Regulation of pathways within cultured epithelial cells for the transcytosis of a basal membrane-bound peroxidase-polylysine conjugate.
A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used as a non-specific adsorptive probe to study transcytosis in MDCK strain I and Caco-2 epithelial cells. As we have shown previously, HRP-PLL transcytosis proceeds via an intracellular, non-lysosomal proteolytic compartment in MDCK cells; yet, this compartment is utilized for transcytosis only in the basal-to-apical direction (Taub, M. E. and Shen, W.-C. J. Cell. Physiol., 150, 283-290, 1992). Using size exclusion chromatography, we demonstrate that the PLL moiety of the conjugate is effectively cleaved during transcytosis, thus releasing free HRP from the apical surface of the cells. Pulse-chase studies indicate that approximately 6% of basolateral surface-associated HRP-PLL conjugate in Transwell-grown cell monolayers enters the basal-to-apical transcytotic pathway. Brief (1 hour) treatment with 160 nM phorbol ester (PMA), a protein kinase C stimulator, elicits a 2-fold increase in the rate and amount of HRP-PLL transcytosis following a 1 hour lag time. Treatment with 1.6 micrograms/ml brefeldin A (BFA) inhibits HRP-PLL transcytosis by approximately 30%; additionally, BFA is able to abolish completely the PMA stimulatory effect. Removal of BFA causes a re-establishment of the normal rate of transcytosis within 2 hours, demonstrating the reversibility of BFA inhibition with respect to HRP-PLL transcytosis. Thus, PMA most likely elicits an increase in the amount of basally internalized conjugate delivered to BFA-sensitive transcytotic compartments.(ABSTRACT TRUNCATED AT 250 WORDS)
Taub
M E
ME
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles 90033.
Shen
W C
WC
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D020126:Brefeldin A; D016764:Cell Polarity / Q000502:physiology*; D003517:Cyclopentanes / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D011107:Polylysine / Q000031:analogs & derivatives* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D003517:Cyclopentanes; D007976:Leupeptins; D011480:Protease Inhibitors; C061731:horseradish peroxidase-polylysine conjugate; D020126:Brefeldin A; D011107:Polylysine; D006735:Horseradish Peroxidase; C032854:leupeptin; D013755:Tetradecanoylphorbol Acetate