ACTH-induced Changes Research Articles

Functional melanocortin-2 receptors are expressed by mouse aorta-derived mesenchymal progenitor cells

A local melanocortin system is active during tissue injury and inflammation. Thus far this system has been described as autocrine in nature where local production of pro-opiomelanocortin (POMC) peptides by leukocytes feeds back on melanocortin receptor (MC-R) expressing immune cells to quell inflammatory cytokine production. Here we present evidence that POMC peptides may generate extracellular matrix (ECM) changes by inducing matrix production by cells of the mesenchymal lineage through activation of the MC2-R. Using immunoblot, we determined that mouse aorta-derived mesenchymal progenitor cells express both MC2-R and MC3-R. These progenitors respond to treatment with ACTH by increasing collagen matrix synthesis as assessed by picrosirius red stain and 3H-proline incorporation. ACTH also induces transient increases in intracellular calcium ([Ca2+]i) as assessed using the fluorescent Ca2+ indicator, fura-2. The ACTH-induced changes in [Ca2+]i are consistent with MC2-R signaling and consist of both an intracellular release and an extracellular influx of Ca2+. Both mouse aortic mesenchymal progenitors and mouse macrophage cells express POMC and the prohormone convertase 1/3 (PC1/3) indicating they have the potential to contribute to the local production of POMC peptides. These data demonstrate functional MC2-R expression in mouse aorta-derived mesenchymal progenitors and implicate both macrophage and mesenchymal cells as relevant sources of local POMC peptides.

ACTH-Induced Changes in Immunity in Infants Treated for West Syndrome

The timing of vaccinations is delayed after ACTH therapy in Japanese infants, because the immune system may be compromised.

Transcriptional and physiological responses to chronic ACTH treatment by the mouse kidney

We investigated the effects on urinary steroid and electrolyte excretion and renal gene expression of chronic infusions of ACTH in the mouse. ACTH caused a sustained increase in corticosteroid excretion; aldosterone excretion was only transiently elevated. There was an increase in the excretion of deoxycorticosterone, a weak mineralocorticoid, to levels of physiological significance. Nevertheless, we observed neither antinatriuresis nor kaliuresis in ACTH-treated mice, and plasma renin activity was not suppressed. We identified no changes in expression of mineralocorticoid target genes. Water turnover was increased in chronic ACTH-treated mice, as were hematocrit and hypertonicity: volume contraction is consistent with high levels of glucocorticoid. ACTH-treated mice exhibited other signs of glucocorticoid excess, such as enhanced weight gain and involution of the thymus. We identified novel ACTH-induced changes in 1) genes involved in vitamin D (Cyp27b1, Cyp24a1, Gc) and calcium (Sgk, Calb1, Trpv5) metabolism associated with calciuria and phosphaturia; 2) genes that would be predicted to desensitize the kidney to glucocorticoid action (Nr3c1, Hsd11b1, Fkbp5); and 3) genes encoding transporters of enzyme systems associated with xenobiotic metabolism and oxidative stress. Although there is evidence that ACTH-induced hypertension is a function of physiological cross talk between glucocorticoids and mineralocorticoids, the present study suggests that the major changes in electrolyte and fluid homeostasis and renal function are attributable to glucocorticoids. The calcium and organic anion metabolism pathways that are affected by ACTH may explain some of the known adverse effects associated with glucocorticoid excess.

Hormonal evaluation and mutation screening for steroid 21-hydroxylase deficiency in patients with unilateral and bilateral adrenal incidentalomas.

The aims of the present study were (a) to examine the occurrence of 21-hydroxylase gene (CYP21) mutations in patients with unilateral and bilateral adrenal incidentalomas and (b) to correlate the results of mutation screening with hormonal parameters of 21-hydroxylase deficiency. The frequency of the eight commonly occurring CYP21 mutations in blood DNA samples of 19 patients with bilateral, as well as in blood and tumoral tissue DNA samples of 31 patients with unilateral adrenal incidentalomas, was determined. In all patients, hormonal evaluation for 21-hydroxylase deficiency was performed using measurements of basal and ACTH-stimulated plasma 17-hydroxyprogesterone (17-OHP) concentrations. Blood and tumoral DNA samples were analyzed by allele-specific PCR for the detection of the eight commonly occurring CYP21 mutations (deletion/large gene conversion, intron 2 splicing, Ile172Asn, exon 6 cluster, Val281Leu, Leu307insT, Gln318Stop and Arg356Trp mutations). Plasma 17-OHP concentrations were measured by radioimmunoassay. Of the 19 patients with bilateral adrenal incidentalomas, one patient had homozygous (Val281Leu) and three patients had heterozygous germline CYP21 mutations (Val281Leu in two cases and Arg356Trp in one case). Heterozygous germline CYP21 mutations were also detected in five of the 31 patients with unilateral adrenal incidentalomas (Ile172Asn in three cases and Val281Leu in two cases). Mutation screening of tumoral DNA in unilateral incidentalomas showed the presence of corresponding germline mutations but no additional somatic mutations were found. ACTH-stimulated plasma 17-OHP concentrations were above 1500 ng/dl in all patients with bilateral incidentalomas who had homozygous and heterozygous CYP21 mutations, but heterozygous carriers with unilateral incidentalomas had highly variable ACTH-stimulated plasma 17-OHP levels (between 111 and 1705 ng/dl). These results suggest a similar frequency of germline CYP21 mutations in patients with bilateral and unilateral adrenal incidentalomas (21.1% and 16.1% respectively). Therefore, it cannot be ruled out that, in at least some patients, CYP21 mutations may play a role in the pathomechanism of bilateral and unilateral adrenal incidentalomas. However, the lack of clear association of CYP21 mutations with increased ACTH-stimulated plasma l7-OHP response, especially in patients with unilateral incidentalomas, suggests that the effect of CYP21 mutations on adrenocortical tumor formation may also involve mechanism(s) independent of ACTH-induced changes in 17-OHP secretion.

A Bovine Adrenocortical Kv1.4 K+ Channel Whose Expression Is Potently Inhibited by ACTH

We have cloned a bovine adrenal cortical (bKv1.4) K(+) channel cDNA whose expression is rapidly inhibited by adrenocorticotropic hormone (ACTH). The 4386-nucleotide cDNA is homologous to other voltage-gated, rapidly inactivating Kv1.4 channels, and includes a 1986-nucleotide coding region and large 5'- and 3'-untranslated regions. Bovine Kv1.4-specific mRNA from adrenal zona fasciculata (AZF) cells was rapidly and potently reduced by ACTH, with a t(12) of approximately 1 h and an IC(50) of 1.2 pm. The membrane-permeable cAMP analog 8-pcpt-cAMP also reduced bKv1.4 mRNA expression with kinetics similar to that observed with ACTH. Reduction of bKv1.4 mRNA expression by ACTH and 8-pcpt-cAMP was only partially inhibited by the selective protein kinase A antagonist H-89. Consistent with their effect on bKv1.4 mRNA, ACTH and 8-pcpt-cAMP both dramatically reduced the expression of bKv1.4-associated A-type current measured over 72 h. These results demonstrate that bovine AZF cells synthesize a Kv1.4-type channel whose expression is inhibited at the pretranslational level by ACTH and 8-pcpt-cAMP by a mechanism that is partially dependent on the activation of protein kinase A. The rapid, potent reduction of bKv1.4 mRNA produced by ACTH and 8-pcpt-cAMP indicates that the expression of this K(+) channel is under tonic inhibitory control of the hypothalamic-pituitary-adrenal axis. The basic electrical properties of AZF cells might be tightly regulated at the transcriptional level by the normal diurnal pattern of ACTH secretion, and altered during bouts of stress by the enhanced release of this pituitary peptide. Under conditions of prolonged stress or adrenal insufficiency, persistent ACTH-induced changes in the electrical properties of AZF cells could be coupled to parallel changes in cortisol secretion.

The effect of breeding facility and sexual stimulation on plasma cortisol in boars.

Nine boars were used to evaluate effects of breeding facility design and sexual activity on plasma cortisol concentrations . In one breeding facility (conventional), boars were housed individually in small pens, and female pigs were mated in those boar pens. In another breeding facility (Detection-Mating Area [DMA] system), boars were housed individually in stalls, and female pigs were mated in a specific mating pen adjacent to the front of stalls where boars were housed. After 51 d of housing treatment, a catheter was surgically implanted in the cephalic vein for collection of blood samples. Daytime profiles (hourly collections from 0900 to 1700) of cortisol did not differ among boars in the two treatment groups. Cortisol was greater (P < .01) in the morning than in the afternoon. Administration of ACTH increased (P < .001) plasma cortisol in boars, but breeding facility did not affect the ACTH-induced changes in cortisol concentrations. There was a treatment x time interaction (P < .02) for cortisol after sexual stimulation, and the magnitude and duration of increase in cortisol were greater (P < .05) in the DMA treatment group. Cortisol was greater (P < .001) after than before mating for both treatment groups. An acute increase in plasma cortisol concentration in boars seems to be a normal biological response to sexual activity. However, magnitude and duration of the increase in cortisol may be influenced by breeding facility design and mating procedure. There is no evidence, based on physiological data, that housing boars in stalls in the DMA system has any adverse effects on their welfare.

The peripheral cytoplasm of adrenocortical cells: zone-specific responses to ACTH.

Differences in the cytoskeletal protein actin in cells from the zona glomerulosa and zona fasciculata would be of considerable interest because there is persuasive evidence that rat corticosteroids are secreted by mechanisms that are somewhat zone-specific. We have previously shown evidence that actin may be involved in steroid secretion, possibly in connection with changes in adrenocortical microvilli. However, the cells upon which the data were based were not separated according to zone of origin. Immunogold electron microscopy and morphometric procedures were used to determine whether ACTH-induced changes in the peripheral cytoplasm of isolated adrenocortical cells occur in both zona fasciculata and zona glomerulosa cells. Actin immunoreactivity was more concentrated in the cytoplasm adjacent to the plasma membrane (including the cytoplasm within the microvilli) than it was in the internal cytoplasm in cells from both zones (4-6 times more concentrated in zona glomerulosa cells and 3-6 times more concentrated in zona fasciculata cells). However, the mean aggregate microvillar surface length (microvillar index) of untreated zona fasciculata cells (previously reported (Loesser and Malamed, 1987)) was 23% greater than that of untreated zona glomerulosa cells. Although ACTH (at a maximal steroidogenic concentration) had no effect on the peripheral cytoplasmic actin concentration of zona glomerulosa cells, there was a 24% increase in the aggregate microvillar length. In contrast, in zona fasciculata cells, ACTH treatment was accompanied by an increase in peripheral cytoplasmic actin concentration of 58-64% and an increase in aggregate microvillar surface length of 40% (previously reported (Loesser and Malamed, 1987)), almost twice that for zona glomerulosa cells. The results suggest that ACTH-induced hormone release from zona fasciculata cells is mediated by increases in peripheral cytoplasmic actin and aggregate microvillar length; in zona glomerulosa cells such changes are small or absent.

Influences of peripheral adrenocorticotropin 1–39 (ACTH) and human corticotropin releasing hormone (h-CRH) on human auditory evoked potentials (AEP)

Hormones of the hypothalamus-pituitary-adrenal (HPA) axis have been considered to form part of an efferent humoral system modulating central nervous stimulus processing. The present experiments were designed to compare the effects of iv bolus administrations of placebo, porcine ACTH 1-39 (1.5 U) and h-CRH (25 micrograms) on auditory evoked potentials (AEPs) in healthy men. Also, cardiovascular parameters, cortisol and self-reported mood were assessed. ACTH significantly reduced the amplitude of the N1 component of the AEP; P1 and P2 remained unchanged. The selective reduction of N1 amplitude defies an interpretation of the changes in terms of a reduced stimulus-induced cortical arousal following ACTH; the ACTH-induced changes may rather indicate an influence on frontocortical functions of directing attention. The effect of ACTH on N1 cannot be attributed to its adrenocorticotropic action or to cardiovascular changes, but appears to represent an intrinsic extraadrenal influence of the hormone. The data do not provide evidence for effects of h-CRH on central nervous stimulus processing in humans, after peripheral administration.

Effects of receptor blockers on ACTH-induced changes in extinction of active avoidance reflex in rat

The actions of blockers of dopaminergic receptors (haloperidol), alpha-receptors (phenoxybenzamine), beta-receptors (propranolol) and muscarinic cholinergic receptor (atropine) on the ACTH-induced delay of the extinction of active avoidance behavior were studied in rats. In the doses used, none of the receptor blockers modified the extinction of active avoidance behavior. ACTH delayed the extinction. However, the dopamine receptor blocker (haloperidol) and the muscarinic cholinergic receptor blocker (atropine) did prevent the action of ACTH in delaying the extinction of active avoidance behavior, whereas the alpha-(phenoxybenzamine) and beta- (propranolol) receptor blockers were ineffective. The results suggest that mainly dopaminergic and cholinergic mediations are involved in the delaying action of ACTH on the extinction of active avoidance behavior.

Adrenocortical function in deoxycorticosterone acetate (DOCA)-hypertensive Yucatan miniature swine.

Adrenocortical function was assessed in six normal and six chronic (greater than 12 weeks), DOCA-hypertensive Yucatan miniature swine; mean arterial pressures were 115.3 +/- 11.7 and 163.6 +/- 27.2 mm Hg, respectively (mean +/- SEM). Adrenocortical function was evaluated in vivo by measuring changes in plasma cortisol and aldosterone in response to exogenous ACTH (0.25 mg, iv), and in vitro by measuring the responses of collagenase-isolated adrenocortical cells to ACTH and angiotensin II. Corticoids were measured by specific radioimmunoassay. Basal plasma cortisol values of conscious DOCA-hypertensive swine were approximately 53% of the values of normotensive swine (P less than 0.05). However, ACTH induced a 419% increase in plasma cortisol values in DOCA-hypertensive swine compared to a 261% increase in the normotensive swine (P less than 0.05). These differences between the two groups were not altered by anesthesia. There were no significant differences in ACTH-induced changes in plasma aldosterone between the normotensive and DOCA-hypertensive swine. Experiments in vitro showed that the corticoid secretory responses of adrenocortical cells from DOCA-hypertensive animals were 6 times more sensitive to ACTH and 3.2 times more sensitive to angiotensin II than those of cells from normotensive swine. Thus, despite the possibility of adrenocortical insufficiency due to suppressed plasma renin activity and the negative feedback of DOCA on the hypothalamic-hypophyseal-adrenal axis, adrenocortical function of DOCA-hypertensive swine was hyperresponsive to trophic hormones. Results from this study suggest that the DOCA-hypertensive swine may be a valuable model in elucidating the relationship between hypertension and adrenocortical function and in investigating nonclassical control of the adrenal cortex, that is, control exerted during the hypertensive state that exists apart from or in addition to that exerted by ACTH and angiotensin II.

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. II. ACTH-induced changes in subcellular corticosterone

Zona fasciculata-reticularis subcellular structures were implicated in corticosterone transport and secretion by noting changes in subcellular corticosterone during a 30-min period following ACTH stimulation. Six decapsulated adrenal homogenate subcellular fractions separated by gradient centrifugation were characterized cytochemically and morphologically. Predominant components in each of six fractions were: (a) floating lipid droplets, (b) 0.125 M sucrose (no organelles), (c) cytosol (0.25 M sucrose supernatant with 0.25 – 1.2 micron electron dense granules), (d) microsomes (interface between 0.5 M and 1.1 M sucrose layers), (e) mitochondria (boundary between 1.1 M and 2.2 M sucrose layers) and (f) nuclei (centrifuge pellet). Whole glands and most subcellular fractions showed peak corticosterone levels 10 to 15, and 30 min after stimulation. Sucrose and cytosolic fractions contained about 75% of the total corticosterone, responded to stimulation most significantly, and were rich in protein. In these two fractions only cytosol contained structures; these consisted of 0.15 – 1.2 micron electron dense granules.

Urinary profile of L-carnitine and its derivatives in starved normal persons and ACTH injected patients with myopathy.

Effect of starvation or ACTH injection on the urinary level and profile of L-carnitine and its derivatives was studied in four healthy adult men or in a normal child and two patients with myopathy, respectively. Mean total L-carnitine level in the control urine sample obtained before starvation was 389 +/- 34 mumol . man . day. The percentage distribution was found to be 46% for free-, 9% for acetyl- and 45% for acyl-L-carnitine. The acyl-L-carnitine fraction contained short-chain (65%) and long-chain acyl-L-carnitine (35%). With 2-day starvation urinary excretion of free-L-carnitine was slightly decreased and, in contrast, that of acetyl-L-carnitine was considerably increased, resulting in a significant increase in urinary total L-carnitine levels. Urinary excretion of acyl-L-carnitine was increased two-folds with starvation, but that of long-chain acyl-L-carnitine was not changed. In a normal child (female, 3.5 yr) and two patients (female, 4.5 yr and male, 23 yr) with myopathy, ACTH injection induced a significant elevation of urinary total L-carnitine levels, being mainly caused by an increased excretion of free-L-carnitine and, in the adult patient, acyl-L-carnitine. Muscle total L-carnitine contents were normal in two children but abnormally low in the adult patient, who had simultaneously very low urinary total L-carnitine level before ACTH injection. Thus, in the adult patient myopathy might be possibly caused in part by carnitine deficiency. Starvation and ACTH-induced changes in urinary level and profile of L-carnitine and its derivatives were discussed in relation to carnitine biosynthesis as well as renal regulation of carnitine clearance.

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.

Acute effects of ACTH on dissociated adrenocortical cells: Quantitative changes in mitochondria and lipid droplets

To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.

Aldosterone Production by Adrenal Cultures Via dbcAMP, and the Reversibility of ACTH and dbcAMP-Induced Changes

Tissue cultures of embryonic adrenal glands were treated with dbcAMP, ACTH and Angio-tensin II. dbcAMP and ACTH caused similar elevation in both aldosterone and corticosterone for 17 days in vitro (DIV) but Angiotensin II treated cultures produced no increase in these steroids above control levels. Mitochondria in dbcAMP and ACTH treated cultures had vesicular cristae and were larger than control mitochondria. These enlarged mitochondria also occupied a greater percentage of cytoplasmic space than controls suggesting hypertrophy or hyperplasia of treated mitochondria and not just a change in their internal configuration. The reversibility of dbcAMP- and ACTH-induced changes was also investigated. If dbcAMP or ACTH were removed from the feeding medium, levels of both aldosterone and corticosterone rapidly fell, and approached control baseline values. At the same time, some mitochondria reverted from a packed vesicle to a plate-like configuration. That ACTH may act via cAMP on aldosterone production and als...

Studies on ACTH-induced changes at the NADP +-binding site of bovine adrenal glucose 6-phosphate dehydrogenase

Adrenal glucose 6-phosphate dehydrogenase ( d-glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49) was inhibited by metal chelating agents and especially by sulfhydryl-inhibiting agents. Sulfhydryl inhibition at the NADP +-binding center by p-chloromercuribenzoate (PCMB) was studied in detail. Compounds having reduced sulfhydryl groups protected the enzyme against PCMB inactivation. Magnesium, barium, or EDTA augmented PCMB inhibition of the enzyme, as did low concentrations of urea, guanidine-HCl, or dodecyl acetate. NADP + and glucose 6-phosphate completely protected the enzyme from PCMB. NAD +, NADH, NADPH, steroid hormones, and fatty acids partially protected the enzyme from PCMB inhibition. ACTH increased the rate of inactivation of the enzyme by PCMB. This inactivation was completely prevented by NADP + and partially prevented by NAD +. Glucose 6-phosphate had no effect on the ACTH augmentation of PCMB inhibition. Amino acid analysis revealed 32 cysteine residues per 238,000 g/mole of enzyme. PCMB titration of the enzyme showed seven to eight sulfhydryl groups in the ureatreated enzyme, four to five sulfhydryl groups in the native enzyme, and only two sulfhydryl groups involved in enzymatic activity. No sulfhydryl groups were titratable when NADP + was present in excess. A mechanism for the binding relationships of substances at the active sites is discussed. The allosteric binding of ACTH to the enzyme induces a change in the NADP +-binding center.

Acth induced changes in tryptophan turnover along induceable pathways in man

Metabolic correlates of glucocorticoid induction of enzymes in man studied in terms of ACTH INDUCED changes in tryptophan turnover along induceable pathways

ACTH-induced changes in purine and carbohydrate metabolism in Dalmatian and mongrel dogs.

ADIABETOGENIC effect of ACTH in normal human subjects has beenreported (Conn, Louis and Johnston, 1949). An attempt to induce diabetes in mongrel dogs weighing 10–12 kg. was unsuccessful in this laboratory when a daily dose of 26 mg. of ACTH (Armour Standard) was used. Houssay and Anderson (1949) injected dogs, in which 83–87% of the pancreas had been removed, with 50 mg. of ACTH (96% Armour Standard) per kg. per day for four days. Hyperglycemia was observed in only one of the four treated, but glucose tolerance tests were not reported. Campbell et al. (1950) showed that ACTH (7.5 mg./kg./day) produced mild hyperglycemia and minimal glycosuria in dogs which were selected for their sensitivity to the diabetogenic activity of anterior pituitary extracts. Overbeek (1950) stated that carbohydrate metabolism was not affected in dogs treated with large doses of ACTH (Organon)