Identification of P2X receptors in cultured mouse and rat parasympathetic otic ganglion neurones including P2X knockout studies

We have used patch-clamp recording from cultured neurones, immunohistochemistry and gene deletion techniques to characterize the P2X receptors present in mouse otic ganglion neurones, and demonstrated the presence of similar receptors in rat neurones. All neurones from wild-type (WT) mice responded to ATP (EC(50) 109 microM), but only 38% also responded to alpha beta-meATP (EC(50) 39 microM). The response to alpha beta-meATP was blocked by TNP-ATP with an IC(50) of 38.6 nM. Lowering extracellular pH and co-application of Zn(2+) potentiated responses to ATP and alpha beta-meATP. In P2X(3)(-/-) mouse otic ganglion, all neurones tested responded to 100 microM ATP with a sustained current, but none responded to alpha beta-meATP. In P2X(2)(-/-) mice, no sustained currents were observed, but 36% of neurones responded to both ATP and alpha beta-meATP with transient currents. In P2X(2)/P2X(3)(Dbl-/-) mice, no responses to ATP or alpha beta-meATP were detected, suggesting that other P2X subunits were not involved. In rat otic ganglia, 96% of neurones responded to both ATP and alpha beta-meATP with sustained currents, suggesting a greater proportion of neurones expressing P2X(2/3) receptors. The maximum response to alpha beta-meATP was 40-60% of that evoked by ATP in the same cell. Immunohistochemistry revealed staining for P2X(2) and P2X(3) subunits in WT mouse otic ganglion neurones, which was absent in knockout animals. In conclusion, we have shown for the first time that at least two distinct P2X receptors are present in mouse and rat otic neurones, probably homomeric P2X(2) and heteromeric P2X(2/3) receptors.     

Competitive antagonism of recombinant P2X(2/3) receptors by 2', 3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP)

TNP-ATP has become widely recognized as a potent and selective P2X receptor antagonist, and is currently being used to discriminate between subtypes of P2X receptors in a variety of tissues. We have investigated the ability of TNP-ATP to inhibit alpha,beta-methylene ATP (alpha,beta-meATP)-evoked responses in 1321N1 human astrocytoma cells expressing recombinant rat or human P2X(2/3) receptors. Pharmacological responses were measured using electrophysiological and calcium imaging techniques. TNP-ATP was a potent inhibitor of P2X(2/3) receptors, blocking both rat and human receptors with IC(50) values of 3 to 6 nM. In competition studies, 10 to 1000 microM alpha,beta-meATP was able to overcome TNP-ATP inhibition. Schild analysis revealed that TNP-ATP was a competitive antagonist with pA(2) values of -8.7 and -8.2. Inhibition of P2X(2/3) receptors by TNP-ATP was rapid in onset, reversible, and did not display use dependence. Although the onset kinetics of inhibition were concentration-dependent, the TNP-ATP off-kinetics were concentration-independent and relatively slow. Full recovery from TNP-ATP inhibition did not occur until >/=5 s after removal of the antagonist. Because of the slow off-kinetics of TNP-ATP, full competition with alpha,beta-meATP for receptor occupancy could be seen only after both ligands had reached a steady-state condition. It is proposed that the slowly desensitizing P2X(2/3) receptor allowed this competitive interaction to be observed over time, whereas the rapid desensitization of other P2X receptors (P2X(3)) may mask the detection of competitive inhibition by TNP-ATP.     

Properties of native P2X receptors in rat trigeminal mesencephalic nucleus neurones: lack of correlation with known, heterologously expressed P2X receptors

Trigeminal mesencephalic nucleus (MNV) neurones express functional P2X receptors. In order to determine the molecular identity of the P2X receptors in this nucleus we have used whole cell patch clamp recording of P2X receptor-mediated currents to determine the pharmacological properties of the receptors, and have compared them with those of cloned P2X receptor subunits. The purine nucleotides ATP (300 microM), ATP-gamma-S (30 microM) and alphabetameATP (300 microM) evoked inward currents in all MNV neurones whereas alphabetameADP (300 microM) did not. betagammame-L-ATP (300 microM) evoked only a small ( approximately 20 pA) current in 3 out of 6 MNV neurones. The P2X receptor antagonist TNP-ATP (10 nM-10 microM) and raised extracellular Ca(2+) (8 and 30 mM) reduced, but did not abolish, the current evoked by ATP-gamma-S. The current remaining in TNP-ATP was insensitive to blockade by raised Ca(2+). These properties suggest that MNV neurones do not express homomeric P2X(3), P2X(4) or P2X(6) receptors. Whilst the TNP-ATP-insensitive ATP-gamma-S-evoked current has many characteristics similar to both homomeric P2X(2) and P2X(5) receptors, its insensitivity to blockade by raised Ca(2+) is difficult to reconcile with the receptor being a P2X(2) or P2X(5) homomeric channel. More likely, the receptor is a heteromer that comprises either or both of these subunits. The TNP-ATP-sensitive component of the ATP-gamma-S-evoked current is dissimilar to known cloned homomeric or heteromeric P2X receptors.     

Sensitization by extracellular Ca(2+) of rat P2X(5) receptor and its pharmacological properties compared with rat P2X(1)

The recombinant rat P2X(5) (rP2X(5)) receptor, a poorly understood ATP-gated ion channel, was studied under voltage-clamp conditions and compared with the better understood homomeric rP2X(1) receptor with which it may coexist in vivo. Expressed in defolliculated Xenopus laevis oocytes, rP2X(5) responded to ATP with slowly desensitizing inward currents that, for successive responses, ran down in the presence of extracellular Ca(2+) (1.8 mM). Replacement of Ca(2+) with either Ba(2+) or Mg(2+) prevented rundown, although agonist responses were very small, whereas reintroduction of Ca(2+) for short periods of time (<300 s) before and during agonist application yielded consistently larger responses. Using this Ca(2+)-pulse conditioning, rP2X(5) responded to ATP and other nucleotides (ATP, 2-methylthio-ATP, adenosine-5'-O-(thiotriphosphate), 2'-&-3'-O-(4-benzoylbenzoyl)-ATP, alpha,beta-methylene-ATP, P(1)-P((4))-diadenosine-5'-phosphate, and more) with pEC(50) values within 1 log unit of respective determinations for rP2X(1). Only GTP was selective for rP2X(5), although 60-fold less potent than ATP. At rP2X(5), lowering extracellular pH reduced the potency and efficacy of ATP, whereas extracellular Zn(2+) ions (0.1-1000 microM) potentiated then inhibited ATP responses in a concentration-dependent manner. However, these modulators affected rP2X(1) receptors in subtly different ways-with increasing H(+) and Zn(2+) ion concentrations reducing agonist potency. For P2 receptor antagonists, the potency order at rP2X(5) was pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) > 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) > suramin > reactive blue 2 (RB-2) > diinosine pentaphosphate (Ip(5)I). In contrast, the potency order at rP2X(1) was TNP-ATP = Ip(5)I > PPADS > suramin = RB-2. Thus, the Ca(2+)-sensitized homomeric rP2X(5) receptor is similar in agonist profile to homomeric rP2X(1)-although it can be distinguished from the latter by GTP agonism, antagonist profile, and the modulatory effects of H(+) and Zn(2+) ions.     

Agonist-dependence of recovery from desensitization of P2X(3) receptors provides a novel and sensitive approach for their rapid up or downregulation

1. Fast-desensitizing P2X(3) receptors of nociceptive dorsol root ganglion (DRG) neurons are thought to mediate pain sensation. Since P2X(3) receptor efficiency is powerfully modulated by desensitization, its underlying properties were studied with patch-clamp recording. 2. On rat cultured DRG neurons, 2 s application of ATP (EC(50)=1.52 microm), ADP (EC(50)=1.1 microm) or alpha,beta-meATP (EC(50)=1.78 microm) produced similar inward currents that fully desensitized, at the same rate, back to baseline. Recovery from desensitization was much slower after ATP and ADP than after alpha,beta-meATP and, in all cases, it had sigmoidal time course. 3. By alternating the application of ATP and alpha,beta-meATP, we observed complete cross-desensitization indicating that these agonists activated the same receptors. This notion was confirmed by the similar antagonism induced by 2', 3'-O-(2,4,6,trinitrophenyl)-adenosine triphosphate (TNP-ATP). 4. Recovery from desensitization elicited by ATP was unexpectedly shaped by transient application of alpha,beta-methylene-adenosine triphosphate (alpha,beta-meATP), and vice versa. Thus, short-lasting, full desensitization produced by alpha,beta-meATP protected receptors from long-lasting desensitization induced by subsequent ATP applications. ATP and ADP had similar properties of recovery from desensitization. 5. Low nm concentrations of alpha,beta-meATP (unable to evoke membrane currents) could speed up recovery from ATP-induced desensitization, while low nm concentrations of ATP enhanced it. Ambient ATP levels were found to be in the pm range (52+/-3 pm). 6. The phenomenon of cross-desensitization and protection was reproduced by rP2X(3) receptors expressed by rat osteoblastic cell 17/2.8 or human embryonic kidney cell 293 cells, indicating P2X(3) receptor specificity. 7. It is suggested that transient application of an agonist that generates rapid recovery from desensitization, is a novel, powerful tool to modulate P2X(3) receptor responsiveness to the natural agonist ATP.     

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.     

Profiling at recombinant homomeric and heteromeric rat P2X receptors identifies the suramin analogue NF449 as a highly potent P2X1 receptor antagonist

P2X receptors are cation channels gated by extracellular ATP and related nucleotides. Because of the widespread distribution of P2X receptors and the high subtype diversity, potent and selective antagonists are needed to dissect their roles in intact tissues. Based on suramin as a lead compound, several derivates have been described that block recombinant P2X receptors with orders of magnitude higher potency than suramin. Here we characterized the suramin analogue 4,4',4',4'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) with respect to its potency to antagonize ATP or alphabeta-methyleneadenosine 5'-trisphosphate-induced inward currents of homomeric rat P2X(1)-P2X(4) receptors or heteromeric P2X(1 + 5) and P2X(2+3) receptors, respectively. NF449 most potently blocked P2X(1) and P2X(1 + 5) receptors with IC(50) values of 0.3 nM and 0.7 nM, respectively. Three to four orders of magnitude higher NF449 concentrations were required to block homomeric P2X(3) or heteromeric P2X(2 + 3) receptors (IC(50) 1.8 and 0.3 microM, respectively). NF449 was least potent at homomeric P2X(2) receptors (IC(50) 47 microM) and homomeric P2X(4) receptors (IC(50) > 300 microM). Altogether, these results characterize NF449 as the so far most potent and selective antagonist of receptors incorporating the P2X(1) subunit such as the P2X(1) homomer and the P2X(1 + 5) heteromer.     

Kinetics of antagonist actions at rat P2X2/3 heteromeric receptors

1. Currents through heteromeric P2X(2/3) receptors were evoked by applying alpha,beta-methylene-ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X(2) and P2X(3) subunits. The concentration of alpha,beta-methylene-ATP were < or =30 microM because higher concentrations can activate homomeric P2X(2) receptors. The kinetics of action of three structurally unrelated antagonists were studied; these were 2', 3'-O-(2,4,6,trinitrophenyl)-ATP (TNP-ATP), pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. The association and dissociation rate constants were determined by pre-applying the antagonist for various periods prior to the co-application of agonist and antagonist, or by changing the solution from one containing only the agonist to one containing both agonist and antagonist. The high affinity of TNP-ATP for the P2X(2/3) receptor (K(D) approximately 2 nM) results from fast binding (k(+1) approximately 100 microM(-1) s(-1)) rather than slow unbinding (k(-1) approximately 0.3 s(-1)). For suramin (K(D) approximately 1 microM) the association rate constant ( approximately 1 microM(-1) s(-1)) was 100 times slower than that of TNP-ATP but the dissociation rate constant was similar (k(-1) approximately 1 s(-1)). PPADS (K(D) approximately 0.1 microM) associated and dissociated some 100 - 10,000 times more slowly than the other antagonists.     

Functional characterization of the P2X(4) receptor orthologues

1. The aim of this study was to functionally characterize the recombinant mouse P2X(4) receptor and to compare its pharmacological properties with those of the human and rat orthologues. 2. Whole cell recordings were made from rafts of HEK-293 cells stably expressing recombinant mouse, rat or human P2X(4) receptors, using Cs-aspartate containing electrodes (3 - 8 MOmega) in a HEPES-buffered extracellular medium. 3. The agonist potency of ATP at the three species orthologues was similar, with mean EC(50) values of 2.3 microM, 1.4 microM and 5.5 microM, respectively. 4. Adenosine-5'-tetraphosphate (AP4) acted as a partial agonist with respect to ATP at the mouse and human P2X(4) receptors (EC(50)=2.6 and 3.0 microM), but was significantly less potent at the rat orthologue (EC(50)=20.0 microM). alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) also acted as a partial agonist, producing 29% of the maximum response at the mouse P2X(4) and 24% at the human P2X(4) receptor. 5. In contrast to the other species orthologues, alpha,beta-meATP failed to elicit a significant agonist response at rat P2X(4) receptors, and was found to act as an antagonist, with an IC(50) of 4.6 microM, against 10 microM ATP. 6. Mouse P2X(4) receptors were found to be sensitive to the antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (IC(50)=10.5 microM), as were human P2X(4) receptors (IC(50)=9.6 microM). The rat receptor however, showed a low sensitivity to PPADS (IC(50)>100 microM). 7. All three orthologues were relatively suramin-insensitive (IC(50)>100 microM) and insensitive to 1-[N, O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62; IC(50)>3 microM). 8. Our results suggest that the pharmacological properties of the mouse receptor are most similar to the human P2X(4) receptor, and differ markedly from the rat receptor.     

Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors

1. The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X(1-4) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5'-adenylic deaminase, and purified using reverse-phase chromatography. 3. Against ATP-responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10-100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP effect. However, the Schild plot was non-linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non-surmountable antagonist. 4. Against ATP-responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6. 0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. 5. Diinosine polyphosphates failed to inhibit ATP-responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1 - 4 receptors. 6. Thus, Ip5I acted as a potent and relatively-selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high-affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (100 nM) concentrations.     

ATP-gated currents in rat primary auditory neurones in situ arise from a heteromultimetric P2X receptor subunit assembly

Spiral ganglion neurones provide the primary afferent innervation to sensory hair cells within the mammalian cochlea. Recent evidence suggests that their function may be modulated by purinergic signalling mechanisms, associated with release of adenosine 5'-triphosphate (ATP). Utilising a newly developed slice preparation of the neonatal rat cochlea, we have investigated the response of neurones in situ, to purinergic agonists and antagonists using whole-cell voltage clamp recordings. In cells identified as type I spiral ganglion neurones on the basis of morphology and voltage-dependent conductances, pressure-applied ATP, alpha,beta-methyleneATP (alpha,beta-meATP), 2-methylthioATP (2-MeSATP) and adenosine 5'-diphosphate (ADP) elicited a consistent phenotype of desensitising, inwardly rectifying current. The ATP-activated currents were reversibly blocked by the P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM), and 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP; IC(50) 407 nM). Neurones were more sensitive to ATP at low pH. The EC(50) value for ATP shifted from 18 microM at pH 7.3, to 1 microM at pH 6.3, with Hill coefficients of approximately 1. The results indicate that ATP-gated ion channels in spiral ganglion neurones arise from a specific heteromultimeric assembly of P2X receptor subunits which has no correspondence with present recombinant P2X receptor models.     

A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors

We have examined the role of ATP-dependent P2X(1) receptors in megakaryocytes (MKs) and platelets using receptor-deficient mice and selective agonists. Alpha,beta-meATP- and ATP- evoked ionotropic inward currents were absent in whole-cell recordings from MKs of P2X(1)(-/-) mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X(1) receptors. P2X(1) receptor deficiency had no effect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development. P2Y receptor-stimulated [Ca(2+)](i) responses were unaffected in MKs from P2X(1)(-/-) mice, however the inward cation current associated with Ca(2+) release was reduced by approximately 50%, suggesting an interaction between the membrane conductances activated by P2X(1) and P2Y receptors. Interaction between P2X(1) and P2Y receptors in human platelets was also examined using [Ca(2+)](i) recordings from cell suspensions. Alpha,beta-meATP (10 microM) evoked a rapid transient P2X(1) receptor-mediated increase in [Ca(2+)](i), whereas ADP-(10 microM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co-application of alpha, beta-meATP and ADP resulted in marked acceleration and amplification of the peak [Ca(2+)](i) response. We conclude that ionotropic P2X(1) receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation.     

Multiple P2X receptors on guinea-pig pelvic ganglion neurons exhibit novel pharmacological properties

1. Application of ATP and alpha,beta-methylene ATP (alpha beta meATP) to voltage-clamped guinea-pig pelvic neurons produced three types of inward currents. A fast-desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly-desensitizing currents, and the remainder had biphasic responses. 2. Slowly-desensitizing responses were characterized pharmacologically. The response to alpha beta meATP 100 microM was 46+/-27% (range 0--100%) of that evoked by ATP 100 microM in the same cell. Cross-desensitization indicated the presence of alpha beta meATP-sensitive and -insensitive receptors. 3. The concentration-response curve for alpha beta meATP had an EC(50) of 55 microM, and a Hill coefficient of 0.99, while at the alpha beta meATP-insensitive receptor, ATP had an EC(50) of 73 microM, with a Hill coefficient of 1.78. 4. The response to alpha beta meATP was blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and Cibacron blue. However, the alpha beta meATP-insensitive receptor was inhibited by PPADS, but not by the other two antagonists. 5. 2'- (or 3'-) O-trinitrophenyl-ATP was 10 times more potent in inhibiting responses to alpha beta meATP than to ATP (at the alpha beta meATP-insensitive receptor). 6. Lowering extracellular pH potentiated responses to alpha beta meATP and ATP, while raising pH attenuated them. 7. Co-application of Zn(2+) (3--300 microM) inhibited the responses to alpha beta meATP and ATP, with IC(50) values of 286 and 60 microM, respectively. 8. In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X(2) homomers, at least three distinct P2X receptors are present in guinea-pig pelvic neurons, probably homomeric P2X(2), P2X(3) and heteromeric P2X(2/3) receptors. However, some of the novel pharmacological properties observed suggest that the guinea-pig P2X receptor subtypes may differ from their rat orthologues.     

Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their desensitization

BACKGROUND AND PURPOSE: The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X(3) receptor-channels. EXPERIMENTAL APPROACH: Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X(3) receptors (HEK293-hP2X(3) cells) and rat dorsal root ganglion (DRG) neurons. KEY RESULTS: In both cell types, the P2Y(1,12,13) receptor agonist, ADP-beta-S, inhibited P2X(3) currents evoked by the selective agonist, alpha,beta-methylene ATP (alpha,beta-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-beta-S, a procedure known to block all G protein heterotrimers. P2X(3) currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X(3) receptor agonist alpha,beta-meATP. In the presence of intracellular GDP-beta-S, ATP- and alpha,beta-meATP-induced currents were identical. Recovery from P2X(3) receptor desensitization induced by repetitive ATP application was slower than the recovery from alpha,beta-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-beta-S, the recovery from the ATP- and alpha,beta-meATP-induced desensitization were of comparable speed. CONCLUSIONS AND IMPLICATIONS: Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X(3) receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X(3) and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.     

2', 3'-O-(2,4,6,trinitrophenyl)-ATP and A-317491 are competitive antagonists at a slowly desensitizing chimeric human P2X3 receptor

(1) Rapid desensitization of ligand-gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast-channel activation difficult. Investigation of the antagonist pharmacology of ATP-sensitive homomeric P2X3 receptors is limited by agonist-evoked fast-desensitization kinetics. (2) In the present studies, chimeric receptors were created using the coding sequence for the N-terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast-desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C-terminus of the other receptor (designated P2X2-3 and P2X3-2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium-imaging methods. (3) Chimeric P2X2-3 and P2X3-2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild-type human P2X2a and P2X3 receptors, respectively. (4) The P2X2-3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild-type receptor being activated by low concentrations of both ATP and alpha,beta-meATP. In contrast, the P2X3-2 chimera had markedly reduced sensitivity to both agonists. (5) The P2X3 receptor antagonists TNP-ATP and A-317491 were shown to be potent, competitive antagonists of the P2X2-3 chimera (Ki=2.2 and 52.1 nm, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild-type homomeric P2X3 receptors.     

Ethanol differentially affects ATP-gated P2X(3) and P2X(4) receptor subtypes expressed in Xenopus oocytes

P2X receptors are cation-selective, ligand-gated ion channels activated by synaptically released, extracellular adenosine 5'-triphosphate (ATP). ATP-gated currents are inhibited by ethanol when tested in dorsal root ganglion and CA1 neurons. Recently, we reported differences in sensitivity to ethanol inhibition between homomeric P2X(2) and P2X(4) receptors expressed in Xenopus oocytes, which suggested that subunit composition of native P2X receptors determines their ethanol sensitivity. The present study extended the investigation to P2X(3) receptors. The effects of ethanol and zinc ions (Zn(2+)) were tested on homomeric P2X(3) and P2X(4) receptors expressed in Xenopus oocytes using two-electrode voltage clamp. Ethanol potentiated ATP-gated P2X(3) receptor currents in a concentration dependent manner. In contrast, ethanol inhibited P2X(4) receptor function. Ethanol did not directly alter receptor function, nor did it alter the Hill coefficient or maximal ATP response (E(max)) in either P2X(3) or P2X(4) receptors. Ethanol increased the maximal response to Zn(2+) ATP-gated currents in P2X3 receptors which suggests that ethanol and Zn(2+) act on different sites. The differences in ethanol response of P2X(3) and P2X(4) receptors set the stage for future investigations that will use chimeric P2X receptors or other molecular manipulations of P2X structure to investigate the molecular sites and mechanisms of action of ethanol.     

Antagonistic properties of the suramin analogue NF023 at heterologously expressed P2X receptors

The suramin analogue 8,8'-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023) antagonizes in a competitive fashion P2X receptor-mediated responses in certain vascular and visceral smooth muscles. In the present study, the effect of NF023 on voltage-clamped Xenopus oocytes heterologously expressing homomultimeric P2X1-P2X4 as well as heteromultimeric P2X2/P2X3 receptors has been characterized. P2X1 receptors were most sensitive to inhibition by NF023 with IC50 values of 0.24 and 0.21 microM for the rat and human homologue, respectively. P2X3 receptors have an intermediate sensitivity with IC50 values of 8.5 and 28.9 microM for rat and human subtypes, respectively and P2X2 was the least sensitive subtype (IC50 > 50 microM). P2X4 receptors were insensitive to NF023 at concentrations up to 100 microM. Coexpression of rat P2X3 with rat P2X2 resulted in receptors whose sensitivity to NF023 was identical to that obtained for homomultimeric rat P2X3 receptors (alphabeta meATP as agonist; IC50 = 1.4 and 1.6 microM, respectively). NF023 inhibited P2X1 receptors in a voltage-insensitive manner. In addition, NF023 (5 and 30 microM) caused a shift of the concentration-response curve to the right without affecting the maximal response to ATP (K(B) = 1.1 +/- 0.2 microM). Our results indicate that NF023 is a subtype-selective and surmountable antagonist at P2X1 receptors heterologously expressed in Xenopus oocytes.     

ADP is not an agonist at P2X(1) receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets

ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X(1), P2Y(1) and P2T(AC). Given the low potency of ADP at P2X(1) receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X(1) receptors by ADP following HPLC and enzymatic purification. Native P2X(1) receptor currents in megakaryocytes were activated by alpha, beta-meATP (10 microM) and commercial samples of ADP (10 microM), but not by purified ADP (10 - 100 microM). Purified ADP (up to 1 mM) was also inactive at recombinant human P2X(1) receptors expressed in XENOPUS: oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca(2+) mobilization in rat megakaryocytes. In human platelets, P2X(1) and P2Y receptor-mediated [Ca(2+)](i) responses were distinguished by their different kinetics at 13 degrees C. In 1 mM Ca(2+) saline, alpha,beta-meATP (10 microM) and commercial ADP (40 microM) activated a rapid [Ca(2+)](i) increase (lag time < or =0.5 s) through the activation of P2X(1) receptors. Hexokinase treatment of ADP shifted the lag time by approximately 2 s, indicating loss of the P2X(1) receptor-mediated response. A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X(1) receptors, whereas ADP is a selective agonist at metabotropic (P2Y(1) and P2T(AC)) receptors.     

Modulatory activity of extracellular H+ and Zn2+ on ATP-responses at rP2X1 and rP2X3 receptors.

1 The modulatory activity of extracellular H+ and Zn2+ was examined on ATP-responses at rat P2X1 (rP2X1) and rat P2X3 (rP2X3) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2 Superfused ATP (0.03-30 microM, at pH 7.5) evoked inward currents at rP2X1 receptors (EC50 value, 300+/-7 nM). ATP potency was reduced 2 fold at pH 6.5, and 6 fold at pH 5.5, without altering the maximum ATP effect. Alkaline conditions (pH 8.0) did not alter ATP activity. 3 Superfused ATP (0.01 - 300 microM, at pH 7. 5) evoked inward currents at rP2X3 receptors (EC50 value, 1.8+/-0.3 microM). ATP activity was affected only at pH 5.5, reducing agonist potency 15 fold without altering the maximum ATP effect. 4 Extracellular Zn2+ inhibited ATP-responses at rP2X1 receptors in a time-dependent manner, a 20 min pre-incubation being optimal (IC50 value, 1.0+/-0.2 microM). However, the Zn2+ effect was pH-independent, suggesting Zn2+- and H+-inhibition of ATP-responses occur through independent processes. 5 Extracellular Zn2+ weakly potentiated ATP-responses at rP2X3 receptors (EC50 value, 11+/-1 microM). The Zn2+ effect was dependent on pre-incubation time and, with 20 min pre-incubation periods, Zn2+ potentiated then inhibited ATP-responses in a concentration-dependent, but pH-independent, manner. 6 In summary, ATP activity at rP2X1 receptors was decreased by both extracellular H+ and Zn2+ and their effects were additive. ATP activity at rP2X3 receptors was less sensitive to H+-inhibition and, in contrast, was potentiated by Zn2+ in a pH-independent manner. These differential effects may help distinguish P2X1 and P2X3 receptors in whole tissues.