Inhibition of cholinergic neurotransmission by β3-adrenoceptors depends on adenosine release and A1-receptor activation in human and rat urinary bladders

Abstract

The direct detrusor relaxant effect of β₃-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of β₃-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A₁ receptors to explain inhibition of cholinergic activity by β₃-adrenoceptors. Isoprenaline (1 µM) decreased [³H]ACh release from stimulated (10 Hz, 200 pulses) human (-47 ± 5%) and rat (-38 ± 1%) detrusor strips. Mirabegron (0.1 µM, -53 ± 8%) and CL316,243 (1 µM, -37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking β₃-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A₁ receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by β₃-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A₁-receptor stimulation in human and rat urinary bladder.

Keywords: acetylcholine release; adenosine A1 receptor; adenosine release; human urinary bladder; β3-adrenoceptor.