Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. II. beta-Lipotropin (beta-LPH)-related peptides

We have studied the post-translational processing of POMC-derived peptides during fetal monkey pituitary development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Whole pituitary glands obtained from Day 50 and 55 fetal monkeys and separated lobes From Day 65 to 155 were extracted, fractionated, and analyzed for beta-melanotropin (beta-MSH), midportion beta-endorphin (beta-EP), and acetylated beta-EP immunoactivity. Separated adult pituitary lobes were analyzed for comparison. At Day 50, POMC-containing cells were located in both the anterior and intermediate pituitary lobes by immunofluorescence staining, the majority of these cells were localized in the anterior lobe. The Day 50 and 55 whole pituitaries contained predominantly beta-lipotropin (beta-LPH), gamma-lipotropin (gamma-LPH), beta-EP(1-31), and 2.2-kda beta-MSH. No acetylated products were found in Day 50 whole pituitary extracts. By Day 55, carboxy-shortened and acetylated beta-EPs were barely detectable in whole pituitary extracts. These forms were more apparent in the Day 65 separated neurointermediate lobe (NIL) extracts, and were similar to adult proportions by Day 80. The adult anterior lobe contained predominantly beta-LPH, beta-EP, and gamma-LPH. Adult NILs contained almost exclusively 2.2-kda beta-MSH, alpha-N-acetyl beta-EP(1-31) and alpha-N-acetyl beta-EP(1-27). The production of 2.2-kda beta-LPH in the monkey NIL indicates that monkey beta-LPH is different from rat beta-LPH in that it must contain the paired-basic cleavage site required for the formation of 2.2-kda beta-MSH that is known to be lacking in rat beta-LPH. Another finding was that monkey beta-EP contains a Tyr residue at position 27 as found in human beta-EP but appears to have the rat Gln substitution at position 31. The post-translational processing patterns characteristic of each lobe were well established by midterm fetal development (Day 80).     

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. I. Adrenocorticotropin (ACTH) and alpha-melanotropins (alpha-MSHs)

We have studied the post-translational processing of POMC-derived peptides during fetal monkey development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Pituitary tissues obtained from fetal monkeys ranging from Gestational Day 50 to 155 were fractionated and analyzed for ACTH- and alpha-MSH-related peptides and compared to adult forms. Extracts of whole pituitary from Fetal Days 50 and 55 contained ACTH(1-39) and very small amounts of CLIP (corticotropin-like intermediate-lobe peptide; ACTH(18-39))-like immunoactivity. Acetylated alpha-MSHs were not detectable at Day 50. alpha-MSHs were barely detectable at Day 55. By Day 65, when pituitary lobes were separable, small amounts of des-, mono-, and diacetyl alpha-MSH were detectable in NIL extracts, but not in anterior lobe extracts. ACTH(1-39) levels were negligible when compared to increasing alpha-MSHs through Fetal Day 80 to 155 in the intermediate lobe. The CLIP immunoactivity was negligible in Day 80 and adult anterior lobe extracts. Thus, lobe-specific proteolytic processing of ACTH-related peptides was well established by midterm gestation. Marked increases of alpha-N- and alpha-N,O-acetylated forms of alpha-MSHs were detected during middle and late stage fetal development. Diacetyl alpha-MSH was the predominant form of alpha-MSH in adult NIL extracts. No acetylated alpha-MSHs were found in anterior lobe tissues, thus adult anterior lobe extracts contained almost exclusively ACTH(1-39). However adult NIL extracts contained two distinct forms of CLIP-related immunoactivity. Therefore changes in post-translational processing patterns of ACTH-related and alpha-MSH-related peptides continued to some extent, postnatally. These data indicate that marked changes in post-translational processing of POMC-derived ACTH-related products occur during the first half of monkey gestation.     

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 pro-opiomelanocortin-derived peptides on plasma levels of glucagon, insulin and glucose

Pro-opiomelanocortin (POMC) is a prohormone for several peptides including corticotropin, melanocyte stimulating hormones and beta-endorphin. POMC-derived peptides have been demonstrated in many tissues, including the hypothalamus and the endocrine pancreas, which play important roles in the control of plasma levels of glucagon, insulin and glucose. This article reviews the present knowledge concerning in vitro and in vivo effects of POMC-derived peptides on glucagon, insulin and glucose levels involving several possible mechanisms: direct effects on the endocrine pancreas (including endocrine, paracrine and peptidergic regulation) and glucose production, and indirect effects involving the hypothalamus, the autonomic nervous system and the adrenal gland.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Synthetic peptides as substrates for processing enzymes in the bovine pituitary

Synthetic peptides, based on sequences of proopiomelanocortin (POMC) cleaved in both the bovine anterior and intermediate pituitaries (-Phe-Pro-Leu-Gly-Phe-Lys-Arg-Glu-Leu-Thr-Gly-) and only in the intermediate lobe (-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-), were used as substrates for the enzymes that process POMC to active hormones in the anterior and intermediate lobes of the pituitary. Cleavage of these peptides at the dibasic pair of residues, the expected cleavage site, was observed with a lysate from bovine pituitary secretory granules. Cleavage occurred optimally at a pH between 4 and 5 and was inhibited with sulfhydryl reagents, pepstatin, and leupeptin. Little specificity for the nature of the basic residues at the cleavage site was observed. An additional cleavage, following glutamic acid residues, was also seen.     

Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.     

Biosynthesis and processing of delta sleep-inducing peptide-like precursors in primary cultures of mouse anterior pituitary cells

The biosynthesis and processing of material resembling delta sleep-inducing peptide (DSIP) have been studied in mouse anterior pituitary primary cell cultures. Cells were pulse/chase incubated with 3H-labelled amino acids (Gly, Arg or Ala) and cell extracts were immunoprecipitated with DSIP antiserum. Labelled DSIP-related proteins were resolved by SDS/PAGE. Multiple forms of DSIP-immunoprecipitable material were observed, including three precursors of molecular mass 50-60 kDa which were processed to two major groups of intermediates of 35-45 kDa and 9-16.5 kDa. These intermediates appear to be processed to a DSIP-related peptide (molecular mass less than 3 kDa), which co-ran on reversed-phase HPLC with an endogenous form of DSIP in mouse anterior pituitary, but not with rabbit DSIP. This less than 3-kDa peptide incorporated [3H]Gly, but not [3H]Arg or [3H]Ala. In addition, it incorporated [3H]glucosamine, indicating that it was a glycopeptide. Secretion studies showed release of the less than 3-kDa DSIP-like glycopeptide and the 9-16.5-kDa group of intermediates into the medium. The present study demonstrates the biosynthesis of a small DSIP-like glycopeptide in mouse anterior pituitary cells, which is not identical with, but has similarities to, rabbit DSIP.     

Biosynthesis and posttranslational processing of site-directed endoproteolytic cleavage mutants of pro-neuropeptide Y in mouse pituitary cells

Although pairs of basic amino acids are common endoproteolytic sites in prohormones, the enzymes responsible for these cleavages have not yet been characterized. To investigate the specificity of these endoproteases, cDNAs encoding pro-neuropeptide Y (pro-NPY) containing all four pairs of basic amino acids were expressed in AtT-20 cells. Pro-NPY was selected as a model substrate because it undergoes a single cleavage at the sequence -Lys-Arg- during posttranslational processing. AtT-20 cells, a mouse anterior pituitary corticotrope line, were selected because they synthesize pro-adrenocorticotropic hormone (pro-ACTH)/endorphin and cleave a well characterized subset of the eight pairs of basic amino acids in the precursor. Altered cDNAs encoding pro-NPY with -Arg-Arg-, -Arg-Lys-, or Lys-Lys- at the cleavage site were used to generate stable cell lines. The production of NPY and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, reversed-phase high performance liquid chromatography, ion-exchange high performance liquid chromatography, tryptic peptide mapping, and microsequencing. Direct amino acid labeling confirmed the identity of the pair of basic amino acids at the cleavage site. Even when the four pairs of basic amino acids were presented in the same structural context, the rate, extent, and type of cleavage was substrate-specific. Pro-NPY(-Arg-Arg-) was cleaved at a rate similar to that observed for the wild-type pro-NPY(-Lys-Arg-). In contrast, pro-NPY(-Arg-Lys-) was cleaved at a much lower rate, and pro-NPY (-Lys-Lys-) was cleaved very poorly. Following endoproteolytic cleavage, the pair of basic amino acids present did not alter the production of mature NPY with a COOH-terminal Tyr-NH2. While two of the three mutant pro-NPY molecules were processed to wild-type carboxyl-terminal peptide, the carboxyl-terminal peptide derived from pro-NPY(-Arg-Lys-) contained an amino-terminal lysine residue, indicating that biosynthetic endoproteolysis occurred in the middle or at the amino terminus of the pair of basic amino acid residues at the cleavage site. Expression of wild-type or mutant pro-NPY inhibited cleavages within the endogenous pro-ACTH/endorphin; poorly cleaved pro-NPY mutants (Lys in the second position of the cleavage site) were the most potent inhibitors of pro-ACTH/endorphin cleavage.     

Secretion of N-terminal pro-opiomelanocortin-derived peptides in response to acute haemorrhagic stress in conscious sheep

To determine whether peptides derived from the N-terminus of the corticotropin/melanotropin/endorphin precursor, pro-opiomelanocortin, are released into blood in response to acute haemorrhagic stress, we examined the effect of haemorrhage on plasma concentrations of immunoreactive gamma 3-melanotropin, beta-endorphin and cortisol. Plasma concentrations of immunoreactive gamma 3-melanotropin (mean +/- SEM) increased within 30 min of haemorrhage from 71.1 +/- 10.4 to 106.8 +/- 6.3 fmol/mL (p less than 0.01) and plasma cortisol increased from 16.2 +/- 3.8 to 85.9 +/- 22.4 pmol/mL (p less than 0.025). The changes in plasma immunoreactive gamma 3-melanotropin and beta-endorphin were positively correlated (p less than 0.025). This study shows that peptides derived from the N-terminus of pro-opiomelanocortin are co-secreted with the C-terminal peptide beta-endorphin during acute haemorrhagic stress in sheep.     

Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development

We developed a simple methodology for labeling sulfated glycosaminoglycans (GAGs) in adult Drosophila melanogaster and studied some aspects of the biosynthesis and metabolism of these polymers during development. Adult D. melanogaster flies were fed with Na(2)(35)SO(4) for 72 h. During this period, (35)S-sulfate was incorporated into males and females and used to synthesize (35)S-sulfate-heparan sulfate (HS) and (35)S-sulfate-chondroitin sulfate (CS). The incorporation of (35)S-sulfate into HS was higher when compared to CS. In a pulse-chase experiment, we observed that (35)S-sulfate incorporated into adult female was recovered in embryos and used for the synthesis of new (35)S-sulfate-GAGs after 2 h of embryonic development. The synthesis of CS was higher than that of HS, indicating a change in the metabolism of these glycans from adult to embryonic and larval stages. Analysis of the CS in embryonic and larval tissues revealed the occurrence of nonsulfated and 4-sulfated disaccharide units in embryos, L1 and L2. In L3, in addition to these disaccharides, we also detected significant amount of 6-sulfated units that are reported here for the first time. Immunohistochemical analysis indicated that HS and CS were present in nonequivalent structures in adult and larval stages of the fly. Overall, these results indicate that (35)S-sulfate-precursors are transferred from adult to embryonic and larval tissues and used to assemble different morphological structures during development.     

Biosynthesis of type IX collagen during chick limb development

We analyzed the collagens synthesized by developing chick limbs (stages 22 to 34). Type IX collagen synthesis started at stage 26, concurrently with the chondrogenic differentiation of limb mesenchyme, and gradually increased during subsequent stages. By stage 34, the central cartilaginous region of the limbs substantially synthesized type IX collagen, in addition to cartilage-specific type II collagen, while the outer non-cartilaginous region of the limbs synthesized predominantly type I collagen. The present study indicates that type IX collagen is cartilage-specific and can be used as a marker for the chondrogenic phenotype.     

Dynorphin and the hypothalamo-pituitary-adrenal axis during fetal development

Although dynorphin has long been considered an endogenous opioid peptide with high affinity for the kappa-opioid receptor, its biological function remains uncertain. The high concentration of dynorphin peptides and kappa-opioid receptors in the hypothalamus suggest a possible role for dynorphin in neuroendocrine regulation. This review will summarize evidence that support a role for dynorphin in regulation of the developing hypothalamo-pituitary-adrenal (HPA) axis. Dynorphin can exert dual actions on adrenocorticotropin (ACTH) release: (i) via activation of hypothalamic kappa-opioid receptors leading to release of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and (ii) via a non-opioid mechanism that involves N-methyl-D-aspartate (NMDA) receptors and prostaglandins, and which is not dependent on CRH or AVP. The primary site of action of dynorphin and NMDA appears to be the fetal hypothalamus or a supra-hypothalamic site. The non-opioid mechanism does not mature until a few days prior to parturition and is active for only the brief perinatal period. In contrast, the opioid mechanism behaves as a constitutive system with sustained activity from prenatal to postnatal life. It is likely that the two mechanisms may respond to different stress stimuli and play a different role during development.