P2X receptors in neuroglia

Abstract

Different types of ionotropic P2X purinoceptors are expressed in all major types of neuroglia, where they mediate a variety of physiological and pathological signaling. Cortical astrocytes express specific P2X_1/5 heteromeric receptors that are activated by ongoing synaptic transmission and can trigger fast local signaling through elevation in cytoplasmic Ca²⁺ and Na⁺ concentrations. Oligodendrocytes express several types of P2X receptors that may control their development and mediate axonal–glial interactions. In microglia, P2X₄ and P2X₇ receptors regulate numerous events associated with microglial activation, motility, and release of proinflammatory factors.

INTRODUCTION: ATP AS A NEUROTRANSMITTER

ATP, its derivatives, and adenosine are ubiquitous and ancient intercellular messengers that mediate cell-to-cell signaling in variety of tissues.^1–3 In the central nervous system (CNS), ATP acts as homocellular (neuronal–neuronal and glial–glial) and heterocellular (neuronal–glial and glial–neuronal) neurotransmitter.^4,5 ATP is physiologically released from many cellular elements of the CNS including neuronal terminals, axons, and astroglia. ATP release pathways include concentration-gradient driven diffusion through plasmalemmal channels with large permeability (as cytosolic concentration of ATP approaches 5–10 mM and extracellular is set at a low nanomolar range, the resulting concentration gradient is arguably one of the highest existing in biological systems); release through ATP-binding cassette transporters and secretion by exocytosis.^4,6,7 The diffusion of ATP can occur through several sets of plasmalemmal channels such as unpaired connexins/hemichannels⁸ or volume-sensitive anion channels.⁹ The latter mechanism was demonstrated to mediate physiological release of ATP associated with axonal firing⁹; although physiological relevance of diffusional ATP release in other CNS structures remains unclear. The most widespread pathway for ATP release in the context of signaling in neural networks is represented by Ca²⁺-regulated exocytosis, which was identified in neuronal terminals^6,7 and in astrocytes.^10–12 Massive release of ATP also accompanies cellular death and destruction; hence ATP acts as a universal ‘damage’ signal and is involved in wide variety of pathological reactions in the nervous system.

P2X RECEPTORS IN ASTROGLIA

Functional properties of P2X receptors (e.g., their agonist sensitivity, desensitization, or Ca²⁺ permeability) are determined by the subunit composition (Table 1). The P2X receptors can be formed through homo- or heteromeric assembly of P2X₁ to P2X₇ subunits (so far homomeric composition was shown for P2X_1–5,7 subunits, whereas P2X₆ subunits apparently cannot oligomerize); heteromeric compositions are represented by P2X_1/2, P2X_1/4, P2X_1/5, P2X_2/3, P2X_2/6, and P2X_4/6 channels.^13,14

TABLE 1.

General Pharmacology of P2X Receptors

Receptor	Kd/EC50	Preferred Substrates	References
P2X1	Kd(ATP) = 2–3 nM	No specific agonists for P2X1 only	102
	EC50(ATP) = 0.07 µM	BzATP > 2meSATP > ATP > αβmeATP	103
	EC50(BzATP) = 0.003 µM
P2X2	EC50(ATP) = 1.2 µM	BzATP > ATP = 2meSATP ≫ αβmeATP	103
	EC50(BzATP) = 0.75 µM
P2X3		2meSATP ≫ ATP > αβmeATP	104
	EC50(ATP) = 0.5 µM	BzATP > 2meSATP > ATP > αβmeATP	103
	EC50(BzATP) = 0.08 µM
P2X4	EC50(ATP) = 10 µM	BzATP > ATP = 2meSATP ≫ αβmeATP	103
	EC50(BzATP) = 7 µM
P2X5	EC50(ATP) = 10 µM	ATP = 2meSATP ≫ αβmeATP > BzATP	103
P2X6 (hetero)	EC50(ATP) = 12 µM	2meSATP ≥ ATP ≫ αβmeATP	103
	EC50(2meSATP) = 9 µM
P2X7	EC50(ATP) = 100 µM	BzATP > ATP = 2meSATP ≫ αβmeATP	103
	EC50(BzATP) = 20 µM

On a transcriptional level, all seven P2X subunits were identified in astrocytes in vitro and in situ.¹⁵ Specific mRNA for P2X_1–5 and P2X₇ receptors was detected in primary cultured rat cortical astrocytes.^16,17 In tissue extracts from rat nucleus accumbens, expression of mRNA for all seven P2X subunits was revealed RT-PCR.¹⁸ Specific mRNA for P2X₃, P2X₄, and P2X₅ subunits was identified in freshly isolated rat retinal Müller cells,¹⁹ the P2X₇ receptor-specific mRNA was identified in human Müller glia.²⁰ In acutely isolated mouse cortical astrocytes, only P2X₁- and P2X₅-specific mRNAs were found.²¹

Expression of P2X subunits in astroglia was also characterized by immunohistochemistry. In nucleus accumbens, immunofluorescence revealed P2X_2–4 receptors co-localized with GFAP-labeled astroglial profiles.¹⁸ Mechanical lesion of the nucleus accumbens resulted in upregulation of P2X_1–4 and P2X₇ immunofluorescence in astrocytes.¹⁸ Immunoreactivity for P2X₁ and P2X₂ receptors was detected in astroglial cells in the cerebellum,^22,23 P2X₂ receptors were visualized in spinal cord astrocytes,²⁴ and P2X₄ receptors were identified in the processes and somatas of the astrocytes from the brainstem.²⁵ In hippocampal astrocytes, immunostaining revealed expression of P2X_1–4, P2X₆, and P2X₇ subunits.²⁶

At a functional level, however, P2X receptors-mediated responses are not ubiquitous property of astroglia. ATP triggered membrane currents, depolarization, and Ca²⁺ signaling in cultured astrocytes,^27,28 although the subunit composition of underlying receptors was not investigated. In hippocampal astrocytes in slices or after acute isolation, no P2X-mediated currents were identified.²⁹ Similarly, ATP-induced currents were not observed in Bergmann glial cells in acute cerebellar slices.³⁰ Nonetheless, absence of ATP-induced currents in the in situ experiments mentioned above cannot be considered conclusive, because complex geometry of glial cells, diffusional barriers, and rapid degradation of ATP in slice tissue may hinder the detection of membrane responses.

P2X_1/5 Receptors in Cortical Astrocytes

Astrocytes in cortex express functional P2X_1/5 heteromeric receptors, which upon activation produce idiosyncratic membrane responses (Figure 1 and Ref 21). Astroglial P2X_1/5 receptor is extraordinary sensitive to ATP (K_D ~ 50 nM), has distinct pharmacology (complete inhibition by 30 µM PPADS and almost complete inhibition by 1 µM TNP-ATP) and biophysical properties (absence of desensitization and activation of ‘rebound’ tail current in response to the washout of the agonist). The P2X_1/5 receptors contribute to fast ion currents triggered in astrocytes in response to stimulation of neuronal afferents in cortical slices.^31,32 The same receptors are involved in generation of spontaneous ‘miniature’ postsynaptic currents measured from astrocytes in cortical slices.^31,33 Astroglial P2X_1/5 receptors have intermediate Ca²⁺ permeability (P_Ca/P_monovalent ~ 2.2) and their activation by exogenous agonists as well as by synaptically released ATP triggers astroglial Ca²⁺ signals (Figure 2 and Ref 32). Expression of P2X_1/5 receptors in astrocytes is age-dependent,³³ being maximal in 3–6 months old mice and substantially declining with an advanced age.

FIGURE 1.

P2X_1/5 receptor-mediated currents in acutely isolated cortical astrocytes. (a) The family of ATP currents evoked by repetitive applications of the agonist. The currents show no apparent desensitization. Current traces have a complex kinetics comprising the peak, the steady-state component, and the ‘rebound’ inward current recorded upon ATP washout as indicated on the graph. (b) Concentration dependence of ATP-induced currents in cortical astrocytes. Membrane currents recorded from a single cell in response to different ATP concentrations are shown on the left. The right panel shows the concentration response curves constructed from nine similar experiments; current amplitudes were measured at the initial peak and at the end of the current, as indicated on the graph. (c) Inhibition of ATP-induced currents by 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP). The graph shows ATP-induced currents measured from the cortical astrocyte in control conditions, in the presence of different concentrations of TNP-ATP and after the washout. Application of TNP-ATP started 2 min before the application of ATP. All recordings were made at holding potential of −80 mV. (Reprinted with permission from Ref 21. Copyright 2008 Society for Neuroscience)

FIGURE 2.

P2X_1/5 receptor-mediated Ca²⁺-signaling in cortical astrocytes. (a) Acutely isolated astrocytes were loaded with Fluo-4 via a patch pipette. Fluorescent images were recorded simultaneously with transmembrane currents evoked by application of 100 µM ATP or 10 µM α,β-methylene ATP. [Ca²⁺]_i-transients are represented as the ΔF/F₀ ratio averaged over the cell soma (green traces). Holding potential was −80 mV. (b) Synaptically induced ionotropic Ca²⁺ signals in protoplasmic cortical astrocytes in situ. Cortical layer II astrocytes were loaded with Fura-2 in situ via patch pipette. Fluorescent images were recorded simultaneously with glial currents evoked by neuronal afferent stimulation in the presence of CNQX (control) and after application of 10 nM of NF-449, selective antagonist of P2X receptors. [Ca²⁺]_i-transients (green traces) are expressed as F₃₄₀/F₃₈₀ ratio. (Reprinted with permission from Ref 32. Copyright 2010 Elsevier)

Astroglial P2X₇ Receptors

The role and functional expression of the cytolitic, pore-forming P2X₇ receptors^14,34 in astroglia remains controversial. Both P2X₇-specific mRNA and P2X₇ receptor protein have been detected in cultured astroglia (reviewed in Refs 15 and 35). Similarly, stimulation of P2X₇ receptors in cultured astrocytes often triggered [Ca²⁺]_i transients, mimicked by P2X₇ receptor agonist 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP) and inhibited by antagonist oxidized ATP (oxATP).^17,36,37 Characteristic P2X₇ receptor currents activated by 1–5 mM ATP or by 0.1 mM Bz-ATP were recorded from cultured astrocytes.^38,39 Likewise, P2X₇ receptor-mediated currents were identified in freshly isolated human retinal Müller glial cells.²⁰ It has to be remembered, however, that Bz-ATP can activate other P2X receptors¹³ and can be degraded to Bz-adenosine, which stimulates P1 receptors.⁴⁰ Similarly, the oxATP also inhibits P2X₁ and P2X₂ receptors.¹³

Stimulation of P2X₇ receptors in astrocytes in vitro affects numerous biochemical process, for example, synthesis of endocannabinoid 2-arachidonoylglycerol, stimulation of production of NO and of lipid mediators of inflammation cysteinyl leukotrienes, regulation of NF-κB signaling, release of gliotransmitters and TNF-α, etc.^41–45 Furthermore, stimulation of P2X₇ receptors in cultured astrocytes induces secretion of various transmitter molecules (‘glio’ transmitters) including glutamate, ATP, and GABA.^38,46,47 Recently, the P2X₇ currents were found in cortical astrocytes in situ, in acutely isolated slices,⁴⁸ and P2X₇ receptor-mediated Ca²⁺ signals were identified in astrocytes from acutely isolated optic nerve.⁴⁹ The role of P2X₇ receptors in astroglial physiology remains unknown, as indeed activation of the receptor requires very high (in excess of 1 mM) ATP concentrations. It was, however, suggested that low ATP concentrations may activate P2X₇ receptor without opening the channel pore; this ‘low-level’ stimulation can be relevant for regulation of a variety of trophic/metabolic processes.⁵⁰ At the same time, astroglial expression of P2X₇ receptors is usually increased in neuropathology (see Ref 15 for review), which may indicate their role in regulation of pathologically relevant astroglial reactions.

P2X RECEPTORS IN OLIGODENDROGLIA

Expression of P2X_1,2,3,4,7 proteins was found in oligodendroglial precursor cells (OPCs) in purified postnatal cultures,^51,52 although P2X-mediated ion currents were not detected in cultured OPCs and mature oligodendrocytes.⁵³ In the isolated optic nerve, application of a broad agonist of P2X receptors α,β-methylene ATP induced relatively small [Ca²⁺]_i elevation, thus suggesting possible involvement of P2X receptors.⁵⁴ In oligodendrocytes in situ in corpus callosum slices, ATP failed to activate transmembrane currents.⁵³

Some evidence indicating expression of functional P2X₇ receptors in cells of oligodendroglial lineage was gathered recently. In cultured OPCs, application of Bz-ATP triggered large Ca²⁺ transients, which were effectively inhibited by oxATP thereby suggesting Ca²⁺ entry though P2X₇ receptors.⁵¹ Immunoreactivity for P2X₇ receptors was detected in oligodendrocytes from the optic nerve and the spinal cord.^55,56 In cultured oligodendrocytes from optic nerve, high concentrations of ATP (EC₅₀ ~ 8.8 mM) and much lower concentrations of BzATP (EC₅₀ ~ 0.5 mM) triggered sustained inward currents. These currents were potentiated in divalent-cations-free extracellular solutions and were inhibited by oxATP. In addition, high concentrations of ATP and BzATP induced a rapid increase in [Ca²⁺]_i, which was almost exclusively dependent on transmembrane Ca²⁺ entry.⁵⁶ Finally, over-stimulation of P2X₇ receptors (with 1 mM ATP or BzATP for 15 min) induced significant oligodendroglial death in culture and in situ in the optic nerve.⁵⁶

The P2X₇-mediated death of oligodendrocytes may be relevant for variety of demyelinating diseases including multiple sclerosis (MS). Treatment of animals suffering from experimental autoimmune encephalitis (EAE, which is generally considered as a model for MS), with P2X₇ antagonists oxATP or brilliant blue G, inhibited demyelination and restored axon conduction velocity.^55,56 Furthermore, expression of P2X₇ receptors increased in white matter of MS patients.⁵⁶

NG2 GLIA

Very little is known about expression of P2X receptors in the NG2-glia, which was recently identified as a separate class of neuroglia. The NG2 glial cells (identifiable by the expression of NG2 chondroitin sulfate proteoglycan⁵⁷) are present throughout the developing and adult brain and may serve as pluripotent stem cells, OPCs or being a specific elements of neural circuitry.^58,59 Recently, it was reported that ATP released from axons and astrocytes in electrically stimulated optic nerve triggers [Ca²⁺]_i transients in NG2-glia, which at least in part can be mediated by P2X₇ receptors.⁶⁰

P2X RECEPTORS CONTROL ACTIVATION OF MICROGLIA

Purinergic signaling system is particularly developed in microglia. Numerous experiments in vitro and in vivo have demonstrated that ATP and its analogues trigger rapid functional responses of microglial cells, which include induction of microglial motility (both of the processes and the soma), the outgrowth of microglial processes, membrane raffling, and the release of cytokines and inflammatory proteins (see Ref 61 for comprehensive review). Effects of purines/pyrimidines on microglia are mediated through all types of purinoceptors (P1 and P2) abundantly expressed in microglial cells throughout the nervous system.⁶¹ Expression of P2X receptor subunits in microglia is developmentally regulated. At embryonic day 16, majority of microglial cells expressed mRNA specific for P2X₁ and P2X₄ subunits, whereas only 30% of these cells expressed P2X₇ receptor transcripts. From postnatal day 7, the P2X₄-expressing microglia concentrated around blood vessels. Finally, at postnatal day 30, microglial cells expressed only P2X₇ and P2X₄ mRNAs; the P2X₇-positive cells were distributed evenly through the forebrain, whereas cells bearing P2X₄ receptors outlined blood vessels and subarachnoid space.⁶²

Expression of different types of purinoceptors in cultured microglia undergoes substantial remodeling while progressing through activation.⁶³ Likewise, expression of purinoceptors changes in various neurological diseases. For example, activation of hippocampal microglia following epileptic seizures (triggered by injections of kainate) resulted in the upregulation of the expression of mRNA specific for P2X_1,4,7 and P2Y_6,12,13 receptors. This upregulation led to an increase in ATP-induced membrane currents and ATP-induced microglial motility.⁶⁴ The upregulation of microglial P2X₄, P2X₇, and P2Y₆ receptors were also observed in the experimental model of amyotrophic lateral sclerosis, in mice expressing mutant superoxide dismutase 1.⁶⁵

The P2X-mediated currents were identified in microglial cultures prepared from several tissues including mouse embryos, newborn rat, and human brains.^66–68 In all these preparations, application of 100 µM ATP triggered desensitizing cationic currents and induced [Ca²⁺]_i elevation produced by Ca²⁺ entry through ionotropic purinoceptors. Similar ATP-induced cationic currents were also detected in microglial cells in acute slices from an adult mouse brain.⁶⁹

P2X₄ Receptors in Pathologically Remodeled Spinal Cord Microglia

Peripheral nerve injury triggers rapid and substantial activation of microglia in the spinal cord, this process being fundamentally important in pathogenesis of neuropathic pain.^70–73 Development of tactile symptoms associated with neuropathic pain (e.g., tactile allodynia) in the rat spinal nerve injury model could be reversed by inhibition of P2X₄ receptors, suggesting their role in neuropathic pain.⁷⁴ This initial observation for further corroborated when it was shown that intraspinal injection of P2X₄ antisense oligodeoxynucleotide inhibited P2X₄ receptors expression and reduced tactile allodynia following nerve injury. The intrathecal injection of activated cultured microglia expressing P2X₄ receptors triggered allodynia without peripheral nerve lesion.⁷⁴

The lesion to the peripheral nerve resulted in a significant upregulation of P2X₄ receptor expression in spinal cord microglia⁷⁴; the P2X₄ protein levels began to increase 1 day after surgery and reached maximal levels after 14 days; this time course matched the development of tactile allodynia. Similarly P2X₄ expression was upregulated following formalin injection into the spinal cord (a common inflammatory pain model) or intraperitoneal injection of LPS^75,76 or mechanical lesion to the spinal cord.⁷⁷ An increase in P2X₄ expression was also observed in rats with experimental autoimmune neuritis.⁷⁸

P2X₇ Receptors in Microglia

Functional P2X₇ receptors were identified in microglial cells in situ, in amoeboid microglia collected from the surface of corpus callosum slices,⁷⁹ and in vitro in freshly isolated mouse microglia.⁸⁰ In cultured microglia, ATP (inmillimolar concentrations) and Bz-ATP evoked Ca²⁺ influx and uptake of ethidium bromide or Lucifer Yellow both being indicative of the formation of P2X₇ receptor associated pore. These effects were blocked by oxATP.^80,81 In the amoeboid microglia from corpus callosum slices of 5–7 days old mice, 1 mM ATP induced classical P2X₇-mediated currents (Figure 3). Sensitivity of receptor was greatly enhanced following removal of extracellular divalent cations.⁷⁹ The P2X₇ receptor-mediated currents were also detected in ramified microglia in acute slices from adult (6–8 weeks old) mice.⁶⁹

FIGURE 3.

ATP triggers P2X₇receptor-mediated currents in amoeboid microglial cells collected from the surface of corpus callosum slices of young (P5–P7) mice. (a–d) Method of patch-voltage clamping of single amoeboid microglial cells from the surface of corpus callosum slice. Combinations of images taken by means of infrared video microscopy (left panels) and schematic drawings showing the method of cell patch-clamping. Video images were captured with an intensified CCD camera (Hamamatsu, Japan) and digitized by a frame grabber connected to the PC. (a) Initial position of amoeboid microglial cells situated on the surface of a corpus callosum slice. (b) A single microglial cell was approached with a micropipette and a whole-cell patch clamp configuration was established. (c) After 2–3 min, the cell partially spread over the pipette, intensifying the cell-to-pipette contact. (d) Finally, the cell was lifted for 200 µm over the slice surface. Scale bar in (d) = 10 µm. (e) P2X₇-mediated membrane currents measured from amoeboid microglial cells in control conditions and Ca²⁺/Mg²⁺-free bath solution. (Reprinted with permission from Ref 79. Copyright 1996 Elsevier)

The P2X₇ receptor seem to be constitutively expressed in resting microglia; in the healthy brain P2X₇-positive microglial cells are homogeneously distributed throughout all brain regions.⁸² Different forms of insults to the brain trigger rapid and substantial increase in the expression of microglial P2X₇ receptors.^35,83,84 Expression of P2X₇ receptors is upregulated after acute ischemic and traumatic attacks^85,86 as well as chronic neuropathologies such as MS⁸⁷ and Alzheimer’s disease.^88,89 A significant increase in expression of P2X₇-specific mRNA and in P2X₇ immunoreactivity was also detected in reactive microglial cells surrounding senile plaques in AD transgenic model.⁹⁰ Microglial expression of P2X₇ receptors was also increased following exposure of cultured microglia to β-amyloid or after intrahippocampal injection of the latter.⁸⁸ An increase in expression of P2X₇ receptors was also reported in microglia from rat model of epilepsy⁹¹ and from brains of scrapie-infected mice.⁹² Pharmacological manipulations with P2X₇ receptors were neuroprotective in ischemia⁸⁶; it also reduced microglial expression and release of proinflammatory factors.⁹³

Activation of P2X₇ receptors regulates various functions associated with microglial activation. The P2X₇ receptors, for example, were obligatory for triggering activation of microglia and inetrleukin-1 production in response to an intrahippocampal injection of Aβ protein; both activation and IL-1 accumulation were absent in P2X₇ knockout mice.⁹⁴ Overexpression of P2X₇ receptors in cultured microglia on its own right triggered microglial activation that could be blocked by oxATP.^95–100 This microglial activation required formation of P2X₇ receptor-associated pore; when microglial cells were transfected with a point mutant P2X₇RG345Y that does not allow pore formation, microglial activation was suppressed.⁹⁵ Microglial P2X₇ receptors also control release of multiple proinflammatory factors. Activation of P2X₇ initiates release of cytokines IL-1α and IL-1β^96,97 that may involve plasmalemmal vesicle formation and shedding.⁹⁸ The P2X₇ receptors also control microglial synthesis and release of CC-chemokine ligand 3 (CCL3)/macrophage inflammatory protein-1α⁹⁹; release of chemokine CXCL2,¹⁰⁰ plasminogen,¹⁰¹ and many other proinflammatory factors (see Ref 61 for comprehensive review).

CONCLUSIONS

P2X receptors are expressed in all types of neuroglial cells. The full regional mapping of glial presence of P2X receptors is far from being complete; however, we already may suggest their roles in various types of signaling in neuronal–glial circuits. In cortical astroglia, P2X_1/5 receptors participate in rapid synaptic-associated signaling producing local [Na⁺]_i and [Ca²⁺]_i transients in glial perisynaptic processes. In oligodendroglia, activation of P2X₇ receptors is somehow involved in pathogenesis of demyelinating diseases. In microglia, P2X₄ and P2X₇ receptors control various aspects of microglial activation and secretion of proinflammatory factors.