The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors

It is not yet clear if the endocannabinoid 2-arachidonoyl-glycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoyl-ethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2-AG pharmacological actions. We have studied simultaneously the facilitated transport of [C-14]AEA and [H-3]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (K-m 11.0 +/- 2.0 and 15.3 +/- 3.1 muM, B-max 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min(-1).mg protein(-1), respectively). Despite these similar K-m values, 2-AG inhibits [C-14]AEA uptake by cells at concentrations (K-i = 30.1 +/- 3.9 muM) significantly higher than those required to either 2-AG or AEA to inhibit [H-3]2-AG uptake (K-i = 18.9 +/- 1.8 and 20.5 +/- 3.2 muM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [C-14]AEA and [H-3]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [H-3]2-AG alone; (b) the uptake of [C-14]AEA and [H-3]2-AG by cells is inhibited with the same potency by AM404 (K-i = 7.5 +/- 0.7 and 10.2 +/- 1.7 muM, respectively) and linvanil (K-i = 9.5 +/- 0.7 and 6.4 +/- 1.2 muM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [C-14]AEA and [H-3]2-AG, thus suggesting that 2-AG action can be regulated through NO release; (d) AEA and 2-AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.