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N L Simmons

Publications and source records attributed to N L Simmons.

At least 55 records · Page 3Linked to original sources
PubMed1995

Angiotensin-converting enzyme (ACE) inhibitor transport in human intestinal epithelial (Caco-2) cells.

1. The role of proton-linked solute transport in the absorption of the angiotensin-converting enzyme (ACE) inhibitors captopril, enalapril maleate and lisinopril has been investigated in human intestinal epithelial (Caco-2) cell monolayers. 2. In Caco-2 cell monolayers the transepithelial apical-to-basal transport and intracellular accumulation (across the apical membrane) of the hydrolysis-resistant dipeptide, glycylsarcosine (Gly-Sar), were stimulated by acidification (pH 6.0) of the apical environment. In contrast, transport and intracellular accumulation of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were low (lower than the paracellular marker mannitol) and were not stimulated by apical acidification. Furthermore, [14C]-lisinopril transport showed little reduction when excess unlabelled lisinopril (20 mM) was added. 3. pH-dependent [14C]-Gly-Sar transport was inhibited by the orally-active ACE inhibitors, enalapril maleate and captopril (both at 20 mM). Lisinopril (20 mM) had a relatively small inhibitory effect on [14C]-Gly-Sar transport. pH-dependent [3H]-proline transport was not inhibited by captopril, enalapril maleate or lisinopril. 4. Experiments with BCECF[2',7',-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]-loaded Caco-2 cells demonstrate that dipeptide transport across the apical membrane is associated with proton flow into the cell. The dipeptide, carnosine (beta-alanyl-L-histidine) and the ACE inhibitors enalapril maleate and captopril, all lowered intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. However, lisinopril was without effect. 5. The effects of enalapril maleate and captopril on [14C]-Gly-Sar transport and pHi suggest that these two ACE inhibitors share the H(+)-coupled mechanism involved in dipeptide transport. The absence of pH-dependent [14C]-lisinopril transport, the relatively small inhibitory effect on [14C]-Gly-Sar transport,and the absence of lisinopril-induced pHi changes, all suggest that lisinopril is a poor substrate for thedi/tripeptide carrier in Caco-2 cells. These observations are consistent with the greater oral availability and time-dependent absorption profile of enalapril maleate and captopril, compared to lisinopril.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1995

D-cycloserine transport in human intestinal epithelial (Caco-2) cells: mediation by a H(+)-coupled amino acid transporter.

1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.

Amino Acid Transport Systems↗
PubMed1995

Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions.

Short-term infection of MDCK II monolayers with Salmonella typhimurium SL1344 caused a progressive decrease in transepithelial electrical resistance concomitant with decreased cation permselectivity and increased paracellular inulin flux. Cytochemical staining of F-actin, E-cadherin, and ZO-1 revealed the concentration of each junctional protein in invaded cells as a result of contraction at their apical poles and resultant distortion of adjacent uninvaded cells.

Actins↗
PubMed1995

Variations in lectin binding properties of intestinal M cells.

Glycoconjugate expression in follicle-associated epithelia has been examined by application of a panel of lectins to fixed, whole tissue preparations of mouse and rabbit Peyer's and caecal patches. Of those examined, only alpha-L-fucose-specific lectins such as Ulex europaeus agglutinin I bound selectively to M cells in murine Peyer's patches and this selectivity did not extend to mouse caecal patch M cells. In common with a variety of other lectins (wheat germ agglutinin, peanut agglutinin and Bandeiraea simplicifolia agglutinin II), Ulex europaeus agglutinin I exhibited selective binding to M cells in rabbit caecal patch and to enterocytes in rabbit Peyer's patch. The marked regional and species variation in M cell surface characteristics documented here has implications for the normal antigen-sampling function of M cells and to the rational design of particulates for transepithelial delivery via the M cell portal.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

L-alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient.

In human Caco-2 intestinal epithelial layers, L-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na(+)-free media, is associated with a saturable net transepithelial absorption of L-alanine. L-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. L-Alanine transport in Na(+)-free media is rheogenic, stimulating an inward short-circuit current in voltage-clamped epithelial monolayers. By measurement of rapid L-alanine influx across the apical membrane, L-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate L-alanine/proton stoichiometry to be 1:0.62 +/- 0.25 (SD) (short-circuit current) or 1:0.73 +/- 0.19 (intracellular acidification). From competition studies, it is likely that L-proline, alpha-aminoisobutyric acid, and beta-alanine, but not L-valine and L-serine, are substrates for proton-linked, substrate transport in the brush border of Caco-2 cells.

Alanine↗
PubMed1994

Junctional uvomorulin/E-cadherin and phosphotyrosine-modified protein content are correlated with paracellular permeability in Madin-Darby canine kidney (MDCK) epithelia.

Strains I and II of Madin-Darby canine kidney (MDCK) cells, which differ markedly in transepithelial resistance (RT) and paracellular permeability, have been used to investigate whether differences in the cellular content of uvomorulin/E-cadherin and phosphotyrosine may be correlated with junctional properties. Using immunocytochemistry, the strain I "tight" epithelia showed significantly stronger uvomorulin staining at regions of cell-cell contact compared with strain II "leaky" MDCK epithelia. In contrast, strain I MDCK cells showed a relatively faint phosphotyrosine staining, distributed evenly throughout the cytoplasm, while strain II MDCK cells displayed intense staining for phosphotyrosine residues in the junctional region and the lateral cell membrane with additional labelling of the cytoplasm. Exposure to vanadate in conjunction with H2O2 (which are potent inhibitors of protein tyrosine phosphatases) resulted in a dramatic increase in phosphotyrosine staining at the intercellular area and, concomitantly, induced changes in cell morphology, a significant decrease in RT, increase in paracellular inulin permeability, and time-dependent disappearance of uvomorulin from the cell-cell contact sites. Moreover, the effects of vanadate/H2O2 treatment were more dramatic in strain II compared with strain I cells, consistent with greater generation of tyrosine-modified protein in strain II cells. An inverse relationship was demonstrated between membrane-associated uvomorulin/E-cadherin and cellular phosphotyrosine content, which varied between the two strains of MDCK cells and when phosphotyrosine was directly manipulated. These data support the hypothesis that regulation of paracellular permeability may result from specific tyrosine phosphorylation of protein components of the junctional complex.

Animals↗
PubMed1994

Heterogenous Na+, K(+)-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissues.

Na+, K(+)-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissue was measured using indirect immunofluorescence and confocal laser scanning microscopy. All four major sites of aggregated lymphoid tissue, i.e. Peyer's patch, sacculus rotundus, caecal patch and appendix, were studied. Na+, K(+)-ATPase expression was localized to the basolateral surface of cells of the follicle-associated epithelium (FAE) and adjacent villous or surface epithelia (non-FAE), where increased expression during enterocyte migration was evident. In the FAE, expression of Na+, K(+)-ATPase appeared to be lower in the specialized M cells than in enterocytic-type cells, although expression in both cell types was lower than in adjacent non-FAE. Quantification of immunofluorescent staining of Na+, K(+)-ATPase by confocal laser scanning imaging showed a reduction of expression in the FAE to approximately 20-60% relative to that in the adjacent non-FAE. These results are consistent with a primary role of the FAE in mucosal immunity with minimal involvement in active solute absorption.

Animals↗
PubMed1994

Preferential interaction of Salmonella typhimurium with mouse Peyer's patch M cells.

We have used a mouse Peyer's patch gut loop model to investigate the role of the intestinal membranous epithelial (M) cells in the pathogenesis of Salmonella typhimurium. These specialized antigen sampling cells are located in the follicle-associated epithelium (FAE) overlying the isolated and aggregated lymphoid follicles in the small and large intestines. Our studies have demonstrated that S. typhimurium adheres more frequently to the Peyer's patch FAE cells than to the villous enterocytes and that, within the FAE, this bacterium preferentially interacts with the M cells. Quantitative light microscopic studies, using the lectin Ulex europaeus 1 (UEA1) to identify M cells, revealed that 34-fold more bacteria bound per unit area of M cells than per unit area of enterocyte. Within a 30-min incubation period, some M cells had clearly been invaded by the Salmonella. We therefore propose that M cells are a major route by which S. typhimurium penetrates the intestinal epithelial barrier. Bacterial adhesion to M cells occurred in a non-uniform pattern, suggesting the existence of M-cell subtypes. The interaction of S. typhimurium with mouse Peyer's patch M cells was accompanied by membrane ruffle formation and polymerized actin redistribution similar to that observed in cultured cell lines infected by this bacterium. This study emphasizes the suitability of Salmonella as an oral vaccine delivery system since, by preferentially interacting with the M cells, these bacteria are targeted to sites where cells of the immune system are concentrated.

Animals↗
PubMed1994

Substrate specificity of the di/tripeptide transporter in human intestinal epithelia (Caco-2): identification of substrates that undergo H(+)-coupled absorption.

1. pH-dependent transepithelial transport and intracellular accumulation of the hydrolysis-resistant dipeptide glycylsarcosine (Gly-Sar) have been demonstrated in the model human intestinal epithelial cell line, Caco-2. 2. Experiments with BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cells demonstrated that dipeptide (Gly-Sar) transport across the apical membrane is coupled to proton flow into the cell. 3. A range of postulated substrates for the intestinal di/tripeptide carrier were tested for their abilities to: (a) inhibit pH-dependent [14C]Gly-Sar apical-to-basal transport and intracellular accumulation and (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers. 4. A range of compounds (including Gly-Gly, Leu-Leu, Gly-Gly-Gly, cefadroxil and cephalexin) caused marked acidification of intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. In contrast leucine and D-Leu-D-Leu failed to induce proton flow. The ability to induce proton-flow across the apical surface by these compounds, in this intestinal epithelium, was directly correlated to the relative inhibitory effects on [14C]-Gly-Sar transport and accumulation. 5. The determination of substrate-induced intracellular pH change in the Caco-2 cell system may provide a useful rapid screen for candidate substrates for absorption via H(+)-coupled transport mechanisms such as the intestinal di/tripeptide carrier in an appropriate physiological context.

Biological Transport↗
PubMed1994

Differential surface characteristics of M cells from mouse intestinal Peyer's and caecal patches.

The mouse caecal patch is located near the blind end of the caecum, and consists of a group of lymphoid follicles. In common with the Peyer's patches, the follicle-associated epithelium overlying these follicles is largely composed of enterocytes, goblet cells and membranous epithelial (M) cells. Each of these types of cell was readily identified by electron microscopy, although caecal patch enterocytes and M cells were morphologically distinct from those of the Peyer's patches. Staining for alkaline phosphatase activity demonstrated that the majority of caecal follicle-associated epithelial cells were alkaline phosphatase-negative, positive cells consisting of a mixture of enterocytes and M cells. In contrast, it has previously been found that Peyer's patch enterocytes are positive for alkaline phosphatase while the M cells are relatively lacking in alkaline phosphatase activity. Lectin histochemistry revealed that surface glycoconjugate expression differs between the caecal and Peyer's patch follicle-associated epithelial cells; in particular, the characteristic staining of Peyer's patch M cells by Ulex europaeus agglutinin 1 was absent on the caecal patch follicle-associated epithelium. These altered surface characteristics indicate that the development of the caecal patch follicle-associated epithelial cells is influenced by the local environment, and these altered properties may be indicative of modified functional roles for the cells at this site.

Alkaline Phosphatase↗
PubMed1994

Active intestinal secretion of the fluoroquinolone antibacterials ciprofloxacin, norfloxacin and pefloxacin; a common secretory pathway?

The transepithelial transport of three fluoroquinolones, norfloxacin, ciprofloxacin and pefloxacin has been compared by using cultured human intestinal Caco-2 cell-layers. Absorptive (apical-basal) fluxes of ciprofloxacin and norfloxacin are small relative to basal to apical fluxes. Norfloxacin and ciprofloxacin are thus subject to active transepithelial secretion. Active net secretion of norfloxacin displays saturation kinetics with Vmax and Km values of 36.2 +/- 6.9 nmol.cm-2.hr-1 and 1.42 +/- 0.79 mM. In contrast, transepithelial pefloxacin fluxes are large, show marked saturation while the direction of net flux is variable and small relative to transepithelial fluxes. Norfloxacin, pefloxacin and ciprofloxacin are all subject to accumulative transport across the basal surface of Caco-2 cell layers. A number of 4-quinolones and fluoroquinolones are capable of inhibition of both net secretion of ciprofloxacin and cellular accumulation across the basal-lateral cell surface. Cinoxacin, a 4-quinolone may selectively inhibit exit from the cell across the apical membrane. Cross-competition studies suggest that fluoroquinolones may compete for a common carrier at the basal-lateral membrane. It is likely that the mechanism of transepithelial secretion involves a common accumulative transport at the basal-lateral membrane followed by facilitated exist across the apical membrane. Pefloxacin may interact with a brush-border carrier for which norfloxacin and ciprofloxacin are poor substrates, enhancing the absorptive flux of this fluoroquinolone.

Biological Transport↗
PubMed1993

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.

Biological Transport↗
PubMed1993

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

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.

Biological Transport↗
PubMed1993

Na(+)-independent, H(+)-coupled transepithelial beta-alanine absorption by human intestinal Caco-2 cell monolayers.

beta-Alanine transport across intact human intestinal epithelial (Caco-2) cell layers has been investigated. In Na(+)-containing solutions, net absorptive flux of beta-alanine from apical-to-basal surfaces is small or absent, despite Na(+)-dependent intracellular beta-alanine accumulation across both apical and basal surfaces. Upon apical acidification (apical pH 6.0, basal pH 7.5), beta-alanine absorptive flux and accumulation across the apical surface are increased. In Na(+)-free conditions, a significant absorptive flux of beta-alanine is observed, which is markedly stimulated upon apical acidification (pH 6.0). Cellular accumulation of beta-alanine across the apical but not basal surface is observed in Na(+)-free conditions, and this is increased by acidic (pH 6.0) solutions. Absorptive beta-alanine flux in Na(+)-free conditions with acidic apical solutions displays saturation kinetics and competitive inhibition by alanine and glycine, but not valine or serine. Addition of 20 mM beta-alanine to the apical solution of epithelial monolayers loaded with the pH indicator 2',7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein) causes a marked decrement in intracellular pH. beta-Alanine transport is also electrogenic, a concentration-dependent increase in an inward short circuit current being observed in voltage-clamped epithelial monolayers. We conclude that a proton-dependent, but Na(+)-independent, amino acid transporter is expressed at the apical membrane of human intestinal Caco-2 cells, and we provide direct evidence for amino acid-stimulated proton influx across the apical membrane in this intact epithelial cell system.

Biological Transport↗