DIFFERENCES BETWEEN FLUID-PHASE ENDOCYTOSIS (PINOCYTOSIS) AND RECEPTOR-MEDIATED ENDOCYTOSIS IN ISOLATED RAT HEPATOCYTES

To characterize possible differences between the fluid-phase endocytos is (pinocytosis) of bovine serum albumin and the receptor-mediated end ocytosis of asialo-orosomucoid (AOM) in isolated rat hepatocytes, both probes were conjugated to radioiodinated tyramine-cellobiose, [I-125] TC. The use of these conjugates made it possible to measure the uptake and intracellular distribution of the intact proteins as well as of t heir acid-soluble, membrane-impermeant degradation products. [I-125]TC -albumin was taken up at a very Bow rate (0.5%/h) compared to [I-125]T C-AOM (45%/h), suggesting that neither membrane adsorption nor membran e permeation compromised its suitability as a fluid-phase marker. Sucr ose 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 [I-125]TC-albumin traversed the endocytic compartmen ts more rapidly than [I-125]TC-AOM, and was partially degraded intraly sosomally already after 15 min. Tile microtubule inhibitor, vinblastin e, had a stronger inhibitory effect on the uptake and degradation of [ I-125]TC-AOM (80% and 95%, respectively) than on the uptake and degrad ation of [I-125]TC-albumin (50% and 70%, respectively). In the presenc e of vinblastine, [I-125]TC-AOM was retained both in light and dense e ndosomes, whereas [I-125]TC-albumin was retained in dense endosomes on ly, suggesting that the early steps of fluid-phase endocytosis mere le ss critically dependent on microtubular function than the early steps of receptor-mediated endocytosis. A perturbant of vacuolar pH, propyla mine, inhibited the degradation of both probes strongly (75-100%), as would be expected from its lysosomotropic effect. Propylamine also inh ibited endocytic uptake, with a stronger effect on [I-125]TC-AOM uptak e (95% inhibition) than on [I-125]TC-albumin uptake (60% inhibition), probably reflecting a reduction in endosomal acidity, reduced receptor -ligand dissociation and diminished recycling of free asialoglycoprote in 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 [I-125]TC-albumin and [I-125]TC-AOM. An inhibitor of lysosomal protei nases, leupeptin, strongly suppressed the degradation of both probes a nd moderately reduced the uptake of [I-125]TC-AOM, whereas tile uptake of [I-125]TC-albumin was unaffected. In contrast, an inhibitor of aut ophagic sequestration, 3-methyladenine, reduced both the uptake and de gradation of [I-125]TC-albumin markedly (55% and 75%, respectively), w ith considerably less effect on [I-125]TC-AOM (25% and 35%, respective ly), An autophagy-inhibitory amino acid mixture did not share these ef fects, suggesting that 3-methyladenine may suppress endocytic fluid-ph ase uptake by an autophagy-independent mechanism. Fluid-phase and rece ptor-mediated endocytosis in hepatocytes thus appear to differ with re spect to uptake mechanisms as well as in the kinetics by which endocyt osed material traverses the endocytic-lysosomal pathway.