Agonists determine the pattern of G-protein activation in μ-opioid receptor-mediated supraspinal analgesia

doi:10.1016/S0361-9230(00)00448-2

Brain Research Bulletin

Volume 54, Issue 2, 15 January 2001, Pages 229-235

https://doi.org/10.1016/S0361-9230(00)00448-2 Get rights and content

Abstract

The opioids heroin, methadone, buprenorphine, and morphine produce supraspinal antinociception in CD-1 mice that is antagonized by Cys², Tyr³, Orn⁵, Pen⁷-amide but not by naltrindole or nor-binaltorphimine. The patterns of GTP-binding regulatory proteins (G-proteins) activation exhibited by these agonists at μ-opioid receptors were characterized. The expression of α-subunits of Gi-protein classes, Gi1, Gi2, Gi3, Go1, Go2 and Gz, and those of the Gq-protein family, Gq and G11, was reduced by administration of antisense oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. The ODN treatments demonstrated differences in the analgesic profiles of these opioids. Though the knock-down of G_i2α or G_zα subunits diminished the analgesic effets of the four opioids, impairment of G_i3α did not modify the potency of morphine. In mice with reduced G_i1α, G_o1α or G₁₁α levels, antinociception induced by heroin and methadone was diminished, but buprenorphine and morphine showed no change in their effects. Also, antinociception induced by heroin and buprenorphine, but neither morphine nor methadone, required intact G_o2α or G_qα levels. Thus, morphine, heroin, methadone, and buprenorphine showed different patterns of G-protein activation in evoking μ-opioid receptor-mediated supraspinal antinociception. Therefore, after binding identical receptors, each agonist determines the classes of GTP-binding regulatory transducer proteins to be activated.

Introduction

Pharmacological studies indicate that the μ opioid receptor is the major site responsible for the antinociception induced by morphine and morphine-like compounds [10]. The opioid receptors couple with a variety of GTP-binding regulatory proteins (G-proteins). In recent years, the identification of the G-protein classes activated by morphine and opioid peptides in the production of supraspinal antinociception has been accomplished by the use of specific antibodies or knock-down of particular G-protein subunits using antisense oligodeoxynucleotides (ODNs) 9, 20, 21, 25. These studies revealed the significance and diversity of G-proteins involved in supraspinal antinociception mediated not only by opioid- but also non-opioid- receptors.

It is known that coupling of receptors to G-proteins is essential for increasing their affinity toward agonists, but not antagonists [2]. The classes of Gα subunits are not homogeneous in the peptide sequences that interact with G-receptors. This fact, plus the finding that not all ligands bind to identical domains in the μ-opioid receptor 3, 27, 29 led to the proposal that opioid agonists not only display different efficacies in activating their receptors when coupled to one or another G-protein, but that they also bind with different affinities these receptor-G-protein complexes. Discernment of conformational coupled states of the μ-opioid receptor is therefore achieved [8]. The possibility of agonist efficacy to depend on the classes of G-proteins activated by the liganded receptor has been suggested in certain expression systems: e.g., the Drosophila octopamine-tyramine receptor in Chinese hamster ovary (CHO) cells [17] the pituitary adenylyl cyclase activating polypeptide receptor transfected into LLCPK1 cells [24]. Patterns of G-protein-dependent agonist-receptor interactions might also account for differences of cyclic adenosine monophosphate (cAMP)-dependent protein kinase phosphorylation of μ-opioid receptors [1].

Considering the capacity of receptors to discriminate among G-proteins and the agonist-dependent binding domains of the receptor, it was hypothesized that certain opioid agonists promote one receptor-G-protein complex while others favor the association of the receptor with distinct G-proteins 5, 7, 8. The present paper substantiates this concept by studying the classes of G-proteins activated after intracerebroventricular (i.c.v.) administration of a series of morphine derivatives used—or abused—by man: heroin, the most widely abused opioid, methadone, a compound useful in substitution therapy for opioid addiction, and buprenorphine, which has a lower abuse potential and is of clinical benefit in treating chronic pain. The expressions of the α-subunits of the Gi1, Gi2, Gi3, Go1, Go2, Gz, Gq, and G11 transducer proteins were reduced by administration of ODNs complementary to sequences of their respective mRNAs. The results from this study further suggest that, after binding to μ-opioid receptors, agonists can promote activation of different G-proteins.

Section snippets

Animals and evaluation of analgesia

Albino male mice CD-1 (Charles River, Barcelona, Spain) weighing 22–25 g were used throughout. Animals were kept at 22°C and a 12-h light/dark cycle (0800 h/2000 h) was established. Food and water were provided ad libitum. Mice were housed and used strictly in accordance with the guidelines of the European Community on the care and use of laboratory animals. To reduce the possibility of interference from spinal events, all substances were i.c.v.-injected into the right lateral ventricle, as

Activity of heroin, methadone and buprenorphine as analgesics in the warm water tail-flick test

These compounds induced a dose-dependent antinociception after i.c.v.-injection into CD-1 mice (Fig. 1). Single doses produced effects that peaked at approximately 10 min. Analgesia lasted 45–60 min. The ED₅₀ values were 14.8 ± 2.2, 80.6 ± 8.3, and 97.5 ± 13.7 (n = 15) nmols per mouse for heroin, methadone, and buprenorphine, respectively. Morphine was the more potent opiate in the test (ED₅₀ 5.9 ± 1.7 nmols per mouse) with a peak effect 30 min after i.c.v. administration (Fig. 1). The

Discussion

This study shows the participation of multiple classes of G-proteins in the supraspinal antinociception induced by heroin, methadone, and buprenorphine. As previously reported for morphine, [D-Ala²,N,Me-Phe⁴,Gly-ol]-enkephalin (DAMGO) and the endogenous opioid peptides endomorphin-1 and endomorphin-2 [22], the agonists in this study exhibited differences in activating the variety of G-proteins regulated by μ-opioid receptors (Fig. 4). Thus, the pattern of G-protein regulation depends on the

Acknowledgements

This work was supported by funding from the Comisión Interministerial de Ciencia y Tecnologı́a (grant CICYT SAF98-0057) and the Programa Sectorial de Promoción General del Conocimiento (grant DGICYT PB98-0649).

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Agonists determine the pattern of G-protein activation in μ-opioid receptor-mediated supraspinal analgesia

Abstract

Introduction

Section snippets

Animals and evaluation of analgesia

Activity of heroin, methadone and buprenorphine as analgesics in the warm water tail-flick test

Discussion

Acknowledgements

Distinct differences between morphine- and [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin-μ-opioid receptor complexes demonstrated by cyclic AMP-dependent protein kinase phosphorylation

J. Neurochem.

A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor

J. Biol. Chem.

Location of regions of the opioid receptor involved in selective agonist binding

J. Biol. Chem.

αN-acetyl derivatives of β-endorphin (1-31) and (1-37) regulate the supraspinal antinociceptive activity of different opioids in mice

Life Sci.

Dissimilar efficacy of opioids to produce mu-mediated analgesiaRole of Gz and Gi2 transducer proteins

Life Sci.

Mu-opioid receptors regulate pertussis toxin insensitive Gz and Gq transducer proteins in the production of supraspinal analgesia in the mouse

Analgesia

Opioids binding mu and delta receptors exhibit diverse efficacy in the activation of Gi2 and Gx/z transducer proteins in mouse periaqueductal gray matter

J. Pharmacol. Exp. Ther.

Influence of Gz and Gi2 transducer proteins in the activity of opioid agonists to μ receptors

Eur. J. Neurosci.

In vivo modulation of G-proteins and opioid receptor function by antisense oligodeoxynucleotides

Meth. Enzymol.

Molecular characterization of opioid receptors

Ann. Rev. Pharmacol. Toxicol.

A comparative study of physiological and subjective effects of heroin and morphine administered intravenously in postaddicts

J. Pharmacol. Exp. Ther.

Blood-brain barrierPenetration of morphine, codeine, heroin and methadone after carotid injection

Science

Mapping of opioid receptors using antisense oligodeoxynucleotidesCorrelating their molecular biology and pharmacology

Trends Pharmacol. Sci.

Pharmacological characterization of buprenorphine, a mixed agonist-antagonist with k3 analgesia

Brain Res.

Distinct differences between morphine- and [D-Ala²,N-MePhe⁴,Gly-ol⁵]-enkephalin-μ-opioid receptor complexes demonstrated by cyclic AMP-dependent protein kinase phosphorylation

^αN-acetyl derivatives of β-endorphin (1-31) and (1-37) regulate the supraspinal antinociceptive activity of different opioids in mice