Rapid Effects Of Glucocorticoids Research Articles

Preextinction Stress Prevents Context-Related Renewal of Fear

Extinction learning, which creates new safety associations, is thought to be the mechanism underlying exposure therapy, commonly used for the treatment of anxiety disorders and posttraumatic stress disorder. The relative strength and availability for retrieval of both the fear and safety memories determine the response in a given situation. While the fear memory is often context-independent and may easily generalize, extinction memory is highly context-specific. “Renewal” of the extinguished fear memory might thus occur following a shift in context. The aim of the current work was to create an enhanced and generalized extinction memory to a discrete stimulus using stress exposure before extinction learning, thereby preventing renewal. In our contextual fear conditioning paradigm, 40 healthy men acquired (Day 1), retrieved and extinguished (Day 2) the fear memories, with no differences between the stress and the control group. A significant difference between the groups emerged in the renewal test (Day 3). A renewal effect was seen in the control group (N = 20), confirming the context-dependency of the extinction memory. In contrast, the stress group (N = 20) showed no renewal effect. Fear reduction was generalized to the acquisition context as well, suggesting that stress rendered the extinction memory more context-independent. These results are in line with previous studies that showed contextualization disruption as a result of pre-learning stress, mediated by the rapid effects of glucocorticoids on the hippocampus. Our findings support research investigating the use of glucocorticoids or stress induction in exposure therapy and suggest the right timing of administration in order to optimize their effects.

Rapid Nongenomic Glucocorticoid Actions in Male Mouse Hypothalamic Neuroendocrine Cells Are Dependent on the Nuclear Glucocorticoid Receptor.

Corticosteroids act classically via cognate nuclear receptors to regulate gene transcription; however, increasing evidence supports rapid, nontranscriptional corticosteroid actions via activation of membrane receptors. Using whole-cell patch clamp recordings in hypothalamic slices from male mouse genetic models, we tested for nongenomic glucocorticoid actions at glutamate and gamma aminobutyric acid (GABA) synapses in hypothalamic neuroendocrine cells, and for their dependence on the nuclear glucocorticoid receptor (GR). In enhanced green fluorescent protein-expressing CRH neurons of the paraventricular nucleus (PVN) and in magnocellular neurons of the PVN and supraoptic nucleus (SON), dexamethasone activated postsynaptic membrane-associated receptors and G protein signaling to elicit a rapid suppression of excitatory postsynaptic inputs, which was blocked by genetic deletion of type I cannabinoid receptors and a type I cannabinoid receptor antagonist. In magnocellular neurons, dexamethasone also elicited a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs. These data indicate a rapid, synapse-specific glucocorticoid-induced retrograde endocannabinoid signaling at glutamate synapses and nitric oxide signaling at GABA synapses. Unexpectedly, the rapid glucocorticoid effects on both excitatory and inhibitory synaptic transmission were lost with conditional deletion of GR in the PVN and SON in slices from a single minded-1-cre-directed conditional GR knockout mouse. Thus, the nongenomic glucocorticoid actions at glutamate and GABA synapses on PVN and SON neuroendocrine cells are dependent on the nuclear GR. The nuclear GR, therefore, is responsible for transducing the rapid steroid response at the membrane, or is either a critical component in the signaling cascade or regulates a critical component of the signaling cascade of a distinct membrane GR.

Non-receptor-tyrosine Kinases Integrate Fast Glucocorticoid Signaling in Hippocampal Neurons

Despite numerous descriptions of rapid effects of corticosterone on neuronal function, the intracellular mechanisms responsible for these changes remain elusive. The present comprehensive analysis reveals that signaling from a membrane-located G protein-coupled receptor activates PKC, Akt/PKB, and PKA, which subsequently trigger the phosphorylation of the tyrosine kinases Pyk2, Src, and Abl. These changes induce rapid cytoskeletal rearrangements (increased PSD-95 co-clustering) within the post-synaptic density; these events are accompanied by increased surface NMDA receptor expression, reflecting corticosterone-induced inhibition of NMDA receptor endocytosis. Notably, none of these signaling mechanisms require de novo protein synthesis. The observed up-regulation of ERK1/2 (downstream of NMDA receptor signaling) together with the fact that c-Abl integrates cytoplasmic and nuclear functions introduces a potential mechanism through which rapid signaling initiated at the plasma membrane may eventually determine the long term integrated response to corticosterone by impacting on the transcriptional machinery that is regulated by classical, nuclear mineralocorticoid, and glucocorticoid receptors.

Critical role of the endocannabinoid system in mediating rapid glucocorticoid effects on memory for emotionally arousing experiences

Critical role of the endocannabinoid system in mediating rapid glucocorticoid effects on memory for emotionally arousing experiences

Glucocorticoids interact with the hippocampal endocannabinoid system in impairing retrieval of contextual fear memory

There is extensive evidence that glucocorticoid hormones impair the retrieval of memory of emotionally arousing experiences. Although it is known that glucocorticoid effects on memory retrieval impairment depend on rapid interactions with arousal-induced noradrenergic activity, the exact mechanism underlying this presumably nongenomically mediated glucocorticoid action remains to be elucidated. Here, we show that the hippocampal endocannabinoid system, a rapidly activated retrograde messenger system, is involved in mediating glucocorticoid effects on retrieval of contextual fear memory. Systemic administration of corticosterone (0.3-3 mg/kg) to male Sprague-Dawley rats 1 h before retention testing impaired the retrieval of contextual fear memory without impairing the retrieval of auditory fear memory or directly affecting the expression of freezing behavior. Importantly, a blockade of hippocampal CB1 receptors with AM251 prevented the impairing effect of corticosterone on retrieval of contextual fear memory, whereas the same impairing dose of corticosterone increased hippocampal levels of the endocannabinoid 2-arachidonoylglycerol. We also found that antagonism of hippocampal β-adrenoceptor activity with local infusions of propranolol blocked the memory retrieval impairment induced by the CB receptor agonist WIN55,212-2. Thus, these findings strongly suggest that the endocannabinoid system plays an intermediary role in regulating rapid glucocorticoid effects on noradrenergic activity in impairing memory retrieval of emotionally arousing experiences.

Endocannabinoids: The silent partner of glucocorticoids in the synapse

Although it has long been recognized that steroid hormones primarily exert their effects on neuronal function through their ability to modulate gene transcription in the nucleus (1), an array of physiological and behavioral effects of glucocorticoids have been documented to occur in a fashion that cannot be explained by genomic regulation (2). These findings have prompted the hypothesis that glucocorticoids (in addition to all other major classes of steroids) possess membrane-associated receptors through which nongenomic signaling may evoke rapid effects on physiology and behavior (2, 3). Research from Miles Orchinik and Frank Moore (4) in the early 1990s clearly demonstrated the presence of glucocorticoid receptors in the neuronal membrane of an amphibian species that evoked observable effects on neuronal signaling and behavior, but progress in this field has been hampered by a lack of comparable discoveries in mammalian species. However, recent data in a study in this issue of PNAS (5) sheds new light on the rapid effects of glucocorticoids in mammals by revealing a putative nongenomic role of glucocorticoids in regulating emotional learning in rodents through a coordinated induction of endocannabinoid signaling.

Rapid glucocorticoid effects on pancreatic beta cells

Glucocorticoids act directly on pancreatic islets suppressing insulin secretion from the beta cells through a genomic mechanism of slow onset. We studied the immediate actions of dexamethasone on two models of insulin secretion: RINm5F and INS-1 beta cell lines. Under normal glucose concentrations, dexamethasone rapidly (within minutes) decreases insulin secretion about 30%. Under hypoglycaemic conditions (glucose concentrations reduced to 50% for 1 hour) dexamethasone increases insulin release. Both these effects are present within 10 minutes and not in longer (up to 1 hour) stimulations. They are completely abolished by preincubation with pertussis toxin, slightly inhibited by the intracellular glucocorticoid receptor (iGR) antagonist mifepristone (RU486) and unaltered by the transcription inhibitor cycloheximide.

Rapid glucocorticoid effects on the expression of hippocampal neurotransmission-related genes

We previously assessed corticosterone mediated gene expression in acute explant hippocampal slices and found over 200 responsive genes 1, 3 and 5 h after glucocorticoid receptor (GR) activation by a brief corticosterone pulse. Interestingly, 1 h after GR activation all genes were downregulated, many of which are involved in hippocampal neurotransmission and plasticity. The aim of the current experiment was 1) to measure the expression of several of these neurotransmission-related genes that were corticosterone-responsive 1 h after GR-activation in an in vivo setting, 2) to elucidate in which hippocampal subregion these expression changes take place and 3) to assess the specificity of regulation by activated GRs. For this purpose, rats were subcutaneously injected with vehicle, corticosterone or corticosterone pretreated with GR-antagonist RU38486. One hour after the corticosterone injections, mRNA expression levels of 5 selected genes were measured using in situ hybridization. The mineralocorticoid receptor (MR), MAO-A, casein kinase 2 and voltage dependent potassium mRNA's, but not dynein mRNA, were rapidly downregulated in vivo after corticosterone administration in hippocampal subregions. Furthermore, RU38486 pretreatment reversed in all cases these effects, illustrating the GR-specificity of transcriptional regulation by corticosterone. The results are important for understanding the role of GR in pleiotropic control of hippocampal neurotransmission and plasticity, which is characterized by recovery of function transiently raised by excitatory input.

Rapid Glucocorticoid Signaling via Membrane-Associated Receptors

Glucocorticoids are secreted into the systemic circulation from the adrenal cortex and initiate a broad range of actions throughout the organism that regulate the function of multiple organ systems, including the liver, muscle, the immune system, the pancreas, fat tissue, and the brain. Delayed glucocorticoid effects are mediated by classical steroid mechanisms involving transcriptional regulation. Relatively rapid effects of glucocorticoids also occur that are incompatible with genomic regulation and invoke a noncanonical mode of steroid action. Studies conducted in several labs and on different species suggest that the rapid effects of glucocorticoids are mediated by the activation of one or more membrane-associated receptors. Here, we provide a brief review focused on multiple lines of evidence suggesting that rapid glucocorticoid actions are triggered by, or at least dependent on, membrane-associated G protein-coupled receptors and activation of downstream signaling cascades. We also discuss the possibility that membrane-initiated actions of glucocorticoids may provide an additional mechanism for the regulation of gene transcription.

Rapid anti-secretory effects of glucocorticoids in human airway epithelium

Glucocorticoids are anti-inflammatory molecules classically described as acting through a genomic pathway. Similar to many steroid hormones, glucocorticoids also induce rapid non-genomic responses. The present paper provides a general overview of the rapid non-genomic effects of glucocorticoids in airway and will be mainly focused on a retrospective of the authors work on rapid effects of glucocorticoids in airway epithelial cell transport. Using fluorescence microscopy, short circuit current measurements in human bronchial epithelial (16HBE14o −) cells, we reported rapid non-genomic effects of dexamethasone on cell signalling and ion transport. Dexamethasone (1 nM) rapidly stimulated Na +/H + exchanger activity and pH i regulation in 16HBE14o − cells. Dexamethasone also produced a rapid decrease of intracellular [Ca 2+] i to a new steady state concentration and inhibited the large and transient [Ca 2+] i increase induced by apical adenosine tri-phosphate (ATP). Dexamethasone also reduced by 1/3 the Ca 2+-dependent Cl − secretion induced by apical ATP. The rapid effects of dexamethasone on intracellular pH and Ca 2+ were not affected by inhibitors of transcription, cycloheximide or by the classical glucocorticoid and mineralocorticoid receptors antagonists, RU486 and spironolactone, respectively. The rapid responses to glucocorticoid were reduced by the inhibitors of adenylated cyclase, cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (ERK1/2). Our results demonstrate, that dexamethasone at low concentrations, rapidly regulates intracellular pH, Ca 2+ and PKA activity and inhibits Cl − secretion in human bronchial epithelial cells via a non-genomic mechanism which neither involve the classical glucocorticoid nor mineralocorticoid receptor.

Rapid Central Corticosteroid Effects: Evidence for Membrane Glucocorticoid Receptors in the Brain

Glucocorticoid secretion occurs in a circadian pattern and in response to stress. Among the broad array of glucocorticoid actions are multiple effects in the brain, including negative feedback regulation of hypothalamic hormone secretion. The negative feedback of glucocorticoids occurs on both rapid and delayed time scales, reflecting different regulatory mechanisms. While the slow glucocorticoid effects are widely held to involve regulation of gene transcription, the rapid effects are too fast to invoke genomic mechanisms. We provide a brief overview of multiple lines of evidence for membrane-associated glucocorticoid receptors in the brain, with an emphasis on our recent findings of a rapid, G protein-dependent glucocorticoid action in the rat hypothalamus. We have observed a novel mechanism of rapid glucocorticoid inhibition of parvocellular neuroendocrine cells of the hypothalamic paraventricular nucleus (PVN) mediated by the retrograde release of endocannabinoids and suppression of synaptic glutamate release. This acute glucocorticoid action may underlie the rapid inhibitory effect of glucocorticoids on hypothalamic neuroendocrine function, and provides a potential model for the rapid glucocorticoid effects that occur in several areas of the brain.

Behavioral specificity of non-genomic glucocorticoid effects in rats: Effects on risk assessment in the elevated plus-maze and the open-field

The rapid effects of glucocorticoids on various behaviors suggest that these hormones play a role in rapidly coping with challenging situations. The variety of behaviors affected in different situations raise, however, questions regarding the specificity and roles of glucocorticoids in controlling behavior. To clarify this issue, we assessed the rapid behavioral effects of glucocorticoids in the elevated plus-maze (EPM) and the open-field (OF) tests in male rats. Both tests measure three different kinds of behavioral responses: locomotion, anxiety-like behaviors (central area and open arm exploration in the OF and EPM tests, respectively), and risk assessment (investigating aversive areas in a stretched attend posture). The acute inhibition of glucocorticoid synthesis by metyrapone decreased risk assessment but did not affect locomotion and anxiety-like behaviors. Corticosterone administration increased risk assessment, without affecting locomotion and anxiety-like behaviors. Moreover, plasma corticosterone levels measured immediately after testing strongly correlated with the intensity of risk assessment. The effects of corticosterone were rapid, as occurred even when the hormone was injected 2 min before behavioral testing. In addition, the effect was resistant to protein synthesis inhibition. These data demonstrate that glucocorticoids are able to increase specifically risk assessment behaviors by non-genomic mechanisms in two different, novelty-related, non-social challenging situations. Thus, glucocorticoids appear to rapidly induce specific behavioral adjustments to meet immediate requirements set by the challenge. These data support earlier assumptions on the role of glucocorticoids in coping, and it can be hypothesized that the rapid activation of the HPA-axis may play a role in forming coping responses.

Specific high-affinity glucocorticoid binding sites on the membrane of the pituitary cell line AtT-20 demonstrated by Fluorescence Correlation Spectroscopy

The fast feedback regulation of ACTH release from the pituitary by glucocorticoids is a physiological rapid steroid effect that has been well documented in vivo and in vitro. The mouse pituitary cell line AtT-20 is a model system for ACTH release and its fast (within 10 minutes) suppression by glucocorticoids. The existence of membrane Glucocorticoid Receptors on this cell line has been suggested by studies on isolated cell membranes. The aim of this study was to identify and characterize such binding sites on the membrane of single living cells using the single molecule technique Fluorescence Correlation Spectroscopy (FCS), a method that allows to monitor the diffusion behaviour of fluorescently labeled molecules with single-molecule sensitivity in a confocal volume measuring 0,35µm in the xy (lateral) and 2,4µm in the z (axial) dimension defined by a microscope objective epi-illuminated by a laser beam. Binding of a ligand to the cell membrane of single living cells can be studied in solution without the need of membrane isolation or other manipulations. We found that fluorescein-labeled dexamethasone binds indeed to the membrane of AtT-20 cells. The binding is specific, since it is washed out by unlabeled dexamethasone or the natural ligand corticosterone, but not estradiol or progesterone in 1000-fold excess. Incubation with different concentrations of dexamethasone resulted in a binding curve with a binding constant of 6 nM. Membrane binding is observed only in a subpopulation of the cells. The fraction of binding-positive cells is significantly reduced upon feeding of the cells. In summary we have shown the existence of specific glucocorticoid binding sites with high affinity on the membrane of living AtT-20 cells. Whether or not these binding sites are related to the rapid glucocorticoid effects on the pituitary remains to be elucidated, but the fact that they are downregulated upon feeding clearly points in the direction of their functionality.

Rapid glucocorticoid effects on immune cells.

Apart from their classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomically mediated effects. Three different mechanisms are currently under discussion as being responsible for these effects: (1) specific interaction with the cytosolic glucocorticoid receptor (cGCR), (2) nonspecific interactions with cellular membranes and (3) specific interactions with membrane-bound glucocorticoid receptors (mGCR). With regard to the first mechanism, there is evidence that although the binding of glucocorticoids to the cGCR-associated multi-protein complex induces the further processes of the classic path, it also leads to a rapid intracellular signalling through other components of the complex (e.g. Src). For the second mechanism, a nonspecific interactive effect with cellular membranes through the intercalation of glucocorticoid molecules is being discussed, which primarily alters cellular functions by influencing cation transport via the plasma membrane and by increasing the proton leak of the mitochondria. With regard to the third, mGCR-mediated mechanism, the first evidence has now been found to suggest a physiological expression of membrane-bound glucocorticoid receptors on human cells, whereas in humans this had previously only been demonstrated on lymphoma cells. The clinical importance and therapeutic relevance of these rapid glucocorticoid effects remains unclear at present, although effects on intracellular signalling, interferences with bioenergetically relevant cell functions and the induction of apoptosis via the mGCR are being discussed. This article gives a detailed presentation of the data available at present concerning rapid glucocorticoid effects on immune cells.

Molecular mechanisms of glucocorticoid-induced osteoporosis

Bone loss resulting from long-term glucocorticoid therapy is common and clinically relevant. A number of different glucocorticoid-mediated effects are responsible for the reduction in bone density: (i) glucocorticoid-induced direct impairment of osteoblast, osteocyte, and osteoclast function leads to reduced bone remodeling and diminished repair of microdamage in bone; (ii) the effects of parathyroid hormone (PTH) might be more pronounced in the presence of glucocorticoids, whereas vitamin D plays a lesser role in the pathogenesis of steroid-induced osteoporosis; (iii) glucocorticoids antagonize gonadal function and inhibit the osteoanabolic action of sex steroids; and (iv) increased renal elimination and reduced intestinal absorption of calcium lead to a negative calcium balance that has been suggested to promote secondary hyperparathyroidism. From a mechanistic point of view, all of the aforementioned effects have long been considered to be mediated at the molecular level exclusively by genomic actions. However, there is now increasing evidence for the existence of rapid glucocorticoid effects that are incompatible with this classical mode of action. These rapid effects, termed nongenomic effects, are mediated by glucocorticoid interactions with biological membranes, either through binding to membrane receptors or by physicochemical interactions. It is possible, but has yet to be shown, that these effects play a role in the pathogenesis of glucocorticoid-induced osteoporosis.

Rapid glucocorticoid effects on excitatory amino acid levels in the hippocampus: a microdialysis study in freely moving rats.

Glucocorticoids can rapidly affect neuronal function and behaviour in mammals. Several studies have suggested the possible existence of rapid, non-genomic effects of glucocorticoids in the hippocampus. To investigate whether glucocorticoids could affect neurotransmission in the hippocampus through rapid, non-genomic mechanisms, we studied the effects of acute glucocorticoid administration on extracellular amino acid levels in the CA1 area of the hippocampus. By means of microdialysis on freely moving rats, we observed that an intraperitoneal injection of corticosterone (2.5 mg/kg) induced a rapid (within 15 min) and transient (returning to basal levels by 35-45 min) increase in extracellular aspartate and glutamate levels ( approximately 155-160%), both in sham-operated and adrenalectomized rats. These effects occurred in parallel with a rise in corticosterone concentration, also detected by microdialysis, in this hippocampal area. Intrahippocampal perfusion of corticosterone by retrodialysis also produced the same fast and reversible effects on excitatory amino acid (EAA) levels. Extracellular concentrations of taurine and gamma-aminobutyric acid (GABA) were unchanged after intrahippocampal glucocorticoid administration. This corticosterone-mediated rise in EAA levels was not inhibited by the presence of specific antagonists for the two types of intracellular corticosteroid receptors, nor by a protein synthesis inhibitor, anisomycin. Perfusion of dexamethasone, a synthetic glucocorticoid, elicited a similar effect to that observed with corticosterone treatment in all studied cases. However, non-glucocorticoid steroids did not affect amino acid transmission in this hippocampal area. These results indicate that glucocorticoids induce a rapid and transient increase in hippocampal EAA levels in vivo that might be exerted through a novel non-genomic mechanism of action.

Rapid Enhancement of High Affinity Glutamate Uptake by Glucocorticoids in Rat Cerebral Cortex Synaptosomes and Human Neuroblastoma Clone SK-N-SH: Possible Involvement of G-protein

The rapid effects of glucocorticoids(GCs) on the Na+dependent, high affinity uptake of3[H]-L-glutamate(Glu) in rat cerebral cortex synaptosomes(4 min incubation) and human neuroblastoma clone SK-N-SH (10min preincubation and 5 min incubation) were investigated. GCs, including corticosterone, corticosterone-sulfate, hydrocortisone-hemisuccinate and dexamethasone 21-phosphate(DEX) were found stimulating Glu uptake. The uptakes in synaptosomes and SK-N-SH cells were increased to 117-126% and 121-137% respectively of the control by 10−6mol/L GCs. The stimulation of GCs was dose-dependent. The maximal effect of DEX in SK-N-SH cells appeared at10−7mol/L, and the least effective dose of DEX was at 10−9mol/L. Guanosine 5-O-(2-thiodiphosphate), an inhibitor of G-protein activation, could block the stimulation of GCs. The results indicated that GCs rapidly enhance the Na+-dependent high affinity Glu uptake in nerve endings and SK-N-SH cells, even at the concentration of physiological conditions, and the G-protein on synaptic membranes or SK-N-SH cell membranes might be involved in the effect of GCs.

Normal glucocorticoid fast feedback following chronic 50% corticosterone pellet treatment

Rapid effects of glucocorticoids on adrenocorticotropin hormone (ACTH) secretion were first demonstrated by Dallman and Jones. Glucocorticoid fast feedback is believed to be dependent upon brain feedback sites, particularly the hippocampus. The current studies are part of a series of studies examining the links between hippocampal adrenal steroid receptor changes and changes in fast feedback. Our previous studies have demonstrated that chronic stress and chronic corticosterone injections are capable of diminishing the fast feedback effect of injected glucocorticoids on ACTH secretion. The current studies explore whether administration of a different form of exogenous corticosterone, a 50% corticosterone pellet, a treatment that leads to constant corticosterone levels of 7-8 micrograms/dl, was capable of affecting fast feedback. Rats were studied both before and after the implantation of the 50% corticosterone pellets. Administration of cortisol at the initiation of restraint stress resulted in decreased ACTH release over the course of 30 min in both the morning and afternoon before implantation of corticosterone pellets. Following the implantation of 50% corticosterone pellets, the overall ACTH stress response was diminished in both the morning and the afternoon. However, cortisol injection at the initiation of restraint stress continued to exert fast feedback effects on ACTH secretion. These studies suggest that constant low plasma corticosterone concentrations of 6-7 micrograms/dl are able to inhibit the overall stress response but not the ability to respond to fast feedback. This contrasts with our previous studies with a corticosterone injection paradigm which produced abnormalities in glucocorticoid fast feedback.

Rapid Corticosterone Inhibition of Corticotropin-Releasing Hormone Binding and Adrenocorticotropin Release by Enriched Populations of Corticotropes: Counteractions by Arginine Vasopressin and Its Second Messengers*

We have demonstrated that anterior pituitary corticotropes can be identified cytochemically by their capacity to bind potent biotinylated analogs of CRH. In addition, 50-80% of corticotropes bind biotinylated arginine vasopressin (AVP). The percentage of CRH-bound cells is rapidly reduced after 1-h exposure to glucocorticoids. However, the rapid effects of glucocorticoids on AVP binding by corticotropes have not been tested. The first aim of this study was to examine the binding capacity of small and large corticotropes enriched to 90% by counterflow centrifugation. Biotinylated analogs of CRH or AVP were detected cytochemically on the cells by avidin-biotin-peroxidase complex protocols. At least 80% of the cells bound CRH after 1 day of culture. More large corticotropes bound AVP (93%) than small corticotropes (80%). AVP pretreatment of large corticotropes stimulated an increase in CRH-bound cells to over 90%, but it had no effect on CRH binding by small corticotropes. Corticosterone pretreatment (100 nM) for 10 min caused a 50% reduction in the percentage of cells that bound CRH and in the levels of ACTH released in response to biotinylated CRH. After 30 and 60 min of pretreatment, the percentages of CRH-bound cells were reduced by 75%, and ACTH levels remained low. No reduction in percentages of AVP-bound cells was evident at any time point after corticosterone pretreatment. These studies stimulated further tests based on previous reports that showed that AVP or its activated second messengers enhanced CRH binding. We reasoned that this potentiation might promote a recovery in CRH binding to corticosterone-inhibited cells. However, 1-h stimulation by AVP or activation of calcium channels (by Bay K 8644) or protein kinase-C by 12-O-tetradecanoyl-phorbol-13-acetate did not restore CRH binding. AVP evoked a partial recovery in ACTH release. Furthermore, Bay K and 12-O-tetradecanoyl-phorbol-13-acetate pretreatment effectively blocked the fast feedback effects of corticosterone on CRH-mediated ACTH release. Thus, these studies demonstrate that glucocorticoids rapidly inhibit CRH-receptor binding in a domain that is not affected by AVP potentiation of ACTH release. Perhaps they immobilize transport processes needed to bring unoccupied CRH receptors to the surface for binding and cytochemical detection.

120. Rapid effects of glucocorticoids on chromatin structure

120. Rapid effects of glucocorticoids on chromatin structure