Gossypol, a component in cottonseed, induced increases in cytosolic Ca²⁺ levels in Chang liver cells

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca²⁺ levels ([Ca²⁺]_i) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca²⁺ indicator. Gossypol (0.2-5μM) increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5μM. The [Ca²⁺]_i response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca²⁺ markedly reduced the [Ca²⁺]_i signals by 80±2%. Preincubation with 0.1mM La³⁺ or 10μM nimodipine abolished the Ca²⁺ influx. Gossypol (5μM)-induced release of intracellular Ca²⁺ was reduced by 75% by pretreatment with 1μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) to deplete the endoplasmic reticulum Ca²⁺. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca²⁺ release. After pretreatment with 5μM gossypol in Ca²⁺-free medium for several min, addition of 3mM Ca²⁺ induced a [Ca²⁺]_i increase of a magnitude nine-fold greater than control. Gossypol (5μM)-induced Ca²⁺ release was not affected by inhibiting phospholipase C with 2μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H- pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca²⁺]_i increases in Chang liver cells by releasing Ca²⁺ from intracellular pools in a phospholipase C-dissociated fashion and by causing La³⁺- and nimodipine-sensitive Ca²⁺ influx.