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Feb 1, 2006 - Mechanisms of Disease: role of purinergic signaling in the pathophysiology of bladder dysfunction. Michael R Ruggieri, Sr. INTRODUCTION.
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Mechanisms of Disease: role of purinergic signaling in the pathophysiology of bladder dysfunction Michael R Ruggieri, Sr

INTRODUCTION

S U M M A RY Although the ‘purinergic nerve hypothesis’ proposed by Burnstock in the early 1970s was met with considerable skepticism, it is now accepted that certain neurons use a purine nucleotide or nucleoside such as ATP or adenosine as a neurotransmitter. Likewise, early studies indicated that the human bladder is devoid of purinergic nerves mediating contraction; however, later studies demonstrated that purinergic nerve-mediated bladder contraction is increased in pathologic conditions such as interstitial cystitis. Cloning and sequencing studies have revealed four subtypes of adenosine receptors and eight subtypes of P2Y receptors, all of which are G-protein-coupled receptors. There are no reports of the cellular location of these receptors in the human bladder. P2X receptors are ligandgated ion channels, and seven subunits have been cloned and sequenced. Immunohistochemical studies have determined that P2X1,2,4 subunits are on detrusor-muscle cells, P2X1–3,5 subunits are on bladder nerves and P2X2,3,5 subunits are on bladder urothelial cells. Development of purinergic antagonist drugs with selectivity for P2X1 receptors on detrusor muscle cells might be useful for treatment of detrusor overactivity. Antagonists with selectivity for P2X3 receptors on bladder sensory nerves might be clinically beneficial for treatment of urinary urgency, and perhaps chronic pelvic pain. KEYWORDS ATP, bladder, bladder diseases, neurogenic bladder dysfunction, purinergic receptors

REVIEW CRITERIA Information for this review was obtained from searches for relevant articles in the Medline database from 1966 through August 2005, as well as searches through the author’s files. Search terms included “bladder”, “bladder, neurogenic”, “bladder diseases”, “adenosine triphosphate”, “receptors, purinergic”. Only articles published in English were included. All papers describing results in human bladder tissue were used and results of animal studies are only included when applicable to a relevant animal model of human disease or when no data for human bladder tissue has been published.

MR Ruggieri, Sr is a Professor and Director of Urologic Research in the Department of Urology, Temple University, and also holds secondary appointments in the Department of Pharmacology and the Department of Computer and Electrical Engineering, Temple University, Philadelphia, PA, USA. Correspondence Temple University School of Medicine, 3400 North Broad Street, 715 OMS, Philadelphia, PA 19140, USA [email protected] Received 14 September 2005 Accepted 1 February 2006 www.nature.com/clinicalpractice doi:10.1038/ncpuro0456

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The definitions used in this review conform to the standards recommended by the International Continence Society,1 except where specifically noted. The term neurogenic bladder dysfunction indicates bladder dysfunction originating in, starting from or caused by the nervous system. This includes bladder dysfunction due to such causes as traumatic or developmental brain or spinal cord injury, multiple sclerosis, and Parkinson’s disease. For the purposes of this review, discussion includes idiopathic bladder dysfunction such as overactive bladder and interstitial cystitis. These terms are not generally included in the definition of neurogenic bladder dysfunction because a definitive neuronal etiology for the urgency, frequency and nocturia symptoms has not been proven, but, nevertheless, many researchers suspect there is an underlying neuronal cause for these bladder disorders. Although it is generally agreed that acetylcholine acting on smooth muscle muscarinic receptors is the primary neurologic mechanism controlling bladder emptying, neural stimulation of the bladder is only partially inhibited by the antimuscarinic agent atropine in many species, including humans.2,3 In keeping with this observation, there is a significant body of evidence for a ‘purinergic’ innervation being responsible for the atropine-resistant component of parasympathetic contraction.4–6 The term purinergic indicates that these nerves use a purine such as ATP as the neurotransmitter. The atropine-resistant response of the bladder is transient, and although it does not substantially contribute to the process of normal bladder emptying, it might be involved in abnormal bladder contractions.5 Although many early reports indicated that nerve-evoked contractions of the human bladder are completely blocked by atropine, true atropine-resistant, tetrodotoxin-sensitive, nerve-evoked contractions are now known to occur in specimens from patients with bladder pathologies

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including hypertrophic unstable bladder,7–9 overactive bladder,7 neurogenic bladder6 and interstitial cystitis.6,10 In cystectomy specimens from bladder-cancer patients, these atropineresistant, purinergic contractions significantly increase with the age of the patient.11 In addition, existing evidence supports the concept that ATP released from the urothelium plays a sensory role in the bladder.12–16 ATP has been shown to induce a large inward current in smooth-muscle cells isolated from human, pig and guinea pig urinary bladders,17 and, despite the absence of atropine-resistant contractions in normal human bladder muscle strips, a substantial contractile response is observed in response to purinergic agonists.6 The mechanism for the relatively smaller contribution of purinergic neurotransmission in human and pig bladders compared to smaller laboratory animals such as the rabbit, rat and guinea pig bladder might, therefore, be related to the closeness of the innervation and the degree of cell to cell coupling.17 Decreased ECTO-ATPASE activity has been shown in bladders from patients with idiopathic detrusor instability;18 this activity might lead to reduced inactivation of ATP released from bladder nerves or the urothelium. This could explain the appearance of atropine resistance, and might provide some explanation for bladder overactivity in this group of patients. It is possible that drugs that selectively inhibit bladder ectoATPase activity could be clinically useful in the treatment of overactive bladder. It is also possible that purinergic signaling could be involved in the brainstem areas that control bladder function. In rats, microinjection of the excitatory neurotransmitter glutamate into or electrical stimulation of the periaquiductal grey matter, or the pontine micturition center of the brain, induces an increase in both pelvic nerve activity and bladder pressure.19 Microinjection of these same brainstem areas with purinergic agonists (ATP or α,β-methyleneATP) also induces a similar increase in pelvic nerve activity and bladder pressure, which can be blocked by previous microinjections of the nonspecific purinergic receptor antagonist suramin.19 PURINERGIC RECEPTORS

The physiologic actions of purine nucleotides and nucleosides, including ATP and adenosine, was described as early as 1929.20 The

basis for distinguishing receptors that respond to adenosine (P1) from those that respond to ATP and ADP (P2) was proposed in 1978.21 Following the recommendation by the nomenclature committee of the International Union of Pharmacology that receptors be named after the preferred endogenous ligand, the term P1 has been largely abandoned, except when referring specifically to older publications.22 The widely accepted nomenclature system first proposed by Burnstock and Kennedy is primarily derived from pharmacologic criteria, and distinguishes between two types of P2 receptors: P2X and P2Y.23 The adenosine receptors and the P2Y receptors are members of the 7 transmembranespanning G-protein-coupled receptor class, whereas the P2X receptors are ligand-gated ion-channel receptors. To date, four subtypes of adenosine receptors (A1, A2A, A2B, and A3) eight subtypes of P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) and seven subunits of P2X receptors (P2X1 to P2X7) have been cloned.24,25

GLOSSARY ECTO-ATPASE An enzyme that hydrolyses extracellular ATP

Bladder adenosine receptors

Using the pharmacology of the relaxation induced by adenosine analogs as a basis, the guinea pig detrusor-muscle adenosine receptor has been classified as A2A;26 however, the highest messenger RNA (mRNA) levels are found for A2B transcripts in the bladder.22 Potent radioligands with sufficient subtype selectivity for quantitative determination of the tissue distribution of A2B adenosine receptors are lacking, and thus the literature reports of tissue distribution are based on mRNA levels, which might or might not correspond to protein levels.27 Bladder P2Y receptors

Immunohistochemistry has identified P2Y1,2,4 subtypes in the urothelium of bladders from healthy cats, and shown that the P2Y2 subtype, localized to the apical urothelial cells, is significantly depleted in cats with feline interstitial cystitis (FIC). The P2Y4 subtype is immunolocalized to nerve bundles both in the submucosal plexus and between the bladder smooth-muscle bundles in healthy and FIC cats, whereas no P2Y receptor staining is evident in the smooth muscle.13 Although adenosine and AMP have been shown to relax the human bladder,28 there are no published reports at this time on the subtype specificity or cellular localization of adenosine and P2Y

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GLOSSARY PAIRWISE AMINO-ACID SEQUENCE IDENTITY When comparing the amino-acid sequence of a pair of receptor subunits, this indicates that the same amino acid is present at the same point in the sequence in both subunits HOMOMULTIMER A functional ligand-gated ion-channel receptor that is composed of multiple subunits of the same subtype, as opposed to a heteromultimer, which is composed of multiple subunits of different subtypes

receptors in the human urinary bladder. This is a significant gap in our knowledge, which is particularly important in light of the proposed active role of the urothelium in bladder sensory mechanisms.29,30 It has been shown that small, spindle-shaped cells (resembling myofibroblasts) isolated from collagen digests of the human urothelium have many of the characteristics of nerve cells, and respond to ATP with an inward current and large, transient increases in intracellular calcium.31 While it is not known which purinergic receptors are present on these cells in the human urothelium, similar cells in the guinea pig urothelium show a pharmacologic profile typical of P2Y receptors (ATP-induced currents mimicked by uridine triphosphate and ADP, but not α,β-methylene-ATP).32 Because these cells show an abundance of the gap junction protein connexin-43, they might serve as signaling cells for the transfer of information between the urothelium and bladder sensory nerves.30 Bladder P2X receptors

Since the initial isolation, in 1994, of complementary DNAs encoding P2X receptor subunits and subsequent development of selective antibodies for each of the seven P2X subunits, as well as knockout mice for several of these subunits, an explosion of information has significantly expanded our knowledge of receptor function in both normal and pathologic bladder physiology. The receptor subunits are 384–595 amino acids long, with 41–55% PAIRWISE AMINO-ACID SEQUENCE IDENTITY in the region of the two transmembrane domains and the intervening extracellular domain. All have two hydrophobic regions of sufficient length to form α-helical transmembrane areas, separated by the bulk of the polypeptide, and considerable evidence indicates that this loop forms the extracellular domain. There are multiple consensus sequences for N-linked glycosylation (ASN-X-Ser/Thr), and some degree of glycosylation is required for channel activity.33 Detailed knowledge of the molecular structure of the ligand-gated ion channel class of receptors has been obtained for the nicotinic acetylcholine receptor from studies into the electric organ of the Torpedo Ray. This specialized organ is almost entirely composed of nicotinic acetylcholine receptors, densely packed in partially crystalline arrays; this arrangement is

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particularly amenable to structural analysis.34 The structure of the nicotinic receptor consists of a pentameric arrangement of five subunits surrounding the ion channel in the central pore. One working hypothesis is that all receptors of this class have this same pentameric structure. Despite some biochemical evidence that P2X subunits might form trimers or hexamers, the structure might still be pentameric. Similar evidence of a hexameric structure was obtained for the mycobacterium tuberculosis largeconductance mechanosensitive channel before its pentameric structure following crystallization was confirmed.33 Coimmunoprecipitation studies of epitopetagged P2X subunits, expressed in pairs in human embryonic kidney cells (HEK293), have indicated which pairs of subunits have the potential to coassemble. Based on this approach, in which other subunits co-immunoprecipitated with an antibody to one subunit are detected with an antibody to the second subunit, P2X1 and P2X2 subunits coassemble with all subunits with the exception of P2X4 or P2X7 subunits (Table 1).33 Patch clamp studies of HEK293 cells that were transfected with each of the individual subunits shows that the ATP-induced current in P2X1 and P2X3 HOMOMULTIMERs desensitizes very rapidly (within milliseconds), while P2X2and P2X4 homomultimers desensitize more slowly (within seconds). In P2X7 homomultimers, currents are continuously induced throughout a 20 s exposure to ATP; in P2X5 homomultimers, the same exposure results in barely detectable currents. No appreciable ATP-induced current is observed with P2X6 transfected cells, which are not believed to form homomultimers.33 BLADDER ATP RELEASE

Neuronal release of ATP in guinea pig bladders was originally shown in 197835 and release of ATP from uroepithelial cells following bladder stretch was first shown in rabbit bladders in 1997.36 In vitro studies using strips of human bladder tissue have shown that ATP is released from the urothelial cells in response to mechanical stretch, and from bladder nerves in response to electric field stimulation.37 In addition, the amount of ATP released by human bladder strips increases with the age of the patient; this correlates with an age-related increase in the purinergic component of contraction and inversely correlates with both acetylcholine

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release and the cholinergic component of contraction.38 In vitro mechanical stretch also promotes ATP release in cultured uroepithelial cells, and ATP release is significantly greater in cells cultured from patients with interstitial cystitis, compared with control patients undergoing other pelvic surgery such as hysterectomy, pelvic floor reconstruction and prostatic brachytherapy.39,40 This stretch-induced ATP release in cultured interstitial cystitis urothelial cells is blocked by exposure to either heparin or dimethyl sulfoxide.41 Cultured uroepithelial cells from patients with benign prostatic hypertrophy also release more ATP than control cells do, and this release can be blocked by doxazosin.42 In conscious, freely moving rats, intravesical infusion of purinergic agonists (ATP or α,β-methylene-ATP) stimulate the micturition reflex,43 indicating that ATP released by the urothelium might play an autocrine role in initiation of the micturition reflex. Studies in rats have shown that urothelial ATP release induced by hypo-osmolarity is increased by both spinal cord injury44 and cyclophosphamide-induced bladder inflammation,44 and these increases can be prevented by pretreatment with botulinum toxin. In rabbit urothelium, purinergic agonists increase and antagonists inhibit pressure-induced membrane trafficking into the umbrella-cell layer.14 This stretch-induced insertion of cytosolic fusiform vesicles into the umbrella-cell apical plasma membrane was not observed by transmission electron microscopy in either P2X2 or P2X3 knockout mice.45 It is possible that development of drugs that inhibit ATP release might find clinical utility in the treatment of pathologic bladder conditions, including interstitial cystitis, and perhaps lowerurinary-tract symptoms resulting from bladder outlet obstruction. LOCALIZATION OF P2X RECEPTORS AND CHANGES WITH DYSFUNCTION

The literature on the localization of each of the seven P2X subunits in the bladder is summarized in Table 2. These studies used immunologic techniques, primarily immunohistochemistry, to define the location of the subunits. Obtaining a large number of completely normal human bladder specimens for these types of studies is not feasible; therefore, most investigators used macroscopically normal-appearing bladder tissue obtained from cancer cystectomy

Table 1 The coassembly potential of P2X subunits. P2X subunit

Subunits that coassemble with the subunit

Subunits that do not coassemble with the subunit

P2X1

P2X1 P2X2 P2X3 P2X5 P2X6

P2X4 P2X7

P2X2

P2X1 P2X2 P2X3 P2X5 P2X6

P2X4 P2X7

P2X3

P2X1 P2X2 P2X3 P2X5

P2X4 P2X6 P2X7

P2X4

P2X4 P2X5 P2X6

P2X1 P2X2 P2X3 P2X7

P2X5

P2X1 P2X2 P2X3 P2X4 P2X5

P2X6 P2X7

P2X6

P2X1 P2X2 P2X4 P2X5

P2X3 P2X6 P2X7

P2X7

P2X7

P2X1 P2X2 P2X3 P2X4 P2X5 P2X6

specimens, biopsies from cystoscopy for surveillance of low-grade malignancy or hematuria investigation, and bladder tissue obtained from pediatric patients during surgical correction of vesicoureteral reflux. Human bladder smooth-muscle stains positively with antibodies to P2X1,2,4 subunits. P2X1 and P2X4 subunits are localized to the periphery of smooth-muscle cells, and the P2X2 subunit is present throughout the cytoplasm.46–48 All seven subunits have been reported to be present in human bladder, colocalized with a proteoglycan, known as SYNAPTIC VESICLE PROTEIN 2 (SV2), found in the varicosities of parasympathetic neurons. P2X1–3,5 subunits, however, are colocalized with over 90% of the SV2 positive nerve varicosities, whereas the other subunits are only observed in less than 20% of SV2 positive nerve varicosities.47,49 Other investigators have

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GLOSSARY SYNAPTIC VESICLE PROTEIN 2 A protein located on the varicosities of peripheral parasympathetic nerves that serves as a marker for parasympathetic nerve varicosities

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Table 2 Immunolocalization of P2X receptor subunits and changes with bladder dysfunction. Receptor subunit

Urothelial cells

Bladder nerves

Smooth muscle cells

P2X1

Present throughout in cat, decreased in FIC13

Present in 98% of SV2-labeled varicosities,47,49 decreased to 3% in SU47

Present in cell periphery in control and DO46 Heavily labeled in areas well removed from parasympathetic nerves47 Strong staining in sarcolemma cell periphery48 Present in cat, decreased in FIC13

P2X2

Detected by Western blots, increased relative to Gα in IC but no change in mRNA50 Present throughout in cat, unchanged in FIC13

Increased in DO compared to control, decreased to control levels by BTX, co-localized with PGP 9.5 in lamina propria and between muscle bundles52,60 Present in 99% of SV2-labeled varicosities,47,49 decreased to 4% in SU47

Present throughout cytoplasm, increased in IDO46 Predominant subunit in cat13

P2X3

Strong labeling in basal region48 Detected by Western blots, increased relative to Gα in IC but mRNA is decreased in IC50 Colocalized with cytokeratin in cultured urothelial cells, increased by in vitro stretch in IC but not normal cells61 Present throughout in cat, unchanged in FIC13

Present in lamina propria and between muscle bundles46,60 Increased in NDO, decreased by RTX in clinical responders53 Present in 94% of SV2 labeled varicosities,47,49 decreased to 2% in SU,47 decreased to 0% in IDO49 Not co-localized with CGRP48

Not observed60 Not observed48 Present in cat13

P2X4

Present throughout in cat, unchanged in FIC13

Present in 15% of SV2-labeled varicosities,47,49 decreased to 4% in SU,47 increase to 36% in IDO49 Present in lamina propria and between muscle bundles in cat13

Present in cell periphery46 Not observed in cat13

P2X5

Present throughout46 Present throughout in cat, unchanged in FIC13

Present in 91% of SV2-labeled varicosities,47,49 decreased to 1% in SU,47 0% in IDO49 Not observed46

Not observed46 Not observed in cat13

P2X6

Not observed46 Present throughout in cat, unchanged in FIC13

Present in 18% of SV2-labeled varicosities,47,49 decreased to 0.4% in SU,47 increased to 33% in IDO49 Not observed46

Not observed46 Not observed in cat13

P2X7

Not observed46 Present throughout in cat, unchanged in FIC13 Present in guinea pig cell nuclei and umbrella-cell apical plasma membrane62

Present in 6% of SV2-labeled varicosities,47,49 decreased to 5% in SU,47 increased to 67% in IDO49 Not observed46

Not observed46 Present in 85% of guinea pig cell nuclei62

Unless specifically indicated, all studies refer to human bladder tissue. BTX, botulinum toxin; CGRP, calcitonin gene related polypeptide; DO, detrusor overactivity; FIC, feline interstitial cystitis; IC, interstitial cystitis; IDO, idiopathic detrusor overactivity; mRNA, messenger RNA; NDO, neurogenic detrusor overactivity; PGP, protein gene product; SU, sensory urgency (urgency symptoms without bladder contractions during filling cystometry); SV2, synaptic vesicle protein 2 (marker for parasympathetic varicosities).

not observed P2X5–7 subunits in human bladder nerves, but have observed P2X3 immunoreactivity in nerve bundles running beneath the urothelium and in between the muscle bundles.46 Immunoreactivity in human bladder urothelial cells has been reported for the P2X2,50 P2X341,48,50 and P2X5 subunits.46 In infants aged 4–9 months, no P2X subunits have been observed colocalized with SV2 on

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the nerve varicosities in the detrusor. Labeling of varicosities for P2X subunits commences in the 10–18-month age group, but is variable, and by 2 years of age the majority of the varicosities are labeled with P2X1–3,5 antisera. In 18 adult female patients with urodynamically demonstrated idiopathic detrusor overactivity (IDO) and incontinence, no P2X3 or P2X5 subunits colocalized with SV2-labeled RUGGIERI APRIL 2006 VOL 3 NO 4

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varicosities, whereas P2X4,6,7 subunits more commonly colocalized with SV2 than control bladder tissue. This pattern of colocalization of P2X subunits in patients with IDO is similar to the pattern found for the 4–6 month old neonatal group that lack bladder control. This prompted the investigators to propose that the pathophysiology of IDO might be manifested as a loss of inhibition of acetylcholine release at the varicosities.49 No direct evidence exists for this, however, and the mechanism for how P2X receptor activation could lead to inhibition of acetylcholine release is unclear. Activation of ligand-gated ion-channel receptors such as P2X receptors, which open a cation channel and thereby depolarize the nerve varicosity, would be expected to increase acetylcholine release from that same varicosity. As tetrodotoxin (which blocks sodium channels and thus most nerve-mediated events that depend on action potentials) potentiates the contractile effect of both α,β-methyleneATP and β,γ-methylene-ATP in human bladder strips,51 it is likely that an inhibitory substance is released by stimulation of bladder purinergic receptors. In patients with sensory urgency (increased bladder sensation during filling cystometry but no overactivity), colocalization of P2X1–6 with SV2 was restricted to less than 2% of varicosities. The authors of this study concluded that since these patients do not have overactive bladders, the loss of P2X1,2,4,6 at the neurotransmitter release sites is associated with a recovery of the inhibitory control over acetylcholine release to the muscle muscarinic receptors.47 Implicit in this is the assumption that the bladder sensory nerves that induce the increased sensation of urgency at lower bladder volumes in these patients with sensory urgency do not use P2X receptors to do so. Other investigators have shown increased P2X2 subunits in the detrusor muscles of patients with detrusor overactivity. Following intradetrusor injections of botulinum toxin, P2X2 immunoreactivity decreases to control values.52 Likewise, in five patients with neurogenic detrusor overactivity who had a positive clinical response to intravesical instillations of resiniferatoxin, bladder P2X3 nerve density also decreased to control values.53 P2X3 immunoreactivity remained elevated in the eight patients who did not have at least a 25% improvement in two or more symptoms (micturition episodes,

urgency episodes, incontinence episodes or bladder capacity).53 In three patients with interstitial cystitis, compared with seven patients that had either prostate or bladder cancer, western blots showed increased urothelial P2X2 subunits (normalized to the G-protein subunit Gα) and greatly increased P2X3 subunits in the patients with interstitial cystitis. The mRNA for P2X2 (normalized to the ribosomal gene L3) was unchanged, but mRNA for P2X3 subunits was actually decreased in the interstitial cystitis patients.50 These studies demonstrate that changes in the cellular location and function of bladder purinergic receptors are probably involved in development of continence during early childhood, and might also play a role in generating symptoms of bladder dysfunction in adulthood, such as incontinence, urgency and perhaps even pelvic pain. Thus, purinergic innervation of the urinary bladder is a prime target for the development of novel pharmacologic agents for the treatment of conditions such as overactive bladder, urinary urgency and painful bladder syndrome (interstitial cystitis and chronic nonbacterial prostatitis). PHARMACOLOGY OF BLADDER PURINERGIC RECEPTORS

There are currently no P2X-specific agonists or antagonists approved for clinical use. This is probably because there are a number of formidable challenges in the development of such drugs. As discussed earlier, it is not known whether three, four, five or six P2X subunits are required to form a functional receptor channel. Nor is it known whether the functional receptors on bladder cells are composed of homomultimers or heteromultimers of two or more of the different subunits that have been localized to bladder muscle, nerve or urothelial cells. Thus, there is no reliable in vitro screening method to search large libraries of chemicals. The pharmacology of the P2X receptors that are present naturally in bladder cells is different from the pharmacology of the recombinant individual subunits expressed as homomultimers in oocytes or other cell types (Table 3). The rank order potency of purinergic agonists for stimulating contraction of strips of human detrusor muscle in vitro is α,β-methylene-ATP > β,γ-methylene-ATP > ATP > ADP.51 Since this rank order does not correspond to any of the rank order potencies listed in Table 3 for

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Table 3 Pharmacology of bladder P2X receptor homomultimers expressed in Xenopus oocytes or other cell types. Subunit

Cellular location

Agonists

Antagonists

Signal transduction

P2X1

Nerve varicosities Smooth muscle periphery

α,βmeATP = ATP = 2meSATP

TNP-ATP IP5I NF023

Intrinsic cation channel Rapid desensitization

P2X2

Urothelium Nerve varicosities Smooth muscle cytoplasm

ATP ATP S 2meSATP>>α,βmeATP

Suramin PPADS

Intrinsic ion channel (particularly Ca++) Slow desensitization

P2X3

Basel urothelial cells Nerve varicosities and bundles

2meSATP ATP α,βmeATP

TNP-ATP Suramin PPADS

Intrinsic cation channel Rapid desensitization

P2X4

Small number of nerve varicosities Muscle cell periphery

ATP>>α,βmeATP



Intrinsic ion channel (particularly Ca++) Slow desensitization

P2X5

Urothelium Small number of nerve varicosities

ATP>>α,βmeATP

Suramin PPADS

Intrinsic ion channel

P2X6

Small number of nerve varicosities

Does not form homomultimer



Intrinsic ion channel

P2X7

Very small number of nerve varicosities

BzATP>ATP 2meATP>>α,βmeATP

KN62 KN04 Coomassie brilliant blue

Intrinsic cation channel large pore Prolonged activation

Adapted from Burnstock.54 α,βmeATP, α,β-methylene-ATP; 2meSATP, 2-methylthioATP; BzATP, 2',3'-O-(4-benzoyl-benzoyl) ATP; CPX, 8-cyclopentyl-1,3-dipropylxanthine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; KN04, N-[1-[N-methyl-p-(5-isoquinolinesulfonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinolinesulfonamide; KN62, 1-[N,O-bis(1,5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; IP5I, diinosine penta phosphate; NF023, [8,8'-[carbonyl bis(imino-3,1phenylenecarbonylamino)] bis-(1,3,5-naphthalenetrisulfonic acid); PPADS, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid; TNP-ATP, 2',3'-O(2,4,6-trinitrophenyl) adenosine 5'-triphosphate; XAC, xanthine amine congener.

homomultimers of P2X subunits transfected in cells, it is possible that detrusor purinergic receptors that mediate bladder contraction are heteromultimers consisting of some combination of P2X1 and P2X2. It is unlikely that these heteromultimers would contain P2X4 subunits, because cotransfection studies indicate that P2X4 subunits do not coassemble with P2X1 or P2X2 subunits. Bladder tissue does, however, contain ectoenzymes that breakdown ATP, rendering it less potent than in cells transfected with the individual subunits where ectoATPases are less important.54 Thus, the potency of ATP might be lower when assessed in bladder tissue than in transfected cells, not because of differences in affinity of the receptors, but because the ATP concentration is reduced by ecto-ATPases during the assay. Adenosine 5’[β-thio]diphosphate induces a much more sustained contraction of human bladder strips than either ATP or α,β-methylene-ATP do. These contractions are blocked by p-chloromercuribenzenesulfonic acid, but α,β-methyleneATP-induced contractions are unaffected. This discovery prompted the investigators to

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propose the existence of two different contractile purinergic receptors in human bladder.55 It is tempting to speculate that this other purinergic receptor that mediates human bladder contractions is a homomultimer of P2X4 subunits. There has been some progress in development of a subunit-selective antagonist for P2X2 homomultimers and P2X2/3 heteromultimers. The compound, A-317491, has been shown to dose dependently and stereo selectively block calcium flux in astrocytoma cells transfected with human or rat P2X3 subunits or with combinations of rat or human P2X2 and P2X3 subunits (but not P2X4, P2X7 or P2Y2 receptors) with 10–100 nM affinity. In several rodent pain assays, A-31749 dose dependently reduced hyperalgesia. These assays included the chronic constriction injury assay, L5/L6 nerve ligation assay, chronic thermal hyperalgesia induced by hind-paw injection of complete Freund’s adjuvant, and the mouse abdominal constriction assay.56 In rats, A-317491 does not readily cross the blood–brain barrier, as evidenced by a brain to plasma concentration ratio of 0.008 1 h

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following subcutaneous injection.57 In addition, the suramin derivative NF449 has been developed to competitively block ATP-induced or α,β-methylene-ATP-induced inward currents in Xenopus cells transfected with rat P2X1 or combinations of P2X1 and P2X5 subunits with subnanomolar potency. In order to to block homomeric P2X3 or heteromeric P2X2 or P2X3 receptors, concentrations of NF449 need to be threefold to fourfold higher than for P2X1 or P2X5.58 CONCLUSION

Because purinergic agonist-induced contractions are completely absent in P2X1 receptor knockout mice,59 it is likely that the primary P2X receptor in human detrusor-muscle cells that mediates contraction is either a P2X1 homomultimer or composed predominately of P2X1 subunits. Thus, development of antagonists selective for P2X receptors that are composed predominately of P2X1 subunits might be clinically useful for treatment of detrusor overactivity. Because there is no reliable method for monitoring sensory nerve activity in human bladder strips in vitro, the rank order potency for stimulation of purinergic receptors in bladder sensory nerves is unknown. This is also true for the purinergic receptors localized to the bladder urothelium whose function is also unknown. Studies in P2X3-knockout mice clearly demonstrate that this subunit is required for bladder sensory response to ATP. Bladder capacity is nearly doubled in P2X3-knockout mice, and there are virtually no micturition contractions in response bladder distension.16 Firing of pelvic-nerve afferents is much less sensitive to bladder distension and does not increase in response to intravesical ATP or α,βmethylene-ATP, but does increase in response to intravesical capsaicin administration (similar to wild-type mice). Bladder distension induces an equivalent ATP release from the urothelium in P2X3 knockout mice as in wild-type controls.15 If these results in knockout-mice models could be extrapolated to humans, and because P2X3 subunits are the predominant subunit found in human bladder nerve profiles, development of antagonists selective for P2X receptors that consist of primarily the P2X3 subunit would probably be clinically beneficial in the treatment of bladder overactivity, urgency and perhaps even chronic pelvic pain syndromes.

KEY POINTS ■ Decreased ecto-ATPase in unstable bladders leading to an increased contractile effect of released ATP might be a mechanism of bladder instability in some patients ■ Between 50 and 80 years of age there is an age-associated increase in purinergicmediated bladder contraction and ATP release and a decrease in cholinergic-mediated bladder contractions and acetylcholine release, which might be one of the mechanisms for the increased incidence of detrusor overactivity in the elderly ■ The functional purinergic receptors on bladder smooth-muscle cells that mediate contraction are either P2X1/P2X2 heteromultimers or P2X1, P2X2 and/or P2X4 homomultimers; development of antagonists to these P2X receptors is likely to be clinically useful in treatment of detrusor overactivity ■ Results from knockout-mice models indicate that the purinergic receptor that mediates bladder sensation and nociception is either a P2X3 homomultimer or a heteromultimer predominantly containing the P2X3 subunit; development of antagonists to this P2X receptor is likely to be clinically useful in the treatment of bladder overactivity, urgency and perhaps even chronic pelvic pain syndromes References 1 Abrams P et al. (2003) The standardisation of terminology in lower urinary tract function: report from the standardisation sub-committee of the International Continence Society. Urology 61: 37–49 2 Cowan WD and Daniel EE (1983) Human female bladder and its noncholinergic contractile function. Can J Physiol Pharmacol 61: 1236–1246 3 Dean DM and Downie JW (1978) Contribution of adrenergic and “purinergic” neurotransmission to contraction in rabbit detrusor. J Pharmacol Exp Ther 207: 431–445 4 Burnstock G (2001) Purinergic signalling in lower urinary tract. In Handbook of Experimental Pharmacology, 423–515 (Eds Abbracchio MP and Williams M) Berlin: Springer–Verlag 5 Levin RM et al. (1986) Functional effects of the purinergic innervation of the rabbit urinary bladder. J Pharmacol Exp Ther 236: 452–457 6 Ruggieri MR et al. (1990) Bladder purinergic receptors. J Urol 144: 176–181 7 Bayliss M et al. (1999) A quantitative study of atropineresistant contractile responses in human detrusor smooth muscle, from stable, unstable and obstructed bladders. J Urol 162: 1833–1839 8 Husted S et al. (1983) Direct effects of adenosine and adenine nucleotides on isolated human urinary bladder and their influence on electrically induced contractions. J Urol 130: 392–398 9 Sjogren C et al. (1982) Atropine resistance of transmurally stimulated isolated human bladder muscle. J Urol 128: 1368–1371 10 Palea S et al. (1993) Evidence for purinergic neurotransmission in human urinary bladder affected by interstitial cystitis. J Urol 150: 2007–2012

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Competing interests The author declared he has no competing interests.

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