. HIF-1 regulates hypoxic induction of NHE1 expression and alkalinization of intracellular pH in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 291: L941-L949, 2006. First published June 9, 2006 doi:10.1152/ajplung.00528.2005.-Vascular remodeling resulting from altered pulmonary arterial smooth muscle cell (PASMC) growth is a contributing factor to the pathogenesis of hypoxic pulmonary hypertension. PASMC growth requires an alkaline shift in intracellular pH (pHi) and we previously showed that PASMCs isolated from mice exposed to chronic hypoxia exhibited increased Na ϩ /H ϩ exchanger (NHE) expression and activity, which resulted in increased pHi. However, the mechanism by which hypoxia caused these changes was unknown. In this study we tested the hypothesis that hypoxia-induced changes in PASMC pH homeostasis are mediated by the transcriptional regulator hypoxiainducible factor 1 (HIF-1). Consistent with previous results, increased NHE isoform 1 (NHE1) mRNA and protein, enhanced NHE activity, and an alkaline shift in pHi were observed in PASMCs isolated from wild-type mice exposed to chronic hypoxia (3 wk at 10% O2). In contrast, these changes were absent in PASMCs isolated from chronically hypoxic mice with partial deficiency for HIF-1. Exposure of PASMCs to hypoxia ex vivo (48 h at 4% O2) or overexpression of HIF-1 in the absence of hypoxia also increased NHE1 mRNA and protein expression. Our results indicate that full expression of HIF-1 is essential for hypoxic induction of NHE1 expression and changes in PASMC pH homeostasis and suggest a novel mechanism by which HIF-1 mediates pulmonary vascular remodeling during the pathogenesis of hypoxic pulmonary hypertension.hypoxia-inducible factor 1; pulmonary arterial smooth muscle cell

(CH), caused by many lung diseases, results in pulmonary hypertension due, in part, to increased muscularity of small pulmonary vessels. Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to growth factors requires increased intracellular pH (pH i) mediated by activation of Na ϩ /H ϩ exchange (NHE); however, the effect of CH on PASMC pH i homeostasis is unknown. Thus we measured basal pH i and NHE activity and expression in PASMCs isolated from mice exposed to normoxia or CH (3 wk/10% O 2). pHi was measured using the pH-sensitive fluorescent dye BCECF-AM. NHE activity was determined from Na ϩ -dependent recovery from NH 4-induced acidosis, and NHE expression was determined by RT-PCR and immunoblot. PASMCs from chronically hypoxic mice exhibited elevated basal pH i and increased NHE activity. NHE1 was the predominate isoform present in mouse PASMCs, and both gene and protein expression of NHE1 was increased following exposure to CH. Our findings indicate that exposure to CH caused increased pH i, NHE activity, and NHE1 expression, changes that may contribute to the development of pulmonary hypertension, in part, via pH-dependent induction of PASMC proliferation. pulmonary hypertension MANY CHRONIC LUNG DISEASES cause prolonged alveolar hypoxia, resulting in the development of pulmonary hypertension. The elevation in pulmonary arterial pressure is due to both active contraction of vascular smooth muscle and structural remodeling (21,24,36,38). Pulmonary vascular remodeling in response to chronic hypoxia has been well characterized and includes pulmonary arterial smooth muscle cell (PASMC) hypertrophy and hyperplasia, intimal thickening, and extension of smooth muscle into previously nonmuscular arterioles (21, 36). The exact cellular mechanisms governing hypoxia-induced PASMC growth are not completely known; however, changes in intracellular pH (pH i ) have been demonstrated to be required for cell growth and/or proliferation in pulmonary (33) and systemic tissue (6,17,22), suggesting a possible pivotal role for H ϩ homeostasis in mediating this response.

Prolonged exposure to decreased oxygen tension causes contraction and proliferation of pulmonary arterial smooth muscle cells (PASMCs) and pulmonary hypertension. Hypoxia-induced inhibition of voltage-gated K(+) (K(v)) channels may contribute to the development of pulmonary hypertension by increasing intracellular calcium concentration ([Ca(2+)](i)). The peptide endothelin-1 (ET-1) has been implicated in the development of pulmonary hypertension and acutely decreases K(v) channel activity. ET-1 also activates several transcription factors, although whether ET-1 alters K(V) channel expression is unclear. The hypoxic induction of ET-1 is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1), which we demonstrated to regulate hypoxia-induced decreases in K(V) channel activity. In this study, we tested the hypothesis that HIF-1-dependent increases in ET-1 lead to decreased K(v) channel expression and subsequent elevation in [Ca(2+)](i). Resting [Ca(2+)](i) and K(v) channel expression were measured in cells exposed to control (18% O(2), 5% CO(2)) and hypoxic (4% O(2), 5% CO(2)) conditions. Hypoxia caused a decrease in expression of K(v)1.5 and K(v)2.1 and a significant increase in resting [Ca(2+)](i). The increase in [Ca(2+)](i) was reduced by nifedipine, an inhibitor of voltage-dependent calcium channels, and removal of extracellular calcium. Treatment with BQ-123, an ET-1 receptor inhibitor, prevented the hypoxia-induced decrease in K(v) channel expression and blunted the hypoxia-induced increase in [Ca(2+)](i) in PASMCs, whereas ET-1 mimicked the effects of hypoxia. Both hypoxia and overexpression of HIF-1 under normoxic conditions increased ET-1 expression. These results suggest that the inhibition of K(v) channel expression and rise in [Ca(2+)](i) during chronic hypoxia may be the result of HIF-1-dependent induction of ET-1.