TY - JOUR T1 - Synthesis and Characterization of a Fluorescent Substrate for the <em>N</em>-Arachidonoylethanolamine (Anandamide) Transmembrane Carrier JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 289 LP - 295 VL - 293 IS - 1 AU - Shanmugam Muthian AU - Kasem Nithipatikom AU - William B. Campbell AU - Cecilia J. Hillard Y1 - 2000/04/01 UR - http://jpet.aspetjournals.org/content/293/1/289.abstract N2 - N-Arachidonoylethanolamine (AEA) is a proposed endogenous ligand of the central cannabinoid receptor (CB1). Previous studies indicate that AEA is translocated across membranes via a process that has the characteristics of carrier-mediated facilitated diffusion. To date, studies of this mechanism have relied on [3H]AEA as a substrate for the carrier. We have synthesized an analog of AEA, SKM 4-45-1, that is nonfluorescent in the extracellular environment. When SKM 4-45-1 is exposed to intracellular esterases, it is de-esterified and becomes fluorescent. We have carried out studies to demonstrate 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. Uptake of SKM 4-45-1 into C6 glioma is inhibited by AEA (IC50=53.8 ± 1.8 μM), arachidonoyl-3-aminopyridine amide (IC50=10.1 ± 1.4 μM), and arachidonoyl-4-hydroxyanilineamide (IC50=6.1 ± 1.3 μM), all of which also inhibit [3H]AEA accumulation. Conversely, [3H]AEA accumulation by cerebellar granule cells is inhibited by SKM 4-45-1 with an IC50 of 7.8 ± 1.3 μM. SKM 4-45-1 is neither a substrate nor inhibitor of fatty acid amide hydrolase, an enzyme that catabolizes AEA. SKM 4-45-1 does not bind the CB1 cannabinoid receptor at concentrations &lt;10 μM. In summary, the cellular accumulation of SKM 4-45-1 occurs via the same pathway as AEA uptake and provides an alternative substrate for the study of this important cellular process. The American Society for Pharmacology and Experimental Therapeutics ER -