Polycation-induced enhancement of epithelial paracellular permeability is independent of tight junctional characteristics.

The nature of polycation-induced change in transepithelial permeability was investigated in strains I (tight) and II (leaky) MDCK epithelial monolayers. Apical exposure to poly(L-lysine) (PLL, mol. wt. (MW) approximately 20,000) induced a dose-dependent increase in transepithelial conductance (GT) in both strains which correlated with increasing transepithelial flux of extracellular markers (thiourea/inulin) indicating that PLL enhanced paracellular permeability in these epithelia. Coincident with the increase in GT, PLL also induced an inward short circuit current (Isc) which was associated with the early phase of the increase in GT and may be responsible for part of it. Morphological studies showed that immunofluorescent staining of the tight junction protein, ZO-1, was abolished following PLL exposure. In addition, F-actin staining in monolayers challenged with PLL demonstrated breaks in the zonulae occludentes at the apical surface. PLL had similar effects on monolayers of T84 and HCT-8 human intestinal cells indicating that polycation action may be general for a range of epithelial types. We conclude that epithelial exposure to polycations results in opening of the paracellular route by mechanisms which are independent of tight junction characteristics.

The effect of heat-stable Escherichia coli enterotoxin, theophylline and forskolin on cyclic nucleotide levels and mucosal surface (acid microclimate) pH in rat proximal jejunum in vivo.

The normally acidic mucosal surface pH of 6.24 +/- 0.02(30) in rat proximal jejunum in vivo is effectively neutralised by 30 min exposure to heat-stable Escherichia coli (STa) enterotoxin (14 micrograms/ml) to 6.80 +/- 0.07 (n = 5) or to a forskolin/theophylline combination (1 mM:20 mM) to 7.10 +/- 0.07(7) while perfusion with Krebs-phosphate buffer alone without glucose left the mucosal surface pH unchanged at a pH of 6.21 +/- 0.02(9). Forskolin alone had no effect, and 20 mM theophylline moderately elevated the surface pH to 6.52 +/- 0.03(5). Theophylline, forskolin and their combination all elevated cAMP levels per mg tissue DNA above control values while STa enterotoxin was without effect. In contrast, all agents elevated cGMP levels per mg tissue DNA above control levels. These findings indicate that surface pH is only moderately affected by changes in cAMP levels and is affected to a much greater extent by altered cGMP levels.

Carbachol stimulates Cl- secretion via activation of two distinct apical Cl- pathways in cultured human T84 intestinal epithelial monolayers.

The mode of action of carbachol in stimulation of transepithelial Cl- secretion in intact human intestinal T84 epithelial monolayers has been investigated in order to determine whether a DIDS-insensitive exit pathway (via CFTR) coexists with a DIDS-sensitive exit pathway at the apical membrane. Carbachol stimulates a transient inward Isc due to Cl- secretion whose magnitude is related to the basal level of inward Isc. The inward current responses to both carbachol and hypo-osmotic media are abolished in nominally Ca(2+)-free media. The action of apical DIDS (100 microM) upon carbachol-stimulated Isc depends on the initial value of the basal Isc. At basal Isc levels < 10 microA cm-2, 100 microM DIDS applied to the apical cell border abolishes the inward Isc following exposure to both carbachol and hypo-osmotic media. In contrast a VIP-stimulated inward Isc is observed in the presence of 100 microM DIDS. After VIP stimulation of inward Isc, or if spontaneous basal values of Isc were > 10 microA cm-2, the carbachol stimulation of inward Isc was largely insensitive to 100 microM DIDS. The data are consistent with the participation of both DIDS-sensitive and DIDS insensitive pathways for Cl- at the apical membrane of human intestinal T84 epithelial cells.

Hypo-osmolar stimulation of transepithelial Cl- secretion in cultured human T84 intestinal epithelial layers.

Intact epithelial monolayers of T84 human colonic adenocarcinoma cells were exposed from the basolateral surfaces to hypo-osmotic media; in responsive tissues this resulted in a transient stimulation of inward short-circuit current (SCC) to a peak of 12.9 +/- 1.5 (S.E., n = 10) microA/cm2 which declined to prestimulation values of SCC (2.1 microA/cm2) within 5 min. Exposure of T84 cells to hypo-osmotic media results in an increase in cytosolic [Ca2+]i, dependent on extracellular Ca2+ influx. The cell-swelling activated SCC is abolished upon medium Cl- replacement and by 100 microM bumetanide applied to the basal-surfaces, consistent with the inward SCC resulting from transepithelial Cl- secretion. 100 microM DIDS (4,4'-diisothiocyanantostilbene-2,2'-disulphonic acid) also abolished the cell-swelling activated increase in SCC; DIDS is without effect upon the VIP-stimulated SCC, suggesting distinct Cl- channels are involved in the two responses.

H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells.

Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid alpha-methylaminoisobutyric acid (MeAIB) across confluent monolayers of the human intestinal epithelial cell line Caco-2 are enhanced by a transepithelial pH gradient (apical pH 6.0, basolateral pH 7.4). In Na(+)-free conditions (apical pH 7.4, basolateral pH 7.4), net absorption (120 +/- 58 pmol/cm2 per h, n = 13) and uptake across the apical membrane (cell/medium ratio 0.56 +/- 0.06, n = 13) are low. However, in Na(+)-free conditions with apical pH 6.0, net absorption (685 +/- 95 pmol/cm2 per h, n = 15) and intracellular accumulation (cell/medium ratio 3.63 +/- 0.29, n = 14) were marked. Continuous monitoring of intracellular pH (pHi) in BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers indicated that apical addition of MeAIB (20 mM) was associated with H(+)-flow across the apical membrane in both Na+ and Na(+)-free conditions. This transport process is rheogenic in Na(+)-free media, stimulating an inward short-circuit current in voltage-clamped Caco-2 cell monolayers. On the basis of competition for MeAIB accumulation and pHi experiments, L-proline, glycine, L-alanine and beta-alanine are also substrates for H(+)-linked transport at the apical membrane of Caco-2 cells but L-valine, L-leucine and L-phenylalanine are not. These data are consistent with the expression, in the apical brush-border membrane of Caco-2 cells, of a H(+)-coupled, Na(+)-independent MeAIB carrier.

Paracellular barrier and junctional protein distribution depend on basolateral extracellular Ca2+ in cultured epithelia.

The polarised nature of the increase in paracellular permeability induced by Ca(2+)-chelation with EGTA was investigated in several cultured epithelial cell lines. In strain I MDCK cells (canine kidney cells), a marked decrease (> 90%) in transepithelial electrical resistance (RT) and increase in mannitol and inulin permeabilities were only observed after addition of EGTA (for 4 h) to either basolateral (basal) or both (apical+basal) bathing solutions; apical Ca(2+)-chelation resulted in significant smaller changes (approximately 30%) in these variables. The increase in paracellular permeability upon basal EGTA addition was significantly lower than that produced by simultaneous apical and basal addition of 2 mM EGTA. A higher concentration of EGTA (20 mM) did not significantly eliminate this difference in potency between basal and apical+basal Ca(2+)-chelation. The polarised Ca(2+)-dependence of the paracellular barrier was associated with polarised effects on the junctional/cytoskeletal protein distribution. Basal or apical+basal EGTA addition induced substantial internalisation of uvomorulin with some cellular redistribution of the perijunctional actin ring and desmosomes and gaps in ZO-1 location between adjacent cells. In addition, polarised Ca(2+)-dependence of the paracellular barrier (assessed by measuring RT) was observed also in strain II MDCK and two human adenocarcinoma intestinal cell lines, Caco-2 and HCT-8, demonstrating generality of the phenomenon. Therefore, the data show a polarity in the ability of EGTA to enhance epithelial permeability and induce cellular redistribution of cytoskeletal/junctional proteins in several epithelia. The basolateral membrane sensitivity to Ca(2+)-chelation might be explained by the polarised distribution of uvomorulin.

Volume-activated Cl- secretion and transepithelial vinblastine secretion mediated by P-glycoprotein are not correlated in cultured human T84 intestinal epithelial layers.

The relationship between the P-glycoprotein-mediated vinblastine secretion and cell-swelling activated Cl- secretion (conductance) in intact epithelial layers of human colonic adenocarcinoma T84 cells has been investigated. Whereas vinblastine secretion is effectively inhibited by 100 microM 1,9-dideoxy-forskolin, volume-stimulated Cl- secretion is unaffected. In contrast, 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) inhibited the volume-stimulated Cl- secretion, but was without effect upon transepithelial vinblastine secretion. In addition, it was noted that some epithelial layers failed to express a volume-stimulated Cl- secretion but maintained a normal level of secretory vinblastine flux.

H(+)-coupled (Na(+)-independent) proline transport in human intestinal (Caco-2) epithelial cell monolayers.

Previously, absorption of L-proline across the apical membrane of the intestinal enterocyte has been attributed to transport via the Na(+)-dependent Imino system. However, net (absorptive) transport of proline across intact Caco-2 cell monolayers was enhanced by acidification of the apical environment, under both Na(+)-containing and Na(+)-free conditions. This Na(+)-independent pH-dependent proline flux was associated with H+ flow across the apical membrane as determined by continuous measurement of intracellular pH. H+/proline symport was associated with an inward Isc in voltage-clamped Caco-2 epithelial layers demonstrating the electrogenic nature of this transport process. In conclusion Caco-2 cells possess an apically-localised, Na(+)-independent, electrogenic H+/imino acid transporter which may play an important role in intestinal proline absorption.

The effect of E. coli STa enterotoxin on the absorption of weakly dissociable drugs from rat proximal jejunum in vivo.

1. The effect of E. coli heat stable (STa) enterotoxin on the absorption of radio-labelled weak electrolytes and their appearance in peripheral blood was assessed in vivo by use of an intestinal recirculation procedure. 2. STa reduced the luminal disappearance (P less than 0.02) and peripheral blood appearance (P less than 0.02) of label from salicylic acid as well as the luminal disappearance (P less than 0.02) of diphenylhydantoin. 3. In contrast, STa increased the appearance in peripheral blood and disappearance from the lumen of label from morphine (P less than 0.05), amphetamine (P less than 0.01) and lignocaine (P less than 0.01). 4. Increased weak base (lignocaine) absorption can also be achieved by a combination of forskolin and theophylline which resembles STa in its ability to neutralise the usually acid surface pH of the proximal jejunum. 5. Increased weak base absorption and hindered weak acid absorption occurs despite a uniform reduction in net fluid absorption after STs exposure, making it unlikely that variations in fluid absorption account for the variations in drug absorption. 6. The ability of STa to elevate the mucosal surface pH (or acid microclimate) to neutral values, thereby altering the proportion of uncharged weak-electrolyte, may explain its different effects on weak acids and bases: neutralisation of the acid microclimate would increase the amount of undissociate weak base available for uptake.

A combined TDDA-PVC pH and reference electrode for use in the upper small intestine.

Incorporation of a ligand into polyvinylchloride allows the production of ion selective electrodes that are soft-bodied and disposable. With features make them especially suitable for clinical, particularly gastroenterological, investigations. We report here on the construction of a combined reference and pH electrode suitable for use at jejunal biopsy. With this type of pH electrode, the pH of the mucosal surface of the jejunum in UK and in Indian subjects, without evidence of upper gastrointestinal disease, was about pH 6.0. This was almost identical to previous values measured using a separate reference electrode. Both the polymer electrode and a suitable data logger can be conveniently produced in the laboratory and compare favourably with commercially available systems, provided the range of pH likely to be encountered is within the operating range of the incorporated ligand.

Direct measurement of mucosal surface pH of pig jejunum in vivo.

The mucosal surface pH of pig jejunum was measured in vivo. When the pH in the bulk phase of the perfusing buffer solution was 7.10, the pH at the mucosa (as measured by a miniaturised glass pH electrode) was 6.19 +/- 0.04 (n = 19). This relatively acidic mucosal surface pH is not an anoxic artefact since anoxia resulted in a significant alkalinisation of the mucosa. This finding, that the pig, like man and the rat, has a jejunal "acid microclimate" has important implications for weak electrolyte absorption from the upper small intestine of this species as well as for any other pH dependent brush border process.

Effect of antisecretory factor on Escherichia coli STa enterotoxin-induced alkalinisation of pig jejunal acid microclimate.

The effect of challenge by Escherichia coli STa enterotoxin on pig jejunal mucosal surface pH was investigated in vivo. Exposure to STa resulted in a rapid and reversible alkalinisation (P less than 0.001) of the jejunal mucosa from 6.27 +/- 0.11 (5) to 6.89 +/- 0.03 (5). This action of STa is probably mediated through cyclic 3'5'-guanosine monophosphate (cGMP) since the 8-bromo analogue of cGMP induced the same effect as that observed after STa challenge. The action of STa on mucosal pH was partially inhibited by pre-administration of an antisecretory factor (ASF) preparation. The action of 8-bromo cGMP was unchanged by the presence of ASF. This implies that ASF inhibition occurs during the early stages of STa action prior to stimulation of guanylate cyclase. This effect of STa on the pig jejunal mucosal surface pH, or acid microclimate, may explain why weak acid supplementation of oral rehydration solutions can be ineffective in certain types of diarrhoeal disease.

Influence of age on antisecretory factor inhibition of enterotoxin action in the pig small intestine.

The effect of age on antisecretory factor (ASF) inhibition of cholera toxin (CT) and E. coli STa enterotoxin-induced fluid secretion in pig jejunum was investigated in vivo. Comparison was made between 2 week and 8 week old animals. ASF inhibited (P less than 0.05) CT-induced fluid secretion by up to 90% in the 8 week animals (from 18.4 +/- 5.87 mg/mg loop dry weight to 0.74 +/- 0.54 mg/mg loop dry weight). There was no effect of ASF on CT-secretion in the 14 day pigs suggesting that there is a minimum age before ASF is effective. ASF had no significant effect on net fluid transport after STa challenge in pigs from either age group. However, the predominant action of STa was to inhibit absorption and this would not be affected by ASF.

Effects of propionate on the acid microclimate of hen (Gallus domesticus) colonic mucosa.

1. Short-chain fatty acid absorption in hen colon is protonated across the apical border coupled to an apical electrogenic proton pump. 2. The surface pH of the isolated colonic epithelium was 6.27 +/- 0.05, when incubated in Krebs-phosphate buffer pH 7.0. 3. Propionate 7 and 40 mmol/l in the incubation medium (pH 7.0) increased microclimate pH to 6.47 +/- 0.04 and 6.56 +/- 0.04. Inhibition of metabolic activity by potassium cyanide 1 mmol/l increased surface pH to 6.66 +/- 0.06. 4. The calculated concentration of propionic acid in the microclimate is near-linearly related to the propionate concentration. Thus, the acid microclimate is not responsible for the Michaelis-Menten like kinetics of propionate transport.

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.

We determined the extent of Na(+)-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na(+)-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na(+)-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, beta-alanine, alpha-aminoisobutyric acid (AIB), alpha-methylaminoisobutyric acid (MeAIB), tau-amino-n-butyric acid and L-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both D-serine and D-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and D-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (Isc) in voltage-clamped Caco-2 cell monolayers in Na(+)-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, D-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pHi and inward Isc. In conclusion, Caco-2 cells express a Na(+)-independent, H(+)-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.

Transepithelial dipeptide (glycylsarcosine) transport across epithelial monolayers of human Caco-2 cells is rheogenic.

Net transepithelial transport (and cellular accumulation) of the dipeptide glycylsarcosine (Gly-Sar), across the apical membrane of human intestinal Caco-2 epithelia, is driven by a proton gradient (Na(+)-free conditions) and displays saturation kinetics (Km 17.4 +/- 5.1 mM, Vmax of 92.8 +/- 15.6 nmol.cm-2.h-1). Net Gly-Sar transport is associated with the stimulation of an inward short-circuit current (Isc). This dipeptide-stimulated Isc is observed in both Na(+)-containing and Na(+)-free conditions, is stimulated by apical acidity, and displays saturation kinetics (in Na(+)-free media at apical pH 6.0, Km of 13.6 +/- 4.5 mM and a Vmax of 284.1 +/- 39.3 nmol.cm-2.h-1). The maximal capacities of Gly-Sar transport and Isc suggest a dipeptide/proton stoichiometry greater than unity (1:3).

The effect of Escherichia coli STa enterotoxin and other secretagogues on mucosal surface pH of rat small intestine in vivo.

The mucosal surface pH of rat small intestine was measured in vivo. The surface pH in the normal jejunum was 6.20 +/- 0.02 (67) and 7.00 +/- 0.05 (5) in the ileum. Escherichia coli STa toxin induced a rapid and reversible alkalinization of both jejunal and ileal mucosae to a pH of 6.91 +/- 0.08 (10) and 7.67 +/- 0.06 (5) respectively. The synthetic ST analogue, STh-(6-19), had an effect identical to native STa toxin on jejunal surface pH. Theophylline (20 mM) maintained the STa-elevated jejunal surface pH after toxin removal but had no effect on untreated tissue. 8-Bromo cyclic GMP resembled STa by causing similar mucosal alkalinization in the jejunum; 8-bromo cyclic AMP, forskolin and cholera toxin individually had considerably smaller effects on surface pH, although combining forskolin or cholera toxin with theophylline resulted in alkalinization of the jejunal mucosa to a pH of 6.92 +/- 0.03 (5) and 6.76 +/- 0.04 (4). These results indicate that cyclic-GMP-dependent secretory processes are more capable of inducing surface pH changes than those dependent on cyclic AMP. The ability of STa to alter mucosal surface pH makes it a useful tool to investigate the microclimate hypothesis for weak electrolyte absorption.

Characterisation of volume-activated ion transport across epithelial monolayers of human intestinal T84 cells.

The effects of hypo-osmolarity upon transepithelial ion transport in human intestinal cell layers have been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (Isc). This transient stimulation of inward current by hypo-osmotic media was abolished by 100 mumol/l 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). After prestimulation of inward Isc by vasoactive intestinal peptide (VIP) or by combinations of carbachol and prostaglandin E1, hypo-osmotic exposure of the basal-lateral surfaces resulted in a further transient stimulation of Isc. The stimulation of Isc in these conditions was largely insensitive to DIDS inhibition. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a stimulation of loop-diuretic-insensitive 86Rb efflux across the basal-lateral surfaces. In addition, hypo-osmotic exposure of T84 cells is also associated with an increase in cytosolic Ca2+. It is concluded that the effects of hypo-osmotic exposure of T84 cells on secretory Isc are consistent with the activation of a DIDS-sensitive apical Cl- conductance and a basal-lateral K+ conductance. With prior activation of inward Isc by VIP via a cAMP-activated DIDS-insensitive apical Cl- conductance, augmentation of the secretory current by hypo-osmotic exposure is likely to result primarily from increased basal-lateral K+ current and loop-diuretic-sensitive Cl- uptake.

Effect of age on the secretory capacity of pig small intestine in vivo and in vitro.

The effect of age on the secretory response of pig small intestine to in vivo challenge by cholera toxin (CT) was investigated. The small intestine of 14-day-old pigs was more sensitive to CT challenge than that of 14-wk-old animals. In the 14-day jejunum CT-induced fluid secretion was five times that observed in the 14-wk tissue. Similarly, the 14-day ileum produced a fourfold higher secretion than the 14-wk ileum, although the magnitude of ileal secretion was markedly lower than that observed in the jejunum at the same CT dose. This reduced response to CT with age was not due to a reduced secretory capacity of the tissue, since supramaximal doses of prostaglandin E2 and theophylline induced a similar response in tissue from both age groups in vitro. We conclude that these results are consistent with the hypothesis that an antisecretory factor, which naturally inhibits fluid losses in enterotoxigenic diarrhea, is produced in older animals.

Stimulation of three distinct guanylate cyclases induces mucosal surface alkalinisation in rat small intestine in vitro.

The absorptive surface of the small intestine is isolated from bulk pH changes in the luminal contents by a zone of maintained low pH, the acid microclimate. The present study set out to compare the effects of stimulation of each of the three guanylate cyclases (GCs) expressed in the intestinal mucosa on the pH microclimate of rat jejunum in vitro. The tissue was exposed to specific ligands for each of the GCs and mucosal surface pH determinations were made by a miniaturised glass pH electrode. The ligands used were E. coli STa enterotoxin, atrial natriuretic peptide (ANP) and nitric oxide (NO, via the donor sodium nitroprusside (SNP)). Challenge from all three agonists resulted in significant alkalinisation of the jejunal mucosa. The actions of SNP were blocked by the soluble GC inhibitor, methylene blue (MB) whereas those of STa were unaffected by MB. The data are consistent with previous observations that cGMP-induced inhibition of brush border Na+/H+ exchange results in elevation of mucosal surface pH. We conclude that all three of the identified GC pathways in the intestinal mucosa are capable of contributing to the control of mucosal acidification in the upper small intestine.