Hitoshi Iwashita

Prostaglandin E2 release from isolated bladder strips in rats with spinal cord injury

Aim:  Recent studies have shown that various factors contribute to the increased excitability into the bladder afferent nerves in spinal cord injury (SCI) rats. It has been reported that prostaglandins (PG) act as local modulators of reflex micturition in pathological conditions. In the present study, we measured the amount of PGE2 release from the bladder of chronic SCI rats.

Pharmacological effects of KRP-197 on the human isolated urinary bladder

KRP-197, 4-(2-methylimidazol-l-yl)-2,2-diphenylbutyramide, is a newly synthesized antimuscarinic drug, developed for the treatment for overactive bladder. For evaluation of pharmacological characteristics of KRP-197, we investigated whether it influenced both prejunctional and postjunctional muscarinic receptors on the isolated human detrusor smooth muscles as compared with the effects of atropine, oxybutynin, and propiverine. Using the muscle bath technique, we investigated the effects of various antimuscarinic drugs on the contractions induced by carbachol, KCl, CaCl2, and electrical field stimulation. Furthermore, using high-performance liquid chromatography with a microdialysis technique, we measured the acetylcholine release from the muscle strips during electrical field stimulation. The effects of various antimuscarinic drugs on acetylcholine releases were also evaluated. Pretreatment with various antimuscarinic drugs caused parallel shifts to the right in carbachol-induced concentration-response curves. The rank order of pA2 values was KRP-197 ≧ atropine > oxybutynin > propiverine. Atropine and KRP-197 did not cause significant inhibition of KCl- and CaCl2-induced contractions. All drugs caused concentration-dependent inhibitions in electrical field stimulation-induced contractions. Pretreatment with atropine and propiverine did not cause significant changes in electrical field stimulation-induced acetylcholine release. However, KRP-197, and oxybutynin caused significant decreases in acetylcholine release. The present study demonstrates that KRP-197 has an inhibitory effect on postjunctional muscarinic receptors as well as on prejunctional muscarinic receptors to modulate acetylcholine release in human detrusor smooth muscles. The findings suggest the usefulness of KRP-197 as a therapeutic drug for an overactive bladder with symptoms of frequency, urgency, and urge incontinence.

Acetylcholine Release from Urinary Bladder Smooth Muscles of Non-Insulin-Dependent Diabetic Rats

Introduction: To investigate the mechanism of voiding dysfunction in non-insulin-dependent diabetes mellitus, we attempted to measure the acetylcholine (ACh) release using an in vivo microdialysis technique and measuring the detrusor pressure after electrical field stimulation (EFS) of the pelvic nerve. Materials and Methods: Eight- and 32-week-old female Goto-Kakizaki (GK) rats (non-insulin-dependent diabetes mellitus model) and age-matched female Wistar rats (controls) were used in this study. The pelvic nerve was exposed on a bipolar platinum electrode to EFS, and a cannula was inserted into the bladder to measure the detrusor pressure. The microdialysis probe was inserted into the bladder wall and was connected to a microinfusion syringe pump. Dialysate was constantly perfused, collected in a microtube, and then injected into the ACh assay system. Histological examinations were performed by staining with hematoxylin and eosin and S-100 immunohistochemical staining in bladder preparations of both GK and control rats. Results: In 8-week-old rats, both detrusor pressures and amounts of ACh release of GK rats were not significantly different from those of control rats. In 32-week-old rats, both detrusor pressures and ACh releases were only significantly increased at 5 and 10 Hz of EFS. In the histological study, the number of nerve fibers or bundles of 32-week-old GK rats was significantly decreased as compared with control rats. Conclusion: The present data suggest that the decrease in EFS-induced ACh release in GK rats, which may be caused by the decreased number of nerve fibers, may contribute to the decrease in bladder contractions.

Delivery of DNA into Bladder via Electroporation

The possibility of in vivo gene transfer into the rat bladder by electroporation (EP) was evaluated. The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of marker genes, green fluorescent protein (GFP) and luciferase, and the neuronal nitric oxide synthase (nNOS) gene were then injected into the subserosal space of the bladder and EP was applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder, and immunohistochemical staining was used to detect nNOS. NOx released from isolated bladder strips was also assessed using microdialysis procedure. From the luciferase assay, 45 V, 1 Hz, 50 ms, and 8 pulses were selected as the optimum conditions for EP. Bladder specimens with GFP genes injected by EP showed numerous bright sites of GFP expression in the smooth-muscle layer. In rats with the nNOS gene injected by EP, there was marked nNOS immunoreactivity, and NOx released from bladder strips was significantly greater than that in the control groups. These results suggest that EP is a useful technique for in vivo gene transfer into rat bladder smooth muscles, and that the nNOS gene transferred by this procedure functionally expresses and contributes to NO production.

Electroporation-mediated muscarinic M3 receptor gene transfer into rat urinary bladder.

Background: Muscarinic M3 (M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M3 receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene.

THIS ARTICLE HAS BEEN RETRACTED Electroporation‐mediated muscarinic M3 receptor gene transfer into rat urinary bladder

Background: Muscarinic M3 (M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M3 receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene.

Effects of castration on nitric oxide-mediated relaxations in male rat corpus cavernosum smooth muscle.

Background: The effects of castration on nitric oxide- mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. Methods: Eight-week-old male rats were assigned to two groups: control (sham operated) and castrated animals. After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS) was applied to the strips precontracted with 30 µM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS and cholinergic stimulation was collected, and the amount of NO–2/NO–3 (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside and carbachol were cumulatively added to the strips precontracted with 30 µM phenylephrine. Results: EFS caused frequency-dependent relaxations and NOx releases in the strips. Pretreatment with Nω-nitro-L-arginine (100 µM) and tetrodotoxin (1 µM) completely inhibited relaxations and NOx releases. The maximum relaxation in the castration group was significantly greater than that in the control group. The release of NOx was significantly greater in the castration group than in the control group. Sodium nitroprusside relaxed the tissues in both groups similarly. Carbachol failed either to relax the tissue or to increase the amount of NOx production in the tissue. Conclusion: The present data suggest that castration enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum.

Erratum: Electroporation-mediated muscarinic M3 receptor gene transfer into rat urinary bladder (International Journal of Urology (2004) 11:11 (1001-1008))

Background : Muscarinic M 3 (M 3 ) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M 3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M 3 receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene. Methods : Plasmids expressing luciferase, a green fluorescence protein and M 3 receptor were injected into the rat bladder and square-wave electric pulses were immediately applied. Two days after gene transfer, we analyzed gene expression. Immunohistochemical staining was performed and the contractile responses from isolated bladder strips, which were induced KCl, carbachol and electrical field stimulation (EFS), were evaluated. Results : The optimal conditions of electroporation were 8 pulses, 45 voltages, 50 milliseconds/ pulses and 1 Hz. Under these conditions, luciferase gene expression was enhanced approximately 300-fold, compared to an injection of DNA only. Regarding immunohistochemistry with an antiM 3 receptor, an increase in immunoactivity was observed in the M 3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the transferred M 3 receptor gene, carbacholand EFS-induced maximum contractile responses of bladder smooth muscle strips significantly increased. Conclusions : These findings suggest that an in vivo EP procedure is an useful method for gene transfer into the bladder and that an overexpression of M 3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity.

THIS ARTICLE HAS BEEN RETRACTED Electroporation-mediated muscarinic M3 receptor gene transfer into rat urinary bladder: M3 gene transfer into rat bladder

Background: Muscarinic M3 (M3) receptor has been recognized as a major muscarinic receptor for smooth muscle contractions of the urinary bladder. Under the hypothesis that overexpression of M3 receptor in the urinary bladder would enhance urinary bladder contractions, we have transferred the M3 receptor gene into rat bladders using electroporation (EP) and evaluated the functional expression of the transferred gene.