Isolated Detrusor Smooth Muscle Research Articles

P.3.e.004 - Effects of long-term treatment with risperidone and clozapine on mice isolated detrusor smooth muscle

Incidence of lower extremity amputations (LEA) in the population with and without diabetes mellitus (DM) was assessed after implementation of a Multidisciplinary Diabetic Foot Unit (MDFU) during 2008.Non-traumatic LEA were analyzed, and those performed before (2001–2007) and after (2008–2014) introduction of the MDFU were compared. LEA were grouped by age and sex. Their incidence was expressed as a rate per 100,000 population per year, adjusted to the standard European population.A total of 664 LEA were performed during the 2001–2014 period, 486 (73%) of them in patients with DM. Total LEA incidence was 11.2/105 population in DM versus 3.9/105 in the population without DM. Incidence of major LEA in patients with DM significantly decreased from 6.1/105 population in the 2001–2007 period to 4.5/105 in the 2008–2014 period (p = 0.03). Joinpoint regression analysis also showed a reduction in the trend of incidence of major LEA in patients with DM, with an annual percentage change of −3.3% [95% CI, −6.2–0.3] (p = 0.025). No significant differences were found for all other incidences and trends in the diabetic and non-diabetic populations.Implementation of a MDFU has been shown to be associated with a significant reduction in major amputation rate in the diabetic population, although the results are not optimal yet. Both results and work at the MDFU should be improved.Evaluamos la incidencia de amputaciones de miembro inferior (AMI) en población con y sin diabetes mellitus (DM) tras la implementación en nuestro centro de una Unidad Multidisciplinar de Pie Diabético (UMPD) durante el año 2008.Analizamos las AMI de causa no traumática y comparamos las realizadas antes del inicio de la UMPD, 2001-2007; vs. las realizadas después, 2008-2014. Las AMI se agruparon por edad y sexo. Su incidencia se expresó por 100.000 habitantes y año, ajustada a la población europea estándar.Se realizaron 664 AMI, 486 (73%) en población con DM durante el período 2001-2014. La incidencia de todas las AMI en DM fue de 11,2/105 habitantes vs. 3,9/105 en población sin DM. La incidencia de AMI mayores en DM se redujo de manera significativa de 6,1/105 habitantes en el período 2001-2007 a 4,5/105 en el período 2008-2014 (p = 0,03). El análisis de regresión jointpoint, también demostró una reducción en la tendencia de la incidencia de AMI mayores en la población con DM, observándose un cambio porcentual anual del -3,3% [IC 95%, -6,2-0,3] (p = 0,025). No encontramos diferencias significativas en el resto de incidencias y tendencias en población con y sin DM.La puesta en funcionamiento de una UMPD en nuestro centro ha demostrado que se asoció a una reducción significativa en la tasa de amputaciones mayores en la población diabética, si bien estos resultados aún no son óptimos. Se precisa mejorar estos resultados y el funcionamiento de la UMPD.

Urinary Bladder Dysfunction in Transgenic Sickle Cell Disease Mice.

BackgroundUrological complications associated with sickle cell disease (SCD), include nocturia, enuresis, urinary infections and urinary incontinence. However, scientific evidence to ascertain the underlying cause of the lower urinary tract symptoms in SCD is lacking.ObjectiveThus, the aim of this study was to evaluate urinary function, in vivo and ex vivo, in the Berkeley SCD murine model (SS).MethodsUrine output was measured in metabolic cage for both wild type and SS mice (25-30 g). Bladder strips and urethra rings were dissected free and mounted in organ baths. In isolated detrusor smooth muscle (DSM), relaxant response to mirabegron and isoproterenol (1nM-10μM) and contractile response to (carbachol (CCh; 1 nM-100μM), KCl (1 mM-300mM), CaCl2 (1μM-100mM), α,β-methylene ATP (1, 3 and 10 μM) and electrical field stimulation (EFS; 1-32 Hz) were measured. Phenylephrine (Phe; 10nM-100μM) was used to evaluate the contraction mechanism in the urethra rings. Cystometry and histomorphometry were also performed in the urinary bladder.ResultsSS mice present a reduced urine output and incapacity to produce typical bladder contractions and bladder emptying (ex vivo), compared to control animals. In DSM, relaxation in response to a selective β3-adrenergic agonist (mirabegron) and to a non-selective β-adrenergic (isoproterenol) agonist were lower in SS mice. Additionally, carbachol, α, β-methylene ATP, KCl, extracellular Ca2+ and electrical-field stimulation promoted smaller bladder contractions in SS group. Urethra contraction induced by phenylephrine was markedly reduced in SS mice. Histological analyses of SS mice bladder revealed severe structural abnormalities, such as reductions in detrusor thickness and bladder volume, and cell infiltration.ConclusionsTaken together, our data demonstrate, for the first time, that SS mice display features of urinary bladder dysfunction, leading to impairment in urinary continence, which may have an important role in the pathogenesis of the enuresis and infections observed the SCD patients.

Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells.

Recent studies have demonstrated the importance of large-conductance Ca(2+)-activated K(+) (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K(+) symmetrical solution (containing 140 mM KCl and ~300 nM free Ca(2+)) showed single-channel conductance of 215-220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca(2+)] from ~300 nM to Ca(2+)-free decreased the P o by ~85 %, whereas elevation to ~800 nM increased P o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca(2+)] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.

Characteristics of Lower Urinary Tract Dysfunction and Bladder Afferent Nerve Properties in Type 2 Diabetic Goto-Kakizaki Rats

We investigated longitudinal changes in lower urinary tract function, especially sensory function, in the type 2 diabetes mellitus model of Goto-Kakizaki rats. We used 16 Wistar and 16 Goto-Kakizaki rats. Body weight, blood glucose, 24-hour voiding frequency-volume data and cardiovascular system data were measured every 3 weeks starting at age 5 weeks until age 14 weeks and then every 6 weeks until age 44 weeks. At ages 10 and 46 weeks conscious cystometry was done, pelvic afferent nerve fiber conduction velocity was measured using urethane anesthesia and isolated detrusor smooth muscle function was determined. Goto-Kakizaki rats showed lower body weight, higher blood glucose and mean voided volume, and less voiding frequency than Wistar rats throughout the observation period. In 46-week-old Goto-Kakizaki rats lower peak micturition pressure, larger bladder capacity and higher bladder compliance were noted on cystometry, while slower conduction velocity and a lesser proportion of Aδ fibers were observed on conduction velocity measurement. Goto-Kakizaki rats showed a lower contractile response to carbachol but not to electrical field stimulation or high K(+) in isolated detrusor smooth muscle. These differences between the 2 groups of rats at age 46 weeks were not noted at age 10 weeks. Cardiovascular system results did not differ between the 2 groups. The results of this longitudinal study in Goto-Kakizaki rats indicate that type 2 diabetes induces bladder sensory dysfunction, manifesting as slower bladder afferent conduction velocity, larger bladder capacity and greater hypocontractility to acetylcholine.

Influence of High Fat Diet Feeding for 20 Weeks on Lower Urinary Tract Function in Mice.

We investigated the possible changes in lower urinary tract function in mice fed a high fat diet (HFD). Male C57BL/6J mice were divided into two different feed groups: normal diet (ND) and HFD (n = 16 in each). The body weight, blood glucose level and voiding frequency/volume (FV) relations (for 24 h) were measured every 4 weeks. At 25 weeks old, blood pressure and heart rate, cystometry and isolated detrusor smooth muscle function were measured. After the experiments, serum fat level was measured. The body weight and blood glucose level of the HFD group were significantly higher than those of the ND group after 9 weeks old. In the FV measurements, the mean voided volume was not significantly different between the two groups, although voiding frequency, total voided volume and water intake volume in the HFD group were significantly lower than those in the ND group. At 25 weeks old, the mean heart rate in the HFD group was significantly higher than that in the ND group, but no significant difference in the blood pressure was observed. None of the cystometric parameters analyzed showed significant differences between the two groups. The contractile response to either carbachol or high K(+) was not significantly different, whereas the contractile response to electrical field stimulation was significantly higher in the HFD group. In the HFD group, the mean total cholesterol level was significantly higher. The present results suggest that HFD-feeding for 20 weeks in mice unlikely affects bladder function even though it induced diabetes, hyperlipidemia and tachycardia.

493 DEVELOPMENT OF A NEUROGENIC BLADDER IN TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 KNOCKOUT MICE

INTRODUCTION AND OBJECTIVES: Bladder pain of unknown etiology has been associated with co-morbid conditions and functional abnormalities in adjacent pelvic organs. This phenomenon is called “crosssensitization” and occurs predominantly via convergent neural pathways connecting distinct pelvic organs. Our previous studies showed that colonic inflammation can cause detrusor instability via activation of transient receptor potential vanilloid 1 (TRPV1) signaling pathways. In this study, we aimed to determine whether cross-sensitization in the pelvis can develop in the absence of TRPV1 receptors and lead to a neurogenic bladder. METHODS: Four groups of adult male mice (N 25) were included in the study: 2 groups of C57BL/6 wild-type (WT) mice with and without acute colitis (induced by intracolonic trinitrobenzene sulfonic acid, TNBS) and 2 groups of TRPV1 knockout mice (TRPV1 / ) mice with and without acute colitis. Mice were sacrificed at day 3 after the induction of pelvic organ cross-sensitization by acute colitis and the function of the detrusor was evaluated in vitro using isolated detrusor smooth muscle (DSM) strips. The level of inflammation in the distal colon and urinary bladder was evaluated by the myeloperoxidase (MPO) assay. The contractility of DSM strips was studied in response to potassium chloride test, electric field stimulation and relaxation of DSM by Rho kinase inhibitor. RESULTS: In WT mice, acute colitis caused 2-fold up-regulation of MPO levels in the colon whereas in TRPV1 / mice an increase reached 1.5 fold (p 0.05 to respective control). MPO levels were unchanged in the bladders from both groups. The contractile response of DSM strips to electric stimulation was significantly increased after colitis in WT mice (85.97 12.27 g/g) when compared to control group (44.39 10.81 g/g, p 0.05). The same changes were observed in the TRPV1 / group with pelvic organ cross-sensitization. The use of Rho kinase inhibitor Y27632 induced DSM relaxation in WT group by 72.87 10.40% whereas it was only 39.38 6.46% in the TRPV / group (N 6, p 0.05 to WT). CONCLUSIONS: Our data provide evidence that the absence of TRPV1 receptors does not eliminate the occurence of cross-sensitization in the pelvis and TRPV / mice develop a neurogenic bladder. However, DSM from TRPV / mice showed decreased relaxation in response to a Rho kinase inhibitor suggesting alterations in DSM signaling pathways.

Characterization of the urinary bladder dysfunction in renovascular hypertensive rats

Association between arterial hypertension and urinary bladder dysfunction has been reported in humans and spontaneously hypertensive rats. However, no study exists evaluating the bladder dysfunction in conditions of renovascular hypertension. The purpose of this study was to characterize the bladder dysfunction in two kidney-one clip (2K-1C) hypertensive rats. A silver clip was placed around the renal artery of male Wistar rats. After 8 weeks, cystometric study, concentration-response curves to contractile and relaxant agents, frequency-dependent contractions, histomorphometry, muscarinic M(2) /M(3) mRNA expression and cyclic AMP measurements were performed. 2K-1C rats showed enhanced bladder volume, wall thickness and smooth muscle density. 2K-1C rats also exhibited increases in bladder capacity and non-void contractions, and decreases in the inter-contraction intervals. In isolated detrusor smooth muscle (DSM), contractions to carbachol and electrical-field stimulation (EFS) were significantly greater in 2K-1C rats. The Rho-kinase inhibitor Y27632 (10 µM) significantly reduced the carbachol-induced contractions in SHAM and 2K-1C rats, but DSM remained overactive in 2K-1C rats in presence of Y27632. Concentration-dependent contractions to the P2X receptor agonist α,β-methylene ATP, KCl and extracellular Ca(2+) did not change between SHAM and 2K-1C groups. In 2K-1C rats, isoproterenol, metaproterenol and BRL 37-344 (non-selective, β(2) - and β(3) -selective adrenoceptor agonists, respectively) produced significantly lower relaxations and decreased cAMP levels, whereas relaxant responses to sodium nitroprusside and BAY 41-2272 remained unchanged. Muscarinic M(3) mRNA expression receptors were higher in 2K-1C group. Renovascular hypertensive rats exhibit bladder dysfunction that involves tissue remodeling and enhanced muscarinic M(3) -mediated contractions associated with reduced β-adrenoceptor-mediated signal transduction.

Evaluation of the relaxant effect of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in isolated detrusor smooth muscle

The nitric oxide (NO)-independent soluble guanylyl cyclase stimulator stimulator BAY 41-2272 was reported to produce relaxant response in different types of smooth muscle. However no study was carried out to investigate the effects of BAY 412282 in detrusor smooth muscle. Thus, this study aimed to evaluate the relaxant effects of BAY 41-2272, in isolated mouse, rat and rabbit detrusor smooth muscle. Mouse, rat and rabbit were anesthetized, and urinary bladder removed. Detrusor smooth muscle was transferred to 10-mL organ baths containing oxygenated and warmed Krebs-Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. BAY 41-2272 (0.001-100 microM) produced concentration-dependent detrusor smooth muscle relaxations in mouse, rat and rabbit with maximal responses of 61.3+/-6.6%, 95.1+/-9.9% and 91.7+/-5.9%, respectively. Sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced detrusor relaxations, but to a much lesser extent than BAY 41-2272. The NO synthesis inhibitor L-NAME and the phosphodiesterase-5 inhibitor sildenafil had no effect in BAY 41-2272-induced responses. However, the soluble guanylyl cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxations. BAY 41-2272 increased the bladder cGMP levels by about of 14- and 20-fold for 10 and 100 microM, respectively, which were markedly reduced by ODQ. The cAMP levels were unaffected by BAY 41-2272. Moreover, BAY 41-2272 significantly reduced the contractile responses to extracellular Ca(2+) in an ODQ-insensitive manner. In conclusion, rabbit detrusor smooth muscle relaxations by BAY 41-2272 involve mainly cGMP production, but an additional mechanism involving Ca(2+) influx blockade independently of cGMP production appears to be involved.

In vitro Effect of Amikacin on Rat and Human Detrusor Muscle Contraction

Introduction: Normal and abnormal bladder contractions are principally mediated by acetylcholine released from postganglionic parasympathetic nerves. Since amikacin was reported to affect neurotransmission by a prejunctional mechanism, we investigated the effect of amikacin on isolated detrusor smooth muscle contraction to further evaluate its potential relaxant properties. Materials and Methods: Detrusor smooth muscle obtained from 15 rats and 8 patients undergoing surgery were studied through measurement of isometric muscular contraction induced with electrical field stimulation (EFS) (10–60 Hz), carbachol (10^–7 to 10^–3M) and nicotine (10^–7 to 10^–3M) in the presence or absence of 1 mM amikacin in a low-Ca medium. Results: Amikacin (1 mM) significantly reduced EFS-induced contraction of isolated rat and human detrusor muscle by 33 ± 6.57% (p < 0.005) and 40 ± 1.14% (p < 0.001), respectively. Contraction was restored after addition of calcium chloride (1 mM). The effect of amikacin was comparable to that of magnesium ions. Rat and human detrusor contractile response to nicotine was inhibited by 70 ± 8.27% (p < 0.001) and 64 ± 14.09% (p < 0.01) after the addition of amikacin (1 mM), while no significant effect was observed on carbachol-induced stimulation. Conclusion: Amikacin significantly inhibited detrusor contraction evoked by prejunctional stimulation in vitro, suggesting a depressant effect on autonomic neurotransmission in urinary bladder.

Melatonin restores impaired contractility in aged guinea pig urinary bladder

Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin-treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca(2+)](i)) was estimated by epifluorescence microscopy of fura-2-loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca(2+)](i) in response to the stimuli, suggesting a reduced Ca(2+)sensitivity. Indeed, the agonist-induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca(2+) sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and with the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca(2+) handling and Ca(2+) sensitizations pathways. Moreover, the indoleamine restored age-induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging-related urinary bladder contractile impairment.

Supersensitivity of muscarinic receptors in rat isolated detrusor smooth muscle (DSM) after chronic nitric oxide inhibition

Address: 1Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas (SP), Brazil, 2Department of Pharmacology, Pontifical Catholic University of Campinas, Campinas (SP), Brazil, 3Department of Physical Education; Institute of Bioscience, Paulista State University, Rio Claro (SP), Brazil, 4Section of Experimental Endocrinology, Department of Pharmacology, UNIFESP (SP), Sao Paulo, Brazil, 5Pharmacology Laboratory, Butanta Institute (SP), Sao Paulo, Brazil, 6Departament of Clinical Pathology, University of Campinas, Campinas (SP), Brazil and 7Faculty of Medicine, Pontifical Catholic University of Campinas, Campinas (SP), Brazil

Relaxant effect of BAY 41–2272 in the rabbit isolated detrusor smooth muscle (DSM): involvement of cGMP-independent mechanisms

Background Overactive bladder (OAB) is described as urgency with or without incontinence, usually accompanied by frequency and nocturia. Overactive bladder reflects involuntary DSM as a consequence of enhanced cholinergic-mediated contractions and decreased b-adrenoceptor-mediated relaxations. The nitric oxide (NO)-cyclic GMP signaling pathway has been described to modulate the muscular tone, neurotransmission and blood flow in DSM, but the exact role of NO in the bladder is not completely understood. It is accepted that NO deficiency contributes to triggering OAB. The compound BAY 41–2272 has been described as a drug that directly activates the soluble guanylate cyclase leading to NO-independent cGMP accumulation. BAY 41–2272 produces potent relaxations in vascular and non-vascular smooth muscle tissues by cGMP-dependent and independent mechanisms, but no studies have been carried out with DSM.

The influence of changes in extracellular and intracellular sodium concentration on detrusor contractility

To determine the role of Na+-Ca2+ exchange in the regulation of isolated detrusor smooth muscle contractility. Isolated guinea-pig detrusor strips were used to record isometric tension generated by; (a) electrical-field stimulation to elicit nerve-mediated responses; and (b) adding carbachol or superfusing with a high-K+ solution. The [Na+] gradient between extracellular and intracellular compartments was altered by: (i) reducing superfusate [Na+] in stages from 140.2 to 10.2 mm; (ii) addition of the cardiac glycoside strophanthidin (200 microm). RESULTS Reducing extracellular [Na+] reversibly reduced the magnitude of nerve-mediated contractions but increased the resting tension and magnitude of carbachol-induced contracture. The mean (sd) [Na+] required for a half-maximum effect on attenuating contractions, at 85.9 (6.2) mm, and developing contracture, at 59.1 (14.3) mm, were significantly different. The time constants of changes to nerve-mediated contractions and carbachol contracture were also significantly different, at 147 (5) vs 1207 (386) s, respectively. These differences suggest that separate mechanisms influence nerve-mediated contraction and contracture in low-Na+ solutions. Exposure to the cardiac glycoside strophanthidin produced a similar effect to low-Na+ solutions for carbachol contracture. Low-Na+ solutions had no significant effect on contractures induced by high extracellular [K+]. CONCLUSION Reducing the transmembrane [Na+] difference increases intracellular [Ca2+]. This increase is largely accommodated in intracellular stores, that can be released by exogenous carbachol. The results are consistent with the presence of a functional Na+-Ca2+ exchanger in the surface membrane. The lack of effect of low-Na+ solutions on contractures evoked by membrane depolarization is consistent with this conclusion. The reduction of the nerve-mediated contraction by low-Na+ solution might result from blockade of the nerve action potential.

The role of Ni2+‐sensitive T‐type Ca2+ channels in the regulation of spontaneous excitation in detrusor smooth muscles of the guinea‐pig bladder

To explore the role of Ni(2+)-sensitive T-type Ca(2+) channels in the generation of spontaneous excitation of detrusor smooth muscles. In isolated detrusor smooth muscle bundles of the guinea-pig bladder, changes in the membrane potential and muscle tension were measured using intracellular microelectrodes and isometric tension recording. Changes in the intracellular Ca(2+) concentration were recorded from bundles loaded with the fluorescent dye fura-PE3. Detrusor smooth muscles had two types of spontaneous electrical activity, i.e. individual and bursting action potentials. Ni(2+) (30 microM), a blocker for T-type Ca(2+) channels, reduced the frequency of individual action potentials without changing their amplitude. Higher concentrations of Ni(2+) (100-300 microM) converted individual action potentials into the bursts, as did apamin (0.1 microM), a blocker of small-conductance Ca(2+)-activated K(+) channels (SK). They also increased the amplitudes of spontaneous Ca(2+) transients and corresponding contractions whilst reducing their frequencies. In preparations which generated bursting action potentials, nifedipine (1 microm) converted action potentials into spontaneous transient depolarizations (STDs), and subsequent applications of Ni(2+) (100 microm) abolished STDs. Gadolinium (100 microM) and SKF96365 (10 microM), blockers for nonselective cation channels, and niflumic acid (100 microm), a blocker for Ca(2+)-activated Cl- channels, had no effect on either the amplitude or frequency of spontaneous action potentials. The T-type Ca(2+) channel may have dual roles in generating spontaneous excitation in detrusor smooth muscles. First, activity of these channels may account for the preceding depolarizations that lead to action potentials. Second, Ca(2+) influx through T-type Ca(2+) channels may couple functionally to SK channels, contributing to the stability of the resting membrane potential in detrusor smooth muscle. Thus, pharmacological manipulation of T-type Ca(2+) channels in detrusor smooth muscles could be of potential value for treating the overactive bladder.

Doxazosin Effects on Cholinergic and Adrenergic Responses in Rat Isolated Detrusor Smooth Muscle Preparations From Obstructed Bladder

We investigated the effect of doxazosin on cholinergic and adrenergic agonists responses in detrusor smooth muscle preparations from sham-operated and 2-week partially obstructed rat bladders. Male Wistar albino rats, 200 – 250 g, were randomly allocated to 4 experimental groups consisting of 12 animals each: sham-operated bladder, sham-operated bladder treated with doxazosin, partially obstructed bladder, and partially obstructed bladder treated with doxazosin. Partial outlet obstruction of the rat was surgically induced. The response to carbachol (10−7 – 10−4), isoproterenol (10−6 – 10−3 M) and 80 mM KCl were recorded. Carbachol caused concentration-dependent contractile responses in the detrusor smooth muscles from sham-operated and partially obstructed bladder. Isoproterenol produced concentration-dependent relaxation responses in the detrusor strips from all groups. Dose-response curves for carbachol and isoproterenol showed a shift to the left in rat detrusor smooth muscles from partially obstructed bladder when compared with the results obtained in detrusor muscles from sham-operated bladder. These responses were reversed to normal by doxazosin treatment in rat detrusor smooth muscles from partially obstructed bladder. KCl produced contractile responses in rat detrusor smooth muscles from all groups. The contractile responses to KCl were not significantly changed in all groups. We have shown that carbachol and isoproterenol responses were shifted to the left in rat detrusor smooth muscles from partially obstructed bladder and these responses were reversed by doxazosin treatment.

THE ELECTROPHYSIOLOGICAL PROPERTIES OF CULTURED AND FRESHLY ISOLATED DETRUSOR SMOOTH MUSCLE CELLS

We generated and characterized a convenient isolated cell model of human detrusor smooth muscle to understand mechanisms that may underlie detrusor instability and provide a suitable model to test potentially useful drugs. The electrophysiological properties of freshly isolated detrusor smooth muscle cells from human and guinea pig biopsies were compared with those undergoing cell culture to document in detail the changes that occur during primary culture and subsequent passages as well as the differences in the 2 species. Resting electrical characteristics were changed in the cultured cells. Membrane potential was less negative (guinea pig -59 versus -42 mV.) and membrane resistance was less (138 versus 124.5 Omegacm.(2)). Regenerative action potentials were recorded in cultured and freshly isolated cells. In guinea pig cells the overall duration and initial rate of depolarization (upstroke) was slower in cultured than in freshly isolated cells, indicative of a decreased magnitude of ionic current in cultured cells. Human cells had a similar prolongation in culture but no decrease in the upstroke rate. Experiments with selective blockers indicated that depolarization is due to influx through L-type Ca2+ channels and repolarization occurred via Ca2+ dependent K+ channels in freshly isolated and cultured cells. No further changes to properties were observed in cells passaged up to 3 times from primary cultured cells. Cell culture qualitatively preserves the electrophysiological properties of detrusor smooth muscle cells, although there is some decrease in channel density.

STUDIES OF THE PATHOPHYSIOLOGY OF IDIOPATHIC DETRUSOR INSTABILITY: THE PHYSIOLOGICAL PROPERTIES OF THE DETRUSOR SMOOTH MUSCLE AND ITS PATTERN OF INNERVATION

Idiopathic detrusor instability (IDI) is a common cause of lower urinary tract storage symptoms, such as urgency, frequency and urge incontinence. We have investigated the in vitro properties and pattern of innervation of the detrusor from patients with this condition. Full thickness bladder specimens were obtained perioperatively from 14 patients with IDI and from 14 cadaveric controls undergoing transplant organ retrieval. Isolated detrusor smooth muscle strips were mounted in organ baths for isometric tension recording. Frequency-response curves to electrical field stimulation (EFS) (1 Hz to 50 Hz) and concentration response curves for carbachol (10(-7) M to 5 x 10(-4) M) and potassium (12 mM to 120 mM) were constructed. Acetylcholinesterase histochemistry and immunohistochemistry for both phosphorylated and non-phosphorylated neurofilaments was carried out on frozen sections of control and IDI bladders. IDI strips developed greater spontaneous tone (0.25 gm./mg. versus 0.12 gm./mg.; p <0.0001) and more spontaneous fused tetanic contractions (16.8% versus 6.8%; p <0.005) during an initial 90 minutes equilibration period. The IDI strips were less responsive than controls to nerve stimulation (max. response to EFS 0.79 gm./mg. versus 1.23 gm./mg.; p <0.0001) and were supersensitive to potassium (EC50 39.7 mM versus 45.7 mM; p = 0.003) but not to carbachol (EC50 7.3 x 10(-6) M versus 6.6 x 10(-6) M; p = 0.48). Morphometric studies revealed reduced staining of presumed cholinergic nerves, with 34.7% of IDI smooth muscle bundles appearing denervated compared with 1.5% of controls (p <0.0001). Our study supports the notion that there is a fundamental abnormality in IDI at the level of the bladder wall, with evidence of altered spontaneous contractile activity consistent with an increased electrical coupling of cells, a patchy denervation of the detrusor and a potassium supersensitivity.

Visco-elastic Properties of Isolated Detrusor Smooth Muscle

Visco-elastic Properties of Isolated Detrusor Smooth Muscle

Visco-elastic Properties of Isolated Detrusor Smooth Muscle

The generation of force by the contractile apparatus and the modulation of that force by the intra- and extracellular matrix are the initial steps in the production of bladder wall tension. The biomechanical components contributing to the observed rise in bladder wall tension may be studied in isolated detrusor tissue and attempts can be made to isolate these. The problem is to determine whether clinically observed alterations in detrusor function are due to changes in the contractile apparatus or in the surrounding matrix. This review discusses the viscoelastic properties of detrusor muscle, concentrates upon the influence of bladder outflow obstruction on the mechanical properties of the detrusor in an attempt to understand changes in contractile function.

Ca2+ mobilization in cultured human bladder smooth muscle cells in response to hypotonic stimuli

The effects of hypotonic solutions on intracellular Ca2+ were determined on primary cultures of human detrusor smooth muscle cells. In the presence of external Ca2+, changing from a solution containing 290 mosmol l-1 to one containing 240 mosmol l-1 (80% normal tonicity) activated a rise in intracellular Ca2+. If the exposure to 240 mosmol l-1 was followed by exposure to 150 mosmol l-1 (50% normal tonicity) a further increase in Ca2+ was noted. Exposure to hypotonic solutions which contained nominally zero Ca2+ also resulted in a rise in intracellular Ca2+, suggesting that mechanical perturbation of the surface membrane can mobilize Ca2+ directly from intracellular stores. Stimulation of cells with a brief exposure to agonist (histamine, 5 microM) in the nominal absence of external Ca2+ produced a release of Ca2+ from intracellular stores. Under these conditions the response to a subsequent hypotonic stimulation was small or absent. These observations suggest that hypotonic stress induces a mobilization of Ca2+ from the same store as that activated by agonist. Detailed analysis of data from individual bladders revealed that there were significant differences between cells cultured from normal bladders and those from bladders with detrusor instability. On exposure to 240 mosmol l-1 cells from unstable bladders gave larger responses than cells from normal bladders. However, exposure of cells from unstable bladders to a subsequent stimulus of 150 mosmol l-1 resulted in a smaller rise in Ca2+ than was observed with normal cells. Overall, these data suggest that isolated detrusor smooth muscle cells are sensitive to membrane stretch by hyposmotic solutions and that the mechanisms involved include the direct mobilization of intracellular Ca2+. The data also suggest that there may be differences in the responsiveness of cells isolated from stable and unstable bladders.