Endothelin‐1 (ET‐1)‐induced reactive oxygen species (ROS) generation requires calcium (Ca2+) influx in pulmonary arterial smooth muscle cells (PASMCs)

Abstract

ET‐1 is a potent endothelium‐derived vasoconstrictor and smooth muscle cell mitogen. During hypoxia, ET‐1 is thought to play a significant role in modulation pulmonary vascular tone. Previous studies in systemic smooth muscle have shown that both an increase in intracellular Ca2+ concentration ([Ca2+]i) and the generation of ROS via nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH oxidase) are major components of ET‐1 signaling. It remains unclear, however, whether ET‐1 causes ROS generation in PASMCs. In this study, we used the ROS‐sensitive dye, DCF, and the Ca2+‐sensitive dye, fura‐2, to determine 1) the effect of ET‐1 on ROS production in transiently cultured (24–48 hr) rat PASMCs and 2) whether this response is Ca2+‐dependent. Measuring both [Ca2+]i and ROS simultaneously, we found that ET‐1 significantly increased both Ca2+ influx and ROS generation, with Ca2+ influx appearing to occur first. Pre‐treating PASMCs with the antioxidant, TEMPOL, or the NADPH oxidase inhibitor, diphenyleneiodonium chloride, had no significant effect on ET‐1‐induced Ca2+ influx but prevented ROS production. In contrast, the removal of extracellular Ca2+ or pretreatment with the Ca2+ chelator, BAPTA‐AM, prevented both the increase in [Ca2+]i and ROS production. These results indicate that, in PASMCs, Ca2+ influx may be necessary for the ET‐1‐induced initiation of ROS production from NADPH oxidase.HL67191