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Effects of morphine in the isolated mouse urinary bladder
Gen. Pharmac. Vol. 17, No. 4, pp. 449--452, 1986 Printed in Great Britain
0306-3623/86 $3.00 + 0.00 Pergamon Journals Ltd
EFFECTS OF MORPHINE IN THE ISOLATED MOUSE URINARY BLADDER* C. G. ACEVEDO, L. TAMAYO and E. CONTRERAS Departamento de Ciencias Fisiol6gicas, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Casilla 2407, Concepci6n, Chile (Received 22 January 1986) Abstract--1. Acute morphine increased the responses to acetylcholine of the isolated mouse urinary bladder. 2. A chronic morphine treatment did not change the responses of the urinary bladder to acetylcholine or ATP. 3. The acute administration of morphine did not modify the contractile response to ATP in the urinary bladders from untreated or chronically morphine treated mice. 4. Methadone and ketocyclazocine decreased the responses to the electrical stimulation of the urinary bladder. These depressant effects were not modified by naloxone. 5. The results suggest the nonexistence of opiate receptors in the mouse urinary bladder and the lack of direct effects of morphine on the neuroeffector junction.
INTRODUCTION An increase in the activity of the autonomieally innervated organs is observed when animals are withdrawn from a chronic morphine treatment. This change in activity may arise from the sustained blockade of transmitter release induced by morphine at the neuroeffector junction (Collier, 1969). Several authors have suggested that the basis of morphine abstinence is the result of an increased release of a neurotransmitter presumptively suppressed by the presence of morphine (Paton, 1957; Sharpless and Jaffe, 1969). Fleming (1976) has given the name supersensitivity to this state, which is attributable to a change in the responsiveness of the cells to a neurotransmitter. Changes in sensitivity have been observed in several tissues, namely the guinea-pig ileum (Paton, 1957), the mouse vas deferens (Rae et al., 1977). These tissues contain several neurotransmitters ineluding enkephalins (Gintzler, 1979); the presence of opiate receptors has also been described in them (Simon, 1984). The present study was designed to study the effects of acute and chronic morphine treatments on the sensitivity of the mouse urinary bladder to acetylcholine and ATP, 2 neurotransmitters involved in the contractile response of this organ (Acevedo and Contreras, 1985). The possible induction of altered responses produced by morphine to the electrical stimulation of the preparation was also investigated.
Acevedo and Contreras (1985) was set up in 12 ml organ bath at 30°C containing Tyrode solution of the following composition (mM) NaC1 194; KCI 4.69; CaCI2 1.90; MgC12 0.5; NaHCO3 25; glucose 5.04 and gased with 95% 02 and 5% CO2. For electrical stimulation the tissue was passed through a pair of platinum electrodes with rings 22 mm apart, and connected to a Grass FT 03 force-displacement transducer connected to a Grass polygraph. The preparation was subjected to 0.5 g basal tension. Drug additions and frequency-response curves were made over frequency of 0.1-50 Hz with pulses of 0.5 msec duration at supramaximal voltage (60 V). In order to avoid preparation fatigue, 15 min time periods were allowed between frequency-response or concentration-response curves. Most experiments were performed under the effect of atropine sulphate (10 -6 M) and guanethidine (3.4 x 10 -6 M).
MATERIALS AND METHODS Male albino mice (26-32 g) of a strain raised in our laboratory were stunned and killed by exanguination; the abdomen was opened by a midline incision and the urinary bladder was removed and placed in a physiological solution. A strip obtained according to the procedure described by *Supported by Direcci6n de Investigaci6n, Proy. 20.33.13, Universidad de Concepci6n. 449
Chronic treatment with morphine Mice received a single injection of morphine in a suspension (morphine as base, 300 mg, sorbital sesquioleate 0.8 ml, liquid paraffin 4.2 ml and saline 5 ml); each animal received 300 mg/kg (s.c.). The animals were killed 72 hr after the injection of morphine. The contractile effects of acetylcholine or ATP were tested in the presence or absence of morphine or other opiate as indicated in Results. Pharmocological responses The contractile effects of acetylcholine on the urinary bladder were tested by cumulative dose-response curves, first in the Tyrode solution without morphine (or the other opiate tested) and afterwards under different concentrations of the analgesic. When ATP was used as the contractile agent the preparation was extensively washed by renewing the bath fluid, and at least 15 min were allowed between each assayed concentration. Frequency-response curves were elicited in the presence or absence of the assayed opiate. The acute effects of morphine were assayed at 2 concentrations: 2.5 x 10 -5 M and 5.0 x 10-5 M. Doses for other drugs are given in Results. Statistical analysis Results of the contractile compounds assayed or the electrical stimulation are expressed % of the maximum response induced by either procedure. The data are ex-
C.G. ACEVEDOet al.
450
pressed as mean + SEM. Student's t-test was used to determine the statistical significance of difference between the means.
/j
120-
Drugs 1 O0 -
The drugs used were the following: noradrenaline hydrochloride, acetylcholine hydrochloride, ATP, all from Sigma Chemical Co., atropine sulphate, morphine hydrochloride and nalorphine hydrochloride from Merck, guanethidine (Ciba), ketocyclazocine (Sterling Winthrop Research), naloxone hydrochloride (Endo Labs Inc.), methadone hydrochloride (May and Baker).
.~_
80-
60. t,O,
RESULTS
Effect of morphine on the cholinergic response of the urinary bladder
20-
The possibility that a chronic morphine treatment might alter the cholinergic response of the mouse urinary bladder was studied after the sustained treatment with the opiate. The treatment did not change the concentration-response curve to acetylcholine. The effects of acute morphine (2.5 x 10 -5 M and 5 × 10 5M) were also tested on tissues from untreated and chronically morphine treated mice. Morphine induced an enhanced response to acetylcholine in both experimental groups (Fig. 1). The augmented responses did not differ in both experimental groups and the effect of both concentrations of the analgesic induced a similar enhancing response. Nalorphine induced a lower potentiating effect when added at similar concentrations to those tested for morphine (not shown). Naloxone (10-rM) did not change morphine or nalorphine effects. Neostigmine (3 × 10 -6 M) suppressed the morphine potentiating effect on the cholinergic response.
are shown in Fig. 2; the chronic morphine treatment did not change the responsiveness of the tissue to ATP. When the effects of this putative neurotransmitter were assayed in the presence of acute morphine in the bathing medium no changes in the responsiveness of the urinary bladders were observed.
Effects of morphine on the contractile response insensitive to atropine
Effects of morphine and other opiates on the neurogenic response of the mouse urinary bladder
ATP was assayed in the urinary bladders from naive and morphine treated mice. The concentration-response curves for the adenine n u cleotide were performed in the presence of atropine and guanethidine in the Tyrode solution. The results
Frequency-response curves ranging from 0.1 to 50 Hz were obtained in the presence of guanethidine and atropine. The chronic morphine treatment did not change the response of the muscle to the electrical stimulation at each different frequency tested.
•//
120
//
,
6 - log
conc
ATP
(M)
Fig. 2. Responses of the mouse urinary bladder to ATP. Untreated mice (O O, n = 6); untreated mice after acute morphine 2.5x 10-SM ( O - - - O , n = 6 ) ; the "washed" preparation from chronically treated mice (x x, n = 6); urinary bladders from chronically treated mice after the acute administration of morphine 2.5 x 10-SM ( x - - - x , n =6).
B 120
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,
/
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~ 80-
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g 60-
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u ax =£ 40-
20-
20-
-------t/, - IO9 CO~ acetylcholine (Id)
-log ¢on¢ acetylchol*ne ( M }
Fig. 1. (A). Responses of the mouse urinary bladder to acetylcholine before (O O, n = 10) and after acute administration of morphine 2.5 x 10-5 M ( x - - - x , n = 10) and 5 x 10-5 M ( 4 A, n = 10). *Values statistically different from those observed before morphine administration. P < 0.01 (paired t-test). (B). As in (A), but the urinary bladders were obtained from mice chronically treated with morphine.
Morphine and mouse urinary bladder
451
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i
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~ 1~
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Fig. 3. (A). Frequency-response curves of the mouse urinary bladder before (O O, n = 10); after the acute administration of methadone 10-5 M (x x, n = 6) and after the acute administration of ketocyclazocine 2.5 x 10-5 M (A A, n = 6). *P < 0.01 (paired t-test). (B) As in (A), but the urinary bladders were obtained from mice chronically treated with morphine.
The presence of acute morphine in the Tyrode solution (2.5 x 10 -5 M or 5 x 10 -5 M) did not alter the neurogenic response of the tissue (not shown). Methadone (5 x 10-6M and 10-SM) and ketocyclazocine (10 -5 M and 2.5 x 10 -5 M) decreased the response to the electrical stimulation of urinary bladders from naive and chronically treated mice. The results are shown in Fig. 3. The depressant effects of both opiates were unchanged by the presence of naloxone in the bath (10 -6 M), which suggests the inexistence of opiate receptors.
DISCUSSION
Guinea-pig ileum (Paton, 1957), mouse vas deferens (Henderson et al., 1972) and rat colon (Huidobro-Toro and Way, 1981) have been shown to have specific opiate receptors and have been used as models for the study of the mechanisms of action of narcotic analgesics. In these tissues morphine effects are readily reversed by naloxone and the sustained contact with opiates results in tolerance, which is an important feature of the interaction of opiates with specific receptors. In a previous work we described changes in the sensitivity to neurotransmitters and catecholamines in mice vas deferens which had been chronically treated with morphine at doses similar to those used in the present work (Contreras et al., 1982). We also showed a dependence like effect when acute morphine was added to the "washed" preparation. Under this condition the analgesic induced a facilitatory action to the contractile effect of catecholamines. A similar effect can be seen to occur in toad skin chronically treated with morphine (Contreras et al., 1980). A dependence like effect was observed on the transepithelial potential and short circuit current when the analgesic was omitted from the bathing solution. The addition of morphine to the skin turned the reduced response to catecholamines to values similar to those obtained in skins from untreated toads. On the other hand, the existence of a purinergic component in the contractile response of the urinary
bladder has been described in several species (Bumstock, 1981). In a previous work (Acevedo and Contreras, 1985) the ability of purine nucleotides to contract the mouse urinary bladder has been established and the evidence suggests that ATP may be the transmitter responsible for the non-cholinergic nonadrenergic response of this organ. Consequently, we thought it of interest to study the effects of acute and chronic morphine on the purinergic responses of this organ. The interaction of opiates on the contractile responses to ATP has not been studied. However, the inhibitory effect of ATP and adenosine have been investigated in the guinea-pig ileum (Gintzler and Musacchio, 1975). In this tissue morphine, ATP and adenosine inhibit the contractions induced by electrical stimulation. The relaxatory responses are potentiated by phosphodiesterase inhibitors and other compounds affecting purinergic mechanisms. The inhibitory responses are thought to be mediated in part by the synthesis of cyclic adenosine 3' 5' monophosphate. Our present results indicate that both the purinergic and the neurogenic responses, resistant to adrenergic and cholinergic blockade, are not modified by the acute administration of morphine. Furthermore, a sustained treatment with morphine also did not change the sensitivity of the mouse urinary bladder to ATP administration, which suggests that morphine has no influence on purinergic contractile mechanisms at the concentrations effective in the mouse vas deferens (Contreras et al., 1982). On the other hand, morphine administration induces inhibition of spontaneous bladder activity in several species (Dray and Metsch, 1984); the site of action of morphine is probably the spinal cord (Dray et al., 1985). Our results show the nonexistence of peripheral opiopeptidergic mechanisms in the contractile responses of the mouse urinary bladder to electrical field stimulation. The suppression by neostigrnine of the increased sensitivity to acetylcholine induced by morphine or nalorphine suggest that these analgesics are acting through inhibition of cholinesterase as it has been reported by Kosterlitz and Waterfield (1975).
452
C . G . ACEVEDOet al. SUMMARY
The effects of acute and chronic morphine administration were studied on the responses of isolated mouse urinary bladder to acetylcholine, A T P and to electrical stimulation. Acute morphine increased the responses to acetylcholine in both urinary bladder from untreated mice and chronically treated mice with the opiate. Nalorphine induced a lower potentiating effect upon acetylcholine responses. These sensitizing effects of the opiates were not modified by the presence of naloxone in the organ bath, but were inhibited by neostigmine. A chronic morphine treatment did not change the responses of the tissue to A T P administered in the presence of atropine and guanethidine. The addition of acute morphine did not change the contractile responses to A T P of the urinary bladder from either untreated or chronically treated mice with morphine. The administration of methadone or ketocyclazocine to the urinary bladders from naive or chronically treated mice with morphine decreased the responses to the electrical stimulation of the tissue. In contrast, morphine did not alter the depressant effects of methadone or ketocyclazocine. These results suggest the nonexistence of opiate receptors in the mouse urinary bladder, and at the same time indicate that morphine does not depress the activity of the autonomic innervation by acting on the neuroeffector junction. Acknowledgements--The authors wish to thank Dr P. Ward for critical reading of the manuscript. REFERENCES
Acevedo C. G. and Contreras E. (1985) Possible involvement of adenine nucleotides in the neurotransmission of the mouse urinary bladder. Comp. Biochem. Physiol. 82, 357-362. Burnstock G. (1981) Neurotransmitters and trophic factors in the autonomic nervous system. J. Physiol., Lond. 313, 1 35. Collier H. O. J. (1969) Humoral transmitters, supersensitivity, receptors and dependence. In Scientific Basis of Drug Dependence. (Edited by Steinberg H.), pp. 49 66. Churchill, London. Contreras E. and Marti M. (1979) Sensitivity of mouse vas deferens to neurotransmitters: changes after morphine treatment. Br. J. Pharmac. 65, 623-628.
Contreras E., Contreras G., Gonz~ilez C. and Concha J. (1980) Acute and chronic effects of morphine on transmembrane potential and short-circuit current in isolated toad skins. Neuropharmacology 19, 1081-1086. Contreras E., Tamayo L., Gaete S. and Juica S. (1982) Effects of denervation on the sensitizing effect to noradrenaline induced by morphine in the vas deferens of mice treated chronically with morphine. Neuropharmacology 21, 751-756. Dray A. and Metsch R. (1984) Inhibition of urinary bladder contractions by spinal action of morphine and other opiates. J. Pharmac. exp. Ther. 231, 254~260. Dray A., Numan L. and Wire W. (1985) Central 6 opioid receptor interactions and the inhibition of reflex urinary bladder contractions in the rat. Br. J. Pharmac. 85, 717-726. Fleming W. W. (1976) Variable sensitivity of excitable cells: possible mechanisms and biological significance. In Reviews of Neuroscience (Edited by Ehrenpreis S. and Kopin I. J.), pp. 43-90. Raven Press, New York. Gintzler A. R. (1979) Serotonin participation in gut withdrawal from opiates. J. Pharmac. exp., Ther. 211, 7-12. Gintzler A. R. and Musacchio J. M. (1975) Interactions of morphine, adenosine, adenosine triphosphate and phosphodiesterase inhibitors on the field-stimulated guineapig ileum. J. Pharmac. exp. Ther. 194, 575-582. Huidobro-Toro J. P. and Way E. L. (198l) Contractile effect of morphine and related opioid alkaloids, fl endorphin and methionine enkephalin on the isolated colon from Long Evans rats. Br. J. Pharmac. 74, 681q594. Henderson G., Hughes J. and Kosterlitz H. W. (1972) A new example of a morphine sensitive neuro-effector junction: adrenergic transmission in the mouse vas deferens. Br. J. Pharmac. 46, 464~466. Kosterlitz H. W. and Waterfield A. A. (1975) An analysis of the phenomenon of acute tolerance to morphine in the guinea-pig isolated ileum. Br. J. Pharmac. 53, 131-138. Paton W. D. M. (1957) The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea-pig ileum. Br. J. Pharmac. Chemother. 11, 119-127. Rae G. A., Neto J. P. and De Moraes S. (1977) Noradrenaline supersensitivity of the mouse vas deferens after long term treatment with morphine. J. Pharm. Pharmac. 29, 310-314. Simon E. J. (1984) Opiate receptors. In Handbook of Neurochemistry. (Edited by Lajtha A.) Vol. 6, 331-352. Plenum Press, New York. Sharpless S. and Jaffe J. (1969) Withdrawal phenomena as manifestations of disuse supersensitivity. In Scientific Basis of Drug Dependence. (Edited by Steinberg H.), pp. 67 76. Churchill, London.
0306-3623/86 $3.00 + 0.00 Pergamon Journals Ltd
EFFECTS OF MORPHINE IN THE ISOLATED MOUSE URINARY BLADDER* C. G. ACEVEDO, L. TAMAYO and E. CONTRERAS Departamento de Ciencias Fisiol6gicas, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Casilla 2407, Concepci6n, Chile (Received 22 January 1986) Abstract--1. Acute morphine increased the responses to acetylcholine of the isolated mouse urinary bladder. 2. A chronic morphine treatment did not change the responses of the urinary bladder to acetylcholine or ATP. 3. The acute administration of morphine did not modify the contractile response to ATP in the urinary bladders from untreated or chronically morphine treated mice. 4. Methadone and ketocyclazocine decreased the responses to the electrical stimulation of the urinary bladder. These depressant effects were not modified by naloxone. 5. The results suggest the nonexistence of opiate receptors in the mouse urinary bladder and the lack of direct effects of morphine on the neuroeffector junction.
INTRODUCTION An increase in the activity of the autonomieally innervated organs is observed when animals are withdrawn from a chronic morphine treatment. This change in activity may arise from the sustained blockade of transmitter release induced by morphine at the neuroeffector junction (Collier, 1969). Several authors have suggested that the basis of morphine abstinence is the result of an increased release of a neurotransmitter presumptively suppressed by the presence of morphine (Paton, 1957; Sharpless and Jaffe, 1969). Fleming (1976) has given the name supersensitivity to this state, which is attributable to a change in the responsiveness of the cells to a neurotransmitter. Changes in sensitivity have been observed in several tissues, namely the guinea-pig ileum (Paton, 1957), the mouse vas deferens (Rae et al., 1977). These tissues contain several neurotransmitters ineluding enkephalins (Gintzler, 1979); the presence of opiate receptors has also been described in them (Simon, 1984). The present study was designed to study the effects of acute and chronic morphine treatments on the sensitivity of the mouse urinary bladder to acetylcholine and ATP, 2 neurotransmitters involved in the contractile response of this organ (Acevedo and Contreras, 1985). The possible induction of altered responses produced by morphine to the electrical stimulation of the preparation was also investigated.
Acevedo and Contreras (1985) was set up in 12 ml organ bath at 30°C containing Tyrode solution of the following composition (mM) NaC1 194; KCI 4.69; CaCI2 1.90; MgC12 0.5; NaHCO3 25; glucose 5.04 and gased with 95% 02 and 5% CO2. For electrical stimulation the tissue was passed through a pair of platinum electrodes with rings 22 mm apart, and connected to a Grass FT 03 force-displacement transducer connected to a Grass polygraph. The preparation was subjected to 0.5 g basal tension. Drug additions and frequency-response curves were made over frequency of 0.1-50 Hz with pulses of 0.5 msec duration at supramaximal voltage (60 V). In order to avoid preparation fatigue, 15 min time periods were allowed between frequency-response or concentration-response curves. Most experiments were performed under the effect of atropine sulphate (10 -6 M) and guanethidine (3.4 x 10 -6 M).
MATERIALS AND METHODS Male albino mice (26-32 g) of a strain raised in our laboratory were stunned and killed by exanguination; the abdomen was opened by a midline incision and the urinary bladder was removed and placed in a physiological solution. A strip obtained according to the procedure described by *Supported by Direcci6n de Investigaci6n, Proy. 20.33.13, Universidad de Concepci6n. 449
Chronic treatment with morphine Mice received a single injection of morphine in a suspension (morphine as base, 300 mg, sorbital sesquioleate 0.8 ml, liquid paraffin 4.2 ml and saline 5 ml); each animal received 300 mg/kg (s.c.). The animals were killed 72 hr after the injection of morphine. The contractile effects of acetylcholine or ATP were tested in the presence or absence of morphine or other opiate as indicated in Results. Pharmocological responses The contractile effects of acetylcholine on the urinary bladder were tested by cumulative dose-response curves, first in the Tyrode solution without morphine (or the other opiate tested) and afterwards under different concentrations of the analgesic. When ATP was used as the contractile agent the preparation was extensively washed by renewing the bath fluid, and at least 15 min were allowed between each assayed concentration. Frequency-response curves were elicited in the presence or absence of the assayed opiate. The acute effects of morphine were assayed at 2 concentrations: 2.5 x 10 -5 M and 5.0 x 10-5 M. Doses for other drugs are given in Results. Statistical analysis Results of the contractile compounds assayed or the electrical stimulation are expressed % of the maximum response induced by either procedure. The data are ex-
C.G. ACEVEDOet al.
450
pressed as mean + SEM. Student's t-test was used to determine the statistical significance of difference between the means.
/j
120-
Drugs 1 O0 -
The drugs used were the following: noradrenaline hydrochloride, acetylcholine hydrochloride, ATP, all from Sigma Chemical Co., atropine sulphate, morphine hydrochloride and nalorphine hydrochloride from Merck, guanethidine (Ciba), ketocyclazocine (Sterling Winthrop Research), naloxone hydrochloride (Endo Labs Inc.), methadone hydrochloride (May and Baker).
.~_
80-
60. t,O,
RESULTS
Effect of morphine on the cholinergic response of the urinary bladder
20-
The possibility that a chronic morphine treatment might alter the cholinergic response of the mouse urinary bladder was studied after the sustained treatment with the opiate. The treatment did not change the concentration-response curve to acetylcholine. The effects of acute morphine (2.5 x 10 -5 M and 5 × 10 5M) were also tested on tissues from untreated and chronically morphine treated mice. Morphine induced an enhanced response to acetylcholine in both experimental groups (Fig. 1). The augmented responses did not differ in both experimental groups and the effect of both concentrations of the analgesic induced a similar enhancing response. Nalorphine induced a lower potentiating effect when added at similar concentrations to those tested for morphine (not shown). Naloxone (10-rM) did not change morphine or nalorphine effects. Neostigmine (3 × 10 -6 M) suppressed the morphine potentiating effect on the cholinergic response.
are shown in Fig. 2; the chronic morphine treatment did not change the responsiveness of the tissue to ATP. When the effects of this putative neurotransmitter were assayed in the presence of acute morphine in the bathing medium no changes in the responsiveness of the urinary bladders were observed.
Effects of morphine on the contractile response insensitive to atropine
Effects of morphine and other opiates on the neurogenic response of the mouse urinary bladder
ATP was assayed in the urinary bladders from naive and morphine treated mice. The concentration-response curves for the adenine n u cleotide were performed in the presence of atropine and guanethidine in the Tyrode solution. The results
Frequency-response curves ranging from 0.1 to 50 Hz were obtained in the presence of guanethidine and atropine. The chronic morphine treatment did not change the response of the muscle to the electrical stimulation at each different frequency tested.
•//
120
//
,
6 - log
conc
ATP
(M)
Fig. 2. Responses of the mouse urinary bladder to ATP. Untreated mice (O O, n = 6); untreated mice after acute morphine 2.5x 10-SM ( O - - - O , n = 6 ) ; the "washed" preparation from chronically treated mice (x x, n = 6); urinary bladders from chronically treated mice after the acute administration of morphine 2.5 x 10-SM ( x - - - x , n =6).
B 120
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i
100~
~_ 80-
,
/
100-
~ 80-
/
g 60-
o60-
X
40-
u ax =£ 40-
20-
20-
-------t/, - IO9 CO~ acetylcholine (Id)
-log ¢on¢ acetylchol*ne ( M }
Fig. 1. (A). Responses of the mouse urinary bladder to acetylcholine before (O O, n = 10) and after acute administration of morphine 2.5 x 10-5 M ( x - - - x , n = 10) and 5 x 10-5 M ( 4 A, n = 10). *Values statistically different from those observed before morphine administration. P < 0.01 (paired t-test). (B). As in (A), but the urinary bladders were obtained from mice chronically treated with morphine.
Morphine and mouse urinary bladder
451
B 100,
1 O0
8O
i
6o
60
~'
,o
o x 2O
20-
O1
05 I $ Iogarilhm of frequency
10 ( HZ )
50
0'1
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~ 1~
Iogorethm of frequency
( Hz )
Fig. 3. (A). Frequency-response curves of the mouse urinary bladder before (O O, n = 10); after the acute administration of methadone 10-5 M (x x, n = 6) and after the acute administration of ketocyclazocine 2.5 x 10-5 M (A A, n = 6). *P < 0.01 (paired t-test). (B) As in (A), but the urinary bladders were obtained from mice chronically treated with morphine.
The presence of acute morphine in the Tyrode solution (2.5 x 10 -5 M or 5 x 10 -5 M) did not alter the neurogenic response of the tissue (not shown). Methadone (5 x 10-6M and 10-SM) and ketocyclazocine (10 -5 M and 2.5 x 10 -5 M) decreased the response to the electrical stimulation of urinary bladders from naive and chronically treated mice. The results are shown in Fig. 3. The depressant effects of both opiates were unchanged by the presence of naloxone in the bath (10 -6 M), which suggests the inexistence of opiate receptors.
DISCUSSION
Guinea-pig ileum (Paton, 1957), mouse vas deferens (Henderson et al., 1972) and rat colon (Huidobro-Toro and Way, 1981) have been shown to have specific opiate receptors and have been used as models for the study of the mechanisms of action of narcotic analgesics. In these tissues morphine effects are readily reversed by naloxone and the sustained contact with opiates results in tolerance, which is an important feature of the interaction of opiates with specific receptors. In a previous work we described changes in the sensitivity to neurotransmitters and catecholamines in mice vas deferens which had been chronically treated with morphine at doses similar to those used in the present work (Contreras et al., 1982). We also showed a dependence like effect when acute morphine was added to the "washed" preparation. Under this condition the analgesic induced a facilitatory action to the contractile effect of catecholamines. A similar effect can be seen to occur in toad skin chronically treated with morphine (Contreras et al., 1980). A dependence like effect was observed on the transepithelial potential and short circuit current when the analgesic was omitted from the bathing solution. The addition of morphine to the skin turned the reduced response to catecholamines to values similar to those obtained in skins from untreated toads. On the other hand, the existence of a purinergic component in the contractile response of the urinary
bladder has been described in several species (Bumstock, 1981). In a previous work (Acevedo and Contreras, 1985) the ability of purine nucleotides to contract the mouse urinary bladder has been established and the evidence suggests that ATP may be the transmitter responsible for the non-cholinergic nonadrenergic response of this organ. Consequently, we thought it of interest to study the effects of acute and chronic morphine on the purinergic responses of this organ. The interaction of opiates on the contractile responses to ATP has not been studied. However, the inhibitory effect of ATP and adenosine have been investigated in the guinea-pig ileum (Gintzler and Musacchio, 1975). In this tissue morphine, ATP and adenosine inhibit the contractions induced by electrical stimulation. The relaxatory responses are potentiated by phosphodiesterase inhibitors and other compounds affecting purinergic mechanisms. The inhibitory responses are thought to be mediated in part by the synthesis of cyclic adenosine 3' 5' monophosphate. Our present results indicate that both the purinergic and the neurogenic responses, resistant to adrenergic and cholinergic blockade, are not modified by the acute administration of morphine. Furthermore, a sustained treatment with morphine also did not change the sensitivity of the mouse urinary bladder to ATP administration, which suggests that morphine has no influence on purinergic contractile mechanisms at the concentrations effective in the mouse vas deferens (Contreras et al., 1982). On the other hand, morphine administration induces inhibition of spontaneous bladder activity in several species (Dray and Metsch, 1984); the site of action of morphine is probably the spinal cord (Dray et al., 1985). Our results show the nonexistence of peripheral opiopeptidergic mechanisms in the contractile responses of the mouse urinary bladder to electrical field stimulation. The suppression by neostigrnine of the increased sensitivity to acetylcholine induced by morphine or nalorphine suggest that these analgesics are acting through inhibition of cholinesterase as it has been reported by Kosterlitz and Waterfield (1975).
452
C . G . ACEVEDOet al. SUMMARY
The effects of acute and chronic morphine administration were studied on the responses of isolated mouse urinary bladder to acetylcholine, A T P and to electrical stimulation. Acute morphine increased the responses to acetylcholine in both urinary bladder from untreated mice and chronically treated mice with the opiate. Nalorphine induced a lower potentiating effect upon acetylcholine responses. These sensitizing effects of the opiates were not modified by the presence of naloxone in the organ bath, but were inhibited by neostigmine. A chronic morphine treatment did not change the responses of the tissue to A T P administered in the presence of atropine and guanethidine. The addition of acute morphine did not change the contractile responses to A T P of the urinary bladder from either untreated or chronically treated mice with morphine. The administration of methadone or ketocyclazocine to the urinary bladders from naive or chronically treated mice with morphine decreased the responses to the electrical stimulation of the tissue. In contrast, morphine did not alter the depressant effects of methadone or ketocyclazocine. These results suggest the nonexistence of opiate receptors in the mouse urinary bladder, and at the same time indicate that morphine does not depress the activity of the autonomic innervation by acting on the neuroeffector junction. Acknowledgements--The authors wish to thank Dr P. Ward for critical reading of the manuscript. REFERENCES
Acevedo C. G. and Contreras E. (1985) Possible involvement of adenine nucleotides in the neurotransmission of the mouse urinary bladder. Comp. Biochem. Physiol. 82, 357-362. Burnstock G. (1981) Neurotransmitters and trophic factors in the autonomic nervous system. J. Physiol., Lond. 313, 1 35. Collier H. O. J. (1969) Humoral transmitters, supersensitivity, receptors and dependence. In Scientific Basis of Drug Dependence. (Edited by Steinberg H.), pp. 49 66. Churchill, London. Contreras E. and Marti M. (1979) Sensitivity of mouse vas deferens to neurotransmitters: changes after morphine treatment. Br. J. Pharmac. 65, 623-628.
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