Adenosine Inhibits Growth of Rat Aortic Smooth Muscle Cells
Possible Role of A2b Receptor
Abstract Abnormal growth of vascular smooth muscle cells (SMC) is frequently associated with hypertension and atherosclerosis, and homeostasis within a normal vessel is maintained by the balanced generation of both vasoconstrictors and vasodilators. Moreover, several endogenous vasoconstricting factors induce SMC growth, whereas several vasodilators inhibit SMC growth. Inasmuch as adenosine is a potent vasodilator, it is possible that it too could inhibit SMC growth. Hence, the effects of adenosine (10−8 to 10−3 mol/L), 2-chloroadenosine (a stable analogue of adenosine; 10−8 to 10−3 mol/L), and 8-bromo-cAMP (10−8 to 10−3 mol/L) on fetal calf serum (FCS; 2.5%)–induced growth of rat aortic SMC were evaluated. Growth was analyzed by assaying DNA synthesis (thymidine incorporation in SMC pulsed for 4 hours with 1 μCi/mL [3H]thymidine) and cell proliferation (change in cell number). Growth-arrested SMC were treated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine, or 8-bromo-cAMP for 24 hours for DNA synthesis or 4 days for cell proliferation. All three substances inhibited DNA synthesis and cell proliferation in a concentration-dependent manner. Compared with adenosine, 2-chloroadenosine was more potent in inhibiting growth. The inhibitory effects of 2-chloroadenosine were reversed by KF17837 (a specific A2 receptor antagonist) but not by DPCPX (a specific A1 receptor antagonist). Furthermore, the inhibitory effects of 2-chloroadenosine were not mimicked by CGS21680 (an A2a receptor agonist), and the effects of N6-cyclopentyladenosine (CPA; an A1 receptor agonist) were not markedly more potent than those of 2-chloroadenosine, suggesting that the inhibitory effects of adenosine are possibly mediated via A2b receptors. These studies provide evidence that adenosine inhibits SMC growth and suggest that a decrease in local levels of adenosine may initiate SMC growth and contribute to the vascular remodeling process observed in hypertension and atherosclerosis.
Vasoocclusive disorders are associated with hypertension, atherosclerosis, and restenosis after angioplasty and bypass surgery.1 2 3 4 Multiple factors and mechanisms may contribute to the pathogenesis of vasoocclusive disorders, including platelet adhesion and aggregation,3 endothelial dysfunction and damage,5 neutrophil-endothelium interactions,3 5 and perhaps most importantly, abnormal growth of vascular SMC.1 2 3 4 5 6 7 The growth processes contributing to pathological vascular structural changes (such as vascular remodeling, medial hyperplasia, and neointima formation) involve accumulation of SMC due to a combination of proliferation and directed migration of arterial SMC from the media to the intima.1 2 3 4 5 A number of autocrine/paracrine factors, present within the vessel wall and circulation,1 2 3 4 5 6 7 as well as physical forces such as pressure, can stimulate SMC growth.8 In normal blood vessels, quiescence is maintained by a balance between circulating and vessel wall–derived growth inhibitors (such as NO and prostaglandins) and growth promoters (such as PDGF, FGF, Ang II, and norepinephrine), which may interact with each other and govern SMC growth.1 2 3 4 5 6 7 Disruption of the balanced generation of vasoconstrictors and vasodilators or growth promoters and growth inhibitors under pathological conditions could trigger a cascade of events leading to increased proliferation and migration of SMC and neointima formation. Therefore, endogenous factors that are generated in substantial amounts locally at the interface between the endothelium and smooth muscle and that are antiproliferative may play a major vasoprotective role.
Adenosine may be another important vasoprotective factor, since this nucleoside is synthesized by the vessel wall9 10 11 12 13 and exerts numerous anti-vasoocclusive actions. Vascular cells have many metabolic pathways responsible for generating adenosine, and it has been shown that endothelial cells synthesize adenosine and have an adenine nucleotide pool that is two to three times higher than that of cardiomyocytes14 or hepatocytes. Furthermore, SMC can also synthesize adenosine via multiple pathways,15 16 17 and we have recently shown that vascular SMC metabolize cAMP to generate adenosine.18 Taken together, these findings suggest that substantial amounts of adenosine are synthesized locally within the vessel wall and more importantly at the interface between endothelial and vascular SMC, thus assuring pharmacologically active levels of vascular adenosine.
Adenosine is a nucleoside that has long been known as a “retaliatory” metabolite,19 particularly in the heart, where it induces cardioprotective effects.19 Furthermore, adenosine has several anti-vasoocclusive properties; for instance, it (1) induces vasodilation,20 (2) inhibits platelet aggregation,21 (3) prevents platelet adhesion,22 (4) abrogates neutrophil-induced endothelial damage,22 and (5) blocks the synthesis of potent vasoocclusive factors such as Ang II and norepinephrine by inhibiting renin release23 24 and noradrenergic neurotransmission.25 26 Although proliferation of SMC is important in neointima formation, less interest has been focused on studying the effects of adenosine on SMC proliferation. To date, only one report, by Jonzon et al,27 has demonstrated that adenosine inhibits PDGF-induced DNA synthesis of vascular smooth muscle; however, the effects of adenosine on cell proliferation (cytokinesis), which is the gold standard for cell growth,1 2 3 have not been studied. Furthermore, no additional studies to date have either confirmed or negated the important observations of Jonzon and colleagues.
The multiple biologic effects of adenosine are mediated via A1, A2a, A2b, A3, and A4 receptors. However, participation of A1 and A2 adenosine receptors appears to be more important in vascular biology, particularly with regard to reducing the risk and consequences of vasoocclusive events associated with hypertension and atherosclerosis.9 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 For example, activation of A1 receptors by adenosine attenuates the sympathetic nervous system by inhibiting the release of norepinephrine25 26 34 and attenuates the renin-angiotensin system by inhibiting renin release from juxtaglomerular cells.23 24 35 Both of these pathways have been implicated and are involved in the vascular remodeling processes associated with hypertension and atherosclerosis. Furthermore, A1 receptor-induced opening of K+ channels has been suggested to protect the heart from myocardial ischemia/reperfusion injury.28 29 30 31 32
Via activation of A2 receptors, adenosine has been shown to induce multiple anti-vasoocclusive effects,9 28 such as vasodilation,20 inhibition of platelet aggregation,21 diminished neutrophil adhesion to vascular endothelial cells,22 attenuation of neutrophil-induced endothelial cell damage,22 and stimulation of NO release from vascular endothelial cells.36 37 When more selective agonists and antagonists are used, it now appears that within the A2 receptor subfamily, the A2a subtype plays the most prominent role in vascular biology; however, A2b receptors mediate vasodilation in selected blood vessels from some species,9 28 and atypical A2 receptors may be involved in mediating adenosine-induced inhibition of platelet aggregation.9 21 28 Additionally, Jonzon et al27 suggested that adenosine inhibits PDGF-induced DNA synthesis by inducing cAMP levels via A2 receptors; however, whether this action involves primarily A2a or A2b receptors is unknown. In addition, whether adenosine similarly inhibits SMC proliferation and DNA synthesis induced by other mitogens has not been investigated.
Although adenosine has multiple receptor-mediated anti-vasoocclusive effects, the actions of adenosine on SMC growth and the mechanism by which adenosine modulates SMC growth are poorly understood. Hence, the aims of the present study were to investigate whether adenosine inhibits growth of vascular SMC and to identify the receptors involved in mediating this effect. To accomplish these aims, it was necessary to stimulate SMC growth, for which purpose FCS was selected. The rationale for choosing FCS was that it is the most potent growth stimulator, containing a battery of circulating and endothelium-derived growth factors, such as PDGF, FGF, Ang II, endothelin, and norepinephrine, that have been implicated in the pathophysiology of neointima formation in hypertension, atherosclerosis, and restenosis.
Phenol red–free DMEM/F-12, Hanks’ balanced salt solution, penicillin, streptomycin, 0.25% trypsin–EDTA solution, and all tissue culture ware were purchased from GIBCO Labs. FCS was obtained from HyClone Labs Inc. Adenosine, 2-chloroadenosine, and 8-bromo-cAMP were purchased from Sigma Chemical Co. CPA, CGS21680, and DPCPX were purchased from Research Biochemicals Int. KF17837 was from Kyowa Hakko Kogyo Co Ltd. [3H]Thymidine (specific activity, 11.8 Ci/mmol) was purchased from ICN Biomedicals. All other chemicals used were of tissue-culture grade or best grade available.
Aortic Smooth Muscle Cell Culture
Sprague-Dawley male rats (Charles River, Wilmington, Mass) weighing 150 to 200 g were fed standard rat chow and tap water ad libitum. Aortic SMC were cultured as explants from the ascending thoracic aortas, obtained from ether-anesthetized rats, after a midline abdominal incision including the diaphragm and as described previously.38 Briefly, the medial layer of the aorta was removed surgically under the microscope, and minced sections of this layer were suspended in primary cell culture medium (DMEM/F-12 supplemented with penicillin [100 U/mL], streptomycin [100 μg/mL], NaHCO3 [13 mmol/L], and HEPES [25 mmol/L]; GIBCO) containing 10% FCS, plated in tissue-culture flasks (75 cm2), and incubated under standard tissue-culture conditions (37°C, 5% CO2/95% air, and 98% humidity). The SMC grew as explants from the medial tissue and were confluent in 12 to 14 days. Confluent monolayers of SMC were dislodged by treatment with 0.25% trypsin–EDTA solution (GIBCO) and passaged further. SMC purity was characterized by immunofluorescence staining with smooth muscle–specific anti–smooth muscle α-actin monoclonal antibodies and by morphological criteria specific for smooth muscle, as described in detail previously.39 SMC between the second and third passages were used for the growth studies.
SMC growth was evaluated by studying the effects of various agents on FCS-induced DNA synthesis and change in cell number.
[3H]Thymidine incorporation studies were done to investigate the effects of adenosine on FCS-induced DNA synthesis. SMC were plated at a density of 2.5×104 cells/well in 24-well tissue-culture dishes and allowed to grow for 48 hours in complete culture medium (DMEM supplemented with NaHCO3 [13 mmol/L] and HEPES [25 mmol/L]; GIBCO) containing 10% FCS under standard tissue-culture conditions. The SMC were then growth-arrested by feeding complete culture medium containing 0.4% FCS for 48 hours. Growth was initiated by treating growth-arrested SMC for 20 hours with complete culture medium supplemented with 2.5% FCS and containing or lacking one of the following agents: adenosine (10−8 to 10−3 mol/L); 2-chloroadenosine, a stable analogue of adenosine that expresses its effects via both A1 and A2 receptors (10−8 to 10−3 mol/L); 8-bromo-cAMP, a stable analogue of the endogenous second messenger cAMP (10−8 to 10−3 mol/L); CPA, an adenosine agonist that expresses its effects selectively via A1 receptors28 (10−12 to 10−5 mol/L); CGS21680, an adenosine agonist that possesses a 170-fold selectivity for A2 versus A1 receptors28 and expresses its effects selectively via A2a receptors28 (10−10 to 10−5 mol/L); 2-chloroadenosine (10−5 mol/L) in the presence of DPCPX (10−8 mol/L), a selective A1 receptor antagonist28 ; or 2-chloroadenosine (10−5 mol/L) in the presence of KF17837 (10−9 mol/L), a selective A2 receptor antagonist.28 After 20 hours of incubation, the treatments were repeated with freshly prepared solutions but supplemented with [3H]thymidine (1 μCi/mL) for an additional 4 hours. The experiments were terminated by washing the cells twice with Dulbecco’s PBS and twice with ice-cold trichloroacetic acid. The precipitate was solubilized in 500 μL of 0.3N NaOH and 0.1% SDS after incubation at 50°C for 2 hours. Aliquots from 4 wells for each treatment were counted in a liquid scintillation counter. Each experiment was repeated three times.
Adenosine is rapidly metabolized by the enzyme adenosine deaminase, which is present in several cell types.9 The presence of adenosine deaminase in SMC would result in the rapid breakdown of exogenous adenosine and possibly reduce its inhibitory effects. Since the rate of catabolism would depend on the number of cells present, we assayed and compared the effects of adenosine on 2.5% FCS–induced thymidine incorporation in SMC plated at high density (2.5×104 cells/well) or lower density (5×103 cells/well), as described above.
Cell Proliferation (Increase in Cell Number)
Trypsinized SMC in third passage were suspended in complete culture medium containing 10% FCS and plated in a 24-well culture dish at a density of 5×103 cells/well. After incubation for 24 hours, the cells were fed complete culture medium containing 0.4% FCS for 48 hours to arrest growth of the cells. To study the effects of adenosine on FCS-induced cytokinesis, growth-arrested SMC were treated every 24 hours for 4 days with complete culture medium containing 2.5% FCS and supplemented with or lacking adenosine (10−5 to 10−3 mol/L) or 2-chloroadenosine (10−5 to 10−3 mol/L). The treatments were terminated on day 5 and cells were dislodged with trypsin-EDTA diluted in Isoton-II. Cells were counted with a hemocytometer-calibrated Coulter counter and aliquots from three wells were counted for each group. Three to five independent experiments were performed for each treatment.
Adenosine Metabolism by Cultured Aortic SMC
To evaluate whether SMC catabolize adenosine, we assayed the levels of adenosine in the medium of confluent monolayers of SMC treated with exogenous adenosine in the presence and absence of the adenosine deaminase inhibitor EHNA. Briefly, SMC (3× passage) were plated in six-well culture plates and grown to confluence by feeding complete culture medium containing 10% FCS. On the day of the experiment, confluent monolayers of SMC were washed twice with Dulbecco’s PBS and then incubated with PBS (Dulbecco’s PBS with 25 mmol/L HEPES and 13 mmol/L NaHCO3) containing or lacking adenosine (10 μmol/L, ie, 6.72 nmol/106 cells) or adenosine (10 μmol/L) plus EHNA (10 μmol/L) under standard tissue-culture conditions. After 4 hours of incubation, the supernatants were withdrawn and transferred into ice-cold microfuge tubes and frozen at −70°C until adenosine levels could be estimated. After the collection of supernatants, the monolayers of cells were inspected microscopically for cellular integrity, and the number of cells was counted.
Adenosine levels in the samples were analyzed by using high-performance liquid chromatography (HPLC) via our previously described method.13 Briefly, samples were thawed and centrifuged at 10 000 rpm for 5 minutes. Supernatant (80 μL) was injected into an Isco HPLC system (pump model 2350, gradient programmer model 2360, V4 absorbance detector, 4.6×250-mm C18 column with 5-μm particle size and ChemResearch Data Management System). Mobile phase A was KH2PO4 (0.1 mol/L; pH 6.1) and mobile phase B was 80% KH2PO4 (0.01 mol/L; pH 3.5) and 20% methanol. Mobile phase A was maintained at 100% for 11 minutes, a 2-minute linear gradient to 50% A was initiated, 50% A was maintained for 21 minutes, a 2-minute linear gradient back to 100% A was initiated, and 100% A was maintained for at least 24 minutes before injecting the next sample. Adenosine levels were quantified as the area under the chromatographic peak, and the absolute amount in each sample was calculated from a standard curve of adenosine analyzed in parallel.
All experiments were performed in triplicate or quadruplicate, with four to five separate cultures. Data for the DNA synthesis and cell number are presented as mean±SEM. Statistical analysis was performed using ANOVA and a paired Student’s t test. Between-group comparisons were made by ANOVA, and Fisher’s least significant difference test was used to determine which treatments were different from the control. A value of P<.05 was considered statistically significant.
Effect of Adenosine, 2-Chloroadenosine, and 8-Bromo-cAMP on FCS-Induced Mitogenesis (DNA Synthesis) and Proliferation (Cell Number)
Compared with the growth-arrested cells treated with 0.4% FCS for 24 hours, treatment with 2.5% FCS stimulated DNA synthesis by 7- to 10-fold (P<.01). Adenosine, as well as 2-chloroadenosine, inhibited FCS-induced [3H]thymidine incorporation in a concentration-dependent manner (Fig 1⇓; P<.05). Compared with adenosine, 2-chloroadenosine was more potent in inhibiting FCS-induced DNA synthesis. In SMC plated at high density, the lowest concentrations of adenosine and 2-chloroadenosine that significantly inhibited FCS-induced DNA synthesis were 250 and 10 μmol/L, respectively. A 50% decrease in FCS (2.5%)-induced thymidine incorporation by adenosine and 2-chloroadenosine was observed at 250 and 20 μmol/L, respectively. The inhibitory effects of adenosine were also mimicked by 8-bromo-cAMP (Fig 1⇓; P<.01).
The inhibitory effects of adenosine on 2.5% FCS-induced DNA synthesis were significantly enhanced when SMC plated at low density were treated with adenosine (Fig 2⇓). Low concentrations of adenosine (10 and 100 μmol/L) inhibited FCS-induced thymidine incorporation by >50% in SMC plated at a low density but not in SMC plated at a high density (Fig 2⇓). To evaluate whether this enhanced effect could be due to the catabolism of adenosine by adenosine deaminase, the levels of adenosine in the media of SMC treated with adenosine (10 μmol/L, ie, 6.72 nmol/106 cells) in the presence and absence of EHNA were measured. As shown in Fig 3⇓, the levels of adenosine in the media of SMC treated with adenosine alone were close to those observed in control cells (cells treated with vehicle). However, in SMC treated with adenosine in the presence of the adenosine deaminase inhibitor EHNA, a dramatic increase in the recovery of adenosine in the media was observed (Fig 3⇓). The mean levels of adenosine in the media of SMC treated with adenosine or adenosine plus EHNA were 6.8 pmol/106 SMC per 4 hours (n=3) and 42 pmol/106 SMC per 4 hours (n=3), respectively. These results are consistent with our recent observation that the inhibitory effects of adenosine on SMC growth are enhanced by EHNA.40
Cell Proliferation (Cell Number)
FCS (2.5%) stimulated proliferation (cell number) of growth-arrested SMC (P>.05; Fig 4⇓). Adenosine and 2-chloroadenosine inhibited FCS-induced increases in cell number in a concentration-dependent manner (Fig 4⇓; P<.05). Similar to the effects on DNA synthesis, 2-chloroadenosine, the stable analogue of adenosine, was more potent in inhibiting cell proliferation than was adenosine (P<.05). The lowest concentration of both adenosine and 2-chloroadenosine that inhibited cell proliferation was 10 μmol/L. The cell number after 4 days of treatment with 2.5% FCS was 1.9×104±0.098 cells/well, and the cell numbers in the presence of 2.5% FCS plus 10 and 100 μmol/L of adenosine or 2-chloroadenosine were adenosine, 1.6×104±0.07 and 1.4×104±0.1 cells/well (n=4), respectively; 2-chloroadenosine, 1×104±0.2 and 0.85×104±0.278 cells/well (n=4), respectively. The inhibitory effects of adenosine on SMC proliferation were also mimicked by 8-bromo-cAMP (P<.05; Fig 4⇓).
To confirm that cell death does not occur during this treatment period and did not contribute to the observed effects, the SMC were examined microscopically for indications of cell toxicity. Additionally, trypan blue exclusion tests were carried out in parallel with the proliferation studies in treated cells. At the concentrations used in this study, there was no loss in viability of cells treated with adenosine or 8-bromo-cAMP, and less than 0.5% took up the dye. In cells treated with 2-chloroadenosine, cell toxicity was observed at the maximal concentration used (10−3 mol/L); however, at low concentrations (10−8 to 10−4 mol/L), no loss in cell viability was evident (data not shown). The cell viability was decreased by 14±3% in SMC treated with 10−3 mol/L of 2-chloroadenosine.
Effect of Receptor-Specific Adenosine Analogues (CPA, CGS21680) on FCS-Induced Mitogenesis (DNA Synthesis)
CGS21680, an adenosine agonist that expresses its effects specifically via activation of A2a receptors, was unable to inhibit FCS-induced DNA synthesis, whereas high (10−5 mol/L) but not low concentrations of CPA, an A1 adenosine receptor agonist, inhibited FCS-induced DNA synthesis (Fig 5⇓). CPA expresses its effects specifically via A1 receptors only at low concentrations (10−9 to 10−8 mol/L), whereas at high concentrations (≥10−7 mol/L), CPA has other nonspecific effects.28
Modulation of 2-Chloroadenosine–Induced Inhibition of DNA Synthesis by Adenosine Receptor Antagonists (DPCPX and KF17837)
Since neither CPA nor CGS21680 inhibited FCS-induced growth, the possible involvement of A1 and A2a receptors in mediating the inhibitory effects of adenosine could be ruled out. Further experiments were conducted using the adenosine receptor antagonists DPCPX and KF17837, which inhibit the effects of adenosine by blocking A1 and A2 receptors, respectively. KF17837 but not DPCPX significantly reversed the inhibitory effects of 2-chloroadenosine (10−5 mol/L) on FCS-induced DNA synthesis (Fig 6⇓; P<.05). Thymidine incorporation in response to 2.5% FCS with or without 2-chloroadenosine (10−5 mol/L) or 2-chloroadenosine (10−5 mol/L) plus KF17837 (10−9 mol/L) (percentage of control; mean±SEM; n=4) was 100% for FCS alone, 41.5±1.2% for FCS plus 2-chloroadenosine, and 66±2% for 2-chloroadenosine plus KF17837 (P<.05).
To confirm that cell death did not occur during this treatment period and did not contribute to the observed inhibitory effects of adenosine and its analogues on FCS-induced DNA synthesis, trypan blue exclusion tests were carried out in parallel with the DNA-synthesis studies in treated cells. At the concentrations used in this study, there was no loss in viability of cells treated with CPA, CGS21680, KF17837, or DPCPX, and less than 0.5% took up the dye (data not shown).
The results of the present study demonstrate that adenosine inhibits FCS-induced growth of aortic SMC. Treatment of SMC with adenosine or its stable analogue 2-chloroadenosine inhibited both FCS-induced DNA synthesis and cell proliferation. The inhibitory effects of adenosine were not mimicked by the adenosine agonists CPA and CGS21680, which are selective A1 and A2a receptor agonists, respectively. Thus, the inhibitory effects of adenosine are not mediated via A1 or A2a receptors. Furthermore, the inhibitory effects of 2-chloroadenosine were significantly reversed by KF17837, a specific A2 receptor antagonist, but not by DPCPX, an A1 receptor antagonist. Taken together, our findings provide the first evidence that adenosine inhibits serum-induced growth via the A2b receptor.
Abnormal growth of vascular smooth muscle contributes to the structural changes and vasoocclusive disorders associated with hypertension, atherosclerosis, and restenosis.1 2 3 4 5 6 7 In a normal vessel, vascular tone, SMC quiescence, and homeostasis are maintained by the simultaneous and balanced release of contracting and relaxing factors as well as growth-promoting and growth-inhibiting factors.1 2 3 4 5 6 7 The fact that SMC quiescence is maintained in a normal vessel suggests that the growth-inhibitory effects dominate and are important for maintaining homeostasis in the vessel wall. Therefore, it is feasible that decreased production of vasodilators/growth inhibitors (such as adenosine) may tilt the balance toward SMC growth, which would result in increased growth of medial SMC, leading to neointima formation and vasoocclusive disorders. Hence, detailed knowledge of the role of different endogenous inhibitors that regulate SMC growth and maintain vascular homeostasis is of great clinical and therapeutic importance. In this regard, the role of the vasodilator adenosine has not been well investigated.
Our findings that adenosine inhibits SMC growth confirm the findings of Jonzon et al,27 who demonstrated that adenosine inhibits PDGF-induced growth of cultured rat aortic SMC. However, structural changes associated with hypertension, atherosclerosis, and restenosis involve not only PDGF but also multiple autocrine/paracrine factors present in the circulation, as well as factors generated by the cells within the vessel wall. FCS contains a battery of growth factors, including PDGF, epidermal growth factor, FGF, Ang II, endothelin, and norepinephrine, which may contribute to the vascular remodeling process. Therefore, we thought it important to evaluate the effects of adenosine on FCS-induced growth of SMC so as to elucidate the growth-regulatory effects of adenosine under more physiological conditions. The fact that adenosine inhibits FCS-induced as well as PDGF-induced SMC growth strengthens the conclusion that adenosine is an important in vivo modulator of vascular SMC growth.
Adenosine has multiple biological effects that are mediated via discrete membrane receptors, and thus far four receptors for adenosine have been identified and cloned, ie, A1, A2a, A2b, and A3 receptors.9 28 Additionally, a fifth adenosine receptor, the A4 receptor, has been characterized pharmacologically.28 A1 and A2 receptors mediate several anti-vasoocclusive effects of adenosine within the vasculature20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 and are coupled to adenylyl cyclase via guanine nucleotide–binding proteins (G proteins). Activation of A1 receptors stimulates Gi, which either inhibits adenylyl cyclase directly or reduces the effectiveness of Gs,28 and results in a fall in tissue levels of cAMP. In contrast, stimulation of A2 receptors results in activation of Gs, which has a stimulatory effect on adenylyl cyclase, leading to a rise in tissue cAMP levels.
Previously, Jonzon et al27 evaluated the influence of adenosine on SMC growth. Using the adenosine analogue NECA, which has an affinity for both A1 and A2 receptors, and l-phenylisopropyladenosine, which is selective for A1 receptors,28 these authors demonstrated that the inhibitory effects of adenosine on PDGF-induced SMC growth were mediated via activation of A2 receptors rather than A1 receptors. To further substantiate the involvement of A2 receptors, they demonstrated that the inhibitory effects of adenosine were accompanied by a significant increase in cAMP levels. However, no additional studies to date have either confirmed or negated these findings. In the present study, using different adenosine agonists and antagonists that express their effects specifically via A1, A2, or A2a receptors, we examined which adenosine receptors are involved in mediating the inhibitory effects of adenosine on FCS-induced growth of SMC.
Our observation that CPA (an adenosine analogue that is highly selective for A1 receptors and expresses its effects at pharmacologically low doses [10−9 mol/L]) was unable to inhibit FCS-induced growth of SMC at low concentrations suggests that the inhibitory effects of adenosine are not mediated via A1 receptors. This conclusion is further supported by our observation that DPCPX, an adenosine receptor antagonist that is 700-fold selective for A1 receptors,28 was unable to block the inhibitory effects of 2-chloroadenosine on FCS-induced growth of SMC.
The observation that high concentrations of CPA inhibited FCS-induced growth of SMC suggests the possible involvement of a receptor with low affinity for CPA. It has recently been shown that although CPA at low concentrations expresses its effects selectively via A1 receptors, high concentrations of CPA can activate A4 adenosine receptors.28 Hence, it is possible that the inhibitory effects of CPA on SMC growth at high concentrations are mediated in part via A4 adenosine receptors. Unlike the A1 and A2 receptors, the A4 receptor is not coupled with adenylyl cyclase or G proteins but rather with K+ channels.28 Although A4 receptors are localized predominantly in the brain, it is possible that the A4 receptor in the aorta may be important in mediating the inhibitory effects of adenosine on vascular SMC via regulation of K+ channels. This possibility needs to be further investigated.
Jonzon et al,27 using NECA, demonstrated that the inhibitory effects of adenosine are mediated via A2 receptors and not via A1 receptors. This conclusion is further supported by our observation that KF17837, a selective inhibitor of A2 adenosine receptors, effectively reversed the inhibitory effects of 2-chloroadenosine at concentrations as low as 10−9 mol/L, and the inhibitory effects of adenosine were mimicked by 8-bromo-cAMP. Moreover, our observation that CGS21680, an adenosine agonist that is highly selective for A2a receptors, is ineffective in mimicking the inhibitory effects of adenosine provides evidence that the effects of adenosine are not mediated via A2a receptors. Rather, our data suggest that the A2b receptors may be involved in mediating the inhibitory effects of adenosine.
Our contention that the inhibitory effects of adenosine are mediated via A2b receptors is further supported by the recently proposed and endorsed subclassification of A2a and A2b receptors.28 Gurden et al41 have recently demonstrated that the relative potencies of CGS21680 and NECA can be used as a reference to differentiate A2a from either A2b or A1 receptors. When the effects of CGS21680 are as potent as those of NECA, the A2a receptor is implicated. However, when CGS21680 is much less potent than NECA, it indicates that the observed effects are mediated via activation of the A2b receptor subtype. Jonzon et al27 have previously demonstrated that very low concentrations (1 μmol/L) of NECA inhibited the growth of SMC cultured from the aorta of Sprague-Dawley rats. We have observed that in aortic SMC, also cultured from Sprague-Dawley rats, CGS21680 was ineffective in mimicking the inhibitory effects of adenosine. Hence, CGS21680 is much less potent than NECA, which substantiates our conclusion that the inhibitory effects of adenosine are mediated via A2b receptors.
A2 receptors are positively coupled with adenylyl cyclase, and their activation results in a significant increase of cAMP levels.28 Stimulation of SMC with adenosine has been shown to elevate cAMP levels, and cAMP in turn inhibits SMC proliferation and migration.27 28 38 42 Although our observation that the inhibitory effects of adenosine were mimicked by 8-bromo-cAMP also suggests that the inhibitory effects of adenosine are largely mediated via the second messenger cAMP, the participation of other mechanism(s) cannot be ruled out. Activation of A2b receptors by adenosine stimulates NO release from endothelial cells,36 37 and we have recently observed that adenosine amplifies lipopolysaccharide-induced NO release from SMC. Since NO inhibits SMC proliferation43 and migration,38 this mechanism provides an additional pathway through which adenosine can inhibit SMC growth.
In contrast to 2-chloroadenosine, adenosine was less potent in inhibiting serum-induced growth of SMC. Since adenosine, but not 2-chloroadenosine, is rapidly metabolized by adenosine deaminase, it is possible that the decreased potency of adenosine is due to its metabolism. Indeed as shown in Fig 3⇑, exogenous adenosine is rapidly metabolized in the absence but not in the presence of EHNA, an adenosine deaminase inhibitor. Moreover, we have recently shown that EHNA enhances the inhibitory effects of exogenous adenosine.40 On the basis of these observations, it could be argued that adenosine deaminase activity plays an important role in governing the inhibitory effects of adenosine.
The concentration of adenosine required to inhibit SMC growth effectively was 10−5 to 10−4 mol/L, whereas the physiological baseline concentration of adenosine within the circulation is 10−8 to 10−6 mol/L. Since adenosine is synthesized in a differential fashion by several cell types, including endothelial and smooth muscle cells, the levels in the blood may not reflect the concentrations present locally within the blood vessel wall. It is possible that the local levels of adenosine within the vessel wall and at the interface of endothelial and smooth muscle cells are much higher than those measured in the circulation, so that circulating levels of adenosine may importantly underestimate the significance of adenosine in inhibiting growth in vivo. Additionally, the level of adenosine within the unstirred water layer adjacent to the cell membrane may be several-fold higher than that observed in the circulation. Finally, we have recently shown that increases in SMC-derived adenosine in response to EHNA and dipyridamole inhibit serum-induced SMC growth,40 thus providing evidence that endogenously synthesized adenosine can inhibit SMC growth and strengthening our contention that adenosine may play a tonic growth-inhibitory role in vivo.
In summary, our data support the hypothesis that local synthesis of adenosine inhibits the mitogenic effects of growth promoters. Therefore, adenosine could play an important role in governing SMC growth and maintaining SMC quiescence in normal vessels. In addition, we provide the first evidence that adenosine inhibits FCS-induced growth of aortic SMC and that the inhibitory effects of adenosine are mediated via activation of A2b receptors. Finally, analogues of adenosine or agents that induce endogenous adenosine levels may be of therapeutic significance in preventing structural changes associated with hypertension, atherosclerosis, and restenosis.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|8-bromo-cAMP||=||8-bromoadenosine 3′:5′-cyclic monophosphate|
|CGS21680||=||2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamino adenosine hydrochloride|
|DMEM/F-12||=||Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12|
|FCS||=||fetal calf serum|
|FGF||=||fibroblast growth factor|
|PDGF||=||platelet-derived growth factor|
|SMC||=||smooth muscle cell(s)|
This work was supported by grants from the National Institutes of Health (HL40319 and HL35909).
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