Cell Physiology

Chronic hypoxia upregulates pulmonary arterial ASIC1: a novel mechanism of enhanced store-operated Ca2+ entry and receptor-dependent vasoconstriction

Nikki L. Jernigan, Lindsay M. Herbert, Benjimen R. Walker, Thomas C. Resta


Acid-sensing ion channel 1 (ASIC1) is a newly characterized contributor to store-operated Ca2+ entry (SOCE) in pulmonary vascular smooth muscle (VSM). Since SOCE is implicated in elevated basal VSM intracellular Ca2+ concentration ([Ca2+]i) and augmented vasoconstriction in chronic hypoxia (CH)-induced pulmonary hypertension, we hypothesized that ASIC1 contributes to these responses. To test this hypothesis, we examined effects of the specific pharmacologic ASIC1a inhibitor, psalmotoxin 1 (PcTX1), on vasoconstrictor and vessel wall [Ca2+]i responses to UTP and KCl (depolarizing stimulus) in fura-2-loaded, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) Wistar rats. PcTX1 had no effect on basal vessel wall [Ca2+]i, but attenuated vasoconstriction and increases in vessel wall [Ca2+]i to UTP in arteries from control and CH rats; normalizing responses between groups. In contrast, responses to the depolarizing stimulus, KCl, were unaffected by CH exposure or PcTX1. Upon examining potential Ca2+ influx mechanisms, we found that PcTX1 prevented augmented SOCE following CH. Exposure to CH resulted in a significant increase in pulmonary arterial ASIC1 protein. This study supports a novel role of ASIC1 in elevated receptor-stimulated vasoconstriction following CH which is likely mediated through increased ASIC1 expression and SOCE.

  • degenerin/epithelial Na+ channel
  • capacitative calcium entry
  • psalmotoxin 1
  • pulmonary hypertension
  • receptor-operated calcium entry
  • L-type voltage-gated calcium entry

many long-term lung diseases associated with chronic hypoxia (CH) can result in pulmonary hypertension and cor pulmonale. An increasing body of evidence supports a role for chronic vasoconstriction as an important mechanism of increased pulmonary vascular resistance in the pathogenesis of pulmonary hypertension. This vasoconstrictor responsiveness is multifaceted including increases in both basal- and agonist-induced tone (2, 4, 16, 26, 49). Additionally, intracellular Ca2+ handling pathways in pulmonary vascular smooth muscle (VSM) cells are markedly altered in pulmonary hypertension, resulting in augmented basal Ca2+ levels, increased store and receptor-operated Ca2+ entry, and enhanced contractile Ca2+ sensitivity, which all contribute to vasoconstriction in this setting (3, 4, 16, 19, 35, 40, 46, 52).

Store-operated Ca2+ entry (SOCE) plays a significant physiological role in regulation of pulmonary vascular resistance in both the normal and pulmonary hypertensive circulation. SOCE has been linked to important vasoregulatory mechanisms such as acute hypoxic pulmonary vasoconstriction and receptor-mediated arterial constriction (11, 23, 33, 48). Furthermore, enhanced SOCE is thought to contribute to elevated basal intracellular Ca2+ concentration ([Ca2+]i) and enhanced vascular tone in CH-induced pulmonary hypertension (19, 46). Our laboratory has recently characterized a unique role for acid- sensing ion channel 1 (ASIC1) in mediating SOCE in pulmonary VSM (14), although contribution of ASIC1 to enhanced Ca2+ entry and vasoreactivity in pulmonary hypertension has not previously been investigated.

ASIC1 belongs to the degenerin/epithelial Na+ channel (Deg/ENaC) family of voltage-insensitive, cationic channels. ASICs are widely distributed in the central and peripheral nervous systems where they participate in neuronal depolarization and action potential generation in response to decreases in extracellular pH (17, 20, 43). More recently, multiple subunits for both ENaC and ASIC have been found in VSM and endothelial cells from a variety of vascular beds where they play important roles in mechanotransduction of the myogenic response, flow-induced shear stress response, exercise pressor response, and cellular migration (5, 7, 9, 10, 13, 24, 47). Although Deg/ENaC family members are known for their high permeability to Na+, ASIC1a can form Ca2+-permeable channels. This Ca2+-permeable form of ASIC1a has been shown to contribute to ischemic/acidosis-induced neuronal injury (44, 50, 51). Consistent with these findings, our laboratory has shown that ASIC1 is an important mediator of Ca2+ influx in pulmonary VSM (14). We therefore hypothesized that ASIC1 contributes to elevated basal [Ca2+]i, SOCE, and receptor-mediated vasoconstriction following CH.


Chronic Hypoxic Exposure Protocol

Male Wistar rats (∼12 wk old, Harlan Industries) were divided into two groups (control and CH) for each experiment. Animals designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ∼380 mmHg for 4 wk. The chamber was opened two to three times per week to provide animals with fresh food, water, and clean bedding. Age-matched control rats were housed at ambient barometric pressure (∼630 mmHg in Albuquerque, NM). All animals were maintained on a 12:12-h light-dark cycle. 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).

Assessment of Polycythemia and Right Ventricular Hypertrophy

Polycythemia was assessed by measuring hematocrit. Blood samples were collected in microcapillary tubes following direct cardiac puncture at the time of lung isolation and centrifuged for 5 min to separate the red blood cells and plasma. To assess right ventricular hypertrophy, the right ventricle (RV) was dissected from the left ventricle plus septum (LV + S), separately weighed, and the ratios of RV:LV+S and RV:body wt (BW) were calculated.

Cannulation and Endothelial Disruption of Small Pulmonary Arteries

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, 6 glucose]. Small intrapulmonary arteries (5th–6th order) of ∼1-mm length and without visible side branches were dissected free and transferred to a vessel chamber (Living Systems, CH-1). The proximal end of the artery was cannulated with a tapered glass pipette, secured in place with a single strand of silk ligature, and gently flushed to remove any blood from the lumen. The vessel lumen was rubbed with a strand of moose mane to disrupt the endothelium. After cannulation of the distal end, the vessel was stretched longitudinally to approximate its in situ length and pressurized with a servo-controlled peristaltic pump (Living Systems) to 12 mmHg. Any vessel that failed to maintain pressure upon switching off the servo-controller was discarded. The vessel chamber was transferred to the stage of a Nikon Eclipse TS100 microscope where the vessel was superfused with PSS. Bright-field images were obtained with an IonOptix CCD100M camera, and dimensional analysis was performed by IonOptix Sarclen software to measure ID. The effectiveness of endothelial disruption was verified by the lack of a vasodilatory response to acetylcholine (1 μM).

Fura-2 Loading of VSM in Isolated Small Pulmonary Arteries

Pressurized arteries were incubated abluminally with the Ca2+-sensitive fluorescent indicator fura-2 AM (2 μM and 0.05% pluronic acid, Molecular Probes) in PSS for 45 min at room temperature. Arteries were rinsed for 20 min with warmed PSS (37°C) to wash out excess dye and to allow for hydrolysis of AM groups by intracellular esterases. Fura-2-loaded vessels were alternately excited at 340 and 380 nm at a frequency of 1 Hz with an IonOptix Hyperswitch dual excitation light source, and the respective 510-nm emissions were detected with a photomultiplier tube. Background-subtracted 340/380 emission ratios were calculated with IonOptix Ion Wizard software and recorded continuously throughout the experiment, with simultaneous measurement of ID from red-wavelength bright-field images for vessels as described above.

Small Pulmonary Artery Vasoconstrictor Reactivity

Receptor- and depolarization-mediated vasoconstriction and changes in vessel wall [Ca2+]i.

The effect of CH exposure on pulmonary vasoreactivity was determined in small pulmonary arteries from control and CH rats. Receptor-mediated vasoconstrictor reactivity was assessed by superfusion (5 ml/min at 37°C) of cumulative concentrations of UTP (10−7 to 10−3.5 M). Depolarization-mediated vasoconstriction was determined using depolarizing concentrations of equal molar KCl (10−1.75 to 10−1.00 M). Similar protocols were conducted in the presence of the specific ASIC1a inhibitor, psalmotoxin 1 (PcTX1) (21 nM), or the L-type voltage-gated Ca2+ channel (VGCC) inhibitor, diltiazem (50 μM). This concentration of PcTX1 was determined in preliminary experiments to maximally inhibit SOCE, and our laboratory has previously demonstrated that this concentration of diltiazem blocks the increase in [Ca2+]i and vasoconstrictor response to a depolarizing concentration of KCl (50 mM) in this preparation (12).

Measurement of VSM SOCE and receptor-operated Ca2+ entry

Ca2+ depletion/repletion.

Pulmonary arteries were superfused (5 ml/min at 37°C) with Ca2+-free PSS containing 3 mM EGTA to chelate any residual Ca2+ and 50 μM diltiazem to prevent Ca2+ entry through L-type VGCCs. In addition, arteries were incubated with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA; 10 μM) to deplete intracellular Ca2+ stores and prevent Ca2+ reuptake. The changes in [Ca2+]i (SOCE) were then determined upon repletion of extracellular Ca2+ (1.8 mM) in the continued presence of diltiazem and CPA. Parallel experiments were performed in the presence of PcTX1 (21 nM).

Receptor-operated Ca2+ entry (ROCE) was induced with UTP as previously described (12). To assess ROCE independently of SOCE and to eliminate other UTP-mediated Ca2+ entry mechanisms, vessels were pretreated with diltiazem (50 μM) and CPA (10 μM), and a stable SOCE response was obtained (described above) before the addition of UTP (100 μM).

Mn2+ quenching of fura-2 fluorescence.

SOCE was additionally quantified by quenching of fura-2 fluorescence with Mn2+, which enters cells as a Ca2+ surrogate and reduces fura-2 fluorescence upon binding to the dye. Fura-2-loaded pulmonary arteries were excited at 360 nm, and emission light was recorded at 510 nm. At the excitation wavelength of 360 nm, fura-2 fluorescence intensity is not influenced by changes in [Ca2+]i; therefore, changes in fluorescence are assumed to be caused by Mn2+ alone. After stable baseline fluorescence was attained, store-operated channels were activated by superfusing the vessel with Ca2+-free PSS (without EGTA) containing diltiazem (50 μM) and CPA (10 μM) for 15 min. MnCl2 (500 μM) was then added to the superfusate for 10 min. Parallel experiments were performed in the presence of PcTX1 (21 nM). Mn2+ quenching of fura-2 fluorescence was calculated as the percent change in intensity (F; 10 min post-MnCl2) from baseline fluorescence intensity at time 0 (F0).

ASIC1 Expression


Intrapulmonary arteries (∼2nd–5th order) from the left lungs of Wistar rats were dissected from accompanying airways and surrounding lung tissue in PSS then stored in RNA Later (Ambion). Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and reverse transcribed to cDNA using the High Capacity Reverse Transcription Kit (Applied Biosystems). To detect transcripts for ASIC1 (Rn00577292_m1) and those of two reference genes, β-actin and cyclophilin A (Rn00690933_m1), TaqMan gene Expression Assays (Applied Biosystems) were used with the 7500 Fast Real-Time PCR system (Applied Biosystems). Relative quantification of gene expression was determined by the 2−ΔΔCT method.

Western blot analysis.

Intrapulmonary arteries from the left and right lungs were dissected from accompanying airways and surrounding lung tissue in PSS and snap frozen in liquid N2. Each sample was homogenized in 10 mM Tris·HCl homogenization buffer containing 255 mM sucrose, 2 mM EDTA, 12 μM leupeptin, 1 μM pepstatin A, 0.3 μM aprotinin, and 1 mM phenylmethylsulfonyl fluoride (all from Sigma). Samples were centrifuged at 10,000 g for 10 min at 4°C to remove insoluble debris. The supernatant was collected, and sample protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay). Pulmonary artery lysates were separated by SDS-PAGE (7.5% 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 (TBS) containing 10 mM Tris·HCl and 50 mM NaCl (pH 7.5). Blots were then incubated overnight at 4°C with rabbit anti-ASIC1 (1:500; Millipore) and 1 hr at room temperature with rabbit anti-β-actin (1:5,000; Abcam). 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 (ECL, Pierce), ASIC1 and β-actin bands were detected by exposing the blots to chemiluminescence-sensitive film (Kodak). Quantification of the bands was accomplished by densitometric analysis of scanned images (SigmaGel software, SPSS).

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 probability of P < 0.05 was accepted as significant for all comparisons.


CH-Induced Polycythemia and Right Ventricular Hypertrophy

Age-matched CH rats weighed significantly less than control animals after 4 wk of exposure (Table 1). Wistar rats also exhibited polycythemia following CH as indicated by a significantly greater hematocrit (Table 1). Both RV and LV + S weights were greater following CH. The RV:LV+S and RV:BW ratios were significantly greater in CH compared with control animals (Table 1), thus demonstrating RV hypertrophy indicative of pulmonary hypertension.

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Table 1.

Age-matched animal body weight, heart weight, and hematocrit following 4-wk exposure to hypobaric hypoxia or ambient control conditions

Basal Vessel Wall [Ca2+]i Is Increased in Isolated Small pulmonary Arteries Following CH

Baseline fura-2 ratios were greater in arteries isolated from CH rats compared with controls (Fig. 1). There was not a statistical significant difference in baseline ratios between vehicle groups among the different treatments. Baseline vessel ID and in situ fura-2 calibrations were not different between control and CH arteries, however, CH arteries displayed more tone compared with control arteries (Table 2). This indicates that the change in fura-2 fluorescence between control and CH arteries is not likely mediated by differences in vessel size or fura-2 loading between groups. In addition, this increased basal vessel wall [Ca2+]i was abolished by incubation in Ca2+-free PSS (Fig. 1). Vessel diameter and basal wall [Ca2+]i were unaffected by the ASIC1a inhibitor, PcTX1, in arteries from control and CH rats (Fig. 1A and Table 2). We additionally examined other potential Ca2+ influx pathways responsible for the observed differences in basal Ca2+ levels. However, inhibition of L-type VGCCs with diltiazem (Fig. 1B), the reverse mode operation of the Na+-Ca2+ exchanger with KB-R7943 (Fig. 1C), or T-type VGCCs with mibefradil (Fig. 1D) had no effect on basal vessel wall [Ca2+]i.

Fig. 1.

Chronic hypoxia (CH)-induced increases in basal vessel wall intracellular Ca2+ concentration ([Ca2+]i) persist after specific acid-sensing ion channel 1a (ASIC1a) inhibition. Paired vessel wall [Ca2+]i ratios (F340/F380) in small pulmonary arteries from control and CH rats are shown. Vessels were superfused with vehicle, Ca2+-free (CF) physiological saline solution and either the ASIC1a inhibitor, psalmotoxin 1 (PcTX1; 21 nM) (A); the L-type voltage-gated Ca2+ channel (VGCC) inhibitor, diltiazem (50 μM) (B); the reverse-mode Na+/Ca2+ exchange inhibitor, KB-R7943 (10 μM) (C); or the T-type VGCC inhibitor, mibefradil (10 μM) (D). Values are means ± SE; n values (no. of animals/group) are indicated in bar parentheses. *P < 0.05 vs. control; #P <0.05 vs. vehicle; τP < 0.05 vs. drug incubation.

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Table 2.

Baseline small pulmonary artery inner diameter for different treatments and in situ fura-2 calibrations following intraluminal pressurization at 12 mmHg

ASIC1 Contributes to Receptor-Mediated, But Not Depolarization-Dependent Vasoconstriction and Vessel Wall [Ca2+]i Following CH

Vasoconstrictor responses (Fig. 2A) and changes in vessel wall [Ca2+]i to UTP (Fig. 2B) were augmented in arteries from CH rats compared with normoxic controls. Similar increases in vasoconstrictor responses were observed in response to endothelin-1 (data not shown). In contrast to receptor-dependent vasoconstriction, depolarization-induced vasoconstriction (Fig. 2C) and changes in vessel wall [Ca2+]i (Fig. 2D) were not different between arteries isolated from control and CH animals (Fig. 2).

Fig. 2.

CH augments receptor-mediated vasoconstriction following CH in small pulmonary arteries. AD: vasoconstriction (% baseline diameter; A and C) and changes (Δ) in vessel wall [Ca2+]i (F340/F380; B and D) to UTP (10−7 to 10−3.5 M; A and B) and depolarizing concentrations of KCl (10−1.75 to 10−1.00 M; C and D) in small pulmonary arteries from control (Con) and CH rats. Veh, vehicle; VSM, vascular smooth muscle. Values are means ± SE; n = 4–11/group. *P < 0.05 vs. control group.

Pretreatment of control and CH rat arteries with the L-type VGCC inhibitor, diltiazem, had little effect on UTP-mediated vasoconstriction (Fig. 3A) or changes in vessel wall [Ca2+]i (Fig. 3B). This is in contrast to the effect of diltiazem to abolish KCl-induced vasoconstriction (50 mM) and changes in vessel wall [Ca2+]i that we have shown previously (12). This suggests that L-type VGCCs do not substantially contribute to agonist-induced Ca2+ entry in small pulmonary arteries and are unlikely responsible for the enhanced receptor-mediated vasoconstriction following CH. On the other hand, inhibition of ASIC1a diminished UTP vasoconstriction (Fig. 4A) in arteries from control and CH rats and changes in vessel wall [Ca2+]i (Fig. 4B) in arteries from CH rats, resulting in similar vasoreactivity and Ca2+ responses between groups. PcTX1 also attenuated vasoconstrictor responses to endothelin-1 in both groups (data not shown). In contrast to receptor-dependent vasoreactivity, KCl-induced vasoconstriction (Fig. 4C) and changes in vessel wall [Ca2+]i (Fig. 4D) were unaffected by PcTX1 in arteries from control or CH animals.

Fig. 3.

Enhanced UTP-mediated vasoconstriction following CH persists following inhibition of L-type voltage-gated Ca2+ channels in rat small pulmonary arteries. A and B: vasoconstriction (% baseline diameter; A) and changes (Δ) in vessel wall [Ca2+]i (F340/F380; B) to UTP (10−7 to 10−3.5 M) in small pulmonary arteries from control and CH rats in the presence of vehicle or diltiazem (50 μM). Vehicle data for vasoconstriction and Δ in vessel wall [Ca2+]i are the same as presented in Fig. 2, A and B. Values are means ± SE; n = 5–11/group. *P < 0.05 vs. control group; #P <0.05 vs. corresponding vehicle.

Fig. 4.

ASIC1 contributes to receptor-, but not depolarization-dependent vasoconstriction and vessel wall [Ca2+]i in small pulmonary arteries following CH. AD: vasoconstriction (% baseline diameter; A and C) and changes (Δ) in vessel wall [Ca2+]i (F340/F380; B and D) to UTP (10−7 to 10−3.5 M; A and B) and depolarizing concentrations of KCl (10−1.75 to 10−1.00 M; C and D) in small pulmonary arteries from control and CH rats in the presence of vehicle or PcTX1 (21 nM). Vehicle data for vasoconstriction and Δ in vessel wall [Ca2+]i are the same as presented in Fig. 2. Values are means ± SE; n = 4–11/group. *P < 0.05 vs. control group, #P <0.05 vs. corresponding vehicle.

Enhanced Store-Operated Ca2+ Entry Following CH Is Prevented by ASIC1 Inhibition in Isolated Small Pulmonary Arteries

Because ASIC1 may contribute to altered SOCE or ROCE to mediate augmented receptor-dependent [Ca2+]i responses following CH, we next examined effects of CH and ASIC1 inhibition on both SOCE and UTP-induced ROCE. While SOCE was largely augmented following CH (Fig. 5B), ROCE was significantly blunted (Fig. 5D). Despite this attenuated ROCE, the associated vasoconstriction to UTP was significantly greater in arteries from CH rats (Fig. 5E), suggesting increased Ca2+ sensitivity as we have previously reported (4, 15, 16). This increased SOCE and diminished ROCE are similar to what we have previously reported in Wistar rats upon stimulating ROCE with ET-1 (39).

Fig. 5.

Store-operated Ca2+ entry (SOCE), but not receptor-operated Ca2+ entry (ROCE) is augmented following CH in isolated small pulmonary arteries. A: representative traces illustrating SOCE- and ROCE-induced changes in vessel wall [Ca2+]i over time. BE: summary data for SOCE- and ROCE- induced changes in vessel wall [Ca2+]i (ΔF340/F380; B and D) and SOCE- and ROCE-induced vasoconstriction (% baseline diameter; C and E). All experiments were performed in the presence of cyclopiazonic acid (CPA; 10 μM) and diltiazem (50 μM). Values are means ± SE; n values are indicated in bar parentheses. *P < 0.05 vs. control group.

We assessed SOCE by two means: Ca2+ depletion/repletion and Mn2+ quenching of fura-2 fluorescence. The specific ASIC1a inhibitor, PcTX1, reduced SOCE and associated vasoconstriction in arteries from both control and CH rats and normalized responses between groups (Fig. 6, A and B). Similar to effects of ASIC1 inhibition on responses to Ca2+ repletion, Mn2+ quenching of fluorescence was markedly decreased in small pulmonary arteries from both groups following pretreatment with PcTX1 (Fig. 6C). These data strongly suggest the involvement of ASIC1 in mediating enhanced SOCE following CH.

Fig. 6.

ASIC1 contributes to augmented SOCE following CH in isolated small pulmonary arteries. AC: SOCE-induced changes in vessel wall [Ca2+]i (ΔF340/F380; A), SOCE-induced vasoconstriction (% baseline diameter; B), and magnitude of Mn2+ quenching 10 min after MnCl2 (500 μM) administration (C). All experiments were performed in the presence of CPA (10 μM) and diltiazem (50 μM); and either vehicle or PcTX1 (21 nM). F, fluorescence intensity; F0, baseline fluorescence intensity at time 0. Values are means ± SE; n values are indicated in bar parentheses. *P < 0.05 vs. control group; #P < 0.05 vs. corresponding vehicle.

CH Increases ASIC1 Protein, But Not mRNA Expression in Small Pulmonary Arteries

Exposure to CH did not significantly change ASIC1 mRNA expression in isolated pulmonary arteries when normalized to either β-actin (Fig. 7A) or cyclophilin A (control =1.01 ± 0.14; CH = 0.91 ± 0.17). In contrast to mRNA expression, ASIC1 protein expression was significantly elevated following CH (Fig. 7, B and C).

Fig. 7.

CH increases ASIC1 protein levels in small pulmonary arteries. A: summary data for qPCR of ASIC1 normalized to β-actin mRNA levels (2−ΔΔCT) in isolated small pulmonary arteries from control and CH rats. B: representative Western blots of ASIC1 and β-actin (50 μg protein/lane). C: summary data for Western analysis of ASIC1/β-actin protein expression. Values are means ± SE; n values are indicated in bar parentheses. *P < 0.05 vs. control.


Our laboratory has recently characterized a novel role for ASIC1 in mediating rat pulmonary arterial smooth muscle SOCE (14). The goal of the present study was to determine the physiological importance of ASIC1 to elevated basal [Ca2+]i and enhanced receptor-mediated vasoconstriction in the hypertensive pulmonary circulation. We found that ASIC1-dependent Ca2+ entry in small pulmonary arteries following CH is independent of elevated basal vessel wall [Ca2+]i, but contributes to augmented receptor-dependent vasoconstriction which is likely mediated through a store-operated mechanism. In addition, we found that CH upregulates pulmonary arterial ASIC1 protein expression. The results from this study have identified a role for ASIC1 in the regulation of pulmonary arterial Ca2+ influx and vasoconstriction. These findings support a potential mechanism by which ASIC1 contributes to pulmonary hypertension: increased ASIC1 expression leads to greater SOCE and augmented vasoconstriction to circulating vasoactive factors resulting in increased vascular resistance.

CH-induced elevations in basal pulmonary vascular [Ca2+]i can elicit vasoconstriction and promote vascular arterial remodeling and thus have important implications in the development of pulmonary hypertension. Indeed, we see both elevated basal vessel wall [Ca2+]i and increased tone in arteries from CH compared with control arteries. Several laboratories have demonstrated that pulmonary VSM membrane potential is depolarized following CH (27, 30, 36, 38, 41, 52) to levels expected to activate VGCC and account for increased basal [Ca2+]i levels. However, previous studies, along with our current observations, show that this increase in [Ca2+]i is due to Ca2+ influx that is independent of VGCCs since 1) removal of extracellular Ca2+ reduces resting [Ca2+]i to a level similar to that of control pulmonary artery smooth muscle cells (PASMCs), and 2) inhibition of VGCC with diltiazem has little effect on resting [Ca2+]i or vessel diameter (19, 35, 37, 46). Instead, there is evidence that enhanced SOCE is responsible for the elevated basal [Ca2+]i levels since putative inhibitors of SOCE (La3+, Ni2+, and SKF-96365) diminish elevated [Ca2+]i in pulmonary arterial myocytes following CH (19, 46). However, these inhibitors are generalized inhibitors of nonselective cation channels and therefore do not necessarily assess a role for SOCE directly or the specific ion channel involved. Our findings that the ASIC1 specific inhibitor, PcTX1, diminishes SOCE but not basal [Ca2+]i levels argues against SOCE, per se, as a mechanism of enhanced basal [Ca2+]i. Consequently it appears that augmented basal PASMC [Ca2+]i following CH is a result of increased activity of nonselective cation channels, possibly one or more of the classical/canonical transient receptor potential (TRPC) family members.

Vasoreactivity to a variety of agonists is augmented following CH, which may contribute to increased vascular resistance and pulmonary hypertension (2, 16, 35, 49). Consistent with this notion, we found vasoconstrictor responses to ET-1 and UTP to be augmented in arteries from CH animals. This enhanced constriction was prevented in the presence of PcTX1, which reduced vasoconstrictor responses to UTP by 18% in control and 28% in CH pulmonary arteries, thereby implicating the physiological importance of ASIC1 in receptor-mediated vasoreactivity and Ca2+ influx. In contrast, members of the ENaC/ASIC family are generally considered to be voltage-insensitive (17), and therefore, would not be expected to respond to depolarizing stimuli (i.e., KCl). These present data illustrate the specificity of ASIC1-dependent Ca2+ entry to contribute to receptor-dependent responses and not to depolarizing stimuli, and further suggest that PcTX1 is not causing a generalized decrease in vasoreactivity.

To determine the mechanism by which ASIC1 contributes to increased vasoconstriction and vessel wall [Ca2+]i following CH, we examined the two possible Ca2+ entry pathways through nonselective cation channels that are activated following G protein-coupled receptor binding. ROCE is thought to be activated directly by diacylglycerol (DAG) independent of protein kinase C (18). SOCE is initiated by intracellular store depletion as a consequence of receptor activation. Our data suggest that CH increases SOCE, but not ROCE, in small pulmonary arteries. These data are at odds with Lin et al. (19), who demonstrated that CH upregulates ROCE in PASMC, but agree with previous reports from our laboratory showing that SOCE is increased, but ET-1-induced ROCE is diminished in isolated pulmonary arteries following CH in Wistar rats (39). SOCE is augmented in several models of pulmonary hypertension, including patients with idiopathic pulmonary hypertension, where it is thought to contribute to increased vasoconstriction and proliferation of pulmonary VSM (8, 19, 22, 32, 45, 46, 53). In the current study, this augmented SOCE appears to be largely dependent on ASIC1, consistent with a recent study from our laboratory that demonstrated a novel contribution of ASIC1 to SOCE and CPA-induced Ca2+ and Na2+ currents in PASMC (14). In addition, CH results in an upregulation of ASIC1 protein expression in pulmonary arteries from CH animals. Interestingly, this increase in ASIC1 protein did not correlate with increased ASIC1 mRNA expression at 4 wk of CH. However, our assay did not discriminate between the ASIC1a and ASIC1b splice variants which could be differentially regulated by CH. Furthermore, it may be possible that ASIC1 mRNA levels are elevated at an earlier time point of CH exposure. Alternatively, CH may alter posttranscriptional and/or posttranslational modification of ASIC1, resulting in stabilization and upregulation of the channel. Future studies are needed to identify the mechanism by which CH upregulates ASIC1 protein.

Several members of the TRPC family of cation channels have been implicated as store- and receptor-operated channels because they form nonselective Ca2+-permeable channels that are activated by receptor-dependent mechanisms and store-depletion (25). However, the role of TRPC in these responses is continually being challenged by a growing body of evidence for the involvement of Orai1 and stromal interaction molecule 1 (STIM1). STIM1 is thought to function as the endoplasmic reticulum Ca2+ sensor protein relaying the signal to the plasma membrane for activation of Orai1, which is reported to be the pore-forming channel that mediates SOCE (6, 21, 34, 42, 54). It is apparent that Ca2+ influx due to store-depletion is not likely mediated by a single molecule/channel, but rather SOCE is a dynamic and highly regulated process produced by several proteins that likely assemble into a macromolecular complex. In pulmonary VSM cells, inhibition of TRPC1, STIM1, or Orai1 with small interfering RNA diminishes SOCE but the effect is not profound, suggesting the involvement of other proteins (19, 28, 29). The data presented here expand the current knowledge regarding the identity of molecules participating in SOCE. Although ASIC1 is known to conduct Ca2+, whether it forms one of the pores conducting Ca2+ influx or functions as a regulator of other Ca2+-permeable channels is unknown since we did not directly examine ASIC1 Ca2+ conductance in this study. Nevertheless, our present findings demonstrate the importance of ASIC1 in regulating Ca2+ influx in both physiological and pathophysiological settings. Further studies are needed to determine how ASIC1 interacts with other SOCE components (TRPC1, Orai1, STIM1), and how such interactions may be altered in the context of pulmonary hypertension.

In conclusion, the current study describes a novel role of ASIC1 in pulmonary vascular Ca2+ homeostasis and vasoregulation. Here we show that CH increases ASIC1 protein expression and augments ASIC1-dependent SOCE and receptor-mediated vasoreactivity. These findings are consistent with the possibility that ASIC1 contributes to the chronic vasoconstriction and increased pulmonary vascular resistance in the pathogenesis of pulmonary hypertension. Our goal for future experiments is to examine the effects of ASIC inhibitors on pulmonary hypertension. There is promising evidence in the literature that amiloride analogs, which also inhibit ASIC, inhibit pulmonary hypertension. However, the focus of these studies was not ASIC, but rather inhibition of the Na+/H+ exchanger and vascular remodeling/proliferation (31). A potential problem with amiloride/amiloride analogs as a treatment is that they also inhibit epithelial Na+ channels (ENaC) which could limit fluid clearance and lead to pulmonary edema (1). Therefore, a challenge of future studies is to target inhibition of ASIC1 specifically to the lung vasculature.


This work was supported by National Heart, Lung, and Blood Institute Grants HL-92598 (to N. L. Jernigan), HL-101351 (to P. G. McGuire), HL-88192 (to T. C. Resta), and HL-95640 (to B. R. Walker).


No conflicts of interest, financial or otherwise, are declared by the author(s).


N.L.J., B.R.W., and T.C.R. conception and design of the research; N.L.J. and L.M.H. performed the experiments; N.L.J. analyzed the data; N.L.J., B.R.W., and T.C.R. interpreted the results of the experiments; N.L.J. prepared the figures; N.L.J. drafted the manuscript; N.L.J., B.R.W., and T.C.R. edited and revised the manuscript; N.L.J., L.M.H., B.R.W., and T.C.R. approved the final version of the manuscript.


The authors thank Britta Beasley and Minerva Murphy for technical support.


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View Abstract