Direct and reversible inhibition of endothelial nitric oxide synthase by nitric oxide

1995 ◽  
Vol 268 (6) ◽  
pp. H2216-H2223 ◽  
Author(s):  
L. V. Ravichandran ◽  
R. A. Johns ◽  
A. Rengasamy
Keyword(s):  
Nitric Oxide ◽  
Enzyme Activity ◽  
Feedback Mechanism ◽  
No Synthase ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Reversible Inhibition ◽  
Endothelial No Synthase ◽  
Endothelial Nitric Oxide

The objective of this study was to investigate the regulation of endothelial nitric oxide (NO) synthase by NO. Partially purified endothelial NO synthase was exposed to authentic NO (10-200 microM) and to the nitrovasodilators sodium nitroprusside (SNP; 10-1,000 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 100-1,000 microM), and enzyme activity was assayed by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in the presence of added cofactors. NO, SNP, and SNAP inhibited NO synthase activity in a dose-dependent manner, NO being the most potent inhibitor. The Michaelis constant for L-arginine was not altered (4.87 microM) by NO (50 microM), whereas the maximal velocity of the enzyme decreased from 784 to 633 pmol.mg-1.min-1. Oxyhemoglobin (10 microM) partially prevented the inhibition of NO synthase by NO (50 microM). The data also suggest that NO inhibits endothelial NO synthase activity by directly interacting with the NO synthase and not by an indirect mechanism such as limitation of cofactor or oxygen availability. Dialysis of NO synthase treated with NO (50 microM) partially restored the enzyme activity. This study demonstrates a direct and reversible inhibition of NO synthase by NO, suggesting a feedback mechanism in vivo.

Characterization and localization of endothelial nitric oxide synthase using specific monoclonal antibodies

1993 ◽  
Vol 265 (5) ◽  
pp. C1379-C1387 ◽  
Author(s):  
J. S. Pollock ◽  
M. Nakane ◽  
L. D. Buttery ◽  
A. Martinez ◽  
D. Springall ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Monoclonal Antibodies ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
No Synthase ◽  
Endothelial No Synthase ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

We have produced specific monoclonal antibodies (MAb) against particulate bovine aortic endothelial nitric oxide synthase. In Western blots, native and cultured bovine aortic endothelial cells as well as cultured bovine microvascular endothelial cells possess immunoreactive NO synthase. In dot blots, MAb H210 and H32 detect 1 ng and 100 pg of purified endothelial NO synthase, respectively. Both antibodies are specific to the endothelial NO synthase and do not cross-react with other known isoforms of NO synthase, namely from the brain, from cytokine/endotoxin-induced macrophages, or from cytokine/endotoxin-induced vascular smooth muscle cells. Immunohistochemical studies demonstrated the specificity of endothelial NO synthase for endothelial cells in various bovine and human tissues. Many types of endothelial cells, macrovascular, microvascular, arterial, and venous were found to possess this specific isoform of NO synthase. Electron microscopy showed the enzyme to be associated with the plasma membrane, membranes of cytoplasmic vesicles, and in the cytoplasm in human umbilical vein endothelial cells. The results demonstrate that particulate endothelial NO synthase is present in a site to act rapidly to produce NO for release into the blood or toward the smooth muscle in many vascular beds.

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin

2008 ◽  
Vol 294 (1) ◽  
pp. C295-C305 ◽  
Author(s):  
James White ◽  
Theresa Guerin ◽  
Hollie Swanson ◽  
Steven Post ◽  
Haining Zhu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Myristic Acid ◽  
Acetylcholine Receptors ◽  
Diabetic Mouse ◽  
No Synthase ◽  
Diabetic Patients ◽  
No Production ◽  
Dependent Manner ◽  
Endothelial No Synthase ◽  
Stimulation Of

In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

scholarly journals Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation

2009 ◽  
Vol 297 (4) ◽  
pp. H1494-H1503 ◽  
Author(s):  
Zeling Cao ◽  
Jeffrey B. Bell ◽  
Joy G. Mohanty ◽  
Enika Nagababu ◽  
Joseph M. Rifkind
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Blood Cells ◽  
Purinergic Receptors ◽  
Atp Release ◽  
Atp Synthesis ◽  
No Synthase ◽  
Endothelial No Synthase ◽  
Rbc Membrane

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by Nω-nitro-l-arginine methyl ester (l-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation.

In vivo lipopolysaccharide pretreatment inhibits cGMP release from the isolated-perfused rat lung

1995 ◽  
Vol 269 (5) ◽  
pp. L618-L624 ◽  
Author(s):  
M. M. Kurrek ◽  
W. M. Zapol ◽  
A. Holzmann ◽  
G. Filippov ◽  
M. Winkler ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Superoxide Dismutase ◽  
Soluble Guanylate Cyclase ◽  
No Synthase ◽  
Rat Lung ◽  
Signal Transduction System ◽  
Endothelial No Synthase ◽  
Cgmp Signal

Administration of bacterial lipopolysaccharide (LPS) to rats stimulates synthesis of nitric oxide (NO), a free radical molecule that activates soluble guanylate cyclase, thereby increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration and inducing systemic vasodilatation. To investigate the effect of endotoxemia on the pulmonary NO/cGMP signal transduction system, we measured the release of cGMP by isolated-perfused lungs of rats that received an intraperitoneal injection of LPS (1 mg/kg) or saline 2 days earlier. Over 90 min, 1.4 +/- 0.78 and 0.079 +/- 0.016 nmol cGMP accumulated in pulmonary perfusates of saline- and LPS-treated rats, respectively (P < 0.05). Despite addition to the perfusate of Zaprinast, superoxide dismutase, or A23187, markedly less cGMP was released from the lungs of rats exposed to LPS than from the lungs of control rats. In contrast, after ventilation with 100 parts per million NO gas, cGMP accumulating in the perfusate of the lungs of both groups of rats was markedly increased, and the quantity of cGMP released from the lungs of LPS-treated rats was similar to that released by control rat lungs (2.8 +/- 0.57 vs. 3.3 +/- 0.88 nmol, P = NS). With the use of immunoblot techniques, equal concentrations of constitutive endothelial NO synthase were detected in the lungs of rats treated with saline or LPS. These results demonstrate that the NO/cGMP signal transduction system is abnormal in the lungs of rats exposed to LPS, at least in part, at the level of endothelial NO synthase activation.

scholarly journals Role of Endothelial Nitric Oxide Synthase in Hypoxia-Induced Pial Artery Dilation

1998 ◽  
Vol 18 (5) ◽  
pp. 531-538 ◽  
Author(s):  
Michael J. Wilderman ◽  
William M. Armstead
Keyword(s):  
Nitric Oxide ◽  
No Synthase ◽  
Methionine Enkephalin ◽  
Cranial Window ◽  
Pial Artery ◽  
Endothelial No Synthase ◽  
Before And After ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Nitric oxide (NO) contributes to hypoxia-induced pial artery dilation, at least in part, through the formation of cGMP and the subsequent release of methionine enkephalin and leucine enkephalin in the newborn pig. In separate studies, these opioids also were observed to elicit NO-dependent pial artery dilation, whereas light/dye endothelial injury reduced hypoxic pial dilation. The current study was designed to investigate the role of the endothelial isoform of NO synthase in hypoxic pial dilation, associated opioid release, and opioid dilation in piglets equipped with a closed cranial window. N-iminoethyl-l-ornithine (l-NIO) (10−6 mol/L), an antagonist that may have greater endothelial NO synthase inhibitory selectivity, had no effect on dilation elicited by hypoxia (Po2 ≈ 35 mm Hg) (24 ± 2 versus 24 ± 2% in the absence and presence of l-NIO, respectively, n = 8). Hypoxic dilation was accompanied by increased CSF cGMP, which also was unchanged in the presence of l-NIO (394 ± 19 and 776 ± 63 versus 323 ± 13 and 739 ± 25 fmol/mL for control and hypoxia in the absence and presence of l-NIO, respectively, n = 6). Additionally, hypoxic pial dilation was associated with increased CSF methionine enkephalin, which also was unchanged in the presence of l-NIO (992 ± 73 and 2469 ± 197 versus 984 ± 18 and 2275 ± 185 pg/mL, respectively, n = 6). In contrast, methionine enkephalin–induced dilation was blocked by l-NIO (6 ± 1, 10 ± 1, and 16 ± 1 versus 1 ± 1, 1 ± 1, and 2 ± 1% for 10−10, 10−8, 10−6 mol/L methionine enkephalin, respectively, before and after l-NIO, n = 8). Substance P–induced pial dilation was blunted by l-NIO, whereas responses to sodium nitroprusside and N-methyl-d-aspartate were unchanged. These data indicate that endothelial NO synthase contributes to opioid-induced pial artery dilation but not hypoxia-induced dilation. Additionally, these data suggest that neuronally derived NO contributes to hypoxic pial dilation.

scholarly journals Changes in the asymmetric dimethylarginine and endothelial nitric oxide synthase levels in pathogenesis of experimental diabetic retinopathy

2020 ◽  
Vol 26 (3) ◽  
pp. 69-74
Author(s):  
Ya.V. Sirman ◽  
I.V. Savytskyi ◽  
N.I. Preys
Keyword(s):  
Nitric Oxide ◽  
Diabetic Retinopathy ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Asymmetric Dimethylarginine ◽  
No Synthase ◽  
Endothelial No Synthase ◽  
White Rats ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelial dysfunction associated with impaired nitric oxide excretion plays an important role in the onset and progression of diabetic retinopathy. It has been proven that a decrease in the activity of endothelial NO-synthase (еNО-S), the inhibitor of which is asymmetric dimethylarginine (ADMA), plays an important role in this. Objective: to study the level of asymmetric dimethylarginine and endothelial nitric oxide synthase at different stages of development of diabetic retinopathy in the experiment. The study was conducted in Wistar white rats of 180-200 g weight. According to the tasks, the animals were separated into 2 groups as follows: group 1 – 60 intact animals, group 2 – 60 animals with simulated diabetic retinopathy without further correction. Type 2 diabetes mellitus and diabetic retinopathy were simulated through intraperitoneal administration of Streptozotocin (Sigma, USA) diluted in 0.1M citrate buffer with pH=4.5. Streptozotocin dose of 55 mg per kg of animal weight was divided into two administrations. The streptozotocin intake was preceded by a 28-day high-fat diet. Our study showed impaired endothelial function in diabetic retinopathy, as evidenced by an increased ADMA level (p<0.001). We have determined a stepwise increase of asymmetric dimethylarginine level in blood of rats with simulated pathology which is apparent in its highest at phase 3. Pathogenetic effect of increased ADMA on еNО-S activity was verified at all experimental stages, Impairment of physiological nitric oxide synthesis in simulated pathology has been proved as evidenced by reduced activity of endothelial NO-synthase yet on the 30th day with further negative dynamics up to the 180th day (p<0.001 compared with the intact group findings).

scholarly journals Decreased Antipyrine Clearance following Endotoxin Administration: In Vivo Evidence of the Role of Nitric Oxide

1999 ◽  
Vol 43 (11) ◽  
pp. 2697-2701 ◽  
Author(s):  
Kiyoyuki Kitaichi ◽  
Li Wang ◽  
Kenji Takagi ◽  
Mitsunori Iwase ◽  
Eiji Shibata ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Enzyme Activity ◽  
Dependent Manner ◽  
Systemic Clearance ◽  
Antipyrine Clearance ◽  
Hepatic Drug ◽  
Drug Metabolizing Enzyme ◽  
Metabolizing Enzyme

ABSTRACT Klebsiella pneumoniae endotoxin has been found to decrease hepatic P450-mediated drug-metabolizing enzyme activity in a time-dependent manner. In this study, we investigated the role of nitric oxide (NO) in the decrease in hepatic drug-metabolizing enzyme activity caused by endotoxin in vivo. We measured in vivo pharmacokinetic parameters of antipyrine in rats treated with endotoxin and/or a selective inhibitor of inducible NO synthase (iNOS),S-methylisothiourea. Intraperitoneal injection of endotoxin (1 mg/kg of body weight) dramatically decreased the systemic clearance of antipyrine, reflecting reduced hepatic drug-metabolizing enzyme activity, and significantly increased the level of nitrite and nitrate (NOx) in the plasma. S-Methylisothiourea (10 mg/kg) reversed this decreasing antipyrine clearance and reduced the level of NOx in plasma. Repeated injections of an NO donor, (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK-409; 10 mg/kg), at a dose which maintained plasma NOx at the same levels as those caused by endotoxin injection, also decreased the systemic clearance of antipyrine. These findings suggest that the overproduction of NO observed in this animal model is at least partially responsible for the significant reduction in the hepatic drug-metabolizing enzyme activity that may happen in a gram-negative bacterial infection.

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 Phaseolin Attenuates Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells and Zebrafish

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 420
Author(s):  
Su-Jung Hwang ◽  
Ye-Seul Song ◽  
Hyo-Jong Lee
Keyword(s):  
Nitric Oxide ◽  
Nuclear Translocation ◽  
Raw 264.7 Cells ◽  
Network Pharmacology ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Bioactive Constituents

Kushen (Radix Sophorae flavescentis) is used to treat ulcerative colitis, tumors, and pruritus. Recently, phaseolin, formononetin, matrine, luteolin, and quercetin, through a network pharmacology approach, were tentatively identified as five bioactive constituents responsible for the anti-inflammatory effects of S. flavescentis. However, the role of phaseolin (one of the primary components of S. flavescentis) in the direct regulation of inflammation and inflammatory processes is not well known. In this study, the beneficial role of phaseolin against inflammation was explored in lipopolysaccharide (LPS)-induced inflammation models of RAW 264.7 macrophages and zebrafish larvae. Phaseolin inhibited LPS-mediated production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), without affecting cell viability. In addition, phaseolin suppressed pro-inflammatory mediators such as cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in a dose-dependent manner. Furthermore, phaseolin reduced matrix metalloproteinase (MMP) activity as well as macrophage adhesion in vitro and the recruitment of leukocytes in vivo by downregulating Ninjurin 1 (Ninj1), an adhesion molecule. Finally, phaseolin inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB). In view of the above, our results suggest that phaseolin could be a potential therapeutic candidate for the management of inflammation.

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