adenosine-5--(n-ethylcarboxamide) and 1-3-dipropyl-8-(4-sulfophenyl)xanthine

Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([(3)H]proline incorporation), total protein synthesis ([(3)H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1, 3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A(2B) receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis following balloon angioplasty.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Adenosine Kinase; Adenosine-5'-(N-ethylcarboxamide); Animals; Anticoagulants; Antihypertensive Agents; Aorta, Abdominal; Becaplermin; Cell Division; Dinucleoside Phosphates; Enzyme Inhibitors; Hyperplasia; Male; MAP Kinase Signaling System; Muscle, Smooth, Vascular; Oligonucleotides, Antisense; Phenethylamines; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta-2; Thymidine; Tritium; Tubercidin; Vasodilator Agents; Xanthines

The behavioral profile of a range of adenosine receptor ligands was examined in rats using a locomotor activity model. Adenosine receptor agonists, including the selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) and the A2A agonist, 2-[(2-aminoethylamino)carbonylethyl-phenylethylamino]- 5'-ethylcarboxa midoadenosine (APEC), reduced spontaneous motor activity in a dose-dependent manner. CPA-induced locomotor depression was attenuated by adenosine A1 receptor selective antagonists, such as 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (R)-1-[(E)-3-(2-phenylpyrazolo[1, 5-a]pyridin-3-yl)-acryloyl]-2-piperidine ethanol (FK453), and (R)-1-[(E)-3-(2-phenylpyrazolo[1, 5-a]pyridin-3-yl)-acryloyl]-piperidin-2-yl acetic acid (FK352), but not by the A2A receptor antagonist, (E)-1,3-dipropyl-8-(3, 4-dimethoxystyryl)-7-methylxanthine (KF17837). By contrast, APEC-induced hypolocomotion was attenuated by KF17837 but not by DPCPX, confirming that adenosine A1 and A2A receptor activation mediates locomotor output independently. It was found that two peripheral adenosine receptor antagonists, 8-(p-sulphophenyl)-1, 3-dipropylxanthine (DPSPX) and 8-(p-sulphophenyl)-1, 3-dimethylxanthine (8-PST), did not alter CPA-induced hypolocomotion. This confirmed that pharmacological reversal of the adenosine A1 receptor-mediated response involved a central site of drug action. The relationship between occupancy of central adenosine A1 receptors and behavioral effect was therefore assessed. Regression analysis on log transformed data confirmed associations between antagonist affinity for brain [3H]DPCPX binding sites and, in order of increasing significance, the equivalent behavioral dose (EBD) for reversal of CPA-induced hypolocomotion (r2 = 0.32), the serum concentration of drug (r2 = 0.65), and most significantly with the brain concentration of drug detected 20 min after administration of the (EBD) (r2 = 0.95). These data suggest that competition between agonists and antagonists, for occupancy of central adenosine A1 receptors, is intrinsic to the pharmacological reversal of CPA-induced hypolocomotion. The validity of the model as a simple predictive screen for the blood/brain barrier permeability of adenosine A1 receptor antagonists was thereby confirmed.

Topics: Adenosine-5'-(N-ethylcarboxamide); Animals; Behavior, Animal; Blood-Brain Barrier; Male; Motor Activity; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Regression Analysis; Vasodilator Agents; Xanthines

Because proliferation of cardiac fibroblasts participates in cardiac hypertrophy/remodeling associated with hypertension and myocardial infarction, it is important to elucidate factors regulating cardiac fibroblast proliferation. Adenosine, a nucleoside abundantly produced by cardiac cells, is antimitogenic vis-à-vis vascular smooth muscle cells; however, the effect of adenosine on cardiac fibroblast proliferation is unknown. The objective of this study was to characterize the effects of exogenous and endogenous (cardiac fibroblast-derived) adenosine on cardiac fibroblast proliferation.. Growth-arrested cardiac fibroblasts were stimulated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), or modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; adenosine deaminase inhibitor); (2) dipyridamole (adenosine transport blocker); and (3) iodotubericidin (adenosine kinase inhibitor). All of these agents inhibited, in a concentration-dependent manner, FCS-induced cardiac fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation) and cell counting. EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 2.3- to 5.6-fold when added separately to cardiac fibroblasts, and EHNA+iodotubericidin or EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by >690-fold. Both KF17837 (selective A2 antagonist) and DPSPX (nonselective A2 antagonist) but not DPCPX (selective A1 antagonist) blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA synthesis, suggesting the involvement of A2A and/or A2B but excluding the participation of A1 receptors. The lack of effect of CGS21680 (selective A2A agonist) excluded involvement of A2A receptors and suggested a major role for A2B receptors. This conclusion was confirmed by the rank order potencies of four adenosine analogues.. Cardiac fibroblasts synthesize adenosine, and exogenous and cardiac fibroblast-derived adenosine inhibits cardiac fibroblast proliferation via activation of A2B receptors. Cardiac fibroblast-derived adenosine may regulate cardiac hypertrophy and/or remodeling by modulating cardiac fibroblast proliferation.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Adenosine-5'-(N-ethylcarboxamide); Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-2 Receptor Antagonists; Animals; Cattle; Cell Division; Culture Media; Dipyridamole; DNA Replication; Enzyme Inhibitors; Fibroblasts; Heart; Male; Myocardium; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-2; Tubercidin; Xanthines

Chemical or surgical sympathetic denervation of blood vessels causes marked changes of the effector cells. Since postganglionic sympathetic cotransmission by noradrenaline and adenosine 5'-triphosphate is well established, the role of these transmitters as putative trophic factors was investigated. Whereas noradrenaline was ineffective in preventing morphological changes due to denervation, both adenosine and N-ethylcarboxamido-adenosine totally prevented them. In conscious rats, the adenosine receptor antagonist dipropylsulphophenylxantine (DPSPX) caused alterations of the blood vessel wall similar to those described for denervation. These results strongly suggest that adenosine is the trophic factor of sympathetic innervation.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Dogs; Inosine; Muscle Denervation; Muscle, Smooth, Vascular; Norepinephrine; Oxidopamine; Purinergic Antagonists; Receptors, Purinergic; Reference Values; Saphenous Vein; Sympathectomy; Sympathectomy, Chemical; Vasodilator Agents; Xanthines

Platelet aggregation and secretion are associated with a rise in intracellular calcium concentration ([Ca2+]i). Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. The antiaggregatory effects of adenosine are related to activation of adenylate cyclase. We studied the effect of adenosine on the rise in [Ca2+]i and platelet aggregation produced by thrombin. Human platelets were obtained from dextrose/citrate-treated plasma. [Ca2+]i was determined by fluorescence-dye techniques (fura-2). Adenosine inhibited the slope of the first phase of aggregation and the rise in [Ca2+]i produced by thrombin, in a dose-dependent manner. The dose that produced 50% inhibition of both aggregation and the rise in [Ca2+]i was approximately 500 nM. The effects of adenosine on [Ca2+]i were shared by its stable analogs, 5'-N-ethylcarboxamidoadenosine being approximately 10-fold more potent than (-)N6-phenylisopropyladenosine, suggesting that these effects were mediated through adenosine A2 receptors. Furthermore, caffeine antagonized the inhibitory effects of adenosine on platelet aggregation and [Ca2+]i. The effects of adenosine on [Ca2+]i appear to be mediated through a rise in intracellular cAMP, because they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1 mM) and were potentiated by phosphodiesterase inhibition with papaverine (1 microM). Adenosine also inhibits the rise in [Ca2+]i produced by thrombin in a calcium-free medium, suggesting that adenosine inhibits both calcium influx and the release of calcium from intracellular stores.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Caffeine; Calcium; Cytosol; Humans; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombin; Xanthines