Introduction»

The general background information on Na,K-ATPase in MDCK cells has been

outlined in Chapter 1, however, additional information on blister formation and Na,K-

ATPase expression in MDCK cells is outlined below.

Leighton et al. (1969) were the first to demonstrate that MDCK cell monolayers viewed under the light microscope formed hemispherical vesicles or blisters. These

blisters were suggested to be comprised of interstitial collections of fluid which had accumulated in the plane between the cell monolayer and the impermeable glass substrate (Leighton et ai, 1969). The properties of the cell monolayers in blistered areas has been shown not to be different from non-blistered areas as the morphology, polarity, and relative ionic permeabilities of the monolayer in these regions were similar (Rabito et al.,

1978). The composition of the fluid contained within blisters has been shown not to be significantly different from culture medium in terms of its sodium and potassium contents (Rabito et al., 1978). The fluid has therefore been suggested to represent reabsorbed components of the growth medium, probably accumulated by the vectoral transport of

salts (Leighton et al., 1969). Blister formation has been shown not to occur on permeable collagen or MilUpore filter substrates (Cereijido et al., 1981a). This was a function of the substrate permeability, rather than its composition, as blisters form on collagen coated glass substrates (Rabito et al., 1978).

A model of blister formation on impermeable substrates has been proposed by Cereijido et al. (1981a). The model predicts that in blistered areas, the tight junctions formed between cells are stronger than the cell-substrate interactions. As fluid accumulates under the monolayer, pressure on both the tight junctions and the cells adhesion to the substrate increases. In some areas, fluid pressure results in the

detachment of the cell monolayer from the substrate and blister formation. While in other regions the tight junctions are burst open allowing the interstitial fluid to return to the

medium. The lateral diffusion of fluid through the interspace between cells , was thought to be negligible, or else blister formation would not occur (Rabito et al., 1978). The formation of blisters on permeable substrates is prevented by diffusion of fluid through the substrate, leaving the cells tight junctions intact and allowing the formation of

electrically resistant cell monolayers. As evidence in support of the model of blister formation, Cereijido et al. (1981a) have shown that [^^^I]-lactoperoxidase labelling of cell surface proteins, was no different in cell monolayers grown on impermeable

substrates, before or after cell tight junctions were dissociated with EGTA. Lactoperoxidase has also been used to label proteins from the apical side of MDCK strain I and II cell monolayers, grown on permeable Millipore filters (Richardson et aU, 1981) or permeable collagen coated nylon substrates (Cereijido et al., 1978), respectively. These experiments have demonstrated that the lactoperoxidase enzyme could not penetrate the tight junctions of MDCK cells, to label proteins on the basolatcral side of the

monolayer, when cells were grown on permeable substrates. Major cell proteins (such as Na,K-ATPase; Caplan et al., 1986; Lamb et al., 1981) are known to be polarised to the

basolateral membrane domain (Na,K-ATPase >90%; Caplan et al., 1986). When cells were grown on impermeable substrates, lactoperoxidase must have had access to the

basolateral domain, because otherwise a larger set of proteins would have been labelled when the tight junctions were dissociated with EGTA. As a consequence of the model of blister formation, only the relatively small percentage of cells associated with blistered areas of the monolayer would have access to their basolateral membranes restricted. Thus the number of [^H]-ouabain binding sites (Na,K-ATPase units) should be determinable

on cells grown on permeable or impermeable substrates, with only a small underestimate

of ouabain binding sites occulting in monolayers with blisters (Cereijido et al., 1981a). There is some evidence from this laboratory (unpublished data; see appendix 4) and others (Kennedy and Lever, 1984; Nakazato et al., 1989) that the amount of cellular

ouabain binding and Na,K-ATPase activity declines as cell cultures approach confluence. The level of pH]-ouabain binding on plastic supports has been reported to be lower than that of cells grown on filter supports (Cereijido et al„ 1981a; Kennedy and Lever, 1984). Wide differences in the number of pH]-ouabain binding sites per cell and hence Na,K-

ATPase units, have been reported in woik on MDCK cells. The numbers estimated range from 2.33 X 10^ (Lamb et al., 1981) to 8 x 10^ sites per cell (Kennedy and Lever, 1984).

Those variations may reflect differences in culturing conditions or cell density when

measurements of ouabain binding were made. However, Nakazato et al. (1989) have shown that different cloned lines of MDCK cells exhibit altered morphologies and Na,K-

ATPase activities during growth. They (Nakazato et al., 1989) have characterised the Na,K-ATPase activity of clonal cell lines which were either motile or non-motile during growth, the two morphological types having different Na,K-ATPase activities. The motile clones, which were judged to be motile during repeated microscopic observations, had extended and flattened cytoplasms and a relatively low level of Na,K-ATPase activity in the first 2 days of growth. This was compared to the non-motile clones, which were

cuboid with few if any cell processes and had a relatively high level of Na,K-ATPase activity. The data determined by Nakazto et al. (1989) suggests that some of the

differences in the number of enzyme units found by several authors may also be due to

the use of different subclones of MDCK cells. In support of this idea, Richardson et al.

(1981) also found differences in the number of enzyme units between two strains of MDCK cells known as Strain I and H.

The puipose of this study was to determine factors affecting the surface expression of Na,K-ATPase units (ouabain binding sites) in MDCK strain I cells, and to correlate alterations in enzyme abundance with changes in Na,K-ATPase a subunit mRNA abundance. In view of the above information, the factors investigated were; 1, changes in the seeding density of cells (ie alteration in the level of cell-ceU contacts), 2, different types of growth surfaces (ie changes in cell-substrate interactions), and 3, the effect of changes in the volume of growth medium. To confirm and extend the work of others (Cereijido et al., 1981a; Rabito et al., 1978), experiments were also performed to test the possible restriction of access of ouabain to the basolateral surface of MDCK strain I cell

monolayers grown on impermeable supports.

6,11, MgthQda*

6.n.i. Cell morphology.

Changes in MDCK cell morphology during culture resulting from different seeding densities was examined by phase contrast light microscopy. The MDCK cells were grown on coverslips at two different seeding densities; a high seeding density of 4.17 x 1 0^ cells/cm^ (the normal seeding density used for cell culture), and a low seeding density of 4.9 x 10^ cells/cm^. Cells were observed after 1,2,4, and 6 days in culture.

Phase contrast light micrographs were also taken of cells growing at high density (as above) on permeable collagen culture plate inserts (ICN Biomedicals; see Chapter 2). Micrograph were taken using a Leitz Dialux 20 microscope and 32 ASA Kodak Pan-X film. An increased number of cells appeared to attach to the central portion of the cover slips and collagen inserts used for the light micrographs. The light micrographs illustrated in figures 1-3 were all taken from the central portion of the cover slip or collagen insert except where otherwise stated.

6.n.ii. Na.K-ATPase e^p.ressiQ.n»

The other methods used were as outlined in Chapters 2. Na,K-ATPase a subunit

mRNA abundance was determined by the dot blotting technique. This was because large numbers of samples were to be blotted, making Northern blotting unsuitable. The rat a isoform-specific DNA probes were unsuitable for use in dot blotting (see section

5.in.iii), as the low stringency hybridisation and washing conditions required for their use, resulted in high level of background signals. As the high stringency conditions used previously with the pHANK probe (partial human a l cDNA; see section 4.I.Ü) showed no signs of background signals on Northern blots and as only a l isoform mRNA was detected in MDCK cell RNA, this probe was chosen in preference to the rat DNA probes for use in dot blotting.

6.IÏI, Rfisults and Plscussion,

6.ni.i. Light micrographs of MDCK cells during growth on glass supports.

Figure 1 shows the effect of high and low seeding density on the subsequent

moiphology of MDCK strain I cells grown in culture. The light micrographs showed that after 24 hours of culture, there was a considerable difference in the morphology between high and low density seeded cultures. As expected, the low density seeded cells appeared mostly as single cells often connected by cell processes (figure la), whereas, the high

density seeded cells were mostly grouped into islands of cells (figure lb). The high

density seeded cells showed evidence of blisters being present well before confluence was established, even after only one day of culture (figure lb). The culture of high

density seeded cells reached confluence after only two days (figure Id), whereas, confluence was only achieved after 4 days culture at the low seeding density (figure le). The MDCK cells exhibited long cellular processes at low density (figure 2a), and often had several cell processes connecting an individual cell to several of the surrounding cells (figure 2b). Micrographs of the cells at low density (figure la, Ic, 2a, and 2b), showed

that many of the cells had cell processes suggesting that the cell population consisted largely of the 'motile* class of MDCK cells, as suggested by Nakazato et al. (1989). However, at the edges of cover slips where the cells were originally less densely populated, the edge of the cell monolayer surrounding large gaps (in the monolayer), had

Figure 1 a-g. Shows phase contrast light micrographs of MDCK cells grown on glass supports. Cells were grown for up to six days after seeding at high or low density (see Chapter 2).

Incubation

(Days). Low dseedensity ed. Higseeh ddensity ed.

J 2 0 0 |im

Figure 2 a-f. Shows phase contrast light micrographs of various morphological of MDCK cells grown on glass supports. Typical cell morphological features include a) single cells connected by a long cell process, b) a cell connected to three other cells by processes, c) the edge of a confluent cell monolayer, where few cell processes were apparent, d) blisters or domes formed on single cells, e) overlapping domes or blisters, f) magnified view of e). The bars illustrated on micrographs represent 20 M-m.

very few cell extensions (see figure 2c). Cells with few extensions were characterised by Nakazato et al. (1989) to be ‘non-motile’ (see introduction). Therefore after seeding at low density, it was possible to find in the population, cells exhibiting both motile and non-motile morphological characteristics. Blisters in this strain of MDCK cells were formed underneath single cells and did not appear to require the formation of a confluent cell monolayer (figure 2d). When blisters were formed in close proximity to each other, they were also capable of overlapping (see figure 2e and 2f). This suggested that cells were capable of vectoral transport before the complete formation of a confluent

monolayer or tight junctions (a marker for the polarisation of cells).