Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes

Endocytosis may originate both in coated pits and in uncoated regions of the plasma membrane. In hepatocytes it has been shown that fluid phase endocytosis (here defined as 'pinocytosis') is unaffected by treatments that arrest coated pit-mediated endocytosis, indicating that pinocytosis is primarily a clathrin-independent process. In this study we have tried to determine possible connections between pinocytosis and clathrin-dependent endocytosis in rat hepatocytes by means of subcellular fractionation, electron microscopy, and by assessing the influence of inhibitors of clathrin-dependent endocytosis on pinocytosis. As marker for clathrin-dependent endocytosis was used asialoorosomucoid (AOM) labelled with [I-125]tyramine cellobiose ([I-125]TC). [I-125]TC-labelled bovine serum albumin ([I-125]TC-BSA) was found to be a useful marker for pinocytosis. Its uptake in the cells is not saturable, and any remnants of[I-125]TC-BSA associated with the cell surface could be removed by incubating the cells with 0.3% pronase at 0 degrees C for 60 min, The data obtained by electron microscopy and by subcellular fractionation suggested that early after initiation of uptake (<15 min) [I-125]TC-BSA and [I-125]TC-AOM were present in different endocytic vesicles. The two probes probably join prior to their entrance in the lysosomal compartment. The relation between endocytosis via coated pits and pinocytosis was also studied with techniques that induced a selective density shift either in the clathrin-dependent pathway (by AOM-HRP) or in the pinocytic pathway (by allowing uptake of AuBSA). Both treatments indicated that the two probes ([I-125]TC-AOM and [I-125]TC-BSA) were early after uptake, at least partly, in separate endocytic compartments. The different distribution of the fluid phase marker and the ligand (internalised via coated pits) was not due to a difference in the rate at which they enter a later compartment, since a lowering of the incubation temperature to 18 degrees C, which should keep the probes in the early endosomes, did not affect their early density distribution. Incubation of cells in a hypertonic medium reduced uptake both of [I-125]TC-AOM and [I-125]TC-BSA; the uptake of [I-125]TC-AOM was, however, reduced much more than that of the fluid phase marker. This finding supports the notion that the two probes enter the cells via different routes. (C) 1999 Published by Elsevier Science B.V. All rights reserved.