Inducible nitric oxide synthase (iNOS) in muscle wasting syndrome, sarcopenia, and cachexia

Muscle atrophy-also known as muscle wasting-is a debilitating syndrome that slowly develops with age (sarcopenia) or rapidly appears at the late stages of deadly diseases such as cancer, AIDS, and sepsis (cachexia). Despite the prevalence and the drastic detrimental effects of these two syndromes, there are currently no widely used, effective treatment options for those suffering from muscle wasting. In an attempt to identify potential therapeutic targets, the molecular mechanisms of sarcopenia and cachexia have begun to be elucidated. Growing evidence suggests that inflammatory cytokines may play an important role in the pathology of both syndromes. As one of the key cytokines involved in both sarcopenic and cachectic muscle wasting, tumor necrosis factor α (TNFα) and its downstream effectors provide an enticing target for pharmacological intervention. However, to date, no drugs targeting the TNFα signaling pathway have been successful as a remedial option for the treatment of muscle wasting. Thus, there is a need to identify new effectors in this important pathway that might prove to be more efficacious targets. Inducible nitric oxide synthase (iNOS) has recently been shown to be an important mediator of TNFα-induced cachectic muscle loss, and studies suggest that it may also play a role in sarcopenia. In addition, investigations into the mechanism of iNOS-mediated muscle loss have begun to reveal potential therapeutic strategies. In this review, we will highlight the potential for targeting the iNOS/NO pathway in the treatment of muscle loss and discuss its functional relevance in sarcopenia and cachexia.     

Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression in fulminant hepatic failure

BACKGROUND/AIMS: Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) have important functions in inflammation and vasoregulation but their role in fulminant hepatic failure (FHF) is not well understood. METHODS: Intrahepatic in situ staining and semi-quantification of iNOS and eNOS by immunohistochemistry in 25 patients with FHF, in 40 patients with chronic liver diseases (CLD) and in ten normal controls (NC). RESULTS: Expression patterns of iNOS and eNOS differed. While in NC only faint iNOS expression was found in some Kupffer cells/macrophages and hepatocytes, eNOS was expressed constitutively in sinusoidal and vascular endothelial cells. In CLD, iNOS expression was induced in Kupffer cells/macrophages and hepatocytes, representing the main iNOS expressing cell types. Additionally, bile ducts, vascular endothelial cells and lymphocytes also expressed iNOS (P = 0.001). In contrast, no differences were found between eNOS expression in CLD and NC (P = 0.64). The same cell types expressed eNOS and iNOS in FHF but numbers of both were significantly enhanced, exceeding the levels seen in CLD (P < 0.001, P = 0.017). CONCLUSIONS: Our data demonstrate that iNOS and eNOS are differently regulated in physiologic conditions and in liver disease. While eNOS seems to be involved in the physiological regulation of hepatic perfusion, strong upregulation of iNOS might contribute to inflammatory processes in FHF.     

Nitrotyrosine,inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) are increased in thyroid tumors from children and adolescents

Nitric oxide (NO) is a reactive cell signal that controls vascular tone and is generated by inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NO synthase (NOS). We hypothesized that NO could be important for growth of thyroid tumors and tested this hypothesis, by staining 41 papillary thyroid carcinoma (PTC), 9 follicular thyroid carcinoma (FTC), and 15 benign thyroid lesions for iNOS, eNOS and nitrotyrosine (N-TYR). Staining intensity was determined by 2 blinded, independent examiners, and quantified from grade 1 (absent) to grade 4 (intense). Average N-TYR staining of benign adenomas (2.5+/-0.42, p=0.009), PTC (3.10+/-0.12, p=0.001), FTC (2.44+/-0.30, p=0.001), and autoimmune lesions (3.25+/-0.48, p=0.019) were greater than that of multinodular goiter (1.0 for all 3) and surrounding normal thyroid (1.1+/-0.1). Average iNOS staining of benign adenomas (2.6+/-0.37), PTC (2.7+/-0.16), FTC (2.4+/-0.26) and autoimmune lesions (3.5+/-0.29) were all greater than that of surrounding normal thyroid (1.1+/-0.1, p<0.008), but there were too few multinodular goiters to achieve a significant difference (no.=2, 3.0+/-1.0). Average eNOS staining of benign adenomas (2.9+/-0.40), multinodular goiters (3.5+/-0.5), PTC (3.24+/-0.18), FTC (3.5+/-0.50), and autoimmune lesions (2.8+/-0.6) were also greater than that of surrounding normal thyroid (mean=1.4+/-0.2, p<0.001). N-TYR staining correlated with that of vascular endothelial growth factor (VEGF, r=0.36, p=0.007) and the number of lymphocytes/high power field (r=0.39, p=0.004). Recurrent disease developed only from carcinoma with moderate-intense N-TYR staining, but there were too few recurrent tumors to achieve statistical significance (p=0.08). We conclude that NO is produced by benign adenomas, PTC and FTC suggesting that NO could be important in vascularization of thyroid tumors and autoimmune thyroid diseases.     

Novel features of nitric oxide,endothelial nitric oxide synthase,and atherosclerosis

There is a complex pathophysiologic scenario involving nitric oxide (NO), endothelial nitric oxide synthase (_eNOS), and the development of atherosclerosis and unstable atheroma. Endothelial damage induced by atherosclerosis leads to the reduction in bioactivity of _eNOS with subsequent impaired release of NO. An important mechanism is local enhanced degradation of NO by increased generation of reactive oxygen species and other free radicals, with subsequent cascade of oxidationsensitive mechanisms in the arterial wall. Novel molecular approaches have resulted in the development of new strains of mice lacking _eNOS. These experimental models will help to understand how to implement NO-based therapies against atherosclerosis. L-arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. The target or goal for new drugs should be the complete restoration of NOmediated signaling pathways in atherosclerotic arteries.     

Novel features of nitric oxide,endothelial nitric oxide synthase,and atherosclerosis

There is a complex pathophysiologic scenario involving nitric oxide (NO), endothelial nitric oxide synthase (eNOS), and the development of atherosclerosis and unstable atheroma. Endothelial damage induced by atherosclerosis leads to the reduction in bioactivity of ENOS with subsequent impaired release of NO. An important mechanism is local enhanced degradation of NO by increased generation of reactive oxygen species and other free radicals, with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Novel molecular approaches have resulted in the development of new strains of mice lacking eNOS. These experimental models will help to understand how to implement NO-based therapies against atherosclerosis. L-arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. The target or goal for new drugs should be the complete restoration of NO-mediated signaling pathways in atherosclerotic arteries.     

In stable lung transplant recipients, exhaled nitric oxide levels positively correlate with airway neutrophilia and bronchial epithelial iNOS

In conditions characterized by airway inflammation, exhaled nitric oxide (eNO) levels are increased. Variable degrees of airway inflammation are present in stable lung transplant recipients (LTR), and may lead to airway remodeling and chronic graft dysfunction. The hypothesis tested is that in stable LTR, eNO concentrations would reflect the expression of inducible (iNOS) (but not constitutive [cNOS] nitric oxide synthase) in the bronchial epithelium as well as the degree of airway inflammation. We determined eNO concentrations in 20 stable LTR, free of infection, rejection, or obliterative bronchiolitis (OB). At routine bronchoscopy, we measured the differential cell count on bronchoalveolar lavage (BAL) and a quantitative assessment of iNOS and cNOS expression in endobronchial biopsies by immunohistochemistry. Mean +/- SEM eNO concentrations in stable LTR were not significantly different from control subjects (13 +/- 0.7 ppb versus 14.2 +/- 0.49; p = 0.42). Percent BAL neutrophils was 11.5 +/- 3.2 which was significantly higher than in a group of local control subjects (1.7 +/- 0.6; p < 0.001). The bronchial epithelium and lamina propria contained abundant iNOS but cNOS was present only in the lamina propria. Using regression analysis, percent BAL neutrophils (r(2) = 0.82; p < 0.0001) and iNOS expression in the bronchial epithelium (r(2) = 0.75; p < 0.0001), but not in the lamina propria (r(2) = 0.16; p = 0.08), were positively predictive of eNO. There was an inverse relationship between cNOS and eNO. We conclude that eNO concentrations although normal for the group, still reflect the degree of airway inflammation in stable LTR. Epithelial iNOS appears to be the major source of eNO and expression of cNOS may be downregulated with increasing iNOS expression.     

Environmental exposures,nitric oxide synthase genes,and exhaled nitric oxide in asthmatic children

Exhaled nitric oxide (FeNO), a measure of airway inflammation, is being explored as a tool to guide asthma management in children. Investigators have identified associations of genetic polymorphisms in nitric oxide synthase genes (NOS1 and NOS3) with FeNO levels; however, none have explored whether these polymorphisms modify the relationship of environmental exposures with FeNO. The objective of this project was to evaluate the association of NOS polymorphisms and environmental exposures with FeNO levels among children with asthma. We conducted a 12‐month prospective cohort study of 225 tobacco‐smoke exposed children (6–12 years) with doctor‐diagnosed asthma. We assessed environmental exposures (tobacco, indoor allergens, & airborne particulates), polymorphisms in NOS1 (an intronic AAT tandem repeat) and NOS3 (G894T), and FeNO levels. There was no association of NOS1 or NOS3 polymorphisms with FeNO levels. There were no significant interactions of environmental exposures and the NOS1 polymorphism with FeNO levels. In contrast, there was an interaction of the NOS3 polymorphism and airborne nicotine concentration with FeNO levels (P = 0.01). Among GG genotype individuals, nicotine exposure did not affect FeNO levels; however, among individuals with at least one T allele, higher nicotine exposure was associated with lower FeNO levels (approximately 5 ppb decrease from the lowest to the highest quartile). We conclude that genetic differences may explain some of the conflicting results in studies of the effects of tobacco smoke exposure on FeNO levels and may make FeNO interpretation difficult for a subset of children with asthma. Pediatr Pulmonol. 2009; 44:812–819. © 2009 Wiley‐Liss, Inc.     

诱导型一氧化氮合酶基因多态性与冠心病的关联性研究

目的探讨一氧化氮合酶(i NOS)基因多态性与冠心病的关联性。方法病例组为291例冠心病患者,年龄、性别匹配的健康体检者487例为对照组。采用Taqman探针荧光定量PCR技术检测rs2779248 C/T、rs1137933 C/T的基因型分布情况。结果经校正传统危险因素后,携带rs2779248 TT基因型的个体患冠心病的风险比携带CC基因型的高2.58倍(95%CI:1.01~6.67);而携带rs1137933 CT+TT基因型的个体患冠心病的风险比携带CC基因型的高1.36倍(95%CI:1.00~1.85),突变T等位基因可能增加冠心病发病风险(OR=1.36,95%CI:1.04~1.77)。各基因型间空腹血糖水平存在明显差异。结论 i NOS基因多态性与中国汉族人群冠心病发病风险相关,i NOS基因可能通过调节血糖影响冠状动脉粥样硬化斑块形成,进而增加冠心病的发病风险。     

Hyperfiltration, nitric oxide, and diabetic nephropathy

Early diabetes is often accompanied by an increased glomerular filtration rate (GFR). This hyperfiltration, which is significantly dependent upon increased nitric oxide activity, is believed to contribute to progression of diabetic nephropathy. In this article, a technique for the measurement of tubular fluid nitric oxide in vivo, in real time, is reviewed, and findings in three commonly used rodent models of diabetes are described. The mechanisms of altered tubuloglomerular feedback (TGF) in diabetes are also reviewed, with emphasis on hyperfiltration and the role of nitric oxide. New findings on the modulation of hyperfiltration in the classic type 2 diabetes db/db mouse are presented, showing suppression of the TGF mechanism and modulation of single-nephron GFR by a specific nitric oxide synthase inhibitor.     

Rho GTPases, statins, and nitric oxide

The lipid-lowering drugs, 3-hydroxy-3-methylgulutaryl-coenzyme A (HMG-CoA) reductase inhibitors or statins, are used in the prevention and treatment of cardiovascular diseases. Recent experimental and clinical studies suggest that statins may exert vascular protective effects beyond cholesterol reduction. For example, statins improve endothelial function by cholesterol-dependent and -independent mechanisms. The cholesterol-independent or "pleiotropic" effects of statins include the upregulation and activation of endothelial NO synthase (eNOS). Because statins inhibit an early step in the cholesterol biosynthetic pathway, they also inhibit the synthesis of isoprenoids such as farnesylpyrophosphate and geranylgeranylpyrophosphate, which are important posttranslational lipid attachments for intracellular signaling molecules such as the Rho GTPases. Indeed, decrease in Rho GTPase responses as a consequence of statin treatment increases the production and bioavailability of endothelium-derived NO. The mechanism involves, in part, Rho/Rho-kinase (ROCK)-mediated changes in the actin cytoskeleton, which leads to decreases in eNOS mRNA stability. The regulation of eNOS by Rho GTPases, therefore, may be an important mechanism underlying the cardiovascular protective effect of statins.     

Caveolae, estrogen and nitric oxide.

Estradiol has numerous physiological and pathophysiological effects on the cardiovascular system, and the ongoing controversy surrounding estrogen replacement therapy clearly illustrates the importance of understanding the molecular mechanisms of estrogen action. Many recent mechanistic studies have focused on the ability of estradiol to stimulate endothelial nitric oxide synthase (eNOS) and the subsequent generation of nitric oxide (NO). NO is centrally involved in many processes such as mitogenesis, cell adhesion, thrombosis, atherosclerosis and hypertension. Consequently, elucidating the mechanisms whereby estradiol influences NO production will directly impact on our understanding of the advantages and disadvantages of estrogen replacement therapy. An exciting aspect of this emerging area of study is that the estrogen, NO and caveolae research fields have merged to identify a novel and clinically relevant molecular process. The goal of this review is to highlight the recent findings in this area and to point out areas of controversy and areas where more studies are needed.     

Endothelial nitric oxide synthase polymorphism, nitric oxide production, salt sensitivity and cardiovascular risk factors in Hispanics

Mutations in the endothelial nitric oxide synthase (eNOS) gene may be associated with abnormal nitric oxide (NO) production and cardiovascular diseases. In this study, we investigated the prevalence of two eNOS polymorphisms, the Glu298Asp variant on exon 7, and the 4a/b variable number of tandem repeats (VNTR) on intron 4, and their association with blood pressure (BP), NO production, salt sensitivity and cardiovascular risk factors in healthy Venezuelans. The prevalence of both polymorphisms in Venezuelans was comparable to that described for Caucasians, but significantly different from that known for African-Americans and Japanese. The 4a/b genotype was associated with reduced levels of NO metabolites (25% decrease), larger BP lowering in response to salt restriction (9.0 vs 4.8 mmHg, P<0.05), greater prevalence of salt sensitivity (39% in 4a/b and 27% in 4b/b; P<0.05) and with higher LDL-cholesterol levels. The Glu298T polymorphism did not affect NO production, nor it was associated with salt sensitivity. Glu298Asp polymorphism was positively associated with higher weight, triglycerides and LDL-cholesterol. Neither polymorphism was associated with changes in fasting or postload serum glucose, BP, obesity and albuminuria. In conclusion, the prevalence of eNOS polymorphisms is strongly determined by ethnic factors. The 4a/b gene polymorphism could be a genetic susceptibility factor for the BP response to salt intake and for the genetic control of NO production. The reduced NO production in subjects with the 4a/b genotype may be responsible for the increased sensitivity of their BP to salt.     

Vascular oxidative stress,nitric oxide and atherosclerosis

In the vascular wall, reactive oxygen species (ROS) are produced by several enzyme systems including NADPH oxidase, xanthine oxidase, uncoupled endothelial nitric oxide synthase (eNOS) and the mitochondrial electron transport chain. On the other hand, the vasculature is protected by antioxidant enzyme systems, including superoxide dismutases, catalase, glutathione peroxidases and paraoxonases, which detoxify ROS. Cardiovascular risk factors such as hypercholesterolemia, hypertension, and diabetes mellitus enhance ROS generation, resulting in oxidative stress. This leads to oxidative modification of lipoproteins and phospholipids, mechanisms that contribute to atherogenesis. In addition, oxidation of tetrahydrobiopterin may cause eNOS uncoupling and thus potentiation of oxidative stress and reduction of eNOS-derived NO, which is a protective principle in the vasculature. This review summarizes the latest advances in the role of ROS-producing enzymes, antioxidative enzymes as well as NO synthases in the initiation and development of atherosclerosis.     

Endothelial mechanotransduction, nitric oxide and vascular inflammation

Numerous aspects of vascular homeostasis are modulated by nitric oxide and reactive oxygen species (ROS). The production of these is dramatically influenced by mechanical forces imposed on the endothelium and vascular smooth muscle. In this review, we will discuss the effects of mechanical forces on the expression of the endothelial cell nitric oxide synthase, production of ROS and modulation of endothelial cell glutathione. We will also review data that exercise training in vivo has a similar effect as laminar shear on endothelial function and discuss the clinical relevance of these basic findings.     

Melatonin and nitric oxide

Melatonin is a product of the amino acid tryptophan in the pineal gland. Once synthesized, the specific mechanisms governing the release of melatonin from the pineal gland and its functions are largely unknown. Besides its regulatory role in circadian rhythms in mammals, because of its widespread subcellular distribution, melatonin contributes to the reduction of oxidative damage in both the lipid and the aqueous environments of the cell. This postulate is widely supported by the experimental observations showing that melatonin protects lipids in membranes, proteins in the cytosol, and DNA in the nucleus and mitochondria from free radical damage. Melatonin thus reduces the severity of disease conditions where free radicals are implicated. The direct free radical scavenging effects of melatonin are receptor independent. It has recently been shown that it has an ability to scavenge free radicals, including hydroxyl radicals, hydrogen peroxide, peroxyl radicals, singlet oxygen and nitric oxide (NO) and peroxynitrite anion. An excessive amount of NO, a free radical which is generated by the inducible form of NO synthase, is known to cause cytotoxic changes in cells. Hence, NO synthase is considered a pro-oxidative enzyme, and any factor that reduces its activity would be considered an antioxidant. Recent studies have shown that melatonin inhibits the activity of NO synthase, beside its NO and peroxynitrite scavenging activity. Thus, inhibition of NO production may be another means whereby melatonin reduces oxidative damage under conditions, such as ischemia-reperfusion, sepsis, etc, where NO seems to be important in terms of the resulting damage.