Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--I. Binding and transport.
To gain insight into the mechanisms governing cellular uptake of glucocorticoids, we studied the binding and membrane transport of corticosterone (B) on a highly purified plasma membrane fraction from rat liver that was homogenized using a gentle, isotonic procedure. The fraction was mostly in the form of right-side out and osmotically active vesicles that were free of intracellular glucocorticoid receptors (GCR), transcortin (CBG) and ATP. Our uptake and binding studies carried out at 22 degrees C with [3H]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [3H]B for uptake by the membrane vesicles; half-maximal competition of specific uptake was achieved with a 10- to 11-fold molar excess of unlabeled B. (2) [3H]B uptake was a saturable process of unusual kinetics (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent Kd-values of 1.3, 4.7 and 17.3 nM, corresponding to free B in the blood of non-stressed rats (4-16 nM). (3) Osmotic shrinkage of the vesicles led to a linear decrease in specific uptake, while non-specific uptake was independent of vesicle volume. Passive diffusion of [3H]B took place in leaky, but not in intact, vesicles. Reversible binding to, and mediated transport through, the membrane were interdependent parts of a strongly linked process. B was accumulated inside the vesicle up a concentration gradient by an active transport that followed first-order kinetics (Kt:3.9 nM); for its statistically reliable mathematical formulation and kinetic analysis, a replot was developed that revealed that relative accumulation increased with decreasing external hormone concentration. (4) Comparative binding studies disclosed that the apparent Kd-values (86.5 +/- 7.3 and 77.0 +/- 14.3 nM, respectively) of the [3H]B interactions with CBG and GCR did not differ (P greater than 0.3). These findings permit the conclusion that a plasma membrane-inserted carrier for B, effectively operating at physiological concentrations in the blood, is involved in a functional and regulatory manner in the biological action of glucocorticoids.
Alléra
A
A
Department of Clinical Biochemistry, University of Bonn, Germany.
Wildt
L
L
D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003345:Corticosterone / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D011485:Protein Binding; D011506:Proteins / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D014156:Transcortin / Q000378:metabolism
D011506:Proteins; D011965:Receptors, Glucocorticoid; D014156:Transcortin; D003345:Corticosterone