Chronic hypoxia elevates intracellular pH and activates Na⁺/H⁺exchange in pulmonary arterial smooth muscle cells

Abstract

(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.

Silencing of Sodium–Hydrogen Exchanger 1 Attenuates the Proliferation, Hypertrophy, and Migration of Pulmonary Artery Smooth Muscle Cells via E2F1

“…We previously reported on the inhibitory effect of reduced NHE activity in the proliferation of PASMCs (6) and on chronic hypoxia-induced pulmonary hypertension and vascular remodeling (7). Increased expression of the NHE1 gene was evident in animals with hypoxia-induced pulmonary hypertension and vascular remodeling (6)(7)(8)(9)(10). Moreover, we recently found that deficiency of the NHE1 gene prevented hypoxia-induced pulmonary hypertension and vascular remodeling in mice (11), accompanied by a significantly reduced proliferation of PASMCs and decreased medial wall thickness of the pulmonary arteries.…”

Na+/H+ Exchange and Hypoxic Pulmonary Hypertension

“…Initial studies demonstrated that PASMCs isolated from chronically hypoxic mice, a widely used model of hypoxic PH, exhibited an alkaline shift in resting pH i that continued to be evident days after the cells had been removed from the animals and the hypoxic stimulus. 27 That the elevation in basal pH i was still observed when the cells were perfused with HCO 3 − -free buffer and could be significantly reduced by addition of an NHE inhibitor was consistent with NHE mediating the hypoxia-induced alkaline shift in pH i . Moreover, the elevation of pH i in PASMCs isolated from chronically hypoxic mice was correlated with augmented NHE activity.…”

“…20,22,23 In contrast to the relatively wide distribution of expression seen with other NHE isoforms, NHEs 6-10 have significantly more restricted tissue and/or intracellular localization. 15,[24][25][26] Examination of the best-characterized NHEs revealed that NHE1, but not NHE2 or NHE3, was present in mouse 27 and rat 28 PASMCs, indicating that NHE1 is the primary isoform responsible for regulating cytosolic pH in this cell type.…”

“…Moreover, the elevation of pH i in PASMCs isolated from chronically hypoxic mice was correlated with augmented NHE activity. 27 Furthermore, NHE1 messenger RNA (mRNA) and protein levels were increased in PASMCs isolated from chronically hypoxic mice, indicating that the PASMC response to prolonged hypoxia includes upregulation of NHE1 expression, leading to intracellular alkalinization. In these studies, the change in basal pH i induced by chronic hypoxia was substantially larger than that observed with acute hypoxia in cat PASMCs, where the alkalinization of PASMCs was found to be due to altered activity of Cl − / HCO 3 − exchange, 6 suggesting that different mechanisms may be responsible for changes in basal pH i during acute and chronic hypoxic conditions.…”

The Na+/H+ Exchanger-1 as a New Molecular Target in Stroke Interventions

“…Activation of NHE-1 has been shown to be a pivotal event in cell damage induced by ischemia and reperfusion in the brain (Horikawa et al, 2001;Hwang et al, 2008;Luo et al, 2005), heart (Liu et al, 1997;Murphy et al, 1991;Wang et al, 2003), liver (Gores et al, 1989), and lungs (Rios et al, 2005). Here we will review recent findings implicating NHE-1 activation as a critical event in the pathogenesis of cellular dysfunction after cerebral ischemia, and the growing evidence supporting the use of NHE inhibitors as neuroprotective agents following cerebral ischemia.…”