Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
Molecular Pharmacology
Forward.
12, 2014
as from
doi:10.1124/mol.114.092528
This article hasFast
not been
copyeditedPublished
and formatted. on
The June
final version
may differ
this version.
MOL #92528
Title Page
Ca2+ influx through P2X1 receptors amplifies P2Y1 receptor-evoked Ca2+
signalling and ADP-evoked platelet aggregation
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
Sarah Jones, Richard J. Evans, Martyn P. Mahaut-Smith
University of Leicester, Department of Cell Physiology and Pharmacology,
Leicester LE1 9HN, UK
1
Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Running Title Page
Running Title: P2X1-dependent amplification of P2Y1 responses
Correspondence: Martyn Mahaut-Smith, Department of Cell Physiology and Pharmacology,
University of Leicester, University Road, PO Box 138, Leicester LE1 9HN. Telephone: +44 116 229
7135; Fax: +44 116 252 5045; email: mpms1@le.ac.uk; Richard Evans, Department of Cell
Physiology and Pharmacology, University of Leicester, University Road, PO Box 138, Leicester LE1
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
9HN. Telephone: +44 116 229 7057; Fax: +44 116 252 5045; email: rje6@le.ac.uk
Text pages: 23
Tables: none
Figures: 8
References: 54
Abstract: 247 words
Introduction: 722 words
Discussion: 1,486
Abbreviations: The abbreviations used are: GF109203X, 2-[1-(3-Dimethylaminopropyl)-1H-indol3-yl]-3-(1H-indol-3-yl)maleimide: Y-27632, trans-4-[(1R)-1-Aminoethyl]-N-4pyridinylcyclohexanecarboxamide dihydrochloride: U0126, 1,4-Diamino-2,3-dicyano-1,4-bis[2aminophenylthio]butadiene: GPCR, G-protein-coupled receptor; MRS2179, 2’-deoxy-N(6)-methyl
adenosine 3’,5’-diphosphate: α,β-meATP: α,β-methyleneATP: HEK, human embryonic kidney:
NF449, 4,4′,4″,4-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3disulfonic acid: ACD, acid citrate dextrose: PRP, platelet-rich plasma: PLC, phospholipase-C:
ERK1/2, extracellular signal-regulated protein kinase1/2: PKC, protein kinase C
2
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Abstract
Many cells express both P2X cation channels and P2Y G-protein-coupled receptors that are costimulated by nucleotides released during physiological or pathophysiological responses. For
example, during haemostasis and thrombosis, ATP-gated P2X1 channels and ADP-stimulated P2Y1
and P2Y12 G-protein coupled receptors play important roles in platelet activation. It has previously
been reported that P2X1 receptors amplify P2Y1-evoked Ca2+ responses in platelets but the
underlying mechanism and influence on function is unknown. In human platelets, we show that
aggregation response to a submaximal concentration of ADP. Co-stimulation of P2X1 and P2Y1
receptors generated a super-additive Ca2+ increase in both human platelets and HEK293 cells via a
mechanism dependent on Ca2+ influx rather than Na+ influx or membrane depolarisation. The
potentiation, due to an enhanced P2Y1 response, was observed if ADP was added up to 60 seconds
after P2X1 activation. P2X1 receptors also enhanced Ca2+ responses when co-stimulated with type 1
protease activated and M1 muscarinic acetylcholine receptors. The P2X1-dependent amplification of
Gq-coupled [Ca2+]i increases was mimicked by ionomycin, and not affected by inhibition of protein
kinase C, Rho-kinase and ERK1/2, suggesting that it results from potentiation of IP3 receptors and/or
phospholipase-C. We conclude that Ca2+ influx through P2X1 receptors amplifies Ca2+ signalling
through P2Y1 and other Gq-coupled receptors. This represents a general form of co-incidence
detection of ATP and co-released agonists, such as ADP at sites of vascular injury or synaptic
transmitters acting at metabotropic Gq-coupled receptors.
3
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
maximally activated P2X1 receptors failed to stimulate significant aggregation, but could amplify the
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Introduction
P2X ligand-gated ion channels and P2Y G-protein-coupled receptors (GPCRs) play important roles in
a variety of excitable and non-excitable tissues (Burnstock and Knight, 2004). In mammals, there are
seven P2X receptor subunits that form a range of homo- and hetero-trimeric channels (Surprenant and
North, 2009), and there are eight genes encoding P2Y receptors. Individual cells normally express
more than one P2 receptor subtype and interactions between their signalling pathways have the
potential to regulate cellular responses. For example, platelets express only P2X1, P2Y1 and P2Y12
P2X1 receptors generate significant transient increases in intracellular Ca2+ leading to shape change
but not aggregation responses (Hechler et al., 2003; Oury et al., 2001; Rolf et al., 2001; Rolf and
Mahaut-Smith, 2002). Previous work has demonstrated interactions between platelet P2X1 and P2Y1
receptors at the level of Ca2+ mobilisation and the activation of P2Y1-evoked non-selective cation
currents (Vial et al., 2002). In contrast, no evidence was found for interactions between P2X1
receptors and Gi-coupled P2Y12 pathways (Rolf and Mahaut-Smith, 2002). Interestingly,
costimulation of Gq-coupled P2Y1 and Gi-coupled P2Y12 receptor pathways is required for full
aggregation responses to ADP (Cosemans et al., 2006; Fabre et al., 1999; Foster et al., 2001; Hechler
et al., 1998; Jin et al., 1998; Jones et al., 2011; Leon et al., 1999). Taken together these studies
highlight the complex interdependency of P2 receptor signalling.
P2X receptors are ATP-gated non-selective cation channels that generate significant direct
Na+ and Ca2+ entry, leading to membrane depolarisation (Surprenant and North, 2009). In excitable
tissues, P2X1-induced depolarisation can therefore lead to activation of additional Ca2+ entry through
stimulation of voltage-gated Ca2+ channels. Furthermore, in both excitable and non-excitable cell
types, Na+ entry has the potential to elevate Ca2+ via reverse Na+/Ca2+ exchange activity (Harper et al.,
2013) and membrane depolarisation has been reported to directly amplify GPCR-evoked Ca2+ release
(Mahaut-Smith et al., 2008). Of note, it has been shown that P2Y1-evoked Ca2+ release is enhanced
by membrane depolarisations of physiologically relevant magnitude in the platelet precursor cell, the
megakaryocyte (Martinez-Pinna et al., 2005; Martinez-Pinna et al., 2004). The ability of P2X1
receptors to modulate Gq-coupled receptor signalling independent of voltage-gated Ca2+ channels
4
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
receptors (Hechler and Gachet, 2011; Kahner et al., 2006; Mahaut-Smith et al., 2011). ATP-gated
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
could have widespread relevance given the ubiquitous occurrence of ATP as a co-transmitter
throughout the peripheral and central nervous system (Burnstock, 2004). The aim of this study was to
use human platelets and a human non-excitable cell line expressing P2X1 and P2Y1 receptors to
investigate the interactions between these two receptors at the level of signalling and function.
Materials and Methods
Reagents ─ Fura2-AM and Fluo3-AM were purchased from Invitrogen (Paisley, UK). GF109203X,
reagents were obtained from Sigma-Aldrich (Poole, UK) unless otherwise stated. ADP was treated
with hexokinase to remove contaminating ATP as reported previously (Mahaut-Smith et al., 2000).
Platelet Preparation ─ Blood was obtained from healthy, aspirin-free, informed consenting
volunteers. The study was approved by the University of Leicester Committee for Research Ethics
concerning human subjects (non-NHS) and carried out in accordance with the Declaration of
Helsinki. Blood was drawn from the forearm by venepuncture into a syringe containing acid citrate
dextrose anticoagulant (ACD: in mM 85 trisodium citrate, 78 citric acid, 111 glucose) 6:1 v/v.
Platelet-rich plasma (PRP) was obtained by centrifugation at 700g for 5 minutes and treated with
apyrase (0.32 U/ml), and where stated aspirin (100 µM). Washed platelet suspensions were prepared
by centrifugation at 350g for 20 min and resuspension in apyrase-containing nominally Ca2+ free
platelet saline (in mM: 145 NaCl, 5 KCl, 1 MgCl2 10 HEPES, 10 glucose, pH 7.35, 0.32U/ml
apyrase). 2mM CaCl2 was added to the platelets immediately prior to each experimental run. For Ca2+
and aggregation experiments, platelets were suspended in a volume of saline equal to twice the
volume of PRP. For intracellular calcium measurements, PRP was incubated with fura2-AM (2 μM),
for 45 min at 37°C prior to preparation of washed suspensions.
Platelet Calcium Measurements ─ Calcium measurements from platelet suspensions were performed
at 37°C under stirring conditions using a Cairn spectrofluorimeter (Cairn Research Limited, Kent,
UK). Fura-2 340/380nm background-corrected fluorescence ratios were calibrated extracellularly
5
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
Y-27632, MRS2179 and NF449 were purchased from Tocris Bioscience (Avonmouth, UK). All other
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
following addition of 50 μM digitonin as described elsewhere using a Kd of 224nM (Rolf and
Mahaut-Smith, 2002).
Platelet Aggregation ─ Aggregation was assessed by turbidimetry in washed platelet suspensions in
the presence of 0.5 mg/ml fibrinogen in response to ADP (1 μM), α,β-meATP (5 μM) or both agonists
at 37°C under stirring conditions using a Model 400 lumi-aggregometer (Chronolog, Havertown, PA,
USA). Responses were measured as the peak or integral of the light transmission increases and
normalised to control responses for the same batch of platelets due to the well-recognised variability
Cell Culture ─ Native human embryonic kidney (HEK293) cells and HEK293 cells stably expressing
recombinant human P2X1 receptors (HEK293-P2X1) (Vial & Evans 2005) were maintained in
minimal essential medium with Earle’s Salts (with GlutaMAX™ I) supplemented with 10% fetal
bovine serum and 1% non-essential amino acids at 37°C in a humidified atmosphere of 95% air, 5 %
CO2. The HEK293-P2X1 line was generated using a plasmid conferring resistance to geneticin, which
was used at 500 μM throughout its culture. Voltage steps from a holding potential of -75 to 5 mV for
>1 s failed to induce intracellular Ca2+ increases in HEK293 cells (Gurung, 2005), indicating the lack
of endogenous functional voltage-gated Ca2+ channels.
HEK293 Calcium Measurements ─ Cells seeded into 96 well black-wall plates 72 hours prior to use
were loaded with Fluo-3AM (2 μM) in normal saline (in mM: 145 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10
HEPES, 10 glucose) containing 1% BSA, 0.16 U/ml apyrase and 0.02% pluronic. After 1 hour at
room temperature, loading buffer was replaced with normal saline and changes in fluorescence
evoked by different agonists were measured using a Flexstation II 96 well fluorimeter (Molecular
Devices, Sunnyvale, California). When required, NF449 (1 μM), GF109203X (10 μM), U0126 (10
μM) or Y27632 (10μM) was added to the cells prior to stimulation. To examine the role of
extracellular Ca2+, experiments were conducted in saline without CaCl2 (nominally Ca2+-free) or
following equimolar substitution with BaCl2. To assess the role of extracellular Na+, NaCl was
replaced with NMDGCl. To compare synergy between different batches of cells, responses were
normalised to the maximal ADP response (at 100 µM ADP).
6
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
between donors and also between samples.
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Derivation of predicted responses and statistical Analysis ─ For both platelet suspensions and
HEK293 cells, predicted calcium responses for co-stimulations were calculated by addition of the
individual calcium increases induced by the two agonists. The integral of the Ca2+ response was
assessed for a period of 60 seconds after agonist addition in the platelet and 90 seconds after agonist
addition in HEK cells. Recordings of calcium and aggregation responses are from individual
experiments with paired controls, representative of 3 to 8 different donors for platelet responses and 3
to 8 separate batches of cells for experiments with cell lines. Averages are reported as the means ±
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
SEM and statistical significance assessed using either paired or unpaired Student’s t-test or 1 way
ANOVA, with Bonferroni multiple comparison test (Microsoft Excel, GraphPad Prism). The level of
significance is indicated in the figures as p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).
7
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Results
Stimulation of platelet P2X1 receptors potentiates ADP-mediated Ca2+ mobilisation and aggregation
Previous studies have provided clear evidence that P2X1 and P2Y1 receptors synergise at the level of
calcium signalling in platelets (Vial et al., 2002) but the impact of this interaction on functional
responses has not been determined. It is established that the coupling of P2Y1 receptors to
aggregation requires co-activation of P2Y12-stimulated pathways (Hechler and Gachet, 2011; Kahner
et al., 2006) and that P2X1 receptors do not directly potentiate P2Y12-evoked aggregation (Rolf and
platelet function, we compared responses to the selective P2X1 agonist α,β-meATP, the physiological
P2Y1 and P2Y12 agonist ADP, and both agonists combined. ADP was used at a concentration (1 µM)
shown previously to stimulate ~60 % of the maximal Ca2+ response via P2Y receptors (MacKenzie et
al., 1996), which in our experiments induced a transient increase in intracellular calcium with an
average peak value of 395 ± 20.2nM (Fig. 1Ai,ii). Maximal stimulation of P2X1 receptors with α,βmeATP (5 μM) evoked a transient Ca2+ response of similar duration and magnitude (peak 277 ± 60.4
nM Fig. 1Ai,ii) to that seen with ADP. Importantly, co-application of α,β-meATP and ADP evoked a
rise in intracellular calcium (907±97 nM, denoted “actual” response), which was 1.69 ± 0.18 fold
greater (p < 0.05) than the response “predicted” by mathematical addition of the Ca2+ responses to the
individual agonists (536±0.9 nM, Fig.1Ai,ii), consistent with previous studies (Vial et al., 2002). The
integral of the Ca2+ response following co-stimulation by α,β-meATP and ADP was also enhanced
compared to the value obtained by summation of the individual agonist responses (Fig. 1Aiii).
However, amplification of the peak increase was more pronounced. In turbidimetric measurements,
1μM ADP evoked a transient aggregation of platelets peaking at 20 % transmission increase. 5μM
α,β-meATP generated a transient shape change but failed to stimulate aggregation (Fig. 1Bi), as
reported previously by our group and others (Hechler et al., 2003; Oury et al., 2001; Rolf et al., 2001;
Rolf and Mahaut-Smith, 2002). However, co-addition of 5μM α,β-meATP and 1 μM ADP evoked an
aggregation response significantly greater than that observed with 1 µM ADP alone (Fig. 1Bi-iii; the
amplitude and integral of the aggregation response were increased 1.95 ± 0.20 fold, p < 0.05, and 2.65
8
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
Mahaut-Smith, 2002). Therefore, in order to explore the consequence of P2X1:P2Y1 interactions on
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
± 0.63 fold, p < 0.01, respectively). Under the conditions of these experiments, the ADP-evoked Ca2+
response is entirely dependent upon P2Y1 receptors as it is blocked by the P2Y1-selective antagonist
MRS2179 and unaffected by the P2Y12 antagonist cangrelor (Fung et al., 2007). Therefore, these
results demonstrate a clear synergistic interaction of P2X1 and P2Y1 receptor signalling at the level of
intracellular Ca2+ mobilisation, leading to enhanced platelet ADP-evoked functional responses. A
major role for extracellular signal-regulated kinase 2 activation (ERK2) has been proposed in the
Ca2+-dependent pathways downstream of P2X1 receptor activation that potentiates collagen-evoked
ADP-stimulated aggregation via the generation of thromboxaneA2 (TXA2) (Garcia et al., 2007;
Stefanini et al., 2009) that reinforces the P2Y-evoked aggregation response (Mustard et al., 1975;
Packham et al., 1989). Inhibition of ERK1/2 activation using U0126 (10 μM) did not affect the α,βmeATP-induced potentiation of P2Y1 mediated calcium mobilisation (1.68 ± 0.15 fold and 1.62 ±
0.12 fold larger than predicted in the presence of vehicle control and U0126, respectively, p < 0.05
Fig. 2A). U0126 also had no significant effect on the aggregation response evoked by 1 μM ADP
alone (not shown). However, U0126 (10μM) completely abolished the enhancement of ADPmediated aggregation by α,β-meATP (aggregation responses 2.1 ± 0.5 and 1.0 ± 0.05 fold for vehicle
control and U0126, respectively; Fig. 2B). Consistent with reports that TXA2 generation is
downstream of ERK phosphorylation (Garcia et al., 2007; Stefanini et al., 2009), potentiation of ADPevoked calcium responses by α,β-meATP was unaffected by treatment with 100 μM aspirin (1.76 ±
0.2 fold increase above predicted Ca2+ response, p <0.05 Fig. 2A), but the enhanced aggregation was
abolished (1.0 ± 0.17 fold change compared to predicted response; Fig. 2B). This effect was not a
result of direct inhibition of the P2Y-evoked response, since the aggregation evoked by 1μM ADP
alone was not significantly altered by 100 μM aspirin (not shown).
Use of HEK293 cells and recombinant P2X1 receptor expression demonstrates that Ca2+
influx through P2X1 receptors potentiates P2Y1-evoked Ca2+ responses ─ To explore further the
mechanism whereby P2X1 and P2Y1 receptors interact at the level of Ca2+ mobilisation, we switched
to a HEK293 cell line in order to avoid the secondary responses that are known to occur in platelets
9
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
aggregation (Oury et al., 2002). Furthermore, Ca2+-dependent ERK2 signalling has been implicated in
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
due to release of nucleotides and other agonists. This also allowed us to assess whether the
P2X1:P2Y1 synergy is a general phenomenon or specific to platelets. In native HEK293 cells, ADP
stimulated a concentration-dependent intracellular Ca2+ increase with an EC50 of 0.04 μM (Fig. 3A,C).
ADP-evoked responses were inhibited by the P2Y1-selective receptor antagonist MRS2179 (Fig. 3B)
and thus are due to endogenously expressed P2Y1 receptors. In contrast, the P2X1 agonist α,βmeATP failed to induce a Ca2+ increase or alter the response to ADP in these native HEK293 cells
(not shown). However, when human P2X1 receptors were stably expressed in HEK293 cells
concentration-dependent inward currents in patch clamp studies (Evans et al., 1996) and increases in
intracellular calcium (Fig. 3D). The HEK293-P2X1 cells therefore provide an ideal model system to
investigate the mechanism underlying synergy between P2X1 and P2Y1 receptors. We assessed the
magnitude of the interaction between these two receptors through calcium responses to ADP (0.01
μM), α,β-meATP (0.05 μM) or combined application of both agonists at these submaximal
concentrations. We reasoned that a lower α,β-meATP concentration was more relevant to study the
synergy in the cell line due to its high P2X1 receptor density. Responses to submaximal α,β-meATP
concentrations were consistently observed in all batches of HEK293-P2X1 cells, in contrast to
platelets where a substantial interdonor variability made it difficult to use low levels of the P2X1
agonist. Fig.4Ai shows the Ca2+ indicator responses from a single experimental run and Fig.4Aii
shows the average peak increase from multiple batches of cells normalised to the response evoked by
a maximal ADP concentration (see methods for further detail). The “actual” peak Ca2+ increase
induced by simultaneous addition of these two agonists was an average of 2.55 ± 0.26 fold greater
than the response “predicted” by summation of the responses to the individual agonists. This
amplification was mediated by P2X1 receptors as (i) α,β-meATP had no effect on ADP responses in
native HEK cells that lack P2X1 receptors (Fig. 5C) and (ii) 1 μM NF449 (a concentration of this
suramin analogue that selectively blocks P2X1 receptors (Fung et al., 2007)) prevented α,β-meATPmediated amplification of ADP-evoked P2Y1 Ca2+ responses (Fig. 5C). In addition, α,β-meATP
(0.05 µM) also caused a similar enhancement of the Ca2+ response to the non-hydrolysable analogue
10
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
(HEK293-P2X1, which did not alter the P2Y1-evoked response; Fig. 3C), α,β-meATP evoked
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
ADPβS (0.1 µM) (2.78 ± 0.18 fold increase above the predicted response, Fig. 4Bi,ii), demonstrating
that the enhanced P2Y1 response does not result from reduced ADP breakdown by inhibition of
ectonucleotidase activity (Jones et al., 2011). The integral of the Ca2+ response for both ADP and
ADPβS also displayed a super-additive response following costimulation of P2X1:P2Y1 receptors but
to a lesser extent than the peak response (Fig. 4Aiii and 4Biii), as observed for platelets. Previous
work has suggested that many Ca2+ dependent functions in platelets require a threshold level of
cytosplasmic Ca2+ to be achieved, thus it is likely that the peak response is more physiologically
experiments that explore the mechanism of the synergy.
The P2X1 receptor is a non-selective cation channel and agonist binding results in channel
opening and membrane depolarisation that results from Na+ and Ca2+ influx (Mahaut-Smith et al.,
2011; Surprenant and North, 2009). It has previously been shown that P2Y1 receptor-mediated Ca2+
mobilisation is sensitive to membrane depolarization (Martinez-Pinna et al., 2005) and that sodium
influx contributes ~90% to the membrane depolarisation upon P2X1 receptor activation (Benham,
1989). To test whether sodium influx and membrane depolarisation are important for the P2X1:P2Y1
synergy we replaced the Na+ in the extracellular solution with NMDG+. NMDG+ is >30 fold less
permeant than Na+ at P2X1 receptors, resulting in a reversal potential of -84 mV for P2X1 receptors
in NMDG+, Na+-free saline and thus hyperpolarization of the membrane potential when these
channels open (Evans et al., 1996). NMDG+ substitution had no effect on the synergy between P2X1
and P2Y1 receptors (Fig. 5A,C) suggesting that neither membrane depolarisation nor Na+ influx
underlie the synergy. To test whether calcium influx through the P2X1 receptor was responsible, we
replaced the external Ca2+ with Ba2+, which is permeable through P2X receptors. Ba2+ substitution
prevented the potentiation of ADP responses by α,β-meATP (Fig. 5B,C). Taken together, these
results show that Ca2+ influx through P2X1 receptor cation channels is required to amplify the ADPevoked calcium responses, with little or no role for influx of Na+ or membrane depolarization.
Inhibition of several Ca2+-dependent or P2X or P2Y1 receptor-dependent pathways, including
ERK1/2, Rho kinase and PKC (using U0126, Y27632, and GF109203X, respectively, all at 10 μM),
had no effect on the synergy between P2X1 and P2Y1 receptors in the HEK-293-P2X1 cell line (p >
11
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
relevant than the integral (Rink et al. 1982). We therefore used the peak increase in subsequent
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
0.05 for all three conditions). This reinforces the conclusion that an increase in cytosolic Ca2+ is the
key signal responsible for the synergy.
To determine whether a general global rise in intracellular calcium could also potentiate P2Y1
receptor mediated responses we compared the P2X1-induced amplification with effects of the Ca2+
ionophore ionomycin. For these experiments an ionomycin concentration was selected (0.1 μM) that
produced a similar peak calcium increase to that observed with 0.05 μM α,β-meATP (8.1 ± 1.1% and
10.2 ± 1.7% of the maximal ADP response, respectively). Co-stimulation with 0.1 µM ionomycin and
that predicted from the sum of the individual responses (see sample traces in Fig. 5D). This
potentiation by ionomycin was not significantly different from that observed by P2X1 receptors (p <
0.05). Together, these experiments suggest that amplification of P2Y1 receptor-mediated Ca2+
mobilisation by P2X1 receptors requires an increase in intracellular Ca2+, which is likely to occur
through a global increase in intracellular calcium concentration rather than via a microdomain specific
to the ionotropic receptor.
Pre-stimulation of P2X1 receptors enhances P2Y1 responses ─ Following activation, P2X1
receptors rapidly desensitise, however the Ca2+ increase they generate extends beyond the period of
channel opening due to the time taken for homeostatic mechanisms to restore the cytoplasmic Ca2+
concentration to resting levels. Therefore, we also investigated whether synergy also occurred if
P2Y1 receptors were stimulated at various times after α,β-meATP in HEK293-P2X1 cells.
Application of ADP (0.01 µM) 15, 30 and 60 seconds after α,β-meATP (0.05μM) resulted in calcium
responses significantly greater than the predicted levels, but of a magnitude that decreased as the
interval was prolonged, consistent with the timecourse of the P2X1-dependent Ca2+ response
(amplifications of 1.77 ± 0.2, 1.84 ± 0.07 % and 1.52 ± 0.14 fold respectively, compared with 2.3 ±
0.18 for co-stimulation; Fig. 6A). In contrast, synergy was absent if ADP was added 5 minutes after
α,β-meATP. As with P2X1-mediated synergy, ionomycin was capable of potentiating the P2Y1
response if ADP was added up to 1 minute later, but this was lost at 5 minutes (Fig. 6B). Therefore,
P2X1-dependent amplification of P2Y1 Ca2+ responses displays a “memory”, allowing synergy
12
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
ADP (0.01 μM) resulted in a response that was significantly greater (1.77 ± 0.33 fold, p < 0.05) than
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
between these two pathways in situations where the GPCR is activated subsequent to the ATP-gated
channel.
P2X1 receptors potentiate other Gq-coupled receptors ─ To determine whether P2X1
receptors have a more general synergistic effect on GPCR-evoked Ca2+ signalling, we examined the
effect of α,β-meATP in combination with activation of other endogenous Gq coupled GPCRs in
HEK293 cells. Type 1 protease activated receptors (PAR1) and M1 muscarinic acetylcholine
receptors (M1AChR) were activated by submaximal concentrations of thrombin (Fig.7A,B) or
P2X1 receptors by 0.05 µM α,β-meATP. Thrombin-stimulated Ca2+ responses were amplified 1.38 ±
0.05 fold, and carbachol 2.27 ± 0.14 fold relative to predicted values. These studies establish the
principle that P2X1 receptors can amplify Ca2+ signals downstream of a range of Gq coupled
receptors.
13
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
carbachol (Fig.7C, D), established from concentration response curves (data not shown), along with
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Discussion
It is well established that ADP-evoked P2Y1 and P2Y12 receptor signals interact to enhance
downstream functional responses in the platelet (Hechler and Gachet, 2011). We now demonstrate
that co-stimulation of ATP-gated P2X1 receptors and ADP-stimulated P2Y receptors also potentiates
platelet aggregation. It is also worth noting that ATP is a partial agonist at P2Y1 and P2Y12 receptors
and unable to generate GPCR-evoked responses in the platelet due to low levels of receptor
expression (reviewed in (Mahaut-Smith et al., 2011)). Furthermore, ADP is not an agonist at P2X1
selectively activated by ATP and ADP, respectively, and the synergy we demonstrate here provides a
means of co-incidence detection of these two nucleotides during haemostasis and thrombosis.
Regarding the underlying mechanism of the synergy, P2X1 receptor-evoked Ca2+ influx clearly
potentiates P2Y1-evoked Ca2+ responses, but additionally ERK2 and TXA2 generation are required
for amplification of aggregation responses. These effects are consistent with the essential involvement
of a cytosolic Ca2+ increase in ADP-evoked aggregation (Garcia et al., 2007; Jin and Kunapuli, 1998;
Varga-Szabo et al., 2009), and also the importance of Ca2+/CalDAG-GEFI-dependent ERK2 in release
of TXA2 that reinforces the P2Y-evoked aggregation response (Garcia et al., 2007; Mustard et al.,
1975; Packham et al., 1989; Stefanini et al., 2009). Although previous work has shown that P2X1
does not directly synergise with P2Y12 to amplify aggregation (Rolf and Mahaut-Smith, 2002), Ca2+dependent ERK2-activation and thus TXA2 release depend upon both P2Y1 and P2Y12 receptors
(Garcia et al., 2007). Therefore P2X1 receptors can be considered to indirectly enhance P2Y12dependent aggregation and thrombosis via an action on P2Y1 receptor-evoked Ca2+ responses.
Our studies in HEK293 cells show that the P2X1-dependent potentiation of P2Y1 receptor
Ca2+ increases is not restricted to platelets, and also occurs between P2X1 and other Gq-coupled
receptors. Therefore, this interaction may have relevance in a range of cell types given both the
widespread expression of P2X1 and the common use of ATP as an extracellular signalling molecule
(Burnstock and Knight, 2004; Surprenant and North, 2009). Ca2+ influx through P2X1 receptors is
necessary and sufficient to explain the enhancement of P2Y1 receptor-dependent Ca2+ signals as the
synergy was abolished in Ca2+-free salines or following treatment with NF449 at a concentration that
14
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
receptors (Mahaut-Smith et al., 2000). Therefore, in the platelet, P2X1 and P2Y receptors are
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
is selective for P2X1 (Fung et al., 2007). Although activation of P2Y1 and other Gq-coupled receptors
leads to enhanced P2X1 receptor responses in cell lines, most likely via phosphorylation of an
accessory protein (Vial et al., 2004), this effect requires a delay of at least 30 seconds and therefore
was not responsible for the P2X1:P2Y1 synergy observed in the present work. We have previously
shown that P2Y1 receptors are directly enhanced by depolarisation, and cation influx through P2X1
receptors will exert a depolarising influence, however replacement of the major permeating ion under
physiological conditions (Na+) with impermeant NMDG+ had not effect on the amplification of P2Y1
exchange (Harper et al., 2013; Sage et al., 1991). Increases in intracellular Ca2+ delivered by
ionomycin mimic the synergistic effect of P2X1 on P2Y1 receptors, further supporting the conclusion
that the P2X1-induced increase in cytosolic Ca2+ is responsible for the synergy. Inhibition of a
number of other pathways (ERK2, Rho kinase or PKC) had no effect. One likely mechanism for the
synergy is enhanced IP3 receptor activation since these intracellular Ca2+ release channels are known
to be potentiated by cytosolic Ca2+ in the range of 10nM to 1μM (Bezprozvanny et al., 1991; Foskett
et al., 2007). Indeed, evidence for amplification of IP3-dependent Ca2+ release following P2X receptor
stimulation has been provided in renal arterial smooth muscle (Povstyan et al. 2011), although
depolarisation leading to activation of voltage-gated Ca2+ channels is responsible for a substantial
component of the P2X-evoked Ca2+ influx thus caution should be taken in directly comparing with
our study on non-excitable cells. Another mechanism by which P2X1 could enhance P2Y1 receptor
Ca2+ responses is via potentiation of phospholipase-C (PLC) since the activity of this enzyme has
been reported to be Ca2+-dependent, including in platelets (Eberhard and Holz, 1988; Watson et al.,
1995). However, using a 3[H]IPX assay we were unable to determine whether P2Y1-evoked PLC
responses were amplified due to the very low levels of IP3 generated in HEK293 cells at agonist
concentrations matching those within the Ca2+ studies1. We have previously observed an increased
activation of a Gq-activated cation channel by P2X1 during whole-cell recordings in the
megakaryocyte (Vial et al., 2002). The underlying channel is most likely TRPC6 (Carter et al., 2006;
Hassock et al., 2002), which could be enhanced by the P2X1-induced increase in [Ca2+]i (Shi et al.,
2004), by stimulation of diacylglycerol (DAG) production (Hassock et al., 2002; Hofmann et al.,
15
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
responses. This experiment also rules out a role for an increase in cytosolic Na+ or reverse Na+/Ca2+
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
1999; Ramanathan et al., 2012) or by a decrease in membrane PIP2 levels (Tolhurst et al., 2005).
Several TRPC channels are endogenously expressed in HEK293 cells, including TRPC3, TRPC6 and
TRPC7 that have been suggested to be DAG-activated (Wu et al., 2000; Zagranichnaya et al., 2005)
and may also contribute to the synergy in this cell line. Greater release of IP3-dependent Ca2+ stores
could also lead to increased Ca2+ entry through Orai1 store-operated Ca2+ channels (Varga-Szabo et al.,
2009). It was interesting to note that the peak of the Ca2+ response was enhanced more than the integral,
which may be explained by an acceleration of one or more of these Ca2+-dependent events by the P2X1-
function in platelets and cells lines (Vial & Evans, 2005; Vial et al. 2006), raising the possibility that a
close association with Gq-coupled receptors in these microdomains may contribute to the synergy that we
describe. However, the comparable amplification of P2Y1 receptor Ca2+ responses by ionomycin and
P2X1 receptors argues against any role for microdomains (Fig. 6). Furthermore, in our previous work we
found no role for lipid rafts in P2Y1 function (Vial et al. 2006). Fig. 8 summarises the mechanism(s)
whereby P2X1 receptors can interact to enhance P2Y1 and P2Y12-dependent signalling leading to
increased aggregation. Consistent with a crucial role for cytosolic Ca2+ increases in P2X1-dependent
amplification of P2Y1 responses, this effect decreased over a timecourse that mirrored the α,βmeATP-stimulated Ca2+ response (Fig. 3D, 6B) and a similar time course was observed for
ionomycin-evoked enhancement of the P2Y1 response (Fig. 6C). This time-dependence of P2Y1
amplification has significance in vivo because ATP released at the site of injury is converted to ADP
by ectonucleotidases present on endothelial cells and leukocytes, and also soluble in plasma, thereby
providing sequential delivery of ATP followed by ADP. Furthermore, when ATP is released from
nerve endings at tissues that co-express P2X1 and P2Y1 receptors, ectonucleotidases will generate
ADP subsequent to P2X1 stimulation (Burnstock and Knight, 2004; Robson et al., 2006).
In conclusion this study demonstrates for the first time that synergy occurs between platelet
P2X1 receptors and P2Y1 receptors at the level of functional responses. The underlying mechanism
involves enhanced Ca2+ responses due to P2X1-dependent Ca2+ influx that amplifies aggregation
through ERK2 and release of TXA2. This represents a form of co-incidence detection of ATP and
ADP released at sites of vascular injury and may contribute to the reported ability of P2X1 receptors
16
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
induced Ca2+ entry. We have previously demonstrated an essential role for lipid raft location for P2X1
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
to amplify thrombosis in small arteries and arterioles (Mulryan et al., 2000). Experiments presented
here also provide evidence that P2X1 receptors are able to amplify Ca2+ mobilisation evoked via
several Gq-coupled receptors which may lead to amplification of responses at neuronal or
neuromuscular junctions when ATP is released as a co-transmitter.
Acknowledgements:
We thank Prof John Challiss and Raj Mistry for conducting IPX assays.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
Authorship contributions:
Participated in research design: Mahaut-Smith, Evans, Jones
Conducted experiments: Jones
Performed data analysis: Jones
Wrote or contributed to the writing of the manuscript: Mahaut-Smith, Evans, Jones
17
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
References
Benham CD (1989) ATP-activated channels gate calcium entry in single smooth muscle cells
dissociated from rabbit ear artery. The Journal of physiology 419:689-701.
Bezprozvanny I, Watras J and Ehrlich BE (1991) Bell-shaped calcium-response curves of
Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature
351(6329):751-754.
Burnstock G (2004) Cotransmission. Current opinion in pharmacology 4(1):47-52.
different systems. International review of cytology 240:31-304.
Carter RN, Tolhurst G, Walmsley G, Vizuete-Forster M, Miller N and Mahaut-Smith MP (2006)
Molecular and electrophysiological characterization of transient receptor potential ion
channels in the primary murine megakaryocyte. The Journal of physiology 576(Pt 1):151-162.
Cosemans JM, Munnix IC, Wetzker R, Heller R, Jackson SP and Heemskerk JW (2006) Continuous
signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in
dynamic thrombus stabilization. Blood 108(9):3045-3052.
Eberhard DA and Holz RW (1988) Intracellular Ca2+ activates phospholipase C. Trends in
neurosciences 11(12):517-520.
Evans RJ, Lewis C, Virginio C, Lundstrom K, Buell G, Surprenant A and North RA (1996) Ionic
permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X
receptors) expressed in mammalian cells. The Journal of physiology 497 ( Pt 2):413-422.
Fabre JE, Nguyen M, Latour A, Keifer JA, Audoly LP, Coffman TM and Koller BH (1999)
Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism
in P2Y1-deficient mice. Nature medicine 5(10):1199-1202.
Foskett JK, White C, Cheung KH and Mak DO (2007) Inositol trisphosphate receptor Ca2+ release
channels. Physiological reviews 87(2):593-658.
Foster CJ, Prosser DM, Agans JM, Zhai Y, Smith MD, Lachowicz JE, Zhang FL, Gustafson E,
Monsma FJ, Jr., Wiekowski MT, Abbondanzo SJ, Cook DN, Bayne ML, Lira SA and
Chintala MS (2001) Molecular identification and characterization of the platelet ADP
18
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
Burnstock G and Knight GE (2004) Cellular distribution and functions of P2 receptor subtypes in
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
receptor targeted by thienopyridine antithrombotic drugs. The Journal of clinical investigation
107(12):1591-1598.
Fung CY, Cendana C, Farndale RW and Mahaut-Smith MP (2007) Primary and secondary agonists
can use P2X1 receptors as a major pathway to increase intracellular Ca2+ in the human
platelet. Journal of thrombosis and haemostasis : JTH 5(5):910-917.
Garcia A, Shankar H, Murugappan S, Kim S and Kunapuli SP (2007) Regulation and functional
consequences of ADP receptor-mediated ERK2 activation in platelets. The Biochemical
Gurung I (2005) Voltage control of Ca2+ signalling via G-protein-coupled receptors. University of
Cambridge PhD Thesis.
Harper MT, Londono JE, Quick K, Londono JC, Flockerzi V, Philipp SE, Birnbaumer L, Freichel M
and Poole AW (2013) Transient receptor potential channels function as a coincidence signal
detector mediating phosphatidylserine exposure. Science signaling 6(281):ra50.
Hassock SR, Zhu MX, Trost C, Flockerzi V and Authi KS (2002) Expression and role of TRPC
proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry
channel. Blood 100(8):2801-2811.
Hechler B and Gachet C (2011) P2 receptors and platelet function. Purinergic signalling 7(3):293303.
Hechler B, Lenain N, Marchese P, Vial C, Heim V, Freund M, Cazenave JP, Cattaneo M, Ruggeri
ZM, Evans R and Gachet C (2003) A role of the fast ATP-gated P2X1 cation channel in
thrombosis of small arteries in vivo. The Journal of experimental medicine 198(4):661-667.
Hechler B, Leon C, Vial C, Vigne P, Frelin C, Cazenave JP and Gachet C (1998) The P2Y1 receptor
is necessary for adenosine 5'-diphosphate-induced platelet aggregation. Blood 92(1):152-159.
Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T and Schultz G (1999) Direct
activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397(6716):259263.
19
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
journal 404(2):299-308.
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Jin J, Daniel JL and Kunapuli SP (1998) Molecular basis for ADP-induced platelet activation. II. The
P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in
platelets. The Journal of biological chemistry 273(4):2030-2034.
Jin J and Kunapuli SP (1998) Coactivation of two different G protein-coupled receptors is essential
for ADP-induced platelet aggregation. Proceedings of the National Academy of Sciences of
the United States of America 95(14):8070-8074.
Jones S, Evans RJ and Mahaut-Smith MP (2011) Extracellular Ca2+ modulates ADP-evoked
P2Y receptor activation. British journal of haematology 153(1):83-91.
Kahner BN, Shankar H, Murugappan S, Prasad GL and Kunapuli SP (2006) Nucleotide receptor
signaling in platelets. Journal of thrombosis and haemostasis : JTH 4(11):2317-2326.
Leon C, Hechler B, Freund M, Eckly A, Vial C, Ohlmann P, Dierich A, LeMeur M, Cazenave JP and
Gachet C (1999) Defective platelet aggregation and increased resistance to thrombosis in
purinergic P2Y1 receptor-null mice. The Journal of clinical investigation 104(12):1731-1737.
MacKenzie AB, Mahaut-Smith MP and Sage SO (1996) Activation of receptor-operated cation
channels via P2X1 not P2T purinoceptors in human platelets. The Journal of biological
chemistry 271(6):2879-2881.
Mahaut-Smith MP, Ennion SJ, Rolf MG and Evans RJ (2000) ADP is not an agonist at P2X1
receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets.
British journal of pharmacology 131(1):108-114.
Mahaut-Smith MP, Jones S and Evans RJ (2011) The P2X1 receptor and platelet function. Purinergic
signalling 7(3):341-356.
Mahaut-Smith MP, Martinez-Pinna J and Gurung IS (2008) A role for membrane potential in
regulating GPCRs? Trends in pharmacological sciences 29(8):421-429.
Martinez-Pinna J, Gurung IS, Vial C, Leon C, Gachet C, Evans RJ and Mahaut-Smith MP (2005)
Direct voltage control of signaling via P2Y1 and other Gaq-coupled receptors. The Journal of
biological chemistry 280(2):1490-1498.
20
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
aggregation through altered agonist degradation: implications for conditions used to study
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Martinez-Pinna J, Tolhurst G, Gurung IS, Vandenberg JI and Mahaut-Smith MP (2004) Sensitivity
limits for voltage control of P2Y receptor-evoked Ca2+ mobilization in the rat megakaryocyte.
The Journal of physiology 555(Pt 1):61-70.
Mulryan K, Gitterman DP, Lewis CJ, Vial C, Leckie BJ, Cobb AL, Brown JE, Conley EC, Buell G,
Pritchard CA and Evans RJ (2000) Reduced vas deferens contraction and male infertility in
mice lacking P2X1 receptors. Nature 403(6765):86-89.
Mustard JF, Perry DW, Kinlough-Rathbone RL and Packham MA (1975) Factors responsible for
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
ADP-induced release reaction of human platelets. The American journal of physiology
228(6):1757-1765.
Oury C, Toth-Zsamboki E, Thys C, Tytgat J, Vermylen J and Hoylaerts MF (2001) The ATP-gated
P2X1 ion channel acts as a positive regulator of platelet responses to collagen. Thrombosis
and haemostasis 86(5):1264-1271.
Oury C, Toth-Zsamboki E, Vermylen J and Hoylaerts MF (2002) P2X1-mediated activation of
extracellular signal-regulated kinase 2 contributes to platelet secretion and aggregation
induced by collagen. Blood 100(7):2499-2505.
Packham MA, Bryant NL, Guccione MA, Kinlough-Rathbone RL and Mustard JF (1989) Effect of
the concentration of Ca2+ in the suspending medium on the responses of human and rabbit
platelets to aggregating agents. Thrombosis and haemostasis 62(3):968-976.
Povstyan OV, Harhun MI and Gordienko DV (2011) Ca2+ entry following P2X receptor activation
induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries. British
journal of pharmacology 162(7):1618-1638.
Ramanathan G, Gupta S, Thielmann I, Pleines I, Varga-Szabo D, May F, Mannhalter C, Dietrich A,
Nieswandt B and Braun A (2012) Defective diacylglycerol-induced Ca2+ entry but normal
agonist-induced activation responses in TRPC6-deficient mouse platelets. Journal of
thrombosis and haemostasis : JTH 10(3):419-429.
Rink TJ, Smith SW and Tsien RY (1982) Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds
and Ca-independent activation for shape-change and secretion. FEBS letters 148(1):21-26.
21
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Robson SC, Sevigny J and Zimmermann H (2006) The E-NTPDase family of ectonucleotidases:
Structure function relationships and pathophysiological significance. Purinergic signalling
2(2):409-430.
Rolf MG, Brearley CA and Mahaut-Smith MP (2001) Platelet shape change evoked by selective
activation of P2X1 purinoceptors with a,b-methylene ATP. Thrombosis and haemostasis
85(2):303-308.
Rolf MG and Mahaut-Smith MP (2002) Effects of enhanced P2X1 receptor Ca2+ influx on functional
Sage SO, Rink TJ and Mahaut-Smith MP (1991) Resting and ADP-evoked changes in cytosolic free
sodium concentration in human platelets loaded with the indicator SBFI. The Journal of
physiology 441:559-573.
Shi J, Mori E, Mori Y, Mori M, Li J, Ito Y and Inoue R (2004) Multiple regulation by calcium of
murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in
HEK293 cells. The Journal of physiology 561(Pt 2):415-432.
Stefanini L, Roden RC and Bergmeier W (2009) CalDAG-GEFI is at the nexus of calcium-dependent
platelet activation. Blood 114(12):2506-2514.
Surprenant A and North RA (2009) Signaling at purinergic P2X receptors. Annual review of
physiology 71:333-359.
Tolhurst G, Vial C, Leon C, Gachet C, Evans RJ and Mahaut-Smith MP (2005) Interplay between
P2Y1, P2Y12, and P2X1 receptors in the activation of megakaryocyte cation influx currents by
ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet
P2 receptor signaling. Blood 106(5):1644-1651.
Varga-Szabo D, Braun A and Nieswandt B (2009) Calcium signaling in platelets. Journal of
thrombosis and haemostasis : JTH 7(7):1057-1066.
Vial C and Evans RJ (2005) Disruption of lipid rafts inhibits P2X1 receptor-mediated currents and
arterial vasoconstriction. The Journal of biological chemistry 280(35):30705-30711.
22
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
responses in human platelets. Thrombosis and haemostasis 88(3):495-502.
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Vial C, Fung CY, Goodall AH, Mahaut-Smith MP and Evans RJ (2006) Differential sensitivity of
human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts. Biochemical and
biophysical research communications 343(2):415-419.
Vial C, Rolf MG, Mahaut-Smith MP and Evans RJ (2002) A study of P2X1 receptor function in
murine megakaryocytes and human platelets reveals synergy with P2Y receptors. British
journal of pharmacology 135(2):363-372.
Vial C, Tobin AB and Evans RJ (2004) G-protein-coupled receptor regulation of P2X1 receptors does
Watson SP, Poole A and Asselin J (1995) Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA) and the tyrphostin ST271 inhibit phospholipase C in human platelets
by preventing Ca2+ entry. Molecular pharmacology 47(4):823-830.
Wu X, Babnigg G and Villereal ML (2000) Functional significance of human trp1 and trp3 in storeoperated Ca2+ entry in HEK-293 cells. American journal of physiology Cell physiology
278(3):C526-536.
Zagranichnaya TK, Wu X and Villereal ML (2005) Endogenous TRPC1, TRPC3, and TRPC7
proteins combine to form native store-operated channels in HEK-293 cells. The Journal of
biological chemistry 280(33):29559-29569.
23
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
not involve direct channel phosphorylation. The Biochemical journal 382(Pt 1):101-110.
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
Footnotes:
This study was funded by the British Heart Foundation [PG/05/014 & PG/06/017].
Address for reprints: Martyn Mahaut-Smith, Department of Cell Physiology and Pharmacology,
University of Leicester, University Road, PO Box 138, Leicester LE1 9HN. Telephone: +44 116 229
7135; Fax: +44 116 252 5045; email: mpms1@le.ac.uk
Sarah Jones : current address, School of Healthcare Science, Manchester Metropolitan University,
Chester Street, Manchester, M1 5GD.
R. Mistry, S. Jones and R.A.J. Challiss, unpublished observations
Legends for Figures
Figure 1. Stimulation of platelet P2X1 receptors potentiates ADP-mediated calcium mobilisation and
aggregation. [Ca2+]i increases (Ai-iii) or aggregation responses (Bi-iii) of human platelets following
stimulation with ADP (1 μM), α,β-meATP (5 μM) or both agonists combined (“actual”). The
“predicted” [Ca2+]i response, either the peak (Aii) or integral (Aiii), is derived by mathematical
addition of the responses to the individual agonists. Note that the “predicted” peak [Ca2+]i increase is
less than that derived from simple summation of the individual increases due to differences in the
timecourse of the P2X1 and P2Y1-evoked peak responses. Aggregation responses (peak, ii or integral,
iii) were normalised to the paired ADP-evoked response for each donor. α,β-meATP alone did not
induce a detectable aggregation response, only a transient shape change (deflection below the zero
aggregation baseline).
Figure 2. Role for ERK and TXA2 production in the P2X1-mediated potentiation of P2Y-induced
aggregation but not Ca2+ mobilisation. Human platelets were stimulated with ADP (1μM), α,βmeATP (5μM) or both agonists combined in the presence of U0126 (10μM), aspirin (100μM), or a
vehicle control. A. Predicted and actual intracellular calcium increases for combined addition of ADP
and α,β-meATP under control conditions compared to the presence of either U0126 or aspirin.
Responses were normalised to the predicted control response. B. Aggregation responses to ADP or
24
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
1
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
combined addition of α,β-meATP and ADP under control conditions or in the presence of either
U0126 or aspirin. Responses were normalised to the ADP-evoked aggregation in each donor.
Figure 3. Calcium responses mediated by endogenous P2Y1 receptors and stably expressed P2X1
receptors in HEK 293 cells. Intracellular Ca2+ responses recorded in fluo-3-loaded native HEK293
cells and HEK293-P2X1 cells following stimulation by a range of concentrations of ADP (0.001-10
μM) (A,C) or α,β-meATP (0.001-10 μM) (D). B. Inhibition of the Ca2+ response to 1μM ADP evoked
by a the P2Y1 antagonist MRS 2179 (0.001-10μM). The percentage response in B and C was
maximal concentration of ADP (100 μM ADP) in the same batch of cells.
Figure 4. Synergy between P2X1 and P2Y1 receptor-evoked calcium mobilisation in HEK293-P2X1
cells. Intracellular Ca2+ responses were recorded in fluo-3-loaded HEK293-P2X1 cells following
selective or combined stimulation of P2X1 and P2Y1 receptors. P2Y1 receptors were stimulated with
either 0.01 μM ADP (Ai-iii) or 0.1 μM ADPβS (Bi-iii)), and P2X1 receptors were stimulated with
0.05 μM α,β-meATP. Ca2+ responses were assessed from the peak (Aii and Bii) or integral (Aiii and
Biii) of the increase in F/F0 fluorescence ratio following addition of agonist and normalised to the
response evoked by a maximal concentration of ADP (100 μM ADP) in the same batch of cells. The
experimental response to combined stimulation of P2X1 and P2Y1 is denoted “actual”, whereas the
“predicted” response is that obtained by mathematical addition of the individual P2X1 and P2Y1
receptor responses.
Figure 5. P2X1 receptors amplify P2Y1-evoked calcium responses via an increase in intracellular
Ca2+ rather than Na+ influx or membrane depolarisation. A,B. HEK293-P2X1 cells were stimulated
with ADP (0.01 μM) and α,β-meATP (0.05 μM), individually or simultaneously and [Ca2+]i responses
measured either in nominally Ca2+-free saline containing Ba2+ (A) or Na+-free (NMDG+) saline (B).
(C) shows the predicted and measured mean [Ca2+]i responses following simultaneous addition of
ADP (0.01 μM) and (0.05 μM) α,β-meATP for (from left to right) HEK293-P2X1 in normal saline,
non-transfected HEK293 cells in normal saline, and HEK293-P2X1 cells exposed to either 1 μM
25
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
calculated from the peak increase in fluo-3 F/F0 ratio and expressed relative to the response to a
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
MOL #92528
NF449 in normal saline, nominally Ca2+ free saline, 1mM Ba2+ (0 Ca2+) saline or Na+-free (NMDG+)
saline. D. Ca2+ responses to 0.1 μM ionomycin and ADP (0.01 μM), individually or in combination.
Figure 6. Time-dependence of the amplification of P2Y1 receptors by P2X1 or ionomycin. HEK293P2X1 cells were stimulated with either 0.05 μM α,β-meATP (A) or 0.1 μM ionomycin (B) and 0.01
μM ADP was added simultaneously or after a delay of between 15 and 300 sec. Predicted and actual
Ca2+ responses are shown at the different intervals, normalised to the maximum peak ADP-evoked
Figure 7. P2X1 stimulation potentiates calcium responses evoked by other Gαq coupled receptors.
HEK293-P2X1 cells were stimulated with α,β -meATP (0.05 μM), thrombin (0.5 U/ml), carbachol (1
μM) and α,β-meATP combined with either thrombin or carbachol. Sample traces are shown in (i) and
the average peak increases shown in (ii) for individual agonists and co-addition of α,β-meATP with
either thrombin (A) or carbachol (B) (“actual”). The “predicted” responses are derived by
mathematical addition of the individual responses to α,β-meATP and either thrombin (A) or carbachol
(B). The peak responses have been normalised to the increase evoked by a maximum concentration
of thrombin (5 U/ml) or carbachol (100 µM)
Figure 8. Proposed mechanism whereby P2X1 enhances P2Y responses in platelets. Ca2+ influx
through P2X1 can potentiate P2Y1-mediated Ca2+ responses by increasing IP3 receptor activation, and
possibly also by increasing PLC activity resulting in increased IP3 production. The elevated cytosolic
Ca2+ increase then leads to an ERK1/2- and TXA2- dependent potentiation of the aggregation
response, possibly by interaction of P2Y1 and P2Y12 receptor pathways
26
Downloaded from molpharm.aspetjournals.org at ASPET Journals on October 1, 2016
response.
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 1.
ii
20
10
0
-10
0
50
100
Time (s)
**
200
100
0
300
*
200
100
0
Actual
Predicted
ADP
0
,β-meATP
Ca2+ Integral (nM)
iii
300
4000
ADP+ α,β-meATP
30
8000
ADP
% Aggregation
ADP
,β-meATP
ADP + α,β-meATP
12000
Aggregation integral (% control)
ii
Bi
Peak Aggregation (%control)
Time (s)
*
16000
Actual
0
100
Predicted
400
,β-meATP
50
800
ADP+ α,β-meATP
0
*
ADP
 Ca2+ (nM)
Actual
1200
ADP
ADP
,β-meATP
Predicted
1000
800
600
400
200
0
iii
Peak Ca2+ (nM)
Ai
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 2.
Predicted
200
*
Actual
**
*
150
100
50
Aspirin
U0126
0
Control
Peak  Ca2+ (% of control)
A
250
*
200
ADP
ADP +
α,β-meATP
150
100
50
Aspirin
U0126
0
Control
Peak aggregation (% of control)
B
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 3
Native HEK293
3.5
F/F0
3
2.5
2
ADP (μM)
10
1
0.1
0.05
0.01
0.001
B
Native HEK293
% Response
A
1.5
1
0.5
Native HEK293
HEK293-P2X1
D
HEK293-P2X1
2.5
F/F0
% Response
C
MRS2179 [µM]
20s
2
1.5
,β-meATP (μM)
10
1
0.1
0.05
0.01
0.001
1
0.5
ADP [µM]
20s
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 4.
Actual
Predicted
ADP
0
,β-meATP
20
**
40
30
20
0
Actual
10
Predicted
20s
40
,β-meATP
0.8
20
0
*
ADPS
1
40
Integral (% maximal [ADP])
1.2
***
60
60
iii
Actual
1.4
ADPβS
,β-meATP
Predicted
Actual
Actual
0
ADPS
1.6
F/F0
20
ii
Peak (% maximal [ADP])
Bi
40
Predicted
20s
60
Predicted
0.8
***
,β-meATP
1.2
80
,β-meATP
F/F0
1.6
ADP
,β-meATP
Predicted
Actual
ADP
2
iii
Integral (% maximal [ADP])
ii
Peak (% maximal [ADP])
Ai
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 5.
0Na+ (NMDG+)
ADP
,β-meATP
Predicted
Actual
1.4
ADP
,β-meATP
Predicted
Actual
1.2
0.8
20s
C
60
***
Predicted
Actual
***
20s
D
F/F0
40
1.6
ADP
Ionomycin
1.4
Predicted
Actual
1.2
20
NF449
Ca2+ free
Native HEK293
0
Ba2+
1
HE293-P2X1
% Peak ADP response
1.6
1
Na+ free
F/F0
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0Ca2+ (Ba2+)
B
F/F0
A
HE293-P2X1
0.8
20s
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 6.
% Peak ADP response
A
***
60
Predicted
Actual
** **
40
*
20
0
0 15 30 60 300
Interval between α,β-meATP
and ADP addition (s)
% Peak ADP response
B
60
*
40
*
**
Predicted
Actual
**
20
0
0 15 30 60 300
Interval between ionomycin
and ADP (s)
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 7.
ii
0.8
20s
ii
1
0.8
20s
80
60
40
20
0
Actual
1.2
**
100
Predicted
1.4
Carbachol
,β-meATP
Predicted
Actual
,β-meATP
1.6
F/F0
0
Carbachol
Bi
10
Actual
1
20
Predicted
1.2
30
,β-meATP
1.4
% Peak carbachol response
F/F0
1.6
**
40
Thrombin
1.8
Thrombin
,β-meATP
Predicted
Actual
% Peak thrombin response
Ai
Molecular Pharmacology Fast Forward. Published on June 12, 2014 as DOI: 10.1124/mol.114.092528
This article has not been copyedited and formatted. The final version may differ from this version.
Figure 8.
ATP
ADP
P2Y12
P2Y1
P2X1
PLC
Na+
Ca2+
+?
DAG
Gq
Gi
PIP2
IP3
+
↑ Ca2+
Ca2+
↓cAMP
2+
Ca2+
Ca
IP3R
Ca2+
Ca2+
Ca2+
PI3K
Rap1b
ERK1/2
TXA2
↑ Aggregation
AKT