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Effects of morphine on adrenaline responses of uteri from progesterone or estradiol treated mice
Camp. Biochem. PhysM. Vol. 8X, No. 2, pp. 425-428, 1987
Printed in Great Britain
0
0306-4492/87 $3.00 + 0.00 1987 PergamonJournals Ltd
EFFECTS OF MORPHINE ON ADRENALINE RESPONSES OF UTERI FROM PROGESTERONE OR ESTRADIOL TREATED MICE ~pa~~ento
C. G. ACEWEDOand E. CONTRERAS de Ciencias Fisiol&,ieas ~Fa~a~lo~a), Faeultad de Ciencias Biol&icas y de Recursos Naturales, Universidad de Conception,
Casilla 2407, Concepcibn, Chile
(Received 14 October 1986) Abstrach-1.
The effect of a chronic morphine treatment on the in oitro contractile responses of the mouse uterus to adrenaline was studied. 2. Chronic morphine treatment induced a su~~ensiti~y state in the uteri from both progesterone and
e&radio1treated mice. 3. The acute administration of morphine to the uteri from morphine tolerant-dependent and progesterone treated mice induced a further increase of the contractile effect of adrenaline. 4. Reserpine administration did not further increase the supersensitivity of the mouse uterus to adrenaline induced by a chronic morphine treatment. 5. Reserpine suppressed the acute effects of morphine in the uteri from tolerant-dependent mice.
INTRODUCTION
Several authors (Pollock et al., 1972; Muir and Pollock, 1973; Gibson and Poilock, 1975; Rae et al., 1977; Rae and de Moraes, 1983) have described an increased sensitivity to neurotrans~tters as a result of a chronic treatment with opiates. This increased sensitivity has been observed in the mouse vas deferens (Rae et al., 1977; Rae and De Moraes, 1983), the anococcygeous muscle and colon of the rat (Pollock et al., 1972; Gibson and Pollock, 1975). Collier (1965, 1969) has suggested that withdrawal reactions from opiates are induced by the long term inhibition of neurotransmitter release induced by these drugs. The inhibition of neurotransmitter release has been observed for acetylcholine in the brain (Beleshin and Polak, 1965; Matthews et al., 1973) and also for noradrenaline (Hughes et aI., 1975; Henderson and Hughes, 1976). In previous works (Contreras and Marti, 1979; Contreras et al., 1982a) we have described a dependence-like effect in the mouse vas deferens. This state occurs when acute morphine is added to the bathing medium containing tissue repeatedly washed by Tyrode solution; in this condition an increased maximum response is observed in the contractile response to noradrenaline. In the present paper we have studied the influence of a chronic morphine treatment on the responsiveness of the mouse uterus to adrenaline. Since the responses to catecholamines change according to the ovarian cycle, the responses of the organ were examined in mice treated with either estradiol or progesterone. MATERIALS AND METHODS Female mice (28-32 g) raised in our laboratory were used. Both uterine horns were set up in an organ bath (12ml) CB.P. 871X-M
containing a solution of the following composition (mM): NaCl 155, KC1 5.67, CaC& 0.272, HaHCO, 5.95, glucose 2.77. The bathing solution was kept at 30°C and continuously gassed with 95% O2 and 5% CO,. The preparation was allowed to equilibrate for 6Omin before drug testing. A tension of 0.5 g was applied to the muscle and the contractions produced by the agonist under test were recorded isometrically. In preliminary experiments we showed that longitudinal strips and unopened uterine horns exhibit similar activity as regards spontaneous or agonist induced contractile force. Uterine contractility was measured by a Grass FT 03 force displacement transducer connected to a Grass Poly~ph. Full dose-response curves were obtained by increasing the concentration of the agonist as described by Van Rossum (1963). Drugs treatment The chronic treatment with morphine was obtained by a single S.C. injection (300mg in the base form) of a slow release preparation (sorbitan sesquioleate 0.8ml, liquid parat& 4.2m1, saline 5.Oml). The volume injected was 10 PI/g body weight. Control mice were injected S.C.with the vehicle of this preparation. Animals were killed 4-7 days later. An acute treatment with morphine was also tested by adding morphine at several concentrations (0.66, 1.32, 2.65 and 3.31 x 10m5M) to the bathing solution. Estradiol 100 .&kg was administered s.e. 24 hr before the experiment. progesterone was given in two doses of 75 n&kg at 24 and I2 hr before the experiment. Reserpine was administered i.p. as indicated in Results. Statistical analysis
The Student’s t-test was used to determine the statistical significance of the difference between the means. All values are the mean of at least 8 experiments. Drugs used The following drugs were used: adrenaline hydrochloride (Matheson, Coleman and Bell) morphine hydrochloride (May and Baker), propranolol chlorhidrate (Sigma Chemical Co.), naloxone (Endo Labs.) reserpine (Nutritional Biochemical Co.), progesterone (Sigma Chemical Co.), estradiol (Sigma Chemical Co.). 425
426
C. G. ACEVEDOand E.
RI?SL!LTS
The chronic morphine treatment induced a supersensitivity to acetylcholine and serotonin, as previously reported (Contreras ef al., 1982b; Acevedo and Contreras, 1984). The effects of acute and chronic morphine administrations on the contractile responses to adrenaline were also studied; for this purpose, propranolol (0.013 mM) was added to the bath in order to avoid relaxatory responses to the catecholamine. The responses to adrenaline were observed in mice treated with either estradiol or progesterone as indicated in Materials and Methods. Figure I shows the effects of adrenaline on the uteri from mice treated with either progesterone or estradiol. Responses to adrenaline in the estrogen treated mice were too small or even absent. However, the chronic morphine treatment induced a supersensitivity to adrenaline responses. The acute administration of morphine did not change the adrenaline responses of the uterine horns from contro1 mice (injected only with the vehicle) or in the uteri from chronically morphinized mice. A striking difference was appreciated in the adrenaline responses of the uterine muscle from progesterone treated mice; namely, the contractile responses were always present and the addition of acute morphine did not change the intensity of the responses. A supersensitivity to the ~t~hoIamine was observed in chronically morphinized mice. Under these conditions the addition of acute morphine to the bathing medium induced a further increase in the contractile response to adrenaline. This effect seems to be dose-related and consisted of a shift in the dosresponse curve to the left together with a simultaneous increase in the maximal
QNTRERAS
response. The highest concentration assayed decreased the maximum. Naloxone (5 x lo-’ M) did not suppress the potentiating effect induced by acute morphine. In experiments in which uterine horns from tolerant mice were set up in a medium containing morphine (10m6M) added to the Tyrode solution in order to avoid its removal by the continuous washing of the preparation, the addition of naloxone (10-7-10-6 M) did not significantly alter the responses to adrenaline observed in the presence of morphine. Effect of reserpine administration on morphine induced supersensit&ty
Groups of mice were treated with one dose of reserpine (3 mg/kg) 24 hr before the in uitrc experiments. Figure 2 shows these results. Reserpine induced a supersensitivity to adrenaline in the uteri from control mice (Fig. 2A). The addition of acute morphine did not change the responses to the catecholamine. The pretreatment with reserpine did not change the responses to adrenaline in the chronically morphinized mice, as can be seen in Fig. 2(B). When acute morphine was added to the bath the opiate did not induce a further increase in the responses to adrenaline in the uteri from reserpine-morphine treated mice. DISCUSSION It is a well known fact that chronic morphine treatment increases the sensitivity of several smooth muscles (Muir and Pollock, 1973; Gibson and Pollock, 197.5; Rae et al., 1977). Fleming (1976) has
A
1600 i 1400 i
3 -bag
8
7
concrntration
6 Adrcnalmo
5
4 (M)
3 -Log
8
7
e~nc~ntrahon
6 Adrenaline
5
L IH)
Fig. 1. (A) Dose-response curves for the contractile effects of adrenaline in the uterus of control mice (animals without chronic morphine). Solid lines represent the results observed in progesterone treated animals. Without morphine in the bath ( x x ); after the addition of morphine 1.32 x 10m5M (m-m); 2.65 x 10es M (e-0) and 3.31 x low5 M (A----A). Broken lines represent the results observed in estradiol treated mice. Without morphine in the bath ( x ------ x ); after the addition of morphine 1.32 x lo-‘M (m------m); 2.65 x 10es M (e------O) and 3.31 x 10m5M (A------& (B) Do&esponse curves for the contractile effects of adrenaline in the uterus of chronically morphine treated mice. Symbols as in (A). Asterisks indicate values statisticallv different (P < 0.01) from those observed before morphine adm&&ation (paired r-test). The curves re&esent the mean of at least 8 preparations.
427
Morphine and adrenaline responses in the mouse uterus
1200-
^, 1000 E 5 I
800-
E 600 -
LOO-
200 -
4, -Log
I
/
8
I
8
8
7
5
5
4
concentration
Adrenaline
IMf
Fig. 2. Effect of reserpine pretreatment in the adrenaline responses of the mouse uterus. (A) Dose-response curves for the contractile effects of adrenaline in progesterone treated mice. (Controls not receiving chronic morphine.) Control mice, without morphine in the bath (O---O); reserpine pretreated mice without morphine in the bath (O------O); reserpine pretreated mice after the addition of acute morphine 2.65 x 10m5M (x ------ x )_ Asterisks indicate a statistically signii%ant difference (P < 0.01) from adrenaline effects in control mice. (B) Results as indicated in (A), but all mice were chronic~y
treated with morphine. The curves represent the mean of at least 10 preparations. extensively studied this enhanced responsiveness of the target cells which develops subsequently to the chronic suppression of the neurotransmitters at its effector cells. This change has been called postjunctional ~~r~nsitivi~ and it can be induced by several procedures, which include chronic denervation, the administration of drugs that deplete the neurotransmitter(s) in the synaptic endings or the chronic prevention of neurotransmitter release. The latter seems to be the mechanism of morphine action at peripheral neuroeffectors, i.e. morphine inhibits noradrenaline reiease induced by electric field stimulation in the mouse vas deferens (Henderson and Hughes, 1976) and acetylcholine in the guinea pig ileum (Paton, 1969). The increased sensitivity described by Fleming is nonspecific since it applies to several agonists as acetylcholine and serotonin responses, which are si~i~c~tly increased in the mouse uterus (Contreras et al., 1982b). Chronic morphinization, similar to the one accomplished in the present paper, also affects the vas deferens of mice. Moreover, when this tissue is obtained from tolerant-dependent mice, the acute administration of morphine induces a further increase in the responses to ~t~ho~~in~ (Contreras and Marti, 1979; Contreras et al., 1982a). This facilitatory response induced by morphine in vas deferens from mice chronically treated with the opiate, was believed to be a form of morphine-dependence, since it is not observed in vas deferens from naive mice. However, no cross dependence was observed when other opiates were admi~stered, e.g. methadone or meperidine. In addition, the presence of naloxone did not change the facilitatory effect of morphine. The permissive effect induced by acute morphine, as described in the mouse vas deferens, was also observed in the uterus from morphine tolerant mice, i.e. the effect seemed to be dose related and was ~o~fied by the presence of naloxone.
The absence of a cross response with other opiates and the inability of naloxone to modify the effect of morphine, makes it difficult to reconcile the facilitatory effect of acute morphine with a dependencelike state in these smooth muscles. The nonspecific nature of the supersensitivity induced by chronic morphine suggests a possible interference of this treatment with the contractile process and calcium movements in the smooth muscle. The interaction of calcium and analgesics on smooth muscle has been studied by Lee and Berkowits (1976, 1977), A stereoselective effect of l-isomers on cal&nndependent contractile action of opiates has been observed (Lee and Berkowitz, 1976) in the rat aortic strip, suggesting that the analgesics increase the rate of calcium entry into the cells when used in small doses. Higher doses seem to inhibit calcium influx in muscle cells since they depress rat aortic contraction (Lee and Berkowitz, 1977). On the other hand, the contractile effect of catechoiamines is known to depend on intracellular calcium (van Breemen et al., 1972). Although chronic morphine effects on calcium disposition in smooth muscle is not fully understood, it can be assumed that the chronic morphine induced changes in the ~nsiti~ty of the mouse uterus is the result of an increased availability of calcium for the contractile process. Reserpine was used on account of its ability to induce a nonspecific supersensitivity in several smooth muscles (Regram and Carrier, 1969). It was then thought of interest to know whether reserpine treatment might change the su~r~nsitivity state induced by the chronic morphine treatment. The inability of reserpine to induce a further increase of this supersensitivity and the inhibition of the facilitatory response induced by acute morphine suggest that both, reserpine and morphine, induced similar effects on calcium localization within the muscle cells. This possibility requires additional studies.
428
C. Cl. ACEVEDOand E. CONTRERAS
Acknowledgement-This work was supported by Direction de Investigation, Universidad de Conception. Proj. 20.33.13.
REFERENCES
Acevedo C. G. and Contreras E. (1984) Effects of morphine in the responses to acetylcholine and adrenaline in the mouse uterus. Ceil. molec. Biol. 30, 239-242. Beleshin D. and Polak R. I. (1965) Depression by morphine and chloralose of acetylcholine release from the cat’s brain. J. Physiol. 117, 411.-419. Collier H. 0. J. (1965) A general theory of the genesis of drug dependence by induction of receptors. Nature 205, 181-182. Collier H. 0. J. (1969) Humordl transmitters, supersensitivity, receptors and dependence. In Scienlifc Busis of Drug Dependence (Edited by Steinberg H.). pp. 49-66. J. and A. 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., Tamayo L., Gaete S. and Juica S. (1982a) 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.
Contreras E., Tamayo L. and Juica S. (1982b) Sensitivity changes after morphine treatment in the mouse uterus. Arch7 int. Pharmacodyn.
ThCr. 255, 3947.
Fleming W. W. (1976) Variable sensitivity of excitable cells: possible mechanisms and biological significance. In Reoiews of Neuroscience (Edited by Ehrenpreis S. and Kopin I. J.), Vol. 2, pp. 43-90. Raven Press, New York. Gibson A. and Pollock D. (1975) The involvement of corticosteroids in the supersensitivity produced in the rat anococcygeous muscie by morphine withdrawal. thyroidectomy or a single dose of reserpine. J. Pharmac. exp. Ther. 192, 390-398. Henderson G. and Hughes J. (1976) The effects of morphine on the release of noradrenaline from the mouse vas deferens. Br. J. Pharmac. 57, 551-557. Hughes J., Kosterlitz H. W. and Leslie F. M. (1975) Effect of morphine on adrenergic transmission in the mouse vas
deferens, assessment of agonist and antagonist potencies of narcotic analgesics. Br. J. Phurmuc. 53, 371-378. Lee C. H. and Berkowitz B. A. (1976) Stereoselective and calcium-dependent contractile effects or narcotic antagonist analgesics in the vascular smooth muscle of the rat. J. Pharmac. exp. Ther. 198, 347 356. LRe C. H. and Berkowitz B. A. (1977) Calcium antagonist
activity of methadone I-acetylmethadol and I-pentazocine in the rat aortic strip. J. Pharmuc. exp. Thu. 2@2. 646653.
Matthews J. D., Ldbrecque G. and Domino E. F. (1973) Effects of morphine, nalorphinc and naloxone on neocortical release of acetylcholine in the rat. Psychopharmacologia 29, 113-120. Muir T. C. and Pollock D. (1973) Morphine induced changes in the responsiveness of autonomic effector organs. In Agonist und Antagonist Actions of Narcotic Analgesic Drugs (Edited by Kosterlitz H. W., Collier H. 6. J. and Villarrea! J. E.). pp. 207-218. University Park Press, Baltimore. Paton W. D. M. (1969) A pharmacological approach to drug dependence and drug tolerance. In Scientific Basis of Drug Dependence (Edited by Steinberg H.) pp. 31-47. J. and A. Churchill, London. Pegram B. L. and Carrier 0. (1969) Change in calcium dependence of isolated arteries after reserpine. Am. J. Physiol. 217, 17361741. Pollock D., Muir T. C., MacDonald A. and Henderson G. (1972) Morphine induced changes in the sensitivity of the isolated colon and vas deferens of the rat. Eur. J. Pharmac. 20, 321-328.
Rae G. A. and De Moraes S. (1983) Supersensitivity to noradrenaline in vas deferens from morphine dependent mice is confirmed. Eur. J. Pharmac. 86, 347 -352. Rae 6. A., Neto 3. P. and De Moraes S. (1977) Noradrenaline supersensitivity of the mouse vas deferens after long term treatment with morphine. J. Pharm. Pharmac. 29, 3IG312.
Van Breemen Farinds B. R., Gerba P. and McNaughton E. D. (1972) Excitationcontraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium ~&IX. Circulation Res. 30, M-54. Van Rossum J. M. (1963) Cumulative doscresponse curves. II. Technique for the making of dose-response curves in isoiated organs and the evaluation of drug parameters. Archs int. Pharmacodyn. Th&. 143,299.330.
Printed in Great Britain
0
0306-4492/87 $3.00 + 0.00 1987 PergamonJournals Ltd
EFFECTS OF MORPHINE ON ADRENALINE RESPONSES OF UTERI FROM PROGESTERONE OR ESTRADIOL TREATED MICE ~pa~~ento
C. G. ACEWEDOand E. CONTRERAS de Ciencias Fisiol&,ieas ~Fa~a~lo~a), Faeultad de Ciencias Biol&icas y de Recursos Naturales, Universidad de Conception,
Casilla 2407, Concepcibn, Chile
(Received 14 October 1986) Abstrach-1.
The effect of a chronic morphine treatment on the in oitro contractile responses of the mouse uterus to adrenaline was studied. 2. Chronic morphine treatment induced a su~~ensiti~y state in the uteri from both progesterone and
e&radio1treated mice. 3. The acute administration of morphine to the uteri from morphine tolerant-dependent and progesterone treated mice induced a further increase of the contractile effect of adrenaline. 4. Reserpine administration did not further increase the supersensitivity of the mouse uterus to adrenaline induced by a chronic morphine treatment. 5. Reserpine suppressed the acute effects of morphine in the uteri from tolerant-dependent mice.
INTRODUCTION
Several authors (Pollock et al., 1972; Muir and Pollock, 1973; Gibson and Poilock, 1975; Rae et al., 1977; Rae and de Moraes, 1983) have described an increased sensitivity to neurotrans~tters as a result of a chronic treatment with opiates. This increased sensitivity has been observed in the mouse vas deferens (Rae et al., 1977; Rae and De Moraes, 1983), the anococcygeous muscle and colon of the rat (Pollock et al., 1972; Gibson and Pollock, 1975). Collier (1965, 1969) has suggested that withdrawal reactions from opiates are induced by the long term inhibition of neurotransmitter release induced by these drugs. The inhibition of neurotransmitter release has been observed for acetylcholine in the brain (Beleshin and Polak, 1965; Matthews et al., 1973) and also for noradrenaline (Hughes et aI., 1975; Henderson and Hughes, 1976). In previous works (Contreras and Marti, 1979; Contreras et al., 1982a) we have described a dependence-like effect in the mouse vas deferens. This state occurs when acute morphine is added to the bathing medium containing tissue repeatedly washed by Tyrode solution; in this condition an increased maximum response is observed in the contractile response to noradrenaline. In the present paper we have studied the influence of a chronic morphine treatment on the responsiveness of the mouse uterus to adrenaline. Since the responses to catecholamines change according to the ovarian cycle, the responses of the organ were examined in mice treated with either estradiol or progesterone. MATERIALS AND METHODS Female mice (28-32 g) raised in our laboratory were used. Both uterine horns were set up in an organ bath (12ml) CB.P. 871X-M
containing a solution of the following composition (mM): NaCl 155, KC1 5.67, CaC& 0.272, HaHCO, 5.95, glucose 2.77. The bathing solution was kept at 30°C and continuously gassed with 95% O2 and 5% CO,. The preparation was allowed to equilibrate for 6Omin before drug testing. A tension of 0.5 g was applied to the muscle and the contractions produced by the agonist under test were recorded isometrically. In preliminary experiments we showed that longitudinal strips and unopened uterine horns exhibit similar activity as regards spontaneous or agonist induced contractile force. Uterine contractility was measured by a Grass FT 03 force displacement transducer connected to a Grass Poly~ph. Full dose-response curves were obtained by increasing the concentration of the agonist as described by Van Rossum (1963). Drugs treatment The chronic treatment with morphine was obtained by a single S.C. injection (300mg in the base form) of a slow release preparation (sorbitan sesquioleate 0.8ml, liquid parat& 4.2m1, saline 5.Oml). The volume injected was 10 PI/g body weight. Control mice were injected S.C.with the vehicle of this preparation. Animals were killed 4-7 days later. An acute treatment with morphine was also tested by adding morphine at several concentrations (0.66, 1.32, 2.65 and 3.31 x 10m5M) to the bathing solution. Estradiol 100 .&kg was administered s.e. 24 hr before the experiment. progesterone was given in two doses of 75 n&kg at 24 and I2 hr before the experiment. Reserpine was administered i.p. as indicated in Results. Statistical analysis
The Student’s t-test was used to determine the statistical significance of the difference between the means. All values are the mean of at least 8 experiments. Drugs used The following drugs were used: adrenaline hydrochloride (Matheson, Coleman and Bell) morphine hydrochloride (May and Baker), propranolol chlorhidrate (Sigma Chemical Co.), naloxone (Endo Labs.) reserpine (Nutritional Biochemical Co.), progesterone (Sigma Chemical Co.), estradiol (Sigma Chemical Co.). 425
426
C. G. ACEVEDOand E.
RI?SL!LTS
The chronic morphine treatment induced a supersensitivity to acetylcholine and serotonin, as previously reported (Contreras ef al., 1982b; Acevedo and Contreras, 1984). The effects of acute and chronic morphine administrations on the contractile responses to adrenaline were also studied; for this purpose, propranolol (0.013 mM) was added to the bath in order to avoid relaxatory responses to the catecholamine. The responses to adrenaline were observed in mice treated with either estradiol or progesterone as indicated in Materials and Methods. Figure I shows the effects of adrenaline on the uteri from mice treated with either progesterone or estradiol. Responses to adrenaline in the estrogen treated mice were too small or even absent. However, the chronic morphine treatment induced a supersensitivity to adrenaline responses. The acute administration of morphine did not change the adrenaline responses of the uterine horns from contro1 mice (injected only with the vehicle) or in the uteri from chronically morphinized mice. A striking difference was appreciated in the adrenaline responses of the uterine muscle from progesterone treated mice; namely, the contractile responses were always present and the addition of acute morphine did not change the intensity of the responses. A supersensitivity to the ~t~hoIamine was observed in chronically morphinized mice. Under these conditions the addition of acute morphine to the bathing medium induced a further increase in the contractile response to adrenaline. This effect seems to be dose-related and consisted of a shift in the dosresponse curve to the left together with a simultaneous increase in the maximal
QNTRERAS
response. The highest concentration assayed decreased the maximum. Naloxone (5 x lo-’ M) did not suppress the potentiating effect induced by acute morphine. In experiments in which uterine horns from tolerant mice were set up in a medium containing morphine (10m6M) added to the Tyrode solution in order to avoid its removal by the continuous washing of the preparation, the addition of naloxone (10-7-10-6 M) did not significantly alter the responses to adrenaline observed in the presence of morphine. Effect of reserpine administration on morphine induced supersensit&ty
Groups of mice were treated with one dose of reserpine (3 mg/kg) 24 hr before the in uitrc experiments. Figure 2 shows these results. Reserpine induced a supersensitivity to adrenaline in the uteri from control mice (Fig. 2A). The addition of acute morphine did not change the responses to the catecholamine. The pretreatment with reserpine did not change the responses to adrenaline in the chronically morphinized mice, as can be seen in Fig. 2(B). When acute morphine was added to the bath the opiate did not induce a further increase in the responses to adrenaline in the uteri from reserpine-morphine treated mice. DISCUSSION It is a well known fact that chronic morphine treatment increases the sensitivity of several smooth muscles (Muir and Pollock, 1973; Gibson and Pollock, 197.5; Rae et al., 1977). Fleming (1976) has
A
1600 i 1400 i
3 -bag
8
7
concrntration
6 Adrcnalmo
5
4 (M)
3 -Log
8
7
e~nc~ntrahon
6 Adrenaline
5
L IH)
Fig. 1. (A) Dose-response curves for the contractile effects of adrenaline in the uterus of control mice (animals without chronic morphine). Solid lines represent the results observed in progesterone treated animals. Without morphine in the bath ( x x ); after the addition of morphine 1.32 x 10m5M (m-m); 2.65 x 10es M (e-0) and 3.31 x low5 M (A----A). Broken lines represent the results observed in estradiol treated mice. Without morphine in the bath ( x ------ x ); after the addition of morphine 1.32 x lo-‘M (m------m); 2.65 x 10es M (e------O) and 3.31 x 10m5M (A------& (B) Do&esponse curves for the contractile effects of adrenaline in the uterus of chronically morphine treated mice. Symbols as in (A). Asterisks indicate values statisticallv different (P < 0.01) from those observed before morphine adm&&ation (paired r-test). The curves re&esent the mean of at least 8 preparations.
427
Morphine and adrenaline responses in the mouse uterus
1200-
^, 1000 E 5 I
800-
E 600 -
LOO-
200 -
4, -Log
I
/
8
I
8
8
7
5
5
4
concentration
Adrenaline
IMf
Fig. 2. Effect of reserpine pretreatment in the adrenaline responses of the mouse uterus. (A) Dose-response curves for the contractile effects of adrenaline in progesterone treated mice. (Controls not receiving chronic morphine.) Control mice, without morphine in the bath (O---O); reserpine pretreated mice without morphine in the bath (O------O); reserpine pretreated mice after the addition of acute morphine 2.65 x 10m5M (x ------ x )_ Asterisks indicate a statistically signii%ant difference (P < 0.01) from adrenaline effects in control mice. (B) Results as indicated in (A), but all mice were chronic~y
treated with morphine. The curves represent the mean of at least 10 preparations. extensively studied this enhanced responsiveness of the target cells which develops subsequently to the chronic suppression of the neurotransmitters at its effector cells. This change has been called postjunctional ~~r~nsitivi~ and it can be induced by several procedures, which include chronic denervation, the administration of drugs that deplete the neurotransmitter(s) in the synaptic endings or the chronic prevention of neurotransmitter release. The latter seems to be the mechanism of morphine action at peripheral neuroeffectors, i.e. morphine inhibits noradrenaline reiease induced by electric field stimulation in the mouse vas deferens (Henderson and Hughes, 1976) and acetylcholine in the guinea pig ileum (Paton, 1969). The increased sensitivity described by Fleming is nonspecific since it applies to several agonists as acetylcholine and serotonin responses, which are si~i~c~tly increased in the mouse uterus (Contreras et al., 1982b). Chronic morphinization, similar to the one accomplished in the present paper, also affects the vas deferens of mice. Moreover, when this tissue is obtained from tolerant-dependent mice, the acute administration of morphine induces a further increase in the responses to ~t~ho~~in~ (Contreras and Marti, 1979; Contreras et al., 1982a). This facilitatory response induced by morphine in vas deferens from mice chronically treated with the opiate, was believed to be a form of morphine-dependence, since it is not observed in vas deferens from naive mice. However, no cross dependence was observed when other opiates were admi~stered, e.g. methadone or meperidine. In addition, the presence of naloxone did not change the facilitatory effect of morphine. The permissive effect induced by acute morphine, as described in the mouse vas deferens, was also observed in the uterus from morphine tolerant mice, i.e. the effect seemed to be dose related and was ~o~fied by the presence of naloxone.
The absence of a cross response with other opiates and the inability of naloxone to modify the effect of morphine, makes it difficult to reconcile the facilitatory effect of acute morphine with a dependencelike state in these smooth muscles. The nonspecific nature of the supersensitivity induced by chronic morphine suggests a possible interference of this treatment with the contractile process and calcium movements in the smooth muscle. The interaction of calcium and analgesics on smooth muscle has been studied by Lee and Berkowits (1976, 1977), A stereoselective effect of l-isomers on cal&nndependent contractile action of opiates has been observed (Lee and Berkowitz, 1976) in the rat aortic strip, suggesting that the analgesics increase the rate of calcium entry into the cells when used in small doses. Higher doses seem to inhibit calcium influx in muscle cells since they depress rat aortic contraction (Lee and Berkowitz, 1977). On the other hand, the contractile effect of catechoiamines is known to depend on intracellular calcium (van Breemen et al., 1972). Although chronic morphine effects on calcium disposition in smooth muscle is not fully understood, it can be assumed that the chronic morphine induced changes in the ~nsiti~ty of the mouse uterus is the result of an increased availability of calcium for the contractile process. Reserpine was used on account of its ability to induce a nonspecific supersensitivity in several smooth muscles (Regram and Carrier, 1969). It was then thought of interest to know whether reserpine treatment might change the su~r~nsitivity state induced by the chronic morphine treatment. The inability of reserpine to induce a further increase of this supersensitivity and the inhibition of the facilitatory response induced by acute morphine suggest that both, reserpine and morphine, induced similar effects on calcium localization within the muscle cells. This possibility requires additional studies.
428
C. Cl. ACEVEDOand E. CONTRERAS
Acknowledgement-This work was supported by Direction de Investigation, Universidad de Conception. Proj. 20.33.13.
REFERENCES
Acevedo C. G. and Contreras E. (1984) Effects of morphine in the responses to acetylcholine and adrenaline in the mouse uterus. Ceil. molec. Biol. 30, 239-242. Beleshin D. and Polak R. I. (1965) Depression by morphine and chloralose of acetylcholine release from the cat’s brain. J. Physiol. 117, 411.-419. Collier H. 0. J. (1965) A general theory of the genesis of drug dependence by induction of receptors. Nature 205, 181-182. Collier H. 0. J. (1969) Humordl transmitters, supersensitivity, receptors and dependence. In Scienlifc Busis of Drug Dependence (Edited by Steinberg H.). pp. 49-66. J. and A. 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., Tamayo L., Gaete S. and Juica S. (1982a) 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.
Contreras E., Tamayo L. and Juica S. (1982b) Sensitivity changes after morphine treatment in the mouse uterus. Arch7 int. Pharmacodyn.
ThCr. 255, 3947.
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