ACTH In Rats Research Articles

Nycterohemeral difference in inhibition of stress-induced ACTH in adrenalectomized rats

To determine the interactions among the determinants of ACTH secretion, we examined the influence of circadian rhythmicity on glucocorticoid suppression of ACTH. Adrenalectomized rats were injected with the same amount of corticosterone at 0900 and 1800 h, and plasma ACTH concentrations were determined under basal conditions and after a standard ether stress. At 0900 h, corticosterone suppressed both basal and stress-induced plasma ACTH concentrations. At 1800 h, the same treatment suppressed basal ACTH secretion but not the stress-induced rise. Although the same amount of corticosterone was injected at both times of day, the plasma corticosterone concentration 5 min after injection was higher at 1800 h than at 0900 h. This study indicates that there is a nycterohemeral difference in feedback suppression of stress-induced ACTH secretion by a given dose of corticosterone. The daily variation in feedback inhibition may be due to the additive effect of the evening surge stimulus and the stress stimulus that together override the feedback signal.

D-Ala2-Met-enkephalinamide, a potent opioid peptide, alters pituitary-adrenocortical secretion in rats.

This study was designed to examine in rats the effects of systemic administration of a potent met-enkephalin analog, D-Ala2-met-enkephalinamide (DALA), on plasma ACTH and corticosterone secretion under basal conditions. Plasma radioimmunoassayable ACTH and corticosterone showed biphasic responses to intraarterial administration of DALA in chronically cannulated, conscious, freely moving, nonstressed animals. Both plasma ACTH and corticosterone increased at 5-10 min and decreased at 45-65 min after the administration of DALA (500 micrograms/kg) compared with either the basal concentrations or the corresponding responses in saline vehicle-injected control rats. Pretreatment with the specific opioid receptor antagonist naltrexone (2 mg/kg, intraarterially) completely prevented the DALA-induced increase in the plasma ACTH concentration and blunted the DALA-induced increase in the plasma corticosterone concentration. Naltrexone also increased plasma concentrations of ACTH and corticosterone above basal levels at a later time in DALA-treated animals. The DALA-induced increase in plasma corticosterone appears to be mediated by ACTH, mice DALA failed to increase plasma corticosterone in either hypophysectomized or dexamethasone-suppressed rats. DALA decreased the adrenocortical responsiveness to ACTH in hypophysectomized, but not in untreated or dexamethasone-suppressed, rats. Naltrexone increased the adrenocortical response to ACTH in DALA-treated rats. These data suggest that endogenous opioid peptides regulate pituitary-adrenocortical secretion under basal conditions by modifying both ACTH secretion and the glucocorticoid response to ACTH.

Synthesis and biological properties of ovine corticotropin-releasing factor (CRF)

The 41-residue sequence of recently identified ovine corticotropin-releasing factor (CRF) was assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptide was purified by gel filtration and reverse-phase, medium pressure, followed by high-performance liquid chromatography (HPLC). In addition to the usual criteria, the homogeneity of the final material, obtained in 7% yield, was assessed by the isolation and examination of cyanogen bromide cleavage and tryptic digestion fragments by HPLC and amino acid analysis. The synthetic 41 amino acid CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column, the responses being related to the log-dose of CRF in the range of 0.05–125 ng/ml. The synthetic peptide also augmented in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal, as assessed by the measurement of serum corticosterone. The data indicates chemical purity and high biological activity of synthetic material.

Sensitivity of the retinal pigmented epithelium to the melanotropic action of acth in normal and mutant rats with hereditary dystrophy of the retina

Sensitivity of the retinal pigmented epithelium to the melanotropic action of acth in normal and mutant rats with hereditary dystrophy of the retina

Impairment of renal compensatory hypertrophy by hypothyroidism in the rat

Renal compensatory hypertrophy (RCH) occurs in hypothyroid rats, but it is impaired when compared to RCH found in euthyroid controls. It is due to cellular hypertrophy as the DNA content does not change and the Protein/DNA ratio increases in the compensating kidney. RCH is enhanced by thyroxine (T4) with a rise in the DNA content of the compensating kidney, but the Protein/DNA ratio does not change indicating that hypertrophy is as important as hyperplasia. Corticotrophin (ACTH) given to eu and hypothyroid rats enhances RCH with an increase in the protein content of the compensating kidney without any change in its DNA content. In the hyperthyroid rats, the enhanced RCH is not further increased by ACTH and the rise in the kidney DNA content elicited by T4 is suppressed by ACTH. The Protein/DNA ratio is increased by ACTH in hypo, eu and hyperthyroid rats. The renotrophic action of ACTH is due to hyper-adrenocorticism: it is related to an increased plasma testosterone level and to a disturbed Na +, K + and glucose metabolism.

Effects of total saponins extracted from several crude drugs on rat adrenocortical hormone secretion

Effects of total saponin preparations extracted from panacis japonici rhizoma, saponariae radix alba, sapindus pericarpium, senegae radix, thea semen, bupleuri radix, hippocastani semen, and styrax pericarpium, which contain oleanane-type aglycone on plasma levels of ACTH, corticosterone, and glucose in rat were determined by the radioimmunoassay method, the competitive protein binding method, and by the glucose oxidase method, respectively. All of saponins tested were found to increase plasma ACTH, corticosterone, and glucose at 30 minutes after the intraperitoneal administration. ED50 Value for the corticosterone secretion-inducing activity of saponins was estimated. Among 8 saponins, styrax saponin was the most effective (0.7 mg/kg) and panacis japonici saponin was the least (54 mg/kg). The increase in plasma glucose by these saponins was similar to that of isolated saikosaponin a or d but not to that of ginseng saponin. Hemolytic activities of these saponins were also determined, and the order of the activity was almost parallel to the order of ED50.

High molecular weight peptide with corticotropin-releasing factor activity from porcine hypothalami.

The presence of a corticotropin-releasing factor (CRF) behaving as a peptide with a molecular weight of about 5000 was established after purification of porcine hypothalamic extracts by gel filtration on Sephadex G-25 and then on Sephadex G-50. Purified CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column. A linear logarithmic dose-response relationship existed between 50 and 200 micrograms of CRF preparations per ml and the total amount of ACTH released by the superfused pituitary cells. The pituitary ACTH response to CRF in the pituitary quarters system was also approximately linearly related to the logarithm of the dose of CRF. CRF also stimulated in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal. CRF activity was labile to digestion with trypsin and chymotrypsin and was partially destroyed by pepsin. The evidence indicates that CRF of porcine origin is a polypeptide of a higher molecular weight than previously assumed.

654 EFFECT OF PERORAL ADMINISTRATION OF ACTH ON PLASMA CORTICOSTERONE LEVELS IN SUCKLING RATS: TIME AND DOSE DEPENDENCY

The presence of ACTH in rats' milk prompted us to explore whether ACTH given perorally (p.o.) to suckling rats has an effect on plasma corticosterone (C). After this was demonstrated in preliminary experiments, we studied the time dependency of the ACTH effect. In suckling Sprague-Dawley rats (11-14 days old), starved for 2 hours and then fed ACTH1-39 (Armour, 0.50 U/g BW in water), plasma C peaked at 30 minutes and remained significantly higher until 180′ when compared with water-fed controls.To determine the lowest effective dose, varying amounts of ACTH were given p.o. (0.06; 0.13; 0.25; 0.50 and 1.00 U/g BW) and plasma C measured 2 hours later. The two lower doses evoked an insignificant change; a significant increase was seen using the 0.25 U dose. Its effect was the same as that of the higher doses used.These studies thus demonstrate the acute effect of p.o. ACTH in suckling rats. Further studies are needed to evaluate the physiologic role of ACTH normally present in rats' milk.

Growth hormone stimulates adrenal steroidogenesis in the fetus.

The 'surge' of corticosteroid in fetal plasma during late gestation has been implicated in the initiation of parturition and the maturation of enzyme systems in organs such as the lung, liver, adrenal medulla and thyroid. But in all species studied, the mechanism responsible for increased secretion in the fetus remains unclear. The hormone adrenocorticotropin (ACTH) is well established as the primary regulator of adrenocortical cellular growth and secretory function during fetal and adult life. However, no increase in fetal plasma ACTH has been observed before the corticosteroid surge in sheep or humans, although differential responsiveness of the fetal adrenal cortex to ACTH at various gestational ages and a possible role of extra-adrenal inhibitory factors have been proposed. The possible steroidogenic effect of alpha-melanocyte-stimulating hormone (alpha MSH) on fetal adrenocortical function is controversial, and we could not demonstrate any such action in fetal lamb or rabbit. Prolactin and growth hormone (GH) potentiate the steroidogenic effect of ACTH in adult rats but prolactin had no such effect in fetal lamb. We have investigated the steroidogenic properties of GH, and report here that it stimulates adrenal steroidogenesis in the fetal but not maternal rabbit in vitro and in fetal but not maternal sheep in vitro as well as in vivo.

Effects of raphe lesions on circadian ACTH, corticosterone and motor activity rhythms in free-running blinded rats

Blinded female rats underwent additional midbrain raphe lesions, in order to explore the role of the raphe in the organization of endogenous circadian rhythms for ACTH, corticosterone (B) and motor activity (MA). Amplitudes and mean levels of rhythms were depressed for ACTH and MA, with persistent free-running circadian periodicity for MA and, in several rats, for ACTH and B as well. Other rats exhibited split circadian and ultradian rhythmicity for ACTH and B, whereas others again displayed no detectable ACTH rhythmicity. These results are discussed in the light of the structure of circadian pacemaker systems.

ACTH in vivo stimulation of the synthesis of a specific mitochondrial protein in the rat

Protein markers induced by hormones are the necessary probes to study hormone regulation of gene expression. We recently showed that ACTH was able to induce one of these markers in the cytosol of the rat adrenal ( Dazord et al, Biochem. J. 176 , 233–239, 1978 ). In this paper we described another protein marker whose localization is mitochondrial and whose MW is 134 K. Maximal stimulation is seen 2 hours after ACTH injection. Actinomycin D injected 30 min before or 1 hour after the hormone blocks the stimulation. In hypophysectomized rats both ACTH and cyclic AMP are able to stimulate the synthesis of this protein.

Effect of escin on adrenocorticotropin and corticosterone levels in rat plasma.

The effect of β-escin on plasma adrenocorticotropin (ACTH) and corticosterone levels in rat plasma was determined by radioimmunoassay and by the competitive protein binding method. Intraperitoneal administration of β-escin induced increases of plasma ACTH and corticosterone in rats. The increase of plasma corticosterone was accompanied by a transient increase of plasma glucose and a transient decrease of plasma immunoreactive insulin. Hexamethonium partially suppressed the escin-induced corticosterone secretion, but diphenhydramine did not.

Serotoninergic system and circadian rhythms of ACTH and corticosterone in rats.

The circadian rhythms of plasma ACTH and corticosterone and of locomotor activity were explored in chronically cannulated female rats, after elimination of serotoninergic (5HT) innervation of the SCN (suprachiasmatic nuclei) either by stereotaxic lesion of the median and dorsal midbrain raphe nuclei (RX) or by local injection of SCN with the neurotoxin 5,7-dihydroxytryptamine (5,7DHT). Completeness of 5HT denervation was checked on serial sections of the hypothalamus either by the Falk-Hillarp technique or by radioautography. Neither lesion eliminated the intrahypothalamic 5HT system, which, however does not take part in the 5HT innervation of the SCN. In both experimental series, the circadian rhythms of the three parameters investigated were maintained in unchanged phase relationships compared to the sham-lesioned controls, and with respect to the photoperiod (12 light-12 dark). However, the estimated amplitudes of the ACTH rhythms dropped by 43% (RX) to 47% (5,7DHT) and their mean levels by 44% (RX) to 60% (5,7DHT), whereas the corticosterone rhythm displayed normal amplitude and its mean level rose by 24% (RX) or 38% (5,7DHT). In regard to locomotor activity rhythm, the most noticeable alteration was a 25-55% increase in the light-phase activity of both experimental groups with a correlative increase in the L/D activity ratio. The essential role of 5HT innervation of the SCN therefore seems to be to facilitate circadian control of the ACTH rhythm.

Effect of various antiinflammatory drugs on plasma acth and cortisol levels in rats

Various antiinflammatory drugs have been injected i.p. to rais at doses equivalent to the maximal doses used in humans and plasma ACTH levels have been measured by a radio-bioassay. After acetylsalicylic acid, diclofenac and ibuprofen administration, a drop in plasma ACTH levels is observed, without a concomitant change in plasma cortisol levels. Small decrease in plasma ACTH is observed with ketoprofen, pirprofen and oxyphenbutazone; no change or a small increase is seen after azapropazone, naproxen, phenylbutazone and benzydamine. Repeated administration (five times 10 mg/kg every 24 hours) of acetylsalicyclic acid brings about a steady decrease of ACTH, which resumes its value after 48 hours. No change in cortisol levels are seen concomitantly to the short term drop in ACTH levels and small changes are seen in repeated administration of acetylsalicylic acid.

Acute Self-Suppression of Corticosteroidogenesis in Isolated Adrenocortical Cells*

The relation between steroidogenesis induced by ACTH and that induced by exogenous concentrations of glucocorticoids was studied in isolated adrenocortical cells. Exogenous corticosterone and cortisol, in concentrations within the production capacity of the adrenal gland, suppressed steroidogenesis induced by ACTH in rat and beef cells, respectively. The precursors pregnenolone and progesterone enhanced steroidogenesis in both rat and beef cells. Aldosterone in rat cells and 17 beta-estradiol in rat and beef cells had little if any effect on steroidogenesis. Either suppression or stimulation by exogenous steroids was acute, that is, after 2-h incubation for rat cells and 1-h incubation for beef cells. A direct suppressive action of end product glucocorticoids is indicated. This observed self-suppression of adrenocortical cells suggests the existence of a mechanism for the find adjustment of steroidogenesis that operates in addition to the classical control exerted by the anterior pituitary.

Adrenocorticotropin and Melanocyte-Stimulating Hormone in the Brain*

We employed several ACTH immunoassays using antisera with affinities for differing regions of the molecule as well as an ACTH bioassay to further study the distribution and character of ACTH in rat, cat, and monkey brain. Acid extracts of brain tissues were examined for N-terminal and midportion ACTH concentrations and MSH content. In each species, ACTH concentrations were greatest in the hypothalamus, with lesser concentrations in more peripheral regions. Hypophysectomy, dexamethasone suppression, or adrenalectomy had only minor effects on ACTH content and distribution. In all species, the concentrations of N-terminal ACTH immunoreactivity were several times greater than those of midportion immunoreactivity. aMSH was present in low concentrations and could account for only a small fraction of N-terminal immunoreactivity. ACTH bioactivity was present only in central areas and correlated well with midportion immunoreactivity. A sample of larger molecular weight ACTH (31,000 daltons) reacted approximately 1...

EVALUATION OF A RADIOIMMUNOASSAY FOR PLASMA ADRENOCORTICOTROPHIN

A radioimmunoassay for plasma ACTH has been described and evaluated. Rabbit antiserum produced by immunization with [Asp25, Ala26, Gly27,]-alphah-corticotrophin-(1--28)-octacosa-peptide (a sequence analogue of alphah1--28-ACTH) bovine gamma globulin conjugate was used. The antiserum is specific for the NH2-terminal portion of the ACTH molecule and cross-reactivity of human, porcine and rat ACTH in the system has been demonstrated. Reasonable agreement was found between estimates obtained by bioassay and radio-immunoassay of the ACTH content of rat pituitary gland incubation media, indicating a close relationship between the sequence of ACTH recognized by the antibodies and the sequence possessing the steroidogenic activity. Measurement of the amount of ACTH in the plasma required the preliminary extraction and concentration of the hormone. Over a range of concentrations between 3.5 and 3600 pg/ml, extraction recovery was independent of the initial concentration of ACTH in the plasma. Extraction gave rise to no changes in the immunological properties of standard ACTH. The concentration of immunoreactive ACTH in rat plasma was 48 +/- 3.6 (S.E.M.) pg/ml in the morning and 106 +/- 9.9 pg/ml in the afternoon. Exposure to either for 5 min and subsequent laparotomy gave rise to a significant increase in the concentration of immunoreactive ACTH in the plasma. The resting level of ACTH and the ACTH response to stress were both significantly higher 1 and 7 days after adrenalectomy. Intravenous injection of a hypothalamic extract elicited a considerable rise in the concentration of immunoreactive ACTH in the plasma, but no response was seen after oral administration of this partially purified extract. The sensitivity, precision and specificity of this ACTH radioimmunoassay make it a useful tool for studying pituitary--adrenal physiology.

Adrenocortical function in response to myocardial necrosis in exercise-trained rats

Plasma corticosterone concentrations and in vitro adrenal secretion of corticosterone was determined in exercise-trained rats. Virgin, male rats, 100 days of age, were trained for an 11-wk period by treadmill running. Following the training program, rats were subjected to two subcutaneous injections of l-isoproterenol 24 h apart and killed 24 h after the second injection. All exercise-trained rats survived isoproterenol treatment, while 44% of the control rats died. Plasma corticosterone concentrations were elevated only in exercise-trained rats treated with isoproterenol. Control rats treated with isoproterenol had plasma corticosterone concentrations similar to that in control and exercise-treated rats given placebo injections. Exercise training reduced adrenocortical responsiveness to ACTH in vitro, but isoproterenol treatment increased in vitro responsiveness to ACTH in exercise-trained and control rats. Total unstimulated corticosterone secretion rates in vitro were similar. The reason for better rat survival in exercise-trained rats is unknown; however, improved energy metabolism, depressed aldosterone secretion, or both are suggested as reasons for the better survival of exercise-trained rats.

Adrenal Steroidogenic Actions of Cyclic Nucleotide Derivatives in the Rat

Fifteen 3',5'-cyclic nucleotides and related compounds were studied for ability to mimic the steroidogenic action of ACTH in rats in which secretion of ACTH and corticosterone were suppressed by treatment with betamethasone, or by hypophysectomy. Subcutaneous administration of 8-chloro-cAMP, at doses of 40 mg/kg or greater, elicited the secretion of corticosterone to normal plasma levels in both betamethasone-treated and hypophysectomized animals. Cyclic AMP, dbcAMP, 8-methylthio-cAMP, 8-hydroxy-cAMP and the 6-chloro-8-aminopurine cyclic ribotide analog of cAMP also displayed steroidogenic activity in the betamethasone-treated rat; cGMP, 8-bromo-cGMP and 8-benzylthio-cGMP were inactive. Each of the steroidogenic derivatives of cAMP also displayed ability to activate steroidogenesis in isolated rat adrenal cells. These experiments demonstrate that various derivatives of cAMP mimic the adrenal steroidogenic action of ACTH, in vivo. Structure-activity comparisons support a steroidogenic mechanism involving direct activation by the nucleotides of cAMP-dependent protein kinase of the adrenal cortex.

ACTH-induced excessive grooming in the rat: Latent activity of ACTH 4–10

Intraventricular injection of ACTH or peptides of the N-terminal part of ACTH in rats induces excessive grooming behavior. The present study deals with the grooming-induction potencies of short peptides sharing sequences with ACTH4–10 (H-Met — Glu — His — Phe — Arg — Trp — Gly-OH). Although ACTH4–10 itself is inactive, ACTH4–7 and [d-Phe7]ACTH4–10 are capable of inducing excessive grooming behavior. From the ineffectiveness of a variety of other oligopeptides within the ACTH4–10 sequence, it was concluded that ACTH4–7 is the shortest sequence to induce this behavioral response as was found in studies on the effect of these peptides on the maintenance of a conditioned avoidance response. The present data underscore the need of detailed information on the configuration of peptides at the CNS receptor site.