Abstract 318: Eicosapentaenoic Acid Improved Nitric Oxide Bioavailability and Reduced Nitroxidative Stress in Human Endothelial Cells in Contrast to Arachidonic Acid In Vitro

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Preston Mason ◽  
Hazem Dawoud ◽  
Samuel Sherratt ◽  
Peter Libby ◽  
Deepak L Bhatt ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Fatty Acid ◽  
Eicosapentaenoic Acid ◽  
Calcium Ionophore ◽  
High Dose ◽  
No Production ◽  
Time Points ◽  
No Release

Treatment with prescription, high dose, stable icosapent ethyl (IPE), which is eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), significantly reduced clinical events in high-risk patients with diabetes and other risk factors or cardiovascular disease (REDUCE-IT). Previous studies suggest that the benefits of EPA correlate positively with its levels and ratio to arachidonic acid (AA) in circulation. Unlike EPA, AA is an omega-6 fatty acid (O6FA) that, along with its metabolites, contributes to inflammation and diabetes. One mechanism of benefit of an increased EPA to AA ratio may be improved endothelial cell (EC) function, as evidenced by increased nitric oxide (NO) release and decreased nitroxidative (ONOO – ) stress. In this study, human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or AA at equimolar levels (10 μM) at various time points (4-24 hr) in 5% FBS. Following treatment, the cells were stimulated with calcium ionophore and assayed for the ratio of NO and ONOO – release, an indicator of eNOS coupling, using tandem porphyrinic nanosensors. ECs treated with EPA had significantly greater NO release following stimulation compared with vehicle at all time points, including 17% and 21% at 4 and 24 hr, respectively (p<0.05 and p<0.01) without changes in eNOS expression. By contrast, AA did not significantly improve NO production. ECs treated with EPA also showed a non-significant reduction in ONOO - release by 10% at 4 hr and 14% at 24 hr. EPA, but not AA, increased NO/ONOO - release ratio by 42% (4.03 ± 0.06 vs 2.83 ± 0.05; p <0.01) by 24 hr. Thus, EPA increased NO bioavailability in human ECs, unlike AA, due to improved eNOS coupling and reduced oxidative stress. These findings support a preferential benefit of EPA on endothelial function as compared to AA and supports further investigation.

scholarly journals Nitric oxide as a second messenger in parathyroid hormone-related protein signaling

2001 ◽  
Vol 170 (2) ◽  
pp. 433-440 ◽  
Author(s):  
L Kalinowski ◽  
LW Dobrucki ◽  
T Malinski
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Parathyroid Hormone ◽  
Smooth Muscles ◽  
Calcium Ionophore ◽  
No Production ◽  
Related Protein ◽  
Parathyroid Hormone Related Protein ◽  
No Release ◽  
Pthrp Receptor

Parathyroid hormone (PTH)-related protein (PTHrP) is produced in smooth muscles and endothelial cells and is believed to participate in the local regulation of vascular tone. No direct evidence for the activation of endothelium-derived nitric oxide (NO) signaling pathway by PTHrP has been found despite attempts to identify it. Based on direct in situ measurements, it is reported here for the first time that the human PTH/PTHrP receptor analogs, hPTH(1--34) and hPTHrP(1--34), stimulate NO release from a single endothelial cell. A highly sensitive porphyrinic microsensor with a response time of 0.1 ms and a detection limit of 1 nmol/l was used for the measurement of NO. Both hPTH(1--34) and hPTHrP(1--34) stimulated NO release at nanomolar concentrations. The peak concentration of 0.1 micromol/l hPTH(1--34)- and 0.1 micromol/l hPTHrP(1--34)-stimulated NO release was 175+/-9 and 248+/-13 nmol/l respectively. This represents about 30%--40% of maximum NO concentration recorded in the presence of (0.1 micromol/l) calcium ionophore. Two competitive PTH/PTHrP receptor antagonists, 10 micromol/l [Leu(11),d -Trp(12)]-hPTHrP(7--34)amide and 10 micromol/l [Nle(8,18),Tyr(34)]-bPTH(3--34)amide, were equipotent in antagonizing hPTH(1--34)-stimulated NO release; [Leu(11),d -Trp(12)]-hPTHrP(7--34)amide was more potent than [Nle(8,18),Tyr(34)]-bPTH(3--34)amide in inhibiting hPTHrP(1--34)-stimulated NO release. The PKC inhibitor, H-7 (50 micromol/l), did not change hPTH(1--34)- and hPTHrP(1--34)-stimulated NO release, whereas the combined effect of 10 micromol/l of the cAMP antagonist, Rp-cAMPS, and 50 micromol/l of the calmodulin inhibitor, W-7, was additive. The present studies show that both hPTH(1--34) and hPTHrP(1--34) activate NO production in endothelial cells. The activation of NO release is through PTH/PTHrP receptors and is mediated via the calcium/calmodulin pathway.

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.

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.

Nitric Oxide Detection and Visualization in Biological Systems. Applications of the FNOCT Method

2000 ◽  
Vol 381 (7) ◽  
pp. 575-582 ◽  
Author(s):  
Petra Meineke ◽  
Ursula Rauen ◽  
Herbert de Groot ◽  
Hans-Gert Korth ◽  
Reiner Sustmann
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Single Cells ◽  
High Sensitivity ◽  
Electron System ◽  
No Production ◽  
No Formation ◽  
Extracellular Release ◽  
No Release ◽  
Spermine Nonoate

Abstract Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs) were applied to specifically trap nitric oxide (NO) with high sensitivity. The fluorescent oquinoid ?electron system of the FNOCTs (? = 460 nm, ? = 600 nm) reacts rapidly with NO to a fluorescent phenanthrene system (? = 380 nm, ? = 460 nm). The cyclic nitroxides thus formed react further to nonradical products which exhibit identical fluorescence properties. Using the acid form of the trap (FNOCT-4), NO release by spermine NONOate and by lipopolysaccharide (LPS) activated alveolar macrophages were studied. A maximum extracellular release of NO of 37.5 nmol h[-1] (10[6] cells)[-1] from the macrophages was determined at 11 h after activation. Furthermore, intracellular NO release by LPSactivated macrophages and by microvascular omentum endothelial cells stimulated by the Ca[2+] ionophore A-23187, respectively, was monitored on the single cell level by means of fluorescence microscopy. After loading the cells with the membranepermeating acetoxymethylester derivative FNOCT-5,which is hydrolyzed to a nonpermeating dicarboxylate by intracellular hydrolases, NO formation by the endothelial cells started immediately upon stimulation, whereas start of NO production by the macrophages was delayed with a variation between 4 and 8 h for individual cells. These results demonstrate that the FNOCTs can be used to monitor NO release from single cells, as well as from NOdonating compounds, with high sensitivity and with temporal and spatial resolution.

scholarly journals 20-HETE-induced nitric oxide production in pulmonary artery endothelial cells is mediated by NADPH oxidase, H2O2, and PI3-kinase/Akt

2010 ◽  
Vol 298 (4) ◽  
pp. L564-L574 ◽  
Author(s):  
Sreedhar Bodiga ◽  
Stephanie K. Gruenloh ◽  
Ying Gao ◽  
Vijay L. Manthati ◽  
Narsimhaswamy Dubasi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Nadph Oxidase ◽  
Pi3 Kinase ◽  
No Production ◽  
Akt Inhibitor ◽  
Akt Phosphorylation ◽  
No Release

We have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) increases both superoxide and nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs). The current study was designed to determine mechanisms underlying 20-HETE-stimulated NO release, and particularly the role of NADPH oxidase, reactive oxygen species, and PI3-kinase in stimulated NO release. Intracellular hydrogen peroxide (H2O2) and NO production were detected by dichlorofluorescein or dihydrorhodamine and diaminofluorescein fluorescence, respectively. Activation of endothelial nitric oxide synthase (eNOS) (Ser1179) and Akt (Ser473) was assessed by comparing the ratio of phosphorylated to total protein expression by Western blotting. Addition of 20-HETE to BPAECs caused an increase in superoxide and hydrogen peroxide, but not peroxynitrite. 20-HETE-evoked activation of Akt and eNOS, as well as enhanced NO release, are dependent on H2O2 as opposed to superoxide in that these endpoints are blocked by PEG-catalase and not PEG-superoxide dismutase. Similarly, 20-HETE-stimulated NO production in BPAECs is blocked by NADPH oxidase inhibitors apocynin or gp91 blocking peptide, and by PI3-kinase/Akt blockers wortmannin, LY-294002, or Akt inhibitor, implicating NADPH oxidase, PI3-kinase, and Akt signaling pathways, respectively, in this process. Together, these data suggest the following scheme: 20-HETE stimulates NADPH oxidase-dependent formation of superoxide. Superoxide is rapidly dismutated to hydrogen peroxide, which then mediates activation of PI3-kinase/Akt, phosphorylation of eNOS, and enhanced release of NO from eNOS in response to 20-HETE in BPAECs.

scholarly journals Nitric oxide production in human endothelial cells stimulated by histamine requires Ca2+ influx

1998 ◽  
Vol 330 (2) ◽  
pp. 695-699 ◽  
Author(s):  
Frédérique LANTOINE ◽  
Lahcen IOUZALEN ◽  
Marie-Aude DEVYNCK ◽  
Elisabeth MILLANVOYE-van BRUSSEL ◽  
Monique DAVID-DUFILHO
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Production ◽  
Nitric Oxide Production ◽  
Human Endothelial Cells ◽  
Indirect Methods ◽  
No Release ◽  
K Concentration ◽  
Internal Side ◽  
Oxide Synthase

The causal relationships between cytosolic free-Ca2+ concentration ([Ca2+]i) increases and production of nitric oxide (NO) have been investigated mostly with indirect methods and remain unclear. Here we demonstrate, by direct real-time measurements of [NO] with a porphyrinic microsensor, that Ca2+ entry, but not an increase in [Ca2+]i, is required for triggering of NO production in human endothelial cells. Histamine, ranging from 0.1 to 100 μM, increased both NO production and [Ca2+]i when given in a single dose. However, histamine caused increased NO release but induced progressively smaller [Ca2+]i changes when cumulatively added. In the absence of a transmembrane Ca2+ gradient, no significant NO release was detectable, despite the marked Ca2+ peak induced by histamine. Inhibition of Ca2+ entry by SK&F 96365 abolished histamine-elicited NO production but only reduced the transient [Ca2+]i rise. The suppression of the sustained [Ca2+]i response under these two conditions suggests that NO release was closely associated with Ca2+ entry from the extracellular space. In addition, membrane depolarization, achieved by increasing the extracellular K+ concentration from 5 to 130 mM, reduced both the amplitude of histamine-induced sustained [Ca2+]i elevation and NO production. These results lead us to propose that the availability of numerous Ca2+ ions around the internal side of the plasma membrane would promote the association between nitric oxide synthase and calmodulin, thereby activating the enzyme.

scholarly journals Cognac polyphenolic compounds increase bradykinin-induced nitric oxide production in endothelial cells

2008 ◽  
pp. 885-892
Author(s):  
AS Diallo ◽  
M Sarr ◽  
HA Mostefai ◽  
N Carusio ◽  
M Pricci ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nadph Oxidase ◽  
Xanthine Oxidase ◽  
Polyphenolic Compounds ◽  
No Production ◽  
Human Endothelial Cells ◽  
Vascular Protection ◽  
Xanthine Oxidase Inhibitor ◽  
No Release

We recently reported that in vitro Cognac polyphenolic compounds (CPC) induce NO-dependent vasorelaxant effects and stimulate cardiac function. In the present study, we aim to investigate the effect of CPC on both nitric oxide (NO) and superoxide anions (O(2)(-)) production in cultured human endothelial cells. In addition, its effect on the bradykinin (BK)-induced NO production was also tested. The role and sources of O(2)(-) in the concomitant effect of BK plus CPC were pharmacologically determined. NO and O(2)(-) signals were measured using electron paramagnetic resonance technique using specific spin trappings. Both, CPC and BK induced an increase in NO production in human endothelial cells. The combination of both further enhanced NO release. The capacity of CPC plus BK to increase NO signal was blunted by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, and was enhanced in the presence either of superoxide dismutase or catalase. Moreover, CPC plus BK response was greater after inhibition of either NADPH oxidase by apocynin or xanthine oxidase by allopurinol but it was not affected by rotenone. CPC did not affect O(2)(-) level either alone or after its increase upon lipopolysaccharide treatment. Finally, the capacity of BK alone to increase NO was enhanced either by apocynin or allopurinol. Altogether, these data demonstrate that CPC is able to directly increase NO production without affecting O(2)(-) and enhances the BK-induced NO production in human endothelial cells. The data highlight the ability of BK to stimulate not only NADPH oxidase- but also xanthine oxidase-inhibitor sensitive mechanisms that reduce its efficiency in increasing NO either alone or in the presence of CPC. These results bring pharmacological evidence for vascular protection by CPC via its potentiating effect of BK response in terms of endothelial NO release.

Abstract 14758: Contrasting Effects of Phospholipid Linked Eicosapentaenoic Acid and Arachidonic Acid on Membrane Structure and Stability

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Samuel Sherratt ◽  
Deepak L Bhatt ◽  
Preston Mason
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Eicosapentaenoic Acid ◽  
Electron Density ◽  
Membrane Structure ◽  
High Dose ◽  
Omega 3 ◽  
Core Electron ◽  
Icosapent Ethyl ◽  
Omega 6

Background: Treatment with prescription, high dose, stable icosapent ethyl (IPE), which is metabolized to eicosapentaenoic acid (EPA), significantly reduced clinical events in high-risk patients with either cardiovascular disease or diabetes plus other risk factors (REDUCE-IT). Previous studies suggest that the benefits of EPA, an omega-3 fatty acid (O3FA), correlate positively with its levels and ratio to arachidonic acid (AA) in circulation and cellular membranes. Unlike EPA, AA is an omega-6 fatty acid (O6FA) that, along with its metabolites, contributes to inflammation and diabetes. Objective: The purpose of this study was to compare the separate and combined effects of phospholipid-linked EPA and AA on membrane structure. Methods: Small angle x-ray diffraction approaches compared the effects of 1-palmitoyl-2-eicosapentaenoyl-sn-glycero-3-phosphocholine (PL-EPA) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PL-AA) at a 1:20 ratio in membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (C) at a 0.3:1 C:PL ratio. As a control, we also evaluated membranes consisting of POPC and C only at the same C:PL ratio. Electron density profiles (electrons/Å 3 vs Å) generated from the diffraction data served to determine membrane structure, including its width or d -space, at 1 Å resolution. Results: Addition of PL-EPA increased membrane hydrocarbon core electron density over a broad area ± 0-10 Å from the membrane center, indicating a stabilizing effect on surrounding phospholipid acyl chains. By contrast, PL-AA caused a smaller, bimodal increase in electron density centered at ± 7 Å from the center of the membrane corresponding to its terminal double bonds. The membrane widths of the PL-EPA and PL-AA containing membranes were similar at 58 ± 0.7 Å and 57 ± 0.5 Å, respectively. The combination of PL-EPA with PL-AA highly attenuated the stabilizing effects of EPA on the membrane. Conclusion: PL-EPA causes membrane stability in a manner that is disrupted by the presence of PL-AA. The contrasting effects of PL-EPA and PL-AA on membrane structure may contribute to differences in biological activity.

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