Faculty Collaboration Database

Synthesis and characterization of a fluorescent substrate for the N-arachidonoylethanolamine (anandamide) transmembrane carrier. J Pharmacol Exp Ther 2000 Apr;293(1):289-95

Date

03/29/2000

Pubmed ID

10734181

Scopus ID

2-s2.0-0034087481 (requires institutional sign-in at Scopus site)   33 Citations

Abstract

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 [(3)H]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 (IC(50)=53.8 +/- 1.8 microM), arachidonoyl-3-aminopyridine amide (IC(50)=10.1 +/- 1.4 microM), and arachidonoyl-4-hydroxyanilineamide (IC(50)=6.1 +/- 1.3 microM), all of which also inhibit [(3)H]AEA accumulation. Conversely, [(3)H]AEA accumulation by cerebellar granule cells is inhibited by SKM 4-45-1 with an IC(50) of 7.8 +/- 1. 3 microM. 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 <10 microM. 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.

Author List

Muthian S, Nithipatikom K, Campbell WB, Hillard CJ

Authors

William B. Campbell PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Cecilia J. Hillard PhD Associate Dean, Center Director, Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Amidohydrolases
Animals
Arachidonic Acids
Brain Neoplasms
Cannabinoids
Carrier Proteins
Cell Membrane
Cerebellum
Cyclohexanols
Endocannabinoids
Esterases
Fluorescent Dyes
Glioma
Humans
Lactones
Microscopy, Fluorescence
Polyunsaturated Alkamides
Rats
Rats, Sprague-Dawley
Tumor Cells, Cultured