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.

Effect of ASP2205 fumarate, a novel 5-HT2C receptor agonist, on urethral closure function in rats.

The pharmacological profile of ASP2205 fumarate (ASP2205), a novel 5-HT2C receptor agonist, was evaluated in vitro and in vivo. ASP2205 showed potent and selective agonistic activity for the human 5-HT2C receptor, with an EC50 of 0.85 nM in the intracellular Ca2+ mobilization assay. Rat 5-HT2C receptor was also activated by ASP2205 with an EC50 of 2.5 nM. Intraduodenal administration (i.d.) of ASP2205 (0.1-1 mg/kg) significantly elevated the leak point pressure (LPP) in anesthetized rats in a dose-dependent manner. This ASP2205 (0.3 mg/kg i.d.)-induced LPP elevation was inhibited by SB242084 (0.3 mg/kg i.v.), a selective 5-HT2C receptor antagonist. Urethral closure responses induced by intravesical pressure loading in rats were enhanced by ASP2205 (0.3 mg/kg i.v.), which was abolished by pretreatment with SB242084 (0.3 mg/kg i.v.) and bilateral transection of the pudendal nerve. In contrast, ASP2205 (0.3 mg/kg i.v.) did not change the resting urethral pressure in rats. These results indicate that ASP2205 can enhance the pudendal nerve-mediated urethral closure reflex via the 5-HT2C receptor, resulting in the prevention of involuntary urine loss.

Urethral function and histopathology in aged female rats as a stress urinary incontinence model.

OBJECTIVE: Stress urinary incontinence (SUI) is a common disease condition in elderly women, suggesting that its etiology may be linked to aging. To investigate the hypothesis that urethral dysfunction and histopathological changes are possible contributors to SUI in elderly women, several parameters of urethral function, as well as histological parameters, were compared between young and aged rats. METHODS: Virgin female rats were examined at 3 different ages, namely 3, 12, and 24 months, corresponding to young, middle-aged, and aged rats, respectively. Urethral function was assessed by measuring the leak point pressure (LPP), pudendal nerve stimulation (PNS)-induced elevation in urethral pressure, and phenylephrine-induced increase in urethral perfusion pressure (UPP). Histopathological assessments were performed following hematoxylin and eosin (HE), Masson's trichrome, and immunofluorescence staining of urethral tissue. RESULTS: LPP of aged rats was significantly reduced compared to that of both young and middle-aged rats. PNS-induced elevation in urethral pressure in aged rats was also significantly lower than that in young rats. In contrast, there were no significant differences in the phenylephrine-induced increase in UPP between young and aged rats. Connective tissue area in the external urethral sphincter (EUS) layer was increased in aged rats, whereas the smooth muscle layer was histologically similar to that in young rats. The number of EUS fibers was significantly reduced in aged rats, whereas the cross-sectional area of EUS fibers increased from differed compared with young rats. CONCLUSION: We have demonstrated age-related changes in EUS function and morphology in the rat urethra, which are considered to be etiological risk factors for SUI in humans.

Actions of cAMP on calcium sensitization in human detrusor smooth muscle contraction.

OBJECTIVES: To clarify the effect of cAMP on the Ca(2+) -sensitized smooth muscle contraction in human detrusor, as well as the role of novel exchange protein directly activated by cAMP (Epac) in cAMP-mediated relaxation. MATERIALS AND METHODS: All experimental protocols to record isometric tension force were performed using α-toxin-permeabilized human detrusor smooth muscle strips. The mechanisms of cAMP-mediated suppression of Ca(2+) sensitization activated by 10 µm carbachol (CCh) and 100 µm GTP were studied using a selective rho kinase (ROK) inhibitor, Y-27632, and a selective protein kinase C (PKC) inhibitor, GF-109203X. The relaxation mechanisms were further probed using a selective protein kinase A (PKA) activator, 6-Bnz-cAMP and a selective Epac activator, 8-pCPT-2'-O-Me-cAMP. RESULTS: We observed that CCh-induced Ca(2+) sensitization was inhibited by cAMP in a concentration-dependent manner. GF-109203X (10 µm) but not Y-27632 (10 µm) significantly enhanced the relaxation effect induced by cAMP (100 µm). 6-Bnz-cAMP (100 µm) predominantly decreased the tension force in comparison with 8-pCPT-2'-O-Me-cAMP (100 µm). CONCLUSIONS: We showed that cAMP predominantly inhibited the ROK pathway but not the PKC pathway. The PKA-dependent pathway is dominant, while Epac plays a minor role in human detrusor smooth muscle Ca(2+) sensitization.

A role for peripheral 5-HT2 receptors in serotonin-induced facilitation of the expulsion phase of ejaculation in male rats.

INTRODUCTION: The urethrogenital reflex (UGR) is used as a physiological animal model of the autonomic and somatic activity that accompanies ejaculatory-like reflexes (ELRs). Serotonin (5-HT) plays an important role in regulating ejaculation. AIM: To examine the effects of intraurethral 5-HT on ELRs and to examine the effects of various 5-HT receptor subtypes on the 5-HT-induced changes in the ELRs. METHODS: The effects of intraurethral infusion of 5-HT on ELRs were examined by monitoring the urethrogenital reflex in male rats. The effects of various 5-HT receptor-specific antagonists on the 5-HT-induced responses were examined. MAIN OUTCOME MEASURES: Main outcome measures were urethral pressure threshold required to elicit the UGR and bulbospongiosus activity or ELRs. RESULTS: Intraurethral infusion of 5-HT (10-1,000 µM) produced a dose-dependent facilitation of the UGR, i.e., decrease in threshold urethral perfusion pressure and an increase in number of ELRs. The 5-HT3 receptor antagonists tropisetron (1 and 3 mg/kg, i.v.) and ramosetron (0.1 and 1 mg/kg, i.v.), the 5-HT7 receptor antagonist SB269970 (3 mg/kg, i.v.), and the 5-HT1 A receptor antagonist WAY-100635 (1 mg/kg, i.v.) all failed to inhibit 5-HT-induced facilitation of the UGR. However, ritanserin (1 mg/kg, i.v.), a nonselective 5-HT2 receptor antagonist, and xylamidine (0.01-1 mg/kg, i.v.), a peripherally restricted nonselective 5-HT2 receptor antagonist, significantly inhibited both the decrease in urethral pressure threshold and the increase in number of ELRs induced by intraurethral infusion of 5-HT. CONCLUSION: These results suggest that in the male rat urethra, peripheral 5-HT2 receptors are involved in the 5-HT-induced facilitation of the expulsion phase of ejaculation.

Transcriptional regulation of neuronal genes and its effect on neural functions: expression and function of forkhead transcription factors in neurons.

Forkhead box transcription factor, class O (FOXO) is a mammalian homologue of DAF-16, which is known to regulate the lifespan of Caenorhabditis elegans and includes subfamilies of forkhead transcription factors such as AFX, FKHRL1, and FKHR. FKHR is phosphorylated on three sites (Thr-24, Ser-256, and Ser-319) in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner, thereby inhibiting death signals. We here documented dephosphorylation of FKHR following transient forebrain ischemia with its concomitant translocation into the nucleus in neurons in gerbil and mouse brains. The activation of FKHR preceded delayed neuronal death in the vulnerable hippocampal regions following ischemic brain injury. The FKHR activation was accompanied by an increase in DNA binding activity for FKHR-responsive element on the Fas ligand promoter. We also defined FKHR-induced downstream targets such as Fas ligand and Bim in brain ischemia. Therefore, we propose a new strategy to rescue neurons from delayed neuronal death by promoting the survival signaling. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, up-regulated Akt activity in the brain and in turn rescue neurons from delayed neuronal death by inhibiting FKHR-dependent or -independent death signals in neurons.