Cyclic AMP-Adenosine Pathway Induces Nitric Oxide Synthesis in Aortic Smooth Muscle Cells
The main purpose of this investigation was to evaluate whether the cyclic AMP-adenosine pathway, ie, the conversion of cAMP to AMP and, hence, to adenosine, is involved in the regulation of nitric oxide (NO) synthesis by vascular smooth muscle cells (SMCs). Treatment of confluent monolayers of SMCs with adenosine, 2-chloroadenosine (stable analog of adenosine), and agents that elevate endogenous (SMC-derived) adenosine (EHNA and iodotubericidin) increased nitrite/nitrate (stable metabolites of NO) levels in the medium and enhanced the conversion of 3H-L-arginine to 3H-L-citrulline by cytosolic extracts obtained from the pretreated SMCs. The stimulatory effects of adenosine were not mimicked by low (1 to 100 nmol/L) concentrations of CGS21680, an A2A receptor agonist, or CPA, a selective A1 receptor agonist. The stimulatory effects of 2-chloroadenosine and EHNA plus iodotubericidin were significantly inhibited by KF17837, a selective A2 receptor antagonist, and by DPSPX, and A1/A2 receptor antagonist, but not by DPCPX, a selective A1 receptor antagonist. DDA (adenylyl cyclase inhibitor) and Rp-cyclic AMP (protein kinase A inhibitor) did not block the effects of adenosine on NO synthesis. Incubation of SMCs with exogenous cyclic AMP, at concentrations previously shown to elevate levels of adenosine in the medium, also increased nitrite/nitrate levels and 3H-L-citrulline formation, and the effects of cyclic AMP on NO synthesis were blocked by DPSPX and KF17837, but not by DPCPX. These findings provide evidence that exogenous and SMC-derived adenosine induce NO synthesis via A2B receptors linked to a pathway not involving adenylyl cyclase/protein kinase A. Moreover, extracellular cyclic AMP induces NO synthesis via conversion to adenosine and activation of A2B adenosine receptors. The cyclic AMP-adenosine pathway may be importantly involved in the vascular production of NO.
- BME = basal medium Eagle’s
- FCS = fetal calf serum
- DMEM = Delbucco’s modified Eagle medium
- HBSS = Hanks balanced salt solution
- IDO = iodotubericidin
- LPS = lipopolysaccharide
- MEM = minimum essential medium
- NO = nitric oxide
- NOS = nitric oxide synthase
- SMC = smooth muscle cells
Vascular endothelial as well as smooth muscle cells (SMCs) are known to synthesize nitric oxide (NO), a labile free radical and intracellular messenger with a half-life of a few seconds.1,2⇓ NO is synthesized by oxidation of the terminal guanidino-nitrogen atoms of L-arginine by a family of enzymes called NO synthase (NOS).1,2⇓ Within the vessel wall, two distinct types of NOS have been identified. The first is a constitutive Ca2+-dependent and calmodulin-dependent membrane-bound form that is present in endothelial cells and plays an important role in the dynamic control of vascular tone. The second is an inducible Ca2+-independent form that is induced by endotoxin and some cytokines and is present in vascular SMCs.1,2⇓ Endothelial cells continuously generate low levels of NO, which maintain homeostasis in normal blood vessels.1–3⇓⇓ In contrast to endothelial cells, SMCs generate large amounts of NO in response to cytokines and endotoxin.
In SMCs, cyclic AMP and agents that increase cellular cyclic AMP levels (eg, forskolin and isopreterenol) induce iNOS expression and NO synthesis, as well as enhance cytokine-induced NO synthesis in SMCs, by stabilizing iNOS mRNA.4–6⇓⇓ Since adenosine stimulates cyclic AMP levels in vascular SMCs via A2 receptors,7 we hypothesize that adenosine may induce NO synthesis in vascular SMCs and enhance cytokine induced NO synthesis (Fig 1).
Recent studies from our laboratory demonstrate that in vascular SMCs cyclic AMP is an important determinant of adenosine production via a biochemical mechanism we refer to as the cyclic AMP-adenosine pathway (Fig 1).8 This pathway involves the conversion of cyclic AMP to AMP, and, hence, to adenosine by the enzymes phosphodiesterase (PDE) and 5′-nucleotidase, respectively. The cyclic AMP-adenosine pathway may have both intracellular and extracellular arms, ie, adenosine might be formed within the cell and transported to the extracellular space, or it might be formed directly in the extracellular space.8 We have recently shown that the inhibitory effects of cyclic AMP on SMC proliferation are blocked by A2 adenosine receptor antagonists,8 thus implying that the cyclic AMP-adenosine pathway regulates SMC growth. Since cyclic AMP is a precursor for adenosine8 and adenosine may induce NO synthesis, we hypothesize that the cyclic AMP-adenosine pathway may be a potential pathway in regulating NO synthesis in vascular SMCs (Fig 1).
Accordingly, the aims of the present study were to determine whether: (1) exogenous adenosine induces NO synthesis by SMCs; (2) specific adenosine receptor subtype(s) are involved in inducing NO synthesis by SMCs; (3) SMC-derived adenosine can effectively increase NO synthesis by SMCs; (4) adenosine enhances LPS-induced NO synthesis by SMCs; and (5) the cyclic AMP-adenosine pathway induces NO synthesis in SMCs.
Adenosine, 2-chloroadenosine (Cl-Ad), erythro-9-(2-hydroxy-3nonyl) adenine hydrochloride (EHNA), NG-nitro-L-arginine methyl ester (L-NAME), nitrate reductase (Aspergillus niger), lipopolysaccharide (LPS), nicotinamide-adenine dinucleotide phosphate (NADPH) and flavine adenine dinucleotide (FAD) were purchased from Sigma Chemical Co. 2′,5′-Dideoxyadenosine (DDA) was obtained from Calbiochem-Novabiochem Corp. (R)-p-Adenosine 3′,5′-cyclic phosphorothioate (Rp-cAMP). N6-cyclopentyladenosine (CPA), 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamide adenosine hydrochloride (CGS21680), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), iodotubericidin (IDO), and 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) were purchased from Research Biochemicals International. Dulbecco’s modified Eagle medium (DMEM), DMEM/F12 medium, nitrite-nitrate free minimum essential medium (MEM), Hanks balanced salt solution (HBSS), penicillin, streptomycin, 0.25% trypsin-EDTA solution, and all tissue culture ware were purchased from GIBCO Laboratories. Fetal calf serum (FCS) was obtained from HyClone Laboratories Inc. 3H-L-arginine (specific activity 68 Ci/mmol) was purchased from Amersham Life Science Inc. KF17837 was generous gift from Dr. Fumio Suzuki, Kyowa Hakko Kogyo Co Ltd, Sunto, Shizuoka, Japan. All other chemicals used were of tissue culture or best grade available.
Aortic Smooth Muscle Cell Culture
Aortic smooth muscle cells (SMCs) were cultured as explants from the ascending thoracic aortas, obtained from ether-anesthetized male Sprague-Dawley rats (Charles River, Wilmington, Mass), after a midline abdominal incision including the diaphragm and as described previously by us.9 SMC purity was characterized by immunofluorescence staining with smooth muscle specific antismooth muscle alphaactin monoclonal antibodies and by morphologic criteria specific for smooth muscle as described in detail previously.9 SMCs were passaged by trypsinization, and cells between the 2nd and 3rd passage were used for NO biosynthesis studies.
Treatment Protocols for NO Synthesis by SMCs
SMCs were cultured in either 24-well tissue culture dishes for the measurement of nitrite+nitrate production or in pertri dishes (100 mm diameter) for the citrulline assay. When the cells reached confluence, the culture medium was aspirated and replaced with serum-free MEM containing 0.25% (wt/vol) of fatty-acid free bovine serum albumin. After overnight incubation, the culture medium was replaced again with fresh basal medium Eagles (BME) supplemented with albumin and treated in the presence or absence of LPS (25 μg/mL) with various combinations of other treatments as follows: (1) Cl-Ad, an analog of adenosine that is not metabolized and mediates its effects via both A1 and A2 receptors;7 (2) adenosine; (3) CPA, an adenosine agonist that mediates its effects selectively via A1 receptors;7 (4) CGS21680, an adenosine agonist that mediates its effects selectively via A2A receptors;7 (5) Cl-Ad plus DPCPX, a selective A1 receptor antagonist;7 (6) Cl-Ad plus DPSPX, and A1/A2 adenosine receptor antagonist;7 (7) Cl-Ad plus KF17837, a selective A2 receptor antagonist;9 (8) EHNA, an inhibitor of adenosine deaminase;9 (9) IDO, an inhibitor of adenosine kinase;10 (10) EHNA plus IDO; (11) DPSPX; (12) EHNA plus IDO plus DPSPX; (13) DPCPX; (14) EHNA plus IDO plus DPCPX; (15) L-NAME, a NOS inhibitor; (16) EHNA plus IDO plus L-NAME; (17) KF17837; (18) EHNA plus IDO plus KF17837; (19) cyclic AMP; (20) cyclic AMP plus DPSPX; (21) cyclic AMP plus DPCPX; (22) cyclic AMP plus KF17837; (23) DDA, an inhibitor of adenylyl cyclase;11 (24) adenosine plus DDA; (25) EHNA plus IDO plus DDA; (26) Rp-cAMP, a preferential inhibitor of cyclic AMP-dependent protein kinase;11 (27) adenosine plus Rp-cAMP; (28) EHNA plus IDO plus Rp-cAMP. For nitrite/nitrate synthesis studies the cells were treated for 30 hours, whereas, for citrulline assay for 20 hours.
Nitrite/Nitrate levels in the conditioned culture medium were measured using the Greiss reagent as previously described.6 Briefly, aliquots (50 μL) of conditioned medium from confluent cells were collected and incubated at room temperature with 25 μL of substrate buffer (imidazole 0.1 mol/L, NADPH 210 μmol/L, flavine adenine dinucleotide 3.8 μmol/L) in the presence of nitrate-reductase (14 mU enzyme/50 μL) from Aspergillus niger to convert NO3 to NO2. Total NO2 was then analyzed by reacting the samples with equal volume of Greiss reagent (1% sulfanilamide, 0.1% naphthalene-ethylene diamine dihydrochloride in 5% H3PO4) for 45 minutes at 27°C and measuring absorbance at 540 nm spectrophotometrically. Conditioned medium from untreated samples with no added Greiss reagent were used as sample blanks. Amounts of NO2 in medium were estimated from a standard curve of NaNO2 obtained by enzymatic conversion of NaNO3 (0-32 μmol/L; >98% conversion achieved; Merck). We did not attempt to differentiate between NO2 and NO3 amounts, but rather enzymatically converted all NO3 to NO2 and therefore report our results as NO2/NO3 All samples were run in duplicates or triplicates.
Confluent monolayers of SMCs were washed three times with BME and once with ice-cold homogenization buffer (50 mmol/L Tris-HCl [pH 7.4], 1.15% KCl, 1 mmol/L EDTA, 5 mM glucose, 0.1 mmol/L DL-dithiothreitol, 200 U/mL superoxide dismutase, 2 mg/L pepstatin A, 10 mg/L trypsin inhibitor, and 44 mg/L phenylmethylsulfonyl fluoride). After adding fresh 0.5 mL of homogenization buffer, the SMCs were scraped and transfered into Eppendorf tubes. The cells were then lysed by sonication and the cell cytosols extracted from the lysates by centrifugation at 2000g for 10 minutes at 4°C, followed by centrifugation at 10 000g for 15 minutes at 4°C. Aliquots (30 μL) of the resulting cytosols were subsequently incubated in the presence or absence of L-NAME (1 mmol/L) or EDTA (1 mmol/L) for 30 minutes at 37°C with a reaction mixture containing 3H-L-arginine (1 μmol/L; 1 μCi), 1 mmol/L NADPH, 15 μmol/L of (6R)-5,6,7,8-tetrahydro-l-biopterin, 1 μmol/L FAD, and 1 μmol/L calmodulin in 50 mmol/l HEPES buffer (pH 5.5) containing 1 mmol/L DL-dithiothreitol, 1 mmol/L EDTA, and 1.25 mmol/L CaCl2 in a final volume of 150 μL. The incubations were terminated by adding 1 mL of ice cold terminating buffer (100 mmol/L HEPES buffer [pH 5.5] containing 10 mmol/L EGTA and 500 mg AG 50W-X8 [counter-ion Na+] cation exchange resin), and incubated for 5 minutes at 4°C, followed by a 5-minute centrifugation at 10 000g. Aliquots of the supernatant were taken, and 3H-L-citrulline levels were quantified by liquid scintillation counting. NO synthase activity was normalized to cell protein and time of incubation and is reported as 3H-L-citrulline formation per minute per milligram of protein. Inhibition of 3H-L-citrulline formation by L-NAME was used to confirm that 3H-citrulline was NOS derived. Levels of protein were analyzed by using Bio-Rad protein assay with bovine serum albumin as a standard.
All treatments for nitrite/nitrate assay were performed in triplicates with three to four separate cultures. For citrulline assays, the treatments were performed in duplicates and with three to four separate cultures. Results are shown as means±SEM. Statistical analysis was performed using ANOVA, paired Student’s t-test, and Fisher’s least significant difference test as appropriate. A value of P<.05 was considered statistically significant.
Compared to monolayers of SMCs treated with culture medium alone, nitrite/nitrate synthesis was increased in a concentration-dependent manner in SMCs treated for 30 hours with Cl-Ad (Fig 2A). Cl-Ad and adenosine, both at 10 μmol/L, induced nitrite/nitrate synthesis from 0.26±0.16 μmol/L in untreated to 2.85±0.35 and 2.16±0.2 μmol/L, respectively, (P<.05 vs control). Treatment with LPS for 30 hours significantly increased nitrite/nitrate levels in the conditioned medium (Fig 2A), and the release of nitrite/nitrate in response to LPS was enhanced in a concentration-dependent manner by Cl-Ad (Fig 2A). In the presence of L-NAME, Cl-Ad, LPS and LPS plus Cl-Ad did not increase nitrite/nitrate levels (Fig 2A). Treatment of confluent monolayers of SMCs with EHNA and IDO for 30 hours increased nitrite/nitrate levels in the conditioned medium, and treatment of SMCs with EHNA plus IDO significantly enhanced nitrite/nitrate formation as compared to SMCs treated with EHNA and IDO alone. Similar to the effects on untreated SMCs, EHNA plus IDO also enhanced LPS-induced nitrite/nitrate formation (Fig 2B). Cotreatment with L-NAME inhibited the increases in nitrite/nitrate levels in response to EHNA plus IDO, LPS, and EHNA plus IDO plus LPS (Fig 2B).
Cytosolic extracts of SMCs treated for 20 hours with Cl-Ad, EHNA, IDO and EHNA plus IDO caused the formation of 3H-L-citrulline from 3H-L-arginine (Fig 3). Exposure of SMCs to LPS also caused the formation of 3H-L-citrulline from 3H-L-arginine by the cytosolic extracts (Fig 3). The formation of 3H-L-citrulline was enhanced significantly in SMCs that had been exposed simultaneously to LPS in combination with either Cl-Ad, EHNA, IDO or EHNA plus IDO (Fig 3). 3H-L-citrulline formation was blocked when cytosolic extracts of SMCs pretreated with Cl-Ad, EHNA, IDO and EHNA plus IDO with and without LPS were incubated with 3H-Larginine in the presence of L-NAME. In contrast, the addition of 2 mM EDTA to cytosolic extracts did not influence 3H-L-citrulline formation (Fig 3).
CGS21680 and CPA only increased nitrite/nitrate levels at extremely high concentrations, whether in the presence or not of LPS (Fig 4). Similarly, only extremely high concentrations of CPA and CGS21680 (10−5 mol/L) induced 3H-L-citrulline formation in LPS treated and untreated SMCs (data not shown).
KF17837 and DPSPX, but not DPCPX, significantly attenuated Cl-Ad induced nitrite/nitrate and 3H-L-citrulline synthesis by SMCs (Fig 5). Similarly, the stimulatory effects of EHNA plus IDO on nitrite/nitrate and 3H-L-citrulline formation by SMCs were significantly attenuated by KF17837 and DPSPX, but not by DPCPX (Fig 5). Treatment of SMCs with exogenous cyclic AMP for 30 hours increased nitrite/nitrate levels in the conditioned medium in a concentration-dependent manner (Fig 6). In contrast to untreated SMCs, cytosolic extracts of SMCs treated for 20 hours with cyclic AMP converted 3H-L-arginine to 3H-L-citrulline, and these effects were abrogated by L-NAME, but not EDTA (Fig 6). Similar to cyclic AMP, treatment of SMCs with 8-bromo-cyclic AMP induced nitrite/nitrate levels and 3H-L-citrulline formation (data not shown). The stimulatory effects of cyclic AMP on nitrite/nitrate as well as 3H-L-citrulline formation were significantly reduced in the presence of KF17837 and DPSPX, but not DPCPX (Fig 6).
The effects of adenosine and EHNA+IDO on nitrite/nitrate and 3H-L-citrulline formation were not blocked by the adenylate cyclase inhibitor, DDA, or the cyclic AMP-dependent kinase inhibitor, Rp-cAMP (Fig 7).
The results of the present study demonstrate that treatment of rat aortic SMCs with adenosine, with a stable adenosine analog (Cl-Ad) and with agents that elevate endogenous adenosine (EHNA and IDO), induce NO synthesis. The stimulatory effects of adenosine are not mimicked by low concentrations of CGS21680, a selective A2A receptor agonist,7 or CPA, a selective A1 receptor agonist.7 Furthermore, the stimulatory effects of Cl-Ad and EHNA plus IDO are significantly attenuated by KF17837, a selective A2 receptor antagonist,9 and by DPSPX, an A1/A2 receptor antagonist,7 but not by DPCPX, a selective A1 receptor antagonist.7 Thus, the stimulatory effects of adenosine are most likely mediated via A2B receptors and not via A1 or A2A receptors. These findings provide evidence that exogenous as well as SMC-derived adenosine induces NO synthesis in an autocrine/paracrine fashion and via the A2B receptor. The present study also establishes that treatment of SMCs with cyclic AMP, a precursor of adenosine, stimulates NO synthesis. Moreover, the stimulatory effects of cyclic AMP are blocked by KF17837 and DPSPX, but not by DPCPX. These findings suggest that cyclic AMP-derived adenosine induces NO-synthesis via A2B receptors. Finally, our studies show that the stimulation of NO synthesis by exogenous and endogenous adenosine is not blocked by DDA, an adenylyl cyclase inhibitor,11 or Rp-cAMP, a preferential inhibitor of cyclic AMP-dependent protein kinase.11 These findings suggest that adenosine induces NO synthesis via a pathway that does not involve adenylyl cyclase/protein kinase A.
Local generation of NO within blood vessels plays a key role in the biology, physiology, and pathophysiology of the vasculature.1–3⇓⇓ NO generated luminally prevents platelet aggregation and adhesion, as well as neutrophil-induced damage to endothelial cells.1–3⇓⇓ When generated abluminally, NO activates soluble guanylyl cyclase in SMCs, and thereby increases cGMP levels, decreases intracellular Ca2+ levels, induces vasodilation, and inhibits proliferation and migration of SMCs.1–3,11⇓⇓⇓ Under basal conditions cNOS within the endothelial cells is the main source for NO synthesis, whereas under pathological conditions, such as sepsis, iNOS within the SMCs plays a key role in inducing hypotension.1–3⇓⇓ In addition to NO, there are several other vasodilatory factors that regulate the vascular function in an autocrine/paracrine fashion.3 Moreover, many of these factors are now known to induce their effects via generation of endothelium-derived NO.1–6⇓⇓⇓⇓⇓ In this regard, recent studies have shown that adenosine, which mimicks most of the anti-vasoocclusive effects of NO,3,12⇓ stimulates NO synthesis by vascular endothelial cells via A2 receptors.13,14⇓
Although the vascular effects of adenosine may, in part, be mediated via endothelial cell-derived NO, adenosine induces vasodilation both in the presence and absence of the endothelium.15 However, the mechanisms for the endothelium-independent dilatory effects adenosine are not fully understood. Recent studies show that several endogenous vasodilatory factors stimulate NO synthesis by SMCs. In this regard, cyclic AMP, as well as agents that elevate cyclic AMP levels, induce NO synthesis by SMCs and mesangial cells via iNOS stimulation.4–6,16⇓⇓⇓ Because treatment of SMCs with adenosine increases intracellular cyclic AMP levels,7 we hypothesized that adenosine induces NO synthesis in vascular SMCs. The findings that adenosine and its stable analog, Cl-Ad, stimulate nitrite/nitrate synthesis and 3H-L-citrulline formation by SMCs and that this stimulation is inhibited by L-NAME provides evidence that adenosine induces NO synthesis in SMCs. Moreover, the observation that EDTA does not inhibit 3H-L-citrulline formation suggests that adenosine induces NO synthesis via iNOS. Consistent with this conclusion, Ikeda et al.17 very recently published a report that Cl-Ad induces iNOS mRNA in rat vascular SMCs, thus confirming our previous18 and present findings that, via iNOS stimulation, adenosine induces NO synthesis in SMCs.
Our observations that CPA (adenosine analog selective for A1 receptors at pharmacologically low doses [<1 nmol/L]7) is unable to induce NO synthesis at low concentrations and DPCPX (selective A1 adenosine receptor antagonist) does not block the stimulatory effects of Cl-Ad on NO synthesis suggest that the stimulatory effects of adenosine are not mediated via A1 receptors. Our findings that KF17837 and DPSPX, both A2 receptor antagonists, but not DPCPX, attenuate the stimulatory effects of C1-Ad suggest that the stimulatory effects of adenosine are A2 receptor mediated. Moreover, our observation that only high, but not low, concentrations of CGS21680, an adenosine agonist which is highly selective for A2A receptors at concentrations of <10 nmol/L, stimulates NO synthesis, provides evidence that the effects of adenosine are not mediated via A2A receptors. Rather, our data suggests that the A2B receptors may be involved in mediating the stimulatory effects of adenosine.
The above findings provide clear evidence that exogenous adenosine stimulates NO synthesis by SMCs; however, whether endogenous adenosine has similar stimulatory effects cannot be inferred from studies with agonists. Elimination of adenosine from the interstitial space is mediated by facilitated transport of adenosine into cells followed by metabolism of adenosine to inosine by adenosine deaminase and to AMP by adenosine kinase.12 Inhibition of adenosine deaminase by EHNA and adenosine kinase by IDO increases endogenous levels of adenosine.12 Moreover, we have shown that EHNA and IDO increase adenosine levels in cultured SMCs5 and cardiac fibroblasts.10 Hence, these two compounds were used in the present study to assess the role of endogenous, ie, SMC-derived, adenosine on NO synthesis.
EHNA and IDO increase NO synthesis in SMCs, and the stimulatory effects of EHNA and IDO are significantly enhanced when SMCs are treated with a combination of these agents. Moreover, the stimulatory effects of EHNA and IDO on NO synthesis are blocked by L-NAME, but not by EDTA, suggesting that SMC NO is derived from iNOS. The A2 receptor blockers, KF17837 and DPSPX, significantly attenuate the stimulatory effects of EHNA plus IDO, supporting our contention that the stimulatory effects of these agents on NO synthesis in SMCs are mediated via generation of adenosine. Moreover, the finding that DPCPX does not reverse the stimulatory effects of EHNA plus IDO on SMCs strongly suggests that the stimulatory effects of endogenous adenosine are mediated via A2 receptors.
Formation of adenosine within and/or near the blood vessel wall occurs via three biochemical pathways: (1) the intracellular ATP pathway, which entails intracellular dephosphorylation of ATP to adenosine; (2) the extracellular ATP pathway, which is a source of extracellular adenosine; (3) the transmethylation pathway, which involves the hydrolysis of S-adenosyl-L-homocysteine (SAH) to L-homocysteine and adenosine.8,12,19⇓⇓ 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. More recently, we have demonstrated the presence of a fourth pathway, the cyclic AMP-adenosine pathway, for adenosine production in vascular SMCs8 that would be more amenable to physiological modulation by hormones.
The cyclic AMP-adenosine pathway begins with the activation of adenylyl cyclase and has both intracellular and extracellular sites of adenosine production.8 Within the cell, cytosolic PDE and cytosolic 5′-nucleotidase metabolize cyclic AMP to AMP and AMP to adenosine, respectively, and the adenosine formed reaches the extracellular space via facilitated transport.8,12⇓ However, due to the competition of cytosolic 5′-nucleotidase and adenylate kinase for AMP and the competition of transport mechanisms with adenosine kinase for adenosine, the intracellular formation of adenosine may be diminished.8,12⇓ Therefore, the extracellular limb of the cyclic AMP-adenosine pathway, which involves the efficient metabolism of cyclic AMP to AMP and adenosine by ecto-PDE and ecto-5′-nucleotidase, respectively, may be quantitatively more important.8,12⇓
Regarding the extracellular cyclic AMP-adenosine pathway, stimulation of adenylyl cyclase results in egress of cyclic AMP,8,12⇓ and relatively modest increases in cyclic AMP production could give rise to significant concentrations of adenosine at the cell surface, since adenosine would be synthesized by a series of spatially linked enzymatic reaction.8,12⇓ We have recently shown that SMCs,8 mesangial cells,20 and cardiac fibroblasts21 express the extracellular limb of the cyclic AMP-adenosine pathway. Moreover, in all these cell types, cyclic AMP-derived adenosine inhibits mitogen-induced cell growth.3,22⇓ These studies led us to hypothesize that the cyclic AMP-adenosine pathway may also induce NO synthesis in SMCs. To test this hypothesis, we evaluated the effects of cyclic AMP on NO synthesis in the presence and absence of the A2 specific and nonspecific receptor antagonists, KF17837 and DPSPX, respectively, and the A1 adenosine receptor antagonist, DPCPX.
Cyclic AMP stimulates NO production by SMCs, and the stimulatory effect of cyclic AMP on NO synthesis is significantly attenuated by the adenosine receptor antagonists KF17837 and DPSPX, but not by DPCPX, suggesting the involvement of A2 receptors. This conclusion is consistent with our observations that the stimulatory effects of exogenous and endogenous adenosine on NO synthesis by SMCs are mediated via A2 receptors and are blocked by KF17837 and DPSPX. Taken together these findings strongly suggest that the cyclic AMP-adenosine pathway may contribute importantly to the regulation of vascular biology and more specifically NO synthesis by SMCs.
Although these findings provide evidence for the role of exogenous, endogenous, and cyclic AMP-derived adenosine in inducing NO synthesis by SMCs, the mechanisms involved in this effect are unclear. Our data provides evidence that the effects of adenosine are mediated via A2 receptors. Since A2 receptors are positively coupled to adenylyl cyclase and cyclic AMP induces NO synthesis in SMCs by increasing iNOS activity, it is feasible that the stimulatory effects of adenosine on NO synthesis by SMCs is mediated largely via the second messenger cyclic AMP. However, neither DDA, an inhibitor of adenylate cyclase that blocks cyclic AMP generation, nor Rp-cAMP, an inhibitor of cyclic AMP-dependent kinase, block the stimulatory effects of either adenosine or EHNA plus IDO on NO synthesis. These findings suggest that the stimulatory effects of exogenous as well as SMC-derived adenosine on NO synthesis are mediated via A2 adenosine receptors linked to a pathway not involving adenylyl cyclase/protein kinase A.
In conclusion, we provide evidence that both exogenous and vascular SMC-derived adenosine induces NO synthesis by vascular SMCs in an autocrine fashion. As the effects of exogenous and endogenous adenosine on 3H-L-citrulline formation are not blocked by EDTA, the stimulatory effects of adenosine are most likely mediated via iNOS. Moreover, our findings provide evidence that adenosine generated from cyclic AMP stimulates NO synthesis by SMCs in an autocrine/paracrine fashion. Since both adenosine and NO induce vasodilation, inhibit platelet aggregation and adhesion, and prevent mitogen-induced proliferation and migration of SMCs,1–3,12⇓⇓⇓ processes importantly involved in vaso-occlusive disorders in various vascular diseases, the current findings suggest that adenosine produced by SMCs may play a vital role as a local anti-vasoocclusive agent, which may induce its effects in part via generation of NO locally within the vessel wall. Moreover, decreased SMC synthesis of adenosine or increased catabolism of adenosine by adenosine deaminase or adenosine kinase may importantly contribute to decreased synthesis of NO. This may in turn be associated with abnormal growth of SMCs and the vascular remodeling and vaso-occlusive disorders observed in hypertension, atherosclerosis, and restenosis following balloon angioplasty.3,12⇓ Agents that elevate endogenous adenosine could be clinically important in preventing vaso-occlusive disorders associated with hypertension, atherosclerosis, and restenosis by inducing NO synthesis within the vasculature, thus exerting beneficial effects on the vessel wall. Finally, it is possible that the cyclic AMP-adenosine pathway may importantly regulate vascular biology and physiology in vivo.
This work was supported by grants from the National Institute of Health (HL 55314 and HL 35909).
- Received September 17, 1997.
- Revision received October 16, 1997.
- Accepted October 29, 1997.
Dubey RK, Mi Z, Gillespie DG, Jackson EK. Cyclic AMP-Adenosine pathway inhibits vascular smooth muscle cell growth. Hypertension. 1996; 28 : 765 –771.
Dubey RK, Gillespie DG, Mi Z, Suzuki F, Jackson EK. Smooth muscle cell-derived adenosine inhibits cell growth. Hypertension. 1996; 27 (suppl II): 766 –773.
Dubey RK, Gillespie DG, Mi Z, Jackson EK. Exogenous and endogenous adenosine inhibits fetal calf serum-induced growth of rat cardiac fibroblasts: role of A2B receptors. Circulation. 1997; 96 : 2656 –2666.
Kunz D, Muhl H, Walker G, Pfeilschifter J. Two distinct signaling pathways trigger the expression of inducible nitric oxide synthase in rat mesangial cells. Proc Natl Acad Sci U S A. 1994; 91 : 5387 –5391.
Ikeda U, Kurosaki K, Ohya K, Shimada K. Adenosine stimulates nitric oxide synthesis in vascular smooth muscle cell. Cardiovas Res. 1997; 35 : 168 –174.
Dubey RK, Gillespie DG, Jackson EK. Adenosine induces nitric oxide synthesis in rat aortic smooth muscle cells. Hypertension. 1996; 28 : 552 (abstract P178).
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 (suppl II): 786 –793.
Dubey RK, Mi Z, Gillespie DG, Jackson EK. Cyclic AMP-Adenosine pathway inhibits glomerular mesangial cell growth. Hypertension. 1997; 30 : 506 (abstract P139).
Dubey RK, Gillespie DG, Mi Z, Jackson EK. Cardiac fibroblasts metabolize cyclic-AMP to generate adenosine. Hypertension. 1996; 28 : 524 (abstract P7).
Dubey RK, Gillespie DG, Jackson EK. Exogenous and cardiac fibroblast cell-derived adenosine inhibits serum-induced collagen and protein synthesis in cardiac fibroblasts. Hypertension. 1996; 28 : 541 (abstract P110).