Third Department of Internal Medicine, Fukui Medical University, Fukui 910-1193, Japan
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ABSTRACT |
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To investigate the effect of extracellular pH on endothelial nitric oxide synthase (eNOS) in human pulmonary arteries, we measured eNOS activity and expression as well as some ion channels in human pulmonary arterial endothelial cells (HPAEC) exposed to various pH levels (6.6-8.0). eNOS activity was found to increase with alkalization and decrease with acidification, while Ca2+ uptake into HPAEC increased with alkalization. The addition of 3',4'-dichlorobenzamil hydrochloride, an inhibitor of the Na+/Ca2+ exchanger (NCX), prevented the increase of eNOS activity with alkalosis. Exposure to alkalosis and acidosis increased eNOS and NCX mRNA levels. These results suggest that an elevation of extracellular pH activates eNOS via the influx of extracellular Ca2+ and that NCX also regulates eNOS activity during alkalosis. Furthermore, NCX may have a tight interaction with eNOS at the level of transcription and might affect pulmonary circulation during alkalosis and acidosis.
calcium influx; sodium calcium exchanger
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INTRODUCTION |
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NITRIC OXIDE SYNTHASE (NOS) is an enzyme found in the pulmonary vascular endothelium that is important for maintaining low pulmonary vascular tone via production of nitric oxide (NO) through its utilization of L-arginine, NADP, calmodulin, tetrahydrobiopterin (BH4), and Ca2+ (8, 11, 16, 17, 30). It has been shown that hypoxic pulmonary vasoconstriction is strengthened by acidosis and attenuated by alkalosis in a very short period (1, 18) and that alkalosis evokes low pulmonary arterial pressure in children with pulmonary hypertensive disorders (2, 20) and causes acute pulmonary vasodilation in animals (9, 12, 24). Inversely, acidosis elevates pulmonary vascular tone. Recent studies have shown that changes in pH affect the production of endothelium-derived relaxing factor (4, 13, 19). Mechanisms by which acidosis and alkalosis affect pulmonary vascular tone have been suggested to be involved with stimulation of NO release via Ca2+ influx into the endothelium by extracellular alkalosis (5, 28, 29), as well as a close relationship between Ca2+ uptake into endothelial cells and activation of the Na+/Ca2+ exchanger (NCX). Additionally, intracellular alkalization is also related to the activation of eNOS in association with Ca2+ influx, which is suppressed by inhibition of the Na+/H+ exchanger (NHE) (10, 23, 25). However, the molecular events in vasodilation due to alkalosis in human pulmonary arteries are incompletely understood and those due to acidosis have rarely been studied.
The purpose of the present study was to investigate the modulatory effects of pH on eNOS and NCX activity and gene and protein expression, by focusing on Ca2+ and Na+ and using cultured human pulmonary artery endothelial cells (HPAEC).
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MATERIALS AND METHODS |
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Chemicals. Humedia EG medium, recombinant human endothelial growth factor (EGF), gentamicin, streptomycin, hydrocortisone, and amphotericin B were obtained from Kurabo (Osaka, Japan). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) and 3',4'-dichlorobenzamil hydrochloride (DCB) were obtained from Molecular Probes (Eugene, OR). L-[14C]arginine, [45Ca2+], and an enhanced chemiluminescence (ECL) system were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Moloney murine leukemia virus reverse transcriptase (RT) was obtained from Toyobo (Osaka, Japan). Taq DNA polymerase was obtained from Promega (Madison, WI), whereas 4-12% Bis-Tris Nupage gels and MES-SDS running buffer were obtained from Invitrogen (Carlsbad, CA). A detergent-compatible (DC) protein assay kit and polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad Laboratories (Richmond, CA). Rabbit anti-eNOS monoclonal antibody was obtained from Cayman Chemical. A mouse anti-NCX was obtained from Affinity Bioreagents, and horseradish peroxidase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology. All other chemicals were obtained from Sigma.
Cell culture. HPAEC from an established cell line (Endocell-PA) were obtained from Kurabo and grown in Humedia EG medium containing 2% fetal bovine serum, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, 10 ng/ml recombinant human epidermal growth factor, and 1 mg/ml hydrocortisone. Cells were cultured in 75-cm2 tissue culture flasks (Corning) and used upon reaching confluence at the fifth to tenth passage after trypsinization.
Cell-free assay (in crude NOS extract). NOS activity was measured by the ability of HPAEC, after homogenization, to convert L-[14C]arginine to L-[14C]citrulline, the coproduct of NO formation, as previously described (6). HPAEC were homogenized on ice in 500 µl of homogenate buffer (50 mM HEPES-NaOH) containing 150 nM L-[14C]arginine, 1 mM dithiothreitol, 1 mM CaCl2, 10 µM BH4, 1 mM NADP, 10 µM FAD, and 10 µg/ml calmodulin, pH adjusted from 5.5 to 9.0 by the addition of NaOH or HCl. Aliquots of these cell homogenates were then incubated for 120 min at 37°C. After incubation, all reactions were stopped by the addition of an ice-cold stop solution (100 mM HEPES, pH 5.1, containing 10 mM EDTA). The newly formed L-citrulline was separated from L-arginine by passing it over AG50W-X8 columns. The eluted material was then measured with a liquid scintillation counter (Aloca LSC-5300, Tokyo, Japan).
Whole cell assay. HPAEC were suspended in Humedia EG medium at a cell density of 2 × 105/ml. Next, 2 ml of the cell suspension was added to each well of a six-well flat-bottom culture plate (Corning). After 24 h, all cells were adherent to the bottom, having reached confluence. Cells were kept in Krebs-Henseleit buffer for 4 h before the assay. A conversion assay using whole intact cells was done by the method introduced by Demura et al. (6) with some modifications. Briefly, the Krebs-Henseleit buffer was exchanged with either Krebs-HEPES buffer (composition in mM: 130 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 1.5 CaCl2, 11.5 glucose, and 10 HEPES, adjusted to pH 7.4 by the addition of NaOH) or with Ca2+-free Krebs-HEPES buffer. Then, L-[14C]arginine was added at a concentration of 0.5 µCi/ml as indicated. The cells were incubated in a CO2-free incubator at 37°C for 3 h, after which, NaOH, HCl, or 10 µM monensin was added to the medium. In some experiments, 30 µM DCB was added to the wells, and incubation was performed for 15 min before NaOH, HCl, or monensin was added. After 30 min, the reaction was terminated by the addition of 0.6 N perchloric acid. The acid-soluble extracts were neutralized with 120 µl of 5 M K2CO3. The neutralized cell extract and culture medium were collected and applied to an AG50W-X8 column and then measured for radioactivity in the same manner described for the cell-free assay.
For adjustment of pH, although use of CO2-HCOCalcium uptake into HPAEC. [45Ca2+] uptake measurements were performed by the method of Demura et al. (7). HPAEC were cultured in a 12-well flat-bottom culture plate until reaching confluence. The adherent cells were washed twice with prewarmed PBS, then placed in Krebs-HEPES buffer medium (with the pH adjusted from 6.6 to 8.0 by the addition of NaOH) containing 1.5 µCi [45Ca2+] and incubated at 37°C in a CO2-free incubator. After 30 min, the incubation was terminated by aspiration of the [45Ca2+] uptake solution, and each well was washed three times with 1 ml of ice-cold PBS containing 3 mM LaCl3. The cells were solubilized with 500 µl of 0.2% SDS, and an aliquot of the SDS lysate was measured for [45Ca2+] radioactivity using a liquid scintillation counter (Aloca LSC-5300).
The protein concentration of the cells was determined per well by the Bradford method. Ca2+ uptake into the cells was calculated as follows: Ca2+ uptake = [45Ca2+]i × [Ca2+]o/[45Ca2+]o, where [45Ca2+]i is the radioactivity of 45Ca2+ retained in the cells, [45Ca2+]o is the radioactivity in the incubation solution, and [Ca2+]o is the amount of nonradioactive Ca2+ in the incubation solution (1.5 mM).Measurement of intracellular pH in HPAEC. HPAEC were cultured in a 12-well flat-bottom culture plate until reaching confluence. The adherent cells were washed twice with prewarmed PBS and then placed in Krebs-HEPES buffer medium containing 10 µM BCECF-AM, with the pH adjusted from 6.6 to 8.0 by the addition of NaOH. The cells were then incubated at 37°C for 30 min in a fluorescence microplate reader (Fluoroscan; Dainippon Pharmaceutical, Osaka, Japan). The fluorescent intensity of the cells (excitation 485 nm and emission 538 nm) was measured, and intracellular pH (pHi) calibration was done by a high K+-nigericin method, as previously described (26). Calibration of the pHi measurements was carried out in 8 µM nigericin containing Na+-free, high-K+, and HEPES-buffered solutions containing (in mM): 140 KCl, 1.0 MgCl2, 1.5 CaCl2, 10 glucose, and 10 HEPES at various pH values, with the pH adjusted from 6.8 to 7.8 by the addition of KOH. The fluorescence intensity was analyzed by linear regression analysis, and the derived equation was used to calculate the pHi values.
RT-PCR. HPAEC were cultured in a 24-well flat-bottom culture plate until reaching confluence. The adherent cells were washed twice with prewarmed PBS and then placed in Krebs-HEPES buffer medium (pH adjusted from 6.6 to 8.0) and subsequently incubated in a CO2-free incubator at 37°C for 90 min. After incubation, cells were harvested by trypsinization, washed three times, and pelleted by centrifugation. Total cellular RNA was obtained from the cells by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture (3). RT was done using 5 µg of total RNA. Briefly, cDNA synthesis was done with 200 units Moloney murine leukemia virus RT, 5 µM oligo(DT), 1 mM dNTPs, and 3 mM Mg2+ in a volume of 20 µl. The temperature profile consisted of annealing at room temperature for 5 min, extension at 44°C for 40 min, and termination at 99°C for 5 min.
PCR was done on the resulting RT product using specific oligonucleotide primers for eNOS and NCX, which we designed using the computer software Primer 3. The sequence of the forward primer for eNOS was 5'-gaccctgtgccctgcttcat-3' and that of the reverse primer was 5'-tacttcgtcccgtgtcccag-3'. The sequence of the forward primer for NCX was 5'-gaaaatggagagaagcgcac-3' and that of the reverse primer was 5'-aaatacaagggacagcacgg-3'. The PCR reactions contained 1 µM primers, 200 µM dNTPs, 1.5 mM Mg2+, 1 unit Taq DNA polymerase, 2 µl cDNA, and reaction buffer in a final volume of 20 µl. PCR reaction tubes were placed in a thermal cycler (model 9600; Perkin-Elmer, Norwalk, CT) and maintained at 95°C for 2 min. The PCR temperature profile consisted of 35 cycles of 95°C for 30 s, 58°C for 20 s, and 72°C for 1 min, followed by a final extension for an additional 5 min at 72°C. The PCR products were visualized by electrophoresis on 1% agarose gels with ethidium bromide staining. The densities of the PCR product bands for eNOS, NCX, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were measured using an IS-4000 (Alpha-Inotec, Osaka, Japan) chemiluminescence imaging analyzer and the computer software Phoretix ID Advanced Version 4.01 (NonLinear Dynamics). The expression level for each target gene was presented as the ratio of the densities of the eNOS and NCX gene bands vs. that of the GAPDH gene band. Each test was performed in triplicate in three independent experiments.Western blot analysis. HPAEC were cultured in a 6-cm dish until reaching confluence. The adherent cells were washed twice with prewarmed PBS and then placed in Krebs-HEPES buffer medium with the pH adjusted from 6.6 to 8.0 and then incubated in a CO2-free incubator at 37°C for 180 min. After incubation, the cells were harvested in ice-cold PBS, pelleted, resuspended in protein lysis buffer (40 mM HEPES, 1% Triton X, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride, pH adjusted to 7.4), and incubated for 30 min on ice. After incubation, cell lysis buffers were centrifuged at 10 000 g for 15 min at 4°C to remove cell fragments, and the supernatants were analyzed for protein content using a DC protein assay kit.
Each sample was quantified, and then 50 µg of protein were loaded on each lane of a 4-12% Bis-Tris Nupage gel with MES-SDS running buffer, according to the manufacturer's protocol. The gel was transferred to a PVDF membrane by electrophoresis at 100 V for 1 h. The membrane was then blocked in PBS, 0.2% Tween 20 (PBS-T), and 5% nonfat milk at room temperature for 1 h. All antibodies were diluted in the same blocking buffer. For Western blot analysis of eNOS, a 1:2,000 dilution of rabbit anti-eNOS monoclonal antibody as the primary antibody was incubated for 1 h at room temperature. For Western blot analysis of NCX, a 1:1,000 dilution of mouse anti-NCX antibody as the primary antibody was incubated for 24 h at 4°C. After incubation, the membrane was washed with PBS-T and then incubated with horseradish peroxidase-conjugated goat anti-rabbit (eNOS) or mouse (NCX) IgG (1:2,000 dilution) for 2 h at room temperature. After washing with PBS-T, the ECL system was used for detection of the protein. After the antibody was removed from the membrane according to the manufacturer's stripping protocol, theStatistical analysis. Results are expressed as means ± SE. Statistical analysis was performed with the use of one-way analysis of variance with Bonferroni's test for multiple comparisons. Comparisons were considered statistically significant at a level of P < 0.05.
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RESULTS |
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Effect of extracellular pH and Ca2+
on L-citrulline synthesis.
The optimal extracellular pH of L-citrulline synthesis from
the crude extract was 7.4 (Fig. 1).
Synthesis activity decreased sharply with acidification and decreased
slightly with rapid alkalization (from pH 7.4 to 8.0). In the whole
cell assay (intact cells), alkalization (up to pH 8.0) strengthened
eNOS activity (Fig. 2A). In
the absence of extracellular Ca2+, L-citrulline
synthesis was decreased by two-thirds at a pH of 7.4 and decreased
further with both acidification and alkalization without changing cell
viability (Fig. 2B). When Ca2+ uptake by HPAEC
was measured, it increased dose dependently according to the elevation
of extracellular pH from 6.6 to 8.0. (Fig.
3).
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Effect of NCX inhibitor on L-citrulline synthesis.
Monensin, which is known as an Na+ ionophore, dose
dependently increased L-citrulline synthesis in the
presence, but not in the absence, of extracellular Ca2+
(Fig. 5).
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Effect of pH on the expression of eNOS and NCX.
Alkalosis and acidosis caused increases of eNOS and NCX mRNA but had no
effect on GAPDH mRNA (Fig. 8).
This increase of mRNA resulted in a slight increase of eNOS and NCX
protein (Fig. 9).
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DISCUSSION |
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Our data obtained by whole cell assay demonstrate that alkalosis increased eNOS gene and protein expression and activity via increased Ca2+ uptake, whereas acidosis suppressed eNOS activity with decreased Ca2+ influx despite increased expression of eNOS mRNA and protein. The result of the pH-dependent changes in cell-free eNOS activity was in agreement with previous a report by Fleming et al. (10), in which methods using human umbilical vein endothelial cells and porcine aortic endothelial cells were used.
If one considers the results of the cell-free (crude) NOS assay, in which a change of pH did not directly upregulate NOS activity but rather suppressed it, it appears that an intact cell membrane and extracellular Ca2+ are required for alkalization to activate the eNOS pathway.
Our results not only confirm that alkalosis activates eNOS (5, 13, 14, 28) but also demonstrate that alkalosis and acidosis increase expression of eNOS mRNA and protein. Interestingly, the nuclear effect of pH on eNOS was not in line with L-citrulline production with acidification, although it fits in the case of alkalization. Although this result contrasts with a previous study from Yamaguchi et al. (31) of isolated perfused rabbit lungs, in which hypercapnic acidosis elevated vascular tone and perfusate nitrite/nitrate, it would appear that acidification can stimulate another unidentified subcellular molecular mechanism in the pretranscriptional phase of eNOS. A recent study relevantly reports that acidosis induced by HCl or hypercapnia increased eNOS mRNA in pig and rat brain vessels (21), although the precise mechanism was not elucidated.
Our second important observation is that alkalosis and acidosis upregulated expression of NCX mRNA and protein. The reasons we used an NCX inhibitor are that NCX, which locates caveolae, may have a tight interaction with eNOS (25) and that addition of monensin, known as a sodium ionophore, evoked resting intracellular Ca2+ concentration ([Ca2+]i) in bovine pulmonary arteries via the NCX (23). Moreover, Fleming et al. (10) reported that intracellular alkalinization induced by bradykinin, known as an activator of NHE, resulted in an increase of [Ca2+]i in porcine aortic endothelial cells and human umbilical endothelial cells. One can thus suspect that activation of NHE may result in an increase of intracellular Na+ and activate Ca2+ influx via the NCX. The result that DCB, a selective inhibitor of NCX, suppressed eNOS activation by alkalization suggests that NCX plays a substantial role during alkalization. However, it is unclear at this time why pH changes upregulate mRNA of NCX and why alkalization increases the protein expression of NCX. Nevertheless, our result indicates that NCX may be more linked with eNOS during alkalization.
Besides the NO story for the vasodilatory mechanism of alkalization, it has been demonstrated that alkalosis enhances prostacyclin synthesis (15, 22, 32). However, we could not detect augmentation of cyclooxygenase-1 mRNA and protein expression during alkalosis in our assay methods (unpublished observation). In this context, Vander Heyden et al. (27) report that the inhibition of either eNOS or prostacyclin alone did not suppress vasodilation in perfused piglet lungs and that combined eNOS and prostacyclin inhibition blunted the response by ~40%. Full blockade needed the addition of tetraethylammonium, suggesting that KCa channels are also involved. Thus it appears that multiple factors are interacting in the regulation of vasodilation during alkalosis.
In summary, the present findings document novel subcellular mechanisms of vasodilation induced by alkalization in HPAEC, in which NCX collaborates for a sustained activation of eNOS. Although our results were obtained from the experiment in which we used a broad extracellular pH range, we believe the result is of importance for understanding NO formation during pathophysiological states such as inflammation, ischemia, and hypoxia.
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ACKNOWLEDGEMENTS |
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This work was supported by grants-in-aid for scientific research (nos. 06670605 and 0744457145) from the Ministry of Education, Science, and Culture, Japan.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Mizuno, Dept. of Internal Medicine, Fukui Medical Univ., 23-3 Matsuoka-cho Yoshida-gun, Fukui 910-1193, Japan.
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.
First published February 22, 2002;10.1152/ajplung.00436.2001
Received 8 November 2001; accepted in final form 14 February 2002.
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