Activation and sensitisation of low and high threshold afferent fibres mediated by P2X receptors in the mouse urinary bladder

Abstract

It has been proposed that extracellular ATP may be involved in visceral mechanosensory transduction by activating ligand-gated ion channels (P2X receptors). In this study, we have investigated the effects of the P2X(3) agonist alpha,beta-methylene ATP (alpha,beta-meATP) and antagonist 2',3'-O-trinitrophenyl-ATP (TNP-ATP) on pelvic afferents innervating the urinary bladder using an in vitro mouse bladder-pelvic nerve preparation. Intravesical application of alpha,beta-meATP (0.03-1 mM) increased multifibre discharges in a concentration-dependent manner. The agonist potentiated, whereas TNP-ATP (0.03 mM) attenuated, the multifibre responses to bladder distensions. Single-unit analysis revealed that both high threshold (HT) fibres (> 15 mmHg; known to be associated with nociception) and low threshold (LT) fibres (< 15 mmHg; probably associated with non-nociceptive events) could be induced to discharge by intravesical alpha,beta-meATP (1 mM, 0.1 ml). The response of the vast majority (21/22, 95.5 %) of HT fibres to bladder distensions was enhanced with a significantly reduced threshold and an increased peak response after exposure to the agonist. On the other hand, 59.7 % (46/77) of LT fibres showed a greater peak and a slightly reduced threshold for response to bladder distension in the presence of alpha,beta-meATP. An additional 11 'silent' fibres became mechanosensitive after exposure to alpha,beta-meATP. TNP-ATP (0.03 mM) did not affect the threshold of LT fibres, but it reduced the peak response of some (22/51, 43.1 %) LT fibres. Conversely, the antagonist resulted in a markedly elevated threshold and reduced peak activity in the majority (13/16, 81.3 %) of HT fibres. The results support the view that P2X(3) receptor-mediated mechanisms contribute to both nociceptive and non-nociceptive (physiological) mechanosensory transduction in the urinary bladder.

Figure 1. Multifibre pelvic afferent activity induced by repeated bladder distensions with Krebs solution (0.2 ml min⁻¹)

Figure 2. Multifibre afferent activity induced by intravesical application of α,β-meATP

A, superimposed traces of the bladder pressure and rate histogram of a multifibre recording following intravesical injection of vehicle and 1 mm α,β-meATP (0.1 ml). B, relative afferent activity following intravesical application of α,β-meATP (0-1000 μM, 0.1 ml) in five multifibre preparations.

Figure 3. The effects of the P2X agonist and antagonist on multifibre afferent response to bladder distension

Traces of bladder pressure and rate for multiunit afferent discharge are superimposed for three consecutive bladder distensions with vehicle, 0.1 mm α,β-meATP and 0.03 mm TNP-ATP, respectively. Note the augmented afferent response in the presence of α,β-meATP and the reduced afferent response in the presence of TNP-ATP.

Figure 4. Activation of single fibre afferents by bladder distension and α,β-meATP

A, afferent discharges evoked by bladder distension. The rates of 3 apparently different units (labelled U1, U2 and U3) are displayed. Note they differ considerably with respect to threshold and discharge rate and can be classified as low threshold (LT, U1) and high threshold (HT, U2 and U3) fibres. B, afferent discharges induced by intravesical application of α,β-meATP (1 mm, 0.1 ml) in the same nerve preparation as in A. Bin = 10 s for A and B. C, superimposed spikes for U1, U2 and U3.

Figure 5. Effects of α,β-meATP on the mechano-sensitive properties of low and high threshold afferent fibres

A, left and right columns show afferent activity in response to bladder distension (Krebs solution, 0.1 ml min⁻¹) after pre-exposure to vehicle (0.1 ml) and α,β-meATP (1 mm, 0.1 ml), respectively. Note that a ‘silent’ fibre is sensitised to become mechano-sensitive by the agonist. B, superimposed action potentials for units 1-3. Bin = 10 s for A and B.

Figure 8. The relationship between bladder pressure and discharge rate for LT and HT fibres

A and B, the rate histogram of a low (A) and a high (B) threshold fibre during bladder distension after pre-exposure to vehicle (0.1 ml) and α,β-meATP (1 mm, 0.1 ml). C and D, the rate histogram of a low (C) and a high (D) threshold fibre during bladder distension with vehicle and 0.03 mm TNP-ATP.

Figure 6. The effects of TNP-ATP on the mechano-sensitive properties of a low threshold afferent fibre

A, single unit activity induced by bladder distension with Krebs solution (0.1 ml min⁻¹). B, single unit activity induced by bladder distension with TNP-ATP (0.03 mm, 0.1 ml min⁻¹). Bin = 10 s for A and B. C, superimposed spikes for the unit.

Figure 7. The effects of TNP-ATP on the mechanosensitive response of a high threshold afferent fibre

A, afferent discharges induced by bladder distension with Krebs solution (0.1 ml min⁻¹). B, afferent discharges induced by distension with 0.03 mm TNP-ATP (0.1 ml min⁻¹). Bin = 10 s for A and B. C, the superimposed action potential for the unit.