Protective Effect of RC-3095, an Antagonist of the Gastrin-Releasing Peptide Receptor, in Experimental Arthritis

Objective. To evaluate the antiinflammatory effects of RC-3095 in 2 experimental models of arthritis, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), and to determine the mechanisms of action involved. Methods. RC-3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin-releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin-17 (IL-17), IL-1 beta, and tumor necrosis factor alpha (TNF alpha) was evaluated in all mouse knees using enzyme-linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA. Results. In mice with AIA, administration of RC-3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC-3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC-3095. Furthermore, arthritic mice treated with RC-3095 showed a significant reduction in the concentrations of IL-17, IL-1 beta, and TNF alpha, and showed a diminished expression of GRPR. Conclusion. These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

The medial forebrain bundle mediates cardiovascular responses to electrical stimulation of the medial prefrontal cortex

The medial prefrontal cortex (MPFC) is involved in cardiovascular control. MPFC electrical stimulation has been reported to cause depressor and bradycardic responses in anesthetized rats. Although the pathway involved is yet unknown, there is evidence indicating the existence of a relay in the lateral hypothalamus (LH). The medial forebrain bundle (MFB) that courses in the lateral portion of the LH carries the vast majority of telencephalic afferent as well efferent projections, including those from the MPFC. To evaluate if the hypotensive pathway originating in the MPFC courses the MFB, we studied the effect of coronal or sagittal knife cuts through the LH and other brain areas on the cardiovascular responses to MPFC electrical stimulation. Knife cuts were performed using blades I to 6 mm wide. Results indicate that the neural pathway descending from the MFB decussates early in the vicinity of MPFC, crossing the midline within the corpus callosurn and yielding two descending pathways that travel rostro-caudally in the lateral portion of the LH, within the MFB. The decussation was confirmed by histological analysis of brain sections processed after the injection of biotinilated dextran amine in the site of the stimulation in the MPFC. Because knife cuts through the LH ipsilateral had minimal effects on the cardiovascular responses and knife cuts performed contralateral to the stimulated MPFC had no effect on the response to MPFC stimulation, data indicate that the contralateral limb of the pathway may be only activated as an alternative pathway when the ipsilateral pathway is blocked. (c) 2009 Elsevier B.V. All rights reserved.

Isoflavone genistein inhibits the angiotensin-converting enzyme and alters the vascular responses to angiotensin I and bradykinin

Genistein produces antihypertensive and beneficial cardiovascular effects, although the mechanisms for these effects are not known. We examined whether genistein inhibits the in vivo responses to angiotensin I or enhances the responses to bradykinin in anaesthetized rats as a result of angiotensin-converting enzyme inhibition. We have also studied the in vitro effects produced by genistein on the angiotensin-converting enzyme activity. We measured the changes in systemic arterial pressure induced by angiotensin I in doses of 0.03 to 10 mu g/kg, by angiotensin II in doses of 0.01 to 3 mu g/kg, and to bradykinin in doses of 0.03 to 10 mu g/kg in anaesthetized rats pretreated with vehicle (controls), or a single i.v. dose of genistein 25 mg/kg, or daily genistein 25 mg/kg i.v for two days, or a single i.v. dose of captopril 2 mg/kg. Plasma angiotensin-converting enzyme activity was determined in controls and genistein-treated rats using a fluorometric method. The effects of genistein (3-300 mu mol/1) on in vitro angiotensin-converting enzyme activity were assessed by adding genistein to plasma samples and measuring angiotensin-converting enzyme activity. We found significant lower angiotensin-converting enzyme activity in plasma samples from rats pretreated with genistein compared with those found in the Control group (77.7 +/- 8.1 his-leu nmol/min/ml and 108.7 +/- 8.4 his-leu nmol/min/ml, respectively; P=0.01). The incubation of genistein with plasma samples showed that genistein decreased the angiotensin-converting enzyme activity in plasma in a concentration-dependent manner (P<0.01). These findings indicate that genistein inhibits the angiotensin-converting enzyme in vivo and in vitro and may explain, at least in part, the antihypertensive and beneficial vascular effects produced by genistein. (C) 2009 Elsevier B.V. All rights reserved.

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.

THE MEDIAL AMYGDALOID NUCLEUS MODULATES CARDIOVASCULAR RESPONSES TO ACUTE RESTRAINT IN RATS

The medial amygdaloid nucleus (MeA) modulates several physiological and behavioral processes and among them, the cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint evokes cardiovascular responses, which are characterized by both elevated blood pressure (BP) and intense heart rate (HR) increase. We presently report effects of MeA pharmacological manipulations on BP and HR responses evoked by acute restraint in rats. Bilateral microinjection of 100 nL of the unspecific synaptic blocker COCl(2) (1 mM) into the MeA increased HR response to acute restraint, without significant effect on the BP response. This result indicates an inhibitory influence of MeA on restraint-evoked HR changes. Injections of the non-selective muscarinic receptor antagonist atropine (3 nmol); the inhibitor of choline uptake hemicholinium (2 nmol) or the selective M(1)-receptor antagonist pirenzepine (6 nmol) caused effects that were similar to those caused by cobalt. These results suggest that local cholinergic neurotransmission and M(1)-receptors mediate the MeA inhibitory influence on restraint-related HR responses. Pretreatment with the M3 receptor antagonist 4-DAMP (4-Diphenylacetoxy-N-methylpiperidine methiodide-2 nmol) did not affect restraint-related cardiovascular responses, reinforcing the idea that M(1)-receptors mediate MeA-related inhibitory influence on restraint-evoked HR increase. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Cannabidiol decreases bone resorption by inhibiting RANK/RANKL expression and pro-inflammatory cytokines during experimental periodontitis in rats

Cannabidiol (CBD) is a cannabinoid component from Cannabis sativa that does not induce psychotomimetic effects and possess anti-inflammatory properties. In the present study we tested the effects of CBD in a periodontitis experimental model in rats. We also investigated possible mechanisms underlying these effects. Periodontal disease was induced by a ligature placed around the mandible first molars of each animal. Male Wistar rats were divided into 3 groups: control animals; ligature-induced animals treated with vehicle and ligature-induced animals treated with CBD (5 mg/kg, daily). Thirty days after the induction of periodontal disease the animals were sacrificed and mandibles and gingival tissues removed for further analysis. Morphometrical analysis of alveolar bone loss demonstrated that CBD-treated animals presented a decreased alveolar bone loss and a lower expression of the activator of nuclear factor-kappa B ligand RANKL/RANK. Moreover, gingival tissues from the CBD-treated group showed decreased neutrophil migration (MPO assay) associated with lower interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha production. These results indicate that CBD may be useful to control bone resorption during progression of experimental periodontitis in rats. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved.

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.

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.

Activation of cannabinoid CB, receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test

There are contradictory results concerning the effects of systemic injections of cannabinoid agonists in anxiety-induced behavioral changes. Direct drug administration into brain structures related to defensive responses could help to clarify the role of cannabinoids in these changes. Activation of cannabinoid CB, receptors in the dorsolateral periaqueductal gray induces anxiolytic-like effects in the elevated plus maze. The aim of this work was to verify if facilitation of endocannabinoid-mediated neurotransmission in this region would also produce anxiolytic-like effects in another model of anxiety, the Vogel conflict test. Male Wistar rats (n = 5-9/group) with cannulae aimed at the dorsolateral periaqueductal gray were water deprived for 24 h and pre-exposed to the apparatus where they were allowed to drink for 3 min. After another 24 h-period of water deprivation, they received the microinjections and, 10 min later, were placed into the experimental box. in this box an electrical shock (0.5 nnA, 2 s) was delivered in the spout of a drinking bottle at every twenty licks. The animals received a first microinjection of vehicle (0.2 mu l) or AM251 (a cannabinoid CB1 receptor antagonist; 100 pmol) followed, 5 min later, by a second microinjection of vehicle, anandamide (an endocannabinoid, 5 pmol), AM404 (an inhibitor of anandamide uptake, 50 pmol) or URB597 (an inhibitor of Fatty Acid Amide Hydrolase, 0.01 or 0.1 nmol). Anandamide, AM404 and URB597 (0.01 nmol) increased the total number of punished licks. These effects were prevented by AM251. The results give further support to the proposal that facilitation of CB1 receptor-mediated endocannabinoid neurotransmission in the dorsolateral periaqueductal gray modulates defensive responses. (C) 2008 Elsevier B.V. All rights reserved.

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.

Leukotriene B(4) is essential for selective eosinophil recruitment following allergen challenge of CD4(+) cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class It expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4(+) (but not CD4(-)) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4(+) cells ex vivo. MK886 blocked induction of CCL17 Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalburnin-restimulated CD4(+) cells initiate eosinophil recruitment which is strictly dependent on LTB4 production. (C) 2008 Elsevier Inc. All rights reserved.

Prelimbic but not infralimbic cortex is involved in the pressor response to chemoreflex activation in awake rats

The ventral portion of the medial prefrontal cortex comprises the prelimbic cortex (PL) and the infralimbic cortex (IL). Several studies have indicated that both the PL and the IL play an important role in cardiovascular control. Chemoreflex activation by systemic administration of potassium cyanide (KCN) evokes pressor and bradycardiac responses in conscious rats, in addition to an increase in respiratory frequency. We report here a comparison between the effects of pharmacological inhibition of PL and IL neurotransmission on blood pressure and heart rate responses evoked by chemoreflex activation using KCN (i.v.) in conscious rats. Bilateral microinjection of 200 nl of the unspecific synaptic blocker CoCl(2) (1 mm) into the PL evoked a significant attenuation of the pressor response, without affecting the chemoreflex-induced heart rate decrease. However, IL local synapse inhibition evoked no changes in cardiovascular responses induced by chemoreflex activation. Thus, our results suggest that the pressor but not the bradycardiac response to chemoreflex activation is, at least in part, mediated by local neurotransmission present in the PL cortex, without influence of the IL cortex.