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Lack of cross-tolerance between morphine and Leu-enkephalin in the mouse vas deferens
260
Bratn Reseal ~tt, 197 ( 19080 260- 203 i , F l s e w e r / N o r t h - H o l l a n d Blomethcal Press
Lack of cross-tolerance between morphine and Leu-enkephalin in the mouse vas deferens
P E T E R 1LLES, W A L T E R Z I E G L G ~ , N S B E R G E R
and ALBERT HERZ
Department oJ Neuropharmacology, Ma.~:-Planclt-ln~tttut fu~ P,sychtatJte, l~raepelm,~t~a,~,~e 2, 1)-80()0 Munchen 40 ( G F R ) (Accepted M a y 8th, 1980)
key wo~ds mouse vas deferens--excltatory Junction potennal,~---opmtereceptors
Morphine reduces the release of noradrenahne from the mouse vas deferens 4. In consequence, the amplitude of excitatory junction potentials (e.j.ps) recorded from single smooth muscle cells 5 and also nerve stimulation evoked contractions of tile organ are depressed 4,6. Tolerance to this action of morphine was evident when the depressson of e.j.p, amplitudes evoked by submaxlmal stimulation was measured in vasa deferentla from morphine treated mice n. The degree of tolerance displayed was inversely related to the intensity of stimulation 7. Thas fact may account for the poor ability of morphine to reduce tolerance when measured by the mhabJnon of the contractile responses using supramaxlmal stamuli ~.:~ The mouse vas deferens has been reported to contain two classes of opiate receptors, one for which morphine (/z-receptor) and another one for which Leu-enkephalin or D-AlaZ-D-LeuS-enkephahn (d-receptor) IS the prototypic agomst ~.~°.t4. In view of these different receptor subtypes, at was of interest to know whether tolerance reduced by long term exposure to an agomst at one receptor was or was not accompamed by tolerance to the effects of an agonlst at the other receptor. The purpose of the present study was to investigate the effect of Leu-enkephalin on e j p, amphtudes of vasa deferentm prepared from morphine treated mice N M R I mice (25-35 g) were killed by decapitation and the vasa deferentm were removed. The preparation was superfused with Krebs solution s gassed w~th 95",, O~/5°(, COo_ and warmed to 37 ~C at a flow rate of 3 mt/min. The intramural sympathetic nerves were stamulated by two platinum ring electrodes. Stimuli were single pulses (1 msec duration) repeated at intervals of 20 sec and were of varlou~ voltages. Stimulataon current was measured over a 10 kf~ resistance placed m series, yielding an essentially constant current stimulus, lntracellular potentials were recorded as in previous experiments s. Some of the mice were amplanted subcutaneously wath morphine containing pellets (75 mg morphine base prepared according to Gibson and Tingstad-'). Half a
261 pellet was i m p l a n t e d o n the first d a y , a n d a w h o l e pellet o n the s e c o n d day. T h e a m m a l s were sacrificed o n the t h i r d day a n d thetr v a s a d e f e r e n t l a were placed m K r e b s s o l u t i o n c o n t a i n i n g 0.4 # M
n o r m o r p h l n e to s i m u l a t e the p l a s m a c o n c e n t r a t i o n o f
m o r p h i n e . T h i s m e t h o d o f p e l l e t - i m p l a n t a t i o n has been p r e v i o u s l y s h o w n to p r o d u c e t o l e r a n c e to m o r p h i n e m the m o u s e vas d e f e r e n s 7. A s e c o n d g r o u p o f v a s a w e r e t a k e n f r o m n a i v e m i c e a n d were s u p e r f u s e d in n o r m a l K r e b s s o l u t i o n and a t h i r d g r o u p o f vasa were t a k e n f r o m n a i v e m i c e a n d s u p e r f u s e d m a K r e b s s o l u t i o n c o n t a i n i n g 0.4 /~M n o r m o r p h m e . T h e d e p r e s s i o n o f the e.j.p, by L e u - e n k e p h a l m was m e a s u r e d by r e p e a t i n g the s t i m u l u s - r e s p o n s e c u r v e in the p r e s e n c e o f the pept~de: the r a t i o o f the s t i m u l u s intensit,es p r o d u c i n g a 20 m V e.j.p, m the a b s e n c e and the p r e s e n c e o f L e u - e n k e p h a h n was called the s t t m u l u s r a t i o (SR). The amplitude
o f e.j.ps m
the vas deferens t a k e n
from
n a i v e m i c e was
p r o p o r t , o n a l to the s t i m u l u s intensity, a n d a plot o f l o g - i n t e n s i t y o f s t t m u l a t ] o n versus
lOOms
20 rnV
A
B
SR-1
10mV 30
B
6
20
10 2-
3
,
,
,
10
30
100 v
L
0
Normorph~ne Leu-enkephahn
04 04
2
1
pM
Fig 1 Inhibition of e j p amphtude by Leu-enkephahn m the vas deferens of naive and morphinetreated mice A" vas deferens prepared from a naive mouse maintained In the presence of 0 4 lCM normorphlne. Plot of log-stimulus intensity against e j.p amplitude yields a straight line over a certain range Leu-enkephahn I /rM shifts the stimulus-response curve to the right, w~thout any apparent change m slope 0 , control, _ , I /iM Leu-enkephahn, A , ~ 2/~M naloxone Each point represents the average of two consecunve measurements. Stimulus parameters, duration, 1 ms; intervals 20 see Inset amplitude of the e j.ps is proportlonal to the strength of stimulation B" the absence of crosstolerance between morphine and leu-enkephahn In the mouse vas deferens F--, naive animals; r morphine-Implanted ammals. Each column represents the mean ~ S E M. of 6-8 experiments. The first pa~r of columns compares the effect of 2/rM leu-enkephahn m vasa deferentla removed from naive mice to the action of 0 4 HM Leu-enkephahn in the same preparation The second pa~r of columns compares Leu-enkephalin effects (I /~M) obtained on vasa deferentla from morphine treated m~ce to results on tissues from naive ammals. *** P O 001
262 e.j.p, amplitude yielded a straight hne (Ftg. I A). Vasa deferentla m a normorphme-free Krebs solution showed a progressive depression of the e.j.p, amplitude w~th increasing Leu-enkephahn concentration (0 4-2 #M); indicated by progressive shifts of the stimulus-response curve (see SR values in Fig. I B). Vasa deferentla from nawe mmc maintained m the presence of 0.4 # M normorphine also showed a depre~smn of the e.j.p, by Leu-enkephahn (1 /ZM). The stimulus-response curve was shifted to the right (the slopes were, controk 40.02 -+ 3 76; 1 # M Leu-enkephalin, 34.06 -~ 4.50; S.E.M, 8; parred t-test, P ~-0.05). Naloxone (2/ZM) reversed the actmn of Leu-enkephahn (Fig. IA). The stimulus-response curve in the presence of naloxone was to the left of the control curve, presumably because the normorphme (0.4 ~M) present m the bath also contributed to the depressmn of the e.j.p. A similar parallel shift m the sttmulus-response curve was observed m vasa deferentm from morphine treated mice which were maintained in 0.4 #M normorphtne (the slopes were, control, 39.53 ~- 2.59: I u M Leu-enkephahn, 36.46 -~ 6.05: S.E.M., prated t-test, P 0.05). The degree of shift produced by 1 # M Leu-enkephahn was the same m vasa deferentm prepared from morphine-treated mice and m t~ssues from control ammals The present results mdncate the lack of any s~gmficant cross-tolerance between morphine and Leu-enkephalin m the mouse vas deferens. Leu-enkephahn shifted the snmulus--response curves m a parallel way to the right both m preparatmns from naive and morphine-treated mice and there was no difference in the stimulus ratios either Waterfield et al.~.~ have shown cross-tolerance between morphme and Met-enkephahn in vasa deferentm obtained from mice implanted w~th morphine-pellets. However, Met-enkephahn Is a less selective ~-receptor stimulant m the mouse vas deferens than Leu-enkephahn (Wtister, Schulz and Herz, to be published). Thus, a long lasting activation of the #-receptor by morphine may render th~s s~te tolerant to the effects of morphine, leaving at the same tm~e unaffected the ~-receptor, where keu-enkephahn almost exclusively exerts its actmn. These results accord with the recent tindmg, that the contractde response of the mouse vas deferens shows marked tolerance with long term in vwo exposure to either sufentanyl (/z-receptor agomst) or i~-Ala'-o-Leu "~enkephahn (c~-receptor agomst) wRhout any sigmficant cross-tolerance between the two agomsts ~' n
-
The authors are grateful to Drs. R A. North and R. Schulz for many helplul suggestions during the preparation of this manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn.
1 Cox, B.M, Multiple mechamsms m opiate tolerance. In J.M. Van Ree and L. leremus (Ed,~), Charactertstics and Functions o f Opioids, Elsevier/North-Holland Amsterdam, 1978, pp. t 3-23 2 G~bson, R D. and Tingstad, J.E, Formulation of a morphine-implantation pellet smtabte for tolerance-phystcal dependence studies in mice, J. Pharm. Sci, 59 (1970) 426-427 3 Gillan, M.G.C,, Kosterlitz, H.W, Robson, L.E. and Waterfield, A.A, The mhabitory effects of presynaptlc rx-adrenoreeeptor agonists on contractmns of guinea-pig ileum and mouse vas deferens m the morphine dependent and withdrawn states produced in vitro, Brtt .t Pharmtwol, 66 (1979) 60l -608
263 4 Henderson, G Hughes, J. and Kosterlitz, H.W., A new example of a morphine-sensitive neuroeflector junction: adrenergic transmission in the mouse vas deferens, Brit J Pharmacol, 46 (19721 764-766. 5 Henderson, G. and North, R.A., Depression by morphine of excitatory junction potentials in the vas deferens of the mouse, Brit. J. Pharmacol, 57 (1976) 341-346 6 Hughes, J., Kosterhtz, H W. and Leshe, F M , Effect of morphine on adrenerglc transmission m the mouse vas deferens Assessment of agonlst and antagonist potencies of narcotic analgesics, BJtt J Phalmacol, 53 (1975) 371-381. 7 Illes, P. and Schulz, R , Inhibition of neuroeffector transmission by morphine in the vas deferens of naive and morphine-treated mice. Br it. J. Pha-macol, in press 8 llles, P , Zleglgansberger, W and Herz, A , Calcium reverses the inhibitory action of morphine on neuroeffector transmission m the mouse vas deferens, BJam Re,search, 191 (1980) 511 522. 9 Kosterhtz, H W , Multiple opiate receptors In J Jacob (Ed), Advances' m Pharmacology and Fhetapeuttcs, Vol l, Pergamon Press, New York, 1978, pp. 15-23 10 Lord, J A H , Waterfield, A . A , Hughes, J and Kosterhtz, H W , Endogenous op~old peptldes multiple agomsts aqd receptors, Natme (Lond), 267 (1977) 495-499 I 1 North, R A and Vltek, L V , The effect of chronic morphine treatment on excitatory junction potentials in the mouse vas deferens, Btit J Pha~macol, 68 (1980) 399-407 12 Schulz, R , Wuster, M , Krenss, H and Herz, A , Selective development of tolerance without dependence m multiple opiate receptors of mouse 'Jas deferens, Natme (Lond), 285 (1980) 242 243 13 Waterfield, A A , Hughes, J. and Kosterlltz, H W , Cross tolerance between morphine and methJomne-enkephalln, Nature (Lond), 260 (1976) 624-625 14 Wuster, M., Schulz, R and Herz, A , Specificity ofoplolds towards the / ¢, ¢) or e-opiate receptors, Neuro~sct Lett, 15 (1979) 193-198
Bratn Reseal ~tt, 197 ( 19080 260- 203 i , F l s e w e r / N o r t h - H o l l a n d Blomethcal Press
Lack of cross-tolerance between morphine and Leu-enkephalin in the mouse vas deferens
P E T E R 1LLES, W A L T E R Z I E G L G ~ , N S B E R G E R
and ALBERT HERZ
Department oJ Neuropharmacology, Ma.~:-Planclt-ln~tttut fu~ P,sychtatJte, l~raepelm,~t~a,~,~e 2, 1)-80()0 Munchen 40 ( G F R ) (Accepted M a y 8th, 1980)
key wo~ds mouse vas deferens--excltatory Junction potennal,~---opmtereceptors
Morphine reduces the release of noradrenahne from the mouse vas deferens 4. In consequence, the amplitude of excitatory junction potentials (e.j.ps) recorded from single smooth muscle cells 5 and also nerve stimulation evoked contractions of tile organ are depressed 4,6. Tolerance to this action of morphine was evident when the depressson of e.j.p, amplitudes evoked by submaxlmal stimulation was measured in vasa deferentla from morphine treated mice n. The degree of tolerance displayed was inversely related to the intensity of stimulation 7. Thas fact may account for the poor ability of morphine to reduce tolerance when measured by the mhabJnon of the contractile responses using supramaxlmal stamuli ~.:~ The mouse vas deferens has been reported to contain two classes of opiate receptors, one for which morphine (/z-receptor) and another one for which Leu-enkephalin or D-AlaZ-D-LeuS-enkephahn (d-receptor) IS the prototypic agomst ~.~°.t4. In view of these different receptor subtypes, at was of interest to know whether tolerance reduced by long term exposure to an agomst at one receptor was or was not accompamed by tolerance to the effects of an agonlst at the other receptor. The purpose of the present study was to investigate the effect of Leu-enkephalin on e j p, amphtudes of vasa deferentm prepared from morphine treated mice N M R I mice (25-35 g) were killed by decapitation and the vasa deferentm were removed. The preparation was superfused with Krebs solution s gassed w~th 95",, O~/5°(, COo_ and warmed to 37 ~C at a flow rate of 3 mt/min. The intramural sympathetic nerves were stamulated by two platinum ring electrodes. Stimuli were single pulses (1 msec duration) repeated at intervals of 20 sec and were of varlou~ voltages. Stimulataon current was measured over a 10 kf~ resistance placed m series, yielding an essentially constant current stimulus, lntracellular potentials were recorded as in previous experiments s. Some of the mice were amplanted subcutaneously wath morphine containing pellets (75 mg morphine base prepared according to Gibson and Tingstad-'). Half a
261 pellet was i m p l a n t e d o n the first d a y , a n d a w h o l e pellet o n the s e c o n d day. T h e a m m a l s were sacrificed o n the t h i r d day a n d thetr v a s a d e f e r e n t l a were placed m K r e b s s o l u t i o n c o n t a i n i n g 0.4 # M
n o r m o r p h l n e to s i m u l a t e the p l a s m a c o n c e n t r a t i o n o f
m o r p h i n e . T h i s m e t h o d o f p e l l e t - i m p l a n t a t i o n has been p r e v i o u s l y s h o w n to p r o d u c e t o l e r a n c e to m o r p h i n e m the m o u s e vas d e f e r e n s 7. A s e c o n d g r o u p o f v a s a w e r e t a k e n f r o m n a i v e m i c e a n d were s u p e r f u s e d in n o r m a l K r e b s s o l u t i o n and a t h i r d g r o u p o f vasa were t a k e n f r o m n a i v e m i c e a n d s u p e r f u s e d m a K r e b s s o l u t i o n c o n t a i n i n g 0.4 /~M n o r m o r p h m e . T h e d e p r e s s i o n o f the e.j.p, by L e u - e n k e p h a l m was m e a s u r e d by r e p e a t i n g the s t i m u l u s - r e s p o n s e c u r v e in the p r e s e n c e o f the pept~de: the r a t i o o f the s t i m u l u s intensit,es p r o d u c i n g a 20 m V e.j.p, m the a b s e n c e and the p r e s e n c e o f L e u - e n k e p h a h n was called the s t t m u l u s r a t i o (SR). The amplitude
o f e.j.ps m
the vas deferens t a k e n
from
n a i v e m i c e was
p r o p o r t , o n a l to the s t i m u l u s intensity, a n d a plot o f l o g - i n t e n s i t y o f s t t m u l a t ] o n versus
lOOms
20 rnV
A
B
SR-1
10mV 30
B
6
20
10 2-
3
,
,
,
10
30
100 v
L
0
Normorph~ne Leu-enkephahn
04 04
2
1
pM
Fig 1 Inhibition of e j p amphtude by Leu-enkephahn m the vas deferens of naive and morphinetreated mice A" vas deferens prepared from a naive mouse maintained In the presence of 0 4 lCM normorphlne. Plot of log-stimulus intensity against e j.p amplitude yields a straight line over a certain range Leu-enkephahn I /rM shifts the stimulus-response curve to the right, w~thout any apparent change m slope 0 , control, _ , I /iM Leu-enkephahn, A , ~ 2/~M naloxone Each point represents the average of two consecunve measurements. Stimulus parameters, duration, 1 ms; intervals 20 see Inset amplitude of the e j.ps is proportlonal to the strength of stimulation B" the absence of crosstolerance between morphine and leu-enkephahn In the mouse vas deferens F--, naive animals; r morphine-Implanted ammals. Each column represents the mean ~ S E M. of 6-8 experiments. The first pa~r of columns compares the effect of 2/rM leu-enkephahn m vasa deferentla removed from naive mice to the action of 0 4 HM Leu-enkephahn in the same preparation The second pa~r of columns compares Leu-enkephalin effects (I /~M) obtained on vasa deferentla from morphine treated m~ce to results on tissues from naive ammals. *** P O 001
262 e.j.p, amplitude yielded a straight hne (Ftg. I A). Vasa deferentla m a normorphme-free Krebs solution showed a progressive depression of the e.j.p, amplitude w~th increasing Leu-enkephahn concentration (0 4-2 #M); indicated by progressive shifts of the stimulus-response curve (see SR values in Fig. I B). Vasa deferentla from nawe mmc maintained m the presence of 0.4 # M normorphine also showed a depre~smn of the e.j.p, by Leu-enkephahn (1 /ZM). The stimulus-response curve was shifted to the right (the slopes were, controk 40.02 -+ 3 76; 1 # M Leu-enkephalin, 34.06 -~ 4.50; S.E.M, 8; parred t-test, P ~-0.05). Naloxone (2/ZM) reversed the actmn of Leu-enkephahn (Fig. IA). The stimulus-response curve in the presence of naloxone was to the left of the control curve, presumably because the normorphme (0.4 ~M) present m the bath also contributed to the depressmn of the e.j.p. A similar parallel shift m the sttmulus-response curve was observed m vasa deferentm from morphine treated mice which were maintained in 0.4 #M normorphtne (the slopes were, control, 39.53 ~- 2.59: I u M Leu-enkephahn, 36.46 -~ 6.05: S.E.M., prated t-test, P 0.05). The degree of shift produced by 1 # M Leu-enkephahn was the same m vasa deferentm prepared from morphine-treated mice and m t~ssues from control ammals The present results mdncate the lack of any s~gmficant cross-tolerance between morphine and Leu-enkephalin m the mouse vas deferens. Leu-enkephahn shifted the snmulus--response curves m a parallel way to the right both m preparatmns from naive and morphine-treated mice and there was no difference in the stimulus ratios either Waterfield et al.~.~ have shown cross-tolerance between morphme and Met-enkephahn in vasa deferentm obtained from mice implanted w~th morphine-pellets. However, Met-enkephahn Is a less selective ~-receptor stimulant m the mouse vas deferens than Leu-enkephahn (Wtister, Schulz and Herz, to be published). Thus, a long lasting activation of the #-receptor by morphine may render th~s s~te tolerant to the effects of morphine, leaving at the same tm~e unaffected the ~-receptor, where keu-enkephahn almost exclusively exerts its actmn. These results accord with the recent tindmg, that the contractde response of the mouse vas deferens shows marked tolerance with long term in vwo exposure to either sufentanyl (/z-receptor agomst) or i~-Ala'-o-Leu "~enkephahn (c~-receptor agomst) wRhout any sigmficant cross-tolerance between the two agomsts ~' n
-
The authors are grateful to Drs. R A. North and R. Schulz for many helplul suggestions during the preparation of this manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn.
1 Cox, B.M, Multiple mechamsms m opiate tolerance. In J.M. Van Ree and L. leremus (Ed,~), Charactertstics and Functions o f Opioids, Elsevier/North-Holland Amsterdam, 1978, pp. t 3-23 2 G~bson, R D. and Tingstad, J.E, Formulation of a morphine-implantation pellet smtabte for tolerance-phystcal dependence studies in mice, J. Pharm. Sci, 59 (1970) 426-427 3 Gillan, M.G.C,, Kosterlitz, H.W, Robson, L.E. and Waterfield, A.A, The mhabitory effects of presynaptlc rx-adrenoreeeptor agonists on contractmns of guinea-pig ileum and mouse vas deferens m the morphine dependent and withdrawn states produced in vitro, Brtt .t Pharmtwol, 66 (1979) 60l -608
263 4 Henderson, G Hughes, J. and Kosterlitz, H.W., A new example of a morphine-sensitive neuroeflector junction: adrenergic transmission in the mouse vas deferens, Brit J Pharmacol, 46 (19721 764-766. 5 Henderson, G. and North, R.A., Depression by morphine of excitatory junction potentials in the vas deferens of the mouse, Brit. J. Pharmacol, 57 (1976) 341-346 6 Hughes, J., Kosterhtz, H W. and Leshe, F M , Effect of morphine on adrenerglc transmission m the mouse vas deferens Assessment of agonlst and antagonist potencies of narcotic analgesics, BJtt J Phalmacol, 53 (1975) 371-381. 7 Illes, P. and Schulz, R , Inhibition of neuroeffector transmission by morphine in the vas deferens of naive and morphine-treated mice. Br it. J. Pha-macol, in press 8 llles, P , Zleglgansberger, W and Herz, A , Calcium reverses the inhibitory action of morphine on neuroeffector transmission m the mouse vas deferens, BJam Re,search, 191 (1980) 511 522. 9 Kosterhtz, H W , Multiple opiate receptors In J Jacob (Ed), Advances' m Pharmacology and Fhetapeuttcs, Vol l, Pergamon Press, New York, 1978, pp. 15-23 10 Lord, J A H , Waterfield, A . A , Hughes, J and Kosterhtz, H W , Endogenous op~old peptldes multiple agomsts aqd receptors, Natme (Lond), 267 (1977) 495-499 I 1 North, R A and Vltek, L V , The effect of chronic morphine treatment on excitatory junction potentials in the mouse vas deferens, Btit J Pha~macol, 68 (1980) 399-407 12 Schulz, R , Wuster, M , Krenss, H and Herz, A , Selective development of tolerance without dependence m multiple opiate receptors of mouse 'Jas deferens, Natme (Lond), 285 (1980) 242 243 13 Waterfield, A A , Hughes, J. and Kosterlltz, H W , Cross tolerance between morphine and methJomne-enkephalln, Nature (Lond), 260 (1976) 624-625 14 Wuster, M., Schulz, R and Herz, A , Specificity ofoplolds towards the / ¢, ¢) or e-opiate receptors, Neuro~sct Lett, 15 (1979) 193-198