Superfast fibres of toadfish swimbladder muscle generate a series of superfast Ca(2+) transients, a necessity for high-frequency calling. How is this accomplished with a relatively low rate of Ca(2+) pumping by the sarcoplasmic reticulum (SR)? We hypothesized that there may not be complete Ca(2+) saturation and desaturation of the troponin Ca(2+) regulatory sites with each twitch during calling. To test this, we determined the number of regulatory sites by measuring the concentration of troponin C (TNC) molecules, 33.8 mumol per kg wet weight. We then estimated how much SR Ca(2+) is released per twitch by measuring the recovery oxygen consumption in the presence of a crossbridge blocker, N-benzyl-p-toluene sulphonamide (BTS). The results agreed closely with SR release estimates obtained with a kinetic model used to analyse Ca(2+) transient measurements. We found that 235 mumol of Ca(2+) per kg muscle is released with the first twitch of an 80 Hz stimulus (15(o)C). Release per twitch declines dramatically thereafter such that by the 10th twitch release is only 48 mumol kg(-1) (well below the concentration of TNC Ca(2+) regulatory sites, 67.6 mumol kg(-1)). The ATP usage per twitch by the myosin crossbridges remains essentially constant at approximately 25 mumol kg(-1) throughout the stimulus period. Hence, for the first twitch, approximately 80% of the energy goes into pumping Ca(2+) (which uses 1 ATP per 2 Ca(2+) ions pumped), but by the 10th and subsequent twitches the proportion is approximately 50%. Even though by the 10th stimulus the Ca(2+) release per twitch has dropped 5-fold, the Ca(2+) remaining in the SR has declined by only approximately 18%; hence dwindling SR Ca(2+) content is not responsible for the drop. Rather, inactivation of the Ca(2+) release channel by myoplasmic Ca(2+) likely explains this reduction. If inactivation did not occur, the SR would run out of Ca(2+) well before the end of even a 40-twitch call. Hence, inactivation of the Ca(2+) release channel plays a critical role in swimbladder muscle during normal in vivo function.
展开▼
