Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue

Abstract

1. Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X(3) mRNA in sensory neurones has led to the hypothesis that activation of P2X(3) and/or P2X(2/3) receptors contributes to nociception. 2. The local administration of the P2X receptor agonist, BzATP (100--1000 nmol paw(-1), s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. 3. The co-administration of the potent P2X receptor antagonist, TNP-ATP (30--300 nmol paw(-1)), but not an inactive analogue, TNP-AMP, with BzATP into the rat hindpaw attenuated BzATP-induced nociception. Similarly, co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin reduced both acute and persistent nociception in this test. 4. Co-administration of cibacron blue (30 and 100 nmol paw(-1)), a selective allosteric enhancer of P2X(3) and P2X(2/3) receptor activation, with BzATP (30 and 100 nmol paw(-1)) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co-administration of cibacron blue (30 and 100 nmol paw(-1)) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. 5. The ability of TNP-ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X(3) containing channels contributes specifically to both acute and persistent nociception in the rat.

Figure 1

(a) Time course for the acute nociceptive effects of intradermal BzATP in the rat. BzATP or 5% formalin (50 μl) was injected into the dorsal surface for the rat hindpaw at the doses indicated (n=6 per group) and nociceptive responses were recorded in 5-min bins for 20 min post-injection. BzATP dose-dependently increased the number of paw flinch responses (F (4,25)=25.9, P<0.05). Values represent mean±s.e.mean, ^*P<0.05 as compared to vehicle (saline, 50 μl)-treated rats. (b) Dose-response determinations for the acute nociceptive effects of P2X receptor agonists following intradermal administration into the rat hindpaw (n=6 per dose group). Values represent mean±s.e.mean for the cumulative nociceptive paw flinching responses occurring 15 min post-injection. ^*P<0.05 as compared to vehicle-treated rats.

Figure 2

(a) Intradermal co-administration of TNP-ATP (F(3,20)=8.20, P<0.05), but not TNP-AMP (F(3,20)=0.30, P>0.05), dose-dependently attenuates BzATP (1000 nmol paw⁻¹) induced acute nociceptive paw flinching in the rat (n=6 per dose group). Values represent mean±s.e.mean for the cumulative nociceptive paw flinching responses occurring 15 min post-injection. ^*P<0.05 as compared to vehicle-treated rats. (b) TNP-ATP (300 nmol paw⁻¹) significantly (P=0.05) attenuates the nociceptive effects of intradermal α,β meATP and BzATP (1000 nmol paw⁻¹) in the rat. Administration of TNP-ATP into the paw contralateral (CONTRA) to P2X agonist injection was ineffective. Values represent mean±s.e.mean, ^*P<0.05 as compared to vehicle-treated rats.

Figure 3

Intradermal co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin attenuated acute nociceptive paw flinching in the rat (n=6 per dose group). Phase I represents cumulative acute nociceptive responses occurring 15 min immediately following intradermal administration (F(2,27)=5.15, P<0.05). Phase II represents cumulative nociceptive responses recorded for a 20-min period beginning 30 min post formalin injection (F(2,27)=6.97, P<0.05). Values represent mean±s.e.mean, ^*P<0.05 as compared to vehicle-treated rats.

Figure 4

Concentration-effect determinations for the effects of cibacron blue on agonist activation of rat P2X₃ and P2X_2/3 receptors. (a) Representative concentration-effect curves for cibacron blue (EC₅₀=0.7 μM) to enhance BzATP (1 μM) and α,β-meATP (10 μM) activation of rat P2X₃ receptors. (b) Representative concentration-effect curves for cibacron blue to enhance BzATP (1 μM) and α,β-meATP (10 μM) activation of rat P2X_2/3 receptors. RFU=relative fluorescence units.

Figure 5

Nociceptive effects of intradermal co-administration of BzATP and cibacron blue into the hindpaw of the rat (F(16,352)=7.30, P<0.05). Vehicle responses indicate the effects of saline (open square) or co-administration of saline and BzATP (filled circles) on acute (cumulative responses for the first 15 min post-injection) paw flinching behaviour. The nociceptive effects of cibacron blue alone are indicated by the open squares and dotted lines. The effects of co-administration of cibacron blue and BzATP are indicated by the filled circles and solid lines. Values represent mean±s.e.mean from three separate experiments (n=6 per dose group), ^*P<0.05 as compared to the nociceptive effects of BzATP alone,+P<0.05 as compared to the nociceptive effects of cibacron blue alone.

Figure 6

Intradermal cibacron blue (CB, 30 and 100 nmol paw⁻¹) significantly (P<0.05) enhances the nociceptive effects of minimally effective doses (30 nmol paw⁻¹) of intradermal BzATP and α,β meATP in the rat. Administration of cibacron blue (CB 100 CONTRA) into the paw contralateral to P2X agonist injection was ineffective. Values represent mean±s.e.mean, ^*P<0.05 as compared to vehicle-treated rats.

Figure 7

Cibacron blue increases nociceptive paw flinching in both Phase I and Phase II of the rat formalin test. Cibacron blue (CB, 30 and 100 nmol paw⁻¹) was co-administered with intradermal formalin (1 – 5%) into the rat hind paw. Phase I nociceptive responses were recorded for the first 15 min following administration. Phase II nociceptive responses were recorded for a 20-min period beginning 30 min after administration. Intradermal administration of increasing doses of formalin alone produced significant increases in nociceptive responses (⁺P<0.05) as compared to vehicle injections. Values represent mean±s.e.mean (n=6 per dose group), ^*P<0.05 as compared to formalin alone.

Figure 8

Reactive orange does not increase nociceptive paw flinching in either Phase I and Phase II of the rat formalin test. Reactive Orange (RO, 30 and 100 nmol paw⁻¹) was co-administered with intradermal formalin (2.5%) into the rat hind paw. Phase I nociceptive responses were recorded for the first 15 min following administration. Phase II nociceptive responses were recorded for a 20-min period begining 30 min after administration. Intradermal administration of increasing doses of formalin alone produced significant increases in nociceptive responses. Values represent mean±s.e.mean (n=6 per dose group), ^*P<0.05 as compared to vehicle alone.