CALCIUM TRANSIENTS EVOKED BY ACTION-POTENTIALS IN FROG TWITCH MUSCLE-FIBERS

Intracellular Ca2+ transients were recorded from frog twitch muscle fibers in response to action potentials and repetitive stimulation, using ionophoretically injected arsenazo III as a Ca2+ monitor. A dual wave-length optical system was used to measure absorbance changes of the injected dye from small areas of single fibers within the cutaneous pectoris muscle. The absorbance spectrum of the injected arsenazo III in a resting fiber was consistent with an intracellular free Mg2+ level of a few hundred micromolar, assuming an intracellular pH of 7.1. The resting free Ca2+ concentration was below the limit of resolution. The wave-length dependence of the arsenazo light absorbance signal during twitches followed that expected for Ca2+ bindig to the dye. Recordings made at wave-lengths where the dye is maximally sensitive to pH or Mg2+ concentration changes indicated that interference from these sources is minimal at the usual wave-length pair (650-700 nm) used for Ca2+ recordings. Over a wide range of intracellular dye concentrations, the size of the arsenazo response to an action potential increased linearly with dye concentration (100-1000 .mu.M), although there were deviations from this relationship at low and high concentrations. An approximate estimate of 8 .mu.M was obtained for the peak free Ca2+ concentration change following a single action potential. Changes in temperature (6.degree.-25.degree. C) did not significantly affect the size of the free Ca2+ transient. During maximal tetanic stimulation, the signal rose to about 3 times higher than the twitch response. An approximate minimum estimate of the increase in total cytoplasmic Ca2+ concentration during a twitch gave a value of 220 .mu.M. A latency of .apprx. 1.5 ms (at 10.degree. C) was observed between the foot of an action potential and the onset of the arsenazo response. Recordings made using a narrow measuring light slit, placed either at the edge or the center of a fiber, suggested that only a small part of this latency could be due to inward conduction of the action potential along the T-tubules. The decay phase of the arsenazo response to an action potential followed an exponential time course, with a time constant of 71 ms at 10.degree. C. This time constant was strongly temperature-dependent, with a Q10 of about 2.4. An Arrhenius plot of the decay time constant gave a straight line. During repetitive stimulation, the arsenazo responses evoked by successive impulses showed 2 changes: a progressive decrease in amplitude and a slowing of the decay. The extent to which successive responses summated during a tetanus depended on the balance between these 2 effects.