Chronic hypoxia limits H2O2-induced inhibition of ASIC1-dependent store-operated calcium entry in pulmonary arterial smooth muscle

Abstract

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.

Keywords: catalase; degenerin/epithelial sodium channel; di-(4-carboxybenzyl) hyponitrite-1; glutathione peroxidase; pulmonary hypertension; superoxide dismutase.

Fig. 1.

Chronic hypoxia (CH) increases pulmonary artery smooth muscle cell (PASMC) O₂^·−. Representative images (A) and summary data (B) showing background-subtracted mean fluorescence intensity (MFI) of dihydroethidium (DHE) in PASMC from control and CH rats treated with vehicle, polyethylene glycol-superoxide dismutase (PEG-SOD) (50 U/ml), or PEG-catalase (250 U/ml). Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. Values are means ± SE; n = 3–5 animals per group; *P < 0.05 vs. control, #P < 0.05 vs. vehicle.

Fig. 2.

Di-(4-carboxybenzyl) hyponitrite (SOTS-1) increases PASMC O₂^·− levels. Representative images (A) and summary data (B) showing background-subtracted MFI of DHE in PASMC from control rats treated with increasing concentrations of SOTS-1 (0.01, 0.1, 1.0 mM) or SOTS-1 (0.01 mM) plus PEG-SOD (50 U/ml) or PEG-catalase (250 U/ml). Fluorescence images were digitally inverted to provide better contrast and visibility of immunofluorescence. Values are means ± SE; n = 3–5 animals per group. **P < 0.001 vs. vehicle; #P < 0.05 vs. 0.01 mM SOTS-1 treatment.

Fig. 3.

CH decreases intrapulmonary arterial H₂O₂ levels. A: Amplex Red relative fluorescence units (RFU) from untreated control and CH intrapulmonary arteries were below the detectable linear range of the background-subtracted standard curve (inset). SOTS-1 increased H₂O₂ levels in a dose-dependent manner. B: summary data of H₂O₂ levels in intrapulmonary arteries from control and CH rats in the absence or presence of SOTS-1 (0.01 mM) and SOTS-1 plus PEG-catalase (250 U/ml). Values are means ± SE; n = 3–6 animals per group; τP < 0.05 vs. untreated pulmonary arteries; *P < 0.05 vs. pulmonary arteries from control rats; #P < 0.05 vs. treatment with 0.01 mM SOTS-1.

Fig. 4.

CH decreases pulmonary arterial SOD expression and activity. Representative Western blots (A) and summary data (B) for SOD1 (18 kDa), SOD2 (25 kDa), and SOD3 (26 kDa) protein expression in isolated pulmonary arteries from control and CH rats. SOD expression was normalized to total protein using the corresponding Coomassie-stained blot. C: summary data for total SOD, MnSOD (SOD 2), and Cu/ZnSOD (SOD 1 and 3) activity in isolated pulmonary arteries from control and CH rats. Values are means ± SE; n = 6–9 animals per group; *P < 0.05 vs. control. MW, molecular weight.

Fig. 5.

Acid-sensing ion channel 1 (ASIC1)-dependent store-operated Ca²⁺ entry (SOCE) is increased in pulmonary arteries from SOD1^−/− mice. A: SOCE responses in isolated pressurized pulmonary arteries from SOD1^+/+ and SOD1^−/− mice in the presence or absence of psalmotoxin-1(PcTX1; 20 nM). Representative Western blots (B) and summary data (C) for ASIC1 (∼65 kDa) and SOD1 (18 kDa) protein expression in isolated pulmonary arteries from SOD1^+/+ and SOD1^−/− mice. ASIC1 expression was normalized to total protein using the corresponding Coomassie-stained blot. Values are means ± SE; n = 4–5 animals per group; *P < 0.05 vs. SOD1^+/+; #P < 0.05 vs. respective vehicle.

Fig. 6.

CH increases pulmonary arterial antioxidant capacity. A: Amplex Red RFU from control intrapulmonary arteries following treatment with PEG-SOD (50 U/ml), the SOD mimetic, tiron (10 mM), or inhibition of catalase and glutathione peroxidase (GPx) with 3-amino-1,2,4-triazole (AT; 5 mM) and mercaptosuccinic acid (MSA; 3 mM) are within the linear range of the background-subtracted standard curve. B: summary data of H₂O₂ levels in intrapulmonary arteries from control and CH rats following above treatments. C: rate of H₂O₂ catalysis (1 μM) by control and CH intrapulmonary arteries measured by Amplex Red fluorescence. Values are means ± SE; n = 6–8 animals per group; τP < 0.05 vs. untreated pulmonary arteries; *P < 0.05 vs. pulmonary arteries from control rats.

Fig. 7.

GPx expression and activity are increased following CH. Representative Western blots (A) and summary data showing protein expression (B) and enzyme activity (C) of catalase and GPx in isolated pulmonary arteries from control and CH rats. Protein expression was normalized to total protein using the corresponding Coomassie-stained blot. Values are means ± SE; n = 6 animals per group; *P < 0.05 vs. control.

Fig. 8.

ASIC1-dependent SOCE in PASMC. A: representative experiment showing SOCE and determination of area under curve (AUC). Representative traces and summary data showing time-control (B) or SOCE (C) responses in the presence of vehicle or PcTX1 separated by 30 min. SOCE responses are expressed as the PcTX1-sensitive component (open bar). Values are means ± SE; n = 3 experiments per group; *P < 0.05 vs. vehicle.

Fig. 9.

H₂O₂ inhibits ASIC1-dependent SOCE. PcTX1-sensitive SOCE in PASMC from control and CH rats in the presence of vehicle (physiological saline solution) (A), PEG-catalase (250 U/ml), or H₂O₂ (25 μM) and vehicle (DMSO) (B), ebselen (30 μM), or mercaptosuccinic acid (3 mM). Values are means ± SE; n = 4–10 experiments from at least 3 animals per group; *P < 0.05 vs. control; #P < 0.05 vs. respective vehicle treatment.

Fig. 10.

Summary diagram showing the effect of CH on H₂O₂ levels and ASIC1-dependent SOCE. A: H₂O₂ inhibits ASIC1-dependent SOCE in PASMC from control animals. B: H₂O₂ levels are decreased following CH as a result of diminished O₂^·− dismutation and increased H₂O₂ catalysis through GPx. This leads to the augmented ASIC1-dependent SOCE in PASMC following CH.