Differential Effect of Intrastriatal Kainic Acid on the Modulation of Dopamine Release by μ- and δ-Opioid Peptides: A Microdialysis Study

The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid.     

Biochemical Evidence of Functional Interaction Between μ- and δ-Opioid Receptors in SK-N-BE Neuroblastoma Cell Line

Abstract: Radioligand binding assays and functional experiments revealed that the SK-N-BE neuroblastoma cell line expresses a similar ratio of μ- and δ-opioid receptors, both negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. Our findings also indicate that some functional interaction occurred between the two opioid subtypes; in fact, long-term exposure to [ d -Ala²- N -methyl-Phe⁴-Gly-ol⁵]enkephalin (DAMGO), a μ-selective agonist, sensitized the functional response of the δ-selective agonist but not vice versa. It is interesting that in acute interaction experiments, we observed a shift to the right of the concentration-effect curve of either DAMGO or [ d -Pen^2,5]enkephalin (DPDPE), a δ-selective agonist, as a result of DPDPE or DAMGO administration, respectively. In addition, low doses of naloxone, an antagonist selective for μ receptors, increased the inhibitory effect of [ d -Ala², d -Met⁵]enkephalinamide (DAME), a mixed μ/δ agonist, on adenylyl cyclase activity. Taken overall, these data support the hypothesis of the existence of a cross talk between μ and δ receptors in the SK-N-BE cell line.     

Differences in Binding Properties of μ and δ Opioid Receptor Subtypes from Rat Brain: Kinetic Analysis and Effects of Ions and Nucleotides

Differences in binding properties of mu and delta opioid receptors were investigated using DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), which occur, respectively, as the most selective mu and delta radioligands available. At high concentration, each agonist is able to interact with its nonspecific sites. Competition experiments indicated that a two-site competitive model was adequate to explain the interactions of DAGO and DTLET with [3H]DTLET and [3H]DAGO binding sites, respectively. The weak cross-reactivity (congruent to 10%) of DTLET for mu sites was taken into account in these experiments. On the other hand, DAGO and DTLET exhibit differential binding kinetics. Thus, at 35 degrees C, the lifetime of DTLET within its receptor site is about 14 times longer than that of the mu agonist. Sodium and manganese ions decrease the maximal number of high affinity mu and delta sites, but the sensitivity of mu receptors is three times higher towards Na+ and 20-fold higher towards Mn2+ than that of delta receptors. GTP reduces similarly the mu and delta binding whereas only the DAGO binding was modified by the nonhydrolyzable analogue guanylylimidodiphosphate [GMP-P(NH)P]. However, in the presence of Na+ ions, GMP-P(NH)P inhibits the DTLET binding in a concentration-dependent manner. The effects of Na+ and GMP-P(NH)P could be explained by a sequential transformation of delta receptors to low-affinity states. This model predicts that Na+, by lowering the affinity of a fraction of sites, produces a decrease in the maximal number of high-affinity delta receptors and that GMP-P(NH)P enhances the Na+ effect. Moreover, the binding kinetic to this high-affinity state was also modified by Na+ and nucleotides. All of these data support the existence of two independent mu and delta binding sites, the properties of which are differentially regulated by these endogenous effectors.     

μ-Opioid Receptors and Not K-Opioid Receptors Are Coupled to the Adenylate Cyclase in the Cerebellum

The putative regulatory effect of opioids on adenylate cyclase was investigated in two different preparations containing, respectively, two different populations of opioid receptors: the rabbit cerebellum (greater than 75% mu-opioid receptors) and the guinea pig cerebellum (greater than 80% kappa-opioid receptors). In the mu-preparation, but not in the kappa-preparation, opioids inhibited the basal and the forskolin-stimulated adenylate cyclase activity in a dose-dependent manner and stereospecifically. The inhibition was in the 20-30% range, required the presence in the assay medium of Mg2+ and of GTP, but was independent of the presence of Na+. Pharmacological characterization of the inhibitory response in the rabbit cerebellum clearly showed that it was under the control of a mu-opioid binding site, with the effect being elicited by non-selective (etorphine and morphine) and mu-selective (Tyr-D-Ala-Gly-Me-Phe-Gly-ol) agonists, whereas delta- and kappa-selective agonists were almost totally ineffective. ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin failed to block the inhibitory effect of opioids, and data presented suggest that this failure is likely to be the consequence of a limited access of the toxin to its substrate in rabbit cerebellum membranes.     

Modulatory Role of Glutathione on μ-Opioid, Substance P/Neurokinin-1, and Kainic Acid Receptor Binding Sites

Reduced glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) is an endogenous tripeptide involved in the formation and maintenance of protein thiol groups as well as in various detoxification reactions. Because multiple receptor types contain thiol groups or disulfide bridges, effects of GSH treatments on mu-opioid, neurokinin-1/substance P, and kainic acid receptor binding sites were investigated and compared with those produced by dithiothreitol (DTT), a potent synthetic reducing agent. GSH inhibited binding more potently than did DTT at all three receptor types in porcine striatal membrane homogenates as well as in CHAPS-solubilized preparations of the mu and neurokinin-1 sites. GSH-induced inhibitory effects were associated with decreases in maximal binding capacity (Bmax) without significant alteration in apparent affinity (KD). Cysteine, the functional moiety of GSH, mimicked GSH effects albeit with lower potencies, whereas oxidized glutathione had no effects at similar concentrations. In CHAPS-solubilized preparations, the combination of low concentrations of GSH and guanylylimidodiphosphate markedly decreased the Bmax values of the binding of [3H][D-Ala2,Gly-ol5]enkephalin and [3H]substance P. This GSH-mediated mechanism may be important to prevent cell overstimulation by accelerating receptor uncoupling, desensitization, and/or internalization. This is in keeping with purported roles of GSH related to the maintenance of cellular integrity.     

Implication of the First and Third Extracellular Loops of the μ-Opioid Receptor in the Formation of the Ligand Binding Site: A Study Using Chimeric μ-Opioid/Angiotensin Receptors

Abstract: Recent studies on chimeric μ/δ-, μ/κ- and δ/κ-opioid receptors have suggested that extracellular loops of the receptors were involved in the discriminatory binding of selective ligands by controlling their entry into the transmembrane binding site. Since homochimeric opioid receptors are mostly informative in terms of selectivity, the role of extracellular loops was examined here by studying heterochimeric μ receptors where the totality or parts of extracellular loops were replaced by the corresponding regions of the receptor for angiotensin II. Chimeric μ receptors with extracellular loop EL1 or EL3 originating from the angiotensin receptor had 100-fold decreased affinities for opioids; the length of the first extracellular loop, which is one residue longer in angiotensin than μ receptors, was shown to be responsible for this situation. Substitution of the μ receptor second extracellular loop by that of the angiotensin receptor diminished by ∼10-fold the affinities for opioids. Since all chimeras had altered affinities for selective and nonselective ligands, we propose that extracellular domains of the μ receptor, particularly the first and third loops, constrain the relative positioning of the connected transmembrane domains where selective as well as nonselective contact points form the opioid binding site.     

Guanine Nucleotide Regulation of [125I]β-Endorphin Binding to Rat Brain Membranes: Monovalent Cation Requirement

The binding of [125I]beta h-endorphin to rat brain membranes was investigated in the presence of GTP and guanylyl-5'-imidodiphosphate. In contrast to the binding of the mu-selective opioid agonist, [3H][D-Ala2,MePhe4,Glyol5]enkephalin, and the delta-selective opioid agonist, [3H][D-penicillamine2, D-penicillamine5]enkephalin, [125I]beta h-endorphin binding was not affected by GTP or guanylyl-5'-imidodiphosphate in a concentration-dependent manner in the absence of cations. However, in the presence of NaCl, the inclusion of either GTP or guanylyl-5'-imidodiphosphate resulted in a concentration-dependent inhibition of [125I]beta h-endorphin binding. This inhibition was significantly greater than the decrease in [125I]beta h-endorphin binding observed in the presence of sodium alone. Although GTP most potently inhibited [125I]beta h-endorphin binding in the presence of sodium, inhibition of [125I]beta h-endorphin binding by GTP was also observed in the presence of the monovalent cations lithium and potassium, but not the divalent cations magnesium, calcium, or manganese. The effect produced by GTP in the presence of NaCl was mimicked by GDP, but not by GMP or other nucleotides. Unlike [125I]beta h-endorphin, the binding of the putative sigma receptor agonist, (+)-[3H]SKF 10,047, was not significantly altered by GTP or guanylyl-5'-imidodiphosphate in the absence or presence of sodium.     

The δ-Opioid Receptor Regulates Activity of Ryanodine Receptors in the Human Neuroblastoma Cell Line SK-N-BE

Abstract: Recent studies have demonstrated that opioid agonists affect the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) either by regulating plasma membrane Ca²⁺-channel activity or by mobilizing intracellular Ca²⁺ stores. The present report documents the [Ca²⁺]_i increase induced by opioid agonists in a human neuroblastoma cell line, SK-N-BE, expressing δ-opioid receptors. In the presence, as well as in the absence, of extracellular Ca²⁺, opioid agonists enhanced significantly [Ca²⁺]_i, whereas carbachol, known to mobilize specifically inositol 1,4,5-trisphosphate-sensitive intracellular Ca²⁺ stores, acted only in the presence of extracellular Ca²⁺. The opioid-induced increase in [Ca²⁺]_i was not affected by treatments modifying the trimeric G_i, G_o, and G_s protein transduction mechanisms or the activity of adenylyl cyclase. The Ca²⁺-ATPase pump-inhibiting sesquiterpene lactone, thapsigargin, did not modify the opioid-induced [Ca²⁺]_i response, whereas it abolished the effects of carbachol. The Ryana speciosa alkaloid, ryanodine, at concentrations known to block endoplasmic reticulum ryanodine receptors, decreased significantly the response to opioids without affecting the effects of carbachol. Thus, our results suggest that, in SK-N-BE cells, δ-opioid receptors mobilize Ca²⁺ from intracellular ryanodine-sensitive stores and the mechanism involved is independent of G_i/G_o and G_s proteins and protein kinase A activation.     

Mutagenesis of a Single Amino Acid in the Rat μ-Opioid Receptor Discriminates Ligand Binding

Abstract: To investigate the role of Asp¹¹⁴ in the cloned rat μ-opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant μ receptor D114N. The wild-type μ receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The μ-selective agonists [ d -Ala²,MePhe⁴,Gly-ol⁵]enkephalin and morphine and the endogenous peptides Met-enkephalin and β-endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild-type μ receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild-type and D114N mutant μ receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N μ receptors to inhibit adenylyl cyclase. Although the Asp¹¹⁴ to Asn¹¹⁴ mutation reduced full-agonist binding, mutation of His²⁹⁷ to Asn²⁹⁷ in the μ receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the μ receptor than do the prototypical full agonists.     

Chronic Activation of Inhibitory δ-Opioid Receptors Cross-Regulates the Stimulatory Adenylate Cyclase-Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108-15) Hybrid Cells

Abstract: The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E₁ (PGE₁) receptor system in neuroblastoma × glioma (NG108-15) hybrid cells. Persistent activation of δ-opioid receptors by morphine (10 µmol/L; 3 days) substantially down-regulates the number of PGE₁ binding sites by ～30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTPγS (100 µmol/L) further revealed that the remaining PGE₁ binding sites are still capable of interacting functionally with their associated stimulatory G proteins, G_s. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the α subunit of G_s (G_sα) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc^? reconstitution experiments, respectively. Evaluation of the functional interaction between PGE₁ receptors and G_s by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of G_sα revealed a significant increase in the ability of PGE₁ receptors to activate G_sα (3.3-fold increase in EC₅₀; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 µmol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 µmol/L) had no further effect on sensitization in PGE₁ receptor/G_s coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE₁ receptor system represents a potential target of chronic δ-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.     

μ-Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine-Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons

The receptors mediating the inhibition of D1 dopamine receptor-stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose-dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50 = 0.05 microM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 microM). SKF 38393 (1 microM)-stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective mu-agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO; EC50 = 0.006 microM) and high concentrations of the selective delta-agonist [D-Ser2(O-tert-butyl), Leu5]-enkephalyl-Thr6 (DSTBU-LET; EC50 = 0.13 microM) but not by the selective delta-agonist [D-penicillamine2, D-penicillamine5]enkephalin (DPDPE). D1 dopamine receptor-stimulated adenylate cyclase activity was also slightly reduced (by approximately 20%) by high concentrations of the kappa-agonist U50,488 (EC50 = 0.63 microM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the mu-opioid receptor-selective antagonist naloxone (EC50 of approximately 0.1 microM). Neither the irreversible delta-ligand fentanyl isothiocyanate (1 microM) nor the reversible delta-antagonist ICI 174864 (1 microM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (greater than 0.1 microM) of the kappa-opioid receptor-selective antagonist norbinaltorphimine. The effect of DAGO (1 microM) was already detectable after 1 day in culture, whereas DPDPE (1 microM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of mu-opioid receptors coupled as inhibitors to D1 dopamine receptor-stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.     

Cloning, Characterization, and Distribution of a μ-Opioid Receptor in Rat Brain

Abstract: We report the isolation and characterization of a rat cDNA clone encoding a μ-opioid receptor. This receptor, a 398 amino acid protein, shares 59% overall identity with the mouse Δ-and K -opioid receptors. Transient expression of the receptor in COS cells revealed high-affinity binding of μ-selective opioid antagonists and agonists, with a K _D for naloxone ∼1.5 n M , and for [D-Ala², N -Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) and morphine at the high-affinity site of 2–4 n M , confirming a μ-opioid pharmacological profile. Northern blotting and in situ hybridization histoohemistry revealed that the μ-opioid receptor mRNA was expressed in many brain regions, including cerebral cortex, caudate putamen, nucleus accumbens, olfactory tubercle, septal nuclei, thalamus, hippocampus, and medial habenular nucleus, in keeping with the known distribution of the μ-opioid receptor.     

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.     

Nociceptin (ORL-1) and μ-Opioid Receptors Mediate Mitogen-Activated Protein Kinase Activation in CHO Cells Through a Gi-Coupled Signaling Pathway: Evidence for Distinct Mechanisms of Agonist-Mediated Desensitization

Abstract: The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to G_i/G_o proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of β-adrenergic receptor kinase (βARKct), which specifically blocks Gβγ-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21^ras, also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing μ-opioid receptor, [d -Ala²,N-Me-Phe⁴,Gly-ol]-enkephalin (DAMGO; a μ-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, βARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and μ-opioid receptors mediate MAP kinase activation via a signaling pathway using the βγ-subunit of G_i, a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and μ-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate μ-opioid receptor signaling in a cellular system.     

Correlating δ13C and δ18O in cellulose of trees

We measured the carbon and oxygen isotopic composition of stem cellulose of Pinus sylvestris, Picea abies, Fagus sylvatica and Fraxinus excelsior. Several sites along a transect of a small valley in Switzerland were selected which differ in soil moisture conditions. At every site, six trees per species were sampled, and a sample representing a mean value for the period from 1940 to 1990 was analysed. For all species, the mean site δ¹³C and δ¹⁸O of stem cellulose are related to the soil moisture availability, whereby higher isotope ratios are found at drier sites. This result is consistent with isotope fractionation models when assuming enhanced stomatal resistance (thus higher δ¹³C of incorporated carbon) and increased oxygen isotope enrichment in the leaf water (thus higher δ¹⁸O) at the dry sites. δ¹⁸ O-δ¹³C plots reveal a linear relationship between the carbon and oxygen isotopes in cellulose. To interpret this relationship we developed an equation which combines the above-mentioned fractionation models. An important new parameter is the degree to which the leaf water enrichment is reflected in the stem cellulose. In the combined model the slope of the δ¹⁸O-δ¹³C plot is related to the sensitivity of the p_i/p_a of a plant to changing relative humidity.     

Bulk leaf δ18O and δ13C reflect the intensity of intraspecific competition for water in a semi-arid tussock grassland

We investigated the extent to which plant water and nutrient status are affected by intraspecific competition intensity and microsite quality in a monodominant tussock grassland. Leaf gas exchange and stable isotope measurements were used to assess the water relations of Stipa tenacissima tussocks growing along a gradient of plant cover and soil depth in a semi-arid catchment of Southeast Spain. Stomatal conductance and photosynthetic rate decreased with increasing intensity of competition during the wet growing season, leading to foliar δ ¹⁸O and δ ¹³C enrichment. A high potential for runoff interception by upslope neighbours exerted strong detrimental effects on the water and phosphorus status of downslope S. tenacissima tussocks. Foliar δ ¹⁵N values became more enriched with increasing soil depth. Multiple stepwise regression showed that competition potential and/or rhizosphere soil depth accounted for large proportions of variance in foliar δ ¹³C, δ ¹⁸O and δ ¹⁵N among target tussocks (57, 37 and 64%, respectively). The results presented here highlight the key role that spatial redistribution of resources (water and nutrients) by runoff plays in semi-arid ecosystems. It is concluded that combined measurement of δ ¹³C, δ ¹⁸O and nutrient concentrations in bulk leaf tissue can provide insight into the intensity of competitive interactions occurring in natural plant communities.     

Structure of α6β3δ GABAA receptors and their lack of ethanol sensitivity

Delta (δ) subunit containing GABA_A receptors are expressed extra‐synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with α₆ subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA_A receptor pentamers by subunit concatenation. These receptors (composed of α₆, β₃ and δ subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one and to 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. α₆‐β₃‐α₆/δ receptors showed a substantial response to GABA alone. Three receptors, β₃‐α₆‐δ/α₆‐β₃, α₆‐β₃‐α₆/β₃‐δ and β₃‐δ‐β₃/α₆‐β₃, were only uncovered in the combined presence of the neurosteroid 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one with GABA. All four receptors were activated by 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the δ subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the δ subunit can assume multiple positions in a receptor pentamer composed of α₆, β₃ and δ subunits.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.