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REVIEW ARTICLE
From the Department of Experimental and Diagnostic
Medicine, Section of General Pathology and Medical Genetics, and Center
of Biotechnology, University of Ferrara, Ferrara, Italy.
Nucleotides are emerging as an ubiquitous family of extracellular
signaling molecules. It has been known for many years that adenosine
diphosphate is a potent platelet aggregating factor, but it is now
clear that virtually every circulating cell is responsive to
nucleotides. Effects as different as proliferation or differentiation, chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species are elicited upon
stimulation of blood cells with extracellular adenosine triphosphate (ATP). These effects are mediated through a specific class of plasma
membrane receptors called purinergic P2 receptors that, according to
the molecular structure, are further subdivided into 2 subfamilies: P2Y
and P2X. ATP and possibly other nucleotides are released from damaged
cells or secreted via nonlytic mechanisms. Thus, during inflammation or
vascular damage, nucleotides may provide an important mechanism
involved in the activation of leukocytes and platelets. However, the
cell physiology of these receptors is still at its dawn, and the
precise function of the multiple P2X and P2Y receptor subtypes remains
to be understood.
(Blood. 2001;97:587-600) In 1978 the existence of plasma membrane receptors
for extracellular nucleotides, the P2 purinergic receptors, was
formally recognized.1 At that time, this identification
was only based on pharmacologic and functional evidence and on the
prophetic intuition of Geoff Burnstock. To date, 12 mammalian P2
receptors have been cloned, characterized, and recognized as
responsible for the diverse cellular responses to stimulation with
extracellular nucleotides.2,3 The P2 receptor family also
includes receptors for extracellular pyrimidines. The new
classification based on the molecular structure is rapidly replacing
the previous one (P2Y, P2X, P2U, P2T, and P2Z) based on the
pharmacologic profile,4 although doubts remain on whether
functional responses of the native P2Z receptor of immune cells can be
entirely explained by the cloned P2X7 subunit. A similar
uncertainty also concerns the platelet P2T receptor, which is likely to
arise from the combination of P2Y and P2X-dependent
responses.2,5 Extracellular effects of nucleotides were
initially recognized in smooth muscle contraction, neurotransmission,
regulation of cardiac function, and platelet aggregation.6
However, over the last 10 years it has become clear that the
intercellular mediator role of these molecules is widespread, and blood
cells have emerged as one of the most interesting targets.
Contrary to a widely held opinion, adenosine triphosphate (ATP) and
possibly also uridine triphosphate (UTP) are often released into the
extracellular environment via nonlytic mechanisms7-12 and
also more frequently as a consequence of cell damage or acute cell death. Furthermore, platelet-dense granules are a relevant source
of secreted ATP.13,14 Once in the pericellular
environment, ATP can serve as a ligand for P2 receptors or be quickly
hydrolyzed by powerful ubiquitous ecto-ATPases and
ectonucleotidases.15-18 ATP can also be used as a
phosphate donor by poorly characterized ectokinases.19
Thus, ATP possesses all the properties of a bona fide fast-acting
intercellular messenger: (a) it is released in a controlled
fashion, (b) ligates specific plasma membrane receptors coupled to intracellular signal transduction, and (c) is
quickly degraded to terminate its action.
Outside excitable tissues, P2 receptors have an obvious relevance in
platelet aggregation, but immunity and inflammation are providing some
of the most exciting developments in this evolving field. A few
reviews covering different aspects of P2 receptor distribution and
function in hemopoietic cells have appeared and have been an invaluable
source of information for the present work.20-26
According to the International Union of Pharmacology (IUPHAR)
Committee on Receptor Nomenclature and Drug
Classification,27 receptors for extracellular nucleotides
are termed P2 receptors (this nomenclature replaces the older
"P2-purinoceptor"). P2 receptors are divided into 2 subfamilies: G protein-coupled (P2Y) and ligand-gated ion channels
(P2X).3,28-30 Current P2Y/P2X nomenclature is based on the
molecular structure and has replaced the previous one based on
pharmacologic and functional criteria. In mammalian cells, 5 P2Y
(P2Y1, P2Y2, P2Y4,
P2Y6, and P2Y11) and 7 P2X (P2X1-7)
receptors have been cloned and characterized
pharmacologically2 (Table 1).
P2Y5, P2Y7, P2Y9, and
P2Y10 have been purged from this sequence because they are
primarily non-nucleotide receptors (although they may also bind
extracellular nucleotides). A p2y3 (lower case to indicate that it has
not been cloned from mammals) receptor has been cloned from chick brain
and suggested to be a homologue of the mammalian
P2Y6.2 P2Y8 has so far only been
cloned from Xenopus neural plate; thus it is not included in
the list of mammalian receptors. The adenosine diphosphate
(ADP)-activated, G protein-coupled receptor of platelets that triggers
inhibition of stimulated adenylate cyclase has not yet been cloned;
thus it is recommended that this receptor should be given in
italics: P2Y ADP.2
P2Y receptors are 7-membrane-spanning proteins, numbering from
328 to 379 amino acids, for a molecular mass of 41 to 53 kd after
glycosylation.2,31,32 The aminoterminal domain faces the
extracellular environment, and the carboxyterminal is on the cytoplasmic side of the plasma membrane (Figure
1). Signal transduction occurs via the
classical pathways triggered by most 7-membrane-spanning receptors:
activation of phospholipase C and/or stimulation/inhibition of
adenylate cyclase. All of the P2Y receptors are activated by ATP, but
at 2 of them, P2Y4 and P2Y6, UTP is more
potent,33-36 and at P2Y2 ATP and UTP are
equipotent.31 At P2Y1, UTP is inactive and ADP
is reported to be equipotent or even more potent than ATP37,38; at P2Y11 ATP is more potent than ADP
and UTP is inactive.39 With respect to the signal
transduction pathway, P2Y1 and P2Y2 are coupled
to stimulation of phospholipase C-
Investigation of P2Y receptors has been severely hindered by the lack
of specific antibodies, whether polyclonal or monoclonal. Likewise, few
selective agonists, besides naturally occurring nucleotides, or
antagonists are available. A widely used P2Y antagonist is
suramin,41 a drug originally developed for the treatment of tripanosomiasis. However, suramin does not discriminate between P2Y
and P2X and has been reported to inhibit other receptors such as the
nicotinic, glutamate, GABA, and 5-hydroxytryptamine receptors as well
as the activity of diverse growth factors.2 Reactive blue
2, trypan blue, and reactive red have also been used as P2Y antagonists, but they also block P2X-dependent responses.2 Recently Harden and coworkers have introduced a number of nucleotide analogues as competitive P2Y1
antagonists.42,43 Pyridoxal phosphate (P5P) and
pyridoxalphosphate-6-azophenyl 2',4'-disulfonic acid (PPADS) are also
sometimes used to inhibit P2Y-dependent responses, but they are more
widely employed to block P2X receptors.
P2X receptors are ATP-gated ion channels
Although still on a limited basis, a few anti-P2X antibodies were made
available over the last 2 years by single laboratories or commercial
sources. Polyclonal antibodies against P2X1,
P2X4, and P2X7 can be obtained from at
least 2 companies; in a few laboratories sera against all the members
of the subfamily have been raised.53,46,49,54 One
monoclonal antibody selective for the human P2X7 receptor has been produced and characterized by Buell and
colleagues.55 Interestingly, this monoclonal antibody,
which recognizes an as yet to be identified epitope on the
extracellular domain, inhibits activation of human macrophages by
3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a P2X7
agonist.55
The unique naturally occurring agonist of P2X receptors is ATP, albeit
diadenosine polyphosphates, such as
P,1P4-diadenosine tetraphosphate
(Ap4A) and P,1P6-diadenosine
hexaphosphate (Ap6A), are active at
P2X1 2, and UTP has been reported to be an
agonist at P2X3 as well as
P2X1.56,57 There is an ongoing debate,
initiated by the pioneeristic experiments of Cockcroft and Gomperts in
mast cells,58,59 on whether P2X receptors recognize the
bianionic (ATP2 Better antagonists, with better-characterized activity, are available
at P2X than at P2Y receptors. PPADS is a noncompetitive inhibitor of
most P2X receptors53 and, depending on the experimental conditions, may act irreversibly. Oxidized ATP (oATP) was introduced 7 years ago as a selective P2Z (P2X7)
inhibitor,64 but it is likely to show the same P2X
antagonist selectivity of PPADS, although no detailed investigation has
been carried out. At the effective concentrations (100-300 µM), oATP
shows little or no inhibitory activity at P2Y receptors and at
ectonucleotidases.64 Action of oATP on ectokinases has not
been tested in depth; thus it cannot be excluded that some effects of
this compound may be due to inhibition of
ectophosphorylation.65 PPADS and oATP likely share the
same mechanism of action. Both compounds have aldehyde groups (1 PPADS, 2 oATPs) that can react with unprotonated lysines to form Schiff's bases. It is assumed that they preferentially modify lysine residues in
the vicinity of the ATP binding site, but this assumption is yet to be
proved. Although PPADS has been used as a P2 blocker for some time, it
was only after the introduction of oATP that attention has been paid to
the time-dependent and irreversible inhibitory effect of this P5P
derivative. Time-dependent and irreversible block is extensively
documented for oATP at the P2X7 receptor: A 1- to
2-hour preincubation with this inhibitor, even if followed by
extensive rinsing, makes all cells so far investigated fully refractory
to ATP stimulation via the P2X7
receptor.64,66-69 Refractoriness lasts several hours,
until new receptors are inserted into the plasma membrane.
More recently, Wiley and colleagues have introduced another powerful
blocker of P2X7, compound
1-[N,0-bis(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62).70 This molecule was originally used as an
inhibitor of the calcium calmodulin-dependent kinase71
and made its first appearance in the purinergic field in a study by
Blanchard et al72 aimed at investigating the role of the
P2Z receptor in cell-mediated cytotoxicity. KN-62 acts as a competitive
inhibitor at nanomolar concentrations and shows a striking species
specificity: It is active only at the human and not at the rat or mouse
P2X7 receptor.73 KN-62 is a very useful tool
for short-term studies, but modification of long-term responses should
be interpreted with caution because of concomitant inhibition of
calcium calmodulin kinase. Surprenant and colleagues74
have recently shown that Coomassie Brilliant Blue G selectively blocks
rat P2X7 with nanomolar affinity.
Early studies by Steinberg and Silverstein showed that the J774
mouse macrophage cell line expressed a plasma membrane receptor selectively activated by ATP and a few analogues.60,75
Stimulation of this receptor triggered the same reversible increase in
plasma membrane permeability to low-molecular-mass solutes originally described by Cockcroft and Gomperts in rat mast
cells.58,59 An intriguing finding of these studies was
that stimulation of the ATP-permeabilizing receptor eventually led to
cell death.75 This incidental observation stirred interest
in the possible physiologic meaning of ATP-dependent cytotoxicity and
fostered subsequent studies on the role of P2 receptors in the immune
system. At about the same time, Greenberg et al demonstrated that J774
macrophages also expressed P2Y-like receptors coupled to
Ca++ mobilization via a mechanism other than the
ATP-permeabilizing receptor.76 This was made possible by
the selection by Steinberg and colleagues75 of
ATP-resistant J774 macrophage clones later shown to lack the
P2X7 receptor.77,78
According to the nomenclature proposed by Gordon,4 the
macrophage-permeabilizing receptor was named P2Z, analogously to the
mast cell and lymphocyte ATP receptor. The receptor responsible for
ATP-dependent permeabilization has been referred to as P2Z until very
recently and, even after the cloning of P2X7 and the demonstration that its transfection confers susceptibility to ATP-dependent permeabilization, some investigators prefer the P2Z
nomenclature to indicate the native ATP-permeabilizing receptor, because it is not clear whether P2X7 is the only
constitutive subunit or, rather, the native P2Z receptor is formed by
the assembly of P2X7 in association with other P2X
subtypes. However, because P2X7 reproduces all known
effects of the native P2Z and cells resisting ATP-mediated
permeabilization lack P2X7, we will assume heretofore that
the macrophage P2Z and P2X7 receptors are the same
molecule. As seen below, the picture is more complex in lymphocytes and
other cells that do not undergo the typical ATP-dependent permeabilization, although they may express P2X7.
All murine macrophage lines so far investigated express P2Y
receptors coupled to release of Ca++ from intracellular
stores and IP3 generation, but the individual subtypes have
not been investigated in detail. Functional and molecular expression of
P2X7 has been shown in some murine cell lines and in mouse
and rat peritoneal macrophages.60,75,79-83 Monocyte-derived human macrophages are susceptible to ATP-mediated permeabilization and express P2X7.66,84,85
Among human macrophage lines, THP-1 and U937 cells express P2Y
receptors (P2Y2, P2Y4, and
P2Y6),5,86-88 but only the THP-1 monocytic
cell line has been reported to express P2X7 to a
significant level.88 However, P2X7 receptor
expression can differ significantly among cell batches propagated in
different laboratories. Monocytes freshly isolated from peripheral
blood express P2Y receptors but lack P2X7, whether investigated at the molecular or at the functional level. Although a
few studies are available, it is generally agreed that, at the most,
15% to 17% of human monocytes undergo the plasma membrane permeability transitions diagnostic of P2X7 expression when
stimulated with ATP.66,84 There appears to be an inverse
correlation between P2Y2 and P2X7 expression
during monocyte to macrophage maturation: P2Y2 messenger
RNA (mRNA) declines while P2X7 mRNA
increases.89 Up-regulation of P2X7 and
acquisition of P2X7-dependent responses are detectable
within 24 hours of seeding human monocytes on plastic dishes.
Up-regulation of P2X7 and down-regulation of
P2Y2 by the inflammatory mediators interferon- The first report on the effect of exogenous nucleotides on
macrophage function was a paper by Cohn and Parks.91 In
this study the authors showed that addition of adenine nucleotides to a
mouse macrophage culture resulted in a dramatic increase in pinocytic
vesicle formation. After this early study, exogenous nucleotides as a
stimulant for macrophages were basically neglected for several years
and resurrected only in 1985 by Silverstein and
coworkers,92 who reported that extracellular ATP inhibited Fc receptor-mediated phagocytosis and at the same time caused influx
of Na+, efflux of K+, and an increase in
[Ca++]i. In this study it was also for the
first time suggested that macrophages expressed receptors specific for
ATP. The possibility that these ATP effects could be due to ATP
hydrolysis by plasma membrane ecto-ATPase was ruled out by subsequent
papers by Steinberg and Silverstein60,75,76 that reported
an in-depth characterization of the macrophage-permeabilizing ATP
receptor. It was also soon clear that the ATP receptor coupled to
release of Ca++ from intracellular stores (P2Y) and the
ATP-permeabilizing (P2Z/P2X7) receptor were 2 separate
entities with widely different nucleotide selectivity and affinity and
likely involved in different responses.76 In J774
macrophages, the concentration of ATP giving one half of the maximal
response (EC50) for Ca++ release from
intracellular stores (and which therefore reflects activation of P2Y)
is in the range of 50 to 70 µM. In microelectrode impalement
experiments, the ATP EC50 for depolarization, presumably reflecting opening of P2X7, was reported to be between 250 and 400 µM,93 but a lower EC50 was reported
for P2X7-triggered Ca++ rise in
thioglycollate-elicited mouse peritoneal macrophages.94 However, determinations based on the measurement of uptake of fluorescent markers give higher EC50 (1.0-1.5 mM ATP) for
the activation of the native mouse P2X7
receptor.76,95 The UTP EC50 for
Ca++ release from intracellular stores is between 300 and
500 nM 76 and thus much lower than the ATP
EC50.76,94 This suggests that macrophages
express P2Y4 or P2Y6 or an endogenous yet to be
identified uridine nucleotide-specific receptor. Therefore, it is
clear that should ATP release occur in a tissue, macrophage P2Y
receptors are likely to be activated more easily and more frequently
than P2X7.
An early and, with hindsight, obvious proposal was that macrophages
and, in general, inflammatory cells, could use P2Y receptors as very
sensitive sensors of cell and tissue damage.76 After all,
mammalian cells contain huge amounts (5-10 mM) of ATP in their cytosol;
thus, any event that causes even a transient break in the plasma
membrane will cause release of ATP into the pericellular environment.
Furthermore, it is becoming apparent that frank cell injury or death
might not even be necessary for ATP release because shear stress forces
and stretching are also powerful stimuli for ATP
leakage.8-12 J774 macrophages chemotact in response to
micromolar concentrations of ADP but, rather intriguingly, not of
UTP.96 Human macrophages in the vicinity of dying K562
cells have been shown in vitro to undergo an increase in
[Ca++]i that can be closely mimicked by the
addition of cell lysate or of ATP at micromolar doses.97
Precedent treatment with the cell lysate made the macrophages
refractory to the subsequent application of ATP, suggesting, although
not proving, that a substance contained in the lysate and ATP might
converge on the same receptor. Thus it can be hypothesized that ATP and
other intracellular nucleotides function as early alarm signals that
alert macrophages of even minor cell and tissue damage (a response
could be elicited with as little as 100 nM ATP) (Figure
3).
The [Ca++]i rise could also be exploited by
the macrophages for the potentiation of antimicrobial defense
mechanisms. Nucleotides by themselves are unable to activate the
macrophage NADPH oxidase but enhance superoxide generation stimulated
by phagocytosable particles.98 It is conceivable that P2
receptors could also be used as an amplification system to spread the
alarm by generating additional inflammatory mediators. In murine and
human macrophages, extracellular ATP triggers release of TNF- Participation of P2 receptors in IL-1
In human monocytes, ATP is a powerful stimulus not only for caspase-1
activation but also for the externalization of mature caspase-1
subunits.112 The meaning of this novel observation is
elusive, but it may point to a possible function of activated caspase-1
either in the extracellular space or on the outer leaflet of the plasma
membrane. In addition, ATP might trigger IL-1 Participation of P2X7 in LPS-dependent activation of immune
cells might have very interesting and far-reaching practical
applications in the treatment of sepsis caused by gram-negative
bacteria. In 1994 Proctor and colleagues115 showed that
the ATP analogue, 2-methylthio-ATP (2-MeS-ATP), inhibited
endotoxin-stimulated release of toxic mediators such as TNF- Stimulation with extracellular nucleotides also switches on the
inducible nitric oxide synthase (iNOS),116-118 a key
enzyme for the bactericidal activity of macrophages. Nucleotides per se
are ineffective, but coexposure to low doses of ATP (or UTP) and LPS produces a much higher stimulation of iNOS activity compared with LPS
alone. In murine Raw 264.7 macrophages a prolonged (18 hours) incubation was needed to elicit nitrite release, suggesting that P2
stimulation acted by increasing iNOS gene expression rather than by
increasing enzyme activity. Other data suggest that P2 receptors are
involved in NO generation in a rather more complex fashion. Denlinger
and coworkers showed that pretreatment with 2-MeS-ATP prevented iNOS
expression and NO generation due to the subsequent addition of
LPS,117 raising the issue of the possible participation of
P2 receptors in LPS-dependent signaling.116,117 In
addition, it has been recently shown that NO production due to
Mycobacterium tuberculosis infection also occurs in
P2X7 knockout mice and it is inhibited by P2
blockers,119 thus pointing to the participation of other
P2X and P2Y receptors. There are an increasing number of papers
suggesting that P2 receptors (namely P2X7) might have a
role in endotoxin- or parasite-mediated macrophage stimulation. Besides
the studies carried out in our laboratory showing that incubation of
macrophages or microglia with oATP or apyrase inhibited LPS-dependent
IL-1 A common event observed in many reactions involving mononuclear
phagocytes is multinucleation: often during chronic inflammatory reactions macrophages differentiate into epithelioid cells that eventually fuse into large polykarions named multinucleated giant cells
(MGCs).120 Furthermore, in the bone, osteoclast precursors normally fuse to generate large elements with increasing bone resorption activity. MGCs are a common finding of widespread infectious diseases such as tuberculosis, but little is known about the molecular mechanism underlying fusion. In 1995, Falzoni et al66
suggested that the P2X7 receptor could be involved in MGC
formation. Monocyte-derived human macrophages can be induced to fuse in
vitro by incubation with concanavalin A or phytohemagglutinin, provided
that contaminating lymphocytes are also present.121
Pretreatment with oATP fully inhibits this process, although other
responses such as concanavalin A-dependent
[Ca++]i changes, chemotaxis, or expression of
plasma membrane molecules thought to take part in cell fusion (eg,
CD11a, CD18, and CD54) are unaffected.66 We have extended
these studies to J774 macrophages and selected several clones that
either express P2X7 to a very high level
(P2X7plus) or lack it altogether (P2X7less).
P2X7plus cells spontaneously fuse in culture to form MGCs
of different size and shape, containing from a few to 20 or more
nuclei.77 A monoclonal antibody raised against the
P2X7 outer domain prevents fusion of human macrophages in
culture.122,123
The participation in ICE activation and IL-1 That extracellular ATP is a potent cytotoxic factor for macrophages was
immediately apparent as soon as a thorough investigation of ATP
receptors was started in these cells, and P2X7 was quickly identified as the culprit. Initially in Silverstein's and later in our
laboratory, murine macrophage clones were selected that showed an
almost absolute refractoriness to ATP-mediated
cytotoxicity.60,75,76,95 These cells showed a normal
mobilization of Ca++ from intracellular stores in response
to ATP, but no permeabilization of the plasma membrane, and accordingly
lacked reactivity to anti-P2X7 antibodies.77</ |
Top
Abstract
Introduction
and inhibition of adenylate
cyclase via Gq/11 and Gi proteins,
respectively.
originally cloned and
characterized in excitable cells
) or tetra-anionic (ATP4
, 
and tumor
necrosis factor (TNF)-
B and
mitogen-associated protein kinase (MAPK) activation are profoundly
inhibited by oATP or by PPADS.