Biomedical subjects

A Aranda

Publications and source records attributed to A Aranda.

At least 73 records · Page 4Linked to original sources

PubMed1993

Repression of c-fos gene expression by thyroid hormone and retinoic acid receptors.

Incubation with 1 nM triiodothyronine (T3) decreased cycloheximide-induced c-fos mRNA levels and the mRNA response to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 100 nM) or to forskolin (15 microM). T3 also reduced the abundance of nuclear proteins that bind to an AP-1 binding site and the levels of c-Fos protein, as determined by Western blot. In transient transfection assays with a c-fos promoter reporter construct T3 decreased basal promoter activity by more than 50-60% and strongly inhibited the TPA and forskolin-induced activity. T3 further suppressed basal promoter activity and led to a drastic decrease of the response to both stimulatory agents after co-transfection of the promoter with an expression vector for the receptor. A truncated receptor that lacks the DNA binding domain abolished the inhibitory effect of the endogenous and transfected T3 receptor. Retinoic acid (RA) had an effect similar to T3 reducing basal CAT activity and the response to TPA and forskolin by 40-50% in cells co-transfected with the c-fos promoter and an expression vector for the RA receptor. The inhibitory effect of the nuclear receptors on c-Fos is reciprocal, since overexpression of c-Fos repressed induction of the activity of the growth hormone promoter by T3 and RA. Co-transfection with an antisense c-fos vector relieved this inhibition and restored the response to both ligands. These results show the antagonism between the nuclear receptors and the membrane signal transduction pathways that converge on the c-fos oncogene.

Animals↗

PubMed1993

Interaction of thyroid hormone and retinoic acid receptors on the regulation of the rat growth hormone gene promoter.

Retinoic acid transcriptionally regulate growth hormone (GH) gene expression through sequences located in the 5'-flanking region of the gene. A partial induction by retinoic acid was obtained with the -181 bp of the rat GH promoter, and sequences up to -209 were required for a full response. These sequences contain the previously identified thyroid hormone responsive element. The retinoid X receptor RXR increased transactivation by T3 and RA. The retinoid was relatively more effective in stimulating the native GH promoter than an heterologous promoter which contains the response element, thus showing the importance of the promoter context on transactivation by the nuclear receptors.

Animals↗

PubMed1993

Nicotinamide analogs and DNA-damaging agents deplete thyroid hormone receptor and c-erbA mRNA levels in pituitary GH1 cells.

Incubation of pituitary GH1 cells with N'-methylnicotinamide, nicotinamide and 3-acetylpyridine which inhibit nuclear ADP-ribosylation and/or the cellular concentration of its substrate NAD+ reduced the amount of nuclear thyroid hormone receptors in a time- and dose-dependent manner without altering the affinity of the receptors for the hormone. A transient activation of poly(ADP-ribose)polymerase by methyl methanesulfonate, ultraviolet irradiation or spermine caused a rapid depletion of cellular NAD+ content and was followed by a strong inhibition of ADP-ribosylation. These agents also produced a very rapid and marked reduction of receptor numbers. The decrease of receptors caused by the different compounds is not secondary to a generalized inhibition of protein synthesis or to an alteration in hormone availability. The abundance of c-erbA alpha and beta mRNAs, which encode thyroid hormone receptors, was reduced in cells treated with the compounds that decrease receptor number, thus suggesting that this effect is caused by a decrease in the expression of c-erbA genes.

Adenosine Diphosphate Ribose↗

PubMed1992

The thyroid hormone response element is required for activation of the growth hormone gene promoter by nicotinamide analogs.

N'-Methylnicotinamide and nicotinamide, which decreased in vitro ADP-ribosylation of nuclear proteins and/or cellular NAD+ content, selectively increased the basal expression of the rat growth hormone (GH) gene promoter and its response to triiodothyronine (T3). This increase was not found when the thyroid hormone response element (TRE) was deleted from the promoter. Transfection with an expression vector for the T3 receptor inhibited basal activity of the TRE-containing promoter and repressed the stimulatory effect of N'-methylnicotinamide. The addition of hormone relieved this inhibition and enhanced transcription above levels found in the absence of the transfected receptors. These results suggest a modulatory role of ADP-ribosylation in hormonal regulation of gene expression.

Adenosine Diphosphate Ribose↗

PubMed1992

Impairment by cyclosporin A of reperfusion-induced arrhythmias.

This study introduces the immunosuppressor, cyclosporin A, as a cardioprotective drug. This effect was analyzed during development of reperfusion/induced arrhythmias after 5-min period of coronary ligation in hearts of rats under anesthesia. The results indicate that cyclosporin, when given before coronary occlusion, at a dose of 20 mg/kg, effectively protects against the high incidence of arrhythmias and the fall in blood pressure induced by reperfusion. In addition, in inhibits the delivery of lactic dehydrogenase and creatine kinase enzymes to the plasma. We propose that the protective effect could be related with its well documented action to restrain Ca(2+)-induced damage of mitochondrial functions.

Animals↗

PubMed1991

Retinoic acid decreases thyroid hormone receptor expression in pituitary GH1 cells.

Retinoic acid produced a time and dose-dependent depletion of thyroid hormone receptors in GH1 cells without modifying their affinity for triiodothyronine (T3). A maximal decrease (50-70%) was obtained after 24-48 h incubation with 5-10 microM retinoic acid. Treatment with 0.8 nM T3 for 24 h caused a similar reduction and did not potentiate the decrease produced by these concentrations of retinoic acid. However, the combination of sub-maximally effective doses of both ligands had an additive effect on receptor levels. The reduction of receptor caused by retinoic acid is accompanied by a decreased expression of c-erbA alpha 1 and alpha 2 mRNAs, but the retinoid did not reduce the abundance of c-erbA beta mRNA. In contrast, T3 decreased the levels of both alpha and beta transcripts.

Animals↗

PubMed1991

Influence of thyroid hormone on ADP-ribosylation of nuclear proteins in cultured GH1 cells.

We present evidence that T3 can alter the ADP-ribosylation of chromatin associated proteins. Nuclei from GH1 cells were incubated with [adenylate-32P]NAD and the radioactivity incorporated into histone and non-histone proteins was quantitated and analyzed by gel electrophoresis and autoradiography. Incubation of GH1 cells for 24 h with T3 lowered by 40-70% the [32P]ADP-ribose incorporated into nuclear proteins. However, incubation for 3 h with T3 resulted in a stimulation instead of a decrease of in vitro [32P]ADP-ribose incorporation. The major ADP-ribosylated component electrophoresed as a 120,000 molecular mass non-histone protein, and radiolabeled histones were also observed. The same protein species were observed for all the experimental groups and T3 affected the extent of ADP-ribosylation but did not alter the sedimentation of the [32P]ADP-ribosylated components excised from chromatin after micrococcal nuclease digestion.

Adenosine Diphosphate Ribose↗

PubMed1991

Expression of the growth hormone gene and the pituitary-specific transcription factor GHF-1 in diabetic rats.

Diabetes in the rat is associated with poor growth and decreased GH in the pituitary. In this study we have examined whether this reduction reflects an impairment of GH gene expression. Diabetes was induced by the administration of streptozotocin (7 mg/100 g BW), and 18 days later, GH content, GH mRNA, and GH transcription rate were determined. GH mRNA levels were reduced by more than 80% in the pituitaries of diabetic rats, which had a similarly reduced GH content. The differences observed in transcription fully account for the changes in mRNA concentration, since the transcription rate of the gene was also reduced by a factor of 10 in the diabetic pituitaries. Insulin therapy (3 U/15 days) partially restored these parameters. The expression of the specific transcription factor GHF-1/Pit-1 in diabetic rats was also analyzed. Both GHF-1 mRNA levels and the binding of nuclear proteins to an oligodeoxynucleotide conforming to the GHF-1 proximal binding site in the promoter of the GH gene were normal in the diabetic pituitaries, thus excluding the possibility that decreased availability of this factor could be responsible for the decreased GH transcription. Since diabetes produced an approximately 3-fold reduction of circulating T3, the potential role of thyroid hormones on GH gene expression was also evaluated in thyroidectomized and thyroidectomized diabetic rats. Thyroidectomy decreased GH and GH mRNA to less than 5% of the values found in intact animals, and a single saturating injection of T3 (250 micrograms/100 g BW) resulted in a 8- to 10-fold induction of GH mRNA after 6 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells.

In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells. The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.

Adenylyl Cyclases↗

PubMed1990

Regulation of thyroid hormone receptor and c-erbA mRNA levels by butyrate in neuroblastoma (N2A) and glioma (C6) cells.

Butyrate produced a biphasic modulation of the thyroid hormone receptor in neuroblastoma N2A cells increasing receptor number by 20-35% at concentrations 0.25-0.75 mM and decreasing receptor levels by 30-55% at 2-4 mM. The half-life of the receptor, as assessed by its disappearance after incubation with 18 microM cycloheximide was 8.4 hr in control cells and 10.3 hr and 5.0 hr in cells incubated with 0.25 and 4 mM butyrate, respectively. This compound increased the abundance of multyacetylated forms of histone H4 from 30% in control cells to almost 70% with butyrate 4 mM. In glioma C6 cells, the fatty acid produced a dose-dependent increase of receptor levels (up to 3-4-fold with 2-5 mM butyrate) and had little effect in increasing multiacetylation (from 30% in controls to 42-46% with 2-5 mM butyrate). Recent studies have shown that the c-erbA proto-oncogen codes for the thyroid hormone receptor. In N2A and C6 cells, 2 c-erbA-related mRNAs, one measuring 2.6 kb and the other 6 kb, were detected. Both forms were differently regulated by butyrate. This compound decreased the abundance of the 2.6 kb forms in both cell types, even at the concentrations at which there was an elevation of receptor levels. Only the largest mRNA correlated with receptor concentration increasing by 2-3-fold after treatment of C6 cells with butyrate, and undergoing a smaller but biphasic change in N2A cells. Our data suggest that modification of chromatin structure probably secondary to acetylation induces changes in thyroid hormone receptor levels in neuroblastoma and glioma cells by affecting both receptor stability and receptor mRNA levels.

Animals↗

PubMed1989

Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: evidence that changes in receptor levels are not associated with changes in receptor distribution.

The association of [125I]T3-receptor complexes with C6 cell chromatin was analyzed after a limited digestion with micrococcal nuclease (MN) or DNase I. Both nucleases solubilized up to 60-70% of receptor and 0.4 M KCl extracted 70% of the non-digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix. With DNase I the receptor was released 2-3-fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN. The digestion of receptor with DNase I and MN occurred 14- and 6-fold faster, respectively, than the appearance of PCA-soluble chromatin. Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases. These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin.

Cell Line↗

PubMed1989

Retinoic acid regulates growth hormone gene expression.

Vitamin A is required for normal growth and development, and retinoic acid (RA) may be the active metabolite in this process. Recent evidence indicates that RA acts through binding to a nuclear receptor which belongs to the steroid/thyroid hormone receptor superfamily. The receptors seem to associate with hormone-response elements in the target genes resulting in the activation (or inhibition) of transcription. Although no interaction of RA-receptor complex with specific DNA sequences has yet been reported, the homology of the different receptors suggests their mechanisms of action are similar. We therefore examined whether the effects of RA on growth could be related to changes in the expression of the growth hormone gene which is known to be transcriptionally regulated by both thyroid and glucocorticoid hormones. Our results show that RA controls growth hormone production in pituitary GH1 cells and that its effect is synergistic with that caused by these hormones.

Dose-Response Relationship, Drug↗

PubMed1989

Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate.

The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells.

Animals↗

PubMed1989

Sodium butyrate induces major morphological changes in C6 glioma cells that are correlated with increased synthesis of a spectrin-like protein.

Butyrate induced flattening and development of cell processes in rat glioma (C6) cells and this change was correlated with an increase in the synthesis of a polypeptide doublet with an apparent molecular weight of about 200 kDa. Blot analysis revealed that at least one of these polypeptides was a spectrin-like protein. Indirect immunofluorescence studies with the spectrin antiserum indicated that the antigen was present in the cell bodies, and also in the cell processes. Thus fodrin may be one the major targets for the action of butyrate on C6 cells.

Autoradiography↗

PubMed1989

Effects of cyclic AMP elevation on the levels of insulin receptors in glial C6 cells.

The regulation of the insulin receptor by cAMP has been examined in glial C6 cells. Incubation for 48 h with dibutyryl cyclic AMP produced a dose-dependent inhibition of 125I-insulin binding to the cells. Other agents that elevate intracellular cAMP levels such as forskolin and cholera toxin mimicked the effect of this cyclic AMP analog. With all compounds the maximal decrease of binding was found between 24 and 48 h and normally varied between 40 and 60%. Forskolin, cholera toxin and dibutyryl cyclic AMP also affected C6 cell proliferation, and the dose-response for decreasing the receptor was very similar to that observed for the inhibition of cell growth, suggesting a relationship between both phenomena. Scatchard analysis showed that cAMP did not produce major changes in the affinity of the receptor for insulin, but rather decreased receptor number. An accumulation of receptors at the cell surface was observed in the absence of de novo protein synthesis, since cycloheximide caused a significant increase in insulin binding to the cells. This inhibitor almost totally blocked the cAMP effect both when added simultaneously to the cells with the agents which increase cAMP and when added to cells pre-treated for 48 h with the same compounds.

Animals↗

PubMed1988

Proliferation and differentiation are not directly related to H1(0) accumulation in cultured glial cells.

A basic nuclear chromatin protein with electrophoretic mobility of H1(0) histone is present in C6 rat glial cells and in primary cultures of rat brain astroglial cells. That this protein is identical to H1(0) is further demonstrated by the finding that it accumulates in C6 cells in a time- and dose-dependent manner in response to butyrate, an agent which is known to induce this protein in other cell types. Other short-chain fatty acids were found to influence H1(0) levels similarly although to a lesser extent than butyrate. There was a very close correlation between the induction of H1(0) and the inhibition of growth induced by different concentrations of short-chain fatty acids which supports the idea that the concentration of this protein is higher in non-proliferating cells. However, when cell growth was inhibited by dexamethasone or agents that increase intracellular cyclic adenosine monophosphate levels, H1(0) levels were not affected, even though these compounds also blocked DNA synthesis and induced morphologic changes in C6 cells. These observations suggest that, at least in glial cells, the accumulation of H1(0) is specifically caused by short-chain fatty acids and that suppression of cell division or commitment to differentiation are not sufficient 'per se' for the induction of this protein.

Animals↗

PubMed1988

Rat growth hormone gene expression. Both cell-specific and thyroid hormone response elements are required for thyroid hormone regulation.

The elements involved in mediating cell-specific and thyroid hormone stimulation of rat growth hormone gene expression have been defined by transfection studies and by nuclease footprinting. 5'-Flanking DNA extending to -104 can mediate cell-specific expression, and this is enhanced 3- to 4-fold with DNA extending to -145. Cell-specific factors, found only in rat growth hormone producing cells, bind within the -137/-107 and -95/-65 regions, and competition studies suggest that the same factor binds to both sites. The sequence A (A or T) TAAAT is found at the center of both footprints at -80 and -122, suggesting that it is a core component of the recognition sequence of the cell-specific factor. Disruption of the spatial and/or distance relationships between the two regions eliminates the enhanced level of cell-specific expression, suggesting a cooperative interaction of the proteins which bind to these elements. Sequences located between -208 and -178 can confer thyroid hormone-regulated expression when linked in either orientation in close proximity to one or both cell-specific elements. The thyroid hormone and cell-specific elements function as an enhancer-like unit and are both required to confer regulated expression to heterologous promoters. We propose that thyroid hormone acts via its receptor to enhance the function of the cell-specific element by forming a more "active" transcription complex which stimulates the level of gene expression.

Animals↗

PubMed1988

Cholera toxin affects nuclear ADP-ribosylation in GH1 cells.

Incubation of GH1 cells with cholera toxin for 24 h inhibits [32P]ADP-ribose incorporation into histones and non-histone nuclear proteins by more than 50%. The toxin produces a generalized decrease of incorporation into all protein acceptors and into the poly(ADP-ribosyl)ated components excised from chromatin after micrococcal nuclease digestion. The cellular levels of NAD were also decreased (40 to 80%) after treatment with cholera toxin. The inhibition of poly(ADP-ribosyl)ation is preceded by an increase of [32P]ADP-ribose incorporation, since incubation with the toxin for 3 h caused an increase instead of a decrease of incorporation. Incubation with dibutyryl cyclic AMP for 24 h also inhibited nuclear poly(ADP-ribosyl)ation, thus showing that the effect of cholera toxin might be mediated by cyclic AMP.

ADP Ribose Transferases↗