Unrelated surface control of commitment to growth and attachment in isolated adult rat hepatocytes.

Abstract

The relation between surface glycoconjugate molecules involved in the control of commitment to growth and attachment was investigated in adult Wistar rat hepatocytes in primary culture. On the one hand, the approach has been to analyse the binding of 3H-labelled concanavalin A ([3H]Con A) to cell surfaces when these two cellular phemona were in turn modulated. The replacement of calf serum (CS) by foetal calf serum (FCS) in culture medium greatly enhanced cell attachment, its effect being about the same as that of FCS adsorbed to glass culture plates, but this change did not significantly affect the growth-stimulatory process. In contrast 2 or 3 microM-cytochalasin D (CD), which is known to act specifically on the inner face of the cell membrane, reversibly decreased the [3H]thymidine (dThd) incorporation in both CS-containing and FCS-containing media, but had only a very moderate effect on cell attachment. The reversible effect of the drug, too, was followed by [3H]leucine incorporation, as well as by measuring the [3H]dThd incorporation in media with CD plus insulin and glucagon. Hepatocytes were assayed for binding either in buffered solution (at 4 or 37 degrees C) or in serum-containing media (at 37 degrees C). The Hill coefficients were calculated to be near 2.2 and 1.9, respectively for CS-stimulated and FCS-stimulated cells, and around 1.6 for CD-treated and glutaraldehyde-fixed cells. We interpret these results as showing that two different types of surface components might be involved in the control of commitment to growth and attachment. On the other hand, evidence was presented to indicate that these components really bind Con A. First, Con A treatment in buffered solution of cells previously attached in the presence of CS or FCS resulted in a progressive inhibition of EGTA-mediated cell detachment from substrata. Secondly, Con A reversibly decreased [3H]dThd incorporation in both CS-containing and FCS-containing media through a specific interaction with the cell surface. We conclude that entrance into S phase and attachment to serum protein substrata are unrelated phenomena in isolated adult rat hepatocytes and seem to be regulated by two species of surface glycoconjugates.