Robert W. Kuhn

The serum transport of steroid hormones.

Publisher Summary This chapter discusses the serum transport of steroid hormones. Steroid hormones are extensively bound to plasma proteins including albumin, corticosteroid binding globulin (CBG), and sex hormone binding globulin (SHBG). Because of its high concentration, albumin binding is important in determining the magnitude of the nonprotein bound or free fraction of a steroid in plasma. The generally accepted model of steroid hormone action suggests that free steroid (in equilibrium with circulating binding proteins) diffuses passively through target cell membranes and binds to a soluble intracellular receptor. The steroid-receptor complex apparently moves into the nucleus where it modifies the chromatin transcriptional activity which results in, among other things, altered levels of protein synthesis. CBG has been differentiated from the intracellular glucocorticoid and progesterone receptors by its inability to bind synthetic glucocorticoids and progestins.

Corticosteroid-binding globulin interactions with target cells and plasma membranes.

It is commonly thought that serum steroid binding proteins such as corticosteroidbinding globulin (CBG), sex hormone-binding globulin ( SBP), thyroid-binding globulin (TBG), and progesterone-binding globulin (PBG) act physiologically to protect the circulating steroid from metabolism, thus serving only as a reservoir to replenish the biologically active free pool. According to this view, the bound fraction remains unavailable for bioactivity. A wide variety of studies from the induction of tyrosine amino transferase (TAT) in cultured hepatoma cells’ to the more precise correlation of clinical response with circulating free (rather than total) steroid levels’ would seem to support this contention. However, other studies have suggested that in at least some instances steroid bound to binding proteins is available to target tissues. Keller et al.’ have found that CBG-corticosterone complexes were as effective as free corticosterone in inducing the production of alanine amino transferase (AAT) activity in tissues with protein-permeable vascular beds (such as the liver). However, bound steroid was ineffective in inducing AAT in the pancreas, a tissue with a relatively impermeable vascular bed. Rosner and Hochberg‘ found that CBG-bound cortisol was at least as active as free cortisol in inducing TAT activity in rat liver and in suppressing peripheral lymphocyte count. These two studies suggest that mechanisms exist for making the bound fraction of steroid available to target cells in at least some tissues. The need for augmentation of the free fraction of steroid has been outlined by Siiteri and co-worker~.”~ They pointed out that based upon the known K,’s of receptors and the plasma levels of unbound hormone for several steroids, the degree of receptor occupancy is not sufficient to account for observed biologic activity if response is directly proportional to receptor occupancy as suggested by Clark and Peck.’ If the only role of CBG is to inactivate circulating steroid and provide a pool for the replenishment of the free fraction, it should be present only in the vascular compartment or that compartment plus lymph (as representative of interstitial fluid). One would expect to find neither intracellular CBG nor specific mechanisms for its interaction with glucocorticoid target cells. Several lines of evidence, however, suggest that CBG may have functions other than the transport of steroid. It has been localized

Comparative structural analyses of corticosteroid binding globulin

Some physicochemical characteristics of corticosteroid binding globulin (CBG) in several species have been determined. Molecular radii were determined from Ferguson plots and were used in conjunction with sedimentation coefficients determined by sucrose density gradient centrifugation to calculate the molecular weights of the CBG. These were found to range from 44,200 (dog) to 60,000 (turtle) for most species. The squirrel monkey was found to have a molecular weight twice that of other species (119,800). Purified CBG was prepared from human, rat, and guinea pig sera. The molecular weights of the purified material, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate, were in excellent agreement with those determined by Ferguson analysis. Careful examination of the purified proteins by electrophoresis at pH 8.3 revealed that each consisted of two closely related electrophoretic variants. Tryptic peptides were prepared from the purified proteins and separated by reversed phase HPLC chromatography. The peptide patterns were identical for the three proteins with the exception of three hydrophilic peptides. Amino terminal sequence analysis of the rat and human proteins revealed no apparent homology, however. The immunologic relatedness of the three purified proteins was also examined, but no crossreactivity was observed. The results obtained suggest that while the molecular size and hydrophobicity of peptides have been conserved across species considerable surface differences must exist.

Corticotropin-releasing factor can stimulate gonadotropin secretion by human fetal pituitaries in superfusion☆

Abstract In previous studies, corticotropin-releasing factor was found to elicit a rise in circulating adrenocorticotropic hormone in human subjects and laboratory animals, but no stimulatory effect of corticotropin-releasing factor on other pituitary hormones was detected. Since stress may be associated with luteinizing hormone changes as well as with those of corticotropin-releasing factor and adrenocorticotropic hormone, we quantified gonadotropin responses to corticotropin-releasing factor and arginine vasopressin in 11 human fetal pituitaries with use of both superfusions and static incubations. Exposure to corticotropin-releasing factor brought about a significant increase in adrenocorticotropic hormone and gonadotropin concentrations in the effluent media by means of the superfusion system. Similar concentrations of corticotropin-releasing factor significantly increased adrenocorticotropic hormone secretion into the medium by dispersed fetal pituitary cells cultured on an extracellular matrix but failed to increase luteinizing hormone and follicle-stimulating hormone secretion. Exposure to 3 mmol/L 8-bromo-cyclic adenosine monophosphate caused an increase in all three peptides, both in superfusion and static incubations. Dose-response studies showed that the effect on gonadotropin secretion occurred at concentrations of 8-bromocyclic adenosine monophosphate two orders of magnitude lower than those affecting adrenocorticotropic hormone secretion. The purity of corticotropin-releasing factor and arginine vasopressin used in these studies was confirmed by high-performance liquid chromatography. These in vitro results are consistent with a paracrine effect of corticotropes acting on gonadotropes. The combination of static incubation and superfusion has proved useful in elucidating the effects of different secretagogues on pituitary cells.

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig.

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.

A Homologous Radioimmunoassay for Guinea Pig Corticosteroid-Binding Globulin

Abstract A rapid, specific, and sensitive (requiring only 20 fmole of antigen equivalent to 0.007 μl of serum) radioimmunoassay (RIA) was developed for the measurement of guinea pig corticosteroid-binding globulin (CBG). CBG was purified to homogeneity from guinea pig serum by affinity chromatography and used for immunization, as the standard and as the radiolabeled trace in the RIA. The antiserum to CBG was raised in rabbits. It was judged specific by immunoelectrophoresis and by comparison of RIA values with steroid-binding assay profiles obtained on serum separated on the basis of size and ion-exchange properties. The results of the radioimmunoassays agree with those of a steroid-binding assay run on identical samples. The sensitivity of the assay allows detection of CBG in serial serum samples, other biologic fluids such as milk, and cell culture supernatants.