GLUCOCORTICOID-RECOGNIZING AND GLUCOCORTICOID-EFFECTOR SITES IN RAT-LIVER PLASMA-MEMBRANE - KINETICS OF CORTICOSTERONE UPTAKE BY ISOLATED MEMBRANE-VESICLES .2. COMPARATIVE INFLUX AND EFFLUX

To elucidate the initial step in the interaction between glucocorticoids (GC) and the hepatocyte, we examined at 22-degrees-C further kinetic properties of active corticosterone (B) transport mediated by a putative, plasma membrane-inserted carrier for GC (GCC) as previously reported [Allera and Wildt, J. Steroid Biochem. Molec. Biol. 42 (1992) 737-756]. We used a purified, well-characterized, osmotically active vesicle fraction of plasma membrane (PM), free of ATP, isolated from rat liver and a method developed by us to describe transport processes mathematically: (1) uptake (U) of 7 nM B into the vesicles (influx, 1) occurred very rapidly whereby T1/2 = 8.3 s, the time (S) required for half maximum transport; the influx velocity (dU/dS = V) decreased degressively with time following second-order kinetics characterized by an initial transport V (V(T0)) of 177.7 fmol/mg membrane protein/s. (2) V(ToI) of B-influx rose with temperature biphasically (P < 0.025): activation energy above and below 15-degrees-C (at PM phase transition) amounted to 9.5 and 26.5 kJ/mol. Neither at 45 nor at 60-degrees-C did transport take place, revealing the high thermolability of GCC. (3) Efflux (E) of 6.5 nM B, i.e. transport out of the vesicles preincubated with the steroid, showed that influx had resulted in a 19.6-fold intravesicular hormone accumulation, indicating active ("uphill") transport. (4) The efflux velocity (dE/dS = V) exhibited almost the same kinetic quality as that of influx: it decreased following mainly second-order kinetics whereby T1/2 = 8.0 s. However, its whole time-course was much slower and the V(T0) of efflux (VIE) was 6.3 lower than V(ToI). (5) Using physics and thermodynamics, we deduced that the affinity (A(F)) between B and GCC is proportional to the square of V(T0). (6) Thus, because A(F) approximately (1/6.3)2, AF of the B-GCC interaction after completion of influx was calculated to be 40 times lower (K(d) = 708 nM; DELTA-G-degrees = -34.9 kJ/mol) than at outset of influx, whereby DELTA-G-degrees = - 44.0 kJ/mol. Concluding from these and previous findings, we present a new hypothesis on B transport into the hepatocyte: There is no difference (P > 0.3) in free enthalpy between transcortin (CBG) and the intracellular GC receptor interacting with B (DELTA-G-degrees = -40.1 and -40.4 kJ/mol). The GCC, however, is characterized by its ability to switch from a high- to lower-affinity when interacting with B (and vice versa due to metabolic energy input). The decrease in free enthalpy of the B-complexed carrier (minimum DELTA-G-degrees = -9.1 to -9.4 kJ/mol) is used as work for an active transport through the PM; there is no passive diffusion of B. Thus, a GCC is a prerequisite to a thermodynamically spontaneous and effective steroid transfer from CBG in the blood to the receptor inside the hepatocyte.