Effect of Gossypol on Intracellular Ca2+

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 µM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 µM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 µM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 µM gossypol was not changed by inhibiting phospholipase C with 2 µM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 µM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx. Key Words: Ca2+ signaling, fura-2, gossypol, HA22/VGH cells, hepatocyte