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.