A1 mRNA Levels Research Articles

Differential adaptation of brain 5-HT 1A and 5-HT 1B receptors and 5-HT transporter in rats treated chronically with fluoxetine

Quantification of receptor binding sites and their encoding mRNAs, and electrophysiological recordings, were used to assess central serotonin (5-HT) neurotransmission in rats 24 h after a 2–3 week treatment with the selective 5-HT reuptake inhibitor fluoxetine (8 mg/kg i.p., daily). Binding studies showed that this treatment affected neither 5-HT 1A nor 5-HT 1B binding sites in all brain areas examined. However, a significant decrease (−38%) in 5-HT 1A mRNA levels in the anterior raphe area (but not forebrain regions) and increases in 5-HT 1B mRNA levels in the striatum (+127%) and the cerebral cortex (+34%) were noted in fluoxetine-treated rats. Electrophysiological recordings in brain slices showed that chronic fluoxetine treatment reduced the potency of the 5-HT 1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin to inhibit neuronal activity in the dorsal raphe nucleus, but did not affect 5-HT 1A-evoked responses of CA1 pyramidal cells in the hippocampus. These data further demonstrate that fluoxetine-induced adaptive changes in 5-HT neurotransmission exhibit marked regional differences. The decrease in 5-HT 1A mRNA levels in the anterior raphe suggests that fluoxetine-induced desensitization of 5-HT 1A autoreceptors involves changes at the transcription level.

Opposite regulation by glucocorticoids of the α 1B- and α 2A-adrenoreceptor mRNA levels in rat cultured anterior hypothalamic slices

In this study we investigated whether the expression of α 1B- and α 2A-adrenoreceptor mRNAs is differently modulated by glucocorticoids in rat cultured anterior hypothalamus slices. Using a semi-quantitative reverse transcription-polymerase chain reaction assay, the level of the α 1B-adrenoreceptor mRNA was significantly reduced in slices cultured in steroid free-medium when compared with that measured in standard medium (i.e. containing basal adrenosteroid plasma concentrations). In contrast, the expression of the α 2A-adrenoreceptor mRNA was markedly increased. Finally, the ratio of α 1B- versus α 2A-mRNA levels was about 1.7 and 0.7 in standard and steroid-free medium, respectively. These responses were completely reversed by supplementation with corticosterone. These findings provide the first evidence that in vitro glucocorticoids may regulate, in an opposite manner, the expression of the α 1B-and α 2A-adrenoreceptor mRNAs in the hypothalamus. This kind of regulation could be related to steroid-dependent changes in the noradrenergic control of neuroendocrine secretions.

The Murine Antiapoptotic Protein A1 Is Induced in Inflammatory Macrophages and Constitutively Expressed in Neutrophils

Myeloid leukocytes are thought to regulate their susceptibility to apoptosis upon migration to a site of inflammation. However, factors that determine survival have not been well characterized in these cells. We have examined the expression of murine A1, an antiapoptotic Bcl-2 relative found in activated myeloid cells, during the course of an acute inflammatory response. Intraperitoneal infection of mice with the virulent RH strain of Toxoplasma gondii led to a 5- to 10-fold increase in A1 mRNA levels in peritoneal cells after several days. Bcl-2 expression was unchanged. The increase in A1 expression depended on the dose of the organism and coincided with a sharp increase in peritoneal cellularity. A1 protein levels were also increased as determined by Western blot analysis and immunohistochemical studies. All neutrophils and approximately half of the macrophages in the inflammatory exudate contained high levels of A1 in cytoplasm. A1 expression did not correlate with intracellular parasitization. Peripheral blood neutrophils from normal mice strongly expressed A1 protein, whereas normal monocytes showed only weak staining. Bax mRNA was induced in parallel with A1 in macrophages. Exudate macrophages and granulocytes that were apoptotic by TUNEL staining occasionally appeared to display A1 throughout the cell nucleus. These studies identify A1 as a potential regulator of apoptosis during acute inflammation.

Serotonin 1A receptor messenger RNA regulation in the hippocampus after acute stress

Background: When rats are subjected to chronic stress for 2 weeks, a significant decrease in hippocampal serotonin (5-HT) 1A messenger RNA (mRNA) is observed. We wanted to investigate whether stress, administered for shorter periods of time, would result in decreases in 5-HT 1A gene expression in hippocampus. Methods: In one experiment, rats were either stressed daily for 1 week or implanted with two corticosterone pellets to produce elevated corticosterone levels. In another experiment, rats were subjected to a severe acute stressor and sacrificed 1 day or 1 week after the stressor. Results: We found that 24 hours after the acute stress, rats showed a significant decrease in 5-HT 1A mRNA levels in CA1 and the dentate gyrus compared to controls. No significant changes in 5-HT 1A mRNA levels were detected in any of the other groups. Conclusions: Although 1 week of chronic stress is not sufficient to cause significant decreases in hippocampal 5-HT 1A mRNA levels, a severe and prolonged acute stress is capable of down-regulating, at least transiently, 5-HT 1A mRNA gene expression in hippocampus.

Alterations of GABA A receptor subunit mRNA levels associated with increases in punished responding induced by acute alprazolam administration: an in situ hybridization study

Changes in the mRNA encoding α1, α2, β2 and γ2 subunits of the GABA A receptor associated with the anxiolytic effects of alprazolam were measured in 20 brain regions using in situ hybridization techniques. Compared to non-punished controls, punishment decreased α1 mRNA levels in two nuclei of the amygdala, the cerebral cortex, and the mediodorsal thalamic nucleus and decreased α2 mRNA levels in the hippocampus. Punishment increased β2 mRNA levels in ventroposterior thalamic nucleus and γ2 mRNA levels in the CA2 area of the hippocampus. All of these effects were reversed when alprazolam increased punished responding, while alprazolam alone had no effect on either non-punished responding or GABA A receptor subunit regulation in these brain regions. Some brain regions that were unaffected by punishment were altered by alprazolam plus punishment. These results demonstrate that punishment and alprazolam can produce reciprocal changes in the mRNA levels for some subunits of the GABA A receptor. These changes may alter GABAergic synaptic inhibition by altering the density of GABA A receptors or their efficacy to bind drugs. They suggest that the underlying mechanisms by which drugs affect behavior can depend upon the conditions under which behavior is assessed.

Acute 17 β-estradiol treatment down-regulates serotonin 5HT 1A receptor mRNA expression in the limbic system of female rats

In situ hybridization histochemistry was used to investigate acute estrogen effects on serotonin 5HT 1A receptor mRNA levels in limbic-related brain areas in the female ovariectomized rat. Acute administration of 17 β-estradiol (10 μg) decreased 5HT 1A receptor mRNA expression levels within the medial amygdala (after 2 and 24 h), piriform cortex (after 2 and 24 h), and perirhinal cortex (after 24 h). No changes in 5HT 1A mRNA levels were observed in hippocampus or retrosplenial cortex. The findings suggest specific regional effects of estrogen on 5HT functions mediated through regulation of the 5HT 1A gene.

Neuronal-type acetylcholine receptors and regulation of ?7 gene expression in vertebrate skeletal muscle

Several neuronal nicotinic acetylcholine receptor (AChR) genes are expressed in chick skeletal muscle during development. One of the most abundantly expressed is alpha 7, which produces a protein capable of binding alpha-bungarotoxin and is physically distinct from muscle AChRs containing the alpha 1 gene product. We show here that the alpha 7-containing species in muscle is indistinguishable pharmacologically from alpha 7-containing AChRs in neurons. In addition, immunologic analysis with subunit-specific muscle antibodies shows that the alpha 7-containing species in muscle lacks the beta 1 and delta muscle AChR gene products as it does the alpha 1. RNase protection experiments measuring alpha 7 mRNA levels indicate that the alpha 1 and alpha 7 genes may, in part, be subject to similar kinds of regulation in the tissue. Surgical denervation of leg muscle in newly hatched chicks caused a small and transient increase in alpha 7 mRNA after 8 days, while alpha 1 transcripts underwent a large and sustained increase in number. Similarly, treating myotube cultures with tetrodotoxin caused a modest increase in alpha 7 transcript levels and a large increase in alpha 1. Calcitonin gene-related peptide (CGRP) increased both kinds of transcripts in myotube cultures equally as did treatment with 8-bromo-cyclic AMP; CGRP is thought to work via a cyclic AMP-dependent pathway in muscle. In at least one respect, however, alpha 7 expression in muscle differs qualitatively from that of alpha 1: AChR-inducing activity (ARIA) increased alpha 1 mRNA levels in culture while slightly depressing alpha 7 mRNA levels. The regulatory pattern of alpha 7 expression in muscle may combine features of both alpha 7 expression in neurons and alpha 1 expression in muscle.

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1B1.

The 4-hydroxy metabolite of 17 beta-estradiol (E2) has been implicated in the carcinogenicity of this hormone. Previous studies showed that aryl hydrocarbon-receptor agonists induced a cytochrome P450 that catalyzed the 4-hydroxylation of E2. This activity was associated with human P450 1B1. To determine the relationship of the human P450 1B1 gene product and E2 4-hydroxylation, the protein was expressed in Saccharomyces cerevisiae. Microsomes from the transformed yeast catalyzed the 4- and 2-hydroxylation of E2 with Km values of 0.71 and 0.78 microM and turnover numbers of 1.39 and 0.27 nmol product min-1.nmol P450-1, respectively. Treatment of MCF-7 human breast cancer cells with the aryl hydrocarbon-receptor ligand indolo[3,2-b]carbazole resulted in a concentration-dependent increase in P450 1B1 and P450 1A1 mRNA levels, and caused increased rates of 2-, 4-, 6 alpha-, and 15 alpha-hydroxylation of E2. At an E2 concentration of 10 nM, the increased rates of 2- and 4-hydroxylation were approximately equal, emphasizing the significance of the low Km P450 1B1-component of E2 metabolism. These studies demonstrate that human P450 1B1 is a catalytically efficient E2 4-hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens.

Atypical cytochrome P450 induction profiles in glomerular mesangial cells at the mRNA and enzyme level: Evidence for CYP1A1 and CYP1B1 expression and their involvement in benzo[A]pyrene metabolism

Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) modulates growth factor-related gene expression and proliferation of renal glomerular mesangial cells (GMCs) in vitro. Because many of the toxic and biochemical effects of this polycyclic aromatic hydrocarbon are mediated through oxidative metabolism, the present studies were conducted to examine the patterns of cytochrome P450IA1 (CYP1A1) and P450IB1 (CYP1B1) inducibility in mesangial cells and the molecular consequences of this response. Exposure of cultured GMCs to BaP (30 μM) or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD, 10 nM) for 24 hr induced CYP1 A1 mRNA levels, a response abolished by cotreatment with 10 μM cycloheximide. The pattern of hydrocarbon inducibility was atypical in that BaP was a more effective inducer of CYP1A1 gene expression than TCDD, and both hydrocarbons induced aryl hydrocarbon hydroxylase (AHH) activity, but not ethoxyresorufin-O-deethylase activity. Cotreatment with α-naphthoflavone (αNF, 1 μM) or ellipticine (ELLIP, 0.1 nM) only partially inhibited the induction of AHH activity by BaP (30 μM). BaP and TCDD also induced expression of the CYP1B1 protein and the pattern of induction was comparable to that observed for CYP1A1. Treatment of GMCs with 30 μM BaP was associated with the formation of eight DNA adducts, and their occurrence could be inhibited by pretreatment with αNF (1 μM), but not ELLIP (0.1 nM). These results demonstrate that CYP1A1 and CYP1B1-related activities are induced in GMCs by BaP and TCDD and this induction is associated with metabolism of BaP to reactive intermediates that bind covalently to DNA.

Chronic alcoholization alters the expression of 5-HT 1A and 5-HT 1B receptor subtypes in rat brain

The expression of central 5-HT 1A and 5-HT 1B receptors was studied in several brain areas of rats subjected to a 2-week period of chronic alcoholization, followed by 18 h withdrawal. Quantitative autoradiography indicated that the ethanol treatment provoked an increase (∼ + 30%) in the labeling by [ 3H]8-hydroxy-2-(di- n-propylamino)tetralin ([ 3H]8-OH-DPAT) and [ 3H] N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-(2-pyridinyl) cyclohexane carboxamide ([ 3H]WAY-100635) of 5-HT 1A autoreceptors in the dorsal raphe nucleus, accompanied by a concomitant decrease in the labeling of postsynaptic 5-HT 1A receptors in the hippocampus (∼ −20%), anterior (∼ −30%) and posterior (∼ −32%) cortices. These changes were associated with a tendency toward an increase and decrease in 5-HT 1A mRNA levels in the anterior raphe area and hippocampus, respectively, suggesting that the changes observed are due to modifications in 5-HT 1A receptor protein synthesis. The autoradiographic labeling of 5-HT 1B receptors by serotonin- O-carboxymethylglycyl[ 125I]iodotyrosinamide ([ 125I]GTI) was found to increase (+55%) in the globus pallidus of alcoholized rats. Interestingly, a significant increase (+57%) in 5-HT 1B receptor mRNA levels was observed in the striatum, which contains cell bodies of neurons projecting into the globus pallidus. These data suggest that altered sensitivity of chronically alcoholized rats to 5-HT 1A and 5-HT 1B receptor ligands may result from alcohol-induced changes in the transcription of the genes encoding these receptors.

Differences in induction by xenobiotics in murine tissues and the Hepa 1c1c7 cell line of mRNAs encoding glutathione transferase, quinone reductase, and CYP1A P450s

Levels of mRNAs encoding class-alpha glutathione transferases, class-mu glutathione transferases, quinone reductase, and cytochrome P450 1A were measured after xenobiotic induction in murine tissues and in the Hepa1c1c7 murine hepatoma cell line. RNA levels in liver and intestinal mucosa were determined after induction with phenobarbital, butylated hydroxyanisole, beta-naphthoflavone, isosafrole, or combinations of these compounds. The tissue culture cells were presented with combinations of butylated hydroxyanisole, tert-butyl-hydroquinone, and beta-naphthoflavone. In murine liver and intestinal mucosa, the greatest induction (5-15-fold) of glutathione transferases and quinone reductase was seen with butylated hydroxyanisole. Administration of phenobarbital or beta-naphthoflavone has only a modest effect (2-3-fold). In contrast, cytochrome P450 1A mRNA levels increase only slightly after BHA induction but are induced dramatically by beta-naphthoflavone. The pattern of induction is different in Hepa1c1c7 cells; there the greatest induction of all mRNAs occurred with beta-naphthoflavone. Administration of antioxidants with other xenobiotics increases mRNA levels only slightly over the levels obtained with BHA in murine tissues, or with beta-naphthoflavone in Hepa1c1c7 cells. mGSTM1 (GT8.7, Yb1), the most abundant glutathione transferase mRNA in murine liver, is also the most abundant glutathione transferase mRNA in both normal and induced Hepa1c1c7 cells. Our results suggest that BHA induction in murine liver and intestinal mucosa of class-mu and class-alpha glutathione transferases may involve regulatory elements and mediators that function poorly in Hepa1c1c7 cells.

Human hnRNP Protein A1 Gene Expression: Structural and Functional Characterization of the Promoter

hnRNP protein A1 (34 kDA, pl 9·5) is a prominent member of the family of proteins (hnRNP proteins) that associate with the nascent transcripts of RNA polymerase II and that accompany the hnRNA through the maturation process and the export to the cytoplasm. New evidence suggests an active and specific role for some of these proteins, including protein A1, in splicing and transport. Contrary to the other hnRNP proteins, the intracellular level of protein A1 was reported to change as a function of proliferation state and cell type. In this work we analyse the A1 gene expression in different cells under different growth and differentiation conditions. Proliferation dependent expression was observed in lymphocytes and fibroblasts while purified neurons express high A1 mRNA levels both in the proliferative (before birth) and in the quiescent (after birth) state. Transformed cell lines exhibit very high (proliferation independent) A1 mRNA levels compared to differentiated tissues.A structural and functional characterization of the A1 gene promoter was carried out by means of DNase I footprinting and CAT assays. The observed promoter features can account for both elevated and regulated mRNA transcription. At least 12 control elements are contained in the 734 nucleotides upstream of the transcription start site. Assays with the deleted and/or mutated promoter indicate a co-operation of multiple transcriptional elements, distributed over the entire promoter, in determining the overall activity and the response to proliferative stimuli (serum).

Developmental changes in the expression of HMG 2a protein

The levels of HMG 2a chromosomal protein and its mRNA change during the post-hatched development of chicks were investigated. The contents of both HMG 2a and 2b proteins of liver, heart, brain, muscle and gizzard were abundant in the newly hatched chicks but their contents decreased significantly in those tissues of the 70-day-old chicks. The HMG 2a mRNA levels of liver, heart and brain in 70-day-old chick decreased to about 40% of those mRNA in the newly hatched chicks while the HMG 2a mRNA levels of muscle and gizzard in the 70-day-old chicks increased 5- and 3-fold, respectively. These results suggest that the decrease in the HMG 2a protein contents of the muscle and gizzard in the 70-day-old chicks may be largely due to the stimulation of HMG 2a protein degradation or the reduction of HMG 2a mRNA translation.

Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes.

Because the metabolic environment can alter gene expression in cultured cells, we examined the effects of change of medium on the levels of several cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on Matrigel. The amounts of P450 1A2, 2B1/2, or 3A1 mRNA were unaffected by changing the medium. In contrast, P450 1A1 mRNA levels were increased 1 to 2 h after media change, reached maximum levels by 6 h, and declined to baseline by 24 h. Supplementing day-old media with components of the medium revealed that only addition of amino acids resulted in 1A1 mRNA induction. From the results of direct additions and omissions, we showed that tryptophan, but not histidine, was largely responsible for the 1A1 mRNA induction. Moreover, mild photoactivation of the tryptophan resulted in a substantially increased magnitude of 1A1 mRNA induction. The time course for 1A1 mRNA induction by treatment with photoactivated tryptophan was identical to that observed after medium change. Treatment of hepatocyte cultures with beta-naphthoflavone, which is metabolized by 1A1, also resulted in a transient 1A1 mRNA induction time-course profile over a 24-h period, whereas treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which is relatively stable to metabolic transformation, produced sustained elevations of 1A1 mRNA, suggesting that the transient response to tryptophan also may involve metabolism of the inducer. Our results extend previous data showing that oxidized products of tryptophan induce 1A1, and suggest that the transient nature of the induction may be due to elimination of the activated tryptophan molecule.