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Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx.
Naunyn Schmiedebergs Arch Pharmacol. 1996 Nov; 354(5):562-71.NS

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 microM), ATP gamma S (EC50 = 50 microM) and 2-meSATP (EC50 = 81 microM) but not alpha beta meATP (1 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 ADP beta S 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 microM) 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 microM), pyridoxal 5-phosphate (3-300 microM) and d-tubocurarine (30-300 microM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100-300 microM) and cibacron blue (30-300 microM) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 microM. 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.

Authors+Show Affiliations

Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, England, UK.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

8938653

Citation

Michel, A D., et al. "Functional Characterisation of P2 Purinoceptors in PC12 Cells By Measurement of Radiolabelled Calcium Influx." Naunyn-Schmiedeberg's Archives of Pharmacology, vol. 354, no. 5, 1996, pp. 562-71.
Michel AD, Grahames CB, Humphrey PP. Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx. Naunyn Schmiedebergs Arch Pharmacol. 1996;354(5):562-71.
Michel, A. D., Grahames, C. B., & Humphrey, P. P. (1996). Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx. Naunyn-Schmiedeberg's Archives of Pharmacology, 354(5), 562-71.
Michel AD, Grahames CB, Humphrey PP. Functional Characterisation of P2 Purinoceptors in PC12 Cells By Measurement of Radiolabelled Calcium Influx. Naunyn Schmiedebergs Arch Pharmacol. 1996;354(5):562-71. PubMed PMID: 8938653.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx. AU - Michel,A D, AU - Grahames,C B, AU - Humphrey,P P, PY - 1996/11/1/pubmed PY - 1996/11/1/medline PY - 1996/11/1/entrez SP - 562 EP - 71 JF - Naunyn-Schmiedeberg's archives of pharmacology JO - Naunyn Schmiedebergs Arch. Pharmacol. VL - 354 IS - 5 N2 - 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 microM), ATP gamma S (EC50 = 50 microM) and 2-meSATP (EC50 = 81 microM) but not alpha beta meATP (1 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 ADP beta S 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 microM) 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 microM), pyridoxal 5-phosphate (3-300 microM) and d-tubocurarine (30-300 microM) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC50. Suramin (100-300 microM) and cibacron blue (30-300 microM) produced a surmountable antagonism while DIDS (4,4'-diisothiocyanatostilbene-2,2'disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 microM. 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. SN - 0028-1298 UR - https://www.unboundmedicine.com/medline/citation/8938653/Functional_characterisation_of_P2_purinoceptors_in_PC12_cells_by_measurement_of_radiolabelled_calcium_influx_ DB - PRIME DP - Unbound Medicine ER -