Pinsky D J, Cai B, Yang X, Rodriguez C, Sciacca R R, Cannon P J
Department of Medicine, Columbia University College of Physicians and Surgeons, New York 10032.
J Clin Invest. 1995 Feb;95(2):677-85. doi: 10.1172/JCI117713.
Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of dilated cardiomyopathies of diverse etiologies.
巨噬细胞产生的诱导型一氧化氮（NO）对入侵生物体具有细胞毒性，在宿主防御中发挥重要作用。最近的研究表明心脏内可产生诱导型NO，且细胞因子诱导的NO介导心脏收缩性改变，但一氧化氮对心脏的细胞毒性潜力尚未明确。为评估诱导型一氧化氮合酶（iNOS）对心肌细胞细胞毒性的作用，我们将成年大鼠心肌细胞单独暴露于细胞因子或与活化的J774巨噬细胞共培养。在用IFNγ和LPS处理的J774巨噬细胞以及用TNF-α、IL-1β和IFNγ处理的心肌细胞中均可见iNOS信使和蛋白表达增加。通过亚硝酸盐生成增加证实了共培养和分离的心肌细胞制剂中NO合成均增加。NO合成增加与心肌细胞死亡的平行增加相关，这通过释放到培养基中的肌酸磷酸激酶（CPK）以及通过台盼蓝染色观察到的膜完整性丧失来衡量。在培养基中添加竞争性NO合酶抑制剂L-NMMA可防止在分离的心肌细胞和共培养实验中细胞因子处理后观察到的亚硝酸盐生成增加和细胞毒性。因为转化生长因子β调节其他细胞类型中的iNOS表达，我们评估了其对心肌细胞iNOS表达和NO介导的心肌细胞毒性的影响。TGF-β平行降低心肌细胞iNOS信使和蛋白的表达、降低亚硝酸盐生成并降低NO介导的细胞毒性。综上所述，这些实验显示了心脏内内源性NO产生的细胞毒性潜力，并提示TGF-β或NO合酶拮抗剂在减轻这些致死效应中发挥作用。这些发现可能有助于解释心脏对败血症或同种异体移植排斥反应的反应，以及各种病因的扩张型心肌病的进展。
