Identification of an Essential Signaling Cascade for Mitogen-activated Protein Kinase Activation by Angiotensin II in Cultured Rat Vascular Smooth Muscle Cells POSSIBLE REQUIREMENT OF G q -MEDIATED p21 ras ACTIVATION COUPLED TO A Ca 2 (cid:49) /CALMODULIN-SENSITIVE TYROSINE KINASE*

In cultured rat vascular smooth muscle cells, angio- tensin II (Ang II) induced a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor, which was insensitive to pertussis toxin but was abolished by the phospholipase C in- hibitor, U73122. The Ang II-induced MAPK activation was not affected by the protein kinase C inhibitor, GF109203X, and was only partially impaired by pretreatment with a phorbol ester, whereas both treatments completely prevented MAPK activation by the phorbol ester. Intracellular Ca 2 (cid:49) chelation by TMB-8, but not extracellular Ca 2 (cid:49) chelation or inhibition of Ca 2 (cid:49) influx, abolished Ang II-induced MAPK activation. The calmod- ulin inhibitor, calmidazolium, and the tyrosine kinase inhibitor, genistein, completely blocked MAPK activa- tion by Ang II as well as by the Ca 2 (cid:49) ionophore A23187. Ang II caused a rapid increase in the binding of GTP to p21 ras , and this was inhibited by genistein, TMB-8, and calmidazolium but not by pertussis toxin or GF109203X. These data suggest that Ang II-induced MAPK activation through the Ang II type 1 receptor could be medi- ated by p21 ras activation through a currently unidentified

In cultured rat vascular smooth muscle cells, angiotensin II (Ang II) induced a rapid increase in mitogenactivated protein kinase (MAPK) activity through the Ang II type 1 receptor, which was insensitive to pertussis toxin but was abolished by the phospholipase C inhibitor, U73122. The Ang II-induced MAPK activation was not affected by the protein kinase C inhibitor, GF109203X, and was only partially impaired by pretreatment with a phorbol ester, whereas both treatments completely prevented MAPK activation by the phorbol ester. Intracellular Ca 2؉ chelation by TMB-8, but not extracellular Ca 2؉ chelation or inhibition of Ca 2؉ influx, abolished Ang II-induced MAPK activation. The calmodulin inhibitor, calmidazolium, and the tyrosine kinase inhibitor, genistein, completely blocked MAPK activation by Ang II as well as by the Ca 2؉ ionophore A23187. Ang II caused a rapid increase in the binding of GTP to p21 ras , and this was inhibited by genistein, TMB-8, and calmidazolium but not by pertussis toxin or GF109203X. These data suggest that Ang II-induced MAPK activation through the Ang II type 1 receptor could be mediated by p21 ras activation through a currently unidentified tyrosine kinase that lies downstream of G q -coupled Ca 2؉ /calmodulin signals.
The peptide hormone angiotensin II (Ang II) 1 evokes diverse physiological responses, including arterial vasoconstriction, stimulation of aldosterone secretion, and renal sodium reabsorption (1). In addition, it is a growth-promoting factor for vascular smooth muscle cells (VSMC) (2)(3)(4), renal mesangial cells (5), cardiomyocytes (6), and cardiac fibroblasts (7). Since Ang II is believed to play a pivotal pathogenic role in the development of cardiovascular diseases such as hypertension and atherosclerosis (8,9), there has been considerable interest in defining its signaling pathways that mediate the growth response of VSMC.
Pharmacological evidence has defined at least two subtypes of Ang II receptors, designated AT 1 and AT 2 (1). Recent molecular cloning has revealed that both receptor subtypes belong to the superfamily of G protein-coupled receptors with seven transmembrane helices (10 -13). In cultured rat VSMC, AT 1 activation by Ang II is initiated by stimulation of a phosphatidylinositol-specific phospholipase C (PI-PLC), leading to the generation of inositol trisphosphate (IP 3 ) and diacylglycerol (14), which are involved in intracellular Ca 2ϩ mobilization (15) and protein kinase C (PKC) activation (16), respectively. In VSMC, Ang II also induces a rapid increase in expression of the growth-associated nuclear proto-oncogenes, c-fos, c-jun, and c-myc (17)(18)(19) and stimulates tyrosine phosphorylation of multiple substrates (20), including mitogen-activated protein kinases (MAPKs) (20 -22).
MAPKs, also known as extracellular signal-regulated kinases, are a family of protein-serine/threonine kinases that are believed to function as integrators for mitogenic signals originating from several distinct classes of cell surface receptors, such as receptor tyrosine kinases and G protein-coupled receptors (23,24). In their activated forms, p44 mapk (ERK1) and p42 mapk (ERK2) transmit extracellular stimuli by phosphorylating a variety of substrates including transcriptional factors and kinases (25). MAPKs are activated by phosphorylation of both threonine and tyrosine residues (26) catalyzed by an MAPK kinase (27) also known as MEK (28). MEK is in turn regulated by serine phosphorylation by several MAPK kinase kinases, including Raf-1 (29,30). Recently, the cascade from growth factor receptor tyrosine kinases to MAPK has been elucidated. The adapter protein Grb2 links the tyrosine-phosphorylated receptor to Sos, which acts as a guanine nucleotide exchange factor for p21 ras (31), and the active GTP-bound p21 ras stimulates Raf-1 kinase activity toward MEK (32,33). However, the pathway originating from G protein-coupled receptors in the activation of MAPK is not clearly defined.
Earlier reports proposed a dominant role of PKC in the mechanism of Ang II-mediated MAPK activation in VSMC (20,21), whereas more recent studies have indicated that calcium signals rather than PKC are critical for MAPK activation by Ang II in cardiac cells (34,35). To define the signal transduction cascades leading to MAPK activation by the AT 1 receptor, we examined the roles of various signaling molecules activated by Ang II through AT 1 in cultured rat aortic VSMC. We found that AT 1 signals to p21 ras and subsequently to MAPK, possibly through Ca 2ϩ /calmodulin-sensitive activation of a protein tyrosine kinase by a G q -coupled pathway. Eagle's medium (DMEM), fetal calf  serum, penicillin, and streptomycin were obtained from Life Technologies, Inc. Ang II was purchased from Peninsula Laboratories. U73122,  GF109203X, BAPTA-AM, TMB-8, nifedipine, W-7, calmidazolium chloride, genistein, and ST638 were purchased from Calbiochem. The AT 1 antagonist DUP753 was a generous gift of DuPont Merck Pharmaceutical Co., and the AT 2 antagonist PD123319 was purchased from Research Biochemicals, Inc. Pertussis toxin (PTX), phorbol 12-myristate 13-acetate (PMA), lysophosphatidic acid (LPA), and EGTA were obtained from Sigma.

Materials-Dulbecco's modified
Cell Culture-VSMC were prepared from the thoracic aorta of 12week-old Sprague-Dawley rats (Charles River Laboratories) by the explant method and cultured in DMEM containing 10% fetal calf serum, penicillin, and streptomycin as described previously (36). Subcultured VSMC from passages 3-15, used in the experiments, showed Ͼ99% positive immunostaining of smooth muscle ␣-actin antibody (Sigma) and were negative for mycoplasma infection by the polymerase chain reaction kit (Stratagene). 2 The expressions of AT 1 receptors 2 and endothelin type A receptors (36) were confirmed on the basis of binding studies with specific receptor antagonists. For the experiments, cells at ϳ80% confluence in culture wells were made quiescent by incubation with serum-free DMEM for 3 days, unless otherwise stated.
MAPK Activity-VSMC grown on a 24-well plate were stimulated with agonists at 37°C in serum-free DMEM for specified durations. The reaction was terminated by the replacement of medium with the icecold lysis buffer (10 mM Tris-HCl, pH 7.4, 20 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin). After brief sonication (10 s), the samples were centrifuged for 5 min at 14,000 ϫ g, and the supernatant was assayed for MAPK activity with an assay kit (Amersham Corp.) that measures the incorporation of [␥-33 P]ATP into a synthetic peptide (KRELVEPLTPAGEAPNQALLR) as a specific MAPK substrate. The reaction was carried out with the cell lysate (ϳ1 g of protein) in 75 mM HEPES buffer, pH 7.4, containing 1.2 mM MgCl 2 , 2 mM substrate peptide, and 1.2 mM ATP, 1 Ci of [␥-33 P]ATP for 30 min at 30°C. The resultant solution was applied to a phosphocellulose membrane and extensively washed in 1% acetic acid and then in H 2 O. The radioactivity trapped on the membrane was measured by liquid scintillation counting.
Immunoblotting-VSMC grown on a 6-well plate were stimulated with agonists at 37°C in serum-free DMEM for specified durations. The reaction was terminated by the replacement of medium with 100 l of SDS-polyacrylamide gel electrophoresis buffer, pH 6.8, containing 62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue. After brief sonication (5 s), samples were boiled for 5 min at 95°C and centrifuged (14,000 ϫ g, 5 min) at 4°C, and the supernatant (25 l) was subjected to SDS-polyacrylamide gel electrophoresis. Proteins in the gel were transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell) by electroblotting. The membrane was treated with rabbit polyclonal phospho-specific MAPK antibodies (New England Biolabs Inc.) that detect p42 mapk and p44 mapk only when catalytically activated by phosphorylation at Tyr-204. After incubation with secondary anti-rabbit antibodies, immunoreactive proteins were detected by the CDP-Star chemiluminescent system (New England Biolabs Inc.).
Analysis of GTP-bound Ras-Detection of guanine nucleotides bound to p21 ras was performed essentially as described previously (37). VSMC grown on a 6-well plate were prelabeled with 0.1 mCi/ml carrier-free 32 P-orthophosphate for 18 h in phosphate-free DMEM. Cells were stimulated with agonists at 37°C for specified durations. The reaction was terminated by aspirating the media, and cells were solubilized in 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 20 mM MgCl 2 , 1 mM Na 3 VO 4 , 0.4 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin. After centrifugation for 5 min at 2000 ϫ g, the supernatant was immunoprecipitated with an anti-Ha-Ras-agarose conjugate (Santa Cruz Biotechnology, Inc.) for 90 min at 4°C. The immune complexes were washed 4 times with solubilization buffer, and p21 ras -associated guanine nucleotides were eluted in 2 mM EDTA, pH 8.0, 2 mM dithiothreitol, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP for 20 min at 65°C. Eluted GTP and GDP were separated on a polyethyleneimine cellulose plate by thin layer chromatography using 1.2 M ammo-nium formate, 0.8 M hydrochloric acid as the solvent. Labeled nucleotides were visualized and analyzed by PhosphorImager (Molecular Dynamics).

Ang II Type 1 Receptor Stimulation Leads to MAPK Activation-
In cultured rat VSMC, the Ang II-induced (100 nM) stimulation of MAPK activity peaked at 5 min and declined in 10 -30 min (Fig. 1A). The MAPK activation was dependent on the concentration of Ang II (Fig. 1B); increased activity was initially detectable at 0.01 nM, half-maximal at approximately 0.2 nM, and maximal at 10 -100 nM. Treatment with 100 nM Ang II for 5 min also resulted in marked tyrosine phosphorylation of p44 mapk and p42 mapk in VSMC, whereas no significant phosphorylation was observed without the stimulation (for examples see Figs. 2, 3, 5, and 7). Therefore, subsequent MAPK experiments were performed with 100 nM Ang II stimulation for 5 min. To determine which Ang II receptor subtype mediates MAPK activation, VSMC were pretreated with either the AT 1 antagonist DUP753 or the AT 2 antagonist PD123319. MAPK activation by Ang II was markedly inhibited by 10 M DUP753 but not by 10 M PD123319 (Fig. 1C), indicating that Ang II-induced MAPK activation is mainly mediated by the AT 1 receptor in VSMC. 2 S. Eguchi and T. Inagami, unpublished data. Ang II-induced MAPK Activation Requires Phospholipase C Activation through a Pertussis Toxin-insensitive G Protein-AT 1 receptors have been reported to be coupled to either G q or G i , which activates PI-PLC or inhibits adenylate cyclase, respectively (1,38). To determine which G protein-mediated signaling is involved in the MAPK activation, the effects of PTX and the specific PI-PLC inhibitor U73122 (39) on Ang II-induced MAPK activation were studied in VSMC. Treatment with PTX (1 g/ml) for 24 h did not affect Ang II-induced MAPK activation, whereas it inhibited MAPK activation induced by LPA that had been reported to use G i for MAPK activation (40, 41) ( Fig. 2A). In contrast, U73122 completely suppressed Ang II-induced MAPK activation in a dose-dependent manner without affecting basal MAPK activity (Fig. 2B). The half-maximal inhibition (ϳ2.5 M) was comparable with that for PI-PLC (39). U73122 (10 M) also inhibited Ang IIinduced tyrosine phosphorylation of p44 mapk and p42 mapk in VSMC (Fig. 2C). These data suggest that PI-PLC activation through a PTX-insensitive G protein (probably G q ) plays a critical role in Ang II-induced MAPK activation in VSMC.
Roles of Protein Kinase C in Ang II-induced MAPK Activation-In cultured VSMC, PI-PLC activation by Ang II leads to production of two second messengers, IP 3 and diacylglycerol (14), that induce the release of Ca 2ϩ from intracellular stores (15) and PKC activation (16). Since PKC activation by a phorbol ester has been reported to stimulate MAPK (42), we examined whether phorbol ester-sensitive PKC is essential for Ang II-induced MAPK activation in VSMC. Although depletion of PKC by a 24-h pretreatment with PMA (10 -1000 nM) moderately increased basal MAPK activity, it completely inhibited MAPK activation by PMA (100 nM) in a dose-dependent manner (Fig. 3A), confirming the completeness of the PKC depletion. However, Ang II-induced MAPK activation was only partially inhibited by the PMA pretreatment, suggesting a dominant role of a PKC-independent mechanism in Ang IIinduced MAPK activation in VSMC.
The partial inhibition of Ang II-induced MAPK activation by prolonged PMA treatment may not necessarily indicate dependence on a phorbol ester-sensitive PKC. It could also be due to the inhibition or desensitization of a signaling mechanism upstream of MAPK because the cells were initially stimulated by a phorbol ester that had been shown to affect the expression of a variety of genes (43). In fact, PKC-mediated desensitization and down-regulation of Ang II receptors have been reported in cultured VSMC (44,45). Therefore, we further examined the effect of the specific PKC inhibitor GF109203X on Ang IIinduced MAPK activation in VSMC. Pretreatment with 2 M GF109203X completely inhibited MAPK activation in response to 100 nM PMA without affecting basal MAPK activity, whereas no significant inhibition was observed in Ang II-induced MAPK activation (Fig. 3B). GF109203X (2 M) also inhibited tyrosine phosphorylation of p44 mapk and p42 mapk induced by PMA but not by Ang II in VSMC (Fig. 3, C and D). These data indicate that Ang II-induced MAPK activation was at least independent of GF109203X-sensitive PKC in VSMC.
Calcium and Calmodulin-dependent MAPK Activation by Ang II-In cultured VSMC, Ang II has been shown to cause a rapid and transient elevation of cytosolic Ca 2ϩ released from the IP 3 -sensitive intracellular stores by the activation of PI-PLC. This is followed by a sustained elevation of cytosolic Ca 2ϩ through its influx mediated by an L-type Ca 2ϩ channel (46,47). Since intracellular Ca 2ϩ elevation has been reported to be a sufficient stimulus for MAPK activation (48), we sought to determine whether the MAPK activation by Ang II was Ca 2ϩdependent. Pretreatment with BAPTA-AM (10 M) or TMB-8 (100 M), drugs commonly used as intracellular Ca 2ϩ chelators, resulted in complete loss of MAPK activation induced by Ang II (Fig. 4, A and B). In contrast, extracellular Ca 2ϩ chelation by EGTA or blockade of L-type Ca 2ϩ channels with nifedipine failed to inhibit Ang II-induced MAPK activation (Fig. 4, C and D) even though these treatments abolished the Ang II-induced sustained phase of Ca 2ϩ elevation. 2 Parallel inhibitory patterns in Ang II-induced tyrosine phosphorylation of p44 mapk and p42 mapk were observed with BAPTA-AM and TMB-8, whereas nifedipine did not affect the tyrosine phosphorylation of MAPKs (Fig. 5). These data indicate that the release of Ca 2ϩ from IP 3 -sensitive stores, rather than Ca 2ϩ influx, may play a major role in Ang II-induced MAPK activation in VSMC.
Elevation of cytosolic Ca 2ϩ is known to activate a variety of enzymes through its interaction with calmodulin (49). To examine whether calmodulin mediates MAPK activation in response to Ang II, VSMC were preincubated with well characterized calmodulin inhibitors, W-7 or calmidazolium. Although pretreatment with high concentrations of W-7 decreased basal MAPK activity, it dose dependently and completely inhibited Ang II-induced MAPK activation (Fig. 6A). Half-maximal inhibition was observed at ϳ25 M, which was comparable with that for Ca 2ϩ /calmodulin-dependent phosphodiesterase (50). Pretreatment with 10 M calmidazolium also completely inhibited both activation of MAPK (Fig. 6B) and tyrosine phosphorylation of p44 mapk and p42 mapk (data not shown) induced by Ang II in VSMC. Treatment with a Ca 2ϩ ionophore, A23187 (10 M), resulted in marked stimulation of MAPK activity that was also inhibited by calmidazolium but not by GF109203X (Fig.  6C). These data suggest that Ang II stimulates MAPK activity through a Ca 2ϩ /calmodulin-dependent mechanism.
Roles of Protein Tyrosine Kinase in Calcium-dependent MAPK Activation-Ang II has been shown to cause a rapid increase in tyrosine phosphorylation of multiple cellular proteins prior to MAPK activation in VSMC (20). To determine whether tyrosine kinase activity is required for the Ca 2ϩ -dependent MAPK activation in response to Ang II, VSMC were pretreated with genistein, a protein kinase inhibitor with a strong preference for tyrosine-specific kinases that acts as a competitive inhibitor of ATP binding (51), and stimulated by either Ang II or A23187. Genistein (100 M) completely abolished both Ang II-and A23187-induced MAPK activations without affecting basal MAPK activity in VSMC (Fig. 7A). A similar inhibitory effect of genistein was observed in tyrosine phosphorylation of p44 mapk and p42 mapk induced by Ang II (Fig.  7B). To confirm the effect of genistein, we tested another specific tyrosine kinase inhibitor (ST638) that acts as a competitive inhibitor of substrate binding (52). Pretreatment with 100 M ST638 for 60 min significantly inhibited MAPK activation induced by Ang II or A23187 in VSMC (data not shown). These data point to the possibility that Ang II-induced MAPK activation is at least in part mediated by a currently unidentified protein tyrosine kinase that lies downstream of a Ca 2ϩ /calmodulin-activated system in VSMC.
Ang II Increases GTP-bound Ras-To further characterize the signaling cascade leading to MAPK activation in response to Ang II, we tested whether Ang II stimulates p21 ras activity in VSMC. Ang II (100 nM) induced a rapid accumulation of GTP-bound p21 ras that reached a maximum in 3-4 min, when it increased approximately 2-fold (Fig. 8, A and B). This response returned to the base-line level in 20 min (Fig. 8B). We also found that treatment with 10 M A23187 increased the GTP-bound p21 ras in VSMC to a similar extent (Fig. 8C). To investigate whether the activation of p21 ras and MAPK by Ang II requires similar upstream signaling, the effects of several signal transduction inhibitors were tested on Ang II-induced p21 ras activation. Pretreatment with genistein (100 M), TMB-8 (100 M), or calmidazolium (10 M), but not with PTX (1 g/ml) or GF109203X (2 M), prevented Ang II-induced p21 ras activation (Fig. 9). Taken together, these data suggest that an AT 1 -mediated Ca 2ϩ /calmodulin signal initiated by G q may activate the sequential cascade from p21 ras to MAPK, a pathway common to the tyrosine kinase receptor, through a tyrosine kinase-dependent mechanism in VSMC.

DISCUSSION
The signaling mechanism leading to MAPK activation from heterotrimeric G protein-coupled receptors has not been clearly defined yet. In the present study, we have proposed a novel signaling pathway from AT 1 to the MAPK cascade involving p21 ras activation mediated by a Ca 2ϩ /calmodulin-dependent tyrosine kinase.
As we demonstrated by specific Ang II receptor antagonists, G protein-coupled AT 1 stimulation leads to MAPK activation in cultured rat VSMC. The cloned AT 1 receptor can couple to either G q or G i (38). Recent evidence suggests that the G icoupled LPA receptor induces PTX-sensitive MAPK activation through p21 ras stimulation (40,41,53). This process is believed to be mediated by the ␤␥ subunit of G i proteins (54 -56). In the present study, control experiments using LPA as the agonist showed that PTX treatment abolished its MAPK activation in VSMC, indicating the presence of this cascade in our VSMC. However, Ang II-induced MAPK activation was PTX-insensi- tive in VSMC. This is in good agreement with a well accepted observation that Ang II couples to a PTX-insensitive G protein (G q ) to activate PI-PLC (57). We further showed that Ang II-induced MAPK activation was suppressed by the PI-PLC inhibitor, U73122. Although the precise site of action of U73122 is unclear, previous studies suggest that it may be at the level of G q or at the link between G protein and the effector enzyme (58). Taken together, these observations indicate that AT 1 transmits its signal through the ␣ subunit of G q (G q ␣) rather than the G␤␥ dissociated from G i in the activation of MAPK in VSMC.
It has been proposed that a G q -coupled receptor can activate MAPK through PKC activation (54,59). PKC is known to activate MAPK presumably by directly phosphorylating Raf-1 (60). Since the present study showed that Ang II-induced MAPK activation was PI-PLC-dependent in VSMC and Ang II has been shown to stimulate PKC activity in VSMC through PI-PLC activation (14,61), Ang II could activate the Raf-1-MAPK system by activated PKC. Indeed, it has been reported that prolonged phorbol ester treatment significantly suppressed Ang II-induced Raf-1 and MAPK activation in cultured rat VSMC (20). However, the present study clearly showed that PKC depletion by prolonged PMA treatment had only a minor effect on MAPK activation by Ang II compared with that by PMA. In addition, we found that the PKC inhibitor GF109203X, which has been shown to inhibit PKC-␣, -␤I, -␤II, and -␥ (62), had no effect on MAPK activation by Ang II, whereas it completely blocked the activation by PMA. Therefore, our data indicate the dominant role of a PKC-independent pathway in Ang II-induced MAPK activation in VSMC; the partial inhibition by prolonged PMA treatment may be due to the down-regulation and/or desensitization of AT 1 in VSMC as reported (44,45). However, we cannot exclude the possibility that GF109203X and phorbol ester-insensitive isoforms of PKC, such as PKC-(63), may play a role in Ang II-induced activation of MAPK.
By contrast, the stimulating effect of the Ca 2ϩ ionophore and the inhibitory effect of intracellular Ca 2ϩ chelators in the present study suggest a critical role of Ca 2ϩ in Ang II-mediated MAPK activation in VSMC. This is in agreement with the recent report demonstrating that in rat cardiac fibroblasts the Ang II analogue [Sar 1 ]Ang II (which is only a weak PKC activator in those cells) as well as the Ca 2ϩ ionophore ionomycin induced MAPK activation even under PKC-depleted conditions (34). Also, a similar Ca 2ϩ -dependent but PKC-independent MAPK activation by Ang II was observed in rat cardiac myocytes (35). In the present study, we obtained further evidence that this Ca 2ϩ -dependent MAPK activation by Ang II was mainly mediated by the release of Ca 2ϩ from IP 3 -sensitive stores, rather than by Ca 2ϩ influx through the L-type Ca 2ϩ channel, by demonstrating that Ang II-induced MAPK activation was eliminated by a PI-PLC inhibitor, whereas it was insensitive to the L-type Ca 2ϩ channel blocker or the extracellular Ca 2ϩ chelation by EGTA.
Although detailed mechanisms by which increased Ca 2ϩ results in the activation of MAPK have not been clearly determined, the involvement of Raf-1 has been suggested (64). The results presented here demonstrated that both Ang II and A23187-induced MAPK activation were blocked by a well known calmodulin inhibitor, calmidazolium, and two different types of tyrosine kinase inhibitors, genistein and ST638. In cultured rat liver epithelial cells (65) and VSMC (66), Ang II-induced activation of tyrosine kinases has been shown to be dependent on Ca 2ϩ . A similar mechanism may operate in the Ang II-induced MAPK activation involving an unidentified Ca 2ϩ /calmodulin-activated tyrosine kinase. Recently, such a Ca 2ϩ /calmodulin-activated tyrosine kinase has been purified from calf uterus (67). Alternatively, other tyrosine kinases such as a Src, acting upstream of Ca 2ϩ /calmodulin signals, have been proposed to mediate Ang II-induced phospholipase C-␥ activation in VSMC (68,69).
Our present study produced evidence that Ang II and A23187 activate p21 ras in cultured rat VSMC. Interestingly, Ang II-induced p21 ras activation was also blocked by either the chelation of intracellular Ca 2ϩ or by the inhibition of calmodulin or tyrosine kinases in VSMC, suggesting a common pathway shared with the MAPK activation as discussed above. It has been shown that the adaptor protein Shc is involved in the coupling of both receptor and non-receptor tyrosine kinases to the Ras/MAPK signaling pathway (70). Tyrosine-phosphorylated Shc can activate p21 ras by binding to the SH2 domain of another adaptor protein, Grb2, that is complexed to the guanine nucleotide exchange factor Sos through its SH3 domains. A recent report showed that Ang II rapidly stimulates p46 and p64 Shc tyrosine phosphorylation in rat cardiac fibloblasts (71). In addition, Ang II-induced Shc phosphorylation resulted in subsequent formation of a complex between Shc and Grb2 in VSMC (72). Collectively, our results suggest that the Ca 2ϩ -dependent MAPK activation by Ang II may use the pathway common to the tyrosine kinase receptor that is known to be initiated by tyrosine phosphorylation-mediated Shc-Grb2-Sos complex formation resulting in the activation of p21 ras . This pathway has been recently shown to contribute also to the MAPK activation mediated by G␤␥ dissociated from G i (73).
The identity of the Ca 2ϩ -dependent tyrosine kinases involved in Ang II-stimulated increases in p21 ras and MAPK activity remains unknown. One of the likely candidates is the recently cloned PYK2 (74), a member of the Fak family of non-receptor tyrosine kinase that has been shown to transmit the calcium signal from a G protein-coupled receptor to the formation of the Shc-Grb2-Sos complex, thereby activating MAPK. However, PYK2 lacks a calmodulin binding motif and is not directly activated by Ca 2ϩ , suggesting the requirement of additional upstream regulatory molecules directly activated by Ca 2ϩ /calmodulin. The other possible candidate is the Ras-GRF exchange factor in response to Ca 2ϩ signals (75). Although Ras-GRF has been shown to be activated by calmodulin binding to its IQ motif, its expression is limited to brain neurons (75). The characterization and identification of the putative effector molecules that transmit Ang II-induced calcium signals to It is likely that other signaling pathways capable of mediating MAPK activation via the AT 1 receptor may exist. The typical G q -coupled ␣ 1B adrenergic receptor and M1 muscarinic cholinergic receptor transfected into COS-7 cells have been shown to induce PKC-dependent and p21 ras -independent MAPK activation that is insensitive to genistein (59), whereas PKC was also reported to induce p21 ras -dependent MAPK activation in PC12 cells (76). The anaphylatoxin C5a receptor, which can be coupled to either G i or G 16 (a homologue of G q ), requires both a G i -mediated G␤␥ signal and G 16 ␣-mediated PKC activation for maximal MAPK activation (77). These data support the concept that the mechanism utilized by a given receptor to stimulate MAPK activation is likely to depend on the class of G proteins and downstream components available in a given cell type (59). Therefore, under physiological conditions, it is possible that additional signaling molecules such as G␤␥ and PKC may contribute synergistically to Ang II-induced MAPK activation in a cell-and tissue-type-dependent manner.
In summary, we have presented several lines of evidence that Ang II-induced p21 ras and MAPK activation in VSMC is mainly mediated by a Ca 2ϩ /calmodulin-dependent tyrosine kinase through PI-PLC-mediated Ca 2ϩ release coupled to G q . The cascade demonstrated here will have to be considered when defining the pathophysiological role of Ang II in the abnormal growth of VSMC observed in cardiovascular diseases.