Analysis of binding domain and function of chimeric mu/kappa opioid receptors to ohmefentanyl stereoisomers.

AIM: To investigate specific domains in mu opioid receptors that accounted for selective binding of three stereoisomers of ohmefentanyl (Ohm9204, Ohm9202, and Ohm9203) and study the function of chimera II. METHODS: Rat mu and kappa opioid receptors (RMOR, RKOR) and four mu/kappa chimeric receptors (chimeras) I, II, III, and IV were transiently expressed in COS-1 cells. The binding ability and binding domain of receptor to ligands were determined by radioactive ligand and receptor binding experiments. Through measuring cellular cAMP levels, we studied the function of chimera II in mediating signal transduction. RESULTS: Binding affinities of four chimeric receptors were similar to wild type opioid receptors (RMOR and RKOR). The binding affinities of Ohm9204 and Ohm9202 to chimera II were similar to that of RMOR. The binding affinities of Ohm9203 to all six receptors were low. U50488 possessed high binding affinity to chimera I, however dynorphie A(1-9) had some binding affinity to chimera II that was similar to RKOR, which indicated the domains of RKOR accounting for selectively binding to peptide ligand dynorphie A(1-9) and nonpeptide ligand U50488 were different. The efficacy of Ohm9204 and Ohm9203 on inhibiting forskolin-stimulated cAMP accumulation in cells transfected with chimera II was similar to that in cells transfected with RMOR. CONCLUSION: Replacing 194-268 residues of RMOR with 185-262 residues of RKOR does not influence the ability of mu opioid receptor to bind Ohm9204 and Ohm9202 and the receptor mediated inhibition of cellular cAMP level.

Analgesic activity and opioid receptor selectivity of stereoisomers of ohmefentanyl isothiocyanate.

Ohmefentanyl is a very potent and highly selective agonist for mu-opioid receptors. We now study analgesia, in vitro activity and opioid receptor affinity of the stereoisomers of ohmefentanyl isothiocyanate. We found that some isomers of ohmefentanyl isothiocyanate had a potent analgesic effect and that all isomers except (3R,4S,2'S)-ohmefentanyl isothiocyanate had a more potent inhibitory action on the electrically evoked contractions of mouse vas deferens than of guinea pig ileum. The inhibitory actions could be antagonized by naloxone. However, compared with the activity of the corresponding stereoisomers of ohmefentanyl, these ohmefentanyl isothiocyanates had significantly reduced analgesia and in vitro activity. They also inhibited the binding of [3H]DPDPE ([D-Pen(2),D-Pen(5)]enkephalin) and [3H]DAGO ([D-Ala(2),Mephe(4),Gly-ol(5)]enkephalin) to opioid receptors in mouse brain membranes. The inhibitory effect of stereoisomers of ohmefentanyl isothiocyanate at mu-opioid receptors was markedly lower than that of their parent compounds. The affinity of stereoisomers of ohmefentanyl isothiocyanate for delta-opioid receptors was, however, greater than or equal to that of their corresponding stereoisomers of ohmefentanyl. The results showed that the introduction of an isothiocyanato group into the phenyl ring in position-1 of ohmefentanyl reduced bioactivity and affinity to mu-opioid receptors but that the selectivity of these compounds for delta-opioid receptors was enhanced. Isomer (3R,4S,2'R)-ohmefentanyl isothiocyanate showed highest selectivity for delta-opioid receptors (K(i)(mu)/K(i)(delta)=13.6) and potent analgesic activity (ED(50)=0.25 mg/kg).

Quantitative comparison of ohmefentanyl isomers induced conditioning place preference in mice.

Differences of analgesia and withdrawal response among ohmefentanyl stereoisomers have been studied. In the present study, Quantitative comparison of reinforcing effects of ohmefentanyl stereoisomers and morphine was performed by using a conditioned place preference design in mice. Results showed that morphine and ohmefentanyl stereoisomers were able to increase significantly the time spent in the drug-paired side with respect to vehicle treated animals. A good linear correlation between doses of drugs and number of mice with place preference was found within a given dose range. On the basis of the dose-response curve analysis, ohmefentanyl stereoisomers displayed a significant difference in place preference ED50. The addictive index (analgesic ED50/place preference ED50) was used to assess the addictive potential of drugs. It was demonstrated that the addictive potential of ohmefentanyl stereoisomers did not exhibit a large difference as addictive index. Among these stereoisomers, the addictive potential of compound F9208 was markedly lower than that of morphine.

Comparison of physical dependence of ohmefentanyl stereoisomers in mice.

Stereo-structural difference of ohmefentanyl stereoisomers on analgesic action and receptor affinity has been studied. To assess the difference of ohmefentanyl stereoisomers in physical dependence, the potency of physical dependence was quantified by estimating the ED50 value of ohmefentanyl stereoisomers in the naloxone-precipitated jumping test in mice. Morphine was used to assess the method and as a drug of comparison. The results indicate that the degree of physical dependence of morphine can been quantified by estimating the ED50 value of morphine withdrawal jumping induced by naloxone. A significant difference was observed in withdrawal jumping ED50 values among ohmefentanyl stereoisomers. Of these isomers, F9202 and F9204 had similarly potent analgesic action, but very significant difference in naloxone precipitated withdrawal response. Dependent potency index of F9204 was 618-fold weaker than that of F9202. It is concluded that a stereo-structural difference in physical dependence is found to exist among ohmefentanyl stereoisomers. Compound F9204 displayed a strong analgesic action and weak physical dependent potency.

Human mu-opioid receptor overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.

Human mu-opioid receptor (HmuOR) with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells. The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by mu-selective agonists [D-Ala2, N-methyl-Phe4, glyol5]enkephalin (DAGO), Ohm, and morphine, but neither by delta nor by kappa selective agonist. Na+ (100 mM) and GTP (50 microM) could reduce HmuOR agonists etorphine and Ohm affinity binding to the overexpressed HmuOR. mu-selective agonists DAGO and Ohm effectively stimulated [35S]GTP-gammaS binding (EC50 = 2.7 nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin (PTX). These results demonstrated that HmuOR overexpressed in Sf9 insect cells functionally coupled to endogenous G(i/o) proteins.

Building 3D-structural model of kappa opioid receptor and studying its interaction mechanism with dynorphin A(1-8).

AIM: To construct the 3D-structural model of human kappa opioid receptor (HKOR) and study its interacting mechanism with dynorphin A(1-8) (Dyn8). METHODS: Comparative molecular modeling was applied to build the 7 transmembrane (TM) helical domain of HKOR using the bovine rhodopsin (OPSD) model as a template. Molecular dynamics was performed to minimize the HKOR model and to simulate the 3D-structure of Dyn8 based on the NMR results of dynorphin A(1-14). The extracellular loops (EL) were built by self-constructed database searching. DOCK4.0 program was performed to construct Dyn8 complex with HKOR. RESULTS: (1) The model of HKOR was obtained and validated by theoretical and experimental data. (2) The Dyn8-HKOR interacting mechanism is reasonably explained: Side chain of residue Asp138 interacts with protonated nitrogen atom at the N-terminal residues of Dyn8 through electrostatic and hydrogen bonding, which play an important role in ligand binding with receptor. (3) Negatively charged amino acids in the second extracellular loop (EL2) as Asp223 and Glu209 interact with the C-terminal positively charged residues in Dyn8, and Glu209 is a likely determinant of peptide ligand specificity. CONCLUSION: Some amino acid residues positioned in EL2, TM3, TM4, and TM5 form the binding site and therefore determine the selectivity of kappa peptide agonist.

Opioid peptide receptor studies, 11: involvement of Tyr148, Trp318 and His319 of the rat mu-opioid receptor in binding of mu-selective ligands.

Previous data obtained with the cloned rat mu opioid receptor demonstrated that the "super-potent" opiates, ohmefentanyl (RTI-4614-4) and its four enantiomers, differ in binding affinity, potency, efficacy, and intrinsic efficacy. Molecular modeling (Tang et al., 1996) of fentanyl derivatives binding to the mu receptor suggests that Asp147, Tyr148, Trp318, and His319 are important residues for binding. According to this model, Asp147 interacts with the positively charged opiate agonist to form potent electrostatic and hydrogen-bonding interactions. In this study, the role of weak electrostatic and hydrogen-bonding "pi-pi" interactions of the O atom of the carbonyl group and the phenyl ring structures of RTI-4614-4 and its four enantiomers with residues Tyr148, Trp318, and His319 were explored via site-directed mutagenesis. Tyr148 (in transmembrane helix 3 {TMH3}), Trp318 (TMH7), and His319 (TMH7) were individually replaced with phenylalanine or alanine. Receptors transiently expressed in COS-7 cells were labeled with [125I]IOXY according to published procedures. Mutation of Tyr148 to phenylalanine reduced the binding affinities of some mu-selective agonists (2-7 fold) but did not alter the affinities of DAMGO, naloxone, and the non-selective opiates etorphine and buprenorphine. In contrast, this mutation significantly increased the binding affinities (decreased the Kd values) of [D-Ala2,D-Leu5]enkephalin, IOXY, and dermorphin. Mutation of Trp318 decreased opioid receptor binding to almost undetectable levels. Substitution of alanine for His319 significantly reduced binding affinities for the opioid ligands tested (1.3- to 48-fold), but did not alter the affinities of naloxone and bremazocine. These results indicate the importance of Tyrl48 and His319 for the binding of fentanyl derivatives to the mu receptor. Functional studies using the mutant receptors will provide additional insight into the mechanism of action of RTI-4614-4 and its four enantiomers.

Leucine-2-alanine enkephalin induced delta opioid receptors internalization expressed stably in CHO cells.

AIM: To characterize the internalization of delta opioid receptors (DOR) stably expressed in Chinese hamster ovary (CHO) cells and the role of the C-terminal in this process. METHODS: Receptor membrane anchoring was shown by immunofluorescence microscopy. Receptor internalization was assessed by measuring the radioligand binding resistant to the acid-buffer wash. RESULTS: Originally, all the wild-type (CHO-W) and C-truncated (CHO-T) DOR expressed were localized to the membrane. Agonist [3H] leucine-2-alanine enkephalin (LAE) but not the antagonist [3H]diprenorphine (Dip) induced rapid receptor internalization. The internalization of C-truncated DOR in CHO-T was similar to that of the wild-type in maximal level, but climbed up more slowly. DOR internalization was extracellular osmolarity- and temperature-sensitive. Pertussis toxin and universal protein kinase inhibitor staurosporine had no effect on it. CONCLUSION: DOR internalization is an agonist and clathrin-coated pits dependent, but post-receptor cellular signal transduction independent process; moreover, the C-terminal of DOR, not engaged in membrane anchoring, affects the initialization of DOR internalization.

Human mu-opioid receptor overexpressed in baculovirus system and its pharmacological characterizations.

AIM: To overexpress human mu-opioid receptor (muOR) with characteristics similar to those of mammalian origin. METHODS: Human muOR with a tag of 6 consecutive histidines at its carboxyl terminus was expressed in recombinant baculovirus infected Sf9 insect cells. Then the pharmacological characterizations of the product were studied by receptor binding assay and cAMP assay. RESULTS: The maximal binding capacity for the [3H]diprenorphine and [3H]ohmefentanyl (Ohm) were 9.1 +/- 0.7 and 6.52 +/- 0.23 nmol/g protein, respectively. The [3H]diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by alpha-selective agonists [D-Ala2, N-methyl-Phe4, glyol5] enkephalin (DAGO), Ohm, and morphine, but neither by the delta-selective agonist [D-Pen2, D-Pen5] enkephalin (DPDPE) nor by the kappa-selective agonist ¿trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]¿ benzacetamide (U50488). NaCl 100 mmol.L-1 and guanosine triphosphate (GTP) 50 mumol.L-1 could reduce mu agonists Ohm and etorphine affinity binding to the expressed muOR. DAGO and Ohm effectively inhibited forskolin-stimulated cAMP accumulation. This agonist-dependent effect was blocked by opioid antagonist naloxone. CONCLUSION: The overexpression of human muOR with a tag of six consecutive histidines at its carboxyl terminus in Sf9 insect cells retained the characteristics of wild-type human muOR.

Molecular modeling of mu opioid receptor and receptor-ligand interaction.

AIM: To construct the 3D structural model of mu opioid receptor (mu OR) and study the interaction between mu OR and fentanyl derivatives. METHODS: The 3D structure of mu OR was modeled using the bacteriorhodopsin (bRh) as a template, in which the alignments of transmembrane (TM) of bRh and mu OR were achieved by scoring the alignment between the amino acid sequence of mu OR and the structure of bRh. The fentanyl derivatives were docked into the 7 helices of mu OR and the binding energies were calculated. RESULTS: (1) The receptor-ligand interaction models were obtained for fentanyl derivatives. (2) In these models, the fundamental binding sites were possibly Asp147 and His297. The negatively charged oxygen of Asp147 and the positively charged ammonium group of ligand formed the potent electrostatic and hydrogen-binding interactions. Whereas the interactions between the positively charged nitrogen of His297 and the carbonyl oxygen of ligand were weak. In addition, there were some pi-pi interactions between the receptor and the ligand. (3) The binding energies of the receptor-ligand complexes had a good correlation with the analgesic activities (-lg ED50) of the fentanyl derivatives. CONCLUSION: This model is helpful for understanding the receptor-ligand interaction and for designing novel mu OR selective ligands.

Binding properties of C-truncated delta opioid receptors.

AIM: To study the role of C-terminal delta opioid receptor involved in ligand binding affinity and selectivity. METHODS: The 31 amino acid residues of C-terminal truncated delta opioid receptors and the wild-type were expressed stably in Chinese hamster ovary (CHO) cells, respectively. Then the ligand binding properties of the products were studied by receptor binding assay. RESULTS: A typical mutated receptor clone CHO-T and a wild-type receptor clone CHO-W were obtained. The Kd values of [3H] diprenorphine (Dip) and [3H]leucine-2-alanine enkephalin (DADLE) bound to CHO-T were similar to CHO-W. Both the specific [3H]Dip bindings of CHO-T and CHO-W were strongly inhibited by delta selective agonists with similar Ki, but neither by mu nor kappa selective agonists. CONCLUSION: The C-terminal of the delta opioid receptor is not involved in the ligands binding affinity and selectivity.

Molecular modeling of interaction between delta opioid receptor and 3-methylfentanylisothiocyanate.

AIM: To construct a 3D structural model of delta opioid receptor (delta OR) and study its interaction with 3-methylfentanylisothiocyanate (SuperFIT). METHODS: Using the bacteriohodopsin as a template, the 3D structure of delta OR was modeled; SuperFIT was docked into its inside. RESULTS: The interaction model between delta OR and (3R, 4S)-SuperFIT was achieved, in which the important binding sites possibly were Asp128, Ser106, Phe104, Tyr308, and Pro315. Asp128 formed the electrostatic and hydrogen-binding interactions with the protonated nitrogen on piperidine of the ligand. Ser106 formed the electrostatic interaction with the N atom of isothiocyano group of the ligand; whereas Phe104, Tyr308, and Pro315 formed the hydrophobic interactions with the S atom of isothiocyano group. In addition, there were some other interactions between delta OR and the ligand. CONCLUSION: The residues Phe104, Tyr308, Pro315, and Ser106 of delta OR are crucial to the delta selectivity of the ligand, which is beneficial for designing novel delta-selective ligand.

[Effects of ohmefentanyl at anesthetic dose on plasma levels of corticosterone, cortisol and antidiuretic hormone in rats].

Ohmefentanyl (OMF) is a new mu opioid receptor agonist with high affinity and selectivity, and possesses anesthetic activity. With radioimmunoassay, the plasma levels of cortisol (C), corticosterone (CS) and antidiuretic hormone (ADH) in rats were measured. The results indicated that no significant differences in the plasma C, CS and ADH levels were observed between the saline control group and the OMF-treated group. Trauma (bone-crush injury) increased significantly the plasma CS level. However, pretreatment with OMF 4.0 micrograms.kg-1 reduced markedly the CS plasma levels in trauma-treated rats. The results suggest that OMF anesthesia itself showed no obvious effect on the plasma concentration of C, CS and ADH, but blocked the hormoral stress responses such as the increment of plasma CS level caused by trauma stimulus.

Analgesic activity and selectivity for opioid receptors of enantiomers of ohmefentanyl.

AIM: To study analgesic activity and selectivity for opioid receptor subtypes of 8 enantomers of ohmefentanyl (Ohm). METHODS: Analgesic activity was evaluated using the hot plate test in mice. Selectivity for opioid receptors was determined in binding assay and bioassay. RESULTS: Among the 8 enantiomers of Ohm, the most potent isomer was F-9204, (+)-cis-(3R, 4S, 2'S)-Ohm, with an analgesic ED50 (i.p.) value of 1.1 (0.8-1.3) micrograms.kg-1. These enantiomers selectively acted on mu opioid receptors. Ki values (mu) of enantiomers F-9204 and F-9202, (-)-cis-(3R, 4S, 2'R)-Ohm were 4.0 +/- 2.0 and 5 +/- 4 pmol.L-1, respectively. Their Ki(delta)/Ki(mu) ratios were 22,500 and 22,800, respectively. On guinea pig ileum and mouse vas deferens F-9202, F-9204, F-9205, F-9206, F-9207, and F-9208 exhibited very potent inhibitons, which were antagonized by naloxone. In rabbit vas deferens these enantiomers had no effect. CONCLUSION: Eight enantiomers of Ohm selectively acted on mu opioid receptors. F-9204 showed the strongest analgesic activity and the highest selectivity for mu opioid receptors.

Molecular modeling of mu opioid receptor and its interaction with ohmefentanyl.

AIM: To build up the structure model of mu opioid receptor, then combined with the receptor model, to investigate the action mechanism of ohmefentanyl on the receptor. METHODS: Using the three-dimensional structure of bacteriorhodopsin as a template, we constructed mu opioid receptor model on computer. Ohmefentanyl was then docked into the supposed receptor binding sites. RESULTS: A good ligand-receptor interaction model was achieved. The possible binding sites were found to be Asp147 and His319. The protonated N atom of ohmefentanyl form potent electrostatic and hydrogen-bonding interactions with residue Asp147 of the receptor, the O atom of the carbonyl group form weak electrostatic and hydrogen-bonding interactions with residue His319, and the two phenyl groups form pi-pi interactions with some aryl residues of the receptor around ligand. CONCLUSION: The ligand-receptor interaction model should be helpful for rational design of novel analgesic.

Stereoisomers of N-[1-hydroxy-(2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide: synthesis, stereochemistry, analgesic activity, and opioid receptor binding characteristics.

N-[1-(2-Hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N-phenylpropanamide (ohmefentanyl,1) is an extremely potent analgesic agent with high affinity and selectivity for opioid mu receptors. There are three chiral carbons in 1, so eight optically active isomers are possible. Respective reaction of optically active 3-methyl-N-phenyl-4 -piperidinamines (5a-d) with (R)- or (S)-styrene oxide produced eight optically active intermediates which were subsequently converted to eight optically active isomers of 1 (1a-h). The absolute configurations of 1a-h were determined by X-ray analysis of (3R,4S,2'R)-(-)-cis-1a and (3R,4R,2'S)-(-)-trans-1g. The analgesic activity (mice, ip, hot plate) revealed their extreme stereodifferences; the ED50 values of (3R,4S,2'R)-(-)-cis-1a and (3R,4S,2'S)-(+)-cis-1b, which are the most potent isomers among eight isomers, were 0.004 65 (2990 times that of morphine) and 0.001 06 mg/kg (13 100 times that of morphine), respectively, while the corresponding antipodes 1d,c were the least potent compounds among the eight isomers. In agreement with pharmacological results, both 1a,b also had the highest receptor affinity and selectivity for the opioid mu receptor. The ratio of K(i)(DPDPE)&K(i)(DAMGO) was 22 800 for 1a and 22 500 for 1b. All isomers except 1c,d strongly inhibited the electrically evoked smooth muscle contraction of GPI and MVD but not that of RVD, and the inhibitory effects could be reversed by naloxone, which indicated that these compounds were potent mu agonists in GPI and MVD. There was a good linear correlation between the analgesic potencies (ED50) and the receptor affinities (K(i)(DAMGO)) or inhibitory effects (IC50) to GPI and MVD. These results suggested that the analgesic effects of ohmefentanyl are mediated by interaction between the agents and opioid mu receptors in the central nervous system and the 3R,4S configuration at the piperidine 3- and 4-carbon atoms and the S configuration at the phenylethyl 2-carbon atom are beneficial for analgesic potency and inhibitory effects in GPI and MVD and the same for an S or R configuration at the phenylethyl 2-carbon atom besides the 3R,4S configuration for receptor mu affinity and selectivity.

Monoclonal antibodies specific for ohmefentanyl.

Ohmefentanyl (Ohm, N[1-(beta-hydroxy-beta-phenylethyl)-3-methyl-4- piperidyl]-N-phenylpro-pronamide), designed and synthesized by our laboratory, is a highly selective mu receptor agonist. After somatic cell fusion between splenocytes of BALB c mice, immunized by Ohm-BSA conjugate and NS-1 myeloma cells, 2 lines of hybridoma (D2 and F4) secreting monoclonal anti-Ohm antibodies (MAb) were obtained. Saturation and competition experiments showed that MAb-D2 and MAb-F4 bound to Ohm-BSA with high affinity and high specificity. Using radioligand binding assay and bioassay, we also found that MAb-D2 and MAb-F4 inhibited [3H] Ohm binding to rat brain opioid receptors in a dose-dependent manner and antagonized the effect of Ohm on the contraction of guinea pig ileum induced by electric field stimulation. These results suggested that MAb-D2 and MAb-F4 were 2 monoclonal antibodies specific for Ohm and could be useful as functional antagonists of Ohm.

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.

Effect of brain lesions on [3H]ohmefentanyl binding site densities in the rat striatum and substantia nigra.

We have recently demonstrated that [3H]ohmefentanyl, a non-peptidergic opioid ligand which was suggested to cross the blood brain barrier in contrast to other peptidergic opioid ligands, bound not only to mu opioid receptor sites but also to sigma sites. In order to examine whether [3H]ohmefentanyl can be used as a marker for mu sites, we investigated the effects of brain lesions on [3H]ohmefentanyl binding site densities, as compared with [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), a selective mu ligand. These binding site densities were measured by quantitative autoradiography in the rat striatum and substantia nigra, two brain structures known to contain a high density of mu receptors, following lesions of the nigro-striatal dopaminergic pathway and striatal intrinsic neurons. Following unilateral nigral lesion with 6-hydroxydopamine, [3H]ohmefentanyl binding site densities were decreased in the patches (-35%) and matrix (-20%) of the ipsilateral striatum and in the lesioned substantia nigra pars compacta (-49%). Unilateral striatal lesion with quinolinic acid induced 72%, 61% and 50% decreases in [3H]ohmefentanyl binding in the patches and matrix of the lesioned striatum and in the ipsilateral substantia nigra pars reticulata, respectively. Similar results were obtained in the binding of [3H]DAGO. Indeed, a significant linear correlation was observed between [3H]ohmefentanyl and [3H]DAGO binding site densities. Therefore, mu opioid receptors may be mainly located on intrinsic neurons in the striatum, dopaminergic cell bodies in the substantia nigra pars compacta and nerve terminals of striatal efferents in the substantia nigra pars reticulata.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ohmefentanyl on CA1 field potentials in rat hippocampus slices.

The effects of ohmefentanyl (OMF), a new opiate agonist with high affinity and high specificity for mu receptors, was examined on CA1 field potentials in the transverse hippocampal slices. OMF showed two effects upon the evoked population spikes (PS) recorded in stratum pyramidale: 1) a concentration-dependent increase in the amplitude of PS, which was largely reversed by naloxone, and 2) production of a naloxone-reversible additional PS at high stimulus intensities. No significant change was seen in field excitatory postsynaptic potential (EPSP) recorded simultaneously in stratum radiatum. The EC50 for OMF and morphine were 6.6 and 3700 nmol.L-1, respectively. Thus OMF was 560 times more potent than morphine. The mechanism of augmentation by OMF of PS could be attributed to disinhibition as judged from the paired-pulse paradigm.