title: NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase.
abstract: The A1/A2 adenosine agonist 5'-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H2O2 (200 microM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0+/-3.2 min in the NECA group versus 29.5+/-2.2 min in untreated cells, P<0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3+/-3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H2O2-induced loss of potential. The direct GSK-3beta inhibitor SB216763 (3 microM) also prolonged the time to fluorescence loss (49.2+/-2.1 min, P<0.001 versus control), and its protection could not be blocked by rapamycin (42.2+/-2.3 min, P<0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0+/-3.8% of the risk zone in control hearts to 11.8+/-2.0% (P<0.001), and rapamycin blocked this NECA-induced protection (38.3+/-3.7%). A comparable protective effect was seen for SB216763 (1 microM) with infarct size reduction to 13.5+/-2.3% (P<0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA's cardioprotection at reperfusion.
authors.affiliation: Department of Cardiology, Ernst-Moritz-Arndt University Greifswald, Loefflerstr. 23, 17487, Greifswald, Germany.
authors.forename: Karina
authors.initials: K
authors.lastname: Förster
authors.forename: Ina
authors.initials: I
authors.lastname: Paul
authors.forename: Natalia
authors.initials: N
authors.lastname: Solenkova
authors.forename: Alexander
authors.initials: A
authors.lastname: Staudt
authors.forename: Michael V
authors.initials: MV
authors.lastname: Cohen
authors.forename: James M
authors.initials: JM
authors.lastname: Downey
authors.forename: Stephan B
authors.initials: SB
authors.lastname: Felix
authors.forename: Thomas
authors.initials: T
authors.lastname: Krieg
mesh_terms: D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects*; D051336:Mitochondrial Membranes / Q000187:drug effects; D000083162:Mitochondrial Permeability Transition Pore; D009203:Myocardial Infarction / Q000473:pathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000517:prevention & control; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000187:drug effects* / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D014665:Vasodilator Agents; D019830:Adenosine-5'-(N-ethylcarboxamide); D006861:Hydrogen Peroxide; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D038362:Glycogen Synthase Kinase 3