β3-Adrenoceptor agonists inhibit purinergic receptor-mediated contractions of the murine detrusor

Abstract

β3-Adrenoceptor (β3-AR) agonists are used to treat overactive bladder syndrome; however, their mechanism of action has not been determined. The aims of this study were to compare the effects of β3-AR agonists on cholinergic versus purinergic receptor-mediated contractions of the detrusor and to examine the mechanisms underlying inhibition of the purinergic responses by β3-AR agonists. Isometric tension recordings were made from strips of murine detrusor and whole cell current recordings were made from freshly isolated detrusor myocytes using the patch-clamp technique. Transcriptional expression of exchange protein directly activated by cAMP (EPAC) subtypes in detrusor strips was assessed using RT-PCR and real-time quantitative PCR. The β3-AR agonists BRL37344 and CL316243 (100 nM) inhibited cholinergic nerve-mediated contractions of the detrusor by 19 and 23%, respectively, but did not reduce contractions induced by the cholinergic agonist carbachol (300 nM). In contrast, BRL37344 and CL316243 inhibited purinergic nerve-mediated responses by 55 and 56%, respectively, and decreased the amplitude of contractions induced by the P2X receptor agonist α,β-methylene ATP by 40 and 45%, respectively. The adenylate cyclase activator forskolin inhibited purinergic responses, and these effects were mimicked by a combination of the PKA activator N6-monobutyryl-cAMP and the EPAC activator 8-pCPT-2′- O-methyl-cAMP-AM (007-AM). Application of ATP (1 μM) evoked reproducible P2X currents in isolated detrusor myocytes voltage-clamped at −60 mV. These responses were reduced in amplitude in the presence of BRL37344 and also by 007-AM. This study demonstrates that β3-AR agonists reduce postjunctional purinergic responses in the detrusor via a pathway involving activation of the cAMP effector EPAC.