ATP (1 mM) induced a biphasic increase in intracellular Ca 2~concentration ([Ca2~]), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[17~-3methoxyestra-1,3, 5(10)-trien -1 7-yl]amino]hexyl]-1Hpyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca2~.We examined the mechanism of the AlP-elicited sustained [Ca2~], increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC 50 of ATP was -~0.75mM. The permeability ratio of various cations carrying this current was Na(defined as 1)> Li(0.92 ± 0.01; n = 5)> K(0.89 ± 0.03; n = 6) > Rb(0.55 ± 0.02; n = 6) > Cs~(0.51 ± 0.01; n = 5) > Ca~(0.22 ± 0.03; n = 3) > N-methyl-n-glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg Abbreviations used: ADP~3S, adenosine 5 '-O-(2-thiodiphosphate); AMPCPP, a~8-methylene-ATP;AMPPCP,~3,y-methylene-ATP; ATPyS, adenosine 5 '-O-(3-thiotriphosphate); BzATP, benzoylbenzoic-ATP; [Ca 2~]~, intracellular Ca2~concentration; I-V, current-voltage; 1P3, inositol 1 ,4,5-trisphosphate; 2MeSATP, 2methylthio-ATP; MS -, methanesulfonic acid; NMG N-methyl-Dglucamine; PLC, phosphoinositide-specific phospholipase C; U73122, 1-[6-[17~3-3-methoxyestra-l,3,5(10)-trien-l7-yl]amino]hexyl ] -lH-pyrrole-2,5-dione.

Extracellular ATP induces a nonselective cation current and elevates intracellular Ca2+ concentration via P2Z receptors in NG108-15 cells. We found that the ATP-induced nonselective cation current became larger in methanesulfonic acid (MS-) than in Cl- external solution. We therefore examined the effects of various external anions on the ATP-induced cation current with the use of the whole cell patch-clamp technique. The concentration-response curves for ATP were obtained in different anionic external solutions. The maximum current density (Imax) and the concentration of agonist that gives 50% of maximum response (EC50) value of ATP were obtained by fitting the curves with the use of the Hill coefficient of 2. The apparent Imax decreased in the order of aspartic acid (Asp-) > MS- > F- > Cl- > Br- > or = I-. The apparent EC50 values for ATP were shifted to the right in the sequence of Asp- < F- < MS- < Br- < Cl-< I-. Thus both Imax and EC50 values were affected by anions, indicating that anions are mixed-type inhibitors of the ATP-induced current. The shift of the EC50 values of ATP indicates that anions interfere with ATP binding to the receptor. External Cl- was a noncompetitive inhibitor with respect to external Na+, a major cation carrying the ATP-induced current. We conclude that extracellular anions inhibit the ATP-induced nonselective cation current at least partly by interfering with ATP binding to the P2Z receptor, which is associated with the nonselective cation channels.

ATP activates a nonselective cation current by stimulating the P2Z receptor in NG1 08-15 cells-a hybrid cell line of the mouse neuroblastoma N18TG-2 cells and the rat glioma C6Bu-1 cells. Recently, the P2X7 receptor was cloned from the rat brain and was found to have electrophysiological properties similar to those of the P2Z receptor. We examined the expression of P2X7 receptor mRNA in NG1O8-15 cells as well as in their parent cell lines, Ni 8TG-2 and C6Bu-1 cells, by reverse transcription-polymerase chain reaction (RT-PCR). The cDNA templates from these cell lines were amplified with primers specific to the P2X7 receptor sequence. Positive signals were detected in the RT-PCR products from NG1O8-15 and Ni 8TG-2 cells but not from C6Bu-i cells. We next examined the effect of ATP on the membrane current in N18TG-2 cells and C6Bu-1 cells by whole-cell voltage clamp. In Ni 8TG-2 cells, ATP induced a sustained current with a reversal potential of 9.3 ± 1.2 mV (n = 22) in a concentration-dependent manner with an EC 50 of 1.76 ± 0.18 mM (n = 36). In contrast, ATP (1 mM) did not induce any current in C6Bu-i cells. The ATP-induced current in Ni 8TG-2 cells resembled that in NG1 08-iS cells in the following points: (a) The currents did not desensitize significantly. (b) EC50 values of ATP are of millimolar order. (c) Benzoylbenzoyl-ATP was a more potent agonist than ATP. (d) The current was larger in methanesulfonate than in C1 external solution. (e) The current was larger at lower external Mg 2concentrations. These results suggest that the hybrid NG1 08-15 cells possess a P2X7 receptor like the P2Z receptor and that the ability of expressing this channel originates from N18TG-2 cells but not from C6Bu-1 cells. Key Words: ATP-P2Z purinoceptor-P2X7 receptor-Nonselective cation channel-N18TG-2 cells. J. Neurochem. 70, 951 -957 (1998).

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