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.     

Expression of inducible nitric oxide synthase in human burn wounds

A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein alpha-sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of alpha-sarcin in its interaction with lipid membranes.     

Evolution of a homopurine-homopyrimidine pentanucleotide repeat sequence upstream of the human inducible nitric oxide synthase gene

The breakdown of membrane phospholipids and subsequent arachidonic acid metabolism to prostanoids is a well-documented brain response to cerebral ischemia. To further elucidate the components of this signal transduction pathway, immunocytochemistry was used to determine the levels of two potentially important enzymes, cytosolic phospholipase A2 (cPLA2) and prostaglandin H synthase-2 (PGHS-2), in the immature rat brain following moderate unilateral hypoxic-ischemia (HI). The CA1 pyramidal cells of the hippocampus which undergo delayed neuronal death on the injured side following HI demonstrated a significant induction of PGHS-2 immunoreactivity 48 h post-insult. However, a consistent increase in PGHS-2 was also evident in the resistant dentate granule cells at an earlier time point. Although PGHS-2 is present in both susceptible and resistant cell populations following HI, the possibility remains that divergence further down-stream in the pathway is responsible for selective vulnerability. In contrast to the neuronal PGHS-2 expression, cPLA2 immunoreactivity appears to be of glial origin with increases in and around the CAI-2 pyramidal cell layer at the 72-168-h time points. These results suggest that prostanoids are likely to serve important roles in HI brain damage and repair in infant brain.     

Selective expression of inducible nitric oxide synthase in human prostate carcinoma

BACKGROUND: Nitric oxide (NO) is synthesized by inducible nitric oxide synthase (iNOS) and plays an important role in tumor growth and angiogenesis. NO generation by iNOS also influences the cytotoxicity of macrophages and tumor-induced immunosuppression. Before now, the expression of iNOS in prostate carcinoma tissue had not been determined. METHODS: In this study, tissue sections from 16 patients with prostate carcinoma were studied immunohistochemically and compared with tissue specimens from 10 patients with benign hyperplasia. RESULTS: Positive iNOS immunostaining was detected in all sections from patients with prostate carcinoma. The malignant epithelial cells were highly positive. The antibody against iNOS also marked round cells, which had the same cell shape as that observed for macrophages. These cells were located in stroma and epithelium adjacent to tumor islets. However, round cells in benign tissue stained negative for iNOS. None of the benign hyperplasia specimens stained positive for iNOS immunohistochemically. CONCLUSIONS: Prostate carcinoma tissue had a high iNOS content, whereas benign tissue did not. The authors suggest that epithelial iNOS expression can be used as a specific immunohistochemical marker for prostate carcinoma. NO generation by iNOS may play multiple roles in the development of this disease.     

Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis

The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.     

Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle

Nitric oxide (NO) is a pluripotent molecule that can be secreted by skeletal muscle through the activity of the neuronal constitutive isoform of NO synthase. To determine whether skeletal muscle and diaphragm might also express the macrophage-inducible form of NO synthase (iNOS) during provocative states, we examined tissue from mice at serial times after intravenous administration of Escherichia coli endotoxin. In these studies, iNOS mRNA was strongly expressed in the diaphragm and skeletal muscle of mice 4 h after intravenous endotoxin and was significantly diminished by 8 h after challenge. Induction of iNOS mRNA was followed by expression of iNOS immunoreactive protein on Western immunoblots. Increased iNOS activity was demonstrated by conversion of arginine to citrulline. Immunochemical analysis of diaphragmatic explants exposed to endotoxin in vitro revealed specific iNOS staining in myocytes, in addition to macrophages and endothelium. These results may be important in understanding the pathogenesis of respiratory pump failure during septic shock, as well as skeletal muscle injury during inflammation or metabolic stress.     

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.     

2-Iminopyrrolidines as potent and selective inhibitors of human inducible nitric oxide synthase

A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase (hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the 2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition of hiNOS (IC50 = 0.25 microM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.     

Failure of L-NAME to cause inhibition of nitric oxide synthesis: role of inducible nitric oxide synthase

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1-1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p < 0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.     

Chlamydial infection in inducible nitric oxide synthase knockout mice

Type 1 CD4+-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4+-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-gamma)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS-/- mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS+/+ and iNOS-/- mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS-/- than in iNOS+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS-/- mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS-/- mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-gamma during CD4+-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-gamma. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.     

Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells

BACKGROUND: Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS: VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS: These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.     

Impact of surgery on nitric oxide in rats: evidence for activation of inducible nitric oxide synthase

We investigated the effect of euvolemic surgical preparation, on chemical indices of activity of the nitric oxide (NO) system, in anesthetized, acutely prepared rats. The urinary excretion of NO2+NO3 (UNOXV) and cGMP (UcGMPV) increased progressively during the experiment. Pretreatment with aminoguanidine or dexamethasone, inhibitors of inducible NO synthase (iNOS), prevented the increase in UNOXV and UcGMPV but had no impact on mean arterial pressure (BP), renal vascular resistance (RVR) or GFR. Since these variables did not change in the conscious rat, the increased UNOXV results from some aspect of the acute surgical preparation. When acutely prepared rats received L-NAME, a non-specific NOS inhibitor, BP and RVR increased but paradoxical increases in UNOXV and UcGMPV were also seen. Nonselective NOS inhibition (+L-NAME) was fatal in 50% of acutely prepared rats, causing cardiac contracture. The same dose of L-NAME produced no deaths in either conscious chronically catheterized rats or in acutely prepared rats, previously subjected to sterile surgery and acute L-NAME in the conscious state. These data indicate that acute, nonsterile surgery induces expression of iNOS, but that the additional NO generated has no obvious cardiovascular/renal actions. Acute UNOXV and UcGMPV do not predict total NO production, or "hemodynamically active" NO. Generalized NO inhibition in rats acutely stressed by surgery/anesthesia can be fatal.