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 [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 <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.
Footnotes
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Send reprint requests to: Cecilia J. Hillard, Ph.D., Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail:chillard{at}mcw.edu
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↵1 This work was supported by National Institute on Drug Abuse Grant DA09155.
- Abbreviations:
- AEA
- N-arachidonoylethanolamine
- CB1
- central cannabinoid receptor
- A3AP
- arachidonoyl-3-aminopyridine amide
- AM404
- arachidonoyl-4-hydroxyanilineamide
- PEA
- N-palmitoylethanolamine
- CGC
- cerebellar granule cell
- FAAH
- fatty acid amide hydrolase
- LCFA
- long- chain fatty acid
- Received November 8, 1999.
- Accepted January 11, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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