Lower Corticosterone Concentrations Research Articles

Effect of neonatal ovariectomy and estradiol treatment on corticosterone release in response to stress in the adult female rat

Although organizational effects of sex hormones in early life on hypothalamic–pituitary–adrenal function have been reported for males, the findings are mixed for females, likely the result of not controlling for adult gonadal status. In experiment 1, females were ovariectomized (OVX) either on postnatal day 1 or as adults and given estradiol benzoate (EB) and progesterone implants or not as adults. Corticosterone release in response to restraint stress was responsive to hormonal replacement irrespective of timing of OVX: without replacement, both OVX groups had lower corticosterone concentrations after restraint stress than did OVX groups with replacement and gonadally intact females. Thus, neonatal OVX did not alter the activational effects of sex hormones in adulthood. In a second experiment, females administered a dose of EB on postnatal day 1 had a more rapid decline of corticosterone concentrations after restraint as adults compared to oil-treated females, irrespective of whether they were OVX as neonates or as adults (all groups were given estradiol replacement as adults). Thus, evidently there are organizational effects of the sex hormonal milieu of the neonatal female, although this is a modest effect compared to previous reports in males.

Corticosterone differences rather than social housing predict performance of T-maze alternation in male CD-1 mice

AbstractThis study examined the effects of social housing manipulations on bodyweight, corticosterone levels, and performance of T-maze alternation in male CD-1 mice. Males that adopted a dominant social rank were heavier than those that adopted a subordinate social rank. Dominant males also had lower corticosterone concentrations than the subordinates. However, there was little to suggest that these physiological indicators of social rank were moderated by housing condition. Indeed, statistical analysis confirmed that the difference in bodyweights was evident before males were socially housed. The mice showed high levels of spatial alternation on the T-maze from the start of testing so performance accuracy was high. Neither social rank nor housing condition had any clear categorical effect on T-maze performance. However, performance did fluctuate over successive blocks of testing and there was a negative association between accuracy on the T-maze and corticosterone levels (consistent with performance impairment because of elevated corticosterone). Therefore, under present conditions, individual differences in corticosterone were a better predictor of T-maze performance than social rank or housing condition. The results of the present study lend further support to the proposition that corticosterone levels measured non-invasively in urine may be used to predict diverse welfare outcomes for laboratory mice, from bodyweight to cognitive performance. Moreover, intrinsic physiological parameters rather than external influences, such as social housing, may have more influence on mouse behaviour.

Corticosteroids modulate Seoul virus infection, regulatory T-cell responses and matrix metalloprotease 9 expression in male, but not female, Norway rats

Human hantaviral disease is mediated by excessive proinflammatory and CD8+ T-cell responses, which can be alleviated by administration of corticosteroids. In contrast with humans, male rats that are infected with their species-specific hantavirus, Seoul virus (SEOV), have reduced proinflammatory and elevated regulatory T-cell responses in tissues where virus persists. To determine the effects of glucocorticoids on SEOV persistence and immune responses during infection, male and female Norway rats received sham surgeries (sham) or were adrenalectomized (ADX0), in some of which corticosterone was replaced at low (ADX10) or high (ADX80) doses. Rats were inoculated with SEOV and serum corticosterone, SEOV RNA, gene expression and protein production were measured at different time points post-inoculation. We observed that SEOV infection suppressed corticosterone in sham males to concentrations seen in ADX0 males. Furthermore, males with low corticosterone had more SEOV RNA in the lungs than either females or males with high corticosterone concentrations during peak infection. Although high concentrations of corticosterone suppressed the expression of innate antiviral and proinflammatory mediators to a greater extent in females than in males, these immunomodulatory effects did not correlate with SEOV load. Males with low corticosterone concentrations and high viral load had elevated regulatory T-cell responses and expression of matrix metalloprotease (MMP)-9. MMP-9 is a glycogenase that disrupts cellular matrices and may facilitate extravasation of SEOV-infected cells from circulation into lung tissue. Suppression of glucocorticoids may thus contribute to more efficient dissemination of SEOV in male than in female rats.

Diurnal and nocturnal differences in hypothalamic–pituitary–adrenal axis function in Galápagos marine iguanas

Temporal modulation of the stress response is a ubiquitous characteristic of animals. Here, we investigate possible mechanisms underlying daily changes in corticosterone release in an ectotherm model system. Earlier work indicated that free-living Galápagos marine iguanas ( Amblyrhynchus cristatus) have lower corticosterone concentrations during the night than during the day. This could result from: (i) a lower circadian secretion of adrenocorticotropic hormone (ACTH) as seen in mammals; (ii) from an increase in corticosterone negative feedback; or (iii) reflect lower metabolic activity during the night when core body temperature falls (from 35 °C during the day to as low as 21 °C during the night). To begin to distinguish between these three possibilities, exogenous ACTH was used to compare diel differences in adrenocortical tissue responsiveness, and dexamethasone was used to compare diel differences in the efficacy of corticosterone negative feedback. Low levels of exogenous ACTH (30 IU/kg body weight) potently stimulated both daytime and nighttime corticosterone release. Dexamethasone (1 mg/kg) inhibited only daytime, but not nighttime endogenous corticosterone release. Because the response to ACTH was similar between day and night we suggest that a simple lowering of core body temperature cannot explain the nighttime reduction in corticosterone release. However, the failure of negative feedback at night suggests that the response is not equivalent to the controlled downregulation seen in mammals.

Impaired action of anxiolytic drugs in mice deficient in cannabinoid CB 1 receptors

The role of cannabinoid CB 1 receptors in the action of anxiolytics was examined. Deletion of CB 1 receptors resulted in increased anxiety-like behaviours in light/dark box, elevated plus maze and social interaction tests. Mutant mice presented basal low corticosterone concentrations and low proopiomelanocortin gene expression in the anterior lobe of the pituitary gland compared to wild-type mice. Ten minutes of restraint stress resulted in a twofold increase in corticosterone concentrations in the plasma of mutant mice, compared to wild-type mice. Bromazepam (50 or 100 μg/kg) markedly increased the time spent in light area in wild-type animals, though both doses were without effect in mutant mice. Administration of buspirone (1 or 2 mg/kg) produced anxiolytic effects in wild-type mice. In contrast, only the highest dose of buspirone had anxiolytic results in mutant mice. Our findings reveal that CB 1 receptors are involved in the regulation of emotional responses, and play a pivotal role in the action mechanism of anxiolytics. They suggest that alterations in the functional activity of the CB 1 receptor may be related to the emergence of anxiety disorders, and may affect treatment with anxiolytics.

Reproductive physiology of free-living White Ibises ( Eudocimus albus) in the Florida Everglades

We measured plasma concentrations of testosterone, estradiol, progesterone, and corticosterone; and recorded changes in gonad size, body condition, molt, and brood patch development of free-living adult White Ibises ( Eudocimus albus) during the breeding season in the Florida Everglades. White Ibises are colonially breeding, long-legged wading birds that inhabit freshwater and estuarine wetlands. They have flexible breeding schedules (nest initiation dates can range from January to September) and onset of nesting is usually associated with increased prey availability caused by concentration of small fish in pools during periods of wetland drying. In this paper, we present the hormonal and physical characteristics of White Ibis reproductive physiology. We classified White Ibis breeding into five stages: pre-breeding, display, copulation/egg production, incubation, and chick rearing. White Ibises showed cyclic gonadal development which corresponded to reproductive stage. Male and female testosterone concentrations increased during the display stage and decreased during copulation, incubation, and chick rearing. Female estradiol concentrations were highest during display and chick rearing and male estradiol concentrations were lowest during copulation. Female progesterone concentrations increased during display and remained high throughout the breeding season. Female ibises had low corticosterone concentrations that increased during incubation and were highest during chick rearing, concomitant with lower body condition and flight muscle-mass scores. Male ibis progesterone and corticosterone concentrations did not show seasonal changes and were more variable than concentrations in female ibises at similar stages. Males and females had elevated body condition scores during the display stage, which were depleted by the onset of incubation. Increased energy stores during display may be used later for fasting in male birds that do not eat during the 10-day copulation/egg production stage, and for egg production in female birds. During incubation, male and female ibises developed brood patches. Ibises molted in all stages of reproduction, indicating that ibis molt and reproductive physiology may not inhibit each other as in most temperate bird species. White Ibises showed similar patterns in reproductive physiology to other monogamous, seasonally breeding bird species in which both sexes incubate and care for the young.

Social stress in laying hens: differential effect of stress on plasma dopamine concentrations and adrenal function in genetically selected chickens

Genetic selection for high or low group productivity and survivability (HGPS, LGPS) has created two phenotypically distinct chicken lines. Each line has unique characteristics in behavioral and physiological adaptability to multiple-bird cage system. The present study was designed to examine whether these differences reflect genetic variation in the control of plasma dopamine (DA) concentrations and adrenal function in response to social stress. Chickens from the HGPS and LGPS lines were randomly assigned to single- or 10-bird cages at 17 wk of age. The 10-bird cages were the same as those used in the development of the two lines. Differences in regulation of DA concentrations and adrenal function in response to different social environments were measured between the two lines when the study was conducted at 24 wk of age. In the 10-bird cages, the HGPS line had lower levels of DA (P < 0.05) and heavier adrenal glands (AG, P < 0.05) than those of the LGPS line, but concentrations of corticosterone (CORT) from the two lines were not significantly different. In the single-bird cages, DA levels in both lines were greater than in that of their siblings in the 10-bird cages, but a greater increase was found in the LGPS line (P < 0.01 and P < 0.05, 405% vs. 293%). Likewise, both lines had lower concentrations of CORT (P < 0.05) in the single- vs. 10-bird cages, but the AG were less heavy in the LGPS line but not in HGPS line in the single-bird cages (P < 0.05). The results indicated that the two strains reacted differently in terms of their stress hormone levels in the two different environments. These differences could contribute to the behavioral and physiological differences existing between the two lines.

Nutritional and metabolic effects of the endotoxin bacterial lipopolysaccharide in orally and parenterally fed rats

Animals treated with tumor necrosis factor alpha (TNF-alpha) developed severe metabolic abnormalities despite receiving sufficient protein and energy by total parenteral nutrition (TPN). We sought to investigate the nutritional and metabolic effects of bacterial lipopolysaccharide (LPS) in rats. Rats were randomly allocated to 5 groups: oral nutrition (ON control; n = 7), TPN control (n = 7), ON+LPS (n = 6), TPN+LPS (n = 9), and pair fed (PF) in relation to ON+LPS (n = 6). Body weight decreased significantly as diet consumption decreased in the ON+LPS and PF groups compared with the ON control group. Relative carcass weights were significantly lower in the TPN+LPS and ON+LPS groups than in their respective control groups. Diaphragm and extensor digitorum longus weights were significantly lower in the ON+LPS and PF rats, but not in the TPN+LPS rats, compared with their respective controls. Biochemical abnormalities and plasma corticosterone concentrations were greater in the TPN+LPS group than in the other groups. These data suggest that provision of sufficient protein and energy by TPN does not prevent general carcass wasting induced by LPS but may protect individual muscles. However, compared with an oral ad libitum diet, TPN providing sufficient protein and energy worsens the biochemical abnormalities induced by LPS. More rapid clearance of TNF-alpha and low corticosterone concentrations in weight-losing animals may help reduce the severity of the metabolic effects of LPS.

Effects of modifying layer cages with solid sides on stress physiology, plumage, pecking and bone strength of hens

Summary. This experiment evaluated the welfare of layer hens housed in cages modified with solid sides. Welfare was assessed on the basis of physiological measures of stress [corticosterone concentrations ‘at rest’ and in response to adrenocorticotrophic hormone (ACTH) and heterophil : lymphocyte ratios)] and immunological responsiveness, feather condition and cover, bone strength, claw length and between-bird pecking behaviour. Factors examined were cage modification (solid sides v. open sides i.e. standard cages), tier (upper v. lower), birds (1 or 2 birds/cage) and age (commencing at 35 v. 60 weeks of age). The cages provided a floor area of 1504 cm2. Floor pens (2.5 by 2.5 m) with 10 birds/pen served as an external control treatment. Solid sides in cages reduced the level of stress, compared with both standard cages and floor pens, based on lower corticosterone concentrations (2.2 v. 3.4 and 4.4 nmol/L, respectively) and lower heterophil : lymphocyte ratios (1.36 v. 1.66 and 1.66, respectively) (P&lt;0.05). The ACTH response data reflected a lower level of stress in the solid side compared with the pen treatment (P&lt;0.05). Feather condition and cover were improved by the presence of solid sides compared with standard cages (P&lt;0.05), were similar to the floor pen treatment and were probably a consequence of reduced pecking behaviour both within (65%) and between cages (86%) (P&lt;0.05). Solid sides had no effects on immunological responsiveness, claw length or bone strength. The experiment showed that solid sides can have a positive effect on welfare by reducing the level of stress, decreasing between-bird pecking and improving feather condition and cover. However, another study showed mortalities increased in cages with solid sides during hot weather.

Corticosterone regulates cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid expression in primary cultures of fetal rat adrenals

Glucocorticoids are known to inhibit growth in many different cell types. Although corticosterone is secreted by the adrenal cortex, its direct effect on the growth of different zones is poorly determined. We studied the effects of corticosterone on cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the rate-limiting step in adrenal steroidogenesis) messenger RNA (mRNA) accumulation in primary cultures of fetal rat adrenals. Adrenocortical cells, grown in the absence of ACTH for 3 weeks, possess typical features of zona glomerulosa cells. These cells differentiate into fasciculata-type cells and undergo biphasic proliferation when stimulated with ACTH. The primary antimitogenic phase of 24 h is followed by rapidly increased bromodeoxyuridine incorporation after 72 h of ACTH treatment. If the treatment is continued, the proliferation decreases again, but remains higher than the proliferation of the untreated cells. Undifferentiated zona glomerulosa-type cells secrete very low amounts of corticosterone. The 10% basal proliferation was not affected if exogenous corticosterone was added. However, if corticosterone was combined with ACTH for 3 days, it blocked the stimulatory growth effect of ACTH dose dependently. Etomidate, an inhibitor of steroidogenic enzymes, completely blocked corticosterone secretion. In our cultures it inhibited 50% of the proliferation of the zona glomerulosa-type cells. However, its effect was totally opposite in long term ACTH-treated cultures; in these fasciculata-type cells, etomidate stimulated the proliferation rate 3-fold. P450scc gene expression was low in undifferentiated zona glomerulosa-like cells. ACTH stimulation increased P450scc mRNA expression 10-fold. Exogenous corticosterone inhibited ACTH-induced P450scc mRNA accumulation by 50%, whereas etomidate doubled it. Our data suggest that a low corticosterone concentration supports the proliferation of undifferentiated zona glomerulosa-type cells, whereas a high corticosterone concentration inhibits the proliferation of differentiated zona fasciculata-type cells. In addition, a high corticosterone concentration may inhibit steroidogenesis by reducing P450scc expression. Thus, corticosterone may be an important modulator of adrenocortical cell proliferation and steroidogenesis in different zones of the adrenal cortex.

Corticosterone regulates cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid expression in primary cultures of fetal rat adrenals.

Glucocorticoids are known to inhibit growth in many different cell types. Although corticosterone is secreted by the adrenal cortex, its direct effect on the growth of different zones is poorly determined. We studied the effects of corticosterone on cell proliferation and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc; the rate-limiting step in adrenal steroidogenesis) messenger RNA (mRNA) accumulation in primary cultures of fetal rat adrenals. Adrenocortical cells, grown in the absence of ACTH for 3 weeks, possess typical features of zona glomerulosa cells. These cells differentiate into fasciculata-type cells and undergo biphasic proliferation when stimulated with ACTH. The primary antimitogenic phase of 24 h is followed by rapidly increased bromodeoxyuridine incorporation after 72 h of ACTH treatment. If the treatment is continued, the proliferation decreases again, but remains higher than the proliferation of the untreated cells. Undifferentiated zona glomerulosa-type cells secrete very low amounts of corticosterone. The 10% basal proliferation was not affected if exogenous corticosterone was added. However, if corticosterone was combined with ACTH for 3 days, it blocked the stimulatory growth effect of ACTH dose dependently. Etomidate, an inhibitor of steroidogenic enzymes, completely blocked corticosterone secretion. In our cultures it inhibited 50% of the proliferation of the zona glomerulosa-type cells. However, its effect was totally opposite in long term ACTH-treated cultures; in these fasciculata-type cells, etomidate stimulated the proliferation rate 3-fold. P450scc gene expression was low in undifferentiated zona glomerulosa-like cells. ACTH stimulation increased P450scc mRNA expression 10-fold. Exogenous corticosterone inhibited ACTH-induced P450scc mRNA accumulation by 50%, whereas etomidate doubled it. Our data suggest that a low corticosterone concentration supports the proliferation of undifferentiated zona glomerulosa-type cells, whereas a high corticosterone concentration inhibits the proliferation of differentiated zona fasciculata-type cells. In addition, a high corticosterone concentration may inhibit steroidogenesis by reducing P450scc expression. Thus, corticosterone may be an important modulator of adrenocortical cell proliferation and steroidogenesis in different zones of the adrenal cortex.

Corticosterone effects on long-term potentiation in mouse hippocampal slices.

Corticosterone (CORT) can alter several electrophysiological properties in the hippocampus. Few studies have reported the effects of CORT on hippocampal long-term potentiation (LTP). In the CA1 region of the hippocampal slice, we studied the relative increase in population spike (PS) amplitude after inducing tetanus (PS-LTP) in control conditions and in the presence of CORT (0.5 nM or 5 microM). Two perifusion mediums were used which differed in calcium concentration (M1:Ca 2.5 mM; M2: Ca 3.1 mM). In the control slices, the PS-LTP amplitude was greater in the M1 medium. With the low CORT concentration (0.5 nM), the PS-LTP amplitude was greater than in control conditions in the M2 medium only. A similar PS-LTP amplitude increase was also observed in the M2 medium after application of 0.5 nM aldosterone (type I receptor analog). With the high CORT concentration (5 microM), PS-LTP amplitude decreased in the two conditions. This effect was reversed by co-application of RU486 (type II receptor antagonist). These results demonstrate that CORT can modulate PS-LTP via its two types of hippocampal CORT receptors, depending on the concentration applied. They also showed that the extracellular calcium concentration may contribute to the functional change induced by CORT.

Hormonal changes in the immature rat after administration of pregnant mare serum gonadotrophin: influence of body weight.

We have shown previously that pregnant mare serum gonadotrophin (PMSG) induces ovulation only in rats weighing over 60 g on the day of injection. The under-60 g rats do not ovulate although they secrete a preovulatory surge of a pleiomorphic form of LH. Presumably this pleiomorph is inactive. Comparisons were made of plasma hormone concentrations in rats treated with PMSG that weighed over and under 60 g. The measurements were made on samples taken between 13.00 and 22.00 h on the day of the expected preovulatory LH surge. Prolactin and corticosterone levels were lower in the lighter group compared with the heavier group. A midday pulse of GH detected in the over-60 g animals did not occur in the under-60 g group. Levels of ACTH were slightly higher in the under-60 g rats and together with the low corticosterone concentrations indicate adrenal insensitivity. Oestradiol, progesterone and TSH concentrations were the same in the two groups. Since progesterone secretion is under LH control, the 'inactive' pleiomorphic form of LH must have steroidogenic activity. There was an indication that the under-60 g rats also secreted a pleiomorphic form of FSH. Reports in the literature indicate that prolactin, corticosterone and GH have a positive modulatory influence on natural puberty. They may also influence precocious puberty induced by PMSG, since in the unresponsive under-60 g rat plasma levels of these three hormones were low. Perhaps the release of one or more of these hormones is dependent upon the physical maturity of the animal as represented by body weight.

19-Hydroxyandrostenedione: evidence for a new class of sodium-retaining and hypertensinogenic steroids.

The biological activities of 19-hydroxyandrostenedione (19-OH-A-dione), which we reported as an amplifier of the action of aldosterone on the basis of the results obtained in bioassays using adrenalectomized rats, were evaluated in rats with the adrenals. The administration of 19-OH-A-dione to the rats caused sodium retention, high blood pressure, suppressed PRA and low plasma aldosterone, corticosterone, and deoxycorticosterone concentrations. The sodium-retaining and hypertensinogenic effects of 19-OH-A-dione were more potent than those of deoxycorticosterone acetate. Spironolactone inhibited the action of 19-OH-A-dione. The results indicate that 19-OH-A-dione, although it is devoid of mineralocorticoid activity, works as a potent sodium-retaining agent and causes a hypertensive state simulating mineralocorticoid excess in the presence of the adrenal cortex.

Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.

EFFECTS OF CORTICOSTERONE ON ADRENOCORTICOTROPHIN-INDUCED MITOCHONDRIAL DIFFERENTIATION WITH SPECIAL REFERENCE TO 11β-AND 18-HYDROXYLATION

The effects of corticosterone in concentrations found in adrenal venous plasma on ACTH-induced changes in cultured cortical cells derived from foetal rat adrenals were studied. Corticosterone at a concentration of 5-7 X 10(-5) mol/l completely inhibited mitochondrial differentiation to fasciculte-like morphology. The same cultures revealed significant inhibition of 11beta- and 18-hydroxylation compared with cultures treated with ACTH only. This was shown by the reduced formation of corticosterone and 18-OH-deoxycorticosterone (48%, P less than 0-001) and simultaneous enhancement of deoxycorticosterone formation (33%, P less than 0-05) from added [4-14C]progesterone. Similar inhibition was observed when dibutyryl cyclic AMP replaced ACTH as an inducer of differentiation. Lower concentrations of corticosterone (1-2 X 10(-5) and 2-4 X 10(-5) mol/l) inhibited ACTH-stimulated formation of corticosterone and 18-OH-deoxycorticosterone from endogenous precursors. The results demonstrate that corticosterone regulates the stage of differentiation in cultured adrenocortical cells. The possible role of corticosterone in the regulation of growth and steroidogenic capacity of the adrenal cortex is discussed.

Relationship Between ACTH Release and Corticosterone Binding by the Receptor Sites of the Adenohypophysis and Dorsal Hippocampus Following Infusion of Corticosterone at a Constant Rate in the Adrenalectomized Rat1

The amount of corticosterone bound to proteins in the adenohypophysis and dorsal hippocampus was studied concurrently with the plasma ACTH concentration in 4-week adrenalectomized male rats under steady-state conditions achieved by infusing the steroid at a constant rate for 45 min. Corticosterone binding was measured by gel chromatography on Sephadex LH-20, using a TE buffer containing 0.4 m NaCl. No saturation was observed in whole homogenates and supernatants with increasing plasma corticosterone concentrations. However, corticosterone binding by the pituitary and by one of two types of hippocampal receptors evidenced saturation within our range of corticosterone infusion rates, indeed. Scatchard plots allowed us to distinguish two types of binding sites in the hippocampus. The first, saturated at a low corticosterone concentration has an association constant of 3.0 times 10-8 M-1 with a number of binding sites estimated at 150 times 10- minus 15 mol/mg protein, whereas the second is associated with non-specific binding. The adenohypophysis shows only one kind of binding site with an association constant of 3.6 times 10-8 m- minus 1 and a number of sites estimated at 989 times 10- minus 15 mol/mg protein. A suggestive relationship was observed between ACTH inhibition by corticosterone and saturation of the pituitary binding sites. Our data are consistent with the possible involvement of corticosterone binding by specific sites in the negative feedback regulation of ACTH release.