Altered l-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells

2002 ◽  
Vol 103 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Raj C. THURAISINGHAM ◽  
Norman B. ROBERTS ◽  
Mark WILKES ◽  
David I. NEW ◽  
A. Claudio MENDES-RIBEIRO ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
In Vivo Model ◽  
No Production ◽  
Arginine Metabolism ◽  
Enos Expression ◽  
Nitric Oxide Release ◽  
Excessive Consumption ◽  
And Control

Results regarding the nitric oxide (NO) system in uraemia are contradictory. l-Arginine, the precursor of NO, is also metabolized by arginase to form ornithine and urea. In the present study, endothelial NO production and arginine metabolism in uraemia were assessed. In addition an in vivo model was used to examine excess consumption of NO in uraemia. NO and amino acid measurements were made from basal and stimulated (by bradykinin) uraemic and control endothelial cells. Reverse-transcriptase PCR was used to assess endothelial NO synthase (eNOS) and inducible NOS (iNOS) expression. Finally, aortae of uraemic rats were stained for nitrotyrosine (a marker of peroxynitrite). Basal uraemic cells produced more NO than the control cells. l-Arginine levels were greater in uraemic (supernatants/cells), but ornithine levels were higher in control (supernatants/cells). Following stimulation, NO levels in supernatants were similar, but the rise in NO production was greater in control compared with uraemic cells; l-arginine levels still remained higher in uraemic supernatants/cells. Differences in ornithine concentration (supernatants/cells) disappeared following bradykinin stimulation, due to a rise in ornithine levels in the uraemic group. There was no difference in eNOS expression, nor was iNOS detected in either group. Only aortae from uraemic rats showed evidence for nitrotyrosine staining. These studies demonstrated increased basal NO release in uraemic endothelial cells, perhaps by inhibition of arginase and hence diversion of arginine to the NO pathway. The increased NO produced under basal conditions may be inactive due to excessive consumption, resulting in peroxynitrite formation. Interestingly, bradykinin appears to restore arginase activity in uraemia, resulting in normalization of NO production.

Reduced perivascular Po2 increases nitric oxide release from endothelial cells

2003 ◽  
Vol 285 (2) ◽  
pp. H507-H515 ◽  
Author(s):  
G. P. Nase ◽  
J. Tuttle ◽  
H. G. Bohlen
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Parenchymal Cell ◽  
Oxygen Availability ◽  
No Production ◽  
Main Site ◽  
Nitric Oxide Release ◽  
No Release ◽  
Parenchymal Cells

Many studies have suggested that endothelial cells can act as “oxygen sensors” to large reductions in oxygen availability by increasing nitric oxide (NO) production. This study determined whether small reductions in oxygen availability enhanced NO production from in vivo intestinal arterioles, venules, and parenchymal cells. In vivo measurements of perivascular NO concentration ([NO]) were made with NO-sensitive microelectrodes during normoxic and reduced oxygen availability. During normoxia, intestinal first-order arteriolar [NO] was 397 ± 26 nM ( n = 5), paired venular [NO] was 298 ± 34 nM ( n = 5), and parenchymal cell [NO] was 138 ± 36 nM ( n = 3). During reduced oxygen availability, arteriolar and venular [NO] significantly increased to 695 ± 79 nM ( n = 5) and 534 ± 66 nM ( n = 5), respectively, whereas parenchymal [NO] remained unchanged at 144 ± 34 nM ( n = 4). During reduced oxygenation, arteriolar and venular diameters increased by 15 ± 3% and 14 ± 5%, respectively: NG-nitro-l-arginine methyl ester strongly suppressed the dilation to lower periarteriolar Po2. Micropipette injection of a CO2 embolus into arterioles significantly attenuated arteriolar dilation and suppressed NO release in response to reduced oxygen availability. These results indicated that in rat intestine, reduced oxygen availability increased both arteriolar and venular NO and that the main site of NO release under these conditions was from endothelial cells.

scholarly journals A theoretical model of nitric oxide transport in arterioles: frequency- vs. amplitude-dependent control of cGMP formation

2004 ◽  
Vol 286 (3) ◽  
pp. H1043-H1056 ◽  
Author(s):  
Nikolaos M. Tsoukias ◽  
Mahendra Kavdia ◽  
Aleksander S. Popel
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Soluble Guanylate Cyclase ◽  
Muscle Tone ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
No Production ◽  
Cgmp Formation

Nitric oxide (NO) plays many important physiological roles, including the regulation of vascular smooth muscle tone. In response to hemodynamic or agonist stimuli, endothelial cells produce NO, which can diffuse to smooth muscle where it activates soluble guanylate cyclase (sGC), leading to cGMP formation and smooth muscle relaxation. The close proximity of red blood cells suggests, however, that a significant amount of NO released will be scavenged by blood, and thus the issue of bioavailability of endothelium-derived NO to smooth muscle has been investigated experimentally and theoretically. We formulated a mathematical model for NO transport in an arteriole to test the hypothesis that transient, burst-like NO production can facilitate efficient NO delivery to smooth muscle and reduce NO scavenging by blood. The model simulations predict that 1) the endothelium can maintain a physiologically significant amount of NO in smooth muscle despite the presence of NO scavengers such as hemoglobin and myoglobin; 2) under certain conditions, transient NO release presents a more efficient way for activating sGC and it can increase cGMP formation severalfold; and 3) frequency-rather than amplitude-dependent control of cGMP formation is possible. This suggests that it is the frequency of NO bursts and perhaps the frequency of Ca2+ oscillations in endothelial cells that may limit cGMP formation and regulate vascular tone. The proposed hypothesis suggests a new functional role for Ca2+ oscillations in endothelial cells. Further experimentation is needed to test whether and under what conditions in silico predictions occur in vivo.

Altered l-arginine metabolism results in increased nitric oxide release from uraemic endothelial cells

2002 ◽  
Vol 103 (1) ◽  
pp. 31 ◽  
Author(s):  
Raj C. THURAISINGHAM ◽  
Norman B. ROBERTS ◽  
Mark WILKES ◽  
David I. NEW ◽  
A. Claudio MENDES-RIBEIRO ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Arginine Metabolism ◽  
Nitric Oxide Release

Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

2005 ◽  
Vol 288 (1) ◽  
pp. H89-H95 ◽  
Author(s):  
Brett G. Zani ◽  
H. Glenn Bohlen
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Blood Flow ◽  
Intestinal Blood Flow ◽  
No Production ◽  
Sodium Ions ◽  
Major Mechanism ◽  
Before And After ◽  
Endothelial Nitric Oxide

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na+-K+-2Cl− cotransporter inhibitor) or amiloride (Na+/H+ exchange channel inhibitor). Suppressing amiloride-sensitive Na+/H+ exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na+-K+-2Cl− channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na+/Ca2+ exchanger extrudes Na+ in exchange for Ca2+, thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na+/Ca2+ exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na+-K+-2Cl− channels. The Na+/Ca2+ exchanger then extrudes Na+ and increases endothelial Ca2+. The increase in endothelial Ca2+ causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.

Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis

2009 ◽  
Vol 296 (6) ◽  
pp. E1440-E1449 ◽  
Author(s):  
Fina Lovren ◽  
Yi Pan ◽  
Praphulla C. Shukla ◽  
Adrian Quan ◽  
Hwee Teoh ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Vein Graft ◽  
Map Kinases ◽  
Enos Expression ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Enos Phosphorylation

Improving endothelial nitric oxide synthase (eNOS) bioactivity and endothelial function is important to limit native, vein graft, and transplant atherosclerosis. Visfatin, a NAD biosynthetic enzyme, regulates the activity of the cellular survival factor, Sirt1. We hypothesized that visfatin may improve eNOS expression, endothelial function, and postnatal angiogenesis. In human umbilical vein (HUVEC) and coronary artery endothelial cells, we evaluated the effects of recombinant human visfatin on eNOS protein and transcript expression and mRNA stability, in the presence and absence of visfatin RNA silencing. We also assessed visfatin-induced protein kinase B (Akt) activation and its association with src-tyrosine kinases, phosphorylation of Ser1177within eNOS in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibition with LY-294002, and evaluated the contributory role of extracellular signal-regulated kinase 1/2. Finally, we determined the impact of visfatin on HUVEC migration, proliferation, inflammation-induced permeability, and in vivo angiogenesis. Visfatin (100 ng/ml) upregulated and stabilized eNOS mRNA and increased the production of nitric oxide and cGMP. Visfatin-treated HUVEC demonstrated greater proliferation, migration, and capillary-like tube formation but less tumor necrosis factor-α-induced permeability; these effects were decreased in visfatin gene-silenced cells. Visfatin increased total Akt and Ser473-phospho-Akt expression with concomitant rises in eNOS phosphorylation at Ser1177; these effects were blocked by LY-2940002. Studies with PP2 showed that the nonreceptor tyrosine kinase, src, is an upstream stimulator of the PI 3-kinase-Akt pathway. Visfatin also activated mitogen-activated protein (MAP) kinase through PI 3-kinase, and mitogen/extracellular signal-regulated kinase inhibition attenuated visfatin-elicited Akt and eNOS phosphorylation. Visfatin-filled Matrigel implants showed an elevated number of infiltrating vessels, and visfatin treatment produced significant recovery of limb perfusion following hindlimb ischemia. These results indicate a novel effect of visfatin to stimulate eNOS expression and function in endothelial cells, via a common upstream, src-mediated signaling cascade, which leads to activation of Akt and MAP kinases. Visfatin represents a translational target to limit endothelial dysfunction, native, vein graft and transplant atherosclerosis, and improve postnatal angiogenesis.

scholarly journals Indolent course of tubulointerstitial disease in a mouse model of subpressor, low-dose nitric oxide synthase inhibition

2008 ◽  
Vol 295 (3) ◽  
pp. F717-F725 ◽  
Author(s):  
Adelina Stoessel ◽  
Alexander Paliege ◽  
Franziska Theilig ◽  
Francesco Addabbo ◽  
Brian Ratliff ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Basement Membrane ◽  
Mouse Model ◽  
Asymmetric Dimethylarginine ◽  
In Vivo Model ◽  
No Production ◽  
Collagen Xviii

Deficiency of nitric oxide (NO) represents a consistent manifestation of endothelial dysfunction (ECD), and the accumulation of asymmetric dimethylarginine occurs early in renal disease. Here, we confirmed in vitro and in vivo the previous finding that a fragment of collagen XVIII, endostatin, was upregulated by chronic inhibition of NO production and sought to support a hypothesis that primary ECD contributes to nephrosclerosis in the absence of other profibrotic factors. To emulate more closely the indolent course of ECD, the study was expanded to an in vivo model with NG-monomethyl-l-arginine(l-NMMA; mimics effects of asymmetric dimethylarginine) administered to mice in the drinking water at subpressor doses of 0.3 and 0.8 mg/ml for 3–6 mo. This resulted in subtle but significant morphological alterations detected in kidneys of mice chronically treated with l-NMMA: 1) consistent perivascular expansion of interstitial matrix components at the inner stripe of the outer medulla and 2) collagen XVIII/endostatin abundance. Ultrastructural abnormalities were detected in l-NMMA-treated mice: 1) increased activity of the interstitial fibroblasts; 2) occasional detachment of endothelial cells from the basement membrane; 3) splitting of the vascular basement membrane; 4) focal fibrosis; and 5) accumulation of lipofuscin by interstitial fibroblasts. Preembedding labeling of microvasculature with anti-CD31 antibodies showed infiltrating leukocytes and agglomerating platelets attaching to the visibly intact or denuded capillaries. Collectively, the data indicate that the mouse model of subpressor chronic administration of l-NMMA is not a robust one (endothelial pathology visible only ultrastructurally), and yet it closely resembles the natural progression of endothelial dysfunction, microvascular abnormalities, and associated tubulointerstitial scarring.

scholarly journals Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase

2006 ◽  
Vol 174 (3) ◽  
pp. 369-377 ◽  
Author(s):  
Carlos Fernández-Hernando ◽  
Masaki Fukata ◽  
Pascal N. Bernatchez ◽  
Yuko Fukata ◽  
Michelle I. Lin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Cdna Clones ◽  
No Production ◽  
Human Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

scholarly journals Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3190-3198 ◽  
Author(s):  
Hongwei Si ◽  
Jie Yu ◽  
Hongling Jiang ◽  
Hazel Lum ◽  
Dongmin Liu
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Enos Expression ◽  
Camp Response Element ◽  
Aortic Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Human Aortic Endothelial Cells

We previously reported that genistein, a phytoestrogen, up-regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats that are independent of estrogen signaling machinery. However, how genistein regulates eNOS expression is unknown. In the present study, we show that genistein enhanced eNOS expression and NO synthesis in primary human aortic endothelial cells. Inhibition of extracellular signal regulated kinase, phosphoinositol-3 kinase, or protein kinase C did not affect genistein-enhanced eNOS expression and NO synthesis. However, chemical inhibition of protein kinase A (PKA) or adenoviral transfer of the specific endogenous PKA inhibitor gene completely abolished PKA activity and genistein-stimulated eNOS expression and NO production. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP response element (CRE)-binding protein (CREB) at Ser133. Suppression of CREB by small interfering RNA transfection abolished genistein-enhanced eNOS expression and NO production. Consistently, deletion of the CRE site within human eNOS promoter eliminated genistein-stimulated eNOS promoter activity. These findings provide the first evidence to our knowledge that genistein may play a beneficial role in vascular function through targeting the PKA/CREB/eNOS/NO signaling pathway.

scholarly journals Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension

2016 ◽  
Vol 310 (11) ◽  
pp. L1199-L1205 ◽  
Author(s):  
Sudakshina Ghosh ◽  
Manveen Gupta ◽  
Weiling Xu ◽  
Deloris A. Mavrakis ◽  
Allison J. Janocha ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Molecular Mechanisms ◽  
Vascular Endothelial Cells ◽  
Vascular Diseases ◽  
No Production ◽  
Pulmonary Arterial

The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by β-blocker also restored NO formation by PAH cells, identifying one mechanism by which β-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions.

scholarly journals Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells

2021 ◽  
Vol 14 (6) ◽  
pp. 577
Author(s):  
Chin-Feng Hsuan ◽  
Thung-Lip Lee ◽  
Wei-Kung Tseng ◽  
Chau-Chung Wu ◽  
Chi-Chang Chang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Western Blot ◽  
Umbilical Vein ◽  
No Production ◽  
Enos Expression ◽  
Human Umbilical Vein ◽  
Whole Plant ◽  
Umbilical Vein Endothelial Cells ◽  
Glossogyne Tenuifolia

The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

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