Des-acetyl alpha-MSH Research Articles

Agouti-related protein (83-132) is a competitive antagonist at the human melanocortin-4 receptor: no evidence for differential interactions with pro-opiomelanocortin-derived ligands.

Interactions between pro-opiomelanocortin (POMC)-derived peptides, agouti-related protein (AGRP) and the melanocortin-4 receptor (MC4-R) are central to energy homeostasis. In this study we have undertaken comprehensive pharmacological analysis of these interactions using a CHOK1 cell line stably transfected with human MC4-R. Our main objectives were (1) to compare the relative affinities and potencies of POMC-derived peptides endogenously secreted within the hypothalamus, (2) to investigate the potency of AGRP(83-132) antagonism with respect to each POMC-derived peptide and (3) to determine whether AGRP(83-132) and POMC-derived peptides act allosterically or orthosterically. We have found that beta melanocyte-stimulating hormone (betaMSH), desacetyl alpha MSH (da-alphaMSH) and adrenocorticotrophic hormone all have very similar affinities and potencies at the MC4-R compared with the presumed natural ligand, alphaMSH. Moreover, even MSH precursors, such as beta lipotrophic hormone, showed significant binding and functional activity. Therefore, many POMC-derived peptides could have important roles in appetite regulation and it seems unlikely that alphaMSH is the sole physiological ligand. We have shown that AGRP(83-132) acts as a competitive antagonist. There was no significant difference in the potency of inhibition by AGRP(83-132) or agouti(87-132) at the MC4-R, regardless of which POMC peptide was used as an agonist. Furthermore, we have found that AGRP(83-132) has no effect on the dissociation kinetics of radiolabelled Nle4,D-Phe7 MSH from the MC4-R, indicating an absence of allosteric effects. This provides strong pharmacological evidence that AGRP(83-132) acts orthosterically at the MC4-R to inhibit Gs-coupled accumulation of intracellular cAMP.

Skin POMC peptides: their actions at the human MC-1 receptor and roles in the tanning response.

The melanocortin 1 (MC-1) receptor is a key control point in the regulation of skin pigmentation. Alpha-MSH is an agonist at this receptor and through its activation regulates melanocyte function. alpha-MSH is cleaved from pro-opiomelanocortin (POMC) in the pituitary, but in humans the skin is a more important source of the peptide. Skin pigmentation is therefore regulated by locally produced alpha-MSH rather than that of pituitary origin. alpha-MSH acts as a paracrine and/or autocrine mediator of UV induced pigmentation. However, the predominant alpha-MSH in human skin is desacetyl alpha-MSH and, compared to the acetylated form, is a relatively weak agonist at the human MC-1 receptor. By acting as a partial agonist desacetyl alpha-MSH may even oppose the actions of acetylated alpha-MSH and other MC-1 receptor agonists. The most abundant MC-1 receptor agonist in human epidermis is ACTH1-17. This POMC peptide, which is produced by keratinocytes, is more potent than acetylated alpha-MSH in stimulating melanogenesis in human melanocytes and, in contrast to the latter, produces a biphasic dose-response curve. This is probably a consequence of its activation of both the cAMP and IP3/DAG signalling pathways. alpha-MSH peptides, on the other hand, selectively activate the cAMP pathway. Compared with alpha-MSH, ACTH1-17 could have the more important role as a paracrine mediator of melanogenesis and other melanocytic processes. However, ACTH1-17 is not the only POMC peptide in the skin and may interact with related peptides at the MC-1 receptor. These interactions are likely to represent important determinants of melanocyte function and skin pigmentation.

Alpha-MSH and the regulation of melanocyte function.

alpha-MSH, has numerous actions in the skin and by activating the MC1 receptor (MC1-R) on melanocytes it stimulates melanogenesis. Rather than producing large increase in melanin production alpha-MSH acts specifically to stimulate eumelanin synthesis. Although this could be important in determining skin color and tanning there is debate as to the pigmentary significance of alpha-MSH in humans. Circulating levels of alpha-MSH are negligible and although it is produced in the skin by different cell types, including melanocytes, the major skin form is desacetyl alpha-MSH, and this is a weak agonist at MC1-R. Certain ACTH peptides, notably ACTH1-17, are more potent agonists at the MC1-R and, since their skin concentrations exceed those of alpha-MSH, they could serve as natural ligands at this receptor and regulate pigmentary responses in humans. Activation of MC1-R does, however, produce other responses in human melanocytes. Thus, alpha-MSH stimulates melanocyte dendricity and attachment to extracellular matrix proteins. It also protects melanocytes from the damaging effects of oxidative stress, and regulates their production of NO by modulating the induction of iNOS--as it does within macrophages. alpha-MSH clearly affects various aspects of melanocyte behavior and its melanogenic effects could be the consequence of a more fundamental role in the melanocyte. The precise nature of this role is unclear, but it could be part of a generic role that alpha-MSH and other POMC peptides have in skin homeostasis.

Characterisation of ACTH peptides in human skin and their activation of the melanocortin-1 receptor.

Alpha-Melanocyte-stimulating hormone (alpha-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 alpha-MSH, is also an agonist at the MC-1 receptor. By using immunocytochemistry, we confirmed the presence of alpha-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 alpha-MSH, respectively. The amounts of immunoreactive ACTH exceeded those of alpha-MSH. Using HPLC we identified for the first time the presence of ACTH1-39, ACTH1-17, ACTH1-10, acetylated ACTH1-10, alpha-MSH, and desacetyl alpha-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 > alpha-MSH > ACTH1-39 > desacetyl alpha-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 alpha-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 alpha-MSH, they may have a role in the regulation of human melanocytes.

Differential acetylation of pro-opiomelanocortin-derived peptides in the pituitary gland of Xenopus laevis in relation to background adaptation

Immunocytochemical analysis revealed the presence of acetylated endorphins in both melanotropes and corticotropes of the pituitary gland of Xenopus laevis. Chemical acetylation studies to determine the steady-state level of acetylated versus non-acetylated endorphins showed that virtually all endorphins are acetylated in both melanotropes and corticotropes. Apparently Xenopus is unique among vertebrates as non-acetylated endorphins are major end-products in the distal lobe of all other vertebrate species studied thus far. The dynamics of endorphin biosynthesis in melanotrope cells using pulse-chase analysis coupled to immunoaffinity chromatography revealed that processing of pro-opiomelanocortin to produce N-terminal-acetylated endorphins is very rapid. To determine the effect of long-term background adaptation on acetylation status of endorphins and alpha-MSH-related peptides, Xenopus laevis were adapted for 3 or 6 weeks to either a black or a white background. In both physiological states the major intracellular form of alpha-MSH-related peptides in melanotropes was desacetyl alpha-MSH while the major endorphin-related peptide was alpha, N-acetyl-beta-endorphin[1-8]. In the medium of superfused neuro-intermediate lobes of black background-adapted animals the major form of secreted melanotropins and endorphins was alpha-MSH and alpha, N-acetyl-beta-endorphin[1-8] respectively. We conclude that there is a marked spatio-temporal difference in acetylation of melanotropin and endorphins, with rapid intracellular acetylation of endorphins while melanotropin is acetylated at the time of its exocytosis. In the medium of superfused neurointermediate lobes of white background-adapted animals the amount of desacetyl alpha-MSH was much higher than in the medium of lobes of black-adapted animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The processing of beta-endorphin and alpha-melanotrophin in the pars intermedia of Xenopus laevis is influenced by background adaptation.

beta-Endorphin- and alpha-melanotrophin (alpha-MSH)-related peptides were extracted from the pars intermedia of Xenopus laevis maintained for 2, 4 or 6 weeks on a white background and for the same periods on a black background. The peptides were resolved under dissociating conditions by gel exclusion chromatography on Sephadex G-50 and they were detected by radioimmunoassay with antibodies to beta-endorphin, alpha,N-acetyl beta-endorphin and alpha-MSH. The beta-endorphin-related peptides separated into two fractions of different molecular size. Further purification of the peptides in each fraction was by ion exchange chromatography on SP-Sephadex C-25 and by high-pressure liquid chromatography. The alpha-MSH-related peptides were resolved by gel exclusion and ion exchange chromatography. The purified beta-endorphin- and alpha-MSH-immunoreactive peptides were identified by comparison of their chromatographic properties with the corresponding peptides from porcine pituitary or by comparison with synthetic peptides. The major form of beta-endorphin in the pars intermedia of the frog adapted to a white background was identified as alpha,N-acetyl beta-endorphin (1-8); it was accompanied by a small quantity of acetylated peptides with molecular size similar to beta-endorphin. In contrast, the pars intermedia of the frogs adapted to a black background contained approximately equal amounts of alpha,N-acetyl beta-endorphin (1-8) and the larger forms of beta-endorphin. The higher molecular weight forms were identified as the alpha,N-acetyl derivatives of beta-endorphin (1-26), (1-27) and (1-31); however after 6 weeks of white adaptation the sole remaining peptide in this group was the 26-residue peptide. An additional beta-endorphin immunoreactive peptide, provisionally identified as beta-endorphin (10-26), was present in both black- and white-adapted animals; the amounts of this peptide increased during white adaptation. Major differences in the processing of alpha-MSH were also observed. In the frogs adapted to a black background des-acetyl alpha-MSH greatly predominated over the acetyl form whereas after 6- weeks adaptation to a white background the acetylated peptide proved to be the principal component. The results demonstrate that the proteolytic processing of beta-endorphin and the acetylation of alpha-MSH in Xenopus laevis are influenced by background adaptation. The formation of beta-endorphin (1-8) appears to reflect the action of an endopeptidase that acts at the single arginine residue present at position 9.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha-melanocyte-stimulating hormone (alpha-MSH) in the brain of the African lungfish, Protopterus annectens: immunohistochemical localization and biochemical characterization.

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) containing neurons and the molecular forms of alpha-MSH-related peptides exhibit substantial differences in the brains of fish and amphibians. Lungfishes, which share similarities with both fishes and tetrapods, represent a valuable group in which to investigate the neuroanatomical and neurochemical facets of evolution. In the present study, we have localized and characterized alpha-MSH-immunoreactive peptides in the central nervous system of the African lungfish Protopterus annectens. Perikarya exhibiting alpha-MSH-like immunoreactivity were observed in two distinct regions of the hypothalamus: the rostral part of the preoptic nucleus and the caudal part of the hypothalamus. In the caudal hypothalamus most alpha-MSH-immunopositive perikarya were located in both the subependymal and deepest layers of the ventral periventricular region. Scattered alpha-MSH-immunopositive cells were occasionally detected in the dorsal side of the caudal hypothalamus. The alpha-MSH-immunoreactive material localized in the brain was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The displacement curves obtained with synthetic alpha-MSH and serial dilutions of brain and pituitary extracts were parallel. HPLC analysis of lungfish hypothalamic extracts showed that the major immunoreactive peak coeluted with synthetic desacetyl alpha-MSH and its sulfoxide derivative. An additional peak coeluted with synthetic sulfoxide alpha-MSH. In contrast, in the pituitary, the predominant form of alpha-MSH-like material coeluted with the N,O-diacetyl alpha-MSH standard. These results provide the first evidence for the presence of alpha-MSH-related peptides in the brain of a lungfish. The distribution of alpha-MSH neuronal systems in the lungfish is very similar to that reported in amphibians, supporting the existence of phylogenetic convergences between these two vertebrate groups.

PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.

A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PC1 and PC2, since in the cell lines analyzed (i) PC1 cleaves POMC into corticotropin and beta-lipotropin, (ii) PC2 cleaves POMC into beta-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either alpha MSH or desacetyl-alpha MSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PC1 and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissue-specific maturation products in the brain and in peripheral tissues.

Interaction between β-Endorphin and a-Melanocyte- Stimulating Hormone in the Control of Prolactin and Luteinizing Hormone Secretion in the Primate*

The ability of alpha MSH, a POMC-derived peptide, to antagonize the effects of beta-endorphin (beta EP) on PRL and LH secretion was studied in the primate. Seven ovariectomized rhesus monkeys bearing chronic indwelling third ventricular catheters for peptide infusion were used for these studies. Peripheral blood samples for PRL and LH RIA were obtained every 15 min during a 3-h control period when saline was infused into the ventricle, followed by a 5-h period of peptide infusion at a rate of 25 microliters/h. When beta EP was infused at a dose of 5 micrograms/h, plasma PRL rose from a mean baseline of 3.5 +/- 0.7 ng/ml to a peak of 21.3 +/- 2.2 ng/ml. When the same animals were infused with 20 micrograms alpha MSH together with 5 micrograms beta EP, the peak concentration of PRL was reduced to 8.2 +/- 1.7 ng/ml (P less than 0.001). When a higher dose of beta EP (20 micrograms/h) was infused, PRL rose to a peak of 38.2 +/- 1.8 ng/ml. This response was again markedly blunted, and the peak PRL response was reduced to 7.3 +/- 2.2 ng/ml when 20 micrograms beta EP were infused together with 80 micrograms alpha MSH (P less than 0.001). Analysis of the area under the plasma PRL concentration curves demonstrated a significant reduction in area during the 5-h infusion with beta EP plus alpha MSH compared to that during infusion of beta EP alone. The mean area was reduced from 3480 +/- 570 ng min/ml after 5 micrograms beta EP alone to 1030 +/- 200 after 5 micrograms beta EP plus 20 micrograms alpha MSH and from 6230 +/- 990 after 20 micrograms beta EP to 1020 +/- 320 ng min/ml after 20 micrograms beta EP plus 80 micrograms alpha MSH (P less than 0.01). Des-acetyl alpha MSH (80 micrograms) was also effective in reducing the PRL response to 830 +/- 380 ng min/ml (P less than 0.05). The suppression of pulsatile LH release by beta EP was also attenuated by alpha MSH. During the 5-h infusion of beta EP, total LH secretion was reduced to 65.9 +/- 3.8% of that measured during the 3-h saline infusion compared to 87.2 +/- 2.7% after infusion of beta EP plus alpha MSH (P less than 0.001) or 91.7 +/- 4.1% after beta EP plus des-acetyl alpha MSH (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha-Melanocyte-Stimulating Hormone Immunoreactivity in Melanoma Cells

Using a radioimmunoassay specific for alpha-melanocyte-stimulating hormone (alpha-MSH), significant levels of immunoreactivity were detected in a range of murine and human melanoma cell lines, including a series of ras-transfected melanocytes. The levels found in the melanoma cell lines tested varied, and overall were higher than in non-melanoma cell lines assayed for comparison. Furthermore the highest levels of immunoreactivity measured tended to be in the least differentiated and most metastatic melanoma lines. High performance liquid chromatography showed a peak of immunoreactivity which co-migrated with a desacetyl alpha-MSH standard. Additional unidentified components of immunoreactivity were found, including a high molecular weight form revealed by Sephadex-G50 gel exclusion. These may represent bound alpha-MSH or fragments of the proopiomelanocortin precursor having in common the C-terminus epitope recognised by the antibody. In view of the known effects of alpha-MSH on anchorage independent growth and metastasis of melanoma cells, our findings raise the possibility that MSH peptides may have an autocrine role in the growth and progression of melanoma. However, further characterisation of the immunoreactive species is required to determine whether these represent biologically active forms.

Demonstration of desacetyl α-melanocyte-stimulating hormone in fetal and adult human anterior pituitary corticotrophs

Immunocytochemistry, radioimmunoassay and high-performance liquid chromatography (HPLC) techniques have been used in combination to investigate the presence of immunoreactive (ir)-alpha-MSH in the normal human pituitary gland, and to investigate the possible origin of these cells from the fetal pars intermedia. Two separate antisera to alpha-MSH were employed in immunocytochemistry to distinguish between authentic alpha-MSH and the desacetyl form. Only desacetyl alpha-MSH was detected in the pituitary gland of fetal and adult man, in both the pars (zona) intermedia and the pars anterior. In the fetus, a large proportion of the ACTH-containing cells of the anterior lobe also contained ir-alpha-MSH, while ir-alpha-MSH containing cells were more sparse in adults. Radioimmunoassay of acid extracts of adult pituitary tissue showed alpha-MSH levels representing less than 0.05% of the ACTH content of the gland. HPLC analysis of these extracts confirmed that only the desacetyl form was present. These results suggest that alpha-MSH peptides are synthesized by anterior lobe cells of the human pituitary gland, which are not derived from the fetal pars intermedia. Possible regulatory mechanisms affecting cells which contain ir-alpha-MSH are discussed, and by comparison with the intermediate lobe of other species it is concluded that there is little evidence for a true intermediate lobe in the human pituitary gland.

A BROMOCRIPTINE‐RESPONSIVE CORTICOTROPH ADENOMA SECRETING α‐MSH IN A PATIENT WITH CUSHING'S DISEASE

Clinical and in-vitro investigations have been performed on a corticotroph adenoma removed from a patient suffering from Cushing's disease. Prior to surgery, the patient's Cushing's disease had been successfully controlled, clinically and biochemically, by long term administration of bromocriptine. After selective adenomectomy, tumour tissue was investigated by a perfused isolated cell column technique. It was shown that the tumour cells secreted immunoreactive- (IR)- ACTH and IR-alpha-MSH and that the release of both peptides was promptly suppressed by dopamine. Chromatographic analysis of the secreted IR-alpha-MSH revealed a high proportion of acetylated alpha-MSH; smaller amounts of desacetyl alpha-MSH and diacetyl alpha-MSH were present. The relevance of these findings to the proposal that certain corticotroph adenomas are derived from the intermediate lobe of the pituitary is discussed. It is concluded that there is little direct evidence for involvement of the residual zona intermedia of the adult human pituitary in the development of Cushing's disease.

Antagonism of beta-endorphin-induced prolactin release by alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide.

The ability of two proopiomelanocortin-derived peptides, alpha MSH and corticotropin-like intermediate lobe peptide (CLIP) [ACTH (18-39)] to antagonize the stimulation of PRL secretion by beta-endorphin (beta EP) was studied in the rat. When 50 ng beta EP were injected into the lateral cerebral ventricle, plasma PRL rose from a mean baseline of 1.87 +/- 0.43 ng/ml (+/- SEM) to a peak of 23.0 +/- 3.67 ng/ml 10 min after the injection. When the same animals received 500 ng alpha MSH together with 50 ng beta EP, the peak concentration of PRL was reduced by 74% to 6.05 +/- 1.43 ng/ml (P less than 0.005). After the injection of 500 ng CLIP together with 500 ng beta EP, the peak concentration of PRL was reduced by 47% to 12.8 +/- 3.09 ng/ml (P less than 0.01). Total PRL release, determined by calculating the areas under the plasma PRL concentration curves, was also significantly reduced by the injection of alpha MSH or CLIP. A dose of 100 ng alpha-MSH or CLIP also antagonized the stimulation of PRL secretion by 50 ng beta EP. PRL release was reduced by 62% after administration of 100 ng alpha MSH (P less than 0.001) and by 43% after 100 ng CLIP (P less than 0.05). When 100 ng alpha MSH and 100 ng CLIP were injected together, there was an additive effect in blocking the stimulation of PRL release by beta EP, and the peak plasma PRL concentration was reduced by 81%. Des-acetyl alpha MSH, the predominant form of alpha MSH in the hypothalamus, was also very effective in antagonizing beta EP-induced PRL release. The peak PRL concentration was reduced by 52% after administration of 100 ng des-acetyl alpha MSH plus 50 ng beta EP compared with that after beta EP alone (P less than 0.005). We conclude that relatively low doses of both alpha MSH and CLIP can effectively antagonize the actions of beta EP on pituitary PRL release. These findings suggest the possibility that differential posttranslational processing of proopiomelanocortin may serve as a regulator of anterior pituitary function.

Stimulation of Adenosine 3′,5′-Monophosphate Production in Rat Sertoli Cells byα-Melanotropin- Stimulating Hormone (αMSH) and Des-AcetylαMSH*

Proopiomelanocortin and its derivative peptides alpha MSH and beta-endorphin are produced by Leydig cells. beta-Endorphin or another testicular opiate is believed to suppress Sertoli cell hypertrophy. The goal of this study was to determine the effects of another proopiomelanocortin-derived peptide on Sertoli cells. The activities of both alpha MSH and des-acetyl alpha MSH have been compared, since this latter peptide has been identified in testicular extracts. Both alpha MSH and des-acetyl alpha MSH stimulated cAMP accumulation in the media of primary Sertoli cell cultures when incubated in the presence of a phosphodiesterase inhibitor, FSH or forskolin. Both peptides shifted the FSH dose-response curve to the left, making the cells more sensitive to this gonadotropin. The apparent potencies of alpha MSH and its des-acetyl derivative, as measured in Sertoli cells, were similar. We conclude that the MSHs are one of a group of modulators regulating Sertoli cells via the cAMP system, and Sertoli cells are equally responsive to alpha MSH and des-acetyl alpha MSH, unlike central nervous system and melanocytes which show differential responses to these peptides.

Three types of alpha-melanocyte-stimulating hormone: bioactivities and half-lives.

Three types of alpha-melanocyte-stimulating hormone (alpha MSH) that differ in the acetyl status of the N-terminal serine have been found in the neurointermediate lobe of the pituitary gland and in the brain: desacetyl alpha MSH, which lacks an acetyl group; monoacetyl alpha MSH, in which the amino group of the serine is acetylated; and diacetyl alpha MSH, in which both amino and hydroxy groups of the serine are acetylated. We compared the lipolytic and melanotropic actions of these three peptides, and their rates of disappearance from plasma, both in vitro and in vivo. The following differences were found. a) For in vitro lipolytic actions on rabbit adipose tissue slices, the potencies differed according to the order diacetyl = monoacetyl greater than desacetyl. On rabbit isolated adipocytes, however, the three peptides were equipotent. b) For in vivo lipolytic action in the rabbit, not only potency but also kinetics differed. Diacetyl alpha MSH had the slowest onset, longest duration, and greatest potency. The desacetyl variant had the quickest onset, shortest duration, and least potency. c) The half-life for elimination from rabbit plasma both in vitro and in vivo was shortest for the desacetyl form and longest for the diacetyl peptide. d) For in vitro melanotropic effect on frog skin, kinetics of action were the same for all three peptides, but potency differed according to the order diacetyl = monoacetyl greater than desacetyl. Thus acetylation of alpha MSH alters lipolytic and melanotropic potencies in vitro and lipolytic potency and kinetics in vivo. These differences result in part from the fact that acetylation slows the degradation of the tridecapeptide both inside and outside the circulation.

Alpha-Melanocyte-stimulating hormone-like peptides in the intermediate lobe of the rat pituitary gland: characterization of content and release in vitro.

Reverse phase high performance liquid chromatography (HPLC) followed by RIA of the chromatographic fractions was used to separate and quantify, respectively, the alpha MSH-like peptides stored in the intermediate lobe (IL) of the rat pituitary gland and released from IL cells in vitro. Immunoreactive material eluting with the same HPLC retention time as N,O-diacetyl alpha MSH accounted for approximately 80% of the total immunoreactive alpha MSH (IR-alpha MSH) in either the neurointermediate lobe or dispersed IL cells. The remainder of the IR-alpha MSH coeluted with either synthetic desacetyl alpha MSH or alpha MSH. Furthermore, the predominant alpha MSH-like compound released in vitro from dispersed IL cells eluted from the HPLC column with the same retention time as synthetic N,O-diacetyl alpha MSH. Treatment of dispersed IL cells with drugs known to enhance (l-isoproterenol or A 23187) or to inhibit (apomorphine or lisuride) the release of IR-alpha MSH revealed that N,O-diacetyl alpha MSH was the primary form released. Finally, an evaluation of the stability of the alpha MSH-like peptides indicated that N,O-diacetyl alpha MSH was readily converted to alpha MSH in the presence of 0.1 N hydrochloric acid.

Characterization of immunoreactive α-melanocyte stimulating hormone ( α-MSH i) in human brain tissue

Adult human hypothalamic tissue was analyzed for the presence of products generated by post-cleavage processing of ACTH 1-13. The displacement curve generated by immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHi) in extracts (5 M acetic acid or acidified ethanol) of adult human hypothalamic tissue was parallel to the alpha-MSH radioimmunoassay standard curve, and alpha-MSHi eluted as a single peak on Sephadex G-10, G-25, and G-50 columns in a manner identical to that of synthetic alpha-MSH. The alpha-MSHi was clearly distinguishable in its mobility on Sephadex G-50 columns from such peptides as beta-MSH, luteinizing hormone releasing hormone, and ACTH. After high performance liquid chromatography of extracts of hypothalamic tissue from young as well as aged men and women, we found that the major peak of alpha-MSHi (75-95% of total alpha-MSHi) coeluted with desacetyl alpha-MSH (ACTH 1-13 amide) rather than alpha-MSH. We suggest that desacetyl alpha-MSH, rather than alpha-MSH, is the predominant alpha-MSHi in adult human brain tissue.

The major immunoreactive alpha-melanocyte-stimulating hormone (alpha MSH)-like substance found in human fetal pituitary tissue is not alpha MSH but may be desacetyl alpha MSH (adrenocorticotropin1-13NH2).

Pituitary glands were obtained from human abortuses during the second half of gestation. Acid extracts were made from the anterior and neurointermediate lobes, and alpha MSH immunoreactivity (alpha MSHi) was quantified by RIA. alpha MSHi was found in both lobes of the pituitary gland, with 20-80% of the total pituitary alpha MSHi being present in extracts of the anterior lobe. Anterior and neurointermediate lobe extracts subjected to gel filtration on Sephadex G-50 revealed one peak of alpha MSHi having an elution profile identical to those of alpha MSH and desacetyl alpha MSH (ACTH1-13NH2). To characterize further the alpha MSHi extracts were subjected to high pressure liquid chromatography. No alpha MSH could be identified in extracts of the anterior lobe, and most of the alpha MSHi had an elution profile identical to that of desacetyl alpha MSH. Although small amounts of alpha MSH might be present in the neurointermediate lobe, most of the alpha MSHi in this lobe coeluted with desacetyl alpha MSH. Since alpha MSH was not converted to desacetyl alpha MSH during the extraction and chromatographic procedures, we hypothesize that the predominant form of alpha MSH-like material in the human fetal pituitary gland may be desacetyl alpha MSH.

Distribution, subcellular localization and identity of immunoreactive alpha-melanotropin in the pituitary gland and brain.

In homogenates of adult human or rat hypothalamic tissue, immunoreactive alpha-MSH (alpha-MSHi) is concentrated in synaptosomes, suggesting that it is localized in neurons. This conclusion is supported by immunohistochemical results on the localization of alpha-MSHi in the brain. The function of alpha-MSHi in the hypothalamus is undefined but the amount of alpha-MSH in this tissue increases in young rats and decreases in old rats. There is little alpha-MSHi in pituitary glands from human abortuses before the 15th gestational week. Thereafter the amount of alpha-MSHi in the anterior and neurointermediate lobes increases appreciably. When acetic acid extracts of each lobe were analysed by high-pressure liquid chromatography the alpha-MSHi in extracts of the anterior lobe had a retention time equal to that of desacetyl alpha-MSH; little if any alpha-MSHi had a retention time identical to that of desacetyl alpha-MSH; alpha-MSH, if present in these extracts, appeared to be a minor component. A similar analysis of extracts of adult human hypothalamic tissue yielded results suggesting that the alpha-MSHi in this tissue is also attributable largely to desacetyl alpha-MSH.