β₃ Adrenoceptor‐induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release

Abstract

Background and Purpose The mechanism by which β3 receptor agonists (e.g. mirabegron) control bladder overactivity may involve adenosine release from human and rat detrusor smooth muscle. Retrograde activation of adenosine A1 receptors reduces ACh release from cholinergic bladder nerves. β3‐Adrenoceptors usually couple to adenylyl cyclase. Here we investigated, which of the cAMP targets, protein kinase A or the exchange protein directly activated by cAMP (EPAC) could be involved in this cholinergic inhibition of the bladder. Experimental Approach [3H]ACh and adenosine release from urothelium‐denuded detrusor strips of cadaveric human organ donors and rats were measured by liquid scintillation spectrometry and HPLC, respectively. In vivo cystometry was also performed in urethane‐anaesthetized rats. Key Results The exchange protein directly activated by cAMP (EPAC) inhibitor, ESI‐09, prevented mirabegron‐ and isoprenaline‐induced adenosine release from human and rat detrusor strips respectively. ESI‐09, but not the PKA inhibitor, H‐89, attenuated inhibition of [3H]ACh release from stimulated (10 Hz) detrusor strips caused by activating β3‐adrenoceptors, AC (forskolin) and EPAC1 (8‐CTP‐2Me‐cAMP). Isoprenaline‐induced inhibition of [3H]ACh release was also prevented by inhibitors of PKC (chelerythrine and Go6976) and of the equilibrative nucleoside transporter 1 (ENT1; dipyridamole and NBTI), but not by PLC inhibition with U73122. Pretreatment with ESI‐09, but not with H‐89, prevented the reduction of the voiding frequency caused by isoprenaline and forskolin in vivo. Conclusion and Implications Data suggest that β3‐adrenoceptor‐induced inhibition of cholinergic neurotransmission in human and rat urinary bladders involves activation of an EPAC1/PKC pathway downstream cAMP production resulting in adenosine outflow via ENT1.

Inhibition of Female and Male Human Detrusor Smooth Muscle Contraction by the Rac Inhibitors EHT1864 and NSC23766

“…As an alternative option, b 3 -adrenoceptor agonists have been recently introduced for treatment of storage symptoms (Nambiar et al, 2018). They may improve storage symptoms by decreasing the bladder smooth muscle tone by inhibition of cholinergic nerve activity via retrograde release of adenosine (D'Agostino et al, 2000;Chapple et al, 2014;Silva et al, 2017;Igawa et al, 2019;Silva et al, 2019). However, it becomes increasingly clear, that their efficacy is not higher than that of anticholinergics (Nambiar et al, 2018), so that the overall situation regarding medical treatment of OAB and storage symptoms still remains inadequate.…”

Selective reduction of neurotransmitter release by cAMP-dependent pathways in mouse detrusor

“…The addition of the EPAC activator, 007-AM, or EPAC inhibitor, ESI-09, had no effect on neuronal ATP release or nerve-mediated contractions. Although inhibiting EPACs reduces the inhibitory effect of forskolin on ACh release ( 35 ), inhibiting PKA or EPACs had no direct effect on neuronal ACh release measured in this study. Modulators of cAMP effectors PKA and EPACs have been shown to regulate P 2 X receptors and consequently atropine-resistant, purinergic-mediated contractions of the detrusor ( 44 , 45 ).…”

“…However, a more complex role for adenosine and A 1 receptors has been suggested in addition to the reduction of nerve-mediated ATP release, namely to regulate nerve-mediated ACh release as judged from colocalization of A 1 receptors with vesicular ACh transporters on cholinergic nerve terminals ( 26 ). In detailed studies, it was proposed that β 3 -adrenoceptor agonists indirectly mediate adenosine release from detrusor smooth muscle, via the equilibrative nucleoside transporter 1, to activate A 1 receptors on cholinergic nerves and reduce neuronal ACh release ( 34 , 35 ). These varying effects of adenosine on nerve-mediated ACh release may be due to the difference in the concentration of adenosine and the different conditions from the experiments reported here.…”

Inhibition of Full Smooth Muscle Contraction in Isolated Human Detrusor Tissues by Mirabegron Is Limited to Off-Target Inhibition of Neurogenic Contractions

“…We assume that inhibition of EFS-induced contractions by 10 µM mirabegron in our study was caused by off-target inhibition of contractile neurotransmission. Inhibition of EFSinduced acetylcholine release in human detrusor tissues by mirabegron has been repeatedly demonstrated, with an EC 50 value of 129 nM (D'Agostino et al, 2015), with reductions of acetylcholine release between 30-55% using 0.01-1 µM mirabegron (Silva et al, 2019), or with a decrease of 57% by 100 nM mirabegron, which was sensitive to a β 3 -adrenoceptor antagonist (Silva et al, 2017). The limited degree of reduced neurotransmission (<60%) may be insufficient to translate to motoric effects, explaining why 1 µM mirabegron failed to inhibit EFS-induced contractions in our experiments.…”