Interaction of cyclopiazonic acid with rat skeletal muscle sarcoplasmic reticulum vesicles. Effect on Ca 2+ binding and Ca 2+ permeability

Abstract

The interaction of cyclopiazonic acid with rat skeletal muscle sarcoplasmic reticulum (SR) vesicles was investigated in order to study the mechanism of cyclopiazonic acid inhibition of the Ca 2+-ATPase (Goeger et al., Biochem Pharmacol 37: 978–981, 1988). Cyclopiazonic acid at 25 μM prevented the binding of Ca 2+ to the high affinity binding site of mixed (light and heavy) SR vesicles and inhibited, in a dose-dependent manner, the Ca 2+-dependent phosphorylation of SR vesicles by ATP. Binding of Ca 2+ to the high affinity site of the Ca 2+-ATPase is necessary for both Ca 2+ transport and for phosphorylation of the Ca 2+-ATPase. We conclude that inhibition of Ca 2+ binding to the high affinity site may be responsible, at least in part, for the activity of cyclopiazonic acid. The mechanism of inhibition remains unclear. The inhibition was not reduced after dialysis and was only partially reversed by gel filtration of SR vesicles treated with cyclopiazonic acid. Neither 1 mM glutathione nor dithiothreitol pretreatment had any effect on the inhibition of the Ca 2+-ATPase. In addition to its inhibition of Ca 2+ uptake and the Ca 2+-ATPase, cyclopiazonic acid had significant effects on Ca 2+ efflux from both passively and actively loaded SR vesicles. Cyclopiazonic acid impeded the efflux of Ca 2+ from passively loaded SR vesicles (in the presence of ruthenium red) when compared to either untreated vesicles or those treated with mersalyl acid, a mercurial which also inhibits the Ca 2+-ATPase and is known to induce Ca 2+ release by both ruthenium red-sensitive and -insensitive pathways. Treatment of actively loaded vesicles with cyclopiazonic acid resulted in a decreased rate of Ca 2+ efflux when compared to SR vesicles in which the Ca 2+-ATPase activity was inhibited by ATP depletion with hexokinase and glucose. The results are consistent with the hypothesis that, in mixed SR vesicles, cyclopiazonic acid inhibits both the Ca 2+ pump and Ca 2+ efflux.