Differences between fluid-phase endocytosis (pinocytosis) and receptor-mediated endocytosis in isolated rat hepatocytes

Abstract

To characterize possible differences between the fluid-phase endocytosis (pinocytosis) of bovine serum albumin and the receptor-mediated endocytosis of asialo-orosomucoid (AOM) in isolated rat hepatocytes, both probes were conjugated to radioiodinated tyramine-cellobiose, [125I]TC. The use of these conjugates made it possible to measure the uptake and intracellular distribution of the intact proteins as well as of their acid-soluble, membrane-impermeant degradation products. [125I]TC-albumin was taken up at a very low rate (0.5%/h) compared to [125I]TC-AOM (45%/h), suggesting that neither membrane adsorption nor membrane permeation compromised its suitability as a fluid-phase marker. Sucrose gradient analysis indicated that both probes sequentially entered light endosomes (1.11 g/ml), dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml), but [125I]TC-albumin traversed the endocytic compartments more rapidly than [125I]TC-AOM, and was partially degraded intralysosomally already after 15 min. The microtubule inhibitor, vinblastine, had a stronger inhibitory effect on the uptake and degradation of [125I]TC-AOM (80% and 95%, respectively) than on the uptake and degradation of [125I]TC-albumin (50% and 70%, respectively). In the presence of vinblastine, [125I]TC-AOM was retained both in light and dense endosomes, whereas [125I]TC-albumin was retained in dense endosomes only, suggesting that the early steps of fluid-phase endocytosis were less critically dependent on microtubular function than the early steps of receptor-mediated endocytosis. A perturbant of vacuolar pH, propylamine, inhibited the degradation of both probes strongly (75-100%), as would be expected from its lysosomotropic effect. Propylamine also inhibited endocytic uptake, with a stronger effect on [125I]TC-AOM uptake (95% inhibition) than on [125I]TC-albumin uptake (60% inhibition), probably reflecting a reduction in endosomal acidity, reduced receptor-ligand dissociation and diminished recycling of free asialoglycoprotein receptors to the cell surface in addition to a general trapping of membrane in swollen vacuoles. A protein phosphatase inhibitor, okadaic acid, strongly (80-100%) inhibited the uptake and degradation of both [125I]TC-albumin and [125I]TC-AOM. An inhibitor of lysosomal proteinases, leupeptin, strongly suppressed the degradation of both probes and moderately reduced the uptake of [125I]TC-AOM, whereas the uptake of [125I]TC-albumin was unaffected. In contrast, an inhibitor of autophagic sequestration, 3-methyladenine, reduced both the uptake and degradation of [125I]TC-albumin markedly (55% and 75%, respectively), with considerably less effect on [125I]TC-AOM (25% and 35%, respectively). As autophagy-inhibitory amino acid mixture did not share these effects, suggesting that 3-methyladenine may suppress endocytic fluid-phase uptake by an autophagy-independent mechanism. Fluid-phase and receptor-mediated endocytosis in hepatocytes thus appear to differ with respect to uptake mechanisms as well as in the kinetics by which endocytosed material traverses the endocytic-lysosomal pathway.