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Am J Physiol Heart Circ Physiol 281: H30-H39, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 1, H30-H39, July 2001

Differential ANG II-induced growth activation pathways in mesenteric artery smooth muscle cells from SHR

Mohammed El Mabrouk, Rhian M. Touyz, and Ernesto L. Schiffrin

Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada H2W 1R7


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiotensin II-induced growth signaling mechanisms were investigated in vascular smooth muscle cells (VSMCs) from mesenteric arteries of sponteneously hypertensive (SHR) and Wistar-Kyoto rats (WKY). In WKY, angiotensin II significantly increased protein synthesis ([3H]leucine incorporation) but not DNA synthesis ([3H]thymidine incorporation). In SHR, angiotensin II increased protein and DNA synthesis. VSMCs from both strains expressed angiotensin type 1 (AT1) and type 2 (AT2) receptors. Losartan (an AT1 receptor antagonist) but not PD-123319 (an AT2 receptor antagonist) attenuated angiotensin II-stimulated protein synthesis in WKY VSMCs. In SHR, losartan and PD-123319 partially inhibited angiotensin II-induced VSMC proliferation. The mitogen-activated protein kinase or extracellular signal-regulated protein kinase (ERK) kinase inhibitor PD-98059 blocked VSMC growth responses to angiotensin II in both strains. Angiotensin II increased ERK1/2 activation more in SHR than WKY, an effect inhibited by losartan but not PD-123319. LY-294002 [a phosphatidylinositol-3 (PI3) kinase inhibitor] blocked angiotensin II-stimulated ERK1/2 activation in SHR but not in WKY, whereas bisindolylmaleimide [a protein kinase C (PKC) inhibitor] was ineffective. In conclusion, angiotensin II stimulates VSMC proliferation via AT1 and AT2 receptors in SHR. In WKY, angiotensin II induces VSMC hypertrophy via AT1 receptors. ERK1/2-dependent pathways regulated by intracellular Ca2+ but not PKC mediate these effects. In SHR VSMCs, PI3 kinase plays a role in augmented angiotensin II-induced ERK1/2 phosphorylation. These angiotensin II-mediated signaling events could contribute to vascular remodeling in SHR.

hypertrophy; renin-angiotensin system; signal transduction; hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALTERED VASCULAR SMOOTH MUSCLE CELL (VSMC) growth may contribute to the vascular remodeling found in small arteries in hypertension. Among many factors implicated in vascular remodeling, angiotensin II (ANG II) appears to be one of the most important. This is supported by in vivo studies (31, 39) demonstrating that angiotensin-converting enzyme inhibitors or angiotensin type 1 (AT1) receptor antagonists lower blood pressure and reverse vascular remodeling in patients with hypertension and in spontaneously hypertensive rats (SHR).

ANG II acts via at least two receptor subtypes, AT1 and AT2. Signaling pathways activated by AT1 receptors in VSMC, which mediate most of the vascular actions of ANG II, include activation of phospholipase C, phospholipase D, phospholipase A2, protein kinase C (PKC), NADH/NADPH oxidase, Src kinase, phosphatidylinositol-3 kinase (PI3 kinase), mitogen-activated protein (MAP) kinases [such as extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinase (JNK), and p38 kinase], Janus tyrosine kinase/signal transducers of activation and transcription, and nuclear factor-kappa B (NF-kappa B) (34, 47). These pathways converge and modulate the expression of certain growth factors such as platelet-derived growth factor, insulin-like growth factor-1, and basic fibroblast growth factor (45). Several studies have reported that AT2 receptors exert growth inhibitory and proapoptotic effects by antagonizing the action of AT1 receptors. These actions are mediated via activation of tyrosine phosphatase(s) such as MAP kinase phosphatase (MKP)-1, anti-Src homology phosphatase (SHP)-1, and protain phosphatase 2A (PP2A) (14) and/or ceramide (34). In contrast, other studies (31, 19, 21, 36, 37, 49) reported a growth and differentiation effect of AT2 receptors. A proinflammatory action of AT2 has also been reported, which is mediated via oxygen free radical- and ceramide-dependent NF-kappa B activation in VSMCs (34).

Activation of ERK1/2-dependent pathways regulates hypertrophy, hyperplasia, and differentiation in many cell types. This intracellular signaling pathway may be altered in hypertension. Small resistance mesenteric arteries from adult SHR exhibit vascular remodeling and enhanced ANG II-induced vascular contractility (10), which appear to be ERK1/2 dependent (45). Furthermore, glomerular activity of ERK and JNK is chronically activated in Dahl salt-sensitive rats (11) and in ANG II-infused rats (12). Activity of aortic ERK and JNK is also increased in Dahl salt-sensitive rats, in stroke-prone SHR, and in ANG II-infused rats (18, 51). Furthermore, cardiac MAP kinase phosphorylation is enhanced in stroke-prone SHR (15). Therefore, altered MAP kinase activation is evident in many tissues in various experimental models of hypertension and may be important in cardiovascular remodeling in hypertension.

Intracellular mechanisms regulating vascular ERKs in hypertension have not been fully identified. We hypothesized that Ca2+, PKC, and PI3 kinase differentially regulate ERK1/2 in SHR and normotensive control Wistar-Kyoto rats (WKY). In the present study, we therefore determined the growth effects of ANG II in VSMCs from mesenteric arteries of WKY and SHR and investigated whether intracellular Ca2+, PKC, and/or PI3 kinase, a nonreceptor tyrosine kinase, regulate vascular ERK in hypertension. Our data demonstrate that the growth-promoting actions of ANG II occur via AT1 and AT2 receptors in SHR VSMCs, whereas they are only mediated via AT1 receptors in WKY VSMCs. Moreover, in SHR, these effects are mediated via ERK-dependent pathways that are regulated by PI3 kinase and intracellular Ca2+ but not by PKC. These novel findings define signaling pathways that may play an important role in altered ANG II-stimulated VSMC growth in hypertension.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. DMEM and Ham's F-12 media were purchased from GIBCO-BRL (Ontario, Canada). [3H]thymidine and [3H]leucine were bought from ICN (Costa Mesa, CA). Losartan (an AT1 receptor antagonist) was a gift from Dr. Ronald D. Smith (Merck; Whitehouse Station, NJ). PD-123319 (an AT2 receptor antagonist) was a gift from Dr. Joan Keiser (Parke-Davis; Ann Arbor, MI). 125I-labeled [Sar1,Ile8]ANG II (125I-[Sar1,Ile8]ANG II) was obtained from Mandel Scientific (Ontario, Canada). The phospho-specific p44/42 ERK (T202/Y204) E10 monoclonal antibody was purchased from New England Biolabs (Ontario, Canada). Antibodies against AT1 and AT2 receptors were obtained from Chemicon International (Temecula, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. PD-98059, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, bisindolylmaleimide, and LY-294002 were from Calbiochem (La Jolla, CA). Seventeen-week-old male WKY and SHR were purchased from Taconic Farms (Germantown, NY). Glass fiber filtmats were from LKB Wallac (Turku, Finland). Polyvinylidene difluoride membranes were from Boehringer-Mannheim (La Jolla, CA). Enhanced chemiluminescence (ECL) reagent, leupeptin, pepstatin, and aprotinin were from Roche Diagnostics (Quebec, Canada). All other reagents were from Sigma (St. Louis, MO).

Cell cultures. VSMCs derived from mesenteric arteries were obtained by enzymatic digestion and cultured as previously described (45). For all experiments, VSMCs were used at the same passages (between passage 3 and 6). VSMCs at 80-90% confluence were maintained in serum-free DMEM for 48 h to render cells quiescent.

[3H]thymidine incorporation. Subconfluent VSMCs in 96-well plates were stimulated with ANG II for 24 h in the absence or presence of 10-5 M losartan (an AT1 receptor antagonist), 10-5 M PD-123319 (an AT2 receptor antagonist), or 10-5 M PD-98059 [a MAPK or ERK kinase (MEK) inhibitor]. [3H]thymidine (1 µCi/ml) was added for the last 4 h of incubation. To stop the reaction, the medium was withdrawn, and the cells were washed three times with cold PBS. Thereafter, cells were trypsinized (0.25 U/ml) and collected with an automatic cell harvester (LKB Wallac) onto glass fiber filtmats (LKB Wallac). The filters were dried and put into a plastic bag, to which 10 ml of scintillation cocktail was added. The radioactivity incorporated into DNA was counted in a beta -plate counter (LKB Wallac 1205 Betaplate).

[3H]leucine incorporation. Subconfluent VSMCs in 24-well plates were stimulated with ANG II for 24 h in the absence or presence of the ANG II receptor antagonists or PD-98059. [3H]leucine (1 µCi/ml) was added at the same time as ANG II. Thereafter, cells were washed three times with PBS and incubated with trichloroacetic acid (TCA; 5%) for 30 min at room temperature. Cells were washed twice with 5% TCA and three times with tap water and then solubilized in 0.2 M NAOH for 30 min at 37°C. The radioactivity incorporated into proteins was determined as described for measurement of [3H]thymidine incorporation.

Western blot analysis of ANG II receptors. Quiescent cells were washed three times with cold PBS and lysed in SDS lysis buffer, which contained 125 mM Tris · HCl (pH 6.8), 2% SDS, 5% glycerol, and 1% beta -mercaptoethanol. The samples were sonicated for 5 s, and equal amounts of proteins (15 and 25 µg for AT1 and AT2 receptors, respectively) were separated by 10% SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane, blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and 3% (wt/vol) casein, and incubated for 1 h at room temperature. The membranes were probed with antibodies against AT1 (1/15,000 dilution) or AT2 receptors (1/500 dilution) overnight at 4°C in TBS-T-1% casein. Thereafter, the membranes were washed in TBS-T buffer five times for 5 min and incubated in TBS-T-1% casein containing horseradish peroxidase-conjugated anti-rabbit IgG for AT1 (1/30,000 dilution) or AT2 receptors (1/7,000 dilution) for 1 h at room temperature. Blots were developed by ECL. Band intensity was analyzed using Image Quant software 5.0 (Molecular Dynamics).

ANG II binding assay. Receptor densities were obtained by performing competition binding experiments in subconfluent VSMCs in 24-well plates as previously described (43). Cells were incubated for 90 min at 22°C with 0.2 nM 125I-[Sar1,Ile8]ANG II in the presence of increasing concentrations of cold [Sar1,Ile8]ANG II, losartan, or PD-123319 in 0.4 ml of DMEM with 0.1% BSA. Thereafter, cells were washed twice with DMEM-BSA and solubilized in 1 M NaOH for 30 min at room temperature. Binding data were analyzed by nonlinear regression (GraphPad Prism 2.01).

Western blot analysis of phosphorylated ERK1/2. Cells were stimulated with ANG II in the absence and presence of BAPTA-AM (10-5 M), bisindolylmaleimide (10-5 M), or LY-294002 (10-5 M) and then washed three times with cold PBS and lysed in lysis buffer, which contained 20 mM HEPES (pH 7.5), 20 mM NaCl, 1 mM orthovanadate, 1 mM sodium fluoride, 5 mM phosphoglycerol, 5 mM EDTA, 1% Triton X-100, 1 µg/ml aprotinin, 0.7 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Lysed cells were centrifuged at 15,000 rpm for 20 min. Proteins (5 µg) were separated by 10% SDS-PAGE. Blots were blocked in TBS-T containing 5% dry milk for 1 h at room temperature. Thereafter, blots were probed with a polyclonal phospho-specific antibody against ERK1/2 (1/2,000 dilution) in blocking buffer at 4°C overnight. Subsequently, membranes were washed in TBS-T buffer five times for 5 min. Detection was carried out using anti-rabbit horseradish peroxidase-conjugated IgG (1/5,000 dilution) in blocking buffer. Blots were developed as described above.

Statistical analysis. Results are reported as means ± SE and were compared by Student's t-test or by ANOVA, followed by a post hoc Tukey-Kramer multiple comparisons test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AT1 and AT2 receptor expression in VSMCs. Western blot analyses demonstrated that both AT1 and AT2 receptors are expressed in low-passage VSMCs from WKY and SHR (Fig. 1A). Bands corresponded to a molecular weight of ~66 kDa rather than ~45 kDa, which suggests that the glycosylated form of the AT1 or AT2 receptor was detected at the same level in both WKY and SHR cells. PC12W cells, which express AT2 but not AT1 receptors, were used as a positive control for the AT2 receptor. The immunoblots from these cell lysates demonstrated a band at ~45 kDa, the native nonglycosylated form of the AT2 receptor. Competition binding assays with losartan and PD-123319 were carried out to measure the density of AT1 and AT2 receptors (Fig. 1B). Nonlinear regression analysis revealed a predominance of AT1 receptors in mesenteric artery smooth muscle cells (SMCs) from WKY and SHR.


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  		Fig. 1.   A: angiotensin II (ANG II) type 1 (AT1) and type 2 (AT2) receptor subtype expression in mesenteric artery smooth muscle cells (SMCs) from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Proteins for Western blotting were extracted from quiescent cells, separated on 10% polyacrylamide gels, and transferred to a polyvinylidene difluoride membrane. Samples were immunoblotted with antibodies against AT1 and AT2 receptors (1/15,000 and 1/500 dilution, respectively). PC12W extracts were used as a positive control for the AT2 receptor. Blots are representative of 3 independent preparations. B: competition binding assays were performed in the presence of 20 nM 125I-labeled [Sar1,Ile8]ANG II and increasing concentrations of [Sar1,Ile8]ANG II, losartan, or PD-123319.

Effects of ANG II on VSMC growth. The basal level of [3H]leucine and [3H]thymidine incorporation in SHR was slightly but not significantly higher in SHR compared with WKY. ANG II induced a concentration-dependent increase in [3H]leucine incorporation in WKY and SHR (Fig. 2, top). In contrast, ANG II, in a concentration-dependent manner, increased [3H]thymidine incorporation in VSMCs from SHR but not from WKY (Fig. 2, bottom). ANG II (10-7 M) induced a 1.5-fold increase in the number of SHR cells relative to unstimulated cells, but no significant change in WKY cell number was observed.


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  		Fig. 2.   Effect of ANG II-stimulated [3H]leucine and [3H]thymidine incorporation in vascular SMCs (VSMCs) from WKY and SHR. Data represent the means ± SE of at least 3 different experiments (each in triplicate).

Neither losartan nor PD-123319 had any significant effect on basal [3H]leucine or [3H]thymidine incorporation (Fig. 3). In WKY, losartan but not PD-123319 inhibited ANG II-stimulated protein synthesis. In contrast, ANG II-stimulated DNA synthesis was decreased by both losartan (39%) and PD-123319 (48%) in SHR.


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  		Fig. 3.   Effect of selective AT1 or AT2 receptor antagonists on ANG II-stimulated VSMC growth. Quiescent VSMCs were pretreated with losartan (10-5 M) or PD-123319 (10-5 M) for 10 min and then stimulated with ANG II (10-7 M) for 24 h. A and B: effects of losartan and PD-123319 on ANG II-stimulated [3H]leucine incorporation in VSMCs derived from WKY (A) and SHR (B), respectively. C: effect of ANG II receptor antagonists on ANG II-stimulated SHR VSMCs. Data represent the means ± SE of at least 3 different experiments (each in triplicate). #P < 0.001 and *P < 0.05 by ANOVA, followed by a post hoc Student-Newman-Keuls multiple comparisons test.

PD-98059 did not alter basal [3H]leucine or [3H]thymidine incorporation. However, it attenuated ANG II-stimulated [3H]leucine incorporation in WKY and SHR and [3H]thymidine incorporation in SHR, indicating that ERK1/2-dependent pathways play a role in protein synthesis in WKY and SHR and DNA synthesis in SHR (Fig. 4).


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  		Fig. 4.   The mitogen-activated protein or extracellular signal-regulated kinase (ERK) kinase inhibitor PD-98059 inhibits ANG II-stimulated VSMC growth. VSMCs were pretreated with PD-98059 (10-5 M) for 30 min and then stimulated with ANG II (10-7 M) for 24 h. A and B: effect of PD-98059 on [3H]leucine stimulated by ANG II in WKY (A) and SHR VSMCs (B). C: effect of PD-98059 on [3H]thymidine stimulated by ANG II in SHR VSMCs. Data represent the means ± SE of at least 3 different experiments (each in triplicate). *P < 0.05 vs. control.

Activation of ERK1/2 by ANG II in VSMCs. Specific phospho-ERK1/2 antibody or ERK1/2 antibodies were used to assess ERK1/2 phosphorylation and expression, respectively. Expression of ERK1/2 was not significantly different in WKY and SHR (data not shown). ERK1/2 phosphorylation was dose dependently increased by ANG II in VSMCs from both strains. However, this response was significantly greater (twofold) in VSMCs from SHR than from WKY (Fig. 5A). Maximum stimulation of ERK1/2 phosphorylation was observed at 10-7 M ANG II, whereas ERK1/2 phosphorylation appeared to be reduced at high concentrations of ANG II in WKY and SHR. ANG II (10-7 M) induced rapid ERK1/2 activation, which was maximal within 5 min of stimulation in WKY and SHR VSMCs (Fig. 5B). This response returned to baseline levels within 10 min in WKY VSMCs. In contrast, in SHR VSMCs, ERK1/2 phosphorylation by ANG II was sustained at suprabasal levels for up to 20 min.


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  		Fig. 5.   ANG II-stimulated ERK1/2 activation in WKY and SHR VSMCs. A: time course of ERK1/2 phosphorylation. Serum-starved WKY and SHR cells were stimulated with ANG II (10-7 M). B: dose response of ANG II-stimulated ERK1/2 phosphorylation of WKY and SHR VSMCs. VSMCs were stimulated with increasing concentrations of ANG II for 5 min. Top: representative Western blots. Bands correspond to the molecular mass of ERK1 (44 kDa) and ERK2 (42 kDa). Bottom: means ± SE of 4 experiments. *P < 0.05 vs. WKY VSMCs. pERK, phosphorylated ERK.

AT1 receptors mediate the effect of ANG II on ERK1/2 activation. To examine the ANG II receptor subtypes whereby ANG II activates ERK1/2, we measured ANG II-induced ERK1/2 phosphorylation in the absence and presence of losartan or PD-123319 (Fig. 6). Treatment of VSMCs with losartan or PD-123319 did not significantly alter basal levels of ERK1/2 phosphorylation. Losartan completely abolished the ERK1/2 phosphorylation in response to ANG II in VSMCs from WKY and SHR after stimulation for 5 min. In contrast, PD-123319 did not affect ERK1/2 activation induced by ANG II during short-term (5 min) or long-term (20 min) stimulation (data not shown).


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  		Fig. 6.   AT1 receptor-mediated ERK1/2 phosphorylation induced by ANG II. Results demonstrate effects of the AT1 and AT2 receptor antagonists losartan (10-5 M) and PD-123319 (10-5 M) on ERK1/2 phosphorylation in WKY (A) and SHR (B) VSMCs. Top: representative Western blots. Bottom: means ± SE of 3 experiments. *P < 0.05, comparison of control and treated cells.

Role of intracellular Ca2+, PKC, and PI3 kinase in ANG II-induced ERK1/2 phosphorylation. Exposure of VSMCs to BAPTA-AM (10-5 M), an intracellular Ca2+ chelator, significantly decreased ANG II-induced ERK1/2 phosphorylation in cells from WKY and SHR (Fig. 7). BAPTA-AM did not influence basal ERK1/2 phosphorylation. These data indicate that intracellular Ca2+ may play a regulatory role in ANG II-stimulated ERK1/2 activation in WKY and SHR.


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  		Fig. 7.   Effect of intracellular Ca2+ chelation on ANG II-stimulated ERK1/2 phosphorylation in WKY and SHR VSMCs. VSMCs were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (10-5 M) for 30 min. Thereafter, cells were stimulated with ANG II (10-7 M) for 5 min. Top: representative Western blots of ERK1/2 phosphorylation. Bottom: means ± SE of 3 experiments. *P < 0.05, comparison of control and treated cells.

To determine whether PKC is required for ANG II-stimulated ERK1/2 phosphorylation, we used the PKC inhibitor bisindolylmaleimide (which at a dose of 10-5 M inhibits most PKC isoforms, including PKC-zeta ) (26). Neither the basal level nor ANG II-stimulated ERK1/2 phosphorylation were affected by this drug in VSMCs from WKY and SHR (Fig. 8A). PKC-mediated activation of the ERK1/2 pathway is functionally intact in VSMCs, because phorbol 12-myristate 13-acetate-stimulated ERK1/2 phosphorylation was abrogated by bisindolylmaleimide (Fig. 8B).


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  		Fig. 8.   Effect of the protein kinase C inhibitor bisindolylmaleimide (Bisindol) on phorbol 12-myristate 13-acetate (PMA)- or ANG II-stimulated ERK1/2 phosphorylation in WKY (A) and SHR VSMCs (B). VSMCs were pretreated with Bisindol (10-5 M) for 30 min. A: cells were stimulated with ANG II (10-7 M) for 5 min. Top: representative Western blots of ERK1/2 phosphorylation. Bottom: means ± SE of 4 experiments. *P < 0.05, comparison of control and treated cells. B: representative Western blot of ERK1/2 phosphorylation stimulated by PMA (10-5 M) for 5 min.

Pretreatment of VSMCs with LY-294002, a specific PI3 kinase inhibitor, did not affect basal ERK1/2 phosphorylation. However, LY-294002 attenuated ANG II-stimulated ERK1/2 phosphorylation in VSMCs from SHR but not from WKY (Fig. 9). These data suggest that PI3 kinase plays a role in ANG II-mediated ERK1/2-dependent pathways in SHR but not in WKY.


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  		Fig. 9.   Effect of the phosphatidylinositol-3 kinase inhibitor LY-294009 (10-5 M) on ANG II-stimulated ERK1/2 phosphorylation in WKY and SHR VSMCs. VSMCs were pretreated with LY-294009 for 30 min. Thereafter, cells were stimulated with ANG II (10-7 M) for 5 min. Top: representative Western blots of phosphorylated and unphosphorylated ERK1/2. The blots were stripped of phospho-ERK1/2 antibody [20 min incubation at 60°C with 2% SDS, 62.5 mM Tris · HCl (pH 6.8), and 0.8% 2-mercaptoethanol] and subsequently incubated with ERK1/2 antibody. Bottom: means ± SE of 4 experiments. *P < 0.05, comparison of control and treated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major novel findings in the present study are that ANG II induces protein synthesis via AT1 receptors in WKY VSMCs, whereas in SHR, ANG II stimulates both protein and DNA synthesis through AT1 and AT2 receptors in the experimental conditions used. These growth effects of ANG II are regulated by ERK1/2-dependent pathways, which are Ca2+ sensitive and PKC independent. In SHR but not in WKY, PI3 kinase plays an important role in ANG II-induced VSMC growth. These data suggest that mechanisms stimulating growth in VSMCs from SHR involve a PI3 kinase-dependent ERK1/2-regulated pathway and are different from those present in WKY VSMCs.

Both AT1 and AT2 receptor subtypes were expressed in VSMCs from WKY and SHR. AT1 and AT2 receptor subtypes are glycoproteins with at least three asparagine motif sites of glycosylation (20, 40). Conflicting data concerning the size of AT1 and AT2 receptor subtypes have been reported. The range of molecular mass values of these receptors varies between 41 and 140 kDa, depending on the level of glycosylation and phosphorylation. The lower molecular mass corresponds to the nonglycosylated form of the receptors, which are located mainly in the endoplasmic reticulum and Golgi apparatus. Moreover, the ligand-binding properties of AT1 and AT2 receptors to ANG II are not affected by the level of glycosylation (20, 40).

Competition binding assays showed the presence but did not allow quantification of the density of AT2 receptors in VSMCs. It is well know that the expression of AT2 receptors is very low in adult VSMCs. The technique was not sensitive enough to precisely quantify their very low density (<30 fmol/mg of proteins) (43). With the use of a specific AT2 radioligand ([125I]-labeled CGP-42112A), Kambayashi et al. (16) found that aortic SMCs expressed ~15 fmol/mg of AT2 receptor binding sites. This amount of receptor was detectable by Western blot (34). We performed Western blot analysis, which revealed the presence of AT2 receptors in mesenteric VSMCs from WKY and SHR, using the same antibody as Ruiz-Ortega et al. (34). Thus mesenteric VSMCs do appear to express low densities of AT2 receptors even if they cannot be quantified by radioligand assay. Although the density of AT2 receptors was much lower than that of AT1 receptors, an effect of AT2 receptors on growth of VSMC from SHR was observed, as demonstrated by the growth-inhibitory effect of the AT2 receptor-selective antagonist. AT2 receptors contribute to VSMC growth in SHR by unknown mechanisms. These findings are supported by results from Ruiz-Ortega et al. (34), who demonstrated that the AT2 receptor mediates ANG II-induced NF-kappa B activation in aortic SMCs.

The AT1 receptor has been linked to vascular remodeling because of its implication in SMC hypertrophy and/or hyperplasia, extracellular matrix deposition, and inflammatory responses. In contrast, the physiological function of AT2 receptors has not been clearly defined, although several studies suggest that they have a proapoptotic effect on VSMCs from normal rats. Other studies (34, 49) suggest that AT2 receptors play a role in differentiation and inflammation. The AT2 receptor is expressed in adult tissues such as the brain, heart, aorta, mesenteric arteries, and renal and skeletal muscle vasculature (3, 28, 31, 41). In this study, ANG II induced hypertrophy in VSMCs from WKY and hypertrophy/hyperplasia in VSMCs in SHR. Hypertrophy and hyperplasia induced by ANG II seem to be mediated via AT1 and AT2 receptors in VSMCs from SHR. To our knowledge, these data demonstrate for the first time the role of the AT2 receptor in VSMC growth from SHR. AT2 receptor-mediated cell growth has been demonstrated in other cells such as A10 smooth muscle cell (37) and endothelial and cardiac fibroblasts after myocardial infarction (19). Previous in vivo studies have revealed controversial results concerning the role of the AT2 receptor on VSMC growth in different rat models of experimental hypertension. Levy et al. (21) and Sabri et al. (36) reported that the AT2 receptor mediated aortic and coronary artery hypertrophy and differentiation, whereas Li et al. (22) found that AT1 receptors mediate vascular hypertrophy in different vascular beds in chronic ANG II-infused Wistar rats. However, Cao et al. (3) demonstrated that both AT1 and AT2 receptors mediated mesenteric vascular hypertrophy and VSMC proliferation in ANG II-infused rats. AT2 receptors have also been shown to induce aortic SMC hypertrophy in SHR (31). These discrepancies may be related to different ages and strains of rats, different models of hypertension, or different experimental conditions. The exact role of AT2 receptors in the regulation of VSMC growth and differentiation in hypertension awaits further clarification.

ANG II induces phosphorylation on tyrosine residues of many proteins, including ERK1/2. These kinases are implicated in cell growth. Results from the present study demonstrate that inhibition of MEK, and subsequently ERK1/2 inhibition, attenuated protein and DNA synthesis induced by ANG II in VSMCs from WKY and SHR, respectively. Kinetic studies reveal that in SHR VSMCs, ERK1/2 phosphorylation was enhanced and sustained compared with WKY VSMCs. These responses were independent of ERK1/2 expression because expression of ERK1/2 was the same in WKY and SHR-derived cells. ERK1/2 activation induced by ANG II or insulin was higher in aortic SMCs derived from SHR than in those derived from WKY (2, 48). Similar results have been observed in VSMCs derived from hypertensive patients (46). Mii et al. (27), who studied the kinetics of ERK1/2 activation, demonstrated that the proliferative potency of an agonist depends on the duration of ERK1/2 activation. The abnormal ERK1/2 activity induced by ANG II in SHR cells may underlie the enhanced proliferative effect of ANG II in hypertension. In addition, this could also be a consequence of activity of phosphatases such as MKP-1, implicated in ERK1/2 dephosphorylation.

ANG II-stimulated ERK1/2 phosphorylation was mediated via AT1 receptors in VSMCs from SHR and WKY. AT2 receptors have been suggested to counteract the AT1 receptor-mediated tyrosine kinase activation by inducing phosphotyrosine phosphatase activation and to subsequently inhibit cell proliferation by modulating ERK1/2 activation (14). Although VSMCs expressed AT2 receptors, ANG II-stimulated ERK1/2 phosphorylation was not exaggerated in the presence of PD-123319, which suggests that the AT2 receptor is not coupled to ERK1/2-dependent signaling pathways.

To clarify the intracellular mechanisms underlying augmented ANG II-stimulated ERK1/2 phosphorylation in SHR cells, we investigated the modulatory role of three different pathways that have been shown to control ERK1/2 activation: intracellular Ca2+, PKC, and PI3 kinase. ANG II increases intracellular Ca2+, leading to VSMC contraction, which may be dependent on ERK1/2 activation (45). Ca2+ modulates various upstream kinases of the ERK1/2 pathway such as Ca2+/calmodulin-dependent protein kinase II (1) and proline-rich tyrosine kinase 2 (6, 35). Eguchi et al. (9) showed a Ca2+-dependent transactivation of the epidermal growth factor (EGF) receptor, leading to ERK1/2 activation induced by ANG II. Our data demonstrated the same dependency of Ca2+-mediated ERK1/2 activation by ANG II in cells derived from WKY and SHR. In contrast, Lucchesi et al. (24) reported a greater dependency on Ca2+ in ANG II-stimulated ERK1/2 in aortic SMCs derived from SHR compared with those derived from WKY. This difference in the control of ANG II signaling pathways confirms that different responses may be found in VSMCs from different vascular beds (42).

The expression pattern of PKC isoforms differs between different vascular beds. Of the 12 known members of the PKC family, rat mesenteric VSMCs express classic (alpha  and gamma ), novel (delta  and epsilon ), and atypical (zeta ) isoforms (29). PKC plays an important role in ANG II signaling pathways and has been implicated in ANG II-induced VSMC contraction (30) and growth (13). In our study, PKC inhibition did not alter ANG II-induced actions, suggesting that ERK1/2-mediated growth effects in mesenteric VSMCs in WKY or SHR are not dependent on PKC, in agreement with previous results (8, 13). However, some studies (25, 48, 52) have demonstrated that PKC downregulation or PKC inhibition decreased ERK1/2 phosphorylation. Some isoforms, such as PKC-epsilon and -zeta , have been implicated in ERK1/2 activation induced by ANG II in rat aortic SMCs (25) and human vein SMCs (23). Thus it seems likely that PKC-dependent ERK1/2 activation is cell type specific.

Intracellular signaling pathways mediated via PI3 kinases regulate several cell functions such as mitogenesis, differentiation, and cell motility and survival (44). ANG II phosphorylates and activates PI3 kinase in VSMCs (38). The mechanism whereby ANG II induced PI3 kinase activation is unclear, but EGF receptor transactivation may be important, because ANG II phosphorylates and activates EGF receptors (9). The EGF receptor is linked to the PI3 kinase pathway. It is also possible that ANG II stimulates PI3 kinase directly, because the beta gamma -complex of heterotrimeric G proteins activates PI3 kinases (17). There is emerging evidence that activation of VSMC ERK1/2 by some agonists is mediated by PI3 kinases in SMCs (4, 5, 32, 33). Because PI3 kinase inhibition affected only ANG II-stimulated ERK1/2 activation in SHR cells, this provides evidence that PI3 kinase-dependent ERK1/2-activated pathways may play a role in SHR but not in WKY. This is supported by another study (50) demonstrating that PI3 kinase activity is increased by ANG II, and this has a modulatory effect on neurons from SHR but not from WKY. ANG II-induced ERK1/2 has also been shown to be independent of PI3 kinase in VSMCs from the Sprague-Dawley rat (7).

In conclusion, the present study demonstrates novel differential growth effects and signaling pathways of ANG II in VSMCs from WKY and SHR. In SHR VSMCs, ANG II induces proliferation via AT1 and AT2 receptors. In contrast, in WKY rats, ANG II induces VSMC hypertrophy exclusively via AT1 receptors. The signal transduction pathways that mediate AT1 receptor-induced proliferation in SHR VSMCs are associated with enhanced and sustained ERK1/2 activation regulated by PI3 kinase-mediated Ca2+-sensitive pathways independent of PKC. The mechanisms through which AT2 receptors contribute to cell growth in SHR VSMCs require further investigation. Our observations may explain, in part, processes whereby ANG II induces its pathological growth effects in VSMCs from SHR, contributing thus to blood pressure elevation in this genetic model of hypertension.


    ACKNOWLEDGEMENTS

This study was supported by a Medical Research Council of Canada (MRC; now the Canadian Institutes of Health Research) Group Grant (to the Multidisciplinary Research Group on Hypertension) and by MRC Grants 13570 and 14080. M. El Mabrouk was the recipient of a studentship from the Canadian Heart and Stroke Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: E. L. Schiffrin, Clinical Research Institute of Montreal, 110 Pine Ave. W, Montreal, Quebec, Canada H2W 1R7 (E-mail: schiffe{at}ircm.qc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 October 2000; accepted in final form 26 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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