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Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

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Abstract

The aim of this study was to determine whether 45Ca2+ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC50 = 45 μM), ATPTS (EC50 = 50 μM) and 2-meSATP (EC50 = 81 μM) but not αβ meATP (I mM) stimulated 45Ca2+ 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 ADPβS did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC50 = 191 μM) 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 45Ca2+ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated 45Ca2+ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency :- guanidinium (EC50 = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose.

A number of P2 purinoceptor antagonists inhibited ATP-stimulated 45Ca2+ influx. Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (3–300 μM), pyridoxal 5phosphate (3–300 μM) and d-tubocurarine (30–300 μM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100–300 μM) and cibacron blue (30–300 μM) produced a surmountable antagonism while DIDS (4,4′-diisothiocyana-tostilbene-2,2′disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 μM. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X2 purinoceptors. These findings suggest that ATP-stimulated 45Ca2+ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.

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  1. Department of Pharmacology, University of Cambridge, Tennis Court Road, CB2 1QJ, Cambridge, England, UK

    Anton D. Michel, Caroline B. A. Grahames & Patrick P. A. Humphrey

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  1. Anton D. Michel
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  2. Caroline B. A. Grahames
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  3. Patrick P. A. Humphrey
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Michel, A.D., Grahames, C.B.A. & Humphrey, P.P.A. Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx. Naunyn-Schmiedeberg's Arch Pharmacol 354, 562–571 (1996). https://doi.org/10.1007/BF00170829

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