Effects of free oxygen radicals on Ca 2+ release mechanisms in the sarcoplasmic reticulum of scallop ( Pecten jacobaeus) adductor muscle

Abstract

In vitro oxyradical effects on SR Ca 2+ regulation were studied by using a SR-containing cell-free preparation from scallop ( Pecten jacobaeus) adductor muscle. Ca 2+ variations were fluorimetrically detected after incubation with Fluo-3 in the presence of ATP. Exposure to Fe 3+/ascorbate produced dose-dependent Ca 2+ release from SR vesicles, eventually leading to massive Ca 2+ loss. Exposure to hypoxanthine/xanthine oxidase also caused Ca 2+ release but at a much slower rate. Pre-incubations with catalase or with the hydroxyl radical scavenger KMBA led to a significant decrease in the Fe 3+/ascorbate-induced Ca 2+ release rate and to a delay of massive Ca 2+ loss. Pre-incubations with GSH or DTT strongly reduced the Ca 2+ release caused by Fe 3+/ascorbate and, moreover, they prevented massive Ca 2+ loss from SR vesicles. Addition of GSH or DTT after Fe 3+/ascorbate promptly reduced the Ca 2+ release rate and delayed massive Ca 2+ release. Pre-incubation with the SR Ca 2+ channel blocker ruthenium red strongly reduced the Ca 2+ release caused by Fe 3+/ascorbate, and also prevented massive Ca 2+ loss. In the presence of ruthenium red, Fe 3+/ascorbate treatments followed by Ca 2+ addition revealed that Ca 2+ uptake inhibition was slower than Ca 2+ release. Taken together, data showed that free radicals and, in particular, hydroxyl radicals, affected the scallop SR Ca 2+ regulation. This mainly occurred through Ca 2+ channel opening, most likely triggered by sulfhydryl oxidation, which eventually led to massive Ca 2+ release from SR vesicles. The demonstration of a specific effect of oxyradicals on SR Ca 2+ channels is in line with their possible involvement in cell signaling.