ATP released to suburothelial layer from the bladder urothelium is suggested to directly activates purinergic receptors that are expressed on primary bladder afferent neurons and induces the micturition reflex. Although ATP is also released to the bladder lumen from the bladder urothelium, the role of ATP in the bladder lumen has not been elucidated in detail. Recently, several clinical studies showed that urinary ATP levels were significantly higher in patients with an overactive bladder (OAB) (1). Another study suggested that urinary ATP levels could be a novel dynamic biomarker of OAB, which reflects disease severity (2). On the basis of these clinical studies, we consider that not only ATP in the suburothelial layer, but also ATP in the bladder lumen, are involved in the micturition reflex. The objective of this study is to examine the role of ATP in the bladder lumen.

First, to investigate the effect of ATP in the bladder lumen on bladder function, we performed intravesical ATP (pH-adjusted 50 mM) instillation in the mouse bladder in vivo and evaluated bladder function with reliable methods using improved cystometry and ultrasonography, which we previously established (3).
Next, to investigate the mechanism of changes of bladder function induced by intravesical ATP instillation, we evaluated neuronal activity of the L6-S1 spinal cord which mainly receives afferent inputs from primary bladder afferent nerves by immunostaining of c-Fos, which is a marker for activation of neurons. Furthermore, to confirm that c-Fos expression in the L6-S1 spinal cord was induced by neural signaling of primary bladder afferent nerves, we injected neuronal tracer cholera toxin subunit B (CTB) into the bladder suburothelial layer and performed c-Fos immunostaining. The location of c-Fos positive cells and bladder afferent nerves visualized by CTB were evaluated.
Then, to investigate whether changes of bladder function induced by intravesical ATP instillation are caused by inflammatory changes of the bladder, we assessed gross and histological inflammatory changes of the mouse bladder. Furthermore, to investigate expression of proinflammatory cytokines, we performed quantitative RT-PCR and evaluated their expression.
Finally, to determine the effects of blockade of bladder luminal ATP receptors on bladder function and spinal neuron activation, we performed intravesical ATP receptor antagonist pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS) instillation in the mouse bladder and assessed bladder function and spinal neuron activation.

Intravesical ATP instillation resulted in less intercontraction intervals (ICIs) compared with controls (p = 0.0016). This decrease began within three bladder contractions after ATP instillation began, and ICIs were mostly stable approximately 40 min after ATP instillation. Maximum voiding pressure, bladder contraction duration, and voiding volume were significantly lower after ATP instillation compared with controls (p = 0.009, p = 0.002, p = 0.002, respectively). There were no significant differences in pressure threshold, resting pressure, and bladder compliance between the two groups. With intravesical saline instillation following ATP instillation for 40 min, ICIs gradually became longer as time proceeded, and they were restored to the pre-administration state within approximately 3 h (Fig.1). In ultrasonography, intravesical ATP instillation resulted in a significantly lower largest cross-sectional area compared with controls (p = 0.012), but did not change the smallest cross-sectional area. These areas reflect maximum bladder volume and post-void residual urine, respectively. 
In the evaluation of the micturition reflex pathway, the number of c-Fos-positive cells in the L6-S1 spinal cord was significantly higher with intravesical ATP instillation compared with controls (p < 0.001). CTB-positive nerves appeared to converge at the deep layer in the L6-S1 spinal cord where almost all c-Fos-positive cells were located. Additionally, CTB-positive nerves appeared to connect directly to c-Fos-positive cells (Fig.2). 
In the evaluation of bladder tissue, there were no differences in edema, hemorrhage, bladder weight, and histological findings in ATP instillation compared with controls. Furthermore, there were no significant differences in expression of proinflammatory cytokines, such as IL-1β and IL-6 between the ATP instillation and control groups. 
In experiments blocking ATP receptors in the bladder lumen, when ATP antagonist PPADS was instillated with ATP to the bladder, the reduction in ICIs and the increase in c-Fos-positive cell number in the L6–S1 spinal cord were inhibited, both of which were induced by intravesical ATP instillation (p = 0.03, p = 0.01, respectively). However, intravesical PPADS alone instillation did not affect ICIs or the number of c-Fos-positive cells in the L6–S1 spinal cord.

Intravesical ATP instillation caused urinary frequency not by an increase in post-void residual urine, but by bladder overactivity, which was triggered by activation of bladder afferent nerves without inflammatory changes in the bladder. These results suggested that ATP in the bladder lumen could enhance the micturition reflex. Interestingly, however, the enhanced micturition reflex induced by intravesical ATP instillation was suppressed by the addition of intravesical PPADS instillation, whereas intravesical PPADS alone instillation showed no change compared to physiological controls. This suggests that ATP in the bladder lumen is not involved in the micturition reflex at low concentrations (physiological state) and may enhance micturition reflex only at high concentrations. Therefore, suppression of ATP in the bladder lumen would not suppress the normal physiological micturition reflex. These results suggest that bladder lumen ATP control may be a novel and safe treatment for frequent urinary disorders with increased urinary ATP such as OAB, without causing increased residual urine volume or urinary retention, which are adverse events associated with existing therapies.

High concentration ATP in the bladder lumen could enhance the micturition reflex. The regulation of ATP in the bladder lumen could be a promising therapeutic target of diseases of bladder overactivity.

V. Kumar, C.R. Chapple, D. Rosario, et al., In vitro release of adenosine triphosphate from the urothelium of human bladders with detrusor overactivity, both neurogenic and idiopathic, Eur Urol 57 (2010) 1087-1092.
M. Silva-Ramos, I. Silva, O. Oliveira, et al., Urinary ATP may be a dynamic biomarker of detrusor overactivity in women with overactive bladder syndrome, PLoS One 8 (2013) e64696.
K. Takezawa, M. Kondo, H. Kiuchi, et al., Combination of bladder ultrasonography and novel cystometry method in mice reveals rapid decrease in bladder capacity and compliance in LPS-induced cystitis, Am J Physiol Renal Physiol 307 (2014) 234-241.

Continence 7S1 (2023) 100966
DOI: 10.1016/j.cont.2023.100966