1 Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; and 2 Serono Pharmaceutical Research Institute, Geneva, Switzerland
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ABSTRACT |
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P2X
receptors function as ATP-gated cation channels. The P2X7
receptor subtype is distinguished from other P2X family members by a
very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated
or prolonged stimulation. Previous studies have indicated that certain
P2X7 receptor-positive cell types, such as human blood
monocytes and murine thymocytes, lack this pore-forming response. In
the present study we compared pore formation in response to
P2X7 receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture.
ATP induced nonselective pores in macrophages but not in freshly
isolated monocytes when both cell types were identically stimulated in
standard NaCl-based salines. However, ion substitution studies revealed
that replacement of extracellular Na+ and Cl
with K+ and nonhalide anions strongly facilitated
ATP-dependent pore formation in monocytes. These ionic conditions also
resulted in increased agonist affinity, such that 30-100 µM ATP
was sufficient for activation of nonselective pores by P2X7
receptors. Comparison of P2X7 receptor expression in blood
monocytes with that in macrophages indicated no differences in
steady-state receptor mRNA levels but significant increases (up to
10-fold) in the amount of immunoreactive P2X7 receptor
protein at the cell surface of macrophages. Thus ability of ATP to
activate nonselective pores in cells that natively express
P2X7 receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+
and Cl
. These mechanisms may prevent adventitious
P2X7 receptor activation in monocytes until these
proinflammatory leukocytes migrate to extravascular sites of tissue damage.
macrophage; extracellular ATP; inflammation; ligand-gated ion channel
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INTRODUCTION |
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THE P2X FAMILY OF NUCLEOTIDE receptors comprises seven genetically distinct subtypes, all of which function as ATP-gated cation channels (6, 23). The P2X7 receptor subtype, which corresponds to the functionally defined P2Z receptor (33), is distinguished by an extraordinarily low affinity for extracellular ATP, the presumed physiological agonist. Activation of P2X7 receptor channels triggers depolarization, Ca2+ influx, and rapid equilibration of Na+ and K+ gradients. However, on repeated or prolonged stimulation by ATP, P2X7 receptors additionally facilitate the induction of a nonselective pore that is permeable to inorganic ions and organic molecules (anionic and cationic) with masses (Mr) as large as 800 Da (29). The structural and functional nature of this nonselective pore is a subject of interest given the ability of extracellular ATP to rapidly induce necrotic or apoptotic death in cells that natively express the P2X7 receptor (4, 32). Recent studies indicate that other P2X receptor subtypes, including P2X2 and P2X4, can also activate a nonselective permeability when overexpressed in heterologous cell types (18, 19, 36).
Because individual P2X7 receptor subunits possess only two transmembrane-spanning segments, it is assumed that functional channels are oligomers of several individual subunits. This assumption is supported by studies showing that recombinant P2X7 receptor subunits can self-assemble during translation and processing into stable, detergent-resistant complexes (34). Although the mechanisms underlying nonselective pore formation by P2X7 receptors are unknown, induction of the pore may involve further multimerization of the P2X7 receptor subunits or a dynamic change in the selectivity filter of existing P2X7 channel complexes (18, 36). However, recent studies by Schilling and colleagues (30, 31) suggest that nonselective pore formation may involve intrinsic membrane proteins that are regulated by, but distinct from, the P2X7 receptor per se.
Functional analyses of natively expressed human P2X7 receptors have largely utilized monocyte-derived macrophages (8, 13), monocytic leukemia cell lines (16), or blood lymphocytes from patients with chronic lymphocytic leukemia (21, 38). Multiple studies have described the ATP-dependent induction of nonselective pores [permeable to ethidium+ (Mr = 314 Da) or YO-PRO2+ (Mr = 375 Da) ion] in human macrophages (3, 8, 14, 32). In contrast, such pores were not observed when human blood monocytes, which are the precursors of macrophages, were similarly stimulated with high concentrations of extracellular ATP under physiological ionic conditions (8, 14). Using a monoclonal antibody suitable for fluorescence-activated cell sorter (FACS) analysis, Buell et al. (2) demonstrated that immunoreactive P2X7 receptors are expressed in >80% of human monocytes. Similar studies have been recently reported by Gu et al. (13). Thus the inability of ATP to efficiently activate the nonselective pore in blood monocytes, despite the expression of plasma membrane P2X7 receptor subunits, indicates that posttranslational mechanisms limit P2X7 receptor-dependent pore formation until monocytes leave the blood, enter proinflammatory tissue spaces, and differentiate into macrophages. A similar lack of nonselective pore formation has been described in murine thymocytes that express P2X7 receptor mRNA and exhibit ATP-gated cation channel activity (16, 31).
Pore formation may require a threshold number of agonist-bound P2X7 receptor subunits, and the surface density of P2X7 receptors may be insufficient on circulating monocytes. Environmental factors such as pH, ionic strength, and ionic composition may additionally limit the ability of P2X7 receptor subunits to undergo the conformational changes required for induction/activation of the nonselective pores. Recent studies have shown that activation of recombinant or native P2X7 receptors by ATP and other nucleotide agonists is exquisitely sensitive to the ionic composition of the extracellular medium (13, 22, 24, 37). In this study, we have used human blood monocytes and monocyte-derived macrophages as a model system to demonstrate critical roles for receptor surface density and allosteric modulation by extracellular ionic composition in the induction of nonselective pores by natively expressed human P2X7 receptors.
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MATERIALS AND METHODS |
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Isolation of blood monocytes. Venous blood (20-30 ml) from healthy volunteers was collected into 4-5 ml of sterile acid-citrate-dextrose containing 100 mM disodium citrate and 128 mM D-glucose, pH 5. The citrated whole blood was diluted 1:1 with PBS, and 30-ml aliquots were layered over 13 ml of Histopaque-1077 (Sigma Chemical, St. Louis, MO) and centrifuged at 400 g for 30 min. Mononuclear leukocytes on top of the Histopaque layer were collected into PBS-citrate and centrifuged at 200 g. The supernatant containing >95% platelets was discarded, while the pellet comprising the peripheral blood leukocytes (PBML) was subjected to two more washes in PBS-citrate and finally resuspended in Iscove's modified DMEM plus 10% iron-supplemented newborn calf serum (Hyclone Laboratories, Logan, UT).
Experimental salt solutions.
PBML, isolated as described above, were resuspended in one of the basic
salt solutions (BSS) described in Table
1. For the experiments involving reduced
concentrations of extracellular divalent cations, each BSS was adjusted
to contain 0.5 mM CaCl2 and no added MgCl2.
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Antibodies. R-phycoerythrin-conjugated anti-CD14 (a monocyte marker) and FITC-conjugated anti-CD14, anti-mouse IgG (FITC conjugated), and anti-mouse IgG (R-phycoerythrin conjugated) were purchased from Sigma Chemical. The monoclonal antibody to the human P2X7 receptor was generated as previously described by Buell et al. (2) and provided by Serono Pharmaceuticals. The exact region of antibody recognition has not yet been determined.
Analysis of P2X7 receptor mRNA by RT-PCR. RT-PCR was carried out exactly as previously described (15). Primers specific for the human P2X7 receptor cDNA sequence [5'-GGCAGTTCAGGGAGGAATCATGG-3' (sense) and 5'-AAAGCGCCAGGTGGCATAGCTC-3' (antisense)] generated a 939-bp product. Commercial primers to human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Stratagene, La Jolla, CA) generated a 600-bp product. Products of PCR were electrophoresed on 1% agarose gels containing ethidium bromide and photographed. In control experiments, standard curves were generated using serial dilutions of the RT reactions as templates for PCR with each primer set. The linear range of the assay was thus determined for each primer set, and products from the original 20-µl RT were diluted (1:100 for P2X7 and 1:500 for GAPDH) into the final PCR volume to ensure nonsaturation of the PCR amplification.
Flow cytometric analysis of P2X7 receptor expression.
PBML were isolated from whole blood as described above but were
pelleted and resuspended (107 cells/ml) in PBS containing
4% BSA (FACS buffer). For experiments involving mature macrophages,
the PBML were first plated and allowed to adhere for 3 h. After
removal of the nonadherent lymphocytes, the monocytes were cultured for
1-9 days in Iscove's DMEM supplemented with 10% calf serum and
1% penicillin-streptomycin. In some experiments (specified below),
recombinant human interferon- (1,000 U/ml; Genentech) was included
in the tissue culture medium. The adherent monocyte-derived macrophages
were nonenzymatically detached by incubation at 37°C for 20 min using
cell dissociation solution (Sigma Chemical). Monocytes or macrophages
were then incubated in FACS buffer supplemented with human IgG (50 µg/ml) for 60 min on ice to block nonspecific binding of specific
antibodies to Fc receptors. After addition of the monoclonal anti-human
P2X7 antibody (1.7 µg/ml), the intact cell samples were
rotated for 2 h at 4°C. The cells were centrifuged and washed
three times in FACS buffer, resuspended in FACS buffer containing
FITC-conjugated anti-mouse IgG (0.17 µg/ml), and incubated for 30 min
at 4°C. They were then rinsed twice in FACS buffer and once in 0.02%
sodium azide in PBS and fixed with 2% paraformaldehyde in PBS for 30 min. The fixed cells were then centrifuged and resuspended in FACS
buffer for analysis. An EPICS XL-mCL flow cytometer (Coulter, Miami,
FL) with an air-cooled argon ion laser operated at 15 mW was used to
analyze fluorescence. The forward scatter was linear, but the side
scatter, as well as the FITC and PE, were acquired by logarithmic
amplification with excitation wavelength of 488 nm and emission
band-pass filters at 530 and 575 nm, respectively. Before the data were
acquired, live gates were set to obtain well-shaped cells and to
exclude cellular debris. A sample of unstained PBML was used to set the
gates for negative values. PBML were identified and gated on the basis
of forward and side scatter and by the CD14-positive marker for
monocytes and CD3/CD19-positive markers for lymphocytes. Acquisition of
data from 10,000 cells was performed in list mode and processed using
XL System 3.0 software with an attached Dell computer. The fluorescence
distribution was displayed as a single histogram. Further analysis was
done using WinList 4.0 software (Verity Software, Topsham, ME).
Analysis of P2X7-dependent pore formation by fluorescence microscopy. For microscopic analysis of dye uptake by human monocytes or macrophages, PBML were plated onto Labtek chambered slides (Nalge-Nunc) and allowed to adhere for 2-3 h at 37°C. The nonadherent cells (mostly lymphocytes) were washed off, and the adherent monocytes were assayed immediately or were tissue cultured for 9 days. For dye uptake assays, the parallel wells containing similar numbers of adherent cells were incubated in complete NaCl BSS containing 2 µM YO-PRO-1 (Molecular Probes, Eugene, OR) for 15 min at 37°C. The YO-PRO-1 medium was variously supplemented with no additions (as a control for basal dye uptake), 3 mM ATP (to test for P2X7-dependent pore formation), or 50 µg/ml digitonin (to nonselectively permeabilize all cells within each sample well). After the 15-min test incubation, the YO-PRO-1-containing BSS was removed and replaced with dye-free NaCl BSS (after 2 washes). The number of YO-PRO-1-positive cells within equivalent optical fields of each separate well was immediately assayed by fluorescence microscopy (Nikon Eclipse E600) using a standard fluorescein filter set.
Analysis of P2X7-dependent pore formation by flow cytometry. For flow cytometric analyses, freshly isolated PBML were resuspended in one of the BSS media containing 2 µM YO-PRO-1. Parallel samples of each cell suspension were stimulated with various concentrations of ATP or no additions (as controls) for 15 min at 37°C. The cells were then centrifuged, washed twice, resuspended in complete NaCl BSS, and immediately subjected to FACS analysis. Monocytes were gated as described for analysis of CD14 and P2X7 receptor expression. YO-PRO-1 uptake was measured using 488-nm excitation and 530-nm emission filters. The distribution and single cell fluorescence intensities were calculated as described above.
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RESULTS |
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ATP-dependent induction of nonselective pores in blood monocytes
vs. monocyte-derived macrophages.
When stimulated with high concentrations of ATP (>1 mM), cells that
express functional P2X7 receptors can become permeable to
large organic dyes, such as YO-PRO2+
(Mr = 375 Da) and ethidium+
(Mr = 314 Da), which form highly
fluorescent complexes on binding to intracellular nucleic acids. We
compared the ATP-dependent accumulation of YO-PRO-1-nucleic acid
complexes in freshly isolated human blood monocytes with that in
monocytes that had differentiated into macrophages after 9 days of in
vitro culture (Fig. 1). The plating
densities were adjusted to contain similar numbers of monocytes or
macrophages per optical field (~350 cells at ×20 magnification).
This was verified by permeabilizing all cells within each well with
digitonin in the presence of YO-PRO-1 and counting the number of
fluorescent nuclei. The labeled nuclei were evenly dispersed in freshly
isolated monocyte cultures. In contrast, macrophage nuclei were
generally clustered within islands of aggregated single cells, while
some nuclei were colocalized within multinucleate giant cells that are
formed by the fusion of single macrophages. Such cell clusters and
multinucleate giant cells typify in vitro cultures of monocyte-derived
macrophages. ATP-dependent pore formation was assayed in parallel wells
of monocytes or macrophages bathed in medium containing physiological concentrations of extracellular Na+, K+,
Cl, Ca2+, and Mg2+. When
incubated for 15 min at 37°C in the absence of extracellular ATP, few
monocytes (~2%) and only modest numbers of macrophages (~10%)
accumulated YO-PRO-1. When 3 mM ATP was included in the YO-PRO-1 assay
medium, >95% of the macrophages within each optical field exhibited
strong cytoplasmic and nuclear fluorescence after the 15-min test
incubation. In contrast, the same ATP challenge produced no significant
change in the numbers of YO-PRO-1-labeled monocytes. Prolonging the
incubation period (up to 30 min) did not increase YO-PRO-1 labeling of
the monocyte samples (data not shown). These observations, which are
similar to those previously reported by Falzoni et al. (8)
and Hickman et al. (14), underscore the significant
quantitative and qualitative differences between the ATP-dependent
induction of nonselective pores in human monocytes and that in the
macrophages derived from these monocytes.
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Expression of P2X7 receptor mRNA and protein in blood
monocytes vs. monocyte-derived macrophages.
Using P2X7 receptor-specific monoclonal antibodies, Buell
et al. (2) and Gu et al. (13) demonstrated
expression of cell surface P2X7 receptors in >80% of
human blood monocytes (2). Given the absence of
ATP-dependent pore formation in freshly isolated human monocytes (Fig.
1) (8, 14), we tested the possibility that
P2X7 receptor gene expression is low in circulating
monocytes but is significantly increased when monocytes are incubated
under in vitro conditions that promote their differentiation into
macrophages. RT-PCR analysis indicated that the steady-state levels of
P2X7 receptor mRNA were similar in freshly isolated blood
monocytes and in monocyte-derived macrophages generated by incubating
monocytes for 3 days in the presence of interferon- (Fig.
2). This suggests that
differentiation of blood monocytes into macrophages does not trigger
significant changes in P2X7 receptor gene transcription or
stability of P2X7 receptor mRNA.
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Inhibitory effects of extracellular
Na+ and Cl on nonselective
pore formation by the P2X7 receptors of blood monocytes.
The results described above suggest that limited expression of cell
surface P2X7 receptor subunits may be one mechanism for the
lack of nonselective pore formation in freshly isolated blood monocytes
stimulated with high concentrations of extracellular ATP. However, pore
formation by the P2X7 receptors natively expressed in
freshly isolated normal or leukemic blood lymphocytes can only be
observed when these cells are stimulated in medium lacking extracellular Na+ and Mg2+ (13, 37,
38). We hypothesized that the absence of ATP-induced pore
formation in human blood monocytes may be similarly repressed by
extracellular ions. This hypothesis was tested by assaying the ability
of 3 mM ATP to stimulate YO-PRO-1 accumulation in freshly isolated
blood mononuclear leukocytes that were suspended in HEPES-buffered
solutions of different ionic composition and analyzed by FACS (Fig.
5). Consistent with the studies using
adherent blood monocytes (Fig. 1), no significant number of monocytes
suspended in standard NaCl-based saline accumulated YO-PRO-1 in
response to ATP (Fig. 5A). Although Mg2+ is
known to strongly modulate P2X7 receptor function (6,
23, 35), removal of extracellular Mg2+ from the
NaCl-based saline did not facilitate ATP-induced YO-PRO-1 uptake (Fig.
5C). In contrast, when NaCl was isosmotically replaced by
potassium glutamate, 3 mM ATP triggered a robust uptake of YO-PRO-1 in
the majority of blood monocytes (~90%) regardless of the presence or
absence of extracellular Mg2+ (Fig. 5, B and
D, respectively). Although extracellular Mg2+
did not alter the percentage of ATP-responsive monocytes, it did affect
the absolute amount of YO-PRO-1 that was accumulated during the 15-min
test incubation. Thus monocytes suspended in Mg2+-free,
potassium glutamate BSS accumulated 4- to 10-fold more YO-PRO-1 dye
(MFI = 2,800 for the experiment in Fig. 5D) than monocytes activated in medium containing 1 mM Mg2+
(MFI = 270 for the experiment in Fig. 5B). Although the
FACS profiles in Fig. 5 illustrate the responses of monocytes from a
single donor, they are representative of similar experiments performed
with different blood donors (n = 9). The averaged
values from these experiments are presented in Fig. 5E. The
results indicate that nonselective pores can be induced in blood
monocytes, despite the relatively low number of P2X7
receptor subunits that are targeted to the surface of these cells.
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Enhanced potency of ATP as an agonist for monocyte P2X7
receptors in the absence of extracellular NaCl.
The experiments in Fig. 5 demonstrate that physiological concentrations
of Na+ and Cl strongly attenuate ATP-mediated
pore formation in human monocytes. Previous studies have shown that
millimolar concentrations of ATP are required for the activation of the
P2X7/P2Z receptor (29, 33). However, Michel et
al. (22) reported that removal of extracellular NaCl can
increase the potency of BzATP as an inducer of pore formation in
HEK-293 cells that express recombinant human P2X7
receptors. We observed that replacement of extracellular Na+ and Cl
similarly affects the potency of
ATP as an agonist for the natively expressed P2X7 receptors
of human blood monocytes (Fig. 6). Thus, although significant YO-PRO-1 accumulation (as assayed by MFI values
from FACS analyses) in response to 100 µM ATP was observed when
monocytes were stimulated in potassium glutamate BSS, even 3 mM ATP
triggered no dye uptake when the same cells were suspended in NaCl BSS.
The same ATP sensitivity was observed in potassium glutamate BSS in the
presence or absence of millimolar Mg2+. The ATP
dose-response relationship in the potassium glutamate media was
biphasic, with an initial rise in the MFI per cell at 30-100 µM
and an apparent maximum between 500 and 1 mM ATP. However, a further
increase in the MFI was observed at >1 mM ATP. This is most likely due
to a reduction in intracellular Mg2+ under these
conditions, resulting in enhanced fluorescence of the YO-PRO-1-nucleic
acid complexes. Mg2+ will permeate the nonselective pore,
and ATP is a strong chelator of Mg2+ (and
Ca2+). Thus, when 3 mM extracellular ATP is in excess over
the concentrations of extracellular Mg2+ (1 mM) and
Ca2+ (1.5 mM), the free concentration of Mg2+
will be reduced to micromolar levels, leading to a transmembrane gradient that will favor efflux of intracellular Mg2+
(given the 0.3-0.5 mM free Mg2+ within the cytosol).
Thus reduction of extracellular Na+ and Cl
increases the potency of ATP as an agonist for P2X7
receptor-dependent pore formation, while removal of extracellular
Mg2+ primarily facilitates an increased rate of dye
influx through these nonselective pores.
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Effects of Na+ vs. Cl
on nonselective pore formation by monocyte P2X7 receptors.
Figure 7 illustrates the independent
effects of Na+ vs. Cl
replacement on
ATP-dependent YO-PRO-1 uptake by blood monocytes using medium
containing physiological concentrations of Mg2+ (1 mM) and
Ca2+ (1.5 mM). Individual substitution of Na+
with K+ (KCl BSS) facilitated significant YO-PRO-1
accumulation (MFI = 40) in 70% of the monocytes (Fig.
7C). Substitution of Cl
with glutamate (sodium
glutamate BSS; Fig. 7B) produced similar changes in
ATP-induced YO-PRO-1 uptake (MFI = 30) in the same percentage of
cells. Replacement of Cl
with gluconate also potentiated
ATP-activated dye accumulation (data not shown). Consistent with
previous studies (Fig. 5), simultaneous replacement of Na+
and Cl
with K+ and glutamate (Fig.
7D) resulted in a synergistic potentiation of ATP-dependent
YO-PRO-1 influx in the same monocytes (MFI = 270). The averaged
data from three independent experiments of this type are illustrated in
Fig. 7E.
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DISCUSSION |
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These studies demonstrate that the P2X7 receptor gene
is transcriptionally active in human blood monocytes and that
P2X7 receptor protein traffics to the plasma membrane of
these cells. However, ATP can induce the nonselective pore that
typifies activated P2X7 receptors only when these blood
leukocytes are assayed in media containing reduced extracellular
Na+ or Cl. This contrasts with the robust
induction of the nonselective pore in macrophages, which are derived
from these human monocytes, when assayed in normal NaCl-containing
medium. These findings suggest that multiple mechanisms have evolved to
minimize the adventitious activation to P2X7 receptors in
circulating monocytes while permitting these receptors to be rapidly
upregulated and activated when monocytes leave the blood and enter
peripheral sites of tissue inflammation.
All members of the P2X receptor family, including P2X7,
function primarily as ATP-gated ion channels that can conduct small, inorganic cations (e.g., Na+, K+, and
Ca2+). Analysis of most P2X family receptors indicates that
the assembled, cation-permeable channels minimally conduct larger
organic molecules such as Tris+ (121 Da),
N-methylglucamine+ (195 Da), or the YO-PRO-1 dye
(375 Da) used in our experiments (24). In contrast, the
sustained activation of P2X7 receptors by ATP results in a
time- and temperature-dependent acquisition of permeability to such
large molecules (7, 24, 29). This delayed change in
permeability has been termed "pore formation" to distinguish it
from the rapidly gated change in conductance to small inorganic
cations. The molecular nature of these nonselective pores remains to be
established but might be consistent with several mechanisms.
1) P2X7 homooligomeric channels with fixed
subunit stoichiometry may have multiple permeability states.
2) P2X7 homooligomers might physically
interact with other P2X7 channels or other membrane proteins to form higher-order pore complexes. 3)
Intracellular signaling cascades activated by P2X7 receptor
channels may trigger covalent or allosteric changes in other proteins
that comprise the nonselective pore (30, 31). Regardless
of mechanism, extracellular Na+ and Cl
clearly exert strong inhibitory effects on the ability of ATP to
activate pore formation via the cell surface P2X7 receptors expressed in circulating monocytes (Figs. 5-7). Isosmotic
replacement of these ions with K+ and nonhalide anions
results in an apparent "unmasking" of the pore-inducing function.
One likely effect of Na+ and Cl
is to inhibit
the binding of ATP to existing P2X7 receptor oligomers and
thereby attenuate rapid gating of the cation channel and subsequent induction of the nonselective pore. Michel et al. (22)
demonstrated that the repressive actions of Na+ and
Cl
on the function of recombinant P2X7
receptors primarily reflect effects on agonist potency, rather than
agonist efficacy, regardless of whether cation channel activity or
YO-PRO-1 accumulation was measured as an index of receptor activation.
We similarly observed that replacement of extracellular NaCl
facilitated pore formation in blood monocytes stimulated with ATP
concentrations as low as 30 µM, even in the presence of
millimolar Mg2+ (Fig. 6).
In addition to reducing the ATP affinity of the P2X7
receptor, Na+ and Cl may directly repress
conformational changes that permit assembly or activation of the
nonselective pore. Pore formation may require a threshold number of
ATP-occupied P2X7 receptor complexes to drive efficient
interaction with other putative pore-forming proteins or to activate
intracellular signaling cascades that regulate such proteins. It should
be stressed that millimolar ATP does activate pore formation in many
P2X7-expressing cell types (8, 15, 29, 32),
including human monocyte-derived macrophages (Fig. 1), bathed in
physiological saline solutions. The inability of millimolar ATP to
increase YO-PRO-1 uptake by human blood monocytes under identical assay
conditions suggests that the number of ATP-occupied P2X7
receptors that can be formed is insufficient to drive formation of the
pore. If a threshold number of ATP-occupied receptors is required for
pore formation, the requisite number of receptors occupied at a given
concentration of ATP may be achieved by increasing ATP affinity (as
acutely induced by reducing extracellular NaCl) or by increasing the
number of cell surface receptors. Consistent with this latter
possibility, our FACS analyses indicated that monocyte-derived human
macrophages express significantly higher numbers (by 3- to 10-fold) of
cell surface P2X7 receptors than circulating monocytes.
Presumably, the number of ATP-occupied P2X7 receptors
required for threshold pore formation can accumulate when such
macrophages are stimulated with millimolar ATP, even in the
presence of high extracellular NaCl.
It is noteworthy that, with some blood donors, 500 µM BzATP [which is more potent and efficacious than ATP as a P2X7 agonist (33, 39)] did stimulate significant YO-PRO-1 accumulation by blood monocytes assayed in NaCl medium (data not shown). This further supports the possibility that pore formation requires a threshold number of agonist-occupied P2X7 receptors. On the basis of similar studies of the P2X7 receptors expressed in human leukemic lymphocytes, Wiley et al. (39) suggested that the rate and extent of pore formation are dependent on the fraction of P2X7 channels held in the open state due to agonist occupancy.
FACS analyses of digitonin-permeabilized blood monocytes using the
monoclonal anti-P2X7 receptor indicated additional pools of
intracellular P2X7 receptors (data not shown). Gu et al.
(13) also described significant intracellular levels of
immunoreactive P2X7 receptor proteins in human lymphocytes,
monocytes, and neutrophils. Such findings suggest that trafficking of
intracellular P2X7 receptors to the cell surface is a
potential mechanism for regulating the pore-forming function. We
considered the possibility that incubation of monocytes in
low-Na+ or low-Cl saline promoted a rapid
redistribution of P2X7 receptors. An acutely induced
increase in the number of cell surface P2X7 receptor channels could underlie the enhanced pore formation observed under such
experimental conditions. This was tested by stimulating monocytes with
3 mM ATP in potassium glutamate BSS for 15 min (optimal conditions for
pore formation) before analysis of cell surface P2X7
receptor by the same FACS protocols used in the experiments illustrated in Figs. 4 and 5. No changes were observed in the anti-P2X7
FACS profiles of these monocytes (data not shown). This suggests that rapid insertion of additional P2X7 receptors into the
plasma membrane is an unlikely mechanism for the potentiation of
ATP-induced pore formation observed when monocyte P2X7
receptors are activated in salines containing low Na+ or
low Cl
.
The physiological roles for these nonselective pores also remain
speculative. However, many biological responses associated with
P2X7 receptor stimulation involve the activation of
protease-based signaling cascades. These include caspase-1-mediated
processing of interleukin-1 in monocytes and macrophages,
caspase-3-dependent apoptosis of macrophages and dendritic
cells (4, 11, 27, 28), and matrix metalloprotease-mediated
shedding of L-selectin and CD23 in lymphocytes (12, 17).
Initiation of these protease-based cascades by ATP-activated
P2X7 receptors appears to involve the sustained collapse of
normal transmembrane gradients for K+, Na+, and
Ca2+. Induction of the nonselective pores may provide a
unique mechanism for ensuring the rapid and complete loss of
intracellular K+ and accumulation of Na+ and
Ca2+. Unlike kinase-based signaling, protease-dependent
signal transduction is not readily reversible. Thus it is not
surprising that activation of the P2X7 receptor and
nonselective pore formation appear to be stringently regulated at
multiple levels.
It is striking that the ATP affinity of the P2X7 receptor
is maximal when the extracellular ionic composition approximates the
cytoplasmic ionic milieu, i.e., a high-K+,
low-Na+ solution wherein organic anions substitute for
Cl. When cells lyse within the immediate vicinity of a
P2X7 receptor-expressing cell, they will not only release
ATP but will also modify the local extracellular ionic composition. The
extracellular NaCl solution will be "diluted" by the intracellular
K+-organic anion solution. Obviously, this sort of ionic
perturbation will be maximal in physiological environments wherein the
ratio of cellular volume to extracellular volume is large
(20). Such conditions may also be operative when monocytes
or macrophages populate inflammatory tissue sites that are
characterized by high cell density and significant numbers of damaged
or dying cells. Thus the ability of extracellular monovalent ions to
allosterically modulate ATP affinity may be a heretofore unrecognized
mechanism for the physiological regulation of the pore-forming
P2X7 receptor.
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ACKNOWLEDGEMENTS |
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We are grateful to Sylvia Kertesy for excellent technical support, R. Michael Sramkoski for assistance with FACS analysis, and Reza Beigi and Karen Parker for helpful discussions.
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FOOTNOTES |
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This study was supported by National Institute of General Medical Sciences Grant GM-36387 (G. R. Dubyak).
Address for reprint requests and other correspondence: G. R. Dubyak, Dept. of Physiology and Biophysics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4970 (E-mail: gxd3{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 August 2000; accepted in final form 26 October 2000.
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