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.     

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.     

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.     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.     

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.     

Functional role of a conserved motif in TM6 of the rat mu opioid receptor: constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp.

Mutations within the "X1BBX2X3B" motif or its variants in the junction of the third intracellular (i3) loop and the sixth transmembrane domain (TM6) have been shown to lead to constitutive activation of several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at the X3 locus of the rat mu opioid receptor was mutated to Lys and Asp, and the mutants were examined for binding and signaling properties. The T6.34(279)K mutant was poorly expressed, and pretreatment with naloxone greatly enhanced its expression. This construct exhibited properties identified previously with constitutive activation: (1) compared with the wild type, it produced much higher agonist-independent [35S]GTPgammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMGO similar to that of the high-affinity state of the wild type, which was not altered by GTPgammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular receptor pool than the wild type. Naloxone inhibited the basal [35S]GTPgammaS binding of the T6.34(279)K mutant, demonstrating inverse agonist activity at this mutant receptor. In contrast, the T6.34(279)D substitution did not increase basal [35S]GTPgammaS binding, greatly reduced agonist-promoted [35S]GTPgammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive states of the receptor. The results were interpreted in the structural context of a model for the mu opioid receptor that incorporates the information from the crystal structure of rhodopsin. The interaction of T6.34(279) with R3.50(165) in the mu opioid receptor is considered to stabilize the inactive conformations. The T6.34(279)K substitution would then disrupt this interaction and support agonist-free activation, while T6.34(279)D mutation should strengthen this interaction which keeps the receptor in inactive states. T6.34(279) may, in addition, interact with the neighboring R6.35(280) to help constrain the receptor in inactive states, and T6.34(279)K and T6.34(279)D mutations would affect this interaction by disrupting or strengthening it, respectively. To the best of our knowledge, the results presented here represent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.     

Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and cannabinoid receptor-activated G proteins in rat brain

Chronic treatment of rats with delta9-tetrahydrocannabinol (delta9-THC) results in tolerance to its acute behavioral effects. In a previous study, 21-day delta9-THC treatment in rats decreased cannabinoid activation of G proteins in brain, as measured by in vitro autoradiography of guanosine-5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding. The present study investigated the time course of changes in cannabinoid-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding in both brain sections and membranes, following daily delta9-THC treatments for 3, 7, 14, and 21 days. Autoradiographic results showed time-dependent decreases in WIN 55212-2-stimulated [35S]GTPgammaS and [3H]WIN 55212-2 binding in cerebellum, hippocampus, caudate-putamen, and globus pallidus, with regional differences in the rate and magnitude of down-regulation and desensitization. Membrane binding assays in these regions showed qualitatively similar decreases in WIN 55212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding (using [3H]SR141716A), and demonstrated that decreases in ligand binding were due to decreases in maximal binding values, and not ligand affinities. These results demonstrated that chronic exposure to delta9-THC produced time-dependent and region-specific down-regulation and desensitization of brain cannabinoid receptors, which may represent underlying biochemical mechanisms of tolerance to cannabinoids.     

Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the mu opioid receptor

We examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the mu opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPgammaS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation.     

(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.     

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.     

Upregulation of mu opioid receptors by voluntary morphine administration in drinking water

Morphine was provided to rats in drinking water for 21 days. Profound analgesic tolerance was detected both in hot-plate and tail-flick tests. The density of [3H]DAMGO binding sites increased by 76% in spinal cord membranes due to morphine exposure compared to those in opioid naive animals. Slightly augmented [3H]DAMGO binding was measured in the synaptic plasma membranes, with a concomitant decrease in the microsomal membranes, of morphine tolerant/dependent brains. These observations suggest that the regulation of spinal mu opioid receptors might be different from those in the brain. It is emphasized that the molecular changes underlying tolerance/dependence are influenced by several factors, such as the tissue or subcellular fractions used, besides the obvious importance of the route of drug administration. Results obtained after voluntary morphine intake further support the growing number of experimental data that chronic morphine does not internalize/downregulate the mu opioid receptors in the central nervous system.     

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.     

Enhanced morphine withdrawal and micro -opioid receptor G-protein coupling in A2A adenosine receptor knockout mice

Much evidence supports the hypothesis that A2A adenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild-type and A2A receptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, micro -opioid and dopamine D2 receptor autoradiography, as well as micro -opioid receptor-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) autoradiography was carried out in brain sections of withdrawn wild-type and knockout mice. No significant changes in D2 and micro -opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of micro receptor-stimulated [35S]GTPgammaS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2A receptor plays a role in opioid withdrawal related to functional receptor activation.     

Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression

The roles of conserved aspartates in the third transmembrane domain of the rat mu opioid receptor (RMOR) were explored with mutations of D3.32(147) and D3.49(164). D3.49(164) in the highly conserved DRY motif was mutated to 13 amino acids. Except for the D3.49(164)E mutant, each mutant displayed little or no detectable [(3)H]diprenorphine binding, and pretreatment with naloxone greatly enhanced binding. D3.49(164)H, -Q, -Y, -M, and -E mutants were further studied. D3.32(147) was substituted with A or N. All seven mutants exhibited similar binding affinities for the antagonist [(3)H]diprenorphine as the wild-type. The D3.49(164)H, -Q, -Y, and -M mutants, but not the D3.49(164)E and D3.32(147) mutants, exhibited enhanced basal [(35)S]GTPgammaS binding which was comparable to the maximally activated level of the wild-type and was related to expression levels. Naloxone, naltrexone, and naloxone methiodide significantly inhibited the basal [(35)S]GTPgammaS binding of the D3.49(164) mutants, indicating inverse agonist activities. Treatment of the D3.49(164)Y mutant with pertussis toxin greatly reduced the basal [(35)S]GTPgammaS binding, demonstrating constitutive activation of Galpha(i)/Galpha(o). The D3.49(164)H, -Y, -M, and -Q mutants had higher affinities for DAMGO than the wild-type, which were not significantly lowered by GTPgammaS. Thus, mutation of D3.49(164) to H, Y, M, or Q in RMOR resulted in receptor assuming activated conformations. In contrast, the D3.49(164)E mutant displayed significantly lower basal [(35)S]GTPgammaS binding and reduced affinity for DAMGO. Upon incubation of membranes at 37 degrees C, the constitutively active D3.49(164)Y mutant was structurally less stable, whereas the inactivated D3.49(164)E mutant was more stable, than the wild-type. Computational simulations showed that the E3.49 side chain interacted strongly with the conserved R3.50 in the DRY motif and stabilized the inactive form of the receptor. Taken together, these results indicate that D3.49 plays an important role in constraining the receptor in inactive conformations.     

Allosteric modulation of adenosine A1 receptor coupling to G-proteins in brain

2-Amino-4,5,6,7-tetrahydrobenzo(beta)thiophen-3-yl 4-chlorophenylmethanone (T62) is a member of a group of allosteric modulators of adenosine A1 receptors tested in animal models of neuropathic pain to increase the efficacy of adenosine. To determine its mechanisms at the level of receptor-G-protein activation, the present studies examined the effect of T62 on A1-stimulated [35S]guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding in brain membranes, and by [35S]GTPgammaS autoradiography using the A1 agonist, phenylisopropyladenosine (PIA), to activate G-proteins. In hippocampal membranes, T62 increased both basal and PIA-stimulated [35S]GTPgammaS binding. The effect of T62 was non-competitive in nature, since it increased the maximal effect of PIA, with no effect on agonist potency. GTPgammaS saturation analysis showed that T62 increased the number of G-proteins activated by agonist but had no effect on the affinity of activated G-proteins for GTPgammaS. [35S]GTPgammaS autoradiography showed that the neuroanatomical localization of T62-stimulated [35S]GTPgammaS binding was identical to that of PIA-stimulated activity. The increase in PIA-stimulated activity by T62 varied between brain regions, with areas of lower A1 activation producing the largest percent modulation by T62. These results suggest a mechanism of allosteric modulators to increase the number of activated G-proteins per receptor, and provide a neuroanatomical basis for understanding potential therapeutic effects of such drugs.     

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.     

Binding of [35S]GTPgammaS stimulated by (+)-pentazocine sigma receptor agonist, is abundant in the guinea pig spleen

Here we measured sigma receptor agonist, [3H](+)-pentazocine binding and (+)-pentazocine-stimulated [35S]GTPgammaS binding throughout brain regions and peripheral organs of mice and guinea pigs to investigate the distribution of G protein-coupled sigma receptors. There was no significant correlationship between both distributions, in which the [3H](+)-pentazocine binding is highest in the liver of each species, while the [35S]GTP-gammaS binding is highest in the guinea pig spleen. The agonist-stimulated [35S]GTPgammaS binding in the spleen was also confirmed by in situ autoradiography using sections. Thus it is suggested that there are at least two subtypes, metabotropic and nonmetabotropic sigma receptors, and the former ones are abundant in the guinea pig spleen.     

Substance P modulation of DAMGO binding in the brain of CXBK and Swiss-Webster mice.

The effects of substance P (SP) on the binding of the selective mu opioid agonist [3H]DAMGO to brain membranes of CXBK and Swiss-Webster (SW) mice were compared. We have previously shown that subnanomolar concentrations of SP and N-terminal fragments of SP modulate DAMGO binding in SW brain membranes and hypothesized that modulation occurs via SP interaction with mu 1 sites. In the present study, binding assays using CXBK mice, a strain deficient in mu receptors including mu 1 sites, were performed to assess the effect of mu receptor deficiency on SP-induced modulation of DAMGO binding. Whereas the addition of 0.1 nM SP to the binding mixtures produced up to 30% increase in the values of Kd and maximum binding capacity (R) for the SW strain, SP produced little or no change in the case of CXBK strain. Maximum binding capacity for DAMGO was 43% less in the brain of CXBK mice than in SW mice. No difference was observed in the estimated binding parameters of the spinal cord for the two strains. Whereas pretreatment of brain membranes of SW mice using beta-funaltrexamine (beta-FNA) increased from 2- to 10-fold the modulatory effect of SP, CXBK brain membranes pretreated with beta-FNA remained nearly insensitive to modulation by SP. The effect of SP on the affinity of DAMGO binding in SW mice, but not in CXBK mice, was reversed by the addition of GTP. It is concluded that mu receptor deficiency can markedly influence SP-induced modulation of DAMGO binding.     

Receptor binding and G-protein activation by new Met5-enkephalin-Arg6-Phe7 derived peptides

Met5-enkephalin-Arg6-Phe7 (Tyr-Gly-Gly-Phe-Met-Arg-Phe, MERF) is a naturally occurring heptapeptide that binds to opioid and non-opioid recognition sites in the central nervous system. Four synthetic analogs with single or double amino acid substitutions were prepared by solid phase peptide synthesis to achieve proteolytically more stable structures: Tyr-D-Ala-Gly-Phe-Met-Arg-Phe (I), Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (II), Tyr-D-Ala-Gly-Phe-L-Nle-Arg-Phe (III) and Tyr-Gly-Gly-Phe-L-Nle-Arg-Phe (IV). In this study receptor binding characteristics and G-protein activation of MERF and its derivatives were compared in crude membrane fractions of frog and rat brain. Synthetic MERF-derived peptides were potent competitors for [3H]MERF and [3H]naloxone binding sites with the exception of analog (II) which turned to be substantially less active. The presence of 100 mM NaCl or 100 microM 5'-guanylylimidodiphosphate, Gpp(NH)p, decreased the affinity of the peptides in [3H]naloxone binding assays, suggesting that these ligands might act as agonists at the opioid receptors. Some of the compounds were also used to stimulate guanosine-5'-O-(3-[gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding in rat and frog brain membranes at concentrations of 10(-9)-10(-5) M. The EC50 values of analog (II) were the highest in both tissues. Analog (I) was as effective as MERF in rat brain membranes, but showed lower maximal stimulation in frog brain preparation. Again, analog (II) seemed to be the least efficacious peptide that stimulated [35S]GTPgammaS binding only by 59%. Specificity of the peptides was further investigated by the inhibition of agonist-stimulated [35S]GTPgammaS binding in the presence of selective antagonists for the opioid receptor types. The mu-selective antagonist cyprodime displayed the lowest potency in inhibiting the effects of the peptides, whereas norbinaltorphimine (kappa-selective antagonist) and naltrindole (delta-selective antagonist) were quite potent in both tissues. We concluded that MERF and its derivatives are able to activate G-proteins mainly via kappa- and delta-opioid receptors.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.