Effect of gossypol on intracellular Ca ²⁺ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca ²⁺ levels ([Ca ²⁺ ] _i ) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca ²⁺ indicator. Gossypol increased [Ca ²⁺ ] _i in a concentration-dependent manner with an EC ₅₀ value of 2 μM. The Ca ²⁺ signal was reduced by removing extracellular Ca ²⁺ or by 10 μM La ³⁺ , but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺ pump inhibitor) to deplete the endoplasmic reticulum Ca ²⁺ partly reduced 10 μM gossypol-induced Ca ²⁺ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca ²⁺ release. The Ca ²⁺ release induced by 10 μM gossypol was not changed by inhibiting phospholipase C with 2 μM U73122 or by depleting ryanodine-sensitive Ca ²⁺ stores with 50 μM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca ²⁺ ] _i increase by causing store Ca ²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca ²⁺ influx.