ABSTRACT
Treatment of rats for 4 days with alpha-methyldopa, 200 mg/kg/day i.p., increases steady state levels of proopiomelanocortin (POMC) mRNA in the mediobasal hypothalamus, as measured by DNA excess solution hybridization. The increase is prevented by parallel treatment with yohimbine, 2 mg/kg/day i.p., but not by naltrexone, 2 mg/kg/day i.p. Treatment with the peripheral vasodilator hydralazine, 2 mg/kg/day, does not affect POMC mRNA levels. In situ hybridization histochemistry with a cRNA probe for POMC indicates that POMC-containing cells are located within the confines of the arcuate nucleus both in control and in alpha-methyldopa-treated rats, and confirms the increase in POMC mRNA in the latter. Microinjection of 2 micrograms of alpha-methylnorepinephrine unilaterally into the arcuate nucleus of urethane-anesthetized rats causes hypotension and bradycardia, which can be inhibited by 200 ng of yohimbine microinjected into the same site, or by 100 ng l-naloxone microinjected into the ipsilateral nucleus tractus solitarii, but not into the arcuate nucleus. These findings are interpreted to indicate that activation of alpha 2-adrenergic receptors located on POMC-containing neurons in the arcuate nucleus causes beta-endorphin release and stimulation of opiate receptors in the NTS, which results in hypotension and bradycardia, and that this mechanism contributes to the hypotensive action of alpha-methyldopa.
Subject(s)
Arcuate Nucleus of Hypothalamus/physiopathology , Bradycardia/chemically induced , Hypotension/chemically induced , Neurons/physiology , Pro-Opiomelanocortin/metabolism , Receptors, Adrenergic, alpha/physiology , Adrenergic alpha-Agonists/administration & dosage , Adrenergic alpha-Agonists/pharmacology , Animals , Arcuate Nucleus of Hypothalamus/drug effects , Bradycardia/physiopathology , Hypotension/physiopathology , In Situ Hybridization , Male , Methyldopa/pharmacology , Microinjections , Nordefrin/administration & dosage , Nordefrin/pharmacology , Pro-Opiomelanocortin/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Adrenergic, alpha/drug effects , Yohimbine/pharmacology , beta-Endorphin/pharmacologyABSTRACT
Two transforming growth factor alpha (TGF alpha) mRNA species with the apparent sizes of 4.5 and 1.6 kb were identified in all human cell lines analysed. The cDNA corresponding to the 4.5-kb species was entirely sequenced, revealing the presence of a 3'-untranslated region (3'-UTR) of 3571 nucleotides, which contained several potential polyadenylation signals. Our results indicate that the 1.6-kb species is derived from the same precursor by alternative polyadenylation. In addition, we present evidence suggesting that TGF alpha-specific mRNAs could be initiated from transcription start points (tsp) located upstream from the tsp previously identified by Jakobovitz et al. [Mol. Cell. Biol. 8 (1988) 5549-5554].
Subject(s)
Transforming Growth Factor alpha/genetics , Amino Acid Sequence , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA , Humans , Molecular Sequence Data , RNA, Messenger/genetics , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
The presence of prolactin (PRL) mRNA in the mammary gland, placenta, and pituitary gland of lactating and pregnant rats was investigated by polymerase chain reaction (PCR). Polyadenylated RNA was prepared from total RNA samples by oligo(dT)-cellulose chromatography, and complementary cDNAs were synthesized. A standardized amount of cDNA from each sample was used as the template in a Taq PCR under high-stringency conditions. PCR amplified a signal with the predicted size of approximately 375 bp in mammary and pituitary glands of lactating and pregnant rats, and in placentae of pregnant rats. This band specifically hybridized with a probe overlapping the entire sequence of the mature rat (r) PRL mRNA in Southern blot analysis. When the rPRL-specific primers were used, PCR revealed no signal in the liver or in lactating mammary gland explants cultured in vitro for 48 h, while the same cDNA preparations gave strong signals for beta-actin. The viability of the mammary gland explants was also suggested by their ability to secrete immunoreactive casein in vitro. PRL mRNA was localized in the epithelium of alveoli and ducts of the lactating mammary gland by in situ hybridization. These data provide evidence that the PRL gene is expressed in the mammary gland of pregnant and lactating rats, and suggest that the mammary gland might contribute to PRL in milk by de novo synthesis. Thus, while the placenta is an exogenous source of PRL-like activities for the fetus in utero, the mammary gland might take over this function after birth.
Subject(s)
Lactation , Mammary Glands, Animal/chemistry , Prolactin/genetics , RNA, Messenger/analysis , Actins/genetics , Animals , Blotting, Northern , Blotting, Southern , Epithelium/chemistry , Ethidium , Female , Placenta/chemistry , Polymerase Chain Reaction , Rats , Rats, Inbred BUF , Rats, Sprague-Dawley , Staining and LabelingABSTRACT
The nucleotide sequence of the ApoH cDNA encoding the bovine apolipoprotein H (ApoH) has been determined. The deduced protein, which contains a 19-amino-acid (aa) signal peptide and the 326-aa mature ApoH, shows 89% and 86% homology with human and rat ApoH, respectively.
Subject(s)
Apolipoproteins/genetics , Glycoproteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , DNA , Humans , Molecular Sequence Data , Rats , Sequence Homology, Amino Acid , beta 2-Glycoprotein IABSTRACT
It is thought that certain actions of ethanol involve an interaction with endogenous opioids, including proopiomelanocortin-derived peptides such as beta-endorphin. To examine this possibility, we used a sensitive and specific assay for proopiomelanocortin mRNA to obtain an estimate of the activity of the endorphinergic system in the mediobasal hypothalamus and the pituitary of rats exposed for 10 days in an inhalation chamber to either ethanol or water. This protocol causes dependence in the ethanol-exposed group, as demonstrated by the presence of withdrawal seizures after cessation of treatment. While ethanol treatment did not affect proopiomelanocortin mRNA levels in the pituitary, the level in hypothalamus was significantly lower in the ethanol-treated animals than in controls. These results suggest that some effect of ethanol may involve the hypothalamic endorphinergic system.
Subject(s)
Alcoholism/genetics , Hypothalamus, Middle/pathology , Pro-Opiomelanocortin/genetics , RNA, Messenger/genetics , Alcohol Withdrawal Delirium/genetics , Alcohol Withdrawal Delirium/pathology , Alcoholism/pathology , Animals , DNA Probes , Male , Rats , Rats, WistarABSTRACT
The regulation of beta 1- and beta 2-adrenergic receptors (beta 1AR and beta 2AR) and receptor gene expression by interleukin-1 alpha (IL-1 alpha) was studied in cultured A549 human lung adenocarcinoma cells. The density and affinity of beta 1 AR and beta 2 AR were analyzed by computerized curve fitting of 125I-pindolol binding and its displacement by subtype selective antagonists. Steady state levels of receptor mRNAs were quantified by DNA excess solution hybridization assays. A549 cells in preconfluent cultures had fewer beta 1AR than beta 2AR (beta 1: 1.9 +/- 0.3 vs beta 2: 4.0 +/- 0.5 fmol/mg protein, means +/- SE), but lost most of their beta 2 AR upon reaching confluency (beta 1: 2.7 +/- 0.4, beta 2: 0.8 +/- 0.3 fmol/mg). Incubation of preconfluent cells for 24 hr with 20 pM of human recombinant IL-1 alpha did not modify the density of either of the beta AR subtypes. Similar incubations of confluent cells increased the density of beta 2 AR from 0.8 +/- 0.3 to 4.2 +/- 0.9 fmol/mg, while the density of beta 1 AR and the antagonist affinities of both receptors remained unaltered. The IL-1 alpha-induced increase in beta 2 AR density in confluent cells was antagonized in a concentration-dependent manner by a recombinant protein antagonist of type I IL-1 receptors (IC50: 0.2 nM). The IL-1 alpha-induced increase in beta 2AR density was preceded by an increase in the steady state level of beta 2AR mRNA, while levels of beta 1AR mRNA remained unchanged. IL-1 alpha increased the stability as well as the rate of transcription of beta 2AR mRNA. These findings demonstrate for the first time that activation of type I IL-1 receptors in A549 cells leads to a cell density-dependent, selective upregulation of beta 2AR, and that the mechanism of this effect involves increased formation and stability of the beta 2AR message.
Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Interleukin-1/pharmacology , Lung Neoplasms/metabolism , Receptors, Adrenergic, beta/genetics , Cell Count , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Adrenergic, beta/metabolism , Receptors, Immunologic/metabolism , Receptors, Interleukin-1 , Transcription, Genetic/drug effects , Tumor Cells, Cultured , Up-RegulationABSTRACT
The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.
Subject(s)
Adrenalectomy , Hypothalamus/physiology , Pro-Opiomelanocortin/genetics , RNA, Messenger/metabolism , Adrenal Glands/physiology , Analysis of Variance , Animals , Arcuate Nucleus of Hypothalamus/physiology , Blotting, Northern , Hypothalamus, Middle/physiology , Male , Nucleic Acid Hybridization , Organ Specificity , Pituitary Gland/physiology , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Rats , Rats, Inbred Strains , Reference ValuesABSTRACT
In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an alpha 1-receptor (alpha 1AR) to a beta 2-receptor (beta 2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time-dependent changes in alpha 1- and beta 2-adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of alpha 1AR and beta 2AR, and the steady state levels of alpha 1BAR and beta 2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in alpha 1AR density, and a 70% decrease in alpha 1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol-induced but not in glucagon- or forskolin-induced cAMP accumulation, no significant change in beta 2AR density, and a twofold increase in beta 2AR mRNA levels. Exposure of cells to cycloheximide, 2 microM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced beta 2AR densities, while the decrease in alpha 1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in alpha 1BAR mRNA and increase in beta 2AR mRNA levels and corresponding inverse changes in the synthesis of alpha 1BAR and beta 2AR which account, at least in part, for the rapid conversion from alpha 1- to beta 2-adrenergic glycogenolysis.
Subject(s)
Liver/cytology , Receptors, Adrenergic, alpha/genetics , Receptors, Adrenergic, beta/genetics , Transcription, Genetic/genetics , Animals , Cells, Cultured , Colforsin/metabolism , Cyclic AMP/metabolism , Cycloheximide/pharmacology , Glucagon/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Isoproterenol/pharmacology , Liver/metabolism , Liver/ultrastructure , Male , Phenylephrine/pharmacology , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats , Rats, Inbred Strains , Receptors, Adrenergic, alpha/analysis , Receptors, Adrenergic, alpha/metabolism , Receptors, Adrenergic, beta/analysis , Receptors, Adrenergic, beta/metabolism , Time FactorsABSTRACT
Steady state levels of the mRNAs for alpha 1B, beta 1- and beta 2-adrenergic receptors (alpha 1BAR, beta 1AR, beta 2AR) were quantified by DNA excess solution hybridization assays in the heart, lungs, and liver of rats. Tissues for RNA extraction were obtained from euthyroid and thyroidectomized rats and from thyroidectomized rats treated with a single dose of thyroxine. Thyroidectomy resulted in significant decreases in beta 1AR and beta 2AR mRNAs in heart and lung and alpha 1BAR mRNA in liver, whereas the levels of beta 2AR mRNA in liver and alpha 1BAR mRNA in heart and lung were significantly increased. All these changes were reversed within 20 hours of a single s.c. injection of 1 mg/kg thyroxine. These findings indicate for the first time that thyroid state regulates mRNA levels for adrenergic receptors, and that this regulation is tissue- and receptor-specific. The changes in adrenergic receptor mRNAs correlate with and probably underlie the well documented, thyroid-dependent changes in the cellular densities and physiological reactivities of adrenergic receptors.