Purine and Pyrimidine (P2) Receptors as Drug Targets

1. P2X Receptors.

Functional P2X receptors are ATP-gated (ligand-gated) ion channels that mediate fast excitatory neurotransmission in excitable tissues including neurons, glia, and smooth muscle cells. ATP can elicit rapid responses (<10 ms) via these ion channels, resulting in selective permeability to Na⁺, K⁺, and Ca²⁺ cations.^4,9 The membrane depolarization resulting from the activation of P2X receptor multimers can lead to activation of voltage-operated ion channels, L-type Ca²⁺ channels, and Ca²⁺-stimulated tyrosine kinases that in turn activate MAP kinases (ERK1 and ERK2) that modulate transcriptional processing.¹⁰³ MAP kinase activation is also involved in P2Y₁-receptor-mediated apotosis.¹⁰⁴

Seven P2X receptor subunits with a two-transmembrane (2TM) motif structurally related to the amiloride-sensitive epithelial Na⁺ channel of approximately 100 amino acids in length with intracellular termini and a cysteine-rich extracellular loop have been cloned and designated P2X_1–7. The degree of sequence identity between these subunits ranges from 26% for P2X₂ and P2X₇ to 47% for P2X₁ and P2X₄.³ These subunits resemble proton-gated channels at a global but not primary structure level¹⁰⁵ and form functional homomeric and heteromeric channels^86–89 that currently available evidence suggests exist as stretched trimers (Figure 2),^106,107 contrasting with the pentameric stoichiometry of other LGICs, e.g., the neuronal nicotinic receptor superfamily.¹⁰⁸ Unlike the latter, very little is known regarding the nature of the ATP binding site(s) on the proposed trimer, of distinct antagonist binding sites, of associated allosteric binding sites, or of ancillary proteins necessary for native receptor function.

P2X subunits, with the exception of the P2X₆ receptor,^4,109 can form functional homomers that are activated by ATP.^86–89 The reason for the inability of P2X₆ receptor subunits to form functional receptors is unclear. Functional heteromers composed of P2X_1/5, P2X_2/3, P2X_2/6, P2X_2/6, and P2X_1/2 subunits have been described.^{86–89,109,110}

While there is convincing evidence for functional heteromers from in vitro studies, in situ analysis studies of P2X receptors have resulted in the identification of additional subunit combinations, e.g., a putative P2X_2/5 heteromer, that are pharmacologically distinct from those described above.¹¹¹ P2X receptors also appear to undergo activation-dependent modifications that include cellular internalization for P2X₁ receptors^89,112,113 or redistribution at synaptic junctions for the P2X₂ receptor.¹¹⁴

On the basis of their kinetic parameters, P2X receptors have been divided into three groups.³ Group 1, which includes P2X₁ and P2X₃ homomers, is potently and rapidly activated by agonists (0.01–0.1 s) and undergoes rapid inactivation (0.1–10 s) in the presence of prolonged agonist activation. Group 2 includes P2X₂, P2X_2/3, P2X_2/6, and P2X₅ multimers that are rapidly activated (0.1–1 s) and show slow inactivation/desensitization (10–100 s) profiles. Group 3 P2X multimers include the P2X_1/5, P2X₄, P2X_4/6, and P2X₇ receptors. These show the same activation kinetics as group 2 multimers but have both a fast and slow desensitization phase. In general, those P2X receptors that undergo rapid desensitization are activated by α,β-meATP, 2-MeSATP, and ATP. P2 receptors that undergo slower desensitization or show no desensitization can be subdivided into two groups, those sensitive to α,β-meATP (P2X_1/5, P2X₄, P2X_4/6) and those that are only weakly sensitive to this agonist (P2X₇).

2. P2Y Receptors.

P2Y receptors are G-protein-coupled receptors (GPCRs) with a typical heptahelical transmembrane (7-TM) motif and are sensitive to activation by both purines (ATP, ADP) and pyrimidines (UTP, UDP).^7,10,171 Other receptors that are closest in sequence homology to P2Y receptors include angiotensin and thrombin peptide receptors, an observation that has led to speculation that there may be an as yet unidentified endogenous peptide ligand for P2Y receptors.

The currently accepted members of this family are the P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors (Table 2). The missing sequence numbers represent species homologues of other receptors, e.g., p2y₃,¹⁷² or receptors that have been misassigned to the P2Y family, e.g., p2y₇,⁷³ which, as already noted, was subsequently cloned as an LTB₄ receptor.⁷⁴ P2Y₂, P2Y₄, and P2Y₆ receptors are activated by pyrimidines.^7,10,171 Most of the P2Y receptors produce their functional effects via G protein coupling to activate phoshoplipase C (PLC), forming IP₃ and mobilizing intercellular calcium.^10,64 This in turn leads to activation of other signaling pathways that include protein kinase C, PLA₂, calcium-dependent K⁺ channels, nitric oxide synthase (NOS), voltage-operated calcium channels, and MAP kinase pathways.¹⁷³ Some P2Y receptors are also linked to inhibition of adenylate cyclase activity.^10,64

On the basis of their structural similarities, P2Y receptors have been divided into two distinct groups: group I consists of P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors; group II consists of P2Y₁₂ and P2Y₁₃ receptors.¹⁷⁴ The UDP-glucose-sensitive GPCR (KIAA0001)⁶⁵ is a potential member of this second group and has been previsionally designated as the P2Y₁₄ receptor.¹⁷⁴

Despite emerging evidence¹⁷⁵ that functional dimerization of GPCRs is a rule rather than an exception, there is currently little information regarding P2Y receptor dimers, although there is evidence for P2Y₁/adenosine A₁ receptor dimerization, the resultant oligomer showing a distinct pharmacology with A₁ agonists and antagonists showing reduced affinity with a concomitant 400-fold increase in affinity for the P2 agonist ADPβS.¹⁷⁶

3. Other Putative P2 Receptors.

As an addition to the list of receptors missasigned to the P2 receptor family already documented, the p2y3 receptor is a nucleotide-sensitive avian receptor for which a mammalian homologue has not yet been described.¹⁷² The p2y5 receptor was identified in activated chick T lymphocytes using radioligand approaches. No functional or structural support for the existence of this receptor has been reported. The P2Y₈ receptor was cloned from Xenopus neural plate and was activated to an equal degree by ATP, UTP, ITP, CTP, and GTP.¹⁵⁰ Mammalian homologues of this receptor have yet to be identified. The receptors cloned as P2Y₉ and P2Y₁₀ are not nucleotide receptors.¹⁷¹ A novel cysteinyl leukotriene receptor 1 (CysLT1) has been identified on human cordblood derived mast cells (hMCs) and is functionally responsive to the cysteinyl leukotrienes LTC₄ and LTD₄ and also to UDP. Interestingly, the effects of both the leukotrienes and UDP can be blocked by the CysLT1 antagonist MK 571.²¹³ This relationship between P2 receptors and CysLT receptors is intriguing especially in light of the misidentification of the LTB₄ receptor⁷⁴ as the putative P2Y₇ receptor,⁷³ suggesting perhaps that there is a close function relationship between the two receptor families. A P_2T-like receptor distinct from that on platelets that shows agonist responses to 2-MeSATP has been identified in brain capillary epithelial cells.²¹⁴

P2X Receptors.

Current evidence^106,107 indicates that functional P2X receptors have a trimeric motif. A model of the generalized P2X receptor subunit (Figure 8) shows the topography of glycosylation sites and the cytoplasmic orientation of both amino and carboxy termini. Features of the extracellular domain include 10 cysteine residues (–S), that are highly conserved among different P2X subtypes and are thought to form disulfide bridges. Four glycosylation sites have been identified on the rat P2X₁ receptor.²¹⁵ However, the number and location of glycosylation sites differ within the different subunits of the P2X family.^215,216 Numerous conserved positively charged residues (K and R) are present in the extracellular loop of P2X receptors, four of which at positions 68, 70, 292, and 309 are important for ATP binding to the human P2X₁ receptor. The neutral residue I67 of the rat P2X₂ receptor defines a critical region of ATP binding.²¹⁷

By use of the “SCAM” technique, in which individual residues were replaced with cysteine and various alkylating reagents were examined for their ability to disrupt ligand recognition by reacting covalently with accessible thiols, the residues within TM2 lining the pore of the rat P2X₂ receptor were identified.²¹⁸ I328, N333, and T336 were at the outer vestibule of the pore, while L338 and D349 lined the cation channel. D349 was solvent-accessible only when ATP was applied. Chimeras of PX₁ and P2X₂ receptor subunits in concert with site-directed mutagenesis of the N terminal of the first transmembrane domain demonstrated a critical role for this domain in agonist effects,²¹⁹ while calcium permeability of the P2X₂ receptor was abolished by replacing polar amino acid residues at Thr339 and Ser340 with tyrosine.²²⁰ A protein kinase C site on the C terminus was responsible for slowing desensitization of the P2X₂ receptor.²²¹

P2Y Receptors.

With information gathered predominately from the P2Y₁ receptor and the low-resolution structure of rhodopsin as a template, the structure of the chick brain P2Y₁ receptor was first modeled using the Quanta/Charmm software (Figure 9).²²² Ligand affinity SAR, sequence analysis of cloned P2Y receptors, and site-directed mutagenesis studies^223–225 have led to the refinement of computer-based models for ligand binding to P2Y receptors.

Recently a model of the human P2Y₁ receptor using the high-resolution structure of rhodopsin as a template resulted in a detailed model of antagonist binding to the P2Y₁ receptor binding.²²⁵ Ligand docking in the P2Y₁ receptor model provided a hypothesis for the coordination of ATP in the TM regions, consistent with site-directed mutagenesis results (see below). The structural similarity between the potent nucleotide antagonist MRS 2179 50 (see below) and nucleotide agonists suggests that receptor activation resulting in a specific conformational change depends on subtle differences between ligands.²²⁶

P2Y₁ receptor models have also been developed in parallel with alanine scanning mutagenic approaches.²²⁷ Modeling of cloned P2Y₁ receptor sequences focused on both the seven-membrane-spanning helical domains (TMs) and the three extracellular loops (ELs). The features of the putative binding site identified within the TM region were consistent with both mutagenesis results and known ligand specificities. A new “cross docking” modeling approach,²²⁴ which simulated the reorganization of the native receptor induced by the ligand, aided in energetically refining the three-dimensional structure of the complex. To ascertain which residues of the human P2Y₁ receptor were involved in ligand recognition, individual residues of both the TMs (3, 5, 6, and 7) and ELs (2 and 3) were mutated to alanine and various charged residues,^227,228 and a cluster of positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7 and, to a lesser extent, TM6 putatively coordinated the phosphate moieties of nucleotide agonists and antagonists. Agonists were inactive at R128A (TM3) and at R310A and S314A (TM7) mutant receptors and had a markedly reduced potency at K280A (TM6) and Q307A (TM7) mutant receptors. Positively charged residues of the human P2Y₂ receptor (H262, R265, and R292 in TM6 and TM7) were similarly found to be critical for activation,²²⁴ suggesting that residues on the exofacial side of TM3 and TM7 were critical determinants of the ATP binding pocket. In contrast, there was no change in the potency or efficacy of agonists in the S317A mutant receptor, and alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant receptors that exhibited a 7- to 18-fold reduction in potency compared to that observed with the wild-type receptor. These residues thus appear to serve a less important modulatory role in ligand binding to the P2Y₁ receptor.

Since changes in the potency of 2-MeSADP 14 and HT-AMP 15 paralleled the changes in potency of 2-MeSATP at the various mutant receptors, it appeared that the β- and γ-phosphates of the adenine nucleotides were less important than the α-phosphate in ligand/P2Y₁ receptor interactions.²²⁷ However, T221A and T222A mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) versus di- or monophosphate potency, a result indicating a greater role of these TM5 residues in γ-phosphate recognition. Taken together, the results suggest that the adenosine and α-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of P2Y receptors.

Two essential disulfide bridges in the extracellular domains of the human P2Y₁ receptor were also identified: one conserved among GPCRs and another conserved between the N-terminal domain and EL3, characteristic of P2Y receptors. Several charged residues in ELs 2 (E209) and 3 (R287) were critical for receptor activation, suggesting that the role of the ELs in ligand recognition was as important as that of the TMs.^227,228 Moreover, energetically favorable “meta binding sites” in the P2Y₁ receptor have been defined, involving the critical residues of the ELs. At these nucleotide docking sites that are postulated to lie distal to the principal TM site, a ligand may bind en route to the principal TM binding site. Such secondary binding sites may then serve to guide the ligand in its approach to the TM binding site and reduce the energy barrier to ligand/receptor complex formation.

Triphosphate Modifications (Figure 2A).

P2X receptors are not activated by AMP or adenosine, although ring-modified adenosine monophosphates (e.g., hexylthioAMP, 15) can activate P2X₁–P2X₄ receptors to varying degrees.^54,232 These 2-thioether 5′-monophosphate derivatives are potent at P2Y₁ receptors (see below).

Modification of the triphosphate group in the form of replacement of the bridging oxygen atoms with methylene units or of the charged oxygen atoms with sulfur has in some cases resulted in potent analogues that are resistant to degradation by nucleotidases. α,β-MeATP (α,β-methylene adenosine 5′-triphosphate, 10) in particular is highly potent at P2X receptors,⁷⁸ being selective for group 1 subtypes (EC₅₀ = 1–10 μM), at which it causes rapid desensitization. α,β-MeATP also activates P2X_2/3 heteromers that are less susceptible to desensitization.¹³² At P2Y receptors, α,β-MeATP is weak or inactive. Owing to the presence of the nonhydrolyzable methylene bridge, α,β-MeATP is more stable than other agonists under most assay conditions and its potency in comparison to other, hydrolyzable nucleotide agonists was therefore overestimated, being previously described as the most potent P2X receptor agonist. Another metabolically stable analogue, β,γ-MeATP, 11, was equipotent to ATP in eliciting vasoconstriction in the rat juxtamedullary afferent arterioles.²³³ The corresponding nucleotide analogue containing the unnatural l-adenosine enantiomer β,γ-Me-l-ATP (l-adenyl 5′-(β,γ-methylene)diphosphonate, l-AMP-PCP 12) is a more potent agonist than ATP at the P2X receptor in the guinea pig bladder^52,234 but is inactive in the guinea pig taenia coli P2Y receptor.

The thio substitution at the terminal phosphate also provides enzymatic stability, leading to such analogues as ATPγS (adenosine 5′-O-(3-thiotriphosphate, 7), ADPβS (adenosine 5′-O-(2-thiodiphosphate, 8), and UTPγS (uridine 5′-O-(3-thiotriphosphate, 9), a potent agonist at P2Y₂ receptors. Compound 7 is a potent agonist at various P2Y subtypes, but not P2X subtypes, and also inhibited ecto-ATPase competitively with micromolar affinity.²³⁵ UTPγS 9 is a potent P2Y₂ receptor agonist that is not readily degraded by nucleotidases.²³⁶ The corresponding β-thiodiphosphate, UDPβS, selectivity activates P2Y₆ receptors.²³⁶

Adenine and Ribose Modifications (Figure 2B).

Dinucleotide Derivatives (Figure 4).

Dinucleotides have both P2 receptor agonist and antagonist activity (see below). The activity and selectivity of diadenosine polyphosphates (Ap_nA, n = 4–6) 59–61 and mixed dinucleotide polyphosphates as agonists has been studied at recombinant P2X receptors and depends on the number of phosphates.^66,242–244 At the rat P2X₁ receptor, Ap₆A is a full agonist, while shorter homologues have increasingly diminished potency and efficacy. Uracil dinucleotides 62–64 function as agonists at P2Y₂ receptors.²⁴⁵

Polysulfonates (Figure 3).

A variety of aryl sulfonates, such as derivatives of the antiparasitic drug suramin (e.g., 23–26) and derivatives of histochemical dyes, have been described as P2 receptor antagonists. These contained anthraquinone (e.g., 27–32), arylazo (e.g., 33), or triphenylmethane (e.g, 35) moieties.

Pyridoxal Phosphate Derivatives (Figure 4A).

Other structural classes of ATP antagonists include derivatives of the coenzyme pyridoxal 5′-phosphate. A diazo derivative of pyridoxal phosphate (PPADS, 36, Figure 4A) was an antagonist at the vas deferens P2X receptor.²⁶³ Although shown to be 10- to 20-fold selective for P2X receptors,²⁶⁸ 36 was a P2Y receptor antagonist. 36 is inactive at the adenylate cyclase coupled P2Y receptor on rat C6 glioma cells. Initially, no distinction was made between 36 and its isomer 37 because of a synthetic error. These compounds may have distinct pharmacological properties, especially with respect to the action of UTP. At P2X receptors, 37 tends to be slightly more potent than 36.²⁶⁹

Analogues of PPADS, 37–41, in which the sulfonate groups on the phenylazo ring were replaced with other functional groups, such as electron-withdrawing nitro groups, as in PPNDS, demonstrated that the sulfonate groups were not essential for antagonist activity.^270–273 Both 38 and 39 were potent antagonists of recombinant rat P2X₁ receptors expressed in oocytes and also completely antagonized the P2X-receptor-mediated contraction of guinea pig vas deferens and urinary bladder. At recombinant rat P2X₂ receptors, 38 (IC₅₀ = 1.1 μM) was more potent than 39.²⁷⁴ Phosphonate analogues of PPADS 42 and 43 were similar in potency to the phosphate derivatives. Compound 42 showed relatively high potency at turkey P2Y₁ receptors and at recombinant rat P2X₂ receptors, with IC₅₀ values of 4.4 and 1.1 μM, respectively. Replacement of the chloro substituent of 42 with a sulfonic acid eliminated binding to P2Y₁ receptors.²⁷¹ The most active analogue at P2X₁ (IC₅₀ = 5 nM) and P2X₃ (IC₅₀ = 22 nM) receptors was 43, being 14-fold and 10-fold more potent than PPADS itself.²⁷³ Compound 43 produced a nonsurmountable inhibition when tested against a range of ATP concentrations, although blockade was reversed by about 85% after 20 min of washout. The diazo linkage of PPADS was also replaced in analogues containing a carbon bridge, a modification that maintained potency at P2X receptors and enhanced chemical stability. Thus, the PPADS template can be altered at the pyridoxal and phenyl moieties to produce P2X₁ and P2X₃ receptor antagonists showing higher potency and a greater degree of reversibility than the parent compound at these group I P2X receptors.

Nucleotide Derivatives (Figure 4B).

Nucleotide derivatives (Figure 4B) have long been used in various modalities to block the effects of ATP. The agonist α,β-MeATP 10 (Figure 2) can block P2X receptor responses via rapid desensitization of the receptor. Other nucleotide derivatives antagonize P2 receptor effects in a more competitive manner.¹⁸¹ A number of nucleotide derivatives, e.g., 44 – 46, have been developed as inhibitors of the platelet P2Y₁₂ receptor. ARL-66096 44 is an ADP receptor antagonist that is a potent inhibitor of platelet aggregation, with the corresponding β,γ-dichloromethylene derivative ARL 67085MX 45²⁷⁵ antagonizing the ADP effects with 30000-fold selectivity for this receptor subtype. However, this compound also potently stimulates the human P2Y₁₁ receptor. ARL 69931MX 46 has exceptionally high potency (IC₅₀ = 0.4 nM) and may be more selective for the P2Y₁₂ subtype.²⁰⁵ Compounds 57a and 57b were derived from ARL 69931MX, and its congeners were found to be potent P2Y₁₂ antagonists lacking a triphosphate group.²³⁵

Trinitrophenyl-ATP (TNP-ATP) 47 and the corresponding di- and monophosphate derivatives are nanomolar antagonists at P2X₁, P2X₃, and P2X_2/3 subtypes expressed in Xenopus oocytes,²⁷⁶ while the potencies are much lower in other functional models.²⁷³

Oxidized ATP (oATP), 48, proposed as an irreversible antagonist for the P2X₇ receptor in the J774 mouse macrophage-like cell line,¹⁶¹ can antagonize the response to ATP acting via P2X₇ receptors without antagonizing coexpressed P2Y receptors.

Naturally occurring nucleotide bisphosphate derivatives, including 49a,b, at micromolar concentrations, act as competitive antagonists or partial agonists of P2Y₁ receptors.^277–280 49b has partial agonist properties at the turkey P2Y₁ receptor but is a pure antagonist at human P2Y₁ receptors. Both 2′- and 3′-deoxy modifications of A3P5P were well tolerated at P2Y₁ receptors, and the removal of the free hydroxyl group decreased agonist efficacy. The introduction of a 2-chloro substituent resulted in 51, which was a selective antagonist at P2Y₁ receptors, e.g., inactive at rat P2X₁ receptors.²⁸⁰ The N⁶-methyl modification resulting in the competitive antagonist MRS 2179 50 enhanced antagonistic potency of 2′-deoxyadenosine 3′,5′-bisphosphate by 17-fold. MRS 2179 had a K_B value at turkey P2Y₁ receptors of 100 nM and was inactive at other P2Y receptor subtypes, including the C6 glioma cell P2Y receptor coupled to inhibition of adenylyl cyclase (J. Boyer, unpublished results). The N⁶-ethyl modification of MRS 2179 was of intermediate potency as an antagonist, while the N⁶-propyl group completely abolished both agonist and antagonist properties. Thus, the N⁶-binding region of the P2Y₁ receptor appears to contain a small hydrophobic pocket. 2-Methylthio and 2-chloro analogues were partial agonists of intermediate potency. A 2′-methoxy group provided intermediate potency as an antagonist while enhancing agonist activity. An N¹-methyl analogue was a weak antagonist with no agonist activity. An 8-bromo substitution and replacement of the N⁶-amino group with methylthio, chloro, or hydroxy groups greatly reduced the ability to interact with P2Y₁ receptors. Benzoylation or dimethylation of the N⁶-amino group also abolished the antagonist activity. MRS 2179 did antagonize electrophysiological effects of ATP at the rat P2X₁ receptor with an IC₅₀ value of 1.2 μM.²⁸⁰

By analogy to acyclic modifications of nucleosides used as antiviral agents, an acyclic modification of the ribose ring of the bisphosphate antagonists also preserved affinity at the P2Y₁ receptor. MRS 2286 52 was a pure antagonist at turkey P2Y₁ receptors (IC₅₀ = 840 nM) and was inactive at P2X₁ receptors.²⁸⁰ All acyclic analogues examined to date are inactive as agonists at P2Y₁ receptors.

Although introducing flexibility in the form of acyclic modifications preserved antagonistic activity, constraining the ribose ring conformationally increased the potency of P2Y₁ receptor antagonists. A 2-Cl-N⁶-methyl-(N)-methanocarba derivative, MRS 2279 53, was a potent antagonist (IC₅₀ = 52 nM at turkey P2Y₁ receptors). In the (N) methanocarba analogues, as with other ribose modifications, e.g., 21, the presence of an N⁶-methyl group in these bisphosphate analogues transformed either a partial or full agonist into a pure antagonist, and the 2-chloro modification enhanced affinity.²⁸¹

Diinosine polyphosphates (Figure 6), e.g., Ip_nI 65, are highly selective antagonists for group 1³ P2X receptors, with Ip₅I blocking P2X₁ receptors in the nanomolar range and with both Ip₄I and Ip₅I blocking P2X₃ receptors in the micromolar range. These polyphosphates were inactive at P2X₂, P2X₄, and P2X_2/3 receptors.⁷⁰

Nonhighly Charged Derivatives (Figure 5).

The isoquinoline derivative KN-62, 54a, is an antagonist of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at micromolar concentrations and a potent antagonist at P2X₇ receptors at even lower concentrations.¹⁶² KN-04 54b has similar potency as a P2X₇ receptor antagonist and is inactive at CaMKII. 54a noncompetitively antagonized P2X₇ receptors in HEK cells with an IC₅₀ value of ~15 nM. In human leukemic B lymphocytes, 54a reduced the rate of permeability increase to larger permeant cations, like ethidium, induced by BzATP with an IC₅₀ value of 13 nM and complete inhibition of flux at 500 nM.²⁸² Compound 54a was inactive at the neutrophil P2Y₂ receptor. Exploration of the SAR of KN-64 resulted in tyrosyl derivatives that were highly potent in mouse and equipotent to KN-62 at human P2X₇ receptors.^283,284

The dihydropyridine nicardipine, 55, and related derivatives can antagonize and/or potentiate P2X receptor responses.²⁸⁵ The pyrimidinedione derivative, 56, is a nonnucleotide antagonist of the P2Y₁₂ receptor in platelets.^260,261 Thienopyridines, such as clopidogrel, have been developed as antagonists of the ADP-induced aggregation of platelets, but the actual receptor antagonist is an active metabolite of the clinically administered compound.²⁸⁶

Miscellaneous Modulators of P2 Receptors (Figure 7).

Pyridylisatogen tosylate, 67 (PIT), is a weak P2 receptor antagonist that also displayed strong receptor-independent vasorelaxant effects.²⁸⁷ At cloned P2Y₁ receptors, PIT acts as an allosteric enhancer of agonist action.²⁸⁸

NBD chloride, 69 (Figure 7), selectively blocked ADP-induced aggregation of platelets through covalent modification of aggregin, a putative (non-P2) ADP receptor, on the cell surface.²⁸⁹

The potent activity of the ω-conotoxin GVIA¹⁴² suggests that other snail-derived peptides may function as P2X antagonists, in line with their broad effects on other ion channels.²⁹⁰ The macrolides, avermectin,⁸⁵ and erythromycin¹⁴⁹ represent additional pharmacophores active at P2X receptors that may bind to sites distinct from those recognizing nucleotides.

Potentiators of the action of ATP at P2X receptors have also been identified. Coumassie Blue²⁶⁴ and the pyridoxal phosphate derivative MRS 2220 72 selectively enhance the effects of ATP at P2X₁ receptors,²⁹¹ and the charged dihydropyridine derivative MRS 2154 73²⁹² selectively enhances the effects of ATP at P2Y₁, P2X₁, and P2X₂ receptors.

Biological Actions and Clinical Targets.

ATP is a key component of every cell in the body and is ubiquitously available in the extracellular medium as a neuromodulatory agent. Its effects on cell function are multifactoral as a distinct ligand, as part of the purinergic cascade,¹¹ and as a source of cellular energy. Thus, modulation of P2-receptor-mediated ATP responses may be anticipated to have profound effects on cellular and tissue function at both the cellular and intracellular levels. However, since ATP appears to be a normal constituent of the extracellular environment, it appears highly probable that functional alterations in extracellular levels of ATP and thus P2 receptor hypoor hyperfunction, or alterations in receptor number associated with discrete disease states, will provide the opportunity for developing novel therapeutic agents that act via P2 receptors. In this context, the therapeutic areas currently of interest are pulmonary (P2Y₂/P2Y₄, Phase III), thrombosis (P2Y₁₂, Phase III), pain (P2X₃, preclinical), and bladder dysfunction (P2X₃, preclinical). Furthermore, as detailed below, evidence is now emerging that in some disorders, there are robust changes in P2 receptor message and/or protein that will dictate tissue responses to extracellular nucleotide concentrations.

Cardiopulmonary Function.

P2X₁, P2X₃, P2X₄, P2Y₂, P2Y₄, and P2Y₆ receptors are present in human fetal heart²⁹³ and mediate distinct effects from those of adenosine acting at P1 receptors.²⁹⁴ ATP is a mediator of vagal reflexes in the heart and lung^295–299 and potently regulates vascular tone²⁹⁷ causing either contraction or relaxation depending on receptor location. Nucleotide-mediated vascular contraction occurs by a direct effect on smooth muscle cells²⁹⁸ involving P2X receptor activation,²⁹⁹ while relaxation involves a P2Y-receptor-mediated, endothelium-dependent mechanism.^300–303 A rat model of ischemic congestive heart failure produced by left coronary artery occlusion shows a selective down regulation of P2X₁ receptor mRNA in resistance vessels³⁰⁴ that can be related to an attenuation of P2X-receptor-mediated pressor effects in vivo.³⁰⁵ ATP may also play a role in the development of vascular disease, e.g., atherosclerosis and hypertension, via its trophic actions.³⁰⁶

In the respiratory system, the nucleotide functions as a homeostatic mediator,³⁰⁷ its presence in the extracellular space modulating normal lung function. Acting via both P2X and P2Y receptors, ATP maintains the patency of the airspaces by modulating the release of phosphatidylcholine as a surfactant and by stimulating mucus secretion, facilitating mucociliary clearance, regulating ciliary beat frequency, and attenuating the inflammation associated with macrophage infiltration following respiratory tract infection, allergen inhalation, and injury. ATP and UTP, acting via P2Y₂ receptors, stimulate chloride secretion in airway epithelium and mucin glycoprotein release from epithelial goblet cells,^308,309 enhancing mucociliary clearance. This represents a potential treatment for cystic fibrosis (CF) and chronic bronchitis. In controlled clinical studies UTP, used in preference to ATP as a P2Y₂ receptor agonist because it does not form cardiovascularly active metabolites such as adenosine, dose-dependently stimulated mucociliary clearance and sputum expectoration in smokers, nonsmokers, and patients with chronic bronchitis.³¹⁰ E-NTPase-resistant analogues of UTP such as INS 365 63,³¹¹ in addition to being used in CF and chronic bronchitis, may act as adjunctive agents to enhance the effectiveness of antibiotics used in the treatment of respiratory infections and thus to reduce the amounts used, potentially avoiding antibiotic resistance phenomena.

ATP may also have a direct role in asthma via its actions on bronchial innervation. P2X receptors in the ventrolateral medulla are involved in cardiorespiratory control networks.³¹² The nucleotide triggers a reflex bronchconstriction via activation of P2X receptors on vagal C fibers,^313,314 and both ATP and UTP can potentiate IgE-mediated mast cell histamine release, effects involving P2Y receptors.³¹⁵ ATP acting via presynaptic P2X receptors in the nucleus tractus solitarius can facilitate glutamate release³¹⁶ as well as modulate renal and adrenal sympathetic nerve activity.³¹⁷

Hemostasis.

ADP as already discussed above is a potent platelet recruiting factor, inducing platelet aggregation.³¹⁸ This involves a complex interplay among three distinct platelet P2 receptors: a P2Y₁ receptor linked to phospholipase C pathways,³¹⁹ the P2Y₁₂ receptor linked to adenylate cyclase inhibition,⁷² and a P2X₁ receptor.³²⁰ In P2Y₁ knockout mice, which showed increased bleeding time and resistance to thromboembolism, ADP was still able to inhibit platelet adenylate cyclase activity, indicating the presence of a second ADP-responsive P2 receptor linked to adenylyl cyclase,^184,185,321 while in P2Y₁₂ knockout mice, there is prolonged bleeding time and reduced sensitivity to ADP, thrombin, and collagen.²¹⁰

Hydrolysis of ADP by the E-NTPase CD39 inhibits platelet aggregation both by removing ADP and by forming adenosine that can also inhibit aggregation.³²² ATP is a competitive ADP antagonist at platelet P2Y receptors and stimulates production of PGI₂ and NO, which can also inhibit platelet aggregation and act as vasodilators. Exogenous ATP can thus act to localize thrombus formation to areas of vascular damage, controlling the relationship among hemostasis, thrombosis, and fibrinolysis. In turn, CD39 appears to act together with the extracellular nucleotides released as a result of tissue damage to modulate blood fluidity and platelet activation. Differentiation of the human promyleocytic leukemia cell line, HL-60, into neutrophils is mediated via ATP effects on the P2Y₁₁ receptor;³²³ thus, agents acting at this receptor may be of interest in relation to the potential treatment of neutropenia and leukemia.^323,324

AR-C 69331-MX 46 is one of a series of systemically active, synthetic P2Y₁₂ receptor antagonists²⁰⁵ that has a safer side effect profile than aspirin and has superior antithrombotic properties compared to other modulators of platelet activity, e.g., GPIIb/IIIa antagonists, which show a narrow margin of safety.²⁰⁷ An orally active P2Y₁₂ receptor antagonist derived from 57b (Figure 5) is reportedly entering Phase I trials as an antithrombotic agent.²³⁵ Both P2Y₁₁ and P2Y₁₂ receptors appear to play a role in hematopoesis.^174,324

ATP and Neuronal Excitability.

The central and peipheral nervous systems contain both P2X and P2Y receptors. ATP acts as fast transmitter in nervous tissue via activation of P2X receptors,^325,326 with other actions being mediated via P2Y receptors. ATP can produce its effects directly via actions on the postsynaptic membrane as well as via an indirect action on presynaptic P2 receptors to modulate the release of a variety of neurotransmitters^8,327 including acetylcholine,^36,328,329 norepinephrine,³³⁰ dopamine,^331,332 serotonin,³³³ and glutamate^316,334 and can enhance GABA,^335,336 vasopressin, and oxytocin release.³³⁷ In the case of GABA, this effect of ATP appears to be mediated via P2X₇ receptors.³³⁸ In nervous tissue, given the co-release of ATP with other transmitters,³ the effects of ATP on transmission can be amplified by modulation of the effects of these other intercellular messengers. ATP also functions to transmit information between neurons and glia.⁸

Microinjection of ATP analogues into the prepiriform cortex can induce generalized motor seizures similar to those seen with NMDA and bicuculline.³³⁹ P2X₂, P2X₄, and P2X₆ receptors are expressed in the prepiriform cortex,¹⁰⁰ suggesting that a P2X receptor antagonist may have potential as an antiepileptic.³³⁹ These receptors are also highly expressed in cerebellum and are colocalized with AMPA-sensitive glutamatergic neurons in hippocampus, suggesting a role in the modulation of long-term potentiation.¹⁴⁶ P2X₇ immunoreactive cells are also increased in the penumbral region around the stroke focus.¹⁵⁴

Auditory and Visual Function.

In the auditory system, ATP, acting via P2Y receptors, depresses sound-evoked gross compound action potentials in auditory nerves and the distortion product otoacoustic emission, the latter a measure of the active process of the outer hair cells.³⁴⁰ P2X and P2Y receptors are present in the vestibular system,^126–129 and P2X₂ receptor splice variants are present in the cochlea. P2X_2–1 and P2X_2–3R receptors are found in the rat, and P2X_2–1, P2X_2–2, and P2X_2–3 receptors are found in the guinea pig.^130,341 In the rat, P2X splice variants are found on the endolymphatic surface of the cochlear endothelium, an area associated with sound transduction.³⁴² P2Y receptors are present in the marginal cells of the stria vascularis, a tissue involved in regulating the ionic and electrical gradients of the cochlea. While little is currently known regarding the pharmacology of hearing and vestibular function, ATP may regulate fluid homeostasis, cochlear blood flow, hearing sensitivity, and development. The parasensory cation flux in cochlear outer sulcus cells and vestibular transitional cells is modulated by ATP acting via a P2X₂ receptor that regulates endolymphatic ion homeostasis and provides protection during intense stimulation.³⁴¹ In a guinea pig model of unilateral peripheral vestibular disorder, intracochlear administration of ATP via a novel delivery system can restore normal function, an effect that preliminary data suggest is mediated by P2Y receptors.³⁴³

In the eye, ATP acting via both P2X and P2Y receptors modulates retinal neurotransmission, affecting retinal blood flow and intraocular pressure ATP.³⁴⁴ In the ocular mucosa, P2Y₂ receptor activation increases salt, water, and mucus secretion and may thus represent a potential treatment for dry eye disease.³¹¹ In the retinal pigmented layer, P2Y₂ receptor activation can promote fluid absorption and may be effective in treating retinal detachment.

P2X receptors are present in bipolar cells of the rat eye^345,346 where they mediate fast, direct neuronal–glial interactions³⁴⁷ and appear to participate in signal transmission processes in the retina.³⁴⁸ There is also emerging evidence that P2-receptor-dependent neurotransmission may play a role in the olfactory and gustatory systems.^348,349

Pain.

ATP is a cotransmitter with norepinephrine (NE) in sympathetic nerves, with acetylcholine (ACh) in parasympathetic nerves supplying the bladder, and in nonadrenergic and noncholinergic (NANC) inhibitory enteric nerves.¹³⁵ The nucleotide has both excitatory and sedative effects in the central nervous system (CNS), with both P2X and P2Y receptors being widely distributed in the central and peripheral nervous systems.³ A specific role for ATP in pain signaling was indicated by seminal work showing that the nucleotide was released from sensory nerves,³⁵⁰ that it produces fast excitatory potentials in dorsal root ganglion neurons,³⁵¹ and that it is a central mediator of primary afferent fiber conduction.³⁵² These actions appear to be physiologically relevant, since exogenous ATP enhances hyperalgesia in a human blister base model³⁵³ and iontophoretic application of ATP to human skin can elicit pain.³⁵⁴ The nucleotide is also a key mediator of neurogenic inflammation via its actions on P2 receptors present on neutrophils, macrophages, monocytes, and microglia, activation of which results in cytokine production and release.⁶⁰ The homomeric P2X₃ receptor and a heteromultimeric combination of P2X₃ and P2X₂ (P2X_2/3) receptors are expressed on a high proportion of isolectin IB4-positive neurons in dorsal root ganglion^{137,355–357} and also human dental pulp.³⁵⁸ Immunohistochemical studies have shown that P2X₃ receptor expression is up-regulated in dorsal root ganglion neurons and in ipsilateral spinal cord following chronic constriction injury (CCI) of the sciatic nerve,³⁵⁹ an effect that results in a specific ectopic sensitivity to ATP, since it is not observed on contralateral (uninjured) nerves.

Many studies have shown that P2 receptor ligands modulate nociceptive behaviors in rodents.^360–363 P2 receptor agonists (e.g., ATP and the more stable analogue α,β-meATP) produce acute paw flinching following intradermal administration^364,365 and enhance the nociceptive effects of formalin³⁶⁶ and carrageenan.³⁶⁷ Furthermore, P2 receptor antagonists, e.g., suramin and PPADS, have antinociceptive effects in rodent models of both acute³⁶⁸ and chronic nociception.^369–371 The potent P2X antagonist TNP-ATP 47 attenuates the nociceptive effects of P2 receptor agonists following intrathecal administration³⁷² and is antinociceptive given intradermally.²⁶⁵ These data suggest that TNP-ATP can provide effective antinociception when this P2X receptor antagonist is administered directly to a relevant site of action. Consistent with these pharmacological data, P2X₃ knockout mice show a loss of rapidly desensitizing inward currents induced by ATP in DRG neurons^138,139 with evidence of a modest but nonsignificant increase in P2X₂ homomers³⁷³ and a significantly reduced, but not elimination of, pain-related behaviors in response to intraplantar ATP or formalin.^138,139 The attenuation, but not elimination, of nociceptive responses in the P2X₃ knockout mice has been related³³ to a residual ATP-mediated nociceptive effect mediated via a P2Y₁-receptor-dependent effect on VR1 receptor function. The ability of ATP to deplete substance P from rat primary sensory neurons³⁷⁴ and the blockade of the faciliatatory effects of ATP on glycine release in neurons from the trigeminal nucleus pars caudalis by Substance P³⁷⁵ suggests a functional interplay between neurokinin-1 (NK-1) and P2 pain-sensing pathways.

Further strengthening the evidence for a role of P2X₃ receptor activation in nociception is the recent demonstration that intrathecal delivery of a P2X₃ receptor gene antisense oligonucleotide reduces P2X₃ receptor expression in dorsal root ganglion and nociception in inflammatory and neuropathic pain models.³⁷⁶ The ability of systemically administered TNP-ATP to fully block acetic acid induced abdominal constrictions in mice³⁷³ further supports a role for P2X₃ receptors in visceral nociception. However, other studies based on the use of P2 antagonists such as suramin and PPADS have questioned the contribution of spinal P2X receptors to pain perception.^377,378

ATP has also been implicated in the pain associated with migraine by virtue of its effects on the neurovsaculature.³⁷⁹ Coadministration of ATP with nitric oxide (NO), the nucleotide probably acting as adenosine following hydrolysis, mimics the effects of the inhalation anesthetic enflurane and also reduces the amount of inhalation anesthetic required for anesthesia.³⁸⁰

P2X₃ receptor mutant mice also show a marked bladder hyporeflexia, suggesting that this P2 receptor subunit plays a preferential role in visceral sensations.¹⁴¹ For visceral pain, a purinergic mechanosensory transduction mechanism has evolved^{135,381–384} where distention of tubes including ureter, gut, salivary and bile ducts, and sacs such as the urinary³⁸⁴ and gall bladders causes ATP release from the lining epithelial cells to act on P2X₃ receptors located on the subepithelial sensory nerve plexus to relay nociceptive signals to the CNS.

Trophic Actions.

The viability and also the regeneration of nervous tissue are sustained by a variety of endogenous polypeptide trophic factors. Neural injury increases growth factor levels, e.g., fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor.^385,386 ATP acts in combination with these growth factors to stimulate astrocyte proliferation contributing to the process of reactive astrogliosis, a hypertrophic/hyperplastic response that is typically associated with nervous system trauma including stroke/ischemia, seizure disorders, and neurodegenerative diseases, e.g., Alzheimer’s and Parkinson’s diseases.

In reactive astrogliosis, astrocytes undergo process elongation and express GFAP (glial fibrillary acidic protein), an astrocyte-specific intermediate filament protein and show an up-regulation of P2X receptors.³⁸⁷ ATP can increase GFAP and AP-1 complex formation in astrocytes, mimicking the actions of FGF. ATP and GTP can induce trophic factor (NGF, NT-3, FGF) synthesis in astrocytes and neurons.³⁸⁸ The effects of GTP are, however, inconsistent with any known P2 receptor. A synthetic purine, Neotrofin (AIT-082 71, Figure 7), has been reported to up-regulate neurotrophin production and also to enhance working memory and restore age-induced memory deficits in mice.³⁸⁸ While initial clinical trials of Neotrofin in Alzheimer’s disease resulted in positive reports, pivotal Phase III placebo-controlled trials failed to show efficacy.

NGF can up-regulate P2X₂ receptor protein and induce neuritogenesis in PC12 cells. The latter effect can be blocked by a number of putative P2 receptor antagonists,³⁸⁹ suggesting a potential role of P2 receptors in NGF signal transduction processes. ATP can also induce TNFα release from rat microglia, albeit at heroic concentrations (1 mM), an effect mediated by P2X₇ receptors involving sustained calcium influx and a MEK/ERK pathway.³⁹⁰ This may play a key role in blunting the inflammatory response following brain trauma³⁸⁷ and in the increase in P2X₇ receptor message in the stroke penumbra.¹⁵⁴ In vascular smooth muscle cells, ATP can induce cell proliferation via modulation of the cell cycle,³⁹¹ acting as a “competence” factor in combination with other growth factors to facilitate tissue repair and regeneration.

The trophic effects of purines also extend to effects on immune cell function. Activation of the macrophage P2X₇ receptor by ATP results in both the activation of the caspase-1 (interleukin-1β convertase; ICE),^{156,163–165} leading to induction of apoptosis,³⁹² and nonselective ion pore formation¹⁶⁶ in mast cells, platelets, macrophages, and lymphocytes.^393,394

Formation of multinucleated giant cells (MGCs) by the fusion of monocytes granulomatous inflammation can be stimulated by IFN-γ and LPS via an up-regulation of P2X₇ receptor expression³⁹⁵ and a concomitant decrease in E-NTPase activity, increasing the susceptibility of MGCs to the cytolytic actions of extracellular ATP. The ATP-induced apoptotic cascade can involve activation of the transcription factor NF-κB³⁹⁶ and is blocked by caspase inhibitors. ATP can also induce cytolysis in macrophages infected with mycobacterium via P2X₇-receptor-mediated apoptotic and necrotic events.³⁹⁷ While the novel antimicrobial activity of ATP was initially thought to have potential utility in the treatment of tuberculosis, studies in P2X₇ receptor knockout mice showed that this receptor, while involved in bacterial killing, was not essential for the antimicrobial effects of ATP.¹⁶⁹ Activation of the P2Y₆ receptor stimulates IL-8 production from monocytes.¹⁹⁹

The involvement of nucleotides in developmental processes,³⁹⁸ where purinergic effects on tissue differentiation precede those of adrenergic signaling, indicates that in many respects, purine-related effects on development parallel the role of purines following tissue trauma, e.g., angiogenesis, cell proliferation, etc.

Bacterial Infection.

The intriguing,³⁹⁷ albeit non-mandatory role¹⁶⁹ of the P2X₇ receptor in macrophage killing of ingested mycobacterium together with the ability of the macrolide antibiotic erythromycin to interact with the P2X₄ receptor at therapeutic concentrations¹⁴⁹ suggests that purinergic signaling mechanisms may be involved in the regulation of bacterial growth. P2X₄ receptors appear to be the site at which erythromycin can block the effects of ATP on calcium influx, thus representing a potentially novel target to identify compounds that suppress fluid secretion in chronic respiratory tract infections.

Bacterial interactions with host cells involve the binding of bacteria to a membrane glycolipid asialoGM1 (ASGM1). Binding of P. aeruginsa flagellin to ASGM1 present on human HM3 epithelial cells promotes the autocrine release of ATP from host cells, activating a P2Y receptor, possibly the P2Y₁₁, on the host cell to elicit a pathogenic defensive response,³⁹⁹ suggesting, despite the evidence from the P2X₇ receptor knockout mouse,¹⁶⁹ that nucleotides may play an important, perhaps “multireceptor”-mediated²⁹⁷ role in the host response to bacterial infection.³⁹⁷

Lower Urinary Tract Function.

Urinary bladder function is regulated by sympathetic and parasympathetic input with ATP mimicking the effects of parasympathetic nerve stimulation, resulting in bladder contraction^400–403 via activation of P2X receptors in the smooth muscle of the urinary bladder detrusor muscle that is involved in bladder emptying.⁴⁰³ Detrusor dysfunction results in urge urinary incontinence (UUI), a major health problem in the aging female population. P2X receptors are also present in the bladder urothelium⁴⁰⁴ with the P2X₁ receptor being the predominant subtype in adult bladder, with the rank order of other P2X receptors being P2X₁ ≫ P2X₄ > P2X₂ > P2X₇ > P2X₅ ≫ P2X₃ = P2X₆.⁴⁰⁵ The P2X₄ receptor shows high levels of expression in the developing, but not adult, bladder.⁴⁰⁶

The urethral relaxation that leads to micturition involves ATP functioning as a cotransmitter with nitric oxide (NO). NO mediates the first stage of relaxation⁴⁰³ with ATP acting via P2 receptors to mediate the second phase of the voiding response. Serosal ATP release occurs in rabbit⁴⁰⁷ and guinea pig³⁸⁴ bladder because of the hydrostatic pressure changes associated with bladder filling. Muscarinic receptors, through which ditropan and tolterodine produce their clinically beneficial effects on UUI, mediate 15% of the neurogenic contraction in rat urinary bladder. An additional 50% of the response appears to be mediated via P2X receptor mechanisms,⁴⁰⁸ suggesting that P2X receptor antagonists may have more potential in the treatment of UUI than cholinergic-based drugs. Partial bladder outlet obstruction in rabbit leads to an increase in purinergic and a decrease in cholinergic components of nerve-mediated detrusir contaction,⁴⁰⁹ a finding that may explain the poor responses to anticholinergic therapy in patients with detrusor instability.

Bladder distension in rats via the use of a 10 μM solution of α,β-MeATP reduces afferent nerve activity by 75% compared to saline controls⁴¹⁰ while P2X₃ receptor knockout mice have a marked bladder hyporeflexia, further supporting a role for P2 receptors in the processing of sensory information in the bladder.¹⁴¹ P2X₃ receptor immunoreactivity has been localized to the urothelium in rat and human bladder, indicating a nonneuronal localization leading to the suggestion,⁴¹¹ despite the knockout data, that P2X₃ receptors may not play a direct role in ATP-mediated sensory responses in the urinary bladder.

Tissue from patients with idiopathic detrusor instability lack P2X₃ and P2X₅ receptor innervation,⁴¹² while P2X₂ receptors in the posterior wall of the bladder were increased with other P2X receptors being decreased.⁴¹³ Furthermore, in unstable bladder tissue but not that from controls, there was a significant purinergic component in nerve-mediated contractions. In tissue from patients with symptomatic outlet obstruction, P2X₁ receptor expression was increased, suggestive of an enhanced purinergic role in the unstable bladder resulting from outlet obstruction.^409,414

Detrusor strips from patients with interstitial cystitis, a chronic, symptomatic disorder of the bladder in females, showed an increased sensitivity to α,β-MeATP with decreased sensitivity to ACh and histamine⁴¹⁵ also indicative of altered purinergic neurotransmission. Both low- and high-threshold fibers in pelvic afferents discharged in response to P2 agonists, indicating that both nociceptive and nonnociceptive mechanasensory responses in the bladder are under purinergic control leading to the suggestion.⁴¹⁶ In male rat genitalia, P2X₁ and P2X₂ subunit antibodies show immunoreactivity in the membranes of the smooth muscle layer of the vas deferens, suggesting an involvement in sperm transport and ejaculation.⁴¹⁷ In male P2X₁ receptor knockout mice, fertility is reduced by approximately 90% with no effect on copulatory performance. This results from a decreased sperm count in the ejaculate due to a 60% reduction in the contraction sensitivity of the vas deferens to sympathetic nerve stimulation.¹¹⁹ The remaining 10% in fertility rate suggests that a minor non-P2X₁-receptor-mediated component is involved in vas deferens smooth muscle tone. While a P2X₁ receptor antagonist may conceptually represent a novel, non-hormonal male contraceptive, some additional attributes will be necessary for it to be a reliable approach to birth control.

In the body of the penis, strong P2X₁ with less P2X₂ subunit immunoreactivity was present in the smooth muscle of blood vessels and the corpus cavernosum, suggestive of a role in erectile function.⁴¹⁸ In adult rat testis, all P2X receptor subtypes are present with the exception of P2X₄ and P2X₆.⁴¹⁸ P2X₁ receptors were found exclusively on blood cells, while P2X₂, P2X₃, and P2X₅ subunits were expressed differentially on germ cells throughout the stages of the cycle of seminiferous epithelium. P2X receptors were present on Sertoli but not Leydig cells. ATP, acting via germ cell P2X receptors, may therefore play a role in controlling germ cell maturation.

ATP has variable effects on cavernosal smooth muscle depending on the basal tone, relaxing corporal smooth muscle at high basal tension and contracting it at low tension.⁴¹⁹ Both adenosine and ATP show an enhanced sensitivity in penile tissue responses in diabetic males, although in diabetic rats, ATP-induced relaxation is decreased while the effects of adenosine, which are thought to be mediated via potassium channels, are enhanced.⁴²⁰

Hepatic Function, Diabetes, and Gastrointestinal Tract Function.

There is a considerable body of data on the role of purines in the control of gut function.⁴²¹ Changes in liver cell volume, e.g., in epithelium following exposure to insulin, and the uptake of amino and bile acids increase ATP release,^41,422 resulting in a change in extracellular concentration from approximately 10 nM to greater than 300 nM.⁴²³ ATP then acts as an autocrine regulator to modulate membrane chloride conductance and thus facilitates cell volume recovery, linking the cellular hydration state to the P2-mediated pathways involved in cellular homeostasis.⁴²⁴ ATP is also involved in bile formation and biliary secretion via activation of cholangiocyte P2Y₂ receptors in the biliary tree.^425,426

ATP acts in the gastrointestinal tract as a paracrine signaling molecule, with ATP released from hepatocytes activating P2 receptor signaling pathways in neighboring hepatocytes and biliary cells.⁴²⁶ ATP may also act as a paracrine mediator in hepatobiliary coupling, a process coordinating the hepatocyte and ductular components of bile formation, thus having clinical potentialin increasing bile flow and in treating prolonged cholestasis.

Purines stimulate glycolysis in isolated perfused rat liver via mechanisms involving both P1 and P2 receptor activation and multiple signaling pathways resulting in an increase in hepatic glucose output.^427,428 ATP also stimulates pancreatic insulin release via a glucose-dependent, P2Y-receptor-mediated mechanism^429,430 and modulates insulin secretion by interactions with ATP-sensitive potassium channels in islet β-cells. ADP thus antagonizes the ATP inhibition of these channels by binding to the second nucleotide binding site on the associated sulfonylurea receptor (SUR),⁴³¹ thus activating K_ATP channels and inhibiting insulin secretion. P2X₁, P2X₄, P2X₇, P2Y₁, and P2Y₂ receptors are found in pancrease from rat and mouse.⁴³² P2X₇ receptors were localized in the outer periphery of the islet in α cells that were neither somatostatin- nor insulin-positive.

Both ATP and adenosine are potent stimulants of fluid and electrolyte (chloride) secretion in the colon and gall bladder and in the pancreatic and bile ducts,⁴²⁴ effects that appear to primarily involve P2Y₂ receptor activation.

Bone Function.

Both P2X and P2Y receptors are present on the two principle types of cell in bone tissue, macrophage-derived osteoclasts and osteoblasts, that originate from mesenchymal stem cells and are responsible for bone formation. ATP, released in response to shear stress^21,135,433 functions as a mechanotransducer in skeletal tissue acting as an osteoblast mitogen, potentiating the effects of growth factors on bone cells.⁴³⁴ ATP, but not adenosine, can stimulate the formation of osteoclasts and their resorptive actions in vitro and can inhibit osteoblast-dependent bone formation.⁴³⁵ The bisphosphonate clodronate, used in the treatment of Paget’s disease and tumor-induced osteolysis, may interact with osteoclast P2 receptors⁴³⁴ ADP, interacting with P2Y₁ receptors on mouse calvarial osteoclasts at nanomolar concentrations, and is a potent osteolytic agent, stimulating bone resorption. It is more potent than ATP being equivalent in potency to PGE₂ via a mechanism involving endogenous prostaglandin synthesis. P2 receptor agonists may thus have potential in the treatment of osteoporosis, rheumatoid arthritis, periodontitis, osteopenia, and inflammatory bone loss.^436–438

Cancer.

While there is a considerable clinical data on the potential use of ATP as a treatment for cancer and the cachexia associated with cancer,⁴³⁹ little progress has been made in advancing the nucleotide to general use or more precisely in understanding its functional role in attenuating metastasis progression. Given the proapoptotic effects of the nucleotide acting via P2X₇ receptors,³⁸⁸ it is likely that ATP effects on cancer growth may be cytokine-dependent because the nucleotide modulates cytokine release.^{60,386,440,441} An alternative mechanism may be the ability of ATP to induce COX-2 expression,¹⁹⁰ inhibitors of the latter enzyme having been demonstrated to suppress colon carcinoma growth in controlled clinical trials.⁴⁴²

The initial reports on the utility of ATP as an antimetastatic agent⁴³⁹ attributed the effects of the nucleotide to inhibition of gluconeogenesis, inhibition of the acute-phase response, and/or the decreased production of the proinflammatory cytokines IL-1 and IL-6.^440,441 In a nonrandomized clinical trial, ATP infusion (50 μg kg^-1 min^-1 for 96 h) at 28-day intervals to patients with advanced non-small-cell lung cancer (NSCLC) increased ATP pools in red blood cells, inhibited weight loss, reduced cachexia, and improved survival.⁴³⁹ In a subsequent randomized trial in patients with advanced (stage IIIB or IV) NSCLC, intravenous ATP infused for 30 h at 2–4 week periods with outcome parameters assessed every month for 7 months resulted in a reduction in mean weight changes per month from a control value of −1.0 kg to a negligible change (0.2 kg) in the ATP-treated group.⁴⁴¹ Serum albumin, which was reduced by −1.3 g/L in the untreated group, and elbow flexor muscle strength, which was decreased by 6% in the untreated group, showed no change, e.g., remained normal, in the ATP-treated group. Negative effects on quality of life measures were also attenuated by ATP, with positive effects on body weight, muscle strength, and albumin concentration being especially marked in cachectic patients. There was no effect on tumor growth or survival.

In nude mice homozygous or heterozygous for the cystic fibrosis transmembrane conductance regulator (CFTR), a decrease in breast tumor implantability was reported that had been ascribed to elevated blood ATP levels.⁴⁴³ Similarly, ATP was found to reduce breast tumor cell growth in vitro, supporting the concept of ATP as an antitumor agent. In cystic fibrosis (CF) patients, there is a reduced incidence of breast cancer and melanoma that is accompanied by an increase in gastrointestinal tract malignancies.⁴⁴⁴

P2Y₂/P2Y₄ receptors have been identified on human colorectal carcinoma cells that may be associated with ATP-mediated control of cell proliferation control.⁴⁴⁵ In prostate carcinoma cells, ATP and BzATP, but not UTP or adenosine, inhibit cell growth, effects ascribed to P2X receptor activation.⁴⁴⁶

ATP has also been reported as inducing malignant tumor growth in brain,⁴⁴⁷ nucleotide infusion eliciting in astrocyte proliferation, reactive astrogliosis, and glioma formation. These in vitro effects contrast with those seen in the clinical situation.^439,441

In stratified epithelium, P2X₅ receptors are associated with proliferating and differentiating cells, while P2X₇ receptors label apoptotic cells.⁴⁴⁸ From these findings, it has been suggested that selective P2X₅ and P2X₇ receptor agonists may have potential in the treatment of skin disorders such as psoriasis, scleroderma, and basal cell carcinoma and for restenosis following angioplasty.