In vitro induction of tyrosinase activity in goldfish (Carassius auratus L.) integument by ACTH and dibutyryl-cAMP.

Tyrosinase was first detected in melanoblasts by the DOPA-oxidase reaction in the presence of catalase in explants of goldfish integument after 12 hr culture with either ACTH (1IU/ml) or DB-cAMP (0.1mM). Melanin did not appear in the new melanocytes until 24 hr. The data indicate that the release of cAMP within the melanoblast in response to ACTH treatment is rapid and the tyrosinase in the melanoblast is released from inhibition and/or activated at least 12 hr prior to melanization of premelanosomes.     

Circadian effect of ACTH 1-17 on mitotic index of the corneal epithelium of BALB/C mice

The objective was to determine the effect of ACTH 1-17, an adrenocorticotropin analogue, on the mitotic index in the corneal epithelium of mice standardized in 12 hr of light alternating with 12 hr darkness. A question asked was whether the time of administration along the 24-hr time scale influenced any response found. The findings showed that ACTH 1-17 could, depending upon when it was administered, bring about a statistically significant decrease, an increase or even no such change in the mitotic index. The greatest responses found were increases, especially when ACTH 1-17 was administered during the dark span. Also the time after injection when the responses occurred varied. The greatest response recorded was at 12 hr after injection when ACTH 1-17 was given at 2 hr into the dark with a 641% and a 718% increase with a low (0.02 IU/kg) and a higher (20 IU/kg) dose, respectively. A 3-way analysis of variance supported the conclusion that the kind-of-treatment, time-of-treatment and treatment-to-kill interval (sampling time) are important factors when determining any response to ACTH 1-17 on the mitotic index.     

Circadian-Stage Dependent Acth1-17 Effect on DNA Synthesis in Murine Duodenum, Colon and Recum

The objective was to determine the effect of adrenocorticotropin (ACTH 1-17) on the incorporation of [³H]TdR into DNA (DNA synthesis) in the duodenum, colon and rectum of CD2F, mice standardised to 12hr of light alternating with 12hr of darkness. A question asked was whether the difference in times of administration along the 24 -hr time scale influenced any response found. The response was complex as ACTH 1-17 was capable of bringing about statistically significant increases in the incorporation of [³H]TdR into DNA at certain times, decreases at other times, or no response at still another time. A generalization that can be made from all these tissues is that ACTH 1-17 had a greater influence in bringing about a decrease in DNA synthesis when it was administered around the time of transition from dark to light. A similar finding was made earlier for the ACTH 1-17 effect upon the tongue, esophagus and stomach.

A 2- and 3-way analysis of variance supports our conclusion that the kind-of-treatment, time-of-treatment and the interval-to-kill (Sampling time) as well as their interactions are important factors when determining any response of ACTH 1-17 or placebo.     

Mutations at the W locus affect survival of neural crest-derived melanocytes in the mouse

The development of melanoblasts in normally pigmented and dominant spotting (W) embryos was followed by in situ hybridisation to TRP-2/DT mRNA, which labels migratory melanoblasts from 10 days post coitum. Numerous melanoblasts migrate to the inner ear around 11 days. In contrast, few migratory melanoblasts are associated with the eye or skin at this stage and melanoblast distribution within the trunk and tail is patchy. The distribution of melanoblasts in 10.5–11-day-old W^v/W^v, W^sh/W^sh and W⁴¹/W⁴¹ mutants was similar to that in controls but melanoblast density was lower and by 12 days was severely reduced. These results suggest that mutations of the c-kit receptor tyrosine kinase encoded at the W locus do not alter early migration or differentiation of melanoblasts but severely affect melanoblast survival.     

Effect of ACTH1-24 and ACTH4-10 on distribution of 3H-noradrenaline in the brain of adrenalectomized rats.

Painful stimuli led to a decrease of the radioactive catecholamine pool in adrenalectomized rats. Intraventricular administration of both tritiated noradrenaline and ACTH produced a greater decrease of the labelled catecholamine pool than in the control adrenalectomized rats in 12 to 18 hr following injection. Blocking of monoamino-oxidase activity or biosynthesis by systemic administration of Pargyline or alpha-methyl-tyrosine did not prevent the effect of ACTH on brain catecholamines. It is concluded that ACTH exerts a direct influence on the brain catecholaminergic system and that this effect might be involved in ACTH dependent behavioural responses.     

Immunoneutralization of corticotropin-releasing hormone prevents the diurnal surge of ACTH

To determine whether CRH is required for the evening rise in plasma ACTH, rats were injected at 0800 hr with CRH antiserum (anti-CRH) or normal rabbit serum (NRS). Blood samples were taken through venous catheters at 0800 hr before treatment and at 1300, 1700, and 2100 hr. Plasma was assayed for immunoreactive ACTH and corticosterone. There was no significant difference in pretreatment values between the two groups. Immunoneutralization of CRH abolished the rise in plasma ACTH seen at 1700 hr in the NRS group but had little effect on earlier levels. The diurnal elevation in plasma corticosterone continued after anti-CRH treatment, but peak levels occurred earlier. Plasma ACTH and corticosterone were significantly correlated at the time of the diurnal surge, but not at 0800 hr or 1300 hr in the NRS controls or at any time point in the anti-CRH group. These results suggest that CRH is required for the diurnal surge of plasma ACTH. They also confirm previous observations by others that the adrenal cortex does not require active CRH or a diurnal surge of ACTH in order to exhibit a significant diurnal increase in secretion of corticosterone, and that factors other than CRH may be relatively more active than CRH in regulation of ACTH secretion during the time of circadian inactivity.     

Plasma adrenocorticotropic hormone and cortisol in pigs: effects of time of day on basal and stressor-altered concentrations

An initial study was conducted to establish the presence in plasma of diurnal rhythms of immunoreactive porcine adrenocorticotropic hormone (pACTH) and cortisol in castrated male pigs (barrows). Fourteen barrows with jugular catheters were bled at 6-hr intervals for 24 hr. Significant changes in plasma pACTH were evident with peak levels (61 +/- 6 pg/ml) at 0100-0700 hr and a trough (38 +/- 4 pg/ml) at 1900 hr. Changes (P less than 0.05) in plasma cortisol were also present in barrows with a peak (44 +/- 6 ng/ml) at 0700 hr and a trough (21 +/- 5 ng/ml) at 1900 hr. Plasma norepinephrine and epinephrine were measured at the same time intervals and did not differ among hours. In these unstressed pigs the ratio cortisol/log10pACTH at 0700 hr (25.3 +/- 3.0) was greater than the ratio at 1900 hr (12.9 +/- 2.7). Sequential blood samples were subsequently taken on four of the barrows 12 and 26 days later. Plasma pACTH was variable among pigs and did not differ among hours. Plasma cortisol on both dates was greater (P less than 0.05) in the morning (0100 or 0700 hr) than at 1900 hr. The ratio cortisol/log10pACTH at 0700 hr was repeatedly greater than at 1900 hr. A second study was conducted to determine whether plasma pACTH and cortisol responses to mild (32 degrees C for 2 hr) or strong (20-min restraint) stressors were dependent on the time of day of stressor application (0800 hr, AM; 1600 hr, PM). Response-associated parameters (maximum concentration, maximum incremental concentration, and integrated response) for pACTH and cortisol did not differ between AM and PM. However, a qualitative difference existed between the AM and PM plasma pACTH responses to restraint +32 degrees C wherein the AM response consisted of a single prolonged surge, and the PM response of an initial major peak followed by a second significant minor peak. A suggested explanation is that the initial 20-min restraint stressor potentiated the hypothalamic-hypophyseal response to 32 degrees C. These studies are the first direct measurements which suggest the presence of diurnal changes in plasma ACTH and cortisol in barrows. The studies also indicate for barrows an absence of diurnal changes in plasma epinephrine and norepinephrine. The responsiveness of the pituitary-adrenocortical axis to stressors did not exhibit quantitative diurnal changes at the time periods measured. However, it is hypothesized that the repeatable AM-PM difference in the ratio cortisol/log10ACTH reflects a diurnal change in adrenal responsiveness to ACTH in unstressed pigs.     

Circadian-Stage Dependent Acth1-17 Effect on DNA Synthesis in Murine Duodenum,Colon and Recum

The objective was to determine the effect of adrenocorticotropin (ACTH 1-17) on the incorporation of [³H]TdR into DNA (DNA synthesis) in the duodenum, colon and rectum of CD2F, mice standardised to 12hr of light alternating with 12hr of darkness. A question asked was whether the difference in times of administration along the 24 -hr time scale influenced any response found. The response was complex as ACTH 1-17 was capable of bringing about statistically significant increases in the incorporation of [³H]TdR into DNA at certain times, decreases at other times, or no response at still another time. A generalization that can be made from all these tissues is that ACTH 1-17 had a greater influence in bringing about a decrease in DNA synthesis when it was administered around the time of transition from dark to light. A similar finding was made earlier for the ACTH 1–17 effect upon the tongue, esophagus and stomach.

A 2- and 3-way analysis of variance supports our conclusion that the kind-of-treatment, time-of-treatment and the interval-to-kill (Sampling time) as well as their interactions are important factors when determining any response of ACTH 1-17 or placebo.     

Dexamethasone suppresses ACTH release without attenuating pituitary cyclic AMP response to stress in vivo

Dexamethasone, a synthetic glucocorticoid, has been shown to decrease basal and stress-elevated levels of the pituitary hormone ACTH. Glucocorticoids are known to bind to multiple sites within the brain and pituitary and it is not known which site(s) is most important in mediating the observed inhibition of ACTH release. At the level of the corticotroph, there is contradictory data from in vitro studies regarding whether dexamethasone acts proximal or distal to the formation of the cyclic AMP second messenger that has been shown to be involved in CRF-stimulated ACTH release. In the present report, we have examined the effects of dexamethasone pretreatment on stress-induced elevations in pituitary cyclic AMP and the release of ACTH in vivo. Acute stress (15 min of intermittent footshock) elevated levels of pituitary cyclic AMP and plasma ACTH consistent with previous studies. Dexamethasone administration (0.4 mg/kg 24 hr prior to sacrifice plus 0.2 mg/kg 2 hr prior to sacrifice) inhibited stress-induced elevations in plasma ACTH but did not affect pituitary cyclic AMP response to acute stress. These findings suggest that dexamethasone inhibits the release of ACTH via an action distal to the generation of cyclic AMP.     

Pharmacological aspects of the influence of melanocortins on the formation of regenerative peripheral nerve sprouts

Adrenocorticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate the initial sprouting response in the crushed rat sciatic nerve. In this report a detailed analysis of the neurotrophic action of Org.2766 [a degradation resistant ACTH(4-9) analog] and alpha-MSH is described. Org.2766 treatment results in enhanced numbers of outgrowing sprouts in the damaged nerve. The growth velocity of the sprouts is not affected. The peptide effect is dose-dependent. A single peptide injection administered immediately following the crush stimulates the formation of sprouts significantly. Continued high blood levels of Org.2766 are probably not critical for the neurotrophic effect of these peptides, since a more moderate dosing protocol (injections given every 48 hr) was more effective than more frequent injections (injections given every 12 hr). The present results further the understanding of the mode of action of ACTH/alpha-MSH-like peptides and underscore the necessity to test a wide range of doses and injection protocols to avoid false negative results in clinical work being planned to start in the near future.     

Effect of dexamethasone on steroidogenesis and on adrenocortical cyclic AMP and cyclic GMP responses to ACTH

The inhibitory action of dexamethasone on the adrenal steroidogenic response to ACTH was confirmed by im administration of graded doses (5, 10 and 30 ng) of synthetic beta 1-24 ACTH to young adult male rats which had received dexamethasone (0.1 mg/100 g bw) 4 hr prior to sacrifice. Following this, kinetic studies were performed by measuring plasma corticosterone, adrenocortical cyclic AMP and cyclic GMP before and 4, 12 and 30 min after administration of either 10 or 30 ng of ACTH. These doses were selected because their effects could be either completely or partially inhibited by dexamethasone. In rats without dexamethasone all the doses of ACTH which were checked induced an increase in both corticosterone and cyclic AMP and a decrease in cyclic GMP. With the smallest dose of ACTH the earlier administration of dexamethasone resulted in complete suppression of both the steroidogenic response and the cyclic AMP response. With the largest dose of ACTH both responses were diminished. In dexamethasone-treated rats the decrease in cyclic GMP was significantly less pronounced 4 min after ACTH than it was in non-treated rats. These results support the view that cyclic AMP and cyclic GMP might both be concerned with the mechanism of acute adrenal steroidogenesis.     

Effects of posttrial hormone injections on memory processes

This experiment examined the effect on memory of posttrial injections of epinephrine, norepinephrine, ACTH, growth hormone, vasopressin and corticosterone. Rats were trained with a weak footshock (0.7 mA, 0.35 sec) in a one-trial inhibitory (passive) avoidance task. The animals received subcutaneous injections of one of the above hormones or saline immediately after training. On a retention test 24 hr after training, animals which received ACTH (0.03 or 0.3 IU/rat), epinephrine (0.1 mg/kg) or norepinephrine (0.1, 0.3 or 1.0 mg/kg) had retention performance which was significantly better than that of saline control animals. A higher posttrial ACTH dose (3.0 I.U./animal) impaired later retention performance. ACTH (0.3 I.U./animal) and norepinephrine (0.3 mg/kg) injections administered 2 hr after training had no significant effect on retention. Immediate posttrial injections of vasopressin (dose range 0.001–1.0 I.U./animal), growth hormone (0.5–1.0 mg/kg), or corticosterone (0.01–4 mg/kg) did not significantly enhance retention. These findings indicate that epinephrine, norepinephrine, and ACTH injections can enhance memory processes if the hormones are injected shortly after training. Such results are consistent with the view that hormonal consequences of an experience, particularly epinephrine, norepinephrine and ACTH release, may normally have a modulatory influence on memory processes in untreated animals. In addition, it is therefore possible that other posttrial treatments which enhance or impair later retention performance may act through hormonal mechanisms.     

The secreted metalloprotease ADAMTS20 is required for melanoblast survival

ADAMTS20 (Adisintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kit(tm1Alf)/+ and bt/bt;Kitl(Sl)/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases.     

Daily profiles of aldosterone levels during normal, high and low sodium intake in patients with essential hypertension.

The episodic secretion of aldosterone depends on the dietary sodium intake, alterations in posture and follows ACTH circadian rhythm. Aldosterone daily profiles have been studied in 23 supine essential hypertensive patients on normal sodium intake. Secretory pulses at a frequency of two to five pulses per 12 hr have occurred, independent of PRA levels. Among 13 patients with normal PRA two lost pulsatility when sodium was loaded (10 g/24 hr) and the same happened with two others on sodium restricted diet (2 g/24 hr). These results suggest a profound effect of dietary sodium intake on the pulsatile pattern of aldosterone secretion, particularly in normal PRA essential hypertension.     

Altered expression of adrenocorticotropic hormone in the epileptic gerbil hippocampus following spontaneous seizure

We investigated the temporal alterations of adrenocorticotropic hormone (ACTH) immunoreactivity in the hippocampus after seizure onset. Expression of ACTH was observed within interneurons in the pre-seizure group of seizure sensitive gerbils, whereas its immunoreactivities were rarely detected in seizure resistant gerbil. Three hr after the seizure, ACTH immunoreactivity was significantly increased in interneurons within all hippocampal regions. On the basis of their localization and morphology through immunofluorescence staining, these cells were identified as GABA_A α1-containing interneurons. At the 12 hr postictal period, ACTH expression in these regions was down-regulated, in a similar manner to the pre-seizure group of gerbils. These findings support the increase in ACTH synthesis that contributes to a reduction of corticotrophin-releasing factor via the negative feedback system which in turn provides an opportunity to enhance the excitability of GABAergic interneurons. Therefore, ACTH may play an important role in the reduction of excitotoxicity in all hippocampal regions. [BMB Reports 2013; 46(2): 80-85]     

Iron-induced accumulation of hepatic metallothionein: the lack of glucocorticoid involvement

The process(es) by which parenteral iron effects the accumulation of hepatic metallothionein (MT) is not known. The present study examined glucocorticoids as potential mediators of this process. Chicks were given either one injection (ip) of iron (+1FE) at 10 mg Fe/kg, two injections of iron (+2FE) given 24 hr apart, or a single injection of saline. Plasma corticosterone was evaluated at various times following the last injection. Plasma corticosterone increased approximately 50% following +1FE but more than 200% at 2 and 4 hr following a second injection of iron (+2FE). Plasma zinc showed a transient increase followed by a considerable depression. Coincidentally, the accumulation (determined at 24 hr) of zinc MT in liver of +2FE chicks was three times higher than that of +1FE chicks. In another experiment, markedly greater changes, at similar time intervals, in plasma corticosterone were effected by multiple subcutaneous injections of adrenocorticotropic hormone (ACTH) (either 5 IU ACTH or 20 IU ACTH/kg). Subsequent analysis of hepatic zinc MT showed only minor changes as a result of ACTH injections. These results indicate that a change in the plasma glucocorticoid corticosterone is not a primary component in the process(es) by which parenteral iron effects an increase in hepatic zinc MT.     

Cocaine-induced stimulation of the rat hypothalamic-pituitary-adrenal axis is progressively attenuated following hourly-interval regimens of the drug.

The role of multiple (iv) injections of cocaine on the rat hypothalamic-pituitary-adrenal (HPA) axis was examined using four different temporal regimens of drug exposure. In intact rats, cocaine (5 mg/kg) consistently stimulated the secretion of adrenocorticotropin hormone (ACTH) and corticosterone over a 6 hr interval regimen. In all experimental groups, administration of the vehicle alone failed to measurably alter the secretion of the aforementioned hormones. When rats where exposed to the drug over a 4 hr interval regimen, a modest attenuation of ACTH, but not corticosterone, secretion was observed following the third and last cocaine injection. To test whether the attenuation of ACTH secretion to cocaine administration was caused by corticosterone-mediated negative feedback, the response of intact and adrenalectomized (ADX) rats over 2 hr and 1 hr interval regimens was compared. In intact rats, both drug interval regimens resulted in a significant attenuation of ACTH secretion following, the second and third injections of the drug. ADX rats, on the other hand, exhibited significant increases in ACTH levels following either interval regimens, though we observed a modest blunting of pituitary responsiveness to the 1 hr regimen. From these results we conclude that in intact rats the activity of the HPA axis is significantly attenuated in response to multiple, acute cocaine injections, and that this decreased response may be at least in part caused by a negative corticoid feedback mechanism.     

Fecal corticosterone reflects serum corticosterone in Florida sandhill cranes

Florida sandhill cranes (Grus canadensis pratensis) were conditioned to confinement 6 hr/day for 7 days. On day 8, each bird's jugular vein was catheterized, blood samples were drawn, and each crane was confined for 6 hr. Using a randomized, restricted cross-over design, cranes were injected intravenously with either 0.9% NaCl solution or ACTH (cosyntropin; Cortrosyn; 0.25 mg). During the 6 hr of confinement, fecal samples (feces and urine) were collected from each of five cranes immediately after defecation. Individual fecal samples were collected approximately at hourly intervals and assayed for corticosterone. We showed previously that serum corticosterone did not vary significantly following saline injection, but peaked significantly 60 min after ACTH injection. Maximal fecal corticosterone concentrations (ng/g) were greater (P < 0.10; median 1087 ng/g) following ACTH stimulation compared to maximal fecal corticosterone concentrations at the end of acclimation (day 7; median 176) and following saline treatment (median 541). In cranes under controlled conditions, fecal corticosterone concentration reflects serum corticosterone levels, fecal corticosterone, Grus canadensis pratensis, sandhill cranes, serum corticosterone levels.     

Plasma immunoassayable ACTH levels during and after hydrocortisone infusion in patients with Cushing's disease.

The plasma ACTH responses to hydrocortisone infusion were compared in patients with Cushing's disease and primary adrenocortical insufficiency. In 4 patients with primary adrenocortical insufficiency, plasma ACTH levels were suppressed exponentially after administration of a relatively large dose of hydrocortisone (1.0 mg/kg/1.5 hr-3.0 mg/kg/2 hr). In patients with post-adrenalectomized Cushing's disease (4, bilateral; 1, unilateral), plasma ACTH suppression was delayed. Plasma ACTH levels, expressed as a percentage of the basal concentrations, were significantly less suppressed in patients with Cushing's disease than in patients with primary adrenocortical insufficiency 90 (p less than 0.05) and 120 (p less than 0.05) min after the beginning of infusion. When 0.5 mg/kg hydrocortisone was infused over a period of 1.5 hr, suppression was also delayed in Cushing's disease, and plasma ACTH levels were less suppressed in 4 patients with Cushing's disease than in 4 patients with primary adrenocortical insufficiency at 30 (p greater than 0.05), 45 (p greater than 0.05) 60 (p less than 0.05) min.     

Effect of adrenergic antagonists and cyclooxygenase inhibitors on the nicotine-induced hypothalamic-pituitary-adrenocortical activity.

Nicotine is a potent stimulus for the hypothalamic-pituitary-adrenal (HPA) axis. Systemic nicotine acts via central mechanisms to stimulate by multiple pathways the release of ACTH from the anterior pituitary corticotrops and corticosterone from the adrenal cortex. Nicotine may stimulate indirectly the hypothalamic paraventricular nucleus, the site of the corticotropin-releasing hormone (CRH) neurons which activates ACTH release. In the present studies an involvement of adrenergic system and prostaglandins synthesized by constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2) in the nicotine-induced HPA response in rats was investigated. Nicotine (2.5-5 mg/kg i.p.) significantly increased plasma ACTH and corticosterone levels measured 1 hr after administration. Adrenergic receptor antagonists or COX inhibitors were injected i.p. 15 min prior to nicotine and the rats were decapitated 1 hr after the last injection. Prazosin (0.01-0.1 mg/kg), an alpha1-adrenergic antagonist, significantly decreased the nicotine-evoked ACTH and corticosterone secretion. Yohimbine (0.1-1.0 mg/kg), an alpha2-adrenergic antagonist, moderately diminished ACTH response, and propranolol (0.1-10 mg/kg), a beta-adrenergic antagonist, did not significantly alter the nicotine-induced hormones secretion. Pretreatment with piroxicam (0.2-2.0 mg/kg), a COX-1 inhibitor, considerably impaired the nicotine-induced ACTH and corticosterone secretion. Compound NS-398 (0.2-5.0 mg/kg), a selective COX-2 blocker did not markedly alter these hormones secretion, and indomethacin (2 mg/kg), a non-selective COX inhibitor significantly diminished ACTH response. These results indicate that systemic nicotine stimulates the HPA axis indirectly, and both adrenergic system and prostaglandins are significantly involved in this stimulation. Noradrenaline, stimulating postsynaptic alpha1-adrenergic receptors, and prostaglandins, synthesized by COX-1 isoenzyme, are of crucial significance in the nicotine-induced ACTH and corticosterone secretion.