Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles—II. Comparative influx and efflux

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To elucidate the initial step in the interaction between glucocorticoids (GC) and the hepatocyte, we examined at 22°C further kinetic properties of active corticosterone (B) transport mediated by a putative, plasma membrane-inserted carrier for GC (GCC) as previously reported [Alléra 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, I) occurred very rapidly whereby T 1 2 = 8.3 s , the time ( S) required for half maximum transport; the influx velocity (d U/d S = 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°C (at PM phase transition) amounted to 9.5 and 26.5 kJ/mol. Neither at 45 nor at 60°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 (d E/d S = V) exhibited almost the same kinetic quality as that of influx: it decreased following mainly second-order kinetics whereby T 1 2 = 8.0 s . However, its whole time-course was much slower and the V T0 of efflux ( V To E ) was 6.3 lower than V To I . (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 ∼; ( 1 6.3 ) 2 , A F of the B-GCC interaction after completion of influx was calculated to be 40 times lower ( K d = 708 nM; ΔG° = −34.9 kJ/mol) than at outset of influx, whereby ΔG° = −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 ( ΔG° = −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 ΔG° = −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.