Introduction

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Diadenosine polyphosphates (Ap_nAs) and adenosine polyphospho guanosines (Ap_nGs) have been detected in human tissues (Schlüter et al., 1994, 1998; Jankowski et al., 1999) where they have been shown in many cases to mediate vasoconstrictor and vasodilator actions via P2X and P2Y receptors, respectively (Schlüter et al., 1994; Ralevic et al., 1995; van der Giet et al., 1997, 1999). Some dinucleotides, for example, diadenosine hexaphosphate (Ap₆A), may also exert negative chronotropic and inotropic effects on the mammalian heart (Vahlensieck et al., 1996). Diadenosine polyphosphates (Ap_nA, n = 3-6) are known to regulate the growth of rat mesangial cells grown in culture, an effect that may have a bearing on renal glomerular function in vivo and, subsequently, on renal control of blood pressure (Heidenreich et al., 1995; Schulze-Lohoff et al., 1995). Additionally, Ap_nAs can exert direct vasoconstrictor effects on renal blood vessels (van der Giet et al., 1997, 1999), as well as Ap_nGs showing vasoconstrictor actions in other blood vessels (e.g., mesenteric artery) where they can elicit fast inward currents in isolated vascular smooth muscle cells (Lewis et al., 2000).

P2X receptors are nucleotide-gated ion channels permeable to monovalent (Na⁺, K⁺) and divalent (Ca²⁺) ions and, in vascular tissues, cause both depolarization and Ca²⁺ influx sufficient to initiate smooth muscle contraction (Benham and Tsien, 1987). Of the P2X subunits cloned from mammalian tissues (Humphrey et al., 1998), transcripts for P2X₁, P2X₂, and P2X₄ have been found in rat cardiovascular tissues (Bogdanov et al., 1998; Nori et al., 1998), while P2X₃ transcripts are present mainly in mammalian sensory nerves but also are found in human cardiac tissue (Chen et al., 1995; Garcia-Guzman et al., 1997; Bogdanov et al., 1998). P2X₁ and P2X₃ receptors belong to group I P2X receptors and are characterized by agonism by ATP and , -meATP, with suramin blockade of agonist-evoked fast-desensitizing inward currents (Humphrey et al., 1998). P2X₂ (group II) and P2X₄ (group III) receptors are sensitive to ATP but not , -meATP, the former evoking slowly desensitizing currents that are blocked by either suramin or pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid at P2X₂ receptors but not P2X₄ receptors (Humphrey et al., 1998).

Dinucleotide activation of P2X receptors in blood vessels has been shown to depend on the length of the polyphosphate chain of these naturally occurring substances, with compounds possessing four or more phosphates causing potent vasoconstriction (Ralevic et al., 1995; van der Giet et al., 1999; Lewis et al., 2000). A recent study has indicated that blood pressure-regulating effects of dinucleotides might involve the activation of more than one type of P2X receptor in renal blood vessels (van der Giet et al., 1999). Thus, a P2X receptor mediating a transient fast constriction in the perfused kidney was tentatively identified as the P2X₁ receptor, while another unidentified yet pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid-sensitive P2X receptor was thought to mediate a sustained vasoconstriction (van der Giet et al., 1999).

The present study focused on the agonist properties at recombinant P2X_1-4 receptors of those Ap_nA and Ap_nG compounds that evoke vasoconstriction in blood vessels. The agonist properties of a recently isolated third group of naturally occurring dinucleotides, diguanosine polyphosphates (Gp_nGs), were also studied at these recombinant P2X receptors, to establish the pharmacological activity of these three families of dinucleotides at molecularly defined P2X receptors and, indirectly, to shed further light on their vasoconstrictor actions in vivo.

	Materials and Methods

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Oocyte Preparation. Xenopus laevis frogs were anesthetized in tricaine (0,4% w/v) and killed by decapitation. The ovarian lobes were removed surgically and stored at 4°C in Barth's solution [pH 7.45; 110 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 7.5 mM Tris-HCl, 0.33 mM Ca(NO₃), 0.41 mM CaCl₂, 0.82 mM MgSO₄, 50 µg/l gentamycine sulfate]. Mature oocytes were taken from ovarian sacs and were defolliculated by a two-step process involving collagenase treatment (type IA, 2 mg/ml in a Ca²⁺-free Ringer's solution, for 2-3 h), after which the follicular cell layer was stripped away using fine forceps. Defolliculated oocytes were stored in Barth's solution (4°C, pH 7.5). Defolliculation was necessary to remove the native purinoceptors found on the follicle cell monolayer enveloping oocytes (King et al., 1996). Cells were injected cytosolically with cRNA (capped RNA) for rP2X₁ (Valera et al., 1994), rP2X₂ (Brake et al., 1994), rP2X₃ (Chen et al., 1995), or rP2X₄ (Bo et al., 1995) (40 nl, 1 µg/µl), and then incubated at 18°C in Barth's solution for 48 h to allow full receptor expression and stored at 4°C in Barth's solution for up to 12 days.

Electrophysiology. Membrane currents were measured from cRNA-injected oocytes using a twin-electrode voltage-clamp amplifier (Axoclamp 2A; Axon Instruments, Foster City, CA). The holding potential (V_h) was 50 mV, unless stated otherwise. The voltage-recording and current-recording microelectrodes (1-5 -tip resistance) were filled with 3 M KCl. Oocytes were placed in an electrophysiological chamber (0.5-ml volume) and superfused (at 5 ml/min) with Ringer's solution containing 110 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 1.8 mM BaCl₂. The pH of the bathing Ringer's solution was adjusted to 7.5 by the addition of either 1.0 N HCl or 1.0 N NaOH. Electrophysiological data were stored on magnetic tape using a DAT recorder (Sony 1000ES; Tokyo, Japan) and displayed using a pen recorder (Gould 2200S; Cleveland, OH).

Solutions. All nucleotides were prepared in Ringer's solution and the pH of stock solutions readjusted to the desired level. Agonists were superfused by gravity flow from independent reservoirs placed above the preparation. All drugs were added for 120 s or until the evoked current reached a peak and then washed off with Ringer's solution for a period of 20 min. Data were normalized to the maximum current (I_max) evoked by the drug. The agonist concentration that evoked 50% of the maximum response (EC₅₀) was taken from Hill plots of the transform log(I/I_max  I), where I is the current evoked by each concentration of agonist. The Hill coefficient (n_H) was also taken from the slope of Hill plots.

Statistics and Graphs. Data are presented as mean ± S.E.M. of four to six (except for ATP, n = 16) sets of data from different oocyte batches. Concentration-response curves were fitted by nonlinear regression analysis using Prism, version 2.0 (GraphPad, San Diego, CA). Significant differences of potencies and efficacies were analyzed by using Kruskal-Wallis test (Instat, version 2.05A; GraphPad).

Drugs. ATP was purchased from Roche Molecular Biochemicals (Mannheim, Germany) and GTP disodium salt was purchased from Sigma Chemical Co. (Poole, Dorset, UK). Diadenosine pentaphosphate (Ap₅A), Ap₆A, adenosine pentaphospho guanosine (Ap₅G), adenosine hexaphospho guanosine (Ap₆G), diguanosine pentaphosphate (Gp₅G), and diguanosine hexaphosphate (Gp₆G) were synthesized and purified as previously described (Heidenreich et al., 1995; Schlüter et al., 1998).

	Results

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Effects at Rat P2X₁ Receptor. Like ATP, Ap₅A, Ap₅G, Ap₆A, and Ap₆G evoked fast inward membrane currents that rapidly inactivated in defolliculated X. laevis oocytes expressing recombinant rat P2X₁ receptors (Fig. 1, A and B, original tracing for Ap₆A not shown). Gp₅G, Gp₆G, and GTP failed to evoke significant membrane currents (Fig. 1C, original traces for Gp₅G and Gp₆G not shown).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Dinucleotide agonism at rP2X1 receptors. Representative traces of Ap5A-activated inward currents compared with activation by ATP (A), Ap5G and Ap6G were only partial agonists at rP2X1 (B), GTP failed to evoke a measurable inward current at rP2X1 (C). All agents were applied at 30 µM for 120 s. Vh = 50 mV.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Nucleotide potency at rat P2X1 receptors. Concentration-response curves for ATP (A), Ap5A (B), and Ap6A (C). Agonist activity was normalized to its own maximal response in each experiment (n = 6). EC50 values and Hill coefficients are given in Table 1. Curves were fitted using the Hill equation, as defined by Prism, version 2.0 (GraphPad); where error bars are not observed, they fall within the symbol size.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Nucleotide efficacy at group I P2X receptors. Maximum responses evoked by dinucleotides at supramaximal concentrations (30 µM) as a percentage of the maximal response evoked by ATP (30 µM) at rP2X1 (A) and rP2X3 (B) receptors. Agonist activity was normalized to ATP response, comparing each dinucleotide with ATP in the same cell (n = 4). *P < 0.05, **P < 0.01, significant differences ATP versus dinucleotide.

Effects at Rat P2X₃ Receptor. Like ATP, Ap₅A, Ap₅G, Ap₆A, and Ap₆G evoked fast inward currents that rapidly inactivated in defolliculated X. laevis oocytes expressing recombinant rat P2X₃ receptors (Fig. 4, A and B, original tracing for Ap₆A not shown). Gp₅G, Gp₆G, and GTP failed to evoke significant membrane currents (Fig. 4C, original tracing for Gp₅G and Gp₆G not shown).

View larger version (8K): [in this window] [in a new window]   Fig. 4.   Dinucleotide agonism at rP2X3 receptors. Representative traces of Ap5A-activated inward currents compared with activation by ATP (A), Ap5G and Ap6G were partial agonists at rP2X3 (B), GTP failed evoke a measurable inward current at rP2X3 (C). All agents were applied at 30 µM for 120 s. Vh = 50 mV.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Nucleotide potency at rat P2X3 receptors. Concentration-response curves for ATP (A), Ap5A (B), and Ap5G (C). Agonist activity was normalized to its own maximal response in each experiment (n = 6). For EC50 values and Hill coefficients are given in Table 2. Curves were fitted using the Hill equation, as defined by Prism, version 2.0 (GraphPad).

Effects at Rat P2X₂ Receptor. Ap₅A, Ap₅G, Ap₆A, Ap₆G, and GTP (all 30 µM) failed to activate membrane currents in defolliculated X. laevis oocytes expressing recombinant rat P2X₂ receptors (data not shown). ATP was a full agonist (EC₅₀ = 18.27 µM; confidence interval, 15.29-21.84 µM).

Effects at Rat P2X₄ Receptor. Ap₅A, Ap₅G, Ap₆A, Ap₆G, and GTP (all 30 µM) failed to evoke inward membrane currents in defolliculated X. laevis oocytes expressing recombinant rat P2X₄ receptors. ATP was a full agonist (EC₅₀ = 3.33 µM; confidence interval, 2.17-5.10 µM).

	Discussion

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The present results confirmed that highly purified Ap₅A and Ap₆A are agonists at P2X₁ and P2X₃ receptors, being relatively selective for these two group I P2X receptors and failing to activate either homomeric P2X₂ or P2X₄ receptors. Like ATP, these two diadenosine polyphosphates evoked fast inward currents that rapidly inactivated in their continued presence. Like ATP, they also required long washout periods of up to 20 min before reproducible inward currents could be evoked by a second challenge. It proved difficult to discriminate between the actions of Ap_nA compounds and ATP at P2X₁ and P2X₃ receptors, since they all yielded broadly similar EC₅₀ values in the low micromolar range. Their maximal responses were also broadly similar and each nucleotide could be considered as a partial agonist. Furthermore, there seemed little opportunity to exploit Ap_nA compounds as a means of distinguishing P2X₁ receptors from P2X₃ receptors, since any differences in their potency and intrinsic efficacy were very minor. It remains the case that the dinucleotidic antagonist diinosine pentaphosphate represents the most effective means to differentiate P2X₁ and P2X₃ receptors (King et al., 1999).

The bis-nucleotides Ap₅G and Ap₆G were also found to be selective agonists of P2X₁ and P2X₃ receptors, since they, too, were quite ineffective at homomeric P2X₂ and P2X₄ receptors. These naturally occurring substances evoked fast inward currents that rapidly inactivated and were thus similar to ATP. Our results showed that the mononucleotide GTP was inactive at P2X₁ and P2X₃ receptors, a finding in keeping with earlier studies of these two P2X receptors (Valera et al., 1994; Chen et al., 1995). Therefore, the observed agonist activity of Ap_nG compounds appeared to depend on the solitary adenine moiety. The EC₅₀ values of Ap_nGs were similar to corresponding Ap_nA compounds, the removal of one adenine moiety having no significant bearing on agonist potency. However, the maximal response (or intrinsic efficacy) evoked by Ap_nGs was significantly reduced at P2X₁ receptors. This observation suggests that the guanine moiety can interfere in some way with the actions of the adenine moiety. This point was borne out where Gp_nGs were tested and found to be quite inactive at P2X₁ and P2X₃ receptors (also P2X₂ and P2X₄ receptors). Thus, group I P2X receptors require the presence of at least one adenine moiety in dinucleotidic compounds to bind to the ligand-docking site and cause the necessary conformational change to open (or keep open for a significant period of time) the intrinsic ion channel.

Our pharmacological data on Ap_nA compounds were comparable with the results of an earlier study of recombinant P2X_1-4 receptors (Wildman et al., 1999), while Ap_nG and Gp_nG have not previously been tested on these ionotropic ATP receptors. The present study had the advantage of using high-performance liquid chromatography-purified dinucleotides, whereas earlier surveys of dinucleotide activity have relied on commercially available compounds. This difference aside, the EC₅₀ values for Ap₅A and Ap₆A fell in the low micromolar range and were indistinguishable statistically from the reported values of Wildman et al. (1999). We did notice that the maximal response to Ap₅A at P2X₁ receptors was significantly greater in our hands than reported earlier for human and rat orthologs of P2X₁ (Evans et al., 1995; Wildman et al., 1999). It should be borne in mind that P2X₁ receptors are rapidly desensitized by both mononucleotides and dinucleotides, and minor differences in rates of recovery from desensitization could account for the observed discrepancies in the intrinsic efficacy of these compounds. Our latest data suggest that Ap₅A and Ap₆A are partial agonists at P2X₁ and P2X₃ receptors.

None of the active dinucleotides evoked sustained inward currents at P2X_1-4 receptors and, accordingly, it remains a difficult prospect to explain how these compounds evoke a sustained vasoconstriction in perfused rat kidney (van der Giet et al., 1999). Of the P2X₁, P2X₂, and P2X₄ messenger RNAs found in arterial tissues (Nori et al., 1998), only P2X₂ and P2X₄ transcripts could result in nondesensitizing homomeric P2X receptors. However, our present results confirmed that the pentaphosphate and hexaphosphate of each dinucleotide were inactive at P2X₂ and P2X₄ receptors. It is possible that heteromeric P2X receptors with nondesensitizing properties could occur in renal vascular tissues, but the stoichiometry of such P2X subunit assemblies remains unknown. It is improbable that P2X₁ and P2X₂ subunits were involved, since an earlier study indicated that their coexpression failed to produce a nondesensitizing heteromer (Lewis et al., 1995). Similarly, biochemical evidence indicates that heteromeric P2X_1/4 and P2X_2/4 receptor assemblies are highly unlikely (Torres et al., 1998). The recently discovered heteromeric P2X_1/5 receptor represents a more interesting candidate, since this receptor produces biphasic , -meATP responses involving transient and sustained inward currents (Haines et al., 1999; Le et al., 1999; Surprenant et al., 2000). Furthermore, a recent study has likened the heteromeric P2X_1/5 receptor to native P2X receptors in guinea pig submucosal arterioles, on the basis of common pharmacological and operational profiles (Surprenant et al., 2000). However, P2X₅-like immunoreactivity has not been reported in renal vasculature (Chan et al., 1998), nor has it been observed in the vascular supply of the adjacent adrenal gland (Afework and Burnstock, 1999). P2X₅ transcripts have been found in rat mesenteric artery (Phillips and Hill, 1999), but Ap_nA and Ap_nG compounds do not elicit nondesensitizing responses in this arterial preparation (Lewis et al., 2000). Thus, it is unlikely that P2X_1/5 receptors could account for the sustained agonism by these dinucleotides in renal vasculature.

Both Ap₅G and Ap₆G, as well as Ap₅A and Ap₆A, have been found in the secretory vesicles of human blood platelets (Schlüter et al., 1994, 1998). Dinucleotide concentrations in the range of 0.5 to 3 µM have been found in the supernatant following platelet aggregation (Schlüter et al., 1998), although the local concentration at the site of release might be 10-fold higher (Beigi et al., 1999). Such concentration levels would saturate the P2X receptor population in the renal vasculature, where agonist-evoked transient and sustained vasoconstrictor responses are maximal in the low micromolar range (van der Giet et al., 1999). Our present results indicate that the fast vasoconstrictor effects of Ap_nGs and Ap_nAs was consistent with the activation P2X₁ receptors since, of the two group I P2X receptors that respond to these compounds, only P2X₁ receptors are present in smooth muscle. The mediator of slow response cannot yet be accounted for, but the occurrence of a nondesensitizing P2X receptor in kidney has important implications in the development and maintenance of essential hypertension by naturally occurring dinucleotides that potentially can be released in physiologically relevant concentrations.