Suppression Of Corticosterone Research Articles

Nongenomic mechanisms of glucocorticoid inhibition of nicotine-induced calcium influx in PC12 cells: involvement of protein kinase C.

Nongenomic mechanisms of corticosterone (B) inhibition of nicotine (Nic)-induced calcium influx were investigated in PC12 cells. Corticosterone could rapidly inhibit the Ca2+ influx induced by Nic, and BSA-conjugated B had a similar inhibitory effect. The inhibition of Nic-induced Ca2+ influx by B could be mimicked by protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and reversed by PKC inhibitors, chelerythrine chloride and Gö6976. When PC12 cells were pretreated with pertussis toxin, the inhibitory effect of B on Nic-induced Ca2+ influx was blocked. Both B and BSA-conjugated B could activate PKC activity, with the maximal responses at 10(-9) and 10(-7) M at 37 C, respectively. The dose-response curve was bell shaped. At 25 C, however, the dose-response curve considerably shifted to the right, and B was most potent at 10(-5) M. The time course showed that PKC activity was highest at 5 min of B's action. The results suggest that B might act via putative membrane receptors and inhibit the Ca2+ influx induced by Nic through the pertussis toxin-sensitive G protein-PKC pathway and that PKC plays an important role in the mechanisms of glucocorticoid nongenomic action.

Systemic alpha-MSH suppresses LPS fever via central melanocortin receptors independently of its suppression of corticosterone and IL-6 release.

Systemically administered alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits endotoxin (lipopolysaccharide; LPS)- or interleukin (IL)-1-induced fever and adrenocortical activation, but the sites of these actions and the mechanisms involved are unknown. The aims of this study were, first, to determine whether melanocortin receptors (MCR) located within the central nervous system mediate the suppressive effects of peripherally administered alpha-MSH on LPS-induced fever and activation of the pituitary-adrenal axis and, second, to determine whether systemic alpha-MSH suppresses the LPS-induced rise in plasma IL-6 levels, potentially contributing to its antipyretic effect. Male rats received Escherichia coli LPS (25 microg/kg ip). Core body temperatures (Tb) were determined hourly by radiotelemetry (0-8 h), and blood was withdrawn via venous catheters for plasma hormone immunoassays (0-2 h) and IL-6 bioassay (0-8 h). alpha-MSH (100 microg/kg ip) completely prevented the onset of LPS-induced fever during the first 3-4 h after LPS and suppressed fever throughout the next 4 h but did not affect Tb in afebrile rats treated with intraperitoneal saline rather than LPS. Intraperitoneal alpha-MSH also suppressed the LPS-induced rise in plasma IL-6, ACTH, and corticosterone (CS) levels. Intracerebroventricular injection of SHU-9119, a potent melanocortin-4 receptor (MC4-R)/MC3-R antagonist, completely blocked the antipyretic effect of intraperitoneal alpha-MSH during the first 4 h after LPS but had no effect on alpha-MSH-induced suppression of LPS-stimulated plasma IL-6 and CS levels. Taken together, the results indicate that the antipyretic effect of peripherally administered alpha-MSH during the early phase of fever is mediated by MCR within the brain. In contrast, the inhibition of LPS-induced increases in plasma CS and IL-6 levels by intraperitoneal alpha-MSH appears to be mediated by a different mechanism(s), and these effects do not contribute to its antipyretic action.

The Ontogeny of Glucocorticoid Negative Feedback: Influence of Maternal Deprivation1

Glucocorticoid feedback can be viewed as having two modes of operation: proactive and reactive. "Proactive" feedback maintains basal activity of the hypothalamic-pituitary-adrenal axis, whereas the termination of stress-induced hypothalamic-pituitary-adrenal activity is facilitated by "reactive" feedback. In the present study we studied the ontogeny of both feedback modes and tested the hypothesis that the development of feedback depends on mother-pup interaction. On postnatal day 9 or 12, pups were deprived (DEP) of the dam for 24 h; nondeprived pups of the same age served as controls. The pups were adrenalectomized (ADX) at the end of deprivation and given corticosterone (CORT) replacement by either injection or pellet implants using the following two designs: first at the time of adrenalectomy (ADX) to test the role of CORT in the maintenance of basal ACTH levels, and then 3 h after ADX, to investigate CORT suppression of elevated ACTH levels induced by prior ADX. Regarding proactive feedback, the results showed that injection of CORT at the time of ADX was only partially effective in preventing ACTH elevations, whereas CORT pellets maintained basal levels of ACTH in all ADX pups. The reactive mode of negative feedback in nondeprived pups was resistant to CORT injection, whereas the CORT pellet resulted in a return to basal levels within 60 min. Maternal deprivation did not affect proactive feedback, but caused a more sustained increase in ACTH levels and a failure to return to basal levels 3 h after ADX despite significantly higher levels of circulating CORT in these DEP pups. It is concluded that 1) proactive and reactive modes of negative feedback are operative, provided the pups are maintained on chronic replacement with CORT; 2) DEP impairs the reactive, rather than the proactive, mode of feedback inhibition in the neonate.

Prenatal alcohol exposure results in hyperactivity of the hypothalamic-pituitary-adrenal axis of the offspring: modulation by fostering at birth and postnatal handling.

Exposure of fetal rats to alcohol results in permanent hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In contrast, postnatal handling or fostering have been reported to restrain HPA activity. Because of the deleterious consequences of a hyperresponsive HPA axis, we thought that the possibility that postnatal manipulations might be able to reverse the influence of prenatal alcohol treatment deserved investigation. To test this hypothesis, we exposed rat dams to alcohol by inhalation during the second week of gestation. At birth, pups were either fostered or remained with their dam. For the first 3 weeks, litters were handled daily for 15 min or left undisturbed. At 22 days of age, male and female pups were decapitated under basal conditions, after 10 min of mild electro-footshock, or 10 min after footshock had been terminated. As expected, prenatal exposure to alcohol induced increased adrenocorticotropin (ACTH) secretion in response to footshock, and postnatal handling of control pups resulted in a suppression of corticosterone and ACTH release, although changes in this latter hormone did not reach statistical significance. Surprisingly, however, pups exposed to alcohol that were also fostered and handled after birth, showed an ACTH response to footshock stress that was significantly larger than all other groups. This unexpected response may be due to alterations in maternal-pup behaviors and may indicate that these manipulations act on different neuronal substrates within the central HPA of young rats. Further studies are needed to determine whether adrenal regulation is also altered in animals exposed to alcohol prenatally and reared in a similar manner.

Corticosterone inhibition of osmotically stimulated vasopressin from hypothalamic-neurohypophysial explants.

Glucocorticoids inhibit and glucocorticoid deficiency increases vasopressin (AVP) release in vivo. To determine whether the effect of glucocorticoids is hypothalamic and mediated via a glucocorticoid receptor, explants of the hypothalamic-neurohypophysial system were used to measure AVP release during agonist and antagonist exposure. Explants from adult rats, which contained AVP neurons of the supraoptic nucleus with axonal projections terminating in the neural lobe but excluded the paraventricular nucleus, were perifused with an osmotic stimulus (increase of 5 mosmol/h over 6 h) in the absence or presence of corticosterone (100 micrograms/dl) or with corticosterone (100 micrograms/dl) in the absence or presence of the glucocorticoid antagonist RU-486 (10 microM). AVP release was not increased during osmotic stimulation in the presence of corticosterone (Cort) and was 20-30% lower than osmotically stimulated release observed in the absence of Cort. RU-486 reversed the inhibitory effect of corticosterone on AVP release. No changes in AVP mRNA content were detected. These results suggest that Cort inhibits osmotically stimulated AVP release by a direct effect within the hypothalamus and/or neurohypophysis. This effect is mediated by the glucocorticoid receptor through either genomic or nongenomic mechanisms.

Localization, identification, and action of galanin in the frog adrenal gland

The distribution of galanin-like immunoreactivity was studied in the adrenal gland of the frog Rana ridibunda using the indirect immunofluorescence technique. A dense network of varicose fibers immunoreactive to galanin was found in the adrenal tissue. A combination of HPLC analysis and RIA detection was used to characterize galanin-like immunoreactivity in frog adrenal gland extracts. The elution profile revealed the existence of a single form of galanin exhibiting the same retention time as synthetic frog galanin. The possible involvement of galanin in the regulation of corticosteroid secretion was investigated in vitro using a perifusion system for frog adrenal slices. For concentrations ranging from 10(-9) to 3 x 10(-6) M, synthetic frog galanin induced a dose-dependent inhibition of corticosterone and aldosterone release. Repeated pulses of galanin (10(-6) M), given at 90-min intervals, resulted in a reproducible inhibition of corticosteroid secretion without any apparent tachyphylaxis. During prolonged administration of galanin (10(-6) M), the steroidogenic effect of ACTH (10(-9) M) was significantly reduced. In contrast, galanin did not attenuate the stimulation of corticosteroid secretion induced by the angiotensin II analog [Sar1,Val5]angiotensin II. These results show the occurrence of galanin in fibers innervating the frog adrenal gland. The data also demonstrate that synthetic galanin inhibits spontaneous and ACTH-induced corticosteroid release. Taken together, these findings suggest that galanin, released by nerve fibers in the adrenal tissue, can act locally as a modulator of steroid secretion.

Localization, identification, and action of galanin in the frog adrenal gland.

The distribution of galanin-like immunoreactivity was studied in the adrenal gland of the frog Rana ridibunda using the indirect immunofluorescence technique. A dense network of varicose fibers immunoreactive to galanin was found in the adrenal tissue. A combination of HPLC analysis and RIA detection was used to characterize galanin-like immunoreactivity in frog adrenal gland extracts. The elution profile revealed the existence of a single form of galanin exhibiting the same retention time as synthetic frog galanin. The possible involvement of galanin in the regulation of corticosteroid secretion was investigated in vitro using a perifusion system for frog adrenal slices. For concentrations ranging from 10(-9) to 3 x 10(-6) M, synthetic frog galanin induced a dose-dependent inhibition of corticosterone and aldosterone release. Repeated pulses of galanin (10(-6) M), given at 90-min intervals, resulted in a reproducible inhibition of corticosteroid secretion without any apparent tachyphylaxis. During prolonged administration of galanin (10(-6) M), the steroidogenic effect of ACTH (10(-9) M) was significantly reduced. In contrast, galanin did not attenuate the stimulation of corticosteroid secretion induced by the angiotensin II analog [Sar1,Val5]angiotensin II. These results show the occurrence of galanin in fibers innervating the frog adrenal gland. The data also demonstrate that synthetic galanin inhibits spontaneous and ACTH-induced corticosteroid release. Taken together, these findings suggest that galanin, released by nerve fibers in the adrenal tissue, can act locally as a modulator of steroid secretion.

Effects of corticosteroid synthesis inhibitors on the sensitization of reward by food restriction

Chronic food restriction sensitizes animals to the rewarding effects of food, drugs and lateral hypothalamic electrical stimulation. The present study employed a curve-shift analysis of lateral hypothalamic self-stimulation (LHSS) to evaluate whether the elevated plasma corticosterone levels that accompany food restriction mediate the sensitization of reward. In Experiment 1, two adrenocorticoid synthesis inhibitors, aminoglutethimide and metyrapone, were administered to food-restricted rats and the magnitude of plasma corticosterone suppression was determined at two post-administration time points. In Experiment 2, these compounds were administered to ad libitum fed and food-restricted rats whose LHSS behavior was evaluated at a time coincident with suppression of corticosterone. It was found that neither compound reversed the sensitizing effect of food-restriction on the rewarding efficacy of brain stimulation. However, aminoglutethimide (50 mg/kg) produced an increase in maximal response rates (a performance factor) across groups while metyrapone (100 mg/kg) produced a decrease. The most interesting result of this study was that 2 h after aminoglutethimide administration, when corticosterone levels had recovered from suppression, the rewarding efficacy of LHSS increased markedly in food-restricted rats. Possible explanations for this effect, including adrenocortical rebound, alterations in neurosteroid synthesis, and exacerbation of metabolic need are discussed.

Effects of corticosteroid synthesis inhibitors on the sensitization of reward by food restriction

Chronic food restriction sensitizes animals to the rewarding effects of food, drugs and lateral hypothalamic electrical stimulation. The present study employed a curve-shift analysis of lateral hypothalamic self-stimulation (LHSS) to evaluate whether the elevated plasma corticosterone levels that accompany food restriction mediate the sensitization of reward. In Experiment 1, two adrenocorticoid synthesis inhibitors, aminoglutethimide and metyrapone, were administered to food-restricted rats and the magnitude of plasma corticosterone suppression was determined at two post-administration time points. In Experiment 2, these compounds were administered to ad libitum fed and food-restricted rats whose LHSS behavior was evaluated at a time coincident with suppression of corticosterone. It was found that neither compound reversed the sensitizing effect of food-restriction on the rewarding efficacy of brain stimulation. However, aminoglutethimide (50 mg/kg) produced an increase in maximal response rates (a performance factor) across groups while metyrapone (100 mg/kg) produced a decrease. The most interesting result of this study was that 2 h after aminoglutethimide administration, when corticosterone levels had recovered from suppression, the rewarding efficacy of LHSS increased markedly in food-restricted rats. Possible explanations for this effect, including adrenocortical rebound, alterations in neurosteroid synthesis, and exacerbation of metabolic need are discussed.

Membrane-mediated inhibition of corticosterone on the release of arginine vasopressin from rat hypothalamic slices

The arginine vasopressin (AVP) released from the hypothalamic slices containing paraventricular and supraoptic nuclei of Sprague-Dawley rats sectioned with vibratome and incubated in static microchambers was measured by radioimmunoassay. The effect of bovine serum albumin-conjugated corticosterone (B-BSA) on the AVP release was investigated. The results were as follows: (1) B-BSA, within 20 min, significantly inhibited AVP release in a dose-dependent manner from 10 −7 to 10 −4 mol/l. (2) RU38486 (10 −4–10 −3 mol/I) could partially block the inhibitory effect of B-BSA although it by itself did not change the AVP release. (3) With the elevation of Ca 2+ concentration in the incubation medium, the AVP release was increased and the inhibitory effect of B-BSA enhanced; while in the absence of Ca 2+, the AVP release decreased and the effect of B-BSA attenuated. (4) The inhibitory effect of B-BSA was enhanced in the presence of neomycin which itself had no influence on AVP release. These results indicated that the inhibitory effect of corticosterone is rapid and membrane-mediated which is non-genomic rather than classical genomic, and that the extracellular Ca 2+ play a role in this rapid inhibitory effect.

Developmental expression and corticosterone inhibition of adenosine deaminase activity in different tissues of mice

The activity expression and corticosterone inhibition of adenosine deaminase (ADA) were studied in the spleen, stomach, and liver of mice at various postnatal ages. The specific activity of ADA is very low in the spleen and stomach of 5- and 10-day-old mice, and increases significantly (2.5- to 3.0-fold) in 20- and 30-day-old animals. Its level shows a further increase in the spleen of 60-day-old mice while stomach increase of ADA is not significant. In contrast, the activity of ADA is significantly higher in the liver of 5- and 10-day-old mice, decreases markedly (2.5-fold) in 20- and 30-day-old animals and shows a sharp increase in the liver of 60-day-old mice. Corticosterone administration brings a marked inhibition in the activity of ADA at all ages studied in the spleen and stomach whereas it inhibits the liver ADA only at 30 and 60 days postnatal age. These findings suggest an age- and tissue-specific expression of ADA activity and also indicate corticosterone as an inhibitory regulator of this enzyme.

Corticosterone circadian secretion differentially facilitates dopamine- mediated psychomotor effect of cocaine and morphine

Studies of intravenous self-administration and psychomotor effects of drugs have recently suggested that stress-induced corticosterone secretion may be an important factor determining vulnerability to drugs of abuse. In this report, we studied if basal physiological corticosterone secretion modulates sensitivity to cocaine and morphine, and if changes in the reactivity of mesolimbic dopaminergic (DA) neurons, one of the principal substrates of drug-reinforcing effects, are involved. For this purpose we determined the psychomotor effects of these drugs in animals in which corticosterone secretion was suppressed by adrenalectomy and in adrenalectomized animals submitted to different corticosterone replacement therapies designed to mimic (1) only the diurnal levels of the hormone, obtained by the subcutaneous implantation of 50 mg corticosterone pellets; (2) only the nocturnal levels, obtained by adding corticosterone (50 micrograms/ml) to the drinking solution during the dark period; and (3) the entire circadian fluctuation, obtained by combining the two previous treatments. Locomotor response to cocaine and morphine was studied after both systemic and central injections, into the nucleus accumbens for cocaine and into the ventral tegmental area for morphine. These sites were chosen because stimulant effects of cocaine and morphine injected in these structures are dopamine dependent. Our results show that suppression of corticosterone by adrenalectomy reduced the locomotor response to cocaine and morphine, injected both systemically and centrally. The reinstatement of diurnal levels of corticosterone totally reversed adrenalectomy's effects on the behavioral response to cocaine, whereas the reestablishment of the entire corticosterone circadian fluctuation (diurnal plus nocturnal levels) was necessary to reverse the response to morphine.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of parathyroid hormone-related peptide in relation to perturbations of gastric motility in the rat

We studied the expression of PTH-related peptide (PTHrP) and its mRNA in rat gastric smooth muscle in relation to various gastric motility states. Male rats were divided into groups subjected to fasting, feeding ad libitum, cold restraint stress, pyloric ligation, and carbachol stimulation. Cold restraint stress induced abnormal contractions. Rhythmic and moderate contractions were produced by carbachol administration, and marked distension was induced by pyloric ligation. PTHrP mRNA expression was weak in the physiological fasting and feeding states, but was markedly increased by pyloric ligation and carbachol stimulation. PTHrP and its mRNA were localized to the proper muscle layer and muscularis mucosa, but not in the mucosa by immunohistochemistry and in situ hybridization. The gene expression of PTHrP receptor in the gastric tissue was confirmed by reverse transcription-polymerase chain reaction, but serum PTHrP levels did not increase in all groups. These findings suggest that PTHrP acts as an autocrine or paracrine factor in gastric smooth muscle that responds to muscle activity caused by distension and cholinergic stimulation. However, PTHrP gene expression was decreased by stress despite the presence of strong contractions, and the sufficient relaxation did not occur. PTHrP suppression by stress is caused by the increase in corticosterone, as pretreatment of metyrapone, an inhibitor of 11 beta-hydroxylation, enhanced PTHrP gene expression in association with serum corticosterone suppression. In conclusion, PTHrP might be an important gastrointestinal peptide that regulates gastric contractile activity and is influenced by the serum corticosterone level.

Expression of parathyroid hormone-related peptide in relation to perturbations of gastric motility in the rat.

We studied the expression of PTH-related peptide (PTHrP) and its mRNA in rat gastric smooth muscle in relation to various gastric motility states. Male rats were divided into groups subjected to fasting, feeding ad libitum, cold restraint stress, pyloric ligation, and carbachol stimulation. Cold restraint stress induced abnormal contractions. Rhythmic and moderate contractions were produced by carbachol administration, and marked distension was induced by pyloric ligation. PTHrP mRNA expression was weak in the physiological fasting and feeding states, but was markedly increased by pyloric ligation and carbachol stimulation. PTHrP and its mRNA were localized to the proper muscle layer and muscularis mucosa, but not in the mucosa by immunohistochemistry and in situ hybridization. The gene expression of PTHrP receptor in the gastric tissue was confirmed by reverse transcription-polymerase chain reaction, but serum PTHrP levels did not increase in all groups. These findings suggest that PTHrP acts as an autocrine or paracrine factor in gastric smooth muscle that responds to muscle activity caused by distension and cholinergic stimulation. However, PTHrP gene expression was decreased by stress despite the presence of strong contractions, and the sufficient relaxation did not occur. PTHrP suppression by stress is caused by the increase in corticosterone, as pretreatment of metyrapone, an inhibitor of 11 beta-hydroxylation, enhanced PTHrP gene expression in association with serum corticosterone suppression. In conclusion, PTHrP might be an important gastrointestinal peptide that regulates gastric contractile activity and is influenced by the serum corticosterone level.

Characterization of dopamine receptors associated with steroid secretion in frog adrenocortical cells.

We investigated the type of receptors involved in the mechanism of action of dopamine on corticosteroid secretion from the frog interrenal (adrenal) gland, using the in-vitro perifusion technique. Exposure of dispersed interrenal cells to 50 microM dopamine for 20 min had a biphasic effect on corticosterone and aldosterone secretion, i.e. a transient stimulation followed by an inhibitory phase. Repeated administration of equimolar pulses of dopamine, given at 150-min intervals, resulted in an enhancement of corticosteroid secretion followed by a subsequent blockade of the stimulatory phase of the response. In contrast, the dopamine-evoked inhibition of corticosteroid release did not show any sensitization or desensitization phenomena. Infusion of repeated pulses of the D1 receptor agonist SKF38393 (32 microM) stimulated corticosteroid release and mimicked the sensitization-desensitization phenomenon induced by dopamine. Repeated administration of the D2 receptor agonist LY171555 (50 microM) resulted in a reproducible inhibition of corticosterone and aldosterone secretion. These results suggested the presence of two different receptors for dopamine, i.e. D1 and D2, on frog adrenocortical cells, responsible respectively for the stimulatory and inhibitory effects of dopamine on steroid secretion. However, bromocriptine (50 microM) and CV205-502 (50 microM), two other D2 receptor agonists, had no effect on corticosteroid release. In addition, several classical D2 receptor antagonists failed to block the effect of dopamine on steroidogenesis. It was also observed that (-)sulpiride, a specific D2 antagonist, did not alter dopamine-induced inhibition of inositol phosphate formation. On the other hand, dopamine and the selective D1 and D2 antagonists SKF38393 and LY171555 did not affect the formation of cyclic AMP by interrenal tissue. Taken together, these data indicate that dopamine directly regulates corticosteroid secretion from frog adrenocortical cells. The effect of dopamine is not coupled to adenylate cyclase activity but is probably mediated through the phosphoinositide-turnover pathway. The pharmacological characteristics of the receptors involved in the mechanism of action of dopamine clearly differ from those of the D1 and D2 subtypes previously described in mammals.

In vitro regulation of pituitary ACTH secretion in inflammatory disease susceptible Lewis (LEW/N) and inflammatory disease resistant Fischer (F344/N) rats.

We have previously shown that susceptibility to inflammatory disease in Lewis (LEW/N) rats is related to their limited hypothalamic-pituitary-adrenal (HPA) axis responses to a variety of inflammatory stimuli, while the relative resistance to inflammatory disease in Fischer (F344/N) rats is related to their potent HPA axis responses to these same stimuli. In vivo studies also showed that LEW/N pituitary ACTH responses to exogenous corticotropin-releasing hormone (CRH) were blunted compared to F344/N. To determine if there is a fundamental difference in pituitary corticotroph function between the two strains, independent of other factors influencing the HPA axis, we compared ACTH responses to a variety of stimuli in LEW/N and F344/N primary pituitary cell cultures. Here we show that in vitro basal ACTH secretion and peak ACTH response to CRH, forskolin and 8-bromo-cAMP are 50% lower in LEW/N than F344/N rats. However, these findings can be explained by other observations: diminished basal ACTH content, POMC mRNA, and a decreased number of corticotrophs, in pituitary cell cultures from LEW/N compared to F344/N rats. In addition, LEW/N corticotrophs were more sensitive to dexamethasone and to corticosterone suppression of CRH-stimulated ACTH secretion compared to F344/N. The data support the possibility of an HPA axis defect in LEW/N rats at the pituitary level which could be secondary to prolonged understimulation by hypothalamic CRH, or could also be partially related to enhanced glucocorticoid feedback inhibition.

Alterations in the binding characteristics of glucocorticoid receptors from obese zucker rats

Obese Zucker rats appear to lack a circadian rhythm of serum corticosterone and maintain relatively high concentrations throughout the 24-h day. The binding characteristics of glucocorticoid receptors in lean and obese Zucker rats were examined in three tissues suggested to be involved in the feedback inhibition of corticosterone: the anterior pituitary, hypothalamus and hippocampus. Hepatic glucocorticoid receptors were also examined to determine if receptor alterations exist in a peripheral tissue. The dissociation constant (Kd) of glucocorticoid receptors in the anterior pituitary of obese rats was 50% greater than the Kd of receptors derived from lean rats. This suggests a decrease in the affinity of these receptors and could indicate a reduced feedback inhibition of corticosterone at the anterior pituitary. Hepatic glucocorticoid receptors of obese rats also showed an increase (150%) in the Kd of binding and a reduction (40%) in the number of receptors. No difference was observed in the Kd or maximal binding of receptors from the hypothalamus or hippocampus of lean and obese rats. It appears that glucocorticoid receptor alterations exist in obese Zucker rats and that these alterations may affect the drive of the pituitary-adrenal axis and possibly the expression of obesity.

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.

Dopamine inhibits corticosteroid secretion in frog adrenocortical cells : Evidence for the involvement of prostaglandins in the mechanism of action of dopamine

We have investigated the possible involvement of arachidonic acid metabolites in dopamine-induced inhibition of adrenocortical steroidogenesis. Administration of dopamine (5 × 10 −5 M) for 20 min to perifused frog adrenal slices caused a marked reduction of the release of both prostaglandin E 2 (PGE 2) and 6-keto-PGF 1α, the stable metabolite of prostacyclin (PGI 2). Dopamine also induced a significant inhibition of corticosterone and aldosterone secretion. A lag period of 20 min was observed between inhibition of prostanoid and corticosteroid releases. Prolonged dopamine infusion did not prevent the stimulatory effect of PGE 1, PGE 2 or arachidonic acid on corticosteroid secretion. These observations indicate that activation of dopaminergic receptors in adrenocortical cells is linked to an inhibition of arachidonic acid metabolism. Our data also suggest that the inhibitory effect of dopamine occurs at a step preceding arachidonic acid formation.

Uptake and metabolism of 3H-(+/-)-noradrenaline in the isolated perfused rat liver.

The influence of inhibitors of metabolism and uptake of noradrenaline on the 3H-noradrenaline removal from the perfusion fluid by the isolated rat liver was studied. Livers were perfused with 3 nmol/l 3H-noradrenaline and 3H-noradrenaline and 3H-metabolites were determined in effluent, liver and bile. After the perfusion with 14,900 +/- 920 dpm.g-1.min-1 during 90 min, cumulative removal of tritium was 323,574 +/- 63,103 dpm/g. 3H-metabolites recovered from the liver after 90 min perfusion represented 71.1 +/- 9.0% of total metabolite formation. Only the OMDA-fraction appeared in the perfusate; its approach to steady state of efflux was slow. The inhibition either of MAO or COMT changed neither the total removal of tritium nor the 3H-metabolites recovered from the liver. Cocaine (10 mumol/l) reduced the accumulation of 3H-noradrenaline in the liver. The uptake2 inhibitor corticosterone (30 mumol/l) diminished total removal of tritium and the 3H-metabolites recovered from the liver without changing the accumulation of 3H-noradrenaline. The hypothesis of two different compartments, one responsible for the metabolism and the other for the accumulation of the amine is discussed.