Endogenous Endothelin‐1 Contributes to Enhanced Basal Pulmonary Arterial Tone Following Chronic Hypoxia: Role of Mitochondria‐Derived Reactive Oxygen Species

Abstract

Previous studies from our laboratory demonstrate the importance of mitochondria-derived reactive oxygen species (mitoROS) in the development of pulmonary hypertension after chronic hypoxia (CH) exposure. Furthermore, we have identified a major role for the endothelium to augment mitoROS-dependent pulmonary vascular tone following CH. However, the mechanism of this response remains unclear. We hypothesized that the endogenous vasoconstrictor peptide, endothelin-1 (ET-1), facilitates mitoROS-dependent increases in basal tone after CH exposure. To test our hypothesis, we measured basal tone by videomicroscopy in isolated, pressurized, small pulmonary arteries [~150 μm inner diameter (i.d.)] from normoxic and CH (4 wk, 0.5 atm) rats with and without the ETA receptor and ETB receptor inhibitors, BQ123 and BQ788 (10 μM each), respectively. Parallel protocols were conducted in the presence of the mitochondria-targeted antioxidant, MitoQ (1 μM). All experiments were performed in the presence of the nitric oxide (NO) synthase inhibitor, Nω-nitro-L-arginine (300 μM), to limit the influence of endogenous NO. Pulmonary arterial tone was calculated as the percent difference in i.d. between Ca2+-free and Ca2+-containing conditions. To evaluate the contribution of mitoROS to ET-1-mediated vasoconstriction, we further assessed vasoconstrictor responses (percentage of baseline i.d.) to serial concentrations of exogenous ET-1 (10-11 to 10-7 M) in the presence or absence of MitoQ in endothelium-disrupted pulmonary arteries from each group. Additional experiments employed confocal microscopy to measure ET-1 (1 nM)-induced mitoROS production in primary cultures of pulmonary arterial smooth muscle cells (PASMCs) from control and CH rats using the fluorescent mitochondrial superoxide indicator MitoSOX (10 μM). Antimycin A (10 µM) treatment, which stimulates mitoROS production from complex III of electron transport chain, was used as a positive control. Both MitoQ and the combined administration of ETA and ETB receptor blockers abolished the effect of CH to increase basal tone (P<0.05). Arteries from CH rats also exhibited greater constriction to exogenous ET-1 (at 1 nM) compared to vessels from normoxic controls, which was prevented by MitoQ (P<0.05). Consistent with these findings, application of ET-1 significantly increased mitoROS production in PASMCs from CH rats (P<0.05), but not control animals. Application of antimycin A led to mitoROS generation in both control and CH groups (P<0.05). We conclude that endogenous ET-1 promotes pulmonary arterial constriction through mitoROS signaling in PASMCs following CH. These findings advance our understanding of the mechanistic basis for the vasoconstrictor component of CH-induced pulmonary hypertension.