Assignment of the proopiomelanocortin (POMC) gene to BTA 11 and RFLPs in cattle and sheep.
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Animals↗
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Cyclic AMP and ethanol interact to control apoptosis and differentiation in hypothalamic beta-endorphin neurons.
In this study we have determined the role of cyclic AMP on the function and differentiation of beta-endorphin (beta-EP) neurons in rat fetal hypothalamic cell cultures. Addition of Bt2cAMP or the cAMP elevating agent, forskolin, in cultures, dose and time dependently increased beta-endorphin secretion. The increased beta-EP secretion after Bt2cAMP or forskolin treatment was associated with proopiomelanocortin gene expression, enhanced neurite growth, and increased neuronal viability. Determination of internucleosomal cleavage of DNA by agarose gel electrophoresis revealed that apoptosis occurred in hypothalamic neurons during the first 6-8 days in culture. Addition of Bt2cAMP during this developmental period inhibited DNA degradation in hypothalamic neurons. Furthermore, incubation with various doses of ethanol, which is known to reduce intracellular levels of Bt2cAMP, increased DNA degradation in these cells. Ethanol-induced DNA degradation was blocked by concomitant incubation with Bt2cAMP. Histochemical identification of apoptotic cells following ethanol and Bt2cAMP treatments further revealed that apoptosis occurred in beta-EP neurons during the developmental period, and that ethanol increased and Bt2cAMP reduced apoptotic beta-EP cell numbers. These results suggest that ethanol neurotoxicity on beta-EP neurons during early neuronal differentiation involves an apoptotic process and that the cAMP signaling system plays an important role in controlling apoptosis and differentiation of the beta-EP neuronal system.
Animals↗
Effect of POMC-derived peptides on corticosterone secretion during the stress hypo-responsive period in rat.
During the stress hyporesponsive period (postnatal days 2-10), the stimulation of corticosterone secretion by ACTH is very low. In our study, we have observed that administration of ACTH during 3 consecutive days is followed by a striking increase in corticosterone response to an acute ACTH test. A comparable potentiation of corticosterone secretion was observed after a similar treatment with Lys gamma 3-MSH. The 3 day-treatment with each peptide induced an increase in adrenal weight. Similar data were obtained with alpha-MSH. However, corticosterone response to ACTH was not significantly altered following the same pattern of alpha-MSH administration.
Adrenal Cortex↗
The proopiomelanocortin (POMC) gene expression of AtT20 mouse pituitary cells is dependent on cell culture conditions.
We have compared the properties of AtT20 cells cultivated in a Dulbecco's modified Eagles medium containing 10% fetal calf serum (FCS) and of AtT20 cells adapted to a chemically better defined medium with transferrin, albumin, insulin, sodium selenit and 0.2% FCS. Our interest was focused on the hypothalamo-pituitary-adrenal axis (HPA) involved adrenocorticotropic hormone (ACTH) and the potent opioid peptide beta-endorphin (beta-END). There were no differences in basal secretion of ACTH and beta-END by cells cultivated in medium containing 10% or 0.2% serum, respectively. In combination to the decreased proliferation activity of AtT20 cells, grown in the serum-reduced medium we found a strongly enhanced ACTH secretion activity stimulated by the corticotropin releasing hormone (CRH) in contrast to normally cultivated AtT20 cells (10% serum). In addition, the proopiomelanocortin (POMC) gene expression was significantly down regulated in serum-reduced medium and was normalized again after further cultivation in a 10% serum containing medium. This leads to the conclusion that under standard conditions (10% serum) the gene transcription is increased by hitherto uncharacterized modulators present in the serum. The unexpected unchanged amounts of ACTH and beta-End could be the result of increased protein convertases activities. These enzymes are responsible for the POMC precursor processing into beta-End and ACTH.
Animals↗
Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2.
AtT-20 mouse corticotrope tumor cell lines overexpressing the prohormone convertases PC1 or PC2 were established and used to examine prohormone and prohormone convertase biosynthetic processing. On a molar basis, wild-type AtT-20 cells synthesize about 20% as much PC1 as the endogenous prohormone, proopiomelanocortin (POMC). Kinetic, oligosaccharide, and temperature blockade analyses established that proPC1 is converted to PC1 in the endoplasmic reticulum at a rate independent of the level of PC1 or PC2 expression. In contrast, proPC2 is converted to PC2 primarily in a post-trans-Golgi compartment. PC1 is further shortened from its COOH-terminal end in a post-trans-Golgi compartment in a step that is accelerated at higher levels of PC1 expression, but unaltered by PC2 overexpression. The initial steps in POMC processing are speeded up by overexpression of PC1, and overexpression of PC1 leads to more extensive cleavage of POMC to smaller products. However, even when the rate of PC1 synthesis exceeds that for POMC by 2-fold, PC1 does not cleave the Lys-Lys or Arg-Lys bonds cleaved upon overexpression of PC2.
Animals↗
Plasma concentrations of immunoreactive proopiomelanocortin peptides and cortisol in clinically normal cats.
We measured immunoreactive (IR) plasma concentrations of the proopiomelanocortin (POMC)-derived peptides (adrenocorticotropic hormone [ACTH]; beta-endorphin/beta-lipotropin [beta END/beta LPH]; and alpha-melanocyte stimulating hormone [alpha MSH]) and of cortisol in 100 clinically normal cats. Median plasma concentration of IR-ACTH was 2.7 pmol/L (range, < or = 1.1 to 22 pmol/L), of beta END/beta LPH was 28 pmol/L (range, 3.8 to 130 pmol/L), of alpha MSH was 36 pmol/L (range, < or = 3.6 to 200 pmol/L), and of cortisol was 35 nmol/L (range, 5 to 140 nmol/L). Plasma concentrations of IR-ACTH, alpha MSH, and beta END/beta LPH were at or below the assay sensitivity in 34, 3, and 0% of the cats, respectively. We did not detect a correlation between plasma concentrations of IR-ACTH and beta END/beta LPH (r = 0.23) or between plasma concentrations of IR-ACTH and alpha MSH (r = 0.19). However, there was a significant (P < 0.001) correlation between plasma concentrations of IR-beta END/beta LPH and alpha MSH (r = 0.81). There was not a significant correlation between plasma concentration of cortisol and plasma concentration of any of the IR-POMC peptides. High plasma concentrations of IR-alpha MSH and beta END, POMC peptides secreted predominantly by melanotrophs in other species, indicate that clinically normal cats have an actively secreting pars intermedia.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenocorticotropic Hormone↗
Post-translational processing of proopiomelanocortin (POMC) in mouse pituitary melanotroph tumors induced by a POMC-simian virus 40 large T antigen transgene.
Mice harboring a transgene composed of proopiomelanocortin (POMC) gene promoter sequences (nucleotides -706 to +64) ligated to the simian virus (SV) 40 early gene encoding large T antigen developed large POMC-expressing pituitary tumors. Histologically the tumors arose from the intermediate lobe, contained nuclear SV40 T antigen and POMC peptides, but did not express other pituitary hormones. POMC processing in the pituitary tumors was indistinguishable from normal mouse intermediate lobe melanotrophs and was characterized by high proportions of acetylated and carboxyl-terminal shortened beta-endorphins, and amino-terminal acetylated alpha-melanocyte-stimulating hormone, and virtually no adrenocorticotropic hormone (ACTH)(1-39), beta-lipotropin, or POMC. The tumors contained abundant levels of mRNA for the prohormone convertase PC2 and undetectable levels of PC1. Normal mouse neurointermediate lobe also has a high ratio of PC2/PC1 expression that is distinct from the relative abundance of PC1 in anterior lobe and AtT-20 corticotroph cells. In contrast, extracts from tumors transplanted subcutaneously in nude mice contained predominantly nonacetylated forms of beta-endorphin(1-31) and -(1-27), very little ACTH(1-39), almost no corticotropin-like intermediate peptide or alpha-melanocyte-stimulating hormone, and higher proportions of intact POMC. Surprisingly, despite the less efficient proteolytic cleavage, a transplanted tumor expressed both PC1 and PC2. These studies are the first biochemical documentation of a melanotroph pituitary tumor in a rodent species and provide a new model for the investigation of pituitary oncogenesis and the molecular basis of tissue-specific prohormone post-translational processing.
Adrenocorticotropic Hormone↗
Proopiomelanocortin in skin: new possibilities for regulation of skin physiology.
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Animals↗
Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin.
We investigated the presence of proopiomelanocortin (POMC) products in sections of skin from normal subjects and patients with neoplastic and non-neoplastic cutaneous disorders. Antibodies specific against adrenocorticotropin, beta-melanotropin, and beta-endorphin were used for detection and characterization of cell types bearing POMC peptides. POMC products were not observed in sections of normal skin from the corporal (non-scalp) areas (six cases), whereas the hair follicles of scalp skin exhibited positive immunostains that were readily apparent (four cases). POMC products were frequently detected in corporal skin affected by diseases (13 of 26 cases), for example, psoriatic keratinocytes, the inflammatory infiltrate in scarring alopecia, nevocytes, the epithelial cell nests of basal cell carcinoma, and melanoma cells. Further tests were performed in keloids, a primary reactive skin disorder, to evaluate whether POMC accumulation represented a disease-related phenomenon or an expression of normal cutaneous reactivity. POMC products were consistently detected (10 of 11 cases) in the keratinocytes and mononuclear cells at keloid lesions. Thus these observations indicate that POMC products may accumulate locally in lesional skin representing, presumably, a novel cutaneous response to injury. The broad spectrum of POMC products detected suggests that these arise from production in situ (expression of the POMC gene itself) by human skin.
Adrenocorticotropic Hormone↗
Properties of AtT-20 cells in serum-reduced medium.
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Animals↗
The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing.
Two subtilisin-like endoproteases called PC1 and PC2 are distributed in a tissue-specific manner in the pituitary and in the brain. AtT-20 cells and corticotropes of the anterior pituitary express primarily PC1 and perform a limited number of cleavages of the proopiomelanocortin (POMC) precursor during biosynthesis. Melanotropes of the intermediate pituitary express both PC1 and PC2 and perform a more extensive set of cleavages during the biosynthetic processing of POMC. To investigate the role of PC2 in the biosynthetic processing of POMC, AtT-20 mouse corticotropes were stably transfected with a full length PC2 cDNA. The AtT-20 cells expressing PC2 acquired the ability to perform all the additional cleavages seen in the intermediate pituitary, but did not acquire the ability to alpha-N-acetylate the product peptides. The kinetics of the earliest steps in biosynthetic processing were unaltered by the expression of PC2, and the changes due to PC2 expression were seen only in the middle and late steps in biosynthetic processing. Thus, both the identity of the final product peptides and the kinetics of the processing steps in the AtT-20 cells expressing PC2 fit the patterns expected for melanotropes of the intermediate pituitary.
Adrenocorticotropic Hormone↗
Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases.
A lysed preparation of isolated insulin secretory granules efficiently cleaved murine proopiomelanocortin (mPOMC) at physiologically important Lys-Arg processing sites. This processing was mostly attributed to an activity that co-eluted with the proinsulin processing type-II endopeptidase from anion exchange chromatography (Lys-Arg-directed; Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96). The principal peptide hormone products generated by the insulin secretory granule lysate were identified by specific radioimmunoassay and NH2-terminal microsequencing analysis of high performance liquid chromatography-separated products as alpha-melanocyte-stimulating hormone, corticotropin-like intermediate, gamma-lipotropin, beta-endorphin-(1-31), 18-kDa NH2-terminal fragment and, to a lesser extent, adrenocorticotrophin and beta-lipotropin. This processing had an acidic pH optimum (pH 5-5.5) and was Ca(2+)-dependent (K0.5 activation = 5-80 microM). With increasing Ca2+ concentrations there was an increase in the extent to which mPOMC was processed. The in vitro processing of mPOMC by the insulin secretory granule endopeptidase activity reported here is in excellent agreement with the in vivo processing of this prohormone by a combination of PC2 and PC3, candidates of prohormone endpeptidase, in gene transfer studies with cells that express the regulated secretory pathway (Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. S., Steiner, D. F., and Thomas, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5297-5301).
Adrenocorticotropic Hormone↗
Pituitary morphology of transgenic mice expressing bovine growth hormone.
BACKGROUND: In mice transgenic for growth hormone (GH), the ectopic production of foreign GH causes gigantism and strong inhibition of endogenous pituitary GH. In human (h) GH transgenics, morphologic changes occurred not only in somatotrophs but in lactotrophs, corticotrophs, and gonadotrophs as well. To distinguish between changes attributed to somatotrophic effect of hGH from those caused by its lactogenic effects, we studied the pituitary morphology of bovine (b) GH transgenics since bGH has no lactogenic activity. EXPERIMENTAL DESIGN: Pituitaries from transgenic mice and nontransgenic siblings were studied by immunocytochemistry for adenohypophysial hormones, in situ hybridization for GH, prolactin (PRL), and proopiomelanocortin mRNAs, and electron microscopy. RESULTS: Transgenic mice had an increased body weight and a significantly decreased pituitary mass. In both sexes, GH immunoreactive cells were markedly reduced in size and moderately decreased in number, and the GH mRNA signal was lower compared with controls; ultrastructurally, in somatotrophs, the cytoplasmic organelles involved in hormone synthesis were inconspicuous. Males were normoprolactinemic, and lactotrophs showed no morphologic changes. In transgenic females, PRL immunoreactive cells were hypertrophic and appeared to be more numerous. In transgenic males, a mild increase in size and number of follicle-stimulating hormone/luteinizing hormone immunoreactive cells was noted. No changes were evident in corticotrophs and thyrotrophs in either sex. In the intermediate and posterior lobes, many corticotrophs and pituicytes were GH immunoreactive, indicating expression of metallothionein-1 (MT)-bGH transgene. CONCLUSIONS: Pituitaries of MT-bGH transgenic mice contained somatotrophs with morphologic features of inhibition. Endogenous GH production was not completely suppressed as indicated by the presence of GH mRNA and GH immunoreactivity. Females had a mild increase in percentage of PRL immunoreactive cells, and previous biochemical data proved that they were hyperprolactinemic. Substantial differences in pituitary morphology between transgenic MT-bGH mice and MT-hGH animals studied previously can be due to the fact that bGH is purely somatotropic, whereas hGH is both somatotropic and lactogenic.
Adrenocorticotropic Hormone↗
"Cryptic" repeating triplets of purines and pyrimidines (cRRY(i)) are frequent and polymorphic: analysis of coding cRRY(i) in the proopiomelanocortin (POMC) and TATA-binding protein (TBP) genes.
Triplets of the form of purine, purine, pyrimidine (RRY(i)) are enhanced in frequency in the genomes of primates, rodents, and bacteria. Some RRY(i) are "cryptic" repeats (cRRY(i)) in which no one tandem run of a trinucleotide predominates. A search of human GenBank sequence revealed that the sequences of cRRY(i) are highly nonrandom. Three randomly chosen human cRRY(i) were sequenced in search of polymorphic alleles. Multiple polymorphic alleles were found in cRRY(i) in the coding regions of the genes for proopiomelanocortin (POMC) and TATA-binding protein (TBP). The highly polymorphic TBP cRRY(i) was characterized in detail. Direct sequencing of 157 unrelated human alleles demonstrated the presence of 20 different alleles which resulted in 29-40 consecutive glutamines in the amino-terminal region of TBP. These alleles are differentially distributed among the races. PCR was used to screen 1,846 additional alleles in order to characterize more fully the range of variation in the population. Three additional alleles were discovered, but there was no example of a substantial sequence amplification as is seen in the repeat sequences associated with X-linked spinal and bulbar muscular atrophy, myotonic dystrophy, or the fragile-X syndrome. The structure of the TBP cRRY(i) is conserved in the five monkey species examined. In the chimpanzee, examination of four individuals revealed that the cRRY(i) was highly polymorphic, but the pattern of polymorphism differed from that in humans. The TBP cRRY(i) displays both similarities with and differences from the previously described RRY(i) in the coding sequence of the androgen receptor. Our data suggest how simple tandem repeats could evolve from cryptic repeats.
Aged↗
Re: G.P. Studzinski and D.C. Moore, sunlight--can it prevent as well as cause cancer? Cancer Res., 55: 4014-4022, 1995.
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Adrenocorticotropic Hormone↗
Pathways to parturition.
Oxytocin is secreted during parturition to stimulate myometrial contractions and birth. Prior to the start of labour, oxytocin neurones undergo changes to prepare for optimal secretion during labour. Thus, during late pregnancy oxytocin secretion is limited by endogenous opioid inhibition. This does not appear to act at the oxytocin nerve terminals in the neural lobe since they in fact become desensitised to opioid inhibition, responding less to either the general opioid antagonist, naloxone, or to the specific kappa-opioid agonist U50,488, and kappa-receptor binding decreases. However, removal of opioid inhibition on oxytocin neurones by naloxone activates oxytocin cell bodies and there is an increase in the number of cells expressing Fos protein in the supraoptic nucleus. This action is mediated via mu- and not kappa-opioid receptors since norBinaltorphimine (kappa-antagonist) is ineffective. Endogenous opioids are likely to act pre-synaptically on inputs to oxytocin neurones, especially those from the brainstem since naloxone potentiates the firing rate response of oxytocin neurones to intravenous cholecystokinin administration which acts via noradrenergic neurones. The endogenous opioid, beta-endorphin may be responsible for inhibition of oxytocin neurones as both the peptide content and its precursor proopiomelanocortin mRNA content increase in the arcuate nucleus during pregnancy, whereas expression of the co-localized opioids, prodynorphin or proenkephalin A, in magnocellular neurones does not alter.
Animals↗
[Bispecific monoclonal antibodies: the isolation and study of their antigen-binding properties].
Bifunctional antibodies (bABs) having a double specificity to alpha-endorphin (alpha-END) and horseradish peroxidase (HRP) were produced by hybridoma technology. The antibodies constituted about 28-29% of all immunologically active IgG secreted by hybrid hybridoma (quadroma). The quadroma was isolated by fusion of two murine hybridomas (anti-HRP and anti-alpha-END) with distinct phenotypes: double mutant AMD(R)/NAT(S) and its wild type. To produce the double mutant phenotype, an actinomycin D-resistant (AMD(R)) mouse myeloma was used to initiate one of the parental hybridomas. bABs were purified from quadroma culture medium and ascitic fluids by sequential HRP-sepharose and alpha-END-sepharose affinity chromatography. With radioimmunoassay, the affinity of the individual anti-alpha-END combining sites of bABs was shown to be identical to that of parental monoclonal antibodies. Binding to the second antigen (HRP) did not affect the binding of bABs to alpha-END. bABs proved to be efficient for the determination of endorphins and their precursor proopiomelanocortin in immunohistology and immunoblotting.
Animals↗
Corticotropin-releasing factor and type 1 corticotropin-releasing factor receptor messenger RNAs in rat brain and pituitary during "binge"-pattern cocaine administration and chronic withdrawal.
Endogenous central corticotropin-releasing factor (CRF) may be involved in the neuroendocrine and behavioral responses to cocaine. In the present study, levels of CRF mRNA were measured in the hypothalamus and in several extrahypothalamic brain regions after different regimens of "binge"-pattern cocaine administration. Male Fischer rats were injected with saline or cocaine (15 mg/kg, 1 hr x 3/day) at the beginning of the light cycle, to approximate the manner in which cocaine is often abused by humans, both in terms of temporal pattern and in relation to circadian rhythm. Cocaine administered in this binge regimen produced time-dependent alterations of CRF mRNA levels in the hypothalamus. There was a significant increase in CRF mRNA levels on day 1, which returned to base-line levels on day 2, with elevated plasma corticosterone levels on both days. CRF receptor type 1 and prooplomeianocortin mRNA levels in the anterior lobe of the pituitary were not significantly altered after acute cocaine injections on day 1 or day 2. On day 14 of chronic binge-pattern cocaine administration, decreased hypothalamic CRF mRNA levels and an attenuated elevation in plasma corticosterone levels were found. After 10 days of withdrawal from 14-day binge cocaine, CRF mRNA returned to basal levels. CRF mRNA levels in the amygdala were also significantly increased on day 1 and returned to basal values on day 2. Chronic (14-day) binge cocaine administration did not alter CRF mRNA levels in the amygdala. These results suggest that the attenuated response in the hypothalamic-pituitary-adrenal axis to chronic binge cocaine administration is coupled to the cocaine-induced decreases in CRF gene expression in the hypothalamus and that activation of CRF gene expression in extrahypothalamic regions may have implications for a molecular understanding of the behavioral responses to cocaine.
Adrenocorticotropic Hormone↗