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. 2003 Jun 17;551(Pt 3):893–903. doi: 10.1113/jphysiol.2003.045914

Urinary bladder instability induced by selective suppression of the murine small conductance calcium-activated potassium (SK3) channel

Gerald M Herrera *, Maria J Pozo ‡, Peter Zvara †, Georgi V Petkov *, Chris T Bond §, John P Adelman §, Mark T Nelson *
PMCID: PMC2343290  PMID: 12813145

Abstract

Small conductance, calcium-activated potassium (SK) channels have an important role in determining the excitability and contractility of urinary bladder smooth muscle. Here, the role of the SK isoform SK3 was examined by altering expression levels of the SK3 gene using a mouse model that conditionally overexpresses SK3 channels (SK3T/T). Prominent SK3 immunostaining was found in both the smooth muscle (detrusor) and urothelium layers of the urinary bladder. SK currents were elevated 2.4-fold in isolated myocytes from SK3T/T mice. Selective suppression of SK3 expression by dietary doxycycline (DOX) decreased SK current density in isolated myocytes, increased phasic contractions of isolated urinary bladder smooth muscle strips and exposed high affinity effects of the blocker apamin of the SK isoforms (SK1–3), suggesting an additional participation from SK2 channels. The role of SK3 channels in urinary bladder function was assessed using cystometry in conscious, freely moving mice. The urinary bladders of SK3T/T had significantly greater bladder capacity, and urine output exceeded the infused saline volume. Suppression of SK3 channel expression did not alter filling pressure, threshold pressure or bladder capacity, but micturition pressure was elevated compared to control mice. However, SK3 suppression did eliminate excess urine production and caused a marked increase in non-voiding contractions. The ability to examine bladder function in mice in which SK3 channel expression is selectively altered reveals that these channels have a significant role in the control of non-voiding contractions in vivo. Activation of these channels may be a therapeutic approach for management of non-voiding contractions, a condition which characterizes many types of urinary bladder dysfunctions including urinary incontinence.

Small conductance Ca2+-activated K+ (SK) channels are important determinants of urinary bladder smooth muscle excitability and contractility. Urinary bladder smooth muscle is characterized by spontaneous action potentials that trigger phasic contractions (Creed, 1971; Creed et al. 1983; Brading, 1992; Heppner et al. 1997). SK channels underlie the action potential afterhyperpolarization in urinary bladder smooth muscle (Creed et al. 1983; Fujii et al. 1990) and blocking SK channels with apamin induces a substantial increase in action potential frequency (Creed et al. 1983; Fujii et al. 1990), and in the amplitude of phasic contractions (Zografos et al. 1992; Herrera et al. 2000; Herrera & Nelson, 2002). Recently, SK2 and SK3 channel mRNAs were identified in mouse urinary bladder smooth muscle using RT-PCR (Ohya et al. 2000). Despite the observations that SK channels are expressed in bladder smooth muscle and that they regulate excitability and contractility of this tissue, the role of urinary bladder SK channels in controlling bladder function in vivo is unknown. Furthermore, the role served by spontaneous phasic contractions observed in isolated bladder smooth muscle preparations in determining bladder function in vivo is also not clear.

One proposed role for phasic bladder smooth muscle contractions is that, when integrated over the entire wall of the bladder, they maintain a level of tone that can be relaxed during filling or synchronized via neuronal input to achieve voiding (Brading, 1997; see also Stevens et al. 1999). Furthermore, during pathological conditions such as outflow obstruction, the characteristic frequent bladder contractions that are not associated with bladder emptying (non-voiding contractions) may result from a increased level of spontaneous phasic contractions of the urinary bladder smooth muscle (Igawa et al. 1992; Brading, 1997). In this way, compounds designed to decrease the excitability and contractility of urinary bladder smooth muscle directly may be beneficial in treating bladder overactivity.

One of the most critical issues in the treatment of bladder overactivity is ascertaining whether the primary defect is due to inappropriate signals to the bladder from nerves, altered sensory signals from the bladder, or altered excitability and contractility of the bladder smooth muscle per se (Brading, 1997; de Groat, 1997; Fowler, 2002). Current therapeutic strategies are aimed at blocking neurotransmission from parasympathetic nerves to detrusor smooth muscle using muscarinic receptor antagonists that prevent the actions of acetylcholine (Chapple et al. 2002). This approach has undesirable side effects such as constipation and dry mouth, causing many patients to discontinue use of anti-cholinergic therapies. Due to the non-specificity of agents that block autonomic neurotransmission, the development of drugs that more specifically diminish the symptoms of bladder overactivity is highly desirable.

Selective targeting of ion channels in urinary bladder smooth muscle to decrease detrusor smooth muscle excitability and contractility is one approach that has showed promise in the treatment of bladder overactivity. In animal models, K+ channel openers that act on ATP-sensitive K+ channels (KATP) (Bonev & Nelson, 1993) have been very effective in treating overactive bladder (Andersson et al. 1988; Igawa et al. 1992; Howe et al. 1995; Petkov et al. 2001). However, in many instances, these compounds have the undesirable action of lowering blood pressure due to direct actions on KATP channels in vascular smooth muscle. There are K+ channel openers being developed, however, that may show bladder selectivity (Brune et al. 2002; Gopalakrishnan et al. 2002; Hewawasam et al. 2002).

In this study, we examined how expression levels of the SK3 channel impact on the function of the urinary bladder. We used transgenic mice in which a tetracycline-based genetic switch was incorporated into the SK3 gene to allow experimental manipulation of SK3 expression (Bond et al. 2000). The homozygous mice (SK3T/T) overexpress SK3 mRNA by ˜3-fold relative to wild-type animals. Treating the animals with doxycycline (DOX) in their drinking water suppresses SK3 expression (Bond et al. 2000). Thus the SK3T/T mice allow us to study the role of SK3 channels in regulating function of the lower urinary tract by manipulating SK3 expression levels. The results demonstrate that suppressing SK3 expression is associated with an increase in the frequency of phasic urinary bladder smooth muscle contractions in vitro, and bladder overactivity in vivo. Targeting SK3 channels may be an effective strategy in the selective treatment of bladder overactivity, since SK3 channels do not seem to be expressed in vascular smooth muscle (Taylor et al. 2002).

METHODS

General

All experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Vermont. The transgenic mice used in this study contained a tetracycline-based genetic switch directing expression of the SK3 gene (see Bond et al. 2000). Homozygous transgenic animals (SK3T/T) express SK3 channels at higher levels than wild-type (SK3+/+) animals, although the distribution pattern of SK3 expression is unaltered (Bond et al. 2000). To suppress SK3 expression, DOX was administered to the animals' drinking water (0.5 g l−1) for a period of 2 weeks (see Bond et al. 2000). Mice were killed with sodium pentobarbital (150 mg kg−1, I.P.) followed by a thoracotomy, and the bladders quickly removed and placed in cold dissection solution (DS, see below for composition). C57BL6 mice, the background strain for the SK3T/T animals, were used as controls for the SK3T/T mice in electrophysiological, histological and functional (myograph) experiments. For in vivo urodynamic measurements, both wild-type (SK3+/+) littermates and C57BL6 were used as controls to illustrate similarities in urodynamic parameters between SK3+/+ and C57BL6 mice.

Immunofluorescence and histology

Fixation of whole bladders was performed to obtain bladder wall cross-sections for immunohistochemical analysis as well as histological measurements of bladder wall thickness. The bladder lumen was rinsed three to four times with small volumes (˜30 μl) of fixative (4 % formaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4)). Luminal contents were then gently emptied, and bladders were left submerged in fixative for 2 h. Fixed bladders were cryoprotected in sucrose (30 % w/v in PBS) overnight at 4 °C. Bladders were frozen in embedding medium (TBS, Triangle Biomedical Sciences, Durham, NC, USA) and cross-sections (10 μm thickness) were obtained with a cryostat.

For immunostaining, cross-sections were washed with PBS, permeabilized with 0.2 % Triton X-100 in PBS at room temperature, and blocked with 2 % bovine serum albumin in PBS. The primary antibody rabbit anti-SK3 (Alomone Labs, Jerusalem, Israel; diluted 1:250 in 2 % BSA/PBS) was applied overnight at 4 °C. The secondary antibody Cy3-anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:500 in 2 % BSA/PBS) was applied for 1 h at room temperature. Nuclei were stained with the fluorescent nucleic acid dye TOTO-3 iodide (Molecular Probes, Inc, Eugene, OR, USA; diluted 1:1500 in PBS). Slides were coverslipped with Citifluor (Citifluor Ltd, Leicester, UK) and kept at 4 °C until use.

Bladder whole-mounts were prepared by fixing with 0.1 M PBS containing 2 % paraformaldehyde and 0.2 % picric acid for 2 h at 4 °C. After washing with PBS, the specimens were dissected to provide urothelium and smooth muscle preparations. Some muscle layers were removed to allow enhanced antibody penetration and improve visualization. For immunostaining, whole-mount preparations were incubated for 1 h at room temperature with PBS + 0.1 % Triton X-100 + 4 % normal horse serum (PBS-Triton-NHS) and the primary and secondary antibodies were diluted in this solution.

Specimens incubated in the absence of primary or secondary antibody were also processed and evaluated for background staining levels. In the absence of primary antibody, no positive immunostaining was observed. The specificity of the SK3 antibody was tested by incubating the samples with the antibody after preabsorption with a control antigen provided by the manufacturer.

Stained preparations were examined with a laser scanning confocal microscope (Bio-Rad MRC 1024ES, Bio-Rad Laboratories, Inc., Life Sciences Division, Hercules, CA, USA). Fluorescence of Cy3 and TOTO-3 was detected using excitation with the 568 and 647 nm lines from a krypton-argon laser at emission wavelengths of 605 and 680 nm, respectively.

Histological analysis of the cross-sections was achieved using the progressive method of haematoxylin and eosin (H&E) staining. Digital images of H&E-stained sections were captured using a MagnaFire camera and an Olympus microscope. Measurements of bladder wall thickness from the H&E-stained slides were performed using Image Tool version 3.00 software (developed at the University of Texas Health Science Center at San Antonio, Texas, USA and available from the Internet by anonymous FTP from maxrad6.uthscsa.edu). Morphometric parameters measured consisted of total bladder wall thickness, mucosal layer thickness and muscular layer thickness. These parameters were measured as distances along a line drawn normal to the long axis of the specimen from the luminal edge to the serosal edge. Total wall thickness was measured as the total length of the line described above. The mucosal thickness was measured as the distance from the luminal edge of the specimen to the serosal surface of the lamina propria. The muscular layer thickness was measured as the distance from the serosal surface of the lamina propria to the serosal edge of the specimen. Measurements on each specimen were performed in triplicate and averaged. Sample sizes reported refer to the total number of cross sections analysed.

Isometric tension recording

The bladder was cut open to expose the urothelial surface and rinsed several times with dissection saline (DS) to remove traces of urine. Small strips of detrusor (2–3 mm wide and 5–7 mm long) were cut from the bladder wall and stored in DS. In most cases the urothelium was removed from the strips. However, some experiments were performed using urothelium-intact bladder strips. Contractility of isolated bladder smooth muscle strips was measured using a MyoMED myograph system (MED Associates, Inc., Georgia, VT, USA). Each strip was mounted in a tissue bath containing aerated PSS (5 ml volume, 95 % O2 and 5 % CO2, 37 °C). Strips were stretched to 10 mN of tension, and allowed to stabilize for 45 min before beginning experimental protocols. Phasic contraction frequency was determined over 5 min periods.

Whole-cell voltage clamp recording

Muscle strips (10–15 pieces) were placed in a vial containing DS with 1 mg ml−1 BSA, 1 mg ml−1 papain (Worthington Biochemical Corporation, Freehold, NJ, USA) and 1 mg ml−1 dithioerythritol (Sigma) for 20–35 min at 37 °C. Next, the tissue was placed in fresh DS (37 °C) containing 1 mg ml−1 BSA, 1 mg ml−1 collagenase (either from Fluka, Milwaukee, WI, USA, or type II from Sigma) and 100 μM CaCl2 for 5–10 min. Following enzyme treatment, the tissue was washed repeatedly with fresh BSA-containing DS, and then stored in this solution on ice. Individual cells were released from the tissue by passing the enzyme-treated tissue bundles through the tip of a fire-polished Pasteur pipette.

Cells plated in an experimental chamber were washed with fresh extracellular solution (see below for composition). The amphotericin-perforated patch technique was used to measure whole-cell currents (Horn & Marty, 1988). Whole-cell currents were recorded during a 100 ms depolarization pulse from a holding potential of −70 to +10 mV. The large-conductance Ca2+-activated K+ channel (BK channel) blocker iberiotoxin (100 nM) was applied to all cells for the duration of recording. The SK channel blocker apamin (1 μM) was applied for 5–10 min in the presence of iberiotoxin. Apamin-sensitive K+ currents were defined as SK currents (see Herrera & Nelson, 2002). Mean currents were measured during all conditions and expressed relative to cell size (capacitance). All experiments were performed at room temperature (22 °C).

Urodynamic measurements in conscious unrestrained mice

Mice were anaesthetized with isoflurane (1–3 % in O2, inhaled). A lower midline abdominal incision was made to expose the urinary bladder and a polyethylene catheter (PE-10) was inserted into the dome of the urinary bladder and secured in place using a purse string suture. The bladder catheter was sealed and routed subcutaneously to the back of the neck, where it was coiled and stored in a skin pouch. Following a 3 day recovery period, the bladder catheter was exteriorized and opened. The animal was placed in a Small Animal Cystometry Lab Station (MED Associates, Inc.) for urodynamic measurements. The catheter was connected to one port of a pressure transducer using a 22 g needle stub and a short piece of PE-50 adapter tubing. The other port of the pressure transducer was connected to a syringe pump. Sterile isotonic saline (0.9 % NaCl; room temperature) was continuously infused into the bladder at a rate of either 25 or 40 μl min−1. An analytical balance beneath the wire-bottom animal cage measured the amount of urine voided during continuous cystometry. A single cystometrogram (CMG) was defined as the simultaneous recording of intravesical pressure, infused volume and voided volume during a single filling-voiding cycle. Data analysis was performed by averaging three to four CMGs which showed reproducibility. Bladder capacity was measured as the amount of saline infused into the bladder at the time when micturition commenced. Residual volume for each CMG was measured as the difference between the infused volume and the voided volume. Minimum pressure was the lowest pressure observed during the filling phase of the CMG. Threshold pressure was the intravesical pressure just before initiation of micturition. Micturition pressure was measured as the peak intravesical pressure during voiding. Non-voiding contractions were defined as rises in intravesical pressure that exceeded 5 mmHg, but were not associated with voiding of urine. Experiments were conducted at similar times of the day to avoid the possibility that circadian variations were responsible for changes in bladder capacity measurements (Dorr, 1992). At the end of the experiment, the mice were killed with sodium pentobarbital (150 mg kg−1, I.P.) followed by a thoracotomy.

Chemicals and solutions

PSS contained (mM): 119 NaCl, 4.7 KCl, 24 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, and 11 glucose. DS was made up of (mM): 80 monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 Hepes, 2 MgCl2 (pH adjusted to 7.3 with NaOH). The standard extracellular (bath) solution used in electrophysiological recordings contained (mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, 10 Hepes (pH adjusted to 7.4 with NaOH). The intracellular (pipette) solution contained (mM): 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 10 Hepes, 0.05 EGTA (pH adjusted to 7.2 with NaOH), plus 200 μg ml−1 amphotericin B. All reagents were from Sigma unless otherwise stated.

Calculations and statistics

Results are summarized as means ± standard errors of the means. Data were compared using Student's t test and one-way or two-way analysis of variance (ANOVA), where appropriate. The Student-Newman-Keuls method was used for all multiple comparisons. The null hypothesis was rejected when P < 0.05.

RESULTS

Distribution of SK3 channels in the mouse urinary bladder

SK3 immunoreactivity was observed in the smooth muscle and urothelial layers of transverse bladder wall sections (Fig. 1A). Pretreatment with excess antigenic peptide abolished SK3 immunoreactivity, demonstrating specificity of the SK3 antibody used (Fig. 1A, inset). Whole mount preparations with and without the urothelium were also examined from control, SK3T/T-overexpressing and SK3T/T-suppressed mice. SK3 immunostaining was increased in both smooth muscle and urothelium of SK3T/T overexpressors compared to control (Fig. 1B and C). Treatment of SK3T/T animals with DOX for 2 weeks substantially decreased SK3 immunostaining compared with SK3T/T overexpressors to levels comparable to or below control (Fig. 1B and C). These data indicate that SK3 channel protein is localized to the smooth muscle and urothelial cells of the bladder wall, and that the level of SK3 protein is elevated in SK3T/T mice relative to wild-type. DOX treatment suppresses expression of SK3 channels in the urinary bladder wall of SK3T/T animals.

Figure 1. Distribution of SK3 immunoreactivity in the urinary bladder wall.

Figure 1

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A, SK3 immunoreactivity in a bladder cross-section from a control (C57BL6) mouse. Positive SK3 immunoreactivity (red) was seen in smooth muscle and urothelium. The inset shows a negative control, in which the tissue slice was pretreated with excess antigenic peptide. B, whole mount urothelial preparations stained for SK3 channels. Immunoreactivity is increased relative to control in SK3T/T overexpressor urothelium, while DOX treatment suppresses SK3 immunostaining. C, whole mount smooth muscle preparations stained for SK3 channels obtained from control, SK3T/T-overexpressing and SK3T/T-suppressed mice.

SK currents in mouse urinary bladder smooth muscle cells

To determine whether SK channel activity was affected as a consequence of manipulating SK3 expression levels, whole-cell SK currents were measured in urinary bladder smooth muscle cells (Fig. 2). Iberiotoxin (100 nM) was applied to suppress BK channel activity. Iberiotoxin-resistant currents were larger in myocytes from SK3T/T-overexpressing mice than in myocytes from SK3T/T-suppressed mice (Fig. 2C). The SK channel blocker apamin (1 μM) reduced the outward current amplitude (Fig. 2A). The apamin-resistant current densities were not different in any of the groups tested (Fig. 2D). This observation suggests that the elevated iberiotoxin-resistant outward current in SK3T/T mice is attributed to SK3 channels. Apamin-sensitive currents (ISK) were elevated 2.4-fold in urinary bladder smooth muscle cells from SK3T/T overexpressors relative to wild-type (Fig. 2B and E). ISK was decreased in SK3T/T animals treated with DOX, relative to SK3T/T overexpressors, consistent with suppression of SK3 channels (Fig. 2C). The remaining apamin-sensitive current in DOX-treated myocytes is likely to reflect SK1 and/or SK2 channel activity that is also apamin sensitive and may be expressed in bladder tissue (Ohya et al. 2000; Herrera & Nelson, 2002).

Figure 2. Whole-cell SK currents recorded from mouse urinary bladder smooth muscle cells.

Figure 2

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A, family of currents elicited by 100 ms depolarization pulses from −70 to +10 mV in bladder myocytes from control (C57BL6), SK3T/T-overexpressing (SK3T/T) and SK3-suppressed (SK3T/T+ DOX) mice in the presence of the BK channel inhibitor iberiotoxin (100 nM) and in the presence of the SK channel inhibitor apamin (1 μM, continued presence of iberiotoxin). B, apamin-sensitive current (ISK) obtained by subtracting the current in the presence of apamin (and iberiotoxin) from the current in the presence of iberiotoxin alone. C, mean outward current density recorded in the presence of iberiotoxin. D, mean outward current density recorded in the presence of apamin (and iberiotoxin). E, ISK density. *P < 0.05 vs. C57BL6, †P < 0.05 vs. SK3T/T (one-way ANOVA).

Apamin sensitivity of phasic contractions

SK3 and SK1 channels are blocked by apamin with EC50 values in the nanomolar range while SK2 channels are blocked more potently by apamin, with an EC50 in the low picomolar range (Köhler et al. 1996; Ishii et al. 1997; Vergara et al. 1998; Bond et al. 1999; Shah& Haylett, 2000; Strøbæk et al. 2000; Hosseini et al. 2001). Selective suppression of SK3 channels should not only illuminate the role of SK3 channels, but may also provide evidence for other apamin-sensitive SK (SK1 and SK2) isoforms.

Inhibiting all subtypes of SK channels with apamin (10−9-10−6 M) increased the amplitude of phasic contractions (Fig. 3C). To determine the sensitivity of bladder strips to apamin, the phasic contraction amplitude under control conditions was subtracted from the phasic contraction amplitude at each apamin concentration, yielding the apamin-induced increase in phasic contraction amplitude. The apamin-induced increase in phasic contraction amplitude at each concentration was normalized to the maximum effect in each strip (100 %; Fig. 3C). Suppressing SK3 channels by DOX treatment resulted in a significant leftward shift in the sensitivity to apamin (Fig. 3C), consistent with the blocking affinity of apamin for SK2 channels. These results support a role for SK3 channels and indicate the participation of other SK isoforms in the regulation of phasic contractions in urinary bladder smooth muscle.

Figure 3. Regulation of mouse urinary bladder smooth muscle phasic contractions by SK3 channels.

Figure 3

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A, recordings of spontaneous phasic contractions in urinary bladder smooth muscle strips isolated from control, SK3T/T-overexpressing and SK3T/T-suppressed mice. B, spontaneous phasic contraction frequency in C57BL6, SK3T/T and SK3T/T+ DOX bladder strips. *P < 0.05 vs. C57BL6; †P < 0.05 vs. SK3T/T (one-way ANOVA). C, apamin-induced increase in phasic contraction amplitude, expressed as a percentage of the maximum apamin-induced effect in each strip. *P < 0.05 vs. C57BL6; †P < 0.05 vs. SK3T/T (two-way ANOVA).

Suppressing SK3 expression elevates phasic contraction frequency

To directly assess the consequences of SK3 suppression on contractility of the urinary bladder smooth muscle, spontaneous phasic contractions were measured in urothelium-denuded bladder smooth muscle strips from control, SK3T/T-overexpressing and SK3T/T-suppressed mice. Bladder tissue from control, SK3T/T-overexpressing and SK3T/T-suppressed mice developed spontaneous phasic contractions (Fig. 3A). In bladder strips from control mice, baseline phasic contraction frequency was 5.7 ± 0.7 min−1 (n = 11; Fig. 3A and B). In strips from SK3T/T mice, phasic contraction frequency was 4.0 ± 0.4 min−1 (n = 12), and was significantly less than that in C57BL6 (Fig. 3B). Suppressing SK3 channels with DOX elevated phasic contraction frequency to 7.8 ± 0.9 min−1 (n = 7; Fig. 3A and B; P < 0.05 vs. C57BL6 and SK3T/T). Thus, suppressing SK3 channel expression increases urinary bladder contractility, consistent with an important influence of SK3 channels on the excitability and contractility of urinary bladder smooth muscle.

To focus on urinary bladder smooth muscle, the above studies were performed in the absence of urothelium. Removal of the urothelium had the opposite effect of suppressing SK3T/T expression with DOX. Phasic contraction frequency was higher in urothelium-intact strips from SK3T/T mice than in denuded strips (8.4 ± 0.6 vs. 4.0 ± 0.4 min−1; P < 0.05). These results suggest that the urothelium exerts a tonic contractile influence on urinary bladder smooth muscle.

SK3 overexpression leads to elevated urine output and bladder capacity

To determine if the increase in bladder contractility seen following SK3 suppression affects lower urinary tract function, continuous filling cystometry was performed in conscious mice. CMGs were recorded in control (C57BL6), wild-type (SK3+/+), SK3T/T-overexpressing and SK3T/T-suppressed mice (Fig. 4A; the CMGs shown for the SK3T/T animal in the presence and absence of DOX were from the same animal). Urodynamic parameters were very similar in C57BL6 and SK3+/+ mice (Fig. 4). Bladder capacity of SK3T/T overexpressors, determined by the infused volume at the point of voiding (red line, Fig. 4A), was 2-fold greater than that of wild-type mice (302 ± 41 vs. 155 ± 24 μl, P < 0.05; Fig. 4B). Suppression of SK3 channels with dietary DOX-treatment did not reduce bladder capacity (Fig. 4B), suggesting that long-term structural remodelling of the urinary bladder had taken place as a consequence of SK3 overexpression and was not reversible by acute SK3 channel down-regulation.

Figure 4. Urodynamic function in control, SK3T/T-overexpressing and SK3T/T-suppressed mice.

Figure 4

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A, cystometrograms (CMGs) from control (C57BL6), wild-type (SK3+/+), SK3T/T-overexpressing (SK3T/T) and SK3T/T-suppressed (SK3T/T+ DOX) mice. The records from the SK3T/T-overexpressing and the SK3T/T-suppressed animal are from the same mouse studied prior to DOX treatment and then again 2 weeks after continuous DOX administration via the drinking water. The red trace shows the infused volume during continuous filling cystometry and the green trace shows the output of urine recorded on an analytical balance beneath the animal cage. Micturition was associated with a sharp rise in intravesical pressure (v). Note the substantial increase in bladder capacity in the SK3T/T animal and the increase in non-voiding contractions (*) after DOX treatment. B, summary showing infused and voided volumes. C, residual volume measured by subtracting the voided volume from the infused volume. A positive value indicates urine remains in the bladder after voiding, whereas a negative value indicates that endogenous urine production was high enough to increase the amount of voided urine to a level greater than the amount of saline infused. D, non-voiding contractions per CMG were defined as increases in intravesical pressure of at least 5 mmHg that were not associated with measurable voiding. *P < 0.05 vs. C57BL6; †P < 0.05 vs. SK3+/+; ‡P < 0.05 vs. SK3T/T (one-way ANOVA).

Consistent with structural remodelling of the bladder in SK3T/T mice, urinary bladder walls were thinner in SK3T/T overexpressors compared to control. Bladder wall thickness in SK3T/T overexpressors, as measured from H&E-stained sections, was 63 % of that of control animals (225 ± 22 vs. 354 ± 28 μm, n = 5 each, P < 0.05). Suppression of SK3 channel expression with dietary DOX for 2 weeks did not restore bladder wall thickness (272 ± 12 μm; n = 7). The bladder-to-body weight ratio (mg g−1), used as an index of bladder size, was 1.03 ± 0.05 in control mice (n = 26), 1.52 ± 0.12 in SK3T/T overexpressors (n = 32; P < 0.05 vs. control) and 1.41 ± 0.22 in SK3T/T-suppressed mice (n = 9). Thus, bladders were larger in SK3T/T overexpressors compared to wild-type, an effect that was not reversed by 2 weeks suppression of SK3 expression.

To determine whether altering expression levels of SK3 channels was associated with dysfunctions such as the inability to completely empty the bladder, the amount of saline voided during each CMG was measured (Fig. 4A, green trace). In wild-type mice, the voided volume corresponded very closely to the amount of saline infused during each CMG (Fig. 4A and B). In contrast, SK3T/T-overexpressing mice had negative residual volumes (−29 ± 8 μl) with each CMG (Fig. 4C), indicating that endogenous urine production contributed to the volume voided during a CMG. In SK3T/T-suppressed mice, bladder capacity remained elevated compared to wild-type (393 ± 41 μl; Fig. 4A and B); however the infused volumes and voided volumes were in close correspondence (residual volume of 0 ± 7 μl; Fig. 4C). This observation indicates that urine production is elevated in SK3T/T-overexpressing mice, and that this effect is reversed by suppressing SK3 expression.

Suppression of SK3 expression results in detrusor overactivity

Intravesical pressure was measured continuously during cystometry to determine whether altering SK3 expression levels would influence the mechanical properties of the urinary bladder in vivo. No significant differences between wild-type, SK3T/T overexpressors or SK3T/T-suppressed mice were observed in minimum or average bladder pressures during filling, or in the threshold or peak micturition pressures (Table 1). There was a significant increase in micturition pressure in DOX-treated mice compared to C57BL6 mice (Table 1). Furthermore, a substantial increase in non-voiding contractions occurred when SK3 channel expression was suppressed with dietary DOX (Fig. 4A and D). Non-voiding contractions, defined as increases in intravesical pressure of at least 5 mmHg that were not associated with voiding, were infrequent in C57BL6, SK3+/+ and SK3T/T-overexpressing mice (Fig. 4D). Following suppression of SK3 channel expression with dietary DOX, non-voiding contraction frequency was elevated 4-fold compared to SK3T/T overexpressors (Fig. 4D). The amplitude of non-voiding contractions was also greater in the SK3T/T-suppressed (+DOX) mice compared to C57BL6 mice (Table 1). Therefore, suppression of SK3 channel expression leads to an elevation in phasic contractions in vitro, and non-voiding contractions in vivo.

Table 1.

Intravesical pressures (mmHg) during continuous filling cystometry in C57BL6, SK3+/+, SK3T/T and SK3T/T+ DOX mice

Minimum pressure Threshold pressure Micturition pressure Non-void amplitude
C57BL6 (n = 9) 2.9 ± 0.7 9.6 ± 1.0 26.3 ± 2.7 6.7 ± 0.3
SK3+/+ (n = 7) 4.6 ± 1.2 14.6 ± 1.5 33.4 ± 2.6 8.0 ± 0.7
SK3+/+ (n = 7) 2.8 ± 0.9 14.6 ± 1.8 36.5 ± 3.3 9.0 ± 0.6
SK3T/T+ DOX (n = 5) 3.2 ± 0.4 13.9 ± 2.5 42.8 ± 4.2* 9.7 ± 0.5*
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* P < 0.05 vs. C57BL6 (one-way ANOVA).

DISCUSSION

Structural and functional changes in the bladder as a consequence of SK3 overexpression

Urinary bladders from SK3T/T-overexpressing mice are dramatically remodelled. The capacities of the bladders from SK3T/T mice were substantially increased, and the bladder walls had undergone thinning compared to control animals. Acute (2 weeks) suppression of SK3 expression with dietary DOX in adult animals did not reverse these effects, indicating that this structural remodelling is likely to reflect the long-term consequences of SK3 overexpression.

SK3T/T-overexpressing mice showed evidence of elevated endogenous urine production rates. The average time between voiding events in wild-type animals was approximately 6 min. Over this time period, urine production did not contribute significantly to each void (see Fig. 4). In contrast, the negative residual volume of approximately −30 μl observed in SK3T/T-overexpressing mice (Fig. 4C), indicates that endogenous urine production contributed to the volume voided during a CMG. Since the amount of time between voiding in SK3T/T-overexpressing mice was about 12 min, this corresponds to an endogenous urine production rate of nearly 2.5 μl min−1. If this rate was the same for wild-type mice, approximately 15 μl of urine would be produced and contribute to the amount collected with each void (a residual volume of −15 μl), an amount within the resolution of this system (˜10 μl). Following suppression of SK3T/T expression with dietary DOX, no evidence for elevated urine production rates was observed, as the infused and voided volumes corresponded closely (Fig. 4B and C).

It is possible that the dilation of the bladder and thinning of the bladder wall in SK3T/T-overexpressing mice occurred as a consequence of chronic over-production of urine and a decrease in the sensitivity of bladder afferent pathways to bladder distension. Similar consequences occur in diabetes (Kudlacz et al. 1988; Malmgren et al. 1992; Tammela et al. 1993). In an experimental model of hereditary diabetes insipidus in rats, Malmgren and colleagues (1992) showed that bladder capacity is elevated compared to control and these diabetic rats have increased 24 h diuresis. Similar changes in bladder structure occur with experimentally induced diuresis (Kudlacz et al. 1988). This is analogous to our finding with urinary bladders from SK3T/T animals (Fig. 4). The fact that basal urine production seems to be elevated in SK3T/T-overexpressing mice (Fig. 4) also indicates that diuresis per se may account for the structural and functional effects in SK3T/T overexpressors.

The basis for increased urine production in SK3T/T-overexpressing mice is not clear. We have noted substantial alterations in blood vessel structure and function in SK3T/T overexpressing mice (Taylor et al. 2002). In mesenteric arteries from SK3T/T mice, there is a tonic vasodilatory influence that is dependent upon SK3 channels in the vascular endothelium (Taylor et al. 2002). It is possible that similar alterations occur in the renal circulation. Increased basal blood flow to the kidneys in SK3T/T animals might result in increased filtration and lead to elevated urine production. It is also possible that SK channels play a direct role in epithelial transport in the nephron (Merot et al. 1989), and overexpression of SK3 channels could result in changes in solute/fluid movement, leading to increased diuresis.

Impaired bladder sensation, or elevated volume at the first desire to void, is also a common symptom of diabetes (Kaplan & Blaivas, 1988). This may reflect a defect in the way that bladder distention is sensed during filling, and over time, could lead to structural and functional changes such as bladder dilation and altered detrusor contractility. It is possible that in mice overexpressing SK3 channels bladder sensation is altered such that the desire to void occurs at higher than normal bladder volumes. Over the long term, this could result in the increase in capacity and thinning of the bladder wall observed in the SK3T/T mice.

SK3 channels are important determinants of urinary bladder contractility

SK channels are important regulators of urinary bladder smooth muscle excitability, and they are involved in generating the action potential afterhyperpolarization. Blocking SK channels increases action potential frequency (Creed et al. 1983; Fujii et al. 1990). Our data support the idea that in addition to SK3 channels, the other SK isoforms are also likely to regulate excitability and contractility (see also Herrera et al. 2002; Herrera & Nelson, 2002). Our results also support an important role of SK3 channels in setting the level of detrusor contractility. Cystometric evaluation of bladder function in conscious mice revealed that suppressing SK3 channels with dietary DOX results in substantial detrusor overactivity as indicated by the increase in non-voiding contractions during each CMG (Fig. 4). The elevation of non-voiding contractions in vivo is likely to reflect an increase in smooth muscle excitability, since phasic contractions of bladder strips from SK3-suppressed mice were also increased (Fig. 3). Alternatively, it is also possible that suppression of SK3 expression in the urothelium leads to an elevation in non-voiding contractions; suppressing urothelial SK3 channel expression may interfere with the way the urothelium responds to bladder distension, resulting in increased release of ATP that would activate sensory fibres and nerve-mediated bladder overactivity (Ferguson et al. 1997; Cockayne et al. 2000). However, our results showing increased phasic contractions in bladder strips from SK3T/T-suppressed mice indicate that suppression of urothelial SK3 channels is not required to observe increased contractility, as these experiments were performed in the absence of urothelium.

It is also conceivable that bladder overactivity seen in the SK3T/T-suppressed mice is related to changes in the expression of SK3 channels in sensory or efferent nerves in the bladder wall. If SK3 channels are expressed in bladder afferent nerves, suppressing SK3 expression would be expected to increase the activity of the bladder afferents. Suppressing SK3 expression in bladder efferent nerves would be expected to lead to enhanced parasympathetic stimulation of the bladder. Either of these situations could lead to neurogenic bladder overactivity. We attempted to localize SK3 immunoreactivity to sensory and efferent nerves in the bladder wall. Various neuronal markers, including PGP 9.5, were used in conjunction with the SK3 antibody. Co-staining of nerves with a neuronal marker and the SK3 antibody was never observed, even in bladders from SK3T/T-overexpressing mice, suggesting that nerves in the bladder wall do not contain SK3 channels.

Summary and conclusion

The transgenic mouse model employed in this study, SK3T/T, overexpresses SK3 channels in all cells that normally express SK3 channels (Bond et al. 2000). One exciting, novel observation made in this context was that SK3 channels are located in both the urothelial and smooth muscle layers of the urinary bladder. Furthermore, we found that urine production is influenced by SK3 channel expression. This result is not likely to reflect changes in SK3 expression in the bladder per se, but changes in urine production could impact on the function of the urinary bladder. Since the targeting of the SK3 gene in SK3T/T mice was not smooth muscle specific, it is difficult to be absolutely certain that the observed changes seen in vivo are due to effects of SK3 expression in bladder smooth muscle. However, when taken together, electrophysiological recordings in isolated bladder myocytes, contractility measurements performed in the absence of urothelium, and in vivo cystometry all support the idea that SK3 channels are important determinants of bladder smooth muscle contractility. In addition, our results support the idea that other SK isoforms (SK1, SK2) are involved in regulating bladder function.

Taken together, the results suggest that modulating SK channel activity in the bladder may be a potent therapeutic approach to treat voiding disorders. Our data suggest that pharmacological activation of SK channels would result in decreased bladder sensation during filling. This would be an attractive avenue for treating disorders such as bladder overactivity and urge incontinence.

Acknowledgments

The authors would like to thank Drs M. F. Gomez and L. Gonzalez Bosc for assistance with immunofluorescence studies, and Ms A. Banever for assistance with urodynamic measurements. Drs L. Gonzalez Bosc, B. Etherton, M. S. Taylor, K. S. Thorneloe, M. K. Wilkerson and Ms L. Nilsson provided comments that were helpful in editing this manuscript. This work was supported by National Institutes of Health Grants DK-53832 to M.T.N., NS-38880 to J.P.A., and by a Training Grant from the National Institutes of Health T32 HL/AR 07944. M.J.P. was partially supported by SEEU from Ministerio Educación Cultura y Deporte.

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