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We studied the long-term effect of neonatal treatment with the synthetic glucocorticoid dexamethasone (DEX) on stress responsivity later in life. It was found that the plasma adrenocorticotropin hormone (ACTH) and corticosterone (CORT) responses induced by novelty or conditioned fear stress were markedly attenuated in adult rats that had been neonatally treated with DEX as compared with saline (SAL)-treated controls. Since there were no differences in the heart rate, body temperature, plasma noradrenaline, plasma adrenaline and behavioral responses to these stressors, this points to a deficit within the hypothalamic-pituitary-adrenal (HPA) axis of DEX rats. We found no differences between DEX and SAL rats in basal plasma CORT concentrations measured throughout the circadian cycle, nor in the fraction unbound of CORT circulating under resting conditions, indicating normal tonic regulation of the HPA axis in DEX rats. Since we also found no differences in the hormonal responses induced by intravenous injection of graded doses of ACTH or corticotropin-releasing hormone (CRH), we investigated the sensitivity of the HPA response to stress for inhibition by glucocorticoids. Pretreatment with a low dose of CORT that did not affect the HPA response of SAL rats markedly inhibited the ACTH and CORT responses induced by novelty stress in DEX rats. This strongly suggests that an enhanced corticosteroid feedback underlies the blunted HPA response to stress in DEX rats. Finally, using quantitative immunocytochemistry, we found an increase in arginine-vasopressin (AVP) but not CRH stores in the external zone of the median eminence, suggesting an altered AVP/CRH ratio in the secretory output of the hypophysiotropic paraventricular nucleus. Taken together, our results show that exposure to DEX during early life leads to hyporesponsivity of the HPA axis to stress most likely due to hypersensitivity of the axis for negative feedback by corticosteroids at the suprapituitary level., (Copyright 2002 S. Karger AG, Basel)

This study describes the effect of antalarmin on basal and stimulated activity of the hypothalamo-pituitary-adrenal (HPA) axis function in the late gestation ovine fetus. Fetuses received antalarmin (15 mg/h i.v.) or vehicle (cremophor El 50% in ethanol) from day 130 gestational age. Antalarmin infusion did not significantly affect immunoreactive corticotropin (ir-ACTH) concentrations, although there was a tendency for ir-ACTH to be lower and cortisol concentrations were lower in the antalarmin-treated fetuses (p < 0.01). The ir-ACTH response to corticotropin-releasing hormone (CRH) challenge was attenuated (p < 0.05) in the antalarmin-treated fetuses, but neither antalarmin- nor vehicle-treated fetuses had significant cortisol responses to CRH. The ir-ACTH response to hypoxia was diminished (p < 0.05) in the antalarmin-treated fetuses while the cortisol responses of antalarmin- and vehicle-treated fetuses were indistinguishable. Deconvolution analysis revealed no effect of antalarmin treatment on ir-ACTH secretory dynamics. In contrast, antalarmin decreased (p < 0.05) basal, mean and integrated cortisol. The plasma cortisol responses of antalarmin- and vehicle-treated fetuses to exogenous ACTH(1-24) were indistinguishable. These data indicate that, while antalarmin inhibits CRH- and stress-induced ir-ACTH secretion, basal ir-ACTH secretion may be less affected by antalarmin treatment. Paradoxically, cortisol secretion is impaired by antalarmin infusion, although adrenal responsiveness to ACTH is not impaired. These results confirm a role for CRH in stress-induced ACTH secretion in the ovine fetus, though its role in the regulation of basal ACTH and cortisol secretion is unclear., (Copyright 2002 S. Karger AG, Basel)

Age related changes in the time courses of response of plasma corticosterone and aldosterone to exogenously applied ACTH were simultaneously studied in old (female and male) Long-Evans rats and compared to both young and adult pentobarbitone-anesthetized and dexamethasone-pretreated control rats. Acute intravenous injection of either 0.5 or 50.0 ng ACTH (1-24)/100 g body weight increased plasma concentrations of the two steroids with a similar time course in all groups of rats. However, we observed a significant age-related attenuation in the plasma corticosteroid response. Thus, in old as compared to young rats there was a decrease of approximately 45, 40 and 30% in plasma corticosterone levels respectively 8 min after the lower dose of ACTH in female and 45 min after the higher dose in female and male rats. Similarly, an attenuated (approximately -38%) response of plasma aldosterone levels, induced 45 min after the higher dose of ACTH, was observed both in old female and male rats. These results suggest that the previously reported age-related decreases of in vivo corticosterone and aldosterone secretion are, at least in part, due to a reduced capacity of adrenocortical cells for steroid biosynthesis and release in response to stimulation by ACTH.

Hormones of the hypothalamo-pituitary-adrenocortical (HPA-) axis are considered to be of physiological and clinical relevance in regulating spontaneous growth hormone (GH) secretion. To further investigate interdependencies between both systems, we studied the effects of adrenocorticotropin [ACTH(1-24)] and human corticotropin-releasing hormone (h-CRH) upon spontaneous GH secretion in 10 male volunteers. Administration of 1 microgram ACTH (1-24), 10 micrograms h-CRH or saline (control: CTL) every hour from 9.00 to 6.00 p.m. resulted in significant differences of cortisol secretion during the entire observation period (8.00 a.m.-3.00 a.m.) between the three groups (p less than 0.001, Friedman two-way ANOVA). Mean area under the time course curve (AUC) values (+/- SEM) for cortisol expressed as ng x 1,000 x min/ml showed also significant differences between the three treatments from 8.00 a.m. to 3.00 a.m.: CTL 64.0 +/- 6.4, ACTH(1-24) 178.5 +/- 9.4 (p less than 0.01, Wilcoxon test), h-CRH 88.5 +/- 5.6 (p less than 0.01). The main portion of cortisol was released during daytime from 8.00 a.m. to 11.00 p.m., where the most significant differences in the AUC values emerged: CTL 59.6 +/- 5.8, ACTH(1-24) 171.5 +/- 8.8 (p less than 0.01, Wilcoxon test), h-CRH 80.2 +/- 5.1 (p less than 0.01). With regard to GH secretion, significant differences became obvious between the three treatments during daytime from 8.00 a.m. to 11.00 p.m. and the sleep-related period from 11.00 p.m. to 3.00 a.m. (p less than 0.01 and p less than 0.02, Friedman two-way ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)

beta-Endorphin and ACTH are secreted concomitantly in baseline conditions and in response to physiological and pharmacological stimuli. However, few and contradictory data are available on their feedback inhibition mechanisms. To investigate this aspect, the effects of exogenous ACTH1-24 and glucocorticoids on endogenous ACTH1-39 and beta-endorphin were tested in 18 patients with Addison's disease. Two main experimental protocols were employed: (1) 7 patients were given ACTH1-24 50 micrograms as an intravenous bolus followed by a 50-micrograms infusion in 90 min. Blood samples for beta-endorphin, ACTH and cortisol were obtained at 0, 15, 30, 45, 60, 90, 120 min. Six other patients were given oCRH 100 micrograms i.v. plus ACTH1-24, as described above. (2) In 5 other patients, hydrocortisone 37.5 mg was administered i.v. in 90 min. Blood samples for beta-endorphin, ACTH and cortisol were drawn at 0, 15, 30, 45, 60, 90, 120 min. One week later, the same patients were given oCRH 100 micrograms i.v. and hydrocortisone 37.5 mg, as described above. ACTH1-24 administration caused a significant (p less than 0.01) decrease in endogenous ACTH but not in beta-endorphin. oCRH injection significantly stimulated both ACTH and beta-endorphin. The response of both ACTH and beta-endorphin was inhibited by exogenous ACTH1-24. There was a potent inhibition by hydrocortisone on both basal and stimulated beta-endorphin, confirming that the feedback mechanism of glucocorticoids concomitantly inhibits ACTH and beta-endorphin. On the other hand, only CRH-stimulated but not basal secretion of beta-endorphin seems affected by ACTH ultrashort feedback, suggesting an intrapituitary regulation.

Central administration of ACTH in rats induces yawning and stretching. In order to study the effects of REM sleep deprivation on ACTH-induced yawning, the peptide was injected immediately after the REM sleep deprivation period or 24 h later. REM sleep deprivation impaired ACTH-induced yawning, but after a 24-hour recovery period, rats displayed a number of yawns similar to those in control animals. Implications for an involvement of dopaminergic and mainly cholinergic systems are discussed.

Tetracosactide (Synacthen, Ciba) increases the concentration of corticosterone in the aqueous humour of the rabbit eye. The maximal increase, about 5-fold of the basal level, occurs with a slight delay of approximately 30 min after the maximally stimulated corticosterone level in blood plasma. Glucocorticoid elevation is paralleled by an increase of protein content and a slight rise of glucose in aqueous. A disruption of the blood-aqueous barrier by ACTH similar to the action of alpha-melanocyte-stimulating hormone is suggested.

The hippocampal electrical activity and behavior of rabbits have been studied in the presence of novel and emotional stimuli. The effects of these stimuli have been recorded in controls and in groups of animals treated with ACTH (4-10)(10 micrograms/100 g, i.m.) and ACTH (1-24) (10 micrograms/100 g, i.m.). Recordings were made immediately and 30 min after injection. ACTH (4-10) injection failed to evoke any significant behavioral or electrical response. Rabbits recorded 30 min after ACTH (1-24) injection showed a reduction of hippocampal RSA (rhythmic slow activity) frequency and behavioral activity. In particular, reduction of exploration, self-grooming, motor activity and approaches to the new object have been observed. Since ACTH is characterized by a corticotropic action these results can be due to corticosteroid stimulation.

Cortisol levels in the serum and cerebrospinal fluid (CSF) were studied in ovariectomized, estrogen-treated monkeys during either prolonged hypercortisolemia or the more transient effects of a bolus injection of cortisol. Control (saline-treated) animals showed the expected diurnal rhythm in serum cortisol, but proportionately more cortisol was present in the CSF when serum levels were high (i.e. in the morning). Prolonged hypercortisolemia for up to 37 days was produced by either thrice daily injections of cortisol itself or single daily injections of ACTH1-24 in the morning. Both treatments produced disproportionately larger amounts of cortisol in the CSF than in the serum, and the CSF/serum cortisol ratio was increased. Furthermore, prolonged ACTH treatment caused a marked elevation in CSF cortisol in afternoon samples taken at 16.30 h compared with those at 10.00 h, in the absence of a similar change in serum cortisol levels. The relative importance of entry and clearance of cortisol in the CSF in these conditions was studied in several ways. 'Free' cortisol levels in serum (determined by equilibrium dialysis) were equal to CSF cortisol levels in control monkeys, but were less than those in the CSF of hypercortisolemic animals. Entry of cortisol into CSF after a bolus injection was rapid, but, unlike serum, CSF cortisol levels did not decline significantly over a 70-min sampling period and the delayed clearance from the CSF could account for some of the effects seen during hypercortisolemia. Neither high levels of prolactin (which is elevated together with cortisol in 'stress'), induced by giving sulpiride, nor treatment with progesterone (which is also bound by corticosteroid binding globulin) altered the distribution of cortisol between blood and CSF. The concentrations of cortisol in the CSF therefore are regulated by factors influencing both its entry and clearance from the cerebral compartment. Neural tissues sensitive to cortisol are thus exposed to levels of this hormone that are both qualitatively and quantitatively different from those expected by direct extrapolation from serum levels.

These studies were designed to: (1) determine the effects of continuous infusion of synthetic ACTH(1-24) on postcastration changes in serum and pituitary LH, FSH and prolactin in the male rat; (2) assess the effects of adrenalectomy on the gonadotropin and prolactin response to ACTH, and (3) test the hypothesis that ACTH may directly (not via adrenal factors) alter gonadotropin secretion at the brain and/or pituitary level. Adult male rats were either orchidectomized (ORX) or orchidectomized-adrenalectomized (ORX-ADX), and were treated continuously for 6 days with ACTH(1-24) (10 micrograms/day) or saline using an osmotic minipump. Animals were killed on day 6 following castration. ACTH treatment reduced serum LH and prolactin levels in ORX rats to mean values +/- SE of 204 +/- 25 and 37 +/- 3 ng/ml respectively, compared to 366 +/- 72 and 62 +/- 7 ng/ml in saline-treated ORX animals. Serum FSH concentrations were not altered by ACTH administration. Pituitary concentrations of LH and FSH, but not prolactin were enhanced by ACTH treatment. Adrenalectomy had no effect on serum and pituitary gonadotropin and prolactin levels, but abolished the effects of ACTH on these parameters. Central (intracerebroventricular) infusion of ACTH(1-24) (6 micrograms/day X 4 days) failed to alter the rise in serum LH in male rats following orchidectomy. Acute treatment with large doses of ACTH of perifused anterior pituitary glands from male rats and chronic treatment with ACTH of enzymatically dispersed anterior pituitary cells from female rats did not influence basal or GnRH-stimulated LH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonatal endocrine status was studied in 14 lambs born 7 days before term, after estrogen injection into the ewes, and in 15 full-term animals. Plasma cortisol and triiodothyronine (T3) levels were depressed during the first hours of life in preterm lambs and plasma reverse T3 levels were significantly higher than in controls. The rise in plasma cortisol levels after Synacthen injection was significantly lowered by prematurity, suggesting reduced sensitivity of the adrenal cortex to ACTH. After ovine TSH injection, plasma thyroxine (T4) levels increased during a shorter period of time in preterm lambs, resulting in a lowered T4 rise; the T3 response was not affected by prematurity. After TRH injection, the rises in plasma T3 and T4 levels were significantly higher in preterm than in full-term lambs, suggesting a pituitary hypersensitivity to TRH linked to prematurity. Moreover it appeared that the response of reverse T3 to TSH or TRH was very weak.

The effect of hypophysectomy on the number of corticosterone receptor sites was investigated in three rat brain regions and was compared with the effect of long-term adrenalectomy. Subsequently, the effect on receptor capacity was measured after the hypophysectomized rats had received as substitution therapy ACTH1-24 and smaller peptide fragments lacking corticotropic activity. All rats (sham and hypophysectomy) were adrenalectomized 24 h prior to sacrifice for depletion of endogenous adrenal hormones and replacement therapy was discontinued at that time. Extensive perfusion with saline was carried out at the time of sacrifice. 3H-corticosterone binding was measured in cytosol by means of an in vitro assay. 2 weeks after hypophysectomy, the apparent maximal binding capacity (Bmax) of the corticosterone receptor system was increased by 60, 36 and 72% in hippocampus, hypothalamus and septum, respectively. The increase in Bmax in the hippocampus of hypophysectomized rats was 30% higher than the increase in animals adrenalectomized 2 weeks previously. Replacement with ACTH1-24 markedly decreased the binding capacity in all brain regions investigated. Replacement with the behaviorally active ACTH4-10 sequence reduced the number of corticosterone receptor sites in the hippocampus by 21%, while the behaviorally inactive ACTH11-24 sequence was ineffective. Des-glycinamide-arginine-vasopressin was also ineffective. There were no alterations in binding affinity for corticosterone in hippocampal cytosol after the surgical procedures or after the different replacement therapies. It is concluded that the neurotropic ACTH4-10 sequence reduces the number of corticosterone receptor sites in the hippocampus of the hypophysectomized rat. The action of ACTH4-10 was specific for the hippocampus and was not observed in other brain regions or plasma transcortin.

ACTH-deprived primary normal adult human adrenocortical cells on the 23rd day in vitro were found to lack any DNA synthetic and proliferative activity, but incorporated uridine-3H and 1-leucine-3H actively. In cortical cells of the same age treated for 2 and 7 days with ACTH1-24 (3 X 10(-6) M) the incorporation of the precursors for RNA and DNA synthesis and the cell proliferation were found to be markedly stimulated. The apparent uptake of leucine-3H was instead reduced in 2-day and, less still, in 7-day treated cells. In parallel with ultrastructural studies, these data suggest that ACTH orderly activates the template activity of chromatin while eliciting the differentiation and hypertrophy of human adrenocortical cells.

The influence of synthetic ACTH1-24 and ACTH1-10 on rat brain RNA was studied. ACTH1-24 (5 IU/100 g s.c.) treatment resulted in a small decrease in labelling of brain stem RNA (-12%), measured 30 min after the injection of [5-3H] uridine (100 muCi; s.c.), without affecting the distribution of radioactivity of the acid-soluble precursor pool. Furthermore, a small and transitional reduction of total brain stem RNA was found. Similar treatment of adrenalectomized rats (36 h after surgery) resulted in a marked increase (+ 40%) in labelling of brain stem RNA, without affecting the distribution of radioactivity in the acid-soluble pool. In both intact and adrenalectomized rats, treatment with ACTH1-24 did not affect RNA of cortex cerebrum and cerebellum. ACTH1-10, a fragment of ACTH without corticotropic activity in vivo, exhibited no effect on brain stem RNA in either intact or adrenalectomized rats, nor did corticosterone (1 mg/100 g) affect adrenalectomized rats.

In the fetal rat, plasma corticosterone concentration (PCC) decreases dramatically on the 20th day of gestation and remains low until birth even if the parturition is delayed until 23.5 days postcoitus. In the normal term newborn, there is an increase of PCC in the 1st h after birth. In postmature newborn rats this increase is not present but PCC can be increased towards normal term values if the adrenal cortex is stimulated with cosyntropin or lysine-vasopressin. This suggests that the lack of elevation of PCC in the postmature newborn is not due to the lack of responsiveness of the adrenal cortex but rather an impairment of the hypophyseal stimulation. The absence of PCC elevation at birth could contribute to the metabolic disorders observed in the postmature newborn rat.

Investigations were carried out on the behavior of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) in essential hypertension (EH) under exogenous administration of synthetic ACTH and insulin. 40 stable EH patients and 21 normal subjects were included in the study. The increase (12-fold basal values) in plasma 18-OH-DOC in normal subjects under Tetracosactide was significantly higher than cortisol (4-fold basal values). Furthermore, insulin hypoglycemia increased 18-OH-DOC levels 5-fold, whilst basal values of cortisol were increased 2-fold. An increase in 18-OH-DOC and cortisol was also observed in EH patients: in the subgroup with normal and low plasma renin activity, however, the rise in these two steroids was significantly lower than in normal subjects both under Tetracosactide and insulin. No significant hormonal modifications were observed after furosemide administration either in the normal subjects or in the EH patients. 18-OH-DOC by itself does not, therefore, appear to play a pathogenetic role in EH.

Tyrosine hydroxylase activity in noradrenergic neurons of the locus coeruleus was found to rise in bilaterally adrenalectomized adult mice, with maximum increase of the enzyme activity (52% above control) occurring 7 days after the surgery. No such increase of tyrosine hydroxylase activity was found in dopaminergic neurons of the substantia nigra. The increase of enzyme activity in the locus coeruleus following adrenalectomy was totally prevented by corticosterone replacement. Moreover, the adrenalectomy effect was abolished by hypophysectomy, indicating the involvement of the pituitary rather than the adrenocortical function. Chronic administration of ACTH (20 IU/kg, i.p., daily) resulted in an increase of tyrosine hydroxylase activity in the locus coeruleus, with a time course and magnitude similar to those found after adrenalectomy. Additionally, the effects of four ACTH analogs were determined. ACTH 1-24 and ACTH 4-10 were as effective as the whole ACTH molecule, whereas ACTH 4-10,7-D-Phe and ACTH 11-24 were ineffective. The effect of ACTH 4-10, a peptide fragment with no adrenocorticotrophic activity, further indicates that glucocorticoids are not involved. From these data, it appears that tyrosine hydroxylase in the locus coeruleus neurons is under the regulatory influence of pituitary ACTH. It remains to be determined whether the hormone can be transported from its pituitary origin to the locus coeruleus and exerts a direct action on the noradrenergic neurons. Regardless of the mechanism, the response of the noradrenergic neurons to pituitary activity may be an important component in physiological adaptation of the central nervous system to chronic stress.

The switch from fetal to adult hemoglobin (Hb) has been investigated in fetal and newborn lambs. In the period between 125 and 145 days gestation, the proportion of fetal cells containing adult Hb increased significantly in catheterized fetuses. This increase paralleled the rise in fetal plasma cortisol under several different experimental conditions. However, infusion of synthetic adrenocorticotropin (ACTH1-24) into the fetus, which increased fetal plasma cortisol concentrations to levels slightly higher than observed at term, did not bring about any increase in the proportion of cells containing adult Hb. Increasing fetal plasma prolactin (PRL) concentration either by infusion of PRL or by stimulating endogenous PRL release with thyrotropin-releasing hormone (TRH) did not influence the time or rate of the switch. The fetal reticulocyte count was significantly higher in blood samples taken at the time of catheterization in fetuses of 103-115 days gestation than at 116-128 days gestation. In one repeatedly sampled fetus of adult type Hb AB, Hb C was observed at the time when the fetus was anemic. At birth the number of cells containing adult Hb was significantly higher in catheterized lambs than in nonoperated controls.