Reactive oxygen species modulate Na⁺-coupled neutral amino acid transporter 1 expression in piglet pulmonary arterial endothelial cells

Abstract

We have previously shown that Na⁺-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in pulmonary arterial endothelial cells (PAECs) from newborn piglets. Specifically, the ability to increase NO production in response to the l-arginine-NO precursor l-citrulline is dependent on SNAT1 expression. Elucidating factors that regulate SNAT1 expression in PAECs could provide new insights and therapeutic targets relevant to NO production. Our major goals were to determine if reactive oxygen species (ROS) modulate SNAT1 expression in PAECs from newborn piglets and to evaluate the role of NADPH oxidase 1 (NOX1) and uncoupled endothelial NO synthase, enzymatic sources of ROS, in hypoxia-induced increases in SNAT1 expression. Treatment with either H₂O₂ or xanthine plus xanthine oxidase increased SNAT1 expression in PAECs from newborn piglets cultured under normoxic conditions. Hypoxia-induced increases in SNAT1 expression were inhibited by treatments with the ROS-removing agents catalase and superoxide dismutase, NOX1 siRNA, and the NO synthase inhibitor N^G-nitro-l-arginine methyl ester. Both tetrahydropbiopterin (BH₄) and l-citrulline, two therapies that decrease ROS by recoupling endothelial NO synthase, reduced the hypoxia-induced increase in SNAT1 expression. BH₄ and l-citrulline treatment improved NO production in hypoxic PAECs despite a reduction in SNAT1 expression. In conclusion, SNAT1 expression is modulated by ROS in PAECs from newborn piglets. However, ROS-mediated decreases in SNAT1 expression per se do not implicate a reduction in NO production. Although SNAT1 may be critical to l-citrulline-induced increases in NO production, therapies designed to alter SNAT1 expression may not lead to a concordant change in NO production.

NEW & NOTEWORTHY Na⁺-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in piglet pulmonary arterial endothelial cells. Factors that regulate SNAT1 expression in pulmonary arterial endothelial cells are unclear. Here, we show that ROS-reducing strategies inhibit hypoxia-induced increases in SNAT1 expression. l-Citrulline and tetrahydropbiopterin decrease SNAT1 expression but increase NO production. Although SNAT1 is modulated by ROS, changes in SNAT1 expression may not cause a concordant change in NO production.

INTRODUCTION

Impairments in nitric oxide (NO) signaling have been shown to contribute to the pathogenesis of a variety of vascular diseases, including chronic hypoxia-induced pulmonary hypertension (9, 17, 29). In part, because of therapeutic potential, delineating means to restore and modulate endogenous NO production is of great interest. We previously identified the role of Na⁺-coupled neutral amino acid transporter 1 (SNAT1) as a novel protein that modulates NO production in pulmonary arterial endothelial cells (PAECs) from newborn piglets (7). In particular, we found that reductions in SNAT1 expression inhibited the ability of PAECs to increase NO production in response to the l-arginine precursor l-citrulline (7). Investigating factors that regulate SNAT1 expression in PAECs might provide new insights and novel targets for manipulating NO production.

Factors regulating SNAT1 expression in PAECs are not yet clear. We have previously shown that exposure to hypoxia increases SNAT1 expression in PAECs from newborn piglets (11). We have also provided evidence that hypoxia increases reactive oxygen species (ROS) production in PAECs at least in part by increasing NADPH oxidase 1 (NOX1) expression (4) and by uncoupling endothelial NO synthase (eNOS) (7). Therefore, we designed experiments to test the hypothesis that ROS contribute to the hypoxia-induced increase in SNAT1 in PAECs from newborn piglets. We also pursued the possibility that NOX1 and uncoupled eNOS are enzymatic sources of ROS that underlie the hypoxia-induced increase in SNAT1. Furthermore, we performed experiments to investigate the impact of treatment with l-citrulline or tetrahydropbiopterin (BH₄), therapies that we have shown to restore uncoupled eNOS and reduce ROS generation (5, 10), on SNAT1 expression in hypoxic PAECs and in pulmonary arteries from piglets with chronic hypoxia-induced pulmonary hypertension.

METHODS

Ethics statement.

Use of animals conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committees of Vanderbilt University Medical Center and University of Utah Health, both of which are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Use.

Pulmonary artery isolation.

Small pulmonary arteries (≤300-μm diameter) that had been previously dissected from lungs of piglets, frozen in liquid nitrogen, and stored at −80°C were used in this study (5, 10). The piglets from which the small pulmonary arteries were isolated had been raised under normoxic or hypoxic conditions until days of life 11–12. Most normoxic control animals were studied on the day of arrival from the vendor at day of life 12. For the hypoxic group, piglets were obtained from the vendor on day of life 2 and raised in a normobaric hypoxic environment until days of life 11–12. Oxygen content was regulated at 10–12% O₂. CO₂ was maintained at 3–6 Torr by absorption with soda lime. Some hypoxic piglets had been treated orally with either l-citrulline (10) or sapropterin dihydrochloride, a synthetic form of BH₄ (5). Treatment with l-citrulline or sapropterin dihydrochloride was started on the third day of hypoxic exposure and continued for an additional 7 days of hypoxia. The total daily dose of l-citrulline was 1–1.5 g·kg⁻¹·day⁻¹. Part of the l-citrulline (0.26 g/kg) was given orally by syringe 2 times/day with an additional 0.5–1.0 g·kg⁻¹·day⁻¹ mixed in milk, which was consumed throughout the day. Piglets treated with sapropterin received 20 mg·kg⁻¹·day⁻¹ on the third day of hypoxic exposure followed by 40 mg·kg⁻¹·day⁻¹ on subsequent days of hypoxia. Sapropterin was given orally by syringe 1 time/day in the morning. On days of life 11–12, animals were given intravenous pentobarbital sodium (50–100 mg/kg) and intravenous heparin (1,000 IU/kg) and exsanguinated.

PAEC isolation.

Using previously published methods (11), 5-day-old mixed breed piglets of both sexes were given intravenous pentobarbital sodium (50–100 mg/kg) and intravenous heparin (1,000 IU/kg) and exsanguinated. The lungs were excised, and the main pulmonary artery was isolated, flushed with PBS, and then filled with 0.25% trypsin-EDTA. To remove endothelial cells, the pulmonary artery was gently flushed with endothelial growth medium (EGM-2; Lonza). Endothelial cells were cultured in EGM-2 in 100-mm plates in a humidified normoxic incubator (21% O₂-5% CO₂) at 37°C. We identified PAECs by their cobblestone morphology and eNOS-positive staining. Cells were subcultured at near confluence and used at passages 4–10.

PAEC protocols.

PAECs were passaged from 100-mm plates to six-well plates and cultured overnight in EGM-2 under normoxic conditions. The next morning, the media were changed, and PAECs were placed in either a normoxic (21% O₂-5% CO₂) or hypoxic (4% O₂-5% CO₂) humidified environment at 37°C. The hypoxic environment was carefully maintained at the desired levels of oxygen using a PRoOX 110 oxygen control system (model 120, Biospherix, Parish, NY). The level of CO₂ was maintained using a 5% CO₂ incubator. To evaluate the effect of exogenous ROS in normoxic PAECs, some PAECs were cultured under normoxic conditions for 48 h, during which time they were treated with either H₂O₂ (0.2, 0.4, or 0.6 mM, Sigma-Aldrich, St. Louis, MO) or xanthine (10⁻⁴ M, Sigma-Aldrich) plus xanthine oxidase (0.005 IU/ml, Sigma-Aldrich). To evaluate the effect of ROS removal in both normoxic and hypoxic PAECs, other PAECs were cultured under normoxic or hypoxic conditions for 48 h while being treated with cell-impermeable or cell-permeable agents to remove either H₂O₂ [250 IU/ml catalase (CAT; Sigma-Aldrich) or 250 IU/ml polyethylene-glycol (PEG)-CAT (Sigma-Aldrich)] or superoxide [250 IU/ml superoxide dismutase (SOD; Sigma-Aldrich) or 50 IU/ml PEG-SOD (Sigma-Aldrich)]. The influence from NO synthase (NOS) as a potential enzymatic source of ROS was evaluated by treating PAECs with N^G-nitro-l-arginine methyl ester (l-NAME, 10⁻³ M, Cayman Chemical) for 48 h while PAECs were cultured under normoxic or hypoxic conditions. The influence from NOX1 as a potential enzymatic source of ROS was evaluated by treating both normoxic and hypoxic PAECs with NOX1-targeting oligonucleotides (see below). Other normoxic and hypoxic PAECs were treated for 48 h with either l-citrulline (1 mM, Sigma-Aldrich), BH₄ (20 μM, Sigma-Aldrich), a combination of l-citrulline (1 mM) plus BH₄ (20 μM), or the NO donor DETA-NONOate (10⁻³ M, Enzo Life Sciences). For each of the preceding protocols, some PAECs were washed with PBS, covered with cell lysis buffer (mammalian protein extraction reagent, Thermo Scientific, Rockford, IL), scraped, and transferred to a tube. After centrifugation at 10,000 revolutions/min for 10 min, aliquots of the supernatant were collected and stored at −80°C for later use in immunoblot analyses.

Modulation of NOX1 expression.

As previously described (7, 8), PAECs were transfected with nontargeting (control) oligonucleotides (siGENOME nontargeting siRNA no. 5) or NOX1-targeting oligonucleotides (sense: 5′-UGGAAUUGCAGAUGAACAAUU-3′ and antisense: 5′-UUGUUCAUCUGCAAUUCCAUU-3′ as well as sense: 5′-CAGCAAUGUUGUUGGUCAUUU-3′ and antisense: 5′-AUGACCAACAACAUUGCUGUU-3′) purchased from Thermo Scientific. For transfection, we replaced EGM-2 with OPTI-MEM (GIBCO). With the use of Lipofectamine (Invitrogen) in OPTI-MEM, PAECs were transfected with a 200- to 400-pmol suspension of either nontargeting or NOX1 oligonucleotides (100–200 pmol for each of the two different NOX1-targeting oligonucleotides). After 4 h, the medium was replaced with EGM-2 and 10% FBS.

NO measurement by electron spin resonance.

PAECs were cultured under normoxic or hypoxic conditions for 48 h in basal media (EGM-2). During the final 90–120 min of the 48-h incubation period, PAECs were treated with either l-citrulline (1 mM), BH₄ (20 μM), or a combination of l-citrulline (1 mM) plus BH₄ (20 μM). Cells were then washed with chilled Krebs-HEPES buffer and incubated with the Ca²⁺ ionophore A-23187 (10 μmol/l, ENZO Life Sciences) and 200 μmol/l Fe(DETC)₂ at 37°C for 1 h. Cells were then scraped in 0.5 ml of Krebs-HEPES buffer, snap frozen in liquid nitrogen, and stored at −80°C until assessed for NO production by electron spin resonance as previously described (8). The amount of detected NO was determined from the calibration curve for integral intensity of the electron spin resonance signal of NO-Fe²⁺(MGD)₂ prepared at various concentrations of the NO donor MAHMA-NONOate (ENZO Life Sciences).

Superoxide measurement using dihydroethidium and a HPLC-based assay.

Superoxide was measured using dihydroethidium (Sigma-Aldrich) and a HPLC-based assay (6). PAECs were cultured under normoxic or hypoxic conditions for 48 h in basal media (EGM-2). During the final 90–120 min of the 48-h incubation period, PAECs were treated with either l-citrulline (1 mM), BH₄ (20 μM), or a combination of l-citrulline (1 mM) plus BH₄ (20 μM). PAECs were washed three times with chilled Krebs-HEPES buffer and then incubated with 1 μmol/l dihydroethidium for 20 min at 37°C in Krebs-HEPES buffer. Cells were scraped in 0.3 ml of methanol, homogenized in a glass Dounce tissue grinder with glass pestle, filtered through 0.22-μm filters, and then stored at −80°C until analyzed by HPLC. Protein measurements by Bradford assay were done in aliquots of homogenates collected before filtration. Separation of ethidium, 2-hydroxyethidium, and dihydroethidium was performed using a Beckman HPLC System Gold model with a C-18 reverse-phase column (Nucleosil 250, 4.5 mm, Sigma-Aldrich) equipped with both UV and fluorescence detectors. Fluorescence detection at 580 nm (emission) and 480 nm (excitation) was used to monitor 2-hydroxyethidium production. UV absorption at 355 nm was used for the detection of dihydroethidium. The mobile phase was composed of a gradient containing 60% acetonitrile and 0.1% trifluoroacetic acid. Dihydroethidium, ethidium, and 2-hydryoxyethidium were separated by a linear increase in acetonitrile concentration from 37% to 47% over 23 min at a flow rate of 0.5 ml/min. Superoxide normalized by protein amount was assessed as the accumulation of 2-hydroxyethidium measured by HPLC. The concentration of 2-hydroxyethidium was determined by using a standard curve of synthetic 2-hydroxyethidium.

Immunoblot analysis of SNAT1, NOX1, eNOS, and eNOS dimers and monomers.

Small pulmonary arteries were frozen in liquid N₂ and then pulverized with a mortar and pestle into a fine powder. The powder was transferred to a tube containing homogenization buffer with protease inhibitors and then sonicated. The homogenate was spun at 10,000 rpm for 10 min, and the supernatant was collected and used for protein concentrations. For PAECs, stored aliquots of frozen supernatant prepared as described above were used for protein concentrations. All protein concentrations were determined by the protein assay (Bradford). For SNAT1, NOX1, and eNOS analysis, using previously described methods (11), supernatants were applied to Tris-glycine precast 4–20% polyacrylamide gels (Invitrogen, Carlsbad, CA) so that equal amounts of protein were loaded. Electrophoresis was carried out, and the proteins were transferred from the gel to a nitrocellulose membrane. The membrane was incubated at room temperature in PBS containing 7.5% nonfat dried milk and 0.1% Tween 20 to block nonspecific protein binding. To detect SNAT1, NOX1, or eNOS, the nitrocellulose membrane was incubated overnight with the primary antibody (SNAT1: 1:500, ab60145, SNAT1 antibody from Abcam; NOX 1: 1:500, sc-5821, NOX1 antibody from Santa Cruz Biotechnology; and eNOS: 1:2,000, 610297, eNOS antibody from BD-Transduction Laboratory) diluted in PBS containing 0.1% Tween 20 and 1% nonfat dried milk (carrier buffer) followed by incubation with horseradish peroxidase-conjugated secondary antibody (Zymed) diluted in carrier buffer (1:2,500–1:4,000). Using nonsonicated and nonboiled lysates and low-temperature SDS-PAGE, eNOS dimers/monomers were immunoblotted (1:2,000, eNOS antibody, 610297, BD-Transduction Laboratory) as previously described (16). Membranes were developed using enhanced chemiluminescence (ECL) reagents (Amersham), and the chemiluminescent signal was captured on X-ray film (ECL Hyperfilm, Kodak). Similar procedures were followed to reprobe the membranes for β-actin (Sigma-Aldrich). Bands for each protein were quantified using densitometry.

Statistical analysis.

Data are presented as means ± SD. Data were compared by an unpaired t-test or one-way ANOVA with Fisher’s protected least-significant-difference post hoc comparison test as appropriate. P values of <0.05 were considered significant.

RESULTS

Treatment with H₂O₂ dose dependently increased SNAT1 expression in PAECs cultured under normoxic conditions (Fig. 1A). Culturing PAECs in the presence of the ROS generator (xanthine + xanthine oxidase) also increased SNAT1 expression (Fig. 1B). These experiments indicate that exposure to exogenous ROS increases SNAT1 expression in piglet PAECs cultured under normoxic conditions.

Fig. 1.

Na⁺-coupled neutral amino acid transporter 1 (SNAT1) expression measured in pulmonary arterial endothelial cells (PAECs) cultured under normoxic conditions in the presence of either H₂O₂ (A) or xanthine plus xanthine oxidase (X + XO) (B). A: culture of normoxic PAECs in the presence of H₂O₂ (n = 6) dose dependently increased expression of the amino acid transporter SNAT1. B: treatment with the reactive oxygen species (ROS) generator X + XO also increased expression of SNAT1 in PAECs from newborn piglets cultured under normoxic conditions (n = 6). *Different from normoxia untreated; ⁺different from normoxia 0.2 mM H₂O₂ treated; ^!different from normoxia 0.4 mM H₂O₂ treated. A: data were compared by one-way ANOVA with Fisher’s protected least-significant difference post hoc comparison test; B: data were compared by unpaired t-test (P < 0.05).

Consistent with our previous findings (7, 11), SNAT1 expression was greater in PAECs cultured under hypoxic conditions compared with PAECs cultured under normoxic conditions (Fig. 2, A–F). Sole (Fig. 2, A–D) or combined (Fig. 2, E and F) treatment with cell-impermeable (Fig. 2, A, C, and E) or cell-permeable (Fig. 2, B, D, and F) agents to remove either H₂O₂ (CAT or PEG-CAT) or superoxide (SOD or PEG-SOD) reduced the hypoxia-induced increase in SNAT1 expression. These findings indicate that endogenously produced ROS contribute to the increased SNAT1 expression in hypoxic piglet PAECs.

Fig. 2.

Na⁺-coupled neutral amino acid transporter 1 (SNAT1) expression measured in pulmonary arterial endothelial cells (PAECs) cultured under normoxic or hypoxic conditions in the presence of cell-impermeable [catalase (CAT), n = 8 (A); superoxide dismutase (SOD), n = 8 (C); or CAT + SOD, n = 6 (E)] or cell-permeable [polyethylene-glycol (PEG)-CAT, n = 8 (B); PEG-SOD, n = 8 (D); or PEG-CAT + PEG-SOD, n = 6 (F)] reactive oxygen species (ROS)-removing agents. A–F: SNAT1 expression was greater in PAECs cultured under hypoxic conditions than under normoxic conditions. All of the ROS-removing treatments, either singly or combined, reduced the hypoxia-induced increase in SNAT1 expression. *Different from normoxia untreated; +different from hypoxia untreated. Data were compared by one-way ANOVA with Fisher’s protected least-significant-difference post hoc comparison test (P < 0.05).

We next evaluated the effects on SNAT1 expression from agents that target potential enzymatic sources of ROS. NOX1, an enzymatic source of ROS, was increased in PAECs cultured under hypoxic conditions (Fig. 3A). Treatment with NOX1 siRNA inhibited the hypoxia-induced increase in NOX1 expression (Fig. 3A) and concomitantly inhibited the elevation in SNAT1 expression found in piglet PAECs cultured under hypoxic conditions (Fig. 3B). The NOS inhibitor l-NAME also inhibited the hypoxia-induced increase in SNAT1 expression (Fig. 3C). These data indicate that NOX1 and eNOS are enzymatic sources of ROS that contribute to the hypoxia-induced increase in SNAT1 expression.

Fig. 3.

NADPH oxidase (NOX1; A) and Na⁺-coupled neutral amino acid transporter 1 (SNAT1) expression (B and C) measured in pulmonary arterial endothelial cells (PAECs) cultured under normoxic or hypoxic conditions in the presence of either NOX1 siRNA (n = 6 for A and B) or N^G-nitro-l-arginine methyl ester [l-NAME; n = 8 (C)]. When transfected with control NOX1 siRNA, both NOX1 (A) and SNAT1 (B) expression was greater in PAECs cultured under hypoxic conditions than under normoxic conditions. NOX1 siRNA inhibited hypoxia-induced increases in expression of both NOX1 (A) and SNAT1 (B). Treatment with the nitric oxide synthase (NOS) inhibitor l-NAME also reduced hypoxia-induced increases in SNAT1 expression (C). A and B: *different from normoxia control siRNA; +different from hypoxia control siRNA. C: *different from untreated normoxia; +different from untreated hypoxia. Data were compared by one-way ANOVA with Fisher’s protected least-significant-difference post hoc comparison test (P < 0.05).

We then considered the possibility that therapies that reduce ROS generation might inhibit the hypoxia-induced increase in SNAT1 expression. We have previously provided evidence that in vivo treatment with either l-citrulline, an l-arginine-NO precursor, or BH₄, an eNOS cofactor, reduces ROS generation by restoring eNOS coupling in pulmonary arteries of piglets with chronic hypoxia-induced pulmonary hypertension (5, 10). Therefore, we next evaluated the impact of in vivo treatment with either l-citrulline or BH₄ on SNAT1 expression in pulmonary arteries isolated from piglets raised in chronic hypoxia. SNAT1 expression was greater in small pulmonary arteries from piglets raised in chronic hypoxia than in those from normoxic control piglets (Fig. 4, A and B). Notably, SNAT1 expression was much less in piglets treated with l-citrulline (Fig. 4A) or BH₄ (Fig. 4B) during exposure to chronic hypoxia compared with SNAT1 expression in untreated chronically hypoxic animals.

Fig. 4.

Na⁺-coupled neutral amino acid transporter 1 (SNAT1) expression in small pulmonary arteries from piglets treated orally with either l-citrulline (A) or sapropterin dihydrochloride, a synthetic form of tetrahydropbiopterin (BH₄, B), during in vivo exposure to chronic hypoxia. A and B: compared with normoxic control piglets, SNAT1 expression was increased in small pulmonary arteries of piglets raised in chronic hypoxia. SNAT1 expression was reduced in small pulmonary arteries of piglets treated with either oral l-citrulline (A) or oral sapropterin dihydrochloride (B) during exposure to chronic hypoxia compared with SNAT1 expression in untreated chronically hypoxic animals. A: representative samples of small pulmonary arteries from n = 7 normoxic control piglets, n = 8 untreated chronic hypoxia piglets, and n = 8 piglets treated with oral l-citrulline during exposure to chronic hypoxia (10). B: representative samples of small pulmonary arteries from n = 10 normoxic control piglets, n = 9 untreated chronic hypoxia piglets, and n = 12 piglets treated with oral sapropterin dihydrochloride during exposure to chronic hypoxia (5). *Different from normoxic control; +different from untreated chronic hypoxia. Data were compared by one-way ANOVA with Fisher’s protected least-significant-difference post hoc comparison test (P < 0.05).

We next performed in vitro experiments with cultured PAECs to evaluate the possibility that a combined therapeutic approach to recouple eNOS, accomplished by improving substrate availability with the l-arginine precursor l-citrulline, and also providing the NOS cofactor BH₄ would have greater impact on recoupling eNOS, reducing generation of the ROS superoxide (O₂^·−) and altering SNAT1 expression compared with treatments with either alone. We found that eNOS dimer-to-monomer ratios (Fig. 5A) and NO production (Fig. 5B) were less and that O₂^·− generation (Fig. 5C) and SNAT1 expression (Fig. 5D) were greater in PAECs cultured under hypoxic conditions than in PAECs cultured under normoxic conditions. Sole treatment of hypoxic PAECs with either l-citrulline or BH₄ increased eNOS dimer-to-monomer ratios (Fig. 5A) and NO production (Fig. 5B) and reduced O₂^·− generation (Fig. 5C) and SNAT1 expression (Fig. 5D). Moreover, the impact on eNOS dimer-to-monomer ratios, NO production, O₂^·− generation, and SNAT1 expression was more marked in hypoxic PAEC incubated with the combination of l-citrulline and BH₄ than with either therapy alone (Fig. 5, A–D).

Fig. 5.

Endothelial nitric oxide (NO) synthase (eNOS) dimers/monomers (n = 4; A), NO production (n = 4; B), superoxide generation, as assessed by the formation of 2-hydroxyethidium (n = 4; C), and Na⁺-coupled neutral amino acid transporter 1 (SNAT1) expression (n = 9; D) in pulmonary arterial endothelial cells (PAECs) cultured under normoxic conditions or under hypoxic conditions in the presence and absence of sole or combined treatment with l-citrulline and tetrahydropbiopterin (BH₄). A and B: eNOS dimer-to-monomer ratios (A) and NO production (B) were less in PAECs cultured under hypoxic conditions than in those cultured under normoxic conditions. All treated groups of hypoxic PAECs had greater levels of eNOS dimer-to-monomer ratios and NO production than those measured in untreated hypoxic PAECs. eNOS dimer-to-monomer ratios and NO production were greater in hypoxic PAECs treated with a combination of l-citrulline and BH₄ than in hypoxic PAECs treated solely with either l-citrulline or BH₄. C and D: superoxide generation (C) and SNAT1 expression (D) were greater in PAECs cultured under hypoxic conditions than in those cultured under normoxic conditions. All treated groups of hypoxic PAECs had lower levels of superoxide generation and SNAT1 expression than untreated hypoxic PAECs. Compared with hypoxic PAECs treated solely with either l-citrulline or BH₄, superoxide generation and SNAT1 expression were less in hypoxic PAECs treated with a combination of l-citrulline and BH₄. *Different from untreated normoxia; +different from untreated hypoxia; ^!different from l-citrulline-treated hypoxia; #different from BH₄-treated hypoxia. Data were compared by one-way ANOVA with Fisher’s protected least-significant-difference post hoc comparison test (P < 0.05).

In addition to reducing O₂^·− generation (Fig. 5A), treatments with l-citrulline and BH₄ increase NO production (Fig. 5B). We therefore considered the possibility that supplying NO might reduce SNAT1 expression. Contrary to this possibility, we found that SNAT1 expression was unchanged by treatment with the NO donor NONOate in normoxic PAECs and was augmented, not diminished, by treatment with NONOate in hypoxic PAECs (Fig. 6).

Fig. 6.

Na⁺-coupled neutral amino acid transporter 1 (SNAT1) expression measured in pulmonary arterial endothelial cells (PAECs) cultured under normoxic or hypoxic conditions in the presence of NONOate (n = 8). SNAT1 expression was the same in normoxic PAECs cultured in the presence versus the absence of NONOate. In contrast, SNAT1 expression was greater in hypoxic PAECs cultured in the presence than in the absence of NONOate. *Different from normoxia untreated; +different from hypoxic untreated. Data were compared by one-way ANOVA with Fisher’s protected least-significant-difference post hoc comparison test (P < 0.05).

DISCUSSION

Consistent with our previous findings, this study shows that hypoxia increases expression of the neutral amino acid transporter SNAT1 in PAECs from newborn piglets (7, 11). A new finding in this study is that, mimicking the impact of hypoxia, ROS exposure increases SNAT1 expression in normoxic PAECs. Moreover, we now provide new evidence that ROS mediate the hypoxia-induced increase in SNAT1 expression in PAECs from newborn piglets.

There are limited data about the effect of either hypoxia or ROS on SNAT1 expression in any tissue or cell type. The few findings available have been conflicting. For example, consistent with our findings, one group of investigators found that oxidative stress induced by exposure to H₂O₂ increased SNAT1 expression in cardiomyocytes isolated from adult rats (15). However, other investigators found that oxidative stress induced by exposure to manganese reduced SNAT1 expression in cultured rat astrocytes (24). SNAT1 expression was reduced in rat pup brains 24 h after hypoxia-ischemia but increased 7 days later. Culturing human trophoblasts under hypoxic conditions was shown to reduce the expression of system A transporters, which include SNAT1 (20). Thus, SNAT1 expression in response to hypoxia or oxidative stress may be cell type or organ specific, may vary with the type of stimulus or ROS that is generated, or may depend on age or developmental stage (23).

Our findings, which implicate the involvement of specific ROS in the hypoxia-induced increase in SNAT1 expression in piglet PAECs, merit some comment. The finding that SOD by itself attenuated the hypoxia-induced increase in SNAT1 suggests that superoxide is involved. Likewise, our findings implicate the involvement of H₂O₂ because CAT by itself also inhibited the hypoxia-induced increase in SNAT1 expression. The finding that increases in SNAT1 are reduced, but not completely inhibited, by combined treatment with SOD plus CAT suggests that mechanisms additional to the ROS O₂^·− and H₂O₂ contribute to the hypoxia-induced increases in SNAT1 protein levels. It is also possible that treatments with SOD or CAT did not completely remove all of the O₂^·− and H₂O₂. Furthermore, consistent with findings of others (27, 28), it is possible that the cell-permeant agents did not penetrate the PAEC cell membrane more effectively than the nonpermeant agents.

To our knowledge, we are the first to provide evidence that SNAT1 expression can be modulated by reducing NOX1 expression. We are also the first to show that pharmacological inhibition of eNOS, in a situation where eNOS is uncoupled, alters SNAT1 expression. It is important to note that we have previously shown that NOX1 (4) and uncoupled eNOS (7) are enzymatic sources of ROS in hypoxic conditions. In fact, as shown by others (18), it is likely that NOX1-derived ROS contribute to the oxidative loss of the eNOS cofactor BH₄ and thereby provide at least one source of ROS that leads to eNOS uncoupling (Fig. 7). Taken together, our findings support the idea that uncoupled eNOS and NOX1 impact SNAT1 expression via generation of ROS.

Fig. 7.

Schematic representation of Na⁺-coupled neutral amino acid transporter 1 (SNAT1) regulation by reactive oxygen species (ROS) in hypoxic piglet pulmonary arterial endothelial cells (PAECs). Hypoxia-induced increases in NADPH oxidase (NOX1) expression and uncoupled endothelial nitric oxide synthase (eNOS) contribute to the generation of superoxide and H₂O₂, which, in turn, induce SNAT1 expression in piglet PAECs. NOX1-derived superoxide likely contributes to eNOS uncoupling by oxidizing the nitric oxide synthase (NOS) cofactor tetrahydropbiopterin (BH₄). l-Citrulline, via metabolism to l-arginine, and BH₄ recouple eNOS, reduce superoxide generation, and hence inhibit hypoxia-induced increases in SNAT1 expression.

Additional findings in our study also point to an important role of ROS in modulating SNAT1 expression. Specifically, we found that treatment with either the l-arginine-NO precursor l-citrulline or the NOS cofactor BH₄ inhibited hypoxia-induced increases in SNAT1 expression while concomitantly reducing hypoxia-induced generation of the ROS O₂^·−. Moreover, the amount of uncoupled eNOS, as reflected by eNOS dimer-to-monomer ratios, an enzymatic source of ROS, was also reduced by l-citrulline and BH₄ treatments. It is noteworthy that the impact on SNAT1 expression was greater with the combination of l-citrulline and BH₄ treatment than with use of either treatment alone, that is, the treatment that caused the greatest changes in ROS generation had the largest impact on SNAT1 expression.

We previously showed that sole treatment with either l-citrulline (10) or BH₄ (5) reduces ROS generation and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets. In this study, we add to these previous findings by showing that SNAT1 expression is reduced in small pulmonary arteries from piglets treated in vivo with either l-citrulline or BH₄ during hypoxic exposure. These findings further support the contention that it is the reductions in ROS achieved with l-citrulline or BH₄ that underlie the decrease in hypoxia-induced elevations in SNAT1 expression.

In addition to reducing ROS generation, NO production is increased by both in vivo and in vitro treatment with l-citrulline or BH₄. Thus, we considered the possibility that the alteration in SNAT1 expression occurred in response to the NO production induced by these treatments. Instead, we found that in vitro treatment with a NO donor increased SNAT1 expression in hypoxic PAECs.

Relevant to the finding that SNAT1 expression was not reduced by a NO donor is the awareness that SNAT1 mediates transport of a number of amino acids, including glutamine (22). Under hypoxic conditions, some cell types become dependent on glutamine to maintain cellular bioenergetics for survival (19). Moreover, glutamine, when converted to glutamate, together with cysteine and glycine, is needed for synthesis of glutathione, a major antioxidant. Thus, it is possible that cellular requirements for amino acids, such as glutamine, that are needed under hypoxic conditions could have overridden any potential for increased NO production to reduce SNAT1 expression.

We know of no other report of the impact of a NO donor on SNAT1 expression in any cell type. NO donors have been shown to induce the transport of amino acids, including glutamate and cysteine, in cultured bovine aortic (cysteine) or pulmonary artery (glutamate) endothelial cells (21, 30). Furthermore, although the transport system was not identified, evidence was provided showing that the increase in cysteine transport was due to a NO-mediated increase in the expression of an amino acid transport system (21). Thus, our findings are in line with these other studies and add to them by identifying SNAT1 as a specific amino acid transporter that can be modulated by supplying exogenous NO.

Alterations in expression of amino acid transporters, such as SNAT1, are of particular interest because of the potential effect on processes downstream of the amino acids they transport. We previously identified l-citrulline as an amino acid whose transport is modulated by SNAT1 expression (11). In turn, alterations in l-citrulline transport are of interest because, as an l-arginine precursor, cellular uptake of this amino acid may have an impact on NO production. Indeed, our interest in l-citrulline transport is in part because of the therapeutic potential to increase NO production and thereby inhibit the development of pulmonary hypertension (12). It should also be noted that, likely via increasing NO production, l-citrulline treatment might improve a variety of other cardiovascular disorders (25), including atherosclerosis (14) and heart failure (2). Moreover, l-citrulline has been reported to have both NO-dependent and non-NO-dependent effects that may be beneficial for muscle protein synthesis and adipose tissue lipolysis (1). Thus, understanding the modulation of l-citrulline transport has implications for a variety of human health issues.

As to its impact on NO production, we previously found that, when SNAT1 expression was reduced by siRNA technique, the ability of PAEC to increase endogenous NO production when supplied with exogenous l-citrulline was diminished (7). One potential interpretation is that NO production will be reduced in all situations where SNAT1 expression is reduced. However, in this study we found that, despite a reduction in SNAT1 expression, NO production was greater, not less, in hypoxic PAEC treated with l-citrulline and/or BH₄ than in untreated hypoxic PAEC. It is likely that the net impact of l-citrulline and/or BH₄ treatment on NO production reflects a greater beneficial effect from a shift in balance of eNOS-generated superoxide toward NO production than any negative effect on NO production from a ROS-related reduction in SNAT1-mediated l-citrulline transport.

Some of our other findings also demonstrate a discordance between SNAT1 expression and NO production. This is illustrated by the finding in both this and our previous study (7) that NO production is less in hypoxic than in normoxic PAECs, despite greater SNAT1 expression in hypoxic compared with normoxic PAECs. It is important to note that we also previously found that the hypoxia-induced increase in SNAT1 expression enhanced the ability of PAECs to transport l-citrulline (11). In other words, even though basal NO production was not positively impacted by hypoxia-induced increases in SNAT1 expression, PAECs were better equipped to transport the l-arginine precursor l-citrulline, improving their ability to increase NO production in response to exogenously administered l-citrulline, suggesting a compensatory mechanism under conditions of hypoxia.

Limitations of our study should be mentioned. For example, we did not determine whether there might be sex-related differences in any of our studies. Another limitation is that the H₂O₂ concentrations used in our experiments with normoxic PAECs exceed plasma levels reported to be physiological in adult humans (1–5 μM) (13). Unfortunately, physiological H₂O₂ plasma levels in newborns are not yet certain. Moreover, evidence that antioxidant capacity is much less in newborns than adults makes it likely that plasma H₂O₂ levels in newborns will be greater than those in adults (3, 26). In addition, hypoxia, the condition we were interested in evaluating, is anticipated to further enhance H₂O₂ levels in newborns above physiological plasma levels measured in adults. These factors guided the choice of H₂O₂ concentrations used in our study. Another issue is that we were unable to find antibodies that detect NOX4 and NOX2 protein in piglet tissues so that we were unable to determine whether NOX1 siRNA impacted these other NOX proteins. Hence, we cannot rule out the possibility that NOX4 and NOX2 are additional sources of ROS that modulate SNAT1 expression nor can we exclude a potential contribution to alterations in SNAT1 expression from other enzymatic and nonenzymatic sources of ROS that were not evaluated in this study.

In summary, findings in this study clearly show that ROS modulate SNAT1 expression in PAEC from newborn piglets. Taken together (Fig. 7), our findings indicate that hypoxia-induced increases in NOX1 expression and eNOS uncoupling generate O₂^·− and H₂O₂, which, in turn, induce SNAT1 expression. Therapies that recouple eNOS, such as l-citrulline and BH₄, inhibit hypoxia-induced increases in SNAT1 expression via their ability to reduce O₂^·− production. We also show that changes in SNAT1 expression may not always lead to a concordant change in direction of NO production. The relationship between SNAT1 and endogenous NO production is nuanced and is affected by factors that are not yet completely understood. Further investigation is needed and must take into consideration cell and organ type, oxygen milieu, and oxidative stress before therapeutic maneuvers to manipulate SNAT1 expression are undertaken as a means of modulating endogenous NO production.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-097566 (to C. Fike).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

A.E.D., Y.Z., M.R.K., and C.D.F. performed experiments; A.E.D., Y.Z., M.R.K., and C.D.F. analyzed data; A.E.D., J.L.A., and C.D.F. interpreted results of experiments; A.E.D., J.L.A., M.R.K., and C.D.F. edited and revised manuscript; A.E.D., J.L.A., Y.Z., M.R.K., and C.D.F. approved final version of manuscript; A.E.D., J.L.A., and C.D.F conceived and designed research; C.D.F. prepared figures; C.D.F. drafted manuscript.