DIFFERENTIAL TARGETING OF GLUCOSYLCERAMIDE AND GALACTOSYLCERAMIDE ANALOGS AFTER SYNTHESIS BUT NOT DURING TRANSCYTOSIS IN MADIN-DARBY CANINEKIDNEY-CELLS

A short-chain analogue of galactosylceramide (6-NBD-amino-hexanoyl-gal actosylceramide, C-6-NBD-GalCer) was inserted into the apical or the b asolateral surface of MDCK cells and transcytosis was monitored by dep leting the opposite cell surface of the analogue with serum albumin. I n MDCK I cells 32% of the analogue from the apical surface and 9% of t he analogue from the basolateral surface transcytosed to the opposite surface per hour. These numbers were very similar to the now of membra ne as calculated from published data on the rate of fluid-phase transc ytosis in these cells, demonstrating that C-6-NBD-GalCer acted as a ma rker of bulk membrane now. It was calculated that in MDCK I cells 155 mu m(2) of membrane transcytosed per cell per hour in each direction. The fourfold higher percentage transported from the apical surface is explained by the apical to basolateral surface area ratio of 1:4. In M DCK II cells, with an apical to basolateral surface ratio of 1:1, tran scytosis of C-6-NBD-GalCer was 25% per hour in both directions. Simila r numbers were obtained from measuring the fraction of endocytosed C-6 -NBD-GalCer that subsequently transcytosed. Under these conditions lip id leakage across the tight junction could be excluded, and the vesicu lar nature of lipid transcytosis was confirmed by the observation that the process was blocked at 17 degrees C. After insertion into one sur face of MDCK II cells, the glucosylceramide analogue C-6-NBD-GlcCer ra ndomly equilibrated over the two surfaces in 8 h. C-6-NBD-GalCer and - GlcCer transcytosed with identical kinetics. Thus no lipid selectivity in transcytosis was observed. Whereas the mechanism by which MDCK cel ls maintain the different lipid compositions of the two surface domain s in the absence of lipid sorting along the transcytotic pathway is un clear, newly synthesized C6NBD-GlcCer was preferentially delivered to the apical surface of MDCK II cells as compared with C-6-NBD-GalCer.