RESUMO
Salt appetite, a conditioning factor for hypertension and cardiovascular diseases, is produced when high doses of mineralocorticoids are given to experimental animals. A commonly used procedure to identify neuronal activation is to determine the number of Fos-immunoreactive cells. In rats with established salt appetite after 8 days of deoxycorticosterone acetate (DOCA) treatment, Fos-positive cells were studied in seven brain areas. Significant increases in Fos activity were recorded in the paraventricular (PVN) and supraoptic (SON) nuclei, median preoptic nucleus (MnPO), organum vasculosum of the lamina terminalis (OVLT), preoptic area (POA), bed nucleus of the stria terminalis (BNST) and amygdala (AMYG). In most of these areas, increased Fos expression was also observed early (2 h) after a single DOCA injection, well before salt appetite develops. Using a mineralocorticoid receptor (MR) antibody, we studied whether Fos-active regions also expressed MR. MR-positive cells were found in the OVLT, MnPO, AMYG and BNST, but not in the POA, PVN and SON. In the PVN and SON, nevertheless, prolonged or single DOCA treatment increased expression of mRNA for arginine vasopressin (AVP). The present demonstration of Fos activation, in conjunction with differential expression of MR and stimulation of AVP mRNA, suggests that a neuroanatomical pathway comprising the AMYG, osmosensitive brain regions and magnocellular nuclei becomes activated during DOCA effects on salt appetite. It is recognized, however, that DOCA effects may also depend on mechanisms and brain structures other than those considered in the present investigation. Since some Fos-positive regions were devoid of MR, a comprehensive view of DOCA-induced salt appetite should consider nongenomic pathways of steroid action, including the role of reduced DOC metabolites binding to GABAergic membrane receptors.
Assuntos
Apetite/fisiologia , Encéfalo/metabolismo , Desoxicorticosterona/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Cloreto de Sódio , Animais , Arginina Vasopressina/genética , Imuno-Histoquímica , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Mineralocorticoides/metabolismo , Distribuição TecidualRESUMO
Opioids have been implicated in sexual differentiation of the brain and in the regulation of reproductive behavior and endocrinology of mammals. Previous studies have indicated that estrogen administration in adults regulates preproenkephalin MRNA levels in several hypothalamic brain nuclei. We have determined preproenkephalin mRNA levels in estrogen-treated juvenile male and female rats to investigate the developmental pattern of estrogenic regulation of enkephalinergic neurons in the medial preoptic area. Rats were treated with estradiol benzoate (20 microgram/kg/day) or oil from day 21 to 23. Sections of the medial preoptic area (mPOA) were studied by in situ hybridization histochemistry at the single cell level and quantified with the assistance of an image analysis system. Our data indicate that males contain higher levels of preproenkephalin mRNA per neuron than females. In addition, our results indicate that estrogen causes an upward shift in the amount of mRNA expressed per cell, females demonstrating a greater response to estrogen than males. An increase in soma cell area following estrogen treatment was observed only in female mPOA enkephalinergic neurons. Taken together, these results indicate a sex difference in total preproenkephalin levels and in estrogenic regulation of preproenkephalin mRNA in the POA of juvenile rats. These results are discussed in relation to the differential role opioids may play in male and female reproductive physiology.
Assuntos
Encefalinas/genética , Estrogênios/fisiologia , Área Pré-Óptica/química , Precursores de Proteínas/genética , Caracteres Sexuais , Maturidade Sexual , Animais , Tamanho Celular/fisiologia , Encefalina Metionina/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Masculino , Neurônios/química , Neurônios/citologia , Neurônios/fisiologia , Área Pré-Óptica/citologia , Área Pré-Óptica/crescimento & desenvolvimento , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
In physiological doses, mineralocorticoids (MC) normalize the high salt intake developed after adrenalectomy. We have studied whether this effect of MC is accompanied by changes in the mRNA of neuronal alpha3 and beta1 subunits of the (Na,K)-ATPase because this enzyme could by a mediator of MC action in target cells. We employed [35S]oligonucleotide probes for the mentioned subunits hybridized to brain sections from adrenalectomized rats and adrenalectomized rats receiving aldosterone (ALDO) during 4 days. Using t-test statistics to measure differences in mean levels of grain density, and the Kolmogorov-Smirnov non-parametric test applied to frequency histograms, we showed that ALDO increased the alpha3 subunit mRNA in the septum medialis, preoptic area medialis, caudate-putamen, periventricular gray substance, amygdala lateralis, hippocampal subfields CA1 to CA4 and the gyrus dentatus. Significant increases for the beta1 subunit mRNA were found in periventricular gray substance, the CA1-CA4 hippocampal subfields and gyrus dentatus. Therefore, the salt-suppression effect of MC was accompanied by coordinate increases in (Na,K)-ATPase alpha3 and beta1 subunit mRNA in the hippocampus, gyrus dentatus and periventricular gray substance, whereas in other regions the stimulatory effect was exclusive of the alpha3 subunit mRNA only. The results suggest that the enzyme could be a target of ALDO action not only in areas related to salt appetite control (amygdala, preoptic area) but also in brain regions subserving other functions of the MC.
Assuntos
Glândulas Suprarrenais/fisiologia , Aldosterona/fisiologia , Encéfalo/metabolismo , Isoenzimas/genética , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Adrenalectomia , Animais , Masculino , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
We have studied the effects of spinal cord transection and of dexamethasone (DEX) treatment on mRNA biosynthesis of the Na+, K(+)-ATPase, a key enzyme necessary for neurotransmission, membrane repolarization and nutrient uptake in the CNS. In situ hybridization analysis revealed a significant reduction in mRNA for the alpha 3 catalytic subunit of the enzyme in medium and large size ventral horn neurones (1000-2300 microns2) but not in small cells (1000 microns2) 24 h after spinal cord transection. DEX treatment significantly reversed the transection effect in medium and large size neurones. It is suggested that up-regulation of mRNA expression for Na+,K(+)-ATPase may constitute an important mechanism by which glucocorticoids help to re-establish neuronal function after spinal cord injury.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Neurônios/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/genética , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Masculino , Regeneração Nervosa/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Regulação para CimaRESUMO
We have studied the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) on salt appetite developed by deoxycorticosterone acetate (DOCA) treated rats. To this end, we measured the effects of DOCA given on alternate days on (1) salt intake; (2) MR and GR in hippocampus (HIPPO), amygdala (AMYG), and hypothalamus (HT); (3) the activity of ornithine decarboxylase (ODC), a GR-mediated response, and (4) the salt intake after treatment with the antiglucocorticoid RU 486 or the antimineralocorticoid ZK 91587. First, we demonstrated that 10 but not 1 mg DOCA induced natriogenesis. Forty-eight hours after adrenalectomy and 24 h after the last DOCA injection, 10 but not 1 mg hormone reduced binding to GR in HIPPO, AMYG, and HT. Both doses of DOCA also reduced the binding to MR in HIPPO, without changes in AMYG; in HT the 1-mg dose was without effect, but the natriogenic dose (10 mg) highly increased binding of [3H]-corticosterone to MR. Scatchard analysis demonstrated increased Bmax and Kd values in the HT of DOCA-treated rats. Occupation of GR by DOCA did not stimulate the ODC activity, in contrast to the four-fold increment effected by the glucocorticoid dexamethasone. Also, administration of RU 486 did not inhibit the sale intake promoted by DOCA, in contrast to ZK 91587 which partly delayed the natriogenic effect of DOCA. It is suggested that brain MR are involved in the natriogenic effect of DOCA, whereas the role of GR is inconclusive.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Apetite/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Desoxicorticosterona/farmacologia , Receptores de Glucocorticoides/fisiologia , Receptores de Mineralocorticoides/fisiologia , Cloreto de Sódio na Dieta/administração & dosagem , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Hipocampo/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Masculino , Mifepristona/farmacologia , Antagonistas de Receptores de Mineralocorticoides , Ornitina Descarboxilase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/antagonistas & inibidores , Espironolactona/análogos & derivados , Espironolactona/farmacologiaRESUMO
The Na+,K(+)-ATPase plays a key role in the regulation of ion fluxes and membrane repolarization in the CNS. We have studied glucocorticoid effects on biosynthesis of the Na+,K(+)-ATPase and on ouabain binding in the ventral horn of the spinal cord using intact rats, adrenalectomized (ADX) rats, and ADX rats receiving dexamethasone (ADX+DEX) during 4 days. Cryostat sections from spinal cords were incubated with a 35S-oligonucleotide coding for the alpha 3-subunit or a 3H-cDNA coding for the beta 1-subunit of the Na+,K(+)-ATPase using in situ hybridization techniques. In ventral horn motoneurons, grain density per cell and grain density per area of soma for both probes were slightly reduced in ADX rats but significantly increased in the ADX+DEX group, using ANOVA and the Bonferroni's test. Statistical analysis of frequency histograms of neuronal densities further indicated a significant shift to the right for intact rats compared with ADX rats for both probes. Concomitantly, [3H]ouabain binding to membrane preparations from ventral horns was reduced in ADX rats and restored to normal by DEX administration. No effect of adrenalectomy or DEX treatment was obtained in the dorsal horn. In conclusion, glucocorticoids positively modulate the mRNA for the alpha 3-subunit and the beta 1-subunit of the Na+,K(+)-ATPase and recover ouabain binding to normal values. The increments of the synthesis and activity of an enzyme affecting membrane repolarization and synaptic neurotransmission are consistent with the alleged stimulatory effect of glucocorticoids on spinal cord function.
Assuntos
Adrenalectomia , Células do Corno Anterior/metabolismo , Dexametasona/farmacologia , Ouabaína/metabolismo , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Análise de Variância , Animais , Células do Corno Anterior/química , Células do Corno Anterior/citologia , Sequência de Bases , Relação Dose-Resposta a Droga , Glucocorticoides/farmacologia , Histocitoquímica , Hibridização In Situ , Masculino , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/análise , Fatores de Tempo , TrítioRESUMO
The effect of glucocorticoids on (Na+K)ATPase mRNA synthesis was studied in 19 brain areas of adrenalectomized (ADX) rats untreated or receiving dexamethasone (DEX). For in situ hybridization, we employed a [35S]oligonucleotide probe for the alpha 3-subunit isoform, and a [3H]cDNA coding for the beta 1-subunit of the enzyme. Mean levels of grain density for the alpha 3 subunit mRNA of DEX-treated rats were significantly higher by a 't' test in medial septum, amygdala lateralis (AL) and medialis (AME), gyrus dentatus, CA4 hippocampal area, substantia nigra and periventricular gray, compared to untreated rats. For the beta 1-subunit, mean levels after DEX were significantly higher in AL and lateral preoptic area. In addition, the Kolmogorov-Smirnov test applied to frequency histograms of neuronal densities indicated a coordinate increase in alpha 3 and beta 1-subunit mRNA expression for the CA2 subfield and preoptic area medialis (POA MED). We conclude that (1) glucocorticoids are positive modulators of (Na+K)ATPase mRNA; (2) analysis of frequency histograms suggests that glucocorticoids promote in a few regions (AL, POA MED, CA2 subfield) a coordinate increase in the biosynthesis of the alpha 3 and beta 1-subunit mRNA. In 11 other areas stimulation occurs for one subunit mRNA only, whereas 5 areas were insensitive to glucocorticoid effects on this enzyme.
Assuntos
Glândulas Suprarrenais/fisiologia , Encéfalo/efeitos dos fármacos , Dexametasona/farmacologia , Fragmentos de Peptídeos/genética , RNA Mensageiro/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/genética , Adrenalectomia , Animais , Sequência de Bases , Encéfalo/metabolismo , Código Genético , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-DawleyRESUMO
Type I corticosteroid receptors were determined in cytosol from hippocampus (HIPPO) and amygdala (AMYG), using [3H]aldosterone (ALDO), [3H]dexamethasone (DEX) or the mineralocorticoid antagonist [3H]ZK 91587 as ligands. Incubations with the first two compounds also contained the pure glucocorticoid RU 28362 to block type II receptors. Binding of the three ligands was comparable in cytosol from HIPPO and it was slightly higher for [3H]DEX in AMYG. However, after heat-induced receptor transformation, binding to DNA-cellulose was observed for [3H]ALDO-receptor complex obtained from HIPPO or AMYG, whereas it was negligible for [3H]ZK 91587. Receptors charged with [3H]DEX or [3H]ALDO showed similar retention on DNA-cellulose columns in the case of the AMYG, while binding to the polynucleotide was higher for [3H]ALDO in the HIPPO. Finally, only [3H]ALDO was taken up to a significant extent in purified cell nuclei prepared from slices of HIPPO and AMYG previously incubated with the three ligands. It is concluded that binding of a natural agonist steroid may be a prerequisite for type I receptor transformation and translocation from the cytoplasm into the nuclear fraction. DEX binding to type I receptors resembles a partial agonist with antagonist properties, whereas antagonists such as ZK 91587 are bound and retained in cytoplasm, without further translocation.
Assuntos
Tonsila do Cerebelo/metabolismo , Hipocampo/metabolismo , Receptores de Glucocorticoides/metabolismo , Aldosterona/química , Aldosterona/metabolismo , Animais , Núcleo Celular/metabolismo , Celulose/análogos & derivados , Celulose/metabolismo , Técnicas de Cultura , DNA/metabolismo , Dexametasona/química , Dexametasona/metabolismo , Masculino , Mineralocorticoides/antagonistas & inibidores , Ratos , Ratos Endogâmicos , Espironolactona/análogos & derivados , Espironolactona/química , Espironolactona/metabolismoRESUMO
We have used three experimental protocols to determine binding parameters for type I and type II glucocorticoid receptors in the spinal cord and hippocampus (HIPPO) from adrenalectomized rats. In protocol A, 0.5-20 nM [3H]dexamethasone (DEX) was incubated plus or minus a 1000-fold excess of unlabeled DEX, assuming binding to a two-site model. In protocol B, [3H]DEX competed with a single concentration of RU 28362 (500 nM), whereas in protocol C, we used a concentration of RU 28362 which varied in parallel to that of [3H]DEX, such as 500 x. Results of protocols A and C were qualitatively similar, in that: (1) Bmax for type I receptors favored the HIPPO, while the content of type II sites was comparable in the two tissues; (2) Kd was consistently lower for type I than for type II sites in both tissues; and (3) type II receptors from the spinal cord showed lower affinity than their homologous sites from HIPPO. This last result was also obtained when using protocol B. In contrast, protocol B yielded binding data indicating that type II sites were of similar or higher affinity than type I sites. Computer simulation of the binding protocols demonstrated that protocols A and C were the most theoretically reliable for estimating the Kd and Bmax of type I sites, and the predicted error was smaller for protocol C, in comparison with protocol B. We suggest that the noted differences in the Kd of type II receptors between the spinal cord and HIPPO could account for a difference in sensitivity of the two systems in the physiological adrenal hormone range.
Assuntos
Hipocampo/metabolismo , Receptores de Glucocorticoides/metabolismo , Medula Espinal/metabolismo , Animais , Ligação Competitiva , Dexametasona/metabolismo , Masculino , Ratos , Ratos EndogâmicosRESUMO
1. We have used in situ hybridization techniques to determine the mRNA for (Na + K)ATPase in 20 brain regions from control rats and rats treated with high doses of deoxycorticosterone (DOC). 2. DOC-treated rats developed a salt appetite following the second hormone administration on alternate days and were used after the fourth DOC administration. 3. DOC treatment did not change the number of silver grains/cell deposited in cells from Ca1, CA2, CA3, and CA4 hippocampal subfields, dentate gyrus, cerebral cortex, medial preoptic area (POA), substantia nigra, and periventricular gray matter. 4. Nonsignificant reductions were detected in lateral POA, medial and lateral septum, caudate-putamen, and three amygdaloid nuclei (cortical, basolateral, and central) from DOC-treated rats. 5. Significant reductions were obtained, after DOC administration, in arcuate and ventromedial hypothalamic nuclei and medial and lateral amygdala. 6. The results suggested that regulation of the beta-subunit mRNA of (Na + K)-ATPase may be related to the central actions of mineralocorticoids in the control of salt intake.