scholarly journals Nitric Oxide Participates in IFN-γ-Induced HUVECs Hyperpermeability

2016 ◽  
pp. 1053-1058 ◽  
Author(s):  
C. T. NG ◽  
L. Y. FONG ◽  
Y. Y. LOW ◽  
J. BAN ◽  
M. N. HAKIM ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Barrier Function ◽  
Cell Monolayer ◽  
Guanosine Monophosphate ◽  
Endothelial Barrier ◽  
No Production ◽  
Endothelial Barrier Function ◽  
No Donor ◽  
Cgmp Production ◽  
Ifn Γ

The endothelial barrier function is tightly controlled by a broad range of signaling cascades including nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway. It has been proposed that disturbances in NO and cGMP production could interfere with proper endothelial barrier function. In this study, we assessed the effect of interferon-gamma (IFN-γ), a pro-inflammatory cytokine, on NO and cGMP levels and examined the mechanisms by which NO and cGMP regulate the IFN-γ-mediated HUVECs hyperpermeability. The flux of fluorescein isothiocyanate-labeled dextran across cell monolayers was used to study the permeability of endothelial cells. Here, we found that IFN-γ significantly attenuated basal NO concentration and the increased NO levels supplied by a NO donor, sodium nitroprusside (SNP). Besides, application of IFN-γ also significantly attenuated both the basal cGMP concentration and the increased cGMP production donated by a cell permeable cGMP analogue, 8-bromo-cyclic GMP (8-Br-cGMP). In addition, exposure of the cell monolayer to IFN-γ significantly increased HUVECs basal permeability. However, L-NAME pretreatment did not suppress IFN-γ-induced HUVECs hyperpermeability. L-NAME pretreatment followed by SNP or SNP pretreatment partially reduced IFN-γ-induced HUVECs hyperpermeability. Pretreatment with a guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), led to a further increase in IFN-γ-induced HUVECs hyperpermeability. The findings suggest that the mechanism underlying IFN-γ-induced increased HUVECs permeability is partly related to the inhibition of NO production.

scholarly journals Modulation of Rac1 Activity by ADMA/DDAH Regulates Pulmonary Endothelial Barrier Function

2009 ◽  
Vol 20 (1) ◽  
pp. 33-42 ◽  
Author(s):  
Beata Wojciak-Stothard ◽  
Belen Torondel ◽  
Lan Zhao ◽  
Thomas Renné ◽  
James M. Leiper
Keyword(s):  
Nitric Oxide ◽  
Actin Cytoskeleton ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Nitric Oxide Synthase Inhibitor ◽  
Rac1 Activity

Endogenously produced nitric oxide synthase inhibitor, asymmetric methylarginine (ADMA) is associated with vascular dysfunction and endothelial leakage. We studied the role of ADMA, and the enzymes metabolizing it, dimethylarginine dimethylaminohydrolases (DDAH) in the regulation of endothelial barrier function in pulmonary macrovascular and microvascular cells in vitro and in lungs of genetically modified heterozygous DDAHI knockout mice in vivo. We show that ADMA increases pulmonary endothelial permeability in vitro and in in vivo and that this effect is mediated by nitric oxide (NO) acting via protein kinase G (PKG) and independent of reactive oxygen species formation. ADMA-induced remodeling of actin cytoskeleton and intercellular adherens junctions results from a decrease in PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and a subsequent down-regulation of Rac1 activity. The effects of ADMA on endothelial permeability, Rac1 activation and VASP phosphorylation are prevented by overexpression of active DDAHI and DDAHII, whereas inactive DDAH mutants have no effect. These findings demonstrate for the first time that ADMA metabolism critically determines pulmonary endothelial barrier function by modulating Rac1-mediated remodeling of the actin cytoskeleton and intercellular junctions.

scholarly journals eNOS-derived nitric oxide regulates endothelial barrier function through VE-cadherin and Rho GTPases

2013 ◽  
Vol 126 (24) ◽  
pp. 5541-5552 ◽  
Author(s):  
Annarita Di Lorenzo ◽  
Michelle I. Lin ◽  
Takahisa Murata ◽  
Shira Landskroner-Eiger ◽  
Michael Schleicher ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Barrier Function ◽  
Rho Gtpases ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function

scholarly journals Evidence for Production and Functional Activity of Nitric Oxide in Seminiferous Tubules and Blood Vessels of the Human Testis1

1997 ◽  
Vol 82 (12) ◽  
pp. 4154-4161 ◽  
Author(s):  
R. Middendorff ◽  
D. Müller ◽  
S. Wichers ◽  
A. F. Holstein ◽  
M. S. Davidoff
Keyword(s):  
Nitric Oxide ◽  
Blood Vessels ◽  
Sertoli Cells ◽  
Cell Function ◽  
Soluble Guanylyl Cyclase ◽  
Guanosine Monophosphate ◽  
Human Testis ◽  
Seminiferous Tubules ◽  
No Production ◽  
No Donor

Previous studies have demonstrated that nitric oxide (NO) influences Leydig cell function. Here we provide evidence for NO production and activity in seminiferous tubules and blood vessels of the human testis. By immunohistochemistry, the soluble guanylyl cyclase (sGC), the intracellular NO receptor, and the second messenger, cyclic guanosine monophosphate (cGMP), were detected in myofibroblasts of the peritubular lamina propria in Sertoli cells, as well as in endothelial and smooth muscle cells of testicular blood vessels. Performed with isolated tubules and blood vessels, the biological activity of sGC could be proved by cGMP generation in response to treatments with the NO donor, sodium nitroprusside. The endothelial and neuronal subtypes of NO synthase (NOS) were localized immunohistochemically to the same cell types that express sGC and cGMP. In isolated tubules and vessels, the presence of endothelial NOS and neuronal NOS was confirmed by immunoblotting, and NOS activity was demonstrated by decreased cGMP production upon incubation with the NOS inhibitor l-nitro arginine methylester. These findings show that peritubular cells, Sertoli cells, and testicular blood vessels may be sites of NO production and activity, possibly involved in relaxation of seminiferous tubules and blood vessels to modulate sperm transport and testicular blood flow, respectively.

scholarly journals eNOS-derived nitric oxide regulates endothelial barrier function through VE-cadherin and Rho GTPases

2014 ◽  
Vol 127 (9) ◽  
pp. 2120-2120 ◽  
Author(s):  
A. Di Lorenzo ◽  
M. I. Lin ◽  
T. Murata ◽  
S. Landskroner-Eiger ◽  
M. Schleicher ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Barrier Function ◽  
Rho Gtpases ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function

scholarly journals Arginase II Downregulates Nitric Oxide (NO) Production and Prevents NO-mediated Apoptosis in Murine Macrophage-derived RAW 264.7 Cells

1999 ◽  
Vol 144 (3) ◽  
pp. 427-434 ◽  
Author(s):  
Tomomi Gotoh ◽  
Masataka Mori
Keyword(s):  
Nitric Oxide ◽  
Raw 264.7 Cells ◽  
No Production ◽  
Raw 264.7 ◽  
Murine Macrophage ◽  
High Concentration ◽  
Expression Plasmid ◽  
No Donor ◽  
Ifn Γ ◽  
Arginase Ii

Excess nitric oxide (NO) induces apoptosis of some cell types, including macrophages. As NO is synthesized by NO synthase (NOS) from arginine, a common substrate of arginase, these two enzymes compete for arginine. There are two known isoforms of arginase, types I and II. Using murine macrophage-like RAW 264.7 cells, we asked if the induction of arginase II would downregulate NO production and hence prevent apoptosis. When cells were exposed to lipopolysaccharide (LPS) and interferon-γ (IFN-γ), the inducible form of NOS (iNOS) was induced, production of NO was elevated, and apoptosis followed. When dexamethasone and cAMP were further added, both iNOS and arginase II were induced, NO production was much decreased, and apoptosis was prevented. When the cells were transfected with an arginase II expression plasmid and treated with LPS/IFN-γ, some cells were rescued from apoptosis. An arginase I expression plasmid was also effective. On the other hand, transfection with the arginase II plasmid did not prevent apoptosis when a NO donor SNAP or a high concentration (12 mM) of arginine was added. These results indicate that arginase II prevents NO-dependent apoptosis of RAW 264.7 cells by depleting intracellular arginine and by decreasing NO production.

Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment

2021 ◽  
pp. 247255522110309
Author(s):  
Oleksii Dubrovskyi ◽  
Erica Hasten ◽  
Steven M. Dudek ◽  
Michael T. Flavin ◽  
Leo Li-Ying Chan
Keyword(s):  
Drug Discovery ◽  
Barrier Function ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Cell Monolayer ◽  
Endothelial Permeability ◽  
Image Cytometry ◽  
Endothelial Barrier ◽  
Published Data ◽  
Endothelial Barrier Function

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.

scholarly journals Nitric Oxide Suppresses the Expression of Bcl-2 Binding Protein BNIP3 in Hepatocytes

2001 ◽  
Vol 276 (50) ◽  
pp. 46887-46895 ◽  
Author(s):  
Ruben Zamora ◽  
Louis Alarcon ◽  
Yoram Vodovotz ◽  
Binnie Betten ◽  
Peter K. M. Kim ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cultured Cells ◽  
Binding Protein ◽  
Soluble Guanylyl Cyclase ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Northern Analysis ◽  
No Production ◽  
No Donor ◽  
Factor Α

Nitric oxide (NO) is not only an important signaling molecule, but it also regulates the expression of a number of genes in the liver. We have previously shown that apoptosis in hepatocytes exposed to tumor necrosis factor-α and actinomycin D is prevented by NO derived from the inducible nitric-oxide synthase (iNOS), by mechanisms that are both dependent on and independent of modulation of cyclic guanosine monophosphate (cGMP) subsequent to activation of soluble guanylyl cyclase (sGC). We hypothesize that one mechanism by which NO exerts these effects is by regulating the expression of genes involved in apoptosis. We used differential display-polymerase chain reaction to isolate NO-regulated genes in hepatocytes fromiNOSknockout mice (to eliminate endogenous inducible NO production). Using this analysis, we identified a NO-suppressed gene fragment homologous with the pro-apoptotic Bcl-2 binding protein BNIP3. Northern analysis confirmed the NO-dependent suppression ofBNIP3in cultured cells. Similarly, the NO donorS-nitroso-N-acetyl-dl-penicillamine (1–1000 μm) down-regulated the expression ofBNIP3in bothiNOSknockout and wild-type hepatocytes. This effect of NO was reversed by the sGC inhibitor 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one (ODQ),suggesting the involvement of the sGC/cGMP pathway in the modulation of BNIP3 by NO. We propose that suppression of BNIP3 expression is one sGC/cGMP-dependent mechanism by which NO might affect the process of hepatocyte apoptosis.

scholarly journals Gycine and GABA interact to regulate the nitric oxide/cGMP signaling pathway in the turtle retina

2005 ◽  
Vol 22 (6) ◽  
pp. 825-838 ◽  
Author(s):  
DOU YU ◽  
WILLIAM D. ELDRED
Keyword(s):  
Nitric Oxide ◽  
Signal Transduction ◽  
Signaling Pathway ◽  
Amacrine Cells ◽  
Guanosine Monophosphate ◽  
No Production ◽  
Double Labeling ◽  
Selective Antagonist ◽  
Cgmp Production ◽  
Cgmp Signaling

Nitric oxide (NO) is a free radical that is important in retinal signal transduction and cyclic guanosine monophosphate (cGMP) is a critical downstream messenger of NO. The NO/cGMP signaling pathway has been shown to modulate neurotransmitter release and gap junction coupling in horizontal cells and amacrine cells, and increase the gain of the light response in photoreceptors. However, many of the mechanisms controlling the production of NO and cGMP remain unclear. Previous studies have shown activation of NO/cGMP production in response to stimulation with N-methyl-d-aspartate (NMDA) or nicotine, and the differential modulation of cGMP production by GABAA and GABAC receptors (GABAARs and GABACRs). This study used cGMP immunocytochemistry and NO imaging to investigate how the inhibitory GABAergic and glycinergic systems modulate the production of NO and cGMP. Our data show that blocking glycine receptors (GLYR) with strychnine (STRY) produced moderate increases in cGMP-like immunoreactivity (cGMP-LI) in select types of amacrine and bipolar cells, and strong increases in NO-induced fluorescence (NO-IF). TPMPA, a selective GABACR antagonist, greatly reduced the increases in cGMP-LI stimulated by STRY, but did not influence the increase in NO-IF stimulated by STRY. Bicuculline (BIC), a GABAAR antagonist, however, enhanced the increases in both the cGMP-LI and NO-IF stimulated by STRY. CNQX, a selective antagonist for α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid hydrobromide/kainic acid (AMPA/KA) receptors, eliminated both the increases in cGMP-LI and NO-IF stimulated by STRY, while MK801, a selective antagonist for NMDA receptors, slightly increased the cGMP-LI and slightly decreased the NO-IF stimulated by STRY. Finally, double labeling of NO-stimulated cGMP and either GLY or GABA indicated that cGMP predominantly colocalized with GLY. Taken together, these findings support the hypothesis that GLY and GABA interact in the regulation of the NO/cGMP signaling pathway, where GLY primarily inhibits NO production and GABA has a greater effect on cGMP production. Such interacting inhibitory pathways could shape the course of signal transduction of the NO/cGMP pathway under different physiological situations.

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