Evidence for increased alpha MSH receptor binding in the F1 variant of B16 melanoma cells grown in dialyzed fetal calf serum

The specific binding of an alpha MSH analogue (Ac-[Nle4, D-Phe7] alpha MSH4-11 NH2) was enhanced in the presence of 10% dialyzed fetal calf serum (FCS) as compared with 10% FCS (nondialyzed) in the F1 variant of B16 melanoma cells. The replenishment of dialyzed serum with adrenocorticotrophic hormone (ACTH) or insulin had no effect on the increased level of alpha MSH receptor binding in these cells. However, 10 nM alpha MSH or 1 microM ACTH under identical conditions significantly decreased the level of alpha MSH binding. Competitive binding studies involving the alpha MSH analogue revealed that the specificity of the receptor was restricted to the complete molecule of alpha MSH, our analogue, beta MSH and ACTH1-24, ACTH4-10, which contains the amino acid sequence responsible for biological activity, showed a very low affinity for the receptor. Furthermore, we observed an interesting phenomenon unique to dialyzed FCS in that once the cells were grown to confluence and melanin was produced, the cells were no longer viable. However, in McCoy's medium, which is deficient in tyrosine, the cells did not produce melanin and remained viable.     

Inhibitory effects on steroid production from isolated adrenal cells of rhesus monkey (Macaca mulatta) of pro-opiomelanocorticotrophic peptides

Adrenal glands from Rhesus monkeys (Macaca mulatta) of 160 days gestation, newborn, 2 months-old infants or 6 months-old infants were excised and prepared, by a collagenase digestion, as a cell suspension. The cells were incubated with 10 pg/ml, 100 pg/ml or 1 ng/ml of a peptide of the ACTH/pro-opiomelanocortin 'family', 57K, 31K, 20K, alpha MSH, ovine-CLIP or gamma LPH either in the presence or absence of 166 pg/ml ACTH1-39. The production by cortisol and androstenedione was measured by radioimmunoassay. Using the steroid production by aliquots of the cell suspension with either no stimulating agent or ACTH1-39 alone as controls, the net influence of these different peptides on basal or ACTH1-39-stimulated production was observed. alpha MSH, ovine-CLIP and gamma LPH had no influence on either basal or stimulated cortisol or androstenedione production. Corticotrophic peptides of 57K, and 20K and pro-opiomelanocortin each had a steroidogenic activity alone, in all age groups. In the fetal and newborn monkeys' adrenal cells, peptides of 57K and 20K at 1 ng/ml had an inhibitory influence on ACTH1-39 stimulated cortisol and androstenedione production. The influence of the 20K peptide is partially inhibitory as the steroidogenic potential of this peptide is not additive with that of ACTH1-39. These results show that, as observed in other species, that the ACTH/pro-opiomelanocortin range of peptides are inhibitory to the action of ACTH1-39 in the developing adrenal.     

The utility of antisera to different synthetic adrenocorticotrophins (ACTH) and melanotrophins (MSH) for immunocytochemical staining of the dog pituitary gland

Using the immunoperoxidase technique and specific antisera to synthetic ACTH beta (1-24), ACTH beta (17-39) and bMSHbeta1, selective immunocytochemical staining was localized in a distinctive cell type in the pars distalis and pars tuberalis of the dog pituitary gland. Except for a rare cell, the pars distalis and pars tuberalis did not stain with an anti-bMSH alpha serum. In the pars intermedia immunoreactive cells containing ACTH beta(1-24), ACTHbetap(17-39), bMSHbeta and/or bMSH alpha were observed. The specificity and validity of the antisera were demonstrated by elimination of their immunostaining capacity after prior absorption with their respective antigens, while absorption with other antigens failed to decrease staining intensity. The cytoplasm of the ACTH/MSH cells showed a positive reaction to periodic-acid-Schiff and assumed a pale aniline blue colour, whilst the granules were stained with carmoisine L and acid alizarine blue. These ACTH/MSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. It is concluded that ACTH and MSH beta were present and most probably produced by the corticomelanotrophs of the pars distalis and pars tuberalis. In addition to corticomelanotrophs analogous to those of the pars distalis and pars tuberalis, the pars intermedia showed many cells which contain MSH alpha alone or together with MSH beta and/or ACTH.     

The prenatal development of alkaline phosphatase activity in the hypothalamus of the rat

The localization of alkaline phosphatase (E.C. 3.1.3.1.) positivity during prenatal development of the hypothalamus of the rat is described. At E12 all layers of the prosencephalon display alkaline phosphatase (AP) positivity. The AP positivity increases from dorsal to ventral. Within the hypothalamic area a second, rostro-ventral gradient exists from E14 onwards. At E18 both gradients have decreased. At E20 almost all AP positivity has disappeared from the hypothalamus, with the exception of some reaction product in the dorsal ventricular matrix of the hypothalamus. The significance of this pattern in relation to the differentiation of the hypothalamus and to the formation of hypothalamic connections is discussed. It is suggested that AP activity is related to the formation of connections.     

Pressor and cardioaccelerator effects of gamma MSH and related peptides

We have recently demonstrated that the hypertensinogenic and natriuretic actions of ACTHI-39 can be found in a non-steroidogenic fragment of ACTH, ACTH4-10. These effects of ACTH or ACTH4-10 may be due to their ability to act as weak agonists of gamma MSH. gamma MSH is found in the 16K N-terminus of pro-opiocortin, and contains a sequence analogous to ACTH4-10, gamma MSH3-9. We investigated the cardiovascular effects of gamma 2MSH, gamma MSH3-9, and sterically restricted analogs of ACTH4-10. The results indicate that gamma MSH3-9, had essentially the same activities as ACTH4-10. The addition of five other amino acid residues to gamma MSH3-9 (gamma 2MSH) resulted in significant enhancement of pressor and cardioaccelerator activity. Steric restriction of the ACTH4-10 sequence by the substitution of a D-Phe in place of an L-Phe residue in position #7, or cyclization of the peptide by a half-Cys4, half Cys10 intramolecular disulfide-bridge derivatization, resulted in increased cardiovascular activities. Based on these data, the cardiovascular actions of ACTH4-10, gamma MSH3-9, and gamma 2MSH are predicted to be due to the assumption of a reverse-turn three-dimensional structure. The additional residues in gamma 2MSH appear to specifically enhance the cardiovascular activities of gamma MSH3-9. The results suggest the existence of a new class of hypophyseal peptides with cardiovascular activities, which require the assumption of a defined three-dimensional structure.     

Neuromuscular response of the immature rat of ACTH/MSH 4--10

The adrenocorticotropin fragment ACTH/MSH 4--10 (0.1 ug/kg IP) effectively modulates the neuromuscular responses of 9 to 15 day old rats. Muscle (extensor digitorum longus) contraction amplitude is increased, fatigue is delayed and muscle half-relaxation time is shortened during 20 min of continuous in situ stimulation of a branch of the deep peroneal nerve (square wave shocks 10 Hz, duration 0.5 msec, strength supermaximal). No effect on contraction time is seen. There is no facilitation or change in any contraction parameter in rats older than two weeks (16 to 40 days) indicating that these older animals, like normal adult rats, are unaffected by the peptide. Immature rats, however, are even more sensitive than hypophysectomized adult rats [29] to the ameliorative action of ACTH/MSH 4-10. This early sensitivity to ACTH/MSH 4--10 corresponds to important developmental changes occurring in nerve and muscle during the most critical period in postnatal development, the first two weeks.     

Localization of alpha1B-adrenergic receptor in female rat brain regions involved in stress and neuroendocrine function.

Activation of alpha1-adrenergic receptors has been linked to the control of blood pressure, neuroendocrine secretion, reproductive behavior and mood. The present study describes the distribution of alpha1B-adrenergic receptor immunoreactivity in female rat brain regions involved in stress and neuroendocrine function. The pattern of immunolabeling seen resembles that obtained in previous in situ hybridization studies. Several hypothalamic areas that control pituitary function showed intense fiber and/or cell immunolabeling, including the paraventricular nucleus of the hypothalamus, the supraoptic nucleus, and the median eminence. Some regions such as the arcuate nucleus, the median eminence, and dorsal hypothalamus exhibit intense labeling of axonal varicosities, while other regions exhibit only perikarya immunolabeling. alpha1B-adrenergic receptor immunoreactivity was also observed in large pyramidal neurons of layer V of the cerebral cortex, the frontal cortex showing a particularly strong immunoreactivity. Virtually all thalamic regions were labeled, especially the lateral and ventral areas. In addition, labeled cells were present in hippocampus, the medial septum, the horizontal and vertical limbs of the diagonal band of Broca, and the caudate putamen. Finally, some midbrain and hindbrain regions important for motor function were immunoreactive. Because ligands specific for alpha1-adrenergic receptor subtypes are not available, the present immunocytochemical study not only addresses the subcellular and regional distribution of alpha1B-adrenergic receptors but may also provide clues about receptor subtype-specific function.     

Immunohistochemical study on adenohypophysial primordia in organ culture

Summary Rathke's pouches isolated from rat fetuses on day 12 were maintained in organ culture for 9 days and investigated immunohistochemically to test whether or not the hypothalamus is involved in the cytodifferentiation of the adenohypophysis. The unlabeled antibody enzyme method demonstrated that the cultured tissue contains different types of glandular cells, i.e., adrenocorticotropin (ACTH)-, growth hormone (GH)-, luteinizing hormone (LH)-, thyrotropin (TSH)-, and prolactin-producing cells. Indirect evidence was also obtained to indicate the presence of melanocyte stimulating hormone (MSH)-cells. These findings suggest that adenohypophysial primordial cells of rats start to synthesize their respective hormones without stimuli from neurosecretory substances of the brain which are known to be essential for the maintenance of the secretory activity of the adult gland.We wish to express our thanks to Dr. A. Kawaoi for providing anti-porcine ^1–39ACTH, to Dr. S.S. Spicer for the supply of anti-porcine ^17–39ACTH and to Dr. P. Petrusz for the gift of antisera against bovine GH, bovine TSH, HCG and rat prolactin. We should also like to thank Mr. Y. Okamura for technical help and Mr. I. Shimada for preparation of the photographs.     

Demonstration of the direct projections of ACTH immunoreactive neurons on the complex of the locus coeruleus in cats

Using a combined retrograde HRP and immunohistochemical technique in the cat, we have demonstrated the existence of direct descending projections of the ACTH immunoreactive neurons in the arcuate nucleus of the hypothalamus to the pontine tegmentum, especially, to the nucleus locus coeruleus alpha.     

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.     

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).     

Microsequencing evidence for the maturation of human proopiomelanocortin into an 18 amino acid beta-melanocyte stimulating hormone [h beta MSH(5-22)] in nonpituitary tissue

Sixty pmoles of a material with molecular size, immunological, and RP-HPLC characteristics identical to that of h beta MSH(5-22) were purified from a bronchial carcinoid tumor responsible for the ectopic ACTH syndrome. The first 16 cycles of microsequencing revealed the following sequence: Asp-Glu-Gly-Pro-Tyr-Arg-Met-Glu-X-Phe-Arg-Trp-Gly-X-Pro- Pro-, identical to the first 16 amino acids of h beta MSH(5-22). Since this material was recognized by an antibody which requires the free COOH-terminal Asp22 residue, it can be assumed that it is indeed h beta MSH(5-22). We also show that neither the 5 N acetic acid nor the 1 N HCl extraction procedure artefactually generated h beta MSH-like material in normal or tumoral human pituitaries and in nonpituitary tumors. We conclude that h beta MSH(5-22) is a normal maturation product of proopiomelanocortin in the human nonpituitary tissues which express its gene, including the hypothalamus and ACTH-secreting tumors.     

Infundibular neurons of the human hypothalamus simultaneously reactive with antisera against endorphins,ACTH, MSH and β-LPH

In man, discrete neurons of the infundibular (arcuate) nucleus contain compounds that can be stained with anti-endorphin (alpha and beta), anti-ACTH, anti-MSH (alpha and beta) and anti-beta-LPH immune sera (I.S.). In the fetus, certain neurons stain with anti-beta-endorphin or anti((17--39)ACTH starting from the 11th week of fetal life. At the ultrastructural level, these neurons contain elementary granules that are immunoreactive with anti-beta-endorphin. In the adult, neurons immunoreactive with anti-beta-endorphin are found in the infundibular nucleus. Their axonal fibers terminate around blood vessels in the neurovascular zone and in the pituitary stalk, or establish contacts with non-immunoreactive perikarya of the infundibular nucleus. These neurons can be stained with anti(17--39)ACTH and anti-beta-endorphin I.S. The most reactive are also stained moderately with anti-alpha-MSH, anti-beta-MSH, anti-beta-LPH, anti-alpha-endorphin, or anti(1--24)ACTH I.S. These results indicate that, in man, compound(s) identical with or immunologically related to endorphins, beta-LPH, ACTH and MSH are secreted by certain hypothalamic neurons. These agents probably originate from a common precursor molecula similar to the so-called pro-opiocortin.     

Presence of adrenocorticotropin and β-Endorphin immunoreactivities in the magnocellular neurosecretory system of the rat hypothalamus

Summary Antisera raised against ACTH (1–39), -endorphin and the 16K proopiocortin were used, in association with the immunoperoxidase reaction, to localize positively-staining cell bodies and nerve fibres in the hypothalamus of the rat. Antigens, cross-reactive against anti-ACTH (1–39) serum were detected in a fibre system in the rostro-dorsal hypothalamus situated between the optic chiasm and the third ventricle while immunoreactive 16K-like material was present in fibres localized in the caudal hypothalamus, dorso-lateral to the arcuate nucleus. This latter system was also associated with the appearance of ACTH (1–39) and ACTH (17–39) immunoreactivity.Cells of the arcuate nucleus stained positively for ACTH (1–39), 16K antigen and -endorphin, and on examining adjacent thin sections it was observed that cells that contained 16K antigen-like material, also gave a positive immunoreaction with ACTH (1–39) and -endorphin antisera. In the magnocellular system, cells of the supraoptic (SON) and paraventricular (PVN) nuclei also gave a positive immunoreaction with anti-ACTH (1–39), 16K antigen and -endorphin serum. As in the case of the arcuate nucleus, common cells stained for these three antigens.On the basis of the precursor theory for the synthesis of ACTH, 16K antigen and -endorphin, it was not unexpected to find these three fragments of pro-opiocortin localized together in cells of the arcuate nucleus. That ACTH (1–39), 16K antigen and -endorphin-like materials are present in the magnocellular neurosecretory system would suggest that cells of the SON and PVN are not only involved in the synthesis of neurophysin and the neurohypophysial hormones, but also of some products of the pro-opiocortin molecule. Whether the biochemical nature of the ACTH and -endorphin in cells of the SON and PVN is identical to that of anterior pituitary origin remains to be established, as does the biosynthetic relationship between neurophysin and oxytocin/ vasopressin and these fragments of pro-opiocortin.Drs. M.M. Wilkes, S.S.C. Yen, G. Pelletier, B.A. Eipper and R. Walter are thanked for supplying some of the antisera and antigens used in this study. Thanks also go to Ciba-Geigy Ltd. and Organon Inc. for supplies of ACTH (17–39) and ACTH (1–24) respectively. This work was financed by The Medical Research Council of New Zealand     

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.     

Peptide influences on the development and regeneration of motor performance

ACTH 1-39 (0.2 U IP daily for up to 18 days) has a beneficial effect on the functional reorganization of regenerating motor units of the extensor digitorum longus (EDL) in the adrenalectomized adult rat following crushing of the peroneal nerve. Motor unit activity (maximum twitch tension amplitude/mean increment in twitch tension as voltage is increased by 0.1 V gradations) and nerve-muscle efficiency (tetanic tension from indirect stimulation/tetanic tension from direct stimulation of EDL) were enhanced by ACTH 1-39. Other electrophysiological and contractile parameters were unaffected by the peptide. Spontaneous motor activity in cold stressed 13 day old rats was prolonged by Org 2766, a substituted analogue of ACTH/MSH 4-9, (0.1 microgram/kg daily) but unaffected by the same dosage of ACTH/MSH 4-10. The responsiveness of developing and regenerating motor systems to neuropeptides indicates a plasticity of neuronal connections, which depends on peptide sequence, dosage and the physiological state of the animal (normal, depressed, regenerating or developing, at rest or stressed).     

Immunocytochemical evidence for anti-LHRH and anti-ACTH activity in the "F" antiserum.

An antiserum which has previously been thought to be specific for LHRH-like immunoreactive material was shown to contain two populations of antibodies, one demonstrating anti-LHRH activity and the other anti-ACTH(1-24) activity. In rat and mouse, ACTH(1-24)-like immunoreactive substance is present in perikarya within the basal hypothalamus and in fibers in arcuate, periventricular and dorsomedial nuclei. LHRH-like immunoreactivity is present in fibers within the median eminence and arcuate nucleus, in a few fibers running along the ventral border of the hypothalamus, and in a small number of cell bodies within the medial basal hypothalamus.     

Effects of alterations in the activity of tuberohypophysial dopaminergic neurons on the secretion of alpha-melanocyte stimulating hormone

Administration of gamma-butyrolactone (GBL), an anesthetic which reduces dopaminergic neuronal activity, decreased the concentration of the dopamine (DA) metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the intermediate lobe of the pituitary gland, and increased alpha-melanocyte stimulating hormone (alpha MSH) concentrations in the serum of male rats. Bilateral electrical stimulation of the rostral arcuate nucleus, which contains perikarya of tuberohypophysial DA neurons, increased DOPAC concentrations in the intermediate lobe and decreased alpha MSH concentrations in the serum of GBL-anesthetized rats. Administration of the DA antagonist haloperidol prevented the decline in serum alpha MSH levels following arcuate nucleus stimulation, but had no effect on serum alpha MSH concentrations in sham-stimulated GBL-treated rats. These results indicate that GBL-induced decreases or stimulation-induced increases in the activity of tuberohypophysial DA neurons are accompanied by corresponding changes in the metabolism of DA in the intermediate lobe of the rat pituitary gland, and by reciprocal changes in the secretion of alpha MSH.     

The bifunctional role of pro-opiomelanocortin derivatives in the mediobasal hypothalamus of the rat

In the present study, a polyclonal antibody against pro-opiomelanocortin derivatives was characterized biochemically. Its immunoreactivity with structures of the arcuate nucleus and the median eminence was investigated by means of the immunogold method and compared with its reaction on adenohypophyseal cells with and without pre-adsorption with pro-opiomelanocortin derivatives. The antiserum detects ACTH and its fragments, in particular alpha-MSH, and beta-endorphin. In the adenohypophysis gold particles are exclusively located on small secretory granules situated in the periphery of branched cells. In the perikarya of the arcuate nucleus gold particles are observed on terminal vesicles abutting from the cis-face of the Golgi apparatus, on granules in its direct vicinity and on small dense core vesicles preferentially located in the cell periphery. Immunoreactive gold-labeled fiber profiles are found in a sub- or intra-ependymal position as well as in the nuclear neuropil proper. Here axodendritic and axosomatic synapses are observed. In both situations the gold particles are mostly restricted to the small dense core vesicles and do not decorate the synaptic vesicles. In the median eminence gold labeled fibers are detected in all layers. The labeled fibers can be closely apposed to tanycytic processes, without, however, forming special contact differentiations. In direction to the perivascular layer of the external zone the labeled profiles are more frequently arranged in groups intermingled with unlabeled fibers. The axons decorated with gold particles can be freely exposed to the perivascular space or are found as single processes in close vicinity to the capillary wall. Subsequent to preincubation of the native antiserum with ACTH1-39 and ACTH18-39 (= CLIP) neither adenohypophyseal cells nor perikarya and fibers in the arcuate nucleus nor axons in the median eminence are decorated with gold particles. Preincubation of the native antiserum with alpha-MSH or beta-endorphin does not change the immunoreaction with the small, peripherally situated granules in the branched adenohypophyseal cells. In neurons of the arcuate nucleus and in fibers of the median eminence, however, the immunoreaction is completely extinguished when the antibody is pre-incubated with alpha-MSH, whereas subsequent to preincubation with beta-endorphin only the amounts of labeled structures are reduced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of tunicamycin on the synthesis, processing, and secretion of pro-opiomelanocortin peptides in mouse pituitary cells

Pro-opiomelanocortin (POMC) is glycosylated and proteolytically cleaved to produce a number of smaller peptide hormones including adrenocorticotropic hormone (ACTH) and endorphin in mammalian pituitary and the mouse pituitary cell line AtT-20/D16v. When glycosylation of POMC is inhibited in AtT-20 cells with the drug tunicamycin, a 26,000-dalton protein appears in place of the glycosylated 29,000- and 32,000-dalton forms of POMC. The 26,000-dalton form found in tunicamycin-treated cells has the same [35S]methionine tryptic peptides as 29,000- and 32,000-dalton POMC, indicating that the decrease in apparent mass is most likely due to loss of carbohydrate and not to changes in the peptide backbone. The 4,500-dalton form of alpha(1-39)ACTH and the 3,000- and 11,000-dalton forms of endorphin are all present in tunicamycin-treated cells. The glycosylated form of alpha(1-39)ACTH, however, is missing and the glycosylated ACTH intermediates are replaced by unglycosylated ACTH intermediates. Pulse-chase studies demonstrate that the 26,000-dalton unglycosylated POMC is the precursor of the smaller ACTH and endorphin molecules in tunicamycin-treated cells. Furthermore, all of the forms of ACTH and endorphin found in tunicamycin-treated cells are secreted. Thus, it appears that glycosylation is not an essential step for correct cleavage or secretion of POMC or its products.