Ketoconazole-Evoked [Ca2+]i Rises and Non-Ca2+-Triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effect of ketoconazole on cytosolic free Ca^{2 +} concentrations ([Ca^{2 +}]_i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca^{2 +} levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca^{2 +}]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 μ M and above increased [Ca^{2 +}]_i in a concentration-dependent manner. The Ca^{2 +} signal was reduced partly by removing extracellular Ca^{2 +}. The ketoconazole-induced Ca^{2 +} influx was insensitive to L-type Ca^{2 +} channel blockers and protein kinase C modulators. In Ca^{2 +}-free medium, after pretreatment with 50 μ M ketoconazole, thapsigargin-(1 μ M)-induced [Ca^{2 +}]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca^{2 +}]_i rises. Inhibition of phospholipase C with 2 μ M U73122 did not change ketoconazole-induced [Ca^{2 +}]_i rises. At concentrations between 5 and 100 μ M, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 μ M ketoconazole was not reversed by prechelating cytosolic Ca^{2 +} with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca^{2 +}]_i rises by causing Ca^{2 +} release from the endoplasmic reticulum and Ca^{2 +} influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca^{2 +}]_i rise.