Local Nitric Oxide Production Research Articles

THE MEASUREMENT OF EXHALED NITRIC OXIDE DURING ORTHOTOPIC LIVER TRANSPLANTATION

S122 INTRODUCTION: The concentration of nitric oxide (NO) in exhaled breath has been demonstrated to be at increased levels in patients with severe liver cirrhosis [1]. This prospective study investigates the variation in levels of exhaled NO in patients undergoing orthotopic liver transplantation. METHOD: After IRB approval and informed consent, 8 patients with severe liver cirrhosis undergoing orthotopic liver transplantation had intraoperative levels of exhaled NO monitored. A chemiluminecence analyzer (Sievers Inc. Boulder, Co) was utilized and samples were measured from the elbow connector attaching the breathing circuit to the endotracheal tube. Nasopharyngeal produced NO was excluded from the sample by the endotracheal tube. Peak concentrations of NO were recorded during exhalation. RESULTS: See Table 1.Table 1: Peak concentrations for exhaled nitric oxide in patients during three stages of orthotopic liver transplantation.DISCUSSION: The NO measured in the exhaled breath of patients with severe liver cirrhosis may be the result of an increased production of NO by hepatocytes and Kuppfer cells in the liver, where it is then transported to the lungs as s-nitrosyl hemoglobin and released, or it may have originated from the local production of NO in the lungs [2-4]. Both of these pathways may exist together. If the liver is a significant source of this NO production, then the anhepatic phase of the transplant procedure should result in a reduction of exhaled NO levels. In this preliminary study, the trend seen in some patients corroborates this hypothesis. If this finding is confirmed by further investigation then exhaled NO analysis may provide an early indication of liver graft function.

The Role of Nitric Oxide and NMDA Receptors in the Development of Motor Neuron Dendrites

Nitric oxide (NO) has been implicated in the establishment of precise synaptic connectivity throughout the neuroaxis in several species. To determine the contribution of NO to NMDA receptor-dependent dendritic growth in motor neurons, we administered the NMDA antagonist MK-801 to wild-type mice and neuronal nitric oxide synthase (nNOS) knock-out mice between postnatal days 7 and 14. Compared to saline-treated wild-type animals the number of dendritic bifurcations was significantly reduced in nNOS knock-out animals and MK-801-treated wild-type animals. There was no significant difference in dendritic bifurcation between MK-801-treated wild-type, MK-801-treated nNOS knock-out, and saline-treated nNOS knock-out animals, suggesting that nNOS knock-out and NMDA receptor block had similar effects. The path of the longest dendrite and the number of primary dendrites was the same in all treatment groups, indicating an effect specific to bifurcation. Sholl analysis revealed that differences in bifurcation numbers occurred between 160 and 320 micrometers from the cell body, the distance at which second, third, and fourth order dendrites are most prevalent. Dendrite order analyses confirmed a significant reduction in numbers, but not lengths, of third and fourth order dendrites in nNOS knock-out and drug-treatment groups. Finally, immunohistochemical examination of the developing spinal cord indicated that NMDA receptors and nNOS are colocalized within interneurons surrounding the motor neuron pool. These results support the view that at least part of NMDA receptor-dependent arborization of motor neuron dendrites is mediated by the local production of NO within the developing spinal cord.

Inducible nitric oxide synthase in skeletal muscle of patients with chronic heart failure

Objectives. The expression and localization of inducible nitric oxide (NO) synthase (NOS II) was evaluated as a source of NO which has been shown to affect muscle contraction.Background. Advanced stages of chronic heart failure are associated with systemic activation of cytokines which have been shown to stimulate the expression of NOS II in various cell types, including myocytes. We hypothesized that systemic cytokine activation could lead to expression of NOS II in skeletal muscle of patients with chronic heart failure.Methods. Skeletal muscle specimens were obtained by percutaneous needle biopsy in six normal volunteers and eight patients with heart failure (New York Heart Association class III). Electron microscopy immunocytochemistry (immunogold labeling) with specific anti-NOS antibodies was utilized to elucidate the intracellular localization of NOS II and neuronal NO synthase (NOS I) in myocytes of skeletal muscle. Reverse transcriptase, competitive polymerase chain reaction (PCR) was applied to quantify NOS II mRNA in skeletal muscle.Results. Inducible nitric oxide synthase was readily expressed in the cytosol of skeletal muscle myocytes; NOS I expression was sparse. Polymerase chain reaction results indicated that NOS II gene expression is increased in patients with chronic heart failure.Conclusions. Inducible NO synthase is expressed in human skeletal muscle and its gene expression is increased in patients with severe heart failure. Given the experimental evidence that NO can attenuate contractile performance of skeletal muscle and can mediate muscle wasting, an increased local production of NO in skeletal muscle by NOS II may have important implications for patients with severe heart failure.

Effect of dietary salt on neuronal nitric oxide synthase in the inner medullary collecting duct.

Nitric oxide (NO) derived from neuronal NO synthase (nNOS) in the kidney inner medulla has been implicated in the regulation of arterial blood pressure. The purpose of the present study was to determine the effect of high dietary NaCl on the expression of nNOS in the rat inner medullary collecting duct (IMCD). After 3 days or 3 wk of high (4.0%)-NaCl diet in rats, urinary NO-2/NO-3 excretion significantly increased. In freshly microdissected IMCD, nNOS was readily detected by immunofluorescence with polyclonal antibody, an effect that was completely blocked by neutralization of antibody with immunizing antigen. In rats fed a 4.0% NaCl diet for 3 days, IMCD nNOS mRNA, detected by RT-PCR, did not change from control values (0.3% NaCl, 19.84 +/- 1.57 x 10(3), vs. 4.0% NaCl, 20.44 +/- 3.14 x 10(3) cpm; P = not significant, n = 3). By Western blotting however, nNOS protein expression significantly increased (0.3% NaCl, 0.51 +/- 0.12, vs. 4.0% NaCl, 0.92 +/- 0.14 arbitrary units; P < 0. 05, n = 5). After 3 wk of 4.0% dietary NaCl, expression of nNOS mRNA and protein in IMCD did not differ significantly from control values. In contrast to these data, renal cortical expression of nNOS mRNA and protein was significantly decreased after 4.0% NaCl diet for 3 days. High dietary NaCl had no significant effect on expression of mRNA for inducible NO synthase (iNOS) in IMCD after either 3 days or 3 wk. In summary, our data indicate that nNOS mRNA and protein are expressed in IMCD and that high dietary NaCl differentially regulates nNOS expression in IMCD and cortex. The early increase in nNOS protein in IMCD may contribute to enhanced local production of NO and thereby represent an adaptive response to salt intake.

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.

High Local Production of Nitric Oxide As a Possible Mechanism by Which Rapamycin Prevents Transplant Arteriosclerosis

High Local Production of Nitric Oxide As a Possible Mechanism by Which Rapamycin Prevents Transplant Arteriosclerosis

In vivo formation of electron paramagnetic resonance-detectable nitric oxide and of nitrotyrosine is not impaired during murine leishmaniasis.

Recent studies have provided evidence for a dual role of nitric oxide (NO) during murine leishmaniasis. To explore this problem, we monitored the formation of NO and its derived oxidants during the course of Leishmania amazonensis infection in tissues of susceptible (BALB/c) and relatively resistant (C57BL/6) mice. NO production was detected directly by low-temperature electron paramagnetic resonance spectra of animal tissues. Both mouse strains presented detectable levels of hemoglobin nitrosyl (HbNO) complexes and of heme nitrosyl and iron-dithiol-dinitrosyl complexes in the blood and footpad lesions, respectively. Estimation of the nitrosyl complex levels demonstrated that most of the NO is synthesized in the footpad lesions. In agreement, immunohistochemical analysis of the lesions demonstrated the presence of nitrotyrosine in proteins of macrophage vacuoles and parasites. Since macrophages lack myeloperoxidase, peroxynitrite is likely to be the nitrating NO metabolite produced during the infection. The levels of HbNO complexes in the blood reflected changes occurring during the infection such as those in parasite burden and lesion size. The maximum levels of HbNO complexes detected in the blood of susceptible mice were higher than those of C57BL/6 mice but occurred at late stages of infection and were accompanied by the presence of bacteria in the cutaneous lesions. The results indicate that the local production of NO is an important mechanism for the elimination of parasites if it occurs before the parasite burden becomes too high. From then on, elevated production of NO and derived oxidants aggravates the inflammatory process with the occurrence of a hypoxic environment that may favor secondary infections.

Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature‐induced periodontitis in the rat

1. Excessive production of nitric oxide (NO), and the generation of peroxynitrite have been implicated in various proinflammatory conditions. In the present study, using mercaptoethylguanidine (MEG), a selective inhibitor of iNOS and a peroxynitrite scavenger, we investigated the role of iNOS and peroxynitrite in a rat model of periodontitis. 2. Periodontitis was produced in rat by a ligature of 2/0 braided silk placed around the cervix of the lower left 1st molar. Animals were then divided into two groups: one group of rats was treated with MEG (30 mg kg(-1), i.p., 4 times per day for 8 days), animals in the other group received vehicle. At day 8, the gingivomucosal tissue encircling the mandibular 1st molars was removed on both sides from ligated and sham operated animals for inducible nitric oxide synthase (iNOS) activity assay and for immunocytochemistry with anti-iNOS serum. Plasma extravasation was measured with the Evans blue technique. Alveolar bone loss was measured with a videomicroscopy. 3. Ligation caused a significant, more than 3 fold increase in the gingival iNOS activity, whereas it did not affect iNOS activity on the contralateral side, when compared to sham-operated animals. Immunohistochemical analysis revealed iNOS-positive macrophages, lymphocytes and PMNs in the connective tissue and immunoreactive layers of epithelium on side of the ligature, and only a few iNOS reactive connective tissue cells on the contralateral side [corrected]. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction compared to the contralateral side. MEG treatment significantly reduced the plasma extravasation and bone destruction. 4. The present results demonstrated that ligature-induced periodontitis increases local NO production and that MEG treatment protects against the associated extravasation and bone destruction. Based on the present data, we propose that enhanced formation of NO and peroxynitrite plays a significant role in the pathogenesis of periodontitis.

Hemagglutinating virus of Japan-liposome–mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff

Purpose: Late graft failure is a critical problem, particularly in the presence of poor runoff vessels. Intimal hyperplasia is considered to be the main cause of graft failure. We have already reported that intimal thickening of experimental vein grafts in dogs with poor runoff vessels is more pronounced than that in dogs with normal vessels. We and others also have reported that production of nitric oxide (NO) in the endothelium of canine vein grafts is impaired. In the present study, we asked whether in vivo gene transfer of endothelial cell NO synthase (ecNOS) would inhibit intimal hyperplasia of autogenous vein grafts implanted in limbs with poor distal runoff in dogs. Methods: After exposing femoral veins, the nuclear-targeted lac Z gene, bovine ecNOS cDNA, or control vector plasmid encapsulated in the hemagglutinating virus of Japan-liposomes was infused intraluminally, followed by incubation for 10 minutes at room temperature under a distending pressure of 100 mm Hg. Twenty reversed vein grafts were implanted under normal runoff conditions, and 4 days later these were used to confirm gene transfer to the vein grafts. Twelve reversed vein grafts were implanted under conditions of poor runoff, and 4 weeks after the operation intimal thickening was evident. Results: In vein grafts under normal runoff conditions, lac Z gene transfer exhibited diffuse and frequent X-Gal-positive signals in both medial and adventitial layers 4 days after implantation (n = 3). In case of the ecNOS gene–transferred vein grafts, bovine ecNOS protein was mainly detected in medial smooth muscle cells and adventitial cells 4 days after implantation, determined using immunohistochemical techniques and bovine ecNOS specific antibody (n = 3). In addition, ecNOS-transferred vessels showed intense purple signals by reduced nicotinamide adenine dinucleotide phosphate diaphorase and nitroblue tetrazolium reaction, in both medial and adventitial layers, whereas weak NOS activity was recognized at the adventitial vasa vasorum of the untreated veins or control vector transferred veins (n = 3, respectively). In vein grafts under poor runoff conditions, the intimal thickness at 4 weeks after implantation was significantly reduced by ecNOS gene transfer (n = 4; 90.0 ± 7.6 μm and 1.18 ± 0.07 mm 2) in comparison with buffer-treated vessels (n = 4; 195.8 ± 25.7 μm and 2.62 ± 0.48 mm 2) or vector vehicle–treated vessels (n = 4; 193.0 ± 15.8 μm and 2.65 ± 0.22 mm 2). Conclusions: Our findings show that gene transfer of ecNOS inhibited intimal hyperplasia of canine vein grafts caused by poor runoff conditions, as a result of an increased local production of NO. Thus ecNOS gene transfer warrants further study as a possible approach to prevent late graft failure. (J Vasc Surg 1998;27:135-44.)

Uterine nitric oxide and prostaglandin E during embryonic implantation in non-insulin-dependent diabetic rats.

In the process of embryo implantation in the rat, both nitric oxide and prostaglandins act as vascular and myometrial regulators. The aim of the present work was to evaluate the effect of diabetes on the synthesis of both agents during embryo implantation. In diabetic rats, uterine activity of the enzyme nitric oxide synthase and prostaglandin E production were increased during peri-implantation compared to the control group (P < 0.05 and P < 0.001, respectively). Both parameters showed a prolonged increase in temporal profile during peri-implantation days. Local production of nitric oxide and prostaglandin E in the implantation sites was higher in diabetic rats (P < 0.05), but the intersite:site ratio was similar to that of the control group. On the other hand, the implantation rate and the timing of the beginning of this process were not altered in the diabetic group. These results suggest that the vasoactive modulators of the implantation process, nitric oxide and prostaglandins, are increased in this diabetic pathology, and that this increase is probably functioning as a compensatory mechanism, so as to allow an unaltered rate of embryo implantation in this model.

L-arginine decreases alveolar macrophage proinflammatory monokine production during acute lung injury by a nitric oxide synthase-dependent mechanism.

Recent clinical reports indicate that inhaled nitric oxide (NO) reduces lung parenchymal inflammation during acute lung injury; however, the mechanism of its protective effects remains incompletely understood. We hypothesized that the provision of substrate for local NO production (L-arginine) would reduce alveolar macrophage proinflammatory monokine production during endotoxin (ETX)-induced acute lung injury. Our purposes were to (1) determine alveolar macrophage tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) production after ETX-induced acute lung injury; (2) determine the effect of L-arginine on alveolar macrophage TNFalpha and IL-1beta production in ETX-induced acute lung injury; and (3) determine whether L-arginine's effects on the alveolar macrophage are mediated by NO. Rats received ETX (0.5 mg/kg intraperitoneal (i.p.)) or vehicle, with or without (1) L-arginine supplementation (300 mg/kg i.p.) and (2) nitric oxide synthase inhibition (N(G)-monomethyl-L-arginine, 30 mg/kg i.p.). Four hours later, alveolar macrophage were harvested by bronchoalveolar lavage and incubated at 10(6) cells/mL + 1 microg/mL phorbol myristase acetate for 24 hours. Cell-free supernatants were collected and assayed (enzyme-linked immunosorbent assay) for TNFalpha and IL-1beta. Sublethal ETX increased alveolar macrophage capacity to produce TNFalpha and IL-1beta (p < 0.05, analysis of variance and Bonferroni/Dunn). L-Arginine decreased alveolar macrophage TNFalpha and IL-1beta release during acute lung injury. Concurrent inhibition of nitric oxide synthase abrogated L-arginine's protective effects, suggesting that L-arginine's anti-inflammatory effects are mediated by NO. (1) L-Arginine is an immunomodulating nutritional supplement; (2) L-arginine decreases alveolar macrophage proinflammatory monokine production during ETX-induced acute lung injury by a nitric oxide synthase-dependent mechanism; and (3) the provision of exogenous substrate for local NO production may reduce inflammation during acute lung injury.

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin.

We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5-2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by N(G)-nitro-L-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5-20 mg/kg, i.v.), but not by an inactive enantiomer, N(G)-nitro-D-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1-100 microg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE2 (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 microg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 microg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE2, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin.

ICAM-1 and iNOS Expression Increased in the Skin of Mice after Vaccination with γ-Irradiated Cercariae ofSchistosoma mansoni

Host responses to migrating schistosomula ofSchistosoma mansoniwere compared in the skin of naive, multiply infected, or vaccinated (with γ-irradiated cercariae) mice during the first 72 hr after cercarial penetration. Cellular response to the migrating parasite was minimal in the skin of naive mice for up to 72 hr after infection. In sharp contrast, the multiply infected or vaccinated animals exhibited a marked inflammatory response in the skin as early as 8 hr after cutaneous penetration of the challenge cercariae. This early inflammatory response in the skin of sensitized animals was characterized by a significant increase in the number of infiltrating cells, predominantly mononuclear cells and neutrophils. Increased exudation of serum proteins was also present in the skin of sensitized animals in areas of cercarial challenge. A time course of analyses revealed that mononuclear cell numbers increased significantly in the skin of vaccinated animals as early as 60 min after a challenge infection and continued to be present at a significantly higher level up to 72 hr after challenge. Peak neutrophil responses occurred in the skin at 24 hr (in multiply infected animals) and at 48 hr (in vaccinated animals) after a challenge infection. Along with the massive cellular infiltration there was an increased tissue expression of ICAM-1 and mRNA for iNOS in the skin of sensitized animals. Further analysis showed that in sensitized animals increased ICAM-1 expression was predominantly found on endothelial cells lining dermal capillaries, especially in areas around schistosomular migration and on cells that surrounded schistosomula in the dermis. In naive animals, however, a similar infection did not induce any ICAM-1 expression or iNOS production in the skin. Thus, an ICAM-1-mediated early accumulation of mononuclear cells in the skin and local production of nitric oxide may be important for the initial cutaneous inflammatory/immune responses to migrating schistosomula ofS. mansoniin vaccinated animals. On the contrary, in naive animals a potential parasite-induced suppression of ICAM-1 may play an important role in reducing cellular reaction in the skin and consequently help the parasite evade immune responses in the skin.

Local production of nitric oxide in acute rejection of rat lung allografts

Local production of nitric oxide in acute rejection of rat lung allografts

The role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis

Purpose: The role of inflammatory cytokines in the pathogenesis of necrotizing enterocolitis (NEC) is still undefined. Elevated levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α have been measured in infants with NEC, while elevated levels of nitric oxide (NO) have been reported in newborn infants with clinical sepsis. However, the cellular source of the NO or cytokines is unknown. The authors hypothesized that local intestinal production of NO induced by cytokines may contribute to the pathogenesis of bowel necrosis in NEC by inducing apoptosis (programmed cell death) or necrosis of the enterocytes. We examined the levels of inflammatory cytokines and NO in the intestine of infants undergoing surgical resection for NEC, and the cellular localization of human inducible NO synthase (NOS-2) in the inflamed gut. Methods: We compared 15 patients undergoing bowel resection for NEC, with six infants (of similar age) undergoing intestinal resection for ileal atresia or stricture, meconium peritonitis, intussusception, or cecal perforation (control). Diagnosis of NEC was confirmed histologically. Representative segments of the surgical specimen were examined for messenger RNA (mRNA) for NOS-2 by Northern blotting and in situ hybridization. Cytokine mRNA was measured by polymerase chain reaction (PCR) because mRNA could not be detected by Northern blotting. The site of NO production was determined by in situ hybridization and immunohistochemistry. Apoptosis was measured using in situ DNA strand break extension (TUNEL). Nitrotyrosine immunoreactivity was assessed to determine if NO mediates cellular injury via peroxynitrite formation. Results: Messenger RNA for NOS-2 was detected in nearly all patients with NEC except for one infant who underwent proximal diverting jejunostomy alone, and who did not have histological evidence of NEC at that site. NOS-2 mRNA was detected less frequently in control patients. In situ hybridization and immunohistochemistry showed that the enterocytes were the predominant source of NOS-2 activity in the intestine of NEC patients. Extensive apoptosis was seen in enterocytes in the apical villi of infants with NEC, and correlated with nitrotyrosine staining. NOS-2 activity was markedly diminished at the time of stoma closure, but remained elevated in infants who died from progressive disease. PCR showed variable cytokine mRNA expression in the intestine. Transforming growth factor (TGF)-β expression was nearly identical in NEC and control. However, interferon (IFN)-γ was present in 9 of 10 NEC, but only in one of six control patients. Conclusion: The data show that NO is produced in large quantity by enterocytes in the intestinal wall of infants with NEC and leads to apoptosis of enterocytes in apical villi through peroxynitrite formation.

Nitric oxide decreases lung injury after intestinal ischemia.

After injury to a primary organ, mediators are released into the circulation and may initiate inflammation of remote organs. We hypothesized that the local production of nitric oxide (NO) may act to limit the spread of inflammation to secondarily targeted organs. In anesthetized rats, 30 min of intestinal ischemia followed by 2 h of reperfusion (I/R) did not increase lung albumin leak. However, after treatment with NG-nitro-L-arginine methyl ester (L-NAME), intestinal I/R led to increased lung leak, suggesting a protective effect of endogenous NO. The site of action of NO appeared to be the lung and not the gut because 1) after treatment with L-NAME, local delivery of NO to the lung by inhalation abolished the increase in intestinal I/R-induced lung leak; 2) L-NAME had no effect on epithelial permeability (51Cr-labeled EDTA clearance) of reperfused small bowel; and 3) after treatment with L-NAME, local delivery of NO to the gut by luminal perfusion did not improve epithelial permeability of reperfused intestines. Furthermore, L-NAME increased, and inhaled NO decreased, the density of lung neutrophils in rats subjected to intestinal I/R, and treatment with the selectin antagonist fucoidan abolished L-NAME-induced lung leak in rats subjected to intestinal I/R. We conclude that endogenous lung NO limits secondary lung injury after intestinal I/R by decreasing pulmonary neutrophil retention.

Elevated nitric oxide in the urinary bladder in infectious and noninfectious cystitis

A role for nitric oxide (NO) has been suggested in inflammation and host defense. At higher concentrations, this gas shows cytotoxic effects that may be directed against microorganisms, tumor cells as well as host cells. The aim of the present study was to study the relationship between bladder mucosal inflammation and local production of NO. We measured NO directly in the urinary bladder in patients with infectious cystitis, interstitial cystitis, irradiation cystitis, and cystitis induced by antitumor treatment with bacillus Calmette-Guérin. NO-free air was introduced into the bladder during cystoscopy. The air was aspirated after 5 minutes of incubation and injected into a chemiluminescence NO analyzer. NO levels were 30 to 50 times higher in all varieties of cystitis as compared to controls. NO may contribute to host-defense mechanisms in the bladder during bacterial infection and antitumor treatment. Direct measurement of gaseous NO in the urinary bladder seems to be an attractive diagnostic method for detection of mucosal inflammation.

Expression of inducible-NOS in human glomerulonephritis: The possible source is infiltrating monocytes/macrophages

Nitric oxide (NO) is a biological mediator which is synthesized from L-arginine by a family of nitric oxide synthases (NOS). In this paper, we have studied the expression of the inducible NO synthase (iNOS) in the tissues of the human kidney at the mRNA level by RT-PCR assay and at the protein level by an immunohistochemistry technique using a specific anti-macrophage NOS monoclonal antibody. Biopsied renal tissues from patients with IgA nephropathy (IgAN; 28 cases) and with non-IgA mesangial proliferative glomerulonephritis (PGN;12 cases), and normal renal tissues obtained from kidneys removed for malignancies (11 cases) were included in this experiment. iNOS message was present in about 73% tissues from IgAN and PGN patients, which was supported by histochemical findings and the iNOS positive cells were predominantly in the tubulointerstitial areas where infiltration of monocytes/macrophages was abundant. The iNOS positive tissues were also strongly positive for CD14, INF-gamma and TNF-alpha mRNA expression. In our in vitro study, iNOS expression was found only in cytokines (INF-gamma and TNF-alpha) stimulated monocytes/macrophages but not in lymphocytes and neutrophils. Normal renal tissues did not show any iNOS expression either at the mRNA level or at the protein level in this study. Clinical and histological data showed that decreased renal function and tubulointerstitial damage were greater in the iNOS expressing patients. This study demonstrates that there is some in vivo induction for iNOS expression, likely to be mediated by cytokines, for local NO production that might be involved in the initiation and/or progression of mesangial proliferative glomerulonephritis.

Intrathecal infusion of the nitric oxide synthase inhibitor N-methyl L-arginine after experimental spinal cord injury in guinea pigs.

The potential role of nitric oxide (NO) production in secondary pathologic processes that follow spinal cord injury was examined in a guinea pig model that shows secondary loss of function for at least 3 days after trauma. Lateral compression injury of the lower thoracic cord was performed under ketamine/xylazine/acepromazine anesthesia. A fine polyethylene cannula was inserted through an incision in the dura rostral to the injury and run along the dorsal subdural space to the lesion level. The tube was connected to an osmotic pump delivering 1 microL/h of a 10 mM solution of either N-methyl-L-arginine or N-methyl-D-arginine in normal saline (pH 7.2). N-Methyl-L-arginine blocks both constitutive and inducible forms of NO synthase (NOS), present in neurons and inflammatory cells, respectively: N-methyl-D-arginine is the inactive stereoisomer. Two groups of 10 animals were used. Behavioral analysis and somatosensory evoked potential measurements were performed daily for 3 days, then the animals were fixed and survival of white matter at the center of the injury was evaluated, using toluidine-blue stained, 1 microns plastic sections. No significant difference was found between treated and control groups in degree or rate of secondary loss of spinal cord function or in the cross-sectional area of surviving white matter. These data do not support the hypothesis that local NO production by phagocytes, neurons, or other cells plays a significant role in secondary pathology of injury in this model.

Role of Nitric Oxide in the Local Regulation of Pulmonary Vascular Resistance in Humans

Endothelium-derived nitric oxide (NO) may be an important mediator of vascular resistance in the pulmonary circulation. We tested the hypotheses that in conscious adults the endothelium, through NO production, is important in maintaining basal pulmonary vascular resistance and that it can increase NO production further in response to receptor-mediated stimulation, leading to further vasodilation. Pulmonary arterial resistance vessel function was studied within the distribution of a segmental lower lobe pulmonary artery in eight conscious adults 37 to 76 years old who were undergoing cardiac catheterization. Segmental blood flow was determined with use of a Doppler-tip guide wire and quantitative angiography. Drugs were administered locally within the segmental artery through an infusion catheter. NG-Monomethyl-L-arginine (L-NMMA) was used as a specific inhibitor of NO production, whereas acetylcholine (ACh) was used to test receptor-mediated vasodilation. To demonstrate that vasodilation to ACh was NO dependent, ACh response was tested alone, in the presence of L-NMMA, and in the presence of a control constrictor phenylephrine. Basal pulmonary vascular resistance was NO dependent because L-NMMA infusion resulted in a dose-dependent decrease in local flow velocity (P < .005), with flow decreasing 33% at the highest dose of L-NMMA. ACh infusion resulted in a dose-dependent increase in flow velocity (P = .001). The ACh response was at least in part NO dependent because it was diminished by the presence of L-NMMA (P < .05). The effect of L-NMMA on the ACh response was not due to nonspecific preconstriction because L-NMMA diminished the ACh response significantly more than did the endothelium-independent constrictor phenylephrine (P < .05) despite comparable preconstriction. In healthy conscious adults, (1) normal basal pulmonary resistance is maintained in part by continuous local production of NO and (2) the local NO production is responsive to receptor-mediated stimulation, leading to further vasodilation, and can be tested with ACh.