Differential binding properties of oripavines at cloned μ- and δ-opioid receptors
Introduction
Opioid receptors are members of the seven transmembrane domain receptor superfamily that are coupled to pertussis toxin sensitive G-proteins (e.g., Dhawan et al., 1996). Evidence from numerous studies shows that the presence of sodium ions and guanine nucleotides reduces agonist binding to opioid receptors while the binding of antagonists is unaffected (Childers and Snyder, 1980; Puttfarcken et al., 1986; Werling et al., 1986; Emmerson et al., 1996). The shift in affinity caused by sodium ions and guanine nucleotides correlates with agonist efficacy (e.g., Childers and Snyder, 1980). These findings support a conformational selection model of agonist action whereby agonist binding to the high affinity state of the receptor shifts the equilibrium in favor of this state, and away from the low affinity form, thereby invoking a response (e.g., Kenakin, 1997).
Buprenorphine, an oripavine derived from thebaine, is a moderate efficacy, μ-opioid agonist with analgesic effects that are 25-times more potent than morphine (Cowan et al., 1977a, Cowan et al., 1977b). From a receptor interaction viewpoint, buprenorphine is of interest because, unlike other opioid agonists, its binding affinity is not affected by the presence of sodium ions (Villiger, 1984; Rothman et al., 1995). However, these conclusions were made from studies performed using rat brain membranes that contain a heterogeneous population of opioid receptors. Consequently it is unclear whether the binding properties of buprenorphine are indicative of its binding only to the μ-opioid receptor. For example, buprenorphine acts as an antagonist at the κ-opioid receptor (Leander, 1987; Negus and Dykstra, 1988; Toll et al., 1998) and has no agonist action at the δ-opioid receptor (Toll et al., 1998). Therefore, antagonist binding of buprenorphine to non-μ-opioid receptors could explain the lack of a Na+ and guanine nucleotide induced shift.
In order to elucidate the binding properties of buprenorphine to specific opioid receptors, cloned μ- and δ-opioid receptors expressed in C6 glioma cells (C6 mu) which do not endogenously express any opioid receptors (Klee and Nirenberg, 1974) were used. During the course of these experiments, it was established that other oripavines with opioid agonist properties also bind to the μ-opioid receptor in a Na+-insensitive fashion. The oripavines examined in addition to buprenorphine were the agonists, dihydroetorphine (Kamei et al., 1995) and etorphine (Blane et al., 1967; Walker et al., 1998), and the antagonist, diprenorphine (Dewey and Harris, 1971; Traynor et al., 1987). The degree of relative agonist efficacy of the compounds was compared in the same cell systems using the []GTPγS binding assay. (Traynor and Nahorski, 1995; Emmerson et al., 1996; Clark et al., 1997; Alt et al., 1998).
The findings of this study show that opioid receptor agonist oripavines bind to the cloned μ-opioid receptor in a fashion that is insensitive to the presence of the non-hydrolysable GTP analog, Gpp(NH)p, while sodium ions produce a slight shift in dihydroetorphine and etorphine binding affinities. This insensitivity may be specific to the μ-opioid receptor since at the cloned δ-opioid receptor expressed in C6 cells (C6 delta) oripavine binding is sensitive to Na+ and guanine nucleotides.
Section snippets
Chemicals
[]Diprenorphine (45 Ci/mmol and 58 Ci/mmol), []naloxone (54.6 Ci/mmol), []naltrindole (33 Ci/mmol) and []GTPγS, (guanosine-5′-O-(3-thio)triphosphate, 1250 Ci/mmol) were purchased from DuPont NEN (Boston, MA). DAMGO ([Tyr-d-Ala2, N-Me-Phe4, Gly-ol5]enkephalin), pertussis toxin, Gpp(NH)p (5′-guanylylimido-diphosphate) and GDP (guanosine diphosphate) were purchased from Sigma (St. Louis, MO). DPDPE ([d-Pen2-d-Pen5]enkephalin) and SNC-80 ((+)-4-((α-R)-α-[(2S,5R
[]Diprenorphine binding
The C6 mu cells used in this study expressed μ receptors at a level of 435±75 fmols mg−1 protein as determined with []diprenorphine, with a Kd for []diprenorphine of 0.13±0.01 nM. The number of receptors in C6 delta cells defined with []naltrindole was 955±54 fmols mg−1 protein, with a Kd for []naltrindole of 0.01±0.003 nM. Wild-type C6 cells demonstrated no specific binding of []diprenorphine at radioligand concentrations of 0.001 to 5 nM, at a protein level which afforded 80%
Discussion
The oripavines dihydroetorphine and etorphine stimulated []GTPγS binding to membranes from C6 mu cells to a higher degree than the highly efficacious agonists DAMGO, fentanyl and morphine. The relative efficacy of moderately efficacious agonists at this receptor was in the order buprenorphine>pentazocine>butorphanol>nalbuphine, while diprenorphine and naloxone were antagonists in these cells confirming and extending previous findings (Emmerson et al., 1996).
Previous studies have shown that
Acknowledgements
This work was supported by National Institute of Drug Abuse Grants DA00254 and DA 02265 and NIGMS (GM07767).
References (42)
- et al.
Receptor-effector coupling by G proteins
Biochim. Biophys. Acta
(1990) - et al.
Regulation of opioid antagonist and mu, kappa or delta agonist binding by guanine nucleotide and sodium
Jpn. J. Pharmacol.
(1984) - et al.
Physical separation of the agonist and antagonist forms of a mu opiate receptor?
Life Sciences
(1983) - et al.
Antinociceptive effect of dihydroetorphine in diabetic mice
Eur. J. Pharmacol.
(1995) - et al.
A single residue, aspartic acid 95, in the δ opioid receptor specifies selective high affinity agonist binding
J. Biol. Chem.
(1993) - et al.
Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via α-adrenergic, cholinergic, and opiate receptors in neuroblastoma×glioma hybrid cells
J. Biol. Chem.
(1983) - et al.
Rate of onset and offset of action of narcotic analgesics in isolated preparations
Eur. J. Pharmacol.
(1975) - et al.
[]etorphine and []diprenorphine receptor binding in vitro and in vivo: differential effect of Na+ and guanylyl imidodiphosphate
Brain Res.
(1982) Buprenorphine has potent kappa opioid receptor antagonist activity
Neuropharmacology
(1987)- et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem.
(1951)
Lipophilicity of opioids determined by a novel micromethod
J. Pharmacol. Toxicol.
Kappa antagonist properties of buprenorphine in the shock titration procedure
Eur. J. Pharmacol.
In vivo receptor binding of oripavines to μ, δ, and κ-sites in rat brain as determined by an ex-vivo radiolabeling method
Eur. J. Pharmacol.
Diprenorphine has agonist activity at opioid kappa-receptors in the myenteric plexus of the guinea pig ileum
Eur. J. Pharmacol.
Binding of buprenorphine to opiate receptors
Neuropharmacology
Sodium regulation of opioid agonist binding is potentiated by pertussis toxin
Biochem. Biophys. Res. Commun.
Stimulation of guanosine-5′-0-(3-[]thio) triphosphate binding by endogenous opioids acting at a cloned mu receptor
J. Pharmacol. Exp. Ther.
Actions of etorphine hydrochloride, (M99): a potent morphine-like agent
Br. J. Pharmacol.
Mutagenesis of a single amino acid in the rat μ-opioid receptor discriminates ligand binding
J. Neurochem.
The physiological relevance of low agonist affinity binding at opioid μ-receptors
Br. J. Pharmacol.
Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction
Biochem. Pharmacol.
Cited by (102)
Thienorphine induces antinociception without dependence through activation of κ- and δ-, and partial activation of μ- opioid receptor
2020, Brain ResearchCitation Excerpt :In contrast to the G protein-mediated pathway, buprenorphine increased pERK1/2 through activation of the β-arrestin-mediated pathway in CHO– κ-PKAcatEGFP cells [Fig. 6B]. At the δ receptor, buprenorphine shows no agonistic effects (Toll et al., 1998; Lee et al., 1999; Romero et al., 1999). In the present study, buprenorphine exhibited a partial agonist activity of δ receptor compared with SNC80 or thienorphine [Fig. 4E].
In vitro profiling of opioid ligands using the cAMP formation inhibition assay and the β-arrestin2 recruitment assay: No two ligands have the same profile
2020, European Journal of PharmacologyCitation Excerpt :Full efficacy was only achieved at 10 μM, a concentration several orders of magnitude higher than its IC50 (0.002 μM) and that is highly unlikely to be achieved in the CNS at clinically administered doses in humans (Elkader and Sproule, 2005; Smith and Houghton, 2013). This observation is consistent with its partial agonist activity at the MOP-receptor when assessed using electrophysiological methods in rat brain slices (Virk et al., 2009), or the [35S]-GTPγS assay in membranes from cultured cells (Lee et al., 1999; Selley et al., 1997; Traynor and Nahorski, 1995), or in rat thalamic membranes (Selley et al., 1997). This partial MOP-receptor agonist activity is thought to underpin the ceiling-effect for buprenorphine in humans and animals, as well as some side-effects (Lutfy et al., 2003).
Molecular Basis of Opioid Action: From Structures to New Leads
2020, Biological PsychiatryPreface
2018, Progress in Brain ResearchModulation of opioid receptor affinity and efficacy via N-substitution of 9β-hydroxy-5-(3-hydroxyphenyl)morphan: Synthesis and computer simulation study
2017, Bioorganic and Medicinal Chemistry