MICROINJECTION OF STRONG CALCIUM BUFFERS SUPPRESSES THE PEAK OF CALCIUM RELEASE DURING DEPOLARIZATION IN FROG SKELETAL-MUSCLE FIBERS

The effects of high intracellular concentrations of various calcium buffers on the myoplasmic calcium transient and on the rate of release of calcium (R(rel)) from the sarcoplasmic reticulum (SR) were studied in voltage-clamped frog skeletal muscle fibers. The changes in intracellular calcium concentration (DELTA[Ca2+]) for 200-ms pulses to 0-20 mV were recorded before and after the injection of the calcium buffer and the underlying R(rel) was calculated. If the buffer concentration after the injection was high, the initial rate of rise of the calcium transient was slower after injection than before and was followed by a slow increase of [Ca2+] that resembled a ramp. The increase in myoplasmic [Mg2+] that accompanies the calcium transient in control was suppressed after the injection and a slight decrease was observed instead. After the injection the buffer concentration in the voltage-clamped segment of the fiber decreased as the buffer diffused away toward the open ends. The calculated apparent diffusion coefficient for fura-2 (D(app) = 0.40 +/- 0.03 x 10(-6) cm2/s, mean +/- SEM, n = 6) suggests that approximately 65-70% of the indicator was bound to relatively immobile intracellular constituents. As the concentration of the injected buffer decreased, the above effects were reversed. The changes in DELTA[Ca2+] were underlined by characteristic modification of R(rel). The early peak component was suppressed or completely eliminated; thus, R(rel) rose monotonically to a maintained steady level if corrected for depletion. If R(rel) was expressed as percentage of SR calcium content, the steady level after injection did not differ significantly from that before. Control injections of anisidine, to the concentration that eliminated the peak of R(rel) when high affinity buffers were used, had only a minor effect on R(rel), the peak was suppressed by 26 +/- 5% (mean +/- SE, n = 6), and the steady level remained unchanged. Thus, the peak component of R(rel) is dependent on a rise in myoplasmic [Ca2+], consistent with calcium-induced calcium release, whereas the steady component of R(rel) is independent of myoplasmic [Ca2+].