Differential Coupling of μ-, δ-, and κ-Opioid Receptors to Gα16-Mediated Stimulation of Phospholipase C

Abstract: The μ-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G₁₆ protein. Given the promiscuous nature of G₁₆ and the high degree of resemblance of signaling properties of the three opioid receptors, both δ- and κ-opioid receptors are likely to activate G₁₆. Interactions of δ- and κ-opioid receptors with G₁₆ were examined by coexpressing the opioid receptors and Gα₁₆ in COS-7 cells. The δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the δ-opioid receptor and Gα₁₆. The δ-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of simeter for quality control of blood units and irradiators. 13.   Transfusion 1993 ; 33 : 898 – 901 . [PubMed link] 14.   Butson MJ , Yu PK , Cheung T , et al . Dosimetry of blood irradiation with radiochromic film. Transfus Med 1999 ; 9 : 205 – 8 . [PubMed link] 15.   Nath R , Biggs PJ , Ling CC , et al . AAPM code of practice for radiotherapy accelerators: Report of AAPM Radiation Therapy Task Group No. 45. Med Phys     

μ-Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin-Sensitive G Protein

Abstract: The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both μ-and δ-opioid receptors), we demonstrated that fentanyl, a μ-preferring opioid, caused a dose-dependent (EC₅₀= 16 n M ) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μ M fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 m M carbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole-(a δ antagonist), reversible. Further studies using [ d -Ala², MePhe⁴, Gly(ol)⁵]enkephalin and [ d -Pen^2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni²⁺ (2.5 m M ) or in Ca²⁺-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μ M fentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis-treated plus 0.1 μ M fentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca²⁺-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca²⁺, could play a part in the cellular mechanisms of opioid action.     

Coupling of the Cloned μ-Opioid Receptor with the ω-Conotoxin-Sensitive Ca2+ Current in NG108-15 Cells

Abstract: Voltage-dependent Ca²⁺ currents were measured in NG108-15 neuroblastoma × glioma hybrid cells transformed to express the rat μ-opioid receptor by the whole-cell configuration of the patch-clamp technique with Ba²⁺ as charge carrier. A μ-opioid receptor-selective agonist, [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin caused significant inhibition of voltage-dependent Ca²⁺ currents in μ-receptor-transformed NG108-15 cells but not in nontransfected or vector-transformed control cells. On the other hand, a δ-opioid receptor-selective agonist, [ d -penicillamine², d -penicillamine⁵]enkephalin, induced inhibition of voltage-dependent Ca²⁺ currents in both control and μ-receptor-transformed cells, which is mediated by the δ-opioid receptor expressed endogenously in NG108-15 cells. The inhibition of voltage-dependent Ca²⁺ currents induced by [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin and [ d -penicillamine², d -penicillamine⁵]enkephalin was reduced by pretreatment of the cells with pertussis toxin or ω-conotoxin GVIA. These results indicate that the μ-opioid receptor expressed from cDNA functionally couples with ω-conotoxin-sensitive N-type Ca²⁺ channels through the action of pertussis toxin-sensitive G proteins in NG108-15 cells.     

Opioid Modulation of Extracellular Signal-Regulated Protein Kinase Activity Is Ras-Dependent and Involves Gβγ Subunits

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([ d -Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin)-, δ- ([ d -Pen², d -Pen⁵]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of G_i/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.     

Regulation of Spontaneous Activity of the δ-Opioid Receptor: Studies of Inverse Agonism in Intact Cells

Abstract: Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [³H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine δ-opioid receptor (clone D2). Basal [³H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin d -Ala², d -Leu⁵ enkephalin (DADLE) produced strong inhibition of forskolin-amplified [³H]cyclic AMP production, whereas the δ-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC₅₀ (9.4 ± 0.6 × 10⁻⁸ M ) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated K _i. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.     

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.     

Activation of Opioid and Muscarinic Receptors Stimulates Basal Adenylyl Cyclase but Inhibits Ca2+/Calmodulin- and Forskolin-Stimulated Enzyme Activities in Rat Olfactory Bulb

Abstract: In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP-dependent and pertussis toxin-sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca²⁺ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu⁵]Enkephalin and selective δ- and μ-, but not κ-, opioid receptors agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK-stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu⁵]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca²⁺/CaM- and FSK-stimulated enzyme activities. These results indicate that in rat olfactory bulb δ- and μ-opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin-sensitive GTP-binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.     

Increase in the Extracellular Histamine Concentration in the Rat Striatum by μ-Opioid Receptor Activation

Abstract: The effects of morphine and selective ligands for μ-, κ-, and δ-opioid receptors on the extracellular histamine (HA) concentration in the striatum of freely moving rats were examined by in vivo microdialysis. On the day after implantation of the dialysis probe, the HA output per 30-min period was measured using HPLC-fluorometry. Morphine (3.8 mg/kg, s.c.) significantly increased the HA output by ∼200% 1–3 h after treatment. This effect was completely antagonized by naltrexone (1.6 mg/kg, s.c.). The HA output decreased to a level below 10% of the basal value by 4 h after treatment with ( S )-α-fluoromethylhistidine (77 mg/kg, s.c.). In such animals, morphine (3.8 mg/kg, s.c.) had no influence on the HA output. [ d -Ala²,MePhe⁴,Gly(ol)⁵]Enkephalin (DAGO; 0.2 µg, i.c.v.), a selective μ-agonist, significantly increased the HA output by ∼150% 0.5–1.5 h after treatment, and this effect was also completely blocked by naltrexone. A selective κ-agonist, U-50,488 (3.8 and 7.6 mg/kg, s.c.), and a selective δ-agonist, [ d -Pen², d -Pen⁵]enkephalin (0.5 and 2 µg, i.c.v.), had no effect on the HA output. These findings suggest that the stimulation of μ-opioid receptors by morphine and DAGO increases the extracellular HA concentration by accelerating HA release from nerve endings.     

Overexpression of δ-Opioid Receptors in Recombinant Baculovirus-Infected Trichoplusia ni"High 5" Insect Cells

Abstract: "High 5" cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse δ-opioid receptor. The maximal binding capacity for the narcotic antagonist [³H]naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [³H][ d -penicillamine², d -penicillamine⁵]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at δ-receptors of NG108-15 hybrid cells and of High 5 and Sf9 insect cells. However, in insect cells the opioid was more than 100-fold less effective in competing with [³H]naltrindole as compared with the mammalian cells. This decline in potency was counteracted in a dose-dependent manner by exposure of High 5 membranes to the exogenous G protein G_o, which increased the binding capacity for DPDPE. Functional studies revealed a dose-dependent inhibition (up to 30%) by opioids on forskolin-stimulated cyclic AMP synthesis, and this effect was potentiated by G_o. Quantification of Gα_o and Gα_i disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned δ-opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of Gα_o/mg of membrane protein, and a 20-fold higher concentration for Gα_i. The distinct G-protein expression in insect cells may be considered an advantage for studying functions of G protein-coupled receptors.     

G Protein independent phosphorylation and internalization of the δ-opioid receptor

Agonist activation of the δ-opioid receptor leads to internalization via Gβγ recruitment of G protein coupled receptor kinase-2, which phosphorylates the receptor at several sites, including Ser363, allowing β-arrestin binding and localization to clathrin coated pits. Using human embryonic kidney cells expressing a δ-opioid receptor we tested the hypothesis that prevention of receptor coupling to G protein by treatment with pertussis toxin (PTX) will block these processes. PTX treatment did not reduce phosphorylation of δ-opioid receptor Ser363 in response to the agonist [ d -Pen2, d -Pen5]enkephalin, or recruitment of β-arrestin 2-green fluorescent protein to the membrane and only slowed, but did not prevent, [ d -Pen2, d -Pen5]enkephalin-induced internalization. Similarly, PTX treatment only partially prevented the ability of the δ-opioid peptide agonists deltorphin II and [Met5]enkephalin and the non-peptide agonist BW373U86 to induce receptor internalization. No internalization was seen with morphine, oxymorphindole or the putative δ₁-opioid agonist TAN-67 in the presence or absence of PTX, even though TAN-67 showed a strong activation of G protein, as measured by guanosine-5'-O-(3-[³⁵S]thio)triphosphate binding. The ability of an agonist to stimulate phosphorylation at Ser363 was predictive of its capacity to induce internalization. The results suggest a role for G protein in δ-opioid receptor internalization, but show that alternative G protein independent pathways exist.     

Pertussis Toxin-Insensitive Signaling of the ORL1 Receptor: Coupling to Gz and G16 Proteins

Abstract: Nociceptin/OFQ is the endogenous ligand for the G protein-coupled opioid receptor-like (ORL₁) receptor. To elucidate the cellular functions of the ORL₁ receptor, we examined its ability to interact with G_z and G₁₆, two pertussis toxin (PTX)-insensitive G proteins that are known molecular partners for the opioid receptors. In HEK 293 cells transiently expressing the ORL₁ and dopamine D₁ receptors, nociceptin/OFQ dose-dependently inhibited dopamine-stimulated cyclic AMP (cAMP) accumulation in a PTX-sensitive manner. However, PTX failed to block the nociceptin/OFQ-induced inhibition of dopamine-stimulated cAMP accumulation in HEK 293 cells co-expressing the α-subunit of G_z. This result indicates functional interaction between the ORL₁ receptor and G_z. A similar result was obtained with retinoic acid-differentiated SH-SY5Y cells, which endogenously express both the ORL₁ receptor and G_z. When the ORL₁ receptor was transiently co-expressed in COS-7 cells with the α-subunit of G₁₆, nociceptin/OFQ dose-dependently stimulated the formation of inositol phosphates. Nociceptin-induced stimulation of phospholipase C was absolutely dependent on the co-expression of α₁₆ and exhibited the appropriate ligand selectivity. In terms of its ability to interact with PTX-insensitive G proteins, the ORL₁ receptor behaves very much like the opioid receptors.     

Functional Coupling of the δ-, μ-, and κ-Opioid Receptors to Mitogen-Activated Protein Kinase and Arachidonate Release in Chinese Hamster Ovary Cells

Abstract: To examine whether the mitogen-activated protein kinase (MAPK) cascade and phospholipase A₂ (PLA₂) are involved in the signal transduction mechanism of the opioid receptor, the δ-, μ-, and κ-opioid receptors were stably expressed from cDNA in Chinese hamster ovary cells. Activation of the δ-, μ-, and κ-receptors by agonists induced a rapid and transient increase in MAPK activity accompanied by reduced electrophoretic mobility of the 42-kDa isoform of MAPK (p42), probably owing to phosphorylation. The opioid receptor-mediated increase in MAPK activity was suppressed not only by pretreatment with genistein, a tyrosine protein kinase inhibitor, but also by prolonged exposure to phorbol 12-myristate 13-acetate and pretreatment with GF 109203X, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of PKC as well as tyrosine protein kinase. Furthermore, stimulation of the δ-, μ-, and κ-receptors with opioid agonists in the presence of A23187, a calcium ionophore, resulted in an increase in arachidonate release, suggesting that PLA₂ is activated by the opioid receptors when the intracellular Ca²⁺ concentration is elevated. Both MAPK activation and increase in arachidonate release mediated by the opioid receptors were abolished by pretreatment with pertussis toxin, suggesting that these responses are mediated by G_i or G_o types of GTP-binding regulatory proteins.     

Characterization of Pituitary Adenylate Cyclase-Activating Polypeptide 38 (PACAP38)-, PACAP27-, and Vasoactive Intestinal Peptide-Stimulated Responses in N1E-115 Neuroblastoma Cells

Abstract: In this study, the effects of three related peptides, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, and vasoactive intestinal peptide (VIP), on cyclic AMP (cAMP) accumulation and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were compared in N1E-115 cells. PACAP38 and PACAP27 stimulated cAMP accumulation up to 60-fold with EC₅₀ values of 0.54 and 0.067 n M , respectively. The effect of VIP on cAMP accumulation was less potent. The binding of ¹²⁵I-PACAP27 to intact cells was inhibited by PACAP38 and PACAP27 (IC₅₀ values of 0.44 and 0.55 n M , respectively) but not by VIP. In fura-2-loaded cells, both PACAP38 and PACAP27 increased [Ca²⁺]_i with EC₅₀ values around 10 n M . The interactions of these three peptides with ionomycin, a Ca²⁺ ionophore, and 4β-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were also determined. Ionomycin increased the cAMP accumulation caused by all three peptides. With low concentrations of PACAP38 or PACAP27, the effect of PMA was inhibitory, whereas at higher concentrations of PACAP (>1 n M ), the effect of PMA was stimulatory. Similar to other agents that elevate cAMP, PACAP38 was an effective stimulator of neurite outgrowth. These results show that (a) PACAP27 and PACAP38 stimulate cAMP accumulation and increase [Ca²⁺]_i through the type I PACAP receptors in N1E-115 cells, (b) ionomycin enhances cAMP accumulation by all three peptides, and (c) activation of protein kinase C has a dose-dependent stimulatory or inhibitory effect on the PACAP38- or PACAP27-stimulated cAMP accumulation.     

Distinct Differences Between Morphine- and [d-Ala2,N-MePhe4,Gly-ol5]-Enkephalin- μ-Opioid Receptor Complexes Demonstrated by Cyclic AMP-Dependent Protein Kinase Phosphorylation

Abstract: The present study demonstrates a conditional, agonist-dependent phosphorylation of the μ-opioid receptor (MOR-1) by cyclic AMP-dependent protein kinase (PKA) in membrane preparations of MOR-1-transfected neuroblastoma Neuro2A cells. Opioid agonist-dependent phosphorylation occurs in a time- and concentration-dependent manner (EC₅₀∼40 n M ) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 µ M morphine and 6 n M PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and β-endorphin) stimulated phosphorylation of MOR-1 by PKA, the potent μ-opioid-selective peptide [ d -Ala², N -MePhe⁴,Gly-ol⁵]-enkephalin (DAMGO) or other enkephalin analogues such as [ d -Ala²]-Met⁵-enkephalinamide (DALA), [ d -Ala², d -Leu⁵]-enkephalin (DADLE), and Met⁵-enkephalin had no effect. The lack of the effect of DAMGO on MOR-1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist-dependent conformations of MOR-1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist-receptor conformations.     

Transmembrane 36CI− Flux Measurements and Desensitization of the γ-Aminobutyric AcidA Receptor

Abstract: Some data on the concentration range of response and the concentration for half-response (EC₅₀) of γ-aminobutyric acid (GABA) for the GABA_A receptor are reviewed and compared. An analysis of the ³⁶CI⁻ flux assay demonstrates that both the EC₅₀ and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the ³⁶CI⁻ concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC₅₀ can be decreased by orders of magnitude by increasing the assay time. The EC₅₀ measured in a finite time is less than the half-response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC₅₀ depends on the rate of receptor desensitization. The computer simulations showed that a GABA_A receptor with a half-response concentration of 100 μ M GABA can give ³⁶CI⁻ flux measurements with an EC₅₀ value 100-fold lower.     

A Subtype of κ-Opioid Receptor Mediates Inhibition of High-Affinity GTPase Inherent in Gi1 in Guinea Pig Cerebellar Membranes

Abstract: The κ-opioid receptor agonists including U-50,488H and dynorphin A (1–17) in ranges of 0.1–100 n M inhibited the hydrolysis of GTP to GDP (P_i release) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 µ M . The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 µg/ml but not with preactivated cholera toxin (30 µg/ml). Similar blockade of κ-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 µ M ) of N -ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in κ-agonist binding. When purified G_i1, but not G_o, was reconstituted into membranes pretreated with NEM, the κ-receptor-mediated inhibition was recovered. These findings suggest that a subtype of κ-opioid receptor is coupled to inhibition of intrinsic activity of G_i1.     

Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.     

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin

Abstract The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E₂ release was studied in monocytes (Mø). Both IL-1α and IL-1β increased the release of PGE₂ in a concentration-dependent manner, with EC₅₀s of 0.48 nM and 0.12 nM, respectively. Intact Mø were prelabelled with [³H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [³H]phosphatidylinositol-4,5,-bisphosphate to aqueous soluble radioactivity by Mø homogenates. IL-1α (5.8 nM) increased the accumulation of IPs within 1–4 minutes and increases in IP₃ and IP₄ occured before the increase in IP₁₊₂ whereas LPS only increased the IPs level after at least 30 min. IL-1α increased PIC activity in Mø homogenates within 15 min with an EC₅₀ of 0.58 nM and IL-1β (0.1 nM) also increased activity. Neither IL-1α nor IL-1β affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in Mø.     

The δ-Opioid Receptor in SK-N-BE Human Neuroblastoma Cell Line Undergoes Heterologous Desensitization

Abstract: The human neuroblastoma cell line SK-N-BE expresses δ-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 n M etorphine leads to an almost complete desensitization (8.2 ± 5.9 vs. 45.8 ± 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, α₂-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 µ M 2-bromo-α-ergocryptine or 1 µ M arterenol resulted in a marked desensitization of D2-dopaminergic and α₂-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the δ-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-bromo-α-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the α₂-adrenergic receptor response, whereas pretreatment with 1 µ M arterenol partly desensitized the δ-opioid receptor response. It is concluded that the δ-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterologous desensitization, and it is suggested that δ-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G_i2 protein, whereas α₂-adrenergic receptor could be coupled to the enzyme via two G proteins, G_i2 and another member of the G_i/G_o family.