Endogenous purinergic control of bladder activity via presynaptic P2X3 and P2X2/3 receptors in the spinal cord

Abstract

P2X(3) and P2X(2/3) receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X(3) and P2X(2/3) antagonist, AF-792 [5-(5-ethynyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine, previously known as RO-5], in addition to less selective purinoceptor ligands, was applied intrathecally in vivo. Cystometry recordings were made to assess changes in the micturition reflex contractions after drug treatments. We found that AF-792 inhibited micturition reflex activity significantly (300 nmol; from baseline contraction intervals of 1.18 +/- 0.07 to 9.33 +/- 2.50 min). Furthermore, inhibition of P2X(3) and P2X(2/3) receptors in the spinal cord significantly attenuated spinal activation of extracellular-signal regulated kinases induced by acute peripheral stimulation of the bladder with 1% acetic acid by 46.4 +/- 12.0% on average. Hence, the data suggest that afferent signals originating from the bladder are regulated by spinal P2X(3) and P2X(2/3) receptors and establish directly an endogenous central presynaptic purinergic mechanism to regulate visceral sensory transmission. Identification of this spinal purinergic control in visceral activities may help the development of P2X(3) and P2X(2/3) antagonist to treat urological dysfunction, such as overactive bladder, and possibly other debilitating sensory disorders, including chronic pain states.

Figure 1.

Sample traces of cystometry recording after intrathecal application of αβmeATP (A), PPADs (B), TNP–ATP (C), and AF-792 (D) and their respective vehicles. Calibration: 5 min, 40 cm H₂O.

Figure 2.

Intrathecal application of αβmeATP (A), PPADs (B), TNP–ATP (C), and AF-792 (D) inhibits isovolumetric bladder contractions in naive anesthetized animals in vivo (n = 5–12 for each dose). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle.

Figure 3.

A, AF-792 (300 nmol, i.t.) significantly reduces pERK-positive cells in the spinal cord after 1% acetic acid stimulation of the bladder (n = 4 for each group). *p < 0.05, ***p < 0.001 compared with 1% acetic acid-stimulated group. Representative photomicrographs of sham group with vehicle (intrathecally) treatment (B), 1% acetic acid-stimulated bladder group with vehicle (intrathecally) treatment (C), and 1% acetic acid-stimulated bladder group with AF-792 (300 nmol, i.t.) treatment (D) with high-power magnification in E and F for the latter two groups, respectively, are shown. White boxes indicate the area of high-power magnification. Scale bars, 100 μm.