André S. Mecawi

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

The respiratory pattern generator modulates the sympathetic outflow, the strength of which is enhanced by challenges produced by hypoxia. This coupling is due to the respiratory-modulated presympathetic neurons in the rostral ventrolateral medulla (RVLM), but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the neural substrates responsible for generation of this respiratory-sympathetic coupling, we combined immunofluorescence, single cell qRT-pCR, and electrophysiological recordings of the RVLM presympathetic neurons in in situ preparations from normal rats and rats submitted to a metabolic challenge produced by chronic intermittent hypoxia (CIH). Our results show that the spinally projected cathecholaminergic C1 and non-C1 respiratory-modulated RVLM presympathetic neurons constitute a heterogeneous neuronal population regarding the intrinsic electrophysiological properties, respiratory synaptic inputs, and expression of ionic currents, albeit all neurons presented persistent sodium current-dependent intrinsic pacemaker properties after synaptic blockade. A specific subpopulation of non-C1 respiratory-modulated RVLM presympathetic neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH. This phenomenon may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory-modulated RVLM presympathetic neurons contribute to the central generation of respiratory-sympathetic coupling as part of a complex neuronal network, which in response to the challenges produced by CIH contribute to respiratory-related increase in the sympathetic activity.

Effects of oversulfated and fucosylated chondroitin sulfates on coagulation. Challenges for the study of anticoagulant polysaccharides.

We report the effects of a chemically oversulfated chondroitin sulfate and a naturally fucosylated chondroitin sulfate on the coagulation system. The former has been recently identified as a contaminant of heparin preparations and the latter has been proposed as an alternative anticoagulant. The mechanism of action of these polymers on coagulation is complex and target different components of the coagulation system. They have serpin-independent anticoagulant activity, which preponderates in plasma. They also have serpin-dependent anticoagulant activity but differ significantly in the target coagulation protease and preferential serpin. Their anticoagulant effects differ even more markedly when tested as inhibitors of coagulation proteases using plasma as a source of serpins. It is possible that the difference is due to the high availability of fucosylated chondroitin sulfate whereas oversulfated chondroitin sulfate has strong unspecific binding to plasma protein and low availability for the binding to serpins. When tested using a venous thrombosis experimental model, oversulfated chondroitin sulfate is less potent as an antithrombotic agent than fucosylated chondroitin sulfate. These highly sulfated chondroitin sulfates activate factor XII in in vitro assays, based on kallikrein release. However, only fucosylated chondroitin sulfate induces hypotension when intravenously injected into rats. In conclusion, the complexity of the regulatory mechanisms involved in the action of highly sulfated polysaccharides in coagulation requires their analysis by a combination of in vitro and in vivo assays. Our results are relevant due to the urgent need for new anticoagulant drugs or alternative sources of heparin.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT₁ receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT₁-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage.

Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress

OBJECTIVES We hypothesized that chronic ethanol intake enhances vascular oxidative stress and induces hypertension through renin-angiotensin system (RAS) activation. METHODS AND RESULTS Male Wistar rats were treated with ethanol (20% v/v). The increase in blood pressure induced by ethanol was prevented by losartan (10mg/kg/day; p.o. gavage), a selective AT1 receptor antagonist. Chronic ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels and serum aldosterone levels. No differences on plasma osmolality and sodium or potassium levels were detected after treatment with ethanol. Ethanol consumption did not alter ACE activity, as well as the levels of ANG I and ANG II in the rat aorta or mesenteric arterial bed (MAB). Ethanol induced systemic and vascular oxidative stress (aorta and MAB) and these effects were prevented by losartan. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was prevented by losartan. Ethanol intake did not alter protein expression of ACE, AT1 or AT2 receptors in both aorta and MAB. Aortas from ethanol-treated rats displayed decreased ERK1/2 phosphorylation and increased protein expression of SAPK/JNK. These responses were prevented by losartan. MAB from ethanol-treated rats displayed reduced phosphorylation of p38MAPK and ERK1/2 and losartan did not prevent these responses. CONCLUSIONS Our study provides novel evidence that chronic ethanol intake increases blood pressure, induces vascular oxidative stress and decreases nitric oxide (NO) bioavailability through AT1-dependent mechanisms.

Oestrogenic influence on brain AT1 receptor signalling on the thirst and sodium appetite in osmotically stimulated and sodium‐depleted female rats

The present work was carried out to investigate the role of angiotensin II type 1 (AT1) receptors in nocturnal thirst and sodium appetite induced by classical models of osmotic and sodium depletion challenges in ovariectomized rats chronically treated with oil or oestradiol benzoate (EB, 20 μg per animal, s.c. daily). In both conditions, the animals were given saline or losartan (108 nmol per animal, i.c.v.), a selective AT1 receptor blocker. Oestrogen therapy significantly reduced the water intake induced by water deprivation, sodium depletion produced by frusemide injected 24 h before, and s.c. acute frusemide plus captopril injection (FUROCAP protocol), with no alteration following s.c. hypertonic saline injection. In contrast, EB therapy decreased the salt intake induced by sodium depletion and FUROCAP protocols, with no alteration following water deprivation and s.c. hypertonic saline injection. Central AT1 blockade inhibited the dipsogenic response induced by water deprivation, osmotic stimulation, chronic sodium depletion and FUROCAP protocols and inhibited the natriorexigenic response induced by sodium depletion in ovariectomized rats. Oestrogen therapy significantly attenuated the losartan‐induced antidipsogenic and antinatriorexigenic actions following sodium depletion and FUROCAP protocols. These results indicate that ovariectomized rats express increased AT1 receptor signalling related to thirst and sodium appetite responses. Oestrogen therapy and brain AT1 receptor blockade weakened or markedly decreased the behavioural responses during the nocturnal period, a time at which brain angiotensinergic activity is expected to be more prominent. Finally, we demonstrated through different experimental protocols a clear‐cut influence of oestrogenic status on the behavioural AT1‐induced signalling response.

A comparison of physiological and transcriptome responses to water deprivation and salt loading in the rat supraoptic nucleus

Salt loading (SL) and water deprivation (WD) are experimental challenges that are often used to study the osmotic circuitry of the brain. Central to this circuit is the supraoptic nucleus (SON) of the hypothalamus, which is responsible for the biosynthesis of the hormones, arginine vasopressin (AVP) and oxytocin (OXT), and their transport to terminals that reside in the posterior lobe of the pituitary. On osmotic challenge evoked by a change in blood volume or osmolality, the SON undergoes a function-related plasticity that creates an environment that allows for an appropriate hormone response. Here, we have described the impact of SL and WD compared with euhydrated (EU) controls in terms of drinking and eating behavior, body weight, and recorded physiological data including circulating hormone data and plasma and urine osmolality. We have also used microarrays to profile the transcriptome of the SON following SL and remined data from the SON that describes the transcriptome response to WD. From a list of 2,783 commonly regulated transcripts, we selected 20 genes for validation by qPCR. All of the 9 genes that have already been described as expressed or regulated in the SON by osmotic stimuli were confirmed in our models. Of the 11 novel genes, 5 were successfully validated while 6 were false discoveries.

Acute restraint stress induces endothelial dysfunction: role of vasoconstrictor prostanoids and oxidative stress

Abstract We hypothesized that acute stress would induce endothelial dysfunction. Male Wistar rats were restrained for 2 h within wire mesh. Functional and biochemical analyses were conducted 24 h after the 2-h period of restraint. Stressed rats showed decreased exploration on the open arms of an elevated-plus maze (EPM) and increased plasma corticosterone concentration. Acute restraint stress did not alter systolic blood pressure, whereas it increased the in vitro contractile response to phenylephrine and serotonin in endothelium-intact rat aortas. NG-nitro-l-arginine methyl ester (l-NAME; nitric oxide synthase, NOS, inhibitor) did not alter the contraction induced by phenylephrine in aortic rings from stressed rats. Tiron, indomethacin and SQ29548 reversed the increase in the contractile response to phenylephrine induced by restraint stress. Increased systemic and vascular oxidative stress was evident in stressed rats. Restraint stress decreased plasma and vascular nitrate/nitrite (NOx) concentration and increased aortic expression of inducible (i) NOS, but not endothelial (e) NOS. Reduced expression of cyclooxygenase (COX)-1, but not COX-2, was observed in aortas from stressed rats. Restraint stress increased thromboxane (TX)B2 (stable TXA2 metabolite) concentration but did not affect prostaglandin (PG)F2α concentration in the aorta. Restraint reduced superoxide dismutase (SOD) activity, whereas concentrations of hydrogen peroxide (H2O2) and reduced glutathione (GSH) were not affected. The major new finding of our study is that restraint stress increases vascular contraction by an endothelium-dependent mechanism that involves increased oxidative stress and the generation of COX-derived vasoconstrictor prostanoids. Such stress-induced endothelial dysfunction could predispose to the development of cardiovascular diseases.

Ethanol withdrawal increases oxidative stress and reduces nitric oxide bioavailability in the vasculature of rats

We analyzed the effects of ethanol withdrawal on the vascular and systemic renin-angiotensin system (RAS) and vascular oxidative stress. Male Wistar rats were treated with ethanol 3-9% (v/v) for a period of 21 days. Ethanol withdrawal was induced by abrupt discontinuation of the treatment. Experiments were performed 48 h after ethanol discontinuation. Rats from the ethanol withdrawal group showed decreased exploration of the open arms of the elevated-plus maze (EPM) and increased plasma corticosterone levels. Ethanol withdrawal significantly increased systolic blood pressure and plasma angiotensin II (ANG II) levels without an effect on plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, or plasma angiotensin I (ANG I) levels. No differences in vascular ANG I, ANG II levels, and ACE activity/expression and AT1 and AT2 receptor expression were detected among the experimental groups. Plasma osmolality, as well as plasma sodium, potassium, and glucose levels were not affected by ethanol withdrawal. Ethanol withdrawal induced systemic and vascular oxidative stress, as evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels and the vascular generation of superoxide anion. Ethanol withdrawal significantly decreased plasma and vascular nitrate/nitrite levels. Major new findings of the present study are that ethanol withdrawal induces vascular oxidative stress and reduces nitric oxide (NO) levels in the vasculature. Additionally, our study provides novel evidence that ethanol withdrawal does not affect the vascular ANG II generating system while stimulating systemic RAS. These responses could predispose individuals to the development of cardiovascular diseases.

Estradiol potentiates hypothalamic vasopressin and oxytocin neuron activation and hormonal secretion induced by hypovolemic shock

Estrogen receptors are located in important brain areas that integrate cardiovascular and hydroelectrolytic responses, including the subfornical organ (SFO) and supraoptic (SON) and paraventricular (PVN) nuclei. The aim of this study was to evaluate the influence of estradiol on cardiovascular and neuroendocrine changes induced by hemorrhagic shock in ovariectomized rats. Female Wistar rats (220-280 g) were ovariectomized and treated for 7 days with vehicle or estradiol cypionate (EC, 10 or 40 μg/kg, sc). On the 8th day, animals were subjected to hemorrhage (1.5 ml/100 g for 1 min). Hemorrhage induced acute hypotension and bradycardia in the ovariectomized-oil group, but EC treatment inhibited these responses. We observed increases in plasma angiotensin II concentrations and decreases in plasma atrial natriuretic peptide levels after hemorrhage; EC treatment produced no effects on these responses. There were also increases in plasma vasopressin (AVP), oxytocin (OT), and prolactin levels after the induction of hemorrhage in all groups, and these responses were potentiated by EC administration. SFO neurons and parvocellular and magnocellular AVP and OT neurons in the PVN and SON were activated by hemorrhagic shock. EC treatment enhanced the activation of SFO neurons and AVP and OT magnocellular neurons in the PVN and SON and AVP neurons in the medial parvocellular region of the PVN. These results suggest that estradiol modulates the cardiovascular responses induced by hemorrhage, and this effect is likely mediated by an enhancement of AVP and OT neuron activity in the SON and PVN.

Oestradiol Potentiates Hormone Secretion and Neuronal Activation in Response to Hypertonic Extracellular Volume Expansion in Ovariectomised Rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 μg/kg, OVX+E group) were subjected to either isotonic (0.15 m NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 m NaCl, 2 ml/100 g b.w., i.v.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c‐Fos‐VP and c‐Fos‐OT double‐labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c‐Fos‐OT and c‐Fos‐VP double‐labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.