Synthesis and characterization of a fluorescent substrate for the N-arachidonoylethanolamine (anandamide) transmembrane carrier

N-Arachidonoylethanolamine (AEA) is a proposed endogenous ligand of the cen tral cannabinoid receptor (CB1). Previous studies indicate that AEA is tran slocated across membranes via a process that has the characteristics of car rier-mediated facilitated diffusion, To date, studies of this mechanism hav e relied on [H-3]AEA as a substrate for the carrier. We have synthesized an analog of AEA, SKM 4-45-1, that is nonfluorescent in the extracellular env ironment. When SKM 4-45-1 is exposed to intracellular esterases, it is de-e sterified and becomes fluorescent. We have carried out studies to demonstra te that SKM 4-45-1 accumulation in cells occurs via the AEA carrier. SKM 4- 45-1 is accumulated by both cerebellar granule cells and C6 glioma cells. U ptake of SKM 4-45-1 into C6 glioma is inhibited by AEA (lC(50)=53.8 +/- 1.8 mu M), arachidonoyl-3-aminopyridine amide(IC50=10.1 +/- 1.4 mu M), and ara chidonoyl-4-hydroxyanilineamide (IC50=6.1 +/- 1.3 mu M), all of which also inhibit [H-3]AEA accumulation. Conversely, [H-3]AEA accumulation by cerebel lar granule cells is inhibited by SKM 4-45-1 with an IC50 of 7.8 +/- 1.3 mu M. SKM 4-45-1 is neither a substrate nor inhibitor of fatty acid amide hyd rolase, an enzyme that catabolizes AEA. SKM 4-45-1 does not bind the CB1 ca nnabinoid receptor at concentrations <10 mu M In summary, the cellular accu mulation of SKM 4-45-1 occurs via the same pathway as AEA uptake and provid es an alternative substrate for the study of this important cellular proces s.