FUNCTIONAL-CHARACTERIZATION OF P2 PURINOCEPTORS IN PC12 CELLS BY MEASUREMENT OF RADIOLABELED CALCIUM INFLUX

The aim of this study was to determine whether Ca-45(2+) influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentia ted PC12 cells, grown in suspension, ATP (EC(50) = 45 mu M), ATP gamma S (EC(50) = 50 mu M) and 2-meSATP (EC(50) = 81 mu M) but not alpha be ta meATP (1 mM) stimulated Ca-45(2+) influx 2-5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADP beta S did not produce any significant effect. Sim ilarly, the effects of ATP were not apparently mediated through activa tion of P2Z purinoceptors since dibenzylATP behaved as a weak (EC(50) = 191 mu M) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells. ATP-stimulated Ca-45(2+) influx was not affected by nifedi pine suggesting that it was not secondary to activation of L-type calc ium channels and rather reflected influx through a P2X purinoceptor pr esent in these cells. The ATP-stimulated Ca-45(2+) influx could be red uced by monovalent cations, presumably as a result of direct competiti on for influx through the cation channel, with the following rank orde r of potency :- guanidinium (EC(50) = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose). A number of P2 purinoceptor antago nists inhibited ATP-stimulated Ca-45(2+) influx. Pyridoxalphosphate-6- azophenyl-2',4'-disulphonic acid (3-300 mu M), pyridoxal 5-phosphate ( 3-300 mu M) and d-tubocurarine (30-300 mu M) produced an insurmountabl e antagonism of responses to ATP, with no marked change in agonist EC( 50). Suramin (100-300 mu M) and cibacron blue (30-300 mu M) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2 'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 mu M. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P 2X(2) purinoceptors. These findings suggest that ATP-stimulated Ca-45( 2+) influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.