Investigation of endosomal compartments involved in endocytosis and transcytosis of polymeric immunoglobulin A by subcellular fractionation of perfused isolated rat liver

Abstract

1. A gamma camera was used to monitor continuously the uptake of radiolabelled polymeric immunoglobulin A (pIgA) into the rat body after intravenous injection. Uptake into liver was fast but, since the peak of liver labelling occurred only after 9-15 min, it was not sufficiently rapid to constitute a pulse dose. A perfused, isolated rat liver system was therefore established which could be given a single pass dose of pIgA; a variety of tests showed such livers remained viable for at least 3 h and could be subsequently fractionated on Ficoll and Nycodenz gradients with normal distributions of marker enzymes. 2. Subcellular fractionation at different times after a single pass dose of pIgA showed that whilst pIgA appeared sequentially in sinusoidal plasma membrane, light endosomes, dense endosomes, very dense endosomes and lysosomes as in vivo, the predominance of pIgA in the light endosome compartment disappeared much earlier than after injection in vivo of pIgA, presumably because this compartment was not being continuously loaded over the first 10-15 min. The time course of appearance of label in bile was unchanged. A large excess of unlabelled asialofetuin did not change these patterns, indicating that the asialoglycoprotein receptor was not involved. 3. Low doses of the microtubule agent colchicine reduced the proportion of pIgA reaching the bile, but subcellular fractionation of treated liver showed that distribution of label amongst liver fractions was little changed, although the overall liver pIgA content had increased. This would suggest that pIgA did not remain in the common compartment which could have supplied bile or lysosomes but rather flowed out of it as rapidly as in untreated liver but towards those compartments supplying the lysosomes. 4. Experiments with nocodazole, which reversibly disrupts microtubules, showed that very little of the pIgA taken into an inhibited liver appeared in the bile after nocodazole was removed 30 min later, even though a second dose of pIgA, given after nocodazole removal, appeared in bile with a normal time course. The first dose of pIgA must therefore have passed beyond the compartments competent to supply the bile before nocodazole was removed. Such compartments were undamaged since the second dose of pIgA appeared in bile normally. We therefore conclude that the bulk of pIgA must be supplied to the bile from light or dense endosomes rather than from very dense endosomes and lysosomes.

5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

“…( PM fraction at 150 min chase was greatly decreased; by 210 min it was almost undetectable. The behavior of plgAR on the domain gradient is consistent with what is known about its localization in liver (Sztul et al, 1985b;Luzio et al, 1986;Perez et al, 1988;Quintart et al, 1989). The appearance of this receptor at the basolateral surface is brief, and internalization into a pool of endosomal/transcytotic vesicles occurs rapidly.…”

“…The distribution of pIgAR in lower density membranes is consistent with its known intracellular distribution at steady state. Most is either in transit through the Golgi complex or resides in transeytotic and endocytic vesicles, all of which are lighter than ER and PM (Perez et al, 1988;Quintart et al, 1989). The time of cellular transit after ligand binding is known to be considerable, since radiolabeled plgAR injected into the blood does not reach a peak in the bile until 40 min later (Hoppe et al, 1985).…”

Expression of a Functional Asialoglycoprotein Receptor in Human Renal Proximal Tubular Epithelial Cells

“…A possible mechanism could be ASGPR on renal tubular cells participating in clearance of (a)sialoglycoprotein from the serum by endocytosis, a function that has been mainly attributed to the liver ASGPR. Transcytosis of (a)sialoglycoproteins into the urine seems unlikely given its resemblance to the hepatic ASGPR [30]. Fibronectin, or its desialylated form, is a possible endogenous ligand for the ASGPR [31].…”

Dissection of Hepatic Receptor-Mediated Endocytic Pathways Using Self-Generated Gradients of Iodixanol (Optiprep)

“…The resolution of the endocytic compartment in sucrose gradients is severely limited by the high osmotic pressure of concentrated sucrose solutions (18, 19). The high viscosity of Ficoll makes handling difficult although it has been used to resolve the intracellular compartments involved in the endocytosis of asialofetuin and polymeric IgA by perfused livers (20,21). Similarly, Percoll (22) and nycodenz (23) have also been used to study hepatic endocytosis by density gradient centrifugation.…”