P2Y-receptors mediating an inhibition of the evoked entry of calcium through N-type calcium channels at neuronal processes.
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 microM) or the N-type calcium channel blocker omega-conotoxin GVIA (0.5 microM) 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) (ADPbetaS) inhibited the electrically evoked entry of calcium without any changes in basal calcium concentrations. 2-Methylthio-ADP was the most potent agonist. Adenosine, P(1),P(4)-di(adenosine-5')-tetraphosphate (Ap4A), UDP, and UTP (30 microM each) had no effect. The effect of ADPbetaS (30 microM) was abolished by the P2-antagonists reactive blue 2 (3 microM), suramin (100 microM), 2-methylthio-AMP (10 microM), p-chloromercuriphenyl sulfonic acid (1 microM), and AR-C 69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene adenosine 5'-triphosphate] (300 nM). In contrast, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (10 microM), the selective P2Y1-receptor antagonist MRS 2179 (N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate; 10 microM), as well as the adenosine A(1)-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 100 nM), caused no change. Pretreatment with pertussis toxin abolished the effect of ADPbetaS. 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 omega-conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.
Kulick
Melanie B
MB
Department of Pharmacology, University of Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany.
von Kügelgen
Ivar
I
D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D005470:Fluorometry; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D016716:PC12 Cells; D037342:Pertussis Toxin / Q000494:pharmacology; D011061:Poly A / Q000096:biosynthesis / Q000235:genetics; D017729:Presynaptic Terminals / Q000187:drug effects; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D005456:Fluorescent Dyes; D009711:Nucleotides; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D011061:Poly A; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000241:Adenosine; D016257:Fura-2