2-chloro-n(6)-(3-iodobenzyl)adenosine-5--n-methyluronamide and Myocardial-Infarction

2-CL-IB-MECA, (A3 adenosine receptor agonist)(A3AR) mediated cardioprotection is well documented although the associated intracellular signalling pathways remain unclear. Here we demonstrate a role of the pro-survival signalling pathways MEK1/2-ERK1/2 and PI3K/AKT and their effect on modifying Caspase-3 activity in A3AR mediated cardioprotection.. Isolated perfused rat hearts or primary adult rat cardiac myocytes were subjected to ischaemia/hypoxia and reperfusion/reoxygenation, respectively. 2-CL-IB-MECA (1 nM) was administered at the onset of reperfusion/reoxygenation in the presence and absence of either the PI3K inhibitor Wortmannin (5 nM) or MEK1/2 inhibitor UO126 (10 μM). Heart tissues were harvested for assessment of p-ERK1/2(Thr202/Tyr204) or p-AKT (Ser-473) status or underwent infarct size assessment. Cardiac myocytes underwent flow-cytometric analysis for apoptosis, necrosis, cleaved-caspase 3/p-BAD (Ser-112 and Ser-136) activity post-reoxygenation.. 2-CL-IB-MECA significantly reduced infarct size compared to non-treated controls, where co-administration with either of the kinase inhibitors abolished the infarct sparing effects. Administration of 2-CL-IB-MECA at reperfusion significantly upregulated the status of p-ERK1/2 and p-AKT compared to time matched controls in a UO126 and Wortmannin sensitive manner respectively. 2-CL-IB-MECA when administered throughout reoxygenation significantly reduced apoptosis, necrosis, cleaved-caspase 3 activity and increased p-BAD (Ser-112) and p-BAD (Ser-136) activity in myocytes subjected to hypoxia/reoxygenation injury. The cytoprotective effect was abolished by co-administration with the kinase inhibitors Wortmannin and/or UO126.. We have described the molecular mechanisms associated with A3AR mediated cardioprotection indicating a role for the pro-survival signalling pathways that decrease caspase-3 activity. These observations provide novel insight into the pharmacological effects of A3ARs in ameliorating myocardial ischaemia/reperfusion injury.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Androstadienes; Animals; Apoptosis; Butadienes; Cardiotonic Agents; Caspase 3; Disease Models, Animal; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitriles; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A3; Signal Transduction; Wortmannin

The goal of this study was to examine whether the A(3) adenosine receptor (A(3)AR) agonist Cl-IB-MECA protects against myocardial ischemia/reperfusion injury when administered at the time of reperfusion in an in vivo mouse model of infarction induced by 30min of coronary occlusion and 24h of reperfusion. Treating B6 wild-type with Cl-IB-MECA during the reperfusion phase (100microg/kg i.v. bolus+0.3microg/kg/min subcutaneously via implantation of Alzet mini-osmotic pumps) reduced myocardial infarct size approximately 37% from 50.1+/-2.5% in vehicle-treated mice to 31.6+/-2.8% in Cl-IB-MECA-treated mice, and significantly reduced the number of leukocytes that infiltrated into the ischemic-reperfused myocardium. Cl-IB-MECA did not reduce infarct size or limit leukocyte accumulation in studies using B6 congenic A(3)AR gene "knock-out" mice or in chimeric mice lacking the expression of A(3)ARs in bone marrow (BM)-derived cells. Subsequent mechanistic studies demonstrated that Cl-IB-MECA inhibited migration of mouse neutrophils isolated from BM towards the chemotactic substance c5a in trans-well migration assays, and inhibited leukocyte migration into the peritoneal cavity in a mouse model of thioglycollate-induced peritonitis. We conclude that treating with the A(3)AR agonist Cl-IB-MECA at the time of reperfusion provides effective protection from ischemia/reperfusion injury in the heart through activation of the A(3)AR expressed in BM-derived cells, potentially by suppressing the robust inflammatory reaction that occurs during reperfusion and neutrophil-mediated tissue injury.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Animals; Blood Pressure; Bone Marrow Cells; Bone Marrow Transplantation; Cell Movement; Histamine; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Neutrophils; Receptor, Adenosine A3

We used pharmacological agents and genetic methods to determine whether the potent A(3) adenosine receptor (AR) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A(3)AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 mug/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A(3)AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A(2A)AR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A(3)AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A(3)AR gene "knock-out" (A(3)KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A(3)KO mice in vivo and did not protect isolated perfused hearts obtained from A(3)KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A(3)AR.

Topics: Adenosine; Adenosine A3 Receptor Agonists; Animals; Blood Pressure; Cardiotonic Agents; Cell Degranulation; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Histamine; In Vitro Techniques; Male; Mice; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; p-Methoxy-N-methylphenethylamine; Pyridines; Radioligand Assay; Receptor, Adenosine A3; Triazines; Triazoles

Ischemia-reperfusion induces both necrotic and apoptotic cell death. The ability of adenosine to attenuate reperfusion-induced injury (RI) and the role played by adenosine receptors are unclear. We therefore studied the role of the A(3) receptor (A(3)R) in ameliorating RI using the specific A(3)R agonist 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxi-N-methyl-b-D-ribofuranuronamide (2-Cl-IB-MECA). Isolated rat hearts and cardiomyocytes were subjected to ischemia or simulated ischemia, followed by reperfusion/reoxygenation. The end points were percent infarction/risk zone and annexin-V (apoptosis) and/or propidium iodide positivity (necrosis), respectively. In isolated hearts, 2-Cl-IB-MECA significantly limited infarct size (44.2 +/- 2.7% in control vs. 21.9 +/- 2.4% at 1 nM and 35.8 +/- 3.3% at 0.1 nM, P < 0.05). In isolated myocytes, apoptosis and necrosis were significantly reduced compared with controls (5.7 +/- 2.6% vs. 17.1 +/- 1.3% and 13.7 +/- 2.0% vs. 23.1 +/- 1.5%, respectively, P < 0.0001). In both models, the beneficial effects were abrogated using the A(3)R antagonist MRS-1191. The involvement of A(2a) receptor activation was also examined. This is the first study to demonstrate that A(3)R activation at reperfusion limits myocardial injury in the isolated rat heart and improves survival in isolated myocytes, possibly by antiapoptotic and antinecrotic mechanisms.

Topics: Adenosine; Age Factors; Animals; Apoptosis; Cardiotonic Agents; Cell Survival; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Muscle Fibers, Skeletal; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; Oxygen; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A3; Receptors, Purinergic P1