Coupling efficacy and selectivity of the human mu-opioid receptor expressed as receptor-Galpha fusion proteins in Escherichia coli

Two constructs encoding the human micro-opioid receptor (hMOR) fused at its C terminus to either one of two Galpha subunits, Galpha(o1) (hMOR-Galpha(o1)) and Galpha(i2) (hMOR-Galpha(i2)), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg). Receptors fused to Galpha(o1) or to Galpha(i2) maintained high-affinity binding of the antagonist diprenorphine. Affinities of the micro-selective agonists morphine, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding were assessed in the presence of added purified Gbetagamma subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [(35)S]GTPgammaS binding. In the presence of Gbetagamma dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-Galpha(i2) than at hMOR-Galpha(o1), whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [(35)S]GTPgammaS binding at hMOR-Galpha(o1) were similar, whereas at hMOR-Galpha(i2), endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to Galpha(o1) and Galpha(i2) and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.     

(35)S]GTPgammaS binding stimulated by endomorphin-2 and morphiceptin analogs

The ability of several mu-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The mu-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal). The results obtained for these peptides in [(35)S]GTPgammaS binding assay were compared with those obtained for a standard mu-opioid agonist DAMGO. [d-1-Nal(3)]Morphiceptin was more potent in G-protein activation (EC(50) value of 82.5+/-4.5 nM) than DAMGO (EC(50)=105+/-9 nM). [d-2-Nal(3)]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either d-1-Nal or d-2-Nal failed to stimulate [(35)S]GTPgammaS binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists.     

Modulating micro-opioid receptor phosphorylation switches agonist-dependent signaling as reflected in PKCepsilon activation and dendritic spine stability

A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKCε pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCε pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKCε pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCε, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCε. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.     

Binding of endomorphin-2 to mu-opioid receptors in experimental mouse mammary adenocarcinoma.

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) binds with high affinity and selectivity to the mu-opioid receptor. In the present study, [125I]endomorphin-2 has been used to characterize mu-opioid-binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [125I]endomorphin-2 (1 nM) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K(d) = 18.79 +/- 1.13 nM, B(max) = 635 +/- 24 fmol/mg protein) and the other shows low affinity and higher capacity (K(d) = 7.67 +/- 0.81 microM, B(max) = 157 +/- 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [125I]endomorphin-2 binding site was [d-1-Nal3]morphiceptin > endomorphin-2 > [d-Phe3]morphiceptin > morphiceptin > [d-1-Nal3]endomorphin-2, indicating binding of these peptides to mu-opioid receptors. The uptake of 131I-labeled peptides administered intraperitoneally to tumor-bearing mice was also investigated. The highest accumulation in the tumor was observed for [d-1-Nal3)morphiceptin, which reached the value of 8.19 +/- 1.14% dose/g tissue.     

G protein activation by endomorphins in the mouse periaqueductal gray matter

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.     

Analgesic effects of endomorphin-1 and endomorphin-2 in the formalin test in mice

Two recently isolated peptides, endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2), are highly selective micro-opioid receptor agonists with analgesic actions in the tail-flick test. To further assess the analgesic properties of these peptides, the effects of endomorphin-1, endomorphin-2, and morphine were examined in the formalin test. Male Swiss Webster mice were injected i.c.v. with endomorphin-1, endomorphin-2, or morphine (0, 1, 3, 10 microg) 5 min before injection of 20 microl of 5% formalin s.c. into the plantar surface of one hind-paw. The mice were observed for 60 min after formalin injection. Endomorphin-1 and endomorphin-2 produced dose-dependent analgesia that was shorter in duration than for morphine. Increased locomotion was observed after morphine, but not after endomorphin-1 or endomorphin-2. These findings extend previous results and suggest that endomorphins may have therapeutic potential for the treatment of acute pain.     

Characterization of the [125I]endomorphin-2 binding sites in the MCF7 breast cancer cell line

In the present study, the expression of the micro-opioid receptor on protein level has been demonstrated in MCF7 breast cancer cells. Binding of the [125I]-labeled micro-opioid receptor selective ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) was examined in vitro using a cross-linking assay followed by a Western blot technique. The radioactive complex had a molecular weight of about 65 kDa and was detectable by anti-micro-opioid receptor antibody, indicating the presence of micro-opioid receptors in MCF7 cell membranes. Characterization of endomorphin-2 binding to the membranes obtained from MCF7 cells was performed. Cold saturation experiments with [125I]endomorphin-2 showed biphasic binding curves in Scatchard coordinates. One component represents a high affinity and low capacity, and the other low affinity and higher capacity binding sites. The obtained Bmax values for [125I]endomorphin-2 binding to MCF7 membranes were much higher than those obtained for mouse brain. Pharmacological characterization of the [125I]endomorphin-2 binding sites was made using endomorphin-2 and two other micro selective ligands, morphiceptin, and [D-1-Nal3]morphiceptin on MCF7 cell membrane preparations and whole MCF7 cells. In both cases, the rank order of potency was [D-1-Nal3]morphiceptin>endomorphin-2>morphiceptin, but in case of whole MCF7 cells the IC50 values were about 40 times higher.     

Inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) on gastrointestinal transit

The inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) (TAPA), a highly selective mu(1)-opioid receptor agonist, on mouse gastrointestinal transit was compared with that of morphine and [D-Ala(2), N-methyl-Phe(4), Gly(5)-ol]-enkephalin (DAMGO). When administered intracerebroventricularly 5 min before the oral injection of charcoal meal, TAPA (10-100 pmol), morphine (0.25-4 nmol), and DAMGO (20-80 pmol) dose-dependently inhibited gastrointestinal transit of charcoal. The inhibitory effect of each mu-opioid receptor agonist was completely antagonized by naloxone, a nonselective opioid receptor antagonist. The inhibitory effects of morphine and DAMGO were significantly antagonized by both beta-funaltrexamine, a selective mu-opioid receptor antagonist, and naloxonazine, a selective mu(1)-opioid receptor antagonist. In contrast, the inhibitory effect of TAPA was not affected at all by beta-funaltrexamine, naloxonazine, nor-binaltorphimine (a selective kappa-opioid receptor antagonist), or naltrindole (a selective delta-opioid receptor antagonist). These results suggest that the inhibitory effect of TAPA on gastrointestinal transit may be mediated through an opioid receptor mechanism different from that of morphine and DAMGO.     

Differential antagonism of endomorphin-1 and endomorphin-2 supraspinal antinociception by naloxonazine and 3-methylnaltrexone

To determine if different subtypes of mu-opioid receptors were involved in antinociception induced by endomorphin-1 and endomorphin-2, the effect of pretreatment with various mu-opioid receptor antagonists beta-funaltrexamine, naloxonazine and 3-methylnaltrexone on the inhibition of the paw-withdrawal induced by endomorphin-1 and endomorphin-2 given intracerebroventricularly (i.c.v.) were studied in ddY male mice. The inhibition of the paw-withdrawal induced by i.c.v. administration of endomorphin-1, endomorphin-2 or DAMGO was completely blocked by the pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine (40 mg/kg), indicating that the antinociception induced by all these peptides are mediated by the stimulation of mu-opioid receptors. However, naloxonazine, a mu1-opioid receptor antagonist pretreated s.c. for 24h was more effective in blocking the antinociception induced by endomorphin-2, than by endomorphin-1 or DAMGO given i.c.v. Pretreatment with a selective morphine-6 beta-glucuronide blocker 3-methylnaltrexone 0.25mg/kg given s.c. for 25 min or co-administration of 3-methylnaltrexone 2.5 ng given i.c.v. effectively attenuated the antinociception induced by endomorphin-2 given i.c.v. and co-administration of 3-methylnaltrexone shifted the dose-response curves for endomorphin-2 induced antinociception to the right by 4-fold. The administration of 3-methylnaltrexone did not affect the antinociception induced by endomorphin-1 or DAMGO given i.c.v. Our results indicate that the antinociception induced by endomorphin-2 is mediated by the stimulation of subtypes of mu-opioid receptor, which is different from that of mu-opioid receptor subtype stimulation by endomorphin-1 and DAMGO.     

C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four mu-receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell- and tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [³⁵S]GTPγS binding in rat brain. [³⁵S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [³⁵S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [³⁵S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [³⁵S]GTPγS binding. Differences in maximal stimulation of [³⁵S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [³⁵S]GTPγS binding revealed a lower apparent B _max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [³⁵S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain.     

Agonist-directed interactions with specific beta-arrestins determine mu-opioid receptor trafficking, ubiquitination, and dephosphorylation

Morphine and other opiates mediate their effects through activation of the μ-opioid receptor (MOR), and regulation of the MOR has been shown to critically affect receptor responsiveness. Activation of the MOR results in receptor phosphorylation, β-arrestin recruitment, and internalization. This classical regulatory process can differ, depending on the ligand occupying the receptor. There are two forms of β-arrestin, β-arrestin1 and β-arrestin2 (also known as arrestin2 and arrestin3, respectively); however, most studies have focused on the consequences of recruiting β-arrestin2 specifically. In this study, we examine the different contributions of β-arrestin1- and β-arrestin2-mediated regulation of the MOR by comparing MOR agonists in cells that lack expression of individual or both β-arrestins. Here we show that morphine only recruits β-arrestin2, whereas the MOR-selective enkephalin [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), recruits either β-arrestin. We show that β-arrestins are required for receptor internalization and that only β-arrestin2 can rescue morphine-induced MOR internalization, whereas either β-arrestin can rescue DAMGO-induced MOR internalization. DAMGO activation of the receptor promotes MOR ubiquitination over time. Interestingly, β-arrestin1 proves to be critical for MOR ubiquitination as modification does not occur in the absence of β-arrestin1 nor when morphine occupies the receptor. Moreover, the selective interactions between the MOR and β-arrestin1 facilitate receptor dephosphorylation, which may play a role in the resensitization of the MOR and thereby contribute to overall development of opioid tolerance.     

Possible involvement of dynorphin A release via mu1-opioid receptor on supraspinal antinociception of endomorphin-2

It has been demonstrated that the antinociception induced by i.t. or i.c.v. administration of endomorphins is mediated through mu-opioid receptors. Moreover, though endomorphins do not have appreciable affinity for kappa-opioid receptors, pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine markedly blocks the antinociception induced by i.c.v.- or i.t.-injected endomorphin-2, but not endomorphin-1. These evidences propose the hypothesis that endomorphin-2 may initially stimulate the mu-opioid receptors, which subsequently induces the release of dynorphins acting on kappa-opioid receptors to produce antinociception. The present study was performed to determine whether the release of dynorphins by i.c.v.-administered endomorphin-2 is mediated through mu-opioid receptors for producing antinociception. Intracerebroventricular pretreatment with an antiserum against dynorphin A, but not dynorphin B or alpha-neo-endorphin, and s.c. pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine dose-dependently attenuated the antinociception induced by i.c.v.-administered endomorphin-2, but not endomorphin-1 and DAMGO. The attenuation of endomorphin-2-induced antinociception by pretreatment with antiserum against dynorphin A or nor-binaltorphimine was dose-dependently eliminated by additional s.c. pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine or a selective mu(1)-opioid receptor antagonist naloxonazine at ultra low doses, which are inactive against mu-opioid receptor agonists in antinociception, suggesting that endomorphin-2 stimulates distinct subclass of mu(1)-opioid receptor that induces the release of dynorphin A acting on kappa-opioid receptors in the brain. It concludes that the antinociception induced by supraspinally administered endomorphin-2 is in part mediated through the release of endogenous kappa-opioid peptide dynorphin A, which is caused by the stimulation of distinct subclass of mu(1)-opioid receptor.     

Opposite Effects of Neuropeptide FF on Central Antinociception Induced by Endomorphin-1 and Endomorphin-2 in Mice

Neuropeptide FF (NPFF) is known to be an endogenous opioid-modulating peptide. Nevertheless, very few researches focused on the interaction between NPFF and endogenous opioid peptides. In the present study, we have investigated the effects of NPFF system on the supraspinal antinociceptive effects induced by the endogenous µ-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2). In the mouse tail-flick assay, intracerebroventricular injection of EM-1 induced antinociception via µ-opioid receptor while the antinociception of intracerebroventricular injected EM-2 was mediated by both µ- and κ-opioid receptors. In addition, central administration of NPFF significantly reduced EM-1-induced central antinociception, but enhanced EM-2-induced central antinociception. The results using the selective NPFF₁ and NPFF₂ receptor agonists indicated that the EM-1-modulating action of NPFF was mainly mediated by NPFF₂ receptor, while NPFF potentiated EM-2-induecd antinociception via both NPFF₁ and NPFF₂ receptors. To further investigate the roles of µ- and κ-opioid systems in the opposite effects of NPFF on central antinociception of endomprphins, the µ- and κ-opioid receptors selective agonists DAMGO and {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593, respectively, were used. Our results showed that NPFF could reduce the central antinociception of DAMGO via NPFF₂ receptor and enhance the central antinociception of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 via both NPFF₁ and NPFF₂ receptors. Taken together, our data demonstrate that NPFF exerts opposite effects on central antinociception of endomorphins and provide the first evidence that NPFF potentiate antinociception of EM-2, which might result from the interaction between NPFF and κ-opioid systems.     

Novel endomorphin-2 analogs with mu-opioid receptor antagonist activity.

A series of position 4-substituted endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) analogs containing 3-(1-naphthyl)-alanine (1-Nal) or 3-(2-naphthyl)-alanine (2-Nal) in L- or D-configuration, was synthesized. The opioid activity profiles of these peptides were determined in the mu-opioid receptor representative binding assay and in the Guinea-Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot-plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the mu-opioid receptor was reduced compared with endomorphin-2. The two most potent analogs were [D-1-Nal(4)]- and [D-2-Nal4]endomorphin-2, with IC50 values 14 +/- 1.25 and 19 +/- 2.1 nM, respectively, compared with 1.9 +/- 0.21 nM for endomorphin-2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot-plate test. Antinociception induced by endomorphin-2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [D-1-Nal4]- and [D-2-Nal4]-endomorphin-2, indicating that these analogs were mu-opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 microg per animal naloxone almost completely inhibited antinociceptive action of endomorphin-2, while [D-1-Nal4]endomorphin-2 in about 46%.     

Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375

Morphine is a poor inducer of micro-opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxy-terminal residue 375 (Ser(375)). Ser(375) phosphorylation was sufficient and required for morphine-induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO-desensitized receptors were rapidly dephosphorylated. In contrast, morphine-desensitized receptors remained at the plasma membrane in a Ser(375)-phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser(375).     

Motility effects of opioid peptides in dog intestine

Six opioid peptides, like morphine, were found to produce dose-dependent contractions of dog isolated intestine when administered as intraarterial boluses. The increases in incidence and amplitude of intestinal contractions were antagonized by naloxone. The rank order of potency of the opioid agonists tested was D-Ala2-met-enkephalinamide greater than D-Ala2-leu-enkephalinamide greater than met-enkephalin greater than beta-endorphin 1-31 greater than morphine greater than morphiceptin greater than dynorphin 1-13. The contractions induced by two opioid agonists displayed differential sensitivity to blockade by tetrodotoxin (TTX). Met-enkephalin was barely affected by concentrations of TTX that markedly reduced responses to morphiceptin. Some portion of the motility effect of metenkephalin may be exerted directly on intestinal smooth muscle.     

Antisense oligodeoxynucleotide targeting distinct exons of the cloned mu-opioid receptor distinguish between endomorphin-1 and morphine supraspinal antinociception in mice.

Antisense oligodeoxynucleotides (ODN) were used to investigate the supraspinal antinociceptive effects of endomorphin-1, an endogenous peptide whose analgesic profile suggests that it is a ligand at the mu-opioid receptor. To selectively restrict the expression of this receptor, five ODN targeting distinct exons of the gene sequence were injected subchronically by the intracerebroventricular route (i.c.v.) into mice. The antinociception induced by endomorphin-1 was greatly reduced in animals receiving the ODN directed to nucleotides 677-697, which code for a sequence located on the second extracellular loop of the mu receptor. ODN-mu(un), one of the two antisense ODN directed to exon 1, also impaired endomorphin-1 antinociception. ODN targeting exons 2 and 4 were totally inactive. In contrast, all five ODN blocked the antinociception induced by morphine and beta-casomorphin. The analgesic potency of endomorphin-1, morphine, and beta-casomorphin remained unaltered by administration of an ODN to nucleotides 29-46 of the murine delta-opioid receptor gene sequence of a random-sequence ODN. This suggest the existence of diverse molecular forms for the mu-opioid receptor that mediate the antinociceptive effects of endomorphin-1 and morphine/beta-casomorphin.     

Dmt1, d-1-Nal3]morphiceptin, a novel opioid peptide analog with high analgesic activity

The morphiceptin-derived peptide [Dmt1, d-1-Nal3]morphiceptin, labeled mu-opioid receptor (MOP) with very high affinity and selectivity in the receptor binding assays. In the mouse hot plate test, [Dmt1, d-1-Nal3]morphiceptin given intracerebroventricularly (i.c.v.) produced profound supraspinal analgesia, being approximately 100-fold more potent than the endogenous MOP receptor ligand, endomorphin-2. The antinociceptive effect of this new analog lasted up to 120min. Thus, [Dmt1, d-1-Nal3]morphiceptin is an interesting and extraordinarily potent analgesic, raising the possibility of novel approaches in the design of clinically useful drugs for pain treatment.     

Heterologous mu-opioid receptor adaptation by repeated stimulation of kappa-opioid receptor: up-regulation of G-protein activation and antinociception

The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride [(-)U-50,488H] on antinociception and G-protein activation induced by mu-opioid receptor agonists in mice. A single s.c. injection of (-)U-50,488H produced a dose-dependent antinociception, and this effect was reversed by a selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Furthermore, a single s.c. pre-treatment with (-)U-50,488H had no effect on the mu-opioid receptor agonist-induced antinociception. In contrast, repeated s.c. administration of (-)U-50,488H resulted in the development of tolerance to (-)U-50,488H-induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (-)U-50,488H significantly enhanced the antinociceptive effect of selective mu-opioid receptor agonists endomorphin-1, endomorphin-2 and [d-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAMGO). Using the guanosine-5'-o-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding assay, we found that (-)U-50,488H was able to produce a nor-BNI-reversible increase in [35S]GTP gamma S binding to membranes of the mouse thalamus, which has a high level of kappa-opioid receptors. Repeated administration of (-)U-50,488H caused a significant reduction in the (-)U-50,488H-stimulated [35S]GTP gamma S binding in this region, whereas chronic treatment with (-)U-50,488H exhibited the increase in the endomorphin-1-, endomorphin-2- and DAMGO-stimulated [35S]GTP gamma S bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of kappa-opioid receptors leads to the heterologous up-regulation of mu-opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of mu-opioid receptor-mediated antinociception.