RESUMEN
Group I metabotropic glutamate (mGlu) receptors (i.e. mGlu1 and mGlu5) coupled to phospholipase C have been widely investigated for their possible role in excitotoxic and post-ischemic neuronal death. Recently, phospholipase C has been shown to directly stimulate the activity of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair that has been proposed to play a key role in necrotic cell death. In this study, we investigated whether the stimulation of group I mGlu receptors leads to an increase in PARP activity, as detected by flow cytometry, immunodot blot and immunocytochemistry, both in baby hamster kidney cells transfected with mGlu1a or mGlu5a receptors and in cultured cortical cells. Our results show that the group I mGlu receptor agonist DHPG elicited a significant increase in PARP activity that was completely abolished by the administration of the mGlu1 antagonist 3-MATIDA and partially prevented, in cortical neurons, by the mGlu5 antagonist MPEP. To evaluate whether this pathway is involved in post-ischemic neuronal death, we used a sublethal model of oxygen-glucose deprivation in mixed cortical cell cultures. DHPG exacerbated neuronal death, and this effect was significantly prevented by the application of the PARP inhibitor DPQ. This novel pathway may contribute to the effects of mGlu1 receptors in the mechanisms leading to post-ischemic neuronal death.
Asunto(s)
Corteza Cerebral/citología , Neuroglía/enzimología , Neuronas/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptores de Glutamato Metabotrópico/fisiología , Animales , Animales Recién Nacidos , Western Blotting/métodos , Células Cultivadas , Cricetinae , Interacciones Farmacológicas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Técnica del Anticuerpo Fluorescente/métodos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Glucosa/deficiencia , Peróxido de Hidrógeno/farmacología , Hipoxia/enzimología , Isoquinolinas/farmacología , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ratones , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Piperidinas/farmacología , Piridinas/farmacología , Tiofenos/farmacología , Factores de Tiempo , Transfección/métodosRESUMEN
Overactivation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) plays a key role in the mechanisms responsible for neuronal death. In the present study, we examined the effects of the PARP-1 inhibitor 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone (DPQ) in two models of N-methyl-d-aspartate (NMDA)-induced neurotoxicity. The exposure of mixed cultured cortical cells to 300 microM NMDA for 10 min induced a caspase-dependent type of apoptotic neuronal death. Conversely, exposure to 2 mM NMDA for 10 min led to the appearance of morphological features of necrosis, with no increase in caspase-3 activity and depletion in adenosine triphosphate (ATP) levels. DPQ (10 microM) reduced the NMDA-induced PARP activation, restored ATP to near control levels and significantly attenuated neuronal injury only in the severe NMDA exposure model. Similar results were obtained when pure neuronal cortical cultures were used. PARP-1 activation thus appears to play a preferential role in necrotic than in caspase-dependent apoptotic neuronal death.
Asunto(s)
Apoptosis/fisiología , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiopatología , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Isoquinolinas/farmacología , Ratones , Modelos Biológicos , N-Metilaspartato/toxicidad , Necrosis , Neuronas/efectos de los fármacos , Neuronas/patología , Neurotoxinas/toxicidad , Piperidinas/farmacología , Inhibidores de Poli(ADP-Ribosa) PolimerasasRESUMEN
Excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme catalyzing the transfer of ADP-ribose units from NAD to acceptor proteins, induces cellular energy failure by NAD and ATP depletion and has been proposed to play a causative role in a number of pathological conditions, including ischemia/reperfusion injury. In this study, we used an in vitro enzyme activity assay to characterize a series of newly synthesized isoquinolinone derivatives as potential PARP-1 inhibitors. Several compounds displayed powerful inhibitory activity: thieno[2,3-c]isoquinolin-5-one (TIQ-A) displayed a submicromolar IC50 of 0.45 +/- 0.1 microM, whereas the 5-hydroxy and 5-methoxy TIQ-A derivatives had IC50 values of 0.39 +/- 0.19 and 0.21 +/- 0.12 microM, respectively. We then examined the neuroprotective effects of the newly characterized compounds in cultured mouse cortical cells exposed to 60 min of oxygen and glucose deprivation (OGD). When PARP-1 inhibitors were present in the incubation medium during OGD and the subsequent 24-h recovery period, they significantly attenuated neuronal injury. TIQ-A provided neuroprotection even when added to the culture 30 min after OGD and was able to reduce the early activation of PARP induced by OGD as detected by flow cytometry. When the IC50 values observed in the PARP-1 activity assay for selected compounds were compared with their IC50 values for the neuroprotective activity, a significant correlation (r = 0.93, P < 0.01) was observed. Our results suggest that TIQ-A and its derivatives are a new class of neuroprotectants that may be helpful in studies aimed at understanding the involvement of PARP-1 in physiology and pathology.
Asunto(s)
Isoquinolinas/farmacología , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Tiofenos/farmacología , Animales , Isquemia Encefálica/patología , Células Cultivadas , Modelos Animales de Enfermedad , Isoquinolinas/química , Ratones , Fenantrenos/química , Fenantrenos/farmacología , Piperidinas/químicaRESUMEN
In order to investigate the involvement of mGlu1 and mGlu5 metabotropic glutamate receptors in the development of postischemic neuronal death, we examined the effects of selective agonists and antagonists in models of cerebral ischemia in vitro and in vivo. In murine cortical cell cultures and rat organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD), the mGlu1 antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA; 300 microM), (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG; 300 microM), 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt; 10-30 microM) and (+)-2-methyl-4-carboxyphenylglycine (LY367385; 30-100 microM) reduced neuronal loss when added to the medium during OGD and the subsequent 24-h recovery period. On the contrary, the potent and selective mGlu5 antagonist methyl-6-(phenylethynyl)-pyridine (MPEP; 0.1-1 microM) did not exhibit neuroprotection in any of these in vitro models. Incubation with the nonselective mGlu1 and mGlu5 agonist 3,5-dihydroxyphenylglycine (3,5-DHPG; 300 microM) but not with the mGlu5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 1 mM) enhanced the severity of OGD-induced neuronal damage. In gerbils subjected to global ischemia, intracerebroventricular administration of AIDA (100 nmol two times) or CBPG (300 nmol, two times) afforded consistent protection against CA1 pyramidal cell death, whereas MPEP (10 pmol i.c.v two times and 10 mg/kg i.p two times) failed to reduce postischemic hippocampal damage. Our results suggest that activation of mGlu1 but not mGlu5 receptor contributes to postischemic neuronal injury.
Asunto(s)
Isquemia Encefálica/patología , Agonistas de Aminoácidos Excitadores/farmacología , Glicina/análogos & derivados , Neuronas/patología , Receptores de Glutamato Metabotrópico/agonistas , Animales , Compuestos Bicíclicos con Puentes/farmacología , Línea Celular , Cricetinae , Antagonistas de Aminoácidos Excitadores/farmacología , Gerbillinae , Glucosa/deficiencia , Glucosa/fisiología , Glicina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Indanos/farmacología , Ataque Isquémico Transitorio/patología , Riñón/efectos de los fármacos , Riñón/metabolismo , Fármacos Neuroprotectores/farmacología , Técnicas de Cultivo de Órganos , Consumo de Oxígeno/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
We examined the pharmacological properties of 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA) by measuring second messenger responses in baby hamster kidney cells stably transfected with mGlu1a, mGlu2, mGlu4a or mGlu5a receptors and ionotropic glutamate receptor agonist-induced depolarizations in mouse cortical wedges. 3-MATIDA was a potent (IC(50)=6.3 microM, 95% confidence limits 3-15) and relatively selective mGlu1 receptor antagonist. When tested on mGlu2, mGlu4 or mGlu5 receptors its IC(50) was >300 microM. When tested in cortical wedges, however, 3-MATIDA was also able to antagonize AMPA or NMDA responses with an IC(50) of 250 microM. When present in the incubation medium of cultured murine cortical cells, 3-MATIDA (1-100 microM) significantly reduced the death of neurons induced by 60 min of oxygen and glucose deprivation (OGD), even when added up to 60 min after OGD. A similar neuroprotective activity was observed when 3-MATIDA was present at 10-100 microM in the medium of rat organotypic hippocampal slice cultures exposed to 30 min OGD. Systemic administration of 3-MATIDA (3-10 mg/kg, immediately and 1 h after the onset of ischemia) reduced the volume of brain infarcts following permanent middle cerebral artery occlusion in rats. Our results show that 3-MATIDA is a potent and possibly selective mGlu 1 receptor antagonist that may be considered as a novel prototype neuroprotective agent.