The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors.
It is not yet clear if the endocannabinoid 2-arachidonoylglycerol (2-AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N-arachidonoylethanolamine, 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 [14C]AEA and [3H]2-AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 +/- 2.0 and 15.3 +/- 3.1 microM, Bmax 1.70 +/- 0.30 and 0.24 +/- 0.04 nmol.min-1.mg protein-1, respectively). Despite these similar Km values, 2-AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 +/- 3.9 microM) significantly higher than those required to either 2-AG or AEA to inhibit [3H]2-AG uptake (Ki = 18.9 +/- 1.8 and 20.5 +/- 3.2 microM, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2-AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2-AG alone; (b) the uptake of [14C]AEA and [3H]2-AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 +/- 0.7 and 10.2 +/- 1.7 microM, respectively) and linvanil (Ki = 9.5 +/- 0.7 and 6.4 +/- 1.2 microM, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]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.
Endocannabinoid Research Group, Istituto per la Chimica di Molecole di Interesse Biologico (VDM, TB), Arco Felice, Napoli, Italy.
T
T
Bisogno
M
M
MacCarrone
L
L
De Petrocellis
A
A
Jarrahian
A
A
Finazzi-Agrò
C
C
Hillard
V
V
Di Marzo
D000818:Animals; D001095:Arachidonic Acids / Q000493:pharmacokinetics* / Q000494:pharmacology; D001693:Biological Transport, Active; D063385:Cannabinoid Receptor Modulators; D002462:Cell Membrane / Q000378:metabolism; D063388:Endocannabinoids; D005910:Glioma / Q000378:metabolism; D005989:Glycerides / Q000493:pharmacokinetics*; D007700:Kinetics; D008956:Models, Chemical; D018377:Neurotransmitter Agents / Q000493:pharmacokinetics*; D009569:Nitric Oxide / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D051381:Rats; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D063388:Endocannabinoids; D005989:Glycerides; D018377:Neurotransmitter Agents; D053284:Polyunsaturated Alkamides; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D009569:Nitric Oxide; C094503:glyceryl 2-arachidonate; C078814:anandamide; C107594:N-(4-hydroxyphenyl)arachidonylamide