. 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

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.

Pisarcik S, Maylor J, Lu W, Yun X, Undem C, Sylvester JT, Semenza GL, Shimoda LA. Activation of hypoxia-inducible factor-1 in pulmonary arterial smooth muscle cells by endothelin-1. Am J Physiol Lung Cell Mol Physiol 304: L549 -L561, 2013. First published February 15, 2013 doi:10.1152/ajplung.00081.2012.-Numerous cellular responses to hypoxia are mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension. Under certain conditions, HIF-1 may utilize feedforward mechanisms to amplify its activity. Since hypoxia increases endothelin-1 (ET-1) levels in the lung, we hypothesized that during moderate, prolonged hypoxia ET-1 might contribute to HIF-1 signaling in pulmonary arterial smooth muscle cells (PASMCs). Primary cultures of rat PASMCs were treated with ET-1 or exposed to moderate, prolonged hypoxia (4% O2 for 60 h). Levels of the oxygen-sensitive HIF-1␣ subunit and expression of HIF target genes were increased in both hypoxic cells and cells treated with ET-1. Both hypoxia and ET-1 also increased HIF-1␣ mRNA expression and decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), which is the protein responsible for targeting HIF-1␣ for O2-dependent degradation. The induction of HIF-1␣ by moderate, prolonged hypoxia was blocked by BQ-123, an antagonist of ET-1 receptor subtype A. The effects of ET-1 were mediated by increased intracellular calcium, generation of reactive oxygen species, and ERK1/2 activation. Neither ET-1 nor moderate hypoxia induced the expression of HIF-1␣ or HIF target genes in aortic smooth muscle cells. These results suggest that ET-1 induces a PASMC-specific increase in HIF-1␣ levels by upregulation of HIF-1␣ synthesis and downregulation of PHD2-mediated degradation, thereby amplifying the induction of HIF-1␣ in PASMCs during moderate, prolonged hypoxia. calcium; prolyl hydroxylase; pulmonary hypertension IN A VARIETY OF CHRONIC lung diseases, the pulmonary circulation is exposed to prolonged periods of hypoxia, often resulting in the development of pulmonary hypertension. Numerous studies have described the structural and functional changes that occur in the pulmonary circulation in response to chronic hypoxia (60). Structural remodeling, characterized by pulmonary arterial smooth muscle cell (PASMC) proliferation, intimal thickening, and extension of muscle into previously nonmuscular arterioles, is commonly observed with pulmonary hypertension (23,40). Changes in the vascular wall are accompanied by active contraction of vessels, evidenced by acute reduction in pulmonary arterial pressure in response to vasodilatory agents (42, 44). These pulmonary vascular changes result, in large part, from altered expression of genes encoding ion channels and transporters that control PASMC ion homeostasis, including increased expression of Na ϩ /H ϩ exchanger isoform 1 (NHE1) and the canonical transient receptor potential (TRPC) family members TRPC1 and TRPC6, as well as reduced levels of mRNA...

Maternal tobacco smoke (TS) exposure is associated with intrauterine growth retardation and low birth weight, both of which increase infant morbidity and mortality. Nicotine, an active component in TS, also decreases birth weight and postnatal weight gain, although the mechanisms involved remain unclear. We hypothesized that in umbilical endothelial cells (ECs), nicotine alters regulation of [Ca2+]i, an important modulator of synthesis and release of endothelial‐derived vasoactive factors and, consequently, umbilical vessel tone and blood flow to and from the fetus. In this study, we used fluorescent microscopy in human umbilical artery ECs (HUAECs) and human umbilical vein ECs (HUVECs) loaded with the Ca2+‐sensitive dye, Fura‐2. In HUAECs, we found that acute (10 min) application of nicotine (100 ng/ml) caused an immediate decrease in [Ca2+]i. Prolonged nicotine treatment (10 or 100 ng/ml; 24 hr) also decreased resting [Ca2+]i. In contrast, both acute (100 ng/ml; 10 min) and prolonged (10 or 100 ng/ml; 24 hr) exposure to nicotine increased [Ca2+]i in HUVECs. Our results indicate that nicotine, at concentrations comparable to plasma levels in smokers and non‐smokers exposed to environmental TS, caused both acute and long‐lasting alterations in [Ca2+]i in HUAECs and HUVECs and suggest that nicotine may differentially regulate vasoactive factor synthesis and vascular tone in umbilical arteries and veins.

No abstract

Increased PASMC contraction and growth during chronic hypoxia (CH) may be due to cytoskeletal rearrangement involving transmembrane and cytosolic proteins. We found that the ion transporter, NHE1, is upregulated in chronically hypoxic rats (10% O2; 3 wk) and is required for development of hypoxic pulmonary hypertension. In addition to regulating pH, NHE1 was recently found to bind phosphorylated ezrin (p‐ezrin), an actin filament binding protein. NHERF1 also binds p‐ezrin, at the same site as NHE1. Since the transmembrane spanning NHE1 can act as an anchor, but cytosolic NHERF1 cannot, we hypothesized that changes in NHE1/ezrin interactions during hypoxia enables actin filaments to be tethered to the cell membrane, promoting PASMC contraction and growth. Confocal images of immunofluorescent stained chronically hypoxic mouse lung sections confirmed co‐localization of p‐ezrin and smooth muscle cell‐specific α‐actin (SM‐actin). Immunoblots showed increased NHE1, increased p‐ezrin and decreased NHERF1 levels in response to hypoxia. Co‐immunoprecipitation studies showed increased NHE1 binding to p‐ezrin and SM‐actin with hypoxia (4% O2; 24 hr), and decreased NHERF1 binding. These results suggest reciprocal regulation of NHERF1 and NHE1 expression in PASMC during hypoxia, with both proteins competing to bind p‐ezrin. Increased NHE1/ezrin interactions may contribute to hypoxic pulmonary hypertension.

Numerous cellular responses to hypoxia are mediated by the transcription factor, HIF‐1. Recent data suggest that, under certain conditions, HIF‐1 may require feed forward for full expression (Peng, et al. J Physiol, 2006). Recently, ET‐1 was found to increase HIF‐1 levels in tumor cells (Spinella, et al. Cancer Res 2007). Since hypoxia increases ET‐1 levels in the lung, we hypothesized that during moderate prolonged hypoxia ET‐1 might feed forward and amplify activation of HIF‐1 in PASMCs. Primary cultures of rat PASMCs were treated with ET‐1 (10−8M; 48 hr) or exposed to hypoxia (4% O2; 60 hrs). Protein levels of the oxygen‐sensitive α subunit of HIF‐1 (HIF‐1α) were markedly increased in nuclear extracts from both hypoxic cells and cells treated with ET‐1 under non‐hypoxic conditions. Time course studies revealed that accumulation of HIF‐1α in response to ET‐1 required greater than 4 hr of exposure. Real‐time PCR revealed that ET‐1 (10−10‐10−8 M; 48 hr) increased Hif1a mRNA expression. ET‐1 also decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), the protein responsible for targeting HIF‐1α for degradation. The induction of HIF‐1α by moderate prolonged hypoxia (4% O2; 60 hr) was blocked by BQ‐123, an ET‐1 receptor subtype A antagonist, whereas BQ‐123 had no effect on the induction of HIF‐1α by severe acute hypoxia (1% O2; 4 hr). These results suggest that ET‐1 induces HIF‐1α by upregulation of HIF‐1α synthesis and downregulation of PHD2‐mediated degradation. Furthermore, the sustained induction of HIF‐1α in PASMCs during moderate prolonged hypoxia may require amplification by ET‐1. Funded by: HL67191

Maternal environmental tobacco smoke (ETS) exposure has been associated with intrauterine growth retardation (IUGR) and low birth weight, both of which increase infant morbidity and mortality. Nicotine, an active component in ETS, is known to decrease birth weight and postnatal weight gain, yet the mechanism remains unclear. We hypothesized that nicotine alters membrane potential (Em)‐dependent regulation of [Ca2+]i and nitric oxide (NO) production in endothelial cells, which could cause umbilical vessel contraction, impairment of blood flow and IUGR. Fluorescent microscopy was used in HUAECs treated with nicotine (10–100ng/ml; 15m–24hr). Fura‐2 was used to measure [Ca2+]i and DiBAC4(3) was used to measure changes in Em. We found that HUAECs treated with 100ng/ml of nicotine showed depolarization and an immediate dramatic decrease in [Ca2+]i. Prolonged nicotine treatment (10 and 100ng/ml; 1–24 hr) also decreased [Ca2+]i . In addition, total nitrite levels in the cell media were measured via Griess reaction following total nitrate reduction. Treatment with nicotine (10ng/ml; 1–4hrs) reduced total nitrite levels in the media in comparison to untreated HUAECs. From these results, we conclude that nicotine, at concentrations comparable to plasma levels in non‐smokers exposed to ETS, caused decreased NO production in HUAECs, perhaps secondary to depolarization and decreased [Ca2+]i. Funded by: HL73859