Mirabegron relaxes urethral smooth muscle by a dual mechanism involving β3-adrenoceptor activation and α1-adrenoceptor blockade

Mirabegron is the first β3-adrenoceptor (AR) agonist approved for treatment of overactive bladder syndrome (OAB). This study aimed to investigate the effects of β3-adrenoceptor (AR) agonist mirabegron in mouse urethra. The possibility that mirabegron exerts α1-AR antagonism was also tested in rat smooth muscle preparations presenting α1A- (vas deferens and prostate), α1D- (aorta) and α1B-AR (spleen). Functional assays were carried out in mouse and rat isolated tissues. Competition assays for the specific binding of [(3) H]Prazosin to membrane preparations of HEK 293 cells expressing each of the human α1-ARs, as well as β-AR mRNA expression and cyclic AMP measurements in mouse urethra were performed. Mirabegron produced concentration-dependent urethral relaxations that were right shifted by the selective β3-AR antagonist L 748,337, but unaffected by β1- and β2-AR antagonists (atenolol and ICI 118,551, respectively). Mirabegron-induced relaxations were enhanced by the phosphodiesterase-4 inhibitor rolipram, and this agonist stimulated cAMP synthesis. Mirabegron also produced rightward shifts in urethral contractions induced by the α1-AR agonist phenylephrine. Schild regression analysis revealed that mirabegron behaves as a competitive antagonist of α1-AR in urethra, vas deferens and prostate (α1A-AR, pA2  ≅ 5.6) and aorta (α1D-AR, pA2  ≅ 5.4), but not in spleen (α1B-AR). The affinities estimated for mirabegron in functional assays were consistent with those estimated in radioligand binding with human recombinant α1A- and α1D-ARs (pKi ≅ 6.0). The effects of mirabegron in urethral smooth muscle are the result of β3-AR agonism together with α1A / α1D-AR antagonism.

Sodium intake, brain c-Fos protein and gastric emptying in cell-dehydrated rats treated with methysergide into the lateral parabrachial nucleus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Toll-Like recepctors (TLRs) and retinoic acid inducible gene – I (RIG-I) activation by viral analogs in bovine endometrial cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Activation of 5-HT2C receptors in the dorsal periaqueductal gray increases antinociception in mice exposed to the elevated plus-maze

Several findings have pointed to the role of the dorsal periaqueductal gray (dPAG) serotonin 5-HT1A and 5-HT2(A-C) receptor subtypes in the modulation of defensive behavior in animals exposed to the elevated plus-maze (EPM). Besides displaying anxiety-like behavior, rodents also exhibit antinociception in the EPM. This study investigated the effects of intra-dPAG injections of 5-HT1A and 5-HT2B/2C receptor ligands on EPM-induced antinociception in mice. Male Swiss mice received 0.1 mu l intra-dPAG injections of vehicle, 5.6 and 10 nmol of 8-OHDPAT, a 5-HT1A receptor agonist (Experiment 1), or 0.01, 0.03 and 0.1 nmol of mCPP, a 5-HT2B/2C receptor agonist (Experiment 2). Five minutes later, each mouse received an intraperitoneal injection of 0.6% acetic acid (0.1 ml/10 g body weight; nociceptive stimulus) and was individually confined in the open (OA) or enclosed (EA) arms of the EPM for 5 min, during which the number of abdominal writhes induced by the acetic acid was recorded. While intra-dPAG injection of 8-OHDPAT did not change open-arm antinociception (OAR). mCPP (0.01 nmol) enhanced it. Combined injections of ketanserin (10 nmol/0.1 mu l), a 5-HT2A/2C receptor antagonist, and 0.01 nmol of mCPP (Experiment 3), selectively and completely blocked the OAR enhancement induced by mCPP. Although intra-dPAG injection of mCPP (0.01 nmol) also produced antinociception in EA-confined mice (Experiment 2), this effect was not confirmed in Experiment 3. Moreover, no other compound changed the nociceptive response in EA-confined animals. These results suggest that the 5-HT2C receptors located within the PAG play a role in this type of environmentally induced pain inhibition in mice. (c) 2012 Elsevier B.V. All rights reserved.

Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)

Antioxidant treatment protects against matrix metalloproteinase activation and cardiomyocyte injury during acute pulmonary thromboembolism

Increased reactive oxygen species (ROS) promote matrix metalloproteinase (MMP) activities and may underlie cardiomyocyte injury and the degradation of cardiac troponin I (cTI) during acute pulmonary thromboembolism (APT). We examined whether pretreatment or therapy with tempol (a ROS scavenger) prevents MMP activation and cardiomyocyte injury of APT. Anesthetized sheep received tempol infusion (1.0 mg kg(-1) min(-1), i.v.) or saline starting 30 min before or 30 min after APT (autologous blood clots). Control animals received saline. Hemodynamic measurements were performed. MMPs were studied in the right ventricle (RV) by gelatin zymography, fluorimetric activity assay, and in situ zymography. The ROS levels were determined in the RV and cTI were measured in serum samples. APT increased the pulmonary arterial pressure and pulmonary vascular resistance by 146 and 164 %, respectively. Pretreatment or therapy with tempol attenuated these increases. While APT increased RV + dP/dt (max), tempol infusions had no effects. APT increased RV MMP-9 (but not MMP-2) levels. In line with these findings, APT increased RV MMP activities, and this finding was confirmed by in situ zymography. APT increased the RV ROS levels and tempol infusion, before or after APT, and blunted APT-induced increases in MMP-9 levels, MMP activities, in situ MMP activities, and ROS levels in the RV. cTI concentrations increased after APT, and tempol attenuated these increases. RV oxidative stress after APT increases the RV MMP activities, leading to the degradation of sarcomeric proteins, including cTI. Antioxidant treatment may prevent MMP activation and protect against cardiomyocyte injury after APT.

Acetic acid- and phenyl-p-benzoquinone-induced overt pain-like behavior depends on spinal activation of MAP kinases, PI3K and microglia in mice

The acetic acid and phenyl-p-benzoquinone are easy and fast screening models to access the activity of novel candidates as analgesic drugs and their mechanisms. These models induce a characteristic and quantifiable overt pain-like behavior described as writhing response or abdominal contortions. The knowledge of the mechanisms involved in the chosen model is a crucial step forward demonstrating the mechanisms that the candidate drug would inhibit because the mechanisms triggered in that model will be addressed. Herein, it was investigated the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal Kinase) and p38, PI3K (phosphatidylinositol 3-kinase) and microglia in the writhing response induced by acetic acid and phenyl-p-benzoquinone, and flinch induced by formalin in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing response over 20 min. The nociceptive response in these models were significantly and in a dose-dependent manner reduced by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI3K (wortmannin) inhibitors. Furthermore, the co-treatment with MAP kinase and PI3K inhibitors, at doses that were ineffective as single treatment, significantly inhibited acetic acid- and phenyl-p-benzoquinone-induced nociception. The treatment with microglia inhibitors minocycline and fluorocitrate also diminished the nociceptive response. Similar results were obtained in the formalin test. Concluding. MAP kinases and PI3K are important spinal signaling kinases in acetic acid and phenyl-p-benzoquinone models of overt pain-like behavior and there is also activation of spinal microglia indicating that it is also important to determine whether drugs tested in these models also modulate such spinal mechanisms. (C) 2012 Elsevier Inc. All rights reserved.

Up-regulation of chemokine C-C ligand 2 (CCL2) and C-X-C chemokine 8 (CXCL8) expression by monocytes in chronic idiopathic urticaria

The disturbed cytokinechemokine network could play an important role in the onset of diseases with inflammatory processes such as chronic idiopathic urticaria (CIU). Our main objectives were to evaluate the relation between proinflammatory chemokine serum levels from CIU patients and their response to autologous skin test (ASST) and basophil histamine release (BHR). We also aimed to assess the chemokine secretion by peripheral blood mononuclear cells (PBMC) upon polyclonal stimulus and to evaluate chemokine CC ligand 2/C-X-C chemokine 8 (CCL2/CXCL8) and Toll-like receptor-4 (TLR-4) expression in monocytes. We observed significantly higher serum levels of the CXCL8, CXCL9, CXCL10 and CCL2 in CIU patients compared to the healthy group, regardless of the BHR or ASST response. The basal secretion of CCL2 by PBMC or induced by Staphylococcus aureus enterotoxin A (SEA) was higher in CIU patients than in the control group, as well as for CXCL8 and CCL5 secretions upon phytohaemagglutinin stimulation. Also, up-regulation of CCL2 and CXCL8 mRNA expression was found in monocytes of patients upon SEA stimulation. The findings showed a high responsiveness of monocytes through CCL2/CXCL8 expression, contributing to the creation of a proinflammatory environment in CIU.

Fructose-Induced Hypothalamic AMPK Activation Stimulates Hepatic PEPCK and Gluconeogenesis due to Increased Corticosterone Levels

Fructose consumption causes insulin resistance and favors hepatic gluconeogenesis through mechanisms that are not completely understood. Recent studies demonstrated that the activation of hypothalamic 5'-AMP-activated protein kinase (AMPK) controls dynamic fluctuations in hepatic glucose production. Thus, the present study was designed to investigate whether hypothalamic AMPK activation by fructose would mediate increased gluconeogenesis. Both ip and intracerebroventricular (icv) fructose treatment stimulated hypothalamic AMPK and acetyl-CoA carboxylase phosphorylation, in parallel with increased hepatic phosphoenolpyruvate carboxy kinase (PEPCK) and gluconeogenesis. An increase in AMPK phosphorylation by icv fructose was observed in the lateral hypothalamus as well as in the paraventricular nucleus and the arcuate nucleus. These effects were mimicked by icv 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside treatment. Hypothalamic AMPK inhibition with icv injection of compound C or with injection of a small interfering RNA targeted to AMPK alpha 2 in the mediobasal hypothalamus (MBH) suppressed the hepatic effects of ip fructose. We also found that fructose increased corticosterone levels through a mechanism that is dependent on hypothalamic AMPK activation. Concomitantly, fructose-stimulated gluconeogenesis, hepatic PEPCK expression, and glucocorticoid receptor binding to the PEPCK gene were suppressed by pharmacological glucocorticoid receptor blockage. Altogether the data presented herein support the hypothesis that fructose-induced hypothalamic AMPK activation stimulates hepatic gluconeogenesis by increasing corticosterone levels. (Endocrinology 153: 3633-3645, 2012)

The adipokine chemerin augments vascular reactivity to contractile stimuli via activation of the MEK-ERK1/2 pathway

Aims: Cytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. Main methods: Male, 10-12 week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5 ng/mL or 5 ng/mL, for 1 or 24 h), and isometric contraction was recorded. Protein expression was determined by Western blotting. Key findings: Constrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1 h with chemerin. Chemerin incubation for 24 h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24 h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. Significance: Chemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases. (c) 2012 Elsevier Inc. All rights reserved.

Effects of diazepam on BOLD activation during the processing of aversive faces

This study aimed to measure, using fMRI, the effect of diazepam on the haemodynamic response to emotional faces. Twelve healthy male volunteers (mean age = 24.83 +/- 3.16 years), were evaluated in a randomized, balanced-order, double-blind, placebo-controlled crossover design. Diazepam (10 mg) or placebo was given 1 h before the neuroimaging acquisition. In a blocked design covert face emotional task, subjects were presented with neutral (A) and aversive (B) (angry or fearful) faces. Participants were also submitted to an explicit emotional face recognition task, and subjective anxiety was evaluated throughout the procedures. Diazepam attenuated the activation of right amygdala and right orbitofrontal cortex and enhanced the activation of right anterior cingulate cortex (ACC) to fearful faces. In contrast, diazepam enhanced the activation of posterior left insula and attenuated the activation of bilateral ACC to angry faces. In the behavioural task, diazepam impaired the recognition of fear in female faces. Under the action of diazepam, volunteers were less anxious at the end of the experimental session. These results suggest that benzodiazepines can differentially modulate brain activation to aversive stimuli, depending on the stimulus features and indicate a role of amygdala and insula in the anxiolytic action of benzodiazepines.

Angiotensin II-induced JNK activation is mediated by NAD(P)H oxidase in isolated rat pancreatic islets

Angiotensin II (All), the active component of the renin angiotensin system (RAS), plays a vital role in the regulation of physiological processes of the cardiovascular system, but also has autocrine and paracrine actions in various tissues and organs. Many studies have shown the existence of RAS in the pancreas of humans and rodents. The aim of this study was to evaluate potential signaling pathways mediated by All in isolated pancreatic islets of rats. Phosphorylation of MAPKs (ERK1/2, JNK and p38MAPK), and the interaction between proteins JAK/STAT were evaluated. All increased JAK2/STAT1 (42%) and JAK2/STAT3 (100%) interaction without altering the total content of JAK2. Analyzing the activation of MAPKs (ERK1/2, JNK and p38MAPK) in isolated pancreatic islets from rats we observed that All rapidly (3 min) promoted a significant increase in the phosphorylation degree of these proteins after incubation with the hormone. Curiously JNK protein phosphorylation was inhibited by DPI, suggesting the involvement of NAD(P)H oxidase in the activation of protein. (C) 2012 Elsevier B.V. All rights reserved.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-kappa B in the rat hippocampus

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-kappa B (NF-kappa B) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-kappa B binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-kappa B activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-kappa B, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-alpha (Tnf-alpha), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF alpha B activation and increased NOS and alpha 2/3-Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-kappa B activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-kappa B activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain. (c) 2011 Wiley Periodicals, Inc.

Kaurenoic Acid from Sphagneticola trilobata Inhibits Inflammatory Pain: Effect on Cytokine Production and Activation of the NO-Cyclic GMP-Protein Kinase G-ATP-Sensitive Potassium Channel Signaling Pathway

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-alpha and IL-1 beta. Furthermore, the analgesic effect of 1 was inhibited by L-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein lcinase G-ATP-sensitive potassium channel signaling pathway.

Stimulation of peripheral Kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3K gamma/AKT/nNOS/NO signaling pathway

Background: In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral mu-opioid receptor (MOR) activation are able to direct block PGE(2)-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE(2)-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated. Results: Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE(2)-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3K gamma/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3K gamma null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3K gamma (congruent to 43%). Conclusions: The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3K gamma/AKT signaling. This study extends a previously study of our group suggesting that PI3K gamma/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.