Purinergic Signalling (2007) 3:317–331

DOI 10.1007/s11302-007-9074-y

ORIGINAL PAPER

New insights into purinergic receptor signaling in neuronal

Received: 16 July 2007 / Accepted: 9 August 2007 / Published online: 12 September 2007
# Springer Science + Business Media B.V. 2007

Abstract Ionotropic P2X and metabotropic P2Y purinergic Keywords ATP . knockout animal . neural stem cells .
receptors are expressed in the central nervous system and neurotransmitter . P19 embryonal carcinoma cells
participate in the synaptic process particularly associated
with acetylcholine, GABA, and glutamate neurotransmis- Abbreviations
sion. As a result of activation, the P2 receptors promote the ACh acetylcholine
elevation of free intracellular calcium concentration as the AD Alzheimer’s disease
main signaling pathway. Purinergic signaling is present in AP alkaline phosphatase
early stages of embryogenesis and is involved in processes ax-2 ataxin-2
of cell proliferation, migration, and differentiation. The use cdks cyclin-dependent kinases
of new techniques such as knockout animals, in vitro CNS central nervous system
models of neuronal differentiation, antisense oligonucleo- DRG dorsal root ganglia
tides to induce downregulation of purinergic receptor gene EC embryonal carcinoma
expression, and the development of selective inhibitors for NTPDase2 ectonucleoside triphosphate
purinergic receptor subtypes contribute to the comprehen- diphosphohydrolase 2
sion of the role of purinergic signaling during neurogenesis. E-NPP ectonucleotide pyrophosphatase
In this review, we shall discuss the participation of phosphodiesterase
purinergic receptors in developmental processes and in EGF epidermal growth factor
brain physiology, including neuron-glia interactions and E-NTPDase ectonucleoside triphosphate
pathophysiology. diphosphohydrolase
ES cells embryonic stem cells
P. Majumder : C. A. Trujillo : C. G. Lopes : K. N. Gomes :
E-5′-NT ecto-5′-nucleotidase
H. Ulrich (*) FGF-2 fibroblast growth factor 2
Departamento de Bioquímica, Instituto de Química, GABA γ-aminobutyric acid
Universidade de São Paulo, GFAP glial fibrillary acidic protein
Av. Prof. Lineu Prestes 748,
IFN-γ interferon-γ
05508-900 São Paulo, SP, Brazil
e-mail: henning@iq.usp.br LIF leukemia inhibitory factor
LTP long-term potentiation
R. R. Resende : L. R. G. Britto MAP-2 microtubule-associated protein-2
Departamento de Fisiologia e Biofísica,
MAPK mitogen-activated protein kinase
Instituto de Ciências Biomédicas,
Universidade de São Paulo, MRF microglial response factor
São Paulo 05508-900 SP, Brazil MRS 2179 2′-deoxy-N6-methyladenosine 3′,
5′-bisphosphate
K. K. Yuahasi
NPC neural progenitor cells
Departamento de Neurologia Clínica,
Universidade Federal de São Paulo, NPC neural stem cells
São Paulo 04023-900 SP, Brazil NA noradrenaline
318 Purinergic Signalling (2007) 3:317–331

NOS nitric oxide synthase The expression of purinergic receptors has been identi-
NTPDase ectonucleoside triphosphate fied during development and differentiation processes [5–
diphosphohydrolase 10]. Nucleotides exert a synergic effect on cell proliferation
NGF nerve growth factor in association with growth factors, chemokines, or cyto-
OP oligodendrocyte kines in early stages of development [11–13] by parallel
oxATP oxidized ATP activation of the MAP kinase pathway and/or by trans-
PPADS pyridoxalphosphate-6-azophenyl-2′, activation of growth factor receptors [14, 15].
4′-disulfonic acid The complete role of ATP action in developmental
SVZ subventricular zone processes still needs to be elucidated. It is known that
TGF transforming growth factor ATP activates purinergic receptors resulting in many cases
TNF-α tumor necrosis factor-α in increases of intracellular free calcium concentration
GAP-43 growth-associated protein 43 [Ca2+]i. Changes in [Ca2+]i are involved in several events
TNAP tissue-nonspecific alkaline phosphatase of differentiation and the embryogenesis process [16, 17].
TNP-ATP 2′3′-O-(2,4,6-trinitrophenyl) adenosine Spitzer et al. [18] showed that naturally occurring patterns
5′-triphosphate of Ca2+ transients encoded neuronal differentiation. Distinct
frequency patterns of [Ca2+]i elevations were sufficient to
promote neuronal differentiation, including physiological
neurotransmitter receptors expression [19]. ATP and UTP
Introduction are the main purinergic agonists activating P2X or P2Y
receptors. These nucleotides can be rapidly degraded in the
During the last two decades, evidence for the participation of extracellular space by ectoenzymes to ADP or UDP,
ATP as neurotransmitter in neuronal signaling was collected subsequently activating distinct P2Y receptors, or be finally
by Drs. Surprenant [1] and Silinsky [2]. Purine-sensitive degraded to adenosine, which is known to induce physio-
receptors were first classified as P1 G-coupled receptors logical responses via activation of P1 G protein-coupled
which are activated by adenosine and P2 receptors, receptors [20] (Fig. 1).
responding to stimulation of ATP [3]. Based on receptor In this review article, we shall discuss the roles of
cloning and studying of receptor-induced signal transduc- purinergic signaling in neurogenesis such as cell cycle
tion, P2 receptors were divided into P2X receptors as ATP- control during neural progenitor proliferation and differen-
gated ion channels and P2Y G protein-coupled receptors [4]. tiation as well as in maintaining physiology of neurons and

Fig. 1 Purine-induced signaling

glial cells and the involvement of purinergic receptors in The neuronal differentiation of EC cells, originated from
pathophysiology. In addition, we shall outline state-of-the- irradiated embryo cells [36], also resembles early neuronal
art approaches used in investigation of P2 receptor function development in vivo. P19 mouse EC cells express stem
in physiological processes such as the use of antisense cell-specific marker proteins and their phenotypic changes
oligonucleotides, generation of knockout animals, and in specific differentiation stages are similar to those of stem
identification of new purinergic receptor subtype-selective cells [37]. Recently, our laboratory [38] has determined
drugs. gene and protein expression of P2 receptor subtypes
throughout in vitro neuronal differentiation of P19 cells as
well as in the undifferentiated cell stage suggesting the
Study of purinergic receptor function during in vitro participation of purinergic signaling in initiating and
differentiation directing differentiation. Differential expression and activity
of P2Y1, P2Y2, P2Y4, P2X2 subtypes and P2X6 subunits
During the development of the mammalian nervous system, were reported during neuronal maturation of P19 cells [38,
neural stem cells and their derivative progenitor cells 39]. As direct evidence for participation of purinergic
generate neurons by asymmetric and symmetric divisions receptors in neuronal differentiation, the presence of the
[21]. P2 receptors were shown to be one of the first antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disul-
functionally active membrane receptors in chick embryo cells fonic acid (PPADS), reactive blue 2, or suramin during
during gastrulation, in which ATP caused rapid accumulation differentiation of P19 neural progenitor cells (NPC) to P19
of inositol triphosphate and Ca2+ mobilization in a similar neurons resulted in reduced activity of cholinergic and
way as acetylcholine (Ach) did via activation of muscarinic glutamate NMDA receptors in differentiated P19 cells,
acetylcholine receptors, whereas other endocrine-acting pointing at a participation of P2Y1, P2Y2, and P2X2
substances such as insulin and noradrenaline (NA) induced receptors.
much weaker effects in terms of intracellular calcium Other in vitro neuronal and glial differentiation models
signaling [22, 23]. The induction of transient fluctuation in used to understand the purinergic signaling are neural stem
[Ca2+]i also denominated as calcium wave signaling allows cells or progenitor cells which are isolated from the
for a coupling of spatial and temporal information. Thus, subventricular region (SVZ) located in the lateral ventricles
calcium waves have been proposed to play a role in mapping (type B cells) or in the subgranular region of the gyrus
of neuronal networks [24] and to modulate neurogenesis dentatus of the hippocampus (residual radial glia) or even
during embryonic cortical development [25]. from the subcortical parenchyma of the cerebral cortex of
Neurotransmitters are prominent candidates for trans- embryonic and adult brain [40–42]. These regions in the
cellular signals that could influence the development of adult brain act as neural stem cell reservoirs. These cells are
embryonic neurons as they surround neural cells throughout already advanced in their differentiation stage when com-
brain development [26–29]. In addition, functional ligand- pared to ES or EC cells. Since NSC and NPC are capable of
gated ionic channel receptors have been identified in neural differentiating in both functional neurons and glial cells, they
progenitor cells prior to establishing cortical and subcortical possess potential therapeutic applications such as ES cells in
synapses [30, 31]. In this context, the extracellular signaling regeneration therapy following neuronal loss.
mechanisms controlling the various transition steps in- These NPC differentiate into olfactory, cerebellar, and
volved in adult neurogenesis are still poorly understood. retinal neurons [40] in the presence of growth factors,
One approach used to identify the function of P2 receptors neurotransmitters, vasoactive peptides in vivo [43], and
during development and differentiation is the use of in vitro growth factors such as epidermal growth factor (EGF),
models for neuronal and glial differentiation such as fibroblast growth factor 2 (FGF-2), and leukemia inhibitory
embryonic and adult neural progenitor cells (NPC), also factor (LIF) in vitro. When exposed to a high concentration
known as neural stem cells (NSC), embryonic stem (ES), of FGF-2 in suspension, proliferating NPC form tridimen-
and embryonal carcinoma (EC) cells. sional cell aggregates denominated as neurospheres, which
ES cells are obtained from the inner mass cell of the following induction to differentiation express neuronal
blastocyst. The differentiation of these cells closely resem- marker proteins such as β-III-tubulin, microtubule-
bles the in vivo process and, therefore, provides stable associated protein-2 (MAP-2), and synaptophysin [44] and
models for embryonic growth and development [32, 33]. express P2X3 and P2X7 receptors which may contribute to
ATP promotes cell proliferation acting through P2X3, P2X4, early [Ca2+]i transients as prerequisites for further differen-
P2Y1, and P2Y2 receptors in murine ES cells [34]. Tissue- tiation [41]. Shukla et al. [45] identified functional P2
nonspecific alkaline phosphatase (TNAP) was also detected receptors in adult mouse hippocampal progenitors in situ
in these cells and used as a marker for their undifferentiated and the nucleoside triphosphate-hydrolyzing ectoenzyme
stage [35]. (NTPDase) in type B cells of the SVZ [46] and in
320 Purinergic Signalling (2007) 3:317–331

hippocampal progenitor cells. In adult murine NPC of SVZ, heteromeric receptors were formed by P2X2/3 receptor
P2Y1 receptor activity mainly contributes to [Ca2+]i subunits. P2X3 receptor immunoreactivity was detected in
transients with some participation of P2Y2 receptors. The cranial motor neurons as early as on E11, when neurons
presence of the specific P2Y1 receptor antagonist MRS exited the cell cycle and started axon outgrowth, as well as
2179 resulted in diminished cell proliferation in neuro- postnatally on days 7 and 14 (P7 and P14) [56, 60].
spheres due to reductions of [Ca2+]i transients. Similar Moreover, expression of P2X3-containing heteromeric
results were obtained with NPC from SVZ of P2Y1 receptor receptors and other subunits was developmentally regulated
knockout mice [47]. P2Y1 receptor-deficient mice are in nucleus ambiguous motoneurons [61]. From E14
viable; however, they have deficits in platelet aggregation onwards P2X7 receptors were also expressed in the
[48]. It is suggested that the purine signaling underlies embryonic brain. For instance, in primary cultures of
autocrine or paracrine mechanisms and P2Y1 and P2Y2 human fetal astrocytes basal levels of P2X7 receptor mRNA
receptors are important for NPC differentiation [47]. These transcription and protein expression were detected [62].
models are useful tools to study the roles of P2 receptor Sperlágh et al. [63] have demonstrated that ATP regulates
signaling in early stages of development and differentiation. glutamate release via activation of P2X7 receptors. P2X7
The importance of ATP release and purinergic signaling has receptor-induced excessive glutamate release alters Ca2+
not only been demonstrated in developmental progenitor homeostasis, subsequently resulting in activation of the
cell expansion and neurogenesis, but also in persistent apoptosis-related caspase cascade [64].
progenitor cells of the adult brain [49]. P2X receptor expression was downregulated in Purkinje
cells and deep cerebellar nuclei at P21 and P66 rat
Expression of purinergic receptors during development embryonic stages, with the exception of P2X5 receptors
of the central nervous system whose immunoreactivity in granular cells was increased
[65]. Evidence for participation of P2X receptors in
Purinergic signaling pathways are also involved in embry- different developmental processes such as neurite out-
onic neurogenesis in much the same way as already growth (involving P2X3 receptors), postnatal neurogenesis
discussed for in vitro differentiation models. ATP mediates (related to P2X4 and P2X5 receptor expression), and cell
elevation of [Ca2+]i and proliferation of immortalized human death (possibly involving P2X7 receptors) was collected.
stem cells from embryonic telencephalon and mouse However, P2X1 and P2X6 receptor subunits may not play a
embryonic neurospheres [50, 51]. Ca2+ waves through radial role in neuronal development [58].
glial cells in slices of the embryonic rat ventricular zone are Neocortical neurons from 2-week-old rats possess a
mediated by P2Y1 receptors. Disrupting Ca2+ waves between quite elaborated purine-triggered signaling system which
embryonic NPC reduced ventricular zone cell proliferation includes both P2Y and P2X receptor activation [66].
during the peak of embryonic neurogenesis [25]. Weissman et al. [25] showed that [Ca2+]i waves and
ATP directly contributes to modulate network-driven subsequent ATP release, with consequent P2Y1 receptor
giant depolarizing potentials in the rat hippocampus during activation, accompanied radial glial cell-derived neuro-
early stages of postnatal development [52]. In the developing genesis in cultured slices of the developing rat forebrain,
hippocampal system a trophic role of ATP and the as mentioned above. Moreover, the importance of calcium
involvement of P2 receptor subtypes in shaping interneuro- signaling for differentiation of NPC has been studied [67,
nal connections during neuronal differentiation have been 68], and direct evidence for the participation of P2Y1
suggested [53]. Alterations of the regulation of embryonic receptor-activated pathways in the early development has
growth by purinergic receptors might be involved in the been provided by Scemes et al. [69]. P2Y receptors
onset of morphological malformations [54]. During rat (particularly the P2Y1 subtype) were widely expressed in
postnatal development ectonucleotidase activity in the the embryonic rat brain as early as on E11 [57]. There was
cerebral cortex steadily increases, reaching maximum values a marked decrease in the concentration of mRNA coding
at 21 days of age [55]. Several P2Y and P2X receptors were for P2Y1 receptors and upregulation of mRNA transcription
shown to be dynamically expressed in the pre- and postnatal coding for P2Y2 receptors in freshly isolated astrocytes of
central and peripheral nervous system [56–59]. ATP developing rat hippocampus [57].
inhibited motor axon outgrowth during early embryonic
neurogenesis, most likely through the P2X3 receptor, and it Functional interactions between neurons and glia:
was speculated that P2X7 receptors might be involved in a physiological overview
programmed cell death during embryogenesis [58].
From all of the studied P2X receptors, homomeric P2X2 An increasing amount of evidence, initiated by the neuron-
receptors were the first expressed in the rat central nervous glia unit idea proposed by Hyden [70], indicates that glial
system (CNS) on embryonic day 14 (E14) [56]. On E14, cells, once referred to as a simple support portion in the
Purinergic Signalling (2007) 3:317–331 321

CNS, are now considered indispensable functional partners rate, instability and low concentration in the extracellular
of neurons [71], both in physiological and pathological environment, and impossibility to cross the plasma mem-
conditions. However, many questions remain unanswered: brane [92, 93]. These properties imply the presence of
(1) how glia detects and interacts with neural function; (2) particular pathways for ATP release that could be associated
does neuron-glia signaling play a significant role in with cellular excitation/response and cell-cell signaling [94,
synaptic transmission and plasticity; and (3) how glial cells 95]. First, ATP may be stored in synaptic vesicles alone or
can communicate with other glial cells. with other neurotransmitters and then released, as a classic
Another important subject related to the interaction synaptic mechanism in the peripheral or central nervous
between glia and neurons emerges in neurogenesis. There system [96, 97]. Second, a nonvesicular mechanism of ATP
is now a general agreement that the adult mammalian release could be observed through gap junction hemi-
nervous system possesses many characteristics of astro- channels, ATP-binding cassette proteins, P2X7 receptor
cytes. The importance of glia in neuronal development was pores in glial cells, and via chloride channels [98–101].
confirmed in a recent study showing that the number of Third, ATP could be released due to cytolysis or cell
GFAP (glial fibrillary acidic protein)-containing cells was damage. While this is not a physiological mechanism, it
reduced following transgenic targeting of adult mouse takes place following biological trauma and contributes to
subependymal and subgranular zones, resulting in an pathological conditions [102].
almost complete loss of neurogenesis [72, 73]. In addition Subsequent to these mechanisms, the metabolism of the
to assisting migration of neurons to their correct position released ATP is regulated by a vast number of different
and managing neurite outgrowth to their final communica- families of ectonucleotidases in the synaptic cleft, including
tion targets [74, 75], glial cells have become an essential the ectonucleoside triphosphate diphosphohydrolase
key for understanding neuronal differentiation by promot- (E-NTPDase) and the ectonucleotide pyrophosphatase
ing initial stem cell proliferation and instructing undiffer- phosphodiesterase (E-NPP) which catalyze the degradation
entiated cells to adopt a neuronal fate [76, 77]. of ATP to ADP or AMP. The degradation to adenosine is
In the mature brain, the proximity of astrocytes to mediated by ecto-5′-nucleotidase (E-5′-NT) and alkaline
neuronal synapses or to the blood-brain barrier makes these phosphatase [91, 103] (Fig. 1). Consequently, the reaction
cells appropriate to control water diffusion and ion products resulting from the ATP hydrolysis may bind to P2
concentration in extracellular spaces [71, 78]. In particular, receptors, in the case of ADP, or to P1 receptors in the case
astrocytes regulate homeostatic environment and neuro- of adenosine [104].
transmitter levels by functional syncytium, in which gap The adenosinergic receptor ligand adenosine is recog-
junctions and specific membrane carriers play an important nized as an important regulator of cellular homeostasis in
role [79–81]. In addition, glial cells produce and release a the CNS and may be involved in the prevention or
vast number of neurotrophins, including fibroblast growth induction of apoptosis [105]. The reduction of ectonucleo-
factor, nerve growth factor, and transforming growth factor, tidase activity in certain pathological conditions provided
which directly interfere in neuron physiology and coordi- additional evidence for the accumulation of ATP in the
nate developmental processes [71, 82–85]. extracellular environment [20]. Therefore, the complexity
of the communication of neural and nonneural cells
ATP release and degradation, connecting adenosinergic expands the functional significance by the interaction of
and purinergic systems the purinergic receptors in association with a variety of
neurotransmitter systems.
As already mentioned, it is well documented that glial cells
may directly alter neuronal activity by releasing neuro- ATP-mediated neuron-glia signaling
trophins and consequently modulating neurotransmitter
release in the synapse [86, 87]. One of the main Novel studies in the purinergic field began to converge with
mechanisms connecting the neuron-glia system is believed glial research as it became more widely accepted that ATP
to be mediated by the release of glutamate from glial cells is released through synaptic vesicles and thus accessible to
[88, 89]. In this context, growing evidence indicates that perisynaptic glial cells, allowing them to detect neuronal
purinergic receptor ligands are widely involved in the cell- activity. In particular, glial cells are responsive to ATP, as
cell signaling mechanism by acting as neurotransmitters or all types of glia, such as astrocytes, oligodendrocytes,
neuromodulators released by glial cells to control synaptic microglia, and Schwann cells, express purinergic receptors
transmission in the CNS, as part of multiple functions of [91]. In Schwann cells and oligodendrocytes, ATP-mediated
astrocytes [22, 90, 91] (Fig. 1). signaling predominantly occurs through P2Y receptors,
ATP is an ideal molecule for cell signaling due to its which in turn trigger intracellular Ca2+ release [106, 107].
intrinsic properties such as its small size, diffusing molecule However, the function of P2X1–6 receptors in astrocytes
322 Purinergic Signalling (2007) 3:317–331

remains unclear, although P2X-mediated currents could be AMP alone do not elicit neurite extensions in PC12 cells
detected in astrocyte cells in culture, and P2X7 receptors are [124].
widespread in these cells with possible contribution to Heine et al. [53] demonstrated that P2 receptor activation
pathological conditions [108, 109]. induced fiber outgrowth in organotypic cocultures in rat
Glial cells express many types of neurotransmitter hippocampus. Fiber outgrowth was inhibited in the pres-
receptors and conventionally are considered to be non- ence of the purinergic antagonist PPADS, suggesting the
excitable [110, 111]. However, a surprising observation was involvement of P2 receptors. In another study, the syner-
reported by Dani et al. [112] that synaptic transmission may gistic interaction between bFGF and ATP was reported on
propagate to glial cells as calcium waves, inducing DNA synthesis in primary cultures of rat cortical astrocytes.
membrane depolarization and regulating neurotransmitter ATP and bFGF induced a twofold and tenfold incorporation
release. These properties of glial cells suggest possible of [3H]thymidine into astrocytes, respectively, but when
rapid communications between neurons and glia during ATP and bFGF were added at the same time a 50-fold
synaptic transmission. This glial communication mecha- increase in [3H]thymidine incorporation was observed [12].
nism allows the released ATP to act onto adjacent
astrocytes and neurons, thus supporting the propagation of Neuroprotection
Ca2+ waves in glial syncytium [113]. For example, in
neuronal-glial cocultures prepared from hippocampus, ATP ATP can activate P2X7 receptors in astrocytes to release
secreted by astrocytes was shown to inhibit glutamatergic glutamate, GABA, and also ATP which might regulate the
synapses through activation of P2Y receptors [114]. excitability of neurons in certain pathological conditions
The glial communication mechanism based on Ca2+ [125]. It has been suggested that astrocytes can sense the
wave propagation could be inhibited by P2 receptor severity of damage in the CNS by the amount of ATP
blockers or enzymes that rapidly hydrolyze extracellular released from damaged cells and that extracellular ATP
ATP [115]. A stimulation applied to a single astrocyte in concentration and the corresponding subtype of activated
cocultures of rat forebrain astrocytes and associated astrocytic P2 receptor modulate the tumor necrosis factor-α
neurons caused an elevation of [Ca2+]i and induced Ca2+ (TNF-α)-mediated inflammatory response [126]. After
wave propagation in dorsal spinal cord through P2Y1 mechanical brain injury, the administration of PPADS
receptor activation [116] and glutamate release [117]. This facilitated the recovery of pathologically changed elec-
new finding provided a parallel mechanism of intercellular troencephalograms [127]. These results suggest that
communication that could allow astrocytes to detect interference with the ATP-induced excitatory responses
synaptic function, propagate the information through could provide neuroprotection and possible therapeutic
neighboring glial cells, and then influence synaptic function consequences.
in a distant part of the nervous system. Evidence for a neuroprotective role was also found for
Purinergic receptor-calcium signaling in glial cells plays the adenosine A1 receptor in hippocampus. This cerebral
important roles during CNS development. P2X1, P2X4, and region is highly sensitive to hypoxia and ischemia. The
P2X7 receptors were expressed in microglia at rat embry- study of the action of hypoxia on synaptic transmission in
onic stage (E16) [59]. Moreover, changes in P2X4 receptor hippocampal slices has suggested that substances being
expression in microglial cells during postnatal development released during hypoxia, such as GABA, ACh, and even
of the rat cerebellum have been reported. P2X5 receptor glutamate, may also play neuroprotective roles. However,
immunoreactivity was also upregulated in microglia and the actions of these neurotransmitters become evident only
granular cells. Both P1 and P2 receptors contribute to the when activation of P1 receptors is impaired, suggesting a
modulation of oligodendrocyte (OP) development, since critical role for this receptor during hypoxic events. These
they have been shown to exert similar effects on OP substances can operate in a redundant or even overprotec-
proliferation and differentiation [118]. tive manner, acting as a substitute for some adenosine
The majority of the studies of ATP action have been actions when the nucleoside is not operative [128].
concerned with the short-term P2 receptor signaling that
occurs in neurotransmission and in secretion [119]. Fur- Neuroimmune interactions
thermore, there is increasing evidence that purines and
pyrimidines can have trophic roles in neuritogenesis [120, Microglia, the immune cells of the CNS, can be activated
121], regeneration [122], and proliferation [123]. However, by purines and pyrimidines to release inflammatory
some purines by themselves have limited trophic effects in cytokines such as IL-1, IL-6, and TNF-α. However,
a few types of cells; they appear to be much more effective hyperstimulation of the immune reaction in the brain may
as neuritogenic agents when they are combined with other accelerate neuronal damage. The P2X7 receptor is consid-
trophic factors, such as NGF. For instance, inosine and 5′ ered to have a potentially pivotal role in the regulation of
Purinergic Signalling (2007) 3:317–331 323

various inflammatory conditions. ATP selectively sup- ipate in neuronal transmission accompanied by GABA-
presses the synthesis of the inflammatory protein microglial mediated actions [139].
response factor through calcium influx via P2X7 receptors
in microglia [129], which also leads to enhancement of Pain The heteromeric channel comprised of P2X2 and
interferon-γ (IFN-γ)-induced type II nitric oxide synthase P2X3 subunits was expressed almost exclusively in a subset
(NOS) activity [130, 131]. P2X7 receptor activity also of primary afferents implicated in nociception [143–145]. It
participated in ATP-induced IL-1 release from macrophages has been observed that mechanical allodynia is reduced in
and microglia that had been primed with substances such as mice with deleted P2X3 receptor genes [146, 147] in
bacterial endotoxin [132] and was shown to stimulate the agreement with data obtained in rats that have been treated
transcription of nuclear factor κB, TNF-α [133], the stress- with intrathecal antisense oligonucleotides reducing expres-
activated protein kinases (SAPK)/JNK pathway [134], and sion of P2X3 receptors [148] or with the selective
the production of 2-arachidonoylglycerol, which is also antagonist for P2X3 and P2X2/3 receptors A-317491 [148,
involved in inflammation induction by microglial cells. 149]. P2X3 receptor knockout mice showed additional
P2Y rather than P2X7 receptors seem to have a major defects in afferent pathways.
role in the IL-6 production by microglial cells [135]. ATP The P2X4 receptor is also implicated in pain sensation.
evoked the release of plasminogen [136] and IL-6 [135]. Activation of dorsal horn microglia and tactile allodynia
The stimulation of microglia by either ATP or BzATP developing several days after ligation of a spinal nerve were
revealed neurotoxic properties and the involvement of the greatly reduced when gene expression of P2X4 receptor in
P2X7 receptor has been reported in excitotoxic/necrotic and the dorsal horn had been inhibited by the presence of
apoptotic degeneration [109]. intrathecal antisense oligonucleotides [150]. Accordingly,
intraspinal administration of microglia following induction
Neurological disorders of expression and activity of P2X4 receptors produced
tactile allodynia in naive rats. Intrathecal administration of
Epilepsy Several anti-epileptic agents reduce the ability of cultured brain microglia produced allodynia, but only when
astrocytes to transmit Ca2+ waves, raising the possibility the cells had been pretreated with ATP [150]. The inhibition
that blockade of ATP-induced [Ca2+]i transients in astro- of P2X4 receptor activity in microglia might be a new
cytes by purinergic receptor antagonists could offer new therapeutic strategy for pain induced by nerve injury.
treatments for epileptic disorders. Antiepileptic effects of
adenosine are mostly due to the well-known inhibitory Alzheimer’s disease Alzheimer’s disease (AD) is caused by
actions of P1 receptors on synaptic transmission in the extracellular deposition of amyloid β-peptide, which can
hippocampus. However, as recently pointed out, adenosine damage neurons, leading to their dysfunction and death
actions are not limited to presynaptic actions on glutamate [151]. ATP and, in particular, aluminum-ATP promoted the
release [137]. The intraventricular injection of high doses of formation of thioflavin T-reactive fibrils of β-amyloid and
ATP in rats evoked severe chronic-tonic convulsions, an unrelated amyloidogenic peptide, which could be
whereas lower doses of ATP or adenosine elicited a kinetic blocked by suramin [152].
state with muscle weakness [138]. P2X2 and P2X4 receptor Microglial cells are believed to contribute to the
expression in the hippocampus of seizure-prone gerbils was progression of AD and are known to release proinflamma-
significantly reduced compared with that of normal gerbils tory neurotoxic substances. Extracellular ATP, acting
[139]. GABAA receptors mediated modulation of expres- through the P2X7 receptor, can alter β-amyloid peptide-
sion of both P2X2 and P2X4 receptors, which may play an induced cytokine secretion from human macrophages and
important role in the regulation of seizure activity in the microglia and thus may be an important modulator of
gerbil hippocampus [139]. P2X7 receptors are thought to neuroinflammation in AD [153]. P2X7 receptors mediate
play a definite, but not yet well defined role in epilepsy. superoxide production in primary microglia, and the
Treatment with the GABAB receptor agonist baclofen and expression of this receptor subtype was specifically
antagonist phaclofen resulted in increased and decreased upregulated around β-amyloid plaques in a transgenic
P2X7 receptor expression in hippocampus, respectively mouse model of AD [154].
[140]. These purinergic receptor responses were interpreted In contrast to the control human brain, the P2Y1 receptor
as compensatory responses to the modulation of GABAB was colocalized with a number of characteristic AD
receptor function [140]. It is noteworthy to mention that structures such as neurofibrillary tangles, neuritic plaques,
this positive relationship between P2X and GABAA and neuropil threads in the hippocampus and cortex [155].
receptors was also reported for the spinal cord [141] and In general, control brain tissue exhibited a greater and more
dorsal root ganglia (DRG) [142]. In these populations of abundant level of P2Y1 receptor immunostaining than AD
neurons, ATP-mediated P2X receptor function may partic- tissue did, probably due to severe neuronal cell degenera-
324 Purinergic Signalling (2007) 3:317–331

tion in most AD brains. The intense P2Y1 receptor staining antagonists (PPADS, oxATP) after acute impact injury
observed over pathological AD structures might imply that significantly improved functional recovery and diminished
this receptor is involved either directly or indirectly in cell death in the peritraumatic zone [168]. The involvement
signaling events mediating neurodegeneration of pyramidal of P2X1 and P2X2 receptors in neuronal reactions after
cells. Alternatively, P2Y1 receptors might have other hemicerebellectomy was also described [169]. Furthermore,
diverse signaling roles, possibly involved in the production neuronal NOS and P2 receptors were colocalized and
of intracellular tau deposits or might even serve to stabilize showed temporal coactivation after cerebellar lesions,
these tangle structures in some way [156]. indicating a close relationship between these two systems
[166]. In addition, in this mixed model of differentiation
Ischemia/hypoxia Under pathological conditions of hypox- and axotomy, the colocalization of ataxin-2 (ax-2, involved
ia or ischemia, extracellular purine nucleotides leak from in resistance to degeneration phenomena, which may be
damaged cells and thereby may reach high concentrations lost after mutation)-immunopositive cells and P2X2 recep-
in the extracellular space [157]. A direct participation of tors was demonstrated in neurons, and post-lesional
extracellular ATP and P2 receptors in ischemic stress has induction of P2X1 receptor and ax-2 immunoreactivity
been reported in various cellular systems [157–160]. For was reported as well [170]. In vivo treatment of P2Y2
example, P2X2 and P2X4 receptor expression in neurons receptor-expressing sciatic nerves with ATP-γS increased
and microglia, respectively, in the hippocampus of gerbils expression levels of the growth-associated protein 43 (GAP-
was upregulated following transient global ischemia [161]. 43) as a marker for axonal growth in wild-type but not in
Increased P2X7 receptor expression in astrocytes, micro- P2Y2 −/− mice [171].
glia, and neurons appears to contribute to the mechanisms Possible therapeutic manipulations to modulate astrocyt-
of cell death caused by in vivo and in vitro ischemia [162, ic proliferation and to diminish glial scar formation in the
163]. Following induction of ischemia P2X7 receptor adult brain and during development include the use of
mRNA transcription and protein expression were elevated drugs known to interfere with nucleotide synthesis. Pekovic
in cultured cerebellar granule neurons and organotypic et al. [172] showed that treatment with the purine
hippocampal cultures [163]. Hence, the P2X7 receptor is nucleoside analogue ribavirin (Virazole; 1-β-D-ribofurano-
apparently an important element in the mechanisms of syl-1,2,4-triazole-3-carboxamide) downregulates the pro-
cellular damage induced by hypoxia/ischemia. In many cell cess of reactive gliosis after sensory motor cortex lesion of
types, the activation of the P2X7 receptor led to rapid the adult brain and facilitates re-establishing synaptic
cytoskeletal rearrangements, such as membrane blebbing connections with the denervated cells at the lesion site.
and cell lysis [164]. P2Y1 receptors are intensely expressed This may be a useful approach for improving neurological
in Purkinje cells in deep layers of the cerebral cortex and in recovery from brain damage. The antiproliferative effect of
ischemia-sensitive areas of the hippocampus [165]. In ribavirin is due to the inhibition of de novo nucleic acid
conclusion, extensive evidence demonstrates a postische- synthesis after depletion of GTP and dGTP pools with
mic time- and region-dependent upregulation of P2X2,4,7 consequent impairment of specific transduction pathways.
and P2Y1 receptor subtypes in neurons and glial cells and
suggests a direct role of P2 receptors in the pathophysiol-
ogy of cerebral ischemia in vitro and in vivo. ATP-induced effects on cell cycle progression

Trauma and axotomy P2 receptors are suggested to be There is evidence showing that extracellular ATP enhances
involved in neuronal reactions after axotomy. Colocaliza- the expression of cell cycle regulating proteins [173, 174].
tion and temporal coactivation of purinergic and nitrergic Progression of the cell cycle is highly controlled. Cyclins
markers support this idea, indicating possible interactions are synthesized and degraded in a synchronous way due to
between these two systems [166]. Following peripheral changing transcription or proteolysis rates, thereby direct-
nerve lesions, P2X3 receptor expression in DRG neurons ing the periods of the cellular cycle. Cyclins interact with
was changed [167]. The increased expression of P2X3 cyclin-dependent kinases (cdks) resulting in activation of
receptor mRNA in intact neurons indicates a role of this their kinase activity, phosphorylating their targets and
subtype in the post-injury pathomechanism in primary themselves, and regulating the specific progression of the
sensory neurons [167]. After spinal cord injury, large cell cycle through checkpoints [175].
regions of the peritraumatic zone were characterized by a Proliferation rates in mammalians are largely determined
sustained process of pathologically high ATP release [168]. during the G1 phase of the cell cycle. The relevant proteins
Spinal cord neurons express P2X7 receptors, and exposure include three D-type cyclins (D1, D2, and D3) that, in
to ATP led to high-frequency spiking, irreversible increases different combinations, bind to and allosterically regulate one
in [Ca2+]i and cell death. The administration of P2 receptor of two cdk subunits, cdk4 and cdk6, as well as the E-type
Purinergic Signalling (2007) 3:317–331 325

cyclins (E1 and E2), which govern the activity of a single It is documented in the literature that purinergic receptor
catalytic subunit, cdk2 [176]. Various combinations of D- inhibitors interfere with the S phase of the cell cycle.
type cyclins are expressed in different cell types, whereas Neurospheres treated with the purinergic receptor antagonists
cyclin E-cdk2 complexes are ubiquitously expressed [177]. reactive blue 2 or suramin are mostly in S phase (5.7±0.3% or
Two families of cdk inhibitors regulate the activity of 8.4±2.3%) when compared to untreated control neurospheres
G1-type cyclins-cdks complexes: the Ink4 family (p16, with 16.4±1.8% of the cells being in S phase. Moreover,
p15, p18, and p19), which blocks the activity of cyclin D- neurosphere cultures treated with suramin or reactive blue 2
cdk4-6 complexes, and the Cip/Kip family (p21, p27, and showed an increase in the expression of the tumor suppressor
p57), which preferentially inhibits cyclin E-cdk2 complexes p27 as a strong regulator of cell division [49].
and also acts as a scaffold for the catalytically active cyclin The discussed findings led to the suggestion that
D-cdk4-6 complexes. In addition to cyclins and cdks, extracellular ATP plays an important physiological role
mitogen-activated protein kinase (MAPK) is also believed during mammalian embryonic development by stimulating
to have a role in induction of cell proliferation. Therefore, proliferation of ES cells, and therefore P2 receptor agonists
cyclin D-dependent kinases may play a role in controlling and antagonists might provide novel and powerful tools for
the cell cycle of embryonic and maybe neural progenitor modulating embryonic cell functions. In conclusion, P2X
cells. In addition MAPK is also believed to have a role in and P2Y purinergic receptors can promote proliferation of
induction of cell proliferation. Extracellular ATP induces ES cells as well as of progenitor cell types by a mechanism
Ca2+-dependent MAPK activation via stimulation of P2 by that ATP induces increases in [Ca2+]i, leading to
receptors in neonatal rat astrocytes [178]. On the other activation of PKC, PI3-kinase/Akt, p38, and p44/42
hand, cell proliferation is associated with activation of MAPK, followed by an alteration in the cdk-cyclin
diverse proteins. Positive regulators include cyclins and complex with p21 and p27, which are involved in
their partners with catalytic activity (cdks), which are stimulation of cell proliferation.
essential for progression of the cells through each phase
of the cell cycle and various cell cycle checkpoints [179, Pharmacological approaches
180]. The regulation of cyclin D1 expression is also
mediated by the Ras/ERK signaling pathways [181, 182]. Most purinergic receptors do not have specific inhibitors.
Raf/MEK/ERK and PI3-K/Akt signaling pathways can act Therefore, P2 receptor agonists and antagonists acting on
in synergy to promote the G1-S phase cell cycle progres- most of the purinergic receptor subtypes are widely used in
sion in both normal and cancer cells [183, 184]. The experimental approaches to study biological functions of
promoter for cyclin D1 contains an AP-1 site, and the these receptors. Such approaches are feasible, since these
ectopic expression of either c-fos or c-jun induces cyclin D1 compounds mostly have higher affinities to some P2
mRNA expression [185, 186]. In many cell types, phos- receptor subtypes than to other ones. As an example, we
phatidylinositol (PI)-3-kinase-dependent signaling path- have used suramin, PPADS, and reactive blue 2 to study the
ways also regulate cyclin D1 expression [187]. It was also participation of P2Y1, P2Y2, and P2X2 receptors in
reported that the control of the cell cycle regulatory proteins neuronal differentiation of P19 EC cells [38].
was dependent on PI3-kinase and p44/42 MAPK pathways, One possible approach towards a subtype-specific
indicating that extracellular ATP alone is sufficient to inhibitor would be based on results from P2 receptor
induce cell cycle progression beyond the G1 phase of the structure determination. Using site-directed mutagenesis it
cell cycle. These findings also suggest that, once P2 has been possible to understand which amino acids are
receptors are activated, protein kinase C (PKC) transmit involved in ATP binding and to identify allosteric sites in
signals to the nucleus through one or more of the MAPK purinergic receptors. The knowledge obtained on location
cascades, which may include Raf-1, MEK, and ERK, and and structural features of ligand and inhibitor binding sites
stimulate transcription factors such as myc, max, fos, and is used in rational based drug design of selective purinergic
jun. Moreover, MAPKs are upstream regulators of cdk2 subtype antagonists. Alternatively, combinatorial libraries
and cdk4 expression. It has been reported that p44/42 formed by vast amounts of possible ligands can be
MAPK phosphorylation is essential and sufficient for the employed for discovery of subtype-specific inhibitors.
increase in cdk2 [188, 189] and decrease in p27Kip1 A-317491 was identified as a specific inhibitor for
expression [190, 191]. However, Delmas et al. [192] P2X2/3 and P2X3 receptors. In the presence of A-317491
provided evidence that p44/p42 MAPK activation triggers both thermal hyperalgesia and mechanical allodynia were
p27Kip1 degradation independently from cdk2/cyclin E in attenuated after chronic nerve constriction injury in which
NIH 3T3 cells. As described above, ATP regulation of the P2X3 homomeric and P2X2/3 heteromeric receptor activi-
MAPK and cdk-cyclin complex has not been elucidated in ties were involved. Although active in chronic pain models,
other types of cells [193]. A-317491 was ineffective in reducing nociception in animal
326 Purinergic Signalling (2007) 3:317–331

models of acute postoperative pain and visceral pain progenitor cells suggest an important role of purinergic
indicating that P2X3 and P2X2/3 receptor activation may signaling in early embryogenesis, especially in cell prolif-
not be a major mediator of acute postoperative or visceral eration, migration, and differentiation, with different sub-
pain [149]. MRS 2179 (2′-deoxy-N6-methyladenosine 3′,5′ types of receptors participating in these processes. Our
-bisphosphate) was discovered as a specific inhibitor of understanding of the biological functions of specific P2
P2Y1 receptor activity [194]. This compound has an receptor subtypes during CNS development and in the adult
efficient antithrombotic action in which P2Y1 receptors brain has increased due to the availability of knockout
are involved [195]. animals and specific inhibition of gene expression or
Based on structure design or combinatorial library activity of purinergic receptor subtypes. The importance
approaches specific agonists or antagonists may be discov- of P2 receptor signaling in neuroprotection, neuroimmunity,
ered for other purinergic receptor subtypes. For instance, and guiding neuronal differentiation, especially in glial and
the SELEX (systematic evolution of ligands by exponential microglial cells, has been related to purinergic receptor
enrichment) technique provides a particularly promising expression. Most importantly, specific agonists and antag-
approach for the discovery of such compounds. This onists for individual P2 receptor subtypes are both needed
technique is based on the reiterative presentation of a for studying their involvement in biological processes. The
partial random RNA or single-stranded DNA library to a discovery of such selective compounds will elucidate yet
protein preparation containing a particular purinergic unknown biological functions of P2 receptor subtypes as
receptor subtype. RNA or DNA molecules bound to a well as open new avenues for therapeutic approaches to
target site on the receptor are displaced from the receptor disease states in which purinergic receptor activity is
and eluted by addition of an excess concentration of an involved.
unspecific purinergic receptor antagonist and amplified by
reverse transcription polymerase chain reaction (PCR) or Acknowledgments H.U. and L.R.G.B. are grateful for grant support
from FAPESP (Fundação de Amparo à Pesquisa do Estado de São
PCR to restore the library used for the next in vitro Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico
selection cycle. Using this approach, it was possible to e Tecnológico), Brazil. K.N.G.’s Ph.D. thesis is supported by
identify inhibitors specific for isoforms of a target protein fellowship from CAPES (Coordenação de Aperfeiçoamento de
[196]. Our group prepared membrane protein fractions of Pessoal de Nível Superior). C.L.G.’s undergraduate research, C.A.
T.’s and K.K.Y.’s Ph.D. theses and R.R.R.’s postdoctoral research are
1321N1 cells stably transfected with rat P2X2 receptors and supported by fellowships from FAPESP. P.M.’s Ph.D. thesis was
coupled them onto an immobilized artificial membrane supported by fellowship from FAPESP.
(IAM) as matrix for affinity chromatography. The equilib-
rium binding to the receptor and competition between ATP
and the purinergic antagonists suramin and 2′3′-O-(2,4,6-
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