Morphological and biochemical changes of LLC-PK1 cells during adaptation to glucose-free culture conditions.

The established renal epithelial cell line LLC-PK1 retained in tissue culture several differentiated properties of renal proximal tubular cells. By adapting LLC-PK1 cells to glucose-free culture conditions, we recently succeeded in isolating a gluconeogenic strain of LLC-PK1 cells capable of growing in the absence of hexoses. In contrast to the parental wild type, the isolated strain expressed fructose-1,6-bisphosphatase activity and was, therefore, designated LLC-PK1-FBPase+. Besides the differences in glucose metabolism, the isolated gluconeogenic substrain differs form the parental wild type with respect to morphological appearance and the expression of apical membrane marker enzymes. LLC-PK1-FBPase+ cells display a drastic accumulation of autophagic vacuoles, disappearance of apical membrane alkaline phosphatase activity, and increased gamma-glutamyltranspeptidase activity. In order to find out whether or not a low alkaline phosphatase activity in combination with the enhanced formation of autophagic vacuoles is related to a change in apical membrane surface, we utilized a combined light and electron microscopic morphometric procedure to determine the absolute amount of organelle volumes and membrane surface areas. This stereologic approach shows that LLC-PK1-FBPase+ cells display a tenfold increase in the volume of autophagic vacuoles and the lysosomal compartment. Analysis of lysosomal enzyme activities, however, revealed no changes as compared to wild-type cells. The apical membrane surface of gluconeogenic cells was found to be increased by 80%. Karyotype analysis revealed that LLC-PK1 wild-type cells were diploid, whereas FBPase+ cells exhibited polyploidy with a high percentage of tetraploid nuclei. Culturing LLC-PK1-FBPase+ cells in the presence of 5 mM glucose does not abolish the morphological and biochemical changes described, indicating the stability of the FBPase+ strain.     

Development of the Na(+)-dependent hexose carrier in LLC-PK1 cells is dependent on microtubules

The Na(+)-dependent hexose carrier, an endogenous apical marker, develops during differentiation of LLC-PK1, an established cell line with characteristics of the proximal tubule. This development was inhibited by the microtubule-disrupting drugs, colchicine and nocodazole, while it was insensitive to lumicolchicine. This strongly suggests that microtubules are involved in the plasma membrane expression of the Na(+)-dependent hexose carrier. We also analyzed the increase in activity of endogenous apical and basolateral membrane proteins during the polarization process. The development of three apical (Na(+)-dependent hexose carrier, gamma-glutamyltransferase and alkaline phosphatase) and one basolateral membrane protein (Na+/K(+)-ATPase) was studied during the reorganization of LLC-PK1 cells into a polarized epithelium. Colchicine inhibited the rapid, transient increase in the expression of the Na(+)-dependent hexose carrier during this polarization process. A similar result was observed for the development of the other apical proteins, while the development of Na+/K(+)-ATPase seemed to be largely insensitive to colchicine. Our results are in agreement with the model that the vesicles containing the apical membrane proteins use microtubules as tracks to reach the plasma membrane. The transport of vesicles containing basolateral membrane proteins clearly occurs by a different pathway which is independent on an intact microtubular network. Since the inhibition by the microtubule-disrupting drugs was complete, it can be concluded that after disruption of microtubules, the apical vesicles do not use the basolateral pathway by default.     

Polarized nature of taurine transport in LLC-PK1 and MDCK cells: Further characterization of divergent transport models

Summary Taurine transport was measured in cultured epithelial cells-LLC-PK1 and MDCK-grown on permeable membrane supports. Taurine transport by LLC-PK1 cells was greater on the apical surface compared to the basolateral surface. MDCK cells exhibited greater taurine uptake from the basolateral side. Transepithelial taurine flux was in the direction of apical to basolateral in the LLC-PK1 monolayers. There was no net transepithelial movement of taurine in the MDCK monolayers. Efflux of taurine from the apical and the basolateral membrane surfaces of LLC-PK1 cell monolayers was stimulated by external-alanine but not L-alanine. Efflux of taurine from MDCK cell monolayers was stimulated by-alanine on the basolateral surface. While the competitive inhibitor guainidinoeithane sulfonate (GES) competitively inhibited taurine uptake to a similar degree on the apical and basolateral surface of LLC-PK1 cell monolayers, GES had a more potent inhibitory effect on the basolateral taurine uptake in MDCK cells when compared to its inhibition of apical taurine transport. We conclude that there are characteristic differences in transport of taurine by apical and basolateral surfaces of LLC-PK1 and MDCK cells which may be the consequence of asymmetric distribution or unique structural properties of the taurine transporter.Supported by a grant from the National Institutes of Health (DK 37223), the American Heart Association (92-004470).     

Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na(+)-dependent hexose carrier in LLC-PK1 cells.

We have analyzed the development of Na(+)-dependent hexose transport during differentiation and during polarization of LLC-PK1, an established cell line with characteristics of the proximal tubule. When cell-cell contact was disturbed by a low extracellular Ca2+ concentration or by a phorbol myristate acetate (PMA) treatment, the development of Na(+)-dependent hexose transport was completely inhibited. The effect of PMA on the development of hexose transport could be uncoupled from its effect on the tight junctions. The PMA concentration needed for the latter effect was approx. 10-fold higher than for the former. As the primary cause of the PMA effect, an influence on the cytoskeleton is suggested. In contrast to PMA, the concentration dependence of both phenomena on the extracellular Ca2+ concentration was almost the same. Moreover, the incorporation of hexose carriers in the plasma membrane could be induced by changing the extracellular CA2+ concentration from low to normal. We conclude that there is a relation between the formation of tight junctions and the development of the Na(+)-dependent hexose carrier, possibly because Ca(2+)-dependent cell adhesion molecules play a role in both phenomena. However, a direct relation between Ca(2+)-dependent elements of the tight junctions and the insertion of the hexose carrier can not be excluded. The Ca(2+)-dependent development seems to be a common characteristic of apical membrane proteins in contrast to the development of the basolateral membrane protein, (Na(+)+K+)-ATPase.     

Morphometry of the galliform cecum: A comparison between Gambel's quail and the domestic fowl

Summary Tissues from the proximal, middle, and distal regions of the ceca of Gambel's quail and domestic fowl were examined by scanning and transmission electron microscopy. Cellular and subcellular structures, including epithelial cell height, mitochondrial volume fraction, microvillous surface area, proportion of goblet cells, and junctional complex characteristics, were quantified by a variety of stereologic procedures and other measurement techniques. The mucosal surface of quail cecum shows a much more highly developed pattern of villous ridges and flat areas than that of fowl cecum. The fowl has significantly greater cell heights than the quail in all cecal regions. The mitochondrial volume fraction does not differ significantly with species or region, but mitochondria are concentrated on the apical side of the nucleus. In both species, the proximal cecal region has the greatest microvillous surface area. All 3 components of junctional complexes, including zonula occludens, zonula adhaerens, and macula adhaerens, are quantified. When all factors are considered, the quail cecum appears to have morphological characteristics consistent with a greater potential capacity for absorption than the fowl cecum.     

Heterogeneity of the Na(+)-H+ antiport systems in renal cells.

This study analyzes the differential characteristics of the Na(+)-H+ antiport systems observed in several epithelial and non-epithelial renal cell lines. Confluent monolayers of LLC-PK1A cells have a Na(+)-H+ antiport system located in the apical membrane of the cell. This system, however, is not expressed during cell proliferation or after incubation in the presence of different mitogenic agents. In contrast, confluent monolayers of MDCK4 express minimal Na(+)-H+ antiport activity in the confluent monolayer state but reach maximal antiport activity during cell proliferation or after activation of the cells by different mitogenic agents. Similar results were obtained with the renal fibroblastic cell line BHK. The system present in MDCK4 cells is localized in the basolateral membrane of the epithelial cell. In LLC-PK1A cells, an increase in the extracellular Na+ concentration produces a hyperbolic increase in the activity of the Na(+)-H+ antiporter. In MDCK4 and BHK cells, however, an increase in external Na+ produces a sigmoid activation of the system. Maximal activation of the system occur at a pHo 7.5 in LLC-PK1A cells and pHo 7.0 in MDCK4 cells. The Na(+)-H+ antiporter of LLC-PK1A cells is more sensitive to the inhibitory effect of amiloride (Ki 1.8 x 10(-7) M) than is the antiporter of MDCK4 cells (Ki 7.0 x 10(-6) M). Moreover, 5-(N-methyl-N-isobutyl)amiloride is the most effective inhibitor of Na(+)-H+ exchange in LLC-PK1A cells, but the least effective inhibitor in MDCK4 cells. Conversely, the analog, 5-(N,N-dimethyl)amiloride, is the most effective inhibitor of Na(+)-H+ exchange in MDCK4 cells, but is the least effective inhibitor in LLC-PK1A cells. These results support the hypothesis that Na(+)-H+ exchange observed in LLC-PK1A and other cell lines may represent the activity of different Na(+)-H+ antiporters.     

Development and polarization of the Na+/H+ antiport system during reorganization of LLC-PK1A cells into an epithelial membrane

Changes in Na+/H+ antiport activity and transepithelial electrical resistance were analyzed in a clone of LLC-PK1 cells as the dispersed cells became organized into an epithelial membrane. The clone designated LLC-PK1A showed a 250% increase in Na+/H+ exchange activity as compared with the parent cell line. Na+ influx induced by an outwardly oriented H+ gradient is almost completely abolished during active cell proliferation or after cell dispersion. The activity of the Na+/H+ antiport system increases after plating the cells at high density. This increase precedes the increase in the transepithelial electrical resistance. The increase in the Na+/H+ antiport activity was not observed when the cells were plated at low density in the presence of an antimitotic agent indicating that close cell contact is an absolute requirement for the development of the system. The increase in Na+ influx correlated with an increase in Vmax, while the Km for Na+ remained essentially unchanged. Unidirectional Na+ influx measured from the apical or basolateral side as the dispersed cells became reorganized into an epithelial membrane indicated that the insertion of the Na+/H+ antiporter proteins occurred directly in the apical membrane of the epithelial cells. This finding is consistent with the hypothesis that the sorting of native proteins occurs intracellularly prior to their insertion in the apical membrane of the epithelial cells. The delay in the increase of transepithelial electrical resistance as compared with the increase in Na+ influx indicates that the settlement of the limits between the apical and basolateral membrane (fence function) precedes the closing of the intercellular space (barrier function) during the development of the occluding junctions. Further, the development of the Na+/H+ antiporter was inhibited by cycloheximide but not by actinomycin D, suggesting that the expression of epithelial cell polarization is a translational or posttranslational event.     

Interaction of N1,N12-diacetylspermine with polyamine transport systems of polarized porcine renal cell line LLC-PK1

LLC-PK(1) cells grown on porous membrane filters were employed as a model system to explore the renal transport of polyamines. The polarity of LLC-PK(1) monolayers was confirmed by the exclusive appearance of a Na(+)-dependent alpha-methylglucoside transport system on the apical surface. The uptake of free polyamines from the basolateral side of monolayers was consistent with the existence of a single class of transport system, while the existence of two kinetically distinct polyamine transport systems with higher and lower affinities on apical membranes was suggested. The results of competition studies indicated that each of these transporters was able to interact with putrescine, spermidine and spermine. LLC-PK(1) cells incorporated monoacetylspermine from the apical surface of monolayers at about half the rate of spermine uptake. Monoacetylspermine inhibited spermidine uptake, indicating that free polyamine transport systems also recognized the monoacetylated derivative. In contrast, N(1),N(12)-diacetylspermine did not inhibit spermidine uptake, nor was it incorporated into the cells, indicating the absence of transport systems that recognize N(1),N(12)-diacetylspermine on the apical membranes of LLC-PK(1) cells. These results may be relevant as to our previous observation that the content of diacetylpolyamines in urine is relatively constant, and may explain the excellence of N(1),N(12)-diacetylspermine as a tumor marker.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

Cadmium (Cd2+) disrupts Ca(2+)-dependent cell-cell junctions and alters the pattern of E-cadherin immunofluorescence in LLC-PK1 cells.

Recent findings from our laboratories have shown that Cd2+ has relatively specific damaging effects on the adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK1. Results of the present studies show that the junction-perturbing effects of Cd2+ in LLC-PK1 cells are more pronounced when Cd2+ is applied to the basolateral cell surface than when it is applied to the apical surface, and that the severity of the effects is inversely related to the concentration of Ca2+ in the medium. Additional results show that exposure to sublethal concentrations of Cd2+ decreases the amount of E-cadherin that is associated with cell-cell contacts. These results suggest that Cd2+ damages Ca(2+)-dependent cell-cell junctions in LLC-PK1 cells by interacting with E-cadherin or a similar Ca(2+)-sensitive site that is oriented toward the basolateral cell surface.     

Developmentally regulated 75-kilodalton protein expressed in LLC-PK1 cultures is a component of the renal Na+/glucose cotransport system

Na+/D-glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75-kD protein in apical membranes isolated from highly differentiated LLC-PK1 cultures, an epithelial cell line of pig renal proximal tubule origin. The 75-kD antigen was enriched from solubilized LLC-PK1 apical membranes by means of high-pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long-term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter-positive cells was dramatically increased after inducer treatment as predicted for differentiation-regulated expression. These results identify a 75-kD protein as a component of a developmentally regulated renal Na+/glucose symporter expressed in cell culture.     

Entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells.

The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract of pigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcine epithelial cell line. To determine the site of viral entry, LLC-PK1 cells were grown on permeable filter supports and infected with TGEV from the apical or basolateral side. Initially after plating, the virus was found to enter the cells from both sides. During further development of cell polarity, however, the entry became restricted to the apical membrane. Viral entry could be blocked by a monoclonal antibody to the viral receptor aminopeptidase N. Confocal laser scanning microscopy showed that this receptor protein was present at both the apical and basolateral plasma membrane domains just after plating of the cells but that it became restricted to the apical plasma membrane during culture. To establish the site of viral release, the viral content of the apical and basolateral media of apically infected LLC-PK1 cells was measured by determining the amount of radioactively labelled viral proteins and infectious viral particles. We found that TGEV was preferentially released from the apical plasma membrane. This conclusion was confirmed by electron microscopy, which demonstrated that newly synthesized viral particles attached to the apical membrane. The results support the idea that the rapid lateral spread of TGEV infection over the intestinal epithelia occurs by the preferential release of virus from infected epithelial cells into the gut lumen followed by efficient infection of nearby cells through the apical domain.     

Differential toxicity as a result of apical and basolateral treatment of LLC-PK1 monolayers with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine

Monolayers of LLC-PK1 cells, a cell line with features typical of proximal tubular epithelial cells, were treated at the apical and basolateral side with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBD-GSH) and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-NAC). Apical treatment with PCBD-GSH (greater than 20 microM) resulted in cytotoxicity, which could be inhibited by acivicin and aminooxyacetic acid (AOAA), inhibitors of gamma-glutamyltranspeptidase (gamma GT) and beta-lyase respectively. In contrast apical treatment with PCBD-NAC was only toxic at high concentrations (greater than 850 microM), and this effect could hardly be inhibited by AOAA. Basolateral treatment of confluent LLC-PK1 monolayers, grown on porous membranes, with PCBD-GSH gave a much smaller response than apical treatment, consistent with the fact that gamma GT is predominantly present at the apical side. Basolateral treatment even with high concentrations of PCBD-NAC (1.1 mM) did not show an increase in cytotoxicity when compared to the effect after apical treatment. These results suggest the absence of an organic anion transporter, by which these conjugates in vivo are transported into the cells from the basolateral side. This supposition was substantiated in a study of transcellular transport of the model ions tetraethyl ammonium (TEA) and para-aminohippurate (PAH), in LLC-PK1 monolayers, grown as indicated above. No active PAH transport could be demonstrated, whereas an active TEA transport was present. The absence of an organic anion transporter limits the usefulness of LLC-PK1 cells for the study of nephrotoxicity of compounds, like PCBD-NAc, needing this transport to enter the cells. However, the finding of an active basolateral organic cation transporter, together with the presence of gamma GT, dipeptidase and beta-lyase, makes this system especially interesting for testing all compounds that use this transporter or these enzymes in order to elicit toxicity.     

Cytomorphometric analysis and surface ultrastructure of developing decidua

A scanning electron microscopic (SEM) and morphometric analysis of the topographical changes occurring in the uterine luminal epithelial layer in association with decidual tissue (DT) formation in guinea pigs was undertaken in order to elucidate the surface ultrastructural characteristics which occur during the process of endometrial differentiation. Experimentally induced decidua formation was promoted by mechanical stimulation of the antimesometrial luminal surface during the period of maximal uterine sensitivity to stromal differentiation. DT-associated remodeling of the uterine epithelial layer was subsequently examined by light and SE microscopic analysis for apical epithelial and luminal contour alterations associated with decidua growth. Cytological changes in the luminal surface associated with DT induction included sparse microvillus growth from the apical epithelial surface, accompanied by the appearance of prominent apical membrane surface protrusions and endometrial gland openings as compared with non-DT-stimulated control samples. Decidua surface growth was characterized by a short, sparse epithelial microvillus pattern present over a highly contoured luminal uterine surface on which contoured gland openings were both numerous and prominent. These surface modifications contrasted with the flat, non-decidualized luminal surface contour which was covered by distinct, microvilli-laden, apical cell membranes, and defined by prominent intercellular membrane borders. The uterine surface at the time of maximal DT formation (i.e. growth) closely resembled that of a uterine luminal surface undergoing apoptosis and subsequent cellular reabsorption, characterized by disrupted cell surface membranes, sparse microvillus surfaces and prominent epithelial contours reflecting stromal tissue and vasculature involution. These data indicate that the alterations in the uterine luminal surface associated with DT formation are reminiscent of the endometrial changes associated with the initiation of early placentation, and may be used as a model for the analysis of the role of epithelial cell surface modifications associated with the induction and support of interstitial blastocyst implantation and early decidua formation.     

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.     

Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.     

Endocytosis and Na+/solute cotransport in renal epithelial cells

Endocytic uptake of [3H]sucrose and lucifer yellow, markers for fluid-phase endocytosis, was studied in cultures of the renal epithelial cell lines LLC-PK1 and OK. Endocytosis in LLC-PK1 cells was inhibited when the cells were grown in the presence of gentamicin (1 mg/ml) for 4 days or when the cells were treated with concanavalin A (1 mg/ml) for 5 h. These changes occurred without perturbation of intracellular Na+ and K+ content, indicating that the cells maintained normal ion gradients. The inhibition of endocytosis was accompanied by marked increases in the apparent Vmax for Na+-dependent cell uptake of solutes such as Pi and L-alanine. The apparent Km was unchanged. In contrast, treatment of OK cells with concanavalin A produced marked stimulation of endocytosis and inhibition of the Na+-dependent uptake of Pi and L-glutamate. These changes occurred in the absence of changes in intracellular Na+ and K+ content. Neither gentamicin nor concanavalin A had a direct effect on Na+/solute cotransport in these cell lines. The changes in Na+/Pi cotransport induced by concanavalin A in both LLC-PK1 and OK cells were blocked by keeping the cells at 4 degrees C during exposure to the lectin, suggesting that endocytosis may be part of the mechanism which mediates the changes in solute uptake. The reciprocal relationship between the changes in endocytosis and the changes in Na+/solute cotransport is consistent with the possibility that the number of Na+/solute cotransporters present in the plasma membrane may be altered by an increase or decrease in the rate of membrane internalization by endocytosis. The Vmax changes in Na+/solute cotransport provide indirect support for this conclusion.     

Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.     

Induction of polarized apical expression and vectorial release of carcinoembryonic antigen (CEA) during the process of differentiation of HT29-D4 cells

HT29-D4 clonal cells can be induced to differentiate by a simple alteration of the culture medium, that is, by the replacement of glucose by galactose [Fantini, J., et al. (1986) J. Cell Sci., 83:235-249] as reported for the nonclonal HT29 cells [Pinto, M., (1982) Biol. Cell, 44:193-196]. An essential property of the HT29-D4 cell line is the fact that no cell loss occurs after the medium change, so that the differentiated cells can be considered as the true counterpart of the undifferentiated one. This model is particularly suitable to study morphological and biochemical events associated with the progressive establishment of the differentiation state. We report here that carcinoembryonic antigen (CEA), a 180 kDa glycoprotein originally described as a colon tumor associated antigen, is faintly expressed at the surface of undifferentiated HT29-D4 cells. These cells release a small amount of CEA (2.5 ng/10(6) cells/24 hr) in the culture medium. Fourty-eight hours after glucose substitution by galactose, both CEA cell surface expression and release are strongly enhanced as demonstrated by immunofluorescence and immunoprecipitation studies. Ten days after the medium change, the amount of CEA released reaches a maximum value of 130 ng/10(6) cells/24 hr, which remains stable for differentiated HT29-D4 cells cultured in glucose-free, galactose-containing medium (Gal-medium) for several months. HT29-D4 cells grown in Gal-medium in porous-bottom culture dishes generate leakproof epithelial monolayers. We have successfully performed an independent radioiodination of the apical and basolateral domains of these cells, followed by immunoprecipitation. We demonstrate that CEA is expressed exclusively at the apical surface of differentiated HT29-D4 cells, since the 180 kDa polypeptide was immunoprecipitated only when the radioiodination was performed at the apical side of the monolayer. Leakproof HT29-D4 monolayers cultured in permeable chambers were also used to demonstrate that CEA was exclusively released in the medium bathing the apical side of the cells. In conclusion, this study of cell surface CEA expression and CEA release during the process of differentiation of HT29-D4 cells demonstrated that 1) CEA cell surface expression and CEA release are correlated with cell differentiation; 2) CEA is expressed in the apical brush border membrane of differentiated HT29-D4 cells; and 3) CEA release is exclusively oriented toward the apical side of the polarized monolayer.