Carvedilol-induced elevation in cytosolic free Ca2+ level and apoptosis in SIRC corneal epithelial cells

Abstract

The effect of the cardiovascular drug carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability was examined in Statens Seruminstitut rabbit cornea (SIRC) corneal epithelial cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1), respectively. Carvedilol at concentrations between 1 and 30 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Carvedilol induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was inhibited by suppression of protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), carvedilol-induced [Ca²⁺]_i rise was reduced; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca²⁺]_i rise. Addition of the phospholipase C inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U73122; 2 μM) did not change carvedilol-induced [Ca²⁺]_i rise. At concentrations between 5 and 70 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 μM carvedilol was not reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Apoptosis was induced by 5—70 μM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺-independent apoptosis in a concentration-dependent manner.

Keywords

apoptosis carvedilol Ca2+cornea SIRC

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