Scatchard Analysis Of Saturation Curves Research Articles

Calcitonin modifies ligand binding to muscarinic receptor in CNS membranes

Calcitonin (CT) is a peptide produced by the thyroid gland, whose best described role is to prevent bone reabsorption, though it also participates in other biological functions through both central and peripheral mechanisms. CT is able to inhibit brain Na +, K +-ATPase activity (Rodrı́guez de Lores Arnaiz, López Ordieres, Peptides 1997;18:613–5) and a relationship between such enzyme activity and cholinergic function has been suggested. Accordingly, we tested CT effect on [ 3H]-quinuclidinyl benzilate ([ 3H]-QNB) binding to rat CNS membranes to determine whether the peptide is able to modify the cholinergic muscarinic receptor as well. It was found that 1×10 −7–1×10 −5 M CT decreased 20–70% ligand binding to hippocampal, cerebellar, cortical and striatal membranes. Scatchard analysis of saturation curves showed that 5×10 −6 M CT significantly modified binding kinetic constants, thus it increased roughly 220% K d values and decreased 20–36% B max values in cerebral cortical and cerebellar membranes. Since the peptide decreases affinity ligand binding and reduces the number of binding sites, CT may well be acting as a cholinergic modulator through a decrease in muscarinic receptor functionality.

Effects of training on the concentration of Na+, K+-ATPase in foal muscle.

We investigated the effect of training on the Na+, K+-ATPase concentration in foal skeletal muscle by measurement of [3H]ouabain binding. From the 7th day after birth, 12 foals were divided in 3 groups: a) staying in a box stall (Box); b) staying in a box stall with a training regimen of an increasing number of sprints per day (Training); and c) staying on pasture (Pasture). Euthanasia was performed after 5 months. In semitendinosus muscle, the concentration of [3H]ouabain binding sites (pmol/g wet wt) was 181 +/- 6 in the Box, 220 +/- 15 in the Training, and 197 +/- 8 in the Pasture group (all n = 6; Box vs. Training, P<0.05). In gluteus medius, the concentration of [3H]ouabain binding sites was 168 +/- 9 in the Box, 219 +/- 12 in the Training, and 175 +/- 4 in the Pasture group (all n = 6; Box or Pasture vs. Training, P<0.02). Scatchard analysis of saturation curves showed that the difference in [3H]ouabain binding sites between the 3 groups could not be ascribed to differences in the Kd for ouabain. Both for semitendinosus and gluteus medius muscle, the concentration of [3H]ouabain binding sites increased in the order Box < Pasture < Training (a total increase of around 20%). This suggests a specific effect of the amount and intensity of exercise on the Na+, K+-ATPase concentration in horse skeletal muscle, and may lead to a better performance during exercise.

Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF 1) and a carboxyl-terminal variant

Corticotropin releasing factor (CRF) is the major neuropeptide regulating the hypothalamo–pituitary–adrenocortical axis in most species. A pituitary receptor for CRF (designated CRF 1) belonging to the seven-transmembrane helix, G-protein-coupled receptor superfamily has been cloned for human, rat, mouse and xenopus. Since ovine CRF shares only 84% identity to human/rat CRF (h/rCRF) we postulated that the sheep pituitary CRF 1 receptor may have similarly diverged from the rodent and human CRF 1. We report the molecular cloning of an ovine pituitary cDNA containing a 1245 bp open reading frame encoding a 415 amino acid sheep CRF 1 receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF 1, respectively. The divergence in primary structure between the sheep CRF 1 and the other mammalian CRF 1s is primarily localized to the extracellular amino terminal domain of the receptor (18 of 22 divergent residues, ovine vs human CRF 1). A variant of the oCRF 1 was also isolated (oCRF 1var) with 133 bp deleted from nucleotide (nt) 1080 to nt 1213 of the open reading frame (ORF) resulting in a new ORF of 1176 nt predicting a 392 residue CRF 1 variant receptor. The 133 bp deletion would cause a frame-shift at residue 358 within the carboxyl-third of the seventh transmembrane domain (TM7) resulting in a shortened cytoplasmic tail with a new amino acid sequence from residue 358 to 392. Scatchard analysis of saturation curves using membrane prepared from Cos 7 cells transfected with oCRF 1 or oCRF 1var indicated that both wild-type and variant receptors were expressed similarly (number of CRF binding sites) and both bound oCRF with high affinity [oCRF 1 ( K d): 2.5+1.6 nM; oCRF 1var: 5.1+2.3 nM]. The non-hydrolyzable GTP analogue (GTP γS) lowered the affinity of both wild-type and variant oCRF 1 receptors to a similar extent (oCRF 1: 18.2 nM; oCRF 1var: 22.4 nM). Both wild-type and variant oCRF 1 receptors exhibited ≈10-fold greater selectivity for oCRF and sauvagine compared to h/rCRF or α-helical [9–41]oCRF. CRF effectively stimulated the accumulation of cAMP (EC 50=51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF 1 receptor. In Cos 7 cells transfected with oCRF 1var, cAMP accumulation was only observed at the highest concentration of oCRF utilized (100 nM). Basal (unstimulated) levels of cAMP in Cos 7 cells transfected with oCRF 1var (in the presence of 2 mM IBMX) were ≈50% lower than for the wild-type oCRF 1. Differences in cAMP accumulation could not be attributed to differences in receptor number since total binding sites in the transfected cells were not different between wild-type or variant oCRF 1 receptors. Agonist-induced receptor internalization, determined as the percent of total [ 125I] Tyr 0-oCRF bound located in the acid-resistant fraction of transfected Cos 7 cells, increased with time (0–60 min at 37°C) for both wild-type and variant oCRF 1. Wild-type CRF 1 internalized ≈2-fold greater percent of total [ 125I] Tyr 0-oCRF bound compared to the variant receptor. In summary, an ovine CRF 1 and a CRF 1 cytoplasmic tail receptor variant displaying high affinity binding to oCRF as well as selectivity for oCRF vs h/rCRF, were cloned from an adult sheep pituitary cDNA library. GTP γS studies indicate that both variant and wild-type receptors couple efficiently to G αs however, only the wild-type oCRF 1 is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF 1var is also reduced compared to the wild-type CRF 1 receptor. We suggest that the oCRF 1var interacts efficiently with G αs but is unable (post-hormonal binding) to effectively stimulate G-protein activation of adenylate cyclase, indicating that the cytoplasmic tail of the CRF 1 can modulate receptor function related to signal transduction. The cytoplasmic carboxyl terminus of the CRF 1 also appears to play a role in agonist-induced internalization of this member of the class II subfamily of G-protein coupled receptors (GPCRs).

Down-regulation of hepatic peripheral-type benzodiazepine receptors caused by acute lead intoxication

In the present study we investigated the influence of acute lead poisoning upon the expression of benzodiazepine receptors. In addition, we examined if administration of PK 11195, an isoquinoline carboxamide derivative, to lead-poisoned rats could modulate the changes in receptor binding properties achieved by lead alone. Lead poisoning was ascertained by determination of urine σ-aminolevulinic acid levels and lead levels in rat livers. Scatchard analysis of saturation curves of [ 3H]PK 11195 binding to liver membranes of rats treated with lead alone or with both lead and PK 11195 showed an approximately two-fold decrease in receptor density in comparison with control groups. Peripheral benzodiazepine receptor density in the kidneys and adrenals of poisoned rats was not changed by lead intoxication per se or by coadministration of PK 11195. Scatchard analysis of saturation curves of [ 3H]Ro 15-1788 binding in rat cerebral cortex tissue showed no difference in the receptor density between the various groups. The K d values of all organs were in the nanomolar range (1–4 nM). We conclude that PK 11195 is not a protective agent of hepatic peripheral benzodiazepine receptors in lead intoxication. Moreover, it causes over-accumulation of lead in hepatocytes in an unknown mechanism of action.

Modulation of human myometrial PGE 2 receptor by GTP characterization of receptor subtype

We studied PGE 2 specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was : PGE 2 ≥ PGE 1 ⪢ PGF 2α > Iloprost ≥ Carbacyclin ⪢ ZK 110841 (PGD 2 analogue). These relative affinities indicated that the receptor was of the EP type. In kinetic experiments GTP, GppNHp and GTPγS increased the rate of PGE 2 binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10 −4 sec −1 (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k−1 = 7.16 10 −4 sec −1 half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 ± 2.8.10 −9 M to 4.96 ± 1.25.10 −9M, but the number of binding sites did not change significantly (310 ± 37 to 350 ± 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10 −4M. GppNHp and GTPγS were effective at 1.10 −5M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE 2 binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla. Competition studies with PGE 2 analogues (sulprostone, 17-phenyl-ω-trinor PGE 2, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP 3 subtype.

Involvement of a Disulfide Bond in the Binding of Flunitrazepam to the Benzodiazepine Receptor from Bovine Cerebral Cortex

The effects of chemical modification of a disulfide bond(s) (-SS-) or sulfhydryl group(s) (-SH) on the [3H]-flunitrazepam ([3H]FNZ) binding to membrane-bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD-R) from bovine cerebral cortex were examined. Reduction of -SS- with dithiothreitol (DTT) brought about a reversible, time- and dose-dependent inhibition of [3H]FNZ binding to the membrane-bound BZD-R. Alkylation of the membranes with the -SH-modifying reagent iodoacetamide (IAA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) produced a slight inhibition of [3H]FNZ binding in a dose-dependent manner. Scatchard analysis of saturation curves of [3H]FNZ binding in the presence and absence of 5 mM DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane-bound and the immunoprecipitated BZD-R led to [3H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of -SS- does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of [3H]FNZ binding by FNZ (an agonist) or by Ro 15-1788 (an antagonist) suggests the presence of -SS- at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with [3H]FNZ revealed a clear-cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 mM DTT/IAA-treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD-R.

Purification and characterization of neurotensin receptor from rat brain with special reference to comparison between newborn and adult age rats

Scatchard analysis of saturation curves was performed to compare newborn and adult rat neurotensin receptor using [ 3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (K d=0.13 nM, B max=710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, K d1=0.13 nM; low affinity site, K d2=20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [ 3H] neurotensin to the purified receptor is saturable and specific, with a K d of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of M r=55,000.

The two forms of the pituitary adenylate cyclase activating polypeptide (PACAP (1–27) and PACAP (1–38)) interact with distinct receptors on rat pancreatic AR 4-2J cell membranes

The existence of specific receptors for the two PACAPs (Pituirary Adenylate Cyclase Activating Peptides of 27 and 38 amino acids) was previously demonstrated on membranes from the pancreatic acinar cell line AR 4-2J (Buscail et al., FEBS Lett. 202, 77–81, 1990) by [125I]PACAP-27 binding. Here we demonstrate, by comparing Scatchard analysis of saturation curves and competition binding curves obtained with [125I]PACAP-27 and [125I]PACAP-38 as radioligands, the coexistence of two classes of receptors : 1/ PACAP-A receptors that recognize PACAP-27 and PACAP-38 with the same high affinity (Kd 0.3 nM) and 2/ PACAP-B receptors that recognize PACAP-38 with a high affinity (Kd 0.3 nM) and PACAP-27 with a lower affinity (Kd 30 nM). These two receptors are coupled to adenylate cyclase but can be clearly distinguished by the ability of PACAP(6–27) to specifically inhibit PACAP-27 adenylate cyclase activation.

Identification and distribution of peripheral benzodiazepine binding sites in male rat genital tract

In the present study we identified and characterized the distribution of high-affinity peripheral benzodiazepine binding sites (PBzS) in male rat vas deferens (whole, and prostatic and epididymal portions), prostate, seminal vesicles, and Cowper's glands. [ 3H]PK 11195, an isoquinoline carboxamide derivative, was used as a radioligand specific for PBzS. Scatchard analysis of saturation curves of [ 3H]PK 11195 binding in the whole vas deferens, the prostatic and epididymal portions of the vas deferens, the prostate, the seminal vesicles, and Cowper's glands yielded mean maximal numbers of binding sites of 1211 ± 158, 1012 ± 311, 1451 ± 156, 1805 ± 86, 865 ± 51, and 2251 ± 135 fmol/mg protein, respectively. The equilibrium dissociation constant values ranged between 1 and 3 mM in all the above tissues. The ability of various drugs to displace the specific binding of [ 3H]PK 11195 from PBzS in Cowper's gland membranes was also tested. The inhibition constants for Ro 5-4864, diazepam, and PK 11195 were 28, 330, and 4 nM, respectively, whereas clonazepam, Ro 15-1788, and testosterone were inefficient in displacing [ 3H]PK 11195. The presence of high densities of PBzS in the male genital tract suggests a functional role in these hormone-dependent organs.

Use of slide-mounted frozen sections to study the autoradiographic localization, characterization and quantification of luteinizing hormone-releasing hormone receptors in the rat pituitary

Slide-mounted frozen sections were used for the autoradiographic localization, characterization and quantification of LHRH receptors. The radioligand was the iodinated LHRH agonist [D-Ser(TBU) 6, des-Gly-NH 2 10]LHRH ethylamide (LHRH-A). Specificity studies demonstrated that specific labeling was concentrated in the anterior pituitary lobe and that the tracer could be displaced with either LHRH or LHRH-A. Scatchard analysis of saturation curves gave one-site interactions with a high affinity (Kd = 0.48 ± 0.1 nM) for the iodinated LHRH-A. In order to evaluate the effects of different treatments on the LHRH receptor concentration, the effects of sex steroids on the density of radioautographic reaction were evaluated using an image analyzer. The results obtained were similar to those previously reported with classical binding assays. Thus, this simple technique appears suitable for the study of localization and characterization of LHRH receptors in different experimental conditions.

Distribution of dopamine D1 receptors in rat cortical areas, neostriatum, olfactory bulb and hippocampus in relation to endogenous dopamine contents.

The tritiated dopamine D1 antagonist SCH23390 was employed to determine the densities of D1 receptors in seven discrete and functionally identified cortical areas (cingulate, frontal, parietal, primary somatosensory, primary visual, retrosplenial and entorhinal-piriform) as well as in the neostriatum, hippocampus and olfactory bulbs. In addition, the tissue levels of the catecholamines NA, AD, DA, the indoleamine 5-HT and their main metabolites (MHPG, DOPAC, HVA, 3-MT, 5-HTP and 5-HIAA) were measured in the different regions by HPLC with electrochemical detection. The Scatchard analysis of saturation curves revealed the highest density of [3H]SCH23390 binding sites for the neostriatum, while the densities were 10-20 times lower for total cerebral cortex and hippocampus respectively. For the olfactory bulb and other cortical areas, D1 receptor densities were determined by equilibrium binding at a fixed radioligand concentration approaching saturation. The distribution of D1 receptors was heterogeneous with the greatest densities in entorhinal-piriform and cingulate cortices. The endogenous DA levels measured for all regions correlated with their metabolite (DOPAC, HVA and 3-MT) contents (r = 0.999; P less than 0.001). There was also a very good correlation (r = 0.981; P less than 0.001) between tissue DA and D1 receptor densities. This quantitative information reflects particular features of the organization of the DA systems and is discussed in relation to turnover and recently established aspects of the DA innervation.

Inhibition of gonadotropin-releasing hormone receptors in rat anterior pituitary monolayer cell cultures by danazol

To study possible cellular antigonadotropic effects of danazol, monolayer cultures of anterior pituitary cells from immature female rats were treated with danazol. Measurements of luteinizing hormone release in response to 10−8 mol/L gonadotropin-releasing hormone challenge and iodine 125-labeled gonadotropin-releasing hormone binding activity were done after exposure to increasing concentrations of danazol and for increasing lengths of time. It was found that luteinizing hormone secreted by pituitary cells in response to gonadotropin-releasing hormone was inhibited after danazol treatment in a dose- and time-dependent manner when compared to controls. Also, a 45% decrease in gonadotropin-releasing hormone receptor binding capacity was observed in pituitary cells cultured in the presence of increasing concentrations of danazol in the range of 10−8 to 10−4 mol/L when compared to controls. Furthermore, exposure to danazol for 25 to 96 hours caused a marked decrease in gonadotropin-releasing hormone binding activity (p < 0.005). Under these experimental conditions danazol treatment decreased the pituitary receptors for gonadotropin-releasing hormone in a dose- and time-dependent manner. Scatchard analysis of saturation curves for the binding of gonadotropin-releasing hormone to cellular gonadotropin-releasing hormone receptors indicated that the observed decrease in gonadotropin-releasing hormone binding in the danazol-treated group was due to a change in the number of gonadotropin-releasing hormone binding sites rather than a change in the affinity. It is therefore concluded that the antigonadotropic activity of danazol appears to be related to a decrease in gonadotropin-releasing hormone receptors in the pituitary.

Localization and characterization of brain somatostatin receptors as studied with somatostatin-14 and somatostatin-28 receptor radioautography

The localization and characterization of receptors for somatostatin-14 (S-14) and somatostatin-28 (S-28) were studied in the rat brain using the iodinated agonists [Tyr 0, d-Trp 8]S-14 and [Leu 8, d-Trp 22,Tyr 25]S-28 as tracers. Slide-mounted frozen sections were used for the autoradiographic localization and biochemical characterization of somatostatin receptors. In the latter case counting was performed on scraped off serial sections from rostral regions of the brain. Specificity studies demonstrated that either tracer could be displaced with S-28, S-14 or their agonists. The N-terminus fragment (1–12) of S-28 as well as a number of unrelated peptides were unable to compete with either tracer, indicating that the binding capacity for ligand-receptor recognition is located in the C-terminal portion of S-28. Scatchard analysis of saturation curves gave a one-site interaction with K d of values of 0.42 ± 0.09nM and0.32 ± 0.04nM for the S-14 and S-28 iodinated agonists, respectively. By radioautoradiography, the distribution of receptors for both S-14 and S-28 appeared very similar with high levels of binding in the deep layers of the cortex, the cingulate cortex, the claustrum, the locus coeruleus and most structures of the limbic system. Treatment with cyteamine, which caused a somatostatin depletion in the brain, was required to observe labeling in the hypothalamus. In some caudal areas of the brain, especially in the cerebellar nuclei, the solitary tract nucleus and the nucleus of the vagus nerve, only labeling with the S-28 agonist could be detected. This S-28 binding could be displaced by native S-14 (10 −6 M). Generally, there was a correlation between the localization of somatostatin receptors and that of immunoreactive somatostatin, as evaluated by immunocytochemistry. However, in some areas, an inverse correlation between receptor and peptide concentrations was observed. These results are in agreement with previous data suggesting that somatostatin could act as a neurotransmitter or neuromodulator in several brain areas.

Localization and characterization of somatostatin-14 and somatostatin-28 receptors in the rat pituitary as studied by slide-mounted frozen sections

The localization and characterization of somatostatin-14 (SS-14) and somatostatin-28 (SS-28) receptors were studied in the rat pituitary using the iodinated agonists [Tyr 0, D-Trp 8]-SS-14 and [Leu 8, D-Trp 22, Tyr 25]-SS-28 as tracers. Slide-mounted frozen sections were used for the autoradiographic localization and biochemical characterization of somatostatin receptors. In the latter case, counting was performed on scraped off serial sections which contained both anterior and posterior pituitary. Specificity studies demonstrated that either tracer could be displaced with SS-28 and SS-14. A number of unrelated peptides were unable to compete with either tracer. Scatchard analysis of saturation curves gave one-site interactions with K D values of 0.53 ± 0.08 nM and 0.22 ± 0.03 nM for the SS-14 and SS-28 iodinated agonists, respectively. Using autoradiography, SS-14 and SS-28 receptors were shown to be distributed in the anterior pituitary only. Using densitometry, displacement and saturation curves could be also obtained.

Receptors for angiotensin II on platelets from man.

Platelets were prepared from peripheral venous blood on iso-osmotic density gradients of Percoll, resulting in a good recovery of cells (50-80%) which were relatively free of contaminating blood cells (erythrocyte less than 0.1%, leucocyte less than 1%). At 22 degrees C, specific binding of 125I-labelled angiotensin II (300 pmol/l) was time and temperature dependent, saturable, reversible and linear with cell concentration. Scatchard analysis of saturation curves revealed a single class of binding sites with Kd 1.5 +/- 0.4 X 10(-10) mol/l and total binding capacity 6.3 +/- 1.2 receptors/platelet. Similar values (Kd 2.4 +/- 0.7 X 10(-10) mol/l and binding capacity 6.5 +/- 1.0 receptors/cell) were obtained by displacement analysis. From kinetic studies the forward and reverse rate constants were 3.1 X 10(8) mol min-1 l-1 and 3.6 X 10(-2)/min giving a Kd of 1.2 X 10(-10) mol/l. The relative binding potencies for angiotensin II and analogues were: [Sar1, Thr8]ANG II greater than ANG II greater than ANG III greater than [Sar1, Ala8]ANG II greater than ANG I. Incubation with an extracellular marker (51Cr-labelled EDTA) demonstrated that binding of angiotensin II to platelets was not due to free fluid endocytosis.

Regulation of glucocorticoid receptors by glucocorticoids in cultured HeLa S3 cells.

HeLa S3 cells contain high affinity, saturable receptors specific for glucocorticoids (congruent to 20,000 per cell). Growth of HeLa S3 cells in media containing dexamethasone (10(-6) M) results in a pronounced (congruent to 70%) reduction in the total cellular level of nuclear or cytoplasmic dexamethasone receptor number without any alteration in steroid-receptor dissociation constant (Kd congruent to 1 X 10(-9) M). This reduction in receptor number is not the result of simple receptor occupation, since this effect also occurs when receptor number is modulated by the direct addition of [3H]dexamethasone. Both control and dexamethasone-treated cells attain equilibrium states of receptor binding by 120 min at 0 C and by 60 min at 37 C, enabling estimation of receptor number and affinity by Scatchard analysis of saturation curves. Down-regulation of glucocorticoid receptor occurs at steroid concentrations as low as 10(-9) M (40% reduction). This alteration in receptor occurs after 24 h of hormone but not after either 2 or 6 h. Only the active glucocorticoids, (dexamethasone, cortisol) down-regulate glucocorticoid receptors. Cortexolone, estradiol, and 5 alpha-dihydrotestosterone have no apparent effect on this process, whereas progesterone (10(-7) M) is partially effective. Subcellular distribution studies indicate that glucocorticoids affect only that population of receptors capable of nuclear translocation at 37 C. We conclude that glucocorticoids can regulate the metabolism of their own receptors, via undefined mechanisms.