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(Hypertension. 1997;29:1265-1272.)
© 1997 American Heart Association, Inc.

Articles

A 90-kD Na⁺-H⁺ Exchanger Kinase Has Increased Activity in Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells

Van N. Phan; Masatoshi Kusuhara; Pamela A. Lucchesi; ; Bradford C. Berk

From the Divisions of Cardiology, Department of Medicine, University of Washington, Seattle, and Department of Physiology and the Cardiovascular Research Institute, Loyal University Medical School, Maywood, Ill (P.A.L.).

	Abstract

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Abstract Increased activity of the Na⁺-H⁺ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype, we measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca²⁺ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.

Key Words: sodium-hydrogen antiporter • signal transduction • muscle, smooth

	Introduction

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Although the genetic basis for hypertension has not been elucidated, several phenotypic differences have been reported to be characteristic of hypertension in individuals with essential hypertension and genetic animal models. Among these differences, increased Na⁺-H⁺ exchange is one of the most common phenotypic differences. The NHE is a member of a multigene family, and four NHE cDNAs (NHE-1 through NHE-4) have recently been cloned.^{1 2 3} The NHE-1 isoform is inhibited by amiloride, expressed ubiquitously, controls cytosolic pH and cell volume, and may also participate in cell growth and differentiation.² The isoforms NHE-2, NHE-3, and NHE-4 are less sensitive to inhibition by amiloride derivatives, distribute in epithelial cells, and are involved in transepithelial Na⁺ transport.²

Because the NHE-1 isoform is activated by both hyperplastic (eg, platelet-derived growth factor) and hypertrophic (eg, Ang II) agents,^{4 5} it has been proposed that abnormal function of this protein may be involved in the pathophysiology of hypertension.^{6 7} Evidence for NHE dysfunction in hypertension is provided by observations that its activity is increased in lymphocytes and skeletal muscle from SHR^{8 9} and in platelets, leukocytes, immortalized lymphoblasts, and skeletal muscle from hypertensive individuals.^{10 11 12 13 14 15} Data from our laboratory as well as others indicate that both cultured VSMCs^{9 16 17 18} and intact mesenteric arteries from SHR¹⁹ have a greater capacity for Na⁺-H⁺ exchange and altered kinetics. Alterations in Na⁺-H⁺ exchange regulation leading to increased activity in hypertension can be theoretically divided into three categories: mutation in the gene, increased expression of the gene product, and altered posttranslational regulation of existing exchangers. By restriction fragment length polymorphism analysis, there is no linkage between the human NHE gene and essential hypertension.^{20 21} Northern blot analysis showed that VSMCs from SHR and WKY expressed only the NHE-1 isoform and the steady-state mRNA levels were similar.^{7 22} Other investigators showed no difference in NHE-1 protein expression in SHR and WKY tissues.¹⁸ However, there is clearly an increase in NHE-1 phosphorylation in cells derived from the SHR, as reported by Siczkowski et al,²³ who showed that the phosphorylation state of the exchanger in growth-arrested SHR VSMCs was 2.2-fold greater than in WKY VSMCs. These findings suggest that the increase in Na⁺-H⁺ exchange in the SHR is caused by an alteration in the regulation of NHE-1 activity. The concept of a pathogenic change in signal transduction is supported by the findings of Rosskopf et al¹³ and Siffert et al,²⁴ who have demonstrated alterations in activation of G protein and Na⁺-H⁺ exchange in individuals with essential hypertension.

NHE activation is likely to be complex, with regulation by several intracellular mediators. NHE-1 consists of two functional subdomains: an amino-terminal domain (amino acids 1 through 505) that contains 10 to 12 transmembrane helices and mediates 1:1 Na⁺-H⁺ exchange, and a carboxyl-terminal domain (amino acids 506 through 815) that is cytosolic and required for intracellular pH sensitivity and activation by growth factors. Analysis of cells transfected with truncated mutants of NHE-1 indicate that the carboxyl-terminal domain is required for expression of its ATP dependence.²⁵ Functional characterization of NHE deletion mutants expressed in fibroblasts revealed that the region between amino acids 567 and 635 is required for both growth factor–induced cytoplasmic alkalinization and maintenance of high intracellular pH sensitivity.²⁶ However, deletion of amino acids 567 through 635 or replacement of any of the serine residues between amino acids 567 and 635 with alanine had no apparent effect on the pattern of growth factor–induced phosphorylation.²⁶ Comparison of phosphopeptide maps for wild-type NHE-1 with those for the internal deletion NHE-1 and the expressed cytoplasmic domain revealed that all major in vivo phosphorylation sites, including growth factor–sensitive ones, map to the cytoplasmic tail (amino acids 636 through 815).²⁶ Deletion of amino acids carboxyl to 635 reduced growth factor–induced cytoplasmic alkalinization by 50%.²⁷ In addition, it has been demonstrated that a novel calmodulin binding site in the exchanger (amino acids 636 through 656) functions as an "autoinhibitory domain."^{28 29} Increases in intracellular Ca²⁺ cause binding of Ca²⁺/calmodulin to the exchanger, activating NHE-1 by binding to this region and abolishing its inhibitory effect. Taken together, these data support three regulatory pathways for "H⁺ sensing" by NHE-1: (1) a regulatory factor or factors interact with a critical cytoplasmic region prior to amino acid 635; (2) phosphorylation occurs on amino acids 636 through 815; and (3) Ca²⁺/calmodulin binding removes a basal state of autoinhibition.

Several protein kinases have been proposed to regulate Na⁺-H⁺ exchange, including Ca²⁺/calmodulin-dependent kinases, cAMP-dependent kinases, PKC, and the mitogen-activated protein (MAP) kinase family (especially ERK1/2). We recently reported that ERK1/2 activation by Ang II differed between WKY and SHR.³⁰ However, maximum activation by Ang II was similar in WKY and SHR, suggesting that ERK1/2 is not responsible for increased Na⁺-H⁺ exchange in the SHR. Recently, Hooley et al³¹ suggested that at least three distinct signaling pathways were responsible for stimulation of NHE-1 activity by Ha-Ras and G₁₃. Although stimulation by Ha-Ras (the likely pathway for growth factor–mediated NHE-1 stimulation) involved Raf-1 and MEK, the downstream pathways used by MEK to stimulate NHE-1 were not determined. Thus, at present, no kinase for which NHE-1 is a specific substrate has been identified.

In this study, we investigated NHE-1 kinase activity in WKY and SHR VSMCs. We report two major findings: (1) a 90-kD NHE-1 kinase is more active in SHR than WKY VSMCs under both growth-arrested and Ang II–stimulated conditions; and (2) activation of the 90-kD NHE-1 kinase by Ang II is Ca²⁺ dependent and PKC independent.

	Methods

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Cell Culture
Primary cultures of VSMCs were obtained from 10- to 12-week-old male SHR and WKY (Harlan Sprague Dawley, Inc, Indianapolis, Ind). All experimentation and animal care conformed to the National Institutes of Health and American Heart Association guidelines for the care and use of animals. The study was approved by the University of Washington Animal Care and Use Committee. VSMCs were isolated from the thoracic aorta by enzymatic dissociation and grown in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, L-glutamine, and 10% fetal bovine serum. Cells were prepared in matched groups and used at the same passage number. Cells were used between passages 2 and 5 because differences in Na⁺-H⁺ exchange are reliably observed during these early passages.¹⁶ For all experiments, cells were plated in 60-mm dishes and were growth-arrested at 70% to 80% confluence by replacing the medium with DMEM containing 0.4% fetal bovine serum for 48 hours. We observed that data varied significantly when cells were growth-arrested for less than 48 hours.

Sprague-Dawley rat VSMCs were isolated by collagenase-elastase digestion as described previously and maintained in DMEM supplemented with 10% calf serum. Cells between passages 5 and 13 were growth-arrested at 70% to 80% confluence by incubation in 0.4% calf serum/DMEM for 48 hours.

Fusion Protein Preparation
Bacterial expression plasmids containing cytoplasmic domains of the human NHE-1 (amino acids 516 through 815) were prepared by subcloning polymerase chain reaction–generated EcoRI and Sac I fragments of human NHE-1 cDNA (cloned in pBluescript) into pGEX-KG to generate a GST fusion protein termed GST–NHE-1. The orientation and reading frames of all constructs were confirmed by sequencing. The homology between rat and human NHE-1 is 92% between amino acids 516 and 815. In this region, there are a total of 37 serine and threonine residues, of which 33 show exact conservation between rat and human. After transformation of GST–NHE-1 constructs into the BL21 strain of Escherichia coli, cultures were grown to sub-log phase and induced for 3 hours at 37°C with 1 mol/L isopropyl -D-thiogalactopyranoside (Sigma Chemical Co). GST–NHE-1 was not synthesized by the Escherichia coli in the soluble fraction, so purification from inclusion bodies was necessary. After induction, cells were collected, sonicated, and centrifuged. The pellet was washed once with 1 mol/L sucrose; resuspended with 10 mmol/L Tris (pH 7.4), 2% Triton X-100, 5 mmol/L EDTA, and 100 mmol/L NaCl; and incubated overnight at 4°C. After centrifugation, the pellet was resuspended with 3% SDS, and the SDS then was removed by chromatography on Extracti-Gel D Detergent Removing gel (Pierce Chemicals). Protein concentrations were determined by Coomassie staining of proteins separated by SDS–polyacrylamide gel electrophoresis (PAGE). GST–NHE-1 represented approximately 90% of the proteins in this preparation (Fig 1). To confirm results obtained with GST-NHE(516-815), we also used the rabbit NHE-1 as a substrate. The fusion protein was a ß-galactosidase construct containing the terminal 178 amino acids of the rabbit NHE-1 (approximately 637 through 815 of the human), kindly provided by Dr Larry Fliegel (University of Alberta). This fusion protein, termed COOH178, was prepared exactly as described previously.³²

View larger version (30K): [in this window] [in a new window]   Figure 1. GST–NHE-1 fusion protein purification. pGEX-KG containing the human NHE-1 (amino acids 516 through 815) was transformed into the BL21 strain of Escherichia coli, and the GST fusion protein was induced with 1 mol/L isopropyl ß-D-thiogalactopyranoside for 3 hours at 37°C. GST–NHE-1 was recovered in inclusion bodies and solubilized with 3% SDS. SDS was removed by chromatography on Extracti-Gel D Detergent Removing gel, and protein purity and concentrations were determined by Coomassie staining of proteins separated by SDS–polyacrylamide gel electrophoresis. Results are representative of five preparations used in the present study. Arrow indicates the approximately 69-kD GST–NHE-1 fusion protein.

Preparation of Cell Lysates for NHE-1 Kinase Experiments and Western Blot Analysis
Cells were incubated at 37°C in HEPES-buffered DMEM containing 100 nmol/L Ang II, 200 nmol/L PMA, or vehicle alone for various times. For experiments in which intracellular Ca²⁺ was chelated, cells were preincubated for 30 minutes in Ca²⁺-free, HEPES-buffered Hanks' balanced salt solution (HBSS) containing 1 mmol/L EGTA and 75 µmol/L acetoxy-methyl ester of BAPTA (BAPTA-AM, Molecular Probes) in 0.1% dimethyl sulfoxide at 37°C as previously described.³⁰ After agonist stimulation, cells were harvested by aspirating the medium and washing with ice-cold phosphate-buffered saline. Cells were lysed by the addition of 0.5 mL ice-cold lysis buffer (50 mmol/L NaCl, 50 mmol/L NaF, 50 mmol/L sodium pyrophosphate, 5 mmol/L EDTA, 5 mmol/L EGTA, 2 mmol/L sodium orthovanadate, 0.1% Triton X-100, 0.5 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 10 mmol/L HEPES, pH 7.4), followed by immediate freezing on ethanol/dry ice. The cell lysates were then thawed on ice, scraped, sonicated, and centrifuged at 14 000g at 4°C for 30 minutes. Supernatants were used immediately or stored at -80°C. Protein concentrations were determined with a Bio-Rad Protein Assay Kit. For Western blot analysis, lysates were subjected to 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine ERK1/2 antibody (New England Biolabs). PD 098059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one] was obtained from Parke Davis.

NHE-1 Kinase Activity Assay
NHE-1 kinase activity was analyzed by an in-gel-kinase assay described previously,³³ with slight modifications. Briefly, cell lysate supernatants (approximately 10 µg protein) were resolved on a 10% SDS-polyacrylamide gel containing 0.15 mg/mL GST–NHE-1. After electrophoresis, the gel was washed twice with buffer A (50 mmol/L HEPES, pH 7.4, and 5 mmol/L ß-mercaptoethanol) containing 20% 2-propanol to remove SDS. The gel was then reequilibrated in buffer A for 1 hour, followed by two 30-minute washes in buffer A containing 6 mol/L guanidine-HCl. Subsequently, proteins were renatured with two changes of buffer A (500 mL each) containing 0.04% Tween-20 at 4°C for 16 hours. The gel was then incubated in buffer B (25 mmol/L HEPES [pH 7.4], 10 mmol/L MgCl₂, 100 µmol/L sodium orthovanadate, 5 mmol/L ß-mercaptoethanol) for 30 minutes at 30°C. The phosphorylation assay was performed by placing the gel in buffer B containing 50 µmol/L ATP and 50 µCi [-³²P]ATP for 1 hour at 30°C. The reaction was terminated by immersing the gel in 5% trichloroacetic acid and 10 mmol/L sodium pyrophosphate. The gel was washed extensively to remove unincorporated radioactivity, dried, and then subjected to autoradiography. Autoradiographic signal intensity was quantified by densitometry in the linear range of film exposure with the National Institutes of Health Image program 1.49.

Data Analysis
Data are reported as mean±SEM. Results were analyzed by one-way ANOVA or the Wilcoxon signed ranks test. Statistics were computed with SYSTAT. For comparison of group means after computation of the ANOVA, a contrast was set up to yield F and P values. Post hoc analysis was performed by the Tukey-Kramer honestly significant difference test of pairwise mean comparisons. A value of P<.05 was considered significant.

	Results

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Several NHE-1 Kinases Are Present in VSMCs
To identify NHE-1 kinases present in rat VSMCs, we used an in-gel-kinase assay with recombinant GST–NHE-1 fusion protein as substrate. Cells were stimulated with 100 nmol/L Ang II or 200 nmol/L PMA for 5 minutes, cell lysates prepared, and in-gel-kinase assays performed. Kinases of molecular weights 42, 44, 56, 60, 62, 70, and 90 kD were activated in VSMCs from aortas of Sprague-Dawley rats (Fig 2, left). The kinase with the greatest increase in activity by both Ang II and PMA was the 90-kD kinase. Of interest, the activity of the 62- and 70-kD kinases was stimulated by Ang II but not by PMA. Finally, the activity of a 50-kD kinase was decreased after stimulation.

View larger version (69K): [in this window] [in a new window]   Figure 2. Several NHE-1 kinases are present in Sprague-Dawley rat VSMCs. Growth-arrested VSMCs were stimulated for 5 minutes with 100 nmol/L Ang II or 200 nmol/L PMA. Cells were harvested in lysis buffer as described in "Methods," and in-gel-kinase assays were performed with GST–NHE-1 (left) or GST (right) as substrate. Solid and open arrows indicate putative kinases (as demonstrated by substrate phosphorylation) with increased and decreased activities, respectively.

To determine the extent to which ³²P incorporation in the in-gel-kinase assay represented protein kinase autophosphorylation versus substrate-specific phosphorylation, we performed an in-gel-kinase assay in gels containing GST only (Fig 2, right). The only kinase that demonstrated phosphorylation was at 90 kD. However, the phosphorylation was only approximately 5% of that observed after Ang II stimulation with GST–NHE-1 as substrate. A similar result was obtained when the in-gel-kinase assay was performed in a gel containing no protein (data not shown). These results indicate that autoradiographic intensity on the in-gel-kinase assay represents predominantly phosphorylation of GST–NHE-1 (95%) rather than phosphorylation of GST or kinase autophosphorylation.

A 90-kD Kinase Has Greater Activity in SHR Than WKY VSMCs
To compare NHE-1 kinase activity in WKY and SHR VSMCs, we stimulated growth-arrested cells with Ang II and identified NHE-1 kinases by in-gel-kinase assay with GST–NHE-1. Similar to Sprague-Dawley VSMCs, Ang II stimulated NHE-1 kinases of molecular weights 42, 44, 56, 62, and 90 kD in both WKY and SHR VSMCs (Fig 3, left). Also similar to Sprague Dawley VSMCs, the kinase with maximal activity was at 90 kD, and it exhibited a small degree of phosphorylation in a GST in-gel-kinase assay (Fig 3, middle). To demonstrate further the reproducibility of this technique, we studied the same cell lysates by in-gel-kinase assay using a ß-galactosidase rabbit NHE-1 fusion protein (COOH178). Similar to results with GST–NHE-1, a prominent 90-kD kinase was identified that showed enhanced activity in growth-arrested and Ang II–stimulated SHR VSMCs (Fig 3, right).

View larger version (48K): [in this window] [in a new window]   Figure 3. Ang II stimulates a 90-kD kinase in SHR VSMCs to a greater extent than in WKY VSMCs. Growth-arrested WKY and SHR VSMCs were stimulated with 100 nmol/L Ang II for 5 minutes. Cell lysates were fractionated by SDS–polyacrylamide gel electrophoresis, and in-gel-kinase assays were performed with GST–NHE-1 (left), GST (middle), and COOH178 (right) as substrate. Longer exposure revealed additional autoradiographic signals with molecular weights similar to those shown in Fig 2. Solid arrows indicate putative kinases.

Further studies focused on the 90-kD kinase for several reasons. First, the activity of this kinase was higher in growth-arrested SHR than WKY VSMCs. This difference in function in growth-arrested cells is similar to previous studies that showed a higher resting pH in SHR VSMCs¹⁶ and greater phosphorylation of the NHE in SHR VSMCs under basal conditions.²³ Second, among several NHE-1 kinases in WKY and SHR VSMCs, the 90-kD kinase showed the greatest increase in activity after Ang II stimulation (3.8±0.9-fold for SHR and WKY combined, P<.01, n=8). Third, the 90-kD kinase was the most active kinase observed by the in-gel-kinase assay.

Comparison of 90-kD kinase activity in WKY and SHR VSMCs revealed the following important results. In growth-arrested cells, 90-kD kinase activity in SHR VSMCs was 1.8±0.2-fold greater than in WKY VSMCs (Fig 4, P<.05, n=5). Stimulation of the 90-kD kinase by Ang II was greater in SHR VSMCs. Specifically, 100 nmol/L Ang II for 5 minutes stimulated the 90-kD kinase (relative to WKY basal 90-kD kinase activity) by 2.6±1.2-fold and 4.5±1.7-fold in WKY and SHR, respectively (Fig 4, P=.07, n=5).

View larger version (19K): [in this window] [in a new window]   Figure 4. Ang II stimulates 90-kD kinase in SHR VSMCs to a greater extent than in WKY VSMCs. GST–NHE-1 in-gel-kinase assays from five experiments were quantified by autoradiographic density with a visible light scanner and National Institutes of Health Image 1.49 software. Results are shown as relative autoradiographic intensity normalized to WKY control 90-kD kinase activity in each experiment. *P<.05 vs WKY control; **P=.07 vs WKY treated with Ang II. Note that relative to the unstimulated WKY and SHR, Ang II stimulated similar increases in 90-kD activity (2.6- and 2.5-fold, respectively, P=NS).

Time Course for Activation of 90-kD Kinase in WKY and SHR VSMCs by Ang II
We previously observed that the time course of ERK1/2 activation by Ang II was significantly different in WKY and SHR VSMCs,³⁰ with ERK1/2 activity being inactivated more rapidly in SHR VSMCs at 20 minutes. To determine the time course for the 90-kD kinase activity, we stimulated growth-arrested VSMCs with 100 nmol/L Ang II. In response to Ang II, there was a rapid stimulation of 90-kD NHE-1 kinase, with peak activity at 5 minutes and inactivation by 20 minutes (Fig 5). At both 5 and 20 minutes, 90-kD kinase activity in SHR VSMCs was higher than in WKY VSMCs, but the time course for inactivation was similar. These results are based on three experiments and so statistical analysis was not performed. Activity of the 90-kD kinase returned to baseline by 60 minutes and showed no reactivation at 180 minutes (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 5. Time course for activation of NHE-1 kinase in WKY and SHR VSMCs by Ang II. Growth-arrested VSMCs were stimulated with 100 nmol/L Ang II for the indicated times. Cells were harvested in lysis buffer as described in "Methods," and in-gel-kinase assays were performed with GST–NHE-1 as substrate. Results are representative of three experiments.

Ca²⁺ Chelation Inhibits 90-kD Kinase Activity in SHR VSMCs
Because activation of the NHE in VSMCs has been reported to be Ca²⁺-dependent,³⁴ we investigated the effect of Ca²⁺ chelation on 90-kD NHE-1 kinase activity. Growth-arrested VSMCs were incubated in Ca²⁺-free HBSS containing 75 µmol/L BAPTA-AM and 1 mmol/L EGTA for 30 minutes before stimulation with 100 nmol/L Ang II. This pretreatment completely inhibits the Ang II–induced rise in intracellular Ca²⁺ concentration.³⁰ Ca²⁺ chelation increased the basal activity of the 90-kD kinase in WKY VSMCs (Fig 6A) but appeared to have little effect on Ang II–stimulated 90-kD kinase activity in WKY VSMCs. It was not possible for us to analyze statistically the effects of BAPTA on Ang II stimulation of the 90-kD kinase in WKY VSMCs because of the effect of BAPTA on baseline activity. In SHR VSMCs, Ca²⁺ chelation also slightly increased the basal activity of the 90-kD kinase (1.2±0.1-fold relative to SHR control, Fig 6, P>.1, n=3). Of interest, Ca²⁺ chelation completely blocked activation of the 90-kD kinase by Ang II in SHR VSMCs (Fig 6B, 1.1±0.1-fold increase, P<.05 versus Ang II–treated SHR without BAPTA-AM, n=3). Thus Ca²⁺ chelation significantly inhibited Ang II stimulation of the 90-kD NHE-1 kinase in SHR VSMCs.

View larger version (28K): [in this window] [in a new window]   Figure 6. Ang II stimulation of 90-kD NHE-1 kinase in SHR VSMCs is Ca2+-dependent. A, Growth-arrested WKY and SHR VSMCs were pretreated in Ca2+-free Hanks' balanced salt solution containing 75 µmol/L BAPTA-AM and 1 mmol/L EGTA for 30 minutes before 100 nmol/L Ang II stimulation. Cells were harvested in lysis buffer as described in "Methods." The 90-kD kinase activity was measured by in-gel-kinase assay with GST–NHE-1 as substrate. Results are representative of five experiments. B, NHE-1 in-gel-kinase assays from five experiments was quantified by autoradiographic density with a visible light scanner and National Institutes of Health Image 1.49 software. Results are shown as relative autoradiographic intensity normalized to 90-kD kinase activity in growth-arrested (control) SHR VSMCs for each experiment. Because Ang II stimulation of the 90-kD kinase was low in these WKY VSMC preparations, no significant effect of Ca2+ chelation was observed (not shown). *P<.01 vs all other groups.

PKC Downregulation Causes Minimal Inhibition of 90-kD Kinase Activity in SHR and WKY VSMCs
Ang II stimulation of Na⁺-H⁺ exchange in VSMCs is both PKC-dependent and PKC-independent.³⁵ Because the 90-kD NHE-1 kinase was activated by PMA in Sprague-Dawley VSMCs (Fig 2), we investigated the role of PKC in Ang II–mediated activation of the 90-kD kinase. For PKC inhibition, PKC was downregulated by pretreatment for 24 hours with 1 µmol/L phorbol 12,13-dibutyrate (PDBU). We have previously shown that PDBU treatment completely inhibits ERK1/2 activation by PMA but not by Ang II in WKY and SHR VSMCs.³⁰ However, PKC downregulation caused little inhibition of Ang II–stimulated 90-kD kinase activity in WKY and SHR VSMCs (Fig 7; 27% and 23% inhibition, respectively; n=2).

View larger version (26K): [in this window] [in a new window]   Figure 7. Effect of PKC downregulation on 90-kD kinase activity in WKY and SHR VSMCs. Growth-arrested VSMCs were treated for 24 hours with 1 µmol/L phorbol 12,13-dibutyrate (PDBU) or vehicle alone. Cells were stimulated for 5 minutes with 100 nmol/L Ang II. Cells were harvested in lysis buffer as described in "Methods," and in-gel-kinase assays were performed with GST–NHE-1 as substrate. Results are representative of two experiments.

PD 098059, an MEK Inhibitor, Inhibits 90-kD Kinase Activity in SHR and WKY VSMCs
A potential role for ERK1/2 in Ang II stimulation of the 90-kD kinase was suggested by the findings that 90-kD activation was Ca²⁺-dependent and PKC-independent, similar to results obtained previously for ERK1/2.³⁰ A recently developed compound, PD 098059, has been shown to inhibit MEK-1 and thereby prevent growth factor stimulation of ERK1/2.³⁶ To determine the role of ERK1/2 in Ang II–stimulated 90-kD kinase activation, we treated growth-arrested WKY and SHR VSMCs with 30 µmol/L PD 098059 for 30 minutes before stimulation with 100 nmol/L Ang II. Treatment with PD 098059 partially inhibited Ang II–stimulated NHE-1 kinase activation in WKY and SHR VSMCs (Fig 8A; 48% and 63% of control, respectively; n=2). Under these conditions, activation of ERK1/2 by Ang II in WKY and SHR VSMCs was inhibited to an even greater extent (Fig 8B; 80% and 85% of control, respectively; n=2). These results suggest that the 90-kD kinase is regulated in part by an ERK1/2-dependent pathway.

View larger version (39K): [in this window] [in a new window]   Figure 8. Effect of PD 098059, an MEK inhibitor, on 90-kD kinase activity in SHR and WKY VSMCs. Growth-arrested VSMCs were treated for 30 minutes with 30 µmol/L PD 098059 or vehicle alone. Cells were stimulated for 5 minutes with 100 nmol/L Ang II. Cells were harvested in lysis buffer as described in "Methods." A, In-gel-kinase assays were performed with GST–NHE-1 as substrate. B, Activation of ERK1/2 was determined by Western blot analysis with anti-ERK1/2 phosphotyrosine antibody. Results are representative of two experiments. The p42 ERK2 is recognized better by the antibody than the p44 ERK1, accounting for the apparent difference in phosphorylation observed.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major finding of the present study is the identification of a 90-kD protein present in VSMCs that has significant kinase activity toward the NHE-1 isoform. This kinase exhibits several features that suggest it may be important in the pathogenesis of hypertension in the SHR. First, activity of this kinase was higher in growth-arrested SHR than WKY VSMCs. This difference in function in growth-arrested cells is similar to previous studies that showed a higher resting pH in SHR VSMCs¹⁶ and greater NHE phosphorylation in growth-arrested SHR VSMCs.²³ Second, among several NHE-1 kinases in WKY and SHR VSMCs, the 90-kD kinase showed the greatest increase in activity after Ang II stimulation, and its maximal stimulated activity was greater in SHR than WKY VSMCs. The relative stimulation of the 90-kD kinase by Ang II was similar in SHR and WKY VSMCs, suggesting that the more important physiological difference is in basal activity of the 90-kD kinase. Third, as discussed below, the signal transduction characteristics of the 90-kD kinase correlate well with the pathways by which Ang II stimulates NHE-1 activity. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of cultured SHR VSMCs and suggest that alterations in the 90-kD NHE-1 kinase and/or the proteins that regulate its activity may be a pathogenic component in SHR hypertension.

A requirement for phosphorylation-dependent pathways in the activation of the NHE is suggested by a large number of studies. Data from Sardet et al³⁷ indicate that the rapid activation of the exchanger by mitogens is associated with increases in its phosphorylation. Phosphorylation of the exchanger is associated with a shift in pH_i dependence toward more alkaline pH values and an increase in maximum activity at acidic pH values.³⁷ More recent work with NHE-1^{2 26 28} has established that the carboxyl portion of the exchanger has both stimulatory and inhibitory domains. As shown in Pouysségur's laboratory for the human NHE-1,²⁶ progressive deletions of the carboxyl portion of the exchanger first cause an increase in activity and then a decrease in activity. Deletion mutational analysis of NHE-1 and comparison of phosphopeptide maps after growth factor stimulation revealed that all major in vivo phosphorylation sites, including growth factor–sensitive ones, map to the cytoplasmic tail (amino acids 636 through 815).²⁶ However, it should be noted that direct phosphorylation of the exchanger by growth factors is unlikely to be the only mechanism of activation.^{26 28} Because the 90-kD kinase phosphorylated both COOH178 and GST–NHE-1, which share amino acids 637 through 815, the present study indicates that this kinase phosphorylates serines and threonines located between amino acids 638 and 815. Future studies with the use of site-directed mutagenesis will be required for determination of which amino acids are phosphorylated and what effect this has on NHE-1 activity.

There are several reasons why we believe the 90-kD NHE-1 kinase represents a novel kinase, distinct from the well-known kinases discussed below. The 90-kD kinase is unlikely to be a member of the PKC family because Ang II–mediated activation was relatively unaffected by PKC downregulation despite the fact that PMA stimulated 90-kD kinase activity. In addition, the 90-kD kinase is larger than most described PKC isozymes, and the calcium-dependent PKC isozymes (, ß, and ) would not be active in the in-gel-kinase assay, which lacks phospholipids and calcium. It is not likely to be a cAMP-dependent kinase because purified cAMP-dependent protein kinase failed to phosphorylate COOH178.³² Finally, although calcium-calmodulin kinase has activity toward COOH178 in vitro,³² its molecular weight is 55 kD, and it would be unlikely to be active in a kinase assay in the absence of calcium and calmodulin. Thus, we believe that the 90-kD kinase may be a novel protein kinase.

The analysis of signal transduction pathways required for Ang II–mediated activation of the 90-kD kinase fits well with previous information regarding Ang II–mediated activation of the NHE in VSMCs. Specifically, we and others have reported a dependence on Ca²⁺, no dependence on PKC, and increased activity in SHR.^{16 18 34 38} Many studies suggest that the PKC-independent pathway in VSMCs involves a Ca²⁺-dependent signaling pathway. For example, exposure of VSMCs to the Ca²⁺ ionophore ionomycin transiently increased pH_i and amiloride-sensitive ²²Na flux, which was abolished in the presence of EGTA.^{28 34} Huang et al³⁹ demonstrated that activation of the NHE by thrombin in PKC-downregulated neonatal rat aortic VSMCs was sensitive to reductions in intracellular Ca²⁺. In VSMCs from adult rats, Berk et al⁴⁰ showed chelation of intracellular Ca²⁺ inhibited thrombin-induced activation of the exchanger, similar to the findings of Huang et al.³⁹ Little et al³⁴ reported that activation of the NHE by exposure to serum could be markedly attenuated by calmodulin antagonists, and Wakabayashi et al²⁸ showed that the exchanger contains a novel binding motif for Ca²⁺/calmodulin. These results suggest that activation of the NHE in VSMCs depends on Ca²⁺/calmodulin-dependent processes. Four mechanisms may be proposed to explain the increased activity of the 90-kD kinase in SHR VSMCs. First, there may be a gain-of-function mutation in the kinase itself. Second, expression of the 90-kD kinase may be increased in SHR VSMCs. Third, there may be increased activity of an upstream regulatory (stimulating) kinase, in particular a Ca²⁺-dependent kinase. Finally, there may be decreased activity of a regulatory (inhibiting) phosphatase in SHR VSMCs. It is of interest that Ca²⁺ chelation with BAPTA-AM increased the activity of the 90-kD kinase in growth-arrested VSMCs (Fig 6), suggesting that a Ca²⁺-dependent phosphatase may be important in regulating its activity.

In summary, the present findings support the simple concept that increased activity of an NHE-1 kinase (or decreased activity of a phosphatase that regulates an NHE-1 kinase) is responsible for increased basal activity of the exchanger in SHR VSMCs. Future work to identify the 90-kD kinase will be required to determine its role in NHE regulation and the pathogenesis of hypertension.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II BAPTA = 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid DMEM = Dulbecco's modified Eagle's medium ERK = extracellular signal regulated kinase GST = glutathione S-transferase MEK = mitogen-activated protein kinase/extracellular signal regulated kinase NHE = Na+-H+ exchanger PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate SDS = sodium dodecyl sulfate SHR = spontaneously hypertensive rat(s) VSMC = vascular smooth muscle cell WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported by National Institutes of Health grant RO1-HL-44721 to B.C.B. and a grant from the American Heart Association, Washington Affiliate to M.K. B.C.B. is an Established Investigator of the American Heart Association. V.N.P. and M.K. contributed equally to this manuscript.

	Footnotes

 
Reprint requests to Bradford C. Berk, Division of Cardiology, 357710, University of Washington, Seattle, WA 98195.

Received March 25, 1996; first decision April 18, 1996; accepted June 4, 1996.

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