TWITCH-DEPENDENT SR CA ACCUMULATION AND RELEASE IN RABBIT VENTRICULAR MYOCYTES

Using caffeine-induced contractures (C(caf) and thapsigargin (TG), we estimated the fraction of sarcoplasmic reticulum (SR) Ca released at one twitch and also the number of twitches required to reload a Ca-depleted SR. Similar results were obtained for twitches or intracellular Ca (Ca(i)) transients with the fluorescent indicator, indo 1. Sustained exposure to 10 mM caffeine completely depletes the SR of Ca in <5 s (as assessed by a second C(caf)). After such Ca depletion, four to five twitches are necessary to reload the SR to the steady-state level (with a twitch constant, tau = 1.6 twitches). We also determined the time required for complete inhibition of the SR Ca-adenosinetriphosphatase (ATPase) by TG. After SR Ca depletion, 5 muM TG was applied for different periods of time before a train of 'reloading'' twitches. A TG exposure period of 90 s was sufficient to completely prevent C(caf) after these reloading twitches. When SR is Ca depleted, the twitch is larger in the presence of TG, indicating that the SR Ca-ATPase can limit the ability of Ca influx to activate contraction. To assess SR Ca released at one twitch in cells with normally Ca-loaded SR, 5 muM TG was applied for 90 s to prevent SR Ca reuptake. Then one or several twitches were activated (causing SR Ca release, but with reuptake completely blocked). After the twitch (or train), a C(caf) was used to assess remaining SR Ca. We found that one twitch released about one-half of the Ca in the SR, but the beat-dependent depletion of SR Ca was biexponential (tau = 0.9 and 60 twitches). The slower phase might represent a caffeine-sensitive pool of Ca not normally released during a twitch.