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RAPID COMMUNICATION
P2X1Receptor Activation in HL60 Cells
By G. Buell, A.D. Michel, C. Lewis, G. Collo, P.P.A. Humphrey, and A. Surprenant
treated with apyrase, which destroys extracellular ATP, or
Recent cloning of the human P2X1 receptor revealed high
with suramin, a P2X receptor antagonist. This implies that
levels of its messenger RNAin differentiated promyelocytes
HL60 cells release ATP, which chronically desensitizes the
(HL60 cells). We found expression of P2Xl receptor protein
receptor. ATP release was detected by direct measurement,
in HL60 cells by radioligand binding,by immunohistochemusing the luciferin-luciferaseassay. It is concludedthat funcistry, using a receptor specific antibody, and by electrophysiin themembrane of differology. The currents elicited by adenosine triphosphate (ATP)tional P2Xl receptors are present
entiated HL60 cells.
had the expected propertiesof P2X1 receptors(rapid desen0 1996 by The American Societyof Hematology.
sitization, mimicked by cY,p-methylene-ATP). However,
these currents were only observed in cells that were pre-
ies, rinsed with physiological buffered saline solution (PBS), and
DENOSINE TRIPHOSPHATE (ATP) is an extracellular
fixed for 30 minutes in 4% paraformaldehye plus 0.2% picric acid
modulator of immune function. Its actions include the
in 0.1 m o m phosphate buffer. They were then rinsed several times
induction of interleukin- 1 (IL-1) release from lipopolysacwith PBS and stored in PBS plus 1% sodium azide. Cells were
charide-primed macrophage' and the mitogenic stimulation
incubated for 18 to 24 hours in anti-P2XI antisera at 1:500 dilution,
of T lymphocytes.2In macrophage, T, and B cells, ATP can
rinsed, and incubated with secondary antibody (rhodamine or fluoinduce calcium entry across the plasma ~nembrane.~"
ATP
rescein-labeled donkey-antirabbit IgG, Jackson Immuno Research,
causes calcium release from intracellular stores as shown for
Westgrove, PA) for 3 hours at 10 pg/mL.
macrophage and mast cell^^.^; mast cells also respond with
Receptor binding for ATP. HL60 cells were differentiated with
elevated ~ecretion.'.~
Prolonged exposure to ATP can induce
0.1 pmol/L phorbol myristate acetate (PMA) for 72 h o ~ r s .Adher'~
ent cells were obtained by incubation in divalent cation-free PBS
cell death in thymocytes, macrophage, and a variety of cell
and collected by centrifugation. Undifferentiated HL60 cells, grown
lines.'"."
in suspension culture, were also procured by centrifugation. Cells
ATP receptors, involved in these actions, are not fully
were homogenized and a well-washed membrane fraction was precharacterized either pharmacologically or in molecular
pared as described previously.2' Membranes were frozen at -80°C
terms. Release of calcium from intracellular stores is generbefore assay. The binding of 0.2 nmoVL [35S]ATPySto HL60 cell
ally thought to result from activation of P2Y receptors that
membranes was performed as described" in a final assay volume of
belong to the G-protein-coupled family of receptors. The
0.25 mL. Incubations for 3 hours at 4°C were terminated by vacuum
direct activation of G proteins with GTPyS can also evoke
filtration. Nonspecific binding was defined using 10 pmoliL ATPyS.
this ele ease.^ A second class of receptor, P2Z, is believed
In these experiments the ATP levels in the membrane preparations
to underlie many of the actions of extracellular ATP on
were assessed using the luciferin-luciferase technique andmemmacrophage, notably the formation of large membrane
branes were diluted to ensure that the ATP level was less than 0.3
nrnom. Data are the mean of three experiments.
'pores' that allow the passage of molecules as large as 900
Quantitation of ATP with luciferin-luciferase. Undifferentiated
daltons. P2Z receptors are particularly sensitive to 3'-0HL60 cells (1 X lo5cells in 1 mL) were incubated in 12-well culture
benzoylbenzoic ATP (BzATP) and show an enhanced action
of ATP by the removal of extracellular m a g n e ~ i u m . ~ ~ ' ~plates
~ ' ~ in the presence of 0.1 p m o m PMA. After 24-, 48-, or 72hour incubations at 37°C the media were removed, the adherent cells
Recently, a third family of ATP receptors, P2X, has been
washed twice with 3 mL of PBS, and 1 mL of RPMI was added.
shown by molecular cloning to compose a new class of
The cells were incubated for 20 minutes at 22°C before measuring
cation-selective ion channel^.'^"^ P2X channels are highly
ATP levels in 50 pL media using the luciferin-luciferase technique.
permeable to calcium and mediate rapid depolarization of
ATP levels calculated on the basis of ATP standards prepared in
the cell when activated by
The full sequence of the
RPMI without serum. Centrifugation of samples had no significant
first member of this family, rat P2X,, was determined by
effect on the ATP levels detected, consistent with the apparent lack
of cell contamination in the media. Data are from three separate
direct expression cloning from smooth-muscle mRNA.I3
HL60 differentiation experiments with mean values representing 4
However, a partial E X , cDNA had previously been identito 6 wells per point for each experiment.
fied as a marker for dexamethasone-induced thymocyte cell
Electrophysiology. Standard wholecell recordingswereobtained
death in vitro." Northern blot analysis with rat P2X, cDNA
shows the presence of P2XI mRNA in thymus and spleen13
and the equivalent human P2XI mRNA occurs in differentiFrom Glaxo Institute for Molecular Biology, Geneva, Switzerland;
ated HL60 cells." These observations raise the question as
and Glmo Institute for Applied Pharmacology, the Department of
to whether this receptor might underlie some of the ATP
Pharmacology, University of Cambridge, Cambridge, UK.
induced actions, noted above. The present work tests for
Submitted November 29, 1995; accepted January 2, 1996.
P2XI receptors in HL60 cells by a range of experimental
Address reprint requests to G. Buell, PhD, Glmo Institute for
Molecular Biology, I228 Plan-les-Ouates, Geneva, Switzerland.
approaches.
A
MATERIALS AND METHODS
Immunohistochemistry. A rabbit polyclonal antibody, directed
against the C-terminus of P2X1,has been characterized?' Cells were
plated onto coverslips for 24 hours as for electrophysiological studBlood, Vol 87,No 7 (April I), 1996:pp 2659-2664
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.
0006-4971/96/8707-0150$3.00/0
2659
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BUELL ET AL
2660
electrophysiologicill studies o n differentiated HL60 cells we
neverdetectedfunctional
P2X responses;undifferentiated
cells gave only occasional, weak ATP-induced currents (see
Fig 3A). We further investigated these cells by testing for
the presence ofP2Xl receptors. independent of their channelforming capacity.
/ r r l r , ~ r r r , o h i . s t o c / ~ ~ ~ rHL60
~ ~ i . cells
~ t ~ . were examined by
ilntnunotltlorescence with antipeptide antisera. raised to the
corboxy terminal 9 amino acids of P2XI.'" Immunoreactive
P2XI protein was observed in differentiated HL60 cells (Fig
IA) andathighermagnificationappearedassociated
with
the outer membrane (Fig ID). Staining of HL60 cells was
undetectable in the absence of differentiation (Fig 1 B).
...
.
A
0 ATP
ap-mATP
A 2-MeSATP
120
h
g
100
S
0
8
80
v
.-F 60
U
S
yi 40
& l
m .
0.0001
B
1
0.01
100
(FM)
concentration
Agonist
T
v -
Fig 1. PZX,-receptor immunoreactivity in HL60 cells, undifferentiated (B and C) and differentiated with PMA for 3 days (A, D, and E).
Right panels show the same field of view under phase-contrast as
for fluorescence panels on the left.
1.2
2
from cells using 4- t o 7-MW patch pipcttcs and an Axopatch ?(W)
amplitier (Axon Instrumcnts. Foslcr City. CA). Internal patch pipette
solution contained (mlnollL):K-aspartate 130. NaCI 20. ECTA IO. and
HEPEY S: extern;tlsaline comprised (tnmol/L):NaCl 117. MgCI: I .
CaCI? 2. KC1 2, HEPES I O . and glucose 12. Agonists were applied by
External solution W:IS supcrfused
fast-flow U-tubc delivery
over cells at a mtc of 2 t o S InIJmin (bath volutnc o f 0.5 1111~).All
rccordings wen' made a t rwn1 tcmpcraturc. Values in lex1 arc indicarctl
mean stantlard ? error. Cells. twatcd with npyr;lsc or sutmnin Ihr I t o
3 hours bcfnrc recording. were placed in the recording chmlbcr. which
was initiolly supcrfuscd with Stlr~tllitl-Conlilillingsolulion: the sunmin
was washed out and actions of agonists examined.
RESULTS
We have shown that differentiation of HL60 cells causes
a 20-fold induction of P2Xl mRNA."' However. in our initial
normal
24h
72 h
48h
C
5
1 1
0.8
0.6
c
a
0.4
0.2
2
0
IC
0
3h
5h
L
Fig 2. (A) Inhibition by cr,pmeATP,ATP,
and 2-MeSATP of the
specific binding of 0.1 nmol/L [35SlATPySt o membranes from differentiated HL60 cells. Total and nonspecific binding were 1,710 and
250 dpm, respectively, with nonspecific binding defined in the presence of 10 p m o l / L ATP$. Each compound competed for
two populations of specific binding sites with ATP possessing PIC,values of
8.38 t 0.07 and 6.67 t 0.17 for 72% ? 5 % and 28%*t 5% of the sites,
respectively. (B)Quantitation of ATP released or metabolized IC)
from differentiated HL60 cells by the luciferinluciferase technique.
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ATP GATED ION CHANNELS IN GRANULOCYTES
HL60 undifferentiated
A
B
g
2661
HL60
2H3 differentiated
RBL
AT_p(30V)
100
ATP (30PM)
C
HL60
80
100
60
80
40
60
Y
RBL
18
W
0
Q
L
cn
-
S
20
34
40
8
1
0
84
20
0
54
differentiated
undifferentiated
Fig 3. ATP-gated currents recordedfrom HL60 and RBL 2H3 cells. (A) Recordings obtained from undifferentiated HL60, differentiated HL60,
and RBL 2H3cells as indicated after preincubation for
1 hour in apyrase 130 UlmL). Each set shows superimposedrecords obtained in response
t o two applications ofATP at 3-minuteintervals; note profound rundown of the
current. ATP applied by fast-flow delivery system for duration
indicated by bar above traces. (B and C) Summary of effects of preincubation with suramin andlor apyrase on response t o ATP in HL60 (B)
and RBL cells IC).Histogram shows percent cells responding t o ATP with no treatment and after 2 hours of treatment with suramin (100
gmollL), apyrase 130 UlmL), or combination of suramin and apyrase; numbers above each value are numbers of cells tested. All recordings
made within 30 minutes of washout of suramin orapyrase.
Ligond-binding. The second technique by which P2Xl
protein could be demonstrated in HL60 cells was receptor
binding. The increase in immunoreactive P2Xl induced by
HL60 differentiation was paralleled by an increase in radioligand binding activity (Fig 2A). Specific binding of 0.2 nmol/
L [."SIATPyS increased from 353 2 220 dpmlmg protein
in undifferentiated cells to 6810 ? 2735 dpm/mg protein in
cells differentiated for 48 hours ( n = 4). From a limited
study of the receptor density and binding site characteristics
in differentiated HL60 cells, ATP, 2-MeSATP, and cr.0meATP competed for S2% to 75% of the [ "SIATPyS binding sites with affinity estimates of 8.38, 7.52, and 7.21. respectively. The plc,,, estimates for ATP,2-MeSATP.and
cr,PmeATP were similar, although not identical to the respective values of 8.93.8.23. and 7. I7 determined for recombinant P2Xl receptor, expressed in CHO-KI cells."
ATP relecrsc.. Because P2Xl receptors on HL60 cells are
capable of binding ATP, the lack of P2Xl channel function
must bc due to somc form of inactivation. We considered a
potcntialmechanism. agonist-mediated desensitization. resulting from thc release of ATP to thc extracellular medium.
The release o f signilicant amounts of ATP from HL60 cells
was tletectcd usinga luciferin-luciferllseassay (Fig 2B). ATP
release.seenforboth
undifferentiated and differentiated
HL6O cells. was maintained during the 72-hour PMA treatment.Cell viability during this period. assessed by trypan
blue exclusion. was greater than 98%. Rcleased ATP was
stable for at least 2 hours at 22°C. suggesting that HL60
cells do not metabolize extracellular ATP at submicromolar
concentrations. This was confirmed in additional studies (Fig
2C). Exogenous ATP (0.9 ptnol/L) was added to differentiated HL60 cells (48 hours. PMA): the measured concentration of ATP was 0.87 ? 0 . I2 pmol/L immediately after ATP
addition. This levelwasunchanged (0.90 % 0.20 pmol/L)
after a 2-hour incubation at room temperature or a further
2-hour incubation at 27°C (0.84 2 0 . I I pmol/L).
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BUELL ET AL
2662
RBL cells
HL60 cells
A
B
C
D
r
400 r
"
t t
300
v
Q)
U
S
c
.E 200
Fig 4. ATP-gated currents in HL60 and RBL 2H3
cells show properties typical of PPX,-purinoceptors.
Superimposedone
currents recorded from
HL60 cell
(A) or RBL 2H3 cell (B) in response to increasing concentration of n,PmATP as indicated; cr,pmATP applied at intervals of 15 minutes withapyrase present
for 13 minutes after each agonist application. (C and
5
Y
OJ
d
100
0
1
10
100
Agonist
concentration (PM)
0.1
0.1
1
10
Agonist
concentration
Electrophysiology. The signals, detected in the luciferinluciferase assay, were eliminated by preincubation of samples with apyrase. We asked whether pretreatment of HMO
cells with apyrase to destroy extracellular ATP would allow
recovery from desensitization and yield cells withfunctional
P2Xl responses. Suramin, a competitive antagonist at
p 2 x , 13.17.23 was likewise tested for its ability to protect the
receptor from inactivation. Ratbasophilicleukemiacells
(RBL) were also treated with apyrase or suramin. As for
HL60 cells, RBL cells expressed P2Xl mRNA but had not
displayed functional P2X ion channel responses (G.B., unpublished observations, November 1994).
Under control conditions, ATP evoked
a very small amplitude (<40 picoamps [PA]) inward current in about 10% of
undifferentiated HL60 cells examined (Fig 3A and B). No
response to ATP was ever
recorded from PMA-differentiated
HL60 cells or RBL cells under control conditions
(Fig 3B
and C). Whencellswere pretreated for40minuteswith
then
suramin (100 ymolL) or apyrase(30U/mL),ATP
evoked large inward currents invirtually all RBL (n = 9
and 14, respectively) and differentiated HL60cells (n =
8 and 12, respectively) although thesetreatmentshad
no
significant effect on undifferentiated HL60 cells (Fig 3). No
cells responded toATP whenpretreatmentwithsuramin
was shortened to 10 minutes (n = 5). We also notedthat
10 to 20 minutes
preincubation of cells with apyrase for only
100
(PM)
D) Concentration-response curves for ATP, 2meSATP,
differentiated
a,pmATP
and
in
HL60 cells
(C) and RBL cells (D). Each point is the mean c SEM
from nine
gave fewer responding cells (3 of 13 HL60 and 0 of 9 RBL
cells).
ATP-evoked currents were characterized in differentiated
HL60 and RBL cells with several purinergic agonists. ATP,
(w,PmATP, and2-MeSATPevoked rapidly desensitizing inward currents whose properties were very similar to those previously described for heterologously expressed P2X, recepIn all cells, repeated application of agonist resulted in
tor~.".'~
marked inactivation of the current; after an initial application
of ATP or (w,,OmATP (30 pmol/L) for l-second duration, a
second application 2 minutes later evoked a current that was
0% to 10% the amplitude of the initial response (Fig 3A).
Reproducible responses were obtained only when the interval
of agonist application was greater than 15 minutes (n = 6).
The amplitude and time course of the initial response showed
little variation from cell to cell. In RBL cells, ATP (30 pmoV
L) evoked a current of 324 t 70 pA with an exponential
onset and desensitization time constant (T,," and T J of 26 i 4
milliseconds and 1 I O ? 13 milliseconds, respectively (n = 11);
similar values in PMA-treated E 6 0 cells were 343 t 31 PA,
19 i I .2 milliseconds, and 205 i 13 milliseconds (n = 18).
ATP currents reversed from inward to outward at about 0 mV
in normal solutions (-1.4 ? 0.8 mV, n = 4 RBL cells and 0.9
t 1.1 mV, n = 5 HL60 cells), indicating the current was caused
by activation of a cation-selective channel.
Agonist concentration-response curves were constructed
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2663
ATP GATED ION CHANNELS IN GRANULOCYTES
either by applying increasing concentrations of agonist at
15-minute intervals while recording from a single cell (Fig
4A and B) or by applying a single concentration of agonist
to many cells and averaging peak amplitudes (Fig 4C and
D). Concentrations producing half-maximal response (ECS0
values) were similar in both cases; ECS0sfor ATP, a,@mATP, and 2-MeSATP in HL60 cells were 3 pmol/L, 1.4
pmol/L, and 3.4 pmol/L, respectively (Fig 4B). ATP, a,@mATP, and 2-MeSATP ECsOvalues for RBL cells were 1.1
pmoVL, 3.2 pmol/L, and 1.4 pmoVL (Fig 4D).
DISCUSSION
We report here the first functional responses for a P2X
receptor in cells with either macrophage (PMA-differentiated HL60 cells) or granulocytic (RBL cells) characteristics.
Several independent observations support the conclusion that
P2XI receptors function in both differentiated HL60 cells
and RBL cells. First, P2Xl immunoreactivity at the outer
membrane is observed in differentiated HL60 cells. Second,
the increase in P2X1 immunoreactivity, seen upon differentiation of HL60 cells, is mirrored by a similar augmentation
in receptor binding for [3sS]ATPyS. Displacement of that
binding with a series of P2Xl ligands was characteristic of
recombinant P2Xl receptors, expressed in CHO-K1 cells.’I
Third, treatment of differentiated HL60 cells or RBL cells
with apyrase, to destroy extracellular ATP, uncovered fully
functional ATP gated ion channels. The properties of those
channels were essentially indistinguishable from recombinant P2XI receptors, cloned from smooth muscle. These
properties included the known selectivity for a range of P2XI
agonists, the rapid kinetics of channel closure (densensitization) and the marked inactivation of current that is observed
upon reapplication of the agonist. P2X1 channels in smooth
muscle are also known to have brief openings and to undergo
rapid desensitization in the presence of ATP.’3,23
The release of ATP that we observed is not without precedence. RBL and peritoneal mast cells have been shown previously to release ATP after ~timulation.~
ATP is a component of mast cell granules and dense platelet granules.24The
release of ATP by cultured cells has been reported for a
variety of other cell types, including the astrocytoma cell
line 132N1.” Epithelial cells may also release ATP although
by a different mechanism that depends on the cystic fibrosis
transmembrane conductance regulator protein.26P2Xl at the
neuromuscular junction receives ATP in discrete quanta, released synaptically by sympathetic neurons. Immune cells
in contrast may be activated by extracellular ATP from some
of the above sources or from damaged endothelium.
Extracellular ATP interacts with receptors on several types
of human granulocytic cells, including natural killer (NK)
cells,27 eosinophils,% and neutrophil^.^ ATP, released by
platelets, is chemotactic for eosinophils, where it raises intracellular calcium and in neutrophils ATP stimulates secretion.
Therefore, it is likely that in vivo, ATP acts on some fully
differentiated granulocytes, modulating their state of activation. Whether ATP-mediated receptor desensitization also
occurs in vivo is unknown. Functional analysis of blood
cells, treated with apyrase or suramin, can presumably address this question. We conclude that such pretreatment may
be essential in fully assessing the capacity of extracellular
ATP in immune modulation.
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1996 87: 2659-2664
P2X1 receptor activation in HL60 cells
G Buell, AD Michel, C Lewis, G Collo, PP Humphrey and A Surprenant
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