scholarly journals Cytokine-induced arginase activity in pulmonary endothelial cells is dependent on Src family tyrosine kinase activity

2008 ◽  
Vol 295 (4) ◽  
pp. L688-L697 ◽  
Author(s):  
Rossana Chang ◽  
Louis G. Chicoine ◽  
Hongmei Cui ◽  
Nancy L. Kanagy ◽  
Benjimen R. Walker ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Tyrosine Kinases ◽  
Critical Role ◽  
Inos Mrna ◽  
No Production ◽  
Appreciable Effect ◽  
Pulmonary Endothelial Cells ◽  
Inflammatory Stimuli ◽  
Arginase Ii

We hypothesized that the Src family tyrosine kinases (STKs) are involved in the upregulation of arginase and inducible nitric oxide synthase (iNOS) expression in response to inflammatory stimuli in pulmonary endothelial cells. Treatment of bovine pulmonary arterial endothelial cells (bPAEC) with lipopolysaccharide and tumor necrosis factor-α (L/T) resulted in increased urea and nitric oxide (NO) production, and this increase in urea and NO production was inhibited by the STK inhibitor PP1 (10 μM). The STK inhibitors PP2 (10 μM) and herbimycin A (10 μM) also prevented the L/T-induced expression of both arginase II and iNOS mRNA in bPAEC. Together, the data demonstrate a central role of STK in the upregulation of both arginase II and iNOS in bPAEC in response to L/T treatment. To identify the specific kinase(s) required for the induction of urea and NO production, we studied human pulmonary microvascular endothelial cells (hPMVEC) so that short interfering RNA (siRNA) techniques could be employed. We found that hPMVEC express Fyn, Yes, c-Src, Lyn, and Blk and that the protein expression of Fyn, Yes, c-Src, and Lyn could be inhibited with specific siRNA. The siRNA targeting Fyn prevented the cytokine-induced increase in urea and NO production, whereas siRNAs specifically targeting Yes, c-Src, and Lyn had no appreciable effect on cytokine-induced urea and NO production. These findings support our hypothesis that inflammatory stimuli lead to increased urea and NO production through a STK-mediated pathway. Furthermore, these results indicate that the STK Fyn plays a critical role in this process.

Regulation of the nitric oxide synthase-nitric oxide- cGMP pathway in rat mesenteric endothelial cells

2001 ◽  
Vol 91 (6) ◽  
pp. 2553-2560 ◽  
Author(s):  
Andreas Papapetropoulos ◽  
Stavroula Andreopoulos ◽  
Carolyn Y. Go ◽  
Azizul Hoque ◽  
Leslie C. Fuchs ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Inos Mrna ◽  
Microtubule Depolymerization ◽  
Inos Protein Expression ◽  
Cgmp Pathway ◽  
Inflammatory Stimuli

Most of the available data on the nitric oxide (NO) pathway in the vasculature is derived from studies performed with cells isolated from conduit arteries. We investigated the expression and regulation of components of the NO synthase (NOS)-NO-cGMP pathway in endothelial cells from the mesenteric vascular bed. Basally, or in response to bradykinin, cultured mesenteric endothelial cells (MEC) do not release NO and do not express endothelial NOS protein. MEC treated with cytokines, but not untreated cells, express inducible NOS (iNOS) mRNA and protein, increase nitrite release, and stimulate cGMP accumulation in reporter smooth muscle cells. Pretreatment of MEC with genistein abolished the cytokine-induced iNOS expression. On the other hand, exposure of MEC to the microtubule depolymerizing agent colchicine did not affect the cytokine-induced increase in nitrite formation and iNOS protein expression, whereas it inhibited the induction of iNOS in smooth muscle cells. Collectively, our findings demonstrate that MEC do not express endothelial NOS but respond to inflammatory stimuli by expressing iNOS, a process that is blocked by tyrosine kinase inhibition but not by microtubule depolymerization.

scholarly journals Regulatory role of arginase I and II in nitric oxide, polyamine, and proline syntheses in endothelial cells

2001 ◽  
Vol 280 (1) ◽  
pp. E75-E82 ◽  
Author(s):  
Hui Li ◽  
Cynthia J. Meininger ◽  
James R. Hawker ◽  
Tony E. Haynes ◽  
Diane Kepka-Lenhart ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Production ◽  
Lacz Gene ◽  
Western Blots ◽  
Enzymatic Assays ◽  
Intracellular Content ◽  
Arginase Ii ◽  
High Level ◽  
Arginase I

Endothelial cells (EC) metabolize l-arginine mainly by arginase, which exists as two distinct isoforms, arginase I and II. To understand the roles of arginase isoforms in EC arginine metabolism, bovine coronary venular EC were stably transfected with the Escherichia coli lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC). Western blots and enzymatic assays confirmed high-level expression of arginase I in the cytosol of AI-EC and of arginase II in mitochondria of AII-EC. For determining arginine catabolism, EC were cultured for 24 h in DMEM containing 0.4 mM l-arginine plus [1-14C]arginine. Urea formation, which accounted for nearly all arginine consumption by these cells, was enhanced by 616 and 157% in AI-EC and AII-EC, respectively, compared with lacZ-EC. Arginine uptake was 31–33% greater in AI-EC and AII-EC than in lacZ-EC. Intracellular arginine content was 25 and 11% lower in AI-EC and AII-EC, respectively, compared with lacZ-EC. Basal nitric oxide (NO) production was reduced by 60% in AI-EC and by 47% in AII-EC. Glutamate and proline production from arginine increased by 164 and 928% in AI-EC and by 79 and 295% in AII-EC, respectively, compared with lacZ-EC. Intracellular content of putrescine and spermidine was increased by 275 and 53% in AI-EC and by 158 and 43% in AII-EC, respectively, compared with lacZ-EC. Our results indicate that arginase expression can modulate NO synthesis in bovine venular EC and that basal levels of arginase I and II are limiting for endothelial syntheses of polyamines, proline, and glutamate and may have important implications for wound healing, angiogenesis, and cardiovascular function.

scholarly journals Nitric Oxide is Produced by Cowdria ruminantium-Infected Bovine Pulmonary Endothelial Cells In Vitro and Is Stimulated by Gamma Interferon

1998 ◽  
Vol 66 (5) ◽  
pp. 2115-2121 ◽  
Author(s):  
Mbithe Mutunga ◽  
Patricia M. Preston ◽  
Keith J. Sumption
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Dependent Manner ◽  
Donor Molecule ◽  
No Donor ◽  
Pulmonary Endothelial Cells ◽  
Cowdria Ruminantium ◽  
Dose Dependent

ABSTRACT Nitric oxide (NO) is a labile inorganic free radical produced by NO synthase from the substrate l-arginine in various cells and tissues including endothelial cells. A substantial elevation of nitrite levels indicative of NO production occurred in cultures ofCowdria ruminantium-infected bovine pulmonary endothelial cells (BPEC) incubated in medium alone. Exposure of the infected cultures to recombinant bovine gamma interferon (BorIFN-γ) resulted in more rapid production of NO, reduced viability of C. ruminantium, and induction of endothelial cell death. Significant inhibition of NO production was noted after addition of the NO synthase inhibitor N-monomethyl-l-arginine (l-NMMA), indicating that the increase in production occurred via the inducible NO synthase pathway. Reduction in the infectivity of C. ruminantium elementary bodies (EBs) occurred in a dose-dependent manner after incubation with the NO donor moleculeS-nitroso-N-acetyl-dl-penicillamine (SNAP) prior to infection of endothelial cells. The level of infection in cultures maintained in SNAP was reduced in a dose-dependent manner with significant negative correlation between the final level of infection on day 7 and the level of SNAP (r = −0.96). It was established that pretreatment and cultivation of C. ruminantium EBs with the NO donor molecule SNAP reduced infectivity to cultures and viability of EBs with the implication that release of NO in vivo following infection of endothelial cells may have an effect upon the multiplication of the agent in the host animal and may be involved in the pathogenesis of heartwater through the effect of this molecule upon circulation.

Regulation of inducible nitric oxide synthase in hepatic sinusoidal endothelial cells

1996 ◽  
Vol 271 (2) ◽  
pp. G260-G267 ◽  
Author(s):  
D. C. Rockey ◽  
J. J. Chung
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Interleukin 1 ◽  
Cell Types ◽  
Tnf Alpha ◽  
Inos Mrna ◽  
No Production ◽  
Sinusoidal Endothelial Cells ◽  
Necrosis Factor Alpha ◽  
Ifn Gamma

Nitric oxide (NO) has many important physiological effects that depend in part on its cellular source(s). In liver, NO is produced by all major cell types, including hepatocytes, Kupffer, stellate, and sinusoidal endothelial cells (SECs). Although endothelial cells have been commonly associated with constitutive NO production, recent evidence suggests that NO is inducible in this cell type. Here, we investigated the regulation of inducible NO synthase (iNOS) in SECs. Interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) as individual compounds induced iNOS mRNA in SECs. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) had no effect when used alone but enhanced iNOS mRNA upregulation by IFN-gamma. iNOS transcription after LPS was present only for 4 h after exposure yet was more sustained after IFN-gamma/TNF-alpha, LPS was unique in that it transiently induced iNOS mRNA, whereas IFN-gamma/TNF-alpha resulted in prolonged increases in iNOS mRNA. Both LPS and IFN-gamma/TNF-alpha caused prolonged elevation of immunoreactive protein. However, when stimulated by LPS, iNOS remained enzymatically active for only 24-48 h. After IFN-gamma or IFN-gamma/TNF-alpha, iNOS activity declined only moderately. LPS added to IFN-gamma alone or IFN-gamma/TNF-alpha did not result in more rapid decay of iNOS enzymatic activity. These data indicate that induction of iNOS by sinusoidal endothelial cells is prominent and that it is regulated both transcriptionally and by its inactivation. Such complex regulation of iNOS has important implications for NO biology in liver disease.

scholarly journals Impairment of autophagy in endothelial cells prevents shear-stress-induced increases in nitric oxide bioavailability

2014 ◽  
Vol 92 (7) ◽  
pp. 605-612 ◽  
Author(s):  
Leena P. Bharath ◽  
Robert Mueller ◽  
Youyou Li ◽  
Ting Ruan ◽  
David Kunz ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Oxidant Stress ◽  
Critical Role ◽  
No Production ◽  
Enos Phosphorylation ◽  
Autophagy Pathway ◽  
No Bioavailability ◽  
Static Conditions

Autophagy is a lysosomal catabolic process by which cells degrade or recycle their contents to maintain cellular homeostasis, adapt to stress, and respond to disease. Impairment of autophagy in endothelial cells studied under static conditions results in oxidant stress and impaired nitric oxide (NO) bioavailability. We tested the hypothesis that vascular autophagy is also important for induction of NO production caused by exposure of endothelial cells to shear stress (i.e., 3 h × ≈20 dyn/cm2). Atg3 is a requisite autophagy pathway mediator. Control cells treated with non-targeting control siRNA showed increased autophagy, reactive oxygen species (ROS) production, endothelial NO synthase (eNOS) phosphorylation, and NO production upon exposure to shear stress (p < 0.05 for all). In contrast, cells with >85% knockdown of Atg3 protein expression (via Atg3 siRNA) exhibited a profound impairment of eNOS phosphorylation, and were incapable of increasing NO in response to shear stress. Moreover, ROS accumulation and inflammatory cytokine production (MCP-1 and IL-8) were exaggerated (all p < 0.05) in response to shear stress. These findings reveal that autophagy not only plays a critical role in maintaining NO bioavailability, but may also be a key regulator of oxidant–antioxidant balance and inflammatory–anti-inflammatory balance that ultimately regulate endothelial cell responses to shear stress.

scholarly journals Comparing the protective effects of resveratrol, curcumin and sulforaphane against LPS/IFN-γ-mediated inflammation in doxorubicin-treated macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Haidy A. Saleh ◽  
Eman Ramdan ◽  
Mohey M. Elmazar ◽  
Hassan M. E. Azzazy ◽  
Anwar Abdelnaser
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Response ◽  
Raw 264.7 Cells ◽  
Inos Mrna ◽  
No Production ◽  
Raw 264.7 ◽  
Mrna Levels ◽  
Protective Effects ◽  
Tnf Α ◽  
Ifn Γ

AbstractDoxorubicin (DOX) chemotherapy is associated with the release of inflammatory cytokines from macrophages. This has been suggested to be, in part, due to DOX-mediated leakage of endotoxins from gut microflora, which activate Toll-like receptor 4 (TLR4) signaling in macrophages, causing severe inflammation. However, the direct function of DOX on macrophages is still unknown. In the present study, we tested the hypothesis that DOX alone is incapable of stimulating inflammatory response in macrophages. Then, we compared the anti-inflammatory effects of curcumin (CUR), resveratrol (RES) and sulforaphane (SFN) against lipopolysaccharide/interferon-gamma (LPS/IFN-γ)-mediated inflammation in the absence or presence of DOX. For this purpose, RAW 264.7 cells were stimulated with LPS/IFN-γ (10 ng/mL/10 U/mL) in the absence or presence of DOX (0.1 µM). Our results showed that DOX alone is incapable of stimulating an inflammatory response in RAW 264.7 macrophages. Furthermore, after 24 h of incubation with LPS/IFN-γ, a significant increase in tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) mRNA levels was observed. Similarly, nitric oxide (NO) production and TNF-α and IL-6 protein levels were significantly upregulated. Moreover, in LPS/IFN-γ-treated macrophages, the microRNAs (miRNAs) miR-146a, miR-155, and miR-21 were significantly overexpressed. Interestingly, upon testing CUR, RES, and SFN against LPS/IFN-γ-mediated inflammation, only SFN was able to significantly reverse the LPS/IFN-γ-mediated induction of iNOS, TNF-α and IL-6 and attenuate miR-146a and miR-155 levels. In conclusion, SFN, at the transcriptional and posttranscriptional levels, exhibits potent immunomodulatory action against LPS/IFN-γ-stimulated macrophages, which may indicate SFN as a potential treatment for DOX-associated inflammation.

scholarly journals Hydrogen Sulfide Increases Nitric Oxide Production and Subsequent S-Nitrosylation in Endothelial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Ping-Ho Chen ◽  
Yaw-Syan Fu ◽  
Yun-Ming Wang ◽  
Kun-Han Yang ◽  
Danny Ling Wang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Hydrogen Sulfide ◽  
Protein Kinase ◽  
Fluorescent Probe ◽  
Acid Methyl Ester ◽  
High Specificity ◽  
Protein S ◽  
No Production ◽  
No Donor

Hydrogen sulfide (H2S) and nitric oxide (NO), two endogenous gaseous molecules in endothelial cells, got increased attention with respect to their protective roles in the cardiovascular system. However, the details of the signaling pathways between H2S and NO in endothelia cells remain unclear. In this study, a treatment with NaHS profoundly increased the expression and the activity of endothelial nitric oxide synthase. Elevated gaseous NO levels were observed by a novel and specific fluorescent probe, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe), and quantified by flow cytometry. Further study indicated an increase of upstream regulator for eNOS activation, AMP-activated protein kinase (AMPK), and protein kinase B (Akt). By using a biotin switch, the level of NO-mediated protein S-nitrosylation was also enhanced. However, with the addition of the NO donor, NOC-18, the expressions of cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase were not changed. The level of H2S was also monitored by a new designed fluorescent probe, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with high specificity. Therefore, NO did not reciprocally increase the expression of H2S-generating enzymes and the H2S level. The present study provides an integrated insight of cellular responses to H2S and NO from protein expression to gaseous molecule generation, which indicates the upstream role of H2S in modulating NO production and protein S-nitrosylation.

Arginase inhibition increases nitric oxide production in bovine pulmonary arterial endothelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L60-L68 ◽  
Author(s):  
Louis G. Chicoine ◽  
Michael L. Paffett ◽  
Tamara L. Young ◽  
Leif D. Nelin
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Urea Production ◽  
Pulmonary Arterial ◽  
Factor Α ◽  
Arterial Endothelial Cells ◽  
Regular Medium ◽  
Necrosis Factor

Nitric oxide (NO) is produced by NO synthase (NOS) from l-arginine (l-Arg). Alternatively, l-Arg can be metabolized by arginase to produce l-ornithine and urea. Arginase (AR) exists in two isoforms, ARI and ARII. We hypothesized that inhibiting AR with l-valine (l-Val) would increase NO production in bovine pulmonary arterial endothelial cells (bPAEC). bPAEC were grown to confluence in either regular medium (EGM; control) or EGM with lipopolysaccharide and tumor necrosis factor-α (L/T) added. Treatment of bPAEC with L/T resulted in greater ARI protein expression and ARII mRNA expression than in control bPAEC. Addition of l-Val to the medium led to a concentration-dependent decrease in urea production and a concentration-dependent increase in NO production in both control and L/T-treated bPAEC. In a second set of experiments, control and L/T bPAEC were grown in EGM, EGM with 30 mM l-Val, EGM with 10 mM l-Arg, or EGM with both 10 mM l-Arg and 30 mM l-Val. In both control and L/T bPAEC, treatment with l-Val decreased urea production and increased NO production. Treatment with l-Arg increased both urea and NO production. The addition of the combination l-Arg and l-Val decreased urea production compared with the addition of l-Arg alone and increased NO production compared with l-Val alone. These data suggest that competition for intracellular l-Arg by AR may be involved in the regulation of NOS activity in control bPAEC and in response to L/T treatment.

scholarly journals Decreased endothelial nitric oxide, systemic oxidative stress, and increased sympathetic modulation contribute to hypertension in obese rats

2014 ◽  
Vol 306 (10) ◽  
pp. H1472-H1480 ◽  
Author(s):  
Natalia Veronez da Cunha ◽  
Phileno Pinge-Filho ◽  
Carolina Panis ◽  
Bruno Rodrigues Silva ◽  
Laena Pernomian ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Arterial Pressure ◽  
Vascular Reactivity ◽  
Monosodium Glutamate ◽  
Saline Control ◽  
Pulse Interval ◽  
No Production ◽  
Obese Rats

We investigated the involvement of nitric oxide (NO) and reactive oxygen species (ROS) on autonomic cardiovascular parameters, vascular reactivity, and endothelial cells isolated from aorta of monosodium glutamate (MSG) obese rats. Obesity was induced by administration of 4 mg/g body wt of MSG or equimolar saline [control (CTR)] to newborn rats. At the 60th day, the treatment was started with NG-nitro-l-arginine methyl ester (l-NAME, 20 mg/kg) or 0.9% saline. At the 90th day, after artery catheterization, mean arterial pressure (MAP) and heart rate were recorded. Plasma was collected to assess lipid peroxidation. Endothelial cells isolated from aorta were evaluated by flow cytometry and fluorescence intensity (FI) emitted by NO-sensitive dye [4,5-diaminofluoresceindiacetate (DAF-2DA)] and by ROS-sensitive dye [dihydroethidium (DHE)]. Vascular reactivity was made by concentration-response curves of acetylcholine. MSG showed hypertension compared with CTR. Treatment with l-NAME increased MAP only in CTR. The MSG induced an increase in the low-frequency (LF) band and a decrease in the high-frequency band of pulse interval. l-NAME treatment increased the LF band of systolic arterial pressure only in CTR without changes in MSG. Lipid peroxidation levels were higher in MSG and were attenuated after l-NAME. In endothelial cells, basal FI to DAF was higher in CTR than in MSG. In both groups, acetylcholine increased FI for DAF from basal. The FI baseline to DHE was higher in MSG than in CTR. Acetylcholine increased FI to DHE in the CTR group, but decreased in MSG animals. We suggest that reduced NO production and increased production of ROS may contribute to hypertension in obese MSG animals.

Temporal dynamics of nitric oxide wave in early vasculogenesis

2021 ◽  
pp. 1358863X2110354
Author(s):  
Saranya Rajendran ◽  
Lakshmikirupa Sundaresan ◽  
Geege Venkatachalam ◽  
Krithika Rajendran ◽  
Jyotirmaya Behera ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Temporal Dynamics ◽  
Quantitative Polymerase Chain Reaction ◽  
Hypoxia Inducible Factor ◽  
Polymerase Chain Reaction Analysis ◽  
Tube Formation ◽  
Nitrite Reduction ◽  
Inos Mrna ◽  
No Production ◽  
Area Vasculosa

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10–HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

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