Maulik N, Engelman D T, Watanabe M, Engelman R M, Maulik G, Cordis G A, Das D K
Department of Surgery, University of Connecticut School of Medicine, Farmington 06030-1110, USA.
Cardiovasc Res. 1995 Oct;30(4):593-601.
Several recent studies have implicated a role of endogenous nitric oxide (NO) in the pathophysiology of myocardial ischemic/reperfusion injury. However, the mechanism by which NO exerts its beneficial/detrimental effects remains unknown. This study examined the intracellular signaling of NO by studying the role of the NO-cGMP signaling pathway on the phospho-diesteratic breakdown and turnover of phosphoinositides during myocardial ischemia and reperfusion.
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 monitored using an on-line amperometric sensor. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [3H]myoinositol and [14C]arachidonic acid. Hearts were then perfused for 10 min in the presence or absence of L-arginine via the Langendorff mode. Ischemia was induced for 30 min followed by 30 min of reperfusion. Experiments were terminated before L-arginine and after L-arginine treatment, after ischemia, and during reperfusion. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis.
The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-Arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. cGMP, which remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The cGMP level persisted up to 10 min of reperfusion and then dropped slightly. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [14C]-arachidonic acid resulted in modest increases in [14C]diacylglycerol and [14C]phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia 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 suggest for the first time that NO plays a significant role in transmembrane 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 breakdown. This signaling effect appears to be reduced as reperfusion progresses. These results, when viewed in the light of free radical chemistry of NO, suggest that such on- and off-signaling of NO may be linked to its interaction with the superoxide radical generated during the reperfusion of ischemic myocardium.
最近的几项研究表明内源性一氧化氮（NO）在心肌缺血/再灌注损伤的病理生理学中起作用。然而，NO发挥其有益/有害作用的机制仍不清楚。本研究通过研究NO - cGMP信号通路在心肌缺血和再灌注期间对磷酸肌醇的磷酸二酯酶分解和周转的作用，来探讨NO的细胞内信号传导。
将离体工作大鼠心脏缺血30分钟，然后再灌注30分钟。另一组心脏在缺血前用3 mM L - 精氨酸预灌注10分钟。使用在线安培传感器监测NO的释放。检测主动脉流量和发展压力，以确定L - 精氨酸对缺血/再灌注损伤的影响。对于信号转导实验，通过用[3H]肌醇和[14C]花生四烯酸灌注离体心脏，对肌膜进行放射性标记。然后通过Langendorff模式在有或没有L - 精氨酸的情况下将心脏灌注10分钟。诱导缺血30分钟，然后再灌注30分钟。在L - 精氨酸处理前、处理后、缺血后和再灌注期间终止实验。对活检组织进行处理，以确定同位素掺入各种磷酸肌醇以及磷脂酸和二酰基甘油的情况。通过放射免疫测定法测定cGMP，通过酶分析测定超氧化物歧化酶（SOD）含量。
缺血和再灌注后NO的释放减少，L - 精氨酸可增强其释放。L - 精氨酸减轻了缺血/再灌注损伤，心肌功能恢复增强证明了这一点。cGMP在缺血和再灌注时未受影响，但在L - 精氨酸处理后显著升高。cGMP水平在再灌注长达10分钟时持续存在，然后略有下降。缺血心肌再灌注导致放射性标记的肌醇磷酸、肌醇二磷酸和肌醇三磷酸显著积累。在再灌注期间，[3H]肌醇掺入磷脂酰肌醇、磷脂酰肌醇 - 4 - 磷酸和磷脂酰肌醇 - 4,5 - 二磷酸的量显著增加。用[14C]花生四烯酸预标记的缺血心脏再灌注导致[14C]二酰基甘油和[14C]磷脂酸适度增加。用L - 精氨酸预处理心脏可显著逆转缺血和早期再灌注期间这种增强的磷酸二酯酶分解。然而，在再灌注结束时，L - 精氨酸对磷酸二酯酶的抑制作用似乎减弱。在L - 精氨酸处理的心脏中，SOD活性随着再灌注时间的延长而逐渐降低。
结果首次表明NO在缺血心肌的跨膜信号传导中起重要作用。该信号似乎通过cGMP传递，并通过抑制缺血/再灌注诱导的磷酸二酯酶分解来对抗磷酸二酯酶的作用。随着再灌注的进行，这种信号作用似乎减弱。从NO的自由基化学角度来看，这些结果表明NO的这种开启和关闭信号可能与其与缺血心肌再灌注期间产生的超氧阴离子的相互作用有关。
