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The effect of gossypol on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Gossypol evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 2 and 20 microM. The response was decreased by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with gossypol nearly abolished the [Ca(2+)](i) increase induced by carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with CCCP and thapsigargin only partly inhibited gossypol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 5 microM gossypol in Ca(2+)-free medium. This Ca(2+) entry was decreased by 25 microM econazole, 50 microM SKF96365 and 40 microM aristolochic acid (a phospholipase A(2) inhibitor). Pretreatment with aristolochic acid inhibited 5 microM gossypol-induced internal Ca(2+) release by 55%, but suppression of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 microM) also increased [Ca(2+)](i) in human bladder cancer cells and neutrophils. Collectively, we have found that gossypol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca(2+) influx from external space.

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