When the urinary bladder is full, activation of parasympathetic nerves causes release of neurotransmitters that induce forceful contraction of the detrusor muscle, leading to urine voiding. The roles of ion channels that regulate contractility of urinary bladder smooth muscle (UBSM) in response to activation of parasympathetic nerves are not well known. The present study was designed to characterize the role of large (BK)- and small-conductance (SK) Ca2+-activated K+ (KCa) channels in regulating UBSM contractility in response to physiological levels of nerve stimulation in UBSM strips from mice. Nerve-evoked contractions were induced by electric field stimulation (0.5–50 Hz) in isolated strips of UBSM. BK and SK channel inhibition substantially increased the amplitude of nerve-evoked contractions up to 2.45 ± 0.12- and 2.99 ± 0.25-fold, respectively. When both SK and BK channels were inhibited, the combined response was additive. Inhibition of L-type voltage-dependent Ca2+ channels (VDCCs) in UBSM inhibited nerve-evoked contractions by 92.3 ± 2.0%. These results suggest that SK and BK channels are part of two distinct negative feedback pathways that limit UBSM contractility in response to nerve stimulation by modulating the activity of VDCCs. Dysfunctional regulation of UBSM contractility by alterations in BK/SK channel expression or function may underlie pathologies such as overactive bladder.
- potassium channel
- large- and small-conductance channels
initiation of urinary bladder smooth muscle (UBSM) contraction by the parasympathetic nervous system is the primary event that triggers voiding of urine under normal circumstances. Upon activation, parasympathetic nerves release the transmitters acetylcholine (ACh) and ATP, which bind to M3 and P2X1 receptors, respectively, to cause bladder smooth muscle contraction (23, 31). In rodents, P2X receptors play a significant role in nerve-initiated urinary bladder contraction (4). In humans, muscarinic ACh receptors (mAChR) may be the more prominent receptors involved in nerve-mediated initiation of micturition (2), whereas an increase in P2X receptors may become significant during pathological conditions such as idiopathic detrusor instability (25).
Small (SK)- and large-conductance (BK) Ca2+-activated K+ (KCa) channels as well as voltage-dependent K+ (KV) channels play important roles to regulate UBSM excitability and contractility in animals and humans (8, 9, 16–20, 26, 28, 30). BK channels mediate action potential repolarization (16), whereas SK and KV channels mediate action potential afterhyperpolarization (8, 9, 30). Given the fundamental importance of nerve-mediated contractions in normal physiological and pathological conditions, we sought to investigate how BK and SK channels regulate contractility of UBSM in response to nerve stimulation. Because there are a number of transgenic mouse models available with relevance to bladder function (1, 20, 26, 32), we performed the present studies in bladder strips isolated from mice.
We found that BK and SK channels are important mediators of bladder contractility in response to nerve stimulation. BK and SK channels comprise two distinct components of negative feedback pathways that limit UBSM contractility in response to nerve stimulation. Pharmacological blockade of BK channels with iberiotoxin and SK channels with apamin resulted in substantial potentiation of nerve-evoked contractions induced by electric field stimulation (EFS) in isolated mouse UBSM strips. Inhibition of voltage-dependent Ca2+ channels (VDCCs) abolished nerve-evoked contractions. The effects of iberiotoxin and apamin were additive, suggesting that these KCa channels act differentially to regulate UBSM excitability and contractility by modulating Ca2+ influx through VDCCs.
All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Vermont. Male C57Bl6 mice were used for all experiments. Mice were euthanized by intraperitoneal injection of euthanasia solution (Sleepaway; Fort Dodge Animal Health), followed by a thoracotomy or decapitation. After euthanasia, urinary bladders were removed and placed in ice-cold dissection solution composed of (in mM) 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 HEPES, and 2 MgCl2, with pH adjusted to 7.3 with NaOH.
The bladder was cut open to expose the urothelial surface and rinsed several times with dissection saline to remove residual traces of urine. The urothelial layer was dissected away from the smooth muscle layer and discarded. Small strips of detrusor (2–3 mm wide and 5–7 mm long) were cut from the bladder wall. Silk threads were attached to each end of the strips, and the strips were transferred to cold (4°C) physiological saline solution (PSS) containing (in mM) 119 NaCl, 4.7 KCl, 24 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, and 11 glucose and aerated with 95% O2-5% CO2 to obtain pH 7.4.
Each strip was mounted in a tissue bath (7-ml volume) containing aerated PSS (95% O2-5% CO2, 37°C; MyoMED myograph system; MED Associates, Georgia, VT). An initial load of 10–15 mN was applied to the bladder strips, and strips were equilibrated for 45 min with bath solution exchanges every 15 min. After the equilibration period, strips were contracted with three successive 5-min applications of 43 mM KCl, each separated by 15 min in PSS. The final application of 43 mM KCl was followed by 30 min of PSS before nerve-mediated bladder contractions were elicited with EFS (see Figs. 1–5) using a pair of electrodes parallel to the muscle strip mounted in the tissue bath. Frequency-response curves were constructed by measuring the EFS-induced contraction amplitude at stimulus frequencies of 0.5, 2, 3.5, 5, 7.5, 10, 12.5, 15, 20, 30, 40, and 50 Hz. Pulse amplitude was 20 V, and polarity was reversed for alternating pulses. Pulse width was 0.2 ms, and stimulus duration was 2 s. Stimuli were given every 3 min using a model PHM-152V stimulator (MED Associates). At the completion of the first frequency-response curve, bladder strips were washed with fresh bath solution three times, followed by a single wash 5 min later. Twenty minutes after the final wash, various pharmacological compounds were added directly to the tissue bath. Strips were incubated in the presence of test compounds for 15 min, and then a second frequency-response curve was generated using the same EFS parameters as before.
In a second set of experiments, the bladder strips were stimulated with carbachol (CCh; 30, 100, 300, and 1,000 nM) added in a cumulative fashion to the tissue bath. These experiments were all performed in the presence of tetrodotoxin (TTX; 1 μM). After the first set of CCh treatments (30 nM for 7 min, 100 nM for 10 min, 300 nM for 15 min, and 1,000 nM for 15 min) was completed, the tissue was washed two times, separated by 5 min. TTX was then added again. After 10 min, K+ channel blockers were added directly to the tissue bath, and after an incubation period of 10 min, CCh was applied as described above. Data from these CCh experiments are shown in Fig. 6. Another set of CCh experiments was performed essentially as described, but the 43 mM KCl step was omitted and a wider range of CCh concentrations was used (in nM) as follows: 1, 10, 100, 1,000, and 10,000. These experiments were used for preparing dose-response curves. CCh dose-response curves were fit to a Boltzmann function to determine the CCh concentration required for half-maximal effect (EC50). EC50 values for each condition were compared using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test for multiple comparisons.
All drugs and reagents were obtained from Sigma except for carbachol, which was obtained from Acros. EFS-induced contractions were analyzed using the MiniAnalysis program (Synaptosoft; www.synaptosoft.com). For statistical comparisons, we used P < 0.05 as a threshold for rejection of the null hypothesis. Frequency-response curves were compared between various groups with a two-way ANOVA or two-way repeated-measures ANOVA, where appropriate. The Student-Newman-Keuls test was used for all multiple comparisons. Normalized frequency-response curves were fit with the Boltzmann equation, and curves between groups were compared using an F-test. CCh-induced mean force was analyzed using MyoViewer software (Med Associates), and CCh-induced phasic contractions were analyzed using MiniAnalysis (Synaptosoft). Statistical analysis was performed with Prism 4.0 (GraphPad Software, San Diego, CA; www.graphpad.com). For all comparisons, two-way ANOVA with Bonferroni post test was performed. Summary data for both sets of experiments are presented as means ± SE.
EFS-induced contractions in mouse UBSM.
UBSM contractions were characterized in response to increasing frequencies of transmural nerve stimulation. The stimulation parameters were chosen to elicit UBSM contractions that were abolished by the Na+ channel inhibitor TTX (Fig. 1). TTX sensitivity indicates the neuronal origin underlying EFS-induced contractions. In the present study, regulation of EFS-induced contractions by KCa channels was assessed by performing a frequency-response curve in each UBSM strip under control conditions and then again in the presence of the BK channel blocker iberiotoxin, the SK channel blocker apamin, or iberiotoxin and apamin combined.
To determine the sensitivity of mouse UBSM strips to repetitive EFS episodes, we performed control experiments in a set of strips in which two successive frequency-response curves were obtained from each strip without the addition of any pharmacological agents. EFS-induced contraction amplitudes increased slightly during the second frequency-response curve compared with the first (P < 0.001 at all frequencies; Fig. 2). The most substantial fractional increase in contraction amplitude during the second frequency response relative to the first frequency response was observed at a stimulation frequency of 0.5 Hz. The amplitude of the 0.5-Hz-induced contraction during the second frequency-response curve was 1.60 ± 0.05-fold greater than the corresponding contraction during the first frequency-response curve (n = 12; Fig. 2C). At stimulation frequencies >10 Hz, EFS-induced contractions were less than 10% greater than the corresponding contractions during the first frequency response (Fig. 2C). The EFS-induced contraction amplitude at the highest stimulation frequency tested (50 Hz) was increased by only 3.3 ± 0.7% during the second frequency response compared with the first (n = 12; Fig. 2C). These increases in EFS-induced contraction amplitudes at each frequency during the second frequency-response curve serve as the baseline from which to compare KCa channel inhibitor-induced effects.
Potentiation of EFS contractions following BK channel inhibition.
BK channels have been shown to play a major role in determining UBSM excitability by regulating repolarization of the action potential (16). In UBSM strips exhibiting spontaneous phasic contractions, blocking BK channels causes a dramatic increase in spontaneous phasic contractility (5, 17, 29). To assess the role of BK channels in nerve-mediated UBSM contractions, we performed a frequency-response curve in mouse UBSM strips, followed by a second frequency-response curve in the presence of the BK channel blocker iberiotoxin (70 nM; Fig. 3).
Iberiotoxin potentiated the amplitude of EFS-induced contractions at all frequencies of nerve stimulation (Fig. 3B). The largest fractional change in EFS-induced contraction amplitude following iberiotoxin treatment was a 2.45 ± 0.12-fold increase at 0.5 Hz. Iberiotoxin-induced increases in EFS-induced contraction amplitudes (Fig. 3C) were significantly greater than those seen in time control experiments (Fig. 2C).
Blocking BK channels also increased the sensitivity of UBSM strips to EFS. EFS-induced contraction amplitudes under control conditions and in the presence of iberiotoxin were normalized to the maximum response during each frequency-response curve. The normalized data were fit with a Boltzmann equation to determine the frequency that yielded a response 50% of that of the maximal response (F50). The F50 under control conditions was 9.0 ± 0.2 Hz (n = 10). Iberiotoxin significantly decreased the F50 to 5.6 ± 0.1 Hz (P < 0.0001; n = 10).
Potentiation of EFS contractions following SK channel inhibition.
SK channels regulate UBSM excitability by contributing to the action potential afterhyperpolarization (8). Blocking SK channels with apamin increases the amplitude of spontaneous phasic UBSM contractions (5, 17, 19). However, the role of SK channels in modulating contractility of UBSM in response to nerve stimulation is not known. To assess the role of SK channels in nerve-mediated UBSM contractions, we performed a frequency-response curve in mouse UBSM strips before and after application of the SK channel inhibitor apamin (230 nM; Fig. 4).
Blocking SK channels with apamin increased the amplitude of EFS-induced contractions at all stimulus frequencies (Fig. 4B). The largest fractional change in EFS-induced contraction amplitude following apamin-treatment was a 2.99 ± 0.25-fold increase over control at 0.5 Hz (Fig. 4C). Apamin-induced increases in EFS-induced contraction amplitudes were significantly greater (Fig. 4C) than those seen in time control experiments (Fig. 2C).
As with BK channel inhibition, blocking SK channels also increased the sensitivity of UBSM strips to EFS. F50 values were determined by fitting the normalized frequency-response curves to a Boltzmann equation in the presence and absence of apamin. Under control conditions, the F50 was 9.5 ± 0.1 Hz (n = 9). Apamin significantly decreased the F50 to 6.6 ± 0.2 Hz (P < 0.0001; n = 9).
Potentiation of EFS contractions following combined BK and SK channel inhibition.
BK and SK channels have significantly different Ca2+ concentration and voltage sensitivities and regulate different aspects of UBSM excitability. Therefore, we predicted that blocking both BK and SK channels would have a greater effect than blocking either BK or SK channel alone. To test this hypothesis, we applied apamin and iberiotoxin simultaneously and examined the effects on EFS-induced contractions (Fig. 5).
Combined BK/SK channel inhibition dramatically increased EFS-induced contraction amplitudes at all frequencies (P < 0.05; Fig. 5B). This increase was significantly greater than the responses to iberiotoxin or apamin alone (P < 0.001; 2-way ANOVA). At a stimulus frequency of 0.5 Hz, combined SK/BK channel inhibition increased the EFS-induced contraction amplitude by 4.54 ± 1.21-fold that of control (n = 8; Fig. 5C).
Combined BK/SK channel inhibition also increased the sensitivity of UBSM strips to nerve stimulation. The BK and SK channel inhibitors did not change baseline force. Fitting the normalized frequency-response curves in the presence and absence of iberiotoxin/apamin showed that the F50 shifted to the left from 10.3 ± 0.2 Hz under control conditions to 4.4 ± 0.3 Hz in the presence of the BK/SK channel inhibitors (n = 8; P < 0.0001). This increase in sensitivity was observed as a leftward shift in the F50 by 5.9 Hz, which was greater than the effect of iberiotoxin or apamin alone. Thus, in terms of regulating EFS-induced UBSM contractions, BK and SK channels have different roles, as expected, in regulating membrane excitability.
Potentiation of CCh-induced contractions following combined BK and SK channel inhibition.
BK and SK channels in UBSM regulate its excitability and contractility. Although it is not known whether BK or SK channels exist in parasympathetic nerves in the urinary bladder, it is conceivable that effects of BK and SK channel inhibition reflect solely actions on nerves. To determine whether the potentiation of EFS-induced contractions following BK and SK channel inhibition is due to effects of the K+ channel blockers on nerves or UBSM, we stimulated the tissue directly with CCh (30, 100, 300, and 1,000 nM) in the presence of TTX (70 nM) to inhibit neuronal activity by blocking voltage-dependent Na+ channels. After the first set of stimulation by CCh (control), a second set of CCh stimulation (treatment) was performed in the presence of apamin, iberiotoxin, or apamin and iberiotoxin (Fig. 6A). Two sets of CCh stimulation without the addition of pharmacological agents but in the presence of TTX were performed as a time control.
Inhibition of BK or SK channels increased the sensitivity of UBSM to CCh. The CCh concentration EC50 was 527 ± 27 nM under control conditions (n = 6 strips from 3 animals). Inhibition of BK channels with iberiotoxin (70 nM) lowered the CCh EC50 to 361 ± 15 nM (P < 0.05 vs. control; n = 6 strips from 3 animals). SK channel inhibition with apamin (230 nM) caused a similar leftward shift in the CCh EC50 to 288 ± 22 nM (P < 0.05 vs. control; n = 6 strips from 3 animals). Similar to the effect on EFS-induced contractions, combined application of apamin and iberiotoxin potentiated the effect on CCh-induced contractions to a greater degree than either toxin applied alone. In the presence of apamin and iberiotoxin, the CCh EC50 was 182 ± 22 nM (P < 0.05 vs. control, apamin alone, and iberiotoxin alone; n = 6 strips from 3 animals).
Combined application of apamin and iberiotoxin also increased the mean force of CCh-induced contractions compared with the corresponding control (Fig. 6B). The difference between apamin/iberiotoxin treatment and the time control is significant for concentrations of CCh of 30, 100, and 300 nM with P < 0.001 (n = 6 strips from 3 animals; Fig. 6B). Combined inhibition of BK and SK channels also increased the amplitude of CCh-induced phasic contractions. The data are expressed as percentages of the corresponding control. The difference between apamin/iberiotoxin treatment and the time control is significant with P < 0.01 for concentrations of CCh of 100 and 300 nM (n = 6 strips from 3 animals; Fig. 6C). Although it is possible that BK and SK channel inhibitors increase neuronal excitability, resulting in increased neurotransmitter release during EFS-induced contractions, these results suggest that a portion of the effect is due to direct actions of the K+ channel inhibitors on UBSM.
Inhibition of EFS-induced contractions by blocking VDCCs.
Potentiation of EFS-induced contractions by blocking BK or SK channels indicates that smooth muscle excitability is involved in nerve-evoked contractions. Blocking K+ channels in UBSM increases contractility by elevating Ca2+ entry through VDCCs. Therefore, the effect of an L-type VDCC inhibitor, diltiazem, on EFS-induced contractions was tested. Nerve-evoked contractions were elicited by stimulating UBSM strips with 2-s pulses (20 Hz) every 30 s (Fig. 7). The VDCC antagonist diltiazem (70 μM) was applied to the tissue upon development of stable EFS-induced contractions. Under control conditions, EFS-induced contraction amplitude was 10.8 ± 1.3 mN and contraction area was 32.8 ± 3.5 mN·s (n = 7). Diltiazem dramatically inhibited EFS induced contractions (Fig. 7B). Contraction amplitude and area were inhibited by 92.3 ± 2.0% and 96.7 ± 0.8%, respectively (n = 7), 20 min after diltiazem application. As well as inhibiting VDCCs, diltiazem at micromolar concentrations previously has been shown to inhibit K+ channels (10, 11). Inhibition of K+ channels, however, would increase UBSM contractility. The potent inhibition of UBSM contractions by diltiazem suggests a central role of Ca2+ entry through VDCCs in mediating UBSM contractility and provides a mechanism by which elevating UBSM excitability with K+ channel blockers has a dramatic impact on nerve-evoked contractility.
Regulation of phasic contractility by BK and SK channels in UBSM.
Previous studies from our laboratory and other laboratories have shown an important role for BK and SK channels in regulating phasic contractions in UBSM (5, 17, 29). In UBSM, phasic contractions are mediated by Ca2+ entry through VDCCs during action potentials (3, 8, 13). In guinea pig UBSM, blocking BK channels increases the amplitude and prolongs the action potential (16, 12). Blocking BK channels in guinea pig UBSM strips substantially increases contractions (17, 12). In guinea pig UBSM strips, blocking SK channels increases the amplitude and duration of phasic contractions (17). In UBSM strips from mice, either SK (20) or BK (26) channel inhibition increases the amplitude of phasic contractions. Other types of K+ channels (e.g., KV channels) also play important roles in the regulation of the UBSM action potential (30).
Negative feedback regulation of nerve-evoked UBSM contractions by BK and SK channels.
In guinea pig and rabbit, parasympathetic nerve-mediated UBSM contractions are thought to be triggered by ACh and ATP (14, 15, 21). P2X1 receptors have been shown to be responsible for ATP-induced excitation-contraction coupling in UBSM of mice (31). M3 muscarinic receptors also are responsible for ACh-induced contractions in UBSM in mice (23). Because neurally released ATP and ACh increase UBSM cell excitability (14), it is likely that pharmacological modulation of UBSM excitability would influence contractility of UBSM in response to nerve stimulation. Although our results are consistent with the observed effects resulting from inhibition of BK and SK channels in the UBSM (Fig. 6), we cannot exclude a participation from blocking BK or SK channels in parasympathetic nerves in UBSM strips if they are present. SK channels have been shown to be expressed at the neuromuscular junction (27), and BK channels have been shown to be expressed in chick choroids and ciliary neurons of the ciliary ganglion (6).
Our results suggest that BK and SK channels form part of a negative feedback loop to limit UBSM excitability and contractility in response to nerve stimulation (Fig. 8). Combined blockade of BK and SK channels with iberiotoxin and apamin, respectively, resulted in an increase in the amplitude of nerve-evoked contractions at stimulation frequencies ranging from 0.5 to 50 Hz. We observed a potentiation in the EFS-induced contractions under control conditions, most prominent at low stimulus frequencies (Fig. 2C). The mechanistic basis for this phenomenon is unclear, but it is possible that the K+ channel blockers potentiate this process of facilitation.
The additive nature of the response to the combined inhibition of BK and SK channels suggests that these channels act differentially to regulate UBSM excitability and contractility in response to nerve stimulation (Fig. 8). In this way, ACh and ATP released from parasympathetic nerve varicosities would trigger UBSM contraction, in part, by causing depolarization that would enhance Ca2+ entry through VDCCs (Fig. 8). Because BK channels are Ca2+ and voltage dependent (7, 24), both the rise in intracellular Ca2+ concentration and the membrane potential depolarization would trigger an increase in BK channel activity, which would limit excitability (Fig. 8). Indeed, activation of BK channels shortens the duration of the UBSM action potential (16). SK channels, which are Ca2+ sensitive and voltage insensitive (22), would be activated solely by the rise in cytosolic Ca2+ levels (Fig. 8) and contribute to the action potential afterhyperpolarization. Furthermore, suppression of SK3 channels in mice leads to an increase in spontaneous phasic contractions, nonvoiding contractions, and micturition pressure in vivo (20), consistent with the results presented in this study.
Our results suggest that alterations of UBSM excitability have a substantial influence on nerve-mediated contractions by altering Ca2+ entry through VDCCs (Fig. 8). We found that nerve-evoked contractions are dramatically reduced by inhibition of VDCCs (Fig. 7). In support of this idea, suppression of expression of the gene for the UBSM VDCCs (CaV 1.2) leads to a substantial reduction in CCh-induced contractions of UBSM and in micturition (32). The present results support a prominent role of BK and SK channels in the regulation of nerve-evoked contractions of UBSM and suggest that dysfunction of either channel type could contribute to urinary bladder pathologies.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants 5R01 DK-053832 and 1R01 DK-065947 (to M. T. Nelson) and National Institutes of Health Training Grant T32 HL/AR-7944 (to G. M. Herrera).
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- Copyright © 2005 the American Physiological Society