Open Journal of Urology, 2011, 1, 60-65
doi:10.4236/oju.2011.13013 Published Online August 2011 (
Copyright © 2011 SciRes. OJU
Differential Effects of K+ Channel Blockers on
Phasic Contractility of Transverse and Longitudinal
Rat Detrusor Strips
Aneira Gracia Hidayat Santoso1, Wan Ning Lo1, Wil lma nn Liang1,2
1School of Biological Sciences, College of Science, Nanyang
Technologi cal University , Singapore City, Singapor e
2Institute of Advanced Studies, Nanyang Technolo gi cal
University, Singapore City, Singapore
Received May 30, 201 1; revised June 28, 2011; accepted July 5, 2011
Spontaneous phasic contractions of detrusor smooth muscle are pivotal to the normal bladder filling process.
The role of K+ channels in mediating phasic contractions has been investigated on different occasions, but
only in detrusor strips isolated longitudinally. In this study, the effects of individual K+ blockers were exam-
ined in both transverse and longitudinal detrusor strips. Detrusor strips were isolated transversely and longi-
tudinally from young adult rat bladders. Tension before and after the introduction of K+ channel blockers
was measured using a myograph. Phasic activity was determined by calculating the integral of tension fluc-
tuations. Phasic activity of transverse strips was increased under tetraethylammonium chloride (TEA),
4-aminopyridine (4-AP) and iberiotoxin (IbTx) treatments. Longitudinal phasic activity was increased under
charybdotoxin (ChTx) treatment. Neither glibenclamide (Glib) nor apamin treatment elicited any significant
effect in both transverse and longitudinal phasic activity. The results indicated that phasic activity was medi-
ated differently depending on the contractile direction. Data from this study reiterate that in addition to the
conventional longitudinal direction, the transverse direction also presents significance when examining the
contractility of a sac-like organ like the bladder.
Keywords: Detrusor Smooth Muscle, Contractility, K+ Channel, Transverse, Longitudinal
1. Introduction
Urine release and storage are major functions of the uri-
nary bladder. Forceful contractions of the detrusor
smooth muscle are essential for urine release. During
bladder filling, detrusor wall tension and intravesical
pressure fluctuate to adjust to the changing urine volume
[1]. The ever changing contractility of the detrusor
smooth muscle contributes to spontaneous phasic con-
tractions frequently seen in whole bladder or isolated
strip experiments. Regulation of phasic contractions can
be of several origins, from nerves, the urothelium or
within the smooth muscle [2]. The importance of phasic
activity in the bladder is implicated in various disease
conditions [2-4]. Indeed many groups have investigated
one aspect or another of phasic contractions, both in
normal and diseased bladders. One group of molecular
candidates believed to mediate phasic contractions is the
K+ channels. Several groups have studied the effects of
K+ channel blockers and reached variable findings attrib-
utable to differences between species and experimental
conditions [5-9]. Although not as extensively used as
other animals, the rat remains a valuable model for stud-
ying bladder physiology. Diseased bladder models have
been established in the rat [4,10]. Findings from the
normal rat bladder therefore provide good references to
identify diseased-induced changes in disease models.
The vast majority of literature on bladder contractility of
the rat (or other animals) reported on longitudinal con-
tractions only, irrespective of contractile differences be-
tween transverse and longitudinal directions [11-15].
These differences point to the importance in considering
contractions in more than one direction. Thus, this study
is conducted to identify differential modulation by K+
channels in detrusor phasic contractions between trans-
verse and longitudinal directions.
2. Materials and Methods
2.1. Tissue Preparation
All procedures were performed according to rules out-
lined by the Institutional Animal Care and Use Commit-
tee at Nanyang Technological University, Singapore
(Project approval No.: ARF SBS/NIE-A 003). Six- to
seven-week-old Sprague-Dawley rats of either gender
were killed by CO2 asphyxiation. Forty-nine rats were
used in total, from which 40 transverse and 30 longitu-
dinal strips were isolated (see below). The whole bladder
was harvested as previously described and immediately
placed in carbogen-aerated ice-cold Krebs’ solution [11].
The bladder base, which made up about one third of the
bladder, was discarded. Only tissues isolated from the
bladder dome (detrusor) were used. The bladder dome
was cut open along the lateral sides and the urothelium
was exposed. Fine pins were used to fix the tissue on a
Sylgard®-coated petri dish. Using a razor blade, two
strips measuring 5 mm by 1 mm each were dissected
from the detrusor. One strip was cut with the longer side
parallel to the longitud inal axis of the detrusor. Th e other
strip was cut with the longer side parallel to the trans-
verse axis of the detrusor. The longer side of the strip
was in line with the direction of contractile force meas-
urements as previously done [12]. The urothelium was
kept intact. All strips were mounted on a tissue myog-
raph system (Danish Myo Technology Model 800MS,
Aarhus, Denmark) containing Krebs’ solution at 37˚C.
Isometric tension was monitored in both transverse and
longitudinal directions and recorded using a Powerlab
interface and the LabChart software (ADInstruments,
Bella Vista, Australia).
2.2. Experimental Protocol
The detrusor strips were allowed to equilibrate for 30
minutes with multiple washouts at 2 g of resting tension.
The viability of the strips was tested using K+-Krebs’
solution bubbled with a mixture of 95% oxygen and 5%
carbon dioxide. After another 30 minutes of continuous
washout, a K+ channel blocker was added. The effective
concentration of each K+ channel blocker in rat detrusor
strips was pre-determined in previous studies [12,13].
Blockers used were as follows (concentrations in paren-
theses): tetraethylammonium chloride (TEA, 10 mM),
4-aminopyridine (4-AP, 3 mM), glibenclamide (Glib,
0.1 µM), iberiotoxin (IbTx, 0.1 µM), charybdotoxin
(ChTx, 0.1 µM) and apamin (0.1 µM). The incubation
period of each K+ channel blocker was 10 minutes.
2.3. Drugs and Chemicals
The composition of Krebs’ solution was as follows (in
mM): NaCl (119), MgCl2 (1.2), NaH 2PO4 (1.2), NaH CO3
(15), KCl (4 .6), CaCl2 (1.5), Glucose (11). For K+-Krebs’
solution, no NaCl was added but 124 mM KCl was used
instead. All constituents remained the same otherwise.
All chemicals and drugs used in this study were pur-
chased from Sigma-Aldrich Co. (Singapore, Singapore).
All drugs were dissolved in Ca2+-free Krebs’ solution
except 4-AP (in 70% ethanol), Glib (in dimethyl sul-
fonide) and apamin (in 0.05 M acetic acid). For drugs not
dissolved in Ca2+-free Krebs’ solution, a maximum of
1-to-1000 dilution from the stock drug solution was per-
formed to prevent nonspecific tissue effects due to the
2.4. Data and Statistical Analysis
Sample tracings were shown in pairs, consisting of con-
trol and K+ blocker-treated readings from the same de-
trusor strip. Fluctuations in tension (F) were expressed
as a percentage of the maximal contraction (F) to
K+-Krebs’ solution. According to the literature, the inte-
gral under the tension recording curve could give an in-
dication of the amount of phasic activity [11,16]. In this
study, phasic activity was quantified this way for a
2-minute period before (i.e. control) and after the addi-
tion of each K+ blocker. For K+ channel blocker-medi-
ated phasic activity, readings were taken starting at 10
minutes after the addition of the blocker. Phasic activity
in the presence of the K+ channel blocker was expressed
as a percent change from the control phasic activity. Sta-
tistical analysis was done using the Prism 4 software
(GraphPad Software Inc., La Jolla, CA, USA). Student’s
t-test was used to determine if the percent change in pha-
sic activity was significantly deviated from zero, which
was the control level by definition. The difference be-
tween the percent change in transverse and longitudinal
phasic activity was also determined by Student’s t-test.
All data shown in graph s were mean ± SEM. P values of
less than 0.05 (P < 0.05) were considered to be statisti-
cally different.
3. Results
3.1. Transverse Phasic Activity Was More
Sensitive to Blockade of Voltage-Sensitive
K+ Channels
Tetraethylammonium chloride (TEA) is a nonselective
Copyright © 2011 SciRes. OJU
blocker of voltage-sensitive K+ (Kv), ATP-sensitive K+
(KATP) and large-conductance Ca2+-activated K+ (BK)
channels. Figure 1(a) shows sample tracings of phasic
activity before (i.e. control) and after adding the K+
channel blockers. In both transverse and longitudinal
directions, phasic activity was significantly increased by
10 mM TEA treatment (Figure 1(b)). Transverse phasic
activity was increased by 110% ± 20%, compared to a
60% ± 10% increase in longitudinal phasic activity (P <
0.05). Since TEA could act at more than one type of K+
channels, namely Kv, KATP and BK channels, selective
blockade of Kv and KATP channels, by 4-aminopyridine
(4-AP) and glibenclamide (Glib) respectively, was ex-
amined next. In the presence of 3 mM 4-AP, transverse
phasic activity was significantly increased (by 50% ±
20%) whereas that of longitudinal remained unchanged
from control (10% ± 10%) (Figure 1(b)). Similar to TEA
treatment, 4-AP elicited a greater effect in transverse
than longitudinal phasic activity (P < 0.05). Under 0.1 M
Glib treatment, there was a small but not significant de-
crease in both transverse and longitudinal phasic activity,
respectively, by 30% ± 10% and 20% ± 20% (Figure
1(b)). The function of KATP channels in mediating phasic
activity was not implicated, whereas that of Kv channels
was demonstrated in the transverse contractile direction.
3.2. Blockade of Ca2+-Activated K+ Channels
Revealed Differential Effects in Transverse
and Longitudinal Phasic Activity
Figure 2(a) shows sample tracings of phasic activity
before and after adding the various Ca2+-activated K+
channel blockers. Aside from Kv and KATP channels, BK
channels are also blocked by TEA. Iberiotoxin (IbTx)
was used here to block BK channel activity selectively.
Only transverse phasic activity was significantly in-
creased (by 80% ± 20% from the control level) under 0.1
M IbTx treatment (Figure 2(b)). As with TEA and
4-AP, IbTx also elicited a greater stimulatory effect in
transverse than longitudinal phasic activity (P < 0.05).
Charybdotoxin (ChTx) is a blocker of intermediate-
conductance Ca2+-activated K+ (IK) channels. In the
presence of 0.1 M ChTx, longitudinal phasic activity
was significantly increased from the control level by
60% ± 20% (Figure 2(b)). The greater effect elicited by
ChTx in longitudinal phasic activity was approaching
statistical significance (P = 0.0765). After adding 0.1 M
apamin, a selective blocker of small-conductance Ca2+-
activated K+ (SK) channels, both transverse and longitu-
dinal phasic activity was only modestly suppressed, by
20% ± 10% and 20% ± 9% (Figure 2(b)). The findings
here suggested that both BK and IK channels mediated
phasic activity, but did so distinctly according to the con-
Figure 1. Transverse and longitudinal phasic contractions
under the influence of nonselective and selective voltage-
sensitive K+ (Kv) and ATP-sensitive K+ (KATP) channel
blockers. (a) Representative tracings of transverse (Tr) and
longitudinal (Lg) phasic contractile activity before (i.e. con-
trol) and after treatment with 10 mM tetraethylammonium
chloride (TEA) (Tr: n = 4; Lg: n = 4), 3 mM 4-amino- pyri-
dine (4-AP) (Tr: n = 8; Lg: n = 5) or 0.1 M glibenclamide
(Glib) (Tr: n = 6; Lg: n = 5); (b) Percent change in trans-
verse (solid columns) and longitudinal (open columns) pha-
sic activity from the control level. Significantly higher pha-
sic activity was present in the transverse direction under
TEA and 4-AP treatments. *denotes P < 0.05 vs. control
level at 0%. #denotes P < 0.05 between transverse and lon-
tractile direction.
4. Discussion
The role of K+ channels in mediatin g spontaneous ph asic
contractions in the detrusor have been studied by differ-
ent groups over the recent years. Both urothelium-intact
and -denuded detrusor tissues from the bladder of dif-
ferent species have been used, often yielding conflicting
results [5,6,8,12,13]. Thus, generalizations, if any, about
K+-channel-mediated phasic contractions in the detrusor
are yet to be made. Using selective K+ channel blockers
at effective concentrations previously established, we
hereby examined phasic contractile activity in both
transverse and longitudinal directions using normal adult
rat urothelium-intact detrusor strips. Despite anatomical
differences between human and rat detrusor, the latter is
still of value due to the numerous established bladder
diseased models in the rat. Examples include the overact-
Copyright © 2011 SciRes. OJU
Figure 2. Transverse and longitudinal phasic contractions
under the influence of selective Ca2+-activated K+ channel
blockers. (a) Representative tracings of transverse (Tr) and
longitudinal (Lg) phasic contractile activity before (i.e. con-
trol) and after treatment with 0.1 M iberiotoxin (IbTx) (Tr:
n = 8; Lg: n = 6), 0.1 M charybdotoxin (ChTx) (Tr: n = 8;
Lg: n = 5) or 0.1 M apamin (Tr: n = 6; Lg: n = 5); (b) Per-
cent change in transverse (solid columns) and longitudinal
(open columns) phasic activity from the control level. Tran-
sverse phasic activity was increased under IbTx treatment
whereas that of longitudinal was increased in the presence
of ChTx. *denotes P < 0.05 vs. control level at 0%. #denotes
P < 0.05 between transverse and longitudinal.
tive bladder model in spontaneously hypertensive rats
and the rat bladder outlet obstruction model [4,10].
Findings in this study would lay the groundwork for
comparisons with the contractility of diseased rat detru-
sor, which would in turn give insights into altered physi-
ology in the human bladder in the future.
Whereas most other studies measured detrusor strip
contractility in the longitudinal direction, we also con-
sidered transverse contractions in our experiments. Dif-
ferences in contractile directions, i.e. transverse vs. lon-
gitudinal, in the detrusor have been reported under dif-
ferent experimental conditions [11-15]. Choice of species
and method of tissue preparation (i.e. urothelium-intact
vs. denuded tissues, or isolated strips vs. whole bladders)
apparently resulted in contrasting findings by different
groups. Nevertheless we demonstrated in the present
study that selected K+ channel blocker treatments could
reveal directional phasic contractile differences. These
differences should be considered when attempting to
draw conclusions from contractile data of a single direc-
tion only.
The stimulatory effect of various K+ channel blockers
in phasic contractions has been well documented, indi-
cating the role of K+ channels in mediating the tension
fluctuations [1]. In the basal or unstimulated state, KATP
and SK channels had no significant functional role in
longitudinal phasic activity of urothelium-intact detrusor
[5,6], findings supported by our data as well. We further
showed that transverse phasic activity was equally unaf-
fected by KATP and SK channel blockade. The distinctive
effects in transverse and longitud inal phasic activity were
demonstrated by blocking Kv, BK and IK channels. The
nonselective K+ channel blocker TEA enhanced phasic
activity in both transverse and longitudinal directions.
The effect in transverse phasic activity was greater due to
possibly larger contribution by Kv and BK channels, both
of which blocked by TEA, in this direction. This was
supported by the results of 4-AP and IbTx treatments
where transverse phasic activity was significantly higher
than the control level. The more prevalent transverse
phasic activity seen under 4-AP treatment has been
demonstrated elsewhere [12]. For IbTx, others have
shown different results depending on tissue origins and
preparation. In the longitudinal guinea-pig detrusor, pha-
sic activity was increased under IbTx treatment [8]. This
was in contrast to the whole rat detrusor where no cha-
nge in phasic activity was detected [9]. It is possible that
phasic activity in individual smooth muscle bundles may
not influence intravesical pressure in the whole bladder
to a great extent. Nevertheless, an intrinsic function in
the greater sensitivity of transverse phasic activity to-
ward BK, and also Kv channel blockade may be impli-
cated. Although ChTx could block BK channels, its use
as an IK channel blocker has been documented. In the
longitudinal but not transverse direction, phasic activity
was enhanced under ChTx treatment, again suggesting
direction-dependent differences in the regulation of pha-
sic contractions.
Spontaneous detrusor phasic contractions occur during
bladder filling to allow maintenance of bladder shape
and wall tension without drastically increasing intravesi-
cal pressure [1]. This prevents immature voiding as well
as ensures efficient micturition when necessary. Other
than K+ channels as demonstrated in this and other stud-
ies, Ca2+ channels [3], gap junctions [3] and the urothe-
lium [5,17,18] also play a part in mediating phasic con-
tractions. The role of the mucosal layer (including the
urothelium and myofibroblasts) is of particular interest
especially in disease conditions [2]. The use of normal
rat detrusor here nevertheless serves as a starting point to
examine disease-induced changes in bladders from es-
tablished rat models. Regardless of species and experi-
mental variations, the urothelium is believed to be an
active participant of normal and diseased bladder physi-
ology. Studies comparing the role of K+ channels in me-
Copyright © 2011 SciRes. OJU
diating urothelium-dependent and urothelium-indepen-
dent detrusor phasic contractions may be useful. It is not
possible to isolate the effects of the urothelium from
those of the smooth muscle if the intravesical pressure of
the whole bladder is measured, although this method
resembles in vivo physi ol o gy more closely.
In summary, the role of individual K+ channel block-
ers in mediating phasic activity in the detrusor was ex-
amined. In measuring phasic contractions in two direc-
tions, transversely and longidutinally, differential sensi-
tivity to the K+ channel blockers was demonstrated. Pha-
sic activity in the transverse direction could be distin-
guished from that in the longitudinal direction by using
selective blockers of Kv, BK and IK channels. The dis-
covery of differential phasic activity highlights the po-
tential importance in considering the physiological func-
tion of contractility in more than one direction.
5. Acknowledgements
This study was supported by the Singapore Ministry of
Education (RG63/06 and RG83/07) and the Institute of
Advanced Studies, Nanyang Technological University,
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