Claudin expression in follicle-associated epithelium of rat Peyer's patches defines a major restriction of the paracellular pathway.

AIM: Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle-associated epithelium covering Peyer's patches (lymphoid follicles). METHODS: Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one-path impedance spectroscopy were performed. Morphometry of HE-stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. RESULTS: Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC-dextran were detected in follicle-associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle-associated epithelium, including claudin-1, claudin-4, claudin-5 and claudin-8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle-associated epithelium vs. neighbouring villous epithelium. CONCLUSION: We demonstrate that follicle-associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions.

Inhibitory effects of Lactobacillus fermentum on microbial growth and biofilm formation.

Beneficial effects of Lactobacilli have been reported, and lactic bacteria are employed for conservation of foods. Therefore, the effects of a Lactobacillus fermentum strain were analyzed regarding inhibitory effects on staphylococci, Candida albicans and enterotoxigenic enterobacteria by transmission electron microscopy (TEM). TEM of bacterial biofilms was performed using cocultures of bacteriocin-producing L. fermentum 97 with different enterotoxigenic strains: Staphylococcus epidermidis expressing the ica gene responsible for biofilm formation, Staphylococcus aureus producing enterotoxin type A, Citrobacter freundii, Enterobacter cloaceae, Klebsiella oxytoca, Proteus mirabilis producing thermolabile and thermostable enterotoxins determined by elt or est genes, and Candida albicans. L. fermentum 97 changed morphological features and suppressed biofilm formation of staphylococci, enterotoxigenic enterobacteria and Candida albicans; a marked transition to resting states, a degradation of the cell walls and cytoplasm, and a disruption of mature bacterial biofilms were observed, the latter indicating efficiency even in the phase of higher cell density.

Claudins of intestine and nephron - a correlation of molecular tight junction structure and barrier function.

A prerequisite of epithelial transport is a paracellular barrier function, which seals the tissue against an uncontrolled leak flux. Moreover, selective paracellular permeability has been shown to be crucial for physiological epithelial transport function. Claudins are tetraspan tight junction proteins which play a major role in paracellular ion permeability across epithelia. The multigene family consists of 24 members and several splice variants which show distinct tissue-specific expression profiles. Moreover, in diseases associated with a loss of barrier function such as forms of inflammatory bowel disease, the expression of claudins is altered. Functional characterization of single claudins revealed specific contribution to barrier properties in epithelia. This review gives an overview on the exploration of molecular structure and barrier function along the intestine and nephron, which not only share mechanisms of selective restriction of the paracellular pathway but also exhibit distinct organ-specific characteristics.

[Barrier properties and tight junction protein expression along the longitudinal axis of rat intestine].

The tight junction (TJ) protein family of claudins is a major determinant of barrier properties in a wide variety of epithelia. Aim of the study was to compare epithelial barrier properties with the presence of TJ proteins in exactly defined intestinal segments. Transepithelial resistance (R(t)) of duodenum, jejunum, ileum and colon tissue preparations was measured in Ussing chambers. In parallel, expression of TJ proteins was analyzed by Western blots. Colon was characterized by higher R(t) than more proximal segments. However, among the small intestinal segments, R(t) was highest in duodenum and lowest in ileum. Along the intestine different claudins were detected by Western blotting with different signal intensities. Colon showed strongest signals for sealing claudins in accordance with R(t), whereas predominant expression of permeability-mediating claudins was observed in small intestinal segments. Along the intestine, claudins show a marked segment-specific expression which is in accordance with respective barrier properties.

TNFalpha up-regulates claudin-2 expression in epithelial HT-29/B6 cells via phosphatidylinositol-3-kinase signaling.

Our aim has been to characterize the molecular mechanisms regulating the expression of the channel-forming tight-junctional protein claudin-2 in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha), which is elevated, for example, in active Crohn's disease. TNFalpha caused an 89% decrease of the paracellular resistance in colonic HT-29/B6 cells, whereas transcellular resistance was unaltered. The claudin-2 protein level was increased by TNFalpha without changes in subcellular tight-junctional protein localization as revealed by confocal laser scanning microscopy. Enhanced gene expression was identified as the source of this increase, since claudin-2-specific mRNA and promoter activity was elevated, whereas mRNA stability remained unaltered. Specific inhibitors and phospho-specific antibodies revealed that the increased gene expression of claudin-2 after TNFalpha treatment was mediated by the phosphatidylinositol-3-kinase pathway. Thus, the up-regulation of claudin-2 by TNFalpha is attributable to the regulation of the expression of the gene, as a result of which epithelial barrier function is disturbed, for example, during chronic intestinal inflammation.

Epithelial transport and barrier function in occludin-deficient mice.

BACKGROUND AND AIMS: This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model. METHODS: Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. RESULTS: In occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic occludin+/- or homozygotic occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin-/- mice. CONCLUSION: Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.

Claudins in the tight junctions of stria vascularis marginal cells.

In the mammalian cochlea, tight junctional strands are visible on freeze fracture images of marginal cells and other inner ear epithelia. The molecular composition of the strial tight junctions is, however, largely unknown. We investigated the expression of integral tight junction-proteins, claudin-1 to -4, and occludin, in stria vascularis of the guinea-pig cochlea, as compared to kidney. Western blot analysis revealed a strong expression of claudin-4 and occludin in strial tissue, and confocal immunofluorescence microscopy demonstrated their presence in the tight junctions of the marginal cells. In addition, a moderate level of claudin-3 and claudin-1 was detected and both were located in the marginal tight junctions. Claudins-1, -3, and -4 are characteristic of epithelia with low paracellular permeability and claudin-4 is known to restrict the passage of cations through epithelial tight junctions. In the marginal cells, these claudins appear to be responsible for the separation of the potassium-rich endolymph from the sodium-rich intrastrial fluid. In contrast, Western blot analysis and confocal microscopy demonstrated that the marginal cell epithelium does not contain claudin-2, which forms a cation-selective pore in tight junctions. Its absence indicates a cation-tight paracellular pathway in the marginal cells.

Tight junction proteins: a novel class of integral membrane proteins. Expression in human epidermis and in HaCaT keratinocytes.

Tight junction proteins comprise a novel group of integral membrane proteins necessary for cell-to-cell contacts and responsible for the barrier function in epithelial and endothelial cells in various tissues. The tight junction membrane domain contains at least three distinct proteins, named occludin, claudin and junctional adhesion molecule. Claudins are products of a gene family consisting of more than 20 members. We investigated mRNA expression of occludin and 13 different claudins in neonatal foreskin, adult skin and cultivated HaCaT keratinocytes by the Northern blot technique, and performed immunohistochemical staining of adult skin for occludin, claudin 1 and claudin 2. Occludin, claudin 1 and claudin 3 mRNAs were expressed in human neonatal and adult keratinocytes as well as in HaCaT keratinocytes. All other tested claudins were negative. Immunohistochemical staining of adult skin was positive for occludin in the intercellular space of the granular layer, and for claudin 1 in the inter-cellular space of the spinosum layer and basal layer, but negative for claudin 2 in all skin layers. Claudin 1 was also positive in the outer root sheath of hair follicles. Our results indicate that occludin, claudin 1 and claudin 3 are involved in cell-to-cell contacts between keratinocytes in human epidermis, although their functional importance remains unknown.

Early aldosterone effect in distal colon by transcriptional regulation of ENaC subunits.

Aldosterone-induced sodium absorption is mediated by the epithelial Na(+) channel (ENaC). It is thought that the "early effect" is not based on genomic regulation of ENaC expression, because ENaC subunit transcription was reported to start later than Na(+) transport. We investigated electrogenic Na(+) absorption (J(Na)) and, in identical tissues, mRNA expression of ENaC subunits in early (EDC) and late (LDC) distal colon of the rat. In both segments, 8-h in vitro incubation with 3 nM aldosterone enhanced expression of beta- and gamma-ENaC mRNA and induced J(Na). J(Na) was 10 times higher in LDC than in EDC. alpha-ENaC mRNA was unchanged in EDC, whereas it decreased in LDC. In LDC, beta- and gamma-ENaC mRNA was induced 1 h after aldosterone addition, whereas J(Na) became apparent >1 h later. Downregulation of alpha-ENaC mRNA did not take part in acute regulation because it started after a lag time of 3 h. Time correlation of beta- and gamma-ENaC induction and J(Na) stimulation suggests that the early aldosterone effect on Na(+) absorption in distal colon is caused by transcriptional upregulation of beta- and gamma-ENaC expression.

Further characteristics of the Ca(2+)-inactivated Cl(-) channel in Xenopus laevis oocytes.

Removal of extracellular Ca(2+) activates ion channels in the plasma membrane of defolliculated oocytes of the South Africa clawed toad Xenopus laevis. At present, there is controversy about the nature of the Ca(2+)-inactivated ion channels. Recently, we identified one of these channels as a Ca(2+)-inactivated Cl(-) channel (CaIC) using single channel analysis. In this work we confirm and extend previous observations on the CaIC by presenting a decisive extension of the regulation and inhibition profile. CaIC current is reversibly blocked by the divalent and trivalent cations Zn(2+) (half-maximal blocker concentration, K(1/2) = 8 muM), and Gd(3+) (K(1/2) = 20 muM). Furthermore, CaIC is inhibited by the specific Cl(-) channel blocker NPPB (K(1/2) approximately 3 muM). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 muM) but insensitive to its high affinity analogue benzamil (100 muM). An investigation of the pH-dependence of the CaIC revealed a reduction of currents in the acidic range. Using simultaneous measurements of membrane current (I(m)), conductance (G(m)) and capacitance (C(m)) we demonstrate that Ca(2+) removal leads to instant activation of CaIC already present in the plasma membrane. Since C(m) remains constant upon Ca(2+) depletion while I(m) and G(m) increase drastically, no exocytotic transport of CaIC from intracellular pools and functional insertion into the plasma membrane is involved in the large CaIC currents. A detailed overview of applicable blockers is given. These blockers are useful when oocytes are utilized as an expression system for foreign proteins whose investigations require Ca(2+)-free solutions and disturbances by CaIC currents are unwanted. We further compare and discuss our results with data of Ca(2+)-inactivated cation channels reported by other groups.

Minimal molecular determinants of substrates for recognition by the intestinal peptide transporter.

Proton-dependent electrogenic transporters for di- and tripeptides have been identified in bacteria, fungi, plants, and mammalian cells. They all show sequence-independent transport of all possible di- and tripeptides as well as of a variety of peptidomimetics. We used the mammalian intestinal peptide transporter PEPT1 as a model to define the molecular basis for its multisubstrate specificity. By employing computational analysis of possible substrate conformations in combination with transport assays using transgenic yeast cells and Xenopus laevis oocytes expressing PEPT1, the minimal structural requirements for substrate binding and transport were determined. Based on a series of medium chain fatty acids bearing an amino group as a head group (omega-amino fatty acids, omega-AFA), we show that electrogenic transport by PEPT1 requires as a minimum the two ionized head groups separated by at least four methylene groups. Consequently, a > 500 pm < 630 pm distance between the two charged centers (carboxylic carbon and amino nitrogen) is sufficient for substrate recognition and transport. Removal of either the amino group or the carboxyl group in omega-AFA maintained the affinity of the compound for interaction with the transporter but abolished the capability for electrogenic transport. Additional groups in the omega-AFA backbone that provide more hydrogen bonding sites appear to increase substrate affinity but are not essential. The information provided here does (a) explain the capability of the peptide carrier for sequence-independent transport of thousands of different substrates and (b) set the molecular basis for a rational drug design to increase the absorption of peptide-based drugs mediated by PEPT1.

Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications.

Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes.

1. To gain information on the mode of operation of the renal proton-coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2. Using differently charged glycyl-dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH-dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2-50 microM) between pH 5.5 and 7.4 and membrane potentials of +/- 0 to -50 mV. 3. Maximal substrate-evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pHo). Potential-dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages. 4. The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from -50 to -150 mV substrate affinities decrease 10- to 50-fold whereas apparent Imax increases almost 10-fold. 5. Even at saturating H+/H3O+ and dipeptide concentrations the I-V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters. 6. These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.

Transport of charged dipeptides by the intestinal H+/peptide symporter PepT1 expressed in Xenopus laevis oocytes.

The cloned intestinal peptide transporter is capable of electrogenic H+-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5-8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H+/H3O+ cotransport may vary depending on pH.

The Ca2+-inactivated Cl- channel at work: selectivity, blocker kinetics and transport visualization.

Removal of extracellular divalent cations activated a Cl- channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca2+-inactivated Cl- channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel patch-clamp techniques. Beside other Cl- channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3' azido-3'deoxythymidine (AZT), a nucleoside analog we used as an antiviral drug, blocked at least partly the CalC-mediated currents. Using the Cl--sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl- from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca2+ removal. In the absence of external Cl- and Ca2+, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl- in the millimolar range. In the presence of external Ca2+, no emission changes could be observed during the same time period. Chelating external Ca2+ in absence of Cl- immediately activated Ca2+-inactivated Cl- channels leading to subsequent emission decrease of SPQ. Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel measurements, we found an anion selectivity sequence I- > Br- > Cl- >> gluconate as it is also typical for maxi Cl- channels. Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated oocytes, also known as eggs, showed the presence of Ca2+-inactivated Cl- channels. However, this egg CaIC revealed an altered sensitivity to external Ca2+ concentrations. All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar among all Cl- channels described up to now.

Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms.

l. Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport. PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes. In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity. In addition, both transporters show differences in pH dependence and substrate specificity. 2. As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1. Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique. 3. Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters. We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region.