Sodium-independent carrier-mediated inositol transport in cultured renal epithelial (LLC-PK1) cells.

In addition to the concentrative, Na(+)-dependent inositol transport system demonstrated in many cell types, carrier-mediated, Na(+)-independent inositol transport is also shown to exist in LLC-PK1 renal epithelia. Inhibition of inositol uptake in Na(+)-free saline by 0.1 mM phloretin, and self-inhibition by net concentrations of inositol exceeding 10 mM, demonstrate the carrier-mediation of the Na(+)-independent uptake and distinguish it from flux through anion channels. The Na(+)-dependent uptake exhibits higher affinity for inositol, as seen by the stronger self-inhibition at lower inositol concentrations in Na+ saline. Kinetic analyses indicate a Km of 178 microM and a Vmax of 2447 pmol/min per microgram DNA for the Na(+)-dependent system, whereas the lower affinity, lower capacity Na(+)-independent system manifests a Km of 5.2 mM and a Vmax of 249 pmol/min per microgram DNA. the Na(+)-independent uptake further differs from the Na(+)-dependent transport by the lack of inhibitory effect of 10 microM glucose, and the greater relative inhibition of phloretin compared to that of phlorizin. Both types of uptake appear to localize predominantly to the basal-lateral cell surface. The Na(+)-independent transport is bidirectional, functioning in efflux as well as influx of inositol.

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.