Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

Abstract

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca2+ concentrations ([Ca2+]i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole-induced Ca(2+) influx was insensitive to L-type Ca2+ channel blockers and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 20 µM econazole, [Ca2+]i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca2+]i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca2+ with BAPTA. This shows that in SIRC cells econazole induces [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca2+]i rise.