Abstract
Urothelial ATP release during bladder filling modulates afferent nerve activity and detrusor contractility to regulate the micturition reflex, yet the upstream mechanosensory mechanism linking distension to ATP release remains unclear. Here we investigated the role of Piezo1 in regulating mechanosensitive ATP release in human urothelial TRT-HU1 cells and in a rat model of bladder outlet obstruction (BOO). In TRT-HU1 cells, mechanical stress increased ATP release 60% above baseline. Bath-applied Yoda1 (1–10 µM) suppressed mechanically stimulated ATP release in a dose-dependent manner, an effect abolished by PIEZO1 knockdown and reproduced across three mechanically distinct stimuli. PIEZO1 knockdown alone, however, did not reduce mechanically stimulated ATP release. PANX1 knockdown reduced baseline but not mechanically stimulated ATP release, whereas carbenoxolone suppressed it, implicating a carbenoxolone-sensitive, PANX1-independent pathway in which connexin hemichannels are the most likely mediator. Removal of extracellular calcium markedly elevated ATP release, and Yoda1 partially suppressed this elevation even in calcium-free conditions, indicating both Ca
2+
-dependent and Ca
2+
-independent inhibitory mechanisms. A 1-min application of Yoda1 evoked a sustained intracellular Ca
2+
rise that was abolished in calcium-free medium and in PIEZO1 knockdown cells. In BOO rats, intravesical ATP was significantly elevated compared to sham animals. Intravesical Yoda1 instillation significantly reduced ATP levels in BOO rats and decreased non-voiding contractions by 40% (p < 0.01) without altering voiding parameters. These findings identify pharmacological activation of Piezo1 as a brake on mechanosensitive purinergic signaling in the bladder and suggest that it warrants further evaluation as a therapeutic approach to BOO-induced lower urinary tract dysfunction.