BLOCKING CENTRAL LEUKOTRIENES SYNTHESIS AFFECTS VASOPRESSIN RELEASE DURING SEPSIS

Recent studies revealed that vasopressinergic neurons have a high content of cys-leukotriene C(4) (LTC(4)) synthase, a critical enzyme in cys-leukotriene synthesis that may play a role in regulating vasopressin secretion. This study investigates the role of this enzyme in arginine vasopressin (AVP) release during experimentally induced sepsis. Male Wistar rats received an i.c.v. injection of 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) (1.0 mu g/kg), a leukotrienes (LTs) synthesis inhibitor, or vehicle, 1 h before cecal ligation and puncture (CLP) or sham operation. In one group of animals the survival rate was monitored for 3 days. In another group, the animals were decapitated at 0, 4, 6, 18 and 24 h after CLP or sham operation, and blood was collected for hematocrit, serum sodium and nitrate, plasma osmolality, protein and AVP determination. A third group was used for blood pressure measurements. The neurohypophysis was removed for quantification of AVP content, and the hypothalamus was dissected for LTC4 synthase analysis by Western blot. Mortality after CLP was reduced by the central administration of MK-886. The increase in plasma AVP levels and hypothalamus LTC4 synthase content in the initial phase of sepsis was blocked, whereas the decrease in neurohypophyseal AVP content was partially reversed. Also the blood pressure drop was abolished in this phase. The increase of serum nitric oxide and hematocrit was reduced, and the decrease in plasma protein and osmolality was not affected by the LTs blocker. In the final phase of sepsis, the plasma AVID level and the hypothalamic LTC4 synthase content were at basal levels. The central administration of MK-886 increased the hypothalamic LTC4 synthase content but did not alter the plasma and neurohypophysis AVID levels observed, or the blood pressure during this phase. These results suggest that the central LTs are involved in the vasopressin release observed during sepsis. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Cardiovascular responses to microinjection of noradrenaline into the medial amygdaloid nucleus of conscious rats result from alpha(2)-receptor activation and vasopressin release

The medial amygdaloid nucleus (MeA) is involved in the modulation of physiological and behavioral processes, as well as regulation of the autonomic nervous system. Moreover, MeA electrical stimulation evokes cardiovascular responses. Thus, as noradrenergic receptors are present in this structure, the present study tested the effects of local noradrenaline (NA) microinjection into the MeA on cardiovascular responses in conscious rats. Moreover, we describe the types of adrenoceptor involved and the peripheral mechanisms involved in the cardiovascular responses. Increasing doses of NA (3, 9, 27 or 45 nmol/100 nL) microinjected into the MeA of conscious rats caused dose-related pressor and bradycardic responses. The NA cardiovascular effects were abolished by local pretreatment of the MeA with 10 nmol/100 nL of the specific alpha(2)-receptor antagonist RX821002, but were not affected by local pretreatment with 10 nmol/100 nL of the specific alpha(1)-receptor antagonist WB4101. The magnitude of pressor response evoked by NA microinjected into the MeA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium (5 mg/kg), and blocked by intravenous pretreatment with the selective V(1)-vasopressin antagonist dTyr(CH(2))(5)(Me)AVP (50 mu g/kg). In conclusion, our results show that microinjection of NA into the MeA of conscious rats activates local alpha(2)-adrenoceptors, evoking pressor and bradycardic responses, which are mediated by vasopressin release.

Role of dexamethasone on vasopressin release during endotoxemic shock

The present study was designed to assess the hypothesis that dexamethasone (DEX) through the control of nitric oxide (NO) synthesis could regulate the release of vasopressin (AVP), which plays an important role in the regulation of arterial pressure and plasma osmolality. Endotoxemic shock was induced by intravenous (i.v.) injection of 1.5 mg/kg lipopolisaccharide (LPS) in male Wistar rats weighing 250-300 g. After LPS administration, a group of animals were treated with DEX (1.0 mg/kg of body weight), whereas saline-injected rats served as controls. The LPS administration induced a significant decrease in mean arterial pressure (MAP) with a concomitant increase in heart rate (HR) (Delta VMAP: -16.1 +/- 4.2 mm Hg; Delta VHR: 47.3 +/- 8.1 bpm). An increase in plasma AVP concentration occurred and was present for 2 h after LPS administration (11.1 +/- 0.9 pg/mL) returning close to basal levels thereafter and remaining unchanged until the end of the experiment. When LPS was combined with i.v. administration of a low dose of DEX, we observed an attenuation in the drop of MAP (Delta VMAP: -2.2 +/- 1.9 mm Hg) and a decrease in NO plasma concentration [NO] after LPS administration (1098.1 +/- 68.1 mu M) compared to [NO] after DEX administration (523.4 +/- 75.2 mu M). However, this attenuation in the drop of MAP was accompanied by a decrease in AVP plasma concentration (3.7 +/- 0.4 pg/mL). These data suggest that AVP does not participate in the recovery of MAP when DEX is administered in this endotoxemic shock model. (C) 2008 Elsevier B.V. All rights reserved.

Central but not systemic inhibition of inducible nitric oxide synthase modulates oxytocin release during endotoxemic shock

Previous studies have shown that immunological challenges as lipopolysaccharide (LPS) administration increases plasma oxytocin (OT) concentration. Nitric oxide (NO), a free radical gas directly related to the immune system has been implicated in the central modulation of neuroendocrine adaptive responses to immunological stress. This study aimed to test the hypothesis that the NO pathway participates in the control of OT release induced by LPS injection. For this purpose, adult male Wistar rats received bolus intravenous (i.v.) injection of LPS, preceded or not by iv. or intracerebroventricular (i.c.v.) injections of aminoguanidine (AG), a selective inducible nitric oxide synthase (iNOS) inhibitor. Rats were decapitated after 2, 4 and 6 h of treatment, for measurement of OT by radioimmunoassay. In a separate set of experiments, mean arterial pressure (MAP) and heart rate (HR) were measured every 15 min over 6 h, using a polygraph. These studies revealed that LPS reduced MAP and increased HR at 4 and 6 h post-injection. LPS significantly increased plasma OT concentration at 2 and 4 h post-injection. Pre-treatment with i.c.v. AG further increased plasma OT concentration and attenuated the LPS-induced decrease in MAP, however, i.v. AG failed to show similar effects. Thus, iNOS pathway may activate a central inhibitory control mechanism that attenuates OT secretion during endotoxemic shock. (C) 2009 Elsevier Inc. All rights reserved.

Hypothermia during endotoxemic shock in female mice lacking inducible nitric oxide synthase

The present study was undertaken to evaluate: (1) whether lipopolysaccharide LPS-incluced hypothermic responses may be altered during two estrous cycle phases, proestrus and diestrus, and after ovariectomy, followed by hormonal supplementation and (2) whether nitric oxide (NO) plays a role on LPS-induced hypothermia responses in female mice. Experiments were performed on adult female wild-type (WT) C57BL and inducible NO synthase knockout (KO) mice weighing 18 to 30 g. Endotoxemia was induced by intraperitoneal LIPS administration from Escherichia coli at a nonlethal dose of 10 mg/kg, and body temperature was measured by biotelemetry. Hormonal replacement was performed in ovariectomized mice through 17 beta-estradiol Silastic capsules (100 mu g) and s.c. injection of progesterone (0.5 mg per animal). We observed that during the diestrus phase, mice presented more intensive hypothermia than during proestrus phase, and hormonal supplementation with 17 beta-estradiol and progesterone attenuated hypothermia in ovariectomized mice. During diestrus and ovariectomy, KO mice had higher hypothermic response when compared with the WT group. During proestrus, the lack of statistical difference between KO and WT mice could be consequent of lower ovarian hormones plasma levels. After hormonal replacement, hypothermia was reverted in KO groups probably because of higher ovarian hormonal levels. In summary, the results demonstrated that NO release by inducible NO synthase has an important thermoregulatory role in LPS-incluced hypothermia in female mice. Besides, this involvement is directly dependent on the presence of ovarian hormones and their respective levels.

The diagonal band of Broca is involved in the pressor pathway activated by noradrenaline microinjected into the periaqueductal gray area of rats

Aims: The dorsal periaqueductal gray area (dPAG) is involved in cardiovascular modulation. Previously, we reported that noradrenaline (NA) microinjection into the dPAG caused a pressor response that was mediated by vasopressin release into the circulation. However, the neuronal pathway that mediates this response is as yet unknown. There is evidence that chemical stimulation of the diagonal band of Broca (dbB) also causes a pressor response mediated by systemic vasopressin release. In the present study, we evaluated the participation of the dbB in the pressor response caused by NA microinjection into the dPAG as well as the existence of neural connections between these areas. Main methods: With the above goal, we verified the effect of the pharmacological ablation of the dbB on the cardiovascular response to NA microinjection into the dPAG of unanesthetized rats. In addition, we microinjected the neuronal tracer biotinylated-dextran-amine (BDA) into the dPAG and looked for efferent projections from the dPAG to the dbB. Key findings: The pharmacologically reversible ablation of the dbB with local microinjection of CoCl(2) significantly reduced the pressor response caused by NA microinjection (15 nmol/50 nL) into the dPAG. In addition, BDA microinjection into the dPAG labeled axons in the dbB, pointing to the existence of direct connections between these areas. Significance: The present results indicate that synapses within the dbB are involved in the pressor pathway activated by NA microinjection into the VAG and direct neural projection from the dPAG to the dbB may constitute the neuroanatomic substrate for this pressor pathway. (C) 2009 Elsevier Inc. All rights reserved.

Mechanisms involved in the pressor response to noradrenaline microinjection into the supraoptic nucleus of unanesthetized rats

We report on the cardiovascular effects of noradrenaline (NA) microinjection into the hypothalamic supraoptic nucleus (SON) as well as the central and peripheral mechanisms involved in their mediation. Microinjections of NA 1, 3, 10, 30 or 45 nmol/100 nL into the SON caused dose-related pressor and bradycardiac response in unanesthetized rats. The response to NA 10 nmol was blocked by SON pretreatment with 15 nmol of the alpha(2)-adrenoceptor antagonist RX821002 and not affected by pretreatment with equimolar dose of the selective alpha(1)-adrenoceptor antagonist WB4101, suggesting that local alpha(2)adrenoceptors mediate these responses. Pretreatment of the SON with the nonselective beta-adrenoceptor antagonist propranolol 15 nmol did not affect the pressor response to NA microinjection of into the SON. Moreover, the microinjection of the 100 nmol of the selective alpha(1)-adrenoceptor agonist methoxamine (MET) into the SON did not cause cardiovascular response while the microinjection of the selective alpha(2)adrenoceptor agonists BHT920 (BHT, 100 nmol) or clonidine (CLO, 5 nmol) caused pressor and bradycardiac responses, similar to that observed after the microinjection of NA. The pressor response to NA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium and was blocked by intravenous pretreatment with the V(1)-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP, suggesting an involvement of circulating vasopressin in this response. In conclusion, our results suggest that pressor responses caused by microinjections of NA into the SON involve activation of local alpha(2)-adrenoceptor receptors and are mediated by vasopressin release into circulation. (c) 2008 Published by Elsevier B.V.

Non-N-Methyl-D-Aspartate Glutamate Receptors in the Paraventricular Nucleus of Hypothalamus Mediate the Pressor Response Evoked by Noradrenaline Microinjected Into the Lateral Septal Area in Rats

The lateral septal area (LSA) is a part of the limbic system and is involved in cardiovascular modulation. We previously reported that microinjection of noradrenaline (NA) into the LSA of unanesthetized rats caused pressor responses that are mediated by acute vasopressin release. Magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) of the hypothalamus synthesize vasopressin. In the present work, we studied which of these nuclei is involved in the pressor pathway activated by unilateral NA injection into the LSA as well as the local neurotransmitter involved. Chemical ablation of the SON by unilateral injection of the nonspecific synapses blocker cobalt chloride (1 mM/100 nl) did not affect the pressor response evoked by NA (21 nmol/200 nl) microinjection into the LSA. However, the response to NA was blocked when cobalt chloride (1 mM/100 nl) was microinjected into the PVN, indicating that this hypothalamic nucleus is responsible for the mediation of the pressor response. There is evidence in the literature pointing to glutamate as a putative neurotransmitter activating magnocellular neurons. Pretreatment of the PVN with the selective non-N-methyl-D-asparate (NMDA) antagonist NBQX (2 nmol/100 nl) blocked the pressor response to NA microinjected into the LSA, whereas pretreatment with the selective NMDA antagonist LY235959 (2 nmol/100 nl) did not affect the response to NA. Our results implicate the PVN as the final structure in the pressor pathway activated by the microinjection of NA into the LSA. They also indicate that local glutamatergic synapses and non-NMDA glutamatergic receptors mediate the response in the PVN. (c) 2008 Wiley-Liss, Inc.

Cardiovascular responses to noradrenaline microinjection in the ventrolateral periaqueductal gray of unanesthetized rats

The periaqueductal gray area (PAG) is a mesencephalic area involved in cardiovascular modulation. Noradrenaline (NA), a neurotransmitter involved in central blood pressure control, is present in the rat PAG. We report here on the cardiovascular effects caused by NA microinjection into the ventrolateral PAG (vlPAG) of unanesthetized rats and the peripheral mechanism involved in their mediation. NA microinjection in the vlPAG of unanesthetized rats evoked dose-related pressor and bradycardiac responses. No significant cardiovascular responses were observed in urethane-anesthetized rats. The pressor response was potentiated by pretreatment with the ganglion blocker pentolinium (5 or 10 mg/kg, intravenously). Pretreatment with the vasopressin antagonist dTyr(CH(2))(5) (Me)AVP (50 mu g/kg, intravenously) blocked the pressor response evoked by the NA microinjection into the vlPAG. Additionally, circulating vasopressin content was found to be significantly increased after NA microinjection in the vlPAG. The results suggest that activation of noradrenergic synapses within the vlPAG modulates vasopressin release in unanesthetized rats. (c) 2007 Wiley-Liss, Inc.

ANGIOTENSIN III MODULATES THE NOCICEPTIVE CONTROL MEDIATED BY THE PERIAQUEDUCTAL GRAY MATTER

Endogenous angiotensin (Ang) II and/or an Ang II-derived peptide, acting on Ang type I (AT(1)) and Ang type 2 (AT(2)) receptors, can carry out part of the nociceptive control modulated by periaqueductal gray matter (PAG). However, neither the identity of this putative Ang-peptide, nor its relationship to Ang II antinociceptive activity was clarified. Therefore, we have used tail-flick and incision allodynia models combined with an HPLC time course of Ang metabolism, to study the Ang III antinociceptive effect in the rat ventrolateral (vi) PAG using peptidase inhibitors and receptor antagonists. Ang III injection into the vIPAG increased tail-flick latency, which was fully blocked by Losartan and CGP 42,112A, but not by divalinal-Ang IV, indicating that. Ang III effect was mediated by AT(1) and AT(2) receptors, but not by the AT(4) receptor. Ang III injected into the vIPAG reduced incision allodynia. Incubation of Ang II with punches of vIPAG homogenate formed Ang III, Ang (1-7) and Ang IV. Amastatin (AM) inhibited the formation of Ang III from Ang II by homogenate, and blocked the antinociceptive activity of Ang II injection into vIPAG, suggesting that aminopeptidase A (APA) formed Ang III from Ang II. Ang III can also be formed from Ang I by a vIPAG alternative pathway. Therefore, the present work shows, for the first time, that: (i) Ang III, acting on AT(1) and AT(2) receptors, can elicit vIPAG-mediated antinociception, (ii) the conversion of Ang II to Ang III in the vIPAG is required to elicit antinociception, and (iii) the antinociceptive activity of endogenous Ang II in vIPAG can be ascribed preponderantly to Ang III. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Paraventricular nucleus mediates pressor response to noradrenaline injection into the dorsal periaqueductal gray area

The dorsal periaqueductal gray area (dPAG) is involved in cardiovascular modulation. In a previous study, we reported that noradrenaline (NA) microinjection into the dPAG of rats caused pressor response that was mediated by vasopressin release. Vasopressin is synthesized by magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In the present study, we verified which nuclei mediated the cardiovascular response to NA as well as the existence of direct neural projection from the dPAG to hypothalamic nuclei. Then, we studied the effect of treating either PVN or SON with the nonselective synaptic blocker cobalt chloride (1 mM) on the cardiovascular response to NA (15 nmol) microinjection into dPAG. Attempting to identify neural projections from dPAG to hypothalamic nuclei, we microinjected the neuronal tracer biotinylated-dextran-amine (BDA) into the dPAG and searched varicosity-containing nerve terminals in the PVN and SON. Unilateral cobalt-induced inhibition of synapses in the SON did not affect the cardiovascular response to NA. However, unilateral inhibition of PVN significantly reduced the pressor response to NA. Moreover, cobalt-induced inhibition of synapses in both PVN blocked the pressor response caused by NA microinjected into the dPAG. Microinjection of BDA into the dPAG evidenced presence of varicosity-containing neuronal fibers in PVN but not in SON. The results from cobalt treatment indicated that synapses in PVN mediate the vasopressin-induced pressor response caused by NA microinjection into the dPAG. In addition, the neuroanatomical results from BDA microinjection into the dPAG pointed out the existence of direct neural projections from the dPAG site to the PVN. (C) 2009 Elsevier B.V. All rights reserved.

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

Microinjection of the cholinergic agonist carbachol into the bed nucleus of the stria terminalis (BST) has been reported to cause pressor response in unanesthetized rats, which was shown to be mediated by an acute release of vasopressin into the systemic circulation and followed by baroreflex-mediated bradycardia. In the present study, we tested the possible involvement of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei in the pressor response evoked by carbachol microinjection into the BST of unanesthetized rats. For this, cardiovascular responses following carbachol (1 nmol/100 nL) microinjection into the BST were studied before and after PVN or SON pretreatment, either ipsilateral or contralateral in relation to BST microinjection site, with the nonselective neurotransmission blocker cobalt chloride (CoCl(2), 1 mM/100 nL). Carbachol microinjection into the BST evoked pressor response. Moreover, BST treatment with carbachol significantly increased plasma vasopressin levels, thus confirming previous evidences that carbachol microinjection into the BST evokes pressor response due to vasopressin release into the circulation. SON pretreatment with CoCl(2), either ipsilateral or contralateral in relation to BST microinjection site, inhibited the pressor response to carbachol microinjection into the BST. However, CoCl(2) microinjection into the ipsilateral or contralateral PVN did not affect carbachol-evoked pressor response. In conclusion, our results suggest that pressor response to carbachol microinjection into the BST is mediated by SON magnocellular neurons, without significant involvement of those in the PVN. The results also indicate that responses to carbachol microinjection into the BST are mediated by a neural pathway that depends on the activation of both ipsilateral and contralateral SON. (C) 2011 Elsevier B.V. All rights reserved.

Involvement of hypothalamic paraventricular nucleus non-N-methyl-d-aspartate receptors in the pressor response to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats

Microinjection of noradrenaline into the bed nucleus of the stria terminalis (BST) has been reported to cause a pressor response in unanesthetized rats, which was shown to be mediated by acute vasopressin release into the systemic circulation. In the present study we verified the involvement of magnocellular neurons of the hypothalamic paraventricular (PVN) or supraoptic (SON) nuclei and the local neurotransmitter involved in the pressor response to noradrenaline microinjection into the BST. The PVN pretreatment with the non-selective neurotransmission blocker CoCl(2) (1 nmol/100 nL) inhibited the noradrenaline-evoked pressor response. However, responses were not affected by SON treatment with CoCl(2). Further experiments were carried out to test if glutamatergic neurotransmission in the PVN mediates the pressor response evoked by noradrenaline microinjection into the BST. Pretreatment of the PVN with the selective N-methyl-d-aspartate (NMDA) receptor antagonist LY235959 (2 nmol/100 nL) did not affect the noradrenaline-evoked pressor response. However, PVN pretreatment with the selective non-NMDA receptor antagonist NBQX (2 nmol/100 nL) significantly reduced the pressor response to noradrenaline microinjection into the BST. In conclusion, our results suggest that pressor responses to noradrenaline microinjection into the BST are mediated by PVN magnocellular neurons without involvement of SON neurons. They also suggest that a glutamatergic neurotransmission through non-NMDA glutamate receptors in the PVN mediates the response.

Central NO-cGMP pathway in thermoregulation and survival rate during polymicrobial sepsis

Sepsis induces production of inflammatory mediators such as nitric oxide (NO) and causes physiological alterations, including changes in body temperature (T(b)). We evaluated the involvement of the central NO cGMP pathway in thermoregulation during sepsis induced by cecal ligation and puncture (CLP), and analyzed its effect on survival rate. Male Wistar rats with a T(b) probe inserted in their abdomen were intracerebroventricularly injected with 1 mu L N(G)-nitro-L-arginine methyl ester (L-NAME, 250 mu g), a nonselective NO synthase (NOS) inhibitor; or aminoguanidine (250 mu g), an inducible NOS inhibitor; or 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.25 mu g), a guanylate cyclase inhibitor. Thirty minutes after injection, sepsis was induced by cecal ligation and puncture (CLP), or the rats were sham operated. The animals were divided into 2 groups for determination of T(b) for 24 h and assessment of survival during 3 days. The drop in T(b) seen in the CLP group was attenuated by pretreatment with the NOS inhibitors (p < 0.05) and blocked with ODQ. CLP rats pretreated with either of the inhibitors showed higher survival rates than vehicle injected groups (p < 0.05), and were even higher in the ODQ pretreated group. Our results showed that the effect of NOS inhibition on the hypothermic response to CLP is consistent with the role of nitrergic pathways in thermoregulation.

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100 mu g/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4,6 or 24 h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24 h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24 h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6 h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24 h after surgery, and in both hypothalamic nuclei only at 6 h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood. (C) 2011 Elsevier B.V. All rights reserved.