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 Ca²⁺ entry (SOCE) that is associated with enhanced agonist-induced vasoconstriction and arterial remodeling. However, this enhanced Ca²⁺ 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 nonselective cation channel, acid-sensing ion channel 1a (ASIC1a), belongs to the degenerin/epithelial sodium channel family and is the major ASIC subtype with Ca²⁺ permeability (53, 59). Our laboratory has recently demonstrated that ASIC1 contributes to the development of hypoxic pulmonary hypertension (32). Following exposure to chronic hypoxia (CH), ASIC1-mediated store-operated Ca²⁺ entry (SOCE) is augmented in pulmonary arterial smooth muscle cells (PASMC), resulting in enhanced agonist-induced vasoconstriction, vascular smooth muscle remodeling, and right ventricular hypertrophy (23, 32). Despite the requirement of ASIC1 for enhanced Ca²⁺ influx, these responses are not dependent on an increased ASIC1 protein expression (32). Therefore, the goal of the present study was to examine alternative regulatory mechanisms of ASIC1 function.

Cellular redox status affects the function of various proteins, including ASIC1a. ASIC subunits are cysteine rich, and the cellular redox status can modify both extracellular and intracellular cysteine residues (11, 60). Reducing agents potentiate ASIC1 activity, acid-induced membrane depolarization, and Ca²⁺ influx. More specifically, reducing agents increase ASIC1 current amplitude and pH sensitivity while decreasing channel inactivation (2, 10, 11, 60). In addition, oxidizing agents like hydrogen peroxide (H₂O₂) may introduce intersubunit disulfide bonds, thereby decreasing the amount of ASIC1a present on the cell surface and reducing acid-evoked currents (60). Together, these studies suggest that redox regulation of ASIC1 is an important factor in determining the overall physiological function of these channels. Furthermore, because cellular redox status can change dramatically under pathological states, this represents a potential mechanism by which ASIC1 contributes to the progression of many diseases (5, 12, 57).

It is well documented that hypoxic pulmonary hypertension is associated with alterations in the cellular redox environment. However, whether the generation of reactive oxygen species (ROS) is enhanced or diminished during hypoxia is still debated (34, 42, 54). Furthermore, the relative importance of changes in ROS levels vs. redox status is unclear. Numerous studies show that ROS, in particular superoxide (O₂^·−), and subsequent oxidative stress contribute to hypoxic pulmonary hypertension (34). Indeed, our laboratory has consistently shown that pulmonary arterial O₂^·− levels increase following exposure to CH (7, 24, 33). In contrast, CH has been shown to decrease H₂O₂ levels in several different species (6, 13, 40). Various models of pulmonary hypertension are associated with a dysregulation of superoxide dismutase (SOD), and possibly other antioxidant pathways, that could lead to lowering of H₂O₂ levels (4, 6, 13, 15, 40). However, neither the mechanisms by which CH mediates this decrease in H₂O₂ nor the importance of this response to regulation of pulmonary arterial smooth muscle calcium homeostasis are clearly defined. Consequently, the present study examined effects of CH on both O₂^·− dismutation and H₂O₂ degradation pathways in pulmonary arterial smooth muscle. In addition, we evaluated the importance of H₂O₂ in regulation of ASIC1, testing the hypothesis that enhanced SOCE following CH results from removal of an inhibitory effect of H₂O₂ on ASIC1.

MATERIALS AND METHODS

Animals and Chronic Hypoxic Exposure

All protocols employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine (Albuquerque, NM). Male Wistar rats (∼12 wk old, Harlan Industries) designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ∼380 mmHg for 4 wk. Age-matched control rats were housed at ambient barometric pressure (∼630 mmHg in Albuquerque, NM). We have previously shown that exposure of rats and mice to CH causes pulmonary hypertension, along with the associated right ventricular hypertrophy, arterial remodeling, and polycythemia (32, 44). Male and female SOD1^−/− and SOD^+/+ mice were obtained from Jackson Laboratory (Sod1 tm1Leb/J, no.002972) and bred on a C56B6/J background. Previous reports from our laboratory indicate that SOD1^−/− mice develop spontaneous pulmonary hypertension (40).

Isolation of Pulmonary Arteries and Generation of PASMC Culture

Rats were anesthetized with pentobarbital sodium (200 mg/kg ip), and the heart and lungs were exposed by midline thoracotomy. The left lung was removed and immediately placed in physiological saline solution (PSS) [pH adjusted to 7.4 with NaOH containing (in mM) 130 NaCl, 4 KC1, 1.2 MgSO₄, 4 NaHCO₃, 1.8 CaC1₂, 10 HEPES, 1.18 KH₂PO₄, and 6 glucose]. Intrapulmonary arteries (∼2nd-5th order) were dissected from surrounding lung parenchyma and either snap-frozen in liquid N₂ or utilized to generate short-term PASMC cultures. To isolate PASMCs, intrapulmonary arteries were enzymatically digested in reduced Ca²⁺ HBSS containing papain (9.5 U/ml), type-I collagenase (1,750 U/ml), and dithiothreitol (1 mM) at 37°C for 30 min. The cell suspension was placed on 25-mm glass coverslips and cultured in Ham's F-12 media supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin for 3–4 days in a humidified atmosphere of 5% CO₂-95% air at 37°C.

Assessment of PASMC Superoxide Levels

To determine the effect of CH on O₂^·− levels, PASMC from control and CH rats were incubated with dihydroethidium (DHE; 5 μM in 0.05% pluronic acid) and TO-PRO-3 (1:2,000, Molecular Probes) at 37°C for 15 min. Cells were subsequently fixed with 2% paraformaldehyde for 10 min at room temperature. To verify the specificity of DHE in this preparation to detect O₂^·−, experiments were repeated with preincubation of either polyethylene glycol (PEG)-SOD (50 U/ml) or PEG-catalase (250 U/ml, Sigma-Aldrich) for 1 h at 37°C. Fluorescence images were acquired with a ×63 objective on a confocal microscope (Leica, TCS SP5). Mean fluorescence intensity was taken per field from 10 images and was averaged from 3–5 animals per group. Each image was thresholded using MetaMorph Imaging software (Molecular Devices) to select for positive-stained areas with fluorescence intensity values above background (i.e., of cells not treated with DHE).

Determination of Pulmonary Arterial H₂O₂ Levels

The effect of CH on pulmonary arterial H₂O₂ levels was determined by an Amplex Red Hydrogen Peroxide/Peroxidase Assay according to manufacturer's protocol (Life Technologies). The left descending intrapulmonary artery from control and CH rats was dissected into 2-mm segments in ice-cold PSS. Pulmonary arterial segments were incubated with Amplex Red reagent for 30 min at 37°C. The supernatant was transferred to a 96-well plate, and Amplex Red fluorescence was measured with a fluorescence microplate reader (Tecan Infinite M200) using excitation at 550 nm and fluorescence detection at 590 nm. Protein concentration was determined for each segment following the assay to verify that equal amounts of sample were used for each group. Additional experiments were performed to evaluate H₂O₂ levels following pretreatment for 30 min with di-(4-carboxybenzyl) hyponitrite (SOTS-1; 10 μM; Cayman Chemicals), PEG-SOD (50 U/ml), the SOD mimetic tiron (10 mM), or the catalase inhibitor 3-amino-1,2,4-triazole (AT; 5 mM) and glutathione peroxidase (GPx) inhibitor mercaptosuccinic acid (3 mM).

Pulmonary arterial antioxidant capacity.

The Amplex Red assay was used to examine the antioxidant capacity of tissue as previously described (50). Pulmonary arteries from control and CH rats were incubated with or without H₂O₂ (1 μM) for 1 h at 37°C. The rate of H₂O₂ catalyzed (R_cat) was determined by R_cat = [H₂O₂ − (Sample_H2O2 − Sample_untreated)]/1 h, where H₂O₂ is the fluorescence value of 1 μM H₂O₂ with no tissue, Sample_H2O2 is the tissue sample incubated with 1 μM H₂O₂, and Sample_untreated is an untreated tissue sample. Sample_untreated was negligible, as it was not detected above background fluorescence (see Fig. 3A, inset).

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.

Western Blotting

Snap-frozen intrapulmonary arteries were homogenized in 10 mM Tris·HCl containing 255 mM sucrose, 2 mM EDTA, 12 μM leupeptin, 4 μM pepstatin A, 1 μM aprotinin (Sigma) and centrifuged at 10,000 g at 4°C to remove insoluble debris. Supernatant was collected, and sample protein concentrations were determined by the Bradford method (Bio-Rad) or spectrophotometer (Nano Drop 2000, Thermo Scientific). Equal protein concentration of pulmonary artery lysates were separated by SDS-PAGE (Tris·HCl gels, Bio-Rad) and transferred to polyvinylidene difluoride membranes. Blots were blocked for 1 h at room temperature with 5% milk and 0.05% Tween 20 (Bio-Rad) in Tris-buffered saline containing 10 mM Tris·HCl and 50 mM NaCl (pH 7.4) before incubation with primary antibody overnight. For immunochemical labeling, blots were incubated for 1 h at room temperature with goat anti-rabbit IgG-horseradish peroxidase (1:3,000, Bio-Rad). After chemiluminescence labeling (Pierce Thermo Scientific), bands were detected by exposure of the blots to chemiluminescence-sensitive film (Bio-Express). Bands were quantified by densitometric analysis using ImageJ (NIH). Blots were normalized to total protein determined by dividing the target protein by the intensity of the corresponding lane in the Coomassie-stained blots because exposure to CH significantly elevates both mRNA and protein expression of the traditional loading controls, β-actin and GAPDH, in pulmonary arterial homogenates (unpublished observations).

SOD Expression, Activity, and Modulation of SOCE

Protein expression.

To determine the effect of CH on SOD expression, Western blot analysis was performed in pulmonary artery lysates from control and CH rats. Blots were incubated with rabbit anti-SOD1 (1:5,000), rabbit anti-SOD2 (1:5,000), and rabbit anti-SOD3 (1:500, Abcam) primary antibodies.

Activity.

The effect of CH on SOD activity was determined by an SOD Assay kit (Cayman Chemical). Intrapulmonary arteries were homogenized in 5 μl buffer (20 mM HEPES pH 7.2 containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose) per milligram of tissue and centrifuged at 1,500 g for 5 min at 4°C. Supernatant was collected, diluted 1:25, and transferred to a 96-well plate, where all samples were incubated in the presence or absence of NaCN (1.8 mM) to inhibit Cu/Zn SOD (SOD1 and SOD3) for 20 min at room temperature. Absorbance was detected at 450 nm using an absorbance microplate reader (ELx800, BioTek Instruments). Cu/Zn SOD activity was calculated by subtracting absorbance from the NaCN-treated samples (Mn SOD activity) from total SOD activity.

Modulation of SOCE.

To determine the influence of SOD1 expression and activity on ASIC1-dependent SOCE, small intrapulmonary arteries from SOD1^+/+ and SOD1^−/− mice were cannulated and pressurized for measurement of [Ca²⁺]_i as described previously (32). Briefly, mice were anesthetized with pentobarbital sodium (200 mg/kg ip). The left lung was removed, and small intrapulmonary arteries (4th-5th order) were dissected free and transferred to a vessel chamber (Living Systems) and secured to tapered glass pipettes with a single strand of silk ligature. After cannulation, the artery was pressurized with a servo-controlled peristaltic pump (Living Systems) to 12 mmHg. Any artery that failed to maintain pressure upon switching off of the servo-controller was discarded. The vessel chamber was superfused with HEPES-based PSS (5 ml/min at 37°C). Arteries were incubated abluminally with fura 2-AM (2 μM and 0.05% pluronic acid in PSS, Molecular Probes) for 45 min at room temperature. Fura 2-loaded arteries were alternately excited at 340 and 380 nm at a frequency of 1 Hz with an IonOptix Hyperswitch dual-excitation light source (IonOptix), and the respective 510-nm emissions were detected with a photomultiplier tube. Following baseline fura 2 measurements, arteries were superfused (5 ml/min at 37°C) with Ca²⁺-free PSS containing 3 mM EGTA to chelate extracellular Ca²⁺, 50 μM diltiazem to prevent Ca²⁺ entry through L-type voltage-gated Ca²⁺ channels, and 10 μM cyclopiazonic acid (CPA) to deplete intracellular Ca²⁺ stores and prevent Ca²⁺ reuptake through the sarcoplasmic reticulum Ca²⁺-ATPase. SOCE was determined by measuring the changes in [Ca²⁺]_i (area under curve) upon repletion of PSS containing 1.8 mM CaCl₂ in the continued presence of diltiazem and CPA. Parallel experiments were performed in the presence of the specific ASIC1 inhibitor, psalmotoxin 1 (PcTX1; 20 nM).

ASIC1 protein expression.

To determine the effect of SOD1 on ASIC1 expression, Western blot analysis was performed in pulmonary artery lysates from SOD1^+/+ and SOD1^−/− mice. Blots were incubated with rabbit anti-ASIC1 (1:500, Millipore) or rabbit anti-SOD1 (1:5,000, Abcam).

Catalase Expression and Activity

Protein expression.

To determine the effect of CH on catalase expression, Western blot analysis was performed as described above in pulmonary artery lysates from control and CH rats. Blots were incubated with rabbit anti-catalase (1:2,000, Pierce Thermo Scientific) primary antibody.

Activity.

The effect of CH on catalase activity was determined by a catalase assay kit (Cayman Chemical) according to the manufacturer's directions. Intrapulmonary arteries were homogenized in 5 μl buffer (50 mM potassium phosphate pH 7.0 containing 1 mM EDTA) per milligram of tissue and centrifuged at 10,000 g for 15 min at 4°C. Supernatant was collected, diluted 1:5, and transferred to a 96-well plate. Absorbance was detected at 540 nm using an absorbance microplate reader (SpectraMax Plus384, Molecular Devices).

GPx Expression and Activity

Protein expression.

To determine the effect of CH on GPx expression, Western blot analysis was performed as described above in pulmonary artery lysates from control and CH rats. Blots were incubated with rabbit anti-GPx-1 (1:5,000, Abcam).

Activity.

The effect of CH on GPx activity was determined by an assay kit (Cayman Chemical). Intrapulmonary arteries were homogenized in 5 μl buffer (50 mM potassium phosphate pH 7.0 containing 1 mM EDTA) per milligram of tissue and centrifuged at 10,000 g for 15 min at 4°C. Supernatant was collected, diluted 1:1 and 1:5, and transferred to a 96-well plate. Absorbance was detected at 340 nm using an absorbance microplate reader (SpectraMax Plus384, Molecular Devices).

H₂O₂ Regulation of ASIC1-dependent SOCE

To assess the role of H₂O₂ on ASIC1-dependent SOCE, a similar protocol to that described above was used to measure SOCE in PASMC from control and CH rats following fura 2 loading (2 μM and 0.05% pluronic acid for 30 min at 32°C). PASMC were treated with PEG-catalase (250 U/ml); H₂O₂ (25 μM); GPx mimetic, ebselen (30 μM); or GPx inhibitor, mercaptosuccinic acid (3 mM) throughout the experiment. The contribution of ASIC1 to SOCE was determined in paired experiments by subsequently repeating the SOCE response following 20-min incubation with PcTX1 (20 nM). Data are expressed as percentage of control vehicle taken as an average of the control vehicle responses.

Calculations and Statistics

All data are expressed as means ± SE. Values of n refer to number of animals in each group unless otherwise stated. A t-test or two-way ANOVA was used to make comparisons when appropriate. If differences were detected by ANOVA, individual groups were compared with the Student-Newman-Keuls test. A Mann-Whitney rank sum t-test or Kruskal-Wallis one-way ANOVA was used to make comparisons for data converted to percentage. A probability of P < 0.05 was accepted as significant for all comparisons.

RESULTS

CH Increases O₂^·− and Decreases H₂O₂ Levels

Exposure to CH resulted in a significant increase in right ventricular hypertrophy expressed as right ventricular/left ventricular plus septum weight as an index of pulmonary hypertension (control: 0.28 ± 0.01 and CH: 0.53 ± 0.02; P < 0.001). Consistent with previous studies from our laboratory in isolated pressurized vessels (7, 24, 33), DHE fluorescence was increased in PASMC from CH rats compared with controls (Fig. 1). Dismutation of O₂^·− by PEG-SOD reduced fluorescence in both groups. DHE fluorescence was not altered by PEG-catalase in PASMC from control animals, suggesting that DHE selectively detects O₂^·− over H₂O₂. However, the augmented DHE fluorescence in PASMC from CH rats was diminished by preincubation with PEG-catalase.

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.

In initial measurements of H₂O₂ in untreated pulmonary artery segments, detection of Amplex Red fluorescence was below the linear range of the standard curve (Fig. 3, A, inset, and B). Therefore, O₂^·− and subsequent H₂O₂ production was stimulated with SOTS-1 in experiments described by Figs. 2 and 3. SOTS-1 is an azo-compound that can be thermally decomposed in aqueous solution to generate O₂^·− at physiological pH and exhibits a half-life of ∼90 min (22). Because there is limited information regarding the use of SOTS-1 to generate O₂^·− in biological systems, we examined DHE and Amplex Red fluorescence to increasing concentrations of SOTS-1 (0.01, 0.1, 1.0 mM). SOTS-1 resulted in a concentration-dependent increase in DHE fluorescence that was abolished by coincubation with PEG-SOD but not PEG-catalase (P = 0.073; Fig. 2). In addition to increasing cellular O₂^·− levels, SOTS-1 caused a concentration-dependent increase in H₂O₂ as measured by Amplex Red fluorescence (Fig. 3). SOTS-1 at 1 mM caused profound PASMC contraction and saturated the Amplex Red fluorescence signal. Therefore, experiments in Fig. 3B were performed using the lowest concentration (0.01 mM SOTS-1) that resulted in a detectable increase in H₂O₂ production. In contrast to effect of CH on O₂^·− levels, CH caused a decrease in pulmonary arterial SOTS-1-induced H₂O₂ production (Fig. 3). PEG-catalase significantly reduced H₂O₂ levels in both groups.

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.

CH Decreases SOD Expression and Activity

One possibility for the reciprocal levels of O₂^·− and H₂O₂ following CH is dysregulation of SOD, which has been shown in several models of pulmonary hypertension (4, 13, 15, 26, 35, 40). Consistent with this possibility, CH decreased Cu/Zn SOD1 (cytosolic) and Cu/Zn SOD3 (extracellular) protein expression (Fig. 4, A and B). This correlated with diminished total SOD and Cu/Zn SOD (SOD1 and SOD3) activity (Fig. 4C). There was no detectable difference in Mn SOD2 (mitochondrial) protein expression or activity between groups.

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.

ASIC1-depedent SOCE is Increased in SOD1^−/− Mice

Our laboratory has previously demonstrated that SOD1^−/− mice develop spontaneous pulmonary hypertension (40). To determine whether the expression and activity of SOD1 influences ASIC1 activity, we examined SOCE in isolated-pressurized pulmonary arteries (average inner diameter: 138 ± 17 μm). SOCE was greater in pulmonary arteries from SOD1^−/− compared with SOD1^+/+ mice (Fig. 5). The specific ASIC1a inhibitor, PcTX1, attenuated SOCE in both groups, and SOCE was not different between groups in the presence of PcTX1. Expression of ASIC1 was similar in pulmonary arteries from SOD1^+/+ and SOD^−/− mice (Fig. 5, B and C). Expression of SOD1 was only present in pulmonary arteries from SOD1^+/+ mice, confirming knockout of this protein (Fig. 5B). These data suggest that a decrease in SOD1 expression and activity is sufficient to increase ASIC1-mediated SOCE, and this is further independent of a change in ASIC1 protein expression, as previously reported (32).

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.

CH Increases Pulmonary Arterial Antioxidant Capacity

If SOD is the rate-limiting step in the formation of H₂O₂, then addition of PEG-SOD should increase H₂O₂ levels to similar levels in pulmonary arteries from control and CH rats. PEG-SOD and the SOD mimetic, tiron, increased Amplex Red fluorescence in both groups above that observed in untreated tissue (Fig. 3, A, inset and Fig. 6A), and, therefore, SOTS-1 was not utilized in these experiments. However, H₂O₂ levels were still less in pulmonary arteries from CH rats (Fig. 6B), suggesting that antioxidant mechanisms other than SOD contribute to the overall H₂O₂ levels. Consistent with this possibility, preventing the catalysis of H₂O₂ with blockers of catalase (AT) and GPx (mercaptosuccinic acid) increased H₂O₂ levels similarly in pulmonary arteries from control and CH rats (Fig. 6B).

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.

To specifically examine differences in the rate of H₂O₂ catalysis, we measured the rate of H₂O₂ decomposition following administration of 1 μM H₂O₂ to the arteries from each group (Fig. 6C). After 1 h, pulmonary arteries from CH rats had degraded significantly more H₂O₂ (637 ± 34 nM) compared with control tissue (373 ± 26 nM). Together, these data suggest that CH increases the rate of H₂O₂ catalysis in pulmonary arteries.

CH Increases GPx Expression and Activity

Next we determined the effect of CH on the expression and activity of the two main enzymes responsible for H₂O₂ degradation in the vasculature, catalase, and GPx-1. Although there was a tendency for catalase expression and activity to be decreased following CH, there was not a statistically significant difference between control and CH groups (Fig. 7). Conversely, GPx-1 expression and activity were augmented in pulmonary arteries from CH rats (Fig. 7).

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.

H₂O₂ Inhibits ASIC1-dependent SOCE

We examined the PcTx1-sensitive component of SOCE to determine the influence of H₂O₂ on ASIC1. In time-control experiments, we found that SOCE was similar between the first and second response (Fig. 8B). Therefore, data are expressed as the PcTX1-sensitive component of SOCE, which is derived from subtracting the SOCE response in the presence of PcTX1 from that of vehicle (Fig. 8C). Consistent with previous data from our laboratory (23, 32), we observed an increase in ASIC1-mediated SOCE in PASMC from CH rats (Fig. 9). Decreasing levels of H₂O₂ with PEG-catalase (Fig. 9A) or the GPx mimetic, ebselen (Fig. 9B), increased SOCE in PASMC from control rats but had no further effect to increase SOCE in PASMC from CH rats. Addition of H₂O₂ largely attenuated SOCE in PASMC from both control and CH rats (Fig. 9A). Likewise, increasing H₂O₂ levels by blocking GPx with mercaptosuccinic acid diminished ASIC1-dependent SOCE (Fig. 9B). Together these data suggest that H₂O₂ negatively regulates ASIC1 activity.

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.

DISCUSSION

Previous studies from our laboratory show that the contribution of ASIC1 to the development of hypoxic pulmonary hypertension is independent of an increase in ASIC1 protein expression (32). Changes in ROS occur following CH that could potentially alter the activity of ASIC1 because ASIC1 is redox sensitive. The goal of the current study was to better understand the changes in ROS that occur following CH and how these changes influence ASIC1-dependent SOCE. The major findings from this study are that 1) CH increased PASMC O₂^·− and decreased pulmonary arterial H₂O₂ production; 2) CH attenuated Cu/Zn SOD expression and activity while increasing GPx expression and activity; 3) H₂O₂ inhibited ASIC1-dependent SOCE in PASMC from both control and CH animals; and 4) catalase and the GPx mimic, ebselen, 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 O₂^·− dismutase and increased H₂O₂ catalysis through GPx, leading to the augmented ASIC1-dependent SOCE (Fig. 10).

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.

Endogenous O₂^·− and H₂O₂ are physiologically important second messengers that regulate a variety of downstream signaling pathways. However, an imbalance in ROS homeostasis has been implicated in the progression of various disease states, including pulmonary hypertension. Several lines of evidence, including studies from our laboratory, suggest that CH increases O₂^·− generation from multiple sources, including mitochondrial electron transport chain, xanthine oxidase, cytochrome P-450, nitric oxide synthase, and NADPH oxidase (34, 38, 43). Our laboratory further demonstrates an important contribution of O₂^·− to induce RhoA activation, myofilament Ca²⁺ sensitization, and enhanced vasoconstriction in hypertensive pulmonary arteries (7, 8, 24, 33). Consistent with our previous studies, which examined O₂^·− generation in whole artery preparations (7, 24, 33), in the current study, we found that O₂^·− is specifically elevated in PASMC from CH rats. Pretreatment of PASMCs with PEG-SOD decreased O₂^·− in both groups and prevented the CH-induced increase in O₂^·−. DHE is expected to selectively detect O₂^·− over H₂O₂, and this is demonstrated in PASMC from control animals. Interestingly, we found that PEG-catalase diminished O₂^·− in PASMC from CH animals. We also observed a tendency for PEG-catalase to decrease SOTS-1-induced O₂^·− (i.e., DHE fluorescence); however, this did not reach statistical significance (Fig. 2; P = 0.073). Although the reason(s) for this effect of PEG-catalase is unclear, it is possible that catalase is removing a H₂O₂-mediated inhibition of SOD (20, 21, 29, 56). Exogenous H₂O₂ has been shown to inactivate Cu/ZnSOD through oxidation of copper at the enzyme active site (20, 21). In theory, PEG-catalase would increase SOD activity. Under conditions such as CH and SOTS-1 where there is more O₂^·− substrate for SOD, the effect of PEG-catalase to enhance SOD activity resulting in increased O₂^·− dismutation may be more apparent compared with control conditions. Although we did not specifically measure the effect of PEG-catalase on SOD activity, important studies from Wedgwood et al. (56) have recently reported this in the fetal pulmonary circulation. Relative to fetal control lambs, DHE fluorescence was increased, and SOD3 activity was decreased in a lamb model of persistent pulmonary hypertension of the newborn (PPHN) (56). This was further exacerbated in fetal PPHN lambs ventilated with 100% O₂. Catalase treatment decreased DHE fluorescence similar to levels observed in fetal control lambs and greatly augmented SOD3 activity in ventilated PPHN lambs (56). In addition, Wedgwood et al. (56) show that exogenous H₂O₂ increases and PEG-catalase decreases SOD3 mRNA expression. This correlation between H₂O₂ and SOD expression is consistent with our current findings, where CH resulted in a decrease in H₂O₂ levels and SOD1 and SOD3 expression.

In contrast to effects of CH to increase O₂^·− levels, we found that CH exposure resulted in a paradoxical decrease in H₂O₂ levels. Although O₂^·− is generally associated with contraction of pulmonary arteries, both contraction and relaxation have been observed in response to H₂O₂ (reviewed in Ref. 25). This varied response to H₂O₂ in the pulmonary circulation appears to be dependent on the level of vascular tone present and the concentration of H₂O₂ applied. At physiological concentrations, H₂O₂ is a vasodilatory and antiproliferative signaling molecule (9, 30). In response to acute hypoxia, basal production of H₂O₂ in the rat pulmonary circulation is diminished (3, 30). This prompted the general notion that, under normoxic conditions, H₂O₂ inhibits smooth muscle contraction, whereas under hypoxia H₂O₂ levels fall, resulting in hypoxic pulmonary vasoconstriction. A decrease in H₂O₂ levels has also been reported following long-term exposure to CH (6, 13, 40, 41), as well as in other experimental models of spontaneous pulmonary hypertension and in humans with pulmonary arterial hypertension (4, 6, 40). This decrease in H₂O₂ is thought to contribute to proproliferative and antiapoptotic effects that are, in part, mediated by hypoxia-inducible factor 1α (HIF-1α) and involve a decrease in K_v1.5 channel expression and elevated [Ca²⁺]_i levels (23).

To achieve a detectable Amplex Red signal in the current study, it was necessary to stimulate H₂O₂ production with the addition of SOTS-1, PEG-SOD, tiron, or mercaptosuccinic acid. Determination of H₂O₂ level was made in dissected intrapulmonary arteries as opposed to whole lung tissue, which may have contributed to the low detection levels. This may be further complicated by the fact that Amplex Red only detects extracellular H₂O₂. To address some of these questions, we examined both the upstream involvement of SOD and downstream involvement of catalase and GPx in the regulation of H₂O₂ levels.

Superoxide is highly reactive and rapidly dismutates to H₂O₂; therefore, an additional means of regulating O₂^·− and H₂O₂ levels is through SOD. Indeed, a number of studies have implicated the downregulation of various SOD isoforms in the development of pulmonary hypertension (4, 6, 13, 36, 40). Although SOD can be regulated by a variety of transcription factors, HIF-1α and HIF-2α directly bind the hypoxia-responsive element of SOD2 and SOD1 promoter, resulting in negative regulation of gene expression (18, 27, 52). Likewise, a loss of SOD1, SOD2, and SOD3 leads to the hypoxic activation of HIF-1α, suggesting that stabilization of HIF-1α is redox sensitive and closely linked to SOD function and the oxidant/antioxidant equilibrium (19, 45, 46, 48). Fawn-hooded rats, which develop spontaneous pulmonary hypertension, have an epigenetic silencing of SOD2 expression/activity resulting in decreased H₂O₂ production (4, 6). Although we did not detect differences in SOD2 expression or activity, we did find that CH decreased SOD1 and SOD3 expression and activity, which is consistent with studies from 3- and 10-day-old CH piglets (13, 15). CH also decreases lung SOD3 protein expression and activity in mice (36), whereas overexpression of SOD3 attenuates both hypoxic- and monocrotaline-induced pulmonary hypertension (1, 26, 36).

Our laboratory has recently shown that SOD1^−/− mice develop spontaneous pulmonary hypertension, and this response is associated with increases in pulmonary arterial O₂^·− and decreases in H₂O₂ levels (40). In the current study, we demonstrate that the loss of SOD1 and subsequent imbalance in O₂^·−/H₂O₂ mimics the effects of hypoxia and leads to enhanced ASIC1-dependent SOCE. Similar to our findings following CH (32), this occurs independently of a significant change in ASIC1 protein expression in pulmonary arteries from SOD^−/− compared with SOD^+/+ mice. Together, these data strongly support the concept that the downregulation of SOD1 and SOD3, and subsequent decrease in H₂O₂, following CH contributes to ASIC1 activation. Additionally, we found that the addition of either PEG-SOD or tiron improved, but did not normalize, H₂O₂ levels in pulmonary arteries from CH rats to those of controls. This suggested that CH may additionally augment the enzymatic decomposition of H₂O₂ by catalase and/or GPx. Consistent with this possibility, we found that expression and activity of GPx-1 was augmented in pulmonary arteries from CH rats. Other studies also indicate that CH exposure in rats results in increased lung GPx-1 (17, 31, 58). Because CH alters the state of various redox couples, it is difficult to determine the physiological impact of increased GPX-1 expression and activity on the overall cellular redox environment. This is further complicated by the fact that redox responses can differ markedly among subcellular compartments (55). Nonetheless, changes to the redox potential represent an important mechanism of ion channel regulation, including regulation of ASIC1a.

Although H₂O₂ has been shown to increase [Ca²⁺]_i and constriction of pulmonary arteries, these responses were unaffected by removal of extracellular Ca²⁺ or by nonselective channel inhibitors, arguing against involvement of store- and receptor-operated Ca²⁺ channels (28, 39). Rather than activation, H₂O₂ seems to cause a profound inhibition of SOCE in vascular smooth muscle and endothelial cells (14, 16, 37, 47), an effect widely observed in a variety of nonvascular cells (49, 51). This is consistent with our current findings that H₂O₂ inhibited ASIC1-dependent SOCE in PASMC from both control and CH rats. Consistently, removing H₂O₂ through the addition of PEG-catalase or the GPx mimetic, ebselen, increased SOCE in PASMC from control animals. In contrast, these inhibitors were without effect on SOCE following CH, suggesting that inhibition of SOCE by endogenous H₂O₂ is absent in PASMC from CH animals. However, whether this is due to a direct effect of decreased H₂O₂ levels on ASIC1 or an indirect effect mediated by a shift in the cytosolic redox environment is currently unknown. Furthermore, whether CH induces an oxidized or reduced shift in the redox potential is unclear, as both have been reported (41, 55).

Reducing agents such as dithiothreitol and glutathione potentiate ASIC1 current, increase pH sensitivity, and decrease channel inactivation (2, 10, 11, 60). In cortical neurons, this leads to enhanced acid-induced membrane depolarization and Ca²⁺ influx (11). Site-directed mutagenesis identified involvement of extracellular cysteine and lysine residues in the modulation of ASICs by oxidizing and reducing agents, respectively (11). Other studies show that intracellular COOH-terminal cysteine residues are important in the intersubunit disulfide bond formation induced by H₂O₂. The formation of intersubunit disulfide bonds inhibits acid-evoked currents and reduces the amount of ASIC1a present on the cell surface (60). It is therefore likely that redox reagents affect both trafficking of ASIC1 to the membrane as well as altering of the kinetics of channel activation.

Oxidative stress reflects an imbalance in the production of free radicals and antioxidant defenses. In lungs, the main enzymatic antioxidants are SOD, catalase, GPx, and thioredoxin (38). In the current study, we show that CH decreases pulmonary arterial SOD1 and SOD3 and increases GPx-1 expression and activity. The resulting decrease in H₂O₂ production and increase in degradation lead to an overall reduction in pulmonary arterial H₂O₂ levels following CH. H₂O₂, either through direct mechanisms or indirectly through redox modification, inhibits ASIC1-dependent SOCE. Loss of endogenous H₂O₂ following CH leads to enhanced ASIC1-mediated SOCE (Fig. 10). Further studies are necessary to specifically address key questions regarding how the redox changes during CH correlate to ASIC1 activity and the mechanisms involved in this response.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-92598 (to N. L. Jernigan), HL-111084 (to N. L. Jernigan), HL-88151 (to L. V. Gonzalez Bosc), HL-88192 (to T. C. Resta), and HL-95640 (to B. R. Walker).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: D.R.P., L.M.H., T.R.Y., and N.L.J. performed experiments; D.R.P. and N.L.J. analyzed data; D.R.P. and N.L.J. prepared figures; D.R.P. and N.L.J. drafted manuscript; D.R.P., L.M.H., T.R.Y., T.C.R., L.V.G.B., B.R.W., and N.L.J. edited and revised manuscript; D.R.P., L.M.H., T.R.Y., T.C.R., L.V.G.B., B.R.W., and N.L.J. approved final version of manuscript; T.C.R. and N.L.J. conception and design of research; T.C.R., L.V.G.B., B.R.W., and N.L.J. interpreted results of experiments.