NKA enhances bladder-afferent mechanosensitivity via urothelial and detrusor activation

Abstract

Tachykinins are expressed within bladder-innervating sensory afferents and have been shown to generate detrusor contraction and trigger micturition. The release of tachykinins from these sensory afferents may also activate tachykinin receptors on the urothelium or sensory afferents directly. Here, we investigated the direct and indirect influence of tachykinins on mechanosensation by recording sensory signaling from the bladder during distension, urothelial transmitter release ex vivo, and direct responses to neurokinin A (NKA) on isolated mouse urothelial cells and bladder-innervating DRG neurons. Bath application of NKA induced concentration-dependent increases in bladder-afferent firing and intravesical pressure that were attenuated by nifedipine and by the NK2 receptor antagonist GR159897 (100 nM). Intravesical NKA significantly decreased bladder compliance but had no direct effect on mechanosensitivity to bladder distension (30 µl/min). GR159897 alone enhanced bladder compliance but had no effect on mechanosensation. Intravesical NKA enhanced both the amplitude and frequency of bladder micromotions during distension, which induced significant transient increases in afferent firing, and were abolished by GR159897. NKA increased intracellular calcium levels in primary urothelial cells but not bladder-innervating DRG neurons. Urothelial ATP release during bladder distention was unchanged in the presence of NKA, whereas acetylcholine levels were reduced. NKA-mediated activation of urothelial cells and enhancement of bladder micromotions are novel mechanisms for NK2 receptor-mediated modulation of bladder mechanosensation. These results suggest that NKA influences bladder afferent activity indirectly via changes in detrusor contraction and urothelial mediator release. Direct actions on sensory nerves are unlikely to contribute to the effects of NKA.

INTRODUCTION

Sensory signaling from the bladder relies on mechanosensitive primary afferents that respond to graded bladder filling and detrusor contraction to initiate micturition (12). Recent data suggest that transient nonvoiding contractions (referred to as micromotions) “tune” afferent sensitivity to augment afferent firing during bladder filling (17). Moreover, such micromotions, which depend upon interactions between muscle, interstitial cells, and the intramural innervation, may become dysregulated and contribute to urinary urgency and detrusor overactivity in patients with overactive bladder (OAB) syndrome (48). Because bladder micromotions result in the generation of bladder afferent activity, it has been hypothesized that pharmacologically reducing bladder micromotions may subsequently decrease afferent activity, thereby providing a novel approach to treat OAB symptoms such as urgency.

Tachykinins have long been known to influence detrusor activity and trigger micturition (21) via activation of the Tachykinin family of G protein-coupled receptors, namely NK1, NK2, and NK3, which show selectivity for substance P, neurokinin A (NKA), and neurokinin B, respectively (1). In this respect, it is NKA that has the greatest potency for stimulating micturition when delivered either close arterially or from the lumen (27). The former provides a powerful stimulus for contraction via NK2 receptors on bladder muscle (38, 46), and the latter potentially acts via the urothelium to influence bladder afferents. Expression profiling of human urothelium has revealed expression of all three neurokinin receptors (35), although the functional significance of this remains unclear.

Bladder afferents are the major source of tachykinins, present in both capsaicin-sensitive (26) and capsaicin-insensitive afferents, that are upregulated in response to bladder injury or inflammation (6, 18, 34). Treatments that desensitize and disrupt the sensory function of capsaicin-sensitive primary afferents (CSPANs) have been shown to produce positive outcomes in both idiopathic and neurogenic bladder disorders (2, 4) through a decrease in urgency and frequency and a corresponding increase in bladder capacity. The presence of tachykinin receptors on muscle, urothelium, interstitial cells, and sensory afferents is the basis for a complex interaction between various cells that modulate sensory signaling from the bladder. We hypothesize that tachykinins influence bladder afferent signaling via NK2 receptors at various sites within the bladder wall. We have employed electrophysiological techniques to directly record the afferent impulse traffic emanating from the bladder wall and examined the interplay between nerve, muscle, and urothelium following tachykinin receptor activation. Our data provide an insight into a signaling cascade that is triggered by tachykinin release, which in turn generates micromotions and untimely nerve activity as a result of urothelial and detrusor interactions. These findings provide clarity on the tachykinin mechanisms that trigger micturition and which may contribute to bladder symptoms in disease states.

METHODS

Animals.

The Animal Ethics Committees of the South Australian Health and Medical Research Institute (SAHMRI) and Griffith University (Australia) and the University of Sheffield Animal Care Committee (UK) approved experiments involving animals under a project license issued by the UK Animals (Scientific Procedures) Act 1986. Male C57BL/6J mice aged 14–16 wk were used in this study. Mice were group-housed (5 mice/cage) in specific housing rooms within a temperature-controlled environment of 22°C and a 12:12-h light-dark cycle. Mice had free access to food and water at all times. Mice were randomly assigned to experimental procedures, and individual mice were used for a single experiment only. All experiments were performed on isolated tissue obtained from mice that were humanely euthanized by cervical dislocation or CO₂ asphyxiation in accordance with the guidelines and laws of the individual countries and ethics committees where the experiments were performed.

Ex vivo bladder afferent nerve recording.

Nerve recording was conducted using a previously described ex vivo model (10). Mice were humanely euthanized, and the entire lower abdomen was removed and submerged in a modified organ bath under continual perfusion with gassed (95% O₂ and 5% CO₂) Krebs-bicarbonate solution (composition in mmol/l: 118.4 NaCl, 24.9 NaHCO₃, 1.9 CaCl₂, 1.2 MgSO₄, 4.7 KCl, 1.2 KH₂PO₄, 11.7 glucose) at 35°C. Excess tissue was removed to expose the bladder, urethra, and ureters. Ureters were tied with perma-hand silk (ETHICON). The bladder was catheterized through the urethra (PE 50) and connected to a syringe pump (NE-1000) to allow a controlled fill rate of 30 µl/min. A second catheter was inserted through the dome of the bladder, secured with silk, and connected to a pressure transducer (NL108T2; Digitimer) to enable recording of intravesical pressure during graded distension. Pelvic nerves were dissected into fine multiunit branches and placed within a sealed glass pipette containing a microelectrode (WPI) attached to a Neurolog headstage (NL100AK; Digitimer). Nerve activity was amplified (NL104), filtered (NL 125/126, band pass 50–5,000 Hz, Neurolog; Digitimer), and digitized (CED 1401; Cambridge Electronic Design, Cambridge, UK) to a PC for offline analysis using Spike2 software (Cambridge Electronic Design). To quantify the magnitude of the afferent response, the number of action potentials crossing a preset threshold was determined per second. The threshold was calculated at twice the baseline nerve activity for all experiments to include only mechanosensitive firing.

Experimental protocols.

At the start of all afferent recording experiments, control bladder distensions were performed with intravesical infusion of isotonic saline (NaCl, 0.9%) at a rate of 30 µl/min to a maximum pressure of 25 mmHg at intervals of 10 min to assess the viability of the preparation and reproducibility of the intravesical pressure and neuronal responses to distension. The volume in the bladder was extrapolated from the known fill rate (30 µl/min) and the time taken (s) to reach the maximum pressure of 25 mmHg, whereas compliance is found from plotting intravesical pressure against the calculated volume. After a stable baseline was maintained, the saline in the infusion pump was replaced by NKA (300 nM) or the NK2 antagonist GR159897 (100 nM) either individually or in combination. For experiments investigating the effect of extravesical tachykinins, agonists/antagonists were dissolved from stock solutions in the Krebs buffer perfusing the organ bath (10 ml/min, bath volume 40 ml).

For contraction studies, once afferent nerve output was stable, bladders were partially filled (12 mmHg) with the outflow tap closed. Infusion was stopped, but the outflow tap remained closed, keeping a set volume within a moderately distended bladder. The preparation was left in this state for 45 min until bladder pressure and baseline nerve activity were once again stable. NKA (1–3,000 nM), Senktide (300 nM), and substance P (300 nM) were applied to the bath by bolus dose (1 ml) and washed out with Krebs buffer. Krebs buffer was continually perfused for 1 h to minimize receptor desensitization before a second administration of experimental compound. In some experiments, GR159897 (100 nM), was perfused in the Krebs solution for the period before the stimulus. Bladder contraction was measured as a change in intravesical pressure (mmHg) and afferent nerve activity as the number of spikes crossing a preset threshold per second.

Bladder mediator release.

Samples for mediator release assays were obtained from whole bladders. Following humane euthanasia, bladders were quickly dissected and placed in a modified organ bath with continual perfusion of gassed (95% O₂ and 5% CO₂) Krebs-bicarbonate solution at 35°C. A three-way catheter attached to a pressure transducer (NL108T2; Digitimer), a syringe pump (NE-1000), and an outflow tap was inserted into the bladder via the urethra and secured with silk (ETHICON). Sterile saline (0.9%; control) or NKA (agonist) was continuously infused into the bladder at 30 µl/min during experiments. Samples of intraluminal contents were taken following a 60-min equilibration period either during a 7-min infusion of vehicle or NKA at 30 µl/min with the outflow tap open (basal; no distension) or following bladder distension to 30 mmHg (Stretch) with vehicle or agonist. Two sets of samples were taken from each preparation. Luminal contents were collected directly onto dry ice to prevent post hoc degradation of substrates. Intravesical contents were assayed for ATP (A22066; Molecular Probes), acetylcholine (Ach) (A12217; Molecular Probes), and prostaglandin E2 (514010; Cayman Chemical) using commercially available kits. For ATP determination, 25 µl of of luminal content was added to 25 µl of standard reaction solution and incubated for 3 min in the dark at room temperature before luminescence measurement. For Ach determination, 25 µl of luminal content was added to 25 µl of the Amplex Red reagent-horseradish peroxidase-choline oxidase-acetylcholinesterase working solution and incubated in the dark for 30 min before fluorescence measurement. Samples were run in duplicate for all assays. Unknowns were calculated from a standard curve of known concentrations and corrected for background fluorescence/luminescence.

Bladder retrograde tracing.

A small aseptic abdominal incision was made in anesthetized (2–4% isoflurane in oxygen) mice. Cholera Toxin subunit B conjugated to AlexaFluor 488 (CTB-488, 0.5% diluted in 0.1 M phosphate buffer; ThermoFisher Scientific) was injected at four sites into the bladder wall (2 µl/injection) using a 5-µl Hamilton syringe attached to a 23-gauge needle (7). The needle is inserted subserosal, parallel with the bladder muscle, to ensure that CTB is not injected into the bladder lumen. The abdominal incision was sutured closed and analgesic (Buprenorphine; 2.7 μg/30g) and antibiotic (Ampicillin; 50 mg/kg) given subcutaneously as mice regained consciousness. Mice were then allowed to recover, housed individually, and monitored for 4 days. After 4 days, animals were humanely euthanized for subsequent lumbosacral (LS; L5-S1) dorsal root ganglion (DRG) removal and isolation to visualize CTB-labeled afferent neurons in the dorsal root ganglia neurons. To avoid any confounding effects of surgical procedure and retrograde tracer injection, the bladders of these mice were not used for any of the other experiments described herein.

Cell culture of bladder DRG neurons.

Pelvic (L5-S1) DRGs were removed from healthy mice 4 days after bladder retrograde tracing with AlexaFluor 488-conjugated cholera-toxin subunit B (CTB-AF488). DRGs were isolated and digested in HBSS (pH 7.4) containing 4 mg/ml collagenase II (Gibco) and 4 mg/ml dispase (Gibco) at 37°C for 30 min. The collagenase-dispase solution was aspirated and replaced with Hanks’ balanced salt solution (HBSS) containing collagenase 4 mg/ml only for 10 min at 37°C. Cells were washed in HBSS (×2) and dissociated in DMEM via trituration of DRGs through fire-polished Pasteur pipettes of descending diameter (36). Neurons were spot-plated (30 µl) onto 13-mm coverslips coated with laminin (20 μg/ml) and poly-d-lysine (800 μg/ml) Coverslips were kept for 2 h in an incubator at 37°C in 5% CO₂ before flooding with DMEM containing 10% FBS and 100 μg/ml penicillin-streptomycin; Invitrogen).

Isolation of mouse urothelial cells.

Following humane euthanasia, bladders were removed, dissected in sterile PBS, pinned urothelial side up, and incubated with modified Eagle’s medium (MEM) media containing 2.5 mg/ml dispase (Gibco) (3 h at 21°C). Cells were collected by gentle scraping of the urothelium with a blunt scalpel and dissociated in 0.025% trypsin EDTA at 37°C (10 min) with gentle trituration. The cell suspension was resuspended in MEM (Gibco) with 10% FBS to deactivate the trypsin before centrifugation (15 min, 1,500 rpm, 4°C). Cells were then resuspended in keratinocyte serum free media (KSFM; Invitrogen) before being plated onto collagen IV-coated coverslips (Sigma-Aldrich). Coverslips were left for 4 h in an incubator at 37°C and 5% CO₂ before flooding with KSFM (2 ml/well) (15). Cells were used for calcium imaging or immunohistochemistry.

Calcium imaging of cultured urothelial and DRG neurones.

Cultured DRG neurones and urothelial cells (20–24 h) were loaded with 2 μM fura-2-acetoxymethyl ester (Fura-2) for 15 min at 37°C and washed with 10 mM HEPES buffer (HEPES (2-hydroxyethyl-1-piperazineethanesulfonic acid), 142 mM NaCl, 2 mM KCl, 10 mM glucose, 2 mM CaCl₂, and 1 mM M_gCl₂, pH 7.4) for 30 min before imaging at room temperature (23°C). Fura-2 was excited at 340 and 380 nm using a Nikon TE300 Eclipse microscope equipped with a Sutter DG-4/OF wavelength switcher, an Omega XF04 filter set for Fura-2, a Photonic Science ISIS-3 intensified CCD camera, and a Universal Interface Card MetaFluor software. Fluorescence images were obtained every 5 s using a ×20 objective. Data were recorded and further analyzed using MetaFluor software. After an initial baseline reading to ensure cell fluorescence was stable, retrogradely traced bladder DRG (identified by the presence of the 488 tracer) and urothelial cells were stimulated with NKA (300 nM), and changes in intracellular calcium [Ca²⁺]_i were monitored in real time. Ionomycin (5 µM) and KCL (40 mM) were applied as positive controls in urothelial and DRG experiments, respectively.

[Ca²⁺]_i is expressed as the ratio between the fluorescence signal at 340/380 nm or normalized to percentage of maximum ionomycin-induced calcium influx to overcome intra-experimental variability.

Immunohistochemistry of cultured urothelial cells.

Urothelial cells were labeled for transitional epithelium using monoclonal antibody cytokeratin 7 (CK7) (OV-TL 12/30; ThermoFisher). The details of the primary antibody used are in Table 1. Coverslips were washed with 0.1 M phosphate-buffered saline (PBS) three times and fixed with ice-cold 4% PFA at room temperature for 20 min. Coverslips were washed with 0.2% Triton-TX 100 (Sigma-Aldrich) in 0.1 M PBS (T-PBS) to remove excess PFA. Nonspecific binding of secondary antibodies was blocked with 3% bovine serum albumin (BSA) diluted in 0.2% Triton-TX 100/ (Sigma-Aldrich) in 0.1 PBS for 1 h. Coverslips were incubated with primary antisera and diluted in T-PBS overnight (18 h) at 4°C. Sections were then washed in T-PBS and incubated for 1 h at room temperature with appropriate secondary antibodies conjugated to Alexa Fluor (Table 1). Sections were then washed in T-PBS before mounting in Prolong Gold Antifade with DAPI (ThermoFisher Scientific) and coverslipped. Slides were allowed to dry for 24 h before visualization.

Table 1.

Primary and secondary antisera details

	Species Raised in	RRID/AF Conjugate	Manufacturer	Dilution
Primary antigen
Cytokeratin 7 (clone OV-TL 12/30)	Mouse	AB_10989596	ThermoFisher	1:50
Secondary antigen
Mouse IgG1	Goat	488	ThermoFisher	0.18055556

AF, Alexa Fluor; RRID, Research Resources Identifier.

Microscopy.

Fluorescence was visualized with a confocal laser scanning microscope (Leica TCS SP8X; Leica Microsystems, Wetzlar, Germany). Images (1,024 × 1,024 pixels) were obtained using a ×63 lens (software zoom ×1.3), ×60 oil immersion (spinal cord slice) objective, and sequential scanning (4- to 5-line average). Separation of fluorophores was achieved using white line laser tuned to 495-nm excitation and 505- to 534-nm emission detection settings for AF-488 and 405-nm excitation and 425- to 475-nm emission detection settings for DAPI. Confocal settings were optimized to reduce background staining by adjusting the white light laser intensity, emission window (as described above), and amplifier gain [726.7 offset: −0.07 (AF488); 10 offset: −0.1 (DAPI)]. These settings were saved and used for all imaging.

Data analysis and Statistics.

Whole nerve multifiber afferent nerve activity was quantified using Spike2 software, which counted the number of action potentials crossing a preset threshold (Cambridge Electronic Design).

Data are presented as means ± SE or means ± 95% confidence interval (CI). Statistical analysis was carried out using either a one- or two-way ANOVA and Bonferroni multiple-comparisons post hoc test, Student’s t-test, or Kruskal-Wallis test with Dunn’s post hoc test, where normal distribution of data could not be confirmed. Significance was confirmed at P < 0.05 using GraphPad Prism 7 software (n = no. of mice).

RESULTS

We explored the relationship between bladder contraction and afferent discharge by applying neurokinin 1, 2, and 3 receptor agonists either to the bath (to evoke muscle responses directly) or into the bladder lumen (to determine any modulating influence from the urothelium).

NKA stimulates bladder afferents secondary to bladder contraction.

Using an ex vivo bladder afferent preparation, which allows the simultaneous recording of both intravesical pressure (a proxy for contraction) and concurrent afferent nerve activity, we found that NKA applied directly into the organ bath elicited a concentration-dependent increase in intravesical pressure and a concomitant increase in afferent nerve firing of a partially distended bladder (Fig. 1A). The contractile response consisted of an initial increase in tone upon which phasic contractile activity was superimposed, especially at the higher concentrations of NKA (Fig. 1A, inset). There was a close correlation between afferent nerve activity and contraction in both magnitude and time course (Fig. 1A, inset), with each phasic contraction accompanied by a phasic increase in afferent firing. To determine whether the NKA-induced increase in afferent firing was through direct activation of afferents or, as was indicated by the pattern of activation, secondary to muscle contraction, the preparation was incubated with the L-type calcium channel blocker nifedipine (1 µM) before NKA application (Fig. 1B, graphs i and ii). Nifedipine significantly attenuated NKA induced bladder contractions and associated bladder afferent firing (Fig. 1B, graph i) but had no effect on baseline afferent activity (Fig. 1B, graph ii). These data confirmed that NKA applied to the bladder serosal surface acts directly on the bladder muscle to subsequently induce an afferent response profile. This effect was specific to the NK2 receptor since substance P and senktide, which show greater selectivity for NK1 and NK3 receptors, respectively, exerted only minor bladder contraction and corresponding spikes in bladder afferent nerve activity (Fig. 1C, graph i). Moreover, when preparations were incubated with the specific NK2 receptor antagonist GR159897 (100 nM) before NKA application, we observed markedly attenuated contractile and afferent responses to NKA (Fig. 1C, graph ii).

Fig. 1.

Neurokinin A (NKA)-induced bladder contraction and afferent firing is inhibited by nifedipine. A: ex vivo experiments revealed a concentration-dependent relationship between increasing concentrations of NKA (10–3,000 nM) bath applied by bolus dose and bladder contractions and afferent discharge. The correlation between bladder contractions and concurrently recorded afferent firing to 300 nM NKA is shown (inset). Overall, NKA induced high-frequency phasic bladder contractions that were able to induce significant afferent nerve activity. B, graph i: the addition of the L-type Ca²⁺ channel blocker nifedipine (1 µM) into the ex vivo bladder preparation organ bath significantly attenuated NKA-induced (300 nM) bladder contraction (***P ≤ 0.001) and abolished NKA-induced afferent firing Data are presented as means ± 95% confidence interval (CI) (##P ≤ 0.01; n = 6 mice; 1-way ANOVA, Bonferroni multiple comparisons post hoc test). B, graph ii: experimental recording shows that NKA-induced (300 nM) bladder contraction and associated afferent firing was consistent when applied sequentially after a washout period. Incubation with nifedipine (1 µM) attenuated bladder contraction and concurrent afferent firing. C, graph i: substance P (300 nM) and the NK3 receptor agonist senktide (300 nM) bath applied by bolus dose to the ex vivo-afferent preparation induced significantly smaller bladder contractions than NKA (300 nM). Data are presented as means ± 95% CI (***P ≤ 0.001) and associated afferent discharge (###P ≤ 0.001) (n = 4 mice; 1-way ANOVA, Bonferroni multiple comparisons post hoc test). C, graph ii: experimental recording shows that NKA-induced (300 nM) bladder contraction and associated afferent firing is almost completely abolished by incubation with the specific NK2 receptor antagonist GR159897 (100 nM).

Because saline was infused into the bladder lumen at a controlled rate, and the volume in the bladder was increased,;there was a predictable increase in intravesical pressure. Initially, when the bladder volume was low, fluid was accommodated with only small increases in pressure (Fig. 2A). However, as the bladder filled, further intravesical pressure increased more steeply, as reflected in the volume/pressure curves (Fig. 2A). This relationship between volume and pressure during ramp distension of the bladder was used to evaluate the effect of NKA on bladder muscle compliance. Bladder compliance was decreased during bath application of NKA (Fig. 2A, graphs i and ii), particularly at lower volumes of distension, indicating that the bladder muscle had generated tone and was less able to stretch, which most likely a result of muscle contraction, as indicated by the effects of NKA in Fig. 1. Interestingly, bladder compliance increased in the presence of GR159897 (100 nM; Fig. 2A, graphs iii and iv), allowing the bladder to accommodate a greater volume during filling, suggesting a role for endogenous tachykinins in modulating bladder tone. Because there is significant variability in the volume of bladders from mice, mechanosensitivity of bladder afferents was determined by correlating afferent responses to intravesical pressure rather than volume. Using this analysis, we found that afferent firing was unchanged during ramp distension in the presence of NKA (300 nM) when normalized to intraluminal pressure (Fig. 2B, graph i). Despite this, when comparing the experimental traces, there was obvious enhancement of the afferent responses to distension at lower volumes (Fig. 2B, graph ii). To provide a more representative analysis of the data, we determined the volume at which afferent activity reached a threshold of 10 imp/s and showed that this occurs at a significantly lower volume in the presence of NKA (Fig. 2B, graph iii), and this volume is a significantly lower proportion of the total bladder volume (Fig. 2B, graph iv), indicating that NKA enhances the bladder afferent response to distension at low volumes.

Fig. 2.

The NK2 receptor mediates bladder compliance. A, graph i: infusion of the bladder ex vivo with saline (control; black line) increased intravesical pressure as the bladder volume increased and the muscle accommodated the increase in volume. Neurokinin A (NKA; 300 nM) added to the organ bath perfusion system (gray line) significantly decreased the ability of the bladder muscle to accommodate an increase in volume. Data are presented as means ± SE (n = 6 mice; 2-way ANOVA, Bonferroni multiple-comparisons post hoc test). A, graph ii: experimental trace from an ex vivo distension experiment showing the pressure/volume relationship inside the bladder before (control) and after the addition of NKA (300 nM). The addition of NKA to the bath perfusate increased the bladder pressure for a given volume. A, graph iii: addition of the specific NK2 receptor antagonist GR159897 (100 nM) to the organ bath perfusion system significantly increased bladder compliance, indicating an increased ability of the bladder to accommodate an increase in volume. Data are presented as means ± SE (n = 5 mice; 2-way ANOVA, Bonferroni multiple comparisons post hoc test). A, graph iv: experimental trace from an ex vivo bladder distension experiment showing the pressure volume relationship before (control) and after the addition of GR159897. The addition of NKA to the external bath perfusion decreased the bladder pressure for a given volume. B, graph i: ex vivo bladder-afferent nerve recordings reveal that neither NKA nor GR159897 had an effect on bladder-afferent sensitivity to ramp distension when normalized to pressure. Data are presented as means ± SE (P ≥ 0.05; n = 5–6 mice; 2-way ANOVA, Bonferroni multiple-comparisons post hoc test). B, graph ii: however, experimental trace of afferent nerve activity before (control; red) and after addition of NKA (blue) to the bath perfusion system shows an enhancement of bladder-afferent activity to distension in the presence of NKA (red) compared with control (blue) at lower volumes, which are overlooked when afferent firing to intravesical pressure is normalized. B, graph iii: analysis of intravesical volume at the point when afferent firing reaches 10 imp/s shows that NKA decreased the intrabladder volume required to elicit afferent firing (**P ≤ 0.01; n = 6 mice; paired t-test). B, graph iv: intrabladder volume as a percentage of total volume required to elicit afferent firing before and after NKA bath perfusion (**P ≤ 0.01; n = 6 mice; paired t-test).

Intravesical NKA alters detrusor compliance and spontaneous muscle contractions.

With bath application of NKA, there will likely be a concentration gradient as the drug diffuses from the serosa in toward the detrusor muscle, where it evokes contraction and from there into deeper layers. However, the powerful influence of NK2 receptors at the level of the muscle may overwhelm any modulating influences on receptors elsewhere in the bladder wall, particularly those expressed in the urothelium and suburothelial innervation. We hypothesized that intraluminal application of tachykinins would preferentially influence receptors in these more superficial locations. With intravesical NKA (300 nM), there was a significant decrease in bladder compliance (Fig. 3A, graph i), an effect that was reversed by prior intravesical infusion with the NK2 receptor antagonist GR159897 (100 nM; Fig. 3A, graph ii). However, unlike with bath application of GR159897 (100 nM), intravesical application of the NK2 antagonist alone had no effect on bladder compliance (Fig. 3A, graph ii), indicating a differential mechanism responsible for changes in compliance when NKA is applied serosally or intravesically. Analysis of mechanosensitive responses to bladder distension showed that neither intravesical NKA nor GR159897 influenced mechanosensitivity, when afferent firing was plotted against intravesical pressure (Fig. 3B, graphs i and ii). However, intravesical NKA had a novel modulating influence on the pressure/volume relationship and associated afferent firing during graded distension (Fig. 3C, graph i), an effect that was masked when the pressure/volume relationship was analyzed directly. The NKA induced phasic oscillations were prevented by GR159897 (Fig. 3C,graph ii), indicating an NK2-dependent mechanism. NKA induced phasic oscillations had increased frequency and amplitude (Fig. 4, A, graphs i and ii, and B) and were accompanied by enhanced bursts of concurrent bladder afferent nerve activity (Fig. 4, B and C) which were not apparent with saline infusion and were prevented by prior instillation of GR159897 (Fig. 4A, graphs i and ii). Analysis of the afferent firing bursts occurring within the more physiological range of distension (≤10 mmHg), showed that NKA enhances the proportion of firing that occurs at lower pressures (Fig. 4C, graph i). During saline distensions, the maximum afferent firing at ≤10mmHg accounted for only 61 ± 7.4% of the maximum firing frequency at 25 mmHg compared with 78 ± 6.1% in the presence of NKA (Fig. 4C, graph ii). These phasic bursts of afferent firing also persisted in the immediate postdistension period rather than returning rapidly to baseline as with control distensions. Because these changes occur at physiological levels of distension, it is feasible that this could impact on sensory mechanotransduction within the bladder, leading to abnormal signaling of bladder volume.

Fig. 3.

Neurokinin A (NKA) infused into the bladder lumen reduces bladder compliance. A, graph i: using the ex vivo bladder-afferent preparation, infusing NKA (300 nM) into the bladder lumen caused a significant reduction in bladder compliance throughout the entire ramp distension compared with infusion with saline (control). Data are presented as means ± SE (n = 6 mice; 2-way ANOVA, Bonferroni multiple-comparisons post hoc test). A, graph ii: prior infusion of the NK2 antagonist GR159897 (100n M) into the bladder lumen prevented NKA-induced (300 nM) changes in bladder compliance during ramp distension. Data are presented as means ± SE (P ≥ 0.05; n = 6 mice; 2-way ANOVA, Bonferroni multiple-comparisons post hoc test. B: perfusion of NKA (300 nM; graph i) or GR159897 (100 nM; graph ii) into the bladder had no effect on the pressure/afferent relationship during ramp distension. C, graph i: experimental trace from an ex vivo bladder preparation shows intravesical pressure before (control; black trace) and after infusion of NKA (red trace) into the bladder lumen. NKA reduced muscle compliance and enhanced spontaneous detrusor contractions during ramp distension. C, graph ii: the effect of NKA on reducing muscle compliance and evoking spontaneous detrusor contractions was blocked in the presence of GR159897 (green line), whereas GR159897 alone had no direct effect on muscle compliance alone (blue line) when infused intravesically into the bladder.

Fig. 4.

Neurokinin A (NKA) perfused into the bladder enhances spontaneous muscle contraction and associated afferent firing. A: analysis of intravesical pressure during graded bladder distension reveals that NKA (300 nM) is able to decrease bladder compliance and also significantly alter the profile of the distension by significantly increasing the amplitude (graph i) and frequency (graph ii) of spontaneous muscle contractions. Data are presented as means ± 95% confidence interval (CI) (*P ≤ 0.05 and **P ≤ 0.01; n = 6 mice; Kruskal-Wallis test, Dunn’s multiple-comparisons post hoc test). B: experimental trace of an ex vivo bladder-afferent preparation showing the potentiation of spontaneous muscle contractions during ramp distension in the presence of NKA (300 nM). The spontaneous contractions were accompanied by dramatic spikes in afferent nerve activity (inset). C, graph i: bursts of afferent firing associated with spontaneous detrusor contractions were enhanced at physiological distension pressures (≤10 mmHg) in the presence of NKA compared with saline (*P ≤ 0.05; n = 6; paired t-test). C, graph ii: although control distensions with saline also induce infrequent bursts of afferent firing, NKA-induced afferent firing bursts contributed a significantly greater percentage of the maximal ramp distension induced afferent response compared with saline (**P ≤ 0.01; n = 6; paired t-test).

NKA activates urothelial cells.

The data described above indicate that activation of NKA receptors induces bursts of mechanosensitive afferent firing secondary to changes in detrusor smooth muscle function, with a potential modulating influence from the urothelium. We further examined the ability of the urothelium to respond directly to NKA using primary mouse urothelial cultures (PMUCs) and compared this sensitivity to isolated DRG neurons retrogradely traced from the bladder. Primary cultures were confirmed to be of urothelial origin through positive staining with the transitional epithelial cell marker cytokeratin 7 (Fig. 5) Application of NKA to PMUCs induced a significant rise in intracellular calcium levels, as reflected by an increase in fluorescent emissions over the course of sustained application (Fig. 6A). The intracellular calcium response to NKA was characterized by a slow increase over the course of the response (60 s), which returned to baseline upon washout. Interestingly, application of NKA at the same concentration to isolated sensory DRG neurons failed to initiate calcium signals (Fig. 6, B and C), suggesting that the effects of NKA perfused into the bladder lumen are not a consequence of direct afferent nerve activation.

Fig. 5.

Cultured urothelial cell stain for cytokeratin 7 (CK7) representative confocal images of urothelial cells. Image i: negative control shows a lack of fluorescence following incubation with secondary antibody only (AF488) at 495-nm excitation and 505- to 534-nm emission. Nuclei staining positive for 4′6-diamidino-2-phenylindole (DAPI)-showing cells are present. Image ii: positive control shows that incubation with both primary (CK7) and secondary antibodies (AF488) emit fluorescence at 505- to 534-nm emission following 495-nm excitation. Image iii: following incubation with both primary (CK7) and secondary antibodies (AF488) and mounted with Prolong Gold Antifade with DAPI. Scale bars, 25 µm.

Fig. 6.

Neurokinin A (NKA) activates urothelial cells and inhibits acetylcholine release. A: calcium imaging of primary mouse urothelial cells reveals that NKA (300 nM) was able to induce sustained intracellular calcium entry in primary mouse urothelial cells (n = 41 cells from n = 5 mice). B: NKA (300 nM) applied to retrogradely traced bladder dorsal root ganglion (DRG) failed to evoke a rise in intracellular calcium (n = 71 cells from n = 5 mice). Neurons were shown to be viable with KCl (40 mM). C, image i: retrogradely traced bladder DRGs are identified under fluorescent microscope (488-nm excitation and 503- to 538-nm emission detection). White arrows indicate traced cells. C, image ii: low fluorescent ratio (F340/380) during continuous perfusion with control external solution indicates that cells are healthy before the start of experiment and neurons show extensive dendrite networks. C, image iii: the addition of NKA (300 nM) had no effect on intracellular calcium. C, image iv: the same neurons showed marked responses to KCl (40 mM). Calcium influx is indicated by an increase in fluorescent ratio (yellow and red color). D: ex vivo bladder distension to 30 mmHg (stretch) with saline (control) increased luminal ATP concentrations over basal levels, whereas NKA (300 nM) had no effect on luminal ATP release from the bladder at rest or during bladder distension. Data are presented as means ± 95% confidence interval (CI) (n = 6 mice; 1-way ANOVA, Bonferroni multiple-comparisons post hoc test). E: acetylcholine (ACh) is released into the bladder lumen at rest and was not significantly influenced by distension. Infusion of NKA (300 nM) into the bladder significantly reduced secreted ACh release both at rest and during bladder distension. Data are presented as means ± 95% CI (n = 6; 1-way ANOVA, Bonferroni multiple-comparisons post hoc test). **P ≤ 0.01

However, activation of urothelial cells could result in the subsequent release of a number of known neurotransmitters and neuromodulators, including acetylcholine (ACh), ATP, and prostaglandin E₂ (PGE₂), that are considered important for bladder signaling during stretch. We hypothesized that NKA would have a modulating influence on the release of such urothelial mediators, and therefore, we measured their concentration in supernatants collected from whole bladders at baseline and following bladder distension with either saline or NKA. Consistent with previous literature, we found that the bladder has a tonic release of ATP and ACh (Fig. 6, D and E) (3, 16, 39, 45). In the case of ATP, significantly higher amounts were released following bladder distension (Fig. 6D), consistent with previous reports (51). NKA had no significant effect on either basal or stretch-induced ATP release (Fig. 6D). In contrast, intravesical perfusion of NKA significantly reduced ACh release at rest and during bladder distension (Fig. 6E). These data imply that NKA, acting through functional NK2 receptors on urothelial cells, has the capacity to attenuate the release of ACh, which in turn may influence afferent firing.

DISCUSSION

These data provide a number of novel findings that have implications for our understanding of the way sensory signals are generated and modulated from within the bladder wall. This includes four major pieces of new evidence: 1) NKA directly influences urothelial cells regulating the release of ACh; 2) NK2 receptors on the urothelium may influence downstream processes to affect both muscle function and mechanosensitive afferent excitability; 3) NK2 receptor effects on muscle serve to modulate phasic motility (often referred to as micromotions), which because of the mechanosensitivity of bladder afferents gives rise to augmented signaling that could in turn alter micturition reflex function and sensations of bladder fullness; and 4) the finding that GR159897 alone alters bladder compliance raises the intriguing possibility that NK2 receptor-mediated mechanisms play a role in setting baseline bladder tone.

We have confirmed that NK2 but not NK1 or NK3 agonists induce significant voltage-gated calcium dependent detrusor contraction (37, 46), as indicated by their sensitivity to nifedipine (37). Moreover, the afferent nerve firing we observed in response to bath applied NKA was also diminished by nifedipine, indicating that the sensory response to NK2 receptor activation was secondary to detrusor contraction, most likely via mechanosensory afferents. This indirect effect of NK2 receptors on afferent function was further demonstrated by the absence of any direct effect of NKA on bladder-innervating DRG neurons in culture. Althiugh this does not directly recapitulate the environment that would be found at the peripheral nerve ending, it provides an important opportunity to study the effects of NKA on neuronal cell bodies in isolation, a condition that would otherwise be unachievable in any other currently developed experimental paradigm. These findings were further confirmed by our results showing that neither NKA nor GR159897 had any direct influence on afferent mechanosensitivity. Despite this, NKA significantly reduced detrusor muscle compliance during ramp distension, which led to an increase in afferent responses at lower distension volumes. These changes are consistent with the alterations in micturition patterns attributed to tachykinins (22, 41) and our own finding that NKA was able to cause robust and concentration-dependent contraction of the detrusor. The NKA-induced decrease in bladder compliance was reversed by GR159897, which, when applied alone, significantly increased bladder compliance during ramp distensions. Therefore, it is possible that tachykinins released from CSPANs in response to muscle stretch (25) are able to provide an endogenous tone to the bladder wall during distension either directly at the level of the muscle or secondary to actions on the urothelium or interstitial cells. A recent study investigating the sensory afferent innervation of the bladder indicates that the majority of afferents innervate the detrusor smooth muscle, but some endings also extend into urothelial layers (43). NK2 antagonists have previously been shown to reduce detrusor tone following distension to a set volume (25) and indirectly via depletion of neuropeptides (24). Furthermore, the majority of mutant mice lacking the preprotachykinin gene exhibit urinary retention and overflow incontinence (23). Therefore, we can hypothesize that an efferent function of mechanosensitive afferents innervating the detrusor smooth muscle is to release NKA, which in turn is able to act on the muscle (33) to control compliance during bladder distension. The importance of such a mechanism is highlighted by the observation that tachykinin immunoreactivity is enhanced during bladder disease/inflammation, with an upregulation of tachykinin-sensitive nerves in animal models of chronic inflammation and idiopathic detrusor overactivity in humans (5, 32, 40, 42). Accordingly, these changes have been considered responsible for the increase in the atropine-insensitive component of detrusor contraction observed in humans with bladder pathologies (53).

Sensory signals generated during distension stimulate the micturition reflex. However, phasic detrusor contractions and associated afferent activity have recently been recognized (17) and in our studies are a feature of physiological distension and are enhanced by NKA infusion into the bladder. A correlation between small detrusor contractions termed “micromotions” and increased sensation during the filling phase of cystometry has been observed in humans (14), and a number of studies have reported increased nonvoiding contractions in animal models of cystitis (8, 52) as well as significant increases in tachykinin-immunoreactive fibers (5). We observed potentiation of spontaneous contractile activity in an NK2-dependent manner similar to that observed in guinea pig whole bladder (21, 27). A striking feature of the current experiments, however, was the concurrent bursts of afferent nerve activity that were coupled to these micromotions, providing a mechanism by which enhanced detrusor contraction can translate to altered sensation. We hypothesize that the phasic bursting of sensory afferents described here is an adequate stimulus to facilitate the micturition reflex observed in previous studies (21, 27). As such, our data are consistent with a mechanism whereby increases in tachykinin levels, which induce enhanced micromotions and associated enhanced afferent output, can result in the development of altered sensory perception in OAB (19).

Although the mechanisms generating and coordinating these phasic bladder micromotions are not clear, they have been described in elegant detail (48) and have also previously been shown to couple to afferent activity (17). Bladder micromotions, as they have been termed (48), show remarkable similarity to the peristaltic waves found in the gastrointestinal tract, which are driven by interstitial cells of Cajal (44). A similar but less complex network of interstitial cells exists within the bladder wall of many species (11, 30, 49, 50) positioned between the urothelium and detrusor, and current research indicates that these cells are able to interact with smooth muscle cells to mediate phasic micromotions (49, 50). Interestingly, bladder interstitial cells express the receptors for neurotransmitters known to be released from the bladder urothelium (29), including acetylcholine and ATP, that could, therefore, have an important role in coordinating mechanosensory signals from afferents and urothelial cells.

In addition to a role of NKA upon smooth muscle, the NK2 receptor is expressed in urothelial cells (47), and NKA directly mediates isolated mucosal contractions via a proposed urothelial mechanism (38). Here, we demonstrate functional NK2 receptor expression in primary mouse urothelial cells by observing elevated intracellular calcium levels in response to NKA, although we were not able to differentiate between the various subtypes of urothelial cells known to make up the urothelial barrier. Primary afferents provide the major source of tachykinins within the bladder (26), whereas nonneuronal sites of tachykinin production have so far not been identified. The short half-life of tachykinins (9, 28) also precludes their presence in urine, and thus a physiological or pathophysiological role for NK2 receptors on the urothelium relies on a close apposition to sensory afferent endings. Indeed, the recent identification of bladder-afferent endings innervating the urothelium provides an anatomic basis for these two cell types to communicate (43). Furthermore, we observed significant changes in urothelial ACh release both at rest and during bladder distension following NKA application, but no changes in ATP release were observed, providing an environment by which NKA released from afferent endings can act upon the urothelium to influence urothelial mediators that have known effects on both the detrusor and afferent function. Previous studies have demonstrated that ATP mediates a positive feedback mechanism on ACh outflow from urothelial cells (16). Our observation that ACh release was changed in the absence of altered ATP release suggests that this feedback mechanism may be uncoupled in the intact bladder and may be due to the differential release mechanism for the two mediators with graded exocytotical release of ATP and ACh released independent of vesicular trafficking (31). An assay of luminal fluid such as in this study, however, reflects only the release of neurotransmitter by the highly differentiated surface umbrella cells and not the basal urothelial cells that have a closer apposition to interstitial cells, nerve endings, and the detrusor. Although we have not examined the link in this study, intrinsic spontaneous activity of the detrusor smooth muscle is shown to be modulated by the presence of the mucosa (13, 20), and it is possible that release of urothelial factors acting locally may contribute to the regulation of micromotions.

We conclude that tachykinins have the potential to modulate sensory signals generated within the bladder. This is in part mediated via an effect on detrusor tone but also secondary to the enhancement of micromotions that cause exaggerated phasic afferent firing during bladder distension. As such, this afferent firing may signal premature reflex voiding or give rise to bladder sensations that are a feature of OAB. Moreover, because tachykinin and CSPAN immunoreactivity levels increase following inflammation and injury (5, 32, 40, 42), it is likely that the effects of NKA are upregulated in human disease such that tachykinins released from peripheral afferent endings could contribute to clinically relevant bladder symptoms.

GRANTS

S. M. Brierley is an NHMRC R.D Wright Biomedical Research Fellow (APP1126378).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.G., R.C.-W., K.H.M., K. Mansfield, R.R., D.J.S., and D.G. conceived and designed research; L.G. and K. Mills performed experiments; L.G. and K. Mills analyzed data; L.G., R.C.-W., S.M.B., K. Mills, K.H.M., K. Mansfield, R.R., and D.G. interpreted results of experiments; L.G. prepared figures; L.G. and R.C.-W. drafted manuscript; L.G., R.C.-W., S.M.B., D.J.S., and D.G. edited and revised manuscript; L.G., R.C.-W., S.M.B., K. Mills, K.H.M., K. Mansfield, R.R., D.J.S., and D.G. approved final version of manuscript.