Species-specific processing of prodynorphin in the posterior pituitary of mammals.

The steady state levels of the prodynorphin-derived opioid peptides, dynorphin A, dynorphin B, and alpha-neo-endorphin, have been extensively studied in the magnocellular/posterior pituitary system of the rat. To determine whether the rat system serves as a general model for prodynorphin processing in the mammalian posterior pituitary, we examined the steady state levels of prodynorphin-derived opioid peptides in the posterior pituitary systems of representatives of three diverse orders of mammals: guinea pig (order Rodentia), pig (order Artiodactyla), and rhesus monkey (order Primates). In each species studied there was evidence for species-specific deviations from the rat model. The most pronounced differences were observed with respect to the conversion of dynorphin A-(1-17) to dynorphin A-(1-8). In rodents, rats, and guinea pigs, under steady state conditions, the molar ratios of these forms are approximately 1:2 and 2.5:1, respectively. However, in the pig and rhesus monkey, the molar ratios of these forms are 10:1 and 14:1, respectively. Thus, under steady state conditions, the conversion of dynorphin A-(1-17) to dynorphin A-(1-8) appears to be a minor event in porcine and rhesus monkey posterior pituitary. Species-specific variations were also observed with respect to the steady state levels of alpha-neo-endorphin and dynorphin B-(1-13). In addition, the results of these studies suggest that the conversion of prodynorphin-derived opioids to leu-enkephalin probably represents a minor event in the species studied.

Detection of adrenocorticotropin-related and alpha-melanocyte-stimulating hormone-related substances in the anterior pituitary of larval and adult Ambystoma tigrinum (class: Amphibia).

Immunohistochemical analysis revealed the colocalization of ACTH-related immunoreactive forms and alpha-MSH-related immunoreactive forms in corticotropic cells of the anterior pituitary of larval Ambystoma tigrinum. Subsequent analysis of acid extracts of anterior pituitaries obtained from larval, neotene, and postmetamorphic adults indicated that the molar ratio of ACTH(1-39) to alpha-MSH was, respectively, 2.5:1, 1:1.5, and 1:1.3. However, in sexually mature adults the molar ratio of ACTH(1-39) to alpha-MSH was 8:1. These results indicate that before and immediately after metamorphosis, both ACTH(1-39) and alpha-MSH are major end products of corticotropic cells in this species. As postmetamorphic animals reach sexual maturity, ACTH(1-39) is a major end product of corticotropic cells and only trace amounts of alpha-MSH are produced. Thus in A. tigrinum the proteolytic processing of ACTH in corticotropic cells is developmentally regulated. The alpha-MSH-related peptide produced by the corticotropic cells appears to be an ACTH(1-13)amide-like form.

Beta-endorphin/ACTH immunocytochemistry in the CNS of the lizard Anolis carolinensis: evidence for a major mesencephalic cell group.

The immunocytochemical distribution of beta-endorphin and other proopiomelanocortin (POMC) peptides in the central nervous system of the lizard Anolis carolinensis was determined. Colchicine pretreatment was used to enhance perikaryal immunoreactivity. A major finding of this study is the localization of a previously undetected mesencephalic cell group which exhibits immunoreactivity to beta-endorphin, ACTH, and alpha-MSH. The perikarya of these neurons are large, bipolar, and situated in the mesencephalic tegmental area. They appear to project to the mesencephalic central gray and other brainstem structures. In contrast, the immunoreactive parvicellular perikarya of the medial-basal hypothalamus, corresponding to the POMC perikarya of the rodent arcuate nucleus, exhibit major rostral projections to various telencephalic and diencephalic structures. The exact extent of fiber projections and innervation patterns arising from either of these two groups is not clear at this time and will require further analyses. Scattered fiber immunoreactivity was also seen in the medial cerebral cortex and the striatal complex, regions which apparently are not innervated by beta-endorphin fibers in the rodent brain. Also, no immunoreactivity was seen to an antiserum to the 16K peptide of POMC. Other similarities and differences in the brain distribution of POMC in reptiles and mammals are discussed.

Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure.

A cDNA containing the sequence of GH was cloned and sequenced from a pituitary cDNA library for the holostean fish Lepisosteus osseus (common name: gar). The gar GH cDNA contained an open reading frame of 633 nucleotides and a 3' untranslated region (including the terminal codon TAG) of 1058 nucleotides. The overall length of the gar GH cDNA including leader sequence, signal sequence, hormone sequence and 3' untranslated region was 1713 nucleotides. Thus, the gar GH cDNA is the largest vertebrate GH cDNA yet cloned. A comparison of GH sequences from ancient (holostean fishes-gar and bowfin; one chondrostean fish-the Russian sturgeon) and more modern (27 species of teleosts) members of class Actinopterygii indicate that members of this class have maintained many of the invariant residues deemed necessary for GH folding motifs (intramolecular relationships) observed in mammals.

Further characterization of the major forms of reptile beta-endorphin.

Biosynthetically labeled reptile intermediate pituitary beta-endorphin-sized material was fractionated by SP-Sephadex ion exchange chromatography into two major opiate-active forms which eluted at 0.28 M NaCl and 0.32 M NaCl, respectively; the 0.32 M form of reptile beta-endorphin (mw = 3500), serves as the precursor for the 0.28 M form of reptile beta-endorphin (mw = 3200), (Dores and Surprenant, 1983). Analysis of tryptic digests of these reptile beta-endorphins by paper electrophoresis at pH 3.5 and gel filtration on a Sephadex G-15 column indicated that there are two tyrosine residues, two arginine residues and one methionine residue in reptile beta-endorphin. Furthermore, the NH2-terminal tryptic peptide of both reptile beta-endorphins is approximately nine amino acids in size and contains tyrosine, methionine and arginine. Analyses of chymotryptic/protease digests of the [3H]tyrosine-labeled NH2-terminal tryptic peptide analyzed by descending paper chromatography revealed that the NH2-terminal tyrosine of reptile beta-endorphin is not alpha-N-acetylated. A second tyrosine-containing tryptic peptide was detected in the COOH-terminal region of reptile beta-endorphin; however this tryptic peptide differs in the two forms of reptile beta-endorphin in terms of size and net charge at pH 3.5. These differences account for the apparent molecular weight differences and distinct ion exchange properties of the 0.28 M and 0.32 M forms of reptile beta-endorphin. Thus in the reptile intermediate pituitary the principal post-translational mechanism for modifying beta-endorphin is COOH-terminal proteolytic cleavage.

Evidence for a common precursor for alpha MSH and beta-endorphin in the intermediate lobe of the pituitary of the reptile Anolis carolinensis.

In order to investigate the biosynthesis of alpha MSH and beta-endorphin in a non-mammalian vertebrate, individual lizard intermediate pituitaries were incubated in complete medium containing a radioactive amino acid, using either a steady label or a pulse/chase protocol. Following incubation, acid extracts of the tissue were immunoprecipitated with either an NH2-terminal ACTH antiserum or a beta-endorphin antiserum and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After a 24 hr steady label in medium containing [3H]tyrosine, multiple molecular weight forms of beta-endorphin-related and NH2-terminal ACTH-related radioactivity were detected. The major peak of beta-endorphin-related radioactivity co-migrated with synthetic beta-endorphin(1-31); minor peaks of beta LPH-sized material and precursor-sized material were also detected. The major peak of NH2-terminal ACTH-related material co-migrated with synthetic alpha MSH; in addition, smaller amounts of material designated ACTH biosynthetic intermediate 1, ACTH biosynthetic intermediate 2, and precursor-sized material were detected. Sequential immunoprecipitation experiments revealed that the precursor-sized material had antigenic determinants for both alpha MSH and beta-endorphin. Pulse/chase experiments established that this material is the common precursor for alpha MSH and beta-endorphin. Based on gel filtration chromatography in 6 M guanidine HCl, the molecular weights of these various peptides are: common precursor, 23,300 daltons; ACTH biosynthetic intermediate 1, 12,200 daltons; ACTH biosynthetic intermediate 2, 4,200 daltons; alpha MSH, 1,500 daltons; beta LPH, 8000 daltons; beta-endorphin, 3,400 daltons. None of the peptides precipitated with either antiserum incorporated [3H]glucosamine; thus glycosylation does not appear to be involved in this biosynthetic pathway in the lizard. The results of the kinetic experiments and molecular weight determinations indicate that the major biosynthetic pathway involves the following events: common precursor is first cleaved to yield ACTH biosynthetic intermediate 1 plus beta LPH; subsequently, beta LPH is cleaved to produce beta-endorphin; ACTH biosynthetic intermediate 1 is cleaved to produce ACTH biosynthetic intermediate 2 which is subsequently cleaved to produce alpha MSH. The pulse/chase experiments indicate minor pathways exist for cleaving beta-endorphin directly from the common precursor or via a high molecular weight form intermediate in size between the common precursor and beta LPH.

Localization of multiple forms of ACTH- and beta-endorphin-related substances in the pituitary of the reptile, Anolis carolinensis.

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, beta-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, alpha MSH-like, beta-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when analyzed by radioimmunoassay, gave displacement curves in ACTH and beta-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an alpha MSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and beta-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and beta-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and beta-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were alpha MSH-sized, CLIP-sized, and beta-endorphin-sized. In both lobes, antisera directed against ACTH and beta-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and beta-endorphin in this species.

Differential N-acetylation of alpha-MSH and beta-endorphin in the intermediate pituitary of the turtle, Pseudemys scripta.

Steady-state analyses of the intermediate pituitary of the turtle, Pseudemys scripta, indicated that alpha-MSH-sized immunoreactive forms and beta-endorphin-sized immunoreactive forms are major end products of melanotropic cells. Three forms of alpha-MSH-related immunoreactivity were detected. The two major forms had the same reversed-phase HPLC properties as synthetic N,O-diacetyl-ACTH(1-13)-NH2 and N-acetyl-ACTH(1-13)-NH2. These forms accounted for 97% of the total alpha-MSH-related immunoreactivity detected. A minor peak of ACTH(1-13)-NH2 was also detected. Multiple forms of beta-endorphin-related immunoreactivity were detected, which varied in net positive charge (+1 to +5), apparent molecular weight (2.4 to 3.5 kDa), and degree of N-terminal acetylation. Although N-acetylated forms of beta-endorphin were detected in the turtle intermediate pituitary, the major forms of turtle beta-endorphin were nonacetylated. These features of the turtle intermediate pituitary POMC-specific N-acetylation mechanism are similar to, yet distinct from, the POMC N-acetylation mechanisms observed for mammals. These data suggest that POMC-specific N-acetylation mechanisms were present in reptiles prior to the divergence of the anapsid and synapsid lines.

Changes in the processing of pro-dynorphin end products in the substantia nigra during neonatal development.

The steady-state levels of pro-dynorphin-related end products were measured in the substantia nigra of the rat at neonatal day 0, 7, 14 and in the adult. At neonatal day 0 there was evidence that pro-dynorphin had undergone posttranslational processing to yield dynorphin A-related products, dynorphin B(1-13) and alpha-neo-endorphin. At this stage the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) was 1:1 and a peak of 4 kilodalton dynorphin A-related immunoreactivity was detected. By neonatal day 7 the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) resembled the adult processing pattern for the substantia nigra. The rapid maturation of the pro-dynorphin system in the substantia nigra is in contrast to the development of the pro-dynorphin system in the posterior pituitary where adult-like processing patterns are not observed until neonatal day 21 (11). Pro-enkephalin products have also been detected in the substantia nigra of the rat (15). At neonatal day 0 and neonatal day 7 the molar ratio of Met-enkephalin to Leu-enkephalin was 4:1. However, in the adult the molar ratio of Met-enkephalin to Leu-enkephalin was 1.4:1 in this terminal field. These results suggest that in the adult or perhaps late in neonatal development some pro-dynorphin end products undergo further proteolytic cleavage to yield Leu-enkephalin.

Isolation of immunoreactive beta-endorphin-related and Met-enkephalin-related peptides from the posterior pituitary of the amphibian, Xenopus laevis.

Acid extracts of the posterior pituitary of the amphibian, Xenopus laevis, were analyzed with two heterologous region specific beta-endorphin RIAs. Following gel filtration chromatography and cation exchange chromatography four peaks of immunoreactivity were detected. All four peaks were detected with a N-acetyl specific beta-endorphin RIA. Peak I represented 92% of the total immunoreactivity isolated following cation exchange chromatography. This peak had a net positive charge at pH 2.5 of +1 and an apparent molecular weight of 1.4 Kd. Following reverse phase HPLC, Peak I fractionated into two peaks: Peak Ia and Peak Ib. Both peaks were detected with the N-acetyl specific beta-endorphin RIA and a Met-enkephalin RIA, however, neither peak co-migrated with either Met-enkephalin or N-acetyl-beta-endorphin(1-16). At present it is not clear whether Peak I is derived from pro-opiomelanocortin or one of the other opioid polyproteins. Peaks II, III, and IV represented 8% of the total immunoreactivity recovered following cation exchange chromatography. These peaks had net positive charges of +3, +4, and +5, respectively, and apparent molecular weights of 2.8, 3.2, and 3.5 Kd, respectively. These apparently N-acetylated beta-endorphin-sized forms are minor end products of the pro-opiomelanocortin biosynthetic pathway.

Detection of Met-enkephalin in the CNS of the teleosts, Anguilla rostrata and Oncorhynchus kisutch.

Acid extracts of the brains of the American eel, Anguilla rostrata, and the coho salmon, Oncorhynchus kisutch, were screened for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 column chromatography, a peak of Met-enkephalin-related immunoreactivity was detected near the total volume of the column for both species. No higher molecular weight forms of Met-enkephalin-related material were detected, nor were any immunoreactive forms with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) or alpha-neo-endorphin detected for either species. The enkephalin-sized immunoreactivity was further analyzed by reverse phase HPLC. For both species, a peak of authentic Met-enkephalin was detected. However, Leu-enkephalin, Met-enkephalin-RGL and Met-enkephalin-RF were not detected by RIA in either species. In addition, no novel C-terminally extended forms of Met-enkephalin were detected in either species. Finally, opiate receptor binding activity was only found associated with the peak of immunoreactive Met-enkephalin.

POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: differential subcellular processing in the Golgi and secretory granules.

In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cosecretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1-13)-NH2-related immunoreactivity was colocalized with N-acetylated beta-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated beta-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH (1-39) to yield ACTH (1-13)-NH2 is a late processing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated beta-endorphin and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of beta-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the beta-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells.

Steady state levels of pro-dynorphin-related end products in the striatum and substantia nigra of the adult rhesus monkey.

Analysis of an acid extract of the striatum of the rhesus monkey revealed that the molar ratio of dynorphin A(1-8)-sized material and dynorphin (A(1-17)-sized material is approximately 1:1. In addition, the molar ratios of the dynorphin A-related end products to both dynorphin B(1-13)-sized material and alpha-neo-endorphin-sized material were approximately 1:1. Fractionation of an acid extract of the substantia nigra by gel filtration and reverse phase HPLC revealed the following molar ratios for pro-dynorphin-related end products. The molar ratio of dynorphin A(1-8) to dynorphin A(1-17) is approximately 6:1. The molar ratios of dynorphin A-related end products to dynorphin B(1-13) and alpha-neo-endorphin were approximately 0.5 and 0.8, respectively. Comparisons between proteolytic processing patterns of pro-dynorphin in the striatum and the substantia nigra of the rhesus monkey are considered. In addition, comparisons between pro-dynorphin processing in the substantia nigra of the rhesus monkey and the substantia nigra of the rat are discussed.

Biosynthesis of multiple forms of beta-endorphin in the reptile intermediate pituitary.

Fractionation of the beta-endorphin-sized material from freshly dissected reptile intermediate pituitaries by ion exchange chromatography on sulfopropyl Sephadex (SP) revealed at least three distinct forms of immunoreactive beta-endorphin. These forms eluted at 0.25 M NaCl, 0.28 M NaCl, and 0.32 M NaCl and represent respectively, 6%, 65% and 29% of the total immunoreactivity. Only the 0.28 M NaCl peak and the 0.32 M NaCl peak exhibited naloxone reversible opiate bioactivity when tested in the isolated guinea pig ileum bioassay system; taking into account the molar amount of immunoreactive peptides the 0.32 M NaCl peak was 6 fold more potent than the 0.28 M NaCl peak. Intermediate pituitaries in culture were incubated with either [3H]tyrosine, [3H]arginine, or [35S]methionine for periods up to 24 hours and beta-endorphin-sized peptides were prepared by immunoprecipitation and gel filtration. Fractionation of the labeled beta-endorphin-sized peptides by ion exchange chromatography yielded profiles nearly identical to the immunoassay analyses of freshly dissected tissue. Further analysis of the major labeled forms of reptile beta-endorphin by chromatography on Sephadex G-50 equilibrated in 6 M guanidine HCl indicated that the 0.32 M NaCl peak had an apparent molecular weight of 3500 +/- 100 and the 0.28 M NaCl peak had an apparent molecular weight of 3200 +/- 100. Furthermore, pulse/chase experiments showed that the 0.32 M NaCl peak was the precursor for the 0.28 M NaCl peak. These results coupled with the relative opiate bioactivities of the major argue that the principal post-translational modification of reptile beta-endorphin is COOH-terminal proteolytic cleavage.

The posttranslational modification of beta-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site.

Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of beta-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus beta-endorphin is N-acetyl-YGGFMTPE. Overall, the steady-state analyses of the minor forms of beta-endorphin indicated that the posttranslational processing of beta-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites.

Strategies for studying opioid peptide regulation at the gene, message and protein levels.

Three opioid peptide precursors have been isolated and characterized in endocrine and nervous tissue: pro-opiomelanocortin, pro-enkephalin, and pro-dynorphin. Since each of those opioid peptide systems have been extensively characterized both biochemically and anatomically, this review will focus on strategies for studying the regulation of these systems at the levels of gene transcription, message translation, post-translational processing, secretion, and target cell receptor interaction.

Proteolytic cleavage of ACTH in corticotropes of sexually mature axolotls (Ambystoma mexicanum).

Immunohistochemical analysis of the pituitary of sexually mature axolotls revealed both ACTH(1-39)-related and alpha-MSH-related immunoreactivity present in corticotropic cells located in the rostral anterior pituitary. Gel filtration analysis indicated that the ACTH(1-39)-sized immunoreactivity and the alpha-MSH-sized immunoreactivity detected in acid extracts of the axolotl anterior pituitary were present in a ratio in a range between 1:1 and 1:0.6. Reversed-phase HPLC analyses indicated that the alpha-MSH-sized immunoreactivity had the same retention time as synthetic ACTH(1-13)-NH2. The corticotropic activity of the ACTH(1-39)-sized immunoreactivity and the purified ACTH(1-13)-NH2 was tested in a heterologous, larval bullfrog adrenal bioassay system. As expected, the ACTH(1-39)-sized immunoreactivity stimulated corticosterone release; however, the purified ACTH(1-13)-NH2 lacked glucocorticoid activity. The proteolytic cleavage of ACTH in corticotropes of sexually mature axolotls was identical to the cleavage events observed in neotenic Ambystoma tigrinum that had not reached sexual maturity. These studies indicate that the transient expression of ACTH cleavage activity is not affected by the reproductive state of the animal. Since axolotls do not undergo metamorphosis, it is possible that events associated with metamorphosis may induce the decline in ACTH cleavage activity observed in amphibian corticotropes.

Detection of N-acetylated forms of beta-endorphin and nonacetylated alpha-MSH in the intermediate pituitary of the toad, Bufo marinus.

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of beta-endorphin and alpha-MSH. Approximately 98% of the immunoreactive beta-endorphin was N-acetylated. The major form of N-acetylated beta-endorphin, which represented 81.5% of the total beta-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of +1 at pH 2.75. Approximately 98% of the immunoreactive alpha-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of alpha-MSH, ACTH(1-13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of alpha-MSH in this species is a cosecretory processing event, whereas the N-acetylation of beta-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of beta-endorphin and alpha-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.