P2Y-Receptors Mediating an Inhibition of the Evoked Entry of Calcium through N-Type Calcium Channels at Neuronal Processes

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Abstract
In the search for P2-receptors modulating the stimulation-evoked entry of calcium at processes of PC12 cells differentiated in the presence of nerve growth factor and neurotrophin-3, electrically evoked increases in free calcium were assessed by fura-2 microfluorimetry. Omission of calcium and addition of cadmium (100 μM) or the N-type calcium channel blocker ω-conotoxin GVIA (0.5 μM) abolished or markedly reduced the evoked responses. The P2Y-receptor agonists 2-methylthio adenosine 5′-diphosphate (2-methylthio-ADP), ADP, and adenosine 5′-O-(2-thiodiphosphate) (ADPβS) inhibited the electrically evoked entry of calcium without any changes in basal calcium concentrations. 2-Methylthio-ADP was the most potent agonist. Adenosine, P1,P4-di(adenosine-5′)-tetraphosphate (Ap4A), UDP, and UTP (30 μM each) had no effect. The effect of ADPβS (30 μM) was abolished by the P2-antagonists reactive blue 2 (3 μM), suramin (100 μM), 2-methylthio-AMP (10 μM),p-chloromercuriphenyl sulfonic acid (1 μM), and AR-C 69931MX [N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-β,γ-dichloromethylene adenosine 5′-triphosphate] (300 nM). In contrast, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (10 μM), the selective P2Y1-receptor antagonist MRS 2179 (N6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate; 10 μM), as well as the adenosine A1-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 100 nM), caused no change. Pretreatment with pertussis toxin abolished the effect of ADPβS. Reverse transcriptase-polymerase chain reaction revealed the presence of mRNA for P2Y12-receptors in nondifferentiated and differentiated PC12 cells. The results indicate that processes of differentiated PC12 cells possess P2Y12-receptors coupling to pertussis toxin-sensitive G-proteins and mediating an inhibition of the stimulation-evoked entry of calcium through ω-conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.