Biomedical subjects

N L Simmons

Publications and source records attributed to N L Simmons.

At least 37 records · Page 2Linked to original sources

PubMed1998

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.

Actins↗

PubMed1997

The intrinsic Cl- conductance of mouse kidney cortex brush-border membrane vesicles is not related to CFTR.

Brush-border membrane vesicles (BBMV) were prepared from whole Balb/c mice kidneys by a Mg2+ precipitation technique. The presence of an intrinsic Cl- conductance co-expressed with Na+/glucose cotransport was inferred by the anion dependence of [14C]glucose uptake and overshoot with inward Na(+)-anion gradients. In Na(+)-equilibrated conditions, an inside-negative membrane potential difference (p.d.) produced by an inward Cl- gradient alone was capable of driving intravesicular [14C]glucose accumulation. The apparent anion conductance had a selectivity of Br- = I- = Cl- > F- > > gluconate, was inhibited by 0.5 mM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) but was unaffected by 0.5 mM 4,4'-diisothiocyanatostilbene 2,2'-disulphonate (DIDS). BBMV were isolated from mice in which the CFTR gene had been disrupted by a termination mutation (-/-) and compared with normal litter mates (+/+) and heterozygotes (-/+)[18]. [14C]Glucose uptake in NaCl media was significantly greater than glucose uptake in Na gluconate media for all three genotypes measured at 20 s: for homozygous -/- animals [14C]glucose uptake was increased by 2.80 +/- 0.53 fold in Cl- media compared to gluconate media, n = 6; for wild-type +/+, by 2.16 +/- 0.53 fold, n = 8; and for heterozygous +/- animals, by 2.17 +/- 0.45 fold, n = 8. The observation of a Cl-(-)dependent component in BBMV isolated from homozygous -/- mutant animals shows that the chloride conductance in these vesicles cannot be due to CFTR expression.

Animals↗

PubMed1997

Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells.

1. Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin. 2. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 microM) or bromosulphophthalein (100 microM). Similarly tetraethylammonium and N-'methylnicotinamide (10 mM) were without effect. 3. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4'-diisothiocyanostilbene-2-2'-disulphonic acid (DIDS, 400 microM). 4. Net secretion of ciprofloxacin was partially inhibited by 100 microM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretin in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK. 5. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na+, Cl- or K+ in the bathing media. 6. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia.

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

PubMed1997

Transepithelial transport of the fluoroquinolone ciprofloxacin by human airway epithelial Calu-3 cells.

Although fluoroquinolone antibiotics such as ciprofloxacin are able to gain access to lung tissue and both pleural and bronchial secretions, the characteristics of transport and cellular uptake of ciprofloxacin in human epithelial lung tissue remain obscure. We have chosen human airway epithelial (Calu-3) cells, reconstituted as functional epithelial layers grown on permeable filter supports, as a model with which to assess both transepithelial transport and cellular uptake of ciprofloxacin. Transepithelial ciprofloxacin fluxes in absorptive (apical-to-basal) and secretory (basal-to-apical) directions were similar throughout the concentration range studied (1.0 microM to 3.0 mM). Transepithelial mannitol fluxes measured concurrently were substantially smaller than ciprofloxacin fluxes in Calu-3 epithelia, suggesting the existence of a mediated transcellular route in addition to a paracellular route for transepithelial permeation. Apical-to-basal ciprofloxacin flux (at 10 microM) was inhibited by a 100-fold excess of unlabelled norfloxacin, enoxacin, and ofloxacin, while secretory flux was unaffected. Cellular uptake of ciprofloxacin, determined as a cell/medium ratio, was greater from the basal compartment (2.7-fold) than apical uptake (1.39-fold) measured at 100 microM ciprofloxacin and showed no saturation up to 3 mM ciprofloxacin. Comparison of the permeation of ciprofloxacin was made with that of lipophilic substrates such as vinblastine and digoxin. There was a linear correlation between transepithelial permeability (Pa-b) and their oil/water partition coefficients with mannitol < ciprofloxacin < digoxin < vinblastine. Comparison of transport of ciprofloxacin across human airway Calu-3 epithelia with that across intestinal Caco-2 epithelia emphasizes the absence of a specific secretory pathway; ciprofloxacin permeation in Calu-3 epithelia appears to be mediated primarily by a transcellular route, with mediated transfer at apical and basal membranes occurring via transporters with low affinity to ciprofloxacin.

Anti-Infective Agents↗

PubMed1996

Proton/solute cotransport in rat kidney brush-border membrane vesicles: relative importance to both D-glucose and peptide transport.

We have determined the relative importance of the transmembrane proton electrochemical gradient to the transport of D-[14C]glucose and [14C]glycylsarcosine (gly-sar) in rat kidney brush-border membrane vesicles (BBMV) from superficial renal cortex. Electrogenic [14C]gly-sar transport was first optimised by imposing a pH gradient (pHo = 5.7, pHi = 8.4) and an interior negative p.d. (using outwardly directed K+ gradient plus valinomycin). Under identical conditions (pHo = 5.7, pHi = 8.4), an acceleration of initial D-[14C]glucose (at 100 microM) transport by 2.0 +/- 0.7-fold was observed compared to no proton gradient (pHo = 8.4, pHi = 8.4). This increase was due primarily to an effect of external protons, since acidic conditions (pHo = pHi = 5.7) also resulted in acceleration of D-glucose influx (2-fold). The increase in D-glucose transport in the presence of external acidity was reduced by the uncoupler FCCP, even in the absence of a proton gradient. Furthermore, the increased D-glucose transport with external acidity in the presence of a proton gradient was insensitive to a K+ gradient-driven diffusion potential in the presence of valinomycin. In no instance was an overshoot accumulation of D-[14C]glucose observed in H+ gradient conditions. H(+)-stimulated D-[14C]glucose transport showed a linear dependence on D-glucose concentration up to 20 mM D-glucose, unlike electrogenic Na(+)-dependent D-glucose transport, whose Km was 1.77 +/- 0.35 mM. In contrast, the initial rate of [14C]gly-sar (100 microM) transport by the renal H+/di-tripeptide transporter was accelerated 15.7 +/- 3.3-fold and stimulated a marked overshoot of 5.1 +/- 0.4-fold over equilibrium values. Conversely, the electrogenic, Na+/glucose transporter could be readily demonstrated, whilst [14C]gly-sar transport could not be energised by an inward Na+ gradient. The absence of electrogenic D-glucose transport in H+ gradient conditions is clear evidence against H+/glucose cotransport in Na(+)-free conditions mediated by SGLT2 (sodium-glucose transporter, renal cortex). Furthermore, since a proton gradient does not increase brush-border membrane D-glucose uptake in Na(+)-rich media, it is unlikely that in vivo renal D-glucose transport mediated via SGLT2 may be energised by the transmembrane proton gradient.

Animals↗

PubMed1996

Adenylate cyclase activity in human pancreatic adenocarcinoma cell lines.

CONCLUSION: BxPC-3, Hs 766T, Capan-2, Panc-1, and Capan-1 cells possess receptors for VIP and beta-adrenergic agonists that are functionally coupled to adenylate cyclase. In this respect, they resemble pancreatic duct cells. However, we speculate that the process of neoplastic transformation has either downregulated the expression of secretin receptors or led to a defect in the receptor itself, placing a question mark over the usefulness of these adenocarcinoma cell lines as models of the pancreatic ductal epithelium BACKGROUND: Because of the importance of ducts in pancreatic disease, we wished to establish which duct cells receptors are functional on adenocarcinoma cell lines. METHODS: We investigated the expression of agonist-stimulated adenylate cyclase activity in six human pancreatic adenocarcinoma cell lines. Known stimulants of pancreatic ductal secretion, VIP, PHI, secretin, beta-adrenergic, and dopamine, were tested. RESULTS: For responsive cell lines, VIP was the most effective stimulant followed by adrenaline, isoprenaline, PHI, and secretin. Dopamine was without effect. Since high concentrations of PHI and secretin were required to stimulate cyclase activity, their effect is probably mediated by VIP receptors. Based on the degree of stimulation observed with the individual agonist, Hs 766T and BxPC-3 were the most responsive cell lines, followed by Capan-2 and Capan-1, and finally Panc-1. MIAPaCa-2 cells did not respond to any of the agonists tested.

Adenocarcinoma↗

PubMed1996

Characterization of whole cell chloride conductances in a mouse inner medullary collecting duct cell line mIMCD-3.

The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144) had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at +/- 60 mV of 66 +/- 5 pA/pF and 44 +/- 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability ratios) of SCN- (2.3), NO3- (1.8), ClO4- (1.7), Br- (1.7), I- (1.6), Cl- (1.0), HCO3- (0.5), gluconate- (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 microM) and DIDS (500 microM) produced varying degrees of block of OR currents with NPPB being the most potent (IC50 of approximately 50 microM) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and were also time- and voltage-independent. The anion selectivity sequence was SCN- (2.5), NO3- (1.9), Br- (1.4), I- (1.1), Cl- (1.0), ClO4- (0.5), HCO3- (0.5), gluconate- (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents. An IC50 of > 100 microM was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular cAMP and were not affected when intracellular Ca2+ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells are discussed.

Animals↗

PubMed1996

An effect of Ca2+ on the Intrinsic Cl(-)-conductance of rat kidney cortex brush border membrane vesicles.

Brush-border membrane vesicles (BBMV) were prepared from superficial rat renal cortex by a divalent(2+)-precipitation technique using either CaCl2 or MgCl2. The dependence of the initial [14C]-D-glucose (or [3H]-L-proline) uptake rate and the extent of the overshoot of D-glucose or L-proline uphill accumulation from solutions containing 100 mM Na+ salt, was found to be dependent upon the precipitating divalent cation. With Mg2+ precipitation the initial uptake and overshoot accumulation of either D-glucose or L-proline were enhanced compared to BBMV prepared by Ca2+ precipitation. When the anion composition of the media was varied (uptake in Cl- media in comparison to gluconate(-)-containing media) it was found that the Cl(-)-dependent component of the initial uptake was markedly depressed with Ca(2+)-prepared BBMV (104.99 +/- 33.31 vs. 13.83 +/- 1.44 pmoles/sec/mg protein for Mg2+ and Ca2+ prepared vesicles respectively). When Ca2+ was loaded into Mg2+ prepared BBMV using a freeze-thaw technique, it was found that the magnitude and Cl- enhancement of D-glucose transport was reduced in a dose-dependent manner. Neomycin, an inhibitor of phospholipase C, had no effect on the reduction of D-glucose uptake by Ca2+ in Mg2+ prepared vesicles. In contrast, phosphatase inhibitors such as vanadate and fluoride were able to partially reverse the Ca2+ inhibition of D-glucose uptake and restore the enhancement due to Cl- media. In addition, inhibitors of protein phosphatase 2B, deltamethrin (50 nM) and trifluoperazine (10 microM), caused partial reversal of Ca2(+)-dependent inhibition of D-glucose uptake. Direct measurement of changes in the bi-ionic (Cl-vs. gluconate-) transmembrane electrical potential differences using the cyanine dye, 3,3'-dipropylthiodicarbocyanine iodide DiSC3-(5) confirmed that Cl- conductance was reduced in Ca(2+)-prepared vesicles. We conclude that a Cl- conductance coexists with Na+ cotransport in rat renal BBMV and this may be subject to negative regulation by Ca2+ via stimulation of protein phosphatase (PP2B).

Amino Acid Sequence↗

PubMed1996

Na+-independent lysine transport in human intestinal Caco-2 cells.

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na+-independent, membrane potential-sensitive uptake. Na+-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na+-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na+-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na+-independent lysine influx (10 micron) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems bo,+, y+ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 micron lysine), normalized to total apical uptake (100%), is apical bo,+ (47%), y+ (27%) and the nonsaturable component (26%), and basal bo,+ (446%), y+ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)+RNA with a human rBAT cDNA probe.

Amino Acid Transport Systems, Basic↗

PubMed1996

Evidence for a rapid, direct effect on epithelial monolayer integrity and transepithelial transport in response to Salmonella invasion.

In cultured monolayers of high-resistance Madin-Darby Canine Kidney (MDCK) cells, infection with Salmonella typhimurium SL1344 resulted in a dose- and time-dependent increase in transepithelial conductance (Gt) and short-circuit current (Isc). There was a direct linear relationship between the S. typhimurium-induced increments in Isc and Gt suggesting that this early change in epithelial parameters is, in part, the result of a cellular conductance change most probably at the apical membrane. An additional wild-type S. typhimurium strain, SR11, and an invasion-deficient isogenic mutant SB111 carrying a non-polar mutation in invA were used to confirm that the S. typhimurium-induced change in epithelial electrical parameters is directly linked to the invasion process. The S. typhimurium-induced change in epithelial electrical parameters was markedly attenuated in Na+-free choline medium. Addition of piretanide (10(-4) M, basal side) failed to affect the increased epithelial conductance and Isc after a 40-min incubation with S. typhimurium. NPPB (5x10(-4) M) added to the apical medium reduced the S. typhimurium-stimulated Isc by 28%, but Gt was not significantly reduced. It is unlikely that the S. typhimurium-induced Isc is due to Cl- secretion. Staining of S. typhimurium-infected MDCK I monolayers with TRITC-phalloidin revealed marked alterations of F-actin; diffuse intracellular accumulations of F-actin corresponding to the presence of invading bacteria were observed by 15 min. After 60 min, prominent extrusions of the apical membrane corresponding to previously described "membrane ruffles" were noted. Marked accumulations of perijunctional F-actin in infected cells corresponded to contraction of the perijunctional actin ring at the apical pole. In adjacent cells marked distortion and stretch of the apical surface was evident. The invasion-deficient invA mutant SB111 failed to induce these morphological changes. These data demonstrate that S. typhimurium invasion induces increased transcellular conductance which does not result from stimulation of Cl- secretion but instead appears to be predominantly due to increased Na+ permeability. The increased membrane conductance is coincident with increased transepithelial inulin permeability indicating that the increment in Gt has an additional "paracellular" component. The S. typhimurium-induced alterations in epithelial parameters may be related to "membrane ruffling" and/or to the accompanying changes in cell shape.

Animals↗

PubMed1996

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 microM 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 microM, whereas at 200 microM, 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 microM). 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 microM digitoxin). 4. The competitive P-glycoprotein inhibitors, verapamil (100 microM), nifedipine (50 microM) and vinblastine (50 microM) 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. I-Chloro-2,4,-dinitrobenzene (10 microM), 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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Targeting to intestinal M cells.

The specialised, antigen-transporting, epithelial M cells in the follicle-associated epithelium (FAE) overlying gut-associated lymphoid tissues constitute the primary target for oral delivery of vaccines. Our studies have shown that polystyrene microspheres selectively bind to, and are efficiently transcytosed by, rabbit Peyer's patch M cells in closed intestinal loops. Binding of biodegradable poly(DL-lactide-co-glycolide) microspheres to rabbit Peyer's patch FAE is an order of magnitude lower than that of polystyrene microspheres. Although poly(DL-lactide-co-glycolide) microspheres are not selectively targeted to M cells, a high proportion of those which bind to M cells are transcytosed, supporting the potential of such microspheres as vehicles for oral vaccine delivery. Comparison of the binding of polystyrene microspheres by murine FAE revealed this to be markedly less extensive than by rabbit FAE. These data demonstrate that microsphere binding by M cells depends on the surface properties of both cells and microspheres and suggest that surface modification may enhance the efficacy of microsphere delivery vehicles. One such approach is the incorporation of molecules with inherent binding specificity for M cells. Lectin-binding studies have revealed that M cells exhibit pronounced regional and species variation in glycoconjugate expression. In murine intestine, certain lectins bind selectively to M cells either in Peyer's patches or caecum, or at both sites. Selective targeting to, and transcytosis of, lectin-conjugates by M cells in ligated segments of murine intestine have also been demonstrated. While several lectins display strong selectivity for rabbit caecal M cells, none to date have been identified with specificity for rabbit or rat Peyer's patch M cells. Knowledge of human M cells is limited and no lectin has yet been identified with specificity for these cells. However, at least one lectin exhibits binding specificity for FAE in the human ileum. In the future, knowledge of the regional patterns of M cell carbohydrate expression within a species may allow lectins to be utilised to target selectively antigenic material to the mucosal immune system at specific locations.

Administration, Oral↗

PubMed1995

Targeted delivery of a substrate for P-glycoprotein to renal cysts in vitro.

Bodipy-verapamil has been tested as a fluorescent substrate for P-glycoprotein-mediated transepithelial secretion in MDCK-C5A epithelia. Net transepithelial secretion (Jnet) of [3H]vinblastine from basal-to-apical surfaces of monolayer epithelia is inhibited by taxotere, verapamil and Bodipy-verapamil primarily by a reduction in basal to apical vinblastine (Jb-a) transport. Bodipy-verapamil is itself subject to transepithelial net secretion by MDCK-C5A epithelia; at 5 microM a Jnet of -310 +/- 32 nmol cm-2 h-1 (n = 3) was observed. When MDCK-C5A cells are grown to form enclosed cysts in hydrated collagen gel. Bodipy-verapamil is accumulated within the cyst lumen showing that epithelial P-glycoprotein function may be used to target substrates to renal cysts.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

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.

Biological Transport↗

PubMed1995

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.

Amino Acids↗

PubMed1995

Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

Actins↗

PubMed1995

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 microns 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 Peyer's 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.

Animals↗

PubMed1995

Enterocytes in the follicle-associated epithelia of rabbit small intestine display distinctive lectin-binding properties.

Glycoconjugate expression in follicle-associated epithelia has been examined by application of a panel of lectins to fixed preparations of rabbit small intestine, including Peyer's patches. Each of the lectins examined (wheat germ agglutinin, peanut agglutinin, Ulex europaeus agglutinin I and Bandeiraea simplicifolia agglutinin II) exhibited a lower affinity for the apical surface of the specialised M cells than to columnar enterocytes within the Peyer's patch follicle-associated epithelium. Peanut agglutinin differed from the other lectins examined in that it displayed a markedly higher affinity for enterocytes within the follicle-associated epithelium than the neighbouring villi. This observation reveals that the specialised development of the follicle-associated epithelium involves expression of distinctive surface properties within the enterocyte population in addition to the more widely documented heterogeneous development of enterocytes and the specialised M cells.

Animals↗