Search

The activation of G protein-coupled receptors results in a cascade of events that include acute signaling, desensitization, and internalization, and it is thought that not all agonists affect each process to the same extent. The early steps in opioid receptor signaling, including desensitization, have been characterized electrophysiologically using brain slice preparations, whereas most previous studies of opioid receptor trafficking have been conducted in heterologous cell models. This study used transgenic mice that express an epitope-tagged (FLAG) micro-opioid receptor (FLAGMOR) targeted to catecholamine neurons by regulatory elements from the tyrosine hydroxylase gene. Brain slices from these mice were used to study tagged MOR receptors in neurons of the locus ceruleus. Activation of the FLAGMOR with [Met5]enkephalin (ME) produced a hyperpolarization that desensitized acutely to the same extent as native MOR in slices from wild-type mice. A series of opioid agonists were then used to study desensitization and receptor trafficking in brain slices, which was monitored with a monoclonal antibody against the FLAG epitope (M1) conjugated to Alexa 594. Three patterns of receptor trafficking and desensitization were observed: 1) ME, etorphine, and methadone resulted in both receptor desensitization and internalization; 2) morphine and oxymorphone caused significant desensitization without evidence for internalization; and 3) oxycodone was ineffective in both processes. These results show that two distinct forms of signaling were differentially engaged depending on the agonist used to activate the receptor, and they support the hypothesis that ligand-specific regulation of opioid receptors occurs in neurons maintained in brain slices from adult animals.

Agonist-selective actions of opioids on the desensitization of mu-opioid receptors (MORs) have been well characterized, but few if any studies have examined agonist-dependent recovery from desensitization. The outward potassium current induced by several opioids was studied using whole-cell voltage-clamp recordings in locus ceruleus neurons. A brief application of the irreversible opioid antagonist beta-chlornaltrexamine (beta-CNA) was applied immediately after treatment of slices with saturating concentrations of opioid agonists. This approach permitted the measurement of desensitization and recovery from desensitization using multiple opioid agonists, including [Met](5)enkephalin (ME), [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), etorphine, fentanyl, methadone, morphine, morphine-6-glucuronide, oxycodone, and oxymorphone. The results indicate that desensitization protects receptors from irreversible antagonism with beta-CNA. The amount of desensitization was measured as the decrease in current during a 10-min application of a saturating agonist concentration and was a good predictor of the extent of receptor protection from irreversible inactivation with beta-CNA. After desensitization with ME or DAMGO and treatment with beta-CNA, there was an initial profound inhibition of MOR-induced current that recovered significantly after 45 min. There was, however, no recovery of MOR-mediated current with time after treatment with agonists that did not cause desensitization, such as oxycodone. These results demonstrate that desensitization prevents irreversible inactivation of receptors by beta-CNA.

Translocation of G protein-coupled receptors (GPCRs) from the cell membrane to cytosol depends on the kind of ligand activating the receptor. This principle is clearly demonstrated for opioid receptors, because diverse opiate agonists rapidly induce receptor internalization, whereas morphine almost fails. We report here the impact of mitogen-activated protein (MAP) kinase isoforms extracellular signal-regulated kinase (ERK)1/2 on the internalization of delta-opioid receptors (DORs) expressed in human embryonic kidney (HEK)293 cells. Receptor activation by etorphine turned out to transiently phosphorylate ERK/MAP kinases and bring about DOR internalization within 20 min. In contrast, prolonged exposure of HEK293 cells to morphine excited persistent phosphorylation of ERK/MAP kinases, and those cells failed to internalize the opioid receptor. When ERK/MAP kinase phosphorylation was blocked by 2'-Amino-3'-methoxyflavone (PD98059), morphine gained the ability to strongly induce DOR endocytosis. The importance of activated MAP kinases for DOR internalization is further demonstrated by glutamate and paclitaxel because these substances induce phosphorylation of ERK1/2 and concomitantly prevent DOR sequestration by etorphine. In addition, receptor internalization by morphine was facilitated by inhibition of protein kinase C and opioid-mediated transactivation of epidermal growth factor receptor (EGFR), both activating ERK/MAP kinases by opioids. The mechanism affording DOR internalization by PD98059 may relate to arrestin, which uncouples GPCRs and thus triggers receptor internalization. Arrestin considerably translocates toward the cell membrane upon DOR activation by morphine in presence of the MAP kinase blocker, but it fails in the absence of PD98059. We conclude that ERK/MAP kinase activity prevents opioid receptor desensitization and sequestration by blocking arrestin 2 interaction with activated DORs.

Deletion of a sequence near the fifth transmembrane domain (258RLSKV262, i3-1 mutant) and a motif residing at the proximal carboxyl tail (344KFCTR348, C-2 mutant) resulted in mu-opioid receptor mutants that were poorly expressed on the surface of transfected human embryonic kidney 293 cells. Treatment with the opioid antagonist naloxone, the agonist etorphine, and other hydrophobic ligands enhanced cell surface expression of i3-1 and C-2 mutants. The observed enhancement was time- and concentration-dependent, required the ligands to be membrane permeable, and was not the result of the reversal of the constitutive activities of the mutant receptors. The binding of the ligands resulted in the trafficking of the mutant receptors retained in the endoplasmic reticulum to the cell surface. The cell surface-expressed mutant C-2, but not i3-1, fully retained ability to mediate inhibition of adenylyl cyclase activity. Furthermore, the Golgi-disturbing agents brefeldin A and monensin completely blocked naloxone-enhanced expression of i3-1 and C-2 mutants. Results of these studies suggest that intracellular interactions of agonist and antagonist with mutant receptors can serve as chaperones in the trafficking of the mutants to the cell surface.

We previously observed that (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488H) promoted internalization and phosphorylation of the FLAG-tagged human kappa opioid receptor (FLAG-hkor) stably expressed in Chinese hamster ovary (CHO) cells. In this study, we compared regulation of the FLAG-hkor expressed in CHO cells by U50,488H, dynorphin A, etorphine, and levorphanol, which were potent full agonists as determined by stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Using fluorescence flow cytometry, we found that dynorphin A(1-17), like U50,488H, promoted internalization of the FLAG-hkor in a time- and dose-dependent manner. The antagonists naloxone and norbinaltorphimine, having no effect on FLAG-hkor internalization, effectively blocked dynorphin A(1-17)- and U50,488H-induced internalization. Interestingly, the full agonists etorphine and levorphanol did not cause internalization of the FLAG-hkor but significantly reduced dynorphin A(1-17)- and U50,488H-induced internalization in a dose-dependent manner. Immunofluorescence staining of FLAG-hkor yielded similar results. Dynorphin A(1-17) and U50,488H enhanced phosphorylation of FLAG-hkor to a greater extent than etorphine, but levorphanol did not increase FLAG-hkor phosphorylation. Etorphine or levorphanol decreased dynorphin- or U50,488H-induced phosphorylation. It is likely that conformations of the hkor required for phosphorylation and initiation of internalization are different from those for activation of G proteins. We also examined whether the four agonists had differential effects on superactivation of adenylate cyclase. Pretreatment with U50,488H, dynorphin A(1-17), or etorphine enhanced forskolin-stimulated adenylate cyclase activity to approximately 200 to 250% of the control, whereas levorphanol pretreatment did not result in significant adenylate cyclase superactivation. Thus, the degree of superactivation caused by an agonist is unrelated to its ability to promote internalization of the hkor.

D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) is a peptide antagonist that demonstrates potent and selective affinity for micro-opioid receptors in radioligand binding assays and in vitro bioassays. However, previous studies indicate that CTAP may possess unusual pharmacology under certain conditions. Therefore, CTAP was evaluated as an antagonist of the antinociceptive effects of a range of structurally diverse high- and low-efficacy peptide and alkaloid opioid agonists and compared with the traditional antagonist naltrexone. Male Sprague-Dawley rats (N = 227) were loosely restrained and the latency for tail withdrawal from 55 degrees C water was measured. Morphine s.c. and i.c.v., buprenorphine s.c., etorphine s.c. and i.c.v., [N-Me-Phe3,D-Pro4]-morphiceptin and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin produced antinociceptive effects. CTAP was at least 10-fold more potent than naltrexone as an antagonist of the antinociceptive effects of all five agonists. High doses of CTAP produced a noncompetitive antagonism of etorphine s.c. and morphine s.c. suggesting that CTAP may interact with additional opioid receptors in vivo or produce insurmountable antagonism at these doses. CTAP was approximately 300-fold more potent as an antagonist of DAMGO than the other agonists, indicating that CTAP may distinguish some peptide agonists such as DAMGO from other agonists based on binding interactions within the micro-opioid receptor or pharmacodynamic properties of these peptides. Naltrexone, however, administered by either s.c. or i.c.v. routes of administration was approximately equipotent as an antagonist of the antinociceptive effects of most agonists. Taken together, these data indicate that the peptide antagonist CTAP possesses a unique pharmacology unlike traditional opioid antagonists such as naltrexone

Chronic opioid agonist treatment produces tolerance and in some cases opioid receptor internalization and down-regulation. Both morphine and etorphine induce tolerance; however, only etorphine produces mu-opioid receptor (muOR) down-regulation. In vitro studies implicate dynamin-2 (DYN-2) and G-protein receptor kinase-2 (GRK-2) in these processes. Therefore, we examined etorphine and morphine effects on regulation of GRK-2 and DYN-2 in mouse spinal cord. Mice were treated for 7 days with etorphine (200 microg/kg/day infusion) or morphine (40 mg/kg/day infusion + one 25-mg implant pellet). Controls were implanted with a placebo pellet. On the 7th day after implantation mice were tested for i.t. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) analgesia. In other mice, spinal cord was removed for [(3)H]DAMGO binding studies or GRK-2 and DYN-2 protein and mRNA abundance were determined. Both etorphine and morphine produced significant tolerance (ED(50) shift = 7.6- and 7.3-fold for morphine and etorphine, respectively). Etorphine decreased spinal muOR density by approximately 30%, whereas morphine did not change muOR density. Etorphine increased ( approximately 70%) DYN-2 protein abundance and decreased its mRNA (31%), whereas it had no effect on GRK-2 protein and mRNA abundance. Morphine had no effect on either DYN-2 or GRK-2 protein or mRNA abundance. These data raise the possibility that unequal receptor regulation by etorphine and morphine might be due to differential regulation of trafficking proteins. Overall, receptor down-regulation associated with chronic etorphine treatment may accelerate dynamin-related activity. Finally, the decrease in DYN-2 mRNA may be related to stabilization of DYN-2 protein abundance, which might inhibit transcription.

To evaluate the pharmacokinetic and pharmacodynamic characteristics of a novel opioid analgesic, dihydroetorphine (DHE), concentrations of DHE and its glucuronide (DG) in plasma and central nervous system (by liquid chromatography-tandem mass spectrometry) and the antinociceptive effect (by tail-immersion test) were measured after intravenous (i.v., 2 microg/kg), intracutaneous (i.c., 2 microg/kg), subcutaneous (s.c., 2 microg/kg), intraperitoneal (i.p., 10 microg/kg), and oral (p.o., 200 microg/kg) administrations in hairless rats. An elimination half-life of plasma DHE concentration was 37.2 min after i.v. injection. Brain DHE concentration reached a maximum within 6 min after i.v. injection, and the concentration ratio in brain to plasma was 5.17. Relative bioavailabilities of DHE to i.v. injection (100%) were 70.8, 79.8, 16.7, and 0.37% after i.c., s.c., i.p., and p.o. administrations, respectively. Area under the plasma concentration-time curve ratios of plasma DG to DHE concentrations after i.v., i.c., s.c., i.p., and p.o. were 1.76, 3.26, 4.74, 14.5, and 290, respectively. Antinociceptive effects appeared rapidly after i.v., i.c., and s.c. administrations but were diminished after i.p. and p.o. administrations, and these effects were closely related to the brain DHE concentrations. DHE was excreted mainly as DG in bile (89.5% of the dose) by 240 min after i.v. injection. Serum protein binding of DHE was 83.4%, which was not influenced by DG. Glucuronidation of DHE was detected in the liver, intestine, and kidney in vitro but was minimal in the skin and brain. In conclusion, DHE was rapidly distributed to the brain in relation to producing the antinociceptive effect, and then it was rapidly metabolized to the pharmacologically inactive DG.

Although NMDA receptor antagonists attenuate the development of morphine tolerance, it is not clear whether NMDA receptor antagonists also prevent tolerance and cross-tolerance to other mu-opioid agonists and, if so, whether prevention is related to the efficacy of the agonist used to examine tolerance. A rat tail-withdrawal procedure was used to test the antinociceptive effects of the mu-opioids etorphine, morphine, and dezocine before and after twice-daily subcutaneous injections with either 0. 003 mg/kg etorphine, 10 mg/kg morphine, or 3.0 mg/kg dezocine, each administered alone or in combination with 3.0 mg/kg of the competitive NMDA antagonist LY235959. After chronic etorphine, the etorphine, morphine, and dezocine curves were shifted rightward 1.0-, 2.2-, and 3.4-fold, respectively. LY235959 prevented cross-tolerance to morphine and dezocine. After chronic morphine, the etorphine and morphine curves were shifted rightward 2.5- and 2. 9-fold, respectively, and the dezocine curve was flattened. LY235959 prevented morphine tolerance and cross-tolerance to etorphine and reduced the magnitude of cross-tolerance to dezocine. After chronic dezocine, the etorphine, morphine, and dezocine curves were shifted rightward 4.1-, 3.5-, and 9.6-fold, respectively. LY235959 did not prevent but reduced the magnitude of tolerance and cross-tolerance. In a separate experiment, the following rank order of efficacy was determined from the magnitudes of rightward shift in each dose-effect curve after administration of 1.0 mg/kg of the irreversible antagonist clocinnamox: etorphine > morphine > dezocine. These data show that differences in tolerance magnitude are related to opioid efficacy and that attenuation of mu-opioid tolerance and cross-tolerance by LY235959 depends upon the magnitude of opioid tolerance.

Previously, we showed that the human kappa-opioid receptor (hkor) stably expressed in Chinese hamster ovary (CHO) cells underwent down-regulation after prolonged U50,488H treatment. In the present study, we determined the mechanisms underlying this process. U50, 488H caused a significant down-regulation of the hkor, although etorphine did not. Neither U50,488H nor etorphine caused down-regulation of the rat kappa-opioid receptor. Thus, similar to internalization, there are agonist and species differences in down-regulation of kappa-opioid receptors. Expression of the dominant negative mutants arrestin-2(319-418) or dynamin I-K44A significantly reduced U50,488H-induced down-regulation of the hkor. Coexpression of GRK2 or GRK2 and arrestin-2 permitted etorphine to induce down-regulation of the hkor, although expression of arrestin-2 or dynamin I alone did not. Expression of the dominant negative mutants rab5A-N133I or rab7-N125I blunted U50,488H-induced down-regulation. Pretreatment with lysosomal enzyme inhibitors [(2S, 3S)trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or chloroquine] or proteasome inhibitors (proteasome inhibitor I, MG-132, or lactacystin) decreased the extent of U50,488H-induced down-regulation. A combination of chloroquine and proteasome inhibitor I abolished U50,488H-induced down-regulation. These results indicate that U50,488H-induced down-regulation of the hkor involves GRK-, arrestin-2-, dynamin-, rab5-, and rab7-dependent mechanisms and receptors seem to be trafficked to lysosomes and proteasomes for degradation. Thus, U50,488H-induced internalization and down-regulation of the hkor share initial common mechanisms. To the best of our knowledge, these results represent the first report on the involvement of both rab5 and rab7 in agonist-induced down-regulation of a G protein-coupled receptor. In addition, this study is among the first to show the involvement of proteasomes in agonist-induced down-regulation of a G protein-coupled receptor.

The opioid agonists morphine, etorphine, buprenorphine and U50,488 were examined alone and in combination with the insurmountable opioid antagonist clocinnamox (C-CAM) in squirrel monkeys responding under a schedule of shock titration. In this procedure, shock intensity increased every 15 sec from 0.01 to 2.0 mA in 30 increments. Five lever presses during any given 15-sec shock period produced a 15-sec timeout, after which shock resumed at the next lower intensity. When given alone, each of these agonists increased the median intensity at which the monkeys maintained shock [median shock level (MSL)]. At the highest dose examined alone, each agonist produced maximal increases in MSL and, except buprenorphine, decreased response rates. C-CAM dose-dependently antagonized the effects of morphine, etorphine and buprenorphine on MSL. In the presence of the higher C-CAM doses, etorphine, morphine and buprenorphine did not produce maximal effects on MSL. The effects of U50,488 were not systematically altered when tested in combination with the highest C-CAM dose. In general, C-CAM was more potent and the duration of antagonism was slightly longer against buprenorphine than against morphine and etorphine. Quantitative analysis of these data according to an extended model of yielded the following apparent affinity and efficacy estimates, respectively: etorphine (0. 085 mg/kg, 117); morphine (49 mg/kg, 24) and buprenorphine (0.62 mg/kg, 7.1). Determination of the individual q values over time indicated that the receptor population recovers more quickly after C-CAM antagonism of etorphine than from C-CAM antagonism of either morphine or buprenorphine. These data suggest that C-CAM functions as a long-lasting antagonist of mu opioid agonist actions in a shock titration procedure and yields estimates of relative intrinsic efficacy with the rank order of etorphine > morphine > buprenorphine.

Opioid analgesics are used extensively in the management of pain. Although the clinically effective opioids bind with high affinity to the mu-opioid receptor, studies have suggested that the delta-opioid agonists might represent more ideal analgesic agents, with fewer side effects. A limitation to opiate effectiveness is the development of tolerance, an event that has been linked to opioid receptor desensitization. To gain a better understanding of delta-receptor agonist regulation, the cloned mouse delta receptor was stably expressed in human embryonic kidney 293 cells, and the functional effects of agonist pretreatment were examined. With a 3-hr pretreatment protocol, the delta-selective agonists [D-Pen2,D-Pen5]enkephalin, [D-Ala2,D-Leu5]enkephalin, and [D-Ser2,Leu5]enkephalin-Thr and the nonselective opioids levorphanol, etorphine, and ethylketocyclazocine were found to desensitize delta receptors. [D-Pen2,D-Pen5]enkephalin, [D-Ser2,Leu5]enkephalin-Thr, [D-Ala2,D-Leu5]enkephalin, and etorphine treatments also caused a pronounced internalization of the epitope-tagged delta receptor, suggesting that the desensitization and internalization may be related. In contrast, levorphanol pretreatment did not internalize the receptor but still resulted in a 400-fold reduction in potency, suggesting that prolonged treatment with levorphanol only uncoupled the delta receptor from adenylyl cyclase. In contrast to the desensitization induced by peptide-selective delta agonists, pretreatment with the delta-selective nonpeptide agonist 7-spiroindanyloxymorphone and morphine sensitized the opioid inhibition of forskolin-stimulated cAMP accumulation. This differential regulation of the delta receptor may be due to variations in the ability of agonists to bind to the receptor. This hypothesis was supported by the finding that a point mutation that converted Asp128 to Asn128 (D128N) diminished the ability of delta-selective agonists to inhibit cAMP accumulation while increasing the potency of morphine to reduce cAMP accumulation. In particular, a lack of desensitization of the delta receptor by morphine may contribute to our understanding of the molecular basis of development of morphine-induced tolerance and dependence.

Opioids have been shown to cause a potent inhibition of neurons in the locus ceruleus (LC) in vivo in brain slices and isolated neurons; however, the kinetics of opioid action have not been described. In this study, we used acutely isolated LC neurons to examine opioid and alpha2-adrenoceptor action on potassium and calcium currents. [Met]Enkephalin (ME), [D-Ser2,Leu5,Thr6]-enkephalin, etorphine, and [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin increased potassium conductance, whereas morphine and naloxone were antagonists. The time constant of potassium channel activation was approximately 0.7 sec and was the same for each agonist. The amplitude of the current and the time constant of decay were dependent on the agonist, suggesting that agonist efficacy and affinity, respectively, determined these parameters. The amplitude of potassium current induced by the alpha2-adrenoceptor agonist UK14304 was not significantly different from that induced by ME, but the time constant of current activation was half that of ME, and the decline was more rapid. When potassium conductances were blocked with the combination of internal cesium and external barium, opioid and alpha2 agonists had no effect at potentials more negative than -50 mV and decreased barium currents at potentials between -40 and +20 mV. Both morphine and clonidine caused a small inhibition of barium current. In dorsal root ganglion cells, morphine alone had small and inconsistent effects on the calcium current, but it always competitively antagonized the inhibition caused by [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin. The results in isolated LC neurons suggest 1) the amplitude and time course of the opioid-induced potassium current depend on agonist efficacy and affinity and 2) the coupling of both mu-opioid and alpha2-adrenoceptors to calcium channels seems to be more efficient than that to potassium channels.

The mitogen-activated protein (MAP) kinase of Chinese hamster ovary cells (CHO mu66 cell line) transfected to express mu-opioid receptors was markedly activated by mu agonists. The rank order of effectiveness of agonists was approximately the same as the rank order of their binding affinities to the mu receptor. The delta and kappa receptor-specific agonists cyclic[D-Pen2, D-Pen5]enkephalin and U69,593 showed a very weak stimulatory effect. The mu agonist-stimulated MAP kinase activity peaked at approximately 4-8 min and lasted almost 1 hr. The stimulatory effect of mu agonists was antagonized by the opioid receptor antagonist naltrexone and inhibited by pretreatment of cells with pertussis toxin. This opioid-induced activation of MAP kinase activity may have a role in the long term effects of opioids.

Opioid binding sites were found in nuclear matrix preparations from NG108-15 neurohybrid cells. Binding parameters of delta-specific radioligands indicated that high-affinity binding sites discovered in purified nuclei were present in nuclear membranes and nuclear matrix fractions. Agonists bind with low affinity, if at all, to nuclear matrix preparations. Neither sensitivity of agonist binding to the GTP analog 5-guanylylimidodiphosphate nor adenylyl cyclase activity were detected in this fraction, suggesting the presence of guanine nucleotide binding regulatory protein/effector uncoupled sites. Opioid inhibition of basal and forskolin-stimulated adenylyl cyclase activity was found in nuclear membrane preparations. Cycloheximide treatment of cells inhibited opioid binding to nuclear membrane fractions to a greater extent than that associated with membranes sedimenting at 20,000 x g (P20) or nuclear matrix. Colchicine, a microtubule disrupter and inhibitor of receptor internalization, caused up-regulation of nuclear membrane and P20 opioid receptors and a loss in nuclear matrix associated sites. Taxol, a microtubule stabilizing agent, prevented the effect of colchicine. Etorphine-elicited down-regulation increased nuclear matrix associated binding while diminishing that in nuclear membranes and P20 fractions. Agonist-induced desensitization completely abolished nuclear matrix binding. In vitro preincubation of nuclear matrix preparations with protein kinase A catalytic subunit mimicked the desensitization effect. Forskolin treatment of cells potentiated nuclear matrix and P20 binding. These data suggest that nuclear membrane opioid receptors represent newly synthesized molecules en route to the cell surface, whereas nuclear matrix contains internalized delta sites.

Six opiate agonists were characterized by in vivo apparent pA2 analysis with respect to their discriminative stimulus, rate-decreasing and analgesic effects, by using the antagonist naltrexone. In drug discrimination experiments, rats were trained to discriminate 3.2 mg/kg of morphine from saline under a fixed-ratio 15 schedule of food reinforcement. In analgesia experiments, rat's tails were immersed into 55 degrees C water and latency for tail withdrawal was measured. Naltrexone (0.01-1.0 mg/kg) antagonized discriminative stimulus effects of all agonists, rate-decreasing effects of etorphine, morphine, fentanyl, buprenorphine and GPA 1657 [(1)-B-2'-hydroxy-2,9-dimethyl-5-phenyl-6,7-benzomorphan] and analgesic effects of etorphine, morphine, buprenorphine and GPA 1657. Analgesic effects of fentanyl and nalbuphine were not tested. Naltrexone apparent pA2 values across the three behavioral measures were etorphine (7.2-7.4 mol/kg), fentanyl (7.3-7.4 mol/kg), morphine (7.5-8.4 mol/kg), GPA 1657 (7.0-7.3 mol/kg), buprenorphine (7.5-7.7 mol/kg) and nalbuphine (7.7 mol/kg). Apparent pA2 values averaged 7.5 mol/kg and slopes of the naltrexone Schild regressions were not different from unity, suggesting that the measured behavioral effects of these agonists are mediated by mu opioid receptors. Nalbuphine also was used as an antagonist in the tail-withdrawal assay. The apparent pA2 values for nalbuphine were etorphine (4.9 mol/kg), morphine (5.9 mol/kg), GPA 1657 (5.7 mol/kg) and buprenorphine (5.5 mol/kg). Slopes of the Schild regressions differed, suggesting that nalbuphine's modest analgesic effects may have prevented proper conditions for an accurate Schild analysis.

We report a novel agonist-induced change in delta-opioid receptor binding properties in NG108-15 cells. Pretreatment of these cells with opioid agonists substantially diminishes the binding of peptide agonists and a peptide antagonist to opioid receptors in intact cells or membrane preparations. However, similar agonist-induced changes in the binding of opiate alkaloid agonists and antagonists were not detected. The change in opioid peptide binding occurs rapidly at 37 degrees (t1/2 approximately 10 min) but is not induced by agonist treatment at 4 degrees. Because of its lability at 37 degrees, the binding change is only detected when equilibrium binding assays are performed at 4 degrees. Both alkaloid and peptide agonists induce the binding change in a dose-dependent manner, with an ED50 for etorphine of approximately 10 nM. The induction of the binding change is completely blocked by the opiate antagonist naloxone. Stimulation of muscarinic receptors (which, like opioid receptors, inhibit adenylate cyclase in these cells) does not induce or block the binding change. These data reveal the operation of a homologous regulation mechanism that rapidly diminishes the interaction of delta-subtype opioid receptors with peptide ligands but does not detectably change the interaction of receptors with alkaloid ligands.

The effect of chronic treatment with opioid agonists and antagonists on mu opioid receptor density and opioid potency was examined in mice. Mice were implanted s.c. with osmotic mini-pumps that infused etorphine (50-500 micrograms/kg/day), fentanyl (0.03-5.0 mg/kg/day) or naloxone (0.1-10.0 mg/kg/day) for 7 to 8 days. Other mice were implanted s.c. with a morphine pellet (75 mg) for 3 or 7 days or were injected s.c. once daily for 7 days with fentanyl (0.3 mg/kg). At the end of treatment, saturation binding studies were conducted ([3H]DAMGO) or antinociceptive tolerance was evaluated using the tail-flick assay. Etorphine produced dose-dependent tolerance as well as down-regulation of mu receptor density. Fentanyl infusions produced upregulation of opioid receptors at lower (0.03 and 1.00 mg/kg/day) doses and down-regulation at the highest dose (5.00 mg/kg/day). The lowest fentanyl infusion dose also produced tolerance. Daily s.c. administration of fentanyl (0.30 mg/kg) increased receptor density and produced tolerance to fentanyl. Morphine pellets increased (3 day) and then had no effect (7 day) on receptor density, although tolerance to morphine was observed at 7 days. Naloxone dose-dependently increased mu opioid receptor density. Receptor affinity was not systematically altered by the drug treatments. Control binding studies indicated that acute etorphine interfered with binding at mu receptors 15 min after administration, but that all drug was eliminated by 16 hr. Thus, binding and tolerance studies using etorphine were conducted 16 hr after the end of infusion, when all drug had been eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)

Buprenorphine is an opiate drug with a mixed agonist-antagonist profile and has therapeutic efficacy in attenuating drug craving and addiction. Because the adenylyl cyclase system has been implicated in the biochemical basis of opiate withdrawal phenomena, we have compared the acute and chronic effects of buprenorphine with the full opiate agonist etorphine on cyclic AMP (cAMP) synthesis in the human neuroblastoma cell SK-N-SH. Both drugs acutely inhibited prostaglandin (PG)E1-stimulated cAMP accumulation; the inhibition caused by either drug was prevented by pretreatment with the opiate antagonist naltrexone or with pertussis toxin. Chronic treatment of the cells with etorphine induced an increase in PGE1-stimulated cAMP synthesis which was observed after withdrawal of the inhibitory drug. Chronic treatment with buprenorphine appeared to have the opposite effect, resulting in an attenuated PGE1 stimulation; additionally, buprenorphine prevented the etorphine-induced enhancement in cAMP synthesis, whether administered before or after prolonged incubation of the cells with etorphine. The attenuating effect of buprenorphine occurred within 5 min and was prevented by a prior application of naltrexone, but could not be reversed by a subsequent treatment with antagonist. These findings suggest that buprenorphine was binding (pseudo)irreversibly to the opiate receptor, resulting in a persistent inhibition of cAMP synthesis which masks the etorphine-induced enhancement of adenylyl cyclase activity. This hypothesis was confirmed by experiments demonstrating that treatment of the cells with buprenorphine significantly reduced available opiate receptor binding sites despite extensive washing of the cells to remove unbound buprenorphine. These pharmacodynamic actions of buprenorphine may be relevant to its therapeutic efficacy in treating drug abuse and addiction.

Etorphine, a potent opioid agonist, has been reported to bind to both mu and epsilon opioid receptors. The present studies were designed to determine what types of opioid receptors and neurotransmitters for descending pain control systems were involved in antinociception induced by etorphine in mice. Morphine, a typical mu opioid receptor agonist, and beta-endorphin, an epsilon opioid receptor agonist, were used for comparison. Antinociceptive response induced by etorphine (20 ng) given i.c.v was blocked by i.c.v administration of D-Phe-Cys-Tyr-D-Tyr-Orn-Thr-Pen-Thr-NH2 (CTOP, 25 ng) and beta-endorphin-(1-27) [beta-EP-(1-27)] (6 micrograms), but not ICI 174,864 (ICI, 5 micrograms) or norbinaltorphimine (N-BNI, 5 micrograms). The antinociception induced by i.c.v. etorphine was also antagonized by the i.c.v. pretreatment of beta-funaltrexamine (beta-FNA, 50 ng, 24 hr). Intracerebroventricular administration of beta-EP-(1-27) (3 micrograms) caused a further attenuation of the i.c.v. etorphine-induced antinociception in mice pretreated with beta-FNA. The antinociceptive response induced by morphine (2 micrograms) given i.c.v. was blocked by i.c.v. administration of CTOP (25 ng) or beta-FNA (50 ng), but not beta-EP-(1-27) (6 micrograms), ICI (5 micrograms) or N-BNI (5 micrograms). These results indicate that the antinociception induced by etorphine given i.c.v. is mediated by the stimulation of both mu and epsilon opioid receptors whereas the antinociception induced by morphine given i.c.v. is mediated by the stimulation of mu, but not epsilon opioid receptors at supraspinal sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C (PKC) activation was examined for its role in delta-opioid receptor down-regulation in the neuroblastoma X glioma hybrid cell line NG108-15. Incubation of NG108-15 cells for 2 hr at 37 degrees with up to 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), a phorbol ester that activates PKC, had no effect on opioid binding to membranes prepared from these cells. However, as little as 3 nM PMA incubated with an opioid agonist and NG108-15 cells potentiated the decrease and the rate of decrease of opioid binding, compared with agonist alone. Scatchard analysis of [3H][D-Ala2,D-Leu5]enkephalin (DADLE) binding revealed that NG108-15 cells incubated for 3 hr with 1 nM DADLE and 30 nM PMA displayed a > 50% reduction in the number of [3H]DADLE binding sites with no affinity change at the remaining sites, compared with cells treated with DADLE alone. The antagonist naloxone blocked both DADLE-induced and PMA-enhanced DADLE-induced down-regulation. The agonists morphine and cyclazocine, which alone were unable to induce delta receptor down-regulation, did so in the presence of PMA. The PKC inhibitor staurosporine and down-regulation of PKC by chronic PMA treatment blocked PMA potentiation of DADLE-induced down-regulation, but not "normal" DADLE-induced down-regulation. The enhancement of down-regulation by PMA was unaffected by either metabolic inhibitor or incubations at 20 degrees, conditions that blocked down-regulation by DADLE alone. NG108-15 cells incubated with [3H]DADLE and PMA retained more [3H]DADLE than cells incubated with [3H]DADLE alone, suggesting that PMA enhanced receptor internalization instead of merely inhibiting membrane binding. The diacylglycerol 1-oleoyl-2-acetyl-glycerol and bradykinin substituted for PMA but not carbachol, indicating that PKC activated physiologically may play a role in delta receptor down-regulation.

Experiments assessed the ability of repeated treatment with morphine to produce tolerance to morphine-like discriminative stimulus effects of buprenorphine, etorphine, methadone, morphine, and nalbuphine. Before treatment, each compound evoked full morphine-like stimulus effects in rats trained to discriminate saline and 3.2 mg/kg morphine. During treatment, training was halted and a dose of 10 mg/kg morphine administered every 12 h for 14-18 days. Repeated treatment with morphine increased the ED50 for stimulus control by etorphine, methadone or buprenorphine 2- to 4-fold and that for morphine 4.5-fold. Repeated treatment produced an insurmountable tolerance to the morphine-like stimulus effects of nalbuphine, so that a dose 150-fold higher than the initial ED50 evoked only 40% generalization. Treatment with a lower dose of morphine (10 mg/kg every 24 h) produced a short-lived surmountable tolerance to stimulus effects of nalbuphine. For etorphine, methadone and morphine, tolerance to morphine-like stimulus effects was accompanied by tolerance to rate suppressing effects. After treatment ended, the ED50 for stimulus control by etorphine, methadone or nalbuphine returned to initial values within 3 days; that for morphine, within 5 days; that for buprenorphine, within 10 days. These results demonstrate that repeated treatment with morphine produces cross-tolerance to compounds that exert morphine-like stimulus effects. Additionally, the results suggest that differences among these compounds in agonist efficacy may be revealed as differences in the degree of tolerance produced by morphine treatment.

Previous reports have shown that [Leu5]enkephalin or [Met5] enkephalin, endogenous delta receptor agonists, or synthetic analogues of these substances, can respectively produce a positive (i.e., increase) or negative (i.e., decrease) modulation of the antinociceptive potency of mu agonists such as morphine; such modulation is believed to be the result of interactions between delta and mu receptors. In spite of these studies showing modulation of mu agonist potency, it is unclear whether delta agonists can similarly modulate the antinociceptive efficacy of mu agonists. This question was addressed by using several levels of nociceptive stimulus intensity in mice. As the nociceptive stimulus intensity increased, the i.c.v. morphine dose-response line was shown to be displaced progressively to the right with decreasing maximal effect (i.e., decreased efficacy) a pattern typical of partial agonists. In contrast, the antinociceptive potency and efficacy of i.c.v. etorphine was unaffected by increasing the stimulus intensity, suggesting that this compound has higher efficacy than morphine in this nociceptive assay. Coadministration of delta opioid agonists produced leftward ([D-Pen2, D-Pen5] enkephalin) or rightward ([Met5]enkephalin) displacement of the morphine dose-response line (i.e., changes in potency). When the delta agonists were coadministered with morphine under conditions of high stimulus intensity, the maximal antinociceptive effects of i.c.v. morphine were increased or decreased from studies with morphine alone (i.e., change in efficacy). Both changes in potency and efficacy produced by the delta agonists, but not the direct antinociceptive effects of morphine, were blocked by the delta antagonist, ICI 174,864, suggesting that modulation occurred via the delta receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Abecarnil (ZK 112119; isopropyl-6-benzyloxy-4-methoxymethyl-beta-carboxylate) is a metabolically stable beta-carboline derivative with potent anxiolytic and few sedative and ataxic effects in rodents. The anticonvulsant and muscle relaxant actions of abecarnil have been evaluated in mice, rats, gerbils and baboons. Abecarnil raised the threshold for tonic electroconvulsions in mice after corneal but not after auricular application, had no effect on maximal electroshock-induced tonic convulsions triggered by either method, protected mice against the tonic hindlimb extension in PTZ-, picrotoxin- and 3-mercaptopropionate-induced seizures and blocked clonus after PTZ, DMCM (methyl-4-ethyl-6,7-dimethoxy-9H-pyrido-(3,4-b)-indol-3-carboxylate) and 3-mercaptopropionate. Abecarnil had no effect on convulsions induced by bicuculline and strychnine. Furthermore, abecarnil blocked kindled seizures after chronic administration of PTZ and FG 7142 (beta-carboline-3-carboxylic acid methylamide) and protected mice and rats against limbic convulsions induced by pilocarpine. Severity and afterdischarge duration of amygdala-kindled seizures were reduced in rats treated with abecarnil. Abecarnil also antagonized selectively convulsions induced by i.c.v. administration of kainate, but not those triggered by N-methyl-D-aspartate or quisqualate. In genetic models of reflex epilepsy, abecarnil was effective against sound-induced convulsions in DBA/2 mice, against air blast-induced generalized seizures in gerbils and against myoclonus in baboons Papio papio. The anticonvulsant effect of abecornil in a PTZ seizure model in mice was potentiated by ethosuximide, whereas no significant potentiation was found with diazepam, clonazepam, diphenylhydantoin, carbamazepine and phenobarbital. Electromyographic monitoring in a etorphine model of muscle rigidity in rats showed no or little muscle relaxant effect of abecarnil.(ABSTRACT TRUNCATED AT 250 WORDS)

These studies were undertaken to determine the effects of morphine and other opiate and opioid agonists on body temperature in the mouse. Mice were lightly restrained, and rectal temperatures were monitored after injection of opiate analgesics at each of three ambient temperatures. The drugs tested were pure agonists representing eight different chemical classes. At 20 degrees C, morphine, hydromorphone, levorphanol, oxymorphone, methadone, etonitazene, fentanyl, etorphine and meperidine produced hyperthermia in low doses and hypothermia as the doses were raised. Codeine produced only hypothermia at 20 degrees C in the doses studied. At 25 degrees C, the hypothermic responses were greatly reduced in magnitude, and most drugs produced biphasic or only hyperthermic responses. At 30 degrees C, dose-related hyperthermia was the usual response with the exception of meperidine which produced only hypothermia, although a temperature increase was observed after anileridine, a closely related phenylpiperidine. There is good correlation between the relative potencies of the agonists with respect to their hypothermic effects in mice and their relative potencies as analgesics in mice. The temperature effects of morphine are complex but appear to be characteristic of opiate agonists as a class. The magnitude and the direction of the temperature responses to opiates are dose-dependent and profoundly influenced by the environmental temperature.

In a recent paper from our laboratory [Mol. Pharmacol 21:538-547 (1982)] evidence was presented which suggested that [3H]leucine enkephalin labels a single class of binding sites (the enkephalin receptor) and that morphine allosterically induces a masking of enkephalin receptors as a consequence of binding to a receptor (the morphine receptor) not labeled by the 3H-peptide. Evidence is presented in this paper that [3H]etorphine can be used to label selectively the morphine receptor and that the inhibitory dissociation constants (KI) of morphine, etorphine, and human beta-endorphin for the [3H]etorphine binding site closely approximate the concentration of these drugs which produce a half-maximal decrease in the number of enkephalin receptors. Furthermore, an examination of the interaction of leucine enkephalin and methionine enkephalin with the morphine receptor has demonstrated that the pentapeptides are not competitive inhibitors of [3H] etorphine binding, and that they have much lower affinities for the morphine receptor than previously thought. On the basis of these data, a working hypothesis has been formulated which postulates that distinct morphine and enkephalin receptors coexist in an opioid-receptor complex.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with the opiate agonist etorphine resulted in a decrease in both opiate receptor density (receptor down-regulation) and opiate ability to inhibit prostaglandin E1 (PGE1)-stimulated increases in cyclic AMP levels (receptor desensitization). Opiate receptor down-regulation and desensitization were homologous as indicated by the lack of apparent change in muscarinic, alpha 2-adrenergic, and PGE1 receptor binding and also retention, albeit modulation, of the ability of carbachol and norepinephrine to inhibit PGE1-stimulated increases in cyclic AMP levels after 24 hr of etorphine treatment. PGE1-stimulated increases in cyclic AMP levels remained identical in etorphine-treated and control cells. Several lines of evidence indicate that receptor desensitization and receptor down-regulation in NG108-15 cells are two separate cellular adaptation processes. (a) With an agonist which appears to be efficiently coupled, i.e., an agonist whose apparent Kd value is much larger than its apparent IC50 value for regulation of cyclic AMP levels (Ki), the concentration of ligand required to produce half-maximal down-regulation is analogous to its Ki value, whereas the concentration of ligand required to produce half-maximal desensitization is related to its Kd value; (b) receptor desensitization precedes receptor down-regulation; (c) only opiate agonists could produce receptor down-regulation, whereas both opiate agonists and partial agonists could desensitize post-receptor occupancy events. Still further evidence for dissociability of these processes was obtained by incubating NG108-15 cells with etorphine at 30 degrees for 2 hr. Under these conditions, there was a decrease in etorphine's ability to regulate adenylate cyclase while [3H]diprenorphine binding remained unaltered. IC50 values of D-Ala2-D-Leu5-enkephalin's competition for [3H]diprenorphine binding to intact cells increased 19.6-fold after etorphine treatment for 90 min, while naloxone IC50 values remained unaltered. This apparent increase in IC50 values was much lower, about 2-fold, when receptor binding was carried out in membranes isolated from cells treated with etorphine chronically. Furthermore, analysis of [3H]etorphine binding to such membranes in the presence of 10 mM Mg2+ indicated a loss of receptor binding sites with no change in apparent affinity, whereas [3H]diprenorphine binding revealed no significant alteration in either Bmax or Kd values. Therefore, during opiate receptor desensitization, a reduction of agonist high-affinity site occurs with no apparent alteration in total receptor number.

Analgesic ED50 values were determined for s.c. morphine, etorphine, heroin and methadone in mice implanted for 3 days with etorphine pellets. Tolerance to morphine (9-fold) was greater than to the other agents (2- to 4-fold). These results agreed with the previously reported unidirectional non-cross-tolerance effect seen after morphine pellet implantation where tolerance to the hydrophilic agent (morphine) was greater than for the more lipophilic agents (etorphine and heroin). As greater tolerance was found to s.c. morphine than for i.c.v. morphine, this manifestation of tolerance was described as a dispositional tolerance. Experimentally, we were able to observe two additional manifestations of tolerance. After pellet removal, the ED50 values for s.c. morphine, heroin and methadone initially increased before returning to control values. Similarly, both morphine and etorphine ED50 values, after i.c.v. administration, initially increased after pellet removal. Because this second manifestation of tolerance occurred during the rapid disappearance of the tolerance-induction drug, it was called withdrawal tolerance. The third manifestation of tolerance, seen after 3 days of pellet implantation, was characterized by the animal's return to a normal nociceptive sensitivity (control tail-flick latency) in the continued presence of the narcotic pellet. Because this adjustment involved an adaptation by the mice to high brain concentrations of narcotic, this third manifestation of tolerance was designated as a physiologic or homeostatic tolerance. The lability of this new homeostatic state (physiologic tolerance) may be associated with an altered sensitivity to naloxone as evidenced by naloxone-induced jumping (precipitated withdrawal).

The effects of naloxone and three oripavine derivatives, diprenorphine, an antagonist, buprenorphine, a mixed agonist-antagonist, and etorphine, an agonist, were examined on food-reinforced responding in squirrel monkeys. Behavior was maintained under a multiple-component 1-min variable-interval schedule in which 12-min periods of unpunished responding alternated with 4-min periods in which each response produced a brief electric shock to the tail. Daily sessions were 1 hr. Responding in the two components was not differentially affected by any of the drugs. Naloxone decreased responding in both components only slightly at high doses (ED50 greater than 10 mg/kg). In contrast, the three oripavines produced prominent dose-related decreases in responding with the following order of potency for the nonpunishment component: etorphine (ED50 = 0.0005 mg/kg) greater than buprenorphine (ED50 = 0.02 mg/kg) greater than diprenorphine (ED50 = 0.11 mg/kg). Etorphine had a short duration of action (approximately 1 hr) whereas the actions of diprenorphine (1.0 mg/kg) and buprenorphine (0.1 mg/kg) persisted for 24 to 48 hr. Concurrent administration of naloxone (0.1 and 1.0 mg/kg) antagonized the response rate-decreasing effects of etorphine and buprenorphine in a dose-dependent manner (i.e., dose-response curves were shifted to the right) but failed to block the effects of diprenorphine. Although all three oripavines produced comparable decreases in food-reinforced responding, there are qualitative as well as quantitative differences between the drugs. Diprenorphine appears to act through a different mechanism from that of buprenorphine and etorphine.

Systemic administration of beta-funaltrexamine (beta-FNA) 24 hr before analgesic testing produced approximately a 10-fold parallel shift in the dose-response curves of the prototypic mu agonists morphine, I-methadone, fentanyl and etorphine in the mouse abdominal constriction test. In contrast, prior administration of beta-FNA produced no appreciable shift in the analgesic dose-response curve of the selective kappa agonist, U-50, 488H. These results suggest that beta-FNA is selective for mu over kappa receptors under the conditions used in this study. The dose-response curves for ethylketazocine and proxorphan were affected only to a small extent by beta-FNA pretreatment, suggesting that these compounds have analgesic actions mediated primarily through nonmu, probably kappa receptors. The dose-response curves for cyclazocine, buprenorphine, butorphanol, nalorphine and nalbuphine were shifted markedly to the right and frequently not in a parallel fashion by the prior administration of beta-FNA. These results seem to indicate a major role for the mu receptor in the analgesic actions of these compounds.

The development of selective tolerance, that is, a loss in the ability of an agonist to exert an effect without concomitant loss in the ability of an agonist which acts through another receptor type to similarly lose its effectiveness, has provided supporting evidence for the existence of multiple opioid receptor types in brain and peripheral tissues. In brain, this phenomenon has generally been demonstrated for agonists which produce different physiological effects. In this study, we describe selective tolerance at two opioid receptor types which converge upon a single function. The effects of chronic treatment for 6 days with the mu agonist morphine (1.7 mg/kg/hr) and the kappa agonist U50, 488H (300 micrograms/kg/hr) on the ability of mu and kappa opioid agonists to inhibit the stimulated release of [3H]norepinephrine from slices of guinea pig cortex were investigated. Mu, delta and kappa selective agonists have been shown previously to be capable of regulating the stimulated release of norepinephrine. Chronic administration of morphine resulted in a diminution in the ability of the mu agonists Tyr-D-Ala-Gly-N(Me)Phe-Gly-ol, morphine and etorphine, but not of U50, 488H, to inhibit the stimulated release of [3H]norepinephrine. Conversely, chronic U50, 488H infusion decreased the ability of U50, 488H to inhibit release, with no change in the effectiveness of Tyr-D-Ala-Gly-N(Me)Phe-Gly-ol. The degree of tolerance observed for mu agonists in tissue from morphinized animals is discussed with regard to their efficacy and selectivity.

The modulatory effect of continued activation of opiate receptors with agonist on the receptor level was examined in current studies. Rats were rendered tolerant to etorphine by s.c. implantation of osmotic minipumps containing 3 mg/ml of etorphine for up to 7 days. During this period, there were a time-dependent increase in the AD50 values of etorphine to inhibit the tail-flick response and an increase in naloxone-precipitated withdrawal signs. When these animals and others were sacrificed and the opiate receptor binding properties were examined, it could be demonstrated that there was also a time-dependent decrease in the amount of [3H]diprenorphine specifically bound, with maximal attenuation reached 3 days after implantation. There was no alteration in alpha 2 adrenergic receptor binding. This observed decrease in [3H]diprenorphine binding was not due to the presence of nonwashed etorphine in the membrane, for acute administration of the same dose of etorphine before sacrificing did not produce an attenuation of the opiate receptor binding. Further examination of opiate receptor binding in the brain regions of cortex, midbrain and striatum revealed the greatest decrease in the amount of [3H]diprenorphine bound in striatal region after chronic etorphine treatment, a 68% decrease. When the relative decrease in mu and delta opioid receptor binding was determined by carrying out [3H]-D-Ala2, DLeu5-enkephalin binding in the presence of 1 microM morphiceptin, it was observed that, 3 days after etorphine treatment, there was a decrease in mu opioid receptor binding, with minimal change in delta opioid receptor binding in both brain regions of striatum and midbrain. Only in cortex was a decrease in binding of both receptor subtypes observed.(ABSTRACT TRUNCATED AT 250 WORDS)

A portion of the specific binding of tritiated SKF-10047 to the guinea-pig brain suspension of the particulate fraction is not inhibited by the strong narcotic analgesic l-etorphine. The binding properties of these etorphine-inaccessible (EI) sites were examined. The specific binding of [3H]SKF-10047 to the EI sites is saturable. Scatchard analysis of the saturation curve revealed a single class of binding sites with apparent Kd of 252 nM and an estimated Bmax of 663 fmol/mg of protein. The EI binding was reduced by heat treatment, trypsin digestion and phospholipase C digestion. The presence of sodium ions slightly increased specific EI binding. Lithium ion increased the EI binding by about 38% at the optimal concentration of 1 mM. Divalent cations such as Mg++, Ca++ and Mn++ reduced EI binding. Morphine-like drugs such as morphine, levorphanol and naltrexone were poor inhibitors for the EI binding, whereas opioid derivatives such as pentazocine, dextrallorphan, cyclazocine, SKF-10047 and dextrorphan were potent inhibitors. Nonopioid drugs such as haloperidol, imipramine, pimozide and propranolol were also potent inhibitors of the EI binding. Distribution of the EI sites in brain was different from that of the mu receptor: highest concentration of EI sites was found in brainstem, midbrain and cerebellum, whereas lower concentrations were found in striatum and cortex. It is suggested that EI sites are not mu receptors but may represent sigma receptors in the central nervous system, mediating psychotomimetic effects of several opioids and other drugs.

Treatment of the isolated mouse was deferens with the enzyme arylsulfatase (E.C. 3.1.6.1) had an effect on the ability of this tissue to respond to various opiates. It increased the IC50 values and slopes of their dose-response curve for enkephalins and their analogs, and shifted to the right the curves for FK33824, levorphanol and normorphine. There was no effect on the action of etorphine, beta-endorphin or dynorphin. With morphine there was a biphasic effect, IC50 values increasing at low enzyme concentrations and decreasing at high enzyme concentrations. A further comparison of arylsulfatase effects on morphine and on D-Ala-2-D-Leu5-enkephalin indicated that the morphine effect, unlike the D-Ala-2-D-Leu5-enkephalin effect, could not be reversed by washing and that morphine, unlike D-Ala2-D-Leu5-enkephalin, became much less sensitive to naloxone antagonism. The observed modifications in the shape of the dose-response curves indicate that the effect of the opiates in the mouse vas deferens is more complex than that expected through the occupation of a simple receptor. There is either more than one type of functional receptor for each agonist or only one receptor, which can interact with every drug in different ways; this complexity is discussed in terms of various possibilities, including fractional occupancy, positive cooperativity and multiple sites.