2-chloro-n(6)-(3-iodobenzyl)adenosine-5--n-methyluronamide and 1-3-dipropyl-8-cyclopentylxanthine

Although adenosine exerts cardio-and vasculoprotective effects, the roles and signaling mechanisms of different adenosine receptors in mediating skeletal muscle protection are not well understood. We used a mouse hindlimb ischemia-reperfusion model to delineate the function of three adenosine receptor subtypes. Adenosine A(3) receptor-selective agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IBMECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 +/- 2.6%, n = 8 mice vs. vehicle-treated 28 +/- 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 +/- 910 U/l, n = 13 vs. vehicle-treated 12,600 +/- 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A(3) receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.07 mg/kg)-induced decrease in skeletal muscle injury was selectively blocked by the A(2A) antagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416; 0.017 mg/kg). The protection induced by the A(3) receptor was abrogated in phospholipase C-beta2/beta3 null mice, but the protection mediated by the A(1) or A(2A) receptor remained unaffected in these animals. The adenosine A(3) receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-beta and represents a new target for ameliorating skeletal muscle injury.

Topics: Adenosine; Animals; Dihydropyridines; Disease Models, Animal; Hindlimb; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Phenethylamines; Phospholipase C beta; Pyrazoles; Pyrimidines; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptor, Adenosine A3; Reperfusion Injury; Signal Transduction; Xanthines

Numerous studies support the concept of impaired postischemic sympathetic neurotransmission in the heart. We hypothesized that postischemic neuronal dysfunction (neuronal stunning) is caused by a transient suppression of exocytotic norepinephrine (NE) release from sympathetic nerve terminals. Furthermore, we assessed the role of presynaptic adenosine-receptors and alpha2-adrenoceptors in neuronal stunning.. Exocytotic NE release was induced by two electrical field stimulations (S(1) and S(2)) in isolated perfused rat hearts. S(1) was performed under baseline conditions and S(2) either during or following intervention. Results are expressed as mean S(2)/S(1) ratios+/-S.E.M. Stepwise increase of global ischemic periods (10, 20, and 30 min) induced a progressive suppression of NE release in the postischemic hearts, which was reversible during reperfusion. Both the degree and duration of NE suppression was dependent on the extent of the preceding ischemic period. Following 10-min ischemia complete recovery of NE release was achieved after 5-min reperfusion (1.07+/-0.12), whereas 5-min reperfusion did not restore NE release after 30 min (0.36+/-0.07) of ischemia. The adenosine-receptor antagonists 8-phenyltheophylline (8-PT; non-selective) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; adenosine A1-receptor subtype selective) significantly increased NE release after 30-min ischemia and 5-min reperfusion (0.78+/-0.06 and 0.64+/-0.07), while in the same experimental protocol blockade of alpha2-adrenoceptors by yohimbine failed to restore the postischemic release (0.24+/-0.06). In non-ischemic hearts the adenosine analogue R(-)N(6)-(2-phenylisopropyl)adenosine (R-PIA) resulted in a marked suppression of NE release (0.61+/-0.07). The inhibitory effect of R-PIA and 2-chloro-N(6)-cyclopentyladenosine (CCPA; adenosine A1-receptor subtype selective agonist) persisted 5 min after cessation of R-PIA (0.62+/-0.05) and CCPA (0.58+/-0.04). Activation of alpha2-adrenoceptors by 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 14,304) also caused a reduction of NE release (0.50+/-0.02), but the release increased to control levels 5 min after cessation of UK 14,304 (0.90+/-0.06).. The results establish the phenomenon of neuronal stunning in terms of a postischemic suppression of exocytotic NE release and provide evidence that neuronal stunning is mediated by endogenous adenosine through activation of presynaptic adenosine A1-receptors.

Topics: Adenosine; Analysis of Variance; Animals; Brimonidine Tartrate; Electric Stimulation; Enzyme Inhibitors; Male; Myocardial Stunning; Myocardium; Norepinephrine; Perfusion; Phenethylamines; Phenylisopropyladenosine; Purinergic Antagonists; Purinergic P1 Receptor Antagonists; Quinoxalines; Rats; Rats, Wistar; Receptors, Purinergic P1; Receptors, Purinergic P2; Sympathetic Nervous System; Theophylline; Time Factors; Xanthines

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A1 and A3 receptors (A1R and A3R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4-5 days in vitro), to an atmosphere of a N2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS 1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS 1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.

Topics: Adenosine; Animals; Animals, Newborn; Cell Hypoxia; Cells, Cultured; L-Lactate Dehydrogenase; Microscopy, Electron; Myocardial Ischemia; Myocardium; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Pyridines; Rats; Receptor, Adenosine A3; Receptors, Purinergic P1; Xanthines

Adenosine modulates hippocampal synaptic plasticity, namely long-term potentiation (LTP) and long-term depression (LTD), through activation of A1 and A2A receptors. We now report a novel role for the recently described adenosine A3 receptor in the regulation of synaptic plasticity in the CA1 area of hippocampal slices. Activation of adenosine A3 receptors by (1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-p-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (Cl-IBMECA) (100 nM) increased the magnitude of theta-burst induced LTP (from 1.2+/-0.6% in the control solution to 25.5+/-0.8% in the presence of Cl-IBMECA) and attenuated LTD (from 30.0+/-5.5% decrease in the control solution to 13.6+/-6.6% decrease in the presence of Cl-IBMECA). The selective adenosine A3 receptor antagonist, MRS 1191 (5-10 microM), prevented the effects of Cl-IBMECA. These findings indicate a functional role for adenosine A3 receptors in the modulation of synaptic plasticity.

Topics: Adenosine; Animals; Excitatory Postsynaptic Potentials; Hippocampus; Long-Term Potentiation; Male; Neuronal Plasticity; Purinergic P1 Receptor Agonists; Rats; Rats, Wistar; Receptor, Adenosine A3; Receptors, Purinergic P1; Xanthines

The A(3) adenosine receptor (A3AR) is one of four receptor subtypes for adenosine and is expressed in a broad spectrum of tissues. In order to study the function of A3AR, a mouse line carrying a mutant A(3) allele was generated. Mice homozygous for targeted disruption of the A3AR gene, A3AR(-/-), are fertile and visually and histologically indistinguishable from wild type mice. The lack of a functional receptor in the A3AR(-/-) mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expression in the A3AR(-/-) mice was demonstrated by lack of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine binding to bone marrow-derived mast cell membranes that were found to express high levels of A3AR in wild type mice. In A3AR(-/-) mice, the density of A(1) and A(2A) adenosine receptor subtypes was the same as in A3AR(+/+) mice as determined by radioligand binding to brain membranes. Additionally, A(2B) receptor transcript expression was not affected by ablation of the A3AR gene. A3AR(-/-) mice have basal heart rates and arterial blood pressures indistinguishable from A3AR(+/+) mice. Functionally, in contrast to wild type mice, adenosine and the A3AR-specific agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2-Cl-IB-MECA), elicit no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells from A3AR(-/-) mice as measured by hexosaminidase release. Also, the ability of 2Cl-IB-MECA to inhibit lipopolysaccharide-induced tumor necrosis factor-alpha production in vivo was decreased in A3AR(-/-) mice in comparison to A3AR(+/+) mice. The A(2A) adenosine receptor agonist, 2-p-(2-carboxyethyl)phenylamino)-5'-N-ethylcarboxamidoadenosine, produced inhibition of lipopolysaccharide-stimulated tumor necrosis factor-alpha production in both A3AR(-/-) and A3AR(+/+) mice. These results show that the inhibition in vivo can be mediated by multiple subtypes, specifically the A(3) and A(2A) adenosine receptors, and A3AR activation plays an important role in both pro- and anti-inflammatory responses.

Topics: Adenosine; Animals; beta-N-Acetylhexosaminidases; Blood Pressure; Gene Targeting; Heart Rate; Inflammation; Lipopolysaccharides; Mast Cells; Mice; Mice, Knockout; Protein Binding; Receptor, Adenosine A3; Receptors, Purinergic P1; RNA, Messenger; Tumor Necrosis Factor-alpha; Xanthines

The possible cardioprotective roles of adenosine A1 and A3 receptors were investigated in a cardiac myocyte model of injury. The adenosine A3 receptor is a novel cardiac receptor capable of mediating potentially important cardioprotective functions. Prolonged hypoxia with glucose deprivation was used to simulate ischemia and to induce injury in cardiac ventricular myocytes cultured from chick embryos 14 days in ovo. When present during the prolonged hypoxia, the adenosine A3 agonists N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA) caused a dose-dependent reduction in the extent of hypoxia-induced injury as manifested by a decrease in the amount of creatine kinase released and the percentage of myocytes killed. The adenosine A1 agonists 2-chloro-N6-cyclopentyladenosine (CCPA), N6-cyclohexyladenosine, and adenosine amine congener were also able to cause a decrease in the extent of myocyte injury. The A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the cardioprotective effect of the A1 but not of the A3 agonists. Conversely, the selective A3 antagonists MRS-1191 and MRS-1097 blocked the protection induced by CI-IB-MECA but had minimal effect on that caused by CCPA. Thus the cardioprotective effects of A1 and A3 agonists were mediated by their respective receptors. This study defines a novel cardioprotective function of the cardiac A3 receptor and provides conclusive evidence that activation of both A1 and A3 receptors during hypoxia can attenuate myocyte injury.

Topics: Adenosine; Animals; Cardiotonic Agents; Cell Hypoxia; Cells, Cultured; Chick Embryo; Dihydropyridines; Heart; Heart Ventricles; Myocardial Ischemia; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptor, Adenosine A3; Receptors, Purinergic P1; Xanthines