RESUMO
Disturbances in the function of the mesostriatal dopamine system may contribute to the development and maintenance of chronic pain, including its sensory and emotional/cognitive aspects. In the present study, we assessed the influence of chronic constriction injury (CCI) of the sciatic nerve on the expression of genes coding for dopamine and opioid receptors as well as opioid propeptides in the mouse mesostriatal system, particularly in the nucleus accumbens. We demonstrated bilateral increases in mRNA levels of the dopamine D1 and D2 receptors (the latter accompanied by elevated protein level), opioid propeptides proenkephalin and prodynorphin, as well as delta and kappa (but not mu) opioid receptors in the nucleus accumbens at 7 to 14 days after CCI. These results show that CCI-induced neuropathic pain is accompanied by a major transcriptional dysregulation of molecules involved in dopaminergic and opioidergic signaling in the striatum/nucleus accumbens. Possible functional consequences of these changes include opposite effects of upregulated enkephalin/delta opioid receptor signaling vs. dynorphin/kappa opioid receptor signaling, with the former most likely having an analgesic effect and the latter exacerbating pain and contributing to pain-related negative emotional states.
Assuntos
Neuralgia/metabolismo , Medição da Dor/métodos , Prosencéfalo/metabolismo , Receptores Dopaminérgicos/biossíntese , Receptores Opioides delta/biossíntese , Receptores Opioides kappa/biossíntese , Animais , Corpo Estriado/metabolismo , Encefalinas/biossíntese , Encefalinas/genética , Expressão Gênica , Masculino , Camundongos , Neuralgia/genética , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Receptores Dopaminérgicos/genética , Receptores Opioides delta/genética , Receptores Opioides kappa/genética , Receptores Opioides mu/biossíntese , Receptores Opioides mu/genéticaRESUMO
Social isolation stress (SIS) as a type of chronic stress could induce depressive- and anxiety-like behaviors. Our study evaluates the role of opioid system on negative behavioral impacts of SIS in male NMRI mice. We investigated effects of morphine, a nonselective opioid receptor (OR) agonist, naltrexone (NLX), an OR antagonist, naltrindole (NLT), a delta opioid receptor (DOR) antagonist, SNC80, a DOR agonist, U-69593, a kappa opioid receptor (KOR) agonist, nor-Binaltorphimine, a selective KOR antagonist and cyprodime hydrochloride a selective mu opioid receptor (MOR) antagonist on depressive- and anxiety-like behaviors. Using RT-PCR we evaluated ORs gene expression in mice brain. Our findings showed that SIS induced anxiety- and depressive-like behavior in the forced swimming test, open field test, splash test and hole-board test. Moreover, administration of SNC-80 significantly mitigated anxiety- and depressive-like behaviors. NLT decreased grooming-activity in the splash test. Excitingly, administration of agents affecting KOR failed to alter the negative effects of SIS. RT-PCR demonstrated that MOR and KOR gene expression decreased in socially isolated mice; however, SIS did not affect DORs expression. Our findings suggest that SIS at least in part, probably via altering endogenous opioids particularly MORs and KORs but not DORs mediated negative impacts on behavior; also, it could be concluded that DORs might be considered as a novel target for studying depression and anxiety.
Assuntos
Receptores Opioides delta/biossíntese , Receptores Opioides kappa/biossíntese , Receptores Opioides mu/biossíntese , Isolamento Social/psicologia , Estresse Psicológico/metabolismo , Estresse Psicológico/psicologia , Analgésicos Opioides/farmacologia , Animais , Ansiedade/metabolismo , Ansiedade/psicologia , Depressão/metabolismo , Depressão/psicologia , Masculino , Camundongos , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidoresRESUMO
There is an important emerging role for the endogenous opioid dynorphin (DYN) and the kappa-opioid receptor (KOR) in the treatment of alcohol dependence. Evidence suggests that the DYN/KOR system in the bed nucleus of the stria terminalis (BNST) contributes to maladaptive behavioral regulation during withdrawal in alcohol dependence. The current experiments were designed to assess dysregulation of the BNST DYN/KOR system by evaluating alcohol dependence-induced changes in DYN/KOR gene expression (Pdyn and Oprk1, respectively), and the sensitivity of alcohol self-administration, negative affective-like behavior and physiological withdrawal to intra-BNST KOR antagonism during acute withdrawal. Wistar rats trained to self-administer alcohol, or not trained, were subjected to an alcohol dependence induction procedure (14â¯h alcohol vapor/10â¯h air) or air-exposure. BNST micropunches from air- and vapor-exposed animals were analyzed using RT-qPCR to quantify dependence-induced changes in Pdyn and Oprk1 mRNA expression. In addition, vapor- and air-exposed groups received an intra-BNST infusion of a KOR antagonist or vehicle prior to measurement of alcohol self-administration. A separate cohort of vapor-exposed rats was assessed for physiological withdrawal and negative affective-like behavior signs following intra-BNST KOR antagonism. During acute withdrawal, following alcohol dependence induction, there was an upregulation in Oprk1 mRNA expression in alcohol self-administering animals, but not non-alcohol self-administering animals, that confirmed dysregulation of the KOR/DYN system within the BNST. Furthermore, intra-BNST KOR antagonism attenuated escalated alcohol self-administration and negative affective-like behavior during acute withdrawal without reliably impacting physiological symptoms of withdrawal. The results confirm KOR system dysregulation in the BNST in alcohol dependence, illustrating the therapeutic potential of targeting the KOR to treat alcohol dependence.
Assuntos
Alcoolismo/metabolismo , Alcoolismo/psicologia , Receptores Opioides kappa/biossíntese , Núcleos Septais/metabolismo , Síndrome de Abstinência a Substâncias/prevenção & controle , Alcoolismo/complicações , Animais , Condicionamento Operante/efeitos dos fármacos , Dinorfinas/biossíntese , Etanol/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , RNA Mensageiro/biossíntese , Ratos , Receptores Opioides kappa/antagonistas & inibidores , Autoadministração , Núcleos Septais/efeitos dos fármacos , Regulação para Cima , Vocalização Animal/efeitos dos fármacosRESUMO
Regulation of µ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48â¯h with mitogenic dose of Con A. The unstimulated lymphocytes did not express µ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both µ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of µ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized µ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [35S]GTPγS binding assay. The up-regulation of µ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gß subunits were unchanged. The level of ß-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, Gi1α/Gi2α proteins and ß-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.
Assuntos
Linfócitos/metabolismo , Receptores Opioides delta/biossíntese , Receptores Opioides kappa/biossíntese , Receptores Opioides mu/biossíntese , Baço/metabolismo , Animais , Concanavalina A/farmacologia , Linfócitos/efeitos dos fármacos , Masculino , Mitógenos/farmacologia , Ratos , Ratos Wistar , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides mu/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Regulação para CimaRESUMO
Dopamine signaling encodes reward learning and motivated behavior through modulation of synaptic signaling in the nucleus accumbens, and aberrations in these processes are thought to underlie obsessive behaviors associated with alcohol abuse. The nucleus accumbens is divided into core and shell sub-regions with overlapping but also divergent contributions to behavior. Here we optogenetically targeted dopamine projections to the accumbens allowing us to isolate stimulation of dopamine terminals ex vivo. We applied 5 pulse (phasic) light stimulations to probe intrinsic differences in dopamine release parameters across regions. Also, we exposed animals to 4weeks of chronic intermittent ethanol vapor and measured phasic release. We found that initial release probability, uptake rate and autoreceptor inhibition were greater in the accumbens core compared to the shell, yet the shell showed greater phasic release ratios. Following chronic ethanol, uptake rates were increased in the core but not the shell, suggesting region-specific neuronal adaptations. Conversely, kappa opioid receptor function was upregulated in both regions to a similar extent, suggesting a local mechanism of kappa opioid receptor regulation that is generalized across the nucleus accumbens. These data suggest that dopamine axons in the nucleus accumbens core and shell display differences in intrinsic release parameters, and that ethanol-induced adaptations to dopamine neuron terminal fields may not be homogeneous. Also, chronic ethanol exposure induces an upregulation in kappa opioid receptor function, providing a mechanism for potential over-inhibition of accumbens dopamine signaling which may negatively impact downstream synaptic function and ultimately bias choice towards previously reinforced alcohol use behaviors.
Assuntos
Alcoolismo/metabolismo , Depressores do Sistema Nervoso Central/toxicidade , Dopamina/metabolismo , Etanol/toxicidade , Núcleo Accumbens/metabolismo , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/efeitos dos fármacos , Optogenética , Técnicas de Cultura de Órgãos , Receptores Opioides kappa/biossíntese , Transmissão Sináptica/efeitos dos fármacosRESUMO
Evidence has demonstrated that dynorphin (DYN) and the kappa opioid receptor (KOR) system contribute to various psychiatric disorders, including anxiety, depression, and addiction. More recently, this endogenous opioid system has received increased attention as a potential therapeutic target for treating alcohol use disorders. In this review, we provide an overview and synthesis of preclinical studies examining the influence of alcohol (ethanol [EtOH]) exposure on DYN/KOR expression and function, as well as studies examining the effects of DYN/KOR manipulation on EtOH's rewarding and aversive properties. We then describe work that has characterized effects of KOR activation and blockade on EtOH self-administration and EtOH dependence/withdrawal-related behaviors. Finally, we address how the DYN/KOR system may contribute to stress-EtOH interactions. Despite an apparent role for the DYN/KOR system in motivational effects of EtOH, support comes from relatively few studies. Nevertheless, review of this literature reveals several common themes: (i) rodent strains genetically predisposed to consume more EtOH generally appear to have reduced DYN/KOR tone in brain reward circuitry; (ii) acute and chronic EtOH exposure typically up-regulate the DYN/KOR system; (iii) KOR antagonists reduce behavioral indices of negative affect associated with stress and chronic EtOH exposure/withdrawal; and (iv) KOR antagonists are effective in reducing EtOH consumption, but are often more efficacious under conditions that engender high levels of consumption, such as dependence or stress exposure. These results support the contention that the DYN/KOR system plays a significant role in contributing to dependence- and stress-induced elevation in EtOH consumption. Overall, more comprehensive analyses (on both behavioral and mechanistic levels) are needed to provide additional insight into how the DYN/KOR system is engaged and adapts to influence the motivation effects of EtOH. This information will be critical for the development of new pharmacological agents targeting KORs as promising novel therapeutics for alcohol use disorders and comorbid affective disorders.
Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Dinorfinas/biossíntese , Etanol/administração & dosagem , Motivação/efeitos dos fármacos , Receptores Opioides kappa/biossíntese , Consumo de Bebidas Alcoólicas/genética , Consumo de Bebidas Alcoólicas/psicologia , Animais , Dinorfinas/genética , Humanos , Motivação/fisiologia , Receptores Opioides kappa/genéticaRESUMO
Anxiety disorders are one of the most common and debilitating mental illnesses worldwide. Growing evidence indicates an age-dependent rise in the incidence of anxiety disorders from adolescence through adulthood, suggestive of underlying neurodevelopmental mechanisms. Kappa opioid receptors (KORs) are known to contribute to the development and expression of anxiety; however, the functional role of KORs in the basolateral amygdala (BLA), a brain structure critical in mediating anxiety, particularly across ontogeny, are unknown. Using whole-cell patch-clamp electrophysiology in acute brain slices from adolescent (postnatal day (P) 30-45) and adult (P60+) male Sprague-Dawley rats, we found that the KOR agonist, U69593, increased the frequency of GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in the adolescent BLA, without an effect in the adult BLA or on sIPSC amplitude at either age. The KOR effect was blocked by the KOR antagonist, nor-BNI, which alone did not alter GABA transmission at either age, and the effect of the KOR agonist was TTX-sensitive. Additionally, KOR activation did not alter glutamatergic transmission in the BLA at either age. In contrast, U69593 inhibited sIPSC frequency in the central amygdala (CeA) at both ages, without altering sIPSC amplitude. Western blot analysis of KOR expression indicated that KOR levels were not different between the two ages in either the BLA or CeA. This is the first study to provide compelling evidence for a novel and unique neuromodulatory switch in one of the primary brain regions involved in initiating and mediating anxiety that may contribute to the ontogenic rise in anxiety disorders.
Assuntos
Envelhecimento/fisiologia , Complexo Nuclear Basolateral da Amígdala/metabolismo , Receptores Opioides kappa/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Benzenoacetamidas/antagonistas & inibidores , Benzenoacetamidas/farmacologia , Ácido Glutâmico/fisiologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Masculino , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Pirrolidinas/antagonistas & inibidores , Pirrolidinas/farmacologia , Ratos , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides kappa/biossíntese , Transmissão Sináptica/efeitos dos fármacosRESUMO
The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non-neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR-Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR-Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR-Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR-Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR-Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR.
Assuntos
Técnicas de Transferência de Genes , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Integrases/química , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Receptores Opioides kappa/biossíntese , Transdução de SinaisRESUMO
Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), µ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists- U-69593, DAMGO and nociceptin- inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure.
Assuntos
Adenilil Ciclases/metabolismo , Analgésicos Opioides/farmacologia , Buprenorfina/farmacologia , Receptores Opioides kappa/biossíntese , Receptores Opioides mu/biossíntese , Inibidores de Adenilil Ciclases/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Técnicas ImunológicasRESUMO
BACKGROUND: Approximately 10 to 15% of women consume alcohol (ethanol [EtOH]) during pregnancy in the United States. Even low amounts of EtOH consumption during pregnancy can elicit long-term consequences. Prenatal experience with as few as 3 drinks has been associated with increase problem drinking in adulthood. Such effects are corroborated in rodents; however, the underlying neural adaptations contributing to this effect are not clear. In the current set of experiments, we investigated whether changes in EtOH responding following prenatal EtOH exposure involved kappa opioid receptor activation and expression. METHODS: Sprague-Dawley rats were prenatally exposed to low levels of alcohol (1.0 g/kg) during late gestation (gestational days 17 to 20 [GD17-20]) via intragastric intubation of pregnant dams. Following birth, EtOH intake, kappa- and mu-opioid-induced place conditioning, and kappa opioid receptor expression in mesolimbic brain regions were assessed in infant rats (postnatal days 14 to 15 [PD14-15]) that were offspring of dams given EtOH, vehicle, or untreated, during pregnancy. RESULTS: Animals exposed to prenatal alcohol drank more alcohol later in life and exhibited significant changes in the kappa opioid system. While control subjects found kappa opioid activation aversive, animals exposed to EtOH prenatally exhibited either no aversion or appetitive responding. Further analysis revealed that synaptosomal kappa opioid receptor expression was significantly decreased in brain areas implicated in responding to EtOH. CONCLUSIONS: Overall, these data suggest that prenatal EtOH affects kappa opioid function and expression and that these changes may be involved in increased drinking later in life.
Assuntos
Etanol/farmacologia , Naltrexona/análogos & derivados , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Pirrolidinas/farmacologia , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Consumo de Bebidas Alcoólicas/tratamento farmacológico , Consumo de Bebidas Alcoólicas/metabolismo , Animais , Química Encefálica/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Etanol/administração & dosagem , Feminino , Expressão Gênica/efeitos dos fármacos , Masculino , Naltrexona/farmacologia , Gravidez , Ratos Sprague-Dawley , Receptores Opioides kappa/biossínteseRESUMO
Cocaine induces neurochemical changes of endogenous prodynorphin-kappa opioid receptor (pDYN-KOP) and pronociceptin/orphaninFQ-nociceptin receptor (pN/OFQ-NOP) systems. Both systems play an important role in rewarding mechanisms and addictive stimulus processing by modulating drug-induced dopaminergic activation in the mesocortico-limbic brain areas. They are also involved in regulating stress mechanisms related to addiction. The aim of this study was to investigate possible changes of gene expression of the dynorphinergic and nociceptinergic system components in the nucleus accumbens (NA) and in medial and lateral caudate putamen (mCPu and lCPu, respectively) of rats, following chronic subcutaneous infusion of cocaine. In addition, the epigenetic histone modifications H3K4me3 and H3K27me3 (an activating and a repressive marker, respectively) at the promoter level of the pDYN, KOP, pN/OFQ and NOP genes were investigated. Results showed that cocaine induced pDYN gene expression up-regulation in the NA and lCPu, and its down-regulation in the mCPu, whereas KOP mRNA levels were unchanged. Moreover, cocaine exposure decreased pN/OFQ gene expression in the NA and lCPu, while NOP mRNA levels appeared significantly increased in the NA and decreased in the lCPu. Specific changes of the H3K4me3 and H3K27me3 levels were found at pDYN, pN/OFQ, and NOP gene promoter, consistent with the observed gene expression alterations. The present findings contribute to better define the role of endogenous pDYN-KOP and pN/OFQ-NOP systems in neuroplasticity mechanisms following chronic cocaine treatment. The epigenetic histone modifications underlying the gene expression changes likely mediate the effects of cocaine on transcriptional regulation of specific gene promoters that result in long-lasting drug-induced plasticity.
Assuntos
Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Dinorfinas/genética , Epigênese Genética/efeitos dos fármacos , Núcleo Accumbens/efeitos dos fármacos , Receptores Opioides kappa/genética , Receptores Opioides/genética , Animais , Corpo Estriado/metabolismo , Dinorfinas/biossíntese , Encefalinas/biossíntese , Encefalinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Masculino , Núcleo Accumbens/metabolismo , Peptídeos Opioides/biossíntese , Peptídeos Opioides/genética , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Ratos , Receptores Opioides/biossíntese , Receptores Opioides kappa/biossíntese , Receptor de Nociceptina , NociceptinaRESUMO
CONTEXT: Our previous study demonstrated that acute and repeated morphine treatment differentially regulated κ-opioid receptor mRNA levels in the rat mesocorticolimbic system. Here, we further investigated the effects of morphine on protein levels of κ-opioid receptor in this reward-related circuitry. METHODS: Three groups of rats received intraperitoneal injection of saline, acute morphine (8.0 mg/kg) and repeated morphine (8.0 mg/kg, once daily for 5 consecutive days) and the κ-receptor protein expression was examined by Western blot analysis. RESULTS: We found that acute morphine treatment did not affect the κ-receptor protein levels in medial prefrontal cortex (mPFC), nucleus accumbens (NAc) and ventral tegmental area (VTA). However, repeated morphine treatment downregulated the κ-receptor protein levels in mPFC and VTA, while there was no significant change in NAc. CONCLUSIONS: These results, along with those reported previously, suggested that morphine dependence may be associated with regionally specific changes in κ-opioid receptor expression in mesocorticolimbic system.
Assuntos
Morfina/administração & dosagem , RNA Mensageiro/efeitos dos fármacos , Receptores Opioides kappa/biossíntese , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
Morphine has been reported to accelerate the progression of chronic kidney disease. However, whether morphine affects slit diaphragm (SD), the major constituent of glomerular filtration barrier, is still unclear. In the present study, we examined the effect of morphine on glomerular filtration barrier in general and podocyte integrity in particular. Mice were administered either normal saline or morphine for 72 h, then urine samples were collected and kidneys were subsequently isolated for immunohistochemical studies and Western blot. For in vitro studies, human podocytes were treated with morphine and then probed for the molecular markers of slit diaphragm. Morphine-receiving mice displayed a significant increase in albuminuria and showed effacement of podocyte foot processes. In both in vivo and in vitro studies, the expression of synaptopodin, a molecular marker for podocyte integrity, and the slit diaphragm constituting molecules (SDCM), such as nephrin, podocin, and CD2-associated protein (CD2AP), were decreased in morphine-treated podocytes. In vitro studies indicated that morphine modulated podocyte expression of SDCM through opiate mu (MOR) and kappa (KOR) receptors. Since morphine also enhanced podocyte oxidative stress, the latter seems to contribute to decreased SDCM expression. In addition, AKT, p38, and JNK pathways were involved in morphine-induced down regulation of SDCM in human podocytes. These findings demonstrate that morphine has the potential to alter the glomerular filtration barrier by compromising the integrity of podocytes.
Assuntos
Albuminúria/metabolismo , Morfina/efeitos adversos , Entorpecentes/efeitos adversos , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Albuminúria/induzido quimicamente , Albuminúria/patologia , Animais , Linhagem Celular Transformada , Proteínas do Citoesqueleto/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/biossíntese , Camundongos , Morfina/farmacologia , Entorpecentes/farmacologia , Podócitos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Opioides kappa/biossíntese , Receptores Opioides mu/biossíntese , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The role of dynorphin A (1-17; Dyn) and its associated kappa opioid receptor (KOR) in nociception represents a longstanding scientific conundrum: Dyn and KOR (Dyn/KOR) have variously been reported to inhibit, facilitate, or have no effect on pain. We investigated whether interactions between sex and pain type (which are usually ignored) influenced Dyn/KOR-mediated antinociception. Blockade of the spinal α(2)-noradrenergic receptor (α(2)-NAR) using yohimbine elicited comparable spinal Dyn release in females and males. Nevertheless, the yohimbine-induced antinociception exhibited sexual dimorphism that depended on the pain test used: in the intraperitoneal acetic acid-induced writhing test, yohimbine produced antinociception only in females, whereas in the intraplantar formalin-induced paw flinch test, antinociception was observed only in males. In females and males, both intrathecal Dyn antibodies and spinal KOR blockade eliminated the yohimbine-induced antinociception, indicating that Dyn/KOR mediated it. However, despite the conditional nature of spinal Dyn/KOR-mediated yohimbine antinociception, both intraplantar formalin and intraperitoneal acetic acid activated spinal Dyn neurons that expressed α(2)-NARs. Moreover, Dyn terminals apposed KOR-expressing spinal nociceptive neurons in both sexes. This similar organization suggests that the sexually dimorphic interdependent effects of sex and pain type may result from the presence of nonfunctional (silent) KORs on nociceptive spinal neurons that are responsive to intraplantar formalin (in females) versus intraperitoneal acetic acid (in males). Our findings that spinal Dyn/KOR-mediated antinociception depends on interactions between sex and pain type underscore the importance of using both sexes and multiple pain models when investigating Dyn/KOR antinociception.
Assuntos
Dinorfinas/metabolismo , Dor Nociceptiva/metabolismo , Medição da Dor , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Opioides kappa/metabolismo , Caracteres Sexuais , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Dinorfinas/biossíntese , Feminino , Injeções Espinhais , Masculino , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Dor Nociceptiva/tratamento farmacológico , Dor Nociceptiva/etiologia , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 2/biossíntese , Receptores Opioides kappa/biossíntese , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Ioimbina/farmacologiaRESUMO
BACKGROUND: Morphine induces adaptive changes in gene expression throughout the reward circuitry of the brain. Here, we investigated the acute and chronic effects of morphine on mRNA levels of κ-opioid receptor in the rat mesocorticolimbic system. METHODS: Three groups of rats received ip injection of saline, acute morphine (8.0 mg/kg) and repeated morphine (8.0 mg/kg, once daily for 5 consecutive days) and the κ-receptor mRNA expression was examined using real-time quantitative PCR method. RESULTS: We found that κ-receptor mRNA in medial prefrontal cortex (mPFC) increased after acute and repeated morphine treatment. However, the mRNA levels in nucleus accumbens (NAc) and ventral tegmental area (VTA) were upregulated after acute morphine treatment and returned to basal levels after repeated morphine treatment. CONCLUSIONS: These results suggest that morphine dependence was associated with regionally specific changes in mRNA levels of κ-opioid receptor in mesocorticolimbic system.
Assuntos
Analgésicos Opioides/farmacologia , Morfina/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , RNA Mensageiro/biossíntese , Receptores Opioides kappa/biossíntese , Área Tegmentar Ventral/efeitos dos fármacos , Analgésicos Opioides/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Masculino , Morfina/administração & dosagem , Dependência de Morfina/metabolismo , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Wistar , Regulação para Cima , Área Tegmentar Ventral/metabolismoRESUMO
Methionine enkephalin (MENK), the endogenous neuropeptide, is known to exert direct effects on the neuroendocrine and the immune systems and participates in regulation of various functions of cells related to both the innate and adaptive immune systems. Dendritic cells (DCs) play important role in initiating and regulating T cell responses. The aim of this work is to investigate the effects of MENK on differentiation, maturation, and function of DCs derived from murine bone marrow progenitors (BM-derived DCs). Our result showed that MENK could induce BM-derived DCs to polarize predominantly to mDC subtype, rather than pDC both in vivo and in vitro, and this was in favor of Th1 response. BM-derived DCs, after treatment with MENK, up-regulated the expressions of MHC class II and key costimulatory molecules. Result by RT-PCR showed MENK could increase expressions of delta and kappa receptors on BM-derived DCs. Also MENK promoted BM-derived DCs to secret higher levels of proinflammatory cytokines of IL-12p70, TNF-α. Furthermore, differentiated BM-derived DCs treated with MENK displayed higher activity to induce allogeneic T cell proliferation and MENK also inhibited tumor growth in vivo and induced apoptosis of tumor cells in vitro. Thus, it is concluded that MENK could be an effective inducer of BM-derived DCs and might be a new therapeutic agent for cancer, as well as other immune handicapped disease. Also we may consider MENK as a potential adjuvant in vaccine preparation.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Encefalina Metionina/farmacologia , Neurotransmissores/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Genes MHC da Classe II/efeitos dos fármacos , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores Opioides delta/biossíntese , Receptores Opioides kappa/biossíntese , Regulação para Cima/efeitos dos fármacosRESUMO
In this study we examined basal and IFN-γ-regulated expression of kappa opioid receptors (KOR) on cells of a murine macrophage cell line, J774. Basal KOR expression was found both at transcriptional and protein levels. KOR protein was predominantly located intracellular (86.4±12.9% positive cells; n=4), and only in minority of J774 cells (9.1±6.4% positive cells; n=4) on plasma membranes, as revealed by Fluorescence-Activated Cell Sorter (FACS) analysis and immunocytochemistry. Proinflammatory cytokine IFN-γ up-regulated KOR expression both at transcriptional (up to 24 times) and protein levels (up to 4.2 times). KOR expressed on J774 cells was functionally active as its ligation with Dynorphin-A(1-17) (100 nM and 1 µM) triggered phosphorylation of ERK1/2. Involvement of KOR in the Dynorphin-A(1-17)-induced triggering of ERK1/2 phosphorylation is suggested since truncated Dynorphin-A(2-17), which does not bind to KOR, was ineffective. Collectively, we have shown for the first time that cells of J774 cell line constitutively express functionally active KOR, which triggers signalization via ERK1/2 phosphorylation and which could be up-regulated by proinflammatory IFN-γ. The data may be relevant for better understanding of the role of KOR and their endogenous ligand Dynorphin-A in regulation of inflammatory and immune responses.
Assuntos
Interferon gama/fisiologia , Macrófagos/imunologia , Receptores Opioides kappa/biossíntese , Regulação para Cima/imunologia , Animais , Linhagem Celular Tumoral , Dinorfinas/farmacologia , Regulação da Expressão Gênica/imunologia , Humanos , Mediadores da Inflamação/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neurotransmissores/farmacologia , Fosforilação/genética , Fosforilação/imunologia , Receptores Opioides kappa/genética , Receptores Opioides kappa/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células U937 , Regulação para Cima/genéticaRESUMO
Both low-frequency electro-acupuncture (EA) and manual acupuncture improve menstrual frequency and decrease circulating androgens in women with polycystic ovary syndrome (PCOS). We sought to determine whether low-frequency EA is more effective than manual stimulation in regulating disturbed oestrous cyclicity in rats with PCOS induced by 5α-dihydrotestosterone. To identify the central mechanisms of the effects of stimulation, we assessed hypothalamic mRNA expression of molecules that regulate reproductive and neuroendocrine function. From age 70 days, rats received 2 Hz EA or manual stimulation with the needles five times per week for 4-5 weeks; untreated rats served as control animals. Specific hypothalamic nuclei were obtained by laser microdissection, and mRNA expression was measured with TaqMan low-density arrays. Untreated rats were acyclic. During the last 2 weeks of treatment, seven of eight (88%) rats in the EA group had epithelial keratinocytes, demonstrating oestrous cycle change (P = 0.034 versus control rats). In the manual group, five of eight (62%) rats had oestrous cycle changes (n.s. versus control animals). The mRNA expression of the opioid receptors Oprk1 and Oprm1 in the hypothalamic arcuate nucleus was lower in the EA group than in untreated control rats. The mRNA expression of the steroid hormone receptors Esr2, Pgr and Kiss1r was lower in the manual group than in the control animals. In rats with 5α-dihydrotestosterone-induced PCOS, low-frequency EA restored disturbed oestrous cyclicity but did not differ from the manual stimulation group, although electrical stimulation lowered serum testosterone in responders, those with restored oestrus cyclicity, and differed from both control animals and the manual stimulation group. Thus, EA cannot in all aspects be considered superior to manual stimulation. The effects of low-frequency EA may be mediated by central opioid receptors, while manual stimulation may involve regulation of steroid hormone/peptide receptors.
Assuntos
Terapia por Acupuntura , Eletroacupuntura , Ciclo Estral/fisiologia , Síndrome do Ovário Policístico/fisiopatologia , Animais , Núcleo Arqueado do Hipotálamo/fisiopatologia , Di-Hidrotestosterona , Modelos Animais de Doenças , Feminino , Síndrome do Ovário Policístico/induzido quimicamente , Progesterona/sangue , Ratos , Receptores Opioides kappa/biossíntese , Receptores Opioides mu/biossíntese , Testosterona/sangueRESUMO
BACKGROUND: The kappa opioid receptor (KOR) system contributes to the prodepressive and aversive consequences of stress and is implicated in the facilitation of conditioned fear and anxiety in rodents. Here, we sought to identify neural circuits that mediate KOR system effects on fear and anxiety in rats. METHODS: We assessed whether fear conditioning induces plasticity in KOR or dynorphin (the endogenous KOR ligand) messenger RNA (mRNA) expression in the basolateral (BLA) and central (CeA) nuclei of the amygdala, hippocampus, or striatum. We then assessed whether microinfusions of the KOR antagonist JDTic (0-10 µg/side) into the BLA or CeA affect the expression of conditioned fear or anxiety. Finally, we examined whether fear extinction induces plasticity in KOR mRNA expression that relates to the quality of fear extinction. RESULTS: Fear conditioning upregulated KOR mRNA in the BLA by 65% and downregulated it in the striatum by 22%, without affecting KOR levels in the CeA or hippocampus, or dynorphin levels in any region. KOR antagonism in either the BLA or CeA decreased conditioned fear in the fear-potentiated startle paradigm, whereas KOR antagonism in the BLA, but not the CeA, produced anxiolytic-like effects in the elevated plus maze. Effective fear extinction was associated with a 67% reduction in KOR mRNA in the BLA. CONCLUSIONS: These findings suggest that fear conditioning and extinction dynamically regulate KOR expression in the BLA and provide evidence that the BLA and CeA are important neural substrates mediating the anxiolytic-like effects of KOR antagonists in models of fear and anxiety.
Assuntos
Tonsila do Cerebelo/metabolismo , Ansiedade/metabolismo , Condicionamento Psicológico/fisiologia , Dinorfinas/fisiologia , Medo/fisiologia , Receptores Opioides kappa/fisiologia , Transdução de Sinais/fisiologia , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Condicionamento Psicológico/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Extinção Psicológica/fisiologia , Medo/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Microinjeções , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Ratos , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides kappa/biossíntese , Reflexo de Sobressalto/efeitos dos fármacos , Reflexo de Sobressalto/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tetra-Hidroisoquinolinas/administração & dosagem , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear-cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator. Using the kappa-opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2-containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.