Corticosterone Binding Sites Research Articles

Corticosterone and cortisol binding sites in plasma, immune organs and brain of developing zebra finches: Intracellular and membrane-associated receptors

Glucocorticoids (GCs) affect the development of both the immune and nervous systems. To do so, GCs bind to intracellular receptors, mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). In addition, GCs bind to membrane-associated corticosteroid receptors (mCR). Two well-known GCs are corticosterone and cortisol. Whereas corticosterone is the primary GC in zebra finch plasma, cortisol is the primary GC in zebra finch lymphoid organs and is also present in the brain and plasma during development. Here, we characterized binding sites for corticosterone and cortisol in plasma, liver, lymphoid organs, and brain of developing zebra finches. In tissues, we examined both intracellular and membrane-associated binding sites. For intracellular receptors, there were MR-like sites and GR-like sites, which differentially bound corticosterone and cortisol in a tissue-specific manner. For mCR, we found little evidence for membrane-associated receptors in immune organs, but this could be due to the small size of immune organs. Interestingly, cortisol, but not corticosterone, showed a low amount of specific binding to bursa of Fabricius membranes. For neural membranes, corticosterone bound to one site with low affinity but a relatively high Bmax, and in contrast, cortisol bound to one site with high affinity but a lower Bmax. Our results indicate that intracellular and membrane-associated receptors differentially bind corticosterone and cortisol suggesting that corticosterone and cortisol might have different roles in immune and nervous system development.

Pharmacological characterization of intracellular, membrane, and plasma binding sites for corticosterone in house sparrows

The diversity and specificity of glucocorticoid effects are dependent on cell-specific receptor mechanisms. Three known corticosteroid receptors mediate tissue effects of glucocorticoids in vertebrates: two intracellular receptors that act primarily as ligand-activated transcription factors, and a membrane-associated receptor. The intracellular receptor sub-types have been well characterized in mammals, however relatively little is known about them across non-mammalian vertebrates. The membrane-associated receptors are poorly characterized in most vertebrate taxa. To explore the basis for glucocorticoid action in birds, we pharmacologically characterized the three putative corticosteroid receptors in the brain, as well as a plasma corticosterone binding globulin, in the house sparrow ( Passer domesticus). We found that house sparrow brain cytosol contained two distinguishable binding sites for corticosterone. A high affinity, mineralocorticoid-like receptor had subnanomolar affinity for corticosterone ( K d ∼ 0.2 nM). However, this ‘MR-like’ high-affinity receptor did not bind RU28318 or canrenoic acid, two compounds that bind mammalian MR with high affinity. A lower-affinity, glucocorticoid-like receptor in brain cytosol bound corticosterone with an average K d = 5.61 nM. This GR-like receptor showed subnanomolar affinity for RU 486. MR- and GR-like receptors were found in equal numbers in whole brain assays (average B max = 69 and 62 fmol/mg protein, respectively). House sparrow brain membranes contain a single binding site specific for glucocorticoids, with characteristics consistent with a steroid/receptor interaction. Corticosterone affinity for this putative membrane receptor was approximately 24 nM, with apparent B max = 177 fmol/mg protein. House sparrow plasma contained a single binding site for [ 3H]corticosterone. Specific binding to plasma sites was inhibited by glucocorticoids, progesterone, and testosterone. Testosterone binding to this corticosteroid binding globulin is noteworthy as sex steroid-specific binding globulins have not been identified in birds. Taken together, these data extend our ability to evaluate the comparative actions of glucocorticoids, increase our understanding of mechanisms behind the tissue specificity of glucocorticoid action, and offer insight into the evolution of glucocorticoid action in vertebrates.

Subtype-Specific Affinity for Corticosterone of Rat Organic Cation Transporters rOCT1 and rOCT2 Depends on Three Amino Acids within the Substrate Binding Region

The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are approximately 150 and approximately 4 microM, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane alpha helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K(m) values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [3H]MPP (0.1 microM) uptake by corticosterone (24 +/- 4 microM) was significantly higher compared with the IC50 value for inhibition of [14C]TEA (10 microM) uptake (5.3 +/- 1.7 microM). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.

Activities of the pituitary–adrenal and gonadal axes during the estrous cycle in adult female rats prenatally exposed to morphine

The present investigation concerns 80–90-day-old female rats born from morphine-exposed mothers (2×10 mg/kg per day from day 11–18 of gestation) or saline-treated ones (controls). The former showed reduced size and activity of the adrenals at birth. At adult stage, they present: (1) higher increase of plasma adrenocorticotrophic hormone level on proestrus; (2) significant rise of plasma corticosterone level on diestrus morning and estrus evening; (3) adrenal atrophy which was significant only on diestrus and estrus morning; (4) more corticosterone binding sites of type I (mineralocorticoid receptors) on proestrus morning in the hippocampus; (5) more corticosterone binding sites of type II (glucocorticoid receptors) in the hippocampus on proestrus morning and in the hypothalamus on estrus morning. In both experimental groups, B max for hypothalamic mineralocorticoid receptors were drastically higher on estrus morning than on the other stages of the estrous cycle. The activity of the pituitary–gonadal axis is poorly affected by prenatal morphine-exposition. In both experimental groups drastic and comparable surges of both plasma progesterone and luteinizing hormone were observed during proestrus. Nevertheless morphine-exposed females showed higher levels of plasma estradiol on diestrus morning but lower levels on metestrus morning. In conclusion, prenatal exposition to morphine has long-term effects mainly on pituitary–adrenal axis as well as on binding sites for corticosterone in the hypothalamus and the hippocampus which are dependent on the estrous cycle stages in adult females.

Novel nuclear corticosteroid binding in rat small intestinal epithelia.

When small intestinal epithelial cells are incubated with [(3)H]corticosterone, nuclear binding is displaced neither by aldosterone nor RU-28362, suggesting that [(3)H]corticosterone is binding to a site distinct from mineralocorticoid receptor and glucocorticoid receptor. Saturation and Scatchard analysis of nuclear [(3)H]corticosterone binding demonstrate a single saturable binding site with a relatively low affinity (49 nM) and high capacity (5 fmol/microg DNA). Competitive binding assays indicate that this site has a unique steroid binding specificity, which distinguishes it from other steroid receptors. Steroid specificity of nuclear binding mirrors inhibition of the low 11beta-dehydrogenase activity, suggesting that binding may be to an 11beta-hydroxysteroid dehydrogenase (11betaHSD) isoform, although 11betaHSD1 is not present in small intestinal epithelia and 11betaHSD2 does not colocalize intracellularly with the binding site. In summary, a nuclear [(3)H]corticosterone binding site is present in small intestinal epithelia that is distinct from other steroid receptors and shares steroid specificity characteristics with 11betaHSD2 but is distinguishable from the latter by its distinct intracellular localization.

A subset of kappa opioid ligands bind to the membrane glucocorticoid receptor in an amphibian brain.

Previous studies demonstrated that a membrane receptor for glucocorticoids (mGR) exists in neuronal membranes from the roughskin newt (Taricha granulosa) and that this receptor appears to be a G protein-coupled receptor (GPCR). The present study investigated the question of whether this mGR recognizes nonsteroid ligands that bind to cognate receptors in the GPCR superfamily. To address this question, ligand-binding competition studies evaluated the potencies of various ligands to displace [3H]corticosterone (CORT) binding to neuronal membranes. Initial screening studies tested 21 different competitors and found that [3H]CORT binding was displaced only by dynorphin 1-13 amide (an endogenous kappa-selective opioid peptide), U50,488 (a synthetic kappa-specific agonist) and naloxone (a nonselective opioid antagonist). Follow-up studies revealed that the kappa agonists bremazocine (BRE) and ethylketocyclazocine (EKC) also displaced [3H]CORT binding to neuronal membranes, but that U69,593 (a kappa specific agonist) and nor-BNI (a kappa specific antagonist) were ineffective. The Ki values measured for the opioid competitors were in the subnanomolar to low micromolar range and had the following rank-order: dynorphin > U50,488 > naloxone > BRE > EKC. Because these ligands displaced, at most, only 70% of [3H]CORT specific binding, it appears that some [3H]CORT binding sites are opioid insensitive. Kinetic analysis of [3H]CORT off-rates in the presence of U50,488 and/or CORT revealed no differences in dissociation rate constants, suggesting that there is a direct, rather than allosteric, interaction with the [3H]CORT binding site. In summary, these results are consistent with the hypothesis that the high-affinity membrane binding site for [3H] CORT is located on a kappa opioid-like receptor.

11 β-Hydroxysteroid dehydrogenase-2 is a high affinity corticosterone-binding protein

In addition to mineralocorticoid and glucocorticoid receptors, a third category of corticosteroid binding sites has been described in the kidney, the Type III binding protein. This intracellular binder has high affinity for corticosterone, but binds neither aldosterone nor synthetic glucocorticoids. Based on similarities in steroid specificity and kinetic parameters, we hypothesized that these corticosterone binding sites belong to the type 2 isoform of 11 β-hydroxysteroid dehydrogenase (11 β-HSD2). The goal of this study was to express the recombinant rabbit 11 β-HSD2 in mammalian cells and test if such cells acquire both NAD-dependent 11 β-HSD2 activity as well as high affinity corticosterone binding sites. Stably transfected CHO cell lines expressed high, NAD-dependent, unidirectional 11 β-HSD2 activity. At the same time, the transfected cells also acquired a large number of corticosterone-specific binding sites (1.21±0.3×10 6), whereas non-transfected cells had no corticosterone binding above background. The K d for corticosterone was 25±8 nM. Neither the glucocorticoid receptor (GR) agonists dexamethasone and RU 28362 nor the mineralocorticoid receptor (MR) agonist aldosterone bound to these sites. The steroid specificity of the binding sites, as determined by competing [ 3H]corticosterone with unlabeled steroids, is identical to that of 11 β-HSD2: corticosterone≫11-hydroxyprogesterone>carbenoxolone>11 dehydrocorticosterone>cortisol>progesterone∼DOC⋙DEX>RU 28362∼aldosterone. These results strongly suggest that the previously described high affinity corticosterone binding sites are 11 β-HSD2. Thus, though Type III binding sites are not corticosteroid receptors as originally thought, they play an important role in regulating the activity of both mineralocorticoid- and glucocorticoid receptors.

Characterization of specific corticosterone binding sites in adrenal cortex plasma membrane and their localization by autoradiographic studies.

Specific corticosterone binding to calf adrenal cortex plasma membrane was measured using the biologically active radioligand [3H]corticosterone. Corticosterone binding was found to be time-dependent, saturable and reversible, and was reduced by more than 70% when membranes were pretreated with proteases. The population of corticosterone binding sites in calf adrenal cortex plasma membrane was homogeneous and displayed the following characteristics: equilibrium dissociation constant Kd = 77 +/- 8 nM and maximum specific binding capacity Bmax = 70,378 +/- 6,385 fmol/mg protein. The relative affinities of several structural analogues of steroids were deduced from competition assays. From these experiments we can conclude that the plasma membrane binding site characterized is selective for corticosterone and progesterone derivatives, and different from nuclear glucocorticoid, mineralocorticoid, estrogen and progestin receptors. Likewise, this corticosterone binding site is independent of mineralocorticoid and Na+, K(+)-ATPase digitalis receptors. From autoradiographic studies we suggest these corticosterone binding sites are located in the whole adrenal cortex.

Is the renal type III corticosteroid-binding site the collecting duct-specific isoform of 11 beta-hydroxysteroid dehydrogenase?

In addition to mineralocorticoid and glucocorticoid receptors, the kidney contains a third high affinity binding site for endogenous glucocorticoids, the type III receptor. These binding sites have been localized to the collecting duct, but their biological function has not yet been identified. We have used immunodissected rabbit cortical collecting duct (CCD) cells to further characterize these binding sites. Experiments using intact cells revealed very high levels of [3H]corticosterone (CS) binding (2.99 +/- 0.38 x 10(6) sites/cell), about 100-fold higher than the number of mineralocorticoid or glucocorticoid receptors in CCD cells. Among the two cell types of the collecting duct, principal cells, the putative targets of aldosterone, contain approximately 10 times more CS-binding sites than intercalated cells. The Kd of the binding sites for CS averaged 54.3 +/- 3.48 nM at 0 C. The relative affinity of unlabeled steroids for the binding sites is CS > carbenoxolone congruent to glycyrrhetinic acid > or = 11-dehydrocorticosterone (11-DHCS) > cortisol congruent to cortisone > deoxycorticosterone > progesterone. Synthetic glucocorticoids (dexamethasone, RU 28362, and RU 486) and aldosterone did not compete for [3H]CS binding. Based on their preferential localization to CCD cells, which are the main targets of aldosterone, we hypothesized that these CS-binding sites are involved in conferring aldosterone specificity on mineralocorticoid receptors in these cells. As 11 beta-hydroxysteroid dehydrogenase (11-OHSD) is thought to play a key role in this process, we studied the relationship between CS-binding sites and the collecting duct isoform of this enzyme (11-OHSD/CD) in purified CCD cells. Freshly isolated cells rapidly converted [3H]CS to 11-DHCS with an apparent Km of about 50 nM, a value close to the Kd of the CS-binding sites for CS in these cells. The rank order of potency of unlabeled steroids to decrease the conversion of [3H]CS to [3H]11-DHCS was identical to their relative affinity for the CS-binding sites. Also, there is a close correlation (r = 0.783; P < 0.0001) between the activity of 11-OHSD and the number of CS-binding sites in different CCD preparations. Based on the similarities between the abundant CS-binding sites and avid CS metabolism in CCD cells, we suggest that these binding sites belong to the collecting duct isoform of 11-OHSD, which, by decreasing the intracellular levels of active glucocorticoids, plays an important role in conferring aldosterone selectivity on mineralocorticoid receptors in CCD cells.

A corticosteroid Receptor in Neuronal Membranes

Steroids may rapidly alter neuronal function and behavior through poorly characterized, direct actions on neuronal membranes. The membrane-bound receptors mediating these behavioral responses have not been identified. [3H]Corticosterone labels a population of specific, high-affinity recognition sites (dissociation constant = 0.51 nanomolar) in synaptic membranes from an amphibian brain. These binding sites were localized by receptor autoradiography in the neuropil, outside the regions of perikarya. The affinities of corticoids for this [3H]corticosterone binding site were linearly related to their potencies in rapidly suppressing male reproductive behavior. Thus, it appears that brain membranes contain a corticosteroid receptor that could participate in the regulation of behavior.

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver

The specific binding of [3H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (KD = 8.8 nM and Bmax = 1477 fmol/mg protein) and the other one has low affinity and high binding capacity (KD = 91 nM and Bmax = 9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (KD = 11.2 nM and Bmax = 1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na+,K(+)-ATPase digitalis receptor. Probably it is of the same nature that the one determinate of [3H]cortisol and [3H]corticosterone in mouse liver plasma membrane. Beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.

Characterization of Specific Binding Sites for Corticosterone in Mouse Liver Plasma Membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.

Characterization of glucocorticoid receptors in whole and cellular subfractions of embryonic chick spinal cord

Steroid-specific binding sites for tritiated corticosterone have been localized, via autoradiography, to motoneurons in the lateral motor columns of the adult rat spinal cord. Binding sites in adult rat spinal cord have been characterized biochemically and shown to possess the properties of a putative glucocorticoid receptor. The presence of receptors for glucocorticoids in embryonic chick spinal cord was determined and their characterization undertaken as a prelude to the study of the functions under regulation by glucocorticoids during development. Assay conditions were defined and binding of [ 3H]dexamethasone ([ 3H]Dex) to cytosols of 6- and 10-day embryonic spinal cord and cellular subfractions of 6-day spinal cord determined. Saturable, high-affinity binding of [ 3H]Dex to cytosols prepared from both whole 6- and 10-day spinal cords and cells of all 3 cellular subfractions of 6-day spinal cords was observed. The binding component in 10-day cytosols was (1) proteinaceous, as binding was eliminated by heating cytosols, and (2) a macromolecular species, as it displayed a sedimentation coefficient of 8S upon centrifugation in sucrose gradients. The putative receptor displayed binding specific for glucocorticoids in a competition assay, with the exception that some inhibition of binding by the androgen ligand, methyltrienolone (R1881) was observed. The binding affinity decreased as the values for K D increased from 3.4 ± 0.9 nM on day 6 to 15.7 ± 1.8 nM on day 10, while the values for B max increased from 270 to 855 fmol/mg protein over the same period. The neuronal fractions I and II displayed fewer sites (60 and 130 fmol/mg protein respectively) of higher affinity (2.1 and 2.5 nM respectively) than the non-neuronal fraction III (B max = 164 fmol/mg protein and K D = 4.2 nM). The number of binding sites per cord increased 4.8-fold from embryonic day 6 to 10. These results demonstrate that specific, high-affinity glucocorticoid receptors are present in chick spinal cord cells, including motoneurons, as early as embryonic day 6, when spontaneous limb movements begin.

Loss of 3H-corticosterone binding capacity in hippocampal slices maintained In vitro

Nuclear uptake of 3H-corticosterone was studied in hippocampal slices subjected to varying periods of preincubation before application of steroid. Uptake capacity declined rapidly toward nonspecific levels over the course of 3 hours from the time of cutting. Glucocorticoid receptors in the cytosol fraction of hippocampal slices also were found to decline and Scatchard analysis indicated a loss of corticosterone binding sites rather than a change in binding affinity. These results are discussed in terms of the reported effects of corticosterone in vitro.

Corticosterone binding sites along the rat nephron

Contents: the correct title for the article on page F504 by S.-M. K. Lee, M. A. Chekal, and A. I. Katz is “Corticosterone binding sites along the rat nephron.”

Corticosterone receptors decline in a site-specific manner in the aged rat brain

Putative glucocorticoid receptors were measured in the brain and pituitary glands of young and aged rats in vivo and in vitro. Adrenalectomized rats were injected with a set dose of radiolabeled corticosterone plus unlabeled corticosterone; 2 h later [ 3H]corticosterone levels were measured in purified nuclear pellets from pituitary and 5 brain regions. Substantial decreases were seen in aged subjects in the maximal number of corticosterone binding sites in hippocampus and amygdala; all other regions showed no age-related changes. In contrast, there were no declines in the nuclear uptake of [ 3H]dexamethasone (DEX) in the aged rat brains. Since DEX interacts selectively with non-neuronal receptors in vivo, the deficit in glucocorticoid binding is selective for neurons. Subsequent studies assessing glucocorticoid receptor levels in cytosol preparations in vitro significant declines in hippocampus and amygdala quantitavely comparable to the decline observed in nuclear binding in these loci. This suggests that the primary deficit leading to nuclear depletion may be the reduction of cytosolic receptor number, rather than other possible factors such as the reduction in receptor affinity or translocation of steroid-receptor complex. This decline may play a role in a number of limbic functions which are influenced by glucocorticoids and shows deficits with age.

Corticosterone binding sites along the rat nephron.

Glucocorticoids influence numerous kidney functions but the precise location of glucocorticoid receptors in the nephron is not known. To identify the renal binding sites of corticosterone, the natural glucocorticoid in the rat, we measured the binding of [3H]corticosterone to discrete nephron segments microdissected from adrenalectomized rats. Highest specific binding capacity at 25 degrees C (expressed as fmol X cm-1 +/- SE) was found in the cortical collecting tubule (9.69 +/- 0.77) followed in decreasing order by the distal convoluted tubule (2.70 +/- 0.49), medullary collecting tubule (2.58 +/- 0.64), proximal convoluted tubule (1.09 +/- 0.10), and pars recta (0.57 +/- 0.08). Binding was lowest in the thick ascending limb of Henle's loop, with comparable values in the medullary (0.27 +/- 0.05) and cortical (0.26 +/- 0.05) portions of this segment. The apparent maximal binding capacity of the cortical collecting tubule for corticosterone exceeded by nearly two orders of magnitude that of aldosterone previously measured by us in this structure, which is in agreement with the observations of other investigators in kidney cytosol. Specific binding of corticosterone can be demonstrated along the entire rat nephron, but binding sites are concentrated in the cortical collecting tubule. This segment appears to be the main target site for corticosterone as it is for aldosterone.

Corticosteroid receptors in the kidney of chick embryo I. Nature and properties of corticosterone receptor

Cytosol from kidney of chick embryo (age 16–18 days) contained a corticosterone-binding site with the features of a putative receptor. This receptor was a thermolabile protein, readily digested by proteolytic enzymes, with a sedimentation coefficient of 7–8 S and with an apparent molecular weight greater than 100,000. Simultaneous studies with transcortin (CBG) revealed several differences between the renal- and serum-binding protein pertaining to the effect of temperature, the sedimentation coefficient, the charcoal “stripping”and, finally, the binding and competition of various steroids for the two proteins. Kinetic analysis showed a rapid association (10 min), which followed second-order reaction kinetics, and a dissociation of pseudo-first-order reaction kinetics with a t 1 2 , of168 min at †. The analysis of the Scatchard plot showed the presence of a single class of binding sites with an association constant ( K A ) of 1.3 × 10 8 M −1 and a binding capacity ( n max) of 500–700 fmoUmg protein. We obtained similar results when we used dexamethasone as a ligand. The association ( k a ) and dissociation ( k d ) rate constants were respectively 2.9 × 10 6 M −1 sec −1 and 6.86 × 10 −5 sec −1. From their ratio a K A value of 4.2 × 10 10 M −1 was obtained. Studies with various steroids demonstrated that only dexamethasone and, to a lesser degree, progesterone competed for the binding site. These data showed that the kidney of chick embryo possessed one type of receptor for the glucocorticoids, which was similar to the type II described in rat kidney.

Aldosterone and corticosterone binding and effects on Na+ transport in cultured kidney cells.

A continuous line of cells (A6) derived from toad kidney has been shown to form epithelia in culture that manifest aldosterone-stimulatable transepithelial sodium transport. In this study an efficient filtration assay for nuclear binding of [3H]aldosterone was validated. Specific high-affinity aldosterone and corticosterone binding sites in the particulate (nuclear-enriched) fraction were characterized in intact epithelia. Despite metabolism of both steroids, two high-affinity binding sites for each were demonstrable: aldosterone, K'd1 = 0.85 (+/- 0.19) X 10(-10) and K'd2 = 1.6 (+/- 0.42) X 10(-8) M; corticosterone, K'd1 = 0.5 (+/- 0.31) X 10(-10) and K'd2 = 0.32 (+/- 0.19) X 10(-8) M. Analogue competition-binding studies indicated a qualitative difference in the two sites and co-occupancy of both sites by the two steroids. The sodium transport response to aldosterone and corticosterone approximated a linear function of occupancy of the lower affinity sites. Although the lower affinity sites resemble mammalian glucocorticoid receptors in terms of relative binding affinities for analogues, we conclude that they are the receptors which mediate the aldosterone and corticosterone stimulation of Na+ transport in these epithelia.

Glucocorticoid and estrogen regulation of corticosteroid-binding globulin production by rat liver.

We have recently demonstrated that rat liver can synthesize and secrete corticosteroid-binding globulin (CBG). The present study extends these observations and examines the hormonal regulation of hepatic CBG production. Male rats were pretreated by adrenalectomy and/or glucocorticoid or estrogen administration and the rate of CBG production was measured in vitro. The production rates were assessed by the generation of specific corticosterone binding sites in both a liver-slice preparation and in an isolated perfused liver. The two techniques showed qualitatively similar results. Adrenalectomy enhanced and glucorticoid administration inhibited the rate of CBG production and secretion. Pretreatment of male rats with estradiol stimulated the rate of CBG production. The production rates were 20- to 40-fold higher in the perfused liver demonstrating its superiority over the liver-slice system. The livers from intact rats secreted CBG binding sites at a rate of approximately 18 pmol/g liver per hours, generating an estimated 20% of the total CBG content of a rat each day. The possible clinical implications of the therapeutic use of glucocorticoids that bind to CBG, yet inhibit CBG production, are discussed.