The alpha-amidation of alpha-melanocyte stimulating hormone in intermediate pituitary requires ascorbic acid

Rat intermediate pituitary cells in primary culture display a time-dependent loss of the ability to produce COOH-terminally alpha-amidated alpha MSH (Glembotski, C.C., Eipper, B.A., and Mains, R.E. (1983) J. Biol. Chem. 258, 7299-7304). Instead of des-, mono-, and diacetyl-adrenocorticotropic hormone(ACTH) (1-13)NH2, the cells produce des-, mono-, and diacetyl-ACTH(1-14)OH. Since the pituitary secretory granule-associated alpha-amidation enzyme requires copper and ascorbic acid for optimal activity (Eipper, B.A., Mains, R.E., and Glembotski, C. C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5144-5148), these cofactors were added to cultures of intermediate pituitary cells in an attempt to reverse the loss of peptide alpha-amidation ability. When the cultures were supplemented with up to 100 microM cooper (II) there was very little change in the ability to alpha-amidate alpha-melanotropin. Ascorbic acid at concentrations of up to 500 microM resulted in a dramatic increase in the ability of the cells to form the alpha-amidated peptide. Various combinations of ascorbic acid and copper additions indicated that a relatively short exposure (hours) to ascorbic acid produced the maximal response. Ascorbic acid displayed a dose-dependent effect on the alpha-amidation ability with a half-optimal concentration of about 25 microM. Pulse-chase labeling experiments demonstrated the ascorbic acid-dependent conversion of labeled ACTH(1-14)OH-related peptides to ACTH(1-13)NH2-related peptides. These results correlate with the ascorbic acid requirement of the pituitary alpha-amidation enzyme and demonstrate that the direct precursors to ACTH(1-13)NH2-related peptides are ACTH(1-14)OH-related peptides. Combined with our previous data, the present studies support the notion that a wide range of neuro- and endocrine peptides become alpha-amidated in a similar ascorbic acid-dependent manner.     

Acetylation of alpha-melanotropin and beta-endorphin in the rat intermediate pituitary. Subcellular localization

The subcellular site of the further processing (NH2-terminal acetylation and COOH-terminal proteolysis) of beta-endorphin-sized molecules in the rat intermediate pituitary has been studied. Rat intermediate pituitary primary cultures that had been incubated in radioactively labeled amino acids were homogenized and the secretory granule fraction was separated from the rough endoplasmic reticulum/Golgi apparatus fraction. The labeled beta-endorphin-sized molecules in each subcellular fraction were analyzed by immunoprecipitation, gel filtration chromatography, and ion exchange chromatography. A large percentage of the labeled beta-endorphin-sized molecules became NH2 terminally acetylated after becoming associated with the secretory granule fraction; most of the further COOH-terminal proteolytic processing of alpha-N-acetyl-beta-endorphin(1-31) to form alpha-N-acetyl-beta-endorphin(1-27) and alpha-N-acetyl-beta-endorphin(1-26) also occurred when the labeled beta-endorphin-sized molecules were associated with the secretory granule fraction. The acetylation of alpha-melanocyte-stimulating hormone (alpha MSH)-sized molecules was also investigated in rat intermediate pituitary primary cell cultures by immunoprecipitation, gel filtration chromatography, and reverse-phase high pressure liquid chromatography. Pulse-chase labeling experiments showed that newly synthesized molecules co-migrating with adrenocorticotropic hormone ((ACTH)(1-13)NH2) were converted first to molecules similar to alpha MSH (alpha-N-acetyl-ACTH(1-13)NH2) and then to molecules similar to alpha-N,O-diacetyl-alpha MSH. These results demonstrate that the enzyme activity(s) responsible for the NH2-terminal acetylation of beta-endorphin alpha MSH-sized molecules is located in the secretory granules of the rat intermediate pituitary.     

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).     

Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.     

Structurally functional organization of corticotropin: lipolytic and steroidogenic activity of some of its fragments]

The influence of ACTH fragments, possessing structural elements, common for certain groups of peptide hormones and kinins--"common" fragments and cluster of basic amino-acids--(Lys 17,18-ACTH 11-18-NH2--I; ACTH 11-13-NH2--II; NH2CO-ACTH18-20-NH2--III) on lipolytic effect of ACTH in rat isolated fat cells and on the steroidogenic effect of ACTH in isolated rat adrenal cells was studied. Fragment I exerts a steroidogenic effect (alpha=0,84) at concentrations of 1--100 microng/ml. At low concentrations (10(-8)--10(-3) microng/ml) fragment I potentiates ACTH-induced steroidogenesis. Fragment I has no effect on the lipolysis;however, it potentiates ACTH-induced lipolysis at concentrations of 10--100 microng/ml. The results obtained support our previous supposition that "common" fragments are essential secondary non-specific active sites of hormones.     

Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate

Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate-dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha-amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose-dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides.     

Amidation of joining peptide, a major pro-ACTH/endorphin-derived product peptide

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.     

Biosynthesis of amidated joining peptide from pro-adrenocorticotropin-endorphin

Joining peptide is the major alpha-amidated product of pro-ACTH/endorphin (PAE) in AtT-20 corticotropic tumor cells. To study intracellular joining peptide synthesis, affinity purified antibodies directed against gamma-MSH, joining peptide, and ACTH were used to immunoprecipitate extracts from biosynthetically labeled AtT-20 cells. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by tryptic peptide mapping on HPLC. In steady labeling experiments, radioactivity in amidated joining peptide (JP) increased roughly linearly with time, in the manner of a final product, whereas radioactivity associated with PAE (1-94)NH2 reached a constant value after 2-4 h, indicating that PAE(1-94)NH2 is an intermediate in the biosynthesis of JP. Radioactivity appeared in ACTH(1-39) well before JP, consistent with a cleavage order in which ACTH is cleaved from PAE(1-95) before JP sequences are cleaved from PAE(1-74). This conclusion was supported by tryptic peptide analyses of immunoprecipitates, which indicated that less than 5% of JP-related material is cleaved from PAE(1-74) before being cleaved from ACTH-related sequences. After a pulse label, radioactivity in PAE(1-94)NH2 reached a peak value after 1 h of chase and declined with a half-life of less than 1 h. Amidated JP increased to a constant level after 2 h of chase. Enough radiolabeled PAE(1-94)NH2 was detected to account for about half of the radioactivity found in amidated JP, indicating that about half of JP-related material is first cleaved from PAE(1-95) before being amidated. This result was corroborated using HPLC purification to determine both amidated and glycine-extended forms of JP.     

The effects of pro-opiomelanocortin peptides on cyclic AMP and tyrosinase in melanoma cells

Des-, mono-, and diacetylated melanotropin (des-, mono-, and di-Ac MSH, respectively) were compared for their dose-related effects on content of adenosine 3':5'-monophosphate (cAMP) and tyrosinase activity in the Cloudman S91 mouse melanoma tumor. Des-Ac MSH was more potent than the acetylated forms of MSH at increasing cellular levels of cAMP; mono- and di-Ac MSHs, however, were more potent than des-Ac MSH at elevating the activity of the enzyme, tyrosinase. Lysine-gamma1 MSH, a melanotropin from the amino terminus of pro-opiomelanocortin, exhibited slight stimulatory effects on tyrosinase and these actions were less than additive to those of mono-Ac MSH. Unlike their actions on amphibian skin-darkening or in mammalian behavior, neither beta-endorphin1-31 nor its derivatives, N-Ac-beta-endorphin1-27 or beta-endorphin30-31 (glycylglutamine), exhibited any influence on tyrosinase activity evoked by mono-Ac MSH in the tumor cells.     

Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary.

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.     

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.     

Proportions of mono- and diacetylated forms of alpha MSH in individual neurointermediate lobe extracts of Cyprinus carpio L

The proportions of the mono- and diacetylated forms of alpha MSH in individual carp neurointermediate lobe (NIL) extracts, as assessed by HPLC and RIA, fluctuate within a narrow range (mono/mono- + diacetyl alpha MSH = 3-14%). These results obtained on individuals of different sex and age show that the diacetylated form predominates in the NIL of all individuals where it represents in the mean 90% of the acetylated forms. The relatively low fluctuation of the proportions of the acetylated forms suggests that the possibility for a physiological modulation of the diacetylation step may be limited in this species of fish.     

Calcitonin is not contained within the common precursor to corticotropin and endorphin in the rat.

The suggestion that calcitonin is contained within the structure of the common precursor to ACTH and endorphin was examined. Immunohistochemical staining demonstrated calcitonin in thyroid parafollicular cells, and ACTH and 16K fragment in ACTH/endorphin cells of pituitary. No 16K fragment immunostaining was detected in thyroid parafollicular cells; no calcitonin staining was detected in pituitary. Immunoprecipitation of [³⁵S]methionine-labeled molecules synthesized by rat intermediate pituitary cells demonstrated that neither 30K precursor, 16K fragment nor any other major labeled cell product was recognized by calcitonin antiserum. Analyses of tryptic peptides of 30K precursor indicated that peptides expected from calcitonin were not present in 30K precursor.     

Regulation of peptide amidation in cultured pituitary cells

The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.     

High molecular weight forms of adrenocorticotropic hormone in the mouse pituitary and in a mouse pituitary tumor cell line.

Denaturing solvents have been used to determine the molecular weight of the adrenocorticotropic hormone (ACTH) activity in mouse pituitary, in an ACTH secreting mouse pituitary tumor cell line (AtT-20/D-16v), and in the tissue culture medium from the pituitary tumor cells. ACTH activity was quantitated by radioimmunoassay and by bioassay. It is possible to utilize guanidine hydrochloride or sodium dodecyl sulfate in characterizing the multiple forms of ACTH because treatment of porcine ACTH (the 39 amino acid polypeptide form of ACTH, alpha(1-39)), pituitary extracts, tumor cell extracts, and tumor cell tissue culture medium with these denaturants does not diminish the immunological ACTH activity. Based on gel filtration in the presence of guanidine hydrocholoride, extracts of the pituitary tumor cells and the mouse pituitary contain three distinct molecular weight classes of ACTH activity. The major form of ACTH has a molecular weight similar to alpha(1-39) (molecular weight 4000-5500), but there are significant amounts of two higher molecular weight forms of ACTH: molecular weight 6500-9000 and molecular weight 20,000-30,000. The 6500-9000 molecular weight form of ACTH is the major form of ACTH in the tissue culture medium; there is no peak of alpha(1-39) size ACTH in the medium. In the radioimmunoasay all three forms of ACTH generate competitive binding curves parallel to that of porcine alpha(1-39); in the bioassay (stimulation of steroidogenesis in a mouse adrenal tumor cell line) the dose response curve for each of the molecular forms of ACTH is parallel to that for porcine alpha(1-39).     

Further characterization of the peptidyl alpha-amidating enzyme in rat anterior pituitary secretory granules

In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha-melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides.     

Regulation of lymphokine (gamma-interferon) production by corticotropin

We have shown that corticotropin (ACTH), alpha-endorphin, and enkephalins can regulate antibody responses, which suggested a role for neuropeptides in a regulatory circuit between the immune and neuroendocrine systems. ACTH and structurally related peptides were examined here for regulation of mitogen induction of the lymphokine gamma-interferon (IFN gamma) in C57BL/6 mouse spleen cell cultures. Synthetic ACTH1-39 and a porcine pituitary extract containing ACTH activity were potent suppressors of the IFN gamma response. Synthetic ACTH1-39 suppressed the response by approximately 62% at 1 to 3 microM, whereas the porcine extract suppressed by greater than 90% at 1 to 3 microM ACTH. The greater potency of the pituitary extract was shown to be due to the presence of an additional peptide of Mr 2100 that was reactive with antibodies to the N-terminal region of ACTH (ACTH1-13), possessed potent anti-cellular activity against L cells and various transformed cells, but lacked ACTH biologic activity. The anti-cellular peptide suppressed the IFN gamma response by greater than 99% at 0.05 microM. The ACTH1-39 cleavage products, alpha-melanocyte stimulating hormone (alpha MSH; acetylated and amidated ACTH1-13), and corticotropin-like intermediate lobe peptide (CLIP; ACTH18-39) had no effect on IFN gamma production. ACTH1-24, like ACTH1-39, has full steroidogenesis activity but also had no effect on IFN gamma production, which suggests a dissociation of the immunoregulatory and steroidogenic properties of ACTH1-39. ACTH1-39, and possibly also the anti-cellular 2100 Mr peptide, is initially synthesized as the precursor polyprotein pro-opiomelanocortin (POMC). Enzymatic processing of POMC, first to the active ACTH1-39 or the anti-cellular peptide and then to the inactive smaller peptides, probably plays an important role in regulation of lymphokine and antibody production by ACTH and ACTH-related neuropeptides. This is consistent with the recent demonstration of the production of ACTH-like peptides by lymphocytes.     

Thymosin fraction 5 (TF5) stimulates secretion of adrenocorticotropic hormone (ACTH) from cultured rat pituitaries

In vivo administration of a partially purified thymic hormone-containing extract of the thymus gland, TF5, causes an increase in serum glucocorticoids. The lack of a direct effect of TF5 on adrenal corticosterone secretion suggests that it is mediated at the level of the pituitary. Cultured rat pituitary monolayers were used to determine if the effect is mediated by stimulation of ACTH secretion from the pituitary. Two lots of TF5, BPP100 and C114080-01, caused a dose dependent secretion of ACTH from cultured pituitary monolayers. There was a synergistic effect when the cells were treated with both TF5 and corticotropin-releasing factor (CRF). Immunoneutralization studies were done in which the cells were treated with TF5 or CRF and an antibody to CRF. The antibody completely blocked CRF induced ACTH release, but had no effect on TF5 stimulated ACTH release, suggesting that the activity is not due to a CRF-like peptide in TF5. A number of peptides isolated from TF5, and certain other peptides produced by the immune system were evaluated for their ability to stimulate ACTH secretion. These included thymosin (TSN) alpha 1, alpha 11, and beta 4, prothymosin alpha (PT alpha, thymopoeitin 5 (TP5), factuer thymique serique (FTS), interferon alpha (INF alpha), INF gamma, interleukin 1 (IL-1), and interleukin 2 (IL-2). None of these factors had any effect on pituitary ACTH secretion. These results demonstrate that some peptide component of TF5 causes an increase in serum corticosteroids by stimulating pituitary ACTH release.     

Steroidogenic activity of high molecular weight forms of corticotropin.

The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules     

Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituaitary

Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20–21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a βLPH-like peptide), and 3.5K (a β-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat antierior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH₂-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20–21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [³H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a β-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a βLPH-like molecule and a β-endorphin like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.