Urinary Bladder Detrusor Research Articles

Modeling and Simulation of Bladder Power Pump Driven by External Electromagnet

ABSTRACTThe clinical urination assist measures are usually taken to overcome the neurogenic bladder difficult to cure, but easily cause patients bladder stones, urinary system infection and other complications. To solve the problem of the urination assist of neurogenic bladder, a bladder power pump (BPP) driven by external electromagnet was proposed in this study according to the structure and micturition mechanism of human urinary bladder. The mathematic models of the BPP were established based on the theories of electromagnetics and fluid dynamics. A simulated experiment system with a bladder physical model for simulating human urinary system was designed according to the similarity criterion of fluid dynamincs. The micturition performance of the BPP was investigated by simulation and experiment. The results showed that the intravesical pressure and urine flow rate of the bladder physical model can be controlled by adjusting electromagnet exciting current. The micturition performance of the BPP is basically accordant with that of human urinary bladder detrusor. The established mathematic models are simple and practicable, which can provide the theoretical guidance for the performance analysis and structural optimization design of the BPP.

Neuronal and extraneuronal release of ATP and NAD+ in smooth muscle

AbstractAdenosine 5′‐triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+) are key intracellular constituents involved in energy transfer and redox homeostasis in the cell. ATP is also released in the extracellular space and in the past half century it has been assumed to be the purinergic neurotransmitter in many systems including smooth muscle. In some smooth muscles (i.e., the human urinary bladder detrusor muscle), ATP does appear to be primarily released from nerves upon action potential firings, but in other smooth muscles (i.e., the human large intestine), ATP does not mimic the endogenous purine neurotransmitter. It was recently found that NAD+, another ubiquitous intracellular adenine nucleotide, also follows a regulated release in neurosecretory cells, vascular and visceral smooth muscles, and the brain. In some cases, NAD+ fulfills presynaptic and postsynaptic criteria for a neurotransmitter better than ATP. Therefore, the purine hypothesis of neural regulation in smooth muscle is in need of reevaluation. This article will briefly review the current understanding of neuronal and extraneuronal release of purines in smooth muscle with emphasis on the roles of extracellular ATP and NAD+ and, further, will discuss more recent information about the likely involvement of multiple purines in smooth muscle neurotransmission. © 2012 IUBMB IUBMB Life, 2012

Neuronal and extraneuronal release of ATP and NAD+ in smooth muscle

Adenosine 5'-triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD(+) ) are key intracellular constituents involved in energy transfer and redox homeostasis in the cell. ATP is also released in the extracellular space and in the past half century it has been assumed to be the purinergic neurotransmitter in many systems including smooth muscle. In some smooth muscles (i.e., the human urinary bladder detrusor muscle), ATP does appear to be primarily released from nerves upon action potential firings, but in other smooth muscles (i.e., the human large intestine), ATP does not mimic the endogenous purine neurotransmitter. It was recently found that NAD(+) , another ubiquitous intracellular adenine nucleotide, also follows a regulated release in neurosecretory cells, vascular and visceral smooth muscles, and the brain. In some cases, NAD(+) fulfills presynaptic and postsynaptic criteria for a neurotransmitter better than ATP. Therefore, the purine hypothesis of neural regulation in smooth muscle is in need of reevaluation. This article will briefly review the current understanding of neuronal and extraneuronal release of purines in smooth muscle with emphasis on the roles of extracellular ATP and NAD(+) and, further, will discuss more recent information about the likely involvement of multiple purines in smooth muscle neurotransmission.

Effects of imidazolines on neurogenic contraction in isolated urinary bladder detrusor strips from rabbit

Moxonidine and clonidine, which are imidazoline compounds, are sympathetic modulators used as centrally acting antihypertensive drugs. Moxonidine, clonidine, and agmatine produce extensive effects in mammalian tissues via imidazoline recognition sites (or receptors) or α(2)-adrenoceptors. To investigate the effects of imidazolines on the function of the urinary bladder, we tested the effects of moxonidine, clonidine, and agmatine on the neurogenic contraction induced by electric field stimulation, and on the post-synaptic receptors in isolated urinary bladder detrusor strips from rabbit. Both moxonidine at 1.0-10.0µmol/L and clonidine at 0.1-10.0µmol/L inhibited electric-field-stimulation-induced contraction in a concentration-dependent manner, but not agmatine (10.0-1000.0µmol/L). Both moxonidine and clonidine failed to affect carbachol or adenosine-triphosphate-induced contractions; however, 1000.0µmol/L agmatine significantly increased these contractions. Our study indicates that (i) moxonidine and clonidine produce a concentration-dependent inhibition of the neurogenic contractile responses to electric field stimulation in isolated detrusor strips from male New Zealand rabbits; (ii) post-synaptic muscarinic receptor and purinergic receptor stimulation are not involved in the responses of moxinidine and clonidine in this study; (iii) the inhibitory effects of these agents are probably not mediated by presynaptic imidazoline receptors.

Unique properties of muscularis mucosae smooth muscle in guinea pig urinary bladder.

The muscularis mucosae, a type of smooth muscle located between the urothelium and the urinary bladder detrusor, has been described, although its properties and role in bladder function have not been characterized. Here, using mucosal tissue strips isolated from guinea pig urinary bladders, we identified spontaneous phasic contractions (SPCs) that appear to originate in the muscularis mucosae. This smooth muscle layer exhibited Ca(2+) waves and flashes, but localized Ca(2+) events (Ca(2+) sparks, purinergic receptor-mediated transients) were not detected. Ca(2+) flashes, often in bursts, occurred with a frequency (∼5.7/min) similar to that of SPCs (∼4/min), suggesting that SPCs are triggered by bursts of Ca(2+) flashes. The force generated by a single mucosal SPC represented the maximal force of the strip, whereas a single detrusor SPC was ∼3% of maximal force of the detrusor strip. Electrical field stimulation (0.5-50 Hz) evoked force transients in isolated detrusor and mucosal strips. Inhibition of cholinergic receptors significantly decreased force in detrusor and mucosal strips (at higher frequencies). Concurrent inhibition of purinergic and cholinergic receptors nearly abolished evoked responses in detrusor and mucosae. Mucosal SPCs were unaffected by blocking small-conductance Ca(2+)-activated K(+) (SK) channels with apamin and were unchanged by blocking large-conductance Ca(2+)-activated K(+) (BK) channels with iberiotoxin (IbTX), indicating that SK and BK channels play a much smaller role in regulating muscularis mucosae SPCs than they do in regulating detrusor SPCs. Consistent with this, BK channel current density in myocytes from muscularis mucosae was ∼20% of that in detrusor myocytes. These findings indicate that the muscularis mucosae in guinea pig represents a second smooth muscle compartment that is physiologically and pharmacologically distinct from the detrusor and may contribute to the overall contractile properties of the urinary bladder.

Effect of the SK/IK channel modulator 4,5‐dichloro‐1,3‐diethyl‐1,3‐dihydro‐benzoimidazol‐2‐one (NS4591) on contractile force in rat, pig and human detrusor smooth muscle

• To investigate the importance of small (SK)- and intermediate (IK)-conductance Ca2(+) -activated K(+) channels on bladder function, by studying the effects of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a new modulator of SK/IK channels, on contractions induced by electrical field stimulation (EFS) and carbachol in rat, pig and human detrusor. • Detrusor biopsies were obtained from rats, pigs and male patients undergoing cystectomy because of bladder cancer. • Force was recorded using myographs. • Intracellular free Ca(2+) was measured in myocytes using microfluorimetry. • In rat bladder rings subjected to EFS, cumulative addition of NS4591 (0.1-30 µM) decreased force by 82 ± 2.9% (n = 6).This effect was reduced by 64 ± 5.2% in the presence of 0.3 µM apamin, a specific inhibitor of SK channels. Apamin increased the force evoked by EFS significantly: force was increased by 14.2 ± 3.4% (n = 5) and 10.1 ± 2.6% (n = 7) in pig and human detrusor strips, respectively (P = 0.04 and P = 0.02). • The cumulative addition of NS4591 (0.3-30 µM) significantly reduced the amplitude of carbachol-induced rhythmic oscillations by 62.0 ± 12.0% (n = 12) and the minimum force between oscillations by 30 ± 5% (n = 9) in pig detrusor strips (P < 0.005). In the presence of 10 µM NS4591, carbachol (1 µM) induced rhythmic contractions with an amplitude and normalized mean power frequency (nmeanPF) of 8.4 ± 5.1% and 0.11 ± 0.06 mN root mean square (rms) Hz (n = 12), respectively, vs. 21 ± 3.4% and 0.17 ± 0.04 mN rms Hz in control strips (n = 13). Apamin induced 6- and 11-fold increases in amplitude and nmeanPF vs. 1.3- and 2-fold increases in control strips. • In human detrusor strips (n = 15), the cumulative addition of NS4591 (1-30 µM) significantly reduced the amplitude by 69 ± 11%, the nmeanPF by 78 ± 6% and the minimum force between carbachol-induced oscillations by 59 ± 5% (P < 0.008). The addition of apamin (0.3 µM) before application of 1 µM carbachol abolished the effects of NS4591 on amplitude and partially abolished its effect on nmeanPF by 41 ± 7%, vs. a 78 ± 6% reduction in the absence of apamin (n = 8). • In spontaneously active detrusor preparations, NS4591 reduced or abolished contractions. • Furthermore, NS4591 (10 µM) decreased the carbachol-induced increase in the fura-2 ratio by 43 ± 3% compared with control (n = 12) (P < 0.03). • The SK/IK channel modulator NS4591 inhibits EFS- and carbachol-induced contractions in rat, pig and human detrusor muscle. • NS4591 may have therapeutic potential for treatment of detrusor overactivity.

Inhibitory effect of anticholinergics on the contraction of isolated caprine urinary bladder detrusor muscle

This study investigated whether four anticholinergics which are not clinically used for relaxing the urinary bladder detrusor muscle inhibit the contraction of isolated caprine (goat) detrusor muscle: cyclopentolate (100 nm), homatropine (5 microm), ipratropium (500 nm) and valethamate (1 microm). The effects of these anticholinergics were compared with tolterodine (3 microm), an anticholinergic clinically used for relaxing the detrusor muscle. The inhibitory effect of each of these five anticholinergics was studied on six strips of caprine detrusor muscle made to contract with 100 mum acetylcholine (ACh) by determining the percent inhibition of height of contraction and the area under the contractile curve (AUC). It was found that all five anticholinergics inhibited the ACh-induced contraction of the caprine detrusor and that this inhibition was reversed by raising the concentration of ACh. Hence, these four anticholinergics, like tolterodine, may be useful in managing clinical conditions that require relaxation of the detrusor muscle.

The Forefront for Novel Therapeutic Agents Based on the Pathophysiology of Lower Urinary Tract Dysfunction: Pathophysiology of Voiding Dysfunction and Pharmacological Therapy

Normal lower urinary tract function consists of voiding and storage. During voiding, the pontine micturition reflex center orders the sacral parasympathetic nucleus to increase parasympathetic activity, resulting in urinary bladder detrusor contraction via activation of post-synaptic muscarinic receptors (M2/3) and in the relaxation of both urethral and prostatic smooth muscle by nitric oxide (NO). In addition, the rhabdosphincter relaxes by inhibition of the pudendal nucleus at the sacral portion. During the storage phase, increase in sympathetic activity relaxes the urinary bladder via activation of post-synaptic β3-receptors and in the contraction of both urethral and prostatic smooth muscles via α1-adrenoceptor. Many factors influence voiding function, including lower urinary tract disorders (benign prostatic hyperplasia in males, urethral stricture) and neurological disorders (central and peripheral). Theories of pharmacotherapy for voiding dysfunction are 1) increase detrusor contractility and 2) decrease urethral resistance. The former includes agonists for muscarinic receptors and cholinesterase inhibitor; and the latter includes α1-adrenoceptor antagonists, NO donors, benzodiazepines, baclofen, dantrolene, and boturinum toxin.

Early fracture callus displays smooth muscle‐like viscoelastic properties ex vivo: Implications for fracture healing

Cells of early, fibrous callus in bone fractures possess much alpha smooth muscle actin. This callus contracts and relaxes; however, active and passive components of its force production have yet to be defined. We aimed to establish whether passive viscoelastic properties of early soft fracture callus are smooth muscle-like in nature. Under anesthesia one rib was fractured in rats and calluses removed 7 days later for analysis. Urinary bladder detrusor muscle and Achilles tendon were also resected and analyzed. Force production in these tissues was measured using a force transducer when preparations were immersed in calcium-free Krebs-Henseleit solution (pH 7.4, 22 degrees C). Viscoelastic responses were measured in each preparation in response to 50 microN increases and decreases in force after achieving basal tissue tension by preconditioning. Callus, bladder, and tendon all displayed varying, reproducible degrees of stress relaxation (SR) and reverse stress relaxation (RSR) (n = 7 for all groups). Hysteresis was observed in callus, with the first SR response significantly larger than that produced in subsequent stretches (p < 0.05). Callus SR responses were greater than tendon (p < 0.001) but less than bladder (p < 0.001). Callus RSR responses were greater than tendon (p < 0.001), but no significant difference was seen between RSR of callus and bladder. We concluded that early, soft callus displayed significant SR and RSR phenomena similar to smooth muscle tissue, and SR and RSR may be important in maintenance of static tension in early callus by promoting osteogenesis and fracture healing.

１．排尿障害の病態と新しい治療

Normal lower urinary tract function consists of voiding and storage. During voiding, the pontine micturition reflex center orders the sacral parasympathetic nucleus to increase parasympathetic activity, resulting in urinary bladder detrusor contraction via activation of post-synaptic muscarinic receptors (M2/3), and the relaxation of both urethral and prostatic smooth muscle by NO. In addition, the rhabdosphincter relaxes by inhibition of pudendal nucleus at the sacral portion. During the storage phase, an increase in sympathetic activity relaxes urinary bladder via activation of post-synaptic β3 receptors, and both the urethral and prostatic smooth muscles are contracted via α1-adrenoceptor. Many factors influence voiding function, including lower urinary tract disorders (benign prostatic hyperplasia in males, urethral stricture), and neurological disorders (central and peripheral). Theories of action of pharmacotherapy for voiding dysfunction are: 1) increase detrusor contractility, and 2) decrease urethral resistance. The former includes agonists for muscarinic receptors, and choline-esterase inhibitor, and the latter includes α1-adrenoceptor antagonists, nitric oxide donors, Benzodiazepines, Baclofen, Dantrolene, and Boturinum toxin.

Comparison of Receptor Binding Characteristics of Commonly Used Muscarinic Antagonists in Human Bladder Detrusor and Mucosa

Recent studies have described muscarinic receptors on the mucosa and the detrusor of the human urinary bladder. Muscarinic receptor antagonists are effective in the treatment of overactive bladder (OAB), but their site(s) of action and actual therapeutic target are unclear. Our aim was to compare, in human bladder mucosa and detrusor, the radioligand binding characteristics of newer, clinically effective agents: darifenacin, its hydroxylated metabolite UK-148,993, fesoterodine, solifenacin, tolterodine, and trospium. Specimens were collected from asymptomatic patients (50-72 years old) undergoing open bladder surgery. Radioligand binding studies with the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) were performed separately on detrusor and mucosal membranes. All antagonists displayed high affinity when competing for [3H]QNB binding in both detrusor and mucosa. Inhibition constants were also obtained for all antagonists against individual muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Here, fesoterodine showed anomalous binding results, suggesting that some conversion to its metabolite had occurred. Global nonlinear regression analysis of bladder binding data with five antagonists demonstrated 82% low-affinity sites in mucosa and 78% low-affinity sites in detrusor, probably representing M(2)/M(4) receptors. There was an excellent correlation (r(2) = 0.99) of low-affinity global estimates between detrusor and mucosa, whereas the corresponding high-affinity estimates ( approximately 20% of sites) were dissimilar. In conclusion, commonly used and clinically effective muscarinic receptor antagonists bind to receptors located on the bladder mucosa and the detrusor, providing support for the hypothesis that muscarinic receptors in the mucosa may represent an important site of action for these agents in OAB.

Partial outlet obstruction of the rabbit urinary bladder induces selective protein oxidation

Oxidation of proteins is presumed to contribute to contractile dysfunction associated with partial outlet obstruction (PBOO) of the urinary bladder. The objective of this study was to determine the acute and chronic effects of PBOO on protein oxidation in urinary bladder detrusor smooth muscle (DSM) and mucosa, and to correlate these findings with in vitro contractile function. Urinary bladders of New Zealand White (NZW) rabbits were obstructed for 1, 3, 7 and 14 days. Proteins were extracted from the bladder tissues and protein carbonyl formation was assessed using immunoblot assays. In vitro contractile response to field stimulation (32 Hz) and carbachol was evaluated in whole bladder strips at the same time points. Significant elevations in oxidation of DSM proteins were observed in the MW range of 29-65 kDa after 3 days and 14 days of obstruction. No changes in the oxidative status of mucosal proteins were detected as a result of short or long term obstruction. The intermediate filament protein, desmin (53-55 kDa) was detected in obstructed DSM samples in the same MW range as oxidized proteins. A significant decrease in contractile response to field stimulation and carbachol was observed after 1 day and 3-days respectively, and continued to deteriorate through 14 days. The increase in protein oxidation at 14-days of obstruction correlates with impaired bladder contractility, suggesting that oxidatively modified proteins may contribute to the contractile and biochemical dysfunction associated with PBOO.

Therapeutic Satisfaction and Dissatisfaction in Patients With Spinal Cord Lesions and Detrusor Sphincter Dyssynergia Who Received Detrusor Botulinum Toxin A Injection

To investigate satisfaction in terms of quality-of-life (QOL) improvement among patients with spinal cord lesions and detrusor sphincter dyssynergia treated with botulinum toxin A (BoNT-A) injections to the detrusor for urinary incontinence and bladder dysfunction. A total of 50 patients with spinal cord lesion and detrusor sphincter dyssynergia were treated with BoNT-A injections (200 U) into the urinary bladder detrusor muscle. The urodynamic parameters, QOL scores, and general satisfaction with the treatment were assessed. Overall satisfactory results were perceived by 78% of patients who received detrusor BoNT-A injections. The maximal bladder capacity had increased significantly (221 +/- 107 vs 432 +/- 115 mL, P = .000), detrusor pressure decreased (43.5 +/- 21.1 vs 12.6 +/- 10.7 cm H(2)O, P = .000), and postvoid residual urine volume increased (121 +/- 107 vs 325 +/- 123 mL, P = .000) by 3 months after BoNT-A treatment. Patients had significant improvement in QOL indexes (Urogenital Distress Inventory 6-item short form 11.7 +/- 2.3 vs 4.1 +/- 2.1, P = .01; and Incontinence Impact Questionnaire, 7-item short form 16.3 +/- 3.5 vs 11.7 +/- 4.1, P = .03) after BoNT-A treatment. Decreases in incontinence grade and urgency episodes contributed the most to satisfaction, and increased difficulty in urination and the need for catheterization contributed to dissatisfaction. Of the 50 patients, 25 (50%) received repeated BoNT-A injections; 4 patients finally decided to undergo augmentation enterocystoplasty because they desired permanent corrections and did not wish to receive repeated injections. The patient satisfaction rate was 78%, and QOL improved among these patients after detrusor BoNT-A injections for urinary incontinence and bladder dysfunction.

Inhibitory effect of nicorandil on the contraction of isolated human urinary bladder detrusor muscle

This study was performed to determine whether the antianginal drug nicorandil relaxes isolated human detrusor muscle. Ten strips of detrusor muscle obtained from 10 pediatric patients who underwent surgery on the urinary bladder were contracted with 80 mM potassium chloride (KCl) before and after incubation with four concentrations of nicorandil (100, 200, 400 and 800 microM). The percent inhibition by nicorandil of the height and area under the curve (AUC) of KCl-induced contractions of the detrusor strips was calculated. The effect of glibenclamide (10 microM) on nicorandil (800 microM)-induced inhibition of KCl-induced detrusor contractions was also studied. Nicorandil caused a concentration-dependent inhibition of KCl-induced contractions of the detrusor strips. The percent inhibition of the height of KCl-induced contractions of the detrusor by nicorandil was significant at concentrations of 200, 400 and 800 microM. The percent inhibition of the AUC for KCl-induced detrusor contractions was significant at all four concentrations of nicorandil used. Glibenclamide reversed the inhibitory effect of 800 microM nicorandil on KCl-induced detrusor contractions. These results suggest that nicorandil inhibits KCl-induced contractions of isolated human detrusor muscle and may therefore be useful in clinical conditions requiring detrusor muscle relaxation.

Assessment of Muscarinic Receptor Subtypes in Human and Rat Lower Urinary Tract by Tissue Segment Binding Assay

Muscarinic receptors in the human and rat lower urinary tract (urinary bladder detrusor muscle and mucosa, and prostate) were identified by intact tissue segment binding assays with two radioligands, and the effects of prolonged receptor activation in vitro on muscarinic receptors were examined. Hydrophilic [3H]-NMS and hydrophobic [3H]-QNB bound to the detrusor muscle segments with the same density, suggesting that the muscarinic receptors were localized at the plasma membrane. While the density of muscarinic receptor was higher in detrusor muscle than in the bladder mucosa and prostate, there was no species-specific difference either in density or in subtype distribution (M1, M2, and M3subtypes in detrusor; M2and M3subtypes in bladder mucosa; and M1and M2subtypes in prostate). Incubation of detrusor strips with carbachol decreased [3H]-NMS binding sites within 20 min, followed by a reduction of [3H]-QNB binding sites after a 60-min lag phase. The loss of the binding sites over 3 h after carbachol treatment was the same (approximately 40%) for both radioligands. The present intact tissue segment binding assay reveals tissue-specific and plasma membrane distribution of distinct muscarinic receptor subtypes and their dynamic changes (internalization and down-regulation) in lower urinary tract of humans and rats.

β-NAD and CD38 provide a novel mechanism of autonomic nervous system control of vascular and visceral smooth muscle

We have recently reported that β-nicotinamide adenine dinucleotide (β-NAD) is released during electrical field stimulation (EFS) of canine isolated mesenteric arteries and veins and urinary bladder detrusor smooth muscle of mouse, rat, guinea-pig, rabbit, monkey, and human (Smyth et al., J Biol Chem 279:48893, 2004; Breen et al., Am J Physiol 290:F 486, 2006). Exogenous β-NAD reduces the release of norepinephrine, causes vasorelaxation in canine mesenteric blood vessels, and inhibits the spontaneous contractions in murine and human bladders.

The effect of intravesical ketoprofen on acetylcholine-evoked urinary bladder contractility and detrusor overactivity in the anesthetized rabbit model

Intraurethral procedures such as the transurethral resection of the prostate can generate detrusor overactivity and bladder irritability. The rabbit model of detrusor overactivity has proven to be an excellent model to study the effects of drugs on detrusor overactivity. Using this model, we evaluated the responses to intravesical ketoprofen. In this model, each rabbit is anesthetized and the right external carotid artery is cannulated for blood pressure monitoring. A catheter is inserted through the femoral artery and is used for administration of acetylcholine (Ach). The bladder is exposed and catheterized for bladder pressure monitoring and drug addition and the proximal urethra is ligated. Cystometry is performed, the bladder drained, and 20 ml buffer placed in the bladder. After 30 min Ach is injected proximal to the vesical artery and the response of the bladder and blood pressure is recorded. Ach administration is repeated at 10-min intervals until three consistent responses are obtained. The bladder is drained and 20 ml of ketoprofen (100 microM final concentration) is placed in the bladder. Ach injections are repeated as given above at 10 min intervals and observed for 4 h. At the end of the experiment, a second cystometry is performed. The following is a summary of the results: Ketoprofen had no effect on either micturition pressure or the intravesical volume at micturition. Ketoprofen administration resulted in a progressive 50% decrease in the response to Ach. Ketoprofen mediated a progressive decrease in detrusor overactivity amplitude and frequency, reaching a maximum at 120-180 min.

Decreased expression of G protein‐coupled receptor kinases in the detrusor smooth muscle of human urinary bladder with outlet obstruction

We examine the expression of mRNA of G protein-coupled receptor kinase (GRK) subtypes and muscarinic acetylcholine receptor (M) subtypes in the detrusor smooth muscle of the human urinary bladder. Furthermore, we confirm the presence and the localization of GRK proteins in the detrusor smooth muscle of the obstructed bladder in comparison with the control bladder. Detrusor smooth muscle tissues of the human urinary bladder were obtained from 12 male patients; 6 patients did not have bladder outlet obstruction, and the other 6 patients had bladder outlet obstruction. Portions of the dome or anterior wall of the urinary bladder were used for the present study. Reverse transcription/polymerase chain reaction for GRK2, M2 and M3 was performed using total RNA extracted from human urinary bladder detrusor. Antibodies to GRK2, GRK3 and GRK4 were used to confirm the presence of the protein product in the human urinary bladder using immunohistochemical staining and the western blotting technique. All complementary DNA (cDNA) transcribed from three different mRNA (M2, M3 and GRK2) were successfully amplified and size-fractionated. The expression of GRK2 protein was strong in the human bladder detrusor, but was significantly weakened by western blotting in obstructed bladder in comparison with control bladder. Failure in desensitization mechanisms of muscarinic acetylcholine receptors might be related to storage symptom elicited by overactivity in obstructed bladder with benign prostatic hyperplasia.

Effects of potassium channel modulators on human detrusor smooth muscle myogenic phasic contractile activity: Potential therapeutic targets for overactive bladder

Objectives Increased urinary bladder detrusor smooth muscle phasic contractility has been suggested to be associated with idiopathic bladder overactivity (OAB). We examined the role of voltage-dependent l-type calcium channels, adenosine triphosphate-sensitive potassium (K ATP) channels, and calcium-activated potassium (BK Ca and SK Ca) channels in the regulation of human detrusor phasic contractile activity. Methods Isolated human bladder strip phasic contractions were measured and quantified as the mean area under the force-time curve, amplitude, and frequency of phasic contractions in 22 bladder samples. Results Human detrusor strips displayed myogenic phasic contractions in the presence of atropine (10 −6 M), phentolamine (10 −6 M), propranolol (10 −6 M), suramin (10 −5 M), and tetrodotoxin (10 −6 M). The l-type calcium channel inhibitor nifedipine (300 nM) abolished the contractile activity. Blockade of K ATP channels by glibenclamide (1 and 10 μM) did not alter myogenic contractions. In contrast, the K ATP channel opener pinacidil (10 μM) markedly inhibited phasic contractility. Iberiotoxin (100 nM) and apamin (100 nM), potent and selective inhibitors of BK Ca and SK Ca channels, respectively, significantly increased the area under the force-time curve and the amplitude of contractions. Conclusions Phasic contractions of human detrusor are dependent on calcium entry through l-type calcium channels. BK Ca and SK Ca channels play a key role in the modulation of human detrusor smooth muscle phasic contractility. Furthermore, these observations support the concept that increasing conductance through K ATP, BK Ca, and SK Ca channels may represent attractive pharmacologic targets for decreasing phasic contractions of detrusor smooth muscle in OAB.

Caveolar nanospaces in smooth muscle cells

Caveolae, specialized membrane nanodomains, have a key role in signaling processes, including calcium handling in smooth muscle cells (SMC). We explored the three-dimensional (3D) architecture of peripheral cytoplasmic space at the nanoscale level and the close spatial relationships between caveolae, sarcoplasmic reticulum (SR), and mitochondria, as ultrastructural basis for an excitation-contraction coupling system and, eventually, for excitation - transcription coupling. About 150 electron micrographs of SMC showed that superficial SR and peripheral mitochondria are rigorously located along the caveolar domains of plasma membrane, alternating with plasmalemmal dense plaques. Electron micrographs made on serial ultrathin sections were digitized, then computer-assisted organellar profiles were traced on images, and automatic 3D reconstruction was obtained using the ‘Reconstruct’ software. The reconstruction was made for 1 μm3 in rat stomach (muscularis mucosae) and 10 μm3 in rat urinary bladder (detrusor smooth muscle). The close appositions (about 15 nm distance) of caveolae, peripheral SR, and mitochondria create coherent cytoplasmic nanoscale subdomains. Apparently, 80% of caveolae establish close contacts with SR and about 10% establish close contacts with mitochondria in both types of SMC. Thus, our results show that caveolae and peripheral SR build Ca2+release units in which mitochondria often could play a part. The caveolae-SR couplings occupy 4.19% of the cellular volume in stomach and 3.10% in rat urinary bladder, while caveolae-mitochondria couplings occupy 3.66% and 3.17%, respectively. We conclude that there are strategic caveolae-SR or caveolae-mitochondria contacts at the nanoscale level in the cortical cytoplasm of SMC, presumably responsible for a vectorial control of free Ca2+ cytoplasmic concentrations in definite nanospaces. This may account for slective activation of specific Ca2+ signaling pathways.