Attenuation of the P2Y receptor-mediated control of neuronal Ca2+ channels in PC12 cells by antithrombotic drugs

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Abstract
1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.