Nicholas L. Simmons

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 µM; 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 µM). 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.

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

We have used a mouse Peyers 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 Peyers 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 Peyers 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.

Substrate upregulation of the human small intestinal peptide transporter, hPepT1

1 Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl‐L‐glutamine (Gly‐Gln) of the proton‐coupled peptide transporter, hPepT1, in the Caco‐2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl‐[14C]sarcosine was increased 1.64 (± 0.34)‐fold after incubation of Caco‐2 cells for 3 days in a peptide‐rich medium (4 mM Gly‐Gln replacing 4 mM Gln). 2 A full‐length Caco‐2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile‐662 to Val. Transcript sizes, on Northern blots of Caco‐2 poly(A)+ RNA probed with a 630 bp 5′ hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide‐induced increase in substrate transport was accompanied by a parallel increase of 1.92 (± 0.30)‐fold (n= 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half‐life from 8.9 ± 1.1 to 12.5 ± 1.6 h (n= 3), but the increase in half‐life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide‐mediated increase in hPepT1 transcription. 3 Anti‐hPepT1‐specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco‐2 cells. Gly‐Gln supplementation of media resulted in a 1.72 (± 0.26)‐fold (n= 5) increase in staining intensity of Caco‐2 cells. 4 We conclude that Caco‐2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly‐Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.

Transport and epithelial secretion of the cardiac glycoside, digoxin, by human intestinal epithelial (Caco‐2) cells

1 Human intestinal epithelial Caco‐2 cells have been used to investigate the transepithelial permeation of the cardiac glycoside, digoxin. 2 Transepithelial basal to apical [3H]‐digoxin flux exceeds apical to basal flux, a net secretion of [3H]‐digoxin being observed. At 200 μm digoxin, net secretory flux (Jnet) was 10.8 ± 0.6 nmol cm−2 h−1. Maximal secretory flux (Jmax) of vinblastine was 1.3 ± 0.1 nmol cm−2 h−1. Cellular uptake of digoxin was different across apical and basal cell boundaries. It was greatest across the basal surface at 1 μm, whereas at 200 μm, apical uptake exceeded basal uptake. 3 Net secretion of [3H]‐digoxin was subject to inhibition by digitoxin and bufalin but was not inhibited by ouabain, convallatoxin, and strophanthidin (all 100 μm). Inhibition was due to both a decrease in Jb‐a and an increase in Ja‐b. Uptake of [3H]‐digoxin at the apical surface was increased by digitoxin and bufalin. All cardiac glycosides decreased [3H]‐digoxin uptake at the basal cell surface (except for 100 μm digitoxin). 4 The competitive P‐glycoprotein inhibitors, verapamil (100 μm), nifedipine (50 μm) and vinblastine (50 μm) all abolished net secretion of [3H]‐digoxin due to both a decrease in Jb‐a and an increase in Ja‐b. Cellular accumulation of [3H]‐digoxin was also increased across both the apical and basal cell surfaces. 1‐Chloro‐2,4,‐dinitrobenzene (10 μm), a substrate for glutathione‐S‐transferase and subsequent ATP‐dependent glutathione‐S‐conjugate secretion, failed to inhibit net secretion of [3H]‐digoxin. The increase in absorptive permeability Pa‐b (= Ja‐b/Ca) and cellular [3H]‐digoxin uptake upon P‐glycoprotein inhibition, showed that the intestinal epithelium was rendered effectively impermeable by ATP‐dependent extrusion at the apical surface. 5 A model for [3H]‐digoxin secretion by the intestinal epithelium is likely to involve both diffusional uptake and Na+‐K+ pump‐mediated endocytosis, followed by active extrusion at the apical membrane.

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 Peyers 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.

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

The interaction between polystyrene microspheres and the follicle-associated epithelium of rabbit Peyers 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 105 microspheres of 0.46 μm diameter across the epithelium overlying each lymphoid follicle dome of rabbit Peyers 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.

Nephrocalcinosis: molecular insights into calcium precipitation within the kidney.

Nephrocalcinosis may be defined as a generalized increase in the calcium content of the kidneys. This renal calcification may occur at a molecular, microscopic or macroscopic level leading to progressive amounts of renal damage. The major causes include those associated with an increase in urinary levels of calcium, oxalate and phosphate. Under these conditions, urine concentration and supersaturation leads to calcium crystal precipitation, which may be an intratubular event or initiate within the renal interstitium. The focus of discussion concerning renal calcification is often limited to factors that lead to renal stones (calculi and nephrolithiasis); however, nephrocalcinosis is a more sinister event, and often implies a serious metabolic defect. This review will discuss the hypotheses concerning initiating lesions of nephrocalcinosis using available laboratory and clinical studies and will examine whether new understanding of the molecular basis of tubulopathies, that lead to nephrocalcinosis, has given further insights.

H+/di-tripeptide transporter (PepT1) expression in the rabbit intestine

In order to examine the intestinal expression of the recently cloned H+/di-tripeptide transporter (PepT1), oligonucleotide probes were synthesized and their specificity confirmed by Northern blot analysis of rabbit jejunal RNA. In situ hybridization studies, using these probes, show that PepT1 is expressed all along the small intestine and at a very much reduced level in the colon. In contrast, PepT1 mRNA was not detected in the stomach, sacculus rotundus or caecum. Microscopic examination of tissue sections showed PepT1 expression to be restricted to intestinal epithelium with no detectable expression in the lamina propria, muscularis mucosae, muscularis or serosa. The accumulation of PepT1 mRNA along the crypt-villus axis was also investigated. In all regions of the small intestine (in duodenum, jejunum and ileum), PepT1 mRNA was undetectable in deeper epithelial cells of the crypts. Expression was first detectable at or near the crypt-villus junction, the amount of PepT1 mRNA increasing rapidly in the lower villus to a maximum approximately 100–200 μm from this point. Along the length of the small intestine PepT1 mRNA was most abundant in duodenal and jejunal enterocytes, with lower levels in the ileal epithelium. PepT1 expression is greatly depressed in the follicle-associated epithelium of the Peyers patch relative to both interfollicular and adjacent “normal” villi. These data are discussed in the context of the known physiological role of PepT1 in the gastrointestinal tract.

Co-expression of vimentin and cytokeratins in M cells of rabbit intestinal lymphoid follicle-associated epithelium

SummaryMembranous epithelial (M) cells within the follicle-associated epithelium which overlies gut-associated lymphoid tissue in Peyers patches and of appendix have been shown by immunocytochemical staining, in rabbit, to contain both vimentin- and cytokeratin-type intermediate filaments. The specificity of vimentin immunostaining has been confirmed by blocking with purified vimentin and by immunoblotting. No evidence was obtained for the expression of vimentin in rat, mouse or human M cells. The possible significance of vimentin-expression in these specialized epithelial cells and the potential use of vimentin as a positive marker for M cells are discussed.

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.