Oxytocinergic and serotonergic systems involvement in sodium intake regulation: satiety or hypertonicity markers?

Previous studies demonstrated the inhibitory participation of serotonergic ( 5-HT) and oxytocinergic (OT) neurons on sodium appetite induced by peritoneal dialysis (PD) in rats. The activity of 5-HT neurons increases after PD- induced 2% NaCl intake and decreases after sodium depletion; however, the activity of the OT neurons appears only after PD-induced 2% NaCl intake. To discriminate whether the differential activations of the 5-HT and OT neurons in this model are a consequence of the sodium satiation process or are the result of stimulation caused by the entry to the body of a hypertonic sodium solution during sodium access, we analyzed the number of Fos-5-HT- and Fos-OT-immunoreactive neurons in the dorsal raphe nucleus and the paraventricular nucleus of the hypothalamus-supraoptic nucleus, respectively, after isotonic vs. hypertonic NaCl intake induced by PD. We also studied the OT plasma levels after PD- induced isotonic or hypertonic NaCl intake. Sodium intake induced by PD significantly increased the number of Fos-5- HT cells, independently of the concentration of NaCl consumed. In contrast, the number of Fos-OT neurons increased after hypertonic NaCl intake, in both depleted and nondepleted animals. The OT plasma levels significantly increased only in the PD- induced 2% NaCl intake group in relation to others, showing a synergic effect of both factors. In summary, 5-HT neurons were activated after body sodium status was reestablished, suggesting that this system is activated under conditions of satiety. In terms of the OT system, both OT neural activity and OT plasma levels were increased by the entry of hypertonic NaCl solution during sodium consumption, suggesting that this system is involved in the processing of hyperosmotic signals.

Endothelial and neuronal nitric oxide synthase inhibitors influences angiotensin II pressor effect in central nervous system

The present study investigated the central role of angiotensin II and nitric oxide on arterial blood pressure (MAP) in rats. Losartan and PD123349 AT 1 and AT 2 (selective no peptides antagonists angiotensin receptors), as well as FK 409 (a nitric oxide donor), N W-nitro-L-arginine methyl ester (L-NAME) a constituve nitric oxide synthase inhibitor endothelial (eNOSI) and 7-nitroindazol (7NI) a specific neuronal nitric oxide synthase inhibitor (nNOSI) were used. Holtzman strain, (Rattus norvergicus) weighting 200-250 g were anesthetized with zoletil 50 mg kg -1 (tiletamine chloridrate 125 mg and zolazepan chloridrate 125 mg) into quadriceps muscle anda stainless steel cannula was stereotaxically implanted into their Lateral Ventricle (LV). Controls were injected with a 0.5 μl volume of 0.15 M NaCl. Angiotensin II injected into LV increased MAP (19±3 vs. control 3±1 mm Hg), which is potentiated by prior injection of L-NAME in the same site 26±2 mm Hg. 7NI injected prior to ANG II into LV also potentiated the pressor effect of ANG II but with a higher intensity than L-NAME 32±3 mm Hg. FK 409 inhibited the pressor effect of ANG II (6±1 mm Hg). Losartan injected into LV before ANG II influences the pressor effect of ANG II (8±1 mm Hg). The PD 123319 decreased the pressor effects of ANG II (16±1 mm Hg). Losartan injected simultaneously with FK 409 blocked the pressor effect of ANG II (3±1 mm Hg). L-NAME produced an increase in the pressor effect of ANG II, may be due to local vasoconstriction and all at once by neuronal NOS inhibition but the main effect is of the 7-NIT an specific nNOS inhibitor. The AT 1 antagonist receptors improve basal nitric oxide (NO) production and release. These data suggest the involvement of constitutive and neuronal NOS in the control of arterial blood pressure induced by ANG II centrally, evolving AT 1 receptor-mediated vasoconstriction and AT 2 receptor-mediated vasodilatation. These results were confirmed by the experiment using FK 409. © 2006 Asian Network for Scientific Information.

Effects of nitric oxide and arginine vasopressin on sodium intake induced by central angiotensin II. Part 2

We study the effects of angiotensin receptors antagonists, arginine vasopressin receptor antagonist, L-arginine and L-NAME, injected into supraoptic nucleus of the hypothalamus (SON) on sodium intake induced by the injection of angiotensin II (ANGII). Holtzman rats weighing 200-250 g with canulae implanted into the SON were used. The drugs were injected in 0.5 μL over 30-60 sec. Sodium intake after injection of saline SAL+SAL 0.15 M NaCl was 0.10±00.1 mL 2 h -1; SAL+ANGII injected into SON increased sodium intake. Losartan injected prior to ANGII into SON decreased sodium intake induced by ANGII. PD123319 injected prior to ANGII produced no changes in sodium intake induced by ANGII. AVPA receptor V 1 antagonist injected prior to ANGII reduced sodium intake with a less intensity than losartan. L-arginine injected prior to ANGII decreases sodium intake at a same intensity than losartan. L-NAME injected prior to ANGII potentiated sodium intake induced by ANGII. Losartan injected simultaneously with L-arginine prior to ANGII blocked the natriorexigenic effect of ANGII. These results confirm the importance of SON in the control of sodium intake. Also suggest that both AT 1 and arginine vasopressin V 1 receptors interact with nitrergic pathways within the SON influencing the sodium metabolism by changing sodium appetite induced by ANGII. © 2007 Asian Network for Scientific Information.

Effects of nitric oxide and arginine vasopressin on water intake induced by central angiotensin II. Part 1

We determined the effects of AT 1 and AT 2 (selective no peptides antagonists angiotensin receptors), arginine vasopressin V 1 receptor antagonist as well as L-arginine, a nitric oxide donor and N W-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, injected into supraoptic nucleus (SON) on water and sodium intake induced by the injection of angiotensin II (ANGII). Male Holtzman rats weighing 200-250 g with canulae implanted into the SON were used. The drugs were injected in 0.5 μL over 30-60 sec. The water intake after injection of saline SAL+SAL 0.15 M NaCl was 0.40±0.1 mL 2 h -1; SAL+ANGII increase water intake. Losartan decreased the water intake induced by ANGII. PD123319 injected prior to produce no change in water intake induced by ANGII. AVPA prior to ANGII reduced the water intake with a less intensity than losartan. L-arginine prior to ANGII decreases the water intake at a same intensity than losartan. L-NAME prior to ANGII potentiated the dipsogenic effect of ANGII. Losartan injected simultaneously with L-arginine prior to ANGII blocked the dipsogenic effect of ANGII. These results confirm the importance of SON in the control of water intake and strongly suggest that AT 1, V 1 receptors interact with nitrergic pathways within the SON influencing the dipsogenic effect of ANGII.

Efeitos da expansão de volume extracelular sobre o conteúdo de catecolaminas e a ativação neuronal de estruturas do hipotálamo e do tronco cerebral de ratos

A regulação fina do volume e osmolaridade dos líquidos corporais é fundamental para a sobrevivência. Qualquer variação na composição do meio interno ativa mecanismos comportamentais, neurais e hormonais compensatórios que controlam a ingestão e excreção de água e eletrólitos a fim de manter a homeostase hidroeletrolítica. Alterações na faixa de 1-2% na osmolaridade sanguínea estimulam a liberação de arginina vasopressina (AVP) que resulta em antidiurese além de ocitocina (OT) e peptídeo natriurético atrial (ANP) que promovem a natriurese. Trabalhos realizados em nosso laboratório utilizando o modelo experimental de expansão do volume extracelular (EVEC) mostraram ativação de neurônios magnocelulares ocitocinérgicos localizados no núcleo paraventricular (PVN) e núcleo supra-óptico (SON) responsáveis pela secreção de OT e AVP, igualmente alteradas em resposta a este estímulo. A participação do sistema nervoso simpático nestas condições tem sido levantada. Projeções medulares e tronco-encefálicas (simpáticas) para o hipotálamo poderiam atuar de forma seletiva inibindo sinalizações para a ingestão e estimulando sinalizações para excreção de água e eletrólitos. O papel de vias noradrenérgicas tronco-encefálicas nesta regulação ainda precisa ser mais bem estabelecido. Assim sendo, objetivamos neste estudo esclarecer o papel do sistema nervoso simpático (via noradrenérgicas) na regulação das alterações induzidas pelo modelo de EVEC, analisando por cromatografia líquida de alta eficácia o conteúdo de noradrenalina (NA), adrenalina (AD) e serotonina (5-HT) em estruturas do tronco cerebral como núcleo do trato solitário (NTS), bulbo rostro-ventro lateral (RVLM), locus coeruleus (LC) e núcleo dorsal da rafe (NDR) e estruturas hipotalâmicas como SON e PVN. Procuramos ainda, através de estudos imunocitoquímicos determinar alterações no padrão de ativação neuronal pela análise de Fos-TH ou Fos-5HT nas estruturas acima mencionadas em condições experimentais nas quais são induzidas alterações do volume do líquido extracelular.

The control of sodium chloride intake: Functional relationship between hypothalamic inhibitory areas and amygdaloid complex stimulating areas

Sodium chloride intake was studied in rats submitted to different neurosurgical procedures. Intake decreased in animals submitted to bilateral destruction of the basolateral amygdaloid complex, and increased after the same animals were submitted to destruction of the anterior lateral hypothalamus, a procedure which is known to cause increased intake in intact rats. In the reverse experiment, where the anterior lateral hypothalamus was destroyed before the basolateral amygdaloid complex, the effect of increased sodium chloride intake induced by destruction of the hypothalamus overcame the decreased expected upon destruction of the amygdaloid complex. These results permit us to conclude that the hypothalamic areas which inhibit sodium chloride intake predominate over the stimulating areas of the amygdaloid complex in the control of sodium chloride intake. © 1981 ANKHO International Inc.

Enhanced Expression of Heme Oxygenase-1 and Carbon Monoxide Excitatory Effects in Oxytocin and Vasopressin Neurones During Water Deprivation

A growing body of evidence indiates that carbon monoxide (CO) acts as a gas neurotransmitter within the central nervous system. Although CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying the actions of CO within the magnocellular neurosecretory system remain largely unknown. In the present study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as the actions of CO on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs), in euhydrated (EU) and 48-h water-deprived rats (48WD). Our results show the expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalisation with OT and VP neurones within the SON and PVN, was significantly enhanced in 48WD rats. Inhibition of HO activity with chromium mesoporphyrin IX chloride (CrMP; 20 mu m) resulted in a slight membrane hyperpolarisation in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarisation, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found that the expression and actions of CO were enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.

Effect of the interaction between food state and the action of estrogen on oxytocinergic system activity

Increased plasma osmolality by food intake evokes augmentation of plasma oxytocin (OT). Ovarian steroids may also influence the balance of body fluids by acting on OT neurones. Our aim was to determine if estrogen influences the activity of OT neurones in paraventricular nucleus (PVN) and supraoptic nucleus (SON) under different osmotic situations. Ovariectomized rats (OVX) were treated with either estradiol (E-2) or vehicle and were divided into three groups: group I was fed ad libitum, group II underwent 48 h of fasting, and group III was refed after 48 h of fasting. On the day of the experiment, blood samples were collected to determine the plasma osmolality and OT. The animals were subsequently perfused, and OT/FOS immunofluorescence analysis was conducted on neurones in the PVN and the SON. When compared to animals which were fasted or fed ad libitum, the plasma osmolality of refed animals was higher, regardless of whether they were treated with vehicle or E-2. We observed neural activation of OT cells in vehicle-or E-2-treated OVX rats refed after 48 h of fasting, but not in animals fed ad libitum or in animals that only underwent 48 h of fasting. Finally, the percentage of neurones that co-expressed OT and FOS was lower in both the PVN and the SON of animals treated with E-2 and refed, when compared to vehicle-treated animals. These results suggest that E-2 may have an inhibitory effect on OT neurones and may modulate the secretion of OT in response to the increase of osmolality induced by refeeding. Journal of Endocrinology (2012) 212, 129-138

Availability of a rich source of sodium during the perinatal period programs the fluid balance restoration pattern in adult offspring

Osmoregulatory mechanisms can be vulnerable to electrolyte and/or endocrine environmental changes during the perinatal period, differentially programming the developing offspring and affecting them even in adulthood. The aim of this study was to evaluate whether availability of hypertonic sodium solution during the perinatal period may induce a differential programming in adult offspring osmoregulatory mechanisms. With this aim, we studied water and sodium intake after Furosemide-sodium depletion in adult offspring exposed to hypertonic sodium solution from 1 week before mating until postnatal day 28 of the offspring, used as a perinatal manipulation model [PM-Na group]. In these animals, we also identified the cell population groups in brain nuclei activated by Furosemide-sodium depletion treatment, analyzing the spatial patterns of Fos and Fos-vasopressin immunoreactivity. In sodium depleted rats, sodium and water intake were significantly lower in the PM-Na group vs. animals without access to hypertonic sodium solution [PM-Ctrol group]. Interestingly, when comparing the volumes consumed of both solutions in each PM group, our data show the expected significant differences between both solutions ingested in the PM-Ctrol group, which makes an isotonic cocktail: however, in the PM-Na group there were no significant differences in the volumes of both solutions consumed after Furosemide-sodium depletion, and therefore the sodium concentration of total fluid ingested by this group was significantly higher than that in the PM-Ctrol group. With regard to brain Fos immunoreactivity, we observed that Furosemide-sodium depletion in the PM-Na group induced a higher number of activated cells in the subfornical organ, ventral subdivision of the paraventricular nucleus and vasopressinergic neurons of the supraoptic nucleus than in the PM-Ctrol animals. Moreover, along the brainstem, we found a decreased number of sodium depletion-activated cells within the nucleus of the solitary tract of the PM-Na group. Our data indicate that early sodium availability induces a long-term effect on fluid drinking and on the cell activity of brain nuclei involved in the control of hydromineral balance. These results also suggest that availability of a rich source of sodium during the perinatal period may provoke a larger anticipatory response in the offspring, activating the vasopressinergic system and reducing thirst after water and sodium depletion, as a result of central osmosensitive mechanism alterations. (C) 2011 Elsevier Inc. All rights reserved.

Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase.

Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. We recently identified a brain-specific protein that associates with huntingtin and is designated huntingtin-associated protein (HAP1). We now describe selective neuronal localizations of HAP1. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy.

Sampling strategies for characterization of neuropeptides using mass spectrometry and functional implications into cell-cell signaling

In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies described here use different extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectrometric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, including 20 unique peptides from known prohormones. This study includes mass spectrometric analysis even from individually isolated magnocellular neuroendocrine cells, where vasopressin and several other peptides are detected. At the same time, it was shown that the same approach could be applied to analyze peptides isolated from a similar hypothalamic region, the suprachiasmatic nucleus (SCN). Although there were some overlaps regarding the detection of the peptides in the two brain nuclei, different peptides were detected specific to each nucleus. Among other peptides, provasopressin fragments were specifically detected in the SON while angiotensin I, somatostatin-14, neurokinin B, galanin, and vasoactive-intestinal peptide (VIP) were detected in the SCN only. Lists of peptides were generated from both brain regions for comparison of the peptidome of SON and SCN nuclei. Moving from analysis of magnocellular neurons in tissue to cell culture, the direct peptidomics of the magnocellular and hippocampal neurons led to the detection of 10 peaks that were assigned to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enabled the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides. The peptides with roles in the development of the pituitary were analyzed using transgenic mice. Hes1 KO is a genetically modified mouse that lives only e18.5 (embryonic days). Anterior pituitaries of Hes1 null mice exhibit hypoplasia due to increased cell death and reduced proliferation and in the intermediate lobe, the cells differentiate abnormally into somatotropes instead of melanotropes. These previous findings demonstrate that Hes1 has multiple roles in pituitary development, cell differentiation, and cell fate. AVP was detected in all samples. Interestingly, somatostatin [92-100] and provasopressin [151-168] were detected in the mutant but not in the wild type or heterozygous pituitaries while somatostatin-14 was detected only in the heterozygous pituitary. In addition, the putative peptide corresponding to m/z 1330.2 and POMC [205-222] are detected in the mutant and heterozygous pituitaries, but not in the wild type. These results indicate that Hes1 influences the processing of different prohormones having possible roles during development and opens new directions for further developmental studies. This research demonstrates the robust capabilities of MS, which ensures the unbiased direct analysis of peptides extracted from complex biological systems and allows addressing important questions to understand cell-cell signaling in the brain.

Hypothalamic supraoptic but not paraventricular nucleus is involved in cardiovascular responses to carbachol microinjected into the bed nucleus of stria terminalis of unanesthetized rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência do FK-506 sobre a expressão de RANKL e OPG na doença periodontal induzida: estudo in vivo e in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Catecholaminergic neurons in the comissural region of the nucleus of the solitary tract modulate hyperosmolality-induced responses

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sodium intake, brain c-Fos protein and gastric emptying in cell-dehydrated rats treated with methysergide into the lateral parabrachial nucleus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)