Permeability Of Hydrophilic Essay, Research Paper

Permeability of Hydrophilic

Supervisors: Vladan Milovic Professor Per Artursson

SUMMARY

Investigations of the integrity and transport characteristics of 2/4/A1 cells

have been done in this report. The cell line was isolated from rat fetal

intestinal epithelial cells and transfected with thermolabile SV40 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/A1 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 paracellular transport characteristics similar to that of the

human intestine. Transmission electron microscopy confirmed the development of

multilayers at 33?C, monolayers at 37?C and defects in the cell layer due to

apoptosis at 39?C.

Different immunostainings of ZO-1, E-cadherin and vinculin confirmed formation

of tight and adherence junctions. Transepithelial resistance reached a plateau

of 25-35 Ohm.cm2, which was similar to the small intestine. In transport studies

2/4/A1 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/A1 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 transcellular route, or across the tight junctions between

the cells – the paracellular 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 endocytosed vesicles.

The hydrophilic and charged drugs are absorbed after passing through the

paracellular route, the water-filled channels between the cells (Artursson

1991). Rates and extent of the paracellular 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 lipophilicity (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 (Audus 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 & 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 (Lennern?s et al., 1995),

although a considerable variability has been reported, being related to

heterogenity, a number of sub populations, and number of passages (Walter &

Kissel, 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.cm2 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/A1) is derived from the rat fetal

intestinal epithelial cells conditionally immortalized with thermolabile SV40

large T antigen, pzipSVtsa58 (Paul et al. 1993). According to the original

report, these cells form more leaky monolayers, with paracellular 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

microvilli; tight junctions are also present (Paul et al. 1993; Hochman,

personal communication).

The 2/4/A1 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 multilayers. 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 paracellular marker

molecules across the 2/4/A1 cell line monolayers and to look at the

characteristics of the cell line.

MATERIALS AND METHODS

Cell culture

2/4/A1 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 geneticin, 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 selenic acid (ITS premixTM, Collaborative

Research), with 5-6% CO2 and 95% humidity.

The cells were seeded on Transwell polycarbonate filter inserts (? 6.5 mm)

coated with ECL extracellular matrix (entactin-collagen IV-laminin; Promega,

Madison, Wisconsin, USA), at a density of 100,000 cm2 in a serum-free RPMI 1640

medium supplemented with 10 mM Hepes, 2 mM L-glutamine, 200 mg/ml geneticin, 1

mg/ml BSA, 2 mg/ml dexamethasone, 20 ng/ml EGF, 10 mg/ml insulin, 10 mg/ml

transferrin and 10 ng/ml selenic acid.

Transport studies

Paracellular markers of different size and molecular weight labelled with 14C 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 Hank’s 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 (C0). 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? & Karlsson,

unpublished results). Development of electrical parameters in 2/4/A1 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 morphology

2/4/A1 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 Aharoni 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 vinculin.

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 SV40

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 vinculin 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/A1 cells

2/4/A1 cells seeded on ECL coated filter supports showed different growth rate

dependent on the temperature. At 33?C 2/4/A1 cells proliferated rapidly, growing

exponentially until day 4 after seeding and forming multilayers 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 SV40 large T

antigen, the presence of the antigen was a prerequisite for growth in 2/4/A1

cells. SV40 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 SV40 large T antigen in 2/4/A1 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/A1 cells

seeded at 33?C, 37?C and 39?C. Bar indicates 5 mm.

Figure 4. Vertical sections of 2/4/A1 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 vinculin (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/A1

cells grown at all temperatures. Its distribution, however was uneven in the

multilayers 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 vinculin 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

multilayers 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 microvilli and without

visible brush borders. These data imply that 2/4/A1 cells may be presumably used

as a model of paracellular transport, in which the influence of brush border

enzymes and transcellular transport systems does not interfere with the

paracellular pathway.

This data indicates that well developed tight and adherence junctions occur when

2/4/A1 cells are grown at 37?C. We therefore decided to evaluate 2/4/A1 cells

grown at 37?C as a model for paracellular transport of hydrophilic drugs across

the small intestine.

Transepithelial resistance

TEER reached a plateau of 25-35 Ohm.cm2 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/A1 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/A1 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/A1

cell line discriminated well between the paracellular 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 4.5×10-6 cm/s and correlated well to the human intestine (Figure 7).

When compared to Caco-2 cell line, 2/4/A1 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

paracellular pathway

Figure 7. Permeability of hydrophilic marker molecules across 2/4/A1 cell line

monolayers. N=6.

Figure 8. Permeability of hydrophilic marker molecules across 2/4/A1 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/A1 cell line

monolayers in Hanks balanced salt solution with (left) and without calcium

(right). N=4. *, p