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.

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. METHODS: Spinal cord was transected at the level of T8-9 in adult female Sprague-Dawley rats. After 10 weeks, specimens of the urinary bladder were obtained from SCI rats and sham-injured control rats, and bladder strips were dissected from the bladder. Using an muscle-bath technique and a microdialysis procedure, the dialysate, containing substance released from bladder strips, was collected. Then the amount of PGE2 in the dialysate was measured by radioimmunoassay. RESULTS: Excretion of urinary PGE2 was significantly higher in SCI rats than in control rats. PGE2 release from bladder strips was significantly higher in SCI rats than in control rats. Removal of urothelium caused significant decreases in PGE2 release in both control and SCI rats. Stretches of the bladder strips caused significant resting tension-dependent increases in PGE2 release from the strips with urothelium. CONCLUSIONS: The present data suggest that bladder urothelium partly contributes to the increase in PGE2 release from the bladder, and that bladder distension may cause increases in PGE2 release in SCI rats.

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. METHODS: Plasmids expressing luciferase, a green fluorescence protein and M3 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 anti-M3 receptor, an increase in immunoactivity was observed in the M3 receptor gene transferred rat bladder, compared to the bladder of the control rat. In rats with the transferred M3 receptor gene, carbachol- and 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 M3 receptor in the rat bladder enhances bladder contractility. This technique may become a new treatment modality for detrusor underactivity.

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.

Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging.

Numerous studies have detailed age-related changes in the structure and function of the bladder that may contribute to the high prevalence of overactive bladder (OAB) in the elderly population, but the relation of these changes to OAB symptoms remains unclear. Physiologic and neurochemical studies have been conducted in human detrusor strips obtained from people of different ages, focusing on potential changes in cholinergic and purinergic neurotransmission, as well as the release and actions of acetylcholine (ACh) from nonneuronal bladder cells. Results from physiologic and microdialysis experiments indicate that purinergic transmission increases with age, whereas cholinergic transmission decreases. These effects are most likely because of decreased release of ACh and increased release of adenosine triphosphate (ATP) from postganglionic parasympathetic axons innervating the bladder. Immunohistochemical experiments showed that choline acetyltransferase in the human detrusor is contained not only in parasympathetic axons, but also in cells of the urothelium. The release of nonneuronal ACh increases with age and detrusor stretch. The age-related increase in purinergic transmission in the detrusor and other data indicating that responses to ATP are increased in detrusor overactivity suggest that purinergic receptor antagonists may provide a useful complement to muscarinic receptor antagonists in the treatment of older patients with OAB. Nonneuronal ACh release may play a key role in the storage phase of the micturition reflex, and this may explain, at least in part, the effectiveness of antimuscarinic agents for the treatment of OAB.

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

INTRODUCTION: We evaluated the effects of diabetes 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 three groups: control (injected with the vehicle), DM (diabetes mellitus, induced by injection with 65 mg/kg streptozotocin), and TES (testosterone, testosterone supplemented after induction of diabetes). After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS, 2-ms pulse duration, 0.3-20 Hz and 3 s train) was applied to the strips precontracted with 30 microM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS was collected, and the amount of NO(-)(2)/NO(-)(3) (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside (0.1 nM to 10 mM) and carbachol (1 nM to 10 mM) were cumulatively added to the strips precontracted with 30 microM phenylephrine. RESULTS: EFS caused frequency-dependent relaxations and NOx releases of the strips. Pretreatment with N(omega)-nitro-L-arginine (100 microM) and tetrodotoxin (1 microM) completely inhibited the relaxations and NOx releases. The maximum relaxation was significantly greater in the DM group than in the control or TES group. The release of NOx was significantly greater in the DM group than in the control or TES group. Sodium nitroprusside, the endothelium-independent vasodilator, relaxed the tissues in all three groups. There were no significant differences among control, DM and TES groups in the maximum relaxation to sodium nitroprusside. CONCLUSION: The present data suggest that diabetes enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum by the reduced testosterone level in the diabetic animals.

Pharmacological effects of darifenacin on human isolated urinary bladder.

Darifenacin [(S)-2--2,2-diphenylacetamide] is a novel antimuscarinic drug currently undergoing phase III trials for the treatment of overactive bladder. We investigated the functional antagonist potency of darifenacin, and the antimuscarinic drugs propiverine, oxybutynin and atropine, on human detrusor smooth muscle. Urinary bladder specimens were obtained from 20 patients who underwent total cystectomy for malignant bladder tumor. Using an organ-bath technique, the effects of the compounds on carbachol-, KCl-, CaCl(2)- or electrical field stimulation (EFS)-induced contractions of the tissues were evaluated. The order of antagonist potency (pA(2 )values) at the muscarinic M(3) receptors was: darifenacin (9.34) > atropine (9.26) > oxybutynin (7.74) > propiverine (7.68). Darifenacin and atropine, at concentrations up to 10(-6) mol/l, did not inhibit the KCl- and CaCl(2)-induced contractions (concentrations 80 and 5 mmol/l, respectively), while propiverine and oxybutynin (10(-5) mol/l) significantly inhibited these contractions. Pretreatment with darifenacin (10(-9)-10(-6) mol/l), propiverine (10(-8)- 10(-5) mol/l), oxybutynin (10(-8)-10(-5) mol/l) and atropine (10(-9)-10(-6) mol/l) significantly inhibited maximum EFS-induced contractions. Darifenacin inhibited contractions of human detrusor smooth muscle only through its antimuscarinic action, while propiverine and oxybutynin had both antimuscarinic and Ca(2+) channel antagonist actions. These findings indicate that darifenacin is a potent antagonist at the M(3) receptor and support its use as a treatment for overactive bladder.

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, CaCl(2), 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 pA(2) values was KRP-197 > or = atropine > oxybutynin > propiverine. Atropine and KRP-197 did not cause significant inhibition of KCl- and CaCl(2)-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.

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 microM 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 microM phenylephrine. RESULTS: EFS caused frequency-dependent relaxations and NOx releases in the strips. Pretreatment with N(omega)-nitro-L-arginine (100 microM) and tetrodotoxin (1 microM) 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.