NMDA‐receptor regulation of muscarinic‐receptor stimulated inositol 1,4,5‐trisphosphate production and protein kinase C activation in single cerebellar granule neurons
Autor: | Stefan R. Nahorski, R. A. John Challiss, Kenneth W. Young, M. Asier Garro |
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Rok vydání: | 2004 |
Předmět: |
Thapsigargin
Recombinant Fusion Proteins Green Fluorescent Proteins Glutamic Acid Inositol 1 4 5-Trisphosphate Muscarinic Agonists Biology Receptors N-Methyl-D-Aspartate Biochemistry Cellular and Molecular Neuroscience chemistry.chemical_compound BAPTA Cerebellum Animals Inositol MARCKS Myristoylated Alanine-Rich C Kinase Substrate Cells Cultured Protein Kinase C Protein kinase C Fluorescent Dyes Feedback Physiological Neurons Phospholipase C Intracellular Signaling Peptides and Proteins Membrane Proteins Proteins Muscarinic acetylcholine receptor M3 Rats Inbred Strains Receptors Muscarinic Rats Cell biology Enzyme Activation Isoenzymes Luminescent Proteins chemistry Type C Phospholipases Calcium Phospholipase C delta Intracellular Signal Transduction |
Zdroj: | Journal of Neurochemistry. 89:1537-1546 |
ISSN: | 1471-4159 0022-3042 |
Popis: | 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. |
Databáze: | OpenAIRE |
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