RESUMO
Visceral pain is a prominent feature of various gastrointestinal diseases. The P2X7 receptor is expressed by multiple cell types including dorsal root ganglion satellite glial cells, macrophages, and spinal microglia, all of which have been implicated in nociceptive sensitization. We have used the selective and CNS penetrant P2X7 receptor antagonist Lu AF27139 to explore this receptor's role in distinct rat models of inflammatory and visceral hypersensitivity. Rats injected with CFA in the hindpaw displayed a marked reduction in hindpaw mechanical threshold, which was dose-dependently reversed by Lu AF27139 (3-30 mg/kg, p.o.). In rats injected with TNBS in the proximal colon, the colorectal distension threshold measured distally was significantly lower than sham treated rats at 7 days post-injection (P < 0.001), indicative of a marked central sensitization. Colonic hypersensitivity was also reversed by Lu AF27139 (10-100 mg/kg) and by the κ-opioid receptor agonist U-50,488H (3 mg/kg, s.c.). Moreover, both Lu AF27139 and U-50,488H prevented a TNBS-induced increase in spinal and brain levels of PGE2 and LTB4, as well as an increase in brain levels of PGF2α and TXB2. Lu AF27139 was well tolerated as revealed by a lack of significant effect on rotarod motor function and coordination at all doses tested up to 300 mg/kg. Thus, P2X7 receptor antagonism is efficacious in a rat model of visceral pain, via a mechanism which potentially involves attenuation of microglial function within spinal and/or supraspinal pain circuits, albeit a peripheral site of action cannot be excluded.
Assuntos
Hipersensibilidade , Dor Visceral , Animais , Ratos , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Doenças do Sistema Nervoso Central , Colo , Hipersensibilidade/metabolismo , Prostaglandinas/metabolismo , Prostaglandinas/farmacologia , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/metabolismo , Dor Visceral/metabolismoRESUMO
Stroke is the leading cause of serious long-term disability and the fifth leading cause of death in the United States. Treatment options for stroke are few in number and limited in efficacy. Neuroinflammation mediated by microglia and infiltrating peripheral immune cells is a major component of stroke pathophysiology. Interfering with the inflammation cascade after stroke holds the promise to modulate stroke outcome. The calcium activated potassium channel KCa3.1 is expressed selectively in the injured CNS by microglia. KCa3.1 function has been implicated in pro-inflammatory activation of microglia and there is recent literature suggesting that this channel is important in the pathophysiology of ischemia/reperfusion (stroke) related brain injury. Here we describe the potential of repurposing Senicapoc, a KCa3.1 inhibitor, to intervene in the inflammation cascade that follows ischemia/reperfusion.
Assuntos
Acetamidas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Microglia/metabolismo , Acidente Vascular Cerebral/metabolismo , Compostos de Tritil/administração & dosagem , Animais , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Microglia/efeitos dos fármacos , Pirazóis/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Over the past decade, glial cells have attracted attention for harboring unexploited targets for drug discovery. Several glial targets have attracted de novo drug discovery programs, as highlighted in this GLIA Special Issue. Drug repurposing, which has the objective of utilizing existing drugs as well as abandoned, failed, or not yet pursued clinical development candidates for new indications, might provide a faster opportunity to bring drugs for glial targets to patients with unmet needs. Here, we review the potential of the intermediate-conductance calcium-activated potassium channels KCa 3.1 as the target for such a repurposing effort. We discuss the data on KCa 3.1 expression on microglia in vitro and in vivo and review the relevant literature on the two KCa 3.1 inhibitors TRAM-34 and Senicapoc. Finally, we provide an outlook of what it might take to harness the potential of KCa 3.1 as a bona fide microglial drug target. GLIA 2016;64:1733-1741.
Assuntos
Reposicionamento de Medicamentos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Microglia/metabolismo , Acetamidas/química , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Animais , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Microglia/efeitos dos fármacos , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Compostos de Tritil/química , Compostos de Tritil/farmacologia , Compostos de Tritil/uso terapêuticoRESUMO
Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of microglia. It is, however, unclear how this "inhibition" is achieved at the molecular and cellular levels. Here, we weigh the evidence whether minocycline is indeed a bona fide microglia inhibitor and discuss how data generated with minocycline should be interpreted. GLIA 2016;64:1788-1794.
Assuntos
Antibacterianos/farmacologia , Microglia/efeitos dos fármacos , Minociclina/farmacologia , Animais , Antibacterianos/uso terapêutico , Bases de Dados Factuais/estatística & dados numéricos , Humanos , Microglia/fisiologia , Minociclina/uso terapêuticoRESUMO
BACKGROUND: P2X7 receptor antagonists have potential for treating various central nervous system (CNS) diseases, including neuropathic pain, although none have been approved for clinical use. Reasons may include insufficient understanding of P2X7 receptor signalling in pain, and the lack of a corresponding preclinical mechanistic biomarker. METHODS: Lu AF27139 is a highly selective and potent small molecule antagonist at rat, mouse and human forms of the P2X7 receptor, with excellent pharmacokinetic and CNS permeability properties. In the current experiments, we probed the utility of previously characterized and novel signalling cascades exposed to Lu AF27139 using cultured microglia combined with release assays. Subsequently, we assessed the biomarker potential of identified candidate molecules in the rat chronic constriction injury (CCI) model of neuropathic pain; study design limitations precluded their assessment in spared nerve injury (SNI) rats. RESULTS: Lu AF27139 blocked several pain-relevant pathways downstream of P2X7 receptors in vitro. At brain and spinal cord receptor occupancy levels capable of functionally blocking P2X7 receptors, it diminished neuropathic hypersensitivity in SNI rats, and less potently in CCI rats. Although tissue levels of numerous molecules previously linked to neuropathic pain and P2X7 receptor function (e.g. IL-6, IL-1ß, cathepsin-S, 2-AG) were unaffected by CCI, Lu AF27139-mediated regulation of spinal PGE2 and miRNA (e.g. rno-miR-93-5p) levels increased by CCI aligned with its ability to diminish neuropathic hypersensitivity. CONCLUSIONS: We have identified a pain-relevant P2X7 receptor-regulated mechanism in neuropathic rats, which could hold promise as a translatable biomarker and by association enhance the clinical progression of P2X7 receptor antagonists in neuropathic pain. SIGNIFICANCE: Sub-optimal translation of preclinical molecules has hindered the clinical development of novel mechanism of action analgesics. We have undertaken a comprehensive in vitro analysis of migroglial signalling mechanisms recruited upon P2X7 receptor activation, a number of which were shown to be modulated by a selective P2X7 receptor antagonist in a well characterized animal model of neuropathic pain. Subject to further confirmation in other neuropathic models, this opens up the possibility to investigate their clinical utility as potential pain biomarkers in patients.
Assuntos
Hipersensibilidade , MicroRNAs , Neuralgia , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X7 , Animais , Hipersensibilidade/metabolismo , MicroRNAs/metabolismo , Microglia/metabolismo , Neuralgia/metabolismo , Prostaglandinas/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/metabolismo , Medula Espinal/metabolismoRESUMO
There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2'(3')-O-(benzoylbenzoyl)adenosine-5'-triphosphate (BzATP)-induced IL-1ß release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.
Assuntos
Sistema Nervoso Central/metabolismo , Antagonistas do Receptor Purinérgico P2X/síntese química , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Sistema Nervoso Central/efeitos dos fármacos , Cães , Feminino , Meia-Vida , Humanos , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microssomos Hepáticos/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Antagonistas do Receptor Purinérgico P2X/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/químicaRESUMO
The release of monoamine neurotransmitters from cell bodies and dendrites has an important role in behavior, but the mechanism (vesicular or non vesicular) has remained unclear. Because the location of vesicular monoamine transporter 2 (VMAT2) defines the secretory vesicles capable of monoamine release, we have studied its trafficking to assess the potential for monoamine release by exocytosis. In neuroendocrine PC12 cells, VMAT2 localizes exclusively to large dense-core vesicles (LDCVs), and we now show that cytoplasmic signals target VMAT2 directly to LDCVs within the biosynthetic pathway. In neurons, VMAT2 localizes to a population of vesicles that we now find undergo regulated exocytosis in dendrites. Although hippocampal neurons do not express typical LDCV proteins, transfected chromogranins A, B, and brain-derived neurotrophic factor (BDNF) colocalize with VMAT2. VMAT2 thus defines a population of secretory vesicles that mediate the activity-dependent somatodendritic release of multiple retrograde signals involved in synaptic function, growth, and plasticity.
Assuntos
Monoaminas Biogênicas/metabolismo , Dendritos/fisiologia , Exocitose/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Motivos de Aminoácidos , Animais , Axônios/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Cromogranina A , Cromograninas/metabolismo , Citoplasma/fisiologia , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/metabolismo , Ratos , Vesículas Secretórias/metabolismo , Transdução de Sinais/fisiologia , Distribuição TecidualRESUMO
Alzheimer's disease (AD) is a large and increasing unmet medical need with no disease-modifying treatment currently available. Genetic evidence from genome-wide association studies (GWASs) and gene network analysis has clearly revealed a key role of the innate immune system in the brain, of which microglia are the most important element. Single-nucleotide polymorphisms (SNPs) in genes predominantly expressed in microglia have been associated with altered risk of developing AD. Furthermore, microglia-specific pathways are affected on the messenger RNA (mRNA) expression level in post-mortem AD tissue and in mouse models of AD. Together these findings have increased the interest in microglia biology, and numerous scientific reports have proposed microglial molecules and pathways as drug targets for AD. Target identification and validation are generally the first steps in drug discovery. Both target validation and drug lead identification for central nervous system (CNS) targets and diseases entail additional significant obstacles compared to peripheral targets and diseases. This makes CNS drug discovery, even with well-validated targets, challenging. In this article, we will illustrate the special challenges of AD drug discovery by discussing the viability/practicality of possible microglia drug targets including cluster of differentiation 33 (CD33), KCa3.1, kynurenines, ionotropic P2 receptor 7 (P2X7), programmed death-1 (PD-1), Toll-like receptors (TLRs), and triggering receptor expressed in myeloid cells 2 (TREM2).
RESUMO
Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC theta and PKC delta, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC theta and delta were expressed in chromaffin cells, mice deficient for PKC theta, but not for PKC delta, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC theta pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC theta mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC theta, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion.
Assuntos
Células Cromafins/enzimologia , Células Cromafins/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Animais , Catecolaminas/metabolismo , Células Cultivadas , Ativação Enzimática/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase C-theta , Ratos , Ratos Sprague-DawleyRESUMO
A key question in understanding mechanisms of neurotransmitter release is whether the fusion pore of a synaptic vesicle regulates the amount of transmitter released during exocytosis. We measured dopamine release from small synaptic vesicles of rat cultured ventral midbrain neurons using carbon fiber amperometry. Our data indicate that small synaptic vesicle fusion pores flicker either once or multiple times in rapid succession, with each flicker releasing approximately 25-30% of vesicular dopamine. The incidence of events with multiple flickers was reciprocally regulated by phorbol esters and staurosporine. Thus, dopamine neurons regulate the amount of neurotransmitter released by small synaptic vesicles by controlling the number of fusion pore flickers per exocytotic event. This mode of exocytosis is a potential mechanism whereby neurons can rapidly reuse vesicles without undergoing the comparatively slow process of recycling.
Assuntos
Dopamina/metabolismo , Exocitose/fisiologia , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Eletrofisiologia/métodos , Técnicas In Vitro , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Modelos Neurológicos , Transporte Proteico/fisiologia , Ratos , Sinapses/fisiologiaRESUMO
α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.
Assuntos
Amiloide/química , Benzoxazóis/química , Benzoxazóis/farmacologia , Agregados Proteicos/efeitos dos fármacos , alfa-Sinucleína/química , Amiloide/antagonistas & inibidores , Amiloide/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/ultraestruturaRESUMO
Dysregulation of dopamine homeostasis and elevation of the cytosolic level of the transmitter have been suggested to underlie the vulnerability of catecholaminergic neurons in Parkinson's disease. Because several known mutations in alpha-synuclein or overexpression of the wild-type (WT) protein causes familial forms of Parkinson's disease, we investigated possible links between alpha-synuclein pathogenesis and dopamine homeostasis. Chromaffin cells isolated from transgenic mice that overexpress A30P alpha-synuclein displayed significantly increased cytosolic catecholamine levels as measured by intracellular patch electrochemistry, whereas cells overexpressing the WT protein and those from knock-out animals were not different from controls. Likewise, catechol concentrations were higher in L-DOPA-treated PC12 cells overexpressing A30P or A53T compared with those expressing WT alpha-synuclein, although the ability of cells to maintain a low cytosolic dopamine level after L-DOPA challenge was markedly inhibited by either protein. We also found that incubation with low-micromolar concentrations of WT, A30P, or A53T alpha-synuclein inhibited ATP-dependent maintenance of pH gradients in isolated chromaffin vesicles and that the WT protein was significantly less potent in inducing the proton leakage. In summary, we demonstrate that overexpression of different types of alpha-synuclein disrupts vesicular pH and leads to a marked increase in the levels of cytosolic catechol species, an effect that may in turn trigger cellular oxyradical damage. Although multiple molecular mechanisms may be responsible for the perturbation of cytosolic catecholamine homeostasis, this study provides critical evidence about how alpha-synuclein might exert its cytotoxicity and selectively damage catecholaminergic cells.
Assuntos
Catecolaminas/metabolismo , Células Cromafins/metabolismo , Citosol/metabolismo , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Transgênicos , Células PC12 , RatosRESUMO
AIM: Lipids such as prostaglandins, leukotrienes and thromboxanes are released as a result of an inflammatory episode in pain (central and peripheral). METHODOLOGY & RESULTS: To measure these lipids as potential mechanistic biomarkers in neuropathic pain models, we developed a higher-throughput LC-MS/MS-based method with simultaneous detection of PGE2, PGD2, PGF2α, LTB4, TXB2 and 2-arachidonoyl glycerol in brain and spinal cord tissues. We also demonstrate that the LC-MS/MS method was more sensitive and specific in differentiating PGE2 levels in CNS tissues compared with ELISA. CONCLUSION: The ability to modify the LC-MS/MS method to accommodate numerous other lipids in one analysis, demonstrates that the presented method offers a cost-effective and more sensitive alternative to ELISA method useful in drug discovery settings.
RESUMO
Neuropathic pain is a debilitating, chronic condition with a significant unmet need for effective treatment options. Recent studies have demonstrated that in addition to neurons, non-neuronal cells such as microglia contribute to the initiation and maintenance of allodynia in rodent models of neuropathic pain. The Ca2+- activated K+ channel, KCa3.1 is critical for the activation of immune cells, including the CNS-resident microglia. In order to evaluate the role of KCa3.1 in the maintenance of mechanical allodynia following peripheral nerve injury, we used senicapoc, a stable and highly potent KCa3.1 inhibitor. In primary cultured microglia, senicapoc inhibited microglial nitric oxide and IL-1ß release. In vivo, senicapoc showed high CNS penetrance and when administered to rats with peripheral nerve injury, it significantly reversed tactile allodynia similar to the standard of care, gabapentin. In contrast to gabapentin, senicapoc achieved efficacy without any overt impact on locomotor activity. Together, the data demonstrate that the KCa3.1 inhibitor senicapoc is effective at reducing mechanical hypersensitivity in a rodent model of peripheral nerve injury.
Assuntos
Acetamidas/farmacologia , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Traumatismos dos Nervos Periféricos/complicações , Bloqueadores dos Canais de Potássio/farmacologia , Compostos de Tritil/farmacologia , Acetamidas/efeitos adversos , Acetamidas/farmacocinética , Acetamidas/uso terapêutico , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Humanos , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Locomoção/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/efeitos adversos , Bloqueadores dos Canais de Potássio/farmacocinética , Bloqueadores dos Canais de Potássio/uso terapêutico , Ratos , Compostos de Tritil/efeitos adversos , Compostos de Tritil/farmacocinética , Compostos de Tritil/uso terapêuticoRESUMO
The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.
Assuntos
Benzo(a)Antracenos/análise , Dopamina/análise , Corantes Fluorescentes/análise , Terminações Pré-Sinápticas/química , Animais , Corpo Estriado/química , Corpo Estriado/metabolismo , Dopamina/metabolismo , Masculino , Camundongos , Plasticidade Neuronal , Terminações Pré-Sinápticas/metabolismo , Receptores de Dopamina D2/metabolismo , Transmissão SinápticaRESUMO
The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. In order to observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.
Assuntos
Benzo(a)Antracenos/metabolismo , Células Cromafins/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Corpo Estriado/citologia , Antagonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Estimulação Elétrica , Exocitose , Corantes Fluorescentes , Camundongos , Camundongos Transgênicos , Plasticidade Neuronal , Receptores de Dopamina D2/metabolismo , Sulpirida/farmacologiaRESUMO
Caffeine and more specific antagonists of the adenosine A(2A) receptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A(2A) antagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP(+), we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP(+) and of MPP(+), suggesting that CSC blocks the conversion of MPTP to MPDP(+) in vivo. In assessing the direct effects of CSC and A(2A) receptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitro with a K(i) value of 100 nm, whereas caffeine and another relatively specific A(2A) antagonist produced little or no inhibition. The A(2A) receptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A(2A) receptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A(2A) receptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A(2A) receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Cafeína/análogos & derivados , Cafeína/farmacologia , Dopaminérgicos/farmacologia , Interações Medicamentosas , Inibidores da Monoaminoxidase/farmacologia , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P1/metabolismo , Animais , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A2A de Adenosina , Fatores de TempoRESUMO
Parkinson's disease (PD) is most commonly a sporadic illness, and is characterized by degeneration of substantia nigra dopamine (DA) neurons and abnormal cytoplasmic aggregates of alpha-synuclein. Rarely, PD may be caused by missense mutations in alpha-synuclein. MPTP, a neurotoxin that inhibits mitochondrial complex I, is a prototype for an environmental cause of PD because it produces a pattern of DA neurodegeneration that closely resembles the neuropathology of PD. Here we show that alpha-synuclein null mice display striking resistance to MPTP-induced degeneration of DA neurons and DA release, and this resistance appears to result from an inability of the toxin to inhibit complex I. Contrary to predictions from in vitro data, this resistance is not due to abnormalities of the DA transporter, which appears to function normally in alpha-synuclein null mice. Our results suggest that some genetic and environmental factors that increase susceptibility to PD may interact with a common molecular pathway, and represent the first demonstration that normal alpha-synuclein function may be important to DA neuron viability.