RESUMO
Anandamide (arachidonoylethanolamide or AEA) is an endocannabinoid that acts at vanilloid (VR1) as well as at cannabinoid (CB1/CB2) and NMDA receptors. Here, we show that AEA, in a dose-dependent manner, causes cell death in cultured rat cortical neurons and cerebellar granule cells. Inhibition of CB1, CB2, VR1 or NMDA receptors by selective antagonists did not reduce AEA neurotoxicity. Anandamide-induced neuronal cell loss was associated with increased intracellular Ca(2+), nuclear condensation and fragmentation, decreases in mitochondrial membrane potential, translocation of cytochrome c, and upregulation of caspase-3-like activity. However, caspase-3, caspase-8 or caspase-9 inhibitors, or blockade of protein synthesis by cycloheximide did not alter anandamide-related cell death. Moreover, AEA caused cell death in caspase-3-deficient MCF-7 cell line and showed similar cytotoxic effects in caspase-9 dominant-negative, caspase-8 dominant-negative or mock-transfected SH-SY5Y neuroblastoma cells. Anandamide upregulated calpain activity in cortical neurons, as revealed by alpha-spectrin cleavage, which was attenuated by the calpain inhibitor calpastatin. Calpain inhibition significantly limited anandamide-induced neuronal loss and associated cytochrome c release. These data indicate that AEA neurotoxicity appears not to be mediated by CB1, CB2, VR1 or NMDA receptors and suggest that calpain activation, rather than intrinsic or extrinsic caspase pathways, may play a critical role in anandamide-induced cell death.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Araquidônicos/farmacologia , Calpaína/metabolismo , Caspases/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Ácidos Araquidônicos/química , Ácidos Araquidônicos/metabolismo , Cálcio/metabolismo , Calpaína/antagonistas & inibidores , Antagonistas de Receptores de Canabinoides , Inibidores de Caspase , Caspases/deficiência , Caspases/genética , Células Cultivadas , Citocromos c/metabolismo , Endocanabinoides , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Alcamidas Poli-Insaturadas , Transporte Proteico , Ratos , Receptores de Canabinoides/metabolismo , Receptores de Droga/antagonistas & inibidores , Receptores de Droga/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de SinaisRESUMO
N-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like serine protease (CSP), prevents DNA fragmentation and apoptotic cell death in certain blood cell lines and was reported to reduce hippocampal neuronal damage caused by cerebral ischemia. We examined the role of CSP on recovery after lateral fluid percussion-induced traumatic brain injury (TBI) in rats, as well as on cell survival in various in vitro models of neuronal cell death. TBI caused significant time-dependent upregulation of CSP activity, but not trypsin-like serine protease activity in injured cortex. Intracerebroventricular administration of TPCK to rats after TBI did not significantly affect deficits of spatial learning but exacerbated motor dysfunction after injury. Moreover, TPCK did not prevent apoptotic neuronal cell death caused by serum/K(+) deprivation or by application of staurosporine or etoposide in cultured rat cerebellar granule cells, rat cortical neurons, or in the human neuroblastoma SH-SY5Y cell line. Instead, at doses from 10 to 100 microM, TPCK was cytotoxic in all cultures tested. Similar results were obtained in cultures treated with another CSP inhibitor, 3,4-dichloroisocoumarin. Cell death caused by CSP inhibitors was neither caspase-dependent nor associated with oligonucleosomal DNA fragmentation. Taken together, these data do not support a neuroprotective role for CSP inhibitors. Rather, they suggest that CSPs may serve an endogenous neuroprotective role, possibly by modulating necrotic cell death.
Assuntos
Apoptose/efeitos dos fármacos , Lesões Encefálicas/tratamento farmacológico , Neurônios/efeitos dos fármacos , Inibidores de Serina Proteinase/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Lesões Encefálicas/patologia , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cumarínicos/administração & dosagem , Meios de Cultura Livres de Soro/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Injeções Intraventriculares , Isocumarinas , Masculino , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Serina Endopeptidases/metabolismo , Comportamento Espacial/efeitos dos fármacos , Estaurosporina/farmacologia , Tosilina Clorometil Cetona/administração & dosagem , Tosilfenilalanil Clorometil Cetona/administração & dosagem , Ferimentos não PenetrantesRESUMO
The effects of selective blockade of group I metabotropic glutamate receptor subtype 1 (mGluR1) on neuronal cell survival and post-traumatic recovery was examined using rat in vitro and in vivo trauma models. The selective mGluR1 antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), and (S)-(+)-alpha-amino-4-carboxy-2-methylbezeneacetic acid (LY367385) provided significant neuroprotection in rat cortical neuronal cultures subjected to mechanical injury, in both pretreatment or posttreatment paradigms. Administration of the antagonists also attenuated glutamate-induced neuronal cell death in the cultures. Coapplication of these antagonists with the N-methyl-d-aspartate (NMDA) receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had additive neuroprotective effects in glutamate injured cultures. Intracerebroventricular administration of AIDA to rats markedly improved recovery from motor dysfunction after lateral fluid percussion induced traumatic brain injury (TBI). Treatment with mGluR1 antagonists also significantly reduced lesion volumes in rats after TBI, as evaluated by MRI. It appears that these compounds mediate their neuroprotective effect through an mGluR1 antagonist action, as demonstrated by inhibition of agonist induced phosphoinositide hydrolysis in our in vitro system. Moreover, AIDA, CPCCOEt, and LY367385, at concentrations shown to be neuroprotective, had no significant effects on the steady state NMDA evoked whole cell current. Taken together, these data suggest that modulation of mGluR1 activity may have substantial therapeutic potential in brain injury.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Glicina/análogos & derivados , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Animais , Benzoatos/administração & dosagem , Lesões Encefálicas/induzido quimicamente , Morte Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Sinergismo Farmacológico , Potenciais Evocados/efeitos dos fármacos , Glicina/administração & dosagem , Glicina/farmacologia , Técnicas In Vitro , Indanos/administração & dosagem , Injeções Intraventriculares , Masculino , Modelos Biológicos , Fármacos Neuroprotetores/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Ferimentos não PenetrantesRESUMO
The effect of selective group I metabotropic glutamate receptor subtype 5 (mGluR5) antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and (E)-2-methyl-6-(2-phenylethenyl)-pyridine (SIB-1893) on neuronal cell survival and post-traumatic recovery was examined using rat in vitro and in vivo trauma models. Treatment with MPEP and SIB-1893 showed significant neuroprotective effects in rat cortical neuronal cultures subjected to mechanical injury. Application of the antagonists also attenuated glutamate- and N-methyl-D-aspartate (NMDA)-induced neuronal cell death in vitro. Intracerebroventricular administration of MPEP to rats markedly improved motor recovery and reduced deficits of spatial learning after lateral fluid percussion-induced traumatic brain injury. Lesion volumes as assessed by magnetic resonance imaging were also substantially reduced by MPEP treatment. Although we show that MPEP acts as a potent mGluR5 antagonist in our culture system, where it completely blocks agonist-induced phosphoinositide hydrolysis, electrophysiological and pharmacological studies indicate that MPEP and SIB-1893 also inhibit NMDA receptor activity at higher concentrations that are neuroprotective. Taken together, these data suggest that MPEP and SIB-1893 may have therapeutic potential in brain injury, although the mechanisms of neuroprotective action for these drugs may reflect their ability to modulate NMDA receptor activity.