Novel effects of gossypol, a chemical contraceptive in man: mobilization of internal Ca~(2+) and activation of external Ca~(2+) entry in intact cells

摘要

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 μM. 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 μM gossypol in Ca~(2+)-free medium. This Ca~(2+) entry was decreased by 25 μM econazole, 50 μM SKF96365 and 40 μM aristolochic acid (a phospholipase A_2 inhibitor). Pretreatment with aristolochic acid inhibited 5 M gossypol-induced internal Ca~(2+) release by 55%, but suppression of phospholipase C with 2 μM 1-(6-((17 β-3-methoxyestra-1,3,5(10-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 μM) 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.