectable A[Ca2+] in the control and wash runs as measured by AP III on the scale of Fig. 1. However, calcium signals recorded simultaneously with Fura-2 for the same pulses to -50 mV and presented in Fig. 1 as Fura-2 saturation (%; bottom row of records) clearly show that there was a definite rise in [Ca2+] for these pulses in the absence of caffeine. Fura-2 percentage saturation records give a distorted representation of [Ca2+] because of kinetic delays and saturation of the dye (Klein et al. 1988). The saturation records are simply presented in Fig. 1 to demonstrate the definite existence of release for these near-threshold pulses in the absence of caffeine. The resting [Ca2+] measured by Fura-2 was also increased in 0-5 mM-caffeine. The Fura-2 percentage saturation in Fig. 1 in the resting fibre before the pulses to -50 mV was 20 in control, 33 in caffeine and 14 after washing caffeine from the chamber (bottom row of records). Using the value of 67 nm obtained for the Fura-2 dissociation constant in this fibre, the saturation values correspond to resting [Ca2+] levels of 14 nm during the control records, 26 nm in the presence of caffeine and 10 nM after washing caffeine from the chamber. In five other fibres in which the voltage dependence of both peak and steady release were determined before, during and after caffeine (fibres used in Table 1, below), the respective mean + S.E.M. values of resting [Ca2+] were 28 + 3, 51 + 14 and 36 + 7 nm. The mean value of the Fura-2 dissociation constant determined in the same fibres (Klein et al. 1988) and used to obtain these
604
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
values was 74 + 7 nM. Previous measurements with calcium-sensitive microelectrodes in intact fibres did not reveal any change in resting [Ca2+] in the presence of 0-5 mmcaffeine (Lopez, Alamo, Caputo, DiPolo & Vergara, 1983, 20-21 0C) perhaps because of limited resolution, whereas measurements of aequorin resting glow did indicate a rise in resting [Ca2+] in intact fibres in the presence of 0 4 mM-caffeine (Konishi & Kurihara, 1987; 17-18 TC). Control
Caffeine
Wash
ACa'+] 2puM V
(MV) + 20
200 ms -20
-40 -50
Fura-2 saturation (
~X
-50
0 ..............................
............ ..................
.
-
..... ............. ........................ ......
100
0
_100i 1m Fig. 1. Effect of 0-5 mM-caffeine on calcium transients measured with AP III (upper four rows) and Fura-2 (lowermost row of records). Left vertical panel, signals measured before exposure of the fibre to caffeine; middle panel, during a 9 min exposure to caffeine; right panel, after washing caffeine from the fibre. The rise in [Ca2+] for the depolarization to -50 mV is shown as measured by AP III (upper) and Fura-2 (lower), the latter given as the percentage saturation of Fura-2. The dotted line below each Fura-2 record indicates the theoretical zero saturation level. The time course of each depolarizing pulse is given at the bottom of each panel, with the membrane potential during the pulse, V, given at the far left. Fibre 485, stretched to a sarcomere spacing of 400 ,m per sarcomere, temperature 9 °C, AP III concentration 872-1421 UM.
Caffeine increases calcium release and shifts its voltage dependence Figure 2 presents records of Rrei calculated from each of the calcium transients in Fig. 1. Note that the time scale in Fig. 2 is expanded twofold compared to Fig. 1. For the pulses to -20 and + 20 mV in Fig. 2 Rrei in caffeine was larger than without caffeine. For these pulses the Rrei waveform in caffeine was generally similar to Rrel in the bracketing control and wash runs, exhibiting a marked early peak followed by a decline towards a maintained level during the pulse (Melzer et al. 1984, 1987). For the pulse to -40 mV Rrei in caffeine was considerably larger and also exhibited a much more prominent peak than the bracketing Rrei records for the same pulse
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
605
without caffeine. The waveform of Rrei for the pulse to -40 mV in caffeine was more similar to the release waveforms for the pulses to -20 mV in the absence of caffeine than to those for the pulse to -40 mV. Similarly, the release waveform for the pulse to -50 mV in caffeine roughly resembled the release waveform for pulses to -40 mV in the absence of caffeine. Thus for the smaller pulses in Fig. 2 caffeine shifted the pulse voltage for occurrence of a given release waveform towards smaller depolarizations. Control
Caffeine
Rate of -1PMM release l0/Mms
V
(mV) +
Wash
20
100 ms
-20
-400X -50
gV -100
Fig. 2. Effect of 0-5 mM-caffeine on the rate of release of calcium from the SR, Rrei. The rate of release was calculated from the calcium transients in Fig. 1 (see text for details). Same format and conditions as in that figure. The time scale is expanded twofold compared to Fig. 1. The parameters of the removal model adjusted in the least-squares fit to the decay of A[Ca2+] after the pulse were the 'off' rate constant of Mg2+ from parvalbumin, koff, Mg-Parv, and maximum pump rate of the SR calcium pump, Vmax. These values were 7-1 s-l and 1809 ,/M s', respectively. The total concentration of Ca2+/Mg2+ sites on parvalbumin, [Parv], was set to 300 /SM, and the 'off'rate constant of Ca2+ from parvalbumin, koff, Ca-Parv' was set to 1-5 sol.
Figure 3A presents the voltage dependence of the values of peak Rrel obtained from the records in Fig. 2 and from two records (control and wash) for pulses to 0 mV in the same fibre not shown in Fig. 2. No pulse to 0 mV was applied to this fibre in caffeine. The data show the increase in peak release at all voltages and also indicate a shift towards more negative voltages in the presence of caffeine. The sets of values of peak Rrei in control, caffeine and wash were each fitted by the cube of a Boltzmann relationship (Melzer et al. 1986a), (1) R = Rmax [1 +exp (V-V)/lk]-3, where R and Rmax denote an individual value and the maximal value of peak Rrel, and
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER V and k are the mid-point voltage and steepness factor respectively for the uncubed Boltzmann. The resulting fits are represented by the curves in Fig. 3A. The parameter values resulting from the fits (Fig. 3 legend) indicate a 65% increase in Rmax and a -112 mV shift in mid-point voltage with little change in steepness in 606
24
A
18-
E12 CO
6
C
o
B
.~1.00 a)
0.7 E 0.50
z// 0.25
0.00 -80
-40 -20 0 20 Membrane potential (mV) Fig. 3. The voltage dependence of the peak rate of release before, during and after exposure to 05 mM-caffeine. A, the symbols represent the peak rate of release from the experiment of Fig. 2. The curves are least-squares fits of text eqn (1) to the data with the following parameter values: control (0), Rm,, = 14-7 #M ms-1, V = -41-3 mV, k = 107 mV; caffeine (M), Rmax = 219/Sm ms-', V=- 53-4 mV, k = 12-5 mV; wash (E), Rmax = 11 9 #M ms-', V = -43-1 mV, k = 12-3 mV. B, the data and theoretical curves from panel A normalized to the value of Rmax determined from the least-squares fit to each data set. 60
caffeine compared to the bracketing control and wash runs without caffeine. The shift in voltage dependence is shown graphically in Fig. 3B, where each data set in Fig. 3A is replotted after being normalized to the value of Rmax for that set obtained from the fit in Fig. 3A. Thus caffeine caused both an increase in peak release for any depolarization and a shift to smaller depolarizations of the voltage dependence of peak rate of release.
Effect of caffeine on peak and steady level of release Figure 4 presents calcium transients (left) recorded from another fibre, with records for depolarizing pulses to the same potential in control, caffeine and wash runs superimposed in each panel. Caffeine (largest A[Ca2+] record in each panel in Fig. 4) caused a marked increase in A[Ca +] compared to the bracketing records without caffeine. The middle column of Fig. 4 presents superimposed rate of release records calculated from each of the corresponding calcium transients on the left.
607 EFFECTS OF CAFFEINE ON CALCIUM RELEASE Peak Rrei was increased in caffeine (largest Rrei record in each panel) compared to the bracketing records without caffeine. For the fibre in Fig. 4 the calcium transients recorded after washing caffeine A [Ca ]
Rrei corrected
Rrel
for depletion
(mv) +
20
Jim= 100 Ms
-20 -45
-60
~
"
-
Fig. 4. The effects of caffeine on the peak and steady-level components of Rrei. Left panel, superimposed A[Ca2+] measured before, during and after exposure of the fibre to caffeine. Middle panel, Rrei calculated from the A[Ca2+] shown to the left. Right panel, Rre, corrected for depletion of calcium from the SR, assuming the initial SR calcium content to be 1200 /iM for all depolarizations in the presence and absence of caffeine. The membrane potential during the pulse is given to the right of the respective A[Ca2+] records, with the relative timing of the pulse shown at the bottom of each panel. The record measured in the presence of caffeine (29 min exposure) is the largest in each set of superimposed records. The wash run is represented by the smallest A[Ca2+] for the depolarizations to -20 and + 20 mV, and by the traces with a smaller peak amplitude and slower time course for decay in the corresponding Rrei records. There was no pulse to -45 mV in the wash run. The adjusted values of the removal model parameters were koff Mg-Parv =6-7 s-l and Vmax = 1974 uM s'l. [Parv] was set to 900/M and koff Ca-Parv to 1-5 s-l. Fibre 489, 4-0 jm per sarcomere, temperature 9 0C, AP III concentration 997-1259/SM.
(smallest amplitude record in each of the two upper panels in the left column of Fig. 4) were smaller than A[Ca2+] before applying caffeine (medium amplitude record in each panel), indicating fibre run-down over the course of the experiment. The peak Rrei after washing out caffeine was considerably smaller than before applying caffeine, confirming the fibre run-down indicated by the calcium transients. The rate constant for the decay of Rrei during a pulse was also considerably slower after washing caffeine from the chamber than before applying caffeine. However, the final level of Rrel during a pulse was not smaller in the wash than in control. Similar observations were made in several other fibres and indicate that peak release was more susceptible to run-down over the course of an experiment than was the steady level of release. These effects were due to fibre run-down and were not related to
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M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
caffeine since they were also seen over the course of other experiments in which fibres were not exposed to caffeine (M. G. Klein, B. J. Simar & M. F. Schneider, unpublished observations). However, there was some indication that run-down may have been accelerated in the presence of caffeine. The release records in the middle column of Fig. 4 indicate that both the peak and the final level of Rrel during a pulse were increased in the presence of caffeine. Previous studies have indicated that Rrei at the end of a 100-200 ms pulse is determined not only by the degree of activation and inactivation of release at that time but also by the extent of depletion of calcium from the SR during the pulse (Schneider, Simon & SzUcs, 1987; Schneider & Simon, 1988). The release records in the middle column of Fig. 4 were therefore corrected for the decline in release due to depletion of calcium from the SR. The resulting release records corrected for depletion are presented in the right column of Fig. 4. The correction was carried out assuming the same calcium content in the SR prior to each pulse in both the presence and the absence of caffeine. The value of the initial SR calcium content was selected so as to make the latter part of the release records for the largest long pulse in Fig. 4 essentially flat after correction for depletion (Schneider et al. 1987; Schneider, Simon & Klein, 1989). Comparison of the middle and right columns in Fig. 4 indicates that the depletion correction had little effect on the relative amplitudes of peak rate of release with and without caffeine. This justifies the use of the peak of uncorrected Rrei records in demonstrating the effect of caffeine on release in Fig. 3. In contrast, the depletion correction did increase the relative difference in final levels of Rrei with and without caffeine. This was because the larger releases in caffeine gave rise to considerably more depletion of calcium from the SR by the end of the pulses in caffeine than for the smaller releases in the absence of caffeine. Equation (1) was fitted to the voltage dependence of both the peak and the steady level of Rrei determined in each of five fibres before applying caffeine, in the presence of 0 5 mM-caffeine and after washing caffeine from the chamber. In all cases the release records were corrected for calcium depletion to obtain the data points fitted by eqn (1). The upper three rows of Table 1 present the mean (+ S.E.M.) value of each parameter obtained from the fit of eqn (1) to the Rrei values in the control, caffeine and wash conditions. The lower two rows of Table 1 compare the value of each parameter in the presence of caffeine with the average of values of the same parameter obtained from the fits in the control and wash conditions in the same fibre. The mean+ S.E.M. increase in maximum value of Rrei was 27+9% for the peak release (denoted by P in Table 1) and was 15 + 13 % for the steady level of release (S in Table 1). 17 was shifted by 8 1 + 1-2 mV to more negative potentials for peak release and by 8-7 + 2-2 mV to more negative voltages for the steady release. The steepness factor was essentially unchanged, showing a mean increase of 3 + 8 % for peak release and 11 + 4 % for the steady release. The increase in the steady level of release at the end of a relatively long (60-120 ms) pulse in caffeine (Table 1 and Fig. 4) was accompanied by the appearance of a component of release that turned off very slowly after the pulse. An example of this can be seen in the two upper right panels of Fig. 4, where the release in caffeine did not turn off completely within a few milliseconds of fibre repolarization. Instead, a small component of release continued after the pulse and decayed very
EFFECTS OF CAFFEINE ON CALCIUM RELEASE 609 slowly towards zero. The size of this 'off pedestal' of release in caffeine was relatively small even after the largest pulse in Fig. 4 (top right), but it was more prominent in other fibres (not shown). For 60-120 ms pulses to -20 to + 20 mV in the five fibres of Table 1 plus two other fibres the mean+ S.E.M. amplitude of the 'off pedestal' 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 caffeine
Rmax (UM ms-') P Control Caffeine Wash
S
27-3+P19
8&4+0-6
28-1+2-1
9-9+0-8 90+06
17A4+1-5
T (mV) P
S -44-3+1P3 -38-3+1 5
-55-2+P18 -510±+3-1
-493+2-3 -45-9+2-2
k (mV) P
S
10O3±+05
12-4+O02
11-2+0-5 11-5+04
16-2+0-8 150+12
Caffeine/no 1-27 +0109 1-15+0 13 1 03+0 08 1t11+0004 caffeine Caffeine-8+1±1-2 -8-7+2 2 no caffeine Rmax is the maximum rate of release, V is the mid-point voltage and k the steepness factor from a least-squares fit of eqn (1) to the data from five fibres. Entries are given as the mean+ s.E.M. of the value. P is the peak value, S is the steady-level component of the rate of release after correction for depletion of Ca2+ from the SR (see text). The two lowermost rows are the ratio and difference, respectively, of the value in caffeine and the average of the control and wash runs in the absence of caffeine (no caffeine).
taken as the average of measured values 11-20 ms after the pulse was 1-7+ 0-4 /M ms-1 greater in 0 5 mM-caffeine than the average of the bracketing control and wash runs in the absence of caffeine measured in the same way. For the same pulses the average increase in steady release during the last 10 ms of the pulse was 2-2 + 0-5 Iw ms-1 in caffeine compared to the bracketing controls. Thus the amplitude of the 'off pedestal' in caffeine was essentially the same as the increase in steady release in caffeine. The fast turn-off of release after the same pulses was calculated as the difference between the steady release at the end of a pulse and the 'off pedestal' 11-20 ms after the pulse. The resulting mean values were 6-2 + 0-9 tM ms-1 before caffeine, 6'0 + 0-7 in the presence caffeine and 5-7 + 0-7 after washing caffeine from the chamber, indicating that the amplitude of the fast turnoff of release was not altered by caffeine. One interpretation of these observations is that the component of increased steady release that appeared in caffeine did not turn off rapidly on fibre repolarization, whereas the component of steady release that was present in control remained unchanged in caffeine and could still turn off rapidly after the pulse in caffeine. Thus the rapid turn-off can be attributed to TT voltage control of release, whereas the slow turn-off might be due to non-inactivatable continued release caused by caffeine. It should be noted that the increase in steady release and the 'off pedestal' in caffeine both arise from the finding that the decline of the calcium transient after a long pulse in caffeine was slower than the rate of decline predicted by the removal model. Since the removal system was assumed to be the same in the presence and absence of caffeine, an alternative interpretation of the slow decline of A[Ca2+] might 20
PHY 425
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER 610 be that caffeine modified the removal system so as to slow calcium removal. The release pedestal and the increase in steady release in caffeine would then be artifacts resulting from use of improper removal parameter values in the presence of caffeine. We therefore tried to calculate release without assuming the removal system to be the same in the presence and absence of caffeine. An independent removal analysis carried out only in the presence of caffeine was not possible because of continued release of calcium after short pulses in caffeine (Simon et al. 1989 and below). Since there is evidence that caffeine does not affect the SR calcium pump (Ogawa, 1970; Batra, 1974; Palade, 1987), we attempted to fit the removal model to the decay of A[Ca2+] after the long pulses in caffeine assuming the pump to be unchanged in caffeine and allowing only the concentration of parvalbumin to change. This procedure was able to reproduce the slowing of the decay of A[Ca2+] and eliminated the pedestal of release after long pulses in caffeine. For the five fibres of Table 1 the parvalbumin concentration was reduced to the mean + s.E.M. value of 271 + 189 #M in caffeine from the mean value of 680+ 143 ,uM obtained in the absence of caffeine from fits of the decay of A[Ca2+] after short and long pulses in the bracketing control and wash runs. Fits to the decay of A[Ca2+] after only long pulses in the bracketing runs in the absence of caffeine gave 624 + 128 and 553 + 165 /M for the parvalbumin concentration in the control and wash runs, indicating little change in removal over the course of the experience. Thus, although a true increase in steady release at the end of a long pulse in caffeine and a pedestal of release after such a pulse seem to be reasonable explanations for the slowed decline of A[Ca2+] after long pulses in caffeine, we cannot rule out a reduction in the effective concentration of parvalbumin as a possible cause of the observed slowed decay of A[Ca2+] in the presence of caffeine.
Caffeine had little effect on charge movement Figure 5 presents intramembrane charge movement currents (IQ) before applying caffeine (left column), in the presence of 0-5 mM-caffeine (middle) and after washing caffeine from the chamber (right), all recorded simultaneously with the calcium transients and release records in Fig. 4. Caffeine had little effect on IQ at any membrane potential even though the calcium transients and calcium release determined simultaneously with the IQ records in Fig. 5 were substantially increased in caffeine (Fig. 4). For the two largest pulses in Figs 4 and 5 the average increase in peak Rrei in caffeine was 55 % compared to the bracketing records in the absence of caffeine whereas 'on' charge movement for the same pulses was increased by only 16 %. Thus the increase in release does not appear to be attributable to an increase in charge movement. Charge movement difference records were formed at each voltage by subtracting the average of the IQ records from the control and wash runs point by point from the IQ measured in the presence of caffeine. The difference records (Fig. 6, left) obtained from the IQ records in Fig. 5 suggest that in caffeine there may have been slightly more ' on' charge during the pulses and slightly less 'off' charge after the pulses than without caffeine in this fibre. However, for pulses to -20 and + 20 mV in the fibre shown in Fig. 6 and in two other fibres the average change in 'on' charge in the presence of caffeine was a 5±10 % (mean+s±.E.M.) reduction and the average change in 'off' charge in caffeine was a 21+6% reduction compared to the average of the bracketing control and wash measurements. For the same three fibres and pulses the
611
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
simultaneously measured mean peak Rrei was increased by 43+14% in caffeine compared to the bracketing runs without caffeine. Since this increase in release occurred when 'on' charge was essentially unchanged, caffeine appeared to increase calcium release by exerting its effects predominantly on steps which occur in excitation-contraction coupling beyond charge movement. The same conclusion was reached previously by Kovacs & Szfics (1983) and would be consistent with caffeine acting directly on the SR calcium release channel by introducing or potentiating calcium-induced calcium release (Endo, 1975, 1985; Rousseau et al. 1988). Control V (mV) + 20-
Caffeine
t
2 .i pF
~.
-4
Wash
(
-20 ---Z^~. ..\'~
2/1A
pF-'l
-45
100 Ms -50
.
,
_
Fig. 5. Intramembrane charge movement currents measured in the presence and absence of caffeine. Charge movement currents were recorded simultaneously with the A[Ca2+] records shown in Fig. 4. Left, middle and right panels, charge movements recorded before, during and after exposure to caffeine, respectively. The relative timing of the pulse is shown at the bottom of each panel, and the membrane potential during each pulse is given at the left of the figure. Same fibre and condition as in Fig. 4.
The right column in Fig. 6 presents the difference in IQ before applying caffeine and after washing caffeine from the chamber. These records indicate that in the fibre in Fig. 6 the decrease in IQ over the course of the experiment was as large or larger than the change in IQ due to the presence of caffeine. Similar changes in charge movement over the course of the experiment were observed in the two other fibres in which charge movement and calcium transients were monitored before, during and after application of caffeine. Thus, our measurements show no marked effect of 0-5 mMcaffeine on charge movement although we cannot absolutely rule out changes due to caffeine of the order of 10-20% considering the change in IQ over the course of the experiments.
Caffeine slows turn-off of release after short pulses The preceding results showed that for relatively long (60-120 ms) depolarizing pulses several changes occurred in calcium release in the presence of caffeine. The voltage dependence of peak and steady Rrei were shifted to more negative voltages. 20-2
612
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
For relatively small pulses the voltage at which a given release waveform occurred was also shifted to more negative values. Such shifts conceivably could be due to quantitative changes in release in caffeine. In contrast, the turn-off of release after very short pulses did appear to be qualitatively different in the presence and absence of caffeine (Simon et al. 1989). Caffeine - no caffeine
i~
_
o A_~~~W.;a
Control - wash
V
v
._
(mV) a\ +
-
20
-20
lpAuF1|
-45
100 ms
-50
-
-60
Fig. 6. The difference of charge movement currents measured in the presence and absence of caffeine. Left panel, the difference currents formed by subtracting point by point the average of the bracketing control and wash records from the caffeine record at each membrane potential shown in Fig. 5. The depolarization to -45 mV is the difference of records measured in caffeine and before exposure to caffeine. Right panel, difference currents formed by subtracting each wash record from the corresponding control record shown in Fig. 5.
Figure 7 illustrates the effect of caffeine on calcium transients and calcium release for short and long pulses. Each upper panel presents superimposed calcium transients for 10 and 60 ms pulses recorded before applying caffeine (panel a), in the presence of 0'5 mM-caffeine (b) and after washing caffeine from the chamber (c). Caffeine increased A[Ca2+] for both the short and long pulses but the relative increase was considerably larger for the short pulse. The lower panels in Fig. 7 present the release records calculated from the corresponding calcium transients in the upper panels. The control and wash Rrei records indicate that release turned off completely within a few milliseconds after both the short and long pulses in the absence of caffeine (panels d and J). There was also a rapid turn-off of release within a few milliseconds after the long pulse in caffeine (panel 3e, long signal). In contrast, after the short pulse in the presence of caffeine release did not turn off abruptly (panel e, short signal) but instead declined to zero with a relatively slow time course that resembled the time course of decline of release during the longer pulse. The slow turnoff of release after short pulses in caffeine was qualitatively different from the abrupt
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
613
turn-off of release after all pulses in the absence of caffeine and after long pulses in the presence of caffeine. Such slow turn-off of release after the short pulse was probably a major factor in the pronounced relative increase in amplitude of A[Ca2+] for the short pulse in caffeine compared to control (Fig. 7, top). b
a
Wash
Caffeine
Control
c
PAM A[Ca']
e
d
f A 10pM PM ms1
JaI
Rate of ~release
~~~~100Jm 100 ms
Fig. 7. Effects of caffeine on A[Ca2+] and Rrei for short and long pulses. a-c, A[Ca2+] for a 10 and a 60 ms depolarization to -20 mV before, during and after exposure to caffeine. df, Rrei calculated from the corresponding A[Ca2+] record shown above. The time course of each depolarization is shown at the bottom of each panel. Same fibre and conditions as in Figs 1-3.
When a fibre is repolarized after a depolarizing pulse calcium release might be turned off by at least two different processes, deactivation and inactivation. Deactivation is the simple voltage-dependent reversal, due to fibre repolarization, of the voltage-dependent process that caused activation of release when the fibre was depolarized. In contrast, inactivation of SR calcium release does not appear to be a voltage-dependent process but instead appears to be due to elevated myoplasmic [Ca2+] (Schneider & Simon, 1988; Schneider, Simon & Klein, 1989). The inactivation process is directly reflected by the decline in release during a pulse. For relatively large pulses that cause large elevations of [Ca2+] the time constant of decline of Rrei during a pulse gives the time constant of the [Ca2+]-independent second step in the development of inactivation, which is rate-limiting at high [Ca2+] (Schneider & Simon, 1988). At lower [Ca2+] the rate of inactivation could be even slower. Figure 7 d andf show that in the absence of caffeine, release turned off more rapidly after both short and long pulses than it declined due to inactivation during the long pulse. This indicates that in the absence of caffeine the turn-off of release after all pulses was too fast to be due to inactivation and must have occurred by deactivation. In caffeine release must also have turned off by deactivation after the long pulse since
614
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
the turn-off was much faster than the decline of release during the long pulse (Fig. 7 e). In contrast, the turn-off of release after the short pulse in caffeine was probably due to inactivation rather than deactivation since the turn-off was relatively slow and followed a time course that was very similar to the decline in release during the long pulse (Fig. 7 e). In order to quantitate the speed of turn-off of release after short pulses a single exponential plus a constant was fitted to the decline of Rrei after 10-20 ms pulses in the absence of caffeine and after 10 ms pulses in the presence of caffeine. The resulting time constant Tas gives the time constant of decay of Rrei 'after short' pulses. Table 2 presents values of ras obtained from eight fibres in control, in caffeine and after washing caffeine from the chamber. The mean + S.E.M. value of 15-3 + 3-3 ms for Tm in these fibres in caffeine was considerably larger than the mean of 3.3 + 0 3 ms in control, confirming the marked slowing of turn-off of release after short pulses in caffeine shown in Fig. 7. The increase in Tas in caffeine was not due to run-down of the fibres during the course of the experiment since in five of the fibres Tas was also measured after washing caffeine from the chamber and was found to be 3-9 + 0 4 ms, essentially the same as the control value. Values of the time constant Tdl obtained from a single exponential plus constant fit to the decline of Rrei 'during long' pulses in the same fibres in which Tas was measured are also presented in Table 2. The mean value of Tdl increased from 9 9 + I 1 ms in control to 13-8 + 1-6 ms in caffeine. This increase is largely attributable to fibre run-down (cf. Figs 2, 4 and 7) since in the five fibres in which Tdl was measured after washing caffeine from the chamber Tdl was increased by an average of 33 % in the wash compared to control. The last three columns of Table 2 give the ratio Tas/Tdl obtained in each fibre in control, in the presence of caffeine and after washing caffeine from the chamber. The mean+ S.E.M. value of Tas/rdl in caffeine was 1-07 + 0-12, demonstrating that in caffeine the turn-off of release after a 10 ms pulse occurred with the same time constant as the decline in release during a longer pulse. Thus in caffeine the turn-off of release after a short pulse could be due to the same inactivation process that is responsible for the decline in release during a long pulse (Schneider & Simon, 1988). In contrast, before applying caffeine the mean value of Tas/Tdl was 0-35 + 0-02 for the fibres in Table 2. Since in the absence of caffeine release turned off about 3 times faster after a pulse than release declined by inactivation during a pulse the turn-off of release after a pulse in the absence of caffeine could not have been due to inactivation but must instead have been due to deactivation. Figure 8a presents the calcium transients for the short pulses of Fig. 7, now scaled to the same peak and superimposed. A[Ca2+] continued to rise for 34 ms after the 10 ms pulse in caffeine but began to decline within 9 ms of the end of the pulse before and after exposure to caffeine. A rising A[Ca2+] implies that calcium release must have exceeded calcium removal. Therefore, even without making any release calculation it can be concluded that release must have continued for at least 34 ms after the 10 ms pulse in caffeine. In contrast, the scaled calcium transients for the 60 ms pulses (Fig. 8 b) show that by the end of the 60 ms pulse A[Ca2+] began to decline within 5 ms of repolarization both with and without caffeine. One possible cause of the slow turn-off of release after short pulses in caffeine might be a modification of intramembrane charge movement. However, the
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
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lower part of Fig. 8 indicates that the continued release of calcium after short pulses in caffeine was not accompanied by any detectable change in the 'off' charge movement after the pulse. Figure 8c presents superimposed charge movement currents for 10 ms pulses recorded simultaneously with the calcium b
a
Normalized A
d
c
[Ca'+]
Superimposed caffeine,
Cq
no caffeine _AuF
6
f
IQ
Caffeine - no caffeine
h
g __
Control - wash
100 ms Fig. 8. A[Ca2+] and charge movements currents for short and long pulses in the presence and absence of caffeine. a and b, superimposed A[Ca2+] for a 10 (a) and 60 ms pulse (b) before, during and after exposure to caffeine, normalized to the same peak amplitude. Same records as Fig. 7. The trace in caffeine is indicated by *. c and d, charge movement currents recorded simultaneously with the A[Ca2+] for 10 (c) and 60 ms pulses (d). The record in caffeine is superimposed on a single record representing the average of the bracketing control and wash. In c, the on charge was taken from the first 10 ms of a 120 ms depolarization to allow determination of the baseline during the pulse. e and f, charge movement difference currents formed by subtracting the average (no caffeine) record from the caffeine record in c and d, respectively. g and A, charge movement difference currents formed by subtracting the wash record from the control for the 10 ms (g) and 60 ms pulse (h). Bottommost traces show the relative timing of the pulse.
transients in Fig. 8a. The IQ records before and after caffeine were averaged and the result is superimposed on the IQ record in caffeine. Caffeine caused no detectable slowing of the 'off' IQ that could account for the delayed turn-off of release. The lack of effect of caffeine on IQ for the short pulse is shown more directly in Fig. 8e, which gives the difference in the IQ records for the 10 ms pulses with and without caffeine. There was no significant difference in the records. Figure 89 gives the difference of the control and wash IQ records to demonstrate the stability of IQ over the course of the experiment. Figure 8df and h are analogous to Fig. 8c, e and 9 but were obtained with the 60 ms pulse. They confirm Figs 4 and 5 in showing that caffeine did not alter IQ for the longer pulses. In the other two fibres in which IQ was measured before,
EFFECTS OF CAFFEINE ON CALCIUM RELEASE 617 during and after caffeine there was a small but consistent decrease in 'off' charge in caffeine for pulses of all durations studied (5-100 ms). However, there was no indication of a slow component of 'off' IQ in caffeine, either when the standard charge movement analysis with sloping baseline subtraction was used or when differences in currents with and without caffeine were directly examined without fitting sloping baselines to remove slow components of ionic current. The slowed turn-off of calcium release after short pulses in caffeine thus does not appear to be due to slowed return of some intramembrane charge after repolarization. Inactivation and recovery of continued release in caffeine The calcium transients and release records in Figs 7 and 8 and those presented by Simon et al. (1989) indicate that in caffeine release continued for a considerable period of time after a 10 ms pulse. However, such continued release after a pulse became less marked with increasing pulse duration and was no longer noticeable after a 60 or 120 ms pulse. Thus, continued release after a pulse in caffeine appeared to inactivate during the pulse, just as total release inactivates during a pulse both with (Simon et al. 1989; present results) and without (Melzer et al. 1984; Schneider & Simon, 1988) caffeine. Studies of the recovery from inactivation following a conditioning pulse provided a convenient means of characterizing inactivation of release in the absence of caffeine (Schneider et al. 1987; Schneider & Simon, 1988). We therefore examined the recovery of both peak rate of release during long test pulses and continued release after short test pulses as a function of the recovery time after a relatively long conditioning pulse that produced near maximal inactivation of release. Figure 9 gives the results of a recovery experiment carried out before applying caffeine to a fibre. Figure 9a presents a family of superimposed calcium transients for a 60 ms conditioning pulse to -20 mV followed after various recovery times, t, by an 80 ms test pulse to the same voltage (pulse protocol at bottom left). Release records were calculated from each of the calcium transients in Fig. 9a and are superimposed in b. During the conditioning pulse release attained its peak level and then declined to close to the steady level representing maximal inactivation. Thus, inactivation of release was nearly complete at the end of the conditioning pulse. The release records for the test pulses in Fig. 9 b demonstrate the previously described marked suppression of the peak in the test release at the shortest recovery time and gradual return of the peak in release as the recovery time was increased (Schneider et al. 1987; Schneider & Simon, 1988). The recovery of peak Rrel that occurs during the initial 1 s of recovery is primarily due to recovery from inactivation of release whereas the slower recovery that occurs over the next 60 s is due to recovery from depletion of calcium from the SR (Schneider et al. 1987; Schneider & Simon, 1988). The records in Fig. 9 c give the family of release records from panel b after correction for any decrease in release due to depletion of calcium from the SR. They thus represent pure recovery from inactivation since any suppression of release due to calcium depletion has been eliminated. The ratio of peak Rrei during the test pulse (P2) to peak Rrei during the pre-pulse (P1) for each recovery time in Fig. 9c thus provides an index of the suppression of release due to inactivation without any effects of depletion. A graphical presentation of the time course of recovery of P2/P1
618
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
in the absence of caffeine as obtained from the records in Fig. 9 c is given in Fig. 11 A
(Z1 and left ordinate scale). Recovery from inactivation of release was complete by 400 ms after the end of the conditioning pulse in the absence of caffeine. Figure 9d presents calcium transients from a recovery run in which the ad [Ca + 1 2 um A2
300 ms
e
b
f
C
Jl
t
'z~~~100 mVn'
1
mM
release 1u
Corrected
20 m
ARate of
ML L =.L t
,lf =s
Fig. 9. Recovery of the peak Rrei after a conditioning pre-pulse in the absence of caffeine. a, A[Ca2"] for a 60 ms conditioning pulse to -20 mV followed after a variable time, t (bottom trace), by a test pulse of 80 ms to the same potential. b, Rrel calculated from the calcium transients in a. c, Rrel from b corrected for depletion of calcium from the SR, assuming the initial contents to be 1200 /tM. d, A[Ca2+] for a 60 ms pre-pulse, followed after a variable time, t, by a 10 ms pulse to -20 mV. e, Rrei calculated from the A[Ca2+] records in d. f, Rre, records corrected for depletion of SR calcium. For the records in d-f the signals during, and for 20 ms after, the conditioning pulses of each trace are superimposed. However, for clarity, only the non-overlapping portion of the record during and after the test pulse is shown. Also in d-f, the rightmost records are A[Ca2+], Rrei and Rrei corrected for depletion, respectively, for a 10 ms pulse with no prior conditioning pulse. The adjusted values of the removal model parameters were koff, Mg-Parv = 4-1 S-l, Vmax = 221 SM s-1. [Parv] and koff, Mg-Parv were set to 800 /IM and 1-5 s'l, respectively. Fibre 471, 4-1 #um per sarcomere, temperature 8 0C, AP III concentration 402-476 ,lM.
conditioning pulse was the same as in Fig. 9a-c but the test pulse duration was decreased to 10 ms (bottom-right pulse protocol). During the experiment the records in Fig. 9d were actually obtained in an alternating sequence with those in a. The calcium transients for all conditioning pulses that were followed by short test pulses are superimposed in Fig. 9d. However, since the calcium transients for the short test pulses were rather small, for clarity only the calcium record for a single test pulse is shown at the time of each test pulse in Fig. 9d. The record at the extreme right of Fig. 9d gives the calcium transient obtained later in the experiment using the short test pulse applied by itself without any conditioning pulse. This represents complete recovery from inactivation. Figure 9e presents the Rrel records obtained from each of the calcium transients in d. The release for the short test pulse was smallest at the
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
619
shortest recovery interval in Fig. 9e and increased with increasing recovery time. Figure 9f gives the release records from e after correcting for suppression of release due to calcium depletion. The small negative component in the release records for the short test pulses during the recovery run probably indicates a slight error in the removal model parameter values. The fact that the release record for the short test pulse applied by itself without any conditioning pulse (Fig. 9 e andf, extreme right)
dt\\>,
al
b
^ 4 fM t~a-'l
e
C
jJ\~ ~ ~I~ ~ r~i
300 ms
Rate of 10p ~~~~~~~f
j
release O1Mms
orrected
-20 mV .Fh~~L~~zVL-100 mV IZZTtf
nZ t HO mV n t | t~~~~~~~~~~~~~~~~~t=
00
Fig. 10. Recovery of the peak Rrei in the presence of caffeine. a-f, same as Fig. 9, after exposing the fibre to 0 5 mM-caffeine. Note the change in vertical scales for A[Ca2+] and Rrei compared to Fig. 9. For the release records corrected for depletion, the initial SR calcium content was assumed to be 1300 pm. Same fibre and removal parameters as Fig. 9, AP III concentration 681-816 um, 30 min exposure to caffeine.
turned off abruptly to zero after the pulse indicates that removal was correctly calculated for the short pulse by itself and that the slight error in the removal parameters was probably related to the predicted rate of calcium dissociation from the parvalbumin sites during the recovery period. Figure 10 gives the results of the same recovery protocol as in Fig. 9 carried out on the same fibre but now in the presence of 0 5 mM-caffeine. Figure lOa presents the superimposed calcium transients for the recovery sequence using long test pulses in caffeine. The calcium transients during the conditioning pulses in Fig. lOa became successively smaller during the sequence, indicating some run-down of the fibre during the sequence in caffeine. Figure lOb presents the release records calculated from the calcium transients in a. The test pulse release records in Fig. lOb show almost complete suppression of peak release for the shortest recovery time and the return of peak release with increasing recovery time. Figure lOc presents the release records from b after correction for calcium depletion. The time course of recovery from inactivation in caffeine is given by P2/P1 from the records in Fig. lOc and is presented graphically in Fig. 1 B as L] and left ordinate.
620
M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
Values of P2/P1 from similar records for two longer recovery times in caffeine not included in Fig. IOa-c are also presented in Fig. 1 B. Comparison of the P2/P1 values (L]) in Fig. 11A and B shows that recovery from inactivation of release was considerably slower in caffeine (Fig. 1 B) than in control (Fig. I IA). Comparison of .21
A Control
1.0
0
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00
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~~~00
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0
0
0-0
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500 1000 1500 Recovery time (ins)
00
of the recovery of the peak Rre, and of th in the presence and absence Fig. 11. Time course of caffeine. A, D and left ordinate, P2/P1 of depletion-corrected B for the 80 ms test pulse (Fig. 9c) versus recovery time. 0 and right ordinate, ti versus recovery time, from depletion-corrected Rrei for the 10 ms test pulse (Fig. 9f). 0, single value of tj from a 10 ms pulse measured after washing caffeine from the fibre. B, El and left ordinate, P2/P1 versus recovery time in the presence of caffeine (from Fig. lOc). 0 and right ordinate, ti versus recovery time from Fig. lOf.
Figs 9a and 10a indicates that the slower recovery from inactivation of release in caffeine could be due to the larger calcium transient during the conditioning pulse in caffeine and the slower decay of [Ca2+] after the conditioning pulse in caffeine compared to control since recovery from inactivation requires the decline of [Ca2+] (Schneider & Simon, 1988). Note that the vertical A[Ca2+] scale is somewhat compressed in Fig. 10a and d compared to Fig. 9a and d. Other experiments indicated that caffeine did not alter the calcium dependence of inactivation of calcium release (M. G. Klein, B. J. Simon & M. F. Schneider, unpublished observations). Figure 10d presents calcium transients for the recovery sequence in caffeine using the short test pulse, with the presentation of records being the same as in Fig. 9d. Comparison of the short-test-pulse calcium transients in control (Fig. 9d) and caffeine (Fig. 1Od) reveals that for long recovery times, or without a conditioning pulse (extreme right), the test-pulse calcium transient began to decline abruptly after
EFFECTS OF CAFFEINE ON CALCIUM RELEASE
621
the test pulse in control but continued to rise after the end of the test pulse in caffeine. Although difficult to discern in Fig. lOd, the test-pulse calcium transients for the shortest recovery times in caffeine did not continue to rise after the test pulse but began to decline abruptly after the pulse. Figure tOe presents the release records calculated from the calcium transients in Fig. 1Od, andf presents the release records corrected for calcium depletion. Figure toe andf shows the recovery of the amplitude of the test release with increasing recovery time. In addition, it demonstrates a systematic slowing of the turn-off of release after the short test pulse release as the recovery time was increased. Slowing of the turn-off of release after short test pulses was seen during recovery in caffeine (Fig. tOe and ]) but not during recovery in control (Fig. 9e and]). The speed of turn-off of release after short test pulses was quantified by determining the time (th) from the peak Rrei to one-fifth of peak for each short test pulse release record after correction for depletion (Figs. 9f and 10f). The values of t obtained in the absence of caffeine are presented as the circles and right ordinate in Fig. I I A. The values of tj in control (O in Fig. I I A) ranged from 4 to 5 ms and were essentially independent of recovery time. In Fig. 1 IA, 0 gives the value of ti obtained with the short test pulse without pre-pulse after washing caffeine from the chamber. It was the same as in control, indicating that any charge of tj in caffeine cannot be attributed to fibre run-down. The circles in Fig. llB give the values of to for the short test pulse in the presence of caffeine. For the shortest recovery time ti was 6 ms, about the same as in the absence of caffeine. However, as the recovery time was increased t1 increased to a mean of 17 + 2 for the four points in Fig. 11 B for no conditioning pulse or recovery times of 1 s or more. The increase in tj with increasing recovery time in caffeine (O in Fig. 1 1 B) occurred in parallel with the recovery of peak release in caffeine (D1 in Fig. 11B). Since a large value of ti represents inability to turn off release rapidly after a short pulse, the data in Fig. 11 demonstrate that the inability to turn off release after a short pulse in caffeine was suppressed by a conditioning pulse and recovered in parallel with the recovery of the inactivatable component of calcium release. The increase in t, with increasing recovery time in caffeine was qualitatively different from the constant relatively small value of ts in the absence of caffeine, which indicates abrupt turn-off of calcium release after short test pulses both without a conditioning pulse and for all recovery times after a conditioning pulse in the absence of caffeine (Fig. lIA).
DISCUSSION
Effects of caffeine on calcium transients and calcium release The present results confirm and extend the results of two previous studies from other laboratories on the effects of 0 5 mM-caffeine on calcium transients in voltageclamped frog muscle fibres. Kovacs & Sziics (1983; 2-4 0C) and Delay et al. (1986; 12 0C) both reported that caffeine caused negative shifts of about 6-10 mV in the voltage dependence of the peak absorbance changes recorded with the calcium indicator dyes Antipyrylazo III or Arsenazo III in cut skeletal fibres. Kovacs & SzUcs (1983) presented AP III absorbance signals for relatively long pulses to -40
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M. G. KLEIN, B. J. SIMON AND M. F. SCHNEIDER
to -60 mV (their Fig. 4) that indicate that the voltage for a given absorbance waveform also was shifted by roughly 10 mV to more negative voltages in the presence of caffeine. Delay et al. (1986) used sufficiently large depolarizations to demonstrate that the maximum absorbance change for large depolarizations was increased in caffeine. The AP III absorbance changes measured in the present study (6-10 0C) and the calcium transients obtained therefrom and presented here confirm each of the above observations on the effects of 0 5 mM-caffeine. In the present study the rate of release of calcium from the SR was calculated from each calcium transient so that the effects of caffeine on the rate of calcium release could also be determined. The calculated release records indicate that for relatively long pulses both the peak and the final level of release were increased in the presence of 0 5 mM-caffeine. The voltage dependence of both peak and steady level were shifted towards more negative voltages in caffeine with little change in steepness. Using short pulses we observed a qualitative change in release in the presence of caffeine. Calcium release continued for an appreciable period after the end of 10-20 ms pulses in the presence of caffeine, but never in its absence (Simon et al. 1989; present results). Continued release after a short pulse in caffeine was suppressed by a relatively long conditioning pulse and recovered after the pulse. Our release calculations show that the recovery of continued release after a conditioning pulse occurred in parallel with the recovery from inactivation of calcium release. Thus continued release after a pulse inactivated and recovered together with the inactivatable release during a pulse. Signs of continued release after the end of short pulses in caffeine were very prominent in the calcium transients from the fibres used in the present study but are not evident in the absorbance signals for short pulses presented by Kovacs & SzUcs (1983). However, using fibres from some other groups of frogs we have also seen little evidence of continued release after the pulse in the calcium transients for short pulses in caffeine, even though the calcium transients were larger in caffeine. A continued rise in A[Ca2+] after a pulse requires that release exceed removal. Preliminary modelling of the calcium-induced calcium release indicates that the occurrence of a continued rise may be quite sensitive to the relative strength of the removal system (M. G. Klein, B. J. Simon & M. F. Schneider, unpublished calculations). Thus the discrepancy between the present results and those of Kovacs & Sziics (1983) may have been due to differences in calcium removal capability, perhaps due to some difference in the physiological state of the frogs or the conditions of the experiments. Also, we have observed in some cases that the continued release after short pulses in caffeine declined with time during exposure to caffeine, even though the amplitudes of the calcium transients were still increased (M. G. Klein, B. J. Simon & M. F. Schneider, unpublished observations). It might appear puzzling that in the present experiments release after a short pulse exhibited qualitatively different behaviour in the presence and absence of caffeine, whereas release during a longer pulse to the same voltage did not appear to be qualitatively different in the presence and absence of caffeine. The explanation for these observations appears to be that release declined by the same calciumdependent inactivation mechanism during each pulse in both the presence and the absence of caffeine. Thus, the control release in the absence of caffeine and the potentiated release in the presence of caffeine both declined during a pulse with the
EFFECTS OF CAFFEINE ON CALCIUM RELEASE 623 same time constant of inactivation, giving both release records the same qualitative appearance. In contrast, the turn-off of release after a short pulse in caffeine was by the relatively slow process of calcium-dependent inactivation, whereas the turn-off of release after all pulses in the absence of caffeine was by the considerably faster process of voltage-dependent deactivation. Thus, the speed of turn-off of release was qualitatively different after short pulses with and without caffeine.
Is calcium-induced calcium release significant in the absence of caffeine? Caffeine is well known to potentiate calcium-induced calcium release in skinned skeletal fibres (Endo, 1985) and in heavy SR vesicles isolated from skeletal fibres (Rousseau et al. 1988). Thus it seems reasonable to assume that all effects of caffeine observed in the present study were produced by caffeine either introducing or increasing a component of calcium-induced release. In this regard it is interesting to note that the changes in calcium release observed here using long pulses in the presence of caffeine were quantitative rather than qualitative. Thus, the present results with long pulses are not inconsistent with the possibility that calcium-induced release was significant during fibre depolarization even in the absence of caffeine. For example, if release in the absence of caffeine involved an initial direct activation of SR calcium channels by TT charge movement followed by a secondary further activation of the same or other SR calcium channels by calcium-induced release, a potentiation of the latter in the presence of caffeine might conceivably lead to the effects of caffeine observed with long pulses. Our observations with short pulses also do not rule out the possibility that calcium-induced calcium release may have contributed significantly to release during depolarization in the absence of caffeine. However, they do indicate that if calciuminduced release were significant in the absence of caffeine, it must have had properties that were different from those of the calcium-induced release introduced by caffeine. For example, caffeine might have potentiated calcium-induced calcium release by increasing the affinity of a calcium binding site that activates release (Endo, 1975, 1985; Rousseau et al. 1988). In that case in the presence of caffeine the myoplasmic free calcium would be sufficiently elevated after repolarization for calcium to remain bound to the activating site. Release would then continue until the calcium removal system reduced the free calcium sufficiently for calcium to dissociate from the activating site and the channel to close. In the absence of caffeine, the much lower calcium affinity of the activating site would preclude binding of calcium at the global level of free calcium measured with the calcium indicators. However, during release large calcium gradients may have existed (Cannell & Allen, 1984). Calcium levels would then be expected to be elevated in the immediate vicinity of the release channel compared with the global [Ca2+], and local [Ca2+] could have been sufficiently high for calcium to bind to the activating site. The local [Ca2+] in the immediate vicinity of a calcium channel could become elevated extremely rapidly after the channel opened and could dissipate just as quickly when the channel closed (Simon & Llinas, 1985). If calcium equilibrated rapidly with the activating site, the channel activation due to bound calcium would also reverse very rapidly when the channel closed. In this case release could turn off rapidly on repolarization in the absence of caffeine despite significant activation by
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calcium-induced calcium release. The channel would then remain closed until activated by a TT voltage sensor or perhaps by calcium ions from a neighbouring calcium channel activated by a voltage sensor. Thus, in the absence of caffeine the contribution of calcium-induced release could be tightly coupled to charge movement.
Control of SR calcium release in the presence and absence of caffeine One possible model for the control of SR calcium release assumes that all SR calcium channels are identical and that each has at least two different types of interaction sites for calcium, activating sites and inactivating sites, in addition to a site for activation by interaction with the TT voltage sensor. Alternatively, there could be two types of channels with some being activated only by the TT voltage sensor and others being activated only by calcium (Rios & Pizarro, 1988), but with all having calcium binding sites for inactivation. A channel would be closed due to inactivation if the calcium inactivating site is occupied by calcium and is in the inactivated conformation (Schneider & Simon, 1988). If the calcium inactivating site is not in the inactivated conformation, the channel could be opened either by interaction with the TT voltage sensor in its activating conformation or by binding calcium at the calcium activating site. Caffeine is assumed to promote the interaction of calcium with the calcium activating site. For present purposes the modulatory binding sites for nucleotides and Mg2" (Endo, 1985; Meissner, Darling & Eveleth, 1986) may be considered to provide a constant influence on channel activation since nucleotide and Mg2+ concentrations would remain relatively constant for the pulses used here. According to the preceding models, the observation that after short pulses in caffeine release turns off with the time constant of inactivation indicates that in caffeine the dissociation of calcium from the calcium activating site must be slow compared to the time constant of inactivation. This could be the case even if calcium equilibrated rapidly with the activating site because [Ca2+] remained above the dissociation constant for the calcium activating site for a relatively long time after repolarization in caffeine. In this regard, it is interesting to note that after long pulses in caffeine there was a fast turn-off of release, presumably by deactivation due to return of the TT voltage to its resting position. This fast but incomplete turn-off was followed by a prolonged small continued release. Since the release remaining at the end of a long pulse is by definition the non-inactivatable component, any part of such release that was associated with occupied calcium activating sites could not turn off by inactivation but would have to turn off by dissociation of calcium from the calcium activating site. Very slow dissociation of calcium from the activating site because of continued high [Ca2+] after the pulse would be consistent with the observed small but prolonged pedestal of release after long pulses in caffeine. In the absence of caffeine the affinity of the calcium activating site would be lower, calcium activation might occur only in response to locally elevated [Ca2+] and reversal of calcium activation could be fast because of rapid dissipation of local [Ca2+] gradients and rapid dissociation of calcium from the calcium activating site (preceding section). Preliminary computer simulations (M. G. Klein, B. J. Simon & M. F. Schneider,
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unpublished) with both the single-channel type of model and the two-channel type of model indicate that both models may be able to account for the voltage shift and increased peak rate of release in the presence of caffeine and for the changes in turnoff of release after short and long pulses in the presence of caffeine. The effect of caffeine was simulated by introducing significant calcium-induced release driven by global [Ca2+] in the presence of caffeine. In the absence of caffeine, both types of model could include significant calcium-induced release and could still reproduce the observed release properties provided that the calcium-induced release was driven by locally elevated [Ca2+] as described above. In all cases, the observed effects of caffeine could be simulated without changes in charge movement. Thus, involvement of calcium-induced calcium release during depolarizations in both the presence and absence of caffeine could be consistent with present observations. However, it remains to be established whether this process is indeed involved in calcium release in the absence of caffeine. We thank Mr Gabe Sinclair and Mr Walt Knapik for custom modification of optical and mechanical apparatus, and Mr Jeff Michael and Mr Chuck Leffingwell for construction of electronic equipment. This work was supported by research grants from the National Institutes of Health (NS 23346) and the Muscular Dystrophy Association. REFERENCES
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