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

The effect of the cardiovascular drug carvedilol on cytosolic free Ca ²⁺ 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 1/4M 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 Ca ²⁺ influx was inhibited by suppression of protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 1/4M 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 [Ca ²⁺]_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 1/4M) did not change carvedilol-induced [Ca ²⁺]_i rise. At concentrations between 5 and 70 1/4M, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 1/4M carvedilol was not reversed by prechelating cytosolic Ca ²⁺ with BAPTA/AM. Apoptosis was induced by 5-70 1/4M carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca ²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca ²⁺ influx via protein kinase C-regulated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺-independent apoptosis in a concentration-dependent manner.