Effect of clomiphene on [Ca²⁺]_i rises and cell viability in rabbit corneal epithelial cells

(Table Presented) The effect of clomiphene a first-line therapy for WHO group II (eu-estrogenic) infertility on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in rabbit corneal epithelial cells (SIRC). This study examined whether clomiphene altered [Ca²⁺]_i levels and caused cell death in SIRC cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Clomiphene at concentrations ≥5 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The clomiphene-induced Ca²⁺ influx was insensitive to blockade of L-type Ca²⁺ channel blockers. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), clomiphene failed to increase [Ca²⁺]_i. Inhibition of phospholipase C with 2 μM U73122 did not change clomiphene-induced [Ca²⁺]_i rises. At concentrations of 0.5-20 μM, clomiphene killed cells in a concentration-dependent manner. The cytotoxic effect of 15 μM clomiphene was not reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in SIRC cells, clomiphene-induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from non-L-type Ca²⁺ channels. Clomiphene-caused cytotoxicity was not mediated by a preceding [Ca ²⁺]_i rise.