N-arachidonoyl L-serine, an endocannabinoid-like brain constituent with vasodilatory properties

Abstract

The endocannabinoid N-arachidonoyl ethanolamine (anandamide), found both in the CNS and in the periphery, plays a role in numerous physiological systems. One might expect that the chemically related N-arachidonoyl-L-serine (ARA-S) could also be formed alongside anandamide. We have now isolated ARA-S from bovine brain and elucidated its structure by comparison with synthetic ARA-S. Contrary to anandamide, ARA-S binds very weakly to cannabinoid CB1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. However, it produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44/42 mitogen-activated protein (MAP) kinase and protein kinase B/Akt in cultured endothelial cells. ARA-S also suppresses LPS-induced formation of TNF-alpha in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB1 or CB2 receptors. Many of these effects parallel those reported for abnormal cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoid-type receptor. Hence, ARA-S may represent an endogenous agonist for this receptor.

Fig. 1.

Identification of ARA-S in bovine brain. (A) Structures of anandamide and ARA-S. (B) Electron impact high-resolution mass spectra of synthetic ARA-S (a) and endogenous ARA-S (b) from bovine brain. Both spectra are after derivatization with N,O-bis(trimethyl-silyl)trifluoroacetamide. (C) Overlay chromatogram of natural and synthetic d- (≈18 min), l- (≈26 min) ARA-S methyl esters obtained with HPLC chiral chromatography.

Fig. 2.

ARA-S-induced vasorelaxation in rat aortic rings (A and B) and mesenteric artery segments (C and D). (A) The aortic relaxant effect of ARA-S (filled squares) is not antagonized by 10 μM O-1918 (open squares) and is nearly eliminated by endothelial denudation (filled circles). (B) Relaxation of aortic rings by ARA-S (filled squares) is blocked by PTX (open triangles). (C) Mesenteric vasorelaxation by ARA-S is inhibited by 10 μM O-1918 (open symbols) in endothelium-intact (squares) and endothelium-denuded (circles) preparations. (D) Mesenteric vasorelaxation by ARA-S (filled squares) is unaffected by PTX (open triangles). Points and vertical bars are means ± SE from four to six experiments.

Fig. 3.

Activation of MAP kinase and protein kinase B/Akt. (A) ARA-S (1 μM) and Abn-CBD (30 μM) increase the phosphorylation of protein kinase B/Akt (Top) and p44/42 MAP kinase (Middle), effects inhibited by PTX (400 ng/ml). Western blotting is described in Materials and Methods. β-Actin (Bottom) was used as loading control. This experiment was replicated two more times with similar results. (B) Concentration-dependent phosphorylation of p44/42 MAP kinase by ARA-S and Abn-CBD.

Fig. 4.

The effect of ARA-S (10 mg/kg), Abn-CBD (20 mg/kg), and HU-210 (50 μg/kg) on LPS-induced plasma TNF-α levels in wild-type (n = 32), CB₁^−/− (n = 5), and CB₂^−/− (n = 4) mice. Basal levels of TNF-α were 30 ± 10, 39 ± 15, and 35 ± 13 pg/ml, and LPS-stimulated levels were 3,407 ± 197, 3,642 ± 326, and 6,877 ± 857 pg/ml in the three groups, respectively. Significant difference from vehicle-treated values is indicated by ∗ (P < 0.05) and ∗∗ (P < 0.001).