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It is well established that ATP serves as an extracellular neurotransmitter to induce various physiological responses in many tissue through the activation of P2 purinoceptors on the membrane of target cells. Using non-differentiated NG108-15 cells as a model system, the inhibitory effects of P2 purinoceptor agonists including ATP, UTP, 2methylthioATP (2MeSATP) and benzoylbezoicATP (BzATP) on forskolin-stimulated cAMP accumlction were first investigated. The rank order of potency in inhibition of cAMP accumulation was UTP > 2MeSATP > BzATP = AMPCPP > AMPPCP > ATP > ADP > ATPgS. Pretreatment of cells with pertussis toxin attenuated the effects of UTP, 2MeSATP, and ATP on cAMP accumulation whereas it had no effect on the BzATP-induced response. P2-purinoceptor mediated inhibition of cAMP accumulation was insensitive to cytosolic Ca2+ concentration ( [Ca2+] i ) although it was elevated by the same agonists. In addition, except for 2MeSATP the breakdown of cAMP was not accelerated by these agonists. The result indicates that the occupancy of P2-purinoceptor is directly coupled to pertussis toxin-sensitive G protein to inhibit cAMP accumulation. After NG108-15 cells have been differentiated, both ATP and BzATP elevated [Ca2+] i by stimulating Ca2+ influx. The EC50 values for ATP4- and BzATP in stimulating of [Ca2+] i incresae were 23 μM and 230 μM, respectively. ATP also triggered ethidium bromide uptake by activating the non-selective pore. Both [Ca2+] i incresae and ethidium bromide uptake induced by ATP or BzATP could be antagonized by extracellular Na+ and Mg2+. Howevere, the inhibitory effect of Na+ was not profound unless the calculated concentration of ATP4- was higher than the EC50 value. P2x7 recptor gene has recently been cloned and displays the functional characteristics of P2z receptor. Using RT-PCR, a cDNA fragment with highly homology to P2x7 is amplified from NG108-15 cells. These data suggest that P2z receptor is the major functional P2 purinoceptor in differentiated NG108-15 cells.
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