RESUMO
In pulmonary hypertension (PHTN), a metabolic shift to aerobic glycolysis promotes a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMC). Enhanced glycolysis induces extracellular acidosis, which can activate proton-sensing membrane receptors and ion channels. We previously reported activation of the proton-gated cation channel, acid-sensing ion channel 1a (ASIC1a), contributes to the development of hypoxic PHTN. Therefore, we hypothesize that enhanced glycolysis and subsequent acidification of the PASMC extracellular microenvironment activates ASIC1a in hypoxic PHTN. We observed decreased oxygen consumption rate and increased extracellular acidification rate in PASMC from chronic hypoxia (CH)-induced PHTN rats, indicating a shift to aerobic glycolysis. Additionally, we found that intracellular alkalization and extracellular acidification occur in PASMC following CH, and in vitro hypoxia, which was prevented by inhibition of glycolysis with 2-deoxy-D-glucose (2-DG). Inhibiting H+transport/secretion through carbonic anhydrase IX, Na+/H+ exchanger 1, or vacuolar-type H+-ATPase did not prevent this pH shift following hypoxia. Although the putative monocarboxylate transporter 1 (MCT1) and -4 (MCT4) inhibitor, syrosingopine, prevented the pH shift; the specific MCT1 inhibitor, AZD3965, and/or the MCT4 inhibitor, VB124, were without effect, suggesting syrosingopine targets the glycolytic pathway independent of H+ export. Furthermore, 2-DG and syrosingopine prevented enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMC from CH rats. These data suggest multiple H+ transport mechanisms contribute to extracellular acidosis and inhibiting glycolysis, rather than specific H+ transporters, more effectively prevents extracellular acidification and ASIC1a activation. Together, these data reveal a novel pathologic relationship between glycolysis and ASIC1a activation in hypoxic PHTN.
RESUMO
Pulmonary hypertension (PH) resulting from chronic hypoxia (CH) occurs in patients with chronic obstructive pulmonary diseases, sleep apnea, and restrictive lung diseases, as well as in residents at high altitude. Previous studies from our group and others demonstrate a detrimental role of reactive oxygen species (ROS) in the pathogenesis of CH-induced PH, although the subcellular sources of ROS are not fully understood. We hypothesized that mitochondria-derived ROS (mtROS) contribute to enhanced vasoconstrictor reactivity and PH following CH. To test the hypothesis, we exposed rats to 4 weeks of hypobaric hypoxia (PB ≈ 380 mmHg), with control rats housed in ambient air (PB ≈ 630 mmHg). Chronic oral administration of the mitochondria-targeted antioxidant MitoQ attenuated CH-induced decreases in pulmonary artery (PA) acceleration time, increases in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary arterial remodeling. In addition, endothelium-intact PAs from CH rats exhibited a significantly greater basal tone compared to those from control animals, as was eliminated via MitoQ. CH also augmented the basal tone in endothelium-disrupted PAs, a response associated with increased mtROS production in primary PA smooth muscle cells (PASMCs) from CH rats. However, we further uncovered an effect of NO synthase inhibition with Nω-nitro-L-arginine (L-NNA) to unmask a potent endothelial vasoconstrictor influence that accentuates mtROS-dependent vasoconstriction following CH. This basal tone augmentation in the presence of L-NNA disappeared following combined endothelin A and B receptor blockade with BQ123 and BQ788. The effects of using CH to augment vasoconstriction and PASMC mtROS production in exogenous endothelin 1 (ET-1) were similarly prevented by MitoQ. We conclude that mtROS participate in the development of CH-induced PH. Furthermore, mtROS signaling in PASMCs is centrally involved in enhanced pulmonary arterial constriction following CH, a response potentiated by endogenous ET-1.