Peter J. Hanson

Differential distribution of nitric oxide synthase between cell fractions isolated from the rat gastric mucosa

Cells isolated from the rat gastric mucosa were resolved into two fractions on a percoll density gradient, and into five fractions using counterflow centrifugation (elutriation). Ca(2+)-dependent nitric oxide synthase (NOS) activity was found in the high density percoll fraction but not in the parietal cell enriched low density fraction. This activity was inhibited by NG-monomethyl-L-arginine with an IC50 of 3.7 microM. Cells in the elutriator fraction rich in mucous-epithelial cells exhibited the highest NOS activity, while the smaller cell fractions had no detectable NOS yet had the highest basal release of prostaglandin E2. The parietal cell enriched elutriator fraction again had low NOS activity. The activity of a constitutive NOS in the mucous-cell fraction may indicate a role of NO in the regulation of epithelial cell integrity or secretion.

Localization of constitutive isoforms of nitric oxide synthase in the gastric glandular mucosa of the rat

Abstract.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.

Absorption and metabolism of chlorogenic acids in cultured gastric epithelial monolayers

Gastric absorption of feruloylquinic acid and di-O-caffeoylquinic acid analogs has never been investigated despite their potential contribution to the proposed beneficial health effects leading to reduced risk of type 2 diabetes. Using a cultured gastric epithelial model, with an acidic apical pH, the relative permeability coefficients (Papp) and metabolic fate of a series of chlorogenic acids (CGAs) were investigated. Mechanistic studies were performed in the apical to basal direction and demonstrated differential rates of absorption for different CGA subgroups. For the first time, we show intact absorption of feruloylquinic acids and caffeoylquinic acid lactones across the gastric epithelium (Papp ∼ 0.2 cm/s). Transport seemed to be mainly by passive diffusion, because good linearity was observed over the incubation period and test concentrations, and we speculate that a potential carrier-mediated component may be involved in uptake of certain 4-acyl CGA isomers. In contrast, absorption of intact di-O-caffeoylquinic acids was rapid (Papp ∼ 2–10 cm/s) but nonlinear with respect to time and concentration dependence, which was potentially limited by interaction with an efflux transporter and/or pH gradient dependence. For the first time, methylation is shown in gastric mucosa. Furthermore, isoferulic acid, dimethoxycinnamic acid, and ferulic acid were identified as novel gastric metabolites of CGA biotransformation. We propose that the stomach is the first location for the release of hydroxycinnamic acids, which could explain their early detection after coffee consumption.

Lipopolysaccharide induces Ca2+-independent nitric oxide synthase activity in rat gastric mucosal cells

Ca2+-independent nitric oxide synthase was detected in gastric mucosal cells isolated from rats injected 4 h previously with Escherichia coli lipopolysaccharide (3 mg/kg i.v.). Induced nitric oxide synthase was located in an elutriated cell fraction of intermediate size which contained epithelial cells, but was absent from the parietal cell fraction. Administration of dexamethasone (2 mg/kg i.p.) 1 h before lipopolysaccharide inhibited the appearance of Ca2+-independent nitric oxide synthase, and prevented the observed reduction in cell viability (trypan blue exclusion). Ca2+-independent nitric oxide synthase activity can thus be induced in certain cells of the gastric mucosa, and may contribute to gastric pathologies where there is activation of the immune system.

Stimulation by carbachol of mucus gel thickness in rat stomach involves nitric oxide

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.

Disparate effects of non‐steroidal anti‐inflammatory drugs on apoptosis in guinea‐pig gastric mucous cells: inhibition of basal apoptosis by diclofenac

Non‐steroidal anti‐inflammatory drugs (NSAIDs) induce apoptosis in gastrointestinal cancer cell lines. Similar actions on normal gastric epithelial cells could contribute to NSAID gastropathy. The present work therefore compared the actions of diclofenac, ibuprofen, indomethacin, and the cyclo‐oxygenase‐2 selective inhibitor, NS‐398, on a primary culture of guinea‐pig gastric mucous epithelial cells. Cell number was assessed by staining with crystal violet. Apoptotic activity was determined by condensation and fragmentation of nuclei and by assay of caspase‐3‐like activity. Necrosis was evaluated from release of cellular enzymes. Ibuprofen (250 μM for 24 h) promoted cell loss, and apoptosis, under both basal conditions and when apoptosis was increased by 25 μM N‐Hexanoyl‐D‐sphingosine (C6‐ceramide). Diclofenac (250 μM for 24 h) reduced the proportion of apoptotic nuclei from 5.2 to 2.1%, and caused inhibition of caspase‐3‐like activity, without causing necrosis under basal conditions. No such reduction in apoptotic activity was evident in the presence of 25 μM C6‐ceramide. The inhibitory effect of diclofenac on basal caspase‐3‐like activity was also exhibited by the structurally similar mefenamic and flufenamic acids (1–250 μM), but not by niflumic acid. Inhibition of superoxide production by the cells increased caspase‐3‐like activity, but the inhibitory action of diclofenac on caspase activity remained. Diclofenac did not affect superoxide production. Diclofenac inhibited caspase‐3‐like activity in cell homogenates and also inhibited human recombinant caspase‐3. In conclusion, NSAIDs vary in their effect on apoptotic activity in a primary culture of guinea‐pig gastric mucous epithelial cells, and the inhibitory effect of diclofenac on basal apoptosis could involve an action on caspase activity.

Induction of heat shock protein 72 by a nitric oxide donor in guinea-pig gastric mucosal cells.

Gastric mucosal cells may be exposed to exogenous nitric oxide (NO) from a variety of sources. The response of primary cultures of guinea-pig gastric mucosal cells to the NO donor S-nitroso-N-acetyl-penicillamine was therefore investigated. Exposure to S-nitroso-N-acetyl-penicillamine for 8 h caused a concentration-dependent induction of heat shock protein 72 (HSP 72). Induction was inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and by blockade of transcription with actinomycin D. Induction of HSP 72 by S-nitroso-N-acetyl-penicillamine was enhanced by diethyl maleate which decreased the intracellular reduced thiol content. By contrast, HSP 72 formation after heat shock was associated with an elevation of reduced thiol. Incubation with S-nitroso-N-acetyl-penicillamine for 18 h increased detachment of cells from the culture plate. The effect of S-nitroso-N-acetyl-penicillamine on detachment was exacerbated by the presence of actinomycin D. In conclusion, exogenous NO induces HSP 72 in guinea-pig gastric mucosal cells and this response may in part protect the cells from the deleterious effects of NO.

Regulation of mucus secretion by cells isolated from the rat gastric mucosa.

1. A suspension of cells, containing about 30% mucous cells, was isolated from the rat fundic mucosa, and was pre‐incubated with D‐[6‐3H]glucosamine. 3H‐labelled material subsequently released into the medium was separated by Fast Protein Liquid Chromatography on a Superose 6 column. 2. A sharp peak of labelled high molecular weight material eluted from the column close to the void volume. This material was identified as mucous glycoprotein by its similar chromatographic behaviour to partially purified rat gastric mucous glycoprotein, by its resistance to complete degradation by papain and by its behaviour on treatment with dithiothreitol. On a caesium chloride density gradient the labelled material was virtually all located between densities of 1.35 and 1.53 g/ml. with the main peak at 1.40 g/ml. 3. A broad peak of lower molecular weight material was also eluted from the column. The release of this unidentified material did not seem to be closely associated with the release of mucous glycoprotein from the cells. 4. Release of mucous glycoprotein was stimulated by secretin (half‐maximally effective concentration 2.3 nM, 84% stimulation above basal release at 100 nM), and by isoprenaline (half‐maximally effective concentration 34 nM, 33% stimulation at 1 microM). Carbachol (0.5 nM) produced a significant (18‐29%) stimulation of mucus secretion, but gastrin (100 nM), histamine (0.5 mM) and epidermal growth factor (200 nM) were without effect. 5. The preparation should prove useful in the identification of the agents which regulate gastric mucus secretion.

Paracellular permeability is increased by basal lipopolysaccharide in a primary culture of colonic epithelial cells; an effect prevented by an activator of Toll-like receptor-2.

Lipopolysaccharide (LPS), which generally activates Toll-like receptor 4 (TLR4), is expressed on commensal colonic bacteria. In a number of tissues, LPS can act directly on epithelial cells to increase paracellular permeability. Such an effect in the colon would have an important impact on the understanding of normal homeostasis and of pathology. Our aim was to use a novel primary culture of colonic epithelial cells grown on Transwells to investigate whether LPS, or Pam3CSK 4, an activator of TLR2, affected paracellular permeability. Consequently, [14C]-mannitol transfer and transepithelial electrical resistance (TEER) were measured. The preparation consisted primarily of cytokeratin-18 positive epithelial cells that produced superoxide, stained for mucus with periodic acid-Schiff reagent, exhibited alkaline phosphatase activity and expressed TLR2 and TLR4. Tight junctions and desmosomes were visible by transmission electron microscopy. Basally, but not apically, applied LPS from Escherichia coli increased the permeability to mannitol and to a 10-kDa dextran, and reduced TEER. The LPS from Helicobacter pylori increased paracellular permeability of gastric cells when applied either apically or basally, in contrast to colon cells, where this LPS was active only from the basal aspect. A pan-caspase inhibitor prevented the increase in caspase activity caused by basal E. coli LPS, and reduced the effects of LPS on paracellular permeability. Synthetic Pam3CSK4 in the basal compartment prevented all effects of basal E. coli LPS. In conclusion, LPS applied to the base of the colonic epithelial cells increased paracellular permeability by a mechanism involving caspase activation, suggesting a process by which perturbation of the gut barrier could be exacerbated. Moreover, activation of TLR2 ameliorated such effects.

Use of Amino Acids as Counterions Improves the Solubility of the BCS II Model Drug, Indomethacin

The number of new chemical entities (NCE) is increasing every day after the introduction of combinatorial chemistry and high throughput screening to the drug discovery cycle. One third of these new compounds have aqueous solubility less than 20µg/mL [1]. Therefore, a great deal of interest has been forwarded to the salt formation technique to overcome solubility limitations. This study aims to improve the drug solubility of a Biopharmaceutical Classification System class II (BCS II) model drug (Indomethacin; IND) using basic amino acids (L-arginine, L-lysine and L-histidine) as counterions. Three new salts were prepared using freeze drying method and characterised by FT-IR spectroscopy, proton nuclear magnetic resonance ((1)HNMR), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA). The effect of pH on IND solubility was also investigated using pH-solubility profile. Both arginine and lysine formed novel salts with IND, while histidine failed to dissociate the free acid and in turn no salt was formed. Arginine and lysine increased IND solubility by 10,000 and 2296 fold, respectively. An increase in dissolution rate was also observed for the novel salts. Since these new salts have improved IND solubility to that similar to BCS class I drugs, IND salts could be considered for possible waivers of bioequivalence.