Vol. 303, Issue 2, 520-526, November 2002

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

Melanie B. Kulick and Ivar von Kügelgen

Department of Pharmacology, University of Bonn, Bonn, Germany

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 -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) (ADPS) 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 ADPS (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)-,-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 ADPS. 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 -conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.