New insights on P2X purinoceptors

Naunyn-Schmiedeberg's Arch Pharmacol (1995) 352:585-596 © Springer-Verlag 1995

Patrick RA. Humphrey - Gary Buell - Ian Kennedy Baljit S. Khakh • Anton D. Michel Annmarie Surprenant • Derek J. Trezise

New insights on P2x purinoceptors

Received: 29 May 1995 / Accepted: 16 August 1995

Abstract Significant advances in understanding of P2x purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becom- ing available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for Pzx receptor heterogeneity, from data with agonists, has recently been reported.

A number of new antagonists at Pzx purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2x purinoceptor.

Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2x purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2x purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2x purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2x purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better un-

ERA. Humphrey (~e) . I. Kennedy • B.S. Khakh • A.D. Michel D.J. Trezise Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK

G. Buell. A. Surprenant Glaxo Institute for Molecular Biology, 14 chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland

derstanding of the physiological and pathological importance of ATP and its activation of P2x purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.

Key words ATP receptors . Structure Function Transduction • recombinant receptors • Cation channels - Electrophysiology

Introduction

It is now recognized that ATP may have important physiological and/or pathological roles as an extracellular humor- al mediator, in addition to its obvious essential role in pro- viding a molecular energy source (see Abbrachio and Burnstock 1994). ATP mediates its various effects on cells throughout the body by activating specific membrane receptors, first postulated by Burnstock in 1972 (Burnstock 1972). It is now known that these receptors, currently called P2 purinoceptors, comprise receptors of the ligand- gated ion channel type as well as of the G-protein linked super-family (Dalziel and Westfall 1994; Fredholm et al. 1994). Historically, these receptors have been termed P2x and P2Y purinoceptors, respectively, but the recent knowledge that there are various types of both receptors raises questions about how they should be characterized pharma- cologically and whether current approaches to classification and nomenclature are appropriate (see reviews by Ab- brachio and Bumstock 1994; Fredholm et al. 1994).

In the last two or three years, some major new insights into the pharmacology of P2 purinoceptors, and in particular on P2x purinoceptors, have been published. The sole focus of this review will be the P2x purinoceptor, which as a ligand-gated cation channel may be of particular importance physiologically as a mediator of fast transmission in both peripheral and central neurones (Edwards et al. 1992; Evans et al. 1992; Silinsky and Gerzanich 1993).

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Two distinct genes for P2x purinoceptors have only recently been cloned, but the characteristics of their respec- tive recombinant receptors are not readily equatable with equivalent receptors in whole tissues (Brake et al. 1994; Valera et al. 1994). This situation has arisen partly because of the problems of characterizing these receptors functionally with the current drag tools (e.g. see Trezise et al. 1994a). The known, but frequently ignored, limitations of the agonists and antagonists available for their characterization have become even more obvious on the basis of recent findings which will be discussed.

Indeed the originally proposed operational characteristics for definition of a Pzx purinoceptor are no longer va- lid (Burnstock and Kennedy 1985). Thus, an agonist action of a,fl-methylene ATP can no longer be considered as diagnostic of the presence or absence of a Pzx purinocep- tot since it is weak or inactive at some P2x purinoceptors (Brake et al. 1994; Khakh et al. 1995a). The desensitiza- tion of a response to ATP by a,fl-methylene ATP no longer seems an appropriate characteristic since some P2x purinoceptor-mediated responses are remarkably prone to tachy- phylaxis, while others are not (Evans and Kennedy 1994; Khakh et al. 1995a, c). Furthermore, there is now evidence to indicate that suramin is not a specific P2 purinoceptor antagonist since it has been shown to block the direct actions of other neurotransmitters (see below).

In the light of such critical observations on P2x purinoceptor pharmacology, conflicting with previous concep- tions of P2x purinoceptor characteristics, it is important to consider the implications. With advances in molecular biology now adding to our knowledge, it would seem pru- dent to consider the characterization of these receptors using an integrated approach involving operational (drug recognition), transductional (electrophysiology) and structural (amino acid sequence) characteristics, an approach which has proved useful in other areas such as for example 5-hydroxytryptamine and prostaglandin receptor characterization (Coleman and Humphrey 1993; Humphrey et al. 1993; Coleman et al. 1994; Hoyer et al. 1994).

Operational characteristics of P2x purinoceptors

Agonists

A major component of the original basis for division of P2 purinoceptors into different groups was the characteristic rank order of agonist potencies obtained for series of purine nucleotides and nucleosides in different multicellular preparations (Burnstock and Kennedy 1985). In certain smooth muscle preparations, such as rabbit ear artery, rat and guinea-pig vas deferens and guinea-pig urinary bladder, contractile responses to purine nucleotides were attributed to activation of P2x purinoceptors. In these tissues the ATP analogue, a,fl-methylene ATR is a far more potent agonist than ATR and has often been used to desensitize responses to other purine nucleotide agonists. 2- Methylthio ATP is only weakly active and this was gener-

ally considered as a negative diagnostic feature of P2x purinoceptors. From the outset (Bumstock and Kennedy 1985), it was recognized that a likely complicating factor in the study of receptors for ATP in this manner was the propensity of some nucleotides to be broken down, and perhaps taken up. However, until recently, the extent to which such factors can distort determinations of agonist potencies was poorly understood, and thus largely over- looked.

Comparisons of results obtained from multicellular preparations containing P2x purinoceptors, with those from single cells, in which problems of agonist removal are largely circumvented, provide circumstantial evidence that the consequences of ATP breakdown for receptor characterization are considerable. In both the guinea-pig urinary bladder and the rabbit ear artery, ATP is much less potent in contracting the whole muscle preparation than in evok- ing inward currents in single dispersed cells (Benham and Tsien 1987; Inoue and Brading 1990; O'Connor et al. 1990). In contrast, a,fl-methylene ATE which is generally resistant to ecto-ATPase activity (see below), is about equipotent under both experimental conditions. This has been studied systematically by both Evans and Kennedy (t994) and Khakh et al. (1995c) who have demonstrated that ATP is highly potent at producing inward currents in dispersed smooth muscle cells, but has low potency in the corresponding whole muscle.

Recently, we compared the potencies of agonists at P2x purinoceptors in rat nodose ganglion cells with those in the corresponding multicellular preparation, the rat vagus nerve (Trezise et al. 1993; Khakh et al. 1995a). When applied by a concentration-clamp method to individual voltage-clamped neuronal cells, ATP evoked marked inward currents with a threshold concentration of 100 nM and an ECs0 value of 1.2 gM (Khakh et al. 1995a), whilst in contrast concentrations of ATP in excess of 10/aM were required to depolarize the vagus nerve (Trezise et al. 1993). Similar concentrations of a,fl-methylene ATP were effective in both systems. In an attempt to rationalize these findings, the agonist effects of ATP and a,fl-methylene ATP were re-determined in the rat vagus nerve in the absence of Ca 2+ and Mg 2+ (+ 1 mM EDTA), experimental conditions under which ATP breakdown is virtually abol- ished (see below). This procedure markedly increased the potency of ATP such that similar concentrations of ATP were effective in both the single cells and the whole nerve trunk (Trezise et al. 1994a; Khakh et al. 1995a). As con- sideration of metabolism predicts, the potency of a,fi- methylene ATP was not modified by the complete removal of all divalent cations (see Fig. 1). These findings largely refute the implication that measurements made in single cells do not underlie the responses observed in the equivalent whole tissue, and clearly demonstrate potent agonist effects of ATP at P2x purinoceptors in a multicellular preparation. In support of this, Left and colleagues have recently described the properties in vitro of an ecto-ATPase inhibitor, FPL67156 (Crack et al. 1995). This agent potentiates the contractile effect of ATE but not that of a,fi-methylene ATR at P2x purinoceptors in the rabbit ear artery.

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Fig. 2 fl,7-Methylene L-ATP discriminates between Pax purinocep- tots on neurones and smooth muscle. The abilities of fi,7-methylene L-ATP (©) and a,fl-methylene ATP (,) to produce depolarization of the rat vagus nerve (upper panel) and to contract the rat isolated vas deferens (lower panel) are compared. Note the potent actions of fl,7- methylene L-ATP in the vas deferens but lack of effect on the vagus nerve (Trezise et al. 1995)

In view of this new appreciation of the profound influence of metabolism, it is crucial to reassess the criteria for identifying and characterizing P2x purinoceptors using agonists as tools. It is evident that, at least at the P2x purinoceptors on smooth muscle and neurones described above, ATP is inherently an equipotent, if not more potent, agonist than a,fl-methylene ATR Perhaps more impor- tantly, 2-methylthio ATP is also a highly potent Pax purinoceptor agonist when breakdown is taken into account (Evans and Kennedy 1994; Trezise et al. 1994a; Khakh et al. 1995a, c). This purine nucleotide was previously believed to be highly selective for P2Y purinoceptors (Bum- stock and Kennedy 1985). Although the available evidence suggests that it remains generally more potent at P2Y than P2x purinoceptors, this compound can no longer be reliably used as sole indicator of the involvement of P2Y purinoceptors. To date, there is little evidence that a,fl- methylene ATP recognizes receptors other than P2x purinoceptors to any degree, so this remains a useful probe when used judiciously. However, there are ligand-gated ion channel purinoceptors in superior cervical ganglia, guinea-pig submucosal plexus and PC12 phaeochromocytoma

cells at which this agonist is weak or inactive (Nakazawa et al. 1990; Cloues et al. 1993; Barajas-Lopez et al. 1994; Khakh et al. 1995a), and so its value is more limited than was first thought.

These observations stress the need to identify and work with nucleotides that are resistant to enzymatic degrada- tion when classifying receptors operationally. Since compounds such as ATP-7-S appear to be metabolized very differently in different tissues, this is not a trivial consid- eration (see below). Recently, several novel synthetic nucleotides have been described which are reportedly resistant to breakdown, and it will be interesting to determine the true value of these agents in purinoceptor characterization (Fischer et al. 1993; Zimmet et al. 1993; Bo et al. 1994).

Despite the caveats outlined, there is reasonable evidence from functional studies for Pzx purinoceptor heterogeneity based on relative agonist potencies. Under conditions where the influence of metabolism is negligible, Pzx

purinoceptors in rat nodose ganglion neurones, tail artery, vas deferens and guinea-pig coeliac ganglion neurones are activated by 2-methylthio ATE ATP and with slightly low-

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Table 1 Potencies of nucleotide agonists at P2x purinoceptors under conditions where ecto-ATPase activity has been largely circumvented or inhibited (NT not tested)

ECs0 (gM) Marked Reference desensitiza-

ATP 2-Methylthio ATP a, fl-Methylene ATP fl, y-Methylene L-ATP tion

Rat vas deferens ~3 ~equal to ATP ~equal to ATP ~equal to ATP Yes Khakh et al. 1995c; Tre- myocyte a zise et al. 1995 Rat vas deferens re- 0.6 0.6 3 NT Yes Valera et al. 1994 combinant receptor a Rabbit ear artery b 0.3 5.6 9.2 NT Yes McKechnie et al. 1995

Rat nodose neurones a 3 0.4 9.2 >300 No Khakh et al. 1995a; Trezise et al. 1995

Guinea-pig coeliac 3.3 2.8 13 NT No Khakh et al. 1995a neurones a Rat vagus nerve c 1.3 0.9 12 >300 No Trezise et al. 1994a, 1995

Rat superior cervical 43 46 >300 NT No Khakh et al. 1995a ganglion neurones a Rat submucous plexus 30 30 >300 NT No Barajas-Lopez et al. 1994 Rat PC12 recombinant 7.7 NT >100 >300 No Evans et al. 1995 receptor a

a The contribution of ecto-ATPase was circumvented by the use of single cells and rapid concentration-clamp applications of agonist b Ecto-ATPase activity was inhibited by the novel inhibitor FPL67156 c The contribution of ecto-ATPase was negated by the removal of divalent cations from the buffer and the addition of 1 mM EDTA

er potency, a,fl-methylene ATP (see Table 1). In contrast, in superior cervical ganglion (SCG) neurones, PC12 cells, parasympathetic cardiac ganglia and some other tissues much higher concentrations of ATP are required, and a,fl- methylene ATP appears to be weak or inactive (Table 1; Nakazawa et al. 1990; Fieber and Adams 1991; Cloues et al. 1993; Barajas-Lopez et al. 1994; Evans and Kennedy 1994; Khakh et al. 1995a; for review see Surprenant et al. 1995). Recent work with the stable ATP analogue, fl,7- methylene L-ATE has provided further good evidence for receptor heterogeneity. Using a variety of experimental approaches it has been shown that fl,7-methylene L-ATP can discriminate between P2x purinoceptors on smooth muscle of vas deferens and those on vagal neurones (Fig. 2; Trezise et al. 1995). Whether or not this selectivity extends to other nucleotides with the L- rather than the D-isomer, configuration awaits the ready availability of such compounds. The supposition of P2x purinoceptor heterogeneity from agonist studies has since been confirmed by the cloning of two distinct genes for P2x purinoceptor types (see below).

Antagonists

The operational characterization of P2 purinoceptors has been hindered by the paucity of specific, selective antagonists. However, a number of molecules, including suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), pyridoxalphosphate-6-azophenyl-2',5'-disulfonic acid (isoPPADS) and pyridoxal-5'-phosphate (P-5-P) do exhibit Pzx purinoceptor blocking properties and, if used cir- cumspectly, are of use in this respect.

Under appropriate conditions, suramin has been shown to behave as a competitive antagonist of P ;x purinoceptors in the mouse vas deferens and in the rabbit ear artery (Left et al. 1990; von Kfigelgen et al. 1990). Concentra- tion-dependent antagonism of P2x purinoceptor-mediated responses to a,fi-methylene ATP in rat vas deferens (Khakh et al. 1994), cat colon (Venkova et al. 1994) and rat vagus nerve has also been described (Trezise et al. 1994b). There are a number of considerations, however, when using this compound. First, it cannot be considered selective for any particular P2 purinoceptor subtype since, as well as Pzx purinoceptors, P2Y and P2u purinoceptors are also suramin-sensitive (Hoyle et al. 1990; Murrin and Boarder 1992; Wilkinson et al. 1993; Vials and Bumstock 1994). Secondly, suramin is often a poor antagonist of ATP itself and its inhibitory effects occur over a narrow concentration range (von Ktigelgen et al. 1990; Bailey and Hourani 1994; Bfiltmann and Starke 1994a; Crack et al. 1994; Trezise et al. 1994b). These phenomena have been attributed to the ability of suramin to inhibit ecto-ATPases (Hourani and Chown 1989; Crack et al. 1994). For example, in rabbit ear artery, occupation of P2x purinoceptors by suramin causes a rightward displacement of the concentration-effect curve to ATR while concomitantly the inhibition of ecto-ATPase shifts the concentration-effect curve leftward (Crack et al. 1994). This 'self-cancellation' is manifest as apparent suramin-resistance. Finally, suramin clearly has other actions attendant to purinoceptor antagonism and inhibition of ecto-ATPases (for review see Voogd et al. 1993). Recent studies demonstrating that suramin antagonizes responses mediated by nicotinic receptors, as well as those mediated by activation of GABA- and glutamate-gated ion channels, further indicate the limitations of

this agent as a tool for characterizing P2x purinoceptors (Allgaier et al. 1995; Nakazawa et al. 1995).

In addition to suramin, several other polyanions, including cibacron blue, trypan blue, Evans blue and congo red, as well as 4,4'-diisothiocyanatostilbene-2,2'disulfonate (DIDS), act as specific surmountable antagonists of the P2x purinoceptor (Btiltmann and Starke 1993, 1994a, b; Btiltmann et al. 1994; Khakh et al. 1994; Dudeck et al. 1995). Interestingly DIDS appears to preferentially block the human bladder recombinant receptor, compared with the rat PC12 recombinant receptor, when expressed in HEK 293 cells (Evans et al. 1995). Cibacron blue 3GA (formerly referred to as reactive blue 2) has been claimed to be a selective P2Y purinoceptor antagonist, but it is an effective antagonist of P2x purlnoceptor-mediated smooth muscle contraction (Btiltmann and Starke 1994a; Khakh et al. 1994) and neuronal depolarization (Trezise et al. 1994b; Khakh et al. 1995d). Like suramin, it also antagonizes responses mediated by GABA- and glutamate-gated ion channels (Nakazawa et al. 1995), although radioligand binding studies confirm that cibacron blue has appreciable affinity for the P2x purinoceptor (Michel and Humphrey 1993; Khakh et al. 1994). In view of such findings its claimed selectivity for P2Y purinoceptors is clearly un- founded (Fredholm et al. 1994).

PPADS and its stereoisomer, isoPPADS, antagonize P2x purinoceptor-mediated responses in certain vascular and visceral smooth muscles and in vagal neurones (Lambrecht et al. 1992; McLaren et al. 1993; Ziganshin et al. 1993; Khakh et al. 1994; Trezise et al. 1994b). PPADS can also antagonize ATP-induced currents mediated via recombinant P2x purinoceptors transiently expressed in HEK cells (Valera et al. 1994). It has been proposed that PPADS is selective for P2x purinoceptors over P2Y purinoceptors, at least over a limited concentration range (Ziganshin et al. 1993; Windschief et al. 1994). At P2Y purinoceptors, which couple via adenylate cyclase in C6 rat glioma cells, PPADS is ineffective as an antagonist (Boyer et al. 1994). However, in the same study, PPADS potently inhibited P2Y purinoceptor-mediated activation of phospholipase C in turkey erythrocytes, suggesting that some, but not all, P2Y purinoceptor types are PPADS-sensitive.

The synthesis precursor of PPADS, P-5-R is also an effective antagonist of P2x purinoceptor-mediated responses in rat vagus nerve and vas deferens (Trezise et al. 1994c), and of P2~ purinoceptor mediated relaxation in guinea-pig aorta (Trezise et al. 1994d). This compound represents the smallest molecule so far identified to exhibit purinoceptor blocking effects. Since P-5-P readily forms Schiff bases with lysine residues in many other proteins (for review see Snell and Dimari 1980), it is tempting to speculate that it may bind to lysine residues on the extracellular domain of the P2x purinoceptor in the same manner. In keeping with this, the effects of P-5-P (and PPADS) are slow to equili- brate and are only partially reversible, suggesting an inter- molecular mechanism involving tight binding to the receptor. Moreover, simple chemical substitutions in the P-5-P molecule that reduce its capacity to form Schiff bases abolish antagonist activity (Trezise et al. 1994c; Trezise,

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unpublished observations). P-5-P, and the other antagonists described above, probably mediate their antagonistic effects by binding to the P2x purinoceptor, since they all dis- place [3H]a,fl-methylene ATP from its binding sites (see below). The molecular structures of P-5-R PPADS and suramin do not contain particularly electron dense regions, which might allow attraction towards the electric field of the pore, and thus are more likely to bind to the extracellular domain of the P2x purinoceptor than the cation channel itself (Nakazawa et al. 1991; Khakh et al. 1995d). In- deed, evidence has been provided that these antagonists have no channel blocking action per se (Nakazawa et al. 1991; Khakh et al. 1995d).

As do suramin and Evans blue, PPADS and P-5-P can attenuate ATP removal by inhibiting ecto-ATPases (Hour- ani and Chown 1989; Bailey and Hourani 1994; Crack et al. 1994; Btiltmann et al. 1995; Khakh et al. 1995b; Zigan- shin et al. 1995). These antagonists inhibit Ca-ATPase activity over a similar concentration range as that for their ability to inhibit specific binding to the P2x purinoceptor (Khakh et al. 1995b). Caution must therefore be exercised when using these compounds as antagonists of hydrolysable agonists such as ATP in isolated tissue studies. How- ever, PPADS and P-5-P are somewhat more potent as receptor antagonists, indicating that the properties of receptor blockade and enzyme inhibition are separable (Khakh et al. 1995b). The challenges for drug discovery will be the design of potent P2 purinoceptor antagonists, lacking the ability to inhibit hydrolytic enzymes for ATE

Radioligand binding

There have been few successful attempts to directly label the Pzx purinoceptor in binding studies, primarily due to the lack of suitable high affinity antagonists and the problems associated with the use of agonist radioligands. The latter are compounded by the difficulty of differentiating between the labelling of a P2x purinoceptor and a nucleotide binding site on one or more of the many ATPase and ATP-dependent enzymes present on the cell surface or in membrane fractions (see Ziganshin et al. 1994). Neverthe- less, some progress has been made with the introduction of [3H]a,fl-methylene ATP as a radioligand for studying the P~x purinoceptor (Bo and Bumstock 1990). This radioligand appears to label both high and low affinity sites with equilibrium dissociation constants reported for the high affinity sites, varying from 0.3 nM to 8 nM, and for the low affinity sites, from 33 nM to 120 nM (Bo and Bumstock 1990; Bo et al. 1992; Michel and Humphrey 1993, 1994). Only the high affinity sites have been amen- able to further analysis, and their binding characteristics have been examined in detail in the rat bladder and vas deferens, where they are present at a relatively high den- sity in the region of 1-10 pmol per mg protein. The rank order of agonist potencies in competing for these high affinity sites is: a,fl-methylene ATP > fi,7-methylene ATP >2- methylthio ATE This is similar to that expected of a Pzx purinoceptor (Bo et al. 1992). Furthermore, Pzx purinocep-

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tor antagonists such as suramin, cibacron blue, PPADS and P-5-P are able to compete for the high affinity binding sites labelled by [3H]a,fl-methylene ATP in rat vas deferens over the same concentration range at which they antagonize P2x purinoceptors in functional studies (Khakh et al. 1994). On the basis of their localization, the rank order of agonist potencies and the affinity estimates for antagonists, it has been concluded that the high affinity sites are P2x purinoceptors (Bo and Burnstock 1990; B o e t al. 1992; Michel and Humphrey 1993).

The existence of [3H]a,fl-methylene ATP binding sites in rat vas deferens and bladder, and their recent demonstration in blood vessels (Bo and Burnstock 1993) and rabbit bladder (Ziganshin et ai. 1993) was expected on the basis of functional studies. However, high affinity [3H]a,fl- methylene ATP binding sites have also been identified in a wide range of other tissues, such as the liver, spleen, heart and kidney (Michel and Humphrey 1993). The binding characteristics of these sites are broadly similar to those of rat vas deferens, but since many of these tissues are not thought to possess functional P2x purinoceptors, their identity is uncertain. High affinity [3H]a,fl-methylene ATP binding sites have also been identified in rat brain (Michel and Humphrey 1993). These sites appear to be distinct from the rat vas deferens receptors, and can be discrimi- nated using fl,7-methylene L-ATP and a,fl-methylene ADR which display selectivity for the vas deferens and brain binding sites, respectively (Michel et al. 1994). The recent demonstration of P2x purinoceptor heterogeneity at the molecular level (see below) and the demonstration of functional P2x purinoceptors in brain (Edwards et al. 1992) may indicate that the brain high affinity [3H]a,fl-methylene ATP binding sites reflect another P2x purinoceptor type.

The use of [3H]a,fi-methylene ATP as a radioligand for the P2x purinoceptor is complicated by a variety of factors, as well as the newly appreciated limitation, that it is unlikely to have sufficient affinity for all P2x purinoceptors (see above). First, the affinity of [3H]a,fl-methylene ATP is very sensitive to divalent cations (Michel and Humphrey 1994), and in their absence it binds with lower affinity (Kd = 18 nM) than in their presence (Ka = 0.7 nM). Secondly, radioligand binding is sensitive to the presence of low concentrations of trivalent cations, which (when present in contaminating dust) can artifactually increase binding markedly in filtration based binding assays, through precipitation of the radioligand (Michel and Hum- phrey 1994). Thirdly, the presence of low affinity sites can be a complicating factor in some tissues such as the brain, while even in rat vas deferens a small proportion of low affinity binding sites are always present (Bo et al. 1992.; Michel and Humphrey 1993). Fourthly, in an endothelial- derived cell [3H]a,fl-methylene ATP binds to high affinity sites which also possess high affinity for a,fl-methylene ADP and probably represent labelling of a 5'-nucleotidase (Michel et ai. 1995).

Another complication, common to the functional studies, is that of agonist lability. Thus, even though the majority of binding studies with [3H]a,fl-methylene ATP have been per- formed at 4°C to avoid nucleotide metabolism, it is now ap-

parent that appreciable metabolism of ATP can occur in the presence of divalent cations at 4°C (Michel, unpublished observation). In the absence of divalent cations, metabolism of ATP is eliminated and the rank order of agonist affinities, ATP = 2-methylthio ATP > a,fl-methylene ATR is found for [3H]a,fl-methylene ATP binding sites in rat vas deferens (Michel, unpublished observation). Interestingly, in the absence of divalent cations ATP-7-S possesses very high affinity for the [3H]a,fi-methylene ATP binding sites in rat bladder (Bo et al. 1994), while in rat vas deferens membranes [35S]ATP-7-S binds to high affinity sites which display the same rank order of agonist affinities as the agonist potencies found for the P2x purinoceptor in rat vagus nerve (Mi- chel and Humphrey 1995). These observations raise the possibility that radiolabelled ATP-7-S, and even ATR which also possesses nanomolar affinity for the [35S]ATP-7-S binding sites, may be used to label the P2x purinoceptor, provided no divalent cations are present.

One important discrepancy which requires explanation is the observation that in P2x purinoceptor binding studies agonist affinity estimates are in the low nanomolar range, whereas in functional studies their potencies are invariably in the low micromolar range. The reason for this discrepancy remains to be elucidated but may reflect binding to a high affinity desensitized state of the receptor (Michel and Humphrey 1993). With the recent cloning of the genes for the rat vas deferens and PC12 cell P2x purinoceptors (see below), it will be possible to investigate this

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Fig. 3 Comparison of [3sS]ATPyS binding sites in rat vas deferens with the sites labelled in CHO-K1 cells infected with the human bladder P2x purinoceptor using the Semliki Forest virus transient expression system. Affinity estimates (pICso values) of nucleotides at the 3s [ S]ATPyS binding sites of rat vas deferens membranes or CHO-K1

cells infected with the human bladder P2x purinoceptor are compared (previously unpublished observations). The dotted line is the line of identity, while the solid line represents the regression line obtained when comparing pICso values in rat vas deferens and the infected CHO-K1 cells (r = 0.953). The nucleotides were: ATP, 2-methylthio ATP (2-meSATP), ATP-))-S (ATP7S), a,fi-methylene ATP (afimeATP), ADP, fl,7-methylene ATP (flTmeATP), L-fl, y-methylene ATP (L- fiTmeATP), a,fl-methylene ADP (ctflmeADP)

possibility together with the specificity of the radioligands, following expression of the different recombinant P2x purinoceptor types in appropriate cell lines. Indeed [35S]ATP- y-S can label high affinity sites in CHO cells infected with the human bladder P2x purinoceptor, and the binding characteristics of these sites are similar to those in rat vas deferens membranes (Fig. 3).

Agonist breakdown

As already emphasized, the problems associated with the breakdown of ATP and its hydrolysable analogues have profound implications for the pharmacology of P2x purinoceptors. The characteristics of the enzymes involved are summarized here in relation to these issues.

It is evident that many, if not all, tissues have cell-surface enzymes that are capable of dephosphorylating ATP (Nagy 1986; Ziganshin et al. 1994). These enzymes se- quentially dephosphorylate ATP to ADR AMP and adenosine. The conversion of AMP to adenosine is catalysed by 5'-nucleotidase (Zimmermann 1992). However, the initial step, the dephosphorylation of ATR appears to involve at least two types of enzyme. Thus, some cell types, such as pig aortic endothelial cells, are reported to possess distinct ecto-enzymes, hydrolysing ATP (ATPase) and ADP (AD- Pase) (Pearson 1986). In others, such as bovine aortic endothelial and smooth muscle cells, it is claimed that a single enzyme, ATP diphosphohydrolase, is responsible for both ATPase and ADPase activities (Yagi et al. 1991). Furthermore, Knowles et al. (1983) have reported that the ATPase and ATP diphosphohydrolase differ in that the latter, but not the former, is inhibited by azide. These work- ers also reported that cell membranes from a variety of mammalian cell types contain varying proportions of these two enzymes.

In contrast to intracellular ATPases, ecto-ATPase enzymes show a broad substrate specificity and are capable of breaking down a range of nucleotide triphosphates (Zi- ganshin et al. 1994). Furthermore, some, but not all, of the synthetic ATP analogues used to define P2x purinoceptors are subject to breakdown by ecto-ATPases. Thus, for example 2-methylthio ATP is broken down as rapidly as ATP itself, whilst phosphate-modified analogues such as a,fl-methylene ATP and fl,?;-methylene ATP are more stable (Welford et al. 1986, 1987). It is, however, note- worthy that there appear to be tissue differences with respect to substrate specificity. Thus for example, a,fl-methylene ATP has been shown to be slowly degraded by guinea-pig taenia coli, but fi, y-methylene ATP was not broken down at all (Welford et al. 1986), whilst the reverse was true on guinea-pig bladder (Welford et al. 1987). The sig- nificance, if any, of these small differences is unclear, but much larger differences have been reported with ATP-y-S. This compound is not broken down at all by porcine aortic endothelial cells (Cusack et al. 1983) or by guinea-pig taenia coli (Welford et al. 1986). However, it is slowly degraded by guinea-pig bladder (Welford et al. 1987) and, at least at low substrate concentrations, it is broken down

591

more rapidly than ATP itself by frog sartorius muscle (Cascalheira and Sebastifio 1992). It is not clear whether these differences reflect different substrate specificities of ecto-ATPases or the presence of a distinct enzyme or enzymes capable of metabolizing ATP-y-S.

An important characteristic of ecto-ATPases is a re- quirement for Ca 2+ and Mg 2+ (Nagy 1986; Ziganshin et al. 1994). In many cases only micromolar concentrations of divalent cations are required (see Juul et al. 1991). In general, either divalent cation will suffice and it is pre- sumed that the Ca 2+ and Mg 2+ ATPase activities reflect the same enzyme. However, this may not always be the case. Thus the ecto-ATPase of porcine aortic endothelial cells requires Mg 2+, but not Ca 2+ (Pearson 1986). Further- more, evidence has been obtained suggesting that the Mg 2+ and Ca ~+ ATPases of human hepatoma Li-7a cells are different enzymes (Knowles 1988).

Undoubtedly, selective ecto-ATPase inhibitors would be valuable tools in functional studies of P2x purinoceptors. Although compounds which inhibit these enzymes are known, until recently none was of sufficient specificity to be useful as pharmacological tools (Ziganshin et al. 1994). Among those compounds known to inhibit ecto-ATPases are a number which also block P2 purinoceptors (see above); clearly this is a potential complication when these compounds are used as tools for receptor classification. Recently Crack et al. (1995) have described the actions of a fi,7-methylene ATP derivative, FPL 67156. This compound is a moderately potent and specific ecto-ATPase inhibitor. This and compounds like it axe obviously poten- tially valuable for use in operational studies on receptor characterization. However, given the evident heterogeneity of ecto-ATPases, it will be important to characterize the enzyme inhibitory activity of such compounds in as wide a range of tissues as possible.

Electrophysiological characteristics of P2x purinoceptors

The transductional characteristics of ligand-gated ion channels, like the P2x purinoceptor, can only be properly eval- uated electrophysiologically. This approach has been used to characterize ATP-gated membrane currents in an in- creasing array of single cells, including dissociated smooth muscle, cardiac muscle, skeletal muscle, autonomic, sensory, enteric and central neurones, as well as glial cells (for review see Surprenant et al. 1995). Under appropriate recording conditions, in all of these cells ATP evokes a rapidly occurring inward current at the resting membrane potential (usually -50 to -70 mV). As expected from this type of direct coupling of agonist to receptor-channel complex, the latency to onset of response is immediate (within 1 ms) and time to reach peak is only several ms (Fig. 4; see Bean 1990; Khakh et al. 1995a). The currents are characteristically inwardly rectifying and are carried by channels that are permeant to both K + and Na + and also larger divalent cations, such a s Ca 2+, as judged by ion sub- stitution and reversal potential experiments. There is evi-

592

native cells a

ATP (3 #M)

/ b

ATP (100 gM)

c~-mATP (3 ~zM)

~ A T P (100 #M)

ATP (3 #M) cq3-mATP (3 gM)

Fig. 4 Ionotropic P2x purinoceptor channels show three distinct phenotypes in native cells (a-c, left hand traces), two of which corre- spond to recombinant Pax purinoceptors from smooth muscle (a, right hand traces) and from PC12 cells (b, right hand traces). Pre- viously unpublished data showing: a a,fl-methylene ATP-sensitive, desensitizing Pax purinoceptor responses recorded from acutely dissociated rat vas deferens smooth muscle and from HEK293 cells expressing the recombinant P2x purinoceptor from rat vas deferens; b a,fl- methylene ATP-insensitive, non-desensitizing P2x purinoceptor re-

dence that the ratio of Ca 2+ to Na + permeability is higher in smooth muscle than in sensory neurones (Bean 1992), although this point is controversial. There have been few attempts to determine single channel conductance, but estimates in the range 12-20 pS have been reported (Cloues 1995; Surprenant et al. 1995). At the present time it is too early to ascertain whether some of the differences in channel conductance properties that have been described relate to P2x purinoceptor heterogeneity, and further studies are required to clarify these issues.

Electrophysiology experiments on single cells have been very useful in delineating the operational characteristics of Pax purinoceptors. The rapid application of nucleotides to single cells effectively circumvents the problems of agonist breakdown, and, in addition, the whole cell pip- ette can be used to abolish effects of activation of P2Y purinoceptors by using appropriate intracellular solutions to dialyse away cytoplasmic components that are necessary for G-protein coupled transduction processes. Moreover, the properties of single molecular species can be readily studied by expressing recombinant receptors in cell lines. To date, electrophysiological experiments have permitted the identification of three distinct phenotypes for ATP- gated cation channels in native cell lines (see Table 1, Fig. 4).

First, an a,/?-methylene ATP-sensitive current which markedly desensitizes and is characteristic of dissociated smooth muscle from vas deferens, bladder and arteries

heterclogous expression of recombinant receptor

ATP (3 gM) c@mATP (3 #M)

V is

(30 gM) cq3-mATP (30 ~tM)

~ O.5 nA

2s

9

sponses recorded from NGF-differentiated PC12 cells and from HEK293 cells expressing the recombinant Pax purinoceptor from PC12 cells; e a,fl-methylene ATP-sensitive, non-desensitizing P2x purinoceptor responses recorded from cultured rat nodose neurones; cDNA encoding a protein exhibiting properties of this type of receptor currently has not been isolated. Each trace shows currents, recorded using whole cell patch clamp methods in response to fast-flow application of ATP or a,fl-methylene ATP for durations indicated by bars. For references see Table 1

(Inoue and Brading 1990; Evans and Kennedy 1994; Khakh et al. 1995b). Secondly, an a,fl-methylene ATP-sensitive current which does not desensitize and has been reported in sensory nodose and dorsal root ganglia, autonomic coeliac and guinea-pig superior cervical ganglia and medial habenular neurones (Bean 1990; Edwards et al. 1992; Khakh et al. 1995a). Thirdly, an a,/?-methylene ATP-insensitive current which does not desensitize and has been observed in the PC12 phaeochromocytoma cell line, parasympathetic cardiac ganglia, rat superior cervical ganglia, tuberomamillary and nucleus tractus solitarius neurones, cochlear hair cells and glia (see Suprenant et al. 1995). The potencies of ATR 2-methylthio ATP and a,fi- methylene ATP are similar for both the desensitizing and non-desensitizing types of a,/?-methylene ATP-sensitive phenotypes with ECs0 concentrations for these agonists falling in the range of 0.5-5 gM (Table 1). Although a,/?- methylene ATP is equally effective at both these receptor types,/?,7-methylene L-ATP is a potent agonist at P2x purinoceptors on vas deferens smooth muscle, but is ineffective at those on vagal neurones (Trezise et al. 1995; see Table 1). The third type of Pax purinoceptor-mediated current seems insensitive to /?,y-methylene L-ATP as well as a,/?-methylene ATP; the agonist ECso values for ATP and 2-methylthio ATP are in the range of 8-60 gM, which is notably higher than for other P2x purinoceptors (Table 1).

Only recently has it been possible to address the question of whether ligand-gated ATP responses recorded in

593

single cells resulted from activation of one or more types of P2x purinoceptor. P2x purinoceptor genes have been cloned from rat vas deferens smooth muscle, human bladder smooth muscle and PC12 cells and the corresponding receptors expressed in oocytes and mammalian cells (Brake et al. 1994; Valera et al. 1994, 1995; see below). Responses recorded from heterologously expressed Pax purinoceptors from smooth muscle or PC12 cells show re- markable similarity to those of native cells (see Fig. 4). The kinetics, high calcium permeability, agonist and antagonist profiles of these recombinant receptors are virtually identical to these properties in native cells. In a recent study, the actions of a wide range of agonists and antagonists on heterologously expressed recombinant receptors from smooth muscle and PC12 cells were compared (Evans et al. 1995). The rank order of agonist potencies for either form of receptor was similar, although ECs0 values for each of these agonists were approximately 10 times higher for the PC12form of the receptor than for the smooth muscle form. As in native cells, a,fi-methylene ATP was a potent and full agonist (ECso of 3 gM) at the recombinant smooth muscle receptor type, but was ineffective at the PC12form. There were only small differences in the antagonist potencies of suramin, PPADs and P-5-P at these receptors, although DIDS was 20 to 30-fold more potent at the smooth muscle form of the receptor than at the PC12 form.

Studies on these heterologously expressed recombinant receptors have shown that functional receptor-channels are formed by homopolymerization of single subunits of the receptor. It would seem likely that P2x purinoceptors with different pharmacological and/or physiological properties might be formed by heteropolymerization of different P2x subunits, in much the same way as differences in nicotinic receptor pharmacology reflect differences in their (penta- meric) subunit composition. In particular, it seems reasonable that heteropolymerization of smooth muscle and PC12forms of the P2x purinoceptor might result in a receptor-channel complex that corresponds to the a,fl-methylene ATP-sensitive, non-desensitizing response, described above. However, it appears that these two P2x purinoceptor types are not able to form functional receptors by com- bination with each other, as co-expression of these two forms results only in the separate formation of the smooth muscle form and the PC12form of the receptor (Surpre- nant and Lewis, unpublished observations). Nevertheless, as molecular biological approaches are certain to yield further members of this multi-gene family, it remains to be seen whether native channels form as a result of heteropolymerization of more than one P2x purinoceptor subunit.

Structural characteristics of P2x purinoceptors

Complementary DNAs, encoding P2x purinoceptors from rat vas deferens and the neuronal cell line, PC12, have been independently isolated by two groups, both using direct expression cloning (Brake et al. 1994; Valera et al.

1994). This recent advance employed the functional expression of these ATP ligand-gated ion channels in oocytes that were injected with poly A + RNA from their re- spective sources. In each experiment the active poly A + RNA was used to synthesize cDNA. Subsequent injection of complementary RNA, prepared from pools of cloned cDNA, resulted in the purification of unique plasmid cDNAs and the definitive identification of the amino acid sequences of two P2x purinoceptor types.

Neither receptor showed significant homology with any other known ligand-gated ion channel or receptor superfa- milies or with any other protein in the Swissprot database. The two types of P2x purinoceptors have amino acid sequences that are only 41% identical with each other, but which display very similar hydropathy profiles. These profiles have provided a structural model in which P2x purinoceptors contain only two transmembrane domains, one near each end of the protein, and each having intraceUular N- and C-termini (see Fig. 5). The majority of the protein, the approximately 280-amino-acid (aa), cysteine-rich din- tral section is believed to be extracellular and to be glyco- sylated. The spacing between these cysteines is absolutely conserved between the two forms of P2x purinoceptors. Comparison of the molecular weight of the native P2x purinoceptor from rat vas deferens, a 60-kDa protein (Bo et al. 1992), with that of the cloned 399-aa protein (45 kDa) supports the prediction that P2x purinoceptors are glycosy- lated and the proposed model.

The P2x purinoceptors are clearly distinguished from the nicotinic acetylcholine superfamily of ligand-gated ion channels, which are larger proteins with four transmembrane spans (Fig. 5). In contrast to P2x purinoceptors, nicotinic acetylcholine channel proteins have putative hydro- phobic leader sequences and long extracellular N-termini. Recently, cDNAs encoding channel-forming proteins that define a third family have been cloned (Canessa et al. 1994). These are the amiloride-sensitive epithelial Na + channel subunits. Although these are larger proteins (716- 734 aa) with no sequence similarity to P2x purinoceptors, they are predicted to have a similar membrane topography. These channels have been shown to be composed of several homologous subunits (a,fl,7). Native P2x purinoceptors may also be heteromeric structures, a question which further cDNA cloning will help to address.

Nicotinic acetyicholine P2X purinoceptor cation channel

Amiloride-sensitive sodium channet

out.or Fig. 5 The schematic diagrams depict the primary amino acid sequences for three different structural types of channel-forming proteins. Broken lines represent putative transmembrane regions. Note that the P2x pufinoceptor topography is more similar to that of amilofide-sensifive sodium channels than that of classical ligand-gated cation channels like the nicotinic receptor

594

The two types of recombinant P2x purinoceptor have one apparent marked structural difference. The P2x purinoceptor isolated from PC12 cells is larger (472 aa), due to an extension of its C-terminus. This extension is very pro- line-rich and may constitute a site of interaction with other proteins. Conspicuous in its absence from either protein was the typical consensus sequence region [G(X4)GK(X7)(I/V)] for ATP binding (Walker et al. 1982). How extracellular ATP interacts with these P2x purinoceptor proteins is presently unknown.

The elucidation of the P2x purinoceptor cDNA sequences revealed a surprising connection between this receptor and the immune system. A partial cDNA complementary to the 3' half of the P2x purinoceptor mRNA had been previously isolated from immature thymocytes (Owens et al. 1991). The thymocytes had been selectively cultured to induce apoptosis and novel sequences were identified by subtractive hybridization. Northern blot analysis confirmed the induction of this abbreviated Pax purinoceptor mRNA in thymocytes treated with dexametha- sone or by ?-irradiation. P2x purinoceptor mRNA has also been identified by northern blotting in some adult tissues, which are known to contain large numbers of immune cells including the thymus, lung and spleen. The functional consequences of these findings remain to be determined.

The human equivalent of the rat P2x purinoceptor mRNA from smooth muscle has also been cloned (Valera et al. 1995). This P2x purinoceptor mRNA is present in the human promyelocytic cell line, HL60 Differentiation of these cells with either phorbol diesters, which induce macrophage-like characteristics, or with dibutryl cAMR which induces granulocytic properties, results in an increase in P2x purinoceptor mRNA. Undoubtedly, such studies will rapidly facilitate our understanding of the potential importance of P2x purinoceptors in disease. Isolation of the human genes for P2x purinoceptors will also be essential in determining whether specific P2x purinoceptors show linkage with known genetic defects

References

Abbrachio MR Burnstock G (1994) Purinoceptors: are there families of P2x and P2Y purinoceptors. Pharmacol Ther 64:445-475

Allgaier C, Wellmann H, Schobert A, Kurz G, yon Kfigelgen I (1995) Cultured chick sympathetic neurons: ATP-induced noradrenaline release and its blockade by nicotinic receptor antagonists. Nan- nyn-Schmiedeberg's Arch Pharmacol 252:25-30

Bailey SJ, Hourani SMO (1994) Differential effects of suramin on P2- purinoceptors mediating contraction of the guinea-pig vas deferens and urinary bladder. Br J Pharmacol 112:219-225

Barajas-Lopez C, Espinosa-Luna R, Gerzanich V (1994) ATP closes and opens a cationic conductance through different receptors in neurons of guinea-pig submucous plexus. J Pharmacol Exp Ther 268:1396-1402

Bean BP (1990) ATP-activated channels in rat and bullfrog sensory neurons: concentration dependence and kinetics. J Neurosci 10:1-10

Bean BP (1992) Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol Sci 13:87-90

Benham CD, Tsien RW (1987) A novel receptor-operated Ca 2+- permeable channel activated by ATP in smooth muscle. Nature 328:275-278

Bo X, Burnstock G (1990) High- and low-affinity binding sites for [3H]-a,fi-methylene ATP in rat urinary bladder membranes. Br J Pharmacol 101:291-296

Bo X, Burnstock G (1993) Heterogeneous distribution of [3H]a,fl- methylene ATP binding sites in blood vessels. J Vasc Res 30:87- 101

Bo X, Simon J, Burnstock G, Barnard EA (1992) Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens. J Biol Chem 267:171581-171587

Bo X, Fischer B, Maillard M, Jacobsen KA, Burnstock G (1994) Com- parative studies on the affinities of ATP derivatives for P2x purinoceptors in rat urinary bladder. Br J Pharmacol 112:1151-1159

Boyer JL, Zohn IE, Jacobson KA, Harden TK (1994) Differential effects of P2-purinoceptor antagonists on phospholipase C- and ade- nylyl cyclase-coupled P2,~purinoceptors. Br J Pharmacol 113:614-620

Brake A, Wagenbach M, Julius D (t994) New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 371:519-523

Bfiltmann R, Starke K (1993) Evans blue blocks Pax-purinoceptors in rat vas deferens. Naunyn-Schmiedeberg's Arch Pharmacol 348:684-687

BiJltmann R, Starke K (1994a) P2-purinoceptor antagonists discriminate three contraction-mediating receptors for ATP in rat vas deferens. Naunyn-Schmiedeberg's Arch Pharmacol 349:74-80

Btiltmann R, Starke K (1994b) Blockade by 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) of P2x-purinoceptors in rat vas deferens. Br J Pharmacol 112:690-694

Bfiltmann R, Trendelenburg M, Starke K (1994) Blockade of P2x-purinoceptors by trypan blue in rat vas deferens. Br J Pharmacol 113:349-354

B~ltmann R, Driessen B, Gongalves J, Starke K (1995) Functional consequences of inhibition of nucleotide breakdown in rat vas deferens: a study with Evans blue. Nannyn-Schmiedeberg's Arch Pharmacol 351:555-560

Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509-581 Burnstock G, Kennedy C (1985) Is there a basis for distinguishing

two types of P2-purinoceptor? Gen Pharmacol 16:433440 Canessa CM, Schild L, Buell G, Thorens B, Gantschi I, Horisberger

J-D, Rossier BC (1994) Amiloride-sensitive epithelial Na + channel is made of three homologous subunits. Nature 367:463-467

Cascalheira JF, Sebasti~o AM (1992) Adenine nucleotide analogues, including y-phosphate-substituted analogues, are metabolised ex- tracellularly in innervated frog sartorius muscle. Eur J Pharmacol 222:49-59

Cloues R (1995) Properties of ATP-gated channels recorded from rat sympathetic neurons: voltage dependence and regulation by Zn 2+ ions. J Neurophysiol 73:312-319

Cloues R, Jones S, Brown DA (1993) Zn 2+ potentiates ATP-activated currents in rat sympathetic neurons. Pflfigers Arch 424:152-158

Coleman RA, Humphrey PPA (1993) Prostanoid receptors: their function and classification. In: Vane JR, O'Grady J (eds) Therapeutic applications of prostaglandins. Edward Arnold, London, pp 15-36

Coleman RA, Smith WL, Narumiya S (1994) Classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 46:205-229

Crack BE, Beukers MW, McKechnie KCW, Ijzerman AP, Left P (1994) Pharmacological analysis of ecto-ATPase inhibition: evidence for combined enzyme inhibition and receptor antagonism in P2x-purinoceptor ligands. Br J Pharmacol 113:1432-1438

Crack BE, Pollard CE, Beukers SM, Roberts SM, Hunt SF, Ingall AH, McKechnie KCW, Ijzerman AR Left P (1995) Pharmacologi- cal and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase. Br J Pharmacol 114:475-481

Cusack NJ, Pearson JD, Gordon JL (1983) Stereoselectivity of ectonucleotidases on vascular endothelial cells. Biochem J 214:975- 981

595

Dalziel HH, Westfall DP (1994) Receptors for adenine nucleotides and nucleosides: subclassification, distribution and molecular characterization. Pharmacol Rev 46:449-466

Dudeck O, Btiltmann R, Starke K (1995) Two relaxation-mediating P2-purinoceptors in guinea-pig taenia caeci. Naunyn-Schmiede- berg's Arch Pharmacol 351:107-110

Edwards FA, Gibb AJ, Colquhoun D (1992) ATP receptor-mediated sy- naptic currents in the central nervous system. Nature 359:144-147

Evans RJ, Kennedy C (1994) Characterization of P2-purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiotogical responses. Br J Pharmacol 113:853-860

Evans RJ, Derkach V, Surprenant A (1992) ATP mediates fast synap- tic transmission in mammalian neurons. Nature 357:503-505

Evans RJ, Lewis C, Buell G, Valera S, North RA, Surprenant A (1995) Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x-purinoceptors). Mol Phar- macol 48:178-183

Fieber LA, Adams DJ (1991) Adenosine triphosphate-evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia. J Physiol (Lond) 434:239-256

Fischer B, Boyer JL, Hoyle CHV, Ziganshin AU, Brizzolara AL, Knight GE, Zimmet J, Burnstock G, Harden TK, Jacobsen KA (1993) Identification of potent, selective P2v-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate. J Med Chem 36:3937-3946

Fredholm BB, Abbrachio MR Bumstock G, Daly JW, Harden TK, Ja- cobson KA, Left R Williams M (1994) VI Nomenclature and classification of pufinoceptors. Pharmacol Rev 46:143-155

Hourani SMO, Chown JA (1989) The effects of some possible inhibitors of ectonucleotidases on the breakdown and pharmacological effects of ATP in the guinea pig urinary bladder. Gen Pharmacol 20:413-416

Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PPA (1994) The IUPHAR Classifica- tion of Receptors for 5-Hydroxytryptamine (Serotonin). Pharmacol Rev 46:157-203

Hoyle CHV, Knight GE, Burnstock G (1990) Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. Br J Phar- macol 99:617-621

Humphrey PPA, Hartig R Hoyer D (1993) A proposed new nomenclature for 5-HT receptors. Trends Pharmacol Sci 14:233-236

Inoue K, Brading AF (1990) The properties of the ATP-induced depolarization and current in single cells isolated from the guinea-pig urinary bladder. Br J Pharmacol 100:619-625

Juul B, LiJscher ME, Aalkjear C, Plesner L (1991) Nucleotide hydrolytic activity of isolated intact rat rnesenteric small arteries. Bio- chim Biophys Acta 1067:201-207

Khakh BS, Michel AD, Humphrey PPA (1994) Estimates of affinities at P2x-purinoceptors in rat vas deferens. Eur J PharmacoI 263:301-309

Khakh BS, Humphrey PPA, Surprenant A (1995a) Electrophysiolo- gical properties of P2x purinoceptors in rat superior cervical, nodose and guinea pig coeliac neurones. J Physiol (Lond) 484:385-395

Khakh BS, Michel AD, Humphrey PPA (1995b) Inhibition of ec- toATPase and Ca-ATPase in rat vas deferens by P2-purinoceptor antagonists. Br J Pharmacol 115:2P

Khakh BS, Surprenant A, Humphrey PPA (1995c) The use of agonists to characterize P2x-purinoceptors mediating electrophysiological and mechanical responses of rat vas deferens. Br J Pharma- col 115:177-185

Khakh BS, Humphrey PPA, Surprenant A (1995d) Actions of antagonists against inward currents induced by ATP in rat nodose neurones. Br J Pharmacol 194:402P

Knowles AF (1988) Differential expression of ectoMg2+-ATPase and 2+ ectoCa -ATPase activities in human hepatoma cells. Arch Bio-

chem Biophys 263:264-271 Knowles AF, Isler RE, Reece JF (1983) The common occurrence of

ATP diphosphohydrolase in mammalian plasma membranes. Bio- chem Biophys Acta 731:88-96

von Kfigelgen I, Btiltmann R, Starke K (1990) Interaction of adenine nucleotides, UTP and suramin in mouse vas deferens: suramin- sensitive and suramin-insensitive components in the contractile effect of ATE Naunyn-Schmiedeberg's Arch Pharmacol 342:198- 205

Lambrecht G, Friebe T, Grimm U, Windshief U, Bungardt E, Hilder- brandt C, Bumert HG, Spatz-Kum-Bel G, Mutschler E (1992) PPADS, a novel functionally selective antagonist of P2 purinocep- tot mediated responses. Eur J Pharmacol 217:217-219

Leff R Wood BE, O'Connor SE (1990) Suramin is a slowly-equili- brating but competitive antagonist at P2x-receptors in the rabbit isolated ear artery. Br J Pharmacol 101:645-649

McKechnie KCW, Crack BE, Dainty I, Left P (1995) The effects of ectoATPase inhibition on agonist potency orders at P2Y and Pzx purinoceptors. Br J Pharmacol 114:104P

McLaren GJ, Sneddon R Kennedy C, Lambrecht G, Burnstock G, Mutschler E, Bumert HG, Hoyle CHV (1993) Investigation of a putative Pzx-purinoceptor antagonist in guinea pig isolated vas deferens. Br J Pharmaeol 109:109P

Michel AD, Humphrey PPA (1993) Distribution and characterization of [3H]a,fi-meATP binding sites in the rat. Naunyn-Schmiede- berg's Arch Pharmacol 348:608-617

Michel AD, Humphrey PPA (1994) Effects of metal cations on [3H]a,fi-methylene ATP binding in rat vas deferens. Naunyn- Schmiedeberg's Arch Pharmacol 350:113-122

Michel AD, Humphrey PPA (1995) High affinity binding sites for [35S]ATP7S in rat vas deferens may be Pzx purinoceptors. Br J Pharmacol 115:1P

Michel AD, Trezise DJ, Grahames CBA, Khakh BS, Humphrey PPA (1994) The selective Pax purinoceptor agonist, fi,7-methylene L- ATE discriminates between smooth muscle and neuronal Pax purinoceptors. Br J Pharmacol 113:58P

Michel AD, Chau N-M, Fan T-PD, Frost EE, Humphrey PPA (1995) Evidence that [3H]c~fi meATP may label an endothelial-derived cell line 5'-nucleotidase with high affinity. Br J Pharmacol 115:767-774

Murrin RJA, Boarder MR (1992) Neuronal "nucleotide" receptor linked to phospholipase C and phospholipase D? Stimulation of PCI2 cells by ATP analogues and UTR Mol Pharmacol 41:561- 568

Nagy A (1986) Enzymatic characteristics and possible role of synap- tosomal eeto-adenosine triphosphatase from mammalian brain. In: Kreutzberg GW (ed) Cellular biology of ectoenzymes. Springer, Berlin Heidelberg New York, pp 49-59

Nakazawa K, Fujimori K, Takanaka A, Inoue K (1990) An ATP activated conductance in phaeochromocytoma cells and its suppres- sion by extracellular calcium. J Physiol (Lond) 428:257-272

Nakazawa K, Inoue K, Fujimori K, Takanaka A (1991) Effects of ATP antagonists on purinoceptor-operated inward currents in rat phaeochromocytoma cells. Pflfigers Arch 418:214-219

Nakazawa K, Inoue K, Ito K, Koizumi K, Inoue K (1995) Inhibition by suramin and reactive blue 2 of GABA and glutamate receptor channels in rat hippocampal neurons. Naunyn-Schmiedeberg's Arch Pharmacol 351:202-208

O'Connor SE, Wood BE, Left P (1990) Characterization of P2~-receptors in rabbit isolated ear artery. Br J PharmacoI 101:640-644

Owens GR Hahn WE, Cohen JJ (1991) Identification of mRNAs associated with programmed cell death in immature thymocytes. Mol Cell Biol 11:4177-4188

Pearson JD (1986) Ectonucleotidases of vascular endothelial cells: characterization and possible physiological roles. In: Kreutzberg GW (ed) Cellular biology of ectoenzymes. Springer, Berlin Hei- delberg New York, pp 17-25

Silinsky EM, Gerzanich V (1993) On the excitatory effects of ATP and its role as a neurotransmitter in coeliac neurones of the guinea-pig. J Physiol (Lond) 464:197-212

Snell EE, Dimari SJ (1980) Schiff base intermediates in enzyme cata- lysis. In: Boyer PD (ed) The enzymes (vol 12). Academic, New York, pp 335-370

Surprenant A, Buell G, North RA (1995) P2x receptor brings new structure to ligand gated ion channels. Trends Neurosci 18:224- 229

596

Trezise DJ, Kennedy I, Humphrey PPA (1993) Characterization of purinoceptors mediating depolarization of rat isolated vagus nerve. Br J Pharmacol 110:1055-1060

Trezise DJ, Bell NJ, Kennedy I, Humphrey PPA (1994a) Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification. Br J Pharmacol 113:463.470

Trezise DJ, Kennedy I, Humphrey PPA (1994b) The use of antagonists to characterize the receptors mediating depolarization of the rat isolated vagus nerve by a,fi-methylene adenosine 5'-triphosphate. Br J Pharmacol 112:282-288

Trezise DJ, Bell NJ, Khakh BS, Michel AD, Humphrey PPA (1994c) P2 purinoceptor properties of pyridoxal-5'-phosphate. Eur J Phar- macol 259:295-300

Trezise DJ, Bell NJ, Humphrey PPA (1994d) P2 purinoceptor antagonist effects of pyridoxal-5-phosphate in the rat vagus nerve and guinea-pig aorta. Br J Pharmacol 112:502P

Trezise DJ, Michel AD, Grahames CBA, Khakh BS, Surprenant A, Humphrey PPA (1995) The selective Pax purinoceptor agonist, fiT-methylene-L-adenosine 5'-triphosphate, discriminates between smooth muscle and neuronal Pax purinoceptors. Nannyn-Schmie- deberg's Arch Pharmacol 351:603-609

Valera S, Hussy N, Evans RJ, Adami N, North A, Surprenant A, Buell G (1994) A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATR Nature 371:516-519

Valera S, Talabot F, Evans RJ, Gos A, Antonarakis SE, Morris MA, Buell G (1995) Characterization and chromosomal localization of a human P2x receptor from urinary bladder. Receptors Channels (in press)

Venkova K, Milne A, Krier J (1994) Contraction mediated by ch- adrenoceptors and P2-purinoceptors in cat colon circular muscle. Br J Pharmacol 112:1237-1243

Vials AJ, Burnstock G (1994) The effect of suramin on vasodilator responses to ATP and 2-methylthio-ATP in the Sprague-Dawley rat coronary vasculature. Eur J Pharmacol 251:299-302

Voogd TE, Vansterkenburg EL, Wilting J, Janssen LH (1993) Recent research on the biological activity of suramin. Pharmacol Rev 45:177-203

Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the a- and fl-subunits of ATP synthase, myosin, ki- nases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1:945-951

Welford LA, Cusack NJ, Hourani SMO (1986) ATP analogues and the guinea-pig taenia coli: a comparison of the structure-activity relationships of ectonucleotidases with those of the P2-purinoceptor. Eur J Pharmacol 129:217-224

Welford LA, Cusack NJ, Hourani SMO (1987) The structure-activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency. Eur J Pharmacol 141:123-130

Wilkinson GF, Purkiss JR, Boarder MR (t993) The regulation of aortic endothelial cells by purines and pyrimidines involves co-exist- ing P2rpurinoceptors and nucleotide receptors linked to phospholipase C. Br J PhamlacoI 108:689-693

Windschief U, Ralevic V, Bumert HG, Mutschler E, Lambrecht G, Burnstock G (1994) Vasoconstrictor and vasodilator responses to various agonists in the rat perfused mesenteric arterial bed: selective inhibition by PPADS of contractions via P2x-purinoceptors. Br J Pharmacol 113:1015-1021

Yagi K, Arai Y, Kato N, Hirota K, Miura Y (1991) ATP diphosphohydrolase is responsible for ecto-ATPase and ecto-ADPase activities in bovine aorta endothelial and smooth muscle cells. Bio- chem Biophys Res Commun 180:1200-1206

Ziganshin AU, Hoyle CHV, Bo X, Lambrecht G, Mutschler E, Bu- G, Bumstock G (1993) PPADS selectively antagonizes P2x-purinoceptor-mediated responses in the rabbit urinary bladder. Br J Pharrnacol 110:1491-1495

Ziganshin AU, Hoyle CHV, Burnstock G (1994) Ecto-enzymes and metabolism of extracellular ATR Drag Dev Res 32:134-146

Ziganshin AU, Ziganshina LE, King BE Burnstock G (1995) Charac- teristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown. Pfltigers Arch 429:412-418

Zimmermann H (1992) 5'-Nucleotidase: molecular structure and functional aspects. Biochem J 285:345-365

Zimmet J, Jrlebark L, Hammarberg T, van Galen PJM, Jacobsen KA, Heilbronn E (1993) Synthesis and biological activity of novel 2- thio derivatives of ATP. Nucleos Nucleot 12:1-20

Note added in proof In the course of publication of this review, a ~ d P2x purinoceptor gene has been cloned and evidence provided for its hetempolymerisa- tion (see Chen et al. 1995, Nature 377:428.431 :and Lewis et al 1995, Nature 377:432.435).

New insights on P2X purinoceptors

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