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Permeability of Hydrophilic Supervisors: Vladan Milovic Professor Per Artursson SUMMARY Investigations of the integrity and transport characteristics of 2 / 4 /A 1 cells have been done in this report. The cell line was isolated from rat fetal intestinal epithelial cells and transfected with thermolabile SV 40 large T antigen. These cells proliferated at 33? C, but eliminated the antigen and ceased proliferating at a non-permissive temperature (39?
C). At 39? C 2 / 4 /A 1 cells started to differentiate but simultaneously the cells also underwent massive cell death. When cultured at 37? C these cells formed confluent and tight monolayers that seemed to have para cellular transport characteristics similar to that of the human intestine. Transmission electron microscopy confirmed the development of multilayer's at 33?
C, monolayers at 37? C and defects in the cell layer due to apoptosis at 39? C. Different immunostaining's of ZO- 1, E-cadherin and vin culin confirmed formation of tight and adherence junctions. Transepithelial resistance reached a plateau of 25 - 35 Ohm. cm 2, which was similar to the small intestine.
In transport studies 2 / 4 /A 1 cell line monolayers selectively restricted the permeation of hydrophilic permeability markers proportional to molecular weight and discriminated more accurately between the molecules of intermediate molecular weight compared to Caco- 2 cells. These results indicated that 2 / 4 /A 1 cells could be used as a model for hydrophilic drug absorption. INTRODUCTION The small intestine plays a crucial role in the absorption of drugs and nutrients. Exogenous substances cross a series of barriers during the process of intestinal absorption: (1) the aqueous boundary / mucus layer, (2) a single layer of epithelial cells, and (3) the lamina propria, which contains the blood and lymph vessels that then transport the absorbed drugs to other parts of the body (Artursson 1991).
The cell monolayer is comprised of two parallel barriers: the cell membrane and the tight junctions. Most drugs are absorbed by a passive diffusion across the cell membrane by the trans cellular route, or across the tight junctions between the cells the para cellular route. Drug transport can also be carrier mediated, when the drug utilizes transporters located in the cellular membrane. Transcytosis is another kind of active transport, in which macromolecules can be transported across the intestinal epithelial cell in endocytosis vesicles. The hydrophilic and charged drugs are absorbed after passing through the para cellular route, the water-filled channels between the cells (Artursson 1991). Rates and extent of the para cellular transport are, therefore, highly influenced by the structure and size of the tight junctions as well as by the size of the molecules.
Only small and hydrophilic drugs can pass between the cells rapidly and completely; permeation of larger molecules can be limited proportionally to their size and lipophilic ity (Hillgren et al. 1995). Simple assay methods are needed for drug absorption studies. Excised intestinal tissue, isolated cells, membrane vesicles and in vivo models have distinct limitations, which have been previously discussed in detail (August et al. 1990; Artursson 1991; Hillgren et al. 1995). The most suitable method for the study of drug intestinal transport appeared to be the use of cultured intestinal epithelial cells.
This model has several advantages over conventional drug absorption models: (a) it is less time-consuming; (b) it enables rapid evaluation of methods for improving drug absorption; (c) it allows an opportunity to use human rather than animal tissues; (d) it can minimize expensive and sometimes controversial animal studies. Human colorectal carcinoma cell line Caco- 2 is nowadays the most widely used and the best explored model for drug intestinal transport (Hidalgo et al. 1989; Artursson 1990; Artursson &# 038; Karlsson 1991). This cell line displays spontaneous enterocytic differentiation in culture and forms a polarized monolayer with apical brush borders and well differentiated tight junctions (Hidalgo, 1989). Drug transport studies across the Caco- 2 cell monolayers showed a satisfactory correlation with other in vitro absorption models, e.
g. rat intestinal segments (Artursson et al. 1993) and in vivo drug absorption (Lenders? s et al. , 1995), although a considerable variability has been reported, being related to heterogeneity, a number of sub populations, and number of passages (Walter &# 038; Kissed, 1995). Caco- 2 cells however, form monolayers that resemble colonic rather than small intestinal epithelial cells. Due to its well-formed tight junctions, Caco- 2 cell monolayers have a transepithelial electrical resistance of 260 Ohm.
cm 2 which is similar to the transepithelial electrical resistance of the colon rather than of the small intestine (Hillgren et al. 1995). Therefore, there is a need to investigate drug intestinal transport in a model which has apparent transport characteristics corresponding to the human intestine, and several studies have been attempted to characterize a cell line that can be used for this purpose. A novel intestinal epithelial cell line (2 / 4 /A 1) is derived from the rat fetal intestinal epithelial cells conditionally immortalized with thermolabile SV 40 large T antigen, pzipSVtsa 58 (Paul et al. 1993). According to the original report, these cells form more leaky monolayers, with para cellular transport characteristics similar to that of the human intestine. When cultured at 32? C these cells continually proliferate and display few markers of intestinal differentiation.
However, after being switched to a non-permissive temperature (39? C), these cells cease proliferating and exhibit a more markedly differentiated phenotype. They form a polarized monolayer covered with a few micro villi; tight junctions are also present (Paul et al. 1993; Hochman, personal communication). The 2 / 4 /A 1 cell line has been preliminary investigated in this laboratory. It appeared that cells grown at 39? C underwent massive apoptotic cell death simultaneously with differentiation, and that those grown at permissive temperature continued proliferating and form multilayer's.
However, when grown at an intermediate temperature (37? C), the cells underwent apoptosis to a lesser extent, but maintained their proliferative capacity sufficiently to form tight and continuous monolayers. The aim of this study was to investigate permeability of para cellular marker molecules across the 2 / 4 /A 1 cell line monolayers and to look at the characteristics of the cell line. MATERIALS AND METHODS Cell culture/A 1 cells were expanded in flasks at 33? C, in RPMI 1640 medium supplemented with 2 % fetal calf serum, 10 mM Hepes, 2 mM L-glutamine, 200 mg / ml genetic, 1 mg / ml BSA, 2 mg / ml dexamethasone, 20 ng / ml EGF, 50 ng / ml IGF-I, 10 mg / ml insulin, 10 mg / ml transferrin and 10 ng / ml splenic acid (ITS premix TM, Collaborative Research), with 5 - 6 % CO 2 and 95 % humidity. The cells were seeded on Transwell polycarbonate filter inserts (? 6. 5 mm) coated with ECL extracellular matrix (ent actin-collagen IV-laminin; Promega, Madison, Wisconsin, USA), at a density of 100, 000 cm 2 in a serum-free RPMI 1640 medium supplemented with 10 mM Hepes, 2 mM L-glutamine, 200 mg / ml genetic, 1 mg / ml BSA, 2 mg / ml dexamethasone, 20 ng / ml EGF, 10 mg / ml insulin, 10 mg / ml transferrin and 10 ng / ml splenic acid.
Transport studies Para cellular markers of different size and molecular weight labelled with 14 C or fluorescein were used: mannitol (MW 182), fluorescein (MW 376), lucifer yellow (MW 450), polyethylene-glycol 4000 (MW 4000), and dextran (MW 50, 000). The experiments were performed at 37? C in Hanks Balanced Salt Solution pH 7. 2 under sink conditions. When PEG 4000 was used unlabelled PEG 4000 was also added to the donor solution to limit possible drug metabolism. The labelled marker molecules, 250 ml, were added to the apical side of the monolayer and after 20, 40, 60 and 80 minutes the inserts were moved to new wells and 500 ml samples taken from the basolateral solution.
Prior to the experiments samples of 50 ml were taken from the apical solutions for measurements of the initial concentration (C 0). All solutions were preheated to 37? C, and a heating plate was used when the wells were moved. Transport was measured over time (days 1 - 10) and compared with the values obtained from Caco- 2 monolayers used as standard.
The radioactivity of the samples was determined using a standard liquid scintillation technique. The apparent permeability coefficient was calculated as described before (Artursson 1990), using a Microsoft Excel 4. 0 software package (Macintosh Power PC computer and Microsoft Office software) and templates modified by K. Palm. Electrophysiological measurements Transepithelial electrical resistance, short circuit current and potential difference were measured by an in-house computer-based automatic system using a single unit Transwell diffusion chamber (Gr? sj? &# 038; Karlsson, unpublished results).
Development of electrical parameters in 2 / 4 /A 1 cells was studied over time (days 1 - 10). The data was processed using a Lab View software package modified by Gr? sj? et al.
Cell morpho log/A 1 cells were routinely monitored under phase-contrast microscope each day. At appropriate time points nuclei were stained with DAPI (4, 6 -diamidino- 2 - phenylindolole, Molecular Probes, Leiden, Holland). The percentage of apoptotic nuclei was quantified according to the method of Aaron et al. (1995). Cells grown on filters at different temperatures were examined by transmission electron microscopy (TEM) after fixation in glutaraldehyde and dehydration with 1 % osmium-tetroxide and 1 % uranyl acetate. The presence of actin was assessed by direct immunofluorescence with rhodamine-conjugated phalloidin.
Development of tight junctions were studied by indirect immunofluorescence to ZO- 1 protein, and adherence junctions by immunostaining to E-cadherin and vin culin. Immunohistology slides were processed under laser scanning confocal microscope (Leica, Heidelberg, Germany) and images were obtained by Silicon graphics software package. Materials If not otherwise indicated, cell culture media and supplements were purchased from Life Technologies AB, T? by, Sweden. Mouse monoclonal antibodies to SV 40 large T antigen were from Oncogene Science, Uniondale, New York, USA, and rhodamine-conjugated phalloidin from Molecular Probes, Leiden, Holland. Rabbit polyclonal antibodies to ZO- 1 were obtained from Zymed Laboratories Inc. , San Francisco, USA, and mouse monoclonal antibodies to human E-cadherin from Transduction Laboratories, Lexington, Kentucky, USA.
Mouse monoclonal antibodies to human and rat vin culin were from Serotec, Oxford, UK. Statistics Numerical data is expressed as the mean + SD of four to six experiments. One-way ANOVA (corresponding to unpaired one-tailed Students t-test) was used to compare means. A 95 % probability was considered significant. RESULTS Growth of 2 / 4 /A 1 cell/A 1 cells seeded on ECL coated filter supports showed different growth rate dependent on the temperature. At 33?
C 2 / 4 /A 1 cells proliferated rapidly, growing exponentially until day 4 after seeding and forming multilayer's consisting of immature enterocytes. Growth was significantly reduced at 37? C and the cells formed monolayers. There was a decrease in cell number at 39?
C and 10 days after seeding only 15 % of the initial number of cells remained attached to the matrix. Apoptosis, as calculated per 1000 cells, was present at 33? C to a negligible extent, although the proportion of apoptotic cells raised steadily at 39? C. After 10 days no nuclei without apoptotic morphology were noted at this temperature. Number of apoptotic cells did not differ at the remaining two temperatures (Figure 1).
As estimated qualitatively by the immunohistochemical detection of SV 40 large T antigen, the presence of the antigen was a prerequisite for growth in 2 / 4 /A 1 cells. SV 40 large T antigen was present in the entire nuclei at 33? C, less prominent at 37? C, and poorly stained in the nuclei at 39? C (Figure 2). Figure 2.
Expression of SV 40 large T antigen in 2 / 4 /A 1 cells seeded at 33? C, 37? C and 39? C. Bar indicates 10 mm. Figure 3.
ZO- 1 (A, B, C), E-cadherin (D, E, F), and actin (G, H, I) in 2 / 4 /A 1 cells seeded at 33? C, 37? C and 39? C. Bar indicates 5 mm. Figure 4.
Vertical sections of 2 / 4 /A 1 cell layers seeded to 33? C (A, C, E) and 37? C (B, D, F) stained to ZO- 1 (A, B), E-cadherin (C, D) and vin culin (E, F). Bar indicates 5 mm. Development of tight and adherence junctions As estimated by the appropriate antibodies, ZO- 1 protein was present in 2 / 4 /A 1 cells grown at all temperatures.
Its distribution, however was uneven in the multilayer's at 33? C, reaching an intensively stained network at 37? C. At the non-permissive temperature the ZO- 1 pattern was discontinuous, indicating loosening of cell-to-cell contact preceding cell death (Figure 3, A-C). Adherence junctions were also present at all temperatures. E-cadherin formed a dotted network distributed diffusely in the cytoplasm at both 33 and 39?
C; the pattern was located more closely near the cellular membrane at 37? C (Figure 3, D-F). Actin filaments were well developed at all three temperatures, showing stress fibers at 33? C and being distributed evenly at 37? C in the cell membrane. At 39?
C the actin network indicated broadening of extracellular spaces and defects in the monolayer (Figure 3, G-I). ZO- 1 protein was located diffusely across the membrane at 33? C. On the contrary, at 37? C ZO- 1 was located exclusively in the upper pole of the cell-to-cell junctions, indicating that normal tight junctions are formed at 37? C.
At 39? C the ZO- 1 formed a discontinuous pattern located at the upper pole of the monolayer, but with clear defects in the staining pattern indicating defects in the cellular layer. E-cadherin and vin culin were located below the ZO- 1 band, forming a dotted network of filaments accumulated around the cell membrane (Figure 4). Transmission electron microscopy confirmed the development of multilayer's at 33? C, monolayers at 37? C, and defects in the layer due to apoptosis at 39?
C (Figure 5). Tight junctions occurred at all temperatures, although those at 37? C were longer and appeared tighter than those at 33? C. At all temperatures, at least within the time interval studied, the brush border membrane surface remained undifferentiated, with few micro villi and without visible brush borders. These data imply that 2 / 4 /A 1 cells may be presumably used as a model of para cellular transport, in which the influence of brush border enzymes and trans cellular transport systems does not interfere with the para cellular pathway.
This data indicates that well developed tight and adherence junctions occur when 2 / 4 /A 1 cells are grown at 37? C. We therefore decided to evaluate 2 / 4 /A 1 cells grown at 37? C as a model for para cellular transport of hydrophilic drugs across the small intestine.
Transepithelial resistance TEER reached a plateau of 25 - 35 Ohm. cm 2 after four days in culture. Resting potential and short circuit current were low throughout the time studied, and were consistent with the cellular morphology (Figure 6). Figure 6.
Transepithelial resistance, resting potential and short circuit current of 2 / 4 /A 1 cell line monolayers seeded at 37? C. Experiments were performed in Hanks balanced salt solution at 37? C.
N = 6. Figure 5. Transmission electron microscopy of 2 / 4 /A 1 cells seeded at (a) 33? C, (b) 37?
C and (c) 39? C. Bar indicates 5 mm. Transport studies Transport experiments were studied 1, 2, 4, 6 and 10 days after seeding. 2 / 4 /A 1 cell line discriminated well between the para cellular markers of increasing molecular weight, maintaining such a selective permeability throughout the investigated period. Papp values for molecules with molecular weight around 400 were about 4. 5 &# 215; 10 - 6 cm / s and correlated well to the human intestine (Figure 7).
When compared to Caco- 2 cell line, 2 / 4 /A 1 cells had 40 to 250 times higher Papp values and discriminated more accurately between the molecules of intermediate molecular weight (Figure 8). Transport of mannitol and PEG- 4000 in a calcium- free medium showed a two-fold increase in comparison to normal values (Figure 9). Since the adherence junctions can not function properly without calcium, this data indicates that the permeation of the markers is restricted mainly to the para cellular pathway Figure 7. Permeability of hydrophilic marker molecules across 2 / 4 /A 1 cell line monolayers. N = 6. Figure 8.
Permeability of hydrophilic marker molecules across 2 / 4 /A 1 cell line monolayers (A) and Caco- 2 cell line monolayers (B). Note that Papp values differ aprox. 100 -fold. N = 6. Figure 9. Permeability of mannitol (MW 182) and PEG- 4000 across 2 / 4 /A 1 cell line monolayers in Hanks balanced salt solution with (left) and without calcium (right). N = 4. , p
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