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(Circulation Research. 1995;76:825-831.)
© 1995 American Heart Association, Inc.

Articles

Na⁺-H⁺ Exchanger Isoform 1 Phosphorylation in Normal Wistar-Kyoto andSpontaneously Hypertensive Rats

Martin Siczkowski, Joan E. Davies, Leong L. Ng

From the Division of Clinical Pharmacology, Department of Medicine and Therapeutics, Leicester Royal Infirmary, United Kingdom.

Correspondence to Dr Martin Siczkowski, Division of Clinical Pharmacology, Department of Medicine and Therapeutics, Clinical Sciences Bldg, Leicester Royal Infirmary, Leicester, LE2 7LX, UK.

	Abstract

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Abstract Increased activity of the cellular Na⁺-H⁺ exchanger (NHE) has been documented in various cell types in essential hypertension and in vascular myocytes of the spontaneously hypertensive rat (SHR). The mechanism underlying this abnormality is unclear. Because the NHE can be activated by phosphorylation, we examined phosphorylation of the Na⁺-H⁺ exchanger isoform 1 (NHE-1) as one possible mechanism for its increased turnover number in cultured vascular myocytes of the SHR. A polyclonal rabbit antibody against a fusion protein consisting of ß-galactosidase and the C-terminus of NHE-1 was used to immunoprecipitate ³²P-labeled NHE-1 from cell extracts of SHR and Wistar-Kyoto (WKY) rat vascular myocytes in the absence and presence of 10% fetal calf serum. Immunoprecipitates were separated by SDS-PAGE, and ³²P-labeled NHE-1 was quantified from autoradiographs. Similar amounts of NHE-1 protein were detected on Western blots of the cultured vascular myocytes from SHR and WKY rats. In quiescent cells, NHE-1 was significantly more phosphorylated in SHR myocytes than in WKY myocytes (2.17±0.06-fold enhancement [mean±SEM]; P<.001, n=8). The addition of fetal calf serum to quiescent cells had no significant effect on the phosphorylation of NHE-1 in SHR myocytes. However, NHE-1 phosphorylation fell transiently in serum-treated WKY myocytes, with recovery to control levels after 20 minutes. Measurement of NHE activity using fluorometry confirmed elevated activity in the quiescent SHR myocytes compared with WKY myocytes. Fetal calf serum led to further enhancement of NHE activity in both cell types. These findings suggest that the increased NHE activity in quiescent SHR myocytes may be correlated with enhanced NHE-1 phosphorylation and that serum stimulates NHE activity in both cell types without a further increase in total NHE-1 phosphorylation, indicating a role for non–phosphorylation-dependent regulatory mechanisms.

Key Words: Na⁺-H⁺ antiport • hypertension • vascular smooth muscle • cells • phosphorylation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hypertension has been associated with abnormalities in a variety of ion transport mechanisms regulating intracellular Na⁺, H⁺, or Ca²⁺. Changes in the activity of the ubiquitous cellular Na⁺-H⁺ exchanger (NHE) in vivo and in vitro have been documented in different cell types from patients with essential hypertension.^{1 2 3 4} Similar findings have been described in the spontaneously hypertensive rat (SHR) both in vivo^{5 6} and in vitro.^{7 8} It has been postulated that this membrane transport abnormality may be relevant to the pathogenesis of hypertension, because there is an association between activation of the Na⁺-H⁺ antiport by agonists and increased vascular medial thickening.⁹ Vascular myocytes from SHR also proliferate more rapidly in vitro than do their normotensive Wistar Kyoto (WKY) counterparts,^{7 10} and both thymidine incorporation and intracellular alkalinization are more responsive in SHR myocytes to stimulation with fetal calf serum (FCS).^{11 12 13}

Since the cloning of the ubiquitous growth factor–sensitive NHE isoform 1 (NHE-1) by Sardet et al,¹⁴ other isoforms of the NHE (NHE-2, NHE-3, and NHE-4) have been recently described in tissues, such as gut and kidney, in which there is transepithelial Na⁺ transport.^{15 16 17} Most of the reports on NHE activity in hypertension have used cells expressing mainly NHE-1 rather than the other isoforms.¹ The reason for the increased activity of NHE-1 in SHR is at present unknown, although we have recently reported that the increased activity of Na⁺-H⁺ exchange in SHR vascular and striated myocytes was not due to an increased number of NHE-1 transporters but to an increased turnover number at each site.¹⁸ Posttranslational processes such as phosphorylation¹⁹ and N-linked glycosylation²⁰ are known to affect NHE activity. However, the similarities in the molecular weight of NHE-1 from SHR and WKY tissue extracts make gross differences in N-linked glycosylation between the strains unlikely.¹⁸ We therefore used NHE-1–specific polyclonal antibodies to determine whether the differences in Na⁺-H⁺ exchange in SHR and WKY vascular myocytes were associated with differences in NHE-1 phosphorylation and whether SHR cells were more responsive to stimulation with FCS. Our findings suggest that NHE-1 from quiescent SHR vascular myocytes is more heavily phosphorylated than that from WKY myocytes and that FCS may stimulate Na⁺-H⁺ exchange in both cell types without any further increase in total NHE-1 phosphorylation.

	Materials and Methods

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Materials
Ham's F-12 medium, DMEM, RPMI 1640 medium, chick embryo extract, and [³²P]orthophosphate were from ICN Flow. FCS was from TechGen International. The Ham's F-12 growth medium contained 15% FCS, 0.5% (wt/vol) chick embryo extract, 2 mmol/L glutamine, 10⁵ U/L penicillin, 100 mg/L streptomycin, and 14 mmol/L NaHCO₃ (pH 7.1 with 5% CO₂ in air). Protein A Sepharose CL4B, glutathione Sepharose 4B, and pGEX-2T were from Pharmacia LKB Biotechnology. Hybond C extra-supported nitrocellulose, horseradish peroxidase–conjugated donkey anti-rabbit antibody, enhanced chemiluminescence Western blotting reagents, ¹⁴C molecular weight markers, and Aurodye were obtained from Amersham International. Molecular weight markers, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate were from Bio-Rad. Protogel acrylamide solution was obtained from National Diagnostics. X-ray film was from Genetic Research Instrumentation Ltd. BCECF-AM was from Cambridge Bioscience. Arginine vasopressin and all other Analar grade chemicals were from Sigma Chemical Co. Ethylisopropylamiloride was a gift from Merck Sharp & Dohme.

Cell Culture and Measurement of NHE Activity
SHR and WKY rats were obtained from the colony maintained at the Biomedical Services Unit, Leicester University. The blood pressure (mean±SD) of 12-week-old rats, measured by the tail-cuff method, was 171±11 mm Hg in SHR and 118±7 mm Hg in WKY rats. These rats were then anesthetized with pentobarbital sodium (50 mg/kg IP), and the thoracic aorta was rapidly dissected free of adventitia. Vascular myocyte lines were obtained from aortic explants, as described previously,⁸ and cultured in Ham's F-12 growth medium. Cultures were studied between passage numbers 5 and 14. NHE activity was measured in SHR and WKY vascular myocytes seeded onto glass coverslips, as described previously,^{7 8 18} and were cultured for 4 days before study. When the cells were confluent, they were rendered quiescent by serum deprivation for 24 hours in DMEM containing 1 g/L bovine serum albumin (BSA), 2 mmol/L glutamine, and antibiotics. Cells adherent on the coverslips were loaded with 5 µmol/L BCECF-AM for 30 minutes in serum-free DMEM at 37°C, washed twice, and then left at room temperature for a further 30 minutes to deesterify the dye. For measurement of the resting pH_i, the coverslips were clamped at an angle of 60° to the incident light in a quartz cuvette containing HBSS composed of (mmol/L) NaCl 130, KCl 5, CaCl₂ 1.8, MgSO₄ 1, glucose 5, and HEPES 20, along with BSA at 1 g/L, pH 7.4, in a 37°C thermostatted sample compartment holder within a dual grating fluorometer (Delta- scan, Photon Technology International Inc), which had dual-wavelength excitation (500 and 439 nm; slit widths, 5 nm) and emission at 530 nm (slit width, 5 nm). The 500/439 ratios were calibrated using nigericin and monensin (5 µmol/L each) in isotonic KCl buffers (replacing the NaCl of HBSS with KCl and omitting the BSA) having various pH values in the range of 6.0 to 8.0, as previously described.⁸ The pH_i was clamped to 6.0 (near the V_max of the Na⁺-H⁺ antiporter⁸ ) by incubation of the cells in isotonic KCl buffer (pH 6.0) containing 5 µmol/L monensin and 5 µmol/L nigericin for 5 minutes at 37°C. The ionophores were then scavenged by addition of BSA. The passive rate of change of pH_i (dpH_i/dt) was measured by addition of Na⁺-free medium (replacing the NaCl in HBSS with N-methyl-D-glucamine chloride, pH 7.4). The total dpH_i/dt was determined in HBSS, and the Na⁺-dependent dpH_i/dt was calculated as the difference between total and passive measurements. The dpH_i/dt values were computed from linear regression of the first 20 seconds of recordings after addition of Na⁺-free or Na⁺-containing buffers, using 100 readings from the recordings (each reading averaged >200 milliseconds). Pearson correlation coefficients always exceeded 0.99 for these regressions. Intrinsic buffering capacity was measured after addition of an NH₄Cl pulse (final concentration, 50 mmol/L), and fluxes mediated by NHE at pH_i 6.0 were calculated by the product of buffering capacity and the Na⁺-dependent dpH_i/dt.⁸ All flux measurements reported were performed on cells after clamping of pH_i to 6.0. Readings were made on cells in the absence of serum and then repeated on similar coverslips after 20 minutes of incubation with 10% FCS at 37°C. These matched experiments in the absence or presence of serum were performed within 30 minutes of each other and in random order.

Production of Polyclonal Antibodies to Human NHE-1
Polyclonal antibodies were raised to the regulatory C-terminus of NHE-1 by using a fusion protein consisting of ß-galactosidase and amino acids 658 to 815 of NHE-1. These nucleotides had been inserted into a PEX 3 vector (EMBL, Genofit), which was a gift from Prof J. Pouyssegur and Dr C. Sardet (University of Nice, France).¹⁹ Induction of the ß-galactosidase–NHE-1 C-terminal fusion protein was by incubation of the bacteria at 42°C for 2 hours as previously described.¹⁹ After two separations on SDS-PAGE, the ß-galactosidase fusion protein was electroeluted from gel slices. Over a period of 6 months, two rabbits were immunized with monthly intravenous injections of 100 µg of the ß-galactosidase fusion protein. The NHE-1–specific immunoglobulin was purified from polyclonal sera of each rabbit by elution from protein A–Sepharose CL4B beads. Antibody G252 was used in Western blots to detect and quantify the amount of NHE-1 present in vascular myocyte extracts,¹⁸ and antibody G253 was used in the immunoprecipitation experiments that are described below.

Another fusion protein was constructed where the nucleotides representing the above amino acids of NHE-1 were inserted into the Sma I and EcoRI sites of a pGEX-2T plasmid. This led to the production of a glutathione S-transferase–NHE-1 C-terminal fusion protein (GST fusion protein) that had a C-terminal amino acid sequence identical to that of the C-terminus of the ß-galactosidase–NHE-1 fusion protein that had been used to raise the antibodies (G252 and G253). Induction of GST fusion protein production was with 1 mmol/L isopropyl ß-D-galactopyranoside. This fusion protein was purified on a glutathione–Sepharose 4B column and eluted with addition of 5 mmol/L glutathione. Thus, the antibodies used in our studies reacted only with the NHE-1 C-terminus of this fusion protein, and the fusion protein could be used to check specificity of antibody reactions on Western blots.

Determination of NHE-1 Density by Western Blotting
We measured the NHE-1 contents of cultures from SHR and WKY myocytes by performing Western blots of these extracts, using a previously described method.¹⁸ This was to ensure that phosphorylation was measured on identical numbers of NHE-1 transporters in the two different strains. In brief, cells were snap-frozen with liquid nitrogen and scraped off the tissue culture flasks into a buffer containing (mmol/L) Tris 50, pH 7.4, NaCl 140, EDTA 5, phenylmethylsulfonyl fluoride 1, o-phenanthroline 1, and iodoacetamide 1. An equal volume of buffer containing 125 mmol/L Tris, pH 6.8, 4% SDS, 20% glycerol, and 0.004% bromophenol blue was added, and the mixture was then boiled for 3 minutes. Extracts were resolved on 7.5% SDS-PAGE gels and electrotransferred onto supported nitrocellulose. Blocking of these membranes overnight with 10% low-fat milk powder (Marvel) in 20 mmol/L Tris, pH 7.4, 137 mmol/L NaCl, and 0.1% Tween 20 (TBS-Tween) was followed by addition of 1 µg/mL G252 antibody in 5% Marvel in TBS-Tween for 2.5 hours. After seven washes in TBS-Tween, horseradish peroxidase–linked donkey anti-rabbit second antibody (1:1500 dilution) was incubated with the membranes for 1 hour, after which detection using enhanced chemiluminescence (Amersham) was performed. The bands corresponding to NHE-1 (molecular weight, 95 kD) were quantified on a Bio-Rad densitometer.¹⁸ The reactivity of antibodies with this band was completely abolished by the inclusion of 2 µg/mL GST fusion protein to neutralize the components of the serum reactive against the C-terminus of NHE-1.

Determination of NHE-1 Phosphorylation
Phosphorylation of NHE-1 was measured in quiescent vascular myocyte cultures by incubating the cells in [³²P]orthophosphate (50 µCi/mL) for 3 hours in phosphate-free HBSS composed of (mmol/L) NaCl 130, KCl 5, CaCl₂ 1.8, MgSO₄ 1, glucose 5, HEPES 20, and glutamine 2, along with BSA at 1 g/L, pH 7.4. The method for immunoprecipitation was adapted from that of Sardet et al.¹⁹ Cells that had been labeled were washed briefly with cold HBSS and snap-frozen with liquid nitrogen. One milliliter of cold (4°C) extraction buffer (containing 10 g/L polyoxyethylene-8-lauryl ether, 30 mmol/L Tris, 130 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L o-phenanthroline, 1 mmol/L iodoacetamide, 100 mmol/L sodium fluoride, 5 mmol/L sodium orthovanadate, 10 mmol/L ATP, 10 mmol/L sodium pyrophosphate, 1 mg/L pepstatin A, and 2 mg/L leupeptin) was then added to the frozen cells, and the monolayers were scraped off the plastic flasks. After sonication, cell debris was removed by centrifugation at 14 000g. The supernatant was then preabsorbed with protein A–Sepharose CL4B beads, and the samples were recentrifuged. The antibody G253 was then added to the supernatant at a final concentration of 100 µg/mL, and the samples were rotated end on end for 2 hours at 4°C. Protein A–Sepharose-CL4B beads that had been pretreated with unlabeled vascular myocyte cell extracts to reduce nonspecific binding were then added for 1 more hour. The beads were washed extensively in extraction buffer containing 1 g/L ovalbumin before solubilization in Laemmli sample buffer for SDS-PAGE analysis on 7.5% gels. Phosphoproteins were detected by autoradiography on preflashed x-ray films. The densities of the ³²P-labeled NHE-1 were determined with a Bio-Rad densitometer and associated software. Readings were automatically corrected for the background density of the same track by using troughs in the density profile adjacent to the NHE-1 band. Values were normalized to an arbitrary value of 1 for the quiescent WKY cell extract.

The effect of FCS on NHE-1 phosphorylation was then investigated by incubating quiescent ³²P-prelabeled myocyte cultures in HBSS containing 10% FCS for periods of up to 20 minutes at 37°C. The phosphate in the FCS had been effectively removed by gel filtration, using Sephadex G25, into buffer containing (mmol/L) NaCl 140, KCl 5, and HEPES 30, pH 7.4.

To ensure that we could detect any increase in NHE-1 phosphorylation, we performed two sets of positive controls. In the first positive control, the effect of serum on NHE-1 phosphorylation was investigated by using human lymphoblasts cultured in RPMI 1640 growth medium containing 10% FCS. Cells (1.5x10⁷) were rendered quiescent by 24-hour serum withdrawal, being incubated with RPMI 1640 medium containing 1 g/L BSA. The cells were then washed three times in phosphate-free HBSS and resuspended in 1 mL HBSS, with the pH adjusted to 7.4 with NaOH. Addition of 2 MBq carrier-free [³²P]orthophosphate to the 1-mL cell suspension was followed by incubation for 3 hours at 37°C. The cells were recovered and split into two aliquots. One was resuspended in HBSS, and the other was treated with 10% FCS in HBSS for 20 minutes. The second positive control involved studying the effect of arginine vasopressin on NHE-induced cellular alkalinization²¹ and NHE-1 phosphorylation of SHR and WKY myocytes. SHR cells have been reported to respond to arginine vasopressin with a greater intracellular alkalinization than WKY cells, a finding that may be related to the greater number of V₁ receptors.²¹ Quiescent cells on coverslips were labeled with BCECF-AM, and a baseline pH_i was determined in HBSS (37°C) as described above. NHE activity was stimulated with 100 nmol/L arginine vasopressin, and the change in pH_i was recorded after 20 minutes. NHE-1 phosphorylation was measured in quiescent SHR or WKY cells prelabeled with [³²P]orthophosphate, as described above, and another flask of cells was then stimulated with the 100 nmol/L arginine vasopressin for 20 minutes. After snap-freezing with liquid nitrogen, the protocol for immunoprecipitating NHE-1 and detection by autoradiography was as described above.

Measurement of Specific Activity of Intracellular [³²P]ATP
To examine whether the difference in NHE-1 phosphorylation between SHR and WKY cultures was due to a difference in the specific activity of intracellular [³²P]ATP, the level of this compound was determined in cell extracts. SHR and WKY myocytes were labeled with 250 nCi/mL carrier-free [³²P]orthophosphate for 3 hours and snap-frozen with liquid nitrogen. The frozen monolayers were scraped into 1 mL cold 10 mmol/L Tris, pH 7.4, containing 5 mmol/L EDTA. Samples were then centrifuged at 13 000g for 10 minutes. The ATP specific activity was measured by high-performance liquid chromatography using a 10SAX Partisil anion exchange column. A linear gradient of 0% to 67% 1.4 mol/L ammonium dihydrogen orthophosphate was used to elute the ATP. Total ATP levels were determined by absorption at 254 nm with reference to known ATP standards. These chromatography peaks were collected, and ³²P incorporation into ATP was measured by Cerenkov counting of these fractions.

Statistics
Results are expressed as mean±SEM, and comparisons were by Student's t test, performed on an OXSTAT statistics package (Microsoft Corporation). Two-tailed P<.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Determination of pH_i using the fluorophore BCECF-AM confirmed earlier findings of an elevated pH_i in quiescent SHR vascular myocytes compared with WKY cells^{7 8} (P<.001; Fig 1). When pH_i was clamped to a value near the V_max of NHE-1,⁷ quiescent myocytes from SHR exhibited increased external Na⁺-dependent dpH_i/dt (P<.001) and also increased NHE activity (P<.001) compared with quiescent WKY myocytes (Fig 1). There were no significant differences in H⁺ efflux in Na⁺-free media or the intrinsic buffering capacity of both cell types (data not shown). A 20-minute incubation with 10% FCS led to significant increases in pH_i, dpH_i/dt, and NHE activity in both SHR and WKY cells (P<.001 for comparisons of all parameters before and after serum incubation in both cell types), with the differences between SHR and WKY myocytes maintained (P<.001 for all three parameters in the presence of serum, Fig 1).

View larger version (27K): [in this window] [in a new window]   Figure 1. Bar graph showing results of fluorometric determinations of pHi (A columns), the passive rate of change of pHi (dpHi/dt) (B columns), and Na+-H+ exchanger activity at pHi 6.0 (C columns) in quiescent spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) myocytes (basal readings). These measurements were repeated after stimulation of cells with 10% fetal calf serum (FCS) for 20 minutes. Values are mean±SEM. Eight to 12 experiments were done in each group. *P<.001 for SHR compared with WKY measurements; *P<.001 for basal compared with serum-stimulated readings.

Cells were prelabeled with [³²P]orthophosphate as described above. Incubation of these extracts with the antibody G253 and subsequently with protein A–Sepharose beads led to immunoprecipitation of a number of phosphoproteins (Fig 2). A 95-kD band was identified as NHE-1 from previous reports of the molecular weight of this exchanger^{17 18} and from reactivity of this particular band with NHE-1–specific antibody (G252) on Western blots. Moreover, this reactivity on Western blots was completely abolished by coincubation of the antibody with GST fusion protein, indicating the specificity of the reaction to NHE-1. In addition, incubation of cell extracts with a rabbit antibody with no immunoreactivity toward NHE-1 extracted a number of phosphoproteins, but no 95-kD ³²P-labeled protein was seen in this region of the gel (Fig 2). Incubation of GST fusion protein with this antibody with no reactivity toward NHE-1 before immunoprecipitation of cell extracts did not alter the phosphoprotein pattern. Both antibodies immunoprecipitated phosphoproteins in the 190-kD region, and this may have obscured a minor amount of dimerized NHE-1 that has been recently reported.²²

View larger version (31K): [in this window] [in a new window]   Figure 2. Autoradiograph showing SDS-PAGE of immunoprecipitates of vascular myocyte extracts using Na+-H+ exchanger isoform 1 (NHE-1)–specific antibody G253 and rabbit immunoglobulin with no immunoreactivity toward NHE-1 (control). Vascular myocytes from spontaneously hypertensive rats were preincubated with [32P]orthophosphate (50 µCi/mL) in phosphate-free medium for 3 hours before extraction.

During subsequent immunoprecipitation experiments with ³²P-labeled cell extracts, we also confirmed that the NHE-1 content per cell in these extracts showed no differences between SHR and WKY myocytes to ensure that differences in phosphorylation of NHE-1 could not be due to differences in total NHE-1 content in the cells. The mean ratio of SHR to WKY NHE-1 content (per cell) amounted to 0.94±0.13 in the Western blotting studies that accompanied the phosphorylation experiments (n=8). A typical Western blot of the NHE-1 content of SHR and WKY vascular myocytes is illustrated in Fig 3 (left). Fig 3 (right) illustrates the equivalent phosphorylation experiment in which a higher density of NHE-1 phosphorylation was demonstrable in quiescent SHR compared with WKY myocytes. The phosphorylation of NHE-1 in quiescent SHR myocytes was enhanced 2.17±0.06-fold compared with WKY cells (P<.001, n=8).

View larger version (29K): [in this window] [in a new window]   Figure 3. Left, Western blot of extracts from spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) vascular myocytes, detected by the Na+-H+ exchanger isoform 1 (NHE-1)–specific antibody G252. Equal numbers of cells (106) were loaded per lane. The 97-kD marker is shown. Right, Autoradiograph of a 7.5% SDS-PAGE gel on which antibody G253 immunoprecipitates of quiescent vascular myocyte extracts from 32P-labeled cells from SHR and WKY are compared. NHE-1 (near the 97-kD marker) is more heavily labeled in SHR extracts than in WKY extracts. After a 20-minute incubation with 10% fetal calf serum, there was no enhancement of NHE-1 phosphorylation in either strain.

The time course of any changes in NHE-1 phosphorylation after stimulation with 10% FCS was studied in both cell types. The data have been normalized to a value of 1 for the zero time point. In three separate experiments on WKY cells, incubation with serum led to a transient reduction in total NHE-1 phosphorylation, which was apparent after 1 minute and reached a nadir by 5 minutes (0.47±0.05, P<.01 compared with the zero time point; Fig 4). NHE-1 phosphorylation then recovered virtually to control levels by 20 minutes (0.85±0.11, P=NS compared with the zero time point) (Fig 4). This transient dephosphorylation of NHE-1 was not seen in serum-stimulated SHR myocytes (mean levels of NHE-1 phosphorylation relative to a value of 1 at the zero time point were 1.02, 0.88, 0.97, 0.83, and 1.08 after 1, 2, 5, 10, and 20 minutes of incubation with 10% FCS).

View larger version (47K): [in this window] [in a new window]   Figure 4. Autoradiograph of a 7.5% SDS-PAGE gel showing time course of phosphorylation of Na+-H+ exchanger isoform 1 from Wistar-Kyoto rat vascular myocytes after incubation with 10% fetal calf serum at 37°C. Immunoprecipitates were obtained from 4x106 cells per time point. A transient reduction of phosphorylation was apparent within a minute, reaching a nadir by 5 minutes and recovering gradually within 20 minutes. Similar results were obtained in two other experiments. The 97-kD marker is shown.

When cells were incubated with 10% FCS for 20 minutes, NHE-1 phosphorylation was thus not enhanced further in either cell type (Figs 3 [right] and 5), even though there were very significant elevations in concomitant measurements of pH_i and NHE activity after 20 minutes of serum stimulation (Fig 1). Thus, stimulation of NHE activity by serum could be dissociated from stimulation of total phosphorylation of NHE-1 in these vascular myocytes. Even after serum stimulation for 20 minutes, the enhanced NHE-1 phosphorylation in SHR cells compared with their WKY counterparts (2.47±0.42 compared with 1.12±0.19, P<.01, n=8) was maintained (Fig 5).

View larger version (21K): [in this window] [in a new window]   Figure 5. Bar graph showing quantification of phosphorylation of Na+-H+ exchanger isoform 1 (NHE-1) from quiescent (basal) and 20-minute fetal calf serum (FCS)–stimulated spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) vascular myocytes. Densitometric results have been normalized to a mean value of 1 in quiescent WKY cultures. Seven to nine experiments were done in each group. ***P<.001, **P<.01 for SHR compared with WKY cells.

The specific activity of intracellular [³²P]ATP was measured in WKY and SHR cells to assess whether changes in labeling efficiency could account for an apparent difference in NHE-1 phosphorylation in quiescent cultures. There were no differences between WKY and SHR [³²P]ATP-specific activities (74.6±2.8 Bq/nmol ATP in WKY compared with 68.3±3.7 Bq/nmol ATP in SHR, P=NS, n=4). Thus, the difference in NHE-1 phosphorylation between strains was not the result of altered ³²P-labeling efficiency of ATP.

Two positive controls were performed for the serum stimulation of NHE-1 phosphorylation to verify the above results on vascular myocytes. First, human lymphoblasts that were quiescent after serum withdrawal for 24 hours showed a basal phosphorylation of NHE-1 (identified as a 100-kD band on the autoradiogram, Fig 6) that was enhanced 2.04±0.10-fold (n=3) after a 20-minute incubation with 10% FCS at 37°C. Second, addition of 100 nmol/L arginine vasopressin to quiescent SHR myocytes on coverslips led to a significant intracellular alkalinization (basal pH_i, 7.27±0.04, rising to 7.45±0.06 at 20 minutes after stimulation; P<.005, n=5), confirming a previous report.²¹ This response was abolished in the presence of 50 µmol/L ethylisopropylamiloride. In contrast, the response of WKY cells was smaller and not significant (basal pH_i, 7.15±0.08, rising to 7.21±0.08 at 20 minutes after stimulation; P=NS, n=5). NHE-1 phosphorylation in the SHR myocytes was elevated to 153±6% of basal values (P<.01, n=3), in contrast to WKY myocytes, in which no significant stimulation of NHE-1 phosphorylation was evident (poststimulation values, 109±14% of basal values; P=NS, n=3).

View larger version (44K): [in this window] [in a new window]   Figure 6. Autoradiograph of an SDS-PAGE gel showing Na+-H+ exchanger isoform 1 immunoprecipitates from human lymphoblasts (1.5x107), labeled with [32P]orthophosphate, that were rendered quiescent by 24-hour serum withdrawal. The cells were divided into two aliquots, and one aliquot was stimulated with 10% fetal calf serum for 20 minutes at 37°C. Na+-H+ exchanger isoform 1 phosphorylation was significantly increased by about twofold with serum incubation. The 97-kD molecular weight marker is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies on NHE activity of vascular myocytes cultured in vitro have shown an elevated transport activity in SHR cells compared with WKY cells.^{7 8} Kinetic analyses of the relation of H⁺ efflux to pH_i in SHR demonstrated that enhanced activity was due to an elevated V_max of the transporter. Because NHE-1 is the ubiquitous isoform of the exchanger and because other isoforms (2, 3, and 4) may be localized to gut and renal epithelia,^{15 16 17} this increased V_max in SHR myocytes could be explained by increased numbers of NHE-1 molecules per cell or by an increased turnover number per NHE-1 site. We have reported that NHE-1 content per cell in cultured SHR and WKY myocytes is very similar and that NHE-1 content in tissues such as brain, heart, kidney, and skeletal muscle ex vivo is also virtually identical between strains.¹⁸ Thus, increased activity cannot be due to increased expression of NHE-1 but to an increased turnover number.¹⁸

Posttranslational processes such as phosphorylation could lead to rapid stimulation of NHE activity, and growth factors such as serum, epidermal growth factor, thrombin, and phorbol esters may increase NHE activity by phosphorylation.^{19 23} Our finding of an increased phosphorylation of NHE-1 in quiescent SHR myocytes may contribute to the increased NHE activity of these cells compared with WKY myocytes. This increased phosphorylation of NHE-1 was not due to differences in the labeling efficiency of intracellular ATP pools. The persistence of these abnormalities despite culture of cells in vitro supports a role for genetic factors in determining this enhanced phosphorylation and NHE activity. These findings are in agreement with the suggestion of Livne et al³ that the NHE of platelets from human hypertensive patients is likely to be more active because of increased phosphorylation rather than an increased transporter number. Furthermore, although NHE-1 has been excluded as a candidate gene in human hypertension²⁴ and there have been no reports of mutations of NHE-1 in SHR, this does not preclude modulation of NHE activity in hypertension by processes such as phosphorylation.

Incubation of SHR and WKY myocytes with serum led to very significant increases in pH_i and enhancement of NHE activity. Previous work has demonstrated that SHR vascular myocytes proliferate more rapidly than WKY cells, and the DNA synthesis and pH_i response to serum and growth factors may be enhanced.^{7 11 12 13 25} In our present studies, although serum increased NHE activity to a greater extent in SHR myocytes than in WKY myocytes, this was not accompanied by any net increase in NHE-1 phosphorylation in either cell type, even after 20 minutes of incubation. Furthermore, treatment of WKY cells with serum led to a transient dephosphorylation of NHE-1 before recovery to prestimulation levels, implying a reduction in kinase or increase in phosphatase activity immediately following stimulation. These findings are in contrast to previous demonstrations that NHE-1 phosphorylation was increased after serum stimulation,¹⁹ although the cells used in that study hyperexpressed epidermal growth factor receptors. Other researchers have shown that NHE-1 activation could be dissociated from increased phosphorylation, eg, during osmotic shrinkage,²⁶ cell differentiation, or stimulation with phorbol esters.²⁷ An additional possibility is that phosphorylation at some sites with dephosphorylation at other sites could have led to stimulation of NHE activity without an increase in net phosphorylation. Recent studies with deletion mutants of NHE-1 have demonstrated the importance of mechanisms not dependent on direct NHE-1 phosphorylation that may have a role in stimulation of transporter activity.^{28 29} When the terminal section of the cytoplasmic tail of NHE-1 (amino acids 636 to 815) that contained the growth factor–sensitive phosphorylation sites was deleted, cytoplasmic alkalinization activated by growth factors was approximately halved. Thus, NHE-1 activity may be modified by direct NHE-1 phosphorylation and other non–phosphorylation-dependent regulatory factors. One possibility is that phosphorylation of NHE-1 may affect the interaction of these proposed regulatory factors with the cytoplasmic domain of NHE-1. However, elucidation of the precise role of the elevated total NHE-1 phosphorylation of quiescent SHR myocytes in mediation of the elevated NHE activity would depend on mapping of the differences in phosphorylation between different sites on NHE-1 of the two rat strains. This would enable a site-directed mutagenesis approach to examination of the relation between phosphorylation at selected sites of the regulatory domain of NHE-1 and NHE activity in SHR cells.

The basis for the enhanced phosphorylation of NHE-1 in quiescent SHR cells may depend on a number of possibilities. Protein kinase inhibitors have been demonstrated to reduce aortic tone and blood pressure in SHR,³⁰ and activators of protein kinase C lead to increased contractile response in SHR aortas.³¹ In addition, NHE activity in vascular myocytes is dependent on calmodulin,³² and there are several putative sites in NHE-1 that are directly phosphorylated by Ca²⁺/cal- modulin–dependent protein kinase II,³³ although no evidence for direct phosphorylation of the NHE-1 C-terminus with protein kinase C or cAMP-dependent protein kinase A was found.³³ The presence of higher concentrations of intracellular Ca²⁺ in vascular myocytes from SHR, especially in the subplasmalemmal zone,³⁴ may be responsible for elevated protein kinase C or Ca²⁺/calmodulin–dependent protein kinase II activity, resulting directly or indirectly in enhanced phosphorylation of NHE-1 in SHR cells. These possibilities remain to be directly tested in this model of hypertension.

In summary, we have provided the first direct evidence that the enhanced activity and turnover number of NHE-1 in SHR cells is associated with increased phosphorylation of NHE-1 rather than a change in the total number of NHE-1 transporters. The mechanism underlying this finding is unknown at present. It is presently unclear whether the increased basal NHE-1 phosphorylation of quiescent SHR cells compared with that in WKY cells is the mechanism leading to enhanced basal transporter activity. Although SHR myocytes exhibit increased responsiveness to FCS with enhanced alkalinization and NHE activity, these changes were not associated with a net increase in NHE-1 phosphorylation, indicating the existence of other regulatory phenomena that may enhance NHE activity.

	Acknowledgments

 
We are grateful for the generous support of the British Heart Foundation. We also thank P. Quinn and J. McDonald for their excellent technical assistance.

Received May 16, 1994; accepted February 1, 1995.

	References

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