No abstract

Stimulated rat peritoneal neutrophils release a platelet inhibitory factor with the pharmacological properties of NO. This release is inhibited by N0-monomethyl-L-arginine and L-canavanine, indicating that it occurs through a mechanism similar to that in vascular endothelial cells and macrophages. As the degree of stimulation increases, the factor released is progressively inactivated by concomitant release of superoxide anions.

1 The effect of endotoxin (E. coli lipopolysaccharide) on the induction of nitric oxide synthase (NOS) and the changes in vascular permeability in the colon and jejunum over a 5 h period have been investigated in the rat. 2 Under resting conditions, a calcium-dependent constitutive NOS, determined by the conversion of radiolabelled L-arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue. 3 Administration of endotoxin (3 mg kg-', i.v.) led, after a 2 h lag period, to the appearance of calcium-independent NOS activity in the colon and jejunum ex vivo, characteristic of the inducible NOS enzyme. 4 Administration of endotoxin led to an increase in colonic and jejunal vascular permeability after a lag period of 3 h, determined by the leakage of radiolabelled albumin. 5 Pretreatment with dexamethasone (1 mg kg-' s.c., 2 h prior to challenge) inhibited both the induction of NOS and the vascular leakage induced by endotoxin. 6 Administration of the NO synthase inhibitor NG-monomethyl-L-arginine (12.5-50mg kg-', s.c.) 3 h after endotoxin injection, dose-dependently reduced the subsequent increase in vascular permeability in jejunum and colon, an effect reversed by L-arginine (300 mg kg-', s.c.). 7 These findings suggest that induction of NOS is associated with the vascular injury induced by endotoxin in the rat colon and jejunum.

SummaryPlatelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) plays an active role in the process of leukocyte migration through cultured endothelial cells in vitro and anti-PECAM-1 antibodies (Abs) inhibit accumulation ofleukocytes into sites of inflammation in vivo. Despite the latter, it is still not clear at which stage of leukocyte emigration in vivo PECAM-1 is involved. To address this point directly, we studied the effect of an anti-PECAM-1 Ab, recognizing rat PECAM-1, on leukocyte responses within rat mesenteric microvessels using intravital microscopy. In mesenteric preparations activated by interleukin (IL)-113, the anti-PECAM-1 Ab had no significant effect on the rolling or adhesion ofleukocytes, but inhibited their migration into the surrounding extravascular tissue in a dose-dependent manner. Although in some vessel segments these leukocytes had come to a halt within the vascular lumen, often the leukocytes appeared to be trapped within the vessel wall. Analysis of these sections by electron microscopy revealed that the leukocytes had passed through endothelial cell junctions but not the basement membrane. In contrast to the effect of the Ab in mesenteric preparations treated with IL-113, leukocyte extravasation induced by topical or intraperitoneal administration of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was not inhibited by the anti-PECAM-1 Ab. These results directly demonstrate a role for PECAM-1 in leukocyte extravasation in vivo and indicate that this involvement is selective for leukocyte extravasation elicited by certain inflammatory mediators. Further, our findings provide the first in vivo indication that PECAM-1 may have an important role in triggering the passage of leukocytes through the perivascular basement membrane.

1 The role of endogenous nitric oxide (NO) in maintaining intestinal vascular integrity following acute endotoxin (E. coli. lipopolysaccharide) challenge was investigated in the anaesthetized rat by use of N0-monomethyl-L-arginine (L-NMMA), a selective inhibitor of NO synthesis. 2 L-NMMA (10-50mgkg-1, i.v.) pretreatment enhanced both the macroscopic and histological intestinal damage and the increases in vascular permeability, measured as the leakage of [125I]-labelled human serum albumen, induced after 15 min by endotoxin (50 mg kg 1, i.v.). 3 The effects of L-NMMA (50mgkg-1, i.v.) were enantiomer specific, as D-NMMA had no effect. Furthermore, these effects were reversed by L-arginine (300mg kg1, i.v.), the precursor of NO synthesis but not by D-arginine (300mg kg-1, i.v.). 4 L-NMMA (10-50mgkg-1, i.v.) increased mean systemic arterial blood pressure but this does not appear to be the mechanism by which endotoxin-induced intestinal damage was enhanced, since similar systemic pressor responses induced by phenylephrine (lOpgkg-1 min-1, i.v.), had no such effect.5 The results suggest that synthesis of NO from L-arginine has a role in maintaining the microvascular integrity of the intestinal mucosa following acute endotoxin challenge.

1 The role of arachidonic acid metabolites and oxygen radicals in carrageenin-induced rat paw oedema and dermal reverse passive Arthus reaction (RPA) have been investigated. 2 Indomethacin (10 mg kg-', p.o.) inhibited carrageenin paw oedema when administered 30 min before, but not 2 h after carrageenin. BWB70C (10 mg kg-', p.o.), a selective inhibitor of 5-lipoxygenase, had no effect whether administered before or after carrageenin. Administration of both indomethacin and BWB70C had no greater anti-inflammatory effect than indomethacin alone. 3 BW755C (20 mg kg-, p.o.), which inhibits the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, or superoxide dismutase-polyethylene glycol conjugate (SOD-PEG, 3000 u, i.v.) inhibited carrageenin paw oedema whether administered either 30 min before, or 2 h after carrageenin.4 Pretreatment with dexamethasone (0.1 mg kg-') or colchicine (2 mg kg-'), likewise suppressed carrageenin paw oedema. 5 BW755C (25-100mgkg-', p.o.) dose-dependently reduced plasma leakage in the RPA, whereas indomethacin (5mgkg-', p.o.) or BWB70C either alone or in combination, did not. 6 SOD-PEG (300-3000 u, i.v.) dose-dependently inhibited plasma leakage in the RPA. In addition, the iron chelator and peroxyl radical scavenger, desferrioxamine (200mgkg-', s.c.) also inhibited plasma leakage. 7 Pretreatment with dexamethasone (0.1 mg kg-') or colchicine (1 mg kg-l) reduced the plasma leakage in RPA, whereas MK-886 (10mgkg-1) had no effect. 8 These results indicate an important role for oxygen radicals but not arachidonic acid metabolites in the maintenance of carrageenin paw oedema and the plasma leakage in RPA. Furthermore, the results suggest that the anti-inflammatory actions of BW755C can be dissociated from its effects on arachidonic acid metabolism and are attributed to its anti-oxidant activity.

The relationship between 14C-arachidonic acid (14C-AA) metabolism, myeloperoxidase activity (MPO) and leukocyte infiltration was studied in a chronic model of inflammatory bowel disease, induced by a single intrarectal application of the hapten, trinitrobenzene sulphonic acid (TNB). The colonic damage produced by TNB was accompanied, after 12-36 hours, by a marked increase in MPO, which was directly correlated to leukocyte infiltration, assessed histologically. There was also a marked increase in the metabolism of 14C-AA, by homogenates of inflamed colon, to 12-, 15-HETE and 6-keto-PGF1 alpha as indices of lipoxygenase and cyclo-oxygenase metabolism respectively. However, a further increase in MPO-cellular infiltration, between 36-72 hours after TNB, was accompanied by a reduction in 12- and 15-HETE formation. The increase in MPO-cellular infiltration was maintained for up to 3 weeks, at which time both 12-, 15-HETE and 6-keto-PGF1 alpha formation had returned to control levels. These results suggest that these AA metabolites have a greater importance in the acute phase of the inflammatory response induced by TNB compared to the later chronic phase.

The discovery of a novel class of nitric oxide synthase (NOS) inhibitors, 2-substituted 1,2-dihydro-4-quinazolinamines, and the related 4'-aminospiro[piperidine-4,2'(1'H)-quinazolin]-4'-amines is described. Members of both series exhibit nanomolar potency and high selectivity for the inducible isoform of the enzyme (i-NOS) relative to the constitutive isoforms in vitro. Efficacy in acute and chronic animal models of inflammatory disease following oral administration has also been demonstrated using these compounds.