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.     

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 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.     

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.     

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.     

TNF-alpha mediates inducible nitric oxide synthase expression in human neuroblastoma cell line by cisplatin

The human neuroblastoma cell line NB-39-nu expressed inducible nitric oxide synthase (iNOS) mRNA following treatment with a combination of interferon-gamma (IFN-gamma) and cis-diamminedichloroplatinum(II) (cisplatin). The level of iNOS mRNA peaked at 48 hr after treatment, and dexamethasone inhibited the induction of iNOS mRNA expression. Cisplatin induced tumor necrosis factor-alpha (TNF-alpha) mRNA expression, and an anti-TNF-alpha neutralizing antibody inhibited the induction of iNOS expression by a combination of cisplatin and IFN-gamma in NB-39-nu cells. Thus, iNOS expression in NB-39-nu cells by a combination of cisplatin and IFN-gamma involves in the TNF-alpha-mediated signal transduction mechanism.     

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.     

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.     

Endogenous opioids regulate the expression of inducible nitric oxide synthase by splenocytes

In the present study, we tested the hypothesis that lipopolysaccharide (LPS)-induced expression of nitric oxide synthase (iNOS) by splenocytes is modulated through the activation of endogenous opioids in the central nervous system. The initial studies determined the parameters of LPS-induced expression of iNOS by splenocytes. Rats were injected with LPS at doses of 0, 1, 10, 100, and 1000 microg/kg, and measures of both iNOS mRNA and protein showed a dose-dependent increase in expression. In a time course study, rats received 100 microg/kg LPS and were killed at 0, 2, 4, 8, and 16 h postinjection. Both iNOS mRNA and protein expression was detectable at the 2-h time point, with peak expression occurring at 8 h. To evaluate the involvement of endogenous opioids, the opioid receptor antagonist naltrexone was administered at 0, 0.1, 1, or 10 mg/kg s.c. in combination with LPS (100 microg/kg), with a second injection of naltrexone at the same dose 4 h after the injection of LPS. Naltrexone induced a pronounced dose-dependent reduction in iNOS mRNA and protein expression by splenocytes. The modulation of iNOS expression occurs via central opioid receptors as intracerebroventricular administration but not peripheral administration of N-methylnaltrexone, the quaternary form of naltrexone that does not readily cross the blood-brain barrier, reduced the expression of iNOS. For all of the manipulations, nitrite/nitrate levels in the plasma showed effects similar to those for iNOS mRNA and protein. Collectively, these findings indicate that central opioid receptors are involved in the in vivo regulation of splenic nitric oxide production.     

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.     

Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. Arabidopsis thaliana seedlings lacking one of the phytochromes, phyB, have elongated hypocotyls and other tissues, suggesting that they may have an alteration in hormone physiology. We have studied the possibility that phyB mutations affect seedling gibberellin (GA) perception and metabolism by testing the responsiveness of wild-type and phyB seedlings to exogenous GAs. The phyB mutant elongates more than the wild type in response to the same exogenous concentrations of GA3 or GA4, showing that the mutation causes an increase in responsiveness to GAs. Among GAs that we were able to detect, we found no significant difference in endogenous levels between wild-type and phyB mutant seedlings. However, GA4 levels were below our limit of detectability, and the concentration of that active GA could have varied between wild-type and phyB mutant seedlings. These results suggest that, although GAs are required for hypocotyl cell elongation, phyB does not act primarily by changing total seedling GA levels but rather by decreasing seedling responsiveness to GAs.     

Azole antifungals: weak inhibitors of inducible nitric oxide synthase in mouse and human cells

The effect of three azole antifungals on inducible nitric oxide (iNOS) activity in different mouse and human cells was evaluated. The iNOS activity was determined by L-citrulline and nitrite measurement. In the murine macrophage cell line RAW 264.7, in mouse peritoneal macrophages (MPM) and in human colorectal adenocarcinoma cells (DLD-1), iNOS activity could be induced with lipopolysaccharides and cytokines. Under similar conditions, no iNOS induction was found in human monocytes/macrophages. The concentration of itraconazole, ketoconazole or miconazole needed to inhibit iNOS activity by 50% in RAW 264.7 cells, MPM and DLD-1 cells was > or = 10 mumol l-1. This is at least 100 times more than the concentrations of these azole antifungals required to produce a 50% inhibition of yeast growth and ergosterol synthesis of, for example, Candida albicans after the same incubation period. These results show that azole antifungals are weak inhibitors of iNOS in intact cells.     

Enhanced expression of inducible nitric oxide synthase in chronic gastritis with intestinal metaplasia

Twelve Standardbred foals (age 3-6 months), with little previous exposure to parasites, were allocated to 2 groups and put onto pasture with low (Group L) or high (Group H) levels of larval contamination of large strongyles and cyathostomes. After 4 weeks grazing in September, the foals were housed indoors until necropsy 15 weeks later. Foals in Group H became clinically more affected than those of Group L in that they showed loss of vigour, weight gain depression, intermittent soft faeces and inappetence. One foal of Group H had persistent diarrhoea and was subjected to euthanasia 12 weeks after housing. Signs of colic were not observed. Faecal egg counts were significantly higher in Group H than in Group L (P<0.05). At necropsy, the mean number of S. vulgaris and cyathostomes was 20 and 18,000, respectively, in Group L, and 167 and 25,000 in Group H. Routine blood chemistry did not specifically reveal presence of S.vulgaris in pre-patency. A transient neutrophilia and eosinophilia, most prominent in Group H, was seen 2-8 weeks after start of exposure and anaemia was observed later in Group H. Serum albumin and albumin/globulin ratio were reduced, particularly in Group H, and a marked hyperbetaglobulinaemia was observed at 16-20 weeks in Group H. In conclusion, heavy infections with strongyles including S. vulgaris may become established in weaned foals after a brief period on pasture. Infections may be expressed clinically as debilitation, inappetence and intermittent diarrhoea without colic, and the need for control is imperative.     

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.     

Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes

In order to determine the effect of kappa-opioid receptor agonist on the beta1-adrenoceptor stimulation in the heart, the effects of norepinephrine (NE), a beta1-adrenoceptor agonist, on contraction and electrically induced intracellular calcium ([Ca2+]i) transient in the single rat ventricular myocyte pretreated with a kappa-opioid receptor agonist, trans-(+/-)-3, 4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide (U50,488H), at 0.01-1 microM were studied with a video edge tracker method and a spectrofluorometric method using fura-2 as calcium indicator, respectively. NE at 0.01-10 microM augmented both twitch amplitude and electrically induced [Ca2+]i transient dose-dependently, which were abolished by propranolol at 1 microM, a beta-adrenoceptor antagonist. The effects of NE on both contraction and [Ca2+]i transient were attenuated in a dose-dependent manner by U50,488H at 0.01-1 microM, which itself had no effect at all. The maximum response ( Emax) was decreased, while the concentration that produces 50% of the maximum response (EC50) was enhanced, by U50, 488H. The inhibitory effects of U50,488H on beta-adrenoceptor stimulation were completely blocked by pretreatment with norbinaltorphimine, a specific kappa-opioid receptor antagonist at 1 microM, or preincubation with pertussis toxin (PTX) at 200 ng/ml for 6 h. On the other hand, the inhibition on NE-induced augmentation in electrically induced [Ca2+]i transient by U50,488H was not affected by pretreatment with U73122, a specific inhibitor of phospholipase C (PLC), at 10 microM for 30 min. U50,488H attenuated the augmentation of the electrically stimulated [Ca2+]i transient induced by forskolin at 0.1 and 0.5 microM. It did not, however, affect the augmentation of the electrically induced [Ca2+]i transient by N6, 2'-O-dibutyryl adenosine cyclic monophosphate (DB-cAMP). The results suggest that kappa-opioid receptor stimulation by U50,488H at 10(-6 )M or lower may inhibit the effects of beta-adrenoceptor stimulation by acting at a PTX-sensitive G-protein and AC, but not via the phosphoinositol pathway.     

Aspirin and salicylate enhances the induction of inducible nitric oxide synthase in cultured rat smooth muscle cells

Aspirin and sodium salicylate enhance to a similar extent the production of nitric oxide (NO) in cultured smooth muscle cells following stimulation by interleukin-1beta (IL-1beta). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the enhancement of NO production. The failure of added prostaglandin E2 (PGE2) or Thromboxane A2 (TXA2) to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The enhancement of NO production occurs dependent of the effects of these agents on induction of inducible nitric oxide synthase (iNOS) expression by IL-1beta. Aspirin and sodium salicylate enhance the induction of iNOS expression by IL-1beta. We previously reported that pretreatment of vascular smooth muscle cells (VSMCs) with high glucose decreased the response of the cells by IL-1beta, that is, the induction of iNOS expression and NO production. We investigated the effect of aspirin and sodium salicylate on the response by IL-1beta of VSMCs pretreated with high glucose (25 mM). Aspirin and sodium salicylate ameliorate the down-regulation of iNOS expression and the decrease of NO production caused by pretreatment with high glucose (25 mM). These results suggest a possible therapeutic role in atherosclerotic disease and diabetes mellitus for aspirin and sodium salicylate by enhancing the level of iNOS expression and NO production.     

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.