Immunoreactive forms of ACTH released by the adenohypophysis of the rat during the perinatal period in vivo and in vitro studies

Immunoreactive ACTH levels were determined in whole plasma, eluate fractions of plasma and perfusates of anterior lobe extracts subjected to chromatography on Sephadex G-50 fine. In the fetal plasma, ACTH levels were higher on day 19 than on days 17, 18, 20 and 21. After birth, ACTH concentrations dropped to reach the lowest values in one week old newborns; thereafter they increased until weaning on day 21. They were then similar to those of non pregnant adult females. Three peaks of immunoreactive ACTH were present in all the chromatograms; the first one eluted near the void volume ("big" ACTH, PM approximately 44,000), the third one coeluted with human ACTH (1-39) ("little" ACTH, PM approximately 4,500) and the second one eluted midway between the 2 previous peaks ("intermediate" ACTH, PM approximately 13,000). During the last days of pregnancy, the proportion of the "little" form of ACTH in the fetal plasma showed a gradual increase whereas that of the "big" one decreased. On days 17, 19 and 21 of gestation the anterior lobes of fetal pituitary glands released in vitro these 3 forms of immunoreactive ACTH in the same proportions as those observed in the anterior lobes. In contrast, the proportions of the circulating forms of ACTH were quite different; the "little" one gradually increased and the "big" one decreased as gestation progressed. In vitro controlled tryptic digestion of the isolated "big" form led to the appearance of "intermediate" and "little" forms suggesting some transformations in the circulation of the ACTH forms released by the fetal hypophysis in vivo.     

Effects of ACTH on the last step of aldosterone biosynthesis

The production of tritiated aldosterone and tritiated SM (a saponifiable 18-hydroxycorticosterone derivative) by rat adrenals were studied at various incubation times in absence or presence of two concentrations of ACTH. Tritiated 18-hydroxycorticosterone or 18-deoxyaldosterone served as precursors. The lower ACTH concentration (150 pM) increased the production of tritiated aldosterone. Whereas, the higher ACTH concentration (1.5 microM) stimulated tritiated aldosterone production at shorter incubation time (30 min), while after 60 min it inhibited. This time dependency would reflect variations in the levels of endogenous steroids. On the other hand, the effects of ACTH on tritiated SM production were opposite to those on tritiated aldosterone. In effect, while 150 pM ACTH inhibited SM production, 1.5 microM ACTH stimulated it. These results suggest that ACTH promotes opposite effects on the productions of aldosterone and SM and therefore both productions would be coordinated under the regulation of ACTH.     

Ovine prolactin potentiates the action of adrenocorticotropic hormone on the secretion of dehydroepiandrosterone sulfate and dehydroepiandrosterone from cultured bovine adrenal cells

To determine the direct effect of prolactin on adrenal androgen secretion, the daily secretions of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), androstenedione and cortisol were determined in monolayer culture of bovine adrenal cells in the presence or absence of adrenocorticotropic hormone (ACTH) and/or prolactin. In the absence of ACTH ovine prolactin alone had no effect on steroid secretion during seven-day culture. Ovine prolactin, when administered in combination with ACTH, significantly potentiated the stimulatory effect of ACTH on DHEA-S and DHEA but not androstenedione secretion on the seventh day in culture. On the first day in culture prolactin showed no synergistic effect with ACTH on DHEA and DHEA-S secretion, although ACTH significantly increased DHEA and cortisol secretion. DHEA-S secretion increased as a function of prolactin concentration in the presence of ACTH. These results indicated that long-term treatment by ovine prolactin with ACTH caused the increase in adrenal androgen secretion from bovine adrenal cells. The site of action of prolactin was suggested to be the partial inhibition of adrenal 3 beta-hydroxysteroid dehydrogenase by the result of increases in DHEA-S and DHEA but not androstenedione secretion.     

Dynamics of adenylate cyclase desensitization during long-term exposure to the hormone

The dependence of adrenal gland adenylate cyclase desensitization on the dose of in vivo injected ACTH, the time of occurrence and duration of the enzyme refractory period and the dependence of desensitization on the number of ACTH injections were analyzed. The experiments were carried out on guinea pigs injected with prolonged action preparations of ACTH (4 and 6 units) daily for 1-6 days. Intramuscular injections of ACTH caused adenylate cyclase refraction to the repeated action of the hormone. The effect of desensitization was the most conspicuous within the first few hours after hormone injection. The decrease of adenylate cyclase sensitivity and the duration of this effect were found to depend on the ACTH dose as well as on the number of injections. It has been shown for the first time that a single in vivo injection of 0.9% NaCl causes short-term desensitization of adenylate cyclase to the repeated action of much higher doses of ACTH in vitro, presumably due to endogenous ACTH release in response to weak stress exposure. The periodicity of changes in adenylate cyclase sensitivity upon prolonged hormone administration is discussed. Sensitization of the enzyme upon daily short-term exposure to physiological doses of ACTH (administration of 0.9% NaCl for 6 days) was revealed.     

Immunocytochemical characterization of ACTH-like immunoreactivity in cerebral nerves and in endocrine cells of the pituitary and gastrointestinal tract by using region-specific antisera

The development and use of region-specific antisera for characterizing pituitary and extrapituitary ACTH immunoreactivity are described. The pituitary corticotrophs and melanotrophs, as well as a system of cerebral nerves, contain antigenic determinants, indistinguishable from those of true, pituitary ACTH [1-39]. The distributional patterns of cerebral nerves, most probably containing ACTH [1-39], is of interest in view of documented behavioral effects of ACTH fragments, as well as the possible interaction between ACTH and certain opioid peptides. Studies on antropyloric gastrin cells, previously reported to contain immunoreactive ACTH-like material indicate that the main form of immunoreactive peptide stored in these cells contains only part of the ACTH [1-39] sequence. Its relation to fragments of the ACTH molecule, as well as to yet unknown (hormonal) peptides, is discussed.     

Effect of neonatally injected ACTH and ACTH analogues on eye-opening of the rat.

Three-day old rats were injected subcutaneously either with natural purified pig ACTH (ACTH 1–39), ACTH 1–24, or ACTH analogues as long-acting zinc-phosphate preparations. ACTH 1–39, ACTH 1–24, ACTH 1–18, ACTH 1–16 accelerated the time of eye-opening, whereas an extract of corticosteroids produced $\text{in}$$\text{vitro}$ by excised adrenals of ACTH-treated three-day old rats was ineffective. Injections with ACTH on the twelfth day of life had no effect on eye-opening. It is concluded that a neonatal injection with ACTH or closely related analogues with markedly less corticotropic activity can accelerate the time of eye-opening. This effect is not mediated by the adrenal cortex. The sensitive period for it appears to be shortly after birth.     

Immunoreactive beta-endorphin and ACTH in the same neurons of the hypothalamic arcuate nucleus in the rat.

Immunoreactive ACTH and beta-endorphin (beta-End) were localized in the brain and pituitaries of normal and colchicine-treated rats, using the immunoperoxidase method at the light microscopic level. On adjacent serial 5-micron paraffin sections of anterior pituitaries, both ACTH and beta-End could be found in the same cells. On adjacent 5-micron paraffin sections of brains of colchicine-treated rats, both ACTH and beta-End could be found in the same perikarya of hypothalamic arcuate nucleus neurons. It appeared that all perikarya containing beta-End contained ACTH as well, suggesting that neurons producing beta-End also produce ACTH. Pathways of ACTH fibers corresponded to pathways of beta-End fibers. These findings suggest that the synthesis, and transport, of ACTH and beta-End are linked in the brain as well as in the pituitary, possibly through a common precursor.     

Corticotropin-induced changes in enzymatic activities of the post-pregnenolone steroidogenic pathway in rabbit adrenocortical cells

In an attempt to delineate the effect of corticotropin (ACTH) on post-pregnenolone steroidogenesis, the activity of enzymatic systems operative in conversion of pregnenolone into glucocorticoids and androgens was studied in adrenocortical cells from control rabbits and from animals treated with ACTH for 12 days (ACTH 1-24, 200 micrograms s.c. daily). The cells from ACTH-treated rabbits exhibited an increased overall steroidogenic capacity and produced much more cortisol (P less than 0.0005) as well as other 17-hydroxylated steroids as a result of increased activity of 17 alpha-hydroxylase; corticosterone generation was concomitantly reduced. The increased conversion of pregnenolone or progesterone into androgens, as a result of previous treatment with ACTH, provides additional evidence for an effect of ACTH on 17 alpha-hydroxylase activity. A stimulatory effect of ACTH on 11 beta-hydroxylase was also evidenced by these cells, since conversion of 11-deoxycortisol into cortisol was enhanced (P less than 0.005). The increased production of androgens from 17-hydroxylated precursors by cells from ACTH-treated rabbits suggests that ACTH also exerts a prolonged stimulatory effect on 17,20-lyase. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was apparently not influenced by chronic treatment with ACTH, judged from unchanged conversion of dehydroepiandrosterone into androstenedione. The activity of 11 beta-dehydrogenase was likewise unchanged in these conditions.     

Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells

The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation.     

Ontogeny of the response of the hypothalamo-pituitary-adrenal axis to maternal immobilization stress in rats.

In this study we investigated the response of the rat fetal hypothalamo-pituitary-adrenal (HPA) axis to an acute maternal stress in late gestation. On day 20 of gestation, pregnant rats were exposed to forced immobilization stress for up to 60 min. In mothers, a significant increase in plasma ACTH and corticosterone(B) was observed at 20 and 60 min. The ACTH content in the maternal pituitary decreased significantly at 60 min. Fetal blood pH was decreased by the maternal stress, showing a hypoxic condition of the fetus. Fetal plasma ACTH increased transiently at 20 min. Fetal plasma B increased at 20 and 60 min. ACTH in the fetal pituitary and the placenta did not show marked changes due to the maternal stress. Pregnant rats on day 18-21 of gestation were subjected to a 20 min maternal stress. In the basal condition without stress, fetal plasma ACTH and B showed parallel ontogenic patterns, having a peak value on day 19 of gestation. Fetal plasma ACTH as well as plasma B were increased significantly by the maternal stress at all points evaluated. These results indicate that fetal hypoxia is important in stress transmission to the fetal HPA axis in this type of maternal stress, and the fetal HPA axis responds to the stress as early as day 18 of gestation.     

A comparative study on the steroidogenic response due to training and ACTH treatment in the adrenal cortex of the male rat

The aim of this study was to ascertain the effects of training and ACTH administration on the steroidogenic in vitro response in the adrenal cortex of the rat when the tissue was incubated with ACTH. ACTH in vivo treatment resulted in a highly significant increase in the steroidogenic response (P less than 0.001) whereas training as such caused only a slight but insignificant increase in the steroidogenic responsiveness (P greater than 0.05). Training furthermore strongly suppressed the ACTH in vivo induced response (P less than 0.001). ACTH as such revealed the smallest effect on adrenal mass but the biggest effect on the steroidogenic response. It would seem that long term exercise resulted in an overall increase in the mass and size of the adrenal glands by either increasing the size of existing cells or by increasing the number of cells or both. The latter exercise-induced proliferation of adrenal tissue may involve an adaptive mechanism whereby larger total quantities of adrenal tissue of lowered steroidogenic efficiencies (on a mass basis) are produced in order to meet the stress resulting from the training program.     

Effect of various ACTH analogs on lordosis behavior in the female rat

The effect of ACTH and various related analogs on lordosis behavior in female rats was compared with that produced by α-MSH. Ovariectomized rats received 2 μg estradiol benzoate on Day 1 and Day 3 either 0.1 or 0.2 mg progesterone. Four hours later the females were placed with sexually experienced male rats and the lordosis quotient (LQ) noted. These particular doses of progesterone were chosen because they were sub-maximal and produced a proportion of both nonreceptive (LQ less than 50%) and receptive (LQ greater than 50%) rats. Treatment with 20 μg α-MSH on Day 2 stimulated lordosis in nonreceptive rats but inhibited lordosis in the receptive rats.Of the other peptides tested only ACTH4–10 was as effective as α-MSH in facilitating and inhibiting lordosis behavior. ACTH1–24 and ACTH4–9 also produced both effects. ACTH1–39 and ACTH1–16, on the other hand, had neither effect but were both effective in stimulating and inhibiting lordosis when administered on Days 1, 2 and 3. It is suggested that ACTH4–10 may contain the essential sequence for these facilitatory and inhibitory effects on female sexual receptivity and that elongation of the peptide chain beyond ACTH 1–13 (α-MSH) may decrease this activity.     

Increased sensitivity of the fatty tissue of rats with spontaneous hypertension to the action of ACTH. The role of calcium]

The action of ACTH on the adipose tissue lypolysis was studied in rats with spontaneous and renal hypertension as well as in normotensive rats of the respective control groups. It was demonstrated that sensitivity of the adipose tissue SHR to ACTH was increased as compared to normotensive controls. The data presented indicate that the increased sensitivity is due to the state or content of intracellular calcium. The rats with renal hypertension did not show such an increased sensitivity of the adipose tissue to ACTH.     

Metabolic clearance rate of adrenocorticotropin in the rat.

The metabolic clearance rate (MCR) of adrenocorticotropin (ACTH) was estimated after the intravenous infusion of graded rates of the hormone (40-2560 muU/min per 100 g body weight) in rats pretreated with chlorpromazine, morphine, and Nembutal, a preparation which proved effective in blocking endogenous ACTH release. The hormone was infused over a period of 45 min, at which time the plasma ACTH concentration had reached a steady state. A specific and sensitive bioassay, based on the corticosterone production of dispersed adrenal cells, was used to measure the plasma ACTH concentration. With increasing infusion rates of ACTH, a threefold decrease in the MCR of ACTH was observed. Previous studies of our group have shown that the MCR of corticosterone increases as a function of the infusion rate of the steroid. It appears, therefore, that the metabolism of these two hormonal links of the hypothalamo-pituitary-adrenocortical axis vary in opposite fashions as a function of the secretion rate of the hormone.     

Effect of adrenocorticotropin fragments on the memory of Tenebrio molitor beetles

Fragments of the adrenocorticotropic hormone ACTH1-24 and ACTH5-7 facilitate memory in the beetle T. molitor, the effect being observed at different stages after learning. An analogue of ACTH5-7 which contains D-phenylalamine, as well as D-phen7-ACTH4-7, inhibit memorization (the results checked 1 day after learning) and induce some other disturbances in the behaviour of beetles. To the 10th day of learning, the effects of the analogues cannot be distinguished from those of L-Phe7-fragments. The results obtained are compared with the known effect of ACTH fragments on memory in vertebrates.     

ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture

It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro. Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes. In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions. Their potencies were compared with the potency of alpha-MSH. After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies. By 14 days, cultures contained mostly pigmented melanocytes. The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39). The length of their peptide chains was inversely proportional to the potency. On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes. In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM). These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely. The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.     

Interactions between CRF, epinephrine, vasopressin and glucocorticoids in the control of ACTH secretion

The secretion of ACTH by corticotrophs in the anterior lobe of the rat pituitary gland is under the stimulatory influence of at least three receptors, namely that for peptidic CRF (corticotropin-releasing factor), vasopressin and alpha 1-adrenergic agents. CRF is a potent stimulator of cyclic AMP accumulation as well as adenylate cyclase activity in the rat adenohypophysis, thus suggesting an important role of cyclic AMP as mediator of CRF action on ACTH secretion. Vasopressin causes a 2-fold increase of the stimulatory effect of CRF on ACTH release in rat anterior pituitary cells in culture. The potentiating effects of vasopressin on CRF-induced ACTH release are accompanied by parallel changes of intracellular cyclic AMP levels. Vasopressin, while having no effect on basal cyclic AMP levels, causes a 2-fold increase in CRF-induced cyclic AMP accumulation without affecting the ED50 value of CRF action. ACTH secretion is also stimulated by a typical alpha 1-adrenergic receptor. Epinephrine causes a marked stimulation of ACTH release which is additive to that of CRF. Epinephrine, in analogy with vasopressin, although having no effect alone on basal cyclic AMP levels, causes a marked potentiation of CRF-induced cyclic AMP accumulation. Glucocorticoids cause a near-complete inhibition of epinephrine-induced ACTH secretion within 4 h with the following order of ED50 values: triamcinolone acetonide (0.2 nM) greater than dexamethasone (1.0 nM) much greater than cortisol (11 nM) greater than corticosterone (22 nM). Similar effects are observed for CRF- and vasopressin-induced ACTH release. Although the activity of the pituitary-adrenocortical axis in the rat is highly dependent upon sex steroids, 17 beta-estradiol, 5 alpha-dihydrotestosterone and the pure progestin R5020 have no detectable effect on basal or epinephrine-induced ACTH release, thus illustrating the high degree of specificity of glucocorticoids in their feedback control of ACTH secretion. Moreover, glucocorticoids have no effect on CRF-induced cyclic AMP accumulation, thus indicating that their inhibitory effect is exerted at a step following cyclic AMP accumulation.     

Characterisation of ACTH Peptides in Human Skin and Their Activation of the Melanocortin-1 Receptor

α-Melanocyte-stimulating hormone (α-MSH) is a proopiomelanocortin (POMC)-derived peptide, which is produced in the pituitary and at other sites including the skin. It has numerous effects and in the skin has a pigmentary action through the activation of the melanocortin-1 (MC-1) receptor, which is expressed by melanocytes. Recent evidence suggests that the related POMC peptides such as adrenocorticotrophin (ACTH), which is the precursor of α-MSH, is also an agonist at the MC-1 receptor. By using immunocytochemistry, we confirmed the presence of α-MSH in human skin where staining was evident in keratinocytes and especially strong in melanocytes and possibly Langerhans cells. ACTH was also present and tended to show the strongest reaction in differentiated keratinocytes. Immunostaining was also observed for the prohormone convertases, PC1 and PC2, which are involved in the formation of ACTH and its cleavage to α-MSH, respectively. The amounts of immunoreactive ACTH exceeded those of α-MSH. Using HPLC we identified for the first time the presence of ACTH1-39, ACTH1-17, ACTH1-10, acetylated ACTH1-10, α-MSH, and desacetyl α-MSH in epidermis and in cultured keratinocytes. The ability of these peptides to activate the human MC-1 receptor was examined in HEK 293 cells that had been transfected with the receptor. All peptides increased adenylate cyclase in these cells with the following order of potency: ACTH1-17 > α-MSH > ACTH1-39 > desacetyl α-MSH > acetylated ACTH1-10 > ACTH1-10. ACTH1-17 also increased the dendricity and melanin content of cultured human melanocytes indicating that the peptide was able to activate MC-1 receptors when present in their normal location. However, as found with α-MSH, not all cultures were responsive and, as we have previously suggested, we suspect that this was the result of changes at the MC-1 receptor. Nevertheless, it would appear that ACTH peptides can serve as natural ligands of the MC-1 receptor on human melanocytes and their presence in the skin suggests that, together with α-MSH, they may have a role in the regulation of human melanocytes.     

Multiple forms of ACTH biological activity in the pars intermedia of the rat adenohypophysis.

Acid extracts of a) acutely dispersed rat pars intermedia (PI) cells, b) media after incubation of PI cells, c) whole nervosa-intermedia, and d) whole pars distalis, were chromatographed on Sephadex G-50 Fine in 1% acetic acid. Three peaks of ACTH biological activity were resolved in all four extracts. Peak I eluted in the void volume of the column, peak III co-eluted with synthetic ACTH^1–39, and peak II eluted in an intermediate position. The predominant ACTH activity derived from the PI tissue was peak I, amounting to over 70% of the total ACTH activity present in that lobe. The positions of PI peaks I and II remained unaltered after rechromatography as well as after treatment with and chromatography in 8 M urea. However, peak I of PI ACTH was further resolved into two separate peaks by chromatography on Sephadex G-100 SF. Thus pars intermedia ACTH activity appears to be composed of four separate entities, with the predominant forms being larger than ACTH^1–39.     

Effect of dexamethasone on the release of pituitary hormones in monolayer cultures of rat pituitary cells

The effect of dexamethasone on the release of ACTH, GH, PRL, LH and TSH was studied in monolayer cultures of rat pituitary cells in 4-hour incubation. With or without the addition of rat hypothalamic extract, the release of GH was significantly inhibited by dexamethasone at concentrations higher than 10(-9) M, although less remarkably than that of ACTH. Intracellular ACTH and GH were unchanged. PRL, LH and TSH were not affected. These results indicate that dexamethasone, when exerted for 4 hours, suppressed the release of GH as well as ACTH, at least in part, at the pituitary level.