Selectivity of diadenosine polyphosphates for rat P2X receptor subunits

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Abstract

The pharmacological activity of diadenosine polyphosphates was investigated at three recombinant P2X receptors (rat P2X1, rat P2X3, rat P2X4) expressed in Xenopus oocytes and studied under voltage-clamp conditions. For the rat P2X1 receptor, only P1,P6-diadenosine hexaphosphate (Ap6A) was a full agonist yet 2–3 folds less potent than ATP. At rat P2X3, P1,P4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A) and Ap6A were full agonists and more potent than ATP. Ap4A alone was equipotent with ATP at rat P2X4, but only as a partial agonist. Compared to known data for rat P2X2 and human P2X1 receptors, our findings contrast with rat P2X2 where only Ap4A is a full agonist although four folds less potent than ATP. At rat and human orthologues of P2X1, Ap5A was a partial agonist with similar potency. These data provide a useful basis for selective agonists of P2X receptor subunits.

Introduction

Diadenosine polyphosphates (ApnA, n=2–6) are naturally-occurring adenine dinucleotides, where two adenine molecules are linked at the 5′ position of their ribose moieties by a chain of phosphates varying from 2 to 6 in length. These adenine dinucleotides possess both intracellular and extracellular actions, are concentrated in central synaptosomes, and are released in a Ca2+-dependent process from brain slices after nerve stimulation (for a review, see Pintor et al., 1997).

Extracellular diadenosine polyphosphates bind to and activate P2X and P2Y receptor subtypes for ATP in a number of mammalian tissues and with a variety of effects (Hoyle, 1990; Abbracchio and Burnstock, 1994; Pintor and Miras-Portugal, 1995; Ogilvie et al., 1996). For the P2X receptor family, seven subtypes (P2X1–7) have been cloned thus far (North and Barnard, 1997), although the pharmacological activity of the diadenosine polyphosphate family has only been studied at human P2X1 (hP2X1) receptors expressed in Xenopus oocytes and human embryonic kidney (HEK 293) cells (Evans et al., 1995) and at rat P2X2 (rP2X2) receptors expressed in Xenopus oocytes (Pintor et al., 1996).

In the present study, we have extended this pharmacological survey of adenine dinucleotide activity to include three recombinant P2X receptors from rat tissue (rP2X1, rP2X3 and rP2X4) and have commented on their agonist and modulatory activities to reveal an emerging picture of selectivity for diadenosine polyphosphates at P2X subunits.

Section snippets

Electrophysiology

Xenopus oocytes were harvested and prepared, as previously described (King et al., 1996, King et al., 1997). Defolliculated oocytes were injected cytosolically with capped ribose nucleic acid (cRNA) encoding either rat P2X1 (Valera et al., 1995), rat P2X3 (Chen et al., 1995) or rat P2X4 receptors (Bo et al., 1995). RNA-injected oocytes and sham-injected control oocytes were incubated for 48 h at 18°C in Barth's solution and kept for 5 to 10 days at 4°C until used in electrophysiological

Agonist activity of adenine dinucleotides at rat P2X1 receptor

ATP (0.01–30 μM) evoked inward membrane currents in defolliculated Xenopus oocytes expressing rP2X1 receptors (EC50 value, 0.30±0.01 μM; Hill coefficient (nH), 1.5±0.1; n=4). Of the dinucleotide series tested, only Ap6A was a full agonist (EC50, 0.72±0.08 μM; nH, 1.2±0.2; n=4) yet 2–3 fold less potent than ATP. Both Ap4A and Ap5A were partial agonists with maximal responses as low as 40% of the maximal ATP effect (Fig. 1A). Ap4A (EC50, 38±11 nM; nH, 1.2±0.1; n=4) was eight fold more potent than

Discussion

In the present study, we found that the diadenosine polyphosphate series (ApnA, n=2–6) showed different patterns of pharmacological activity at three rat P2X receptors (rP2X1, rP2X3, rP2X4). These data, when compared to like studies of human P2X1 (Evans et al., 1995) and rat P2X2 receptors (Pintor et al., 1996), reveal agonist selectivity for dinucleotides at the P2X1–4 subunits (see Table 1).

Only Ap6A was a full agonist at rP2X1, although the potency order of dinucleotides showing agonist

Conclusion

In conclusion, it is evident that there is some selectivity in the actions of the adenine dinucleotides for those P2X subunits tested thus far (hP2X1, rP2X1, rP2X2, rP2X3 and rP2X4 receptors). A picture is emerging that these P2X subunits are affected by certain adenine dinucleotides in different ways. Of the dinucleotides tested, Ap6A seems the best choice as an agonist for rP2X1 although Ap6A will also fully activate rP2X3. However, Ap5A and Ap4A are full agonists at rP2X3 but not rP2X1 and,

Acknowledgements

This work was supported by the British Heart Foundation, Roche Bioscience (Palo Alto, USA) and Gilead Sciences (Foster City, CA). We are grateful to Dr. G. Buell (formerly Glaxo, Geneva) for the gift of cDNA encoding the rat P2X1 receptor, Professor J. Wood (UCL, London) for cDNA encoding the rat P2X3 receptor, and Dr. X. Bo and Dr. R. Schoepfer (UCL, London) for cDNA encoding the rat P2X4 receptor. We thank Dr. Charles Hoyle (UCL, London) and Dr. J. Pintor (Madrid, Spain) for their critical

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