Anandamide uptake by human endothelial cells and its regulation by nitric oxide

Anandamide (AEA) has vasodilator activity, which can be terminated by cellu lar re-uptake and degradation. Here we investigated the presence and regula tion of the AEA transporter in human umbelical vein endothelial cells (HUVE Cs), HUVECs take up AEA by facilitated transport (apparent K-m = 190 +/- 10 nM and V-max = 45 +/- 3 pmol min(-1) . mg(-1) protein), which is inhibited by alpha-linolenoyl-vanillyl-amide and N-(4-hydroxyphenyl) arachidonoylami de, and stimulated up to 2.2-fold by nitric oxide (NO) donors. The NO scave nger hydroxocobalamin abolishes the latter effect, which is instead enhance d by superoxide anions but inhibited by superoxide dismutase and N-acetylcy steine, a precursor of glutathione synthesis. Peroxynitrite (ONOO-) causes a 4-fold activation of AEA transport into cells. The HUVEC AEA transporter contributes to the termination of a typical type 1 cannabinoid receptor (CB 1) -mediated action of AEA i.e. the inhibition of forskolin-stimulated aden ylyl cyclase, because NO/ONOO- donors and alpha-linolenoyl-vanillyl-amide/N -(4-hydroxyphenyl)-arachidonoylamide were found to attenuate and enhance, r espectively, this effect of AEA. Consistently, activation of CB, cannabinoi d receptors by either AEA or the cannabinoid HU-210 caused a stimulation of HUVEC inducible NO synthase activity and expression up to 2.9- and 2.6-fol d, respectively. Also these effects are regulated by the AEA transporter, H U-210 enhanced AEA uptake by HOVECs in a fashion sensitive to the NO syntha se inhibitor N omega-nitro-L-arginine methyl ester, These findings suggest a NO-mediated regulatory loop between CB1 cannabinoid receptors and AEA tra nsporter.