Detrusor Smooth Muscle Tissue Research Articles

Anti-inflammation properties of resveratrol in the detrusor smooth muscle of the diabetic rat.

In this paper, we aimed to prove that resveratrol can inhibit inflammation in the detrusor smooth muscle of diabetic rats, which may provide a new direction for diabetic cystopathy (DCP) treatment. We induced a Sprague-Dawley (SD) rat model of type 1 diabetes by intraperitoneal injections of streptozotocin (STZ). Then, we separated the SD rats into four groups: (1) an excipient-treated control group; (2) a resveratrol-treated control group; (3) an excipient-treated streptozotocin (STZ)-injected group; and (4) a resveratrol-treated STZ-injected group. We administered the resveratrol or excipient by intragastric administration. After 12weeks of diabetes induction, we measured the blood-sugar concentrations and bladder weights, and we took the bladder tissues of each group of rats for hematoxylin-eosin staining to observe the histological changes. We used real-time quantitative polymerase chain reaction (qPCR) and Western blotting to analyze the expression levels of tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), interleukin (IL)-6, and IL-1β. The bodyweights of the diabetic rats were appreciably reduced, while the bladder weights and blood-glucose concentrations were substantially increased. Oral resveratrol could not improve the changes in the bodyweights and blood-glucose concentrations, but it had a certain effect on the bladder weights. In a macroscopic evaluation, the bladder walls of the STZ-induced diabetes rats were thickened, and, from the H&E staining, we could see that the bladder tissues of the diabetic rats had inflammatory cell infiltration, edema, and the capillary congestion of the mucosa and lamina propria. After resveratrol treatment, the bladder-wall thickening was reduced, and the tissue damage and inflammation were significantly ameliorated. We could associate all these changes with markedly heightened expressions of TNF-α, IL-1β, IL-6, and NF-κB in the detrusor smooth muscle (DSM) tissues of the diabetic rats. Oral treatment with resveratrol alleviated the expressivity of the inflammatory cytokines in the DSM tissues. Resveratrol treatment ameliorated the histological changes in the bladder and inhibited the expressions of DSM-tissue inflammatory factors in diabetes rats. Resveratrol may provide a new direction of research for the treatment of diabetic cystopathy.

The phosphodiesterase type 4 inhibitor roflumilast suppresses inflammation to improve diabetic bladder dysfunction rats.

To demonstrate that phosphodiesterase type 4 (PDE4) inhibitors could potentially treat diabetic bladder dysfunction (DBD) through modulation of the systemic inflammatory response. In this 6-week study, 60 female Sprague-Dawley rats were divided into three groups: (i) vehicle-treated control rats; (ii) vehicle-treated streptozocin (STZ)-injected rats; and (iii) roflumilast-treated STZ-injected rats. Oral roflumilast (5mg/kg/day) was administered during the last 4 weeks of STZ injection to induce diabetes in the test group. At 6 weeks, a urodynamic study was performed in each group. The expression of nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-1β in detrusor smooth muscle (DSM) was analyzed using quantitative reverse transcription-polymerase chain reaction and Western blotting. A significant decrease in bodyweight and significant increases in bladder weight and blood glucose level were observed in the diabetic rats and were not ameliorated by roflumilast treatment. Cystometry showed the increased bladder capacity, voiding volume, residual urine volume, and voiding interval in the diabetic rats and the prevention of these changes by roflumilast. These changes were accompanied by significantly enhanced expression of NF-κB, TNF-α, IL-6, and IL-1β in DSM tissue from diabetic rats. Furthermore, roflumilast attenuated the expression of inflammatory factors in DSM tissue. Oral treatment with roflumilast in diabetic rats improves bladder function and inhibits the expression of inflammatory factors in DSM tissue, indicating that PDE4 is a potential therapeutic target for DBD.

Effect of high-fat diet-induced obesity on the small-conductance Ca2+-activated K+ channel function affecting the contractility of rat detrusor smooth muscle.

Obesity usually induces overactive bladder (OAB) associated with detrusor overactivity, which is related to increased contractility of the detrusor smooth muscle (DSM). Small-conductance Ca2+-activated K+ (SK) channels play a constitutive role in the regulation of DSM contractility. However, the role of SK channels in the DSM changes in obesity-related OAB is still unknown. Here, we tested the hypothesis that obesity-related OAB is associated with reduced expression and activity of SK channels in DSM and that SK channels activation is a potential treatment for OAB. Female Sprague-Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD) and weighed after 12 weeks. Urodynamic studies, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and isometric tension recording were performed. Increased average body weights and urodynamically demonstrated OAB were observed in HFD rats. qRT-PCR experiments revealed a decrease in the mRNA expression level of SK channel in DSM tissue of the HFD rats. Isometric tension recordings indicated an attenuated relaxation effect of NS309 on the spontaneous phasic and electrical field stimulation-induced contractions that occurred via SK channel activation in HFD DSM strips. Reduced expression and activity of SK channels in the DSM contribute to obesity-related OAB, indicating that SK channels are a potential therapeutic target for OAB.

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

ABSTRACTWe recently reported key physiologic roles for Ca2+-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca2+-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca2+, inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum represents a potential Ca2+ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release in human DSM. With in situ PLA, we demonstrated 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 potential Ca2+-mediated TRPM4-IP3R regulatory mechanism. 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, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM.

Constitutively active PKA regulates neuronal acetylcholine release and contractility of guinea pig urinary bladder smooth muscle.

Autonomic and somatic motor neurons that innervate the urinary bladder and urethra control the highly coordinated functions of the lower urinary tract, the storage, and the emptying of urine. ACh is the primary excitatory neurotransmitter in the bladder. Here, we aimed to determine whether PKA regulates neuronal ACh release and related nerve-evoked detrusor smooth muscle (DSM) contractions in the guinea pig urinary bladder. Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation (EFS)- and carbachol-induced DSM contractions with a combination of pharmacological tools. The colorimetric method was used to measure ACh released by the parasympathetic nerves in DSM isolated strips. The pharmacological inhibition of PKA with H-89 (10 μM) increased the spontaneous phasic contractions, whereas it attenuated the EFS-induced DSM contractions. Intriguingly, H-89 (10 μM) attenuated the (primary) cholinergic component, whereas it simultaneously increased the (secondary) purinergic component of the nerve-evoked contractions in DSM isolated strips. The acetylcholinesterase inhibitor, eserine (10 μM), increased EFS-induced DSM contractions, and the subsequent addition of H-89 attenuated the contractions. H-89 (10 μM) significantly increased DSM phasic contractions induced by the cholinergic agonist carbachol. The inhibition of PKA decreased the neuronal release of ACh in DSM tissues. This study revealed that PKA-mediated signaling pathways differentially regulate nerve-evoked and spontaneous phasic contractions of guinea pig DSM. Constitutively active PKA in the bladder nerves controls synaptic ACh release, thus regulating the nerve-evoked DSM contractions, whereas PKA in DSM cells controls the spontaneous phasic contractility.

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 Novel KV7.2/KV7.3 Channel Opener ICA-069673 Reveals Subtype-Specific Functional Roles in Guinea Pig Detrusor Smooth Muscle Excitability and Contractility.

The physiologic roles of voltage-gated KV7 channel subtypes (KV7.1-KV7.5) in detrusor smooth muscle (DSM) are poorly understood. Here, we sought to elucidate the functional roles of KV7.2/KV7.3 channels in guinea pig DSM excitability and contractility using the novel KV7.2/KV7.3 channel activator ICA-069673 [N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide]. We employed a multilevel experimental approach using Western blot analysis, immunocytochemistry, isometric DSM tension recordings, fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp electrophysiology. Western blot experiments revealed the protein expression of KV7.2 and KV7.3 channel subunits in DSM tissue. In isolated DSM cells, immunocytochemistry with confocal microscopy further confirmed protein expression for KV7.2 and KV7.3 channel subunits, where they localize within the vicinity of the cell membrane. ICA-069673 inhibited spontaneous phasic, pharmacologically induced, and nerve-evoked contractions in DSM isolated strips in a concentration-dependent manner. The inhibitory effects of ICA-069673 on DSM spontaneous phasic and tonic contractions were abolished in the presence of the KV7 channel inhibitor XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride]. Under conditions of elevated extracellular K(+) (60 mM), the effects of ICA-069673 on DSM tonic contractions were significantly attenuated. ICA-069673 decreased the global intracellular Ca(2+) concentration in DSM cells, an effect blocked by the L-type Ca(2+) channel inhibitor nifedipine. ICA-069673 hyperpolarized the membrane potential and inhibited spontaneous action potentials of isolated DSM cells, effects that were blocked in the presence of XE991. In conclusion, using the novel KV7.2/KV7.3 channel activator ICA-069673, this study provides strong evidence for a critical role for the KV7.2- and KV7.3-containing channels in DSM function at both cellular and tissue levels.

MP17-05 POTENTIAL NOVEL TARGET FOR TREATMENT OF OVERACTIVE BLADDER: TRANSIENT RECEPTOR POTENTIAL MELASTATIN 4 CHANNEL IN HUMAN URINARY BLADDER

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology1 Apr 2014MP17-05 POTENTIAL NOVEL TARGET FOR TREATMENT OF OVERACTIVE BLADDER: TRANSIENT RECEPTOR POTENTIAL MELASTATIN 4 CHANNEL IN HUMAN URINARY BLADDER Kiril Hristov, Shankar Parajuli, Amy Smith, John Malysz, Eric Rovner, and Georgi Petkov Kiril HristovKiril Hristov More articles by this author , Shankar ParajuliShankar Parajuli More articles by this author , Amy SmithAmy Smith More articles by this author , John MalyszJohn Malysz More articles by this author , Eric RovnerEric Rovner More articles by this author , and Georgi PetkovGeorgi Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.526AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Transient receptor potential melastatin 4 (TRPM 4) channels have been recently identified as potential regulators of detrusor smooth muscle (DSM) excitability and contractility in rodents; however, their expression and function in human DSM remains largely unexplored. Here, we provide a novel insight into a functional role of TRPM4 channels in human DSM. METHODS We used a multidisciplinary experimental approach including: RT-PCR, Western blot, immunohistochemistry and immunocytochemistry, amphotericin-perforated patch-clamp electrophysiology on native freshly-isolated human DSM cells, as well as functional studies on DSM contractility of DSM isolated strips from donor patients, and the novel TRPM4 channel inhibitor 9-phenathrol. RESULTS RT-PCR experiments detected mRNA message for TRPM4 channels in whole DSM tissue and single DSM cells. Western blot analysis and immunohistochemical staining revealed TRPM4 protein expression in whole DSM tissue. Immunocytochemical experiments further detected TRPM4 protein expression in isolated single DSM cells. Voltage-clamp experiments on freshly-isolated native human DSM cells showed that inhibition of TRPM4 channels with 9-phenathrol significantly decreased the transient inward cation currents (TICCs) activity, when recorded at -70 mV. Pretreatment of DSM cells with xestospongin C, an inositol-3-phosphate receptor inhibitor, abolished the TICCs. Current-clamp experiments demonstrated that inhibition of TRPM4 channels with 9-phenathrol hyperpolarized DSM cell resting membrane potential by ∼11 mV. In vitro functional studies on DSM contractility revealed that TRPM4 channel inhibitor 9-phenantrol significantly attenuated the spontaneous phasic, carbachol-induced, and nerve-evoked contractions in human DSM isolated strips in a concentration-dependent manner. CONCLUSIONS Our results demonstrate the novel finding that TRPM4 channels are expressed in human DSM and support the concept that they regulate human DSM excitability and contractility. Therefore, TRPM4 channels may be considered as novel targets for treatment of overactive bladder. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e136-e137 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kiril Hristov More articles by this author Shankar Parajuli More articles by this author Amy Smith More articles by this author John Malysz More articles by this author Eric Rovner More articles by this author Georgi Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Molecular Expression and Pharmacological Evidence for a Functional Role of Kv7 Channel Subtypes in Guinea Pig Urinary Bladder Smooth Muscle

Voltage-gated Kv7 (KCNQ) channels are emerging as essential regulators of smooth muscle excitability and contractility. However, their physiological role in detrusor smooth muscle (DSM) remains to be elucidated. Here, we explored the molecular expression and function of Kv7 channel subtypes in guinea pig DSM by RT-PCR, qRT-PCR, immunohistochemistry, electrophysiology, and isometric tension recordings. In whole DSM tissue, mRNAs for all Kv7 channel subtypes were detected in a rank order: Kv7.1~Kv7.2Kv7.3~Kv7.5Kv7.4. In contrast, freshly-isolated DSM cells showed mRNA expression of: Kv7.1~Kv7.2Kv7.5Kv7.3~Kv7.4. Immunohistochemical confocal microscopy analyses of DSM, conducted by using co-labeling of Kv7 channel subtype-specific antibodies and α-smooth muscle actin, detected protein expression for all Kv7 channel subtypes, except for the Kv7.4, in DSM cells. L-364373 (R-L3), a Kv7.1 channel activator, and retigabine, a Kv7.2-7.5 channel activator, inhibited spontaneous phasic contractions and the 10-Hz electrical field stimulation (EFS)-induced contractions of DSM isolated strips. Linopiridine and XE991, two pan-Kv7 (effective at Kv7.1-Kv7.5 subtypes) channel inhibitors, had opposite effects increasing DSM spontaneous phasic and 10 Hz EFS-induced contractions. EFS-induced DSM contractions generated by a wide range of stimulation frequencies were decreased by L-364373 (10 µM) or retigabine (10 µM), and increased by XE991 (10 µM). Retigabine (10 µM) induced hyperpolarization and inhibited spontaneous action potentials in freshly-isolated DSM cells. In summary, Kv7 channel subtypes are expressed at mRNA and protein levels in guinea pig DSM cells. Their pharmacological modulation can control DSM contractility and excitability; therefore, Kv7 channel subtypes provide potential novel therapeutic targets for urinary bladder dysfunction.

Neurogenic Detrusor Overactivity Is Associated with Decreased Expression and Function of the Large Conductance Voltage- and Ca2+-Activated K+ Channels

Patients suffering from a variety of neurological diseases such as spinal cord injury, Parkinson’s disease, and multiple sclerosis often develop neurogenic detrusor overactivity (NDO), which currently lacks a universally effective therapy. Here, we tested the hypothesis that NDO is associated with changes in detrusor smooth muscle (DSM) large conductance Ca2+-activated K+ (BK) channel expression and function. DSM tissue samples from 33 patients were obtained during open bladder surgeries. NDO patients were clinically characterized preoperatively with pressure-flow urodynamics demonstrating detrusor overactivity, in the setting of a clinically relevant neurological condition. Control patients did not have overactive bladder and did not have a clinically relevant neurological disease. We conducted quantitative polymerase chain reactions (qPCR), perforated patch-clamp electrophysiology on freshly-isolated DSM cells, and functional studies on DSM contractility. qPCR experiments revealed that DSM samples from NDO patients showed decreased BK channel mRNA expression in comparison to controls. Patch-clamp experiments demonstrated reduced whole cell and transient BK currents (TBKCs) in freshly-isolated DSM cells from NDO patients. Functional studies on DSM contractility showed that spontaneous phasic contractions had a decreased sensitivity to iberiotoxin, a selective BK channel inhibitor, in DSM strips isolated from NDO patients. These results reveal the novel finding that NDO is associated with decreased DSM BK channel expression and function leading to increased DSM excitability and contractility. BK channel openers or BK channel gene transfer could be an alternative strategy to control NDO. Future clinical trials are needed to evaluate the value of BK channel opening drugs or gene therapies for NDO treatment and to identify any possible adverse effects.

Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology

Members of the transient receptor potential (TRP) channel superfamily, including the Ca(2+)-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca(2+) imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca(2+) imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca(2+) levels. 9-Phenanthrol (0.1-30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1-7 μM and 70-80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5-50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling.

TRPM4 channel: a new player in urinary bladder smooth muscle function in rats

The TRPM4 channel is a Ca(2+)-activated, monovalent cation-selective channel of the melastatin transient receptor potential (TRPM) family. The TRPM4 channel is implicated in the regulation of many cellular processes including the immune response, insulin secretion, and pressure-induced vasoconstriction of cerebral arteries. However, the expression and function of the TRPM4 channels in detrusor smooth muscle (DSM) have not yet been explored. Here, we provide the first molecular, electrophysiological, and functional evidence for the presence of TRPM4 channels in rat DSM. We detected the expression of TRPM4 channels at mRNA and protein levels in freshly isolated DSM single cells and DSM tissue using RT-PCR, Western blotting, immunohistochemistry, and immunocytochemistry. 9-Hydroxyphenanthrene (9-phenanthrol), a novel selective inhibitor of TRPM4 channels, was used to examine their role in DSM function. In perforated patch-clamp recordings using freshly isolated rat DSM cells, 9-phenanthrol (30 μM) decreased the spontaneous inward current activity at -70 mV. Real-time DSM live-cell Ca(2+) imaging showed that selective inhibition of TRPM4 channels with 9-phenanthrol (30 μM) significantly reduced the intracellular Ca(2+) levels. Isometric DSM tension recordings revealed that 9-phenanthrol (0.1-30 μM) significantly inhibited the amplitude, muscle force integral, and frequency of the spontaneous phasic and pharmacologically induced contractions of rat DSM isolated strips. 9-Phenanthrol also decreased the amplitude and muscle force integral of electrical field stimulation-induced contractions. In conclusion, this is the first study to examine the expression and provide evidence for TRPM4 channels as critical regulators of rat DSM excitability and contractility.

SK but not IK channels regulate human detrusor smooth muscle spontaneous and nerve-evoked contractions.

Animal studies suggest that the small (SK) and intermediate (IK) conductance Ca(2+)-activated K(+) channels may contribute to detrusor smooth muscle (DSM) excitability and contractility. However, the ability of SK and IK channels to control DSM spontaneous phasic and nerve-evoked contractions in human DSM remains unclear. We first investigated SK and IK channels molecular expression in native human DSM and further assessed their functional role using isometric DSM tension recordings and SK/IK channel-selective inhibitors. Quantitative PCR experiments revealed that SK3 channel mRNA expression in isolated DSM single cells was ∼12- to 44-fold higher than SK1, SK2, and IK channels. RT-PCR studies at the single-cell level detected mRNA messages for SK3 channels but not SK1, SK2, and IK channels. Western blot and immunohistochemistry analysis further confirmed protein expression for the SK3 channel and lack of detectable protein expression for IK channel in whole DSM tissue. Apamin (1 μM), a selective SK channel inhibitor, significantly increased the spontaneous phasic contraction amplitude, muscle force integral, phasic contraction duration, and muscle tone of human DSM isolated strips. Apamin (1 μM) also increased the amplitude of human DSM electrical field stimulation (EFS)-induced contractions. However, TRAM-34 (1 μM), a selective IK channel inhibitor, had no effect on the spontaneous phasic and EFS-induced DSM contractions suggesting a lack of IK channel functional role in human DSM. In summary, our molecular and functional studies revealed that the SK, particularly the SK3 subtype, but not IK channels are expressed and regulate the spontaneous and nerve-evoked contractions in human DSM.

Expression and function of KV2-containing channels in human urinary bladder smooth muscle

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β(1)-, and β(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, β(1), and β(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.

DIABETES INDUCED DECREASE IN DETRUSOR SMOOTH MUSCLE FORCE IS ASSOCIATED WITH OXIDATIVE STRESS AND OVERACTIVITY OF ALDOSE REDUCTASE

Bladder dysfunction is one of the complications of diabetes. We determined whether diabetic induced bladder dysfunction is associated with decreased detrusor smooth muscle contractility, hyperglycemia induced over expression of aldose reductase (AR) and increased sorbitol production. In addition, we compared oxidative stress in the detrusor smooth muscle in diabetic rabbits with that in normal rabbits by estimating lipid peroxidation. Diabetes was induced in New Zealand White, age matched male rabbits by intravenous injection of alloxan (100 mg/kg body weight). Normal and sucrose drinking rabbits served as controls. Six months after the induction of diabetes rabbits with a blood glucose level of 400 mg/dl or higher were sacrificed and detrusor smooth muscle tissue was isolated. Detrusor was analyzed for force generation, lipid peroxidation products using malondialdehyde as a biomarker, and AR expression and function by reverse transcriptase-polymerase chain reaction and sorbitol levels, respectively. The mean maximum force +/- SE produced by detrusor muscle strips in response to 125 mM KCl was 17.50 +/- 1.66, 17.56 +/- 1.23 and 7.51 +/- 2.56 gm/100 mg tissue in normal, sucrose drinking and diabetic rabbits, respectively, representing a 57% force decrease in diabetic subjects. Bethanechol elicited force decreased 40% (26.52 +/- 3.21, 27.3 +/- 2.87 and 16.32 +/- 1.67 gm/100 mg tissue, respectively, in normal, sucrose drinking and diabetic rabbits) in diabetic vs control subjects. Concomitant with the force decrease, the expression of AR, sorbitol content and lipid peroxidation products were increased. Diabetes induced a decrease in detrusor smooth muscle force. This was associated with an increase in lipid peroxides and sorbitol concomitant with over expression of AR and polyol pathway activation. Our data suggest that these changes might contribute to oxidative stress and decreased contractility of detrusor smooth muscle, leading to bladder dysfunction.