Muscarinic M3 positive allosteric modulator ASP8302 enhances bladder contraction and improves voiding dysfunction in rats.

OBJECTIVES: Muscarinic M3 (M3 ) receptors mediate cholinergic smooth muscle contraction of the bladder. Current drugs targeting bladder M3 receptors for micturition disorders have a risk of cholinergic side effects due to excessive receptor activation and insufficient selectivity. We investigated the effect of ASP8302, a novel positive allosteric modulator (PAM) of M3 receptors, on bladder function in rats. METHODS: Modulation of carbachol-induced increases in intracellular Ca2+ was assessed in cells expressing rat muscarinic receptors. Potentiation of bladder contractions was evaluated using isolated rat bladder strips and by measuring intravesical pressure in anesthetized rats. Conscious cystometry was performed to investigate the effects on residual urine volume and voiding efficiency in rat voiding dysfunction models induced by the α1 -adrenoceptor agonist midodrine and muscarinic receptor antagonist atropine, and bladder outlet obstruction. To assess potential side effects, the number of stools and tracheal insufflation pressure were measured in conscious and anesthetized rats, respectively. RESULTS: ASP8302 demonstrated PAM effects on the rat M3 receptor in cell assays, and augmented cholinergic bladder contractions both in vivo and in vitro. ASP8302 improved voiding efficiency and reduced residual urine volume in two voiding dysfunction models as effectively as distigmine bromide, but unlike distigmine bromide did not affect the number of stools or tracheal insufflation pressure. CONCLUSIONS: Our results in rats indicate that ASP8302 improves voiding dysfunction by potentiating bladder contraction with fewer effects on cholinergic responses in other organs, and suggest a potential advantage over current cholinomimetic drugs for treating micturition disorders caused by insufficient bladder contraction.

Potentiation of Muscarinic M3 Receptor Activation through a New Allosteric Site with a Novel Positive Allosteric Modulator ASP8302.

Muscarinic M3 (M3) receptors mediate a wide range of acetylcholine (ACh)-induced functions, including visceral smooth-muscle contraction and glandular secretion. Positive allosteric modulators (PAMs) can avoid various side effects of muscarinic agonists with their spatiotemporal receptor activation control and potentially better subtype selectivity. However, the mechanism of allosteric modulation of M3 receptors is not fully understood, presumably because of the lack of a potent and selective PAM. In this study, we investigated the pharmacological profile of ASP8302, a novel PAM of M3 receptors, and explored the principal site of amino-acid sequences in the human M3 receptor required for the potentiation of receptor activation. In cells expressing human M3 and M5 receptors, ASP8302 shifted the concentration-response curve (CRC) for carbachol to the lower concentrations with no significant effects on other subtypes. In a binding study with M3 receptor-expressing membrane, ASP8302 also shifted the CRC for ACh without affecting the binding of orthosteric agonists. Similar shifts in the CRC of contractions by multiple stimulants were also confirmed in isolated human bladder strips. Mutagenesis analysis indicated no interaction between ASP8302 and previously reported allosteric sites; however, it identified threonine 230 as the amino acid essential for the PAM effect of ASP8302. These results demonstrate that ASP8302 enhances the activation of human M3 receptors by interacting with a single amino acid distinct from the reported allosteric sites. Our findings suggest not only a novel allosteric site of M3 receptors but also the potential application of ASP8302 to diseases caused by insufficient M3 receptor activation. SIGNIFICANCE STATEMENT: The significance of this study is that the novel M3 receptor positive allosteric modulator ASP8302 enhances the activation of human M3 receptor by interacting with a residue distinct from the reported allosteric sites. The finding of Thr230 as a novel amino acid involved in the allosteric modulation of M3 receptors provides significant insight into further research of the mechanism of allosteric modulation of M3 and other muscarinic receptors.

Effects of neurokinin 3 receptor antagonist fezolinetant on hot flash-like symptoms in ovariectomized rats.

The majority of women experience vasomotor symptoms (VMS), such as hot flashes and night sweats, during the menopausal transition. Recent evidence strongly suggests a connection between neurokinin 3 (NK3) receptor signaling and VMS associated with menopause. The NK3 receptor antagonist fezolinetant is currently in phase 3 development for treatment of moderate to severe VMS associated with menopause. We investigated the pharmacological effects of repeated administration of fezolinetant on levels of sex hormones and gonadotropins, neuronal activity in the hypothalamus, and skin temperature as an index of hot flash-like symptoms in ovariectomized rats as a model of menopause. Ovariectomized rats exhibited several typical menopausal symptoms: hyperphagia, increased body weight, significantly decreased plasma estradiol levels, increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, and significantly increased skin temperature. Increased c-Fos expression (an indirect marker of neuronal activity) in median preoptic nucleus (MnPO) hypothalamic neurons was also observed in ovariectomized rats. Repeated oral administration of fezolinetant (1-10 mg/kg, twice daily) for 1 week dose-dependently reduced plasma LH levels without affecting estradiol or FSH levels, inhibited the activation of MnPO neurons, and attenuated hot flash-like symptoms. In addition, fezolinetant dose-dependently reduced hyperphagia and weight gain in ovariectomized rats. These preclinical findings suggest that fezolinetant attenuates hot flash-like symptoms via inhibition of neuronal activity in the MnPO of ovariectomized rats and provides further support for the ongoing clinical development of fezolinetant for the treatment of VMS associated with menopause.

Modulation of urinary frequency via type 1 lysophosphatidic acid receptors: Effect of the novel antagonist ASP6432 in conscious rats.

Bladder dysfunctions associated with benign prostatic hyperplasia are not sufficiently alleviated by current pharmacotherapies. Lysophosphatidic acid (LPA) is a phospholipid with diverse biological effects. LPA modulates prostate and urethral contraction via the type 1 LPA (LPA1) receptor, suggesting the potential of the LPA1 receptor as a therapeutic target. However, the role of LPA and the LPA1 receptor in bladder function has not been studied in vivo. We investigated the effects of LPA and the novel LPA1 receptor antagonist ASP6432 (potassium 1-(2-{[3,5-dimethoxy-4-methyl-N-(3-phenylpropyl)benzamido]methyl}- 1,3-thiazole-4-carbonyl)- 3-ethyl-2,2-dioxo-2λ6-diazathian-1-ide) on the micturition reflex in conscious rats using cystometry. Intravenous infusion of LPA decreased the micturition interval and threshold pressure with no apparent changes in baseline pressure or maximum intravesical pressure. ASP6432 inhibited the LPA-induced decrease in MI. In contrast, ASP6432 had no effect on the LPA-induced decrease in threshold pressure. Similarly, ASP6432 had no effect on either baseline pressure or maximum intravesical pressure. We also evaluated the effect of ASP6432 on the urinary frequency induced by the nitric oxide synthase inhibitor L-Nω-nitro arginine methyl ester (L-NAME). Intravenous L-NAME administration decreased the micturition interval. ASP6432 dose-dependently reversed the L-NAME-induced decrease in micturition interval. Our findings demonstrate for the first time that LPA causes bladder overactivity in rats. ASP6432 inhibited the LPA- and L-NAME-induced decrease in micturition interval, suggesting a significant role for the LPA1 receptor in regulating the functional capacity of the bladder. Our results also suggest the potential of ASP6432 as a novel therapy for the treatment of bladder dysfunction associated with lower urinary tract diseases.

ASP6432, a type 1 lysophosphatidic acid receptor antagonist, reduces urethral function during urine voiding and improves voiding dysfunction.

Current pharmacotherapies for voiding dysfunctions are in need of improvement. Lysophosphatidic acid (LPA) is a phospholipid that contracts the urethra by activating type 1 LPA receptors (LPA1). However, the role of LPA1 in regulating urethral tonus during urine voiding which primarily affects the voiding function has not been investigated. To elucidate the role of LPA1 in the regulation of urethral tonus during urine voiding, we investigated the effects of ASP6432, a novel LPA1 antagonist, and the α1-adrenoceptor antagonist tamsulosin on urethral perfusion pressure (UPP) at the filling phase (UPPbase) and the minimum UPP at the voiding phase (UPPnadir) in anesthetized rats under isovolumetric conditions. We further evaluated the effects of ASP6432 and tamsulosin on voiding dysfunction characterized by changes in post-void residual urine (PVR) and voiding efficiency (VE) induced by the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) in conscious rats using single cystometry. ASP6432 dose-dependently decreased UPPbase and UPPnadir, while tamsulosin reduced UPPbase but did not change UPPnadir. ASP6432 dose-dependently suppressed the L-NAME-induced increase in PVR and decrease in VE, whereas tamsulosin did not affect either PVR or VE. We demonstrate that ASP6432 reduced UPPnadir and ameliorated L-NAME-induced voiding dysfunction, neither of which were affected by tamsulosin. Our study results suggest that LPA1 has a significant role in regulating urethral tonus during urine voiding, and highlight the potential of ASP6432 for improving voiding dysfunctions associated with various lower urinary tract diseases.

Effect of ASP6432, a Novel Type 1 Lysophosphatidic Acid Receptor Antagonist, on Urethral Function and Prostate Cell Proliferation.

Current pharmacotherapies for lower urinary tract symptoms associated with benign prostate hyperplasia (LUTS/BPH) are in need of improvement. Lysophosphatidic acid (LPA) is a phospholipid with various biologic functions. However, its exact role in the lower urinary tract and its target receptor subtype have not been fully elucidated. We investigated the role of LPA and the type 1 LPA receptor (LPA1) in urethral/prostatic contractile function and prostate cell proliferation by pharmacologically characterizing ASP6432 (potassium 1-(2-{[3,5-dimethoxy-4-methyl-N-(3-phenylpropyl)benzamido]methyl}-1,3-thiazole-4-carbonyl)-3-ethyl-2,2-dioxo-2λ6-diazathian-1-ide), a novel LPA1 antagonist. ASP6432 exhibited potent and selective antagonistic activity against LPA1 in cells expressing LPA receptor subtypes. In isolated rat tissue strips and anesthetized rats, ASP6432 concentration-/dose-dependently inhibited LPA-induced urethra and prostate contractions. In addition, in anesthetized rats, ASP6432 maximally decreased the urethral perfusion pressure (UPP) in the absence of exogenous LPA stimulation by 43% from baseline, whereas tamsulosin, an α1-adrenoceptor antagonist, reduced UPP by 22%. Further, in human prostate stromal cells, ASP6432 significantly and concentration-dependently suppressed LPA-induced bromodeoxyuridine incorporation. These results demonstrate a pivotal role for LPA and LPA1 in the regulation of urethral tonus and prostate cell proliferation. The potent urethral relaxation and inhibition of prostatic stromal cell growth indicate the potential of ASP6432 as a novel therapeutic agent for LUTS/BPH.

Pharmacological profile of the selective ß3-adrenoceptor agonist mirabegron in cynomolgus monkeys.

Mirabegron is a novel ß3-adrenoceptor agonist developed for the treatment of overactive bladder. To clarify the relationship between the pharmacological effects of mirabegron in monkeys and the clinical efficacy in patients with overactive bladder, the effect of mirabegron on bladder function was evaluated using cynomolgus monkeys. Quantitative PCR revealed that mRNA expression of ß3-adrenoceptors was most abundant (98 %) among ß-adrenoceptor subtypes in the bladder of cynomolgus monkeys. Mirabegron, which showed selective and potent agonistic activity on monkey ß3-adrenoceptors expressed in Chinese hamster ovary cells with EC50 value of 32 nmol/L and intrinsic activity of 0.8, induced concentration-dependent relaxation of bladder smooth muscle strips isolated from cynomolgus monkeys with EC50 values of 120 nmol/L in 20 mmol/L KCl stimulation and 43 nmol/L under 9.81 mN resting tension. In conscious cynomolgus monkeys, mirabegron decreased micturition frequency at oral doses of 1 and 3 mg/kg and increased mean volume voided per micturition at an oral dose of 3 mg/kg. Plasma concentration at which bladder function improved in the cynomolgus monkeys was similar to that at the clinically effective dose in patients with overactive bladder. These data suggest that the relaxant function in monkey bladder is mainly mediated by ß3-adrenoceptors similar to that in the human bladder and mirabegron showed efficacy on the bladder functions of the same parameters in clinical evaluation endpoints.

Effect of mirabegron, a novel ß3-adrenoceptor agonist, on bladder function during storage phase in rats.

Mirabegron, a selective ß(3)-adrenoceptor agonist, facilitates urine storage function by exerting a relaxing effect on bladder smooth muscle. Here, we investigated the effect of mirabegron on bladder function during the storage phase. We assessed the effect of mirabegron on the resting intravesical pressure in anesthetized rats and also tested antimuscarinics (oxybutynin and tolterodine) under the same experimental conditions. Mirabegron dose-dependently decreased the resting intravesical pressure, while oxybutynin and tolterodine showed no statistically significant effects on resting intravesical pressure. We also investigated the effect of mirabegron on bladder function using cystometry technique in conscious rats with bladder outlet obstruction. While mirabegron dose-dependently decreased the frequency of nonvoiding contractions, considered an index of abnormal response in bladder storage, no significant effects were noted on the amplitude of nonvoiding contractions, micturition pressure, threshold pressure, voided volume, residual volume, or bladder capacity. Neither oxybutynin nor tolterodine affected the frequency of nonvoiding contractions; however, oxybutynin increased residual volume and tended to decrease voided volume in a dose-dependent manner, and tolterodine dose-dependently decreased voided volume. Taken together, these results shed light on the suggestion of mirabegron as a therapeutic agent, compared with antimuscarinics, with its most prominent effect being the facilitation of bladder storage.

In vitro and in vivo pharmacological profile of the selective ß3-adrenoceptor agonist mirabegron in rats.

To investigate the pharmacological properties of mirabegron in in vitro and in vivo, the effects on cAMP accumulation in Chinese hamster ovary (CHO) cells expressing rat ß-adrenoceptors, the relaxant activity in isolated rat bladder smooth muscle, and the voiding effects in cerebral infarcted rats were evaluated. Mirabegron increased cAMP accumulation with EC(50) value and intrinsic activity of 19 nmol/L and 1.0, respectively, in CHO cells expressing rat ß(3)-adrenoceptors. The EC(50) values and the intrinsic activities of mirabegron were 610 nmol/L and 0.6 for rat ß(1)-adrenoceptors and were sumless and 0.1 for ß(2)-adrenoceptors, respectively. Mirabegron showed concentration-dependent relaxant and full agonistic effects in rat bladder strips under passive tension with EC(50) value of 290 nmol/L. The concentration-response curve of mirabegron was affected neither by the ß(1)-adrenoceptor selective antagonist CGP-20712A nor by the ß(2)-adrenoceptor selective antagonist ICI-118,551. In in vivo studies with cerebral infarcted rats, a significant decrease in the volume voided per micturition compared with sham-operated rats was observed. Mirabegron dose-dependently increased the volume voided per micturition. In conclusion, we have extended the selectivity profile of mirabegron to rats and demonstrated that it is effective via stimulation of ß(3)-adrenoceptors in a rat cerebral infarction model of detrusor overactivity.

Effects of α1-adrenoceptor antagonists on phenylephrine-induced salivary secretion and intraurethral pressure elevation in anesthetized rats.

α(1)-Adrenoceptor antagonists are widely used for the treatment of voiding dysfunction associated with benign prostatic hyperplasia. Activation of α(1)-adrenoceptors is reported to induce salivary secretion in rats and humans. However, the effects of α(1)-adrenoceptor antagonists on salivary secretion remain unknown. Here, we investigated the effects of the α(1)-adrenoceptor antagonists prazosin, silodosin, tamsulosin and urapidil on phenylephrine-induced salivary secretion and compared the results with the effects on phenylephrine-induced intraurethral pressure (IUP) elevation in anesthetized rats. All antagonists inhibited phenylephrine-induced salivary secretion and IUP elevation in a dose-dependent fashion. Comparison of DR(10) values (the dose required to shift the dose-response curve 10-fold to the right) in both tissues showed that the inhibitory effect of silodosin was significantly more potent in the salivary gland than in the urethra (18-fold), but tamsulosin (2.3-fold), prazosin (1.7-fold) and urapidil (1.1-fold) did not show comparable tissue selectivity. These results suggest that α(1)-adrenoceptor antagonists inhibit not only urethral contraction but also salivary secretion, and that high tissue selectivity for the salivary gland over the urethra as shown by silodosin may contribute to the incidence of dry mouth.

Tamsulosin potently and selectively antagonizes human recombinant α(1A/1D)-adrenoceptors: slow dissociation from the α(1A)-adrenoceptor may account for selectivity for α(1A)-adrenoceptor over α(1B)-adrenoceptor subtype.

We determined the binding affinity of tamsulosin, a selective α(1)-adrenoceptor antagonist, for human α(1)-adrenoceptor subtypes in comparison with those of other α(1)-adrenoceptor antagonists including silodosin, prazosin, 5-methylurapidil, terazosin, alfuzosin, nafopidil, urapidil and BMY7378. The association and dissociation kinetics of [(3)H]tamsulosin for recombinant human α(1)-adrenoceptor subtypes were compared with those of [(3)H]prazosin. Tamsulosin competitively inhibited [(3)H]prazosin binding to human α(1A)-, α(1B)- and α(1D)-adrenoceptors (pK(i) values were 10.38, 9.33, 9.85) indicating 11 and 3.4-fold higher affinities for human α(1A)-adrenoceptor than those for α(1B)- and α(1D)-adrenoceptors, respectively. The affinity of tamsulosin for the human α(1A)-adrenoceptor was, respectively, 5, 9.9, 38, 120, 280, 400, 1200 and 10000 fold higher than those of silodosin, prazosin, 5-methylurapidil, terazosin, alfuzosin, naftopidil, urapidil and BMY7378, respectively. [(3)H]Tamsulosin dissociated from the α(1A)-adrenoceptor slower than from the α(1B)- and α(1D)-adrenoceptors (α(1B)>α(1D)>α(1A)). Moreover, [(3)H]tamsulosin dissociated slower than [(3)H]prazosin from the α(1A)-adrenoceptor and faster from the α(1B)- and α(1D)-adrenoceptors. In conclusion, tamsulosin potently and selectively antagonized α(1A/1D)-adrenoceptor ligand binding, and slowly dissociated from the α(1A)-adrenoceptor subtype.

Synergistic Effect by Co-Administration of Tamsulosin and Solifenacin on Bladder Activity in Rats.

OBJECTIVES: We examined the effects of alpha1-adrenoceptor antagonist (tamsulosin hydrochloride) and antimuscarinic agent (solifenacin succinate) alone or in combination on the urinary adenosine triphosphate (ATP) level and cystometric parameters before and after bladder stimulation. METHODS: Female rats were administered tamsulosin hydrochloride (0.5 or 3 µg/kg/h) and/or solifenacin succinate (20 or 100 µg/kg/h) via a subcutaneously implanted osmotic minipump. Rats receiving distilled water were used as control. After 2 weeks, continuous cystometry with physiological saline or 0.1% acetic acid solution was performed. Urinary ATP level was also measured before and after stimulation by 0.1% acetic acid solution. RESULTS: During cystometry with bladder stimulation, the interval between voiding became shorter and the maximum voiding pressure (MVP) became higher in the control group. In the high-dose tamsulosin and solifenacin groups, the inhibition of urinary frequency was observed. The MVP also became higher in the high-dose tamsulosin group, but such a change was not seen in the high-dose solifenacin group. In case of low-dose administration, either agent alone did not inhibit the increase of urinary frequency and MVP due to bladder stimulation. However, co-administration of these ineffective low doses of tamsulosin and solifenacin resulted in the inhibition of urinary frequency. The high-dose or low-dose solifenacin group and the co-administration group showed similar inhibition of the increase of urinary ATP after bladder stimulation. CONCLUSION: Tamsulosin may have a different effect on the bladder and/or the neuronal pathways that is unrelated to ATP, so the combination of tamsulosin and solifenacin may synergistically inhibit urinary frequency after bladder stimulation.

Effect of tamsulosin on bladder blood flow and bladder function in a rat model of bladder over distention/emptying induced bladder overactivity.

PURPOSE: Decreased bladder blood flow was the subject of a recent study as a pathophysiological cause of bladder overactivity. We developed a rat model of bladder over distention/emptying induced bladder overactivity and investigated the effect of the α(1)-adrenoceptor antagonist tamsulosin on bladder blood flow and bladder function in this model. MATERIALS AND METHODS: The bladder was distended with 2 ml saline using anesthesia for 2 hours (over distention) and then emptied. Bladder blood flow was measured using a perfusion imager. Micturition behavior and parameters were observed using a metabolic cage and a cystometry method, respectively, from 2 hours after bladder emptying. After model establishment was confirmed we examined the participation of afferent C-fibers and the effects of tamsulosin in rats pretreated with capsaicin (Sigma-Aldrich®) (125 mg/kg) and tamsulosin (1 µg/kg per hour), respectively, using a metabolic cage. RESULTS: Decreased bladder blood flow was observed upon over distention with partial recovery at emptying. Bladder over distention/emptying increased micturition frequency and decreased mean voided volume in the micturition recording study, and decreased the intercontraction interval and voided volume without affecting micturition pressure, threshold pressure or post-void residual volume in the cystometry study. Capsaicin pretreatment did not affect bladder overactivity. However, 1-week continuous treatment with tamsulosin increased bladder blood flow after bladder emptying, resulting in decreased micturition frequency and increased voided volume. CONCLUSIONS: Bladder over distention/emptying induced bladder blood flow decrease/partial recovery and caused bladder overactivity via a mechanism other than capsaicin sensitive C-fiber activation. Findings in tamsulosin treated rats confirmed the potency of tamsulosin to increase bladder blood flow and ameliorate bladder overactivity.

Effect of tamsulosin on bladder microcirculation in a rat ischemia-reperfusion model, evaluated by pencil lens charge-coupled device microscopy system.

OBJECTIVES: To investigate the effect of tamsulosin hydrochloride on bladder microcirculation in a rat ischemia-reperfusion model using a pencil lens charge-coupled device microscopy system (PLCMS). METHODS: Changes in blood flow through a submucosal capillary of the rat bladder were measured during bladder filling using the PLCMS. One week after starting infusion of either physiological saline or tamsulosin, blood flow in the bladder was halted by bladder overdistention via an infusion of physiological saline. The bladder was then emptied to be reperfused with blood. Changes in blood flow through a submucosal capillary of the bladder during ischemia and reperfusion were measured using a PLCMS, and the data obtained for the control group and tamsulosin group were compared. RESULTS: As the bladder was distended, the velocity of red blood cell flow in a submucosal capillary of the bladder slowed and stopped altogether when the bladder became overdistended. In the control group, capillary blood flow improved over time after release from overdistention but failed to return to the baseline level, demonstrating that reperfusion injury to bladder microcirculation was caused by bladder overdistention and emptying. In the tamsulosin group, capillary blood flow rapidly returned to baseline after release from overdistention. CONCLUSIONS: Using a PLCMS, bladder microcirculation was able to be visualized and quantitatively assessed by measuring the velocity of blood flow in a submucosal capillary of the bladder. Findings from the present study suggest that tamsulosin hydrochloride exerts a protective effect on blood flow in ischemia-reperfusion injury of the bladder.

The forefront for novel therapeutic agents based on the pathophysiology of lower urinary tract dysfunction: ameliorative effect of solifenacin succinate (Vesicare), a bladder-selective antimuscarinic agent, on overactive bladder symptoms, especially urgency episodes.

Overactive bladder (OAB) syndrome is a common condition that is most often observed in the elderly. Pharmacological treatment with muscarinic receptor antagonists has been most widely used for OAB. An antimuscarinic agent, solifenacin, showed the highest affinity for the muscarinic M(3) receptor, which mediates urinary bladder contraction. In preclinical studies, solifenacin exhibited a highly bladder-selective profile compared with other antimuscarinic agents. Solifenacin was also shown to increase bladder capacity without affecting residual urine in an OAB model of rats. Urgency is now considered to result from overactivation of afferent nerves from the urinary bladder. It has been reported that afferent nerves are located adjacent to the urothelium, and stimulation of muscarinic receptors expressed on the urothelium may contribute to the activation of afferent nerves via non-neuronal ATP release. Solifenacin produces its inhibitory effect on bladder afferent activity partly via the suppression of non-neuronal ATP release. Clinically, solifenacin ameliorates all symptoms in OAB patients; and in particular, it produces a significant decrease in urgency episodes, which is the principal symptom of OAB. The pharmacological profile of solifenacin is therefore considered to contribute to its beneficial effects of high efficacy against OAB symptoms with good tolerability.

Zolpidem increases bladder capacity and decreases urine excretion in rats.

AIMS: To clarify the effects of zolpidem, a gamma-aminobutyric acid (GABA)(A) receptor agonist, on bladder function, and urine production, we investigated the effects of zolpidem administration on bladder overactivity induced by cerebral infarction (CI) and on urine excretion increased by water overloading in Wistar rats. METHODS: CI was induced by left middle cerebral artery occlusion. The effects on bladder function of zolpidem alone or in combination with the GABA(A) receptor antagonist bicuculline, were then examined in the CI rats using cystometry. The antidiuretic effect of zolpidem was investigated in water-loaded and Brattleboro rats (genetically vasopressin-deficient). Blood samples were collected from water-loaded rats to determine the aldosterone level 1 and 6 hr after zolpidem administration. RESULTS: Zolpidem increased bladder capacity dose-dependently, but had no significant effect on bladder contraction pressure in CI rats. Bicuculline dose-dependently inhibited zolpidem-induced increases in bladder capacity without affecting bladder contraction pressure. Zolpidem dose-dependently decreased the volume of urine excreted in water-loaded and Brattleboro rats. Compared with the control group, zolpidem significantly increased the aldosterone concentration in the plasma of water-loaded rats 1 hr after administration. CONCLUSIONS: Zolpidem increased bladder capacity via a GABAergic mechanism in CI rats, and suppressed urine excretion via a pathway that was not through activation of vasopressin V(2) receptors in water-loaded and Brattleboro rats. These results suggest that zolpidem may improve nocturia via an increase in bladder capacity and a decrease in urine excretion.

Effects of tamsulosin on bladder blood flow and bladder function in rats with bladder outlet obstruction.

OBJECTIVES: To investigate the mechanism underlying the ameliorating effect of tamsulosin, an alpha(1)-adrenoceptor antagonist, on storage symptoms associated with benign prostatic hyperplasia, the effects of tamsulosin on bladder blood flow (BBF) and bladder function was evaluated in rats with bladder outlet obstruction (BOO). METHODS: BOO was produced by partial ligature of the proximal urethra, which was maintained for 2 weeks. Tamsulosin was subcutaneously administered via an osmotic pump for 2 weeks immediately after the BOO surgery. The BBF in the sham-operated rats, the control BOO rats, and the tamsulosin-treated BOO rats was measured using the fluoromicrosphere method. Each rat was kept in a metabolic cage for observation of micturition behavior. Expression of the alpha(1)-adrenoceptor subtype mRNA in the vesical artery was measured by reverse transcriptase-polymerase chain reaction. RESULTS: BBF was significantly reduced in BOO rats compared with sham-operated rats, and tamsulosin significantly increased the BBF in BOO rats. Tamsulosin ameliorated the decrease in mean voided volume in BOO rats with bladder masses < 500 mg. Expression of the alpha(1)-adrenoceptor subtype in the vesical artery was alpha(1a)- > alpha(1d)-adrenoceptors; almost no expression was observed of alpha(1b)-adrenoceptors in either sham-operated or BOO rats. CONCLUSIONS: Tamsulosin increased BBF in BOO rats via an antagonistic effect, presumably on the alpha(1A)- and/or alpha(1D)-adrenoceptor in the vesical artery mainly, and improved the decrease in mean voided volume. Therefore, the results of this study suggest that tamsulosin improves bladder overactivity via improvement of BBF.

Comparison of muscarinic receptor selectivity of solifenacin and oxybutynin in the bladder and submandibular gland of muscarinic receptor knockout mice.

Solifenacin is a novel selective antagonist of M(3) muscarinic receptor developed for the treatment of overactive bladder. The current study was undertaken to characterize in vivo muscarinic receptor subtype selectivity of solifenacin in the bladder and submandibular gland by using muscarinic receptor subtype knockout (KO) mice. Muscarinic receptors in the bladder and submandibular gland of wild type, M(2)R KO and M(3)R KO mice under in vitro and after oral administration of solifenacin and oxybutynin were measured by radioligand binding assay using [N-methyl-(3)H]scopolamine ([(3)H]NMS). There was little difference between the bladder and submandibular gland of M(2)R KO mice in the receptor binding activities of oxybutynin and solifenacin in vitro, suggesting equal affinity for residual (predominantly M(3) subtype) muscarinic receptors in both tissues. In contrast, compared with oral oxybutynin, oral administration of solifenacin exerted a significantly greater activity to bind muscarinic receptors in the bladder of M(2)R KO mice, while exhibiting a significantly less activity to bind those in the submandibular gland. In the bladder and submandibular gland of M(3)R KO mice, the binding activity of solifenacin and oxybutynin showed no significant difference. Plasma concentrations of solifenacin and oxybutynin after oral administration differed little among wild type, M(2)R KO and M(3)R KO mice. The results indicate that oral solifenacin, unlike oral oxybutynin, may selectively bind to the muscarinic M(3) subtype in the bladder compared with such receptors in the submandibular gland in vivo. Oral solifenacin may be advantageous for the treatment of overactive bladder, in terms of high affinity for M(3) receptors in the bladder.

In vitro and in vivo effects of endothelin-1 and YM598, a selective endothelin ET A receptor antagonist, on the lower urinary tract.

We investigated the contractile response of the lower urinary tract to endothelin-1 in vitro (rabbits) and in vivo (dogs). We also assessed the effects of a selective endothelin ET A receptor antagonist, (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2, 2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), on endothelin-1-induced contractile responses. In the in vitro study, endothelin-1 induced contractile responses in isolated rabbit bladder base, urethra, and prostate tissues. YM598 (10(-7)-10(-5) M) antagonized these endothelin-1-induced contractile responses without affecting the maximal responses. In the in vivo study, endothelin-1 induced the elevation of non-prostatic urethral pressure as well as prostatic urethral pressure even in the presence of tamsulosin (10 microg/kg, i.v.) in anesthetized male dogs. YM598 (0.1-3 mg/kg, i.v.) inhibited these endothelin-1-induced contractile responses in a dose-dependent fashion. These results suggest that endothelin ET A receptors play an important role in the lower urinary tract contraction, and that the selective endothelin ET A receptor antagonist YM598 has ameliorating effects on various urinary dysfunctions, including benign prostatic hyperplasia.

In vivo study on the effects of alpha1-adrenoceptor antagonists on intraurethral pressure in the prostatic urethra and intraluminal pressure in the vas deferens in male dogs.

Alpha-adrenoceptor antagonists are used worldwide for the treatment of voiding dysfunction associated with benign prostatic hyperplasia. Recently, abnormal ejaculation, an adverse effect associated with their use, has attracted attention. Here, we simultaneously investigated the effects of alpha(1)-adrenoceptor antagonists on intraurethral pressure in the prostatic urethra and intraluminal pressure in the vas deferens in anesthetized male dogs, and compared their tissue selectivity. Phenylephrine, an alpha(1)-adrenoceptor agonist, induced simultaneous increases in intraurethral and intraluminal pressure. Alfuzosin, naftopidil, prazosin, silodosin and tamsulosin dose-dependently inhibited both responses. Comparison of ED(50) values in both tissues showed that silodosin had the highest selectivity for the vas deferens (7.5-fold), followed by naftopidil (4.3-fold), alfuzosin (3.8-fold), tamsulosin (2.6-fold) and prazosin (2.5-fold). These results suggest that alpha(1)-adrenoceptor antagonists inhibit contraction of not only the urethra but also the vas deferens in a dose-dependent fashion, and that their high tissue selectivity for the vas deferens over the urethra may contribute to the incidence of abnormal ejaculation.