Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM-1 expression, and neutrophil accumulation during endotoxemia

The roles of nitric oxide derived from either the constitutive endothelial NO synthase (eNOS or NOS3) or the inducible NOS (iNOS or NOS2) in hepatic injury during endotoxemia remain controversial. To investigate this further, rats received a bolus of lipopolysaccharide (LPS) following implantation of osmotic pumps containing one of two nonselective NOS inhibitors (NMA or NAME), one of two inducible NOS inhibitors (NIL or AG), or saline. The inhibitors were infused continuously into the liver via the portal vein. Treatment of LPS-injected rats with NMA and NAME resulted in 106 and 227% increases, respectively, in circulating hepatic enzyme levels compared to LPS-treated control rats. In contrast, infusion of the iNOS-selective inhibitors had no effect on the LPS-induced hepatic necrosis. In rats receiving NAME, LPS induced greater neutrophil infiltration and ICAM-1 expression than in the LPS + saline group, whereas NIL infusion did not. The increased hepatic necrosis and PMN infiltration in the LPS + NAME group was partially prevented by a simultaneous infusion of a liver-selective NO donor. Inhibition of PMN accumulation using an anti-ICAM-1 antibody or by PMN depletion using vinblastine pretreatment, however, did not reverse the increased necrosis with NAME infusion during endotoxemia. In contrast to the assessment for necrosis, increased apoptosis was observed in the livers of LPS-treated rats receiving infusions of either NAME or NIL, but not with LPS alone. These data indicate that NO produced by eNOS may be adequate to prevent necrosis by a mechanism independent of PMN, while induced NO appears to prevent apoptosis.     

Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension

Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1-28 days of hyperoxia (FIO2 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13-16% in distal alveolar vessels of the normal lung to 73-86% and 40-59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Endothelial nitric oxide synthase gene polymorphism and hypertension

Nitric oxide (NO) is produced by endothelial cells and serves as a potent vasodilator. Several lines of evidence have shown that NO plays an important role in the regulation of blood pressure and regional blood flow. Endothelial NO synthase (eNOS) gene polymorphisms exhibit a positive association in myocardial infarction and smoking-dependent risk of coronary artery disease. However, the relationship between eNOS gene polymorphisms and hypertension is controversial. To examine the possible involvement of the eNOS gene in the genetic basis for hypertension, we identified genotypes for 2 eNOS gene polymorphisms in 166 hypertensive and 174 normotensive populations in Aomori prefecture, in northern Japan. The specific genotypes for Glu298Asp missense variant and variable numbers of tandem repeats in intron 4 (eNOS 4b/4a) were isolated using allele specific gene amplification and restriction fragment length polymorphism. The 298Asp variant was significantly correlated to hypertension in these groups (odds ratio, 1.8; 95% confidence interval, 1.1-3.2). The allelic frequencies of 298Asp for Glu298 in hypertensive patients were significantly higher than those in normotensive subjects (0.136 vs 0.083, p < 0.05). However, disequilibrium of eNOS4b/4a was absent between these 2 groups. These results suggest that Glu298Asp is a genetic susceptibility factor for hypertension.     

Altered mRNA expression of the neuronal nitric oxide synthase gene in Hirschsprung''s disease

Deoxyribonuclease I (DNase I) is an actin monomer-sequestering actin binding protein (ABP) that inhibits the rate and extent of actin polymerisation in vitro by forming a high affinity, stoichiometric 1:1 complex. Using capillary zone electrophoresis (CZE), we have studied the interaction between G-actin and DNase I to evaluate the capability of CZE to determine the dissociation constant (K(d) value) for this interaction. We used (i) an uncoated fused-silica capillary and ultraviolet (UV) detection at 214 nm; (ii) a hydrophilic-coated capillary with UV detection at 214 nm; and (iii) a hydrophilic-coated capillary with laser-induced fluorescence (LIF) detection. Using procedure (ii), a K(d) value of approximately 0.03 microM was obtained by simulation of binding data. We conclude that CZE combined with a LIF detector has the capacity to extend the determination of K(d) values from the micromolar range to the nanomolar range. Subsequent determination of K(d) values for other actin-binding proteins should provide information on interactions between the binding sites on actin for these proteins and their spatial relationship.     

Histochemical demonstration of neuronal nitric oxide synthase during development of mouse respiratory tract

Several studies, including histochemical ones, have indicated that nitric oxide (NO) of endothelial origin may be related to the pulmonary vasodilation that occurs at birth. Since no histologic studies have been done of the possible parallel perinatal increase in production of neuronal NO synthase (nNOS) by pulmonary nerve plexuses, we investigated the distribution of nNOS in fetal, neonatal, and adult mouse lung. Lungs from mice aged 13 d gestation to 6 d after birth and lungs of adults were studied through histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity and immunocytochemistry. Both techniques gave almost similar results in relation to time of appearance, distribution, and frequency of neural structures positive for NADPH-d and NOS. NADPH-d staining was also applied to whole mounts of developing and adult tracheae. Staining was found from gestational days 13 to 15 onward in a small portion of the neuronal population. In all stages studied, NADPH-d/NOS staining was found in neuron cell bodies in the hilar region and bronchiolar wall, as well as in neuronal processes. Labeled terminal nerve fibers with varicosities were more frequent in pulmonary blood vessels than in airways. In tracheae, similar NADPH-d/NOS-positive nerve plexuses were found. The presence of nNOS in fetal and neonatal mouse respiratory tract suggests that neurally derived NO must play a role in developing lung physiology. However, because no perinatal increase in the number or intensity of staining of nNOS-positive nerve structures was seen, no apparent relation between neural NO and vasodilation can be established at birth.     

Genetic contribution of the endothelial constitutive nitric oxide synthase gene to plasma nitric oxide levels

Nitric oxide (NO) has an important physiological role in regulating vascular tone and is also relevant to many pathological processes including hypertension and atherosclerosis. Endothelial constitutive nitric oxide synthase (ecNOS) is the key enzyme in determining basal vascular wall NO production. We used a combination of maximum-likelihood-based statistical genetic methods to explore the contributions of the ecNOS gene and other unmeasured genes to basal NO production measured by its metabolites (NOx: nitrite and nitrate) in 428 members of 108 nuclear families. Our initial quantitative genetic analysis estimated that approximately 30% of the variance in fasting NOx levels is due to genes (chi 2(1) = 16.04, P = .000062). Complex segregation analysis detected the effects of both a single locus and residual polygenes on NOx levels, and measured genotype analysis showed that plasma NOx levels in those homozygous for the rare allele (64.9 +/- 7.8 mumol/L) were significantly higher (P = .000242) than those homozygous for the common allele (30.2 +/- 3.1 mumol/L). The results of the variance component linkage analysis were consistent with linkage of a quantitative trait locus in or near the ecNOS gene to variation in plasma NOx levels (P = .0066). While many environmental factors have been shown to alter transiently plasma NOx levels, our study is the first to identify a substantial effect of the ecNOS locus on the variance of plasma NOx, i.e. basal NO production. This finding may be relevant to atherogenesis and NO-related disorders.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

S-2-amino-5-azolylpentanoic acids related to L-ornithine as inhibitors of the isoforms of nitric oxide synthase (NOS)

To characterize the importance of a highly conserved region of the class II beta chain, we introduced an amino acid substitution that is predicted to eliminate a hydrogen bond formed between the class II molecule and peptide. We expressed the mutated beta chain with a wild-type alpha chain in a murine L cell by gene transfection. The mutant class II molecule (81betaH-) assembles normally in the endoplasmic reticulum and transits the Golgi complex. When invariant chain (Ii) is coexpressed with 81betaH-, the class II-Ii complex is degraded in the endosomes. Expression of 81betaH- in the absence of Ii results in a cell surface expressed molecule that is susceptible to proteolysis, a condition reversed by incubation with a peptide known to associate with 81betaH-. We propose that 81betaH- is protease sensitive because it is unable to productively associate with most peptides, including classII-associated invariant chain peptides. This model is supported by our data demonstrating protease sensitivity of peptide-free wild-type I-Ad molecules. Collectively, our results suggest both that the hydrogen bonds formed between the class II molecule and peptide are important for the integrity and stability of the complex, and that empty class II molecules are protease sensitive and degraded in endosomes. One function of DM may be to insure continuous groove occupancy of the class II molecule.     

Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.     

Gadolinium chloride inhibits Kupffer cell nitric oxide synthase (iNOS) induction

Kupffer cells (KC) are the phagocytic macrophages of the liver. The rare earth metal, gadolinium (GdCl3), is a lanthanide, which, after phagocytosis by the KC, has been found to alter various aspects of KC physiology. In this study, we describe for the first time that the in vivo administration of GdCl3 to rats decreases the release of NO by isolated rat KC in response to lipopolysaccharide. Western blot analysis shows decreased expression of both inducible nitric oxide synthase as well as total cellular calmodulin after GdCl3 treatment. Possible mechanisms for this phenomenon are suggested.     

Non-enzymatic production of nitric oxide (NO) from NO synthase inhibitors

In an attempt to evaluate the role of thrombopoietin (TPO) in the pathobiology of aplastic anaemia (AA), we have examined TPO levels in sera from 54 AA patients and 119 healthy controls. A total of 92 samples were collected from AA patients: 43 samples were harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples when patients were in partial (n=10) or complete remission (n=16) following immunosuppressive treatment. TPO serum levels were assessed by a sandwich-antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal. Serum samples from normal donors revealed a mean TPO level of 95.3 +/- 54.0 pg/ml (standard deviation). Mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728 +/- 1074 pg/ml (P<0.001 compared to normal controls: mean platelet count at that time: 27x10(9)/l). TPO serum levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P<0.001). However, despite normal platelet counts (mean 167x10(9)/l), TPO levels remained significantly elevated in complete remission (mean TPO 1009 +/- 590 pg/ml, P<0.001 compared to normal controls). There was a significant inverse correlation between serum TPO levels and platelet counts in AA patients who were not transfused for at least 2 weeks prior to sample collection (coefficient of correlation (r) = -0.70, P<0.0001). In summary, TPO levels were highly elevated in sera of patients with AA. Thus there is no evidence to suggest an impaired TPO response contributing to thrombocytopenia in AA. Thrombopoietin did not return to normal levels in remission, indicating a persisting haemopoietic defect in remission of AA. We hypothesize that elevated levels of TPO may be required to maintain normal or near normal platelet counts in remission of AA.     

Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the nos caveolin binding domain in vivo

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.     

Regulation of inducible nitric oxide synthase (iNOS) and GTP cyclohydrolase I (GTP-CH I) gene expression by ox-LDL in rat vascular smooth muscle cells

The generation of nitric oxide is regulated by several factors, including the substrates and cofactors supplementation. Decreased expression and activity of nitric oxide synthase as well as diminished amount of L-arginine or enzyme cofactors results in the inhibition of nitric oxide generation in vascular wall cells. GTP cyclohydrolase 1 is a key enzyme involved in the synthesis of tetrahydrobiopterin, one of the most important cofactors of NO synthases. We have demonstrated that oxidized LDL inhibit not only inducible nitric oxide synthase gene expression but also GTP cyclohydrolase I gene expression in interleukin-1 beta activated rat vascular smooth muscle cells in vitro. It is postulated that diminished availability of tetrahydrobiopterin may additionally impair the generation of nitric oxide in atherosclerosis.     

An unprocessed pseudogene of inducible nitric oxide synthase gene in human

Over 4.5 years, 32 patients with spinal epidural metastases were decompressed and stabilized. Median survival was 9.5 months. Myelopathy was the predominant indication (41%) for the operation, intractable pain (microinstability) the second most important. The type of tumor spreading and biomechanics necessitated ventral decompression and stabilization in 65%. Corporectomy or extensive laminectomy was always combined with internal fixation and bone cement. With the exception of six patients (5 early deaths), all patients were able to walk after surgery. The Karnofsky index was improved significantly from 35 to 66%. The longest survival time was found in breast carcinomas and myelomas. Preoperative radiological embolization was a keystone in the treatment. Indication for surgery in spinal metastases is critical and needs an interdisciplinary approach. When the patient is suffering from higher degrees of paresis or even paralysis, he/she is no longer an ideal candidate for the operation. The same applies in the presence of uncontrolled primary tumors and neoplastic disease of the GI tract and the bronchus.     

Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes

BACKGROUND: Plasma levels of adrenomedullin are increased in patients with congestive heart failure, but there has been no report concerning the effects of adrenomedullin on the heart. We investigated the effects of adrenomedullin on NO synthase activity in cardiac myocytes. METHODS AND RESULTS: We measured the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes with the Griess reagent. Inducible NO synthase mRNA and protein expression were assayed by Northern and Western blotting, respectively. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite accumulation. Adrenomedullin significantly augmented nitrite production by interleukin-1 beta-stimulated but not by unstimulated cardiac myocytes in a dose-dependent manner (10(-10) to 10(-6) mol/L). The adrenomedullin-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by increased inducible NO synthase mRNA and protein expression. In the presence of dibutyryl cAMP, the interleukin-1 beta-induced nitrite accumulation was increased further, but the stimulatory effect of adrenomedullin on nitrite production was abolished. Adrenomedullin dose-dependently increased intracellular cAMP levels in cardiac myocytes. Addition of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP[8-37] to the culture dose-dependently inhibited both cAMP and NO generation stimulated by adrenomedullin. CONCLUSIONS: These results indicate that adrenomedullin acts on cardiac myocytes and augments NO synthesis in these cells under cytokine-stimulated conditions, at least partially through a cAMP-dependent pathway.     

Depressed renal and vascular nitric oxide synthase expression in cyclosporine-induced hypertension

With the longer life expectancy of the population, calcific aortic stenosis has become a common cardiac problem in the elderly. When patients with moderate to severe aortic stenosis become symptomatic, the prognosis is usually poor in absence of valve replacement and sudden death is a feared complication. It has been hypothesized that malignant ventricular arrhythmias could be responsible for the high incidence of sudden death in symptomatic patients with aortic stenosis. The purpose of this review is to analyze the prevalence, the electrophysiologic mechanisms, and the possible role of ventricular arrhythmias in the development of symptoms and in the outcome of adult subjects with aortic stenosis.