A recent study revealed that administration of OEtA counteracted the alterations in urodynamic variables provoked by intravesical PGE 2 in rats 18 . To the best of our knowledge, there is no data regarding the relationship between peripherally restricted endocannabinods and PGE 2 ‐induced nociceptive response of the bladder. Thus, in the present study, we investigated if URB937 could inhibit the changes in urodynamic variables and bladder afferent activities induced by intravesical PGE 2 instillation.

Prostaglandin E 2 (PGE 2 ) is an eicosanoid associated with fever, inflammation, pain, blood circulation and gastric mucosal protection via the activation of EP1–4 receptor subtypes 11 . For the LUT, elevated urinary levels of PGE 2 have been reported in patients with overactive bladder 12 . Intravesical administration of PGE 2 stimulates the micturition reflex in humans and rats, while administration of an EP1 receptor antagonist improves bladder storage function in rats and mice 13 - 15 , suggesting the involvement of PGE 2 via the EP1 receptor in bladder sensory function. In addition, i.v. administration of an EP1 antagonist relieved bladder pain in rats with cyclophosphamide‐induced cystitis 16 . Moreover, we have previously shown that PGE 2 significantly increases bladder mechanosensitive afferent activity of C‐fibres, but not Aδ‐fibres, in the rat 17 .

Cannabinoid receptors (CB 1 and CB 2 ) are physiologically activated by endogenous ligands such as N‐arachidonoylethanolamine (anandamide), which is degraded by fatty acid amide hydrolase (FAAH) 1 , 2 . As a key regulator of anandamide and related FAAs turnover in vivo , FAAH is involved in the control of endocannabinoid tone, which can modulate pain perception 1 - 4 . URB937 is a peripherally restricted FAAH inhibitor 5 , and findings that peripheral actions of the endocannabinoids system (ECS) are involved in the regulation of bladder function and dysfunction have generated an interest in targeting the ECS for the treatment of LUTS 6 - 8 . FAAH has been located in human, mouse, and rat bladder mucosa, and oleoyl ethyl amide (OEtA), a FAAH inhibitor that can pass the blood–brain barrier, altered bladder sensory functions in the rat 9 . We previously showed that inhibiting peripheral FAAH with URB937 depresses both Aδ‐ and C‐fibre activities of primary bladder afferents via CB 1 and CB 2 receptors in normal rats 10 .

PS was purchased from Sigma‐Aldrich (St. Louis, MO, USA). PGE 2 and URB937 [3′‐carbamoyl‐6‐hydroxy‐[1,1′‐biphenyl]‐3‐yl cyclohexylcarbamate] were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Tocrisolve was purchased from Tocris Bioscience (Bristol, UK). The stock solution of PGE 2 (10 −2 m) was prepared in absolute ethanol (stored at −80 °C) and then diluted in saline just before use. URB937 was dissolved in Tocrisolve ® at the dose of 10 mg/mL as a stock solution, and was diluted in saline just before use. Tocrisolve diluted in saline was used as the vehicle. URB937 and its vehicle were administered i.v. through a PE‐50 catheter placed in the right external jugular vein at a dose of 1 mg/kg. The doses of drug investigated were selected based on previous studies 4 , 5 , 10 , 17 , 25 .

The L6 dorsal root ganglion (DRG) was bilaterally dissected from four rats and immediately immersion‐fixed in 4% paraformaldehyde in PBS for 1 h. Specimens were then washed in PBS, frozen, cryosectioned (12 μm) and co‐incubated overnight (4 °C) with a mouse FAAH (1:200; Santa Cruz Biotech, Santa Cruz, CA, USA), or a rabbit CB 1 or a rabbit CB 2 (1:200; AbCam, Cambridge, UK) primary antibody. After rinsing in PBS, species‐directed secondary Green or Red Alexa antibodies (1:600, Santa Cruz, TX, USA) were applied to the sections to distinguish between primary antigens. The sections were examined under a fluorescence microscope (Leica DMR, Solms, Germany) with epi‐illumination and filter settings for Alexa Red, Alexa Green, and a dual filter. Images were captured with Leica DC 200 camera (IM50 image manager V 1.20). Control experiments without primary antibodies were performed. The specificity of the antibodies have previously been described 9 , 21 - 24 . The percentage of cell bodies that displayed co‐expression of investigated antigens were calculated as follows: (the amount of FAAH‐positive cell bodies that also expressed CB 1 or CB 2 /the total amount of FAAH‐positive cell bodies) × 100.

At the beginning of the experiments, recording was repeated three consecutive times at 5‐min intervals to evaluate reproducibility. The third recording served as the control (before) value, subsequently PGE 2 (100 μm) was instilled 3 min after i.v. administration of URB937 (1 mg/kg) or vehicle. The recording was repeated three times with 5‐min intervals and all the three cycle recordings were used to evaluate the time dependency.

To facilitate PGE 2 ‐penetration into the bladder urothelium, protamine sulphate (PS) solution (10 mg/mL, 0.3 mL) was instilled intravesically and kept in the bladder for 60 min just before the measurement. SAA activities were recorded during constant filling CMG with saline at a rate of 0.08 mL/min. Filling continued until an intravesical pressure of 30 cmH 2 O was reached, and bladder compliance was calculated between the start and end of this bladder filling. The SAA caused by pelvic nerve stimulation was also recorded before and after bladder filling, and confirmed to correspond with that caused by bladder filling.

In separate experiments, other rats were anesthetised with urethane (1.2 g/kg, i.p.) for SAA measurements. Body temperature was maintained by a heated blanket at 38 °C. SSA measurements were performed as previously described 10 , 17 . In brief, the left pelvic nerve was dissected from the surrounding tissue proximal to the major pelvic ganglion. A pair of silver electrodes was placed around the pelvic nerve. A PE‐50 catheter (Clay‐Adams) was inserted in the bladder. Both L6 dorsal roots were cut near their entrance into the spinal cord after the laminectomy. Fine filaments were dissected from the left L6 dorsal root and placed across shielded bipolar silver electrodes. Clearly different unitary action potentials of afferent fibres originating from the bladder were identified by electrical stimulation of the left pelvic nerve and bladder distention with saline. These action potentials were discriminated by the Spike2 (CED, Cambridge, UK) impulse shape recognition program, and action potentials of a maximum of three fibres were investigated at the same time during a single bladder filling. Conduction velocity of the identified action potential was calculated from the latency of response to electrical stimulation and the conduction distance between stimulation and recording sites, which was based on our anatomical data. Fibres were grouped based on conduction velocity; those with a conduction velocity of <2.5 m/s were considered to correspond to unmyelinated C‐fibres and those with a conduction velocity of ≥2.5 m/s to myelinated Aδ‐fibres.

CMG was performed as previously described 18 . In brief, 3 days after bladder catheter [polyethylene (PE)‐50, Clay Adams, Parsippany, NJ, USA] implantation, the rats were placed without restraint in metabolic cages and CMG performed with constant bladder instillation of saline (10 mL/h). After control registrations (30–45 min), PGE 2 (50 μm) was instilled (30 min) to induce experimental bladder overactivity (BO), then URB937 (0.1 or 1 mg/kg) or its vehicle was administered i.v. and recording continued for a further 30 min. The following CMG parameters were analysed: micturition pressure (MP; maximum bladder pressure during micturition), basal pressure (the lowest bladder pressure during filling), threshold pressure (bladder pressure immediately before the micturition contraction starts), flow pressure (bladder pressure at the time of start of flow of urine from the urethra), bladder capacity (BC; residual volume at the most recent previous micturition plus the volume of infused saline at micturition), micturition volume (MV; volume of expelled urine), residual volume (BC–MV), the micturition interval (MI; the period between two micturitions calculated from MP to MP), and the area under the pressure curve (AUC; the area under the intravesical pressure curve divided by the time from start to stop for the area analysis) 18 - 20 . When convenient, the percentage changes of above the variables were also compared within each study group by analysing CMG endpoints before and after treatment with vehicle or drug.

In all, 40 adult female Sprague–Dawley rats weighing 190–350 g [250–350 g for cystometry (CMG) and 190–249 g for single‐unit afferent activity (SAA) measurements] were used. These are standard sized rats that have previously produced reliable urodynamic and SAA responses 9 , 10 , 18 . The rats were maintained under standard laboratory conditions with a 12:12 h light:dark cycle, and free access to food and water. The protocol was approved by the Institutional Animal Care and Use Committees of the University of Tokyo and San Raffaele University, and conformed to National Institutes of Health (NIH) guidelines for the care and use of experimental animals.

Double stainings of the rat L6 DRG. ( A ) Alexa Green immunoreactivity (‐IR) for FAAH in nerve cell bodies. ( B ) Same section as in A describing Alexa Red CB 1 ‐IR in nerve cell bodies. ( C ) Merged plate for A and B depicting co‐expression (yellow) of FAAH‐IR and CB 1 ‐IR in nerve cell bodies of the L6 DRG. ( D ) Alexa Green‐IR for FAAH in nerve cell bodies. ( E ) Same section as in A describing Alexa Red CB 2 ‐IR in nerve cell bodies. ( F ) Merged plate for A and B depicting co‐expression (yellow) of FAAH‐IR and CB 2 ‐IR in nerve cell bodies of the L6 DRG ×200.

When compared with the control registration (before vehicle or drug administration), intravesical PGE 2 reduced MI, MV, and BC, and increased basal pressure and the AUC of all rats (Table 1 ). Whereas neither vehicle nor 0.1 mg/kg URB937 had significant effects on any of the urodynamic variables, 1 mg/kg of URB937 increased MI by 28 (1)%, MV by 34 (1)% and BC by 28 (1)%, and reduced the basal pressure by 39 (2)% and the AUC by 23 (1)%.

Discussion

In the present study, we used PS‐pretreatment and a higher dose of PGE 2 instillation (100 μm) to facilitate PGE 2 ‐penetration into the bladder urothelium for the SAA investigations. This is because of the time limitation (within 1 h) for preserving adequate conditions of afferent nerve fibres isolated for recording. It has been reported that PS‐exposure affects only epithelial cells while sparing the underlying layers 26, and we have found no relevant influences of PS‐exposure itself on the bladder afferent activities 27. Under these conditions, intravesical PGE 2 ‐instillation (100 μm) facilitated the mechanosensitive afferent activities of C‐fibres but not Aδ‐fibres in rats. The increased C‐fibre activity was abolished by URB937 pretreatment. The dose of URB937 was set at 1 mg/kg based on our findings that URB937 at 1 mg/kg, but not at 0.1 mg/kg, counteracted PGE 2 ‐induced BO at CMG. In addition, URB937 had an inhibitory effect on the Aδ‐fibre afferent activity during PGE 2 ‐instillation (100 μm). Our previous studies using the same in vivo procedure as the present study demonstrated that PGE 2 (100 μm) significantly increased activities of C‐fibres but not Aδ‐fibres 17, and that URB937 inhibited both Aδ‐ and C‐fibre afferent activities via CB 1 and CB 2 receptors in normal rats 10. Thus, the present results are consistent with the previous findings and additionally reveal that inhibition of peripheral FAAH by URB937 suppresses the PGE 2 ‐induced (100 μm) BO and mechanosensitive C‐fibre hyperactivity. Our present CMG measurements in conscious rats showed that PGE 2 instillation (50 μm) reduced MI, MV, and BC, and that 1 mg/kg of URB937 reversed these changes towards control values. We have recently reported that OEtA, another FAAH inhibitor, counteracted alterations of CMG variables (including MI, MV, and BC) provoked by intravesical PGE 2 (50 μm) 18. The present findings on afferent C‐fibres support the present and previous CMG results, and suggest that the peripheral ECS can act not only during normal bladder mechanosensation but also in abnormal situations, which are possibly accompanied with chemosensation or nociception.

Pathophysiological roles of PGE 2 and its receptors in bladder sensory disorders remain to be established. Ponglowhapan et al. 28 using immunohistochemistry reported that the expression of all EP receptors (EP1–4) were intense in the urothelium, and intermediate to low in the muscle and suburothelial layers regardless of gonadal status or gender in the canine bladder. Among the EP1–4 receptor subtypes, the EP1 receptor is presumed to be most responsible for the PGE 2 ‐induced BO. Mice lacking EP1 receptors had normal CMG results, but did not react to intravesical PGE 2 ‐instillation, which caused BO in wild‐type mice 29. PGE 2 is unlikely to act as a direct effector messenger along the efferent pathway of the micturition reflex, but rather acts as a neuromodulator of efferent or afferent neurotransmission 30. Ishizuka et al. 14 suggested that PGE 2 ‐instillation into the bladder stimulated release of tachykinins from nerves in the urothelium and suburothelial space of the rat bladder. Interestingly, Wang et al. 13 suggested that the urothelial EP1 receptor could be activated by PGE 2 to evoke ATP‐release from urothelium and to facilitate bladder mechanosensitive afferent activity, and the micturition reflex in mice. In addition, Nile et al. 31 using strips of urothelium lamina propria of the guinea‐pig bladder reported that acetylcholine (ACh) was released by PGE 2 ‐exposure, whereas cholinergic agonists induced a concentration‐dependent production of PGE 2 , which was inhibited by the presence of a nitric oxide (NO) donor. These previous observations suggest that complex signal interactions occur within the urothelial and suburothelial layers involving tachykinins, ATP, ACh, NO, and PGE 2 . Regarding the ECS, co‐expression of CB 1 and P2X 3 receptors in the urothelium and nerve structures of the bladder has been reported in the mouse bladder 32. Another previous study showed partial co‐localisation of CB 1 and calcitonin gene‐related peptide (CGRP) fluorescence in nerve fibres and terminals of rat and mouse bladders 33. CB 2 receptors have been localised on sensory nerves and urothelium, and on terminal varicosities that co‐express transient receptor potential vanilloid 1 (TRPV1) or CGRP 34. In addition, some CB 2 ‐positive nerve fibres had coinciding profiles with vesicular ACh transporter (VAChT)‐containing nerves 34, 35. Moreover, cannabinoids have been shown to suppress nociceptive transmission at a peripheral level including the DRG or at the spinal level in numerous pain investigations, and depending on agents or models used both CB 1 and CB 2 receptors involvement has been suggested 36, 37. We previously reported that CGRP‐positive L6 DRG neurones showed strong FAAH, CB 1 and CB 2 staining 10. In the present study, we found that 77% or 87% FAAH‐positive nerve cell bodies in DRG co‐expressed CB 1 or CB 2 immunoreactivity, respectively. Thus, it is conceivable that both CB receptors could be involved in the inhibitory actions of URB937 on PGE 2 ‐facilitated C‐fibre activity.

Our present study did not fully address the mechanisms involved in the counteraction of the FAAH inhibitor on BO and hypersensitivity induced by PGE 2 . Neuromodulators such as CGRP, ATP, and tachykinins in the bladder or DRG may be related to the PGE 2 ‐induced hyperactivity and its inhibition. We will investigate these undetermined possibilities in further studies.