Effect of tetanus duration on the free calcium during the relaxation of frog skeletal muscle fibres.

Single fibres were dissected from the tibialis anterior muscle of the frog and injected with the photoprotein aequorin. Tension and the light emission of the injected aequorin (a function of the free intracellular calcium concentration) were recorded both at rest and during tetanus relaxation. The level of light emission from resting single fibres corresponded to a free intracellular calcium concentration (Ca2+i) of 100 nM (+/- 40 nM, n = 4). The time course of the decline in light was examined during the three periods of muscle relaxation: period 1 during the slow phase of tension relaxation, period 2 during the exponential phase of relaxation and period 3 after the completion of force relaxation. The time course of the decline in light (after a correction for the kinetics of the aequorin reaction) showed that Ca2+i declines exponentially with a rate constant of 25 s-1 (+/- 1.7, n = 3) after a single stimulus at 10 degrees C. With increasing tetanus duration, the rate of decline of Ca2+i decreased during period 1. It is suggested that this decrease in the rate of decline of Ca2+i results from an intracellular calcium buffer (which takes up calcium in parallel with the sarcoplasmic reticulum) becoming loaded with calcium during the tetanus. Throughout period 2 Ca2+i was elevated above resting levels. The level of Ca2+i during this period varied from fibre to fibre but could be as high as 1 microM. The mean level of Ca2+i during this period also depended on the tetanus duration. A long-lasting elevation in Ca2+i was observed during period 3, Ca2+i returning towards resting levels with an approximately exponential time course. During this period the level of Ca2+i (at a given time after the last stimulus) depended on the tetanus duration. It is suggested that this long-lasting elevation in Ca2+i reflects the release of calcium from the intracellular calcium buffer described above. The results suggest that the rate of decline of Ca2+i after a few seconds of tetanic stimulation can be explained by the rate of calcium sequestration by the sarcoplasmic reticulum. The increased rate of decline of Ca2+i after shorter periods of stimulation may be explained by the presence of a buffer that takes up calcium in parallel with the sarcoplasmic reticulum. The later release of calcium from this buffer gives rise to the long-lasting elevation in Ca2+i during period 3. The slow kinetics of calcium binding and release by this buffer appear compatible with published data on the kinetic properties of parvalbumin.