[Isolation and study of the physical chemical properties of transcortin from human retroplacental plasma].
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Amino Acids↗
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[Clinical significance of determining transcortin-bound hydrocortisone in chronic liver diseases].
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Chronic Disease↗
Dialysance of adrenocorticoids during continuous ambulatory peritoneal dialysis.
We postulated that significant quantities of both protein-bound and unbound adrenocorticoids are lost during continuous ambulatory peritoneal dialysis (CAPD). To test this hypothesis we measured the dialysate removal rates (DRR) of adrenocorticoids in six CAPD patients. The distribution of the adrenocorticoids among unbound, albumin-bound, and transcortin-bound fractions in dialysate effluent was determined. The distribution of cortisol among unbound, albumin-bound, and transcortin-bound fractions in plasma was determined in six other CAPD patients. The mean DRR of cortisol was 193.8 +/- 20.3 (+/- SE) nmol/day. Smaller quantities of 11-deoxycorticosterone, corticosterone, aldosterone, 18-hydroxy-11-deoxycorticosterone, and 18-hydroxycorticosterone were removed during CAPD. The mean DRR values for total protein, albumin, and transcortin were 11.2 +/- 2.1, 6.0 +/- 2.2, and 0.087 +/- 0.018 g/day, respectively. The distribution of cortisol among unbound, albumin-bound, and transcortin-bound fractions was normal in plasma from CAPD patients. Plasma transcortin had a normal affinity (2 x 10(7) mol/L-1) and a normal binding capacity (559 nmol/L) for cortisol. In contrast, dialysate transcortin had a low affinity (1.4 x 10(7) mol/L-1) for cortisol and a low cortisol-binding capacity (11.5 nmol/L). The fractional occupancy rates of high affinity cortisol-binding sites on transcortin were 52.0 +/- 3.3% and 3.3 +/- 0.6% in plasma and dialysate effluent, respectively (P less than 0.001). The transcortin to cortisol molar concentration ratio in dialysate (6.3 +/- 0.6) was significantly higher than that in plasma (1.6 +/- 0.2; P less than 0.001). These results demonstrate that cortisol is the major adrenocorticoid lost during CAPD. However, the amount of cortisol removed in the dialysate is less than 1% of the normal daily secretion rate. Significant quantities of other adrenocorticoids are also lost during CAPD. The adrenocorticoids present in dialysate effluent are principally unbound, in contrast to their state in plasma. However, small fractions of the respective steroids are bound to transcortin and albumin.
Adrenal Cortex Hormones↗
[Effects of changes of body temperature on serum cortisol fractions (author's transl)].
Most of the cortisol in circulating blood is bound to proteins (transcortin and albumin), and the rest is protein-unbound. It has generally been accepted that transcortin-bound cortisol (Tr-F) and albumin-bound cortisol (Al-F) are physiologically inactive, and that protein-unbound cortisol (U-F) is active. It is also known that the binding capacity of transcortin to cortisol is lower at 37 degrees C than at 4 degrees C. However, no investigations concerning the influence of temperature changes the physiological range have been reported, probably because of the lack of proper methods. In this report, the influence of temperature changes the physiological range on serum protein-cortisol binding is assessed and its physiological roles are discussed. Cortisol binding of transcortin and albumin was determined by a newly developed isocolloidosmolar equilibrium dialysis method. The total cortisol concentration was determined by the CPB assay method described by Murphy. In the beginning, experiments were performed using pooled human serum. This serum was dialyzed at 35, 37, 39 and 41 degrees C. U-F and Al-F concentrations increased in accordance with the increment of incubation temperature, whereas Tr-F concentration decreased. The association constant of transcortin to cortisol decreased with the rise of incubation temperature. It was 3.43, 2.92, 2,32 and 1.66 X 10(7)M-1 at 35, 37, 39 and 41 degrees C, respectively. However, the association constant of albumin to cortisol was 3.04 X 10(3)M-1 and stayed unchanged under these conditions. These results suggest that the transcortin-cortisol complex dissociates with temperature rises and that a part of freed cortisol is bound to albumin while the other part remains protein-unbound cortisol. When various amounts of cortisol were added to the pooled human serum, which was dialyzed at 35, 37, 39 and 41 degrees C; U-F, Al-F and Tr-F concentrations increased corresponding to the amount of added cortisol. The increase of U-F, Al-F concentrations and decrease of Tr-F concentration induced by the rise of temperature was still significant at each total coritsol concentration. Cortisol fractions in the serum of healthy subjects were compared with those of 39 febrile patients consisting of A group (body temperature 37 approximately 38 degrees C), B group (38 approximately 39 degrees C), and C group (39 approximately 40 degrees C). Total cortisol concentrations increased with the rise of body temperature. They were 7.62 (normal), 10.64 (A), 14.84 (B) and 21.37 (C) microgram/100ml, respectively. U-F% and Al-F% increased parallel with the total cortisol concentration. Tr-F%, binding capacity of transcortin. These results reveal that the rise of body temperature elevates the total cortisol concentration, and the greater part of increased cortisol becomes U-F and Al-F, while a relatively small part of it is bound to transcortin. The percent change of the total cortisol concentration in comparison with healthy subjects was 150 (A), 209 (B) and 593 (C), respectively...
Binding, Competitive↗
Tissue differences in the up-regulation of glucocorticoid-binding proteins in the rat.
The magnitude of increase in glucocorticoid receptor concentration and transcortin-like binding was examined in a variety of peripheral tissues and brain structures after adrenalectomy. Glucocorticoid binding was assayed in liver, heart, kidney, pituitary, hippocampus, cerebral cortex, amygdala-entorhinal area, and hypothalamus. Glucocorticoid receptor concentration, measured using [3H]dexamethasone as ligand, increased in all eight tissues, but the magnitude of this increase varied 30-fold among tissues. The largest increase was shown by kidney cytosol, followed by amygdala-entorhinal cortex, hippocampus, liver, cerebral cortex, hypothalamus, pituitary, and heart. These increases were not due to a selective enhancement of mineralocorticoid receptors. The increase in transcortin in peripheral tissues was variable and exceeded the increase in plasma transcortin by an order of magnitude. It was concluded that up-regulation of the glucocorticoid receptor after adrenalectomy is a response common to most, if not all, glucocorticoid target tissues. However, the magnitude of this response was tissue specific and was not directly related to initial receptor density. The marked increase in tissue transcortin ([3H]corticosterone binding in the presence of excess dexamethasone) suggested that plasma transcortin is sequestered by peripheral tissues in substantial amounts in the acutely adrenalectomized rat. The increase in transcortin uptake by tissues and the increases in cytosolic receptor number are apparently subject to different regulatory control.
Adrenalectomy↗
Strain differences in corticosteroid receptor efficiencies and regulation in Brown Norway and Fischer 344 rats.
During the dark phase of the diurnal cycle, and during recovery from restraint stress, Brown Norway (BN) rats secrete less corticosterone than Fischer 344 (F344) rats. These strains also display different levels of corticosteroid receptors in the hippocampus, and of plasma transcortin. Because corticosteroid receptors, plasma transcortin and corticosterone secretion are mutually regulated, we examined brain and pituitary mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression and some of the parameters modulated by these receptors (i.e. body and thymus weight, fluid intake, plasma transcortin) in BN and F344 rat strains, by comparing the effects of either hormone deprivation by long-term (21 days) adrenalectomy (ADX), or chronic elevation of corticosterone given in drinking fluid to ADX rats. In BN rats, body weight gain and fluid intake were insensitive to corticosterone deprivation, suggesting that MR-related mechanisms are constitutively active in this strain. Body weight (b.w.) gain, plasma transcortin and thymus weight were reduced to a greater extent by chronic corticosterone in BN rats than in F344 rats, possibly as a consequence of higher free, active fraction of plasma corticosterone due to lower plasma transcortin concentrations and/or a greater efficiency of GR-related mechanisms in BN rats. F344 rats displayed twofold higher brain and pituitary MR levels than BN rats, whereas tissue-and strain-specific regulations were observed for GR levels. The differences in MR levels observed between BN and F344 strains cannot completely explain the differences in corticosterone actions, suggesting that strain differences in response to ADX or corticosterone treatment result from variable receptor efficiencies.
Adrenalectomy↗
Distribution of 18-hydroxycorticosterone between red blood cells and plasma.
Plasma 18-hydroxycorticosterone (18-OH-B) to aldosterone (aldo) concentration ratios reflect adrenal corticosterone methyloxidase type II activity. This ratio is determined not only by the relative secretion rates of the two steroids but also by differences in binding, distribution, and metabolism. Plasma cortisol alters the distribution of aldo between red blood cells (RBC) and plasma. We postulated that the distribution of 18-OH-B, like that of aldo, is determined by the availability of high affinity binding sites on plasma transcortin. Double equilibrium dialyses demonstrated that 18-OH-B, aldo, and cortisol compete for binding sites on transcortin. Increasing amounts of each of the three unlabeled steroids produced progressive decrements in the binding of all three labeled steroids to transcortin. The affinity of 18-OH-B (2 X 10(6) M-1) for transcortin was intermediate between those of cortisol (3 X 10(7) M-1) and aldo (0.9 X 10(6) M-1). Heat treatment of plasma decreased the binding of 18-OH-B and cortisol to transcortin by 82% and 75%, respectively. Gel filtration of plasma revealed that protein-bound [3H]18-OH-B and [14C]cortisol eluted in the same fractions. The addition of increasing quantities of unlabeled cortisol to whole blood in vitro produced similar increments in RBC to plasma concentration ratios of [3H]18-OH-B and [14C]aldo. The ratio of the percentage of circulating 18-OH-B in plasma to the percentage of circulating aldo in plasma was constant in blood containing low and high cortisol concentrations. Therefore, changes in plasma cortisol have similar effects on the distribution of 18-OH-B and aldo between RBC and plasma.
18-Hydroxycorticosterone↗
[3H] Corticosterone binding in rat superior cervical ganglion.
Binding of [3H]corticosterone was examined in the soluble fraction (100,000 g) from superior cervical ganglia dissected from adrenalectomized and perfused rats. A large binding capacity (2100 fmol/mg protein) was found. However, no binding for [3H]-dexamethasone and [3H]triamcinolone was detected. The binding protein present in the ganglion preparation was characterized by DEAE-cellulose and DNA-cellulose chromatography and by isoelectric focusing. The corticosterone binding protein from the ganglia was found to be different from the cytoplasmic glucocorticoid receptor, but similar to plasma transcortin. A large portion (approximately 75%) of the transcortin-like [3H]corticosterone binding protein was located in the interstitial space between the sheath and the ganglionic cells. However, 25% of the binding protein was retained in the desheathed ganglion even after extensive washing with saline. Our findings indicate that cytoplasmic glucocorticoid receptors are not present in the superior cervical ganglion. It is possible that the transcortin-like protein associated with the desheathed ganglion may be intracellular, as other investigators have suggested similar intracellular presence of a transcortin-like protein in the liver, uterus, and pituitary.
Animals↗
Metabolism of asialotranscortin by hepatocytes and cortisol binding to hepatocytes.
Binding and catabolism of iodinated human transcortin and asialotranscortin were studied in isolated rat hepatocytes. Human transcortin binding to hepatocytes was very low (less than 3%) while 40-60% of the asialotranscortin bound to the hepatocytes. Asialofetuin inhibited asialotranscortin binding to hepatocytes, producing 50% inhibition of binding at 5 x 10-9M, but had no effect on transcortin binding. The lysosomal inhibitors leupeptin (0.05 mM) and ammonium chloride (10 mM) had no effect on asialotranscortin binding but prevented the latter's catabolism, suggesting lysosomal involvement in asialotranscortin degradation. Both transcortin and asialotranscortin inhibited the binding of [3H]cortisol to the hepatocytes in a dose-dependent manner.
Ammonium Chloride↗
Effects of testosterone mediated or modulated by pituitary factors.
Adult rats of both sexes were either gonadectomized or hypophysectomized and gonadectomized. Three to eight weeks later they were treated for 14 consecutive days with oil or with 75 or 200 mug testosterone propionate (TP) per 100 g body weight. The animals were killed and for each sex the gonadectomized animals were compared with the hypophysectomized-gonadectomized animals as far as their NADPH- and NADH-dependent 3alpha-hydroxy-steroid dehydrogenases (3alpha-HSD) in renal microsomes, transcortin levels in serum and five organ weights relative to total body weight were concerned. For two of the latter, i.e. the relative kidney and prostatic weights, no significant differences were found. Transcortin levels, relative adrenal weights and renal NADPH-dependent 3alpha-HSD activities were higher in oil-treated gonadectomized animals than in oil-treated hypophysectomized-gonadectomized animals. The opposite was found for the relative weights of uterus and seminal vesicles and renal NADH-dependent 3alpha-HSD activities. These differences between gonadectomized and hypophysectomized-gonadectomized animals disappeared after TP treatment as far as transcortin levels were concerned but remained for the five other parameters. After gonadectomy sexual differences subsisted for all parameters studied. But whereas intact male rats had higher NADH-dependent 3alpha-HSD activities than female rats the opposite was found after gonadectomy. After gonadectomy plus hypophysectomy the between sex differences disappeared as far as transcortin levels were concerned but remained in the other parameters studied.
Adrenal Glands↗
Protein binding of plasma cortisol in the foetal lamb near term.
Binding of cortisol to plasma proteins was studied in the foetal lamb by equilibrium dialysis at 37 degrees C. At 122 days of pregnancy the mean level of transcortin expressed as cortisol-binding capacity was 28 +/- 6 (S.D.) ng cortisol/ml plasma. During the last 14 days of pregnancy there was a progressive increase in transcortin-binding capacity to 85 +/- 14 ng cortisol/ml plasma. A sharp increase in the concentration of both protein-bound and unbound cortisol was observed over the same period. A rise in the concentration of total cortisol from around 3 to 42 ng/ml was associated with an increase in unbound cortisol from 0-2 to a maximum of 2-1 ng/ml. The concentration of albumin-bound cortisol was approximately equal to that of unbound cortisol. The mean value for the transcortin-cortisol affinity constant was 1-15 x 10(8) l/mol. It is concluded that an increase in transcortin-binding capacity is partly responsible for the prepartum increase of corticosteroid levels observed in normal foetal lambs.
Animals↗
Testosterone-dependent variations in plasma and intrapituitary corticosteroid binding globulin and stress hypothalamic-pituitary-adrenal activity in the male rat.
Hypothalamic-pituitary-adrenal (HPA) activity is governed by glucocorticoid negative feedback and the magnitude of this signal is determined, in part, by variations in plasma corticosteroid-binding globulin (CBG) capacity. Here, in gonadectomized male rats we examine the extent to which different testosterone replacement levels impact on CBG and HPA function. Compared with gonadectomized rats with low testosterone replacement ( approximately 2 ng/ml), plasma adrenocorticotropin and beta-endorphin/beta-lipotropin responses to restraint stress were reduced in gonadectomized rats with high testosterone replacement ( approximately 5 ng/ml). Plasma CBG levels also varied negatively as a function of testosterone concentration. Moreover, glucocorticoid receptor binding in the liver was elevated by higher testosterone replacement, suggesting that testosterone acts to enhance glucocorticoid suppression of CBG synthesis. Since pituitary intracellular CBG (or transcortin) is derived from plasma, this prompted us to examine whether transcortin binding was similarly responsive to different testosterone replacement levels. Transcortin binding was lower in gonadectomized rats with high plasma testosterone replacement ( approximately 7 ng/ml) than in gonadectomized rats with low testosterone replacement ( approximately 2 ng/ml). This testosterone-dependent decrease in pituitary transcortin was associated, in vitro, with an enhanced nuclear uptake of corticosterone. These findings indicate that the inhibitory effects of testosterone on corticotrope responses to stress may be linked to decrements in plasma and intrapituitary CBG. This could permit greater access of corticosterone to its receptors and enhance glucocorticoid feedback regulation of ACTH release and/or proopiomelanocortin processing.
Adrenocorticotropic Hormone↗
Studies on the interaction between human serum protein fractions and 18O-labeled oxosteroids.
The nature of the interaction between progesterone or testosterone and human albumin as well as the interaction between progesterone and partially purified human transcortin has been studied. Modification of lysine residues of albumin with maleic anhydride resulted in a decreased binding of the steroid as judged from equilibrium dialysis experiments. This suggested that lysine residues in albumin interact with the oxosteroids. In order to check this hypothesis, steroids labeled with 18O in their oxo function (testosterone and progesterone) were synthesized for use as probes of the interactions. However, no loss of label was noted when testosterone or progesterone specifically 18O-labeled in their oxo functions were incubated with albumin. This suggested that no covalent interaction between the steroidal oxo group and albumin took place. This was in contrast to the results obtained with 3,20-18O-labeled progesterone and partially purified transcortin, where a complete loss of 18O label in the protein-bound steroid was found. The nonbound steroid showed an almost complete retention of label. These results indicate a participation of steroid oxo groups in the binding of progesterone to transcortin. Of the possible mechanisms discussed, imine bonds between the steroid and transcortin seem most likely although other types of interactions cannot be ruled out.
Blood Proteins↗
[Reversible precipitation of the glucocorticoid receptor from rat liver by La3+ ions].
It was shown that La3+ ions are capable of precipitating the glucocorticoid receptor from rat liver. Treatment of cytosol containing either free receptor or the hormonereceptor complex with 0,005 M La(NO3)3 results in recptor precipitation. The pellet is readily dissolved in buffer with EDTA. Transcortin and transcortin-like proteins are not affected by La3+ ions. The lanthanium-treated receptor does not lose its ability to bind DNA and chromatin in vitro. It is suggested that precipitation by La3+ ions can be used for separation of the receptor from transcortin and transcortin-like proteins as well as for evaluation of binding parameters for steroid-receptor complexes in rat liver cytosol.
Animals↗
Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus.
In cytosol of the hippocampus corticosterone displays highest affinity for the sites that remain available for binding in the presence of excess RU 26988, which is shown to be a "pure" glucocorticoid. A rather high affinity (greater than or equal to 25%) was found for 11 beta-hydroxyprogesterone, 21-hydroxyprogesterone, 5 alpha-corticosterone, 19-nor-deoxycorticosterone, 11-deoxycorticosterone and cortisol. A moderate affinity (greater than 5% and less than 25%) was displayed by about 14 steroids among which progesterone, aldosterone, 9 alpha-fluorocortisol and dexamethasone. Corticosterone also shows highest affinity to plasma transcortin and thymus cytosol in the presence of RU 26988. However, the rank-order in affinity by the competing steroids was distinctly different from that observed in the hippocampus; cf. aldosterone and dexamethasone displaced [3H]corticosterone from sites unoccupied by RU 26988 in the hippocampus but not from transcortin or sites in thymus cytosol. In thymus cytosol some potent glucocorticoids have higher affinity for the [3H]dexamethasone labeled sites than dexamethasone. The binding of [3H]dexamethasone in thymus cytosol is completely abolished in the presence of a 100-fold excess of RU 26988. We conclude that our data support the evidence for RU 26988 as a selective ligand for glucocorticoid receptors. RU 26988 leaves binding sites available with highest affinity for corticosterone in hippocampus cytosol that are distinct from transcortin-like sites as found in thymus cytosol or from plasma transcortin.
Androstanols↗
Conditions affecting AtT-20 cell glucocorticoid uptake.
Glucocorticoid uptake by intact AtT-20/D-1 cells was studied to determine if the extent of uptake was enhanced or retarded by binding components in serum. The results demonstrate that the uptake of corticosterone, which binds to transcortin, was reduced by addition of serum while uptake of triamcinolone acetonide, which is not bound by transcortin, was unaffected. Neither heat denatured serum nor bovine serum albumin affected corticosterone uptake, further emphasizing the specificity of the inhibition. The presence of serum also affected the apparent binding specificity, since steroids able to bind to transcortin became less effective competitors when serum was present in the incubation medium. In the absence of serum, the specificity of glucocorticoid uptake was qualitatively similar to that of the isolated cytosol receptor. These results emphasize that the selective inhibitory effect of serum transcortin on whole cell uptake of certain steroids should be considered when assaying steroid potency in intact cellular systems.
Animals↗
[Characterization of human retroplacental blood plasma proteins isolated by biospecific chromatography on immobilized cortisol].
Human retroplacental blood plasma proteins with affinity for cortisol were isolated by biospecific chromatography and identified by electrophoretic and immunochemical methods as alpha1- and beta1-globulins and IgG. IgM and IgA immunoglobulins. A high specific affinity for cortisol (Kas = 1,5 . 10(8) M-1 at 23 degrees C) and progesterone (Kas = 2,0 . 10(8) M-1 at 23 degrees C) was observed only for alpha-globulin; other proteins had a low affinity for cortisol. The molecular weight of alpha1-globulin (transcortin) was found to be 50,000-55,000. The amino acid and monosaccharide compositions of this glycoprotein were studied. Its N-terminal and C-terminal amino acid sequences are: Met-Asx-Pro-Asx-Ala- and (Val, Gln)-Leu, respectively. It was concluded that under normal physiological conditions and during pregnancy transcortin is the only specific corticosteroid-binding plasma protein. A complete removal of bound cortisol from the protein mixture and subsequent hydroxylapatite chromatography resulted in homogeneous transcortin retaining more than 90% of its binding capacity. The formation of the transcortin-steroid complex and its complete dissociation are accompanied by conformational changes of the protein globule. Significant changes of the spectral properties of the tryptophane residue of protein and the steroid delta4-3-keto group are indicative of the possibility of their direct interaction.
Amino Acid Sequence↗
The macromolecular binding of prednisone in plasma of healthy volunteers including pregnant women and oral contraceptive users.
The macromolecular binding of prednisone has been studied in the plasma of eight healthy human volunteers. The subjects included two control males, two control females, two females taking estrogen-containing oral contraceptives, and two females in the third trimester of pregnancy. All volunteers exhibited the expected nonlinear plasma binding of prednisolone with free fraction increasing as the total prednisolone concentration was increased. Both the oral contraceptive and pregnant subjects had increased transcortin binding capacity over the control volunteers as evidenced by their increased binding of prednisolone. Prednisone macromolecular binding, however, was not altered by either changing total prednisone concentration or transcortin binding capacity. The mean prednisone free fraction was 0.250 +/- 0.027 in the eight subjects over the concentration range 0-2500 ng/ml. The addition of prednisolone in up to a 25-fold excess did not alter prednisone's free fraction. Prednisone apparently does not bind to transcortin with the same strong affinity that characterizes prednisolone's binding, and due to albumin's extensive binding capacity, prednisone macromolecular binding is not saturable over the pharmacological drug concentration range.
Adult↗