Long-term interactions between opioid and cannabinoid agonists at the cellular level: cross-desensitization and downregulation

doi:10.1016/S0006-8993(02)03842-8

Brain Research

Volume 960, Issues 1–2, 17 January 2003, Pages 190-200

https://doi.org/10.1016/S0006-8993(02)03842-8 Get rights and content

Abstract

In the present study we investigated long-term interactions between opioid and cannabinoid drugs at several steps along their cellular signal transduction pathways. For this purpose we co-transfected HEK-293 and COS-7 cells with δ-opioid (DOR) and CB1-cannabinoid receptors, and examined the effect of prolonged exposure to either opioid (etorphine) or cannabinoid (DALN) agonists on DOR and CB-1 receptor density and on the ability of subsequent application of the agonists to activate G-proteins (as measured by [³⁵S]GTPγS binding) and to inhibit cAMP production. In HEK-293 cells, etorphine induced both homologous and heterologous desensitization, while DALN induced only homologous desensitization. This asymmetric cross-desensitization coincided with asymmetric cross downregulation: etorphine downregulated the binding of the cannabinoid ligand [³H]CP55,940, while DALN failed to reduce the binding of the opioid ligand [³H]diprenorphine. In contrast to the asymmetric desensitization in HEK-293 cells, COS-7 cells presented a two-way cross-desensitization between opioid and cannabinoid agonists, and DALN downregulated the binding of [³H]diprenorphine in these cells. Thus, a complete correlation was found between downregulation and reduction in cell responsiveness (‘desensitization’). Moreover, when opioid downregulation in HEK-293 cells was inhibited by either hypertonic sucrose solution or protein kinase inhibitors, desensitization was suppressed to the same extent. These results suggest that, under the present experimental conditions, the reduction in cell responsiveness resulted primarily from downregulation of the receptors.

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)

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Research reportLong-term interactions between opioid and cannabinoid agonists at the cellular level: cross-desensitization and downregulation

Abstract

Introduction

Section snippets

Cell culture and transfection

Results

Discussion

Acknowledgements

Biochem. Pharmacol.

Pharmacol. Biochem. Behav.

Life Sci.

Peptides

Chem. Phys. Lipids

J. Biol. Chem.

Neurosci. Lett.

J. Biol. Chem.

Eur. J. Pharmacol.

Methods Enzymol.

J. Neuroimmunol.

Brain Res.

Mol. Brain Res.

Eur. J. Pharmacol.

Pharmacol. Biochem. Behav.

Eur. J. Pharmacol.

Pharmacol. Biochem. Behav.

Drug Alcohol Depend.

Brain Res.

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.

Research report
Long-term interactions between opioid and cannabinoid agonists at the cellular level: cross-desensitization and downregulation

A comparison of some pharmacological actions of morphine and Δ⁹-tetrahydrocannabinol in the mouse