Pulmonary, gastrointestinal and urogenital pharmacology
Modulation of urinary frequency via type 1 lysophosphatidic acid receptors: Effect of the novel antagonist ASP6432 in conscious rats

https://doi.org/10.1016/j.ejphar.2019.03.011Get rights and content

Abstract

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.

Introduction

Maintenance of continence and control of the timing of urination by proper storage of urine in the bladder are fundamental to leading a normal personal and social life. The micturition center located in the pons and the autonomic nervous system cooperatively regulate the normal reservoir function of the bladder. Noradrenaline released from sympathetic nerve endings activates β3-adrenoceptors expressed in the bladder to distend the detrusor smooth muscle and α1-adrenoceptors to contract the urethral smooth muscle (de Groat et al., 2015).

Dysregulation of bladder urine storage function causes a series of clinical conditions called storage symptoms, characterized by urgency (“the complaint of a sudden compelling desire to pass urine which is difficult to defer”), increased daytime frequency, nocturia (“the complaint that the individual has to wake at night one or more times to void”), and incontinence (Abrams et al., 2002). Storage symptoms are commonly observed in patients with lower urinary tract symptoms (LUTS) associated with benign prostate hyperplasia (BPH). Although patients typically find storage symptoms the most bothersome (Chapple et al., 2014), treatment of LUTS associated with BPH tends to focus on improving voiding symptoms characterized by slow stream hesitancy, incomplete bladder emptying, and terminal dribbling. Alpha1-adrenoceptor antagonists, current first-choice pharmacotherapies, primarily relax contractions of the prostatic part of the urethra and relieve bladder outlet obstruction (BOO), but do not always sufficiently alleviate storage symptoms (van Kerrebroeck et al., 2013). An agent that potently improves bladder dysfunction in addition to BOO may be a better treatment option for LUTS associated with BPH.

Lysophosphatidic acid (LPA) is a small glycerophospholipid that mediates diverse biological actions and demonstrates medicinal relevance (Yung et al., 2014). In the lower urinary tract, LPA contracts urethral strips isolated from rats, and increases intraurethral pressure in rats and dogs via the type 1 LPA (LPA1) receptor (Terakado et al., 2017). In cultured bladder smooth muscle cells, LPA induces contraction (Kropp et al., 1999) and is suggested to mediate stretch-induced cellular activation possibly via the LPA1 receptor, the most prominently expressed receptor subtype in the bladder among the six known LPA receptors (Kawashima et al., 2015). To our knowledge, however, no study has yet investigated the effect of LPA on bladder function in vivo. We previously demonstrated that LPA induced contraction of the prostate and urethra via the LPA1 receptor using the potent and selective 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) (Sakamoto et al., 2018, Sakamoto et al., 2019). However, the effect of ASP6432 on bladder function remains to be elucidated.

To clarify the role of LPA and the LPA1 receptor in the regulation of bladder function, we examined the effects of LPA and the LPA1 receptor antagonist ASP6432 on the micturition reflex in conscious rats using continuous cystometry. In addition, we evaluated the effect of ASP6432 on the decrease in micturition interval (MI, a cystometry parameter reflective of urinary frequency) induced by Nω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor.

Section snippets

Test reagents

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) was synthesized at Astellas Pharma Inc. (Tokyo, Japan). ASP6432 was dissolved and serially diluted with either distilled water containing 0.025 mol/L NaOH or with saline (0.9% NaCl) containing 5% N,N-dimethylformamide (DMF). LPA (1-linolenoyl-2-hydroxy-sn-glycero-3-phosphate, 1-linolenoyl LPA) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) was

LPA caused bladder overactivity in conscious rats

Under intravenous infusion of vehicle for LPA (saline containing 0.1% BSA), there were no differences in the baseline parameters measured (MI, max IVP, BP, or TP; Fig. 1A) between the groups. After switching the infusion from vehicle to LPA (1, 5, 10, or 15 mg/kg/h), MI was dose-dependently decreased compared to baseline at 5 mg/kg/h and greater. TP was also statistically significantly decreased compared to baseline at 10 mg/kg/h. No significant changes were observed in BP or max IVP (Fig. 1B).

LPA-induced urinary frequency was reversed by the LPA1 receptor antagonist ASP6432

Discussion

This study demonstrated that LPA decreased MI and TP in conscious rats, and that the LPA1 receptor antagonist ASP6432 inhibited the LPA- and L-NAME-induced decreases in MI. Despite recent studies demonstrating a significant role for the LPA-LPA1 receptor signaling axis in urethral contraction (Saga et al., 2014, Sakamoto et al., 2018, Terakado et al., 2017), the effect of LPA and the LPA1 receptor in the bladder has not been clarified in vivo. To our knowledge, this is the first study to show

Conclusions

The present study demonstrated for the first time that LPA is a bioactive phospholipid capable of causing bladder overactivity characterized by decreases in MI and TP, and that the LPA1 receptor antagonist ASP6432 reverses LPA- and L-NAME-induced decreases in MI. Our results suggest a significant role for the LPA1 receptor in regulating the functional capacity of the bladder, and the potential of ASP6432 as a novel therapy for the treatment of bladder dysfunction caused by lower urinary tract

Acknowledgement

We thank Dr. Eiji Kawaminami and other medicinal chemists at Astellas Pharma Inc. for providing ASP6432.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

Authors are employees of the Astellas group. All studies were conducted at Astellas Pharma Inc.

(A) Micturition interval, maximum intravesical pressure (IVP), baseline pressure, and threshold pressure were examined during cystometry. (B) Parameters at baseline and after LPA infusion (1, 5, 10, and 15 mg/kg/h) were measured. Each column represents the mean ± S.E.M. of four

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  • Cited by (1)

    1

    Also belongs to: School of Integrative and Global Majors, University of Tsukuba.

    2

    Present affiliation: Regulatory Affairs, Astellas Pharma Inc.

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