Urinary bladder instability induced by selective suppression of the murine small conductance...

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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 afterhyper-

polarization in urinary bladder smooth muscle (Creed etal. 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

Urinary bladder instability induced by selective suppressionof the murine small conductance calcium-activatedpotassium (SK3) channelGerald M. Herrera*, Maria J. Pozo‡, Peter Zvara†, Georgi V. Petkov*, Chris T. Bond§, John P. Adelman§and Mark T. Nelson*

Departments of *Pharmacology and †Surgery, University of Vermont, Burlington, VT, USA, ‡Department of Physiology, Nursing School, Universityof Extremadura, Cáceres, Spain and §The Vollum Institute, Oregon Health Sciences Center University, Portland, OR, USA

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.

(Received 28 April 2003; accepted after revision 12 June 2003; first published online 17 June 2003)

Corresponding author M. T. Nelson: Department of Pharmacology, University of Vermont, Burlington, VT 05405-0068, USA.Email: [email protected]

J Physiol (2003), 551.3, pp. 893–903 DOI: 10.1113/jphysiol.2003.045914

© The Physiological Society 2003 www.jphysiol.org

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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 etal. 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).

METHODSGeneralAll experiments were reviewed and approved by the InstitutionalAnimal 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 (seeBond 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 isunaltered (Bond et al. 2000). To suppress SK3 expression, DOXwas administered to the animals’ drinking water (0.5 g l_1) for aperiod of 2 weeks (see Bond et al. 2000). Mice were killed withsodium pentobarbital (150 mg kg_1, I.P.) followed by athoracotomy, and the bladders quickly removed and placed incold dissection solution (DS, see below for composition). C57BL6mice, the background strain for the SK3T/T animals, were used ascontrols for the SK3T/T mice in electrophysiological, histologicaland functional (myograph) experiments. For in vivo urodynamicmeasurements, both wild-type (SK3+/+) littermates and C57BL6were used as controls to illustrate similarities in urodynamicparameters between SK3+/+ and C57BL6 mice.

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

For immunostaining, cross-sections were washed with PBS,permeabilized with 0.2 % Triton X-100 in PBS at roomtemperature, 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 at4 °C. The secondary antibody Cy3-anti-rabbit IgG (JacksonImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:500in 2 % BSA/PBS) was applied for 1 h at room temperature. Nucleiwere 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 PBScontaining 2 % paraformaldehyde and 0.2 % picric acid for 2 h at4 °C. After washing with PBS, the specimens were dissected toprovide urothelium and smooth muscle preparations. Somemuscle layers were removed to allow enhanced antibodypenetration and improve visualization. For immunostaining,whole-mount preparations were incubated for 1 h at roomtemperature with PBS + 0.1 % Triton X-100 + 4 % normal horseserum (PBS–Triton–NHS) and the primary and secondaryantibodies were diluted in this solution.

Specimens incubated in the absence of primary or secondaryantibody were also processed and evaluated for backgroundstaining levels. In the absence of primary antibody, no positiveimmunostaining was observed. The specificity of the SK3antibody was tested by incubating the samples with the antibody

G. M. Herrera and others894 J Physiol 551.3

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after preabsorption with a control antigen provided by themanufacturer.

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

Histological analysis of the cross-sections was achieved using theprogressive method of haematoxylin and eosin (H&E) staining.Digital images of H&E-stained sections were captured using aMagnaFire camera and an Olympus microscope. Measurementsof bladder wall thickness from the H&E-stained slides wereperformed using Image Tool version 3.00 software (developed atthe University of Texas Health Science Center at San Antonio,Texas, USA and available from the Internet by anonymousFTP from maxrad6.uthscsa.edu). Morphometric parametersmeasured consisted of total bladder wall thickness, mucosal layerthickness and muscular layer thickness. These parameters weremeasured as distances along a line drawn normal to the long axisof the specimen from the luminal edge to the serosal edge. Totalwall thickness was measured as the total length of the linedescribed above. The mucosal thickness was measured as thedistance from the luminal edge of the specimen to the serosalsurface of the lamina propria. The muscular layer thickness wasmeasured as the distance from the serosal surface of the laminapropria to the serosal edge of the specimen. Measurements oneach specimen were performed in triplicate and averaged. Samplesizes reported refer to the total number of cross sections analysed.

Isometric tension recordingThe bladder was cut open to expose the urothelial surface andrinsed several times with dissection saline (DS) to remove traces ofurine. 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 theurothelium was removed from the strips. However, someexperiments were performed using urothelium-intact bladderstrips. Contractility of isolated bladder smooth muscle strips wasmeasured using a MyoMED myograph system (MED Associates,Inc., Georgia, VT, USA). Each strip was mounted in a tissue bathcontaining aerated PSS (5 ml volume, 95 % O2 and 5 % CO2,37 °C). Strips were stretched to 10 mN of tension, and allowed tostabilize for 45 min before beginning experimental protocols.Phasic contraction frequency was determined over 5 min periods.

Whole-cell voltage clamp recordingMuscle strips (10–15 pieces) were placed in a vial containing DSwith 1 mg ml_1 BSA, 1 mg ml_1 papain (Worthington BiochemicalCorporation, Freehold, NJ, USA) and 1 mg ml_1 dithioerythritol(Sigma) for 20–35 min at 37 °C. Next, the tissue was placed infresh 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 mM CaCl2 for 5–10 min. Following enzyme treatment, thetissue was washed repeatedly with fresh BSA-containing DS, andthen stored in this solution on ice. Individual cells were releasedfrom the tissue by passing the enzyme-treated tissue bundlesthrough the tip of a fire-polished Pasteur pipette.

Cells plated in an experimental chamber were washed with freshextracellular solution (see below for composition). Theamphotericin-perforated patch technique was used to measurewhole-cell currents (Horn & Marty, 1988). Whole-cell currentswere recorded during a 100 ms depolarization pulse from aholding 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 SKchannel blocker apamin (1 mM) was applied for 5–10 min in thepresence of iberiotoxin. Apamin-sensitive K+ currents weredefined as SK currents (see Herrera & Nelson, 2002). Meancurrents were measured during all conditions and expressedrelative to cell size (capacitance). All experiments were performedat room temperature (22 °C).

Urodynamic measurements in conscious unrestrained miceMice were anaesthetized with isoflurane (1–3 % in O2, inhaled). Alower midline abdominal incision was made to expose the urinarybladder and a polyethylene catheter (PE-10) was inserted into thedome of the urinary bladder and secured in place using a pursestring suture. The bladder catheter was sealed and routedsubcutaneously to the back of the neck, where it was coiled andstored in a skin pouch. Following a 3 day recovery period, thebladder catheter was exteriorized and opened. The animal wasplaced in a Small Animal Cystometry Lab Station (MEDAssociates, Inc.) for urodynamic measurements. The catheter wasconnected to one port of a pressure transducer using a 22 g needlestub and a short piece of PE-50 adapter tubing. The other port ofthe pressure transducer was connected to a syringe pump. Sterileisotonic saline (0.9 % NaCl; room temperature) was continuouslyinfused into the bladder at a rate of either 25 or 40 ml min_1. Ananalytical balance beneath the wire-bottom animal cage measuredthe amount of urine voided during continuous cystometry. Asingle cystometrogram (CMG) was defined as the simultaneousrecording of intravesical pressure, infused volume and voidedvolume during a single filling–voiding cycle. Data analysis wasperformed by averaging three to four CMGs which showedreproducibility. Bladder capacity was measured as the amount ofsaline infused into the bladder at the time when micturitioncommenced. Residual volume for each CMG was measured as thedifference between the infused volume and the voided volume.Minimum pressure was the lowest pressure observed during thefilling phase of the CMG. Threshold pressure was the intravesicalpressure just before initiation of micturition. Micturition pressurewas measured as the peak intravesical pressure during voiding.Non-voiding contractions were defined as rises in intravesicalpressure that exceeded 5 mmHg, but were not associated withvoiding of urine. Experiments were conducted at similar times ofthe day to avoid the possibility that circadian variations wereresponsible for changes in bladder capacity measurements (Dorr,1992). At the end of the experiment, the mice were killedwith sodium pentobarbital (150 mg kg_1, I.P.) followed by athoracotomy.

Chemicals and solutionsPSS 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): 80monosodium glutamate, 55 NaCl, 6 KCl, 10 glucose, 10 Hepes, 2MgCl2 (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, 10NaCl, 1 MgCl2, 10 Hepes, 0.05 EGTA (pH adjusted to 7.2 withNaOH), plus 200 mg ml_1 amphotericin B. All reagents were fromSigma unless otherwise stated.

Calculations and statisticsResults 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

SK3 channels in the urinary bladderJ Physiol 551.3 895

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Student-Newman-Keuls method was used for all multiplecomparisons. The null hypothesis was rejected when P < 0.05.

RESULTSDistribution of SK3 channels in the mouse urinarybladderSK3 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.

SK currents in mouse urinary bladder smoothmuscle cellsTo 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 mM) 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. 2Band 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 1. Distribution of SK3 immunoreactivity in the urinary bladder wallA, SK3 immunoreactivity in a bladder cross-section from a control (C57BL6) mouse. Positive SK3immunoreactivity (red) was seen in smooth muscle and urothelium. The inset shows a negative control, inwhich the tissue slice was pretreated with excess antigenic peptide. B, whole mount urothelial preparationsstained for SK3 channels. Immunoreactivity is increased relative to control in SK3T/T overexpressorurothelium, while DOX treatment suppresses SK3 immunostaining. C, whole mount smooth musclepreparations stained for SK3 channels obtained from control, SK3T/T-overexpressing and SK3T/T-suppressedmice.

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Apamin sensitivity of phasic contractionsSK3 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_6M) 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.

Suppressing SK3 expression elevates phasiccontraction frequencyTo 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.


Figure 2. Whole-cell SK currents recorded from mouse urinary bladder smooth muscle cellsA, family of currents elicited by 100 ms depolarization pulses from _70 to +10 mV in bladder myocytes fromcontrol (C57BL6), SK3T/T-overexpressing (SK3T/T) and SK3-suppressed (SK3T/T + DOX) mice in thepresence of the BK channel inhibitor iberiotoxin (100 nM) and in the presence of the SK channel inhibitorapamin (1 mM, continued presence of iberiotoxin). B, apamin-sensitive current (ISK) obtained by subtractingthe 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 densityrecorded in the presence of apamin (and iberiotoxin). E, ISK density. * P < 0.05 vs. C57BL6, † P < 0.05 vs.SK3T/T (one-way ANOVA).

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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 outputand bladder capacityTo 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 ml, 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.

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 mm, 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 mm; 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.


Figure 3. Regulation of mouse urinary bladder smooth muscle phasic contractions by SK3channelsA, recordings of spontaneous phasic contractions in urinary bladder smooth muscle strips isolated fromcontrol, SK3T/T-overexpressing and SK3T/T-suppressed mice. B, spontaneous phasic contraction frequency inC57BL6, SK3T/T and SK3T/T + DOX bladder strips. * P < 0.05 vs. C57BL6; † P < 0.05 vs. SK3T/T (one-wayANOVA). C, apamin-induced increase in phasic contraction amplitude, expressed as a percentage of themaximum apamin-induced effect in each strip. * P < 0.05 vs. C57BL6; † P < 0.05 vs. SK3T/T (two-wayANOVA).

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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 ml) 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 ml; Fig. 4A and B); however the infused volumes

and voided volumes were in close correspondence

(residual volume of 0 ± 7 ml; 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.


Figure 4. Urodynamic function in control, SK3T/T-overexpressing and SK3T/T-suppressed miceA, 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 aftercontinuous DOX administration via the drinking water. The red trace shows the infused volume duringcontinuous filling cystometry and the green trace shows the output of urine recorded on an analytical balancebeneath the animal cage. Micturition was associated with a sharp rise in intravesical pressure (v). Note thesubstantial 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 bysubtracting the voided volume from the infused volume. A positive value indicates urine remains in thebladder after voiding, whereas a negative value indicates that endogenous urine production was high enoughto increase the amount of voided urine to a level greater than the amount of saline infused. D, non-voidingcontractions per CMG were defined as increases in intravesical pressure of at least 5 mmHg that were notassociated with measurable voiding. * P < 0.05 vs. C57BL6; † P < 0.05 vs. SK3+/+; ‡ P < 0.05 vs. SK3T/T (one-way ANOVA).

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Suppression of SK3 expression results in detrusoroveractivityIntravesical 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.

DISCUSSIONStructural and functional changes in the bladder as aconsequence of SK3 overexpressionUrinary 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 ml 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 ml min_1. If this rate was the same for wild-type

mice, approximately 15 ml of urine would be produced

and contribute to the amount collected with each void (a

residual volume of _15 ml), an amount within the

resolution of this system (~10 ml). 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. 4Band 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


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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 urinarybladder contractilitySK 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 conclusionThe 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, electro-

physiological 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.

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Acknowledgements The authors would like to thank Drs M. F. Gomez and L. GonzalezBosc for assistance with immunofluorescence studies, andMs 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 werehelpful in editing this manuscript. This work was supported byNational Institutes of Health Grants DK-53832 to M.T.N., NS-38880 to J.P.A., and by a Training Grant from the NationalInstitutes of Health T32 HL/AR 07944. M.J.P. was partiallysupported by SEEU from Ministerio Educación Cultura yDeporte.


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