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At least 19 recordsLinked to original sources
PubMed2005

Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis.

There is a complex pathophysiologic scenario involving nitric oxide (NO), endothelial nitric oxide synthase (eNOS), and the development of atherosclerosis and unstable atheroma. Endothelial damage induced by atherosclerosis leads to the reduction in bioactivity of eNOS with subsequent impaired release of NO. An important mechanism is local enhanced degradation of NO by increased generation of reactive oxygen species and other free radicals, with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Novel molecular approaches have resulted in the development of new strains of mice lacking eNOS. These experimental models will help to understand how to implement NO-based therapies against atherosclerosis. L-arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. The target or goal for new drugs should be the complete restoration of NO-mediated signaling pathways in atherosclerotic arteries.

Arginine↗
PubMed2004

Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis.

There is a complex pathophysiologic scenario involving nitric oxide (NO), endothelial nitric oxide synthase (eNOS), and the development of atherosclerosis and unstable atheroma. Endothelial damage induced by atherosclerosis leads to the reduction in bioactivity of ENOS with subsequent impaired release of NO. An important mechanism is local enhanced degradation of NO by increased generation of reactive oxygen species and other free radicals, with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Novel molecular approaches have resulted in the development of new strains of mice lacking eNOS. These experimental models will help to understand how to implement NO-based therapies against atherosclerosis. L-arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. The target or goal for new drugs should be the complete restoration of NO-mediated signaling pathways in atherosclerotic arteries.

Animals↗
PubMedDate not supplied

Effects of pulsatile shear stress on signaling mechanisms controlling nitric oxide production, endothelial nitric oxide synthase phosphorylation, and expression in ovine fetoplacental artery endothelial cells.

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm2, then were exposed to 10, 15, or 25 dynes/cm2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p < .05). Production of NO by OFPAE cells exposed to pulsatile shear stress was de novo; i.e., inhibited by L-NMMA (N(G)-monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm2 (8-fold) exceeded (p < .05) that seen for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). Acute rises in NO production with shear stress occurred by eNOS activation, whereas prolonged NO rises were via elevations in both eNOS expression and enzyme activation. The authors therefore used Western analysis to investigate the signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by "flow-adapted" OFPAE cells. Increasing shear stress from 3 to 15 dynes/cm2 very rapidly increased eNOS Ser1177, ERK1/2 (extracellular signal-regulated kinase 1 and 2) and Akt, but not p38 MAPK (p38 mitogen-activated protein kinase) phosphorylation by Western analysis. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by PI-3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and LY294002, but not the MEK (MAPK kinase) inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels induced by 15 dynes/cm2 shear stress. Blocking of either signaling pathways or p38 MAPK did not change the shear stress-induced increase in eNOS protein levels. Therefore, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibiting MEK. Prolonged shear stress-stimulated increases in eNOS protein levels were not affected by inhibition of MEK- or PI-3K-mediated pathways. In conclusion, pulsatile shear stress greatly induces NO production by OFPAE cells through the mechanisms of both PI-3K-mediated eNOS activation and elevations in eNOS protein levels; bFGF does not further stimulate eNOS expression under flow condition.

Animals↗
PubMed2005

The effect of cardiopulmonary bypass on the expression of inducible nitric oxide synthase, endothelial nitric oxide synthase, and vascular endothelial growth factor in the internal mammary artery.

OBJECTIVE: Endothelial function of a vessel may be impaired by local or systemic inflammation initiated by cardiopulmonary bypass (CPB) during coronary artery bypass graft (CABG) surgery. The present study was designed to investigate the early effects of CPB on nitric oxide production and vascular endothelial growth factor (VEGF) expression in internal mammary artery (IMA). DESIGN: Prospective study. SETTING: University hospital. PARTICIPANTS: Twenty patients who were scheduled for elective CABG with CPB. INTERVENTIONS: IMA sections were studied immunohistochemically from these patients. The samples were taken from the distal end of the IMA before the institution of CPB and just before the construction of the IMA-left anterior descending artery anastomosis. MEASUREMENT AND MAIN RESULTS: After CPB, VEGF and endothelial nitric oxide synthase immunoreactivity increased significantly when compared with baseline values in the endothelium (p = 0.0156, p = 0.0313) and adventitia (p = 0.0313, p = 0.0001), respectively. No significant change was observed in inducible nitric oxide synthase immunoreactivity. CONCLUSIONS: The increase in eNOS expression may have been induced by the inflammation caused by CPB.

Cardiopulmonary Bypass↗
PubMedDate not supplied

mRNA expression of inducible nitric oxide synthase, endothelial nitric oxide synthase and vascular endothelial growth factor in esophageal mucosa biopsy specimens from patients with reflux esophagitis.

BACKGROUND/AIMS: Nitric oxide (NO) production is elevated in the intestine and may contribute to intestinal injury during inflammation. However, how the expression of inducible NO synthase (iNOS) mRNA and endothelial NO synthase (eNOS) mRNA in the esophageal mucosa contribute to mucosal damage caused by reflux esophagitis remains unknown. Since vascular endothelial growth factor (VEGF) exerts its action on microcirculation, contributing to angiogenesis and inflammation, we examined the role of VEGF together with iNOS and eNOS on development of reflux esophagitis. METHODOLOGY: The mRNA expression levels of iNOS, eNOS and VEGF were measured in biopsy specimens from 25 patients with reflux esophagitis, using TaqMan PCR and reverse transcription PCR. RESULTS: The expression of iNOS mRNA in the esophageal mucosa increased parallel to the severity of the esophagitis. There were no significant differences between both eNOS and VEGF mRNA expression levels and the severity of the esophagitis. The existence of gastric mucosal atrophy, hiatus hernia, therapy and Helicobacter pylori infection did not affect the levels of mRNA expression. CONCLUSIONS: The accumulation of NO, produced by iNOS, was considered to be related to the exacerbation of reflux esophagitis. Therapeutic intervention that reduces NO production may thus be of use in preventing development of esophageal mucosal injury in patients with reflux esophagitis.

Adult↗
PubMed2000

Myocardial glucose uptake is regulated by nitric oxide via endothelial nitric oxide synthase in Langendorff mouse heart.

Although the role of nitric oxide (NO) in the modulation of vascular tone has been studied and well understood, its potential role in the control of myocardial metabolism is only recently evident. Several lines of evidence indicate that NO regulates myocardial glucose metabolism; however, the details and mechanisms responsible are still unknown. The aim of this study was to further define the role of NO in the control of myocardial glucose metabolism and the nitric oxide synthase (NOS) isoform responsible using transgenic animals lacking endothelial NOS (ecNOS). In the present study, we examined the regulation of myocardial glucose uptake using isometrically contracting Langendorff-perfused hearts from normal mice (C57BL/6J), mice with defects in the expression of ecNOS [ecNOS (-/-)], and its heterozygote [ecNOS (+/-)], and wild-type mice [ecNOS (+/+)] (n=6, respectively). In hearts from normal mice, little myocardial glucose uptake was observed. This myocardial glucose uptake increased significantly in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Similarly, in the hearts from ecNOS (-/-), glucose uptake was much greater than in normal mice, whereas myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice was not different from normal mice. In addition, myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice increased significantly in the presence of L-NAME. At a workload of 800 g. beats/min, L-NAME increased glucose uptake from 0.1+/-0.1 to 3+/-0.4 microg/min x mg in ecNOS (+/-) mice and from 0.2+/-0.1 to 2.7+/-0.7 microg/min x mg in ecNOS (+/+) mice. Furthermore, in the hearts from ecNOS (-/-) mice, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog or S-nitroso-N-acetylpenicillamine (SNAP), a NO donor essentially shut off glucose uptake, and in hearts from ecNOS (+/-) mice, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), an inhibitor of cGMP, increased the glucose uptake significantly. These results indicate clearly that cardiac NO production regulates myocardial glucose uptake via a cGMP-dependent mechanism and strongly suggest that ecNOS plays a pivotal role in this regulation. These findings may be important in the understanding of the pathogenesis of the diseases such as ischemic heart disease, heart failure, diabetes mellitus, hypertension, and hypercholesterolemia, in which NO synthesis is altered and substrate utilization by the heart changes.

Animals↗
PubMed2005

A defect of neuronal nitric oxide synthase increases xanthine oxidase-derived superoxide anion and attenuates the control of myocardial oxygen consumption by nitric oxide derived from endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) plays an important role in the control of myocardial oxygen consumption (MVO2) by nitric oxide (NO). A NOS isoform is present in cardiac mitochondria and it is derived from neuronal NOS (nNOS). However, the role of nNOS in the control of MVO2 remains unknown. MVO2 in left ventricular tissues from nNOS-/- mice was measured in vitro. Stimulation of NO production by bradykinin or carbachol induced a significant reduction in MVO2 in wild-type (WT) mice. In contrast to WT, bradykinin- or carbachol-induced reduction in MVO2 was attenuated in nNOS-/-. S-methyl-L-thiocitrulline, a potent isoform selective inhibitor of nNOS, had no effect on bradykinin-induced reduction in MVO2 in WT. Bradykinin-induced reduction in MVO2 in eNOS-/- mice, in which nNOS still exists, was also attenuated. The attenuated bradykinin-induced reduction in MVO2 in nNOS-/- was restored by preincubation with Tiron, ascorbic acid, Tempol, oxypurinol, or SB203850, an inhibitor of p38 kinase, but not apocynin. There was an increase in lucigenin-detectable superoxide anion (O2-) in cardiac tissues from nNOS-/- compared with WT. Tempol, oxypurinol, or SB203850 decreased O2- in all groups to levels that were not different from each other. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOS-/- compared with WT mice. These results indicate that a defect of nNOS increases O2- through the activation of xanthine oxidase, which is mediated by the activation of p38 kinase, and attenuates the control of MVO2 by NO derived from eNOS.

Acetophenones↗
PubMed2002

Effect of uterine blood flow occlusion on shear stress-mediated nitric oxide production and endothelial nitric oxide synthase expression during ovine pregnancy.

During normal pregnancy, uterine blood flow (UBF) is increased in association with elevations of endothelial nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Shear stress increases endothelial-derived NO production to reduce vasomotor tone. We hypothesized that decreasing in vivo UBF, and thus shear stress, will decrease NO and/or eNOS levels. In this experiment, one of the main uterine arteries of chronically instrumented late pregnant sheep (125 +/- 1 days' gestation [mean +/- SEM]; n = 15) was occluded for 24 h. Cardiovascular parameters (systemic and uterine arterial pressure, heart rate [HR], and ipsilateral and contralateral UBF) and NO(2)/NO(3) (NO(x)) levels were evaluated. Although UBF measured using Transonic flow probes was reduced unilaterally 41.5% +/- 2.1%, uterine perfusion pressure only fell 12.2% +/- 4.5%. Systemic arterial blood pressure and HR were unaltered. Using radioactive microspheres, ipsilateral UBF was reduced approximately 28% during occlusion. The redistribution of UBF to other reproductive tissues suggests that collateral circulation develops in response to occlusion. Systemic arterial and uterine venous NO(x) levels were reduced 22.1% +/- 6.7% and 22.6% +/- 7.6%, respectively, during occlusion. Treatment with microspheres produced an unexpected initial ( approximately 2.5 h) increase in systemic arterial and uterine venous NO(x) levels by 116% +/- 30% and 97% +/- 49%, respectively. Despite a decline in NO(x) levels after 6 h, no significant differences versus preocclusion NO(x) levels were detected by 24 h of occlusion in this experimental group. In contrast, NO(x), UBF, and uterine perfusion pressure levels unexpectedly failed to return to baseline values following release of occlusion. No differences in uterine artery eNOS expression were demonstrated by Western analysis from occlusion. Thus, our data suggest that shear stress may mediate in vivo vasomotor tone via production of NO(x).

Animals↗
PubMed2006

Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanol-induced liver injury.

AIM: To study the expression and activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha) expression in the liver. METHODS: Female Sprague-Dawley rats were given fish oil (0.5 mL) along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase (ALT) activity and pathological analysis. Liver malondialdehyde (MDA), nitric oxide contents, iNOS and eNOS activity were determined. NF-kappaB p65ìiNOS, eNOS and TNF-alpha protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration (6 wk) enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-kappaB p65, iNOS and TNF-alpha protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB, and TNF-alpha mRNA expression. CONCLUSION: iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-kappaB and TNF-alpha expression. eNOS activity is reduced, but its mRNA expression is not affected.

Animals↗
PubMed2003

Effect of high glucose on nitric oxide production and endothelial nitric oxide synthase protein expression in human glomerular endothelial cells.

BACKGROUND: Hyperglycemia directly contributes to the development of diabetic nephropathy. Nitric oxide (NO), a potent endothelium-derived vasodilator, has been suggested to participate in the regulation of renal blood flow, glomerular filtration rate, and mesangial matrix accumulation. Human vascular endothelial cells are known to exhibit functional heterogeneity, this prompted us to do the first study of NO bioavailability in human glomerular endothelial cells (HGECs), in response to high glucose exposure. METHODS: NO release was examined by detecting nitrite generation by the Griess assay in HGECs exposed to control-level (5.5 mM) and high-level (15, 30 and 60 mM) glucose solutions at various time periods (24, 48 and 72 h) in the presence or absence of L-arginine (1 mM), or superoxide dismutase (SOD) (250 U/ml). In addition, we evaluated the effect of glucose on the expression of endothelial nitric oxide synthase (eNOS) in HGECs by Western blotting. RESULTS: Final levels of nitrite generated in HGECs were reduced significantly, in a time- and concentration-dependent manner, after high glucose exposure. However, Western blot analysis revealed that eNOS protein expression was significantly upregulated at 12 h after exposure to high glucose concentrations (30 mM), reaching a peak at 48 h (twofold increase over baseline levels). The inhibitory effect of high glucose on NO production was restored by the addition of SOD. Addition of L-arginine (1 mM) to external media also reversed the inhibitory effect of high glucose on NO production of HGECs as well. CONCLUSIONS: The present study demonstrated that high glucose increased eNOS protein expression, but decreased NO release finally. Decreased NO bioavailability seems to be associated with overproduction of superoxide and L-arginine deficiency. These findings provide an important clue in clarifying the molecular basis of the mechanisms by which elevated glucose leads to an imbalance between NO and superoxide, resulting in impaired endothelial function. In addition, restoration of NO function by both administration of L-arginine and adequate intake of antioxidants suggests a potential supportive treatment for patients with diabetic nephropathy.

Arginine↗
PubMed2003

Beyond lipid-lowering: effects of statins on endothelial nitric oxide.

Endothelial dysfunction is now recognised as an important process in the pathogenesis of atherosclerosis. Nitric oxide (NO) release by the endothelium regulates blood flow, inflammation and platelet aggregation, and consequently its disruption during endothelial dysfunction can decrease plaque stability and encourage the formation of atherosclerotic lesions and thrombi. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) are often utilised in the prevention of coronary heart disease due to their efficacy at lowering lipid levels. However, statins may also prevent atherosclerotic disease by non-lipid or pleiotropic effects, for example, improving endothelial function by promoting the production of NO. There are various mechanisms whereby statins may alter NO release, such as inhibiting the production of mevalonate and important isoprenoid intermediates, thereby preventing the isoprenylation of the small GTPase Rho, which negatively regulates the expression of endothelial nitric oxide synthase (eNOS). Furthermore, statins may also increase eNOS activity via post-translational activation of the phosphatidylinositol 3-kinase/protein kinase Akt (PI3 K/Akt) pathway and/or through an interaction with the molecular chaperone heat-shock protein 90 (HSP90). Data suggest that statins may vary in their efficacy for enhancing the release of NO, and the mechanisms dictating these differences are not yet clear. By increasing NO production, statins may interfere with atherosclerotic lesion development, stabilise plaque, inhibit platelet aggregation, improve blood flow and protect against ischaemia. Therefore, the ability of statins to improve endothelial function through the release of NO may partially account for their beneficial effects at reducing the incidence of cardiovascular events.

Arteriosclerosis↗
PubMed1998

Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs.

To examine the effect of chronic hypoxia on nitric oxide (NO) production and the amount of the endothelial isoform of nitric oxide synthase (eNOS) in lungs of newborn piglets, studies were performed using 1- to 3-day-old piglets raised in room air (control) or 10% O2 (chronic hypoxia) for 10-12 days. Exhaled NO output and plasma nitrites and nitrates (collectively termed NOx-) were measured in anesthetized animals. NOx- concentrations were measured in the perfusate of isolated lungs. eNOS amounts were assessed in whole lung homogenates. In the intact piglets, exhaled NO outputs and plasma NOx- were lower in the chronically hypoxic (exhaled NO output = 0.2 +/- 0.1 nmol/min; plasma NOx- = 10.3 +/- 3.7 nmol/ml) than in control animals (exhaled NO output = 0.8 +/- 0.2 nmol/min; plasma NOx- = 22.3 +/- 4.3 nmol/ml). In perfused lungs, the perfusate accumulation of NOx- was lower in chronic hypoxia (1.0 +/- 0.3 nmol/min) than in control (2.6 +/- 0.6 nmol/min) piglets. The amount of whole lung homogenate eNOS from the chronic hypoxia piglets was 40 +/- 8% less than that from the control piglets. The reduced NO production observed in anesthetized animals or perfused lungs of chronically hypoxic newborn piglets is consistent with the finding of reduced lung eNOS protein amounts. Decreased NO production might contribute to the development of chronic hypoxia-induced pulmonary hypertension in newborns.

Animals↗
PubMed2004

Hepatocyte growth factor stimulates nitric oxide production through endothelial nitric oxide synthase activation by the phosphoinositide 3-kinase/Akt pathway and possibly by mitogen-activated protein kinase kinase in vascular endothelial cells.

Hepatocyte growth factor (HGF) has recently been the focus of attention due to its angiogenic effects, which are similar to those of vascular endothelial growth factor (VEGF); because of these effects, HGF is considered to be a novel therapeutic agent against vascular disorders, including atherosclerotic angiopathies. Although nitric oxide (NO), which is derived from vascular endothelial cells (ECs), is also involved in angiogenesis, little is known regarding the interactions between HGF and NO. We therefore examined the effects of HGF on NO production as well as endothelial NO synthase (eNOS) phosphorylation, and investigated their mechanisms. In bovine aortic ECs, HGF induced a rapid (5 min) increase of NO production measured by diaminofluorescein-2 diacetate. Moreover, HGF rapidly (2.5 min) stimulated eNOS phosphorylation (Ser-1179) as determined by Western immunoblot analyses. Both of these effects were almost completely suppressed by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and were partially suppressed by the mitogen-activated protein kinase (MAPK) kinase 1/2 inhibitor U0126. HGF also stimulated Akt phosphorylation (Ser-473), which was completely suppressed by LY294002 and was partially suppressed by U0126. Moreover, HGF stimulated extracellular signal-regulated kinase 1/2 phosphorylation (Thr-202/Tyr-204), which was completely suppressed by U0126 and was partially suppressed by LY294002. Taken together, these results indicate that HGF not only phosphorylates eNOS through the PI3K/Akt pathway, but also partially through the MAPK pathway, and that these two pathways may interact. Compared with VEGF, HGF was more potent in both NO production and eNOS phosphorylation. Our study thus demonstrates a novel activity of HGF-the stimulation of NO production-which occurs via eNOS phosphorylation that may in turn be mediated by cross-talk between the PI3K/Akt and MAPK pathways.

Animals↗
PubMedDate not supplied

The Akt kinase signals directly to endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

Amino Acid Sequence↗
PubMed1994

Identification of covalently bound amino-terminal myristic acid in endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) is unique among the nitric oxide synthase family of proteins due to the presence of an N-myristoylation consensus sequence elucidated from the cloning of its cDNA. Although eNOS was metabolically labeled with [3H]myristic acid and mutation of glycine 2 in the N-myristoylation consensus sequence changed the particulate localization of the enzyme to a cytosolic form, the definitive characterization of eNOS as an N-myristoylprotein has not been demonstrated. Therefore, the purpose of the present study was to determine the nature of the fatty acid incorporated into eNOS. Wild-type or G2A mutant (mutation of glycine 2, the myristic acid acceptor site, to alanine) eNOS-transfected COS cells and bovine aortic endothelial cells (BAEC) were metabolically labeled with [3H]myristic acid for 5 h. The radiolabel was primarily incorporated into membrane-associated eNOS from wild-type transfected COS cells and cultured BAEC but not into the mutant eNOS from G2A-transfected COS cells. Qualitatively similar amounts of immunoreactive protein were found in wild-type and G2A-transfected cells. In addition, linkage of the radiolabel to eNOS was insensitive to hydroxylamine treatment, and incorporation of the radiolabel into eNOS was abolished by cyclo-heximide. Chemical analysis of the fatty acid released by acid methanolysis of labeled eNOS verified the 3H-labeled fatty acid as protein-bound myristic acid. These results unequivocally demonstrate that eNOS incorporates myristic acid via an amide linkage with the amino-terminal glycine of the enzyme as a co-translational modification.

Alanine↗
PubMed2006

Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) plays a crucial role in the regulation of a variety of cardiovascular functions. Many studies have shown that dietary n-3 polyunsaturated fatty acids (PUFAs) have beneficial effects on coronary atherosclerosis. However, the mechanisms of n-3 PUFAs regulation in eNOS activation remain unknown. In the present study we investigated the effects of eicosapentaenoic acid (EPA, 20:5 n-3) on subcellular distribution of eNOS and lipid composition of caveolae. We demonstrated for the first time that EPA treatment profoundly altered lipid composition and fatty acyl substitutions of phospholipids in caveolae. We found that caveolin-1 was solely located in caveolae fractions in control cells, and EPA treatment displaced caveolin-1 from caveolae. eNOS was detected in the caveolin-enriched fractions and noncaveolae fractions in control cells. EPA treatment induced the translocation of eNOS from caveolae fractions to soluble fractions. P-eNOS was also distributed in both fractions. After EPA treatment, the level of p-eNOS in each fraction was increased but the distribution of which was unaffected. Moreover, the results of immunofluorescence confirmed that EPA could redistribute caveolin-1 and eNOS in plasma membrane. eNOS activity in HUVEC cells was increased after EPA treatment, which was in a dose dependent manner. And incubation with 50 microM EPA had the maximum effect on eNOS activity. Our results suggested that eNOS translocation was paralleled by a stimulated capacity for NO production in the cells. We found that total Akt and p-Akt were primarily presented in heavy membranes in control cells, and the relative level of p-Akt increased but the distribution did not change after EPA treatment. The distribution of CaM was slightly changed after EPA treatment. Our results indicated that n-3 PUFAs profoundly altered caveolae microenvironment, thereby modifying location and function of proteins in caveolae. EPA-induced alterations of lipid and proteins in caveolae may be an important mechanism in the pathophysiologic process of atherosclerosis.

Blotting, Western↗
PubMed1995

Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) is a calmodulin (CaM)-dependent, membrane-associated, myristoylated enzyme, which has an important role in regulation of vascular tone and platelet aggregation. In this study, wild-type and mutant forms of bovine eNOS were overexpressed in a baculovirus/Sf9 insect cell system and examined for interactions with membrane phospholipids. Purified wild-type eNOS binds to pure anionic phospholipid vesicles but not to neutral phospholipid vesicles, demonstrating that eNOS attachment to lipid bilayers requires electrostatic as well as hydrophobic interactions. Moreover, catalytic activity of the enzyme is potently inhibited by anionic phospholipids, notably phosphatidylserine (PS), but not by neutral phospholipids. eNOS activity is also significantly inhibited upon enzyme binding to biological membranes isolated from cultured cells. Binding of eNOS to PS vesicles prevents subsequent binding of the enzyme to CaM-Sepharose. Interactions of eNOS with PS are not affected by site-specific mutation of the myristic acid acceptor site in the enzyme. Deletional mutation of the eNOS CaM-binding domain, however, results in loss of binding capacity of the enzyme not only for CaM-Sepharose but also for PS vesicles. Furthermore, removal of the CaM-binding domain converts eNOS from a membrane to a cytosolic protein when the enzyme is expressed in Sf9 cells. These data demonstrate that electrostatic interactions between anionic membrane phospholipids and basic residues in the eNOS CaM-binding domain are important for enzyme membrane association. Membrane association can thus function to inhibit eNOS catalytic activity by interfering with the interaction of the enzyme with calmodulin.

Amino Acid Oxidoreductases↗