Nitric oxide in the rat gastrointestinal tract

This study concerns the nature of nitric oxide synthase (NOS) and the role of nitric oxide (NO) in the rat gastrointestinal tract. The major objectives were (i) to characterise NOS isoforms in the gastric glandular mucosa, (ii) to localise NOS isoforms in the rat gastric glandular mucosa, (iii) to investigate the role of NO in carbachol-stimulated gastric mucus secretion, (iv) to investigate the nature of NOS and small intestine. Immunoblotting was performed using polyclonal antisera raised against two peptides found in the rat brain NOS sequence and commercial monoclonal antibodies directed against neuronal and endothelial isoforms of NOS. A160kDa band was detected in brain and gastric mucosal samples with antibodies and antisera directed against neuronal NOS sequences, and a 140kDa band was detected in gastric mucosal samples using an anti-endothelial NOS antibody. An intense 160kDa neuronal NOS band was detected in a high-density fraction of gastric mucosal cells separated on a Percoll gradient. Detection of neuronal NOS by a carboxyl-terminal antiserum in samples of brain, but not of gastric mucosa, could be blocked by the peptide (20g/ml) against which the antibody was raised. After affinity purification, recognition of gastric mucosal NOS was blocked by peptide. Particulate neuronal NOS was found in the brain by immunoblotting while 94% of gastric mucosal enzyme was soluble. Gastric mucosal endothelial NOS was 95% particulate. 95% of NOS activity in the gastric mucosa was due to neuronal NOS. Paraformaldehyde- and acetone-fixed gastric mucosal sections were subject to immunocytochemistry using the above antibodies. Neuronal NOS was localised to the surface mucosal epithelial cells while endothelial NOS was associated with microvessels at the base of the mucosa and to larger vessels in the submucosa. Intragastric administration of carbachol or 16, 16-dimethyl prostaglandin E2 increased the thickness of the rat gastric mucus layer. The NOS inhibitor NG-nitro-L-arginine methyl ester dose-dependently, and selectively, prevented the stimulatory effect of carbachol. Ca2+-independent NOS activity in rat ileal, jejunal and colonic muscle was increased after LPS induction. Ca2+-dependent activity was not affected. Distribution of inducible NOS protein paralleled Ca2+ -independent activity. LPS treatment did not affect the content of neuronal NOS in colonic muscle.

Generation of nitric oxide and its protective and toxic actions in the gastrointestinal tract

Nitric oxide is a free-radical gas which can exert both protective and damaging effects. The objectives of the thesis were: (i) to investigate arginine metabolism in isolated rat gastric mucosal cells, (ii) to investigate the role of NO in the induction of ornithine decarboxylase in the rat gastric mucosa damaged by hypertonic saline in vivo, (iii) to expose primary cultures of guinea-pig gastric mucosal cells to oxidative challenge and an NO donor, and to investigate the response in terms of heat shock protein 72 (HSP 72) induction, and (iv) to investigate the induction of iNOS and the role of potential modulators of activity in gastric cell lines. Isolated rat gastric mucosal cells converted exogenous arginine to ornithine and citrulline. This metabolism of arginine was not affected by a range of NO synthase inhibitors, but was reduced by the arginase inhibitors NG-hydroxy-L-arginine and L-ornithine. Thus, the predominant pathway of arginine metabolism involves arginase and ornithine transcarbamoylase, not NO synthase. Pretreatment of rats with NG-nitro-L-arginine promoted activation of ornithine decarboxylase after intragastric hypertonic saline, but did not increase acid phosphatase release (damage). NO may therefore restrict activation of ornithine decarboxylase in response to damage. Exposure of primary cultures of guinea-pig gastric mucosal cells to S-nitroso-N-acetyl-penicillamine (SNAP) caused a concentration dependent induction of HSP 72, which was inhibited by an NO scavenger and blockade of transcription. The effect of SNAP was enhanced by decreasing the intracellular reduced thiol content with diethyl maleate, which itself also induced HSP 72 formation. Substantial amounts of NO may induce defensive responses in cells. Induction of iNOS was not detected in HGT-1 or AGS cells exposed to cytokines. Conclusions An arginase pathway may restrict availability of arginine for NO synthase in gastric mucosa or may be present to supply ornithine for polyamine synthesis. NO may modulate the response to damage of the stomach epithelium in vivo. Exogenous NO may induce a defensive response in gastric mucosal cells.

Aspects of signal transduction in the gastrointestinal tract

This study was undertaken to increase knowledge of the mechanisms of inter- and intracellular signalling in the gastrointestinal tract. Specific aims were: to use cell lines to elucidate factors affecting growth of gastric cells, to investigate the distribution and aspects of function of isoforms of protein kinase C in a gastric cell line and in the rat gastrointestinal tract and to determine the presence and regulation of nitric oxide synthase in gastrointestinal tissues from the rat and in cell lines. The gastric cancer cell line HGT-1 was used to investigate control of growth. Increases in cell number were found to be dependent on the seeding density of the cells. In cells plated at low density insulin, epidermal growth factor and gastrin all increased cell number. Gastrin produced a bell-shaped dose response curve with a maximum activity at 5nM. No effect of gastrin was apparent in cells plated at high density. α and β isoforms of protein kinase C were found, by immunoblotting procedures, to be widespread in the gastrointestinal tract of the rat, but protein kinase Cε was confined to the gastric mucosa and gastrointestinal smooth muscle. HGT-1 cells contained protein kinase C α and ε but β or γ were not detected. Preincubation of HGT-1 cells for 24h with 1μM phorbol-12,13-dibutyrate down-regulated protein kinase C α but not ε. The inhibition by the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) of the histamine-stimulated increase in cAMP in HGT-1 cells was down regulated by phorbol-12,13-dibutyrate. Inhibition of histamine-stimulation of adenylate cyclase by TPA was Ca2+-dependent and inhibited by the addition of an antibody to protein kinase C α. A role for protein kinase C α in modulating the effect of histamine on adenylate cyclase in HGT-1 cells is suggested. No nitric oxide synthase activity was detected in the gastrointestinal cell lines HGT-l, MKN-45 or CaCo-2. Ca2+-dependent nitric oxide synthase activity was observed in the gastric mucosa and the gastrointestinal smooth muscle from stomach to colon. The gastric: mucosal enzyme was soluble and showed half-maximal activity at 400nM Ca2+. Pretreatment of rats with endotoxin (3mg/kg body weight) induced nitric oxide synthase activity in both jejunal, ileal and colonic mucosa and muscle. A major portion of the induced activity in ileal and colonic mucosa was Ca2+-independent. Nitric oxide synthase activity in a high-density fraction of gastric mucosal cells was inhibited in a dose-dependent fashion by L-nitroarginine, NG-monomethyl-L-arginine, trifluoperazine and L-canavanine (in descending order of potency). Preincubation with okadaic acid and addition of ATPlMg2+ to the homogenisation buffer inhibited enzyme activity, which implies that phosphorylation inhibits gastric mucosal nitric oxide synthase.

Metformin and the gastrointestinal tract

Metformin is an effective agent with a good safety profile that is widely used as a first-line treatment for type 2 diabetes, yet its mechanisms of action and variability in terms of efficacy and side effects remain poorly understood. Although the liver is recognised as a major site of metformin pharmacodynamics, recent evidence also implicates the gut as an important site of action. Metformin has a number of actions within the gut. It increases intestinal glucose uptake and lactate production, increases GLP-1 concentrations and the bile acid pool within the intestine, and alters the microbiome. A novel delayed-release preparation of metformin has recently been shown to improve glycaemic control to a similar extent to immediate-release metformin, but with less systemic exposure. We believe that metformin response and tolerance is intrinsically linked with the gut. This review examines the passage of metformin through the gut, and how this can affect the efficacy of metformin treatment in the individual, and contribute to the side effects associated with metformin intolerance.

Density and cross-sectional areas of axons in the olfactory tract in control subjects and Alzheimer's disease:an image analysis study

Objective. Using an image analysis system to determine whether there is loss of axons in the olfactory tract (OT) in Alzheimer’s disease (AD). Design. A retrospective neuropathological study. Patients Nine control patients and eight clinically and pathologically verified AD cases. Measurements and Results. There was a reduction in axon density in AD compared with control subjects in the central and peripheral regions of the tract. Axonal loss was mainly of axons with smaller (<2.99 µm2) myelinated cross-sectional areas. Conclusions. The data suggest significant degeneration of axons within the OT involving the smaller sized axons. Loss of axons in the OT is likely to be secondary to pathological changes originating within the parahippocampal gyrus rather than to a pathogen spreading into the brain via the olfactory pathways.

Elevated left and reduced right orbitomedial prefrontal fractional anisotropy in adults with bipolar disorder revealed by tract-based spatial statistics

Context - Diffusion tensor imaging (DTI) studies in adults with bipolar disorder (BD) indicate altered white matter (WM) in the orbitomedial prefrontal cortex (OMPFC), potentially underlying abnormal prefrontal corticolimbic connectivity and mood dysregulation in BD. Objective - To use tract-based spatial statistics (TBSS) to examine WM skeleton (ie, the most compact whole-brain WM) in subjects with BD vs healthy control subjects. Design - Cross-sectional, case-control, whole-brain DTI using TBSS. Setting - University research institute. Participants - Fifty-six individuals, 31 having a DSM-IV diagnosis of BD type I (mean age, 35.9 years [age range, 24-52 years]) and 25 controls (mean age, 29.5 years [age range, 19-52 years]). Main Outcome Measures - Fractional anisotropy (FA) longitudinal and radial diffusivities in subjects with BD vs controls (covarying for age) and their relationships with clinical and demographic variables. Results - Subjects with BD vs controls had significantly greater FA (t > 3.0, P = .05 corrected) in the left uncinate fasciculus (reduced radial diffusivity distally and increased longitudinal diffusivity centrally), left optic radiation (increased longitudinal diffusivity), and right anterothalamic radiation (no significant diffusivity change). Subjects with BD vs controls had significantly reduced FA (t > 3.0, P = .05 corrected) in the right uncinate fasciculus (greater radial diffusivity). Among subjects with BD, significant negative correlations (P < .01) were found between age and FA in bilateral uncinate fasciculi and in the right anterothalamic radiation, as well as between medication load and FA in the left optic radiation. Decreased FA (P < .01) was observed in the left optic radiation and in the right anterothalamic radiation among subjects with BD taking vs those not taking mood stabilizers, as well as in the left optic radiation among depressed vs remitted subjects with BD. Subjects having BD with vs without lifetime alcohol or other drug abuse had significantly decreased FA in the left uncinate fasciculus. Conclusions - To our knowledge, this is the first study to use TBSS to examine WM in subjects with BD. Subjects with BD vs controls showed greater WM FA in the left OMPFC that diminished with age and with alcohol or other drug abuse, as well as reduced WM FA in the right OMPFC. Mood stabilizers and depressed episode reduced WM FA in left-sided sensory visual processing regions among subjects with BD. Abnormal right vs left asymmetry in FA in OMPFC WM among subjects with BD, likely reflecting increased proportions of left-sided longitudinally aligned and right-sided obliquely aligned myelinated fibers, may represent a biologic mechanism for mood dysregulation in BD.

Design, synthesis and evaluation of dipeptides as probes for studies of gastrointestinal tract dipeptide transport system

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

Design, synthesis and evaluation of dipeptides as probes for studies of gastro-intestinal tract dipeptide transport

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

Effect of nitric oxide on apoptotic activity in the rat gastrointestinal tract

The effect of nitric oxide (NO) on apoptosis in the gastrointestinal mucosa was investigated. Experiments involved long-term exposure of rat gastric mucosal cells in vitro to exogenous NO delivered from the NO, donor S-nitroso-N-acetyl-penicillamine, and the effect of intravenous administration of lipopolysaccharide in vivo, in the presence and absence of the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl) acetamidine (1400 W). S-nitroso-N-acetyl-penicillamine produced a dose-related inhibition of caspase 3-like activity and DNA fragmentation in isolated gastric mucosal cells. Caspase 3-like activity and DNA fragmentation in gastric, ileal and colonic mucosa were increased both 5 and 24 h after injection of lipopolysaccharide (3 mg/kg, i.v.) to rats in vivo. Administration of 1400 W (5 mg/kg, i.v.) immediately after lipopolysaccharide enhanced caspase 3-like activity and DNA fragmentation above that found with lipopolysaccharide alone. In conclusion, data obtained both in vitro and in vivo suggest that NO exerts an anti-apoptotic effect on rat gastrointestinal mucosal cells. © 2001 Elsevier Science B.V.

Role of hydrogen sulfide in the female reproductive tract

It is well established that hydrogen sulfide (H 2S) has a signaling role in the body. So far it has been shown that H 2S is produced by intra-uterine tissues in the pregnant rat and the human placenta. Two main enzymes responsible for H 2S production, cystathionine- synthase and cystathionine-lyase, have been demonstrated in the pregnant and nonpregnant uterus, fetal membranes and placenta in the rat, and in human placenta. H 2S donors have been shown to inhibit contraction of the pregnant rat uterus. H 2S could play a role in maintaining uterine quiescence during pregnancy, as an oxygen sensor and vasodilator in the placenta, or as an anti-inflammatory. More research is required in this area to elucidate the roles of H 2S in the female reproductive tract and its mechanisms of action. © 2010 Expert Reviews Ltd.