RESUMO
Inositol 1,4,5-trisphosphate (InsP(3)) production in single cerebellar granule neurons (CGNs) grown in culture was measured using the PH domain of phospholipase C delta1 tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta1)). These measurements were correlated with changes in intracellular free Ca2+ determined by single cell imaging. In control CGNs, intracellular Ca2+ stores appeared replete. However, the refilling state of these stores appeared dependent on the fluorophore used to measure Ca2+-release. Thus, methacholine (MCH), acting via muscarinic acetylcholine-receptors (mAchRs), mobilised intracellular Ca2+ in cells loaded with fluo-3 and fura-4f, but not fura-2. Confocal measurements of single CGNs expressing eGFP-PH(PLCdelta1) demonstrated that MCH stimulated a robust peak increase in InsP(3), which was followed by a sustained plateau phase of InsP(3) production. In contrast, glutamate-induced InsP(3) signals were weak or not detectable. MCH-stimulated InsP(3) production was reduced by chelation of intracellular Ca2+ with BAPTA, and emptying of intracellular stores with thapsigargin, indicated a positive feedback effect of Ca2+ mobilisation onto PLC activity. In CGNs, NMDA- and KCl-mediated Ca2+-entry significantly enhanced MCH-induced InsP(3) production. Furthermore, mAchR-mediated PLC activation appeared sensitive to the full dynamic range of intracellular Ca2+ increases stimulated by 100 microm NMDA. This dynamic regulation was also observed at the level of PKC activation indicated by an enhanced translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate (MARCKS) protein in cells stimulated with MCH. Thus, NMDA-mediated Ca2+ influx and PLC activation may represent a coincident-detection system whereby ionotropic and metabotropic signals combine to stimulate InsP(3) production and PKC-mediated phosphorylation events in CGNs.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Receptores Muscarínicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Cerebelo/citologia , Ativação Enzimática/fisiologia , Retroalimentação Fisiológica/efeitos dos fármacos , Corantes Fluorescentes , Ácido Glutâmico/farmacologia , Proteínas de Fluorescência Verde , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Agonistas Muscarínicos/farmacologia , Substrato Quinase C Rico em Alanina Miristoilada , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosfolipase C delta , Proteínas/genética , Ratos , Ratos Endogâmicos , Receptores Muscarínicos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/genéticaRESUMO
The phosphoinositide signal transduction system, and particularly, phospholipase Cbeta isozymes, are relevant in the etiopathogeny of human neuropsychiatric pathologies such as depression. Stimulation of phospholipase Cbeta activity by muscarinic receptors and G proteins was determined in crude and synaptosomal membrane preparations from nine postmortem human frontal cortices (postmortem delay range 8 to 50 h). Thus, the phospholipase Cbeta activity was determined by measuring the hydrolysis of exogenous [3H]-phosphatidylinositol 4,5-bisphosphate. There was a postmortem delay-mediated decrease in the PIP2 hydrolysis irrespective of the membrane preparation used (P < 0.05). Moreover, there were statistically significant differences for exponential decay curve parameters (K factor and Span) of PLCbeta activity induced by agonist-mediated activation between crude and synaptosomal membrane preparations. These results show that the postsynaptic component of the PLCbeta activity is more sensible to the postmortem delay effect.
