Violeta N. Mutafova-Yambolieva

Objective: To explore molecular mechanisms underlying excessive extracellular adenosine (ADO) accumulation in the bladder lamina propria (LP) of mice with type 2 diabetes (T2D) induced by a high‐fat, high‐sucrose diet (HFHSD), conceivably leading to bladder underactivity. Methods: Performing automated, non‐invasive, continuous monitoring in metabolic cages, we evaluated 24‐h water intake and urine output of male C57Bl/6 nondiabetic (ND) and HFHSD‐T2D mice. Using a detrusor‐free bladder model and HPLC with fluorescence detection, we assessed availability of ATP, ADP, AMP, and ADO as well as the degradation of 1 N ⁶ ‐etheno‐ATP (eATP, 2 µM) in extraluminal solutions (ELS) interfacing with the LP of both nondistended and distended bladders from ND and HFHSD‐T2D mice. We also investigated the effect of ENT1 inhibition on transurothelial eADO transport and quantified AMPKα expression, its phosphorylation status, and ENT1 protein levels in ND and T2D bladder mucosa using the Jess Protein Western nano‐immunoassay. Glass's ∆ and Hedges' g effect size indicators were used to compare non‐diabetic and HFHSD‐T2D groups. Results: In the HFHSD‐T2D mice, the number of voids per 24 h was significantly decreased (Glass's ∆ 3.694) whereas urine volume per void was significantly increased (Glass's ∆ 1.554) in comparison with ND mice. Purines were released in ELS/LP of ND and diabetic bladders. T2D bladders exhibited a marked reduction in ATP (Glass's ∆ 12.772) and a substantial elevation in ADO levels within the ELS of distended preparations (Glass's ∆ 4.472). Hydrolysis of eATP to eADP, eAMP, and eADO was accelerated in the T2D ELS/LP. AMPKα2 expression and T172 phosphorylation were notably diminished in T2D bladder mucosa (Glass's ∆ = 2.453 and 2.572, respectively). Pharmacological inhibition of ENT1 reduced the transport of eADO from the lumen to the ELS. ENT1 protein levels were significantly lower in T2D bladder mucosa (Glass's ∆ = 12.824). Conclusions: Excessive accumulation of the inhibitory purine ADO in the LP of T2D bladders is driven by two convergent mechanisms: (1) accelerated degradation of ATP possibly linked to AMPK dysregulation, and (2) impaired cellular uptake of ADO due to diminished ENT1 expression. Together, these disruptions contribute to elevated extracellular ADO levels, which likely inhibit detrusor muscle contractility and promote the development of an underactive bladder.

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.

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.

The urinary bladder mucosa (urothelium and suburothelium/lamina propria) functions as a barrier between the content of the urine and the underlying bladder tissue. The bladder mucosa is also a mechanosensitive tissue that releases signaling molecules that affect functions of cells in the bladder wall interconnecting the mucosa with the detrusor muscle and the CNS. Adenosine 5ʹ-triphosphate (ATP) is a primary mechanotransduction signal that is released from cells in the bladder mucosa in response to bladder wall distention and activates cell membrane-localized P2X and P2Y purine receptors on urothelial cells, sensory and efferent neurons, interstitial cells, and detrusor smooth muscle cells. The amounts of ATP at active receptor sites depend significantly on the amounts of extracellularly released ATP. Spontaneous and distention-induced release of ATP appear to be under differential control. This review is focused on mechanisms underlying urothelial release of ATP in response to mechanical stimulation. First, we present a brief overview of studies that report mechanosensitive ATP release in bladder cells or tissues. Then, we discuss experimental evidence for mechanosensitive release of urothelial ATP by vesicular and non-vesicular mechanisms and roles of the stretch-activated channels PIEZO channels, transient receptor potential vanilloid type 4, and pannexin 1. This is followed by brief discussion of possible involvement of calcium homeostasis modulator 1, acid-sensing channels, and connexins in the release of urothelial ATP. We conclude with brief discussion of limitations of current research and of needs for further studies to increase our understanding of mechanotransduction in the bladder wall and of purinergic regulation of bladder function.

Purinergic signaling is important for normal bladder function, as it is thought to initiate the voiding reflex and modulate smooth muscle tone. The availability of adenine nucleotides and nucleosides (aka purines) at receptor sites of various cell types in the bladder wall is regulated by ectonucleotidases (ENTDs). ENTDs hydrolyze purines such as adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP) with varying preference for the individual substrate. Therefore, the end effect of extracellular purines may depend significantly on the type of ENTD that is expressed in close proximity to the target cells. ENTDs likely have distinct cellular associations, but the specific locations of individual enzymes in the bladder wall are poorly understood. We used RNAscope™, an RNA in situ hybridization (ISH) technology, to visualize the distribution and measure the levels of gene expression of the main recognized ectonucleotidases in large high-resolution images of murine bladder sections. The relative gene expression of ENTDs was Entpd3 > Alpl >> Enpp1 = Entpd2 >> Enpp3 > Entpd1 (very low to no signal) in the urothelium, Entpd1 ≥ Entpd2 >> Enpp3 > Enpp1 = Alpl ≥ Nt5e (very low to no signal) in the lamina propria, and Entpd1 >> Nt5e = Entpd2 >> Enpp1 > Alpl = Enpp3 in the detrusor. These layer-specific differences might be important in compartmentalized regulation of purine availability and subsequent functions in the bladder wall and may explain reported asymmetries in purine availability in the bladder lumen and suburothelium/lamina propria spaces.

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 bladder urothelium releases ATP into the lamina propria (LP) during filling, which can activate P2X receptors on afferent neurons and trigger the micturition reflex. Effective ATP concentrations are largely dependent on metabolism by membrane-bound and soluble ectonucleotidases (s-ENTDs), and the latter are released in the LP in a mechanosensitive manner. Pannexin 1 (PANX1) channel and P2X7 receptor (P2X7R) participate in urothelial ATP release and are physically and functionally coupled, hence we investigated whether they modulate s-ENTDs release. Using ultrasensitive HPLC-FLD, we evaluated the degradation of 1,N⁶-etheno-ATP (eATP, substrate) to eADP, eAMP, and e-adenosine (e-ADO) in extraluminal solutions that were in contact with the LP of mouse detrusor-free bladders during filling prior to substrate addition, as an indirect measure of s-ENDTS release. Deletion of Panx1 increased the distention-induced, but not the spontaneous, release of s-ENTDs, whereas activation of P2X7R by BzATP or high concentration of ATP in WT bladders increased both. In Panx1^−/− bladders or WT bladders treated with the PANX1 inhibitory peptide ¹⁰Panx, however, BzATP had no effect on s-ENTDS release, suggesting that P2X7R activity depends on PANX1 channel opening. We concluded, therefore, that P2X7R and PANX1 are in complex interaction to regulate s-ENTDs release and maintain suitable ATP concentrations in the LP. Thus, while stretch-activated PANX1 hinders s-ENTDS release possibly to preserve effective ATP concentration at the end of bladder filling, P2X7R activation, presumably in cystitis, would facilitate s-ENTDs-mediated ATP degradation to counteract excessive bladder excitability.

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.

Adenosine 5′-triphosphate (ATP) is released in the bladder lumen during filling. Urothelial ATP is presumed to regulate bladder excitability. Urinary ATP is suggested as a urinary biomarker of bladder dysfunctions since ATP is increased in the urine of patients with overactive bladder, interstitial cystitis or bladder pain syndrome. Altered urinary ATP might also be associated with voiding dysfunctions linked to disease states associated with metabolic syndrome. Extracellular ATP levels are determined by ATP release and ATP hydrolysis by membrane-bound and soluble nucleotidases (s-NTDs). It is currently unknown whether s-NTDs regulate urinary ATP. Using etheno-ATP substrate and HPLC-FLD detection techniques, we found that s-NTDs are released in the lumen of ex vivo mouse detrusor-free bladders. Capillary immunoelectrophoresis by ProteinSimple Wes determined that intraluminal solutions (ILS) collected at the end of filling contain ENTPD3 > ENPP1 > ENPP3 ≥ ENTPD2 = NT5E = ALPL/TNAP. Activation of adenylyl cyclase with forskolin increased luminal s-NTDs release whereas the AC inhibitor SQ22536 had no effect. In contrast, forskolin reduced and SQ22536 increased s-NTDs release in the lamina propria. Adenosine enhanced s-NTDs release and accelerated ATP hydrolysis in ILS and lamina propria. Therefore, there is a regulated release of s-NTDs in the bladder lumen during filling. Aberrant release or functions of urothelial s-NTDs might cause elevated urinary ATP in conditions with abnormal bladder excitability.

Prior studies suggest that urothelium-released adenosine 5′-triphosphate (ATP) has a prominent role in bladder mechanotransduction. Urothelial ATP regulates the micturition cycle through activation of purinergic receptors that are expressed in many cell types in the lamina propria (LP), including afferent neurons, and might also be important for direct mechanosensitive signaling between urothelium and detrusor. The excitatory action of ATP is terminated by enzymatic hydrolysis, which subsequently produces bioactive metabolites. We examined possible mechanosensitive mechanisms of ATP hydrolysis in the LP by determining the degradation of 1,N⁶-etheno-ATP (eATP) at the anti-luminal side of nondistended (empty) or distended (full) murine (C57BL/6J) detrusor-free bladder model, using HPLC. The hydrolysis of eATP and eADP was greater in contact with LP of distended than of nondistended bladders whereas the hydrolysis of eAMP remained unchanged during filling, suggesting that some steps of eATP hydrolysis in the LP are mechanosensitive. eATP and eADP were also catabolized in extraluminal solutions (ELS) that were in contact with the LP of detrusor-free bladders, but removed from the organ chambers prior to addition of substrate. The degradation of both purines was greater in ELS from distended than from nondistended preparations, suggesting the presence of mechanosensitive release of soluble nucleotidases in the LP. The released enzyme activities were affected differently by Ca²⁺and Mg²⁺. The common nucleotidase inhibitors ARL67156, POM-1, PSB06126, and ENPP1 Inhibitor C, but not the alkaline phosphatase inhibitor (-)-p-bromotetramisole oxalate, inhibited the enzymes released during bladder distention. Membrane-bound nucleotidases were identified in tissue homogenates and in concentrated ELS from distended preparations by Wes immunodetection. The relative distribution of nucleotidases was ENTPD1 >> ENPP1 > ENTPD2 = ENTPD3 > ENPP3 = NT5E >> ENTPD8 = TNAP in urothelium and ENTPD1 >> ENTPD3 >> ENPP3 > ENPP1 = ENTPD2 = NT5E >> ENTPD8 = TNAP in concentrated ELS, suggesting that regulated ectodomain shedding of membrane-bound nucleotidases possibly occurs in the LP during bladder filling. Mechanosensitive degradation of ATP and ADP by membrane-bound and soluble nucleotidases in the LP diminishes the availability of excitatory purines in the LP at the end of bladder filling. This might be a safeguard mechanism to prevent over-excitability of the bladder. Proper proportions of excitatory and inhibitory purines in the bladder wall are determined by distention-associated purine release and purine metabolism.