Calcium dependence of purinergic subtype P2Y1 receptor modulation of C6 glioma cell migration

We have examined activation of purinergic P2Y1 receptor-dependent Ca2+-signaling pathways in mediating C6 glioma cell migration. The administration of 2-methylthioadenosine 5′-diphosphate (2MeSADP), a selective agonist for P2Y1R, induced marked increases in patterns of glioma migration in both scratch wound and Boyden chamber assays. Antagonism of P2Y1R with either the broad spectrum purinergic blocker, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS) or the specific P2Y1R antagonist, 2′-deoxy-N6-methyladenosine-3′,5′-bisphosphate (MRS2179), significantly inhibited C6 cell migration. Calcium-sensitive spectrofluorometry showed 2MeSADP stimulation of glioma cells caused a biphasic change in intracellular Ca2+ ([Ca2+]i). The rapid transient phase was unchanged in Ca2+-free solution reflecting a [Ca2+]i component due to intracellular stores release subsequent to activation of a metabotropic P2Y subtype receptor. The secondary prolonged phase of [Ca2+]i was abolished in Ca2+-free solution or in glioma cells treated with the store-operated channel (SOC) blocker, SKF96365. Treatment of glioma with either MRS2179 or PPADS significantly attenuated both the rapid and prolonged phases of [Ca2+]i. These results suggest critical roles for activation of P2Y1R in mediating glioma cell mobility and migration with changes in [Ca2+]i contributing as a mechanistic link between activated receptor and functional response. Our findings suggest that pharmacological modulation of metabotropic P2Y1R-dependent signaling pathways may serve as a novel therapeutic procedure to slow glioma progression.

► Purinergic P2Y1 receptor mediates glioma cell migration. ► Changes in intracellular Ca2+ links activation of P2Y1R with glioma cell migration. ► Ca2+ mobilization includes influx and intracellular release components. ► Block of P2Y1R inhibits Ca2+ responses and reduces glioma cell migration. ► Modulation of P2Y1R could be a novel strategy to slow glioma development.