n(6)-cyclopentyladenosine and Myocardial-Ischemia

The cardioprotective effects of a novel adenosine A1 receptor agonist N6-(2,2,5,5-tetramethylpyrrolidin-1-yloxyl-3-ylmethyl) adenosine (VCP28) were compared with the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in a H9c2(2-1) cardiac cell line-simulated ischemia (SI) model (12 hours) and a global ischemia (30 minutes) and reperfusion (60 minutes) model in isolated rat heart model. H9c2(2-1) cells were treated with CPA and VCP28 at the start of ischemia for entire ischemic duration, whereas isolated rat hearts were treated at the onset of reperfusion for 15 minutes. In the H9c2(2-1) cells SI model, CPA and VCP28 (100 nM) significantly (P < 0.05, n = 5-6) reduced the proportion of nonviable cells (30.88% +/- 2.49% and 16.17% +/- 3.77% of SI group, respectively) and lactate dehydrogenase efflux. In isolated rat hearts, CPA and VCP28 significantly (n = 6-8, P < 0.05) improved post-ischemic contractility (dP/dt(max), 81.69% +/- 10.96%, 91.07% +/- 19.87% of baseline, respectively), left ventricular developed pressure, and end diastolic pressure and reduced infarct size. The adenosine A1 receptor antagonist abolished the cardioprotective effects of CPA and VCP28 in SI model and isolated rat hearts. In conclusion, the adenosine A1 receptor agonist VCP28 has equal cardioprotective effects to the prototype A1 agonist CPA at concentrations that have no effect on heart rate.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; Cell Line; Heart Rate; In Vitro Techniques; Myocardial Contraction; Myocardial Ischemia; Myocardium; Pyrrolidines; Rats; Reperfusion Injury

Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 microM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10(-7)M) and N6-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA; 10(-7)M) reduced the proportion of nonviable cells to 30.87 +/- 2.49% and 35.18 +/- 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 +/- 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 +/- 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 +/- 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection.

Topics: Acetamides; Adenosine; Adenosine A1 Receptor Agonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine A3 Receptor Agonists; Adenosine A3 Receptor Antagonists; Aminopyridines; Animals; Apoptosis; Cardiotonic Agents; Cell Line; Cell Survival; Dihydropyridines; Imidazoles; Myocardial Ischemia; Phenethylamines; Purines; Rats; Triazines; Triazoles; Xanthines

Herein we report the synthesis and biological evaluation of some potent and selective A(1) adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N(6)-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (K(i)=50nM) and good selectivity (A(3)/A(1) > or = 400) for the A(1) adenosine receptor. N(6)-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (K(i)=7 nM), but lower selectivity (A(3)/A(1)= approximately 3). All compounds bind weakly (K(i)>1 microM) to A(2A) and A(2B) receptors. The combination of A(1) agonist activity and antioxidant activity has the potential to produce cardioprotective effects.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; Antioxidants; Binding Sites; Cardiotonic Agents; Cell Line; Indicators and Reagents; Isoindoles; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Pyrrolidines; Rats; Structure-Activity Relationship; Xanthines

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A(1), A(2A) and A(3)) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A(1) selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A(2A) selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O(2)) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A(1) and A(3) receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G(i)/G(o)-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A3 Receptor Agonists; Animals; Animals, Newborn; Cardiotonic Agents; Cell Hypoxia; Cells, Cultured; GTP-Binding Protein alpha Subunits, Gi-Go; Mitogen-Activated Protein Kinase 3; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Receptor, Adenosine A1; Receptor, Adenosine A3

To characterize the role that cardiac sensory P(1) purinergic (adenosine A(1) or A(2)) receptors play in transducing myocardial ischemia.. Porcine nodose ganglion cardiac sensory neuron adenosine A(1) or A(2) receptor function was studied in situ during control states as well as in the presence of the peptides bradykinin and substance P or focal ventricular ischemia. The responses of porcine nodose ganglion cardiac and non-cardiac afferent neuronal somata to adenosine were also studied in vitro.. Local application of A(1) or A(2) adenosine receptor agonists modified the activity generated by ventricular sensory neurites associated with 70 and 74% of identified nodose ganglion cardiac afferent somata in situ, respectively, exciting most neurons. In contrast, adenosine reduced the excitability of nodose ganglion cardiac afferent neuronal somata in vitro. Bradykinin and substance P affected 56 and 63%, respectively, of tested afferent neurons. The capacity of ventricular sensory neurites to transduce signals relating to these peptides was virtually eliminated by the presence of P(1) purinergic receptor antagonists. So was their capacity to transduce focal ventricular ischemia. Since most cardiac sensory neurites responded differently to adenosine in vivo than did cardiac afferent neuronal somata in vitro, it appears that the transduction properties of cardiac afferent neurons need to be characterized in situ.. Most ventricular sensory neurites associated with nodose ganglion afferent neurons possess adenosine A(1) and/or A(2) receptors that play a primary role in transducing myocardial ischemic events to central neurons. These data support clinical observations implicating cardiac sensory purinoceptors in transducing myocardial ischemic events.

Topics: Adenosine; Animals; Bradykinin; Cells, Cultured; Electrophysiology; Female; Heart; Heart Ventricles; Male; Myocardial Ischemia; Neurons, Afferent; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Signal Transduction; Substance P; Swine

1. The goal of this study was to investigate the effects of the delayed pharmacological preconditioning produced by an adenosine A(1)-receptor agonist (A(1)-DPC) against ventricular arrhythmias induced by ischaemia and reperfusion, compared to those of ischaemia-induced delayed preconditioning (I-DPC). 2. Eighty-nine instrumented conscious rabbits underwent a 2 consecutive days protocol. On day 1, rabbits were randomly divided into four groups: 'Control' (saline, i.v.), 'I-DPC' (six 4-min coronary artery occlusion/4-min reperfusion cycles), 'A(1)-DPC(100)' (N(6)-cyclopentyladenosine, 100 microg kg(-1), i.v.), and 'A(1)-DPC(400)' (N(6)-cyclopentyladenosine, 400 microg kg(-1), i.v.). On day 2, i.e., 24 h later, the incidence and severity of ventricular arrhythmias during a 30-min coronary artery occlusion and subsequent reperfusion were analysed in all animals, using an arrhythmia score. 3. I-DPC, A(1)-DPC(100) and A(1)-DPC(400) significantly reduced the infarct size (34+/-5, 42+/-3 and 43+/-7% of the area at risk, respectively) as compared to Control (55+/-3% of the area at risk). 4. During both ischaemia and reperfusion, neither the incidence nor the severity of ventricular arrhythmias were altered by A(1)-DPC(100), A(1)-DPC(400) or I-DPC as compared to Control. 5. Thus, despite reduction of infarct size induced by delayed preconditioning, A(1)-DPC as well as I-DPC failed to exert any anti-arrhythmic effect in the conscious rabbit model of ischaemia-reperfusion.

Topics: Adenosine; Animals; Arrhythmias, Cardiac; Blood Pressure; Coronary Disease; Dose-Response Relationship, Drug; Electrocardiography; Heart Rate; Heart Ventricles; Hemodynamics; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion; Purinergic P1 Receptor Agonists; Rabbits

Activation of myocardial A1 adenosine receptors (A1AR) protects the heart from ischemic injury. In this study transgenic mice were created using the cardiac-specific alpha-myosin heavy chain promoter and rat A1AR cDNA. Heart membranes from two transgene positive lines displayed approximately 1,000-fold overexpression of A1AR (6,574 +/- 965 and 10,691 +/- 1,002 fmol per mg of protein vs. 8 +/- 5 fmol per mg of protein in control hearts). Compared with control hearts, transgenic Langendorff-perfused hearts had a significantly lower intrinsic heart rate (248 beats per min vs. 318 beats per min, P < 0. 05), lower developed tension (1.2 g vs. 1.6 g, P < 0.05), and similar coronary resistance. The difference in developed tension was eliminated by pacing. Injury of control hearts during global ischemia, indexed by time-to-ischemic contracture, was accelerated by blocking adenosine receptors with 50 microM 8-(p-sulfophenyl) theophylline but was unaffected by addition of 20 nM N6-cyclopentyladenosine, an A1AR agonist. Thus A1ARs in ischemic myocardium are presumably saturated by endogenous adenosine. Overexpressing myocardial A1ARs increased time-to-ischemic contracture and improved functional recovery during reperfusion. The data indicate that A1AR activation by endogenous adenosine affords protection during ischemia, but that the response is limited by A1AR number in murine myocardium. Overexpression of A1AR affords additional protection. These data support the concept that genetic manipulation of A1AR expression may improve myocardial tolerance to ischemia.

Topics: Adenosine; Analysis of Variance; Animals; Cell Membrane; Coronary Vessels; Female; Heart; Heart Rate; In Vitro Techniques; Male; Mice; Mice, Transgenic; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Myosin Heavy Chains; Promoter Regions, Genetic; Radioligand Assay; Rats; Receptors, Purinergic P1; Recombinant Fusion Proteins; Theophylline; Vascular Resistance

Adenosine has been implicated in the pathogenesis of cardiac pain through activation of cardiac sympathetic afferents. The present study was performed to assess directly the contribution of adenosine in activating ischemically sensitive cardiac sympathetic afferents. Single-unit activity of ischemically sensitive afferents located in both ventricles was recorded from the left thoracic sympathetic chain or rami communicantes of anesthetized cats during 5 min of myocardial ischemia. Intracardiac injection (5 mg) or epicardial application (1-5 mg/ml) of adenosine onto the receptive fields failed to activate 31 ischemically sensitive A delta- and C fiber afferents, which were responsive to topical application of bradykinin (10 micrograms/ml). Intracardiac injection (5 mg) or topical application (1-5 mg/ml) of an adenosine A1 receptor agonist, N6-cyclopentyladenosine, also did not increase the discharge activity of 13 other ischemically sensitive C fiber afferents. Treatment with dipyridamole (1 mg/kg iv) to inhibit the cellular uptake of adenosine did not significantly potentiate the response of 10 separate C fiber afferents to 5 min of myocardial ischemia. Furthermore, blockade of adenosine receptors with aminophylline (5 mg/kg iv) did not significantly attenuate the response of 10 other C fiber afferents to 5 min of myocardial ischemia. The results of the present study demonstrate that exogenous and endogenous adenosine do not contribute to activation of ischemically sensitive cardiac sympathetic afferents. The findings of the present study fail to support a substantial role for adenosine and its A1 receptors in activation of cardiac sympathetic afferents during myocardial ischemia.

Topics: Adenosine; Aminophylline; Animals; Bradykinin; Cats; Dipyridamole; Electrophysiology; Heart Conduction System; Myocardial Ischemia; Neurons, Afferent; Sympathetic Nervous System

1. The effects of myocardial ischaemia/reperfusion were tested on the coronary vasorelaxant responses to agonists selective for the A1 and A2 adenosine receptor subtypes in the dog. The left anterior descending (LAD) coronary artery was occluded distal to the first diagonal branch. The occlusion was maintained for 1 h, followed by 1 h of reperfusion. 2. In the first series of experiments, LAD and circumflex arteries were excised and contracted with prostaglandin F2 alpha (PGF2 alpha). Ischaemia/reperfusion did not significantly alter the vasorelaxation produced by either sodium nitroprusside (endothelium-independent) or acetylcholine (endothelium-dependent). The A1 selective agonist, cyclopentyladenosine (CPA), produced coronary vasorelaxation in both normally perfused vessels and vessels subjected to ischaemia/reperfusion. In contrast, the relaxation produced by the A2-selective agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl) ethyl] adenosine (DPMA) was significantly attenuated by ischaemia/reperfusion (14 fold shift in EC50). 3. In the second series of experiments, coronary blood flow was increased by administration of the A1 and A2 agonists before and after ischaemia/reperfusion of the LAD in anaesthetized dogs. Both compounds dose-dependently increased coronary blood flow. The slopes of the dose-response functions to CPA or DPMA were not significantly altered in the normally perfused circumflex vascular bed. Similarly, the CPA dose-response function in the LAD was unaltered by ischaemia/reperfusion. However, the slope of the coronary vasodilator response to the A2 agonist was significantly reduced following ischaemia/reperfusion of the LAD. 4. We conclude that ischaemia/reperfusion reduces responsiveness to an adenosine A2 receptor subtype agonist, but not an A1 receptor subtype agonist. These data confirm the independent nature of A1- and A2-mediated coronary vasodilatation.

Topics: Adenosine; Animals; Coronary Vessels; Dinoprost; Dogs; In Vitro Techniques; Myocardial Ischemia; Myocardial Reperfusion; Receptors, Purinergic P1; Vasodilation