Inhibition of an equilibrative nucleoside transporter by cannabidiol: A mechanism of cannabinoid immunosuppression

Abstract

The plant-derived cannabinoids ⌬ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) both have immunosuppressive effects; although some effects of THC are mediated by the CB2 receptor, CB2 binds CBD weakly. In examining the effects of THC and CBD on microglial proliferation, we found that these compounds potently inhibit [ 3 H]thymidine incorporation into a murine microglial cell line with no effect on cell cycle. Because adenosine agonists have antiinflammatory effects, and because uptake of adenosine is a primary mechanism of terminating adenosine signaling, we tested the hypothesis that CBD is immunosuppressive because it enhances endogenous adenosine signaling. In vivo treatment with a low dose of CBD decreases TNF␣ production in lipopolysaccharide-treated mice; this effect is reversed with an A2A adenosine receptor antagonist and abolished in A2A receptor knockout mice. These studies demonstrate that CBD has the ability to enhance adenosine signaling through inhibition of uptake and provide a non-cannabinoid receptor mechanism by which CBD can decrease inflammation.adenosine ͉ lipopolysaccharide ͉ tetrahydrocannabinol ͉ thymidine ͉ tumor necrosis factor-␣ T he marijuana-derived cannabinoids ⌬ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD) each have immunosuppressive effects (1) and are currently in clinical trials for treatment of multiple sclerosis (2). Although the CB 2 cannabinoid receptor partially mediates the antiinflammatory effects of THC (3), CBD does not bind well to the known cannabinoid receptors (4); this low affinity results in the inability of CBD to produce the subjective ''high'' and cognitive effects that are characteristic of marijuana and THC (5, 6). To date, the mechanism by which CBD decreases inflammation is unknown, although micromolar concentrations of CBD have been shown to inhibit lipoxygenase activity (7).In addition to multiple sclerosis, CBD has shown promise in several rodent models of inflammation. Oral treatment with CBD decreases edema and hyperalgesia in a rat paw model of carrageenan-induced inflammation (8). A single dose of CBD also decreases serum TNF␣ production in lipopolysaccharide (LPS)-treated mice (9). CBD improves arthritis symptoms and joint pathology in murine collagen-induced arthritis while inhibiting IFN-␥ production and lymph node cell proliferation, as measured by [ 3 H]thymidine incorporation (9). Treatment with THC also decreases proliferation of lymph node cells as well as splenocytes (10).Given the potential of THC and CBD to decrease proliferation in immune cells, we initially examined the effects of several natural and synthetic cannabinoids on microglial cell proliferation by using [ 3 H]thymidine incorporation. Microglia are the resident immune cells of the brain, and their proliferation has been linked to a number of neurodegenerative diseases (11). However, CBD and THC decreased [ 3 H]thymidine incorporation into EOC-20 microglial cells with no effect on cell cycle. Further experiments demonstrate that this decrease in [ 3 H]thymidine incor...

Molecular Targets of Cannabidiol in Neurological Disorders

“…In previous studies, CBD has been shown to exert anti-inflammatory effects, but a distinct mechanism of action for this effect remains unresolved [106], despite hypotheses that CBD exerts anti-inflammatory effects by facilitating adenosine transmission [10,106]. To test this hypothesis, the effect of CBD on the uptake of [ 3 H]-adenosine was investigated (see Table 4).…”

“…Further studies, which examined the affinity of CBD for the equilibrative nucleoside transporter 1 (ENT1) showed that CBD displaced radiolabelled 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine ([ 3 H]-NMBPR) with a K i value of 237 nM, consistent with the result obtained from RAW264.7 macrophages. These findings indicate that CBD can inhibit adenosine uptake by binding ENT1 [106]. Thus, while it is clear that CBD can modulate adenosine signaling at both the receptor (see BReceptors^) and transporter levels, the contribution of these effects to the in vivo pharmacology of CBD still requires definitive study.…”

“…CBD can inhibit cellular uptake of adenosine via inhibition of the ENT1 carrier at nanomolar concentrations, and it is now thought that adenosinergic signaling could play a role in neuroprotection [10,106,228,235]. Although possible evidence that CBD might act, in part, via this system was suggested by the observation that in brain slices taken from a newborn mouse hypoxia model that A 2A and A 1A receptor antagonists inhibited a protective CBD effect, the most significant effect was only observed at 100 μM-a physiologically implausible concentration [31].…”

Cannabinoids and Epilepsy

“…CBD inhibits uptake of adenosine by blocking the equilibrative nucleoside transporter [118,119]. Increased levels of adenosine activate A2 receptors, which regulate striatal CB 1 Rs [120].…”

The adenosine kinase hypothesis of epileptogenesis

“…Human nucleoside transporters are classified into three known isoforms of concentrative nucleoside transporters (CNT) that are coupled to a Na + gradient (Gray et al, 2004;Smith et al, 2007) and into four known isoforms of equilibrative nucleoside transporters (ENT), which rapidly equilibrate pools of extra-and intracellular adenosine (Baldwin et al, 2004). Under physiological conditions ENTs appear to play the predominant role in adjusting extra-and intracellular pools of adenosine in the brain, since the extracellular concentration of adenosine can be enhanced by inhibition of ENTs with dipyridamole, dilazep, nitrobenzylthioinosine, or cannabidiol (Carrier et al, 2006;Kong et al, 2004).…”