Mechanosensitive Hydrolysis of ATP and ADP in Lamina Propria of the Murine Bladder by Membrane-Bound and Soluble Nucleotidases

Abstract

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,N ⁶ -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.

Keywords: ADP; ATP hydrolysis; ATP-adenosine triphosphate; bladder; lamina propria (LP); nucleotidases; purine nucleotides; urothelium.

FIGURE 1

Principal pathways of extracellular ATP hydrolysis. ATP is hydrolyzed to ADP and then ADP is hydrolyzed to AMP by ENTPD1, 2, 3, and 8. ATP can be degraded directly to AMP by alkaline phosphatase (ALPL/TNAP) and ENPP1 and 3. AMP, in turn, is degraded to ADO by NT5E and ALPL/TNAP.

FIGURE 2

Schematics of experimental procedures for evaluation of eATP degradation in extraluminal solutions from nondistended and distended detrusor-free bladder preparations. (A) Substrate (e.g., eATP or eADP) was added to KBS bathing the bladder preparation. Decrease of substrate and increase of product was measured using HPLC-FLD. (B) Bladder was incubated in KBS. After incubation time equivalent to the time for bladder filling, an aliquot of the bath solution (aka extraluminal solution, ELS) was transferred to an empty chamber. Substrate (e.g., eATP or eADP) was added to the transferred ELS and substrate hydrolysis was assessed by HPLC. (C) Bladder was incubated in KBS as described in panel (B). Then an aliquot of ELS was placed in a filtration unit (MWCO 10 kDa) and centrifuged. The supernatant (aka concentrated ELS, cELS) was transferred to an Eppendorf tube to which the substrate was added and substrate decrease and product increase were evaluated using HPLC. (D) Bladder was incubated with KBS. Then, an aliquot of ELS was concentrated as described in panel (C). Next, 2.9-ml of modified KBS (mKBS) (Table 1) was added in the centrifugal unit with EL supernatant in KBS and centrifuged. Then, 2.9-ml of mKBS was added to the centrifugal unit containing the EL supernatant in mKBS and centrifuged. The resulting supernatant was transferred to an Eppendorf tube and the volume of cELS was brought to 200 µl with mKBS (37°C). Substrate was added to the Eppendorf tube and substrate decrease and product increase was measured by HPLC.

FIGURE 3

Hydrolysis of eATP, eADP, and eAMP in contact with the LP of nondistended and distended detrusor-free bladders. (A) Original chromatograms of eATP in beaker (blue) and at 60 min of contact of eATP with the basolateral/anti-luminal side of nondistended (red) and distended (green) bladder preparations. Note the greater decrease of eATP and greater increase in eADP, eAMP, and eADO in distended than in nondistended preparations. (B) Summarized data showing the degradation of eATP in EL samples collected for 1 h after addition of eATP to ELS in the presence of bladder preparation (Protocol 1). eATP, eADP, eAMP, and eADO are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in ELS samples from nondistended (dotted connecting lines) and distended (solid connecting lines) preparations. (C) Original chromatograms of eADP in beaker (blue) and at 60 min of contact of eADP with the anti-luminal side of nondistended (red) and distended (green) bladder preparations. The chromatograms demonstrate greater decrease of eADP and greater increase in eAMP and eADO in distended than in nondistended preparations. (D) Summarized data showing the degradation of eADP in EL samples collected at different time points during 1 h after addition of eADP to ELS in the presence of bladder preparation (Protocol 1). eADP, eAMP, and eADO are presented as percentages of total purines (eADP + eAMP + eADO) present in ELS samples from nondistended (dotted connecting lines) and distended (solid connecting lines) preparations. (E) Original chromatograms of eAMP in beaker (blue) and at 60 min of contact of eAMP with the anti-luminal side of nondistended (red) and distended (green) bladder preparations. The chromatograms demonstrate similar decrease of eAMP and increase in eADO in distended and nondistended preparations. (F) Summarized data showing the degradation of eAMP in EL samples collected during 1 h after addition of eAMP in ELS in the presence of bladder preparation. eAMP and eADO are presented as percentages of total purines (eAMP + eADO) present in ELS samples from nondistended (dotted connecting lines) and distended (solid connecting lines preparations). Statistical significance is described in main text Results.

FIGURE 4

Degradation of eATP in the suburothelium evaluated by microdialysis of the bladder wall. (A) Microdialysis probe implanted between suburothelium/LP and detrusor muscle of nondistended (left panel) and distended (right panel) mouse urinary bladder. m, microdialysis membrane outlined with black dots; white arrows show the direction of microdialysis perfusion. *, air bubbles inserted in bladder to show transparency of the wall. (B) Original chromatograms of eATP substrate in absence of tissue (beaker and blue) and in dialysate from nondistended bladder (red). Addition of eATP in the solution perfusing the microdialysis probe resulted in an eADP increase and the appearance of eAMP and eADO. (C) Summarized data showing formation of product (eADP + eAMP + eADO) from eATP after microdialysis of nondistended and distended bladder preparations with eATP. Data are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in dialysate. *P < 0.05, ns, non-significant difference.

FIGURE 5

Degradation of eATP and eADP by soluble enzymes released in the LP at rest and during bladder filling. (A) Summarized data showing the degradation of eATP in diluted (i.e., 2.5-ml) EL samples collected at different time points during 1 h after addition of eATP in the absence of bladder preparation (Protocol 2). eATP, eADP, eAMP, and eADO are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in EL solution samples from nondistended (dotted connecting lines) and distended (solid connecting lines) preparations. (B) Summarized data showing the degradation of eADP in diluted (i.e., 2.5-ml) EL samples collected at different time points during 1 h after addition of eADP in the absence of bladder preparation (Protocol 2). eADP, eAMP, and eADO are presented as percentages of total purines (eADP + eAMP + eADO) present in EL solution samples from nondistended (dotted connecting lines) and distended (solid connecting lines) preparations. (C) Original chromatograms of eATP in beaker (blue) and at 60 min of addition of eATP to concentrated EL samples (Protocol 3) collected from nondistended (red) and distended (green) bladder preparations. The chromatograms demonstrate greater decrease of eATP and increase in eADP, eAMP, and eADO in EL solutions from distended than from nondistended preparations. (D) Original chromatograms of eADP in beaker (blue) and at 60 min of addition of eADP to concentrated EL samples (Protocol 3) collected from nondistended (red) and distended (green) bladder preparations. The chromatograms demonstrate greater decrease of eADP and increase in eAMP and eADO in EL solutions from distended than from nondistended preparations. (E) Summarized data showing the degradation of eATP in EL samples collected at different time points during 1 h after addition of eATP in concentrated EL samples collected from nondistended (dotted connecting lines) and distended (solid connecting lines) (Protocol 3). eATP, eADP, eAMP, and eADO are presented as percentages of total purines (eATP + eADP + eAMP + eADO) present in the EL solution samples. (F) Summarized data showing the degradation of eADP in EL samples collected at different time points during 1 h after addition of eADP in concentrated EL samples collected from nondistended (dotted connecting lines) and distended (solid connecting lines) preparations (Protocol 3). eADP, eAMP, and eADO are presented as percentages of total purines (eADP + eAMP + eADO) present in the EL solution samples. Statistical significance is described in main text Results.

FIGURE 6

Effects of extracellular Ca²⁺ and Mg²⁺ on activities of released ATPases and ADPases. (A–D) eATP hydrolysis by soluble enzymes in controls (KBS) and mKBS: panel (A) shows the decrease of eATP and panels (B–D) show the increase in eADP, eAMP, and eADO formed from eATP, respectively. (E–G) eADP hydrolysis by soluble enzymes in control (KBS) and mKBS: panel (E) shows eADP decrease and panels (F,G) show the increase in eAMP and eADO formed from eADP. KBS contains normal Ca²⁺ (1.8 mM) and Mg²⁺ (1.2 mM). mKBS-A contains normal Ca²⁺ (1.8 mM) and no Mg²⁺ (0 mM); mKBS-B contains 0 mM Ca²⁺, normal Mg²⁺ (1.2 mM), and EGTA (5 mM); mKBS-C contains 0 mM Ca²⁺, 0 mM Mg²⁺, and EGTA (5 mM); mKBS-D contains 0 mM Ca²⁺, normal Mg²⁺ (1.2 mM), EGTA (5 mM), and EDTA (5 mM); mKBS-E contains 0 mM Ca²⁺, 0 mM Mg²⁺, EGTA (5 mM), and EDTA (5 mM). Asterisks denote significant difference from controls (KBS). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7

Effects of ARL67156 and POM-1 on hydrolysis of eATP by soluble enzymes in LP. (A–D) eATP in the presence of vehicle (KBS) or ARL67156 (100 µM) or POM-1 (100 µM). (E–G) eADP hydrolysis by soluble enzymes in the presence of vehicle (KBS) or ARL67156 (100 µM). Asterisks denote significant difference from vehicle controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 8

Effects of PSB06126, ENPP1-Inh-C and L-p-bromotetramisole (L-p-BT) on hydrolysis of eATP by soluble enzymes in LP. (A–D) eATP hydrolysis by soluble enzymes in the presence of vehicle (0.1% DMSO) or PSB06126 (10 µM), ENPP1-Inh-C (50 µM) or L-p-bromotetramisole (L-p-BT) (100 µM). Asterisks denote significant difference of eATP decrease in the presence of ENPP1-Inh-C from vehicle controls. *p < 0.05, **p < 0.01, ****p < 0.0001. Open circles denote significant difference of eATP decrease in the presence of PSB06126 and vehicle control (panel A). ^oP<0.05, ^ooP<0.01.

FIGURE 9

Validation of anti-nucleotidase antibodies used for enzyme identification. ProteinSimple Wes was used to specifically detect the indicated nucleotidases in (A) mouse brain homogenates (positive controls) and in (B) mouse tissue homogenates in which the indicated nucleotidase has not been detected (negative controls). Panel (Ba–Bi) show representative blot images (left) and immunoelectropherograms (right) of negative control tissue homogenates. Each antibody was tested in duplicate. The antibodies, dilutions, corresponding amounts of homogenate per well, and expected molecular weight (from vendor) used were: rabbit anti-ENTPD1 (1:200, 15 μg, and 80 kDa), sheep anti-ENTPD2 (1:500, 7.5 µg, and 80 kDa); rabbit anti-ENTPD3 (1:500, 3.0 µg, and 80 kDa); rabbit anti-ENTPD8 (1:200, 15 μg, and 60 kDa); rabbit anti-ENPP1 (1:500, 15 μg, and 100 kDa); rabbit anti-ENPP3 (1:500, 15 μg, and 100 kDa); rabbit anti-NT5E (1:500, 15 μg, 7and 0 kDa); rabbit anti-TNAP (1:200, 30 μg, and 60 kDa; rabbit anti-NT5C1A (1:100, 30 μg, and 40 kDa)).

FIGURE 10

Protein expression levels in urothelium homogenates prepared from detrusor-free bladder preparations. Representative immunoelectropherograms (duplicates) of nucleotidases detected in urothelium using ProteinSimple Wes (A–J). Each antibody was diluted 100-fold and each well contained 6 µg of urothelium homogenate sample. The antibodies used are described in Figure 9 and in main text Antibodies. (K) Scatter plots of AUC of chemiluminescence (CL) signals normalized per µg loaded urothelium sample. Each symbol represents a single loading from 5 urothelium samples, loaded in triplicates. Statistical significance is described in main text Results.

FIGURE 11

Protein expression levels in cELS collected from distended detrusor-free bladder preparations. Representative immunoelectropherograms (duplicates) of nucleotidases detected in cELS using ProteinSimple Wes (A–J). Each antibody was diluted 100-fold and each well contained 3 µl of cELS sample. The antibodies used are described in Figure 9 and in main text Antibodies. (K) Scatter plots of AUC of chemiluminescence (CL) signals normalized per µL loaded cELS sample. Each symbol represents a single loading from 3 cELS samples, loaded in triplicates. Statistical significance is described in main text Results.

FIGURE 12

A model depicting mechanisms of purinergic signaling in the lamina propria during bladder filling. Stretch of the bladder wall during filling causes release of ATP from the urothelium into the suburothelium (Birder and Andersson, 2013; Burnstock, 2014; Dalghi et al., 2020). ATP activates P2X (e.g., P2X2/X3) receptors (P2XR) on afferent nerve terminals in urothelium and suburothelium/lamina propria (SubU/LP) and triggers a voiding reflex (Cockayne et al., 2000; Vlaskovska et al., 2001). ATP that is released in the LP is hydrolyzed to ADP, AMP, and adenosine (ADO) by four families of membrane-bound nucleotidases (Zimmermann et al., 2012). Bladder excitability during filling is regulated by excitatory (ATP and ADP) and inhibitory (ADO) purine mediators in the LP that activate specific purinergic receptors. ATP activates ligand-gated P2XR and G protein-coupled P2Y receptors (P2YR), ADP activates P2YR, and ADO activates G-protein coupled adenosine receptors (AR) (Burnstock, 2014). P2XR, P2YR, and AR are ubiquitously expressed in the bladder wall, including in cells in the detrusor, the urothelium, and the LP (Dalghi et al., 2020). Cell types that express purinergic receptors in the LP include afferent neurons (AN), interstitial cells (IC), fibroblasts (Fb), and blood vessels (BV). Nucleotidases have the ability to terminate P2XR or P2YR responses initiated by ATP and to favor the activation of AR or ADP-responding receptors. In addition to the membrane-bound nucleotidases, enzymes that metabolize ATP are released in the LP spontaneously and during distention of the bladder wall during bladder filling. Released enzymes degrade ATP to ADP, AMP, and ADO. The activity of released enzymes is greater in distended LP than in nondistended LP indicating mechanosensitive release of enzymes. Soluble nucleotidases in the LP identify with several membrane-bound nucleotidases and are possibly released in the LP by distention-induced ectodomain shedding (Lichtenthaler et al., 2018). Distention-dependent degradation of ATP by membrane-bound and soluble nucleotidases diminishes the presence of ATP in the LP at the end of bladder filling (Durnin et al., 2019b) to prevent abnormal excitability of the bladder. The proper availability of excitatory and inhibitory purines in the bladder wall is determined by distention-associated purine release and purine metabolism.