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Opioid dependence and cross-dependence in the isolated guinea-pig ileum
European Journal of Pharmacology, 84 (1982) 33-40
33
Elsevier Biomedical Press
O P I O I D D E P E N D E N C E A N D C R O S S - D E P E N D E N C E IN T H E I S O L A T E D G U I N E A - P I G I L E U M RI~DIGER SCHULZ, EVA SEIDL, MICHAEL WI~STERand ALBERT HERZ Department of Neuropharmacology, Max-Planck-lnstitut fi~r Psychiatrie, Kraepelinstrasse2, D-8000 Miinchen 40, F.R.G.
Received 1 April 1982, revised MS received 25 June 1982, accepted 5 July 1982
R. SCHULZ, E. SEIDL, M. W~STER and A. HERZ, Opioid dependence and cross-dependence in the isolated guinea-pig ileum, European J. Pharmacol. 84 (1982) 33-40.
The development of opioid dependence and tolerance attributed to selective types of opiate receptors was studied in the isolated ileum of guinea pigs chronically exposed to specific opioids. These investigations were based on reports that in this preparation highly tolerant opiate receptors may coexist with opiate receptors of almost unchanged sensitivity. Thus, the ilea were set up in vitro and tested for tolerance and dependence. Apparently precipitation of the withdrawal contracture, indicating dependence, proved a more sensitive parameter than the phenomenon of tolerance. Maximal dependence was determined at rather low degrees of tolerance (5 to 10 fold). The intensity of the withdrawal contracture failed to increase as opiate ~olerance did. Furthermore, the experiments failed to present evidence for the existence of selective dependence at specific opiate receptor types. These findings may suggest multiple adaptational mechanisms upon chronic activation of opiate receptors. One mechanism may be responsible for the development of dependence and a low degree of tolerance, whilst a further increase of tolerance may be associated with changes at the opiate binding site level. Opioids
Dependence Cross-dependence Tolerance Guinea-pigileum
1. Introduction A reliable approach for the differentiation of opiate receptors that is, binding site as well as affector, relates to the phenomenon of tolerance development (Martin et al., 1976; Schulz et al., 1981a; Wi~ster et al., 1981a; Clark and Bernardini, 1982). In fact, isolated tissues such as the mouse vas deferens and the guinea-pig ileum (GPI) proved of particular interest since extremely tolerant opiate receptors may coexist in the same preparation with opiate receptors of almost unchanged sensitivity (Schulz et al., 1980; Schulz et al., 1981b). Assuming that a single mechanism accounts for the development of tolerance as well as for dependence ('unitary theory'; Goldstein and Goldstein, 1968; Shuster, 1961; Collier, 1968; 1980), then the above findings would imply that highly tolerant opiate receptors are also highly dependent, and less tolerant receptors are less dependent. Accordingly, tolerance induction for a selective type of opiate 0014-2999/82/0000-0000/$02.75 © Elsevier Biomedical Press
receptors should be complemented by selective dependence. However, such an association may be invalid if tolerance/dependence does not relate to a common mechanism. In fact, multiple adaptational mechanisms upon chronic opiate receptor activation have been reported, involving alterations either at the post recognition stage of the receptor binding site or at the binding site itself (Collier et al., 1981; Sharma et al., 1975; Cox, 1978; Davis et al. 1979; Hazum et al., 1980). To investigate the issue of opiate dependence at specific opiate receptor types, the isolated G P I was chosen for two reasons. First, a high degree of tolerance for highly selectively acting opioids has been demonstrated, suggesting the separate existence of functional /~- and r-receptors in this preparation (Schulz et al., 1981b; Wtister et al., 1981b). Second, the response of a tolerant G P I to naloxone is confined to a single symptom, that is, the withdrawal contracture (Ehrenpreis et al., 1972; Schulz and Herz, 1976a). This makes the G P I
34 highly preferable to the opiate-dependent central nervous s y s t e m which initiates a diversity of withdrawal symptoms upon naloxone challenge (Bl~sig et al., 1978). The availability of narcotic agonists combining preferentially with specific types of receptors allows the demonstration of tolerance for specific types of opiate receptors. However, an analogous demonstration of dependence is complicated by the fact that a receptor-specific narcotic antagonist is not available. Therefore, use was made of the principle that precipitation of an abstinence symptom by the antagonist naloxone requires - besides other conditions - the occupation of opiate receptors by an opioid. In addition only those receptors made tolerant/dependent respond, after they have been withdrawn and reexposed to the opiate, by an intensive withdrawal contracture upon naloxone challenge (Schulz and Herz, 1976a). Thus, the GPI made tolerant/dependent in vivo was intensively washed in vitro to unmask all opiate receptors. Thereafter, the preparations were re-exposed to an opioid combining with a specific type of receptor. The withdrawal contracture precipitated under these conditions by naloxone was assumed to present a demonstration of dependence for a specific type of opiate receptor.
2, Materials and methods
Male guinea pigs (300-350 g) were decapitated and the ileum removed. The longitudinal musclemyenteric plexus (strip) was prepared as described by Kosterlitz et al. (1970). A strip (2cm) was mounted in a 5 ml organ bath in Krebs-Ringer-bicarbonate solution for electrical field stimulation (60 V, 0.5 ms, 0.1 Hz; Schulz and Goldstein, 1972). The preparations were equilibrated by changing the bathing fluid at 5-10 rain intervals. The drug effects evoked were expressed as percentage change of the electrically induced twitches. The ICs0 refer to drug concentrations causing a 50% inhibition of twitch tension. The ilea of guinea pigs were rendered tolerant and dependent by subcutaneous (s.c.) administration (6 days) of opioids dissolved in water using osmotic minipumps (model 2001 and 2 ML1, re-
spectively; Alza Corp., Palo Alto, U.S.A.). The pumps were implanted s.c. under ether anesthesia. To prevent loss of tolerance/dependence of the preparations outside the animals, the strips were exposed in vitro to a defined concentration of the opioid infused in vivo. The opioids used to render the myenteric plexus tolerant were the /~-agonist morphine and the ~agonist benzomorphans ethylketazocine (EK) and MRZ 2549 (Schulz et al., 1981b). In order to obtain a variety of degrees of tolerance/dependence, the guinea pigs were infused chronically with different concentrations of the opioid and the strips were set up in vitro in the presence of the opiate agonist. The infusion rates for morphine were 10, 50 and 100 /~g/h for 6 days. The concentrations used for the in vitro exposure of strips from these animals ranged between 100 and 600 nM normorphine. These concentrations were tolerated with respect to the ability of the preparations to exhibit electrically evoked twitch tension similar to that displayed by naive strips (see Schulz et al., 1981b). The conditions employed for EK were: infusion of 1, 2, 10, 50 ~ g / h for 6 days. The in vitro incubations were conducted with 0.1-4 nM EK. The data for MRZ were 1 and 2 ~ g / h for 6 days, and the in vitro concentrations ranged between 0.1 and 1.0 nM MRZ. Assessment of opiate tolerance: The ICs0 values served for the quantification of tolerance and cross-tolerance. The assays were conducted in the presence of the maintenance opioid (see above). Failure of tolerance development is defined by the quotient ( Q ) - - 1 , that is, the IC.s0 value of this compound in a preparation chronically exposed to an opioid is identical to that observed in a naive preparation. Thus, Q increases as the degree of tolerance increased. Assessment of opiate dependence: Strips of guinea pigs chronically exposed to an opioid were challenged in vitro with naloxone in the absence of electrical stimulation. Two experimental designs were used. First, opioid tolerance/dependence was maintained in vitro from the beginning by exposure of the tissues to a specific opioid. Electrical stimulation was applied continuously. These strips served for the determination of tolerance followed by the test for dependence. Morphine was sub-
35 stituted by normorphine in vitro because of the more hydrophilic characteristics of this derivative. Second, strips were withdrawn in vitro by repeated washing for 1 h. The unmasking of opiate receptors by this procedure was documented by the failure of naloxone (5 /~M) to precipitate a withdrawal contracture. Thereafter, the strips were re-exposed in vitro for 30 min to a specific opioid. This re-exposure period was adapted from experiments conducted with morphine tolerant strips (Schulz and Herz, 1976a). Electrical stimulation was applied during this period and the bathing fluid renewed at 3-5 rain intervals. Subsequently, naloxone (5/tM) was added to the bath to test for dependence. The withdrawal contracture was quantified by calculating the percentage of the intensity of the withdrawal contracture in relation to the twitch tension evoked by electrical stimulation either during exposure to the opioid to which tolerance had developed or at the end of the 1 h withdrawal period (see also Schulz and Herz, 1976a). Chronic clonidine exposure: Guinea pigs were infused with clonidine (50 /~g/h, 6 days). The strips were set up in vitro under two different experimental conditions: first, they were maintained at 100 nM clonidine, and second, they were kept in clonidine (100 nM) simultaneously with 500 nM normorphine. Dependence was tested by challenge of these preparations with phentolamine (1/~M) and naloxone (5#M), respectively. The quantification of the withdrawal symptom followed that described above for opioid dependent strips. Statistical analysis: each quotient (Q) represents the mean ---S.E.M. of at least 4 independent experiments. One experiment reflects the Q computed from the mean ICs0 values of each of 3 preparations from a naive and from a chronically treated animal. The regression lines of fig. 1 (Q 1-10 and Q 50-200) were fitted by the method of least squares, and sums of the products of deviations from the regression gave the regression coefficients. The curves expressing the relation between tolerance and dependence were obtained by best-fit computer analysis. The following substances were used: Morphine
HC1 (Merck, Darmstadt, F.R.G.); normorphine, NORM (Bios Products, Brussels, Belgium), aneoendorphin-(1-8), ANE; dynorphin-(1-13), DYN (Peninsula Labs., San Carols, U.S.A.); ethylketazocine, EK (Sterling-Winthrop Research Institute, New York, U.S.A); MRZ 2549, (5,9-dimethyl,2',5)-5-9-dimethyl-2'-hydroxy-2-(methoxypropyl)-6, 7-benzomorphan, clonidine (Boehringer, Ingelheim, F.R.G.).
3. Results
Different concentrations of naloxone are required to antagonize the action of/t- or ~-agonists in the GPI. To determine the optimal naloxone concentration to precipitate a maximal abstinence sign for any opioid under investigation, strips were rendered tolerant to EK (100/~g/h, 6 days, set up in vitro at 10 nM EK). The K e value of naloxone in the GPI for the r-agonist EK is several fold higher than for a ~-agonist (Hutchinson et al., 1975). Under these conditions, precipitation of a maximal withdrawal sign required a concentration of at least 2/~M naloxone. Hence, 5/.tM naloxone was employed throughout to assay opioid dependence. Fig. 1 shows the relation between the development of dependence, as expressed by the withdrawal contracture, and tolerance, as expressed by the quotient 'Q' for the /~-agonist morphine (panel A) as well as for the r-agonists EK (panel B) and MRZ (panel C). The dotted line in each panel indicates the best-fit computer analysis between the two parameters. Apparently, the shape of the tolerance/dependence response curves is very similar for the various drugs under investigation. A very steep slope of the curves at low degrees of tolerance is followed by an extremely flat slope at high degrees of tolerance. This is documented by the regression coefficients for the initial phase (b~) of the curves (Q = 1-10) as well as for the flat component (b 2) (Q = 50-200): panel A: b~ =4.6, b z - - - 0 . 0 2 ; panel B: b~=4.0, b2--0.05; panel C; bj = 5.7, b 2 = --0.01. EK showed a difference from the other drugs used. Apparently, in these experiments the plateau was lower than that for morphine and MRZ. The most plausible
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Fig. 1. Correlation between tolerance and dependence in the isolated GPI. Guinea pigs were infused with different concentrations of morphine, EK or MRZ. The strips were maintained in vitro at different concentrations of the opioid which had been infused in vivo, that is, normorphine (panel A), EK (panel B) and MRZ (panel C). Each dot represents data of a single strip which was used once to assay tolerance and dependence. The dotted line indicates the tolerance/dependence curve obtained by best-fit computer analysis. The solid lines indicate the regression lines for the degree of tolerance (Q) ranging from 1 to 10 and 50 to 200. b t and b 2 reflect the respective regression coefficients. The mean values obtained for Q>200 are based on a variety of single data indicated by 'n'. Abscissa: degree of tolerance (Q) calculated from IC50 values of tolerant and naive preparations. Ordinate: intensity of withdrawal contracture expressed as percent of the preceding electrical stimulation. e x p l a n a t i o n for this p h e n o m e n o n takes i n t o account the offset time of the action of EK which g o v e r n s t h e s t r e n g t h o f t h e w i t h d r a w a l sign ( S c h u l z a n d H e r z , 1976b).
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reincubation (n~) Fig. 2. Opioid dependence and cross-dependence in the isolated GPI. Strips were rendered dependent by infusing guinea pigs with morphine, EK or MRZ. The dosages per hour were infused for 6 days as indicated in the corresponding panels. After withdrawal of the preparations, they were reincubated with opioids with high receptor selectivity, such as normorphine (NORM) for #-receptors, and ethylketazoeine (EK), dynorphin-(I-13) (DYN~.~3) and a-neo-endorphin-(l-8) (ANEI_s) for ~-receptors. The reincubation concentrations of the drugs are indicated on the abscissa. The preparations were challenged with naloxone and the resulting strength of the withdrawal contractions is given on the ordinate. The columns indicate means --+S.E.M. of at least 6 different preparations. The incubation concentrations selected for the strips of guinea pigs infused with lower drug concentrations were the ICs0 values determined m naive preparations.
38 additional contracture could be evoked at the peak of the contracture by either electrical stimulation or by the addition of serotonin (100 nM) to the bath. This drug concentration manifests its actions at the myenteric plexus and in turn causes the release of acetylcholine (Adam-Vizi and Vizi, 1978). Fig. 2 presents data from experiments on crossdependence in preparations chronically exposed to specific receptor agonists like morphine (upper panel), EK and MRZ (lower panels). The guinea pigs received different infusion concentrations per h (see fig.) in order to induce different degrees of dependence. The upper panel demonstrates the withdrawal contractures of preparations made tolerant by infusion of 10 and 100 # g / h of the /~-agonist morphine. These preparations were withdrawn in vitro and reincubated with the /~agonist normophine as well as with DYN-(1-13) or ANEI(1-8), both interacting with ~-receptors in this preparation (Wtister et al., 1981b). The drug concentrations selected corresponded to the ICs0 values (determined on naive preparation) for the low infusion concentration. The re-exposure concentration was increased 5 fold for strips taken from guinea pigs infused with 100 /~g/h for 6 days. Surprisingly, naloxone challenge of such preparations revealed no difference in the intensity of the withdrawal contracture within an identical experimental design, regardless of the opiate receptor agonist used for reincubation. As expected, a more intense withdrawal sign was precipitated in preparations taken from guinea pigs infused with 100/~g/h morphine. In principle, this result obtained with strips made previously dependent to a /~-agonist was also observed with preparations made tolerant to the ~-agonists EK or MRZ (see the two lower panels of fig. 2). The demonstration of dependence to a nonopioid drug, clonidine, in the GPI (Collier et al., 1981) caused us to test for cross-dependence between this o~z-adrenergic agonist and the t~-receptor agonist NORM. Clonidine-tolerant strips exhibited dependence upon challenge with phentolamine. The withdrawal contracture amounted to 23 -+ 17% (n = 12). The preparations set up in the presence of clonidine and normorphine failed to display a withdrawal contracture upon challenge with naloxone.
4. Discussion The naloxone-precipitated withdrawal contracture in the GPI has been used repeatedly as an indication of opioid dependence (Schulz, 1978). Moreover, theoretical considerations as well as experimental evidence have intimately linked the phenomenon of dependence with that of tolerance, claiming a common underlying biochemical mechanism (Goldstein and Goldstein, 1968; Shuster, 1961; Collier, 1980; Bl~sig and Herz, 1978). In principle, part of the data communicated here are not in conflict with these theoretical considerations. However, an interpretation of all the findings reported here would imply multiple adaptational mechanisms. A discrepancy between the rate of development of dependence and that of tolerance in the GPI becomes obvious when the phenomena are compared. The degree of dependence, as indicated by the intensity of the withdrawal contracture, is already considerable while tolerance is hardly detectable or very low. In fact, an almost maximal degree of dependence is exhibited at about a 5 fold degree of tolerance. As tolerance increases, dependence does not increase further. Apparently, this failure of tolerant/dependent preparations to display progressively increasing withdrawal contractures seems not to be due to exhaustion of the ability to contract. Withdrawal contracture thus reflects an extremely sensitive parameter for the detection of adaptational processes i.e., dependence, upon chronic opiate receptor activation. It is more easily detected than tolerance. This parallels results obtained in the central nervous system, where dependence is already detectable in the apparent absence of tolerance (Johnson and Duggan, 1981). A further aspect of the dependence studies relates to the phenomenon of cross-dependence. The assumption that highly tolerant opiate receptors are also highly dependent and that such receptors coexist with low tolerant/dependent receptors was not confirmed by the present investigations. Their experimental design showed that specific types of opiate receptors failed to develop dependence. That is, dependence of/~-receptors caused by their chronic activation was associated with the
39
same degree of dependence of g-receptors. It is of interest in this context that induction of 50 to 80 fold tolerance in a specific receptor type, e.g. kt-receptors, was always associated with some degree of cross-tolerance (5-10 fold) in other opiate receptors, e.g. r-receptors (Schulz et al., 1981b). As shown in fig. 1, a maximal degree of withdrawal contracture is precipitated by naloxone at a relatively low level of tolerance (5-10 fold) of this preparation. Thus a similar degree of cross-dependence for the different opiate receptor types would even be expected on the basis of this result. These findings, together with those published earlier (Schulz et al., 1981b), would imply that chronic activation of a specific opiate receptor population may result in a high degree of tolerance for this specific population with a low degree of crosstolerance to other opiate receptor types. Under these conditions almost maximal cross-dependence is demonstrated at the different types of opiate receptors. The remaining question is to integrate the present data into a concept explaining tolerance and dependence in the GPI. As has been stated above, a 'unitary theory' can hardly account for all data reported here. Assuming, however, that adenylate cyclase exerts a critical function in the expression of tolerance and dependence (Collier and Roy, 1974; Collier and Francis, 1975; Sharma et al., 1975), then the appearance of the minor degree of cross-tolerance as well as of cross-dependence between different types of opiate receptors could be explained. Such an interpretation surmise s , however, access of all opiate receptors to the same adapted adenylate cyclase either directly or by chance contact as proposed in the 'mobile-receptor hypothesis' (Hazum et al., 1981). Provided that the prototype a2-adrenoreceptor agonist clonidine also brings about its action via adenylate cyclase, and that the az-receptor as well as the opiate receptors are located at the same part of the neurone, then different pools of cyclase have to be considered. This may be the theoretical consequence of the fact that cross-dependence between a2-receptors and opiate receptors is lacking (Collier et al., 1981), a finding confirmed here. Thus, adenylate cyclase could account for both the low degree of crosstolerance (up to 10 fold) as well as for the corn-
plete degree of dependence observed. The further increase of tolerance without associated increase of dependence may be related to a different mechanism such as changes at the binding site level, a phenomenon already described (Davis et al., 1979; Hazum et al., 1981; Chang, personal communication). This would be in line with data reported for the hormonal regulation of peptide receptors (Cart et al., 1979). Incidentally, all endogenous ligands of the opiate receptors are in fact neuropeptides.
Acknowledgements We wish to thank Dr. H. Merz (Boehringer, Ingelheim) for providing the x-agonists MRZ 2549, and H. Rohde for the computer analysis. We also thank Dr. G. Shearman for stylistic correction of the mansucript. This work was supported by Deutsche Forschungsgemeinschaft.
References Adam-Vizi, V. and E.S. Vizi, 1978, Direct evidence of acetylcholine releasing effect of serotonin in the Auerbach Plexus, J. Neural Transm. 42, 127. Bl~isig, J., G. Meyer and M. St~dele, 1978, Factors influencing the manifestation of dependence in rats, in: Factors affecting the Action of narcotics, eds. M.L. Adler, L. Manara and R. Samanin (Raven Press, New York) p. 133. Cart, K.J., J.P. Harwood, G. Aguilera and M.L. Dufau, 1979, Hormonal regulation of peptide receptors and target cell responses, Nature 280, 109. Clark, W.G. and G.L. Bernardini, 1982, Morphine-induced hyperthermia: lack of cross-tolerance with enkephalin analogs, Brain Res. 231, 231. Collier, H.O.J., 1968, Supersensitivity and dependence, Nature 220, 228. Collier, H.O.J., 1980, Cellular site of opiate dependence, Nature, 283, 625. Collier, H.O.J., N.J. Cuthbert and D.L. Francis, 1981, Clonidine dependence in the guinea-pig isolated ileum, Br. J. Pharmacol. 73, 443. Collier, H.O.J., N.J. Cuthbert and D.L. Francis, 1981, Model of opiate dependence in the guinea-pig isolated ileum, Br. J. Pharmacol. 73, 921. Collier, H.O.J. and D.L. Francis, 1975, Morphine abstinence is associated with increased brain cyclic AMP, Nature 255, 159. Collier, H.O.J. and A.C. Roy, 1974, Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate, Nature 248, 24. Cox, B.M., 1978, Multiple mechanisms in opiate tolerance, ini Characteristics and Function of Opioids, eds. J.M. van R~e
40 and L. Terenius (Elsevier/North-Holland Biomedical Press, Amsterdam) p. 13. Davis, M.E., T. Akera and T.M. Brody, 1979, Reduction of opiate binding tO brain stem glia associated with the development of tolerance to morphine in rats, J. Pharmacol. Exp. Ther. 211, 112. Ehrenpreis, S., J. Light and G.H. Schonbuch, 1972, Use of the electrically stimulated guinea-pig ileum to study potent analgesics, in: Drug Addiction: Experimental Pharmacology, eds. J.M. Singh, L.H. Miller and H. Lal (Futura, Mount Kisco, New York) p. 319. Goldstein, D.B. and A. Goldstein, 1968, Enzyme expansion theory of drug tolerance and physical dependence, Assoc. for Res. of Nervous and Mental Diseases, Res. Publications 46, 265. Hazum, E., K.-J. Chang and P. Cuatrecases, 1980, Receptor redistribution induced by hormones and neurotransmitters: possible relationship to biological functions, Neuropeptides 1,217. Hutchinson, M., H.W. Kosterlitz, F.M. Leslie and A.A. Waterfield, 1975, Assessment in the guinea-pig ileum and mouse vas deferens of benzomorphans which have strong antinociceptive activity but do not substitute for morphine in the dependent monkey, Br. J. Pharmacol. 55,541. Johnson, S.M. and A.W. Duggan, 1981, Importance of opiate receptors of the substantia gelatinosa to morphine tolerance and dependence of cat dorsal horn neurones, in: Advances in Endogenous and Exogenous Opioids, eds. H. Takagi and E.J. Simon (Elsevier Biomedical Press, Amsterdam) p. 431. Kosterlitz, H.W., R.J. Lydon and A.J. Watt, 1970, The effects of adrenaline, noradrenaline and isoprenaline on inhibitory alpha- and beta-adrenoceptors in the longitudinal muscle of the guinea-pig ileum, Br. J. Pharmacol. 39, 398. Martin, W.R., C.G. Eades, J.A. Thompson, R.E. Huppler and P.E. Gilbert, 1976, The effects of morphine- and nalorphine-like drugs in the nondependent and morphine dependent chronic spinal dog, J. Pharmacol. Exp. Ther. 197, 517.
Sharma, S.K., W.A. Klee and M. Nirenberg, 1975, Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance, Proc. Nat. Acad. Sci. 72, 3092. Shuster, L., 1961, Repression and de-repression of enzyme synthesis as a possible explanation of some aspects of drug action, Nature 189, 314. Schulz, R., 1978, The use of isolated organs to study the mechanisms of action of narcotic analgesics, in: Development in Opiate Research, ed. A. Herz (Marcel Dekker, New York and Basle). p. 241. Schulz, R. and A. Goldstein, A., 1972, Inactivity of narcotic glucoronides or analgesics and on guinea-pig ileum~ J. Pharmacol. Exp. Ther. 183, 404. Schulz, R. and A. Herz, 1976a, Aspects of opiate dependence in the meyenteric plexus of the guinea-pig, Life Sci. 19, 1117. Schulz, R. and A. Herz, 1976b, The guinea-pig ileum as an in vitro model to analyse dependence liability of narcotic drugs, in: Opiates and Endogenous Opioid Peptides, ed. H.W. Kosterlitz (Elsevier/North-Holland Biomedical Press, Amsterdam) p. 319. Schulz, R., M. Wiister, H. Krenss and A. Herz, 1980, Selective development of tolerance without dependence in multiple opiate receptors of mouse vas deferens, Nature 285, 242. Schulz, R., M. Wi~ster and A. Herz, 1981a, Differentiation of opiate receptors in the brain by the selective development of tolerance, Pharmacol. Biochem. Behav. 14, 75. Schulz, R., M. W~ister, P. Rubini and A. Herz, 1981b, Functional opiate receptors in the guinea-pig ileum: their differentiation by means of selective tolerance development, J. Pharmacol. Exp. Ther. 219, 547. Wi~ster, M., R. Schulz and A. Herz, 1981a, Multiple opiate receptors in the peripheral tissue preparations, Biochem. Pharmacol. 30, 1883. Wiister, M., P. Rubini and R. Schulz, 1981b, The preference of putative pro-enkephafins for different types of opiate receptors, Life Sci. 29, 1219.
33
Elsevier Biomedical Press
O P I O I D D E P E N D E N C E A N D C R O S S - D E P E N D E N C E IN T H E I S O L A T E D G U I N E A - P I G I L E U M RI~DIGER SCHULZ, EVA SEIDL, MICHAEL WI~STERand ALBERT HERZ Department of Neuropharmacology, Max-Planck-lnstitut fi~r Psychiatrie, Kraepelinstrasse2, D-8000 Miinchen 40, F.R.G.
Received 1 April 1982, revised MS received 25 June 1982, accepted 5 July 1982
R. SCHULZ, E. SEIDL, M. W~STER and A. HERZ, Opioid dependence and cross-dependence in the isolated guinea-pig ileum, European J. Pharmacol. 84 (1982) 33-40.
The development of opioid dependence and tolerance attributed to selective types of opiate receptors was studied in the isolated ileum of guinea pigs chronically exposed to specific opioids. These investigations were based on reports that in this preparation highly tolerant opiate receptors may coexist with opiate receptors of almost unchanged sensitivity. Thus, the ilea were set up in vitro and tested for tolerance and dependence. Apparently precipitation of the withdrawal contracture, indicating dependence, proved a more sensitive parameter than the phenomenon of tolerance. Maximal dependence was determined at rather low degrees of tolerance (5 to 10 fold). The intensity of the withdrawal contracture failed to increase as opiate ~olerance did. Furthermore, the experiments failed to present evidence for the existence of selective dependence at specific opiate receptor types. These findings may suggest multiple adaptational mechanisms upon chronic activation of opiate receptors. One mechanism may be responsible for the development of dependence and a low degree of tolerance, whilst a further increase of tolerance may be associated with changes at the opiate binding site level. Opioids
Dependence Cross-dependence Tolerance Guinea-pigileum
1. Introduction A reliable approach for the differentiation of opiate receptors that is, binding site as well as affector, relates to the phenomenon of tolerance development (Martin et al., 1976; Schulz et al., 1981a; Wi~ster et al., 1981a; Clark and Bernardini, 1982). In fact, isolated tissues such as the mouse vas deferens and the guinea-pig ileum (GPI) proved of particular interest since extremely tolerant opiate receptors may coexist in the same preparation with opiate receptors of almost unchanged sensitivity (Schulz et al., 1980; Schulz et al., 1981b). Assuming that a single mechanism accounts for the development of tolerance as well as for dependence ('unitary theory'; Goldstein and Goldstein, 1968; Shuster, 1961; Collier, 1968; 1980), then the above findings would imply that highly tolerant opiate receptors are also highly dependent, and less tolerant receptors are less dependent. Accordingly, tolerance induction for a selective type of opiate 0014-2999/82/0000-0000/$02.75 © Elsevier Biomedical Press
receptors should be complemented by selective dependence. However, such an association may be invalid if tolerance/dependence does not relate to a common mechanism. In fact, multiple adaptational mechanisms upon chronic opiate receptor activation have been reported, involving alterations either at the post recognition stage of the receptor binding site or at the binding site itself (Collier et al., 1981; Sharma et al., 1975; Cox, 1978; Davis et al. 1979; Hazum et al., 1980). To investigate the issue of opiate dependence at specific opiate receptor types, the isolated G P I was chosen for two reasons. First, a high degree of tolerance for highly selectively acting opioids has been demonstrated, suggesting the separate existence of functional /~- and r-receptors in this preparation (Schulz et al., 1981b; Wtister et al., 1981b). Second, the response of a tolerant G P I to naloxone is confined to a single symptom, that is, the withdrawal contracture (Ehrenpreis et al., 1972; Schulz and Herz, 1976a). This makes the G P I
34 highly preferable to the opiate-dependent central nervous s y s t e m which initiates a diversity of withdrawal symptoms upon naloxone challenge (Bl~sig et al., 1978). The availability of narcotic agonists combining preferentially with specific types of receptors allows the demonstration of tolerance for specific types of opiate receptors. However, an analogous demonstration of dependence is complicated by the fact that a receptor-specific narcotic antagonist is not available. Therefore, use was made of the principle that precipitation of an abstinence symptom by the antagonist naloxone requires - besides other conditions - the occupation of opiate receptors by an opioid. In addition only those receptors made tolerant/dependent respond, after they have been withdrawn and reexposed to the opiate, by an intensive withdrawal contracture upon naloxone challenge (Schulz and Herz, 1976a). Thus, the GPI made tolerant/dependent in vivo was intensively washed in vitro to unmask all opiate receptors. Thereafter, the preparations were re-exposed to an opioid combining with a specific type of receptor. The withdrawal contracture precipitated under these conditions by naloxone was assumed to present a demonstration of dependence for a specific type of opiate receptor.
2, Materials and methods
Male guinea pigs (300-350 g) were decapitated and the ileum removed. The longitudinal musclemyenteric plexus (strip) was prepared as described by Kosterlitz et al. (1970). A strip (2cm) was mounted in a 5 ml organ bath in Krebs-Ringer-bicarbonate solution for electrical field stimulation (60 V, 0.5 ms, 0.1 Hz; Schulz and Goldstein, 1972). The preparations were equilibrated by changing the bathing fluid at 5-10 rain intervals. The drug effects evoked were expressed as percentage change of the electrically induced twitches. The ICs0 refer to drug concentrations causing a 50% inhibition of twitch tension. The ilea of guinea pigs were rendered tolerant and dependent by subcutaneous (s.c.) administration (6 days) of opioids dissolved in water using osmotic minipumps (model 2001 and 2 ML1, re-
spectively; Alza Corp., Palo Alto, U.S.A.). The pumps were implanted s.c. under ether anesthesia. To prevent loss of tolerance/dependence of the preparations outside the animals, the strips were exposed in vitro to a defined concentration of the opioid infused in vivo. The opioids used to render the myenteric plexus tolerant were the /~-agonist morphine and the ~agonist benzomorphans ethylketazocine (EK) and MRZ 2549 (Schulz et al., 1981b). In order to obtain a variety of degrees of tolerance/dependence, the guinea pigs were infused chronically with different concentrations of the opioid and the strips were set up in vitro in the presence of the opiate agonist. The infusion rates for morphine were 10, 50 and 100 /~g/h for 6 days. The concentrations used for the in vitro exposure of strips from these animals ranged between 100 and 600 nM normorphine. These concentrations were tolerated with respect to the ability of the preparations to exhibit electrically evoked twitch tension similar to that displayed by naive strips (see Schulz et al., 1981b). The conditions employed for EK were: infusion of 1, 2, 10, 50 ~ g / h for 6 days. The in vitro incubations were conducted with 0.1-4 nM EK. The data for MRZ were 1 and 2 ~ g / h for 6 days, and the in vitro concentrations ranged between 0.1 and 1.0 nM MRZ. Assessment of opiate tolerance: The ICs0 values served for the quantification of tolerance and cross-tolerance. The assays were conducted in the presence of the maintenance opioid (see above). Failure of tolerance development is defined by the quotient ( Q ) - - 1 , that is, the IC.s0 value of this compound in a preparation chronically exposed to an opioid is identical to that observed in a naive preparation. Thus, Q increases as the degree of tolerance increased. Assessment of opiate dependence: Strips of guinea pigs chronically exposed to an opioid were challenged in vitro with naloxone in the absence of electrical stimulation. Two experimental designs were used. First, opioid tolerance/dependence was maintained in vitro from the beginning by exposure of the tissues to a specific opioid. Electrical stimulation was applied continuously. These strips served for the determination of tolerance followed by the test for dependence. Morphine was sub-
35 stituted by normorphine in vitro because of the more hydrophilic characteristics of this derivative. Second, strips were withdrawn in vitro by repeated washing for 1 h. The unmasking of opiate receptors by this procedure was documented by the failure of naloxone (5 /~M) to precipitate a withdrawal contracture. Thereafter, the strips were re-exposed in vitro for 30 min to a specific opioid. This re-exposure period was adapted from experiments conducted with morphine tolerant strips (Schulz and Herz, 1976a). Electrical stimulation was applied during this period and the bathing fluid renewed at 3-5 rain intervals. Subsequently, naloxone (5/tM) was added to the bath to test for dependence. The withdrawal contracture was quantified by calculating the percentage of the intensity of the withdrawal contracture in relation to the twitch tension evoked by electrical stimulation either during exposure to the opioid to which tolerance had developed or at the end of the 1 h withdrawal period (see also Schulz and Herz, 1976a). Chronic clonidine exposure: Guinea pigs were infused with clonidine (50 /~g/h, 6 days). The strips were set up in vitro under two different experimental conditions: first, they were maintained at 100 nM clonidine, and second, they were kept in clonidine (100 nM) simultaneously with 500 nM normorphine. Dependence was tested by challenge of these preparations with phentolamine (1/~M) and naloxone (5#M), respectively. The quantification of the withdrawal symptom followed that described above for opioid dependent strips. Statistical analysis: each quotient (Q) represents the mean ---S.E.M. of at least 4 independent experiments. One experiment reflects the Q computed from the mean ICs0 values of each of 3 preparations from a naive and from a chronically treated animal. The regression lines of fig. 1 (Q 1-10 and Q 50-200) were fitted by the method of least squares, and sums of the products of deviations from the regression gave the regression coefficients. The curves expressing the relation between tolerance and dependence were obtained by best-fit computer analysis. The following substances were used: Morphine
HC1 (Merck, Darmstadt, F.R.G.); normorphine, NORM (Bios Products, Brussels, Belgium), aneoendorphin-(1-8), ANE; dynorphin-(1-13), DYN (Peninsula Labs., San Carols, U.S.A.); ethylketazocine, EK (Sterling-Winthrop Research Institute, New York, U.S.A); MRZ 2549, (5,9-dimethyl,2',5)-5-9-dimethyl-2'-hydroxy-2-(methoxypropyl)-6, 7-benzomorphan, clonidine (Boehringer, Ingelheim, F.R.G.).
3. Results
Different concentrations of naloxone are required to antagonize the action of/t- or ~-agonists in the GPI. To determine the optimal naloxone concentration to precipitate a maximal abstinence sign for any opioid under investigation, strips were rendered tolerant to EK (100/~g/h, 6 days, set up in vitro at 10 nM EK). The K e value of naloxone in the GPI for the r-agonist EK is several fold higher than for a ~-agonist (Hutchinson et al., 1975). Under these conditions, precipitation of a maximal withdrawal sign required a concentration of at least 2/~M naloxone. Hence, 5/.tM naloxone was employed throughout to assay opioid dependence. Fig. 1 shows the relation between the development of dependence, as expressed by the withdrawal contracture, and tolerance, as expressed by the quotient 'Q' for the /~-agonist morphine (panel A) as well as for the r-agonists EK (panel B) and MRZ (panel C). The dotted line in each panel indicates the best-fit computer analysis between the two parameters. Apparently, the shape of the tolerance/dependence response curves is very similar for the various drugs under investigation. A very steep slope of the curves at low degrees of tolerance is followed by an extremely flat slope at high degrees of tolerance. This is documented by the regression coefficients for the initial phase (b~) of the curves (Q = 1-10) as well as for the flat component (b 2) (Q = 50-200): panel A: b~ =4.6, b z - - - 0 . 0 2 ; panel B: b~=4.0, b2--0.05; panel C; bj = 5.7, b 2 = --0.01. EK showed a difference from the other drugs used. Apparently, in these experiments the plateau was lower than that for morphine and MRZ. The most plausible
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Fig. 1. Correlation between tolerance and dependence in the isolated GPI. Guinea pigs were infused with different concentrations of morphine, EK or MRZ. The strips were maintained in vitro at different concentrations of the opioid which had been infused in vivo, that is, normorphine (panel A), EK (panel B) and MRZ (panel C). Each dot represents data of a single strip which was used once to assay tolerance and dependence. The dotted line indicates the tolerance/dependence curve obtained by best-fit computer analysis. The solid lines indicate the regression lines for the degree of tolerance (Q) ranging from 1 to 10 and 50 to 200. b t and b 2 reflect the respective regression coefficients. The mean values obtained for Q>200 are based on a variety of single data indicated by 'n'. Abscissa: degree of tolerance (Q) calculated from IC50 values of tolerant and naive preparations. Ordinate: intensity of withdrawal contracture expressed as percent of the preceding electrical stimulation. e x p l a n a t i o n for this p h e n o m e n o n takes i n t o account the offset time of the action of EK which g o v e r n s t h e s t r e n g t h o f t h e w i t h d r a w a l sign ( S c h u l z a n d H e r z , 1976b).
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reincubation (n~) Fig. 2. Opioid dependence and cross-dependence in the isolated GPI. Strips were rendered dependent by infusing guinea pigs with morphine, EK or MRZ. The dosages per hour were infused for 6 days as indicated in the corresponding panels. After withdrawal of the preparations, they were reincubated with opioids with high receptor selectivity, such as normorphine (NORM) for #-receptors, and ethylketazoeine (EK), dynorphin-(I-13) (DYN~.~3) and a-neo-endorphin-(l-8) (ANEI_s) for ~-receptors. The reincubation concentrations of the drugs are indicated on the abscissa. The preparations were challenged with naloxone and the resulting strength of the withdrawal contractions is given on the ordinate. The columns indicate means --+S.E.M. of at least 6 different preparations. The incubation concentrations selected for the strips of guinea pigs infused with lower drug concentrations were the ICs0 values determined m naive preparations.
38 additional contracture could be evoked at the peak of the contracture by either electrical stimulation or by the addition of serotonin (100 nM) to the bath. This drug concentration manifests its actions at the myenteric plexus and in turn causes the release of acetylcholine (Adam-Vizi and Vizi, 1978). Fig. 2 presents data from experiments on crossdependence in preparations chronically exposed to specific receptor agonists like morphine (upper panel), EK and MRZ (lower panels). The guinea pigs received different infusion concentrations per h (see fig.) in order to induce different degrees of dependence. The upper panel demonstrates the withdrawal contractures of preparations made tolerant by infusion of 10 and 100 # g / h of the /~-agonist morphine. These preparations were withdrawn in vitro and reincubated with the /~agonist normophine as well as with DYN-(1-13) or ANEI(1-8), both interacting with ~-receptors in this preparation (Wtister et al., 1981b). The drug concentrations selected corresponded to the ICs0 values (determined on naive preparation) for the low infusion concentration. The re-exposure concentration was increased 5 fold for strips taken from guinea pigs infused with 100 /~g/h for 6 days. Surprisingly, naloxone challenge of such preparations revealed no difference in the intensity of the withdrawal contracture within an identical experimental design, regardless of the opiate receptor agonist used for reincubation. As expected, a more intense withdrawal sign was precipitated in preparations taken from guinea pigs infused with 100/~g/h morphine. In principle, this result obtained with strips made previously dependent to a /~-agonist was also observed with preparations made tolerant to the ~-agonists EK or MRZ (see the two lower panels of fig. 2). The demonstration of dependence to a nonopioid drug, clonidine, in the GPI (Collier et al., 1981) caused us to test for cross-dependence between this o~z-adrenergic agonist and the t~-receptor agonist NORM. Clonidine-tolerant strips exhibited dependence upon challenge with phentolamine. The withdrawal contracture amounted to 23 -+ 17% (n = 12). The preparations set up in the presence of clonidine and normorphine failed to display a withdrawal contracture upon challenge with naloxone.
4. Discussion The naloxone-precipitated withdrawal contracture in the GPI has been used repeatedly as an indication of opioid dependence (Schulz, 1978). Moreover, theoretical considerations as well as experimental evidence have intimately linked the phenomenon of dependence with that of tolerance, claiming a common underlying biochemical mechanism (Goldstein and Goldstein, 1968; Shuster, 1961; Collier, 1980; Bl~sig and Herz, 1978). In principle, part of the data communicated here are not in conflict with these theoretical considerations. However, an interpretation of all the findings reported here would imply multiple adaptational mechanisms. A discrepancy between the rate of development of dependence and that of tolerance in the GPI becomes obvious when the phenomena are compared. The degree of dependence, as indicated by the intensity of the withdrawal contracture, is already considerable while tolerance is hardly detectable or very low. In fact, an almost maximal degree of dependence is exhibited at about a 5 fold degree of tolerance. As tolerance increases, dependence does not increase further. Apparently, this failure of tolerant/dependent preparations to display progressively increasing withdrawal contractures seems not to be due to exhaustion of the ability to contract. Withdrawal contracture thus reflects an extremely sensitive parameter for the detection of adaptational processes i.e., dependence, upon chronic opiate receptor activation. It is more easily detected than tolerance. This parallels results obtained in the central nervous system, where dependence is already detectable in the apparent absence of tolerance (Johnson and Duggan, 1981). A further aspect of the dependence studies relates to the phenomenon of cross-dependence. The assumption that highly tolerant opiate receptors are also highly dependent and that such receptors coexist with low tolerant/dependent receptors was not confirmed by the present investigations. Their experimental design showed that specific types of opiate receptors failed to develop dependence. That is, dependence of/~-receptors caused by their chronic activation was associated with the
39
same degree of dependence of g-receptors. It is of interest in this context that induction of 50 to 80 fold tolerance in a specific receptor type, e.g. kt-receptors, was always associated with some degree of cross-tolerance (5-10 fold) in other opiate receptors, e.g. r-receptors (Schulz et al., 1981b). As shown in fig. 1, a maximal degree of withdrawal contracture is precipitated by naloxone at a relatively low level of tolerance (5-10 fold) of this preparation. Thus a similar degree of cross-dependence for the different opiate receptor types would even be expected on the basis of this result. These findings, together with those published earlier (Schulz et al., 1981b), would imply that chronic activation of a specific opiate receptor population may result in a high degree of tolerance for this specific population with a low degree of crosstolerance to other opiate receptor types. Under these conditions almost maximal cross-dependence is demonstrated at the different types of opiate receptors. The remaining question is to integrate the present data into a concept explaining tolerance and dependence in the GPI. As has been stated above, a 'unitary theory' can hardly account for all data reported here. Assuming, however, that adenylate cyclase exerts a critical function in the expression of tolerance and dependence (Collier and Roy, 1974; Collier and Francis, 1975; Sharma et al., 1975), then the appearance of the minor degree of cross-tolerance as well as of cross-dependence between different types of opiate receptors could be explained. Such an interpretation surmise s , however, access of all opiate receptors to the same adapted adenylate cyclase either directly or by chance contact as proposed in the 'mobile-receptor hypothesis' (Hazum et al., 1981). Provided that the prototype a2-adrenoreceptor agonist clonidine also brings about its action via adenylate cyclase, and that the az-receptor as well as the opiate receptors are located at the same part of the neurone, then different pools of cyclase have to be considered. This may be the theoretical consequence of the fact that cross-dependence between a2-receptors and opiate receptors is lacking (Collier et al., 1981), a finding confirmed here. Thus, adenylate cyclase could account for both the low degree of crosstolerance (up to 10 fold) as well as for the corn-
plete degree of dependence observed. The further increase of tolerance without associated increase of dependence may be related to a different mechanism such as changes at the binding site level, a phenomenon already described (Davis et al., 1979; Hazum et al., 1981; Chang, personal communication). This would be in line with data reported for the hormonal regulation of peptide receptors (Cart et al., 1979). Incidentally, all endogenous ligands of the opiate receptors are in fact neuropeptides.
Acknowledgements We wish to thank Dr. H. Merz (Boehringer, Ingelheim) for providing the x-agonists MRZ 2549, and H. Rohde for the computer analysis. We also thank Dr. G. Shearman for stylistic correction of the mansucript. This work was supported by Deutsche Forschungsgemeinschaft.
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