Roles of IL-1 and TNF-alpha in endotoxin-induced activation of nitric oxide synthase in cultured rat brain cells

1996 ◽  
Vol 270 (2) ◽  
pp. R326-R332 ◽  
Author(s):  
L. I. Romero ◽  
J. B. Tatro ◽  
J. A. Field ◽  
S. Reichlin
Keyword(s):  
Nitric Oxide ◽  
Interleukin 1 ◽  
Primary Cultures ◽  
Neonatal Rat ◽  
Tnf Alpha ◽  
Brain Cells ◽  
Nitrite Accumulation ◽  
No Production ◽  
Necrosis Factor Alpha ◽  
Oxide Synthase

In astrocytes and microglia, bacterial lipopolysaccharide (LPS) stimulates production and release of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO). Although IL-1 beta and TNF-alpha are themselves capable of inducing NO synthase (NOS) in glia, the specific factors mediating LPS induction of NOS in brain have not been identified. To determine whether LPS induction of NOS in brain cells is mediated by IL-1 or TNF-alpha, acting alone or in concert, the effects of IL-1-receptor antagonist (IL-1Ra) and of TNF-soluble receptor (TNFsRp55), presented individually and in combination, on LPS-induced NOS activity were tested. In glial-enriched mixed primary cultures of neonatal rat telencephalic cells, LPS (0.1-100 ng/ml), IL-1 beta (0.01-10 nM), and TNF-alpha (0.1-100 nM) each concentration dependently stimulated accumulation of nitrite, an indicator of NO production. Induction of nitrite accumulation by LPS and by IL-1 was blocked by N omega-nitro-L-arginine methyl ester and N omega-monomethyl-L-arginine, indicating that it was mediated by NOS. TNF-alpha alone induced NO production weakly as compared with IL-1, but combined submaximal concentrations of IL-1 beta (1 nM) and TNF-alpha (10 nM) induced NOS synergistically. Furthermore, TNFsRp55 and IL-1Ra each produced a dose-dependent partial inhibition of the NO response to LPS, and the effect of TNFsRp55 was equal to or greater than that of IL-1Ra. TNFsRp55 and IL-1Ra in combination were not significantly more effective than TNF-sRp55 alone. The results indicate that LPS induction of NOS activity in brain cells is mediated in part by both IL-1 beta and TNF-alpha.

scholarly journals Intraperitoneal injection of tetracyclines protects mice from lethal endotoxemia downregulating inducible nitric oxide synthase in various organs and cytokine and nitrate secretion in blood.

1997 ◽  
Vol 41 (1) ◽  
pp. 117-121 ◽  
Author(s):  
S Milano ◽  
F Arcoleo ◽  
P D'Agostino ◽  
E Cillari
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Intraperitoneal Injection ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

We have tested whether tetracyclines (TETs) are able to protect mice from lipopolysaccharide (LPS)-induced shock, a cytokine-mediated inflammatory reaction. Mice, injected with a single dose of tetracycline base (TETb; 1.5, 10 and 20 mg/kg of body weight) or doxycycline (DOXY; 1.5 mg/kg), were significantly protected from a lethal intraperitoneal injection of LPS (500 micrograms per mouse). TETs acted in early events triggered in response to LSP; in fact, they were no longer significantly protective if injected more than 1 h after the injection of endotoxin. LPS-treated mice protected by TETs showed a significant inhibition of tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and nitrate secretion in the blood, events that were directly related with the survival. In mice treated with TETs a significant decrease of inducible nitric oxide synthase (iNOS) activity was observed in spleen and peritoneal cells compared with that detected in mice treated with LPS alone. Furthermore, TETs were found to inhibit NO synthesis by peritoneal macrophages stimulated in vitro with LPS. On the contrary, TETs were unable to decrease the ability of the macrophages to synthesize IL-1 alpha and TNF-alpha in vitro. These results indicate that TETs are not able to act directly on the synthesis of these cytokines, but they may modulate other pathways that could in turn be responsible for the inhibition of IL-1 alpha and TNF-alpha synthesis. Altogether, these results indicate that TETs are advantageous candidates for the prophylaxis and treatment of septic shock in mice, having both antimicrobial activity and the ability to inhibit endogenous TNF-alpha, IL-1 alpha, and iNOS, hence, exerting, potent anti-inflammatory effects.

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 Inducible Nitric Oxide Synthase Is Not Essential for Control of Trypanosoma cruzi Infection in Mice

2004 ◽  
Vol 72 (7) ◽  
pp. 4081-4089 ◽  
Author(s):  
Kara L. Cummings ◽  
Rick L. Tarleton
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Trypanosoma Cruzi ◽  
Inducible Nitric Oxide Synthase ◽  
Interleukin 1 ◽  
Mouse Strains ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Immune control of many intracellular pathogens, including Trypanosoma cruzi, is reported to be dependent on the production of nitric oxide. In this study, we show that mice deficient in inducible nitric oxide synthase (iNOS or NOS2) exhibit resistance to T. cruzi infection that is comparable to that of wild-type mice. This is the case for two iNOS-deficient mouse strains, Nos2tm1Lau and Nos2 N5, infected with the Brazil or Tulahuen strain of T. cruzi. In all cases, blood parasitemia, tissue parasite load, and survival rates are similar between wild-type and iNOS-deficient mice. In contrast, both wild-type and Nos2tm1Lau mice died within 32 days postinfection when treated with the nitric oxide synthase inhibitor aminoguanidine. Increased transcription of NOS1 or NOS3 is not found in iNOS-knockout (KO) mice, indicating that the absence of nitric oxide production through iNOS is not compensated for by increased production of other NOS isoforms. However, Nos2tm1Lau mice exhibit enhanced expression of tumor necrosis factor alpha, interleukin-1, and macrophage inflammatory protein 1α compared to that of wild-type mice, and these alterations may in part compensate for the lack of iNOS. These results clearly show that iNOS is not required for control of T. cruzi infection in mice.

scholarly journals Effects of Resveratrol on Inflammatory Biomarkers in Glaucomatous Human Trabecular Meshwork Cells

Nutrients ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 984 ◽  
Author(s):  
Selom Avotri ◽  
Danita Eatman ◽  
Karen Russell-Randall
Keyword(s):  
Nitric Oxide ◽  
Trabecular Meshwork ◽  
Interleukin 1 ◽  
Inos Expression ◽  
No Production ◽  
Enos Expression ◽  
Average Value ◽  
Beneficial Effects ◽  
Trabecular Meshwork Cells ◽  
Oxide Synthase

Purpose: Resveratrol (RSV), an antioxidant polyphenol, has demonstrated beneficial effects in various ocular diseases including glaucoma. Our study was designed to evaluate the effects of RSV on nitric oxide synthase (NOS) enzymes, nitric oxide (NO) and interleukin-1 alpha (IL-1 α), in human glaucomatous trabecular meshwork (TM) cells. Methods: Western blot was utilized to determine endothelial and inducible NOS (eNOS, iNOS) expression. The concentration-related effects of RSV on IL-1 α and NO levels were assessed using the respective ELISA kits. Results: Densitometry data showed concentration-related increases in eNOS, and reduction in iNOS expression at high RSV concentrations. RSV treatment (0.1, 1, 10 and 100 µM) resulted in increased NO levels (6 ± 0.7, 7 ± 0.8, 7.3 ± 0.7 and 9.5 ± 1 nM/mg protein, respectively). The average value obtained for control was 4.8 ± 0.6 nM/mg protein. Significant increases in IL-1α levels were observed with lower concentrations of RSV. However, at higher RSV concentrations (10–100 μM), IL-1 levels decreased. Conclusions: Resveratrol increased NO in glaucomatous TM cells, possibly by increasing eNOS expression. Thus, RSV-induced NO production supports the beneficial effects of this antioxidant in glaucoma. Furthermore, our results showing a reduction in iNOS, a contributor to oxidative stress expression, further support RSV’s antioxidant capabilities in vision.

Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells

1995 ◽  
Vol 268 (4) ◽  
pp. F770-F777 ◽  
Author(s):  
M. G. Mohaupt ◽  
J. Schwobel ◽  
J. L. Elzie ◽  
G. S. Kannan ◽  
B. C. Kone
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Cytokines ◽  
Collecting Duct ◽  
Tnf Alpha ◽  
Inos Mrna ◽  
No Production ◽  
Inos Gene ◽  
Medullary Collecting Duct ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis.

TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells

1995 ◽  
Vol 269 (2) ◽  
pp. F212-F217 ◽  
Author(s):  
K. S. Lau ◽  
O. Nakashima ◽  
G. R. Aalund ◽  
L. Hogarth ◽  
K. Ujiie ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Nitric Oxide Synthase ◽  
Vascular Smooth Muscle ◽  
Guanylyl Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
Tnf Alpha ◽  
No Production ◽  
Ifn Gamma ◽  
Oxide Synthase

Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription-polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN-gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC.

Hydrogen peroxide downregulates IL-1-driven mesangial iNOS activity: implications for glomerulonephritis

1997 ◽  
Vol 272 (6) ◽  
pp. F721-F728 ◽  
Author(s):  
E. A. Jaimes ◽  
K. A. Nath ◽  
L. Raij
Keyword(s):  
Nitric Oxide ◽  
Hydrogen Peroxide ◽  
Mesangial Cells ◽  
Interleukin 1 ◽  
Inhibitory Effect ◽  
Inos Mrna ◽  
No Production ◽  
Glomerular Injury ◽  
Inos Activity ◽  
Oxide Synthase

In glomerulonephritides, autacoids such as nitric oxide (NO), reactive oxygen species, and prostanoids are produced in increased amounts in response to cytokines such as interleukin-1 (IL-1). These autacoids influence the expression of glomerular injury by their direct as well as interactive actions. We studied the effect of hydrogen peroxide (H2O2) on NO production in rat mesangial cells. We demonstrate that transient exposure of mesangial cells to H2O2 prior to sustained exposure to IL-1 decreased extracellular accumulation of NO2/NO3 and cellular guanosine 3,'5'-cyclic monophosphate (cGMP) content. H2O2 markedly impaired inducible nitric oxide synthase (iNOS) activity induced by IL-1 directly measured by the conversion of L-[14C]arginine to L-[14C]citrulline. Such impairment in iNOS activity was accompanied by a parallel reduction in iNOS protein abundance but not by a reduced expression of iNOS mRNA. The inhibitory effect of H2O2 on NOS activity was further supported by peroxide-induced impairment in IL-1-driven, NO-dependent synthesis of prostaglandin E2. Our studies thus provide the first direct evidence of a posttranscriptional inhibitory effect of H2O2 on iNOS activity. Additionally, our studies uncover the existence of a previously unrecognized effect of H2O2 on the production of NO that may exert influence on the severity of glomerular injury during glomerular inflammation.

Abstract 1333: Modulation Of Cardiac Beta-adrenergic Responsiveness By The Neuronal Nitric Oxide Synthase/Sarcolemmal Calcium Pump Complex

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Tamer M Mohamed ◽  
Delvac Oceandy ◽  
Nasser Alatwi ◽  
Florence Baudoin ◽  
Elizabeth J Cartwright ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Neonatal Rat ◽  
No Production ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Adrenergic Response ◽  
Oxide Synthase

The pivotal role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function has only recently been unveiled. Notably, others have shown that responsiveness to β-adrenergic stimulation is dependent on nNOS activity. In a cellular model, we showed that the Ca 2+ /calmodulin-dependent nNOS activity is reduced by overexpression of isoform 4b of the plasma membrane Ca 2+ /Calmodulin-dependent Ca 2+ -pump (PMCA4b), which binds to nNOS. We demonstrated that PMCA4b overexpression in the heart reduced β-adrenergic responsiveness in vivo via an nNOS dependent mechanism (Oceandy et al, Circulation 2007). Here we investigated the cellular mechanisms of the regulation of the β-adrenergic response by PMCA4b. We used an adenoviral system to overexpress PMCA4b (PMCA4b cells) or LacZ (control, C) in neonatal rat cardiomyocytes. PMCA4b cells showed an 18±5% and 24±5% reduction in nitric oxide (DAF-FM fluorescence) and cGMP levels, respectively (n=6, p<0.05 each) compared to C demonstrating the regulation of NO production by the PMCA4b in this system. Since nNOS has been shown to regulate phospholamban (PLB) phosphorylation, we examined phosphorylation of PLB at Ser16. PMCA4b cells showed a significant increase in Ser16-PLB at baseline (66±17%, p<0.05) compared to C. As a result of increased baseline Ser16-PLB in PMCA4b cells, β-adrenergic stimulation of PMCA4b cells using 2μM isoproter-enol (IP) showed reduced relative induction in Ser16-PLB (23±10% vs. 78±19% in C; n=5, p<0.05). Further analysis in adult cardiomyocytes isolated from our PMCA4b transgenic mice (PMCA4b TG) demonstrated that PMCA4b TG showed 3-fold higher Ser16-PLB phosphorylation at baseline compared to wild type (WT) myocytes and the relative response following β-adrenergic stimulation was significantly reduced (1.2±0.2 fold induction after IP treatment in PMCA4b TG, vs. 3.1±0.7 in WT, n=5, p<0.05). Thus, PMCA4b regulates NO production from nNOS, which in turn modulates cGMP levels and PLB phosphorylation. These findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4b and place this Ca 2+ -pump upstream of the recently described pathway linking nNOS and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).

Protein kinase A activation is required for IL-1-induced nitric oxide production by cardiac myocytes

1996 ◽  
Vol 271 (1) ◽  
pp. C429-C434 ◽  
Author(s):  
C. V. Oddis ◽  
R. L. Simmons ◽  
B. G. Hattler ◽  
M. S. Finkel
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cardiac Myocytes ◽  
Interleukin 1 ◽  
Neonatal Rat ◽  
Inos Mrna ◽  
No Production ◽  
Neonatal Rat Cardiac Myocytes ◽  
Kinase A

We have previously reported that interleukin-1 beta (IL-1) alone induced the transcription of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production by isolated neonatal rat cardiac myocytes (CM). The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. The addition of IL-1 to CM resulted in a peak rise in both adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) activities by 10 min followed by rapid declines and return to basal levels within 60 min. The PKA inhibitor KT-5720 completely blocked NO-2 production by IL-1-stimulated CM (P < 0.01; n = 12). The protein kinase C (PKC) inhibitor, calphostin C, had no effect on NO2- production by IL-1 stimulated CM [P = not significant (NS); n = 12]. The addition of PKA+cAMP to cytosols derived from IL-1-treated CM did not directly enhance iNOS enzyme activity (P = NS; n = 3). CM treated with IL-1 alone stained positively for iNOS protein by immunohistochemistry. iNOS staining was absent in CM treated with IL-1+KT-5720. KT-5720 resulted in an earlier disappearance of iNOS mRNA from IL-1-treated CM, as detected by semiquantitative reverse transcriptase-polymerase chain reaction. We report for the first time that PKA (but not PKC) activation is required for IL-1-induced NO production by CM.

Adenosine stimulates nitric oxide synthesis in rat cardiac myocytes

1997 ◽  
Vol 273 (1) ◽  
pp. H59-H65 ◽  
Author(s):  
U. Ikeda ◽  
K. Kurosaki ◽  
M. Shimpo ◽  
K. Okada ◽  
T. Saito ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Receptor Antagonist ◽  
Cardiac Myocytes ◽  
Interleukin 1 ◽  
Neonatal Rat ◽  
Nitrite Accumulation ◽  
Dependent Manner ◽  
Interleukin 1 Beta ◽  
Nitrite Production ◽  
Rat Cardiac Myocytes

We investigated the effects of adenosine on nitric oxide (NO) synthesis by measuring the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes. Incubation of cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite production. The interleukin-1 beta-induced nitrite production by cardiac myocytes was significantly increased by adenosine or its stable analog 2-chloroadenosine in a dose-dependent manner (10(-7)-10(-4) M). The adenosine A2-receptor antagonist KF-17837 (10(-6) M), but not the A1-receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (10(-6) M), significantly inhibited 2-chloroadenosine-mediated nitrite production. The 2-chloroadenosine-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by inducible NO synthase mRNA and protein accumulation. In the presence of N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) (10(-3) M) or isoproterenol (10(-5) M), interleukin-1 beta-induced nitrite accumulation was further increased, but the effect of 2-chloroadenosine was not additive or synergistic. The protein kinase C inhibitor calphostin C did not inhibit the effect of 2-chloroadenosine. These results indicate that adenosine acts on A2 receptors and augments NO synthesis in interleukin-1 beta-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

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