Independent effects of the broccoli-derived compound sulforaphane on Ca²âº influx and apoptosis in Madin-Darby canine renal tubular cells.

This study explored whether sulforaphane changed basal [Ca²âº]i levels in suspended Madin-Darby canine kidney (MDCK) cells by using fura-2 as a Ca²âº-sensitive fluorescent dye. Sulforaphane at concentrations between 2.5-10 microM increased [Ca²âº]i in a concentration-dependent manner. This Ca²âº influx was inhibited by phospholipase A2 inhibitor aristolochic acid but not by Ca²âº channel blockers such as nifedipine, nimodipine, nicardipine, diltiazem, verapamil, econazole and SK&F96365. The Ca²âº signal was abolished by removing extracellular Ca²âº. In Ca²âº-free medium, pretreatment with sulforaphane did not alter the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin-induced Ca²âº release suggesting sulforaphane did not induce slow Ca²âº release from endoplasmic reticulum. At concentrations between 1 and 20 microM, sulforaphane induced concentration-dependent decrease in cell viability which was not affected by pre-chelation of cytosolic Ca²âº with BAPTA/AM. Flow cytometry data suggest that 20 (but not 5 and 10) microM sulforaphane induced significant increase in sub G1 phase indicating involvement of apoptosis. Collectively, in MDCK cells, sulforaphane induced [Ca²âº]i rises by causing Ca²âº entry through phospholipase A2-sensitive pathways without inducing Ca²âº release from the endoplasmic reticulum. Sulforaphane also induced Ca²âº-independent cell death that might involve apoptosis.

Carvedilol-induced elevation in cytosolic free Ca(2+) level and apoptosis in SIRC corneal epithelial cells.

The effect of the cardiovascular drug carvedilol on cytosolic free Ca(2+) concentrations ([Ca( 2+)](i)) and viability was examined in Statens Seruminstitut rabbit cornea (SIRC) corneal epithelial cells. [Ca(2+)](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 microM increased [Ca( 2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Carvedilol induced Mn(2+) quench of fura-2 fluorescence implicating Ca(2+) influx. The Ca(2+) influx was inhibited by suppression of protein kinase C activity. In Ca(2+)-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca( 2+) pump inhibitor), carvedilol-induced [Ca(2+)](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 microM) did not change carvedilol-induced [Ca(2+)](i) rise. At concentrations between 5 and 70 microM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 microM carvedilol was not reversed by prechelating cytosolic Ca(2+) with BAPTA/AM. Apoptosis was induced by 5-70 microM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca( 2+) influx via protein kinase C-regulated Ca(2+) channels. Carvedilol-caused cytotoxicity was mediated by Ca(2+)-independent apoptosis in a concentration-dependent manner.