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Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation.

ABSTRACT: 1 The role of nitric oxide (NO) derived from constitutive and inducible nitric oxide synthase (cNOS and iNOS) and its relationship to oxygen-derived free radicals and prostaglandins (PG) was investigated in a carrageenan-induced model of acute hindpaw inflammation. 2 The intraplantar injection of carrageenan elicited an inflammatory response that was characterized by a time-dependent increase in paw oedema, neutrophil infiltration, and increased levels of nitrite/nitrate (NO2-/NO3-) and prostaglandin E2(PGE2) in the paw exudate. 3 Paw oedema was maximal by 6 h and remained elevated for 10 h following carrageenan administration. The non-selective cNOS/iNOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) given intravenously (30-300 mg kg-1) 1 h before or after carrageenan administration, inhibited paw oedema at all time points. 4 The selective iNOS inhibitors, N-iminoethyl-L-lysine (L-NIL) or aminoguanidine (AG), failed to inhibit carrageenan-induced paw oedema during the first 4 h following carrageenan administration, but inhibited paw oedema at subsequent time points (from 5-10 h). iNOS mRNA was detected between 3 to 10 h following carrageenan administration using ribonuclease protection assays. iNOS protein was first detected 6 h and was maximal 10 h following carrageenan administration as shown by Western blot analysis. Administration of the iNOS inhibitors 5 h after carrageenan (a time point where iNOS was expressed) inhibited paw oedema at all subsequent time points. Infiltrating neutrophils were not the source of iNOS since pretreatment with colchicine (2 mg kg-1) suppressed neutrophil infiltration, but did not inhibit the iNOS mRNA expression or the elevated NO2-/NO3- levels in the paw exudate. 5 Inhibition of paw oedema by the NOS inhibitors was associated with attenuation of both the NO2-/NO3- and PGE2 levels in the paw exudate. These inhibitors also reduced the neutrophil infiltration at the site of inflammation. 6 Recombinant human Cu/Zn superoxide dismutase coupled to polyethyleneglycol (PEGrhSOD; 12 x 10(3) u kg-1), administered intravenously either 30 min prior to or 1 h after carrageenan injection, inhibited paw oedema and neutrophil infiltration, but had no effect on NO2-/NO3- or PGE2 production in the paw exudate. The administration of catalase (40 x 10(3) u kg-1), given intraperitoneally 30 min before carrageenan administration, had no effect on paw oedema. Treatment with desferrioxamine (300 mg kg-1), given subcutaneously 1 h before carrageenan, inhibited paw oedema during the first 2 h after carrageenan administration, but not at later times. 7 These results suggest that the NO produced by cNOS is involved in the development of inflammation at early time points following carrageenan administration and that NO produced by iNOS is involved in the maintenance of the inflammatory response at later time points. The potential interactions of NO with superoxide anion and PG is discussed.

SUBMITTER: Salvemini D

PROVIDER: S-EPMC1909531 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation.

Salvemini D D Wang Z Q ZQ Wyatt P S PS Bourdon D M DM Marino M H MH Manning P T PT Currie M G MG

British journal of pharmacology 19960601 4

1 The role of nitric oxide (NO) derived from constitutive and inducible nitric oxide synthase (cNOS and iNOS) and its relationship to oxygen-derived free radicals and prostaglandins (PG) was investigated in a carrageenan-induced model of acute hindpaw inflammation. 2 The intraplantar injection of carrageenan elicited an inflammatory response that was characterized by a time-dependent increase in paw oedema, neutrophil infiltration, and increased levels of nitrite/nitrate (NO2-/NO3-) and prostagl ...[more]

PMID: 8799551

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Project description:Endothelial nitric oxide synthase (eNOS) is a critical modulator of vascular tone and blood flow and plays major roles in liver physiology and pathophysiology. Nitric oxide (NO) is widely recognized as one of the key humoral factors important for the initiation of liver regeneration in response to partial hepatectomy. Liver regeneration in response to partial hepatectomy is dependent on the efficiency of growth factor-mediated cell-cycle progression. Epidermal growth factor receptor (EGFR) is a critical mediator of multiple hepatic mitogens, such as epidermal growth factor (EGF), transforming growth factor alpha, amphiregulin, and heparin-binding EGF in regenerating livers. However, the functional significance of endothelial nitric oxide synthase (eNOS) expressed in hepatocytes, and its potential role in EGFR-mediated hepatocyte proliferation, remains unexplored. We sought to determine whether eNOS is essential for hepatocyte proliferation in response to partial hepatectomy (PH). Our studies with eNOS knockout (eNOS(-/-) ) mice suggest that eNOS activation is essential for the efficient induction of early events and elicitation of a robust hepatocyte proliferative response to PH. Moreover, eNOS expression is essential for the efficient early induction of matrix metalloprotease-9, a known mediator of extracellular matrix remodeling and growth factor activation in regenerating livers. Our in vitro studies suggest that eNOS is a critical mediator of EGF-induced hepatocyte proliferation, potentially via its influence on the induction of early growth response-1 (Egr-1) and phosphorylation of c-Jun--known mediators of cell-cycle progression. EGF-induced eNOS phosphorylation at Ser 1177 is dependent on the phosphorylation and activation of EGFR/PI3 kinase/AKT signaling in hepatocytes.Collectively, these results highlight a hitherto unrecognized role for eNOS activation in hepatocyte proliferation with implications for targeted therapies to enhance liver regenerative response in chronic disorders.

Project description:Osteoarthritis (OA) is an aging-associated disease of diarthrodial joints that involves changes in the bone, cartilage and soft tissue. Chondrocytes are the only cell type in cartilage and play a pivotal role in tissue homeostasis and cartilage remodeling in OA. Age- or OA-dependent metabolic changes affecting extracelluar matrix composition as well as variations in the response to cytokines and growth factors have been investigated in human chondrocytes. However, the molecular mechanisms causing age-dependent variation of the chondrocyte phenotype are poorly characterized. The proinflammatory mediator nitric oxide (NO) is produced when chondrocytes are exposed to cytokines such as IL-1. NO affects the energy metabolism of cells through interfering with mitochondrial respiration and glycolysis and has been implicated in extracellular matrix synthesis and degradation and chondrocyte death. NO may contribute to the aging process by compromising the energy balance ! in chondrocytes through the generation of an oxidizing environment and this may in turn affect matrix gene expression. In human chondrocytes NO effects on the expression of extacelluar matrix genes or genes related to matrix structure have not been addressed comprehensively. In chondrocytes NO has profound effects on extracellular matrix: Interleukin-1 induced the production of nitric oxide in human articular chondrocytes in alginate culture and simultaneously inhibited the biosynthesis of proteoglycans. NG-monomethyl-L-arginine (L-NMMA) inhibited nitric oxide formation and the suppression of proteoglycan synthesis. The nitric oxide donor, S-nitrosyl-acetyl-D,L- penicillamine (SNAP) also inhibited proteoglycan biosynthesis suggesting that interleukin-1 suppresses matrix synthesis through mechanisms involving the production of NO. Slices of rabbit articular cartilage synthesized large quantities of nitric oxide (NO) following exposure to IL-1 and strongly suppressed the incorporation of 35SO4 into the glycosaminoglycans (GAGs) of cartilage proteoglycans. Simultaneous treatment of cartilage fragments with IL-1 and L-NMMA inhibited NO synthesis in response to IL-1 and restored proteoglycan synthesis. SNAP reversibly mimicked the effect! of IL-1 on proteoglycan synthesis. These data suggest that endogenously synthesized NO is the mediator which reduces cartilage proteoglycan synthesis in response to cytokines such as IL-1. Aggrecan and type II collagen synthesis are also affected by NO. Our hypothesis entails that in human chondrocytes endogenous NO modulates the expression of matrix genes as well as enzymes involved in extracellular matrix remodelling. Experimental approach: In this experiment cultured chondrocytes from 3 donors was left unstimulated (control) or stimulated with IL-1, L-NMMA or IL-1 + L-NMMA for a period of 8 days in order to achieve sufficiently high levels of intracellular NO and stable expression of a subset of genes. Total RNA was isolated using the Trizol procedure. Gene expression will be probed using the Glycov2 chip.

Dataset Information

Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation.

Publications

Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation.

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