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)