Carvedilol stimulates nitric oxide synthesis in rat cardiac myocytes

The purpose of this study was to investigate the effects of the beta-adrenergic blocker carvedilol on nitric oxide (NO) synthesis in cardiac myocytes. We measured the accumulation of nitrite, a stable oxidation product of NO, and the expression of inducible NO synthase (iNOS) protein in cultured neonatal rat cardiac myocytes. Incubation of the cultures with interleukin 1 beta (IL-1 beta; 10 ng/ml) caused a marked increase in nitrite production. Although carvedilol alone showed no effect on nitrite accumulation, it significantly enhanced IL-1 beta-induced nitrite production by cardiac myocytes. The effect of carvedilol was completely abolished in the presence of aminoguanidine or actinomycin D. The nitrite production enhanced by carvedilol was accompanied by increased iNOS protein expression. Unlike carvedilol, other beta-blockers, namely propranolol, atenolol and arotinolol, did not enhance IL-1 beta-induced nitrite production. Addition of isoproterenol significantly increased nitrite production by IL-1 beta-stimulated cardiac myocytes. Atenolol suppressed this isoproterenol-induced nitrite accumulation, while carvedilol further increased the nitrite accumulation. These findings indicate that carvedilol increases NO synthesis in IL-1 beta-stimulated rat cardiac myocytes by a beta-adrenoceptor-independent mechanism.     

Lethal effect of cytokine-induced nitric oxide and peroxynitrite on cultured rat cardiac myocytes

We examined the cytotoxic effect of iNOS-generated NO in cultured cardiac myocytes treated with IL-1 beta, IFN- gamma and LPS. Treatment of the myocytes with cytokines for 48 h resulted in a marked NO production, a significant decline in cellular ATP content, and a significant increase in myocyte death with morphological characteristics of necrosis. Moreover, immunohistochemical examination showed that the cytokines caused nitrotyrosine formation in the injured myocytes. Uric acid and L-cysteine which have the ability to quench peroxynitrite significantly attenuated these cytokine-induced effects, although they did not alter NO production or the decline in cellular ATP. These data suggest that NO production induced by cytokines can not only cause deleterious effects in the myocardial energy balance but also induce myocytes necrosis, through the formation of peroxynitrite.     

Intracellular signaling pathways in cardiac myocytes induced by mechanical stress

Mechanical stress is a major cause for cardiac hypertrophy. Although the mechanisms by which mechanical load induces cardiomyocyte hypertrophy have long been a subject of great interest for cardiologists, the lack of a good in vitro system has hampered the understanding of the biochemical mechanisms. For these past several years, however, an in vitro neonatal cardiocyte culture system has made it possible to examine the biochemical basis for the signal transduction of mechanical stress. Passive stretch of cardiac myocytes cultured on silicone membranes activates phosphorylation cascades and induces the expression of specific genes as well as the increase in protein synthesis. Although an important question regarding how mechanical stimulus is converted into biochemical signals remains unanswered, cultured cardiac myocytes may be a good model to examine the signal transduction pathways of mechanical stress.     

Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes

The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.     

Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes

OBJECTIVES: The present study examined whether nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) can directly inhibit aerobic energy metabolism and impair cell function in interleukin (IL)-1beta,-stimulated cardiac myocytes. BACKGROUND: Recent reports have indicated that excessive production of NO induced by cytokines can disrupt cellular energy balance through the inhibition of mitochondrial respiration in a variety of cells. However, it is still largely uncertain whether the NO-induced energy depletion affects myocardial contractility. METHODS: Primary cultures of rat neonatal cardiac myocytes were prepared, and NO2-/NO3- (NOx) in the culture media was measured using Griess reagent. RESULTS: Treatment with IL-1beta (10 ng/ml) increased myocyte production of NOx in a time-dependent manner. The myocytes showed a concomitant significant increase in glucose consumption, a marked increase in lactate production, and a significant decrease in cellular ATP (adenosine 5'-triphosphate). These metabolic changes were blocked by co-incubation with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis. Sodium nitroprusside (SNP), a NO donor, induced similar metabolic changes in a dose-dependent manner, but 8-bromo-cyclic guanosine 3',5'-monophosphate (8-bromo-cGMP), a cGMP donor, had no effect on these parameters. The activities of the mitochondrial iron-sulfur enzymes, NADH-CoQreductase and succinate-CoQreductase, but not oligomycin-sensitive ATPase, were significantly inhibited in the IL-1beta, or SNP-treated myocytes. Both IL-1beta and SNP significantly elevated maximum diastolic potential, reduced peak calcium current (I(Ca)), and lowered contractility in the myocytes. KT5823, an inhibitor of cGMP-dependent protein kinase, did not block the electrophysiological and contractility effects. CONCLUSIONS: These data suggest that IL-1beta-induced NO production in cardiac myocytes lowers energy production and myocardial contractility through a direct attack on the mitochondria, rather than through cGMP-mediated pathways.     

L-精氨酸对离体心肌细胞和心脏微血管内皮细胞一氧化氮合酶活性的影响

目的 :探讨 L-精氨酸对体外培养的心肌细胞和内皮细胞一氧化氮合酶 (NOS)活性的影响。方法 :体外培养 SD大鼠的心肌细胞和心脏微血管内皮细胞 ,72 h后分组 ,实验组在生长液中加入 1 mol/L的 L-精氨酸 ,分别在加入 L-精氨酸后 1 /2、1、2、4h取细胞 ,反复冻融 3次使细胞破裂 ,用分光光度法测细胞内 NOS活性。结果 :加入 1 mol/L的 L-精氨酸后 ,心肌细胞的 NOS活性在 1 /2 h升高至对照组的 1 2 8% (P <0 .0 5) ,2 h时升高至 1 37% (P <0 .0 5) ,4h后下降 ;而内皮细胞在加入 L-精氨酸后 1 /2、1、2、4h时的 NOS活性分别为对照组的 1 2 2 %、1 38%、1 2 8%和1 0 3% ,且内皮细胞 NOS活性较心肌细胞为高。结论 :L-精氨酸可升高心肌细胞和心脏微血管内皮细胞的 NOS活性     

Negative effect of nitric oxide on shorteningfrequency relationship in cardiac myocytes is diminished after simulated ischemia-reperfusion

Increasing stimulation rate increases function in cardiac myocytes and nitric oxide and cyclic GMP inhibit this effect. We tested the hypothesis that myocyte stunning would blunt both the effects of increases in rate and of nitric oxide and cyclic GMP. Ventricular myocytes from 11 rabbits were used to determine maximum rate of shortening (R_max, µm/s) and %shortening during control and after simulated ischemia [15 min 95% N₂- 5% CO₂] and reperfusion [reoxygenation]. Measurements were obtained at 1–4 Hz with vehicle, 1H[1,2,4]oxadiazolo[4,3,alpha] quinoxaline-1-one (ODQ) 10^–6 M, soluble guanylyl cyclase inhibitor, or N^G-nitro-L-arginine methyl ester, nitric oxide synthase inhibitor (L-NAME) 10^–5 M. In control, increases in rate increased R_max from 69 ± 3 to 254±12 and %shortening from 5.3 ± 0.3 to 8.7 ± 0.5. Both ODQ and L-NAME shifted values higher. With stunning, the effects of pacing on Rmax and %shortening were blunted and ODQ and L-NAME failed to alter these values. Cyclic GMP was 322±37 pmol/10⁵ myocytes at baseline and these values were lowered by ODQ (244 ± 31) and LNAME (207 ± 23), and similar changes were observed in stunned myocytes. Increasing frequency increased function, and reducing nitric oxide/cyclic GMP enhanced this relationship. The effect of nitric oxide was diminished by stunning, but this was not related to altered cyclic GMP levels. This suggested changes in effects of cyclic GMP downstream to its production during myocardial stunning.     

Cytokine-stimulated nitric oxide production inhibits adenylyl cyclase and cAMP-dependent secretion in cholangiocytes

BACKGROUND & AIMS: The biliary epithelium is involved both in bile production and in the inflammatory/reparative response to liver damage. Recent data indicate that inflammatory aggression to intrahepatic bile ducts results in chronic progressive cholestasis. METHODS: To understand the effects of nitric oxide on cholangiocyte secretion and biliary tract pathophysiology we have investigated: (1) the effects of proinflammatory cytokines on NO production and expression of the inducible nitric oxide synthase (NOS2), (2) the effects of NO on cAMP-dependent secretory mechanisms, and (3) the immunohistochemical expression of NOS2 in a number of human chronic liver diseases. RESULTS: Our results show that: (1) tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, synergically stimulate NO production in cultured cholangiocytes through an increase in NOS2 gene and protein expression; (2) micromolar concentrations of NO inhibit forskolin-stimulated cAMP production by adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP)-dependent fluid secretion, and cAMP-dependent Cl(-) and HCO(3)(-) transport mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and anion exchanger isoform 2, respectively; (3) cholestatic effects of NO and of proinflammatory cytokines are prevented by NOS-2 inhibitors and by agents (manganese(III)-tetrakis(4-benzoic acid)porphyrin [MnTBAP], urate, trolox) able to block the formation of reactive nitrogen oxide species (RNOS); (4) NOS2 expression is increased significantly in the biliary epithelium of patients with primary sclerosing cholangitis (PSC). CONCLUSIONS: Our findings show that proinflammatory cytokines stimulate the biliary epithelium to generate NO, via NOS2 induction, and that NO causes ductular cholestasis by a RNOS-mediated inhibition of AC and of cAMP-dependent HCO(3)(-) and Cl(-) secretory mechanisms. This pathogenetic sequence may contribute to ductal cholestasis in inflammatory cholangiopathies.     

Cross-talk between cyclooxygenase and nitric oxide pathways: prostaglandin E2 negatively modulates induction of nitric oxide synthase by interleukin 1.

The inflammatory cytokine interleukin 1 beta (IL-1 beta) induces both cyclooxygenase (COX) and nitric oxide synthase (NOS) with increases in the release of prostaglandin (PG) and nitric oxide (NO) by mesangial cells. Recently, activation of the COX enzyme by NO has been described. However, the effects of COX products (PGs) on the NO pathway have not been fully clarified. Thus we determined the effect of COX inhibition and exogenous PGs on NO production and NOS induction in rat mesangial cells. A COX inhibitor, indomethacin, enhanced IL-1 beta-induced steady-state level of the inducible NOS (iNOS) mRNA and nitrite production. The effect of indomethacin was dose dependently reversed by the replacement of endogenous PGE2 with exogenous PGE2, which is the predominant product of the COX pathway in rat mesangial cells. In contrast to PGE2, a stable analog of PGI2, carba prostacyclin, enhanced IL-1 beta-induced iNOS mRNA levels and nitrite production. Forskolin, an activator of the adenylate cyclase, mimicked the effect of carba prostacyclin but not PGE2. These data suggest that (i) endogenous PGE2 downregulates iNOS induction, (ii) this inhibitory effect of PGE2 on iNOS induction is not mediated by activation of adenylate cyclase, and (iii) exogenous PGI2 stimulates COX induction possibly by activation of adenylate cyclase.     

辛伐他汀对脓毒症大鼠心肌细胞核因子-κB、诱导型一氧化氮合酶表达的影响及意义

目的 观察核因子(NF)-κB和诱导型一氧比氮合酶(iNOS)在脓毒症大鼠心肌细胞的表达,同时探讨辛伐他汀对其表达的影响,以及他汀类药物的心肌保护性作用.方法 雌性Wistar大鼠30只随机分为三组:正常组(N组),盲肠结扎穿孔术组(CLP组)和辛伐他汀组(S组),处理方法:①S组:喂养辛伐他汀20 mg/kg每天一次共2w.②2w后S组与CLP组大鼠行盲肠结扎穿孔术.③术后48 h取各组大鼠心肌组织制成石蜡切片,采用HE染色和免疫组织化学染色的方法检测心肌纤维iNOS和NF-κB的蛋白表达.结果 HE染色显示CLP组大鼠心肌纤维水肿,疏松,间质充血水肿,而S组心肌的组织病理学改变较CLP组轻.免疫组织化学染色显示CLP组大鼠心肌细胞iNOS与NF-κB蛋白表达于术后48 h较N组明显增加(P＜0.05),S组大鼠心肌细胞iNOS与NF-κB蛋白表达与CLP组相比明显减弱(P＜0.05),与N组相比无明显差异(P＞0.05).结论 辛伐他汀可以减弱脓毒症大鼠心肌细胞iNOS和NF-κB的蛋白表达,改善脓毒症时的心脏功能.