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The uptake of cortisol by isolated rat liver cells was studied. Cortisol was taken up rapidly; the uptake increased with increasing temperature and reached a plateau after 45 s at 37 degrees, C, after 60 s at 27 degrees C, and after 90 s at 22 degrees C; at 5 degrees C the uptake increased linearly with time. The uptake was linear up to 1.5 mg of cell protein. Analysis of uptake as a function of increasing concentration of cortisol in the external medium indicated the presence of two saturable systems: a high-affinity system with an apparent Km value of 190 +/- 25 nM and a low-affinity system with an apparent Km value of 2200 +/- 180 nM. Above 600 nM, the rate of uptake of cortisol increased almost linearly with increasing cortisol concentration. Treatment of cells with KCN or 2,4-dinitrophenol inhibited the two saturable components, leaving the nonsaturable system unaffected. The affinity constants, Ka, were 6 X 10(6) M-1 and 0.6 X 10(6) M-1 for the high and low affinity components, respectively. These values increased approximately two-fold when uptake rates were corrected for diffusion. Cortisone and corticosterone inhibited the uptake of cortisol by liver cells competitively; dexamethasone inhibited cortisol uptake noncompetitively. Similarly, oestrone, oestradiol and testosterone decreased the uptake of cortisol, at a concentration of 2000 nM in the external medium, by 20, 49 and 35 percent, respectively; the inhibition was noncompetitive. p-Chloromercuribenzoate, N-ethylmaleimide and 1-fluoro-2,4-dinitrobenzene decreased the uptake of cortisol. Ouabain did not influence the uptake of cortisol; varying the external sodium concentration also did not affect uptake of cortisol. Cyclic-3',5'-adenosine monophosphate had a stimulatory effect. The results show that the first step, before cortisol is bound to intracellular binding proteins, is the uptake of cortisol by proteins in the plasma membrane. At lower concentrations of cortisol, uptake takes place by saturable processes; at higher concentrations saturation is not achieved, indicating that simple diffusion becomes the major route of transport into the cell. The proteins in the plasma membrane probably function as carriers to transport the glucocorticoid into the cell.

The isolated rat liver was perfused with a haemoglobin-free, albumin containing salt solution at 28 degrees and 37 degrees C, respectively, with [4-14C]oestrone as substrate. During perfusion, the functional state of the liver was checked by continuously measuring the oxygen pressure and hydrogen ion concentration in the perfusion medium, flow rate, oxygen tension at various areas of the liver surface, and oxygen consumption; in addition, the following biochemical parameters were determined: ATP, ADP, lactate and pyruvate in liver tissue, and lactate and pyruvate in the perfusion medium. After 30 min of perfusion, free steroids, steroid glucuronides, steroid sulphates and the remaining water-soluble metabolites, present in liver tissue, perfusion medium and bile, were separated from each other and characterised by thin-layer and paper chromatography. It was found that, during perfusion at 37 degrees C, less hydroxylated metabolites were formed than at 28 degrees C. In contrast, metabolites whose formation is not directly oxygen-dependent, such as glucuronides and sulphates, arose in higher amounts at 37 degrees C than at 28 degrees C. It may be concluded that the relative shortage of oxygen at 37 degrees C leads to a selective impairment of metabolic pathways requiring a sufficient supply of molecular oxygen. Since oxidative processes play an important role in the biogenesis and metabolism of steroid hormones, it is evident that results, obtained in perfusion experiments with haemoglobin-free media at 37 degrees C, must be treated with reserve.