P2Y 1 and P2X 7 receptors induce calcium/calmodulin‐dependent protein kinase II phosphorylation in cerebellar granule neurons

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), α,β‐methylene adenosine 5′‐triphosphate (α,β‐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.