scholarly journals Nitric oxide formation by lymphatic bulb and valves is a major regulatory component of lymphatic pumping

2011 ◽  
Vol 301 (5) ◽  
pp. H1897-H1906 ◽  
Author(s):  
H. Glenn Bohlen ◽  
Olga Yu. Gasheva ◽  
David C. Zawieja
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Stroke Volume ◽  
No Production ◽  
Dependent Manner ◽  
Nitric Oxide Formation ◽  
Valve Housing ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Microscopic lymphatics produce nitric oxide (NO) during contraction as flow shear activates the endothelial cells. The valve leaflets and bulbous valve housing contain a large amount of endothelial nitric oxide synthase (eNOS) due both to many endothelial cells and increased expression of eNOS. Direct NO measurements indicate the valve area has a 30–50% higher NO concentration ([NO]) than tubular regions although both regions generate equivalent relative increases in [NO] with each contraction. We hypothesize that 1) the greater eNOS and [NO] of the bulb region would have greater effects to lower pumping activity of the overall lymphatic than occurs in tubular regions and 2), the elevated [NO] in the bulb region may be because of high NO production in the valve leaflets that diffuses to the wall of the bulb. Measurement of [NO] with a micropipette inside the lymphatic bulb revealed the valve leaflets generate ∼50% larger [NO] than the bulb wall in the in vivo rat mesenteric lymphatics. The valves add NO to the lymph that quickly diffuses to the bulb wall. Bradykinin locally released iontophoretically from a micropipette on both bulbs and tubes increased the [NO] in a dose-dependent manner up to ∼50%, demonstrating agonist activation of the NO pathway. However, pumping output determined by contraction frequency and stroke volume decreased much more for the bulb than tubular areas in response to the bradykinin. In effect, NO generation by the bulb area and its valves limits the pumped flow of the total lymphatic by lowering frequency and stroke volume of individual contractions.

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.

Effects of homocysteine on endothelial nitric oxide production

2000 ◽  
Vol 279 (4) ◽  
pp. F671-F678 ◽  
Author(s):  
Xiaohui Zhang ◽  
Hong Li ◽  
Haoli Jin ◽  
Zachary Ebin ◽  
Sergey Brodsky ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Redox State ◽  
Calcium Ionophore ◽  
No Production ◽  
Cellular Redox ◽  
A Cell ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Peroxynitrite Scavengers

Hyperhomocysteinemia (HHCy) is an independent and graded cardiovascular risk factor. HHCy is prevalent in patients with chronic renal failure, contributing to the increased mortality rate. Controversy exists as to the effects of HHCy on nitric oxide (NO) production: it has been shown that HHCy both increases and suppresses it. We addressed this problem by using amperometric electrochemical NO detection with a porphyrinic microelectrode to study responses of endothelial cells incubated with homocysteine (Hcy) to the stimulation with bradykinin, calcium ionophore, or l-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 μM) resulted in a gradual decline in responsiveness of endothelial cells to the above stimuli. Hcy did not affect the expression of endothelial nitric oxide synthase (eNOS), but it stimulated formation of superoxide anions, as judged by fluorescence of dichlorofluorescein, and peroxynitrite, as detected by using immunoprecipitation and immunoblotting of proteins modified by tyrosine nitration. Hcy did not directly affect the ability of recombinant eNOS to generate NO, but oxidation of sulfhydryl groups in eNOS reduced its NO-generating activity. Addition of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme nor formation of nitrotyrosine-modified proteins. In addition, a scavenger of peroxynitrite or a cell-permeant superoxide dismutase mimetic reversed the Hcy-induced suppression of NO production by endothelial cells. In conclusion, electrochemical detection of NO release from cultured endothelial cells demonstrated that concentrations of Hcy >20 μM produce a significant indirect suppression of eNOS activity without any discernible effects on its expression. Folates, superoxide ions, and peroxynitrite scavengers restore the NO-generating activity to eNOS, collectively suggesting that cellular redox state plays an important role in HCy-suppressed NO-generating function of this enzyme.

scholarly journals Cilostazol Induces eNOS and TM Expression via Activation with Sirtuin 1/Krüppel-like Factor 2 Pathway in Endothelial Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10287
Author(s):  
Chih-Hsien Wu ◽  
Yi-Lin Chiu ◽  
Chung-Yueh Hsieh ◽  
Guo-Shiang Tsung ◽  
Lian-Shan Wu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Umbilical Vein ◽  
Sirtuin 1 ◽  
No Production ◽  
Beneficial Effects ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

scholarly journals Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase

2008 ◽  
Vol 86 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Syamantak Majumder ◽  
Ajit Muley ◽  
Gopi Krishna Kolluru ◽  
Samir Saurabh ◽  
K. P. Tamilarasan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Egg Yolk ◽  
Cellular Migration ◽  
No Production ◽  
Enos Phosphorylation ◽  
Low Levels ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.

scholarly journals Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation

2011 ◽  
Vol 286 (22) ◽  
pp. 20100-20108 ◽  
Author(s):  
Zhihong Xiao ◽  
Tingting Wang ◽  
Honghua Qin ◽  
Chao Huang ◽  
Youmei Feng ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Nitric Oxide Synthase ◽  
Protein Phosphorylation ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca2+ and protein phosphorylation, but the interrelationship between intracellular Ca2+ mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca2+ release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca2+ release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca2+ release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca2+ and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca2+ release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca2+/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca2+ release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca2+ release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca2+ is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca2+-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.

scholarly journals Endothelial nitric oxide synthase activation is required for heparin receptor effects on vascular smooth muscle cells

2020 ◽  
Vol 318 (3) ◽  
pp. C463-C475
Author(s):  
Yaqiu Li ◽  
Leanna M. Talotta-Altenburg ◽  
Kayli A. Silimperi ◽  
Grace O. Ciabattoni ◽  
Linda J. Lowe-Krentz
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Nitric Oxide Synthase ◽  
Vascular Smooth Muscle ◽  
Endothelial Nitric Oxide Synthase ◽  
Dependent Manner ◽  
Heparin Treatment ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Published studies indicate that TMEM184A is a heparin receptor that interacts with and transduces stimulation from heparin in vascular cells. Previous studies have indicated that heparin increases endothelial nitric oxide synthase (eNOS) activity in bovine endothelial cells. However, the precise mechanism remains unknown. In this study, we investigated the impact of heparin treatment and TMEM184A on eNOS’s activation and the role of eNOS in heparin signaling in the cloned A7r5 rat vascular smooth muscle cell line and confirmed results in endothelial cells. We employed a combination of TMEM184A knockdown A7r5 cells along with transient eNOS knockdown and enzyme inhibitor strategies. The results indicate that heparin induces phosphorylation of eNOS. eNOS can be immunoprecipitated with TMEM184A and is internalized to the perinuclear region in a TMEM184A-dependent manner in response to heparin. We also examined how heparin treatment leads to phosphorylation of eNOS and confirmed that TMEM184A and Ca2+ were required to mediate heparin-elicited eNOS phosphorylation. Evidence supporting the involvement of transient receptor potential cation channel subfamily V member 4 with TMEM184A in this eNOS activation process is also presented.

scholarly journals The loss of sustained Ca2+ signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy

2013 ◽  
Vol 305 (7) ◽  
pp. H969-H979 ◽  
Author(s):  
Jennifer Krupp ◽  
Derek S. Boeldt ◽  
Fu-Xian Yi ◽  
Mary A. Grummer ◽  
Heather A. Bankowski Anaya ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Normal Subjects ◽  
Fura 2 ◽  
Intracellular Ca ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca2+ concentration ([Ca2+]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 μM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 μM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca2+]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca2+]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca2+]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.

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.

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

2006 ◽  
Vol 291 (6) ◽  
pp. C1240-C1246 ◽  
Author(s):  
Erin Lepic ◽  
Dylan Burger ◽  
Xiangru Lu ◽  
Wei Song ◽  
Qingping Feng
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Brdu Incorporation ◽  
No Production ◽  
Dependent Manner ◽  
Cardiomyocyte Proliferation ◽  
Cell Counts ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS−/−) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes ( P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS−/− cardiomyocytes ( P < 0.05). Inhibition of nitric oxide synthase activity using NG-nitro-l-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes ( P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner ( P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS−/− cardiomyocytes ( P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS−/− hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development.

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