Anandamide uptake by human endothelial cells and its regulation by nitric oxide.
Anandamide (AEA) has vasodilator activity, which can be terminated by cellular re-uptake and degradation. Here we investigated the presence and regulation of the AEA transporter in human umbelical vein endothelial cells (HUVECs). 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)-arachidonoylamide, and stimulated up to 2.2-fold by nitric oxide (NO) donors. The NO scavenger hydroxocobalamin abolishes the latter effect, which is instead enhanced by superoxide anions but inhibited by superoxide dismutase and N-acetylcysteine, 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 adenylyl cyclase, because NO/ONOO(-) donors and alpha-linolenoyl-vanillyl-amide/N-(4-hydroxyphenyl)-arachidonoylamide were found to attenuate and enhance, respectively, this effect of AEA. Consistently, activation of CB(1) cannabinoid 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-fold, respectively. Also these effects are regulated by the AEA transporter. HU-210 enhanced AEA uptake by HUVECs in a fashion sensitive to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester. These findings suggest a NO-mediated regulatory loop between CB(1) cannabinoid receptors and AEA transporter.
Maccarrone
M
M
Department of Experimental Medicine, University of Rome Tor Vergata, Via di Tor Vergata 135, I-00133 Rome, Italy.
Bari
M
M
Lorenzon
T
T
Bisogno
T
T
Di Marzo
V
V
Finazzi-Agrò
A
A
D001095:Arachidonic Acids / Q000378:metabolism*; D001692:Biological Transport; D002186:Cannabinoids / Q000378:metabolism; D002478:Cells, Cultured; D063388:Endocannabinoids; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D011869:Radioligand Assay
D001095:Arachidonic Acids; D002186:Cannabinoids; D063388:Endocannabinoids; D020030:Nitric Oxide Donors; D053284:Polyunsaturated Alkamides; D009569:Nitric Oxide; C078814:anandamide