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An ATP-Activated Channel Is Involved in Mitogenic Stimulation of Human
T Lymphocytes
By Olavio R. Baricordi, Davide Ferrari, Loredana Melchiorri, Paola Chiozzi, Stefania Hanau, Elisabetta Chiari,
Michele Rubini, and Francesco Di Virgilio
We investigated the effect of pharmacologic modulation of
the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs)
and purified T lymphocytes. Extracellular ATP(ATP.) triggered in these cells an increase in the cytoplasmic CaZ+concentration ([Ca2+li) and plasma membrane depolarization.
Whereas both CBz+release from intracellular stores and influx across the plasma membrane weredetected in the
whole PBL population, only Ca2+influx was observed in T
cells. In the presence of near physiologic extracellular Na+
concentrations (125 mmol/L), Caz+permeability through the
ATP.-gated channel was very low, suggesting a higher selectivity formonovalent over divalent cations. The selective Pz2
agonist benzoylbenzoic ATP (BzATP) increased [Ca2+liin the
presence but not theabsence of extracellular Ca2+and also
caused plasma membrane depolarization. The covalent
blocker oxidized ATP (OATPI, an inhibitor of Paxand P= receptors, prevented Ca2+influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores.
Stimulation with ATP. alone had no significant effects on
PBL 'H-thymidine incorporation. On the contrary, ATP. or
BzATP had a synergistic effect on DNA synthesis stimulated
by selective T-cell mitogens such as phytohemagglutinin,
anti-CD3 monoclonal antibody, or allogeneic PBLs (mixed
lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation bythese receptor-directed agents but not
by the combined application of the Ca2+ ionophore ionomycin and phorbol myristateacetate. Interleukin-2 partially
relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel
gated by ATP,. that is involved in mitogenic stimulation.
0 1996 by The American Society of Hematology.
E
rinergic receptors in lymphoid cells, very little isknown
about their possible physiologic role. It has been suggested
that they may be involved in the modulation of mitogenic
responses'.'' and cytotoxic rea~tions,6"~.~~
but the cellular
mechanism and the in vivo significance of these actions are
still dubious.
A relevant obstacle to understanding the role ofATP,
receptors is the lack of specific agonists or antagonists. In
recent years, we have characterized a compound, periodateoxidized ATP (oATP), also known as dialdehyde ATP, that
showed a good selectivity for the ATPe-gated channellpore
of mouse macrophage and human lymphoblastoid
This reagent turned out to be very interesting because it
(1) inhibited plasma membrane ion fluxes or permeability
changes without decreasing Ca2' mobilization from intracellular stores, ( 2 ) abolished all cytotoxic effects due to ATP,
stimulation of macrophage or lymphoblastoid cells, and (3)
was devoided of any toxic effects even after prolonged (2
to 3 days) in vitro culture. Furthermore, because oATP forms
a Schiff base or a dihydromorpholino derivative with unprotonated lysins in the vicinity of the ATP, binding site,16 it
allows covalent (irreversible) modification of the ATP,-gated
channel.
In this report, we show that human T lymphocytes from
PBLs express an ATPe-gatedplasma membrane channel and
investigate the effect of pharmacologic modulation of this
channel on T-cell-specific mitogenic Stimulation.
Our results suggest that the ATP receptor expressed by
human T lymphocytes maybe involved inthe control of
mitogenic stimulation by different stimuli.
XTRACELLULAR ATP (ATP,)
can
modulate responses of diverse lymphocyte cell populations via
specific cell membrane purinergic receptors."6 At least two
functionally distinct receptor subtypes have been described
in lymphocytes: (1) a G-protein-coupled receptor linked to
inositol 1, 4 ,5-trisphosphate generation and Ca2' mobilization from intracellular stores, also known as Piy; and (2) an
ion channel permeable to Na+ and Ca'' directly gated by
ATP, without involvement of known intracellular second
messengers, tentatively identified as a P2X/P2r
r e ~ e p t o r .The
~.~
Pzy receptor has been described in human B lymphocytes
and has been reported to be absent from T cells.'.* The nucleotide-activated ion channel has been shown to be expressed
to a low level in normal human B lymphocyte^^,^ and to be
upregulated in resting mouse T and B lymphocytes, leukemic
peripheral blood lymphocytes, and B-lymphoblastoid cells
isolated from individuals affected by Duchenne muscular
dystrophy!-6.10." Whether receptors for ATP, are also expressedby normal human T lymphocyte cells is an open
question.
Despite compelling evidence for the presence of pu-
From the Institutes of Medical Genetics and General Pathology,
the Department of Biochemistry and Molecular Biology, andthe
Biotechnology Center, University of Ferrara, Ferrara, Italy.
Submitted January 17, 1995; accepted August 24, 1995.
Supported in part by grants from the National Research Council
of Italy (Target Projects BTBS and ACRO, Special Project Biology
and Pathology of Calcium), the Ministry of Scient$c Research
(MURST 40% and 60%), the Italian Association for Cancer Research (AIRC), the VII AIDS Project, and Telethon of Italy.
Address reprint requests to Olavio R. Baricordi, PhD, Institute
of Medical Genetics, University of Ferrara, Via Borsari, 46, 1-44100
Ferrara, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8702-0038$3.00/0
682
MATERIALSANDMETHODS
Cells and solutions. PBLs were isolated by Ficoll gradient from
samples withdrawn from healthy volunteers. Plastic nonadherent
cells were passed through a nylon-wool column to isolate purified
T cells. CD4' and CD8' cells were separated by positive selection
with the Dynabead-Detachabead technique (Dynal AS., Oslo, Norway). In brief, PBLs were resuspended in RPM1 medium at a concentration of 107/mLand incubated with mouse antihuman CD4 or CD8
monoclonal antibody (MoAb)-coated magnetic beads at a ratio of
Blood, Vol 87, No 2 (January 15), 1996: pp 682-690
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ATP-ACTIVATED CHANNELS IN
T LYMPHOCYTES
5:l (beads:lymphocytes). The bead:cell suspension was gently rotated for 60 minutes at 4°C. Rosetted beads were selected on a
magnet, carefully washed 5 times to remove unbound cells, and
resuspended in RPMI medium at a concentration of lo7 beadd100
pL. Selected cells were released from the beads by coincubation
with goat-antimouse Ig detachabead solution at a concentration of
1 U/107 beads. The suspension was gently rotated for 45 minutes at
room temperature, the beads were isolated with a magnet, and the
cells were pelleted. Viability was greater than 90%. The purity of
the T cells obtained was assessed by indirect immunofluorescence
by using a FACS Vantage cytofluorimeter (Becton Dickinson,EGTA
San
Jose, CA) and MoAb anti-CD3 (kindly provided by Dr Fabio Malavasi, University of Torino, Torino, Italy) or anti-CD4 and CD8
MoAbs (Dakopatts, Copenhagen, Denmark) and was found to be in
all cases greater than 95%. Jurkat cells were cultured in RPMI 1640
medium supplemented with10% heat-inactivated fetal calf serum
(FCS) and 2 mmol/L glutamine, hereafter abbreviated as RPMIlOF.
['HIThymidine incorporation was performed in RPMIlOF medium.
Fluorimetric measurements were performed in saline or sucrose solution. Saline solution hadthe following composition: 125 mmol/L
NaCI, 5 m m o K KCI, 1 mmol/L MgSO,, 1 mmol/L Na2HP04, 5.5
mmol/L glucose, 5 mmol/L NaHC03, 1 mmol/LCaCI,,and
20
mmol/L HEPES (pH 7.4). Sucrose solution contained 300 mmol/L
sucrose, 1 m o V L MgS04, 1 mmom K,HPO,, 5 mmoVL KHC03,
1 mmoVL CaCI,, 5.5 mmol/L glucose, and 20 mmol/L HEPES and
the pH was adjusted to 7.4 with Tris-HCI.
Measurement of plasma membrane potential and [Ca"],.
Changes in plasma membrane potential were measured withthe
fluorescent dye bis 1, 3-diethylthiobarbiturate trimethineoxonal (bisoxonol; Molecular Probes, Inc, Eugene, OR) at the wavelength pair
540/580 nm, as previously described." Changes in [Ca"], were
measured withthe fluorescent indicator fura-2/AM, as described
previously.'8 Briefly, cells were loaded with 2 pmom fura-2/AM
and incubated in a thermostat-controlled (37°C)and magnetically
stirred fluorometer cuvette (Perkin-Elmer LS50; Perkin-Elmer Ltd,
Beaconsfield, Buckinghamshire, UK). Sulfynpirazone (250 pmoY
L; Sigma-Aldrich Srl, Milano, Italy) was present throughout the
experiment to prevent fura-2 leakage and/or sequestration." [Ca2+Ii
levels were determined with the 340/380 excitation ratio, at an emission wavelength of 500 nm. When indicated, cells were also pretreated with apyrase (Sigma) at the concentration of 0.2 U/mL.
Tyrosine phosphorylation. PBLs (1 X 106/mL) were incubated
in 10% FCS Iscove's modified Dulbecco's medium in the presence
and absence of 200 pmol/L oATP. After 2 hours of incubation, they
were stimulated with the anti-CD3 MoAb CBT3 (1 pg/mL) and 5
minutes later rinsed three times with cold Hank's medium and lysed
1
in a buffer containing 150 mmol/L NaCI,1.5mmoVLMgCI,,
mmoVL EGTA, 10% glycerol, 1% Triton X-100, 100 mmol/L NaF,
0.2 mmoVL Na3V04,10 mmol/L phenilmethylsulphonyl fluoride
(PMSF), 0.1 mg/mL aprotinin, 50 mmol/L HEPES, pH 7.5. The
lysates were placed in ice for 4 minutes, collected, and centrifuged
(2 minutes at 13,000 rpm in Beckman microfuge; Beckman Instruments, Inc, Fullerton, CA) to sediment the nuclear fraction. The
supernatants containing the lysates (400 pg) were then diluted with
HNTG (150 mmoVL NaCI,10% glycerol, 1% Triton X-100, 100
mmol/L NaF, 0.2 mmol/L Na3VO4, 10 mmol/L PMSF, 0.1 mg/mL
aprotinin, and 20 mmol/L HEPES, pH 7.5) supplemented with 100
pL anti-CD3 MoAb CBT3 and 20 pL of agarose-conjugated proteinA (Oncogene Science, Uniondale, NY) in a total volume of 1.5 mL.
Ab/Ag complexes were allowed to form for 4 hours at 4°C and were
then precipitated by centrifugation in the cold for 4 minutes at 13,OOO
rpm (Beckman microfuge). Immunoprecipitated complexes were
washed three times in HNTG and resuspended in 20 pL of Laemli
buffer (20% glycerol, 3% sodium dodecylsulfate, 3% P-mercaptoethanol, 10 mmol/L EDTA, and 0.05% bromophenol blue). Samples
683
200 r
ATP
'
150
r;-
2
min
AT P
'
EGTA
100
t
t
AT P
UT P
Fig 1. ATP. increases[Ca2'liin human PBLs. PBLs were loaded
with 2 pmol/L fura-UAM as describedin the Materials and Methods
and incubated in the fluorimeter cuvette at a concentration of 5 x
105/mL. Trace a, saline solution; trace b. sucrose solution; traces c
and d. Caz+-freesaline solution. The ATP. and UTP. concentration
was 1 mmol/L; the EGTA concentrationwas 0.5 mmol/L. Intracellular
fura-2 concentrationwas 56 pmol/106 cells.
were boiled for 4 minutes and run on a 4% to 20% gradient polyacrylamide gel (BioRad, Hercules, CA). Proteins were electroblotted
onto a nitrocellulose filter. Phosphorylated proteins were detected
by western immunoblot analysis by standard technique with an antiphosphotyrosine antibody (PY20) conjugated with horseradish peroxidase (Transduction Lab, Lexington, KY) and visualization by
enhanced chemiluminescence (ECL) detection system (Amersham,
Little Chalfont, Buckinghamshire, UK).
Thymidine incorporation. For ['Hlthymidine incorporation,
PBLs were resuspended in RPMIlOF medium at a concentration of
106/mL;100 pL was plated in Falcon 96-well 3072 plates and either
challenged with the different stimulants (phytohemagglutinin [PHA],
anti-CD3, or allogeneic cells), or left untreated (controls). After 72
(when the challenge was PHA or anti-CD3) or 120 hours (when the
challenge was allogeneic cells), [3H]thymidine (1 pCi/well) was
added to the culture medium andthe incubation was performed
for an additional 16 to 24 hours. Allogeneic cells were previously
irradiated at 2,500 Rads in a volume of 100 pL. After mitogenic
stimulation, cells were harvested and radioactivity was determined
by liquid scintillation counting.
RESULTS
Characterization of ATP,-dependent ion changes in human PBLs. Few data are currently available onATP,-dependent [Ca2+Iiresponses of PBLs and human T lymphocytes. In fact, it has been suggested that human lymphocytes
from healthy individuals lack Ca2+mobilization in response
to this nucle~tide.~.~
A recent study of ours in B-lymphoblastoid cells obtained
from Duchenne patients showed that extracellular Ca2+and
Na+ compete for entry through the ATP,-gated receptor/
channel"; thus, under physiologic extracellular Na+ and
Ca" concentrations, Caz+ influx is obliterated by the simultaneous Na' influx. Figure l shows that ATP, stimulation
of fura-2-loaded PBLs suspended in standard saline (trace
a) triggered a small but clearly detectable increase in [Ca2+li,
whereas a much larger increase was observed in Na'-free,
sucrose-supplemented medium (trace b). Although the sus-
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684
'
I
BARlCORDl ET AL
200
100.
0
L_
EGTA
150.
50
-
a
"
P
t
ATP
t
Ca'+
2
t
AT P
Fig 2. ATP. increases [Ca2'li in human T lymphocytes. T lymphocytes were separated on nylon wool as described in the Materiels
and Methods, loaded with 4 pmol/L fura-ZIAM in the presence of
250 pmolIL sulfinpyrazone and incubated in the fluorimeter cuvette
at a concentrationof 5 x 106/mL. In trace a, lymphocyteswere incubated in Ca*+-frw saline solution; in trace b. lymphocyteswere incubated in Na+-free sucrose solution. ATP., EGTA, and CB'+ additions
were 1, 0.5, and 2 mmol/L, respectively.
tained phase of the [Caz+liincrease was fully dependent on
influx across the plasma membrane, as it was obliterated by
chelation of extracellular Ca", the fast initial [Ca"], spike
likely reflected release of Ca2+from intracellular stores (trace
c). Besides ATP,,UTP, also released a small but clearly
detectable amount of Ca2+from intracellular stores (trace d).
Ca2+ release from intracellular stores was likely due to
activation of B lymphocytes or residual contaminating
monocytes, because these cells were previously shown to
mobilize Ca2+from intracellular stores in response to extracellular nucleotide^'*^; therefore, we investigated the response to ATP, in a purified T-cell population. As shown in
Fig 2, trace a, ATP, caused no mobilization of intracellular
Ca2+in T cells incubated in the absence of this extracellular
cation and in the presence of EGTA. Nonetheless, this nucleotide activated a plasma membrane channel, because re-addition of Ca" triggered a rapid increase in [Ca2+Ii.A similar
increase was triggered by stimulation of T cells in Ca2+containing medium (trace b). Preliminary experiments in purified resting CD4+ and CD8+ lymphocytes suggests that an
ATP-gated plasma membrane channel is present in both cell
populations (O.R. Baricordi and F. Di Virgilio, manuscript
in preparation).
A [Ca2+Iiincrease, larger in Na+-free medium, was also
caused in PBLs by the ATP analog BzATP, an agonist specific for purinergic receptors of the P2JPZxsubtype" (Fig 3,
traces a and c). Pretreatment with oATP blocked the [Ca2+l,
increase (trace b), thus suggesting involvement of a P2JPZx
purinergic receptor subtype. As shown in trace c, the kinetics
of the [Ca2+Iiincrease triggered by BzATP was slower than
that caused by ATP, (see Fig 1, trace a), suggesting that the
fast initial discharge of Ca2+ from intracellular stores was
lacking in response to stimulation with this ATP analog. To
confirm this suggestion, PBLs were stimulated with BzATP
in the absence of extracellular Ca2+ and in the presence of
EGTA. Under these conditons, BzATP triggered no [Ca"],
increase, whereas UTP, was still active (trace d). These results show that BzATP and oATP allow selective pharmacologic manipulation of the ATP,-gated channel (P2xE'2zreceptor) expressed by T lymphocytes while leaving unaltered
responses mediated by the nucleotide receptor of B lymphocytes. To further strengthen this finding, we investigated the
ability of BzATP to trigger a [Ca2+Iiincrease in oATP-
inhibited PBLs. As shown in trace e, incubation in the presence of oATP prevented the BzATP- butnottheUTP,dependent [Ca"Ii increase.
As previously reported in mouse lymphocytes and human
Duchenne B lymphocytes,'*"ATP,
caused PBLplasma
membrane depolarization that was potentiated by chelation
of extracellular Ca2+ (Fig 4, upper panel, traces a and b).
Like the Ca2+response, membrane depolarization was also
triggered by BzATP (trace c) and blocked by preincubation
with oATP (trace d). Quite interestingly, UTP, was ineffective (trace c), suggesting that depolarization was mainly, if
not entirely, due to activation of the ATP,-gated channel of
T lymphocytes. To confirm this suggestion, T lymphocytes
were examined. Trace e shows that ATP, caused a nearmaximal depolarization in T lymphocytes. Depolarization
was also triggered by BzATP but not by UTP (trace f, and
was inhibited by pretreatment with oATP (trace g). ATP,
dose-dependency for activation of the ATP,-gated channel,
measured as plasma membrane depolarization, was close to
that reported for mouse thymocytes, showing a threshold
and an ECSoof 100 and 400 pmol/L, respectively (Fig 4,
lower panel).2' Besides plasma membrane depolarization, we
did not observe a non selective increase in plasma membrane
permeability due to ATP, (data not shown).
Effects of ATP, on T lymphocyte proliferation. ATP, has
been shown to modulate diverse lymphocyte functional responses, such as DNA synthesis, blastogenesis, and cellmediated killing, but a clear-cut physiologic role (if any) has
never been identified. In our hands, ATP,, at concentrations
ranging from 100 to 300 pmoYL, was unable to stimulate
PBL proliferation (data not shown). However, this nucleotide
showed a strong costimulatory effect when added together
with PHA or anti-CD3 at suboptimal concentrations (Table
1). The costimulatory effect was stronger at 100 pmol/L than
300 pmol/L ATP, both in the case of PHA- and anti-CD3driven DNA synthesis. In agreement with the [Ca"], in-
c
t
BZATP
BzATP
2
' min '
5
EGTA
Fig 3. A [Caz'li increase is also triggered by BzATP and inhibited
by oATP. Experimental conditions are as in Fig 1. Traces a and b.
sucrose solution; trace c and e, saline solution; trace d,Caz'-free
saline solution. In traces b and e, lymphocytes were preincubated
for 2 hours in the presence of 300 pmol/L oATP before stimulation
with BzATP. The &ATP concentration was 100 pmol/L. UTP was at
1 mmol/L, and EGTA was at 0.5 mmol/L.
r
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ATP-ACTIVATED CHANNELS IN T LYMPHOCYTES
z
0
5
N
t
ATP
z
0
<
N
2
-.f
t
ArP
OD1
a1
1
10
Fig 4. PBLsand Tlymphocytesaredepolarizedby
ATP..PBLs
(upper panel) and T lymphocytes (middle panel) were incubated in
the fluorimeter cuvetteat a concentration of 5 x lO*/mL in the presence of 200 nmol/L biaoxonol. Traces b. c, and d. standard saline;
traces a, e, f, and g, Ca*+-free,0.5 mmol/L EGTA-supplemented saline.
In traces d and g, cells were incubated for 2 hours in the presence
of 300 pmol/L oATP before being stimulated. ATP., UTP., and BzATP
were at 1.1, and 0.1 mmol/L, reswively. KC1was usedto depolarize
the plasma membrane and allow intarnal calibration
of ATP.-dependent potentialchanges. EachKC1 addition w w 30 mmol/L. The ATP.
dossdependenca shown in lower panel was performed
in Ca*+-free,
0.5 mmol/L EGTA-supplemented saline solution.In lower panel, depolarization is expressed as the percentage of maximal depolarization induced by3 mmol/L ATP..
creasing activity showed in Fig 1, BzATP also turned out
to be a costimulatory factor at lower concentrations than
ATP,. We also observed that synergistic effect developed
rapidly because the increase in 3H-thymidine incorporation
did not significantly change whether BzATP was added together with anti-CD3 at time 0 or during the last 48 or 24
hours of incubation in the presence of the mitogen. In further
support of a role for the ATPe-gated channel in mitogenic
stimulation, LJTP, a nucleotide that does not activate this
channel, showed no costimulatory effect and, in fact, was
slightly inhibitory.
685
These experiments suggested that a P2 receptor, probably
of the P2f12, subtype, could play a role in mitogen-induced
proliferation of human T lymphocytes. However, although
100 pmoVL was an optimal ATP, concentration as a comitogenic stimulus, it caused only a barely detectable activation
of the ATP,-gated channel, even in the absence of Ca2+as
a complexing cation (see dose-dependency curve in Fig 4).
One possible reason for this discrepancy could be that, during the in vitro incubation, ATP, receptors are desensitized
by the continuous leakage of endogenously released ATP."
To test this hypothesis, we treated purified T lymphocytes
with the ATP-hydrolysing enzyme apyrase before the challenge with ATP,. Figure 5A shows that 100 pmol/L ATP,
triggered in apyrase-treated T cells incubated in Ca" and
Mg2+-containingsaline solution a clear and sustained plasma
membrane depolarization that was further stimulated by a
subsequent 300 pmoVL ATPe addition. AnATP, dose-dependency curve performed under these conditions shows that
the ECS0was not significantly shifted with respect that determined in Ca2+-freemedium (Figs 4 and 5), but the threshold
for receptor activation was lowered to approximately 30
p m o m ATP,.
To further test the involvement of P2f12, receptors in
mitogenic stimulation, we examined the effect of the specific
receptor blocker oATP. Figure 6 shows that oATP inhibited
in a dose-dependent fashion T-lymphocyte stimulation by
three different stimuli in PBL cultures, ie, PHA, anti-CD3,
and allogeneic cells. Stimulation by anti-CD3 was extremely
sensitive to inhibition because, at a concentration of 25
pmol/L oATP, an 80%reduction of 'H-thymidine incorporation was observed. On the contrary, PHA stimulation was
inhibited by only about 40% at the same oATP concentration. Even less sensitive to inhibition was stimulation by
allogeneic cells, as DNA synthesis was decreased by only
5%. However, near full inhibition of DNA synthesis irrespectively of the stimulant used was achieved at 300 pmoV
L oATP, an inhibitor concentration that we have previously
shown to fully block the ATP receptor of mouse15 and human2' macrophages and human B-lymphoblastoid cells."
We think that the different sensitivity to oATP inhibition
observed with the three different stimuli might depend on
two reasons. On the one hand, CD3 is known to be a weaker
stimulus than PHA, thus presumably more susceptible to
inhibitory agents. On the other hand, in the experiments
in which allogeneic cells were a stimulus, a double cell
concentration was present in the sample ( lo6 stimulator plus
lo6 responder cells), thus increasing the rate of degradation
of the oxidized n~cleotide.~'
oATP also inhibited DNA synthesis when added after the
mitogenic stimulus, albeit witha lesser efficiency (Table
2). The addition of oATP 24 or 72 hours after mitogenic
stimulation reduced 'H-thymidine incorporation by about
80% and 50%, respectively. Quite interestingly, sensitivity
to stimulation by PHA, but not anti-CD3, was partially restored by supplementation of the lymphocyte growth factor
interleukin-2 (L-2) to the culture medium.
It cannot be excluded that oATP, besides blocking the
ATP,-gated channel, also caused a nonspecific inhibition of
early events associated with TCR activation. To rule out this
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686
BARlCORDl ET AL
Table 1. Synergistic Effect of Extracellular ATP and PHA or Anti-CD3 on PBL Proliferation
ATP
45,489
PHA
Anti-CD3
Control
100 NrnoliL
t 2,022
41,304 t 3,266
75,478 t 504 ( P i ,001)
55,966 -t 2,111 ( P i,011
300 urnoliL
61,913 t 762 ( P i ,051
41,064 t 2,392 (NS)
UTP
Control
PHA
Anti-CD3
100 prnoliL
44,471 t 1,854
10,324 t 602
300 prnol/L
36,568 t 1,968
7,867 2 650
39,260 5 1,297
9,508 If- 690
BzATP (IO prnol/L)
Control
Anti-CD3
13,458 t 2,908
22,124
0
t 2,256 ( P i25,882
.5)
24 h
t 3,711
48h
( Pi.02)
23,935 t 4,546 ( P < ,051
PHA and anti-CD3 were added ata concentration of 0.2 wg/mL. In the experiments withATP, or UTP, the nucleotide was added together with
PHA or anti-CD3, whereas in those withBzATP, this analog was either added together with anti-CD3 (time
0)or 24 or 48 hours after. Data (cpm)
are the means 2 SD of triplicate determinations from one experiment representative of three others that gave very similar results. Statistical
significance (Student's t-test) is expressed towards controls in the absence of nucleotide.
Abbreviation: NS, not significant.
possibility, we investigated the pattern of tyrosine phosphorylation and [Ca2'li changes in T lymphocytes challenged
with anti-CD3 MoAbs in the absence and presence of oATP.
Figure 7A shows that treatment with oATP did not alter
the pattern of protein tyrosine phosphorylation triggered by
activation of the TCWCD3 complex; in fact, the oxidized
nucleotide, if anything, slightly increased phosphate incorporation. Figure 7B shows that [Ca2'], changes due to TCW
CD3 activation were also unmodified by oATP treament.
Note that the Ca2+ experiments were performed in Jurkat
cells, because it is known that anti-CD3 MoAbs are a weak
stimulus for [Ca"], mobilization in peripheral T lymphocyte~.~~
Because oATP is a substrate for plasma membrane ectoATPases, it is likely that oxidized adenosine is generated
during incubation of cell cultures in the presence of oATP.
To rule out a possible inhibitory effect of oxidized adenosine,
control experiments were performed in the presence of this
compound (300 pnol/L) without observing any decrease in
thymidine incorporation (data not shown). As a further proof
that inhibition of DNA synthesis by oATP was not due to
its degradation products, we tested the effect of oxidized
UTP (oUTP), a compound that is also known to block plasma
membrane ATP,-gatedchanneLZ5 oUTP turnedoutto
be
almost as good an inhibitor as oATP (O.R. Baricordi and F.
Di Virgilio, unpublished observation).
Di-aldehyde functions of oATP react with unprotonated
lysines in the vicinity of ATP binding sites, thus forming a
covalent Schiff base or a dihydromorpholino derivative."
Therefore, the ATP receptor should be permanently inactivated by oATP and functional ATP receptors should only
be restored by insertion into the plasma membrane of novel
receptors. Were this hypothesis true, oATP-treated PBLs
should remain refractory to mitogenic stimulation once
washed free of the inhibitor. Table 3 shows that this is in
fact the case, as a short pretreatment of 2 hours is sufficient
to inhibit by 50% and 60% DNA synthesis stimulated by
PHA and anti-CD3, respectively. Furthermore, it can be anticipated that, provided PBLs are allowed enough recovery
time in culture, block by oATP should be completely reversible. Table 3 shows that this anticipation is fulfilled because,
72 hours after oATP labeling and wash-out, PBLs recover
near complete sensitivity to mitogenic stimulation. Rather
surprisingly, oATP-pretreated PBLs showed a slightly larger
DNA synthesis with respect to control cells, especially when
the stimulant was anti-CD3. oATP-treated lymphocytes also
recovered the Ca2+response (data not shown).
PBL DNA synthesis can be stimulated by agents that bypass plasma membrane receptors, such as phorbol esters and
the Ca2+ionophore ionomycin. Figure 8 shows that 2 hours
of incubation in the presence of oATP did not block 3Hthymidine incorporation stimulated by the combined addition
of these stimuli, although an inhibitory effect was present
when PMA orionomycin were added alone. We think that it
is of particular interest that oATP also blocked 3H-thymidine
incorporation due to PMA alone (the effect on ionomycindependent stimulation is of difficult interpretation because
this ionophore alone was a veryweak stimulus), because
this might suggest that, during in vitro culture, endogenously
released ATP might play a stimulatory or costimulatory role.
Lack of sensitivity to oATP inhibition of PMA plus ionomycin, stimuli that bypass surface receptors, is a further
demonstration that the ATP receptor modulates the mitogenic signal at a site proximal to the plasma membrane.
DISCUSSION
ATP, has been variably implicated in the modulation of
different lymphocyte responses such as gene expre~sion,~
proliferation,l'12 and c y t o t o ~ i c i t y ~ , ' through
~ " ~ , ~ ~interaction
with specific plasma membrane receptors. Most of these
studies were performed in mouse lymphocytes, butmore
recently human B and T lymphocytes have also been investi-
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ATP-ACTIVATEDCHANNELS IN T LYMPHOCYTES
687
gated.4*7'10"l
It is thought that many of the effects of ATP,
are mediated through purinergic receptors of the PZYsubtype,
coupled to a G protein-regulated phospholipase C and to
Ca2+release from intracellular stores.' However, freshly isolated T lymphocytes do not show any detectable Ca2+mobilization from intracellular stores in response to ATP,, suggesting the lack of PZy(or PZU)type of purinergic receptor.
On the contrary, ATP, causes Ca2+influx from the extracellular medium and plasma membrane depolarization, sug-
ALLOGENEIC
CELLS
50
.
0-
A
1
50
2
' min
'
0
I
k
a
300
Fig 6.oATP inhibits 'H-thymidine incorporation in mitogenically
stimulated PBLs. PBLs were incubated in 96-well plates as described
in theMaterials and Methods. At time 0, the three dflerent stimuli
were added at the following concentrations: PHA, 1 pg/mL; antiCD3.1 pglml; allogeneic cells,lO%nL. Furthermore, increasing concentrations of oATP were also added at the same time. After 72 to
120 hours,1pCilwell of 'H-thymidine was addedand the incubation
was performedfor eddWional16to 18 hours. *H-thymidine incorporation of control cells in the absence of the inhibitor l100%1was 23,775
f 1,806,14,008
2 198, and 9,334 f 234 when PHA,anti-CD3, and
used as stimulus. Dataara means
allogeneic cells, reapectively, were
? SD of quadruplicate determinationsfrom a single experiment representative of three others. (AI anti-CD3; (0)PHA; IO) allogeneic
cells.
N
-.
U
a
A
0
a
W
n
A
gesting the presence of a nucleotide-activated plasma membrane ion channel.
The ATP,-activated channel of human T lymphocytes
shares many features in common with the channel previously
described in B lymphocytes isolated from patients affected
by B-cell chronic lymphocytx leukemia or lymphoblastoid
cell lines established from Duchenne muscular dystrophy
patients. The channel is permeable to both Na+ and Ca*',
although permeability to Ca2+is low in Na+-containing (125
mmol/L) media; thus, ATP,, under physiologic conditions,
triggers membrane depolarization and Ca2+influx in the absence of permeabilization to low (<900 Daltons) molecular
weight hydrophylic solutes.
100pM
100
-
c
a
N
K
a
-l
2
200
100
[oATP] p M
z
z
0
25
50-
W
0
-l
a
-
I
X
a
P
bp
Table 2. Time Course of oATP-Dependent Inhibition
of Mitogenic Stimulation
O-
~~
No Stimulus
0.01
B
0.1
l
~ T P J
mM
Fig 5. Effect of apyrase treatment on ATP.-dependentplasma
membrane depolarization.PurifiedT lymphocyteswere preincubated
in standard d i n e (37°C) for 1 hour at a concentration of lO'/mL in
the presence of 0.2 U/mL of apyrase. After this incubation time, cells
were centrifuged and suspended at the concentration of 5 x 10' in
standard saline (in the absence of apyrase) containing 200 nmol/L
bisoxonol. Each KCL addition in (AI was 30 mmol/L. Depolarization
in IBI is expressed asthe percentage of maximaltriggered by 3 mmoll
L ATP. All experiments were performed in the presence of 1 mmol/
L Ca2+.
No oATP
oATP time 0
oATP 24 h
oATP 48 h
oATP 72 h
oATP + IL-2
198 t 16
48 t 3
84 t 4
82 t 4
116 t 9
181 ? 2
~~
_____
~~
PHA
31,582
499
5,248
5,935
13,819
5,906
2 1,406
t 31
2 466
t 21 1
t 328
t 214
Anti-CD3
19,073
355
1,399
2,354
9,846
857
t 326
2 14
t 27
t 76
2 176
t 63
PBLs were incubated as described in Fig 6. PHA and anti-CD3 MoAb
were 1 pg/mL. oATP (300pnol/L) was either added at time 0 or 24,48,
or 72 hours after the addition of the stimuli
and kept in the incubation
medium throughout the experiment. IL-2 (20 U/mL), when present,
was added at time 0. Cells were harvested after 96 hours. Data (cpm)
are the means -c SD of quadruplicate determinations from a single
experiment representative of three others.
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BARICORDIET AL
688
A
B
a b c
250,
- 200
- 97
- 68
."
I
f
Fig 7. Lack of effect of oATP on TCR-dependent
intracellular early events. (A) Lymphocytes were incubated at37°C in RPMllOF medium and stimulated
with 1 pg/mL of anti-CD3 MoAbs. After 5 minutes,
cells were lysed and processed as described in the
Materials and Methods. (B) Jurkat cells were incubated in standard saline at a concentration of 5 x
105/mL and stimulated with 1 pg/mL of anti-CD3
MoAbs. Trace a, controls; trace b. cells treated for 2
hours with 300 pmol/L oATP before stimulation.
- 43
The ATP, threshold for cation influx is about 100 pmoV
L, similar to that reported for tumoral B lymphocytes and
B-lymphoblastoid Duchenne cells, and about 2 log units
higher than that reported for the nucleotide-gated channel
described in rat PC12 cell^.^*"^*^ An additional feature in
common with lymphoblastoid cells is the lack of significant
inactivation during a prolonged exposure to ATP,. Furthermore, BzATP is more potent than ATP, and oATP is a very
good inhibitor. These characteristics suggest that the ATP,
channel of human T cells belongs to the Pz subtype, although lack of perrneabilization would rather be consistent
with a P2x subtype. However, we feel that, until purinergic
receptors expressed in different tissues will be cloned and
fully biochemically characterized, attribution to a given subtype only onthe basis of functional and pharmacologic criteria can be misleading. In fact, it cannot be excluded that the
same receptor subtype (ie, a P= receptor) may show different
features in different tissues.
The role of purinergic receptors in lymphocytes has remainedmostly elusive. LowATP, concentrations are reported to stimulate DNA synthesis in mouse lymphocytes'.I2
and early gene expression in B-humanlymphocytes:but
there is no direct indication for a role of purinergic receptors
in the mitogenic response of human lymphocyte cells. Our
data suggest thatATP, receptors maybeinvolved
in the
mitogenic response to different stimulants such as PHA, antiCD3, and allogeneic cells. In fact, oATP, a covalent and
[IONOMYCIN] ,M
0
0.5
8
1.0
1
100 c
z
-l-
0
a
a
2
50-
a
0
Table 3. Reversibility of oATP-Induced Inhibition of Proliferation
No Stimulus
Time 0
no oATP
+ oATP
48 h
no oATP
+ oATP
72 h
no oATP
+ oATP
PHA
Anti-CD3
V
2
bp
566 t 36
283 -c 12
32,195 2 714
16,584 f 326
16,732 2 361
6,201 2 511
671 2 14
1,106 2 189
21,122 2 291
17,077 2 614
18,200 -c 167
23,060 t 671
1,282 2 78
947 2 36
36,861 f 1,415
38,791 2 915
26,742 2 1,211
28,856 2 2,004
Experimental conditions are as in Fig 6. PBLs were incubated in the
presence of 300 pmol/L oATP for 2 hours, rinsed, and then either
immediately (time 0)stimulated with PHA or anti-CD3 or allowed to
recover for 48 or 72 hours before the addition of the mitogenic stimulus. Control cells were incubated in the absence of oATP for 0,48, or
72 hours before being stimulated with PHA or anti-CD3. Data (cpm)
are the means f SD of quadruplicate determinations from a single
experiment representative of three others.
Oh
l
I
I
0
25
50
[PMA] nM
Fig 8. Lack of effect of oATP on DNA synthesys ,dimulated by the
combined application of PMA and ionomycin. Experimental conditions are as in Fig 6. PBLs were stimulated with ionomycin (A, A),
PMA (0.0).
or both stimuli (U, m) in the absence (open symbols) or
presence (solid symbols) of
300 pmol/L oATP. 3H-thymidine incorporation of PBLs stimulated with PMA and ionomycin in the absence
of the inhibitor(100%)was 18,946 2 698 cpm. Data are means f SD
of quadruplicate determinations froma single experiment representative of three others.
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689
ATP-ACTIVATED CHANNELS IN T LYMPHOCYTES
specific inhibitor of p H z x receptors, powerfully inhibited
DNA synthesis stimulated by these various agonists. Inhibition of DNA synthesis could be overcome by mitogens that
bypass plasma membrane receptors, such as the Ca” ionophore ionomycin plus PMA, showing that oATP blocked a
pathway associated to plasma membrane receptors and did
not nonspecifically inhibit cell responses. Furthermore, the
block could also be partially overcome by supplementing
PBL cultures with L-2.
oATP is a covalent reagent that irreversibly binds to and
inactivates ATP, receptors. However, inhibition of ATP, receptor-linked responses would be expected not to be irreversible because newly synthesized receptors are expressed on
the plasma membrane. This i s in fact the case, as shown by
the observation that T cells became again susceptible to
mitogenic stimulation 2 to 3 days after oATP wash-out. Full
reversibility of mitogenic block, together with lack of biochemical and morphologic signs of cell damage, clearly
shows that oATP had no untoward effects on T-cell viability.
It can be argued that O A P , being a dialdheyde reagent,
might form Schiff bases with unprotonated lysin residues
present on other membrane proteins unrelated to ATP, receptors. However, we do not think that this is the case under
our experimental conditions. Previous studies in human
monocyte/macrophage cells have shown that oATP does not
alter chemiotactic response and membrane adhesion molecule expression in these cells.23Furthermore, oATP did not
block [Ca2+Iimobilization triggered by anti-CD3 MoAbs or
the asociated tyrosine phosphorylation. Finally, the inhibitory effect of oATP is prevented by coincubation in the
presence of excess ATP, (F. Di Virgilio, unpublished observation), a clear indication that it is due to specific interaction
with an ATP, binding site.
Requirement for a functional ATP, receptor during mitogenic stimulation suggests that this receptor might beinvolved at some postreceptor step crucial for lymphocyte activation. Mitogenic or comitogenic effects of ATPewere
described in several cell model^^'^^^ as well as lymphocytes.”
Although these effects are commonly believed to be mediated by Pzytype receptors, a mitogenic role for ATP,-gated
plasma membrane channels has been recently suggested in
cultured vascular smooth muscle cell^.^' The mechanism
whereby the ATP, channel exerts its comitogenic effect deserves further investigation. This nucleotide triggers both
Na+ and Ca2+ fluxes, thus it cannot be excluded that the
increase in the intracellular Na+ concentration might be an
important permissive factor for volume changes that are well
known to occur in mitogenically stimulated lymphocyte^.^'
These observations stress the very complex role of purinergic receptors in the regulation of lymphocyte responses.
We and others have shown that prolonged exposures to ATPe
are cytotoxic to mouse T and B lymphocytes, Duchenne
human B-lymphoblastoid cells, and mouse and human macrop~ages~6.1~,14.Z6.33.~
Very recently, the potential cytotoxic
role of ATP, receptors has been reinforced by the molecular
cloning of two P2x subtypes that showed striking homologies
with membrane receptors known to be involved in apoptotic
cell death.35*36
This contrasting dual role of ATP, receptors is not surpris-
ing because it is conceivable that a transient activation of
the channel by low ATP, concentrations preferentially leads
to cell activation, whereas a sustained stimulation by high
concentrations leads to a drastic perturbation of ion homeostasis and cell death. Thus, the ATP, receptor of T lymphocytes shares interesting similarities with receptors for excitatory amino acids in central neurons. In the central nervous
system, receptors for excitatory amino acids mediate Ca”
and Na+ fluxes in the absence of generation of Ca2+-releasing
intracellular messengers and are involved in cell death (excitotoxi~ity).~’
It is tempting to speculate that ATPe has in the
immune system a role similar to that of excitatory aminoacids in the central nervous system, ie, a mediator of cellto-cell communication under normal physiologic conditions
and an effector of cell death under pathologic conditions.
One last unsolved issue is the origin of ATP, responsible
for activation of Pulpzx receptors. A number of cell types
have been shown to release ATP upon stimulation (see. Dubyak and El-Moatassim’ for a recent review), most notably
blood platelets that are known to store millimolar ATP concentrations within their dense gran~les.~’
However, it should
not be overlooked that, besides controlled release via exocytotic secretion, another important source ofATP, is from
damaged or dead cells that are commonly encountered at
sites of inflammatory or immune reactions.
REFERENCES
1. Ikehara S , Pahwa RP, Lunzer DG, Good RA, Modak MJ:
Adenosine 5’-triphosphate (ATP)-mediated stimulation and suppression of DNA synthesis in lymphoid cells. J Immunol 127:1834, 1981
2. Schmidt A, Ortaldo JR, Herberman RH: Inhibition of human
natural killer cell ractivity by exogenous adenosine 5’- triphosphate.
J Immunol 132:146, 1984
3. Lin J, Krishnaraj R, Kemp RG: Exogenous ATP enhances
calcium influx in intact thymocytes. J Immunol 135:3403, 1985
4. Wiley J, Dubyak GR:Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes. Blood 73:1316, 1989
5. El-Moatassim C, Bernard N, Mani J-C, Domand J: Extracellular ATP induces a nonspecific permeability of thymocyte plasma
membranes. Biochem Cell Biol 67:495, 1989
6. Di Virgilio F, Bronte V, Collavo D, Zanovello P: Responses
of mouse lymphocytes to extracellular adenosine 5’- triphosphate
(ATP). Lymphocytes with cytotoxic activity are resistant to the permeabilizing effects of ATP. J Immunol 143:1955, 1989
7. Padeh S , Cohen A, Roifman CM: ATP-induced activation of
human B lymphocytes via P,-purinoceptors. J Immunol 146:1626,
1991
8. Dubyak GR,El-Moatassim C: Signal transduction via €2-purinergic receptors for extracellular ATP and other nucleotides. Am
J Physiol 265:C577, 1993
9. Bretschneider F, Klapperstuck M, Lohn M, Markwardt F Nonselective cationic currents elicited by extracellular ATP in human
B-lymphocytes. Eur J Physiol 429:691, 1995
10. Wiley JS, Chen R, Wiley JM, Jamieson JP: The ATP- receptor-operated ion channel of human lymphocytes: Inhibition of ion
fluxes by amiloride analogs and by extracellular sodium ions. Arch
Biochem Biophys 292:411, 1992
11. Ferrari D, Munerati M, Melchiom L, Hanau S , Di Virgilio
F, Baricordi OR: Responses to extracellular ATP of lymphoblastoid
cell lines from Duchenne muscular dystrophy patients. Am J Physiol
267:C886, 1994
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
690
12. Gregory SH, Kern M: Adenosine and adenine nucleotides are
mitogenic for mouse thymocytes. Biochem Biophys Res Commun
83:111I , 1978
13. Di Virgilio F, Pizzo P, Zanovello P, Bronte V, Collavo D:
Extracellular ATP as a possible mediator of cell-mediated cytotoxicity. Immunol Today 11:274, 1990
14. Filippini A, Taffs RE, Agui T, Sitkovsky MV: Ecto-ATPase
activity in cytolytic lymphocytes. Protection from the cytolytic effects of extracellular ATP. J Biol Chem 265:334, 1990
IS. Murgia M, Hanau S, Pizzo P, Rippa M, Di Virgilio F: Oxidized ATP. An irreversible inhibitor of the macrophage purinergic
receptor. J Biol Chem 268:8199, 1993
16. Colman RF: Site-specific modification of enzyme sites, in
Sigman DS, Boyer PD (eds): The Enzymes, v01 19. San Diego, CA,
Academic, 1982, p 283
17. De Togni P, Cabrini G , Di Virgilio F: Cyclic AMP inhibition
of fMet-Leu-Phe-dependent metabolic responses in human neutrophils is not due to its effects on cytosolic Ca2+.Biochem J 224:629,
1984
18. Di Virgilio F, Fasolato C, Steinberg TH: Inhibitors of membrane transport system for organic anions block fura-2 excretion
from K 1 2 and N2A cells. Biochem J 256:959, 1988
19. Di Virgilio F, Steinberg TH, Silverstein SC: Inhibition of
fura-2 sequestration and secretion with organic anion transport
blockers. Cell Calcium 1157, 1990
20. Nuttle LC, El-Moatassim C, Dubyak GR: Expression of the
pore-forming PZzpurinoceptor in Xenopous oocytes injected with
poly(A)+ RNA from murine macrophages. Mol Pharmacol 44:93,
1993
21. Pizzo P, Zanovello P, Bronte V, Di Virgilio F: Extracellular
ATP causes lysis of mouse thymocytes and activates a plasma membrane ion channel. Biochem J 274:139, 1991
22. Grierson J, Meldolesi J: Sheare-stress-induced [Ca”], transients and oscillations in mouse fibroblasts are mediated by endogenously-released ATP. J Biol Chem 270:4451, 1995
23. Falzoni S , Munerati M, Ferrari D, Spisani S, Moretti S, Di
Virgilio F: The purinergic PZzreceptor of human macrophage cells:
Characterization and possible physiological role. J Clin Invest
95:1207, 1995
24. Murgia M,Mion M, Veronese L, Panozzo M, Coppola V,
Rizzuto R, Brini M, Malavasi F, Amadori A, Chieco-Bianchi L,
Pozzan T: Cytosolic free calcium concentration in the mitogenic
stimulation of T lymphocytes by anti-CD3 monoclonal antibodies.
Cell Calcium 16:167, 1994
BARlCORDl ET AL
25. Thomas SA, Zawisa M, Lin X, Hume RI: A receptor that is
highly specific for extracellular ATP in developing chick skeletal
muscle in vitro. Br J Pharmacol 103:1963, 1991
26. Zanovello P, Bronte V, Rosato A, Pizzo P, Di Virgilio F:
Responses of mouse lymphocytes to extracellular ATP. 11. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation.
J Immunol 145:154S, 1990
27. Fasolato C, Pizzo P, Pozzan T: Receptor-mediated calcium
influx in PC12 cells. ATP and bradykinin activate two independent
pathways. J Biol Chem 265:20351, 1990
28. Wang D-J, Huang N-N, Heppel LA: Extracellular ATP and
ADP stimulate proliferation of porcine aortic smooth mucle cells. J
Cell Physiol 153:221, 1992
29. Huang N-N, Wang D-J, Heppel LA: Role of adenosine 3’:s’monophosphate-dependent protein kinase and CAMPlevels in ATPdependent mitogenesis in Swiss 3T3 cells. J BiolChem 269548,
1994
30. Popper LD, Batra S: Calcium mobilization and cell proliferation activated by extracellular ATP in human ovarian tumor cells.
Cell Calcium 14:P209, 1993
31. Erlinge D, Yo0 H, Edvinsson L, Reis DJ, Wahlestedt C:
Mitogenic effects of ATP on vascular smooth muscle cells vs other
growth factors and sympathetic cotransmitters. Am J Physiol
26S:H1089,1993
32. Deutsch C, Slater L, Goldstein P: Volume regulation of human peripheral blood lymphocytes and stimulated proliferation of
volume-adapted cells. Biochim Biophys Acta 721:262, 1982
33. Steinberg TH, Newman AS, Swanson JA, Silverstein SC:
AT€“ permeabilizes the plasma membrane of mouse macrophages
to fluorescent dyes. J Biol Chem 2623884, 1987
34. Blanchard DK, McMillen S, Djeu JY: IFN-gamma enhances
sensitivity of human macrophages to extracellular ATP-mediated
lysis. J Immunol 147:2579, 1991
35. Valera S , Hussy N, Evans RI, Adami N, North A, Surprenant
A, Buell G: A new class of ligand-gated ion channel defined by P,,
receptor for extracellular ATP. Nature 371516, 1994
36. Brake AJ, Wagenbach MJ, Julius D: New structural motif for
ligand-gated ion channels defined byan ionotropic ATP receptor.
Nature 371519, 1994
37. Choi DW: Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623, 1988
38. Colman RW: Aggregin: A platelet ADP receptor that mediates activation. FASEB J 4:1425, 1990
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1996 87: 682-690
An ATP-activated channel is involved in mitogenic stimulation of
human T lymphocytes
OR Baricordi, D Ferrari, L Melchiorri, P Chiozzi, S Hanau, E Chiari, M Rubini and F Di Virgilio
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