Regulation Of Nitric Oxide Synthase Research Articles

Role of constitutive nitric oxide synthase in regulation of <i>Helicobacter pylori</i>-induced gastric mucosal cyclooxygenase-2 ac-tivation through S-nitrosylation: mechanism of ghrelin action

Gastric mucosal inflammatory responses to H. pylori lipopolysaccharide (LPS), are characterized by the excessive NO and prostaglandin (PGE2) generation due to the disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) systems. Here, we report that the LPS-induced enhancement in gastric mucosal inducible (i) iNOS) activity and up-regulation in PGE2 production was associated with the suppression in Akt kinase activity and the impairment in constitutive (c) cNOS activation. The stimulatory effect of the LPS on PGE2 production, furthermore, was susceptible to suppression by COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400 W. Further, we show that the countering effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in up-regu- lation in Akt activity and the increase in cNOS activation through phosphorylation, and accompanied by the suppression in iNOS expression and the reduction in COX-2 activity associated with the loss in COX-2 protein S-nitrosylation. Moreover, the effect of ghre-lin on the LPS-induced COX-2 S-nitrosylation was subject to repression by Akt inhibition. Our findings demonstrate that induction in iNOS with H. pylori in- fection leads to COX-2 activation through S-nitro- sylation and up-regulation in PGE2 generation, and that ghrelin counters these untoward consequences of the LPS through Akt-mediated up-regulation in cNO- S activation required for the iNOS gene repression.

The Transcriptional Response in Human Umbilical Vein Endothelial Cells Exposed to Insulin: A Dynamic Gene Expression Approach

BackgroundIn diabetes chronic hyperinsulinemia contributes to the instability of the atherosclerotic plaque and stimulates cellular proliferation through the activation of the MAP kinases, which in turn regulate cellular proliferation. However, it is not known whether insulin itself could increase the transcription of specific genes for cellular proliferation in the endothelium. Hence, the characterization of transcriptional modifications in endothelium is an important step for a better understanding of the mechanism of insulin action and the relationship between endothelial cell dysfunction and insulin resistance.Methodology and principal findingsThe transcriptional response of endothelial cells in the 440 minutes following insulin stimulation was monitored using microarrays and compared to a control condition. About 1700 genes were selected as differentially expressed based on their treated minus control profile, thus allowing the detection of even small but systematic changes in gene expression. Genes were clustered in 7 groups according to their time expression profile and classified into 15 functional categories that can support the biological effects of insulin, based on Gene Ontology enrichment analysis. In terms of endothelial function, the most prominent processes affected were NADH dehydrogenase activity, N-terminal myristoylation domain binding, nitric-oxide synthase regulator activity and growth factor binding. Pathway-based enrichment analysis revealed “Electron Transport Chain” significantly enriched.Results were validated on genes belonging to “Electron Transport Chain” pathway, using quantitative RT-PCR.ConclusionsAs far as we know, this is the first systematic study in the literature monitoring transcriptional response to insulin in endothelial cells, in a time series microarray experiment. Since chronic hyperinsulinemia contributes to the instability of the atherosclerotic plaque and stimulates cellular proliferation, some of the genes identified in the present work are potential novel candidates in diabetes complications related to endothelial dysfunction.

Regulation of inducible nitric oxide synthase activity/expression in rat hearts from ghrelin-treated rats

The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible nitric oxide (NO) synthase (iNOS; for gene Nos2) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 μl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO, L-arginine (L-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of Nos2 mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of L-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased Nos2 gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin.

Functional Proteomic Analysis for Regulatory T Cell Surveillance of the HIV-1-Infected Macrophage

Regulatory T cells (Treg) induce robust neuroprotection in murine models of neuroAIDS, in part, through eliciting anti-inflammatory responses for HIV-1-infected brain mononuclear phagocytes (MP; macrophage and microglia). Herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that Treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected MP, and inducing phenotypic cell switches. Changes in individual Treg-induced macrophage proteins were quantified by iTRAQ labeling followed by mass spectrometry identifications. Reduction in virus release paralleled the upregulation of interferon-stimulated gene 15, an ubiquitin-like protein involved in interferon-mediated antiviral immunity. Treg killed virus-infected macrophages through caspase-3 and granzyme and perforin pathways. Independently, Treg transformed virus-infected macrophages from an M1 to an M2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. Taken together, Treg affects a range of virus-infected MP functions. The observations made serve to challenge the dogma of solitary Treg immune suppressor functions and provides novel insights into how Treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection.

Is tetrahydrobiopterin a therapeutic option in diabetic hypertensive patients?

Nitric oxide (NO) is an important regulator of vascular tone, and is also an antithrombotic, anti-inflammatory, antiproliferative, and antiatherogenic factor. Endothelial function is altered in patients with coronary artery disease, stroke, and peripheral artery disease, and endothelial dysfunction correlates with the risk factor profile for a patient. Hypertension and type 2 diabetes are risk factors for vascular disease, and are both pathologies characterized by loss of NO activity. Indeed, endothelial dysfunction is usually present in diabetic and/or hypertensive patients. Tetrahydrobiopterin is an essential cofactor for the NO synthase enzyme, and insufficiency of this cofactor leads to uncoupling of the enzyme, release of superoxide, endothelial dysfunction, progression of hypertension, and finally, proatherogenic effects. Tetrahydrobiopterin is also an important mediator of NO synthase regulation in type 2 diabetes and hypertension, and may be a rational therapeutic target to restore endothelial function and prevent vascular disease in these patients. The aim of this paper is to review the rationale for therapeutic strategies directed to biopterins as a target for vascular disease in type 2 diabetic hypertensive patients.

Chronic inhibition of farnesyl pyrophosphate synthase improves endothelial function in spontaneously hypertensive rats

Farnesyl pyrophosphate synthase (FPPS, EC 2.5.1.10), an essential enzyme in the mevalonate pathway, catalyzes the synthesis of isoprenoid intermediates. The isoprenoid intermediates are needed for protein isoprenylation of RhoA for its function on regulation of endothelial nitric oxide synthase (eNOS). We previously reported that FPPS were upregulated in spontaneously hypertensive rats (SHR) when compared with Wistar–Kyoto rats (WKY), and this was accompanied by development of endothelial dysfunction. Five-week-old male rats were daily gavaged with vehicle or an FPPS inhibitor (alendronate, 1 or 10 mg/kg). After 12-week administration of alendronate, endothelium-dependent and -independent vasorelaxation were measured in isolated aortic rings. Twelve-week of alendronate (10 mg/kg/day) treatment restored the impaired endothelium-dependent vasodilation in SHR. Furthermore, long-term treatment with an FPPS inhibitor significantly suppressed RhoA activation and increased phospho-eNOS/eNOS ratio. In conclusion, chronic treatment with an FPPS inhibitor improves the endothelial function in SHR, and the upregulation of phospho-eNOS/eNOS ratio with inhibition of RhoA activation may be an important mechanism.

Regulation of NF-κB activity and inducible nitric oxide synthase by regulatory particle non-ATPase subunit 13 (Rpn13)

Human Rpn13, also known as adhesion regulating molecule 1 (ADRM1), was recently identified as a novel 19S proteasome cap-associated protein, which recruits the deubiquitinating enzyme UCH37 to the 26S proteasome. Knockdown of Rpn13 by siRNA does not lead to global accumulation of ubiquitinated cellular proteins or changes in proteasome expression, suggesting that Rpn13 must have a specialized role in proteasome function. Thus, Rpn13 participation in protein degradation, by recruiting UCH37, is rather selective to specific proteins whose degradation critically depends on UCH37 deubiquitination activity. The specific substrates for the Rpn13/UCH37 complex have not been determined. Because of a previous discovery of an interaction between Rpn13 and inducible nitric oxide synthase (iNOS), we hypothesized that iNOS is one of the substrates for the Rpn13/UCH37 complex. In this study, we show that Rpn13 is involved in iNOS degradation and is required for iNOS interaction with the deubiquitination protein UCH37. Furthermore, we discovered that IkappaB-alpha, a protein whose proteasomal degradation activates the transcription factor NF-kappaB, is also a substrate for the Rpn13/UCH37 complex. Thus, this study defines two substrates, with important roles in inflammation and host defense for the Rpn13/UCH37 pathway.

Post-translational Regulation of Endothelial Nitric Oxide Synthase (eNOS) by Estrogens in the Rat Vagina

Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17beta (15 microg). Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS-caveolin-1 interaction.

Proteomic Profiling of Perturbed Protein Sulfenation in Renal Medulla of the Spontaneously Hypertensive Rat

Protein sulfenic acids have been proposed as potential biochemical switches for redox signaling. This post-translational modification (PTM) is readily reversible, in contrast to some other types of oxidative PTM. Enhanced oxidative stress has been reported as a feature of hypertension, and renal function has been implicated in the development and progression of the disease in animal models such as the spontaneously hypertensive rat (SHR). However, reactive oxygen species (ROS) are also signaling molecules and may play a role in vascular function. To investigate protein sulfenation under hypertensive conditions, we examined protein extracts of SHR kidney medulla in comparison to medulla from normotensive Wistar rats. Total free thiol content of the SHR medulla was significantly lower than that of Wistar medulla, indicating enhanced oxidation of sulfhydryls. Protein sulfenation was also significantly greater in the medulla of hypertensive animals. Thioredoxin reductase activity was also reduced in SHR medulla and this may account, in part, for enhanced protein sulfenation. Purification of sulfenated proteins from SHR medulla revealed several proteins involved in processes such as metabolism, antioxidant defense, and regulation of nitric oxide synthase. Enhanced sulfenation may represent perturbed redox signaling in SHR medulla, or simply enhanced ROS generation.

Effects of exercise training on cellular mechanisms of endothelial nitric oxide synthase regulation in coronary arteries after chronic occlusion

Exercise training enhances agonist-mediated relaxation in both control and collateral-dependent coronary arteries of hearts subjected to chronic occlusion, an enhancement that is mediated in part by nitric oxide. The purpose of the present study was to elucidate exercise training-induced adaptations in specific cellular mechanisms involved in the regulation of endothelial nitric oxide synthase (eNOS) in coronary arteries of ischemic hearts. Ameroid constrictors were surgically placed around the proximal left circumflex coronary artery (LCX) of adult female Yucatan miniature swine. Eight weeks postoperatively, animals were randomized into sedentary (pen-confined) or exercise training (treadmill run; 5 days/wk; 14 wk) protocols. Coronary artery segments ( approximately 1.0 mm luminal diameter) were isolated from collateral-dependent (LCX) and control (nonoccluded left anterior descending) arteries 22 wk after ameroid placement. Endothelial cells were enzymatically dissociated, and intracellular Ca(2+) responses (fura 2) to bradykinin stimulation were studied. Immunofluorescence and laser scanning confocal microscopy were used to quantify endothelial cell eNOS and caveolin-1 cellular distribution under basal and bradykinin-stimulated conditions. Immunoblot analysis was used to determine eNOS, phosphorylated (p)-eNOS, protein kinase B (Akt), pAkt, and caveolin-1 protein levels. Bradykinin-stimulated nitrite plus nitrate (NOx; nitric oxide metabolites) levels were assessed via HPLC. Exercise training resulted in significantly enhanced bradykinin-mediated increases in endothelial Ca(2+) levels, NOx levels, and the distribution of eNOS-to-caveolin-1 ratio at the plasma membrane in endothelial cells of control and collateral-dependent arteries. Exercise training also significantly increased total eNOS and phosphorylated levels of eNOS (pSer(1179)) in collateral-dependent arteries. Total eNOS protein levels were also significantly increased in collateral-dependent arteries of sedentary animals. These data provide new insights into exercise training-induced adaptations in cellular mechanisms of nitric oxide regulation in collateral-dependent coronary arteries of chronically occluded hearts that contribute to enhanced nitric oxide production.

Insulin Coordinately Regulates Nitric Oxide Synthase and Argininosuccinate Synthase to Maintain Vascular Endothelial Function

The pathogenesis of diabetes includes impairment of vascular endothelial cell nitric oxide (NO) production. In this report, we demonstrate that insulin not only maintains endothelial NO production through regulation of endothelial nitric oxide synthase (eNOS), but also via the regulation of argininosuccinate synthase (AS), which is the rate‐limiting step of the citrulline nitric oxide cycle. First, using serum starved, cultured vascular endothelial cells, we show that insulin coordinately up‐regulates both eNOS and AS expression, supporting NO production. Second, using a rat model, we show that diminished eNOS and AS expression in rats with streptozotocin‐induced diabetes is reversed by insulin treatment, delivered via slow release pellets. Impaired endothelium‐dependent vasorelaxation is also reversed with insulin treatment. Thus, insulin supports endothelial function not only through regulation of eNOS, but also through the coordinate regulation of AS. (Supported by the James and Esther King Biomedical Research Program, DOH Florida; NIH RO1 HL083153‐01A2).

The regulation of endothelial nitric oxide synthase by caveolin: a paradigm validated in vivo and shared by the ‘endothelium-derived hyperpolarizing factor’

The endothelium plays a crucial role in the regulation of cardiovascular homeostasis through the release of vasoactive autacoids such as nitric oxide, prostacyclin, and a third factor or pathway termed 'endothelium-derived hyperpolarizing factor' (EDHF). Although the functional influence of NO and EDHF is sometimes reciprocal (i.e., their effects decrease or increase with the reduction in vessel diameter, respectively), recent insights led to the identification of caveolae and caveolin as common regulators of their production. In this review, we will first focus on the current understanding of the caveolin/eNOS paradigm and will then detail the most recent findings on the role of caveolae in driving EDHF-signaling pathways.

Apocynin restores endothelial dysfunction in streptozotocin diabetic rats through regulation of nitric oxide synthase and NADPH oxidase expressions

Aim Increased production of reactive oxygen species (ROS) in the diabetic vasculature results in the impairment of nitric oxide (NO)-mediated relaxations leading to impaired endothelium-dependent vasodilation. An important source of ROS is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the inhibition of this enzyme is an active area of interest. This study aimed to investigate the effects of apocynin, an NADPH oxidase inhibitor, on endothelial dysfunction and on the expression of NO synthase (NOS) and NADPH oxidase in thoracic aorta of diabetic rats. Method Streptozotocin (STZ)-diabetic rats received apocynin (16 mg/kg per day) for 4 weeks. Endothelium-dependent and -independent relaxations were determined in thoracic aortic rings. Western blotting and RT-PCR analysis were performed for NOSs and NADPH oxidase in the aortic tissue. Results Acetylcholine-induced relaxations and l-NAME-induced contractions were decreased in diabetic aorta. The decrease in acetylcholine and l-NAME responses were prevented by apocynin treatment without a significant change in plasma glucose levels. Endothelial NOS (eNOS) protein and mRNA expression exhibited significant decrease in diabetes, while protein and/or mRNA expressions of inducible NOS (iNOS) as well as p22 phox and gp91 phox subunits of NADPH oxidase were increased, and these alterations were markedly prevented by apocynin treatment. Conclusion NADPH oxidase expression is increased in diabetic rat aorta. NADPH oxidase-mediated oxidative stress is accompanied by the decreased eNOS and increased iNOS expressions, contributing to endothelial dysfunction. Apocynin effectively prevents the increased NADPH oxidase expression in diabetic aorta and restores the alterations in NOS expression, blocking the vicious cycle leading to diabetes-associated endothelial dysfunction.

Propofol induces endothelial nitric oxide synthase phosphorylation and activation in human umbilical vein endothelial cells by inhibiting protein kinase Cδ expression

Protein kinase Cs (PKCs) are involved in the regulation of endothelial nitric oxide synthase (eNOS). The purpose of this study is to evaluate the role of PKC delta in mediating the effects of chronic treatment with propofol on eNOS activation in human umbilical venous endothelial cells. Human umbilical venous endothelial cells were treated chronically with propofol (1-100 micromol l(-1)) to induce phosphorylation of eNOS activation, which was assessed by immunoblot and cyclic GMP accumulation assay. The involvement of the phosphoinositide 3-kinase/Akt pathway was examined by an immunoblot assay. In addition, the role of PKC delta in regulating propofol-induced activation of eNOS was explored. Finally, cells were treated with a combination of ceramide (10 nmol l(-1)) and propofol (50 micromol l(-1)), and the role of protein phosphatase 2A in mediating drug-induced eNOS activation was evaluated. Cells treated with propofol showed an increase in phosphorylation of eNOS at serine(1177) in a dose-dependent manner. Cyclic GMP accumulation was also increased accordingly. Akt was not phosphorylated in response to propofol (50 micromol l(-1)) at serine(473). In addition, a phosphoinositide 3-kinase inhibitor, LY294002 (10 micromol l(-1)), did not affect eNOS phosphorylation and nitric oxide production by propofol. Chronic treatment with propofol induced a downregulation of PKC delta, which was associated with eNOS activation. Moreover, ceramide inhibited the effect of propofol on eNOS activation. Propofol induces eNOS activation through a PKC delta inhibition-dependent, protein phosphatase 2A-coordinated, but phosphoinositide 3-kinase/Akt-independent pathway.

β-Actin Association with Endothelial Nitric-oxide Synthase Modulates Nitric Oxide and Superoxide Generation from the Enzyme

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.

Differential regulation of inducible and endothelial nitric oxide synthase by kinin B1 and B2 receptors

Kinins are vasoactive peptides that play important roles in cardiovascular homeostasis, pain and inflammation. After release from their precursor kininogens, kinins or their C-terminal des-Arg metabolites activate two distinct G protein-coupled receptors (GPCR), called B2 (B2R) or B1 (B1R). The B2R is expressed constitutively with a wide tissue distribution. In contrast, the B1R is not expressed under normal conditions but is upregulated by tissue insult or inflammatory mediators. The B2R is considered to mediate many of the acute effects of kinins while the B1R is more responsible for chronic responses in inflammation. Both receptors can couple to Galphai and Galphaq families of G proteins to release mediators such as nitric oxide (NO), arachidonic acid, prostaglandins, leukotrienes and endothelium-derived hyperpolarizing factor and can induce the release of other inflammatory agents. The focus of this review is on the different transduction events that take place upon B2R and B1R activation in human endothelial cells that leads to generation of NO via activation of different NOS isoforms. Importantly, B2R-mediated eNOS activation leads to a transient ( approximately 5min) output of NO in control endothelial cells whereas in cytokine-treated endothelial cells, B1R activation leads to very high and prolonged ( approximately 90min) NO production that is mediated by a novel signal transduction pathway leading to post-translational activation of iNOS.

Cytosolic phospholipase A2 S-nitrosylation in ghrelin protection against detrimental effect of ethanol cytotoxicity on gastric mucin synthesis ——Ghrelin in gastric mucosal protection

Ghrelin, a peptide hormone produced mainly in the stomach, has emerged recently as an important regulator of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems, the products of which play direct cytoprotective function in the maintenance of gastric mucosal integrity. In this study, using gastric mucosal cells, we report on the role of ghrelin in countering the cytotoxic effect of ethanol on mucin synthesis. We show that the countering effect of ghrelin on mucin synthesis was associated with the increase in NO and PGE2 production, and characterized by a marked up-regulation in cytosolic phospholipase A2 (cPLA2) activity. The ghrelin-induced up-regulation in mucin synthesis, like that of cPLA2 activity, was subject to suppression by Src inhibitor, PP2 and ERK inhibitor, PD98059, as well as ascorbate. Moreover, the loss in countering effect of ghrelin on the ethanol cytotoxicity and mucin synthesis was attained with cNOS inhibitor, L-NAME as well as COX-1 inhibitor SC-560. The effect of L-NAME was reflected in the inhibition of ghrelin-induced mucosal cell capacity for NO production, cPLA2 S-nitrosylation and PGE2 generation, whereas COX-1 inhibitor caused only the inhibition in PGE2 generation. Our findings suggest that the activation of gastric mucosal cPLA2 through cNOS-induced S-nitrosylation plays an essential role in the countering effect of ghrelin on the disturbances in gastric mucin synthesis caused by ethanol cytotoxicity.

Regulation of endothelial nitric oxide synthase by agmatine after transient global cerebral ischemia in rat brain

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) plays a protective role in cerebral ischemia by maintaining vascular permeability, whereas NO derived from neuronal and inducible NOS is neurotoxic and can participate in neuronal damage occurring in ischemia. Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane if brain vessels to promote cell death and tissue injury. We previously reported that agmatine, synthesized from L-arginine by arginine decarboxylase (ADC) which is expressed in endothelial cells, has shown a direct increased eNOS expression and decreased MMPs expression in bEnd3 cells. But, there are few reports about the regulation of eNOS by agmatine in ischemic animal model. In the present study, we examined the expression of eNOS and MMPs by agmatine treatment after transient global ischemia in vivo. Global ischemia was induced with four vessel occlusion (4-VO) and agmatine (100 mg/kg) was administered intraperitoneally at the onset of reperfusion. The animals were euthanized at 6 and 24 hours after global ischemia and prepared for other analysis. Global ischemia led severe neuronal damage in the rat hippocampus and cerebral cortex, but agmatine treatment protected neurons from ischemic injury. Moreover, the level and expression of eNOS was increased by agmatine treatment, whereas inducible NOS (iNOS) and MMP-9 protein expressions were decreased in the brain. These results suggest that agmatine protects microvessels in the brain by activation eNOS as well as reduces extracellular matrix degradation during the early phase of ischemic insult.

Shear stress regulation of nitric oxide production in uterine and placental artery endothelial cells: experimental studies and hemodynamic models of shear stresses on endothelial cells

Hemodynamic shear stress is the most powerful physiological regulator of endothelial Nitric Oxide Synthase (eNOS), leading to rapid rises in nitric oxide (NO). The substantial increases in uterine and placental blood flows throughout gestation rely heavily on the action of NO. We and others have investigated endothelial function in response to shear stress with cell culture models of shear stress. In order to apply the results of these studies more effectively, we need a more complete understanding of the origin and coupling of the hemodynamic forces and vascular tissue behavior. For example, equations commonly used to calculate in vivo shear stress incorporate assumptions of steady (non-pulsatile) blood flow and constant viscosity of blood (Newtonian fluid). Using computational models, we can estimate a waveform of shear stress over a cardiac cycle and the change in blood viscosity with shear rate and hematocrit levels, two variables that often change with size of vessel and location within a vascular tree. This review discusses hemodynamics as they apply to blood flow in vessels, in the hope that an integration of these fields can lead to improved in vitro shear stress experiments and understanding of NO production in uterine and placental vascular physiology during gestation.

Shear stress-induced ATP-mediated endothelial constitutive nitric oxide synthase expression in human lymphatic endothelial cells

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/cm(2) increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from 10(-9) to 10(-6) M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by 10(-6)M ATP was significantly reduced by 10(-5) M suramin. Suramin (10(-5) M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, 10(-4) M apamin, 10(-9) M iberiotoxin, 10(-5) M 2-aminoethoxydephenyl borate, or 10(-5)M xestospongin C, but not 10(-5) M glybenclamide or 10(-5) M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small- (SK(Ca)) and big-conductance (BK(Ca)) Ca(2+)-activated K(+) channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphate-mediated release of intracellular Ca(2+) ions and the activation of Ca(2+)-activated K(+) channels in LEC.