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.