A P2 purinoceptor activated by ADP in rat medullar neurons

ADP evoked outwardly rectifying potassium currents with a latency of 0.6 s in cultured rat medullar neurons. Purinoceptor agonists, such as 2-methylthio ATP (2-MeSATP), ATP, AMP, alpha,beta-methylene ATP (alpha,beta-MeATP), and UTP, produced similar outward currents with the order of their potencies for current amplitudes: 2-MeSATP > ADP > ATP > or = alpha,beta-MeATP > or = AMP > UTP. This order corresponds to that for a subtype of P2Y purinoceptors. ADP-evoked currents were fully blocked by a broad G-protein inhibitor, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), whereas a G(i)/G(o)-protein inhibitor, pertussis toxin (PTX) had no effect. The currents were not affected by a phospholipase C (PLC) inhibitor, neomycin. Furthermore, a selective protein kinase C inhibitor, GF109203X or a selective cAMP-dependent protein kinase inhibitor, H-89 showed no effect on the currents. These results suggest that ADP activates the potassium channel via a P2Y purinoceptor linked to a PTX-insensitive G-protein and its channel regulation may be due to a direct action of the G-protein beta gamma subunits regardless of second messenger signaling cascades. Additionally, ADP enhanced intracellular free Ca2+ concentration ([Ca2+]i) both in the presence and absence of extracellular calcium, and this [Ca2+]i increase was not inhibited by neomycin. This provides an additional evidence that ADP binds to a subtype of P2Y purinoceptors, which is not involved in PLC stimulation.