European Journal of Pharmacology, 211 (1992) 177-182 © 1992 Elsevier Soence Pubhshers B.V All rights reserved 0014-2999/92/$05 00

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EJP 52274

Modulation of acute inflammation by endogenous nitric oxide A r m a n d o Ialenti, A n g e l a I a n a r o , S a l v a d o r M o n c a d a 1 a n d M a s s i m o Di R o s a Department of Experimental Pharmacology, Umverszty of Naples Federlco II, Via Domemco Montesano 49, 80131 Napoh, Italy and 1 Wellcome Research Laboratorws, Langley Court, Beckenham, Kent BR3 3BS, U K Recewed 8 August 1991, revised MS received 22 October 1991, accepted 12 November 1991

The role of endogenous nitric oxide (NO) in acute inflammation was investigated using two inhibitors of NO synthase (NG-nitro-L-arginine methyl ester(L-NAME) and NG-monomethyl-L-argInlne (L-NMMA)) as well as L- or D-arginine. The effect of test compounds was studied on the carrhgeenin-lnduced increase in vascular permeability in rat skin and in dextran- and carrageenin-induced paw oedema. Both L-NAME and L-NMMA dose dependently inhibited the increase in vascular permeability and oedema formation. L- but not D-arginine increased these inflammatory responses and reversed the inhibitory effects of L-NAME and L-NMMA. In dexamethasone-treated rats L-arginlne enhanced the dextran-induced oedema and the early phase of carrageenin-lnduced oedema but did not modify the inhibition by dexamethasone of the late phase of carrageenin-induced oedema. These results suggest that endogenous NO is released at the site of acute inflammation and modulates oedema formation. Depending on the time course or on the type of inflammation, NO may be predominantly generated by the constitutive or by the inducible NO synthase. Carrageenin, Dextran; Nitric oxide (NO); Oedema; Vascular permeability

I. Introduction

Acute inflammation depends on the release of chemical mediators which bring about oedema formation as a result of extravasation of fluid and proteins from the local microvasculature and accumulation of polymorphonuclear leucocytes (PMNs) at the inflammatory site. Nitric oxide (NO) is a labile humoral substance which accounts for the activity of endothelium-derived relaxing factor ( E D R F ) and causes vasodilatation by relaxing vascular smooth muscle via stimulation of the soluble guanylate cyclase (for review see Moncada et al., 1989). NO is released from vascular endothelial cells in vitro in response to a variety of substances such as bradykinin (Palmer et al., 1987), histamine (Sakuma and Levi, 1988) and 5-hydroxytryptamine (Richard et al., 1990). NO is also formed by macrophages activated by lipopolysaccharide (LPS) or cytokines (for review see Hibbs et al., 1990). Furthermore, phlogogenic agents (oyster glycogen, carrageenin) and inflammatory mediators (leukotriene B 4) induce or enhance the formation and release of E D R F / N O from PMNs (Rimele

Correspondence to: M. DI Rosa, Department of Experimental Pharmacology, Unwerslty of Naples Federlco II, Via Domenico Montesano 49, 80131 Napoh, Italy. Tel 39 81.748 6414, fax 39 81 748 6403

et al., 1988; Sturm et al., 1989; McCall et al., 1989). Thus at the site of an acute inflammatory reaction all the conditions are met for the generation of NO and for a role of this compound as an inflammatory mediator. NO is synthesized from the terminal guanidino nitrogen atom(s) of the amino acid L-arginine by an enzyme, NO synthase (Palmer et al., 1988). At least two types of NO synthase have so far been identified (Moncada et al., 1991). One is constitutive, C a 2 + / calmodulin-dependent and releases NO for short periods in response to receptor stimulation. The other enzyme, which has b e e n f o u n d in activated macrophages and PMNs, is inducible, CaZ+-indepen dent and, once expressed, synthesizes NO for long periods. Both enzymes are inhibited in vivo and in vitro by certain L-arginine analogues (Rees et al., 1990; McCall et al., 1991). In addition, glucocorticoids inhibit the induction of the m a c r o p h a g e / P M N enzyme but do not affect the endothelial/brain NO synthase (Di Rosa et al., 1990; Radomski et al., 1990). Furthermore, in several systems the effects of inhibitors of the NO synthase can be reversed by L- but not D-arginine, while those of dexamethasone cannot (O'Connor and Moncada, 1991). The identification and characterization of these inhibitors allows the formation of NO from Larginine to be studied indirectly.

178 In this study we have investigated the effect of the NO synthase inhibitors, NC-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (LNAME), as well as L- or D-arginine, on the increase in skin vascular permeability induced by carrageenin and on paw oedema formation induced by carrageenin and dextran in the rat. We have also studied the effect of L- or D-arginine on these inflammatory reactions in rats treated with dexamethasone in order to evaluate the possible role of the Ca2+-independent NO synthase in acute inflammation.

2. Materials and methods

2.1. M a ~ n a ~ Dextran (molecular weight 70000) was obtained from Pharmacia and fluorescein isothiocyanate was from Fluka. L-NMMA was synthesized by Wellcome Research Laboratories. All other reagents were obtained from Sigma. 2. 2. Vascular permeability Local increases in vascular permeability were measured in the shaved dorsal skin of male Wistar rats (180-220 g) as the extravasation of fluorescein isothiocyanate-conjugated bovine serum albumin (F-BSA) accumulated at skin sites in response to intradermal injections of test agents made up in saline, as previously described (Watanabe et al., 1986). Fluorescein isothiocyanate was conjugated to bovine serum albumin according to the procedure described by McKinney et al. (1964). The agents used were carrageenin, L-arginine, Darginine, L-NAME and L-NMMA. Each combination of test agents was injected intradermally (0.1 ml) according to a balanced site pattern. After 150 min the rats were injected i.v. with 1 m l / k g 10% F-BSA solution in saline. After a 30-min accumulation period the rats were killed with ether, the dorsal skin was removed and skin sites of 20 mm diameter were punched out. The skin samples were processed as described (Watanabe et al., 1986) for the extraction of the fluorescent tracer which was measured with a spectrofluorimeter (Perkin-Elmer, LS-5B) with 490 nm for excitation and 521 nm for emission. Vascular permeability was measured as F-BSA extravasation and expressed as /xg F-BSA accumulated at the injection site. The amount of F-BSA in each skin sample was calculated from a calibration curve obtained from skin samples of rats injected intradermally with various amounts of F-BSA.

The 30-rain accumulation period between 150-180 rain was selected since we had found in preliminary experiments that the peak increase in vascular permeability induced by carrageenin occurs about 3 h after its intradermal injection. 2.3. Paw oedema Paw oedema was induced in male Wistar rats (140160 g) by subplantar injection of 0.1 ml saline containing 1% A-carrageenin or 3% dextran. Each phlogogenic agent was given alone or in combination with L-arginine, D-arginine, L-NAME or L-NMMA. In each case the test agents were solubilized in saline and the injected volume was 0.1 ml. The volume of the paw was measured by plethysmometry (Basile, Milano, Italy) immediately after the injection as previously described (Di Rosa and Willoughby, 1971). Subsequent readings of the volume of the same paw were carried out at 30or 60-min intervals and compared to the initial readings. In gome experiments paw oedema was induced in rats treated with dexamethasone sodium phosphate (0.1 m g / k g ) given s.c. 2 h prior to the phlogogenic agents.

3. Results

3.1. Vascular permeabtlity In preliminary experiments we established that LN A M E or L-NMMA at doses up to 25 ~g per site had no effect on basal vascular permeability (the F-BSA extravasation measured at saline-injected sites). The effects of various test agents on F-BSA extravasation induced by intradermal injection of carrageenin are shown in fig. 1. Both L-NAME and L-NMMA, given at 1, 5 and 25 ~g, caused a dose-related inhibition of carrageenin-induced F-BSA extravasation in rat skin. Thus both agents given at 1 /zg inhibited the carrageenin-induced F-BSA extravasation by about 7%, at 5 ~ g by about 20% (P < 0.05) and at 25 /xg by about 35% (P < 0.05). In contrast, L-arginine, but not Darginine, 1 and 3 mg, increased F-BSA extravasation by 8 and 28% (P < 0.05), respectively. The inhibitory effects of 25 /~g of both L-NAME and L-NMMA were completely reversed by the concomitant administration of 3 mg L-arginine but not D-arginine. 3.2. Paw oedema Rat paw oedema whether induced by dextran or by carrageenin, was inhibited by L-NAME or L-NMMA and increased by L- but not D-arginine. The time

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Modulation of acute inflammation by endogenous nitric oxide.

The role of endogenous nitric oxide (NO) in acute inflammation was investigated using two inhibitors of NO synthase (NG-nitro-L-arginine methyl ester(...
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