RESUMEN
We investigated the role of p38 mitogen-activated protein kinase (MAPK) phosphorylation and opening of the mitochondrial ATP-sensitive K(+) [(K(ATP))(mito)] channel in the adenosine A(1) receptor (A(1)AR)-induced delayed cardioprotective effect in the mouse heart. Adult male mice were treated with vehicle (5% DMSO) or the A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.1 mg/kg ip). Twenty-four hours later, hearts were subjected to 30 min of global ischemia and 30 min of reperfusion in the Langendorff mode. Genistein or SB-203580 (1 mg/kg i.p.) given 30 min before CCPA treatment was used to block receptor tyrosine kinase or p38 MAPK phosphorylation, respectively. 5-Hydroxydecanoate (5-HD; 200 microM) was used to block (K(ATP))(mito) channels. CCPA produced marked improvement in left ventricular function, which was partially blocked by SB-203580 and 5-HD and completely abolished with genistein. CCPA caused a reduction in infarct size (12.0 +/- 2.0 vs. 30.3 +/- 3.0% in vehicle), which was blocked by genistein (29.4 +/- 2.3%), SB-203580 (28.3 +/- 2.6%), and 5-HD (33.9 +/- 2.4%). CCPA treatment also caused increased phosphorylation of p38 MAPK during ischemia, which was blocked by genistein, SB-203580, and 5-HD. The results suggest that A(1)AR-triggered delayed cardioprotection is mediated by p38 MAPK phosphorylation. Blockade of cardioprotection with 5-HD concomitant with decrease in p38 MAPK phosphorylation suggests a potential role of (K(ATP))(mito) channel opening in phosphorylation and ensuing the late preconditioning effect of A(1)AR.
Asunto(s)
Adenosina/farmacología , Precondicionamiento Isquémico Miocárdico , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/enzimología , Canales de Potasio/metabolismo , Vasodilatadores/farmacología , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Masculino , Ratones , Ratones Endogámicos ICR , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por Mitógenos/análisis , Infarto del Miocardio/metabolismo , Isquemia Miocárdica/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Función Ventricular Izquierda , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
A 28 kDa inhibitory protein was purified from rat testis cytosol by sequential 40-65% ammonium sulfate precipitation, cation exchange chromatography, anion exchange chromatography, and preparative SDS-polyacrylamide gel electrophoresis. The heat-stable, trypsin-labile protein exhibited nonenzymatic, concentration-dependent inhibition of testicular and pancreatic cholesteryl ester hydrolases at all stages of purification. Copurifying at each stage was a 26.5 kDa protein which comprised 25% of the mass of the two proteins. Polyclonal antibodies raised to either or both 28 kDa and 26.5 kDa proteins by direct injection of excised electrophoretic bands cross-reacted with both proteins on western blots, immunoprecipitated both proteins, and neutralized inhibitory activity. Amino acid compositions of the individual proteins electroeluted from SDS-polyacrylamide gels were different from those of other surface-active proteins of similar molecular weights. Both proteins exhibited identical pl of 4.8 on chromatofocusing columns and two-dimensional gel electrophoresis. Although the subcellular distribution of the 28 kDa protein is unknown, its testicular cytosolic concentration, calculated from the purified protein mass, was 8 X 10(-9) mols/L, which probably underestimates the actual concentration by an order of magnitude. This is greater than the minimum concentration required for in vitro inhibition (10(-9) mols/L), consistent with a physiological role for this protein.