The use of BeWo cells as an in vitro model for placental iron transport

BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 +/- 0.5% of the total dose was transported after 8 h, equivalent to 38.8 +/- 2.1 pmol.cm(-2).h(-1). Transfer of Tf across the cell layer was much more limited; 2.4 +/- 0.2% of the total dose was transported after 8 h, equivalent to 5.0 +/- 0.4 pmol.cm(-2).h(-1). Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.     

Pachytene spermatocyte proteins influence Sertoli cell function

Isolated Sertoli cells were cultured on MatrigelTM-coated Millipore filters in bicameral chambers. The Sertoli cells form confluent epithelial sheets that, by virtue of the Sertoli cell tight junctions, form transepithelial permeability barriers between the apical and basal domains of the cells. These Sertoli cells secrete metabolically labeled proteins in a polarized manner. Three peptides, P1 (pI = 4.5-5.0, MW = 70,000), P2 (pI = 4.5-5.0, MW = 50,000), and P3 (pI = 4.0-4.7, MW = 34,000) are secreted apically from the epithelial sheets of Sertoli cells and are not found in basal secretions from the same Sertoli cells. Pachytene spermatocyte-conditioned medium contains proteins released from the germ cells that are uniquely different from the Sertoli cell-secreted proteins. Addition of the pachytene spermatocyte-conditioned medium to the apical reservoir of the bicameral chambers over an epithelial sheet of Sertoli cells stimulated the synthesis and secretion of total protein, transferrin, and specifically induced peptides S1 and S2 from Sertoli cells. As controls, conditioned medium from 3T3 fibroblasts and round spermatids did not stimulate the Sertoli cells. Hence, the ability of pachytene spermatocyte proteins to induce specific Sertoli cell secretion indicates that the pachytene spermatocytes are able to influence their surrounding milieu, and provides further support to the concept of a paracrine interaction between germ cells and Sertoli cells during spermatogenesis.     

Dual compartment (bicameral) culture: role of basement membrane in epithelial differentiation

A number of years ago we reported that tight junctions between adjacent Sertoli cells subdivide the seminiferous epithelium into two compartments, basal and adluminal, thus forming the morphological basis of the blood-testis barrier. It is now generally believed that the special milieu created by the Sertoli cells in the adluminal compartment is essential for germ cell differentiation. In order to duplicate the compartmentalization that occurs in vivo, Sertoli cells were cultured in bicameral chambers on Millipore filters impregnated with a reconstituted basement membrane. Confluent monolayers of these cells were tall columnar (40–60 µ in height) and highly polarized. These Sertoli cell monolayers established electrical resistance that peaked when the Sertoli-Sertoli tight junctions developed in culture. In addition, the monolayers formed a permeability barrier to ³H-inulin and lanthanum nitrate. The bicameral chambers were utilized in a number of studies on protein secretion, and it was revealed that numerous proteens are secreted in a polarized manner. In another study, hormone- stimulated aromatase activity was measured in Sertoli cells grown on plastic culture dishes, plastic dishes coated with laminin or Matrigel, and in the bicameral chambers. Cell culture on basement membrane substrate decreased the FSH-dependent estrogen production. No estrogen production was observed when the Sertoli cells were cultured in the bicameral chambers. These results are in accord with the hypothesis that differentiated Sertoli cells lose their ability to metabolize androgen to estrogen in an hormone-dependent manner, whereas undifferentiated cells in culture, or in vivo, have a very active FSH-dependent aromatase activity. This bicameral culture system could serve as an important model system to examine various functions of Sertoli cells including interactions of Sertoli cells with germ, Leydig, and myoid cells.     

The chick retinal pigment epithelium grown on permeable support demonstrates functional polarity

The retinal pigment epithelium (RPE) from the chick embryo was cultured on permeable support. Using confluent cultures and analysis of the incubation medium, the present study demonstrates that RPE cells cultured on permeable membrane retain functional polarity, a characteristic of the RPE in vivo. The degree of intercellular permeability in the confluent RPE cultures was estimated by following [3H]inulin movement from the apical side to the basal side of the cultures. Twenty-four hours after exposure of the apical side of the culture to [3H]inulin, the 3H concentration in the apical medium remained at 3.4 to 4.4 times of that in the basal medium. The barrier function of RPE disappears in the presence of EDTA. Net unidirectional fluid movement from the apical side of the cultures to the basal side of the cultures is regularly observed in confluent RPE cultures. The rate varies among different preparations of cultures and the highest is 1.60-1.84 microliters/cm2/h. When cultures are given 26 h of [35S]methionine, more than 20 bands with molecular weights ranging from 20,000 to greater than 250,000 Da can be detected in the medium as assessed by autoradiography of SDS-polyacrylamide gels. While six macromolecules appear to be equally concentrated in the basal medium and the apical medium, the majority are in higher concentration in the basal medium. Analysis of the 10% TCA-precipitable fraction of the medium showed that the specific activities in the apical medium and basal medium were 24.0 +/- 0.4 X 10(6) and 46.4 +/- 0.2 X 10(6) (mean +/- SEM, N = 8) cpm/ml/mg RPE protein, respectively. When cultures react with VIP (vasoactive intestinal peptide), the elevated intracellular cyclic AMP is extruded into the medium bathing the cells. However, the rate of extrusion into the basal medium is twice as fast as that into the apical medium. Electron microscopy of the confluent RPE cultures shows morphological polarization of the cells. The intercellular spaces appear to be closed at the apical side of the cells by junctional complexes consisting of tight junctions, zonular adherens junctions, and gap junctions.     

Identification and characterization of monolayer cultures of sheep trophoblast cells maintained in bicameral culture chambers

Enriched epithelial cell and fibroblast fractions were isolated from ovine placentomes by isopycnic centrifugation of collagenase/DNAse-dispersed cells through a density gradient of 45% Percoll. The epithelial cells formed confluent monolayers when plated onto filters impregnated with a 50-microns layer of Matrigel in medium containing 10% fetal bovine serum. These cells were maintained in dual environment culture chambers in serum-free medium for at least 12 days. The epithelium had a polarized appearance similar to that found in vivo only when cells were plated at high density (10(7)/cells/cm2). The epithelial monolayer consisted predominantly of a single population of uninucleate cells with intracellular features similar to those previously described for ovine trophoblast both in vivo and in vitro. These cells stained positively with an antiserum to alpha-keratin, a marker specific to epithelial cells, and no staining was observed with antisera raised against binucleate cells or leucocyte-common antigen. Binucleate cells were detected by microscopy and immunostaining in the pellet of cells obtained from the Percoll gradient but were rarely seen in the epithelium. The epithelial monolayer excluded 3H-inulin, added to the basal chamber, from the apical chamber, thus demonstrating the formation of a permeability barrier similar to that found in vivo. The maintenance of a monolayer of pure ovine trophoblast cells in vitro, which retain the characteristics of the epithelium in vivo, will enable the study of many cellular functions of the trophoblast.     

Polarized expression of an apical membrane glycoprotein is established before functional tight junctions have developed in MCF-7 cells

A milk-fat globule membrane antigen, designated MAM-6 and detected immunocytochemically by the monoclonal antibody 115D8, is expressed apically in confluent MCF-7 monolayer cultures. Immediately after preparation of a single-cell suspension, MAM-6 appears on the entire cell surface. However, polarized apical expression of MAM-6 is restored as early as 2-6 hr after plating of unpolarized cells, before functional tight junctions are established, as judged by freeze-fracture and ruthenium red permeability. Quantitative immunogold cytochemistry reveals that the apical:basal ratio of MAM-6 expression was about 17:1 after 6 hr. Tight junctions developed as late as 12-24 hr after plating. At this time the apical:basal MAM-6 ratio was about 30:1 (as compared to about 50:1 in control monolayers).     

Pachytene spermatocyte protein(s) stimulate sertoli cells grown in bicameral chambers: Dose-dependent secretion of ceruloplasmin,sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin

Summary Interactions between pachytene spermatocytes and Sertoli cells were investigated using the bicameral culture chamber system. Pachytene spermatocytes were isolated from adult rats with a purity in excess of 90% by centrifugal elutriation. The pachytene spermatocytes were cultured in a defined media and pachytene spermatocyte protein prepared from the conditioned media by dialysis and lyophilization. This pachytene spermatocyte protein was reconstituted at various concentrations and incubated with confluent epithelial sheets of immature Sertoli cells cultured in bicameral chambers. Pachytene spermatocyte protein stimulated secretion of total [³⁵S]methionine-labeled protein from Sertoli cells in a dose-dependent manner predominantly in an apical direction. This stimulatory effect of pachytene spermatocyte protein was domain specific from the apical surface of Sertoli cells, and seemed specific for secretion because total intracellular protein did not increase under the influence of pachytene spermatocyte protein. Pachytene spermatocyte protein and follicle-stimulating hormone additively stimulated Sertoli cell secretion. The physicochemical characteristics of the stimulatory pachytene spermatocyte protein are indicative of heat stability, whereas the stimulatory pachytene spermatocyte protein exhibit acid, dithiothreitol and trypsin sensitivity, and partial urea sensitivity. Furthermore, Sertoli cell secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin in response to various concentrations of pachytene spermatocyte protein were determined by immunoprecipitate of these [³⁵S]methionine-labeled proteins with polyclonal antibodies. Maximal stimulation of ceruloplasmin and sulfated glycoprotein-1 secretion from Sertoli cells was observed at a dose of 50 μg/ml pachytene spermatocyte protein, whereas maximal stimulation of sulfated glycoprotein-2 and transferrin secretion from Sertoli cells was observed at a dose of 100 μg/ml of pachytene spermatocyte protein. These results suggest that pachytene spermatocytes modulate Sertoli cell secretory function of at least four proteins in the regulation of spermatogenesis. Supported by grant #DCB-8915930 (D. D.) from the National Science Foundation, Washington, DC.     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.     

Iodide transport in primary cultured thyroid follicle cells: evidence of a TSH-regulated channel mediating iodide efflux selectively across the apical domain of the plasma membrane.

The transport of iodide was studied in porcine thyroid follicle cells cultured in bicameral chambers. The continuous layer of polarized follicle cells, joined by tight junctions, formed a diffusion barrier between the two compartments (apical and basal) of the culture chamber. Uptake and efflux of 125I- at either surface (apical and basolateral) of the cells were thus possible to determine. Protein binding of iodide was inhibited by methimazole (10(-3) M) in all experiments. Radioiodide was taken up by the cells from the basal medium in a thyroid-stimulating hormone (TSH)-dose dependent manner with a maximal cell/medium ratio of 125I- of about 50 in cultures prestimulated with 0.1 to 1 mU/ml for 2 days. This uptake was inhibited by perchlorate and ouabain. In contrast, 125I- was not taken up from the apical medium. In preloaded cells, iodide efflux was rapidly (within 1-2 min) and dose-dependently (0.1-10 mU/ml) stimulated by TSH. Bidirectional measurements revealed that TSH stimulated iodide efflux in apical direction, leaving efflux in basal direction unchanged. In experiments with continuous uptake of label from the basal compartment, the TSH-stimulated efflux in apical direction had a duration of 4 to 6 min and resulted in a reduction in the cellular content of radioiodide by up to 80%. Decreased levels of cellular 125I- remained for at least 15 min after TSH addition. From our observations we conclude that the TSH-regulated uptake and efflux of iodide take place at opposite surfaces of the porcine thyroid follicle cell. Acutely stimulated iodide efflux is not the result of an increased permeability for iodide in the entire plasma membrane but only in the apical domain of this membrane. This implicates the presence of an iodide channel mediating TSH-stimulated efflux across the apical plasma membrane of the follicle cell. The mechanism is suggested to facilitate a vectorial transport of iodide in apical direction, i.e., to the lumen of the intact follicle.     

Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm² with 50 μM PC, and 17.0 and 23.0 nmol/h/cm² with 100 μM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 μM DHA-enriched PC, and to 25 and 30% with 50 and 100 μM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 μM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells. (Mol Cell Biochem xxx: 1–9, 2005)     

Ion transport by primary cultures of canine tracheal epithelium: Methodology,morphology, and electrophysiology

Summary Canine tracheal epithelial cells were isolated by enzymatic and mechanical dispersion and cultured on permeable supports. The cells formed confluent monolayers and retained most of the morphologic characteristics of the intact epithelium, including apical microvilli, apical tight junctions, and a moderately interdigitated lateral intercellular space. The cells also retained the functional properties of the epithelium. The monolayer responded to addition of isoproterenol with the characteristic changes in cellular electrical properties expected for stimulation of Cl secretion: isoproterenol increased transepithelial voltage, depolarized apical membrane voltage, and decreased both transepithelial resistance and the ratio of apical-to-basolateral membrane resistance. Examination of the cellular response to ion substitutions and inhibitors of Cl secretion indicate that the cultured monolayers retain the same cellular mechanisms of ion transport as the intact epithelium. Thus, primary cultures of tracheal epithelium may provide a useful preparation for future studies of the mechanism and regulation of Cl secretion by airway epithelia.     

Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane- associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.     

Apical trehalase expression associated with cell patterning after inducer treatment of LLC-PK1 monolayers

Trehalase, a differentiation-specific marker of renal proximal tubule brush border membrane, is expressed in confluent long-term cultures of the renal epithelial cell line LLC-PK1. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membrane-associated differentiated functions (Yoneyama and Lever: J. Cell. Physiol. 121: 64-73, 1984). In the present study, we utilize a polyclonal antibody specific for renal trehalase to demonstrate that trehalase expression induced in LLC-PK1 cultures after HMBA treatment is localized in cells forming a three-dimensional network of strands across the confluent monolayer. The antitrehalase antibody recognized an apical membrane antigen of apparent molecular weight 100-110 kD both in LLC-PK1 cultures and in the corresponding pig renal brush border membranes. Strand formation and total trehalase activity increased in parallel as a function of inducer concentration and duration of exposure. Strand formation and trehalase expression were also greatly enhanced in monolayers grown on a Nuclepore filter support even in the absence of inducer. Strand formation was not a prerequisite for induced trehalase expression in culture, since strands did not develop in cultures treated with N, N'-dimethylformamide (DMF) and equally potent inducer of trehalase expression. In this case, cells which expressed increased levels of trehalase were dispersed at random over the monolayer. Induction of strand formation and trehalase expression by HMBA required a minimum exposure period of 48 hr and persisted up to a week after removal of inducer. By contrast, the response to DMF required continuous presence of inducer. Levels of trehalase declined even in the continuous presence of inducer in local regions of low cell density created by wound-repair of the monolayer. In addition to the membrane-bound form, trehalase activity was also recoverable from the culture medium, but release of trehalase was not affected by inducers. These observations are consistent with the view that a cell type committed to express a program of differentiation after HMBA treatment or growth on a permeable support is organized in specific cell patterns visible as strands over the confluent cell monolayer.     

A biphasic chamber system for maintaining polarity of differentiation of culture respiratory tract epithelial cells

Summary A simple, disposable, biphasic cultivation chamber has been developed for respiratory tract epithelial cells. This chamber, the Whicutt chamber, contains a movable, transparent, permeable gelatin membrane that can be employed either submerged in the culture medium, thereby feeding the cells by the traditional immersion method, or raised to the surface of the culture medium, to bring the apical surfaces of the cells into contact with air and provide nutrients only from below (basal feeding). The effects of biphasic cultivation on the growth and differentiation of respiratory tract epithelial cells from different sources have been studied in Whitcutt chambers. Primary hamster tracheal epithelial (HTE) cells grown to confluence with basal feeding developed a ciliated columnar morphology, with differentiated features (cilia and mucous granules) located in the apical region of the epithelial layer. These cells secreted mucinlike molecules from the apical surface (i.e. the surface in contact with air). Although the apical localization of differentiation features was greater, mucous cell differentiation achieved by basal feeding was quantitatively not greater than that achieved by continuous immersion feeding. Similarly, basal feeding did not alter the degree of epithelial cell differentiation in cultures derived from rat, rabbit, and monkey tracheas or from human bronchial and nasal tissues. In contrast, the differentiation of guinea pig tracheal epithelial cells in culture was significantly influenced by the feeding method employed. When fed basally, guinea pig tracheal epithelial cell cultures expressed various mucociliary functions with resemblance to mucociliary layers in vivo, whereas constantly immersed cultures seemed stratified and squamous. These results suggest that, at least for guinea pigs, the combination of feeding methods provided by the Whitcutt chamber can be used to achieve differentiated cultures of tracheal epithelial cells with a polarity of differentiation that is similar to that observed in intact airways in vivo. Editor's statement Supported in part by grants from NCI (CA42097) and American Cancer Society (BC-465) to R. W., and grants from The Council for Tobacco Research-USA, and Cystic Fibrosis Foundation to K.B.A.     

Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells.

Since most in vitro studies exploring the action of Clostridium perfringens enterotoxin (CPE) utilize either Vero or CaCo-2 cells, the current study directly compared the CPE responsiveness of those two cell lines. When CPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of approximately 135, approximately 155, and approximately 200 kDa. However, confluent Transwell cultures of either cell line CPE-treated for 20 min formed only the approximately 155-kDa complex. Since those Transwell cultures also exhibited significant (86)Rb release, approximately 155-kDa complex formation is sufficient for CPE-induced cytotoxicity. Several differences in CPE responsiveness between the two cell lines were also detected. (i) CaCo-2 cells were more sensitive when CPE-treated on their basal surface, whereas Vero cells were more sensitive when CPE-treated on their apical surface; those sensitivity differences correlated with CPE binding the apical versus basolateral surfaces of these two cell lines. (ii) CPE-treated Vero cells released (86)Rb into both Transwell chambers, whereas CaCo-2 cells released (86)Rb only into the CPE-containing Transwell chamber. (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form TJs. The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming TJs. Confluent CaCo-2 Transwell cultures CPE-treated for >1 h formed the approximately 200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their TJs. Collectively, these results identify CPE as a bifunctional toxin that, in confluent polarized cells, first exerts a cytotoxic effect mediated by the approximately 155-kDa complex. Resultant damage then provides CPE access to TJs, leading to approximately 200-kDa complex formation, internalization of some TJ proteins, and TJ damage that may increase paracellular permeability and thereby contribute to the diarrhea of CPE-induced gastrointestinal disease.     

Establishment of a peritubular myoid-like cell line and interactions between established testicular cell lines in culture

Established cell lines and primary cultures derived from somatic cells of the testis have been used to study cell-cell interactions. Primary cultures of Sertoli cells or Sertoli-derived cell lines from the mouse (TM4) and rat (TR-ST) will aggregate when plated on monolayers of primary cultures of peritubular myoid cells or a rat (TR-M) cell line which has many properties of peritubular myoid cells. Time-lapse cinematography and scanning and transmission electron microscopy reveal that Sertoli cells formed aggregates after 1 day in coculture, display surface activity and move on the monolayer. When these aggregates touch one another, they rapidly combine. By the 4th day of culture, spherical aggregates are composed of 50 to 200 cells. They do not display surface activity or movement on the myoid monolayer. On the 5th and 6th day of culture most spherical aggregates have flattened to form dome-shaped aggregates in close association with the monolayer. Cells in the aggregates are characterized by long microvilli and some ruffles. In large aggregates, cells sometimes form close associations within the aggregates although junctions are seldom observed. Sertoli-derived cell lines will not aggregate on monolayers of Leydig-derived (TM3) or testicular endothelial-derived (TR-1) cell lines. Neither TM3 nor TR-1 cells will aggregate when plated on myoid monolayers. The TR-M cells produced an extensive extracellular matrix beneath the cells which contains collagen, an amorphous globular material resembling elastin and a fibrous noncollagenous component. Sertoli cells plated on this matrix will not aggregate. Thus the aggregation of Sertoli cells on myoid cell monolayers is cell type, but not species dependent and not determined solely by extracellular matrix components produced by TR-M cells.