title: Glutamate release and synapsin-I phosphorylation induced by P2X7 receptors activation in cerebellar granule neurons.
abstract: The present work reports that activation of P2X7 receptor induces synaptic vesicle release in granule neurons and phosphorylation of synapsin-I by calcium-calmodulin-dependent protein kinase II (CaMKII), which in turn modulates secretory event. ATP, in absence of magnesium, induced a concentration-dependent glutamate release with an EC50 value of 1.95 microM. The involvement of P2X7 receptor was suggested when maximal secretory response was significantly reduced by the selective P2X7 antagonist Brilliant Blue G (BBG; 100 nM) and abolished by removing extracellular Ca2+. The involvement of P2X7 receptor on synaptic vesicle release was confirmed by measuring the release of FM 1-43 dye. In this case, pharmacological activation of P2X7 was achieved with the more selective agonist 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 microM) showing a significant FM 1-43 release that was blocked by BBG (100 nM), by Zn2+ ions (100 microM), both P2X7 blockers, but not by suramin (100 microM), antagonist of P2X1, P2X2, P2X3 and P2X5. In addition, BzATP, through P2X7 receptor activation, significantly increased the phosphorylation of synapsin-I, the main presynaptic target of CaMKII. Both effects mediated by BzATP were inhibited by the CaMKII inhibitors KN-62 (10 microM) and KN-93 (10 microM). These results suggest, therefore, that Ca2+ entrance mediated by P2X7 receptor induces glutamate release and in parallel synapsin-I phosphorylation.
authors.affiliation: Department of Biochemistry, Veterinary Faculty, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: León
authors.forename: Jesús
authors.initials: J
authors.lastname: Sánchez-Nogueiro
authors.forename: Patricia
authors.initials: P
authors.lastname: Marín-García
authors.forename: M A Teresa
authors.initials: MA
authors.lastname: Miras-Portugal
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001557:Benzenesulfonates / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D011726:Pyridinium Compounds / Q000493:pharmacokinetics; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D013498:Suramin / Q000494:pharmacology; D016704:Synapsins / Q000187:drug effects / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism
chemical_list: D001557:Benzenesulfonates; D004791:Enzyme Inhibitors; C073804:FM1 43; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D016704:Synapsins; D018698:Glutamic Acid; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D013498:Suramin; D000255:Adenosine Triphosphate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C006796:brilliant blue