Cyclic AMP inhibition of thrombin-induced growth in vascular smooth muscle cells correlates with decreased JNK1 activity and c-Jun expression.

Thrombin is a potent modulator of vascular tone and vascular smooth muscle cell (VSMC) mitogenesis. Early studies from other laboratories demonstrated that cyclic AMP (cAMP) antagonizes the mitogenic effects of platelet-derived growth factor and epidermal growth factor by inhibiting the extracellular signal-regulated protein kinases (ERKs; p42, p44) group of mitogen-activated protein kinases (MAPKs) in several cell types. This report examines the role of ERKs and Jun N-terminal kinase 1 (JNK1) groups of mitogen-activated protein kinases in thrombin-induced DNA synthesis in VSMCs using agents such as forskolin and dibutyrylcyclic AMP that increase intracellular cAMP levels. Both agents significantly inhibited thrombin-stimulated DNA synthesis in VSMCs. These agents, however, had no effect on thrombin induction of ERKs activation and c-Fos expression, suggesting divergence of the latter two events from the growth-signaling events of thrombin that are sensitive to inhibition by cAMP. Thrombin activated JNK1 and induced c-Jun expression in VSMCs in a time-dependent manner. In contrast to ERKs and c-Fos, thrombin-induced JNK1 activation and c-Jun expression were sensitive to inhibition by forskolin, suggesting an association of these events with thrombin-stimulated growth in these cells. Thrombin also increased AP-1 activity, and this response was significantly blunted by forskolin. Together, these results demonstrate a correlation between JNK1 activation and c-Jun expression by thrombin and their association with the mitogenic signaling events of thrombin in VSMCs.

Besides activating platelet aggregation, thrombin also induces growth in VSMCs 1 and fibroblasts (1)(2)(3). The coagulant and mitogenic effects of thrombin appear to be mediated by a seven-transmembrane G protein-coupled receptor that has been cloned and characterized in recent years (4,5). Thrombin activates its receptor by a unique mechanism involving cleavage of the N terminus of the receptor, generating a new N terminus, which, in turn, acts as a tethered ligand (4). G protein-coupled receptors, including the thrombin receptor, do not possess intrinsic tyrosine kinase activity (6). Nonetheless, their ligands, such as thrombin, require protein tyrosine kinase activity to produce mitogenic effects in VSMCs (1,6).
Protein tyrosine phosphorylation events play important roles in cell proliferation and differentiation (8,9). Upon binding, peptide growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), cause their cognate receptor tyrosine kinases to undergo autophosphorylation on tyrosine residues (8,9). The autophosphorylated receptors, by interacting with Src homology 2 and Src homology 3 domaincontaining proteins such as growth factor receptor-bound protein 2 and SHC, activate Ras (10 -12). Ras, in turn, stimulates a cascade of kinase activities leading to activation of ERKs (13)(14)(15)(16). Activated ERKs translocate to the nucleus and influence gene transcription by phosphorylating transcriptional factors such as ternary complex factor/Elk1 (17,18). In a parallel pathway, Ras also activates a kinase cascade that leads to JNK activation (19 -21). JNKs like ERKs modulate gene expression by translocating to the nucleus and phosphorylating transcriptional factors such as c-Jun/AP-1 and ATF2 (22). Although agonists of receptor tyrosine kinases preferentially activate the "ERK" pathway (23,24), cytokines such as tumor necrosis factor ␣ and cellular stresses such as UV irradiation have been shown to potently stimulate the "JNK" pathway (23,24).
A large body of evidence indicates that ERKs play an important role in integrating receptor tyrosine kinase-initiated mitogenic signaling events in many cell types (8,9,13). Because thrombin, a G protein-coupled receptor agonist, requires protein tyrosine kinase activity for its mitogenic effects in VSMCs, we have previously studied the role of ERKs in the transmission of thrombin-induced protein tyrosine phosphorylation events to cause nuclear effects in these cells. We observed a dissociation between ERKs activation and induced protein tyrosine phosphorylation events leading to DNA synthesis in VSMCs in response to thrombin (7). To understand the role of MAPKs in thrombin-induced growth in VSMCs, we have studied here the effect of cAMP on thrombin-induced DNA synthesis and ERKs, JNK1, and p38 groups of MAPKs activation in these cells. Earlier reports from several laboratories have demonstrated that cAMP inhibits the mitogenic effects of PDGF and EGF by blocking the ERK pathway at the Raf-1 level in many cell types (25,26). In the present study, we report that forskolin and dcAMP, two agents that increase intracellular cAMP levels, significantly inhibit thrombin-stimulated DNA synthesis in VSMCs without compromising the effect of thrombin on ERKs activation and c-Fos expression. On the other hand, both thrombin-induced activation of JNK1 and expression of c-Jun demonstrated significantly blunted effects by the above agents in these cells. Forskolin also decreased thrombininduced AP-1 activity. These findings thus show a correlation between JNK1 activation and growth in thrombin-treated VSMCs and demonstrate that agents that increase intracellular cAMP levels may affect G protein-coupled receptor agonistinduced growth by inhibiting JNK1 activity and c-Jun expression, but not ERKs activities and c-Fos expression, at least in this cell type. These results also reveal a correlation between JNK1 activation and c-Jun expression in thrombin-treated VSMCs.
Cell Culture-VSMCs were isolated from the thoracic aortae of 200 -250-g male Sprague-Dawley rats by enzymatic digestion as described earlier (7). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained at 37°C in a humidified 5% CO 2 atmosphere.
DNA Synthesis-VSMCs were plated onto 60-mm dishes, allowed to grow to 70 -80% confluence, and then growth-arrested by incubation in Dulbecco's modified Eagle's medium containing 0.1% calf serum for 72 h. Growth-arrested VSMCs were exposed to thrombin (0.1 unit/ml) in the presence and absence of the indicated agents for 24 h. Cells were pulse-labeled with 1 Ci/ml [ 3 H]thymidine for 2 h just before the end of the incubation period and harvested by trypsinization, followed by centrifugation. The cell pellet was resuspended in cold 10% trichloroacetic acid and vortexed vigorously to lyse the cells. The mixture was allowed to sit on ice for 20 min and then passed through a GF/F glass microfiber filter. The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% ethanol, dried, and placed in a liquid scintillation vial containing the mixture; then the radioactivity was measured in a liquid scintillation counter (Beckman LS 3801).
Western Blot Analysis-Growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) in the presence and absence of appropriate agents for the indicated time periods at 37°C. Medium was aspirated, cells were rinsed with cold phosphate-buffered saline and frozen immediately in liquid nitrogen. Two hundred fifty l of lysis buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml PMSF, 100 g/ml aprotinin, 1 g/ml leupeptin, 20 mM ␤-glycerophosphate, 2 mM sodium fluoride, 2 mM sodium pyrophosphate, and 1 mM sodium orthovanadate) was added to the frozen monolayers, thawed on ice for 15 min, and scrapped into 1.5-ml Eppendorf tubes. The cell lysates were cleared by centrifugation at 12000 rpm for 30 min at 4°C. Protein content of the supernatants was determined using the Bradford reagent from Bio-Rad. Cell lysates containing equal amounts of protein were resolved by electrophoresis on a 0.1% SDS and 10% polyacrylamide gel under reducing conditions. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Corp.). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with appropriate peroxidase-conjugated secondary antibodies. The antigen-antibody complexes were detected using chemiluminescence reagent kit (Amersham Corp.).
In-Gel Kinase Assay-Cell lysates containing equal amounts of protein (50 g/lane) from each treatment were resolved on a 0.1% SDS and 10% polyacrylamide minigel that was copolymerized with 350 g/ml MBP (7). The gel was washed twice for 30 min with 150 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 20% isopropanol and twice for 30 min with 150 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM ␤-mercaptoethanol (buffer A). After incubation for 1 h in 150 ml of 6 M guanidine hydrochloride in buffer A at room temperature, the gel was renatured by repeated washings with buffer A containing 0.04% Tween 20 at 4°C. The kinase reaction was performed by incubating the gel in 30 ml of 40 mM Hepes buffer, pH 8.0, containing 10 mM MgCl 2 , 0.5 mM EGTA, 2 mM DTT, 50 M ATP, and 5 Ci/ml [␥-32 P]ATP for 1 h at room temperature. The gel was washed several times with 200 ml of 5% trichloroacetic acid and 1% sodium pyrophosphate until the cpm were at background levels, dried, and subjected to autoradiography. To measure p38 MAPK activity, cell lysates containing equal amounts of protein (500 g) from each condition were immunoprecipitated with anti-p38 antibodies, and the immunoprecipitates were subjected to in-gel kinase assay using MBP as a substrate as described above.
Immunocomplex Kinase Assay-Cell lysates containing equal amounts of protein (500 g) from each treatment were incubated with 4 g of anti-JNK1 antibodies overnight at 4°C. Forty l of protein A-agarose beads were added to each, and incubation continued for another 2 h on ice with gentle rocking. The beads were collected by centrifugation at 8000 rpm for 1 min and were washed three times with lysis buffer and once with kinase buffer (20 mM Tris-HCl, pH 7.4, 20 mM NaCl, and 10 mM MgCl 2 ). The beads were suspended in 25 l of kinase buffer containing 1 mM DTT, 20 M ATP, 5 Ci [␥-32 P]ATP, and 100 ng of glutathione S-transferase-c-Jun and incubated for 30 min at 30°C. Reactions were stopped by adding an equal volume of 2 ϫ Laemmli Sample buffer and heating for 5 min. The mixtures were separated on 0.1% SDS and 10% polyacrylamide gel electrophoresis. The gel was dried and exposed to Kodak X-Omat AR x-ray film with an intensifying screen at Ϫ70°C for 6 -24 h.
Nuclear Extract Preparation and Gel Mobility Shift Assay-Growtharrested VSMCs were treated for 4 h with and without thrombin (0.1 unit/ml) in the presence and absence of various concentrations of forskolin and washed once with phosphate-buffered saline. Cells were then harvested by scraping into Eppendorf tubes in 1 ml of phosphatebuffered saline. Cells were pelleted by centrifugation at 3500 rpm for 4 min at 4°C, and nuclear extracts were prepared according to the method described by Dignam et al. (27). In brief, the cell pellet was suspended in 500 l of buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM DTT, 1 mM PMSF, 10 g/ml leupeptin, and 10 g/ml aprotinin) and repelleted by centrifugation as described above. The pelleted cells were resuspended in 80 l of buffer A containing 0.1% (w/v) Triton X-100 and incubated on ice for 10 min. Nuclei were pelleted by centrifugation as described above. The nuclear pellet was then suspended in 50 l of buffer C (20 mM Hepes, pH 7.9, 1 mM DTT, 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin, and 25% glycerol) and incubated for 30 min on ice. Cell debris was removed by centrifugation at 14,000 rpm for 10 min at 4°C, and the protein concentration of the nuclear extract was determined as described above. Nuclear extracts were stored at Ϫ70°C. Protein⅐DNA complexes were formed by incubating 5 g of nuclear protein in a total volume of 20 l consisting of 15 mM Hepes, pH 7.9, 3 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM PMSF, 1 mM DTT, 4.5 g of bovine serum albumin, 2 g of poly(dI-dC), 15% glycerol, and 100,000 cpm of 32 P-labeled oligonucleotide probe for 20 min at 30°C. Protein⅐DNA complexes were resolved on a 4% polyacrylamide gel using 0.25 ϫ TBE buffer (1 ϫ TBE ϭ 50 mM Tris borate, pH 8.3, and 1 mM EDTA). Double-stranded oligonucleotide (AP-1, 5Ј-CGCTTGATGAGTCAGCCGGAA-3Ј) was labeled with [␥-32 P]ATP using a T4 polynucleotide kinase kit per the supplier's protocol (Promega). Unincorporated nucleotides were removed by chromatography in a G-25 spin column (Bio-Rad).

RESULTS AND DISCUSSION
To determine whether forskolin and dcAMP inhibit thrombin-induced growth in VSMCs, growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) in the presence and absence of forskolin (10 M) or dcAMP (1 mM) for 24 h, and DNA synthesis was measured by [ 3 H]thymidine incorporation into trichloroacetic acid-precipitable material. Thrombin stimulated VSMC DNA synthesis 12-fold compared to untreated cells, and this response was significantly blunted by both forskolin and dcAMP (Fig. 1). Forskolin inhibited thrombin-induced VSMC DNA synthesis in a dose-dependent manner with 40 and 100% inhibition at 1 and 10 M concentration, respectively (data not shown). Earlier studies from other laboratories have demonstrated that forskolin and other agents such as prostaglandin E 2 , which increase intracellular cAMP levels, antagonize the mitogenic effects of PDGF and EGF by inhibiting the ERK pathway at Raf-1 level in various cell types including human arterial smooth muscle cells (25,26). Raf-1 is an upstream serine/threonine kinase, the activity of which is required for phosphorylation and activation of MEK1 and MEK2 (13)(14)(15)(16). MEKs (also called MKKs) are a group of dual specificity enzymes and activate, although with differential specificities, ERKs, JNKs, and p38 groups of MAPKs by phosphorylating threonine and tyrosine residues (13,15,28). To investigate whether forskolin and dcAMP decrease thrombininduced VSMC growth by a mechanism similar to that reported for receptor tyrosine kinase agonists (25,26), growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) for various periods of time in the presence and absence of forskolin (10 M) or dcAMP (1 mM), and cell lysates were prepared. ERKs activities in the cell lysates were determined by in-gel kinase assay using MBP as a substrate. As shown in Fig. 2, thrombin activated ERKs in a biphasic manner with a first and highest peak of activity at 5 min (20-fold), followed by a second and more sustained lower peak of activity at 2 h (5-fold). Forskolin and dcAMP, surprisingly, had no effect on thrombin activation of ERKs (Fig. 2), a result that indicates that forskolin and dcAMP inhibit thrombin-induced growth by interfering with a mitogenic signaling event that is distinct from the ERKs.
Recent studies have shown that G protein-coupled receptor agonists, such as carbachol and angiotensin II, potently activate JNKs in fibroblasts and epithelial cells, respectively (29,30). This suggests further that JNKs play an important role in signaling events elicited by G protein-coupled receptor agonists in these cells. To determine if thrombin activates JNKs and their role in the mitogenic effect of thrombin, growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) for various periods of time as well as in the presence and absence of forskolin (10 M), and cell lysates were prepared. JNK1 activity in the cell lysates was determined by an immunocomplex kinase assay using recombinant glutathione S-transferase-c-Jun as a substrate. Thrombin activated JNK1 in a time-dependent manner (Fig. 3). Thrombin activation of JNK1 was observed at 5 min, reached maximum (7-fold) by 10 min, and dropped almost to basal levels by 1 h (Fig. 3). Inter-estingly, thrombin-induced activation of JNK1 was significantly blocked by forskolin (Fig. 3). To find out whether thrombin activates the p38 group of MAPKs, and if so, its responsiveness to elevation of intracellular cAMP levels, growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) in the presence and absence of forskolin (10 M) for various time periods, and cell lysates were prepared. Cell lysates containing equal amounts of protein (500 g) from each condition were immunoprecipitated with anti-p38 antibodies, and the immunoprecipitates were subjected to in-gel kinase assay using MBP as a substrate. In comparison to levels in untreated cells, no significant changes were observed in p38 MAPK activities in response to thrombin in the presence or absence of forskolin (data not shown).
ERKs via phosphorylation activate transcriptional factors such as ternary complex factor/Elk1 (17,18), whereas JNK1 activates c-Jun/AP-1 and ATF2 (22). The role of these transcriptional factors in regulating the expression of c-Fos and c-Jun in many cell types in response to a wide variety of stimuli has been documented (13,31). To relate the activation of ERKs and JNK1 to c-Fos and c-Jun expression by thrombin, growtharrested VSMC were treated with and without thrombin (0.1 unit/ml) for various periods of time as well as in the presence and absence of forskolin (10 M) or dcAMP (1 mM). Cell extracts were prepared, and equal amounts of protein (40 g) from each condition were analyzed for c-Fos and c-Jun by Western blotting using appropriate antibodies. Thrombin stimulated expression of c-Fos and c-Jun in a time-dependent manner in VSMCs (Fig. 4). Induced expression of both c-Fos (50-fold) and c-Jun (15-fold) occurred at 2 h and persisted for at least 4 h. In addition, whereas forskolin and dcAMP had no effect on thrombin-induced c-Fos expression, both these agents significantly attenuated the stimulation of c-Jun expression by thrombin

FIG. 2. Thrombin activation of ERKs is insensitive to inhibition by forskolin and dcAMP.
Growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) in the presence and absence of forskolin (10 M) or dcAMP (1 mM) for the indicated time periods, and cell extracts were prepared. Fifty g of protein from each treatment was resolved on a mini-SDS-polyacrylamide gel that was copolymerized with MBP, and ERKs activities were measured by in-gel kinase assay as described under "Experimental Procedures." Results shown here are representative of one experiment and were reproduced in three separate experiments. (Fig. 5). To test whether decreased expression of c-Jun also results in reduced AP-1 activity, growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) for 4 h in the presence and absence of various concentrations of forskolin, nuclear proteins were prepared, and AP-1 activity was measured. Thrombin induced AP-1-DNA binding activity by 5-fold as compared to control, and this response was dose-dependently inhibited by forskolin (Fig. 6).
The important findings of this study are that: 1) thrombin activates JNK1 in growth-arrested VSMCs; and 2) this response is linked to the mitogenic effect of thrombin in these cells. The involvement of JNK1 in the mitogenic signaling FIG. 6. Thrombin induces AP-1-DNA binding activity in VSMCs, and forskolin inhibits this response. Growth-arrested VSMCs were treated for 4 h with and without thrombin (0.1 unit/ml) in the presence and absence of the indicated concentrations of forskolin, and nuclear extracts were prepared. Five g of nuclear protein from each condition was then incubated with radiolabeled AP-1 oligonucleotide probe, and the AP-1⅐DNA complex was separated as described under "Experimental Procedures." These results were reproduced in two independent experiments.

FIG. 3. Thrombin activates JNK1, and forskolin inhibits this response.
Growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) for the indicated time periods or for 10 min in the presence and absence of 10 M forskolin, and cell extracts were prepared. Five hundred g of protein from each treatment was immunoprecipitated with anti-JNK1 antibodies, and JNK1 activity in the immunocomplexes was measured using recombinant glutathione S-transferase-c-Jun protein as a substrate. Results shown here are representative of one experiment, and these results were reproduced in three separate experiments.

FIG. 4. Thrombin induces expression of c-Fos and c-Jun.
Growth-arrested VSMCs were treated with and without thrombin (0.1 unit/ml) for the indicated time periods, and cell extracts were prepared. Forty g of protein from each treatment was separated on SDS-polyacrylamide gel and subjected to Western blot analysis using anti-c-Fos or c-Jun antibodies. These results were confirmed in four separate experiments. events of thrombin in VSMCs is supported by our finding that agents that increase cAMP levels blocked both JNK1 activation and DNA synthesis induced by thrombin. A variety of growth stimulants, including the receptor tyrosine kinase agonists PDGF and EGF, have been shown to preferentially activate the ERK pathway in many cell types (13)(14)(15)(16)23). In addition, several studies have shown that cAMP abrogates PDGF-and EGF-induced growth in smooth muscle cells and fibroblasts, respectively, by inhibiting the ERK pathway at the Raf-1 level (25)(26). On the other hand, our results indicate that thrombin, a G protein-coupled receptor agonist, potently activates both ERKs and JNK1. Nonetheless, forskolin inhibited only thrombin-stimulated JNK1 activation but not ERKs activation. These findings, along with others, suggest that cAMP inhibits receptor tyrosine kinase-and G protein-coupled receptor-mediated growth via different mechanisms. It is possible that cAMP attenuates the receptor tyrosine kinase-and G protein-coupled receptor-mediated DNA synthesis by activating different protein kinase A isozymes: one for antagonizing Raf-1 activity and the other for inhibiting the MEK kinase activity. MEK kinase is a serine/threonine kinase and activates SEK/JNKK in the JNK pathway (16,20). SEK/JNKK, in turn, by phosphorylating threonine and tyrosine residues, activates stress-activated protein kinases/JNKs (19,24,32,33). At least two types of protein kinase A isozymes, types I and II, have been isolated, and differential activation of these isozymes in response to different hormones has been demonstrated (34,35). In this regard, it is important to note that type I, but not type II, protein kinase A isozyme activation inhibited T-cell replication (36), and cAMP blocked T-cell activation by inhibiting JNK1 activity but not ERKs activity (37). These observations are consistent with our present findings. Future studies should address whether thrombin activates the upstream kinases of JNK1, i.e. SEK/ JNKK, and MEK kinase in the JNK pathway and at what level cAMP blocks this pathway to inhibit growth. It is also possible that cAMP inhibition of VSMC growth is in part due to its effects on additional as yet undiscovered or unstudied kinase cascades.
c-Fos and c-Jun dimerize to form the transcriptional factor AP-1 (31,38,39). c-Jun and its related family of proteins (Jun-B and Jun-D) alone by homodimerization can form AP-1, whereas c-Fos and its related family of proteins (Fos-B, Fra-1, and Fra-2) cannot (31, 38 -40). AP-1 plays an important role in cell proliferation and differentiation (31,38,39). Since thrombin induced expression of the AP-1 constituents, c-Fos and c-Jun, in growth-arrested VSMCs, it is likely that this transcriptional factor is an important mediator of the growth-related nuclear events of thrombin in these cells. In fact, our finding that inhibition of thrombin-induced VSMC DNA synthesis by cAMP, preceded by down-regulation of c-Jun and AP-1 activity, supports a role for this transcriptional factor in the mitogenic signaling events of thrombin in VSMCs. A similar dose-dependent effect of forskolin on inhibition of AP-1 activity and DNA synthesis in VSMCs also suggests that the latter two events are related. Although AP-1 activity can be modulated by increased expression of its constituents, c-Fos and c-Jun, posttranslational modifications such as phosphorylation of these proteins also play a significant role in the regulation of AP-1 in response to a wide variety of growth stimuli (31,38,39). For example, in response to both growth factors and cytokines, JNKs are activated (23,33,41), and in turn, these modulate gene expression by phosphorylating and activating c-Jun transactivating activity (19,24). The role of c-Jun in its autoregulation has been demonstrated (31). These findings, along with the fact that cAMP-inhibited JNK1 activation was associated with decreased expression of c-Jun in thrombin-treated VSMCs, suggest that JNK1 mediates thrombin-induced c-Jun expression in these cells. Several studies have demonstrated that in response to a variety of growth factors, ERKs phosphorylate and activate ternary complex factor/Elk1 (17,18,(42)(43)(44), which, in turn, by association with serum response factor binds to the serum response element and induces c-Fos transcription (43,45). Although cAMP did not block thrombin-stimulated ERKs activation and c-Fos expression in VSMCs, it remains to be studied whether ERKs play any role in thrombin-induced c-Fos expression and DNA synthesis in these cells. Nevertheless, the present findings show differential mechanisms of regulation of expression of c-Fos and c-Jun by thrombin in growth-arrested VSMCs. In addition, these results also show a correlation between JNK1 activation, c-Jun expression, AP-1 activity, and growth in VSMCs in response to this agonist.