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.
Footnotes
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This work was supported by the Doktor Robert Pfleger Stiftung (Bamberg, Germany).
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DOI: 10.1124/jpet.102.037960
- Abbreviations:
- fura-2 AM
- fura-2 acetoxymethyl ester
- F 340 nm/F 380 nm
- ratio of fluorescence emission due to excitation at 340 nm/fluorescence emission due to excitation at 380 nm
- RT-PCR
- reverse transcriptase-polymerase chain reaction
- ADPβS
- adenosine 5′-O-(2-thiodiphosphate)
- 2-methylthio-ADP
- 2-methylthio adenosine 5′-diphosphate
- Ap4A
- P1,P4-di(adenosine-5′)-tetraphosphate
- MRS 2179
- N6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate
- DPCPX
- 8-cyclopentyl-1,3-dipropylxanthine
- CMPS
- p-chloromercuriphenyl sulfonic acid
- 2-methylthio-AMP
- 2-methylthio adenosine 5′-monophosphate
- AR-C 69931MX
- N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-β,γ-dichloromethylene adenosine 5′-triphosphate
- RB2
- reactive blue 2
- PPADS
- pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid
- DMSO
- dimethyl sulfoxide
- Received April 23, 2002.
- Accepted July 15, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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