Ca2+ effluxes from the sarcoplasmic reticulum vesicles of frog muscle: Effects of cyclopiazonic acid and thapsigargin

Ca2+ efflux from frog muscle sarcoplasmic reticulum (SR) vesicles was studied by measuring external free [Ca2+] using Fluo-3 fluorescence. Light SR vesicles were preloaded with Ca2+ in the presence of ATP and inorganic phosphate (P-i). Calcium pump reversal was activated by either depletion of the medium ATP by apyrase in the presence of 20 mM P-i, or resuspending preloaded vesicles in an ATP-free solution containing 1 mM ADP and 20 mM Pi. Cyclopiazonic acid (CPA) and thapsigargin (TG), at concentrations of 2.5 mu M, which completely inhibit Ca2+ uptake, both inhibited the pump reversal efflux almost completely. When active Ca2+ uptake was stopped by either ATP-depletion or addition of CPA, a leak efflux of 6-7 nmole/mg/min was recorded. TG (2.5 mu M) reduced this leak by over 50%, suggesting that TG, but not CPA, can slow the passage of calcium ions through the Ca2+-ATPase passive channel.