Urinary bladder smooth muscle is innervated by both sympathetic and parasympathetic nerves. Acetylcholine released from postganglionic parasympathetic nerve terminals activates postjunctional muscarinic receptors in urinary bladder, which modulate urinary bladder contraction during the voiding phase and control detrusor tone during the filling phase. Five muscarinic receptor subtypes (M 1 -M 5 ) have been identified by both molecular biological and pharmacological investigations.1) The urinary bladder smooth muscle contains a mixed population of muscarinic M 2 and M 3 receptors.2) Although muscarinic M 2 receptors are numerically predominant, muscarinic M 3 receptors are considered to predominate in the mediation of bladder contraction.3,4) An important functional role of the muscarinic M 3 receptor in mediating bladder contraction has also been suggested in experiments using mutant mice lacking the muscarinic M 3 receptor gene. 5)Overactive bladder is characterized by symptoms of urgency and urinary frequency with or without urge incontinence. It has a profoundly negative effect on the quality of life of those affected. Muscarinic receptor antagonists are the most widely used therapy for overactive bladder.6-8) Solifenacin succinate [YM905; (3R)-1-azabicyclo[2.2.2]oct-3-yl(1S)-1-phenyl-3,4-dihydroisoquinoline-2(1H)-carboxylate monosuccinate] is a new muscarinic receptor antagonist developed for the treatment of overactive bladder. Affinity constants (K i values) of this drug for human muscarinic M 1 , M 2 and M 3 receptors only have been reported, along with its antagonism of the contractile effect of carbachol in isolated guinea pig urinary bladder.9) The present study was therefore undertaken to investigate the affinity of solifenacin for all human muscarinic receptor subtypes (M 1 -M 5 ) and its functional muscarinic M 3 receptor antagonism in rats, and to compare the results with those for tolterodine, oxybutynin, darifenacin, propiverine and atropine. Additionally, we also investigated the effect of solifenacin on voiding function in anesthetized rats. MATERIALS AND METHODS MaterialsSolifenacin succinate (YM905, Vesicare ® ), tolterodine tartrate, darifenacin and propiverine hydrochloride were prepared by Astellas Pharma Inc. (Tokyo, Japan). Oxybutynin chloride, atropine sulfate and carbachol (carbamylcholine chloride) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Darifenacin was dissolved in dimethyl sulfoxide and the others were dissolved in dimethyl sulfoxide, Krebs-Henseleit solution or physiological saline.Animals Male Wistar rats and male Sprague-Dawley rats were purchased from Charles River Laboratories Japan (Kanagawa, Japan) and Japan SLC (Shizuoka, Japan), respectively. In in vitro studies, rats were sacrificed by exsanguination under ether anesthesia. All animal experiments were performed in compliance with the regulations of the Institutional Animal Ethical Committee of Astellas Pharma Inc.Radioligand Receptor Binding Assay Membranes of Chinese hamster ovary (CHO)-K1 cells expressi...

Purpose-A new animal model that can evaluate bladder function and nociceptive behavior concurrently was developed using freely moving, non-catheterized conscious rats to assess nociceptive behavior responses induced by intravesical instillation of RTx and its relationship with bladder dysfunction.Materials and Methods-In female SD rats, RTx (0, 0.3 and 3μM) was instilled via a catheter temporarily inserted into the bladder through the urethra. Then, after removing the catheter, the incidence of nociceptive behavior (lower abdominal licking and freezing) was scored. Voided urine was collected continuously for the measurement of bladder capacity. In some animals, the pudendal nerves were transected bilaterally (PNT rats) in order to eliminate activation of urethral afferents by RTx.Results-Intravesical instillation of RTx induced decreased bladder capacity and increased nociceptive behaviors such as licking and freezing, which were blocked by BCTC, a TRPV1 antagonist. In PNT rats, the early phase of RTx-induced licking was decreased without affecting the RTx-induced reduction in bladder capacity and late-phase licking behavior, and RTx-induced latephase licking in the water-unloaded group was observed to a lesser extent compared with the waterloaded diuresis group.Conclusions-The intravesical instillation of RTx, which decreases bladder capacity, acts by at least three distinct mechanisms to induce licking behavior; (1) the immediate response mediated by activation of urethral afferents in the pudendal nerve, (2) a late occurring response evoked by direct stimulation of C-fiber afferents in the bladder and (3) gradual facilitation of the response elicited by bladder wall distension induced by rapid bladder filling.

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The contractile responses to capsaicin and anandamide, exogenous and endogenous agonists for transient receptor potential vanilloid receptor 1 (TRPV1), respectively, were investigated in muscle strips isolated from the rat urinary bladder. Capsaicin and anandamide produced concentration-dependent contractions of the muscle strips. The contractile response induced by capsaicin disappeared within approximately 20 min. In contrast, anandamide produced contractile responses lasting at least for 30 min. Capsaicin produced additive contractile responses in anandamide-treated muscle strips. The contractile response to anandamide was attenuated, but not abolished in strips desensitized by capsaicin. The response to capsaicin was abolished in the presence of a TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chlorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), but not altered in the presence of either tetrodotoxin, atropine or indomethacin. In the presence of SR140333, a tachykinin NK(1) receptor antagonist or SR48968, an NK(2) receptor antagonist, the response to capsaicin was attenuated. The response to anandamide was partially attenuated in the presence of ONO8130, a prostanoid EP(1) receptor antagonist, URB597, a fatty-acid amide hydrolase inhibitor, BCTC, SR140333 or SR48968, and almost completely abolished by indomethacin. Neither tetrodotoxin, atropine, a cannabinoid CB(1) receptor antagonist, AM251, nor a cannabinoid CB(2) receptor antagonist, AM630, had any effect on the response to anandamide. These results indicate that capsaicin produces muscle contractions by stimulating the TRPV1 receptor, followed by release of neuropeptides that can activate tachykinin NK(1) and/or NK(2) receptors in the bladder and that the contractile response to anandamide is mediated at least in part by activation of prostanoid EP(1) receptors due to production of prostaglandins in addition to TRPV1 receptor activation.