Characterization of cytochalasin B binding to adult rat liver parenchymal cells in primary culture.
The characterization of cytochalasin B binding and the resulting effect on hexose transport in rat liver parenchymal cells in primary culture were studied. The cells were isolated from adult rats by perfusing the liver in situ with collagenase and separating the hepatocytes from the other cell types by differential centrifugation. The cells were established in primary culture on collagen-coated dishes. The binding of [4-3H]cytochalasin B and transport of 3-O-methyl-D-[14C]glucose into cells were investigated in monolayer culture followed by digestion of cells and scintillation counting of radioactivity. The binding of cytochalasin B to cells was rapid and reversible with association and dissociation being essentially complete within 2 min. Analysis of the kinetics of cytochalasin B binding by Scatchard plots revealed that binding was biphasic, with the parenchymal cell being extremely rich in high-affinity binding sites. The high-affinity site, thought to be the glucose-transport carrier, exhibited a KD of 2.86 x 10(-7) M, while the low-affinity site had a KD of 1.13 x 10(-5) M. Sugar transport was monitored by 3-O-methyl-D-glucose uptake and it was found that cytochalasin B (10(-5) M) drastically inhibited transport. However, D-glucose (10(-5) M) did not displace cytochalasin B, and cytochalasin E, which does not inhibit transport, was competitive for cytochalasin B at only the low-affinity site, demonstrating that the cytochalasin B inhibition of sugar transport occurs at the high-affinity site but that the inhibition is non-competitive in nature. Therefore, the liver parenchymal cells may represent an unusually rich source of glucose-transport system which may be useful in the isolation of this important membrane carrier.
D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000378:metabolism*; D003572:Cytochalasins / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008757:Methylglucosides / Q000378:metabolism; D051381:Rats
D003572:Cytochalasins; D008757:Methylglucosides; D003571:Cytochalasin B; D005947:Glucose