Activation of β3‐adrenoceptors inhibits purinergic‐mediated contractions of the murine detrusor

Abstract

β3‐adrenergic receptor (β3‐AR) agonists are used for treatment of overactive bladder, however the mechanisms underlying their effect are unclear. β3‐AR agonists inhibit cholinergic‐mediated responses of the detrusor, however the concentration required to exert this effect exceed the plasma levels achieved through therapeutic dosing of these drugs1. ATP is regarded as an excitatory neurotransmitter in the detrusor, and upregulation of purinergic signaling is considered as a causative factor in the pathogenesis of overactive bladder2. The purpose of the present study was 1) to examine the effect of β3‐AR activation on purinergic‐evoked contractions of the murine detrusor & on P2XR1 currents recorded from isolated murine detrusor myocytes and 2) to investigate if purinergic responses in the detrusor were modulated by agents that activate exchange factor directly activated by cAMP (EPAC), a novel cAMP effector.Mice were killed by cervical dislocation and their bladder removed, in accordance with the DKIT Animal Care & Use Committee and EU legislation (EU Directive 2010/63/EU). Using fine dissection, the urothelium was removed leaving the remaining detrusor layer. Isometric tension recordings were made from strips of detrusor muscle. Detrusor smooth muscle cells (DSMCs) were isolated by enzymatic digestion (Collagenase Type II, 5 minutes at 37°C) of detrusor tissue cut into fine pieces. The perforated patch clamp configuration was used to investigate inward currents in isolated DSMCs. Perforation was achieved using the antibiotic amphotericin B (600 μg/ml).In the presence of the muscarinic antagonist atropine (1 μM), electric field stimulation (EFS; 4 Hz) induced robust purinergic‐mediated contractions of murine detrusor strips. Therapeutic‐relevant concentrations (100 nM) of the β3‐AR agonists BRL37344 & CL316,243, reduced the amplitude of EFS‐evoked detrusor contractions by 59% (n=8, p<0.01) and 50% (n=6, p<0.05), respectively. These effects were partially reversed by the β3‐AR antagonist, L748,337 (1 μM). The adenylyl cyclase stimulant forskolin and the PDE4 inhibitor rolipram, similarly inhibited EFS‐evoked purinergic contractions. Under voltage clamp conditions (‐60 mV), ATP (1 μM) evoked inward currents in isolated DSMCs. These currents were reduced from −1803 ± 418 pA to −514 ± 168 pA (n=6, p<0.05) by BRL37344 (100 nM). Quantitative PCR experiments revealed that EPAC1 & 2 subtypes were expressed in murine detrusor, with EPAC1 ~10‐fold more abundant. Application of the EPAC activator, 007‐AM, reduced the amplitude of purinergic contractions by ~48%, (n=6, p<0.01), and this effect was partially reversed in the presence of the EPAC1 blocker, CE3F4.These data show that β3‐AR agonists inhibit purinergic‐mediated responses of the murine detrusor by inhibiting P2XR1 currents in detrusor myocytes, possibly by a mechanism involving activation of EPAC1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.