Synergic Suppressive Effect of Silodosin and Imidafenacin on Non-Voiding Bladder Contractions in Male Rats with Subacute Bladder Outlet Obstruction.

OBJECTIVES: To investigate single or combined effect of silodosin, an α1A-adrenoceptor antagonist, and imidafenacin, an antimuscarinic agent, on bladder function in a subacute bladder outlet obstruction (BOO) model of male rats. METHODS: Partial BOO was created in male Wistar rats by ligating the urethra with a steel rod. On day 10 after surgery, when frequent voiding was most remarkable on voiding behavior measurement in a metabolic cage, cystometric investigations in a conscious restrained condition were conducted with cumulative intravenous administration of silodosin alone (0.1, 1, 10, and 100 µg/kg), imidafenacin alone (1, 3, and 10 µg/kg), or a combination of the two drugs. RESULTS: When compared with the Sham rats, the BOO rats showed an increase in bladder capacity, residual volume, mean amplitude and the number of non-voiding contractions (NVCs), accompanied with an increase in bladder weight. In the BOO rats, silodosin alone at 100 µg/kg significantly decreased the number of NVCs, whereas imidafenacin alone at 3 and 10 µg/kg significantly decreased both the number and mean amplitude of NVCs. The combination administration with lower doses (silodosin at 10 µg/kg and imidafenacin at 1 µg/kg) significantly suppressed both the number and mean amplitude of NVCs. CONCLUSIONS: The present results indicate a suppressive effect of silodosin or imidafenacin alone and a synergic effect of the combination of these two drugs on NVCs in a subacute BOO model of male rats.

Effects of Sildenafil, a Phosphodiesterase Type 5 Inhibitor, on the Primary Single Afferent Activity of the Rat Bladder.

OBJECTIVES: We investigated the direct effects of sildenafil, a phosphodiesterase type 5 inhibitor, on the single-unit mechanosensitive afferent activities (SAAs) primarily originated from the bladder in the rat. METHODS: Female Sprague-Dawley rats were anesthetized with urethane. SAAs were recorded from the left L6 dorsal roots and classified by conduction velocity as Aδ- or C-fibers. A catheter was inserted into the bladder dome, and a separate catheter was placed in the carotid artery and external iliac vein for monitoring of blood pressure and sildenafil-administration, respectively. After measuring control SAA during constant filling cystometry with saline, the procedure was repeated with cumulative intravenous administrations of sildenafil (1, 3 and 10 mg/kg). RESULTS: Thirteen single units were isolated (Aδ-fibers: n = 6, C-fibers: n = 7) from 11 rats. After sildenafil-administrations, SAAs of Aδ-fibers significantly decreased in a dose-dependent manner, whereas SAAs of C-fibers decreased significantly only at the highest dose used. In addition, blood pressure significantly decreased after sildenafiladministration even at the lowest dose used. Bladder compliance significantly increased after sildenafil administration at higher doses. CONCLUSIONS: These results indicate that sildenafil can inhibit Aδ-fibers (partly also C-fibers) of the primary bladder mechanosensitive afferents of the rat although these effects may be partially influenced by systemic hypotension. The present results support the view that the NO/cGMP signaling pathway plays an inhibitory role in the bladder afferent transduction, and thus improves storage symptoms.

Functional roles of bladder α1-adrenoceptors in the activation of single-unit primary bladder afferent activity in rats.

OBJECTIVES: To clarify the involvement of bladder α1-adrenoceptors (α1-ARs) in afferent pathways by investigating the effects of silodosin and BMY7378, selective α1A- or α1D-AR antagonists, respectively, on single-unit afferent nerve fibre activity (SAA) of the primary bladder afferent nerves and their relationship with bladder microcontractions in rats. MATERIALS AND METHODS: A total of 63 female Sprague-Dawley rats were anaesthetized with urethane. The SAA of Aδ and C fibres generated from the left L6 dorsal roots was determined using electrical stimulation of the left pelvic nerve and bladder distension. After measuring baseline SAA during constant filling cystometry, the procedure was repeated with i.v. (0.3-30 µg/kg) or intravesical (10 µm) administration of each antagonist. In separate rats, the bladder was filled with saline until the intravesical pressure reached 30 cmH2 O, and was kept under isovolumetric conditions, then the recording was performed with i.v.-administered vehicle and silodosin (0.3 µg/kg). RESULTS: A total of Aδ fibres and 33 C fibres were isolated from 63 rats. The SAA of Aδ fibres, but not C fibres, were dose-dependently decreased after both i.v. and intravesical administrations of each of the antagonists. In the experiments under bladder isovolumetric conditions, silodosin administration significantly decreased the SAA of Aδ fibres, but not C fibres, compared with vehicle administration. There were no significant effects on either the mean basal bladder pressure or microcontractions. CONCLUSION: The present study suggests that both α1A- or α1D-ARs in the rat bladder are involved in the activation of the bladder mechanosensory Aδ fibres during bladder filling, and that this activation may not be related to bladder microcontractions.

Functional role of the transient receptor potential melastatin 8 (TRPM8) ion channel in the urinary bladder assessed by conscious cystometry and ex vivo measurements of single-unit mechanosensitive bladder afferent activities in the rat.

OBJECTIVE: To evaluate the role of the transient receptor potential melastatin 8 (TRPM8) channel on bladder mechanosensory function by using L-menthol, a TRPM8 agonist, and RQ-00203078 (RQ), a selective TRPM8 antagonist. MATERIALS AND METHODS: Female Sprague-Dawley rats were used. In conscious cystometry (CMG), the effects of intravesical instillation of L-menthol (3 mm) were recorded after intravenous (i.v.) pretreatment with RQ (3 mg/kg) or vehicle. The direct effects of RQ on conscious CMG and deep body temperature were evaluated with cumulative i.v. administrations of RQ at 0.3, 1, and 3mg/kg. Single-unit mechanosensitive bladder afferent activities (SAAs) were monitored in a newly established ex vivo rat bladder model to avoid systemic influences of the drugs. Recordings were performed after cumulative intra-aortic administration of RQ (0.3 and 3 mg/kg) with or without intra-vesical L-menthol instillation (3 mm). RESULTS: Intravesical L-menthol decreased bladder capacity and voided volume, which was counteracted by RQ-pretreatment. RQ itself increased bladder capacity and voided volume, and lowered deep body temperature in a dose-dependent manner. RQ decreased mechanosensitive SAAs of C-fibres, and inhibited the activation of SAAs induced by intravesical L-menthol. CONCLUSION: Our results suggest that TRPM8 channels have a role in activation of bladder afferent pathways during filling of the bladder in the normal rat. This effect seems, at least partly, to be mediated via mechanosensitive C-fibres.

Selective inhibitory effect of imidafenacin and 5-hydroxymethyl tolterodine on capsaicin sensitive C fibers of the primary bladder mechanosensitive afferent nerves in the rat.

PURPOSE: Imidafenacin and fesoterodine are used to treat overactive bladder. Imidafenacin, fesoterodine and its active metabolite 5-hydroxymethyl tolterodine are muscarinic receptor antagonists. It is believed that these agents act on afferent nerves in addition to smooth muscle. We investigated the effects of imidafenacin and 5-hydroxymethyl tolterodine on single unit afferent activity of mechanosensitive capsaicin sensitive and insensitive primary bladder afferent nerve fibers in rats. MATERIALS AND METHODS: Female Sprague Dawley® rats were anesthetized. Single unit afferent activity was recorded from the L6 dorsal roots and classified by conduction velocity as that of Aδ or C fibers. After measuring control single afferent activity during constant filling cystometry the procedure was repeated with intravenous administration of imidafenacin (0.3 to 30 µg/kg) or 5-hydroxymethyl tolterodine (0.01 to 1 mg/kg) at cumulative doses with or without intravesical capsaicin or oxotremorine-M instillation. RESULTS: A total of 139 single unit afferent fibers were isolated from 111 rats, including 19 Aδ and 120 C fibers. Neither imidafenacin nor 5-hydroxymethyl tolterodine significantly affected the overall single unit afferent activity of Aδ or C fibers. Based on capsaicin sensitivity C fibers were divided into capsaicin sensitive and insensitive groups. Each antimuscarinic inhibited the single unit afferent activity of capsaicin sensitive C fibers but not of capsaicin insensitive C fibers at the highest dose. Moreover, oxotremorine-M facilitated single unit afferent activity in a proportion of C fibers. The facilitated single unit afferent activity was significantly attenuated by the highest dose of imidafenacin. CONCLUSIONS: These findings demonstrate that imidafenacin and 5-hydroxymethyl tolterodine can selectively inhibit capsaicin sensitive C fibers among mechanosensitive bladder afferents by antagonizing bladder muscarinic receptors.

Influence of urethane-anesthesia on the effect of resiniferatoxin treatment on bladder function in rats with spinal cord injury.

AIMS: We investigated the effect of resiniferatoxin (RTX)-treatment on cystometric parameters in the spinal cord injury (SCI) rats in both conscious and urethane-anesthetized conditions and evaluated the influence of urethane-anesthesia on the effect of RTX on lower urinary tract (LUT) function in SCI rats. METHODS: Female Sprague-Dawley rats were used. SCI was created by transection of the T8-T9 spinal cord. Four weeks after the transection, the animals were placed in a restraint cage for the first cystometric measurements in a conscious state. Secondary cystometric measurements were performed in a conscious condition following the 1 day after RTX-(0.3 mg/kg) or vehicle-subcutaneous injection. Then the animals were injected with urethane (1.5 g/kg, subcutaneously), and cystometric measurements were repeated four times every 1 hr-interval. RESULTS: After the RTX-treatment in a conscious condition, urinary retention was observed in three out of five animals. In addition, the number of non-voiding contractions (NVCs) significantly decreased although their amplitude did not change significantly. After the urethane-injection, all of the animals treated with RTX developed urinary retention. The amplitude of NVCs significantly decreased, whereas the number of NVCs did not change significantly in the RTX-treated group. No cystometric parameters significantly changed after either vehicle- or urethane-injection in the vehicle-treated group. CONCLUSIONS: The present results indicate that the suppressive effects of RTX on NVCs as well as voiding contractions in SCI rats can be enhanced by urethane-anesthesia. Such suppressive effect of urethane-anesthesia itself should be taken into consideration when we evaluate a drug-effect on LUT function in rats with SCI.

Defect of suppression of inflammasome-independent interleukin-8 secretion from SW982 synovial sarcoma cells by familial Mediterranean fever-derived pyrin mutations.

Familial Mediterranean fever (FMF) is a recessive inherited autoinflammatory syndrome. Patients with FMF have symptoms such as recurrent fever and abdominal pain, sometimes accompanied by arthralgia. Biopsy specimens have revealed substantial neutrophil infiltration into synovia. FMF patients have a mutation in the Mediterranean fever gene, encoding pyrin, which is known to regulate the inflammasome, a platform for processing interleukin (IL)-1ß. FMF patients heterozygous for E148Q mutation, heterozygous for M694I mutation, or combined heterozygous for E148Q and M694I mutations, which were found to be major mutations in an FMF study group in Japan, suffer from arthritis, the severity of which is likely to be lower than in FMF patients with M694V mutations. Expression plasmids of wild-type (WT) pyrin and mutated pyrin, such as E148Q, M694I, M694V, and E148Q+M694I, were constructed, and SW982 synovial sarcoma cells were transfected with these expression plasmids. IL-8 and IL-6 were spontaneously secreted from the culture supernatant of SW982 cells without any stimulation, whereas IL-1ß and TNF-α could not be detected even when stimulated with lipopolysaccharide. Notably, two inflammasome components, ASC and caspase-1, could not be detected in SW982 cells by Western blotting. IL-8 but not IL-6 secretion from SW982 cells was largely suppressed by WT pyrin, but less suppressed by mutated pyrin, which appeared to become weaker in the order of E148Q, M694I, E148Q+M694I, and M694V mutations. As for IL-8 and IL-6, similar results were obtained using stable THP-1 cells expressing the WT pyrin or mutated pyrins, such as M694V or E148Q, when stimulated by LPS. In addition, IL-8 secretion from mononuclear cells of FMF patients was significantly higher than that of healthy volunteers when incubated on a culture plate. Thus, our results suggest that IL-8 secretion from SW982 synovial sarcoma cells suppressed by pyrin independently of inflammasome is affected by pyrin mutations, which may reflect the activity in FMF arthritis.