Effects of chronic treatment with vardenafil,a phosphodiesterase 5 inhibitor,on female rat bladder in a partial bladder outlet obstruction model

OBJECTIVES

To investigate whether vardenafil, a phosphodiesterase 5 (PDE‐5) inhibitor, would protect the bladder from decompensatory changes in a 4‐week rat bladder outlet obstruction (BOO) model, as evidence has been accumulating that PDE‐5 inhibitors improve lower urinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia (BPH).

MATERIALS AND METHODS

In all, 50 12‐week‐old female Sprague‐Dawley rats were divided into five equal groups; group 1, sham operated vehicle control rats; group 2, BOO vehicle rats; group 3–5, BOO rats given oral vardenafil at 5, 20, 80 mg/L, respectively. Vardenafil was given in drinking water from the day of surgery. At 4‐weeks after the introduction of BOO, vardenafil was washed‐out by giving water for 24–48 h, and then the bladder was excised and dissected into four longitudinal strips for isometric organ‐bath assay. Contractile responses of bladder strips to electrical field stimulation (EFS), carbachol and KCl was determined for each group.

RESULTS

BOO induced a significant increase in bladder weight in group 2 compared with group 1. Bladder weights of groups 3–5 were not significantly different from that of group 2. The contractile forces in response to EFS, carbachol and KCl in group 2 were 30.7–51.7% of those in group 1. Vardenafil treatment in groups 3–5 generally did not block the BOO‐induced reduction of contractile force in the bladder strips. However, treatment with a high dose of vardenafil resulted in a significant increase in the contractile response to carbachol (78.4% group 5 vs 51.7% group 2).

CONCLUSION

Chronic treatment with a high dose of vardenafil protected the rat bladder from BOO‐induced contractile dysfunction to carbachol.     

Alterations in purinergic and cholinergic components of contractile responses of isolated detrusor contraction in a rat model of partial bladder outlet obstruction

OBJECTIVE: To study the effect of 3 weeks of partial bladder outlet obstruction (BOO), compared to a sham operation, on the cholinergic and purinergic components of detrusor contractile responses to agonists and to electrical field stimulation (EFS); the expression of P2X receptor subtypes was also examined. MATERIALS AND METHODS: Partial BOO was induced in female Sprague-Dawley rats by surgically applying a jeweller's silver 'jump' ring around the urethra, such that the urethra was constricted but not closed. Sham-operated female rats underwent an identical procedure without placement of a ring. RESULTS: After 3 weeks of partial BOO the rat bladders became significantly hypertrophied, doubling in weight. Spontaneous activity was markedly increased, but the contractile response to a single bolus of KCl (120 mM) was unaltered. The neurogenic-induced contractile responses of strips of detrusor from obstructed bladders were significantly greater than those from sham-operated bladders, and the responses of strips of detrusor from obstructed bladders to EFS showed a significantly greater atropine-sensitive component than sham-operated detrusor. However, the response of detrusor strips to EFS that was susceptible to desensitization by alpha,beta-methylene ATP was not significantly changed in obstructed bladders. The sensitivity of the strips from obstructed bladders to carbachol, ATP and beta,gamma-methylene ATP was less than in sham-operated detrusor. Immunohistochemical studies showed no difference in the P2X receptor subtypes expressed on detrusor smooth muscle from obstructed and sham-operated rats. CONCLUSION: In the rat, after moderate bladder hypertrophy, the atropine-sensitive component was significantly up-regulated, but the ATP-sensitive component was marginally reduced, although not significantly. These results suggest that up-regulation of the P2X component of bladder contraction seen in humans with bladder instability, and in other species models of BOO, is not mirrored in the rat, or occurs later in the pathological process of bladder hypertrophy.     

Comparisons of the responses of anterior and posterior human adult male bladder neck smooth muscle to in vitro stimulation

OBJECTIVE

To evaluate differing methods of stimulation on strips of human bladder neck smooth muscle and compare muscle taken from the anterior and posterior aspects.

MATERIALS AND METHODS

Samples of adult human male bladder neck muscle were obtained from patients undergoing open radical prostatectomy. Muscle was taken from either the anterior or posterior (nine and six patients, respectively) aspects of the bladder neck. Muscle strips dissected from these samples were suspended in the Brading‐Sibley organ bath. The strips were superfused with 100 mm KCl‐enriched Krebs’ solution for 4 min to determine viability. This allowed experimentation on 17 strips from the anterior aspect of the bladder neck and 13 from the posterior bladder neck. These remaining strips were then superfused either with various concentrations (×10^?7 to ×10^?3m ) of carbachol or noradrenaline in Krebs’ solution, for 15 s. A further set of strips (eight from anterior, six from posterior) was suspended and responses to electrical field stimulation (EFS) with varying parameters were measured. Each EFS experiment was repeated after a 15 min exposure to 10^?3m atropine, and again after a 15 min exposure 10^?7m tetrodotoxin (TTX). Tension responses produced in these series of experiments were measured using strain gauges and analysed using data acquisition software. Student’s t‐test was used for the statistical analysis.

RESULTS

All muscle strips included in the study responded to EFS. The magnitude of this contraction is frequency dependent. The contractions were abolished by superfusion of the muscle strips with atropine. There was no further suppression of the contractile response on addition of TTX. Posterior bladder neck samples had a greater mean contractile response per unit mass than anterior strips at all frequencies of >1 Hz, and significantly more at 20 and 30 Hz. There was a concentration‐dependent response in bladder neck contraction to carbachol but only in the strips from the anterior bladder neck at concentrations of <10^?3m . Posterior bladder neck strips did not significantly contract upon application of carbachol. Similarly, there was a concentration‐dependent response to noradrenaline. Responses to noradrenaline were not uniform around the bladder neck, but not significantly different. Carbachol was the more ‘potent’ stimulator in anterior smooth muscle strips, but again the differences between agonists were not statistically significant.

CONCLUSION

These experiments show physiological variability around the circumference of the human male bladder neck. The posterior bladder neck shows significantly stronger contraction to α‐adrenergic agonists compared with cholinergic agonists; the anterior bladder neck does not have a similarly significant differential response. The uniform response to noradrenaline may underlie the bladder neck’s role in the prevention of retrograde ejaculation. The differential responses to carbachol may reflect differences in the embryological derivation of the anterior and posterior bladder neck fibres or in their innervation. Some of these differences may have clinical importance through the action of therapeutic agents.     

Effects of local anesthetics on human bladder contractility

AIMS: We investigated the invitro effects of local anesthetics on the contractility of the human bladder. METHODS: By measuring the invitro isometric contractions of human bladder strips, we determined the effects of tetracaine, bupivacaine, lidocaine, and ropivacaine on the basal spontaneous contractions and contractions induced by various stimuli, namely, KCl (60 mM), carbachol (CCh), and electrical field stimulation (EFS). The effect of local anesthetic agents on Ca(2+)-independent sustained tonic contraction (SuTC) of the detrusor was also investigated. RESULTS: Local anesthetics increased phasic and tonic spontaneous contractile activity dose dependently in the concentration range 1-500 muM, but abolished phasic activity at higher concentrations. Local anesthetic agents inhibited nerve-mediated contraction (EFS, 0.8 msec) in a concentration-dependent manner (ropivacaine > tetracaine = bupivacaine > lidocaine), and inhibited non-nerve mediated contractions induced by KCl, long pulse EFS (direct muscle stimulation, 100 msec), and CCh. Inhibitory potency on non-nerve mediated contraction was for long pulse EFS: ropivacaine = tetracaine > bupivacaine = lidocaine and for KCl- and CCh-induced contractions: ropivacaine > tetracaine > bupivacaine = lidocaine. Higher concentrations of local anesthetics were needed to inhibit non-nerve-mediated bladder contraction than nerve-mediated contraction. SuTC was suppressed by all local anesthetics concentration dependently. CONCLUSIONS: Our study demonstrates that local anesthetics have inhibitory effects on the contraction of human bladder as induced by different stimulants and concentrations. Their effects and differences suggest that they may be considered potentially useful as diagnostic and therapeutic agents for bladder dysfunction.     

TAC‐302 promotes neurite outgrowth of isolated peripheral neurons and prevents bladder denervation related bladder dysfunctions following bladder outlet obstruction in rats

Aims

To evaluate the ability of TAC‐302, a cyclohexenoic fatty alcohol derivative, to enhance neurite outgrowth in cultured rat dorsal root ganglion (DRG) neurons, and the preventive effects of TAC‐302 on bladder denervation‐related storage and voiding dysfunctions in rats with bladder outlet obstruction (BOO).

Methods

Rat DRG neurons were cultured in the presence of TAC‐302. Cell numbers and neurite lengths were quantified after a 24 h culture. BOO was achieved by partial ligature of the proximal urethra in female rats. BOO rats were divided into three groups and orally treated with vehicle of 3 or 30 mg/kg TAC‐302 twice a day for 4 weeks. Cystometry was performed under conscious conditions. Immunohistochemical staining using anti‐PGP9.5 of the bladder muscle layer was performed, and the innervation area was scored.

Results

TAC‐302 significantly and dose‐dependently increased neurite outgrowth in cultured DRG neurons. BOO rats showed a decreased innervation area in the urinary bladder compared to sham‐operated rats. BOO‐induced denervation of the urinary bladder was partially prevented by oral treatment with TAC‐302. TAC‐302 significantly reduced the frequency of non‐voiding contraction (NVC) and residual urine volume (RUV) compared with the BOO vehicle group (P < 0.05). The innervation area score exhibited significant negative correlations with NVC and RUV, indicating that they increased according to the progression of denervation.

Conclusions

Our data indicate that TAC‐302 promotes neurite outgrowth in vitro. In addition, TAC‐302 prevents BOO‐induced bladder dysfunction in rats, and has a protective effect on bladder denervation.     

The effects of a type 4 phosphodiesterase inhibitor and the muscarinic cholinergic antagonist tolterodine tartrate on detrusor overactivity in female rats with bladder outlet obstruction

OBJECTIVE

To investigate the effects of the selective phosphodiesterase (PDE) type 4 inhibitor IC485 and the widely used antimuscarinic drug tolterodine tartrate on bladder activity in rats with bladder outlet obstruction (BOO), as inhibition of PDE4 leads to elevation of intracellular cAMP levels and relaxation of smooth muscle.

MATERIALS AND METHODS

BOO was induced in female Sprague‐Dawley rats by tying a silk ligature around the urethra. Six weeks after inducing BOO, conscious rats were assessed by cystometry with the urethral ligature intact. The effects of IC485 (5, 10 and 50 mg/kg intravenous, i.v.) were examined and compared with those of tolterodine (0.01, 0.1 and 1 mg/kg i.v.).

RESULTS

IC485 (5–50 mg/kg i.v.) decreased the number and amplitude of non‐voiding contractions during the storage phase by 63–88% and 49–83%, respectively; IC485 also increased bladder capacity by 28–37%. There was no change in blood pressure after applying IC485. Tolterodine tartrate (0.1 and 1.0 mg/kg) significantly decreased the number and amplitude of non‐voiding contractions by 38–74% and 29–44%, respectively, and increased bladder capacity by 19–51%. Whereas voiding efficiency and maximum voiding pressure were not altered by IC485 at any dose, tolterodine significantly reduced both, by 35–67% and 19–34%, respectively.

CONCLUSION

Both IC485 and tolterodine tartrate reduced detrusor overactivity in rats with BOO. In addition, doses of IC485 that suppressed non‐voiding contractions had no effect on voiding function. Therefore, selective PDE4 inhibitors deserve further study as potential agents for treating detrusor overactivity in patients with BOO.     

Effects of doxycycline on voiding behaviour of rats with bladder outlet obstruction

OBJECTIVE

To examine the voiding behaviour changes in rats with bladder outlet obstruction (BOO) while inhibiting matrix metalloproteinase (MMP) activity with doxycycline, as increased MMP activity may be involved in obstruction‐induced bladder hypertrophy.

MATERIALS AND METHODS

Female Sprague‐Dawley were divided into eight groups (three rats in each group): normal control (NC) ± doxycycline, 3 weeks partial BOO (3WPBOO) ± doxycycline, 6 weeks PBOO ± doxycycline, and 3 weeks PBOO followed by 3 weeks de‐obstruction (3WOD) ± doxycycline. All rats received the same food and water and were on the same 12 h dark/light cycle housed in metabolic cages. Treatment groups were given doxycycline 15 mg/kg/day subcutaneously twice daily. The voiding variables measured were average voided volume (AV V) and voiding frequency (VF) in 24 h. After completion of the voiding behaviour studies, the rats were killed and their bladders were excised and weighed.

RESULTS

The AV Vs were significantly increased (P < 0.05) in all study groups compared with the NC group except for the 3WPBOO‐doxycycline and 3WOD‐doxycycline groups. The VF was significantly increased (P < 0.05) only in the 3WOD‐doxycycline group. The bladder weights were significantly increased after PBOO in all the study groups (P < 0.05), except for the 3WOD group.

CONCLUSION

These data show that MMP inhibition may affect voiding behaviour during the response to BOO or its relief. This is the first clinical demonstration that interfering with a principal target of bladder muscle wall remodelling may have a direct effect on bladder function.     

Effect of urothelium on bladder contractility in diabetic rats

AIM: It is known that physiopathological changes in diabetes affect the function of the bladder. In this study, we aimed to demonstrate the possible effects of diabetes on the urothelium during this physiopathological process. METHODS: Diabetes was induced in rats by tail vein injection of 35 mg/kg streptozotocin. Eight weeks later, intact and denuded bladder strips were prepared from these rats. Electrical field stimulation (EFS; 0.5-32 Hz), carbachol (10(-8)-10(-3) mol/L; cumulative dosage-response curves) and KCl (120 mmol/L) were used for the evaluation of the contractile responses. All responses were expressed as mg tension developed per mg of bladder tissue. Weights of rats and of their bladders, blood glucose levels, and frequency- and concentration-response curves were compared using anova, the paired t-test and the independent t-test. Differences were considered significant at P<0.05. RESULTS: Although no differences related to the weight of bladders of the control and diabetic groups were observed, there were differences in blood glucose levels and body weights between the two groups. Similarly, although there were no differences between the data obtained with EFS and KCl from tissues with intact and denuded strips in the control group, carbachol responses significantly differed between intact and denuded strips in the non-diabetic group. These differences were not observed in the diabetic group. In the control groups, in the presence of additional strips with intact urothelium placed in the medium containing denuded tissue, the differences in contractile responses between the intact control strip and the denuded strip disappeared. CONCLUSIONS: Diabetes possibly changes the interaction between the relaxant factors that are released from urothelium and muscarinic stimulation, but these interactions are not completely understood yet. Consequently, the response of the bladder to contractile stimulants is also affected. Further studies are required to reveal the mechanism by which diabetes influences the urothelium.     

Pharmacological effects of propiverine and its active metabolite, M-1, on isolated human urinary bladder smooth muscle, and on bladder contraction in rats

Objective:   To investigate the effects of M-1, a major active metabolite of propiverine on the bladder.
Methods:   We have evaluated the effects of M-1 on the contractions induced by carbachol, KCl, CaCl₂, and electrical field stimulation (EFS) in human detrusor smooth muscles, and pelvic nerve stimulation-induced bladder contractions in rats. The effects of M-1 were also compared with the effects of propiverine and tolterodine.
Results:   Pretreatment with propiverine and tolterodine caused parallel shifts to the right of the concentration-response curves to carbachol. M-1 caused concentration-dependent reduction in the maximum contractile responses induced by carbachol. Although tolterodine did not inhibit the KCl- and CaCl₂-induced contractions, M-1 and propiverine significantly inhibited these contractions. In the presence of atropine, M-1 and propiverine significantly inhibited the atropine resistant part of the contraction induced by EFS. On the other hand, tolterodine did not have significant inhibitory effects on atropine resistant contractions. Pelvic nerve stimulation induced bimodal phasic and tonic contractions in the rat bladder. M-1 mainly inhibited the phasic contraction. Tolterodine caused a significant inhibition in the tonic contraction, and propiverine had inhibitory effects on both contractions.
Conclusions:   The present results suggest that M-1 has inhibitory effects on the bladder smooth muscles through calcium antagonistic action. It is possible that the clinical effects of propiverine on the human bladder are based not only on the action of propiverine itself but also on one of its active metabolites, M-1.     

Up-regulation of endothelin-B (ETB) receptors and ETB receptor-mediated rabbit detrusor contraction in partial bladder outlet obstruction.

OBJECTIVE: To investigate, in a rabbit model of bladder outlet obstruction (BOO), whether ETB receptors initiate any contractile activity, and to assess the density of these receptors. MATERIALS AND METHODS: Partial BOO was produced in male New Zealand White rabbits, with age-matched sham-operated rabbits acting as controls. One and 3 weeks later, the detrusor and bladder neck strips were incubated in organ baths with either BQ788 (an ETB antagonist), BQ123 (an ETA antagonist) or vehicle. Concentration-response curves were constructed using IRL-1620 (a selective ETB agonist). Low-resolution autoradiography was performed on serial detrusor and bladder neck sections from control and partial BOO (3-week) rabbits using radioligands for ETA and ETB. RESULTS: In strips from controls and after 1 week of partial BOO, IRL-1620 induced no contractions, but after 3 weeks of BOO, IRL-1620 induced significant concentration-dependent detrusor contractions (producing 12%, 25% and 70% of the KCl response at 10-8, 10-7 and 10-6 mol/L, respectively). The ETA antagonist had no effect on IRL-1620-mediated contractions. In contrast, the ETB antagonist completely abolished these contractions. Autoradiography showed the presence of ETA and ETB receptors in the detrusor and bladder neck of normal and obstructed animals, and a significant up-regulation of ETA and ETB receptors only in the obstructed detrusor smooth muscle. CONCLUSIONS: In BOO, ETB receptors initiate detrusor contractile activity. This is a time-dependent process that may depend on the up-regulation of ETB receptors in the detrusor. Therefore, ETB receptors may play a role in the pathophysiology of partial BOO.     

Signal transduction pathways of muscarinic receptor mediated activation in the newborn and adult mouse urinary bladder

OBJECTIVE

To study the role of M₂ and M₃ muscarinic receptor subtypes, sources of activator Ca²⁺, and mechanisms involved in increased force oscillations in muscarinic contractions in the bladders of newborn and adult mice, as in the adult bladder muscarinic M₃ receptors are considered to mediate the main part of bladder contraction, and this has not been established in the newborn bladder.

MATERIALS AND METHODS

Bladder preparations from newborn (0–2 days) and adult (10–12 weeks) mice were mounted for in vitro force registration and activated with carbachol and high‐K⁺ solution in the presence of M₃ (4‐DAMP 30 nm ) or M₂ (methoctramine, 100 nm ) receptor antagonists. Thapsigargin (1 µm ) or ryanodine (10 µm ) were used to inhibit sarcoplasmic reticulum Ca²⁺ release. L‐NAME (300 µm ) and 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 10 µm ) were used to inhibit nitric oxide synthase and guanylyl cyclase, respectively. Gap‐junction function was inhibited with by 18‐β‐glycyrrhetinic acid (18‐β‐GA; 0.1–100 µm ). Big‐conductance (BK) and small‐conductance (SK) K⁺ channels were inhibited by apamine and charybdotoxin (0.3 µm ), respectively.

RESULTS

Concentration–response relations for carbachol in the presence of 4‐DAMP and methoctramine showed that M₃ receptors are the main activating pathway also in the newborn bladder. Neither thapsigargin nor ryanodine influenced the muscarinic responses of the newborn and adult bladders. Carbachol‐induced contractions were not influenced by L‐NAME or ODQ. The 18‐β‐GA inhibited carbachol‐induced contractions in both newborn and adult tissue in a similar manner. Apamine and charybdotoxin slightly increased the amplitude of the contractile responses.

CONCLUSION

These results suggest that in the newborn mouse bladder, as in adult bladders, the M₃ muscarinic receptor subtype is mainly responsible for carbachol‐induced contractile responses. The main mechanism for muscarinic receptor‐induced activation is influx of Ca²⁺ from the extracellular medium, and there seems to be no major contribution of Ca²⁺ release from intracellular stores. The phasic contractile activity induced by carbachol in the newborn bladder is not influenced by gap junction inhibition and does not involve SK and BK channels.     

Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivo

OBJECTIVE

To investigate the primary pharmacology of fesoterodine (a novel antimuscarinic drug developed for treating overactive bladder) and SPM 7605 (its active metabolite, considered to be the main pharmacologically active principle of fesoterodine in man) against human muscarinic receptor subtypes, and to investigate in vitro and in vivo functional activity of these agents on the rat bladder compared with existing standard agents.

MATERIALS AND METHODS

The displacement of radioligand binding by fesoterodine, SPM 7605 and standard agents in membrane preparations of Chinese hamster ovary (CHO) cells expressing the different human muscarinic receptors (M1–M5) was characterized. Agonistic and antagonistic activities were studied using different CHO cell lines stably expressing the human recombinant muscarinic receptor subtypes. The effects of fesoterodine and SPM 7605 on isolated bladder strips contracted by carbachol or electrical field stimulation (EFS) were investigated. In vivo the effects of fesoterodine and SPM 7605 on micturition variables were assessed using continuous cystometry in conscious female Sprague‐Dawley rats, and compared to those of oxybutynin and atropine.

RESULTS

In vitro SPM 7605 potently inhibited radioligand binding at all five human muscarinic receptor subtypes with equal affinity across all five. Fesoterodine had a similar balanced selectivity profile but was less potent than SPM 7605. Both substances were competitive antagonists of cholinergic agonist‐stimulated responses in human M1‐M5 cell lines and had a similar potency and selectivity profile to the radioligand‐binding studies. In rat bladder strips, fesoterodine and SPM 7605 caused a rightward shift of the concentration‐response curve for carbachol with no depression of the maximum, and concentration‐dependently reduced contractions induced by EFS. The potency of both drugs was similar to that of atropine and oxybutynin. In the presence of the esterase inhibitor neostigmine, the concentration‐response curve of fesoterodine was shifted to the right, suggesting that part of the activity was caused by metabolism to SPM 7605 by tissue enzymes. In vivo, low doses (0.01 mg/kg) of fesoterodine and SPM 7605 reduced micturition pressure and increased intercontraction intervals and bladder capacity, but did not affect residual volume.

CONCLUSIONS

Fesoterodine and its active metabolite, SPM 7605, are nonsubtype selective, competitive antagonists of human muscarinic receptors, but SPM 7605 has greater potency than the parent compound. Pharmacodynamic studies in the rat bladder in vitro confirm the competitive muscarinic antagonist profile of these agents in a native tissue preparation, and in vivo studies in the rat showed effects on bladder function consistent with a muscarinic antagonist profile.     

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.     

Oxidative stress reduces the muscarinic receptor function in the urinary bladder

AIMS: Several pathophysiological conditions in the urinary bladder, for example, ischemia/reperfusion and inflammation are characterized by the formation of reactive oxygen species (ROS). The ROS are highly toxic because they can destroy proteins, DNA, and lipids. The aim of this study was to investigate the effect of oxidative stress on excitation-contraction coupling of detrusor smooth muscle. MATERIALS AND METHODS: Smooth muscle strips were dissected from pig urinary bladder and mounted in organ baths. Oxidative stress was mimicked by the addition of Cumene hydroperoxide (CHP), a lipophilic hydroperoxide, to the organ baths. Contractile responses to electrical field stimulation (EFS: 4-32 Hz), carbachol (10(-8)-3 x 10(-5) M), potassium (65.3 mM), and ATP (1 mM) were monitored before and after the addition of CHP. RESULTS: Responses of detrusor strips to EFS were for the greater part based on neurogenic stimulation and the release of acetylcholine. CHP diminished contractile responses to EFS and carbachol to the same extent. The pD(2) value of the carbachol concentration-response curve decreased significantly after exposure to 0.1 mM, 0.4 mM, 0.8 mM CHP. Furthermore the maximal effect obtained with carbachol was significantly reduced after 0.1 mM, 0.4 mM, and 0.8 mM CHP treatment. Contractions induced by potassium and ATP were significantly less affected by oxidative stress compared to EFS- and carbachol-induced responses of comparable amplitude. CONCLUSIONS: The results of our study demonstrate that oxidative stress induced by CHP affects pig bladder contractility. The muscarinic receptor signaling system is severely damaged. L-type calcium channels and the contractile system are less affected and cholinergic nerves remain largely unaffected.     

Altered bladder function in elastin-deficient mice at baseline and in response to partial bladder outlet obstruction

Study Type – Aetiology (case control) Level of Evidence 3b What's known on the subject? and What does the study add? As one of the major components of the extracellular matrix, elastic fibres are believed to enhance tissue compliance. However, the role of elastic fibres in normal bladder function and dysfunction remains speculative. Although transgenic mice overexpressing elastin showed increased bladder compliance, the findings in patients with non‐compliant bladders are inconsistent. Using transgenic elastin‐deficient mouse models, this study provides the first direct evidence that sufficient elastin content is critical for healthy bladder function, and elastin is involved in the detrusor response to partial bladder outlet obstruction.

OBJECTIVE

• ? To examine functional and molecular changes of the bladders from elastin‐haploinsufficient mice (Eln^+/?) at baseline as well as in response to partial bladder outlet obstruction (pBOO).

MATERIALS AND METHODS

• ? Female Eln^+/? and wild type (Wt) mice (3–4 months old) were studied.
• ? The bladder elastin content was quantified by measuring desmosine.
• ? Mice were divided into two groups to undergo surgery to create pBOO or to undergo sham surgery. Three days after surgery, bladder function was evaluated by in vivo cystometry, and the contractile response of bladder strips exposed to electrical field stimulation (EFS) and carbachol was examined by ex vivo myography.

RESULTS

• ? The Eln^+/?‐sham mice had a 33.6% decrease in bladder elastin compared with Wt‐sham mice.
• ? Cystometry showed significantly decreased bladder compliance and capacity in Eln^+/?‐sham vs Wt‐sham mice; pBOO increased bladder compliance and capacity to a greater extent in Eln^+/? mice compared with Wt mice.
• ? Bladder strips from Eln^+/?‐sham mice showed a significantly heightened contractile response to both EFS and carbachol compared with Wt‐sham mice.
• ? A significantly increased contractile response to carbachol was detected in Wt‐pBOO vs Wt‐sham but not between Eln^+/?‐pBOO and Eln^+/?‐sham mice.

CONCLUSION

• ? The results that elastin‐deficient mice had decreased bladder compliance and capacity and increased bladder contractility; and that Wt‐pBOO mice showed an enhanced contractile response to carbachol, but Eln^+/?‐pBOO mice did not, suggest that elastin is critical for normal bladder function and is involved in bladder response to pBOO.

     

Changes to the contractile function of ureter smooth muscle after partial infravesical obstruction in fetal sheep

OBJECTIVE

To measure spontaneous contractile activity, and the responses to agonists using in vitro preparations of sheep ureter after a period of bladder outlet obstruction (BOO) initiated at mid‐gestation.

MATERIAL AND METHODS

Date‐mated Romney Marsh ewes, bearing fetuses of 70–75 days of gestation (term in this breed is 145 days) were used. Five fetuses underwent urachal obstruction and partial urethral constriction for 30 days. Six fetuses at the same gestational time underwent a sham operation (control group). Small strips of mid‐ureter were cut in the longitudinal axis, and in the cross‐sectional (transverse) plane of the ureter. Spontaneous contractile activity and the response to carbachol, high‐K⁺ (120 mm ) solution and α,β‐methylene ATP (ABMA) were characterized by measuring the magnitude of evoked responses and the magnitude and frequency of spontaneous activity.

RESULTS

The ureters from fetuses with BOO were significantly larger in diameter and had expanded lumens. The proportion of smooth muscle was not significantly different between the BOO and control groups. Spontaneous contractile activity in all preparations was resistant to atropine and the neurotoxin, tetrodotoxin. With transverse sections, the magnitude of spontaneous contractions was smaller in the BOO group, but the frequency was greater. The response to carbachol was also smaller in the BOO group, but the response to high‐K⁺ solution was similar to that of the control group. ABMA did not generate a response in any preparation. With longitudinal ureteric preparations, spontaneous or agonist‐induced activity was negligible in the control group, while preparations from the BOO sheep had spontaneous activity and responded to carbachol and high‐K⁺ solutions.

CONCLUSION

These results show that in fetuses with BOO and dilated ureters absolute ureteric contractile activity is diminished. However, there is functional reorganization of the muscle layers that generates more force in the longitudinal rather than the transverse axis. This would contribute to a reduced ability of the ureter to propel urine and contribute to the development of raised upper tract pressures.     

CHANGE IN BLADDER CONTRACTILITY ASSOCIATED WITH BLADDER OVERACTIVITY IN RATS WITH CEREBRAL INFARCTION

Purpose

To evaluate the contractile properties of overactive bladder from rats in the chronic stage of experimental cerebral infarction.

Materials and Methods

Cystometry was performed in conscious male S-D rats after inducing occlusion of the left middle cerebral artery. Bladder muscle strips were evaluated for force development in response to field stimulation, acetylcholine and KCl. By measuring the contractile response to field stimulation after adding atropine and alpha, beta-methylene-ATP, contributions of cholinergic and purinergic transmission were determined.

Results

Bladder capacity of cerebral-infarcted rats was <50% of the capacity of sham-operated rats and significantly less than that of sham-operated rats even 4 months after surgery. There was no significant difference in bladder weight between sham-operated rats and cerebral-infarcted rats. No differences in the contractile response of detrusor strips to field stimulation and acetylcholine, or in the relative contribution of cholinergic and purinergic transmission to the contractile response, were observed over time or between strips from sham-operated rats and cerebral-infarcted rats. KCl induced significantly less contraction in strips from 4 month infarcted rats than in strips from 4 month sham-operated rats, 2 week infarcted rats and 2 month infarcted rats.

Conclusions

This animal model will be useful for chronic studies on the mechanism of detrusor hyperactivity (DH).     

Increased intra-abdominal pressure alters the contractile properties of rabbit bladder

OBJECTIVE: To evaluate the effects of increased intra-abdominal pressure (IAP) on the contractility of the rabbit bladder, as the dynamics of the bladder may be impaired in conditions associated with a high IAP, e.g. constipation and pregnancy. Material and methods The study comprised 22 adult male New Zealand rabbits; six served as the control group, eight had an IAP of 7 cmH2O imposed for 10 days by instilling air into the abdominal cavity and this IAP was maintained for 60 days in a further eight rabbits. After treatment, the rabbits were killed, and the bladders removed and cut into 3 x 12 mm strips. The contractile activity of the muscle strips was then recorded isometrically. Electrical field stimulation (EFS) was applied using a pair of platinum ring electrodes in trains of 3 s duration every 100 s (1 ms, 100 V, 2-100 Hz). Contractile responses to carbachol and isotonic KCl were also evaluated. RESULTS: EFS induced a frequency-dependent increase in contractile activity in all bladder strips. Ten days of high IAP resulted in an increased responsiveness to EFS, but high IAP for 60 days reduced the EFS-induced responses to the control levels. Carbachol (10-9-10-3 mol/L) elicited concentration-dependent contractions in all groups. From the concentration-response curves of carbachol, the log EC50 values (the concentration producing half the maximum effect) of the control and 60-day treated animals were comparable, at -6.24 (0.05) and -6.25 (0.04), respectively. However, the log EC50 of the 10 day-treated group was -4.97 (0.08) and significantly (P < 0.01) lower than that of both groups. Isotonic KCl produced contractions in all preparations; these contractions in the control and 60-day treated animals were similar, while the 10 day-treated group had significantly (P < 0.05) higher contraction amplitudes. CONCLUSION: Increased IAP alters the contractile properties of the bladder and its responsiveness to carbachol and KCl. As the intravesical pressure closely reflects the IAP, both should be increased in the present experimental design.     

Bladder function in 17β-estradiol-induced nonbacterial prostatitis model in Wister rat

Objectives

To investigate bladder function in a model of nonbacterial prostatitis (NBP) induced in castrated rats by 17β-estradiol injection.

Methods

Ten-month-old male Wistar rats were divided into two groups, sham and NBP (both N = 8). NBP was induced by castration followed by daily subcutaneous injection of 17β-estradiol for 30 days. On the 31st day after surgery, we investigated (1) voiding behavior, (2) bladder blood flow (BBF), (3) prostate and bladder weight, and proinflammatory cytokines (TNF-α and CXCL1) levels and (4) bladder contractile responses to electrical field stimulation (EFS), carbachol and KCl.

Results

(1) Voiding behavior (average micturition volume, total urine volume and number of micturitions) and (2) BBF were not significantly different between the sham and NBP groups. (3) NBP led to a significant decrease in prostatic weight and increase in proinflammatory cytokine levels in the prostate, but NBP did not cause a significant change in bladder weight or proinflammatory cytokine levels in the bladder. (4) Bladder contractile forces in response to EFS, carbachol and KCl were not significantly affected by NBP.

Conclusions

In this rat model, NBP did not cause a significant change in the level of proinflammatory cytokines in the bladder and affect bladder function.     

Changes of bladder activity and glycine levels in the lumbosacral cord after partial bladder outlet obstruction in rats

Objectives: We investigated the time course of changes in bladder activity as well as in spinal and serum levels of glutamate and glycine after partial bladder outlet obstruction (BOO) in rats. Methods: A total of 36 female rats were divided into six groups: sham operation (control); 3 days, 14 days, and 28 days after BOO; 3 days and 28 days after relief of BOO. Under urethane anesthesia, isovolumetric cystometry was carried out in each group. Then, spinal and serum levels of glutamate and glycine were measured. Results: The interval between bladder contractions was shorter in all of the groups compared with the control group. The amplitude and duration of bladder contractions was decreased at 3 days, 14 days, and 28 days after BOO, and at 3 days after relief of BOO. Spinal and serum glutamate levels showed no changes. However, the spinal glycine level was decreased at 14 days and 28 days after BOO, and at 28 days after relief of BOO. Serum glycine level was also decreased at 28 days after BOO and 28 days after relief of BOO. Conclusions: Detrusor overactivity during the chronic phase of partial BOO is partly caused by a decrease of glycinergic neuronal activity in the lumbosacral cord. A 3‐day period of BOO produces detrusor overactivity, which might be due to an irreversible decrease of spinal glycinergic neuronal activity.