Inhibition of autophagy and multiple steps in asialoglycoprotein endocytosis by inhibitors of tyrosine protein kinases (tyrphostins).
In isolated rat hepatocytes, several tyrosine protein kinase inhibitors (tyrphostins) reduced the autophagic sequestration of electroinjected [3H]raffinose by 40-75% at doses that did not significantly affect cellular ATP levels or plasma membrane integrity. Tyrphostin 46 specifically inhibited autophagy, whereas tyrphostins 1, 25 and 51 also suppressed the receptor-mediated endocytic uptake of 125I-tyramine-cellobiose-asialoorosomucoid, 125I-TC-AOM, by 20-30% and its degradation by 70-90%. Tyrphostins 1 and 51, and the microtubule inhibitor vinblastine, inhibited an early endocytic step (endosome maturation/multivesiculation?), causing accumulation of endocytosed 125I-TC-AOM in a recycling compartment that corresponded to light endosomes (1.10-1.11 g/ml) in sucrose density gradients. In the electron microscope, these endosomes could be recognized as small, peripheral endocytic vesicles and tubules accumulating endocytosed AOM-gold. The serine/threonine protein phosphatase inhibitor okadaic acid inhibited an intermediate endocytic step (detachment of multivesicular endosomes from the tubulovesicular network?), causing accumulation of 125I-TC-AOM in a recycling compartment corresponding to light endosomes (1.10-1.11 g/ml), but with a multivesicular rather than a tubulovesicular morphology. Tyrphostin 25 inhibited endocytosis at a late step (endosome-lysosome fusion?), causing accumulation of 125I-TC-AOM in a non-recycling compartment corresponding to dense, multivesicular endosomes (1.14 g/ml) that had probably detached from the light endosomal network.
Holen
I
I
Department of Tissue Culture, Norwegian Radium Hospital, Oslo.
Strømhaug
P E
PE
Gordon
P B
PB
Fengsrud
M
M
Berg
T O
TO
Seglen
P O
PO
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism*; D001343:Autophagy / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000502:physiology / Q000648:ultrastructure; D004988:Ethers, Cyclic / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D008247:Lysosomes / Q000502:physiology / Q000648:ultrastructure; D008297:Male; D019319:Okadaic Acid; D015388:Organelles / Q000502:physiology* / Q000648:ultrastructure; D014176:Protein Biosynthesis / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D051381:Rats; D017208:Rats, Wistar; D014617:Vacuoles / Q000502:physiology / Q000648:ultrastructure; D014638:Vanadates / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D001212:Asialoglycoproteins; D004988:Ethers, Cyclic; D019319:Okadaic Acid; D014638:Vanadates; D014747:Vinblastine; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases