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1.
J Neurochem ; 70(6): 2637-40, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9603231

RESUMO

The neurotoxin 6-hydroxydopamine (6-OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6-OHDA-induced apoptosis is morphologically indistinguishable from serum deprivation-induced apoptosis. Exposure of PC12 cells to a low concentration of 6-OHDA (25 microM) results in apoptosis, whereas an increased concentration (50 microM) results in a mixture of apoptosis and necrosis. We investigated the involvement of caspases in the apoptotic death of PC12 cells induced by 6-OHDA, using a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), and compared this with serum deprivation-induced apoptosis, which is known to involve caspases. We show that zVAD-fmk (100 microM) completely prevented the apoptotic morphology of chromatin condensation induced by exposure to either 6-OHDA (25 and 50 microM) or serum deprivation. Furthermore, cell lysates from 6-OHDA-treated cultures showed cleavage of a fluorogenic substrate for caspase-3-like proteases (caspase-2, 3, and 7), acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin, and this was inhibited by zVAD-fmk. However, although zVAD-fmk restored total cell viability to serum-deprived cells or cells exposed to 25 microM 6-OHDA, the inhibitor did not restore viability to cells exposed to 50 microM 6-OHDA. These data show the involvement of a caspase-3-like protease in 6-OHDA-induced apoptosis and that caspase inhibition is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6-OHDA neurotoxicity.


Assuntos
Apoptose/fisiologia , Cisteína Endopeptidases/metabolismo , Necrose , Oxidopamina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/farmacologia , Meios de Cultura Livres de Soro , Inibidores de Cisteína Proteinase/farmacologia , Microscopia de Fluorescência , Oligopeptídeos/farmacologia , Células PC12 , Ratos
2.
Brain Res Mol Brain Res ; 40(2): 271-4, 1996 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-8872311

RESUMO

Several studies have shown marked changes in the regional expression pattern of N-methyl-D-aspartate (NMDA) receptor subunit composition in vivo in the developing brain. Similar developmental changes may also occur in vitro. Thus, displacement of [3H]MK-801 binding by the subunit-selective NMDA antagonist, ifenprodil, in membrane homogenates prepared from cultured neurones, has been shown to comprise two components, the relative proportion of which changed with time in culture [11]. In the present experiments we have used electrophysiological methods to determine the influence of time in culture on NMDA receptor subtype expression pattern in individual neurones. Shortly after plating (13 days in vitro (DIV)), approximately 70% of total NMDA-induced inward current in voltage-clamped rat cortical neurones is antagonised by ifenprodil with relatively high affinity (approximately 1 microM). By 65 DIV, however, the high-affinity component contributed to only approximately 20% of the overall antagonism. Cerebellar granule cells also appear to undergo a similar change in their NMDA receptor expression with the exception that, in general, they appear initially to show considerably less of the ifenprodil high-affinity component and this disappears completely by 15 DIV. These experiments suggest that individual cortical and granule cells express at least two different NMDA receptor subtypes and that their relative proportion changes with time in culture.


Assuntos
Encéfalo/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Patch-Clamp , Ratos , Fatores de Tempo
3.
Neuroreport ; 5(14): 1763-5, 1994 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-7827326

RESUMO

The non-competitive N-methyl-D-aspartate (NMDA) antagonist, ifenprodil, discriminates two receptor populations, each of which shows a reciprocal abundance in cultured cortical and cerebellar granule cells. Thus approximately 70% of NMDA-gated membrane current was antagonized with high affinity (IC50 = 1.4 +/- 0.9 microM) in cortical neurones whereas only approximately 20% was antagonized with high affinity (IC50 = 1.3 +/- 0.3 microM) in granule cells. Inhibition curves for CGS 19755 appeared relatively monophasic: this competitive NMDA antagonist had a significantly higher affinity for the granule cell receptor (Ki = 0.8 +/- 0.2 microM) compared with that on cortical neurones (Ki = 2 +/- 0.6 microM). The data suggest that these two antagonists may be of value in identifying the expression of subpopulations of native NMDA receptors in other brain regions.


Assuntos
Química Encefálica/fisiologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Encéfalo/citologia , Células Cultivadas , Glicina/farmacologia , N-Metilaspartato/antagonistas & inibidores , N-Metilaspartato/farmacologia , Técnicas de Patch-Clamp , Ácidos Pipecólicos/farmacologia , Piperidinas/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
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