Basolateral Pole Research Articles

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.

Interactions of Escherichia coli strains of non-EPEC serogroups that carry eae and lack the EAF and stx gene sequences with undifferentiated and differentiated intestinal human Caco-2 cells

Escherichia coli strains of non-EPEC serotypes that carry eae and lack the EAF and the Shiga toxin ( stx) gene sequences have been found in acute diarrhea. Both the cell association and the cell entry of these strains in human intestinal epithelial cells were studied as a function of cell differentiation and polarization. The eae+/EAF−/ stx− non-EPEC E. coli strains invaded undifferentiated Caco-2 cells more efficiently than differentiated cells. In contrast, prototype EPEC strain E2348/69 did not show significative differences from invasion rates of undifferentiated and differentiated cells. The uptake of these strains was greatly enhanced by pretreatment of differentiated Caco-2 cells with EGTA. These results suggest that the eae+/EAF−/ stx− non-EPEC E. coli invasion of intestinal cells may be dependent on receptors expressed on the surface of undifferentiated cells and the basolateral pole of differentiated cells.

Interactions of Escherichia coli strains of non-EPEC serogroups that carry eae and lack the EAF and stx gene sequences with undifferentiated and differentiated intestinal human Caco-2 cells.

Escherichia coli strains of non-EPEC serotypes that carry eae and lack the EAF and the Shiga toxin (stx) gene sequences have been found in acute diarrhea. Both the cell association and the cell entry of these strains in human intestinal epithelial cells were studied as a function of cell differentiation and polarization. The eae+/EAF-/stx- non-EPEC E. coli strains invaded undifferentiated Caco-2 cells more efficiently than differentiated cells. In contrast, prototype EPEC strain E2348/69 did not show significative differences from invasion rates of undifferentiated and differentiated cells. The uptake of these strains was greatly enhanced by pretreatment of differentiated Caco-2 cells with EGTA. These results suggest that the eae+/EAF-/stx- non-EPEC E. coli invasion of intestinal cells may be dependent on receptors expressed on the surface of undifferentiated cells and the basolateral pole of differentiated cells.

Exploitation of host factors for efficient infection by Shigella

Abstract Shigellosis is a worldwide endemic ulcerating disease of the large intestine caused by enteroinvasive bacteria. Shigella takes the route via M-cells and macrophages to access the basolateral pole of enterocytes. After invasion of and cell-to-cell spread within the epithelial cell layer, the bacterium multiplies within the cytoplasm of enterocytes. Induced by a limited number of bacterial effector proteins, Shigella makes use of established signaling pathways of the host cell to achieve internalization, transcytosis, apoptosis or cell-to-cell spread. This review addresses the host factors required for efficient infection focusing on Shigella-induced cytoskeletal rearrangements and associated signaling.

The function of alkaline phosphatase in the liver: Regulation of intrahepatic biliary epithelium secretory activities in the rat

We studied the effects of alkaline phosphatase (AP) on the secretory processes of the rat intrahepatic biliary epithelium as well as the role of the intrahepatic biliary epithelium in the uptake and biliary secretion of exogenous AP. The effects of acute and chronic administration of AP on bile secretory parameters were investigated in vivo in normal and bile duct ligated (BDL) rats and in vitro in isolated rat bile duct units (IBDU).In vivo, acute AP administration decreased bile flow and biliary bicarbonate excretion and abolished secretin choleresis in BDL rats but not in normal rats. On the contrary, the AP inhibitor, levamisole, increased in BDL rat bile flow and biliary bicarbonate excretion. In vitro, basal and secretin-stimulated Cl−/HCO3− exchanger activity in IBDU was immediately inhibited by AP intraluminal microinjection (apical exposure) but only after a prolonged exposure to the basolateral pole. Levamisole increased the Cl−/HCO3− exchanger activity of IBDU. A significant basolateral uptake of AP occurs in IBDU with a progressive transport to the apical domain. AP chronic treatment increased AP and γ-glutamyltranspeptidase (γ-GT) activities in the intrahepatic bile ducts and hepatocyte canalicular pole, promoted enlargement of bile canaliculi, and decreased bile flow and biliary bicarbonate excretion. In conclusion, the intrahepatic biliary epithelium plays a role in the uptake and biliary secretion of serum AP. AP inhibits the secretory processes of the intrahepatic biliary epithelium and induces features of intrahepatic cholestasis after chronic administration. These findings indicate that AP plays an active role in down-regulating the secretory activities of the intrahepatic biliary epithelium. (Hepatology 2000;32:174-184.)

Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA.

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.

A Monocarboxylate Transporter MCT1 is Located at the Basolateral Pole of Rat Jejunum

We have functionally expressed and identified a monocarboxylate transporter (MCT1) from rat jejunal enterocyte and we provide evidence for its basolateral localization. Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of a proton-lactate symporter was investigated by means of L-[14C]lactate uptake. The existence of an endogenous capacity for L-lactate transport was demonstrated; when, however, oocytes were injected with jejunal mRNA, an expressed L-lactate uptake was seen which differed from the endogenous transporter since it was significantly pH dependent. After sucrose density gradient fractionation, the highest expression of the pH-dependent lactate uptake was detected with the mRNA size fraction of about 2-3 kb in length. The substrate specificity, stereoselectivity and sensitivity to pCMBS (an organomercurial thiol reagent that modifies cysteine residues) of the expressed transport were in good agreement with results previously obtained using isolated jejunal basolateral membranes. Using the reverse transcriptase-polymerase chain reaction, the presence of mRNA coding for the MCT1 isoform was demonstrated in jejunal enterocytes. These data, together with previous results, suggest that MCT1 is a major route for lactate efflux across the basolateral membrane of rat jejunum; this is in contrast to current opinion which restricts the presence of MCT1 to the apical membrane of the whole small intestine.

Modulation of intracellular growth of Listeria monocytogenes in human enterocyte Caco-2 cells by interferon-gamma and interleukin-6: role of nitric oxide and cooperation with antibiotics.

The influence of interferon (IFN)-gamma and interleukin (IL)-6 on the intracellular growth of Listeria monocytogenes phagocytosed from the apical pole was examined in polarized Caco-2 cells. IFN-gamma (from the apical pole) and IL-6 (from the basolateral pole) considerably reduced the bacterial intracellular growth, an effect largely abolished by l-monomethyl arginine. Both cytokines caused overexpression of inducible nitric oxide synthase. IL-6, but not IFN-gamma, caused a partial restriction of L. monocytogenes in phagosomes and largely prevented the cytosolic forms from being surrounded by actin. Ampicillin was bacteriostatic in unstimulated cells but modestly bactericidal in cells treated with IFN-gamma and IL-6. Azithromycin (a macrolide) was fairly bactericidal and sparfloxacin (a fluoroquinolone) highly bactericidal in all situations. IFN-gamma and IL-6 may therefore be important determinants in the protection of epithelial cells from intracellular multiplication of L. monocytogenes. Ampicillin may fail in their absence, requiring the use of other antibiotics such as the fluoroquinolones.

Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain.

In this study we investigated the distribution of a recently cloned polyspecific organic anion transporting polypeptide (Oatp2) in rat brain by nonradioactive in situ hybridization histochemistry and immunofluorescence microscopy. The results demonstrate that Oatp2 is expressed in brain capillary and in plexus epithelial cells. At the blood-brain barrier (BBB), Oatp2 expression could be co-localized with the endothelial marker vWF (von Willebrand factor) but not with the astrocyte marker GFAP (glial fibrillary acidic protein). In choroid plexus epithelial cells, Oatp2 could be localized to the basolateral cell pole, whereas the first member of the Oatp gene family of membrane transporters to be cloned (Oatp1) co-localized with the alpha(1)-subunit of Na,K-ATPase at the apical plasma membrane domain. Because Oatp1 and Oatp2 have been previously shown to mediate transmembrane transport of a wide variety of amphipathic organic compounds, including many drugs and other xenobiotics, the histochemical localization of Oatp2 at the BBB and of Oatp1 and Oatp2 in the choroid plexus imply a role for these transporters in the active exchange of amphipathic solutes between the blood, brain, and cerebrospinal fluid compartments. (J Histochem Cytochem 47:1255-1263, 1999)

Mucosal immunity and inflammation. V. Innate mechanisms of mucosal defense and repair: the best offense is a good defense.

Well-coordinated mechanisms have evolved that provide both innate protection against gastrointestinal mucosal injury and facilitation of rapid mucosal repair following mucosal damage. Generic protection from injury is provided by intrinsic structural features of the epithelium that form a highly competent barrier and a complex formed at the apical surface by trefoil peptides that comprise the interface between mucosa and lumen. When the epithelial barrier has been broken, regardless of the nature of the injury, epithelial surface continuity is rapidly reestablished through restitution as cells migrate and elongate. This process is promoted by trefoil peptides at the apical surface and a large array of cytokines and growth factors acting at the basolateral pole. Many of these regulatory peptides are products of the immune and other lamina propria cell populations, which are activated following disruption of the mucosal barrier. Thus efforts to repair the epithelium follow inherently from inflammatory effects after initial damage; the repair process in turn may allow abrogation of further inflammation. Ultimate repair of injury requires both proliferative replacement of damaged epithelial cells and remodeling of extracellular matrix and deeper cell populations to restore normal architecture and a fully functional mucosa.

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Potassium conductances in isolated single cells from Xenopus laevis colonic epithelium.

The patch-clamp technique was employed in whole cells to analyze K+ conductances of amphibian colonic cells. Xenopus laevis colonic epithelium was dissected, and single epithelial cells were isolated using Ca(2+)-free solution and mild enzyme treatment. Vital epithelial cells had a round shape, and a distinction between apical and basolateral poles was no longer possible. Their epithelial origin was, however, verified by antibodies against keratin. The average resting potential of the colonocytes was -37.6 +/- 1 mV (n = 220) and the resulting membrane current was strongly potassium selective. Further characterization of this conductance was achieved by current-voltage relationship in the presence and absence of various K+ channel blockers. Barium and cesium showed pronounced voltage-dependent blockage, with interaction at about 35% inside the pores. Lidocain, as well as quinine and quinidine also blocked, but with different kinetics and binding charactertics. Both TEA and verapamil were ineffective. We also explored the effects of extra- (pHo) and intracellular pH (pHi) on the K+ conductance. An increase of pHo, as well as pHi, caused membrane hyperpolarization, and the shift of the current-voltage relationship indicates a stimulation of K+ channels by decreasing external and/or internal H+ concentration. The results provide the first whole-cell measurements on isolated amphibian colonic epithelial cells and demonstrate the presence of various K+ channel types in this preparation.

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells.

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

The actin-binding proteins adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine

To understand the distinct functions of the closely related actin-severing proteins adseverin and gelsolin, we examined the expression of these proteins in detail during mouse and human development using a new highly sensitive and specific set of antibody reagents. Immunoblot analysis demonstrated that adseverin was highly expressed in mouse kidney and intestine at all stages of development and in human fetal and adult kidney. In contrast and as reported previously, gelsolin was expressed much more widely in both murine and human tissues. Immunohistochemistry on murine kidney sections revealed a predominantly differential localization of adseverin and gelsolin. Adseverin was expressed in peripolar cells, thin limbs, thick ascending limbs, and principal cells of cortical and medullary collecting ducts where it was diffusely localized in the cytoplasm. Gelsolin was expressed in the distal convoluted tubule, intercalated cells and principal cells of cortical and medullary collecting ducts, and in ureter. In the distal convoluted tubule, gelsolin showed a diffuse distribution and in principal cells of collecting ducts a localization at the basolateral pole. In intercalated cells, gelsolin localization was heterogeneous, either at the apical pole or diffusely in the cytoplasm. In human fetal and adult kidney, adseverin was expressed only in collecting ducts whereas gelsolin was expressed in thick ascending limbs and collecting ducts. In mouse and human intestine adseverin was expressed in enterocytes with a gradient of increasing expression from the duodenum to the colon, and from the crypt to the villus. The observations indicate high level expression of adseverin in specific cells of the kidney and colon, and suggest a previously unrecognized function of adseverin in epithelial cell function.

Expression of α-and β-subunits and activity of Na +K +ATPase in pig thyroid cells in primary culture: modulation by thyrotropin and thyroid hormones

Na +K +ATPase located at the basolateral pole of thyroid epithelial cells, contributes to thyroid hormone synthesis by generating the driving force for the uptake of the substrate, iodide. We have investigated whether the expression of the α- and β-subunits and activity of Na +K +ATPase were subjected to variations in response, (a) to TSH, that controls the expression of differentiation in thyroid cells and (b) to thyroid hormones as potential autocrine factors. Studies were carried out on pig thyroid cells cultured (a) without TSH to obtain thyroid cell monolayers (TCM) in basal state or (b) with TSH in the form of cell monolayers (TCM-T) or as reconstituted thyroid follicles (RTF). Iodide uptake activity, thyroperoxidase protein and thyroglobulin mRNA taken as parameters of thyroid cell differentiation were 6 to 25-fold higher in RTF and TCM-T than in TCM. Western blot analyses of Na +K +ATPase subunits revealed that the α-subunit (105 kDa) content of TCM-T and RTF was similar but 8-fold higher than that of TCM. In contrast, the β-subunit (50 kDa) content of TCM-T and RTF was only about twice that of TCM. Similar relative variations were observed at the mRNA level for both α- and β-subunits. Na +K +ATPase activity was only 40% higher in RTF and TCM-T than in TCM. A 48 h treatment of RTF by either T4 or T3 (1–100nM) induced a 3-fold increase of the α-subunit but did neither alter the β-subunit nor the Na +K +ATPase activity. In conclusion, Na +K +ATPase activity and the level of expression of its β-subunit, known to control the assembly and targetting of α– β oligomers and thus the amount of functional sodium pump at the plasma membrane, are only moderately altered when thyroid cells undergo major changes in their differentiation status. Our data show that the expression of the α-subunit of Na +K +ATPase by thyroid cells is up-regulated by TSH and thyroid hormones.

Regulation of basolateral plasma membrane recycling by protein kinase C: Role of F-actin and marcks

Certain membrane proteins are rapidly regulated by endocytotic retrieval or exocytotic insertion. We have shown that phorbol myristate acetate (PMA), acting via protein kinase C (PKC), downregulates multiple basolateral transport sites in intestinal epithelia. AIM: to examine whether PKC enhances membrane retrieval and to define a possible role for the actin cytoskeleton and the PKC-regulated actin crosslinker MARCKS (__myristolated ~lanine-_rich _C_ kinase substrate). METHODS: Endocytosis was estimated by uptake of FITC-dextran (FD) from either the apical or basolateral side of confluent T84 monolayers grown on permeable supports. Exocytosis was estimated by efflux of FD from pre-loaded cells. *P 100, 0.1-1000 nM PMA, 20-120 min exposure; e.g., 6 min basolateral uptake of FD=245 +-24 (% control) after 1 hr exposure to 10nM PMA, n=9*. Two additional phorbol esters, as well as bryostatin-1 and the diacylglycerol analogue nAG (which all activate PKC) also increased basolateral endocytosis (*), but the PKC-inactive 4et-PMA did not (n=3-12). PMA decreased basolateral exocytosis to 75 +--1% control and stimulated apical exocytosis to 127 ± 5% (n=23). PMA's effect on basolateral endocytosis was blocked by putative PKC inhibitors including staurosporine, calphostin C, rottlerin, and a PKCo~ pseudosubstrate peptide (*).Role of F-actin: PMA remodeled basolateral F-actin in a manner resembling previous findings with the actin disassembler cytochalasin D (CD). Like PMA, CD enhanced basolateral endocytosis (329-+ 56% control, n=3*) and stimulated apical exocytosis (156 +-2%, n=3*). PMA-stimulated endocytosis was blocked by the F-actin stabilizer jasplakinolide (n=3*). Role ofMARCKS: MARCKS was fiuorescently immunolocalized in a punctate distribution at the basolateral pole of monolayers grown on optically clear filters and, by immunoprecipitation, was membrane-associated. In response to PMA, MARCKS visually and biochemically redistributed to the cytosolic compartment/fraction. A myristolated peptide corresponding to the phosphorylation site domain of MARCKS inhibited the PMA effect on endocytosis (n=3*). CONCLUSIONS: PKC selectively enhances basolateral membrane retrieval in model polarized intestinal epithelia by an F-actin dependent mechanism that may involve MARCKS. This may represent a general mechanism whereby PKC regulates surface expression of proteins that participate in membrane recycling pathways. supported by NIH DK48010 and DK51630.

Cl/HCO3 Antiport Affects H-Lactate Symport Activity at the Basolateral Pole of Jejunum Enterocyte

Background/Aims: Under normal conditions the jejunal tract of the rat intestine absorbs HCO₃. A basolateral Cl/HCO₃ exchange, evidenced by means of membrane vesicles, could be involved in this process. Aim of this study was to investigate the anion exchange activity in the whole jejunal tract, where various transport systems could interact. Methods: In the jejunal tract of rat intestine everted and incubated in vitro, the experimental conditions set up minimized loss of CO₂ from the serosal solution, where pH and pCO₂ were determined together with fluid and electrolyte transintestinal transport. Results: The serosal pCO₂ increase and pH decrease, evident during the experiment, could be antagonized by enhancing the 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) concentrations in the serosal fluid. Moreover high DIDS concentration affected fluid, sodium, lactate, bicarbonate and, although in the opposite direction, chloride transport, whilst they were ineffective on K flux. Conclusion: These results give evidence that in the basolateral membrane the inhibition of Cl/HCO₃ antiport causes a diminution of lactic acid movement. Therefore we can hypothesize that Cl/HCO₃ antiport facilitates basolateral H-lactate symport in order to carry endogenous lactic acid towards the blood stream.

Mammary gland membrane transport systems.

The secretion of milk depends on the activity of a large number of membrane transport systems located on the apical and basolateral membranes of mammary secretory cells. It follows that a thorough knowledge of individual mammary tissue membrane transport systems is required if we are to fully understand the process of milk secretion. The distribution of the transporters between the apical and basolateral poles of the mammary epithelium must be asymmetrical given that the mammary gland is capable of vectorial transport. This is particularly evident in the case of glucose and amino acid transport systems: the transport mechanisms for these compounds are predominantly situated in the blood-facing aspect of the secretory cells. In addition. it is apparent that there is a polarized distribution of transport systems (carriers and channels) which accept sodium, potassium, chloride, phosphate, and calcium as substrates.

Role of lipopolysaccharide in signaling to subepithelial polymorphonuclear leukocytes.

Polymorphonuclear leukocyte (PMN) infiltration and migration across colonic intestinal epithelia is a hallmark of inflammation in Shigella flexneri-mediated dysentery. To identify bacterial signals associated with this process, potential stimulatory factors mediating initial PMN association with the epithelium and subsequent transepithelial migration were examined in an in vitro model system. Quantitative analyses revealed that purified S. flexneri lipopolysaccharide (LPS) deposited at the apical surface of polarized intestinal epithelial cells transcytosed to the basolateral pole, a process dependent on the stage of epithelial cell differentiation. Transcytosed LPS in the presence of normal human serum (NHS), a source of LPS binding protein and soluble CD14, mediated both interleukin-8 secretion at the basolateral pole and enhanced PMN adherence. In addition, LPS stimulated a significant degree of directed transepithelial migration of PMNs, an event that was further enhanced in the presence of NHS. These results implicate LPS in signaling subepithelial PMN emigration and enhancing PMN-epithelium interactions prior to and during subsequent Shigella-induced transepithelial migration.

Mg2+ transport in plasma membrane vesicles of renal epithelium of the Mozambique tilapia (Oreochromis mossambicus)

To elucidate the mechanisms involved in Mg2+ transport at the apical and basolateral poles of the renal tubular epithelium, apical and basolateral plasma membrane vesicle preparations were derived from kidney tissue of freshwater- and seawater-adapted Mozambique tilapia Oreochromis mossambicus. Brush-border preparations were enriched 15.8-fold in alkaline phosphatase activity and consisted almost exclusively of right-side-out membrane vesicles. Basolateral membrane preparations were enriched 7.5-fold in Na+/K+-ATPase activity and contained resealed vesicles and leaky membrane fragments. Mg2+ association with brush-border and basolateral plasma membranes, traced using radioactive 27Mg, occurred in an osmotically active space. In all instances, Mg2+ binding to the vesicular membrane was low compared with the vesicular uptake. Mg2+ equilibration across the vesicular membrane of brush-border preparations was rapid and sensitive to the presence of extravesicular Ca2+, suggesting that the apical membrane of the renal epithelium contains a transport pathway for divalent cations. Application of various ionic gradients did not affect vesicular Mg2+ transport in apical and basolateral membrane preparations, suggesting the presence of an ion-coupled transport mechanism. ATP or ATP--S did not stimulate Mg2+ fluxes, indicating that Mg2+ transport does not proceed via an ATP-driven or activated transporter. In these aspects, vesicular Mg2+ transport was similar in seawater and freshwater preparations. These results suggest that the apical membrane of renal epithelial cells lacks an active secretory Mg2+ transport mechanism. We propose that the Mg2+ conductivity of the apical membrane reflects a route for downhill Mg2+ entry and is involved in renal Mg2+ reabsorption.