2-(4-(2-carboxyethyl)phenethylamino)-5--n-ethylcarboxamidoadenosine and Necrosis

Signaling in adenosine-mediated preconditioning is controversial. We examined roles of mitochondrial (mito) K(ATP) channels, protein kinase C (PKC) and nitric oxide (NO).. Langendorff perfused C57/Bl6 mouse hearts were subjected to 20 min ischemia and 45 min reperfusion. Effects of adenosine-mediated preconditioning were assessed in the absence and presence of signaling inhibitors.. Control hearts recovered 70+/-2 mmHg ventricular pressure, and released 18.1+/-2.0 IU/g lactate dehydrogenase (LDH). Preconditioning with 10 microM adenosine limited necrosis (10.6+/-1.4 IU/g) without modifying contractility (72+/-2 mmHg) whereas 50 microM adenosine reduced necrosis (10.3+/-1.6 IU/g) and contractile dysfunction (91+/-2 mmHg). All protective effects of 10 and 50 microM adenosine were abrogated by mito K(ATP) channel blockade with 100 microM 5-hydroxydecanoate (5-HD) during the 'trigger' phase, but unaltered by PKC or NO synthase inhibition with 3 microM chelerythrine or 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), respectively. Protection against necrosis was eliminated by 5-HD but unaltered by chelerythrine or L-NAME during the 'mediation' phase (ischemia-reperfusion). Reduced contractile dysfunction with 50 microM adenosine was partially sensitive to 5-HD and chelerythrine, and only eliminated by co-infusion of the inhibitors.. Adenosine-mediated preconditioning is dose-dependent with high level stimulation reducing contractile dysfunction in addition to necrosis. Preconditioning is triggered by a mito K(ATP) channel dependent process independently of PKC and NO. Subsequent protection against necrosis is also mediated by a mito K(ATP) channel dependent process independent of PKC and NO. In contrast, functional protection may be mediated by parallel mito K(ATP) and PKC dependent paths.

Topics: Adenosine; Adenosine Diphosphate; Alkaloids; Animals; Benzophenanthridines; Calcium Channel Blockers; Decanoic Acids; Diazoxide; Enzyme Inhibitors; Hydroxy Acids; Hypoxanthine; Inosine; Ischemic Preconditioning, Myocardial; L-Lactate Dehydrogenase; Male; Mice; Mice, Inbred C57BL; Mitochondria, Heart; Myocardial Contraction; Myocardial Infarction; Necrosis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Perfusion; Phenanthridines; Phenethylamines; Potassium Channels; Protein Kinase C; Receptors, Purinergic P2; Signal Transduction; Xanthine

This study tests the hypothesis that infarct reduction with adenosine (Ado) is associated with inhibition of apoptotic cell death by modulating expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins and reducing neutrophil accumulation. In three groups of dogs, the left anterior descending coronary artery was occluded for 60 min and reperfused for 6 h. Either saline (Control, n=8), Ado (140 microg/kg/min, n=8) or CGS21680, an adenosine A2A receptor analogue, (0.2 microg/kg/min, n=7) were infused during the first 2 h of reperfusion. Myocardial apoptosis was detected by histological TUNEL staining and DNA laddering. Expression of Bcl-2 and Bax proteins was analyzed using Western blot assay. Neutrophil localization was detected by immunohistochemistry with monoclonal anti-neutrophil CD18 antibody. There was no group difference in collateral blood flow (colored microspheres) during ischemia. Intra-left atrial administration of Ado and CGS21680 significantly decreased infarct size from 26+/-2% in Control to 13+/-1%* and 16+/-3%*, respectively. TUNEL positive cells in the peri-necrotic zone of the ischemic myocardium were also significantly reduced from 16+/-2% in Control group to 9+/-1%* and 10+/-2%*, respectively, consistent with the absence of DNA laddering in these two groups. Densitometrically, Ado and CGS21680 at reperfusion significantly increased the expression (% of normal myocardium) of downregulated Bcl-2 from 45+/-6% in Control group to 78+/-12%* and 69+/-10%*, respectively, and attenuated expression of upregulated Bax from 198+/-16% in Control group to 148+/-10%* and 158+/-12%*, respectively. Furthermore, the number of positive CD18 cells (mm(2) myocardium), which was significantly correlated with TUNEL positive cells in peri-necrotic zone, was significantly reduced from 403+/-42 in Control group to 142+/-18* in Ado group and 153+/-20%* in CGS21680 group, respectively. In conclusion, the present study suggests that inhibition of apoptosis by Ado at reperfusion involves alterations in anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins and neutrophil accumulation, primarily mediated by an adenosine A2A receptor. * P<0.05 v Control group.

Topics: Adenosine; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; CD18 Antigens; Coronary Circulation; DNA Fragmentation; Dogs; Drug Evaluation, Preclinical; Female; Gene Expression Regulation; Genes, bcl-2; Hemodynamics; Injections, Intra-Arterial; Male; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Necrosis; Neutrophils; Phenethylamines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Purinergic P1