PROTEOGLYCANS IN POLARIZED EPITHELIAL MADIN-DARBY CANINE KIDNEY-CELLS

Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polariz ed epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown t o confluency and labelled with [S-35]sulphate for 24 h. The apical and basolateral media and the cell frictions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [S-35]sulp hate-labelled macromolecules bound strongly to the ion-exchange column s, and could be eluted in three distinct peaks. The latest eluting pea k was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by usi ng chondroitinase ABC and nitrous acid (pH 1.5) respectively to depoly merize the [S-35]glycosaminoglycan chains. Peak I contained negligible amounts of proteoglycans. Large differences could be observed in prot eoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67 % of the proteoglycans to the apical side and 17 % to the basolateral side. The cell fraction contained 17 % of the proteoglycans after 24 h of labelling. In contrast, 19 % of the proteoglycans were sorted to t he apical side of MDCK II cells and 61 % to the basolateral side, wher eas the cell fraction contained 20 %. Furthermore, the level of [S-35] proteoglycan biosynthesis (apical and basolateral media and cell fract ion total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid res pectively, and the total amounts of [S-35]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized appro x. 56 % chondroitin sulphate and 44 % heparan sulphate. In contrast, t he MDCK II strain synthesized 69 % heparan sulphate and 31 % chondroit in sulphate. To further identify the [S-35]proteoglycans synthesized b y MDCK I and II cells, antibodies against perlecan, versican and synde can were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MD CK I and II cells expressed perlecan; 57-61 % could be recovered from the basolateral fractions and 18-34 % from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53 %) was found in the cell fractio ns.