The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in PC12 cells

We studied the effects of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells using fura-2 fluorescence imaging. Carmustine (100 microM) caused a delayed increase in [Ca(2+)](i) that developed within approximately 3 h. This effect was enhanced in cells that were pretreated with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BSO, 200 microM, 24 h), and was suppressed in cells that were treated with an antioxidant deferoxamine (50 microM). The carmustine-induced increase in [Ca(2+)](i) was absolutely dependent on the presence of extracellular Ca(2+) and could be inhibited by dihydropyridine blockers of L-type voltage-gated Ca(2+) channels (nimodipine or nitrendipine, 10 microM). The increase in [Ca(2+)](i) was also suppressed in Cl(-)-free solution and in the presence of the Cl(-) channel blockers, indanyloxyacetic acid 94 (IAA-94, 100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM). The inhibition was complete when the blockers were applied simultaneously with carmustine and was partial when the blockers were applied after the initial increase in [Ca(2+)](i). We conclude that carmustine induces an influx of extracellular Ca(2+) through L-type Ca(2+) channels and that this effect is mediated by oxidative stress that results from the depletion of GSH following the inhibition by carmustine of glutathione reductase.