Activity Of Detrusor Smooth Muscle Research Articles

In Silico Electrophysiological Investigation of Transient Receptor Potential Melastatin-4 Ion Channel Biophysics to Study Detrusor Overactivity.

Enhanced electrical activity in detrusor smooth muscle (DSM) cells is a key factor in detrusor overactivity which causes overactive bladder pathological disorders. Transient receptor potential melastatin-4 (TRPM4) channels, which are calcium-activated cation channels, play a role in regulating DSM electrical activities. These channels likely contribute to depolarizing the DSM cell membrane, leading to bladder overactivity. Our research focuses on understanding TRPM4 channel function in the DSM cells of mice, using computational modeling. We aimed to create a detailed computational model of the TRPM4 channel based on existing electrophysiological data. We employed a modified Hodgkin-Huxley model with an incorporated TRP-like current to simulate action potential firing in response to current and synaptic stimulus inputs. Validation against experimental data showed close agreement with our simulations. Our model is the first to analyze the TRPM4 channel's role in DSM electrical activity, potentially revealing insights into bladder overactivity. In conclusion, TRPM4 channels are pivotal in regulating human DSM function, and TRPM4 channel inhibitors could be promising targets for treating overactive bladder.

Autophagy Alters Bladder Angiogenesis and Improves Bladder Hyperactivity in the Pathogenesis of Ketamine-Induced Cystitis in a Rat Model.

Simple SummaryLong-term ketamine abuse may increase urinary frequency, nocturia, urgency, bladder pain, dysuria, and sometimes hematuria. Evaluation of the pathophysiological mechanism of bladder voiding dysfunction in ketamine-induced cystitis (KIC) patients is a critical step for therapy. This study uses autophagy inducer (rapamycin, mTOR inhibitor) and inhibitor (wortmannin, PI3K-III inhibitor) to identify the role of autophagy in bladder angiogenesis alteration and bladder hyperactivity improvement.The present study attempts to elucidate whether autophagy alters bladder angiogenesis, decreases inflammatory response, and ameliorates bladder hyperactivity—thereby influencing bladder function in ketamine-induced cystitis (KIC). In our methodology, female Sprague-Dawley (S-D) rats were randomly divided into the control group, the ketamine group, the ketamine+rapamycin group, and the ketamine+wortmannin group. The bladder function, contractile activity of detrusor smooth muscle, distribution of autophagosome and autolysosome, total white blood cells (WBCs) and leukocyte differential counts, the expressions of autophagy-associated protein, angiogenesis markers, and signaling pathway molecules involved in KIC were tested, respectively. The data revealed that treatment with ketamine significantly results in bladder overactivity, enhanced interstitial fibrosis, impaired endothelium, induced eosinophil-mediated inflammation, swelling, and degraded mitochondria and organelles, inhibited angiogenesis, and elevated the phosphorylation of Akt. However, treatment with rapamycin caused an inhibitory effect on vascular formation, removed ketamine metabolites, decreased the eosinophil-mediated inflammation, and ameliorated bladder hyperactivity, leading to improve bladder function in KIC. Moreover, wortmannin treatment reduced basophil-mediated inflammatory response, improved bladder angiogenesis by increasing capillary density and VEGF expression, to reverse antiangiogenic effect to repair KIC. In conclusion, these findings suggested that autophagy could modulate inflammatory responses and angiogenesis, which improved bladder function in KIC.

MP28-17 SPATIAL AND FUNCTIONAL INTERACTIONS OF THE TRANSIENT RECEPTOR POTENTIAL MELASTATIN-4 CHANNELS AND INOSITOL TRISPHOSPHATE RECEPTORS: NOVEL REGULATORY MECHANISM IN HUMAN DETRUSOR SMOOTH MUSCLE FUNCTION

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-17 SPATIAL AND FUNCTIONAL INTERACTIONS OF THE TRANSIENT RECEPTOR POTENTIAL MELASTATIN-4 CHANNELS AND INOSITOL TRISPHOSPHATE RECEPTORS: NOVEL REGULATORY MECHANISM IN HUMAN DETRUSOR SMOOTH MUSCLE FUNCTION Aaron Provence, Kiril Hristov, Eric Rovner, and Georgi V. Petkov Aaron ProvenceAaron Provence More articles by this author , Kiril HristovKiril Hristov More articles by this author , Eric RovnerEric Rovner More articles by this author , and Georgi V. PetkovGeorgi V. Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1067AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Transient receptor potential melastatin-4 (TRPM4) channels are emerging as key regulators of human detrusor smooth muscle (DSM) function. However, the cellular mechanisms regulating TRPM4 channel activity in human DSM is virtually unexplored. As a potential cellular mechanism regulating TRPM4 channel activity in human DSM, we have directed our attention to the inositol trisphosphate (IP3) receptors (IP3Rs), which facilitate the release of Ca2+ from the sarcoplasmic reticulum (SR). Since the TRPM4 channels are activated by Ca2+, IP3R-mediated Ca2+ release of the SR represents a potential Ca2+ source for TRPM4 channel activation. Using clinically-characterized human tissues, we investigated the molecular and functional interactions of the TRPM4 channels and IP3Rs in human DSM cells. METHODS In accordance with the approved IRB Pro00045232 of the Medical University of South Carolina, we examined human DSM tissues from 6 donor-patients. Using in situ proximity ligation assay (PLA) and amphotericin-B perforated patch-clamp electrophysiology, we sought to test the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release of the SR in human DSM. RESULTS As determined by in situ PLA, we demonstrated close co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a TRPM4-IP3R regulatory mechanism in human DSM function. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, the selective IP3R inhibitor xestospongin C significantly decreased TICCs. These findings suggest that the SR IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. CONCLUSIONS In conclusion, this study provides novel insight into the cellular mechanisms regulating TRPM4 channel activity in human DSM function. The study reveals that the TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM, information that is critical for further evaluating the potential role of the TRPM4 channels as novel therapeutic targets for overactive bladder. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e378 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Aaron Provence More articles by this author Kiril Hristov More articles by this author Eric Rovner More articles by this author Georgi V. Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Role of Rac1 on Carbachol-induced Contractile Activity in Detrusor Smooth Muscle from Streptozotocin-induced Diabetic Rats.

This study was designed to determine the role of the small GTPase Rac1 on carbachol-induced contractile activity in detrusor smooth muscle using small inhibitor NSC 23766 in diabetic rats. Rac1 expression in bladder tissue was also evaluated. In the streptozotocin (STZ)-induced diabetic rat model, three study groups were composed of control, diabetic and insulin-treated diabetic subjects. The detrusor muscle strips were suspended in organ baths at the end of 8-12 weeks after STZ injection. Carbachol (CCh) (10(-9) -10(-4) M) concentration-response curves were obtained both in the absence and in the presence of Rac1 inhibitor NSC 23766 (0.1, 1 and 10 μM). Diabetes-related histopathological changes and Rac1 expressions were assessed by haematoxylin and eosin staining and immunohistochemical staining, respectively. CCh caused dose-dependent contractile responses in all the study groups. Rac1 inhibitor NSC 23766 inhibited CCh-induced contractile responses in all groups, but this inhibition seen in both diabetes groups was greater than in the control group. Histological examination revealed an increased bladder wall thickness both in the diabetes and in the insulin-treated diabetes groups compared to the control group. In immunohistochemical staining, expression of Rac1 was observed to be increased in all layers of bladder in both diabetic groups compared to the control group. In the diabetic bladders, increased expression of Rac1 and considerable inhibition of CCh-induced responses in the presence of NSC 23766 compared to those of the control group may indicate a specific role of Rac1 in diabetes-related bladder dysfunction, especially associated with cholinergic mediated detrusor overactivity.

Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

792 EFFECT OF BOTULINUM NEUROTOXIN-A ON T-TYPE VOLTAGE-GATED CA2+ CURRENT IN DETRUSOR SMOOTH MUSCLE

INTRODUCTION AND OBJECTIVES: Botulinum neurotoxin-A (BoNT/A) is one of novel therapeutic agents for urinary dysfunction including OAB. The inhibitory mechanism of BoNT/A in the bladder is considered mainly as that it cleaves synaptosome-associated protein of 25,000 daltons (SNAP-25), resulting in failure of acetylcholine exocytosis from efferent nerve terminals. On the other hand, we reported that BoNT/A had inhibitory effects on muscle contraction and L-type voltage-gated Ca channel (VGCC) activity in detrusor smooth muscle (AUA 2009). In this study, we further investigated the effects of BoNT/A on T-type VGCC in rat detrusor smooth muscle. METHODS: Bladder smooth muscle strips were obtained from female rats. Single myocytes were obtained by enzymatic digestion, and VGCC was investigated using barium as a charge carrier in whole-cell patch-clamp methods. We administrated BoNT/A light chain (BoNT/A-LC) or a peptide that designed as C-terminus 9 amino-acids of SNAP-25 (SNAP-25C) directory into the cytosol through a patch pipette. RESULTS: In control condition with normal pipette solution, T-type inward currents were evoked by depolarization to 40 mV from the holding potential of 90 mV, and the currents were not inhibited by nifedipine 5 M, a L-type VGCC blocker but abolished by NiCl2 500 M. We monitored the time course of peak amplitude of T-type inward currents. After 16 min intracellular perfusion of control pipette solution, the peak amplitude of T-type current did not change (relative value to control, 1.1 0.12, n 6). However, after 16 min perfusion of BoNT/ A-LC 100 nM, the peak amplitude of T-type current was significantly increased (relative value to control, 1.6 0.082, n 6). Similar result was demonstrated by intracellular perfusion of SNAP-25C 1 M (relative value to control, 1.4 0.16, n 6). Time to peak of T-type current was prolonged after intracellular perfusion of BoNT/A-LC or SNAP25C. In the analysis of steady-state inactivation of T-type VGCC in the presence of nifedipine 5 M, the Vh value in BoNT/A-LC 100 nM or SNAP-25C 1 M intracellular administrated group was significantly deeper than that in control group ( 52 2.5 mV (n 6) in control group, 62 0.66 mV (n 6) in BoNT/A-LC group and 66 3.6 mV (n 5) in SNAP-25C 1 M group). CONCLUSIONS: These results suggest that BoNT/A changes the ion channel kinetics of T-type VGCC in detrusor smooth muscle, and this effect seems to be mediated by cleaved SNAP-25 Cterminus. The negative shift of Vh values of T-type VGCC after BoNT/A treatment might contribute to a BoNT/A-induced reduction in detrusor muscle contractility in OAB patients.

Role of K+ Channels in Regulating Spontaneous Activity in Detrusor Smooth Muscle In Situ in the Mouse Bladder

We investigated the functional role of K(+) channels for regulating spontaneous activity in mouse bladder detrusor smooth muscle. The effects of different K(+) channels blockers on spontaneous changes in membrane potential and intracellular Ca(2+) dynamics were examined using intracellular recording techniques and Ca(2+) imaging with fluo-4 fluorescence, respectively. Detrusor smooth muscle generated spontaneous action potentials and Ca(2+) transients. Iberiotoxin (0.1 microM), charybdotoxin (0.1 microM) or tetraethylammonium (1 mM) increased the amplitude of action potentials and prolonged their repolarizing phase without inhibiting their after-hyperpolarization. Tetraethylammonium (10 mM) but not stromatoxin (0.1 microM) suppressed after-hyperpolarization and further increased the amplitude and half duration of action potentials. Apamin (0.1 microM) increased the frequency of action potentials but had no effect on their configuration. Spontaneous Ca(2+) transients were generated in individual detrusor smooth muscle cells and occasionally propagated to neighboring cells to form intercellular Ca(2+) waves. Transmural nerve stimulations invariably initiated synchronous Ca(2+) transients within and across muscle bundles. Charybdotoxin (0.1 microM) increased the amplitude of spontaneous Ca(2+) transients, while the subsequent application of tetraethylammonium (10 mM) increased their half duration. In addition, tetraethylammonium increased the synchronicity of Ca(2+) transients in muscle bundles. These results suggest that large and intermediate conductance Ca(2+) activated K(+) channels contribute to action potential repolarization and restrict the excitability of detrusor smooth muscle in the mouse bladder. In addition, the activation of voltage dependent K(+) channels is involved in repolarization and after-hyperpolarization, and it has a fundamental role in stabilizing detrusor smooth muscle excitability.

Altered distribution of interstitial cells in the guinea pig bladder following bladder outlet obstruction

We investigated the effects of bladder outlet obstruction (BOO) on the distribution of interstitial cells (ICs) in the guinea-pig bladder. Bladder overactivity of BOO animals was validated with urodynamic studies. Immunohistochemical analyses for Kit and vimentin as markers for ICs were performed on both BOO and control bladders. Morphological and functional properties of detrusor smooth muscle (DSM) were examined with alpha-smooth muscle actin staining and intracellular recording, respectively. Electron microscopy was also carried out to characterize ultrastructural morphology of ICs. Two weeks after surgery, BOO animals showed an increased voiding frequency and a reduced voiding volume. Filling cystometry demonstrated a frequent incidence of non-voiding contractions, a reduced interval between voiding contractions and an increased voiding pressure in BOO bladders. In BOO bladders, the thickness of suburothelial and subserosal connective tissue layers was increased, whilst that of detrusor smooth muscle (DSM) layer was less affected. Population of Kit or vimentin immunoreactive ICs was increased in subserosal layers, and their distribution was altered in suburotherial layer in BOO bladders. Neither alpha-actin immunoreactivity nor spontaneous electrical activity of DSM was altered in BOO bladders. ICs were characterized by their numerous mitochondria and caveolae, and had a close contact with each other and with neighboring DSM or nerves. These results demonstrated the increased population of ICs in the BOO guinea-pig model for the first time, and suggest that the altered distribution of ICs may contribute to the pathophysiology of bladder overactivity.

985: Rho-Kinase Inhibitor Decreases Spontaneous Activity in Detrusor Smooth Muscle with Partial Bladder Outlet Obstruction

985: Rho-Kinase Inhibitor Decreases Spontaneous Activity in Detrusor Smooth Muscle with Partial Bladder Outlet Obstruction

Correlation between spontaneous electrical, calcium and mechanical activity in detrusor smooth muscle of the guinea-pig bladder.

1. To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea-pig urinary bladder, isometric tension was measured in muscle bundles while recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca(2+)](i); calcium transients) were recorded in strips loaded with the fluorescent dye, fura-PE3. 2. In 40% of preparations, individual action potentials and contractions, which were abolished by nifedipine (1 microm), were generated. In the remaining preparations, bursting action potentials and contractions were generated. Contractions were again abolished by nifedipine (1 microm), while higher concentrations of nifedipine (10-30 microm) were required to prevent the electrical activity. 3. Carbachol (0.1 microm) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 microm) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nm) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 microm) reduced the frequency of action potentials, action potential bursts and the size of the contractions. 4. Forskolin (0.1 microm), 8-bromoguanosin 3', 5' cyclic monophosphate (8Br-cGMP, 0.1 mm) and Y-26763 (10 microm) suppressed contractions without reducing the amplitude of either action potentials or Ca transients. 5. This paper confirms that action potentials and associated calcium transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea-pig bladder. However, in parallel with the excitation-contraction coupling, the sensitivity of the contractile proteins for Ca(2+) may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase.

Role of Mitochondria in the Generation of Spontaneous Activity in Detrusor Smooth Muscles of the Guinea Pig Bladder.

The rhythmic electrical activity of gastrointestinal smooth muscles is associated with mitochondrial Ca2+ handling. We examined the role of mitochondria in the generation of spontaneous activity in detrusor smooth muscles. Changes in the membrane potential and intracellular Ca2+ concentration ([Ca2+]i) were measured in detrusor smooth muscles of the guinea pig using conventional microelectrode techniques and Fura-PE3 (Calbiochem, San Diego, California) fluorescence, respectively. Detrusor smooth muscle cells showed spontaneous action potentials and associated transient increases in [Ca2+]i (Ca transients). The mitochondrial protonophore CCCP (carbonyl cyanide m-chlorophenyl hydrazone) (10 microM) depolarized the membrane, increased [Ca2+]i and caused activation followed by suppression of action potentials and Ca transients. High K solution potassium concentration ([K+]o = 30 mM) depolarized the membrane and increased [Ca2+]i to levels similar to those produced by 10 microM CCCP but this depolarization did not suppress action potentials. Nifedipine (10 microM) decreased the amplitude of CCCP induced increases in [Ca2+]i by about 50%. CCCP induced increases in [Ca2+]i were further reduced by about 70% in Ca2+-free solution and by about 30% in the presence of 10 microM SKF96365, a blocker for store operated Ca entry. In the presence of 10 microM nifedipine and 10 microM cyclopiazonic acid, CCCP induced [Ca2+]i responses were suppressed to about 25% of control values. Under these conditions repetitive applications of 10 microM acetylcholine chloride successively decreased [Ca2+]i responses and finally failed to increase [Ca2+]i. Subsequent CCCP failed to elevate [Ca2+]i. These results suggest that mitochondria have an important role in Ca2+ buffering in bladder smooth muscles. Mitochondrial Ca2+ is presumably supplied by Ca2+ transport from internal stores and also by capacitative calcium entry through nonselective cation channels. Mitochondrial Ca2+ handling may also be critical for the generation of spontaneous activity in detrusor smooth muscle.

A NEW APPROACH TO RECORDING THE ELECTROMYOGRAPHIC ACTIVITY OF DETRUSOR SMOOTH MUSCLE

Extracellular recordings from the whole intact mammalian bladder of the electrical events leading to contraction of the organ have been elusive for almost 50 years despite the widespread potential applications of such a technique. The principal problem is the need to isolate the small real signals reflecting membrane depolarization from the large electromechanical artifact generated as the organ contracts. In this preliminary study we determined whether electrical signals may be isolated and verified as biological using extracellular bipolar reversible suction electrodes. Six whole excised guinea pig bladders were mounted in an especially constructed organ bath. Electrical activity resulting from nerve stimulation of the organ was recorded using a novel 10 bipolar Pt/PtCl suction electrode simultaneously with changes in intravesical pressure. Mechanical and pharmacological control experiments were performed to determine the true origin of these signals. A predominantly biphasic electrical signal of a mean amplitude plus or minus standard deviation of 647 plus or minus 301 microV. and a mean duration of 293 plus or minus 51 milliseconds was consistently recorded from the serosal surface of all guinea pig bladders. In all cases the electrical signal and mechanical response to stimulation were completely abolished by 1 microM. tetrodotoxin. The signal always preceded any change in intravesical pressure. It was sensitive to changes in the CaCl2 concentration of the superfusate, abolished by purinergic but not cholinergic neuromuscular blockade and independent of electromechanical artifact. In this preliminary report we describe a novel technique by which nerve mediated detrusor electrical activity leading to contraction of the whole intact guinea pig bladder may be isolated from artifact and verified as real. We hope that development of this technique may enable its application to the in situ human bladder. However, to our knowledge whether electromyographic activity may be recorded from human detrusor remains to be determined.

Energy turnover and lactate dehydrogenase activity in detrusor smooth muscle from rats with streptozotocin-induced diabetes.

Force generation and tissue glucose metabolism were measured in the urinary bladder smooth muscle from rats with streptozotocin-induced diabetes (7-8 wk duration). Bladder wet wt was almost 4-fold higher in the diabetic animals compared with the untreated controls. Morphological analysis showed that the growth was associated with hypertrophy of the smooth muscle component in the bladder wall. Force generation of isolated bladder strip preparations was measured in vitro at different ambient oxygen tensions. Activation of intramural nerves, with electrical field stimulation, induced contractions that were unaffected by reduction of oxygen tension down to PO2 100 mmHg for both control and diabetic muscle strips. At zero PO2 force was reduced by approximately 10-20%, in both groups. High-K+ solution induced 'tonic' contractions that were slightly more inhibited by lowering PO2. At intermediate PO2 (between 100 and 20 mmHg) the diabetic muscle gave slightly higher force. At zero PO2 no significant difference could be detected between strips from control and diabetic animals. Oxygen consumption and lactate production in the preparations were determined at a PO2 of 290 mmHg and related to the volume of smooth muscle. At zero PO2, lactate formation increased 3- to 4-fold. The metabolic tension cost was lower at zero PO2. No differences in basal and contraction related metabolic rates could be detected between the two groups under normoxic and anoxic conditions. The maximal activity of lactate dehydrogenase (LDH) determined in tissue samples was about 2-fold higher in the diabetic bladder muscle.(ABSTRACT TRUNCATED AT 250 WORDS)