What's known on the subject? and What does the study add?
In the urinary bladder, histological studies suggest a network of functionally connected interstitial cells of Cajal (ICCs) are located between the urothelium and sensory nerve endings, which might transfer signals directly between them. Purinergic P2X3 receptors may play roles in the micturition reflex pathway, and its expression profiles in ICCs could be altered in urinary bladder dysfunction.
The present experiments showed a novel time-dependent P2X3 receptor up-regulation in ICCs in an experimental rat model of partial bladder outlet obstruction.
To investigate the expression and electrophysiological characteristics of purinergic P2X3 receptors in interstitial cells of Cajal (ICCs) at different time points after partial bladder outlet obstruction (PBOO) in rats.
Materials and Methods
In all, 48 female Sprague-Dawley rats were randomly divided into four groups: 4, 6 and 8 week PBOO groups and sham-operated controls. At 4 weeks after surgery, cystometry was performed to investigate bladder function in vivo. Subsequently, the rats were humanely killed at 4, 6 or 8 weeks and the bladders were harvested for measurements.
P2X3 expression in ICCs of bladder was investigated by immunofluorescent staining.
The level of P2X3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR).
Inward currents in corresponding ICCs after PBOO were investigated electrophysiologically.
Cystometrography showed a valid increase in maximum detrusor pressure in rats subjected to PBOO. The bladder weight in the PBOO group was significantly higher than that in the control group.
In contrast to sham rats, there was a significant increase in the intensity of P2X3 staining in the ICCs in all PBOO groups. C-kit labelled isolated ICCs were strongly stained with the P2X3 antibody.
RT-PCR showed that the expression of P2X3 mRNA was significantly up-regulated at 4, 6 and 8 weeks in the ICCs from the PBOO rats.
In the ICCs, the mean peak amplitude of inward currents was significantly increased in the PBOO groups compared with the control group.
The expression of P2X3 receptors showed a time dependent up-regulation in the ICCs of the bladder in rats with PBOO.
PBOO induced the potentiation of P2X3 receptors function, as evidenced by α, β-methylene ATP-enhanced inward currents in the ICCs of the rat bladder.
Partial BOO (PBOO) results in changes in bladder structure and function that include detrusor hypertrophy, elevated detrusor contractile pressure, and detrusor instability, which can lead to an overactive bladder (OAB) . In recent years, the role of the interstitial cells of Cajal (ICCs) in urgency and the pathophysiology of OAB has become the focus of intense interest. These special mesenchymal cells, named myofibroblasts, ICCs, or interstitial cells (ICs) were first recognised in the gut . Myofibroblasts are a unique group of smooth-muscle-like fibroblasts that have similar morphological characteristics and function regardless of their tissue location . The selective identification and localisation of the ICCs, has been greatly facilitated by the discovery of their expression of the C-kit receptor. These cells play important physiological roles in the gastrointestinal tract, including the generation of electrical slow-wave activity, the facilitation of active propagation of electrical phenomena and the mediation of neurotransmission between enteric nerves and smooth muscle cells (SMCs) [4, 5]. In the urinary bladder, histological studies suggest that a network of functionally connected ICCs are located between the urothelium and sensory nerve endings, and might transfer signals directly between them [6-8]. Kubota et al.  reported an increased population of ICCs in guinea-pigs with PBOO, which showed bladder overactivity on cystometry. Studies have shown that the numbers of ICCs in patients with OAB are significantly higher than normal. Inhibition of ICCs activity can reduce the spontaneous activity of the detrusor muscle in patients with OAB, and can improve capacity and compliance in guinea-pig bladders . In addition, ultrastructural features of ICCs changed in the PBOO model. The quantitative or qualitative changes in ICCs may account for the pathologically increased signal transmission between either homogenous or heterogeneous populations of cells in the bladder wall.
Adenosine triphosphate (ATP) was identified as a motor neurotransmitter in the urinary bladder of the guinea-pig in 1972 and recently recognised as a sensory neurotransmitter in the rabbit bladder [11-13]. There is substantial evidence supporting a functional role for ATP in mechanotransduction in the urinary bladder [14, 15]. ATP is produced and released from the urothelium when it is exposed to stretch by physical or chemical methods [16, 17]. There are seven subtypes of purinergic (P2X) ATP receptor . P2X3 is most closely involved in the primary sensory effects of ATP, as it plays a key role in the pain signalling mechanism. P2X3 receptor-knockout mice exhibit bladder hyporeflexia on cystometry with decreased voiding frequencies, increased bladder capacities and voided volumes, but normal bladder pressures . Numerous studies have shown that P2X3 receptors play a crucial role in nociception and mechanosensory transduction . But few methods have been available to define the relative contribution of these receptors to specific sensory behaviours in the ICCs. ATP and α, β-methylene ATP (α, β-meATP) can directly stimulate the micturition reflex in conscious rats . P2X3 receptors are rapidly desensitising, activated by α, β-meATP . Thus, P2X3 receptors may play roles in the micturition reflex pathway, and its expression profiles could be altered in urinary bladder dysfunction. Our previous work showed that a network of suburothelial ICCs had close associations with afferent nerve fibres, and that P2X3 receptor immunoreactivity was extensively distributed in the bladder suburothelial ICCs . The ICCs that are present in cases of bladder overactivity are likely to produce abnormal sensory signals mediated by the P2X3 receptors. However, the underlying detailed mechanisms remained to be elucidated. In the present study, we investigated the changes in expression and function of P2X3 receptors in the bladder ICCs of rats with PBOO. And the relationship between PBOO and changes in P2X3 receptors was determined.
Materials and Methods
The studies were performed on 48 female Sprague–Dawley rats weighing 240–260 g. They were obtained from the Laboratory Animal Centre of our university (Xi'an, China). The rats were housed in a temperature-controlled room under a 12-h light/dark cycle and allowed free access to food and water. The experimental protocols were approved by the Local Ethics Committee. The rats were divided randomly into four groups: 36 underwent surgery (4-, 6- and 8-week PBOO groups), and 12 were sham-operated controls. PBOO was induced as described previously . Briefly, the rats were sedated with ketamine (15 mg/kg) and xylazine (5 mg/kg), administered i.p., and then prepared under sterile conditions. The abdomen was opened through a midline suprapubic incision and the bladder and the proximal urethra were exposed. Urethral obstruction was produced by tying a ligature of 3-0 silk around the urethra and a catheter was inserted with an external diameter of 1.10 mm; the catheter was then removed. Sham surgery was performed in an identical manner, except no ligature was placed around the urethra.
At 4 weeks after surgery, cystometry was performed under urethane anaesthesia (1.0 g/kg, s.c.) to investigate bladder function in vivo. A suprapubic midline laparotomy was made to expose the bladder, and a 25-G needle connected to polyethylene tubing was inserted into the bladder through the bladder dome. The tubing was connected to a pressure transducer and a Harvard syringe pump via a three-way stopcock to record intravesical pressure and to infuse saline into the bladder. After the bladder had been emptied, cystometrography was performed, with saline infused at a rate of 0.04 mL/min. The contraction interval and contraction pressure were recorded in each rat using a polygraph (Grass 7D; Grass Institute Co, Xi'an, China). In each rat, three to five voiding cycles were recorded and the means of the voids were calculated. After the experiments, the rats were killed by cervical dislocation. The bladders were excised at the level of ureteric orifices and weighed.
The bladders were removed and placed in a Ca2+-free medium (mm): NaCl 105.4; NaHCO3 22.3, KCl 3.6, MgCl2 0.9, NaH2PO4 0.4, HEPES 19.5, glucose 5.4, sodium pyruvate 4.5, pH 7.4. Full-thickness, longitudinal strips were dissected from one half of the bladder, placed in embedding medium (Cryo-m-Bed, Bright, UK) and stored in liquid N2. For the tissue immunofluorescence analysis, 15-μm frozen sections were cut, collected on poly-l-lysine-coated slides and fixed with 4% (w/v) paraformaldehyde in PBS for 30 min at room temperature.
ICCs were isolated from rat detrusor muscle tissue as described previously . The cells were dispersed from 1-mm3 pieces of fresh rat detrusor in Ca2+-free Hanks solution. The solution had the same ionic composition as above, with the following additions: 10 mg collagenase, 10 mg trypsin inhibitor, 10 mg BSA and 1 mg protease per 5 mL for 15 min at 35 °C. Cells that were healthy in appearance were washed in enzyme-free Ca2+-free Hanks solution. The residual cells were transferred to an uncoated culture dish and re-suspended in RPMI-1640 culture medium (Invitrogen, UK) with 4% (w/v) fetal bovine serum, and allowed to settle for 2 h at 37 °C in a 5% CO2 atmosphere. This final step was important to allow the cells to adhere to the culture plate so that they could be washed and exposed to antibody solutions without being washed away. For the immunofluorescence analysis, the culture medium was discarded after the 2-h incubation, and the cells fixed with 4% (w/v) paraformaldehyde in PBS for 30 min at room temperature.
Freshly dispersed cells from the bladder were placed in separate glass-bottomed dishes on a Nikon Diaphot inverted microscope equipped with fluorescence and phase-contrast optics. In all, 40–60 ICCs were selected for reverse transcription (RT)-PCR analyses. The cell selection procedure was performed as described previously .
To perform the patch-clamp recordings, the detrusor layer was disrupted to yield isolated SMCs. Other processes were mentioned above. Two main cell types could be seen under a light microscope: (i) large, round urothelial cells and (ii) a layer of ovoid or spindle-shaped cells, with or without one or more spindle-like structures. The latter cells were used for experimental recording. Some of the latter cells were stained for C-kit .
For immunofluorescence, tissue slices and samples of cells that would otherwise have been used for experimental recording were washed in PBS and nonspecific sites blocked by adding PBS with 1% BSA at room temperature for 1 h. Double-labelling for purinoceptors and C-kit was performed by applying primary rabbit polyclonal antibodies for P2X3 (1:500 dilution; Sigma, USA) and mouse monoclonal antibody to C-kit (1:100 dilution; Sigma, USA) in PBS with 1% BSA for 48 h at 4 °C. A thorough wash in PBS secondary labelling was carried out with Alexa 568 or 488-labeled antibodies (1:500 dilution; Molecular Probes) conjugate for purinoceptors and C-kit were applied at room temperature for 2 h. Negative controls were obtained by omitting the primary antibody from the first incubation stage. All cells were subsequently examined with a confocal laser scanning microscope (FV1000; Olympus Corp). C-kit and P2X3 immunoreactivity in controls and experimental groups was evaluated using a confocal laser scanning microscope (488 nm excitation and 510 nm emission filter) with a ×40 objective. Images of immunofluorescence labelling from tissue slices were taken with a Leica DC 200 digital camera (Leica, Switzerland) attached to a Zeiss Axioplan microscope (Zeiss, Germany).
For RT-PCR, total RNA was isolated from ICCs using a TRIzol isolation method. The total RNA was not contaminated when the ratio of A260 to A280 exceeded 1.8. Complementary DNA (cDNA) was reverse transcribed from 10 μL of total RNA in a reaction volume of 25 μL. The following were added: diethylpyrocarbonate (DEPC) 4.875 μL, 5×buffer 5 μL, RNasin 0.625 μL, deoxyribonucleotide triphosphate (dNTP) 1.5 μL, oligo-dT 2 μL and Moloney Murine Leukaemia Virus (MMLV) 1 μL. RT reactions were conducted at 37 °C for 1 h. PCR was performed in a 25 μL reaction volume using 4 μL cDNA, 1 μL each of P2X3 sense primer 5′-CTGTATATCAGACTTCTTCACCTACGA-3′ and antisense primer 5′-GATTGGAGTGGCTGTTCCTGTATT-3′, and 1 μL of β-actin sense primer 5′-TAAAGACCTCTATGCCAACACAGT-3′ and antisense primer 5′-CACGATGGAGGGGCCGGACTCATC-3′. Amplification took place in a PCR thermocycler using the following protocol: denaturation at 94 °C for 5 min, repeated cycles of denaturation at 94 °C, annealing at 53 °C and extension at 72 °C, each for 45 s, with final extension at 72 °C for 10 min. A total of 35 cycles were used. The amplification products (8 μL) were examined by electrophoresis on a 1% agarose gel and visualised using ethidium bromide under ultraviolet light. We used the ratio of P2X3 absorbance to β-actin absorbance as the final result. All chemicals were from Sigma Co, unless otherwise stated.
To investigate whether P2X3 receptors function was altered after PBOO, we measured the electrophysiological responses to α, β-meATP, an agonist for P2X1 and P2X3 receptors . Cells were continuously superfused with control solution containing (in mm): 152 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES; pH 7.4 adjusted with NaOH. Recordings were made using patch-type pipettes (3–4 MΩ) made from borosilicate glass and filled with the following solution (in mm): 140 KCl, 2 MgCl2, 0.5 CaCl2, 2 ATP-Mg, 2 GTP-Li, 20 HEPES, 5 ethylene glycol tetraacetic acid (EGTA); pH 7.2 adjusted with KOH. An Axopatch 700-B system (Axon Instruments) was used for experiments: data were recorded via an analogue-digital converter (Digidata 1200, Axopatch version 9, Axon Instruments) at 4 kHz and filtered with a low-pass filter with a threshold frequency of 3 kHz. Cells were maintained at a holding potential of –60 mV for experiments, as this is close to the average resting potential . Ionic currents were normalised to cell capacitance. The agonist α, β-meATP (10 μm) was applied for 5 s to record the current responses in ICCs by rapid solution changer system (Perfusion Fast-Step System SF-77B, Warmer Instruments, Xi'an, China). The results were analysed using pClamp and GraphPad Prism (version 2.01) software.
All in vitro experiments were done in triplicate. Numerical data were expressed as the mean (sd). ANOVA was used to determine the differences in the means among the various treatment groups followed by post hoc Dunnett-t test. A P < 0.05 was considered to indicate statistical significance.
Two rats died as a result of UTI. At 4 weeks after the induction of PBOO, cystometrography showed a significantly higher maximum detrusor pressure in the PBOO group rats compared with the control rats, at 13.1 (2.9) and 10.2 (2.7) mmHg, respectively (P < 0.05). The contraction interval was significantly shorter in the PBOO groups, at 4.1 (1.8) min, than in the control group, at 8.6 (2.4) min (P < 0.05). The bladder weight in the PBOO group was significantly higher than that in the control group, at 234.8 (69.9) vs 91.6 (7.1) mg (P < 0.05).
A layer of C-kit-positive staining ICCs were found double-labelled with P2X3 and these spindle-shaped ICCs were distributed mainly in the suburothelium of the bladder. The P2X3 receptors were mainly expressed in the nucleus of the ICCs. P2X3 immuno-labelling was concentrated in a reticular meshwork of spindle-like C-kit-positive cells, which extended the branching processes within the matrix and along the basal margins of the urothelium. Tissue samples collected from PBOO rats showed a consistent pattern of P2X3 receptors expression. In contrast to normal rats, there was a significant increase in P2X3 staining in PBOO ICCs in all tissues sampled, being more obvious at 6 and 8 weeks (Fig. 1A). In addition, the immunoreactivity of P2X3 receptors increased in a time-dependent manner (Fig. 1B). P2X3 immunoreactivity was quantified with an image-analysis system.
Samples of cells used for P2X3 receptors immuno-labelling showed labelling for C-kit ligand and were double-labelled for P2X3 and C-kit using fluorescent probes as mentioned above. Cells labelled with the antibody to C-kit had intense staining in the entire cell, except for the nucleus. P2X3 was expressed in the entire cell and was more highly expressed in the nucleus than in other locations. Overlay shows that the two markers were not exactly co-located, as they occurred at different locations in the cell, but were present throughout the cell images (Fig. 2A). In addition, higher resolution views of the 8-week PBOO bladders showed remarkable P2X3 expression in the cell membrane of ICCs (Fig. 2B, arrow indicated). There was a higher level of P2X3 receptor immunoreactivity in the ICCs of the PBOO groups than in those of the control group (Fig. 2C). P2X3 immunoreactivity was quantified with an image-analysis system; 10 cells were used for each sample.
A time-dependent increase in P2X3 mRNA was examined using RT-PCR and found to occur in the PBOO groups (Fig. 3A,B).
Under a voltage clamp at a holding potential of –60 mV, the ICCs generated spontaneous inward currents. Treatment with 10 μm α, β-meATP in cultured ICCs increased the amplitude of inward currents in a dose-dependent manner (Fig. 4A). As shown in Fig. 4B, the mean peak amplitude of inward currents was significantly increased in the PBOO compared with the control group. To determine whether the increased α, β-meATP affinity for P2X3 receptors lead to increased ICCs responses to α, β-meATP in PBOO ICCs, the current level of the α, β-meATP-enhanced peak inward currents was plotted as a function of concentration (Fig. 4C). The Hill equation was used to fit a curve to the means to obtain a concentration–response function. The dose–response curves were constructed by averaging α, β-meATP-enhanced peak amplitudes from ICCs from control and PBOO rats, respectively. The α, β-meATP half maximal effective concentration (EC50) values were 9.93 μm in control ICCs and 9.12, 8.37, 8.15 μm in PBOO 4-, 6-, 8-week ICCs and not significantly different. Thus, the changes in α, β-meATP affinities for P2X3 receptors in ICCs were not significant.
Interest in purinergic receptors in both the normal and the pathological bladder has increased, as they may provide new drug targets to relieve bladder symptoms. To our knowledge, this is the first study to show that the P2X3 receptors are up-regulated in the ICCs of the rat bladder with PBOO. ICCs have been identified in the urinary tract, including the renal pelvis, ureter, bladder and urethra, by their immunoreactivity against filaments of vimentin, a marker for cells of mesenchymal origin, and the proto-oncogene C-kit . In the bladder, ICCs are distributed throughout the bladder wall, in the suburothelial region, in the lamina propria and at the margins of the detrusor smooth muscle bundles . The ICCs in the suburothelial layer are located close to both the urothelium and nerves. Their potential ability to act as a functional electrical syncytium, and their response to ATP, place them in an ideal setting to act as modulators of mechanosensory process .
A hypothesis has been proposed for differential purine-mediated mechanosensory transduction in the visceral organs. Endogenous ATP is released during distension from epithelial cells and acts on P2X3 or P2X2/3 receptors on subepithelial sensory nerves initiating impulses via the spinal cord to pain centres in the brain. Supporting evidence for this comes from studies of gut , ureter , and bladder . P2X receptor-mediated transduction mechanisms in bladder afferent pathways seem to play important roles in the control of bladder function under normal and pathological conditions . The mechanism by which ATP elicits detrusor responses has not been fully elucidated. It has been suggested that the purinergic system is important in initiating detrusor contraction and voiding. Furthermore, exogenous ATP activates several types of bladder afferent and sensitises their mechanosensory responses [33, 34]. The up-regulation of P2X3 receptors has been shown in cultured urothelial cells from patients with interstitial cystitis during in vitro stretching . An increased density of P2X3-expressing nerve fibres has also been found in the pathophysiology of human neurogenic detrusor overactivity . Thus, P2X3 receptors may play a crucial role in peripheral pain responses and afferent pathways that control urinary bladder volume reflexes, as well as those that regulate micturition reflex excitability .
Several studies have shown that the afferent nerve fibres in the suburothelial plexus in the mouse and human bladder are immunoreactive to P2X3 antibody [13, 37]. However, Elneil et al.  showed that P2X3 receptors were not found on calcitonin gene-related peptide (CGRP)-containing nerves in human and rat bladder. Moreover, based on our previously published paper, immunoreactivity of P2X3 receptors was not distributed on any of the suburothelial afferent nerve fibres including CGRP, substance P and isolectin B4-containing nerves in the bladder. In addition, that study showed that there was a strong immunoreactivity for P2X3 receptors in the suburothelial ICCs . In the present study, we characterised the expression of P2X3 in ICCs from a rat model of surgically induced PBOO. P2X3 receptor immunoreactivity was greater in the ICCs from the PBOO groups than in those from the control group. We found that the level of P2X3 mRNA was increased in ICCs after 4, 6 and 8 weeks in the PBOO model. These results suggest that PBOO induced a time-dependent increase in P2X3 receptors expression. To our knowledge, there is no report on a time-dependent increase in the expression of this receptor subtype in ICCs, and the significance of this finding is unknown at present.
In addition to an increase in the P2X3 gene transcription level or number of P2X3 receptors-labelled ICCs, PBOO induced the potentiation of P2X3 receptors function, as evidenced by α, β-meATP-enhanced inward currents in the ICCs. The mean peak amplitude of inward currents was significantly increased in the PBOO compared with the control group. As the EC50 value for α, β-meATP does not significantly change in ICCs, the potentiation cannot be attributed to an increased in the affinity of α, β-meATP for its receptors. This enhanced response could lead to the sensitisation of ICCs under pathological conditions. α, β-meATP is an agonist for P2X1, P2X3 receptors . AS there is no expression of P2X1 receptors in ICCs , the ICCs inward currents enhanced by α, β-meATP, was thought to be mediated by P2X3 receptors.
It was reported that P2X3 receptors were involved in the normal physiological regulation of afferent pathways controlling volume reflexes in the urinary bladder . Cockayne et al.  reported a significantly decreased micturition frequency and increased bladder capacity in mice that lacked P2X3 receptors. They used cystometry to analyse voiding reflexes in wild-type and P2X3-knockout mice. There was no accompanying change in baseline or voiding bladder pressures or in the density of sensory neurone innervation. These results led investigators to propose a generalised role for P2X3 receptors in sensing internal organ distension .
As P2X3 purinoceptor immunoreactivity was shown to be located on ICCs, a possible role for suburothelial ICCs might be to act as an intermediary stage in the ATP-induced sensory transduction process of bladder filling [6, 40]. Cheng et al.  reported that P2X3 receptors were involved in ATP signalling in suburothelial ICCs. Furthermore, these cells were able to register bladder fullness very sensitively, which predisposes them for modulating afferent bladder signalling in normal and pathological conditions. Adult patients with OAB were treated with injections of the resiniferatoxin (RTX), which lead to the reduction of P2X3 expression in the suburothelial space . The reduced level of P2X3 receptors correlated with improvement in urgency sensations. These findings underline that P2X3 receptors play important roles in the mechanosensory regulation of urinary bladder function . Biers et al. reported increased numbers of C-kit-positive ICCs in overactive human bladder samples compared with normal tissues and showed a greater inhibitory effect of imatinib mesylate, a selective inhibitor of C-kit receptor tyrosine kinase, on detrusor contractions in samples from overactive bladder . However, the changes in P2X3 receptors in ICCs in the pathogenesis of PBOO-induced and idiopathic OAB has seldom been studied. It also remains to be determined whether changes in P2X3 receptor expression occur in ICCs exclusively. The present data showed an increase in P2X3 in ICCs mainly distributed in the suburothelium, which might suggest that P2X3 regulates its functional role via receptors located in the suburothelium. Although further investigation is essential, these results also suggest that abnormal P2X3 transmission in the bladder might explain the development of symptoms in patients with OAB.
In conclusion, these studies show a novel observation of P2X3 receptors up-regulation in a time-dependent manner in ICCs in an experimental rat model of PBOO. The P2X3 receptor agonist, α, β-meATP, enhanced the inward currents in the PBOO ICCs. The mean peak amplitude of ICC inward currents was significantly increased in the PBOO compared with the control group. The results indicate that the up-regulation of P2X3 in bladder ICCs may contribute to the pathophysiology of bladder overactivity. Nevertheless, it remains to be determined whether changes in P2X3 receptor expression in PBOO play a role in the maturation of the voiding process. We will conduct further experiments to investigate the mechanism that underlies the regulation of P2X3 expression in ICCs.
The authors gratefully acknowledge Song Zhang for his skilful technical assistance. This work was supported by grants from the National Natural Science Foundation of China (30801142/30873005).