Increased iNOS Activity Research Articles

Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes.

To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes. Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease. Chondrocytes were isolated, plated at a high density (10(5) cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination with bacterial lipopolysaccharide (LPS) or recombinant human interleukin-1alpha (rhIL-1alpha). Nitric oxide release was measured indirectly by use of the Griess reaction and was expressed as micromol of nitrite in the supernatant/microg of protein in the cell layer. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the conversion of radiolabeled arginine to radiolabeled citrulline. Treatment of chondrocytes with adenosine alone had no significant effect on NO release. However, adenosine and NECA inhibited LPS- and rhIL-1alpha-induced NO release. This response was mimicked by forskolin, which acts to increase adenylate cyclase activity, but not by the calcium ionophore A23187 Treatment of chondrocytes with phorbol myristate acetate, which acts to increase protein kinase C activity, potentiated LPS-induced NO release. Adenosine treatment also significantly inhibited the LPS-induced increase in iNOS activity. Adenosine and the nonspecific adenosine receptor agonist NECA inhibited inflammatory mediator-induced release of NO from equine articular chondrocytes. Modulation of adenosine receptor-mediated pathways may offer novel methods for treatment of inflammation in horses with joint disease.

The Protective Role of Endogenous Estrogens in Carrageenan-Induced Lung Injury in the Rat

We have recently demonstrated that 17beta-estradiol (E2) inhibits the increase of inducible nitric oxide synthetase (iNOS) activity in selected model systems such as macrophages, microglia, smooth muscle cells, and proposed that this effect might be associated with an anti-inflammatory activity of this hormone. Here we investigate the effects of endogenous estrogens in rats subjected to carrageenan-induced pleurisy. Adult female rats were ovariectomized 3 weeks before the experiments to deplete circulating estrogens. Selected inflammatory markers, landmarks of the delayed phase of carrageenan-induced pleurisy, were measured in intact (N-OVX), and ovariectomized (OVX) female rats. In addition, the effect of hormone replacement was evaluated in ovariectomized rats with intraperitoneal injection of 17beta-estradiol (E2; 50 microg/kg) 1 hr before carrageenan treatment (OVX + E2). Ovariectomy enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and lipid peroxidation were significantly increased in estrogens-deprived rats. The iNOS in lung samples was significantly increased by the surgery. The increase of iNOS activity was correlated with a marked enhancement in the production of TNF-alpha and IL-1beta. Immunohistochemical analysis for P-selectin and ICAM-I, as well as nitrotyrosine and poly (ADP-ribose) synthetase (PARS) revealed a positive staining in lungs from carrageenan-treated rats, which was markedly enhanced in ovariectomized rats when compared to cycling rats, particularly in the estrous phase of the cycle. Estrogen replacement counteracted the effect of surgery on all of the above indicators of lung inflammation, suggesting that in the cycling rat this hormone plays a key role in the increased sensitivity to inflammatory injury observed in the OVX rat. This study demonstrates that endogenous estrogens production plays an important protective role against carrageenan-induced acute inflammation by decreasing the expression of specific markers of the delayed phase of this well-known model of acute inflammation.

INOS and nitrotyrosine expression after spinal cord injury.

Secondary tissue damage after spinal cord injury (SCI) may be due to inflammatory mediators. After SCI, the nuclear factor-kappaB (NF-kappaB) transcription factor can activate many pro-inflammatory genes, one of which is inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a key inflammatory mediator, which in turn reacts with superoxide to generate peroxynitrite. Peroxynitrite is a strong oxidant that can damage cellular enzymes, membranes, and subcellular organelles through the nitration of tyrosine residues on proteins. The presence of nitrotyrosine (NT) is an indirect chemical indicator of toxic NO and peroxynitrite-induced cellular damage. Using a New York University (NYU) impactor to induce SCI in adult rats, we examined the temporal and cellular expression of iNOS and NT. We observed a progressive increase in iNOS expression in the injured cord starting at day 1 with maximal expression occurring at day 7, as determined by Western blot analysis. iNOS expression corresponded temporally to an increase in iNOS enzyme activity after SCI. In parallel with the progressive increase in iNOS activity, NT expression also increased with time after SCI. The iNOS and NT immunoreactivity was localized in neurons, astrocytes, endothelial cells and ependymal cells at the epicenter and adjacent to the region of spinal cord impact and injury. Results from the present study suggest that increased iNOS and peroxynitrite anion, as reflected by the progressive accumulation of NT in the injured impacted spinal cord, may contribute to the secondary injury process after SCI.

Effects of nitric oxide synthase blockers on renal function.

Three isoforms of nitric oxide synthase (NOS) [neuronal NOS (bNOS), inducible NOS (iNOS) and endothelial NOS (eNOS)] are expressed in the kidney. The use of pharmacological inhibitors of these enzymes has been a major experimental tool to determine the role of nitric oxide (NO) in renal physiology and pathophysiology. Studies performed in both human and experimental animals demonstrate that NOS blockade increases renal vascular resistances and decreases the glomerular ultrafiltration coefficient. These studies also support the presence of an important interaction between NO, angiotensin and renal nerves in the control of renal function. Renal iNOS activity is significantly increased in various pathophysiological conditions including autoimmune tubulointerstitial nephritis and sepsis. Interestingly, recent evidence suggests that high NO levels secondary to increased iNOS activity may inhibit eNOS activity and through this mechanism lead to renal vasoconstriction and reductions in glomerular filtration rate. The use of NOS blockers has generated a great deal of information on the role of NO in the control of renal function and has also allowed us to begin to understand the high level of complexity of this system.

Time Course of Inducible Nitric Oxide Synthase Activity Following Endotoxin Administration in Dogs

An increased production of nitric oxide (NO) via the inducible isoform of NO synthase (iNOS) has been incriminated in the pathogenesis of septic shock. Since the time course of iNOS activity is not known during endotoxic shock in dogs, we measured iNOS activity, estimated by the rate of conversion of 14C-arginine to 14C-citrulline in the absence of calcium, in the heart, lung, liver, kidney, and gut at 1, 2, 3, 4, and 6 h after a bolus of Escherichia coli endotoxin (2 mg/kg, iv), in the dog. This model, including generous fluid administration, is associated with typical features of human septic shock, including low systemic vascular resistance, altered myocardial function and limited oxygen extraction capability. An increase in iNOS activity was observed at 4 h in the liver (0.24 vs 0.04 mU/mg/min) and at 6 h in the heart (0.26 vs 0.09 mU/mg/min). These findings may contribute to a better delineation of the involvement of NO in endotoxic shock, and to the evaluation of the therapeutic effects of NO inhibitors.

Aberrantly glycosylated IgA molecules downregulate the synthesis and secretion of vascular endothelial growth factor in human mesangial cells

To gain insight into the glomerular capillary repair mechanisms in immunoglobulin A (IgA) nephropathy, we focused on vascular endothelial growth factor (VEGF-A) and nitric oxide (NO). Because abnormal glycosylation of serum IgA has been shown in IgA nephropathy, we examined whether VEGF-A and NO production by mesangial cells (MCs) could be modulated by aberrantly glycosylated (desialylated or degalactosylated) IgA. VEGF-A and NO synthase (NOS) gene expression were examined by reverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot analysis, and VEGF-A peptide, by capture enzyme-linked immunosorbent assay and NOS activity as production of tritium ([3H]) citrulline from [3H] arginine. Semiquantitative densitometric analysis of RT-PCR experiments showed a significant downregulation of VEGF-A messenger RNA (mRNA) in MCs incubated with aberrantly glycosylated IgA. This resulted in decreased release of VEGF-A in culture medium (P < 0.01). NOS activity and inducible NOS (iNOS) mRNA were enhanced by aberrantly glycosylated IgA (both P < 0.01). No modulation of constitutive NOS mRNA was found. The depression of the VEGF-A production induced by aberrantly glycosylated IgA was mediated by NO because it was completely reversed by the NOS inhibitor, N ω-nitro-l -arginine methyl ester. The NO donor, sodium nitroprusside, induced a bimodal modulation of VEGF; although low concentrations (0.0001 nmol/L) increased VEGF-A synthesis, greater concentrations (1,000 nmol/L) depressed it. In conclusion, we report negative control of VEGF-A synthesis in MCs by aberrantly glycosylated IgA, mediated by enhanced iNOS activity. We speculate that both increased iNOS activity and depressed VEGF-A synthesis might have a role in impairing vascular repair and favor sclerosis in IgA nephropathy.

INOS inhibitor 1400W improves microcirculation of denervated cremaster muscle following ischemia/reperfusion

Introduction: Nitric oxide (NO) may play a predominant role in ischemia/reperfusion (I/R) injury, both in microcirculation and function. This study was designed to observe the effects of the selective inducible NO synthase (iNOS) inhibitor, 1400W, on the microcirculation of denervated I/R skeletal muscles. Methods: Under anesthesia, the left cremaster muscles of 96 rats weighing 90 ‐110 g were isolated, opened, denervated, and spread onto a transparent acrylic microscope stage. 1400W was administered subcutaneously 10 min prior to reperfusion. Results: Following 3 hrs of ischemia, the mean blood flow of the muscle measured by using laser Doppler flowmetry was 30.2 6 16% of baseline in controls at 10 min and gradually increased to 67.4 6 32% by the end of 90 min reperfusion. In the 1400W group, mean blood flow reached 76.3 6 14% at 10 min and a maximum of 106.3% 6 18% at 50 min of reperfusion, with a significantly (p , 0.01) greater flow than those in controls at all time points with the exclusion of that at 60 and 90 min. Intravital microscopy examination showed the average diameters of 10 ‐70 mm arteries in controls were between 54% and 62% of baseline at 10 min and gradually increased to a maximum of 79 6 11% in 10 ‐20 mm, 73 6 14% in 21‐ 40 mm, and 85 6 3% in 41‐70 mm arteries at 90 min of reperfusion. In contrast, the diameter of 3 vessel categories in the 1400W group sharply increased to over baseline at 10 min and remained at this level throughout 90 min of observation. There was a significant (p , 0.001) difference in mean vessel diameter between the two groups at every time point. Data analyzed using ANOVA. Conclusions: 1) I/R results in an increase of iNOS activity and/or production in the reperfused tissues. 2) Selective inhibition of iNOS improves microcirculation in the reperfused tissue, thereby reducing I/R injury and improving the ‘no-reflow‘ phenomenon. Our data support an iNOS-mediated mechanism in I/R injury and indicate that the inhibition of iNOS in I/R injured muscle could have potential clinical benefit.

Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-γ-treated RAW 264.7 cells

The roles of PKC in iNOS induction by IFN-γ have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-γ-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-γ-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-γ-treated RAW 264.7 cells. PMA together with IFN-γ increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-γ-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-κB and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-γ in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-κB. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-γ-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-γ-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages.

Nitric oxide synthesis and TNF-α secretion in RAW 264.7 macrophages: Mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-γ (IFN-γ) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-α (TNF-α) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-γ in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-γ, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-γ. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-γ, both FPP fractions stimulated TNF-α secretion. However in non-stimulated macrophages, TNF-α secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-α secretion independently of lipopolysaccharides.

Bifunctional role of protein tyrosine kinases in late preconditioning against myocardial stunning in conscious rabbits.

Although protein tyrosine kinases (PTKs) have been implicated in late preconditioning (PC) against infarction, their role in late PC against stunning is unknown. Furthermore, it is unknown whether PTK signaling is necessary only to trigger late PC on day 1 or also to mediate it on day 2. Thus, conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In the control group (group I, n=7), the recovery of systolic wall thickening after the 6 occlusion/reperfusion cycles was markedly improved on days 2 and 3 compared with day 1, indicating the development of late PC against stunning. Administration of the PTK inhibitor lavendustin-A (LD-A, 1 mg/kg IV) before the first occlusion on day 1 (group II, n=7) completely prevented the late PC effect against stunning on day 2. Late PC against stunning was also abrogated when LD-A was given before the first occlusion on day 2 (group III, n=7); however, in these rabbits, the late PC effect became apparent on day 3, indicating that LD-A itself did not have any delayed deleterious actions on myocardial stunning. In group V (n=5), the sequence of 6 occlusion/reperfusion cycles resulted in a robust increase in the activity of inducible NO synthase (iNOS [assessed as Ca(2+)-independent L-citrulline formation]) and nitrite+nitrate (NO(x)) tissue levels 24 hours later (on day 2), with no concomitant change in Ca(2+)-dependent NO synthase (endothelial NO synthase and/or neuronal NO synthase) activity. Similar results were obtained on day 3 (group VIII, n=6), indicating sustained upregulation of iNOS. Administration of LD-A either on day 1 (group VI, n=5) or on day 2 (group VII, n=6) abrogated the increase in iNOS activity and NO(x) levels on day 2. LD-A had no effect on iNOS activity or NO(x) levels in the absence of PC (group X, n=5). This study demonstrates that in conscious rabbits, PTK activity is necessary not only to trigger late PC against stunning on day 1 but also to mediate the protection on day 2. This investigation also provides the first direct evidence that cardiac iNOS activity is upregulated during the late phase of ischemic PC in rabbits. Furthermore, the data indicate that PTK signaling is essential for the augmentation of iNOS activity and that PTKs modulate this enzyme at two distinct levels: at an early stage on day 1 and at a late stage on day 2. This bifunctional role of PTKs in late PC has broad implications for the signaling mechanisms that underlie the response of the heart to ischemic stress and, possibly, other stresses.

ROLES OF ENTEROBACTERIA, NITRIC OXIDE AND NEUTROPHIL IN PATHOGENESIS OF INDOMETHACIN-INDUCED SMALL INTESTINAL LESIONS IN RATS

Roles of enterobacteria, nitric oxide (NO) and neutrophil in indomethacin-induced small intestinal lesions were examined in rats. Indomethacin (10 mg kg−1), administered s.c. as a single injection, caused haemorrhagic lesions in the small intestine, mostly in the jejunum and ileum. The lesions were first observed 6 h after administration of indomethacin, the severity increasing progressively with time up to 24 h later. Following indomethacin, the enterobacterial numbers, inducible NO synthase (iNOS) activity and NO production in the intestinal mucosa were also increased with time, and changes in the former preceded those in the latter two as well as the occurrence of intestinal damage. Treatment of the animals with both NG- nitro- l -arginine methyl ester (l -NAME) and aminoguanidine prevented intestinal lesions induced by indomethacin, with suppression of NO production. Both dexamethasone and FR167653 (an inhibitor of interleukin-1β/tumour necrosis factor-α production) also reduced the severity of intestinal lesions as well as the increase in iNOS activity following administration of indomethacin. Likewise, the occurrence of intestinal lesions was attenuated by pretreatment of the animals with anti-neutrophil serum (ANS). None of these treatments, however, affect the translocation of enterobacteria in the mucosa. By contrast, ampicillin (an anti-bacterial agent) suppressed the increase in mucosal iNOS activity as well as the enterobacterial numbers invaded in the mucosa and inhibited the occurrence of intestinal lesions after administration of indomethacin. These results strongly suggest that enterobacterial translocation in the mucosa is the first step required for activation of various factors such as iNOS/NO and neutrophils, all involved in the pathogenesis of indomethacin-induced intestinal lesions.pc 1999 Academic Press@p$hr

Modulation of stress-induced gastric mucosal lesions by exogenous L-arginine

L-arginine, a nitric oxide (NO) precursor, can exert both ameriolative and deteriorative effects on gastric mucosal lesions. This study was designed to determine whether exogenous L-arginine modulates stress-induced gastric mucosal lesions through NO production by either constitutive NO synthase (cNOS) or inducible NO synthase (iNOS) in gastric mucosal tissues. In rats subjected to water immersion restraint stress over a 6-hour period, the concentration of gastric mucosal nitrite/nitrate, breakdown products of NO, increased with the development of gastric mucosal lesions and a decrease in cNOS activity and a drastic increase in INOS activity in the gastric mucosal tissue. Preadministration of L-arginine (150 to 600 mg/kg intraperitoneally) attenuated the lesion development with prevention of increases in gastric mucosal nitrite/nitrate concentration and INOS activity. In contrast, postadministration of L-arginine (150 to 600 mg/kg intraperitoneally) enhanced the lesion development with further increase in gastric mucosal nitrite/nitrate concentration. This deteriorative action of postadministration of L-arginine (300 mg/kg intraperitoneally) was prevented by pretreatment with aminoguanidine (100 mg/kg subcutaneously), a selective iNOS inhibitor, with inhibition of increases in gastric mucosal iNOS activity and nitrite/nitrate concentration. These results indicate that preadministered L-arginine protects against water immersion restraint stress-induced gastric mucosal lesions, possibly through restricted NO production by cNOS in gastric mucosal tissues, whereas postadministered L-arginine aggravates the stress-induced gastric mucosal lesions, possibly through excessive NO production by iNOS increasing in gastric mucosal tissues.

Mechanism of adipose tissue iNOS induction in endotoxemia.

The aim of the present study was to investigate the mechanism of adipose tissue inducible nitric oxide synthase (iNOS) induction in endotoxemia. Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) to rats for </=8 h markedly increased iNOS mRNA and protein levels in white and brown adipose tissues. This effect was comparable to or greater than the induction of iNOS in liver, kidney, or skeletal muscle. iNOS activity was also found to be greatly enhanced in both white and brown adipose tissues of LPS-treated rats (an approximately 12- to 20-fold increase). Treatment of cultured 3T3-L1 adipocytes with LPS, tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone failed to induce iNOS activity. However, when used in combination, TNF-alpha, IFN-gamma, and LPS markedly and synergistically increased iNOS activity in these cells. In conclusion, these results suggest that adipose tissue is a major site of iNOS expression in endotoxemia. Our data further indicate that iNOS induction can be reproduced in vitro in cultured adipocytes and that a concerted action of cytokines and endotoxin is needed for maximal activation of the enzyme.

Growth of rat mesangial cells on oxidized extracellular matrix increases inducible nitric oxide synthase activity.

Biochemical modification of extracellular matrix (ECM) proteins can alter the function in overlying cells. We tested the hypothesis that metal-catalyzed oxidation of native ECM and individual matrix proteins modulates the activity of inducible nitric oxide synthase (iNOS) in cultured rat mesangial cells (RMC). Oxidized modification of native ECM resulted in a 32% increase in iNOS activity (P<0.01) without influencing the response to supplemental L-arginine or to the addition of the iNOS inhibitor, L-NAME. Immunoblot analysis indicated that enhanced iNOS activity was not associated with a parallel rise in the cytosolic content of iNOS. Synthesis of type IV collagen was unaffected by growth of RMC on oxidized native ECM. Oxidation of three normal constituents of the mesangial matrix - type IV collagen, laminin, and fibronectin - also stimulated iNOS activity in overlying RMC by 18-32% (P<0.05). Growth of RMC on oxidized type I collagen or Vitrogel had no effect on NO production. We conclude that oxidized modification of the mesangial matrix promotes increased iNOS activity and NO production by mesangial cells. Further work is required to determine whether this response limits glomerular injury or promotes damage to the mesangium in oxygen free radical-mediated diseases such as chronic renal failure, atherosclerosis and diabetes.

Changes in gut mucosal nitric oxide synthase (NOS) activity after thermal injury and its relation with barrier failure.

This study was designed to investigate changes in mucosal NOS activity after burns and its relation to barrier failure. In Experiment 1, female specific pathogen free (SPF) Sprague-Dawley rats underwent 35% total body surface area (TBSA) burn. One to six days after burn, intestinal permeability was determined from the plasma leakage of fluorescein isothiocyanate (FITC)-dextran 4400, intestinal mucosal cNOS and iNOS activity were assayed using Griess' reagent, and the cellular localization of iNOS was examined using immunostaining. In Experiment 2, S-methylisothiourea (SMT) was given (5 mg/kg, i.p. every 12 h) for 2 days to suppress inducible NOS (iNOS) activity after thermal injury. On postburn Day 2, the effect of SMT on gut mucosal NOS activity, intestinal permeability, and barrier function were evaluated. The activity of iNOS increased 24 h after the injury and up to a maximum of twofold on postburn Day 2, and decreased thereafter. The increase in iNOS activity in gut mucosa correlated well with the increase in intestinal permeability, an index for barrier failure (r = .776, p = .0002). Results from iNOS immunostaining showed that changes in mucosal iNOS activity after the burn occurred mainly in the enterocytes rather than in the macrophages. Administration of SMT decreased mucosal iNOS activity, intestinal permeability, and bacterial translocation incidence to mesenteric lymph node concurrently. In conclusion, thermal injury induces intestinal mucosal iNOS, which is principally in the enterocytes. The increased intestinal iNOS activity was closely related to barrier failure. SMT inhibited intestinal mucosal iNOS activity and prevented barrier failure as demonstrated by a decrease in BT occurrence and intestinal permeability.

Induction of iNOS in a rat model of acute colitis.

Induction of colitis by 2,4,6-Trinitrobenzenesulphonic acid (TNB) in the rat is a widely used experimental model of inflammatory bowel disease. Action of TNB as a hapten, induces colitis involving infiltration of colonic mucosa by neutrophils and macrophages and increased production of inflammatory mediators. The aim of the present study was to measure nitric oxide synthase (NOS) activity and characterize relations between inducible NOS (iNOS) activity and other signs of inflammation in TNB-induced colitis. A profound and sustained increase in the activity of iNOS was found in the colon. The activity of NOS in the spleen was also increased, but remained at low levels as compared to those in colon. No increases in plasma nitrite + nitrate concentrations were found suggesting local rather than systemic induction of iNOS. The increase in iNOS activity in the colon was preceded by macroscopic inflammatory lesions, like hyperemia, ulcerations and edema formation as well as neutrophil accumulation in the gastric mucosa and increased circulating concentrations of PGE2 metabolite (PGEM). Concentrations of PGEM in the plasma and myeloperoxidase activity (MPO; marker of neutrophil infiltration) in the gut declined in 48h whereas increased iNOS activity and the macroscopic inflammatory lesions remained over the 72h follow-up period. The results demonstrate increased local iNOS activity in TNB-Induced colitis mimicking the situation in human inflammatory bowel disease.

Hemodialysis hypotension: interaction of inhibitors, iNOS, and the interdialytic period.

Hypotension during hemodialysis in end-stage renal disease (ESRD) not explained by excessive ultrafiltration has been linked to an apparent increase in the synthesis of nitric oxide (NO). The authors tested whether the induction of NO synthase (iNOS) by cytokines or differences in the concentrations of inhibitors of NOS or both could account for variability in the amount of NO synthesized during hemodialysis. Plasma levels of an inhibitor of NOS, asymmetric dimethylarginine (ADMA), L-arginine, the substrate for NOS, the end-products N02+N03, iNOS activity in circulating buffy coat cells, and their interdialytic changes were measured in 10 patients during three treatments. Predialysis (0') levels of ADMA were markedly elevated with a mean of 0.008+/-0.002 micromol/mL of deproteinized plasma, compared to controls where ADMA is present in trace amounts. ADMA levels from 30 minutes to the end of dialysis correlated directly with the drop in blood pressure (BP), with levels being much higher in patients with severe hypotension. Postdialysis ADMA levels correlated directly with the 0' systolic BP and the drop in BP at the next dialysis treatment. NOS activity was detected in two thirds of the predialysis buffy coat samples, and appeared to increase as dialysis progressed. 0' iNOS activity correlated inversely with the 0' BP, but activities did not differ based on percent drop in BP. iNOS activity in the 0' samples correlated inversely with the time since the last dialysis, reflecting the greater accumulation of dialyzable inhibitors of NOS as the interval is prolonged. The interdialytic change in iNOS activity correlated inversely with the drop in BP. The isoform detected immunochemically in the buffy coat samples had an Mr of 130 kDa and was reactive with antihuman iNOS. Thus, iNOS is already induced in the cells of the buffy coat in many intermittently hemodialyzed ESRD subjects, but its expression may be masked by inhibitors. After 60 minutes of dialysis (too brief a time for the de novo induction of iNOS,) the appearance of or increase in iNOS activity suggests that an inhibitor had been removed. Because ADMA levels are associated with higher predialysis systolic BPs that result in a greater severity of hypotension, reduction in ADMA concentrations would appear to play a major role in the resumption of NO synthesis by various isoforms.

Hemodialysis Hypotension: Interaction of Inhibitors, iNOS, and the Interdialytic Period

Hypotension during hemodialysis in end-stage renal disease (ESRD) not explained by excessive ultrafiltration has been linked to an apparent increase in the synthesis of nitric oxide (NO). The authors tested whether the induction of NO synthase (iNOS) by cytokines or differences in the concentrations of inhibitors of NOS or both could account for variability in the amount of NO synthesized during hemodialysis. Plasma levels of an inhibitor of NOS, asymmetric dimethylarginine (ADMA), L-arginine, the substrate for NOS, the end-products N02 + N03, iNOS activity in circulating buffy coat cells, and their interdialytic changes were measured in 10 patients during three treatments. Predialysis (0') levels of ADMA were markedly elevated with a mean of 0.008 ± 0.002 μmol/mL of deproteinized plasma, compared to controls where ADMA is present in trace amounts. ADMA levels from 30 minutes to the end of dialysis correlated directly with the drop in blood pressure (BP), with levels being much higher in patients with severe hypotension. Postdialysis ADMA levels correlated directly with the 0' systolic BP and the drop in BP at the next dialysis treatment. NOS activity was detected in two thirds of the predialysis buffy coat samples, and appeared to increase as dialysis progressed. 0' iNOS activity correlated inversely with the 0' BP, but activities did not differ based on percent drop in BP. iNOS activity in the 0' samples correlated inversely with the time since the last dialysis, reflecting the greater accumulation of dialyzable inhibitors of NOS as the interval is prolonged. The interdialytic change in iNOS activity correlated inversely with the drop in BP. The isoform detected immunochemically in the buffy coat samples had an Mr of 130 kDa and was reactive with antihuman iNOS. Thus, iNOS is already induced in the cells of the buffy coat in many intermittently hemodialyzed ESRD subjects, but its expression may be masked by inhibitors. After 60 minutes of dialysis (too brief a time for the de novo induction of iNOS,) the appearance of or increase in iNOS activity suggests that an inhibitor had been removed. Because ADMA levels are associated with higher predialysis systolic BPs that result in a greater severity of hypotension, reduction in ADMA concentrations would appear to play a major role in the resumption of NO synthesis by various isoforms.

Expression of inducible nitric oxide synthase in human gastric cancer.

Inducible nitric oxide synthase (iNOS) is an enzyme that catalyzes the formation of nitric oxide (NO) from L-arginine. iNOS expression and activity results in the production of high levels of NO[1]. The generation of physiological levels of NO is important for mucosal function and it also exerts a cytoprotective effect on the gastrointestinal mucosa. However, increased iNOS expression has been observed in patients with chronic inflammatory diseases of the gastrointestinal tract, such as ulcerative colitis[2,3], and gastritis[4] and it has been speculated that increased NO may induce DNA damage[5,6] and angiogenesis[7]. Nonetheless, the role of iNOS in human GI neoplasia is largely unkown. Previous studies have demonstrated increased iNOS expression in breast cancer[8,9], and increased iNOS activity and protein levels have been demonstrated in colorectal cancer[10] and adenocarcinoma of the esophagus[11]. However, to date, the role of iNOS in gastric carcinogenesis has not been elucidated.

Role of nitric oxide in inflammation-induced suppression of secretion in a mouse model of acute colitis.

The role of nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) in epithelial transport dysfunction was studied in a model of colitis induced in mice by intrarectal 2,4, 6-trinitrobenzenesulfonic acid in 30% ethanol. Expression of iNOS mRNA was determined by RT-PCR. Electrolyte transport studies were conducted in Ussing chambers in which segments of inflamed colon were incubated with or without the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL). Seven days after the induction of colitis, colonic tissue exhibited increased myeloperoxidase activity compared with saline controls. There was a detectable basal expression of iNOS mRNA, but expression was increased 3.7-fold in inflamed colons. Inflammation also caused an increase in iNOS activity and a concomitant decrease in constitutive NOS activity. In Ussing chamber experiments, there was a decreased response to electrical field stimulation in inflamed tissue, which was partially reversed by pretreatment of the tissue with L-NIL. The short-circuit current response to the muscarinic agonist carbachol was also reduced in inflammation, but this was not reversed by L-NIL. In summary, NO derived from iNOS mediates, in part, inflammation-induced suppression of neurally evoked electrolyte transport.