Korean J Physiol Pharmacol.  2018 Sep;22(5):577-584. 10.4196/kjpp.2018.22.5.577.

The change of signaling pathway on the electrical stimulated contraction in streptozotocin-induced bladder dysfunction of rats

• Han JS
• Min YS
• Kim GH
• Chae SH
• Nam Y
• Lee J
• Lee SY
• Sohn UD

• KMID: 2419007
• DOI: http://doi.org/10.4196/kjpp.2018.22.5.577

Abstract

Bladder dysfunction is a common complication of diabetes mellitus (DM). However, there have been a few studies evaluating bladder smooth muscle contraction in DM in the presence of pharmacological inhibitors. In the present study, we compared the contractility of bladder smooth muscle from normal rats and DM rats. Furthermore, we utilized pharmacological inhibitors to delineate the mechanisms underlying bladder muscle differences between normal and DM rats. DM was established in 14 days after using a single injection of streptozotocin (65 mg/kg, intraperitoneal) in Sprague-Dawley rats. Bladder smooth muscle contraction was induced electrically using electrical field stimulation consisting of pulse trains at an amplitude of 40 V and pulse duration of 1 ms at frequencies of 2-10 Hz. In this study, the pharmacological inhibitors atropine (muscarinic receptor antagonist), U73122 (phospholipase C inhibitor), DPCPX (adenosine A₁ receptor antagonist), udenafil (PDE5 inhibitor), prazosin (α₁-receptor antagonist), verapamil (calcium channel blocker), and chelerythrine (protein kinase C inhibitor) were used to pretreat bladder smooth muscles. It was found that the contractility of bladder smooth muscles from DM rats was lower than that of normal rats. In addition, there were significant differences in percent change of contractility between normal and DM rats following pretreatment with prazosin, udenafil, verapamil, and U73122. In conclusion, we suggest that the decreased bladder muscle contractility in DM rats was a result of perturbations in PLC/IP₃-mediated intracellular Ca²âº release and PDE5 activity.

Keyword

Bladder; Contractility; Diabetes; Phospholipase C; Smooth muscle

MeSH Terms

Animals
Atropine
Diabetes Mellitus
Muscle, Smooth
Phosphotransferases
Prazosin
Rats*
Rats, Sprague-Dawley
Streptozocin
Type C Phospholipases
Urinary Bladder*
Verapamil
Atropine
Phosphotransferases
Prazosin
Streptozocin
Type C Phospholipases
Verapamil

Figure

• 

  Fig. 1 Representative traces and tension comparison for electrical field stimulation-induced contraction of bladder smooth muscle from normal and diabetic rats. (A) Electrical field stimulation (EFS)-induced contraction. The left and right sides are NM and DM rats, respectively. (B) Average tension of EFS contraction for muscles from normal (NM) and rats with diabetes mellitus (DM rats). (A, B) EFS stimulation was administered at 2, 4, 6, 8, and 10 Hz. Each point represents the mean±standard error of the mean (SEM), where n=13. *Indicates statistically significant (p<0.05) when evaluated by Student's t-test and ### indicates statistically significant (p<0.001) when evaluated by two-way ANOVA.

• 

  Fig. 2 Changes in contraction of smooth bladder muscles from normal and DM rats following atropine treatment. EFS-induced contractions of muscles from normal and DM rats. Atropine was added at 10−6 M. EFS was administered using 2, 4, 6, 8, and 10 Hz. Each point represents the mean±SEM, where n=6.

• 

  Fig. 3 Changes in contraction of bladder smooth muscles from normal and DM rats following prazosin treatment. EFS-induced contractions of muscles from normal and DM rats. Prazosin was added at 10−6 M. EFS was administered at 2, 4, 6, 8, and 10 Hz. Each point represents the mean±SEM, where n=6. ### indicates statistically significant (p<0.001) based on two-way ANOVA.

• 

  Fig. 4 Changes in contraction of bladder smooth muscles from normal and DM rats following DPCPX treatment. EFS-induced contractions in muscles from normal and DM rats. DPCPX was added at 10−6 M. EFS was administered at 2, 4, 6, 8, and 10 Hz. Each point represents the mean±SEM, where n=6.

• 

  Fig. 5 Changes in contractions of bladder smooth muscles from normal and DM rats following verapamil and U73122 treatment. (A) EFS-induced contractions following 10−6 M verapamil treatment. (B) EFS-induced contractions following 10−6 M U73122 treatment. EFS was administered at 2, 4, 6, 8, and 10 Hz. Each point represents the mean±SEM, where n=6. ## and ### indicate statistical significance (p<0.01 and p<0.001, respectively) based on two-way ANOVA.

• 

  Fig. 6 Changes in contractions of bladder smooth muscles from normal and DM rats following udenafil and NaHS treatment. (A) EFS-induced contractions following 10−6 M udenafil treatment. (B) EFS-induced contractions following 10−6 M NaHS treatment. EFS was administered at 2, 4, 6, 8, and 10 Hz. Each point represents the mean±SEM, where n=6. ### indicates statistically significant (p<0.001) based on two-way ANOVA.

• 

  Fig. 7 Changes in contractions of bladder smooth muscles from normal and DM rats following chelerythrine treatment. EFS-induced contractions following addition of 10−6 M chelerythrine. EFS was administered at 2, 4, 6, 8, and 10 Hz. Each point represents the mean±SEM, where n=6.

• 

  Fig. 8 Summary of mechanistic changes in smooth muscle bladder contraction in DM rats. Both G protein-coupled receptor- and nitric oxide-mediated intracellular signaling pathways are involved in changes in the contraction or relaxation of bladder smooth muscle. Grey symbols () indicate signaling pathway differences between normal and DM rats. Phospholipase C generates the intracellular second messenger IP3 from the membrane lipid PIP2. IP3 then induces Ca2+ release and the released Ca2+ binds to calmodulin. Ca2+/calmodulin activates MLCK, which phosphorylates myosin light chain, thus leading to contraction. In bladder smooth muscles from DM rats, changes in G protein-coupled receptor-mediated phospholipase C signaling affects IP3-dependent Ca2+ release and changes in PDE5 affect contraction or relaxation. These factors are expected to result in a decrease in bladder contractility. DM, diabetes mellitus; EFS, electrical field stimulation; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; DAG, diacylglycerol; IP3, inositol trisphosphate; CaM, calmodulin; MLCK, myosin light chain kinase; MLC-P, myosin light chain phosphatase; NO, nitric oxide; PKG, protein kinase G; PDE5, phosphodiesterase 5.

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