Our laboratory shows that acid-sensing ion channel 1 (ASIC1) contributes to the development of hypoxic pulmonary hypertension by augmenting store-operated Ca(2+) entry (SOCE) that is associated with enhanced agonist-induced vasoconstriction and arterial remodeling. However, this enhanced Ca(2+) influx following chronic hypoxia (CH) is not dependent on an increased ASIC1 protein expression in pulmonary arterial smooth muscle cells (PASMC). It is well documented that hypoxic pulmonary hypertension is associated with changes in redox potential and reactive oxygen species homeostasis. ASIC1 is a redox-sensitive channel showing increased activity in response to reducing agents, representing an alternative mechanism of regulation. We hypothesize that the enhanced SOCE following CH results from removal of an inhibitory effect of hydrogen peroxide (H2O2) on ASIC1. We found that CH increased PASMC superoxide (O2 (·-)) and decreased rat pulmonary arterial H2O2 levels. This decrease in H2O2 is a result of decreased Cu/Zn superoxide dismutase expression and activity, as well as increased glutathione peroxidase (GPx) expression and activity following CH. Whereas H2O2 inhibited ASIC1-dependent SOCE in PASMC from control and CH animals, addition of catalase augmented ASIC1-mediated SOCE in PASMC from control rats but had no further effect in PASMC from CH rats. These data suggest that, under control conditions, H2O2 inhibits ASIC1-dependent SOCE. Furthermore, H2O2 levels are decreased following CH as a result of diminished dismutation of O2 (·-) and increased H2O2 catalysis through GPx-1, leading to augmented ASIC1-dependent SOCE.

The development of chronic hypoxia (CH)-induced pulmonary hypertension is associated with increased pulmonary arterial smooth muscle cell (PASMC) Ca(2+) influx through acid-sensing ion channel-1 (ASIC1) and activation of the Ca(2+)/calcineurin-dependent transcription factor known as nuclear factor of activated T-cells isoform c3 (NFATc3). Whether Ca(2+) influx through ASIC1 contributes to NFATc3 activation in the pulmonary vasculature is unknown. Furthermore, both ASIC1 and calcineurin have been shown to interact with the scaffolding protein known as protein interacting with C kinase-1 (PICK1). In the present study, we tested the hypothesis that ASIC1 contributes to NFATc3 nuclear translocation in PASMC in a PICK1-dependent manner. Using both ASIC1 knockout (ASIC1(-/-)) mice and pharmacological inhibition of ASIC1, we demonstrate that ASIC1 contributes to CH-induced (1 wk at 380 mmHg) and endothelin-1 (ET-1)-induced (10(-7) M) Ca(2+) responses and NFATc3 nuclear import in PASMC. The interaction between ASIC1/PICK1/calcineurin was shown using a Duolink in situ Proximity Ligation Assay. Inhibition of PICK1 by using FSC231 abolished ET-1-induced and ionomycin-induced NFATc3 nuclear import, but it did not alter ET-1-mediated Ca(2+) responses, suggesting that PICK1 acts downstream of Ca(2+) influx. The key findings of the present work are that 1) Ca(2+) influx through ASIC1 mediates CH- and ET-1-induced NFATc3 nuclear import and 2) the scaffolding protein PICK1 is necessary for NFATc3 nuclear import. Together, these data provide an essential link between CH-induced ASIC1-mediated Ca(2+) influx and activation of the NFATc3 transcription factor. Identification of this ASIC1/PICK1/NFATc3 signaling complex increases our understanding of the mechanisms contributing to the vascular remodeling and increased vascular contractility that are associated with CH-induced pulmonary hypertension.

Our laboratory has demonstrated an important role for acid‐sensing ion channel 1 (ASIC1) in mediating increased pulmonary arterial smooth muscle cell (PASMC) Ca2+ influx and enhanced agonist‐induced vasoconstriction following chronic hypoxia (CH)‐induced pulmonary hypertension. ASIC1 activity is highly sensitive to changes in the redox state of the cell; in particular, hydrogen peroxide (H2O2) has been shown to inhibit ASIC1 function. Furthermore, alterations in reactive oxygen species are known to contribute to the pathogenesis of pulmonary hypertension. Therefore, we hypothesize the increased ASIC1‐mediated store‐operated calcium entry (SOCE) in PASMCs following CH is due to decreased H2O2, attributable to a down regulation of superoxide dismutase (SOD). We found that PEG‐catalase augmented ASIC1‐dependent SOCE in PASMCs from control rats, but was without effect on SOCE in PASMCs from CH rats (4 wks @ 380 Torr). However, the addition of H2O2 inhibited SOCE in both groups. Additionally, SOD1 and SOD3 protein expression were decreased in isolated pulmonary arteries from CH rats compared to control. Together these data suggest that downregulation of SOD1 and SOD3 following CH results in a loss of endogenous H2O2‐induced inhibition of ASIC1‐dependent SOCE in PASMC. This work is supported by NIH HL‐09258 (NLJ) and HL‐07736 (BRW).

Acid sensing ion channel 1 (ASIC1) mediates enhanced store operated calcium entry (SOCE) in pulmonary arterial smooth muscle cells (PASMC) and contributes to the development of chronic hypoxia (CH)‐induced pulmonary hypertension. ASIC1 is a redox sensitive ion channel, such that reducing agents increase and oxidizing agents decrease channel activity. Recent studies from our laboratory have shown that 1) hydrogen peroxide (H2O2) inhibits ASIC1‐dependent SOCE in PASMC and 2) pulmonary arterial H2O2 levels are decreased following CH which may contribute to the enhanced SOCE. Considering that H2O2 has additionally been shown to decrease ASIC1 cell surface expression in neurons, we hypothesized that H2O2 inhibits plasma membrane localization of ASIC1 in PASMC. Using a cell surface biotinylation assay, we found a 2.9 ± 0.1 fold increase in plasma membrane ASIC1 expression in PASMC from CH animals compared to control (two‐tailed P value=0.0002). In PASMC from control rats pretreatment with PEG‐catalase (250 U/ml; 1 hr at 37°C) increased and H2O2 (25 µM; 1 hr at 37°C) decreased ASIC1 surface expression compared to vehicle treated PASMC (one way ANOVA P value<0.001). We conclude that ASIC1 plasma membrane localization is inhibited by H2O2 in PASMC and this represents one potential mechanism by which the loss of H2O2 following CH enhances ASIC1‐dependent Ca2+ influx in PASMC. Grant Funding Source: Supported by T32‐HL07736; R01‐HL111084; K01 HL092598

The development of chronic hypoxia (CH)‐induced pulmonary hypertension is associated with 1) the activation of the Ca2+‐dependent transcription factor, nuclear factor of activated T‐cells isoform c3 (NFATc3), and 2) enhanced acid sensing ion channel 1 (ASIC1)‐dependent Ca2+ influx in pulmonary arterial smooth muscle cells (PASMC). Therefore, we hypothesize that ASIC1‐mediated Ca2+ influx increases NFATc3 nuclear translocation in pulmonary vascular smooth muscle. To test this hypothesis we first examined the effect of ASIC1 inhibition on NFATc3 nuclear translocation in PASMC in response to endothelin‐1 (ET‐1; 10‐7 M). Inhibition of ASIC1 with psalmotoxin‐1 (20 nM) diminished ET‐1 induced Ca2+ influx and NFATc3 nuclear import. In addition, ET‐1 mediated pulmonary arterial vasoconstriction and Ca2+ influx following CH (1 wk @ 380 mmHg) were blunted in ASIC1 knockout mice (ASIC1‐/‐) compared to the ASIC1 wildtype mice (ASIC1+/+). Similarly, CH increased pulmonary vascular smooth muscle NFATc3 nuclear translocation in ASIC1+/+ mice, but not in ASIC1‐/‐ mice. These CH‐induced responses were additionally associated with decreased right ventricular systolic pressure and right ventricular hypertrophy in ASIC1‐/‐ mice. Taken together, these data suggest that ASIC1‐dependent Ca2+ influx is required for the activation and nuclear translocation of NFATc3 during the development of pulmonary hypertension. Grant Funding Source: Supported by R01 HL92598, R01 HL111084, T32 HL07736, R01HL088151, R01 HL088192