Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function and in the pathophysiology of detrusor overactivity. ATP mediates nonadrenergic-noncholinergic detrusor contractions in overactive bladders. In vitro studies have demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladder samples (OAB) release more ATP than controls. Here, we compared the urinary ATP concentration in samples collected non-invasively from OAB women with detrusor overactivity and age-matched controls. Patients with neurologic diseases, history of malignancy, urinary tract infections or renal impairment (creatinine clearance <70 ml/min) were excluded. All patients completed a 3-day voiding diary, a 24 h urine collection and blood sampling to evaluate creatinine clearance. Urine samples collected during voluntary voids were immediately freeze-preserved for ATP determination by the luciferin-luciferase bioluminescence assay; for comparison purposes, samples were also tested for urinary nerve growth factor (NGF) by ELISA. The urinary content of ATP, but not of NGF, normalized to patients’ urine creatinine levels (ATP/Cr) or urinary volume (ATP.Vol) were significantly (P<0.05) higher in OAB women with detrusor overactivity (n = 34) than in healthy controls (n = 30). Significant differences between the two groups were still observed by boosting urinary ATP/Cr content after water intake, but these were not detected for NGF/Cr. In OAB patients, urinary ATP/Cr levels correlated inversely with mean voided volumes determined in a 3-day voiding diary. A high area under the receiver operator characteristics (ROC) curve (0.741; 95% CI 0.62–0.86; P<0.001) is consistent with urinary ATP/Cr being a highly sensitive dynamic biomarker for assessing detrusor overactivity in women with OAB syndrome.

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 A₁receptors 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 A₁receptors 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.

Deregulation of purinergic bladder signaling may contribute to persistent detrusor overactivity in patients with bladder outlet obstruction. Activation of uridine diphosphate sensitive P2Y6 receptors increases voiding frequency in rats indirectly by releasing adenosine triphosphate from the urothelium. To our knowledge this mechanism has never been tested in the human bladder. We examined the role of the uridine diphosphate sensitive P2Y6 receptor on tetrodotoxin insensitive nonneuronal adenosine triphosphate and [(3)H]acetylcholine release from the human urothelium with the lamina propria of control organ donors and patients with benign prostatic hyperplasia. The adenosine triphosphate-to-[(3)H]acetylcholine ratio was fivefold higher in mucosal urothelium/lamina propria strips from benign prostatic hyperplasia patients than control men. The selective P2Y6 receptor agonist PSB0474 (100 nM) augmented by a similar amount adenosine triphosphate and [(3)H]acetylcholine release from mucosal urothelium/lamina propria strips from both groups of individuals. The facilitatory effect of PSB0474 was prevented by MRS2578 (50 nM) and by carbenoxolone (10 μM), which block P2Y6 receptor and pannexin-1 hemichannels, respectively. Blockade of P2X3 (and/or P2X2/3) receptors with A317491 (100 nM) also attenuated release facilitation by PSB0474 in control men but not in patients with benign prostatic hyperplasia. Immunolocalization studies showed that P2Y6, P2X2 and P2X3 receptors were present in choline acetyltransferase positive urothelial cells. In contrast to P2Y6 staining, choline acetyltransferase, P2X2 and P2X3 immunoreactivity decreased in the urothelium of benign prostatic hyperplasia patients. Activation of P2Y6 receptor amplifies mucosal adenosine triphosphate release underlying bladder overactivity in patients with benign prostatic hyperplasia. Therefore, we propose selective P2Y6 receptor blockade as a novel therapeutic strategy to control persistent storage symptoms in obstructed patients.