Effects of endothelin-1 antagonist BQ610 on hypoxia-induced injury and [Ca2+]i changes in cultured neonatal rat cardiomyocytes

Hypoxia is a strong stimulus for endothelin-1 (ET-1) synthesis, which is involved in myocardial ischemia. To shed light on the role and calcium mechanism of enclogenous ET-1 in myocardial ischemia, the effects of the ET-1 antagonist BQ610 on hypoxia-induced injury and [Ca2+]i changes were observed in cultured neonatal rat cardiomyocytes. Hypoxia-induced injury was assessed by determining supernatant activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in cardiomyocytes exposed to hypoxia produced by a 3% O-2, 5% CO2 atmosphere. [Ca I i was measured with the Ca sensitive dye Fluo-3 under a confocal microscope. The hypoxia model used for [Ca2+](i) measurement was prepared by perfusing cells with 95% N-2, 5% CO2 Saturated DMEM solution containing 1 mM Na2S2O4. The results showed that: 1) Hypoxia significantly increased ET-1 content in culture supernatant. 2) The supernatant LDH was significantly increased after 12- or 24-h hypoxia. On the other hand, SOD activity was decreased significantly. BQ610 0.2-5 muM dose-dependently reduced LDH release and enhanced SOD activity. 3) [Ca2+](i) transient was observed in the normal cultured beating cardiomyocytes. Exposure to hypoxia caused termination of [Ca2+](i) transient in 3-5 min and a continuous rapid increase in [Ca2+](i) after about 20 min. In the presence of BQ610, the termination of [Ca2+](i) transient was postponed to 15-20 min into hypoxia; the [Ca2+](i) increase was rather slow until approximately 45 min into hypoxia. These results indicate that enclogenous ET-1 contributed to hypoxia-induced injury in the cultured neonatal rat cardionnyocytes and that the injury was associated with a [Ca2+](i) mechanism partially mediated by ET-1. (C) 2003 Wiley-Liss, Inc.