Prolonged exposure to decreased oxygen tension, as occurs with many pulmonary diseases, results in pulmonary hypertension, significantly worsening prognosis. The mechanism underlying the pathogenesis of this process remains unknown. Pulmonary arterial smooth muscle cell (PASMC) contraction associated with chronic hypoxia (CH) may be caused by elevated intracellular Ca2+ concentration ([Ca2+]i). In PASMCs, depolarization is observed with CH, fueling speculation that [Ca2+]i is increased due to activation of voltage-gated Ca2+ channels or enhanced Na+/Ca2+ exchange. The endothelium-derived constricting factor, endothelin-1 (ET-1), may contribute to the pathogenesis of CHPH, since ET-1 increases [Ca2+]i. Following exposure to CH, the ET-1-induced rise in [Ca2+]i is reduced but contraction is maintained, suggesting activation of Ca2+-independent contractile pathways. This may be due to ET-1-induced activation of tyrosine kinases (TK). Hypoxic induction of ET-1 occurs via activation of the transcription factor, HIF-1, in systemic endothelium and in mice partially deficient for HIF-1, CH-induced pulmonary hypertension is markedly reduced. Therefore, we hypothesize induction of HIF-1 is an initiating step in the development of pulmonary hypertension, leading to elevated ET-1 levels. ET-1 then diffuses to PASMCs, activating three contractile mechanisms. First, ET-1 decreases voltage-gated K+ channel expression, leading to depolarization-driven activation of Na+/Ca2+ exchange and elevation of resting [Ca2+]i. Second, ET-1 causes TK-mediated Ca2+ influx through L-type Ca2+ channels. Both of these mechanisms increase phosphorylation of myosin light chains (MLCs). Finally, ET-1 causes changes in Ca2+-sensitivity of the contractile apparatus via TK-mediated regulation of actin binding proteins. This final step allows actin to interact with the phosphorylated MLCs generated in steps 1 and 2, and results in contraction. To test these hypotheses, we will use a combination of techniques in our model of hypoxic pulmonary hypertension, including isometric tension recording in arterial segments, Northern and Western blot analysis, whole-cell patch-clamp and microfluorescence measurements, to accomplish the following Specific Aims: 1) confirm that HIF-1 regulates hypoxic induction of ET-1 in the pulmonary vasculature and identify the cell type(s) involved; 2) determine the mechanisms responsible for the CH-induced increase in resting [Ca2+]i and 3) determine the mechanisms by which ET-1 causes contraction during CH.
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