MODULATION OF GLIAL P2Y RECEPTORS BY ALGOGENIC MEDIATORS IN THE TRIGEMINAL GANGLIA: A ROLE FOR THE PURINERGIC SYSTEM IN THE MECHANISM OF ACTION OF KNOWN ANTI-MIGRAINE DRUGS?

Aim of the investigation: to study the role of the purinergic system in the neuron-to-glial cell communication within the trigeminal ganglion, and its cross-talk with known pro-algogenic systems (such as bradikynin, BK, calcitonin gene-related peptide, CGRP, and prostaglandins). The final goal is the identification of new cellular and molecular players in the onset and maintenance of trigeminal-associated pain, for the development of new effective therapeutic strategies for migraine. Methods: primary mixed neuron-glia or purified glial cultures from trigeminal ganglion (TG) were prepared from C57BL6 mice. G protein-coupled P2Y receptor function was evaluated by single cell calcium imaging, and the extracellular concentrations of CGRP were measured by an ELISA assay. Western blot experiments on P2Y1 and P2Y2 receptors subtypes were also performed. Results: exposure of mixed-neuron glia cultures to BK induced 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. A 30-min pre-treatment with the anti-migraine drug sumatriptan completely inhibited both BK-mediated CGRP release from TG neurons, and the potentiation of P2Y receptors. Acetylsalicylic acid (1 mM) and ibuprofen (50 µM) were also able to inhibit CGRP release from neurons, suggesting that prostaglandins are also involved in the complex network of events leading to the potentiation of glial P2Y receptors. To clearly identify the P2Y receptor subtypes involved, we utilized purified glial cultures where CGRP increased ADP- or UTP-induced intracellular calcium concentrations. The selective P2Y1 receptor antagonist MRS2179 completely inhibited ADP-mediated responses, while UTP-mediated responses were almost completely inhibited by a selective P2Y2 receptor antagonist, both under control condition and after exposure to CGRP. After application of the selective P2Y2 antagonist, about 14% of cells showed a residual response to UTP in control cultures, suggesting a contribution of the P2Y4 receptor subtype to UTP-mediated responses. On the other hand, in CGRP-treated cells, no residual UTP-mediated increases of intracellular calcium concentrations were detected, suggesting that only the P2Y2 subtype is potentiated by CGRP exposure. Western blot analysis using commercial polyclonal antibodies against P2Y1 and P2Y2 receptors showed specific protein bands for both receptor subtypes with a significant increase in P2Y1 receptor protein and a trend to increase in P2Y2 receptor protein expression after CGRP treatment. 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 COX inhibitors, 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 for the different P2Y receptor subtypes, and to test whether innovative and more selective pharmacological approaches can be identified for the sake of those migraineurs who are insensitive to currently available drugs.