Effect of temperature and divalent cations on the substratum attachment of rat hepatocytes in vitro

The attachment of rat hepatocytes to polystyrene-adsorbed serum protein is relatively insensitive to inhibitors such as dextran sulphate, cycloheximide, colchicine and cytochalasin B, and enzymes like trypsin and neuraminidase, but it is strongly dependent on divalent cations. Mg2+ supports attachment better than Ca2+, but a combination of both is required for maximal attachment. The attachment is very temperature-sensitive, with a biphasic Arrhenius plot indicating an activation energy of 123 kJ/mol above 34 degrees C and 374 kJ/mol below 34 degrees C. The adsorbed attachment-promoting serum factor is inactivated by trypsin, or by Ca2+-dependent proteases which contaminate commercial preparations of collagenase. The adsorbed factor is resistant to treatment with glutaraldehyde, neuraminidase and heating to 90 degrees C, whereas the same factor in the unadsorbed state (in serum) is destroyed by heating to 70 degrees C. The factor in serum is unable to compete with the adsorbed factor for cell binding, hence it would appear that adsorption to polystyrene induces the active, heat-resistant conformation of the factor.