Basolateral 3-O-methylglucose transport by cultured kidney (LLC-PK1) epithelial cells.
In previous work we demonstrated the similarity of basolateral sugar transport of LLC-PK1 renal epithelia to basolateral kidney sugar transport using 2-deoxy-D-glucose as a substrate. In this study we first examine a central limitation to use of 2-deoxyglucose for basolateral sugar transport study in LLC-PK1 epithelia, namely, a shift of the rate-limiting step in uptake from transport to phosphorylation. Use of 3-O-methylglucose avoids this complication because it is not phosphorylated. However, use of 3-O-methylglucose requires much shorter incubation periods to examine linear rates of uptake (steady state is reached by 60 s at 22 degrees C for 0.1 mM 3-O-methylglucose). As was true for 2-deoxyglucose, apical uptake of 3-O-methylglucose was only a fraction of total uptake. Basolateral uptake was characteristically more sensitive to phloretin and cytochalasin B inhibition, relative to phlorizin. Inhibition studies indicate a requirement for a free hydroxyl on C-1 carbon of the pyranose ring, as is characteristic for renal basolateral sugar transport. Kinetic analysis indicates a single transport system with a Km of 10.9 mM and Vmax of 17.2 pmol.micrograms DNA-1.15 s-1. Subconfluent, undifferentiated LLC-PK1 cells show a similar Km (12.7 mM) but a ninefold higher Vmax (166.2 pmol.micrograms DNA-1.15 s-1). Stimulation of 3-O-methylglucose transport rate in confluent cultures by phorbol ester is relatively small (less than 100%) compared with effects on other somatic cells. The uptake rate of 3-O-methylglucose is not affected by glucose starvation, but subsequent refeeding with glucose-containing medium does significantly stimulate uptake.
Mullin
J M
JM
Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096.
Kofeldt
L M
LM
Russo
L M
LM
Hagee
M M
MM
Dantzig
A H
AH
D019325:3-O-Methylglucose; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002250:Carbon Radioisotopes; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology; D003847:Deoxyglucose / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006601:Hexoses / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D008757:Methylglucosides / Q000378:metabolism*; D010693:Phloretin / Q000494:pharmacology; D010695:Phlorhizin / Q000494:pharmacology; D011865:Radioisotope Dilution Technique; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium
D002250:Carbon Radioisotopes; D006601:Hexoses; D008757:Methylglucosides; D014316:Tritium; D019325:3-O-Methylglucose; D003571:Cytochalasin B; D008353:Mannitol; D003847:Deoxyglucose; D010695:Phlorhizin; D013755:Tetradecanoylphorbol Acetate; D010693:Phloretin