Transepithelial secretion, cellular accumulation and cytotoxicity of vinblastine in defined MDCK cell strains.

Transepithelial vinblastine secretion in two defined MDCK strains displays saturation kinetics; (Strain 1) Km = 2.8 +/- 0.6 microM (six experiments), Vmax 35.9 +/- 1.93 pmol/cm2 per h (six experiments), Strain 2 Km 0.78 +/- 0.36 microM (three experiments), Vmax 12.1 +/- 4.5 pmol/cm2 per h (three experiments). Concentrations of vinblastine > 1 microM are associated with an increased passive vinblastine permeability (PA-B). This correlates with an increased transepithelial conductance/decreased permselectivity, suggesting that this may in part result from increased paracellular conductance. Verapamil inhibits vinblastine secretion, half-maximal inhibition of basal-to-apical flux (JB-A) is observed at 3.4 +/- 0.3 and 1.7 +/- 0.05 microM verapamil for Strain-1 and Strain-2 epithelial layers, respectively. Cellular accumulation of vinblastine across the apical membrane is small with respect to that across the basolateral surfaces. This polarity is unaffected by verapamil. The apical membranes, therefore, possess a low intrinsic permeability to vinblastine. Inhibition of cell growth by vinblastine is enhanced by verapamil. Both the effect of vinblastine, and its enhancement by verapamil, upon cell growth are reduced as initial cell seeding density increases.

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.

H(+)-coupled dipeptide (glycylsarcosine) transport across apical and basal borders of human intestinal Caco-2 cell monolayers display distinctive characteristics.

Transepithelial transport and intracellular accumulation of the dipeptide glycylsarcosine (Gly-Sar) were studied using intact monolayers of the human intestinal epithelial cell line, Caco-2. Gly-Sar transport was demonstrated in both absorptive (apical-to-basal) and secretory (basal-to-apical) directions. In both directions, transport and accumulation were enhanced in the presence of a pH gradient (pHo < pHi). Under conditions similar to those found at the intestinal membrane in vivo (apical pH 6.0, basolateral pH 7.4), net absorption (145.2 pmol/cm2 per h) was observed, although experimental conditions could also be manipulated (apical pH 7.4, basolateral pH 6.0) so that net secretion was observed. Transport and accumulation (in both directions) were inhibited in the presence of either 20 mM (unlabelled) Gly-Sar or 20 mM cephalexin (an aminocephalosporin antibiotic). When added to either the apical or basolateral surface of BCECF (2',7',-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers Gly-Sar (20 mM), at pH 6.0, caused a marked intracellular acidification, demonstrating that dipeptide absorption is accompanied by H(+)-flow into the cells. Cephalexin (20 mM) had similar effects (as Gly-Sar) when presented at the apical surface but also caused a marked intracellular acidification when perfused into the basolateral chamber at pH 7.4. In contrast, addition of Gly-Sar (20 mM) to the basolateral chamber (at pH 7.4) had no effect. Transepithelial absorption of dipeptides (Gly-Sar) and beta-lactam antibiotics (cephalexin) at low concentrations is predominately via a transcellular route mediated by carrier mechanisms located at both apical and basolateral membranes. Interestingly, Gly-Sar and cephalexin transport across the basolateral membrane (and, therefore, exit from the cell) display both common and distinct characteristics suggesting that more than one mechanism may be responsible for exit into the basolateral space.

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.

The use of chimeric gene constructs to express a bacterial endoglucanase in mammalian cells.

The synthesis and secretion of a truncated Clostridium thermocellum endoglucanase (EGE') encoded by the celE' gene was investigated in Chinese hamster ovary (CHO) cells. Fusion genes consisting of the human growth hormone (hGH) gene and celE', transcribed from the SV40 early enhancer/promoter, were constructed and stably transfected into CHO cells. A gene consisting of celE' inserted into the first exon of the hGH gene resulted in the synthesis of truncated proteins (less than or equal to 22 kDa) lacking endoglucanase activity. Cloning celE' into the second exon of the hGH gene, resulted in the synthesis and secretion of a 50 kDa protein with endoglucanase activity. A 50 kDa protein was also synthesised by cells transfected with celE' cloned into the fifth exon of the hGH gene. However, despite a 5-fold increase in enzyme activity compared to the exon 2 transfected cell line less than 40% of the protein was secreted. Constructs devoid of introns, in which celE' was fused to the SV40 early promoter and to the rabbit beta-globin polyadenylation sequence resulted in a 2-18-fold increase in endoglucanase activity compared to the constructs containing introns. In addition more than 75% of the synthesised protein was secreted. Analyses of EGE' encoded mRNA from the transfected cell lines suggests that the presence of introns results in the aberrant splicing of message by the use of cryptic splice sites in the celE' gene. These results demonstrate that introns are not required for the efficient expression of a bacterial endoglucanase in mammalian cells, rather introns appear to reduce expression of the encoded protein.

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.

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.

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.

The rat mucosal mast cell chymase, RMCP-II, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and occludin.

Mucosal mast cells undergo hyperplasia in a variety of inflammatory bowel diseases including nematode infection in man and animals. The intra-epithelial localization of these cells make their soluble mediators prime candidates for modulators of epithelial function. In particular previous in vivo and ex vivo studies have established a link between the release of the highly soluble mast cell granule chymases and increased mucosal permeability. The hypothesis that the rat mast cell protease, RMCP-II, directly increases permeability to macromolecules via the paracellular route is tested in this study. Monolayers of epithelial cells (Madin-Darby canine kidney cell line) were exposed to varying concentrations of RMCP-II in vitro, in the absence of other cell types or mediators, and the effect on permeability and tight junction associated proteins was investigated. Basolateral, but not apical, exposure of polarized MDCK monolayers on porous supports to RMCP-II led to concentration- (> 100 microg/ml) and time-dependent increases in electrical conductance and permeability to mannitol (MW182) and inulin (MW5000), which was accompanied by decreases in the immunostaining of the tight junction-associated proteins occludin and ZO-1. Furthermore, prolonged exposure to RMCP-II (> 12 hours) resulted in the formation of identifiable gaps separating adjacent epithelial cells, in the absence of evidence of cytotoxicity. Inhibition of RMCP-II with Soya bean trypsin inhibitor completely abrogated the response, demonstrating that proteolysis was required. These data provide direct evidence that the rat mast cell chymase RMCP-II can, in the absence of other inflammatory mediators, increase epithelial permeability via an effect on the paracellular route.

Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia.

Polarized monolayers of strain II Madin-Darby canine kidney cells (MDCK II) were treated with vanadate/H2O2, known inhibitors of protein tyrosine phosphatase activity. Vanadate/H2O2 treatment resulted in a rapid increase in paracellular permeability as revealed by decreased transepithelial resistance and increased permeability to inulin. These alterations in epithelial barrier function coincided with increased phosphotyrosine immunofluorescence in the vicinity of intercellular junctions and with redistribution of F-actin, the adherens junction protein E-cadherin and the tight junction protein ZO-1. The effects of vanadate/H2O2 on intercellular junction permeability and protein distribution were completely blocked by the specific protein tyrosine kinase (PTK) inhibitor tyrphostin 25 and partially inhibited by the alternative PTK inhibitor genistein. The relative potency of these two inhibitors in blocking the effects of vanadate/H2O2 on intercellular junctions correlated with their abilities to inhibit tyrosine phosphorylation. The potent ser/thr protein kinase inhibitor staurosporine had only a small influence on the vanadate/H2O2-induced increase in paracellular permeability and did not affect the observed redistribution of intercellular junction proteins or phosphotyrosine immunofluorescence. The relative potencies of these distinct protein kinase inhibitors in reversing the effects of vanadate/H2O2 indicate that these effects are directly related to tyrosine phosphorylation. In conclusion, our data provide evidence that enhanced tyrosine phosphorylation of intercellular junction proteins in MDCK epithelia increases paracellular permeability and can also induce prominent reorganization of the junctional complex.