Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:866-873

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bouchie, J. L.
Right arrow Articles by Feener, E. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bouchie, J. L.
Right arrow Articles by Feener, E. P.
Related Collections
Right arrow Fibrinolysis
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:866.)
© 2000 American Heart Association, Inc.

Thrombosis

P2Y Receptor Regulation of PAI-1 Expression in Vascular Smooth Muscle Cells

Julie L. Bouchie; Hong-Chi Chen; Rebbeca Carney; J. Courtney Bagot; Peter A. Wilden; Edward P. Feener

From the Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Mass (J.L.B., H.-C.C., R.C., E.P.F.), and the Department of Pharmacology and Molecular Biology Program, University of Missouri, School of Medicine, Columbia, Mo (J.C.B., P.A.W.).

Correspondence to Edward P. Feener, PhD, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail edward.feener{at}joslin.harvard.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—P2Y-type purine and pyrimidine nucleotide receptors play important roles in the regulation of vascular hemostasis. In this article, the regulation of plasminogen activator inhibitor-1 (PAI-1) expression in rat aortic smooth muscle cells (RASMCs) by adenine and uridine nucleotides was examined and compared. Northern analysis revealed that RASMCs express multiple P2Y receptor subtypes, including P2Y1, P2Y2, and P2Y6. Treatment of RASMCs with UTP increased PAI-1 mRNA expression and extracellular PAI-1 protein levels by 21-fold (P<0.001) and 7-fold (P<0.001), respectively. The ED50 for the effect of UTP on PAI-1 expression was {approx}1 µmol/L, and its maximal effect occurred at 3 hours. UDP stimulated a 5-fold increase (P<0.005) in PAI-1 expression. In contrast to these potent stimulatory effects of uridine nucleotides, ATP and 2-methylthioadenosine triphosphate (2-MeSATP) caused a small and transient increase in PAI-1 mRNA at 1 hour, followed by a rapid decrease to baseline levels. ADP produced only an inhibitory effect, reducing PAI-1 mRNA levels by 63% (P<0.05) at 3 hours. The relative nucleotide potency in stimulating PAI-1 expression is UTP>UDP>ATP=2-MeSATP, consistent with a predominant role of the P2Y6 receptor. Further studies revealed that exposure of RASMCs to either ATP or ADP for 3 hours inhibited both UTP- and angiotensin II–stimulated PAI-1 expression by up to 90% (P<0.001). Thus, ATP induced a small and transient upregulation of PAI-1 that was followed by a strong inhibition of PAI-1 expression. These results show that extracellular adenine and uridine nucleotides exert potent and opposing effects on vascular PAI-1 expression.


Key Words: purinoceptors • nucleotides • plasminogen activator inhibitor • vascular smooth muscle cells • rats


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Plasminogen activator inhibitor-1 (PAI-1) is the primary regulator of plasminogen activation. Elevated levels of PAI-1 shift the fibrinolytic balance toward thrombosis and may impair the turnover of extracellular matrix proteins.1 2 Reports from our laboratory and others have shown that vasoactive hormones may play an important role in regulating PAI-1 expression in vascular cells. Vasopressor hormones, including angiotensin II (Ang II) and arginine8-vasopressin, are potent stimulators of PAI-1 expression in cultured vascular smooth muscle cells (VSMCs).3 4 Evidence for an important physiological role of the renin-angiotensin system in the regulation of PAI-1 has been provided by studies showing that inhibition of the angiotensin system reduces aortic PAI-1 expression and circulating PAI-1 levels.5 6 7 In addition, vasodilatory hormones, including nitric oxide and natriuretic factors, appear to reduce PAI-1 expression in VSMCs and endothelial cells.8 9 These findings suggest that the fibrinolytic balance may be coordinately regulated with vascular tone and blood pressure.

Extracellular purine and pyrimidine nucleotides regulate vascular tone and hemostasis by activating cell-surface P2-type receptors.10 11 Exogenous delivery of adenine nucleotides has been shown to increase forearm and cochlear blood flow12 13 and induce endothelium-dependent vasorelaxation in isolated coronary and cerebral arteries.14 15 16 In contrast, uridine nucleotides, acting primarily on smooth muscle cells, induce vasoconstriction in pulmonary and coronary arteries.17 18 The vascular actions of these nucleotides are mediated via 2 P2 receptor families, including the P2X ligand-gated cation channels and the P2Y G protein–coupled receptors.10 11 Multiple subtypes of these P2X and P2Y receptors are expressed by vascular cells, and specific receptor subtypes differ in their tissular distribution, regulation, and sensitivity to nucleotide agonists.11 19 20 21 22 These vascular P2 receptors are activated in an autocrine or paracrine manner by nucleotides that are released to the extracellular milieu from perivascular nerves, mechanically strained cells, and activated platelets.23 24 25 26 In addition to modulating vascular tone and blood flow, extracellular nucleotides have been shown to stimulate VSMC growth and endothelial permeability and chemotaxis and to inhibit glucose transport in cardiomyocytes.27 28 29 30 31 32 33

To examine the potential role of P2-type receptors in the regulation of the plasminogen system, we examined the effects of extracellular adenine and uridine nucleotides in the regulation of PAI-1 expression in rat aortic smooth muscle cells (RASMCs). These studies have revealed that uridine nucleotides are highly potent stimulators of PAI-1 expression. In contrast, adenine nucleotides exert a combination of both stimulatory and inhibitory effects on PAI-1 expression. A role of the P2Y6 receptor subtype in the regulation of PAI-1 expression in RASMCs is implicated.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture
RASMCs were isolated from Sprague-Dawley rats, cultured in DMEM/100 mg/dL D-glucose (Gibco-BRL) with 10% FBS (Gibco BRL) as described previously,4 and used between passages 8 and 15. Confluent monolayers of cells were deprived of serum in DMEM containing 0.1% (wt/vol) BSA for 18 hours before stimulation. Cells were stimulated with uridine and adenine nucleotides (Sigma) in the absence or presence of additional stimulation with Ang II (Sigma), PD098059 (Biomol), and GF109203X (Calbiochem) as described.

Reverse Transcription–Polymerase Chain Reaction of P2Y Receptors From RASMCs
Total RNA was treated with RNase-free DNase, and cDNA was synthesized from total RNA by use of a random hexamer primer and a first-strand cDNA Synthesis Kit (Clontech). Then cDNA was used as template in the following polymerase chain reaction (PCR) reactions. The rat P2Y1 receptor sequence was amplified with primers corresponding to 755 to 779 and 1506 to 1482 nucleotides, yielding a 751-bp product. The rat P2Y2 receptor sequence was amplified with primers corresponding to 78 to 103 and 855 to 831 nucleotides, yielding a 777-bp product. The rat P2Y6 receptor sequence was amplified with primers corresponding to 37 to 61 and 907 to 883 nucleotides, yielding an 870-bp product. The PCR was performed with 2 U of Taq DNA polymerase and 20 pmol of each primer in a 50-µL reaction volume. The PCR was 1 cycle of 1 minute at 95°C followed by 30 cycles of 1 minute at 95°C, 1 minute at 60°C, and 1 minute at 72°C. The resultant amplified PCR products were resolved on a 1% agarose ethidium bromide gel. Fragments generated from PCR of P2Y1 receptor cDNA and products generated by reverse transcription–PCR of RASMC RNA (P2Y2 and P2Y6) were cloned with TOPO TA cloning vector (Invitrogen) and sequenced. The sequences of these products matched the P2Y receptor subtype previously reported.22 34 35

RNA Isolation and Northern Blot Analysis
Total RNA was isolated with TRI reagent (Molecular Research Center). Poly A–enriched mRNA was obtained from total RNA with an Oligotex mRNA mini kit (Qiagen). RNA was separated by agarose gel electrophoresis, and PAI-1 mRNA was probed with a cDNA probe against rat PAI-1, as described previously.4 Cloned P2Y inserts were excised with EcoR1, agarose gel–purified, labeled with the Multiprime DNA labeling system (Amersham Corp), purified with a NICK column (Pharmacia LKB Biotechnology Inc), and used for Northern blot analysis of P2Y receptor mRNA. Levels of mRNA were visualized and quantified by PhosphorImager analysis (Molecular Dynamics Inc). RNA loading was normalized to acidic ribosomal phosphoprotein (36B4) with a 32P end-labeled oligonucleotide probe.

Western Blot Analysis
PAI-1 protein levels were measured as described previously.4 Conditioned medium from RASMCs treated with UTP and ATP in the absence or presence of Ang II was collected. Equal aliquots of conditioned medium were separated by 10% SDS-PAGE and transferred to nitrocellulose (Novex). Western blotting was performed with an anti–rat PAI-1 antibody (American Diagnostica), followed by detection with enhanced chemiluminescence (ECL) (New England Biolabs). PAI-1 was visualized and quantified with ImageQuant software (Molecular Dynamics). PAI-1 levels were normalized to total protein from cell lysate by the Bradford method (Bio-Rad). Mitogen-activated protein (MAP) kinase (ERK-1 and ERK-2) phosphorylations at T202/Y204 were measured by immunoblot analyses with phosphospecific antibodies. RASMCs were treated with 50 µmol/L UTP or ATP, rapidly washed with ice-cold PBS containing 2 mmol/L Na3VO4, and lysed in Laemmli sample buffer. Cell lysates were separated by SDS/PAGE, transferred to nitrocellulose (Novex), and immunoblotted with antiphospho T202/Y204 ERK-1 and ERK-2 antibodies (New England Biolabs), followed by detection by ECL. Results were quantified with ImageQuant.

Statistics
All statistical analyses were performed by 1-way ANOVA with SigmaStat (Jandel Scientific). Values of P<0.05 were considered significantly different.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Northern blot analysis was performed to examine and compare P2Y1, P2Y2, and P2Y6 receptor expression in cultured RASMCs, aorta, and heart left ventricle from Sprague-Dawley rats. This study revealed P2Y1 expression in total RNA from rat heart and poly A–enriched RNA from RASMCs (Figure 1Down). P2Y2 expression was detected in total RNA from heart and aorta and both total and poly A–enriched RNA from RASMCs. P2Y6 mRNA expression was detected in total RNA from aorta and poly A–enriched RNA from RASMCs. These results demonstrate that RASMCs express low levels of P2Y1 mRNA and comparably higher levels of P2Y2 and P2Y6 transcripts.



View larger version (61K):
[in this window]
[in a new window]
  		Figure 1. P2Y subtype receptor expression in rat heart, aorta, and RASMCs. Northern blot analysis of P2Y1, P2Y2, and P2Y6 expression in 20 µg total RNA from rat heart, aorta, and RASMCs or 12 µg poly A–enriched RNA from RASMCs.

Because P2Y2 and P2Y6 are activated by both adenine and uridine nucleotides, the effects of these nucleotides on the regulation of PAI-1 expression in RASMCs were examined. Cells were grown to confluence, incubated for 18 hours in medium containing 0.1% BSA, and treated from 1 to 4 hours with 50 µmol/L ATP, UTP, or 2-methylthioadenosine triphosphate (2-MeSATP). After these treatments, RNA was harvested and PAI-1 mRNA levels were measured by Northern blot analysis. This study revealed that the effects of uridine and adenosine nucleotide triphosphates on PAI-1 mRNA differed in both kinetics and magnitude. UTP caused a sustained increase in PAI-1 mRNA, reaching 21-fold induction (P<0.001, ANOVA) after 3 hours (Figure 2ADown). In contrast, ATP and 2-MeSATP induced a small and transient increase in PAI-1 levels, reaching {approx}2-fold after 1 hour, followed by a return toward baseline after 3 to 4 hours (Figure 2BDown). The effects of adenine and uridine nucleotide diphosphates on PAI-1 expression were also examined. PAI-1 mRNA levels were measured in cells stimulated with 50 µmol/L ADP or UDP for 1 to 3 hours (Figure 2CDown). UDP increased PAI-1 levels by up to 5-fold at 2 hours. In contrast, ADP reduced PAI-1 mRNA levels after 2 and 3 hours by 58% and 63%, respectively (P<0.05). These results indicate that the relative potency of nucleotides on stimulating PAI-1 expression in RASMCs is UTP>UDP>ATP=2-MeSATP. The dose-response effect of UTP on PAI-1 expression was examined over a nucleotide concentration range from 100 nmol/L to 100 µmol/L. This study revealed that the ED50 for the effect of UTP was {approx}1 µmol/L, and its maximal effect was observed at 10 µmol/L (not shown). Treatment of cells with 50 µmol/L GTP did not alter PAI-1 expression (not shown).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Effects of purine and pyrimidine nucleotides on PAI-1 mRNA expression in RASMCs. Cells were treated with 50 µmol/L of UTP (A), ATP or 2-MeSATP (B), and ADP or UDP (C) over the time courses indicated. After these treatments, total RNA was harvested, and PAI-1 and 36B4 mRNA levels were examined by Northern blot analysis. Results were visualized and quantified by PhosphorImager (Molecular Dynamics). Representative blots of PAI-1 and 36B4 mRNA expression and bar graph quantification of PAI-1 mRNA levels normalized to 36B4 mRNA from >=3 separate experiments are shown. Significant differences from controls were determined by ANOVA and are indicated as *P<0.05, **P<0.005, or ***P<0.001. Con indicates control.

Because cells may be simultaneously exposed to adenine and uridine nucleotides, the combined effect of these nucleotides on PAI-1 expression in RASMCs was examined. Cells were treated with 50 µmol/L UTP in the absence or presence of a concentration range of 500 nmol/L to 50 µmol/L ATP or ADP for 3 hours. Treatment of cells with UTP alone induced a 10-fold increase in PAI-1 expression (Figure 3Down), as described in Figure 2AUp. Interestingly, addition of either ATP or ADP inhibited UTP-stimulated PAI-1 expression in a concentration-dependent manner (Figure 3Down). The IC50s for the adenine nucleotides were {approx}5 µmol/L, and inhibitory effects at 50 µmol/L of ATP and ADP reduced PAI-1 levels by 93% and 90%, respectively (P<0.001).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. Effects of adenine nucleotides on UTP-stimulated PAI-1 mRNA expression in RASMCs. Cells were stimulated for 3 hours with 50 µmol/L UTP in the absence or presence of the indicated concentrations of ATP or ADP. Representative blots showing PAI-1 and 36B4 mRNA expression and bar graph quantification of PAI-1 mRNA levels normalized to 36B4 mRNA are shown. Significant differences between UTP and UTP plus ATP or ADP are indicated as *P<0.05 and ***P<0.001. Con indicates control.

To determine whether ATP can also inhibit PAI-1 expression induced by other agonists of PAI-1 expression, the effect of ATP on angiotensin II (Ang II)–stimulated PAI-1 was examined. Previous reports have shown that Ang II is a potent stimulator of PAI-1 mRNA expression in RASMCs and that the effect of Ang II is maximal at 3 hours.3 4 8 Cells were stimulated with 100 nmol/L Ang II for 3 hours either alone or with 50 µmol/L of either ATP or UTP. Stimulation of cells with Ang II alone increased PAI-1 mRNA levels by 13-fold (Figure 4Down). Treatment of cells with a combination of Ang II and ATP for 3 hours was 86% less effective than Ang II treatment alone in stimulating PAI-1. This inhibitory effect of ATP was also observed when ATP was added 1 hour before or 1 hour after the addition of Ang II (Figure 4Down). Thus, treatment of RASMCs with ATP for >=2 hours inhibits both Ang II–stimulated and UTP-stimulated PAI-1 expression in RASMCs (Figures 3Up and 4Down). In contrast to these inhibitory effects of ATP, treatment of cells with a combination of UTP and Ang II was 2-fold (P<0.001) more effective than Ang II alone in increasing PAI-1 expression (not shown).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 4. Effects of ATP on Ang II–stimulated PAI-1 expression in RASMCs. Cells were stimulated with Ang II for 3 hours with or without concurrent stimulation with ATP for the times indicated. Representative blots and bar graph quantification from 4 experiments. Significant differences (P<0.001, ANOVA) are indicated as ***. Con indicates control.

The effects of UTP and ATP on PAI-1 protein production were examined. Cells were stimulated with UTP or ATP (50 µmol/L) either alone or in combination with Ang II (100 nmol/L) for 18 hours, and PAI-1 protein released to the conditioned medium was measured by immunoblot analysis, as described previously.4 8 Conditioned media were separated by SDS-PAGE, and PAI-1 protein was quantified by immunoblot analysis with an anti–rat PAI-1 antibody. This study revealed that UTP increased PAI-1 protein production by 7-fold, which was similar to the 8-fold increase in PAI-1 induced by Ang II (Figure 5Down). The combined effect of Ang II and UTP on PAI-1 levels was 34% (P<0.05) greater than that observed with UTP alone. Treatment of cells with ATP did not significantly alter PAI-1 protein levels either alone or in the presence of Ang II.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 5. Effects of ATP, UTP, and Ang II on PAI-1 protein released by RASMCs. Cells were treated with 50 µmol/L nucleotides and/or 100 nmol/L Ang II for 18 hours. PAI-1 protein in the conditioned medium, normalized to total protein from cell monolayers, was measured by Western blot analysis with an anti–rat PAI-1 antibody. Results were visualized by ECL and quantified with ImageQuant (Molecular Dynamics). Representative blot and bar graph quantification from 3 experiments are shown. Significant differences compared with untreated control are indicated as **P<0.005 and ***P<0.001. Significant difference between treated groups is indicated as *P<0.05. Con indicates control.

Previous studies have shown that ATP and UTP stimulation of P2Y receptors leads to the activation of the MAP kinase (ERK-1, -2) pathway28 36 and that activation of this pathway can increase PAI-1 transcription.37 To determine whether signaling differences of these nucleotides through the MAP kinase pathway may contribute their differential effects on PAI-1 expression, the abilities of ATP and UTP to activate the ERK-1, -2 were examined and compared. Activation of ERK-1 and -2 was assessed by measurement of phosphorylation at T202/Y204. Although both nucleotides activated this MAP kinase pathway, this study revealed that UTP was {approx}2-fold (P<0.001) more potent than ATP in increasing ERK-2 phosphorylation (Figure 6Down). To examine the role of the MAP kinase pathway in UTP-stimulated PAI-1 expression, cells were pretreated with MEK inhibitor PD098059 for 15 minutes, followed by stimulation with 50 µmol/L UTP for 3 hours. Northern blot analysis of PAI-1 mRNA expression showed that 30 and 100 µmol/L PD098059 inhibited UTP-induced PAI-1 expression by 34% and 51% (P<0.05), respectively (not shown). These results suggest that the MAP kinase (ERK-1, -2) pathway partially contributes to UTP-stimulated PAI-1 expression in RASMCs. Thus, the greater stimulation of the MAP kinase pathway by UTP, compared with ATP, may contribute to its more potent effect in increasing PAI-1 expression.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 6. Effects of ATP and UTP on ERK-1, -2 phosphorylation. RASMCs were stimulated with 50 µmol/L ATP or UTP for the times indicated. ERK phosphorylation was determined by immunoblot blot analysis. Results were visualized by ECL and quantified by PhosphorImage analysis. Representative blots and bar graph quantification from 3 experiments are shown. Significant differences from untreated controls are indicated as *P<0.05 or ***P<0.001.

To further characterize the mechanism of UTP-induced PAI-1 expression, the role of PKC was examined. Previous reports have shown that P2Y2 and P2Y6 receptors are G protein–coupled to phospholipase C,36 38 39 40 which leads to the generation of diacylglycerol and the elevation of cytosolic calcium, cofactors for protein kinase C (PKC). Because we have shown that PKC regulates PAI-1 expression in RASMCs,4 the effect of PKC inhibition on UTP-stimulated PAI-1 expression was examined. Pretreatment of cells with the PKC inhibitor GF109203X reduced UTP-stimulated PAI-1 expression by 58% (P<0.001) (not shown). These results suggest that PKC contributes to the induction of PAI-1 by UTP.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This report demonstrates that extracellular adenine and uridine nucleotides exert potent effects on PAI-1 expression in RASMCs. Exposure of these cells to UTP increased PAI-1 mRNA levels and extracellular PAI-1 protein by 20-fold and 7-fold, respectively. The magnitude of this induction of PAI-1 expression by UTP is comparable to that of other strong agonists of PAI-1, such as platelet-derived growth factor and Ang II.3 4 8 The time course of UTP-stimulated PAI-1 expression showed that elevation of PAI-1 mRNA levels is rapid, being elevated by 10-fold after 1 hour and sustained over the 4-hour study. UDP also induced a rapid upregulation of PAI-1 expression, but its effect was less than that of UTP. The ED50 of UTP-stimulated PAI-1 was between 1 and 10 µmol/L, which is comparable to the other signaling and biological effects of UTP.33 34 36 In addition, stimulatory effects of UTP on PAI-1 mRNA and protein expression appeared to be additive to that of Ang II. These findings suggest that these uridine nucleotides, which induce VSMC vasoconstriction,17 18 are also potent stimulators of PAI-1 expression in RASMCs.

In contrast to the pronounced induction of PAI-1 by uridine nucleotides, both ATP and the P2Y-agonist 2-MeSATP produced only small increases in PAI-1 mRNA after 1 hour, which were followed by a rapid decline to basal levels. Treatment of cells with ADP reduced PAI-1 levels without stimulating the transient increase in PAI-1 observed with ATP. Coincubation of cells with UTP and ATP or ADP revealed that both adenine nucleotides suppress PAI-1 expression in a concentration-dependent manner. Because ATP similarly inhibited Ang II–stimulated PAI-1 expression, the inhibitory effects of ATP are not due to antagonistic competition for pyrimidinoceptors. The ED50 of ATP and the inhibitory effects of ADP on PAI-1 expression were {approx}5 µmol/L, which is similar to the ED50 of other actions of these nucleotides.27 34 41 These results suggest that ATP exerts both stimulatory and inhibitory effects on PAI-1 expression, which are separated, at least in part, by kinetics.

Examination of P2Y receptor subtype expression in RASMCs derived from Sprague-Dawley rats revealed P2Y1, P2Y2, and P2Y6 receptors. The expression of P2Y1 receptors in RASMCs was detected in poly A–enriched RNA but not in total RNA, indicating that this receptor subtype is expressed at low levels in these cells. Although a previous report demonstrated that this receptor subtype is present in VSMCs, particularly in its synthetic phenotype,19 another report did not detect P2Y1 in VSMCs derived from spontaneously hypertensive rats (SHR).42 Thus, expression of P2Y1 may depend on both VSMC phenotype and species. Expression of P2Y2 in RASMCs is consistent with a previous report showing expression of P2Y2 in medial smooth muscle cells in rat aorta from Wistar rats and SHR-derived VSMCs.19 22 43 Detection of P2Y6 in RASMCs is consistent with previous reports of this receptor in VSMCs.18 34 Comparison of the levels of P2Y subtype expression in RASMCs with that in rat aorta shows that P2Y2 and P2Y6 expression in vivo is similar to or higher than that observed in cultured cells.

The P2Y receptor subtypes described above differ in regard to their sensitivity to nucleotide agonists. P2Y1 receptors are activated primarily by adenine nucleotides, with UTP having little or no effect.11 P2Y2 and P2Y4 receptors in rat are equally activated by ATP and UTP.44 45 46 The P2Y6 receptor is the only P2Y receptor subtype in rat that is preferentially activated by uridine nucleotides compared with adenine nucleotides.34 Although the effects of uridine and adenine nucleotides on PAI-1 expression in RASMCs is most likely the result of the combined effect of P2Y receptors, the more potent effects of uridine nucleotides compared with adenine nucleotides is consistent with a predominant role of P2Y6 in the regulation of PAI-1 in these cells. Because the P2Y6 receptor is strongly expressed in aorta, this receptor may also contribute to the regulation of vascular PAI-1 expression in vivo. It is possible, however, that the potential effect of ATP-activated P2Y receptors on stimulating PAI-1 expression in RASMCs is underestimated because of the secondary inhibitory effects of ATP breakdown products, such as ADP and adenosine. Further characterization of the relative contributions of specific P2Y receptors in the regulation of vascular PAI-1 will require the development of subtype-specific receptor antagonists.

We have shown that UTP is a potent stimulator of ERK phosphorylation and that inhibition of PKC or MEK1,2 kinase partially inhibits UTP-induced PAI-1 expression. Because activation of PKC and the MAP kinase pathway has been shown to increase PAI-1 transcription,37 47 it is likely that UTP signaling through these elements similarly increases PAI-1 transcription. However, because inhibition of PKC and MEK1,2 was only partially effective in blocking UTP-stimulated PAI-1, it appears likely that additional signal processes contribute to P2Y-mediated upregulation of PAI-1 expression. Because ATP also activates the ERK pathway, it is likely that this pathway also contributes to the transient upregulation of PAI-1 mRNA by ATP. Comparison of the signaling effects of ATP and UTP showed that UTP was {approx}2-fold more effective in increasing ERK phosphorylation. In contrast, UTP was 20-fold more effective than ATP in upregulating PAI-1 expression. These results suggest that the difference between the effects of ATP and UTP on PAI-1 is due to a combination of a smaller stimulation of RASMCs by ATP as well as a secondary inhibitory effect of ATP on PAI-1 expression. A possible mechanism for the inhibitory effects of ATP on PAI-1 expression could be related to its extracellular dephosphorylation to ADP and adenosine. VSMCs express ATP diphosphohydrolase and ecto-5'-nucleotidase,48 49 which convert adenine nucleotides to adenosine and thereby may lead to the activation of adenosine P1 receptors.50 We have found that treatment of cells with cyclopentyladenosine, a selective A1 adenosine receptor agonist, reduced PAI-1 expression without affecting PAI-1 mRNA half-life (not shown). Because ATP and ADP exert greater inhibitory effects on UTP-stimulated PAI-1 than basal PAI-1 expression, it appears likely that adenosine nucleotides act, at least in part, by reducing PAI-1 transcription. Consistent with this, we observed that the rate of decrease in PAI-1 mRNA levels in ADP-treated cells (58% decrease in 2 hours) was similar to the decrease in PAI-1 mRNA we reported in actinomycin D–treated RASMCs (54% decrease in 2 hours).8

The potent effects of uridine and adenine nucleotides on PAI-1 expression in RASMCs suggest that P2Y receptors may play an important role in the regulation of vascular PAI-1 expression. There are a number of physiological and pathophysiological conditions that regulate extracellular nucleotide levels in the vascular milieu. In cultured RASMCs, cyclic mechanical stretch has been shown to rapidly elevate extracellular ATP levels, which act in an autocrine manner via P2Y receptors to activate MAP kinases.25 This autocrine/paracrine activation of P2Y receptors in VSMCs may contribute to the rapid activation of MAP kinases and the elevation of PAI-1 expression reported in vessels after balloon-catheter stretch.5 51 In addition, endothelium disruption at sites of vascular injury may facilitate platelet adhesion and subsequent adenine and uridine nucleotide release, which may locally stimulate VSMCs.11 In vivo studies have shown that interstitial ATP levels in rat heart increase by 10-fold after regional ischemia,52 and interstitial ATP and ADP levels have been shown to range from 2 to 6 µmol/L in exercising skeletal muscle.53 These reports indicate that extracellular levels of ATP and ADP are dynamically regulated and can reach micromolar concentrations that encompass the ED50 of the inhibitory effects of ATP and ADP on PAI-1. Because interstitial ATP is rapidly dephosphorylated to ADP and adenosine,52 it is likely that this pool of nucleotides predominantly suppress PAI-1 expression.

In summary, we have shown that extracellular uridine and adenine nucleotides regulate PAI-1 expression in RASMCs. Uridine nucleotides induce a prolonged induction of PAI-1 that is comparable to the effects of hormones and growth factors, such as Ang II and platelet-derived growth factor. In contrast, extracellular ATP had a bimodal effect that included a small transient upregulation of PAI-1 mRNA followed by a potent inhibitory effect. These results suggest that P2-type purinoceptors and pyrimidinoceptors may play an important role in the regulation of PAI-1 expression in the vasculature.


*    Acknowledgments
 
This work was supported in part by National Institutes of Health grants DK-48358 and DK-36836 (Joslin’s Diabetes and Endocrinology Research Center Grant) and grants from the Juvenile Diabetes Foundation International (to E.P. Feener) and the American Diabetes Association (to P.A. Wilden).

Received July 29, 1999; accepted November 4, 1999.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Kruithof EK. Plasminogen activator inhibitors: a review. Enzyme. 1988;40:113–121.[Medline] [Order article via Infotrieve]
  2. Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest. 1991;88:1067–1072.
  3. van Leeuwen RT, Kol A, Andreotti F, Kluft C, Maseri A, Sperti G. Angiotensin II increases plasminogen activator inhibitor type 1 and tissue-type plasminogen activator messenger RNA in cultured rat aortic smooth muscle cells. Circulation. 1994;90:362–368.[Abstract/Free Full Text]
  4. Feener EP, Northrup JM, Aiello LP, King GL. Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells. J Clin Invest. 1995;95:1353–1362.
  5. Hamdan AD, Quist WC, Gagne JB, Feener EP. Angiotensin-converting enzyme inhibition suppresses plasminogen activator inhibitor-1 expression in the neointima of balloon-injured rat aorta. Circulation. 1996;93:1073–1078.[Abstract/Free Full Text]
  6. Vaughan DE, Rouleau JL, Ridker PM, Arnold JM, Menapace FJ, Pfeffer MA, HEART Study Investigators. Effects of ramipril on plasma fibrinolytic balance in patients with acute anterior myocardial infarction. Circulation. 1997;96:442–447.[Abstract/Free Full Text]
  7. Goodfield NE, Newby DE, Ludlam CA, Flapan AD. Effects of acute angiotensin II type 1 receptor antagonism and angiotensin converting enzyme inhibition on plasma fibrinolytic parameters in patients with heart failure. Circulation. 1999;99:2983–2985.[Abstract/Free Full Text]
  8. Bouchie JL, Hansen H, Feener EP. Natriuretic factors and nitric oxide suppress plasminogen activator inhibitor-1 expression in vascular smooth muscle cells: role of cGMP in the regulation of the plasminogen system. Arterioscler Thromb Vasc Biol. 1998;18:1771–1779.[Abstract/Free Full Text]
  9. Yoshizumi M, Tsuji H, Nishimura H, Kasahara T, Sugano T, Masuda H, Nakagawa K, Nakahara Y, Kitamura H, Yamada K, Yoneda M, Sawada S, Nakagawa M. Atrial natriuretic peptide inhibits the expression of tissue factor and plasminogen activator inhibitor 1 induced by angiotensin II in cultured rat aortic endothelial cells. Thromb Haemost. 1998;79:631–634.[Medline] [Order article via Infotrieve]
  10. Boarder MR, Hourani SM. The regulation of vascular function by P2 receptors: multiple sites and multiple receptors. Trends Pharmacol Sci. 1998;19:99–107.[Medline] [Order article via Infotrieve]
  11. Kunapuli SP, Daniel JL. P2 receptor subtypes in the cardiovascular system. Biochem J. 1998;336:513–523.
  12. Rongen GA, Smits P, Thien T. Characterization of ATP-induced vasodilation in the human forearm vascular bed. Circulation. 1994;90:1891–1898.[Abstract/Free Full Text]
  13. Munoz DJ, McFie C, Thorne PR. Modulation of cochlear blood flow by extracellular purines. Hear Res. 1999;127:55–61.[Medline] [Order article via Infotrieve]
  14. Hansmann G, Ihling C, Pieske B, Bultmann R. Nucleotide-evoked relaxation of human coronary artery. Eur J Pharmacol. 1998;359:59–67.[Medline] [Order article via Infotrieve]
  15. You J, Johnson TD, Childres WF, Bryan RMJ. Endothelial-mediated dilations of rat middle cerebral arteries by ATP and ADP. Am J Physiol. 1997;273:H1472–H1477.[Abstract/Free Full Text]
  16. Marrelli SP, Khorovets A, Johnson TD, Childres WF, Bryan RMJ. P2 purinoceptor-mediated dilations in the rat middle cerebral artery after ischemia-reperfusion. Am J Physiol. 1999;276:H33–H41.
  17. Matsumoto T, Nakane T, Chiba S. UTP induces vascular responses in the isolated and perfused canine epicardial coronary artery via UTP-preferring P2Y receptors. Br J Pharmacol. 1997;122:1625–1632.[Medline] [Order article via Infotrieve]
  18. Hartley SA, Kato K, Salter KJ, Kozlowski RZ. Functional evidence for a novel suramin-insensitive pyrimidine receptor in rat small pulmonary arteries. Circ Res. 1998;83:940–946.[Abstract/Free Full Text]
  19. Erlinge D, Hou M, Webb TE, Barnard EA, Moller S. Phenotype changes of the vascular smooth muscle cell regulate P2 receptor expression as measured by quantitative RT-PCR. Biochem Biophys Res Commun. 1998;248:864–870.[Medline] [Order article via Infotrieve]
  20. Communi D, Raspe E, Pirotton S, Boeynaems JM. Coexpression of P2Y and P2U receptors on aortic endothelial cells: comparison of cell localization and signaling pathways. Circ Res. 1995;76:191–198.[Abstract/Free Full Text]
  21. Webb TE, Boluyt MO, Barnard EA. Molecular biology of P2Y purinoceptors: expression in rat heart. J Auton Pharmacol. 1996;16:303–307.[Medline] [Order article via Infotrieve]
  22. Seye CI, Gadeau AP, Daret D, Dupuch F, Alzieu P, Capron L, Desgranges C. Overexpression of P2Y2 purinoceptor in intimal lesions of the rat aorta. Arterioscler Thromb Vasc Biol. 1997;17:3602–3610.[Abstract/Free Full Text]
  23. Ralevic V, Burnstock G. Roles of P2-purinoceptors in the cardiovascular system. Circulation. 1991;84:1–14.[Abstract/Free Full Text]
  24. Lazarowski ER, Homolya L, Boucher RC, Harden TK. Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation. J Biol Chem. 1997;272:24348–24354.[Abstract/Free Full Text]
  25. Hamada K, Takuwa N, Yokoyama K, Takuwa Y. Stretch activates Jun N-terminal kinase/stress-activated protein kinase in vascular smooth muscle cells through mechanisms involving autocrine ATP stimulation of purinoceptors. J Biol Chem. 1998;273:6334–6340.[Abstract/Free Full Text]
  26. Fukami MH, Salganicoff L. Human platelet storage organelles: a review. Thromb Haemost. 1977;38:963–970.[Medline] [Order article via Infotrieve]
  27. Fischer Y, Becker C, Loken C. Purinergic inhibition of glucose transport in cardiomyocytes. J Biol Chem. 1999;274:755–761.[Abstract/Free Full Text]
  28. Wilden PA, Agazie YM, Kaufman R, Halenda SP. ATP-stimulated smooth muscle cell proliferation requires independent ERK and PI3K signaling pathways. Am J Physiol. 1998;275:H1209–H1215.
  29. Wang DJ, Huang NN, Heppel LA. Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells. J Cell Physiol. 1992;153:221–233.[Medline] [Order article via Infotrieve]
  30. Erlinge D, Yoo H, Edvinsson L, Reis DJ, Wahlestedt C. Mitogenic effects of ATP on vascular smooth muscle cells vs. other growth factors and sympathetic cotransmitters. Am J Physiol. 1993;265:H1089–H1097.[Abstract/Free Full Text]
  31. Noll T, Holschermann H, Koprek K, Gunduz D, Haberbosch W, Tillmanns H, Piper HM. ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i. Am J Physiol. 1999;276:H1892–H1901.[Abstract/Free Full Text]
  32. Satterwhite CM, Farrelly AM, Bradley ME. Chemotactic, mitogenic, and angiogenic actions of UTP on vascular endothelial cells. Am J Physiol. 1999;276:H1091–H1097.[Abstract/Free Full Text]
  33. Miyagi Y, Kobayashi S, Ahmed A, Nishimura J, Fukui M, Kanaide H. P2U purinergic activation leads to the cell cycle progression from the G1 to the S and M phases but not from the G0 to G1 phase in vascular smooth muscle cells in primary culture. Biochem Biophys Res Commun. 1996;222:652–658.[Medline] [Order article via Infotrieve]
  34. Chang K, Hanaoka K, Kumada M, Takuwa Y. Molecular cloning and functional analysis of a novel P2 nucleotide receptor. J Biol Chem. 1995;270:26152–26158.[Abstract/Free Full Text]
  35. Tokuyama Y, Hara M, Jones EM, Fan Z, Bell GI. Cloning of rat and mouse P2Y purinoceptors. Biochem Biophys Res Commun. 1995;211:211–218.[Medline] [Order article via Infotrieve]
  36. Soltoff SP, Avraham H, Avraham S, Cantley LC. Activation of P2Y2 receptors by UTP and ATP stimulates mitogen-activated kinase activity through a pathway that involves related adhesion focal tyrosine kinase and protein kinase C. J Biol Chem. 1998;273:2653–2660.[Abstract/Free Full Text]
  37. Mucsi I, Skorecki KL, Goldberg HJ. Extracellular signal-regulated kinase and the small GTP-binding protein, Rac, contribute to the effects of transforming growth factor-ß1 on gene expression. J Biol Chem. 1996;271:16567–16572.[Abstract/Free Full Text]
  38. Murthy KS, Makhlouf GM. Coexpression of ligand-gated P2X and G protein-coupled P2Y receptors in smooth muscle. Preferential activation of P2Y receptors coupled to phospholipase C (PLC)-ß1 via G{alpha}q/11 and to PLC-ß3 via Gß{gamma}i3. J Biol Chem. 1998;273:4695–4704.[Abstract/Free Full Text]
  39. Soltoff SP. Related adhesion focal tyrosine kinase and the epidermal growth factor receptor mediate the stimulation of mitogen-activated protein kinase by the G-protein-coupled P2Y2 receptor: phorbol ester or [Ca2+] elevation can substitute for receptor activation. J Biol Chem. 1998;273:23110–23117.[Abstract/Free Full Text]
  40. Nicholas RA, Watt WC, Lazarowski ER, Li Q, Harden K. Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol Pharmacol. 1996;50:224–229.[Abstract]
  41. Jin J, Daniel JL, Kunapuli SP. Molecular basis for ADP-induced platelet activation, II: the P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem. 1998;273:2030–2034.[Abstract/Free Full Text]
  42. Harper S, Webb TE, Charlton SJ, Ng LL, Boarder MR. Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP. Br J Pharmacol. 1998;124:703–710.[Medline] [Order article via Infotrieve]
  43. Hou M, Möller S, Edvinsson L, Erlinge D. MAPKK-dependent growth factor-induced upregulation of P2Y2 receptors in vascular smooth muscle cells. Biochem Biophys Res Commun. 1999;258:648–652.[Medline] [Order article via Infotrieve]
  44. Chen ZP, Krull N, Xu S, Levy A, Lightman SL. Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor. Endocrinology. 1996;137:1833–1840.[Abstract]
  45. Bogdanov YD, Wildman SS, Clements MP, King BF, Burnstock G. Molecular cloning and characterization of rat P2Y4 nucleotide receptor. Br J Pharmacol. 1998;124:428–430.[Medline] [Order article via Infotrieve]
  46. Webb TE, Henderson DJ, Roberts JA, Barnard EA. Molecular cloning and characterization of the rat P2Y4 receptor. J Neurochem. 1998;71:1348–1357.[Medline] [Order article via Infotrieve]
  47. Arts J, Grimbergen J, Bosma PJ, Rahmsdorf HJ, Kooistra T. Role of c-Jun and proximal phorbol 12-myristate-13-acetate-(PMA)- responsive elements in the regulation of basal and PMA-stimulated plasminogen-activator inhibitor-1 gene expression in HepG2. Eur J Biochem. 1996;241:393–402.[Medline] [Order article via Infotrieve]
  48. Picher M, Sevigny J, D’Orleans-Juste P, Beaudoin AR. Hydrolysis of P2-purinoceptor agonists by a purified ectonucleotidase from the bovine aorta, the ATP-diphosphohydrolase. Biochem Pharmacol. 1996;51:1453–1460.[Medline] [Order article via Infotrieve]
  49. Gordon EL, Pearson JD, Dickinson ES, Moreau D, Slakey LL. The hydrolysis of extracellular adenine nucleotides by arterial smooth muscle cells: regulation of adenosine production at the cell surface. J Biol Chem. 1989;264:18986–18995.[Abstract/Free Full Text]
  50. Cheng DY, DeWitt BJ, Suzuki F, Neely CF, Kadowitz PJ. Adenosine A1 and A2 receptors mediate tone-dependent responses in feline pulmonary vascular bed. Am J Physiol. 1996;270:H200–H207.[Abstract/Free Full Text]
  51. Hu Y, Cheng L, Hochleitner BW, Xu Q. Activation of mitogen-activated protein kinases (ERK/JNK) and AP-1 transcription factor in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol. 1997;17:2808–2816.[Abstract/Free Full Text]
  52. Kuzmin AI, Lakomkin VL, Kapelko VI, Vassort G. Interstitial ATP level and degradation in control and postmyocardial infarcted rats. Am J Physiol. 1998;275:C766–C771.
  53. Hellsten Y, Maclean D, Radegran G, Saltin B, Bangsbo J. Adenosine concentrations in the interstitium of resting and contracting human skeletal muscle. Circulation. 1998;98:6–8.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 DiabetesHome page
D. J. Friedman, H. G. Rennke, E. Csizmadia, K. Enjyoji, and S. C. Robson
The Vascular Ectonucleotidase ENTPD1 Is a Novel Renoprotective Factor in Diabetic Nephropathy
Diabetes, September 1, 2007; 56(9): 2371 - 2379.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bouchie, J. L.
Right arrow Articles by Feener, E. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bouchie, J. L.
Right arrow Articles by Feener, E. P.
Related Collections
Right arrow Fibrinolysis

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2000 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.