Anxiogenic and antinociceptive effects induced by corticotropin-releasing factor (CRF) injections into the periaqueductal gray are modulated by CRF1 receptor in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peripheral kappa and delta opioid receptors are involved in the antinociceptive effect of crotalphine in a rat model of cancer pain

Cancer pain is an important clinical problem and may not respond satisfactorily to the current analgesic therapy. We have characterized a novel and potent analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in a rat model of cancer pain induced by intraplantar injection of Walker 256 carcinoma cells. Intraplantar injection of tumor cells caused the development of hyperalgesia and allodynia, detected on day 5 after tumor cell inoculation. Crotalphine (6 μg/kg), administered p.o., blocked both phenomena. The antinociceptive effect was detected 1 h after treatment and lasted for up to 48 h. Intraplantar injection of nor-binaltorphimine (50 g/paw), a selective antagonist of κ-opioid receptors, antagonized the antinociceptive effect of the peptide, whereas N,N-diallyl-Tyr-Aib-Phe-Leu (ICI 174,864, 10 μg/paw), a selective antagonist of δ-opioid receptors, partially reversed this effect. On the other hand, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP, 20 g/paw), an antagonist of μ-opioid receptors, did not modify crotalphine-induced antinociception. These data indicate that crotalphine induces a potent and long lasting opioid-mediated antinociception in cancer pain. © 2013 Elsevier Inc.

Efeitos comportamental, clínico e antinociceptivo da crotalfina em eqüinos: comparação com morfina, U50-488H e fenilbutazona

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Erythrina mulungu Alkaloids Are Potent Inhibitors of Neuronal Nicotinic Receptor Currents in Mammalian Cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Assessment of micronucleus frequency in the peripheral blood of female rats in persistent estrus treated with selective estrogen receptor modulators

The aim of this study was to evaluate micronucleus (MN) frequency in polychromatic erythrocytes (PCE) of female rats in persistent estrus (a model developed to mimic polycystic ovary syndrome) treated with selective estrogen receptor modulators (SERMs, tamoxifen, and raloxifene). Forty female Wistar-Hannover rats were divided into four groups of 10 animals each: Group I (normally cycling rats) and Group II (persistent estrus) both received only vehicle, while Group III (persistent estrus) was treated with tamoxifen (250 mu g/animal/day) and Group IV (persistent estrus) was treated with raloxifene (750 mu g/animal/day). Tamoxifen and raloxifene were given by oral gavage beginning on postnatal day 90 and continuing for 30 consecutive days. Peripheral blood samples were collected from tails 1 day following the last exposure. Blood smears were made on glass slides and stained with 10% Giemsa solution. ANOVA and a Tukey post-hoc test were used for data analysis. Mean percentages of MN were 1.82 +/- 0.13, 5.20 +/- 0.24, 3.32 +/- 0.13, and 3.04 +/- 0.12 in Groups I, II, III, and IV, respectively. The results indicate that tamoxifen and raloxifene similarly reduced the formation of MNPCE of female rats in persistent estrus (P < 0.0001 for Groups III and IV vs. Group II), using the dosages and time periods applied in the present study. The data suggest possibly antimutagenic effects of SERMs under high levels of estrogens. The findings also suggest that this is an interesting animal model for studying the genotoxicity of estrogens. Environ. Mol. Mutagen. 2012. (C) 2011 Wiley Periodicals, Inc.

Impact of Glucocorticoid Receptor Gene Polymorphisms on the Metabolic Profile of Adult Patients with the Classical Form of 21-Hydroxylase Deficiency

Background: CAH patients have an increased risk of cardiovascular disease, and it remains unknown if lifelong glucocorticoid (GC) treatment is a contributing factor. In the general population, glucocorticoid receptor gene (NR3C1) polymorphisms are associated with an adverse metabolic profile. Our aim was to analyze the association between the NR3C1 polymorphisms and the metabolic profile of CAH patients. Methodology: Sixty-eight adult patients (34SV/34SW) with a mean age of 28.4 +/- 9 years received dexamethasone (mean 0.27 +/- 0.11 mg/day) to obtain normal androgen levels. SW patients also received fludrocortisone (50 mu g/day). Metabolic syndrome (MetS) was defined by the NCEP ATPIII criteria and obesity by BMI >= 30 kg/m(2). NR3C1 alleles were genotyped, and association analyses with phenotype were carried out with Chi-square, t-test and regression analysis. Results: Obesity and MetS were observed in 23.5% and 7.3% of patients, respectively, and were not correlated with GC doses and treatment duration. BMI was positively correlated with blood pressure (BP), triglycerides (TG), LDL-c levels and HOMA-IR and inversely correlated with HDL-c levels. BclI and A3669G variants were found in 26.4% and 9.6% of alleles, respectively. Heterozygotes for the BclI polymorphism presented with higher BMI (29 kg/m(2) +/- 5.3 vs. 26 kg/m(2) +/- 5.3, respectively) and waist circumference (89 cm +/- 12.7 vs. 81 cm +/- 13, respectively) compared to wild-type subjects. Hypertension was found in 12% of patients and heterozygotes for the BclI polymorphism presented higher systolic BP than wild type subjects. Low HDL-c and high TG levels were identified in 30% and 10% of patients, respectively, and were not associated with the NR3C1 polymorphisms. A3669G carriers and non-carriers did not differ. Conclusion: In addition to GC therapy, the BclI GR variant might play an important role in obesity susceptibility in CAH patients. Genotyping of GR polymorphisms could result in the identification of a subgroup at risk patients, allowing for the establishment of personalized treatment and the avoidance of long-term adverse consequences.

Expression of the P2X(2) receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM: To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. METHODS: The study examined the distribution of the P2X(2) receptor (P2X(2)R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X(2)R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm(2)) and area profile (mu m(2)) of P2X(2)R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. RESULTS: In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X(2)R, nNOS, ChAT and CaIR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X(2)R-IR was observed to co-localize 100% with that for nNOS, ChAT and CaIR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm(2)) of P2X(2)R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CaIR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (mu m(2)) of nNOS-IR, ChAT-IR and CaIR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the nnyenteric ganglia revealed an overall similarity between the two groups. CONCLUSION: We demonstrate increases in P2X(2)R expression and alterations in nNOS, ChAT and CaIR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls. (c) 2012 Baishideng. All rights reserved.

mu(1)- and 5-HT1-dependent mechanisms in the anterior pretectal nucleus mediate the antinociceptive effects of retrosplenial cortex stimulation in rats

Aim: This study examines if injection of cobalt chloride (CoCl2) or antagonists of muscarinic cholinergic (atropine), mu(1)-opioid (naloxonazine) or 5-HT1 serotonergic (methiothepin) receptors into the dorsal or ventral portions of the anterior pretectal nucleus (APtN) alters the antinociceptive effects of stimulating the retrosplenial cortex (RSC) in rats. Main method: Changes in the nociceptive threshold were evaluated using the tail flick or incision pain tests in rats that were electrically stimulated at the RSC after the injection of saline, CoCl2 (1 mM, 0.10 mu L) or antagonists into the dorsal or ventral APtN. Key findings: The injection of CoCl2, naloxonazine (5 mu g/0.10 mu L) or methiothepin (3 mu g/0.10 mu L) into the dorsal APtN reduced the stimulation-produced antinociception from the RSC in the rat tail flick test. Reduction of incision pain was observed following stimulation of the RSC after the injection of the same substances into the ventral APtN. The injection of atropine (10 ng/0.10 mu L) or ketanserine (5 mu g/0.10 mu L) into the dorsal or ventral APtN was ineffective against the antinociception resulting from RSC stimulation. Significance: mu(1)-opioid- and 5-HT1-expressing neurons and cell processes in dorsal and ventral APtN are both implicated in the mediation of stimulation-produced antinociception from the RSC in the rat tail flick and incision pain tests, respectively. (c) 2012 Elsevier Inc. All rights reserved.

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 mu g/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-alpha-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-alpha-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-alpha-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'. (C) 2011 Elsevier Ltd. All rights reserved.

Toll-like receptor 4 contributes to blood pressure regulation and vascular contraction in spontaneously hypertensive rats

Activation of TLRs (Toll-like receptors) induces gene expression of proteins involved in the immune system response. TLR4 has been implicated in the development and progression of CVDs (cardiovascular diseases). Innate and adaptive immunity contribute to hypertension-associated end-organ damage, although the mechanism by which this occurs remains unclear. In the present study, we hypothesize that inhibition of TLR4 decreases BP (blood pressure) and improves vascular contractility in resistance arteries from SHR (spontaneously hypertensive rats). TLR4 protein expression in mesenteric resistance arteries was higher in 15-week-old SHR than in age-matched Wistar controls or in 5-week-old SHR. To decrease the activation of TLR4, 15-week-old SHR and Wistar rats were treated with anti-TLR4 (anti-TLR4 antibody) or non-specific IgG control antibody for 15 days (1 mu g per day, intraperitoneal). Treatment with anti-TLR4 decreased MAP (mean arterial pressure) as well as TLR4 protein expression in mesenteric resistance arteries and IL-6 (interleukin 6) serum levels from SHR when compared with SHR treated with IgG. No changes in these parameters were found in treated Wistar control rats. Mesenteric resistance arteries from anti-TLR4-treated SHR exhibited decreased maximal contractile response to NA (noradrenaline) compared with IgG-treated SHR. Inhibition of COX (cyclo-oxygenase)-1 and COX-2, enzymes related to inflammatory pathways, decreased NA responses only in mesenteric resistance arteries of SHR treated with IgG. COX-2 expression and TXA(2) (thromboxane A(2)) release were decreased in SHR treated with anti-TLR4 compared with IgG-treated SHR. Our results suggest that TLR4 activation contributes to increased BP, low-grade inflammation and plays a role in the augmented vascular contractility displayed by SHR.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

Maternal immune activation in late gestation enhances locomotor response to acute but not chronic amphetamine treatment in male mice offspring: Role of the D1 receptor

Exposure to elevated levels of maternal cytokines can lead to functional abnormalities of the dopaminergic system in the adult offspring, including enhanced amphetamine (AMPH)-induced locomotion. Therefore, it seems reasonable to consider that offspring of challenged mothers would behave differently in models of addictive behavior, such as behavioral sensitization. Thus, we sought to evaluate the effects of prenatal exposure to lipopolysaccharide (LPS) on the locomotor response to acute and chronic AMPH treatment in male mice offspring. For this purpose, LPS (Escherichia coli 0127:B8; 120 mu g/kg) was administered intraperitoneally to pregnant Swiss mice on gestational day 17. At adulthood, male offspring were studied under one of the following conditions: (1) locomotor response to acute AMPH treatment (2.5 or 5.0 mg/kg) in an open field test; (2) behavioral sensitization paradigm, which consists of a daily injection of AMPH (1.0 mg/kg) for 10 days and observation of locomotion in the open field on days 1, 5, 10 (development phase), 15 and 17 (expression phase). The LPS stimulated offspring showed enhancement of the locomotor-stimulant effect after an acute AMPH challenge in comparison to baseline and saline pre-treated mice. They also showed development of behavioral sensitization earlier than the saline pre-treated group, although no changes between saline and LPS pre-treated groups were observed on development or expression of locomotor behavioral sensitization to AMPH. Furthermore, there was up-regulation of D1 receptor protein level within striatum in the LPS-stimulated offspring which was strongly correlated with increased grooming behavior. Taken together, our results indicate that motor and dopaminergic alterations caused by maternal immune activation are restricted to the acute AMPH challenge, mostly due to up-regulation of the D1 receptor within the mesolimbic and nigrostriatal pathways, but no locomotor differences were observed for behavioral sensitization to AMPH. (C) 2012 Elsevier B.V. All rights reserved.

Effects of Continuous Erythropoietin Receptor Activator in Sepsis-Induced Acute Kidney Injury and Multi-Organ Dysfunction

Background: Despite advances in supportive care, sepsis-related mortality remains high, especially in patients with acute kidney injury (AKI). Erythropoietin can protect organs against ischemia and sepsis. This effect has been linked to activation of intracellular survival pathways, although the mechanism remains unclear. Continuous erythropoietin receptor activator (CERA) is an erythropoietin with a unique pharmacologic profile and long half-life. We hypothesized that pretreatment with CERA would be renoprotective in the cecal ligation and puncture (CLP) model of sepsis-induced AKI. Methods: Rats were randomized into three groups: control; CLP; and CLP+CERA (5 mu g/kg body weight, i.p. administered 24 h before CLP). At 24 hours after CLP, we measured creatinine clearance, biochemical variables, and hemodynamic parameters. In kidney tissue, we performed immunoblotting-to quantify expression of the Na-K-2Cl cotransporter (NKCC2), aquaporin 2 (AQP2), Toll-like receptor 4 (TLR4), erythropoietin receptor (EpoR), and nuclear factor kappa B (NF-kappa B)-and immunohistochemical staining for CD68 (macrophage infiltration). Plasma interleukin (IL)-2, IL-1 beta, IL-6, IL-10, interferon gamma, and tumor necrosis factor alpha were measured by multiplex detection. Results: Pretreatment with CERA preserved creatinine clearance and tubular function, as well as the expression of NKCC2 and AQP2. In addition, CERA maintained plasma lactate at normal levels, as well as preserving plasma levels of transaminases and lactate dehydrogenase. Renal expression of TLR4 and NF-kappa B was lower in CLP+CERA rats than in CLP rats (p<0.05 and p<0.01, respectively), as were CD68-positive cell counts (p<0.01), whereas renal EpoR expression was higher (p<0.05). Plasma levels of all measured cytokines were lower in CLP+CERA rats than in CLP rats. Conclusion: CERA protects against sepsis-induced AKI. This protective effect is, in part, attributable to suppression of the inflammatory response.

Kinin-B2 Receptor Mediated Neuroprotection after NMDA Excitotoxicity Is Reversed in the Presence of Kinin-B1 Receptor Agonists

Background: Kinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-Daspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Principal Findings: Bradykinin at 10 nM and 1 mu M concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059,showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. Conclusions: Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.