P2Y1 and P2X7 receptors induce calcium/calmodulin-dependent protein kinase II phosphorylation in cerebellar granule neurons

Abstract

The activation of nucleotide receptors-- both ionotropic, P2X, and most of metabotropic, P2Y-- increases intracellular calcium concentration, resulting in calcium/calmodulin-dependent protein kinase II (CaMKII) activation. Stimulation of cerebellar granule neurons in culture-- with different P2X and P2Y agonists and their effect on CaMKII phosphorylation-- was studied using immunocytochemical and microfluorimetrical techniques. P2X agonist: 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP) and diadenosine pentaphosphate (Ap(5)A); and P2Y agonists: 2-(methylthyo)-adenosine diphosphate (2MeSADP) and uridine 5'-bisphosphate (UDP); tested induced a CaMKII phosphorylation but with a different immunostaining pattern in each group. Stimulation with 2MeSADP induced a Ca(2+) release from intracellular stores and a significant CaMKII phosphorylation in cell somas and neurites. This agrees with the subcellular distribution of P2Y(1). MRS 2179, a specific P2Y(1) inhibitor, antagonized the 2MeSADP effect. On the other hand, cerebellar granule neuron stimulation with BzATP, in Mg(2+)-free conditions, produced extracellular calcium entrance and, as a result, a significant increase in CaMKII phosphorylation mostly in fibres, which correspond with P2X(7) subdistribution. Immunocytochemical and microfluorimetrical experiments, using Zn(2+) and Brilliant Blue G (BBG), as a specific P2X(7) antagonist, confirmed that BzATP was acting through the P2X(7) receptor. These results indicate that P2Y(1) and P2X(7) produce a significant increase in CaMKII phosphorylation, but show important differences in subcellular distribution and in effect duration. P2X(7) activation in granule neurons is not associated with pore formation, according to the absence of YO-PRO-1 fluorescence. The abundant presence of P2X(7) at the synaptic structures suggests a relevant role played by this receptor in synaptic plasticity.