Diinosine pentaphosphate: an antagonist which discriminates between recombinant P2X(3) and P2X(2/3) receptors and between two P2X receptors in rat sensory neurones

Abstract

1. We have compared the antagonist activity of trinitrophenyl-ATP (TNP-ATP) and diinosine pentaphosphate (Ip(5)I) on recombinant P2X receptors expressed in Xenopus oocytes with their actions at native P2X receptors in sensory neurones from dorsal root and nodose ganglia. 2. Slowly-desensitizing responses to alpha,beta-methylene ATP (alpha,beta-meATP) recorded from oocytes expressing P2X(2/3) receptors were inhibited by TNP-ATP at sub-micromolar concentrations. However, Ip(5)I at concentrations up to 30 microM was without effect. 3. Nodose ganglion neurones responded to alpha,beta-meATP with slowly-desensitizing inward currents. These were inhibited by TNP-ATP (IC(50), 20 nM), but not by Ip(5)I at concentrations up to 30 microM. 4. In DRG neurones that responded to ATP with a rapidly-desensitizing inward current, the response was inhibited by TNP-ATP with an IC(50) of 0.8 nM. These responses were also inhibited by Ip(5)I with an IC(50) of 0.1 microM. Both antagonists are known to inhibit homomeric P2X(3) receptors. 5. Some DRG neurones responded to alpha,beta-meATP with a biphasic inward current, consisting of transient and sustained components. While the transient current was abolished by 1 microM Ip(5)I, the sustained component remained unaffected. 6. In conclusion, Ip(5)I is a potent antagonist at homomeric P2X(3) receptors but not at heteromeric P2X(2/3) receptors, and therefore should be a useful tool for elucidating the subunit composition of native P2X receptors.

Figure 1

Comparison of the action of TNP-ATP and Ip₅I on recombinant P2X₂, P2X₃ and P2X_2/3 receptors. (a) Response of three different Xenopus oocytes expressing P2X₃, P2X₂ and P2X_2/3 receptors, to ATP or α,β-meATP alone, and in the presence of 30 μM diinosine pentaphosphate (Ip₅I). (b) Concentration-effect curves for the inhibition of P2X₃, P2X₂ and P2X_2/3 receptors by Ip₅I. ATP was used as the agonist at 3 μM and 10 μM for P2X₃ and P2X₂ receptors respectively, while 1 μM α,β-meATP was used in experiments on the heteromeric receptor. Points represent the mean±s.e.mean from five cells. Antagonists were given for 60 s before, and during the agonist application. In the presence of Ip₅I, the peak amplitude of agonist evoked responses at P2X₂ and P2X_2/3 receptors were not significantly different from control (P>0.05). (c) Responses to α,β-meATP recorded from an oocyte expressing P2X_2/3 receptors in the presence of increasing concentrations of TNP-ATP. Comparable results were obtained in a further three cells. Oocytes were voltage-clamped at a holding potential of −50 mV. Antagonists were present for 60 s before and during the agonist applications. In oocytes co-injected with P2X₂ and P2X₃ transcripts, the transient current due to activation of homomeric P2X₃ receptors was abolished by a condition application of α,β-meATP (10 μM, 20 s) given 60 s before the test response, leaving only the sustained response mediated by P2X_2/3 receptors.

Figure 2

The effect of Ip₅I and TNP-ATP on composite responses at recombinant P2X receptors. (a) Response of an oocyte, co-injected with rP2X₂ and rP2X₃ transcripts and expressing both P2X₃ and P2X_2/3 receptors, to 1 μM α,β-meATP, alone and in the presence of 10 and 100 nM TNP-ATP. (b) Response of another oocyte to 1 μM α,β-meATP alone, and in the presence of 1 and 10 μM Ip₅I. Similar effects of TNP-ATP and Ip₅I were observed in three and five cells respectively. Oocytes were voltage-clamped at a holding potential of −50 mV. Antagonists were present for 60 s before and during the agonist application.

Figure 3

Inhibition of P2X receptors in sensory neurones by TNP-ATP. (a) Response of a nodose ganglion neurone to 10 μM α,β-meATP before, in the presence of, and following washout of 30 nM TNP-ATP. (b) Response of a dorsal root ganglion neurone to 10 μM ATP before, in the presence of and following washout of 3 nM TNP-ATP. Cells were voltage-clamped at a holding potential of −60 mV. Responses were recorded at 3.5 min intervals and antagonists were present for 3 min before, and during the second agonist application. (c) Concentration-effect curves for the inhibition of the response of DRG neurones and nodose ganglion neurones by TNP-ATP. The agonist used was 10 μM ATP and 10 μM α,β-meATP for DRG and nodose ganglion neurones respectively. Each point represents the mean±s.e.mean from 3–12 cells. Solid lines show least squares fit of the Hill equation to the data which yielded IC₅₀ values of 0.77±0.12 μM and 20.6±2.8 nM and Hill coefficients of 0.6 and 1.3 for DRG and nodose ganglion neurones respectively. (d) Concentration-effect curves for the inhibition by Ip₅I of the response of DRG neurones and nodose ganglion neurones to 10 μM ATP and 10 μM α,β-meATP, respectively. Each point represents the mean±s.e.mean from 3–6 cells. The solid curve show least squares fit of the Hill equation to the data for DRG neurones which yielded an IC₅₀ value of 0.07±0.007 μM and a Hill coefficient of 0.73. Responses of nodose ganglion neurones in the presence of Ip₅I at concentrations up to 30 μM were not significantly different from control responses (P>0.05).

Figure 4

The nature of the antagonism produced by TNP-ATP and Ip₅I. (a) Concentration-response curves for α,β-meATP evoked inward currents recorded from nodose ganglion neurones in the absence or presence of 0.1 μM TNP-ATP. Cells were voltage-clamped at −60 mV. Responses were normalized with respect to that produced by 100 μM α,β-meATP in the absence of antagonist recorded from the same cell. Points represent the mean±s.e.mean from 3–6 cells. The curves show least squares fit of the Hill equation to the data, which gave EC₅₀ values of 39.2±2.5 μM and 167±22 μM and Hill coefficients of 1.6 and 1.1 in the absence and presence of antagonist respectively. (b) Concentration-response curves for the rapidly-desensitizing inward currents produced by ATP in DRG neurones in the absence and presence of 0.3 μM Ip₅I. Points represent the mean±s.e.mean from 4–6 cells. Responses were normalized with respect to that produced by 10 μM ATP in the same cell, in the absence of antagonist.

Figure 5

Effect of Ip₅I on mixed responses in dorsal root ganglion neurones. The traces show membrane currents evoked by 30 μM α,β-meATP in a DRG neurone voltage-clamped at −60 mV, alone and in the presence of 1 μM Ip₅I. While the transient component of the response was abolished, the sustained component was unaffected by the antagonist. Similar results were observed in a total of four neurones.