Contraction Of Urinary Bladder Smooth Muscle Research Articles

Intracellular signaling pathways of muscarinic acetylcholine receptor-mediated detrusor muscle contractions.

Acetylcholine plays an essential role in the regulation of detrusor muscle contractions, and antimuscarinics are widely used in the management of overactive bladder syndrome. However, several adverse effects limit their application and patients' compliance. Thus, this study aimed to further analyze the signal transduction of M2 and M3 receptors in the murine urinary bladder to eventually find more specific therapeutic targets. Experiments were performed on adult male wild-type, M2, M3, M2/M3, or Gαq/11 knockout (KO), and pertussis toxin (PTX)-treated mice. Contraction force and RhoA activity were measured in the urinary bladder smooth muscle (UBSM). Our results indicate that carbamoylcholine (CCh)-induced contractions were associated with increased activity of RhoA and were reduced in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632 in UBSM. CCh-evoked contractile responses and RhoA activation were markedly reduced in detrusor strips lacking either M2 or M3 receptors and abolished in M2/M3 KO mice. Inhibition of Gαi-coupled signaling by PTX treatment shifted the concentration-response curve of CCh to the right and diminished RhoA activation. CCh-induced contractile responses were markedly decreased in Gαq/11 KO mice; however, RhoA activation was unaffected. In conclusion, cholinergic detrusor contraction and RhoA activation are mediated by both M2 and M3 receptors. Furthermore, whereas both Gαi and Gαq/11 proteins mediate UBSM contraction, the activation at the RhoA-ROCK pathway appears to be linked specifically to Gαi. These findings may aid the identification of more specific therapeutic targets for bladder dysfunctions.NEW & NOTEWORTHY Muscarinic acetylcholine receptors are of utmost importance in physiological regulation of micturition and also in the development of voiding disorders. We demonstrate that the RhoA-Rho-associated kinase (ROCK) pathway plays a crucial role in contractions induced by cholinergic stimulation in detrusor muscle. Activation of RhoA is mediated by both M2 and M3 receptors as well as by Gi but not Gq/11 proteins. The Gi-RhoA-ROCK pathway may provide a novel therapeutic target for overactive voiding disorders.

Pharmacological study on the enhancing effects of U46619 on guinea pig urinary bladder smooth muscle contraction induced by acetylcholine and α,β-methylene ATP and the possible involvement of protein kinase C

We examined whether U46619 (a prostanoid TP receptor agonist) could enhance the contractions of guinea pig urinary bladder smooth muscle (UBSM) in response to acetylcholine (ACh) and an ATP analog (α,β-methylene ATP (αβ-MeATP)) through stimulation of the UBSM TP receptor and whether protein kinase C (PKC) is involved. U46619 (10−7 M) markedly enhanced UBSM contractions induced by electrical field stimulation and ACh/αβ-MeATP (3 × 10−6 M each), the potentiation of which was completely suppressed by SQ 29,548 (a TP receptor antagonist, 6 × 10−7 M). PKC inhibitors did not attenuate the ACh-induced contractions enhanced by U46619 although they partly suppressed the U46619-enhanced, αβ-MeATP-induced contractions. While phorbol 12-myristate 13-acetate (PMA, a PKC activator, 10−6 M) did not enhance ACh-induced contractions, it enhanced αβ-MeATP-induced contractions, an effect that was completely suppressed by PKC inhibitors. αβ-MeATP-induced contractions, both with and without U46619 enhancement, were strongly inhibited by diltiazem. U46619/PMA enhanced 50 mM KCl-induced contractions, the potentiation of which was partly/completely attenuated by PKC inhibitors. These findings suggest that U46619 potentiates parasympathetic nerve-associated UBSM contractions by stimulating UBSM TP receptors. PKC-increased Ca2+ influx through voltage-dependent Ca2+ channels may partially play a role in purinergic receptor-mediated UBSM contractions enhanced by TP receptor stimulation.

Pharmacological Studies on the Ca2+ Influx Pathways in Platelet-Activating Factor (PAF)-Induced Mouse Urinary Bladder Smooth Muscle Contraction.

Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10-6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10-5 M), diltiazem (10-5 M), and nifedipine (10-7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10-5 M) was strongly suppressed by SKF-96365 (3 × 10-5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10-5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.

The mast cell stimulator compound 48/80 causes urothelium-dependent increases in murine urinary bladder contractility.

Mast cells and degranulation of preformed inflammatory mediators contribute to lower urinary tract symptoms. This study investigated pathways by which the mast cell stimulator compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that 1) mast cell degranulation causes spontaneous urinary bladder smooth muscle contractions and 2) these contractions are caused by urothelium-derived PGE2. Urothelium-intact and -denuded urinary bladder strips were collected from mast cell-sufficient (C57Bl/6) and mast cell-deficient (B6.Cg-Kitw-sh) mice to determine if compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were used to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell-sufficient and -deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blockade of P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished compound 48/80 responses. However, only combined blockade of PGE2 (EP1), PGF2α (FP), and thromboxane A2 (TP) receptors inhibited compound 48/80-induced responses. Thus, the effects of compound 48/80 are urothelium dependent but independent of mast cells. Furthermore, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory nonneurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using compound 48/80 to determine mast cell-dependent responses in the urinary bladder.NEW & NOTEWORTHY Urothelial cells are first responders to noxious contents of the urine. Our study demonstrates that the urothelium is not only a barrier but also a modulator of urinary bladder smooth muscle phasic activity and contractility independent of immune cell recruitment in response to an inflammatory insult.

Dimethyl Sulfoxide Enhances Acetylcholine-Induced Contractions in Rat Urinary Bladder Smooth Muscle by Inhibiting Acetylcholinesterase Activities.

Dimethyl sulfoxide (DMSO) has been used not only as an experimental solvent, but also as a therapeutic agent for interstitial cystitis. The therapeutic effects of DMSO on interstitial cystitis are presumed to involve anti-inflammatory and analgesic effects. However, the effects of DMSO on urinary bladder smooth muscle (UBSM) have not been fully investigated. Thus, in this study, we investigated the effects of DMSO on rat UBSM contractions, and these effects were compared with those of acetone, which has a structure in which the sulfur of DMSO is replaced with carbon. DMSO (0.5-5%) enhanced acetylcholine (ACh)-induced contractions, whereas acetone (3 and 5%) suppressed them. Additionally, DMSO (5%) suppressed carbachol-induced contractions. DMSO/acetone (0.5-5%) inhibited 80 mM KCl-induced contractions in a concentration-dependent manner; however, the inhibitory effects of DMSO were weaker than those of acetone. The enhancing/suppressing effects of DMSO and acetone were almost completely abolished by wash out. DMSO and acetone (0.5-5%) inhibited recombinant human acetylcholinesterase (rhAChE) activity in a concentration-dependent manner. At 0.5 and 1%, the inhibitory effects of DMSO on rhAChE activity were more potent than those of acetone. These findings suggest that DMSO can enhance ACh-induced UBSM contractions and promote urinary bladder motility by inhibiting acetylcholinesterase (AChE), although DMSO also inhibits Ca2+ influx-mediated UBSM contractions. In addition, the sulfur atom in DMSO might play an important role in its enhancing effect on ACh-induced contractions by inhibiting AChE, as acetone did not enhance these contractions.

Inhibitory Effects of Antipsychotics on the Contractile Response to Acetylcholine in Rat Urinary Bladder Smooth Muscles.

The clinical applications of antipsychotics for symptoms unrelated to schizophrenia, such as behavioral and psychological symptoms, in patients with Alzheimer's disease, and the likelihood of doctors prescribing antipsychotics for elderly people are increasing. In elderly people, drug-induced and aging-associated urinary disorders are likely to occur. The most significant factor causing drug-induced urinary disorders is a decrease in urinary bladder smooth muscle (UBSM) contraction induced by the anticholinergic action of therapeutics. However, the anticholinergic action-associated inhibitory effects of antipsychotics on UBSM contraction have not been sufficiently assessed. In this study, we examined 26 clinically available antipsychotics to determine the extent to which they inhibit acetylcholine (ACh)-induced contraction in rat UBSM to predict the drugs that should not be used by elderly people to avoid urinary disorders. Of the 26 antipsychotics, six (chlorpromazine, levomepromazine (phenothiazines), zotepine (a thiepine), olanzapine, quetiapine, clozapine (multi-acting receptor targeted antipsychotics (MARTAs))) competitively inhibited ACh-induced contractions at concentrations corresponding to clinically significant doses. Further, 11 antipsychotics (perphenazine, fluphenazine, prochlorperazine (phenothiazines), haloperidol, bromperidol, timiperone, spiperone (butyrophenones), pimozide (a diphenylbutylpiperidine), perospirone, blonanserin (serotonin-dopamine antagonists; SDAs), and asenapine (a MARTA)) significantly suppressed ACh-induced contraction; however, suppression occurred at concentrations substantially exceeding clinically achievable blood levels. The remaining nine antipsychotics (pipamperone (a butyrophenone), sulpiride, sultopride, tiapride, nemonapride (benzamides), risperidone, paliperidone (SDAs), aripiprazole, and brexpiprazole (dopamine partial agonists)) did not inhibit ACh-induced contractions at concentrations up to 10-5 M. These findings suggest that chlorpromazine, levomepromazine, zotepine, olanzapine, quetiapine, and clozapine should be avoided by elderly people with urinary disorders.

The mast cell activator Compound 48/80 causes phasic urinary bladder smooth muscle contractions independent of histamine release

Inflammatory mediators released from mast cells cause both transient and phasic contractions of urinary bladder smooth muscle (UBSM). These contractions are viewed as initial disruptors of normal bladder function and can lead to profound bladder pathologies. However, the mechanisms responsible for each of these contractions has not been thoroughly investigated. Previously, we discovered that histamine-mediated contractions rapidly desensitize, whereas the mast cell activator compound 48/80 (10 µg/ml) caused both a long-lasting increase in the amplitude and frequency of phasic contractions in UBSM strips as well as a transient increase in baseline tension. Thus, we hypothesized that phasic contractions caused by compound 48/80 depended on the release of histamine from mast cells. Isometric contractility was performed with urothelium-intact UBSM strips from C57BL/6 mice in the presence or absence of the following drugs: the cholinergic antagonist atropine (2 µM); the 5HT2A receptor antagonist MDL 11939 (1 µM); the histamine H1 receptor antagonist fexofenadine (10 µM), the histamine H2 receptor antagonist roxatidine (25 µM), the H2 antagonist cimetidine (10 µM); or the mast cell stabilizer cromolyn (100 µM). None of the compounds tested significantly reduced the amplitude and duration of the spontaneous phasic contractions caused by compound 48/80. However, both MDL 11939 and cromolyn significantly reduced the baseline contraction. Our data suggest that responses to the mast cell activator compound 48/80 are a superimposition of 2 components: (1) a slowly desensitizing increase in baseline tension caused by release of 5-HT from mast cells, and (2) large-amplitude phasic contractions that are histamine- and 5HT-independent. However, the mechanism responsible for increases in the amplitude of phasic contractions remains unclear.

Mechanism of the Long-lasting Potentiating Effect of Distigmine on Urinary Bladder Motility

Distigmine bromide (distigmine) is a carbamate cholinesterase (ChE) inhibitor, which is mainly used for the treatment of myasthenia gravis. Distigmine is also used in Japan for the treatment for underactive bladder and glaucoma. The effectiveness of distigmine for underactive bladder treatment has been confirmed by many clinical reports, and this effect is thought to be caused by potentiating urinary bladder smooth muscle contraction due to inhibition of acetylcholine degradation during micturition. However, the pharmacological effects of distigmine on urinary bladder smooth muscle have not been well studied. The most distinctive pharmacological feature of distigmine is that it shows long-lasting effects than other ChE inhibitors; however, few studies have investigated the persistence of the enhancing effect of distigmine on the contractile function of urinary bladder smooth muscle. Moreover, this mechanism remains unclear. In this review, we present our findings on the mechanism of the potentiating effect of distigmine on isolated guinea pig urinary bladder smooth muscle contraction. We also discuss the long-lasting potentiating effect of distigmine on urinary bladder motility and the mechanism of these effects using guinea pig urinary bladder smooth muscle in vivo and in vitro. In addition, we present our investigations on the long-lasting mechanism of distigmine using recombinant human acetylcholinesterase.

Ketamine‑induced bladder dysfunction is associated with extracellular matrix accumulation and impairment of calcium signaling in a mouse model.

Due to the rising abuse of ketamine usage in recent years, ketamine-induced urinary tract syndrome has received increasing attention. The present study aimed to investigate the molecular mechanism underlying ketamine-associated cystitis in a mouse model. Female C57BL/6 mice were randomly divided into two groups: One group was treated with ketamine (100 mg/kg/day of ketamine for 20 weeks), whereas, the control group was treated with saline solution. In each group, micturition frequency and urine volume were examined to assess urinary voiding functions. Mouse bladders were extracted and samples were examined for pathological and morphological alterations using hematoxylin and eosin staining, Masson's trichrome staining and scanning electron microscopy. A cDNA microarray was conducted to investigate the differentially expressed genes following treatment with ketamine. The results suggested that bladder hyperactivity increased in the mice treated with ketamine. Furthermore, treatment with ketamine resulted in a smooth apical epithelial surface, subepithelial vascular congestion and lymphoplasmacytic aggregation. Microarray analysis identified a number of genes involved in extracellular matrix accumulation, which is associated with connective tissue fibrosis progression, and in calcium signaling regulation, that was associated with urinary bladder smooth muscle contraction. Collectively, the present results suggested that these differentially expressed genes may serve critical roles in ketamine-induced alterations of micturition patterns and urothelial pathogenesis. Furthermore, the present findings may provide a theoretical basis for the development of effective therapies to treat ketamine-induced urinary tract syndrome.

Effect of distigmine on the contractile response of guinea pig urinary bladder to electrical field stimulation

Distigmine bromide (distigmine) is a reversible carbamate group cholinesterase (ChE) inhibitor. Although mainly used clinically for the treatment of myasthenia gravis, distigmine is also indicated for detrusor underactivity in Japan. According to the pharmacological classification of distigmine, its therapeutic effect against detrusor underactivity appears to be produced by enhanced urinary bladder smooth muscle (UBSM) contractility due to an increased concentration of acetylcholine between parasympathetic nerve endings and UBSM cells. However, ATP as well as acetylcholine is also released from parasympathetic nerve endings that dominate UBSM. The present study was thus carried out to investigate the potentiating effects of distigmine on the two UBSM contractile components in response to parasympathetic nerve stimulation induced by electrical field stimulation (EFS). In isolated guinea pig UBSM tissues, EFS (1–16Hz) produced tetrodotoxin-sensitive, frequency-dependent contractions. The contractile responses to EFS were largely diminished by atropine (10−6M), and the remaining contractile components in the presence of atropine were virtually abolished by α,β-methylene adenosine triphosphate (α,β-mATP) (10−4M). Distigmine (10−6M) significantly potentiated EFS-induced contractile components generated in the presence of α,β-mATP (10−4M), but did not potentiate EFS-induced contractile components generated in the presence of atropine (10−6M). These findings clearly indicate that distigmine strongly potentiates UBSM contraction selectively induced by parasympathetic nerve-derived acetylcholine, suggesting a potential mechanism by which distigmine restores detrusor underactivity.

Effects of Distigmine on Electrical Field Stimulation-Induced Contraction of Mouse Urinary Bladder Smooth Muscles

Distigmine bromide (distigmine), a reversible carbamate cholinesterase inhibitor, is used in Japan for treating detrusor underactivity, myasthenia gravis, and glaucoma. Although there is clinical evidence about the effectiveness of distigmine in the treatment of detrusor underactivity, mechanisms by which distigmine restores impaired urinary bladder smooth muscle (UBSM) contractility have not been fully investigated. The aims of this study were to investigate the potentiating effects of distigmine on UBSM contractions in response to parasympathetic nerve stimulation induced by electrical field stimulation (EFS) in mice. In isolated mouse UBSM, EFS (1-16 Hz) produced tetrodotoxin-sensitive, frequency-dependent contractions. The contractile responses to EFS were largely attenuated by atropine (10^-6 mol/l). UBSM contractility that occurred in the presence of atropine was nearly eliminated by the addition of α,β-methylene adenosine triphosphate (α,β-mATP, 10^-4 mol/l). Distigmine (3 × 10^-7 mol/l) significantly potentiated EFS-induced contractile responses engendered in the presence of α,β-mATP (10^-4 mol/l), but not atropine (10^-6 mol/l). These findings indicate that distigmine powerfully potentiates UBSM contractions selectively induced by parasympathetic nerve-derived acetylcholine, thereby demonstrating a potential mechanism by which it stimulates detrusor contractile function.

Pharmacological Opening of KV7 Channels by the Novel Activator ML‐213: Role in Guinea Pig Urinary Bladder Smooth Muscle Function

It has been recently suggested that voltage‐gated KV7 channels (KV7.1‐KV7.5) regulate urinary bladder smooth muscle (UBSM) function. Despite emerging developments, the physiological role of individual KV7 channel subtypes remains less clear. Here, we utilized the novel compound N‐(2,4,6‐Trimethylphenyl)‐bicyclo[2.2.1]heptane‐2‐carboxamide (ML‐213), a potent activator of KV7.2, KV7.4, and KV7.5 channels, to elucidate their physiological roles in guinea pig UBSM function. Using isometric UBSM tension recordings, Ca2+ imaging, and amphotericin‐B perforated patch‐clamp electrophysiology, we elucidated the role of ML‐213‐sensitive KV7 channels in regulating UBSM excitability and contractility. In functional studies of UBSM contractility, ML‐213 concentration‐dependently (100 nM‐30 μM) inhibited spontaneous phasic, pharmacologically‐induced, and nerve‐evoked contractions in UBSM isolated strips. In UBSM strips loaded with the ratiometric fluorescence probe fura 2, ML‐213 (10 μM) decreased the global intracellular Ca2+ concentration and inhibited spontaneous Ca2+ transients, which is consistent with the inhibitory effects on UBSM contractility. ML‐213‐induced attenuation of global Ca2+ levels was abolished in the presence of the L‐type voltage‐gated Ca2+ channel inhibitor nifedipine (1 μM) and the KV7.1–KV7.5 channel inhibitor XE991 (10 μM). These data suggests that ML‐213 decreases the global intracellular Ca2+ concentration by inhibiting L‐type voltage‐gated Ca2+ channels through an indirect mechanism downstream from KV7 channel activation. In current‐clamp mode of the perforated patch‐clamp technique, ML‐213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in UBSM cells. ML‐213‐induced hyperpolarization of the UBSM cell membrane potential was reversible by washout of the compound. We next aimed to examine the effects of ML‐213 on voltage‐step depolarization‐induced whole cell KV7 currents using the perforated patch‐clamp technique in voltage‐clamp mode. To isolate KV7 currents, the extracellular bath solution contained the large conductance voltage‐ and Ca2+‐activated K+ channel inhibitor paxilline (1 μM) and gadolinium chloride (GdCl3, 100 μM), which blocks L‐type voltage‐gated Ca2+ channels and non‐selective cation channels. Under these experimental conditions, ML‐213 (10 μM) enhanced whole cell KV7 currents. These findings suggest that the modulation of K+ transport through ML‐213‐sensitive KV7 channels underlies ML‐213‐induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and UBSM contractility. These combined results, using the novel compound ML‐213, suggest that KV7.2‐, KV7.4‐, and KV7.5‐containing channels are essential regulators of the excitability and contractility of UBSM by virtue of their control of the resting membrane potential. In addition, these studies provide a foundational basis for further studies investigating KV7 channel functional roles in human UBSM excitability and contractility to confirm their potential as novel therapeutic targets for bladder dysfunction.Support or Funding InformationSupported by NIH grant R01‐DK106964 to Georgi V. Petkov and F31‐DK104528 to Aaron Provence.

Novel mechanism of hydrogen sulfide-induced guinea pig urinary bladder smooth muscle contraction: role of BK channels and cholinergic neurotransmission.

Hydrogen sulfide (H2S) is a key signaling molecule regulating important physiological processes, including smooth muscle function. However, the mechanisms underlying H2S-induced detrusor smooth muscle (DSM) contractions are not well understood. This study investigates the cellular and tissue mechanisms by which H2S regulates DSM contractility, excitatory neurotransmission, and large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in freshly isolated guinea pig DSM. We used a multidisciplinary experimental approach including isometric DSM tension recordings, colorimetric ACh measurement, Ca(2+) imaging, and patch-clamp electrophysiology. In isolated DSM strips, the novel slow release H2S donor, P-(4-methoxyphenyl)-p-4-morpholinylphosphinodithioic acid morpholine salt (GYY4137), significantly increased the spontaneous phasic and nerve-evoked DSM contractions. The blockade of neuronal voltage-gated Na(+) channels or muscarinic ACh receptors with tetrodotoxin or atropine, respectively, reduced the stimulatory effect of GYY4137 on DSM contractility. GYY4137 increased ACh release from bladder nerves, which was inhibited upon blockade of L-type voltage-gated Ca(2+) channels with nifedipine. Furthermore, GYY4137 increased the amplitude of the Ca(2+) transients and basal Ca(2+) levels in isolated DSM strips. GYY4137 reduced the DSM relaxation induced by the BK channel opener, NS11021. In freshly isolated DSM cells, GYY4137 decreased the amplitude and frequency of transient BK currents recorded in a perforated whole cell configuration and reduced the single BK channel open probability measured in excised inside-out patches. GYY4137 inhibited spontaneous transient hyperpolarizations and depolarized the DSM cell membrane potential. Our results reveal the novel findings that H2S increases spontaneous phasic and nerve-evoked DSM contractions by activating ACh release from bladder nerves in combination with a direct inhibition of DSM BK channels.

Functional roles for K V 7.2 and K V 7.3 channels in guinea pig urinary bladder smooth muscle revealed by the novel K V 7.2/K V 7.3 channel opener ICA‐069673

We elucidated the physiological role of KV7.2/KV7.3 channels in guinea pig urinary bladder smooth muscle (UBSM) in vitro using the novel KV7.2/KV7.3 channel opener ICA-069673 and a multidisciplinary experimental approach. Western blot revealed protein expression for KV7.2 and KV7.3 channels in UBSM. In isolated UBSM cells, immunocytochemistry detected protein expression of KV7.2 and KV7.3 channels with localization at the cell membrane. ICA-069673 caused a concentration-dependent (100 nM-30 µM) inhibition of spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in UBSM isolated strips. In solution with elevated K+ concentration (60 mM), the inhibitory effects of ICA-069673 on UBSM contractility were attenuated. ICA-069673 decreased the global intracellular Ca2+ concentration in UBSM cells, an effect blocked by the L-type voltage-gated Ca2+ channel inhibitor nifedipine (1 µM). ICA-069673 significantly hyperpolarized the membrane potential and inhibited spontaneous action potentials in UBSM cells measured with perforated patch-clamp. XE991 (10 µM), a Kv7 channel inhibitor, abolished the effects of ICA069673 on USBM contractility and membrane potential. These results establish KV7.2/KV7.3 channels as critical regulators of UBSM excitability and contractility. Supported by NIH R01DK04284 to Georgi V. Petkov & NIH F31DK104528 to Aaron Provence.

Large conductance voltage‐ and Ca 2+ ‐activated K + channels mediate the 17β‐estradiol inhibitory effects on human urinary bladder smooth muscle cell excitability and contractility

Estrogens are critical regulators of smooth muscle function, including urinary bladder smooth muscle (UBSM) contractility. However, the mechanism underlying estrogen's regulatory role in UBSM has not been fully elucidated. Here, we explored whether 17β-estradiol,a physiologically active estrogen, activates large conductance voltage- and Ca2+-activated K+ (BK) channels in human UBSM. We obtained UBSM samples from 18 patients to examine the physiological role of 17β-estradiol in regulating UBSM function. 17β-estradiol inhibited UBSM spontaneous phasic contractions in a concentration-dependent manner (100 nM-1 µM). These 17β-estradiol inhibitory effects were prevented in the presence of paxilline (1 µM), a selective inhibitor of BK channels. 17β-estradiol (100 nM) also inhibited nerved-evoked contractions of UBSM isolated strips. Perforated patch-clamp data showed that 17β-estradiol increased the depolarization-induced whole cell outward currents, effects that were blocked by paxilline (1 µM). 17β-estradiol (100 nM) also increased the frequency of the transient BK currents in human UBSM cells. 17β-estradiol (100 nM) increased the BK channel open probability in inside-out excised patches from UBSM cells, supporting the concept that 17β-estradiol activates BK channels directly. Our results suggest that 17β-estradiol has a direct stimulatory effect on BK channel activity, thus decreasing the human UBSM excitability and contractility. Supported by NIH R01DK04284 to G.V. Petkov & NIH F31DK104528 to A. Provence.

Constitutive PKA activity is essential for maintaining the excitability and contractility in guinea pig urinary bladder smooth muscle: role of the BK channel.

The elevation of protein kinase A (PKA) activity activates the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in urinary bladder smooth muscle (UBSM) cells and consequently attenuates spontaneous phasic contractions of UBSM. However, the role of constitutive PKA activity in UBSM function has not been studied. Here, we tested the hypothesis that constitutive PKA activity is essential for controlling the excitability and contractility of UBSM. We used patch clamp electrophysiology, line-scanning confocal and ratiometric fluorescence microscopy on freshly isolated guinea pig UBSM cells, and isometric tension recordings on freshly isolated UBSM strips. Pharmacological inhibition of the constitutive PKA activity with H-89 or PKI 14-22 significantly reduced the frequency and amplitude of spontaneous transient BK channel currents (TBKCs) in UBSM cells. Confocal and ratiometric fluorescence microscopy studies revealed that inhibition of constitutive PKA activity with H-89 reduced the frequency and amplitude of the localized Ca(2+) sparks but increased global Ca(2+) levels and the magnitude of Ca(2+) oscillations in UBSM cells. H-89 abolished the spontaneous transient membrane hyperpolarizations and depolarized the membrane potential in UBSM cells. Inhibition of PKA with H-89 or KT-5720 also increased the amplitude and muscle force of UBSM spontaneous phasic contractions. This study reveals the novel concept that constitutive PKA activity is essential for controlling localized Ca(2+) signals generated by intracellular Ca(2+) stores and cytosolic Ca(2+) levels. Furthermore, constitutive PKA activity is critical for mediating the spontaneous TBKCs in UBSM cells, where it plays a key role in regulating spontaneous phasic contractions in UBSM.

Cocaine- and amphetamine-regulated transcript peptide (CARTp): distribution and function in rat urinary bladder.

We investigated the distribution of CARTp(55-102) in rat lower urinary tract and evaluated its effect on urinary bladder function in vitro. Immunohistochemistry and a vertical isolated tissue bath system were used. Neurons, clusters of nonneuronal endocrine cells, and nerve fibers stained positive for CARTp(55-102) in young adult rat urinary bladder. The CARTp-expressing neuronal elements were nitric oxide synthase (NOS)- and tyrosine hydroxylase (TH)-IR, whereas all nonneuronal CARTp-IR elements stained positively only for TH (100%). In isolated bladder strips, CARTp significantly increased the amplitude of electric field stimulation (EFS)-induced detrusor contractions at stimulation frequencies ≤12.5Hz (p ≤ 0.001) as well as amplitude and frequency of spontaneous phasic urinary bladder smooth muscle (UBSM) contractions (p ≤ 0.05). The responses to CARTp stimulation were dose-dependent and increased in the presence of the urothelium. To determine if the CARTp increase in nerve-mediated contractions may involve an action of CARTp on specific neural pathways, we blocked cholinergic, purinergic, and adrenergic pathways and determined CARTp actions on EFS-medicated contractions. CARTp enhancement of EFS-mediated contractions does not involve alteration in purinergic, adrenergic, or cholinergic pathways. The study demonstrates that CARTp(55-102) is highly expressed in rat urinary bladder. CARTp increased the amplitude of EFS-induced detrusor contractions as well as the amplitude and frequency of spontaneous phasic urinary bladder smooth muscle contractions. We conclude that CARTp may alter the release of compounds from the urothelium that leads to an enhancement of UBSM contractility/excitability.

Prostaglandin E2 inhibits the large‐conductance voltage‐ and Ca2+‐activated K+ channels in guinea pig urinary bladder smooth muscle (865.7)

Prostaglandin E2 (PGE2) is an important signaling molecule that has been shown to be involved in the regulation of urinary bladder smooth muscle (UBSM) contractility in both rodents and humans. However, the underlying mechanism by which PGE2 evokes UBSM contractions is not well understood. Here, we investigated whether large conductance voltage‐ and Ca2+‐activated K+ (BK) channels mediate the stimulatory effects induced by PGE2 on guinea pig UBSM contractility. We used a combined experimental approach including patch‐clamp electrophysiology, live‐cell Ca2+ imaging, and isometric UBSM tension recordings to investigate BK channel regulation by PGE2 at both cellular and tissue levels. PGE2 increased the spontaneous phasic contractions of UBSM isolated strips in a concentration‐dependent manner (10 nM‐10 µM). BK channel inhibition with paxilline attenuated the PGE2‐induced UBSM phasic contractions, suggesting that BK channels are among the targets of PGE2. Our data also indicate that PGE2 (10 µM) caused an inhibition of the amplitude and frequency of spontaneous transient BK currents and increased the intracellular Ca2+ levels in freshly‐isolated UBSM cells. Thus, for the first time we have revealed a novel mechanism by which BK channels mediate the PGE2 stimulatory effects on guinea pig UBSM contractions.Grant Funding Source: Supported by NIH R01 DK084284 grant to Georgi V. Petkov.

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility (865.4)

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility Wenkuan Xin1, Ning Li1, Eric S. Rovner2, Qiuping Cheng1, and Georgi V. Petkov1,2 1Drug Discovery & Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 and 2Medical University of South Carolina, Charleston, SC 29425 Activation of muscarinic M3 receptors (M3Rs) leads to an inhibition of the large conductance Ca2+‐activated K+ (BK) channels and an increase in human urinary bladder smooth muscle (UBSM) excitability. M3R‐mediated Ca2+ influx activates phosphodiesterase‐1 (PDE1) and reduces cellular cAMP levels. We investigated the mechanism by which the integrated PDE1 and MR signaling pathways stimulate human UBSM excitability and contractility. Pharmacological activation of M3Rs with carbachol (1 µM) decreased the frequency and amplitude of transient BK currents (TBKCs) in UBSM cells and subsequent inhibition of PDE1 with 8MM‐IBMX (10 µM) recovered TBKC activity. In the absence of M3R stimulation, inhibition of PDE1 increased TBKCs frequency, and subsequent inhibition of protein kinase A (PKA) with H‐89 (10 µM) reduced TBKCs frequency. 8MM‐IBMX (10 µM) hyperpolarized UBSM membrane potential, an effect which was blocked upon PKA inhibition. 8MM‐IBMX suppressed spontaneous phasic contractions and nerve‐evoked contractions of UBSM isolated strips. In conclusion, PDE1 enhances the M3R‐mediated inhibition of BK channels. Inhibition of PDE1 attenuates UBSM excitability by increasing BK channel activity via a PKA‐dependent mechanism. A combination of a PDE1 inhibitor and a MR antagonist can be an effective novel approach for the treatment of bladder overactivity.Key words: phosphodiesterase, muscarinic receptor, detrusorGrant Funding Source: NIH R01 DK084284 to Georgi V. Petkov

Novel BK channel regulatory mechanism by protein kinase C in guinea pig urinary bladder smooth muscle (865.1)

Large conductance Ca2+‐activated K+ (BK) channels are critical regulators of urinary bladder smooth muscle (UBSM) function. Here, we provide a novel mechanistic insight into BK channel regulation by protein kinase C (PKC) in UBSM. Voltage‐clamp experiments showed that pharmacological activation of PKC with phorbol 12‐myristate 13‐acetate (PMA), inhibited the spontaneous transient BK currents (TBKCs) in native freshly‐isolated guinea pig UBSM cells. Current‐clamp recordings revealed that PMA significantly depolarized UBSM membrane potential, and reduced the spontaneous transient hyperpolarizations in UBSM cells. The PMA inhibitory effects on UBSM membrane potential were abolished by the selective BK channel inhibitor paxilline and were not observed with its inactive analog, 4‐alpha‐PMA. PMA did not affect the amplitude of the whole cell steady‐state BK current or single BK channel open probability (recorded in cell‐attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PMA elevated the intracellular Ca2+ levels in UBSM cells, and increased spontaneous phasic and nerve‐evoked contractions of UBSM isolated strips. The results support the concept that PKC activation leads to a reduction in BK channel activity in UBSM via a Ca2+‐dependent mechanism, thus increasing UBSM contractility.Grant Funding Source: Supported by R01 DK084284 to G. V. Petkov