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(Circulation Research. 1996;78:962-970.)
© 1996 American Heart Association, Inc.

Articles

Ca²⁺-Dependent Mitogen-Activated Protein Kinase Activation in Spontaneously Hypertensive Rat Vascular Smooth Muscle Defines a Hypertensive Signal Transduction Phenotype

Pamela A. Lucchesi, Jeremy M. Bell, Laura S. Willis, Kenneth L. Byron, Marshall A. Corson, Bradford C. Berk

From the Departments of Physiology (P.A.L., J.M.B.) and Medicine (K.L.B.), Loyola University Medical School, Maywood, Ill, and the Department of Medicine (L.S.W., M.A.C., B.C.B.), Division of Cardiology, University of Washington, Seattle.

Correspondence to Dr Pamela A. Lucchesi, Department of Physiology and the Cardiovascular Research Institute, Loyola University Medical School, 2160 S First Ave, Maywood, IL 60153. E-mail plucche@wpo.it.luc.edu.

	Abstract

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Abstract The mechanisms responsible for altered vascular smooth muscle cell (VSMC) function in hypertension remain unknown. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, there are multiple abnormalities in VSMC function, including increased growth, Na⁺-H⁺ exchange, and increased signal transduction by protein kinase C. The family of kinases termed mitogen-activated protein (MAP) kinases has recently been shown to be essential mediators of growth factor signal transduction. In the present study, alterations in MAP kinase function in the hypertensive phenotype were investigated using early-passage SHR and Wistar-Kyoto (WKY) VSMCs stimulated with angiotensin II (Ang II, 100 nmol/L) or platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Two differences between SHR and WKY rats were observed for Ang II–mediated MAP kinase activation: (1) Inactivation after Ang II stimulation was more rapid in SHR than WKY VSMCs. (2) Activity in SHR VSMCs showed a greater dependence on Ca²⁺ mobilization, since chelation of intracellular Ca²⁺ with BAPTA inhibited maximal activity by 95% in SHR VSMCs but by only 50% in WKY VSMCs. In contrast to the results with Ang II, no differences in PDGF-stimulated MAP kinase activity were observed. These findings establish activation of MAP kinase by Ang II as a feature that distinguishes SHR VSMCs from WKY VSMCs and suggest that differences in regulation of MAP kinase signaling may alter cellular events that are increased in the SHR genetic model of hypertension.

Key Words: vascular smooth muscle • hypertension • kinase • phosphatase • signal transduction

	Introduction

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The pathogenesis of hypertension is multifactorial, as evidenced by studies showing polygenic inheritance.¹ The cellular and molecular mechanisms that contribute to hypertension remain unknown. Among candidate cellular mechanisms are alterations in VSMC growth and function. Recent evidence indicates that the MAP kinase family of protein kinases is a critical mediator of growth factor signal transduction. Our laboratory^{2 3} and others^{4 5 6} have shown that the p42 and p44 MAP kinases (ERK2 and ERK1, respectively) are rapidly activated by Ang II and PDGF in VSMCs. Specific growth-related events that are regulated by MAP kinases include phosphorylation and activation of pp90^rsk (the ribosomal S6 kinase⁷ ), phosphorylation of p62^TCF (an essential transcription factor in the serum response element⁸ ), and induction of mRNAs for proto-oncogenes (c-fos and c-myc⁹ ). In addition, a dominant negative mutant of MAP kinase caused a 50% reduction in cell growth.¹⁰ These data indicate that regulation of MAP kinase activity is critical for cell growth.

A pathogenic feature of human essential hypertension is increased VSMC growth (both hypertrophy and hyperplasia). The SHR model is an appropriate one for studying differences in cell growth, as many investigators have shown increased VSMC growth compared with the normotensive WKY model.¹¹ Several mechanisms have been proposed to account for this abnormality in cell growth. Foremost among these abnormalities have been differences in signal transduction mediators related to growth. These have included kinases activated by growth factors such as PKC^{12 13 14} and receptor-coupling molecules such as G proteins.¹⁵ In addition to these signal transduction mediators, many abnormalities in ion transporters that regulate both Ca²⁺ and Na⁺ have been described.^{16 17 18}

Among the ion transporters proposed as pathogenic in human and SHR hypertension, the NHE-1 isoform of the Na⁺-H⁺ exchanger has been most extensively studied.¹⁹ NHE-1 participates in signal transduction pathways by which vasoactive agents modulate vascular tone and regulate VSMC proliferation and growth.^{11 20 21 22 23} A generalized dysfunction of the Na⁺-H⁺ exchanger in hypertension is apparent on the basis of observations that its activity is increased in mesenteric arteries, VSMCs, and skeletal muscle from the SHR^{11 18 21} and in leukocytes and platelets from hypertensive patients.^{18 24 25} The increased Na⁺-H⁺ exchanger activity in hypertension appears to be caused by alterations in the regulatory mechanisms responsible for activation of the exchanger rather than an abnormality of the exchanger itself.²⁶ Our laboratory has recently demonstrated that steady state levels of the NHE-1 mRNA in tissue and cells are not different in SHR and WKY rats and that hypertension does not induce the expression of the other known Na⁺-H⁺ exchanger isoforms.²⁷ Siczkowski et al²⁸ showed that there was no change in NHE-1 protein expression. Recently, increased phosphorylation of NHE-1 under basal conditions has been observed in SHR VSMCs and in immortalized lymphocytes from hypertensive patients.^{29 30} However, it appears that growth factor–mediated activation of the Na⁺-H⁺ exchanger does not involve direct phosphorylation of the exchanger but, instead, requires phosphorylation of an associated regulatory protein.³¹ The kinases responsible for activating the Na⁺-H⁺ exchanger have not yet been identified. Thus, alterations in the MAP kinase signaling pathway may contribute to the observed increase in Na⁺-H⁺ exchange in SHR VSMCs.

In the present studies, we compared the activation of MAP kinase by Ang II and PDGF in cultured SHR and WKY VSMCs. Our results demonstrate that both agonists activate 42-kD and 44-kD MAP kinases (ERK2 and ERK1, respectively), but with different time courses. The activation by PDGF did not differ between SHR and WKY VSMCs. However, MAP kinase activation by Ang II in SHR VSMCs ceased more rapidly and displayed a greater dependence upon changes in intracellular Ca²⁺ when compared with WKY VSMCs.

	Materials and Methods

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Cell Culture
Primary cultures of VSMCs were obtained from 10- to 12-week-old SHR and WKY rats (Harlan Sprague-Dawley, Inc, Indianapolis, Ind). VSMCs were isolated from thoracic aorta by enzymatic dissociation.¹¹ Animal procedures conformed to the Guide for Care and Use of Laboratory Animals, issued by the US Institute for Laboratory Resources. For all experiments, VSMCs from three different paired preparations of SHR and WKY aortas were used. Cells were grown in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, L-glutamine, and 10% FBS. 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 and growth are reliably observed during these early passages.¹¹ For all experiments, cells were plated in 60-mm dishes and growth-arrested at 70% to 80% confluence for 48 hours by replacing the media with DMEM containing 0.4% FBS. For measurement of [Ca²⁺]_i, cells were grown on 9x22-mm glass coverslips.

Preparation of Cell Lysates for MAP Kinase Experiments
Cells were incubated at 37°C in HEPES-buffered DMEM containing 100 nmol/L Ang II (Sigma), 10 ng/mL PDGF-BB (human recombinant, R&D), 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 HBSS containing 1 mmol/L EGTA and the acetoxymethyl ester of BAPTA (BAPTA-AM, 50 µmol/L in 0.1% dimethyl sulfoxide, 37°C). Control cells were preincubated with vehicle (0.1% dimethyl sulfoxide). After agonist stimulation, cells were harvested by aspirating the medium and washing with ice-cold PBS. Cells were lysed by the addition of 0.5 mL of ice-cold lysis buffer containing (mmol/L) NaCl 50, NaF 50, sodium pyrophosphate 50, EDTA 5, EGTA 5, Na₃VO₄ 2, phenylmethylsulfonyl fluoride 0.5, and HEPES 10 at pH 7.4, along with 0.1% Triton X-100 and 10 µg/mL leupeptin, 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 using a bicinchoninic acid protein assay kit from Pierce.

Western Blot Analysis
Cell lysates (30 µg) were subjected to electrophoresis on a 7.5% SDS–polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked with a commercially available blocking solution (GIBCO-BRL), and Western blot analysis was performed using ERK1- and ERK2-specific (Santa Cruz) primary antibodies at a 1:1500 dilution and a horseradish peroxidase–conjugated goat anti-rabbit IgG (Fisher) at a 1:2000 dilution. Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Amersham).

In-Gel Kinase Assay
MAP kinase activity was analyzed by the in-gel kinase assay previously described by Chao et al,³² as modified by Duff et al.³

Northern Blot Analysis
Total RNA was extracted from VSMCs by the guanidine isothiocyanate–cesium chloride gradient procedure as described previously.³³ Equal amounts of RNA were size-fractionated by electrophoresis in 1% agarose gels containing 25 mmol/L MOPS buffer (pH 7.8), 1 mmol/L EDTA, and 2% formaldehyde. Capillary transfer of RNA to Nytran membranes was performed overnight using 10x SSC (1x SSC contains 0.15 mol/L NaCl and 0.015 mol/L sodium citrate). RNA was cross-linked to the membrane using UV irradiation (Stratagene). Radiolabeling of the probes, MKP-1 cDNA (a full-length rat MKP-1 cDNA subcloned into pGEM)³⁴ or rat GAPDH (a full-length cDNA pRGAPD-13),³⁵ was performed with a Bethesda Research Laboratories random primer labeling kit according to the manufacturer's protocol using [-³²P]dCTP (specific activity, 3000 Ci/mmol; Du Pont-New England Nuclear). Hybridization on Nytran membranes was performed as previously described.³⁶ Blots were washed once in 1x SSC plus 1% SDS (30 minutes, room temperature) and once in 0.1x SSC plus 0.1% SDS (30 minutes, 55°C). The membrane was exposed to Kodak X-OMAT AR x-ray film with intensifying screens at -70°C for 2 to 24 hours.

Measurement of [Ca²⁺]_i
Cells were loaded with fura 2 by incubating for 2 hours with the acetoxymethyl ester form (fura 2-AM, 4 mmol/L) in HBSS supplemented with 0.1% BSA and 0.25% Pluronic F127. The cells were then washed twice and incubated (1) for 30 minutes in HBSS followed by 30 minutes in Ca²⁺-free HBSS containing 50 µmol/L BAPTA-AM, (2) for 1 hour in Ca²⁺-free HBSS, or (3) for 1 hour in HBSS, before measurement of fluorescence in a Perkin-Elmer LS50B fluorescence spectrophotometer. The coverslip was inserted into a 4.5-mL optical methacrylate cuvette on a 30° angle to the light beam.³⁷ The solution bathing the cells was changed by perfusing fresh solution from gravity-fed reservoirs into the bottom of the cuvette while aspirating continuously from just above the coverslip. At the perfusion rates used (5 to 10 mL/min), the half-time for mixing in the cuvette was 20 seconds, and complete exchange occurred within 50 seconds. The cells were excited alternately with 340- and 380-nm light every 0.02 second using a rotating filter wheel in the path of the excitation light. To correct for background fluorescence, the cells were treated for at least 10 minutes with ionomycin (1 mmol/L) and MnCl₂ (6 mmol/L) in Ca²⁺-free HBSS to quench the fura 2 fluorescence. The remaining fluorescence at each wavelength was then subtracted from the experimental traces. An integrated ratio (340 nm/380 nm) of the light emitted at 510 nm was then determined at 1-second intervals.

Data Analysis
Autoradiograms exposed in the linear range of film density were scanned using an LKB laser densitometer, and densitometric analysis was performed with NIH Image 1.49 software. All experiments were performed at least three times, and results are expressed as mean±SE. For Western blot analysis, "percent MAP kinase activation" was defined as the autoradiographic density (measured in arbitrary units) of phosphorylated MAP kinase (p42 or p44) divided by the total autoradiographic density of both the unphosphorylated and phosphorylated MAP kinases (p42+42 or p44+44) times 100%. For in-gel kinase assays, percent activation of MAP kinases was expressed relative to the autoradiographic density of the p42 and p44 MAP kinases in the unstimulated control condition. Statistical analysis was performed by a paired Student's t test (two-tailed) with significant differences determined at P<.05. For figure preparation, autoradiograms were scanned using a Hewlett-Packard scanner (ScanJet IIcx) and Adobe Photoshop software for image creation.

	Results

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Time Course for MAP Kinase Activation by PDGF and Ang II in SHR and WKY VSMCs
To compare MAP kinase signaling in SHR and WKY VSMCs, the time course of MAP kinase activation by Ang II and PDGF was determined in cells at early passage numbers. MAP kinase activation was measured using three techniques (Figs 1 through 3): (1) Western blots with antibodies that recognize the 42-kD and 44-kD MAP kinases demonstrated a "band shift" to an apparently higher molecular weight due to increased phosphorylation (p42 and p44 in Figs 1 and 2). (2) Western blots with an anti-phosphotyrosine antibody identified tyrosine-phosphorylated proteins at molecular weights consistent with the 42-kD and 44-kD MAP kinases (Fig 1, bottom). Tyrosine phosphorylation of MAP kinase is a marker for its activation, as evidenced by the finding that only the p42 and p44 MAP kinases contain tyrosine. (3) In-gel kinase assays using myelin basic protein as substrate demonstrated increased phosphotransferase activities of 42-kD and 44-kD kinases (Fig 3). The 42-kD and 44-kD proteins identified as MAP kinases by all three techniques exhibited activation kinetics with similar time courses and magnitude. The data in Fig 1 suggest that there may be increased amounts of 42-kD and 44-kD MAP kinases in control WKY VSMCs compared with SHR VSMCs. However, analysis of the pooled data from 10 Western blots indicated that there was no significant difference in MAP kinase protein expression between SHR and WKY VSMCs.

View larger version (43K): [in this window] [in a new window]   Figure 1. Time course for activation of MAP kinase in SHR and WKY VSMCs by Ang II or PDGF: Western blots. Top, Growth-arrested SHR and WKY VSMCs were stimulated with 100 nmol/L Ang II or 10 ng/mL PDGF for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and equal protein concentrations (25 µg per lane) were analyzed by Western blots using anti-ERK1 and -ERK2 antibodies (Santa Cruz) at a dilution of 1:1500 in GIBCO blocking buffer. Bottom, The blot in the top panel was stripped and reprobed with an anti-phosphotyrosine (P-tyr) antibody (UBI) at a dilution of 1:2000. Results shown are representative of 10 separate experiments.

View larger version (17K): [in this window] [in a new window]   Figure 2. Time course for activation of MAP kinase in SHR and WKY VSMCs stimulated by Ang II and PDGF: Western blots. Growth-arrested SHR and WKY VSMCs were stimulated with 100 nmol/L Ang II or 10 ng/mL PDGF for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and Western blot analysis was performed using anti-ERK1 and -ERK2 antibodies (Santa Cruz). Top, Western blots from SHR and WKY VSMCs stimulated with 10 ng/mL PDGF-BB were analyzed by densitometry and normalized to percent activation as described in "Materials and Methods." Bottom, Results shown are mean±SE of 10 experiments. Western blots from SHR and WKY VSMCs stimulated with 100 nmol/L Ang II were analyzed by densitometry and normalized to percent activation as described in "Materials and Methods."

View larger version (49K): [in this window] [in a new window]   Figure 3. Time course for activation of MAP kinase in SHR and WKY VSMCs by Ang II: in-gel kinase assays. Growth-arrested SHR and WKY VSMCs were stimulated with 100 nmol/L Ang II for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and in-gel kinase assays were performed using myelin basic protein incorporated in the gel. Results shown are representative of 10 separate experiments.

PDGF stimulated MAP kinase phosphorylation (Figs 1 and 2) and phosphotransferase activity (Fig 3), with a time course that was similar in SHR and WKY VSMCs. Maximal activation occurred at 5 minutes and persisted for at least 20 minutes (Figs 1 through 3). However, as shown in Fig 2, there was a trend toward greater magnitude MAP kinase phosphorylation in SHR VSMCs than in WKY VSMCs at 5 minutes (71±21% versus 56±6% of maximum) and 20 minutes (78±20% versus 55±9% of maximum), which was not statistically significant (P=.12, n=10).

Ang II also stimulated MAP kinase phosphorylation (Figs 1 and 2) and phosphotransferase activity (Fig 3) in SHR and WKY VSMCs, with maximal effect at 5 minutes (Figs 1 through 3). In comparison to PDGF, Ang II–mediated phosphorylation was more rapid, more transient, and of greater magnitude in both SHR and WKY VSMCs. Specifically in SHR VSMCs, at 2 minutes MAP kinase phosphorylation was 77±5% of maximum in response to Ang II (Fig 2) versus 6±5% of maximum in response to PDGF. At 20 minutes, MAP kinase phosphorylation had returned to 3±3% of maximum in the Ang II–stimulated SHR VSMC lysates but was still at 78±20% of maximum in the PDGF-stimulated lysates. In terms of the extent of MAP kinase phosphorylation, at the time of maximal phosphorylation (5 minutes), Ang II–stimulated MAP kinase phosphorylation was 97±5% of maximum, whereas PDGF-stimulated phosphorylation was 71±21% of maximum.

The time course for Ang II stimulation of MAP kinase differed significantly between SHR and WKY VSMCs, in contrast to PDGF stimulation. At 20 minutes, MAP kinase was almost completely dephosphorylated in SHR VSMCs (3±3% of maximum, n=10), whereas it remained significantly phosphorylated (48±10% of maximum, n=10) in WKY VSMCs (Figs 1 and 2). A similar difference was observed in MAP kinase phosphotransferase activity (Fig 3). Thus, the 42-kD and 44-kD MAP kinases are more rapidly inactivated in SHR VSMCs than in WKY VSMCs after stimulation by Ang II.

There was no significant difference in the concentration-response curves for MAP kinase activation by Ang II or PDGF in SHR and WKY VSMCs (data not shown). In both cell types, maximal stimulation of MAP kinase activation was observed at 100 nmol/L Ang II and 10 ng/mL PDGF. These data indicate that the differences in the kinetics of MAP kinase activation between SHR and WKY VSMCs are not due to differences in receptor efficacy, since MAP kinase was maximally stimulated at the same concentrations of agonists in the two cell types.

Role of Ca²⁺ and PKC in the Phosphorylation of MAP Kinase by Ang II
The different kinetics of MAP kinase activation by Ang II in SHR and WKY VSMCs suggest that several mechanisms may regulate MAP kinase in VSMCs. In comparison to PDGF, Ang II has been reported to cause greater maximal increases in intracellular Ca²⁺ in VSMCs obtained from Sprague-Dawley³⁸ and SHR¹⁵ aortas. To evaluate the role of Ca²⁺ mobilization in the stimulation of MAP kinases by Ang II, cells were pretreated with the Ca²⁺ chelator, BAPTA-AM (50 µmol/L for 30 minutes in nominally Ca²⁺-free HBSS). This treatment chelates intracellular Ca²⁺ and completely prevents Ang II–induced increases in intracellular Ca²⁺ in both SHR and WKY VSMCs without significantly affecting basal intracellular Ca²⁺ levels (Fig 4, bottom). MAP kinase phosphorylation by Ang II was inhibited to some extent by Ca²⁺ chelation at all time points in both SHR and WKY VSMCs (Figs 4 and 5). However, the degree of inhibition was significantly greater in SHR VSMCs compared with WKY VSMCs. Specifically, at 5 minutes, Ca²⁺ chelation inhibited Ang II–stimulated MAP kinase activity by 90% in SHR VSMCs but only by 50% in WKY VSMCs. These results indicate that activation of MAP kinase by Ang II is mediated in part by changes in intracellular Ca²⁺, and this activation exhibits a greater dependence on Ca²⁺ in SHR VSMCs compared with WKY VSMCs. As shown in Fig 4, both resting [Ca²⁺]_i and Ang II–induced increases in [Ca²⁺]_i were not different between SHR and WKY VSMCs. This suggests that the greater Ca²⁺ dependence for MAP kinase activation in SHR is not related to differences in mobilization of [Ca²⁺]_i. To prove that BAPTA-AM pretreatment was not toxic, the effect of Ca²⁺ chelation was reversed by incubating cells in 1.5 mmol/L Ca²⁺–containing HBSS for 10 minutes before exposure to Ang II. Under these conditions, there was no difference in Ang II–stimulated MAP kinase phosphorylation in BAPTA-treated cells compared with untreated control cells (data not shown).

View larger version (70K): [in this window] [in a new window]   Figure 4. Effect of Ca2+ chelation on MAP kinase activity in SHR and WKY VSMCs stimulated with Ang II. Top, Effect of BAPTA pretreatment on Ang II–induced Ca2+ transients. Cells on coverslips were preloaded with 2 µmol/L fura 2 and incubated for 30 minutes in HEPES-buffered HBSS, Ca2+-free HBSS (with 1 mmol/L EGTA), or Ca2+-free HBSS with 50 µmol/L BAPTA-AM. Cells were then perfused in the appropriate buffer to establish basal [Ca2+]i. Ang II (100 nmol/L) was added at the indicated time. Traces shown are representative of three separate experiments performed in duplicate. In all experiments, [Ca2+]i in BAPTA-AM–pretreated cells was measured for the duration of the Ang II–induced Ca2+ transient observed in control cells. Bottom, In-gel kinase assay. Growth-arrested WKY or SHR VSMCs were pretreated in HBSS or Ca2+-free HBSS containing 50 µmol/L BAPTA-AM and 1 mmol/L EGTA for 30 minutes before stimulation with 100 nmol/L Ang II for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and in-gel kinase assays were performed using myelin basic protein incorporated in the gel. Results shown are representative of six separate experiments.

View larger version (16K): [in this window] [in a new window]   Figure 5. Effect of Ca2+ chelation on MAP kinase activation in WKY and SHR VSMCs stimulated with Ang II: cumulative results from Western blot analysis. Growth-arrested WKY or SHR VSMCs were pretreated in HBSS or Ca2+-free HBSS containing 50 µmol/L BAPTA-AM and 1 mmol/L EGTA for 30 minutes before stimulation with 100 nmol/L Ang II for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and Western blot and densitometric analyses were performed as described in Fig 2. Results shown are mean±SE of six experiments.

Many Ang II–stimulated signal events, including c-fos induction³⁹ and Na⁺-H⁺ exchange activation,⁴⁰ are dependent on PKC activity. To assess the role of PKC in Ang II–stimulated MAP kinase activation, the effect of PKC downregulation (24-hour pretreatment with 2 µmol/L PDBU) was studied. PDBU pretreatment completely inhibited PMA-stimulated MAP kinase phosphorylation in both SHR and WKY VSMCs (data not shown). However, PDBU pretreatment only partially inhibited Ang II–stimulated MAP kinase phosphorylation (Fig 6) and phosphotransferase activity (Fig 7). There was a trend for a greater inhibition of MAP kinase activation by PDBU pretreatment in WKY VSMCs compared with SHR VSMCs, but this difference was not statistically significant.

View larger version (18K): [in this window] [in a new window]   Figure 6. Effect of PKC downregulation on MAP kinase activity in WKY and SHR VSMCs stimulated with Ang II. Growth-arrested WKY or SHR VSMCs were pretreated with DMEM containing 0.4% calf serum and 1 µmol/L PDBU or vehicle (0.1% dimethyl sulfoxide) for 24 hours before stimulation with 100 nmol/L Ang II for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and Western blot and densitometric analyses were performed as described in Fig 2. Results shown are mean±SE of four experiments.

View larger version (38K): [in this window] [in a new window]   Figure 7. Effect of PKC downregulation on MAP kinase activity in WKY and SHR VSMCs stimulated with Ang II: in-gel kinase assays. Growth-arrested WKY or SHR VSMCs were pretreated with DMEM containing 0.4% calf serum and 1 µmol/L PDBU or vehicle (0.1% dimethyl sulfoxide) for 24 hours before stimulation with 100 nmol/L Ang II for the indicated times. Cells were harvested in lysis buffer as described in "Materials and Methods," and in-gel kinase assays were performed using myelin basic protein incorporated in the gel. Results shown are representative of four separate experiments.

Induction of the MAP Kinase Phosphatase, MKP-1, in SHR and WKY VSMCs
To elucidate the mechanism for the more rapid inactivation of Ang II–stimulated MAP kinases in SHR, the induction of MKP-1 was determined. MKP-1 is a protein tyrosine and threonine phosphatase that is transcriptionally regulated and has high activity toward phosphorylated MAP kinase.^{36 41} We have previously shown that Ang II rapidly induces the expression of MKP-1 in Sprague-Dawley VSMCs.³⁶ In addition, we have shown that inhibiting MKP-1 expression with antisense oligonucleotides prolongs MAP kinase activity, suggesting an important role for MKP-1 in regulating MAP kinase activity.³ To determine whether differences in MKP-1 expression in response to Ang II accounted for differences in MAP kinase activity present in SHR and WKY VSMCs, steady state MKP-1 mRNA levels were measured by Northern blot analysis of total RNA prepared from cells exposed to 100 nmol/L Ang II (Fig 8). Preliminary experiments indicated that MKP-1 mRNA levels were present 5 minutes after Ang II treatment, maximal at 30 minutes, and undetectable by 2 hours (data not shown). The time course for MKP-1 mRNA expression after Ang II was similar in SHR and WKY VSMCs, and the magnitude of MKP-1 expression was also similar. Treatment with BAPTA completely abolished MKP-1 expression induced by Ang II at 30 minutes in both cell types (Fig 8). GAPDH mRNA expression was monitored as a control and showed no difference. Thus, the more rapid decrease in MAP kinase activity in SHR VSMCs cannot be explained by more rapid or greater expression of MKP-1.

View larger version (68K): [in this window] [in a new window]   Figure 8. Stimulation of MKP-1 mRNA expression by Ang II in WKY and SHR VSMCs. Growth-arrested SHR and WKY VSMCs were stimulated with 100 nmol/L Ang II for 30 minutes with or without a 30-minute pretreatment with 50 µmol/L BAPTA-AM. Total RNA was prepared, and 5 µg was size-fractionated for Northern blot analysis. Nytran membranes were hybridized with a 32P-labeled MKP-1 probe and then rehybridized with a GAPDH probe. Results are representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of the present study is that regulation of MAP kinase activity by Ang II differs in SHR VSMCs compared with WKY VSMCs. Two differences in MAP kinase signal transduction were identified. First, there was more rapid inactivation of MAP kinase in SHR stimulated by Ang II. Second, there was a requirement for Ca²⁺ mobilization in SHR cells to a much greater extent than in WKY cells. Thus, we propose that alterations in the regulation of MAP kinase activity by Ang II define a signal transduction event characteristic of the hypertensive phenotype. The fact that this difference is maintained for many passages in tissue culture (at least five) suggests that the difference in MAP kinase activation is genetically determined. Importantly, there is no difference in the relative abundance of either the 42-kD or 44-kD MAP kinase in SHR and WKY VSMCs, indicating that the difference in MAP kinase activity is due to changes in the regulation of MAP kinase phosphorylation and dephosphorylation.

The time course for inactivation of MAP kinase (Figs 1 through 3) differed significantly between SHR and WKY VSMCs, with nearly complete inactivation in SHR VSMCs 20 minutes after Ang II treatment. Since our laboratory has previously demonstrated that Ang II stimulates expression of the transcriptionally regulated MAP kinase phosphatase, MKP-1,³⁶ which is an important regulator of MAP kinase activity,³ it is possible that increased MKP-1 expression in SHR VSMCs could explain the present results. However, there was no difference in steady state MKP-1 mRNA levels between SHR and WKY cells (Fig 8). On the basis of these results, we propose a model that explains several possible mechanisms for the more rapid inactivation of MAP kinase in SHR VSMCs (Fig 9, left). The most likely explanation is that a MAP kinase phosphatase different from MKP-1 is more active in SHR than WKY VSMCs. Alternatively, MKP-1 activity may be greater in SHR than WKY VSMCs. Although there was no difference in MKP-1 mRNA expression, it is possible that protein stability or the ability of MKP-1 to interact with MAP kinase is greater in SHR than WKY VSMCs. Finally, it is possible that there is more rapid inactivation of a kinase upstream from MAP kinase (eg, MEK or Raf) in SHR.

View larger version (22K): [in this window] [in a new window]   Figure 9. Hypothetical model for the regulation of MAP kinase activation by Ang II in SHR VSMCs. Kinases and phosphatases affect MAP kinase activation by shifting the equilibrium between the unphosphorylated inactive species and the active phosphorylated (P) species. As indicated by the larger font, we postulate that the more rapid inactivation of MAP kinase in SHR stimulated by Ang II is due to an increase in the activity of a MAP kinase phosphatase. We also postulate that the greater Ca2+ dependence for MAP kinase activation in SHR is due to the loss of a Ca2+-independent MAP kinase kinase.

The second major difference in MAP kinase regulation was a requirement for Ca²⁺ mobilization in SHR cells to a much greater extent than in WKY cells (Figs 4 and 5). There appear to be at least four kinases that may account for the upstream Ca²⁺ dependence of MAP kinase activation in SHR: (1) Among the upstream kinases proposed to activate MAP kinases, a modulatory role for cytosolic Ca²⁺ has been implicated, most clearly in Raf activation. For example, Chao et al⁴² demonstrated that MAP kinase activation by the Ca²⁺-mobilizing agent thapsigargin was inhibited in a Balb/c-derived cell line expressing a dominant-negative mutant of Raf but was not affected by PKC downregulation. On the basis of these and other data, they concluded that Ca²⁺ may regulate MAP kinase activation at the level of Raf. (2) Ang II–stimulated tyrosine phosphorylation has been shown to be Ca²⁺ dependent by several investigators.^{5 43} Although the nature of the Ca²⁺-dependent tyrosine kinase remains to be determined, it is possible that receptor-associated tyrosine kinases may be activated by Ang II, as indicated by the finding that the Ang II type 1 receptor can be tyrosine-phosphorylated in vitro by members of the src family of tyrosine kinases⁴⁴ and in response to Ang II in vivo.⁴⁵ The importance of intracellular Ca²⁺ for Ang II activation of MAP kinases has also been demonstrated in rat ventricular myocytes.⁴⁶ (3) PKC is activated by Ang II in VSMCs⁴⁰ and stimulates MAP kinase in VSMCs and other cell types.⁴⁷ However, Ang II activation of MAP kinase in both WKY and SHR VSMCs appears to be independent of typical and novel PKC isozymes, since prolonged exposure to PDBU to downregulate PKC did not have any significant effect on activation in either SHR or WKY VSMCs. We (unpublished data, 1995) and others⁴⁴ have found similar results in cultured VSMCs from Sprague-Dawley rats, although Molloy et al⁵ reported a partial PKC dependence of Ang II–stimulated tyrosine phosphorylation of the 42-kD and 44-kD MAP kinases. (4) Recently, Lev et al⁴⁸ described a novel Ca²⁺-dependent tyrosine kinase termed PYK2 that is able to activate the MAP kinase pathway in PC12 cells. Future work will be required to assess specifically the Ca²⁺ dependence of Raf and other tyrosine kinases in SHR and WKY VSMCs.

There are several reports suggesting that abnormalities in signal transduction contribute to the pathogenesis of human disease. For example, mutations in G proteins are responsible for several diseases, including precocious puberty, pituitary tumors, pseudohypoparathyroidism, and McCune-Albright syndrome (reviewed in Reference 49⁴⁹ ). The possibility that alterations in signal transduction play an important role in the pathogenesis of hypertension has previously been suggested.^{11 15 19 26} Alterations in the MAP kinase signaling pathway may play a pathogenic role in hypertension, since this pathway has been shown to be associated with activation of the Na⁺-H⁺ exchanger^{11 19 26} and enhanced cell proliferation. It has been shown recently that increased phosphorylation of NHE-1 was associated with an increase in exchanger activity in quiescent SHR VSMCs compared with WKY VSMCs.³⁰ Findings from the present study suggest that MAP kinase does not account for the increased phosphorylation of NHE-1 in quiescent SHR VSMCs, since there was no difference in MAP kinase activity between unstimulated WKY and SHR VSMCs (Fig 3). However, there is strong data to suggest that a Ca²⁺-calmodulin–dependent kinase may be a critical regulator of the exchanger.^{31 50 51} Binding experiments with calmodulin-Sepharose, as well as fluorescence measurements with dansylated calmodulin, revealed that the NHE-1 cytoplasmic domain strongly binds calmodulin in a Ca²⁺-dependent manner.⁵⁰ Mutations that prevent calmodulin binding to the high-affinity binding region rendered NHE-1 constitutively active.³¹ These data suggest that the high-affinity calmodulin-binding region functions as an "autoinhibitory domain" and that Ca²⁺-calmodulin activates NHE-1 by relieving this autoinhibition. There is also evidence that Ca²⁺-calmodulin–dependent kinase phosphorylates the exchanger directly,⁵² but the functional significance is unclear. Thus, alterations in the Ca²⁺-dependent activation of MAP kinase in SHR may be part of a generalized abnormality in Ca²⁺-dependent signal transduction in SHR that is also manifested by increased Na⁺-H⁺ exchange. Future research directed toward understanding the interactions between Ca²⁺-dependent signal events and MAP kinase activity may provide important insights into understanding the mechanisms responsible for alterations in cell growth and Na⁺-H⁺ exchanger activity that have been observed in SHR VSMCs.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II ERK = extracellular-regulated signal transduction kinase HBSS = Hanks' balanced salt solution MAP = mitogen-activated protein MEK = mitogen-activated protein kinase/ERK kinase MEKK = mitogen-activated protein/ERK kinase MKP-1 = mitogen-activated protein kinase phosphatase 1 NHE-1 = Na+-H+ exchanger isoform 1 PDBU = phorbol 12,13-dibutyrate PDGF = platelet-derived growth factor PKC = protein kinase C SHR = spontaneously hypertensive rat(s) VSMC = vascular smooth muscle cell WKY = Wistar-Kyoto

	Acknowledgments

 
This study was supported in part by grants from the National Institutes of Health, Heart, Lung, and Blood Institute (HL-03282 to Dr Corson and HL-44721 to Dr Berk). Dr Berk is an Established Investigator of the American Heart Association.

	Footnotes

 
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received August 14, 1995; accepted March 11, 1996.

	References

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				D. Zhao, A. C. Keates, S. Kuhnt-Moore, M. P. Moyer, C. P. Kelly, and C. Pothoulakis Signal Transduction Pathways Mediating Neurotensin-stimulated Interleukin-8 Expression in Human Colonocytes J. Biol. Chem., November 21, 2001; 276(48): 44464 - 44471. [Abstract] [Full Text] [PDF]

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