Urinary ATP Levels Are Controlled by Nucleotidases Released from the Urothelium in a Regulated Manner

Abstract

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.

Keywords: ATP; ATP hydrolysis; CD73; bladder; bladder lamina propria; bladder lumen; nucleotidases; purine nucleotides; purinergic; urothelium.

Figure 1

Hydrolysis of eATP in bladder lumen of detrusor-free bladder preparations by membrane-bound and soluble nucleotidases. Original chromatograms of eATP in beaker (blue, 0′, no enzymes present) and at 10 s (red) and 60 min (green) after starting the enzymatic reactions in the bladder lumen (a), in ILS collected at the end of bladder filling (b), and in ILS filtered through 10 kDa MWCO membranes (c). Note the decrease of eATP substrate and the increase or appearance of the e-products eADP, eAMP and eADO. Summarized data showing time-courses of the degradation of eATP in the bladder lumen, n = 4–6 (d), in ILS collected at the end of bladder filling, n = 6–14 (e), and in ILS collected at the end of bladder filling and filtered through 10 kDa MWCO membranes, n = 3 (f); n, number of bladders preparations. eATP, eADP, eAMP and eADO are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in reaction solutions at each time point for the duration of 1 h.

Figure 2

Effects of ARL67156 and POM-1 on the hydrolysis of eATP by soluble enzymes released in bladder lumen during filling. Changes in eATP (a), eADP (b), eAMP (c), and eADO (d) in ILS in the presence of vehicle (KBS, n = 6–14)) or ARL67156 (100 µM, n = 5) or POM-1 (100 µM, n = 3). eATP, eADP, eAMP and eADO are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in reaction solutions; n, number of bladders preparations. Asterisks denote significant differences from vehicle controls. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 2 way ANOVA with Tukey’s multiple comparisons tests.

Figure 3

Effects of PSB06126 and ENPP1-Inhibitor-C on the hydrolysis of eATP by soluble enzymes released in bladder lumen during filling. Changes in eATP, eADP, eAMP, and eADO in ILS in the presence of vehicle (DMSO 0.1%, n = 7) or PSB06126 (10 µM, n = 3) (a) or ENPP-Inh-C (50 µM, n = 4) (b); n, number of bladder preparations. eATP, eADP, eAMP and eADO are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in reaction solutions. * p < 0.05 vs. control, 2 way ANOVA with Sidak’s multiple comparisons test.

Figure 4

Effect of L-p-BT on the hydrolysis of eATP by soluble enzymes released in bladder lumen during filling. Changes in eATP (a), eADP (b), eAMP (c), and eADO (d) in ILS in the presence of vehicle (DMSO 0.1%, n = 7) or L-p-BT (100 µM, n = 3); n, number of bladder preparations. eATP, eADP, eAMP and eADO are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in reaction solutions. Asterisks denote significant difference from vehicle controls. ** p < 0.01, *** p < 0.001, **** p < 0.0001. 2 way ANOVA with Sidak’s multiple comparisons tests.

Figure 5

Hydrolysis of eAMP by soluble nucleotidases in the lumen of bladder preparations from WT and Nt5e^−/− mice. Original chromatograms of eAMP in beaker (blue, 0′, no enzymes present), at 10 min (red), and at 60 min (green) after starting the enzymatic reactions in ILS collected at the end of bladder filling from WT controls (a), Nt5e^−/− controls (b) and Nt5e^−/− preparations treated with levamisole (1 mM) (c). Summarized data showing time-courses of eAMP decrease and eADO increase in ILS from WT bladders (n = 4–12) and from bladders isolated from Nt5e^−/− mice (n = 4–7) (d), in Nt5e^−/− preparations filled with either vehicle (KBS) or levamisole (n = 3) (e) or in Nt5e^−/− bladders filled either with vehicle (DMSO 0.1%, n = 4) or L-p-BT (100 µM, n = 3) (f); n, number of bladder preparations. Note that the eAMP decrease and eADO increase were the greatest in WT preparations and that the eAMP hydrolysis was significantly inhibited in the presence of either levamisole or L-p-BT. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. controls. 2 way ANOVA with Sidak’s multiple comparisons tests.

Figure 6

Protein expression levels in ILS collected at the end of filling of detrusor-free bladder preparations. (a–j) Representative immunoelectropherograms (duplicates) of nucleotidases evaluated using ProteinSimple Wes. Each antibody was diluted 100-fold and each well contained 3 µL of ILS sample. (k) Scatter plots of AUC of chemiluminescence (CL) signals normalized per µL loaded ILS sample (n = 8); n, number of bladder preparations. Statistical significance is described in Section 3.5.

Figure 7

Effect of forskolin (FSK) on the hydrolysis of eATP by soluble enzymes released in bladder lumen during filling. Decrease of eATP (a) and increase in eADP (b), eAMP (c), and eADO (d) after addition of eATP in ILS from bladder preparations filled with vehicle (DMSO 0.1%, n = 7) or FSK (n = 4 each); n, number of bladder preparations. Note that the decrease of eATP and the formation of eADP and eADO were enhanced in the presence of FSK. Asterisks denote significant differences from vehicle controls. * p < 0.05, ** p < 0.01. 2 way ANOVA with Tukey’s multiple comparisons tests.

Figure 8

Effect of FSK on the hydrolysis of eATP by soluble enzymes released in the bladder lamina propria. Original chromatograms of eATP in beaker (blue, 0′), at 10 min (red), and at 60 min (green) after addition of eATP to concentrated ELS collected at the end of bladder filling in the presence of vehicle (DMSO 0.1%) (a) or FSK (25 µM) (b). Summarized data showing time-courses of eATP decrease (c), eADP increase (d), eAMP increase (e), and eADO increase (f) in the presence of vehicle (DMSO 0.1%, n = 7) or FSK (n = 4 each); n, number of bladder preparations. Note that the degradation of eATP was reduced in the presence of FSK. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. vehicle controls. 2 way ANOVA with Tukey’s multiple comparisons tests.

Figure 9

Effect of SQ22536 on the hydrolysis of eATP by soluble nucleotidases released in the bladder lumen during filling. The decrease in eATP and increase in eADP, eAMP, and eADO were not significantly different in the presence of vehicle (DMSO 0.2%, n = 4) or SQ22536 (100 µM, n = 3).

Figure 10

Effect of SQ22536 on the hydrolysis of eATP by soluble nucleotidases released in the lamina propria of nondistended denuded bladder preparations. Time courses of eATP decrease (a), eADP increase (b), eAMP increase (c), and eADO increase (d) after addition of eATP to concentrated ELS collected from nondistended (empty) preparations. Note that the degradation of eATP was enhanced in the presence of SQ22536 (100 µM, n = 3) in comparison to DMSO 0.2% control (n = 4); n, number of bladder preparations. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. vehicle controls. 2 way ANOVA with Sidak’s multiple comparisons tests.

Figure 11

Effect of SQ22536 on the hydrolysis of eATP by soluble nucleotidases released in the lamina propria of distended denuded bladder preparations. Original chromatograms of eATP in beaker (blue, 0′), at 10 min (red), and at 60 min (green) after addition of eATP to concentrated ELS collected at the end of bladder filling in the presence of vehicle (DMSO 0.2%) (a) or SQ22536 (100 µM) (b). Summarized data of time courses of eATP decrease (c), eADP increase (d), eAMP increase (e), and eADO increase (f) after addition of eATP to concentrated ELS collected at the end of bladder filling. Note that the degradation of eATP was enhanced in the presence of SQ22536 (n = 3) in comparison with DMSO 0.2% control (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 2 way ANOVA with Sidak’s multiple comparisons tests.

Figure 12

Effects of adenosine (ADO) on the release of soluble nucleotidases in the bladder lumen. Time courses of eATP decrease (a), eADP increase (b), eAMP increase (c), and eADO increase (d) after addition of eATP to concentrated ILS collected at the end of bladder filling with either vehicle (KBS, n = 10) or ADO (n = 4); n, number of bladder preparations. Note that the degradation of eATP was enhanced in ILS from bladders filled with ADO. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. vehicle control. 2 way ANOVA with Sidak’s multiple comparisons tests.

Figure 13

Effects of adenosine (ADO) on the release of soluble nucleotidases in lamina propria of nondistended denuded bladder preparations. Time courses of eATP decrease (a), eADP increase (b), eAMP increase (c), and eADO increase (d) after addition of eATP to ELS collected from nondistended bladder preparations treated with either vehicle (KBS, n = 9) or ADO (n = 4 each) for a time period equivalent to the time for bladder filling; n, number of bladder preparations. Note that the degradation of eATP was enhanced in concentrated ELS collected from preparations treated with ADO. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. vehicle controls. 2 way ANOVA with Tukey’s multiple comparisons tests.

Figure 14

Effects of adenosine (ADO) on the release of soluble nucleotidases in lamina propria of distended denuded bladder preparations. eATP decrease and eADP, eAMP, and eADO increase after addition of eATP to concentrated ELS collected at the end of bladder filling. Note that there is no significant difference between eATP degradation in ELS from bladder preparations treated with vehicle (KBS, n = 9) and adenosine (n = 4 each); n, number of bladder preparations.