Yohimbine reduces morphine tolerance in guinea-pig ileum

Life Sciences, Vol. 40, pp. Printed in the U.S.A.

YOHIMB!NE

155-160

REDUCES

Luis F. Alguacil,

Pergamon

MORPHINE

Cecilio

Alamo,

TOLERANCE Carmen

IN GUINEA-PIG Santos

(Received

in final

form October

ILEUM

and Eduardo

D e p a r t a m e n t o de Fisiol. y Farmacologia, Fac. Universidad de Alcal~ de Henares Aleal~ de Henares, Madrid, Spain.

Journals

Cuenca

Medicina

7, 1986)

Summary Opiates are known to inhibit electrically-evoked twitches of longitudinal muscle-myenterie plexus strips from guinea-pig ileum. When this preparation was incubated with morphine for i h tolerance developed to the inhibitory effect, since dose-response curves were shifted to the right. In the present study, the effects of alpha-2 adrenergic agents on the tolerance induced by morphine in this preparation was investigated. Addition of yohimbine i0 ~M (but not 0.i or i ~M) to the incubating medium reduced the magnitude of opiate tolerance. This effect did not appear in the presence of the alpha-2 agonists clonidine or guanfacine (i0 ~M). Our results provide evidence of the longitudinal muscle-myenteric plexus as a useful model for the study of the relationship between morphine tolerance and alpha-2 adrenergic mechanisms. Acute and chronic effects of opiates can be altered by drugs acting on alpha-2 adrenoceptors in several conditions. Thus, the alpha-2 antagonist yohimbine reduces m o r p h i n e - i n d u c e d analgesia (i), hypothermia (2) and inhibition of intestinal transit (3) in rodents, and is able to abolish hypotension induced by enkephalins in dogs (4). Withdrawal from a chronic exposure to opiates shows a variety of experimental signs which can be inhibited by alpha-2 agonists such as elonidine or guanfacine (5); this effect constitudes the basis for the therapeutic use of elonidine and analogues on opiate dependence (6,7,81 Guinea-pig ileum (GPI) and strips of longitudinal muscle-myenteric plexus of the g u i n e a - p i g ileum (LM-MP) are widely used to investigate opiate actions. Both alpha-2 agonists and opiates inhibit e l e c t r i c a l l y - e v o k e d twitches of GPI and LM-MP by decreasing acetylcholine release (9,10). Tolerance to this opiate effect can appear after a chronic exposure to opiates "in vivo" or "in vitro"; so, "in vitro" incubation of LM-MP with morphine induces a loss of opiate inhibition when an assessment is made after this period. Tolerance achieved by these means closely resembles tolerance to other effects of opiates, i.e. analgesia: it is a stereospeeific effect, it can be blocked by naloxone coadministration, it is inhibited by cycloheximide, etc. (ii). The aim of this work has been to determine whether tolerance to morphine in LM-MP can be modified by simultaneous administration of alpha-2 adrenergie agents. Methods LM-MP preparation. Albino guinea-pig of both sexes weighing about used. Animals received water and food "ad libitum" until at least

Copyright

0024-3205/87 $3.00 + .00 (c) 1987 Pergamon Journals

Ltd.

500 g were iS h before

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the experiment, and were s a c r i f i c e d by a b l o w on the neck and exsanguination. lleum was r a p i d l y e x c i s e d and kept in Ringer's m o d i f i e d solution of the f o l l o w i n g c o m p o s i t i o n (mM): NaCI, 154; KCI, 5.66; CaCI 2, 2.54; glucose, 2.77; NaHCO 3, 5.95 and choline chloride, 0.002. The last 15 cm of the terminal ileum were s y s t e m a t i c a l l y discarded. The l o n g i t u d i n a l muscle with the a t t a c h e d m y e n t e r i c plexus was o b t a i n e d from 3 cm segments of ileum a c c o r d i n g to Puig et al. (12). Each strip was m o u n t e d in a 7 ml organ bath c o n t a i n i n g Ringer's solution at 37 °C and b u b b l e d with 95% 0 2 - 5 % CO 2. The tissues were s u s p e n d e d under a r e s t i n g tension of 0.5 g and were a l l o w e d to e q u i l i b r a t e for a 60 min period. Field s t i m u l a t i o n was effected with Grass and SRI s t i m u l a t o r s through two p l a t i n u m rings s e r v i n g as electrodes, using r e p e a t e d pulses of 70 V of 5 msec in duration and at a frequency of O.15 Hz. Twitches were r e c o r d e d by Ugo basile isotonic t r a n s d u c e r s attached to an Ugo basile poligraph. D e v e l o p m e n t and a s s e s s m e n t of t o l e r a n c e to morphine. Induction and a s s e s s m e n t of m o r p h i n e t o l e r a n c e were based on the technique described by Rezvani et al. (ii). Once e q u i l i b r a t i o n period transcurred, electric s t i m u l a t i o n was applied until a steady a m p l i t u d e was obtained. A d d i t i o n of morphine to the bath resulted in a rapid twitch i n h i b i t i o n r e a c h i n g a m a x i m u m within 2 min after application. When i n h i b i t i o n stabilized, the LM-MP was washed and allowed to equilibrate before a second trial. Three to five i n c r e a s i n g c o n c e n t r a t i o n s of m o r p h J ne were applied. A plot was then made of the degree of twitch inhibition (percentage) a g a i n s t the c o n c e n t r a t i o n of the opiate (-log M), and the concentration to inhibit o n e - h a l f the maximal a m p l i t u d e of the twitch response (IC50) was d e t e r m i n e d by interpolation. Segments were r e n d e r e d tolerant by i n c u b a t i o n with Ringer c o n t a i n i n g theirspecific IC50 of m o r p h i n e for i h. Bath solution was e x c h a n g e d at 20 min intervals. At the end of the i n c u b a t i o n time, the IC50 of morphine was r e d e t e r m i n e d as d e s c r i b e d above while m a i n t a i n i n g the i n c u b a t i n g c o n c e n t r a t i o n of morphine to p r e v e n t opiate w i t h d r a w a l responses. The ratio of the morphine IC50 determined post and p r e i n c u b a t i o n with morphine (IC50POST and IC50PRE, respectively) p r o v i d e d an index of the degree of tolerance developed. A separate group of e x p e r i m e n t s were p e r f o r m e d to verify that loss of s e n s i t i v i t y to m o r p h i n e was not due to s p o n t a n e o u s d e s e n s i t i z a t i o n of LM-MP in our e x p e r i m e n t a l conditions. In these cases, the ratio I C 5 0 P O S T / I C 5 0 P R E was s t a b l i s h e d after i n c u b a t i o n with Ringer free of morphine for i h. Effect of y o h i m b i n e on m o r p h i n e tolerance. To assess tolerance, y o h i m b i n e (0.i, 1 and i0 ~M) was included and washed before ICsoPOST determination.

its effect on m o r p h i n e in the i n c u b a t i n g s o l u t i o n

Four e x p e r i m e n t s were made to i n v e s t i g a t e the effect of i n c u b a t i o n with y o h i m b i n e (i0 ~M) alone for i h on the i n h i b i t o r y effect of morphine. Again, y o h i m b i n e was washed before IC50POST determination. R e v e r s i o n of y o h i m b i n e effect by a l p h a - 2 agonists. In these groups of experiments, either c l o n i d i n e or g u a n f a c i n e (iO ~M) were p r e s e n t in the i n c u b a t i o n m e d i u m together with morphine and y o h i m b i n e (iO UM). All drugs except morphine were w i t h d r a w n to c a l c u l a t e IC50POST. Influence of y o h i m b i n e on the acute effect of morphine. The i n h i b i t o r y effect of m o r p h i n e 0.2 ~M in LM-MP was d e t e r m i n e d in absence and in presence of y o h i m b i n e (0.01-I0 ~M). The c o n c e n t r a t i o n of m o r p h i n e used for these experiments is the mean IC50 obtained in 59 d e t e r m i n a t i o n s (0.20±0.01).

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Effect

Statistics. Statistical using Student t-test.

comparison

of Y o h i m b i n e

between

on Opiate

different

Tolerance

treatments

157

was made

Results Incubation with morphine provoked a shift to the right of the doseresponse curve to the opiate, thus showing tolerance development (Fig. I,A). IC 5 ~ O S T / I C 5 ~ R E ratio quantified this effect with a value of 5.25 ± 0.45 (mean £ s.e., Table I). The ratio obtained by incubation with Ringer free of morphine showed no desensitization of LM-MP to the opiate effect (Table I). Addition of yohimbine together with morphine attenuated the tolerance ratio induced by the opiate. This effect was statistically significant when the concentration of yohimbine used was i0 pM (Table I). As expected, in this last case the shift of the postincubation curve was also attenuated (Fig. I,B). On the other hand, incubation with yohimbine i0 ~M alone did not modifie the sensitivity to morphine, as the tolerance ratio was similar to that obtained in the group incubated with Ringer (Table I). The combined incubation with clonidine (i0 WM), yohimbine (i0 pM) and morphine increased the degree of tolerance up to values not significantly different from those observed with opiate incubation (Table I. Fig. I,C). Similar results were achieved with guanfacine (i0 pM), yohimbine (i0 pM) and morphine (Table I, Fig. I,D). The acute effect of morphine concentrations used (Fig. 2).

(0.2 pM) was not altered

TABLE

by yohimbine

at the

I

Degree of Tolerance to Morphine in LM-MP after Different Incubation Procedures

INCUBATION

MOR RINGER YOH(IO

pM)

MOR+YOH(O.I pM) MOR+YOH(I.O pM) MOR+YOH(IO ~M) MOR+¥OH(IO ~M)~CL(IO MOR+YOH(IO ~M)~GU(Io

pM) pM)

n

I C 5 0 PBE ( pM )

I C 5 0P O S T ( pM )

TOLERANCE

RATIO

i0 7 4

0.19 ± 0.03 0.24 ± 0.04 0.28 ± 0.03

1.10 ± 0.29 0.22 ± 0.04 0.23 ± 0.08

5.25 ± 0 . 4 5 0.91 ± 0.i0" 0.99 ± 0.26*

7 8 i0

0.26 ± 0.08 0.28 f 0.05 0.]4 ± 0.02

1.21 ± 0.49 0.92 ± 0.20 0.23 ± 0.07

4.24 ± 0.76 3.68 ± 0.79 1.54 ± 0.22*

9 8

0.15 ± 0.02 0.19 ± 0.03

0.74 ± 0.21 1.02 ± 0.32

4.34 4.99

± 0.73 ± 1.04

IC50 of morphine was determined before (IC50PRE) and after (IC50POST) incubation of each LM-MP strip. MOB: IC50PRE of morphine; RINGER: Ringer free of morphine; YOH: yohimbine; CL: clonidine; GU: guanfacine. Data are expressed as mean + s.e. of (n) experiments. Tolerance ratio is expressed as mean + s.e. 9f ICsoPOST/ICsoPRE values. (~), p < 0.001 with respect to the tolerance ~atio obtained in the group incubated with morphine.

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Effect

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A. MOR

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40, No.

2, 1987

B. MOR+YOH (iO IJM)

80 z o

~60

m

60

T

g

4O

4O

H

~f"

/

POST

2O

~20

I

I

J

L

I

I

I

I

.5

7

6.5

6

7.5

7

6.5

6

1ogM

[MORPHII~E],

C. MOR+YOH

(iO WM)+CL

(iO ~M)

D. MOR+YOH

80

/f

o

Z

~, 60 =

=

(iO WM)+GU (10 ~ )

80

4O

20

I

.5

I 7

I

m

60

m

40

~

20

I 6

.5

[MORPHINE],

logM

6.5

I 7

i

i

6.5

6

FIG. i D o s e - r e s p o n s e curves to morphine before (PRE,Q) and after (POST,O) d i f f e r e n t incubations (A, B, C and D). NOR: IC50PRE of morphine; YOH: yohimbine; CL: clonidine; GU: guanfaeine. Values r e p r e s e n t mean (+ s.e.) from at least 4 trials.

60

£

5o

T x

oo

T

40

FIG.

3O

i

S 2o 10

MOR

MoR!+JMOR YoH ! IYOH

°'°ii Io.i

MOR

MOR

+ YOH

+ YOH

1.0

i0

2

Influence of y o h i m b i n e (YOH, pM) on the acute effect of morphine (MOR, 0.2 DM) in the LM-MP. The data are e x p r e s s e d as the mean~s.e. from at least five experiments.

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Discussion Incubation for i h of LM-MP with morphine produced a marked degree of tolerance to the opiate inhibition of e l e c t r i c a l l y - i n d u c e d twitches; indeed, more than a 5-fold increase in morphine concentration was necessary to get a 50% twitch inhibition. These results are in agreement with those reported by the original authors of the technique and confirm the validity of the method to study tolerance in this preparation. In addition, "in vitro" induction of tolerance in LM-MP has some advantages to study the effect of alpha-2 agents on opiate tolerant tissues. When clonidine action is assayed in LM-MP from guinea-pigs made tolerant "in vivo", profound differences may be found depending on the presence or absence of morphine in the bath medium (13); so, this kind of experiments depends on an arbitrary concentration of morphine added to the bath to prevent "withdrawal" that could vary the tolerance obtained by chronic "in vivo" administration and the sensitivity of alpha-2 adrenoceptors. The technique used in this work seems to be superior in this way since the concentration of morphine remains constant from the start of the incubation. It can be also realized that "in vitro" tolerance is faster than chronic "in vivo" administration of the opiate and it is easily reproducible. In our experimental conditions, "in vitro" tolerance to morphine was reduced when yohimbine was added to the incubation medium. This effect was dose-dependent and statistically significant when the higher concentration of yohimbine (i0 ~M) was assayed. It can be thought that this high dose of yohimbine would exert its action by removing morphine from its receptor, since the alpha-2 antagonist is able to displace opiates from their specific b i n d i n g sites in the rat brain at the range of concentrations used (i). Nevertheless, this possibility seems improbable because the acute effect of a single dose of morphine similar to that employed for incubations (IC50) was not modified oy yohimbine. Browning et al. (I) showed a lack of antagonism of the acute effect of morphine by yohimbine in GPI (i), which supports our conclusion. On the other hand, concomitant incubation with alpha-2 agonists prevented the inhibition of opiate tolerance observed with yohimbine; this fact let us ascribe that yohimbine action implies a blockade of alpha-2 adrenoeeptors rather than other pharmacological aspects of the drug, i.e. "in vitro" serotonin antagonism (14). It should be pointed out that the concentration of yohimbine required to inhibit opiate tolerance significantly is much higher than the concentration corresponding to its pAn value reDorted a~ainst norepinephrine (14) and clonidine (9) in GPI; this fact suggests that an intense alpha-2 adrenoceptor blockade is needed to antagonize opiate tolerance. We have also found that incubation with yohimbine alone was ineffective to vary the effect of morphine. This finding suggests that incubation with yohimbine restores the sensitivity to morphine in tolerant LM-MP by interferring with some biological process absent in naive LM-MP. One could explain these results on the basis of an increased alpha-2 adrenergie funeionalism which would be involved in morphine tolerance development. This increase c o u l d be caused, for instance, by an enhanced release of endogenous agonists. Such a possibility is in agreement with the findings of Alfaro et al. (15); these authors reported that morphine tolerance in LM-MP can be maintained in absence of the opiate if elonidine is administered to the animals. An "in vivo" cross-tolerance has been also observed when other effects as analgesia (16) or modifications of operant behavior (17) were studied. Ramaswamy et al. (18) have reported opposite results in mouse ileum: they showed that clonidine was effective to reduce opiate tolerance assessed by supersensitivity to exogenous acetylcholine. Such a s u p e r s e n s i t i v i t y has not

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been found in GPI made tolerant "in vivo" or "in vitro" (11,19). The differences with respect to our work may be explained therefore on the basis of the species used; the c r o s s - t o l e r a n c e reported between clonidine and morphine that has been m e n t i o n e d above argue against a similar effect in LM-MP. Acknowledgements This work was supported by grants from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica (C.A.I.C.Y.T.) and from the Caja de Ahorros de Madrid. We thank Dr. M. Vallejo and Dr. E. Morcillo for helpful discussions, as well as Sandoz and B o e h r i n g e r Sohn laboratories for the generous gift of guanfacine and elonidine. References i. S. BROWNING, D. LAWRENCE, A. LIVINGSTON and B. MORRIS, Br. J. Pharmac. 77 487-491 (1982). 2. D. LAWRENCE and A. LIVINGSTON, J. Physiol. 319 52-53 (1981). 3. C.L. WONG, Clin. Exp. Pharmacol. Physiol. ii 605-610 (1984). 4. S. KOYAMA, N. TERADA, Y. SHIOJIMA and T. TAKEUCHI, Jpn. J. Physiol. 34 995-1002 (1984). 5. J.J. BUCCAFUSCO, D.C. MARSHALL and R.M. TURNER, Life Sci. 35 1401-1408

(1984). 6. M.S. 7. A.M.

GOLD, D.E. REDMOND Jr. and H.D. KLEBER, Lancet II 599-602 WASHTON, R.B. RESNICK, J.F. PERZEL and J. GARWOOD, Lancet

(1978). I 991-992

(1981). 8. H. SCHUBERT, W.W. FLEISCHHACKER, U. MEISE and C. THEOHAR, Am. J. Psychiat. 141 1271-1273 (198a). 9. G.M. DREW, Br. J. Pharmac. 64 293-300 (1978). i0. R.A. NORTH and J.T. WILLIAMS-~, Trends in Neurosci. 6 337-339 (1983). ii. A. REZVANI, J.P. HUIDOBRO-TORO, J. HU and E.L. WAY, J. Pharmacol. Exp. Ther. 225 251-255 (1983). 12. M.M. PUIG, P. GASCON and J.M. MUSACCHIO, J. Pharmacol. Exp. Ther. 206 289-301 (1978). 13. M.G.C. GILLAN, H.W. KOSTERLITZ, L.E. ROBSON and A.A. WATERFIELD, Br. J. Pharmac. 66 601-608 (1979). 14. M.R. G O L D B E R G and D. ROBERTSON, Pharmacol. Rev. 35 143-180 (1983). 15. M.J. ALFARO, M.I. COLADO, M.I. MARTIN and P. LORENZO, Actas VIII Reuni6n de la A s o e i a c i 6 n Espa~ola de Farmac61ogos, Jaca, Spain (1984). p. 97. 16. G. PAALZOW, N a u n y n - S c h m i e d e b e r g ' s Arch. Pharmacol. 304 1-4 (1978). 17. J.W. McKEARNEY, Pharmacol. Biochem. Behav. 22 5 7 3 - Z 7 6 (1985). 18. S. RAMASWAMY, N. PADMANABHA PILLAI, V. G O P A L A K R I S H N A N and M.N. GHOSH, Life Sci. 33 1167-1172 (1983). 19. B.M. COX and R. PADHYA, Br. J. Pharmac. 61 271-278 (1977).