Effects of free oxygen radicals on Ca2+ release mechanisms in the sarcoplasmic reticulum of scallop (Pecten jacobaeus) adductor muscle

In vitro oxyradical effects on SR Ca2+ regulation were studied by using a SR-containing cell-free preparation from scallop (Pecten jacobaeus) adductor muscle, Ca2+ variations were fluorimetrically detected after incubation with Fluo-3 in the presence of ATP. Exposure to Fe3+/ascorbate produced dose-dependent Ca2+ release from SR vesicles, eventually leading to massive Ca2+ loss. Exposure to hypoxanthine/xanthine oxidase also caused Ca2+ 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 Fe3+/ascorbate-induced Ca2+ release rate and to a delay of massive Ca2+ loss, Pre-incubations with GSH or DTT strongly reduced the Ca2+ release caused by Fe3+/ascorbate and, moreover, they prevented massive Ca2+ loss from SR vesicles, Addition of GSH or DTT after Fe3+/ascorbate promptly reduced the Ca2+ release rate and delayed massive Ca2+ release. Pre-incubation with the SR Ca2+ channel blocker ruthenium red strongly reduced the Ca2+ release caused by Fe3+/ascorbate, and also prevented massive Ca2+ loss. In the presence of ruthenium red, Fe3+/ascorbate treatments followed by Ca2+ addition revealed that Ca2+ uptake inhibition was slower than Ca2+ release. Taken together, data showed that free radicals and, in particular, hydroxyl radicals, affected the scallop SR Ca2+ regulation. This mainly occurred through Ca2+ channel opening, most likely triggered by sulfhydryl oxidation, which eventually led to massive Ca2+ release from SR vesicles. The demonstration of a specific effect of oxyradicals on SR Ca2+ channels is in line with their possible involvement in cell signaling.