Calcium transients in frog skeletal muscle fibres following conditioning stimuli

1. Intracellular Ca(2+) transients were recorded from frog twitch muscle fibres, using arsenazo III as a Ca(2+) monitor. When fibres were stimulated by two action potentials, the arsenazo signal to the second stimulus was smaller than the first, for stimulus intervals of up to several seconds.2. The recovery of the amplitude of the second response followed two exponential time courses; a fast one with a time constant of about 70 msec giving recovery to about 90% of the control value, followed by a slow recovery to 100%, with a time constant of about 12 sec (at 10 degrees C).3. The time constant of the fast recovery component was strongly temperature-dependent, with a Q(10) of approximately 2.7, whilst the Q(10) of the slow component was about 1.4.4. Removal of Ca(2+) in the bathing medium lengthened the time constant of the slow recovery component by a factor of three, but had little effect on the fast recovery component. The lengthening of the slow component was not reversed by addition of Mg(2+), but Sr(2+) ions could substitute for Ca(2+).5. The influence of membrane potential on the recovery time-course was investigated after blocking action potentials with tetrodotoxin, using a voltage clamp to control membrane potential. Paired depolarizing stimuli were used, with the potential held to either low (-60 or -80 mV) or high (-110 or -140 mV) potentials between stimuli. No differences were apparent in either the fast or slow recovery components at these holding potentials.6. The arsenazo response elicited by an action potential following a conditioning tetanus was reduced in size even more strongly than following a single action potential. The time course of recovery of the response following a tetanus again comprised two exponential components. After a 20 Hz tetanus for 0.5 sec, the fast component had a time constant of about 400 msec, and gave a recovery to about 60% of the control value. Subsequent recovery to 100% occurred with a time constant of about 12 sec.7. The time constant of the fast recovery component increased markedly with increasing frequency or duration of the conditioning tetanus. The time constant of the slow component was not appreciably altered by conditioning tetani varying between one impulse and sixty impulses. However, the reduction in response size due to the slow component, extrapolated to zero stimulus interval, increased with increasing number of impulses in the tetanus.8. The time constant of the fast recovery component corresponded closely with the decay time constant of the arsenazo response to the conditioning stimulus. This correspondence held over a nearly fifty-fold range of time constants, and for two different conditions which affected the decay time constant (temperature, and frequency of tetanic stimulation).9. The decay time constant of the arsenazo response elicited by an action potential was slowed by a preceding impulse or tetanus. Following a 20 Hz tetanus for 0.5 sec, recovery of the half decay time appeared to follow an exponential time course, with a time constant of about 12 sec.10. These results suggest that the fast recovery component reflects the re-filling of release stores in the sarcoplasmic reticulum by Ca(2+) ions taken up from the cytoplasm. The origin of the slow component is less clear, but it may arise from inactivation of the excitation-contraction (e-c) coupling process between T-tubule depolarization and Ca(2+) release from the sarcoplasmic reticulum.