Molecular forms of α-melanocyte-stimulating hormone in the canine pituitary anterior and intermediate lobe

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of α-melanocyte-stimulating hormone (α-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of α-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-α-MSH, while monoacetyl-α-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-α-MSH was the major form of α-MSH. The profile of α-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-α-MSH and diacetyl-α-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of α-MSH predominate in PI tissue, while nonacetylated α-MSH is the major form in AL tissue. It appears, however, that acetylation of α-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.     

Pro-opiomelanocortin-derived peptides in the testis: evidence for a possible role in Leydig and Sertoli cell function

Pro-opiomelanocortin (POMC)-derived peptides such as beta-endorphin, ACTH, and MSHs were identified in the testis where they were exclusively localized in Leydig cells. Examination of testicular extracts by a variety of physicochemical and immunological techniques indicates that the processing of the POMC in the testis is very similar to that in the brain. By using a cDNA probe, the POMC-like mRNA present in total testis and cultured Leydig cells was 150-200 bases shorter than that in the hypothalamus and pituitary. In addition, POMC mRNA was localized to Leydig cells using in situ hybridization. The expression of the POMC-like gene and the accumulation of POMC-derived peptides in Leydig cell were shown to be under the control of gonadotropin. As the testis contains low concentrations of POMC-derived peptides, we suggested that they may be implicated in local regulatory events within this organ. This postulate was supported by results from in vivo and in vitro experiments suggesting that different portions of the POMC-molecule may have opposite effects on Sertoli cell functions. For example, MSHs increased cAMP accumulation and aromatase activity in these cells, while opioids inhibited Sertoli cell proliferation and androgen binding protein (ABP) secretion. Furthermore, following intratesticular administration of opiate antagonists, testosterone production was reduced, suggesting that Leydig cell function may be also modulated by beta-endorphin and/or other related peptides. Taken together, these studies support the hypothesis of a possible role of POMC-derived peptides in testicular function.     

Gamma-endorphin-like peptides in pituitary tissue: evidence for their existence in vivo

Beta and gamma endorphin-like peptides were measured by radioimmunoassay in whole pituitary. Boiling of acetic acid extracts prior to tissue disruption increased the concentration of both beta E- and gamma E-like peptides. The gamma E-like immunoreactivity from the neurointermediate lobe of the pituitary co-eluted with synthetic gamma E upon gel permeation chromatography. Immunoreactivity for beta E-like and gamma E-like peptides in the intermediate lobe of the pituitary was also shown by immunoperoxidase staining. The results suggest that gamma E-like peptides are present primarily in the pars intermedia in vivo and do not arise as artifacts of acid extraction of pituitary tissue.     

Particular processing of pro-opiomelanocortin in Xenopus laevis intermediate pituitary. Sequencing of alpha- and beta-melanocyte-stimulating hormones

alpha- and beta-melanocyte-stimulating hormones (alpha-MSH and beta-MSH) have been isolated from Xenopus laevis neurointermediate pituitary and microsequenced. Intracellular alpha-MSH is not N-acetylated after proteolytic processing of pro-opiomelanocortin in contrast to mammalian alpha-MSHs. There is a high preservation of the melanotropic amino acid sequence common to all MSHs although in Xenopus beta-MSH a histidine residue replaces the glutamic acid residue found in position 8 of mammalian beta-MSHs.     

A multicatalytical protease complex from pituitary that forms enkephalin and enkephalin containing peptides

The pituitary contains a high molecular weight (M.W.～700,000) neutral endopeptidase complex that behaves in solution as a single protein, but on polyacrylamide gel electrophoresis under dissociating conditions shows the presence of five components with molecular weights of 24,000 to 28,000. The complex exhibits trypsinlike, chymotrypsinlike and peptidylglutamyl-peptide bond hydrolysing activities. Experiments indicate that each of these activities is associated with a separate component of the complex. The chymotrypsinlike and trypsinlike activities efficiently generate Leu-enkephalin, Leu-enkephalin-Arg⁶ and Leu-enkephalin-Arg⁶-Phe⁷ from a single synthetic precursor. Selective inhibition of the chymotrypsinlike activity enhances the trypsinlike activity and alters the relative proportion of opioid peptides formed.     

Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers

On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQwl, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DRI-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1 . In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQwl-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.     

Update: brain and pituitary hormones of lampreys

Lampreys and hagfish of the class Agnatha are of particular importance in understanding endocrinological relationships since they represent the oldest lineages of extant vertebrates which evolved over 550 million years ago. This review briefly summarizes the latest findings on the reproductive endocrinology of the sea lampreys. Since the First International Symposium of Fish Endocrinology in 1988, when virtually little was known of the hypothalamic-pituitary-gonadal axis, substantial new biochemical, molecular, physiological and immunological evidence has now clearly shown that lamprey reproduction is controlled by the neuroendocrine axis. In addition, five brain and six pituitary hormones of lampreys have been identified mainly by Sower and Kawauchi and colleagues between 1986 and 2000. We now hypothesize that lamprey reproduction is a highly synchronized process that is initiated or mediated by a coordination of complex integration of environmental cues and hormonal mechanisms which is broadly similar to that exhibited by gnathostome vertebrates.     

Adrenocorticotrophic and melanocyte-stimulating peptides in the human pituitary

The adrenocorticotrophic and melanocyte-stimulating peptides of the human pituitary were investigated by means of radioimmunoassay, bioassay and physicochemical procedures. Substantial amounts of adrenocorticotrophin and a peptide resembling beta-lipotrophin were identified in pituitary extracts, but alpha-melanocyte-stimulating hormone, beta-melanocyte-stimulating hormone and corticotrophin-like intermediate lobe peptide, which have been identified in the pars intermedia of pituitaries from other vertebrates, were not found. The absence of beta-melanocyte-stimulating hormone appears to contradict previous chemical and radioimmunological studies. Our results suggest, however, that it is not a natural pituitary peptide but an artefact formed by enzymic degradation of beta-lipotrophin during extraction.     

Immunohistochemical localization of hormones and peptides in the human pituitary cells in a case of hypercortisolism by ACTH secreting microadenoma

This study assesses the action of hypercortisolism on the hormone and peptide periadenoma region of removed ACTH-producing microadenoma. Our findings show that cortisol excess affects both ACTH and GH production, with no immunoreaction for these hormones. The remaining pituitary hormones (TSH, FSH and PRL) and POMC-derived peptides (betaEnd, alphaMSH and betaMSH) were not modified. Likewise, we observed pituitary immunoreactive cells for Neurotensin (NT), Intestinal vasoactive peptide (VIP), Substance P (SP) and Angiotensin-II (Ang-II). The colocalization demonstrated that NT was expressed in thyrotrope and gonadotrope cells, VIP in gonadotrope cells and SP in corticotrope cells. The results about Ang-II were inconclusive. On the other hand, immunoreaction for the NPY and Gal peptides were not present. In the adenomatous cells, the peptide NT is present in ACTH cells as well as SP. These results suggest a peptide regulation of pituitary cells in the pathological state that can differ between normal and tumoural cells of the same pituitary.     

Structure-activity relationships for glycine-extended peptides and the alpha-amidating enzyme derived from medullary thyroid CA-77 cells

A peptidyl alpha-amidating enzyme has been partially purified from conditioned medium derived from cultured medullary thyroid CA-77 cells. The interactions of this enzyme with a series of tripeptides, pentapeptides, and mature glycine-extended prohormones has now been studied using a competition assay that features the enzymatic alpha-amidation of N-dansyl-Tyr-Val-Gly. While a peptide C-terminal glycine was obligatory for tight binding to the alpha-amidating enzyme, other peptide structural elements modulated the interaction. Thus, a greater than 1300-fold range in apparent inhibitor constants was observed by substitution at the -1 (penultimate) position in a C-terminal glycine-containing tripeptide with each of the 20 common L-amino acids. Peptide inhibitory potency decreased through the following amino acid groupings: sulfur containing greater than aromatic greater than or equal to histidine greater than nonpolar greater than polar greater than glycine greater than charged. This pattern was qualitatively dissimilar to that observed for a more limited series of substitutions at the -2 position, demonstrating the positional selectivity of these structural requirements. The structure-activity relationships observed with the tripeptides at the -1 position were consistent with the apparent inhibitor constants obtained for a collection of prohormones and their pentapeptide mimics. Finally, selected prohormones and their pentapeptide mimics were equipotent inhibitors, demonstrating that the peptide structural elements important for alpha-amidating enzyme recognition are located entirely within the C-terminal pentapeptide segment.     

Thymosin alpha 1-like peptides: localization and biochemical characterization in the rat brain and pituitary gland

Using a radioimmunoassay for thymosin alpha 1, endogenous thymosin-like peptides were characterized in the rat brain and pituitary gland. Thymosin alpha 1-like peptides were present in high concentrations in hypothalamus and pituitary extracts. These peptides were characterized using gel filtration techniques and the main peak of immunoreactive thymosin had a molecular weight similar to that of thymosin alpha 1 (3108 daltons). Using HPLC techniques, one main peak of immunoreactivity was present in brain extracts, whereas two peaks were present in pituitary extracts, one of which coeluted with thymosin alpha 1. The discrete regional distribution of thymosin alpha 1-like peptides was investigated and the highest densities of immunoreactive thymosin were present in the median eminence and arcuate nucleus of the hypothalamus, as well as the neurointermediate lobe of the pituitary. Due to the anatomical proximity of immunoreactive thymosin to loci containing known releasing factors and hormones, thymosin alpha 1-like peptides may function as neuroendocrine regulatory agents.     

Pro-opiomelanocortin peptides in the human fetal pituitary

Levels of immunoreactive pro-opiomelanocortin (POMC) peptides (N- and C-terminal ACTH, N- and C-terminal LPH and α-MSH) have been measured in pituitary extracts from human fetuses of 12–22 weeks gestation. The levels of ACTH were 30–200 times higher than α-MSH in all fetuses studied. Sephadex G-75 and G-25 chromatography of 8 extracts showed peaks of 34 kilodaltons (K) POMC, 22K ACTH, β-LPH, γ-LPH, β-endorphin, approximately 8K ACTH, 1–39 ACTH, α-MSH and CLIP. The 8K and 22K forms of ACTH are both partly glycosylated.In vitro culture of pituitaries from 2 fetuses (22 and 26 weeks gestation) gave a detectable basal output of ACTH but not of α-MSH. Stimulation of these pituitary cells with human fetal and rat hypothalamic extracts and with synthetic ovine CRF-41 produced a significant increase in ACTH release, and either small or undetectable amounts of α-MSH.These results demonstrate the presence of POMC-related peptides in early gestation human fetal pituitaries and suggest that ACTH, and not α-MSH, is the major corticotrophic hormone at this stage of gestation.     

Effects of glycine-extended and serine13-phosphorylated forms of peptide YY on food intake in rats

The gut hormone peptide YY(3-36)-amide [PYY(3-36)-NH₂] is significantly more potent than PYY(1-36)-NH₂ in reducing food intake in rats and humans. Other Gly-extended and Ser¹³-phosphorylated PYY forms have been detected or predicted based upon known cellular processes of PYY synthesis and modification. Here we compared the effects of 3-h IV infusion of PYY(1-36)-NH₂, PYY(3-36)-NH₂, PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser¹³(PO₃)-PYY(1-36)-NH₂, Ser¹³(PO₃)-PYY(3-36)-NH₂, Ser¹³(PO₃)-PYY(1-36)-Gly-OH, and Ser¹³(PO₃)-PYY(3-36)-Gly-OH during the early dark period on food intake in freely feeding rats. PYY(3-36)-NH₂ and Ser¹³(PO₃)-PYY(3-36)-NH₂ reduced food intake similarly at 50 pmol/kg/min, while only PYY(3-36)-NH₂ reduced food intake at 15 pmol/kg/min. PYY(1-36)-NH₂ and Ser¹³(PO₃)-PYY(1-36)-NH₂ reduced food intake similarly at 50 and 150 pmol/kg/min. In contrast, PYY(1-36)-Gly-OH, PYY(3-36)-Gly-OH, Ser¹³(PO₃)-PYY(3-36)-Gly-OH, and Ser¹³(PO₃)-PYY(1-36)-Gly-OH had no effect on food intake at doses of 50 or 150 pmol/kg/min. Taken together, these results indicate that (i) PYY(3-36)-NH₂ is significantly more potent than PYY(1-36)-NH₂ in reducing food intake, (ii) Gly-extended forms of PYY are significantly less potent than non-extended forms, and (iii) Ser¹³-phosphorylation of PYY(3-36)-NH₂ decreases the anorexigenic potency PYY(3-36)-NH₂, but not PYY(1-36)-NH₂. Thus, PYY(3-36)-NH₂ appears to be the most potent PYY form for reducing food intake in rats.     

alpha-Melanocyte-stimulating-hormone precursors in the pig pituitary.

The occurrence of intermediates from the processing of ACTH-(1-39) [adrenocorticotropic hormone-(1-39)] to alpha-melanocyte-stimulating hormone was investigated in normal pig pituitaries by the use of sensitive and specific radioimmunoassays for ACTH-(1-13), ACTH-(1-14), ACTH-(1-13)-NH2 and ACTH-(1-39). Fractionation by reverse-phase h.p.l.c. revealed ACTH(1-17) and their acetylated analogues. The intermediate lobe contained NO-diacetyl-ACTH-(1-13)-NH2, N-acetyl-ACTH-(1-13)-NH2 and ACTH-(1-13)-NH2. In addition, the corresponding ACTH-(1-14) peptides (the glycine-extended precursor of the amidated peptides) were detected in lower amounts in both the intermediate lobe and the anterior lobe. ACTH-(1-17), ACTH-(1-13) and their acetylated analogues could not be detected in the anterior lobe or the intermediate lobe. The results suggest that an endopeptidase initially cleaves ACTH-(1-39) at the Lys-16-Arg-17 bond. ACTH-(1-16) is then processed by a pituitary carboxypeptidase to ACTH-(1-14) and ACTH-(17-39) by the aminopeptidase to ACTH-(18-39).