Modulation of spontaneous activity in the overactive bladder: the role of P2Y agonists

Abstract

Spinal cord transection (SCT) leads to an increase in spontaneous contractile activity in the isolated bladder that is reminiscent of an overactive bladder syndrome in patients with similar damage to the central nervous system. An increase in interstitial cell number in the suburothelial space between the urothelium and detrusor smooth muscle layer occurs in SCT bladders, and these cells elicit excitatory responses to purines and pyrimidines such as ATP, ADP, and UTP. We have investigated the hypothesis that these agents underlie the increase in spontaneous activity. Rats underwent lower thoracic spinal cord transection, and their bladder sheets or strips, with intact mucosa except where specified, were used for experiments. Isometric tension was recorded and propagating Ca²⁺ and membrane potential (E_m) waves were recorded by fluorescence imaging using photodiode arrays. SCT bladders were associated with regular spontaneous contractions (2.9 ± 0.4/min); ADP, UTP, and UDP augmented the amplitude but not their frequency. With strips from such bladders, a P2Y₆-selective agonist (PSB0474) exerted similar effects. Fluorescence imaging of bladder sheets showed that ADP or UTP increased the conduction velocity of Ca²⁺/E_m waves that were confined to regions of the bladder wall with an intact mucosa. When transverse bladder sections were used, Ca²⁺/E_m waves originated in the suburothelial space and propagated to the detrusor and urothelium. Analysis of wave propagation showed that the suburothelial space exhibited properties of an electrical syncitium. These experiments are consistent with the hypothesis that P2Y-receptor agonists increase spontaneous contractile activity by augmenting functional activity of the cellular syncitium in the suburothelial space.

the overactive bladder in patients is associated with spontaneous transient increases in pressure that occur particularly during filling and, if sufficiently severe, contribute to lower urinary tracts symptoms of frequency, urgency, and incontinence (1). The origins of this common pathology are unclear but can be mimicked in animals in which the bladder outflow is artificially obstructed or the spinal cord is transected (SCT; T8–T9) (12). Whole rat bladders from an SCT model demonstrate large, regular contractions (∼2/min) that coincide with waves of raised intracellular Ca²⁺ and depolarization (E_m) propagating across the surface but originating from one or very few foci. This is in contrast to normal animals where more frequent (5–10/min), low-amplitude contractions are associated with multiple Ca²⁺/E_m foci (12, 14). The detrusor layer of the bladder wall contributes the bulk of tissue and hence most of the Ca²⁺/E_m signals, but an intact mucosa (urothelium and suburothelium) is required to generate large propagating waves, as well as to enhance spontaneous contractile activity (13, 25).

A cellular target for an understanding of such activity is a population of suburothelial interstitial cells (ICs) (24). In response to physiological stimuli such as mechanical stretch, the urothelium releases transmitters such as ATP and acetylcholine (9, 29), and it is proposed that ICs are intermediates in the pathways upon which these transmitters act. The overactive bladder is associated with an increase in IC number in animal and human tissue (12, 21); agents that target c-kit receptors on ICs, such as Glivec, reduce spontaneous activity (3), and ICs generate large depolarizing responses to transmitters, such as ATP (28). Excitatory responses from suburothelial ICs to ATP are via P2Y agonists; ADP and UTP generate similar responses to ATP itself (28), and the most readily labeled purinoceptor is the P2Y₆ subtype (26). P2Y agonists induce relaxation of the detrusor muscle itself (16). Thus one paradigm for studying the influence of the mucosa and ICs on spontaneous activity is to characterize the action of ADP and other P2Y agonists, such as UDP and UTP, on the contractile function and intercellular signaling of bladder wall tissue with an intact mucosa from SCT animals.

METHODS

Animal preparations.

Bladders were isolated from adult Sprague-Dawley rats that had SCT at level T8–T9 as described previously (12). Briefly, rats were operated under anesthesia with an isoflurane/O₂ mixture (2%-98%), and after a laminectomy the dura and spinal cord were cut. Gelfoam was packed between the cut ends, the muscle and skin were sutured, and the animals were allowed to recover with prophylactic antibiotics (100 mg ampicillin or 2 mg·kg⁻¹·day⁻¹ gentamycin for 2 wk). For these first 2 wk following the operation, bladders were emptied two to three times per day by manual abdominal compression, until the animals' own micturition reflexes recovered. Sham-operated animals received the same operative preparation but without the laminectomy and did not have postoperative abdominal compression. Animals were used between 4 and 6 wk after the operation. All procedures conformed to institutional guidelines and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee or followed European Community Council Directive 86/609/EEC and the UK Animal Act (1986) as appropriate.

Optical imaging and force measurements.

SCT rats that were to be used for combined optical imaging and force measurements were euthanized by CO₂ inhalation and exsanguination. The bladders were removed following a laparotomy and placed immediately in Tyrode's solution (below). Bladders were loaded with the Ca²⁺- and E_m-sensitive fluorochromes Rhod-2A and di-4-ANEPPS, respectively, and two types of preparation were prepared. A sheet preparation was generated by cutting the bladder longitudinally, from base to dome, along the ventral surface containing the mucosa or with the mucosa removed from half the area by blunt dissection. The preparation was placed in a recording chamber, impaled at one end with pins, and at the other end tied to an isometric force transducer. The preparation was superfused at 1–2 ml/min with Tyrode's solution at 37°C. Intracellular Ca²⁺ and E_m were measured by an optical imaging system that used 16 × 16 photodiode arrays to record 256 separate images that mapped the preparation surface at 500 Hz. Isochronal maps were generated to record the progression of Ca²⁺/E_m waves, centered on transients with the shortest delays as the initiating points. The second preparation imaged transverse sections of the bladder wall. The bladder wall was sliced with a sharp razor, and the exposed edge was placed uppermost in a superfusion chamber to permit optical imaging of the edge. The methods have been described in detail previously (13, 14).

Isolated detrusor strips (5 mm length, <1 mm diameter) from SCT rat bladders were also used. The bladder was opened as a sheet, and the mucosa was removed or left intact before strips were cut from the dorsal wall of the dome. Preparations were tied between a fixed hook and an isometric force transducer and superfused at 37°C with Tyrode's solution, as above. Spontaneous contractions were recorded continuously. At the end of each experiment, the wet weight of the preparation was recorded to normalize tension measurements.

Solutions.

The Ca²⁺-free solution contained (mM) 114 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, and 11.7 glucose, pH 7.4 gassed with 5% CO₂-95% O₂. Tyrode's solution contained (mM): NaCl 118, KCl 4.0, MgCl₂ 1.0, NaH₂PO₄ 0.4, NaHCO₃ 24, CaCl₂ 2.5, glucose 6.1, Na pyruvate 5.0, pH 7.4 gassed with 5%CO₂-95%O₂. Intervention agents were added from aqueous 10 mM stock solutions to achieve the desired concentration in Tyrode's solution.

Immunofluorescence and confocal imaging.

Bladders from adult male Sprague-Dawley rats (350 g) were quickly removed, washed with PBS, and the mid-cross sections were embedded in OCT and then snap frozen in liquid nitrogen. Cryosections (10 μm) were then placed on poly-l-lysine-coated slides and stored at −20°C until use. Cryosections were fixed with 4% paraformaldehyde for 4 min at room temperature (RT), washed with PBS, and then incubated with a blocking buffer (PBS, 1% BSA, 0.1% Triton-X) for 1 h at RT followed by overnight incubation at 4°C in the primary antibody (rabbit anti-P2Y₆, 1:200 Sigma-Aldrich). Sections were then washed with PBS and incubated for 1 h at RT with the secondary antibody (goat anti-rabbit IgG Alexa Fluor 594, 1:200, Millipore). To identify cellular nuclei, the fluorescent nucleic acid binding dye TO-PRO-3 Iodide (1:300, Invitrogen) was added with the secondary antibody. After a final step of washing, slides were air dried and mounted with Vectashield (Vector Laboratories). Slides were viewed at ×60 magnification (oil-immersion lens) using a Zeiss inverted laser-scanning confocal fluorescent Axiovert 135 microscope. Images were acquired using Zen 9000 software and analyzed with Zeiss axovision and Image-J software.

Data presentation, analysis, and calculations.

Data are shown as means ± SD; n is the number of preparations. Differences between data sets were tested by Student's paired or unpaired t-tests; the null hypothesis was rejected at P < 0.05. Spontaneous activity, frequency, and amplitude in detrusor strip preparations were counted using a custom-designed macro after digitization of the primary data.

Intracellular resistivity, R_i, of the suburothelial cellular network was calculated from the one-dimensional cable equation for a uniformly conducting electrical response (7)

$$R_{\text{i}}=\frac{a}{2C_{\text{m}}\tau *{\theta }^2}{\left(1+\frac{\tau *}{{\tau }_{\text{m}}}\right)}^{-1}$$ 1

where a is the cell radius, C_m is the specific membrane capacitance (1 μF/cm²), τ_m is the membrane time constant of the cell propagating the signal, and τ* is the time constant of the exponential base of the propagating signal (see Fig. 7).

Fig. 7.

Individual propagating membrane potential transients from a transverse section of a SCT rat: A: in baseline conditions. B: in the presence of 30 μM ADP. Insets: basal regions of the transients used to calculate the variable τ*. Shaded boxes show the regions from which the insets were analyzed.

RESULTS

Spontaneous contractile activity in SCT rat preparations: effect of purines.

All bladder preparations from SCT rats exhibited spontaneous contractions; the mean tension was 21.5 ± 5.7 mN/g, at a frequency of 2.9 ± 0.4/min (SE, n = 8). Figure 1A shows the effect of 30 μM ADP on spontaneous activity with several consistent observations: 1) a large initial contraction; 2) an increase in contraction magnitude during the exposure; and 3) on removal of ADP, a transient reduction of spontaneous activity before a return to preintervention activity. Table 1 shows the amplitude and frequency of contractions before (baseline conditions) and during ADP, as well as the coefficient of variation (mean/SD) for the two variables. ADP significantly increased the contraction amplitude but had no effect on frequency. Furthermore, the coefficient of variation of these variables was unaffected by ADP, which indicates that the coordination of the pathways responsible for generating the contraction within the tissue mass was not altered by ADP. Figure 1B shows the response to UTP (10 μM), and Table 1 shows that similar conclusions were derived compared with ADP. Contraction amplitude was increased, but frequency, or the variability of these characteristics, was unaltered. Figure 1 shows that ADP and UTP also generated a significant rise in baseline tension (8.7 ± 6.3 mN/g, n = 9), and thus a final contractile characteristic was the tension integral, measured for 10 min before and during the intervention from a baseline of the preintervention tension. ADP and UTP both significantly increased the tension integral (Table 1). Two experiments each with ADP and UDP also showed increased amplitude and tension integral, but no effect on frequency of spontaneous contractions.

Fig. 1.

Spontaneous contractile activity from spinal cord transected (SCT) rat bladder sheets from an intact mucosa. A: exposure to 30 μM ADP. B: exposure to 10 μM UTP for durations as indicated by the lengths of the horizontal bars.

Table 1.

Effect of 30 μM ADP or UTP on spontaneous contractions in mucosa-intact bladder preparations from SCT rats

	Amplitude, mN/g	Amplitude, Coefficient of Variation	Frequency, no./min	Frequency, Coefficient of Variation	Integral, mN·min−1·g−1
Baseline	30.6 ± 21.1	0.35 ± 0.16	2.8 ± 1.0	0.51 ± 0.21	14.5 ± 3.9
ADP (n = 7)	42.9 ± 18.7*	0.57 ± 0.20	3.1 ± 1.2	0.39 ± 0.14	23.1 ± 4.0*
Baseline	23.8 ± 19.0	0.58 ± 0.34	2.6 ± 1.2	0.53 ± 0.20	11.9 ± 3.4
UTP (n = 8)	50.1 ± 32.5*	0.62 ± 0.52	2.7 ± 0.7	0.41 ± 0.18	34.6 ± 24.7*

Values are means ± SD. SCT, spinal cord transected. The amplitude and frequency of the contractions as well as their coefficients of variation (= SD/mean) are quoted. The integrals of these contractions above the baseline are also given.

^*P < 0.05.

Experiments using ADP with small strips of detrusor from SCT animals from which the mucosa had been removed are shown in Fig. 2A. With 30 or 100 μM ADP, there was a large contraction on initial exposure (arrowhead) but then a reduction of baseline tension and the amplitude of spontaneous contractions. The tension integral was also reduced, illustrated for 30 μM ADP. There was no significant effect on the frequency of spontaneous contractions generated by these strips (2.5 ± 0.3 vs. 1.9 ± 0.2/min; baseline vs. ADP, n = 3).

Fig. 2.

A: Spontaneous contractile activity from detrusor strips, with mucosa removed, of SCT rat bladders. Top traces show response to 30 and 100 μM ADP added for the duration shown in the grey boxes. The bar chart shows the tension integral in baseline conditions and in the presence of 30 μM ADP. B: P2Y₆ receptor labeling (red) in the rat mucosa; nuclei are labeled with TO-PRO-3 (blue). C: P2Y₆ and TO-PRO-3 labeling in the detrusor layer. D: detrusor strips with intact mucosa. Top trace shows response to 10 μM PSB0474, added for the duration of the thick bar above the trace. The bar chart shows the tension integral in baseline conditions and in the presence of 10 μM PSB0474.

Suburothelial ICs in guinea pigs were readily labeled by P2Y₆ immunofluorescent markers (26). We sought to verify that similar labeling was present in the rat bladder wall. Figure 2B shows that labeling was observed in the suburothelial space as well as the urothelium. In the suburothelium, P2Y₆ labeling was observed near TO-PRO-3-labeled nuclei and thus was considered to have a cellular distribution; more intense labeling was also detected in the urothelium, similar to that in guinea pig bladders (26). In the detrusor layer, labeling was very sparse (Fig. 2C); only a few punctate labels were observed. Similar observations were made in sections from three different bladders. Therefore, the effects of a P2Y₆-selective agonist (PSB0474) on basal tone and spontaneous contractions were measured in detrusor strips from SCT rats with an intact mucosa. PSB0474 (10 μM) significantly increased basal tension, as well as the amplitude of spontaneous contractions (Fig. 2D) but had no significant effect on their frequency (2.9 ± 0.4 vs. 2.8 ± 0.3/min; baseline vs. PSB0474, n = 3). The tension integral was also increased significantly by PSB0474 (Fig. 2D). These effects of PSB0474 were similar to those of ADP (tension integral increased from 7.8 ± 13 to 10.9 ± 1.4, n = 6) and to ADP or UTP on the bladder sheets with an intact mucosa (Fig. 1), and unlike the effect of ADP on mucosa-denuded muscle strips. Note that removal of the mucosa reduced the baseline amplitude of the spontaneous contractions (1.4 ± 0.05 vs. 0.50 ± 0.09 mN/mg; n = 6; P < 0.001), and increased frequency (2.2 ± 0.6 vs. 3.5 ± 0.2; n = 6; P < 0.05).

Action of ADP and UTP on Ca²⁺/E_m waves in bladder sheets.

Spontaneous contractions in bladders from SCT rats are associated with propagated Ca²⁺ and E_m waves originating from one or very few focal sites (12, 14). These experiments aimed to determine whether ADP and UTP 1) enhanced these waves and 2) required an intact mucosa. Figure 3A shows Ca²⁺ waves from a SCT rat bladder sheet with an intact mucosa. The left-hand map was obtained in baseline conditions; the lightest areas correspond to where Ca²⁺ transients occurred with the shortest delay and darker areas to successively longer delays. A single focal area was observed near the base of the bladder (arrow). In four preparations, Ca²⁺ waves propagated in the base-dome axis at 4.8 ± 1.7 mm/s in baseline conditions; the corresponding velocity for E_m waves was 5.6 ± 1.6 mm/s. Conduction in the lateral plane was generally slower, as seen by the closer isochrones, with velocities of 0.8 ± 0.3 and 1.3 ± 0.9 mm/s for Ca²⁺ and E_m waves, respectively. The right-hand map shows a corresponding Ca²⁺ wave map in the presence of 30 μM UTP. The tension trace between the maps corresponds to the experiment from which they were obtained; the two arrows correspond to contractions in baseline conditions (Fig. 3A, left) and in the presence of 30 μM UTP (Fig. 3A, right). UTP increased basal contractile tone and the amplitude of spontaneous contractions; the Ca²⁺ wave map was taken when enhancement of contractile activity was maximum and at steady state. The focal initiation site in the Ca²⁺ wave map was enhanced, particularly in the base-dome axis to a wider area of the preparation, although it did not alter the origin. In this example, conduction velocity was increased from 4.5 to 6.5 mm/s. The acceleration of conduction velocity may be expected to coordinate a larger area of the bladder surface and thus increase the magnitude of the subsequent contraction.

Fig. 3.

Ca²⁺ wave maps from bladder sheets with an intact mucosa (isochronal interval 100 ms). The base and dome regions of the sheets are indicated in the left-hand maps. A: SCT rat bladder. Maps show sheets in the baseline condition (left) and in the presence of 30 μM UTP (right). The tension trace corresponding to the experiment from which the maps were constructed is shown. Arrows mark the spontaneous contractions corresponding to the 2 maps. UTP was added for the duration of the bar below the trace. The arrows indicate the origins of the propagating wave. B: correspondence between the time course of a Ca²⁺ transient from a single pixel and contractile activity in a bladder preparation during exposure to 30 μM ADP.

Figure 3B shows that Ca²⁺ transients were indeed associated with contractile activity. The example, obtained in the presence of 30 μM ADP, shows Ca²⁺ transients from one pixel in an optical imaged array near to the focal point of Ca²⁺ wave propagation and contractile activity of the preparation. The similarity of the traces suggests contraction is associated with these Ca²⁺ waves.

Spontaneous Ca²⁺ waves and contractile activity in normal rat preparations.

Figure 4 shows an example of Ca²⁺ waves and spontaneous contractions in a bladder sheet from an unoperated animal with an intact mucosa, demonstrating multiple foci (arrows). Addition of ADP did not coalesce the small, individual foci but preserved the pattern; the same observation was made in three preparations. The corresponding tension trace shows that ADP generated a rise in basal tension overlain throughout by small spontaneous contractions. ADP (30 μM) had no significant effect on the frequency (8.0 ± 1.8 vs. 9.0 ± 1.2/min; n = 3, P > 0.05) or amplitude (1.08 ± 0.20 vs. 1.07 ± 0.06 mN/g; n = 3, P > 0.05) of spontaneous contractions; however, basal tone increased significantly (4.27 ± 1.91 mN/g; n = 3). Data were also obtained from small mucosa-attached detrusor strips of six sham-operated animals. ADP (30 μM) again had no effect on the frequency (9.4 ± 0.5 vs. 8.7 ± 0.4/min, n = 9, P > 0.05) or amplitude (0.98 ± 0.16 vs. 0.76 ± 0.24 mN/mg; n = 6, P > 0.05) of spontaneous contractions, but did increase basal tension by a small amount 0.17 ± 0.01 mN/mg, n = 6, i.e., 17.3 ± 2.4% of the baseline spontaneous contraction amplitude.

Fig. 4.

Ca²⁺ wave maps for a bladder sheet from an unoperated animal. Maps show the sheet in baseline solution (left) and in the presence of 30 μM ADP (right) and the corresponding tension trace. ADP was added for the duration of the bar below the trace. Arrows mark focal origins of activity.

Role of the mucosa in Ca²⁺/E_m waves.

Previous experiments have shown that an intact mucosa enhances spontaneous detrusor contractile activity (25). Its importance in the generation of Ca²⁺/E_m waves, and any role of ADP, was also investigated. Figure 5 shows an experiment using SCT rat bladders from which half the mucosa had been removed; the intact portion is denoted by the boxes on the left side of each of the Ca²⁺ and E_m maps. In baseline conditions (top maps) prominent Ca²⁺ and E_m foci were generated on the half with an intact mucosa, although there was a smaller, secondary wave in the denuded half of the preparation in both maps. ADP increased the distance between isochrones in both the Ca²⁺ and E_m maps (bottom maps), although the position of the focal points did not greatly alter. This indicates that in the presence of ADP, conduction of Ca²⁺ and E_m waves was accelerated in the region with an intact mucosa, although the spread to the denuded portion remained slow. In this example, the Ca²⁺ wave conduction velocity was enhanced from 4.8 to 8.6 mm/s and E_m wave conduction velocity from 5.6 to 9.3 mm/s in the mucosa-intact region.

Fig. 5.

Ca²⁺ (left) and membrane potential (E_m; right) maps from a SCT rat bladder sheet. The mucosa was removed from the right-hand side of the sheet; the box marks the area of intact mucosa. Maps were obtained before (top) and during (bottom) the application of 30 μM ADP. Arrows on the tension trace mark the spontaneous contractions corresponding to the maps. ADP was added for the duration of the bar below the trace.

Action of ADP and UTP on Ca²⁺/E_m waves in bladder transverse sections.

When bladder sections were imaged, Ca²⁺ and E_m waves originated from the mucosal surface and propagated eventually to the detrusor layer. Figure 6 shows Ca²⁺ isochronal maps in baseline conditions (left) and in the presence of 30 μM UTP (middle) or UDP (right). In this example, under baseline conditions two small areas of focal activity (arrows) were observed on the mucosa/detrusor boundary. Propagation of signals was quicker in the longitudinal direction (top to bottom), along the suburothelial layer and slower in the transverse direction, especially in the direction toward the detrusor, as seen by the higher density of isochrones. It was clear that the wave origins are submucosal (i.e., not at the extreme right of the maps). This observation was preserved when activity was enhanced by UTP or UDP and is consistent with the hypothesis that spontaneous activity arises within the suburothelial region of the bladder wall. Similar observations were made in the presence of ADP (not shown). Under baseline conditions, longitudinal and transverse conduction velocities (CV_L and CV_T) were 0.5 ± 0.2 and 0.09 ± 0.02 mm/s, respectively (n = 4). CV_L was increased significantly (P < 0.05, n = 4) to 2.3 ± 1.1, 1.2 ± 0.5 and 1.9 ± 0.5 mm/s in the presence of ADP, UDP, and UTP, respectively. Similarly, CV_T was increased significantly to 0.5 ± 0.1, 0.5 ± 0.2 and 0.5 ± 0.2 mm/s in the presence of ADP, UDP, and UTP.

Fig. 6.

Ca²⁺ maps from a transverse section of a SCT rat bladder in the baseline condition (left) and during the subsequent application of 30 μM UTP and 30 μM UDP. The positions of 2 loci for Ca²⁺ waves are indicated by the black arrow on the baseline map. The UTP map was obtained immediately after the baseline map. The UDP map was obtained after 15-min washout of UTP.

Electrical characteristics of the suburothelial space.

In many preparations, E_m waves spread along the suburothelial space from a focal point (Fig. 6) at a velocity much greater (>10-fold) than in the transverse plane. Under such circumstances, signals were considered to be one-dimensional within a functional syncitium, and this permitted estimation of the resistance, R_i, of the cellular network (ICs): Eq. 1. These cells have fine projections from a central body with a radius, a, of ∼0.25 μm (27) and a value for τ_m of 70 ms (28). Figure 7 shows typical individual E_m transients from the area of suburothelial propagation under baseline conditions (Fig. 7A) and in the presence of 30 μM ADP (Fig. 7B). The value of τ* (Eq. 1) was estimated from the basal region of the transient (shaded boxes, Fig. 7) and had a value of 0.45 ± 0.16 s under baseline conditions (n = 11, 4 preparations) and decreased to 0.27 ± 0.07 s (n = 12, 4 preparations) in the presence of ADP or UTP (data combined). Values for a longitudinal conduction velocity of 0.27 ± 0.097 cm and 0.37 ± 0.099 cm/s (n = 4) in the absence and presence of ADP/UTP allowed calculation of R_i and yielded values of 1,095 ± 357 and 903 ± 121 Ω·cm, respectively, and were not significantly different. Thus R_i was similar in both conditions, and the increase in conduction velocity was due to a decrease in the variable τ* (see below).

DISCUSSION

Spontaneous activity in spinal cord injury.

SCT at the T8–T9 level generates an overactive bladder phenotype of large, fairly regular bladder contractions. The objective of this study was to gain insight into the cellular and tissue pathways contributing to this phenotype. This overactive behavior was mirrored in isolated bladder preparations by increased spontaneous contractile activity, suggesting that at least part of the increased response is myogenic (6, 12). With bladders from unoperated animals, spontaneous activity consisted of smaller and more frequent contractions and was mirrored by optical imaging as multiple focal points of activity. Although increased spontaneous activity in bladders from SCT rats may be due in part to altered properties of detrusor muscle, this and other studies (13) suggest that an intact mucosa is important for full expression of increased spontaneous activity. Moreover, optical mapping of transverse sections of the bladder wall showed here that Ca²⁺ and E_m activity originated in the mucosa and propagated to the detrusor. The concordance between contractile activity and optical signals of propagating Ca²⁺ and E_m waves suggests a causal relationship, and we propose that such activity originates in the suburothelial space. It is not possible to determine whether mucosal-detrusor interaction is due to a diffusible agent released from the mucosa or whether it requires a physical connection. However, there was a considerable delay of signal at the mucosa-detrusor border, which may be interpreted as a requirement for a diffusible factor at least at this site. In addition, these experiments also showed that activity originated not at the apical edge of the mucosa but rather in the suburothelial layer and spread both toward the urothelium as well as to the detrusor. In principle, Ca²⁺ an E_m signals could spread across the urothelium, as this region labels strongly for the gap junction protein Cx26 (10); however, these experiments lacked the spatial resolution to confirm this fact.

Action of purines and pyrimidines on spontaneous activity.

The contractile responses in SCT preparations were significantly augmented by exogenous purines (ADP and ATP) and pyrimidines (UTP and UDP). They were mirrored by an increase in Ca²⁺ and E_m signaling in bladder sheets with an intact mucosa and in the mucosal layer of transverse sections, all suggesting an involvement of P2Y receptors (19). These agents all ultimately produce relaxation of isolated detrusor smooth muscle (2, 5, 16), and preservation of the relaxatory response to ADP in detrusor from SCT animals was confirmed in these experiments. Thus it is unlikely that the increased contractile and Ca²⁺/E_m activity is due to a direct action on the detrusor layer. No single P2Y receptor is likely to be responsible for the augmentation of activity considering that a range of purine and pyrimidines produced similar changes to spontaneous activity. P2Y₁ receptors are activated most effectively by ADP, whereas UTP is a potent activator of P2Y₂ and P2Y₄ receptors (19). The P2Y₆ receptor is most potently activated by UDP, and the role of this receptor was corroborated by an increase in spontaneous activity by the selective agonist PSB0474. P2Y₁, P2Y₂, and P2Y₄ receptors have all been localized to the urothelium in the cat bladder (4). Furthermore, strong labeling for P2Y₆ receptors was found in suburothelial ICs in the guinea pig bladder, with weaker labeling of P2Y₂ and P2Y₄ receptors but none for P2Y₁ (26).

The ability of P2Y agonists to augment spontaneous activity, especially in the overactive bladder, could arise from ATP that is released from the urothelium/suburothelium during stresses such as bladder wall stretch and raised transmural pressure. ATP is readily broken down to ADP and other metabolites by endonucleotidases present in the bladder wall (22) that could act on ICs to generate excitatory responses. Moreover, there is evidence that such a system is upregulated in the overactive bladder to produce the larger and better coordinated spontaneous contractions, including an increase in IC number (25) and augmented ATP release (15, 17). We propose that this P2Y-mediated pathway represents a target for the reduction of overactive bladder spontaneous contractions.

Syncitial properties of the suburothelial cellular network.

Immunohistochemical studies have demonstrated that a major site for the gap junction protein Cx43 is suburothelial ICs, suggesting that they are capable of transmitting intercellular signals (24). These experiments provide direct evidence for signal propagation along the suburothelial layer, more quickly than transversely to the detrusor or urothelium. In a subset of experiments, longitudinal transmission was more than 10-fold greater than in transverse dimensions, and thus it was possible to treat this layer as a one-dimensional multicellular syncitium with only ∼10% error (20). Using a solution of the cable equation for signals of constant propagation velocity (methods), a value for intracellular resistivity of ∼1,000 Ω·cm was calculated both in baseline conditions and with P2Y agonists. This value is comparable to 600–800 Ω·cm in smooth muscle (18, 23) but greater than that in better-coupled syncitia, such as ventricular myocardium (226 Ω·cm) (8). The important conclusion is that the suburothelial space has the required electrical properties to permit relatively rapid propagation of signals over distances of many cell lengths and thus permit coordination of significant areas of the bladder wall.

Increased conduction velocity with P2Y agonists was not due to a reduction in R_i but rather to a decrease in the value of τ*. The parameter τ* represents the initial depolarization rate of a propagating wave and is determined by the strength of local circuits from adjacent regions undergoing electrical activity; the magnitude of local circuits in turn depends largely on the density of inward currents generating the electrical response (11). Because P2Y agonists increase inward currents in ICs (28), this will accelerate the propagation rate of electrical signals within the suburothelium to enhance the coordination of signals. This analysis gives further weight to the hypothesis that ICs in the suburothelial space act as a functional syncitium whose coordinating role can be augmented by locally produced exogenous agents.

Conclusion.

Bladders from SCT rats generate large spontaneous contractions and propagating waves of intracellular Ca²⁺ and membrane depolarization, which require an overlay of mucosa on the detrusor smooth muscle. These activities are augmented by P2Y receptor agonists such as ADP, UTP, and UDP. The Ca²⁺ and E_m waves originate in the suburothelium of the mucosa and spread to the detrusor, a phenomenon accelerated by P2Y receptor agonists. The mucosa may be modeled as a functional synctium to account for wave propagation. This study shows the crucial importance of the mucosa in determining the contractile properties of bladders with an overactive phenotype and the influence of purinergic modulators that are generated by the urothelium.

GRANTS

This work was funded by grants to C. H. Fry, R. I. Jabr (EU FP7 INComb, Pfizer), J. S. Young (AgeUK), and A. J. Kanai (NIH/NIDDK DK 057284).

DISCLOSURES

C. H. Fry is a member of an advisory board to Eli Lilly and is carrying out a joint project with Y. Takeda.

AUTHOR CONTRIBUTIONS

Author contributions: C.H.F., J.S.Y., R.I.J., C.M., Y.I., and A.J.K. provided conception and design of research; C.H.F., J.S.Y., R.I.J., C.M., and Y.I. performed experiments; C.H.F., J.S.Y., and R.I.J. analyzed data; C.H.F., J.S.Y., R.I.J., C.M., Y.I., and A.J.K. interpreted results of experiments; C.H.F. and R.I.J. prepared figures; C.H.F. drafted manuscript; C.H.F., J.S.Y., R.I.J., C.M., Y.I., and A.J.K. edited and revised manuscript; C.H.F., J.S.Y., R.I.J., C.M., and A.J.K. approved final version of manuscript.