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Departments of Pediatrics and Medicine, Duke University Medical Center, Durham, North Carolina 27710
Submitted 2 June 2003 ; accepted in final form 2 July 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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). Because the short-lived anion cannot diffuse across plasma membranes, its release from vascular cells requires specialized mechanisms that have not been well delineated in the pulmonary circulation. We have shown that the bicarbonate 
anion exchange protein (AE2) expressed in the lung also exchanges for 
. Thus we determined whether release involved in pulmonary vascular tone depends on extracellular . We assessed endothelium-dependent vascular reactivity and release in the presence or absence of in pulmonary artery (PA) rings isolated from normal rats and those exposed to hypoxia for 3 days. Lack of extracellular in normal PA rings significantly attenuated endothelial release, opposed hypoxic vasoconstriction, and enhanced acetylcholine-mediated vasodilation. Release of was also inhibited by an AE2 inhibitor (SITS) and abolished in normoxia by an NO synthase inhibitor (NG-nitro-L-arginine methyl ester). In contrast, hypoxia increased PA AE2 protein expression and release; the latter was not affected by NG-nitro-L-arginine methyl ester or other inhibitors of enzymatic generation. Enhanced release by uncoupling NO synthase with geldanamycin was attenuated by hypoxia or by elimination. These results indicate that produced by endothelial NOS in normoxia and unidentified sources in hypoxia regulate pulmonary vascular tone via AE2.
anion exchange protein; nitric oxide; endothelial nitric oxide synthase; hypoxia
The endothelium-derived relaxing factor NO·, generated from L-arginine by nitric oxide (NO) synthase (NOS) activity, contributes to basal vascular tone in normoxia and counteracts hypoxic pulmonary vasoconstriction (HPV) (28, 44). Superoxide (), a short-lived ROS formed by the univalent reduction of molecular O2, also influences vascular tone. causes vasoconstriction in part by reacting rapidly with NO·, which generates peroxynitrite (OONO–) and inactivates NO· as a functional endothelium-derived relaxing factor (6, 13, 21). Vasoconstriction in severe hypoxia is partially mediated by decreased NOS activity, and decreased NO bioavailability may be exacerbated by increased production of in hypoxia (12, 20, 37, 44). is particularly important in the extracellular compartment and vascular tissues of the lung where a specialized extracellular antioxidant enzyme, extracellular superoxide dismutase (EC-SOD) that inactivates extracellular , is abundant. EC-SOD contributes to low pulmonary vascular tone under physiological conditions by scavenging extracellular and increasing NO bioactivity (31).
Because is an anion, it does not readily cross cell membranes, and transport systems are required for it to leave the cell. For example, produced within erythrocyte membrane vesicles reduces cytochrome c in the extracellular medium. The appearance of in the extracellular milieu could be inhibited by 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS), a potent and relatively specific inhibitor of 
exchanger proteins (anion exchange proteins or AE). Therefore, transport through this anion channel was proposed (23).
AE proteins are found in many tissues where they function as exchangers (1, 18). AE1 is found in erythrocytes, and three tissue isoforms have been characterized (1, 18, 38). In the lung, AE2 is the predominant isoform (8, 19, 27, 30, 42), and inhibition of 
AE activity blocks release of extracellular in the lung (30). In this study, we tested the hypothesis that release of contributes to the NO·-mediated regulation of pulmonary vascular tone. We performed experiments in the rat to examine the role of 
release on NO·-mediated pulmonary vasodilation and hypoxic vasoconstriction in pulmonary artery (PA) rings and correlated the expression of AE2 in PAs of rats exposed to hypoxia with increases in release and decreased NO-mediated vasodilation. We report the novel finding that release of by AE2 opposes NO-mediated pulmonary vasodilation under normoxic and hypoxic conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Isolated PA segments weighing 7–15 mg were obtained after the chests of anesthetized rats were opened and exsanguination of the animals. The left and right arteries were dissected quickly from the main PA and placed in physiological buffer (see buffers below). Rings (
2 mm width) were obtained from first branch PAs for use in PA ring bioassays.
Composition of buffers. 
Krebs buffer (K-B) contained (in mM) 82.8 NaCl, 4.7 KCl, 2.4 KH2PO4, 1.2 MgSO4, 2.7 CaCl2, 11.1 dextrose, and 25 NaHCO3 at pH 7.4. 
Krebs buffer (K-H) replaced NaHCO3 with 6 mM NaHEPES. NaCl was increased to 107 mM to maintain osmolarity, and pH was adjusted to 7.4 before use.
Detection of extracellular 
. Two methods were used to detect extracellular in isolated rat lungs and PA segments: 1) reduction of nitroblue tetrazolium (NBT) in perfused lungs, and 2) SOD-inhibitable reduction of cytochrome c (Cyt c), which is highly specific for in isolated PA segments (2, 30). Isolated rat lungs were inflated with air and perfused at 10 ml/min with 60 ml or K-B with NBT (5 mg, n = 2) at room temperature. NBT, a large divalent cation that does not cross cell membranes, detected the vascular appearance by a strong blue color developing in the lungs over 30 min. The lungs were inflation fixed at 20 mmHg with 4% paraformaldehyde, and lobes were photographed.
The PA segments were incubated in 2.4 ml buffer containing 50 µM Cyt c, a concentration that remained stable in the buffer because it does not enter the cell in significant amounts. Samples of 400 µl were removed at 5, 10, 15, and 20 min for differential spectrophotometry. Reduction of Cyt c was expressed per 10 mg wet weight of tissue as the change in absorbance at 550–540 nm relative to buffer-containing reference solution [50 µM, Cyt c (90% oxidized)]. The 550-nm wavelength measures the peak of the reduced Cyt c heme, whereas the 540-nm wavelength does not change between reduced and oxidized forms and serves as a reference for light scattering. This method was chosen because it is one of the few that reliably detects extracellular superoxide release almost exclusively. SOD inhibition was determined by using Cu,Zn SOD (500 units, Sigma Chemical). Reduction and oxidation of Cyt c were tested under a wide range of experimental conditions in vessels of control rats and rats exposed to 3 days of hypobaric hypoxia (n = 4–6 per group). Control experiments were performed with all experimental agents in vitro to exclude independent effects on Cyt c reduction. The AE2 inhibitor DIDS interfered with the Cyt c assay in vitro, most likely because its strong yellow color interfered with the optical readings. Therefore, Cyt c reduction was measured in the presence of another stilbene AE2 inhibitor SITS, which demonstrated much less interference with the Cyt c signal. All studies involving Cyt c were conducted at the same PO2 (150 mmHg).
PA ring bioassay. Rat PA rings were mounted in 15-ml tissue baths filled with K-B buffer and bubbled with 21% O2-5% CO2-balance N2 at 37°C. Isometric tension was measured. All rings were suspended at similar baseline levels of tension (1 g). Tissue baths were thoroughly rinsed with fresh buffer between interventions. In all rings, a dose-response curve to phenylephrine (PE) was performed to test vascular responsiveness (10–8–10–6 M) followed by acetylcholine (ACh) (10–6 M) to confirm endothelial integrity.
NO-mediated vasodilation was tested with a dose response to ACh (10–8–10–6 M) in rings maximally preconstricted with PE (10–6 M). The response to ACh was compared in rings bathed in K-B buffer or K-H buffer and expressed as the percentage of maximal ring tension with 10–6 M PE for each ring. The response to ACh was also measured in the rings of animals exposed to hypobaric hypoxia for 3 days as outlined below. The 3-day time point was based on data demonstrating consistent increases in pulmonary vascular AE2 expression and minimal pulmonary vascular remodeling.
Acute HPV was produced in rings preconstricted with PE at half-maximal constriction based on the initial dose-response curve. The selected PE dose was between 10–8 and 5 x 10–8 M in each experiment. Hypoxia was created by changing the gas mixture bubbled into the bath to 5% CO2-balance N2. The surface of the chamber was covered tightly with a plastic sheet (Parafilm) to minimize exposure to room air, and a second source of anoxic gas mixture was delivered to the air space above the buffer. Buffer samples (5 ml) collected after 30 min in gas-tight glass syringes were measured on a calibrated blood gas analyzer (IL model 1640) to ensure hypoxic conditions (PO2 20–25 mmHg). Rings exhibiting the well-described triphasic response to hypoxia were tested (4, 35). The response to hypoxia in individual rings was repeated with both K-B buffer and K-H buffer with half of the rings tested initially with each buffer. The data were expressed as the change in ring tension in grams from the baseline preconstricted tension for the initial constriction within seconds (phase I), subsequent vasodilation toward baseline tension (phase II), and late constriction 25 min after initiation of hypoxia (phase III).
Western analysis, RT-PCR, and immunohistochemistry (IHC). For Western blot analysis, lung tissue was homogenized in 3% Nonidet-40, 150 mM NaCl, 1 mM MgCl2, 5 mM EDTA, and 50 mM Tris (pH 7.6). Lysis buffer included protease inhibitors (at 1:20): 2 mM 1,10 phenanthroline, 2 mM 3,4 diisocoumarin, and 0.4 mM E-64. Protein samples were separated by electrophoresis on a 7.5% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore). Membranes were probed with a primary polyclonal AE2 peptide antibody (1:1,000) and secondary goat anti-mouse antibody conjugated to horseradish peroxidase with detection by enhanced chemiluminescence (Amersham Pharmacia Biotech). Total RNA was isolated using a TRIzol kit, and RT-PCR was conducted on the rat lung and PA in control and exhypoxic conditions using gene-specific primers for AE2 and GAPDH based on the GenBank DNA database. Optical densities for AE2 mRNA bands were normalized against those of the GAPDH bands. Rat AE2 primers were the following: AE2 sense 5'-TGTAGCAGCAACCACCTGGAGT-3' and AE2 Antisense 5'-GCAGGAAGAAGGCGATGAAGAA-3' (39). For immunohistochemistry, lungs were inflation fixed with 4% paraformaldehyde at 20 cmH2O pressure and paraffin embedded. Tissue sections were blocked with normal goat serum in 1% bovine albumin in PBS. Sections were then incubated with the primary polyclonal AE2 antibody (1:100 in 1% BSA/PBS) overnight at 4°C followed by a biotinylated goat anti-mouse IgG antibody (1: 1,000) and labeled with horseradish peroxidase-conjugated streptavadin (Innogenex). Slides were developed with 3,3'-diaminobenzidine and counterstained with hematoxylin. Negative controls were performed with normal mouse serum and antibody coincubated with AE2 peptide. Tissue sections were examined by light microscopy and photographed at x132.
The primary AE2 antibody was developed in mouse ascites fluid against the rat AE2 COOH-terminal amino acids 1224–1237 (CEGVDEYNEMPMPV-COOH) (29, 47). Peptide sequences were synthesized, purified, and confirmed by HPLC (Bio Synthesis). The AE2 antibody was affinity purified over a protein S sepharose column and characterized using immunoprecipitated AE2 protein Protein G IP kit (K&L Labs, Gaithersburg, MD) to confirm the identity of the single 95-kDa band and peptide competition (1:10) to inhibit the signal.
Statistical analysis. Grouped data are expressed as means ± SE as indicated. Comparisons were made by analysis of variance followed by Fisher's least-square protected difference test using Statview software (SAS Institute; Cary, NC). P values are provided where statistical tests were performed.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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in the buffer was required for the extracellular release of . The extracellular reduced NBT, indicated by a strong visible blue color in lungs infused with K-B buffer (Fig. 1A, left). In contrast, when lungs were infused with K-H buffer, NBT was not reduced, indicating that extracellular was not present (Fig. 1A, right). Similarly, release from PA segments in K-B buffer, measured by SOD-inhibited reduction of Cyt c (50 µM), increased over time. In K-H buffer, a small amount of reduced Cyt c originally present in the mixture was oxidized, shown by the negative signal (n = 18) (Fig. 1B). Oxidant release by PA segments in K-H buffer was found to involve H2O2 because oxidation of Cyt c was eliminated by active but not inactive catalase (500 units) (n = 4, P < 0.05) (Fig. 1B).
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In the PA ring bioassay, the presence of strongly influenced the responses to both NO·-mediated pulmonary vasodilation by ACh and acute HPV. The dose-response curve to ACh (10–8–10–5 M) produced a characteristic decrease in ring tension in K-B buffer expressed as percent maximum constriction. This is well characterized as NO dependent. The decrease in ring tension with ACh was significantly pronounced when isoosmotic K-H buffer (pH 7.3) was substituted for buffer (K-B) (n = 4–6, P < 0.05) (Fig. 2A), suggesting that release is opposing NO bioactivity. Conversely, with acute hypoxia (5% CO2-balance N2), initial and late vasoconstriction (phase I and III) were greatly attenuated K-H buffer (n = 3, P < 0.05) (Fig. 2B), whereas vasodilation in phase II was not affected. A representative tracing (Fig. 2C) shows the blunted immediate response of PA rings to hypoxia in K-H buffer in phase I. These results confirmed the importance of extracellular for release as well as NO-dependent vasodilation by ACh and acute HPV. Because removal of inhibits function of AE proteins, further experiments were designed to test the potential role of AE in release.
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To evaluate the role of AE2 in release from pulmonary vessels more specifically, reduction of Cyt c was analyzed in rings pretreated with SITS, a specific AE inhibitor. SITS (100 µM) abolished reduction of Cyt c by vessels in K-B buffer (Fig. 3A). The requirement for intact vascular endothelium was studied in rubbed vessels. After the endothelium was removed, the reduction of Cyt c by was virtually eliminated, implicating endothelial NOS (eNOS) as a source of (Fig. 3B). eNOS is a known source of under certain conditions associated with uncoupling of enzyme oxygenase and reductase activities. In PA segments, both Cyt c reduction and oxidation were prevented by the eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 µM), but not its inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME), further supporting eNOS as the source of (Fig. 4A). Loss of oxidation after L-NAME suggests that originating from eNOS can also be reduced to H2O2 in the PA, e.g., within endothelial cells. In rat PA segments, production by eNOS was due in part to L-arginine limitation because supplemental L-arginine (100 µM) attenuated but did not eliminate Cyt c reduction (Fig. 4B) (n = 4, P < 0.05). Uncoupling eNOS activity by administration of the heat shock protein-90 (HSP-90) inhibitor geldanamycin (10 µM) increased production in K-B threefold, further implicating production by eNOS (Fig. 4B). In PA segments in K-H buffer, geldanamycin also increased release but its effect was nearly fivefold less than in K-B buffer due partly to the lack of baseline leak under these conditions (data not shown).
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The effects of hypoxia on AE2 protein and mRNA expression were evaluated in the rat pulmonary vasculature. The mRNA was present constitutively but did not change significantly after 1, 3, or 7 days of hypoxia (data not shown). However, after 1, 3, or 7 days of hypobaric hypoxia at 17,000 ft. altitude, lung AE2 expression by Western blot analysis was strongly increased (Fig. 5A). By immunochemical staining, AE2 was present in both PA endothelial and vascular smooth muscle cells (Fig. 5B). Specificity was determined using immunoprecipitated AE2 protein to confirm the identity of the single 95-kDa band and peptide competition (1:10) to inhibit the signal (data not shown). We have not yet determined why the protein expression increased, whereas the mRNA did not.
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Reduction of Cyt c was increased significantly in K-B buffer in PA segments of rats treated with 3 days of hypoxia compared with control vessels (Fig. 6A). Increased Cyt c reduction corresponded to diminished NO-mediated vasodilation. In PA rings of hypoxia-exposed rats, ACh vasodilation was attenuated significantly compared with the responses of rings from control rats. To measure the vascular response to ACh, rings were preconstricted with PE (10–6 M), and the decrease in vascular tone with ACh (10–5 M) was expressed relative to the peak constriction with PE (10–6 M) (% maximum constriction). Vasorelaxation to ACh was significantly less in rings from hypoxia-treated rats (41.7 ± 0.04% in rings of control rats vs. 70.3 ± 0.04%, in rings of hypoxia-treated rats; n = 4, P < 0.05) (Fig. 6B). In rings from hypoxia-exposed animals, treatment with SOD increased the response to ACh by twofold, similar to control rings, whereas incubation with catalase did not restore the response to ACh (Fig. 6B). These findings indicate that extracellular in PAs increased following exposure to hypoxia and contributed to decreased NO-mediated vasodilation by ACh.
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In rings from hypoxia-exposed rats studied at the same PO2 as control rings, the reduction of Cyt c in K-B buffer remained dependent. However, Cyt c reduction was no longer inhibited by L-NAME (Fig. 6C). This loss of L-NAME effect indicated that the release was derived not from eNOS but from a different source. In addition, removal of the endothelium by rubbing the vessels no longer eliminated the ability of the vessel to reduce Cyt c (Fig. 7A). This indicated that vascular smooth muscle, not endothelium, was the source of .
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Experiments were conducted to investigate the source of production in the posthypoxic vessels. Pretreatment with oxypurinol (100 µM) to inhibit xanthine oxidase, 1-aminobenzatriazole (20 µM) to inhibit cytochrome P-450, or rotenone (10 µM) to block complex I of mitochondrial electron transport did not inhibit Cyt c reduction, excluding these enzymes as the predominant sources of extracellular in these pulmonary vessels (P > 0.05, n = 3 for each) (Fig. 8, A–C). In addition, Cyt c reduction was not inhibited by any of the three concentrations of diphenyleneidonium chloride (DPI, 5, 25, and 50 µM), which was used to inhibit NADPH oxidase, an important vascular source of (Fig. 8D). The DPI and rotenone studies were repeated after 3 days of hypoxia in rubbed PA segments to facilitate uptake of the compounds by smooth muscle cells (Fig. 7, A–C). Neither of these inhibitors interfered with Cyt c reduction in exhypoxic vessels after removal of the endothelium (P > 0.05, n = 3).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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release of extracellular in regulating NO-mediated pulmonary vascular responses in the rat. We first demonstrated that the appearance of extracellular in both the intact rat lung and rat PA, detected by NBT reduction and SOD-inhibitable Cyt c reduction, requires the presence of . These findings are consistent with our previous reports of release of in isolated perfused rabbit lungs and cultured human bronchial epithelial cells (HBEC) (30) (9). Diminished extracellular release in buffer was not attributable to differences in buffer osmolarity or extracellular pH, which were equivalent in both buffers and stable throughout the experimental period. Decreased NBT or Cyt c reduction in buffer was not likely due to changes in intracellular pH because pH is predominantly maintained by intracellular proteins, whose ionization should not be greatly altered by extracellular buffer substitution experiments (11). Small changes in Na+ concentration in the two buffers could have a small impact in the function of other cation transport proteins but would not likely explain our results because this would not account for differences in extracellular release in the presence of stilbene inhibitors.
In the intact lung, the strong qualitative decrease in measured by NBT reduction was not attributable to the difference in buffer composition because potassium superoxide placed on the surface of the lung reduces NBT in HEPES buffer (30). In PA segments, the small amount of reduced Cyt c present in the preparation was instead oxidized by H2O2 in buffer because H2O2 formed within cells by dismutation of diffuses freely across plasma membranes. Therefore, the reaction of H2O2 with Cyt c outside the vessel was not eliminated by the prevention of transport.
influenced the vascular response in isolated PA rings to both NO-mediated pulmonary vasodilation with ACh and acute HPV. In conditions, vasorelaxation by ACh, which is mediated by release of endothelium-derived NO, was enhanced in accordance with increased NO bioactivity that occurs when less extracellular is available to react with NO. conditions also blunted acute hypoxic vasoconstriction, which could also be mediated by increased NO bioactivity. The mechanisms of acute HPV are complex and incompletely understood, although there is substantial evidence that NO influences vascular tone in hypoxia. For example, severe hypoxia limits NOS activity (12, 20), and NOS inhibition accentuates HPV (44). Vasoconstriction in severe hypoxia may be further exacerbated by increased production of under hypoxic conditions (15).
The decreased vascular tone in conditions correlated with decreased extracellular , which supports the hypothesis that generation modulates NO-mediated pulmonary vasodilation. These findings are consistent with published work on hyperoxia in which buffer substitution and AE protein inhibitors protected isolated perfused rabbit lungs against pulmonary hypertension, decreased extracellular , and increased NO (30). The simplest interpretation of these findings is that at the cell membrane exchanges rather than chemically competes for with the near-diffusion limited reaction of with NO. Although alkalosis and acidosis are well known to affect tone in the PA, this is the first evidence that directly regulates vascular tone at physiological pH and normal CO2 tension (25, 34, 40, 46).
A large body of observations by our group and others strongly implicates the lung AE protein in the transport of to the extracellular compartment. The removal of from buffers or media and incubations with stilbene inhibitors have been useful to block 
exchange activity by AE (22, 24, 30). In this study, Cyt c reduction by the PA was decreased by substitution with buffer and after pretreatment with the AE inhibitor SITS. This requirement in the rat is consistent with our data in perfused rabbit lungs in which buffers and the related AE inhibitor DIDS prevented both Cyt c reduction and NBT reduction (30). Also in cultured HBEC, loss of AE2 function in media or SITS prevented generation of extracellular (9). Studies in erythrocytes, the brain, and the lung have used AE inhibitors to block transport of across cell membranes, but these are the first studies to link AE2 and vascular transport (10, 14, 17, 23, 30).
SOD-inhibitable Cyt c reduction in oxidation in and its buffers were eliminated in rubbed PA and by L-NAME, identifying eNOS as the predominant source of extracellular . eNOS generates under restriction of tetrahydrobiopterin (BH4) or L-arginine (41, 45); (16, 36). In this study, L-arginine availability played a role ex vivo in generation because supplementation with L-arginine partially attenuated release. BH4 was not evaluated specifically, but it should be available in fresh tissue. Release of by eNOS also involves HSP-90, which if uncoupled from eNOS activity increases production by eNOS (33). The specific HSP-90 inhibitor geldanamycin significantly increased release, further implicating eNOS-containing cells as the source. Geldanamycin can participate in redox reactions to generate independently (7), but release by the PA after geldanamycin was greatly attenuated in buffer.
In this study, continuous hypoxia altered the source of generation in the rat PA; eNOS no longer was the primary source of extracellular . In exhypoxic vessels, neither L-NAME nor removal of endothelium blocked Cyt c reduction, indicating that another site, presumably in the vascular smooth muscle cell, was producing the bulk of the extracellular . However, other well-known sources of could not be implicated in the generation of extracellular , including the mitochondrial electron transport chain, NAD(P)H oxidase, xanthine oxidase, cyclooxygenase, or P450 enzymes. NADPH oxidase and mitochondrial electron transport have been identified as important sources of in some hypoxia experiments (6, 26, 43), but generally these studies have not distinguished intracellular from extracellular . In any event, increased extracellular correlated with decreased NO-mediated vasodilation, and the source of extracellular in PAs after exposure to prolonged hypoxia still requires elucidation. The increase in in hypoxia was associated with a decreased vasodilator response to ACh. These measures were performed in normoxia after prolonged hypoxia and, therefore, may reflect smooth muscle alterations of hypoxia and/or responses to reoxygenation.
The increase in release of in the PA after alveolar hypoxia suggests that hypoxia, which is associated with ROS, might increase AE2 protein expression. We found that hypoxia increases AE2 protein expression in PA smooth muscle, which suggests AE2 is important not only as previously believed to regulate intracellular pH and cell volume (24), but to regulate transport in hypoxia. The increase in AE2 expression in vascular smooth muscle correlated with endothelium-independent increases in released (in rubbed vessels). Computer analysis of the AE2 promoter sequence indicates consensus sequences for transcription factors such as AP-1 and HSF, which may provide mechanisms for its regulation by oxygen tension (39) (Transcription Factor Search). The novel function for AE2 in the transport of 
in the rat PA and a report that NO export by red blood cells is mediated by AE1 suggests a general role for AE proteins in vasoregulation by NO and (32).
In summary, we report a novel mechanism by which generated by eNOS is released from the PA endothelium in the presence of to regulate pulmonary vascular tone. The efflux appears to be mediated by an AE2 transport mechanism. Such a transport mechanism may also facilitate during hypoxia from smooth muscle sources when intracellular production is increased. The findings also suggest a new avenue of research into 
mechanisms of vasoconstriction, which may lead to novel therapies for diseases associated with alveolar hypoxia.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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exchanger isoforms in proximal and distal human airways. Am J Physiol Lung Cell Mol Physiol 276: L971–L978, 1999. transporter superfamily is an apical anion exchanger of beta-intercalated cells in the kidney. J Biol Chem 276: 8180–8189, 2001.
exchanger are encoded by mRNAs transcribed from alternative promoters. J Biol Chem 271: 7835–7843, 1996.This article has been cited by other articles:
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P < 0.05 vs. K-H).
