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

Michel, Anton D.; Grahames, Caroline; Humphrey, P. P. A.

doi:10.1007/bf00170829

Cited by 43 publications

(49 citation statements)

References 39 publications

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“…A nervous system-specific P2X receptor subtype, P2X2, was initially cloned from PC12 cells and exhibits a distinctive pharmacological profile (10) and has been implicated in mediating many of the biological actions of ATP. Michel et al (11) have recently demonstrated that the P2X2 receptor appears to be principally responsible for the ATPstimulated calcium influx in PC12 cells, extending and confirming a number of earlier reports of the involvement of the P2X subclass in mediating the action of ATP.…”

supporting

confidence: 66%

“…The ATP-stimulated calcium influx (11) and MAP kinase activation in PC12 cells is a consequence of the activa- ). B, the two PC12 cell lines, the line used in this study (left panels), and the line obtained from Dr. S. Soltoff (right panels) were deprived of serum for 16 h and then treated with 100 mM ATP, UTP for 2 min or 50 ng/ml NGF for 5 min.…”

Section: Discussionmentioning

confidence: 99%

“…ATP has been demonstrated to depolarize PC12 cells (6), raise intracellular calcium levels (7,11,24), and elevate phosphoinositide levels (25). The principal biological effect of ATP is its action as a secretogogue, causing the release of catecholamines (5-7, 8, 9).…”

Section: Discussionmentioning

confidence: 99%

“…The effect of ATP on P2X purinoreceptors is selectively antagonized by suramin and reactive blue 2 (8,9,11,20). Pretreatment of PC12 cells with either of these agents at concentrations that block ATP-driven calcium influx (11) for 30 min prior to stimulation with ATP abolished the ability of ATP to stimulate the MAP kinases (Fig.…”

Section: Extracellular Atp Activates the Map Kinases Erk1 And Erk2 Inmentioning

confidence: 99%

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ATP-stimulated Activation of the Mitogen-activated Protein Kinases through Ionotrophic P2X2 Purinoreceptors in PC12 Cells

Swanson¹,

Reigh

Landreth

1998

Journal of Biological Chemistry

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Extracellular purine nucleotides elicit a diverse range of biological responses through binding to specific cell surface receptors. The ionotrophic P2X subclass of purinoreceptors respond to ATP by stimulation of calcium ion permeability; however, it is unknown how P2X purinoreceptor activation is linked to intracellular signaling pathways. We report that stimulation of PC12 cells with ATP results in the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 and was wholly dependent upon extracellular calcium ions. Treatment of the cells with adenosine, AMP, ADP, UTP, or ␣,␤-methylene ATP was without effect; however, MAP kinase activation was abolished by pretreatment with suramin and reactive blue 2. The calcium-activated tyrosine kinase, Pyk2, acts as an upstream regulator of the MAP kinases and became tyrosine phosphorylated following treatment of the cells with ATP. We have ruled out the involvement of depolarization-mediated calcium influx because specific blockers of voltage-gated calcium channels did not affect MAP kinase activation. These data provide direct evidence that calcium influx through P2X2 receptors results in the activation of the MAP kinase cascade. Finally, we demonstrate that a different line of PC12 cells respond to ATP through P2Y2 purinoreceptors, providing an explanation for the conflicting findings of purine nucleotide responsiveness in PC12 cells.Adenine nucleotides act through specific cell surface receptors to provoke a variety of biological responses (1, 2). In the nervous system, extracellular ATP has been postulated to serve as a classical neurotransmitter and to also act as a co-transmitter through its coordinate packaging and release with norepinephrine and acetylcholine (3). The analysis of these roles of ATP has been complicated by its catabolism into other biologically active species and the unexpected diversity of cell surface purinoreceptors. Purinoreceptors have been classified into two primary classes, the P1 receptors are responsive to adenosine, whereas the P2 class receptors respond to a variety of purine nucleotides, including ATP. Presently, twelve P2 subtypes have been identified and assigned to two mechanistically distinct subclasses of purinoreceptors (1). The metabotrophic purinoreceptors of the P2Y subclass (formerly P2u, P2t, and P2y) initiate their biological actions by G-protein-dependent activation of phospholipase C and subsequent elevation of intracellular calcium levels through liberation of calcium from internal stores (4). The P2X purinoreceptors comprise a distinct subclass of receptors that are ligand-gated calcium channels functionally related to glutamate and nicotinic acetylcholine receptors. The ionotrophic P2X receptors regulate intracellular calcium levels through the ligand-stimulated increase in calcium permeability; thus their actions are dependent upon extracellular calcium ions. Although the ability of the P2X receptors to evoke a rise in intracellular calcium levels is well documented, it is entirely unclear how these rec...

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supporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Extracellular Atp Activates the Map Kinases Erk1 And Erk2 Inmentioning

confidence: 99%

See 2 more Smart Citations

ATP-stimulated Activation of the Mitogen-activated Protein Kinases through Ionotrophic P2X2 Purinoreceptors in PC12 Cells

Swanson¹,

Reigh

Landreth

1998

Journal of Biological Chemistry

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“…It has also been suggested that RB-2 inhibits ectonucleotidase activity in X. laevis oocytes and ATP-induced inflammation of the mouse hind paw . The inhibitory effects of RB-2 were also observed in currents and calcium measurements in single PC12 cells (Nakazawa et al, 1991;Michel et al, 1996) as well as in experiments with cells expressing recombinant rP2X1R and P2X2R (Surprenant, 1996). For a more detailed description of the antagonistic actions of RB-2, see Ralevic and Burnstock (1998).…”

Section: A P2x Receptor Agonism and Antagonismmentioning

confidence: 86%

Activation and Regulation of Purinergic P2X Receptor Channels

et al. 2011

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Lack of specificity of [35S]-ATP?S and [35S]-ADP?S as radioligands for ionotropic and metabotropic P2 receptor binding

Bianchi

Metzger

et al. 1999

Drug Dev. Res.

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Adenosine‐5′‐O‐3‐thio[35S]triphosphate ([35S]‐ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X1, P2X2, P2X3, and P2X4. Similarly, adenosine‐5′‐O‐2‐thio[35S]diphosphate ([35S]‐ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [35S]‐ATPγS and [35S]‐ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X4 transfected 1321N1 astrocytoma cells. [35S]‐ATPγS (0.2 nM) and [35S]‐ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [35S]‐ATPγS and [35S]‐ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y1 and P2Y2 receptor subtypes, to 1321N1 cells stably transfected with the human P2X4 receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X4 receptor, as measured by cytosolic calcium flux. These data indicate that [35S]‐ATPγS and [35S]‐ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. Drug Dev. Res. 48:84–93, 1999. © 1999 Wiley‐Liss, Inc.

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

Cited by 43 publications

References 39 publications

ATP-stimulated Activation of the Mitogen-activated Protein Kinases through Ionotrophic P2X2 Purinoreceptors in PC12 Cells

ATP-stimulated Activation of the Mitogen-activated Protein Kinases through Ionotrophic P2X2 Purinoreceptors in PC12 Cells

Activation and Regulation of Purinergic P2X Receptor Channels

Lack of specificity of [35S]-ATP?S and [35S]-ADP?S as radioligands for ionotropic and metabotropic P2 receptor binding

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