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