NITRIC-OXIDE SIGNALING IN ISCHEMIC HEART

Objective: Several recent studies have implicated a role of endogenous nitric oxide (NO) in the pathophysiology of myocardial ischemic/reper fusion injury. However, the mechanism by which NO exerts its beneficia l/detrimental effects remains unknown. This study examined the intrace llular signaling of NO by studying the role of the NO-cGMP signaling p athway on the phospho-diesteratic breakdown and turnover of phosphoino sitides during myocardial ischemia and reperfusion. Methods: Isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L -arginine for 10 min prior to ischemia. The release of NO was monitore d using an on-line amperometric sensor. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ische mic/reperfusion injury. For signal transduction experiments, sarcolemm al membranes were radiolabeled by perfusing the isolated hearts with [ H-3] myoinositol and [C-14]arachidonic acid. Hearts were then perfused for 10 min in the presence or absence of L-arginine via the Langendor ff mode. Ischemia was induced for 30 min followed by 30 min of reperfu sion. Experiments were terminated before L-arginine and after L-argini ne treatment, after ischemia, and during reperfusion. Biopsies were pr ocessed to determine the isotopic incorporation into various phosphoin ositols as well as phosphatidic acid and diacylglycerol. cGMP was assa yed by radioimmunoassay and SOD content was determined by enzymatic an alysis. Results: The release of NO was diminished following ischemia a nd reperfusion and was augmented by L-arginine. L-Arginine reduced isc hemic/reperfusion injury as evidenced by the enhanced myocardial funct ional recovery. cGMP, which remained unaffected by ischemia and reperf usion, was stimulated significantly after L-arginine treatment. The cG MP level persisted up to 10 min of reperfusion and then dropped slight ly. Reperfusion of ischemic myocardium resulted in significant accumul ation of radiolabeled inositol phosphate, inositol bisphosphate, and i nositol triphosphate. Isotopic incorporation of [H-3]inositol into pho sphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidyli nositol-4,5-bisphosphate was increased significantly during reperfusio n. Reperfusion of the ischemic heart prelabeled with [C-14]-arachidoni c acid resulted in modest increases in [C-14]diacylglycerol and [C-14] phosphatidic acid. Pretreatment of the heart with L-arginine significa ntly reversed this enhanced phosphodiesteratic breakdown during ischem ia and early reperfusion. However, at the end of the reperfusion the i nhibitory effect of L-arginine on the phosphodiesterases seems to be r educed. In L-arginine-treated hearts, SOD activity was progressively d ecreased with the duration of reperfusion time. Conclusions: The resul ts suggest for the first time that NO plays a significant role in tran smembrane signaling in the ischemic myocardium. The signaling seems to be transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic bre akdown. This signaling effect appears to be reduced as reperfusion pro gresses. These results, when viewed in the light of free radical chemi stry of NO, suggest that such on- and off-signaling of NO may be linke d to its interaction with the superoxide radical generated during the reperfusion of ischemic myocardium.