Opioid agonist efficacy predicts the magnitude of tolerance and the regulation of mu-opioid receptors and dynamin-2.

It has been proposed that opioid agonist efficacy may play a role in tolerance and the regulation of opioid receptor density. To address this issue, the present studies estimated the in vivo efficacy of three opioid agonists and then examined changes in spinal mu-opioid receptor density following chronic treatment in the mouse. In addition, tolerance and regulation of the trafficking protein dynamin-2 were determined. To evaluate efficacy, the method of irreversible receptor alkylation was employed and the efficacy parameter tau estimated. Mice were injected with the irreversible mu-opioid receptor antagonist clocinnamox (0.32-25.6 mg/kg, i.p), and 24 h later, the analgesic potency of s.c. morphine, oxycodone and etorphine were determined. Clocinnamox dose-dependently antagonized the analgesic effects of morphine, etorphine and oxycodone. The shift to the right of the dose-response curves was greater for morphine and oxycodone compared to etorphine and the highest dose of clocinnamox reduced the maximal effect of morphine and oxycodone, but not etorphine. The order of efficacy calculated from these results was etorphine>morphine>oxycodone. Other mice were infused for 7 days with oxycodone (10-150 mg/kg/day, s.c.) or etorphine (50-250 microg/kg/day, s.c.) and the analgesic potency of s.c. morphine determined. The low efficacy agonist (oxycodone) produced more tolerance than the high efficacy agonist (etorphine) at equi-effective infusion doses. In saturation binding experiments, the low efficacy opioid agonists (morphine, oxycodone) did not regulate the density of spinal mu-opioid receptors, while etorphine produced approximately 40% reduction in mu-opioid receptor density. Furthermore, etorphine increased spinal dynamin-2 abundance, while oxycodone did not produce any significant change in dynamin-2 abundance. Overall, these data indicate that high efficacy agonists produce less tolerance at equi-effective doses. Furthermore, increased efficacy was associated with mu-opioid receptor downregulation and dynamin-2 upregulation. Conversely, lower efficacy agonists produced more tolerance at equi-effective doses, but did not regulate mu-opioid receptor density or dynamin-2 abundance. Taken together, these studies indicate that agonist efficacy plays an important role in tolerance and regulation of receptors and trafficking proteins.

A previously unidentified acepromazine metabolite in humans: implications for the measurement of acepromazine in blood.

High-performance liquid chromatography-diode-array detection results obtained during the investigation of two cases involving acepromazine prompted us to study the stability of the drug in blood. It was found that acepromazine can undergo in vitro conversion by human red blood cells to 2-(1-hydroxyethyl)promazine, a product that has been reported as a minor urinary metabolite in horse urine but not previously identified in humans. Further, our analytical findings in the two cases examined suggest that 2-(1-hydroxyethyl)promazine may be the major unconjugated metabolite of acepromazine in humans. These findings have important implications for the analytical toxicology of acepromazine.

Analysis of etorphine in postmortem samples by HPLC with UV diode-array detection.

Etorphine is a synthetic narcotic analgesic usually used in veterinary medicine. It possesses an analgesic potency up to 1000 times greater than morphine and is therefore used in low doses, primarily for tranquilising large animals. For veterinary use, etorphine is usually available in its commercial formulation as Immobilon, when in combination with acepromazine or methotrimeprazine. Due to the potency of etorphine, only very low doses are required to produce adverse or fatal effects. This paper describes a method for detecting and quantifying etorphine using HPLC with UV diode array detection (HPLC-DAD) and demonstrates the advantage of the technique for the detection of Immobilon at low doses. In a forensic case involving Immobilon, the etorphine concentrations measured in postmortem femoral vein and heart blood specimens were 14.5 and 23.5 micrograms/l, respectively. No etorphine was detected in the urine. To our knowledge this is the first time postmortem etorphine concentrations have been reported.

Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.

The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.

Identification of serine 356 and serine 363 as the amino acids involved in etorphine-induced down-regulation of the mu-opioid receptor.

Agonist-induced internalization of G protein-coupled receptors is influenced by many structural determinants including the carboxyl tail. To investigate the role of serine and threonine residues within the carboxyl tail, several mutants were constructed by truncating the carboxyl tail of the hemagglutinin-tagged mu-opioid receptor, thereby removing serines and threonines systematically. Neuro2A cells stably expressing the truncated receptors did not exhibit a significant alteration in the affinity of [3H]diprenorphine or etorphine for the receptor or the potency of etorphine to inhibit forskolin-stimulated adenylyl cyclase activity. Chronic etorphine treatment resulted in a time-dependent down-regulation of all the truncated receptors, except MOR1TAG355D, thus revealing the importance of the four amino acids between Ser355 and Glu359 (STIE). Surprisingly, deletion of the STIE sequence resulted in a receptor that down-regulated the same as the wild-type receptor. The involvement of multiple amino acids within the carboxyl tail was demonstrated by combining alanine substitutions of several putative G-protein-coupled receptor kinase phosphorylation sites. Systematic analysis of these receptors indicated that mutation of Ser356 and Ser363 to alanine attenuated agonist-mediated down-regulation. The magnitude of etorphine-induced phosphorylation of this mutant receptor, however, was similar to that of the wild-type mu-opioid receptor. Thus, phosphorylation of the carboxyl tail of the mu-opioid receptor is not an obligatory event for etorphine-induced down-regulation of the receptor.

Agonist-induced signaling and trafficking of the mu-opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C-terminal domain.

The human mu-opioid receptor and a mutant form, muS/ T[i3+Cter]A, in which all Ser and Thr residues from the third cytoplasmic loop and C-terminal domain were changed to Ala, were studied after expression in CHO-K1 cells. Although the mutant receptors had similar affinities for agonists and EC50 values for inhibition of adenylyl cyclase as compared to wild-type receptors, the Emax were almost 2-fold decreased, suggesting a role of the mutated residues in G-protein coupling. After chronic morphine or etorphine, the EC50 values of the agonists were about 5-fold increased at both receptors but the Emax values were not altered; upon agonist withdrawal forskolin-stimulated cAMP levels were increased to almost 200% of control levels. Sequestration and rapid down-regulation of the mu-opioid receptor were induced by DAGO and etorphine but not morphine. In contrast, the muS/T[i3+Cter]A receptor was not sequestered and was up-regulated (150-380%) after treatment with agonists. The results indicate that the Ser and Thr residues in the third cytoplasmic loop and C-terminus of the mu-opioid receptor are not involved in the limited desensitization or in the adenylyl cyclase superactivation promoted by agonists but that their integrity and/or their phosphorylation is required in the intricate and coordinately regulated pathways involved in receptor signaling and trafficking.

Replacement of Gln280 by His in TM6 of the human ORL1 receptor increases affinity but reduces intrinsic activity of opioids.

The ORL1 (Opioid Receptor-Like) receptor is the G protein-coupled receptor whose amino acid sequence is closest to those of opioid receptors. Residues that are conserved in ORL1 and the three types of opioid receptor, but also a residue, His in the sixth putative transmembrane (TM6) helix, which is present in all opioid receptor types but absent in ORL1, appear to play a key role in receptor recognition and/or activation. Here we have sought to create an opioid binding pocket in the non-opioid ORL1 receptor by replacing residue Gln280 in its TM6 by the corresponding His residue of opioid receptors. The mutation affects neither the affinity of nociceptin - the natural ORL1 agonist - for the receptor, nor the potency of nociceptin to inhibit adenylyl cyclase via ORL1. In contrast, we find that a few opioid ligands, the agonists lofentanil, etorphine and dynorphin A, and especially the antagonists diprenorphine and nor-BNI, bind the mutant Q280H receptor with substantially (5- to > 100-fold) higher apparent affinity than they do the wild-type receptor. Moreover, lofentanil and etorphine no longer act as pure agonists, as they do at the native ORL1 receptor, but are endowed with clear antagonist properties at the mutant receptor. The mutation Q280H, which increases affinity while decreasing intrinsic activity of opioids at ORL1, emphasizes the importance of the His residue for opioid recognition and activation.

[Binding of [3H] dihydroetorphine to opioid receptors in rat brain membrane].

AIM: To study the binding characteristics of dihydroetorphine (DHE) to opioid receptors. METHODS: The binding of [3H] DHE to rat brain membrane opioid receptors was observed by radioligand binding study. RESULTS: Saturation study showed the binding of [3H] DHE to rat brain membrane presented a single high-affinity class of binding sites with Kd = 0.19 nmol.L-1 and Bmax = 115 pmol/g protein. Kinetics study showed the association rate of [3H] DHE to opioid receptor was very rapid and dissociation very slow. NaCl 100 mmol.L-1 + guanosine triphosphate (GTP) 50 mumol.L-1 inhibited the binding of [3H] DHE and made Kd increase to 7.87 nmol.L-1, without effect on Bmax. Competitive inhibition of agonists and antagonists indicated that the affinity of [3H]DHE to mu opioid receptor was higher than to delta and kappa opioid receptors. CONCLUSION: DHE is a selective agonist of mu opioid receptors.

Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.

Further characterization of [3H]U69593 binding sites in the rat heart.

K binding sites in the crude membrane preparation of the rat heart homogenate were further characterized by a displacement binding assay of [3H]-U69593 with specific kappa ligands and a direct binding assay with [3H]-etorphine. Scatchard analysis of specific [3H]-U69593 binding showed that the Kd and Bmax were 6.4 +/- 1.0 nM and 97 +/- 8 fmol/mg protein. respectively. The binding of [3H]-U69593 was effectively displaced by the selective kappa 1 ligands, U-69593 and U-50488H, but only weakly displaced by Met5-enkephalin-Arg6-Phe7, a selective kappa 2 ligand, which showed only 11 +/- 3% inhibition of [3H]-U69593 binding at the concentration of 1 microM. In addition, there was no binding site for [3H]-etorphine, known to bind to mu, delta and kappa 2 binding sites, but not kappa 1 binding sites. The findings suggest that the kappa binding sites in the rat heart most likely belong to the kappa 1 subtype. The binding sites have high and low affinity components as nonlinear regression analysis of the competition curves is best fit by two components with IC50 values of 11 +/- 2 and 62 +/- 7 nM for U-69593, and 9.9 +/- 1.5 and 414 +/- 108 nM for U-50488H. Furthermore, the binding of [3H]-U69593 were inhibited by both monovalent cations (Na+, Li+) and divalent cations (Mg2+, Mn2+ and Ca2+).

Dihydroetorphine is a mu-receptor-selective ligand.

The selectivity to opioid receptors of dihydroetorphine, a potent analgesic with only mild physical dependence, was investigated using radioligand binding assay and its analgesic activity in mice determined. The relative affinity ratio of dihydroetorphine to mu-, delta- and kappa- opioid receptors was 333:1:1. The analgesic effect of intracerebro-ventricular injection in mice could be antagonized by the mu-antagonist beta-funaltrexamine but could not be antagonized by delta- and kappa-selective antagonists naltrindole and norbinaltorphimine. We conclude that dihydroetorphine is a selective ligand for the mu-opioid receptor.

Different distributions of opioid receptors in spontaneously hypertensive rats and Wistar-Kyoto rats.

AIM: To compare the densities of opioid receptors in spontaneously hypertensive rats (SHR) with those of normotensive Wistar-Kyoto (WKY) rats in central nervous system which are related to the regulation of BP. METHODS: [3H] Etorphine, a nonspecific opioid ligand, was used to determine the distributions of opioid receptors in 16-wk-old SHR and WKY rats by quantitative autoradiography. RESULTS: The densities of [3H]etorphine in hippocampus (P < 0.01), periaqueductal gray, nucleus of the solitary tract, and thoracic (T4-6) spinal cord (P < 0.05) of SHR were lower than those of WKY rats. But in basolateral amygdaloid nucleus (P < 0.01), habenular nuclei (P < 0.05), and hypothalamic nuclei including arcuate nucleus (P < 0.01), higher densities of opioid receptors were found in SHR. No difference existed in interpeduncular nuclei between the 2 groups. CONCLUSION: The difference in distributions of opioid receptors is related to the hypertension in SHR.

Opioid receptor interaction and adenylyl cyclase inhibition of dihydroetorphine: direct comparison with etorphine.

To find out the reason of weak addiction property of dihydroetorphine, we compared the affinities of dihydroetorphine to the type selective opioid receptor and inhibition effect on the adenylyl cyclase activity with those of etorphine. Dihydroetorphine and etorphine have almost the same binding affinities to all types (mu, delta, and kappa) of opioid receptors and antagonist binding sites, and have similar inhibition activities to forskolin stimulated adenylyl cyclase. However, dihydroetorphine showed significantly smaller value of DTNB-index compared with that of etorphine. This differentiation may explain partly the high analgesic with low dependence properties of dihydroetorphine.

[The potentiation effect of haloperidol on the binding of etorphine to brain membranes in acupuncture analgesia].

[3H]-etorphine was used in receptor binding studies on membrane preparation from rabbit brain. Scatchard analysis revealed that rabbit brain had two different affinity binding sites. In the high affinity site, the dissociation constant (Kd1) of the control group was 2.57 +/- 0.33 nmol/L. When analgesia was induced by haloperidol or acupuncture, the Kd1 values decreased respectively (P < 0.05) to 1.44 +/- 0.03 nmol/L and 1.53 +/- 0.05 nmol/L. These Kd1 values further decreased to 1.25 +/- 0.03 nmol/L when acupuncture analgesia was potentiated by combined action of acupuncture and i.v. haloperidol (P < 0.01). In case of autoradiographic analysis of brain slices showed that the density of opioid receptors in many brain regions, such as N. Caudatus, preoptic lateral area, N. Paraventricularis, N. Centromedianus, Periaqueductal gray, showed significant increase, a results indicating that the upregulation of opioid receptors in rabbit brain may be one of the mechanisms in the potentiating action of haloperidol on acupuncture analgesia.

Receptor-mediated regulation of neuropeptide gene expression in astrocytes.

One of the functions of glial receptors is to regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn act on neurons or other glia. Because of the potential importance of these interactions in injured brain, we have examined the role of two different receptors in the regulation of astrocyte neuropeptide synthesis. Stimulation of beta-adrenergic receptors on type 1 astrocytes resulted in increased mRNA and protein for the proenkephalin (PE) and somatostatin genes. This receptor also increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The potential role of opiate receptors was examined in several ways. Treatment of newborn rats for 7 days with the opiate antagonist naltrexone, prior to preparation of astrocytes, had no effect on PE mRNA or met-enkephalin content but resulted in a significant increase in NGF content. However, treatment of astrocytes in culture with met-enkephalin, morphine, or naltrexone had no effect on any of these parameters. No opiate binding could be detected, using either etorphine or bremazocine, to membranes of astrocytes prepared from cortex, cerebellum, striatum, or hippocampus of 1-day, 7-day, or 14-day postnatal rats. Thus we conclude that type 1 astrocytes do not express opiate receptors and that the in vivo effects of naltrexone are mediated indirectly via some other cell type/receptor.

SK-N-BE: a human neuroblastoma cell line containing two subtypes of delta-opioid receptors.

A human neuroblastoma cell line, SK-N-BE, was shown to express a substantial amount of opioid receptors (200-300 fmol/mg of protein). A ligand binding profile of these receptors revealed that they could belong to two distinct subtypes of delta-opioid receptors. Results from sucrose-gradient sedimentation experiments were compared with similar data obtained with the mu-opioid receptor of the rabbit cerebellum and the delta-opioid receptor of the hybrid NG108-15 cell line and have shown that the opioid receptor of the SK-N-BE cell line behaved hydrodynamically as an intermediate between mu- and delta-opioid receptors. Taken together, pharmacological and hydrodynamic studies suggest that the opioid receptors present in the SK-N-BE cell membranes could belong to two delta-opioid receptor subtypes interacting allosterically. Functional experiments suggest that at least one of these subtypes of delta-opioid receptor is negatively coupled to the adenylate cyclase via a Gi protein and that the opiate receptors of the SK-N-BE neuroblastoma cell line undergo a rapid down-regulation when preincubated in the presence of the high-affinity opioid, etorphine.

Stereochemical requirements for pseudoirreversible inhibition of opioid mu receptor binding by the 3-methylfentanyl congeners, RTI-46144 and its enantiomers: evidence for different binding domains.

Fentanyl and its congeners are of interest not only because of their clinical applications, but also because certain members of this series of opioid analgesics exhibit unique properties, such as acting as pseudoirreversible inhibitors of mu receptor binding, both in vitro and in vivo. Previous studies showed that pretreatment of membranes with (+)-cis-3-methylfentanyl resulted in a lower affinity interaction of [3H]ohmefentanyl with the mu binding site, as well as an increased dissociation rate. The present study was undertaken to determine the stereochemical requirements for pseudoirreversible inhibition of mu receptor binding using the methylfentanyl congeners, (+-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl (RTI-4614-4) and its four resolved enantiomers. AR configuration of the 2-hydroxy group was essential for high affinity binding and pseudoirreversible inhibition. The two enantiomers with this configuration, 1b((2R,3R,4S)-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide oxolate) and 1c 1c ((2R,3S,4R)-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl), acted as pseudoirreversible inhibitors of the mu receptor as labeled with [3H][D-Ala2-MePhe4,Gly-ol5]enkaphalin, [3H]fentanyl or [3H]etorphine. RTI-4614-4, 1b, and 1c decreased the Bmax of [3H][D-Ala2-MaePhe4,Gly-ol5]enkepalin binding sites without altering the dissociation rate. These drugs had a lesser effect on steady-state [3H]fentanyl and [3H]etorphine binding but did produce statistically significant changes in the parameters of the two-component dissociation model, which accurately described the dissociation of these [3H]ligands. Viewed collectively, these data indicate that the mechanism of the pseudoirreversible inhibition appears to depend on the radioligand used to label the mu receptor. To explain these data, a pseudoallosteric model is proposed that postulates that certain mu ligands bind to different domains of the drug recognition site of the mu receptor and that the prebinding of pseudoirreversible inhibitors to the recognition site changes the domains available to a radioligand, leading to alterations in steady-state binding levels and dissociation kinetics.

Characterization and distribution of mu opioid receptors in rabbit cerebellum.

The binding characteristics and distributions of [3H]ohmefentanyl ([3H]OMF), [3H]etorphine, [3H] N-methyl-N-[7-(I-pyrrolidinyl)-1-oxaspiro(4,5)dec-7-yl] benzeneacetamide [3H]U-69593), and [3H] (D-penicillamine2, D-penicillamine5) enkephalin ([3H]DPDPE) in rabbit cerebellum were studied. In saturation experiment, [3H]OMF bound to a single population of sites with Kd = 2.2 +/- 1.3 nmol.L-1 and Bmax = 69 +/- 13 fmol/mg protein. The results of [3H]etorphine (Kd = 1.0 +/- 0.4 nmol.L-1 and Bmax = 16 +/- 6 fmol/mg protein) were similar to those of [3H]OMF. There were no specific bindings of [3H]U-69593 and [3H]DPDPE in rabbit cerebellar membranes. In autoradiographic study, the gray scales of specific binding for [3H]OMF, [3H]etorphine, [3H]U-69593, and [3H]DPDPE were 20.2 +/- 4.6, 8.5 +/- 2.7, 2.0 +/- 0.7, and 3.2 +/- 4.3, respectively. The gray matter of rabbit cerebellum was intensely labeled by [3H]OMF and [3H]etorphine, but not by [3H]U-69593 or [3H]DPDPE. These results suggested that the rabbit cerebellum contained mu opioid receptors.

Altered transition between agonist- and antagonist-favoring states of mu-opioid receptor in brain membranes with modified microviscosity.

In unmodified synaptosomal brain membranes the presence of NaCl inhibited the binding to mu receptors of the tritiated opioid agonists etorphine, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and sufentanil by 53, 43, and 37%, respectively, and increased that of the antagonist [3H]naltrexone by 54%. On the other hand, in membranes whose microviscosity was increased by incorporation of cholesteryl hemisuccinate (CHS) the effects of sodium on opioid agonist and antagonist binding were abolished and strongly reduced, respectively. Furthermore, in the modified membranes the ability of sodium to protect the opioid receptor from inactivation by the sulfhydryl-reactive agent N-ethylmaleimide (NEM) was diminished. In CHS-treated membranes whose elevated microviscosity was reduced by the incorporation of oleic acid, the effectiveness of sodium in modulating opioid binding and attenuating receptor inactivation by NEM was restored. The results implicate membrane microviscosity in the mechanism by which sodium modulates the conversion between agonist- and antagonist-favoring states of mu opioid receptor.

Solubilization of high-affinity, guanine nucleotide-sensitive mu-opioid receptors from 7315c cell membranes.

High-affinity mu-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [3H]-etorphine with high affinity (KD = 0.304 +/- 0.06 nM; Bmax = 154 +/- 33 fmol/mg of protein). Of the membrane-associated [3H]etorphine binding sites, 40 +/- 5% were recovered in the solubilized fraction. Both mu-selective and non-selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, delta- and kappa-opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of < 1 microM. The mu-selective ligand [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin also bound with high affinity (KD = 0.79 nM; Bmax = 108 +/- 17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin binding sites, 43 +/- 3% were recovered in the solubilized material. Guanosine 5'-O-(3-thiotriphosphate), GTP, and guanosine 5'-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [3H][D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin binding in a concentration-dependent manner. Finally, mu-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate.