INHIBITION OF AUTOPHAGY AND MULTIPLE STEPS IN ASIALOGLYCOPROTEIN ENDOCYTOSIS BY INHIBITORS OF TYROSINE PROTEIN-KINASES (TYRPHOSTINS)

In isolated rat hepatocytes, several tyrosine protein kinase inhibitor s (tyrphostins) reduced the autophagic sequestration of electroinjecte d [H-3]raffinose by 40-75% at doses that did not significantly affect cellular ATP levels or plasma membrane integrity, Tyrphostin 46 specif ically inhibited autophagy, whereas tyrphostins 1, 25 and 51 also supp ressed the receptor-mediated endocytic uptake of I-125-tyramine-cellob iose-asialoorosomucoid, I-125-TC-AOM, by 20-30% and its degradation by 70-90%. Tyrphostins 1 and 51, and the microtubule inhibitor vinblasti ne, inhibited an early endocytic step (endosome maturation/multivesicu lation?), causing accumulation of endocytosed I-125-TC-AOM in a recycl ing compartment that corresponded to light endosomes (1.10-1.11 g/ml) in sucrose density gradients. In the electron microscope, these endoso mes could be recognized as small, peripheral endocytic vesicles and tu bules 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 I-125-TC-AOM in a recycling compar tment corresponding to light endosomes (1.10-1.11 g/ml), but with a mu ltivesicular rather than a tubulovesicular morphology, Tyrphostin 25 i nhibited endocytosis at a late step (endosome-lysosome fusion?), causi ng accumulation of I-125-TC-AOM in a non-recycling compartment corresp onding to dense, multivesicular endosomes (1.14 g/ml) that had probabl y detached from the light endosomal network.