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

Abstract

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 μM gossypol-induced internal Ca 2+ release by 55%, but suppression of phospholipase C with 2 μM 1-(6-((17β-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.