The contradictory effects of nitric oxide in caerulein-induced acute pancreatitis in rats

This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 microg/kg caerulein, L-arginine used for NO induction and N(omega)-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.     

The contradictory effects of nitric oxide in caerulein-induced acute pancreatitis in rats

This study was planned to observe the effects of nitric oxide synthesis on the antioxidative defense enzymes and pancreatic tissue histology in caerulein-induced acute pancreatitis. Acute pancreatitis was induced by intraperitoneal injections of 50 µg/kg caerulein, L-arginine used for NO induction and N^ω-nitro-L-arginine methyl ester (L-NAME) used for NO inhibition. In the caerulein group acinar cell degeneration, interstitial inflammation, oedema and haemorrhage were detected. Pancreatic damage scores were decreased with both NO induction and inhibition (p<0.05). MDA, GSH-Px, CAT, GSH and SOD activities were significantly changed in the caerulein group and indicated increased oxidative stress. Both NO induction and inhibition decreased this oxidative stress. It is concluded that both nitric oxide induction and inhibition ameliorated caerulein-induced acute pancreatitis. The findings indicate that a certain amount of NO production has beneficial effects in experimental acute pancreatitis, but uncontrolled over-production of NO may be detrimental.     

Endothelial nitric oxide synthase is myristylated.

The enzyme responsible for the synthesis of endothelium-derived relaxing factor and/or nitric oxide in the endothelium has been described as a particulate enzyme, whereas other isoforms of nitric oxide synthase are soluble enzymes. Here we are reporting that endothelial cells metabolically incorporate myristate (C14), but not palmitate (C16), into nitric oxide synthase. We are postulating that the endothelial-derived nitric oxide synthase is a particulate enzyme because of the fatty acid acylation of the protein which 'anchors' the enzyme into the membrane either directly or via another membrane-bound protein.     

Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2–3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser⁶⁰² lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr⁴⁶ and Ser⁵⁸ are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.     

Endothelial nitric oxide synthase in the amphibian, Xenopus tropicalis

Nitric oxide (NO) is generated by NO synthase (NOS) of which there are three isoforms: neuronal NOS (nNOS, nos1), inducible NOS (iNOS, nos2), and endothelial NOS (eNOS, nos3). This study utilised the genome of Xenopus tropicalis to sequence a nos3 cDNA and determine if eNOS protein is expressed in blood vessels. A nos3 cDNA was sequenced that encoded a 1177 amino acid protein called XteNOS, which showed closest sequence identity to mammalian eNOS protein. The X. tropicalis nos3 gene and eNOS protein were determined to be an orthologue of mammalian nos3 and eNOS using gene synteny and phylogenetic analyses, respectively. In X. tropicalis, nos3 mRNA expression was highest in lung and skeletal muscle and lower in the liver, gut, kidney, heart and brain. Western analysis of kidney protein using an affinity-purified anti-XteNOS produced a single band at 140kDa. Immunohistochemistry showed XteNOS immunoreactivity in the proximal tubule of the kidney and endocardium of the heart, but not in the endothelium of blood vessels. Thus, X. tropicalis has a nos3 gene that appears not to be expressed in the vascular endothelium.     

Effects of S-propargyl-cysteine (SPRC) in caerulein-induced acute pancreatitis in mice

Hydrogen sulfide (H(2)S), a novel gaseous messenger, is synthesized endogenously from L-cysteine by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). S-propargyl-cysteine (SPRC) is a slow H(2)S releasing drug that provides cysteine, a substrate of CSE. The present study was aimed to investigate the effects of SPRC in an in vivo model of acute pancreatitis (AP) in mice. AP was induced in mice by hourly caerulein injections (50 μg/kg) for 10 hours. Mice were treated with SPRC (10 mg/kg) or vehicle (distilled water). SPRC was administered either 12 h before or 3 h before the induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and processed to measure the plasma amylase, plasma H(2)S, myeloperoxidase (MPO) activities and cytokine levels in pancreas and lung. The results revealed that significant reduction of inflammation, both in pancreas and lung was associated with SPRC given 3 h prior to the induction of AP. Furthermore, the beneficial effects of SPRC were associated with reduction of pancreatic and pulmonary pro-inflammatory cytokines and increase of anti-inflammatory cytokine. SPRC administered 12 h before AP induction did not cause significant improvement in pancreatic and lung inflammation. Plasma H(2)S concentration showed significant difference in H(2)S levels between control, vehicle and SPRC (administered 3 h before AP) treatment groups. In conclusion, these data provide evidence for protective effects of SPRC in AP possibly by virtue of its slow release of endogenous H(2)S.     

Endothelial nitric oxide synthase modulates gastric ulcer healing in rats

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3-10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N(6)-(iminoethyl)-L-lysine did not affect angiogenesis or ulcer healing, while N(G)-nitro-L-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.     

Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury

Previous studies indicate that deficiency of endothelial nitric oxide (NO) synthase (eNOS)-derived NO exacerbates myocardial reperfusion injury. We hypothesized that overexpression of eNOS would reduce the extent of myocardial ischemia-reperfusion (MI/R) injury. We investigated two distinct strains of transgenic (TG) mice overexpressing the eNOS gene (eNOS TG). Bovine eNOS was overexpressed in one strain (eNOS TG-Kobe), whereas the human eNOS gene was overexpressed in the other strain (eNOS TG-RT). Non-TG (NTG) and eNOS TG mice were subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion, and the extent of myocardial infarction was determined. Myocardial infarct size was reduced by 33% in the eNOS TG-Kobe strain (P < 0.05 vs. NTG) and by 32% in the eNOS TG-RT strain (P < 0.05 vs. NTG). However, postischemic cardiac function (cardiac output, fractional shortening) was not improved in the eNOS TG-Kobe mouse at 24 h of reperfusion [P = not significant (NS) vs. NTG]. In additional studies, eNOS TG-Kobe mice were subjected to 30 min of myocardial infarction and 7 days of reperfusion. Fractional shortening and the first derivative of left ventricular pressure were measured in eNOS TG-Kobe and NTG mice, and no significant differences in contractility were observed (P = NS) between the eNOS TG mice and NTG controls. Left ventricular end-diastolic pressure was significantly (P < 0.05 vs. NTG) reduced in the eNOS TG-Kobe strain at 7 days of reperfusion. The cardioprotective effects of eNOS overexpression on myocardial infarct size were ablated by Nomega-nitro-l-arginine methyl ester (300 mg/kg) pretreatment. Thus genetic overexpression of eNOS in mice attenuates myocardial infarction after MI/R but fails to significantly protect against postischemic myocardial contractile dysfunction in mice.     

Endothelial nitric oxide synthase is a molecular vascular target for the Chinese herb Danshen in hypertension

Danshen, a Chinese herb, reduces hypertension in Oriental medicine. We hypothesized that Danshen acts partially through endothelial nitric oxide synthase (eNOS) signaling mechanisms. We tested the hypothesis using tanshinone II(A), an active ingredient of Danshen, and the two-kidney, one-clip renovascular hypertension model in hamsters. Oral tanshinone (50 microg/100 g body wt) reduced mean arterial pressure (MAP) from 161.2 +/- 6.9 to 130.0 +/- 7.8 mmHg (mean +/- SE; P < 0.05) in hypertensive hamsters. MAP in sham-operated hamsters was 114.3 +/- 9.2 mmHg. Topical tanshinone at 1 microg/ml and 5 microg/ml increased normalized arteriolar diameter from 1.00 to 1.25 +/- 0.08 and 1.57 +/- 0.11, respectively, and increased periarteriolar nitric oxide concentration from 87.1 +/- 11.3 to 146.9 +/- 23.1 nM (P < 0.05) at 5 microg/ml in hamster cheek pouch. N(G)-monomethyl-L-arginine inhibited tanshinone-induced vasodilation. Hypertension reduced eNOS protein relative to sham-operated control. Tanshinone prevented the hypertension-induced reduction of eNOS and increased eNOS expression to levels higher than sham-operated control in hamster cheek pouch. Topical tanshinone increased normalized arteriolar diameter from 1.0 to 1.47 +/- 0.08 in the cremaster muscle of control mice and to 1.12 +/- 0.13 in cremasters of eNOS knockout mice. In ECV-304 cells transfected with eNOS-green fluorescent protein, tanshinone increased eNOS protein expression 1.35 +/- 0.05- and 1.85 +/- 0.07-fold above control after 5-min and 1-h application, respectively. Tanshinone also increased eNOS phosphorylation 1.19 +/- 0.07- and 1.72 +/- 0.20-fold relative to control after 5-min and 1-h application. Our data provide a basis to understand the action of a Chinese herb used in alternative medicine. We conclude that eNOS stimulation is one mechanism by which tanshinone induces vasodilation and reduces blood pressure.     

Inducible nitric oxide synthase protection against coxsackievirus pancreatitis.

Coxsackievirus infection causes myocarditis and pancreatitis in humans. In certain strains of mice, Coxsackievirus causes a severe pancreatitis. We explored the role of NO in the host immune response to viral pancreatitis. Coxsackievirus replicates to higher titers in mice lacking NO synthase 2 (NOS2) than in wild-type mice, with particularly high viral titers and viral RNA levels in the pancreas. Mice lacking NOS have a severe, necrotizing pancreatitis, with elevated pancreatic enzymes in the blood and necrotic acinar cells. Lack of NOS2 leads to a rapid increase in the mortality of infected mice. Thus, NOS2 is a critical component in the immune response to Coxsackievirus infection.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Endothelial nitric oxide synthase gene polymorphisms and the risk of acute myocardial infarction in a South Indian population

Myocardial infarction (MI) is a complex multi-factorial, polygenic disorder which results from an interaction between a person’s genetic makeup and various environmental factors. Nitric oxide (NO), a potent vasodilator produced by endothelial cells, plays an important role in the regulation of blood pressure, regional blood flow and also inhibits platelet aggregation, vascular smooth muscle cell proliferation and leukocyte adhesion to vascular endothelium. Our aim was to analyze the association of NOS3 (endothelial nitric oxide synthase 3) 894G>T and ?786T>C gene polymorphisms and MI risk in the South Indian population. A total of 287 MI patients, 279 risk control patients and 321 healthy controls were recruited for the retrospective study. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There was no significant association observed between NOS3 894G>T, ?786T>C polymorphisms and MI. A significant difference was observed in the distribution of GT genotype of the NOS3 894G>T polymorphism between the cases and the risk controls (p = 0.05) but the odds ratio (0.6) did not show risk for MI. The present study showed lack of association between NOS3 gene polymorphisms and MI in South Indian population.     

Inducible nitric oxide synthase is involved in acid-induced gastric hyperemia in rats and mice

The role of different isoforms of nitric oxide synthase (NOS) in the gastric mucosal hyperemia, induced by 155 mM luminal hydrochloric acid (pH approximately 0.8) without a barrier breaker, was investigated. Rats were anesthetized with Inactin (120 mg/kg ip), and mice were anesthetized with Forene (2.2% in 40% oxygen gas at 150 ml/min); the gastric mucosa was exteriorized. Gastric mucosal blood flow was measured with laser-Doppler flowmetry (LDF) in rats treated with Nomega-nitro-l-arginine (l-NNA; unspecific NOS inhibitor), l-N6-(1-iminoethyl)lysine [l-NIL; inducible (i) NOS inhibitor], or S-methyl-l-thiocitrulline [SMTC; neuronal (n) NOS inhibitor], 10 mg/kg, followed by 3 mg. kg-1. h-1 iv, in iNOS-deficient (-/-) and nNOS(-/-) mice. mRNA was isolated from the gastric mucosa in iNOS(-/-) and wild-type (wt) mice, and real-time RT-PCR was performed. The effect of 155 mM acid on gastric mucosal permeability was determined by measuring the clearance of 51Cr-EDTA from blood to lumen. LDF increased by 48 +/- 13% during 155 mM HCl luminally, an increase that was abolished by l-NNA, SMTC, or l-NIL. In iNOS wt mice, LDF increased by 33 +/- 8% during luminal acid. The blood flow increase was attenuated substantially in iNOS(-/-) mice. RT-PCR revealed iNOS mRNA expression in the gastric mucosa in the iNOS wt groups. The blood flow increase in response to acid was not abolished in nNOS(-/-) mice (nNOS-sufficient mice, 39 +/- 18%; heterozygous mice, 25 +/- 19%; -/- mice, 19 +/- 7%). Mucosal permeability was transiently increased during 155 mM HCl. The results suggest that iNOS is constitutively expressed in the gastric mucosa and is involved in acid-induced hyperemia, suggesting a novel role for iNOS in gastric mucosal protection.     

The role of neutral endopeptidase in caerulein-induced acute pancreatitis

Substance P (SP) is well known to promote inflammation in acute pancreatitis (AP) by interacting with neurokinin-1 receptor. However, mechanisms that terminate SP-mediated responses are unclear. Neutral endopeptidase (NEP) is a cell-surface enzyme that degrades SP in the extracellular fluid. In this study, we examined the expression and the role of NEP in caerulein-induced AP. Male BALB/c mice (20-25 g) subjected to 3-10 hourly injections of caerulein (50 μg/kg) exhibited reduced NEP activity and protein expression in the pancreas and lungs. Additionally, caerulein (10(-7) M) also downregulated NEP activity and mRNA expression in isolated pancreatic acinar cells. The role of NEP in AP was examined in two opposite ways: inhibition of NEP (phosphoramidon [5 mg/kg] or thiorphan [10 mg/kg]) followed by 6 hourly caerulein injections) or supplementation with exogenous NEP (10 hourly caerulein injections, treatment of recombinant mouse NEP [1 mg/kg] during second caerulein injection). Inhibition of NEP raised SP levels and exacerbated inflammatory conditions in mice. Meanwhile, the severity of AP, determined by histological examination, tissue water content, myeloperoxidase activity, and plasma amylase activity, was markedly better in mice that received exogenous NEP treatment. Our results suggest that NEP is anti-inflammatory in caerulein-induced AP. Acute inhibition of NEP contributes to increased SP levels in caerulein-induced AP, which leads to augmented inflammatory responses in the pancreas and associated lung injury.     

Neuronal nitric oxide synthase is necessary for elimination of Giardia lamblia infections in mice

NO produced by inducible NO synthase (NOS2) is important for the control of numerous infections. In vitro, NO inhibits replication and differentiation of the intestinal protozoan parasite Giardia lamblia. However, the role of NO against this parasite has not been tested in vivo. IL-6-deficient mice fail to control Giardia infections, and these mice have reduced levels of NOS2 mRNA in the small intestine after infection compared with wild-type mice. However, NOS2 gene-targeted mice and wild-type mice treated with the NOS2 inhibitor N-iminoethyl-L-lysine eliminated parasites as well as control mice. In contrast, neuronal NOS (NOS1)-deficient mice and wild-type mice treated with the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester and the NOS1-specific inhibitor 7-nitroindazole all had delayed parasite clearance. Finally, Giardia infection increased gastrointestinal motility in wild-type mice, but not in SCID mice. Furthermore, treatment of wild-type mice with NG-nitro-L-arginine methyl ester or loperamide prevented both the increased motility and the elimination of parasites. Together, these data show that NOS1, but not NOS2, is necessary for clearance of Giardia infection. They also suggest that increased gastrointestinal motility contributes to elimination of the parasite and may also contribute to parasite-induced diarrhea. Importantly, this is the first example of NOS1 being involved in the elimination of an infection.     

Endothelial dysfunction does not require loss of endothelial nitric oxide synthase

Whereas altered nitric oxide (NO.) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO. formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO. synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO. formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by the L-[(14)C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO. generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO. synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.     

Ventilator-induced lung injury is reduced in transgenic mice that overexpress endothelial nitric oxide synthase

Although mechanical ventilation (MV) is an important supportive strategy for patients with acute respiratory distress syndrome, MV itself can cause a type of acute lung damage termed ventilator-induced lung injury (VILI). Because nitric oxide (NO) has been reported to play roles in the pathogenesis of acute lung injury, the present study explores the effects on VILI of NO derived from chronically overexpressed endothelial nitric oxide synthase (eNOS). Anesthetized eNOS-transgenic (Tg) and wild-type (WT) C57BL/6 mice were ventilated at high or low tidal volume (Vt; 20 or 7 ml/kg, respectively) for 4 h. After MV, lung damage, including neutrophil infiltration, water leakage, and cytokine concentration in bronchoalveolar lavage fluid (BALF) and plasma, was evaluated. Some mice were given N(omega)-nitro-L-arginine methyl ester (L-NAME), a potent NOS inhibitor, via drinking water (1 mg/ml) for 1 wk before MV. Histological analysis revealed that high Vt ventilation caused severe VILI, whereas low Vt ventilation caused minimal VILI. Under high Vt conditions, neutrophil infiltration and lung water content were significantly attenuated in eNOS-Tg mice compared with WT animals. The concentrations of macrophage inflammatory protein-2 in BALF and plasma, as well as plasma tumor necrosis factor-alpha and monocyte chemoattractant protein-1, also were decreased in eNOS-Tg mice. L-NAME abrogated the beneficial effect of eNOS overexpression. In conclusion, chronic eNOS overexpression may protect the lung from VILI by inhibiting the production of inflammatory chemokines and cytokines that are associated with neutrophil infiltration into the air space.     

Expression of endothelial nitric oxide synthase is suppressed in the renal vasculature of angiotensinogen-gene knockout mice

We have attempted to elucidate the mechanism by which endothelial-type nitric oxide synthase (eNOS) is regulated in the kidney, with special reference to the role of renal hemodynamics and angiotensin II (Ang II). We compared angiotensinogen gene knockout (Atg−/−) mice, which lacked Ang II (resulting in sodium/water depletion and severe hypotension), with wild-type (Atg+/+) mice. Using Western blot analysis and the NADPH diaphorase histochemical reaction, we found that the expression and activity of eNOS were markedly lower in the renal vessels of Atg−/− mice compared with wild-type (Atg+/+) mice. Dietary salt loading significantly enhanced renal eNOS levels and increased blood pressure in Atg−/− mice, but severe hypotension almost abolished the effects of salt loading. In contrast, in Atg+/+ mice, altered salt intake or hydralazine had no effect on renal eNOS levels. These results suggest that perfusion pressure plays an essential role in maintaining renal vascular eNOS activity, whereas Ang II plays a supportive role, especially when renal circulation is impaired. This study was supported by Grants-in-Aid for Scientific Resarch 2001–2003, Japan Society for Promotion of Science (grant no. 13670735).