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(Hypertension. 1998;31:1151-1156.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Role of Mitogen-Activated Protein Kinase in the Angiotensin II–Induced DNA Synthesis in Vascular Smooth Muscle Cells

Stefan Seewald; Claudia Seul; Ralf Kettenhofen; Dirk Bokemeyer; Yon Ko; Hans Vetter; ; Agapios Sachinidis

From the Medizinische Universitäts-Poliklinik, Bonn, Germany.

Correspondence to Agapios Sachinidis, PhD, Medizinische Universitäts-Poliklinik, Wilhelmstrasse 35-37, 53111 Bonn, Germany. E-mail umm501{at}ibm.rhrz.uni-bonn.de

	Abstract

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Abstract—The activation of mitogen-activated protein (MAP) kinase and increase in intracellular free calcium concentration ([Ca²⁺]_i) are discussed in reference to activation of different protein kinases and growth of vascular smooth muscle cells (VSMCs). The aim of the present study was to investigate the role of angiotensin (Ang) II–induced increase in [Ca²⁺]_i for activation of 44-kD/42-kD MAP kinase (p44^mapk/p42^mapk) and DNA synthesis in VSMCs. Experiments were performed by chelation of [Ca²⁺]_i by the intracellular chelator 1,2-bis-(o-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). Ca²⁺ was measured by the fura 2 method. MAP kinase activation was determined by the Western blotting method. DNA synthesis was determined by measurement of [³H]thymidine incorporation into the cell DNA. Treatment of VSMCs with 20 µmol/L MAPTAM for 30 minutes resulted in a complete abolishment of the maximal Ang II–induced increase at 10 seconds. Ang II phosphorylated the p44^mapk/p42^mapk in a time-dependent manner, showing a maximum at 3 minutes. In MAPTAM-treated cells, the maximal phosphorylation of MAP kinase isoforms was shifted to 5 minutes, and dephosphorylation was delayed compared with untreated cells. In concordance with this finding, the induction of the MAP kinase phosphatase-1 was markedly impaired in MAPTAM-treated cells. Ang II induced a 2.3-fold increase in [³H]thymidine incorporation into DNA synthesis in untreated cells. This effect was not reduced in MAPTAM-treated cells. Treatment of the cells with PD 98059 (10 µmol/L), a MAP kinase kinase inhibitor, caused 85% inhibition of the Ang II–induced activation of MAP kinases but did not inhibit the Ang II–induced DNA synthesis. In conclusion, the Ang II–induced stimulation of the MAP kinase is a Ca²⁺-dependent process. Furthermore, blockade of the Ang II–induced stimulation of the early intracellular events, such as increase in [Ca²⁺]_i or phosphorylation of the MAP kinase, is not accompanied by an inhibition of the Ang II–induced DNA synthesis.

Key Words: angiotensin II • kinase • calcium • muscle, smooth, vascular

	Introduction

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Vascular smooth muscle cell growth plays an important role in the pathogenesis of cardiovascular disease.^{1 2} It is widely believed that Ang II plays a pivotal role in the development of hypertension and atherosclerosis by promoting VSMC growth.^{3 4 5} Thus, elucidation of the intracellular signaling pathway of Ang II in VSMCs that mediates the growth response of VSMCs is crucial for understanding the vascular biology of cardiovascular disease. In this context, it has been demonstrated that Ang II induces hypertrophy and hyperplasia.^{4 6 7 8 9 10} After binding to the Ang II type 1 receptor (AT₁), Ang II stimulates the phosphoinositide signaling system, leading to an increase in [Ca²⁺]_i,¹¹ activation of protein kinase C, and activation of MAP kinase.¹² Activation of MAP kinases, in particular the 44-kD and the 42-kD (p44^mapk/p42^mapk) isoforms, appears to be an important step in growth-signal transduction of several growth-promoting factors.^{13 14} It is established that sequentially activated protein kinases such as the MAP kinase mediate further transmission of growth signals to the nucleus by expression of immediate-early growth response gene c-fos. In this context, it is assumed that activation of the MAP kinase by classic growth factors such as platelet-derived growth factor occurs by the MEK via threonine and tyrosine phosphorylation and requires the activation of p21^ras, p21^ras guanosine triphosphate binding protein (GAP), Raf-1 kinase, and MEK.^{13 14} It is believed that activation of Raf-1 kinase occurs via phosphorylation of several serine and threonine residues of the molecule by the activated c-Ras via an unknown mechanism. Recently, it has been shown that Ang II activates MAP kinase and c-Raf-1.¹² Activation of c-Raf-1 was associated with membrane translocation, and binding to H-Ras and Ang II–stimulated MAP kinase occurred via a c-Raf-1–independent pathway. Activation of MAP kinase appears to be important for cell growth, and its activity is mainly regulated by MKP-1.¹⁵ MKP-1 (also known as 3CH134) is an immediate-early gene product that possesses a dual-specificity phosphatase and dephosphorylates MAP kinase.¹⁵ Similar to CL100, MKP-1 belongs to a family of vaccinia virus–like phosphatases and is the mouse homologue of CL100 (97% identity).^{15 16 17} It is widely believed that [Ca²⁺]_i plays an important regulatory role in the control of cell growth.¹⁸ Therefore, it was of interest to examine whether the Ang II–induced elevation in [Ca²⁺]_i is implicated in the process of the MAP kinase activation via modulation of the induction of MKP-1 protein. Furthermore, to examine the role of the MAP kinase pathway in Ang II–induced DNA synthesis, the effect of Ang II on the phosphorylation of p42^mapk and p44^mapk isoforms in VSMCs and cell DNA synthesis in the presence and absence of the selective MEK inhibitor [2–2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] (PD 98059)¹⁹ was investigated.

	Methods

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Isolation and Culture of VSMCs
Rat aortic VSMCs were isolated from thoracic aortas from Wistar-Kyoto rats (6 to 8 weeks old, Charles River Wiga GmbH) by enzymatic dispersion using a slight modification of the method of Chamley et al²⁰ as described previously.²¹ Cells were cultured in DMEM supplemented with 10% fetal calf serum, nonessential amino acids, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C in the Steri-cult incubator from Forma Scientific in a humidified atmosphere of 95% air and 5% CO₂. Cells were grown to confluence in 75-cm² flasks over 4 to 5 days. The purity of VSMC cultures was confirmed by immunocytochemical localization of smooth muscle–specific -smooth muscle actin using monoclonal anti–-smooth muscle actin plus FITC-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulins. Experiments were performed using cells between passages 5 and 20.

Measurement of [Ca²⁺]_i
VSMCs were cultured on round glass microscope slides (diameter, 12 mm) under normal tissue culture conditions until confluence. After 24 hours in serum-free medium, for experiments in which intracellular Ca²⁺ was chelated, cells were preincubated for 30 minutes with 20 µmol/L MAPTAM. Control cells were preincubated with vehicle (0.1% DMSO). Medium was then replaced with HEPES buffer (in mmol/L: 20 HEPES, 16 glucose, 130 NaCl, 1 MgSO₄ · 7 H₂O, 0.5 CaCl₂, Tris-base, pH 7.4) containing 2 µmol/L fura 2 pentaacetoxymethyl ester and 1% BSA (wt:vol). Measurements were performed in HEPES buffer containing 1 mmol/L CaCl₂. The Ca²⁺–fura 2 fluorescence was measured at 37°C in a Perkin-Elmer LS50 fluorescence spectrofluorometer at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 505 nm. After calibration of fluorescence signals, [Ca²⁺]_i was calculated according to the method of Grynkiewicz et al.²²

Gel Electrophoresis and Immunostaining
Confluent cells in 3-cm-diameter culture dishes were incubated in serum-free medium consisting of a mixture of DMEM and Ham's F-10 medium (1:1) for 24 hours before addition of 20 µmol/L MAPTAM or 10 µmol/L PD 98059 for 30 minutes. Cells were then stimulated for different time periods with 100 nmol/L Ang II. After removal of the medium, cells were lysed with SDS sample buffer containing 62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS (wt:vol), 10% glycerol, and 50 mmol/L DTT. Aliquots were used for protein determinations using the Bio-Rad protein assay according to the method of Bradford.²³ Then 0.1% bromphenol blue (wt:vol) was added to the aliquots. Protein (10 µg) was analyzed with SDS-PAGE in a 12.5% acrylamide gel with a thickness of 0.75 mm using the Mini Gel Protean system (Bio-Rad). Proteins were transferred to a PVDF membrane overnight at 100 mA with a buffer containing 25 mmol/L Tris-HCl, 192 mmol/L glycin, and 20% methanol, pH 8.3. The protein transfer was checked using Ponseau S staining. MAP kinase protein analysis was performed with the chemiluminescence Western blotting method as described in the instructions of the PhosphoPlus MAPK Antibody Kit (New England BioLabs) using a phosphospecific mapk rabbit polyclonal IgG primary antibody and the alkaline phosphatase–conjugated anti-rabbit secondary antibody. The primary antibody recognized p42^mapk and p44^mapk only when catalytically activated by phosphorylation at Tyr204.²⁴

Immunoprecipitation and detection of MKP-1, the mouse homologue of CL100 (97% identity), were performed as previously described.¹⁶ Briefly, confluent cells in 10-cm-diameter culture dishes were incubated in serum-free medium consisting of a mixture of DMEM and Ham's F-10 medium (1:1) for 24 hours before addition of 20 µmol/L MAPTAM for 30 minutes. Cells were then stimulated for different time periods with 100 nmol/L Ang II. After removal of the medium, cells were lysed in 400 µL Triton X-100 lysis buffer containing 50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 mmol/L PMSF for 5 minutes at 4°C. Insoluble material was removed by centrifugation at 10 000g for 15 minutes at 4°C. VSMC lysates (500 µg protein) were incubated for 2 hours with 2 µL polyclonal anti-CL100 antibody. The anti-CL100 antibody was produced by immunizing rabbits with a synthetic peptide, CALSYLQSPITTSPS (bold letters correspond to residues 353 to 366 in the carboxy-terminal segment of CL100).¹⁶ Immunocomplexes were adsorbed to protein A–sepharose and washed three times with lysis buffer. Proteins were resolubilized by addition of an equal volume of 2x sample buffer. Then SDS-PAGE was performed using 4% and 10% acrylamide for stacking and resolving gels, respectively. Protein was transferred to nitrocellulose and probed with polyclonal antibodies against CL100. Detection of MKP-1 was performed using horseradish peroxidase–conjugated protein A and the ECL chemiluminescence system from Amersham.

Determination of DNA Synthesis
The effect of Ang II on DNA synthesis was measured as described previously.²¹ VSMCs were seeded in 24-well culture plates and cultivated in culture medium until confluent. Medium was then replaced by serum-free medium consisting of a mixture of DMEM and Ham's F-10 medium (1:1). After 24 hours of cultivation in serum-free medium for experiments in which intracellular Ca²⁺ was chelated, cells were preincubated for 30 minutes with 20 µmol/L MAPTAM or with 10 µmol/L PD 98059 for inhibition of MAP kinase activity. Control cells were preincubated with vehicle (0.1% DMSO). Medium then was replaced with serum-free medium, and cells were stimulated with 100 nmol/L Ang II. After 20 hours of incubation, 3 µCi/mL [³H]thymidine was added to the serum-free medium. Four hours later, experiments were terminated, and [³H]thymidine incorporation was quantified as described previously.²¹

Materials
Ang II was obtained from Sigma Chemical Co, and MAPTAM and [2-2'-(amino-3'-methoxyphenyl)-oxanaphthalen-4-one] (PD 98059) were obtained from Calbiochem. Anti–-smooth muscle actin was obtained from Sigma. FITC-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulins was obtained from Dako GmbH. DMEM, Ham's F-10, and Dulbecco's PBS were from Gibco BRL. Hybond N⁺ membranes and ECL Western blotting detection system were obtained from the PhosphoPlus MAPK Antibody Kit from New England BioLabs. Hyperfilm MP, 18x24 cm, was obtained from Amersham. PVDF membranes were obtained from Millipore.

Statistics
Values are expressed as mean±SD. Statistical analysis of the data was performed using the Mann-Whitney U test. Triplicate wells were analyzed for each [³H]thymidine incorporation experiment, and each experiment was performed independently a minimum of three times. Data presented are from representative experiments unless otherwise indicated. A value of P<.05 was considered statistically significant.

	Results

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Effect of MAPTAM on Ang II–Induced Increase in [Ca²⁺]_i
Ang II (100 nmol/L) induced a rapid maximal increase in [Ca²⁺]_i from 60±10 to 427±55 nmol/L within 10 seconds (mean±SD, n=3, P<.05) (Fig 1a). In cells pretreated with MAPTAM (20 µmol/L) for 30 minutes, no increase in [Ca²⁺]_i after stimulation with Ang II could be observed (Fig 1b).

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of Ang II on [Ca2+]i in MAPTAM-treated VSMCs. Confluent VSMCs on slides were precultured for 24 hours in serum-free medium. Cells were then preincubated in the presence and absence of 20 µmol/L MAPTAM for 30 minutes. After loading of the cells with fura 2, Ang II (100 nmol/L) was applied to VSMCs (a) or MAPTAM-treated cells (b), and changes in fluorescence were monitored. After subtraction of autofluorescence, changes in 340/380-nm excitation wavelength ratio by the emission wavelength of 505 nm were converted into corresponding levels of [Ca2+]i.

Effect of MAPTAM on Ang II–Induced MAP Kinase Activation
Stimulation of the cells with Ang II (100 nmol/L) resulted in a time-dependent increase of phosphorylated p42^mapk and p44^mapk detected with the phosphospecific MAP kinase antibodies, which recognized the Tyr204-phosphorylated isoforms. As demonstrated, in untreated VSMCs derived from cell line A, Ang II caused a time-dependent phosphorylation of p44^mapk/p42^mapk, showing a maximum at 3 minutes (Fig 2A). Chelation of the Ang II–induced [Ca²⁺]_i by MAPTAM resulted in a shift of the maximal phosphorylation from 3 to 5 minutes. A striking finding was that the amount of the phosphorylated p42^mapk/p44^mapk at 30 minutes was enhanced by 250% in MAPTAM-treated VSMCs compared with untreated cells (100%). This interesting finding was confirmed after MAPTAM treatment and stimulation of VSMCs derived from cell line B (Fig 2B). Again, the maximal phosphorylation of the p44^mapk/p42^mapk in untreated cells or in MAPTAM-treated cells occurred 3 minutes and 5 minutes after stimulation with Ang II, respectively. Furthermore, the amount of the phosphorylated p44^mapk/p42^mapk at 30 minutes in MAPTAM-treated cells was enhanced by 500% compared with that in the untreated VSMCs.

View larger version (61K): [in this window] [in a new window]   Figure 2. Effect of Ang II on the phosphorylation of the p44mapk and p42mapk at Tyr204 in MAPTAM-pretreated VSMCs. A, VSMCs from cell line A were seeded in culture dishes (diameter, 3 cm) and cultivated in culture medium until confluence. The medium then was replaced by serum-free medium. After 24 hours of cultivation, VSMCs were preincubated with 20 µmol/L MAPTAM for 30 minutes before stimulation with Ang II for different time periods. Cells were lysed, and 30 µg protein was analyzed with SDS-PAGE. MAP kinase was detected after blotting on PVDF membranes by a specific MAP kinase antibody that recognizes the catalytically activated p42mapk and p44mapk. B, Another independent experiment using cells from cell line B.

Effect of MAPTAM on Ang II–Induced Expression of MKP-1 Protein
Stimulation of the VSMCs with 100 nmol/L Ang II resulted in a time-dependent induction of MKP-1 protein (apparent molecular weight, 39 kD) that was first detectable at 30 minutes (Fig 3). Maximal induction occurred 1 hour after stimulation. As demonstrated in Fig 3, the effect of Ang II on MKP-1 protein induction was markedly impaired in MAPTAM-treated VSMCs compared with that in untreated cells. Quantification of the bands by scanning densitometry shows that the MKP-1 protein amount in MAPTAM-treated cells at 30 minutes, 1 hour, and 2 hours was estimated to be 15%, 14%, and 20%, respectively, of that induced by Ang II in untreated cells (100%).

View larger version (62K): [in this window] [in a new window]   Figure 3. Effect of Ang II on the MKP-1 protein induction in MAPTAM-treated VSMCs. VSMCs were seeded in culture dishes (diameter, 10 cm) and cultivated in culture medium until confluence. The medium then was replaced by serum-free medium. After 24 hours of cultivation, VSMCs were preincubated with 20 µmol/L MAPTAM for 30 minutes before stimulation with Ang II for different time periods. Cells were lysed, and MKP-1 protein was immunoprecipitated using anti-CL100 polyclonal antibodies. After SDS-PAGE, detection of MKP-1 was performed by chemiluminescence Western blotting method. The dominant band with an apparent molecular weight of 53 kD correlates to the heavy chain of the antibodies used for immunoprecipitation.

Effect of PD 98059 on Ang II–Induced Phosphorylation of p44^mapk and p42^mapk in VSMCs
As shown in Fig 4, treatment of VSMCs with the specific MEK inhibitor PD 98059 remarkably inhibited the Ang II–induced phosphorylation of both MAP kinase isoforms (Fig 4). Densitometric analysis of three blots derived from three independent experiments revealed that treatment of the cells with 10 µmol/L PD 98059 resulted in a 85±6% inhibition of the phosphorylation of p44^mapk/p42^mapk.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of PD 98059 on Ang II–induced phosphorylation of the p44mapk and p42mapk at Tyr204. VSMCs were seeded in culture dishes (diameter, 3 cm) and cultivated in culture medium until confluence. The medium then was replaced by serum-free medium. After 24 hours of cultivation, VSMCs were preincubated with 10 µmol/L PD 98059 for 30 minutes before stimulation with Ang II for 3 minutes. Cells were lysed, and 30 µg protein was analyzed with SDS-PAGE. MAP kinase was detected after blotting on PVDF membranes by a specific MAP kinase antibody that recognizes the catalytically activated p42mapk and p44mapk.

Effect of MAPTAM and PD 98059 on Ang II–Induced DNA Synthesis
Ang II (100 nmol/L) induced an increase in [³H]thymidine incorporation into cell DNA from 100% (basal value) to 230% (2.3-fold increase over the basal value) (Fig 5). Treatment of the VSMCs with MAPTAM had no significant effect on the basal value or the Ang II–induced increase in DNA synthesis. Treatment of the cells with PD 98059 per se resulted in a 75% decrease of the basal value. Stimulation of the PD 98059–treated cells with Ang II caused an increase in [³H]thymidine incorporation from 25% to 100% (4-fold increase over the value of PD 98059–treated cells).

View larger version (59K): [in this window] [in a new window]   Figure 5. Effect of Ang II on cell DNA synthesis in MAPTAM- and PD 98059–treated VSMCs. Confluent VSMCs were precultured in 24-well plates for 24 hours in serum-free medium. Then cells were precultured with 20 µmol/L MAPTAM or 10 µmol/L PD 98059 for 30 minutes before stimulation with 100 nmol/L Ang II. After another 20 hours of incubation, cells were exposed to 3 µCi/mL [3H]thymidine. Four hours later the reaction was terminated, and cell protein and [3H]thymidine incorporation into cell DNA were quantified. *P<.05 for Ang II and MAPTAM+Ang II vs control, n=3; **P<.05 for Ang II vs PD 98059+Ang II, n=3; ***P<.05 for PD 98059 vs control, n=3.

	Discussion

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It is well established that MAP kinases are a family of serine/threonine kinases activated by both tyrosine kinase and G protein–linked receptor agonists. MAP kinase activation seems to be an important step in the intracellular transmission of growth signals to the nucleus. Ang II stimulates tyrosine phosphorylation and activation of MAP kinase. Stimulation of protein kinase C by Ang II is not involved in this process.¹² Our study further elucidates the role of [Ca²⁺]_i in the Ang II–induced signaling cascade leading to activation of MAP kinase and growth of VSMCs.

Different MEK and MAP kinase phosphatases seem to be responsible for the equilibrium between the unphosphorylated inactive and the active phosphorylated MAP kinases.^{13 14 15 16 17} In this context, Ca²⁺-dependent and -independent MEK and MAP kinase phosphatases are discussed. It is believed that MKP-1 dephosphorylates and inactivates MAP kinases.¹⁵ Our results show that pretreatment of the cells with 20 µmol/L MAPTAM resulted in a shift of maximal stimulation of the MAP kinase isoforms. This phenomenon is also observed in MAPTAM-treated VSMCs stimulated by low-density lipoprotein.²⁵ Another striking finding of the present study was that subsequent inactivation of p44^mapk/p42^mapk was delayed in MAPTAM-treated cells in comparison to untreated VSMCs. From these find-ings, we supposed that delayed inactivation of MAP kinase in MAPTAM-treated cells occurs because of an inhibition of MKP-1 and that expression of MKP-1 protein may be controlled by [Ca²⁺]_i. To confirm our hypothesis, the expression of MKP-1 in MAPTAM-treated cells was examined. Our findings clearly demonstrated that chelation of the Ang II–induced increase in [Ca²⁺]_i by MAPTAM resulted in a marked attenuation of the expression of MKP-1. Therefore, we suggest that delay of inactivation of p44^mapk/p42^mapk in MAPTAM-treated cells occurred because of attenuation of the MKP-1 induction in these cells.

Until now, reports have been inconsistent on the role of the Ang II–induced elevation in [Ca²⁺]_i in Ang II–mediated p44^mapk/p42^mapk activation. In this context, Lucchesi et al²⁶ reported that chelation of the Ang II–induced [Ca²⁺]_i by BAPTA-AM caused a 50% attenuation of the Ang II stimulation of p42^mapk at 5 minutes in Wistar-Kyoto VSMCs. In contrast, Eguchi et al²⁷ reported that intracellular chelation of Ca²⁺ in VSMCs by BAPTA-AM or by 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), but not extracellular chelation or inhibition of Ca²⁺ influx, completely abolished the Ang II–induced MAP kinase activation determined after 5 minutes of stimulation of the cells.

We found that chelation of the Ang II–induced increase in [Ca²⁺]_i by MAPTAM resulted in a shift of the maximal stimulation from 3 minutes (untreated cells) to 5 minutes and a remarkable enhancement of the phosphorylation of the MAP kinases induced by Ang II at 30 minutes (Fig 2A and 2B). Similar to the findings of Lucchesi et al,²⁶ who demonstrated a 50% reduction of the MAP kinase activity in BABTA-AM–treated Wistar-Kyoto VSMCs at 5 minutes (data at 3 minutes are not available in this report²⁶), we observed a 40% attenuation of the phosphorylated (active) MAP kinase isoforms in MAPTAM-treated VSMCs at 3 minutes. Moreover, we found that chelation of the Ang II–induced increase in [Ca²⁺]_i by MAPTAM resulted in a marked elevation of the phosphorylation of the MAP kinases induced by Ang II at 30 minutes. In both reports, no data are available showing the MAP kinase activity in BABTA-AM–treated cells at 30 minutes.^{26 27} We also observed a marked decrease of the expression of MKP-1 protein in MAPTAM-treated cells, explaining the increased level of phosphorylated MAP kinase isoforms at 30 minutes compared with that in untreated cells. From these results, we suggest that induction of MKP-1 and activation of the MAP kinase isoforms in VSMCs by Ang II are Ca²⁺-dependent processes. The inconsistencies among our results, the results of Lucchesi et al, and the results of Eguchi et al²⁷ most likely reflect differences in the experimental procedures used. For instance, in contrast to our group, MAP kinase activity at 30 minutes was not determined by the other two groups.^{26 27} Authors used BAPTA-AM instead MAPTAM for chelation of the Ang II–induced [Ca²⁺]_i, and stimulation of the MAP kinase isoforms occurred in a Ca²⁺-free medium containing EGTA. In our study, stimulation of the VSMCs was performed in the presence of extracellular Ca²⁺ (without EGTA). Because MAPTAM was able to completely block the Ang II–induced increase in [Ca²⁺]_i even in the presence of extracellular Ca²⁺, we avoided the use of EGTA, which might in some degree be responsible for the complete attenuation of the MAP kinase activity obtained by Eguchi et al. Also, "side effects" of TMB-8 (eg, it has been reported that TMB-8 inhibits Na⁺ influx in fibroblasts²⁸) may be responsible in some degree for the complete inhibition of MAP kinase activity at 5 minutes seen by Eguchi et al.²⁷ In general, inconsistencies among all three studies also may reflect differences in factors such as isolation procedure, strain and age of the animal, cultivation conditions, number of passages, cell-seeding density, and culture time.

Moreover, we showed that treatment of VSMCs with MAPTAM resulted in an abolishment of the Ang II–induced transient increase in [Ca²⁺]_i but failed to inhibit the Ang II–induced increase in DNA synthesis, determined 24 hours after stimulation. From this finding, we may suppose that the Ang II–induced transient increase in [Ca²⁺]_i alone is not sufficient for generating DNA synthesis and that induction of DNA synthesis by Ang II may occur in conjunction with other signaling components such as MAP kinase. Similarly, we found that chelation of the LDL-induced increase in [Ca²⁺]_i did not influence the LDL-induced DNA synthesis.²⁵

Finally, to investigate the role of the MAP kinase pathway in the mitogenic effect of Ang II, the effect of Ang II on the phosphorylation of MAP kinases and DNA synthesis in PD 98059–treated VSMCs was examined. Treatment of the cells with PD 98059 resulted in a 75% reduction of the basal DNA synthesis in untreated cells. Stimulation of the PD 98059–treated cells with Ang II caused an increase of DNA synthesis from 25% to 100% (4-fold increase). These results demonstrate that treatment of VSMCs with 10 µmol/L PD 98059 failed to inhibit the mitogenic effect of Ang II. This may be explained by the observation that PD 98059 at a concentration of 10 µmol/L failed to inhibit the MAP kinase activity completely (85% inhibition), and the remaining 15% of the phosphorylated MAP kinase isoforms may be sufficient for the mitogenic response of the cells to Ang II. On the other hand, because activation of MAPTAM-treated cells with Ang II resulted in a marked increase of Ang II–induced stimulation of MAP kinase phosphorylation at 30 minutes, one should expect an enhancement of Ang II–induced DNA synthesis in these cells. However, treatment of the cells with MAPTAM did not result in an enhancement of Ang II–induced DNA synthesis. From these findings, we may suppose that the Ang II–induced stimulation of the early intracellular events such as an increase in [Ca²⁺]_i or activation of the MAP kinase may be essential but not sufficient for generating DNA synthesis. Induction of DNA synthesis by Ang II may occur in conjunction with other signaling components.

Hypertension in animals and humans is associated with an increase in smooth muscle cell mass and cardiac hypertrophy.³ The renin-angiotensin system represents a cascade of biochemical events leading to the generation of Ang II. Local generation and autocrine and paracrine actions of Ang II have been shown in the vascular system and the heart.³ In vivo studies demonstrated that AT₁ receptor antagonists^{29 30} as well as angiotensin-converting-enzyme inhibitors³¹ significantly reduced intimal lesions in rats after balloon injury (approximately 50% to 80% reduction). Using cultured VSMCs, we previously demonstrated that AT₁ receptor antagonists at a pharmacologically relevant concentration completely blocked the Ang II–induced stimulation of early events such as elevation in [Ca²⁺]_i,³² c-fos³³ and egr-1³⁴ mRNA expression, as well as DNA synthesis.³⁵ Now, we show that although the Ang II–induced increase in [Ca²⁺]_i was completely blocked and the phosphorylation of MAP kinase was inhibited by 85%, the Ang II–induced DNA synthesis was not inhibited.

Therefore, up to the present, our in vitro findings demonstrate that AT₁ receptor antagonists may be more beneficial agents for preventing an increase in vascular mass or intimal lesions than Ca²⁺-lowering agents or inhibitors of MAP kinase activity. Furthermore, much more efficient inhibitors for MAP kinase activity must be developed for a new therapeutic concept for prevention of cardiovascular disease.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1 = angiotensin II type 1 receptor BAPTA-AM = 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester [Ca2+]i = cytosolic free Ca2+ concentration DMEM = Dulbecco's modified Eagle's medium MAP = mitogen-activated protein MAPTAM = 1,2-bis-(o-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester MEK = MAP kinase kinase MKP-1 = MAP kinase phosphatase-1 PVDF = polyvinylidene difluoride SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Sa 568/1–2). The excellent technical assistance of Marion Lindemann is greatly appreciated.

Received November 14, 1997; first decision December 3, 1997; accepted January 6, 1998.

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