The paraventricular nucleus of the hypothalamus is involved in cardiovascular responses to acute restraint stress in rats

The paraventricular nucleus of the hypothalamus (PVN) has been implicated in several aspects of cardiovascular control. Stimulation of the PVN evokes changes in blood pressure and heart rate. Additionally, this brain area is connected to several limbic structures implicated in behavioral control, as well as to forebrain and brainstem structures involved in cardiovascular control. This evidence indicates that the PVN may modulate cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint is an unavoidable stressor that evokes marked and sustained cardiovascular changes, which are characterized by elevated mean arterial pressure (MAP) and an intense heart rate (HR) increase. We report on the effect of inhibition of PVN synapses on MAP and HR responses evoked by acute restraint in rats. Bilateral microinjection of the nonspecific synaptic blocker cobalt (CoCl2, 1mM/100nl) into the PVN did not change the HR response or the initial peak of the MAP response to restraint stress, but reduced the area under the curve of the MAP response. Moreover, bilateral microinjection of cobalt in areas surrounding the PVN did not change the cardiovascular response to restraint. These results indicate that synapses in the PVN are involved in the neural pathway that controls blood pressure changes evoked by restraint.

Role of the bed nucleus of the stria terminalis in the cardiovascular responses to acute restraint stress in rats

The aim of this work was to test the hypothesis that the bed nucleus of the stria terminalis (BST) and noradrenergic neurotransmission therein mediate cardiovascular responses to acute restraint stress in rats. Bilateral microinjection of the non-specific synaptic blocker CoCl2 (0.1nmol/100nl) into the BST enhanced the heart rate (HR) increase associated with acute restraint without affecting the blood pressure increase, indicating that synapses within the BST influence restraint-evoked HR changes. BST pretreatment with the selective 1-adrenoceptor antagonist WB4101 (15nmol/100nl) caused similar effects to cobalt, indicating that local noradrenergic neurotransmission mediates the BST inhibitory influence on restraint-related HR responses. BST treatment with equimolar doses of the 2-adrenoceptor antagonist RX821002 or the -adrenoceptor antagonist propranolol did not affect restraint-related cardiovascular responses, reinforcing the inference that 1-adrenoceptors mediate the BST-related inhibitory influence on HR responses. Microinjection of WB4101 into the BST of rats pretreated intravenously with the anticholinergic drug homatropine methyl bromide (0.2mg/kg) did not affect restraint-related cardiovascular responses, indicating that the inhibitory influence of the BST on the restraint-evoked HR increase could be related to an increase in parasympathetic activity. Thus, our results suggest an inhibitory influence of the BST on the HR increase evoked by restraint stress, and that this is mediated by local 1-adrenoceptors. The results also indicate that such an inhibitory influence is a result of parasympathetic activation.

Activation of the ET-1/ETA Pathway Contributes to Erectile Dysfunction Associated with Mineralocorticoid Hypertension

The cavernosal tissue is highly responsive to endothelin-1 (ET-1), and penile smooth muscle cells not only respond to but also synthesize ET-1. Considering that ET-1 is directly involved in end-organ damage in salt-sensitive forms of hypertension, we hypothesized that activation of the ET-1/ET(A) receptor pathway contributes to erectile dysfunction (ED) associated with mineralocorticoid hypertension. Wistar rats were uninephrectomized and submitted to deoxycorticosterone acetate (DOCA)-salt treatment for 5 weeks. Control (Uni [uninephrectomized control]) animals were uninephrectomized and given tap water. Uni and DOCA-salt rats were simultaneously treated with vehicle or atrasentan (ET(A) receptor antagonist, 5 mg/Kg/day). Cavernosal reactivity to ET-1, phenylephrine (PE), ET(B) receptor agonist (IRL-1620) and electric field stimulation (EFS) were evaluated in vitro. Expression of ROCK alpha, ROCK beta, myosin phosphatase target subunit 1 (MYPT-1), and extracellular signal-regulated kinase 1/2 (ERK 1/2) were evaluated by western blot analysis. ET-1 and ET(A) receptor mRNA expression was evaluated by real-time reverse-transcriptase polymerase chain reaction. Voltage-dependent increase in intracavernosal pressure/mean arterial pressure (ICP/MAP) was used to evaluate erectile function in vivo. ET(A) receptor blockade prevents DOCA-salt-associated ED. Cavernosal strips from DOCA-salt rats displayed augmented preproET-1 expression, increased contractile responses to ET-1 and decreased relaxation to IRL-1620. Contractile responses induced by EFS and PE were enhanced in cavernosal tissues from DOCA-salt hypertensive rats. These functional changes were associated with increased activation of the RhoA/Rho-kinase and ERK 1/2 pathways. Treatment of rats with atrasentan completely prevented changes in cavernosal reactivity in DOCA-salt rats and restored the decreased ICP/MAP, completely preventing ED in DOCA-salt rats. Activation of the ET-1/ET(A) pathway contributes to mineralocorticoid hypertension-associated ED. ET(A) receptor blockade may represent an alternative therapeutic approach for ED associated with salt-sensitive hypertension and in pathological conditions where increased levels of ET-1 are present. Carneiro FS, Nunes KP, Giachini FRC, Lima VV, Carneiro ZN, Nogueira EF, Leite R, Ergul A, Rainey WE, Webb RC, and Tostes RC. Activation of the ET-1/ETA pathway contributes to erectile dysfunction associated with mineralocorticoid hypertension. J Sex Med **;**:**-**.

Murine and rat cavernosal responses to endothelin-1 and urotensin-II Vasoactive Peptide Symposium

Endothelin-1 (ET-1) and urotensin-II (U-II) are the most potent constrictors of human vessels. Although the cavernosal tissue is highly responsive to ET-1, no information exists on the effects of U-II on cavernosal function. The aim of this study was to characterize ET-1 and U-II responses in corpora cavernosa from rats and mice. Male Wistar rats and C57/BL6 mice were used at 13 weeks. Cumulative concentration-response curves to ET-1, U-II, and IRL-1620, an ET(B) agonist, were performed. ET-1 increased force generation in cavernosal strips from mice and rats, but no response to U-II was observed in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME), or in strips prestimulated with 20 mM KCI. IRL-1620 did not induce cavernosal contraction even in presence of L-NAME, but induced a cavernosal relaxation that was greater in rats than mice. No relaxation responses to U-II were observed in cavernosal strips precontracted with phenylephrine. mRNA expression of ET-1, ET(A), ET(B), and U-II receptors, but not U-II was observed in cavernosal strips. ET-1, via ET(A) receptors activation, causes contractile responses in cavernosal strips from rats and mice, whereas ET(B) receptor activation produces relaxation. Although the cavernosal tissue expresses U-II receptors, U-II does not induce contractile responses in corpora cavernosa from mice or rats. J Am Soc Hypertens 2008;2(6): 439-447. Published by Elsevier Inc. on behalf of the American Society of Hypertension.

P2X(7) receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1 beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1 beta into mature IL-1 beta, which is then secreted. Because both LPS (in vivo) and IL-1 beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1 beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco`s modified Eagle`s medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1 beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1 beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-L-arginine methyl ester or N-([3-(aminomethyl) phenyl] methyl) ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1 beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1 beta induced iNOS protein expression. Thus, activation of P2X 7 receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1 beta-mediated release of nitric oxide by iNOS.

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.

Aldosterone and angiotensin II synergistically stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways

Objective - Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). Methods and Results - VSMCs from WKY rats were studied. At low concentrations (10(-10) mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone ( Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 ( inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. Conclusions - Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

Involvement of the lateral habenula in the regulation of generalized anxiety- and panic-related defensive responses in rats

Recently obtained evidence points to the involvement of the lateral habenular nuclei (LHb) in the mediation of coping defensive responses to threatening/stressful stimuli. Nevertheless, the role of this brain area in the regulation of defensive responses that have been associated with specific subtypes of anxiety disorders recognized in clinical settings is presently unknown. To address this question, we investigated the effects of either electrolytic lesions or chemical stimulation of the LHb on the defensive behaviors generated in rats by the elevated T-maze. This experimental model allows the measurement, in a same rat, of two defensive behaviors, inhibitory avoidance and escape, that have been related in terms of psychopathology to generalized anxiety and panic disorders, respectively. Bilateral electrolytic lesions of the LHb (1 mA, 10 s) impaired inhibitory avoidance acquisition and facilitated escape performance. On the other hand, chemical stimulation of the LHb by bilateral microinjection of kainic acid (30-60 pmol/0.2 mu L) had the opposite effect, i.e., facilitated inhibitory avoidance and impaired escape. The present results indicate that the LHb exerts an opposed regulatory control on generalized anxiety- and panic-related defensive responses in rats. (c) 2008 Elsevier Inc. All rights reserved.

DOCA-salt treatment enhances responses to endothelin-1 in murine corpus cavernosurn

The penis is kept in the flaccid state mainly via a tonic activity of norepinephrine and endothelins (ETs). ET-1 is important in salt-sensitive forms of hypertension. We hypothesized that cavernosal responses to ET-1 are enhanced in deoxycorticosterone acetate (DOCA)-salt mice and that blockade of ETA receptors prevents abnormal responses of the corpus cavernosum in DOCA-salt hypertension. Male C57BL/6 mice were unilaterally nephrectomized and treated for 5 weeks with both DOCA and water containing 1% NaCl and 0.2% KCl. Control mice were uninephrectomized and received tap water with no added salt. Animals received either the ETA antagonist atrasentan (5 mg.day(-1).kg(-1) body weight) or vehicle. DOCA-salt mice displayed increased systolic blood pressure (SBP), and treatment with atrasentan decreased SBP in DOCA-salt mice. Contractile responses in cavernosal strips from DOCA-salt mice were enhanced by ET-1, phenylephrine, and electrical field stimulation (EFS) of adrenergic nerves, whereas relaxations were not altered by IRL-1620 (an ETB agonist), acetylcholine, sodium nitroprusside, and EFS of nonadrenergic noncholinergic nerves. PD59089 (an ERK1/2 inhibitor), but not Y-27632 (a Rho-kinase inhibitor), abolished enhanced contractions to ET-1 in cavernosum from DOCA-salt mice. Treatment of DOCA-salt mice with atrasentan did not normalize cavernosal responses. In summary, DOCA-salt treatment in mice enhances cavernosal reactivity to contractile, but not to relaxant, stimuli, via ET-1/ETA receptor-independent mechanisms.

Adenosine actions are preserved in corpus cavernosum from obese and type II diabetic db/db mouse

Introduction. Erectile dysfunction (ED) in diabetes is associated with autonomic neuropathy and endothelial dysfunction. Whereas the nonadrenergic-noncholinergic (NANC)/neurogenic nitric oxide pathway has received great attention in diabetes-associated ED, few studies have addressed sympathetic overactivity. Aim. To test the hypothesis that adenosine-induced inhibition of adrenergic-mediated contractile responses in mouse corpus cavernosum is impaired in the presence of diabetes. Methods. The db/db (obesity and type II diabetes caused by a leptin receptor mutation) mouse strain was used as a model of obesity and type II diabetes, and standard procedures were performed to evaluate functional cavernosal responses. Main Outcome Measures. Increased cavernosal responses to sympathetic stimulation in db/db mice are not associated with impaired prejunctional actions of adenosine. Results. Electrical field stimulation (EFS)-, but not phenylephrine (PE)-, induced contractions are enhanced in cavernosal strips from db/db mice in comparison with those from lean littermates. Direct effects of adenosine, 2-chloro-adenosine, A(1) receptor agonist C-8031 (N6 cyclopentyladenosine), and sodium nitroprusside are similar between the strips from lean and db/db mice, whereas relaxant responses to acetylcholine and NANC stimulation are significantly impaired in the cavernosal strips from db/db mice. 5`-Iodotubercidin (adenosine kinase inhibitor) and dipyridamole (inhibitor of adenosine transport), as well as the A(1) agonist C-8031, significantly and similarly inhibit contractions induced by stimulation of adrenergic nerves in the cavernosal strips from lean and db/db mice. Conclusions. Results from this study suggest that corpora cavernosa from obese and diabetic db/db mice display altered neural-mediated responses that would favor penile detumescence, i.e., increased contractile response to adrenergic nerve stimulation and decreased relaxant responses upon activation of NANC nerves. However, increased cavernosal responses to adrenergic nerve stimulation are not due to impaired negative modulation of sympathetic neurotransmission by adenosine in this diabetic model.

Effects of reversible inactivation of the dorsal hippocampus on the behavioral and cardiovascular responses to an aversive conditioned context

In rats, conditioned fear to context causes freezing immobility and cardiovascular changes. The dorsal hippocampus (DH) has a critical role in several memory processes, including conditioning fear to contextual information. To explore a possible involvement of the DH in contextual fear conditioning-evoked cardiovascular (mean arterial pressure and heart rate increases) and behavioral (freezing) responses, DH synaptic transmission was temporarily inhibited by bilateral microinjections of 500 nl of the nonselective synapse blocker, cobalt chloride (COCl2, 1 mmol/l), at different periods of the experimental procedure. During re-exposure to the foot shock chamber in which conditioning had taken place, bilateral DH inhibition 10 min before the conditioning session had no effect on either behavioral or cardiovascular responses. Bilateral DH inhibition immediately after the conditioning session (110 min) decreased both behavioral and cardiovascular responses during the context test. Finally, 48 h after the conditioning session, bilateral DH inhibition 10 min before re-exposure to the foot shock chamber significantly reduced cardiovascular responses but not freezing responses. These results suggest that contextual fear conditioning acquisition does not depend on the DH. This structure, however, is crucial for the consolidation of contextual fear. Moreover, although the DH appears to be less important for the behavioral (freezing) changes induced by re-exposure to the aversive conditioned context, it may play an important role on the cardiovascular responses generated by this model.

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.

Both alpha 1 and alpha 2-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the bed nucleus of the stria terminal of rats

Background and purpose: We have previously shown that noradrenaline microinjected into the bed nucleus of stria terminalis (BST) elicited pressor and bradycardiac responses in unanaesthetized rats. In the present study, we investigated the subtype of adrenoceptors that mediates the cardiovascular response to noradrenaline microinjection into the BST. Experimental approach: Cardiovascular responses following noradrenaline microinjection into the BST of male Wistar rats were studied before and after BST pretreatment with different doses of the selective alpha(1)-adrenoceptor antagonist WB4101, the alpha(2)-adrenoceptor antagonist RX821002, the combination of WB4101 and RX821002, the non-selective beta-adrenoceptor antagonist propranolol, the selective beta(1)-adrenoceptor antagonist CGP20712 or the selective beta(2)-adrenoceptor antagonist ICI118,551. Key results: Noradrenaline microinjected into the BST of unanaesthetized rats caused pressor and bradycardiac responses. Pretreatment of the BST with different doses of either WB4101 or RX821002 only partially reduced the response to noradrenaline. However, the response to noradrenaline was blocked when WB4101 and RX821002 were combined. Pretreatment with this combination also shifted the resulting dose-effect curve to the left, clearly showing a potentiating effect of this antagonist combination. Pretreatment with different doses of either propranolol or CGP20712 increased the cardiovascular responses to noradrenaline microinjected into the BST. Pretreatment with ICI118,551 did not affect cardiovascular responses to noradrenaline. Conclusion and implications: The present results indicate that alpha(1) and alpha(2)-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the BST. In addition, they point to an inhibitory role played by the activation of local beta(1)-adrenoceptors in the cardiovascular response to noradrenaline microinjected into the BST.

Effects of intracisternal administration of cannabidiol on the cardiovascular and behavioral responses to acute restraint stress

Systemic administration of cannabidiol (CBD), a non-psychotomimetic compound from Cannabis sativa, attenuates the cardiovascular and behavioral responses to restraint stress. Although the brain structures related to CBD effects are not entirely known, they could involve brainstem structures responsible for cardiovascular control. Therefore, to investigate this possibility the present study verified the effects of CBD (15.30 and 60 nmol) injected into the cisterna magna on the autonomic and behavioral changes induced by acute restraint stress. During exposure to restraint stress (1 h) there was a significant increase in mean arterial pressure (MAP) and heart rate (HR). Also, 24 h later the animals showed a decreased percentage of entries onto the open arms of the elevated plus-maze. These effects were attenuated by CBD (30 nmol). The drug had no effect on MAP and HR baseline values. These results indicate that intracisternal administration of CBD can attenuate autonomic responses to stress. However, since CBD decreased the anxiogenic consequences of restraint stress, it is possible that the drug is also acting on forebrain structures. (C) 2011 Elsevier Inc. All rights reserved.

BED NUCLEUS OF THE STRIA TERMINALIS alpha(1)- AND alpha(2)-ADRENOCEPTORS DIFFERENTIALLY MODULATE THE CARDIOVASCULAR RESPONSES TO EXERCISE IN RATS

Dynamic exercise evokes sustained blood pressure and heart rate (HR) increases. Although it is well accepted that there is a CNS mediation of cardiovascular adjustments during dynamic exercise, information on the role of specific CNS structures is still limited. The bed nucleus of the stria terminalis (BST) is involved in exercise-evoked cardiovascular responses in rats. However, the specific neurotransmitter involved in BST-related modulation of cardiovascular responses to dynamic exercise is still unclear. In the present study, we investigated the role of local BST adrenoceptors in the cardiovascular responses evoked when rats are submitted to an acute bout of exercise on a rodent treadmill. We observed that bilateral microinjection of the selective alpha 1-adrenoceptor antagonist WB4101 into the BST enhanced the HR increase evoked by dynamic exercise without affecting the mean arterial pressure (MAP) increase. Bilateral microinjection of the selective alpha 2-adrenoceptor antagonist RX821002 reduced exercise-evoked pressor response without changing the tachycardiac response. BST pretreatment with the nonselective beta-adrenoceptor antagonist propranolol did not affect exercise-related cardiovascular responses. BST treatment with either WB4101 or RX821002 did not affect motor performance in the open-field test, which indicates that effects of BST adrenoceptor antagonism in exercise-evoked cardiovascular responses were not due to changes in motor activity. The present findings are the first evidence showing the involvement of CNS adrenoceptors in cardiovascular responses during dynamic exercise. Our results indicate an inhibitory influence of BST alpha 1-adrenoceptor on the exercise-evoked HR response. Data also point to a facilitatory role played by the activation of BST alpha 2-adrenoceptor on the pressor response to dynamic exercise. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.