Activation of P2X(7) receptors stimulates the expression of P2Y(2) receptor mRNA in astrocytes cultured from rat brain

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X(7) ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X(7) receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y(2) in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y(2) expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y(2) sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y(2) mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X(7)-antagonists oATP and BBG. The BzATP-evoked P2Y(2) receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y(2)siRNA, thus supporting evidence of increased P2Y(2) activity. To further investigate the mechanisms by which P2X(7) receptors mediated the P2Y(2) mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y(2) mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X(7)-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X(7) receptors in astrocytes enhances P2Y(2) mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways.     

Apparent species differences in the kinetic properties of P2X(7) receptors

1. Apparent species differences in the responses of recombinant P2X(7) receptors to repeated application of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) have been investigated. 2. Repeated application of 100 microM BzATP resulted in a progressive increase in current magnitude (current growth) at mouse and human, but not rat P2X(7) receptors. 3. Current growth was thought to reflect progressive dilation of the P2X(7) ion-channel to a pore permeable to large molecules (MW<900), suggesting that channel dilation was not occurring at the rat P2X(7) receptor. However, 100 microM BzATP produced a rapid influx of YO-PRO-1 (MW375) in cells expressing rat or human P2X(7) receptors. 4. There were, however, species differences in agonist potency such that 100 microM BzATP was a supra-maximal concentration at rat, but not human or mouse, P2X(7) receptors. Importantly, when sub-maximal concentrations of BzATP or ATP were examined, current growth occurred at rat P2X(7) receptors. 5. The rate of current growth and YO-PRO-1 accumulation increased with agonist concentration and appeared more rapid at rat and human, than at mouse P2X(7) receptors. 6. The potency of BzATP and ATP was 1.5 - 10 fold lower in na?ve cells than in cells repeatedly exposed to ATP. 7. This study demonstrates that current growth occurs at mouse, rat and human P2X(7) receptors but only when using sub-maximal concentrations of agonist. Previously, current growth was thought to reflect the progressive increase in pore diameter of the P2X(7) receptor ion channel, however, the results of this study suggest a progressive increase in agonist potency may also contribute.     

Pharmacological characterization of the P2X7 receptor on human macrophages using the patch-clamp technique

Whole-cell patch-clamp recordings were made from macrophages derived from human monocytes that had been cultured for 5-7 days. The P2X agonists ATP (100 microM) and 2',3'-(4-benzoyl)-benzoyl ATP (BzATP, 100 microM) induced inward currents. A second application of the agonists was characterized by strong desensitisation of the maximum current. Pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a non-specific P2X antagonist, and 1-( N, O- bis[5-isoquinolinesulphonyl]- N-methyl- L-tyrosyl)-4-phenylpiperazine (KN62), a potent P2X(7) antagonist at the human receptor, both reduced the ATP-induced inward current. KN62 also inhibited the BzATP-induced current. The P2X(7) antagonist Coomassie Brilliant Blue G (BBG), believed to be potent at the human but even more so potent at the rat receptor, did not reduce the BzATP-induced inward current significantly. These results indicate that the native P2X(7) receptor subtype is expressed in human macrophages and that this receptor subtype is involved in the ATP-mediated inward current. Our experiments suggest that other P2X receptors also appear to be involved in the ATP-mediated current in human monocyte-derived macrophages.     

P2X7 receptor activation in rat brain cultured astrocytes increases the biosynthetic release of cysteinyl leukotrienes

Astrocytes have been recognized as important elements in controlling inflammatory as well as immune processes in the central nervous system (CNS). Recently, glial cells have been shown to produce cysteinyl leukotrienes (CysLTs) which are known lipid mediators of inflammation and whose extracellular concentrations rise under different pathological conditions in the brain. In the same conditions also extracellular concentrations of ATP dramatically increase reaching levels able to activate P2X7 ionotropic receptors for which an emerging role in neuroinflammation and neurodegeneration has been claimed. RTPCR analysis showed that primary cultures of rat brain astrocytes express P2X7 receptors. Application of the selective P2X7 agonist benzoyl benzoly ATP (BzATP) markedly increased [Ca2+]i which was mediated by a calcium influx from the extracellular milieu. The P2X7 antagonist, oATP, suppressed the BzATP-induced calcium increase. Consistent with the evidence that increased calcium levels activate the leukotriene biosynthetic pathway, challenge of astrocytes with either the calcium ionophore A23187 or BzATP significantly increased CysLT production and the cell pre-treatment with EGTA abolished these effects. Again the P2X7 antagonist prevented the BzATP-mediated CysLT efflux, whereas the astrocyte pretreatment with MK-571, a CysLT1 receptor antagonist, was ineffective. The astrocyte pre-treatment with a cocktail of inhibitors of ATP binding cassette (ABC) proteins reduced the BzATP-mediated CysLT production confirming that ABC transporters are involved in the release of CysLTs. The astrocyte P2X7- evoked rise of CysLT efflux was abolished in the presence of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP) whose expression, along with that of 5-lipoxygenase (5-LO) was reported by Northern Blot analysis. The stimulation of P2X7 induced an up-regulation of FLAPmRNA that was reduced by the antagonist oATP. These data suggest that in rat brain cultured astrocytes P2X7ATP receptors may participate in the control of CysLT release thus further supporting a role for extracellular ATP as an integral component of the inflammatory brain response.     

Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP

Agonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 microM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.     

Cloning and pharmacological characterization of the guinea pig P2X7 receptor orthologue

BACKGROUND AND PURPOSE: The human, rat, and mouse P2X(7) receptors have been previously characterized, and in this study we report the cloning and pharmacological properties of the guinea pig orthologue. EXPERIMENTAL APPROACH: A cDNA encoding for the guinea pig P2X(7) receptor was isolated from a guinea pig brain library. The receptor was expressed in U-2 OS cells using the BacMam viral expression system. A monoclonal antibody was used to confirm high levels of cell surface expression and the functional properties were determined in ethidium bromide accumulation studies. KEY RESULTS: The predicted guinea pig protein is one amino acid shorter than the human and rat orthologues and over 70% identical to the rat and human receptors. In contrast to human and rat P2X(7) receptors, 2'-&3'-O-(4benzoylbenzoyl)ATP (BzATP) was a partial agonist of the guinea pig P2X(7) receptor when compared to ATP and acted as an antagonist in some assays. However, as at other species orthologues, BzATP was more potent than ATP. The guinea pig P2X(7) receptor possessed higher affinity for 1-[N,O-bis(5-isoquinoline sulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62), suramin and Coomassie Brilliant Blue than human or rat P2X(7) receptors suggesting that it is pharmacologically different to other rodent or human P2X(7) receptors. CONCLUSIONS AND IMPLICATIONS: The guinea pig recombinant P2X(7) receptor displays a number of unique properties that differentiate it from the human, rat and mouse orthologues and this structural and functional information should aid in our understanding of the interaction of agonists and antagonist with the P2X(7) receptor.     

Stimulation of Ca(2+) influx through ATP receptors on rat brain synaptosomes: identification of functional P2X(7) receptor subtypes

1. ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons. 2. In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca(2+) influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2-me-S-ATP and ATPgammaS were much less active. alpha,beta-me- ATP and beta,gamma-me-ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca(2+) influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X(7) receptors. 3. Influx was unaffected by the VSCC inhibitors omega-CTx-MVIIC and (-) 202 - 791, indicating that ATP induced Ca(2+) influx occurred primarily through P2X receptors. 4. P2X(7) receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX-42 but contained greatly enriched amounts of syntaxin and SNAP 25. 5. The various pharmacological and biochemical studies were all consistent with the presence of functional P2X(7) receptors.     

Species- and agonist-dependent differences in the deactivation-kinetics of P2X7 receptors

In this study we have expressed recombinant P2X7 receptors in HEK293 cells and examined the reasons for the species- and agonist-dependent differences in the time taken for the closure of the P2X7 receptor ion-channels after agonist removal. Channel closure times, measured in electrophysiological studies or by measuring cellular permeability to ethidium cations, were slower at rat than at human or mouse P2X7 channels following washout of the P2X7 agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP). In contrast, there were no species differences in channel closure times when ATP was the agonist. BzATP was more potent than ATP at the three species homologues and exhibited highest potency for rat P2X7 receptors suggesting that channel closure time was related to agonist potency. Furthermore, BzATP potency for the P2X7 receptor could be modified by changing extracellular ionic concentrations or by mutating the receptor and modifications which increased agonist potency also increased the time taken for channel closure. The dependence of channel closure time on agonist potency suggests it reflects agonist dissociation from the P2X7 receptor rather being an intrinsic property of the ion-channel. Consistent with this, our previous studies have shown that agonist potency increases after repeated agonist applications and in this study channel closure time at rat P2X7 receptors increased after repeated agonist applications. Overall these results suggest that the species differences in channel closure times reflect differences in agonist dissociation rates which arise as a consequence of the marked species differences in agonist potency.     

Potentiation of native and recombinant P2X7-mediated calcium signaling by arachidonic acid in cultured cortical astrocytes and human embryonic kidney 293 cells

In the brain, arachidonic acid (AA) plays a critical role in the modulation of a broad spectrum of biological responses, including those underlying neuroinflammation. By using microfluorometry, we investigated the action of extracellular AA in the modulation of the purinoceptor P2X7-mediated elevation of [Ca(2+)](i) in cultured neocortical type-1 astrocytes and P2X7-, P2X2-transfected human embryonic kidney (HEK) 293 cells. We report that in cultured astrocytes, AA-induced [Ca(2+)](i) elevation is coupled to depletion of intracellular Ca(2+) stores and to a sustained noncapacitative Ca(2+) entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated [Ca(2+)](i) rise evoked by the selective agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP). Pharmacological studies demonstrate that the selective P2X7 antagonists oxidized ATP and Brilliant Blue G abrogated the AA-mediated potentiation of BzATP-evoked [Ca(2+)](i) elevation. Fluorescent dye uptake experiments showed that the AA-induced increase in [Ca(2+)](i) was not due to a switch of the P2X7 receptor from channel to the pore mode of gating. The synergistic effect of AA and BzATP was also observed in HEK293 cells stably expressing rat and human P2X7 but not in rat P2X2. Control HEK293 cells responded to AA exposure only with a transient [Ca(2+)](i) elevation, whereas in those expressing the P2X7 receptor, AA elicited a potentiation of the BzATP-induced [Ca(2+)](i) rise. Together, these findings indicate that AA mediates a complex regulation of [Ca(2+)](i) dynamics also through P2X7-mediated Ca(2+) entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of [Ca(2+)](i) signaling in astroglial cells.     

Species and agonist dependent zinc modulation of endogenous and recombinant ATP-gated P2X7 receptors

Zinc (Zn2+) and copper (Cu2+) are key signalling molecules in the immune system and regulate the activity of many ion channels. Both Zn2+ and Cu2+ potently inhibit rat P2X7 receptors via a binding site identified by mutagenesis. Here we show that extracellular Cu2+ also potently inhibits mouse P2X7 receptors. By contrast, the receptor expression system and agonist strongly influence the action of extracellular Zn2+ at mouse P2X7 receptors. Consistent with previous reports, Zn2+ inhibits recombinant rat P2X7 receptors. However, recombinant mouse P2X7 receptors are potentiated by Zn2+ when activated by ATP4- but inhibited when stimulated with the ATP analogue BzATP4-. Endogenous murine macrophage P2X7 receptors are not modulated by Zn2+ when stimulated by ATP4- however Zn2+ inhibits BzATP4- mediated responses. In summary, these findings provide a fundamental insight into the differential actions of Zn2+ and Cu2+ between different P2X7 receptor species.     

Serum constituents can affect 2'-& 3'-O-(4-benzoylbenzoyl)-ATP potency at P2X(7) receptors

1. 2'-& 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) is the prototypic agonist for P2X(7) receptors. In this study we demonstrate that bovine serum albumin (BSA) can affect the potency of BzATP at P2X receptors. 2. BzATP potency (pEC(50)) to stimulate ethidium accumulation in cells expressing recombinant P2X7 receptors varied between 6.5 and 4, depending upon the species orthologue studied and ionic conditions employed. BSA (0.1 - 1 mg ml(-1)) and foetal bovine serum (FBS, 1 - 10% v v(-1)) inhibited responses to BzATP but only when the BzATP pEC(50) exceeded 5. 3. BSA did not block ATP-stimulated ethidium accumulation, suggesting its effects were independent of P2X(7) receptor blockade. 4. BSA did not cause breakdown of nucleotides, although FBS (10% v v(-1)) exhibited appreciable nucleotidase activity and caused significant breakdown of ATP. 5. In the presence of BSA, lipids such as 11-((5-dimethylaminonaphthalene-1-sulphonyl)amino)undecanoic acid (DAUDA) and arachidonic acid (AA) markedly increased BzATP potency. Lipids had no affect on ATP potency in the presence of BSA and had little effect on responses to BzATP in the absence of BSA. 6. These results suggested that the reduction in BzATP potency by BSA was due to BzATP binding to BSA and that lipids prevented this binding. Consistent with this hypothesis, BzATP inhibited binding of the fluorescent lipid, DAUDA, to BSA. 7. In conclusion, BSA and lipids can markedly affect BzATP potency at P2X(7) receptors but this is probably a consequence of BzATP binding to BSA. This finding has important implications when using BzATP in vivo or in the presence of albumin.     

Effects of extracellular nucleotides and nucleosides on prostate carcinoma cells

1. The purpose of this work was to characterize the receptors involved in the action of nucleotides on the human prostate carcinoma cell lines LNCaP, PC-3 and DU145. 2. Northern blotting revealed the presence of P2Y(2), P2Y(6) and P2Y(11) messengers in the three cell lines. P2Y(1) mRNA was only observed in the DU145 cells. In both PC-3 and DU145 cells, ATP and UTP stimulated inositol phosphate accumulation in an equipotent, equiactive and non-additive way, suggesting the involvement of P2Y(2) receptors. ATP also increased cyclic AMP, but this effect is likely to result from degradation into adenosine and activation of A(2) receptor. A(2) receptor activation led to a synergistic enhancement of prostate-specific antigen secretion induced by vasoactive intestinal peptide. 3. RT - PCR experiments detected the expression of the P2X(4) and P2X(5) receptors in the DU145 cells and the P2X(4), P2X(5) and P2X(7) receptors in the PC-3 cells. The calcium influx induced by BzATP confirmed the functional expression of P2X receptors. 4. ATP inhibited the growth of PC-3 and DU145 cells. This effect was mimicked neither by UTP nor by adenosine, indicating that it does not result from phospholipase C or adenylyl cyclase activation. On the contrary, in PC-3 cells, BzATP reproduced the effect of ATP, which was associated to a moderate decrease of proliferation and an increase of apoptosis. In DU145 cells, ATP was more potent than BzATP and growth inhibition was mainly associated with necrosis. We suggest that P2X receptors might be involved in the inhibition by nucleotides of prostate carcinoma cell growth.     

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue

1. Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X(3) mRNA in sensory neurones has led to the hypothesis that activation of P2X(3) and/or P2X(2/3) receptors contributes to nociception. 2. The local administration of the P2X receptor agonist, BzATP (100--1000 nmol paw(-1), s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. 3. The co-administration of the potent P2X receptor antagonist, TNP-ATP (30--300 nmol paw(-1)), but not an inactive analogue, TNP-AMP, with BzATP into the rat hindpaw attenuated BzATP-induced nociception. Similarly, co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin reduced both acute and persistent nociception in this test. 4. Co-administration of cibacron blue (30 and 100 nmol paw(-1)), a selective allosteric enhancer of P2X(3) and P2X(2/3) receptor activation, with BzATP (30 and 100 nmol paw(-1)) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co-administration of cibacron blue (30 and 100 nmol paw(-1)) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. 5. The ability of TNP-ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X(3) containing channels contributes specifically to both acute and persistent nociception in the rat.     

Characterization of a selective and potent antagonist of human P2X(7) receptors, AZ11645373

BACKGROUND AND PURPOSE: The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized. EXPERIMENTAL APPROACH: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). KEY RESULTS: AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - 20 nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM. CONCLUSIONS AND IMPLICATIONS: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.     

Electrophysiological classification of P2X7 receptors in rat cultured neocortical astroglia

Background and purpose:

P2X7 receptors are ATP-gated cation channels mediating important functions in microglial cells, such as the release of cytokines and phagocytosis. Electrophysiological evidence that these receptors also occur in CNS astroglia is rare and rather incomplete.

Experimental approach:

We used whole-cell patch-clamp recordings to search for P2X7 receptors in astroglial–neuronal co-cultures prepared from the cerebral cortex of rats.

Key results:

All the astroglial cells investigated responded to ATP with membrane currents, reversing around 0 mV. These currents could be also detected in isolated outside-out patch vesicles. The results of the experiments with the P2X [α,β-methylene ATP and 2′-3′-O-(4-benzoyl) ATP] and P2Y receptor agonists [adenosine 5′-O-(2-thiodiphosphate), uridine 5′-diphosphate, uridine 5′-triphosphate (UTP) and UDP-glucose] suggested the involvement of P2X receptors in this response. The potentiation of ATP responses in a low divalent cation or alkaline bath, but not by ivermectin, made it likely that a P2X7 receptor is operational. Blockade of the ATP effect by the P2X7 antagonists Brilliant Blue G, calmidazolium and oxidized ATP corroborated this assumption.

Conclusions and implications:

Rat cultured cortical astroglia possesses functional P2X7 receptors. It is suggested that astrocytic P2X7 receptors respond to high local ATP concentrations during neuronal injury.     

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.     

Presence of diverse functional P2X receptors in rat cerebellar synaptic terminals

Studies in individual synaptic terminals have demonstrated the presence of diverse functional P2X receptors in rat cerebellum. No immunolabelling for P2X1, P2X4, P2X5 and P2X6, and scarce presence of P2X2 were found at the cerebellar synaptic terminals. P2X3 immunolabelling was present in 28% of isolated synaptosomes. At these synaptic terminals, nucleotides as ATP or alpha,beta-meATP induced Ca2+ transients in the presence of extracellular Ca2+, showing homologous and heterologous receptor desensitization in 60% of cases. Ip5I 10 nM did not block responses to alpha,beta-meATP, but inhibition occurred when antagonist concentrations were equal or higher than 100 nM. These data agree with the presence of abundant P2X3 homomeric receptors. P2X7 immunolabelling was present in 60% of terminals and P2X7 receptor hallmarks in Ca2+ responses have been found. BzATP was more potent than ATP and responses were potentiated when assayed in Mg2+-free medium. EC50 values were, respectively, 39.4+/-0.4 and 0.3+/-0.1 microM for ATP in the presence or absence of Mg2+. Maximal values of synaptosomal calcium transients, in the presence or absence of Mg2+, were, respectively, 91.6+/-11.9 and 132.9+/-12.9 nM for ATP; and 104.3+/-9.4 and 169.7+/-17.1 nM for BzATP. In addition, Zn2+ inhibited ATP responses in the absence of Mg2+ and the P2X7 specific antagonist Brilliant Blue G completely blocked these responses in one half of synaptosomes. This study reports the presence of functional P2X3 and P2X7 receptors at synaptic sites, which provides complexity and regulatory possibilities to the cerebellar neurotransmission.     

Possible involvement of P2X7 receptor activation in microglial neuroprotection against focal cerebral ischemia in rats

Microglia play important roles in the pathogenic cascade following cerebral ischemia, since they express growth factors, chemokines and regulatory cytokines as well as free radicals and other toxic mediators. P2X7 receptor, a subtype of a family of P2 purinoceptors, is primarily expressed in microglia and macrophages, suggesting that it regulates immune function and inflammatory responses. However, the involvement of ATP in such microglial responses after cerebral ischemia is not yet understood. In this study, we investigated the possible involvement of ATP, especially through the P2X7 receptors, in a rat model of focal cerebral ischemia. In immunohistochemical analysis, P2X7 receptor-like immunoreactivity was predominantly detected in microglia, and then activated microglia accumulated in the ischemic region, in rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Intracerebroventricular injection with P2X7 receptor agonist 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) improved behavioral dysfunction accessed by rota-rod test and ischemic neural injury induced by MCAO. In contrast, P2X7 receptor antagonist adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP) exacerbated ischemic brain damage. These results suggest that microglia play an important role in neuroprotection against rat cerebral ischemia, which is regulated by a P2X7 receptor-mediated ATP signal.     

Decavanadate, a P2X receptor antagonist, and its use to study ligand interactions with P2X7 receptors

In this study we have studied decavanadate effects at P2X receptors. Decavanadate competitively blocked 2'- and 3'-O-(4benzoylbenzoyl) ATP (BzATP) stimulated ethidium accumulation in HEK293 cells expressing human recombinant P2X7 receptors (pK(B) 7.5). The effects of decavanadate were rapid (minutes) in both onset and offset and contrasted with the much slower kinetics of pyridoxal 5-phosphate (P5P), Coomassie brilliant blue (CBB) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62). Decavanadate competitively blocked the slowly reversible, or irreversible, blockade of the P2X7 receptor produced by P5P and oxidised ATP suggesting competition for a common binding site. However, the interaction between decavanadate and KN62 was non-competitive. Decavanadate also blocked P2X2 and P2X4 receptors but with slightly lower potency. These data demonstrate that decavanadate is the first reversible and competitive antagonist of the P2X7 receptor and is a useful tool for studying the mechanism of interaction of ligands with the P2X7 receptor.     

Mammalian P2X7 receptor pharmacology: comparison of recombinant mouse,rat and human P2X7 receptors

Background and purpose:

Acute activation of P2X7 receptors rapidly opens a non-selective cation channel. Sustained P2X7 receptor activation leads to the formation of cytolytic pores, mediated by downstream recruitment of hemichannels to the cell surface. Species- and single-nucleotide polymorphism-mediated differences in P2X7 receptor activation have been reported that complicate understanding of the physiological role of P2X7 receptors. Studies were conducted to determine pharmacological differences between human, rat and mouse P2X7 receptors.

Experimental approach:

Receptor-mediated changes in calcium influx and Yo-Pro uptake were compared between recombinant mouse, rat and human P2X7 receptors. For mouse P2X7 receptors, wild-type (BALB/c) and a reported loss of function (C57BL/6) P2X7 receptor were also compared.

Key results:

BzATP [2,3-O-(4-benzoylbenzoyl)-ATP] was more potent than ATP in stimulating calcium influx and Yo-Pro uptake at rat, human, BALB/c and C57BL/6 mouse P2X7 receptors. Two selective P2X7 receptor antagonists, A-740003 and A-438079, potently blocked P2X7 receptor activation across mammalian species. Several reported P2X1 receptor antagonists [e.g. MRS 2159 (4-[(4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl}-2-pyridinyl)azo]-benzoic acid), PPNDS and NF279] blocked P2X7 receptors. NF279 fully blocked human P2X7 receptors, but only partially blocked BALB/c P2X7 receptors and was inactive at C57BL/6 P2X7 receptors.

Conclusions and implications:

These data provide new insights into P2X7 receptor antagonist pharmacology across mammalian species. P2X7 receptor pharmacology in a widely used knockout background mouse strain (C57BL/6) was similar to wild-type mouse P2X7 receptors. Several structurally novel, selective and competitive P2X7 receptor antagonists show less species differences compared with earlier non-selective antagonists.