NMDA-receptor regulation of muscarinic-receptor stimulated inositol 1,4,5-trisphosphate production and protein kinase C activation in single cerebellar granule neurons.
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.
Young
Kenneth W
KW
Department of Cell Physiology and Pharmacology, University of Leicester, Medical Sciences Building, University Road, Leicester LE1 9HN, UK. KWY1@le.ac.uk
Garro
M Asier
MA
Challiss
R A John
RA
Nahorski
Stefan R
SR
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D004789:Enzyme Activation / Q000502:physiology; D025461:Feedback, Physiological / Q000187:drug effects; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins; D018721:Muscarinic Agonists / Q000494:pharmacology; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D054803:Phospholipase C delta; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D010738:Type C Phospholipases / Q000235:genetics
D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D018721:Muscarinic Agonists; D011506:Proteins; D011976:Receptors, Muscarinic; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054803:Phospholipase C delta; C518026:Plcd1 protein, rat; D002118:Calcium