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Background and purpose Beraprost is a prostacyclin analogue and IP receptor agonist which is approved to treat pulmonary arterial hypertension (PAH) in Asia. The beraprost-314d isomer (esuberaprost) is one of four stereoisomers contained within the racemic mixture of beraprost. The pharmacological profile of esuberaprost is now evaluated to determine how stereoisomer separation affects its potency and mode of action in functional assays. Experimental approach Vascular tone was assessed using wire myography in rat and human distal pulmonary arteries (PAs) pre-contracted with U46619 (100 nM). HEK-293 cells stably expressing the human IP receptor (HEK-293-IP) and pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients were used to assess cyclic AMP (cAMP) generation and cell proliferation, respectively. Key results Esuberaprost relaxed rat PAs with a 5-fold greater potency compared with beraprost, and effects were strongly inhibited by RO3244794 (IP receptor antagonist) or L-NAME (NO synthase inhibitor). Esuberaprost caused EP3 receptor-dependent vasoconstriction at high concentrations ≥ 1000 nM, but contractions were 50% lower compared to beraprost. In HEK-293-IP cells, esuberaprost was 26-fold more potent (EC50 0.4 nM) at increasing cAMP than beraprost. In human PASMCs, esuberaprost was 40-fold more potent than beraprost at inhibiting cell proliferation (EC50 3 nM versus 120 nM), contrasting the 5-fold potency difference for cAMP elevation. Antiproliferative effects of esuberaprost appeared more dependent on NO than on the IP receptor. In PAs from patients with pulmonary hypertension, esuberaprost, caused some relaxation whereas beraprost instead produced a weak contraction. Conclusions and implications Stereoisomer separation of beraprost has a significant effect on the pharmacology of the individual isomer, esuberaprost, identified in vitro as a highly potent prostanoid IP receptor agonist.

The involvement of endogenous vasopressin in the actions of indomethacin following the concurrent administration of the nitric oxide (NO) synthase inhibitor, NG -nitro-l-arginine methyl ester (l-NAME), on acute intestinal microvascular permeability has been investigated in the rat. Administration of indomethacin (10mg/kg, s.c.) or L-NAME (10mg/kg, s.c.) alone did not affect jejunal and ileal vascular permeability after 1 h, as determined by the leakage of radiolabelled serum albumin. In contrast, when indomethacin (10mg/kg, s.c.) was injected concurrently with L-NAME (2-10mg/kg, s.c), significant dose-dependent plasma leakage occurred in the jejunum. Pretreatment with l-arginine (300 mg/kg s.c.) 15 min prior to l-NAME prevented these changes in microvascular permeability. Moreover, pretreatment with the vasopressin pressor-receptor antagonist, d(CH2 )5 Tyr(Me)AVP (0.01-0.2mg/kg, s.c.) dose-dependently attenuated such damage. These findings suggest that following indomethacin administration, the early inhibition of NO synthase leads to acute microvascular injury involving vasopressin in the rat jejunum. This suggests a protective role of NO, formed by constitutive NO synthase, counteracting effectively the deleterious actions of endogenous vasopressin.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme situated downstream of cyclo-oxygenase-2, promoting the excessive PGE2 production in inflammation. Dexamethasone is known to suppress mPGES-1 but the mechanisms regulating mPGES-1 expression remain poorly known. MKP-1 is a phosphatase controlling the proinflammatory MAP kinase pathways p38 and JNK, thus limiting the inflammatory responses. We have now investigated the role of MKP-1 and MAP kinases p38 and JNK in the regulation of mPGES-1 expression by dexamethasone. Dexamethasone increased MKP-1 and decreased mPGES-1 expression in J774 macrophages and in peritoneal macrophages from wild-type but not from MKP-1 deficient mice. Dexamethasone also reduced p38 and JNK phosphorylation along with enhancement of MKP-1, while inhibition of JNK reduced mPGES-1 expression. These findings were also translated to in vivo conditions as dexamethasone downregulated mPGES-1 expression in paw inflammation in wild-type but not in MKP-1 deficient mice. In conclusion, dexamethasone was found to downregulate mPGES-1 expression through enhanced MKP-1 expression and reduced JNK phosphorylation in inflammatory conditions. The results extend the understanding on the regulation of mPGES-1 expression and highlight the potential of MKP-1 as an anti-inflammatory drug target.

The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated.

Prostacyclins are extensively used to treat pulmonary arterial hypertension (PAH), a life-threatening disease involving the progressive thickening of small pulmonary arteries. Although these agents are considered to act therapeutically via the prostanoid IP receptor, treprostinil is the only prostacyclin mimetic that potently binds to the prostanoid EP2 receptor, the role of which is unknown in PAH. We hypothesised that EP2 receptors contribute to the anti-proliferative effects of treprostinil in human pulmonary arterial smooth muscle cells (PASMCs), contrasting with selexipag, a non-prostanoid selective IP agonist. Human PASMCs from PAH patients were used to assess prostanoid receptor expression, cell proliferation, and cyclic adenosine monophosphate (cAMP) levels following the addition of agonists, antagonists or EP2 receptor small interfering RNAs (siRNAs). Immunohistochemical staining was performed in lung sections from control and PAH patients. We demonstrate using selective IP (RO1138452) and EP2 (PF-04418948) antagonists that the anti-proliferative actions of treprostinil depend largely on EP2 receptors rather than IP receptors, unlike MRE-269 (selexipag-active metabolite). Likewise, EP2 receptor knockdown selectively reduced the functional responses to treprostinil but not MRE-269. Furthermore, EP2 receptor levels were enhanced in human PASMCs and in lung sections from PAH patients compared to controls. Thus, EP2 receptors represent a novel therapeutic target for treprostinil, highlighting key pharmacological differences between prostacyclin mimetics used in PAH.

Background and purpose: Leukotriene B4 (LTB4), formed by the sequential actions of the 5-lipoxygenase (5-LO) and leukotriene A4 hydrolase (LTA4H), is a pro-inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease. However, inhibitors of 5-LO have not proved to be consistent in their therapeutic efficacy in colitis. Another approach to inhibiting LTB4 synthesis is through the use of inhibitors of LTA4H, such as the novel, potent and selective compound, JNJ 26993135.

Reactive oxygen species, suggested to be involved in inflammatory bowel disease, may be modulated by endogenous anti-oxidant products of heme oxygenase-1 (HO-1). In the present work, HO-1 expression in trinitrobenzene sulphonic acid (TNBS)-induced colitis in the rat and the effects of HO-1 modulation, particularly by the HO-1 inducer, heme, were further evaluated. Colitis was induced by intracolonic challenge with TNBS and assessed macroscopically and by myeloperoxidase (MPO) assay. Heme oxygenase activity was determined by measurement of bilirubin formation and HO-1 protein expression was determined by Western blotting. TNBS challenge led to an early and substantial induction of HO-1 protein expression and heme oxygenase activity in the colon that peaked after 48-72 h and declined over 10 days. Heme (30 micromol/kg/day, s.c) increased colonic HO-1 protein expression and enzyme activity and decreased colonic damage and myeloperoxidase activity. Short-term administration of cadmium chloride (2 mg/kg, s.c.), another known HO-1 inducer, also reduced the colonic injury and myeloperoxidase levels. In contrast, the HO-1 inhibitor, zinc protoporphyrin (50 micromol/kg/day, s.c) significantly increased the colonic damage and myeloperoxidase activity over 10 days, as did tin protoporphyrin (30 micromol/kg/day, s.c). These results support the proposal that induction of HO-1 provides a protective mechanism in this model under both acute and more-chronic conditions, and that its selective up-regulation could thus be of therapeutic potential in colitis.

The effects of the inhibitors of glycogen synthase kinase-3β (GSK-3β), TDZD-8 and SB 415286, which can substantially reduce the systemic inflammation associated with endotoxic shock in vivo, have now been investigated on the acute colitis provoked by trinitrobenzene sulphonic acid (TNBS) in the rat. Administration of the GSK-3β inhibitor TDZD-8 (0.1, 0.33 or 1.0 mg kg−1, s.c., b.i.d., for 3 days) caused a dose-dependent reduction in the colonic inflammation induced by intracolonic TNBS assessed after 3 days, both as the area of macroscopic involvement and as a score using 0–10 scale. Likewise, following administration of the GSK-3β inhibitor SB 415286 (0.1, 0.33 or 1.0 mg kg−1, s.c., b.i.d., for 3 days), the extent and degree of the TNBS-provoked colonic inflammation was reduced. Administration of either TDZD-8 or SB 415286 reduced the fall in body weight following challenge with TNBS at each dose level studied. The increase in myeloperoxidase activity, an index of neutrophil infiltration into the TNBS-induced inflamed colon, was significantly inhibited by both TDZD-8 and SB 415286 at each dose level. The increase in the levels of the proinflammatory cytokine, TNF-α, in the inflamed colon was also significantly inhibited by either compound at the highest doses evaluated. The elevated levels of the transcription factor NF-κB subunit p65, as determined by Western blot in the nuclear extracts from the TNBS-provoked inflamed colonic tissue, were dose-dependently reduced by TDZD-8 or SB 415286 treatment. These findings demonstrate that two chemically distinct selective inhibitors of the activity of GSK-3β reduce the inflammation and tissue injury in a rat model of acute colitis. The mechanisms underlying this anti-inflammatory action may be related to downregulation of NF-κB activity, involved in the generation of proinflammatory mediators. British Journal of Pharmacology (2006) 147, 575–582. doi:10.1038/sj.bjp.0706509

Almost 20 years after the identification of the biological role of nitric oxide (NO), the full therapeutic potential of novel agents that mimic the activity of NO or interfere with its synthesis has yet to be realised for utilities involving the gastrointestinal tract. New utilities for classical NO donors, which were used as vasodilators for decades, in the treatment of motility disorders have been explored and a product for treating anal fissure was recently launched. New classes of compounds incorporating a NO-donating moiety into standard non-steroidal anti-inflammatory drugs, the NO-non-steroidal anti-inflammatory drugs (NO-NSAIDs) or COX-inhibiting nitric oxide donors (CINODs) have also been developed. These have been shown to exhibit reduced gastrointestinal injury in experimental models, and reports on their efficacy and safety in Phase I and II studies are now available. Modulation of the inducible NO synthase isoform that generates excessive NO that can lead to subsequent cytotoxic moieties, such as peroxynitrite, may have therapeutic possibilities in a range of inflammatory diseases of the gut. Likewise, agents that promote the decomposition of peroxynitrite or removal of its other component, superoxide, may also prove to be of use. Further targets for pharmaceutical exploitation are likely to come from both genomic and molecular insights into the processes that regulate the NO system.

The histamine H(4) receptor is a G-protein coupled receptor with little homology to the pro-inflammatory histamine H(1) receptor, expressed on cells of the immune system with hematopoietic lineage such as eosinophils and mast cells. The effects of the recently described highly selective histamine H(4) receptor antagonists JNJ 10191584 and JNJ 7777120 have now been investigated on the acute colitis provoked by trinitrobenzene sulphonic acid over 3 days in the rat. Treatment with JNJ 10191584 (10-100 mg/kg p.o., b.i.d.) caused a dose-dependent reduction in macroscopic damage, inhibition of the TNBS-provoked elevation of both colonic myeloperoxidase and tumour necrosis factor-alpha (TNF-alpha), and a reduction in the histologically assessed increase in mucosal and submucosal thickness and neutrophil infiltration. JNJ 7777120 (100 mg/kg p.o., b.i.d.) likewise reduced the macroscopic injury and the increases in colonic myeloperoxidase and TNF-alpha levels. These findings indicate a pro-inflammatory role for the histamine H(4) receptor in this model and suggest a novel pharmacological approach to the treatment of colitis.

Nitric oxide (NO) can protect the gastrointestinal tract from injury, including that provoked by non-steroidal anti-inflammatory drugs (NSAIDs). This protective profile of NO, which predominantly reflects actions on the microcirculation, is mimicked by NO donors. Moreover, the NO-donating agents know as the NO-NSAIDs or CINODs (cyclo-oxygenase-inhibiting nitric oxide-donating drugs) exhibit reduced gut injury in experimental models, which is considered to reflect these local beneficial actions of NO. NSAIDs cause chronic inflammatory lesions in the small intestine in experimental models. This injury results from initial COX inhibition and other local events, with translocation of indigenous luminal bacteria, leading to induction of NO synthase isoform, iNOS, and subsequent production of the cytotoxic moiety, peroxynitrite from NO and superoxide. Agents that inhibit iNOS or superoxide production can attenuate such intestinal injury. In the absence of reactive oxygen moieties, NO may play a beneficial role in the resolution of inflammatory damage to the gut, thus reconciling the potential opposing properties of NO in tissue inflammation and injury.

Non-steroidal anti-inflammatory drugs (NSAIDs) causes extensive damage to the gastrointestinal (GI) tract. The underlying mechanisms of gastric injury include topical irritant actions that disrupt the epithelial barrier, as well as the inhibition of cyclo-oxygenase (COX), which is predominantly the COX-1 isoform in the mucosa. This damage can be attenuated by antisecretory agents or by mucosal protective agents such as the synthetic prostanoids or nitric oxide (NO) donors. Compounds designed to attenuate topical irritancy, or have protective agents incorporated, such as NO-containing NSAIDs, the CINODs (cyclo-oxygenase-inhibiting NO-donating drugs) show reduced mucosal injury. NSAIDs also cause injury in the small intestine, which appears to result from initial COX inhibition, with subsequent translocation of indigenous bacteria, induction of NO synthase and production of the cytotoxic moiety, peroxynitrite. The COX-2 selective agents, the coxibs, which inhibit prostanoid biosynthesis at inflammatory sites, but not the endogenous protective prostanoids in the gut formed by COX-1, have proved so far to be a successful therapeutic approach to reducing NSAIDs GI damage. The clinical outcome of the use of the second generation of coxibs, and the newer NO NSAIDs is now awaited.

The role of nitric oxide (NO) formed by inducible NO synthase (iNOS), superoxide and the lipopolysaccharide from luminal bacteria in non-steroidal anti-inflammatory drug-induced intestinal injury was investigated in the rat. Administration (s.c. or p.o.) of indomethacin (10 mg kg(-1)), flurbiprofen (40 mg kg(-1)) or diclofenac (40 mg kg(-1)) increased the vascular leakage of radiolabelled albumin in the jejunum, determined after 24 h, associated with the induction of iNOS, assessed by the conversion of radiolabelled L-arginine. Pre-treatment with ampicillin (200 mg kg(-1) day(-1), p.o.), metronidazole (200 mg kg(-1) day(-1), p.o.), or polymixin B (15 mg kg(-1) day(-1), s.c.), inhibited indomethacin-induced lesion formation, reduced microvascular leakage and prevented the expression of iNOS activity. Administration of the highly selective iNOS inhibitor, GW273629 ((R)-2-amino-4,4-dioxo-6(1-iminioethylamino)-4-thiahexanoic acid; 5 mg kg(-1), s.c.), 18 h after indomethacin, likewise prevented the intestinal lesions and attenuated the microvascular leakage. Superoxide dismutase conjugated with polyethylene glycol (3000 U kg(-1), i.v.), inhibited the indomethacin-induced lesions and microvascular leakage, but not the expression of iNOS activity. These findings suggest that non-steroidal anti-inflammatory drugs compromise mucosal integrity, leading to luminal bacterial translocation. This provokes iNOS induction, leading to microvascular injury involving both NO and superoxide.

The actions of a purified Helicobacter pylori lipopolysaccharide (3 mg x kg(-1), i.v.) on rat gastric antral and duodenal microvascular integrity (determined as radiolabelled albumin leakage) and the expression of the inducible nitric oxide (NO) synthase (iNOS; assessed by the citrulline assay) were investigated 4 h after challenge. Significant increases of albumin leakage and expression of iNOS in both antral and duodenal tissues were observed following challenge. Concurrent administration of the selective iNOS inhibitor, 1400W (N-(8-(aminomethyl)benzyl)-acetamidine; 0.2-1 mg x kg(-1), s.c.), with lipopolysaccharide, caused a dose-dependent attenuation of the gastric and duodenal albumin leakage. Thus, H. pylori lipopolysaccharide can initiate the expression of iNOS in the stomach and duodenum following systemic challenge, which can provoke gastroduodenal microvascular dysfunction.

BACKGROUND—The inducible isoform of nitric oxide synthase (iNOS) may be involved in the mucosal injury associated with inflammatory bowel disease (IBD). In contrast with iNOS, the inducible heme oxygenase 1 (HO-1) is considered to act as a protective antioxidant system. AIMS—To evaluate the effects of the known HO-1 inducers, cadmium and bismuth salts, heme, and nitric oxide (NO) donors, on iNOS activity, and expression in the human intestinal epithelial cell line DLD-1. METHODS—iNOS activity was assessed by the Griess reaction and the radiochemical L-arginine conversion assay. iNOS mRNA and iNOS protein expression were determined by northern and western blotting, respectively. RESULTS—Cytokine exposure led to induction of iNOS activity, iNOS mRNA, and iNOS protein expression. Preincubation of DLD-1 cells with heme (1-50 µM) inhibited cytokine induced iNOS activity in a concentration dependent manner. This inhibitory effect was abolished by the HO-1 specific inhibitor tin protoporphyrin. Preincubation with NO donors sodium nitroprusside (SNP 1-1000 µM) or S-nitroso-acetyl-penicillamine (SNAP 1-1000 µM), or with the heavy metals cadmium chloride (10-40 µM), bismuth citrate, or ranitidine bismuth citrate (10-3000 µM) inhibited iNOS activity in a concentration dependent manner. Moreover, SNP and heme abolished cytokine induced iNOS protein as well as iNOS mRNA expression, whereas cadmium chloride did not modify iNOS protein expression. CONCLUSIONS—Heme, the heavy metals cadmium and bismuth, as well as NO donors, are potent inhibitors of cytokine induced iNOS activity. Heme and NO donors act at the transcriptional level inhibiting iNOS mRNA expression. Such findings suggest the potential for interplay between the iNOS and HO-1 systems, which may modulate the progress of IBD. Keywords: inducible nitric oxide synthase; nitric oxide; colonic epithelial cells; cytokines; heme oxygenase-1; bismuth citrate

The involvement of nitric oxide (NO) formed by the inducible isoform of NO synthase (iNOS) has been investigated in the development of rat intestinal lesions following indomethacin administration. Over a 72-h period, indomethacin (10 mg kg −1 , s.c.) provoked a time-dependent increase in expression of iNOS (assessed by the conversion of radiolabelled l -arginine to citrulline) and enhancement of vascular leakage of radiolabelled human serum albumin in the jejunum which commenced 18 h after indomethacin. Similar effects were not observed in the ileum, colon or caecum. In addition, macroscopic lesions were detectable and myeloperoxidase activity (an index of neutrophil recruitment) were increased in the rat jejunum 18–24 h after indomethacin, but remained at basal levels in the ileum and colon. These findings suggest that indomethacin provokes a site-selective expression of iNOS in the rat jejunum which correlates with lesion formation and vascular leakage, whereas both the ileum and colon are spared.

The role of nitric oxide (NO) in the maintenance of microvascular integrity during minor surgical manipulation has been evaluated in the rat. The NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME, 5 mg kg−1, s.c.) and NG-monomethyl-L-arginine (L-NMMA, 50 mg kg−1, s.c.) had no effect on microvascular leakage of radiolabelled albumin over 1 h in the stomach, duodenum, jejunum, colon, lung and kidney in the un-operated conscious or pentobarbitone-anaesthetized rat. In contrast, in anaesthetized rats with a midline abdominal laparotomy (5 cm), L-NAME (1–5 mg kg−1, s.c.) or L-NMMA (12.5–50 mg kg−1, s.c.) dose-dependently increased gastrointestinal, renal and pulmonary vascular leakage, effects reversed by L-arginine pretreatment (300 mg kg−1, s.c., 15 min). These actions were not observed in anaesthetized rats that had only received a midline abdominal skin incision (5 cm). Pretreatment with a rabbit anti-rat neutrophil serum (0.4 ml kg−1, i.p.), 4 h before laparotomy, abolished the plasma leakage induced by L-NAME in all the organs investigated. These results indicate that the following abdominal laparotomy, inhibition of constitutive NO synthase provokes vascular leakage in the general microcirculation, by a process that may involve neutrophils. Such effects could thus confound studies on the microvascular actions of NO synthase inhibitors using acute surgically prepared in vivo models. The findings thus suggest that constitutively-formed NO has a crucial role in the maintenance of acute microvascular integrity following abdominal surgical intervention. British Journal of Pharmacology (1999) 126, 515–521; doi:10.1038/sj.bjp.0702322

1 Helicobacter pylori (Hp) infection, which involves the gastric antrum and duodenal mucosa, may be involved in peptic ulceration by stimulating the local release of cytoxic or pro-inflammatory factors. 2 Nitric oxide (NO) is known to be cytotoxic at high concentration. The aim of the present study was therefore to investigate the ability of a water soluble extract of Hp to induce NO synthase in duodenal mucosa and epithelial cells following its administration in vivo in rats and determine its association with cell damage. 3 Administration of Hp water extract (4 ml kg−1) led to the expression of the calcium-independent inducible nitric oxide synthase (iNOS) after 4 h in the duodenum, determined as [14C]-arginine conversion to citrulline. 4 This iNOS activity was not reduced by pretreatment with anti-neutrophil serum (0.4 ml kg−1, i.p., 3 h before challenge). However, dexamethasone pretreatment (1 mg kg−1, i.v., 2 h before the extract), or administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 5 mg kg−1, i.v., 2.5 h after the extract) reduced this activity. 5 Furthermore, iNOS was expressed in duodenal isolated epithelial cells 4 h after the i.v. challenge with the extract, at a time when the cellular viability was also reduced, as assessed by trypan blue exclusion. 6 Dexamethasone pretreatment, administration of L-NAME, or pretreatment with polymyxin B (1 mg kg−1, i.v.) which binds endotoxin, reduced both the iNOS activity and epithelial cell damage. 7 The induction of NO synthase by the Hp extract thus results in duodenal epithelial cell injury and such actions could play a role in pathogenesis of peptic ulcer disease. British Journal of Pharmacology (1998) 123, 1073–1078; doi:10.1038/sj.bjp.0701706

The time-dependent actions following pretreatment or delayed administration of the nitric oxide (NO) synthase inhibitor, NG-nitro- l -arginine methyl ester ( l -NAME) on colonic inflammation and inducible NO synthase activity following the intrarectal administration of trinitrobenzene sulphonic acid (TNBS) were evaluated in the rat. Intracolonic instillation of TNBS (30 mg in 0.25 ml of 50% ethanol) led to macroscopic injury, an increase of mucosal myeloperoxidase activity and the expression of the Ca2+-independent inducible NO synthase over 8 days. The inflammatory response following TNBS reached maximum levels between 12 and 72 h and then it declined until 14 days. Oral administration of l -NAME (25 mg kg−1 per 24 h in the drinking water) 2 days before TNBS augmented macroscopic damage and increased colonic inducible NO synthase activity 6, 12, 24 and 72 h after TNBS administration. In contrast, when l -NAME was administered 6 h after TNBS instillation, at time of expression of inducible NO synthase, the macroscopic lesions were reduced, as well as the enhanced inducible NO synthase activity, determined, over 72 h. Delayed (6 h after TNBS) administration of l -NAME also attenuated the colonic myeloperoxidase activity provoked by TNBS, after 24 h. This activity was not affected by pretreatment (2 days before TNBS) with l -NAME. These findings indicate that the timing of administration of non-selective NO synthase inhibitors such as l -NAME, in models of colitis is critical to the eventual outcome. Thus, pretreatment with l -NAME, which will inhibit constitutive NO synthase, exacerbates the subsequent damage following challenge. In contrast, delayed administration of l -NAME at the time of inducible NO synthase expression, has a beneficial action on the colonic injury and inflammation.

Nitric oxide (NO) synthase activity, which converts arginine to citrulline and NO, is present in homogenates of rat gastric mucosal cells. The aims of this study were to identify the form of NO synthase expressed in gastric cells isolated from fed rats, and to investigate the metabolism of arginine by suspensions of intact mucosal cells. Antibodies directed against the neuronal form of NO synthase recognised a protein of 160 kDa on immunoblots of extracts of gastric cells, and stained isolated cells of approx. 8 microm in diameter. NO synthase was enriched in a cell fraction which banded at high-density in a Percoll gradient, and was inhibited (IC50) by N(G)-nitro-L-arginine (0.8 microM), N(G)-monomethyl-L-arginine (12.6 microM), L-canavanine (147 microM), trifluoperazine (140 microM) and by phosphorylation involving protein kinase C. Intact gastric cells converted exogenous arginine to ornithine and citrulline. Arginase was present in the cells, and was predominantly responsible for arginine metabolism because formation of ornithine and citrulline was reduced by the arginase inhibitors, N(G)-hydroxy-L-arginine and L-ornithine, but not by NO synthase inhibitors such as N(G)-nitro-L-arginine. In conclusion, NO synthase that resembles the neuronal isoform is present in gastric mucosal cells, but a pathway involving arginase seems to be largely responsible for citrulline formation from exogenous arginine in intact mucosal cells.

Moderate somatic stress inhibits gastric acid secretion. We have investigated the role of endogenously released NO in this phenomenon. Elevation of body temperature by 3°C or a reduction of 35 mmHg (1 mmHg = 133 Pa) in blood pressure for 10 min produced a rapid and long-lasting reduction of distension-stimulated acid secretion in the rat perfused stomach in vivo . A similar inhibitory effect on acid secretion was produced by the intracisternal (i.c.) administration of oxytocin, a peptide known to be released during stress. Intracisternal administration of the NO-synthase inhibitor, N G -nitro- l -arginine methyl ester ( l -NAME) reversed the antisecretory effect induced by all these stimuli, an action prevented by intracisternal coadministration of the NO precursor, l -arginine. Furthermore, microinjection of l -NAME into the dorsal motor nucleus of the vagus nerve reversed the acid inhibitory effects of mild hyperthermia, i.v. endotoxin, or i.c. oxytocin, an action prevented by prior microinjection of l -arginine. By contrast, microinjection of l -NAME into the nucleus tractus solitarius failed to affect the inhibitory effects of hyperthermia, i.v. endotoxin, or i.c. oxytocin. Immunohistochemical techniques demonstrated that following hyperthermia there was a significant increase in immunoreactivity to neuronal NO synthase in different areas of the brain, including the dorsal motor nucleus of the vagus. Thus, our results suggest that the inhibition of gastric acid secretion, a defense mechanism during stress, is mediated by a nervous reflex involving a neuronal pathway that includes NO synthesis in the brain, specifically in the dorsal motor nucleus of the vagus.

Nitric oxide has been implicated in the regulation of blood flow, mucosal integrity and mucus secretion in the gastric mucosa. An antiserum directed against the C-terminal hexadecapeptide of rat brain nitric oxide synthase (NOS) and monoclonal antibodies to the neuronal and endothelial forms of NOS were used to establish the location of isoforms of NOS in rat gastric glandular mucosa. Antibodies to the neuronal form of NOS reacted with a band of 160 kDa on immunoblots of brain and gastric mucosa, and the addition of the hexadecapeptide inhibited recognition by the antipeptide antiserum. The antibody to endothelial NOS detected a band of 140 kDa on protein blots of samples of intestinal mesentery and gastric mucosa. Immunohistochemistry using these antibodies demonstrated that material related to neuronal NOS was present in surface cells of the gastric mucosa, and showed a similar localization to intense NADPH diaphorase activity. The antibody to endothelial NOS did not stain the surface of the gastric mucosa but recognized blood vessels in the lower region of the gastric glands and in the sub-mucosa. This study suggests that nitric oxide might act both as an intra- and inter-cellular messenger to regulate mucus release, and that the NOS present in surface cells is related more closely to the neuronal than to the endothelial isoform.

Original article can be found at: http://www.sciencedirect.com/science/journal/14714892 Copyright Elsevier B.V. DOI: 10.1016/j.coph.2004.09.003 [Full text of this article is not available in the UHRA]

1. The effects of aminoguanidine on neutrophil adherence to venules and on the diameter of arterioles in the mesenteric vascular bed of the pentobarbitone-anaesthetized rat have been compared with those of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). 2. Administration of L-NAME (1-10 mg kg-1, i.v.) caused a dose-dependent increase in leukocyte adherence and a reduction in leukocyte rolling velocity in postcapillary venules of the rat mesentery over 1 h. 3. Likewise, aminoguanidine (10-100 mg kg-1, i.v.) dose-dependently increased leukocyte adherence and decreased leukocyte rolling velocity over 1 h. 4. Both L-NAME and aminoguanidine caused a dose-dependent reduction in mesenteric arteriolar diameter and an increase in systemic arterial blood pressure. 5. The effects of aminoguanidine (50 mg kg-1, i.v.) on leukocyte adherence, arteriolar diameter and on blood pressure were significantly reversed by pretreatment with L-arginine (300 mg kg-1, i.v.). 6. These findings indicate that, like L-NAME, aminoguanidine can acutely promote leukocyte adherence to the mesenteric venular wall and reduce arteriolar diameter. Moreover, these acute effects were reversed by L-arginine, suggesting they are mediated through inhibition of constitutive NO synthase.

Nitric oxide (NO) can exert a multitude of biological actions. NO, formed froml-arginine by a calcium-dependent enzyme (NO synthase) plays a key physiological role in regulating vascular tone and integrity. NO, formed by a constitutive neuronal isoform of NO synthase, likewise plays an important neuromodulator role. By contrast, high levels of NO can be generated following induction of a calcium-independent isoform of NO synthase. This excessive production of NO can provoke hypotension such as that observed in septic shock, and can exert cytotoxic actions leading to tissue injury and inflammation. Selective inhibitors of this inducible isoform thus have therapeutic potential in a number of disease states.

Endotoxin (Escherichia coli lipopolysaccharide 0111:B4, 3 mg/kg i.v.) induced the expression of a calcium-independent nitric oxide (NO) synthase, determined after 5 h in cardiac, hepatic, pulmonary and renal tissues, as assessed by the conversion of radiolabelled L-arginine to L-citrulline. This widespread induction of NO synthase in these conscious rats was associated with microvascular injury, as assessed by the vascular leakage of radiolabelled human serum albumin. Concurrent administration of the NO synthase inhibitor. NG-nitro-L-arginine methyl ester (L-NAME, 1-5 mg/kg s.c.) with endotoxin, provoked acute vascular leakage within 2 h in the various organs. By contrast, the delayed injection of L-NAME (1-5 mg/kg s.c.) or NG-monomethyl-L-arginine (12.5-50 mg/kg s.c.) until 3 h after endotoxin challenge inhibited the subsequent microvascular leakage in these organs. These effects of NO synthase inhibitors were reversed by L-arginine (300 mg/kg s.c.) pretreatment. These results support a protective role of constitutive NO synthase in the early phase of endotoxin shock. Such actions contrast with the aggressive actions of the products of inducible NO synthase in the development of widespread microvascular injury in endotoxemic states.

1. Nitric oxide (NO) and the superoxide anion can interact to form the cytotoxic moiety, peroxynitrite. The involvement and potential source of superoxide in the gastric mucosal damage induced by local infusion of NO donors, has now been investigated in the pentobarbitone-anaesthetized rat. 2. Local intra-arterial infusion of the NO donor, sodium nitroprusside (40 micrograms kg-1 min-1) for 10 min induced macroscopically apparent gastric mucosal injury. 3. This mucosal damage was dose-dependently reduced by prior administration of a systemically acting form of superoxide dismutase conjugated with polyethylene glycol (500-2000 iu kg-1, i.v.). 4. Likewise, the mucosal damage induced by nitroprusside was dose-dependently reduced by prior administration of the xanthine oxidase inhibitor, allopurinol (20-100 mg kg-1, i.p. or 100 mg kg-1, p.o.). 5. Pretreatment with allopurinol (100 mg kg-1, i.p.) also reduced the mucosal injury induced by local intra-arterial infusion of the nitrosothiol, S-nitroso-N-acetyl-penicillamine (40 micrograms kg-1 min-1), but not that induced by local infusion of endothelin-1 (5 pmol kg-1 min-1), indicating specificity of action. 6. Prior administration (4h) of rabbit anti-rat neutrophil serum (0.4 ml kg-1, i.p.), which reduced circulating neutrophils by 90%, did not significantly protect against mucosal injury induced by nitroprusside. 7. Intravenous administration of the platelet-activating factor receptor antagonists, WEB 2086 (1 mg kg-1) or BN 52021 (10 mg kg-1), or the thromboxane synthase inhibitor, OKY 15181 (25 mg kg-1), did not modify mucosal damage induced by nitroprusside, showing lack of involvement of these neutrophil-derived mediators. 8. These findings indicate the involvement of superoxide in the injurious actions of the NO donors, implicating a cytotoxic role of peroxynitrite. Xanthine oxidase, but not neutrophils, appears to be a source of the superoxide.

Pretreatment (1 h) with low doses (5-40 micrograms/kg i.p.) of Escherichia coli endotoxin dose dependently reduced the gastric mucosal damage induced by a 10 min challenge with 1 ml ethanol (50% and 100%) in conscious rats. Treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 5 and 10 mg/kg i.p.), significantly inhibited the protective effects of endotoxin (40 micrograms/kg i.p.). The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.p.). The protective effects of endotoxin were not influenced by pretreatment with dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg s.c.). However, ablation of sensory afferent neurons by capsaicin pretreatment (20, 30 and 50 mg/kg s.c.) abolished the mucosa protective effects of endotoxin (40 micrograms/kg). These findings suggest that the protection elicited by low doses of endotoxin against ethanol-induced mucosal damage involves synthesis of nitric oxide and activation of sensory neurones.

1. The involvement of nitric oxide in the acute inhibitory effects of low doses of endotoxin, following intracerebroventricular (i.c.v.) or intravenous (i.v.) administration, on gastric acid secretion stimulated by distension or i.v. infusion of pentagastrin has been investigated in the continuously perfused stomach of the anaesthetized rat. 2. The i.c.v. administration of E. coli endotoxin (800 ng kg-1) abolished the acid secretory response induced by gastric distension (20 cm water intragastric pressure) within 30 min of administration. 3. By contrast, submaximal rates of acid secretion induced by i.v. infusion of pentagastrin (8 micrograms kg-1 h-1) were not inhibited by i.c.v. administration of endotoxin (800 ng kg-1). 4. Prior i.c.v. administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 800 micrograms kg-1) restored the acid secretory responses to distension in rats treated with endotoxin (i.c.v.). 5. Likewise, i.v. administration of endotoxin (5 micrograms kg-1) abolished the acid secretory response induced by gastric distension within 30 min of administration. Prior i.c.v. injection of L-NAME (800 micrograms kg-1) or its i.v. administration (10 mg kg-1) restored acid secretory responses in rats receiving i.v. endotoxin. 6. The reversal by L-NAME (i.v.) of the acid inhibitory effects of endotoxin (i.v.) was prevented by L-arginine (12 mg kg-1, i.c.v. or 100 mg kg-1, i.v.), but not by its enantiomer D-arginine. 7. The present results imply the existence of an acute response to endotoxin involving NO synthesis in the brain. NO may act as a neuromodulator or neurotransmitter in a nervous reflex leading to the inhibition of acid secretion stimulated by gastric distension.

The effects of aminoguanine on the intestinal vascular permeability following endotoxin administration in vivo has been compared to those of the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) in the rat. Concurrent administration of aminoguanidine. (12.5-50 mg/kg, s.c.) with endotoxin (E. coli lipopolysaccharide, 3 mg/kg, i.v.), dose dependently increased vascular leakage of radiolabelled albumin in the ileum and colon after 1 h, an effect reversed by the pretreatment with L-arginine (300 mg/kg, s.c.). Aminoguanidine (50 mg/kg, s.c.) also elevated arterial blood pressure over the 1 h investigation period. Similar acute potentiation of endotoxin-provoked vascular injury was observed 1 h following L-NMMA (50 mg/kg s.c.) which also increased blood pressure, indicating the inhibition of constitutive NO synthase. By contrast, administration of aminoguanidine (12.5-50 mg/kg, s.c.) 3 h after endotoxin, at the time of the expression of the inducible NO synthase, reduced the subsequent endotoxin-induced vascular leakage, as did L-NMMA (50 mg/kg). In homogenates of rat ileal or colonic tissue, aminoguanidine inhibited both the constitutive and inducible NO synthase activity showing only 2-fold selectivity for the inducible isoform. Thus, although aminoguanidine inhibits these isoforms of NO synthase, it is not a selective inhibitor of the inducible isoform in the intestinal microvasculature in vivo.

Recent experimental studies may undermine our understanding of the gastrointestinal side effects of non-steroidal anti-inflammatory drugs and cast a shadow on the original concept that underpins the development of the recent addition to the clinical anti-inflammatory armamentarium, the COX-2 selective inhibitors. But is this just a passing cloud or a total eclipse of the COX theory?

Why publish this series on clinical research methods in gastroenterology now, when all of the key targets for research and development of new drugs in gastroenterology have presumably been achieved from a clinical pharmacologist's perspective? Well, as this innovative series of review articles will demonstrate, we are far from that the state of therapeutic Nirvana in this area. Moreover, evaluation of the side-effect profile of a range of new chemical entities on the gastrointestinal tract has become an issue of ever-increasing importance and more rigorous assessment in appropriately designed studies is becoming a necessity for several new classes of drug.

Instillation of carbachol (150 micrograms/kg) into the gastric lumen in vivo increased the thickness of the mucus gel layer. Intravenous administration of the inhibitor of nitric oxide (NO) synthase, NG-nitro-L- arginine methyl ester (L-NAME, 0.4-5 mg/kg) dose-dependently reduced the stimulation by carbachol, the half-maximal inhibitory dose being 0.57 mg/kg. This effect of L-NAME was abolished by administration of L-arginine but not by D-arginine (100 mg/kg i.v.). By contrast L-NAME (5 mg/kg) did not reduce the stimulatory effect of intraluminal 16,16-dimethyl prostaglandin E2 (50 micrograms/kg) on mucus gel thickness. These results implicate NO in the cholinergic activation of gastric mucus secretion.

This study investigates the effects of inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME), the inhibition of prostaglandin synthesis with indomethacin and the combined effects on gastric mucosal hyperemia of ketamine-anesthetized rats with portal hypertension induced by partial portal vein ligation. The hydrogen gas-clearance technique was used for measurement of gastric mucosal blood flow. Blood pressure increased with L-NAME administration in a similar manner in portal-hypertensive and sham-operated rats. Low doses of L-NAME (1 and 3 mg/kg, intravenously) caused a significant and dose-dependent reduction in gastric mucosal blood flow in portal-hypertensive rats but had no effect on sham-operated animals. With a higher dose of L-NAME (13 mg/kg, intravenously), a significant decrease in gastric mucosal blood flow was observed in both portal-hypertensive and sham-operated rats. Indomethacin pretreatment (5 mg/kg, subcutaneously) caused a significant decrease in basal gastric mucosal blood flow of portal-hypertensive rats but did not modify this parameter in sham-operated animals. In sham-operated rats pretreated with indomethacin, the lower dose of L-NAME (3 mg/kg) did not significantly modify basal gastric mucosal blood flow. Likewise, pretreatment with indomethacin in sham-operated rats did not augment the significant reduction in gastric mucosal blood flow produced by the higher dose of L-NAME. In portal-hypertensive rats the significant dose-dependent reduction in gastric mucosal blood flow induced by L-NAME (3 and 13 mg/kg) was not significantly altered by pretreatment with indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study determined the presence of constitutive and inducible nitric oxide (NO) synthase activities in intestinal isolated epithelial cells and the effects of NO induction on intestinal epithelial cell viability. Epithelial cells were isolated from rat proximal small intestine by dispersion using citrate and EDTA. Constitutive NO synthase activity, determined by [14C]arginine conversion to citrulline that was inhibited by in vitro incubation with the arginine analogue NG-monomethyl-L-arginine (L-NMMA; 300 microM) or ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 1 mM), was observed in these cells. Administration of Escherichia coli lipopolysaccharide (LPS; 3 mg/kg iv) significantly augmented NO synthase activity (determined 4 h later), which was inhibited in vitro by incubation with L-NMMA but not by EGTA. The highest level of constitutive and inducible NO synthase activity occurred in intestinal villus cells, and the lowest was in crypt cells. Induction of NO synthase activity was associated with a decrease in cellular viability as assessed by a decrease in trypan blue exclusion. Dexamethasone pretreatment (1 mg/kg iv 2 h before LPS administration) significantly reduced both induction of NO synthase activity and the reduction in cellular viability. Likewise concurrent administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg sc) ameliorated the reduction in cell viability induced by LPS administration, an effect abolished by pretreatment with the NO substrate L-arginine (350 mg/kg sc). Whereas constitutively formed NO may have a physiological role in these cells, the results in this study suggest that induction of NO synthase in epithelial cells may represent a mechanism of local intestinal damage.

1. The protective or damaging actions on the gastric mucosa, of locally infused nitrovasodilators that donate nitric oxide (NO), have been investigated in the pentobarbitone-anaesthetized rat. 2. Local intra-arterial infusion of endothelin-1 (ET-1; 5 pmol kg-1 min-1 for 10 min) induced extensive, macroscopically apparent, haemorrhagic injury to the rat gastric mucosa. This damage was dose-dependently reduced by concurrent local intra-arterial infusion of glyceryl trinitrate (GTN; 10-40 micrograms kg-1 min-1) which liberates NO on metabolic transformation, or the nitrosothiol, S-nitroso-N-acetyl-penicillamine (SNAP, 2.5-10 micrograms kg-1 min-1) which spontaneously liberates NO. 3. Local infusion of higher doses of SNAP (20 and 40 micrograms kg-1 min-1, i.a.) did not, however, significantly protect against mucosal injury induced by ET-1. 4. Furthermore, local infusion alone of these higher doses of SNAP, as well as sodium nitroprusside (10-40 micrograms kg-1 min-1, i.a.) which also spontaneously liberates NO, induced significant mucosal injury, as assessed macroscopically and confirmed by histology. 5. Local infusion of these higher doses of SNAP and nitroprusside reduced systemic arterial blood pressure (BP), but this was not correlated with the extent of mucosal injury. 6. Furthermore, local infusion of GTN (10-40 micrograms kg-1 min-1, i.a.) alone, which also reduced BP, failed to induce gastric mucosal damage. 7. These findings suggest that exogenous NO can protect the rat gastric mucosa from damage induced by the vasoconstrictor peptide ET-1, which may reflect local microcirculatory interactions. However, the unregulated release of high levels of NO within the microvasculature induces mucosal injury.

We studied the actions of purified Helicobacter pylori endotoxin (3 mg kg(-1), i.v.) on rat intestinal vascular permeability (assessed by the radiolabelled human serum albumin leakage technique) and on nitric oxide synthase induction (assessed by the citrulline assay) 4 h later. We found increased albumin leakage and expression of the inducible nitric oxide synthase in jejunum and colon, effects reversed by a selective inducible nitric oxide synthase inhibitor N-(8-(aminomethyl)benzyl)-acetamidine (1400W; 0.2-1 mg kg(-1), s.c., concurrently with endotoxin). Thus, H. pylori endotoxin seems to be capable of provoking an inflammatory response in the rat intestinal tissue. Systemic liberation of H. pylori endotoxin might possibly attenuate jejunal and colonic mucosal barrier function, a process mediated by the expression of the inducible nitric oxide synthase.

A prevalent unwanted action of cyclo-oxygenase (COX) inhibitors, as exemplified by the non-steroidal anti-inflammatory drugs (NSAIDs), is their potential to produce gastrointestinal side effects in clinical use. The injury provoked by such agents includes rapid superficial disruption to the surface layer of the gastric mucosa, the production of acute gastric erosions in the corpus region and the formation of ulcers in the antral region of the stomach. The small intestine is also adversely affected, with a developing enteropathy over a more protracted period that causes lesions and inflammation in the gut. From experimental work, the interactive mechanisms of such damage in the stomach differ distinctly from those that underlie the intestinal injury, yet the damage in both regions involves the inhibition of both COX-1 and COX-2 isoforms. This chapter outlines the in vivo methods that can be used to identify the potential for novel NSAIDs and selective COX-inhibitors to produce acute gastric corpus lesions and more-chronic antral ulcers in the rat, as well as causing small intestinal enteropathy. Such methods can also be utilized to evaluate the ability of novel agents to prevent the gastrointestinal injury provoked by NSAIDs or COX-inhibitors.

Administration of E. coli endotoxin (1 mg/kg i.v.) abolished the acid secretory response induced by a bolus injection of pentagastrin (100 micrograms/kg i.v.) in the continuously perfused stomach of the anaesthetized rat. Endotoxin administration did not modify mean systemic arterial blood pressure. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 5-20 mg/kg i.v.), but not dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg i.m.), substantially restored the secretory responses to pentagastrin. The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.v.), but not by its enantiomer D-arginine (100 mg/kg i.v.). L-NAME (10 mg/kg i.v.) increased blood pressure but this does not seem to be the mechanism by which endotoxin-induced acid inhibition was prevented, since similar systemic pressor responses induced by noradrenaline (15 micrograms/kg per min i.v.) had no such effect. The platelet-activating factor (PAF) receptor antagonist, WEB 2086 (2 mg/kg), induced a partial reversal of the inhibition by endotoxin of acid responses to pentagastrin. In endotoxin-treated rats, the combined administration of L-NAME (10 mg/kg) and WEB 2086 (2 mg/kg) completely restored the degree of H+ output induced by pentagastrin to levels similar to those of control, vehicle-treated animals. These findings suggest that endotoxin-induced acute inhibition of acid responses to pentagastrin involves NO synthesis and the release of PAF.

The actions of nitric oxide (NO) on the acute gastrointestinal damage induced by platelet-activating factor (PAF) have been investigated in the rat.S-nitroso-N-acetyl penicillamine, which spontaneously generates NO, dose-dependently inhibited PAF-induced gastrointestinal plasma leakage, a measure of the initiation of vascular damage. The inhibitor of NO synthase,NG-monomethyl-l-arginine substantially potentiated gastrointestinal damage and plasma leakage induced byE. coli endotoxin, but had no effect on that induced by intravenous infusion of PAF.

1. The effects of inhibiting endogenous nitric oxide (NO) synthesis with NG-monomethyl-L-arginine (L-NMMA) on the systemic and splanchnic circulation have been investigated in rats with experimental chronic portal hypertension, anaesthetized with ketamine. 2. Portal hypertension was induced by partial portal vein ligation, 2 weeks prior to study. This procedure induced a reduction in systemic arterial blood pressure (MAP), an increase in cardiac output as measured by radiolabelled microspheres, a reduction in peripheral and splanchnic vascular resistance and an increased portal venous inflow (PVI) and portal pressure, as compared to control non-ligated rats. 3. L-NMAA (6.25 and 50 mg kg-1, i.v.) dose-dependently increased MAP, reduced cardiac output and PVI, and increased peripheral and splanchnic vascular resistance. With L-NMMA (50 mg kg-1), PVI and the vascular resistances returned to values comparable to those determined in control non-ligated anaesthetized rats under resting conditions. 4. Porto-collateral resistance was also increased by these doses of L-NMMA, whereas portal pressure was unchanged. The increase in renal blood flow and decrease in renal vascular resistance also seen in portal-hypertensive rats was reversed by L-NMMA (50 mg kg-1). 5. These effects of L-NMMA (50 mg kg-1) were inhibited by prior administration of L-arginine (300 mg kg-1, i.v.). 6. These findings indicate that the chronic hyperdynamic circulatory characteristics following portal vein stenosis can be attenuated by L-NMMA. Thus, the excessive formation of endogenous NO may be implicated in the pathogenesis of the haemodynamic disturbances and splanchnic vasodilatation associated with chronic portal hypertension.

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 micrograms 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.

Macroscopic jejunal damage and plasma leakage induced within 15 min by E. coli lipopolysaccharide (LPS 50 mg kg −1 i.v.) in the rat was enhanced by the inhibitor of nitric oxide (NO) formation, N G -monomethyl-L-arginine (L-NMMA 50 mg kg −1 i.v.). The nitro-vasodilator, S-nitroso-N-acetyl-pencillamine (SNAP; 10 μg kg −1 min −1 i.v.), which generates NO, attenuated both LPS-induced intestinal damage and the enhancement of such damage and plasma leakage produced by L-NMMA. Endogenous NO may thus have a protective role in the intestinal vasculature that can be mimicked by generators of NO.

The mechanism of action of 5-aminosalicylic acid (5-ASA), the active therapeutic moiety of a number of clinically used anti-colitic agents, is unclear. The present study investigates whether the beneficial effects in vivo could involve induction of the heat shock protein, heme oxygenase-1 (HO-1), known to provide endogenous anti-oxidant and anti-inflammatory moieties which can modulate colonic inflammation. The effects of 5-ASA on the colonic expression and activity of HO-1 along with its effect on the inflammatory damage have been evaluated in the colitis provoked by instillation of trinitrobenzene sulphonic acid (TNBS) over 48 h in the rat. Intracolonic administration of 5-ASA (8, 25 and 75 mg/kg/day) dose-dependently reduced the TNBS-provoked macroscopic colonic inflammatory injury, myeloperoxidase (MPO) activity and TNF-alpha levels, while also dose-dependently increasing colonic heme oxygenase enzyme activity. Colonic HO-1 protein expression, determined by Western blot analysis in this colitis model, was likewise further induced by 5-ASA. Intracolonic administration of 5-ASA alone under unchallenged conditions also induced colonic HO-1 protein expression and stimulated heme oxygenase enzyme activity. Administration of zinc protoporphyrin (50 micromol/kg/day, s.c.), which prevented the increase in colonic heme oxygenase activity, abolished the anti-colitic effect of 5-ASA. These results suggest that 5-ASA may exert its colonic anti-oxidant and anti-inflammatory effects in vivo in part through the up-regulation of HO-1 enzyme expression and activity.

After a decade of intense pharmacological and drug development activity by the pharmaceutical industry, compounds derived from two key strategies for reducing gastrointestinal effects of non-steroidal anti-inflammatory drugs (NSAIDs) have been subjected to rigorous clinical appraisal. Despite the undoubted therapeutic and commercial success of the selective cyclooxygenase (COX)-2 inhibitors, known as the coxibs, with second-generation compounds already approved and launched, some concerns over their full gastrointestinal profile still linger, while the cardiovascular safety of this class has become a key issue. Likewise, Phase II evaluation of compounds incorporating a nitric oxide (NO)-donating moiety into standard NSAIDs (the NO-NSAIDs or CINODs) has created recent controversy over the full clinical profile of these compounds. Other approaches such as NO-COX-2 inhibitors and dual COX-lipoxygenase inhibitors are already warranting interest. It might therefore be too early to predict the eventual winning strategy for safer anti-inflammatory drugs.