Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy

2001 ◽  
Vol 281 (6) ◽  
pp. C1819-C1824 ◽  
Author(s):  
Yao Song ◽  
Jay L. Zweier ◽  
Yong Xia
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Heat Shock Protein 90 ◽  
Tryptophan Fluorescence ◽  
Hsp90 Inhibitor ◽  
Resonance Spectroscopy ◽  
No Production ◽  
Dependent Manner ◽  
Intact Cells

Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the K d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.

scholarly journals Nitric oxide and superoxide generation from endothelial NOS: modulation by HSP90

2007 ◽  
Vol 293 (6) ◽  
pp. L1444-L1453 ◽  
Author(s):  
Neetu Sud ◽  
Shruti Sharma ◽  
Dean A. Wiseman ◽  
Cynthia Harmon ◽  
Sanjiv Kumar ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Hypertension ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
No Production ◽  
Superoxide Generation ◽  
Hsp90 Expression ◽  
Enos Uncoupling ◽  
Pulmonary Arterial

Previously, we have shown that pulmonary arterial endothelial cells (PAECs) isolated from fetal lambs produce significant levels of nitric oxide (NO) but minimal superoxide upon stimulation, whereas PAECs isolated from 4-wk-old lambs produce significant amounts of both NO and superoxide. These data indicated that a certain degree of uncoupling of endothelial NO synthase (eNOS) occurs in PAECs during postnatal development. In this study, we sought to extend these studies by investigating the potential role of heat shock protein 90 (HSP90) in eNOS coupling. Western blot analyses revealed higher HSP90 expression in PAECs isolated from fetal compared with 4-wk-old lambs, whereas the analysis of recombinant human eNOS activation in vitro in the presence of HSP90 indicated that HSP90 significantly augmented NO production while inhibiting superoxide generation from eNOS. To further investigate whether HSP90 could be involved in uncoupling of eNOS in PAECs isolated from 4-wk-old lambs, we utilized an adenovirus to overexpress HSP90. We found that overexpression of HSP90 significantly increased the shear-stimulated association of HSP90 with eNOS and led to significant increases in NO production and reduced NOS-dependent superoxide generation. Conversely, the exposure of PAECs isolated from fetal lambs to the HSP90 inhibitor radicicol led to significant decreases in eNOS-HSP90 interactions, decreased shear-stimulated NO generation, and increased NOS-dependent superoxide production indicative of eNOS uncoupling. Finally, we examined eNOS-HSP90 interactions in our lamb model of pulmonary hypertension associated with increased pulmonary blood flow (shunt). Our data indicate that HSP90-eNOS interactions were decreased in shunt lambs and that this was associated with decreased NO generation and an increase in eNOS-dependent generation of superoxide. Together, our data support a significant role for HSP90 in promoting NO generation and inhibiting superoxide generation by eNOS and indicate that the disruption of this interaction may be involved in the endothelial dysfunction associated with pulmonary hypertension.

scholarly journals Nitric oxide interactions with cobalamins: biochemical and functional consequences

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1857-1864 ◽  
Author(s):  
M Brouwer ◽  
W Chamulitrat ◽  
G Ferruzzi ◽  
DL Sauls ◽  
JB Weinberg
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
No Formation ◽  
Iron Nitrosyl ◽  
Wide Range ◽  
Functional Consequences

Abstract Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.

scholarly journals ANTI-INFLAMMATORY ACTIVITY OF TINOCRISPOSIDE BY INHIBITING NITRIC OXIDE PRODUCTION IN LIPOPOLYSACCHARIDES-STIMULATED RAW 264.7 CELLS

2018 ◽  
Vol 11 (4) ◽  
pp. 149 ◽  
Author(s):  
Adek Zamrud Adnan ◽  
Muhammad Taher ◽  
Tika Afriani ◽  
Annisa Fauzana ◽  
Dewi Imelda Roesma ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Diseases ◽  
Positive Control ◽  
Inflammatory Activity ◽  
Raw 264.7 Cells ◽  
No Production ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Anti Inflammatory

 Objective: The aim of this study was to investigate in vitro anti-inflammatory activity of tinocrisposide using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cells. Tinocrisposide is a furano diterpene glycoside that was isolated in our previous study from Tinospora crispa.Methods: Anti-inflammatory effect was quantified spectrometrically using Griess method by measuring nitric oxide (NO) production after the addition of Griess reagent.Results: The sample concentrations of 1, 5, 25, 50, and 100 μM and 100 μM of dexamethasone (positive control) have been tested against the LPS-stimulated RAW 264.7 cells, and the results showed NO level production of 39.23, 34.00, 28.9, 20.25, 16.3, and 13.68 μM, respectively, and the inhibition level of 22.67, 33.00, 43.03, 60.10, 68.00, and 73%, respectively.Conclusions: From the study, it could be concluded that tinocrisposide was able to inhibit the formation of NO in the LPS-stimulated RAW 264.7 cells in concentration activity-dependent manner, with half-maximal inhibition concentration 46.92 μM. It can be developed as anti-inflammatory candidate drug because NO is a reactive nitrogen species which is produced by NO synthase. The production of NO has been established as a mediator in inflammatory diseases.

Activation of Nitric Oxide Release and Oxidative Metabolism by Leukotrienes B4, C4, and D4 in Human Polymorphonuclear Leukocytes

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1399-1405 ◽  
Author(s):  
Gerd Lärfars ◽  
Frédérique Lantoine ◽  
Marie-Aude Devynck ◽  
Jan Palmblad ◽  
Hans Gyllenhammar
Keyword(s):  
Nitric Oxide ◽  
Rank Order ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Kinetic Properties ◽  
No Production ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cysteinyl Leukotrienes ◽  
Mediators Of Inflammation

Abstract Because arachidonate metabolites are potent mediators of inflammation, we have studied the effects of leukotriene B4(LTB4) and the cysteinyl leukotrienes C4 and D4 (LTC4 and LTD4) on the release of nitric oxide (NO), in vitro, by human polymorphonuclear granulocytes (PMN). Two independent and highly sensitive real-time methods were used for these studies, ie, the NO-dependent oxidation of oxyhemoglobin (HbO2) to methemoglobin and a NO-sensitive microelectrode. When activated with LTB4, LTC4, or LTD4, but not with other lipoxygenase products such as 5S-HETE, 5-oxo-ETE or 5S,12S-diHETE, PMN produced NO in a stimulus- and concentration-dependent manner. The rank order of potency was LTB4 = LTC4 &gt; LTD4, corresponding to 232 ± 50 pmol of NO/106 PMN for 100 nmol/L LTB4 after 30 minutes. The kinetic properties of the responses were similar for all three leukotrienes with a maximum response at 13 ± 3 minutes. Cysteinyl leukotriene and LTB4 antagonists inhibited the agonist-induced NO production by 70%, and treatment with Bordetella pertussis toxin, or chelation of cytosolic Ca2+, [Ca2+]i, also efficiently inhibited this response. In contrast, treatment of PMN with cytochalasin B (5 μg/mL) enhanced the LTB4-induced NO formation by 86%. Thus, this is the first demonstration that the cysteinyl leukotrienes LTC4 and LTD4, as well as LTB4, activate NO release from human PMN by surface receptor, G-protein and [Ca2+]i-dependent mechanisms. This effect differs from activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, for which only LTB4is an activator.

scholarly journals Epithelial Invasion by Escherichia coli Bearing Dr Fimbriae Is Controlled by Nitric Oxide-Regulated Expression of CD55

2004 ◽  
Vol 72 (5) ◽  
pp. 2907-2914 ◽  
Author(s):  
Li Fang ◽  
Bogdan J. Nowicki ◽  
Petri Urvil ◽  
Pawel Goluszko ◽  
Stella Nowicki ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Epithelial Cell Line ◽  
No Production ◽  
Dependent Manner ◽  
E Coli ◽  
Regulated Expression ◽  
Ishikawa Cells ◽  
Epithelial Invasion

ABSTRACT We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr+). Epithelial binding and invasion by Dr+ E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr+ E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr+ E. coli and a Dr− E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr+ E. coli. Invasion by Dr+ E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection.

scholarly journals Monoterpenoids from the Fruits of Amomum tsao-ko Have Inhibitory Effects on Nitric Oxide Production

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 257
Author(s):  
Seong Su Hong ◽  
Ji Eun Lee ◽  
Yeon Woo Jung ◽  
Ju-Hyoung Park ◽  
Jung A. Lee ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Diseases ◽  
Interleukin 1 ◽  
Resonance Spectroscopy ◽  
No Production ◽  
Dependent Manner ◽  
Interleukin 1 Beta ◽  
Inhibitory Effects ◽  
Concentration Dependent Manner ◽  
First Time

In our search for novel plant-derived inhibitors of nitric oxide (NO) with potential for treating inflammatory diseases, the phytochemicals of Amomum tsao-ko fruits were investigated, leading to the isolation of one bicyclic nonane (1), three menthene skeleton monoterpenoids (2–4), and two acyclic monoterpenoids (5 and 6). Their structures were identified using one- and two-dimensional nuclear magnetic resonance spectroscopy, and mass spectrometry. To the best of our knowledge, compounds 2–5 were obtained from the genus Amomum for the first time. All isolates were tested for their ability to inhibit lipopolysaccharide-stimulated NO overproduction in RAW264.7 cells. Compound 4 was found to inhibit NO production. Western blotting analysis indicated that active compound 4 can regulate inducible NO synthase expression. In addition, lipopolysaccharide-induced interleukin 1 beta and interleukin-6 overproduction was reduced in a concentration-dependent manner.

Nitric oxide induces apoptosis in renal tubular epithelial cells through activation of caspase-8

2006 ◽  
Vol 290 (5) ◽  
pp. F1044-F1054 ◽  
Author(s):  
Caigan Du ◽  
Qiunong Guan ◽  
Hong Diao ◽  
Ziqin Yin ◽  
Anthony M. Jevnikar
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Proinflammatory Cytokines ◽  
Mononuclear Cells ◽  
Caspase 8 ◽  
No Production ◽  
Dependent Manner ◽  
Tubular Epithelial Cells ◽  
Inhibitory Protein

The susceptibility or resistance of tubular epithelial cells (TEC) to apoptosis is pivotal to the long-term maintenance of kidney function following episodes of inflammation, such as graft rejection. TEC apoptosis can occur with ischemia as well as with proinflammatory cytokines and nitric oxide (NO), produced by infiltrating mononuclear cells. TEC can also produce abundant amounts of NO during inflammation but the role and regulation of NO-induced injury of TEC are not well understood. Apoptosis in TEC in vitro was determined by FACS analysis with annexin-V and propidium iodide staining. NO in culture supernatants was measured by Greiss reagent, and protein expression of inducible NO synthetase (NOS2/iNOS) and caspase-8 was examined by Western blot analysis. Here, we showed that murine TEC produced abundant amounts of NO in response to proinflammatory cytokines (IFN-γ/TNF-α) through upregulation of NOS2, and inhibition of endogenous NO production by l-NMMA reduced TEC apoptosis in cytokine-stimulated cultures. Addition of exogenous NO (sodium nitroprusside) induced TEC apoptosis as well as caspase-8 activation in a dose-dependent manner. The key role of caspase-8 in NO-induced TEC apoptosis was demonstrated by that NO-induced TEC apoptosis can be blocked by caspase-8 inhibition using z-IETD-fmk, caspase-8 silencing with shRNA or by overexpressing the endogenous caspase-8 inhibitor c-FLIP (cellular Flice-inhibitory protein). In conclusion, endogenous NO from NOS2 activity as well as exogenous NO can contribute to renal injury through apoptosis of TEC. Activation of caspase-8 plays a central role in NO-induced apoptosis and caspase-8 inhibition may be an important therapeutic target during renal inflammation.

The Role of Nitric Oxide in Regulation of Red Blood Cell Deformability.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3730-3730
Author(s):  
Xiaojun He ◽  
Ivan Azarov ◽  
Beth Gordon ◽  
Daniel B. Kim-Shapiro ◽  
Samir K. Ballas
Keyword(s):  
Nitric Oxide ◽  
Sodium Nitroprusside ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Flow Channel ◽  
Resonance Spectroscopy ◽  
No Production ◽  
Cell Deformability ◽  
No Donor ◽  
Physiol Heart Circ ◽  
Rbc Deformability

Abstract Nitric Oxide (NO) has been suggested to modulate the deformability of red blood cells (RBCs). Bor-Kucukatay (Bor-Kucukatay et al. Am J Physiol Heart Circ Physiol284: H1577, 2003) found that cells incubated with 1 μM of the NO donor sodium nitroprusside lead to a small but significant increase RBC deformability as measured by ektacytometry. However, no significant effect was seen at lower or higher concentrations of sodium nitroprusside or for any concentration of another NO donor, diethylenetriamine NONOate. Kleinbongard (Kleinbongard et al. Blood10; 3992, 2005) found large increases in red cell deformability as a function of added arginine (the substrate for Nitric Oxide Synthase) by measuring the flow rate through filters. On the other hand, using cell aspiration techniques, Bateman (Bateman et al. Am J Physiol Heart Circ Physiol 280; H2848 H2001) found that NO production during sepsis causes a decrease in RBC deformability. Clearly more work is needed to determine the effects of NO on RBC deformability. The present work was undertaken to further investigate the effect of NO on normal and sickle RBC deformability. ProLi NONOate, arginine, and nitrite (which can be reduced to NO by hemoglobin (Hb), were incubated with blood at various concentrations over a period of 2 hours. Nitrosyl Hb and MetHb formed due to the interaction between NO and RBCs were quantified by electron paramagnetic resonance spectroscopy. The deformability was measured using a flow channel laser diffraction similar to ektacytometry (Huang et al. Am J Hematol67; 151, 2001, Biophys J85; 2374, 2003) with a stress range from 0 to 1,000 Pa. Diffraction patterns produced by deformed cells were analyzed by Matlab®. The deformability coefficients were compared to the control (n=6 per experiment condition). Our results suggested that ProLi NONOate did not significantly effect the deformability of normal RBCs. In a single case, ProLi NONOate improved the deformability of poorly deformable sickle red cells and this result is being studied further. Using our flow channel assay, we did not find any significant affects of arginine on RBC deformability. In addition, our studies involving nitrite, performed under both oxygenated and deoxygenated conditions, suggested that nitrite has no significant effect on RBC deformability. In summary, NO didn’t significantly affect the deformability of normal RBCs, and its potential effects on sickle RBCs needs to be further investigated.

scholarly journals Erythrocyte deformability is a nitric oxide-mediated factor in decreased capillary density during sepsis

2001 ◽  
Vol 280 (6) ◽  
pp. H2848-H2856 ◽  
Author(s):  
Ryon M. Bateman ◽  
Justin E. Jagger ◽  
Michael D. Sharpe ◽  
Mary L. Ellsworth ◽  
Sanjay Mehta ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Cecal Ligation ◽  
Capillary Density ◽  
Erythrocyte Deformability ◽  
Resonance Spectroscopy ◽  
No Production ◽  
Nitrite Nitrate ◽  
Plasma Nitrite ◽  
Rbc Deformability

Erythrocyte deformability has been recognized as a determinant of microvascular perfusion. Because nitric oxide (NO) is implicated in the modulation of red blood cell (RBC) deformability and NO levels increase during sepsis, we tested the hypothesis that a NO-mediated decrease in RBC deformability contributes to decreased functional capillary density (CD) in remote organs. With the use of a peritonitis model of sepsis in the rat [cecal ligation and perforation (CLP)] and aminoguanidine (AG) to prevent increases in NO, we measured CD in skeletal muscle (intravital microscopy), mean erythrocyte membrane deformability ([Formula: see text]; micropipette aspiration), systemic NO production [plasma nitrite/nitrate (NOx) chemiluminescence], and NO accumulation in RBC [NO bound to hemoglobin (HbNO) detected by electron paramagnetic resonance spectroscopy]. In untreated CLP animals relative to sham, NOx increased 254% ( P < 0.05), stopped flow capillaries increased 149% ( P < 0.05), and [Formula: see text] decreased 12.7% ( P < 0.05), with a subpopulation (5%) of RBC with deformabilities below the normal range. AG prevented increases in NOx, accumulation of HbNO, and decreases in both [Formula: see text]and functional CD. We found no evidence of leukocyte plugging postcapillary venules. Our findings suggest that decreased functional CD during sepsis resulted from a NO-mediated decrease in erythrocyte deformability.

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