Fluid phase endocytosis by cultured rat hepatocytes and perfused rat liver: implications for plasma membrane turnover and vesicular trafficking of fluid phase markers.

Abstract

Hepatocytes take up a variety of ligands via receptor-mediated endocytosis, yet little is known regarding either the volume of fluid or the amount of membrane internalized via endocytosis in liver cells. In these studies, we have utilized radiolabeled inulin to characterize fluid phase endocytosis by rat hepatocytes in primary culture and perfused rat liver. Uptake of inulin by cultured hepatocytes was nonlinear with time, occurring most rapidly during the first 2 min. Inulin uptake and efflux in cultured hepatocytes and inulin uptake by perfused rat liver were kinetically compatible with the entry of inulin into a rapidly (t1/2, 1-2 min) turning-over (presumably endosomal) compartment that exchanged contents with the extracellular space and comprised -3% of hepatocyte volume, as well as entry into and concentration of inulin within slowly (t1/2, >1 hr) turning-over storage compartments. Based on inulin uptake, it is estimated that cultured hepatocytes endocytosed the equivalent of 20% or more of their volume and 5 or more times their plasma membrane surface area each hour. Neither chloroquine (1 mM) nor taurocholate (200 ,uM) affected inulin handling by cultured cells, whereas colchicine (10 ,uM) inhibited transfer to storage compartments by >50%. In conjunction with our previous observations, the present findings suggest that inulin endocytosed across the basolateral membrane is largely (Q80%) regurgitated back into plasma, with smaller amounts transported to intracellular storage compartments ("18%) or to bile (-2%). Transport of inulin via these pathways is unaffected by taurocholate and does not require vesicle acidification, whereas intact microtubular function is required for transfer to storage compartments or biliary secretion.The ability to internalize extracellular material appears to be a property shared by most, if not all, cells. Hepatocytes are actively endocytic and internalize a variety of ligands by receptor-mediated endocytosis (1, 2), yet relatively little is known regarding the overall rate at which hepatocytes internalize extracellular fluid or fluid phase markers (3, 4).We have recently reported evidence that the intact perfused rat liver transports a variety of fluid phase markers from perfusate to bile via a transcellular vesicular mechanism (5). In the present study, we have extended these observations in perfused liver and also characterized fluid phase endocytosis by rat hepatocytes in primary culture. In addition to measuring the rate at which hepatocytes endocytose extracellular fluid, our principal objectives were as follows. First, several recent reports indicate that fluid phase markers are internalized initially into a compartment that exchanges with extracellular fluid (6-8); however, the kinetics of this process, which presumably reflect membrane recycling, remain incompletely characterized. We therefore sought evidence of such a process in mammalian liver and characterized it kinetically. Our second objective was to integrate observations in perfused liver and culture...

A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells

“…It is therefore evident that a high proportion of the internalized membrane and a portion of the endocytosed components must be returned to the plasma membrane and extracellular space, respectively, by exocytosis. Our estimate of membrane internalization agrees closely with that calculated by Scharschmidt et al [6] for cultivated rat hepatocytes.…”

“…Based on the current data, the uptake of extracellular fluid was 0.27 ,al/h per 106 cells. This is in agreement with earlier reports on fluid-phase endocytosis in vivo [6,22], as well as in vitro [5,6,15]. Assuming that the initial vesicles formed during fluidphase endocytosis are 0.2,m in diameter [1] and the diameter of the parenchymal cells is 20 ,um [1], this rate of uptake reflects an internalization of membrane corresponding to the whole cell surface every 10 min.…”

“…In the present model, the rate constant for recycling of tracer from early endosomes was predicted to be 0.08 min-1, corresponding to a transit time of 12 min or a ti of 9 min, which is within the range of values published 2previously (t1 between 1 and 20 min) [6,[23][24][25][26] Vol. 262 compartment 3 to the medium was estimated to be 67 min.…”

“…A compartment with slow turnover similar to our compartment 3 was demonstrated by Besterman et al [23] in macrophages and fibroblasts. Entry of a fluid-phase marker into a slowly turning-over compartment has also been demonstrated in cultured rat hepatocytes [6]. Direct evidence for transfer of endocytosed protein from lysosomes to the medium has been obtained with mouse peritoneal macrophages.…”

“…Some newly formed endosomes will rapidly (within minutes) fuse with the plasma membrane [5,6]. The rest will probably fuse with each other to form larger endosomes, the so-called CURL (compartment for uncoupling of receptor and ligand) [7].…”