Change of Cytosolic Ca 2+ Mobility in Cultured Bovine Corneal Endothelial Cells by Endothelin-1

Abstract

The effect of endothelin-1 (ET-1) on the intracellular free Ca²⁺ ([Ca²⁺]_i) in cultured bovine corneal endothelial cells was studied after loading with fura-2-AM. In Ca²⁺-containing buffer and Ca²⁺-free buffer, ET-1 induced a significant rise in [Ca²⁺]_i at concentrations from 10^-9 to 10^-7 M. In Ca²⁺-free buffer, pretreatment of the cells with ET-1 inhibited thapsigargin-induced [Ca²⁺]_i increase and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca²⁺ release by 99% and 62%, respectively. Pretreatment of the cells with thapsigargin or CCCP also inhibited ET-1-induced [Ca²⁺]_i rise by 36% and 92%, respectively. In Ca²⁺-containing buffer, the ET_A receptor antagonist (BQ123) and ET_B receptor antagonist (BQ788) partially inhibited ET-1-induced [Ca²⁺]_i by 92% and 98%, respectively. Nifedipine and La³⁺ also inhibited ET-1-induced [Ca²⁺]_i increase by 26% and 91%, respectively. The intracellular calcium release caused by ET-1 was partially inhibited by phospholipase C inhibitor (U73122). After incubation of the cells with ET-1 in Ca²⁺-free buffer, the addition of 5 mM CaCl₂ increased Ca²⁺ influx, implying that release of Ca²⁺ from internal stores caused by ET-1 further induced capacitative Ca²⁺ entry. These data suggest that ET-1-induced [Ca²⁺]_i rise in bovine corneal endothelial cells are mediated by ET_A receptor, ET_B receptor, La³⁺-sensitive Ca²⁺ pump and L-type voltage-operated Ca²⁺ channel, leading to Ca²⁺ influx. ET-1 also increased the internal Ca²⁺ release from endoplasmic reticulum and mitochondria Ca²⁺ stores followed by capacitative Ca²⁺ entry. ET-1-induced intracellular Ca²⁺ release was modulated by phospholipase C-coupled events.