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N L Simmons

Publications and source records attributed to N L Simmons.

At least 73 records · Page 4Linked to original sources
PubMed1993

Direct assessment of dipeptide/H+ symport in intact human intestinal (Caco-2) epithelium: a novel method utilising continuous intracellular pH measurement.

Direct demonstration of intact peptide transport by the intestinal H+/dipeptide carrier is limited both by luminal/cytosolic hydrolysis and the availability of suitable radiolabelled substrates. Perfusion of Val-Val (20mM) at the apical surface of human intestinal epithelial (Caco-2) cell monolayers resulted in a marked intracellular acidification, due to dipeptide-induced H(+)-flow across the apical membrane. Val-Val (20mM) also inhibited both the pH-dependent apical-to-basal transport and intracellular accumulation of [14C]Gly-Sar. Valine (20mM) had no effect on [14C]Gly-Sar transport (and intracellular accumulation) or pHi. We conclude that this novel method for studying H(+)-coupled transport clearly differentiates between the mechanisms responsible for absorption of an intact substrate and products of hydrolysis.

Biological Transport↗
PubMed1993

Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators.

The functional expression of P-glycoprotein has been studied in confluent epithelial layers of human Caco-2 cells, a polarized, highly differentiated cell line demonstrating an intestinal absorptive cell phenotype. Expression of P-glycoprotein was localized, by indirect immunofluorescence with monoclonal antibody MRK16, to the apical brush-border, approximately 20 microns above the base of the cells. Functional, high capacity expression of P-glycoprotein in Caco-2 cell layers was demonstrated by the saturable secretion of vinblastine, a typical substrate, from basolateral to apical surfaces: Km 18.99 +/- 5.55 microM, Vmax 1285.9 +/- 281.2 pmol.cm-2 h-1. The direct correlation of apical P-glycoprotein expression with vinblastine net secretory flux was demonstrated by the reduction of this flux after treatment with MRK16 antibodies. Vinblastine secretory flux was also reduced by treatment with verapamil (R- and S-isomers with equal affinity), nifedipine, taxotere, and 1,9-dideoxyforskolin. Kinetic analyses suggest that the inhibition of vinblastine secretory flux by verapamil and nifedipine was competitive, while that by dideoxyforskolin was non-competitive, in nature. The polarized expression and activity of P-glycoprotein in Caco-2 cells is direct evidence for its secretory detoxifying function in the intestine, subserving at least one role of the gastrointestinal epithelial barrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

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.

Actins↗
PubMed1993

Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by expression of H(+)-coupled carriers at both apical and basal membranes.

Glycylsarcosine (Gly-Sar) transport in human intestinal epithelial (Caco-2) cells has been investigated. Gly-Sar transport, from apical to basal surfaces (Ja-b), and intracellular accumulation are greatest when the apical medium is acidified (apical pH 6.0, basal pH 7.4). Both transport and accumulation are susceptible to saturation and competition. Similarly, Gly-Sar transport, from basal to apical surfaces (Jb-a), is increased with acidification of the basal medium (basal pH 6.0, apical pH 7.4). Apical addition of 20 mM Gly-Sar (pH 6.0) to Caco-2 cell monolayers loaded with the pH indicator BCECF (2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein) caused a marked cytosolic acidification. Basolateral application of 20 mM Gly-Sar (pH 6.0) also caused a fall in intracellular pH. These observations are consistent with the expression of H(+)-coupled dipeptide transporters at both membrane faces of the human intestinal epithelial Caco-2 cell line. We also provide direct evidence for dipeptide-stimulated H(+)-influx, across both apical and basolateral membranes, in this intact epithelial cell system.

Adenocarcinoma↗
PubMed1993

Epithelial M cells in the rabbit caecal lymphoid patch display distinctive surface characteristics.

The follicle-associated epithelium (FAE) in the rabbit caecal lymphoid patch is characterized by the presence of membranous (M) cells, which are believed to be functionally equivalent to those present at other sites of gut-associated lymphoid tissue (GALT). Caecal patch M cells display distinctive features compared with those of other GALT sites, despite similar general morphology and expression of the M cell marker vimentin, suggesting marked heterogeneity in the apical surface of M cells at discrete GALT sites. Electron microscopy reveals that rabbit caecal patch M cells differ from those in the small intestinal Peyer's patch FAE: the former have a prominent aspect within the epithelium and possess microvilli which are longer than those of adjacent enterocytes. Many of the M cells in peripheral regions of the caecal patch FAE are not associated with leucocytes and may thus represent an immature M cell population. The M cells are also histochemically distinct from adjacent enterocytes and from Peyer's patch M cells, showing greater expression of brush-border alkaline phosphatase activity and affinity for certain lectins (peanut and wheat germ agglutinins, Bandeiraea simplicifolia agglutinin II). The differences in the brush-border morphology and glycocalyx structure between M cells at different GALT sites may affect their function at these sites by influencing the interaction of luminal antigens and microorganisms with the M cell surface. The present data also support the hypothesis that M cells arise directly from differentiation of crypt stem cells and not from the transformation of existing fully differentiated enterocytes.

Alkaline Phosphatase↗
PubMed1993

Identification of M cells and their distribution in rabbit intestinal Peyer's patches and appendix.

The distribution of intestinal membranous (M) cells has been studied within the follicle-associated epithelium of rabbit Peyer's patches and appendix. Vimentin expression has been assessed as a primary criterion to identify rabbit M cells in tissue sections and in whole tissue preparations. This criterion has been compared to the use of the absence of alkaline phosphatase which, due to its heterogeneous distribution within the enterocyte population, is less reliable than vimentin expression as a marker for rabbit M cells. The pattern of vimentin immunostaining revealed that the majority of M cells are located in the periphery of the follicle-associated epithelium, the dome apex being largely free of M cells. This distribution was confirmed by scanning electron microscopy. Vimentin is also expressed by follicle-associated epithelial cells in the vicinity of crypts which lack the typical lymphocyte-containing pocket of M cells. Cytoplasmic peanut agglutinin binding coincides with vimentin-expression throughout the follicle-associated epithelium but is absent from vimentin-negative enterocytes. The co-localisation of these two phenotypic markers in both M cells and epithelial cells adjacent to crypts, which lack the typical morphology of fully developed rabbit M cells, suggests that they correspond to immature M cells which by their location appear to derive directly from undifferentiated crypt stem cells and not from mature columnar enterocytes.

Alkaline Phosphatase↗
PubMed1993

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

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).

Biological Transport↗
PubMed1993

Co-expression of an anion conductance pathway with Na(+)-glucose cotransport in rat renal brush-border membrane vesicles.

Brush-border membrane vesicles were prepared from superficial rat renal cortex by a Mg(2+)-precipitation technique. The initial (20 s) [14C]glucose uptake rate from solutions containing 100 mmol/l Na (salt) was found to be dependent upon the anion composition of the medium; in comparison to gluconate-containing medium (0.46 +/- 0.05 nmol/mg protein), Cl- accelerated the initial rate to 1.47 +/- 0.21 nmol/mg protein (n = 4 preparations, +/- SEM). This enhancement was reduced by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.5 mmol/l), but was unaffected by 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS, 0.5 mmol/l). When membrane vesicles were pre-equilibrated with 100 mmol/l K (salt) and 100 mmol/l mannitol and glucose uptake was measured from a solution containing 100 mmol/l Na gluconate and 100 mmol/l mannitol in the presence of 80 mumol/l valinomycin (to generate an outward K+ diffusion electrical p. d.), it was found that intravesicular KCl depressed the initial glucose uptake compared to K gluconate. NPPB (0.5 mmol/l) increased the initial glucose uptake with intravesicular KCl towards values seen in K gluconate vesicles. In conditions where the only driving force for glucose uptake was established by an inward anion gradient (Nao = Nai) it was found that inward Cl- gradients could drive uphill glucose transport and that this was sensitive to NPPB (0.5 mmol/l), but insensitive to DIDS. We conclude that a Cl- conductance co-exists with Na-cotransport in rat renal brush-border membrane vesicles prepared from superficial renal cortex and this may function to regulate the activity of electrogenic transport systems at this membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Selective binding and transcytosis of latex microspheres by rabbit intestinal M cells.

The interaction between polystyrene microspheres and the follicle-associated epithelium of rabbit Peyer's patches has been examined. Microspheres bind selectively to, and are transcytosed by, membranous or microfold (M) cells within the follicle-associated epithelium. M cells are able to transport, in 45 min, approximately 10(5) microspheres of 0.46 micron diameter across the epithelium overlying each lymphoid follicle dome of rabbit Peyer's patches. The high capacity of M cells for particulate transcytosis and the subsequent delivery of these particulates to the mucosal immune system highlights the potential importance of this portal in the delivery of antigens and drugs. In addition, the selective binding and uptake of microspheres may be utilised as a functional marker in the identification and isolation of M cells.

Animals↗
PubMed1993

Passive transepithelial absorption of thyrotropin-releasing hormone (TRH) via a paracellular route in cultured intestinal and renal epithelial cell lines.

Transport studies using intestinal brush-border membrane vesicles isolated from rats and rabbits have failed to demonstrate proton- or Na(+)-dependent carrier-mediated transport of thyrotropin-releasing hormone (TRH), despite a pharmacologically relevant oral bioavailability. To examine the hypothesis that reported levels of oral bioavailability reflect predominantly a paracellular rather than transcellular route for transepithelial transport of TRH, we have studied TRH transport in cultured epithelial cell types of intestinal (Caco-2 and T84) and renal (MDCK I, MDCK II, and LLC-PK1) origin, whose paracellular pathways span the range of permeability values observed in natural epithelia. Transport of TRH across monolayers of intestinal Caco-2 cells was similar to the flux of mannitol (approximately 1-4% per 4 hr), and unlike other putative substrates for the di-/tripeptide carrier, apical-to-basolateral transport was not increased by the presence of an acidic pH in the apical chamber. TRH transport did not show saturation, being uneffected in the presence of 20 mM cold TRH. In each cell type studied TRH and mannitol transport were similar and positively correlated with the conductance of the cell layers, consistent with a passive mechanism of absorption. This evidence suggests that, providing that a peptide is resistant to luminal hydrolysis, small but pharmacologically significant amounts of peptide absorption may be achieved by passive absorption across a paracellular route.

Absorption↗
PubMed1993

Drug absorption limited by P-glycoprotein-mediated secretory drug transport in human intestinal epithelial Caco-2 cell layers.

The hypothesis was tested that the operation of an ATP-dependent export pump localized at the apical (brush border) surface of the intestinal epithelium may limit substrate absorption kinetics. Human intestinal Caco-2 cell-layers display saturable secretion of vinblastine from basal to apical surfaces (Km, 18.99 +/- 5.55 microM; Vmax, 1285.9 +/- 281.2 pmol cm-2 hr-1) that is inhibited by verapamil, consistent with the expression of the ATP-dependent P-glycoprotein drug efflux pump at the apical brush border membrane. Inhibition of P-glycoprotein by a variety of modulators (verapamil, 1,9-dideoxyforskolin, nifedipine, and taxotere) is associated with an increased vinblastine absorptive permeability. Vinblastine absorption displayed a nonlinear dependence upon luminal (apical) vinblastine concentration, and vinblastine absorption increased markedly at concentrations where vinblastine secretory flux was saturated (> 20 microM). Upon inhibition of P-glycoprotein by verapamil and 1,9-dideoxyforskolin, vinblastine absorption increased and was linearly dependent on vinblastine concentration. The limitation of P-glycoprotein substrate absorption by active ATP-dependent export via P-glycoprotein is discussed, together with the possibility that other classes of substrate may be substrates for different ATP-dependent export pumps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Thyrotropin-releasing hormone (TRH) uptake in intestinal brush-border membrane vesicles: comparison with proton-coupled dipeptide and Na(+)-coupled glucose transport.

The mechanism of thyrotropin-releasing hormone (pGlu-His-Pro-NH2; TRH) uptake across the luminal membrane of intestinal enterocytes was investigated using brush-border membrane vesicles (BBMV) from rabbit duodenum and jejunum and rat upper small intestine. [14C]Glucose accumulated within the intestinal vesicles (at 10 sec), in the presence of an inwardly directed Na+ gradient, 7- to 14-fold higher than equilibrium values (65 min). The vesicles also accumulated the dipeptide [14C]Gly-Sar. Dipeptide uptake was greatest in the presence of both an inwardly directed proton gradient and an inside negative membrane potential. The H(+)-dependent Gly-Sar transport was not affected by the presence of an excess (46-fold) of cold TRH. In contrast to the observations with glucose and Gly-Sar, the uptake of [3H]TRH after 10 or 60 sec (in each of the vesicle preparations) was not enhanced by either Na+ or H+ gradient conditions. The absence of vesicular accumulation of TRH was not due to peptide hydrolysis. For example, after a 60-sec incubation with rabbit jejunal BBMV no degradation of the tripeptide was evident. After 65 min, 6% of [3H]TRH had undergone degradation, by deamidation, to form TRH-OH. These studies provide no evidence for the oral absorption of TRH by a Na(+)- or H(+)-dependent carrier system in the brush-border membrane. Previous observations of TRH absorption in vivo may be accounted for by passive absorption of the peptide combined with its relative resistance to luminal hydrolysis.

Animals↗
PubMed1993

Active secretion of the fluoroquinolone ciprofloxacin by human intestinal epithelial Caco-2 cell layers.

The bidirectional transepithelial fluxes of ciprofloxacin, an antibacterial fluoroquinolone, across the human intestinal epithelial Caco-2 cell-line show marked asymmetry. Basal-to-apical flux of ciprofloxacin (10 microM) exceeds apical-to-basal flux indicating net secretion. Net ciprofloxacin secretion is abolished by azide/2-deoxy-D-glucose treatment, displays saturation kinetics (Km = 0.89 +/- 0.23 mM, Vmax 44.3 +/- 4.9 nmol cm-2.h) and competition by other fluoroquinolones. A specific, active secretion in Caco-2 epithelia may explain the transintestinal elimination of ciprofloxacin observed in pharmacokinetic studies in man.

Azides↗
PubMed1993

Actin accumulation at sites of attachment of indigenous apathogenic segmented filamentous bacteria to mouse ileal epithelial cells.

Selective attachment of indigenous, apathogenic, segmented filamentous bacteria to follicle-associated epithelia of terminal ileal Peyer's patches involves both the specialized M cells and normal enterocytes. A striking, highly localized cytoplasmic accumulation of polymerized actin, which resembles that associated with adherence of enteropathogenic Escherichia coli to cultured epithelial cells, occurs at attachment sites of segmented filamentous bacteria. The epithelial response to the attachment of microorganisms may thus involve mechanisms which are the same for diverse microorganisms.

Actins↗
PubMed1993

Differential expression of lectin-binding sites defines mouse intestinal M-cells.

We investigated the binding of four lectins to the follicle-associated epithelium (FAE) overlying fixed mouse small intestinal Peyer's patches to identify M-cell-specific surface markers. Wheat germ agglutinin and peanut agglutinin displayed heterogeneous staining patterns, binding most avidly to the intestine goblet cells. In contrast, the lectins Ulex europaeus 1 (UEA 1) and Psophocarpus tetragonolobus (winged bean; WBA) were almost exclusively M-cell specific. When confocal laser scanning images of tissues stained with fluorescein isothiocyanate (FITC)-conjugated UEA1 or WBA were compared with the appearance of the same tissues under the scanning electron microscope (SEM), UEA1 strongly stained 97.2% (106/109) of M-cells, 0.6% (3/516) enterocytes, and 0% (0/28) goblet cells, whereas WBA stained 100% (83/83) M-cells, 1.7% (6/361) enterocytes, and 5.3% (1/19) goblet cells. The M-cell specificity of the lectin binding was further demonstrated by localization of horseradish peroxidase (HRP)-conjugated lectins under the transmission electron microscope (TEM). This is the first demonstration of carbohydrates in the glycocalyx of M-cells that are not expressed elsewhere on the FAE surface. These carbohydrates not only provide a means to identify mouse M-cells by LM but may also contribute to the occurrence of specific interactions between microorganisms and the M-cell apical membrane.

Animals↗
PubMed1993

Comparison of poly(DL-lactide-co-glycolide) and polystyrene microsphere targeting to intestinal M cells.

The interaction of poly(DL-lactide-co-glycolide) (PLG) and polystyrene microspheres with the follicle-associated epithelium (FAE) of rabbit Peyer's patches was compared. Binding by PLG microspheres to the FAE was an order of magnitude lower than that of polystyrene microspheres of equivalent size (0.5-0.6 microns diameter). Although PLG microspheres are not selectively targeted to the M cell surface, as is the case with polystyrene microspheres, a high proportion of those that bind to M cells are transcytosed, resulting in the transepithelial delivery of 1.5 x 10(4) PLG microspheres/mm2 FAE. This represents the first direct demonstration of transepithelial delivery of PLG microspheres by M cells, which is crucial to the potential use of such vehicles for the oral delivery of drugs and vaccines. As native PLG microspheres are not optimally targeted to the M cell surface, there is scope for the further improvement of their efficacy by surface modifications.

Animals↗