Carbachol potentiates cholera toxin-induced secretion in a colonic epithelial cell line (HT29-19A) and rat ileal mucosa in vitro.

Recent studies show that enteric nerves are involved in the action of cholera toxin, both in vivo and in vitro. The aim of this study was to investigate in vitro the influence of carbachol, a cholinergic agonist, on the action of cholera toxin. Cultured HT29-19A cell lines and rat ileal mucosa were used in an Ussing chamber for the measurement of short-circuit current induced by cholera toxin. Cyclic AMP was measured from HT29-19A cell lines by standard radio-immunoassay. Pre-treatment of the HT29-19A cell lines with carbachol potentiated cholera toxin-induced secretory response, and enhanced accumulation of cAMP. Carbachol also potentiated the cholera toxin-secretory response in the rat ileal mucosa, but only following pretreatment with the prostaglandin synthesis inhibitor, indomethacin. There was synergistic interaction between cholera toxin and cholinergic neurotransmitter carbachol on the intestinal epithelium. Cholinergic agonists may play a role in regulating the secretory response to the toxin. Such interaction is masked in the intact tissues in vitro due to the release of prostaglandins during isolation.

Platelet activating factor: release from colonic mucosa in patients with ulcerative colitis and its effect on colonic secretion.

Inflammatory mediators have been implicated in the pathophysiology of ulcerative colitis. They may stimulate intestinal secretion and contribute to the production of diarrhoea. Platelet activating factor (PAF) may be responsible for a high proportion of this secretory response. Biopsy specimens from inflamed and quiescent mucosa of patients with ulcerative colitis and normal human colonic mucosa were cultured or co-cultured. The release of PAF, prostaglandin E2, and leukotriene D4 into the culture medium was measured and the ability of this culture medium, from inflamed and normal tissues, to influence secretion in rat colonic mucosa was assessed. PAF was liberated by inflamed tissue. Its release from quiescent but not normal tissue was stimulated by medium in which inflamed mucosal biopsy tissues had been cultured and by exogenous bradykinin and 5-hydroxytryptamine, but not by histamine. PAF stimulated eicosanoid production. The rise in short circuit current produced in vitro by inflamed tissue culture medium was inhibited by the PAF receptor antagonist (CV 6209) (46%) (32.4 (2.9) v 17.5 (1.19) muA.cm-2, p < 0.005) and further by combined cyclooxygenase and lipoxygenase inhibition (indomethacin plus ICI 207968) (58%) (32.4 (2.9) v 13.6 (1.9) muA.cm-2, p < 0.005). Mepacrine and hydrocortisone attenuated considerably the electrical response evoked by medium from inflamed mucosa to a similar extent (32.4 (2.9) v 6.3 (1.2) v 5.1 (0.9) muA.cm-2, p < 0.001). These data suggest that PAF accounted for 46% of the culture medium secretory effect. Thus, any attempt to block its release in patients with ulcerative colitis may have only a partial effect on their symptoms.

Na-K-Cl cotransport in villus and crypt cells from rat duodenum.

The aim of the present study was to examine the possibility that Cl- uptake into both villus and crypt epithelial cells of rat duodenum occurs via an electroneutral Na-K-Cl coupled-transport mechanism. Sheets of villus cells and whole crypts were isolated using a Ca2+ chelation technique combined with continuous vibration at low temperatures. Structurally intact, viable epithelia from defined regions along the villus-crypt axis were produced. Uptake of 86Rb+ (as a proxy for K+) into both villus and crypt cells appeared to depend on a coupled process, as evidenced by the inhibition of 86Rb+ uptake by bumetanide and by the removal of either Na+ or Cl- from the bathing media. We report an improved method of isolation of viable enterocytes from defined regions along the villus-crypt axis. We demonstrate the presence of Na-K-Cl cotransport in both villus and crypt duodenal enterocytes.

Potential role for interleukin-1 in the pathophysiology of ulcerative colitis.

1. Biopsies of colonic mucosa from patients with ulcerative colitis liberated more interleukin-1 beta, prostaglandin E2, leukotriene C4 and platelet-activating factor into the medium in which they were cultured than biopsies from patients with irritable bowel syndrome and histologically normal mucosa. 2. Addition of interleukin-1 stimulated release of greater quantities of all these inflammatory mediators, including interleukin-1 itself, from inflamed and normal mucosa. 3. Blockade of cyclo-oxygenase with indomethacin or of lipoxygenase with ICI 207968 or of phospholipase A2 with mepacrine inhibited release of prostaglandin E2 or leukotriene C4 or both of these plus platelet-activating factor, respectively. 4. Interleukin-1 stimulated the short-circuit current across isolated rat colonic mucosa mounted in flux chambers in a dose-dependent manner (Km 2 x 10(-11) mol/l). This stimulation was markedly inhibited by the removal of chloride from the bathing media. 5. Indomethacin or ICI 207968 inhibited the short-circuit current response to interleukin-1 and a combination of these antagonists produced a greater inhibition. Mepacrine caused an even greater inhibition whereas tetrodotoxin plus mepacrine inhibited the current completely. 6. These data indicate that interleukin-1, released in excess from inflamed colonic mucosa, stimulates the release of a range of inflammatory mediators as well as of more interleukin-1. It probably acts by stimulating phospholipase A2 in inflammatory cells, probably lymphocytes, and can do so in normal and inflamed mucosa. Since, in rat colonic mucosa it stimulated an electrical response in very low concentrations, it is feasible that it is involved in the chloride secretion, and hence the diarrhoea, which may occur in inflammatory reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple G-protein-dependent pathways mediate the antisecretory effects of somatostatin and clonidine in the HT29-19A colonic cell line.

Using the functionally differentiated colonic cell line, HT29-19A, we have examined sites at which inhibitory G-proteins mediate the antisecretory actions of somatostatin (SST) and the alpha 2-adrenergic agonist, clonidine (CLON) at the epithelial level. Both agents caused a dose-dependent inhibition (EC50:SST 35 nM; CLON 225 nM) of Cl- secretion (assessed by changes in short circuit current) activated by cAMP-mediated agonists, PGE2 and cholera toxin. Inhibition was accompanied by a reduction in intracellular cAMP accumulation and could be blocked by pretreatment with pertussis toxin at a concentration (200 ng/ml) which activated ADP-ribosylation of a 41-kD inhibitory G protein in HT29-19A membranes. Secretion stimulated by the permeant cAMP analogue, dibutyryl cAMP, was also inhibited by SST and CLON (30-50%; P < 0.005), indicating additional inhibitory sites located distal to cAMP production. Both agents were effective inhibitors of secretion mediated through the Ca2+ signaling pathway. SST (1 microM) and CLON (10 microM) reduced the Isc response to the muscarinic agonist, carbachol, by 60-70%; inhibition was reversed in pertussis toxin-treated cells. These effects did not, however, involve inhibition of the carbachol-induced increase in cellular inositol 1,4,5-trisphosphate levels or the rise in cytosolic calcium, [Ca]i. Inhibition by SST of secretion induced by phorbol 12,13 dibutyrate but not by the calcium agonist, thapsigargin, suggests that SST may act at a distal inhibitory site in the Ca(2+)-dependent secretory process activated by protein kinase C. We conclude that SST and alpha 2-adrenergic agonists can act directly on intestinal epithelial cells to exert a comprehensive inhibition of Cl- secretion mediated through both cAMP and Ca2+/protein kinase C signaling pathways. Inhibition is mediated via pertussis toxin-sensitive G-proteins at sites located both proximal and distal to the production of second messengers.

Inter-relationships between inflammatory mediators released from colonic mucosa in ulcerative colitis and their effects on colonic secretion.

Metabolites of arachidonic acid have been implicated in the pathophysiology of ulcerative colitis-they can stimulate intestinal secretion, increase mucosal blood flow, and influence smooth muscle activity. The influence on the mucosal transport function of culture medium in which colonic mucosal biopsy specimens had been incubated was investigated using rat stripped distal colonic mucosa in vitro as the assay system. Colonic tissue from patients with colitis and from control subjects was cultured. Medium from inflamed tissue contained more prostaglandin E2 (PGE2) and leukotriene D4 (LTD4) and evoked a greater electrical (secretory) response in rat colonic mucosa than control tissue medium. In inflamed tissue, cyclo-oxygenase inhibition (indomethacin) attenuated PGE2 but increased LTD4 production; conversely lipoxygenase inhibition (ICI 207968) inhibited LTD4 production but enhanced PGE2 output. Each inhibitor alone enhanced the electrical response in the rat colon. Inhibition of both enzymes (indomethacin plus ICI 207968) caused a fall in both PGE2 (82%) and LTD4 (89%) production and in the electrical response (57%). Inflamed tissue treated with a phospholipase A2 inhibitor (mepacrine) produced less PGE2, LTD4, and electrical responses when compared with inflamed tissue, either untreated (91%, 92%, and 79% respectively) or treated with cyclo-oxygenase and lipoxygenase inhibition. Incubation with bradykinin stimulated eicosanoid release and electrical response, while a bradykinin antagonist caused a modest inhibition. Analysis of these observations suggests that a combination of arachidonic acid derivatives accounts for about half the secretory response. Other products of phospholipase A2 activity are probably responsible for much of the remainder, leaving up to 20% the result of types of mediator not determined in this study.

Use of coculture of colonic mucosal biopsies to investigate the release of eicosanoids by inflamed and uninflamed mucosa from patients with inflammatory bowel disease.

Eicosanoid production was measured in cultured biopsies of colonic mucosa from control patients, with the irritable bowel syndrome, and from patients with proctosigmoiditis and with colonic Crohn's disease. Cultured inflamed colonic mucosa from patients with proctosigmoiditis and Crohn's disease produced more prostaglandin E2 and leukotrienes C4 than control tissues. In addition, eicosanoid production by macroscopically uninflamed or 'quiescent' mucosa from the right colon was examined in patients with proctosigmoiditis and between skip lesions in Crohn's disease patients. In the proctosigmoiditis group quiescent mucosa produced eicosanoids in similar quantities to control tissue. Coculture of quiescent plus inflamed tissue however, generated a marked increase in eicosanoid output in 12 of 20 of the patients and this was similar to the quantity obtained from two pieces of inflamed tissue. In the Crohn's disease group, quiescent mucosa produced more eicosanoids than control mucosa but production was markedly stimulated by coculture with inflamed mucosa in all patients. These findings suggest that in some patients with proctosigmoiditis and in all patients with Crohn's disease quiescent mucosa appears to be sensitised. A small but significant increase in the macrophage population may be partly responsible but it is likely that these and other cells are primed to release eicosanoids, and may be induced to do so by soluble mediators produced by actively inflamed tissue.

Absorption of polar drugs following caecal instillation in healthy volunteers.

We have investigated colonic drug absorption in man by the caecal instillation of a multi-component solution of atenolol, cimetidine, frusemide, hydrochlorothiazide and salicylic acid. We found that salicylic acid absorption from this solution was delayed but complete whereas the absorption of atenolol, cimetidine, frusemide and hydrochlorothiazide was four- to five-fold lower than expected from oral bioavailability studies.

A Cl- conductance sensitive to external pH in the apical membrane of rat duodenal enterocytes.

1. The pH dependence of a chloride conductance in the apical membrane of rat duodenal enterocytes was examined. 2. A stepwise reduction of both internal and external pH from 7.4 to 6.8 resulted in a significant stimulation of 36Cl flux driven by an inside-positive membrane potential. 3. A stepwise reduction in pH had no significant effect upon other parameters such as the initial rate of D-[3H]glucose or voltage-independent 36Cl uptake, suggesting a specific effect upon the chloride conductance. 4. The pH-dependent stimulation of 36Cl uptake exhibited saturation kinetics, with an apparent Vmax (maximum velocity) of 5.5 nmol (mg protein)-1 (4 s)-1 and an apparent Km (Michaelis-Menten constant) of 88 nM H+ ions. 5. To determine the site of action of protons upon the conductance the effect of asymmetrically reducing either the internal or external pH was examined. 6. A step reduction of extracellular pH from 7.8 to 6.8 significantly stimulated the rate of 36Cl uptake. In contrast, a step reduction of internal pH from 7.8 to 6.8 was without effect upon the rate of 36Cl uptake. 7. These results suggest that the chloride conductance on the apical membrane of rat duodenal enterocytes is allosterically regulated by protons at an external site.

The influence of gastrointestinal transit on drug absorption in healthy volunteers.

1. The effect of variability of gastric emptying and oro-caecal transit on the absorption of a multicomponent solution of frusemide, atenolol, hydrochlorthiazide and salicylic acid has been studied in six healthy subjects. Each subject was studied on five separate occasions: three times under basal conditions, once following metoclopramide and once following codeine pretreatment in an attempt to speed and slow transit respectively. 2. Inter-subject variability of gastric emptying, oro-caecal transit and the rate and extent of drug absorption was considerable. 3. The absorption of salicylic acid appeared rate-limited by gastric emptying but the rate and extent of frusemide, atenolol and hydrochlorthiazide absorption were unrelated to measures of gastric emptying or oro-caecal transit. 4. Codeine phosphate caused a two-fold delay in oro-caecal transit but did not influence gastric emptying while metoclopramide had no significant effect on either function. 5. Metoclopramide and codeine had no significant effect on the rate or extent of absorption of any of the study drugs. 6. Within the limits of this experiment, oro-caecal transit time did not appear to be an important determinant of frusemide, atenolol, hydrochlorothiazide or salicylic acid absorption. Other factors must account for the observed variability in drug absorption.

Effects of a non-absorbable osmotic load on drug absorption in healthy volunteers.

1. We have studied the effects of a non-absorbable osmotic load on the absorption of a multicomponent solution of frusemide, atenolol, hydrochlorothiazide and salicylic acid in six healthy volunteers. 2. Each subject was studied on up to four separate occasions. The drugs were administered in one of four solutions: a) a mannitol/electrolyte solution, b) a double-strength mannitol/electrolyte solution, c) a glucose/electrolyte solution and d) water. Lactulose or sulphasalazine were added as oro-caecal transit markers. Lactulose was included in the mannitol- and glucose-based solutions, adding a further non-absorbable osmotic load, and sulphasalazine was added to the water, adding little osmotic load. 3. The absorption of atenolol and hydrochlorothiazide was two- to three-times less from all lactulose-containing solutions than from the sulphasalazine-containing solution. The absorption of frusemide and salicylic acid was similar from all four solutions. 4. The largest non-absorbable osmotic load impaired the absorption of atenolol and hydrochlorothiazide most and the incorporation of glucose only partly restored absorption. 5. These results suggest that transmucosal water movement is an important determinant of atenolol and hydrochlorothiazide absorption but is less relevant for the absorption of frusemide and salicylic acid. Furthermore, these data demonstrate a previously unrecognised interaction between a commonly prescribed laxative--lactulose, and atenolol and hydrochlorothiazide.

Studies of luminal and mucosal pH in reflux esophagitis and antral gastritis.

Luminal and mucosal pH were measured endoscopically in patients with reflux esophagitis and antral gastritis and in control subjects. In all subjects, significant lumen-to-mucosa gradients were observed in the esophagus, stomach and acidified proximal duodenum. In the reflux patients luminal pH was lower in the fundus (mean +/- SEM, control vs. reflux esophagitis: 2.01 +/- 0.17 vs. 1.32 +/- 0.18; p less than 0.02) and antrum (3.51 +/- 0.35 vs. 2.13 +/- 0.24; p less than 0.01) and, in the gastritis patients, in the fundus (2.01 +/- 0.17 vs. 1.3 +/- 0.17; p less than 0.02). In both patient groups, mucosal pH was lower in the fundus (control vs. reflux vs. gastritis: 4.84 +/- 0.37 vs. 3.37 +/- 0.61 vs. 3.12 +/- 0.6; p less than 0.05) and acidified duodenal cap (6.74 +/- 0.13 vs. 6.09 +/- 0.24 vs. 5.73 +/- 0.46; p less than 0.03). Mucosal pH profiles at the various sites showed less resistance of the gradient to a highly acidic environment in both the lower esophagus and antrum than in fundus and duodenum, and this was the case in the patient and control groups. Though associated with a more acid environment, neither esophagitis nor antral gastritis exhibits a specific deficit in the 'mucus-bicarbonate barrier', suggesting that the pathogenesis of these disorders may depend more on abnormal 'attack' rather than impaired defense.

Demonstration of bicarbonate secretion in rat duodenal brush border membrane vesicles.

This study was designed to characterize the mechanisms available for bicarbonate transport across the apical membrane using rat duodenal brush border membrane vesicles (BBMVs), including the morphological findings. BBMVs were purified using a double Mg2+ precipitation technique. Electron microscopy of the final pellet showed only membrane material without contaminating organelles of cytoskeletal aggregates. BBMVs were enriched 15.1 +/- 3.2-fold in A-lp and 12.5 +/- 2.4-fold in LAP. The time course of 36Cl uptake into BBMVs demonstrated a good response curve and equilibrated to the intravesicular space over 120 min. In this present study, these BBMVs were purified and showed a stabilized structure. Therefore, these results suggest that this model was useful for studying HCO3- secretion and ulcer etiology.