Nobiletin, a flacone form shekwasha, induces relaxant effects via cAMP signaling-dependent in detrusor smooth muscle.

Ito Y1, Woo J2, Yamada S1

Research Type

Basic Science / Translational

Abstract Category

Pharmacology

Abstract 745
Non Discussion Abstract
Scientific Non Discussion Abstract Session 37
Detrusor Overactivity Animal Study Basic Science
1.University of Shizuoka, 2.Okinawa Research Center Co., Ltd.
Links

Abstract

Hypothesis / aims of study
Phototherapeutic agents are very popular in many European countries as herbal remedies and represent up to 80% of all drugs prescribed for these disorders. Nobiletin, is a polymethoxy flavonoid abundantly present in citrus fruits, including shekwasha (citrus depressa) produced in southern parts of Japan such as Okinawa. Current studies have shown that nobiletin exhibits anti-inflammatory, antiallergenic, antiatherosclerotic and antitumor activities. Previously we reported that nobiletin has binding potential to muscarinic receptor and alleviates acetic acid- induced hypertensive bladder response in rats. To understand the mechanism of effect of nobiletin on urodynamic functions, we performed in vitro organ bath study and measured intracellular cAMP concentration on rat bladder strips.
Study design, materials and methods
Female Sprague-Dawley rats were anesthetized by isoflurane. The bladder was immediately removed and prepared for the organ bath study.  Bladder smooth muscle stirps were placed in organ baths containing 10 mL of an aerated Krebs-Henseteit solution, and contractions were recorded using isometric force displacement transducers. In order to verify the responsiveness of the bladder strips, an 80 mM KCl-induced contraction was produced at the beginning of each experiment. Isotonic 80 mM KCl-Krebs solution was prepared by replacement of NaCl with an equimolar amount of KCl. After the bladder strips had been fully recovered by washing with fresh bath solution, the bladder strips were treated with 1 mM acetylcholine (ACh) or 80 mM KCl-Krebs solution. When the contraction reached a steady-state level, 3 µM forskolin or vehicle (DMSO, 30 µL) was applied to the bath solution in order to increase the production of cAMP in bladder smooth muscle cells. A five minutes later 100 µM nobiletin or vehicle (DMSO, 10 µL) was applied to the bath solution. When the relaxation induced by forskolin and/or nobiletin reached a steady-state level, the strips were frozen in liquid nitrogen for measuring intracellular cAMP concentration. The concentration of cAMP was measured using a Cyclic AMP EIA kit (Cayman Chemical, Ann Arbor, MI, U.S.A.). cAMP level was normalized by weight of the bladder strips. Results are presented as mean ± standard error of the mean. Statistical comparisons were evaluated with One-way ANOVA followed by Bonferroni’s multiple comparisons test.
Results
For the strips of applying of vehicle, steady-state contraction of 1 mM ACh reached 41.96 ± 1.26 % of 80 mM KCl peak contraction (n=10). Single applying of nobiletin or forskolin significantly inhibited ACh-induced contraction on the rat bladder strips compared with applying of vehicle. Coapplying with nobiletin and forskolin synergistically inhibited ACh-induced contraction. We further measured intracellular cAMP level using an ELISA kit. In rat bladder smooth muscle cells, forskolin (3 µM), an adenylate cyclase activator, drastically increased intracellular cAMP levels, whereas nobiletin (100 µM) had no significant effect on these levels under the current experimental conditions. However, coapplying with nobiletin and forskolin significantly increased intracellular cAMP concentration compared with single applying of forskolin.
For the strips of applying of vehicle, steady-state contraction of 80 mM KCl reached 44.05 ± 3.74 % of 80 mM KCl peak contraction (n=10). Single applying of nobiletin or forskolin significantly inhibited 80 mM KCl-induced contraction on the rat bladder strips compared with applying of vehicle. Coapplying with nobiletin and forskolin synergistically inhibited 80 mM KCl-induced contraction.  In rat bladder smooth muscle cells, forskolin drastically increased intracellular cAMP levels, whereas nobiletin had no significant effect on these levels under the current experimental conditions. However, coapplying with nobiletin and forskolin significantly increased intracellular cAMP concentration compared with single applying of forskolin.
Interpretation of results
Nobiletin has been shown to increase intracellular cAMP levels via the inhibition of phosphodiesterase (PDE) in PC12D cells [1]. Thus, the effect of nobiletin on intracellular cAMP levels of bladder smooth muscle cell was investigated. Forskolin (3 μM) caused a slight increase in intracellular cAMP levels of bladder smooth muscle cells. In the presence of nobiletin (100 μM) a larger and significant increase in intracellular cAMP levels by forskolin was seen. While nobiletin alone did not increase cAMP levels, nobiletin enhanced the increased cAMP induced by forskolin, indicating that nobiletin increases cAMP levels by inhibiting PDE.
Concluding message
Nobiletin improved urodynamic symptoms in hyperactive bladder through the cAMP pathway. Thus, the current results may support the clinical efficacy of nobiletin in the treatment of lower urinary tract symptoms accompanying overactive bladder.
Figure 1 Effects of nobiletin and forskolin, on 1 mM ACh-induced contraction of bladder strips (A) and intracellular cAMP level of bladder smooth muscle cells (B).
Figure 2 Effects of nobiletin and forskolin, on 80 mM KCl-induced contraction of bladder strips (A) and intracellular cAMP level of bladder smooth muscle cells (B).
References
  1. H. Nagase, T. Yamakuni, K. Matsuzaki, et al. Mechanism of neurotrophic action of nobiletin in PC12D Cells. Biochemistry, 44 (42) (2005), pp. 13683-13691
Disclosures
Funding No Clinical Trial No Subjects Animal Species Rat Ethics Committee animal care and use committee of University of Shizuoka