Isolated Detrusor Smooth Muscle Cells Research Articles

Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents.

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2–3 mm x 4–6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 °C for 30–45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

Chloride Single Channel Activity in Freshly‐Isolated Guinea Pig Detrusor Smooth Muscle Cells

Ion channels in detrusor smooth muscle (DSM) control excitability and contractility of the urinary bladder. Several ion channel types have already been detected and characterized in DSM cells, especially voltage‐gated cationic channels. However, little is known about Cl− channels in DSM cells. Specifically, it is unknown what Cl− channel subtypes are expressed in the plasma membrane of DSM cells and how they control DSM cell excitability. Earlier molecular and fluorometric studies suggested the presence of Ca2+‐activated Cl− channels in DSM cells. Our Agilent Human GE 4x44K v2 microarray data analysis on single DSM cell mRNA detected a number of candidates underlying Cl− conductance in DSM cells including plasmalemmal TMEM16A/ANO1, CLCN3, CLCN6, and CLCN7. Thus, detailed biophysical analyses are needed to characterize these Cl− channels. Here, guinea pig DSM cells were freshly isolated by an enzymatic digestion method with papain and collagenase type II, plated at the glass bottom of a recording chamber, and electrophysiologically tested within 6 hours after the isolation. Inside‐out single channel patch clamp recording technique was used to detect electrical activity of Clchannels in the plasma membrane of isolated DSM cells. To separate Cl− from cationic currents, all experiments were done using asymmetrical Cl− and symmetrical Na+ solutions. The bath/intracellular solution contained (in mM): 110 Na‐glutamate, 5 NaCl, 60 mannitol, 10 HEPES, 1 CaCl2, 1 CoCl2, 0.0005 phorbol 12‐myristate 13‐acetate, pH 7.2; the pipette/extracellular contained (in mM): 110 NaCl, 1.5 MgCl2, 2 CaCl2, 60 mannitol, 10 HEPES, 0.001 paxilline, 0.01 nifedipine, pH 7.4. Cl− single channel activity detection frequency varied from zero to 26.7 % within a single DSM cell preparation with an average frequency for detection of 7.4 % (13 out of 175 patches, 8 guinea pigs). Cl− channels displayed unique gating with multiple subconductive states and a fully opened single‐channel conductance in a range from 140 pS to 193 pS with an average value of 163 ± 15 pS (12 patches, 8 guinea pigs, mean ± SD). A linear fitting of single channel current amplitude – membrane potential relationships yielded a reversal potential of − 41.3 ± 5.5 mV (12 patches, 8 guinea pigs, mean ± SD) for this Cl− channel, close to the theoretical ECl of − 65 mV and confirming that this channel is permeable to Cl−. The Cl− channel demonstrated strong voltage‐dependence of activation with virtually no activity below the measured reversal potential and always robust prolonged openings at depolarizing voltages. The channel had similar gating when exposed to the intracellular Ca2+‐free (buffered with 5 mM EGTA in nominal Ca2+) and 1 mM Ca2+ solutions. The Cl− channel was not blocked by 9‐phenanthrol (30 μM), a transient receptor potential melastatin 4 channel inhibitor that also has been reported to block the activity of TMEM16A Ca2+‐activated Cl− channels in vascular smooth muscle cells. In conclusion, our data reveal for the first time the presence of Ca2+‐insensitive voltage‐dependent Cl− channel in guinea pig DSM cells at the single channel level. This channel may be a key regulator of DSM excitability.Support or Funding InformationSupported by NIH R01DK106964 to Georgi V. Petkov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nongenomic modulation of the large conductance voltage- and Ca2+-activated K+ channels by estrogen: A novel regulatory mechanism in human detrusor smooth muscle.

Estrogens have an important role in regulating detrusor smooth muscle (DSM) function. However, the underlying molecular and cellular mechanisms by which estrogens control human DSM excitability and contractility are not well known. Here, we used human DSM specimens from open bladder surgeries on 27 patients to elucidate the mechanism by which 17β‐estradiol regulates large conductance voltage‐ and Ca2+‐activated K+ (BK) channels, the most prominent K+ channels in human DSM. We employed single BK channel recordings on inside‐out excised membrane patches, perforated whole‐cell patch‐clamp on freshly isolated DSM cells, and isometric tension recordings on DSM‐isolated strips to investigate the mechanism by which 17β‐estradiol activates BK channels. 17β‐Estradiol (100 nmol/L) rapidly increased depolarization‐induced whole‐cell K+ currents in DSM cells. The 17β‐estradiol stimulatory effects on whole‐cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 μmol/L), clearly indicating that 17β‐estradiol specifically activates BK channels. 17β‐Estradiol also increased the frequency of ryanodine receptor‐mediated transient BK currents. Single BK channel recordings showed that 17β‐estradiol (100 nmol/L) significantly increased the BK channel open probability of inside‐out excised membrane patches, revealing that 17β‐estradiol activates BK channels directly. 17β‐Estradiol reduced spontaneous phasic contractions of human DSM‐isolated strips in a concentration‐dependent manner (100 nmol/L‐1 μmol/L), and this effect was blocked by paxilline (1 μmol/L). 17β‐Estradiol (100 nmol/L) also reduced nerve‐evoked contractions of human DSM‐isolated strips. Collectively, our results reveal that 17β‐estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.

Myorelaxant action of fluorine-containing pinacidil analog, flocalin, in bladder smooth muscle is mediated by inhibition of L-type calcium channels rather than activation of KATP channels.

Flocalin (FLO) is a new ATP-sensitive K(+) (KATP) channel opener (KCO) derived from pinacidil (PIN) by adding fluorine group to the drug's structure. FLO acts as a potent cardioprotector against ischemia-reperfusion damage in isolated heart and whole animal models primarily via activating cardiac-specific Kir6.2/SUR2A KATP channels. Given that FLO also confers relaxation on several types of smooth muscles and can partially inhibit L-type Ca(2+) channels, in this study, we asked what is the mechanism of FLO action in bladder detrusor smooth muscle (DSM). The actions of FLO and PIN on contractility of rat and guinea pig DSM strips and membrane currents of isolated DSM cells were compared by tensiometry and patch clamp. Kir6 and SUR subunit expression in rat DSM was assayed by reverse transcription PCR (RT-PCR). In contrast to PIN (10μM), FLO (10μM) did not produce glibenclamide-sensitive DSM strips' relaxation and inhibition of spontaneous and electrically evoked contractions. However, FLO, but not PIN, inhibited contractions evoked by high K(+) depolarization. FLO (40μM) did not change the level of isolated DSM cell's background K(+) current, but suppressed by 20% L-type Ca(2+) current. Determining various Kir6 and SUR messenger RNA (mRNA) expressions in rat DSM by RT-PCR indicated that dominant KATP channel in rat DSM is of vascular type involving association of Kir6.1 and SUR2B subunits. Myorelaxant effects of FLO in bladder DSM are explained by partial blockade of L-type Ca(2+) channel-mediated Ca(2+) influx rather than by hyperpolarization associated with increased K(+) permeability. Thus, insertion of fluorine group in PIN's structure made the drug more discriminative between Kir6.2/SUR2A cardiac- and Kir6.1/SUR2B vascular-type KATP channels and rendered it partial L-type Ca(2+) channel-blocking potency.

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.

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.

BK Channel-Mediated Relaxation of Urinary Bladder Smooth Muscle: A Novel Paradigm for Phosphodiesterase Type 4 Regulation of Bladder Function

Elevation of intracellular cAMP and activation of protein kinase A (PKA) lead to activation of large conductance voltage- and Ca(2+)-activated K(+) (BK) channels, thus attenuation of detrusor smooth muscle (DSM) contractility. In this study, we investigated the mechanism by which pharmacological inhibition of cAMP-specific phosphodiesterase 4 (PDE4) with rolipram or Ro-20-1724 (C(15)H(22)N(2)O(3)) suppresses guinea pig DSM excitability and contractility. We used high-speed line-scanning confocal microscopy, ratiometric fluorescence Ca(2+) imaging, and perforated whole-cell patch-clamp techniques on freshly isolated DSM cells, along with isometric tension recordings of DSM isolated strips. Rolipram caused an increase in the frequency of Ca(2+) sparks and the spontaneous transient BK currents (TBKCs), hyperpolarized the cell membrane potential (MP), and decreased the intracellular Ca(2+) levels. Blocking BK channels with paxilline reversed the hyperpolarizing effect of rolipram and depolarized the MP back to the control levels. In the presence of H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride], a PKA inhibitor, rolipram did not cause MP hyperpolarization. Rolipram or Ro-20-1724 reduced DSM spontaneous and carbachol-induced phasic contraction amplitude, muscle force, duration, and frequency, and electrical field stimulation-induced contraction amplitude, muscle force, and tone. Paxilline recovered DSM contractility, which was suppressed by pretreatment with PDE4 inhibitors. Rolipram had reduced inhibitory effects on DSM contractility in DSM strips pretreated with paxilline. This study revealed a novel cellular mechanism whereby pharmacological inhibition of PDE4 leads to suppression of guinea pig DSM contractility by increasing the frequency of Ca(2+) sparks and the functionally coupled TBKCs, consequently hyperpolarizing DSM cell MP. Collectively, this decreases the global intracellular Ca(2+) levels and DSM contractility in a BK channel-dependent manner.

Stimulation of M3 but not M2 muscarinic receptor type inhibits large‐conductance Ca2+ activated K+ channel in rat detrusor smooth muscle

Large conductance Ca2+‐activated K+ (BK) channel is a major regulator of detrusor smooth muscle (DSM) contractility. Whether the regulation of BK channel function is mediated by muscarinic receptors (M‐Rs) remains unexplored. We studied the functional link between BK channels and M‐Rs in freshly isolated DSM cells using the perforated patch‐clamp technique. Carbachol (CCh) initially elicited large outward BK currents followed by inhibition of the amplitude and frequency of transient BK currents (TBKCs). CCh also inhibited the spontaneous transient hyperpolarizations and depolarized the cell membrane potential (MP). CCh inhibitory effects on the BK channels were suppressed by selectively blocking the M3‐Rs with 4‐DAMP, but not M2‐Rs with methoctramine. Blocking the IP3 receptors with xestospongin C attenuated CCh‐evoked initial effects on the large outward BK currents without altering the TBKCs suggesting an involvement of Ca2+ release from IP3 receptors in the initiation of the large outward BK currents. In the presence of thapsigargin, ryanodine, and nifedipine – CCh did not change the steady‐state BK currents suggesting requirement of intracellular Ca2+in the BK channel inhibition by cholinergic pathways. In conclusion, our results revealed that stimulation of M3‐Rs, but not M2‐Rs inhibits TBKCs, depolarized MP, and promotes DSM contractions. Supported by NIH DK08424 to GVP and AUA Fellowship to SPP.

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

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

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.

Selective inhibition of phosphodiesterase 1 relaxes urinary bladder smooth muscle: role for ryanodine receptor-mediated BK channel activation

The large conductance voltage- and Ca(2+)-activated K(+) (BK) channel is a major regulator of detrusor smooth muscle (DSM) excitability and contractility. Recently, we showed that nonselective phosphodiesterase (PDE) inhibition reduces guinea pig DSM excitability and contractility by increasing BK channel activity. Here, we investigated how DSM excitability and contractility changes upon selective inhibition of PDE type 1 (PDE1) and the underlying cellular mechanism involving ryanodine receptors (RyRs) and BK channels. PDE1 inhibition with 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX; 10 μM) increased the cAMP levels in guinea pig DSM cells. Patch-clamp experiments on freshly isolated DSM cells showed that 8MM-IBMX increased transient BK currents and the spontaneous transient hyperpolarization (STH) frequency by ∼2.5- and ∼1.8-fold, respectively. 8MM-IBMX hyperpolarized guinea pig and human DSM cell membrane potential and significantly decreased the intracellular Ca(2+) levels in guinea pig DSM cells. Blocking BK channels with 1 μM paxilline or inhibiting RyRs with 30 μM ryanodine abolished the STHs and the 8MM-IBMX inhibitory effects on the DSM cell membrane potential. Isometric DSM tension recordings showed that 8MM-IBMX significantly reduced the spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and tone of DSM isolated strips. The electrical field stimulation-induced DSM contraction amplitude, muscle force integral, and duration were also attenuated by 10 μM 8MM-IBMX. Blocking BK channels with paxilline abolished the 8MM-IBMX effects on DSM contractions. Our data provide evidence that PDE1 inhibition relaxes DSM by raising cellular cAMP levels and subsequently stimulates RyRs, which leads to BK channel activation, membrane potential hyperpolarization, and decrease in intracellular Ca(2+) levels.

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.

Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca2+-activated K+ channel

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ∼3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ∼2-fold and hyperpolarized the DSM cell resting membrane potential by ∼6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca(2+) sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.

Pharmacological Activation of Small Conductance Calcium-Activated Potassium Channels with Naphtho[1,2-d]thiazol-2-ylamine Decreases Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

Small conductance Ca²⁺-activated K⁺ (SK) and intermediate conductance Ca(2+)-activated K⁺ (IK) channels are thought to be involved in detrusor smooth muscle (DSM) excitability and contractility. Using naphtho[1,2-d]thiazol-2-ylamine (SKA-31), a novel and highly specific SK/IK channel activator, we investigated whether pharmacological activation of SK/IK channels reduced guinea pig DSM excitability and contractility. We detected the expression of all known isoforms of SK (SK1-SK3) and IK channels at mRNA and protein levels in DSM by single-cell reverse transcription-polymerase chain reaction and Western blot. Using the perforated patch-clamp technique on freshly isolated DSM cells, we observed that SKA-31 (10 μM) increased SK currents, which were blocked by apamin (1 μM), a selective SK channel inhibitor. In current-clamp mode, SKA-31 (10 μM) hyperpolarized the cell resting membrane potential, which was blocked by apamin (1 μM) but not by 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), a selective IK channel inhibitor. SKA-31 (10 nM-10 μM) significantly inhibited the spontaneous phasic contraction amplitude, frequency, duration, and muscle force in DSM isolated strips. The SKA-31 inhibitory effects on DSM contractility were blocked by apamin (1 μM) but not by TRAM-34 (1 μM), which did not per se significantly affect DSM spontaneous contractility. SK channel activation with SKA-31 reduced contractions evoked by electrical field stimulation. SKA-31 effects were reversible upon washout. In conclusion, SK channels, but not IK channels, mediate SKA-31 effects in guinea pig DSM. Pharmacological activation of SK channels reduces DSM excitability and contractility and therefore may provide a novel therapeutic approach for controlling bladder dysfunction.

KV2.1 and electrically silent KV channel subunits control excitability and contractility of guinea pig detrusor smooth muscle

Voltage-gated K(+) (K(V)) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric K(V) channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of K(V)2.1 and electrically silent K(V) channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric K(V)2.1, K(V)2.2, and K(V)4.2 as well as the heterotetrameric K(V)2.1/6.3 and K(V)2.1/9.3 channels, was used to examine the role of these K(V) channels in DSM function. RT-PCR indicated mRNA expression of K(V)2.1, K(V)6.2-6.3, K(V)8.2, and K(V)9.1-9.3 subunits in isolated DSM cells. K(V)2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the K(V) current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for K(V) current. However, ScTx1 (100 nM) decreased the activation time-constant of the K(V) current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric K(V)2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric K(V)2.1/silent K(V) channels. Current-clamp recordings showed that ScTx1 (100 nM) did not change the DSM cell resting membrane potential. ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and muscle tone as well as the amplitude of the electrical field stimulation-induced contractions of isolated DSM strips. Collectively, our data revealed that K(V)2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility.

SK channel activation with SKA‐31 decreases excitability and contractility in Guinea pig urinary bladder smooth muscle

Small (SK) and intermediate (IK) conductance Ca2+-activated K+ channels have been suggested to be involved in determining the excitability and contractility of detrusor smooth muscle (DSM). We used SKA-31, a novel SK/IK channel activator to probe the therapeutic potential of SK/IK channel activation to control bladder function. Isometric tension recordings of isolated Guinea pig DSM strips indicated that SKA-31 (100 nM-10 μM) significantly inhibited the spontaneous contraction amplitude, muscle force, frequency, and duration. This inhibitory effect was blocked by apamin (1 μM), a selective SK channel inhibitor. SK channel activation with SKA-31 (3 μM) also reduced the contractions evoked by electrical field stimulation. TRAM-34 (30 μM), an IK channel selective inhibitor, did not suppress the inhibitory effect of SKA-31, indicating a role only for the SK channel in detrusor contractility. Using the perforated patch-clamp technique, we observed that SKA-31 (10 μM) increased the voltage-step induced SK currents in freshly isolated DSM cells. In current clamp mode, SKA-31 (10 μM) hyperpolarized the resting membrane potential by ~3 mV. These effects were blocked by apamin (1 μM). Collectively, our data indicate that activation of SK channels may provide a novel therapeutic approach to control bladder function. Supported by NIH DK084284 & DK083687 to GVP. SKA-31 is a gift from Dr. Heike Wulff.

1-[1-Hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone, a Potent and Highly Selective Small Molecule Blocker of the Large-Conductance Voltage-Gated and Calcium-Dependent K+Channel

The large-conductance voltage-gated and calcium-dependent K(+) (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels (alpha subunit, alpha+beta1 and alpha+beta4 subunits). HMIMP blocked all of these channels with an IC(50) of approximately 2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na(V)1.5 (1 microM), Ca(V)3.2, L-type Ca(2+), human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K(+) or voltage-dependent K(+) channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.

Melatonin restores impaired contractility in aged guinea pig urinary bladder

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

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

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