N-arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium-activated potassium channels

Abstract

Background and purpose: N-arachidonoyl glycine (NAGly) is an endogenous lipid that is structurally similar to the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). While NAGly does not activate cannabinoid receptors, it exerts cannabimimetic effects in pain regulation. Here, we have determined if NAGly, like anandamide, modulates vascular tone.

Experimental approach: In rat isolated small mesenteric arteries, the relaxant responses to NAGly were characterized. Effects of N-arachidonoyl serine and N-arachidonoyl gamma-aminobutyric acid were also examined.

Key results: In endothelium-intact arteries, NAGly-induced relaxation (pEC(50%)= 5.7 +/- 0.2; relaxation at 30 microM = 98 +/- 1%) was attenuated by l-NAME (a nitric oxide synthase inhibitor) or iberiotoxin [selective blocker of large conductance Ca(2+)-activated K(+) channels (BK(Ca))], and abolished by high extracellular K(+) concentration. Endothelial removal reduced the potency of NAGly, and the resultant relaxation was inhibited by iberiotoxin, but not l-NAME. NAGly responses were sensitive to the novel cannabinoid receptor antagonist O-1918 independently of endothelial integrity, whereas pertussis toxin, which uncouples G(i/o) proteins, attenuated NAGly relaxation only in endothelium-intact arteries. Treatments with antagonists for CB(1), CB(2) and TRPV1 receptors, or inhibitors of fatty acid amide hydrolase and COX had no effect. The two other arachidonoyl amino acids also induced iberiotoxin- and L-NAME-sensitive relaxations.

Conclusion and implications: NAGly acts as a vasorelaxant predominantly via activation of BK(Ca) in rat small mesenteric arteries. We suggest that NAGly activates an unknown G(i/o)-coupled receptor, stimulating endothelial release of nitric oxide which in turn activates BK(Ca) in the smooth muscle. In addition, NAGly might also activate BK(Ca) through G(i/o)- and nitric oxide-independent mechanisms.

Figure 1

Structures of the endocannabinoid, anandamide (A) and three endogenous conjugates of arachidonic acid and amino acids; N-arachidonoyl glycine (NAGly; B), N-arachidonoyl serine (C), N-arachidonoyl γ-aminobutyric acid (D).

Figure 2

Original traces of relaxation to NAGly in endothelium-intact (A) and endothelium-denuded (B) mesenteric arteries. The experiments were conducted in separate vessels obtained from the same rat. The vertical lines indicate addition of drug at the concentrations indicated. MO, methoxamine; NAGly, N-arachidonoyl glycine.

Figure 3

Effects of inhibitors of nitric oxide signalling on relaxation to NAGly in mesenteric arteries. In endothelium-intact (A) and endothelium-denuded (B) vessels, relaxation was elicited by NAGly alone, or after treatment with l-NAME (300 µM) or l-NAME and apamin (50 nM) plus charybdotoxin (50 nM). (C) Relaxation was elicited by NAGly alone, or after treatment with ODQ (10 µM) in endothelium-intact vessels. n= 5–7. Values are shown as means, and vertical bars represent SEM.

Figure 4

Effects of K⁺ channel blockade on relaxation to NAGly in mesenteric arteries. (A) Relaxation was elicited by NAGly alone, or after treatment with iberiotoxin (50 nM), or iberiotoxin (50 nM) plus l-NAME (300 µM) in endothelium-intact vessels. Relaxation was also elicited by NAGly alone in vessels precontracted with 60 mM KCl, instead of 10 µM methoxamine. (B) Relaxation was elicited by NAGly alone, or after treatment with iberiotoxin (50 nM) in endothelium-denuded vessels. n= 4–6. Values are shown as means, and vertical bars represent SEM.

Figure 5

Effects of O-1918 (3 µM) or pertussis toxin (400 ng·mL⁻¹) on relaxation to NAGly in endothelium-intact (A) and endothelium-denuded (B) mesenteric arteries. Control NAGly responses have been pooled for clarity in these graphs. n= 4–7. Values are shown as means, and vertical bars represent SEM.

Figure 6

Effects of URB597 (1 µM) and indomethacin (10 µM) on relaxation to NAGly in endothelium-intact mesenteric arteries. n= 4–6. Values are shown as means, and vertical bars represent SEM.

Figure 7

Effects of iberiotoxin (50 nM) and O-1918 (3 µM) on relaxation to SNP in endothelium-denuded mesenteric arteries. n= 5–8. Values are shown as means, and vertical bars represent SEM.

Figure 8

Effects of iberiotoxin (50 nM) or l-NAME (300 µM) on relaxation to N-arachidonoyl serine (A) and N-arachidonoyl γ-aminobutyric acid (B) in endothelium-intact mesenteric arteries. n= 5–7. Values are shown as means, and vertical bars represent SEM.