Adrenocorticotropic hormone and corticosterone responses to acute hypoxia in the neonatal rat: effects of body temperature maintenance

The corticosterone response to acute hypoxia in neonatal rats develops in the 1st wk of life, with a shift from ACTH independence to ACTH dependence. Acute hypoxia also leads to hypothermia, which may be protective. There is little information about the endocrine effects of body temperature maintenance during periods of neonatal hypoxia. We hypothesized that prevention of hypothermia during neonatal hypoxia would augment the adrenocortical stress response. Rat pups separated from their dams were studied at postnatal days 2 and 8 (PD2 and PD8). In one group of pups, body temperature was allowed to spontaneously decrease during a 30-min prehypoxia period. Pups were then exposed to 8% O(2) for 3 h and allowed to become spontaneously hypothermic or externally warmed (via servo-controlled heat) to maintain isothermia. In another group, external warming was used to maintain isothermia during the prehypoxia period, and then hypoxia with or without isothermia was applied. Plasma ACTH and corticosterone and mRNA expression of genes for upstream proteins involved in the steroidogenic pathway were measured. Maintenance of isothermia during the prehypoxia period increased baseline plasma ACTH at both ages. Hypothermic hypoxia caused an increase in plasma corticosterone; this response was augmented by isothermia at PD2, when the response was ACTH-independent, and at PD8, when the response was ACTH-dependent. In PD8 rats, isothermia also augmented the plasma ACTH response to hypoxia. We conclude that maintenance of isothermia augments the adrenocortical response to acute hypoxia in the neonate. Prevention of hypothermia may increase the stress response during neonatal hypoxia, becoming more pronounced with increased age.     

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. I. Clearance profiles

Marked differences were observed between the clearance profiles of immunoreactive plasma gonadotropins in gonadectomized and intact male bullfrogs (Rana catesbeiana). The disappearance patterns of endogenously secreted follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from plasma of intact animals following chronic (1-4 days) infusion with gonadotropin releasing hormone (GnRH) showed multiple components, but the initial few half-lives were relatively short (less than 1 h) and about 90% of both gonadotropins were cleared from the plasma within 6 h. Hypophysectomy had no effect on gonadotropin clearance rates following the termination of GnRH infusion. Clearance profiles of exogenous gonadotropins after chronic (6 h) infusion of bullfrog pituitary extract were similar to those observed after GnRH infusion. Gonadectomized frogs also cleared these infused pituitary gonadotropins at the same rate as intact animals, confirming that gonadectomy did not impair peripheral clearance mechanisms. Relatively rapid clearance rates were also observed for endogenous FSH and LH in normal untreated frogs. By comparison, the disappearance rates of FSH and LH from plasma of six long-term gonadectomized males following hypophysectomy were extremely slow: first half-lives for FSH and LH were 25.6 h and 17.2 h, respectively, and subsequent half-lives were even longer. Several weeks were required to clear fully the FSH and LH from the circulation in these males. Thus, a significant change in the physicochemical form of the circulating gonadotropins after gonadectomy in the male bullfrog is postulated; the corresponding changes in clearance rates were considerably greater than have been observed in any other species.     

ACTH increases adrenal medullary PNMT activity in neonatal rats

Levels of plasma corticosterone and the activities of adrenomedullary dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) were measured in the 7-day-old rat following the administration of adrenocorticotropic hormone (ACTH) for 7 consecutive days beginning with day 1. ACTH led to significant adrenal hypertrophy and a concomitant elevation (10 to 15 fold) of plasma corticosterone concentration. Whereas DBH activity remained unchanged, adrenal PNMT activity was increased significantly following ACTH-induced elevation of plasma corticosterone levels. These results indicate that the pituitary-adreno-cortical-adrenomedullary axis is functional in the neonatal rat. Furthermore, since the transsynaptic control mechanisms are known to be non-functional or immature in the 7-day-old rat, our data suggest that neonatal rat adrenal catecholamine biosynthesis may be largely controlled by the pituitary-adrenocortical axis via glucocorticoids.     

Daily fluctuations of peripheral metabolism of corticosterone in male Japanese quails: a dynamic approach to the development of plasma corticosterone daily rhythm

An ultradian rhythm was found in the daily variation of the parameters concerning the peripheral metabolism of corticosterone in sexually mature male Japanese quails. The period of this rhythm was eight hours; three peaks and minima were found in the daily fluctuation of metabolic clearance rata and an antiphysed rhythm in the biological half-life as well as in tetrahydrocorticosterone/corticosterone conversion rata. The findings emphasize the importance of the peripheral metabolism in developing of corticosterone plasma levels; and analyze the circadian rhythm of corticosterone plasma level as a result of secretion rate and peripheral disappearance.     

Sex differences in adrenal function in the lizard Cnemidophorus sexlineatus: II. Responses to acute stress in the laboratory.

Circulating concentrations of plasma corticosterone and gonadal steroids were measured in intact and gonadectomized male and female lizards (Cnemidophorus sexlineatus) following acute stress (handling) in the laboratory. There was a significant increase in plasma corticosterone after stress. Whereas intact females exhibited greater concentrations of corticosterone relative to intact males, ovariectomized females exhibited lower concentrations of corticosterone relative to castrated males. In addition to sex differences in corticosterone responses to gonadectomy, progesterone was elevated by stress in both intact and ovariectomized females but not in males. Corticosterone adjusted for castration and handling in males was negatively correlated with the plasma androgen level. The adrenal responsiveness of males to acute stress may be attenuated by androgens presumably secreted by the testis. Not only does adrenal function influence reproduction, but adrenal responses differ between males and females, and appear to be influenced by the gonadal axis. The sex differences in adrenal responses to stress likely reflect different reproductive strategies and nutritional requirements of males and females during the breeding season.     

Elevated corticosterone and inhibition of ACTH responses to CRH and ether in the neonatal rat: effect of hypoxia from birth

Hypoxia is a common cause of neonatal morbidity and mortality. We have previously demonstrated a dramatic ACTH-independent activation of adrenal steroidogenesis in hypoxic neonatal rats, leading to increases in circulating corticosterone levels. The purpose of the present study was to determine if this ACTH-independent increase in corticosterone inhibits the ACTH response to acute stimuli. Neonatal rats were exposed to normoxia (control) or hypoxia from birth to 5 or 7 days of age. At the end of the exposure, plasma ACTH and corticosterone were measured before and after either ether vapors were administered for 3 min or CRH (10 microg/kg) was given intraperitoneally. Thyroid function, pituitary pro-opiomelanocortin (POMC) mRNA and ACTH content, and hypothalamic corticotropin-releasing hormone (CRH), neuropeptide Y (NPY), and AVP mRNA were also assessed. Hypoxia led to a significant increase in corticosterone without a large increase in ACTH, confirming previous studies. The ACTH responses to ether or CRH administration were almost completely inhibited in hypoxic pups. Hypoxia did not affect the established regulators of the neonatal hypothalamic-pituitary-adrenal axis, including pituitary POMC or ACTH content, hypothalamic CRH, NPY, or AVP mRNA (parvo- or magnocellular), or thyroid function. We conclude that hypoxia from birth to 5 or 7 days of age leads to an attenuated ACTH response to acute stimuli, most likely due to glucocorticoid negative feedback. The neural and biochemical mechanism of this effect has yet to be elucidated.     

Production of corticosterone in male homozygous Brattleboro rats

Plasma concentration, metabolic clearance rate and in vitro adrenal production of corticosterone were measured in Brattleboro male rats homozygous for diabetes insipidus (DI) and in Long-Evans male rats (LE) as controls in resting conditions, under stress caused by pentobarbitone anesthesia and surgery and after three days water deprivation. In resting animals, plasma concentrations and in vitro adrenal production of corticosterone were higher in DI rats than in LE rats. Under pentobarbitone anesthesia and surgery, plasma concentrations and metabolic clearance rate of corticosterone were slightly but not statistically lower in DI rats; however, the in vivo production rate of corticosterone was significantly lower. After three days water deprivation, increasing plasma corticosterone level was consistently higher in DI than in Le rats. These results are not in favour of a reduced glucocorticoid activity of the adrenal of DI rats and of an important role played by vasopressin on the stimulation of the hypothalamopituitary adrenal activity at least in resting conditions; its role may depend upon stressful circumstances.     

Adrenal function in female rats before puberty: the effects of gonadectomy

The following study was designed to test whether the change in the amplitude of the adrenal rhythm and the response to stress seen at puberty in female rats is dependent upon stimulation of the adrenal system by gonadal steroids. Rats were gonadectomized either at two days of age or at 21 days of age and periodic blood samples were taken by cardiac puncture and assayed for corticosterone (B) by a fluorometric procedure. Ovariectomy at weaning age (21 days) had no effect on adrenal function until the time of normal puberty onset. In intact animals, a rise in resting levels of corticosterone and an increase in the incremental response to ether stress was noted at 35 days of age. (Puberty in females was 35.3 ± 1.2 days as indicated by vaginal opening). Ovulation occurred the following day in 9/10 rats. Gonadectomy at two days of age had essentially the same effect as later gonadectomy in females. By 70 days of age, resting corticosterone values and stress responses in gonadectomized females reached levels similar to intact females. It is concluded that an independent adrenarche can occur in females but that gonadal steroids present at the time of puberty modulate the timing of this process.     

Effect of neonatal hypoxia on leptin, insulin, growth hormone and body composition in the rat.

The purpose of the present study was to evaluate the effect of exposure to hypoxia from birth to 7 days of age on leptin, insulin, growth hormone (GH), insulin-like growth factor-1 (IGF-1), glucose, corticosterone, body weight, and body composition in rats studied at 7 days of age and then after return to normoxia. Hypoxia for the first 7 days of life resulted in a significant decrease in plasma leptin, body weight, and an increase in corticosterone and insulin with no change in plasma glucose, GH or IGF-1. There was no significant effect of hypoxia on % lean body mass, but a small but significant increase in % body fat. Bone mineral density (BMD) was lower in 7-day-old hypoxic rats as compared to normoxic controls. All hormonal variables and BMD had normalized by 7 days after return to normoxia. However, body weight remained lower even 5 weeks after return to normoxia. We conclude that leptin is decreased during neonatal hypoxia despite no change in adiposity. Furthermore, insulin is increased probably to overcome the effects of increased counterregulatory hormones (such as corticosterone).     

Adrenal lipid profiles of chemically sympathectomized normoxic and hypoxic neonatal rats.

Neonatal hypoxia is a common condition that elicits a coordinated endocrine response. In the neonatal rat, hypoxia induces an ACTH-independent increase in corticosterone which can be partially blocked by chemical sympathectomy. The present study sought to characterize the effects of sympathectomy on the adrenal lipid profile, since previous work suggested that augmented plasma corticosterone during hypoxia may be due to changes in adrenal lipid metabolism. Newborn rats were exposed to normoxia or hypoxia from birth to seven days of age, and guanethidine was used to produce the sympathectomy. Plasma epinephrine and norepinephrine were not significantly affected by hypoxia, while guanethidine decreased plasma norepinephrine in normoxic and hypoxic pups. Hypoxia alone increased the concentration of cholesterol esters in the adrenal gland; this increase was due to increases in cholesterol ester-associated oleic (18:1n9), docosahexaenoic (22:6n3), arachidonic (20:4n6), and adrenic (22:4n6) acids. Hypoxia also increased diglyceride-associated adrenic acid. Guanethidine treatment attenuated the hypoxia-induced increase in cholesterol ester-bound arachidonic and adrenic acids. Guanethidine also decreased saturated fatty acid concentrations and increased n3 fatty acid-enriched triglycerides. The results support the idea that the ACTH-independent corticosterone response to hypoxia in the neonatal rat is mediated by specific, sympathetically driven alterations in the adrenal lipid profile.     

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100μg/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4, 6 or 24h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24h after surgery, and in both hypothalamic nuclei only at 6h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood.     

Resilience and vulnerability are dose-dependently related to neonatal stressors in mice

Early life experiences have been shown to adjust cognitive abilities, stress reactivity, fear responses and immune activity in adult mammals of many species. However, whereas severe stressors have been generally associated with the emergence of hypothalamic pituitary adreno-cortical (HPA)-mediated pathology, mild neonatal stressful experiences have been traditionally associated with ‘positive’ effects or resilience. External stressors stimulate the HPA axis to induce a corticosterone secretion in mouse dams, which, in turn is directly transmitted to the progeny through lactation. Such corticosteroid transfer may offer a unitary mechanism whereby early low corticosterone exposure may favor resilience in the offspring and high corticosterone increase vulnerability to pathology. In this study we further investigated this hypothesis by evaluating the long-term effects of a neonatal exposure to low (33 mg/l) and high (100 mg/l) doses of corticosterone during the first 10 days of life in outbred CD-1 mice through supplementation in the maternal drinking water. Offspring attentional set-shifting abilities, central neurotrophic regulation and levels of natural auto-antibodies (na-Abs) directed to serotonin (SERT) and dopamine (DAT) transporters were assessed in adulthood. While low levels of neonatal corticosterone improved adult cognitive abilities and increased na-Abs levels directed to SERT, high doses of neonatal corticosterone reduced hippocampal BDNF levels and na-Abs directed to DAT. These findings confirm and extend our previous findings, supporting the view that both adaptive plasticity and pathological outcomes in adulthood may depend on circulating neonatal corticosterone levels and that these effects follow a U-shaped profile.     

Corticosterone secretion induced by chronic isolation in neonatal rats is sexually dimorphic and accompanied by elevated ACTH

Rat pups repeatedly subjected to brief periods of isolation during the stress hyporesponsive period (SHRP) exhibit varied neuroendocrine and behavioral changes as neonates and as adults. For example, neonatal rats exhibit increased circulating corticosterone after 1-h isolation on postnatal day 9 (P9) only if they were isolated daily from P2 to P8 [McCormick, C.M., Kehoe, P., Kovacs, S., 1998. Corticosterone release in response to repeated, short episodes of neonatal isolation: evidence of sensitization. Int. J. Dev. Neurosci. 16, 175-185]. It is not known if the increase in adrenocortical response on P9 following repeated isolation is mediated by increased pituitary ACTH secretion. The present study examined the responsivity of the hypothalamic-pituitary-adrenal (HPA) axis during the SHRP following brief, repeated isolation or acute pharmacological manipulation. Removal from the nest for 1 h daily on P4-8 increased circulating corticosterone after 1-h isolation on P9 by approximately twofold. Neither unhandled nor handled controls showed a corticosterone response to 1-h isolation on P9. The increased corticosterone was sexually dimorphic, with only females showing the sensitization response. Other findings suggest that the hormonal response is centrally mediated; chronically isolated pups of both sexes exhibit increased plasma ACTH following 1-h isolation on P9. While we could not detect an increase in Fos immunoreactivity (IR) on P9 in the hypothalamic paraventricular nucleus (PVN) of chronically isolated pups, acute pharmacological activation of serotonin 2A/2C receptors produced robust activation of ACTH and corticosterone secretion as well as expression of Fos in the PVN on P9. We conclude that chronic isolation stress limited to the SHRP stimulates the neonatal HPA axis, and that the adrenal response is sexually dimorphic. In addition, PVN neurons can express Fos IR on P9 in response to a very potent activation of the HPA axis.     

Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats

Previous studies have indicated that neonatal handling influences development of hypothalamic-pituitary-adrenal (HPA) control of corticosterone. In addition, corticosterone influences memory consolidation processes in contextual fear conditioning. Therefore, neonatal handling may affect hippocampal-dependent memory processes present in contextual fear conditioning by influencing the development of HPA control of corticosterone. To investigate the effects of neonatal handling on early learning, rat pups were either handled (15-min removal from home cage) on the first 15 days after birth or left undisturbed in their home cage. Handled rats and nonhandled rats were fear conditioned at 18, 21, or 30 days of age and then tested at two time points--24 h following conditioning and at postnatal day 45. Subsequently, at approximately postnatal day 60, rats were exposed to restraint stress and corticosterone levels were assessed during restraint and recovery. Handled and nonhandled rats did not differ significantly in their freezing response immediately following footshock on the conditioning day. However, when tested for contextual fear conditioning at 24 h following conditioning and at postnatal day 45, handled rats showed more freezing behavior than nonhandled rats. When exposed to restraint stress, handled rats had a more rapid return of corticosterone to basal levels than nonhandled rats. These results indicate that neonatal handling enhances developmentally early memory processes involved in contextual fear conditioning and confirms previously reported effects of neonatal handling on HPA control of corticosterone.     

Development of the ACTH and corticosterone response to acute hypoxia in the neonatal rat

Acute episodes of severe hypoxia are among the most common stressors in neonates. An understanding of the development of the physiological response to acute hypoxia will help improve clinical interventions. The present study measured ACTH and corticosterone responses to acute, severe hypoxia (8% inspired O(2) for 4 h) in neonatal rats at postnatal days (PD) 2, 5, and 8. Expression of specific hypothalamic, anterior pituitary, and adrenocortical mRNAs was assessed by real-time PCR, and expression of specific proteins in isolated adrenal mitochondria from adrenal zona fascisulata/reticularis was assessed by immunoblot analyses. Oxygen saturation, heart rate, and body temperature were also measured. Exposure to 8% O(2) for as little as 1 h elicited an increase in plasma corticosterone in all age groups studied, with PD2 pups showing the greatest response ( approximately 3 times greater than PD8 pups). Interestingly, the ACTH response to hypoxia was absent in PD2 pups, while plasma ACTH nearly tripled in PD8 pups. Analysis of adrenal mRNA expression revealed a hypoxia-induced increase in Ldlr mRNA at PD2, while both Ldlr and Star mRNA were increased at PD8. Acute hypoxia decreased arterial O(2) saturation (SPo(2)) to approximately 80% and also decreased body temperature by 5-6 degrees C. The hypoxic thermal response may contribute to the ACTH and corticosterone response to decreases in oxygen. The present data describe a developmentally regulated, differential corticosterone response to acute hypoxia, shifting from ACTH independence in early life (PD2) to ACTH dependence less than 1 wk later (PD8).     

低氧暴露对大鼠下丘脑CRF分泌的影响

目的和方法：利用人工模拟低氧及放射免疫测定（RIA）法,观察不同低氧（5km,7km）暴露对大鼠下丘脑分泌促肾上腺皮质激素释放激素（C orticotropin-releasing factor,CRF）的作用和血浆皮质酮水平的变化。比较低氧暴露不同时间（2h,24h,5d,15d）后,大鼠下丘脑CRF分泌和血浆皮质酮的变化。结果：低氧暴露2h,24h后下丘脑的CRF分泌明显增加,血浆皮质酮水平显著升高,这种变化随着低氧程度的加深而增强,随着低氧暴露时间的延长（5d）上述变化减弱。至慢性低氧暴露15d后,下丘脑CRF分泌及血浆皮质酮水平与对照组相比已无显著差异,基本恢复到对照水平。结论：动物暴露于低氧环境后,下丘脑CRF分泌及血浆皮质酮水平变化为：低氧暴露2h,24h后CRF分泌和皮质酮分泌被激活;低氧暴露5d后CRF分泌和皮质酮分泌活动减弱,并开始恢复;至低氧暴露15d后,这种分泌活动基本恢复,进入恢复期。     

Degradation of CCK-8 by rat synaptosomal peptidases

By use of an antiserum raised against conjugated ovine corticotropin releasing factor (CRF_1–41), nerve fibres can be stained immunocytochemically in the external zone of the median eminence of rats. The presence of CRF-immunoreactive (CRF_i) nerve fibres and the plasma corticosterone response to ether stress were studied in rats 6–7 days after making various types of lesions in the hypothalamus. Complete anterolateral deafferentation of the mediobasal hypothalamus caused complete disappearance of CRF_i fibres from the median eminence and blocked the corticosterone response to stress. Incomplete anterolateral hypothalamic deafferentation did not prevent the stress-induced increase of corticosterone and in these rats, part of the CRF_i nerve fibres remained intact. A horizontal cut placed ventral to the paraventricular nuclei, completely prevented the corticosterone response in those rats that showed a complete disappearance of CRF_i nerve fibres from the median eminence. Some rats however, still exhibited CRF_i nerve fibres and these animals responded to stress with increased corticosterone levels. A similar horizontal cut made just dorsal to the paraventricular nuclei affected neither the corticosterone response to stress nor the appearance of CRF_i nerve fibres in the median eminence. We conclude that the presence of CRF_i nerve fibres in the median eminence is a prerequisite for rats to show a pituitary-adrenal response to ether stress and therefore represents the first functional evidence for the role of these hypothalamic CRF_i-neurons.     

EFFECTS OF PRE- OR POSTNATAL DEXAMETHASONE, ADRENOCORTICOTROPHIC HORMONE AND ENVIRONMENTAL STRESS ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE ACTIVITY AND CATECHOLAMINES IN SYMPATHETIC GANGLIA OF NEONATAL RATS

Abstract— Injections of dexamethasone (0.1 mg/kg/day, s.c.) on the first 2–3 days of life increased the phenylethanolamine- N -methyltransferase (PNMT) activity and epinephrine content of the superior cervical ganglion (SCG) and stellate ganglion of neonatal rats; the dopamine content was unaltered while norepinephrine was slightly reduced in these ganglia. Dexamethasone did not alter the PNMT activity or epinephrine content of the salivary glands or heart. The PNMT activity and epinephrine content of the SCG remained elevated for 10–14 days. Pretreatment with 6-hydroxydopamine did not alter the dexamethasone effects.
Injections of adrenocorticotrophic hormone (ACTH) (25 munits/rat twice a day) or exposure to a cold stress (4°C, 3 times a day) on the first 2–3 days of life, elevated the plasma concentration of corticosterone, and also increased the PNMT activity and epinephrine content in SCG of neonatal rats. Injecting pregnant rats with dexamethasone or ACTH, or exposing them to cold or restraint stress on the last 3 days of gestation increased the PNMT activity and epinephrine content in the SCG of their pups. These results indicate that the actions of dexamethasone on neonatal sympathetic ganglia may be mimicked by increasing the plasma concentration of endogenous adrenocortical steroids.     

The neonatal glucocorticoid treatment-produced long-term changes of the pituitary-adrenal function and brain corticosteroid receptors in rats.

Two distinct periods of sensitivity to elevated glucocorticoid hormone levels during postnatal development of the pituitary-adrenal axis were studied. Wistar rats were injected subcutaneously (s.c.) with cortisol (1 mg/kg) on postnatal days 1-5 or 14-18. The steroid treatment during the first postnatal week resulted in a decrease of the morning basal and stress-induced plasma corticosterone levels in 30 day-old male rats, as well as in rats that were injected with cortisol on the third postnatal week. Stress-induced corticosterone levels in 90-day old cortisol-treated rats were determined in blood samples drawn from the tail vein before the restraint stress, immediately after the 20-min long stress, then 60 and 180 min afterwards. Only the rats treated with cortisol during the third week showed a prolonged stress-induced corticosterone secretion, with the highest corticosterone level in 180 min after the restraint stress. The early neonatal cortisol treatment had no effect on (3)H-corticosterone binding in all studied brain areas of the 90-day old rats. The rats treated with cortisol at the 14-17th postnatal days showed a significantly lower (3)H-corticosterone binding in the frontal cortex, hippocampus, and hypothalamus. These findings suggest that the third week of life in rats is more sensitive to elevated levels of corticosterone than the first one. The high level of glucocorticoids at this period has long-term effects on the efficiency of the negative feedback mechanisms provided by hypothalamus-pituitary-adrenal axis.     

Determination of occupied cytoplasmic glucocorticoid receptor sites by an exchange assay in rat muscles

We have established an assay to determine total cytoplasmic glucocorticoid receptor sites in heart, vastus and levator-ani-bulbocavernosus muscles from rats. We have tested the exchange of endogenously bound hormone with labeled triamcinolone acetonide by performing long incubation at 0-4 degrees C. This procedure appeared to produce an adequate exchange of exogenously added unlabeled corticosterone in the range of 16 nM (normal unstressed concentration of corticosterone in plasma) with vastus and levator ani-bulbocavernosus muscles. Beyond that level, the displacement of unlabeled corticosterone by labeled triamcinolone acetonide was less efficient. By contrast, in heart and especially in liver cytosols, the exchange was adequate even in the presence of high concentrations of corticosterone (64 and 460 nM respectively). Using the exchange assay conditions (20 h of incubation at 0-4 degrees C with 16 nM of labeled triamcinolone acetonide), we found no significant difference between glucocorticoid receptor levels in intact male and female rats. In both sexes, these glucocorticoid receptor values were not affected by gonadectomy, but increased more than 2-fold after adrenalectomy. No seasonal variations were recorded in intact or adrenalectomized rats.