NITRIC-OXIDE - A RETROGRADE MESSENGER FOR CARBON-MONOXIDE SIGNALING IN ISCHEMIC HEART

To examine the intracellular signaling mechanism of NO in ischemic myo cardium, isolated working rat hearts were made ischemic for 30 min fol lowed by 30 min of reperfusion. A separate group of hearts were pre-pe rfused with 3 mM L-arginine in the presence or absence of 650 mu M of protoporphyrin, a heme oxygenase inhibitor for 10 min prior to ischemi a. The release of NO was monitored using an on-line amperometric senso r placed into the right atrium. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/repe rfusion injury. Induction for the expression of heme oxygenase was stu died by Northern hybridization. For signal transduction experiments, s arcolemmal membranes were radiolabeled by perfusing the isolated heart s with [H-3] myoinositol and [C-14] arachidonic acid. 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. The release of NO was diminished following ischemia and reperf usion and was augmented by L-arginine. L-arginine reduced ischemic/rep erfusion injury as evidenced by the enhanced myocardial functional rec overy. Protoporphyrin modulated the effects of L-arginine. cGMP, which was remained unaffected by ischemia and reperfusion, was stimulated s ignificantly after L-arginine treatment. The NO-mediated augmentation of cGMP was reduced by protoporphyrin suggesting that part of the effe cts may be mediated by CO generated through the heme oxygenase pathway . Reperfusion of ischemic myocardium resulted in significant accumulat ion of radiolabeled inositol phosphate, inositol bisphosphate, and ino sitol triphosphate. Isotopic incorporation of [H-3] inositol into phos phatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylin ositol-4,5-bisphosphate was increased significantly during reperfusion . Reperfusion of the ischemic heart prelabeled with [C-14] arachidonic acid resulted in modest increases in [C-14] diacylglycerol and [C-14] phosphatidic acid. Pretreatment of the heart with L-arginine signific antly reversed this enhanced phosphodiesteratic breakdown during ische mia and early reperfusion. However, at the end of the reperfusion the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. The results suggests for the first time that NO plays a significant role in transmembrane s ignaling in the ischemic myocardium. This signaling appears to be on- and off- nature, and linked with SOD content of the tissue. The signal ing is transmitted via cGMP and opposes the effects of phosphodiestera ses by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. Our results also suggest that NO activates heme oxygenase w hich further stimulates the production of cGMP presumably by CO signal ing. Thus, NO not only potentiates cGMP mediated intracellular signali ng, it also functions as a retrograde messenger for CO signaling in he art.