Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP

D. Kyle Hogarth,1 Nathan Sandbo,1 Sebastien Taurin,1 Vladimir Kolenko,2 Joseph M. Miano,3 and Nickolai O. Dulin1

1Section of Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, Illinois 60637; 2Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; and 3University of Rochester Medical Center, Rochester, New York 14642

Submitted 4 December 2003 ; accepted in final form 7 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular ATP is released from activated platelets and endothelial cells and stimulates proliferation of vascular smooth muscle cells (VSMC). We found that ATP stimulates a profound but transient activation of protein kinase A (PKA) via purinergic P2Y receptors. The specific inhibition of PKA by adenovirus-mediated transduction of the PKA inhibitor (PKI) attenuates VSMC proliferation in response to ATP, suggesting a positive role for transient PKA activation in VSMC proliferation. By contrast, isoproterenol and forskolin, which stimulate a more sustained PKA activation, inhibit VSMC growth as expected. On the other hand, the activity of serum response factor (SRF) and the SRF-dependent expression of smooth muscle (SM) genes, such as SM-{alpha}-actin and SM22, are extremely sensitive to regulation by PKA, and even transient PKA activation by ATP is sufficient for their downregulation. Analysis of the dose responses of PKA activation, VSMC proliferation, SRF activity, and SM gene expression to ATP, with or without PKI overexpression, suggests the following model for the phenotypic modulation of VSMC by ATP, in which the transient PKA activation plays a critical role. At low micromolar doses, ATP elicits a negligible effect on DNA synthesis but induces profound SRF activity and SM gene expression, thus promoting the contractile VSMC phenotype. At high micromolar doses, ATP inhibits SRF activity and SM gene expression and promotes VSMC growth in a manner dependent on transient PKA activation. Transformation of VSMC by high doses of ATP can be prevented and even reversed by inhibition of PKA activity.

adenosine triphosphate; purinergic receptors; protein kinase A; serum response factor; proliferation; {alpha}-actin; SM22


EXTRACELLULAR NUCLEOTIDES ATP, ADP, UTP, and UDP are released from sympathetic neurons, activated platelets, inflammatory cells, and injured endothelial cells. They modulate contraction and stimulate proliferation of vascular smooth muscle cells (VSMC) via P2 purinergic receptors (3, 11, 31, 40). The P2 receptors are classified as P2X and P2Y types. The P2X receptors constitute a family of ATP-gated ion channels (19, 28), whereas the P2Y receptors constitute a family of G protein-coupled receptors (GPCR) whose signaling is mediated by heterotrimeric G proteins (39). The mitogenic responses of VSMC to ATP are mediated by P2Y receptors (3, 11, 40).

The expression of P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors in VSMC was shown at the mRNA and protein levels (29, 41). P2Y1, P2Y2, P2Y4, and P2Y6 receptors stimulate inositol 1,4,5-trisphosphate (IP3) production and Ca2+ release (20). P2Y11 receptors are linked to both IP3 and cAMP pathways (4, 5). The downstream signaling of P2Y receptors includes various protein kinases (PKC, MAPKs, PKB/Akt) whose activation is critical for P2Y-induced VSMC proliferation (11). P2Y1, P2Y2, P2Y4, and P2Y6 receptors were recently linked to the activation of a small GTPase RhoA and to the stress fiber formation in VSMC (33). We (7) found that ATP stimulates the activity of serum response factor (SRF), a transcription factor commonly activated by a RhoA-dependent mechanism (18) and implicated in the induction of many smooth muscle (SM)-specific genes in VSMC (25, 35).

In the present study, we found that ATP stimulates a profound but transient activation of protein kinase A (PKA), an effector enzyme commonly activated by cAMP (37). In VSMC, the cAMP-raising agents inhibit cell proliferation (2) and SRF activity (7). Therefore, we sought to determine how PKA is implicated in ATP-induced VSMC proliferation and SM gene expression. Our data suggest that the functional significance of the transient PKA activation by ATP is at least twofold: 1) it is positively implicated in ATP-induced VSMC proliferation, and 2) it negatively regulates the SRF-dependent expression of SM genes.


    METHODS
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 METHODS
 RESULTS
 DISCUSSION
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Cell culture. The WKY7 vascular smooth muscle cell line maintaining endogenous receptors to various vasoactive ligands was described previously (7). Briefly, primary aortic smooth muscle cells derived from Wistar-Kyoto (WKY) rats were subcloned by serial dilution. Clone 7 (WKY7) was selected for the responsiveness to various vasoactive ligands, subcloned again to ensure its purity, and characterized for the expression of SM-specific proteins. WKY7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml streptomycin, 250 ng/ml amphotericin B, and 100 U/ml penicillin. The cells were serum deprived using DMEM containing 0.2% calf serum and 2 mM L-glutamine. All stimulations were performed in DMEM containing 0.1% bovine serum albumin (BSA) and 2 mM L-glutamine.

Adenovirus-mediated gene transduction. The cells were plated on a 12-well plate at a density of 100,000 cells per well and were grown for 24 h, followed by serum deprivation for 24 h. The cells were then incubated with adenovirus at desired concentrations (see figure legends) for 24 h in the media containing 0.1% BSA.

Nonradioactive in vitro assay for PKA activity. After stimulation with desired agonists, the cells (grown in 12-well plates) were lysed in 0.15 ml/well lysis buffer containing 25 mM HEPES (pH 7.5), 0.5% Nonidet P-40, protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mM PMSF), and phosphatase inhibitors (1 mM NaF and 200 µM Na-orthovanadate). The lysates were cleared from insoluble material by centrifugation at 20,000 g for 10 min, and 5 µl of cleared lysates were subjected to a kinase reaction with the fluorescence-labeled PKA substrate kemptide (Promega), according to the manufacturer's protocol. The reaction was stopped by boiling the samples for 10 min. The phosphorylated kemptide was separated from nonphosphorylated kemptide by 0.8% agarose electrophoresis. The fluorescent images were taken with the use of a luminescent image analyzer LAS-3000 (Fujifilm) and were analyzed quantitatively.

[3H]thymidine uptake assay. The WKY7 cells were plated on 48-well plates at a density of 5,000 cells per well, grown for 24 h, and serum starved for 48 h. The cells were then stimulated with desired agonists for 24 h. [3H]thymidine (1 µCi/ml) was added 6 h after cell stimulation for 18 h. The cells were then washed twice with ice-cold PBS, precipitated with 10% trichloroacetic acid (TCA) for 30 min, washed once with 10% TCA, and lysed in a solution containing 0.1% NaOH and 0.1% SDS for 15 min. The lysates were analyzed for radioactivity by using scintillation spectrometry.

Western blot analysis. After stimulation with desired agonists, cells were lysed in modified radioimmunoprotection assay buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Triton-X100, 0.1% SDS, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1 mM NaF, 200 µM Na-orthovanadate, and protease inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF). The lysates were cleared from insoluble material by centrifugation at 20,000 g for 10 min, boiled in Laemmli buffer, subjected to polyacrylamide gel electrophoresis, analyzed by Western blotting with desired primary antibodies followed by horseradish peroxide-conjugated secondary antibodies (Calbiochem), and developed using an enhanced chemiluminescence reaction (Pierce).

Luciferase assay. The cells grown in 24-well plates at a density of 50,000 cells per well were transfected with 100 ng/well luciferase reporter plasmid and 100 ng/well cytomegalovirus (CMV)-driven LacZ plasmid under the serum-free conditions for 24 h, using Lipofectamine Plus reagent (Invitrogen). Cells were then stimulated with desired agonists for 6 h, washed twice with PBS, and lysed in protein extraction reagent. The cleared lysates were assayed for luciferase and {beta}-galactosidase activity using the corresponding assay kits (Promega, Madison, WI). To account for differences in transfection efficiency, we normalized the luciferase activity of each sample to the {beta}-galactosidase activity and expressed relative to the control.

Reagents. An E1, E3 replication-deficient adenovirus (Ad5) vector encoding the complete sequence of the PKA inhibitor (PKI) gene under the control of CMV promoter (AdPKI) was described previously (23) and was kindly provided by Dr. Richard Green (University of Illinois at Chicago, Chicago, IL). The control adenovirus encoding CMV-driven LacZ gene was a gift from Dr. Michael Dunn (Medical College of Wisconsin, Milwaukee, WI) (12). The adenoviruses encoding cDNAs for the dominant negative mutant of SRF (dnSRF) and for the LacZ gene under the control of SM22 promoter was described previously (26). The SRF-luciferase reporter plasmid (SRE.L-Luc) was described previously (7, 18). The polyclonal anti-SM22 antibodies (15) were kindly provided by Dr. Julian Solway (University of Chicago). Anti-ERK1/2 was a gift from Dr. Michael J. Dunn (Medical College of Wisconsin); anti-phospho-ERK1/2 was obtained from Cell Signaling; and anti-SM-{alpha}-actin and anti-{beta}-actin were obtained from Sigma. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S), ADP, and UTP also were obtained from Sigma.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of PKA by ATP. Figure 1A shows the effect of ATP on PKA activity in cell extracts prepared from stimulated VSMC, as measured using a nonradioactive in vitro PKA assay. The assay is based on the differential electrophoretic migration of the PKA-specific fluorescence-labeled peptide substrate (kemptide) toward the cathode and anode, dependent on its phosphorylation state. Because ATP can be hydrolyzed to adenosine by cellular ecto-ATPases, the possibility existed that PKA activation by ATP could be mediated by adenosine receptors coupled to a cAMP pathway. To address this possibility, we examined the effect of the A1/A2-receptor antagonist 1,3-dipropyl-8-sulfophenylxanthine (DPSPX) on ATP-induced PKA activation. As shown in Fig. 1A, DPSPX had little or no effect on ATP-induced PKA activity, whereas it blocked the adenosine-induced PKA activation. Confirming its specificity, DPSPX had no effect on ATP-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2), as determined by electrophoretic mobility shift assay of phosphorylated ERK1 and ERK2 (Fig. 1B). The effect of ATP was mimicked by the nonhydrolyzable ATP analog ATP{gamma}S and was as strong as that of the {beta}-agonist isoproterenol (ISO), whereas UTP stimulated PKA activity to a lesser extent (Fig. 1C).



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Fig. 1. Activation of protein kinase A (PKA) by ATP in vascular smooth muscle cells (VSMC). A and B: VSMC were preincubated for 1 h with or without the A1/A2-receptor antagonist 1,3-dipropyl-8-sulfophenylxanthine (DPSPX; 10 µM), followed by stimulation with 100 µM ATP or adenosine (Ade) for 5 min. Cell lysates were analyzed for PKA activity by nonradioactive in vitro PKA assay (A) or for ERK1/2 phosphorylation by electrophoretic mobility shift of phosphorylated ERK1 and ERK2 (B). Kemptide, fluorescence-labeled PKA substrate; P-kemptide, phosphorylated kemptide. C: relative activation of PKA by a 5-min stimulation of cells with 10 µM isoproterenol (ISO), 100 µM ATP, 100 µM UTP, or 100 µM adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S). Shown are representative data from at least 3 independent experiments with similar results.

 
Despite having a lesser efficacy, UTP had a similar potency to ATP in PKA activation, reaching maximum at 10–100 µM (Fig. 2, A and B). This suggests that PKA activation by ATP is partially mediated by UTP-sensitive purinergic receptors (P2Y2, P2Y4; Ref. 39). Interestingly, both ATP and UTP stimulated a complete phosphorylation of ERK1/2 at much lower concentrations (1 µM; Fig. 2C), suggesting that ERK1/2 pathway is more sensitive than PKA pathway to purinergic stimulation. To assess the role of other purinergic receptors in the remaining UTP-insensitive component of PKA activation by ATP, we examined the effect of ADP, which stimulates P2Y1, P2Y12, and P2Y13 receptors with higher potency compared with ATP (24, 27, 39). As shown in Fig. 2, ADP elicited negligible effect on PKA activation at all doses tested, and it stimulated ERK1/2 phosphorylation only at high concentration (100 µM). Furthermore, the P2Y1-selective antagonist MRS-2179 had no effect on PKA activation or ERK1/2 phosphorylation induced by various concentrations of ATP (data not shown).



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Fig. 2. Dose response of PKA activation to ATP, UTP, and ADP. VSMC were stimulated with increasing concentrations of ATP, UTP, and ADP for 5 min. Cell lysates were analyzed for PKA activity by in vitro PKA assay (A and B). A: representative fluorescent image of phosphorylated kemptide (P-kempt). B: quantified fluorescence of P-kemptide, expressed relative to control. C: the same lysates were analyzed for ERK1/2 phosphorylation (electrophoretic mobility shift assay) by immunoblotting with ERK1/2 antibodies.

 
To summarize the results shown in Figs. 1 and 2, 1) ATP stimulates PKA activity as profoundly as isoproterenol (Fig. 1C); 2) PKA activation by ATP is not mediated by adenosine receptors (Fig. 1A); 3) PKA activation by ATP is partially mediated by UTP-sensitive P2Y2 or P2Y4 receptors but not by UTP-insensitive P2Y1, P2Y12, or P2Y13 receptors (Fig. 2); and 4) the UTP-insensitive component of PKA activation by ATP is mediated by a yet unknown mechanism.

Inhibition of PKA by adenovirus-mediated transduction of PKI. To examine the role of PKA in cell responses to ATP, it is necessary to inhibit PKA effectively and specifically in the total population of cells. The pharmacological agents H-89 and KT5720 are widely used for PKA inhibition. However, recent evidence suggests that these inhibitors elicit nonspecific effects on several other protein kinases (6) that are critical for VSMC proliferation and gene expression. We (7) have previously used the PKA dominant negative mutant (dnPKA) cDNA by transient cotransfection with the desired reporter constructs. However, because of moderate transient transfection efficiency in VSMC (~10%), this approach cannot be used in the total cell population assays. Therefore, we accommodated another approach, a high-efficiency (close to 100%) adenovirus-mediated expression of the PKA inhibitor PKI (AdPKI), which was previously used for PKA inhibition in a total population of human microvascular endothelial cells (23). As shown in Fig. 3A, the transduction of AdPKI resulted in a dose-dependent inhibition of ATP-induced PKA activity in VSMC. The transduction of a control adenovirus carrying the LacZ gene (AdLacZ) had no effect on PKA activity (Fig. 3B). Neither AdPKI nor AdLacZ affected the ATP-induced ERK1/2 phosphorylation, as determined by Western blotting of cell lysates with phospho-specific ERK1/2 antibodies (Fig. 3C) or by electrophoretic mobility shift assay for phosphorylated ERK1 and ERK2 (Fig. 3D). Together, these data demonstrate the effectiveness and the specificity of AdPKI as a tool for PKA inhibition in intact VSMC.



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Fig. 3. Inhibition of PKA by adenovirus-mediated transduction of PKI. VSMC were transduced with increasing concentrations (viral particles per milliliter, vp/ml) of AdPKI (A) or with fixed concentration (30 vp/ml) of AdLacZ or AdPKI (B–D) for 24 h. Cells were then stimulated with 100 µM ATP for 5 min, and cell lysates were analyzed for PKA activity by in vitro PKA assay (A and B), for ERK1/2 phosphorylation by Western blotting with P-ERK antibodies (C), or by gel retardation assay with ERK1/2 antibodies (D). Shown are representative data from at least 3 independent experiments with similar results.

 
Positive role of PKA in ATP-induced proliferation of VSMC. Figure 4A shows a dose-dependent increase of [3H]thymidine incorporation in response to ATP, which is consistent with previously published mitogenic effects of ATP on VSMC (11, 40). The dose response of [3H]thymidine uptake (Fig. 4A) was similar to the dose response of PKA activation (Fig. 4B) but not to that of ERK1/2 phosphorylation (Fig. 4C), suggesting that on its own, ERK1/2 phosphorylation may not be sufficient for a profound VSMC proliferation.



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Fig. 4. Dose responses of [3H]thymidine uptake, PKA activation, and ERK1/2 phosphorylation to ATP. A: VSMC were stimulated by increasing doses of ATP as indicated, followed by [3H]thymidine incorporation assay as described in METHODS. Data are expressed as the radioactivity of incorporated [3H]thymidine (counts per min, cpm) per well. Shown is a representative plot from at least 3 independent experiments performed in quadruplicate. B and C: cells were stimulated by increasing doses of ATP for 5 min, followed by PKA assay (B) or ERK1/2 phosphorylation shift assay (C) of cell lysates. Shown are representative blots from 2 independent experiments with similar results.

 
Having confirmed the mitogenic effect of ATP, we next examined the role of PKA in the ATP-induced DNA synthesis using AdPKI. As shown in Fig. 5A, PKI overexpression significantly attenuated the DNA synthesis induced by 30 µM ATP, whereas the control AdLacZ had no effect. At concentrations of 3 µM, ATP stimulated a moderate (2- to 3-fold) increase in [3H]thymidine uptake without inducing PKA activity (Fig. 4). Overexpression of PKI did not inhibit DNA synthesis induced by 3 µM ATP (Fig. 5A). The DNA synthesis induced by either 3 or 30 µM ATP was completely blocked by the MEK1/2 inhibitors PD-98059 (Fig. 5A) and U0126 (data not shown), as expected.



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Fig. 5. Dual role of PKA in proliferation of VSMC. A: VSMC were transduced with 30 vp/ml AdLacZ or AdPKI for 24 h, followed by preincubation with 30 µM PD-98059 for 1 h, as indicated. Cells were then stimulated with 30 µM ATP or 3 µM ATP, followed by [3H]thymidine incorporation assay. Data are expressed relative to control conditions (AdLacZ transduction, no inhibitor, no stimulation). Transduction of AdLacZ had no significant effect on basal or ATP-induced [3H]thymidine uptake compared with "no virus" conditions (data not shown). The concentration of PD-98059 was chosen on the basis of our previous dose-response experiments (10). B: VSMC were transduced with AdLacZ or AdPKI as in A, stimulated with 30 µM ATP, 30 µM ATP{gamma}S, or UTP, followed by [3H]thymidine uptake assay. Data are expressed relative to control conditions, as in A. C: VSMC were pretreated with 10 µM ISO or 3 µM forskolin (FSK) for 1 min, followed by stimulation with 30 µM ATP as indicated. The [3H]thymidine incorporation assay was then performed as described in METHODS. Shown are representative data from at least 3 experiments performed in quadruplicate.

 
To address the possibility of ATP hydrolysis during long-term treatments in the [3H]thymidine uptake assays, we examined the effect of the nonhydrolyzable ATP analog ATP{gamma}S. As shown in Fig. 5B, ATP{gamma}S stimulated DNA synthesis even more profoundly than ATP, and its effect was attenuated by AdPKI to a similar extent. In addition, the A1/A2-receptor antagonist DPSPX, which blocked PKA activation by adenosine but not by ATP (Fig. 1A), had no significant effect on ATP-induced DNA synthesis (data not shown). UTP also stimulated [3H]thymidine uptake to an extent similar to that of ATP (Fig. 5B). However, in contrast to ATP and ATP{gamma}S, the effect of UTP on DNA synthesis was much less dependent on PKA activity, because overexpression of PKI only marginally decreased UTP-induced [3H]thymidine uptake. The lower PKA dependency of DNA synthesis induced by UTP (Fig. 5B) parallels its lower efficacy of PKA activation compared with that of ATP (Fig. 2). Together, these results suggest that 1) PKA activation promotes VSMC proliferation induced by high doses of ATP or UTP; and 2) ERK1/2 phosphorylation is required, but not sufficient, for a profound DNA synthesis induced by these ligands.

Because our results were not in accord with the commonly accepted view of PKA as a negative regulator of VSMC growth (2), we next examined the effect of the {beta}-agonist ISO and of the adenylyl cyclase activator forskolin (FSK), whose regulatory effects on VSMC proliferation are well established. As shown in Fig. 5C, isoproterenol and FSK inhibited the ATP-induced [3H]thymidine uptake, which was expected. Together, these data suggest that the role of PKA in VSMC proliferation is agonist specific: PKA activation by ATP or UTP promotes VSMC proliferation, whereas PKA activation by ISO or FSK plays a regulatory role.

To begin to understand the agonist-dependent role of PKA in VSMC proliferation, we compared the time course of PKA activation in response to ATP with the time courses induced by ISO or FSK. As shown in Fig. 6A, ATP stimulated a transient PKA activation, reaching its maximum at 5 min and declining to the basal level by 30 min. By contrast, the effects of ISO (Fig. 6B) and FSK (Fig. 6C) were more sustained and lasted for up to 2 h. This suggested that PKA might play a dual role in the proliferation of VSMC dependent on the duration of its activity; a transient PKA activation (induced by ATP) might promote VSMC proliferation (cooperating with MAPK and other signaling pathways), whereas a sustained PKA activation (induced by ISO or FSK) might play a negative role.



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Fig. 6. Time course of PKA activation in response to ATP, ISO, and FSK. Serum-deprived cells were stimulated with 30 µM ATP (A), 10 µM ISO (B), or 3 µM FSK (C) for indicated time periods, followed by in vitro PKA assay of cell lysates as described in METHODS. Note the differential time scale for ATP vs. ISO and FSK stimulation. Shown are representative data from 3 independent experiments with similar results.

 
One way to confirm this hypothesis was to mimic the effect of high ATP concentration (30 µM) by using a combination of low ATP concentration (3 µM), which stimulates ERK1/2 phosphorylation but not PKA activation (Fig. 4), together with another agent stimulating a profound but transient PKA activation. To find the conditions for a transient PKA activation, we used two approaches: 1) we examined the effect of various concentrations of FSK, hoping that at low doses FSK would elicit a transient response; and 2) we examined whether removing FSK after short-term treatment (10 min) would result in a decline of PKA activity by the 30 min time point, as occurs in the case of ATP stimulation (Fig. 6A). As shown in Fig. 7A, the reduction of FSK concentration from 10 µM to 1 µM resulted in a decreased PKA activation at 5 min, but this did not provide a transient response. However, the washout of FSK after 10 min of stimulation resulted in a decline of PKA activity to a basal level by 30 min, regardless of the FSK concentration used (Fig. 7A). On the basis of these experiments, we chose to use 10 µM FSK to examine how its removal would affect the DNA synthesis induced by 3 or 30 µM ATP. FSK was removed, after 10 min of simultaneous stimulation with ATP, by two washes with the media containing the corresponding ATP concentrations. As shown in Fig. 7B, FSK, when present in the media, inhibited ATP-induced [3H]thymidine uptake as expected. By contrast, the FSK washout abrogated its inhibitory effect. However, the removal of FSK did not promote the DNA synthesis induced by 3 µM ATP, as we anticipated (Fig. 7B). Taken together, these results allow us to make two important conclusions: 1) inhibition of VSMC proliferation by PKA requires its sustained activation; and 2) the positive role of PKA in ATP-induced DNA synthesis cannot be explained solely by the transient duration of its activity in response to this agonist.



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Fig. 7. Sustained PKA activation is required for inhibition of VSMC proliferation. A: VSMC were stimulated with increasing concentrations of FSK for 5 or 30 min, or for 10 min followed by a dual wash with warm DMEM and incubation for an additional 20 min. Cells were then lysed, and cell lysates were analyzed for PKA activity. B: VSMC were stimulated with a combination of 10 µM FSK together with 3 or 30 µM ATP, as indicated. After 10-min incubation, cells were either left untouched (no wash) or were washed twice with warm DMEM containing the corresponding concentrations of ATP (wash after 10 min). The [3H]thymidine incorporation assay was then performed as described in METHODS.

 
Regulation of smooth muscle gene expression by PKA. We previously reported (7) that the activity of SRF is negatively regulated by PKA. Figure 8A shows that ATP stimulates the activity of SRF reporter at low micromolar concentrations, whereas at higher doses of ATP, which coincide with PKA activation (Fig. 4), the SRF activity is downregulated. In VSMC, SRF is implicated in the transcription of several SM genes, such as SM-{alpha}-actin and SM22, by binding a conserved DNA target, called a CArG box, in their promoters (25, 35). The SM-{alpha}-actin promoter contains two CArG boxes at positions –62 and –112, mutation of which completely abolishes the angiotensin II-induced increases in promoter activity (17). We previously showed (7) that inhibition of SRF by overexpression of the dnSRF mutant attenuates ATP-induced activity of SM-{alpha}-actin promoter region. The SM22 promoter also contains two CArG boxes, at positions –150 and –273, and their deletion abrogates its activity in VSMC (36). Therefore, we examined how the biphasic activation of SRF by increasing doses of ATP (Fig. 8A) translated to the regulation of SM-{alpha}-actin and SM22 expression at the protein level. Figure 7, B and C, shows that, similarly to the SRF activity, the expression of SM-{alpha}-actin and SM22 proteins is induced by low doses of ATP (1–3 µM), whereas at high concentrations of ATP (30–100 µM), their expression is decreased to, or even below, the basal level. To confirm that the expression of SM-{alpha}-actin and SM22 proteins is dependent on SRF activity, we transduced the cells with adenovirus encoding the DNA binding-deficient SRF mutant (30), whose dominant negative properties we previously confirmed (26). As shown in Fig. 9, overexpression of dnSRF attenuated SM-{alpha}-actin and SM22 protein expression induced by 3 µM ATP without affecting the {beta}-actin protein levels.



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Fig. 8. Dose response of serum response factor (SRF) activation and smooth muscle (SM)-{alpha}-actin and SM22 protein expression to ATP. A: VSMC were transiently transfected with SRF luciferase reporter plasmid (SRE.L-Luc) and CMV-driven LacZ plasmid as described in METHODS. After stimulation with indicated concentrations of ATP for 6 h, the luciferase activity was measured, normalized for {beta}-galactosidase ({beta}-Gal) activity, and expressed relative to corresponding basal levels. Shown are representative data from 1 of 3 experiments performed in quadruplicate. B–D: serum-deprived cells were stimulated with increasing concentrations of ATP for 24 h. The equal amounts (per protein) of cell lysates were analyzed for the expression of SM-{alpha}-actin (B), SM22 (C), and {beta}-actin (D) by Western blotting. Shown are representative blots from 3 independent experiments.

 


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Fig. 9. SRF-dependent expression of SM-{alpha}-actin and SM22 proteins. VSMC were transduced with 100 plaque-forming units per cell of AdLacZ or AddnSRF for 24 h, as described previously (26), followed by stimulation with 3 µM ATP for 24 h. The equal amounts (per protein) of cell lysates were analyzed for the expression of SM-{alpha}-actin (B), SM22 (C), and {beta}-actin (D) by Western blotting. Shown are representative blots from 2 independent experiments.

 
If the activation of PKA by high doses of ATP is responsible for the downregulation of SM-{alpha}-actin and SM22 protein expression, then 1) the stimulatory effect of low ATP should be inhibited by the additional activation of PKA with other agents; and 2) the regulatory effect of high ATP should be reversed by inhibition of PKA activity. Figure 10 shows that costimulation of cells with increasing concentrations of ISO results in a dose-dependent inhibition of SM-{alpha}-actin and SM22 protein expression induced by 3 µM ATP, without affecting the levels of {beta}-actin. Similar inhibition of SM-{alpha}-actin and SM22 expression was observed after treatment of cells with FSK (data not shown). Figure 11 shows that the transduction of AdPKI, but not of AdLacZ, results in an upregulation of SM-{alpha}-actin and SM22 protein levels in response to 30 µM ATP, without altering the {beta}-actin expression. Similar results were obtained by using 30 µM ATP{gamma}S and 30 µM UTP as stimuli (Fig. 11), except that the PKA dependency of the effects of UTP were less pronounced, which, again, is in accord with its lower efficacy on PKA activation (Fig. 2). Together, these data demonstrate that 1) dependent on concentration, ATP can either induce or inhibit the expression of SM-{alpha}-actin and SM22; and 2) the inhibitory effects of high doses of ATP, as well as of ATP{gamma}S and, to a lesser extent, UTP, are mediated by PKA.



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Fig. 10. Regulation of ATP-induced SM-{alpha}-actin and SM22 expression by ISO. VSMC were treated with increasing concentrations of ISO for 1 min, followed by stimulation with 3 µM ATP for 24 h. The equal amounts (per protein) of cell lysates were analyzed for the expression of SM-{alpha}-actin (A), SM22 (B), and {beta}-actin (C) by Western blotting. Shown are representative blots from 3 independent experiments. Similar results were obtained when ISO was added simultaneously with ATP.

 


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Fig. 11. Effect of AdPKI transduction on SM-{alpha}-actin and SM22 expression in response to ATP, ATP{gamma}S or UTP. VSMC were transduced with AdLacZ or AdPKI as in Fig. 4, B and C, followed by stimulation with 30 µM ATP, 30 µM ATP{gamma}S, or 30 µM UTP for 24 h, as indicated. The equal amounts (per protein) of cell lysates were analyzed for the expression of SM-{alpha}-actin (A), SM22 (B), and {beta}-actin (C) by Western blotting. Shown are representative blots from at least 3 experiments with similar results.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular ATP is continually present in plasma and the pericellular space. It is released from many sources, including sympathetic nerves, activated platelets, inflammatory cells, endothelial cells, and smooth muscle cells (13). The normal bulk levels of ATP in plasma average between high nanomolar and low micromolar concentrations (38). Under pathological conditions, such as atherosclerosis and restenosis following vascular injury, which are accompanied by platelet aggregation, endothelial cell activation, and inflammatory cell infiltration, the levels of ATP increase dramatically and reach a concentration of 20–50 µM. Together with the increased endothelial permeability, VSMC become targets of high ATP concentrations (reviewed in Ref. 8).

The vascular wall is represented by at least two phenotypes of VSMC: the contractile and the synthetic ones (1). The contractile phenotype is characterized by a profound expression of SM-cytoskeletal proteins (34, 35), whereas the synthetic phenotype is characterized by a decrease in their expression and by an increased rate of proliferation and migration (9). The relative amount of VSMC with synthetic phenotype is increased in the intimal thickening during atherosclerosis and restenosis (1, 32).

Our results, summarized in Table 1, suggest that VSMC phenotypic responses to ATP are dependent on the concentration of the agonist. At low micromolar concentrations (1–3 µM), corresponding to normal levels in plasma (38), ATP stimulates SRF activity and SRF-dependent expression of SM cytoskeletal proteins, such as SM-{alpha}-actin and SM22 (Figs. 8 and 9), thus fostering the contractile phenotype of VSMC. The low concentrations of ATP also stimulate a maximal phosphorylation (and, presumably, activation) of the MAPKs ERK1/2 (Figs. 2C and 4C). However, the latter does not result in a profound DNA synthesis (Fig. 4A). In contrast, at high micromolar concentrations, which may mimic pathological conditions such as atherosclerosis and restenosis (8), ATP elicits a dual effect on VSMC. First, it stimulates the proliferation of VSMC, the response reported by several laboratories (3, 11, 40) and confirmed by us (Fig. 4A). Second, it stops stimulating the expression of SM cytoskeletal proteins (Fig. 8). Together, these two responses may lead to a shift of VSMC from the contractile to the proliferative phenotype. According to our results, PKA plays a critical role in such a phenotypic transformation of VSMC by high ATP concentrations.


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Table 1. Summary of VSMC responses to low ATP, high ATP, high ATP + AdPKI transduction, and isoproterenol or forskolin

 
Our data suggest that PKA promotes ATP-induced VSMC proliferation, because its inhibition results in attenuated DNA synthesis in response to ATP (Fig. 5, A and B). The positive role of PKA in ATP-induced VSMC proliferation does not contradict the established regulatory role of PKA induced by other stimuli, because, under the same experimental conditions, ISO and FSK inhibited the DNA synthesis as expected (Fig. 5C). Our results suggest that a sustained duration of PKA activity, induced by ISO and FSK, is required for inhibition of VSMC growth by these agonists. Thus the removal of FSK after short-term treatment, providing a transient PKA activation (Fig. 7A), ablates the ability of FSK to inhibit DNA synthesis (Fig. 7B). However, such a transient PKA activation by FSK does not promote VSMC proliferation in response to low ATP concentration (Fig. 7). Thus the positive role of PKA in ATP-induced DNA synthesis cannot be explained solely by the transient duration of PKA activity induced by this agonist. It is noteworthy that despite the transient PKA activation (Fig. 6A), ATP stimulates a sustained phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein (VASP), as we previously demonstrated (7). Thus it is possible that the duration of PKA substrate(s) phosphorylation could be critical for the overall role of PKA. We are currently evaluating the mechanisms that may provide an agonist-specific role of PKA in modulation of VSMC growth.

The regulatory role of PKA in ATP-induced expression of SM-{alpha}-actin and SM22 proteins is consistent with our previous publication (7) reporting the negative regulation of SRF activity and SM-{alpha}-actin expression by PKA at the promoter level. Our present results indicate that at low doses, ATP stimulates a profound SRF activity, whereas at high doses, this effect is negated (Fig. 8). Furthermore, such a biphasic activation of SRF by ATP parallels the regulation of SM-{alpha}-actin and SM22 expression at the protein level (Fig. 8, B and C). The initial increase in SM-{alpha}-actin and SM22 protein levels by low doses of ATP is mediated by SRF (Fig. 9), which is consistent with the SRF dependency of SM-{alpha}-actin and SM22 gene transcription (17, 36). The decrease of SRF activity and of SM-{alpha}-actin and SM22 protein levels at higher doses of ATP (Fig. 8) is mediated by PKA activation, as judged by 1) comparison of the dose responses of PKA activation (Fig. 4) to those of SRF activity and SM-{alpha}-actin and SM22 protein levels (Fig. 8); 2) downregulation of low ATP-induced SM-{alpha}-actin and SM22 protein expression by ISO (Fig. 10); and 3) upregulation of SM-{alpha}-actin and SM22 protein levels in response to high doses of ATP, when PKA is inhibited by PKI overexpression (Fig. 11).

Several important questions arise from this study and are currently being addressed in our laboratory. First, what is the mechanism by which ATP stimulates PKA activity, and what determines such a transient response? Several types of purinergic receptors are expressed in VSMC (29, 41), providing a number of possibilities. Our data suggest that ATP-induced PKA activation is partially (but not entirely) mediated by P2Y2 or P2Y4 receptors, but not by P2Y1, P2Y12, or P2Y13 receptors, because 1) UTP is as potent as ATP in PKA activation (Fig. 2), implicating P2Y2 or P2Y4 receptors (39); 2) at high doses, UTP is less effective than ATP in PKA activation (Figs. 1C and 2, A and B), suggesting that the other ATP-selective receptors are recruited; 3) ADP, which is a potent agonist for P2Y1, P2Y12, and P2Y13 receptors (24, 27, 39), has little effect on PKA activation; and 4) the P2Y1-selective antagonist MRS-2179 has no effect on PKA activation induced by various concentrations of ATP (data not shown). In addition, ATP may partially mediate the effect of UTP, because the latter can be used by ectonucleoside diphosphokinase to convert ADP (present in the media) to ATP by transphosphorylation (22). Regarding the UTP-insensitive, ATP-selective component of PKA activation, the P2Y11 receptors, which are sensitive to ATP but not to ADP or UTP and which are coupled to cAMP and IP3 signaling (4, 5), could be likely candidates. However, this possibility is still open, because 1) no P2Y11-selective antagonists are available; and 2) the rodent (in our case, rat) P2Y11 receptor cDNAs have not been cloned. Alternatively, it is possible that the ATP-selective component of PKA activation is mediated by P2X receptors (19, 28).

The second important question relates to the mechanism by which the transient PKA activation promotes VSMC proliferation. We previously demonstrated (7) that cAMP response element (CRE)-dependent gene transcription was profoundly activated by ATP, entirely in a PKA-dependent manner. Our additional data also suggest that PKA is partially involved in the stimulation of the activator protein-1 (AP-1) by ATP (data not shown). Given that these transcription factors are implicated in the induction of early response genes such as c-fos, cyclin D1, and others, it is conceivable that these genes are upregulated by ATP in a PKA-dependent manner. Finally, the mechanism by which PKA regulates the SRF activity and the expression of SM genes remains to be determined. SRF activation is mediated by the small GTPase RhoA (18), whose membrane localization is regulated by its PKA-dependent phosphorylation (21). It is likely that the inhibition of SRF activity by PKA occurs as a result of RhoA regulation, but other possibilities also exist. VASP is implicated in SRF activation by promoting polymerization of actin (14). Phosphorylation of VASP by PKA results in its release from the filamentous actin (16) and may ablate its ability to promote SRF activation. We previously showed (7) that ATP stimulates a complete, sustained, and PKA-dependent phosphorylation of VASP, suggesting an additional mechanism for SRF regulation and SM gene expression by PKA. In addition, the regulation of SM gene expression by PKA may occur at a posttranscriptional level. These possibilities are currently being evaluated in our laboratory.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by American Heart Association Grant AHA0235405Z (to N. O. Dulin), the Louis B. Block Fund (to N. O. Dulin), and a GlaxoSmithKline Pulmonary Fellowship Award (to D. K. Hogarth).


    ACKNOWLEDGMENTS
 
We thank Dr. Richard Green for providing the PKI adenovirus, Dr. Julian Solway for providing anti-SM22 antibodies, Dr. Michael Dunn for providing anti-ERK1/2 antibodies, and Krista Hogarth for editorial assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Dulin, Section of Pulmonary and Critical Care Medicine, Univ. of Chicago Dept. of Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637 (E-mail: ndulin{at}medicine.bsd.uchicago.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Bochaton-Piallat ML, Ropraz P, Gabbiani F, and Gabbiani G. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol 16: 815–820, 1996.[Abstract/Free Full Text]

2. Bornfeldt KE and Krebs EG. Crosstalk between protein kinase A and growth factor receptor signaling pathways in arterial smooth muscle. Cell Signal 11: 465–477, 1999.[CrossRef][ISI][Medline]

3. Burnstock G. Purinergic signaling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol 22: 364–373, 2002.[Abstract/Free Full Text]

4. Communi D, Govaerts C, Parmentier M, and Boeynaems JM. Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272: 31969–31973, 1997.[Abstract/Free Full Text]

5. Communi D, Suarez-Huerta N, Dussossoy D, Savi P, and Boeynaems JM. Cotranscription and intergenic splicing of human P2Y11 and SSF1 genes. J Biol Chem 276: 16561–16566, 2001.[Abstract/Free Full Text]

6. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][ISI][Medline]

7. Davis A, Hogarth K, Fernandes D, Solway J, Niu J, Kolenko V, Browning D, Miano JM, Orlov SN, and Dulin NO. Functional significance of protein kinase A activation by endothelin-1 and ATP: negative regulation of SRF-dependent gene expression by PKA. Cell Signal 15: 597–604, 2003.[CrossRef][ISI][Medline]

8. Di Virgilio F and Solini A. P2 receptors: new potential players in atherosclerosis. Br J Pharmacol 135: 831–842, 2002.[Abstract/Free Full Text]

9. Dilley RJ, McGeachie JK, and Prendergast FJ. A review of the proliferative behaviour, morphology and phenotypes of vascular smooth muscle. Atherosclerosis 63: 99–107, 1987.[ISI][Medline]

10. Dulin NO, Orlov SN, Kitchen CM, Voyno-Yasenetskaya TA, and Miano JM. G-protein-coupled-receptor activation of the smooth muscle calponin gene. Biochem J 357: 587–592, 2001.[CrossRef][ISI][Medline]

11. Erlinge D. Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen Pharmacol 31: 1–8, 1998.[Medline]

12. Foschi M, Chari S, Dunn MJ, and Sorokin A. Biphasic activation of p21ras by endothelin-1 sequentially activates the ERK cascade and phosphatidylinositol 3-kinase. EMBO J 16: 6439–6451, 1997.[Abstract/Free Full Text]

13. Gordon JL. Extracellular ATP: effects, sources and fate. Biochem J 233: 309–319, 1986.[ISI][Medline]

14. Grosse R, Copeland JW, Newsome TP, Way M, and Treisman R. A role for VASP in RhoA—Diaphanous signalling to actin dynamics and SRF activity. EMBO J 22: 3050–3061, 2003.[Abstract/Free Full Text]

15. Halayko AJ, Camoretti-Mercado B, Forsythe SM, Vieira JE, Mitchell RW, Wylam ME, Hershenson MB, and Solway J. Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am J Physiol Lung Cell Mol Physiol 276: L197–L206, 1999.[Abstract/Free Full Text]

16. Harbeck B, Huttelmaier S, Schluter K, Jockusch BM, and Illenberger S. Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem 275: 30817–30825, 2000.[Abstract/Free Full Text]

17. Hautmann MB, Thompson MM, Swartz EA, Olson EN, and Owens GK. Angiotensin II-induced stimulation of smooth muscle alpha-actin expression by serum response factor and the homeodomain transcription factor MHox. Circ Res 81: 600–610, 1997.[Abstract/Free Full Text]

18. Hill CS, Wynne J, and Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81: 1159–1170, 1995.[ISI][Medline]

19. Khakh BS, Burnstock G, Kennedy C, King BF, North RA, Seguela P, Voigt M, and Humphrey PP. International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol Rev 53: 107–118, 2001.[Abstract/Free Full Text]

20. Kunapuli SP and Daniel JL. P2 receptor subtypes in the cardiovascular system. Biochem J 336: 513–523, 1998.[ISI][Medline]

21. Lang P, Gesbert F, Delespine-Carmagnat M, Stancou R, Pouchelet M, and Bertoglio J. Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J 15: 510–519, 1996.[Abstract]

22. Lazarowski ER, Homolya L, Boucher RC, and Harden TK. Identification of an ecto-nucleoside diphosphokinase and its contribution to interconversion of P2 receptor agonists. J Biol Chem 272: 20402–20407, 1997.[Abstract/Free Full Text]

23. Lum H, Jaffe HA, Schulz IT, Masood A, RayChaudhury A, and Green RD. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction. Am J Physiol Cell Physiol 277: C580–C588, 1999.[Abstract/Free Full Text]

24. Marteau F, Le Poul E, Communi D, Labouret C, Savi P, Boeynaems JM, and Gonzalez NS. Pharmacological characterization of the human P2Y13 receptor. Mol Pharmacol 64: 104–112, 2003.[Abstract/Free Full Text]

25. Miano JM. Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol 35: 577–593, 2003.[CrossRef][ISI][Medline]

26. Miano JM, Carlson MJ, Spencer JA, and Misra RP. Serum response factor-dependent regulation of the smooth muscle calponin gene. J Biol Chem 275: 9814–9822, 2000.[Abstract/Free Full Text]

27. Nicholas RA. Identification of the P2Y12 receptor: a novel member of the P2Y family of receptors activated by extracellular nucleotides. Mol Pharmacol 60: 416–420, 2001.[Free Full Text]

28. North RA and Surprenant A. Pharmacology of cloned P2X receptors. Annu Rev Pharmacol Toxicol 40: 563–580, 2000.[CrossRef][ISI][Medline]

29. Pillois X, Chaulet H, Belloc I, Dupuch F, Desgranges C, and Gadeau AP. Nucleotide receptors involved in UTP-induced rat arterial smooth muscle cell migration. Circ Res 90: 678–681, 2002.[Abstract/Free Full Text]

30. Prywes R and Zhu H. In vitro squelching of activated transcription by serum response factor: evidence for a common coactivator used by multiple transcriptional activators. Nucleic Acids Res 20: 513–520, 1992.[Abstract]

31. Ralevic V and Burnstock G. Roles of P2-purinoceptors in the cardiovascular system. Circulation 84: 1–14, 1991.[Abstract]

32. Sanders M. Molecular and cellular concepts in atherosclerosis. Pharmacol Ther 61: 109–153, 1994.[CrossRef][ISI][Medline]

33. Sauzeau V, Le Jeune H, Cario-Toumaniantz C, Vaillant N, Gadeau AP, Desgranges C, Scalbert E, Chardin P, Pacaud P, and Loirand G. P2Y1, P2Y2, P2Y4, and P2Y6 receptors are coupled to Rho and Rho kinase activation in vascular myocytes. Am J Physiol Heart Circ Physiol 278: H1751–H1761, 2000.[Abstract/Free Full Text]

34. Shanahan CM and Weissberg PL. Smooth muscle cell heterogeneity : patterns of gene expression in vascular smooth muscle cells in vitro and in vivo. Arterioscler Thromb Vasc Biol 18: 333–338, 1998.[Abstract/Free Full Text]

35. Sobue K, Hayashi K, and Nishida W. Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation. Mol Cell Biochem 190: 105–118, 1999.[CrossRef][ISI][Medline]

36. Solway J, Seltzer J, Samaha FF, Kim S, Alger LE, Niu Q, Morrisey EE, Ip HS, and Parmacek MS. Structure and expression of a smooth muscle cell-specific gene, SM22. J Biol Chem 270: 13460–13469, 1995.[Abstract/Free Full Text]

37. Taylor SS, Buechler JA, and Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem 59: 971–1005, 1990.[CrossRef][ISI][Medline]

38. Traut TW. Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140: 1–22, 1994.[ISI][Medline]

39. Von Kugelgen I and Wetter A. Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol 362: 310–323, 2000.[CrossRef][ISI][Medline]

40. Wang DJ, Huang NN, and Heppel LA. Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells. J Cell Physiol 153: 221–233, 1992.[ISI][Medline]

41. Wang L, Karlsson L, Moses S, Hultgardh-Nilsson A, Andersson M, Borna C, Gudbjartsson T, Jern S, and Erlinge D. P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol 40: 841–853, 2002.[CrossRef][ISI][Medline]