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.
Bisogno
T
T
Endocannabinoid Research Group, Istituto per la Chimica di Molecole di Interesse Biologico (VDM, TB), Arco Felice, Napoli, Italy.
MacCarrone
M
M
De Petrocellis
L
L
Jarrahian
A
A
Finazzi-Agrò
A
A
Hillard
C
C
Di Marzo
V
V
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