AB295. SPR-22 Functional relevance of purinergic P2X4R in bladder smooth muscle

Objective: Under physiologic conditions in animals and pathologic conditions in humans, purinergic mechanisms contribute significantly to detrusor contractions. Although the activation of P2X1 receptors (P2X1R) accounts for the largest portion of bladder smooth muscle (BSM) responses to ATP, previous reports have shown that P2X1R antagonists do not completely abolish the purinergic component of neurogenic contractions, suggesting the presence of other P2XR subtypes on BSM. P2X4R has been identified in bladder tissue, however whether this receptor is functionally relevant warrants investigation. The aim of this study was to examine the extent of P2X4R expression in BSM tissue and to investigate its pharmacological contribution to ATP-mediated detrusor contractions. Methods: P2X4R mRNA and protein expression was investigated in mouse bladder tissue (without mucosa) and in cultured BSM cells by real-time RT-PCR and western blotting respectively. In vitro isometric tension studies were performed in mouse BSM strips without mucosa. Purinergic detrusor contractions were elicited by administration of α-β-methylene-ATP (αβmATP), and the purinergic component of neurogenic contractions induced by electrical field stimulations (EFS) was isolated by pre-treatment with the muscarinic receptor antagonist atropine. The inhibitory effect of two P2X4R selective antagonists, 5-BDBD and BX430, on the αβmATPand EFS-induced contractions was investigated in the presence of P2X1R antagonist NF449. In addition, the effect of the P2X4R positive modulator ivermectin (IVC) on αβmATP responses was investigated. Data were analyzed by a repeated measures analysis of variance. Results: P2X4R mRNA was detected in smooth muscle from both bladder tissue and cultured BSM cells, although its expression was significantly lower than P2X1R expression. Immunoreactivity for P2X4R was detected in lysates from both mouse BSM tissue and smooth muscle cells. Functional studies indicated that although P2X1R activation is predominantly responsible for purinergic contractions in mouse detrusor, a significant portion of the contractile response to both αβmATP (22.3±7%) and EFS (27.5±4% of purinergic component of EFS) was resistant to P2X1R inhibition. This NF449-resistant component was abolished by administration of P2X4R antagonists 5-BDBD or BX430. In addition, responses to αβmATP increased significantly upon administration of IVC. Conclusions: The expression of P2X4R in detrusor smooth muscle together with the identification of a P2X4Rsensitive component of bladder contractions suggest that the activation of this P2X receptor subtype could significantly contribute to ATP-mediated BSM responses. P2X4R may thus potentially represent a novel target for the management of detrusor dysfunctions associated with alterations in purinergic signaling. Funding Source(s): Department of Veterans Affairs, Research Service BX001790; BX002806