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.
Similar content being viewed by others
References
Albus U, Habermann E, Ferry DR, Glossman H (1984) Novel 1,4-dih9idropyridine (Bay K 8644) facilitates calcium-dependerr [3H]noradrenaline release from PC 12 cells. J Neurochem 42:1186–1189
Barry VA, Cheek TR (1994) Extracellular ATP triggers two functionally distinct calcium signalling pathways in PC12 cells. J Cell Sci 107:451–462
Beukers MW, Pirovano IM, van Weert A, Kerkhof CJ, Ijzerman AP, Soudijn W (1993) Characterization of ecto-ATPase on humar blood cells. A physiological role in platelet aggregation? Biochem Pharmacol 46:1959–1966
Bo X, Zhang Y, Nassar M, Burnstock G, Schoepfer R (1995) A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett 375:129–133
Brake AJ, Wagenbach MJ, Julius D (1994) New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371:519–523
Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, Wood JN (1995) A P2X purinoceptor expressed by a subset of sensory neurons. Nature 377:428–431
Collo G, North RA, Kawashima E, Merlo-Pich E, Neidhart S, Surprenant A, Buell G (1996) Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J Neurosci 16:2495–2507
el-Moatassim C, Dubyak GR (1992) A novel pathway for the activation of phospholipase D by P2z purinergic receptors in BACL2F5 macrophages. J Biol Chem 267:23664–23673
Erb L, Lustig KD, Ahmed AH, Gonzalez FA, Weisman GA (1990) Covalent incorporation of 3′-O-(4-benzoyl)benzoyl-ATP into a P2 purinoceptor in transformed mouse fibroblasts. J Biol Chem 265:7424–7431
Evans RJ, Lewis C, Buell G, Valera S, North RA, Surprenant A (1995) Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol 48:178–183
Fasolato C, Pizzo P, Pozzan T (1990) Receptor-mediated calcium influx in PC12 cells. ATP and bradykinin activate two independent pathways. J Biol Chem 265:20351–20355
Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, Williams M (1994) Nomenclature and classification of purinoceptors. Pharmacol Rev 46:143–156
Gollasch M, Haller H (1995) Multiple pathways for ATP-induced intracellular calcium elevation in pheochromocytoma (PC12) cells. Renal Physiol Biochem 18:57–65
Gomperts BD (1983) Involvement of guanine nucleotide-binding protein in the gating of Ca2+ by receptors. Nature 306:64–66
Grahames CBA, Michel AD, Humphrey PPA (1996) ATP-stimulated 45Ca2+ uptake as a means of measuring P2X purinoceptor activation in PC12 cells. Br J Pharmacol 117:106p
Housley GD, Greenwood D, Bennett T, Ryan AF (1995) Identification of a short form of the P2xRl-purinoceptor subunit produced by alternative splicing in the pituitary and cochlea. Biochem Biophys Res Commun 212:501–508
Inoue K, Nakazawa K, Fujimori K, Takanaka A (1989) Extracellular adenosine 5′-triphosphate-evoked norepinephrine secretion not relating to voltage-gated Ca channels in pheochromocytoma PC12 cells. Neurosci Lett 106:294–299
Khakh BS, Michel A, Humphrey PP (1994) Estimates of antagonist affinities at P2X purinoceptors in rat vas deferens. Eur J Pharmacol 263:301–309
Kim WK, Rabin RA (1994) Characterization of the purinergic P2 receptors in PC12 cells. Evidence for a novel subtype. J Biol Chem 269:6471–6477
Lewis C, Neidhart S, Holy C, North RA, Buell G, Surprenant A (1995) Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature 377:432–435
Majid MA, Okajima F, Kondo Y (1992) Characterization of ATP receptor which mediates norepinephrine release in PC12 cells. Biochim Biophys Acta 1136:283–289
Majid MA, Okajima F, Kondo Y (1993) UTP activates phospholipase C-Ca2+ system through a receptor different from the 53-kDa ATP receptor in PC12 cells. Biochem Biophys Res Commun 195:415–421
Michel AD, Humphrey PPA (1996) High affinity P2X purinoceptor binding sites for [35S]adenosine 5′-O-[3-thiotriphosphate] in rat vas deferens membranes. Br J Pharmacol 117:63–70
Michel AD, Lundstrom K, Buell GN, Surprenant A, Valera S, Humphrey PPA (1996a) The binding characteristics of a human bladder recombinant P2x purinoceptor, labelled using [3H]]αβmeATP, [35S]ATPγS or [33P]ATP. Br J Pharmacol 117:1254–1260
Michel AD, Lundstrom K, Buell GN, Surprenant A, Valera S, Humphrey PPA (1996b) A comparison of the binding characteristics of recombinant P2X1 and P2X2 purinoceptors. Br J Pharmacol 118:1806–1812
Murayama T, Oda H, Watanabe A, Nomura Y (1995) ATP receptor-mediated increase of Ca ionophore-stimulated arachadonic acid release from PC12 pheochromocytoma cells. Jpn J Pharmacol 69:43–51
Murrin RJ, Boarder MR (1992) Neuronal “nucleotide” receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP. Mol Pharmacol 41:561–568
Nakazawa K, Inoue K (1992) Roles of Ca2+ influx through ATP-activated channels in catecholamine release from pheochromocytoma PC12 cells. J Neurophysiol 68:2026–2932
Nakazawa K, Fujimori K, Takanaka A, Inoue K (1990) An ATP-activated conductance in pheochromocytoma cells and its suppression by extracellular calcium. J Physiol (Lond) 428:257–272
Nakazawa K, Inoue K, Fujimori K, Takanaka A (1991) Effects of ATP antagonists on purinoceptor-operated inward currents in rat phaeochromocytoma cells. Pflügers Arch 418:214–219
Nikodijevic B, Sei Y, Shin Y, Daly JW (1994) Effects of ATP and UTP in pheochromocytoma PC12 cells: evidence for the presence of three P2 receptors, only one of which subserves stimulation of norepinephrine release. Cell Mol Neurobiol 14:27–47
Nuttle LC, Dubyak GR (1994) Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes. J Biol Chem 269:13988–13996
Raha S, de Souza LR, Reed JK (1993) Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells. J Cell Physiol 154:623–630
Ransom JT, Cherwinski HM, Delmendo RE, Sharif NA, Eglen RM (1991) Characterisation of the M4 muscarinic receptor Ca2+ response in a subclone of PC-12 cells by single cell flow cytometry: inhibition of the response by bradykinin. J Biol Chem 266:11738–11744
Sela D, Ram E, Atlas D (1991) ATP receptor. A putative receptor-operated channel in PC-12 cells. J Biol Chem 266:17990–17994
Soltoff SP, McMillian MK, Talamo BR (1989) Coomassie Brilliant Blue G is a more potent antagonist of P2 purinergic responses than Reactive Blue 2 (Cibacron Blue 3GA) in rat parotid acinar cells. Biochem Biophys Res Commun 165:1279–1285
Soltoff SP, McMillian MK, Talamo BR, Cantley LC (1993) Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds. Biochem Pharmacol 45:1936–1940
Surprenant A, Rassendren F, Kawashima E, North RA, Buell G (1996) The cytolytic P2z receptor for extracellular ATP identified as a P2x receptor (P2X7). Science 272:735–738
Tatham PE, Cusack NJ, Gomperts BD (1988) Characterisation of the ATP4-receptor that mediates permeabilisation of rat mast cells. Eur J Pharmacol 147:13–21
Trezise DJ, Bell NJ, Khakh BS, Michel AD, Humphrey PA (1994a) P2 purinoceptor antagonist properties of pyridoxal-5-phosphate. Eur J Pharmacol 259:295–300
Trezise DJ, Kennedy 1, Humphrey PP (1994b) The use of antagonists to characterize the receptors mediating depolarization of the rat isolated vagus nerve by alpha, beta-methylene adenosine 5'-triphosphate. Br J Pharmacol 112:282–288
Valera S, Hussy N, Evans RJ, Adami N, North RA, Surprenant A, Buell G (1994) A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature 371:516–519
Wiley JS, Chen R, Jamieson GP (1993) The ATP4-receptor-operated channel (P2Z class) of human lymphocytes allows Ba2+ and ethidium+ uptake: inhibition of fluxes by suramin. Arch Biochem Biophys 305:54–60
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00170829