Research reportLong-term interactions between opioid and cannabinoid agonists at the cellular level: cross-desensitization and downregulation
Introduction
Opioids and cannabinoids are two separate groups of psychoactive drugs that share a similar pharmacological profile. Both opioid and cannabinoid agonists induce analgesia, hypothermia, sedation, hypotension, inhibition of intestinal motility, modulation of the immune system, and motor depression. The two groups of drugs activate different receptors, opioid (MOR, DOR and KOR) and cannabinoid (CB1 and CB2) receptors. These receptors belong to the super-family of seven-transmembrane G-protein-coupled receptors (GPCR), they activate Gi/Go GTP-binding proteins, and modulate similar intracellular systems, including the cAMP-protein kinase cascade, the MAPK cascade, and voltage-dependent potassium and calcium channels [18], [21].
A prolonged exposure to either opioid or cannabinoid agonists results in a reduction in the responsiveness of the receptors termed ‘desensitization’ or ‘tolerance’. At the cellular level, the reduction in function begins immediately after the application of the agonist and occurs in two separate, but closely related, steps: (a) desensitization due to uncoupling of the receptors from their G-proteins and (b) reduction in receptor density on the cell surface (‘downregulation’). A continuous exposure to either opioid or cannabinoid agonists was found to reduce the activation of G-proteins and the ability of agonists to inhibit cAMP production in several brain regions as well as in various cell lines [5], [12], [19], [36], [37]. Similarly, downregulation of opioid and cannabinoid receptors was evident in most studies examining long term effects of opioid and cannabinoid agonists in the brain and in cell lines [5], [19], [29], [32], [41].
The similarity between the effects of opioid and cannabinoid agonists, both in vivo and in vitro, raises the possibility of interactions between these two groups of drugs. Indeed, several in vivo studies revealed acute as well as chronic interactions. Opioid antagonists were found to block the effect of cannabinoids [27], [39], [44], and synergism between opioids and cannabinoids has been reported in studies using sub-effective doses of these compounds [8], [39]. Chronic exposure to opioid agonists induced tolerance to the antinociceptive effect of the cannabinoid 9Δ-THC [4], [17], [40], [42]. Similarly, 9Δ-THC induced tolerance to the antinociceptive effects of opioids [40], [45]. In other studies, however, no cross-tolerance was observed [22], [23]. In most cases, these in vivo interactions have been explained as resulting from a sequential activation of the endogenous opioid and cannabinoid systems [43], [46]. Nevertheless, since opioid and cannabinoid receptors are co-expressed by the same neurons [7], [10], [28], interactions can take place at the cellular level as well. Indeed, we [33] and others [11] previously demonstrated cross-desensitization between opioid and cannabinoid agonists in N18TG2 murine neuroblastoma and its hybrid NG108,15 cell-line.
The goal of the present study was to investigate long-term interactions between opioid and cannabinoid agonists along the intracellular signal transduction pathway. For that purpose we transfected COS-7 and HEK-293 cells with both δ-opioid and CB1-cannabinoid receptors, and measured the chronic effect of opioid and cannabinoid drugs on receptor density, G-proteins activation and inhibition of cAMP production. As expected, opioid and cannabinoid agonists induced homologous desensitization in both COS-7 and HEK-293 cell lines. We further found that opioids induced heterologous desensitization and reduced the responsiveness of both cell lines to cannabinoids, while cannabinoids induced heterologous desensitization in COS-7 cells, but not in HEK-293 cells. In all cases, a correlation was found between the ability of an agonist to induce desensitization and its ability to downregulate the receptors, suggesting that, under the present experimental conditions, desensitization resulted primarily from receptor downregulation. This hypothesis was further supported by the findings that agents which interfered with opioid receptor downregulation also interfered with opioid desensitization.
Section snippets
Cell culture and transfection
COS-7 and HEK-293 cells were grown in 10-cm petri dishes at 37 °C in a humidified atmosphere of 5% CO2/95% air. The cells were maintained in DMEM medium supplemented with either 5% (COS-7) or 10% (HEK-293) fetal calf serum (FCS), 40 units/ml penicillin and 40 μg/ml streptomycin for 3–7 days until reaching confluence and then split 1:10 to new 10-cm petri dishes using Trypsin–EDTA.
COS-7 cells were co-transfected with 2 μg/ml of mouse δ-opioid receptor (DOR-1) in pcDM8 vector [14], and SKR6 cDNA
Results
In HEK-CBDOR cells, forskolin (10 μM) elevated basal cAMP production more than 20-fold. The cannabinoid agonist desacetyllevonantradol (DALN; 1 μM) inhibited forskolin-stimulated cAMP production by 32±2.1% (P<0.001; n=19), and the opioid agonist etorphine (1 μM) inhibited production by 70±3.5% (P<0.001; n=16). Similar inhibition was achieved by a 100 nM concentration of each agonist (29±4% and 72±3% inhibition by DALN and etorphine, respectively; P<0.001; n=8), suggesting that a maximal effect
Discussion
A continuous use of either opioid or cannabinoid drugs leads to the development of tolerance [3], [15]. Several in vivo studies reported that cross-tolerance between these two groups of drugs also occur [4], [17], [40], [42]; however, the mechanism underlying cross-tolerance is not clear. Since cannabinoids activate the endogenous opioid system [43], [46], a simple explanation might be that the observed cross-tolerance represents a homologous desensitization of the activated opioid system.
Acknowledgements
This study was supported by The Israel Science Foundation founded by The Israel Academy of Sciences and Humanities (grant #184-99)
References (47)
- et al.
Isolation and expression of a mouse CB1 cannabinoid receptor gene—comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells
Biochem. Pharmacol.
(1997) - et al.
Development of behavioral tolerance to delta 9-THC without alteration of cannabinoid receptor binding or mRNA levels in whole brain
Pharmacol. Biochem. Behav.
(1993) - et al.
Regulation of delta opioid receptors by Delta(9)-tetrahydrocannabinol in NG108-15 hybrid cells
Life Sci.
(1998) - et al.
Opiate tolerance and dependence: receptors, G-proteins, and antiopiates
Peptides
(1998) - et al.
Cellular signal transduction by anandamide and 2-arachidonoylglycerol
Chem. Phys. Lipids
(2000) - et al.
Deltorphin II-induced rapid desensitization of delta-opioid receptor requires both phosphorylation and internalization of the receptor
J. Biol. Chem.
(2000) - et al.
Two distinctive antinociceptive systems in rats with pathological pain
Neurosci. Lett.
(2000) - et al.
Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization
J. Biol. Chem.
(1999) - et al.
A role for central cannabinoid and opioid systems in peripheral Delta(9)-tetrahydrocannabinol-induced analgesia in mice
Eur. J. Pharmacol.
(1996) Cellular responsiveness to hormones and neurotransmitters: conversion of [3H]adenine to [3H]cAMP in cell monolayers, cell suspensions, and tissue slices
Methods Enzymol.
(1991)
Long-term regulation of opioid receptors in neuroblastoma and lymphoma cell lines
J. Neuroimmunol.
Independence of, and interactions between, cannabinoid and opioid signal transduction pathways in N18TG2 cells
Brain Res.
Diverse pathways mediate delta-opioid receptor down regulation within the same cell
Mol. Brain Res.
Differences in G-protein activation by mu- and delta-opioid, and cannabinoid, receptors in rat striatum
Eur. J. Pharmacol.
The enhancement of morphine antinociception in mice by Delta(9)-tetrahydrocannabinol
Pharmacol. Biochem. Behav.
Evidence for a bidirectional cross-tolerance between morphine and delta(9)-tetrahydrocannabinol in mice
Eur. J. Pharmacol.
Blockade of cannabinoid-induced antinociception by naloxone benzoylhydrazone (Nalbzh)
Pharmacol. Biochem. Behav.
Characterization of anandamide-induced tolerance: comparison to Delta(9)-THC-induced interactions with dynorphinergic systems
Drug Alcohol Depend.
Synergistic interactions of endogenous opioids and cannabinoid systems
Brain Res.
Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization
J. Biol. Chem.
Molecular-cloning and expression of a delta-opioid receptor from rat-brain
J. Neurosci. Res.
A comparison of some pharmacological actions of morphine and Δ9-tetrahydrocannabinol in the mouse
Psychopharmacology
Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and cannabinoid receptor-activated G proteins in rat brain
J. Neurochem.
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