Administration of a nitric oxide synthase inhibitor does not suppress low-dose streptozotocin-induced diabetes in mice

Summary Nitric oxide (NO) has been reported as being a key mediator of the autoimmune destruction of B-cells in type I diabetes, and studies have described a suppression of low-dose streptozotocin-induced (LDS) diabetes in mice after the use of NO synthase inhibitors. However, these studies disagree with regard to the outcome of hyperglycemia and insulitis after treatment with thesel-arginine analogs. The present study tries to clarify this topic by administeringN-nitro-l-arginine-methylester (NAME) (15 mg/d/mouse/15 d) after an LDS treatment in 108 male C57BL6/J mice. Glycemia measured at the end of the NAME treatment did show a slight, but significant, reduction when compared to LDS control animals (p<0.001), but values returned to diabetic levels 2 wk after withdrawal of NAME. Morphological observations demonstrated that the degree of infiltration and islet B-cell damage was absolutely not inhibited by NAME. In conclusion, treatment withl-arginine analogs is not capable of protecting mice from LDS-induced diabetes.     

Increased expression of constitutive nitric oxide synthase III,but not inducible nitric oxide synthase II,in human heart failure

Objectives. The purpose of the present study was to examine the expression of the endothelial-type nitric oxide synthase (NOS III) and the inducible-type NOS (NOS II) in human myocardium and their regulation in heart failure from patients with different etiologies.Background. In heart failure, plasma levels of nitrates were found to be elevated. However, data on myocardial NOS expression in heart failure are conflicting.Methods. Using RNase protection analysis and Western blotting, the expression of NOS III and NOS II was investigated in ventricular myocardium from nonfailing (NF) hearts (n = 5) and from failing hearts of patients with idiopathic dilated cardiomyopathy (dCMP, n = 14), ischemic cardiomyopathy (iCMP, n = 9) or postmyocarditis cardiomyopathy (mCMP, n = 7). Furthermore, immunohistochemical studies were performed to localize NOS III and NOS II within the ventricular myocardium.Results. In failing human hearts, NOS III mRNA levels were increased to 180% in dCMP, 200% in iCMP and to 210% in mCMP as compared to NF hearts. Similarly, in Western blots (using constitutively expressed beta-tubulin as a reference) NOS III protein expression was increased about twofold in failing compared to NF hearts. Immunohistochemical studies with a selective antibody to NOS III showed no obvious differences in the staining of the endothelium of cardiac blood vessels from NF and failing human hearts. However, NOS III-immunoreactivity in cardiomyocytes was significantly more intense in failing compared to NF hearts. Low expression of NOS II mRNA was detected in only 2 of 30 failing human hearts and was not found in NF hearts. Inducible-type NOS protein was undetectable in either group.Conclusions. We conclude that the increased NOS III expression in the ventricular myocardium of failing human hearts may contribute to the contractile dysfunction observed in heart failure and/or may play a role in morphologic alterations such as hypertrophy and apoptosis of cardiomyocytes.     

Functional properties of failing human ventricular myocytes.

Reduced peak systolic Ca2+ and slow decay of the Ca2+ transient are common features of the end-stage failing human ventricular myocyte and are thought to underlie abnormal ventricular contractility in congestive heart failure (CHF). Individual changes in the expression or activity of Ca2+ transport proteins of the sarcoplasmic reticulum (SR Ca2+ ATPase, SERCa) or the sarcolemmal (sodium-calcium exchanger, NCX) have not always been observed in CHF and cannot per se consistently explain these Ca2+ transient defects. We review recent data that suggests that the normal balance of transport activities of SERCa and NCX is deranged in failing human myocytes. We hypothesize that an increase in the NCX/SERCa transport capacity in failing myocytes can explain the abnormal Ca2+ homeostasis of the failing human ventricular myocyte.     

Lack of inducible nitric oxide synthase does not prevent aging-associated insulin resistance

Inducible nitric oxide synthase (iNOS) is involved in obesity-induced insulin resistance. Since aging is accompanied by increased iNOS expression, the effect of iNOS gene deletion on aging-associated insulin resistance was investigated in 7-month-old (adult) and 22-month-old (old) iNOS knockout and wild-type mice using the hyperinsulinemic–euglycemic clamp. While body weight and fat mass were increased, muscle mass was reduced with aging in wild-type mice. However, body composition was not changed with aging in iNOS knockout mice due to increased locomotor activity. NO metabolites in plasma, and protein levels of iNOS and nitrotyrosine in skeletal muscle increased with aging in wild-type mice. Deletion of iNOS gene attenuated NO metabolites and nitrotyrosine with aging in iNOS knockout mice. Glucose uptake in whole body and skeletal muscle was reduced with aging in both wild-type and iNOS knockout mice and there was no difference between two groups. Plasma level of tumor necrosis factor-α and gene expression of proinflammatory cytokines in peripheral tissues were increased with aging in both groups, and that was more heightened in iNOS knockout mice. These results suggest that lack of iNOS does not prevent aging-associated insulin resistance in mice and heightened production of proinflammatory cytokines may be involved.     

Increased neuronal nitric oxide synthase-derived NO production in the failing human heart

Experimental data suggest that nitric oxide (NO) generated from neuronal NO synthase (nNOS) modulates the myocardial inotropic state. To assess the contribution of NO, derived from endothelial and neuronal isoforms, to the pathophysiology of congestive heart failure in human beings, we compared expression, localisation, and specific activity of NOS isoforms in myocardium from patients with dilated cardiomyopathy with those in controls who had died from head trauma or intracranial bleeds. Diseased hearts had a significant increase in nNOS mRNA and protein expression, and activity associated with the translocation of nNOS to the sarcolemma through interactions with caveolin 3. Enhanced nNOS activity counteracted a decrease in eNOS expression and activity. Our results provide evidence of increased nNOS-derived NO in the failing human heart. Such altered regulation may be important in the pathophysiology of cardiac dysfunction in human congestive heart failure.     

Ventricular assist device therapy normalizes inducible nitric oxide synthase expression and reduces cardiomyocyte apoptosis in the failing human heart

OBJECTIVES: We examined the effect of mechanical unloading with ventricular assist device (VAD) therapy on myocardial inducible nitric oxide synthase (iNOS) expression and cardiomyocyte apoptosis in patients with end-stage heart failure (HF). BACKGROUND: Despite advances in medical therapy, HF continues to be a progressive and ultimately fatal disorder. High levels of iNOS expression are present in the myocardium of failing hearts, suggesting a potential role for iNOS in HF progression. METHODS: Inducible NOS protein expression was analyzed by Western blotting and cardiomyocyte apoptosis by terminal deoxynucleotidyltransferase dUTP nick end-labeling (TUNEL) in myocardial samples from failing hearts. Included in these analyses were tissues from 9 patients at the time of transplantation (HF-transplant group), 10 patients at the time of VAD insertion (pre-VAD group), and 11 patients undergoing transplant after VAD support (post-VAD group). Seven control samples were obtained at autopsy. RESULTS: Low or undetectable levels of iNOS were present in controls (0.005 +/- 0.002). The HF-transplant and pre-VAD myocardial specimens exhibited a marked increase in iNOS expression (1.48 +/- 0.34 and 1.29 +/- 0.26, respectively; p < 0.01 for both vs. controls). The increase in iNOS expression was reversed in the post-VAD group (0.36 +/- 0.16; p < 0.01 vs. HF-transplant and pre-VAD groups). The rate of TUNEL-positive cardiomyocytes was high in the pre-VAD group and significantly lower in the post-VAD group (0.64 +/- 0.15% in pre-VAD group and 0.16 +/- 0.07% in post-VAD group; p < 0.01). The iNOS levels correlated significantly with cardiomyocyte apoptosis (r = 0.66, p < 0.01). CONCLUSIONS: Therapy with VAD normalizes iNOS expression in association with diminished cardiomyocyte apoptosis in the failing heart. Further work is required to define whether a causal relationship exists between iNOS and cardiomyocyte apoptosis.     

Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure

Inducible nitric oxide synthase (iNOS) protein is expressed in cardiac myocytes of patients and experimental animals with congestive heart failure (CHF). Here we show that iNOS expression plays a role in pressure overload-induced myocardial chamber dilation and hypertrophy. In wild-type mice, chronic transverse aortic constriction (TAC) resulted in myocardial iNOS expression, cardiac hypertrophy, ventricular dilation and dysfunction, and fibrosis, whereas iNOS-deficient mice displayed much less hypertrophy, dilation, fibrosis, and dysfunction. Consistent with these findings, TAC resulted in marked increases of myocardial atrial natriuretic peptide 4-hydroxy-2-nonenal (a marker of lipid peroxidation) and nitrotyrosine (a marker for peroxynitrite) in wild-type mice but not in iNOS-deficient mice. In response to TAC, myocardial endothelial NO synthase and iNOS was expressed as both monomer and dimer in wild-type mice, and this was associated with increased reactive oxygen species production, suggesting that iNOS monomer was a source for the increased oxidative stress. Moreover, systolic overload-induced Akt, mammalian target of rapamycin, and ribosomal protein S6 activation was significantly attenuated in iNOS-deficient mice. Furthermore, selective iNOS inhibition with 1400W (6 mg/kg per hour) significantly attenuated TAC induced myocardial hypertrophy and pulmonary congestion. These data implicate iNOS in the maladaptative response to systolic overload and suggest that selective iNOS inhibition or attenuation of iNOS monomer content might be effective for treatment of systolic overload-induced cardiac dysfunction.     

一氧化氮和一氧化氮合酶与肿瘤放疗敏感性的关系

一氧化氮(nitricoxide,NO)的生物学作用具有复杂性和多样性,在基础条件下诱导型一氧化氮合酶(induciblenitricoxidesynthase,iNOS)活性很低,当机体遭受微生物内外毒素、炎症介质等刺激时iNOS可诱导合成大量的NO.肿瘤生物学上一般认为高水平的NO对肿瘤细胞具有直接的细胞毒作用,而较低水平的NO具有生长刺激作用.多种试验显示NO的供体能增加肿瘤的放疗敏感性.研究认为,NO的生物学作用可能是通过p53依赖途径介导的.调节NO杀灭肿瘤或促进肿瘤生长,p53起到关键性的作用.已有多种药品作为放射敏化剂,NO供体药物在体内给药可能导致系统低血压,增加肿瘤血液灌注和氧合作用,具有潜在的促进肿瘤生长的作用,限制了其临床使用.直接将iNOS基因转染入肿瘤细胞内,肿瘤内的乏氧环境,可降低iNOS的活性而影响NO的产量.携带iNOS基因的腺病毒(adenoviralvectorcarryingtheiNOScDNA,AdiNOS)转染靶细胞导致iNOS过表达,产生大量NO,有望成为一种增加肿瘤放疗敏感性有效可行的方法.     

Atopic sensitization to common allergens without symptoms or signs of airway disorders does not increase exhaled nitric oxide

Background: Elevated fractional exhaled nitric oxide (FENO) associates positively with symptomatic atopy among asthmatics and in the general population. It is, however, unclear whether sensitization to common allergens per se– as verified with positive skin prick tests – affects FENO in healthy individuals. Objective: The aim of this study was to examine the association between FENO and sensitization to common allergens in healthy nonsmoking adults with no signs or symptoms of airway disorders. Methods: FENO measurements (flow rate: 50 mL/s), skin prick tests to common inhalant allergens, structured interviews, spirometry, bronchodilatation tests and bronchial histamine challenges were performed on a randomly selected population of 248 subjects. Seventy‐three of them (29%) were nonsmoking asymptomatic adults with no history of asthma, persistent or recurrent upper or lower airway symptoms and no signs of airway disorders in the tests listed above. Results: FENO concentrations were similar in skin prick test positive (n = 32) and negative (n = 41) healthy subjects, with median values of 13.2 and 15.5 ppb, respectively (P = 0.304). No correlation appeared between FENO and the number of positive reactions (r = ?0.138; P = 0.244), or the total sum of wheal diameters (r = ?0.135; P = 0.254). The nonparametric one‐tailed 95% upper limits of FENO among skin prick positive and negative healthy nonsmoking subjects were 29 and 31 ppb, respectively. Conclusions: Atopic constitution defined as positive skin prick test results does not increase FENO in healthy nonsmoking adults with no signs or symptoms of airway disorders. This suggests that same reference ranges for FENO can be applied to both skin prick test positive and negative subjects. Please cite this paper as: Rouhos A, Kainu A, Karjalainen J, Lindqvist A, Piirilä P, Sarna S, Haahtela T and Sovijärvi ARA. Atopic sensitization to common allergens without symptoms or signs of airway disorders does not increase exhaled nitric oxide. The Clinical Respiratory Journal 2008; 2: 141–148.     

Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.     

L-arginine availability is not limiting for nitric oxide generation from recombinant endothelial nitric oxide synthase

L-arginine (L-Arg) may be limiting for inducible nitric oxide synthase (NOS) activity and under certain circumstances, such as increased concentrations of a NOS inhibitor, may also be limiting for endothelial NOS activity. It is unknown if L-Arg is limiting for recombinant eNOS activity in the vascular wall after adenoviral mediated gene transfer. Our aim was to examine, if L-Arg is limiting for recombinant eNOS activity in the normal or atherosclerotic vessel wall. Rings of rabbit aorta from chow or cholesterol fed animals were transduced with adenovirus vector encoding eNOS (AdeNOS) or beta-galactosidase (AdbetaGal). After 24 h, transgene expression was confirmed and vasomotor studies were performed in the absence or presence of L-Arg. During maximal contractions to phenylephrine (10(-5) M), L-Arg (3 mM) was added to the organ chamber for 30 min. Subsequently, relaxations to acetylcholine during half-maximal contractions were obtained. In the chow- and cholesterol-fed animals, relaxations were significantly enhanced in the NOS and NOS + L-Arg groups compared to the betaGal and betaGal + L-Arg groups. There was no difference between NOS and NOS + L-Arg or betaGal and betaGal + L-Arg rings from chow- or cholesterol-fed animals. While gene transfer of eNOS enhances endothelium-dependent vasorelaxation in the normal and atherosclerotic vessel wall, L-arginine is not limiting for recombinant eNOS activity.     

A small amount of inhaled nitric oxide does not increase lung diffusing capacity.

The aim of the present study was to determine: 1) whether 40-50 ppm nitric oxide (NO) increases diffusing capacity of the lung for NO (D(L,NO)) and carbon monoxide (D(L,CO)), membrane diffusing capacity for CO (D(m,CO)) and pulmonary capillary blood volume (V(c)); 2) the actual number of tests required to provide a reasonable estimate of D(L,NO), D(L,CO), D(m,CO) and V(c); and 3) repeatability of these parameters using the single-breath D(L,NO)-D(L,CO) method. In total, 31 subjects performed five single-breath hold manoeuvres at rest, inhaling 43+/-3 ppm NO together with a standard diffusion mixture. D(L,NO) (D(m,CO)) remained unchanged from the first to fifth trial. However, compared with the first trial, D(L,CO) and V(c) had decreased by the fourth (-4+/-5%; 95% confidence interval (CI) = -5- -2%) and third trial (-5+/-7%; 95% CI = -7- -2%), respectively. Repeatability over five trials was 17, 3 and 7 mL.min(-1).mmHg(-1) for D(L,NO), D(L,CO) and D(m,CO), respectively, and 13 mL for V(c) when D(m,CO) = D(L,NO)/2.42. In conclusion, nitric oxide inhaled during sequential single-breath manoeuvres has no effect on diffusing capacity of the lung for nitric oxide and, thus, membrane diffusing capacity for carbon monoxide. Since more than two and three trials will lower pulmonary capillary blood volume and diffusing capacity of the lung for carbon monoxide, respectively, the average value of only two properly performed trials is suggested.     

An inhibitor of inducible nitric oxide synthase decreases forearm blood flow in patients with congestive heart failure.

OBJECTIVES: The functional activation of inducible nitric oxide synthase (iNOS) was evaluated as a source of nitric oxide (NO) in the forearm of patients with heart failure. BACKGROUND: Although endogenous NO is normally produced by constitutive NO synthase (cNOS) in patients with congestive heart failure (CHF), expression of iNOS provides an additional source of NO. However, there are no in vivo studies showing functional activation of iNOS in humans. METHODS: A nonselective NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), and a selective inhibitor of iNOS, aminoguanidine, were administered intra-arterially in graded doses into the brachial arteries of 13 patients with CHF and 10 normal control subjects. Forearm blood flow (FBF) was measured simultaneously in the infused and noninfused arms by plethysmography. Arterial and venous plasma concentrations of nitrite/nitrate (NOx) were measured at baseline and at the highest dose of each drug. RESULTS: L-NMMA significantly reduced the FBF ratio between the infused and noninfused arms in both the control and patient groups (35 +/- 12% and 34 +/- 10%, respectively; both p < 0.001). Aminoguanidine at the same concentration significantly reduced the ratio in the patient group (15 +/- 9%, p < 0.01), with no change in the control group. The arterial NOx concentration was not affected by either drug; however, venous NOx concentrations were significantly decreased in both the control and patient groups by L-NMMA (18 +/- 5% and 18 +/- 17%, respectively; both p < 0.05) and in the patient group only by aminoguanidine (7 +/- 6%, p < 0.05). CONCLUSIONS: These findings suggest that NO production in the forearms of patients with CHF is induced partly by iNOS activation, whereas in normal subjects, it can be ascribed to cNOS activation.     

Cellular basis of abnormal calcium transients of failing human ventricular myocytes

Depressed contractility is a central feature of the failing human heart and has been attributed to altered [Ca2+]i. This study examined the respective roles of the L-type Ca2+ current (ICa), SR Ca2+ uptake, storage and release, Ca2+ transport via the Na+-Ca2+ exchanger (NCX), and Ca2+ buffering in the altered Ca2+ transients of failing human ventricular myocytes. Electrophysiological techniques were used to measure and control V(m) and measure I(m), respectively, and Fluo-3 was used to measure [Ca2+]i in myocytes from nonfailing (NF) and failing (F) human hearts. Ca2+ transients from F myocytes were significantly smaller and decayed more slowly than those from NF hearts. Ca2+ uptake rates by the SR and the amount of Ca2+ stored in the SR were significantly reduced in F myocytes. There were no significant changes in the rate of Ca2+ removal from F myocytes by the NCX, in the density of NCX current as a function of [Ca2+]i, ICa density, or cellular Ca2+ buffering. However, Ca2+ influx during the late portions of the action potential seems able to elevate [Ca2+]i in F but not in NF myocytes. A reduction in the rate of net Ca2+ uptake by the SR slows the decay of the Ca2+ transient and reduces SR Ca2+ stores. This leads to reduced SR Ca2+ release, which induces additional Ca2+ influx during the plateau phase of the action potential, further slowing the decay of the Ca2+ transient. These changes can explain the defective Ca2+ transients of the failing human ventricular myocyte.     

Imbalance between xanthine oxidase and nitric oxide synthase signaling pathways underlies mechanoenergetic uncoupling in the failing heart

Inhibition of xanthine oxidase (XO) in failing hearts improves cardiac efficiency by an unknown mechanism. We hypothesized that this energetic effect is due to reduced oxidative stress and critically depends on nitric oxide synthase (NOS) activity, reflecting a balance between generation of nitric oxide (NO) and reactive oxygen species. In dogs with pacing-induced heart failure (HF), ascorbate (1000 mg) mimicked the beneficial energetic effects of allopurinol, increasing both contractility and efficiency, suggesting an antioxidant mechanism. Allopurinol had no additive effect beyond that of ascorbate. Crosstalk between XO and NOS signaling was assessed. NOS inhibition with N(G)-monomethyl-L-arginine (L-NMMA; 20 mg/kg) had no effect on basal contractility or efficiency in HF, but prevented the +26.2+/-3.5% and +66.5+/-17% enhancements of contractility and efficiency, respectively, observed with allopurinol alone. Similarly, improvements in contractility and energetics due to ascorbate were also inhibited by L-NMMA. Because of the observed NOS-XO crosstalk, we predicted that in normal hearts NOS inhibition would uncover a depression of energetics caused by XO activity. In normal conscious dogs, L-NMMA increased myocardial oxygen consumption (MVO2) while lowering left ventricular external work, reducing efficiency by 31.1+/-3.8% (P<0.005). Lowered efficiency was reversed by XO inhibition (allopurinol, 200 mg) or by ascorbate without affecting cardiac load or systemic hemodynamics. Single-cell immunofluorescence detected XO protein in cardiac myocytes that was enhanced in HF, consistent with autocrine signaling. These data show that both NOS and XO signaling systems participate in the regulation of myocardial mechanical efficiency and that upregulation of XO relative to NOS contributes to mechanoenergetic uncoupling in heart failure.     

Inhibition of neuronal nitric oxide synthase activity does not alter vasopressin secretion in septic rats

Background/Purpose

During the early phase of sepsis, hypotension is accompanied by increase of plasma vasopressin hormone (AVP) levels, which decline during the late phase. This hypotension is due in part to increase of nitric oxide (NO) synthesis by nitric oxide synthase (NOS) enzyme. Neuronal isoform of this enzyme (nNOS) is present in vasopressinergics neurons of hypothalamus, but its role in vasopressin secretion during sepsis is unknown.

Methods

We evaluated the role of nNOS in NO production and vasopressin secretion during sepsis. Wistar rats received 7-nitroindazole (50 mg/kg, i.p.), an inhibitor of nNOS activity, or vehicle and were submitted to septic stimulus by cecal ligation and puncture (CLP). At the time points 0, 4, 6, 18 and 24 h after sepsis induction the animals were decapitated and neurohypophysis and hypothalamus were removed for analysis of vasopressin content and NOS activity, respectively. Hematocrit, serum sodium, osmolality, proteins and plasmatic AVP were quantified.

Results

Mortality was not affected by 7-nitroindazole (7-NI). Sodium and plasma proteins levels decreased after CLP and the treatment anticipated the protein loss, and delayed serum sodium decrease. Septic animals treated with 7-NI showed decrease of osmolality 4 h after CLP. Nitric oxide synthase activity in hypothalamus increased at 4 and 24 h after CLP and was reduced with 7-NI. Neurohypophysis content of AVP diminished after CLP and 7-NI did not alter this parameter. Plasma AVP levels increased at 6 h and decreased 18 and 24 h after CLP. Treatment with 7-NI did not alter plasma vasopressin levels.

Conclusion

We concluded that nNOS does not have a substantial role in vasopressin secretion during experimental sepsis.