16K Fragment Research Articles

The Role of the Melanocortin 3 Receptor in Mediating the Effects of Gamma‐MSH Peptides on the Adrenal

The pro‐opiomelanocortin (POMC)‐derived peptides, pro‐γ‐MSH (16K fragment), and Lys‐γ3‐MSH, have been shown to potentiate the steroidogenic action of corticotrophin (ACTH) on the adrenal cortex. Using a continuously perfused adrenal cell column system, we have tested the hypothesis that γ‐MSH peptides exert their effect through the Melanocortin 3 Receptor (MC3‐R), since this is the only known receptor to have high affinity for γ‐MSH peptides and has been suggested to be expressed in the rat adrenal. To investigate this hypothesis we tested whether the MC3‐R agonist MTII and antagonist SHU9119 could mimic or block the actions of pro‐γ‐MSH. We found that MTII could not mimic, and SHU9119 could not block pro‐γ‐MSH mediated potentiation of ACTH‐induced steroidogenesis. These results suggest that the MC3–R is not involved in mediating the potentiation effect, adding further evidence to the argument that another melanocortin receptor exists.

Proteolysis of human prolactin: resistance to cathepsin D and formation of a nonangiostatic, C-terminal 16K fragment by thrombin.

The N-terminal 16K fragments of rat and human PRLs possess angiostatic activity. 16K PRL has also been detected in vivo in both humans and rats. Based on an in vitro study, cathepsin D, an acid protease, has been implicated in the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme that is capable of forming an angiostatic human 16K PRL. To confirm the angiostatic action of rat 16K PRL, the fragment was generated by incubating 23K PRL with rat mammary microsomal fraction at pH 3.2. Upon incubation with human umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal- and basic fibroblast growth factor-stimulated cell proliferation. Intact rat and human PRLs were then incubated with cathepsin D or acidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were incubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both human and rat PRL by thrombin resulted in the formation of 16K fragments. PRL contained within human amniotic fluid was also cleaved by thrombin. Enzyme specificity was supported by prevention of cleavage by the thrombin inhibitor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angiostatic activity. The activity of this fragment in the Nb2 lymphoma bioassay was 10- to 15-fold lower than that of 23K PRL. Mass spectrometry revealed that the fragment has a mass of 16,878.30+/-15.8 Daltons. Subsequent N-terminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-terminal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, human PRL is resistant to cleavage by cathepsin D. Thrombin at a physiological pH can generate a C-terminal 16K fragment of human PRL that is not angiostatic and retains little mitogenic activity. We suggest that the precise nature of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined.

Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin

Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin

Heparin-binding property of human prolactin: a novel aspect of prolactin biology.

Prolactin (PRL) shares several characteristics with growth factors and cytokines, many of which are known to bind to heparan sulfate proteoglycans. In this study we examined the heparin-binding properties of selected members of the PRL/GH family, using heparin affinity columns followed by gel electrophoresis/Western blotting. Purified human PRL and its cleaved 16K fragment, but not human GH or placental lactogen, were retained on the heparin column and were displaced by 0.5 M NaCl. Native PRL in human pituitary extracts and amniotic fluid showed a similar binding affinity to heparin as the purified hormone. None of the other hormones tested, e.g., rat, ovine and bovine PRL, glycosylated ovine PRL or rat GH, bound to heparin. Two consensus heparin-binding sequences are present in human PRL but not in the other hormones included in this study. We postulate that the heparin-binding capability of PRL affects its biological activity as a growth factor and the angiostatic actions of its 16K fragment.

Effects of pro-opiomelanocortin-derived peptides on adrenal steroidogenesis in guinea-pig adrenal cells in vitro

The specific control of adrenal androgen secretion is unclear. This study was undertaken to investigate the effects of peptides derived from the ACTH precursor molecule pro-opiomelanocortin (POMC) on cortisol and androgen production using isolated guinea-pig adrenal cells. ACTH 10 −13−10 −9M, stimulated steroid production in a dose dependent manner, reaching a maximum of 12, 10 and 7 times basal levels for cortisol, androstenedione (A4) and dehydroepiandrosterone (DHEA), respectively, measured by specific radioimmunoassays. β-Lipotropin (β-LPH), 10 −10−10 −8M, also stimulated steroid production, reaching 6, 5 and 5 times basal levels of cortisol, A4 and DHEA, respectively. The N-terminal 16K fragment, λ 3- and β-MSH stimulated steroid production which reached statistical significance ( P < 0.05) only in the case of cortisol. Joining peptide, α-, β- and γ-endorphin resulted in no significant change in steroid production. Met- and leu-enkephalin resulted in significant inhibition of DHEA production. POMC-derived peptides altered the steroid response to ACTH. β-LPH and 16K fragment significantly increased the cortisol and A4 responses to a low concentration of ACTH. β-LPH reduced the maximum cortisol and A4 responses to a high concentration of ACTH. This study suggests that β-LPH may have a role in modulation of adrenal steroidogenesis but yielded no evidence to support a role for POMC-derived peptides in preferential stimulation of adrenal androgen production in guinea-pig adrenal cells.

Interaction between the heavy and the regulatory light chains in smooth muscle myosin subfragment 1

The interaction between the heavy and the regulatory light chains within chicken gizzard myosin heads was investigated by using a zero-length chemical cross-linker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC). The chicken gizzard subfragment 1 (S-1) used was treated with papain so that the heavy chain was partly cleaved into the NH2-terminal 72K and the COOH-terminal 24K fragments and the regulatory light chain into the 16K fragment. S-1 was reacted with EDC either alone or in the presence of ATP or F-actin. In all cases, the 16K fragment of the regulatory light chain formed a covalent cross-link with the 24K heavy chain fragment but not with the 72K fragment. The 38K cross-linked peptide, which was the product of cross-linking between the 16K light chain and the 24K heavy chain fragments, was isolated and further cleaved with cyanogen bromide and arginylendopeptidase. Smaller cross-linked peptides were purified by reverse-phase HPLC and then characterized by amino acid analysis and sequencing. The results indicated that cross-linking occurred between Lys-845 in the heavy chain and Asp-168, Asp-170, or Asp-171 in the regulatory light chain. The position of the cross-linked lysine was only three amino acid residues away from the invariant proline residue mapped as the S-1-rod hinge by McLachlan and Karn [McLachlan, A. D., & Karn, J. (1982) Nature (London) 299, 226-231]. We propose that the COOH-terminal region of the regulatory light chain is located in the neck region of myosin and that this region and the phosphorylation site of the regulatory light chain together may play a role in the phosphorylation-induced conformational change of gizzard myosin.

Cytochemical Studies of Responses of Corticotropes and Thyrotropes to Cold and Novel Environment Stress

Cold stress stimulates the release of both ACTH and TSH from the pituitary. More striking changes in ACTH content have been seen in the intermediate lobe after cold stress. Therefore, this study was designed to test responses of individual anterior lobe corticotropes to cold exposure. Male rats were exposed to either 30 min of cold (+3-5 C), 30 min of a novel, temperate environment (+24 C) or were unstressed (+24 C). Pituitaries were fixed and embedded in preparation for immunolabeling for ACTH or TSH-beta at the light (semithin sections) and electron microscopic levels. The semithin sections were used to measure areas of corticotropes and thyrotropes with Bioquant image analysis equipment. Separate groups of pituitaries were dissociated and the cells were cultured for 2 or 15 h. Then the cells were stimulated for 5-10 min with biotinylated analogs of corticotropin-releasing hormone (bio-CRH) or arginine vasopressin (bio-AVP) to detect the target cells cytochemically. A third group of dissociated cells were fixed for immunolabeling for ACTH, 16K fragment of pro-opiomelanocortin, beta-endorphin, or TSH-beta. Cold exposure resulted in a 1-4-fold increase in the levels of serum ACTH over that of unstressed rats. This was correlated with a 40% increase in the percentage of cells that contained 16K fragment and a 30-40% increase in percentages of cells storing ACTH or beta-endorphin. Cold stress also increased the percentage of cells that bound bio-CRH or bio-AVP by 45%. Analyses of semithin sections showed that areas of corticotropes increased by 21% following cold stress. The number of rows of immunolabeled (ACTH) secretion granules also increased in corticotropes from cold-stressed rats. Exposure to a novel environment for 30 min resulted in no significant increase in serum ACTH over that of unstressed rats. There was, however, a 20% increase in percentages of cells that stored 16K fragment, beta-endorphin, or target cells that bound bio-CRH. However, the corticotropes were not significantly larger. Many of the cells exhibited reduced numbers of immunolabeled secretory granules. Other corticotropes resembled those from cold-stressed rats. When TSH cells were studied, their percentages increased from 8 +/- 3% to 15.8 +/- 4% and their areas increased by 22% following exposure to cold. After exposure to a novel environment, percentages of cells that stored TSH-beta increased to 11 +/- 2%, however, no changes in areas of TSH cells were measured. These studies demonstrated that the anterior lobe corticotrope is clearly activated by exposure to both cold and novel environment.(ABSTRACT TRUNCATED AT 400 WORDS)

Cleaved prolactin and its 16K fragment are generated by an acid protease.

A cleaved form of rat PRL (cldPRL), which resolves after reduction of the disulfide bonds into two fragments of 16K and 8K mol wt, has been reported in the pituitary gland. To further understand the genesis of this cleaved PRL and its related 16K fragment (cldPRL/16K), pituitary proteins were labeled with [35S]methionine and then dialyzed and lyophilized before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pulse-chase experiments did not show a precursor-product relationship between PRL and cldPRL. However, if proteins were concentrated by trichloroacetic acid instead of dialysis, no cldPRL/16K was observed. Other experiments demonstrated that during dialysis cldPRL/16K is generated in vitro at the expense of intact PRL in a time-dependent manner. Labeled proteins dialyzed against water previously adjusted to different pHs showed after electrophoresis that cldPRL/16K was generated only at acidic pH. In the presence of the protease inhibitors phenylmethylsulfonylfluoride, tosylamidelysyl chloromethylketone, or tosylphenylalanine chloromethylketone or when the incubation medium was heated at 65 C for 45 min, the acid pH was unable to generate cldPRL/16K. Trypsin inhibitors did not prevent cldPRL generation. In conclusion, an acid protease, possibly of lysosomal origin, cleaves PRL in vitro. cldPRL/16K form was not found in the pituitary or incubation medium under physiological conditions. This PRL variant is generated as a preparative artifact unless experimental conditions are controlled.

Binding studies with intact rat prolactin and a 16K fragment of the hormone.

A cleaved form of PRL has been found in the pituitary of rodents and humans and in human blood. A 16K fragment derived from cleaved rat (r) PRL has mitogenic and lactogenic activities in PRL bioassays. In this report we analyzed whether 16K rPRL acts through PRL and/or specific 16K receptors. The 16K PRL displaced 23K PRL label with a potency that varied among different tissues. With liver membranes the fragment had only 5-6% the potency of intact PRL, but in whole brain its potency was similar to that of the 23K form. By contrast, 16K PRL was 30 times more effective at displacing [125I]23K rPRL from the kidney membranes than was intact PRL. [125I]16K PRL bound specifically to membranes of kidney, liver, brain, and skeletal muscle, and it was not displaced by intact or cleaved 23K rPRL, intact ovine PRL, or rat or bovine GH. The specific binding of 16K PRL to the kidney changed with incubation time, temperature, and pH and was significantly higher than that of 23K PRL to brain and kidney membranes. However, the specific binding of the 16K was lower in liver and skeletal muscle. The binding sites for 16K PRL in the kidney had a higher affinity than did those for 23K PRL (Kd = 6.9 x 10(-9) vs. 0.8 x 10(-8), respectively), but the opposite was found in the other tissues. The specific 16K PRL-binding sites in different tissues may represent receptors that mediate actions of the fragment that are unshared by intact PRL. Furthermore, 16K PRL binds to 23K receptors with an activity that varies among the target tissues. These results support the notion of distinct PRL isoreceptors in different tissues.

Identification of functional antigenic segments of toxic shock syndrome toxin 1 by differential immunoreactivity and by differential mitogenic responses of human peripheral blood mononuclear cells, using active toxin fragments.

When toxic shock syndrome toxin 1 was subjected to papain hydrolysis, two serologically active fragments of 16.3 kilodaltons (16K fragment) and 12.4 kilodaltons (12K fragment) were generated, whereas a third fragment of 9.7 kilodaltons (10K fragment) was inactive. The biologic activities of the fragments were evaluated in vitro by determining their ability to promote nonspecific proliferation of human peripheral blood mononuclear cells. The 12K fragment was significantly (P less than or equal to 0.013) more stimulatory than the 16K fragment. When human peripheral blood mononuclear cells were preincubated for a period of 24 h with various concentrations of the 16K fragment, followed by incubation with a constant amount (2 x 10(-2) ng/ml) of whole toxin, the level of DNA synthesis induced by the holotoxin was reduced by approximately 60% when compared with that of controls exposed to whole toxin alone. The 12K fragment did not demonstrate a similar blocking effect. Immunoblots of the toxic shock syndrome toxin 1 digest, which were exposed to monoclonal antibodies (MAbs) developed against native toxin, depicted the presence of two different antigenic regions (epitopes). One MAb, 8-5-7, which has been shown previously to inhibit the biologic activity of the holotoxin in vitro and in vivo, reacted primarily with the 12K fragment. A second MAb, 10-6-1, that did not neutralize interleukin-1 production reacted primarily with the 16K fragment. On the basis of the differential mitogenic responses and the identification of heterologous epitopes, it was concluded that the functional region of the holotoxin can be partitioned into at least two functional segments encompassed between amino acid residues 53 and 87 and between amino acid residues 88 and 194 on the polypeptide chain.

Analysis of the proteolytic cleavage of prolactin by the mammary gland and liver of the rat: characterization of the cleaved and 16K forms.

A cleaved form of PRL (CPRL) has been reported to exist naturally in the pituitary and to be the main product of PRL proteolysis by its target tissues. A 16K fragment derived from CPRL has mammary mitogenic activity. To analyze the possible functional significance of the cleavage of PRL generated by incubation of unlabeled rat (r) PRL with a 25,000 generated by incubation of unlabeled rat (r) PRL with a 25,000 x g pellet from the mammary gland and liver of rats. The optimum pH for cleavage was 3.4. PRL and the cleaved products were easily identified and separated from other proteins in the sample. The mammary gland pellet from lactating rats was significantly more potent at cleaving the rPRL molecule than were those of virgin or pregnant rats. However, the livers of all rats showed a similar PRL-cleaving potency. Thus, target tissues may differ in their PRL-cleaving potencies in different physiological states. The PRL-cleaving activity was also present in rat milk. While cleaved and 16K PRLs were the only proteolytic products obtained from rPRL, the PRLs (ovine and human) or the GHs (rat, bovine, and human) of other species were processed into a variety of low mol wt forms. Human placental lactogen was not modified. Accordingly, the processing of rPRL by target tissue enzymes appears to be hormone and species specific. Cleaved and 16K PRLs had 100% and 6.5% of the binding activity for rat liver PRL receptors as did intact rPRL, respectively. Nevertheless, the 16K form may still be of physiological significance.

Use of ion-exchange sep-pak cartridges in the batch fractionation of pituitary peptides

Silica-based ion-exchange Sep-Pak cartridges, packed with either carboxymethyl (CM) cation-exchanger or a quaternary methyl ammonium (QMA) anion exchanger, are now available. The feasibility of using ion-exchange Sep-Pak cartridges for the fractionation of pituitary peptides was investigated. Extracts of bovine posterior pituitaries were fractionated at either pH 5 or pH 7 by pairs of cation and anion exchangers, connected in series. The capacity to bind peptides was well correlated with the theoretical charge calculated for a variety of peptides. At pH 5 the entire tissue extract could be fractionated into either basic or acidic pools. In contrast, at pH 7 only the more basic or acidic peptides were retained by the respective ion exchangers. The rest of the peptides passed through both ion exchangers and were recovered in the neutral pool. The ion-exchange fractionation principle was used to facilitate the purification of 35S-labelled intermediate pituitary glycopeptides, prepared by incubating mouse intermediate lobes in explant culture with 35S-labelled sulphate. 35S-labelled glycosylated forms of Lys 1γ 3MSH, corticotropin-like intermediate lobe peptide, and the amino terminal or 16K fragment of pro-opiomelanocortin ( i.e. 16K 1–74) were fractionated into separate pools such that they could be purified to homogeneity in a single step by reversed-phase high-performance liquid chromatography (RP-HPLC). Purification by conventional means would require at least two RP-HPLC steps. Thus, radiolabelled peptides can be purified with the minimum of chromatographic manipulation, thereby ensuring maximal recoveries.

Proopiomelanocortin peptide immunocytochemistry in rhesus monkey brain

The immunocytochemical distribution of Proopiomelanocortin (POMC) peptides (β-endorphin, ACTH, α-MSH, 16K fragment) was studied in the brain of the rhesus monkey ( Macaca mulatta). Some animals were administered colchicine intracerebroventricularly prior to sacrifice to enhance the visualization of perikaryal immunoreactivity. Immunoreactive perikarya are localized to hypothalamic infundibular nucleus, giving rise to several distinct projections. Rostral projections extend through midline diencephalic and preoptic areas, and enter the telencephalon. Along this course, immunoreactive fibers are seen in midline hypothalamic and preoptic nuclei, nucleus of the diagonal band, olfactory tubercle, nucleus accumbens, bed nucleus of stria terminalis, septum, and other limbic structures in telencephalon. Caudal to the anterior commissure, some fibers ascend dorsally to enter the midline thalamus, which they innervate. Lateral projections of the infundibular perikarya course through the medial-basal hypothalamus, dorsal to the optic tracts, and enter the amygdala region where they innervate more medially situated amygdaloid nuclei. Caudal projections of the POMC neurons also extend through midline diencephalon, some coursing along a periventricular path to innervate midline hypothalamic and thalamic nuclei. This projection extends into the mesencephalic substantia grisea centralis and may also contribute to the innervation of more dorsally situated nuclei in the pons and medulla, such as the parabrachial nuclei and nucleus tractus solitarius. Other caudal projections originating in the hypothalamus course through the ventral tegmentum of mesencephalon and pons and may contribute to the innervation of midline raphe and other ventrally situated nuclei in the pons and medulla. The distribution of immunoreactive perikarya and fibers in the brain of rhesus monkey is strikingly similar to that found in the rat brain. However, subtle differences appear to exist in the innervation patterns of particular brain regions.

Big form of γ-melanotropin-like immunoreactivity in normal human plasma

Using a radioimmunoassay for γ 3-melanotropin ( γ 3-MSH), γ-melanotropin-like immunoreactivity (γ-MSH-LI) was detected in plasma extracts of normal subjects before and after metyrapone administration. Plasma γ-MSH-LI from four normal men rose significantly after a single oral administration of metyrapone (30 mg/kg of body weight). Gel chromatographic study of plasma extract after metyrapone administration showed a single peak of γ-MSH-LI near the elution position of mouse 16K fragment, however smaller forms of γ-MSH-LI were not detected.

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

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH 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 β-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 β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-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 β-endorphin in this species.

Characterization of gamma-melanotropin-like immunoreactivity and its secretion in an adrenocorticotropin-producing mouse pituitary tumor cell line.

gamma MSH, a putative hormone in the N-terminal region of the ACTH/beta-endorphin (beta-EP) precursor protein, was studied by RIA with an antiserum against gamma 3MSH in ACTH-producing mouse pituitary tumor cells, AtT-20/D16v. Serial dilution of the culture medium or the cell extract gave parallel lines to the standard curve in the RIA for gamma MSH. Rat median eminence extracts enhanced the release of gamma MSH-like immunoreactivity (gamma MSH-LI) concomitant with ACTH-like immunoreactivity (ACTH-LI) and beta-EP-like immunoreactivity (beta-EP-LI). Dexamethasone suppressed the release of gamma MSH-LI as well as ACTH-LI and beta-EP-LI. Gel exclusion chromatography of the culture medium and the cell extract has revealed that gamma MSH-LI consists of two peaks; one eluted near the elution position of beta-lipotropin and the other near the elution position of beta-EP. There was no peak corresponding to the elution position of synthetic gamma 3MSH. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has demonstrated that gamma MSH-LI migrated at five positions with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, respectively. The 31K gamma MSH coincided with the migration position of 31K ACTH of 31K beta-EP, and 21-23K gamma MSH coincided with the position of 21-23K ACTH on SDS-PAGE. The 16-17K gamma MSH coincided with the mouse 16K fragment (reported by Eipper and Mains) of ACTH-beta-lipotropin precursor protein in the migration in SDS-PAGE and in immunoreactivity to anti-gamma MSH antiserum. [3H]Glucosamine was incorporated into 16K, 13K, and 3.8K gamma MSH. These results suggest that AtT-20/D16v cells produce gamma MSH-LIs with molecular weights of 31K, 21-23K, 16-17K, 13-14K, and 3.8K, and they are secreted concomitantly with ACTH-LI and beta-EP-LI.

RESPONSES OF ISOLATED GUINEA-PIG ADRENAL CELLS TO ACTH AND PRO-OPIOCORTIN-DERIVED PEPTIDES.

A comparison of the responses of isolated guinea-pig adrenal cells to ACTH and pro-opiocortin-derived peptides was carried out by measuring cortisol, aldosterone, androstenedione and dehydroepiandrosterone production. With concentrations below 10,000 pg/ml, no steroidogenic activity was found in response to either beta-LPH, gamma-LPH, gamma 3-MSH or the 16K fragment, whether assayed alone or in association with ACTH. At concentrations above 10,000 pg/ml, gamma-LPH (100 ng), the 16K fragment (100 ng) and beta-endorphin (500 ng) proved to be totally inactive. beta-LPH from 25 to 250 ng, however, exhibited a significant though slight stimulatory effect on cortisol, aldosterone and androstenedione production. Its effectiveness on aldosterone production was especially marked, but the extent of the response was modest in view of the concentrations used.

Ontogenesis of Adrenocorticotropin-Related Peptide Determinants in the Hypothalamus and Pituitary Gland of the Rat*

The time of appearance of the antigenic determinants of ACTH-related peptides (16K fragment, ACTH, gamma-lipoprotein, and beta-endorphin) was determined immunohistochemically in the brain and pituitary gland of the fetal rat. Major antigenic determinants of the pro-ACTH/endorphin precursor first appear in basally located cells of the hypothalamus on day 12 of gestation. Long axonal fibers are evident at day 13. Laterally located cells give rise to lateral, superficial fibers, whereas medially located cells give rise to periventricular fibers. The antigenic determinants appear simultaneously in the anterior lobe of the pituitary gland on day 16 and in the intermediate lobe on day 17. The intensity of immunostaining with anti-16K fragment antiserum is stronger than with the other antisera in the brain but is equal with other antisera in the pituitary. Neuronal perikarya always stain less intensely than pituitary cells. It is concluded that 1) ACTH-related antigenic determinants are produced within the central nervous system, and 2) cells containing ACTH-related antigenic determinants in pituitary gland and brain develop independently of one another.

Immunoreactive gamma-melanotropin in rat pituitary and plasma: a partial characterization.

A RIA for the gamma MSH region of proopiomelanocortin has been established and validated. The antiserum was raised to synthetic bovine gamma MSH, and it cross-reacts well with larger polypeptides, including gamma MSH and 16K fragment, which contain the gamma MSH sequence. Gel filtration chromatography of extracts prepared from the anterior and neurointermediate lobes of rat pituitary and from rat plasma reveal two heterogeneous immunoreactive (IR-) forms of gamma MSH in each venue which elute in molecular weight peaks of approximately 11,000 (11K) and 6,000 (6K). Addition experiments indicate that the smaller material is not an artifact generated from the larger form during preparation. By the criterion of retention on affinity columns of Concanavalin A-agarose, these peptides are glycosylated. The 6K form represents 8-17% of the total IR-gamma MSH in the anterior lobe and about 30% of that in the neurointermediate lobe. IR-gamma MSH are released by dispersed rat pituitary cells in response to several of the same secretagogues which modulate the secretion of other proopiomelanocortin-derived peptides. Changes in the plasma and anterior lobe content of IR-gamma MSHs and ACTH resulting from perturbation of the pituitary-adrenal axis are concordant. In particular, the plasma concentration of 6K IR-gamma MSH increases dramatically after imposed stress.

Gamma-melanotrophin-like immunoreactivities in human pituitaries, ACTH-producing pituitary adenomas, and ectopic ACTH-producing tumours: evidence for an abnormality in glycosylation in ectopic ACTH-producing tumours.

Using gel exclusion chromatography on Bio-Gel P-60, gamma-melanotropin-like immunoreactivity (gamma-MSH-LI) in three human pituitary glands, two ACTH-producing pituitary adenomas, and three ectopic ACTH-producing tumours (two medullary thyroid carcinomas and one thymoma) was divided into one or two molecular weight classes. The largest component eluted near the position of mouse 16K fragment and was designated big gamma-MSH-LI. This big gamma-MSH-LI was present in all samples. The second one, designated intermediate gamma-MSH-LI, eluted between the position of mouse 16K fragment and human ACTH, and was demonstrated only in two ectopic ACTH-producing tumours. No gamma-MSH-LI emerged at the elution position of synthetic gamma 3-MSH. Affinity chromatography on concanavalin A-agarose revealed that a significant fraction (52-68%) of gamma-MSH-LI from human pituitary glands, ACTH-producing pituitary adenomas, and one ectopic ACTH-producing tumour bound to the column and was eluted with alpha-methyl-D-mannopyranoside. In two ectopic ACTH-producing tumours which contained big and intermediate gamma-MSH-LI, a relatively small fraction (27-35%) of gamma-MSH-LI bound to the column and was similarly eluted. These observations suggest that human gamma-MSH-LI is glycosylated and that there is an abnormality in the glycosylation of gamma-MSH-LI in some ectopic ACTH-producing tumours.