Independent Nitric Oxide Synthase Activity Research Articles

Distant effects of nitric oxide inhalation in lavage‐induced lung injury in anaesthetised pigs

Inhalation of nitric oxide (INO) exerts both local and distant effects. INO in healthy pigs causes down-regulation of endogenous nitric oxide (NO) production and vasoconstriction in lung regions not reached by INO, especially in hypoxic regions, which augments hypoxic pulmonary vasoconstriction. In contrast, in pigs with endotoxemia-induced lung injury, INO causes increased NO production in lung regions not reached by INO. The aim of this study was to investigate whether INO exerts distant effects in surfactant-depleted lungs. Twelve pigs were anaesthetised, and the left lower lobe (LLL) was separately ventilated. Lavage injury was induced in all lung regions, except the LLL. In six pigs, 40 ppm INO was given to the LLL (INO group), and the effects on endogenous NO production and blood flow in the lavage-injured lung regions were studied. Six pigs served as a control group. NO concentration in exhaled air (ENO), NO synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) in lung tissue, and regional pulmonary blood flow were measured. The calcium (Ca(2+) )-dependent NOS activity was lower (P < 0.05) in the lavage-injured lung regions in the INO group than in the control group. There were no measurable differences between the groups for Ca(2+) -independent NOS activity, cGMP, ENO, or regional pulmonary blood flow. Regional INO did not increase endogenous NO production in lavage-injured lung regions not directly reached by INO, but instead down-regulated the constitutive calcium-dependent nitric oxide synthase activity, indicating that NO may inhibit its own synthesis.

The effect of N-nitro-L-arginine and aminoguanidine treatment on changes in constitutive and inducible nitric oxide synthases in the spinal cord after sciatic nerve transection

Ca2+-dependent and Ca2+-independent nitric oxide synthase (NOS) activity, and neuronal and inducible NOS immunoreactivity (nNOS-IR and iNOS-IR), were investigated in the rabbit lower lumbar spinal cord after i) sciatic nerve transection and survival of experimental animals for 2 weeks, ii) treatment of animals with N-nitro-L-arginine (NNLA), an inhibitor of nNOS dosed at 20 mg/b.w. for 12 days, and iii) after treatment of animals with the inducible NOS (iNOS) inhibitor, aminoguanidine, dosed at 100 mg/b.w. for 4 and 12 days. Our attention was focused on the dorsal part of L4-L6 segments receiving sensory inputs from the sciatic nerve, and on the ventral part consisting of sciatic nerve motor neurons. Sciatic nerve transection increased Ca2+-dependent NOS activity and the density of nNOS in the dorsal part of the spinal cord on the ipsilateral side. NNLA treatment effectively reduced nNOS-IR in both the dorsal horn and the dorsal column, and decreased Ca2+-dependent NOS activity in the lower lumbar segments. Immunocytochemical analysis disclosed the up-regulation of iNOS immunoreactive staining after peripheral axotomy in alpha-motoneurons. The changes in iNOS expression and Ca2+-independent NOS activity were not significantly corrected by aminoguanidine treatment for 4 days. Long-lasting iNOS inhibition decreased Ca2+-independent NOS activity, but caused motor neuron degeneration and mediated small necrotic foci in the ventrolateral portion of the ventral horn. The results of the present study provide evidence that constitutive NOS inhibition by NNLA is more effective than specific long-lasting inhibition of iNOS by aminoguanidine treatment.

Time-related changes in constitutive and inducible nitric oxide synthases in the rat striatum in a model of Huntington's disease

Excitotoxicity and oxidative stress are mechanisms involved in the neuronal cell death induced by the intrastriatal injection of quinolinic acid (QUIN) as a model of Huntington's disease. Production of nitric oxide by nitric oxide synthase (NOS) has been proposed to participate in QUIN-induced neurotoxicity; however, the precise role of NOS in QUIN-induced toxicity still remains controversial. In order to provide further information on the role of NOS isoforms in QUIN toxicity, we performed real time RT-PCR and immunohistochemistry of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) and determined Ca 2+-dependent and Ca 2+-independent NOS activity in a temporal course (3–48 h), after an intrastriatal injection of QUIN to rats. NOS isoforms exhibited a transitory expression of mRNA and protein after QUIN infusion: eNOS increased between 3 and 24 h, iNOS between 12 and 24 h, while nNOS at 35 and 48 h. Ca 2+-independent activity (iNOS) did not show any change, while Ca 2+-dependent activity (constitutive NOS: eNOS/nNOS) exhibited increased levels at 3 h. Our results support the participation of Ca 2+-dependent NOS isoforms during the toxic events produced at early times after QUIN injection.

Differential regulation of the nitric oxide–cGMP pathway exacerbates postischaemic heart injury in stroke‐prone hypertensive rats

Using a working perfused heart model, we investigated the hypothesis that alterations in the NO-cGMP pathway may exacerbate postischaemic mechanical dysfunction in the hypertrophied heart. Ischaemia for 25 min followed by reperfusion for 30 min produced marked cardiac mechanical dysfunction in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Exogenous treatment with S-nitroso-N-acetyl-dl-penicillamine (SNAP), a NO donor, had beneficial effects on the cardiac dysfunction induced by ischaemia-reperfusion (I/R) in the WKY heart, but the cardioprotective effect of SNAP was eliminated by guanylyl cyclase inhibitor. Cardiac cGMP levels were increased by SNAP or ischaemia in WKY. In contrast, in SHRSP hearts, SNAP could not alleviate the cardiac dysfunction caused by I/R. Pre-ischaemia, the cardiac cGMP level was significantly higher in SHRSP than in WKY; however, no significant difference was found after SNAP and ischaemia. The myocardial Ca(2+)-dependent NO synthase (NOS) activity increased at the end of ischaemia in WKY. Conversely, the Ca(2+)-independent NOS activity and protein levels were upregulated by I/R in the SHRSP myocardium. In the SHRSP hearts, non-selective NOS and selective Ca(2+)-independent NOS inhibitors or antioxidant treatment alleviated cardiac dysfunction caused by I/R. Moreover, mRNA expression and Western blotting analysis of cGMP-dependent protein kinase type I showed more deterioration of SHRSP hearts compared with WKY. These results suggest that: (1) the NO-dependent cardioprotective effect is depressed; and (2) overproduction of NO derived from Ca(2+)-independent NOS contributes to postischaemic heart injury in the hypertrophied heart of hypertensive status.

Regional differences in nitric oxide‐mediated vasorelaxation in porcine pulmonary arteries

Several previous investigations have shown improved oxygenation when ventilator-treated patients with acute lung injury are turned prone. In a previous human study, we demonstrated higher Ca(2+)-dependent nitric oxide synthase (NOS) activity in dorsal than in ventral parts of the lung. The current investigation was designed to determine whether Ca(2+)-dependent NOS activity was different in dorsal and ventral porcine lung regions. In addition, possible differences in vascular responses to nitroprusside or secondary to acetylcholine- or bradykinin-stimulated NO production were studied in dorsal and ventral pulmonary arteries. In the study, 20 pigs were used. Lung biopsies and pulmonary arterial rings were harvested from ventral and dorsal lung regions. NOS activity was determined by citrulline assay in the presence and absence of the calcium chelator ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EDTA) to discriminate between Ca(2+)-dependent and Ca(2+)-independent NOS activity. In organ baths, in submaximally contracted arterial rings, vasorelaxation induced by acetylcholine, bradykinin and nitroprusside was measured. Ca(2+)-dependent NOS activity was higher in dorsal parts (87.2 +/- 9.1 citrulline units) than in ventral parts (62.2 +/- 10.1 citrulline units, P < 0.05) of porcine lung. There was a greater relaxation in dorsal than in ventral pulmonary arterial rings induced by both acetylcholine and bradykinin. Nitroprusside relaxed both sites equally. Our results show that endothelial-derived NO is an important factor influencing the differences between dorsal and ventral lung regions in vasorelaxing activity in porcine pulmonary arteries. This finding provides an explanation for the improved oxygenation when patients with severe acute lung insufficiency are turned prone.

Airway hyperresponsiveness induced by repetitive intraperitoneal injection of lipopolysacharide and the involvement of inflammation and nitric oxide in guinea pigs

Airway hyperresponsiveness (AHR) is involved in bronchial asthma and chronic obstructive pulmonary disease (COPD) and produces respiratory symptoms. Lipopolysaccharide (LPS) has been found to be significantly related to the severity of asthma. However, its clinical mechanism still remains controversial. This study investigated the in vivo effect of repetitive intraperitoneal administration of lipopolysaccharide (LPS) on airway hyperresponsiveness (AHR) in guinea pigs and the possible involvement of inflammation, nitric oxide (NO) and nitric oxide synthase (NOS). There were two exposure groups for intraperitoneal LPS injection: (1) LPS was given at a dose of 1 mg x kg(-1), followed by sterile saline (NS) 1 ml x kg(-1) 8 h later every 24 h; (2) LPS was given at a dose of 0.5 mg x kg(-1) two times with an interval of 8 h every 24 h. Each exposure regime was repeated 4 times. Control animals were given NS and 6 naive guinea pigs were used as baseline control. Determinations were made 24 h after each exposure. Persistent AHR occurred 24 h after the third and fourth exposures to LPS in the first exposure group (at one dose), but occurred earlier after the exposures to LPS in the second exposure group (at divided doses). The numbers of total cells and neutrophils were elevated initially but subsided subsequently in LPS-treated groups. No evidence of morphological changes in the small airways was found 24 h after any of the exposures. The Ca(2+)-dependent and Ca(2+)-independent NOS activities (mainly produced by iNOS) in the BALF, as well as the production of NO, were significantly elevated 24 h after any of third and fourth exposures in LPS-treated groups. Our results demonstrate that repetitive intraperitoneal LPS can induce persistent AHR which occurs earlier when the frequency of injection increase, and an elevation of NO production and iNOS activity may be involved in is systemic-LPS-induced AHR.

Demonstration of nitric oxide synthase activity in crustacean hemocytes and anti-microbial activity of hemocyte-derived nitric oxide

We determined the biochemical characteristics of nitric oxide synthase (NOS) in hemocytes of the crayfish Procambarus clarkii and investigated the roles of hemocyte-derived NO in host defense. Biochemical analysis indicated the presence of a Ca 2+-independent NOS activity, which was elevated by lipopolysaccharide (LPS) treatment. When bacteria ( Staphylococcus aureus) and hemocytes were co-incubated, adhesion of bacteria to hemocytes was observed. NO donor sodium nitroprusside (SNP) significantly increased the numbers of hemocytes to which bacteria adhered. Similarly, LPS elicited bacterial adhesion and the LPS-induced adhesion was prevented by NOS inhibitor N G-monomethyl- l-arginine ( l-NMMA). Finally, plate count assay demonstrated that addition of LPS to the hemocytes/bacteria co-incubation resulted in a significant decrease in bacterial colony forming unit (CFU), and that l-NMMA reversed the decreasing effect of LPS on CFU. The combined results demonstrate the presence of a Ca 2+-independent LPS-inducible NOS activity in crayfish hemocytes and suggest that hemocyte-derived NO is involved in promoting bacterial adhesion to hemocytes and enhancing bactericidal activity of hemocytes.

Chronic administration of aminoguanidine reduces vascular nitric oxide production and attenuates liver damage in bile duct‐ligated rats

Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. This study investigated the activity of nitric oxide synthase (NOS) in cirrhosis induced by bile duct-ligation (BDL) with NOS inhibitors. Three days after operation, rats were randomized to receive aminoguanidine (AG, 25 mg/kg/day) or L-N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg/day) for 21 days. Vascular NO production, which was increased in BDL cirrhotic rats, was reduced by 75% with AG but not L-NAME chronic administration. AG treatment attenuated liver damage, while L-NAME aggravated it. AG significantly suppressed inducible NOS (iNOS) expression in aorta of BDL rats at both mRNA and protein level, but much less efficient in reducing it in liver. In contrast, endothelial NOS (eNOS) expression was not markedly affected. Calcium-independent NOS activity, which was dramatically increased in aorta of BDL rats, was abolished by AG treatment. In liver, however, both calcium-dependent and -independent NOS activity were increased by AG treatment. Chronic administration of AG could reduce systemic NO levels as well as suppress iNOS expression and activity in aorta of BDL rats. It also improved liver function, possibly because of its ability to increase hepatic NOS activity, and to correct the systemic hemodynamic disorders by decreasing vascular NO production.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca 2+-independent NOS activity

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca 2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca 2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca 2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca 2+-independent and underestimation of Ca 2+-dependent NOS activities. The revised Ca 2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca 2+-independent enzymatic activity in cerebral tissue after a brain insult.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca2+-independent NOS activity

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.

Differential roles of neuronal and endothelial nitric oxide synthases during carrageenan-induced inflammatory hyperalgesia

The present study investigated the role of neuronal nitric oxide synthase (nNOS) in carrageenan-induced inflammatory pain by combining genomic and pharmacological strategies. Intrathecal injection of the nNOS inhibitor 7-nitroindazole dose-dependently inhibited carrageenan-induced thermal hyperalgesia in both early and late phases in wild-type mice. However in nNOS knockout mice, carrageenan-induced thermal hyperalgesia remained intact in the early phase but was reduced in the late phase. Spinal Ca 2+-dependent nitric oxide synthase (NOS) activity in nNOS knockout mice was significantly lower than that in wild-type mice. Following carrageenan injection, although the spinal Ca 2+-dependent NOS activity in both wild-type and knockout mice increased, the enzyme activity in nNOS knockout mice reached a level similar to that in wild-type mice. On the other hand, no significant difference in spinal Ca 2+-independent NOS activity was noted between wild-type and nNOS knockout mice before and after carrageenan injection. Furthermore, intrathecal administration of the endothelial NOS (eNOS) inhibitor l- N 5-(1-iminoethyl)-ornithinein nNOS knockout mice inhibited the thermal hyperalgesia in both early and late phases, though this inhibitor had no effect in wild-type mice. Meanwhile, Western blot showed that eNOS expression in the spinal cord of nNOS knockout mice was up-regulated compared with wild-type mice; immunohistochemical staining showed that the spinal eNOS was mainly distributed in superficial laminae of the dorsal horn. Finally, double staining with confocal analysis showed that the enhanced spinal eNOS was expressed in astrocytes, but not in neurons. Our current results indicate that nNOS plays different roles in the two phases of carrageenan-induced inflammatory pain. In this model, enhanced spinal eNOS appears to compensate for the role of nNOS in nNOS knockout mice.

Hyperlipidemia induced by a cholesterol-rich diet leads to enhanced peroxynitrite formation in rat hearts.

We investigated the influence of experimental hyperlipidemia on the formation of cardiac NO, superoxide, and peroxynitrite (ONOO(-)) in rat hearts. Wistar rats were fed 2% cholesterol-enriched diet or normal diet for 8 weeks. Separate groups of normal and hyperlipidemic rats were injected twice intraperitoneally with 2 x 20 micromol/kg FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), a ONOO(-) decomposition catalyst, 24 h and 1 h before isolation of the hearts. A cholesterol diet significantly decreased myocardial NO content, however, myocardial Ca(2+)-dependent and Ca(2+)-independent NO synthase activity and NO synthase protein level did not change. Myocardial superoxide formation and xanthine oxidase activity were significantly increased; however, cardiac superoxide dismutase activity did not change in the cholesterol-fed group. Dityrosine in the perfusate, a marker of cardiac ONOO(-) formation, and plasma nitrotyrosine, a marker for systemic ONOO(-) formation, were both elevated in hyperlipidemic rats. In cholesterol-fed rats, left ventricular end-diastolic pressure (LVEDP) was significantly elevated as compared to controls. Administration of FeTPPS normalized LVEDP in the cholesterol-fed group. We conclude that cholesterol-enriched diet-induced hyperlipidemia leads to an increase in cardiac ONOO(-) formation and a decrease in the bioavailability of NO which contributes to the deterioration of cardiac performance and may lead to further cardiac pathologies.

Low endotoxemia prevents the reduction of gastric blood flow induced by NSAIDs: role of nitric oxide.

1 The role of nitric oxide (NO) in the effects of low endotoxemia on gastric damage and blood flow has been evaluated in indomethacin-treated rats. 2 Pretreatment (-1 h) with endotoxin (40 micro g kg(-1)) reduced gastric damage induced by indomethacin (20 mg kg(-1)) in conscious rats. 3 Endotoxin prevented the reduction in gastric blood flow (laser Doppler flowmetry) induced by indomethacin in pentobarbital-anaesthetised rats. 4 Pretreatment with an NO-synthase (NOS) inhibitor (L-NAME, 1 mg kg(-1)) reversed the protective effect of endotoxin on gastric blood perfusion. 5 Endotoxin did not modify the expression of mRNA for endothelial NOS or inducible NOS in the gastric corpus when evaluated 1 h postinjection. However, a 3.8-fold increase in inducible NOS mRNA and a 61% reduction in endothelial NOS mRNA were observed in the gastric corpus 4 h after endotoxin administration. 6 Evaluation of both total and Ca(2+)-dependent NOS activity by analysing the rate of conversion of L-arginine to L-citrulline in gastric corpus homogenates showed no differences between animals treated with endotoxin and those treated with saline 1 or 4 h beforehand. Ca(2+)-independent NOS activity was almost non-apparent in control as well as in endotoxin-treated rats at all the time points analysed. 7 Low endotoxemia preserves blood perfusion and protects the gastric mucosa against the deleterious effects of indomethacin through the endogenous NO release. NO synthesis in response to endotoxin does not involve the inducible NOS, but probably depends on the post-translational/biochemical regulation in vivo of a Ca(2+)-dependent NOS, most probably endothelial NOS.

Increased matrix metalloproteinase activity after canine cardiopulmonary bypass is suppressed by a nitric oxide scavenger

Objectives: We tested whether nitric oxide scavenging with a ruthenium-based compound (AMD6221) would improve hemodynamics and alter nitric oxide synthase and matrix metalloproteinase activities in a canine model of cardiopulmonary bypass. Methods: Dogs were randomized to either cardiopulmonary bypass (n = 12) or control (n = 12) groups. They were further randomized to receive a continuous infusion of AMD6221 or placebo. Cardiopulmonary bypass was maintained for 90 minutes, and then, 4 hours later, dogs were killed. Cardiac, lung, and brain sections were snap frozen in liquid nitrogen for determination of nitric oxide synthase, matrix metalloproteinase 2, and matrix metalloproteinase 9 activities. Results: After cardiopulmonary bypass, 3 of 6 placebo-treated (cardiopulmonary bypass-placebo) and 0 of 6 AMD6221-treated (cardiopulmonary bypass-6221) animals required phenylephrine infusion to maintain a predetermined blood pressure (P <.05). Total fluid administration was lower in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (983 ± 134 vs 1617 ± 254 mL, respectively; P <.005). After cardiopulmonary bypass, matrix metalloproteinase 2 and matrix metalloproteinase 9 activities in the lung, left ventricle, and left atrium were decreased in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (P <.05). Ca2+-independent nitric oxide synthase activity and matrix metalloproteinase 2 activity in the brain were also lower (P <.05) in the cardiopulmonary bypass-SCV group. Finally, neutrophil expression of CD18, an adhesion complex, was lower at 4 hours after cardiopulmonary bypass in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (38 ± 27 vs 81 ± 11; P <.05). Conclusions: We found that (1) infusion of an nitric oxide scavenger, AMD6221, was associated with improved predefined hemodynamics; (2) cardiopulmonary bypass increased activities of Ca2+-independent nitric oxide synthase and matrix metalloproteinases in multiple organs; and (3) AMD6221 could ameliorate the increased generation of nitric oxide and increased matrix metalloproteinase activities.J Thorac Cardiovasc Surg 2003;125:661-8

Distribution of nitric oxide synthase in normal and cirrhotic human liver.

Chronic liver disorders represent a serious health problem, considering that 300 million people worldwide are hepatitis B virus carriers, and 8,000-10,000 patients per year, in the U.S. alone, die as a result of liver failure caused by hepatitis C infection. Nitric oxide synthase (NOS) regulates hepatic vasculature; however, the patterns of expression and activity of NOS proteins in healthy and diseased human livers are unknown. Sections of diseased (n = 42) and control livers (n = 14) were collected during orthotopic liver transplants and partial hepatectomy. The diseased sections included alcoholic cirrhosis, viral hepatitis, cholestasis, acute necrosis, and uncommon pathologies including alpha(1)-anti-trypsin disorder. The endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were studied by using the citrulline assay, Western immunoblot, immunohistochemistry, and in situ hybridization. The systemic generation of plasma NO metabolites was measured by HPLC. In control livers, Ca(2+)-dependent and -independent NOS activities were identified by Western analysis as eNOS and iNOS, respectively. The eNOS was uniformly distributed in the hepatocytes and also detected in the endothelium of hepatic arteries, terminal hepatic venules, sinusoids, and in biliary epithelium. The iNOS was detected in hepatocytes and localized mainly in the periportal zone of the liver acinus. This pattern of distribution of eNOS and iNOS in normal liver was confirmed by in situ hybridization. In diseased livers, there was a significant increase in Ca(2+)-independent NOS with the corresponding strong appearance of iNOS in the cirrhotic areas. The eNOS was translocated to hepatocyte nuclei. Thus, eNOS and iNOS proteins are differentially expressed in healthy human liver, and this expression is significantly altered in cirrhotic liver disorders.

Downregulation of nNOS and synthesis of PGs associated with endotoxin-induced delay in gastric emptying.

A single intraperitoneal injection of endotoxin (40 microg/kg) significantly delayed gastric emptying of a solid nutrient meal. Blockade of nitric oxide synthase (NOS) with 30 mg/kg ip N(G)-nitro-L-arginine methyl ester or 20 mg/kg ip 7-nitroindazole [neuronal NOS (nNOS) inhibitor] significantly delayed gastric emptying in control animals but failed to modify gastric emptying in endotoxin-treated rats. Administration of 2.5, 5, and 10 mg/kg ip N(6)-iminoethyl-L-lysine [inducible NOS (iNOS) inhibitor] had no effect in either experimental group. Indomethacin (5 mg/kg sc), NS-398 (cyclooxygenase-2 inhibitor; 10 mg/kg ip), and dexamethasone (10 mg/kg sc) but not quinacrine (20 mg/kg ip) significantly prevented delay in gastric emptying induced by endotoxin but failed to modify gastric emptying in vehicle-treated animals. Ca(2+)-dependent NOS activity in the antrum pylorus of the stomach was diminished by endotoxin, whereas Ca(2+)-independent NOS activity was not changed. In addition, decreased nNOS mRNA and protein were observed in the antrum pylorus of endotoxin-treated rats. Our results suggest that downregulation of nNOS in the antrum pylorus of the stomach and synthesis of prostaglandins mediate the delay in gastric emptying of a solid nutrient meal induced by endotoxin.

Increased activity and expression of iNOS in human duodenal enterocytes from patients with celiac disease.

The activity of nitric oxide synthase (NOS) was assayed in enterocytes isolated from human duodenal biopsies to determine its role in celiac disease. Patients were categorized into groups with irritable bowel syndrome, iron-deficiency anemia, B(12)/folate deficiency, and treated and untreated celiac disease. Enterocytes isolated from all groups showed 1400W-inhibitable Ca2+-independent NOS activity with a pH level and temperature optimum of 9.4 and 37 degrees C, respectively. Western blotting showed that enterocytes expressed the inducible NOS protein and proteins with nitrated tyrosine residues, the latter being indicative of nitric oxide-driven peroxynitrite and/or free-radical damage. Endothelial NOS was seen only in the lamina propria. Patients with celiac disease had higher NOS activity than other patient groups. Treatment of the condition led to a fall in activity. Enzyme-linked immunosorbent assay demonstrated cGMP production by the enterocyte fraction, but cGMP levels did not correlate with NOS activity. These results suggest that inducible NOS is constitutively expressed in human duodenal enterocytes, is increased in patients with untreated celiac disease, and is partially corrected when such patients are treated. We found no evidence to support a role for nitric oxide in the formation of cGMP within the small intestine. Furthermore, we were unable to demonstrate a role for peroxynitrite/free radical damage in the pathophysiology of celiac disease.

Nitric oxide and nitric oxide synthases in the fetal cerebral cortex of rats following transient uteroplacental ischemia

The effect of transient uteroplacental ischemia on nitric oxide (NO) levels, enzymatic activity, and expression of NO synthase (NOS) isoforms was studied in fetal rat brains. Fetuses were subjected to ischemia by clamping the uterine arteries for 5 min on gestational day 17 (GD17). At different times after ischemia, fetuses were delivered by Cesarean section under anesthesia to obtain the brains. Transient uteroplacental ischemia produced a time dependent increase in nitrite levels in the brain, reaching a maximum value (300±25% of baseline) 24 h after uterine artery occlusion and remaining elevated as long as 48 h. Significantly increased nitrite levels were found in the cerebral cortex but not in the mesencephalon and cerebellum. The ischemia-induced increment in nitrite levels was totally blocked by either l-NAME (10 mg/kg) or AMT (0.65 mg/kg) administered i.p. 1 h before uterine artery occlusion. Both Ca 2+-dependent and Ca 2+-independent NOS activities in the cerebral cortex remained significantly increased with respect to controls after 24 h following the ischemia. Reverse transcriptase-polymerase chain reaction showed augmented levels of mRNAs for both nNOS and iNOS when compared with controls at 8 h after ischemia. At 36 h, nNOS mRNA returned to basal levels whereas eNOS mRNA levels increased and iNOS mRNA remained elevated. Our results show that the three NOS isoforms participate in increasing NO levels after transient ischemia and suggest a biphasic and differential regulation of the expression of constitutive NOS isoforms in the rat cerebral cortex.

Expression and activity of nitric oxide synthase isoforms in rat brain during the development of experimental allergic encephalomyelitis

The activity and expression of nitric oxide synthase (NOS) isoforms and protein nitrotyrosine (NT) residues were investigated in whole encephalic mass (WEM) homogenates during the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. EAE stages (0–III) were daily defined by clinical evaluation, and in the end of each stage, WEMs were removed for analysis of NOS activity, protein NT residues and mRNA for the different NOS isoforms. In the presence of NADPH, WEMs from EAE-III rats showed lower Ca 2+-dependent NOS activity than those from control group. These differences disappeared in the presence of exogenous calmodulin, flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH 4) and NADPH. Of all the cofactors, just the omission of FAD caused comparable decrease of Ca 2+-dependent NOS activity from both groups. Ca 2+-independent NOS activity from EAE-III animals was insensitive to the omission of any of the cofactors, while in control animals this activity was significantly inhibited by the omission of either FAD or BH 4. Increased levels of both iNOS mRNA and protein NT expression were observed in animals with EAE, which also showed lower levels of a thermolabile NOS inhibitor in WEM homogenates and sera than controls. In conclusion, during late EAE stages, constitutive Ca 2+-dependent NOS activity decreases concomitantly with iNOS upregulation, which could be responsible for the high protein NT levels. The differential dependence of iNOS activity on cofactors and the absence of an endogenous thermolabile NOS inhibitor in animals with EAE could reflect additional control mechanisms of NOS activity in this model of multiple sclerosis.

BACILLUS CALMETTE-GUERIN INDUCES LONG-TERM LOCAL FORMATION OF NITRIC OXIDE IN THE BLADDER VIA THE INDUCTION OF NITRIC OXIDE SYNTHASE ACTIVITY IN UROTHELIAL CELLS

Bladder instillation of bacillus Calmette-Guerin (BCG) is effective therapy for recurrent superficial bladder cancer and carcinoma in situ. BCG induces nitric oxide synthase activity in the bladder. Nitric oxide is formed from L-arginine by nitric oxide synthase. We investigated nitric oxide formation and its localization in bladder cancer patients treated with intravesical BCG instillation. The L-citrulline conversion assay was done to assess nitric oxide synthase activity in BCG treated T24 human bladder cancer cells and cultured normal human urothelial cells. Nitrite and nitrate in cell culture medium, urine and plasma were measured by capillary electrophoresis. Nitric oxide formation in the bladder was measured by chemiluminescence. A 24-hour treatment with BCG induced calcium independent nitric oxide synthase activity in T24 cells in a dose dependent manner. Nitrite and nitrate production by T24 cells also increased in a dose dependent manner after 24-hour BCG treatment. BCG treatment of cultured normal human urothelial cells resulted in the induction of calcium dependent and independent nitric oxide synthase activity. Nitrite in the urine of patients receiving BCG for the first time was increased 5-fold 24 hours after instillation. Furthermore, BCG increased luminal nitric oxide in the bladder. The increase was noted after a single treatment and sustained for 6 months. No changes in plasma nitrite or nitrate were observed after BCG treatment. BCG induces the local formation of nitric oxide in the bladder, whereas no evidence for systemic nitric oxide formation was noted. Increased nitric oxide production in the bladder is likely due to the induction of nitric oxide synthase activity in urothelial cells.