Brendan J.R. Whittle

Role of nitric oxide in maintaining vascular integrity in endotoxin‐induced acute intestinal damage in the rat

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 NG‐monomethyl‐l‐arginine (l‐NMMA), a selective inhibitor of NO synthesis. 2 l‐NMMA (10–50 mg kg−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 (50 mg kg−1, i.v.) were enantiomer specific, as d‐NMMA had no effect. Furthermore, these effects were reversed by l‐arginine (300 mg kg−1, i.v.), the precursor of NO synthesis but not by d‐arginine (300 mg kg−1, i.v.). 4 l‐NMMA (10–50 mg kg−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 (10 μg kg−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.

Comparison of the effects of prostacyclin (PGI2), prostaglandin E1 and D2 on platelet aggregation in different species.

The activity of prostacyclin (PGI2), PGE1 or PGD2 as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD2 was a weak inhibitor in dog, rabbit and rat plasma whereas PGE1 and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE1 or PGD2. Compound N-0164 abolished the inhibition by PGD2 of human platelet aggregation but did not inhibit the effects of PGE1 or prostacyclin. The results suggest that prostacyclin and PGE1 act on similar sites on platelets which are distinct from those for PGD2.

The induction of nitric oxide synthase and intestinal vascular permeability by endotoxin in the rat

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 radiolabeled l‐arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue. 3 Administration of endotoxin (3 mg kg−1, 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−1 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 (12.5–50 mg kg−1, 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−1, 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.

The vasodilator role of endogenous nitric oxide in the rat gastric microcirculation.

The role of endogenous nitric oxide (NO) in the gastric microcirculation of the anaesthetised rat was investigated using the selective inhibitor of NO synthesis, NG-monomethyl-L-arginine (L-NMMA). L-NMMA (12.5-50 mg kg-1 i.v.) induced a dose-dependent increase in systemic arterial blood pressure (BP) and fall in resting gastric mucosal blood flow (MBF), as estimated by hydrogen-gas clearance. The effects of L-NMMA on BP and MBF were abolished by concurrent administration of L-arginine. The enantiomer D-NMMA had no effect on resting BP or MBF. These findings indicate that endogenous NO, derived from L-arginine, plays a local vasodilator role in the gastric mucosal microvasculature.

Potent ulcerogenic actions of platelet-activating factor on the stomach.

Platelet-activating factor (PAF) is an endogenous phospholipid which has been implicated as a mediator of allergic and inflammatory processes1. It is synthesized and released by neutrophils, platelets, macrophages, monocytes, basophils and endothelial cells2–8, and is a potent platelet-aggregating agent, a vasodilator, increases vascular permeability, stimulates neutrophil aggregation and degranulation9 and induces release of lysosomal enzymes10. A role for PAF in the hypotension associated with endotoxin shock11,12 and in necrotizing enterocolitis13 has recently been suggested. As there is an association between septic shock and acute gastric damage14, we propose that PAF is an endogenous mediator of ulceration in the stomach. Indeed, as reported here, intravenous (i.v.) infusion of PAF to rats, at doses of 20–200 pmol per kg per min, resulted in the formation of extensive haemorrhagic erosions in the gastric mucosa. The ulcerogenic actions of PAF are not attributable solely to its hypotensive actions and were not mediated via effects on platelets or cyclooxygenase products, nor via histamine H1, H2 or α-adrenergic receptors. PAF is the most potent gastric ulcerogen yet described and its endogenous release may underlie or contribute to certain forms of gastric ulceration.

Biosynthesis of lipoxygenase and cyclo-oxygenase products from [14C]-arachidonic acid by human colonic mucosa.

The human colon synthetises several prostanoids which may have a role in inflammatory bowel diseases. As lipoxygenase products of arachidonate metabolism have been implicated in inflammatory processes, we have now investigated the formation of both lipoxygenase and cyclo-oxygenase metabolites from [14C]-arachidonic acid [(14C]-AA) by human colonic tissue. Homogenates of human colonic mucosa were incubated with [14C]-AA and after extraction into diethyl ether, separated by thin layer chromatography using two solvent systems that allowed resolution of cyclo-oxygenase and lipoxygenase products. The predominant cyclo-oxygenase products, as identified by their chromatographic mobility, were PGE2 greater than PGF2 alpha greater than PGD2 greater than TXB2 greater than 6-keto-PGF1 alpha. The formation of these products was inhibited both by indomethacin (1-10 microM) and the dual pathway inhibitor, BW755C (1-30 microM). The predominant lipoxygenase products formed, which had the chromatographic mobility of 11-, 12-, 15-HETE (which ran together) were inhibited by BW755C (19 microM) but not by indomethacin (3 microM). Further resolution of this TLC band, performed using normal phase HPLC, indicated that both 12-HETE and 15-HETE were major lipoxygenase products formed by human colonic homogenate. The present findings indicate that human colonic tissue can convert [14C]-AA into lipoxygenase as well as cyclo-oxygenase products and support the suggestion that lipoxygenase products may have a role in inflammatory bowel disease.

Nitric oxide donors increase mucus gel thickness in rat stomach

Instillation of the nitric oxide (NO) generator isosorbide dinitrate (0.1-1 mM) into the rat gastric lumen in vivo produced a dose-related increase in mucus gel thickness that was prevented by coadministration of oxyhaemoglobin (10 microM). Isosorbide dinitrate did not induce epithelial cell damage. S-Nitroso-N-acetyl-penicillamine (0.3 mM) and dibutyryl cyclic GMP (1 mM) also increased mucus thickness. These findings, along with the presence of NO synthase in the gastric mucosa, imply a role for NO in vivo in mediation of gastric mucus release.

Actions of prostacyclin (PGI2) and its product, 6-oxo-PGF1α on the rat gastric mucosa in vivo and in vitro

The effects of prostacyclin (PGI2) and its breakdown product 6-oxo-PGF1alpha on various aspects of gastric function were investigated in the rat. PGI2 increased mucosal blood flow when infused intravenously. PGI2 was a more potent inhibitor of gastric acid secretion in vivo than PGE2. Like PGE2, PGI2 inhibited acid secretion from the rat stomach in vitro. PGI2 had comparable activity to PGE2 in inhibiting indomethacin-induced gastric erosions. Thus prostacyclin shares several of the activities of PGE2, and may be involved in the regulation of gastric mucosal function.

Evidence for Platelet-Activating Factor as a Mediator of Endotoxin-Induced Gastrointestinal Damage in the Rat

The potential role of platelet-activating factor (PAF) as a mediator of gastrointestinal ulceration associated with septic shock was examined in the rat. The damaging effects of both PAF and Escherichia coli endotoxin in the stomach and small intestine were compared, as were their effects on plasma leakage into the lumen of the gastrointestinal tract. Intravenous administration of either endotoxin or PAF produced extensive necrosis and vascular congestion in the stomach and small intestine, but not the distal colon. With either agent, the duodenum and jejunum were the tissues most susceptible to damage and in which the greatest plasma leakage was observed. The prolonged hypotension and gastrointestinal damage induced by PAF or endotoxin were significantly inhibited by three structurally dissimilar PAF antagonists (CV-3988, BN-52021, and Ro-193704). CV-3988 (10 mg/kg) significantly (p less than 0.05) reduced both endotoxin- and PAF-induced plasma leakage in the stomach and small intestine. Of the three antagonists, only CV-3988 significantly reduced ethanol-induced gastric mucosal damage, perhaps reflecting actions of this compound unrelated to antagonism of PAF receptors. These studies support the hypothesis that PAF is an important mediator of the hypotension and plasma leakage observed during endotoxic shock and its endogenous release may contribute to the gastrointestinal ulceration associated with this syndrome. Thus, PAF receptor antagonists may be useful for prevention of such ulceration.

Neuronal and endothelium‐derived mediators in the modulation of the gastric microcirculation: integrity in the balance

J.H. Gaddum began his long and most productive career in pharmacology in 1925, when he joined the Wellcome Research Laboratories in Beckenham, under the guidance of J.W. Trevan. It was here that he was exposed to problems in bioassay, the generation of quantitative data and its subsequent mathematical and statistical evaluation. Three years later, when he joined Sir Henry Dale at the National Institute of Medial Research in Hampstead, he retained his enthusiasm for such analytical pharmacology, while developing interests in many other areas including peripheral neuronal regulatory mechanisms and the production and actions of novel local mediators. Of particular relevance to the theme of this review was his detection in 1931, along with U.S. von Euler, of an unknown depressor substance in extracts of various tissues, particularly the brain and intestine (Euler & Gaddum, 1931). This factor, which they called substance P, for no better reason than this was the letter on the label used on the original standard preparations of the extracts, was more fully characterized in collaboration with H.O. Schild (Gaddum & Schild, 1934). These studies by Gaddum thus initiated the field of pharmacological study on sensory neuropeptides. Only more recently, however, has there been appropriately detailed research on the actions and role of this diverse group of peptides with potent biological properties. As will be discussed later, the sensory neuropeptide, calcitonin gene-related peptide (CGRP), plays an important role in the regulation of gastric mucosal integrity, interacting with other local mediators. During his time at the College of the Pharmaceutical Society in Bloomsbury, Gaddum also developed and refined techniques to determine the release of neurotransmitters into the perfused vasculature of isolated tissues (Gaddum et al., 1939). It is therefore fitting, in view of his interest in neuronal modulatory processes and the release of local mediators, to describe in this Thirteenth Gaddum Memorial Lecture, our current understanding of the mechanisms that regulate microvascular blood flow in the gastric mucosa. It will become apparent that local neuronal and endotheliumderived factors that affect the microcirculation have a crucial influence on the processes that allow the gastric mucosa to