Investigation of endosomal compartments involved in endocytosis and transcytosis of polymeric immunoglobulin A by subcellular fractionation of perfused isolated rat liver.
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.
Perez
J H
JH
Department of Clinical Biochemistry, University of Cambridge, U.K.
Branch
W J
WJ
Smith
L
L
Mullock
B M
BM
Luzio
J P
JP
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D001646:Bile / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects; D002458:Cell Fractionation; D002500:Centrifugation, Isopycnic; D003078:Colchicine / Q000494:pharmacology; D004705:Endocytosis; D007070:Immunoglobulin A / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects; D015739:Nocodazole; D011108:Polymers / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000276:immunology / Q000378:metabolism
D001562:Benzimidazoles; D007070:Immunoglobulin A; D011108:Polymers; C034639:polymeric IgA; D015739:Nocodazole; D003078:Colchicine