1 The antagonist activity of a series of diinosine polyphosphates (Ip n I, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X 1 ± 4 receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2 Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap 5 A into Ip 5 I) using 5'-adenylic deaminase, and puri®ed using reverse-phase chromatography. 3 Against ATP-responses at rP2X 1 receptors, the potency order for antagonism was (pIC 50 ): Ip 5 I (8.5)4Ip 4 I (6.3)4Ip 3 I (44.5). Ip 5 I (10 ± 100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP e ect. However, the Schild plot was non-linear which indicated Ip 5 I is not a competitive antagonist. Blockade by micromolar concentrations of Ip 5 I was not surmountable. Ip 4 I also behaved as a nonsurmountable antagonist. 4 Against ATP-responses at rP2X 3 receptors, the potency order for antagonism was (pIC 50 ): Ip 4 I (6.0)4Ip 5 I (5.6)4Ip 3 I (44.5). Blockade by Ip 4 I (pA 2 , 6.75) and Ip 5 I (pA 2 , 6.27) was surmountable at micromolar concentrations. 5 Diinosine polyphosphates failed to inhibit ATP-responses at rP2X 2 receptors, whereas agonist responses at rP2X 4 were reversibly potentiated by Ip 4 I and Ip 5 I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X 1 ± 4 receptors. 6 Thus, Ip 5 I acted as a potent and relatively-selective antagonist at the rP2X 1 receptor. This dinucleotide pentaphosphate represents a high-a nity antagonist for the P2X 1 receptor, at which it acts in a competitive manner at low (4100 nM) concentrations but has more complex actions at higher (4100 nM) concentrations.

1 Whole-cell patch-clamp recording was used to determine the functional expression and pharmacological properties of P2X receptors in chroma n cells dissociated from adrenal medullae of rats and guinea-pigs. 2 In rat chroma n cells maintained in culture for 1 ± 7 days, ATP and UTP failed to evoke any detectable response. 3 Guinea-pig chroma n cells responded to ATP (100 mM) with a rapidly activating inward current. The amplitude of the response to ATP increased over the period cells were maintained in culture and so did the number of cells giving a detectable response, with 69% of cells responding after 54 days of culture. 4 The response to ATP desensitized slowly, and had a reversal potential of 2.5 mV. The EC 50 for ATP was 43 mM. The potency order for ATP analogues was 2-MeSATP4ATP4ADP. Adenosine, UTP and a,b-meATP were inactive. 5 Suramin (100 mM) and Cibacron blue (50 mM) inhibited the ATP (100 mM)-activated current by 51 and 47%, respectively. PPADS antagonized the response to ATP (100 mM) with an IC 50 of 3.2 mM. 6 The ATP concentration-response curve shifted to the left at pH 6.8 (EC 50 , 19 mM) and right at pH 8.0 (EC 50 , 96 mM), without changing the maximal response. Zn 2+ inhibited the response to ATP (100 mM) with an IC 50 of 48 mM. 7 This study indicates that expression of ATP-gated cation channels in chroma n cells is species dependent. The P2X receptors in guinea-pig chroma n cells show many characteristics of the P2X 2 receptor subtype.