Maulik N, Engelman D T, Watanabe M, Engelman R M, Rousou J A, Flack J E, Deaton D W, Gorbunov N V, Elsayed N M, Kagan V E, Das D K
Department of Surgery, University of Connecticut School of Medicine, Farmington 06030-1110, USA.
Circulation. 1996 Nov 1;94(9 Suppl):II398-406.
In heart, NO is produced from L-arginine catalyzed by NO synthase, and CO is formed during the conversion of bilirubin from heme by the action of heme oxygenase. NO, which exerts its biological actions through cGMP and heme, has recently been implicated in myocardial protection during ischemia and reperfusion. We hypothesized that the intracellular signaling by NO may be modulated by heme oxygenase.
To test this hypothesis, isolated rat hearts were perfused for 10 minutes with one of the following: (1) buffer alone; (2) 3 mmol/L L-arginine, a precursor for NO; (3) 650 mumol/L zinc protoporphyrin, a heme oxygenase inhibitor; (4) 3 mmol/L L-arginine plus 650 mumol/L zinc protoporphyrin; (5) 15 mumol/L methylene blue, a cGMP inhibitor; or (6) 3 mmol/L L-arginine plus 15 mumol/L methylene blue. Hearts were then made ischemic for 30 minutes, followed by 30 minutes of reperfusion. L-Arginine afforded significant myocardial protection, as evidenced by increased developed pressure (DP) (53.3 +/- 4.3 versus 35.4 +/- 1.8 for control), dP/dtmax (2405 +/- 125 versus 1758 +/- 117 for control), aortic flow (23 +/- 1.5 versus 9.4 +/- 1.6 for control), and coronary flow (CF) (23.0 +/- 0.8 versus 19.0 +/- 1.6 for control) at the end of reperfusion. Protoporphyrin tended to reduce these values compared with L-arginine alone (DP, 27.5 +/- 1.4; dP/dtmax, 1400 +/- 78; CF, 17 +/- 0.5), suggesting a contribution of heme oxygenase in addition to NO for myocardial preservation. Increased mRNAs for the heme oxygenase were noticed in the ischemic reperfused myocardium. Contents of cGMP, the second messenger for NO signaling, increased in the L-arginine group (1.6 +/- 0.1 versus 1.1 +/- 0.1 for control) and were reduced by protoporphyrin. cGMP was completely inhibited by methylene blue, which also retarded postischemic myocardial functional recovery. Malonaldehyde formation, a presumptive marker for free radical generation, was decreased in the L-arginine group (0.053 +/- 0.003) compared with control (0.089 +/- 0.005) but was increased in the protoporphyrin group (0.09 +/- 0.003) compared with the L-arginine group. In vitro studies demonstrated that NO was able to reduce the reactive oxygen species produced by myoglobin, especially oxoferrylmyoglobin, which either are present in heart or are formed in high concentrations during the reperfusion of ischemic myocardium.
The results suggest that NO contributes to myocardial preservation by both cGMP-dependent and cGMP-independent mechanisms, the former being modulated by CO signaling and the latter by virtue of its antioxidant action.
在心脏中，一氧化氮（NO）由一氧化氮合酶催化L-精氨酸生成，而一氧化碳（CO）则在血红素加氧酶将胆红素从血红素转化的过程中形成。NO通过环磷酸鸟苷（cGMP）和血红素发挥其生物学作用，最近已被证明在缺血再灌注期间具有心肌保护作用。我们推测NO的细胞内信号传导可能受到血红素加氧酶的调节。
为验证这一假设，将离体大鼠心脏用以下溶液之一灌注10分钟：（1）单独的缓冲液；（2）3 mmol/L L-精氨酸，NO的前体；（3）650 μmol/L锌原卟啉，一种血红素加氧酶抑制剂；（4）3 mmol/L L-精氨酸加650 μmol/L锌原卟啉；（5）15 μmol/L亚甲蓝，一种cGMP抑制剂；或（6）3 mmol/L L-精氨酸加15 μmol/L亚甲蓝。然后使心脏缺血30分钟，随后再灌注30分钟。L-精氨酸提供了显著的心肌保护作用，再灌注结束时，发展压力（DP）增加（对照组为35.4±1.8，L-精氨酸组为53.3±4.3）、最大dp/dt（对照组为1758±117，L-精氨酸组为2405±125）、主动脉流量（对照组为9.4±1.6，L-精氨酸组为23±1.5）以及冠状动脉流量（CF）（对照组为19.0±1.6，L-精氨酸组为23.0±0.8）均证明了这一点。与单独使用L-精氨酸相比，原卟啉倾向于降低这些值（DP为27.5±1.4；最大dp/dt为1400±78；CF为17±0.5），这表明除了NO之外，血红素加氧酶对心肌保存也有作用。在缺血再灌注心肌中发现血红素加氧酶的mRNA增加。cGMP作为NO信号传导的第二信使，其含量在L-精氨酸组增加（对照组为1.1±0.1，L-精氨酸组为1.6±0.1），并被原卟啉降低。亚甲蓝完全抑制了cGMP，这也阻碍了缺血后心肌功能的恢复。丙二醛的形成是自由基产生的推测性标志物，与对照组（0.089±0.005）相比，L-精氨酸组（0.053±0.003）降低，但与L-精氨酸组相比，原卟啉组（0.09±0.003）增加。体外研究表明，NO能够减少肌红蛋白产生的活性氧，特别是氧合高铁肌红蛋白，它们要么存在于心脏中，要么在缺血心肌再灌注期间大量形成。
结果表明，NO通过cGMP依赖性和cGMP非依赖性机制对心肌保存有作用，前者由CO信号传导调节，后者凭借其抗氧化作用。
