The localization of labelled fish prolactin in various tissues of rainbow trout (Salmo gairdneri).

Rostral lobes of the pars distalis from rainbow trout, Salmo gairdneri, were incubated in vitro in a medium containing 14C-labelled lysine. The labelled proteins in these lobes and medium were separated by polyacrylamide gel electrophoresis, the prolactin eluted from the appropriate band, and injected into intact trout. Following the injections, various tissues were dissected out and observed autoradiographically. There was no binding of labelled prolactin to tissues of pituitary gland, thyroid, pyloric caecum, stomach, pancreas or muscle. There was, however, significant labelling in liver, intestine, kidney, bladder, skin and gill. The binding of labelled chum salmon prolactin to these latter tissues in vitro was significantly reduced when unlabelled hormone was also added.

Regulation of melanotropin release from the pars intermedia of the amphibian Xenopus laevis: evaluation of the involvement of serotonergic, cholinergic, or adrenergic receptor mechanisms.

Melanophore-stimulating hormone (MSH) release from the pars intermedia of the pituitary gland is probably regulated by multiple factors of hypothalamic origin. We have examined a number of potential regulatory factors for their effects on MSH release from the amphibian Xenopus laevis. Serotonin and acetylcholine have no effect on MSH release. Both adrenaline and noradrenaline inhibit release of MSH in a dose-dependent manner. Studies with specific receptor agonists and antagonists reveal that these neurotransmitters exert their in vitro effects primarily through a dopamine D-2 receptor, although an alpha-adrenergic receptor could not be excluded. We further conclude that the pars intermedia of X. laevis lacks a beta-adrenergic receptor for the regulation of MSH secretion from the pars intermedia. In mammals, this receptor activates the adenylate cyclase system. Our studies reveal that despite the lack of beta-adrenergic receptors, cyclic-AMP is likely an intracellular factor involved in the stimulation of MSH release.

The pituitary adrenocorticotropes originate from neural ridge tissue in Xenopus laevis.

A series of grafting experiments was conducted to determine pituitary origins prior to brain tube closure in Xenopus laevis. Extirpation experiments indicated that the ventral neural ridge (VNR) tissue of stage-18+ embryos was essential for pituitary development. Bolton-Hunter reagent was used to label stage-18+ VNR tissue with 125I, and this tissue was then returned to the donor and its subsequent ontogenesis followed. Labelled tissue was ultimately found in the ventral hypothalamus, the ventral retina, and the anterior pituitary. Using immunocytochemical techniques with antisera to adrenocorticotropin (ACTH), it was found that some of the VNR-derived cells were corticotropes. A region of the nucleus infundibularis which was radioactive labelled also gave ACTH-positive immunoreaction. This might indicate that some ACTH-containing neurones of the hypothalamus are VNR in origin. We suggest that stage-18+ VNR is the site of attachment of brain and anterior pituitary ectoderm. Part of this adherence point is eventually incorporated into the anterior pituitary and will form corticotropes. It is concluded that the ventral retina, the preoptic region of the hypothalamus, some hypothalamic ACTH-immunoreactive cells, and the most anterior portion of the adenohypophysis are all ventral neural ridge in origin.

Acetylation of melanocyte-stimulating hormone and beta-endorphin in the pars intermedia of the perinatal pituitary gland in the mouse.

This report concerns ontogenetic aspects of the production and in vitro release of NH2-terminally acetylated forms of melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin by the pars intermedia of the pituitary gland of the mouse. In vitro biosynthetic analysis and radioimmunoassay revealed that approximately 12 h before birth most of the MSH in the fetal pars intermedia is present as des-N alpha-acetyl alpha-MSH. The same non-acetylated peptide is at this stage also the major release form of melanotropin. In 1-day-old mice the level of alpha-MSH and diacetylated alpha-MSH had increased considerably, although des-N alpha-acetyl alpha-MSH remained the major form. Five days after birth alpha-MSH and its diacetylated form constitute the major tissue and release form of the peptide, a situation very similar to that in adult mice. Acetylation of beta-endorphin appeared to occur earlier in development, N alpha-acetyl beta-endorphin (1-31) being the major form of endorphin already in the fetal pars intermedia. It is concluded that in the mouse acetylation of melanotropin and acetylation of beta-endorphin are not necessarily concomitant events. It could be established that the ability of the pars intermedia cells for cleaving N alpha-acetyl beta-endorphin (1-31) to yield C-terminally shortened forms of beta-endorphin develops after birth.

The functional significance of glycosylation of pro-opiomelanocortin in melanotrophs of the mouse pituitary gland.

Pro-opiomelanocortin (POMC) is a glycoprotein precursor for a number of neuropeptides and peptide hormones. The functional significance of the glycosylation of POMC has never been established. Using the antibiotic tunicamycin to block glycosylation of the prohormone in the mouse pars intermedia, we have compared processing of non-glycosylated prohormone with that of glycosylated prohormone in pulse-chase experiments. The peptides produced from non-glycosylated prohormone were shown to be correct cleavage products. Therefore it was concluded that, with the possible exception of peptides from the N-terminal region of the prohormone, the carbohydrate on POMC plays no role in directing cleavage or in protecting the prohormone from random proteolysis. Tunicamycin treatment retarded N-terminal acetylation of melanotrophin but had no apparent effect on acetylation of beta-endorphin. The mouse pars intermedia synthesizes two forms of POMC which differ in their degree of glycosylation. Our results indicated that, during secretion, the melanotrophs make no distinction between peptides derived from the two prohormones.

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.

The development of the pars intermedia and its role in the regulation of dermal melanophores in the larvae of the amphibian Xenopus laevis.

The ontogenesis of biosynthesis of pro-opiomelanocortin (POMC)-related peptides in the pars intermedia of Xenopus laevis tadpoles was studied. The results were related to the capacity of the animal to adapt to background color through regulation of pigment dispersion in dermal melanophores. Using immunocytochemical techniques with antisera to alpha-melanophore-stimulating hormone (alpha-MSH), it was revealed that this peptide first appeared at developmental stage 37/38, just prior to the animal's ability to adapt to background. It was shown that pigment dispersion in melanophores between stages 33 and 39 was not dependent on melanotropins of pituitary origin. Using in vitro biosynthetic studies it was possible to follow POMC biosynthetic activity, its processing and the release of peptides from stage 48 onward. Among the newly synthesized peptides observed were a gamma 3-MSH-like peptide, des-N-alpha-acetyl-alpha-MSH, alpha-MSH, and two endorphin-like peptides. By stage 57 a biosynthetic pattern almost identical to that of the adult pars intermedia had evolved. It was concluded that stage 39/40 is a critical stage in the simultaneous development of a number of the components involved in the neuroendocrine control of background adaptation.

Biosynthesis of pro-opiomelanocortin-derived peptides in the mouse neurointermediate lobe.

This report concerns biosynthetic studies conducted with neurointermediate lobes of the mouse pituitary gland. High performance liquid chromatography was used to resolve newly synthesized peptides after in-vitro incubation of lobes with radioactive amino acids. Among the newly synthesized peptides identified were alpha-MSH, des-N alpha-acetyl-alpha-MSH, two forms of corticotrophin-like intermediate lobe peptide and beta-endorphin. The biosynthesis of a glycosylated gamma 3-MSH-like peptide was also demonstrated. While no newly synthesized beta-MSH could be identified, a peptide designated gamma-lipotrophin was found. Pulse-chase analysis revealed that the major biosynthetic pathway leading to the production of alpha-MSH involved the acetylation of the des-acetyl form of this peptide. Furthermore, it was evident that newly synthesized beta-endorphin was largely converted to modified forms of this peptide; most of the terminal product was probably N-acetylated endorphin.

The frog pars intermedia contains only the non-acetylated form of alpha-MSH: acetylation to generate alpha-MSH occurs during the release process.

Pulse-chase experiments revealed that the frog pars intermedia synthesizes the desacetyl form of alpha-MSH. Its structure was shown to be similar, if not identical, to the mammalian structure. During release two additional peptides derived from desacetyl alpha-MSH appeared, one being alpha-MSH. We conclude that the N-acetylation of newly synthesized MSH is associated with release of the hormone. Radioimmunoassays and bioassays showed that the non-acetylated peptide is the only tissue form of MSH and confirmed that acetylation is linked to release.

In vivo biosynthesis of melanotropins and related peptides in the pars intermedia of Xenopus laevis.

To study in vivo biosynthesis of pars intermedia peptides in Xenopus laevis, [3H]lysine was administered by an osmotic minipump via a cannula inserted near the pituitary gland. Following extraction of the neurointermediate lobe, high-performance liquid chromatography was used to separate the newly synthesized peptides. In black-background adapted animals, [3H]lysine was incorporated into a number of peptides. The elution characteristics of these peptides corresponded exactly with those of peptides synthesized during in vitro incubation of neurointermediate lobes, and which were identified as des-N alpha-acetyl-alpha-MSH, a gamma-MSH-like peptide, two corticotropin-like intermediate lobe peptides, and two forms of endorphin. In white-background adapted Xenopus, practically no synthesis of pars intermedia peptides occurred. Transfer of black-adapted toads to a white background at the beginning of infusion led to storage of newly synthesized peptides. When such animals were maintained on a white background for 10 days, des-N alpha-acetyl-alpha-MSH, but not alpha-MSH, was present in the pars intermedia; this supports the notion that des-N alpha-acetyl-alpha-MSH constitutes the "storage form" of alpha-MSH.

Biosynthesis of two structurally different pro-opiomelanocortins in the pars intermedia of the amphibian pituitary gland.

This study reports the biosynthesis of two forms of pro-opiomelanocortin in the pars intermedia of the pituitary gland of the African clawed toad Xenopus laevis. The two forms could be resolved by dodecyl sulphate gel electrophoresis on a 9-16% acrylamide gradient and their molecular weights were 38200 and 37300. Incubation of neurointermediate lobes with [3H]glucosamine followed by tryptic digestion of the newly synthesized glycoproteins, revealed that both prohormones have only one glycosylated site, namely within the region corresponding to gamma 3-melanotropin. Biosynthesis of proteins in lobes treated with tunicamycin to prevent glycosylation again resulted in the production of two pro-opiomelanocortins (Mr 35000 and 34200), indicating that the two forms differ in their primary structure. This notion was corroborated by the results of tryptic mapping of the newly synthesized prohormones. The maps showed that the primary structures of the two forms of pro-opiomelanocortin differ in at least two parts of the molecules, one part concerning the endorphin region.

Biosynthesis of a gamma 3-melanotropin-like peptide in the pars intermedia of the amphibian pituitary gland.

This study reveals the biosynthesis of a gamma 3-melanotropin-like peptide in the pars intermedia of the pituitary gland of the aquatic toad Xenopus laevis. Pulse-chase experiments in vitro showed that this product is synthesized through processing of a prohormone, pro-opiomelanocortin, and that it is released into the incubation medium. The peptide immunoprecipitated with antiserum to gamma 3-melanotropin, appeared to be a glycopeptide and displayed melanotropic activity. This last observation together with the results of tryptic and chymotryptic peptide mapping of the newly synthesized product indicates that Xenopus gamma 3-melanotropin is structurally different from the proposed mammalian gamma-melanotropins.

Biosynthesis of pairs of peptides related to melanotropin, corticotropin and endorphin in the pars intermedia of the amphibian pituitary gland.

This study concerns the biosynthesis of a number of peptides in the neurointermediate lobe of the pituitary gland of the aquatic toad, Xenopus laevis. Using pulse-chase incubations in vitro and high-performance liquid chromatographic analysis, it could be shown that these peptides are synthesized through processing of a prohormone, pro-opiomelanocortin; all peptides were released into the incubation medium. On the basis of electrophoretic analysis, selective amino acid incorporation and immunoprecipitation, as well as peptide mapping by high-performance liquid chromatography, the peptides were classified into three distinct groups: two related to melanocyte-stimulating hormone (melanotropin), two related to adrenocorticotropic hormone (corticotropin) and two endorphin-like peptides. Using tryptic and chymotryptic maps of synthetic alpha-melanotropin and des-Ac alpha N-alpha-melanotropin as references, one of the melanotropin-like peptides was identified as des-Ac alpha N-alpha-melanotropin; the other one represents neither alpha-melanotropin nor any other known melanotropic peptide. The two peptides that were immunologically related to corticotropin had characteristics consistent with a structures resembling a peptide previously named 'corticotropin-like intermediate lobe peptide', corticotropin-(18-39). The two endorphin-like peptides, although highly related, do not have the same primary structure. In view of the apparent structural differences between the two peptides in each group, the possible occurrence of two prohormones is discussed.

Biosynthesis of MSH and related peptides in the pars intermedia of the mouse: a pulse-chase analysis.

Protein biosynthesis in neurointermediate lobes of mouse pituitaries was investigated using pulse and pulse-chase techniques with [3H]lysine. Electrophoretic analysis of lobe homogenates on acid-urea gels resolved 11 labeled products. One was a large protein which was rapidly synthesized during pulse-incubations and disappeared during chase incubations. Three of the products increased during chase incubations, suggesting a precursor-product mode of biosynthesis for these chasde peptides. One of these three products co-migrated with synthetic alpha-MSH and also corresponds to the major peak of mouse neurointermediate lobe MSH bioactivity and immunoactivity on electrophoretograms. Another case of these peptides has electrophoretic properties similar to those of ACTH.