Scott S.P. Wildman

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y4 (hP2Y4) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y4 receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2‐MeSATP=Ap4A>UDP(pure). ADP, ATPγS, 2‐MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y4 is more like the P2U receptor subtype. The rP2Y4 receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y2 receptor (a known P2U receptor). Thus, rP2Y4 and the P2Y2 subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi‐active) but can be distinguished as suramin‐insensitive and suramin‐sensitive P2U receptors, respectively.

Effects of extracellular pH on agonism and antagonism at a recombinant P2X2 receptor

Under voltage‐clamp conditions, the activity of agonists and antagonists at a recombinant P2X2 receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pHe). In normal Ringer (Mg2+ ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pHe (8.0–5.5). ATP‐responses reached a maximum at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.05±0.05. Receptor affinity for ATP was lowered when extracellular Ca2+ ions were replaced with equimolar Mg2+ ions. However, the amplitude of the ATP‐responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP‐affinity; the apparent pKa was 7.35±0.05. ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X2 receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. The potency order for agonists of P2X2 receptors was: ATP>2‐MeS‐ATPATPγS> ATPαS>>CTPBzATP, while other nucleotides were inactive. EC50 and nH values for full agonists were determined at pH 7.4 and re‐examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2‐MeSATP, ATPγS and ATPαS), without altering the potency order. The potency order for antagonists at P2X2 receptors was: Reactive blue‐2>trinitrophenol‐ATPPalatine fast blackCoomassie brilliant bluePPADS>suramin (at pH 7.4). IC50 values and slopes of the inhibition curves were re‐examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (IC50 values: 10.4±2 μM, at pH 7.4; 78±5 nM, at pH 6.5; 30±6 nM, at pH 5.5). In summary, a lowered pHe enhanced the activity of all agonists at P2X2 receptors but, with the exception of suramin, not antagonists. Since a lowered pHe is also known to enhance agonist activity at P2X receptors on sensory neurones containing P2X2 transcripts, the sensitization by metabolic acidosis of native P2X receptors containing P2X2 subunits may have a significant effect on purinergic cell‐to‐cell signalling.

Extended pharmacological profiles of rat P2Y2 and rat P2Y4 receptors and their sensitivity to extracellular H+ and Zn2+ ions.

Two molecularly distinct rat P2Y receptors activated equally by adenosine‐5′‐triphosphate (ATP) and uridine‐5′‐triphosphate (UTP) (rP2Y2 and rP2Y4 receptors) were expressed in Xenopus oocytes and studied extensively to find ways to pharmacologically distinguish one from the other. Both P2Y subtypes were activated fully by a number of nucleotides. Tested nucleotides were equipotent at rP2Y4 (ATP=UTP=CTP=GTP=ITP), but not at rP2Y2 (ATP=UTP>CTP>GTP>ITP). For dinucleotides (ApnA, n=2–6), rP2Y4 was only fully activated by Ap4A, which was as potent as ATP. All tested dinucleotides, except for Ap2A, fully activated rP2Y2, but none were as potent as ATP. ATPγS and BzATP fully activated rP2Y2, whereas ATPγS was a weak agonist and BzATP was inactive (as an agonist) at rP2Y4 receptors. Each P2Y subtype showed different sensitivities to known P2 receptor antagonists. For rP2Y2, the potency order was suramin>>PPADS= RB‐2>TNP‐ATP and suramin was a competitive antagonist (pA2, 5.40). For rP2Y4, the order was RB‐2>>suramin>PPADS> TNP‐ATP and RB‐2 was a competitive antagonist (pA2, 6.43). Also, BzATP was an antagonist at rP2Y4 receptors. Extracellular acidification (from pH 8.0 to pH 5.5) enhanced the potency of ATP and UTP by 8–10‐fold at rP2Y4 but did not affect agonist responses at rP2Y2 receptors. Extracellular Zn2+ ions (0.1–300 μM) coapplied with ATP inhibited agonist responses at rP2Y4 but not at rP2Y2 receptors. These two P2Y receptors differ significantly in terms of agonist and antagonist profiles, and the modulatory activities of extracellular H+ and Zn2+ ions. These pharmacological differences will help to distinguish between rP2Y2 and rP2Y4 receptors, in vivo.

Zn2+ modulation of ATP‐responses at recombinant P2X2 receptors and its dependence on extracellular pH

1 Using recombinant P2X2 receptors expressed in Xenopus oocytes, the modulatory effects of zinc (Zn2+) on ATP‐responses were studied under voltage‐clamp conditions and at different levels of extracellular pH. 2 Zn2+ (0.3–300 μM) added to the bathing medium potentiated ATP‐activated membrane currents, increasing ATP‐responses by up to 20 fold. This potentiating effect was reversed on washout. Zn2+‐potentiation was reduced in an exponential manner (decaying 1/e in 42 s) as the interval was lengthened between adding Zn2+ then ATP to the superfusate. 3 The potentiating effect of Zn2+ was progressively diminished by acidic shifts in extracellular pH (pHe) which, of itself, also potentiated ATP‐responses at P2X2 receptors. The maximal potentiating effects of Zn2+ and H+ were not additive. 4 Neither Zn2+ nor H+ potentiation of ATP‐responses was abolished by diethylpyrocarbonate (DEPC, 0.3–3 mM), which irreversibly denatures histidyl residues. Nine histidyl residues are present in the extracellular loop of P2X2 receptors. 5 Zn2+ also enhanced the blocking activity of the P2 receptor antagonist suramin at P2X2 receptors. Therefore, Zn2+ also mimics H+ in increasing suramin‐activity at P2X2 receptors. 6 In summary, Zn2+ and H+ potentiate agonist and antagonist activity at P2X2 receptors but their effects are not wholly alike for receptor agonism. There, the potentiating effects of Zn2+ are time‐dependent and gradually convert to inhibition while those of H+ are time‐independent, persistent and more potent, suggesting that either these modulators interact in a different way with a single allosteric site or with different allosteric sites.

Molecular cloning, functional characterization and possible cooperativity between the murine P2X4 and P2X4a receptors

We have cloned and functionally characterised the mouse orthologue of the P2X4 receptor, mP2X4, and a splice variant of this receptor, mP2X4a. mP2X4 is 388 amino acids in length and shares 94% and 87% identity with the rat and human P2X4 receptors, respectively, while mP2X4a is 361 amino acids in length and lacks a 27-amino acid region in the extracellular domain corresponding to exon 6 of the known P2X receptor gene structures. When expressed in Xenopus laevis oocytes, mP2X4 produces a rapid inward current in response to ATP with an EC50 of 1.68+/-0.2 microM, consistent with the affinity of the rat and human P2X4 receptors for ATP. This agonist response is potentiated by the P2X receptor antagonists suramin, Reactive blue 2 and, over a limited concentration range, by PPADS. Although mP2X4a forms a poorly functional homomeric receptor, it appears able to interact with the full-length mP2X4 subunit to result in a functional channel with a reduced affinity for ATP. These results suggest a possible role for splice variants of P2X receptors in the formation of functional heteromeric ion channels.

Modulation of ATP-responses at recombinant rP2X4 receptors by extracellular pH and zinc

The modulatory effects of extracellular H+ and Zn2+ were tested against ATP‐responses at rat P2X4 (rP2X4) receptors expressed in Xenopus oocytes under voltage‐clamp conditions. ATP (0.1–100 μM, at pH 7.5), evoked inward currents via rP2X4 receptors (EC50 value, 4.1±0.98 μM; nH, 1.2±0.1). ATP potency was reduced 2 fold, at pH 6.5, without altering maximal activity. ATP potency was reduced by a further 4 fold, at pH 5.5, and the maximal activity of ATP was also reduced. Alkaline conditions (pH 8.0) had no effect on ATP‐responses. Zn2+ (100 nM–10 μM) potentiated ATP‐responses at the rP2X4 receptor by 2 fold, whereas higher concentrations (30 μM–1 mM) inhibited ATP‐responses. Zn2+ potentiation was due to an increase in ATP potency, whereas its inhibitory action was due to a reduction in ATP efficacy. Zn2+ modulation of ATP‐responses was pH‐dependent. At pH 6.5, the bell‐shaped curve for Zn2+ was shifted to the right by 1 log unit. At pH 5.5, Zn2+ potentiation was abolished and its inhibitory effect reduced considerably. Suramin (50 μM) also potentiated ATP‐responses at rP2X4 receptors. Neither H+ (pH 6.5 and 5.5), Zn2+ (10–100 μM) or a combination of both failed to reveal an inhibitory action of suramin at rP2X4 receptors. In conclusion, H+ and Zn2+ exerted opposite effects on the rP2X4 receptor by lowering and raising agonist potency, respectively. H+ (3 μM) and Zn2+ (30 μM) also reduces agonist efficacy by lowering the number of rP2X4 receptors available for activation. The striking differences between the modulatory actions of H+ and Zn2+ at rP2X4 and rP2X2 receptors are discussed.

Selectivity of diadenosine polyphosphates for rat P2X receptor subunits.

The pharmacological activity of diadenosine polyphosphates was investigated at three recombinant P2X receptors (rat P2X1, rat P2X3, rat P2X4) expressed in Xenopus oocytes and studied under voltage-clamp conditions. For the rat P2X1 receptor, only P1,P6-diadenosine hexaphosphate (Ap6A) was a full agonist yet 2-3 folds less potent than ATP. At rat P2X3, P1,p4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A) and Ap6A were full agonists and more potent than ATP. Ap4A alone was equipotent with ATP at rat P2X4, but only as a partial agonist. Compared to known data for rat P2X2 and human P2X1 receptors, our findings contrast with rat P2X2 where only Ap4A is a full agonist although four folds less potent than ATP. At rat and human orthologues of P2X1, Ap5A was a partial agonist with similar potency. These data provide a useful basis for selective agonists of P2X receptor subunits.

Potentiation of ATP-responses at a recombinant P2x2 receptor by neurotransmitters and related substances.

The modulator effects of a series of neurotransmitters and related substances were tested on responses to adenosine 5′‐triphosphate (ATP) at a recombinant P2X2 receptor expressed in defolliculated Xenopus oocytes. Nicotine, 5‐hydroxytryptamine (5‐HT), noradrenaline, adenosine, bradykinin and histamine (all 100 μm) potentiated the responses to ATP (3 μm), an effect found due to acidification of the bathing solution by these drugs. Arachidonic acid, met‐enkephalin, substance P, calcitonin gene‐related peptide (CGRP) (all 100 μm) and nerve growth factor (NGF; 50 ng ml−1) potentiated the responses to ATP (3 μm) through a largely or wholly pH‐independent effect. Small acidic and alkaline shifts, as little as 0.03 pH‐units, enhanced or diminished the responses to ATP, respectively. A linear relationship existed between the degree of potentiation of the ATP‐induced responses caused by nicotine, 5‐HT, noradrenaline, adenosine, bradykinin and histamine and the potentiation of these responses induced by the addition of acid to the superfusate. Since P2X receptors on sensory neurones include P2X2 subunits, the attendant acidosis and ATP‐release associated with tissue injury may play a role in sensitizing sensory nerve fibres.

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

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. Superfused ATP (0.03–30 μM, 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. Superfused ATP (0.01–300 μM, at pH 7.5) evoked inward currents at rP2X3 receptors (EC50 value, 1.8±0.3 μM). ATP activity was affected only at pH 5.5, reducing agonist potency 15 fold without altering the maximum ATP effect. 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 μM). However, the Zn2+ effect was pH‐independent, suggesting Zn2+‐ and H+‐inhibition of ATP‐responses occur through independent processes. Extracellular Zn2+ weakly potentiated ATP‐responses at rP2X3 receptors (EC50 value, 11±1 μM). 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. 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.

Sodium-Dependent Regulation of Renal Amiloride-Sensitive Currents by Apical P2 Receptors

The epithelial sodium channel (ENaC) plays a major role in the regulation of sodium balance and BP by controlling Na(+) reabsorption along the renal distal tubule and collecting duct (CD). ENaC activity is affected by extracellular nucleotides acting on P2 receptors (P2R); however, there remain uncertainties over the P2R subtype(s) involved, the molecular mechanism(s) responsible, and their physiologic role. This study investigated the relationship between apical P2R and ENaC activity by assessing the effects of P2R agonists on amiloride-sensitive current in the rat CD. Using whole-cell patch clamp of principal cells of split-open CD from Na(+)-restricted rats, in combination with immunohistochemistry and real-time PCR, we found that activation of metabotropic P2R (most likely the P2Y(2) and/or (4) subtype), via phospholipase C, inhibited ENaC activity. In addition, activation of ionotropic P2R (most likely the P2X(4) and/or (4/6) subtype), via phosphatidylinositol-3 kinase, either inhibited or potentiated ENaC activity, depending on the extracellular Na(+) concentration; therefore, it is proposed that P2X(4) and/or (4/6) receptors might function as apical Na(+) sensors responsible for local regulation of ENaC activity in the CD and could thereby help to regulate Na(+) balance and systemic BP.