The urinary bladder requires adequate concentrations of extracellular adenosine 5′-triphosphate (ATP) and other purines at receptor sites to function properly. Sequential dephosphorylation of ATP to ADP, AMP and adenosine (ADO) by membrane-bound and soluble ectonucleotidases (s-ENTDs) is essential for achieving suitable extracellular levels of purine mediators. S-ENTDs, in particular, are released in the bladder suburothelium/lamina propria (LP) in a mechanosensitive manner. Using 1,N⁶-etheno-ATP (eATP) as substrate and sensitive HPLC-FLD methodology, we evaluated the degradation of eATP to eADP, eAMP and eADO in solutions that were in contact with the LP of ex vivo mouse detrusor-free bladders during filling prior to substrate addition. The inhibition of neural activity with tetrodotoxin and ω-conotoxin GVIA, of PIEZO channels with GsMTx4 and D-GsMTx4 and of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1) with PACAP6-38 all increased the distention-induced but not spontaneous release of s-ENTDs in LP. It is conceivable, therefore, that the activation of these mechanisms in response to distention restricts the further release of s-ENTDs and prevents excessive hydrolysis of ATP. Together, these data suggest that afferent neurons, PIEZO channels, PAC1 receptors and s-ENTDs form a system that operates a highly regulated homeostatic mechanism to maintain proper extracellular purine concentrations in the LP and ensure normal bladder excitability during bladder filling.

Bladder urothelium and suburothelium/lamina propria (LP) have prominent sensory and transducer functions with the active participation of afferent neurons and urothelium-derived purine mediators such as adenosine 5’-triphosphate (ATP), adenosine 5’-diphosphate (ADP), and adenosine (ADO). Effective concentrations of purines at receptor targets depend significantly on the extracellular degradation of ATP by ectonucleotidases (ENTDs). We recently reported the regulated release of soluble ENTDs (s-ENTDs) in the LP and the consequent degradation of ATP to ADP, AMP, and ADO. Afferent neurons in the LP can be activated by urothelial ATP and release peptides and other transmitters that can alter the activity of cells in their vicinity. Using a murine decentralized ex vivo detrusor-free bladder model, 1,N⁶-etheno-ATP (eATP) as substrate, and sensitive HPLC-FLD methodologies, we found that exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (Sub P), neurokinin A (NKA), and pituitary adenylate cyclase-activating polypeptide [PACAP (1-38)] all increased the degradation of eATP by s-ENTDs that were released in the LP spontaneously and/or during bladder filling. Using antagonists of neuropeptide receptors, we observed that endogenous NKA did not modify the ATP hydrolysis by s-ENTDs, whereas endogenous Sub P increased both the constitutive and distention-induced release of s-ENTDs. In contrast, endogenous CGRP and PACAP (1-38) increased the distention-induced, but not the spontaneous, release of s-ENTDs. The present study puts forward the novel idea that interactions between peptidergic and purinergic signaling mechanisms in the LP have an impact on bladder excitability and functions by regulating the effective concentrations of adenine purines at effector cells in the LP.

The urothelium and lamina propria (LP) contribute to sensations of bladder fullness by releasing multiple mediators, including prostaglandins (PGs) and adenosine 5′-triphosphate (ATP), that activate or modulate functions of cells throughout the bladder wall. Mediators that are simultaneously released in response to bladder distention likely influence each other’s mechanisms of release and action. This study investigated whether PGs could alter the extracellular hydrolysis of ATP by soluble nucleotidases (s-NTDs) released in the LP of nondistended or distended bladders. Using an ex vivo murine detrusor-free bladder model to access the LP during bladder filling and a sensitive HPLC-FLD detection methodology, we evaluated the decrease in ATP and the increase in adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), and adenosine by s-NTDs released in the LP. Endogenous PGE₂ increased the spontaneous but not the distention-induced release of s-NTD via EP2 and EP3 prostanoid receptors, whereas exogenous PGE₂ increased the spontaneous s-NTD release via EP3, EP4, and FP receptors and the distention-induced s-NTD release via EP1-4 and FP receptors. Endogenous PGF_2α, PGD₂, and PGI₂ did not change the s-NTD release. Exogenous PGD₂ increased the spontaneous s-NTD release via DP2 receptors and the distention-induced s-NTD release via DP1 and DP2 receptors. Exogenous PGF_2α increased the spontaneous but not the distention-induced release of s-NTD via FP receptors. It is possible that higher concentrations of PGE₂, PGF_2α, and PGD₂ (as expected in inflammation, bladder pain syndrome, or overactive bladder) potentiate the release of s-NTDs and the consecutive degradation of ATP as a safeguard mechanism to prevent the development of excessive bladder excitability and overactivity by high amounts of extracellular ATP.

Distention of the urinary bladder wall during filling stretches the urothelium and induces the release of chemical mediators, including adenosine 5′-triphosphate (ATP) and prostaglandins (PGs), that transmit signals between cells within the bladder wall. The urothelium also releases soluble nucleotidases (s-NTDs) that control the availability of ATP and its metabolites at receptor sites in umbrella cells and cells deeper in the bladder wall, as well as in the urine. This study investigated whether PGs regulate the intravesical breakdown of ATP by s-NTDs. Using a murine decentralized mucosa-only bladder model and an HPLC technology with fluorescence detection, we evaluated the decrease in ATP and increase in ADP, AMP, and adenosine (ADO) in intraluminal solutions (ILS) collected at the end of physiological bladder filling. PGD₂, PGE₂, and PGI₂, but not PGF_2α, inhibited the conversion of AMP (produced from ATP) to ADO, likely due to a suppressed intravesical release of s-AMPases. The effects of exogenous PGD₂, PGE₂, and PGI₂ were mediated by DP1/DP2, EP2, and IP prostanoid receptors, respectively. Activation of either DP1 or DP2 receptors by endogenous PGD₂ also led to AMP increase and ADO decrease in ILS-containing ATP substrate. Finally, PGs produced by either COX-1 or COX-2 inhibited the hydrolysis of AMP to ADO. Together, these observations suggest that (1) endogenous PGs (chiefly PGD₂, and to lesser degree PGE₂ and PGI₂) allow release of s-NTDs like s-ATPases and s-ADPases but impede the formation of ADO from intravesical ATP by inhibiting the release of s-NTDs/s-AMPases; (2) it is possible that high concentrations of PGD₂, PGE₂ and PGI₂, as anticipated in inflammation or bladder pain syndrome, delay the ADO production and prolong the action of excitatory purine mediators; and (3) either COX-1 and COX-2 are constitutively expressed in the mouse bladder mucosa or COX-2 is induced by distention of the urothelium during bladder filling.