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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Synthesis and Characterization of a Fluorescent Substrate for the N-Arachidonoylethanolamine (Anandamide) Transmembrane Carrier

Shanmugam Muthian, Kasem Nithipatikom, William B. Campbell and Cecilia J. Hillard
Journal of Pharmacology and Experimental Therapeutics April 2000, 293 (1) 289-295;
Shanmugam Muthian
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Kasem Nithipatikom
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William B. Campbell
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Cecilia J. Hillard
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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

  • 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

  • ↵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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Journal of Pharmacology and Experimental Therapeutics: 293 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 293, Issue 1
1 Apr 2000
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Synthesis and Characterization of a Fluorescent Substrate for the N-Arachidonoylethanolamine (Anandamide) Transmembrane Carrier

Shanmugam Muthian, Kasem Nithipatikom, William B. Campbell and Cecilia J. Hillard
Journal of Pharmacology and Experimental Therapeutics April 1, 2000, 293 (1) 289-295;

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Synthesis and Characterization of a Fluorescent Substrate for the N-Arachidonoylethanolamine (Anandamide) Transmembrane Carrier

Shanmugam Muthian, Kasem Nithipatikom, William B. Campbell and Cecilia J. Hillard
Journal of Pharmacology and Experimental Therapeutics April 1, 2000, 293 (1) 289-295;
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