Anales RANF

P.95 β 3 -ADRENOCEPTOR COUPLING TO EPAC1 AND PKC PROMOTES ADENOSINE RELEASE VIA ENT1 LEADING TO INHIBITION OF CHOLINERGIC NEUROTRANSMISSION IN THE HUMAN BLADDER I. Silva 1,2 , M.T. Magalhães-Cardoso 1,2 , F. Ferreirinha 1,2 , S. Moreira 1,2 , A.F. Costa 1,2 , D. Silva 1,2 , C. Vieira 1,2 , M. Silva-Ramos 1,2,3 & Paulo Correia-de-Sá 1,2, 1 Laboratório de Farmacologia e Neurobiologia, 2 Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, and 3 Serviço Urologia, Centro Hospitalar do Porto (CHP), Porto, Portugal. Selective β 3 -adrenoceptor agonists, such as mirabegron, are increasingly prescribed for the treatment of overactive bladder. Our group demonstrated that activation of G s - protein- coupled β 3 -adrenoceptors favors the release of adenosine in urothelium-denuded human and rat detrusor strips, via equilibrative nucleoside transporters 1 (ENT1; Silva et al., 2017, Am J Physiol 313: F388- F403). Thus, cholinergic inhibition by β 3 - adrenoceptor agonists may be indirectly mediated by adenosine released from the detrusor leading to retrograde activation of inhibitory A 1 receptors on cholinergic nerve terminals (Silva-Ramos et al., 2015, Purinergic Signal 11: 595-606). Here, we investigated the cyclic AMP responsive element, protein kinase A (PKA) and the exchange pro tein directly activated by cAMP (EPAC), most likely participating in β 3 - induced cholinergic inhibition of the urinary bladder obtained from human organ donors and Wistar rats. Using immunofluorescence confocal microscopy and Western blot analysis, we show that the human and the rat detrusor exhibit β 3 -adrenoceptor, EPAC1 and ENT1 immunoreactivity, but express very small amounts of β 2 -adrenoceptor and EPAC2 proteins. The EPAC inhibitor, ESI- 09, prevented β 3 -induced adenosine release from human and rat detrusor strips caused by mirabegron and isoprenaline, respectively. ESI-09, but not the PKA inhibitor, H-89, attenuated inhibition of [ 3 H]ACh release from stimulated (10 Hz) detrusor strips caused by drugs activating β 3 -adrenoceptors, adenylylcyclase (forskolin), and EPAC1 (8-CTP-2Me-cAMP). Isoprenaline-induced inhibition of [ 3 H]ACh release was also prevented by inhibitors of protein kinase C (chelerythrine and Go6976) and ENT1 (dipyridamole and NBTI). Pretreatment with ESI-09, but not with H-89, prevented the reduction of the voiding frequency caused by isoprenaline and forskolin in urethane- anaesthetized rats. Data suggest that β 3 - adrenoceptor-induced inhibition of cholinergic neurotransmission in human and rat urinary bladders involves activation of an EPAC1/PKC pathway downstream cyclic AMP production resulting in adenosine outflow via ENT1. Work supported by FCT (FEDER funding, project UID/BIM/4308/2016); IS was in receipt for a PhD Studentship from FCT (SFRH/BD/88855/2012).