Modulation of glial P2Y receptors by algogenic mediators in the trigeminal ganglion: a role for the purinergic system in the mechanism of action of known anti-migraine drugs?

Aim: to study the role of the purinergic system in the neuron-to-glial cell communication within the trigeminal ganglion (TG), and its cross-talk with known pro-algogenic systems (such as bradykinin, BK, calcitonin gene-related peptide, CGRP, and prostaglandins). The final goal is to identify new cellular and molecular players in the onset and maintenance of trigeminal-associated pain, for the development of innovative therapies for migraine. Methods: primary mixed neuron-glia or purified glial cultures from mouse TG were used. G protein-coupled P2Y receptor function was evaluated by single cell calcium imaging; the extracellular concentrations of CGRP and PGE2 were measured by ELISA assays. Western blot experiments on P2Y1, P2Y2 and P2Y4 receptor subtypes were also performed. The effect of known analgesic drugs on P2Y receptor function and CGRP and PGE2 release was also tested. Results: exposure of mixed-neuron glia cultures to BK induced the neuronal release of CGRP, which, in turn, significantly potentiated P2Y receptor-mediated calcium responses to their cognate ligands ADP and UTP on satellite glial cells. The anti-migraine drug sumatriptan completely inhibited both BK-mediated CGRP release, and the potentiation of P2Y receptors. Acetylsalicylic acid and ibuprofen also inhibited CGRP and PGE2 release from neurons, suggesting that prostaglandins are involved in the complex network of events leading to the potentiation of glial P2Y receptors. Single cell calcium imaging experiments by using selective and non-selective P2Y receptors antagonists suggested that CGRP exposure potentiates P2Y1 and P2Y2 receptor subtypes, as confirmed by western blot analysis. Methyl-β-cyclodextrin, which removes cholesterol from cultured cells and disrupts lipid rafts, significantly inhibits the increase in the [Ca2+]i and in the % of cells responding to the P2Y1 agonist ADP with no effects on UTP-mediated responses through the P2Y2 receptor subtype, suggesting the localization in membrane lipid rafts of P2Y1 receptors only. Conclusions: our data suggest that a complex cross-talk between neuronal and glial cells takes place in the TG, involving pain mediators and extracellular nucleotides. Modulation of this network by known anti-migraine drugs, such as triptans and NSAIDs, suggests that it might play an important role in the development of migraine pain. Further studies are in progress to understand the pro- or anti-algogenic role of the different P2Y receptor subtypes.