Adenosine triphosphate (ATP) is a key mediator of urothelial mechanosensory signalling, and purinergic signalling has been suggested to play a role in lower urinary tract dysfunction, including bladder outlet obstruction (BOO). Piezo1, a stretch-activated cation channel highly expressed in the urothelium, has been suggested to promote urothelial ATP release upon activation. This study investigated whether the selective Piezo1 agonist Yoda1 similarly stimulates urothelial ATP release and whether this contributes to detrusor overactivity in BOO.

In-vitro experiments used human urothelial TRT-HU1 cells. ATP release was quantified by luciferin-luciferase bioluminescence assay under baseline and mechanically stimulated conditions (repetitive pipetting). Piezo1 and Pannexin1 were silenced by siRNA. Yoda1 (10 µM) and the pannexin/connexin inhibitor carbenoxolone (CBX; 10 µM) were applied pharmacologically. Calcium-free conditions (0 mM CaCl₂ + 1 mM EGTA) were used to probe Ca²⁺-dependent mechanisms. Intracellular Ca²⁺ was monitored by Fura-2 ratiometric imaging. In vivo, male Sprague–Dawley rats underwent partial urethral ligation to model BOO (n = 6) or sham surgery. Awake cystometry was performed one week post-surgery before and after intravesical Yoda1 (10 µM). Intravesical ATP concentrations were measured under 30 cmH₂O hydrostatic pressure. Statistical analyses used two-way ANOVA with Sidak post-hoc testing; p < 0.05 was considered significant. All animal procedures conformed to institutional guidelines and were approved by the Institutional Animal Care and Use Committee.

In-vitro study: Under control conditions, mechanical stress increased ATP release 60% above baseline. Yoda1 suppressed mechanically stimulated ATP release in a dose-dependent manner, an effect abolished by Piezo1 knockdown, confirming that Yoda1 acts through Piezo1 (Fig. 1A). CBX, but not Pannexin1 knockdown alone, significantly suppressed mechanically stimulated ATP, suggesting the possible involvement of connexin hemichannels in mechanically stimulated ATP release (Fig. 1B). Under calcium-free conditions, both baseline and mechanically stimulated ATP were markedly elevated compared to control (Fig. 1C). In the presence of Yoda1, baseline ATP was similarly increased; however, mechanically stimulated ATP was significantly attenuated compared to calcium-free conditions without Yoda1. These findings suggest that Yoda1 exerts regulatory effects through both Ca²⁺-dependent and Ca²⁺-independent mechanisms. Fura-2 imaging demonstrated that Yoda1 induced a rapid, sustained (> 10 min) intracellular Ca²⁺ rise from extracellular sources, abolished in Ca²⁺-free medium and in Piezo1 knockdown cells (Fig. 1D).
In-vivo study: Intravesical ATP was significantly higher in BOO rats (55 ± 12 nM) versus sham controls (12.5 ± 7.0 nM) (Fig. 2A). Intravesical Yoda1 instillation significantly reduced ATP levels in BOO rats. Awake cystometry showed that Yoda1 reduced non-voiding contractions (NVCs) by 40% (p < 0.01) without altering voided volume, bladder capacity, or post-void residual (Fig. 2B, C).

Contrary to expectations, in-vitro experiments using the human urothelial cell culture system revealed that the Piezo1 agonist Yoda1 suppressed mechanosensitive urothelial ATP release through multiple mechanisms. Sustained Ca²⁺ influx may lead to connexin hemichannel closure, and Ca²⁺-independent pathways may also contribute to this suppression. These results indicate that Piezo1 channel activation in human urothelial cells exerts inhibitory effects on urothelial ATP release, thereby attenuating bladder sensory and detrusor activities. In a rat model of BOO, intravesical Yoda1 reduced elevated ATP and attenuated detrusor overactivity evident as a reduction in NVCs, suggesting that Piezo1-mediated suppression of BOO-induced detrusor overactivity is driven by decreased urothelial ATP release.

Intravesical Yoda1 suppresses urothelial ATP release and attenuates BOO-induced detrusor overactivity, supporting Piezo1 agonism as a novel therapeutic target for lower urinary tract dysfunction.