RESUMO
Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.
Assuntos
Proteínas Hemolisinas/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Neurônios/metabolismo , Análise de Célula Única/métodos , Permeabilidade da Membrana Celular , Células HEK293 , Calefação , Humanos , Microscopia Confocal , Neurônios/citologiaRESUMO
Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure-function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.
Assuntos
Dispositivos Lab-On-A-Chip/métodos , Proteólise , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ativação do Canal Iônico , Modelos Moleculares , Conformação Proteica , Propriedades de Superfície , Tripsina/metabolismoRESUMO
BACKGROUND: Substantial clinical data support the addition of low doses of atypical antipsychotic drugs to selective serotonin reuptake inhibitors (SSRIs) to rapidly enhance the antidepressant effect in treatment-resistant depression. Preclinical studies suggest that this effect is at least partly explained by an increased catecholamine outflow in the medial prefrontal cortex (mPFC). METHODS: In the present study we used in vivo microdialysis in freely moving rats and in vitro intracellular recordings of pyramidal cells of the rat mPFC to investigate the effects of adding the novel atypical antipsychotic drug asenapine to the SSRI escitalopram with regards to monoamine outflow in the mPFC and dopamine outflow in nucleus accumbens as well as glutamatergic transmission in the mPFC. RESULTS: The present study shows that addition of low doses (0.05 and 0.1 mg/kg) of asenapine to escitalopram (5 mg/kg) markedly enhances dopamine, noradrenaline, and serotonin release in the rat mPFC as well as dopamine release in the nucleus accumbens. Moreover, this drug combination facilitated both N-methyl-d-Aspartate (NMDA)- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced currents as well as electrically evoked excitatory postsynaptic potentials in pyramidal cells of the rat mPFC. CONCLUSIONS: Our results support the notion that the augmentation of SSRIs by atypical antipsychotic drugs in treatment-resistant depression may, at least in part, be related to enhanced catecholamine output in the prefrontal cortex and that asenapine may be clinically used to achieve this end. In particular, the subsequent activation of the D1 receptor may be of importance for the augmented antidepressant effect, as this mechanism facilitated both NMDA and AMPA receptor-mediated transmission in the mPFC. Our novel observation that the drug combination, like ketamine, facilitates glutamatergic transmission in the mPFC may contribute to explain the rapid and potent antidepressant effect obtained when atypical antipsychotic drugs are added to SSRIs.
Assuntos
Antipsicóticos/farmacologia , Monoaminas Biogênicas/metabolismo , Citalopram/farmacologia , Ácido Glutâmico/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Benzazepinas/farmacologia , Bicuculina/farmacologia , Dibenzocicloeptenos , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/citologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Muscle fiber force production is determined by the excitation frequency of motor nerves, which induce transient increases in cytoplasmic free Ca2+ concentration ([Ca2+]i) and the force-generating capacity of the actomyosin cross-bridges. Previous studies suggest that, in addition to altered cross-bridge properties, force changes during dynamic (concentric or eccentric) contraction might be affected by Ca2+-dependent components. Here we investigated this by measuring [Ca2+]i and force in mouse muscle fibers undergoing isometric, concentric, and eccentric contractions. Intact single muscle fibers were dissected from the flexor digitorum brevis muscle of mice. Fibers were electrically activated isometrically at 30-100 Hz and after reaching the isometric force plateau, they were actively shortened or stretched. We calculated the ratio (relative changes) in force and [Ca2+]i attained in submaximal (30 Hz) and near-maximal (100 Hz) contractions under isometric or dynamic conditions. Tetanic [Ca2+]i was similar during isometric, concentric and eccentric phases of contraction at given stimulation frequencies while the forces were clearly different depending on the contraction types. The 30/100 Hz force ratio was significantly lower in the concentric (44.1 ± 20.3%) than in the isometric (50.3 ± 20.4%) condition (p = 0.005), whereas this ratio did not differ between eccentric and isometric conditions (p = 0.186). We conclude that the larger force decrease by decreasing the stimulation frequency during concentric than during isometric contraction is caused by decreased myofibrillar Ca2+ sensitivity, not by the decreased [Ca2+]i.
Assuntos
Citoesqueleto de Actina , Fibras Musculares Esqueléticas , Animais , Camundongos , Actomiosina , Citoplasma , CitosolRESUMO
KRAS belongs to a family of small GTPases that act as binary switches upstream of several signalling cascades, controlling proliferation and survival of cells. Mutations in KRAS drive oncogenesis, especially in pancreatic, lung, and colorectal cancers (CRC). Although historic attempts at targeting mutant KRAS with small molecule inhibitors have proven challenging, there are recent successes with the G12C, and G12D mutations. However, clinically important RAS mutations such as G12V, G13D, Q61L, and A146T, remain elusive drug targets, and insights to their structural landscape is of critical importance to develop novel, and effective therapeutic concepts. We present a fully open, P-loop exposing conformer of KRAS G13D by X-ray crystallography at 1.4-2.4 Å resolution in Mg2+-free phosphate and malonate buffers. The G13D conformer has the switch-I region displaced in an upright position leaving the catalytic core fully exposed. To prove that this state is druggable, we developed a P-loop-targeting monoclonal antibody (mAb). The mAb displayed high-affinity binding to G13D and was shown using high resolution fluorescence microscopy to be spontaneously taken up by G13D-mutated HCT 116 cells (human CRC derived) by macropinocytosis. The mAb inhibited KRAS signalling in phosphoproteomic and genomic studies. Taken together, the data propose novel druggable space of G13D that is reachable in the cellular context. It is our hope that these findings will stimulate attempts to drug this fully open state G13D conformer using mAbs or other modalities.
Assuntos
Anticorpos Monoclonais , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/química , Cristalografia por Raios X , Biologia Computacional/métodos , Mutação , Modelos Moleculares , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismoRESUMO
The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
Assuntos
Colesterol/deficiência , Canais de Cátion TRPV/metabolismo , Animais , Células CHO , Capsaicina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colesterol/farmacologia , Cricetinae , Cricetulus , Temperatura Alta , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Porosidade , Prótons , beta-Ciclodextrinas/farmacologiaRESUMO
Here, we provide a protocol for isolation of mouse primary skeletal muscle fibers using two alternative approaches-enzymatic dissociation or mechanical microdissection. We describe the procedures for surgical removal of muscle of interest and isolation of intact single-muscle fibers by either collagenase digestion or mechanical microdissection. We then detail intracellular calcium measurements by microinjecting or loading the isolated muscle fibers with membrane permeable calcium dyes. Finally, we outline steps for intracellular calcium quantification by fluorescent measurement. For complete details on the use and execution of this protocol, please refer to Gineste et al.1.
RESUMO
Antidepressant drugs are frequently used to treat affective symptoms in schizophrenia. We have recently shown that escitalopram, but not citalopram or R-citalopram, increases firing rate and burst firing of midbrain dopamine neurons, potentiates cortical N-methyl-D-aspartate (NMDA) receptor-mediated transmission and enhances cognition, effects that might influence the outcome of concomitant antipsychotic medication. Here, we studied, in rats, the behavioral and neurobiological effects of adding escitalopram, citalopram, or R-citalopram to the second-generation antipsychotic drug risperidone. We examined antipsychotic efficacy using the conditioned avoidance response (CAR) test, extrapyramidal side effect (EPS) liability using a catalepsy test, dopamine outflow in the medial prefrontal cortex (mPFC) and nucleus accumbens using in vivo microdialysis in freely moving animals, and NMDA receptor-mediated transmission in the mPFC using intracellular electrophysiological recording in vitro. Only escitalopram (5 mg/kg), but not citalopram (10 mg/kg), or R-citalopram (10 mg/kg), dramatically enhanced the antipsychotic-like effect of a low dose of risperidone (0.25 mg/kg), without increasing catalepsy. Given alone, escitalopram, but not citalopram or R-citalopram, markedly enhanced both cortical dopamine output and NMDA receptor-mediated transmission. Addition of escitalopram and to some extent R-citalopram, but not citalopram, significantly enhanced both cortical dopamine output and cortical NMDA receptor-mediated transmission induced by a suboptimal dose/concentration of risperidone. These results suggest that adjunct treatment with escitalopram, but not citalopram, may enhance the effect of a subtherapeutic dose of risperidone on positive, negative, cognitive, and depressive symptoms in schizophrenia, yet without increased EPS liability.
Assuntos
Antipsicóticos/administração & dosagem , Encéfalo/efeitos dos fármacos , Citalopram/administração & dosagem , Risperidona/administração & dosagem , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Dopamina/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Masculino , Microdiálise , Técnicas de Patch-Clamp , Ratos , Ratos WistarRESUMO
Escitalopram, the S-enantiomer of citalopram, possesses superior efficacy compared to other selective serotonin reuptake inhibitors (SSRIs) in the treatment of major depression. Escitalopram binds to an allosteric site on the serotonin transporter, which further enhances the blockade of serotonin reuptake, whereas R-citalopram antagonizes this positive allosteric modulation. Escitalopram's effects on neurotransmitters other than serotonin, for example, dopamine and glutamate, are not well studied. Therefore, we here studied the effects of escitalopram, citalopram, and R-citalopram on dopamine cell firing in the ventral tegmental area, using single-cell recording in vivo and on NMDA receptor-mediated currents in pyramidal neurons in the medial prefrontal cortex using in vitro electrophysiology in rats. The cognitive effects of escitalopram and citalopram were also compared using the novel object recognition test. Escitalopram (40-640 µg/kg i.v.) increased both firing rate and burst firing of dopaminergic neurons, whereas citalopram (80-1280 µg/kg) had no effect on firing rate and only increased burst firing at high dosage. R-citalopram (40-640 µg/kg) had no significant effects. R-citalopram (320 µg/kg) antagonized the effects of escitalopram (320 µg/kg). A very low concentration of escitalopram (5 nM), but not citalopram (10 nM) or R-citalopram (5 nM), potentiated NMDA-induced currents in pyramidal neurons. Escitalopram's effect was antagonized by R-citalopram and blocked by the dopamine D(1) receptor antagonist SCH23390. Escitalopram, but not citalopram, improved recognition memory. Our data suggest that the excitatory effect of escitalopram on dopaminergic and NMDA receptor-mediated neurotransmission may have bearing on its cognitive-enhancing effect and superior efficacy compared to other SSRIs in major depression.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Citalopram/farmacologia , Cognição/fisiologia , Dopamina/metabolismo , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Área Tegmentar Ventral/citologia , Análise de Variância , Animais , Benzazepinas/farmacologia , Dopaminérgicos/farmacologia , Relação Dose-Resposta a Droga , Masculino , Testes Neuropsicológicos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Reconhecimento PsicológicoRESUMO
Excess of brain kynurenic acid (KYNA), a neuroactive metabolite of the kynurenine pathway, is known to elicit cognitive dysfunction. In the present study, we investigated spatial working memory in mice with elevated levels of KYNA, induced by targeted deletion of kynurenine 3-monooxygenase (KMO), as well as long-term potentiation (LTP) of field excitatory postsynaptic potentials (fEPSPs) in hippocampal brain slices from these mice. The KMO knock-out (KMO-/-) mice performed more poorly in the spatial working memory task as compared to their wild-type (WT) counterparts, as reflected by fewer correct choices in a T-maze. Both fEPSPs, or LTP, did not significantly differ between the 2 mouse strains. However, administration of PF-04859989, a kynurenine aminotransferase (KAT) II inhibitor, limiting the production of KYNA, facilitated fEPSP and enhanced LTP to a greater extent in hippocampal slices from KMO-/- mice compared to WT mice. The results of the present study point to an essential role for KYNA in modulating LTP in the hippocampus of KMO-/- mice which may account for their dysfunctional spatial working memory.
RESUMO
Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.
Assuntos
Anticorpos Monoclonais , Antígenos , Anticorpos Monoclonais/química , Sítios de Ligação de Anticorpos , Epitopos , HumanosRESUMO
Compared to both first- and second-generation antipsychotic drugs (APDs), clozapine shows superior efficacy in treatment-resistant schizophrenia. In contrast to most APDs clozapine possesses high affinity for alpha2-adrenoceptors, and clinical and preclinical studies provide evidence that the alpha2-adrenoceptor antagonist idazoxan enhances the antipsychotic efficacy of typical D2 receptor antagonists as well as olanzapine. Risperidone has lower affinity for alpha2-adrenoceptors than clozapine but higher than most other APDs. Here we examined, in rats, the effects of adding idazoxan to risperidone on antipsychotic effect using the conditioned avoidance response (CAR) test, extrapyramidal side-effect (EPS) liability using the catalepsy test, brain dopamine efflux using in-vivo microdialysis in freely moving animals, cortical N-methyl-D-aspartate (NMDA) receptor-mediated transmission using intracellular electrophysiological recording in vitro, and ex-vivo autoradiography to assess the in-vivo alpha2A- and alpha2C-adrenoceptor occupancies by risperidone. The dose of risperidone needed for antipsychotic effect in the CAR test was approximately 0.4 mg/kg, which produced 11% and 17% in-vivo receptor occupancy at alpha2A- and alpha2C-adrenoceptors, respectively. Addition of idazoxan (1.5 mg/kg) to a low dose of risperidone (0.25 mg/kg) enhanced the suppression of CAR, but did not enhance catalepsy. Both cortical dopamine release and NMDA receptor-mediated responses were enhanced. These data propose that the therapeutic effect of risperidone in schizophrenia can be enhanced and its EPS liability reduced by adjunctive treatment with an alpha2-adrenoceptor antagonist, and generally support the notion that the potent alpha2-adrenoceptor antagonistic action of clozapine may be highly important for its unique efficacy in schizophrenia.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Antipsicóticos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Idazoxano/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Risperidona/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Antagonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Animais , Antipsicóticos/administração & dosagem , Antipsicóticos/metabolismo , Aprendizagem da Esquiva/efeitos dos fármacos , Catalepsia/tratamento farmacológico , Catalepsia/metabolismo , Córtex Cerebral/metabolismo , Agonistas de Dopamina/administração & dosagem , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/administração & dosagem , Fármacos Atuantes sobre Aminoácidos Excitatórios/metabolismo , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Idazoxano/administração & dosagem , Idazoxano/metabolismo , Masculino , Microdiálise , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Risperidona/administração & dosagem , Risperidona/metabolismoRESUMO
Asenapine is a novel psychopharmacologic agent being developed for schizophrenia and bipolar disorder. Like clozapine, asenapine facilitates cortical dopaminergic and N-methyl-D-aspartate (NMDA) receptor-mediated transmission in rats. The facilitation of NMDA-induced currents in cortical pyramidal cells by clozapine is dependent on dopamine and D(1) receptor activation. Moreover, previous results show that clozapine prevents and reverses the blockade of NMDA-induced currents and firing activity in the pyramidal cells by the noncompetitive NMDA receptor antagonist phencyclidine (PCP). Here, we investigated the effects of asenapine in these regards using intracellular electrophysiological recording in vitro. Asenapine (5 nM) significantly facilitated NMDA-induced currents (162 ± 15% of control) in pyramidal cells of the medial prefrontal cortex (mPFC). The asenapine-induced facilitation was blocked by the D(1) receptor antagonist SCH23390 (1 µM). Furthermore, the PCP-induced blockade of cortical NMDA-induced currents was effectively reversed by 5 nM asenapine. Our results demonstrate a clozapine-like facilitation of cortical NMDA-induced currents by asenapine that involves prefrontal dopamine and activation of D(1) receptors. Asenapine and clozapine also share the ability to reverse functional PCP-induced hypoactivity of cortical NMDA receptors. The ability of asenapine to increase both cortical dopaminergic and NMDA receptor-mediated glutamatergic transmission suggests that this drug may have an advantageous effect not only on positive symptoms in patients with schizophrenia, but also on negative and cognitive symptoms.
Assuntos
Antipsicóticos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/citologia , Receptores de Dopamina D1/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Benzazepinas/farmacologia , Dibenzocicloeptenos , Interações Medicamentosas , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp/métodos , Fenciclidina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/antagonistas & inibidoresRESUMO
Treatment with topiramate may improve negative symptoms in schizophrenia when added to typical antipsychotic drugs (APDs) but not to clozapine. Both dopaminergic and glutamatergic transmissions in the medial prefrontal cortex (mPFC) are facilitated by atypical, but not typical, APDs, which is thought to improve negative symptoms and cognitive dysfunction in schizophrenia. Our previous results show that topiramate increases prefrontal dopamine (DA) outflow when added to the D(2/3) receptorantagonist raclopride. Here, using intracellular recording in vitro, we investigated the effects of topiramate on glutamatergic neurotransmission in the rat mPFC, both when given alone and in combination with raclopride or clozapine. Neither topiramate nor raclopride alone had any effect on N-methyl-D-aspartate (NMDA)-induced currents in pyramidal cells of the mPFC. However, the combination of topiramate and raclopride facilitated the NMDA-induced currents, and this effect was blocked by the D1 receptor antagonist SCH23390. Topiramate also facilitated the effect of a submaximal, but inhibited the effect of a maximal, concentration of clozapine on these currents. The effect of combined topiramate and a submaximal concentration of clozapine could be blocked by SCH23390. In addition, combined topiramate and raclopride facilitated excitatory postsynaptic potentials. In contrast, topiramate inhibited clozapine's facilitating effect on these potentials. These data may help explain the improvement of negative symptoms when topiramate is used as adjunctive therapy in schizophrenic patients receiving typical APDs, but they may also shed light on the observed deterioration of symptoms when topiramate is added to full dose clozapine.
Assuntos
Clozapina/farmacologia , Antagonistas de Dopamina/farmacologia , Frutose/análogos & derivados , Ácido Glutâmico/metabolismo , Fármacos Neuroprotetores/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Racloprida/farmacologia , Antagonistas da Serotonina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Frutose/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp/métodos , Córtex Pré-Frontal/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , TopiramatoRESUMO
Rheumatoid arthritis-associated joint pain is frequently observed independent of disease activity, suggesting unidentified pain mechanisms. We demonstrate that antibodies binding to cartilage, specific for collagen type II (CII) or cartilage oligomeric matrix protein (COMP), elicit mechanical hypersensitivity in mice, uncoupled from visual, histological and molecular indications of inflammation. Cartilage antibody-induced pain-like behavior does not depend on complement activation or joint inflammation, but instead on tissue antigen recognition and local immune complex (IC) formation. smFISH and IHC suggest that neuronal Fcgr1 and Fcgr2b mRNA are transported to peripheral ends of primary afferents. CII-ICs directly activate cultured WT but not FcRγ chain-deficient DRG neurons. In line with this observation, CII-IC does not induce mechanical hypersensitivity in FcRγ chain-deficient mice. Furthermore, injection of CII antibodies does not generate pain-like behavior in FcRγ chain-deficient mice or mice lacking activating FcγRs in neurons. In summary, this study defines functional coupling between autoantibodies and pain transmission that may facilitate the development of new disease-relevant pain therapeutics.
Assuntos
Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Artralgia/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Cartilagem/imunologia , Neurônios/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Artralgia/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Autoanticorpos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Proteína de Matriz Oligomérica de Cartilagem/imunologia , Colágeno Tipo II/imunologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Receptores de IgG/deficiência , Receptores de IgG/genéticaRESUMO
RATIONALE: Asenapine is a novel psychopharmacologic agent being developed for the treatment of schizophrenia and bipolar disorder. MATERIALS AND METHODS: The present study was undertaken to investigate the effects of asenapine using animal models predictive of antipsychotic efficacy (conditioned avoidance response [CAR]) and extrapyramidal side effects (EPS; catalepsy). In parallel, the effects of asenapine on regional dopamine output using in vivo microdialysis in freely moving rats, dopamine output in the core and shell subregions of nucleus accumbens (NAc) using in vivo voltammetry in anesthetized rats, and N-methyl-D: -aspartate (NMDA)-induced currents in pyramidal neurons of the medial prefrontal cortex (mPFC) using the electrophysiological technique intracellular recording in vitro were assessed. RESULTS: Asenapine (0.05-0.2 mg/kg, subcutaneous [s.c.]) induced a dose-dependent suppression of CAR (no escape failures recorded) and did not induce catalepsy. Asenapine (0.05-0.2 mg/kg, s.c.) increased dopamine efflux in both the mPFC and the NAc. Low-dose asenapine (0.01 mg/kg, intravenous [i.v.]) increased dopamine efflux preferentially in the shell compared to the core of NAc, whereas at a higher dose (0.05 mg/kg, i.v.), the difference disappeared. Finally, like clozapine (100 nM), but at a considerably lower concentration (5 nM), asenapine significantly potentiated the NMDA-induced responses in pyramidal cells of the mPFC. CONCLUSIONS: These preclinical data suggest that asenapine may exhibit highly potent antipsychotic activity with very low EPS liability. Its ability to increase both dopaminergic and glutamatergic activity in rat mPFC suggests that asenapine may possess an advantageous effect not only on positive symptoms in patients with schizophrenia, but also on negative and cognitive symptoms.
Assuntos
Antipsicóticos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Esquizofrenia/tratamento farmacológico , Animais , Antipsicóticos/administração & dosagem , Antipsicóticos/efeitos adversos , Aprendizagem da Esquiva , Catalepsia/induzido quimicamente , Condicionamento Clássico , Dibenzocicloeptenos , Modelos Animais de Doenças , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Compostos Heterocíclicos de 4 ou mais Anéis/efeitos adversos , Injeções Subcutâneas , Masculino , Microdiálise , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Ratos , Ratos WistarRESUMO
Brexpiprazole (Rexulti®), a novel D2/3 receptor (R) partial agonist, was recently approved as monotherapy for schizophrenia, demonstrating effectiveness against both positive and negative symptoms, and also approved as add-on treatment to antidepressant drugs, inducing a potent antidepressant effect with a faster onset compared to an antidepressant given alone. Moreover, brexpiprazole has demonstrated pro-cognitive effects in preclinical studies. To explore whether the observed effects may be mediated via modulation of prefrontal glutamatergic transmission, we investigated the effect of brexpiprazole, alone and in combination with the SSRI escitalopram, on prefrontal glutamatergic transmission using in vitro electrophysiological intracellular recordings of deep layer pyramidal cells of the rat medial prefrontal cortex (mPFC). Nanomolar concentrations of brexpiprazole potentiated NMDAR-induced currents and electrically evoked EPSPs via activation of dopamine D1Rs, in similarity with the effect of the atypical antipsychotic drug clozapine. The effect of an ineffective concentration of brexpiprazole was significantly potentiated by the addition of escitalopram. When combined with escitalopram, brexpiprazole also potentiated AMPAR-mediated transmission, in similarity with the clinically rapid acting antidepressant drug ketamine. The effect on the AMPAR-mediated currents was also D1R dependent. In conclusion, our data propose that brexpiprazole exerts a clozapine-like potentiation of NMDAR-mediated currents in the mPFC, which can explain its efficacy on negative symptoms of schizophrenia and the pro-cognitive effects observed preclinically. Moreover, add-on brexpiprazole to escitalopram also potentiated AMPAR-mediated transmission, which may provide a neurobiological explanation to the faster antidepressant effect of add-on brexpiprazole in major depression.
Assuntos
Agonistas de Dopamina/farmacologia , Ácido Glutâmico/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Quinolonas/farmacologia , Receptores de Dopamina D1/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Citalopram/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Masculino , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Sprague-Dawley , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute ß-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: ß-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to ß-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after ß-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute ß-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.
Assuntos
Cardiomiopatias/metabolismo , Síndrome Metabólica/metabolismo , Miócitos Cardíacos/metabolismo , Obesidade/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Animais , Cálcio/metabolismo , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Dieta Hiperlipídica/efeitos adversos , Estimulação Elétrica , Peróxido de Hidrogênio/farmacologia , Isoproterenol/farmacologia , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Obesidade/etiologia , Obesidade/patologia , Técnicas de Cultura de Órgãos , Cultura Primária de CélulasRESUMO
Nicotine has been found to improve cognition and reduce negative symptoms in schizophrenia and a genetic and pathophysiological link between the α7 nicotinic acetylcholine receptors (nAChRs) and schizophrenia has been demonstrated. Therefore, there has been a large interest in developing drugs affecting the α7 nAChRs for schizophrenia. In the present study we investigated, in rats, the effects of a selective α7 agonist (PNU282987) and a α7 positive allosteric modulator (PAM; NS1738) alone and in combination with the atypical antipsychotic drug risperidone for their utility as adjunct treatment in schizophrenia. Moreover we also investigated their utility as adjunct treatment in depression in combination with the SSRI citalopram. We found that NS1738 and to some extent also PNU282987, potentiated a subeffective dose of risperidone in the conditioned avoidance response test. Both drugs also potentiated the effect of a sub-effective concentration of risperidone on NMDA-induced currents in pyramidal cells of the medial prefrontal cortex. Moreover, NS1738 and PNU282987 enhanced recognition memory in the novel object recognition test, when given separately. Both drugs also potentiated accumbal but not prefrontal risperidone-induced dopamine release. Finally, PNU282987 reduced immobility in the forced swim test, indicating an antidepressant-like effect. Taken together, our data support the utility of drugs targeting the α7 nAChRs, perhaps especially α7 PAMs, to potentiate the effect of atypical antipsychotic drugs. Moreover, our data suggest that α7 agonists and PAMs can be used to ameliorate cognitive symptoms in schizophrenia and depression.
Assuntos
Antidepressivos/farmacologia , Antipsicóticos/farmacologia , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Compostos de Fenilureia/farmacologia , Esquizofrenia/tratamento farmacológico , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Citalopram/farmacologia , Depressão/tratamento farmacológico , Depressão/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Masculino , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacologia , Agonistas Nicotínicos/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Risperidona/farmacologia , Esquizofrenia/metabolismo , Psicologia do Esquizofrênico , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Preclinical studies indicate that the rapid antidepressant effect of ketamine is dependent on activation of AMPA receptors in the medial prefrontal cortex (mPFC) resulting in a prolonged enhancement of glutamatergic transmission in the mPFC. In similarity, addition of atypical antipsychotic drugs (APDs) to SSRIs has also been found to induce a rapid and potent antidepressant effect. Using intracellular recordings in layer V/VI pyramidal cells of the rat mPFC in vitro, we found that a combination of low, clinically relevant concentrations of the atypical APD olanzapine and the SSRI fluoxetine facilitated NMDA and AMPA-induced currents in pyramidal cells via activation of dopamine D1 receptors. A single ketamine injection (10mg/kg, 24h before the experiment) enhanced AMPA-and apparently to some extent also NMDA-induced currents. Our results propose that the rapid and potent antidepressant effects of both treatments may be related to a common mechanism of action, namely facilitation of glutamatergic, in particular AMPA receptor-mediated transmission, in the mPFC.