BJU International (1999), 84, 343–349

Carbachol-induced sustained tonic contraction of ratdetrusor muscleS.J . OH, S.C. AHN*, S.J . KIM*, K.W. KIM*, A. LEE, K.M. KIM and H. CHOIDepartments of Urology and *Physiology & Biophysics, Seoul National University College of Medicine, Seoul, Korea

Objective To investigate the underlying contractile Moreover, a similar SuTC was induced even after thecomplete elimination of extracellular Ca2+ by addingmechanism of the sustained tonic contraction (SuTC)

induced by repetitive carbachol application in rat 2 mmol/L EGTA to the Ca2+-free Tyrode solution. Toexclude intracellular Ca2+ sources related to the sarco-detrusor muscles.

Materials and methods Longitudinal muscle strips with plasmic reticulum (SR), the eCects of SR Ca2+ pumpinhibitors, cyclopiazonic acid (CPA, 10 mmol/L) andno mucosa were obtained from the anterior wall of

the urinary bladder in 12-week-old Sprague–Dawley thapsigargin (0.5 mmol/L) were tested. The carbachol-induced SuTC was insensitive to pretreatment withrats. Carbachol (5 mmol/L) was applied repetitively to

induce SuTC. The carbachol-induced SuTC was assessed CPA and/or thapsigargin. To deplete the ryanodine-sensitive Ca2+ pool, muscle strips were repetitivelyin the presence of various Ca2+-channel blockers and

drugs aCecting intracellular Ca2+ concentration. stimulated with caCeine (10 mmol/L) in the presenceof 10 mmol/L ryanodine, which did not aCect theResults The first application of carbachol elicited a large

phasic contraction followed by a tonic contraction carbachol-induced SuTC.Conclusions Although the characteristics of the carba-(TC); the carbachol-induced contraction was com-

pletely reversed by washing out the solution. However, chol-induced SuTC have not been defined, these resultsshow that a significant proportion of the carbachol-the initial phasic contraction was not reproduced after

a second or further application of carbachol. There induced contraction in rats is contributed by the SuTC,which is present even in the complete absence ofwas consistently only a SuTC with no phasic contrac-

tion. The amplitude of the SuTC was 85% of the TC external Ca2+. The SuTC was not aCected by limitingthe contributions of internal Ca2+ sources. This sug-induced by the first carbachol application. The appli-

cation of atropine (1 mmol/L) to the bath completely gests that the SuTC in rat bladders is unrelated toknown Ca2+ mobilization mechanisms.blocked SuTC. The carbachol-induced SuTC was insen-

sitive to nicardipine (5 mmol/L) and extracellular poly- Keywords Urinary bladder, calcium, carbachol, contrac-tion, smooth musclevalent cations (1 mmol/L, La3+ , Co2+, Cd2+, Ni2+).

Muscarinic stimulation is known to play an importantIntroduction

role in detrusor muscle contraction [9]. The mechanismof muscarinic stimulation-induced muscle contractionThe contractions of smooth muscles distributed in many

organs are modulated by various cellular signals; gener- has been widely investigated in gastrointestinal smoothmuscles [10]. However, in detrusor muscle, the mechan-ally, Ca2+ is recognized as the most important regulator

in smooth muscle contraction [1]. The Ca2+ sources to isms of muscarinic-stimulated contraction are not fullyunderstood. In the present study, we used a rat model,raise intracellular Ca2+ concentration are intracellular

or extracellular; Ca2+ influx from the extracellular space one of the most widely used animals for bladder disease,to investigate the characteristics and Ca2+ sources inoccurs through, e.g. voltage-operated Ca2+ channels [2],

voltage-insensitive, receptor-operated Ca2+ channels muscarinic stimulation-induced contraction of ratdetrusor muscle.[2,3], and nonselective cation channels [4,5]; the major

intracellular Ca2+ sources are known to be mobilized byInsP

3-induced calcium release (IICR) [6] or Ca2+-induced

Materials and methodsCa2+ release (CICR) [7,8] from sarcoplasmic reticulum(SR). Sprague–Dawley rats of either sex (mean body weight

380 g, sd 23) were exsanguinated after ether anaes-thesia. The whole bladder was excised and placed in aAccepted for publication 9 March 1999

343© 1999 BJU International

344 S.J . OH et al.

bath containing oxygenated phosphate-buCered Tyrode (4.1)% of that at the first application. The amplitude oftonic contractions was also reduced to 85 (6.0)% of thesolution at room temperature. Strips of the muscle coat

were obtained by removing the mucosal layer in phos- baseline tonic contraction at the first application.However, it was maintained at that level even afterphate-buCered Tyrode solution. Longitudinal muscle

strips (0.7 mm thick, 1.1 mm wide and 5.7 mm long) repeated applications. To investigate whether it is acharacteristic phenomenon in rat detrusor muscle, awere dissected from the bladder anterior wall.

Using vertical chambers (20 mL), one end of the similar experiment was performed under the same con-ditions but using rat gastric fundic circular muscle strips.muscle strip was tied to a glass hook attached to the

muscle-holder and the other connected to a Grass force In this experiment, 1 mmol/L carbachol (rather than5 mmol/L) was used as the maximal contraction occurredtransducer (FT03, Grass Instruments, Quincy, MA),

which was connected to an amplifier (P-122, Grass). at this concentration. Rat gastric muscle strips showedonly tonic contractions and the amplitudes were main-The analogue signal obtained from the force transducer

was converted to a digital signal (Polyview, Grass) at a tained even after 10 or more trials (eight strips). In thisexperiment, the amplitude of the sustained tonic contrac-sampling rate of 3 Hz and stored on a computer. Using

these stored data, contraction responses were re-plotted tions (SuTCs) observed after repeated application ofcarbachol was used to monitor the reproducibility.against time and analysed using suitable software

(Microcal Origin V4.1, SAS V6.04). The maximum con-traction was obtained from the muscle strips stretched

Extracellular Ca2+ and SuTCto #215% of resting length (in preliminary experiments)and that length was maintained through the experi- The contribution of Ca2+-influx through voltage-

operated Ca2+ channels to the SuTC was initiallyments. Before starting the study experiments, the musclestrips were equilibrated in CO

2/bicarbonate-buCered assessed using nicardipine. As shown in Fig. 2, treatment

with nicardipine before or after carbachol-induced con-Tyrode solution (36.5°C) for 40–50 min; this solutionwas used throughout except for experiments with extra- traction did not aCect the amplitude of the SuTC. Another

Ca2+-channel blocker, verapamil, gave similar resultscellular heavy metal ions, where 100% O2

/HEPES-buC-ered Tyrode solution was used. In some experiments, rat (data not shown).

The eCect of external polyvalent cations on SuTC wasgastric circular muscle strips were prepared and used asdescribed. Results were expressed as the mean (sem). also assessed. As 1 mmol/L of LaCl

3was reported to

eCectively inhibit the nifedipine-resistant, carbachol-Phosphate-buCered Tyrode solution contained(in mmol/L); NaCl 145, KCl 5.4, MgCl

21, CaCl

21.5, induced contractions in human urinary bladder [11],

the eCect of LaCl3

was determined; it had no eCect onNaH2PO

40.42, Na

2HPO

41.81 and glucose 5 (pH 7.35,

gassed with 100% O2

at room temperature). the SuTC at 100–1000 mmol/L (Fig. 2c, 17 strips). Otherinorganic Ca2+-channel blockers, e.g. Co2+ , Cd2+ andCO

2/bicarbonate-buCered Tyrode solution contained

(in mmol/L); NaCl 116, KCl 5.4, CaCl2

1.5, MgCl2

1, Ni2+ had no significant eCect on SuTC (data not shown).NaHCO

324, glucose 5 (pH 7.3–7.4, bubbled with

5% CO2/95% O

2). All drugs used in this study were

Contractions independent of external Ca2+purchased from Sigma, St. Louis, MO, USA.

Carbachol elicited SuTC even after repeated washoutwith Ca2+-free solution containing EGTA. However,

Resultsunder Ca2+-free conditions, 60 mmol/L K+ elicited nocontractions (Fig. 3a), suggesting that carbachol-induced

Characterization of carbachol-induced contraction in ratSuTC were independent of external Ca2+ concentration.

detrusorIn contrast, in rat gastric smooth muscle, both stimula-tions failed to elicit contractions (Fig. 3b). The amplitudeThe application of 5 mmol/L carbachol elicited a contrac-

tion which was sensitively inhibited by atropine (Fig. 1a). of the SuTC obtained under the Ca2+-free conditionswas 77 (3.0)% of the tonic contraction at the initialThere were two components to the contraction; an initial

phasic contraction lasting 93 (15) s (107 strips), followed application under normal Tyrode solution, and 85 (4)%when Ca2+ was supplied.by tonic contraction. On the first application of carba-

chol, small phasic contractions were superimposed onthe tonic contractions. Repeated application of carbachol

The role of intracellular Ca2+ on SuTCfor 10 min, with recovery periods of 25 min, attenuatedboth the phasic and tonic contractions to leave only the In the presence of 5 mmol/L nicardipine, the amplitude

of SuTC was changed little by applying inhibitors oftonic (Fig. 1b). The mean amplitude of the initial phasiccontraction at the third application was reduced to 31.2 the sarcoplasmic reticulum (SR) Ca2+-ATPase, i.e.

© 1999 BJU International 84, 343–349

CONTRACTION OF RAT DETRUSOR MUSCLE 345

Fig. 1. The characteristics of carbachol-induced bladder contractions: a, firstapplication of 5 mmol/L carbachol into thebath for 10 min; b, subsequent applicationsof carbachol.

Carbachol 5 µmol/L

b

a

Fourth

Third

Second

First

3 min

0.3 g

3 min

0.3 g

3 min

0.3 g

3 min

0.3 g

Atropine 1 µmol/L

3 min

0.3 g

cyclopiazonic acid (CPA) or thapsigargin (data not external Ca2+ , a similar experiment was conducted inCa2+-free solution containing 2 mmol/L EGTA. Undershown). To deplete Ca2+ from the SR completely, repeti-

tive electrical field stimulations (EFS, 60 Hz, 0.8 ms pulse these conditions also, CPA or thapsigargin did not changethe amplitude of SuTC (Fig. 4b), suggesting that SuTC isduration, for 2.5 s) (S48, Grass) were given for >30 min,

with repeated washouts in the presence of 10 mmol/L also independent of known internal Ca2+ mobilizationmechanisms.caCeine and 5 mmol/L ryanodine. CaCeine releases Ca2+

from intracellular stores by increasing the probability ofopen ryanodine-receptor type Ca2+-release channels

Discussion(RyR), and ryanodine locks RyR in a subconductingstate at micromolar concentrations [5,7]. Therefore, it The initial phasic contractions of rat detrusor muscle

were not reproducible during repeated applications ofwas expected that such combined treatment woulddeplete the stored Ca2+ of the SR. However, even after carbachol, while the amplitude of SuTC was maintained

relatively well. Although we cannot explain thisthis treatment, the amplitude of the SuTC was similar tothat of the control (Fig. 4a). To exclude the eCect of phenomenon fully, it did not result from the lack of

© 1999 BJU International 84, 343–349

346 S.J . OH et al.

Fig. 2. The eCects of nicardipine andpolyvalent heavy metal ions on thecarbachol-induced SuTC: a, treatment with10 mmol/L nicardipine after carbachol (n=18). b, pretreatment with 5 mmol/Lnicardipine. c, The eCect of extracellularLa3+ on the carbachol-induced nicardipine-

Carbachol5 µmol/L

La3+

Nicardipine5 µmol/L

0.1 g

3 min

a

c

1 mmol/L100 µmol/L

Carbachol5 µmol/L

Nicardipine5 µmol/L

0.1 g

3 min

Carbachol5 µmol/L

Nicardipine

0.1 g

2 min

10 µmol/L5 µmol/L

1 µmol/L

b

resistant SuTC of the rat bladder.

recovery periods between each carbachol trial, as there taken from the same animal and assessed under thesame experimental conditions.was a similar attenuation of amplitude of the initial

phasic contractions even after a 1-h rest (data not Ca2+ influx through voltage-operated Ca2+ channelshas been reported to contribute to bladder contractionshown). It might be unrelated to the concentration of

carbachol, because it occurred at 0.05–100 mmol/L car- during carbachol stimulation [12–16]. However, theextent of this contribution diCers depending on thebachol (data not shown). However, it could be related

to diCerences between organs; as shown in Fig. 1c, there species. In pig detrusor muscle [15], 0.3 mmol/Lnifedipine completely inhibited the carbachol-inducedwas no such an attenuation in the gastric muscle strip

© 1999 BJU International 84, 343–349

CONTRACTION OF RAT DETRUSOR MUSCLE 347

Fig. 3. External Ca2+-independentcontractions: a, Washing out the normalbicarbonate solution three times with Ca2+-free solution containing 2 mmol/L EGTA;the high K+-induced contraction wascompletely abolished in Ca2+-free mediacontaining EGTA. The baseline decreasedafter replacing normal bath solution withCa2+-free solution plus 2 mmol/L EGTA. b,

Under the same Ca2+-free conditions,5 mmol/L carbachol caused no contractileresponse in fundic circular strips of ratstomach muscle.

Tyrode solution

Carbachol5 µmol/L

Calcium-free solution+ 2 mmol/L EGTA

0.5 g

3 minb

a

K 60 mmol/L

Tyrode

Carbachol5 µmol/L

Calcium-free solution + 2 mmol/L EGTA

K 60 mmol/L

0.1 g

3 min

contraction. However, in rat detrusor muscle [12], a large extracellular sources are probably not a major contribu-tor to the Ca2+ increase.component (20–40%) of the carbachol-induced contrac-

tion was resistant to nifedipine at up to 1–3 mmol/L. Also, In rat urinary bladder, Ca2+ mobilization from theinternal Ca2+ store has been reported to play a role inin human bladder [11], it was reported that carbachol-

induced contraction was relatively insensitive to nicardip- the carbachol-induced contraction [12]. In that report,carbachol elicited a transient contraction even afterine, while nicardipine-resistant contraction was suscep-

tible to LaCl3

, a nonspecific blocker of Ca2+ movement prolonged perfusion with Ca2+-free medium containingEDTA. However, in the present study, the role of Ca2+through the plasma membrane.

In the present study, most of the carbachol-induced released from internal store was ambiguous in main-taining the SuTC; the latter was not aCected by CPA orcontraction was resistant to nicardipine and polyvalent

cations, indicating the possible presence of unknown thapsigargin, both inhibitors of the SR Ca2+-ATPase.Also, the combined treatment with ryanodine andCa2+ influx pathways in rat urinary bladder smooth

muscle. However, as complete removal of external Ca2+ caCeine did not aCect the amplitude of SuTC [5]. Thus,Ca2+ release from the internal Ca2+ stores by previouslyonly slightly decreased the amplitude of the SuTC, the

© 1999 BJU International 84, 343–349

348 S.J . OH et al.

Fig. 4. a, In the presence of 5 mmol/Lryanodine and 10 mmol/L caCeine, theinternal Ca2+ store was abolished byrepetitive EFS. However, after undergoingthis procedure three times, there was nosignificant decrease in the amplitude of theSuTC. b, In Ca2+-free solution containing2 mmol/L EGTA, the SuTC still occurredafter pretreatment for 20 min withthapsigargin (0.5 mmol/L). The baselinetone decreased after changing from Ca2+-free solution containing 2 mmol/L EGTA.The SuTC was completely abolished by1 mmol/L atropine. The response of theSuTC to pre-incubation with 10 mmol/Lcyclopiazonic acid produced the same

Tyrode solution

Carbachol5 µmol/L

Calcium-free solution+ 2 mmol/L EGTA

Thapsigargin 0.5 µmol/L

Atropine1 µmol/L

0.1 g10 min

b

Before EFS After EFS

Carbachol5 µmol/L

Nicardipine5 µmol/L

0.1 g

3 min

Caffeine 10 mmol/LRyanodine 5 µmol/L

EFS

a Washout WashoutWashout

20 min

0.5 g

response (data not shown).

known mechanism might have no role in the mainten- can be mobilized from other storage sites. Ca2+ could bereleased from the ‘tightly bound intracellular Ca2+ pool’ance of SuTC.

The present SuTC was similar to that reported by [11]. The candidates for such sites might be mitochondria[17] and caveolae [18], although the role of these twoMaggi et al. [11]; in that study, they concluded that

nifedipine-resistant contraction resulted from the mobiliz- components in the contraction has not been elucidatedclearly.ation of intracellular Ca2+ because it was inhibited by

tetracaine or caCeine. However, in the present study, the The tonic phase of bladder contraction, rather thanthe initial phasic phase, is reportedly important in uri-SuTC was not aCected by pretreatment with CPA, thapsi-

gargin and ryanodine, more selective inhibitors of SR nary emptying, according to in vitro whole-bladderexperiments [19], and cholinergic stimulation is theCa2+ release. The reason for the discrepancy between

the present results and those of Maggi et al. could not main contributor. The SuTC assessed here was resistantto those conditions that modified intra- or extracellularbe determined.

These results indicate that the SuTC is independent of Ca2+ mobilization. We have confirmed that such contrac-tions are present in humans, even in the absence ofknown Ca2+ mobilization mechanisms; however, Ca2+

© 1999 BJU International 84, 343–349

CONTRACTION OF RAT DETRUSOR MUSCLE 349

in smooth muscle. In Kao CY, Carsten ME, eds, Cellularknown Ca2+ mobilization mechanisms from external orAspects of Smooth Muscle Function. Cambridge: Cambridgeinternal sources (unpublished observation), and furtherUniversity Press, 1997: 132–68study is currently underway. From these results, it is

11 Maggi CA, Giuliani S, Patacchini R et al. Multiple sourcessuggested that the carbachol-induced SuTC is indepen-of calcium for contraction of the human urinary bladderdent of known mechanisms of intra- or extracellularmuscle. Br J Pharmacol 1989; 98: 1021–31

Ca2+ mobilization, and the eCect on such SuTC may12 Maggi CA, Manzini S, Parlani M, Conte B, Giuliani S, Meli

have to be considered in the development of therapeutic A. The eCect of nifedipine on spontaneous, drug-inducedagents against the overactive bladder. and reflexly-activated contractions of the rat urinary

bladder: evidence for the participation of an intracellularcalcium store to micturition contraction. Gen PharmacolAcknowledgements1988; 19: 73–81

13 Bhat MB, Mishra SK, Raviprakash V. DiCerential susceptibil-This work was supported by grant no. 03–1998–042ity of cholinergic and noncholinergic neurogenic responsesfrom the Seoul National University Hospital Researchto calcium channel blockers and low Ca2+ medium in ratFund. We give our best appreciation to the devotingurinary bladder. Br J Pharmacol 1989; 96: 837–42eCorts of Miss So-Young Lee.

14 Lowe VC, Noronha-Blob L. ECect of extracellular Ca2+ oncholinergic, KCl and phorbol ester-mediated phosphoinosi-tide turnover and guinea pig urinary bladder contraction.

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