ABSTRACT
Adrenomedullin (AM) is a multifunctional regulatory peptide with important angiogenic and mitogenic properties. Here we identify a region of stable secondary structure in the 5'-untranslated region (5' UTR) of human AM mRNA. Reverse transcriptase-polymerase chain reaction of the 5' UTR consistently resulted, in addition to the product with the expected size of 155 base pair (bp), in a second product with an approximately 65-bp deletion from the central region of the 5' UTR, suggesting the presence of a secondary structure. The presence of a stem-loop structure was confirmed by probing the 5' UTR with RNases with selectivity for single- or double-stranded RNA. We investigated the role of this stem-loop structure in expression of luciferase reporter gene in cultured cell lines. Reporter assays using a chimeric mRNA that combined luciferase and the 5' UTR of AM mRNA demonstrated a dramatic decrease of the reporter activity owing to a decreased translation, whereas the deletion of the stem-loop structure localized between nt +31 and +95 from the cap site led to the recovery of activity. Gel migration shift assays using cytosolic extracts from mammalian cell lines demonstrate a specific binding of a cytosolic protein to riboprobes containing the 5' UTR of AM but not to riboprobes either corresponding to other areas of the message or containing the 5' UTR but lacking the region of secondary structure. Although we conclude that the 5' UTR of the human AM mRNA can modulate the translation of AM mRNA in vivo, and that the predicted stem-loop structure is necessary for this inhibition, the functional consequences of the cis element-binding activity remain to be determined.
Subject(s)
5' Untranslated Regions/chemistry , Peptides/chemistry , Protein Biosynthesis/physiology , RNA, Messenger/chemistry , Adrenomedullin , Base Sequence , Cells, Cultured , DNA, Complementary/chemistry , Genes, Reporter , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Peptides/metabolism , RNA-Binding Proteins/metabolism , Sequence AlignmentABSTRACT
Insulin-induced hypoglycemia (IIH) is a strong stimulator of pituitary ACTH secretion. The mechanisms by which IIH activates the corticotrophs are still controversial. Indeed, in rats the variations of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) secretion in hypophysial portal blood (HPB) during IIH have been diversely appreciated. This may be due to the stressful conditions required for portal blood collection in rats. We studied the effects of IIH on the secretion of CRF and AVP in HPB and on the release of ACTH and cortisol in peripheral plasma in conscious, unrestrained, castrated rams. After the injection of a low (0.2 IU/kg) or high dose (2 IU/kg) of insulin, ACTH and cortisol levels in peripheral plasma increased in a dose-related manner. After injection of the low dose of insulin, CRF and AVP secretion in HPB were equally stimulated. After injection of the high dose of insulin, CRF secretion was further stimulated, while AVP release was dramatically increased. These results suggest that when the hypoglycemia is moderate, CRF is the main factor triggering ACTH release, and that the increased AVP secretion potentiates the stimulatory effect of CRF. When hypoglycemia is deeper, AVP secretion becomes predominant and may by itself stimulate ACTH release.
Subject(s)
Arginine Vasopressin/metabolism , Corticotropin-Releasing Hormone/metabolism , Hypoglycemia/physiopathology , Insulin/pharmacology , Adrenocorticotropic Hormone/blood , Animals , Blood Glucose/metabolism , Hydrocortisone/blood , Male , Pituitary Gland/blood supply , Secretory Rate/drug effects , SheepABSTRACT
OBJECTIVE: Adrenomedullin (AM), a potent vasodilatator and antioxidative peptide, was shown recently to be expressed by adipose tissue. The aim of our study was to investigate the precise localization of AM within human adipose tissue, and to examine AM regulation in obesity. DESIGN: Subcutaneous (SC) and omental (OM) adipose tissues from 9 lean and 13 obese women were profiled for AM expression changes. Preadipocytes from human adipose tissue were isolated and differentiated under defined adipogenic conditions. METHODS: AM expression was analyzed by immunohistochemistry, in situ hybridization and quantitative RT-PCR. RESULTS: A strong AM expression was observed in vessel walls, stromal cell clusters and isolated stromal cells, some of them being CD 68 positive, whereas mature adipocytes were not labeled. Calcitonin receptor-like receptor and receptor activity-modifying proteins (RAMP) 2 and RAMP 3 were expressed in vessel walls. In vitro, preadipocytes of early differentiation stages spontaneously secreted AM. No difference in AM localization was found between SC and OM adipose tissue. AM levels in SC tissue did not differ between lean and obese subjects. By contrast, AM levels in OM tissue were significantly higher in obese as compared with lean women. Moreover, we found a positive relationship between OM AM and tumor necrosis factor alpha mRNA levels and AM-immunoreactive area in OM tissue followed the features of the metabolic syndrome. CONCLUSION: Stromal cells from human adipose tissue, including macrophages, produce AM. Its synthesis increased in the OM territory during obesity and paralleled the features of the metabolic syndrome. Therefore, AM should be considered as a new member of the adipokine family.
Subject(s)
Adipose Tissue/metabolism , Obesity/metabolism , Peptides/metabolism , Adrenomedullin , Adult , Anthropometry , Blood Chemical Analysis , Body Weight/physiology , Cell Differentiation/physiology , Female , Hemodynamics/physiology , Humans , Immunohistochemistry , In Situ Hybridization , Middle Aged , Peptides/genetics , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/metabolism , Stromal Cells/metabolism , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
After therapeutic hormone deprivation, prostate cancer (CaP) cells often develop androgen-independent growth through not-well-defined mechanisms. The presence of neuroendocrine (NE) cells is often greater in prostate carcinoma than in normal prostate, and the frequency of NE cells correlates with tumor malignancy, loss of androgen sensitivity, increase of autocrine-paracrine activity, and poor prognosis. In some CaPs, neuropeptides have been previously implicated as growth factors. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme producing alpha-amidated bioactive peptides from their inactive glycine-extended precursors. In the present work, we demonstrate that androgen-independent PC-3 and DU145 cell lines, derived from human CaP, express PAM in vitro and in xenografts implanted in athymic nude mice, indicating that they are able to produce alpha-amidated peptides. Contrarily, barely detectable levels of PAM were found in the androgen-sensitive LNCaP cell line. We also show that whereas PC-3 and DU145 cells produce and secrete adrenomedullin (AM), a multifunctional amidated peptide, no expression was found in LNCaP cells. We further demonstrate that AM acts as a growth factor for DU145 cells, which suggests the existence of an autocrine loop mechanism that could potentially drive neoplastic growth. PAM mRNA levels were found to be 3-fold higher in prostate adenocarcinomas compared with that of human benign prostate hyperplasia (BPH) as demonstrated by real-time quantitative reverse transcription-PCR. The analysis of AM message expression in BPH and CaP (Gleason's score, 6-9) shows a clear distinction between benign and CaP. The expression was detected only in adenocarcinomas tissues with a marked increase in samples with a high Gleason's score. Immunocytochemically, AM was localized in the carcinomatous epithelial compartment. NE phenotype, assessed after the immunocytochemical localization of neuron-specific enolase (NSE), was found in both the epithelial and the stromal compartments of cancers; in BPH, only some spare basal cells were NSE-labeled. Cancer progression could be accelerated by peptides secreted by a population of cells capable of inducing androgen-independent tumoral growth via autocrine-paracrine mechanisms.
Subject(s)
Adenocarcinoma/metabolism , Mixed Function Oxygenases/metabolism , Multienzyme Complexes , Peptides/metabolism , Prostatic Neoplasms/metabolism , Adenocarcinoma/enzymology , Adrenomedullin , Animals , Cell Division/drug effects , Humans , Immunohistochemistry , In Situ Hybridization , Male , Mice , Mice, Nude , Mixed Function Oxygenases/biosynthesis , Mixed Function Oxygenases/genetics , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Neoplasms, Hormone-Dependent/enzymology , Neoplasms, Hormone-Dependent/metabolism , Peptides/genetics , Peptides/pharmacology , Phosphopyruvate Hydratase/metabolism , Prostatic Hyperplasia/enzymology , Prostatic Hyperplasia/metabolism , Prostatic Neoplasms/enzymology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Peptidyl-glycine alpha-amidating monooxygenase (PAM) is a posttranslational processing enzyme which catalyzes the formation of biologically active alpha-amidated peptides. The two major neuropeptides involved in the regulation of ACTH secretion [CRF and arginine vasopressin (AVP)], synthesized in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), are amidated, and their synthesis and/or release is negatively regulated by glucocorticoids. In this study, using in situ hybridization, we have shown that PAM mRNA is abundantly expressed in the hypothalamic paraventricular and supraoptic nucleus. Surgical adrenalectomy (ADX) induced increases in PAM, CRF, and AVP mRNA in the parvocellular part of the PVN, while corticosterone treatment normalized these values. PAM and AVP gene expression were not changed in the magnocellular part of the PVN or in the supraoptic nucleus. These observations suggest that in addition to stimulation of CRF and AVP synthesis, ADX induces an increase in PAM synthesis in the PVN and, thus, support the hypothesis of increased secretion of both CRF and AVP after ADX.
Subject(s)
Glucocorticoids/pharmacology , Mixed Function Oxygenases/genetics , Multienzyme Complexes , Paraventricular Hypothalamic Nucleus/enzymology , Adrenalectomy , Animals , Arginine Vasopressin/biosynthesis , Arginine Vasopressin/genetics , Corticotropin-Releasing Hormone/biosynthesis , Corticotropin-Releasing Hormone/genetics , Gene Expression Regulation , Male , Mixed Function Oxygenases/biosynthesis , Paraventricular Hypothalamic Nucleus/drug effects , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Supraoptic Nucleus/drug effects , Supraoptic Nucleus/enzymologyABSTRACT
Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. Growing evidence suggests that the metabolism of PAM messenger RNAs (mRNAs) can be regulated within the cytoplasm. To understand the mechanisms controlling the metabolism of PAM mRNAs, we sought to identify cis elements of the 3'-untranslated region (3'-UTR) of PAM mRNA that are recognized by cytoplasmic factors. From gel retardation assays, one sequence element is shown to form a specific RNA-protein complex. The protein-binding site of the complex was determined by ribonuclease T1 mapping, by blocking the putative binding site with antisense oligonucleotide, and by competition assays. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 20-nucleotide cis element that interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) to form a 60-kDa PAM mRNA-binding protein complex. The binding activity was redox sensitive. Tissue distribution of the protein in the rat showed a marked tissue-specific expression, with ovary, testis, lung, heart septum, anterior pituitary and hypothalamus containing large amounts compared with liver, ventricle, atrium, and neurointermediate lobe. No binding activity was detectable in pancreas, intestine, or kidney extracts. Northwestern blot analysis of AtT-20 (mouse corticotrope tumor cell line) cytoplasmic extracts revealed a protein of 46 kDa. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 3'-UTR of PAM mRNA from many animal species. Although these data suggest that cis element-binding activity could be a cytoplasmic regulator of PAM mRNA metabolism, the functional consequences of this binding remain to be determined.
Subject(s)
Mixed Function Oxygenases/genetics , Multienzyme Complexes , RNA, Messenger/metabolism , RNA-Binding Proteins/analysis , Animals , Base Sequence , Binding Sites , Conserved Sequence , Molecular Sequence Data , RatsABSTRACT
Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyze the conversion of glycine-extended peptides into COOH-terminal amidated peptides. We have previously shown that PAM messenger RNA (mRNA) levels in the anterior pituitary of intact cycling adult female rats showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17beta-estradiol was accompanied by a 4.5 +/- 0.5-fold decrease in total PAM mRNA and a 2-fold decrease in PAM activity in the anterior pituitary gland. To investigate the cellular site at which 17beta-estradiol acts to affect the PAM mRNA, we made parallel measurements of the relative levels of PAM mRNA and nuclear precursor RNA and the relative rate of gene transcription after treatments designed to alter the estrogen status. The transcription rate experiments indicated that these 17beta-estradiol effects were not due to reduced PAM gene activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. Primary rat pituitary cell cultures from OVX and OVX-17beta-estradiol-treated rats in the presence of actinomycin D showed that 17beta-estradiol treatment decreased the half-life of PAM mRNA from 15-16 h to 8-9 h. There was no effect of 17beta-estradiol on PAM mRNA poly(A) tail length or site of polyadenylation. However, in this study the down-regulation of PAM was identified as a nuclear event. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that decreased PAM expression after 17beta-estradiol treatment was largely due to intranuclear destabilization of the primary transcript. The levels of nuclear precursor RNA were decreased roughly 5- to 6-fold in OVX + 17beta-estradiol compared with OVX rats. The decrease in PAM mRNA is blocked by cycloheximide, indicating that its requires new protein synthesis. Mechanisms that would generate such an effect include altered stability of unprocessed message in the nucleus. The proportional changes observed in the nuclear precursor and mRNA levels suggest that the site of control is at the level of stability of the nuclear precursor RNA for PAM mRNA.
Subject(s)
Cell Nucleus/metabolism , Estrogens/physiology , Mixed Function Oxygenases/genetics , Multienzyme Complexes , Protein Processing, Post-Translational/physiology , RNA, Messenger/metabolism , Animals , Culture Techniques , Cytoplasm/metabolism , Drug Stability , Estradiol/pharmacology , Female , Half-Life , Ovariectomy , Pituitary Gland, Anterior/metabolism , Poly A/metabolism , RNA Precursors/metabolism , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/chemistry , Rats , Rats, Sprague-Dawley , Transcription, Genetic/drug effectsABSTRACT
Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. The regulatory mechanism(s) involved in the tissue-specific induction of PAM messenger RNA (mRNA) by thyroid status have been investigated in rat anterior pituitary gland. In this tissue, cellular PAM mRNA increases in response to hypothyroidism (4- to 7-fold above basal levels). To gain further insight into this pretranslational control, nuclear in vitro run-on transcription assays were performed. Using PAM complementary DNAs and intronic probe, we showed that the transcriptional rate of rat pituitary PAM gene in isolated nuclei was not altered by thyroid status. Primary rat pituitary cells cultures from hypo- and euthyroid rats in the presence of actinomycin D showed that hypothyroidism increased the half-life of PAM mRNA from 9-10 h to 15-17 h. Taken together, these data suggest that hypothyroidism induces PAM mRNA levels by increasing its stability in the cytoplasm.
Subject(s)
Gene Expression Regulation , Mixed Function Oxygenases/genetics , Multienzyme Complexes , Pituitary Gland, Anterior/physiology , Thyroid Hormones/physiology , Animals , Cells, Cultured , Drug Stability , Half-Life , Hyperthyroidism/metabolism , Hypothyroidism/metabolism , Male , Mixed Function Oxygenases/metabolism , Pituitary Gland, Anterior/cytology , Protein Processing, Post-Translational , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Transcription, GeneticABSTRACT
The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase catalytic domains necessary for the two-step formation of alpha-amidated peptides from their COOH-terminal glycine extended precursors. Expression of PAM was evaluated in the anterior pituitary of intact cycling adult female rat and after experimental manipulation of estrogen status. PAM messenger RNA (mRNA) levels showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17 beta-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. In situ hybridization of anterior pituitary sections using 35S-labeled full length RNA antisense transcripts of rat PAM-1 complementary DNA showed that 17 beta-estradiol treatment induced an overall decrease of the hybridization signal, as compared with OVX rats. Progesterone treatment did not change PAM mRNA levels both in OVX or OVX + E2 rats. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by RT-PCR, it was found that estrogen status does not affect the distribution of PAM mRNA among its various alternatively spliced forms. In OVX 17 beta-estradiol treated rats, the specific activity of PAM in the anterior pituitary decreased in both soluble and particulate fractions compared with OVX animals. Western blot analysis demonstrated a 105-kDa PAM protein in particulate fractions prepared from OVX and OVX-17 beta-estradiol treated animals. The soluble fraction from OVX animals contained major PAM proteins of 105, 95, 84, 75, and 45 kDa, and 17 beta-estradiol treatment caused a decrease in the prevalence of these proteins. These results indicate that estrogens are involved, either directly or indirectly, in regulating the expression of PAM in several cell types in the anterior pituitary gland.
Subject(s)
Estradiol/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Mixed Function Oxygenases/genetics , Multienzyme Complexes , Pituitary Gland, Anterior/enzymology , Alternative Splicing , Animals , Estrus/metabolism , Female , In Situ Hybridization , Ovariectomy , Progesterone/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
When gestating rats were injected iv with an antiserum to somatostatin (SRIF-AS) during the last week of gestation, serum GH levels in fetuses and 6-h-old newborn rats were not significantly different from controls. Similarly, 2 h after the ip administration of SRIF-AS, no change in serum GH concentration was observed in 2-h-old rats. However, under the same conditions, a significant increase in serum GH was observed in 24-h-old rats and in 2- to 60-day-old rats. The injection of SRIF-AS neither changed basal serum TSH levels during the neonatal development nor in the adult stage. A significant increase in TRH-induced TSH release was observed after the third postnatal day. It is concluded that endogenous SRIF plays a physiological role in GH release by 24 h of age in the rat and that the fall in GH secretion that normally occurs during the first week of life is due to the development of inhibitory mechanisms mediated by hypothalamus SRIF. Additionally the results suggest that the influence of SRIF upon TSH secretion is present before that of TRH.
Subject(s)
Growth Hormone/metabolism , Immune Sera , Somatostatin/physiology , Thyrotropin/metabolism , Aging , Animals , Animals, Newborn , Female , Growth Hormone/blood , Pregnancy , Rats , Rats, Inbred Strains , Somatostatin/immunology , Thyrotropin/blood , Thyrotropin-Releasing Hormone/pharmacologyABSTRACT
Brattleboro rats which lack endogenous vasopressin have been used to study the role of vasopressin as a corticotropin-releasing factor. Plasma ACTH, beta-endorphin, and corticosterone were measured by RIA in male and female Long-Evans and Brattleboro rats under the following conditions: unstressed, after ether stress, after nicotine injection, and after adrenalectomy. A significant reduction in the ACTH, beta-endorphin, and corticosterone responses to the different experimental procedures was observed in the Brattleboro rats. However, in this strain of rats, a significant increase in the release of all three hormones was obtained, suggesting that vasopressin has only a synergistic role in the regulation of their secretion.
Subject(s)
Adrenocorticotropic Hormone/blood , Corticosterone/blood , Endorphins/blood , Rats, Inbred Strains/metabolism , Adrenal Glands/physiology , Animals , Female , Hypothalamo-Hypophyseal System/drug effects , Male , Nicotine/pharmacology , Rats , Sex Factors , Stress, Physiological/metabolism , beta-EndorphinABSTRACT
ACTH and cortisol diurnal variations and responses to two types of stress (insulin-induced hypoglycemia and isolation-restraint stress) and to an acute injection of CRF were determined in intact as well as in actively antiarginine vasopressin (AVP)-immunized rams. All immunized sheep developed antibodies to AVP, displayed diabetes insipidus, and looked healthy in spite of their lower gain weight. Basal secretion and diurnal variations of ACTH and cortisol were unaltered in the group of anti-AVP-immunized animals. In contrast, ACTH and cortisol responses to both types of stress and CRF injection were significantly reduced compared to those in controls. These results suggest that endogenous AVP plays a physiological role in the corticotropic response to stress. However, endogenous AVP does not appear to affect basal secretion and diurnal variations of ACTH and cortisol.
Subject(s)
Adrenocorticotropic Hormone/metabolism , Arginine Vasopressin/physiology , Circadian Rhythm , Corticotropin-Releasing Hormone/pharmacology , Hydrocortisone/metabolism , Immunization, Passive , Pituitary-Adrenal System/physiology , Adrenocorticotropic Hormone/blood , Analysis of Variance , Animals , Arginine Vasopressin/immunology , Blood Glucose/metabolism , Body Weight , Diabetes Insipidus/physiopathology , Drinking Behavior , Hydrocortisone/blood , Hypoglycemia/chemically induced , Hypoglycemia/physiopathology , Insulin/pharmacology , Male , Pituitary-Adrenal System/drug effects , Pituitary-Adrenal System/physiopathology , Restraint, Physical , Sheep , Stress, Physiological/physiopathology , Stress, Psychological/physiopathologyABSTRACT
ACTH and cortisol diurnal variations and responses to two types of stress (insulin-induced hypoglycemia and isolation-restraint stress) or to an acute injection of lysine-vasopressin were studied in intact and anti-corticotropin-releasing factor (CRF) actively immunized rams. Immunization was obtained by the injection of synthetic ovine CRF coupled to BSA with carbodiimide. All animals developed antibodies anti-CRF and displayed an alteration of their general condition and a body weight reduction. The mean basal ACTH and cortisol secretion as well as the number and mean amplitude of diurnal pulses of these hormones was significantly reduced in the group of anti-CRF immunized rams. However, the reduction in all three parameters was much more pronounced for cortisol than for ACTH. No ACTH and cortisol response to insulin-induced hypoglycemia and isolation-restraint stress was observed. The stimulating action of lysine-vasopressin on ACTH release was significantly reduced as compared to controls. These results indicate that CRF is a major physiological component of the ovine hypothalamo-hypophysial-adrenal axis and participates in the events that regulate ACTH and cortisol diurnal variations and response to stress.
Subject(s)
Adrenocorticotropic Hormone/metabolism , Corticotropin-Releasing Hormone/physiology , Hydrocortisone/metabolism , Pituitary-Adrenal System/physiology , Adrenocorticotropic Hormone/blood , Animals , Circadian Rhythm , Corticotropin-Releasing Hormone/immunology , Hydrocortisone/blood , Hypoglycemia/metabolism , Immune Sera/immunology , Immunization , Lypressin/pharmacology , Male , SheepABSTRACT
To examine if down-regulation of CRH-induced ACTH release occurs in corticotroph adenoma cells as well as CRH-glucocorticoid interactions in these cells, we established primary cultures of pituitary adenoma cells obtained by transphenoidal surgery from five patients with Cushing's disease. To prevent binding of glucocorticoids by serum proteins, we used a serum-free medium containing insulin, transferrin, selenium, and epidermal growth factor. The latter was found to be essential for both basal and CRH-stimulated ACTH secretion. CRH acutely stimulated, in a dose-dependent manner, ACTH release by all adenomas studied, with an IC50 of 0.5 X 10(-9) mol/L. Prolonged exposure (10 days) to a half-maximal stimulatory concentration of CRH led to continuous stimulation of ACTH secretion. A 4-day incubation with cortisol induced a dose-dependent decrease in both basal and long term CRH-stimulated ACTH release, with no difference in the IC50 (1 X 10(-8) mol/L). These data suggest that long term exposure to CRH does not desensitize corticotroph adenoma cells. Thus, it is unlikely that long-acting analogs of CRH will be useful in the treatment of Cushing's disease. ACTH secretion from corticotroph adenomas is restrained by glucocorticoids; the sensitivity of these cells to the negative effect of glucocorticoids is not modified by long term stimulation with CRH.
Subject(s)
Adenoma/metabolism , Adrenocorticotropic Hormone/metabolism , Corticotropin-Releasing Hormone/pharmacology , Hydrocortisone/pharmacology , Pituitary Neoplasms/metabolism , Adenoma/pathology , Adolescent , Adult , Drug Interactions , Female , Humans , Pituitary Gland, Anterior/metabolism , Pituitary Gland, Anterior/pathology , Pituitary Neoplasms/pathology , Tumor Cells, CulturedABSTRACT
Specific receptors for CRH were identified in five freshly excised pituitary adenomas causing Cushing's disease. Their kinetic properties and mean affinity constant [1.45 +/- 0.38 (+/- SE) nmol/L] were comparable to the characteristics of rat and monkey anterior pituitary CRH receptors. No correlation was found between the immediate preoperative plasma and urinary cortisol levels and the number of pituitary adenoma CRH receptors, which ranged from 6-96 fmol/mg protein, unlike in rats, in which corticosterone modulates the number of anterior pituitary CRH receptors. The lack of correlation between the concentration of CRH receptors and plasma cortisol levels may reflect the inability of glucocorticoids to down-regulate CRH receptors in these tumors. Thus, corticotroph adenomas are resistant not only to the feedback actions of glucocorticoids on proopiomelanocortin synthesis and secretion but also to their actions on CRH receptors.
Subject(s)
Adenoma/analysis , Glucocorticoids/physiology , Pituitary Neoplasms/analysis , Receptors, Neurotransmitter/analysis , Adrenocorticotropic Hormone/metabolism , Adult , Corticotropin-Releasing Hormone/physiology , Cushing Syndrome/etiology , Female , Humans , Hydrocortisone/analysis , Middle Aged , Pituitary Gland, Anterior , Receptors, Corticotropin-Releasing HormoneABSTRACT
Preproenkephalin metabolism, in the rat, was studied in primary striatal neurons maintained in a chemically defined medium. Acute treatment (30 min) with forskolin (10(-5) M) or phorbol 12 myristate 13 acetate (10(-7) M) resulted, respectively, in a two- and seven-fold increase in methionine-enkephalin secretion. Chronic treatment with forskolin or phorbol 12 myristate 13 acetate (24 h) induced a 100% increase in methionine-enkephalin content (forskolin) and on the other hand a 50% decrease in methionine-enkephalin (phorbol 12 myristate 13 acetate). Both treatments increased preproenkephalin mRNA levels in a time-dependent manner, this augmentation being observable after 180 min by Northern blot analysis and in situ hybridization. These data indicate that under chronic stimulation, with either forskolin or phorbol 12 myristate 13 acetate, proenkephalin turnover is accelerated. However, after stimulation with phorbol 12 myristate 13 acetate, the more potent methionine-enkephalin secretagogue, increased peptide synthesis is not sufficient to replenish methionine-enkephalin intracellular stores. Preproenkephalin gene transcription was analysed by introducing the preproenkephalin gene promoter fused to the bacterial acetyl chloramphenicol transferase reporter gene into primary neurons. Chronic stimulation (48 h) by forskolin (10(-5) M) or phorbol 12 myristate 13 acetate (10(-7) M) of striatal neurons transfected with this fusion gene increased chloramphenicol acetyltransferase activity six-fold and the two effects were additive. These data suggest that the cyclic AMP and the protein kinase C pathways directly activate preproenkephalin gene transcription.
Subject(s)
Corpus Striatum/metabolism , Cyclic AMP/physiology , Enkephalins/metabolism , Protein Kinase C/physiology , Protein Precursors/metabolism , Transcription, Genetic , Animals , Cells, Cultured , Colforsin/pharmacology , Enkephalin, Methionine/metabolism , Enkephalins/biosynthesis , Female , Immunohistochemistry , Neural Pathways/physiology , Neurons/drug effects , Nucleic Acid Hybridization , Pregnancy , Protein Precursors/biosynthesis , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Receptors, Neurotransmitter/metabolism , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
Corticotrophin-releasing factor-41 (CRF-41) immunoreactivity has been measured in hypothalamic extracts of fetal (on days 17, 19 and 21 of gestation), neonatal (1, 2, 3 and 4 weeks of age) and adult rats with a specific radioimmunoassay developed for synthetic rat CRF-41. The hypothalamic content (fmol) and concentration (fmol/mg protein) of immunoreactive CRF-41 gradually increased with age. Chromatography of hypothalamic extracts on Sephadex G-50 Fine showed one single peak of immunoreactive CRF-41 which co-eluted with synthetic rat CRF-41. The retention time of hypothalamic CRF-41 during high-performance liquid chromatography was identical to that of synthetic rat CRF-41 at all stages investigated. These results are consistent with the development of neurones containing CRF-41-like molecules in both the hypothalamus and the median eminence of the fetus, as well as with the hypothalamic control of the cortico-stimulating function of the pituitary gland as early as day 19 of gestation.
Subject(s)
Animals, Newborn/metabolism , Corticotropin-Releasing Hormone/analysis , Fetus/analysis , Hypothalamus/embryology , Rats, Inbred Strains/metabolism , Aging/metabolism , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Hypothalamus/analysis , Radioimmunoassay , RatsABSTRACT
Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyse the alpha-amidation of neuroendocrine peptides. Previous studies have demonstrated that alpha-adrenergic stimulation results in an increase in intracellular volume and protein content of cultured neonatal rat myocardial cells. The present study examined the regulated expression of PAM during alpha-adrenergic stimulation. Alpha1-adrenergic stimulation activates the expression and release of PAM from myocytes. Following phenylephrine treatment, myocardial cells displayed a several fold increase in PAM activity, and a 2-4-fold increase in the steady state levels of PAM mRNA. This effect of alpha-adrenergic stimulation was dependent on the concentration and duration of exposure to the agonist, and displayed alpha1-adrenergic receptor specificity. The transcription rate experiments indicated that these alpha-adrenergic effects were not due to increased PAM gene activity, suggesting that a post-transcriptional mechanism was involved. The most common mechanism of post-transcriptional regulation affects cytoplasmic mRNA stability. Cardiomyocytes cultures from atria and ventricles in the presence of 5,6 dichloro-1-beta ribofuranosyl benzamidazole (DRB) showed that phenylephrine treatment increased the half-life of PAM mRNA from 13 +/- 1 to 21 +/- 1 h in atrial cells and from 8 +/- 1 to 12 +/- 1 h in ventricle cells. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that increased PAM expression after phenylephrine treatment was not due to intranuclear stabilisation of the primary transcript. Protein kinase C inhibitors H7 and GF109203x, completely blocked the phenylephrine stimulated PAM expression. These results suggest that alpha-adrenergic agonist induces PAM mRNA levels by increasing its stability in the cytoplasm. They indicate that PAM gene expression augments through a H7 and GF109203x sensitive pathway, involving the activation of protein kinase C.
Subject(s)
Adrenergic alpha-1 Receptor Agonists , Mixed Function Oxygenases/genetics , Multienzyme Complexes , Myocardium/metabolism , Receptors, Adrenergic, alpha-1/genetics , Sulfonamides , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Isoquinolines/pharmacology , Kinetics , Mixed Function Oxygenases/metabolism , Myocardium/cytology , Phenylephrine/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , RNA Stability , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Transcription, Genetic/drug effectsABSTRACT
In order to elucidate the physiological role of the 41 amino-acid residue corticotropin-releasing factor (41-CRF) on the secretion of ACTH, B-Endorphin and alpha-MSH, plasma levels of these peptides were measured by radioimmunoassay in intact and adrenalectomized rats, two hours after the injection of either 41-CRF antiserum (CRF-AS) or normal rabbit serum for controls. The administration of CRF-AS strikingly lowered the plasma ACTH levels in both intact and adrenalectomized rats. A statistically significant reduction of plasma levels of B-Endorphin was also observed in the same rats. However, the effect of CRF-AS on B-Endorphin release was less pronounced than the effect on ACTH release. No changes in plasma alpha-MSH levels were observed after passive immunization with CRF-AS. We conclude that, in the rat, 41-CRF plays a physiological role in the regulation of ACTH and B-Endorphin secretion, but is not involved in the regulation of alpha-MSH release from the pituitary gland.
Subject(s)
Adrenocorticotropic Hormone/metabolism , Corticotropin-Releasing Hormone/physiology , Endorphins/metabolism , Melanocyte-Stimulating Hormones/metabolism , Animals , Antibodies , Corticotropin-Releasing Hormone/immunology , Hypothalamus/physiology , Immunization, Passive , Male , Pituitary Gland/metabolism , Rats , Rats, Inbred Strains , beta-EndorphinABSTRACT
Passive immunization of pregnant rats with a specific antiserum to rat GRF (GRF-AS) is followed by a decrease in fetal serum GH on the 19th day of gestation. A significant reduction in serum GH is still observed in older fetuses and newborn rats. Pituitary GH content increases in 19- and 20-day-old fetuses after GRF-AS administration to their mothers. These results suggest that endogenous fetal hypothalamic GRF (or placenta GRF) play a physiological role in the secretion of pituitary GH as early as the 19th day of fetal life and may be responsible for the peak of GH release that occurs in fetuses at the end of gestation.