Thiol oxidation by 2,2′-dithiodipyridine induced calcium mobilization in MG63 human osteosarcoma cells

2,2′-dithiodipyridine (2,2′-DTDP), a reactive disulphide that mobilizes Ca²⁺ in muscle, induced an increase in cytoplasmic free Ca²⁺concentrations ([Ca²⁺]_i) in MG63 human osteosarcoma cells loaded with the Ca²⁺-sensitive dye fura-2. 2,2′-DTDP acted in a concentration-independent manner with an EC₅₀ of 50 μM. The Ca²⁺ signal comprised an initial spike and a prolonged increase. Removing extracellular Ca²⁺ did not alter the Ca²⁺ signal, suggesting that the Ca²⁺ signal was due to store Ca²⁺ release. In Ca²⁺-free medium, the 2,2′-DTDP-induced [Ca²⁺]_i increase was not changed by depleting store Ca²⁺ with 50 μM bredfeldin A (a Golgi apparatus permeabilizer), 2 μM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺pump inhibitor) or 5 μM ryanodine. Conversely, 2,2′-DTDP pretreatment abolished CCCP and thapsigargin-induced [Ca²⁺]_i increases. 2,2′-DTDP-induced Ca²⁺ signals in Ca²⁺-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 2,2′-DTDP-induced Ca²⁺ release was inhibited by a thiol-selective reducing reagent, dithiothreitol (5-25 μM) in a concentration-dependent manner. Collectively, this study shows that 2,2′-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 2,2′-DTDP-induced store Ca²⁺ release appeared to be dependent on oxidation of membranes.