La expresión génica hipotalámica de la enzima Citocromo P450scc en ratas hembras es regulada positivamente por estrógeno y negativamente por progesterona

Los neuroesteroides son potentes moléculas modulatorias sintetizadas en sistema nervioso central y cuya actividad se restringe a dicho ámbito. En su síntesis participan enzimas del grupo del citocromo P450 (CP450) enzimas que no pertenecen a dicho grupo. En este trabajo se estudia la expresión génica de CP450scc a nivel de hipotálamo medio basal (HMB) de ratas hembra en diferentes condiciones hormonales, y se lo relaciona con su eventual regulación a través de esteroides gonadales, particularmente estrógenos y progesterona. Se proporciona evidencia de que existe una expresión diferencial en HMB, y que dicha expresión se encuentra positivamente modulada por estrógenos, y negativamente modulada por progesterona.

Evidence of high expression of peptidylglycine α-amidating monooxygenase in the rat uterus: Estrogen regulation

In the present study, high levels of peptidylglycine α-amidating monooxygenase (PAM), which catalyzes the two-step formation of bioactive α-amidated peptides from their glycine-extended precursors, have been found in the uterus. Expression of PAM was evaluated in the uterus of intact cycling adult female rats and after experimental manipulation of the estrogen status of the rats. During the estrous cycle, PAM mRNA levels exhibited striking changes inversely related to the physiological variations of plasma estrogen levels. The levels of PAM transcripts changed markedly during the estrous cycle, reaching the highest levels at metestrus. There was a 15-fold increase in the abundance of PAM mRNA between metestrus and proestrus. Chronic treatment of ovariectomized rats with 17β-estradiol decreased PAM mRNA levels to values comparable with those found in intact rats at proestrus. Progesterone was without effect on PAM mRNA levels, indicating that the effect was specific for estradiol. In situ hybridization studies were conducted to determine the tissue disposition and cell types expressing PAM. High levels of PAM mRNA were localized in the endometrium at the level of luminal and glandular cells. A weak signal was observed in stromal cells, and the myometrium cells were negative. 17β-Estradiol treatment induced an overall decrease of the hybridization signal, as compared with ovariectomized rats. These results demonstrate the presence of high levels of PAM in the uterus and indicate that estrogens are involved in regulating the expression of the enzyme in this tissue. However, the present study provides no information regarding whether this regulation takes place at the level of transcription or influences mRNA stability.

Nitric oxide in the contractile action of bradykinin, oxytocin, and prostaglandin F2 α in the estrogenized rat uterus

Experiments were performed on uteri from estrogen-primed female rats. Bradykinin (BK) (10−8 M) significantly augmented biosynthesis of prostaglandin F2 α (PGF2α) and prostaglandin E2 (PGE2), and this synthesis was completely blocked by NG-monomethyl l-arginine (NMMA) (300 μM), a competitive inhibitor of nitric oxide synthase (NOS). Blockade of prostaglandin synthesis by indomethacin caused rapid dissipation of isometric developed tension (IDT) induced by BK. Blockade of NOS with NMMA had similar but less marked effects. Combining the two inhibitors produced an even more rapid decay in IDT, suggesting that BK-induced NO release maintains IDT by release of prostanoids. The decline of frequency of contraction (FC) was not significantly altered by either indomethacin or NMMA but was markedly accelerated by combination of the inhibitors, which suggests that PGs maintain FC and therefore FC decline is accelerated only when PG production is blocked completely by combination of the two inhibitors of PG synthesis. The increase in IDT induced by oxytocin was unaltered by indomethacin, NMMA or their combination indicating that neither NO nor PGs are involved in the contractions induced by oxytocin. However, the decline in FC with time was significantly reduced by the inhibitor of NOS, NMMA, suggesting that FC decay following oxytocin is caused by NO released by the contractile process. In the case of PGF2α, NMMA resulted in increased initial IDT and FC. The decline in FC was rapid and dramatically inhibited by NMMA. Receptor-mediated contraction by BK, oxytocin, and PGF2α is modulated by NO that maintains IDT by releasing PGs but reduces IDT and FC via cyclic GMP.

Oxytocin releases atrial natriuretic peptide by combining with oxytocin receptors in the heart

Previous studies indicated that the central nervous system induces release of the cardiac hormone atrial natriuretic peptide (ANP) by release of oxytocin from the neurohypophysis. The presence of specific transcripts for the oxytocin receptor was demonstrated in all chambers of the heart by amplification of cDNA by the PCR using specific oligonucleotide primers. Oxytocin receptor mRNA content in the heart is 10 times lower than in the uterus of female rats. Oxytocin receptor transcripts were demonstrated by in situ hybridization in atrial and ventricular sections and confirmed by competitive binding assay using frozen heart sections. Perfusion of female rat hearts for 25 min with Krebs–Henseleit buffer resulted in nearly constant release of ANP. Addition of oxytocin (10−6 M) significantly stimulated ANP release, and an oxytocin receptor antagonist (10−7 and 10−6 M) caused dose-related inhibition of oxytocin-induced ANP release and in the last few minutes of perfusion decreased ANP release below that in control hearts, suggesting that intracardiac oxytocin stimulates ANP release. In contrast, brain natriuretic peptide release was unaltered by oxytocin. During perfusion, heart rate decreased gradually and it was further decreased significantly by oxytocin (10−6 M). This decrease was totally reversed by the oxytocin antagonist (10−6 M) indicating that oxytocin released ANP that directly slowed the heart, probably by release of cyclic GMP. The results indicate that oxytocin receptors mediate the action of oxytocin to release ANP, which slows the heart and reduces its force of contraction to produce a rapid reduction in circulating blood volume.

Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth hormone

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: A serial electron-microscopic study

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.

Excitatory versus inhibitory GABA as a divergence point in steroid-mediated sexual differentiation of the brain

Whereas adult sex differences in brain morphology and behavior result from developmental exposure to steroid hormones, the mechanism by which steroids differentiate the brain is unknown. Studies to date have described subtle sex differences in levels of proteins and neurotransmitters during brain development, but these have lacked explanatory power for the profound sex differences induced by steroids. We report here a major divergence in the response to injection of the γ-aminobutyric acid type A (GABAA) agonist, muscimol, in newborn male and female rats. In females, muscimol treatment primarily decreased the phosphorylation of cAMP response element binding protein (CREB) within the hypothalamus and the CA1 region of the hippocampus. In contrast, muscimol increased the phosphorylation of CREB in males within these same brain regions. Within the arcuate nucleus, muscimol treatment increased the phosphorylation of CREB in both females and males. Thus, the response to GABA can be excitatory or inhibitory on signal-transduction pathways that alter CREB phosphorylation depending on the sex and the region in developing brain. This divergence in response to GABA allows for a previously unknown form of steroid-mediated neuronal plasticity and may be an initial step in establishing sexually dimorphic signal-transduction pathways in developing brain.

Estrogen and thyroid hormone interaction on regulation of gene expression.

Estrogen receptor (ER) and thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that can bind to an identical half-site, AGGTCA, of their cognate hormone response elements. By in vitro transfection analysis in CV-1 cells, we show that estrogen induction of chloramphenicol acetyltransferase (CAT) activity in a construct containing a CAT reporter gene under the control of a minimal thymidine kinase (tk) promoter and a copy of the consensus ER response element was attenuated by cotransfection of TR alpha 1 plus triiodothyronine treatment. This inhibitory effect of TR was ligand-dependent and isoform-specific. Neither TR beta 1 nor TR beta 2 cotransfection inhibited estrogen-induced CAT activity, although both TR alpha and TR beta can bind to a consensus ER response element. Furthermore, cotransfection of a mutated TR alpha 1 that lacks binding to the AGGTCA sequence also inhibited the estrogen effect. Thus, the repression of estrogen action by liganded TR alpha 1 may involve protein-protein interactions although competition of ER and TR at the DNA level cannot be excluded. A similar inhibitory effect of liganded TR alpha 1 on estrogen induction of CAT activity was observed in a construct containing the preproenkephalin (PPE) promoter. A study in hypophysectomized female rats demonstrated that the estrogen-induced increase in PPE mRNA levels in the ventromedial hypothalamus was diminished by coadministration of triiodothyronine. These results suggest that ER and TR may interact to modulate estrogen-sensitive gene expression, such as for PPE, in the hypothalamus.

Mixed synapses discovered and mapped throughout mammalian spinal cord.

Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system.

A simple test of the vicarious trial-and-error hypothesis of hippocampal function.

Vicarious trial-and-error (VTE) is a term that Muenzinger and Tolman used to describe the rat's conflict-like behavior before responding to choice. Recently, VTE was proposed as a mechanism alternative to the concept of "cognitive map" in accounts of hippocampal function. That is, many phenomena of impaired learning and memory related to hippocampal interventions may be explained by behavioral first principles: reduced conflicting, incipient, pre-choice tendencies to approach and avoid. The nonspatial black-white discrimination learning and VTE behavior of the rat were investigated. Hippocampal-lesioned and sham-lesioned animals were trained for 25 days (20 trials per day) starting at 60 days of age. Each movement of the head from one discriminative stimulus to the other was counted as a VTE instance. Lesioned rats had fewer VTEs than sham controls, and the former learned much more slowly or never learned. After learning, VTE frequency declined. Male and female rats showed no significant differences in VTE behavior or discrimination learning.

Influência da deficiência ou suplementação com selênio durante o período gestacional de ratas na suscetibilidade da progênie feminina à carcinogênese mamária

Fatores dietéticos como o selênio (Se) são apontados como importantes moduladores do risco de desenvolvimento do câncer de mama. Essa neoplasia pode apresentar sua origem no início do desenvolvimento e, assim, a alimentação materna poderia ter importantes repercussões na programação fetal da doença. A fim de verificar se diferentes concentração de selênio na dieta materna poderiam programar o risco da progênie feminina ao câncer de mama, ratas foram alimentadas com ração contendo 0,15 (CO), 1,0 (SUP) ou 0,05 (DEF) ppm de Se durante a gestação e sua progênie feminina iniciada com DMBA. A progênie do grupo SUP apresentou menor suscetibilidade à carcinogênese, indicado pelo menor número médio e multiplicidade de adenocarcinomas mamários (p< 0,05), enquanto a do grupo DEF apresentou maior suscetibilidade à carcinogênese, indicado pela maior incidência dos mesmos (p< 0,05). Mães do grupo DEF apresentaram menor concentração de Se no sangue (p< 0,05) e sua prole apresentou menor atividade da enzima GPx1 (p< 0,05). Além disso, observou-se na glândula mamária da progênie de 50 dias menor expressão (western blot e qPCR) de ERα, Her-2, EGFR e Ras no grupo SUP em comparação aos grupos CO e DEF (p< 0,05). Analisou-se, ainda, o padrão de metilação global do DNA (HPLC-DAD), expressão das enzimas DNMT1, 3a e 3b (qPCR), o padrão global de modificações pós traducionais em histonas (western blot) e o padrão de metilação da região promotora do gene Erα (modificação com bissulfito e pirossequenciamento) na glândula mamária da progênie de 50 dias. Não houve diferença no padrão de metilação global do DNA e expressão das enzimas DNMTs (p>0,05). Houve aumento na expressão de H4K16 acetilada nos grupos SUP e DEF (p< 0,05). Finalmente, em comparação a progênie do grupo DEF, a do grupo SUP apresentou região promotora de Erα com aumento marginal (p=0,07) na metilação de dois dinucleotídeos CpG. Conclui-se que o consumo de diferentes concentrações de Se na dieta materna tem impacto sobre a suscetibilidade da progênie ao câncer de mama na vida adulta através da modulação da expressão de receptores e oncogenes relacionados ao desenvolvimeto dessa neoplasia, além da influência em processos epigenéticos. Tais resultados apontam para a existência de uma \"janela de programação\" no início do desenvolvimento sensível a ação do Se, resultando em diminuição do risco de câncer de mama quando suplementado na dieta materna e o inverso quando de sua deficiencia.

Influência do LPS e do zinco na interação mãe e filhote

O comportamento materno consiste em um conjunto de mudanças comportamentais e fisiológicas, exercidas pelos indivíduos adultos em torno dos indivíduos reprodutivamente imaturos, garantindo sua sobrevivência e a propagação de sua espécie. A interação mãe e filhote é tida tipicamente como simbiótica. Os filhotes quando separados da mãe sinalizam para serem recolhidos através de dicas olfativas, visuais e da vocalização que representa uma forma de comunicação filhote e mãe. O modelo de febre clássico e amplamente empregado envolve a utilização do lipopolissacarídeo (LPS), principal componente da parede celular de bactérias Gram-Negativas. Além da febre, as infecções apresentam uma cadeia de respostas não especificas do hospedeiro que se sabe estarem envolvidos em muitas das funções vitais, incluindo a resposta imune estas incluem a hipozinquemia. Sendo assim, fêmeas virgens, gestantes e lactantes receberam LPS (100 µg/kg, i.p.) e foram tratadas com zinco (2 mg/kg, s.c.) O peso corporal, consumo de água, ração, e a temperatura corporal foram medidas por noventa e seis horas, duas horas após a administração do LPS. No quinto dia de lactação foram observados o comportamento maternal, a atividade geral em campo aberto e a vocalização ultrassônica nos filhotes. No dia do desmame os filhotes dessas fêmeas receberam um desafio com LPS (50 µg/kg, i.p.) e duas horas após a administração, foram observados a atividade geral em campo aberto, e o burst e fagocitose de neutrófilos. Observamos que: 1) Em ratas virgens, gestantes e lactantes, a exposição ao LPS e o tratamento com zinco modificou de forma específica a temperatura e peso corporal, consumo de água e ração e a atividade geral observadas em campo aberto; 2) No período de lactação, houve redução da latência para busca do primeiro filhote. Na prole das fêmeas lactantes verificou-se que: 3) Houve alteração no padrão de vocalização dos filhotes; 4) houve alteração na atividade geral observada em campo aberto e no burst e fagocitose de neutrófilos no vigésimo primeiro dia pós natal, após um desafio com a endotoxina, Assim, os resultados indicam que a administração de LPS e o tratamento com zinco têm seus efeitos modulados conforme o estágio fisiológico em que a fêmea se encontra, e interfere com a interação mãe/filhote, resultando em efeitos de curto e longo prazo sobre o comportamento dos filhotes. A partir deste trabalho, a possibilidade da exposição de mães à endotoxina bacteriana e da modulação de seus efeitos pelo zinco programar as respostas inflamatórias dos filhos torna-se factível

Efeitos da hiperprolactinemia sobre a inflamação alérgica pulmonar em ratos Wistar

Objetivo: Foi investigada a hipótese da hiperprolactinemia modular a resposta inflamatória alérgica pulmonar em ratos machos e em fêmas lactantes sem tratamento de domperidona. Métodos: Em ratos machos, a hiperprolactinemia foi de curta duração (5 dias) induzida pela domperidona (5,1 mg.kg-1 por dia, i.p). A resposta alérgica foi gerada por sensibilização e desafios inalatórios com ovoalbumina. Foi feita contagem de leucócitos totais e diferenciados do lavado bronco alveolar (BAL), lavado medular femoral (BFL) e sangue; a percentagem de produção de muco e colageno no pulmão, níveis de corticosterona e prolactina e citocinas TNF-α, IL-4, IL-6, IL-10, em explantes de pulmão e IFNg no BAL, foram medidos. Pela citometria foram avaliadaos os receptores de prolactina; Resultados: Hiperprolactinemia de curta duração feita antes do desafio inalatório disminuiu a resposta alérgica pulmonar na contagem de leucócitos no lavado broncoalveolar. Esse tratamento reduziu a celularidade no BFL e a percentagem de muco e aumentou a expressão de citocinas IL-4, IL-6, IL-10, TNFα e da expressão do IFNg. Níveis altos de prolactina diminuiram o número de eosinófilos ao pulmão no BAL. Pela citometria revelou-se que além de ter menor número de granulócitos migrados ao pulmão, estes apresentaram maior expressão do número de receptores por granulócito para prolactina no grupo tratado com domperidona. Alterações similares foram reveladas em fêmeas lactantes como foi a diminuição nos leucócitos do BAL, e no número de células do BFL. O tratamento profilático diminuiu a resposta alérgica tanto no grupo hiperprolactinêmico como no grupo veículo. O tratamento feito após o desafio inalatório não evidenciou alterações relevantes nas variáveis medidas. Conclusões: A hiperprolactinemia de curta duração, feita após a sensibilização e antes da inalação diminui a resposta inflamatória no pulmão em ratos. Os resultados deste estudo demonstram que a hiperprolactinemia induzida antes do desafio antigênico diminue a inflamação alérgica pulmonar. Assim, é provável que a prolactina endógena tenha um papel relevante como um imunomodulador da asma. Este estudo aponta a possibilidade futura do uso da domperidona para pacientes asmáticos. Durante a primavera muitas espécies de mamíferos têm seus filhotes e ocorre abundância de fatores alergenos no ar. Logo, um fator endógeno que favoreça a proteção de fêmeas durante a lactação, tal como a hiperprolactinemia, tem elevado valor adaptativo

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on express ion/activity of the main DDS phase-II- metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxiclation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.