Role of chain termini in selective steroidogenic effect of ACTH/MSH(4-10) on isolated adrenocortical cells

To investigate the role of charged chain ends in the corticosteroidogenic effect of ACTH/MSH(4-10), acetyl and amide derivatives of ACTH/MSH(4-10) were synthesized and tested in isolated zona glomerulosa and zona fasciculata cells. ACTH/MSH(4-10)-NH2, Ac-ACTH/MSH(4-10) and Ac-ACTH/MSH(4-10)-NH2 (10 microM to 1 mM) stimulated the aldosterone production of zona glomerulosa cells, whereas these peptides did not stimulate the corticosterone production of zona fasciculata cells, even at 1 mM concentration. As ACTH/MSH(4-10) has been shown to have a steroidogenic effect on both types of adrenocortical cells, both charged chain termini seem to be essential for triggering of the corticosterone production of zona fasciculata cells, but for aldosterone production their presence appears not to be important.     

Adrenocorticotropin is a specific mitogen for mammalian myogenic cells

Peptides derived from proopiomelanocortin (POMC) have been found to stimulate the proliferation of murine myogenic cells. Among these peptides, adrenocorticotropin (ACTH) and alpha-, beta-, and gamma-melanocyte-stimulating hormones (MSH) were found to be active, whereas the opioid peptides were not. At clonal density, both ACTH and MSH caused a three- to fourfold increase in the average number of cells per clone in myogenic but not in fibroblast colonies. At high cell density, ACTH and MSH caused a three- to fourfold increase in proliferation of myogenic cells, reflected by an increased accumulation of skeletal myosin. On the other hand mouse embryo skin or muscle fibroblasts or vertebral chondroblasts did not increase proliferation in response to POMC-derived peptides. The half-maximal dose at which ACTH stimulated myoblast proliferation was around 5 nM, and the mitogenic effect was doubled by suboptimal doses of fibroblast growth factor. The possible physiological significance of the mitogenic effect of ACTH on myogenic cells is discussed.     

Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3'': 5''-Cyclic Monophosphate in Cultured Murine Brain Cells

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.     

Pressor and cardioaccelerator effects of gamma MSH and related peptides

We have recently demonstrated that the hypertensinogenic and natriuretic actions of ACTHI-39 can be found in a non-steroidogenic fragment of ACTH, ACTH4-10. These effects of ACTH or ACTH4-10 may be due to their ability to act as weak agonists of gamma MSH. gamma MSH is found in the 16K N-terminus of pro-opiocortin, and contains a sequence analogous to ACTH4-10, gamma MSH3-9. We investigated the cardiovascular effects of gamma 2MSH, gamma MSH3-9, and sterically restricted analogs of ACTH4-10. The results indicate that gamma MSH3-9, had essentially the same activities as ACTH4-10. The addition of five other amino acid residues to gamma MSH3-9 (gamma 2MSH) resulted in significant enhancement of pressor and cardioaccelerator activity. Steric restriction of the ACTH4-10 sequence by the substitution of a D-Phe in place of an L-Phe residue in position #7, or cyclization of the peptide by a half-Cys4, half Cys10 intramolecular disulfide-bridge derivatization, resulted in increased cardiovascular activities. Based on these data, the cardiovascular actions of ACTH4-10, gamma MSH3-9, and gamma 2MSH are predicted to be due to the assumption of a reverse-turn three-dimensional structure. The additional residues in gamma 2MSH appear to specifically enhance the cardiovascular activities of gamma MSH3-9. The results suggest the existence of a new class of hypophyseal peptides with cardiovascular activities, which require the assumption of a defined three-dimensional structure.     

Cultured Human Melanocytes Respond to MSH Peptides and ACTH

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α-MSH and its synthetic analogue Nle⁴DPhe⁷α-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.     

Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA.

Melanocytes and melanoma cells are known to possess receptors for melanocyte stimulating hormone (MSH). A cDNA clone, designated 11D, has been isolated from human melanoma cells and encodes a MSH receptor. The cloned cDNA encodes a 317 amino acid protein with transmembrane topography characteristics of a G-protein-coupled receptor, but it does not show striking similarity to already published sequences of other G-protein-coupled receptors. When 11D cDNA is expressed in COS-7 cells, it binds an 125I-labelled MSH analogue (NDP-MSH) in a specific manner. The bound ligand could be displaced by melanotropic peptides, alpha-MSH, beta-MSH, gamma-MSH and ACTH (adrenocorticotropic hormone), but not by the non-melanotropic peptide, beta-endorphin. This is the first report of the cloning of the receptor gene of the melanotropin receptor family.     

Inhibitory effects on steroid production from isolated adrenal cells of rhesus monkey (Macaca mulatta) of pro-opiomelanocorticotrophic peptides

Adrenal glands from Rhesus monkeys (Macaca mulatta) of 160 days gestation, newborn, 2 months-old infants or 6 months-old infants were excised and prepared, by a collagenase digestion, as a cell suspension. The cells were incubated with 10 pg/ml, 100 pg/ml or 1 ng/ml of a peptide of the ACTH/pro-opiomelanocortin 'family', 57K, 31K, 20K, alpha MSH, ovine-CLIP or gamma LPH either in the presence or absence of 166 pg/ml ACTH1-39. The production by cortisol and androstenedione was measured by radioimmunoassay. Using the steroid production by aliquots of the cell suspension with either no stimulating agent or ACTH1-39 alone as controls, the net influence of these different peptides on basal or ACTH1-39-stimulated production was observed. alpha MSH, ovine-CLIP and gamma LPH had no influence on either basal or stimulated cortisol or androstenedione production. Corticotrophic peptides of 57K, and 20K and pro-opiomelanocortin each had a steroidogenic activity alone, in all age groups. In the fetal and newborn monkeys' adrenal cells, peptides of 57K and 20K at 1 ng/ml had an inhibitory influence on ACTH1-39 stimulated cortisol and androstenedione production. The influence of the 20K peptide is partially inhibitory as the steroidogenic potential of this peptide is not additive with that of ACTH1-39. These results show that, as observed in other species, that the ACTH/pro-opiomelanocortin range of peptides are inhibitory to the action of ACTH1-39 in the developing adrenal.     

Therapy with bromocriptine and behavior of various hormones in psoriasis patients

The activity of bromocriptin has been clinically tested on 18 patients suffering from psoriasis. The plasma levels of Human Growth Hormone (HGH), Human Adrenocorticotropic Hormone (ACTH), Thyroid Stimulating Hormone (TSH), Prolactin (PL), Aldosterone and Cortisol were investigated in these 18 patients along with 35 untreated psoriatic patients and 19 normal subjects. Bromocriptin was shown to be effective in 13 of our 18 psoriatic patients. Plasma levels of HGH, ACTH, and Aldosterone, measured in all 53 psoriatic patients, were found to be higher than normal in 11, 26 and 40 patients respectively (HGH and Aldosterone: p less than 0,005; ACTH: p less than 0,001).     

Characterization of MSH/ACTH-like immunoreactivity in sciatic nerves of Xenopus laevis by immunocytochemistry, western blotting and radioimmunoassay

Previous studies have indicated that rat neurofilament protein may contain an endogenous MSH-like epitope with neuroregenerative properties. The presence of such an epitope has now been studied in nerve tissue from Xenopus laevis. Western blot analyses of sciatic nerve tissue using an assortment of sequence-specific MSH/ACTH antisera revealed the presence of two major immunoreactive protein bands of 52 and 50 kDa, which contained a mid-region MSH-like epitope. Weaker staining occurred in another protein band at 135 kDa. Immunocytochemistry revealed the immunoreactivity to reside in the axis cylinders of the nerve fibers. Other antisera, recognizing other regions of MSH/ACTH produced strong staining of Xenopus intermediate lobes, but failed to stain sciatic nerves. Thus, the proteins detected have no clear relation to either Xenopus neurofilament proteins or proopiomelanocortin.     

Effect of gamma 3 or gamma 2 melanocyte stimulating hormone on steroidogenesis in the fetal sheep during late gestation

We have measured circulating concentrations of gamma 3 Melanocyte Stimulating Hormone (MSH) in fetal sheep between 111 and 145 days gestation. There was no significant effect of gestational age on the fetal plasma concentrations of gamma 3 MSH throughout this period. We have examined the role of gamma-MSH related peptides in the control of fetal adrenal steroidogenesis and found no significant change in fetal plasma cortisol or pregnenolone concentrations during a 60-72 h infusion of saline, gamma 2 MSH or gamma 3 MSH in sheep between 130 and 135 days gestation. Therefore although we have demonstrated the presence of gamma MSH related peptides in fetal sheep plasma during late gestation we have failed to demonstrate a role for gamma 3 or gamma 2 MSH in the changes in fetal steroid concentrations which occur prepartum.     

The effect of MSH/ACTH 4-10 on delayed response performance and post-test locomotor activity in rats

Delayed response performance was measured in male, Long-Evans rats 1 hr after IP administration of various doses of MSH/ACTH 4-10 or control in a Hunter delayed reaction apparatus. Additional treatments consisting of naloxone 500 micrograms/kg (IP) and naloxone 500 micrograms/kg in conjunction with MSH/ACTH 4-10 95 micrograms/kg were also administered. Directly after delayed response performance was assessed, gross locomotor activity was determined. MSH/ACTH 4-10, at a dose of 95 micrograms/kg, significantly enhanced retention of a visual stimulus, while MSH/ACTH 4-10, at doses of 195 and 285 micrograms/kg, significantly impaired delayed response performance. Naloxone treatment resulted in significantly impaired delayed response performance when compared to control. However, naloxone plus MSH/ACTH 4-10 treatment failed to produce a significant difference from control in the delayed response performance paradigm. In post-test locomotor activity determination, an apparent dose-response existed for MSH/ACTH 4-10 with the two highest doses (190 and 285 micrograms/kg) resulting in significantly increased locomotor activity. The observed delayed response performance data support theories implicating MSH/ACTH peptides in attentional processes involving visual stimuli. The fact that large doses of MSH/ACTH 4-10 disrupt delayed response performance while increasing post-test activity suggest that an optimum level of effect caused by the MSH/ACTH peptide exists in this paradigm.     

Regulation of lymphokine (gamma-interferon) production by corticotropin

We have shown that corticotropin (ACTH), alpha-endorphin, and enkephalins can regulate antibody responses, which suggested a role for neuropeptides in a regulatory circuit between the immune and neuroendocrine systems. ACTH and structurally related peptides were examined here for regulation of mitogen induction of the lymphokine gamma-interferon (IFN gamma) in C57BL/6 mouse spleen cell cultures. Synthetic ACTH1-39 and a porcine pituitary extract containing ACTH activity were potent suppressors of the IFN gamma response. Synthetic ACTH1-39 suppressed the response by approximately 62% at 1 to 3 microM, whereas the porcine extract suppressed by greater than 90% at 1 to 3 microM ACTH. The greater potency of the pituitary extract was shown to be due to the presence of an additional peptide of Mr 2100 that was reactive with antibodies to the N-terminal region of ACTH (ACTH1-13), possessed potent anti-cellular activity against L cells and various transformed cells, but lacked ACTH biologic activity. The anti-cellular peptide suppressed the IFN gamma response by greater than 99% at 0.05 microM. The ACTH1-39 cleavage products, alpha-melanocyte stimulating hormone (alpha MSH; acetylated and amidated ACTH1-13), and corticotropin-like intermediate lobe peptide (CLIP; ACTH18-39) had no effect on IFN gamma production. ACTH1-24, like ACTH1-39, has full steroidogenesis activity but also had no effect on IFN gamma production, which suggests a dissociation of the immunoregulatory and steroidogenic properties of ACTH1-39. ACTH1-39, and possibly also the anti-cellular 2100 Mr peptide, is initially synthesized as the precursor polyprotein pro-opiomelanocortin (POMC). Enzymatic processing of POMC, first to the active ACTH1-39 or the anti-cellular peptide and then to the inactive smaller peptides, probably plays an important role in regulation of lymphokine and antibody production by ACTH and ACTH-related neuropeptides. This is consistent with the recent demonstration of the production of ACTH-like peptides by lymphocytes.     

Characterization of MSH/ACTH-like immunoreactivity in sciatic nerves of Xenopus laevis by immunocytochemistry,Western blotting and radioimmunoassay

Summary Previous studies have indicated that rat neurofilament protein may contain an endogenous MSH-like epitope with neuroregenerative properties. The presence of such an epitope has now been studied in nerve tissue from Xenopus laevis. Western blot analyses of sciatic nerve tissue using an assortment of sequence-specific MSH/ACTH antisera revealed the presence of two major immunoreactive protein bands of 52 and 50 kDa, which contained a mid-region MSH-like epitope. Weaker staining occurred in another protein band at 135 kDa. Immunocytochemistry revealed the immunoreactivity to reside in the axis cylinders of the nerve fibers. Other antisera, recognizing other regions of MSH/ACTH produced strong staining of Xenopus intermediate lobes, but failed to stain sciatic nerves. Thus, the proteins detected have no clear relation to either Xenopus neurofilament proteins or proopiomelanocortin.     

The MSH/ACTH(4-9) analog Org2766 counteracts isolation-induced enhanced social behavior via the amygdala.

MSH/ACTH-like peptides influence social behavior induced by isolation It has been previously demonstrated that changes in locomotor activity as a result of isolation can be counteracted by Org2766 via the amygdala. The present study investigates whether isolation-induced changes in social behavior can also be affected by this peptide via the amygdala. A fully automated observation system was applied for detailed registration and analysis of movements of group-housed and 7-day isolated rats in a social interaction test. Administration of the MSH/ACTH(4-9) analog into the central nucleus of the amygdala elicited decreased locomotion, approach, and avoidance behaviors after isolation as compared to placebo-treated controls. However, general activity and social interest of group-housed rats were not affected by the MSH/ACTH(4-9) fragment. It is hypothesized that the amygdala is a site of action for neuropeptides in modulating social behavior.     

Formaldehyde-ozone-induced fluorescence in pituitary ACTH cells: Effect of adrenalectomy

Summary ACTH and MSH cells of the pituitary are rich in peptides with NH₂-terminal tryptophan, as revealed by fluorescence histochemistry. Adrenalectomy stimulates the ACTH cells but not the MSH cells. As a result, ACTH as well as tryptophyl-peptides disappear from the ACTH cells but not from the MSH cells. It is concluded that the tryptophyl-peptides are stored together with the respective hormone in the ACTH and MSH cells and that tryptophyl-peptides in the ACTH cells are released together with the hormone.     

Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary.

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.     

Seasonal, circadian and episodic variations of human immunoreactive beta-MSH, ACTH and cortisol

The 24-hour profile of plasma levels of immunoreactive beta-MSH (IR-beta MSH), ACTH and cortisol was obtained at 15-min intervals in six normal males in summer and winter. In the radioimmunoassay used, dilution curves of human beta-MSH and human beta-LPH were not parallel. A seasonal variation in basal pituitary-adrenal secretion, with higher levels in winter than in summer, was demonstrated. A circadian rhythm was found to be significant for ACTH, IR-beta MSH and cortisol in all investigations. Whereas ACTH and cortisol patterns were largely concordant in all studies, there was a significant desynchronization of the circadian rhythm of IR-beta MSH as compared to ACTH in five cases. Eighty-three percent of the secretory spikes of cortisol but only 68% of the IR-beta MSH spikes were concomitant with an ACTH spike. Correlations between maximal levels of concomitant spikes were higher during the quiescent period of pituitary-adrenal secretion (22.00--04.00) for ACTH-cortisol whereas for ACTH-IR-beta MSH, highest correlations were found during the active early morning secretory phase (04.00--10.00). For the three plasma constituents studied, longer apparent half-lives were found to occur when the basal level before spiking was already elevated, suggesting that ACTH, beta-LPH and cortisol are secreted in bursts superimposed on a continuous basal secretion. Absolute increments of concentration appeared to be relatively independent of the level before spiking. It is suggested that the dissociations between ACTH and IR-beta MSH fluctuations in plasma observed here result from in vitro proteolysis of beta-LPH.     

Corticotropin-releasing factor (CRF) induced anorexia is not influenced by a melanocortin 4 receptor blockage.

CRF and melanocortin (MSH/ACTH) peptides share a number of central effects including anorexia and grooming. The effects of CRF may be secondary, due to CRF's effects on melanocortin peptide release. We investigated if the newly discovered selective melanocortin 4 receptor antagonist HS014 could influence CRF induced anorexia and grooming. The data show that ICV administration of CRF (3 mg/rat), significantly reduced food intake, feeding time and feeding episodes whereas it increased grooming time and grooming episodes. HS014 (5 mg/rat), that previously has been shown to antagonize the anorectic effect and the excessive grooming induced by alpha-MSH, did however not influence any of the behavioral effects induced by CRF when the peptides were administered together. The data indicate that the anorectic and grooming effects of CRF are independent of pathways involving the MC4 receptors. These data suggest that the anorectic and grooming effect of CRF are not due to a secondary effect caused by increase in release of melanocortins acting on the central MC receptors.     

Melanocortins and fast axonal transport in intact and regenerating sciatic nerve

The effect of ACTH/MSH peptides on fast axonal transport along intact or regenerating sciatic nerve was examined following injection of tritiated leucine into the rat lumbar spinal cord. The rate of fast axonal transport was not significantly changed by treatment with ACTH/MSH(4-10), the ACTH(4-9) analog ORG 2766, hypophysectomy, or adrenalectomy. Fast axonal transport was unchanged in regenerating nerves and in regenerating, ACTH(4-10)-treated nerves. However, treatment with ORG 2766 in dosages of either 1 or 10 micrograms/kg/day IP for seven days significantly reduced (62% and 64%, respectively) the crest height of the fast axonal transport curve of intact sciatic nerve. The results suggest that the reported peptide-induced enhancement of nerve regeneration is not due to changes in the rate of fast axonal transport.     

In situ melanin assay for MSH using mouse B16 melanoma cells in culture

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with alpha-MSH, [Nle4, D-Phe7]-alpha-MSH, [3'-iodo-Tyr2]-alpha-MSH, adrenocorticotropin (ACTH)(1-24), and ACTH(1-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of alpha-MSH was 27 pM, i.e., about five- to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.