Pharmacology & Toxicology 199 I , 68, 34-38.

Motor Effects of Loperamide on Rat Urinary Bladder: An in Vitro Study

A. Berggren, U. S i l l h and A. Rubenson

Department of Pediatric Surgery, b t r a sjukhuset. S-41685 Goteborg, Sweden

(Received February 6, 1990; Accepted July 19, 1990)

Abstract: Motility recordings in muscle strips from rat urinary bladder were performed and the effect of the opiate agonist loperamide on motor activity was studied. Loperamide induced a concentration-dependent ( 10-7-10-3M) inhibition of the contractile response of the detrusor strip of the same order of magnitude after activation of intramural nerves, stimulation of cholinergic receptors with acetylcholine and after direct depolarisation of the cell with potassium. Pretreat- ment with the opiate-antagonist naloxone ( IO-’M) did not antagonise the inhibitory action of loperamide on bladder motility regardless of the type of activation. Naloxone per se. however, facilitated the nerve-mediated motor response. The inhibitory action of loperamide on the potassium-induced contraction could partly be counteracted by elevation of the calcium concentration in the medium. It is suggested that the demonstrated inhibitory effect of loperamide on bladder motility is a calcium-dependent direct smooth muscle action. without any significant opiate-receptor-mediated action in the present in vitro preparation.

Besides their well documented central nervous inhibitory effects on bladder motility (Sillen & Rubenson 1986; Dray & Metsch 1984), opioid substances have recently been sug- gested to participate as endogenous inhibitory mediators in peripheral autonomic pathways to the bladder (Kawatani et ul. 1983; deGroat et al. 1984; Vaalasti et al. 1985). In agreement with these findings are the results of a recent experimental in vivo study in which the peripherally active opioid agonist loperamide was shown to inhibit bladder motility (Sillen et al. unpublished results). The demonstrated inhibitory effect of loperamide seemed, however, to be me- diated only partially by peripheral opioid receptors, since the opioid-antagonist naloxone could only partly counteract the effect of loperamide. The origin of this main non-opioid part of the depressive effect of loperamide remains unclear but there are indications that loperamide might act directly on the smooth muscle in the detrusor.

In order to analyse further the mechanism of action of the inhibitory effect of loperamide on detrusor motility, an experimental in vitro set-up has been used and the posibility of receptor-mediated actions, as well as direct smooth muscle actions, has been evaluated.

Materials and Methods Male Spraque-Dawley rats (20C350 g), anaesthetised with pento- barbitone (60 mg/kg intraperitoneally), were used in all experiments. The abdomen was opened by midline incision and the urinary bladder was removed subtotally by a transverse incision just above the trigone. From the removed bladder, four detrusor strips includ- ing all layers (2 x 5 mm) were prepared by incisions from the trigonal end of the bladder towards the fundus. This procedure was carried out under a microscope. After preparation, the strips were mounted vertically in a 20 ml tube containing Krebs solution, connected at the bottom of the tube to a rigid support and at the other end to a force displacement transducer (Grass FT IOC). The detrusor strips were initially loaded with a tension of 5 mN according to studies by e.g. Mackenzie & Burnstock (1984). and the mechanical activity

was recorded isometrically and displayed on a Grass polygraph. The strips were allowed to equilibrate in the organ bath for I hr before the start of the experiment, in order to achieve a steady basal tone.

All test drugs were added to the bath in increments of I ml and were maintained there until the detrusor strip reached the maximal tension response or for at most 5 min., and then washed out until the tension of the strip returned to the level observed before adminis- tration. With this rinsing procedure, the interval between drug ad- ministration was 15-20 min. All blocking agents (atropine, loperam- ide, naloxone) were added to the bath about 10 min. before any kind of stimulation was performed.

To investigate the effect of loperamide on excitatory motor rc- sponses of the detrusor muscle, the influence of loperamide on responses due to A) cholinergic receptor stimulation. B) direct smooth muscle activation and C) stimulation of intramural nerves, was tested.

A . Cholinergic stimulation. Stimulation of cholinergic receptors in the detrusor strip was achieved by administration of acetylcholine (ACh) in increasing concentrations ( 10-7-10-’M). In some experi- ments, only one concentration of ACh (10-’M) was used. This concentration evoked a contractile response of 33 8 mN (n = 26). corresponding to 50% of the maximal contractile response (EC50, fig. 2). In control experiments, ACh (10- ’M) was administered at 10 min. intervals for 2 hr with rinsing in between. No fatigue of the contractile response was seen. In some experiments, atropine was used to achieve muscarinic receptor blockade and a concentration of 10-6M was found to give total muscarinic receptor blockade (fig. 6).

B. Smooth muscle activation. To obtain postsynaptic direct smooth muscle stimulation, a membrane depolarisation procedure was used (Bolton 1979). including a muscle cell contraction by increasing the extracellular K+-concentration. This ionic change in the organ bath was accomplished by changing the normal Krebs solution to a medium containing a tenfold higher potassium concentration (4.73 to 50 mM) and simultaneously lowering the Na concentration. The K +-concentration (50 mM) used in these experiments corresponded to the EC5O value of potassium, obtained from a concentration- response curve of K+-induced contractions.

MPERAMIDE AND RAT URINARY BLADDER 35

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C. fn/ramural nerve stimulation, Intramural nerve stimulation was performed by creating an electrical field between two parallel plati- num plates ( 5 x 5 mm), fixed on either side of the detrusor strip at a distance of 7-8 mm. The stimulation was obtained by means of a constant-current stimulator (Hassle AB, Sweden), delivering square wave pulses with a constant current alternating directions.

In the light of data from others (Krell e/ al. 1981; Milne e/ al. 1978; Creed er al. 1983) and the results from our own studies of the parameters, we have chosen to use 8 Hz, 0.5 msec., 6&90 mA in trains of 5-6 sec. for the electrical field stimulation. The contractile response induced by these parameters was TTX-sensitive and thus abolished by addition of TTX I .5 x 10-6M to the organ bath.

Soluiions. The normal Krebs solution had the following compo- sition (mM): NaCl 133, NaHCO, 26.3, glucose 8.5, KCI 4.73, CaCI, 3.31, NaH2P0, 1.92, MgSO, 1.25. The pH was 7.4. The irrigation solution was gassed with 95% O2 and 5% C 0 2 and the temperature maintained at 37°C.

In some experiments the Cat +-concentration in the solution was altered (0.25-30 mM). Since C a t + would precipitate in the higher calcium-concentrations, the Krebs solution was changed to a buf- fered tromethamol solution (TRIS). This TRIS-solution was gassed with 100% O2 and the pH was rigorously kept at 7.4.

Drugs. The following drugs were used in the experiments acetylcho- line chloride (Sigma, 10-8-10-’M), naloxone (Sigma, 10-8-10-3M), loperamide (Imodium@, Janssen, 10-8-10-’M), tetrodotoxin (TTX, Sigma, 1.6 x 10-6M). All substances were dissolved in saline except loperamide, which had to be dissolved in a small amount of propyle- ne glycol (0.5 ml per mg loperamide). The concentration of propyle- ne glycol used did not have any effect on the detrusor muscle (n=8). All solutions and drugs were prepared on the day of the experiments.

Siatistical analysis. Student’s t-test for paired and un-paired data were used for comparing means. All results in the figures and the text are given withkS.E.M.

Results

Effect of loperamide on cholinergically induced contractions. The ACh ( 10-3M = EC5O)-induced contraction of the prepa- ration was concentration-dependently suppressed after pre- treatment with loperamide 10-7-10-3M) n=24, fig. 1A). Loperamide 10-7M had no significant effect, whereas the ACh response was totally abolished at a loperamide concen- tration of 10-3M. Loperamide itself was found to have no significant inhibitory or facilitatory action on the spon- taneous activity of the detrusor muscle in concentrations

Acetylcholine Field stimulation Potassim 50 mM A R c

7 8 5 4 3 - m 7 6 5 4 3 a p 7 6 5 4 3 - @

Loperarmde c m (M) Lwrarmde corn (M) Loperamide corn (MI

Fig. I . Concentration-dependent inhibition (k S.E.M.) by loperami- de of contraction induced by A. acetylcholine (ACh lO-’M), n = 24). B. electrical field stimulation (FS, n = 18) and C. depolarisation with potassium (K+ 50 mM, n=8) in isolated rat detrusor.

Acetylcholine + Atropine 1 6 % P T Acetylcholine

Acetylcholine + Loperamide 10-5M

8 7 6 5 4 3 2 t -Iog Acetylcholine concentration (U)

Fig. 2. Effect of atropine (IO-’M, n = 12) and loperamide (IO-’M, n= I2,iS.E.M.) on the contraction-response curve for acetylcho- line (control, n=26) in isolated rat detrusor.

between and 10-3M. To evaluate a possible anticholin- ergic action of loperamide, the concentration-response curve of ACh (10-’-10-’M) was compared with the curves ob- tained when atropine (10-5M) and loperamide (IO-jM), respectively, were added. Atropine displaced the ACh con- centration-response curve to the right without changing its shape or depressing the maximum (fig. 2), indicating a com- petitive inhibition. Loperamide ( 10-5M), on the other hand, changed the slope and the maximum of the ACh concen- tration-response curve and a so-called miniature curve was obtained (fig. 2), indicating a non-competitive antagonism of loperamide.

Effect of loperamide on intramural nerve stimulation-induced contractions. Detrusor strip contractions induced by electrical field stim- ulation (8 Hz, 0.5 msec. 3S100 mA) were effectively re- duced in a concentration-dependent manner by loperamide ( 10-7-10-3M, n = 18), administered cumulatively at 2S30 min. intervals (fig. 1B). No reduction of the nerve-induced contractile response was seen at a loperamide concentration of 10-7M (107 7.6% of control, p = NS). A loperamide concentration of lO-’M reduced the contractile response to 53 f 6.9% of control, whereas the response was totally abolished at loperamide concentrations above lOP3M. After repeated rinses, the detrusor strip response recovered within 30-60 min. after administration of loperamide concen- trations up to 10-4M, whereas the highest loperamide con- centration ( 10-3M) caused an irreversible inhibition.

The inhibitory effect of loperamide on detrusor strip contrac- tility: Interaction with naloxone. In experiments where the nerve-mediated contractility was inhibited concentration-dependently by loperamide (10- 7-10-3M), the remaining detrusor response could be facili- tated by addition of naloxone lO-’M (fig. 3). The enhance- ment of the detrusor activity induced by naloxone was, however, not a reversal of the loperamide-induced inhibition

36 A. BERGGREN ET AL.

*** wow1 ** P<O01

7 6 5 4 3-log 7 6 5 4 -log

LOperamide cmentration (MI Loperamide concentration (M)

Fig. 3. Effect of loperamide (10-7-10-3M,+S.E.M.) on contractions in isolated rate detrusor induced by A. electrical field stimulation (FS) with (n= 11) and without naloxone (10-’M, n = 10) pretreat- ment and B. acetylcholine (ACh 10-3M) with (n= 16) and without naloxone (IO-’M, n = 10) pretreatment. Values without dots are not significantly different from each other.

since a facilitation of the field stimulation response by naloxone was also seen in experiments without loperamide (naloxone iO-8-iO-3, fig. 4).

The above-described potentiation by naloxone of the elec- trically induced contraction was not obtained when muscar- inic receptors were blocked with atropine IO-’M (fig. 4). Furthermore, naloxone 1 O-8-10-3M did not significantly affect the motor response induced by ACh 10-’M either without (fig. 4) or with (fig. 3) simultaneous loperamide administration.

Effect of loperamide on potassium-induced contractions. Potassium-induced detrusor strip contractions could be con- centration-dependently reduced by pretreatment with loper-

200

160

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Atropine 10%4

Acetylcholine 10-3M

*+* pco.001

** pco.01

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8 7 6 5 4 3-log Naloxone concentration (MI

Fig. 4. Effect of naloxone (10-8-10-’M) on contractions in isolated rat detrusor induced by A. electrical field stimulation (FS, n = 16), B. electrical field stimulation with atropine (IO-’M, n=28) pretreatment and C. acetylcholine (ACh IO-’M, n = 16). The given significances for the curves are in comparison with controls and values without dots are not significant. The arrows represent S.E.M.

amide (10-7-10-3M, fig. 1C). The lowest loperamide con- centration ( 10-7M) did not significantly change the potas- sium-induced contraction, whereas pretreatment with loperamide 10-3M could reduce the contraction to 5 f 2.4% of control (n = 8).

The inhibitory effect of loperamide on potassium-induced de- trusor contractility - Interaction with calcium fluxes. The effect of loperamide lo-’ and lOP4M) on potas- sium (50 mM)-induced contraction of detrusor strips in different calcium concentrations (0.25-30 mM) in the organ bath is shown in fig. 5. Only one concentration of loperami- de was tested in one preparation. As depicted in fig. 5, the inhibitory effect of loperamide IO-’M could be counteract- ed by increasing the C a t + content of the solution in the bath. The pronounced inhibitory effect of the highest con- centration of loperamide tested ( 10-4M) could only be mini- mally counteracted, and then only by high Cat +-concen- trations (> 5 mM, fig. 5). After pretreatment with the lowest loperamide concentration tested ( 10-6M), no significant in- hibition of the contraction was registered even in media with low Cat +-concentrations where no counteraction could be expected. Instead, a non-significant facilitation of the con- tractile response was observed at this loperamide concen- tration in media with Ca+ +-concentrations above 2.5 mM.

The inhibitory effect of loperamide on the detrusor strip contractility was also compared with the effect of terodiline, a calcium channel blocking agent with antimuscarinic prop- erties. As shown in fig. 6, it was found that terodiline (10-7-10-3M) had similar effects to loperamide ( 10-7-10- 3M) on the contractile response induced by 1) cholinergic stimulation, 2) electrical nerve stimulation and 3) K + -in- duced contractions (n= 12). Furthermore, in the experi- ments with terodiline a facilitatory effect of all three tested contractile responses was seen after the lowest concentration of the drug (lO-?M), beside the concentration-dependent inhibition seen after higher doses ( 10-6-10-3M).

0.25 1.0 2.5 5.0 15 30 Calciumconcentration (mM)

Fig. 5 . Effect of loperamide 10-6M (n=12), lO-$M (n=30) and lO-’M n = 12) on contractions in isolated rat detrusor induced by potassium in calcium concentrations from 0.25 to 30 mM. In the Ca2+ control curve 100% of control was chosen as the potassium induced contraction in a medium with a calcium concentration of 30 mM. Arrows represent S.E.M.

LOPERAMIDE AND RAT URINARY BLADDER 31

50 -

0-

Loperamide A :I4\. 0 A Field Ace;l K * 50mM sti-RIIation khohe 10%

Terodiline

Fig. 6. Comparison of inhibitory effects of loperamide 10-’-10-’M and terodiline IO-’-IO-’M on acetylcholine (n = 8 and n = 12, resp.), electrical field stimulation (n= 16 and n = 12, resp.) and potassium- induced contractions (n = 12).

Discussion

Our in vitro data demonstrate a clear-cut inhibitory action of the opioid agonist loperamide on bladder motor activity, probably via a direct action on the detrusor muscle and to a minor extent via interaction with opioid mechanisms. Evidence for such a direct interference with the smooth muscle cell on a post-receptor level in the urinary bladder is given by the finding that the drug reduces the contractile response of the detrusor to about the same extent regardless of the type of activation of the muscle strip. The reduction of the motor response is thus similar after activation of intramural nerves, stimulation of cholinergic receptors with acetylcholine and direct depolarisation of the cell with pot- assium. Further support for a post-receptor action of loper- amide is the fact that the drug seems not to possess specific antagonistic actions on the main contractile system of the urinary bladder i.e. anticholineric effects, since the bladder response to cholinergic receptor stimulation with acetylcho- line could not be competitively inhibited with loperamide.

The suggested direct inhibitory effect of loperamide on the detrusor strip appears to be dependent on the ambient calcium concentration, indicating a calcium-antagonistic ac- tion of the drug, since the depressive effect of loperamide can be significantly reduced by elevation of the calcium concentration in the solution.

A further indication of an interaction with calcium-ion channels is the fact that loperamide and the well-known calcium channel blocking agent, terodiline (Husted et al. 1980) exert a similar depressive action on the detrusor con- tractions induced by activation of intramural nerves, stim- ulation of cholinergic receptors and direct depolarisation of the cell. The above-described findings suggesting a calcium- blocking action of loperamide as being responsible for the inhibitory effect on the bladder strip contractility are also in line with the reports by Kenakin & Beek (1982) and Reynolds et al. (1984), in which a calcium-antagonistic ac- tion of the drug in the gut was demonstrated. Calcium-

antagonistic properties of loperamide in the gut have also been reported by Javezc el al. (1982), who suggested binding of the drug to calmodulin, a calcium-binding, regulatory protein.

We recently also demonstrated an inhibitory effect of loperamide on detrusor motor activity in an in vivo experi- mental study in the rat (Sillen et al. unpublished results). In this latter investigation, the drug appeared to induce inhibition not only by direct activation of the smooth muscle membrane, as described in this study, but also by stimula- tion of opioid mechanisms.

In the present in vitro preparation, however, there are no valid indications of such an opioid part of the inhibitory action of loperamide on motor activity of the bladder strip. The partial blockade of the inhibitory action of loperamide on the nerve-induced contraction seen after pretreatment with the specific opioid antagonist naloxone can be inter- preted as specific, but since naloxone per se appears to increase the nerve-mediated bladder contractility to about the same extent, this conclusion cannot be drawn. The dif- ference in mechanism of action of loperamide, with respect to stimulation of opioid receptors, on detrusor muscle con- tractility in vivo as compared to in vitro might reflect the fact that in virro bladder preparation is devoid of autonomic ganglia (Alm & Elmer 1975), which is considered to be the target area for opioid mechanisms (deGroat er al. 1984).

On the other hand, the finding that naloxone per se influences the nerve-mediated bladder response in the pres- ent in vitro preparation, reflected by a facilitating effect of the drug on the electrically induced contractility, might indicate that opioid mechanisms peripheral to the pelvic ganglia are involved in the nervous control of detrusor motor activity as well. Hitherto, however, there are no indi- cators that opioid receptors are present in the detrusor muscle of the rat, peripheral to the autonomic ganglia, although it has been shown in human detrusor muscle (Vaal- asti et al. 1986; Cairou el al. 1985).

Thus, further investigation are needed to clarify these relationships. Moreover, the described facilitating effect of naloxone on the nerve-induced contraction seems to interact with muscarinic receptors since pretreatment with atropine reduced the effect of naloxone on the nerve-induced re- sponse. The muscarinic interaction of naloxone is probably not on the cholinergic end-receptor, since the contractions induced by acetylcholine are unaffected by naloxone.

Acknowledgements

Research Council (€386-1 9X-05450-08B). This work was supported by grants from the Swedish

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