Novel Mitogenic Effect of Adenosine on Coronary Artery Smooth Muscle Cells
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循环研究杂志 2005年第5期
The Department of Medical Pharmacology and Physiology (J.S., S.H., M.S., P.W.), Department of Internal Medicine (M.S.), Center for Diabetes and Cardiovascular Health (J.S., M.S., P.W.), University of Missouri-Columbia, School of Medicine, Columbia, Mo
Department of Cellular and Integrative Physiology (M.S.), Indiana University School of Medicine, Indianapolis, Ind.
Abstract
Adenosine is a vascular endothelial cell mitogen, but anti-mitogenic for aortic smooth muscle cells and fibroblasts when acting via the A2B adenosine receptor. However, we show that adenosine increases porcine coronary artery smooth muscle cell (CASMC) number, cellular DNA content, protein synthesis, and PCNA staining. RT-PCR analysis indicates that porcine CASMC express A1, A2A, A3, and barely detectable levels of A2B receptor mRNAs. The mitogenic effect of adenosine is mimicked by NECA, CCPA, and R-PIA, but not by CGS21680and 2-Cl-IB-MECA, and is inhibited by DPCPX, indicating a prominent role for the A1 receptor. This interpretation is supported by the finding that adenosine- and CCPA-induced DNA synthesis is significantly inhibited by pertussis toxin, but substantially potentiated by PD81723, an allosteric enhancer of the A1 receptor. When a cDNA encoding the porcine A1 receptor was cloned and expressed in COS-1 cells, A1 receptor pharmacology is confirmed. Anti-sense oligonucleotides to the cloned sequence dramatically suppress the mitogenic effect of adenosine and CCPA. Conversely, over-expression of the cloned A1 receptor in CASMC increases adenosine- and CCPA-induced DNA synthesis. Furthermore, stimulation with adenosine or CCPA of intact coronary arteries in an organ culture model of vascular disease increases cellular DNA synthesis, which was abolished by DPCPX. We conclude that adenosine acts as a novel mitogen in porcine CASMC that express the A1 adenosine receptor, possibly contributing to the development of coronary artery disease.
Key Words: adenosine receptors coronary artery smooth muscle cells proliferation molecular cloning porcine
Introduction
The diverse cellular actions of adenosine are mediated by a family of adenosine receptors (ARs), of which 4 subtypes (A1R, A2AR, A2BR, and A3R) have been cloned and pharmacologically characterized.1,2 In general, the A1R and A3R are coupled with Gi/o proteins, whose activation causes a decrease of intracellular cAMP. In contrast, activation of the Gs-coupled A2AR and A2BR increases intracellular cAMP.1,2 Thus, the end biological action of adenosine in a particular organ or cell population may depend on the relative expression level and signaling efficiency of the individual AR subtypes.3
Adenosine, like many other vasodilators, historically has been thought to act as an inhibitor of the proliferation of vascular smooth muscle cells (VSMCs). This contention was supported by recent studies in cultured aortic VSMCs, showing that adenosine was antimitogenic through its activation of the A2BR.4eC8 To the best of our knowledge, however, current studies of the long-term effects of adenosine on VSMC proliferation have been limited to aortic smooth muscle. Given the heterogeneity of VSMCs9 and the diversity of the expression profile of ARs in cells from different blood vessels,10 it remains to be shown whether the antiproliferative action of adenosine can be extended to other VSMCs, particularly coronary artery smooth muscle cells (CASMCs).
In porcine CASMCs, previous work in our laboratory has demonstrated that ATP, a major precursor of adenosine, induced a proliferative response via activation of several intracellular signaling pathways, involving activation of the P2Y2 receptor.11eC13 In the process of identifying the P2Y-independent receptor mechanism(s) responsible for ATP-induced CASMC proliferation, we unexpectedly found that adenosine, but not most other nucleotides/nucleosides, mimicked the effects of ATP. This observation led us to propose that, in contrast to the previous observation in aortic VSMCs, adenosine may behave as a mitogen in CASMCs by a receptor mechanism not involving A2BR. Indeed, ligand-binding assays14 and histological studies15 indicated that porcine coronary arteries express A1R. We therefore hypothesized that A1R activation causes CASMC proliferation.
Because no porcine AR has been cloned, in the present study, we have cloned and pharmacologically characterized the porcine A1R and explored its role in CASMC proliferation. Results presented here demonstrate that adenosine acts via the A1R as a mitogen in cultured porcine CASMCs and in intact coronary arteries.
Materials and Methods
CASMC Isolation and Cell Culture
Porcine CASMCs dispersed from right coronary arteries of farm pigs (Jennings Premium Meats, New Franklin, Mo) were cultured as previously described in our laboratory.11eC13 Smooth muscle cell lineage was confirmed by -smooth muscle actin immunocytochemistry. Stock CASMC cultures were maintained in a subconfluent state and used between passage 4 and 10.11eC13
[3H]-Thymidine Incorporation Assays
CASMCs (35 000/well) were seeded in 12-well plates in DMEM/high-glucose medium containing 10% FBS for 48 hours, and then starved for 24 hours in serum-free DMEM containing 0.1% BSA before stimulation. Incorporation of [3H]-thymidine into DNA was performed as previously described.11eC13
Protein Synthesis Assays
CASMCs were seeded and starved as stated earlier. [3H]-leucine incorporation was used for measuring cellular total protein synthesis as previously described.13
Determination of Cell Numbers
CASMCs were seeded in 96-well plates (2000 cells/well) in DMEM containing 10% FBS for 48 hours, and then serum-starved for 48 hours, after which the culture medium was replaced with fresh serum-free starvation medium and CASMCs were stimulated with agonists for 4 days. Cell numbers were determined by MTT Cell Growth Assay Kit (Chemicon) as previously described.13
Immunocytochemistry for Proliferating Cell Nuclear Antigen
CASMCs at 40% confluence were starved for 24 hours before stimulation with adenosine for 24 hours. Standard immunocytochemistry for proliferating cell nuclear antigen (PCNA) was performed using rabbit anti-PCNA (Santa Cruz Biotechnology).
Receptor cDNA Cloning and RACE Experiments
The full cDNA sequence of the porcine A1R and partial cDNA coding sequences for porcine A2AR, A2BR, and A3R were cloned by PCR using a Smart Race cDNA amplification kit (Clontech).
Heterologous Expression
The pCR3.1 plasmids containing the porcine A1R sequence were transfected into COS-1 cells using Effectene Transfection Reagent (Qiagen).
Intracellular [Ca2+] Measurements
Intracellular free Ca2+ concentration ([Ca2+]i) was measured with the InCa2+ calcium imaging system (Intracellular Imaging Inc) as previously described.13,16
Intracellular cAMP Measurements
Intracellular cAMP levels were determined by using the Direct cAMP Correlate-EIA Kit (Assay Designs, Inc).
Radioligand Binding Assays
Binding assays were performed with crude cell membranes using [3H]-DPCPX (130 Ci/mmol; Amersham) as the radioligand as previously described.17
A1R Overexpression in CASMCs
CASMCs were transfected in 12-well plates using the Effectene Transfection Reagent according to the manufacturer’s instruction.
Antisense Oligonucleotide Experiments
Phosphorothioate-modified oligonucleotide (OGN) (antisense, 5'-GAGATGGAGGGCGGCATGGT-3'; sense, 5'-ACCATGCCGCCCTCCATCTC-3') were used for A1R knockdown as previously documented.13
Western Blot Analysis
Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc). Membranes were stripped and reprobed with anti-actin antibody (1:1000; Cytoskeleton).11eC13
Ex Vivo Coronary Artery Organ Culture and DNA Synthesis Assays
Pig hearts were isolated and arteries organ cultured as described in our laboratory.13,16 Incorporation of [3H]-thymidine into coronary arterial rings was performed as described previously.18
Reverse-Transcription Polymerase Chain Reaction Analysis
Primers for reverse-transcription polymerase chain reaction (RT-PCR) were designed based on our original cloned sequences for the 4 porcine ARs (GenBank accession nos. AY772411, AY772412, AY772413, and AY772414).
Materials
The following compounds were from TOCRIS: 5'-N-ethylcarboxamidoadenosine (NECA); 2-chloro-N6-cyclopentyladenosine (CCPA); 2-[p-(2-carboxyethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2-Cl-IB-MECA); 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}-triazin-5-yl-amino]ethyl)phenol (ZM241385); N-(4-acethyl-phenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1706); N-(2-methoxyphenyl)-N-[2-(3-pyridyl) quinazolin-4-yl]urea (VUF5574). Reagents from Sigma were as following: 3,7-dimethyl-1-propargylxanthine (DMPX); (eC)-N6-(2-phenylisopropyl)adenosine (R-PIA); platelet-derived growth factor (PDGF); and lysophosphatidic acid (LPA).
Analysis of Data
Data are expressed as mean±SEM. Means of 2 groups were compared using Student t test (unpaired, two tailed), and one-way ANOVA was used for comparison of more than 2 groups with P<0.05 considered to be statistically significant.
An expanded Materials and Methods can be found in the online data supplement available at http://circres.ahajournals.org.
Results
Effect of Adenosine on CASMC Proliferation
Stimulation of cultured porcine CASMCs with adenosine induced increases of the cellular DNA content, protein synthesis, and cell number in a concentration-dependent manner, with an apparent EC50 10 eol/L (Figure 1A through 1C). On average, adenosine, at 100 eol/L, induced 5-fold and 2.5-fold increases in cellular DNA content and protein synthesis respectively (Figure 1A and 1B). Consistent with this observation, a corresponding 30% increase in the total cell number was induced after a 4-day incubation of CASMCs with adenosine (Figure 1C). In addition, adenosine stimulation (100 eol/L, 24 hours) nearly doubled the number of PCNA-positive cells (Figure 1D through 1F). These results clearly indicate that adenosine is a CASMC mitogen.
AR mRNA Expression in Cultured CASMCs and Intact Coronary Arteries
Figure 2 shows that cultured CASMCs expressed high levels of the mRNAs for A1R, A2AR, and A3R, with a barely detectable mRNA level for A2BR. A similar expression pattern was found in native CASMCs isolated from pig right coronary arteries denuded of endothelium (Figure 2). Of note, although our RT-PCR approach only was able to amplify very faint bands for the A2BR in CASMCs, the same experimental condition resulted in a robust amplification for the A2BR mRNA in primary cultured porcine coronary endothelial cells (Figure 2B). Thus, our RT-PCR analysis suggests that porcine CASMCs primarily express A1R, A2AR, and A3R but not A2BR.
Effect of AR Agonists on CASMC Proliferation
Stimulation of CASMCs with NECA, a nonselective AR agonist, fully mimicked the mitogenic effect of adenosine as shown by increases in the cellular DNA/protein synthesis and cell number (Figure 3). As expected, NECA exhibited a similar efficacy to adenosine, yet it was more potent than adenosine in triggering cell proliferation as evidenced by one log-unit left shift of its dose-response curves (Figure 3A and 3B). In addition, less efficacious, but more potent, mitogenic effects were observed for R-PIA or CCPA, two A1R-selective agonists (Figure 3). In contrast, CGS21680and 2-Cl-IB-MECA, A2AR and A3R-selective agonists, respectively, were essentially inactive up to 1 eol/L (Figure 3). These results suggest a major role for A1R in mediating the mitogenic action of adenosine.
Effect of AR Antagonists on CASMC Proliferation
Figure 4 shows that adenosine-, NECA-, and CCPA-induced DNA/protein synthesis were dramatically inhibited in a concentration-dependent manner by DPCPX, a highly selective A1R antagonist. Interestingly, although CCPA-induced DNA/protein synthesis were nearly abolished by 0.1 eol/L DPCPX, the effects of adenosine and NECA were only inhibited by 70% to 90% (Figure 4A and 4B). In addition, treatment of the cells with MRS1706 or VUF5574, A2BR- and A3R-selective antagonists, respectively, had no significant impact on the synthesis of DNA (Figure 4C) and protein (Figure 4D) induced by adenosine, NECA, or CCPA. This indicates that A2BR and A3R may not be critical in the mitogenic action of adenosine. In analysis of A2AR, however, we observed an apparent conflicting result in that DMPX. A broad A2-selective antagonist even at 10 eol/L, did not affect the DNA/protein synthesis induced by adenosine, NECA, or CCPA, whereas ZM241385, an A2AR-selective antagonist, unexpectedly inhibited not only the effects of adenosine and NECA, but also the effect of CCPA by 30% at the DNA level and 50% at the protein level (Figure 4C and 4D). Therefore, we question the selectivity of ZM241385 on the porcine A2AR versus A1R.
Molecular Cloning and Pharmacological Characterization of the Porcine A1R
To overcome the potential drawback of our pharmacological analysis that relied on the ligand-selectivity inferred from the A1R of other species, we cloned the porcine A1R and characterized its pharmacological properties. By degenerate PCR and RACE approaches, we successfully isolated the porcine A1R cDNA with an open reading frame of 981 bp (GenBank accession no. AY772411). Hydrophobicity analysis showed a deduced primary sequence of 326 amino acids containing 7 hydrophobic domains characteristic of G proteineCcoupled receptors (Figure 5A). Sequence alignment revealed that the porcine A1R had the highest amino acid identity with human A1R (94.2%), and like the A1R of other species,19 displayed very short N- and C-termini (Figure 5A). In addition, a possible palmitoylation site,20 2 putative N-glycosylation sites, and several protein kinase phosphorylation sites were conserved (Figure 5A).
The cloned cDNA was stably transfected into COS-1 cells devoid of ARs,21 and its binding properties were compared with the native receptor in CASMCs. Figure 5B shows membranes from the transfected COS-1 cells displayed high-affinity binding for [3H]-DPCPX (Kd=0.58±0.06 nmol/L) with a Bmax of 290.6±44.3 fmol/mg. A similar binding site was also determined in CASMC membranes (Kd=0.59±0.08 nmol/L) with a Bmax of 28.77±6.45 fmol/mg. No specific binding to [3H]-DPCPX was detected in the membranes of nontransfected COS-1 cells (data not shown). In addition, competition binding assays revealed the rank order of potency for the agonists was CCPA R-PIA > NECA > 2-Cl-IB-MECA > CGS21680 and of the antagonists was DPCPX > ZM241385 > MRS1706 > DMPX VUF5574 (Figure 5C), reflecting characteristics of typical A1R.
Figure 5D shows that in A1R-transfected COS-1 cells, CCPA inhibited forskolin-induced cAMP elevation, and increased [Ca2+]i in a concentration-dependent manner. CCPA also inhibited forskolin-induced cAMP accumulation in cultured CASMCs, albeit in a lower efficacy (Figure 5D). We, however, did not observe any Ca2+ signaling on CCPA stimulation of CASMCs. Consistent with the literature,1 the A1R-mediated Ca2+ response in COS-1 cells was completely prevented by pertussis toxin (PTX) pretreatment (data not shown), indicative of coupling to Gi/o proteins.
Effects of A1R Antisense OGN and Overexpression on CASMC Proliferation
To extend our pharmacological analysis, we designed antisense OGN to the porcine A1R and tested their effects on cell proliferation. Figure 6A shows antisense, but not sense, OGN substantially decreased A1R protein expression as evaluated by Western blotting. Reduction of the A1R by antisense, but not sense, OGN decreased adenosine- and NECA-induced DNA synthesis by more than 80% and also nearly abolished the action of CCPA, with no effect on LPA-induced DNA synthesis (Figure 6B).
Figure 6D shows that A1R overexpression had no significant effect on the basal DNA synthesis as compared with mock transfection (Figure 6D). However, A1R overexpression potentiated adenosine- and CCPA-induced DNA synthesis by 53% and 61%, respectively, whereas the response to LPA was not affected (Figure 6D). A moderate increase of the transfected A1R protein expression was confirmed by Western blotting (Figure 6C).
Effects of PTX and PD81723 on CASMC Proliferation
Pretreatment of CASMCs with PTX (Gi protein inhibitor, 100 ng/mL, 24 hours) significantly suppressed, but did not abolish, both adenosine- and NECA-induced DNA synthesis, whereas the action of CCPA was completely inhibited (Figure 7A). These results are consistent with our antagonist and antisense results.
PD81723 can allosterically enhance A1R binding and signaling.1 Treatment of the cells with PD81723 (3 eol/L) significantly potentiated adenosine- and CCPA-induced cellular DNA synthesis, with no effect on the basal or LPA-induced DNA synthesis (Figure 7B). The opposite effects of PTX and PD81723 provided additional evidence that A1R can mediate CASMC proliferation.
Dual Effects of Adenosine and CCPA on DNA Synthesis in Organ-Cultured Coronary Arteries
We next extended our observation in cell culture into the ex vivo model of vessel cultures. Treatment of the endothelium-denuded porcine coronary artery rings with adenosine or CCPA induced a stimulatory effect on DNA synthesis at low concentrations and an inhibitory action at high concentrations (Figure 8A). Specifically, adenosine at 10 eol/L and CCPA at 10 nmol/L significantly increased DNA synthesis by 34.7% and 40.3%, respectively, whereas at the highest concentrations, both inhibited DNA synthesis by 38.9% and 15.8%, respectively (Figure 8A). Strikingly, the magnitude of increase in DNA synthesis triggered by CCPA was comparable to the maximal action of PDGF (Figure 8A).
Finally, we examined whether the stimulatory effects of adenosine and CCPA are because of A1R activation. Figure 8B shows that pretreatment of the artery rings with DPCPX (0.1 eol/L) abolished adenosine- and CCPA-induced increases of DNA synthesis, without affecting the basal and PDGF-induced DNA synthesis, indicative of a predominant role for the A1R.
Discussion
In the present study, we have demonstrated for the first time that adenosine can stimulate the proliferation of porcine CASMCs in culture and in intact coronary arteries. We also have demonstrated that the mitogenic effect of adenosine on CASMCs is predominately mediated by the A1R that has been cloned and pharmacologically characterized.
Historically, it has been thought that contractile vasoactive substances such as TxA2, angiotensin II, and endothelin-1 have stimulatory effects on VSMC growth, whereas smooth muscle dilators, eg, prostacyclin and nitric oxide, inhibit VSMC proliferation.7 In line with this concept, Jonzon et al22 observed nearly 2 decades ago that in cultured rat aortic VSMCs, adenosine inhibited cellular DNA synthesis. Recently, using the same model of cultured aortic VSMCs, Dubey et al reported a series of studies supporting the antimitogenic effect of adenosine and further highlighting a specific role for the A2BR.4eC8 In addition, an A2BR-mediated proapoptotic effect of adenosine in cultured human aortic VSMCs has also been reported.23 Despite these consistent observations, it remained unknown whether the growth-inhibitory effect of adenosine on aortic VSMCs can be generalized to VSMCs from other locations. Raising this point is important because adenosine is mitogenic in vascular endothelial cells via activation of the same A2BR.24,25 In the present study, we found that stimulation of cultured porcine CASMCs with adenosine increased cellular DNA content, protein synthesis, cell number, and PCNA staining, indicating a mitogenic action of adenosine. Importantly, our data further showed that this mitogenic effect of adenosine extends to intact coronary arteries in an organ culture model of vascular disease. Thus, our results are in sharp contrast to the aforementioned studies and challenge the conceptual view that vasodilators can only function as antimitogens in VSMCs. In this regard, it is tempting to postulate that at least in the porcine conduit coronary arteries, adenosine may be a unique vasodilator that also functions as a mitogen in smooth muscles.
How can we reconcile the contradictory results between our study and those of others The precise reasons for this discrepancy are unclear, but several factors could potentially account for the difference, eg, the difference in experimental methodology and animal species. However, the most important factor that could explain such a difference is a possible differential receptor expression mechanism. The hypothesis we propose here is that VSMC A1R mediates mitogenesis, whereas A2BR mediates antimitogenic effects. Therefore, the end functional readout in response to adenosine with respect to proliferation versus antiproliferation would highly depend on the balance of the expression levels and the signaling efficiency of these 2 receptors. In the present study, we found porcine CASMCs expressed a high level of A1R as demonstrated by the high mRNA level, detectable ligand binding, positive immunoblotting, and potent cAMP modulation. In contrast, a barely detectable message for A2BR, together with the fact that the A2BR-selective antagonist did not affect adenosine-induced cell proliferation, strongly indicate that porcine CASMCs did not express a significant number of functional A2BR. Thus, lack of the growth-inhibitory A2BR could render porcine CASMCs proliferative in response to adenosine via activation of the highly expressed A1R. Evidence supporting this view includes the following: (1) A1R-selective agonists mimicked the mitogenic action of adenosine; (2) A1R-selective antagonist blocked adenosine-, NECA- and CCPA-induced DNA/protein synthesis; (3) adenosine- and CCPA-induced DNA synthesis was inhibited by PTX, but potentiated by PD81723, an allosteric A1R enhancer; (4) antisense knockdown of A1R decreased adenosine- and CCPA-induced DNA synthesis; and (5) conversely, A1R overexpression increased the magnitude of the responses to adenosine and CCPA. Thus, we have provided several lines of compelling data demonstrating that A1R plays a key role in adenosine-mediated CASMC proliferation, a finding that is consistent with a recent study showing stimulation of angiogenesis by A1R agonists.26 On the other hand, it should be noted that the antimitogenic effect of adenosine on rat aortic VSMCs was observed in the presence of serum or PDGF,4eC8 both of which are potent mitogens that could mask the relatively weak growth-promoting signals from the A1R that could be in a lower expression level than the A2BR. In addition, it should also be emphasized that the antimitogenic effect of adenosine on rat aortic VSMCs was observed at low, but not standard, cell seeding density.7,8 Therefore, the contradictory results between our study and others could be reconciled by a differential receptor expression mechanism and perhaps also by the difference in the experimental design. We, however, favor the differential receptor expression mechanism, because we found the following: (1) adenosine increased the DNA synthesis of CASMCs seeded in standard and low density, and further increased serum- and PDGF-induced DNA synthesis (see Figures I and II in the online data supplement); (2) in contrast, adenosine has no or minor effects on the DNA synthesis of aortic VSMCs in the absence of serum (online Figure III); and (3) importantly, overexpression of A1R in aortic VSMCs changed the effect of adenosine from antimitogenic to mitogenic (online Figures V and VI).
Although there were no significant growth-inhibitory signals generated by adenosine in CASMCs, it should be noted that the A2AR are highly expressed in porcine CASMCs and intact coronary arteries, and it has been shown that adenosine-induced relaxation of coronary arteries from pig27,28 and mouse29 are mediated by A2AR, as opposed to A2BR in rat coronary arteries.30 Although A2AR and A2BR may similarly increase cAMP, perhaps the A2AR and A2BR are more strongly coupled to relaxation and antiproliferation, respectively. Thus, adenosine-induced relaxation and proliferation in porcine CASMCs could be 2 unrelated cellular events driven by different receptors. From this point of view, adenosine is unique, because virtually all other vasodilators inhibit VSMC growth, perhaps because of their targeting on either one receptor, enzyme or ion channel in VSMCs.
The A1R have remarkable species differences in pharmacology. The bovine, canine,31 and guinea pig1 homologs are the most divergent as compared with human, rabbit, rat, and mouse with respect to the agonist binding affinity. Porcine CASMCs have been postulated to express A1R.14,15 However, the molecular and pharmacological properties of this receptor have not been conclusively documented. We now report the cloning of the cDNA for this receptor. Heterologous expression and functional analysis of this receptor demonstrated CCPA and DPCPX are the most potent agonist and antagonist, respectively. In addition, our ligand binding assay revealed the rank order of potency for the agonists/antagonists was similar to the A1R cloned from humans, rabbits, and rodents.1 The compound ZM241385, however, exhibits a moderate affinity to the porcine A1R that is much higher than in other species. Therefore, although this compound has been used as a highly selective A2AR antagonist, caution must be taken when used in pig tissues/cells to differentiate the A1R versus A2AR.
The fact that our results differ from results of prior work in VSMCs of other sites exemplifies the prediction made by Ross32 that VSMCs of different embryonic origins could respond differently to stimuli that generate atherosclerotic lesions in an artery segmenteCdependent manner. Although it is too early to conclude that adenosine is involved in the etiology of coronary artery diseases, several aspects of consideration pertaining to adenosine and the A1R point to that potential: (1) the A1R is upregulated in diabetes,33 hypertension,34 and by oxidative stress,35 all of which contribute to the development of coronary artery disease; (2) the A1R has higher affinity for adenosine than A2R, and the desensitization of A1R is much slower than A2R (t1/2: 10 hours versus 20 minutes),36 both properties of which could imply a role for A1R in chronic effects on CASMC proliferation; and (3) adenosine is directly released from endothelial cells, stressed myocardial cells, and can also accumulate after catabolism of ATP/ADP released from activated platelets and inflammatory cells surrounding CASMCs after arterial injury.1 From these points of view, together with the fact that VSMC proliferation is involved in the development of hypertension and atherosclerosis as well as restenosis after angioplasty and bypass surgery,7,8,32 it is conceivable that identification of a novel mitogenic effect of adenosine and defining its receptor mechanism on CASMC will not only change the paradigm of our current view on the role of adenosine in the coronary circulation and disease, but also imply potential clinical applications via pharmacological intervention such as A1R antagonism for limiting the abnormal growth of CASMCs under the aforementioned diseases.
In summary, we report a novel mitogenic action of adenosine in cultured porcine CASMCs and in ex vivo coronary arteries. In addition, we demonstrate that this effect is predominantly mediated by the A1R. These results suggest that adenosine may make a previously unrecognized contribution in vascular diseases related to CASMC proliferation, thereby highlighting the A1R as a new potential target for further mechanistic study and pharmacological intervention.
Acknowledgments
This work was supported by NIH grants RR13223 and HL62552 (M.S.), an American Diabetes Association grant (P.A.W.), and a MU Life Science Fellowship (J.S.). The authors would like to thank Meifang Wang, Christy Hanna, and Marlene Vaught for their technical assistance.
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Tucker AL, Robeva AS, Taylor HE, Holeton D, Bockner M, Lynch KR, Linden J. A1 adenosine receptors: two amino acids are responsible for species differences in ligand recognition. J Biol Chem. 1994; 269: 27900eC27906.
Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999; 340: 115eC126.
Liu IM, Tzeng TF, Tsai CC, Lai TY, Chang CT, Cheng JT. Increase in adenosine A1 receptor gene expression in the liver of streptozotocin-induced diabetic rats. Diabetes Metab Res Rev. 2003; 19: 209eC215.
Fahim M, Mustafa SJ. Evidence for the presence of A1 adenosine receptors in the aorta of spontaneously hypertensive rats. Br J Pharmacol. 2001; 134: 1760eC1766.
Nie Z, Mei Y, Ford M, Rybak L, Marcuzzi A, Ren H, Stiles GL, Ramkumar V. Oxidative stress increases A1 adenosine receptor expression by activating nuclear factor kappa B. Mol Pharmacol. 1998; 53: 663eC669.
Stiles GL. Adenosine receptors. J Biol Chem. 1992; 267: 6451eC6454.(Jianzhong Shen, Stephen P)
Department of Cellular and Integrative Physiology (M.S.), Indiana University School of Medicine, Indianapolis, Ind.
Abstract
Adenosine is a vascular endothelial cell mitogen, but anti-mitogenic for aortic smooth muscle cells and fibroblasts when acting via the A2B adenosine receptor. However, we show that adenosine increases porcine coronary artery smooth muscle cell (CASMC) number, cellular DNA content, protein synthesis, and PCNA staining. RT-PCR analysis indicates that porcine CASMC express A1, A2A, A3, and barely detectable levels of A2B receptor mRNAs. The mitogenic effect of adenosine is mimicked by NECA, CCPA, and R-PIA, but not by CGS21680and 2-Cl-IB-MECA, and is inhibited by DPCPX, indicating a prominent role for the A1 receptor. This interpretation is supported by the finding that adenosine- and CCPA-induced DNA synthesis is significantly inhibited by pertussis toxin, but substantially potentiated by PD81723, an allosteric enhancer of the A1 receptor. When a cDNA encoding the porcine A1 receptor was cloned and expressed in COS-1 cells, A1 receptor pharmacology is confirmed. Anti-sense oligonucleotides to the cloned sequence dramatically suppress the mitogenic effect of adenosine and CCPA. Conversely, over-expression of the cloned A1 receptor in CASMC increases adenosine- and CCPA-induced DNA synthesis. Furthermore, stimulation with adenosine or CCPA of intact coronary arteries in an organ culture model of vascular disease increases cellular DNA synthesis, which was abolished by DPCPX. We conclude that adenosine acts as a novel mitogen in porcine CASMC that express the A1 adenosine receptor, possibly contributing to the development of coronary artery disease.
Key Words: adenosine receptors coronary artery smooth muscle cells proliferation molecular cloning porcine
Introduction
The diverse cellular actions of adenosine are mediated by a family of adenosine receptors (ARs), of which 4 subtypes (A1R, A2AR, A2BR, and A3R) have been cloned and pharmacologically characterized.1,2 In general, the A1R and A3R are coupled with Gi/o proteins, whose activation causes a decrease of intracellular cAMP. In contrast, activation of the Gs-coupled A2AR and A2BR increases intracellular cAMP.1,2 Thus, the end biological action of adenosine in a particular organ or cell population may depend on the relative expression level and signaling efficiency of the individual AR subtypes.3
Adenosine, like many other vasodilators, historically has been thought to act as an inhibitor of the proliferation of vascular smooth muscle cells (VSMCs). This contention was supported by recent studies in cultured aortic VSMCs, showing that adenosine was antimitogenic through its activation of the A2BR.4eC8 To the best of our knowledge, however, current studies of the long-term effects of adenosine on VSMC proliferation have been limited to aortic smooth muscle. Given the heterogeneity of VSMCs9 and the diversity of the expression profile of ARs in cells from different blood vessels,10 it remains to be shown whether the antiproliferative action of adenosine can be extended to other VSMCs, particularly coronary artery smooth muscle cells (CASMCs).
In porcine CASMCs, previous work in our laboratory has demonstrated that ATP, a major precursor of adenosine, induced a proliferative response via activation of several intracellular signaling pathways, involving activation of the P2Y2 receptor.11eC13 In the process of identifying the P2Y-independent receptor mechanism(s) responsible for ATP-induced CASMC proliferation, we unexpectedly found that adenosine, but not most other nucleotides/nucleosides, mimicked the effects of ATP. This observation led us to propose that, in contrast to the previous observation in aortic VSMCs, adenosine may behave as a mitogen in CASMCs by a receptor mechanism not involving A2BR. Indeed, ligand-binding assays14 and histological studies15 indicated that porcine coronary arteries express A1R. We therefore hypothesized that A1R activation causes CASMC proliferation.
Because no porcine AR has been cloned, in the present study, we have cloned and pharmacologically characterized the porcine A1R and explored its role in CASMC proliferation. Results presented here demonstrate that adenosine acts via the A1R as a mitogen in cultured porcine CASMCs and in intact coronary arteries.
Materials and Methods
CASMC Isolation and Cell Culture
Porcine CASMCs dispersed from right coronary arteries of farm pigs (Jennings Premium Meats, New Franklin, Mo) were cultured as previously described in our laboratory.11eC13 Smooth muscle cell lineage was confirmed by -smooth muscle actin immunocytochemistry. Stock CASMC cultures were maintained in a subconfluent state and used between passage 4 and 10.11eC13
[3H]-Thymidine Incorporation Assays
CASMCs (35 000/well) were seeded in 12-well plates in DMEM/high-glucose medium containing 10% FBS for 48 hours, and then starved for 24 hours in serum-free DMEM containing 0.1% BSA before stimulation. Incorporation of [3H]-thymidine into DNA was performed as previously described.11eC13
Protein Synthesis Assays
CASMCs were seeded and starved as stated earlier. [3H]-leucine incorporation was used for measuring cellular total protein synthesis as previously described.13
Determination of Cell Numbers
CASMCs were seeded in 96-well plates (2000 cells/well) in DMEM containing 10% FBS for 48 hours, and then serum-starved for 48 hours, after which the culture medium was replaced with fresh serum-free starvation medium and CASMCs were stimulated with agonists for 4 days. Cell numbers were determined by MTT Cell Growth Assay Kit (Chemicon) as previously described.13
Immunocytochemistry for Proliferating Cell Nuclear Antigen
CASMCs at 40% confluence were starved for 24 hours before stimulation with adenosine for 24 hours. Standard immunocytochemistry for proliferating cell nuclear antigen (PCNA) was performed using rabbit anti-PCNA (Santa Cruz Biotechnology).
Receptor cDNA Cloning and RACE Experiments
The full cDNA sequence of the porcine A1R and partial cDNA coding sequences for porcine A2AR, A2BR, and A3R were cloned by PCR using a Smart Race cDNA amplification kit (Clontech).
Heterologous Expression
The pCR3.1 plasmids containing the porcine A1R sequence were transfected into COS-1 cells using Effectene Transfection Reagent (Qiagen).
Intracellular [Ca2+] Measurements
Intracellular free Ca2+ concentration ([Ca2+]i) was measured with the InCa2+ calcium imaging system (Intracellular Imaging Inc) as previously described.13,16
Intracellular cAMP Measurements
Intracellular cAMP levels were determined by using the Direct cAMP Correlate-EIA Kit (Assay Designs, Inc).
Radioligand Binding Assays
Binding assays were performed with crude cell membranes using [3H]-DPCPX (130 Ci/mmol; Amersham) as the radioligand as previously described.17
A1R Overexpression in CASMCs
CASMCs were transfected in 12-well plates using the Effectene Transfection Reagent according to the manufacturer’s instruction.
Antisense Oligonucleotide Experiments
Phosphorothioate-modified oligonucleotide (OGN) (antisense, 5'-GAGATGGAGGGCGGCATGGT-3'; sense, 5'-ACCATGCCGCCCTCCATCTC-3') were used for A1R knockdown as previously documented.13
Western Blot Analysis
Cells were lysed and lysates Western blotted for the A1R (rabbit polyclonal antibody, 1:1000 dilutions, Novus Biologicals, Inc). Membranes were stripped and reprobed with anti-actin antibody (1:1000; Cytoskeleton).11eC13
Ex Vivo Coronary Artery Organ Culture and DNA Synthesis Assays
Pig hearts were isolated and arteries organ cultured as described in our laboratory.13,16 Incorporation of [3H]-thymidine into coronary arterial rings was performed as described previously.18
Reverse-Transcription Polymerase Chain Reaction Analysis
Primers for reverse-transcription polymerase chain reaction (RT-PCR) were designed based on our original cloned sequences for the 4 porcine ARs (GenBank accession nos. AY772411, AY772412, AY772413, and AY772414).
Materials
The following compounds were from TOCRIS: 5'-N-ethylcarboxamidoadenosine (NECA); 2-chloro-N6-cyclopentyladenosine (CCPA); 2-[p-(2-carboxyethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2-Cl-IB-MECA); 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo-{2,3-a}{1,3,5}-triazin-5-yl-amino]ethyl)phenol (ZM241385); N-(4-acethyl-phenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1706); N-(2-methoxyphenyl)-N-[2-(3-pyridyl) quinazolin-4-yl]urea (VUF5574). Reagents from Sigma were as following: 3,7-dimethyl-1-propargylxanthine (DMPX); (eC)-N6-(2-phenylisopropyl)adenosine (R-PIA); platelet-derived growth factor (PDGF); and lysophosphatidic acid (LPA).
Analysis of Data
Data are expressed as mean±SEM. Means of 2 groups were compared using Student t test (unpaired, two tailed), and one-way ANOVA was used for comparison of more than 2 groups with P<0.05 considered to be statistically significant.
An expanded Materials and Methods can be found in the online data supplement available at http://circres.ahajournals.org.
Results
Effect of Adenosine on CASMC Proliferation
Stimulation of cultured porcine CASMCs with adenosine induced increases of the cellular DNA content, protein synthesis, and cell number in a concentration-dependent manner, with an apparent EC50 10 eol/L (Figure 1A through 1C). On average, adenosine, at 100 eol/L, induced 5-fold and 2.5-fold increases in cellular DNA content and protein synthesis respectively (Figure 1A and 1B). Consistent with this observation, a corresponding 30% increase in the total cell number was induced after a 4-day incubation of CASMCs with adenosine (Figure 1C). In addition, adenosine stimulation (100 eol/L, 24 hours) nearly doubled the number of PCNA-positive cells (Figure 1D through 1F). These results clearly indicate that adenosine is a CASMC mitogen.
AR mRNA Expression in Cultured CASMCs and Intact Coronary Arteries
Figure 2 shows that cultured CASMCs expressed high levels of the mRNAs for A1R, A2AR, and A3R, with a barely detectable mRNA level for A2BR. A similar expression pattern was found in native CASMCs isolated from pig right coronary arteries denuded of endothelium (Figure 2). Of note, although our RT-PCR approach only was able to amplify very faint bands for the A2BR in CASMCs, the same experimental condition resulted in a robust amplification for the A2BR mRNA in primary cultured porcine coronary endothelial cells (Figure 2B). Thus, our RT-PCR analysis suggests that porcine CASMCs primarily express A1R, A2AR, and A3R but not A2BR.
Effect of AR Agonists on CASMC Proliferation
Stimulation of CASMCs with NECA, a nonselective AR agonist, fully mimicked the mitogenic effect of adenosine as shown by increases in the cellular DNA/protein synthesis and cell number (Figure 3). As expected, NECA exhibited a similar efficacy to adenosine, yet it was more potent than adenosine in triggering cell proliferation as evidenced by one log-unit left shift of its dose-response curves (Figure 3A and 3B). In addition, less efficacious, but more potent, mitogenic effects were observed for R-PIA or CCPA, two A1R-selective agonists (Figure 3). In contrast, CGS21680and 2-Cl-IB-MECA, A2AR and A3R-selective agonists, respectively, were essentially inactive up to 1 eol/L (Figure 3). These results suggest a major role for A1R in mediating the mitogenic action of adenosine.
Effect of AR Antagonists on CASMC Proliferation
Figure 4 shows that adenosine-, NECA-, and CCPA-induced DNA/protein synthesis were dramatically inhibited in a concentration-dependent manner by DPCPX, a highly selective A1R antagonist. Interestingly, although CCPA-induced DNA/protein synthesis were nearly abolished by 0.1 eol/L DPCPX, the effects of adenosine and NECA were only inhibited by 70% to 90% (Figure 4A and 4B). In addition, treatment of the cells with MRS1706 or VUF5574, A2BR- and A3R-selective antagonists, respectively, had no significant impact on the synthesis of DNA (Figure 4C) and protein (Figure 4D) induced by adenosine, NECA, or CCPA. This indicates that A2BR and A3R may not be critical in the mitogenic action of adenosine. In analysis of A2AR, however, we observed an apparent conflicting result in that DMPX. A broad A2-selective antagonist even at 10 eol/L, did not affect the DNA/protein synthesis induced by adenosine, NECA, or CCPA, whereas ZM241385, an A2AR-selective antagonist, unexpectedly inhibited not only the effects of adenosine and NECA, but also the effect of CCPA by 30% at the DNA level and 50% at the protein level (Figure 4C and 4D). Therefore, we question the selectivity of ZM241385 on the porcine A2AR versus A1R.
Molecular Cloning and Pharmacological Characterization of the Porcine A1R
To overcome the potential drawback of our pharmacological analysis that relied on the ligand-selectivity inferred from the A1R of other species, we cloned the porcine A1R and characterized its pharmacological properties. By degenerate PCR and RACE approaches, we successfully isolated the porcine A1R cDNA with an open reading frame of 981 bp (GenBank accession no. AY772411). Hydrophobicity analysis showed a deduced primary sequence of 326 amino acids containing 7 hydrophobic domains characteristic of G proteineCcoupled receptors (Figure 5A). Sequence alignment revealed that the porcine A1R had the highest amino acid identity with human A1R (94.2%), and like the A1R of other species,19 displayed very short N- and C-termini (Figure 5A). In addition, a possible palmitoylation site,20 2 putative N-glycosylation sites, and several protein kinase phosphorylation sites were conserved (Figure 5A).
The cloned cDNA was stably transfected into COS-1 cells devoid of ARs,21 and its binding properties were compared with the native receptor in CASMCs. Figure 5B shows membranes from the transfected COS-1 cells displayed high-affinity binding for [3H]-DPCPX (Kd=0.58±0.06 nmol/L) with a Bmax of 290.6±44.3 fmol/mg. A similar binding site was also determined in CASMC membranes (Kd=0.59±0.08 nmol/L) with a Bmax of 28.77±6.45 fmol/mg. No specific binding to [3H]-DPCPX was detected in the membranes of nontransfected COS-1 cells (data not shown). In addition, competition binding assays revealed the rank order of potency for the agonists was CCPA R-PIA > NECA > 2-Cl-IB-MECA > CGS21680 and of the antagonists was DPCPX > ZM241385 > MRS1706 > DMPX VUF5574 (Figure 5C), reflecting characteristics of typical A1R.
Figure 5D shows that in A1R-transfected COS-1 cells, CCPA inhibited forskolin-induced cAMP elevation, and increased [Ca2+]i in a concentration-dependent manner. CCPA also inhibited forskolin-induced cAMP accumulation in cultured CASMCs, albeit in a lower efficacy (Figure 5D). We, however, did not observe any Ca2+ signaling on CCPA stimulation of CASMCs. Consistent with the literature,1 the A1R-mediated Ca2+ response in COS-1 cells was completely prevented by pertussis toxin (PTX) pretreatment (data not shown), indicative of coupling to Gi/o proteins.
Effects of A1R Antisense OGN and Overexpression on CASMC Proliferation
To extend our pharmacological analysis, we designed antisense OGN to the porcine A1R and tested their effects on cell proliferation. Figure 6A shows antisense, but not sense, OGN substantially decreased A1R protein expression as evaluated by Western blotting. Reduction of the A1R by antisense, but not sense, OGN decreased adenosine- and NECA-induced DNA synthesis by more than 80% and also nearly abolished the action of CCPA, with no effect on LPA-induced DNA synthesis (Figure 6B).
Figure 6D shows that A1R overexpression had no significant effect on the basal DNA synthesis as compared with mock transfection (Figure 6D). However, A1R overexpression potentiated adenosine- and CCPA-induced DNA synthesis by 53% and 61%, respectively, whereas the response to LPA was not affected (Figure 6D). A moderate increase of the transfected A1R protein expression was confirmed by Western blotting (Figure 6C).
Effects of PTX and PD81723 on CASMC Proliferation
Pretreatment of CASMCs with PTX (Gi protein inhibitor, 100 ng/mL, 24 hours) significantly suppressed, but did not abolish, both adenosine- and NECA-induced DNA synthesis, whereas the action of CCPA was completely inhibited (Figure 7A). These results are consistent with our antagonist and antisense results.
PD81723 can allosterically enhance A1R binding and signaling.1 Treatment of the cells with PD81723 (3 eol/L) significantly potentiated adenosine- and CCPA-induced cellular DNA synthesis, with no effect on the basal or LPA-induced DNA synthesis (Figure 7B). The opposite effects of PTX and PD81723 provided additional evidence that A1R can mediate CASMC proliferation.
Dual Effects of Adenosine and CCPA on DNA Synthesis in Organ-Cultured Coronary Arteries
We next extended our observation in cell culture into the ex vivo model of vessel cultures. Treatment of the endothelium-denuded porcine coronary artery rings with adenosine or CCPA induced a stimulatory effect on DNA synthesis at low concentrations and an inhibitory action at high concentrations (Figure 8A). Specifically, adenosine at 10 eol/L and CCPA at 10 nmol/L significantly increased DNA synthesis by 34.7% and 40.3%, respectively, whereas at the highest concentrations, both inhibited DNA synthesis by 38.9% and 15.8%, respectively (Figure 8A). Strikingly, the magnitude of increase in DNA synthesis triggered by CCPA was comparable to the maximal action of PDGF (Figure 8A).
Finally, we examined whether the stimulatory effects of adenosine and CCPA are because of A1R activation. Figure 8B shows that pretreatment of the artery rings with DPCPX (0.1 eol/L) abolished adenosine- and CCPA-induced increases of DNA synthesis, without affecting the basal and PDGF-induced DNA synthesis, indicative of a predominant role for the A1R.
Discussion
In the present study, we have demonstrated for the first time that adenosine can stimulate the proliferation of porcine CASMCs in culture and in intact coronary arteries. We also have demonstrated that the mitogenic effect of adenosine on CASMCs is predominately mediated by the A1R that has been cloned and pharmacologically characterized.
Historically, it has been thought that contractile vasoactive substances such as TxA2, angiotensin II, and endothelin-1 have stimulatory effects on VSMC growth, whereas smooth muscle dilators, eg, prostacyclin and nitric oxide, inhibit VSMC proliferation.7 In line with this concept, Jonzon et al22 observed nearly 2 decades ago that in cultured rat aortic VSMCs, adenosine inhibited cellular DNA synthesis. Recently, using the same model of cultured aortic VSMCs, Dubey et al reported a series of studies supporting the antimitogenic effect of adenosine and further highlighting a specific role for the A2BR.4eC8 In addition, an A2BR-mediated proapoptotic effect of adenosine in cultured human aortic VSMCs has also been reported.23 Despite these consistent observations, it remained unknown whether the growth-inhibitory effect of adenosine on aortic VSMCs can be generalized to VSMCs from other locations. Raising this point is important because adenosine is mitogenic in vascular endothelial cells via activation of the same A2BR.24,25 In the present study, we found that stimulation of cultured porcine CASMCs with adenosine increased cellular DNA content, protein synthesis, cell number, and PCNA staining, indicating a mitogenic action of adenosine. Importantly, our data further showed that this mitogenic effect of adenosine extends to intact coronary arteries in an organ culture model of vascular disease. Thus, our results are in sharp contrast to the aforementioned studies and challenge the conceptual view that vasodilators can only function as antimitogens in VSMCs. In this regard, it is tempting to postulate that at least in the porcine conduit coronary arteries, adenosine may be a unique vasodilator that also functions as a mitogen in smooth muscles.
How can we reconcile the contradictory results between our study and those of others The precise reasons for this discrepancy are unclear, but several factors could potentially account for the difference, eg, the difference in experimental methodology and animal species. However, the most important factor that could explain such a difference is a possible differential receptor expression mechanism. The hypothesis we propose here is that VSMC A1R mediates mitogenesis, whereas A2BR mediates antimitogenic effects. Therefore, the end functional readout in response to adenosine with respect to proliferation versus antiproliferation would highly depend on the balance of the expression levels and the signaling efficiency of these 2 receptors. In the present study, we found porcine CASMCs expressed a high level of A1R as demonstrated by the high mRNA level, detectable ligand binding, positive immunoblotting, and potent cAMP modulation. In contrast, a barely detectable message for A2BR, together with the fact that the A2BR-selective antagonist did not affect adenosine-induced cell proliferation, strongly indicate that porcine CASMCs did not express a significant number of functional A2BR. Thus, lack of the growth-inhibitory A2BR could render porcine CASMCs proliferative in response to adenosine via activation of the highly expressed A1R. Evidence supporting this view includes the following: (1) A1R-selective agonists mimicked the mitogenic action of adenosine; (2) A1R-selective antagonist blocked adenosine-, NECA- and CCPA-induced DNA/protein synthesis; (3) adenosine- and CCPA-induced DNA synthesis was inhibited by PTX, but potentiated by PD81723, an allosteric A1R enhancer; (4) antisense knockdown of A1R decreased adenosine- and CCPA-induced DNA synthesis; and (5) conversely, A1R overexpression increased the magnitude of the responses to adenosine and CCPA. Thus, we have provided several lines of compelling data demonstrating that A1R plays a key role in adenosine-mediated CASMC proliferation, a finding that is consistent with a recent study showing stimulation of angiogenesis by A1R agonists.26 On the other hand, it should be noted that the antimitogenic effect of adenosine on rat aortic VSMCs was observed in the presence of serum or PDGF,4eC8 both of which are potent mitogens that could mask the relatively weak growth-promoting signals from the A1R that could be in a lower expression level than the A2BR. In addition, it should also be emphasized that the antimitogenic effect of adenosine on rat aortic VSMCs was observed at low, but not standard, cell seeding density.7,8 Therefore, the contradictory results between our study and others could be reconciled by a differential receptor expression mechanism and perhaps also by the difference in the experimental design. We, however, favor the differential receptor expression mechanism, because we found the following: (1) adenosine increased the DNA synthesis of CASMCs seeded in standard and low density, and further increased serum- and PDGF-induced DNA synthesis (see Figures I and II in the online data supplement); (2) in contrast, adenosine has no or minor effects on the DNA synthesis of aortic VSMCs in the absence of serum (online Figure III); and (3) importantly, overexpression of A1R in aortic VSMCs changed the effect of adenosine from antimitogenic to mitogenic (online Figures V and VI).
Although there were no significant growth-inhibitory signals generated by adenosine in CASMCs, it should be noted that the A2AR are highly expressed in porcine CASMCs and intact coronary arteries, and it has been shown that adenosine-induced relaxation of coronary arteries from pig27,28 and mouse29 are mediated by A2AR, as opposed to A2BR in rat coronary arteries.30 Although A2AR and A2BR may similarly increase cAMP, perhaps the A2AR and A2BR are more strongly coupled to relaxation and antiproliferation, respectively. Thus, adenosine-induced relaxation and proliferation in porcine CASMCs could be 2 unrelated cellular events driven by different receptors. From this point of view, adenosine is unique, because virtually all other vasodilators inhibit VSMC growth, perhaps because of their targeting on either one receptor, enzyme or ion channel in VSMCs.
The A1R have remarkable species differences in pharmacology. The bovine, canine,31 and guinea pig1 homologs are the most divergent as compared with human, rabbit, rat, and mouse with respect to the agonist binding affinity. Porcine CASMCs have been postulated to express A1R.14,15 However, the molecular and pharmacological properties of this receptor have not been conclusively documented. We now report the cloning of the cDNA for this receptor. Heterologous expression and functional analysis of this receptor demonstrated CCPA and DPCPX are the most potent agonist and antagonist, respectively. In addition, our ligand binding assay revealed the rank order of potency for the agonists/antagonists was similar to the A1R cloned from humans, rabbits, and rodents.1 The compound ZM241385, however, exhibits a moderate affinity to the porcine A1R that is much higher than in other species. Therefore, although this compound has been used as a highly selective A2AR antagonist, caution must be taken when used in pig tissues/cells to differentiate the A1R versus A2AR.
The fact that our results differ from results of prior work in VSMCs of other sites exemplifies the prediction made by Ross32 that VSMCs of different embryonic origins could respond differently to stimuli that generate atherosclerotic lesions in an artery segmenteCdependent manner. Although it is too early to conclude that adenosine is involved in the etiology of coronary artery diseases, several aspects of consideration pertaining to adenosine and the A1R point to that potential: (1) the A1R is upregulated in diabetes,33 hypertension,34 and by oxidative stress,35 all of which contribute to the development of coronary artery disease; (2) the A1R has higher affinity for adenosine than A2R, and the desensitization of A1R is much slower than A2R (t1/2: 10 hours versus 20 minutes),36 both properties of which could imply a role for A1R in chronic effects on CASMC proliferation; and (3) adenosine is directly released from endothelial cells, stressed myocardial cells, and can also accumulate after catabolism of ATP/ADP released from activated platelets and inflammatory cells surrounding CASMCs after arterial injury.1 From these points of view, together with the fact that VSMC proliferation is involved in the development of hypertension and atherosclerosis as well as restenosis after angioplasty and bypass surgery,7,8,32 it is conceivable that identification of a novel mitogenic effect of adenosine and defining its receptor mechanism on CASMC will not only change the paradigm of our current view on the role of adenosine in the coronary circulation and disease, but also imply potential clinical applications via pharmacological intervention such as A1R antagonism for limiting the abnormal growth of CASMCs under the aforementioned diseases.
In summary, we report a novel mitogenic action of adenosine in cultured porcine CASMCs and in ex vivo coronary arteries. In addition, we demonstrate that this effect is predominantly mediated by the A1R. These results suggest that adenosine may make a previously unrecognized contribution in vascular diseases related to CASMC proliferation, thereby highlighting the A1R as a new potential target for further mechanistic study and pharmacological intervention.
Acknowledgments
This work was supported by NIH grants RR13223 and HL62552 (M.S.), an American Diabetes Association grant (P.A.W.), and a MU Life Science Fellowship (J.S.). The authors would like to thank Meifang Wang, Christy Hanna, and Marlene Vaught for their technical assistance.
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