RESUMO
BACKGROUND AND PURPOSE: Targeting the prostaglandin I2 prostanoid (IP) receptor to reduce stroke injury has been hindered by the lack of selective drugs. MRE-269 is the active metabolite of selexipag showing a high selectivity toward the IP receptor. Selexipag has been recently approved for clinical use in pulmonary hypertension. We hypothesized that postischemic treatment with MRE-269 provides long-lasting neuroprotection with improved neurological outcomes in a clinically relevant rat stroke model. METHODS: Aged male Sprague-Dawley rats underwent transient middle cerebral artery occlusion and were randomly selected to receive either vehicle or MRE-269 (0.25 mg/kg) intravenously starting at 4.5 hours post ischemia. Accelerating rotarod and adhesive removal tests were conducted before and at 3, 7, 14, and 21 days after stroke. Infarct volume was quantified by magnetic resonance imaging at 48 hours and 21 days post middle cerebral artery occlusion. In parallel experiments, cerebral cortex samples from stroke and nonstroke sides from vehicle- and MRE-269-treated groups were collected at 18 hours post middle cerebral artery occlusion for molecular biology analyses. RESULTS: Quantitative magnetic resonance imaging data showed that postischemic MRE-269 treatment significantly reduced infarct volume compared with vehicle-treated rats at both 48 hours and 3 weeks after stroke. MRE-269 treatment resulted in a significant long-term recovery in both locomotor and somatosensory functions after middle cerebral artery occlusion, which was associated with a reduced weight loss in animals receiving the IP receptor agonist. Postischemic MRE-269 treatment reduced proinflammatory cytokines/chemokines and oxidative stress. Damage to the blood-brain barrier, as assessed by extravasation of immunoglobulin G to the ischemic brain, was significantly reduced by MRE-269, which was associated with a reduction in matrix metalloproteinase-9 activity in the brain of stroked aged rats given the IP agonist at 4.5 hours after ischemia onset. CONCLUSIONS: Our data suggest that targeting the IP receptor with MRE-269 is a novel strategy to reduce cerebral ischemia injury and promote long-term neurological recovery in ischemic stroke.
Assuntos
Acetatos/farmacologia , Isquemia Encefálica/tratamento farmacológico , Epoprostenol/análise , Pirazinas/farmacologia , Receptores de Prostaglandina/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico , Acetatos/administração & dosagem , Fatores Etários , Animais , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/imunologia , Infarto da Artéria Cerebral Média , Masculino , Pirazinas/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/imunologiaRESUMO
Tobacco addiction is characterized by a lack of control over smoking and relapse after periods of abstinence. Smoking cessation leads to a dysphoric state that contributes to relapse to smoking. After the acute withdrawal phase, exposure to stressors increases the risk for relapse. Blockade of melanocortin 4 (MC4 ) receptors has anxiolytic and antidepressant-like effects in animal models. The aim of these studies was to investigate the role of MC4 receptors in the dysphoria associated with nicotine withdrawal and stress-induced reinstatement of nicotine seeking. To study stress-induced reinstatement, rats self-administered nicotine for 16 days and then nicotine seeking was extinguished by substituting saline for nicotine. Nicotine seeking was reinstated by intermittent footshock stress. The intracranial self-stimulation (ICSS) procedure was used to assess the negative mood state associated with nicotine withdrawal. Elevations in the ICSS thresholds are indicative of a dysphoric state. The selective MC4 receptor antagonists HS014 and HS024 prevented stress-induced reinstatement of extinguished nicotine seeking. Drug doses that prevented stress-induced relapse did not affect responding for food pellets, which indicates that the drugs did not induce sedation or motor impairments. In the ICSS experiments, the nicotinic acetylcholine receptor antagonist mecamylamine elevated the ICSS thresholds of the nicotine-dependent rats. Pre-treatment with HS014 or HS024 did not prevent the elevations in ICSS thresholds. These studies indicate that MC4 receptors play a critical role in stress-induced reinstatement of nicotine seeking, but these receptors may not play a role in the dysphoria associated with acute nicotine withdrawal.
Assuntos
Comportamento de Procura de Droga/fisiologia , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Receptor Tipo 4 de Melanocortina/metabolismo , Estresse Psicológico/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Tabagismo/metabolismo , Animais , Comportamento de Procura de Droga/efeitos dos fármacos , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Peptídeos Cíclicos/farmacologia , Ratos , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Recidiva , Síndrome de Abstinência a Substâncias/etiologiaRESUMO
BACKGROUND: Acute nicotine administration potentiates brain reward function and enhances motor and cognitive function. These studies investigated which brain areas are being activated by a wide range of doses of nicotine, and if this is diminished by pretreatment with the nonselective nicotinic receptor antagonist mecamylamine. METHODS: Drug-induced changes in brain activity were assessed by measuring changes in the blood oxygen level dependent (BOLD) signal using an 11.1-Tesla magnetic resonance scanner. In the first experiment, nicotine naïve rats were mildly anesthetized and the effect of nicotine (0.03-0.6 mg/kg) on the BOLD signal was investigated for 10 min. In the second experiment, the effect of mecamylamine on nicotine-induced brain activity was investigated. RESULTS: A high dose of nicotine increased the BOLD signal in brain areas implicated in reward signaling, such as the nucleus accumbens shell and the prelimbic area. Nicotine also induced a dose-dependent increase in the BOLD signal in the striato-thalamo-orbitofrontal circuit, which plays a role in compulsive drug intake, and in the insular cortex, which contributes to nicotine craving and relapse. In addition, nicotine induced a large increase in the BOLD signal in motor and somatosensory cortices. Mecamylamine alone did not affect the BOLD signal in most brain areas, but induced a negative BOLD response in cortical areas, including insular, motor, and somatosensory cortices. Pretreatment with mecamylamine completely blocked the nicotine-induced increase in the BOLD signal. CONCLUSIONS: These studies demonstrate that acute nicotine administration activates brain areas that play a role in reward signaling, compulsive behavior, and motor and cognitive function.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Animais , Mapeamento Encefálico , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Comportamento Compulsivo/fisiopatologia , Relação Dose-Resposta a Droga , Imageamento por Ressonância Magnética , Masculino , Mecamilamina/farmacologia , Antagonistas Nicotínicos/farmacologia , Oxigênio/sangue , Ratos Wistar , RecompensaRESUMO
Potassium channel interacting proteins (KChIPs) are members of a family of calcium binding proteins that interact with Kv4 potassium (K(+)) channel primary subunits and also act as transcription factors. The Kv4 subunit is a primary K(+) channel pore-forming subunit, which contributes to the somatic and dendritic A-type currents throughout the nervous system. These A-type currents play a key role in the regulation of neuronal excitability and dendritic processing of incoming synaptic information. KChIP3 is also known as calsenilin and as the transcription factor, downstream regulatory element antagonist modulator (DREAM), which regulates a number of genes including prodynorphin. KChIP3 and Kv4 primary channel subunits are highly expressed in hippocampus, an area of the brain important for learning and memory. Through its various functions, KChIP3 may play a role in the regulation of synaptic plasticity and learning and memory. We evaluated the role of KChIP3 in a hippocampus-dependent memory task, contextual fear conditioning. Male KChIP3 knockout (KO) mice showed significantly enhanced memory 24 hours after training as measured by percent freezing. In addition, we found that membrane association and interaction with Kv4.2 of KChIP3 protein was significantly decreased and nuclear KChIP3 expression was increased six hours after the fear conditioning training paradigm with no significant change in KChIP3 mRNA. In addition, prodynorphin mRNA expression was significantly decreased six hours after fear conditioning training in wild-type (WT) but not in KO animals. These data suggest a role for regulation of gene expression by KChIP3/DREAM/calsenilin in consolidation of contextual fear conditioning memories.
Assuntos
Condicionamento Clássico/fisiologia , Medo , Regulação da Expressão Gênica/fisiologia , Proteínas Interatuantes com Canais de Kv/fisiologia , Proteínas Repressoras/fisiologia , Análise de Variância , Animais , Comportamento Animal , Nucléolo Celular/metabolismo , Sinais (Psicologia) , Encefalinas/genética , Comportamento Exploratório/fisiologia , Reação de Congelamento Cataléptica/fisiologia , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Imunoprecipitação/métodos , Proteínas Interatuantes com Canais de Kv/deficiência , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/genética , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Teste de Desempenho do Rota-Rod , Limiar Sensorial/fisiologia , Canais de Potássio Shal/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A4 (LXA 4) is an anti-inflammatory, pro-resolution lipid mediator that reduces neuroinflammation in stroke. Since LXA 4 is rapidly inactivated, potent analogs have been synthesized, including BML-111. We hypothesized that post-ischemic, intravenous treatment with BML-111 for 1 week would provide neuroprotection and reduce neurobehavioral deficits at 4 weeks after ischemic stroke in rats. Additionally, we investigated the potential protective mechanisms of BML-111 on the post-stroke molecular and cellular profile. METHODS: A total of 133 male Sprague-Dawley rats were subjected to 90 min of transient middle cerebral artery occlusion (MCAO) and BML-111 administration was started at the time of reperfusion. Two methods of week-long BML-111 intravenous administration were tested: continuous infusion via ALZET ® osmotic pumps (1.25 and 3.75 µg µl-1 hr-1), or freshly prepared daily single injections (0.3, 1, and 3 mg/kg). We report for the first time on the stability of BML-111 and characterized an optimal dose and a dosing schedule for the administration of BML-111. RESULTS: One week of BML-111 intravenous injections did not reduce infarct size or improve behavioral deficits 4 weeks after ischemic stroke. However, post-ischemic treatment with BML-111 did elicit early protective effects as demonstrated by a significant reduction in infarct volume and improved sensorimotor function at 1 week after stroke. This protection was associated with reduced pro-inflammatory cytokine and chemokine levels, decreased M1 CD40+ macrophages, and increased alternatively activated, anti-inflammatory M2 microglia/macrophage cell populations in the post-ischemic brain. CONCLUSION: These data suggest that targeting the endogenous LXA 4 pathway could be a promising therapeutic strategy for the treatment of ischemic stroke. More work is necessary to determine whether a different dosing regimen or more stable LXA 4 analogs could confer long-term protection.
Assuntos
Encéfalo/imunologia , Ácidos Heptanoicos/farmacologia , Infarto da Artéria Cerebral Média , Inflamação/imunologia , Lipoxinas , Acidente Vascular Cerebral , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/imunologia , Lipoxinas/agonistas , Lipoxinas/imunologia , Masculino , Microglia/efeitos dos fármacos , Fatores de Proteção , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/imunologia , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/terapia , TempoRESUMO
Activation of the angiotensin II type 2 receptor (AT2R) by administration of Compound 21 (C21), a selective AT2R agonist, induces neuroprotection in models of ischemic stroke in young adult animals. The mechanisms of this neuroprotective action are varied, and may include direct and indirect effects of AT2R activation. Our objectives were to assess the long-term protective effects of post-stroke C21 treatments in a clinically-relevant model of stroke in aged rats and to characterize the cellular localization of AT2Rs in the mouse brain of transgenic reporter mice following stroke. Intraperitoneal injections of C21 (0.03mg/kg) after ischemic stroke induced by transient monofilament middle cerebral artery occlusion resulted in protective effects that were sustained for up to at least 3-weeks post-stroke. These included improved neurological function across multiple assessments and a significant reduction in infarct volume as assessed by magnetic resonance imaging. We also found AT2R expression to be on neurons, not astrocytes or microglia, in normal female and male mouse brains. Stroke did not induce altered cellular localization of AT2R when assessed at 7 and 14 days post-stroke. These findings demonstrate that the neuroprotection previously characterized only during earlier time points using stroke models in young animals is sustained long-term in aged rats, implying even greater clinical relevance for the study of AT2R agonists for the acute treatment of ischemic stroke in human disease. Further, it appears that this sustained neuroprotection is likely due to a mix of both direct and indirect effects stemming from selective activation of AT2Rs on neurons or other cells besides astrocytes and microglia.
Assuntos
Fármacos Neuroprotetores/farmacologia , Receptor Tipo 2 de Angiotensina/agonistas , Acidente Vascular Cerebral/fisiopatologia , Animais , Feminino , Masculino , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Tobacco addiction is characterized by a negative mood state upon smoking cessation and relapse after periods of abstinence. Clinical studies indicate that negative mood states lead to craving and relapse. The partial α4/α6/ß2* nicotinic acetylcholine receptor (nAChR) agonists varenicline and cytisine are widely used as smoking cessation treatments. Varenicline has been approved in the United States for smoking cessation and cytisine is used in Eastern European countries. Despite the widespread use of these compounds, very little is known about their effects on mood states. These studies investigated the effects of varenicline, cytisine, and the cytisine-derivative 3-(pyridin-3'-yl)-cytisine (3-pyr-Cyt) on brain reward function in nicotine-naive and nicotine-withdrawing rats. The cytisine-derivative 3-pyr-Cyt is a very weak α4ß2* nAChR partial agonist and like cytisine and varenicline has antidepressant-like effects in animal models. The intracranial self-stimulation (ICSS) procedure was used to investigate the effects of these compounds on brain reward function. Elevations in ICSS thresholds reflect a dysphoric state and a lowering of thresholds is indicative of a potentiation of brain reward function. It was shown that acute administration of nicotine and varenicline lowered ICSS thresholds. Acute administration of cytisine or 3-pyr-Cyt did not affect ICSS thresholds. Discontinuation of chronic, 14 days, nicotine administration led to elevations in ICSS thresholds that lasted for about 2 days. Varenicline and cytisine, but not 3-pyr-Cyt, diminished the nicotine withdrawal-induced elevations in ICSS thresholds. In conclusion, these studies indicate that varenicline and cytisine diminish the dysphoric-like state associated with nicotine withdrawal and may thereby prevent relapse to smoking in humans.
Assuntos
Alcaloides/administração & dosagem , Benzazepinas/administração & dosagem , Nicotina/administração & dosagem , Nicotina/efeitos adversos , Agonistas Nicotínicos/administração & dosagem , Quinoxalinas/administração & dosagem , Síndrome de Abstinência a Substâncias/prevenção & controle , Animais , Azocinas/administração & dosagem , Bombas de Infusão Implantáveis , Masculino , Quinolizinas/administração & dosagem , Ratos , Ratos Wistar , Síndrome de Abstinência a Substâncias/psicologia , VareniclinaRESUMO
The majority of smokers relapse during the acute withdrawal phase when withdrawal symptoms are most severe. The goal of the present studies was to investigate the role of corticotropin-releasing factor (CRF) and noradrenergic transmission in the central nucleus of the amygdala (CeA) in the dysphoria associated with smoking cessation. It was investigated if blockade of CRF1 receptors, blockade of α1-adrenergic receptors, or stimulation of α2-adrenergic receptors in the CeA diminishes the deficit in brain reward function associated with nicotine withdrawal in rats. Nicotine dependence was induced by implanting minipumps that delivered a nicotine solution. Withdrawal was precipitated with the nicotinic acetylcholine receptor antagonist mecamylamine. A discrete-trial intracranial self-stimulation procedure was used to assess the negative affective aspects of nicotine withdrawal. Elevations in brain reward thresholds are indicative of a deficit in brain reward function. In all the experiments, mecamylamine elevated the brain reward thresholds of the rats chronically treated with nicotine and did not affect the brain reward thresholds of the saline-treated control rats. Intra-CeA administration of the CRF1 receptor antagonist R278995/CRA0450 completely prevented the mecamylamine-induced elevations in brain reward thresholds in the nicotine-treated rats and did not affect the brain reward thresholds of the saline-treated control rats. R278995/CRA0450 has also been shown to block sigma-1 receptors but there is no evidence that this could affect negative mood states. Intra-CeA administration of the α1-adrenergic receptor antagonist prazosin or the α2-adrenergic receptor agonist clonidine did not affect the brain reward thresholds of the nicotine or saline-treated rats. These studies suggest that CRF1 receptor antagonists may diminish the dysphoria associated with smoking cessation by blocking CRF1 receptors in the CeA.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Abandono do Hábito de Fumar/psicologia , Síndrome de Abstinência a Substâncias/psicologia , Tabagismo/psicologia , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Benzenossulfonatos/farmacologia , Clonidina/farmacologia , Eletrodos Implantados , Masculino , Mecamilamina/farmacologia , Antagonistas Nicotínicos/farmacologia , Prazosina/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Wistar , Recompensa , Síndrome de Abstinência a Substâncias/tratamento farmacológicoRESUMO
RATIONALE: Tobacco withdrawal is characterized by a negative mood state and relatively mild somatic symptoms. Increased noradrenergic transmission has been reported to play an important role in opioid withdrawal, but little is known about the role of noradrenergic transmission in nicotine withdrawal. OBJECTIVES: The aim of these experiments was to investigate the effects of prazosin, clonidine, and propranolol on the negative mood state and somatic signs associated with nicotine withdrawal in rats. METHODS: A discrete-trial intracranial self-stimulation procedure was used to assess the negative affective state of nicotine withdrawal. Elevations in brain reward thresholds are indicative of a deficit in brain reward function. RESULTS: In all the experiments, the nicotinic acetylcholine receptor antagonist mecamylamine (3 mg/kg) elevated the brain reward thresholds of the nicotine-treated rats and did not affect those of the control rats. The α1-adrenergic receptor antagonist prazosin (0.0625 and 0.125 mg/kg) dose-dependently attenuated the elevations in brain reward thresholds associated with precipitated nicotine withdrawal. The α2-adrenergic receptor agonist clonidine (10-40 µg/kg) and the nonselective ß-adrenergic receptor antagonist propranolol (2.5-10 mg/kg) did not attenuate the elevations in brain reward thresholds associated with nicotine withdrawal. Furthermore, mecamylamine (2 mg/kg) induced more somatic signs in the nicotine-treated rats than in the control rats. Clonidine and propranolol, but not prazosin, decreased the total number of somatic signs associated with nicotine withdrawal. CONCLUSION: Blockade of α1-adrenergic receptors attenuates the deficit in brain reward function associated with nicotine withdrawal. Antagonism of ß-adrenergic receptors or stimulation of α2-adrenergic receptors attenuates the somatic symptoms of nicotine withdrawal.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Comportamento Animal/efeitos dos fármacos , Clonidina/farmacologia , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Prazosina/farmacologia , Propranolol/farmacologia , Recompensa , Síndrome de Abstinência a Substâncias/tratamento farmacológico , Tabagismo/tratamento farmacológico , Animais , Relação Dose-Resposta a Droga , Masculino , Antagonistas Nicotínicos/farmacologia , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacos , Reforço Psicológico , Síndrome de Abstinência a Substâncias/etiologia , Síndrome de Abstinência a Substâncias/psicologia , Tabagismo/psicologiaRESUMO
Small conductance, Ca2+-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 microm) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxyl-terminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser465 (minor site) and three amino acid residues Ser568, Ser569, and Ser570 (major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression.