Prostaglandins Regulate Urinary Purines by Modulating Soluble Nucleotidase Release in the Bladder Lumen

Abstract

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.

Keywords: ATP; bladder; nucleotidases; prostaglandin D2; prostaglandin E2; prostaglandin I2; prostaglandins; urothelium.

Figure 1

Time course of eATP degradation to eADP, eAMP, and eADO in ILS. Original HPLC chromatograms showing the hydrolysis of eATP and formation of eADP, eAMP, and eADO at 0′ (Bk, substrate), 10 s, 2′, 4′, 6′, 8′, 10′, 20′, 30′, 40′, and 60′ of contact of eATP with s-NTDs released in the bladder lumen during filling of the bladder with vehicle DMSO 0.2% (a). LU, luminescence units. Summarized results demonstrating the patterns of eATP decrease and eADP, eAMP, and eADO increase in ILS of bladders filled with vehicle, n = 6. Each purine is expressed as µmol/L detected in ILS at each time point of enzymatic reaction (b). Mean area under the curve (AUC) for time courses of eATP, eADP, eAMP, and eADO (c). Total e-purines (eATP + eADP + eAMP + eADO) in reaction solutions for the duration of enzymatic reactions (d).

Figure 2

Effects of exogenous prostaglandins on the intravesical eATP hydrolysis. Time courses of the decrease of eATP (a–d) and the increase of eADP (e–h), eAMP (i–l), and eADO (m–p) in the presence of vehicle (n = 6) or PGE₂ (n = 8) (a,e,i,m), PGF_2α (n = 6) (b,f,j,n), PGI₂ (n = 4) (c,g,k,o), and PGD₂ (n = 8) (d,h,l,p); n, number of bladder preparations. Each purine is expressed as µmol/L detected in ILS at each time point of enzymatic reaction. AUC for time courses of eATP, eADP, eAMP, and eADO (q–t). Asterisks denote significant differences from vehicle control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Sum of eATP, eADP, eAMP, and eADO (total e-purines) at each time point of enzymatic reactions in ILS of bladders treated with exogenous PGs (u–x).

Figure 3

eATP hydrolysis by s-NTDs released in IL solutions of bladder preparations treated with exogenous PGE₂ in the absence or presence of EP prostanoid receptor antagonists. Summarized results demonstrating time courses of eATP decrease (a–d) and the increase of eADP (e–h), eAMP (i–l), and eADO (m–p) by PGE₂ (10 µM) in the presence of vehicle (n = 8) or of the EP1 antagonist SC51322 (1 µM, n = 6) (a,e,i,m), the EP2 receptor antagonist PF04418948 (1 µM, n = 6) (b,f,j,n), the EP3 receptor antagonist L-798,106 (0.25 µM, n = 10) (c,g,k,o), and the EP4 antagonist L-161,982 (1 µM, n = 4) (d,h,l,p); n, number of bladder preparations. AUC for time courses of eATP, eADP, eAMP, and eADO (q–t). Asterisks denote significant difference from vehicle control (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Open circle denotes significant differences in eATP degradation in PGE₂ alone vs. EP receptor antagonist + PGE₂. ^o p < 0.05. Two-way ANOVA with Tukey’s multiple comparisons test.

Figure 4

eATP hydrolysis by s-NTDs released in IL solutions of bladder preparations treated with PGI₂ in the absence or presence of an IP prostanoid receptor antagonist. Original chromatograms showing the eATP degradation after 60 min of contact with IL solutions (a). LU, luminescence units. Summarized results demonstrating time courses of eATP decrease (b) and the increase of eADP (c), eAMP (d), and eADO (e) in ILS of bladders treated with vehicle (n = 6), PGI₂ (n = 4) or RO1138452 + PGI₂ (n = 4); n, number of bladder preparations. Asterisks denote significant differences vs. vehicle controls. * p < 0.05, ** p < 0.01. Open circle denotes significant differences in eATP degradation in PGI₂ alone vs. RO1138452 + PGI₂. ^o p < 0.05. Two-way ANOVA with Tukey’s multiple comparisons test.

Figure 5

eATP hydrolysis by s-NTDs released in IL solutions of bladder preparations treated with PGD₂ in the absence or presence of DP prostanoid receptor antagonists. Original chromatograms showing the eATP degradation after 60 min of contact with the ILS of bladders treated with PGD₂ in the absence and presence of the DP1 receptor antagonist S-5751 (a) or of the DP2 receptor antagonist OC000459 (b). LU, luminescence units. Summarized results demonstrating time courses of eATP decrease (c,d) and the increase of eADP (e,f), eAMP (g,h), and eADO (i,j) in ILS of bladders treated with vehicle (n = 6), PGD₂ (n = 8), S-5751 + PGD₂ (n = 4) or OC000459 + PGD₂ (n = 4); n, number of bladder preparations. AUC for time courses of eATP, eADP, eAMP, and eADO (k,l). Asterisks denote significant differences vs. vehicle controls. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns means not significant. Open circles denote significant differences in eATP degradation in PGD₂ alone vs. DP receptor antagonist + PGD₂. ^o p < 0.05, ^oo p < 0.01, ^ooo p < 0.001, ^oooo p < 0.0001. Two-way ANOVA with Tukey’s multiple comparisons test.

Figure 6

Effects of DP prostanoid receptor antagonists on the intravesical eATP hydrolysis. Original HPLC chromatograms showing the hydrolysis of eATP and formation of eADP, eAMP, and eADO after 60 min of contact of eATP with s-NTDs released in ILS of preparations treated either with vehicle (i.e., DMSO 0.2%) or with the DP1 receptor antagonist S-5751 and the DP2 receptor antagonist OC000459 (a) or S-5751 + OC000459 (b). LU, luminescence units. Summarized results demonstrating time courses of the eATP decrease (c–e) and the increase of eADP (f–h), eAMP (i–k), and eADO (l–n) in the presence of vehicle (n = 6) and S-5751 (n = 4) (c,f,i,l), OC000459 (n = 4) (d,g,j,m) or S-5751 + OC000459 (n = 4) (e,h,k,n); n, number of bladder preparations. Each purine is expressed as a percentage of the total amount of purines detected in ILS at each time point of enzymatic reaction. AUC for time courses of eATP, eADP, eAMP, and eADO (o–q). Asterisks denote significant differences from vehicle control ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

Effects of COX inhibitors on the intravesical eATP hydrolysis by s-NTDs. An original HPLC chromatogram of eATP substrate in the absence of enzymes (beaker, Bk) (a). Original chromatograms showing the hydrolysis of eATP and formation of eADP, eAMP, and eADO after 60 min of contact of eATP with ILS of bladders treated with vehicle, SC-560, NS-398 (b), or SC-560 + NS-398 (c). LU, luminescence units. Summarized results demonstrating time courses of the eATP decrease (d–f) and the increase in eADP (g–i), eAMP (j–l), and eADO (m–o) in the presence of vehicle (n = 6), SC-560 (n = 4) (d,g,j,m), NS-398 (n = 4) (e,h,k,n), or SC-560 + NS-398 (n = 4) (f,i,l,o); n, number of bladder preparations. Each purine is expressed as a percentage of the total amount of purines detected in ILS at each time point of enzymatic reaction. AUC for time courses of eATP, eADP, eAMP, and eADO (p–r). Asterisks denote significant differences from vehicle control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 8

A model depicting regulation of s-NTDs release by endogenous and exogenous PGs. Distention of the bladder wall during bladder filling causes release of ATP (1), s-NTDs (e.g., s-ATPases, s-ADPases, s-AMPases) (2), and PGs (e.g., PGI₂, PGD_2, and PGE₂ that are produced locally by COX-1 and COX-2) (3) in the bladder lumen. S-NTDs sequentially break down ATP to ADP and AMP, and then, AMP is hydrolyzed to ADO. Released PGD₂, PGE₂, and PGI₂, as well as exogenous PGs (or high concentrations of PGs released in the urine during inflammation), activate their G-protein coupled receptors (4) on membranes of umbrella cells and other urothelial cells. Activation of EP2 receptors by PGE₂, of DP1 or DP2 receptors by PGD₂, and of IP receptors by PGI₂ triggers intracellular signaling mechanisms that inhibit the release of enzymes converting AMP to ADO (e.g., AMPases) (5). Diminished release of AMPases (e.g., NT5E and/or ALPL) leads to increased AMP and decreased ADO (6), likely causing prolonged excitation due to suppressed inhibition of bladder function. PG inhibition of s-NTDs/s-AMPases release in the bladder lumen is a novel mechanism of regulation of bladder function and can be targeted in disease states characterized by bladder overactivity or underactivity (Figure generated with BioRender).