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

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 [Ca2+]i rise. Drug Dev Res 69:272–278, 2008 ©2008 Wiley‐Liss, Inc.