Enhancement of galactose/N-acetylgalactosamine receptor activity on the surface of freshly isolated rat hepatocytes: evidence for masking of receptor sites by inhibitors derived from collagenase preparations.

Rat hepatocytes prepared by collagenase perfusion of the liver are known to exhibit increased binding of asialoorosomucoid (ASOR) after prior treatment with EDTA or after warming at 37 degrees C. The cause of the apparent increase in the surface binding activity of the galactose/N-acetylgalactosamine (Gal/GalNAc) receptor on freshly isolated rat hepatocytes was investigated. Binding experiments using three different galactose-terminated ligands revealed up to a 2- to 6-fold increase in the level of surface receptor sites on rat hepatocytes upon prior incubation at 4 degrees C with 10 mM GalNAc or 10 mM EDTA or at 37 degrees C compared to untreated cells. With digitonin-permeabilized cells, it was shown that the newly exposed receptor sites most likely originated from masked surface receptor sites, as no alteration in the size of the internal pool of receptor was observed. Collagenase preparations were found to inhibit the binding of 125I-labeled ASOR to the Gal/GalNAc receptor. Exposure of hepatocytes to collagenase resulted in a significant decrease in 125I-labeled ASOR binding, which was reversible upon treatment with GalNAc or EDTA at 4 degrees C or upon warming at 37 degrees C. Perfusion of EDTA through the isolated whole liver at 0-2 degrees C to remove any possible bound endogenous ligands did not result in a significant increase in the level of 125I-labeled ASOR binding, while perfusion of collagenase caused a marked decrease in the binding activity of the liver. We conclude that the enhancement of Gal/GalNAc receptor activity on the surface of freshly isolated hepatocytes by temperature and EDTA is potentially an artifact of the collagenase perfusion method.