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

Role of Kⴙ Channels in Regulating Spontaneous Activity in Detrusor Smooth Muscle In Situ in the Mouse Bladder Masa Hayase, Hikaru Hashitani,* Kenjiro Kohri and Hikaru Suzuki From the Departments of Nephro-Urology (MH, KK) and Cell Physiology (MH, HH, HS), Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Purpose: We investigated the functional role of K⫹ channels for regulating spontaneous activity in mouse bladder detrusor smooth muscle. Materials and Methods: The effects of different K⫹ channels blockers on spontaneous changes in membrane potential and intracellular Ca2⫹ dynamics were examined using intracellular recording techniques and Ca2⫹ imaging with fluo-4 fluorescence, respectively. Results: Detrusor smooth muscle generated spontaneous action potentials and Ca2⫹ transients. Iberiotoxin (0.1 ␮M), charybdotoxin (0.1 ␮M) or tetraethylammonium (1 mM) increased the amplitude of action potentials and prolonged their repolarizing phase without inhibiting their after-hyperpolarization. Tetraethylammonium (10 mM) but not stromatoxin (0.1 ␮M) suppressed after-hyperpolarization and further increased the amplitude and half duration of action potentials. Apamin (0.1 ␮M) increased the frequency of action potentials but had no effect on their configuration. Spontaneous Ca2⫹ transients were generated in individual detrusor smooth muscle cells and occasionally propagated to neighboring cells to form intercellular Ca2⫹ waves. Transmural nerve stimulations invariably initiated synchronous Ca2⫹ transients within and across muscle bundles. Charybdotoxin (0.1 ␮M) increased the amplitude of spontaneous Ca2⫹ transients, while the subsequent application of tetraethylammonium (10 mM) increased their half duration. In addition, tetraethylammonium increased the synchronicity of Ca2⫹ transients in muscle bundles. Conclusions: These results suggest that large and intermediate conductance Ca2⫹ activated K⫹ channels contribute to action potential repolarization and restrict the excitability of detrusor smooth muscle in the mouse bladder. In addition, the activation of voltage dependent K⫹ channels is involved in repolarization and after-hyperpolarization, and it has a fundamental role in stabilizing detrusor smooth muscle excitability. Key Words: urinary bladder; calcium channels; muscle, smooth; mice, inbred BALB C; muscle contraction DETRUSOR smooth muscle strips from the bladders of many species, including humans, develop spontaneous contractions.1 These contractions occur locally and do not propagate over longer distances to increase intravesical pressure. However, if enhanced spontaneous activity of DSM

occurs during filling, it might generate increases in pressure and result in overactive bladder.2 Spontaneous DSM contractions generally occur upon action potential discharge, which results in Ca2⫹ influx through L-type Ca2⫹ channels and associated Ca2⫹ transients.3 The regener-

0022-5347/09/1815-2355/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 181, 2355-2365, May 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.01.013

Abbreviations and Acronyms AHP ⫽ after-hyperpolarization ATP ⫽ adenosine triphosphate BK ⫽ large conductance Ca2⫹ activated K⫹ CTX ⫽ charybdotoxin DSM ⫽ detrusor smooth muscle IbTX ⫽ iberiotoxin IK ⫽ intermediate conductance Ca2⫹ activated K⫹ Kv ⫽ voltage dependent K⫹ PSS ⫽ physiological salt solution SK ⫽ small conductance Ca2⫹ activated K⫹ STD ⫽ spontaneous transient depolarization STX ⫽ stromatoxin TEA ⫽ tetraethylammonium TTX ⫽ tetrodotoxin Submitted for publication August 6, 2008. Study received ethics committee approval. Supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) 19390418 (HH). * Correspondence: Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan (telephone: 81-52-8538131; FAX: 81-52-8421538; e-mail: [email protected]).

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ative nature of L-type Ca2⫹ channel opening is fundamental in the initiation of the action potential discharge and its intercellular propagation.4 K⫹ channels are particularly important for controlling the excitability of DSM by decreasing the likelihood of L-type Ca2⫹ channel opening. Decreased activation or a decreased number of K⫹ channels would enhance DSM excitability and, therefore, K⫹ channels are a potential target for pharmacological and genetic treatment for bladder overactivity. The mouse bladder is a fruitful model in which to study bladder function in physiology and pathophysiology since various genetically modified mice have become available. Slo⫺/⫺ mice, in which the mSlo1 gene encoding the pore forming subunit of BK channels has been knocked out, show increased urination frequency and voiding pressure, and oscillations in intravesical pressure.5,6 A mouse model that conditionally over expresses the SK channel isoform SK3 has greater bladder capacity, while selective suppression of SK3 expression results in a marked increase in nonvoiding contractions.7 Kv channels

have also been identified in the mouse DSM and they are suggested to contribute to action potential repolarization and AHP because of their slow deactivation characteristics.8 Although there have been extensive investigations of K⫹ channels using DSM myocytes as well as contractile studies of bladder function in normal and genetically modified mice, to our knowledge there is no information on the role of individual K⫹ channel populations in regulating the action potential in situ. We pharmacologically investigated the role of different types of K⫹ channels in action potential configurations. In addition, their role in regulating the initiation and propagation of Ca2⫹ transients was examined by imaging fluo-4 fluorescence.

MATERIALS AND METHODS Tissue Preparation Six to 8-week-old male BALB/c mice were sacrificed by the procedure approved by the Physiological Society of Japan animal experimentation ethics committee. The bladder

Figure 1. Mouse bladder DSM generated spontaneous action potentials and STDs (A). Most action potentials were preceded by slow depolarization (green trace) but often had steeper foot-like depolarization (red trace) (B). ms, milliseconds. Spike-like action potentials (blue trace) were also seen. STDs with varying amplitudes were composed of rapid rising phase and slower falling phase (C). Averaged traces of 72 STDs show that these phases could be fitted by single exponential (blue line) (D).

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Figure 2. In DSM preparations that generated spontaneous action potentials 0.1 ␮M IbTX increased action potential amplitude and frequency (A). Overlaid averaged traces of 25 to 30 action potentials each show IbTX effects on action potential configuration (B). ms, milliseconds. Black line represents control. Red line represents IbTX. In another DSM generating spontaneous action potentials 0.1 ␮M CTX increased action potential frequency and amplitude (C). Overlaid averaged traces of 25 to 30 action potentials each show effects of CTX on action potential configuration (D). Red line represents CTX.

was removed and the outer circular DSM layer was removed, leaving the inner longitudinal DSM layers.

Intracellular Recording DSM sections (3 mm2) were superfused with PSS warmed to 36C. Individual DSM cells in muscle bundles were impaled with glass capillary microelectrodes and membrane potential changes were recorded.3

Intracellular Ca Imaging DSM preparations loaded with fluo-4 (5 ␮M) were illuminated at 495 nm and fluorescence emission above 515 nm was detected.4 The relative amplitude of Ca2⫹ transients is expressed as F/F0, where F represents the fluorescence generated by an event and F0 represents baseline fluorescence.

Effects of K⫹ channel blockers on parameters of spontaneous action potential parameters in mouse bladder DSM Mean ⫾ SD Resting Membrane Potential (mV)

Mean ⫾ SD Frequency (mins⫺1)

Mean ⫾ SD Amplitude (mV)

Mean ⫾ SD Half Duration (msecs)

Mean ⫾ SD R dV/dtmax (mV 䡠 ms⫺1)

Mean ⫾ SD F dV/dtMax (mV 䡠 ms⫺1)

Mean ⫾ SD AHP (mV)

Control 100 nM IbTX

⫺41.8 ⫾ 2.4 ⫺42.2 ⫾ 2.6

4.7 ⫾ 1.6 6.9 ⫾ 1.9*

46.9 ⫾ 4.8 55.1 ⫾ 3.6*

6.9 ⫾ 0.9 9.1 ⫾ 1.7*

10.1 ⫾ 2.7 11.6 ⫾ 2.0*

⫺13.3 ⫾ 3.1 ⫺10.8 ⫾ 1.9*

10.1 ⫾ 2.2 9.1 ⫾ 2.1

Control 100 nM CTX

⫺42.3 ⫾ 1.5 ⫺43.3 ⫾ 1.4

6.3 ⫾ 2.3 8.6 ⫾ 2.1*

47.0 ⫾ 2.2 64.3 ⫾ 4.5*

7.9 ⫾ 1.1 19.7 ⫾ 2.3*

9.5 ⫾ 1.6 11.4 ⫾ 1.3*

⫺11.4 ⫾ 1.7 ⫺7.3 ⫾ 1.6*

8.4 ⫾ 2.7 9.6 ⫾ 2.6

Control 100 nM apamin

⫺42.3 ⫾ 1.8 ⫺42.1 ⫾ 2.5

5.7 ⫾ 2.1 8.3 ⫾ 2.3*

46.1 ⫾ 3.8 45.9 ⫾ 3.2

7.1 ⫾ 1.6 7.4 ⫾ 1.8

9.6 ⫾ 2.4 9.1 ⫾ 3.6

⫺12.4 ⫾ 2.1 ⫺12.7 ⫾ 2.2

12.2 ⫾ 2.0 12.7 ⫾ 2.1

Control 10 mM TEA

⫺42.7 ⫾ 1.8 ⫺45.9 ⫾ 1.9*

7.2 ⫾ 2.1 3.3 ⫾ 1.5*

47.6 ⫾ 3.6 73.6 ⫾ 3.4*

8.8 ⫾ 1.4 53.1 ⫾ 21.6*

10.1 ⫾ 2.3 10.6 ⫾ 1.8*

⫺11.2 ⫾ 2.1 ⫺5.4 ⫾ 1.1*

Control 1 mM TEA

⫺43.0 ⫾ 2.3 ⫺43.6 ⫾ 2.1

5.5 ⫾ 1.2 7.4 ⫾ 1.8*

46.4 ⫾ 3.0 58.4 ⫾ 2.3*

9.6 ⫾ 2.0 13.8 ⫾ 2.1*

9.9 ⫾ 1.4 11.8 ⫾ 0.9*

⫺11.7 ⫾ 1.2 ⫺10.0 ⫾ 0.9*

No. Preparations (blockers) 6:

8:

8:

8: 9.8 ⫾ 1.6 1.7 ⫾ 0.9*

7:

* Significantly different vs control (p ⬍0.05).

11.0 ⫾ 1.3 10.5 ⫾ 1.2

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For transmural nerve stimulation the preparations were stimulated by passing brief pulses of constant current (duration 50 microseconds) between a pair of platinum electrodes. Neural selectivity was confirmed by sensitivity to TTX (1 ␮M).

age of 3 to 5 minutes of recording. The maximum rate of rise or R dV/dtMax and fall or F dV/dtMax was also measured for action potentials, where V represents voltage and t represents time.

Solutions

RESULTS

The composition of PSS was 137.5 mM Na⫹, 4.7 mM K⫹, 2.5 mM Ca2⫹, 1.2 mM Mg2⫹, 15.5 mM HCO3⫺, 1.2 mM H2PO4⫺, 134 Cl⫺ mM and 15 mM glucose. PSS pH was 7.2 to 7.3 when bubbled with 95% O2 and 5% CO2. The drugs used were apamin, CTX, IbTX, ␣,␤ methylene-ATP, atropine, STX, TEA chloride, TTX and nicardipine. Drugs were dissolved in distilled water except nicardipine, which was dissolved in dimethyl sulfoxide.

Calculations and Statistics Measured values are expressed as the mean ⫾ SD. Statistical significance was tested using the paired t test and considered significant at p ⬍0.05. Electrical events were captured and averaged using the template search and averaging functions of Clampfit 10 software (Molecular Devices®). The synchronicity of Ca2⫹ signals between a pair of cells was analyzed using the cross-correlation function of Clampfit 10. The parameters of action potentials and Ca2⫹ transients that were measured were peak amplitude, which was measured as the value from the resting level to the peak of events, half duration, which was measured as the time between 50% peak amplitude on the rising and falling phases, and frequency, which was defined as an aver-

General Observations In 52 preparations DSM cells showed spontaneous action potentials and had a mean ⫾ SD resting membrane potential of ⫺43.5 ⫾ 3.6 mV (fig. 1, A). Action potentials often had an initial depolarizing phase or steeper foot-like depolarization and were called pacemaker type (fig. 1, B).9 Of the action potentials 19 (mean 14.8% ⫾ 10.4%) rose abruptly from resting membrane potential without any preceding depolarization and were called spike type (fig. 1, B).9 They lacked or had much smaller AHPs than pacemaker type action potentials. Resting membrane potential was characterized by the presence of random STDs (fig. 1, A).9 STDs had a mean peak amplitude of 5.9 ⫾ 1.3 mV at a cutoff threshold of 2 mV in 8 preparations (fig. 1, C). Their decay could be fitted by single exponential, giving a time constant of a mean of 49.6 ⫾ 5.4 milliseconds (fig. 1, D). In preparations pretreated with ␣,␤ methylene-ATP (10 ␮M for 30 minutes) STDs were abolished and, thus, they were assumed to

Figure 3. Continuous traces show that apamin increased action potential frequency without changing resting membrane potential (A and B). Overlaid averaged traces of 25 to 30 action potentials each show no effects of apamin on action potential configuration (C). s, seconds. Black line represents control. Red line represents apamin.

K⫹ CHANNELS AND SPONTANEOUS BLADDER DETRUSOR ACTIVITY

be purinergic spontaneous excitatory junction potentials. In contrast, ␣,␤ methylene-ATP pretreatment decreased the action potential discharge to a mean frequency 4.2 ⫾ 1.3 minutes⫺1 vs 6.8 ⫾ 2.1 in controls in 6 preparations each (p ⬍0.05), suggesting that spontaneous action potentials are basically myogenic in origin. Spontaneous Action Potentials Role of BK and IK channels. IbTX (0.1 ␮M), which is a blocker for BK channels, increased action potential amplitude and frequency without changing the membrane potential (fig. 2, A). IbTX increased the amplitude and half duration of action potentials but did not suppress their AHPs (see table and fig. 2, B). TEA (1 mM) had effects on action potentials that were similar to those of IbTX (0.1 ␮M) (see table). CTX (0.1 ␮M), which is a blocker of BK and IK channels, increased action potential amplitude and frequency without changing the membrane potential (fig. 2, C and D). CTX also increased the amplitude and half duration of action potentials but did not suppress AHPs (see table and fig. 2, D). The half duration was increased by a mean of 32.4% ⫾ 17.7% with IbTX in 6 preparations and by 137.6% ⫾ 28.0% with CTX in 8 (unpaired t test p ⬍0.05). In 3 preparations pretreated with atropine (3 ␮M) and ␣,␤

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methylene ATP (10 ␮M) for 30 minutes CTX caused effects on action potentials similar to those in the absence of neurotransmitter blockers. Role of SK channels. Regardless of the presence or absence of TTX (1 ␮M) in 4 preparations each apamin (0.1 ␮M), which is a blocker of SK channels, increased action potential frequency without changing the membrane potential (fig. 3, A and B). However, it did not change the action potential configuration (see table and fig. 3, C). Role of Kv channels. TEA (10 mM) initially increased action potential frequency with or without transient depolarization (fig. 4, A). Eventually it decreased their frequency with a small hyperpolarization (fig. 4, B). With TEA (10 mM) action potentials had an increased amplitude and half duration, and AHPs were strongly suppressed (see table and fig. 4, C). The effects of TEA (10 mM) were not affected by pretreatment with ␣,␤ methylene-ATP (10 ␮M) and atropine (3 ␮M) in 3 preparations each or with TTX (1 ␮M) in 2. Since TEA blocks not only Kv channels, but also BK channels, the selectivity of the effects of TEA (10 mM) on Kv channels was assessed in preparations exposed to ␣,␤ methylene-ATP (10 ␮M) and atropine (3 ␮M) for 30 minutes. In BK channel blocked prep-

Figure 4. In DSM generated spontaneous action potentials continuous trace demonstrates that 10 mM TEA increased action potential frequency and amplitude, and caused transient depolarization (A). Continuous trace shows that TEA subsequently decreased action potential frequency and hyperpolarized membrane (B). Dotted lines indicate resting membrane potentials. Overlaid averaged traces of 25 to 30 action potentials each show TEA effects on action potential configuration (C). s, seconds. Black line represents control. Red line represents TEA.

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arations TEA (10 mM) evoked massive contractions, such that the maintenance of microelectrode impalement was not possible. Therefore, 2 preparations each were first exposed to TEA with IbTX (0.1 ␮M) or CTX (0.1 ␮M) for 20 minutes before impalement and the response of tissues was examined upon removing TEA. Upon removal action potential amplitude was decreased and AHP was restored in the continuous presence of CTX or IbTX (fig. 5, A and B). STX (0.1 ␮M), which is a blocker of Kv2.1 channels, showed no additional effect on action potentials in 3 preparations treated with CTX (0.1 ␮M) for greater than 20 minutes (fig. 5, C and D). Spontaneous and Nerve Evoked Changes in Intracellular Ca2ⴙ DSM demonstrated spontaneous Ca2⫹ transients that remained within individual cells and seldom propagated to neighboring cells to create propagating Ca2⫹ waves (fig. 6, A and B).10 As a consequence, cross-correlation factors of Ca2⫹ transients in neighboring cells did not produce a prominent peak near lag period zero, indicating a low temporal correlation among cells in a muscle bundle (fig. 6, C). Single transmural nerve stimulation initiated synchronous Ca2⫹ transients within and across

muscle bundles where previously asynchronous spontaneous Ca2⫹ transients were generated (fig. 7), suggesting that neuroeffector transmission is an absolute requirement for synchronizing DSM in the mouse bladder. Effects of CTX and TEA on Spontaneous Ca2ⴙ Transients Experiments were also done in preparations exposed to ␣,␤ methylene-ATP (10 ␮M), atropine (3 ␮M) and TTX (1 ␮M) for 30 minutes. All Ca2⫹ signals under these conditions were abolished by nicardipine (1 ␮M) in 4 preparations each. Compared to control values CTX (0.1 ␮M) increased Ca2⫹ transient amplitude and frequency in 8 preparations each (6.6 ⫾ 1.2 vs 8.5 ⫾ 1.8 minutes-1, p ⬍0.05). It also increased their half duration (fig. 8, A and C). Subsequent TEA (10 mM) addition caused a transient increases in baseline Ca2⫹ that was associated with a summation of Ca2⫹ transients (fig. 8, B). Increased Ca2⫹ levels gradually returned to baseline and the frequency of Ca2⫹ transients was also decreased (mean 4.6 ⫾ 1.4 minutes⫺1 in 9 CTX plus TEA preparations). Ca2⫹ transients in TEA preparations had a larger amplitude and much longer duration that those in the presence of CTX alone (figs. 8, C and 9).

Figure 5. In DSM treated with 10 mM TEA and 0.1 ␮M IbTX large action potentials lacking AHPs were generated (A). Upon removal of 10 mM TEA action potential amplitude was decreased and AHPs were restored. Dotted line indicates resting membrane potential. Overlaid averaged traces of 25 to 30 action potentials each demonstrate additional effects of TEA on action potential configuration (B). Black line represents IbTX. Red line represents IbTX plus TEA. In DSM treated with 0.1 ␮M CTX addition of 0.1 ␮M STX had no effect on spontaneous action potential frequency (C). Averaged traces of 25 to 30 action potentials each reveal that STX (red trace) did not have any additional effects on action potential configuration of CTX (black trace) treated preparations (D). ms, milliseconds.

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Figure 6. Asynchronous spontaneous Ca2⫹ transients were generated in 4 cells (1 to 4) in DSM bundle (A). s, seconds. Series of frames at 0.1-second intervals demonstrate Ca2⫹ transient generated in cell 3 that remained within cell and did not propagate to neighboring cells (B). Cross-correlation of 4 cells in muscle bundle did not reveal any peak near lag period zero (C).

In addition to the reinforcement of individual Ca2⫹ transients, TEA (10 mM) increased synchronicity in muscle bundles (fig. 10, A and C). Thus, it increased cross-correlation factors to form a prominent peak near lag period zero, suggesting close

temporal correlation among the cells in 8 muscle bundles (fig. 10, B and D). However, in another 3 muscle bundles TEA did not improve synchronicity among cells. Thus, properties of gap junction channels may dominantly determine cell-to-cell coupling.

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K⫹ CHANNELS AND SPONTANEOUS BLADDER DETRUSOR ACTIVITY

Figure 7. Single transmural nerve stimulations (triple arrowheads) initiated synchronous Ca2⫹ transients in 3 cells (1 to 3) in muscle bundle and train of stimuli (arrowhead) at 10 Hz and 1 second (s) evoked larger synchronous Ca2⫹ transients (A). Series of frames at 0.1-second intervals reveal nerve evoked Ca2⫹ transient generated in DSM bundle (B). Single electrical stimuli (triple arrowheads) initiated synchronous Ca2⫹ transients in 3 muscle bundles (1 to 3), while trains of stimuli (arrowhead) at 10 Hz and 1 second evoked larger, prolonged Ca2⫹ transients (C). Bar represents 10 Hz and 5 seconds. Series of frames at 0.1-second intervals demonstrate nerve evoked Ca2⫹ transient generated in DSM bundles (D).

DISCUSSION BK channels have a dominant role in regulating spontaneous action potentials in the DSM of many species, including humans.11 In the mouse bladder blockade of these channels with IbTX, or decreased expression of their pore forming ␣-subunit5,6 or regulatory ␤1-subunit12 results in increased contractility of DSM strips in vitro and bladder overactivity in

vivo. In guinea pig bladder DSM IbTX and CTX increased the amplitude and half duration of action potentials, and suppressed AHPs.13 However, in the current study CTX caused significantly more pronounced effects on action potential duration than IbTX, suggesting that BK and IK channels contribute to action potential repolarization. IK channels have been identified as the SK4 subtype on reverse

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Figure 8. CTX (0.1 ␮M) increased amplitude and frequency of spontaneous Ca2⫹ transients (A). Subsequent application of 10 mM TEA caused transient increase in baseline Ca2⫹ that was associated with dramatic increase in Ca2⫹ transient frequency (B). s, seconds. Averaged traces of 15 to 20 Ca2⫹ transients each reveal that during continuous exposure to CTX plus TEA baseline Ca2⫹ returned to original level and Ca2⫹ transient frequency was decreased (C). CTX increased Ca2⫹ transient amplitude (red trace) and TEA dominantly increased their half duration (blue trace).

transcriptase-polymerase chain reaction.14 Nevertheless, each blocker failed to suppress AHPs, as did CTX in the DSM of human and pig bladders.11 SK2 and SK3 isoforms have been shown to regulate the excitability of mouse bladder DSM.7,15 In the guinea pig bladder apamin converted individual

Figure 9. Averaged traces of 15 to 20 Ca2⫹ transients each reveal that 0.1 ␮M CTX increased Ca2⫹ transient amplitude of action potentials (A) and TEA dominantly increased their half duration (B). Asterisk indicate p ⬍0.05 vs control. Pound sign indicates p ⬍0.05 vs CTX.

action potentials into bursts.13 In human and pig bladders SK channels have a more dominant role in regulating DSM excitability, such that apamin abolishes the fast AHP, muscarinic inhibitory junction potentials and the clustering of action potential discharge.11 In the current experiments apamin increased action potential frequency but failed to alter the action potential configuration, although it has previously been shown to enhance spontaneous contractions in DSM strips.15 SK3 channels are expressed not only in DSM cells, but also in urothelium, which is now considered an important source of biologically active substances.7 In addition, SK3 was reported to be expressed on interstitial cells of Cajal in the gastrointestinal tract.16 Although the physiological significance of interstitial cells of Cajal-like cells in bladder function is not yet well established,17,18 the effects of apamin on nonsmooth muscle cells that may alter the excitability of DSM should be considered. A striking result in the current study is the significant effects of the blockade of Kv channels with high concentrations of TEA. In the human bladder a blockade of Kv1 channels by 3,4-diaminopyridine

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Figure 10. In preparations exposed to 0.1 ␮M CTX for 20 minutes asynchronous Ca2⫹ transients were generated in 2 DSM cells in bundle (A). Cross-correlogram of this pair of cells does not show peak near lag period zero (B). s, seconds. Subsequently 10 mM TEA increased Ca2⫹ transient synchronicity between 2 cells (C). Cross-correlogram of this pair of cells shows peak near lag period zero (D).

results in an increased amplitude of spontaneous contractions.19 On the other hand, Kv2.1 channels have been shown to be a dominant channel forming isoform in the mouse bladder and the lack of Kv current inhibition by 4-aminophenol suggests that co-assembly of the Kv 5 or Kv 6 family alters the original properties of Kv2.1 channels.8 The failure of STX to alter action potential configurations in BK channel blocked preparations may also be the case. Thus, further investigation of the heteromultimetric expression of Kv channels may well be a fruitful avenue in the development of selective pharmacological and genetic therapies for bladder overactivity. DSM generally has less negative membrane potentials than other smooth muscles, presumably because of background inward currents20 as well as relatively smaller sustained potassium currents. Therefore, the inhibition of inward currents, for example by increasing intracellular Ca⫹, may account for TEA induced hyperpolarization, if any. In the guinea pig bladder spontaneous action potentials and Ca2⫹ transients readily propagate to neighboring cells through gap junctions to form intercellular Ca2⫹ waves in DSM bundles.4 In the

current study spontaneous Ca2⫹ transients seldom propagated into neighboring cells. However, transmural nerve stimulation invariably initiated synchronous Ca2⫹ transients not only within, but also across DSM bundles. Thus, there must be some mechanism(s) that prevent the propagation of spontaneous activity. Since the propagation of spontaneous activity requires the transmission of regenerative action potentials across gap junctions,4 TEA induced increases in the synchronicity of Ca2⫹ transients suggest that the activation of Kv channels may decrease L-type Ca2⫹ channel opening. Increased synchronicity would result in enhanced contractile activity. Thus, Kv channels may have a critical role, not only in the action potential configuration, but also in cell-to-cell coupling.

CONCLUSIONS In the mouse bladder DSM, BK and IK channels contribute to action potential repolarization and regulate the amplitude of Ca2⫹ transients. Kv channels may have a critical role in regulating action potential repolarization and AHP, and they dominantly

Role of Kⴙ Channels in Regulating Spontaneous Activity in Detrusor Smooth Muscle In Situ in the Mouse Bladder Masa Hayase, Hikaru Hashitani,* Kenjiro Kohri and Hikaru Suzuki From the Departments of Nephro-Urology (MH, KK) and Cell Physiology (MH, HH, HS), Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Purpose: We investigated the functional role of K⫹ channels for regulating spontaneous activity in mouse bladder detrusor smooth muscle. Materials and Methods: The effects of different K⫹ channels blockers on spontaneous changes in membrane potential and intracellular Ca2⫹ dynamics were examined using intracellular recording techniques and Ca2⫹ imaging with fluo-4 fluorescence, respectively. Results: Detrusor smooth muscle generated spontaneous action potentials and Ca2⫹ transients. Iberiotoxin (0.1 ␮M), charybdotoxin (0.1 ␮M) or tetraethylammonium (1 mM) increased the amplitude of action potentials and prolonged their repolarizing phase without inhibiting their after-hyperpolarization. Tetraethylammonium (10 mM) but not stromatoxin (0.1 ␮M) suppressed after-hyperpolarization and further increased the amplitude and half duration of action potentials. Apamin (0.1 ␮M) increased the frequency of action potentials but had no effect on their configuration. Spontaneous Ca2⫹ transients were generated in individual detrusor smooth muscle cells and occasionally propagated to neighboring cells to form intercellular Ca2⫹ waves. Transmural nerve stimulations invariably initiated synchronous Ca2⫹ transients within and across muscle bundles. Charybdotoxin (0.1 ␮M) increased the amplitude of spontaneous Ca2⫹ transients, while the subsequent application of tetraethylammonium (10 mM) increased their half duration. In addition, tetraethylammonium increased the synchronicity of Ca2⫹ transients in muscle bundles. Conclusions: These results suggest that large and intermediate conductance Ca2⫹ activated K⫹ channels contribute to action potential repolarization and restrict the excitability of detrusor smooth muscle in the mouse bladder. In addition, the activation of voltage dependent K⫹ channels is involved in repolarization and after-hyperpolarization, and it has a fundamental role in stabilizing detrusor smooth muscle excitability. Key Words: urinary bladder; calcium channels; muscle, smooth; mice, inbred BALB C; muscle contraction DETRUSOR smooth muscle strips from the bladders of many species, including humans, develop spontaneous contractions.1 These contractions occur locally and do not propagate over longer distances to increase intravesical pressure. However, if enhanced spontaneous activity of DSM

occurs during filling, it might generate increases in pressure and result in overactive bladder.2 Spontaneous DSM contractions generally occur upon action potential discharge, which results in Ca2⫹ influx through L-type Ca2⫹ channels and associated Ca2⫹ transients.3 The regener-

0022-5347/09/1815-2355/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 181, 2355-2365, May 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.01.013

Abbreviations and Acronyms AHP ⫽ after-hyperpolarization ATP ⫽ adenosine triphosphate BK ⫽ large conductance Ca2⫹ activated K⫹ CTX ⫽ charybdotoxin DSM ⫽ detrusor smooth muscle IbTX ⫽ iberiotoxin IK ⫽ intermediate conductance Ca2⫹ activated K⫹ Kv ⫽ voltage dependent K⫹ PSS ⫽ physiological salt solution SK ⫽ small conductance Ca2⫹ activated K⫹ STD ⫽ spontaneous transient depolarization STX ⫽ stromatoxin TEA ⫽ tetraethylammonium TTX ⫽ tetrodotoxin Submitted for publication August 6, 2008. Study received ethics committee approval. Supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) 19390418 (HH). * Correspondence: Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan (telephone: 81-52-8538131; FAX: 81-52-8421538; e-mail: [email protected]).

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