Novel Opiate Binding Sites Selective for Benzomorphan Drugs

Home , Benzomorphan, Diprenorphine, Morphiceptin

Proc. NatL Acad. Sci. USA Vol. 78, No. 7, pp. 4141-4145, July 1981 Biochemistry

Novel opiate binding sites selective for benzomorphan drugs (enkephalins/morphine/diprenorphine/K and a receptors/morphiceptin) KWEN-JEN CHANG, ELI HAZUM, AND PEDRO CUATRECASAS Department of Molecular Biology, The Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709 Communicated by George H. Hitchings, March 30, 1981 ABSTRACT The simultaneous addition of [D-Ala2, D-Leu5]- present studies identify such benzomorphan-selective binding enkephalin and morphiceptin at concentrations at which 98% of sites by using [3H]diprenorphine and unlabeled enkephalins enkephalin (8) and morphine (it) receptors are occupied only par- and morphiceptin to quench the 8 and u receptors, respectively. tially inhibits the binding of [3H]diprenorphine to rat brain membranes. These conditions, furthermore, do not affect the curves for displacement of [3H]diprenorphine binding by unlabeled di- MATERIALS AND METHODS prenorphine. These data suggest that [3H]diprenorphine binds to [3H]Diprenorphine (9 Ci/mmol; 1 Ci = 3.7 x 10'° becquerels) a third subtype of opiate binding site, which has high affinity for was purchased from Amersham. Other opiates, benzomor- iliprenorphine but verylowaffinityfor ,i and8agonists. The [3H]- phans, and enkephalins were available as for previous studies diprenorphine binding observed in the presence of morphiceptin (1, 3, 7). UM 909, 2-(2-methyl-3-furylmethyl)-2'-hydroxyl-5, and [D-Ala2, D-Leu5lenkephalin exhibits high affinity for several 9a-dimethyl-6,7-benzomorphan methanesulfonate; UM 911, benzomorphan drugs in the chemical family of6,7-benzomorphan 2-(3-methylfurfuryl)-2'-hydroxyl-5,9a-dimethyl-6,7-benzomor- (e.g., cyclazocine, ethylketocyclazocine, SKF 10047, UM 1072, oxilorphan, etc). Because ofits selectivity for most benzomorphan phan methanesulfonate; UM 1070, (+)-(LR/S, 5R/S, 9S/R, drugs, this putative receptor site is tentatively referred to as a 9S/R, 5'R/S)-5,9,(3dimethyl-2'-hydroxyl-2-tetrahydrofurfur- benzomorphan binding site. Its regional distribution in rat brain yl-6,7-benzomorphan hydrochloride and UM 1072, the 9- is similar to that of morphine (#) receptors but differs from that methyl steric conformer ofUM 1070, were very kindly provided for enkephalin (8) receptors. The content ofbenzomorphan bind- by James Woods, University of Michigan. ing sites in rat brain is only one-half to one-third that ofmorphine Preparation of rat (Sprague-Dawley) brain membranes and receptors. The relative affinities of various opioids to morphine, procedures for determining distribution have been described enkephalin, andUenzomorphan binding sites are also described. (1, 3). Brain sections were homogenized in 50 mM Tris HCl (pH 7.7) and centrifuged at 40,000 x g for 30 min. The pellets Recent receptor binding studies in brain membranes indicate were subjected to a 0.1 M NaCl-containing buffer for 1 hr to that there are at least two major subtypes of opiate receptors dissociate endogenous enkephalins and washed twice with (1-6). Enkephalin (8) receptors bind enkephalins and some of Tris HCI buffer. The membranes (2 ml) were incubated with their analogues preferentially, and morphine (IL) receptors bind [3H]diprenorphine for 60-90 min in the absence and presence morphine and related alkaloids with affinities greater than those ofunlabeled ligands, quickly filtered through GF/C glass filters for enkephalins. Naloxone binds to morphine receptors with an (Whatman), and washed twice with cold buffer (10 ml). Non- affinity that is about 20 times greater than for enkephalin re- specific binding was determined with the presence of 1 uM ceptors (1, 3). Diprenorphine binds equally well to both recep- diprenorphine. Under the same conditions, no specific binding tors (1, 4, 7). Very recently, the synthetic amide of a tetrapep- can be obtained without the addition ofbrain membranes. tide fragment of 3casein, Tyr-Pro-Phe-Pro-NH2, was found to be a potent and highly specific agonist for morphine receptors, RESULTS and was thus referred to as "morphiceptin" (8). Morphine receptors are localized predominantly in the hypothalamus and Apparent Homogeneity ofDiprenorphine Binding. Equilib- thalamus, whereas enkephalin receptors are highest in the stria- rium saturation binding of [3H]diprenorphine (0.1-14 nM) tum, limbic structures, and cerebral cortex (3). In vitro light shows apparent homogeneity ofbinding with a dissociation con- microscopic autoradiographic studies further confirm and ex- stant, Kd, value ofabout 0.23 nM and a maximal binding, Bmax tend these regional distributional differences (9). of 530 fmol/mg ofprotein (Fig. 1). These results are consistent On the basis of the distinctive central pharmacologic actions with otherdataindicating that diprenorphine binds to morphine of benzomorphans, Martin and colleagues proposed the possi- and enkephalin receptors with an affinity of about 0.2 nM (4, ble existence of discrete a, K, and a receptors (10, 11). The 7). The homogeneity of diprenorphine binding was confirmed occurrence of separate K and a receptors was recently ques- by displacement experiments of diprenorphine against [3H]di- tioned on the basis of binding studies using 3H-labeled ethyl- prenorphine, which show Hill coefficients (n) of unity (Fig. 2; ketocyclazocine (7, 12) and N-allylnormetazocine (SKF 10047) Hill plots are not shown). Furthermore, ifa receptor other than (12, 13). These binding studies also indicated that both the pu- the morphine (,u) or enkephalin (8) types should exist, its affin- tative K and or agonists bind with high affinities to both mor- ity for diprenorphine must exhibit a similar, if not identical, phine and enkephalin receptors (5, 7). However, these studies value. did not exclude the possible existence of a small population of Apparent Heterogeneity of Competition of [3H]Dipre- benzomorphan-selective receptor sites, possibly with affinities norphine Binding by Morphiceptin. Morphice tin is quite po- lower than those for morphine and enkephalin receptors. The tent in inhibiting the binding ofthe pu agonist [ H]dihydromor- phine, but it is weak in inhibiting the binding of the 8 agonist The publication costs ofthis article were defrayed in part by page charge '"I-labeled [D-Ala2, D-Leu5]enkephalin (Fig. 3). The 50% in- payment. This article must therefore be hereby marked "advertise- hibitory concentration (ICw) values obtained by using these la- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. beled pu and 8 agonists are 20 nM and 50 p.M, respectively. The 4141 Downloaded by guest on September 26, 2021 4142 Biochemistry: Chang et al. Proc. Natl. Acad. Sci. USA 78 (1981)

-4 0 0.5 Q E._ Q ve) 0.44 8 80 0 - 0 bB a, c- 0.3 0 0-- 40 a 20- 0.2 0.4 0.6 ._v 0.1 B, pmol/mg 10-8 10-7 10-6 10-5 10-4 Morphiceptin, M 1 2 3 4 5 6 7 FIG. 3. Binding competition curves of morphiceptin against 0.12 Free [3H]diprenorphine, nM nM [3Hldihydromorphine (o), 0.1 nM 1251-labeled [D-Ala2, D-Leu5]enkephalin (n), and 0.5 nM [3H]diprenorphine (o). The results are ex- FIG. 1. Equilibrium saturation binding of [3Hldiprenorphine to pressed as the mean of duplicate incubations, which are ± 10% of the rat brain membranes. The binding was carried out in 2 ml of 50 mM mean. Tris-HCl buffer (pH 7.7) for 90 min at room temperature. The concentration of [3Hldiprenorphine varied between 0.1 and 14 nM. Values are the mean of duplicate incubations. Inset is the Scatchard plot. B and Biphasic Competition Curve of Enkephalin Against [3H]- F, bound and free [3H]diprenorphine. The calculated apparent Kd is Diprenorphine Binding in the Presence of Morphiceptin. 0.23 nM and the Bmax is 530 fmol/mg of protein. The protein concentration was 0.45 mg/ml. Binding competition studies ofenkephalins against [3H]diprenorphine were carried out in the absence and presence of10 ,uM morphiceptin (Fig. 4). The biphasic characteristic is readily ap- in two assays is difference the IC50 values ofthese binding about parent in the presence of and suggests that there of of morphiceptin 2500-fold. The small inhibition binding "2I-labeled [D- may be two subtypes of opiate binding sites that are labeled by Ala2, D-Leu5]enkephalin observed with low concentrations of [3H]diprenorphine under these conditions. The first phase is morphiceptin is due to the slight crossreactivity of enkephalin nearly completely inhibited at 0.1 ,uM [D-Ala2, D-Leu5]enkeph- with receptors 3). morphine (Fig. alin with an apparent Ki value of 1.8 nM. This probably re- Binding competition curves of morphiceptin against [3H]di- flects binding to enkephalin (8) receptors, because this value are About 60% of the prenorphine biphasic (Fig. 3). [3H]di- is similar to its affinity for enkephalin receptors (1, 3). The sec- prenorphine binding can clearly be inhibited by concentrations ond phase begins above 1 AM and suggests the presence of a 1 the remaining is inhibited below puM, whereas binding only third binding site with an affinity for [D-Ala2, D-Leu5]enkeph- by concentrations greater than 20 p.M. These data suggest that alin about micromolar in value. When the binding is carried only 60% of the [3H]diprenorphine-labeled sites are morphine out in the presence of10 ,uM morphiceptin and 0.1 p.M [D-Ala2, (,u) receptors. Because 10 p.M morphiceptin is 500 times above D-Leu5]enkephalin, to eliminate the binding of [3H]diprenor- its value for morphine receptors, and it does not interact with phine to both morphine and enkephalin receptors, the remain- enkephalin receptors, 10 p.M concentrations were subse- ing binding can be inhibited only by very high [Leu]enkephalin quently used to inhibit the binding of [3H]diprenorphine to concentrations (i.e., about 5 Similarly, the agonists receptors and to its IC50 ,uM). morphine independently explore possible morphine and Sandoz FK 33824 are also very ineffective in in- to sites. binding other binding hibiting the remaining [3H]diprenorphine binding in the presence of morphiceptin and [D-Ala2, D-Leu5]enkephalin (Fig. 5). A Diprenorphine Competition Curve Unaffected by Mor- bB phiceptin and Enkephalin. Morphiceptin at 10 p.M decreases .8

bB ;V 60 i e- 40-

0 60- 20-

40 *-~ 0.~~~~~~~~~~~0 lo- 10 10-9 lo.-8 10-7 Diprenorphine, M

FIG. 2. Effect of morphiceptin (10 pM) and [D-Ala2, DLeu5]enkow 10 9 lo-8 10-7 10-6 10-5 phalin (0.1 u&M) on the competition of [3Hldiprenorphine binding by diprenorphine. The competition by diprenorphine for the binding of Enkephalin, M [3H]diprenorphine to rat brain membranes was carried out in the absence (e) and presence of 10 1uM mophiceptin (o) and 10 pM mor- FIG. 4. Binding competition curves of enkephalin against [3H]di- phiceptin plus 0.1 AM [D-Ala, D-Leu lenkephalin (A). Specific [3H]- prenorphine (0.5 nM) in the absence (e) and presence of 10 pM mor- diprenorphine binding was 1922 cpm with no addition. The [3H1- phiceptin (o) or 10 IAM morphiceptin plus 0.1 /AM [D-Ala2, D- diprenorphine binding was reduced to 1116 cpm (58%) in the presence Leu']enkephalin (A). For the first two experiments (o and *), of 10 pM morphiceptin and was further reduced to 517 cpm (27%) with competition experiments were studied for [D-Ala2, D-Leu5lenkephalin. the further addition of 0.1 pM [D-Ala2, D-Leu5lenkephalin. In the last experiment (A) [Leulenkephalin was used as the inhibitor. Downloaded by guest on September 26, 2021 IBiochemistry: Chang et al. Proc. Natl Acad. Sci. USA 78 (1981) 4143

Table 1. Comparison of affinities of opioids for morphine, enkephalin, and benzomorphan binding sites 80 Apparent Ki for binding sites, nM Receptor Mor- Enkeph- Benzo- affinity 60 Ligand phine alin morphan ratio 40 ,u agonists Morphine 0.4 50 110 1:125:275 Levorphanol 0.3 4 76 1:13:253 20 FK 33824 1.2 14 165 1:12:138 Morphiceptin 20 >20,000 >10,000 1:2000:2000 6 agonists 10O9 108 lo-, -106 1j-5 [Leu]Enkephalin 20 3.0 *835 1:0.15:42 Ligand, M [Met]Enkephalin 8 4.4 835 1:0.55:104 [D-Ala2, D-Leu5]- FIG. 5. Competition by opioids for the binding of [3H]diprenor- Enkephalin 4 1.6 500 1:0.4:125 phine to benzomorphan receptors in ratbrain membranes. The binding Mixed agonist- of [3H]diprenorphine (0.5 nM) to benzomorphan receptors was carried antagonists out in the presence of 10 ,uM morphiceptin and 0.1 ,uM [-Ma2, Butorphanol OA 1.7 10 1:4:25 Leu5]enkephalin to prevent the binding of [3H]diprenorphine to morphine and enkephalin receptors. Sandoz FK 33824 (m), [Metlenke- Oxilorphan 0.3 1.0 1.6 1:3:5 phalin (0), naloxone (n), ethylketocyclazocine (A), SKF 10047 (A), UM Cyclazocine 0.3 1.2 1.5 1:4:5 1072 (e), diprenorphine (Q), and [Leulenkephalin (v) were studied. K agonists UM 909 12 66 22 1:5.5:1.8 UM 911 7 16 10 1:2.3:1.5 the [3H]diprenorphine binding to 58% and further addition of UM 1070 1.8 10 6 1:5.5:3.3 O. 1 UM [D-Ala2, D-Leu5]enkephalin further decreases the bind- UM 1072 0.8 6 3 1:7.5:3.8 ing to 27%. The subsequent competition curves with dipren- Ethylketocyla- orphine are shifted to the right only slightly despite the pres- zocine 1.0 3.2 16 1:3.2:16 ence of morphiceptin and enkephalin under conditions that Ketocyclazocine 2.2 7.5 60 1:3.4:27 occupy more than 98% of the morphine and enkephalin recep- a agonist tors (Fig. 2). Thus, [3H]diprenorphine, in addition to binding N-Allylnormet- to enkephalin and morphine receptors, appears capable of la- azocine 0.7 1.2 15 1:1.7:22 beling a third biochemically distinct type of binding site. The Antagonists 15 16 1:15:16 slight shift in the curve (about 2-fold in the presence of mor- Naloxone 1.0 Diprenorphine 0.20 0.18 0.47 1:0.9:2.3 phiceptin and enkephalin) probably reflects the weak crossreactivity of enkephalin with this third binding site. This factor Nalorphine 0.3 8 16 1:27:53 will be considered in the following section in the calculations The apparent Ki values were obtained from measurements of IC50 of the apparent K, values. values obtained by measuring the binding of "2I-labeled [n-Ala2, Na- Selectivity of [3H]Diprenorphine Binding Sites for Benzo- MePhe4, Met(O)ol5]enkephalin (0.05 nM) and of I251-labeled [D-Ala2, The of and D-Leu5]enkephalin (0.05 nM) for morphine and enkephalin receptors, morphan Drugs. ability many opiates enkephalins respectively. The apparentKi values for benzomorphan receptors were to compete with the binding of [3H]diprenorphine to the third calculated according to Cheng and Prusoff (14), using the binding as- opiate binding site was demonstrated in the presence of 10 IIM say of [3H]diprenorphine in the presence of 10 ,uM morphiceptin plus morphiceptin and 0.1 MM [D-Ala2, D-Leu5]enkephalin (Fig. 5). 0.1 pM [D-Ala2, D-Leu5]enkephalin. Ki = IC50/[1 + L/Kd] in which L The ranking of potency observed is diprenorphine > oxilor- is the concentration of labeled [3H]diprenorphine (0.5 nM), Kd is the phan, cyclazocine > UM 1072 > SKF 10047, naloxone, and dissociation constant of [3H]diprenorphine (0.23 nM), and IC50 values ethylketocyclazocine > Sandoz FK33-824, morphine > [Leul- are the concentrations that inhibit the binding of labeled ligand by 50%. The factor of 2 is also usedto correct the IC50 values due to a slight and [Met]enkephalin and [D-Ala2, D-Leu5]enkephalin. Other crossreactivity of enkephalin (Fig. 2) with benzomorphan receptors. enkephalin analogs such as [D-Ala2, Met5]enkephalinamide and Receptor affinity ratio is the ratio of the apparent Ki values for mor- [D-Met2, Pro5]enkephalin also have low potency (Y4 1 MuM). phine, enkephalin, and benzomorphan receptors. The apparent K1 values calculated according to the Cheng and Prusoff equation (14), and corrected by a factor of 2 as men- prenorphine has very high (and equal) affinity to all three bind- tioned above, are listed in Table 1. These results suggest that, ing sites.Naloxone and nalorphine bind to morphine receptors in the presence of morphiceptin and enkephalin, [3H]dipren- with high affinity, and are about 1/50th to 1/15th as strong for orphine binds to receptor sites selective for agonists with a both enkephalin and benzomorphan receptors. Interestingly, structure in the chemical family of 6,7-benzomorphan-11-ol levorphanol binds to benzomorphan binding sites with an af- (Fig. 6). Thus, we prefer, tentatively, to call them benzomor- finity similar to that of morphine. Dextrorphan is about 1/ phan receptors. For comparison, the affinities of these com- 100th as strong as levorphanol. pounds for all three opiate receptors are also listed in Table 1. Differences in Regional Receptor Distributions. [3H]Di- The relative affinities for these three receptor subtypes are ex- prenorphine binding to different brain regions was carried out pressed as ratios ofmorphine receptors to enkephalin recep- in the presence of 10 ,uM morphiceptin, 10 MM morphiceptin tors to benzomorphan binding sites. All agonists show high plus 0.1 uM [D-Ala2, D-Leu5]enkephalin, or 1 ,uM diprenor- selectivity for morphine receptors. agonists bind to enkepha- phine (Table 2). The results with respect to morphine and en- lin receptors with highest affinity, to morphine receptors with kephalin receptors are similar to those of previous studies (3) slightly lower affinity, and to benzomorphan binding sites with using other markers, which show a basic division into three a further lower, 1/100th, affinity. Most mixed agonist-antag- groupings. The hypothalamus and thalamus show the highest onists and benzomorphan drugs show much less selectivity. relative content of morphine receptors, with intermediate val- Some preference for morphine receptors is still obvious. Di- ues for brain stem, hippocampus, and the limbic system, and Downloaded by guest on September 26, 2021 4144 Biochemistry: Chang et al. Proc. Natl. Acad.- Sci. USA 78 (1981) -CH3 -KQ>

Butorphmnol Cyclazocine Oxi lorphan

9o -N-CH2 -< N-CH2-CH=CH2. /X~ CH3 CH3 -H C2H5 -=/ CH3 OH OH UM 1072 EthylI ketocyclazoc ine N-al lylnormetozocine (SKF 10047)

841

6-7- Benzomorphan- 1-oal FIG. 6. Structures of various benzomorphan drugs. the lowest relative content for striatum and cerebral cortex. In binding sites. The quantity of benzomorphan binding sites in general, the relative morphine receptor content (i.e., ratio) is rat brain is also less than that of morphine and enkephalin re- slightly higher than obtained greviously (3) by using '25I-[D- ceptors (Table 2). These may be the reasons why previous stud- Ala2, D-Leu5]enkephalin and I-labeled [D-Ala2, Na-MePhe4, ies failed to detect K and oreceptors by using 3H-labeled ethyl- Met(O)ol5]enkephalin, probably because of slight overestima- ketocyclazocine (7, 12) and SKF 10047 (13). tion ofenkephalin receptor content due to some crossreactivity Some evidence suggesting the possible existence of a third of the enkephalin label with morphine receptors. subtype of opiate binding site in the guinea pig brain was de- The distribution of benzomorphan binding sites is very dif- scribed by Kosterlitz and Paterson (15). However, the limited ferent from that of enkephalin receptors but resembles closely data presented in that report preclude accurate assessment of that of morphine receptors. The absolute content of morphine whether these represent the same binding sites as are de- receptors is, however, about 2 to 3 times greater throughout. tected in the present studies. Preliminary studies with The content of benzomorphan binding sites is highest in the [3H]ethylketocyclazocine binding to guinea pig brain mem- striatum and is intermediate in the hypothalamus and thalamus. branes in the presence of morphiceptin and [D-Ala2, D-Leu5] enkephalin indicate a similar relative potency ofopioids in com- DISCUSSION peting with the residual [3H]ethylketocyclazocine binding. This suggests that residual [3H]ethylketocyclazocine binding after The data point to the existence in rat brain membranes ofa sub- quenching the ,u and 8 receptors is similar, if not to type of sites that is and identical, opiate binding biochemically topograph- the binding at benzomorphan sites in rat brain. ically distinct from the now well-characterized enkephalin and The relative affinities of many opiates and enkephalins for morphine receptors. Excluding opiate antagonists (naloxone, the three are nalorphine, and diprenorphine), thus far all agonists and mixed identifiable binding sites listed in Table 1. All of, the and 8 re- agonist-antagonists tested (cyclazocine, oxilorphan, UM 1072, ,u agonists show little affinity for benzomorphan ethylketocyclazocine, and SKF 10047) that have high affinity ceptors. However, the so-called mixed agonist-antagonists, K agonists, and cr agonists have much greater affinity compared for this third subtype ofbinding site are in the chemical family to and 8 UM are of 6,7-benzomorphan-11ol (Fig. 6) with bulky substitutions at A agonists. 1072, cyclazocine, and oxilorphan the nitrogen atom. Although butorphanol and oxilorphan have the best ligasds for the benzomorphan binding sites. Naloxone and to an been classified as derivatives of morphinan, they can also be nalorphine bind this receptor with apparent value classified chemically as benzomorphan drugs (Fig. 6). There- of 16 nM, which is similar to their affinity for enkephalin re- fore, this binding site is tentatively referred to as a benzomor- ceptors but is about 1/50th to 1/15th of that for morphine re- phan binding site. Although benzomorphan drugs are the opiate ceptors. Diprenorphine binds to all three subtypes ofreceptors ligands that bind to this binding site with the highest affinity, with relatively high affinity (1K about 0.2 nM). Martin and col- it should be appreciated that these drugs also bind to morphine leagues (10, 11) postulated a K receptor forthese benzomorphan and enkephalin receptors with equal or even greater affinities drugs. Ethylketocyclazocine and ketocyclazocine are typical K (5, 7) (Table 1). The affinities of ethylketocyclazocine and SKF agonists. Oxilorphan and diprenorphine are potent antagonists 10047 for these three binding sites are lowest for benzomorphan at putative K receptors (10). The potency for naloxone in antag- Downloaded by guest on September 26, 2021 Biochemistry: Chang et al. Proc. Natd Acad. Sci. USA 78 (1981) 4145

Table 2. Regional distribution of morphine, enkephalin, and morphine-dependent animals (10, 11). They are also not self- benzomorphan binding sites in rat brain administered by monkeys (17). The possible reasons for the ap- Specific [3H]diprenorphine binding, parent discrepancies between the results ofin vivo central ner- cpm/mg protein vous system pharmacology and in vitro binding assays remain obscure. It is possible that interactions in the central nervous [D-Ala2, D- system among neurons containing these three subtypes of op- Morphicep- Leu5]Enke- Remain- iate receptors could lead to unusual pharmacologic effects (5, tin-displace- phalin-dis- ing bind- ofspecific ligands for the various receptor able (mor- placeable ing (ben- 7). The development Brain phine (enkephalin zomorphan Binding subtypes may help in the ultimate resolution of this problem. receptor) receptor) receptor) ratio region We thank Mark A. Collins for excellent technical assistance. Hypothalamus 2880 ± 280 550 ± 160 1250 ± 320 5:1:2.3 Thalamus 3060 ± 340 310 ± 20 990 ± 310 10:1:3.6 1. Chang, K.-J. & Cuatrecasas, P. (1979) J. Biol Chem. 254, 2610- Brain stem 1180 ± 150 270 ± 40 660 ± 170 4.3:1:2.4 2618. Hippocampus 1780 ± 80 440 ± 20 550 ± 160 4:1:1.2 2. Lord, J. A. H., Waterfield, A. A. Hughes, J. S. & Kosterlitz, H. Limbic system 1570 ± 90 690 ± 120 800 ± 270 2.3:1:1.2 W. (1977) Nature (London) 267, 495-500. Striatum 4930 ± 820 2680 ± 640 2030 ± 730 1.8:1:0.7 3. Chang, K.-J., Cooper, B. R., Hazum, E. & Cuatrecasas, P. Sensomotor (1979) Mol. Pharmacol 16, 91-104. cortex 1840 ± 230 1180 ± 110 790 ± 210 1.5:1:0.7 4. Childers, S. R., Snowman, A. M. & Snyder, S. H. (1978) Eur. J. ± ± ± Pharmacol 55, 11-18. Frontal cortex 1970 170 1340 190 800 100 1.5:1:0.6 5. Chang, K.-J., Hazum, E. & Cuatrecasas, P. (1980) Trends Neu- The rat brain regions were dissected as described (3). The binding rosci. 3, 160-162. assays were carried out with 0.5 nM [3H]diprenorphine in the presence 6. Smith, J. R. & Simon, E. J. (1980) Proc. Natl Acad. Sci. USA 77, of 10 ,.M morphiceptin, 10 MM morphiceptin plus 0.1 MM [D-Ala2, D- 281-284. Leu5]enkephalin or 1 uM diprenorphine. The results are expressed as 7. Chang, K.-J., Hazum, E. & Cuatrecasas, P. (1980) Proc. Natl cpm per mg of protein, mean ± SEM of three separate experiments. Acad. Sci. USA 77, 4469-4473. Binding ratio is the ratio of the values for morphine enkephalin, and 8. Chang, K.-J., Killian A., Hazum, E., Cuatrecasas, P. & Chang, benzomorphan receptors. J.-K. (1981) Science 212, 75-77. 9. Goodman, R. R., Snyder, S. H., Kuhar, M. J. & Young, W. S. (1980) Proc. Natl Acad. Sci. USA 77, 6239-6243. onizing the K agonist effects is about 1/30th of that for the Au 10. Martin, W. R., Eades, C. G., Thompson, J. A. Huppler, R. E. agonists effects (10, 11). Cyclazocine, nalorphine, and SKF & Gilbert, P. E. (1976) J. Pharmacol Exp. Ther. 197, 517-532. 10047 (10, 18) behave as potent mixed agonists-antagonists at 11. Gilbert, P. E. & Martin, W. R. (1976) J. Pharmacol Exp. Ther. 198, 66-82. putative K receptors. Woods et al. (16-18) showed that the fu- 12. Hiller, J. M. & Simon, E. J. (1979) Eur. J. Pharmacol 60, 389- ryl-substituted benzomorphans such as UM 909, UM 911, UM 391. 1070, and UM 1072 have K agonists activity with a ranking po- 13. Snyder, S. H. & Goodman, R. R. (1980)J. Neurochem. 35, 5-15. tency of UM 1072 > UM 1070 > UM 911 > UM 909. All of 14. Cheng, Y. C. & Prusoff, W. H. (1973) Biochem. Pharmacol 22, these considerations are consistent with their binding potency 3099-3108. to benzomorphan binding sites. However, because of the dif- 15. Kosterlitz, H. W. & Paterson, S. J. (1980) Proc. R. Soc. London Ser. B 210, 113-126. ficulty in quantitating the pharmacological effects of K agonists, 16. Woods, J. H., Fly, C. L. & Swain, H. H. (1978) in Characteris- definitive conclusions cannot be made at the present time. tics and Function of Opioids, eds. Van Ree, J. & Terenius, L. Nonetheless, the discovery of benzomorphan binding sites (Elsevier/North-Holland, New York), pp. 403-411. may help explain some of the pharmacologic effects of these 17. Woods, J. H., Smith, C. B., Medzihradsky, F. & Swain, H. H. drugs that differ from those of morphine, such as the temper- (1979) in Mechanism of Pain and Analgesic Compounds, eds. ature, cardiovascular, respiratory (19), sedative (10, 11), loco- Beers, R. F., Jr. & Bassett, E. G. (Raven, New York), pp. 429- 445. motor (16), hallucinatory, and behavioral effects (17, 18). De- 18. Hein, D. W., Young, A. M., Herling, S. & Woods, J. H. (1981) spite the fact that benzomorphans bind to morphine and J. Pharmacol Exp. Ther., in press. enkephalin receptors with high affinity (7), they neither pre- 19. Pickworth, W. B. & Sharpe, L. G. (1979) Neuropharmacology cipitate abstinence nor suppress the withdrawal symptoms in 18, 617-622. Downloaded by guest on September 26, 2021