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Total RNA was extracted from Mustella vison pituitary gland, and cDNA for proopiomelanocortin mRNA was synthesised and cloned. A 600 b. p. insert encoding for total ACTH, beta LPH and 3'-nontranslated end of the mRNA was sequenced using the Maxam-Gilbert technique.
Cloning of DNA and complementary mRNA of bovine, rat and human proopiomelanocortin (POMC) was carried out. A structural analysis of the cloned cDNA of POMC was performed. Using restriction fragments of bovine, rat and human POMC cloned cDNA, probes for molecular hybridization based on one-chain bacteriophage M13 were made. Using the dot-hybridization technique with labeled [32P] POMC cDNA, the effect of 17 beta-estradiol and adrenalectomy on the POMC mRNA level in rat hypophysis was studied. The hormone was shown to significantly decrease the POMC mRNA content at a concentration of 4 and 8 micrograms per 100 g of body weight 4 hours after injection. Adrenalectomy caused a 2-3-fold increase in the POMC mRNA level in rat hypophysis after a period of 5-7 days. The possibility of polyhormonal control of POMC genome transcription is discussed.
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Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.
In situ hybridization is a method for detecting specific nucleotide sequences by using a labeled complementary nucleic acid probe. We have characterized in situ hybridization in rat pituitary using a cDNA probe directed against a portion of proopiomelanocortin (POMC) mRNA to show its specificity in tissue sections. This characterization includes the size of the target mRNA from tissue processed for in situ hybridization, the dissociation pattern of the cDNA:mRNA hybrid, and quantitation of changes in POMC mRNA levels in the rat pituitary intermediate lobe detected both by in situ hybridization and "dot blot" techniques.
The cells of the frog pars intermedia synthesize a 36 000 (36K) protein called proopiomelanocortin (POMC). After [3H]glucosamine incorporation, separation of newly synthesized products by SDS-polyacrylamide gel electrophoresis showed that this 36K protein was glycosylated. Tryptic mapping revealed only one site of glycosylation and showed that the carbohydrate side-chain was located in the N-terminal region of POMC. The 36K protein was not released by the melanotrophs, but it generated, through specific intracellular proteolytic cleavage, a number of smaller peptides which were subsequently released. These peptides were identified by various methods including selective amino-acid incorporation, HPLC purification, acid-urea gel electrophoresis, tryptic and chymotryptic mapping, assay of melanotropic activity, radioimmunoassays and immunoprecipitations. Some of the newly synthesized N-terminal (18K) fragment of the POMC was secreted intact while a portion of it was further processed, via an intermediate peptide, to give mature gamma-MSH. All three of these peptides were glycosylated. In addition, the mature peptide (gamma-MSH) exhibited a low but significant melanotropic activity. The C-terminal portion of the prohormone was very rapidly processed to give des N alpha-acetyl alpha-MSH, corticotropin-like-intermediate lobe peptide (CLIP) and beta-endorphin. Authentic alpha-MSH was always absent in cellular extracts: acetylation to give rise to alpha-MSH was a late enzymatic process strictly linked to hormonal release. Since acetylation of alpha-MSH is required for full biological activity of this peptide, it is possible to conceive that this later step could be under neuroendocrine control. Using the perifusion technique we have been able to show the complexity of the control mechanisms regulating amphibian melanotrophs. It is generally accepted that the aminergic innervation of the intermediate lobe of the pituitary is involved in the hypothalamic control of melanotropin release. We have demonstrated that, in amphibians, dopamine inhibits alpha-MSH secretion through D2-type dopaminergic receptors whereas norepinephrine and (or) epinephrine stimulate alpha-MSH secretion via beta-adrenergic receptors. The existence of peptidergic fibers within parenchymal cells of the pars intermedia has been demonstrated. Evidence for TRH-containing fibers has been obtained by immunohistochemistry. Using a specific radioimmunoassay for TRH, we have confirmed the presence of TRH in the neurointermediate lobe of the frog. We have shown that TRH is a powerful MSH-releasing factor in these animals.(ABSTRACT TRUNCATED AT 400 WORDS)
The immunocytochemical study of the bovine placenta has demonstrated the presence of cells containing alphamelanotropic hormones, in the decidual epithelium, since the 4th month of pregnancy. The number and the staining intensity of those cells grow up with the placental development. Since the 6th month, we observed the immunoreactional presence of cells in the foetal chorionic epithelium.
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Intravenous (iv) bolus injection of 320 and 640 micrograms/Kg of lipopolysaccharides (LPS) of S. sonnei (either in phase I or in phase II) resulted in a dose-dependent decrease in mean arterial pressure, when given in freely moving rats. No changes were observed in plasma beta-endorphin (beta-EP), ACTH and corticosterone levels. Pituitary beta-EP content was not modified 1 h after iv injection of LPS. Data suggest that doses of LPS, which are able to produce reversible hypotension, do not alter pro-opiocortin cleavage and corticosterone release.
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Although more and more is known about the nature of neuropeptides, there are two basic questions which need to be looked at in order for the neurophysiologists and the neuropharmacologists to have all the tools necessary for their studies. The first question is the site of biosynthesis of neuropeptides and the second is their biosynthetic pathway. We believe and propose that all neuropeptides will be formed from a larger molecular weight precursor similar to beta-LPH for beta-endorphin. All models of peptide hormones (pro-insulin, pro-PTH, pro-glucagon, beta-lipotropin etc.,) show the same characteristic preferential site of cleavage made of a double basic residue. This is also the type of cleavage recently described by Nakanishi et al. (1979). Using the rat pars intermedia, we showed that this tissue preferentially makes alpha-MSH and beta-endorphin from a large precursor, while beta-LPH and ACTH are short lived intermediates. All products of maturation were identified by chemical analyses leaving no doubt about their nature and identity. We strongly believe that the brain will biosynthesize beta-endorphin and possibly alpha-MSH in similar fashion. We also predict that our results will apply to the other neuropeptides which wil be formed from large precursors. These studies could be very useful in the interpretation of the intricate relationship between neuropeptides and behavioral effects.
The subcellular localization of the post-translational processing steps which occur in the conversion of pro-adrenocorticotropic hormone (ACTH)/endorphin into beta-endorphin-sized molecules in rat intermediate pituitary has been studied. Primary cell cultures were incubated in radioactively labeled amino acids, and a subcellular fraction containing secretory granules was separated from a subcellular fraction containing rough endoplasmic reticulum and Golgi apparatus by centrifugation of homogenates on gradients on Percoll (Pharmacia Fine Chemicals). The radiolabeled beta-endorphin-related material in the granule and rough endoplasmic reticulum/Golgi apparatus fractions was quantitated by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. A pulse-chase labeling experiment demonstrated that newly synthesized beta-endorphin-related material first appeared in the rough endoplasmic reticulum/Golgi apparatus fraction and after longer incubations (chase) appeared in the secretory granule fraction. After 2 h of chase incubation, about 85% of the beta-endorphin-related material synthesized during the 30-min pulse incubation had been transferred from the rough endoplasmic reticulum/Golgi apparatus to the secretory granule fraction. The conversion of most of the newly synthesized pro-ACTH/endorphin into beta-lipotropin occurred in the rough endoplasmic reticulum/Golgi apparatus fraction, whereas the conversion of most of the beta-lipotropin into beta-endorphin-sized molecules occurred in the secretory granule fraction.