José Antunes Rodrigues

Interaction between supraoptic nucleus and septal area in the control of water, sodium intake and arterial blood pressure induced by injection of angiotensin II.

We investigated the effects of injection into the supraoptic nucleus (SON) of losartanand PD 123319 (nonpeptide AT(1) and AT(2)-angiotensin II [ANG II] receptor antagonists, respectively); d(CH(2))(5)-Tyr(Me)-AVP (AVPA; an arginine-vasopressin [AVP] V(1) receptor antagonist), FK 409 (a nitric oxide [NO] donor), and N(W)-nitro-l-arginine methyl ester (l-NAME; an NO synthase inhibitor) on water intake, sodium chloride 3% (NaCl) intake and arterial blood pressure induced by injection of ANG II into the lateral septal area (LSA). Male Holtzman rats (250-300 g) were implanted with cannulae into SON and LSA unilaterally. The drugs were injected in 0.5 microl over 30-60 s. Controls were injected with a similar volume of 0.15 M NaCl. ANG II was injected at a dose of 10 pmol. ANG II antagonists and AVPA were injected at doses of 80 nmol. FK 409 and l-NAME were injected at doses of 20 and 40 microg, respectively. Water and NaCl intake was measured over a 2-h period. Prior administration of losartan into the SON decreased water and NaCl intake induced by injection of ANG II. While there was a decrease in water intake, ANG II-induced NaCl intake was significantly increased following injection of AVPA. FK 409 injection decreased water intake and sodium intake induced by ANG II. l-NAME alone increased water and sodium intake and induced a pressor effect. l-NAME-potentiated water and sodium intake induced by ANG II. PD 123319 produced no changes in water or sodium intake induced by ANG II. The prior administration of losartan or AVPA decreased mean arterial pressure (MAP) induced by ANG II. PD 123319 decreased the pressor effect of ANG II to a lesser degree than losartan. FK 409 decreased the pressor effect of ANG II while l-NAME potentiated it. These results suggest that both ANG II AT(1) and AVP V(1) receptors and NO within the SON may be involved in water intake, NaCl intake and the pressor response were induced by activation of ANG II receptors within the LSA. These results do not support the involvement of LSA AT(2) receptors in the mediation of water and NaCl intake responses induced by ANG II, but influence the pressor response.

Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus

BackgroundArginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression.ResultsThe effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress.ConclusionHere we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.

The Increase in Signaling by Kisspeptin Neurons in the Preoptic Area and Associated Changes in Clock Gene Expression That Trigger the LH Surge in Female Rats Are Dependent on the Facilitatory Action of a Noradrenaline Input

In rodents, kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) of the preoptic area are considered to provide a major stimulatory input to the GnRH neuronal network that is responsible for triggering the preovulatory LH surge. Noradrenaline (NA) is one of the main modulators of GnRH release, and NA fibers are found in close apposition to kisspeptin neurons in the RP3V. Our objective was to interrogate the role of NA signaling in the kisspeptin control of GnRH secretion during the estradiol induced LH surge in ovariectomized rats, using prazosin, an α1-adrenergic receptor antagonist. In control rats, the estradiol-induced LH surge at 17 hours was associated with a significant increase in GnRH and kisspeptin content in the median eminence with the increase in kisspeptin preceding that of GnRH and LH. Prazosin, administered 5 and 3 hours prior to the predicted time of the LH surge truncated the LH surge and abolished the rise in GnRH and kisspeptin in the median eminence. In the preoptic area, prazosin blocked the increases in Kiss1 gene expression and kisspeptin content in association with a disruption in the expression of the clock genes, Per1 and Bmal1. Together these findings demonstrate for the first time that NA modulates kisspeptin synthesis in the RP3V through the activation of α1-adrenergic receptors prior to the initiation of the LH surge and indicate a potential role of α1-adrenergic signaling in the circadian-controlled pathway timing of the preovulatory LH surge.

Neurohypophyseal response to fluid resuscitation with hypertonic saline during septic shock in rats

•  What is the central question of this study? Small‐volume resuscitation with hypertonic saline (HS) has been proposed to restore physiological haemodynamics during haemorrhagic and endotoxic shock. Hypertonic solutions rapidly increase intravascular volume by generating an osmotic gradient that pulls water from the intracellular and interstitial space into the intravascular compartment. Infusion of HS increases plasma sodium levels and osmolality, which may stimulate hormone release from the neurohypophysis. In the present study, we sought to investigate the effects of HS administration during septic shock caused by caecal ligation and perforation. •  What is the main finding and its importance? The present study demonstrated that HS infusion into rats subjected to caecal ligation and perforation induced neurohypophyseal hormone secretion, as well as a transient increase in blood pressure that was mediated by the V1 receptor.

Reduced collagen accumulation and augmented MMP-2 activity in left ventricle of old rats submitted to high-intensity resistance training.

Progressive fibrosis is a hallmark of the aging heart. Age-related fibrosis is modulated by endurance exercise training; however, little is known concerning the influence of resistance training (RT). Therefore we investigated the chronic effects of high-intensity RT on age-associated alterations of left ventricle (LV) structure, collagen content, matrix metalloproteinase-2 (MMP-2), and extracellular matrix-related gene expression, including transforming growth factor-β (TGF-β). Young adult (3 mo) and aged (21 mo) male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), three times a week for 12 wk. Forty-eight hours posttraining, arterial systolic and diastolic pressure, LV end-diastolic pressure (LVEDP) and dP/dt were recorded. LV morphology, collagen deposition, and gene expression of type I (COL-I) and type III (COL-III) collagen, MMP-2, tissue inhibitor of metalloproteinases-1 (TIMP-1), and TGF-β1 were analyzed by quantitative reverse transcriptase-PCR. MMP-2 content was assessed by zymography. Increased collagen deposition was observed in LV from aged rats. These parameters were modulated by RT and were associated with increased MMP-2 activity and decreased COL-I, TGF-β1, and TIMP-1 mRNA content. Despite the effect of RT on collagen accumulation, there was no improvement on LVEDP and maximal negative LV dP/dt of aged rats. Cardiomyocyte diameter was preserved in all experimental conditions. In conclusion, RT attenuated age-associated collagen accumulation, concomitant to the increase in MMP-2 activity and decreased expression of COL-I, TGF-β1, and TIMP-1 in LV, illustrating a cardioprotective effect of RT on ventricular structure and function.NEW & NOTEWORTHY We demonstrated the beneficial resistance-training effect against age-related left ventricle collagen accumulation in the left ventricle, which was associated with decreased type I collagen (COL-I), transforming growth factor-β1 (TGF-β1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) gene expression and matrix metalloproteinase-2 (MMP-2) activity. Our findings suggest for the first time the potential effects of resistance training in modulating collagen accumulation and possibly fibrosis in the aging heart.

Nitric Oxide Modulates HCN Channels in Magnocellular Neurons of the Supraoptic Nucleus of Rats by an S-Nitrosylation-Dependent Mechanism

The control of the excitability in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus has been attributed mainly to synaptic inputs from circunventricular organs. However, nitric oxide (NO), a gaseous messenger produced in this nucleus during isotonic and short-term hypertonic conditions, is an example of a modulator that can act directly on MNCs to modulate their firing rate. NO inhibits the electrical excitability of MNCs, leading to a decrease in the release of vasopressin and oxytocin. Although the effects of NO on MNCs are well established, the mechanism by which this gas produces its effect is, so far, unknown. Because NO acts independently of synaptic inputs, we hypothesized that ion channels present in MNCs are the targets of NO. To investigate this hypothesis, we used the patch-clamp technique in vitro and in situ to measure currents carried by hyperpolarization-activated and nucleotide-gated cation (HCN) channels and establish their role in determining the electrical excitability of MNCs in rats. Our results show that blockade of HCN channels by ZD7288 decreases MNC firing rate with significant consequences on the release of OT and VP, measured by radioimmunoassay. NO induced a significant reduction in HCN currents by binding to cysteine residues and forming S-nitrosothiol complexes. These findings shed new light on the mechanisms that control the electrical excitability of MNCs via the nitrergic system and strengthen the importance of HCN channels in the control of hydroelectrolyte homeostasis. SIGNIFICANCE STATEMENT Cells in our organism live in a liquid environment whose composition and osmolality are maintained within tight limits. Magnocellular neurons (MNCs) of the supra optic nucleus can sense osmolality and control the synthesis and secretion of vasopressin (VP) and oxytocin (OT) by the neurohypophysis. OT and VP act on the kidneys controlling the excretion of water and sodium to maintain homeostasis. Here we combined electrophysiology, molecular biology, and radioimmunoassay to show that the electrical activity of MNCs can be controlled by nitric oxide (NO), a gaseous messenger. NO reacts with cysteine residues (S-nitrosylation) on hyperpolarization-activated and nucleotide-gated cation channels decreasing the firing rate of MNCs and the consequent secretion of VP and OT.