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.