GLUCOCORTICOID-RECOGNIZING AND GLUCOCORTICOID-EFFECTOR SITES IN RAT-LIVER PLASMA-MEMBRANE - KINETICS OF CORTICOSTERONE UPTAKE BY ISOLATED MEMBRANE-VESICLES .1. 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 [H-3]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [H-3]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) [H-3]B uptake was a saturable process of unusual kinetics (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent K(d)-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 [H-3]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 (K(t): 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 K(d)-values (86.5 +/- 7.3 and 77.0 +/- 14.3 nM, respectively) of the [H-3]B interactions with CBG and GCR did not differ (P > 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.