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.
Shieh
Pochuen
P
Department of Pharmacy, Tajen University, Pingtung, Taiwan. shea7166@hotmail.com
Lee
Chih-Hung
CH
Yi
Ng Ling
NL
Jan
Chung-Ren
CR
D000319:Adrenergic beta-Antagonists / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020013:Calcium Signaling / Q000187:drug effects*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002227:Carbazoles / Q000633:toxicity*; D000077261:Carvedilol; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D005456:Fluorescent Dyes; D007476:Ionophores / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009994:Osmolar Concentration; D011412:Propanolamines / Q000633:toxicity*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011817:Rabbits; D013997:Time Factors; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D000319:Adrenergic beta-Antagonists; D002227:Carbazoles; D002614:Chelating Agents; D005456:Fluorescent Dyes; D007476:Ionophores; D011412:Propanolamines; D000077261:Carvedilol; D008345:Manganese; D011493:Protein Kinase C; D010738:Type C Phospholipases; D000252:Calcium-Transporting ATPases