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.     

(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.     

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.     

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.     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

Detection of a novel immunoreactive endomorphin 2-like peptide in rat brain extracts

To pursue further the possible de novo biosynthetic pathway of endomorphins in rat brain we raised antibodies to endomorphin-2 conjugate in rabbits. Antiserum R1 recognized endomorphin-2 with good selectivity as compared to endomorphin-1 with a median detection value of 65.5+/-7.5 pg/tube (n=7), whereas R4 antiserum recognized both endomorphins with similar sensitivity. Neither antisera recognized YP-related di- or tripeptides or YGGF-related opioid sequences (enkephalins, beta-endorphin, dynorphin). Using the same rat brain extraction-RP-HPLC-gradient separation paradigm as previously, antisera detected 144.6+/-40.0 (n=3) pg/g wet brain weight endomorphin-2-like immunoreactivity in the fraction corresponding to standard endomorphin-2 retention time and also in the fraction matching endomorphin-2-OH standard retention time (179.1+/-30.1 pg/g). Since R1 failed to recognize authentic endomorphin-2-OH, the second immunoreactive species must be different from both endomorphin-2 and endomorphin-2-OH. Possible biosynthetic intermediates to endomorphins, synthetic YPFFG and YPWFG had retention times close to the parent endomorphin standards in RP-HPLC gradient separation profile. The former was a mu-opioid receptor agonist of medium potency in the in vitro assays (rat brain RBA>P gamma S binding and mouse vas deferens), whereas the latter was a weak mu-opioid receptor agonist with a significant delta-opioid receptorial action as well and a definite indication of partial agonism.     

Partial and full agonism in endomorphin derivatives: comparison by null and operational model

The partial mu-opioid receptor pool inactivation strategy in isolated mouse vas deferens was used to determine partial agonism of endomorphins and their analogs (endomorphin-1-ol, 2',6'-dimethyltyrosine (Dmt)-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2) using morphine, normorphine, morphiceptin, (D-Ala2,MePhe4,Gly5-ol)-enkephalin (DAMGO) and its amide (DAMGA) as reference opioid agonists. Agonist affinities (KA) and efficacies were assessed both by the "null" and the "operational" method. The KA values determined by the two methods correlated significantly with each other and also with the displacing potencies against 3H-naloxone in the receptor binding assay in the presence of Na+. DAMGO and DAMGA were full agonist prototypes, morphine, endomorphin-1, endomorphin-1-ol, Dmt-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2 were found by both methods to be partial agonists whereas the parameters for normorphine, morphiceptin and endomorphin-2 were intermediate.     

Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.     

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.     

Mu-opioid receptors are involved in the tolerance to nicotine antinociception

Several studies have shown the participation of the endogenous opioid system on the antinociceptive effects and addictive properties of nicotine. The aim of the present study was to explore the involvement of the mu-opioid receptors in the development of tolerance to nicotine antinociception. Chronic treatment of C57BL/6 mice with nicotine (5 mg/kg s.c., three times daily during 12 days) resulted in tolerance to its antinociceptive responses in the tail-immersion test. We investigated the possible existence of adaptive changes in the expression and/or functional activity of mu-opioid receptors in these tolerant mice by using autoradiography of [(3)H]D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H]DAMGO) binding and DAMGO-stimulated guanosine [(35)S]5'-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding. The density of mu-opioid receptors in the spinal cord was not modified in nicotine-tolerant mice, whereas a decrease was found in the caudate-putamen, as well as in the core and the shell of the nucleus accumbens. However, the functional activity of these receptors was significantly increased in the spinal cord as a consequence of nicotine treatment. To further investigate the role of mu-opioid receptors in the tolerance to nicotine-induced antinociception, we evaluated this response in C57BL/6 mu-opioid receptor knockout mice. Chronic nicotine treatment produced tolerance in both wild-type and knockout animals, but tolerance developed faster in mice lacking mu-opioid receptors. These results indicate that mu-opioid receptors play an important role in the development of tolerance to nicotine antinociceptive effects.     

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.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

4-Phenyl-4-[1H-imidazol-2-yl]-piperidine derivatives, a novel class of selective delta-opioid agonists

A novel series of 4-phenyl-4-[1H-imidazol-2-yl]-piperidine derivatives has been prepared and their synthesis described herein. In vitro affinities for delta-, mu-, and kappa-opioid receptors, as well as the functional activity in the [(35)S]GTPgammaS assay are reported. The most potent and selective delta-opioid agonist 18a exhibited a K(i) of 18 nM, and was >258-fold and 28-fold selective over mu- and kappa-receptors, respectively; the compound is a full agonist with an EC(50) value of 14 nM.     

Endomorphins interact with the substance P (SP) aminoterminal SP(1-7) binding in the ventral tegmental area of the rat brain

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP(1-7) in the rat spinal cord. This site appeared very specific for SP(1-7) as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP(1-7) from this site. In the present work using a [(3)H]-labeled derivative of the heptapeptide we have identified and characterized [(3)H]-SP(1-7) binding in the rat ventral tegmental area (VTA). Similarly to the [(3)H]-SP(1-7) binding in the spinal cord the affinity of unlabeled SP(1-7) to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP(1-7) site was 4-5 times weaker than that for SP(1-7) but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. It was concluded that the specific site identified for SP(1-7) binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.     

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.     

Increased brain neuropeptide Y1 and Y2 receptor binding in NPY knock out mice does not result in increased receptor function

The brain neuropeptide Neuropeptide Y (NPY) is an important modulator of a number of centrally mediated processes including feeding, anxiety-like behaviors, blood pressure and others. NPY produces its effects through at least four functional G-protein coupled receptors termed Y1, Y2, Y4 and Y5. In the brain, the Y1 and Y2 receptor subtypes are the predominant receptor population. To better understand the roles of NPY, genetically modified mice lacking NPY were produced but lacked the expected phenotypes. These mice have previously been reported to have a marked increase in Y2 receptor binding. In the present study, we found an upregulation of both Y1 and Y2 receptor binding and extended these findings to the female. These increases were as large as 10-fold or greater in many brain regions. To assess functional coupling of the receptors, we performed agonist-induced [(35)S]GTPgammaS autoradiography. In the mouse brain, the Y1/Y4/Y5 agonist Leu(31),Pro(34)-NPY increased [(35)S]GTPgammaS binding with a regional distribution consistent with that produced when labeling adjacent sections with [(125)I]-Leu(31),Pro(34)-PYY. In a few brain regions, minor increases were noted in the agonist-induced binding when comparing knock out mice to wild type. The Y2 agonist C2-NPY stimulated [(35)S]GTPgammaS binding in numerous brain areas with a regional distribution similar to the binding observed with [(125)I]-PYY3-36. Again, no major increases were noted in the functional activation of Y2 receptors between knock out and wild type mice. Therefore, the increased Y1 and Y2 binding observed in the NPY knock out mice does not represent an increase in NPY receptor mediated signaling and is likely due to an increase in spare (uncoupled) receptors.     

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%.     

Affinity labeling of delta opioid receptors by an enkephalin-derivative alkylating agent, DSLET-Mal

Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [(3)H][Ile(5,6)]deltorphin II (K(i) = 8 nM), whereas its binding to the mu ([(3)H]DAMGO) and kappa ([(3)H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [(3)H][Ile(5,6)]deltorphin II binding sites (96% blocking at 10 microM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile(5, 6)]deltorphin II-stimulated [(35)S]GTPgammaS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [(35)S]GTPgammaS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors.     

Side chain modifications change the binding and agonist properties of endomorphin 2.

Side chain modifications were introduced to endomorphin 2 (E2) to improve its binding properties and biological activity. A number of C-terminal modifications decreased the binding affinity to the mu-opioid receptor and the intrinsic activity in rat brain membranes. The exception was E2-ol, which showed increased binding affinity to MOR and higher potency in stimulating [(35)S]GTPgammaS binding. N-methylation of Phe(3) (MePhe(3)) attenuated the binding affinity and produced a rightward shift of [(35)S]GTPgammaS binding curves. All derivatives had lower intrinsic activity than E2. Some of the modified peptides partially inhibited, while YPF-benzyl-allyl-amide fully inhibited, the E2 or [d-Ala(2),MePhe(4),Gly(5)ol]enkephalin stimulated [(35)S]GTPgammaS binding. Marked differences were found between the results obtained using tritiated E2, tritiated naloxone, and [(35)S]GTPgammaS binding, indicating the possible involvement of multiple binding sites. The data presented demonstrate that the C-terminal amide group has an essential role in the regulation of the binding and the agonist/antagonist properties of E2.