Cyclic AMP–Adenosine Pathway Inhibits Vascular Smooth Muscle Cell Growth
In this study we determined whether cAMP is metabolized to adenosine in vascular smooth muscle cells and whether cAMP-derived adenosine modulates vascular smooth muscle cell growth. Confluent smooth muscle cells were exposed to cAMP (0.01 to 30 μmol/L) in the presence and absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/L; an inhibitor of both extracellular and intracellular phosphodiesterase), α,β-methyleneadenosine 5′-diphosphate (AMP-CP, 100 μmol/L; an ecto-5′-nucleotidase inhibitor), and 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, 100 μmol/L; a xanthine that can inhibit extracellular phosphodiesterase) for 0 to 60 minutes. Medium was then sampled and assayed for AMP, adenosine, and inosine. cAMP increased the amount of AMP, adenosine, and inosine in the medium in a time- and concentration-dependent manner. The conversion of cAMP to adenosine and inosine was inhibited by blockade of phosphodiesterase with IBMX, of ecto-phosphodiesterase with DPSPX, and of ecto-5′-nucleotidase with AMP-CP. To evaluate the physiological relevance of cAMP-derived adenosine in vascular smooth muscle cell proliferation, we studied the inhibitory effects of cAMP (10−4 mol/L) and 8-bromo-cAMP (10−4 mol/L) on fetal calf serum–induced DNA synthesis ([3H]thymidine incorporation) in the presence and absence of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, an inhibitor of adenosine deaminase), dipyridamole (a blocker of adenosine transport), KF17837 (a selective A2 adenosine receptor antagonist), and DPSPX (a nonselective adenosine receptor antagonist). cAMP inhibited DNA synthesis, and both EHNA and dipyridamole enhanced this effect. Both KF17837 and DPSPX significantly reduced the inhibitory effects of cAMP on DNA synthesis; however, they did not reduce the inhibitory effects of 8-bromo-cAMP on DNA synthesis. These results indicate that vascular smooth muscle cells metabolize cAMP to adenosine via the sequential action of ecto-phosphodiesterase and ecto-5′-nucleotidase and provide the first evidence that cAMP-derived adenosine can inhibit vascular smooth muscle cell growth. Hence, this cAMP-adenosine pathway may importantly contribute to the regulation of vascular biology.
Adenosine has multiple actions, mediated primarily by A1 and A2 adenosine receptors, that may serve important roles in vascular biology, particularly with regard to reducing the risk and consequences of vaso-occlusive diseases. Activation of A1 receptors attenuates both the sympathetic nervous system1 2 and the renin-angiotensin system,3 4 two extremely important regulatory systems in mammalian cardiovascular physiology that are involved in the pathophysiology of vascular disease. Moreover, A1 receptor–induced opening of K+ channels appears to protect the heart from myocardial ischemia/reperfusion injury.5 6 A2 receptor activation also evokes antivaso-occlusive mechanisms, including direct vasodilation,7 inhibition of platelet aggregation,8 diminished neutrophil adhesion to vascular endothelial cells,9 attenuation of neutrophil-induced endothelial cell damage,9 and stimulation of nitric oxide release from vascular endothelial cells.10 11 In addition, we have demonstrated recently that the A2 receptor in aortic vascular SMCs stimulates basal nitric oxide release, augments lipopolysaccharide-induced nitric oxide production, and inhibits vascular SMC proliferation.12 13
Although adenosine may play an important role in vascular biology, the biochemical mechanisms regulating adenosine levels in or near the blood vessel wall are poorly understood. Recently, we proposed the hypothesis that cAMP may be an important determinant of adenosine production via a biochemical mechanism we refer to as the cAMP-adenosine pathway.14 15 This pathway, outlined in Fig 1⇓, involves the conversion of cAMP to AMP and hence to adenosine by the enzymes PDE and 5′-nucleotidase, respectively. Also as shown in Fig 1⇓, the cAMP-adenosine pathway may have both intracellular and extracellular arms; ie, adenosine might be formed within the cell and transported to the extracellular space or might be formed directly in the extracellular space.
Our purpose in the present study was to determine in cultured vascular SMCs whether the extracellular limb of the putative cAMP-adenosine pathway is a viable metabolic mechanism for adenosine production. To test this hypothesis, we examined the ability of rat aortic vascular SMCs in culture to convert exogenous cAMP to AMP, adenosine, and inosine and determined whether this conversion is time and concentration dependent. In addition, we examined whether IBMX (a PDE inhibitor that penetrates cell membranes16 ), AMP-CP (an ecto-5′-nucleotidase inhibitor17 ), and DPSPX (a xanthine that can inhibit PDE but is restricted to the extracellular compartment18 ) alter the conversion of exogenous cAMP to AMP, adenosine, and inosine. Furthermore, to address the physiological significance of the cAMP-adenosine pathway with regard to vascular SMC growth, we examined whether the inhibitory effects of cAMP on FCS-induced DNA synthesis were reduced by KF17837 and DPSPX (adenosine receptor antagonists19 20 ) and increased by EHNA and dipyridamole, which differentially increase adenosine levels in vivo.21 22
Aortic Vascular SMC Culture
Male Sprague-Dawley rats (n=10) weighing 150 to 200 g were obtained from Charles River Laboratories (Wilmington, Mass) and received standard rat chow and tap water ad libitum. Aortic SMCs were cultured by our previously described explant technique.23 Briefly, thoracic aortas were obtained surgically from ether-anesthetized rats after a midline abdominal incision including the diaphragm. The medial layer of the aorta was removed surgically under a microscope. Minced sections of this layer, devoid of adventitial tissue, were suspended in complete culture medium (Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 mmol/L HEPES; GIBCO Laboratories) containing 10% FCS (HyClone Laboratories Inc), plated in tissue culture flasks (75 cm2, Sigma Chemical Co), and incubated under standard tissue culture conditions (37°C, 5% CO2/95% air, 98% humidity). The SMCs grew as explants from the medial tissue and were confluent in 12 to 14 days. Confluent monolayers of SMCs 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 monoclonal antibodies against smooth muscle α-actin24 and by morphological criteria specific for smooth muscle, as we previously described in detail.24 SMCs in second and third passages were used for assay of cAMP metabolism. All other chemicals used were of tissue culture or the best grade available.
Experimental Protocol for the Metabolism of cAMP by SMCs
SMCs in the second or third passage were plated in 12-well tissue culture plates and grown to confluence under standard tissue culture conditions. Once cells were confluent, the culture medium was removed, and the culture dish was washed twice with HEPES (25 mmol/L)–buffered Hanks' balanced salt solution (GIBCO). After the washing, SMC monolayers were treated with 0.5 mL PBS buffered with HEPES (25 mmol/L) and NaHCO3 (13 mmol/L) in the presence and absence of cAMP (30 μmol/L), IBMX (1 mmol/L, Sigma), IBMX (1 mmol/L) plus cAMP (30 μmol/L), AMP-CP (100 μmol/L, Sigma), AMP-CP (100 μmol/L) plus cAMP (30 μmol/L), DPSPX (100 μmol/L, Research Biochemicals International), or DPSPX (100 μmol/L) plus cAMP (30 μmol/L). After 1 hour of incubation under standard tissue culture conditions, the supernatant was collected, transferred immediately into ice-cold tubes, and frozen at −70°C until adenosine, inosine, and AMP levels were estimated. The remaining cells were solubilized in 0.5N NaOH, and protein content was measured by the method of Lowry et al.25 Furthermore, we used trypan blue exclusion assays to evaluate the viability of SMCs treated in parallel and to ensure that the various treatments caused no toxic effects or cell death.
To evaluate whether the conversion of cAMP to AMP, adenosine, and inosine depends on cAMP concentration, we assayed the formation of these metabolites in the supernatant of cells incubated for 60 minutes with different concentrations (0.01, 1, 10, and 30 μmol/L) of cAMP. Furthermore, to assess whether the conversion of cAMP to AMP, adenosine, and inosine is time dependent, we quantified the formation of these metabolites in the incubation medium of SMCs treated with 30 μmol/L cAMP for 0, 5, 10, 20, 40, and 60 minutes.
Adenosine, AMP, and Inosine Analysis
We analyzed adenosine, AMP, and inosine levels in the samples with an HPLC system using our previously described method.15 Briefly, samples (supernatant) were thawed and centrifuged at 3000 rpm for 15 minutes. Eighty microliters of each sample 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, ChemResearch Data Management System). Mobile phase A was 0.1 mol/L KH2PO4 (pH 6.1) and mobile phase B was 80% of 0.01 mol/L KH2PO4 (pH 3.5) and 20% methanol. Mobile phase A was maintained at 100% for 11 minutes; a 2-minute linear gradient to 50% mobile phase A was initiated; 50% mobile phase A was maintained for 21 minutes; a 2-minute linear gradient back to 100% mobile phase A was initiated; and 100% mobile phase A was maintained for at least 24 minutes before the next sample was injected. The eluant was monitored at a wavelength of 254 nm, and AMP, adenosine, and inosine were measured as the area under the chromatographic peak; the amount of each substance in the samples is presented as nanomoles per liter per milligram protein.
To evaluate the physiological significance of cAMP metabolism to adenosine in regulating SMC growth, we investigated the inhibitory effects of cAMP on FCS-induced DNA synthesis in the presence and absence of KF17837 (an A2 receptor antagonist19 ; a generous gift from Dr Fumio Suzuki, Kyowa Hakko Kogyo Co Ltd, Sunto, Shizuoka, Japan), DPSPX (a nonselective adenosine receptor antagonist20 ), EHNA (an adenosine deaminase inhibitor12 13 21 ), and dipyridamole (a nucleoside transport inhibitor).21 22
We performed [3H]thymidine incorporation studies to investigate the effects of cAMP-derived adenosine on FCS-induced DNA synthesis. SMCs (8×103 cells per well) were plated in 24-well tissue culture dishes and allowed to grow for 48 hours in complete culture medium (Dulbecco's modified Eagle's medium, 13 mmol/L NaHCO3, 25 mmol/L HEPES) containing 10% FCS for 48 hours under standard tissue culture conditions. SMC monolayers were then growth arrested with complete culture medium containing 0.25% FCS for 36 hours. Growth-arrested SMCs were treated for 20 hours with complete culture medium supplemented with 2.5% FCS in the presence and absence of cAMP (10−4 mol/L), 8-bromo-cAMP (10−4 mol/L), KF17837 (10−9 mol/L), cAMP (10−4 mol/L) plus KF17837 (10−9 mol/L), 8-bromo-cAMP (10−4 mol/L) plus KF17837 (10−9 mol/L), DPSPX (10−8 mol/L), cAMP (10−4 mol/L) plus DPSPX (10−8 mol/L), 8-bromo-cAMP (10−4 mol/L) plus DPSPX (10−8 mol/L), EHNA (10−5 mol/L), cAMP (10−4 mol/L) plus EHNA (10−5 mol/L), dipyridamole (10−7 mol/L), and cAMP (10−4 mol/L) plus dipyridamole (10−7 mol/L). After 20 hours, the treatments were repeated with freshly prepared solutions but supplemented with [3H]thymidine (1 μCi/mL) for an additional 4 hours. 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% sodium dodecyl sulfate after incubation at 50°C for 2 hours. Aliquots from four wells for each treatment, with 10 mL scintillation fluid, were counted in a liquid scintillation counter. Each experiment was conducted in triplicate or quadruplicate and repeated three to four times.
Results are presented as mean±SE of SMC preparations; n indicates the number of rat aortas. Statistical analyses were performed with ANOVA and paired or unpaired Student's t test as appropriate. A value of P<.05 was considered statistically significant.
cAMP Metabolism by SMCs and AMP, Adenosine, and Inosine Formation
Compared with SMCs treated with PBS (vehicle) alone, the extracellular (medium) levels of AMP, adenosine, and inosine increased significantly in SMCs treated with 30 μmol/L cAMP (P<.05, Fig 2⇓). AMP, adenosine, and inosine levels in cultured SMCs were near or below the assay detection limit, whereas in cAMP-treated cells, the levels were 337±59, 118±25, and 742±109 nmol/L per milligram protein, respectively (n=5-10, Fig 2⇓, P<.05 compared with vehicle).
IBMX (1 mmol/L), a PDE inhibitor that crosses cell membranes,16 significantly inhibited cAMP metabolism into AMP, adenosine, and inosine (Fig 2⇑). Adenosine and AMP levels were below the detection limit in the medium of SMCs treated with IBMX (n=3) alone and not significantly increased in SMCs treated with IBMX plus cAMP (30 μmol/L, n=7, Fig 2⇑). Although inosine levels were detectable in the media, the levels in IBMX-treated cells were not significantly different from the levels found in the IBMX plus cAMP–treated cells (177±44 [n=3] and 203±82 nmol/L per milligram protein, respectively; n=7, P=.97, Fig 2⇑).
In contrast to IBMX, treatment of SMCs with cAMP in the presence of the ecto-5′-nucleotidase inhibitor AMP-CP17 (100 μmol/L) inhibited cAMP metabolism to adenosine and inosine but not to AMP (Fig 2⇑). Adenosine and inosine levels in SMCs treated with AMP-CP alone (n=5) or with AMP-CP plus cAMP (n=8) were below the detection limits (Fig 2⇑), whereas AMP levels were 658±99 (n=5) and 7696±1029 nmol/L per milligram protein (n=8, P<.05 compared with SMCs treated with AMP-CP alone), respectively (Fig 2⇑).
Compared with IBMX, DPSPX (100 μmol/L), a xanthine that can inhibit PDE but is restricted to the extracellular compartment,18 only partially inhibited cAMP metabolism to AMP, adenosine, and inosine. In SMCs treated with DPSPX alone, AMP, adenosine, and inosine levels were below the detection limits (Fig 2⇑). However, in SMCs treated with cAMP in the presence of DPSPX, AMP, adenosine, and inosine levels were 74±18 (n=4, P<.05), 81±10 (n=7, P<.05), and 342±41 nmol/L per milligram protein (n=7, P<.05, Fig 2⇑), respectively (P values compared with SMCs treated with DPSPX alone). Furthermore, compared with SMCs treated with cAMP (30 μmol/L) alone, AMP, adenosine, and inosine levels were lowered by approximately 50% in SMCs treated with cAMP plus DPSPX (P<.05, Fig 2⇑), indicating that DPSPX partially inhibits the metabolism of cAMP to AMP, adenosine, and inosine.
To confirm that the effects of IBMX, AMP-CP, and DPSPX on cAMP metabolism to AMP, adenosine, and inosine were due to their inhibitory effects on specific biochemical pathways and not to cell toxicity, we conducted viability tests using trypan blue exclusion in SMCs treated similarly and in parallel to the metabolic studies. The viability in SMCs incubated with PBS alone was greater than 97%, and no loss in cell viability was observed in cells treated with IBMX, AMP-CP, or DPSPX in the absence and presence of cAMP (data not shown).
We also evaluated whether the cAMP metabolism to AMP, adenosine, and inosine was time and concentration dependent. The cAMP metabolism to AMP and inosine was time dependent, but the metabolism to adenosine was so rapid that the levels were maximally elevated after only 5 minutes of incubation (Fig 3⇓). Compared with SMCs not treated with cAMP, the adenosine levels increased significantly after 5 minutes of incubation and remained unchanged in samples incubated for 10, 20, 40, and 60 minutes. In contrast to adenosine, the increase in the AMP levels reached a plateau after 20 minutes, whereas the increase in inosine levels did not reach a plateau even after 60 minutes of incubation (Fig 3⇓).
The cAMP metabolism to AMP, adenosine, and inosine was concentration dependent (Fig 4⇓). Compared with untreated controls, AMP, adenosine, and inosine levels were not significantly different in SMCs incubated for 60 minutes with 0.01 to 0.1 μmol/L cAMP. However, in SMCs treated with 1 μmol/L and higher, a concentration-dependent increase in AMP, adenosine, and inosine levels was observed (Fig 4⇓). At concentrations of 1 μmol/L and higher, inosine levels were greater than those for AMP and adenosine, and AMP levels were greater than those for adenosine.
Effects of cAMP on FCS-Induced DNA Synthesis in the Absence and Presence of EHNA and Dipyridamole
Treatment of growth-arrested SMCs with exogenous cAMP (10−4 mol/L) inhibited 2.5% FCS–induced thymidine incorporation by 62±1.2% (Fig 5⇓, P<.001). EHNA (10−5 mol/L), which elevates endogenous adenosine levels by inhibiting adenosine deaminase,12 13 21 inhibited FCS-induced [3H]thymidine incorporation by 20±2% (P<.01). Dipyridamole (10−7 mol/L), which increases endogenous adenosine by blocking its transport,21 22 also inhibited FCS-induced [3H]thymidine incorporation by 39±1.2% (P<.001). The inhibitory effects of cAMP on 2.5% FCS–induced thymidine incorporation were significantly enhanced in the presence of EHNA and of dipyridamole (P<.01). [3H]Thymidine incorporation (percentage of control; four experiments conducted in quadruplicate) was 100% in controls (2.5% FCS), 37±1.2% in the presence of cAMP (P<.01 versus control), 80±2% in the presence of EHNA (P<.01 versus control), 61±1.2% in the presence of dipyridamole (P<.001 versus control), 32±1% in the presence of cAMP plus EHNA (P<.05 versus cAMP and EHNA), and 26±3% in the presence of cAMP plus dipyridamole (P<.05 versus cAMP and dipyridamole, Fig 5⇓).
Effects of cAMP on FCS-Induced DNA Synthesis in the Absence and Presence of Adenosine Receptor Antagonist (KF17837 and DPSPX)
Using KF17837, which represents a prototype for a new class of selective A2 receptor antagonist,19 we have recently shown that A2 receptors mediate the inhibitory effects of adenosine on SMC growth.12 13 To confirm that adenosine generation partly mediated the effects of cAMP on DNA synthesis, we evaluated the effects of cAMP in the presence and absence of KF17837 as well as DPSPX, a nonselective adenosine receptor antagonist.20 26 The inhibitory effects of cAMP on FCS-induced DNA synthesis were significantly reduced in the presence of both KF17837 (10−9 mol/L) and DPSPX (10−8 mol/L, Fig 6⇓). Similar to cAMP, treatment of SMCs with 8-bromo-cAMP (10−4 mol/L), a nonmetabolizable analogue of cAMP, inhibited FCS-induced DNA synthesis (Fig 6⇓). However, in contrast to cAMP, the inhibitory effects of 8-bromo-cAMP on FCS-induced DNA synthesis were not reduced in the presence of KF17837 or DPSPX (Fig 6⇓). Thymidine incorporation (percentage of control) in SMCs treated with 2.5% FCS and in the presence of 8-bromo-cAMP, KF17837, 8-bromo-cAMP plus KF17837, DPSPX, and 8-bromo-cAMP plus DPSPX was 100% for FCS, 54±4.3% for 8-bromo-cAMP (P<.01 compared with 2.5% FCS), 94±3% for KF17837 (P>.05 compared with 2.5% FCS), 55±1% for 8-bromo-cAMP plus KF17837 (P<.01 compared with FCS or FCS plus KF17837; P>.05 compared with 8-bromo-cAMP), 97±2% for DPSPX (P>.05 compared with 2.5% FCS), and 55.6±2% for 8-bromo-cAMP plus DPSPX (P<.01 compared with FCS or FCS plus DPSPX; P>.05 compared with 8-bromo-cAMP).
It is widely held that the adenosine formation within and/or near the blood vessel wall occurs via three biochemical pathways.27 The intracellular ATP pathway entails intracellular dephosphorylation of ATP to adenosine in the event that energy demand exceeds energy supply. In contrast to the intracellular ATP pathway, the extracellular ATP pathway is a source of extracellular adenosine regardless of the metabolic demands of the tissue. Adenine nucleotides (ATP, ADP, and AMP) can be released from a variety of sources, including chromaffin cells, sympathetic nerve terminals, endothelial cells, platelets, and vascular SMCs, thus providing substrates for ectoenzymes (ecto-ATPases, ecto-ADPases, and ecto-5′-nucleotidase) that convert ATP to adenosine. The third traditional pathway for adenosine formation is the transmethylation pathway involving the hydrolysis of S-adenosyl-l-homocysteine to l-homocysteine and adenosine by the enzyme S-adenosyl-l-homocysteine hydrolase. Since the intracellular and extracellular ATP pathways of adenosine production require crisis events and the transmethylation pathway is mostly constitutive, the three traditional routes of adenosine biosynthesis are not well suited for physiological modulation.
We have proposed a fourth pathway, the cAMP-adenosine pathway, for adenosine production in vascular SMCs that would be more amenable to physiological modulation by hormones.14 15 As outlined in Fig 1⇑, the cAMP-adenosine pathway begins with the activation of adenylyl cyclase and has both intracellular and extracellular sites of adenosine production. Within the cell, metabolism of cAMP to AMP and AMP to adenosine is catalyzed via cytosolic PDE and cytosolic 5′-nucleotidase, respectively, and the adenosine thus formed reaches the extracellular space by way of facilitated transport. However, intracellular formation of adenosine may be diminished by the competition of cytosolic 5′-nucleotidase and adenylate kinase for AMP and by the competition of transport mechanisms with adenosine kinase for adenosine.28 Therefore, the extracellular limb of the cAMP-adenosine pathway may be quantitatively more important.
Regarding the extracellular limb of the cAMP-adenosine pathway, ecto-5′-nucleotidase is a ubiquitous enzyme tethered to the extracellular face of the plasma membrane via a lipid-sugar linkage.29 This enzyme efficiently metabolizes AMP to adenosine, and vascular endothelial cells30 31 and SMCs32 33 are well equipped with ecto-5′-nucleotidase. Stimulation of adenylyl cyclase always causes egress of cAMP into the extracellular space.34 35 Therefore, provided that sufficient levels of ecto-PDE exist, activation of adenylyl cyclase would trigger the extracellular metabolism of cAMP to AMP and hence to adenosine. Since these reactions would occur in a highly localized environment, this newly formed adenosine could then act in an autocrine and/or paracrine fashion to adjust (amplify, inhibit, and/or expand) the local response to hormonal stimulation of adenylyl cyclase. It is important to note that relatively modest increases in cAMP production could give rise to significant adenosine concentrations at the cell surface because adenosine would be synthesized by a series of spatially linked enzymatic reactions.
Recently, we conducted a number of experiments in the perfused rat renal vascular bed in vitro to test the putative cAMP-adenosine pathway.15 We added cAMP to the Tyrode's solution used to perfuse the renal vasculature and measured by HPLC the renal secretion rates of AMP, adenosine, and inosine. In some experimental groups, kidneys were perfused with Tyrode's solution containing both cAMP and either IBMX (a PDE inhibitor), AMP-CP (an ecto-5′-nucleotidase inhibitor), or DPSPX (an ecto-PDE inhibitor at high concentrations). Exogenous cAMP caused a concentration-dependent increase in the renal secretion rates of AMP, adenosine, and inosine, and IBMX and DPSPX inhibited the increases in AMP and adenosine secretion, whereas AMP-CP blocked the increase in adenosine, but not in AMP, secretion. The results of our experiments in isolated perfused kidneys were consistent with the cAMP-adenosine pathway, but because we conducted those experiments in intact kidneys, we could make no inferences regarding cell types that participated in the metabolism of exogenous cAMP to adenosine.
Vascular SMCs contain both receptor-activated adenylyl cyclase and ecto-5′-nucleotidase; thus, it is possible that vascular SMCs represent one cell type that supports a cAMP-adenosine pathway. In the present study, we tested this hypothesis in rat aortic vascular SMCs by incubating the cells with cAMP in the absence and presence of IBMX, AMP-CP, and DPSPX. In vascular SMCs incubated with exogenous cAMP, extracellular levels (ie, levels in the medium) of AMP, adenosine, and inosine were increased severalfold. As with the perfused rat kidney, increases in AMP, adenosine, and inosine were blocked by inhibition of PDE with IBMX and ecto-PDE with DPSPX. Also, AMP-CP blocked the metabolism of exogenous cAMP to adenosine and inosine but not to AMP. These data are consistent with the hypothesis that the cAMP-adenosine pathway exists in vascular SMCs and contributes to the vascular production of adenosine.
The increase in AMP, adenosine, and inosine induced by addition of cAMP and the inhibition of this phenomenon by blockade of PDE with IBMX are highly consistent with the existence of the cAMP-adenosine pathway. Several lines of evidence strongly suggest that the metabolism of exogenous cAMP to adenosine occurs mainly in the extracellular compartment. First, since cAMP is hydrophilic, exogenous cAMP should not penetrate cell membranes, so its conversion to adenosine most likely occurs extracellularly. Second, since AMP-CP only inhibits ecto-5′-nucleotidase, not endo-5′-nucleotidase,17 the blockade of cAMP metabolism to adenosine by AMP-CP is also consistent with an extracellular site of metabolism. Finally, since DPSPX has a negative charge at physiological pH, it is restricted to the extracellular space.18 Therefore, inhibition by DPSPX of the conversion of exogenous cAMP to AMP and adenosine further supports an extracellular site of metabolism.
The finding that cAMP is rapidly metabolized to adenosine and attains a constant rate of production, together with the fact that inosine synthesis continues to increase linearly, provides evidence that cAMP is continuously metabolized by SMCs to adenosine and adenosine in turn to inosine. cAMP metabolism to AMP, adenosine, and inosine was concentration dependent, and significant increases in AMP, adenosine, and inosine levels were observed when SMCs were incubated with cAMP concentrations of 1 μmol/L and higher. The above findings suggest that if the production rate of cAMP is increased in response to hormonal stimulation, SMCs can effectively increase the extracellular levels of adenosine. It is possible that the increased generation of adenosine locally at the surface of SMCs may be an important mechanism by which SMCs protect themselves against factors that induce vaso-occlusive disorders such as abnormal growth of SMCs and neointima formation.
We have recently shown that SMCs synthesize adenosine and that exogenous and endogenous (SMC-derived) adenosine inhibit FCS-induced growth in an autocrine/paracrine fashion.12 13 On the basis of these observations, we hypothesized that the cAMP-adenosine pathway may importantly contribute to the regulation of vascular biology. To test this hypothesis, we evaluated the effects of cAMP on SMC growth in the presence and absence of EHNA and dipyridamole, which would increase cAMP-derived adenosine levels by blocking adenosine metabolism and uptake, respectively. cAMP inhibited SMC growth, and both EHNA and dipyridamole significantly enhanced the inhibitory effects of cAMP on FCS-induced SMC growth. The increased inhibitory effects of cAMP in the presence of EHNA, which prevents the metabolism of adenosine to inosine,12 13 21 as well as in the presence of dipyridamole, which increases adenosine levels by inhibiting the transport of the nucleoside,21 22 suggest that cAMP-derived adenosine inhibits SMC growth.
Direct proof regarding the role of cAMP-derived adenosine in inhibiting SMC growth comes from our observation that the inhibitory effects of cAMP, but not of 8-bromo-cAMP, on SMC growth were significantly reversed in the presence of the adenosine receptor antagonists KF17837 and DPSPX. We have recently shown that the inhibitory effects of exogenous as well as endogenous adenosine on SMC growth are mediated via A2 receptors12 13 and are blocked by KF17837 and DPSPX. Therefore, the observations that KF17837 and DPSPX block the inhibitory effects of cAMP on SMC growth, whereas they do not reverse the inhibitory effects of 8-bromo-cAMP, strongly suggest that the cAMP-adenosine pathway may contribute importantly to the regulation of vascular biology and more specifically to SMC growth.
In summary, the present experiments confirm that rat aortic vascular SMCs are capable of metabolizing cAMP to adenosine via the extracellular limb of the cAMP-adenosine pathway shown in Fig 1⇑. Moreover, our findings provide evidence that adenosine generated from cAMP inhibits FCS-induced growth of SMCs in an autocrine/paracrine fashion. Although these results only apply to vascular SMCs in a cultured cell system, they suggest that the role of the cAMP-adenosine pathway in vascular biology should be investigated in vivo and that the cAMP-adenosine pathway may play an important role in vascular biology.
Selected Abbreviations and Acronyms
|FCS||=||fetal calf serum|
|HPLC||=||high-performance liquid chromatography|
|SMC||=||smooth muscle cell|
This work was supported by grants from the National Institutes of Health (HL-55314).
Reprint requests to Dr Edwin K. Jackson, Center for Clinical Pharmacology, Department of Medicine and Pharmacology, 623 Scaife Hall, 200 Lothrop St, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-2582.
- Received September 20, 1995.
- Revision received November 29, 1995.
- Revision received May 24, 1996.
Churchill PC, Churchill MC. A1 and A2 adenosine receptor activation inhibits and stimulates renin secretion of rat renal cortical slices. J Pharmacol Exp Ther. 1985;232:589-594.
Murray RD, Churchill PC. Concentration dependency of the renal vascular and renin secretory responses to adenosine receptor agonists. J Pharmacol Exp Ther. 1985;232:189-193.
Auchampach JA, Gross GJ. Adenosine A1 receptors, KATP channels, and ischemic preconditioning in dogs. Am J Physiol. 1993;264:H1327-H1336.
Headrick JP, Berne RM. Endothelium-dependent and -independent relaxations to adenosine in guinea pig aorta. Am J Physiol. 1990;259:H62-H67.
Cronstein BN. Adenosine, an endogenous anti-inflammatory agent. J Appl Physiol. 1994;76:5-13.
Li J-M, Fenton RA, Cutler BS, Dobson JG Jr. Adenosine enhances nitric oxide production by vascular endothelial cells. Am J Physiol. 1995;269:C519-C523.
Dubey RK, Gillespie DG, Osaka K, Suzuki F, Jackson EK. Adenosine inhibits growth of rat aortic smooth muscle cells: possible role of A2b receptor. Hypertension. 1996;27(part 2):786-793.
Dubey RK, Gillespie DG, Mi Z, Suzuki F, Jackson EK. Smooth muscle cell-derived adenosine inhibits cell growth. Hypertension. 1996;27(part 2):766-773.
Mi Z, Jackson EK. Metabolism of exogenous cyclic AMP to adenosine in the rat kidney. J Pharmacol Exp Ther. 1995;273:728-733.
Zimmermann H. 5′-Nucleotidase: molecular structure and functional aspects. Biochem J. 1992;285:345-365.
Tofovic SP, Branch KR, Oliver RD, Magee WD, Jackson EK. Caffeine potentiates vasodilator induced renin release. J Pharmacol Exp Ther.. 1991;256:850-860.
Feoktistov I, Biaggioni I. Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells: an enprofylline-sensitive mechanism with implications for asthma. J Clin Invest. 1995;96:1979-1986.
Wang T, Mentzer RM, Van Wylen DG. Interstitial adenosine with dipyridamole: effect of adenosine receptor blockade and adenosine deaminase. Am J Physiol. 1992;263:H552-H558.
Dubey RK, Jackson EK, Luscher TF. Nitric oxide inhibits angiotensin II-induced migration of rat aortic smooth muscle cell: role of cyclic nucleotides and angiotensin 1 receptors. J Clin Invest.. 1995;96:141-149.
Dubey RK, Roy A, Overbeck HW. Culture of renal arteriolar smooth muscle cells: mitogenic responses to angiotensin II. Circ Res. 1992;71:1143-1152.
Lowry OH, Rosebrough NJ, Farr LA, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
Kroll K, Decking UK, Dreikorn K, Schrader J. Rapid turnover of the AMP-adenosine metabolic cycle in the guinea pig heart. Circ Res.. 1993;73:846-856.
Misumi Y, Ogata S, Hirose S, Ikehara Y. Primary structure of rat liver 5′-nucleotidase deduced from the cDNA. J Biol Chem. 1990;265:2178-2183.
Pearson JD, Carleton JS, Gordon JL. Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth-muscle cells in culture. Biochem J. 1980;190:421-429.
Pearson JD, Coade SB, Cusack NJ. Characterization of ectonucleotidases on vascular smooth muscle cells. Biochem J.. 1985;230:503-507.
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.
Barber R, Butcher RW. The egress of cyclic AMP from metazoan cells. Adv Cyclic Nucleotide Res. 1983;15:119-138.
Barber R, Butcher RW. The quantitative relationship between intracellular concentration and egress of cyclic AMP from cultured cells. Mol Pharmacol. 1981;19:38-43.