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

The effect of oxidants on Ca²⁺ movement in osteoblasts is unclear. In this study, we show that 4,4′-dithiodipyridine (4,4′-DTDP), a reactive disulphide that mobilizes Ca²⁺ in muscle, induces an increase in cytoplasmic free-Ca²⁺ concentrations ([Ca²⁺]_i) in MG63 human osteosarcoma cells loaded with the Ca²⁺-sensitive dye fura-2. 4,4′-DTDP acted in a concentration-dependent manner with an EC₅₀ of 10 μM. Removing extracellular Ca²⁺ reduced the Ca²⁺ signal by 35%. In Ca²⁺-free medium, the 4,4′-DTDP-induced [Ca²⁺] _i increase was not changed by depleting store Ca²⁺ with 50 μM brefeldin A (a Golgi apparatus permeabilizer), by 2 μM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 μM thapsigargin (an inhibitor of the endoplasmic reticulum Ca²⁺ pump) or by 5 μM ryanodine. Ca²⁺ signals induced by 4,4′-DTDP in Ca²⁺-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4′-DTDP-induced Ca²⁺ 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 [Ca²⁺]_i increases in human osteosarcoma cells via releasing store Ca²⁺ from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca²⁺ 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.