Behavioral Effects of Opioid Peptides Selective for Mu or Delta Receptors. I. Morphine-Like Discriminative Stimulus Effects1

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1 /86/ $02.OO/O THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright C 1986 by The American Society for Pharmacologj and Experimental Therapeutics VoL 238, No.3 Printed in USA. Behavioral Effects of Opioid Peptides Selective for Mu or Delta Receptors. I. Morphine-Like Discriminative Stimulus Effects1 KENNETH W. LOCKE2 and STEPHEN G. HOLTZMAN Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia Acceptedforpubl,cationJune 13, 1986 ABSTRACT The morphine-like discriminative stimulus effects of opiold peptides with selectivity for the mu- or defta-opioid receptors were examined in rats trained to discriminate 3.0 mg/kg of morphine (s.c.) from saline in a two-choice discrete-thai avoidance paradigm. The mu-selective peptides D-A1a-NMePhe4- Gl(o1)enkephalin, FK 33,824 and morphiceptin, the defta-selective peptides D-Ala2-D-Leu5enkephalin and metkephamid and fiendorphin (mu- and de/ta-selective) produced morphine-like stirnulus effects after administration into the lateral ventricle. Generalization with the morphine cue was dose-dependent and occuffed over a wide range of doses ( ag), depending upon peptide. On a molar basis, the order of relative potency of the peptides as morphine-like discilminative stimuli was: D-A1a2- Drug discrimination techniques have proven to be of value for distinguishing among opioid alkaloids that have been proposed (Martin et al, 1976; Gilbert and Martin, 1976) to interact with the several types of opioid receptors. Differences in the patterns of stimulus generalization to novel compounds in animals trained with prototypic opioids and differences in the relative potency of naloxone or naltrexone for antagonizing the stimulus effects of prototypic opioids have provided a basis for categorizing opioid alkaloids in a manner consistent with models of multiple receptors (for reviews, see Herling and Woods, 1981; Holtzman, 1982, 1983). The existence of distinct mu and delta-opioid receptors was first suggested by results obtained in bioassays of peripheral tissues and receptor binding assays conducted in vitro (i.e., Lord et a!., 1977). Peptides that display a relative selectivity for mu- or delta-opioid receptors have subsequently been synthesized. However, there is a paucity of in vivo data comparing the central actions of such peptides, particularly with respect to whether or not selectivity for mu- or delta-opioid receptors Received for publication December 12, Thiswork was supported in part by Grant DA00541 andby Research Scientist Award DA00008, both from the National Institute on Drug Abuse. A preliminary report of this work appeared in Soc. Neurosci. Abet. (9: 289, 1983). 2Present address: American Hoechst Corporation, Route North, Somerville, NJ NMePhe4-Gl(ol)enkephalin = FK 33,824 > fl-endorphin > D- A1a2-D-Leu5enkephalin = metkephamid > morphiceptin. The discriminative effects of D-Ala2-NMePhe4-Gl(oi)enkephalin, o-a1a2- D-Leu5enkephalin and $-endorphin were antagonized by low doses of s.c. naltrexone ( mg/kg). Furthermore, the stimulus effects of s.c. morphine were antagonized by 24-hr pretreatment of rats with the irreversible mu-antagonist fl-funaltrexamine(5.0 g i.c.v.). Based upon the order of relative potency of the peptides and the relative potency for antagonism of their discriminative effects by naltrexone and fl-funaltrexamine, muopk,kl receptors in the brain appear to be an important eiement in the genesis of morphine-like discriminative effects by opiold -S. in vitro results in differential effects on the behavior of subjects in vivo. Drug discrimination techniques may afford an in vivo approach for defining the receptor types involved in the actions of the opioid peptides. A number of natural and synthetic opioid peptides have been shown to share discriminative effects with morphine-like opiate agonists (Chipken et a!., 1978; Colpaert et a!., 1978; van Ree et a!., 1979). For example, the i.c.v. injection of 1.0 to 2.5 g of - endorphin produced primarily drug-appropriate responding in rats trained to discriminate between the s.c. administration of saline and either etonitazene (2.5.ig/kg) or morphine (2.5 mgi kg) (Browne and Fondren, 1978). The putative delta-receptor agonist DADL generalized with the discriminative stimulus effects ofthe mu-receptor agonist fentanyl (0.04 mg/kg) in rats, but not with those of the kappa-receptor agonist ethylketocyclazocine (0.32 mg/kg) (Shearman and Hers, 1982). Metabolically stable opioid peptides have also been found to have morphine-like stimulus properties when given peripherally. The stable Met-enkephalin analogs, FK 33,824 (D-Ala2- NMePhe4-Met(O)5o1 enkephalin) and metkephamid (D-A1a2- NMeMet5-NH2 enkephalin), shared stimulus properties with the morphine-like agonists etorphine and codeine when administered s.c. to rhesus monkeys (Woods et a!., 1980a,b, 1981; Young et a!., 1983). However, the discriminative stimulus effects of a series of receptor-selective opioid peptides have not ABBREVIATIONS: DADL, D-AIa2-D-Leuenkephaiin; DAGO, D-Ala-NMePhe4-Gly(OI)eflkephalin; fl-fna, fl-funaltrexamine. 990

2 1986 Oploid Peptides: DiscrIminatIve Effects 991 been evaluated systematically in a single study. Moreover, the sensitivity of the discriminative effects of the opioid peptides to antagonism by naloxone or naltrexone has not been exammed with the same degree of rigor that has been applied so successfully to the nonpeptide opiates. The objective of this study was therefore to characterize systematically the roles of mu- and delta-opioid receptors in mediating the morphine-like discriminative stimulus effects of a number of opioid peptides in rats trained to discriminate 3.0 mg/kg of morphine (s.c.) from saline. Several approaches to this problem were used. The first was to determine the morphine-like stimulus effects of peptides with relative selectivity for mu- or delta-opioid receptors. The effects of DAGO (Handa et at., 1981), morphiceptin (Tyr-Pro-Phe-Pro-NH2) (Chang et a!., 1981) and FK 33,824 (Roemer et al, 1977), mu-agonists, fiendorphin (Cox et a!., 1976; Lee et a!., 1982), a mu- and deltareceptor agonist, and DADL (Chang and Cuatrecasas, 1979) and metkephamid (Frederickson et a!., 1981), delta-receptor agonists, were tested over a broad range of doses administered i.c.v. for generalization to the morphine stimulus. A second approach was to compare the potency of the opiate antagonist naltrexone for blocking the morphine-like stimulus effects of DAGO, DADL and fl-endorphin. Third, the irreversible mureceptor antagonist -FNA (Portoghese et a!., 1980) was tested in this procedure for its ability to block the stimulus effects of morphine administered systemically. 9-FNA has been shown to produce a long-lasting antagonism of the effects of morphine in the guinea pig ileum, mouse vas deferens and acetic acidinduced writhing assays (Takemori et a!., 1981; Ward et a!., 1982a; Takemori and Portoghese, 1985). Methods Subjects. The subjects were male Sprague-Dawley-derived rats (Holtzman Co., Madison, WI or Charles River Breeding Laboratories, Wilmington, MA) weighing 250 to 350 g at the start of discrimination training. The rats were housed individually in a ventilated cabinet in which they had continuous access to food and water. The lights in the cabinet were illuminated between 6:00 A.M. and 6:00 P.M. Apparatus. The apparatus has been described in detail elsewhere (Shannon and Holtzman, 1976a,b; 1977). Briefly, a one-lever rat chamher (model 1110-L, Grason-Stadler Co., Bolton, MA) was modified by adding two choice response levers on the wall opposite the original lever, which was designated the observing lever. The choice response levers were separated by a clear Plexiglas partition 5.0 cm wide that extended from the ceiling of the test chamber to 1.0 cm above the grid floor. A constant current shock generator (model 700, Grason-Stadler Co.) delivered a scrambled electric shock to the grid floor of the chamber, which was housed within a ventilated, lightproof and soundattenuating enclosure. Automatic relay programming equipment was used to control the schedule contingencies. Discrimination training. Rats were trained to discriminate between 3.0 mg/kg of morphine and saline in a two-choice discrete-trial avoidance paradigm (Shannon and Holtzman, 1976a,b; 1977). The onset of a trial was signalled by the simultaneous illumination of the house light and the presentation of white noise. At this time, the rat was required to press the observing lever on one wall of the chamber, turn and press one of two choice levers on the opposite wall of the chamber. The first observing response ofthe trial terminated the white noise and the appropriate choice response extinguished the house light and ended the trial. Beginning 5.0 sec after the onset of the trial, a 1.0 ma-shock was delivered to the grid floor of the chamber every 3.0 sec in 0.5-sec pulses until the two-response chain was completed. The intertrial interval was 50 sec during which time the chamber was dimly illuminated by a red light. Experimental sessions ended after 21 trials or 30 mm, whichever came first. The first trial of each session was considered a warm up and was not included in the data analysis. Training sessions were conducted 5 days/week, often two sessions per day at least 3 hr apart. Either morphine (3.0 mg/kg) or saline was injected s.c. 30 mm before each training session. When two training sessions were conducted on the same day, saline was always administered before the first, whereas either saline or morphine was administered before the second. Half of the rats were trained to press the right choice lever after morphine administration and the left choice lever after saline injection; designation of choice levers was reversed for the other rats. Training continued until rats could complete reliably at least 18 of 20 trials (i.e., 90% exclusive of the first trial) on the appropriate choice lever under both conditions. Once this criterion was met, a cannula was implanted in the left lateral cerebral ventricle of the rat. Drug testing. After recovery from surgery, training sessions were resumed until stimulus control of behavior was re-established (i.e., 18 of 20 trials completed on the appropriate choice lever). Drug test sessions were conducted on Tuesdays and Fridays provided the rats satisfied the performance criterion in training sessions on intervening days. During test sessions, both choice levers were activated so that a response on either choice lever after the observing response terminated the trial. Test sessions and training sessions were identical in all other aspects. Drug injections were given s.c. or i.c.v. 15 to 30 mm before the test session. When two drugs were administered concurrently by different routes, the s.c. injection immediately preceded the i.c.v. injection. An initial stimulus generalization curve for i.c.v. morphine was determined for each rat in order to assess the sensitivity of the animal to morphine before further drug testing. Stimulus generalization curves for one to four peptides were determined in each rat in a nonsystematic order. A cumulative dosing procedure (Bertalmio et al, 1982) was used to test the ability of fi-fna to block the stimulus effects of morphine. Animals were pretreated i.c.v. with either saline or 5.0 g of fi-fna 24 hr before the determination of the cumulative dose-response curve for morphine administered systemically. On the next day, rats were injected with saline (s.c.) and placed in the test chamber 15 mm later. After the completion of the 30-mm te8t session, rats were removed to their home cage and injected with morphine s.c. before the start of the next test session 15 mm later. Test sessions were conducted in this manner with increasing doses of morphine until the rats completed at least 18 of 20 trials on the morphine-appropriate lever. The cumulative dosing procedure was used because the long duration of action of - FNA (i.e., T#{189} > 48 hr Portoghese et al, 1980) would have interrupted the normal training procedure between test sessions conducted at 3- to 4-day intervals. The dose of -FNA (5.0 gig) was selected on the basis of the results of studies conducted by Ward et al (1982a,b,c) and on the basis of the ability of 9-FNA to block morphine-induced analgesia in the rat (K. W. Locke and S. G. Holtzman, unpublished observations). Surgical procedure. Rats were pretreated with atropine sulfate (1.0 mg/kg s.c.) and anesthetized with sodium pentobarbital (55 mg/kg i.p.) or a mixture of ketamine (100 mg/kg i.m.) and acepromazine (0.9 mg/kg i.m.). The rats were placed in a stereotaxic instrument (David Kopf Co., Tujunga, CA) and a 22-gauge stainless steel guide cannula (No. C313G, Plastic Products Co., Roanoke, VA) was inserted in the left lateral cerebral ventricle (AP, -0.5 mm relative to bregma; L, +1.8 mm; V, -2.0 mm relative to dura) as described previously (Locke and Holtzman, 1985). Cannula patency was assessed by measuring the drinking response produced by the i.c.v. administration of 10 ng of angiotensin II (Severs and Summy-Long, 1976). Rats were required to consume at least 5.0 ml of water in 15 mm after the injection of angiotensin II before being used in experiments. Periodic tests were performed subsequently with angiotensin II to ensure continued pat. ency of the cannula. Drugs. The following peptides were used -endorphin (Beckman Instruments, Inc., Palo Alto, CA): DADL, morphiceptin and FK 33,824 (Bachem, Inc., Torrance, CA); DAGO (Cambridge Research Biochemicals, New York, NY); and metkephamid (Eli Lilly and Co., Indianap-

3 992 Locke and Holtzman Vol. 238 ohs, IN). Peptide solutions for s.c. or i.c.v. injection were prepared by dissolving the acetate salts in sterile distilled water, aliquots of the solution were frozen for subsequent use. fi-fna (National Institute on Drug Abuse, Rockville, MD) was prepared similarly for i.c.v. administration. Doses of the peptides and fl-fna are expressed in terms of the salt. Naltrexone hydrochloride (National Institute on Drug Abuse) and morphine sulfate (Penick Corp., Newark, NJ) were dissolved in sterile 0.9% saline for s.c. injection. Doses of these drugs are expressed in terms of the free base. All s.c. injections were made in a volume of 1.0 mi/kg b.wt. All i.c.v. injections were made in a volume of 2.0 to 5.0 il/rat. Intracerebroventricular injections were made by first backioading saline or the drug solution into an injection cannula connected to a microsyringe by PE- 10 tubing and then administering the solution at a rate of 1.0 d/20 sec. The injection cannula was left in place for at least 30 sec after injection. Data analysis. The data were analyzed in terms of the number of trials completed on the morphine-appropriate choice lever. Trials completed on the saline-appropriate choice lever were recorded but are not shown in the figures. All animals completed all trials of every session. A dose of test drug was considered to produce discriminative stimulus effects comparable to those produced by the training dose of morphine (3.0 mg/kg) if a rat completed at least 18 of 20 trials on the morphineappropriate lever. Results Discriminative effects ofpeptides selective for the mureceptor. The i.c.v. administration of peptides that are relatively selective agonists at the mu-opioid receptor (DAGO, FK 33,824 and morphiceptin) produced dose-related increases in the number of trials completed on the morphine-appropriate lever (fig. 1). Complete generalization to DAGO and FK 33,824 occurred in a dose range of 0.01 to 0.1 ig (fig. 1, A and B). Stimulus generalization curves for these peptides showed intermediate levels of morphine-appropriate responding (i.e., more than 2 but less than 18 trials to the morphine lever) in at least half of the animals at one or more doses of peptide (fig. 1). In three rats tested with FK 33,824 administered s.c., complete generalization occurred at a minimum dose of 3.0 mg/kg (data not shown). Morphiceptin was the least potent of all peptides tested. A dose of 30 to 100 ig i.c.v. was required to produce trials To IRPHII( B morphine-appropriate responding comparable to that engendered by the training dose of morphine (fig. 1C). These results were obtained with 15-mm pretreatment; the results between animals were inconsistent with a 30-mm interval was used (data not shown). Discriminative effects of fl-endorphin and peptides selective for the delta-receptor. The i.c.v. administration of peptides that are relatively selective agonists at the deltaopioid receptor (DADL and metkephamid) also produced doserelated increases in the number of trials completed on the morphine-appropriate lever (fig. 2, A and B). Over a 30-fold dose range ( ig), DADL engendered morphine-appropriate responding comparable to that produced by the systemic training dose (fig. 2A). However, four of seven animals generalized to DADL at the 1.0 ig dose. Metkephamid was approximately equipotent to DADL in producing morphine-like stimulus control ofbehavior when administered i.c.v. Generalization with the morphine cue was produced by 1.0 to 3.0 g of this peptide (fig. 2B). In contrast, when injected s.c. in doses up to 30 mg/kg, metkephamid engendered only saline-appropriate responding (data not shown). A limited supply of the drug precluded testing higher doses. /3-Endorphin completely generalized at a dose of 1.0 to 10 g after i.c.v. administration (fig. 2C). The results of the stimulus generalization tests of the opioid peptides are summarized in table 1, which shows median doses expressed both in micrograms and nanomoles. Antagonism of the morphine-like discriminative effeets of opioid peptides by naltrexone. DAGO (mu-selective), DADL (delta-selective), and fl-endorphin (mu- and deltaselective) were chosen as representative peptides for antagonism experiments with naltrexone administered s.c. In order to ensure consistent responding among rats, the dose of peptide tested in each animal was 0.5 log dose greater than the minimum dose of that peptide that was generalized completely to the training dose of morphine. In a few cases, the minimum effective dose for generalization was used because the higher dose (0.5 log dose greater) suppressed responding completely. The s.c. administration of naltrexone blocked the morphinelike discriminative effects of all three peptides in a dose-related C o.iu 113 :- t o. oi ah o i o i DA ImSEFK 33,824 (ti) ImSEMORPHICEPTIN (ug) Fig. 1. Dose-dependent morphine-like discilminative stimulus effects of the mu-selective peptides DAGO (A), FK 33,824 (B) and morphiceptin (C) after i.c.v. administration in individual rats trained to discriminate between saline and 3.0 mg/kg of morphine administered s.c. Data are reported as the number of trials of a 20-trial session completed on the morphine-appropriate lever. The remaining trials were completed on the saline-appropriate lever and are not shown. The dashed horizontal lines at 18 and 2 responses represent the criteria for morphine- and saline-appropriate responding, respectively, during training sessions. The dose of peptide administered i.c.v. is in micrograms. Each point represents a single observation in one rat. Different symbols denote different rats. Each rat always completed 18 or more trials on the saline-appropriate lever in test sessions with i.c.v. saline that were interspersed among tests of the various peptide doses (data not shown).

4 1986 OId Peptidos: Discriminative Effects 993 C TRIALS TO NORPHItE LEVER II U DOSEDAft (ug) DOSEtkEPHAMlO (ug) C cmsb-oiooiipwn (ug) Fig. 2. Dose-dependent morphine-like discriminative stimulus effects of the delta-selective peptides DADL (A), metkephamid (B) and -endorphin (C) after i.c.v. administration in individual rats trained to discriminate between saline and 3.0 mg/kg of morphine administered s.c. Other details as in figure 1. TABLE 1 Potency comparison of the discriminative stimulus effects of centrally administered oplold peptides In rats trained to discriminate 3.0 mg/kg of morphine from saline 0 Relative 0 fls_ Medisi Dosefic.v.) Produciig Sms conpor Mg/nP np DAGO Mu ( ) FK 33,824 Mu ( ) 9-Endorphin Mu and delta DADL Delta ( ) Metkephamid Delta Morphinec Mu ( ) Morphceptin Mu Dose resulting in ocmpletion of 18 or more trials on the morphine-appropriate chcce lever. a interval for medians based UPOfl at 1685t Sfl observations (Zar. 1984). C From Locke and Holtzman, 1985 (n = 24). manner, with complete antagonism of DAGO at 0.03 to 1.0 mgi kg (fig. 3A), of DADL at 0.01 to 0.3 mg/kg (fig. 3B) and of fiendorphin at 0.01 to 0.03 mg/kg (fig. 3C). Although there was considerable variation in the dose of naltrexone required for complete antagonism of the stimulus effects of DAGO, DADL and $-endorphin, dose-response -_ curves for individual animals 1$ TRIALS 16 A RIl 30.01) 14 U To #{163} satan 12 0 tl0.1) 10 JRPHIIE LEVER 0 sian 6 were steep and blockade generally occurred within a dose range of 1.0 log unit. Antagonism of the discriminative effects of morphine by -FNA. The i.c.v. administration of 5.0 g of -FNA 24 hr before the determination of the cumulative morphine doseresponse curve resulted in a 10-fold shift to the right of the curve as compared to a morphine curve determined after 24-hr pretreatment with i.c.v. saline (fig. 4). Discussion The results of this study provide the first systematic characterization of the discriminative stimulus effects of a series of opioid peptides administered centrally over a wide range of doses. All ofthe peptides tested produced dose-related increases in the number of trials completed on the morphine-appropriate choice lever in rats trained to discriminate s.c. injections of 3.0 mg/kg of morphine from saline. Stimulus control of behavior comparable to that produced by the training dose of morphine occurred over a 10,000-fold range of peptide doses ( ag). Moreover, the discriminative effects of the opioid peptides were antagonized by relatively low doses ( mg/kg) of naltrexone. Thus, all of the opioid peptides tested possess morphine-like discriminative stimulus properties in the rat. \ \\ #{149}10.0I 0. B1(3.0) 0 R1(10) A I2I(I0) 2 0 tot 0.i o.bi 0.b3 IXJSENAUREXOIF(.g/kg) ImSENAITREXOIC(ig/kg) cmsenaltrexoi(ig/kg) Fig. 3. Dose-related antagonism by s.c. naltrexone of the morphine-like discriminative stimulus effects of DAGO (A), DADL (B) and fi-endorphin (C) administered i.c.v. in individual rats trained to discriminate s.c. injections of 3.0 mg/kg of morphine from saline. Each symbol represents the doseresponse curve for an individual animal; the dose of peptide antagonized by naltrexone is given in parentheses and is expressed in micrograms. The dose of naltrexone is shown in milligrams per kilogram along the abscissa. Other details as in figure 1.

5 994 Locke and Holtzman Vol. 238 TRIALS To N0RPHIl LEVER A U I 8-ENA SAl IXISE*JRPHIIE (.g/k) Fig. 4. Cumulative dose-response curves of s.c. morphine constructed 24 hr after i.c.v. administration of either saline (SAL) or 5.0 g of fi-fna in rats trained to discriminate 3.0 mg/kg of morphine from SAL The stimulus generalization curve for each condition represents the mean of the dose-response curves of four ($-FNA) or five (SAL) animals. The dose of morphine is shown in milligrams per kilogram along the abscissa. Other details as in figure 1. The order of relative potency of the opioid peptides on a molar basis as morphine-like discriminative stimuli was: DAGO = FK 33,824 > 3-endorphin > DADL = metkephamid > morphiceptin. The mu-selective peptides, DAGO and FK 33,824, were extremely potent as morphine-like discriminative stimuli. Generalization to these peptides occurred at a median effective dose of only 0.01 &g (approximately 20 pmol). In contrast, peptides with significant affmity for the delta-opioid receptor generalized with the morphine training dose at a median effective dose one (/9-endorphin)- to two (DADL and metkephamid)-orders of magnitude higher than that observed with the mu-selective peptides. The order of potency of this series of peptides relative to morphine for producing morphinelike discriminative effects is similar to the order of potency for producing analgesia (Ronai, 1983). The potency order also parallels the relative selectivity ofthe peptides for mu receptors vs. delta receptors as determined in bioassays of activity in vitro on preparations of the guinea pig ileum (mu) and mouse vas deferens (delta) (Chang et a!., 1983; Ronai, 1983). It is tempting to conclude that high doses of DADL and metkephamid engender morphine-like discriminative effects through actions at the mu receptor. However, the possibility that delta receptors can also mediate morphine-like discriminative effects cannot be ruled out simply on the basis of relative potencies of agonists, especially ones that are receptor-selective rather than specific. Differences in the stability of the peptides in vivo cannot account for their order of relative potency for producing morphine-like stimulus control of behavior. DADL (Chang and Cuatrecasas, 1979) and metkephamid (Frederickson et al, 1981) both are as biologically stable as DAGO (Handa et al, 1981) and FK 33,824 (Roemer et a!., 1977). Differences in the distribution of the peptides after i.c.v. administration also seem unlikely to account for their order of potency. Four of the compounds, DAGO, FK 33,824, DADL and metkephamid, are pentapeptides that share many similarities with one another in terms of amino acid content and molecular weight. In contrast, the pharmacokinetic properties of morphiceptin probably influenced its potency as a morphine-like discriminative stimulus. Although it is a highly selective mu-receptor agonist, morphiceptin has a relatively low affinity for the mu receptor and a relatively low potency in binding and peripheral tissue assays, apparently because it is degraded rapidly (Chang et a!., 1983; Kreil et a!., 1983). Thus, the potency of morphiceptin as a morphine-like discriminative stimulus in this study is consistent with its reported receptor selectivity and biological instability. The morphine-like stimulus effects of the opioid peptides, like those of morphine itself, are centrally mediated. Support for this conclusion is based upon a comparison of the stimulus effects of FK 33,824 and metkephamid administered s.c. and i.c.v. FK 33,824 was 100,000 times more potent as a discriminative stimulus when administered centrally than when injected systemically. Metkephamid failed to occasion morphineappropriate responding when up to 30 mg/kg was injected s.c., a dose 10,000 times higher than doses that were effective after central administration. Morphine was 1000 times more potent by the i.c.v. than by the s.c. route of administration (Shannon and Holtzman, 1977; Locke and Holtzman, 1985). The lower i.c.v./s.c. potency ratio of morphine compared to the two peptides may be indicative of the greater lipophilicity ofthe former (Herz and Tesehemacher, 1971). This fact, in turn, could explain why morphine, the prototypic mu-receptor agonist, was less potent than the delta-receptor agonists in engendering stimulus control. We have proposed that periventricular brain sites are involved intimately in mediating morphine-like discriminative effects (Locke and Holtzman, 1985). If this is indeed the case, after infusion into the ventricles, morphine, by virtue of its greater lipophilicity, would diffuse out of the ventricles and away from its site(s) of action more rapidly than would the peptides, resulting in a reduced apparent potency. This supposition remains to be tested definitively. This study represents the first attempt to determine the sensitivity of the discriminative stimulus effects of the opioid peptides to antagonism by graded doses of naltrexone. Relatively low doses of naltrexone ( mg/kg) antagonized completely the stimulus effects of three representative opioid peptides, DAGO, DADL and -endorphin, in a dose-dependent manner. The morphine-appropriate responding engendered by DAGO and DADL was blocked completely by a median dose of 0.1 to 0.2 mg/kg s.c. of naltrexone; that engendered by j3- endorphin was blocked by a 3-fold lower dose. These doses are comparable to the dose of naltrexone required to block the discriminative effects of 10 g of i.c.v. morphine (Locke and Holtzman, 1985), as well as the 3.0 mg/kg systemic training dose of morphine (Shannon and Holtzman, 1976b). It is not clear why the dose of naltrexone necessary for antagonism of the discriminative effects of /3-endorphin was lower than that for DADL, DAGO or morphine. It may be that the dose of - endorphin used in this experiment was not equally discriminable to that of the other two peptides or of morphine. The differences in the dose of naltrexone required for antagonism of the discriminative stimulus effects of the opioid peptides may also be due to the large interanimal variability in responsiveness to each of the compounds administered i.c.v. In addition to inherent differences in sensitivity to the drugs (i.e., normal biological variability), interanimal variability could reflect small animal-to-animal differences in cannula placement or in perfusion of the ventricular system by the drug solution. Naltrexone has a higher affinity for mu receptors than for delta receptors (Wood, 1982). The similar range oflow doses

6 1986 Old Peptides: Discriminative EffeCts 995 over which naltrexone blocks the discriminative stimulus effects ofcentrally administered morphine (Locke and Holtzman, 1985) and opioid peptides is consonant with an interaction at the mu receptor. The antagonism ofthe discriminative effects of s.c. morphine by the irreversible mu-selective antagonist, -FNA, is also consistent with the concept of mu-receptor involvement in the stimulus effects of morphine. Using a cumulative dosing procedure, $-FNA produced a 10-fold shift to the right of the morphine dose-response curve. The shift induced by the 24-hr pretreatment with 5.0 g of 9-FNA was comparable in magnitude to the shift produced by the acute administration of 0.03 mg/kg s.c. of naltrexone (Holtzman, 1983). Thus, the effects of $-FNA in this paradigm are consistent with its reported action at mu-receptors (Takemori et a!., 1981; Ward et al, 1982a,b). However, these experiments do not address directly the opioid receptor selectivity of fi-fna which, in fact, may not differentiate mu and delta receptors well (Hayes et a!., 1985; Locke and Holtzman, 1986). These experiments also do not address the irreversibility of 9-FNA as an antagonist. The shift of the morphine dose-response curve to the right 24 hr after treatment with fl-fna was comparable to that produced by a reversible antagonist such as naltrexone. However, an irreversible antagonist may appear reversible if there are spare receptors in the system or if an adequate number of new receptors become available by the time that tests are performed. Limitations of pharmacologic tools not withstanding, each of the opioid peptides tested in this study showed graded morphine-like discriminative stimulus effects over a range of doses. This fact, the relative order of potency of the peptides, and the sensitivity of their morphine-like discriminative effects to blockade by competitive and by nonequilibrium antagonists suggest that interactions of the peptides with the mu-opioid receptor is a major element in the genesis of morphine-like discriminative effects. Acknowledgments Metkephamid was provided generously by Dr. Martin D. Hynes (Eli Lilly and Company). References BERTALMIO, A. J., HERLING, S., HAMPTON, R. Y., WINGER, G. AND WooDs, J. H.: A procedure for rapid evaluation of the discriminative stimulus effects of drugs. J. Pharmacol. Methods 7: , BROWNE, R. G. AND FONDREN, B.: f3-endorphin and the narcotic cue. In Stimulus Properties of Drugs: Ten Years of Progress, ed. by F. C. Colpaert and J. A. Rosecrans, pp , Elsevier/North Holland, Amsterdam, CHANG, K.-J. AND CUATRECASAS, P.: Multiple opiate receptors: Enkephalins and morphine bind to receptors of different specificity. J. Biol. 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7 996 L.ocle and Holtzman Vol. 238 of Drug Dependence 1979, National Institute on Drug Abuse Research Mono- the reinforcing and discriminative stimulus properties of opioids and opioid graph 27, Department of Health, Education and Welfare Publication No. peptides. In The Neurobiology of Opiate Reward Processes, ed. by J. E. Smith (ADM) , ed. by L S. Harris, pp , US. Government Printing and J. D. Lane, pp , Elsevier Press, New York, Office, Washington, DC, 1980b. ZAR, J. H.: Biostatistical Analysis, Prentice-Hall, Engelwood Cliffs, WooDs, J. H., HEELING, S. AND YOUNG, A. M.: Comparison of discriminative and reinforcing stimulus characteristics ofmorphine-like opioids and two met- Send reprint requests to: Stephen G. Holtzman, Ph.D., Department of PRierenkephalin analogues. Neuropeptides 1: , macology, Emory University School of Medicine, Atlanta, GA YOUNG, A. M., WooDs, J. H., HERLING, S. AND HEIN, D. W.: Comparison of