The role of inducible nitric oxide synthase in cardiac allograft rejection

Rats were exposed to either a footshock stimulus (FS) or emotional stimulus (ES, forced perception of another rat receiving footshocks) during a daily 10-min session for 5 consecutive days. The consequences of FS and ES on their behavioural responsiveness were assessed at different post-stress intervals using a small open-field. FS induced a decrease in ambulation, rearing and sniffing and an increased immobility in the small open field. These effects were present in rats tested immediately after the last session and remained present for at least 15 days. In contrast, ES induced a transient decrease in ambulation and rearing immediately after the last session, but in the period from half an hour until at least 15 days after the stimulus experience, an increase in ambulation, rearing and sniffing was observed. Exposure to one footshock per session for 5 consecutive days or to 10 footshocks in a single session also resulted in a long-lasting reduction in ambulation and sniffing and an increase in immobility. The former regime did not influence the behavioural response of ES rats, but the latter resulted in an increase in ambulation, rearing and sniffing in ES rats. Naloxone (1 mg/kg s.c.) pretreatment antagonized the increased behavioural activity of the ES rats whereas the activity of control and FS animals was not affected, suggesting an involvement of endogenous opioid systems in the behavioural responses observed in ES rats. It is suggested that the behavioural responses of the ES and FS animals are regulated by different mechanisms.     

Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury

The role of nitric oxide (NO) in lung injury remains unclear. Both beneficial and detrimental roles have been proposed. In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury. Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later. Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation. In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells. This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity. By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining. These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice.     

Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

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.     

Endothelin enhances lipopolysaccharide-induced expression of inducible nitric oxide synthase in rat glial cells

Lipopolysaccharide is known to stimulate production of nitrite via expression of inducible nitric oxide (NO) synthase in not only macrophages but also glial cells. We found that in glial cell cultures lipopolysaccharide-stimulated inducible NO synthase expression and nitrite accumulation were synergistically enhanced by pretreatment with endothelin, whereas endothelin itself did not induce these responses. Pretreatment with endothelin-1, endothelin-3, and the selective endothelin type B (ETB) receptor agonist IRL 1620 caused the same effect with similar potencies, suggesting that the synergism was mediated via the endothelin ETB receptor. A protein kinase C inhibitor, calphostin C, suppressed endothelin-3-enhanced inducible NO synthase expression. Pretreatment with either endothelin-3 or phorbol ester enhanced lipopolysaccharide-induced production of tumor necrosis factor-alpha (TNF-alpha). Simultaneous addition of TNF-alpha increased lipopolysaccharide-stimulated inducible NO synthase expression. These results suggest that the increase in inducible NO synthase expression by endothelin was due to the elevated TNF-alpha production via protein kinase C. Our findings present the possibility that endothelin is implicated in neurotoxicity via enhancement of inducible NO synthase expression.     

Alterations in mRNA for inducible and endothelial nitric oxide synthase and plasma nitric oxide with rejection and/or infection of allotransplanted lungs

BACKGROUND: Experiments were designed to determine expression of type II (iNOS) and type III (ecNOS) nitric oxide synthase in lung parenchyma and systemic endothelial cells with rejection and/or infection of single lung allografts. METHODS: After single lung allotransplantation, dogs were maintained on standard triple immunosuppressive therapy for 5 days and then placed into one of three groups. Group I (n=4) was maintained on immunosuppressants, group II (n=7) immunosuppression was withdrawn to allow acute rejection of the allograft, and group III (n=6) infection was induced by bronchoscopic inoculation of Escherichia coli. RESULTS: At postoperative days 7-9, no histological evidence of rejection or infection was observed in transplanted lungs of group I. In lungs of group II, rejection ranged from mild to severe; in lungs of group III, infection was severe. Some animals had both rejection and infection (n=8) and were studied separately. Plasma levels of nitric oxide increased comparably with rejection and/or infection compared to preoperative values. Expression of mRNA for ecNOS decreased significantly in lung parenchyma but not in aortic endothelial cells from dogs of groups II and III. However, expression of mRNA for iNOS increased with both rejection and/or infection in both lung parenchyma and aortic endothelial cells. CONCLUSIONS: iNOS is induced locally within the graft and systemically in aortic endothelial cells with rejection and/or infection of lung allografts. Plasma levels of nitric oxide are elevated with both rejection and infection and may not be useful in the differential diagnosis of these processes after lung transplantation.     

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.     

Apoptosis and expression of inducible nitric oxide synthase are mutually exclusive in renal mesangial cells

Nitric oxide (NO) is a multipurpose messenger molecule, important for blood vessel relaxation, neuronal communication, and antimicrobial activities. The generation of NO from L-arginine is catalyzed by NO synthase (NOS). An inducible form of NOS, iNOS, was first characterized in macrophages and then in many other tissues and cells, including renal mesangial cells. Mesangial cells play a crucial role in the regulation of the glomerular filtration rate as well as in the pathophysiology of certain forms of glomerulonephritis in which mesangial cells and macrophages produce NO in high amounts. Because reports have associated NO production with apoptotic cell death in macrophages and we recently demonstrated NO-mediated apoptosis in mesangial cells, we searched for the relationship between in situ iNOS induction and apoptosis by iNOS immunocytochemistry and terminal desoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. RAW 264.7 macrophages exhibited homogeneous iNOS expression and apoptotic nuclei in the iNOS-containing cells upon stimulation with interferon-gamma and lipopolysaccharide. In contrast, stimulated rat mesangial cells stained heterogeneously for iNOS, depending on cell passage and iNOS-stimulating pathway. Mesangial cells expressing iNOS did not display signs of apoptosis and, vice versa, cells showing characteristic features of apoptosis did not stain for iNOS. Thus, our study suggests that mesangial cells react to stimulation by interleukin-1 and/or cAMP-elevating compounds with mutually exclusive responses, either by expression of iNOS or by undergoing programmed cell death.     

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.     

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.     

Temporal expression of pro-inflammatory cytokines and inducible nitric oxide synthase in experimental acute Chagasic cardiomyopathy

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease resulting from mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Distinguishing features of PMD include pleiotropy and a range of disease severities among patients. Previously, we demonstrated that, when expressed in transfected fibroblasts, many naturally occurring mutant PLP alleles encode proteins that accumulate in the endoplasmic reticulum and are not transported to the cell surface. In the present communication, we show that oligodendrocytes in an animal model of PMD, the msd mouse, accumulate Plp gene products in the perinuclear region and are unable to transport them to the cell surface. Another important aspect of disease in msd mice is oligodendrocyte cell death, which is increased by two- to threefold. We demonstrate in msd mice that this death occurs by apoptosis and show that at the time oligodendrocytes die, they have differentiated, extended processes that frequently contact axons and are expressing myelin structural proteins. Finally, we define a hypothesis that accounts for pathogenesis in most PMD patients and animal models of this disease and, moreover, can be used to develop potential therapeutic strategies for ameliorating the disease phenotype.     

Expression of inducible nitric oxide synthase in the brains of scrapie-infected mice

Thoracic trauma in the elderly population constitutes a major challenge for both thoracic and trauma surgeons as their presentation and outcomes differ from the adult population in addition to their high morbidity and mortality. One hundred and one patients, 60 years of age or older, with thoracic trauma were treated at Dicle University School of Medicine during a 6-year period. Eighty-five per cent were male and 15% were female with a mean age of 64.5 years. The cause of thoracic injury was blunt in 77.2% and penetrating in 22.8% of the patients. Sixty-two patients (61.4%) had isolated thoracic injuries. The median Injury Severity Score (ISS) was 23. The morbidity rate was 23.8%. The mortality rate was 16.8%. Seven of 10 patients (70%) who had an ISS greater than 25 died, whereas six of 24 (25%) patients with an ISS between 17 and 25, and four of 67 (5.9%) patients with an ISS less than 16 died. In the elderly the morbidity and mortality rates were higher for blunt trauma compared with penetrating trauma. For ISS greater than 25 the mortality rate was 71.4% for blunt and 66.6% for penetrating trauma. As the morbidity and mortality rate are significantly higher in the elderly patients the approach to these patients should include recognition of their high risk for morbidity and mortality, especially for those who had an ISS greater than 25.     

Location of an inducible nitric oxide synthase mRNA in the normal kidney

An inducible nitric oxide synthase (iNOS) mRNA was found primarily in the outer medulla of normal rat kidney. Identification of the mRNA was based upon the specificity of the oligonucleotide primers used for PCR amplification, PCR-Southern blot analysis and the nucleic acid sequence of the cloned PCR product. In addition to the outer medulla, glomeruli prepared from normal rat kidney contained significant amounts of an iNOS mRNA. These results suggest that there may be tonic influences in the outer medulla of the normal rat kidney resulting in the "steady-state" presence of an iNOS mRNA. Cortical tubules and the inner medulla were found to contain detectable but lesser amounts of the iNOS mRNA. The outer medulla was microdissected into proximal straight tubule (PST), medullary thick ascending limb (MTAL), medullary collecting duct (MCD) and vasa recta bundle (VRB). The iNOS mRNA was found primarily in the MTAL with minor amounts in the MCD and VRB of normal rat kidney. Animals were injected with lipopolysaccharide (LPS) and sacrificed 24 hours later. Treatment with LPS caused at least a 20-fold increase in the amount of iNOS mRNA in the liver or in macrophages isolated from the peritoneum. Endotoxin treatment led to over a 10-fold increase in iNOS mRNA content in glomeruli and the inner medulla. The iNOS mRNA level of the outer medulla was increased two- to threefold due to LPS treatment.     

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.     

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.     

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.