Maulik N, Engelman D T, Watanabe M, Engelman R M, Das D K
Department of Surgery, University of Connecticut School of Medicine, Farmington 06030, USA.
Mol Cell Biochem. 1996;157(1-2):75-86. doi: 10.1007/BF00227883.
To examine the intracellular signaling mechanism of NO in ischemic myocardium, 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 in the presence or absence of 650 microM of protoporphyrin, a heme oxygenase inhibitor for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor placed into the right atrium. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. Induction for the expression of heme oxygenase was studied by Northern hybridization. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [3H] myoinositol and [14C] arachidonic acid. 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. Protoporphyrin modulated the effects of L-arginine. cGMP, which was remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The NO-mediated augmentation of cGMP was reduced by protoporphyrin suggesting that part of the effects may be mediated by CO generated through the heme oxygenase pathway. 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 suggests for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. This signaling appears to be on- and off- nature, and linked with SOD content of the tissue. The signaling is transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. Our results also suggest that NO activates heme oxygenase which further stimulates the production of cGMP presumably by CO signaling. Thus, NO not only potentiates cGMP mediated intracellular signaling, it also functions as a retrograde messenger for CO signaling in heart.
为研究一氧化氮（NO）在缺血心肌中的细胞内信号传导机制，将离体工作大鼠心脏进行30分钟缺血处理，随后再灌注30分钟。另一组心脏在缺血前10分钟，于存在或不存在650微摩尔原卟啉（一种血红素加氧酶抑制剂）的情况下，用3毫摩尔L - 精氨酸进行预灌注。使用置于右心房的在线安培传感器监测NO的释放。检测主动脉流量和心脏收缩压，以确定L - 精氨酸对缺血/再灌注损伤的影响。通过Northern杂交研究血红素加氧酶表达的诱导情况。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。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甘油的情况。通过放射免疫分析法测定环磷酸鸟苷（cGMP），并通过酶分析法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，而L - 精氨酸可使其增加。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。原卟啉调节了L - 精氨酸的作用。cGMP在缺血和再灌注时未受影响，但在L - 精氨酸处理后显著增加。原卟啉降低了NO介导的cGMP增加，这表明部分作用可能由通过血红素加氧酶途径产生的一氧化碳（CO）介导。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]花生四烯酸预先标记的缺血心脏再灌注后，[14C]二酰甘油和[14C]磷脂酸有适度增加。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。这种信号传导似乎具有开启和关闭的特性，并与组织中的SOD含量相关。该信号通过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。我们的结果还表明，NO激活血红素加氧酶，后者可能通过CO信号进一步刺激cGMP产生。因此NO不仅增强cGMP介导的细胞内信号传导，还在心脏中作为CO信号的逆行信使发挥作用。
