Reversible Thiol-Dependent Activation of Ryanodine-Sensitive Ca2+ Release Channel by Etoposide (VP-16) Phenoxyl Radical

Many phenolic compounds can act as antioxidants by donating a proton to peroxyl radicals and quenching peroxidation. Phenoxyl radicals produced this way or from metabolism by peroxidases, tyrosinase, or mixed-function oxidases, however, may react with sulfhydryl groups of proteins and other endogenous thiols. In this regard, phenolic compounds may have cytotoxic potential instead of antioxidant effects. W e employed the anticancer drug, etoposide (VP-16), as a model phenolic compound to study the sensitivity of ryanodine-sensitive Ca2+ channel (RyR) to VP-16 phenoxyl radicals. The combination of VP-16 and tyrosinase, used to generate the etoposide phenoxyl radical, produced marked Ca2+ release from Ca2+-loaded RyR-rich vesicles prepared from terminal cisternae fraction of sarcoplasmic reticulum (SR). This effect was reversed by the SH-reagent, dithiothreitol (DTT), suggesting that cysteines within the RyR-protein complex were targets for modification by VP-16 phenoxyl radicals. VP-16/tyrosinase-induced release of Ca2+ was attenuated in vesicles prepared from longitudinal SR, which contain relatively little RyR. The effects of the VP-16 phenoxyl radical on Ca2+-ATPase in SR vesicles resembled those observed with caffeine or 4,4'-dithiodipyridine, both of which activate RyR Ca2+ release and lead to activation of Ca2+-ATPase via prolonged Ca2+ cycling. The addition of ruthenium red returned Ca2+-ATPase to its original level. Thus, under these conditions Ca2+-ATPase was not directly affected by VP-16 phenoxyl radical. The hypersensitive SH-groups on RyR are shown to be targets for oxidation of VP-16 phenoxyl radical, and suggest that other phenolic compounds could similarly disrupt Ca2+ homeostasis. Antiox. Redox Signal. 2, 73-82.