Elsevier

Biochemical Pharmacology

Volume 81, Issue 10, 15 May 2011, Pages 1248-1254
Biochemical Pharmacology

Distinct pharmacological properties of morphine metabolites at Gi-protein and β-arrestin signaling pathways activated by the human μ-opioid receptor

https://doi.org/10.1016/j.bcp.2011.03.001Get rights and content

Abstract

Morphine and several other opioids are important drugs for the treatment of acute and chronic pain. Opioid-induced analgesia is predominantly mediated by the μ-opioid receptor (MOR). When administered to humans, complex metabolic pathways lead to generation of many metabolites, nine of which may be considered major metabolites. While the properties of the two main compounds, morphine-6-glucuronide and morphine-3-glucuronide, are well described, the activity of other morphine metabolites is largely unknown. Here we performed an extensive pharmacological characterization by comparing efficacies and potencies of morphine and its nine major metabolites for the two main signaling pathways engaged by the human MOR, which occur via Gi-protein activation and β-arrestins, respectively. We used radioligand binding studies and FRET-based methods to monitor MOR-mediated Gi-protein activation and β-arrestin recruitment in single intact 293T cells. This approach identified two major groups of morphine metabolites, which we classified into “strong” and “weak” receptor ligands. Strong partial agonists morphine, morphine-6-glucuronide, normorphine, morphine-6-sulfate, 6-acetylmorphine and 3-acetylmorphine showed efficacies in the nanomolar range, while the weak metabolites morphine-N-oxide, morphine-3-sulfate, morphine-3-glucuronide and pseudomorphine activated MOR pathways only in the micromolar range. Interestingly, three metabolites, normorphine, 6-acetylmorphine and morphine-6-glucuronide, had lower potencies for Gi-protein activation but higher potencies and efficacies for β-arrestin recruitment than morphine itself, suggesting that they are biased towards β-arrestin pathways.

Introduction

Morphine is an important alkaloid from opium poppy and serves as the prototypical analgesic drug. Morphine and some other opioids, for example 3,6-di-acetylmorphine (heroin) are also relevant for drug abuse. Analgesic effects of opioids are achieved largely via activation of μ-opioid receptors (MOR) [1], [2]. MOR is a G-protein-coupled receptor (GPCR), which can activate several cellular signaling pathways. The first pathway is mediated by the activation of inhibitory pertussis-toxin sensitive G-proteins (Gi/o-proteins). The second pathway is induced by the recruitment of β-arrestins to the receptor, which leads to subsequent activation of other, so-called “non-classical” signaling cascades such as the mitogen activated protein kinase pathway [3], [4].

When administered to humans, morphine and other opiate alkaloids undergo extensive biotransformation by various metabolic pathways. The predominant metabolic pathway involves conjugation with glucuronic acid, which leads to the major metabolites morphine-3-glucuronide and morphine-6-glucuronide [5]. Other metabolic pathways include conjugation with sulfonic acid, leading to morphine-6-sulfates and morphine-3-sulfates, acetylation to 3- and 6-acetylmorphines, oxidation to morphine-N-oxide and demethylation to normorphine [6], [7] (Fig. 1). Among all known morphine metabolites, the pharmacological properties have been best studied for morphine-6-glucuronide and morphine-3-glucuronide, which are generally considered as active and inactive metabolites, respectively [8]. Much less is known about the activity of other metabolites, since no systematic pharmacological analysis has so far been performed, and only rare single reports on these compounds are available. Lack of information about opiate metabolites does not allow definitive conclusions about their roles in analgesia and in adverse effects of morphine [6], [7].

To study the pharmacological properties of various ligands for G protein-coupled receptors, including MOR, we have developed a new real-time approach to monitor Gi-protein activation in intact cells by using biosensors which are based on Förster resonance energy transfer (FRET) [9], [10], [11]. In addition, we have developed another technique to monitor β-arrestin recruitment to various receptors by measuring FRET between a receptor and β-arrestin [12], [13]. These approaches allow us to study pharmacological properties of different receptors and their ligands in single intact cells with high temporal and spatial resolution [14], [15].

Here we used these FRET assays together with radioligand binding studies to perform a systematic analysis of the pharmacological properties of all major morphine metabolites at the human MOR. We found that morphine metabolites exert either strong or weak signaling effects, often comparable to morphine itself, and that some metabolites show a previously unrecognized β-arrestin-biased behavior and are more efficacious in recruiting β-arrestin to the receptor than morphine itself.

Section snippets

Substances

Morphine, morphine-6-glucuronide, morphine-3-glucuronide and DAMGO ([d-Ala2, N-MePhe4, Gly-ol]-enkephalin) were purchased from Sigma–Aldrich (Deisenhofen, Germany). Normorphine, 3-acetylmorphine and 6-acetylmorphine were from Lipomed GmbH (Weil am Rhein, Germany).

Morphine-N-oxide, morphine-3-sulfate, morphine-6-sulfate and pseudomorphine were synthesized as follows. Morphine-N-oxide and pseudomorphine were prepared as described by Garrido et al. [16]. Morphine-3-sulfate was prepared via

Results

We analyzed the pharmacological properties of morphine and its nine major metabolites in three types of assays, namely radioligand binding studies, G-protein activation measurements and an assay to monitor β-arrestin recruitment to the opiate receptor. These experiments were designed to compare the action of morphine metabolites and their efficacies and potencies for two distinct signaling pathways engaged by human MOR.

Discussion

In this study, we performed a systematic analysis of the pharmacological properties of morphine metabolites at the human MOR. We analyzed the ability of these substances to bind to the receptor and to activate the two major independent downstream signaling pathways, which are activation of Gi-proteins and β-arrestin recruitment. The information obtained in these assays allows to predict, which of these metabolites might play a role in MOR signaling in vivo.

To analyze MOR activation, we used

Acknowledgements

The study was supported by the Deutsche Forschungsgemeinschaft (SFB487), the European Research Council (TOPAS) and the National Institutes of Health (NIH) (5R21DA024418-02).

References (33)

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