Amplitude Of Spontaneous Phasic Contractions Research Articles

Effect of DA-9701 on the Gastrointestinal Motility in the Streptozotocin-Induced Diabetic Mice.

Background: Compared to the general population, diabetic patients experience more frequent episodes of gastrointestinal (GI) motility dysfunction, owing to the disruption of functional innervations. DA-9701 is a new prokinetic agent formulated from the extracts of Pharbitidis semen and Corydalis tuber. Aim: To investigate the effect of DA-9701 on GI motility in an animal model of streptozotocin (STZ)-induced diabetes. Methods: Diabetes was induced in mice by intraperitoneal injection of STZ (40 mg/kg of body weight in 0.1 M citrate buffer) for 3 days. Diabetic mice were divided into four groups and administered DA-9701 in different doses (1, 3, and 10 mg/kg) or placebo for 2 weeks. Intestinal transit was assessed using charcoal meal movement. GI isometric contraction was measured by applying an isometric force transducer on a circular muscle strip of the antrum, ileum, and proximal colon of sacrificed mice. Gastric emptying rate was evaluated by measuring the dye percentage remaining in the stomach relative to the total dye amount recovered in a standardization group of mice. Results: Body weight and antral and small intestinal motility were less in diabetic mice than in control mice, and colonic motility was similar in both. DA-9701 showed a dose-dependent increase in the amplitude of spontaneous phasic contractions in the antrum, ileum, and colon in diabetic mice without influencing body weight or blood glucose levels. The degree of improvement was comparable between diabetic and control mice. Intestinal transit was significantly more delayed in diabetic mice than in controls (43 ± 7% vs. 67 ± 8%, p < 0.05); however, DA-9701 restored the delayed intestinal transit more effectively compared to placebo (75% vs. 50%). The gastric emptying rate was significantly more delayed in diabetic mice than in controls (43 ± 10% vs. 62 ± 12%, p < 0.05), and was improved by DA-9701 in a dose-dependent manner (50%, 55%, and 60% in mice treated with 1, 3, and 10 mg/kg of DA-9701, respectively, vs. 43% in placebo-treated and 60% in control mice). Conclusions: DA-9701 improved GI contractility without affecting blood sugar and body weight in diabetic mice. DA-9701 could improve the decreased GI motility and clinical symptoms in progressive diabetic patients.

Diabetes attenuates urothelial modulation of detrusor contractility and spontaneous activity.

To investigate the effect of diabetes on urothelial modulation of bladder contractility. Bladder strips (urothelium intact or denuded) were prepared from 8-week-old streptozotocin-induced diabetic (n = 19) and non-diabetic control rats (n = 10). The effect of modulators of MaxiK (iberiotoxin and tetraethylammonium) and Kv7 (XE991 and retigabine) potassium channel activity were investigated for their effects on both carbachol-induced force generation and spontaneous contractile activity. In bladder strips from non-diabetic animals, the presence of the urothelium resulted in marked sensitivity to carbachol-induced force generation by modulators of MaxiK and Kv7 channel activity, whereas in the diabetic animal urothelial sensitivity to these agents was significantly diminished. Urothelial-intact bladder strips from non-diabetic animals were more sensitive to modulators of Kv7 activity in reducing the amplitude of spontaneous phasic contractions than urothelial-denuded bladder strips, whereas in diabetic animals the presence or absence of the urothelium did not alter the sensitivity to modulators of Kv7 activity. Spontaneous activity in the presence of tetraethylammonium was not affected by the urothelium in bladder strips from either diabetic or non-diabetic animals. The presence of the urothelium in bladders from non-diabetic animals modulates the activity of potassium blockers to affect bladder contractility, whereas in the diabetic bladder this effect is attenuated. These findings could help to explain the lack of success of pharmaceutical treatments targeting potassium channels to treat bladder pathology in patients with diseases imparing urothelial function.

Functional implications of Kv7 channels expression in guinea pig urinary bladder smooth muscle contractility

The physiological roles of Kv7 channel family have been well studied in various tissues. However, knowledge about the functional role of Kv7 channels in the urinary bladder smooth muscle (UBSM) is limited. We investigated Kv7 channels molecular expression in guinea pig UBSM and further assessed their functional role using isometric DSM tension recordings and Kv7 channels modulators. RT‐PCR experiments revealed mRNA expression for Kv7.1, Kv7.2, Kv7.3, Kv7.4, and Kv7.5 channel subunits in guinea pig UBSM tissue. Quantitative PCR analysis showed that in UBSM tissue, Kv7 channel subunits relative mRNA expression level was as follows: Kv7.1 ≈ Kv7.2 &gt;; Kv7.3 ≈ Kv7.5 &gt;; Kv7.4. Retigabine, a Kv7 channel activator, and L‐364,373, a selective Kv7.1 channel activator, induced relaxation of spontaneous phasic contraction amplitude, force integral, duration, and frequency as well as the 10 Hz electrical field stimulation (EFS)‐induced contractions amplitude and force of UBSM isolated strips (IC50 = 3 – 6 μM). Linopiridine and XE991, two well‐established Kv7 channel inhibitors, increased the spontaneous phasic contraction amplitude and force as well as the 10 Hz EFS‐induced contractions amplitude and force of UBSM isolated strips (IC50 = 3 – 4 μM). In summary, we revealed the expression and the regulatory role of Kv7 channels in the spontaneous and nerve‐evoked contractions of guinea pig UBSM. Supported by NIH DK084284 to GVP.

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca(2+) -activated K(+) (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function. We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function. We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions. Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity.

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.

Constitutively active phosphodiesterase activity regulates urinary bladder smooth muscle function: critical role of KCa1.1 channel

Pharmacological blockade of cyclic nucleotide phosphodiesterase (PDE) can relax human urinary bladder smooth muscle (UBSM); however, the underlying cellular mechanism is unknown. In this study, we investigated the effects of PDE pharmacological blockade on human UBSM excitability, spontaneous and nerve-evoked contractility, and determined the underlying cellular mechanism mediating these effects. Patch-clamp electrophysiological experiments showed that 3-isobutyl-1-methylxanthine (10 μM), a nonselective PDE inhibitor, caused ∼3.6-fold increase in the transient K(Ca)1.1 channel current frequency and ∼2.5-fold increase in the spontaneous transient hyperpolarization frequency in UBSM-isolated cells. PDE blockade also caused ∼5.6-mV hyperpolarization of the UBSM cell membrane potential. Blocking the K(Ca)1.1 channels with paxilline abolished the spontaneous transient hyperpolarization and the hyperpolarization effect of PDE blockade on the UBSM cell membrane potential. Live cell Ca(2+)-imaging experiments showed that PDE blockade significantly decreased the global intracellular Ca(2+) levels. Attenuation of PDE activity significantly reduced spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and muscle tone of human UBSM isolated strips. Blockade of PDE also significantly reduced the contraction amplitude, muscle force integral, and duration of the nerve-evoked contractions induced by 20-Hz electrical field stimulation. Pharmacological inhibition of K(Ca)1.1 channels abolished the relaxation effects of PDE blockade on both spontaneous and nerve-evoked contractions in human UBSM-isolated strips. Our data provide strong evidence that in human UBSM PDE is constitutively active, thus maintaining spontaneous UBSM contractility. PDE blockade causes relaxation of human UBSM by increasing transient K(Ca)1.1 channel current activity, hyperpolarizing cell membrane potential, and decreasing the global intracellular Ca(2+).

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.

IBMX Relaxes Guinea Pig Urinary Bladder Smooth Muscle via Activation of Large Conductance Ca2+-Activated K+ Channels

Stimulation of cyclic nucleotide signaling pathways leads to activation of the large conductance voltage- and Ca2+-activated K+ (BK) channels, thereby inhibiting urinary bladder smooth muscle (UBSM) contractility. We tested the hypothesis that nonselective inhibition of phosphodiesterases (PDEs) with 3-isobutyl-1-methylxanthine (IBMX) can reduce guinea pig UBSM phasic contractions by stimulating the BK channel activity.In UBSM isolated strips, IBMX (10 μM) significantly reduced the spontaneous phasic contraction amplitude, muscle force integral, duration, frequency, and tone by 74%, 95%, 50%, 55%, and 32%, respectively (n=8; P 0.05).In conclusion, nonselective inhibition of PDEs with IBMX effectively reduces guinea pig UBSM excitability and contractility through direct BK channel activation, increase in TBKCs frequency, and cell membrane hyperpolarization.Supported by NIH DK084284 & DK083687 to Georgi V. Petkov

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.

Do β3-adrenergic receptors play a role in guinea pig detrusor smooth muscle excitability and contractility?

In many species, β3-adrenergic receptors (β3-ARs) have been reported to play a primary role in pharmacologically induced detrusor smooth muscle (DSM) relaxation. However, their role in guinea pig DSM remains controversial. The aim of this study was to investigate whether β3-ARs are expressed in guinea pig DSM and to evaluate how BRL37344 and L-755,507, two selective β3-AR agonists, modulate guinea pig DSM excitability and contractility. We used a combined experimental approach including RT-PCR, patch-clamp electrophysiology, and isometric DSM tension recordings. β3-AR mRNA message was detected in freshly isolated guinea pig DSM single cells. BRL37344 but not L-755,507 caused a slight decrease in DSM spontaneous phasic contraction amplitude and frequency in a concentration-dependent manner. In the presence of atropine (1 μM), only the spontaneous phasic contractions frequency was inhibited by BRL37344 at higher concentrations. Both BRL37344 and L-755,507 significantly decreased DSM carbachol-induced phasic and tonic contractions in a concentration-dependent manner. However, only BRL37344 inhibitory effect was partially antagonized by SR59230A (10 μM), a β3-AR antagonist. In the presence of atropine, BRL37344 and L-755,507 had no inhibitory effect on electrical field stimulation-induced contractions. Patch-clamp experiments showed that BRL37344 (100 μM) did not affect the DSM cell resting membrane potential and K(+) conductance. Although β3-ARs are expressed at the mRNA level, they play a minor to no role in guinea pig DSM spontaneous contractility without affecting cell excitability. However, BRL37344 and L-755,507 have pronounced inhibitory effects on guinea pig DSM carbachol-induced contractions. The study outlines important DSM β3-ARs species differences.

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.

Voltage-gated K+ channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle

Members of the voltage-gated K(+) (K(V)) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits K(V)2.1, K(V)2.2, K(V)4.2, and K(V)2.1/9.3 channels. The objective of this study was to investigate whether K(V) channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 microM). This indicates that, under conditions of sustained membrane depolarization, the K(V) channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells 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. Protein expression of K(V)2.1 and K(V)2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that K(V)2.1 and K(V)2.2, but not K(V)4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions.

The large conductance Ca2+‐ and voltage‐activated K+ channel is the primary K+ channel that controls cell excitability and contractility in human urinary bladder smooth muscle

We examined the large conductance Ca2+‐activated K+ (BK) channel expression, electrophysiological properties, and possible physiological roles in human urinary bladder smooth muscle (UBSM). RT‐PCR with freshly isolated human UBSM cells shows mRNA expression of BK channel pore‐forming α, and regulatory β1/β4 subunits. The expression of all three subunits was confirmed by specific antibodies and immunocytochemistry on single cells. The majority of the whole‐cell outward current induced by voltage‐step depolarizations (‐40 to +80 mV) was inhibited by iberiotoxin (IBTX), a BK channel selective inhibitor. Voltage‐dependent spontaneous transient outward currents (STOCs) were recorded from ‐40 to +40 mV. STOCs were blocked by IBTX or ryanodine. In current‐clamp mode, IBTX caused ~3 mV depolarization. In whole cell configuration, in the absence of STOCs, single channels of 139.5 pS conductance with voltage‐dependent amplitude and open probability were recorded. These channels were blocked by IBTX, consistent with the BK channel. In isolated strips, IBTX increased spontaneous phasic contraction amplitude, force, and tone as well as the amplitude of electrical field induced contractions. In conclusion, the BK channel in the human bladder consists of α, β1 and β4 subunits and is the primary K+ channel that controls cell excitability and contractility of human UBSM.Supported by NIH DK‐070909 to G.V. Petkov.

Stromatoxin‐1‐sensitive voltage‐gated K+ channels regulate rat urinary bladder smooth muscle contractility

Recent studies report that stromatoxin‐1 (ScTx1), a 34‐amino acid peptide, selectively inhibits the voltage‐gated K+ (Kv) channel members, Kv2.1, Kv2.2, Kv4.2, and Kv2.1/9.3 heteromultimers with high affinity. The objective of this study was to identify if any voltage‐gated Kv channels sensitive to ScTx1 participate in the regulation of rat urinary bladder smooth muscle (UBSM) contractility. Freshly isolated rat UBSM strips and single UBSM cells were studied using molecular, immunological, and physiological techniques. RT‐PCR experiments on isolated UBSM cells show mRNA expression of Kv2.1, Kv2.2, and Kv9.3 but not Kv4.2 subunits (n=3). Protein expression of Kv2.1 and Kv2.2 was further confirmed in single UBSM cells by applying immunological methods with specific antibodies (n=3). Isometric UBSM tension recordings of isolated UBSM strips showed that ScTx1 (100 nM) increased spontaneous phasic contraction amplitude and muscle force by about 20% without any effect on contraction frequency or muscle tone (n=5). The ScTx1 (100 nM) effect on UBSM contraction amplitude was observed even after the UBSM strips were depolarized with 20 mM K+ (n=3). Our studies indicate that Kv2.1 and Kv2.2 members of the Kv family play a key role in shaping rat UBSM contractility. New drugs targeting specific Kv channel subunits in the UBSM may prove useful for overactive bladder.Supported by NIH DK‐070909 to G.V. Petkov.

Spasmolytic effect of Psidium guajava Linn. (Myrtaceae) leaf aqueous extract on rat isolated uterine horns

Globally, primary dysmenorrhoea is one of the most frequent gynaecological disorders in young women. It is associated with increased uterine tone, and exaggerated contractility of uterine smooth muscles. In many rural African communities, a number of medicinal plants, including Psidium guajava Linn. (family: Myrtaceae), are used traditionally for the management, control and/or treatment of primary dysmenorrhoea. The present study was, therefore, undertaken to examine the spasmolytic effect of Psidium guajava leaf aqueous extract (PGE) on isolated, spontaneously-contracting and oestrogen-dominated, quiescent uterine horns of healthy, young adult, female Wistar rats. Graded, escalated concentrations of PGE (0.5-4.0 mg/ml) produced concentration-dependent and significant inhibitions of the amplitude of spontaneous phasic contractions of the isolated rat uterine horn preparations. In a concentration-related manner, PGE also significantly inhibited or abolished contractions produced by acetylcholine (ACh, 0.5-8.0 microg/ml), oxytocin (0.5-4.0 microU), bradykinin (2.5-10 ng/ml), carbachol (CCh, 0.5-8.0 microg/ml) or potassium chloride (K+, 10-80 mM) in quiescent uterine horn preparations isolated from the oestrogen-dominated rats. The spasmolytic effect of PGE observed in the present study lends pharmacological support to the traditional use of ;guava' leaves in the management, control and/or treatment of primary dysmenorrhoea in some rural African communities.

Functional and electrophysiological identification of large conductance voltage‐ and Ca2+‐activated K+ (BK) channels in human urinary bladder smooth muscle

The large conductance voltage‐ and Ca2+‐activated K+ (BK or KCa1.1) channel is a key player in controlling excitability and contractility of animal urinary bladder smooth muscle (UBSM), however its role in human UBSM is unknown. Here, using the BK channel specific inhibitor‐iberiotoxin, functional studies and the perforated patch‐clamp technique, we identified the BK channel in human UBSM. In isolated UBSM strips iberiotoxin increased spontaneous phasic contraction amplitude and force (n=10). In isolated UBSM cells, voltage‐step depolarizations caused large K+ currents (∼1 nA; n=15). About 80% of these K+ currents were inhibited by iberiotoxin. In the voltage range of −40 to +40 mV, these cells also revealed spontaneous transient outward currents (STOCs). STOCs amplitude and frequency increased with increasing the holding potential toward positive values (n=20). STOCs were inhibited by iberiotoxin (n=4). In the presence of ryanodine, when the STOCs were blocked, single channels with large single channel conductance (120.6 pS) were observed (n=10). Their amplitude and open probability were voltage‐dependent. In current‐clamp mode, iberiotoxin caused membrane potential depolarization (n=4). The data reveal that the BK channel is the major voltage‐dependent K+ channel expressed in human UBSM cells that plays a key role in determining cell excitability and contractility. Supported by DK070909 to G.V.P.

PACAP Enhances Mouse Urinary Bladder Contractility and Is Upregulated in Micturition Reflex Pathways after Cystitis

Pituitary adenylate cyclase-activating polypeptide (PACAP) elicits a transient contraction, sustained increase in the amplitude of spontaneous phasic contractions, and significantly increases the amplitude of nerve-mediated contractions in mouse urinary bladder smooth muscle (UBSM) strips. PACAP immunoreactivity (IR) is increased in micturition reflex pathways following cystitis. PACAP may contribute to altered sensation and bladder overactivity in the chronic bladder inflammatory syndrome, interstitial cystitis.

Effect of Ethanol on Spontaneous Phasic Contractions of Cat Gastric Smooth Muscle

Background: Ethanol is generally believed to inhibit extracellular Ca 2+ influx, thereby inhibiting gastric muscle contraction. Recently, we observed that verapamil inhibited only the amplitude of spontaneous phasic contractions, whereas ethanol inhibited both amplitude and frequency. In our objective to investigate the mechanism of ethanol's inhibition of gastric motility, the involvement of various protein kinases in ethanol-inhibited spontaneous phasic contractions of the stomach muscle strips was tested. Methods: Circular muscle strips (2.0 × 0.2 cm) were prepared from the corpus of cat stomach in order to measure isometric contraction in a chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36 °C) bubbled with 5% CO 2 in O 2. Results: Spontaneous phasic contraction was not affected by various receptor antagonists (1 μM atropine, 1 μM hexamethonium, 1 μM phentolamine and 1 μM propranolol) or 1 μM tetrodotoxin. EGTA and verapamil dose-dependently inhibited only the amplitude of spontaneous phasic contractions and not the frequency. Ethanol dose-dependently inhibited both the amplitude and frequency of phasic contractions. The amplitude and frequency of spontaneous phasic contractions were significantly inhibited by protein kinase C and tyrosine kinase inhibitors. However, neither protein kinase C activator nor various phosphatase inhibitors blocked the inhibitory effect of ethanol. Conclusions: Ethanol appears to inhibit spontaneous phasic contractions by a mechanism other than the inhibition of protein kinase C or tyrosine kinase or the inhibition of extracellular Ca 2+ influx.

Mice lacking the dopamine transporter display altered regulation of distal colonic motility.

The mechanisms by which dopamine (DA) influences gastrointestinal (GI) tract motility are incompletely understood and complicated by tissue- and species-specific differences in dopaminergic function. To improve the understanding of DA action on GI motility, we used an organ tissue bath system to characterize motor function of distal colonic smooth muscle segments from wild-type and DA transporter knockout (DAT -/-) mice. In wild-type mice, combined blockade of D(1) and D(2) receptors resulted in significant increases in tone (62 +/- 9%), amplitude of spontaneous phasic contractions (167 +/- 24%), and electric field stimulation (EFS)-induced (40 +/- 8%) contractions, suggesting that endogenous DA is inhibitory to mouse distal colonic motility. The amplitudes of spontaneous phasic and EFS-induced contractions were lower in DAT -/- mice relative to wild-type mice. These differences were eliminated by combined D(1) and D(2) receptor blockade, indicating that the inhibitory effects of DA on distal colonic motility are potentiated in DAT -/- mice. Motility index was decreased but spontaneous phasic contraction frequency was enhanced in DAT -/- mice relative to wild-type mice. The fact that spontaneous phasic and EFS-induced contractile activity were altered by the lack of the DA transporter suggests an important role for endogenous DA in modulating motility of mouse distal colon.