Voiding Dysfunction

In Vitro Release of Adenosine Triphosphate from the Urothelium

of Human Bladders with Detrusor Overactivity, Both Neurogenic

and Idiopathic

Vivek Kumar a,*, Christopher R. Chapple b, Derek Rosario c, Paul R. Tophill d,Russell Chess-Williams e

a Department of Reconstructive Urology, Royal Hallamshire Hospital, Sheffield, United Kingdomb Royal Hallamshire Hospital, University of Sheffield and Sheffield Hallam University, Sheffield, United Kingdomc Royal Hallamshire Hospital, University of Sheffield, Sheffield, United Kingdomd Princess Royal Spinal Injury Unit, Northern General Hospital, Sheffield, United Kingdome Faculty of Health Sciences and Medicine, Bond University, Queensland, Australia

E U R O P E A N U R O L O G Y 5 7 ( 2 0 1 0 ) 1 0 8 7 – 1 0 9 2

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Article info

Article history:

Accepted November 25, 2009Published online ahead ofprint on December 3, 2009

Keywords:

ATP

OAB

Purinergic neurotransmission

Purinergic receptors

Neurogenic bladder

Abstract

Background: There is increased evidence to suggest a role for nonadrenergic–noncho-

linergic neurotransmission in the pathogenesis of bladder dysfunction.

Objective: In this set of experiments, we have assessed the contribution of the urothelium

to purinergic activity by quantifying the amount of adenosine triphosphate (ATP) released

from the urothelium of patients with idiopathic detrusor overactivity (IDO) and with

neurogenic detrusor overactivity (NDO) and comparing these releases to those of controls.

Design, setting, and participants: Bladder tissue with urodynamically and clinically

proven NDO (n = 8) and IDO (n = 8) were included in this study. The carefully dissected

urothelium was stimulated by mechanically stretching as well as electrically stimulating

and the ATP; thus, release was quantified.

Measurements: We used a Lucy Anthos 1 luminometre (Anthos Labtec Instruments

GmBH, Wals, Austria) to perform the assay. The results were analysed using Stingray

software (Dazdaq Ltd, Brighton, UK).

Results and limitations: Both mechanical stretch and electric field stimulation (EFS)

led to increased ATP release in both sets of tissues with overactivity compared to

the controls; this rise was even more significant for the IDO urothelium (2416.7 �479.8 pmol/g [p < 0.005]) than for the NDO urothelium (133.1 � 22.4 pmol/g [p < 0.01]);

values for the controls were 77.6 � 16.2 pmol/g. ATP release following mechanical stretch

was more sensitive to tetrodotoxin in bladders with NDO compared to those with IDO as

well as to the controls, with ATP levels falling from 233.5 � 20.7 pmol/g to 107.2 � 11.6

pmol/g, expressed as percentage of basal levels ( p < 0.002). The experiments were

performed in vitro, and the female patients were a mix of peri- and postmenopausal states.

Conclusions: These experiments suggested a significant rise in ATP release from the

urothelium of bladders with NDO as well as those with IDO in comparison to controls.

Most of the ATP released from bladders with NDO is primarily from neuronal sources.

# 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.

* Corresponding author. Department of Reconstructive Urology, Royal Hallamshire Hospital,Glossop Road, Sheffield, South Yorkshire, S10 2JF, UK.

E-mail address: v.kumar@sheffield.ac.uk (V. Kumar).

0302-2838/$ – see back matter # 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.eururo.2009.11.042

E U R O P E A N U R O L O G Y 5 7 ( 2 0 1 0 ) 1 0 8 7 – 1 0 9 21088

1. Introduction

Overactive bladder (OAB) is a storage symptom complex

that refers to the symptoms of urinary urgency associated

with incontinence in up to one-third of cases, usually with

increased urinary frequency and nocturia [1]. Prevalence

rates range between 12% and 17% in North America and

Europe and are comparable among men and women [2–4].

OAB symptoms are suggestive of urodynamically de-

monstrable detrusor overactivity (DO) that may be sponta-

neous or provoked. DO may be further characterised as

neurogenic DO (NDO) or idiopathic DO (IDO). Common

neurogenic causes include stroke, Parkinson’s disease,

multiple sclerosis (MS), and spinal injury. Thus, the term

is applied to a wide range of conditions that may have a

common final pathophysiologic pathway [5].

The suburothelial layer of the bladder is rich in sensory

(afferent) nerves [6,7], and increased sensitivity of these

suburothelial sensory nerves may trigger the overactive

detrusor contraction. In 1997, Ferguson and associates [8]

demonstrated distension-evoked adenosine triphosphate

(ATP) release from the bladder urothelium. Since then,

evidence has been accumulating to support a role for

urothelial-derived ATP release in both autocrine and

paracrine signalling [9]. Furthermore, the purinergic

receptors (ionotropic purinergic receptor [P2X] and meta-

botropic purinergic receptors [P2Y]) are expressed in

various types of cells located at or near the urothelium of

the urinary bladder [10]. Local inflammation and stimula-

tion by the P2X receptor agonist can lead to increased

bladder afferent firing [11]. Thus, the stretch-evoked,

urothelial-derived ATP may stimulate the local sensory

mechanism, triggering sensations of fullness and pain that

induce changes in bladder activity [12].

When the bladder is stretched during the filling phase, it

releases several neurotransmitters that may act on recep-

tors present on the sensory nerves in the suburothelium,

thereby conveying information to the central nervous

system and initiating voiding. ATP is one such putative

neurotransmitter that contributes to the bulk of the

nonadrenergic–noncholinergic (NANC) activity, which is

responsible for the atropine-resistant detrusor contraction.

Whereas this NANC contribution in a normal human

bladder contraction is small [13], it can become significant

in functionally abnormal and aging bladders [14].

In this set of experiments, we assessed the contribution

of the urothelium to purinergic activity by quantifying the

amount of ATP released from the urothelium of patients

with IDO and NDO and comparing these releases to those of

controls.

2. Patients and methods

The experiments were performed using tissue from the dome of human

urinary bladder. Multiple full-thickness bladder tissues were extracted

from patients with urodynamically and clinically proven NDO (n = 8)

undergoing clam ileocystoplasty at the Spinal Injury Unit, Northern

General Hospital, Sheffield, United Kingdom. Those with MS (n = 6 [all

women]) and suprasacral spinal cord injury (n = 2 [one man]) were

included in this study (age range: 32–46 yr [mean: 37.5 yr]). In addition,

eight women (age range: 36–45 yr [mean: 41.5 yr]) who were clinically

and urodynamically diagnosed with IDO were also included in this

study. These patients had refractory symptoms of urinary frequency,

urgency, and urge incontinence along with DO demonstrated on video

urodynamics and had the full-thickness tissue samples taken at the time

of their clam cystoplasty.

Urothelium and suburothelial tissue were macroscopically dissected

off the samples for purposes of the experiments. The mean maximum

cystometric capacity (plus or minus standard error) of the patients with

NDO was 283 � 40 ml (range: 255–375 ml); for those patients with IDO,

capacity was 315 � 55 ml (range: 230–420 ml). Control bladders were

obtained from patients with urodynamically proven stable bladders

undergoing surgery for stress urinary incontinence (n = 4) using a cold

cup biopsy forceps (5 mm) and from patients undergoing cystectomy for

cancer (full-thickness sample) who had no history of receiving radiation

therapy and no lower urinary tract symptoms that would suggest a

functional bladder disorder (n = 5). The tissue was taken from a site distant

to the tumour from a normal-looking bladder area. The mucosa was

again macroscopically dissected off the tissues and immediately placed in

ice-cold Krebs bicarbonate solution (composition: sodium chloride,

118.4 mM/l; sodium bicarbonate, 24.9 mM/l; potassium chloride,

4.7 mM/l; calcium chloride, 21.9 mM/l; magnesium sulphate, 1.15 mM/l;

potassium dihydrogen phosphate, 1.15 mM/l; glucose 11.7 mM/l). All of the

tissues were obtained with full informed consent and with approval from

the local ethics committee.

Four tissue strips from each patient (5 � 3 mm) were placed between

two platinum-wire loop electrodes; using suture silk, the bottom end of

the tissues were secured to a tissue holder. The upper ends were

attached (with suture silk) to the UFI model 1030 transducer (UFI, Morro

Bay, CA, USA). The transducers were connected to the NeuroLog data

acquisition system (Digitimer, Welwyn Garden City, UK), allowing

changes in the tension to be recorded. Tissues were placed under 1 g of

constant resting tension in a 2.5-ml organ bath containing Krebs

bicarbonate solution at 37 8C and gassed with a mixture of 95% oxygen

and 5% carbon dioxide for 1 h, with washes at 15-min intervals.

The urothelium was mechanically stretched to 130%, and then,

following a period of rest (resting tension of 1 g), to a further 150% of the

original length. Samples (50 ml) were taken following the stretch for

quantification of ATP release. The tissues were also subjected to electric

field stimulation (EFS) at 10 Hz (40 V, 0.2 ms pulse width for 60 s) using a

Digitimer PowerStim stimulator, and ATP release was quantified using

luminometry. The ATP reagent HS luciferin–luciferase enzyme used was

obtained from BioTherma (BioTherma Luminescent Assays, Haninge,

Sweden). A Lucy Anthos 1 luminometre (Anthos Labtec Instruments

GmBH, Wals, Austria) was used to perform the assay, and the results

were analysed using Stingray software (Dazdaq Ltd, Brighton, UK).

Tetrodotoxin (5 mM) exposure to the urothelium for 20 min was used to

block the neuronal source of ATP release, thereby enabling us to measure

the non-neuronal component.

All of the tissues were weighed at the end of each experiment, and

ATP levels were calculated, as picomole per gram of tissue, as well as

relative rise, expressed as percentage of the basal level. Statistical

analysis was performed using paired t test.

3. Results

The basal release of ATP from the urothelium of control and

NDO bladders was similar (Table 1). Mechanical stretch led

to increased levels of ATP in both sets of tissues, but this

increase was significantly greater for the NDO tissues

(Table 1; Fig. 1). EFS also elicited a higher level of ATP; again,

the magnitude of this response was greater in the urothelial

Table 1 – The amount of adenosine triphosphate released, in picomoles per gram of tissue, from idiopathic and neurogenic detrusoroveractive urothelium with mechanical stretch (130% and 150%) and electric field stimulation (10 Hz)

ATP release with mechanical stretch (pmol/g tissue � SEM) ATP release with electrical stimulation(pmol/g tissue � SEM)

Baseline 130% 150% Baseline 10 Hz

Control urothelium (n = 9) 45.7 � 4.9 67.5 � 13.2 77.6 � 16.2* 45.8 � 5.1 61.7 � 10.1*

IDO urothelium (n = 8) 1064.2 � 238.9z 2103.2 � 456z* 2416.7 � 479.8z* 578.7 � 62.9z 1654.5 � 177.5z*NDO urothelium (n = 8) 57.0 � 7.9y 102.3 � 16.9* 133.1 � 22.4* 56.5 � 11.6y 124.5 � 20.7z*

ATP = adenosine triphosphate; SEM = standard error of the mean; IDO = idiopathic detrusor overactivity; NDO = neurogenic detrusor overactivity.

* p < 0.01 compared to the baseline ATP release.

y p is not significant, compared with control urothelium.

z p < 0.005 compared with control urothelium.

Table 2 – The effect of 5 mM of tetrodotoxin on the urothelium from neurogenic and idiopathic detrusor overactive bladders comparedwith normal urothelium**

ATP release with mechanical stretch(150% elongation) (% of baseline � SEM)

ATP release with electrical stimulation(10 Hz) (% of baseline � SEM)

TTX absent TTX present TTX absent TTX present

Control urothelium (n = 9) 175.4 � 21.7 147.4 � 20.1 137.9 � 4.4 30.2 � 2.2

IDO urothelium (n = 8) 227.1 � 17.9z 209.9 � 16.7z 286.6 � 33.5z 159.8 � 7.0*zNDO urothelium (n = 8) 233.5 � 20.7z 107.2 � 11.6* 221.8 � 32.4z 38.3 � 9.6*

ATP = adenosine triphosphate; SEM = standard error of the mean; TTX = tetrodotoxin; IDO = idiopathic detrusor overactivity; NDO = neurogenic detrusor

overactivity.

* p = 0.002 compared with TTX absent.

z p < 0.02 compared with control urothelium.

** The amount of ATP released is expressed as a percentage of the basal levels.

E U R O P E A N U R O L O G Y 5 7 ( 2 0 1 0 ) 1 0 8 7 – 1 0 9 2 1089

tissues taken from patients with NDO (Table 1). These

increases in ATP release were statistically significant,

whether expressed in absolute values (Table 1) or as a

percentage of the basal levels (Table 2).

In contrast, the basal release of ATP from bladders with

IDO without any stimulation was remarkably raised com-

pared to both the control and NDO bladders. In addition, this

urothelium showed significantly greater ATP release with

both mechanical stretch (130% and 150% stretch) as well as

EFS (10 Hz) when compared to the controls (Tables 1 and 2;

Fig. 2).

The ATP release from the urothelium of both NDO

bladders and controls following EFS showed a marked

sensitivity to the neurotoxin tetrodotoxin ( p = 0.002).

Fig. 1 – Amounts of adenosine triphosphate (ATP) released withmechanical stretch of the urothelium from bladders with neurogenicdetrusor overactivity (NDO) compared to controls following a basalrelease at a resting tension of 1 g.

The level of ATP release fell by 83% in NDO bladders and

by 78% in normal bladders following exposure to tetrodo-

toxin at a concentration of 5 mM (Table 2), suggesting that

EFS results in primarily neuronal ATP release. In compari-

son, the urothelial ATP release following mechanical

stretch, which may be more physiologic, was more sensitive

to tetrodotoxin in bladders with NDO compared to controls.

Tetrodotoxin reduced stretch-induced ATP release by 54% in

urothelium from bladders with NDO ( p = 0.005) and by only

16% in urothelium from controls.

Similarly, the ATP release from the urothelium of IDO

bladders following EFS showed a marked sensitivity to

tetrodotoxin ( p < 0.05), resulting in an overall reduction

of ATP levels by 45% when exposed to tetrodotoxin

(Table 2). This again confirmed that EFS predominantly

results in neuronally released ATP. However, the ATP

release following mechanical stretch of IDO bladders was

not sensitive to tetrodotoxin, with a small but similar fall

to that of the controls in the level of ATP release (8%

for idiopathic overactive bladders [p = not significant

(ns)] and 16% for controls [p = ns]) from the basal level

(Table 2).

4. Discussion

Brading and Turner [15] have emphasised that basic

myogenic changes (regardless of aetiology) underlie the

pathophysiology of DO. Furthermore, evidence of denerva-

tion is consistently found in detrusor biopsy specimens

from patients with various forms of DO [16,17], suggesting

Fig. 2 – Amounts of adenosine triphosphate (ATP) released withmechanical stretch expressed as a percentage of basal release at a restingtension of 1 g from the urothelium of bladders with idiopathic detrusoroveractivity (IDO).

E U R O P E A N U R O L O G Y 5 7 ( 2 0 1 0 ) 1 0 8 7 – 1 0 9 21090

that partial denervation of the detrusor may also alter the

properties of smooth muscle, leading to increased excit-

ability and coupling between cells. Under normal circum-

stances, local contraction (activity) that occurs somewhere

in the detrusor is thought to spread throughout the bladder

wall, resulting in a coordinated myogenic contraction of the

whole bladder. Although the exact mechanism that would

trigger this local reflex is not clear, Drake et al. [18,19] have

postulated that the bladder is modular in nature, consisting

of several tiny modules that can communicate with each

other via a myovesical plexus, thereby resulting in a

coordinated contraction of the detrusor.

In normal bladder, afferent activity is mediated largely

by small myelinated afferent fibres (Ad-fibres) that pass

through spinal tracts to the brain stem and from there to the

periaquaeductal gray matter, where they alert the higher

centres about the state of bladder fullness [20]. After spinal

disruption, a different type of afferent pathway emerges

that is mediated by unmyelinated C-fibres that are sensitive

to capsaicin, which might be responsible for development of

NDO [21]. Gosling et al. [22] postulated that the urothelium

is innervated and demonstrated a complex sensory network

in the suburothelial layer consisting of vesicle-packed

naked axons, some penetrating the urothelial basal

epithelium. The unmyelinated nerves have been demon-

strated to contain a wide variety of active agents, including

calcitonin gene-related peptide, substance P, vasoactive

intestinal peptide (VIP), substance Y, and acetylcholine;

they may also release ATP [21]. Recently, evidence has been

emerging in support of a role for the suburothelial

myofibroblasts in detrusor contraction. The urothelial

ATP release may act on the P2X and P2Y receptors present

on these myofibroblasts, thereby modulating the bladder

contraction [23].

It is clear that the urothelium is involved in sensory

perception in response to thermal, mechanical, and chemical

stimuli and can release chemical mediators. Afferent nerves

next to the urothelium could be targets for neurotransmitters

released by the urothelium and suburothelium. Identified

sensory–motor neurotransmitters include nitric oxide,

urothelium-derived inhibitory factor, ATP, substance P,

neurokinin-A, VIP, and acetylcholine [24].

It has been hypothesised that ATP released by urothelial

stretch acts on P2X purinoceptor 2 (P2X2)/P2X purinocep-

tor 3 (P2X3) receptors on the suburothelial sensory nerves,

thereby initiating bladder voiding [25]. This theory was

further supported following the development of P2X3-

deficient mice that exhibited a reduced urinary frequency

and increased urinary volume and bladder capacity with

normal detrusor pressures [26]. Also, intravesical instilla-

tion of ATP in unanaesthetised and freely moving rats

induces DO [27]. This effect is counteracted by the P2X3

receptor antagonist, 20,30-O-trinitrophenyl-ATP (TNP-ATP).

O’Reilly et al. [28] have demonstrated a significant increase

in P2X2 receptors in patients with IDO, emphasising

the importance of the urothelial purinergic system in a

functionally abnormal bladder and the enormous potential

for drug development in his area.

In this set of experiments, the NDO bladder tissue

showed significantly greater ATP release on stretching

compared to control bladder. The enhancement was evident

when ATP release was expressed both as absolute values or

as the percentage increase from basal levels. In addition, the

ATP release from the urothelium of bladders with IDO

showed an even greater release from unstretched as well as

stretched urothelium. This increase was statistically signif-

icant, regardless of the methods used to express the

amounts: direct quantification or the rise above basal

levels. The ATP release was far greater for IDO bladders

compared to NDO bladders, and it is difficult to explain this

in terms of the pathophysiology of the disorder. ATP is a

well-accepted neurotransmitter, and this increased release

may stimulate the P2X3 receptors present on the urothelial

and suburothelial sensory nerves, possibly the unmyelinat-

ed C-fibres, stimulating the afferent arm of the micturition

reflex and thereby contributing to the DO that is character-

istic of both idiopathic and neuropathic overactive bladders.

This phenomenon provides direct evidence of an increased

purinergic activity in human bladders with DO. The

importance of purinergic neurotransmission is further

supported by the observed increase in the expression of

P2X3 receptors in the urothelium and the suburothelial

nerves [29,30].

The experiments were performed following equilibra-

tion the tissues under 1 g of resting tension, which can

itself influence ATP release. However, the tissues were

tested and retested for ATP release to minimise the

artefact.

Tetrodotoxin (5 mM) blocked 54% of ATP release follow-

ing mechanical stretch of neurogenic overactive bladders,

whereas in IDO bladders, it only inhibited ATP release by

8%, which was not significant and similar to the controls.

Thus, the NDO bladders showed a 3-fold increase in the

amount of neuronally released ATP compared to controls,

with the ratio of neuronal to non-neuronal sources of ATP

release rising from 1 in 6 to 1 in 1. This would suggest that in

bladders with NDO, the peripheral nerves release greater

amounts of ATP in the bladder, and this release may play a

vital role in the pathogenesis of DO. This is in contrast with

IDO bladders, in which the ATP released is predominantly

from non-neuronal sources.

E U R O P E A N U R O L O G Y 5 7 ( 2 0 1 0 ) 1 0 8 7 – 1 0 9 2 1091

5. Conclusions

These experiments suggest an increased purinergic

activity in the form of a significant rise in ATP release

from the urothelium of bladders with NDO as well as

those with IDO compared to controls. Most of the ATP

released from bladders with NDO is primarily from

neuronal sources. This observation further supports the

view that the unmyelinated C-fibres in the suburothelium

take up a more prominent role in bladders with NDO. In

bladders with IDO, ATP is being released from non-

neuronal sources, and this increased ATP release may act

as a sensory neurotransmitter and stimulate the afferent

arm of the micturition reflex, leading to a symptomatic

bladder with DO.

Author contributions: Vivek Kumar had full access to all the data in the

study and takes responsibility for the integrity of the data and the

accuracy of the data analysis

Study concept and design: Chess-Williams.

Acquisition of data: Kumar.

Analysis and interpretation of data: Kumar.

Drafting of the manuscript: Kumar, Chapple.

Critical revision of the manuscript for important intellectual content:

Chapple, Chess-Williams.

Statistical analysis: Kumar.

Obtaining funding: None.

Administrative, technical, or material support: Rosario, Tophill.

Supervision: Chapple.

Other (specify): None.

Financial disclosures: I certify that all conflicts of interest, including

specific financial interests and relationships and affiliations relevant

to the subject matter or materials discussed in the manuscript

(eg, employment/affiliation, grants or funding, consultancies, honoraria,

stock ownership or options, expert testimony, royalties, or patents filed,

received, or pending), are the following: None

Funding/Support and role of the sponsor: None.

Acknowledgement statement: The authors acknowledge Professor Ann-

Marie Surprenant of the Biomedical Department, University of Sheffield,

for her support and guidance.

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