Sulfhydryl modification by 4,4'-dithiodipyridine induces calcium mobilization in human osteoblast-like cells

The effect of oxidants on Ca(2+) movement in osteoblasts is unclear. In this study, we show that 4,4'-dithiodipyridine (4,4'-DTDP), a reactive disulphide that mobilizes Ca(2+) in muscle, induces an increase in cytoplasmic free-Ca(2+) concentrations ([Ca(2+)](i)) in MG63 human osteosarcoma cells loaded with the Ca(2+)-sensitive dye fura-2. 4,4'-DTDP acted in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 35%. In Ca(2+)-free medium, the 4,4'-DTDP-induced [Ca(2+)](i) increase was not changed by depleting store Ca(2+) with 50 microM brefeldin A (a Golgi apparatus permeabilizer), by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) pump) or by 5 microM ryanodine. Ca(2+) signals induced by 4,4'-DTDP in Ca(2+)-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4'-DTDP-induced Ca(2+) release was inhibited by a thiol-selective reducing reagent, dithiothreitol (0.05-2.5 mM), in a concentration-dependent manner. Collectively, this study shows that 4,4'-DTDP induced [Ca(2+)](i) increases in human osteosarcoma cells via releasing store Ca(2+) from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca(2+) release induced by 4,4'-DTDP appears to be associated with thiol oxidation. Furthermore, overnight incubation with 4,4'-DTDP inhibited cell activity in a concentration-dependent manner.