Na+-sugar cotransport system as a polarization marker during organization of epithelial membrane

1988 ◽  
Vol 255 (6) ◽  
pp. C745-C753 ◽  
Author(s):  
C. Lasheras ◽  
J. A. Scott ◽  
C. A. Rabito
Keyword(s):  
Electrical Resistance ◽  
Apical Membrane ◽  
Sugar Transport ◽  
Cell Polarization ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Binding Studies ◽  
Occluding Junctions ◽  
Basolateral Side ◽  
Epithelial Membranes

The present study analyzed the changes in Na+-dependent sugar transport and transepithelial electrical resistance as LLC-PK1 cells reorganize into epithelial membranes. Sugar influx increased to reach a maximum 9 h after plating. The increase in the transepithelial electrical resistance, however, showed a significant delay, reaching steady state 15 h after plating. No changes in the electrochemical Na+ gradient were observed during the reorganization of the epithelial membranes. Kinetic analysis and [3H]phlorizin-binding studies showed that the increase in sugar influx resulted from an increase in the number of carriers. Unidirectional sugar influx measurements indicated that the sugar transporters were primarily located at the apical surface of the epithelial cells. These observations are consistent with the hypothesis that the sorting of native proteins occurs intracellularly before their insertion in the apical membrane, or as an alternative that they are randomly inserted, but then immediately sorted such as any carrier could be detected in the basolateral side during the reorganization process. In addition, the results suggest that the functional development of the apical membrane may occur before the complete sealing of the intercellular space during the development of the occluding junctions. Furthermore, development of the sugar transport system and occluding junctions was inhibited by cycloheximide and puromycin but not by actinomycin D, suggesting that the expression of epithelial cell polarization is probably a posttranslational event in the protein synthesis.

Transport properties of toad kidney epithelia in culture

1981 ◽  
Vol 241 (3) ◽  
pp. C154-C159 ◽  
Author(s):  
F. M. Perkins ◽  
J. S. Handler
Keyword(s):  
Electrical Resistance ◽  
Hormonal Regulation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Osmotic Water ◽  
Sodium Flux ◽  
Regulation Of Transport ◽  
Osmotic Water Flow

The characteristics of a continuous line of toad kidney epithelial cells (A6) are described. These cells form a monolayer epithelium of high transepithelial electrical resistance (about 5,000 omega . cm2). The cells generate a transepithelial potential difference (apical surface negative) of about 9 mV. The short-circuit current is equivalent to net sodium flux. Net sodium flux is stimulated by aldosterone and by analogues of cAMP. The stimulation is readily reversible. Neither urea permeability nor osmotic water flow is altered by analogues of cAMP. Amiloride eliminates 90% of the short-circuit current. Thus A6 cells form an epithelium with several differentiated properties including hormonal regulation of transport.

scholarly journals Trafficking of Shigella Lipopolysaccharide in Polarized Intestinal Epithelial Cells

1999 ◽  
Vol 145 (4) ◽  
pp. 689-698 ◽  
Author(s):  
Wandy L. Beatty ◽  
Stéphane Méresse ◽  
Pierre Gounon ◽  
Jean Davoust ◽  
Joëlle Mounier ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Fluid Phase ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Electron Microscopic ◽  
Basolateral Side ◽  
Endocytic Compartments

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.

scholarly journals Occluding junctions and paracellular pathways studied in monolayers of MDCK cells

1983 ◽  
Vol 106 (1) ◽  
pp. 205-215 ◽  
Author(s):  
M. Cereijido ◽  
L. Gonzalez-Mariscal ◽  
L. Borboa
Keyword(s):  
Electrical Resistance ◽  
Intercellular Space ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Apical Surface ◽  
Leaky Epithelia ◽  
Occluding Junctions ◽  
Freeze Fracture Electron Microscopy ◽  
Current Flows ◽  
High Conductance

MDCK cells (epithelioid, derived from the kidney of a normal dog) cultured in monolayers on a permeable support, exhibit properties of natural transporting epithelia. Comparisons of the electrical resistance across the plasma membrane of MDCK cells (as studied with microelectrodes) and the resistance across the whole monolayer, (mounted as a flat sheet between two chambers) indicate that most of the current flows through an extracellular pathway. Scanning of the electrical field over the apical surface shows that this pathway is located at the intercellular space. Yet conductance is not evenly distributed along the intercellular space as in leaky epithelia, but is restricted to sites scattered irregularly along the intercellular space. Studies of freeze fracture electron microscopy indicate that the number of strands of the junctions is also distributed irregularly, varying from 1 to 10 in a few nanometers. This suggests that regions with few strands would correspond to spots with high conductance and vice versa. However, in this preparation the sealing property of the junction bears little relationship to its structure. Thus by changing the temperature from 37 to 3 degrees C and back, the electrical resistance increases reversibly by 306%, while the number and arrangement of the strands show no significant modification. The resistance of the monolayer varies also with the age of the cells, suggesting that sealing and ion-permeating components of the junction may be dynamic entities that are not permanently installed, but can be accommodated to the requirements of the tissue.

scholarly journals Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression

2012 ◽  
Vol 23 (16) ◽  
pp. 3229-3239 ◽  
Author(s):  
Takao Yasuda ◽  
Chika Saegusa ◽  
Sachiko Kamakura ◽  
Hideki Sumimoto ◽  
Mitsunori Fukuda
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Apical Surface ◽  
Dependent Manner ◽  
Signaling Molecule ◽  
Downstream Target ◽  
Junction Protein ◽  
Polarized Trafficking

Most cells in tissues are polarized and usually have two distinct plasma membrane domains—an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin–Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a–knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.

Occluding junctions in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells

1986 ◽  
Vol 250 (4) ◽  
pp. F734-F743 ◽  
Author(s):  
C. A. Rabito
Keyword(s):  
Electrical Resistance ◽  
Electrical Potential ◽  
Sugar Transport ◽  
Bathing Solution ◽  
Chloride Permeability ◽  
Occluding Junctions ◽  
Current Voltage ◽  
Direct Measurements ◽  
Proximal Tubular ◽  
Renal Cell Line

At confluency, LLC-PK1 monolayers develop a transepithelial electrical resistance of 127 omega X cm2 and a spontaneous electrical potential that very seldom exceeds 1 mV. The monolayer shows a linear current-voltage relation in symmetrical solutions. The total conductance increases linearly with increases in the electrolyte concentration of the bathing solution. These characteristics indicate that the membrane that controls the permeability properties of the monolayer is a single membrane that it does not contain very weakly charged sites and it is sufficiently thick to obey the principle of microscopic electroneutrality. The sodium-to-chloride permeability determined from dilution potentials or from direct measurements of unidirectional Na+ and Cl- flux are 0.30 and 0.38, respectively, almost identical to the ratio obtained in the straight segment of the renal proximal tubule. The steady-state value of the electrical resistance depends on the Ca2+ concentration in the incubation medium with an apparent Km of 0.1 mM. A transitory opening of the occluding junctions results in a more uniform distribution of the Na+-dependent sugar transport system, which is normally confined to the apical membrane of the epithelial cell. This result indicates that the occluding junctions in LLC-PK1 monolayers act as a mechanical barrier, preventing the intermixing of extrinsic as well as intrinsic membrane proteins.

Protamine increases the permeability of cultured epithelial monolayers

1990 ◽  
Vol 68 (1) ◽  
pp. 220-227 ◽  
Author(s):  
M. W. Peterson ◽  
D. Gruenhaupt
Keyword(s):  
Actin Cytoskeleton ◽  
Electrical Resistance ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Type Ii ◽  
Epithelial Monolayers ◽  
Organ Specific ◽  
Darby Canine Kidney

Polycations, including protamine, have been reported to decrease the barrier integrity of cultured rat pulmonary type II epithelial monolayers. In contrast, protamine has been reported to increase the transepithelial electrical resistance of gallbladder epithelium. The present study was done using Madin Darby canine kidney epithelial cells (MDCK) to determine whether the effect of protamine on type II epithelial monolayers was species or organ specific or was dependent on the presence of nonepithelial cells and to investigate the effect of protamine on the actin cytoskeleton. Exposure of MDCK monolayers to protamine resulted in decreased transepithelial electrical resistance (Rt), increased short-circuit current (Isc) across the monolayers, and increased mannitol permeability (Pmann) of the monolayers. The decrease in Rt and increase in Isc was seen only after the addition of protamine to the apical surface of the cells. The importance of charge in this action was supported by the fact that exposure of the monolayer to the polycation poly-L-lysine also resulted in increased Pmann, and both the decreased Rt and increased Pmann seen after the addition of protamine were prevented if the monolayers were exposed in the presence of the polyanions heparin or sulfated dextran. The increase in Pmann appeared to be the result of increased permeability in the paracellular pathway, because increased mannitol uptake by the cells represented only a fraction of the increase in Pmann. Subtle changes in the actin cytoskeleton were seen after exposure of the monolayers to protamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Reassembly of the occluding junctions in a renal cell line with characteristics of proximal tubular cells

1986 ◽  
Vol 251 (6) ◽  
pp. F978-F987 ◽  
Author(s):  
C. A. Rabito
Keyword(s):  
Cell Growth ◽  
Cell Density ◽  
Exponential Growth ◽  
Electrical Resistance ◽  
Mdck Cells ◽  
Intercellular Junctions ◽  
Transepithelial Electrical Resistance ◽  
Transcriptional Level ◽  
Intrinsic Properties ◽  
Occluding Junctions

LLC-PK1 cells from trypsin-treated confluent cultures formed a continuous monolayer when plated at high cell density on collagen-coated Nuclepore filters. These monolayers developed a significant transepithelial electrical resistance that reached a maximum at 20 h. At 48 h, the resistance decreased to a value usually one-half the value obtained at 20 h. These changes were associated with an increase in the cell density of the monolayers. The drop in electrical resistance at 48 h was not observed when cell growth was arrested with excess thymidine. A hyperbolic relationship was demonstrated between cell density and electrical resistance. Although the increase in cell density was associated with an increase in the unidirectional flux of mannitol across the monolayers, selectivity studies indicated that the intrinsic properties of the occluding junctions were similar in the high and low cell density monolayers. These results indicate that, when cell growth is not arrested, changes in transepithelial electrical resistance observed after plating correspond to an increase in cell density and not to changes in the intrinsic properties of the occluding junctions. The development of transepithelial electrical resistance was delayed when the cells were in exponential growth. No such delay was observed, however, when exponential growth was synchronized. These findings and results obtained with the antimicrotubular agent Nocodazole indicate that the delay in the development of transepithelial electrical resistance is due to the asynchronous transit of the cells through the mitotic phase of the cell cycle: a time when most of the intercellular junctions are probably disrupted. Further investigation revealed that inhibition of protein but not mRNA synthesis blocked the development of occluding junctions in cells from confluent and exponentially growing cultures alike. These results indicate that, in contrast to MDCK cells, regulation of the occluding junctions in exponentially growing LLC-PK1 cells occurs at the translational not at the transcriptional level of protein synthesis.

scholarly journals Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells.

1990 ◽  
Vol 110 (4) ◽  
pp. 987-998 ◽  
Author(s):  
E Schaerer ◽  
F Verrey ◽  
L Racine ◽  
C Tallichet ◽  
M Reinhardt ◽  
...  
Keyword(s):  
Cell Surface ◽  
Electrical Resistance ◽  
Apical Membrane ◽  
Apical Cell ◽  
Cell Surface Expression ◽  
Apical Surface ◽  
Basal Cells ◽  
Half Time ◽  
Cytokeratin 7 ◽  
Additional Time

A cDNA for the rabbit low Mr polymeric immunoglobulin (poly-Ig) receptor was expressed in an immortalized rabbit mammary cell line. The intracellular routing of the receptor and its cell surface expression was analyzed in stably transfected cells grown on permeable supports. Initially the cells formed a monolayer with no transmural electrical resistance. All monolayer cells expressed the poly-Ig receptor and cytokeratin 7 filaments characteristic of luminal mammary cells but absent in myoepithelial cells. Within 7 d in culture, the cells underwent cytodifferentiation and formed a bilayer with a transepithelial electrical resistance of approximately 500 omega x cm2. Upper layer cells formed tight junctions with adjacent cells and gap junctions with basal cells. Expression of the poly-Ig receptor and cytokeratin 7 was restricted to the cells from the upper layer. The kinetics of receptor biosynthesis and processing was similar to that reported for rabbit mammary gland and rat liver. The receptor was cleaved at the apical cell surface and release of secretory component into the apical medium occurred with a half-time of approximately 2 h. Selective cell surface trypsinization combined with pulse-chase experiments served to determine at which cell surface domain newly synthesized receptor appeared first. The receptor was digested with a half-time of approximately 60 min with trypsin present in the basolateral medium and 90 min with apical trypsin. These data are consistent with selective targeting of newly synthesized receptor to the basolateral surface. The results indicate that transcytosis of the receptor from basolateral to apical membrane in the presence or the absence of its ligand requires approximately 30 min. Cleavage of the receptor by endogenous protease is not concomitant with its appearance at the apical surface, but requires additional time, thus explaining the presence of intact receptor on the apical membrane.

scholarly journals The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

2013 ◽  
Vol 200 (5) ◽  
pp. 635-650 ◽  
Author(s):  
Junya Hayase ◽  
Sachiko Kamakura ◽  
Yuko Iwakiri ◽  
Yoshihiro Yamaguchi ◽  
Tomoko Izaki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Apical Membrane ◽  
Transmembrane Protein ◽  
Cyst Formation ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Apical Surface ◽  
And Function

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.

Heme transport exhibits polarity in Caco-2 cells: evidence for an active and membrane protein-mediated process

2004 ◽  
Vol 287 (6) ◽  
pp. G1150-G1157 ◽  
Author(s):  
Aliye Uc ◽  
John B. Stokes ◽  
Bradley E. Britigan
Keyword(s):  
Apical Membrane ◽  
Heme Oxygenase 1 ◽  
Cellular Injury ◽  
Apical Surface ◽  
Oxygen Species ◽  
Heme Transport ◽  
Basolateral Side ◽  
Living Organisms ◽  
Prosthetic Groups ◽  
Metabolic Transport

Heme prosthetic groups are vital for all living organisms, but they can also promote cellular injury by generating reactive oxygen species. Therefore, intestinal heme absorption and distribution should be carefully regulated. Although a human intestine brush-border heme receptor/transporter has been suggested, the mechanism by which heme crosses the apical membrane is unknown. After it enters the cell, heme is degraded by heme oxygenase-1 (HO-1), and iron is released. We hypothesized that heme transport is actively regulated in Caco-2 cells. Cells exposed to hemin from the basolateral side demonstrated a higher HO-1 induction than cells exposed to hemin from the apical surface. Hemin secretion was more rapid than absorption, and net secretion occurred against a concentration gradient. Treatment of the apical membrane with trypsin increased hemin absorption by threefold, but basolateral treatment with trypsin had no effect on hemin secretion. Neither apical nor basolateral trypsin changed the paracellular pathway. We conclude that heme is acquired and transported in both absorptive and secretory directions in polarized Caco-2 cells. Secretion is via an active metabolic/transport process. Trypsin applied to the apical surface increased hemin absorption, suggesting that protease activity can uncover a process for heme uptake that is otherwise quiescent. These processes may be involved in preventing iron overload in humans.

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