Measurement of saturable and non-saturable components of anandamide uptake into P19 embryonic carcinoma cells in the presence of fatty acid-free bovine serum albumin

https://doi.org/10.1016/j.chemphyslip.2004.12.010Get rights and content

Abstract

There is considerable controversy at present concerning the mechanisms responsible for the cellular uptake of anandamide. One particular issue concerns whether fatty acid-free bovine serum albumin should be used in the assays, it having been argued that such a presence effectively prevents the specific uptake of anandamide. In the present study, it has been demonstrated that in the presence of a low (0.1%, w/v) concentration of fatty acid-free bovine serum albumin, a temperature-dependent and saturable (Km  1 μM) uptake of anandamide into P19 embryonic carcinoma cells can be demonstrated using an incubation time of 4 min. Under these conditions, the uptake of anandamide at 4 °C is low at a substrate concentration of 100 nM. The uptake at 37 °C was not significantly reduced following treatment of the cells with either methyl-β-cyclodextrin (50 μM) or mevinolin (1 μM), but was reduced by the FAAH inhibitor URB597 (1 μM) and inhibited by the transport inhibitor cum FAAH substrate AM404 with an IC50 value of 12 μM. When a 45 s incubation time was used, the uptake of anandamide was not saturable at 37 °C over the concentration range tested (0.1–1 μM). Analysis of the data at 37 °C obtained with 45 s, 4 min and 15 min incubation times revealed a very rapid (i.e. complete by 45 s) non-saturable component followed by a slower saturable (Km  1 μM) component of the uptake. It is concluded that the presence of a low concentration of fatty acid-free bovine serum albumin at a suitable concentration reduces non-specific binding (and release) of anandamide to cell culture wells, greatly reduces the cellular accumulation seen at 4 °C, and allows the visualisation of both non-saturable and saturable components of the uptake to be seen at 37 °C.

Introduction

It is now well established that endocannabinoids are synthesised upon demand and act as agonists at plasma membrane cannabinoid receptors (review, see De Petrocellis et al., 2004). The most well studied endocannabinoid, annadamide (arachidonoylethanolamide, AEA), has a short duration of action, due to effective metabolic pathways involving cellular uptake and metabolism. The main metabolic enzyme, fatty acid amide hydrolase, is located primarily in the mitochondria and endoplasmic reticulum (Schmid et al., 1985), and catalyses the hydrolysis of AEA to give arachidonic acid and ethanolamine (Deutsch and Chin, 1993).

In contrast to the well-characterised metabolic pathways for AEA, the mechanism(s) responsible for AEA uptake are a matter of considerable controversy. It was originally suggested that AEA uptake was brought about by a process of facilitated diffusion (Di Marzo et al., 1994, Hillard et al., 1997), although no protein responsible for this process has yet been identified. In contrast, Glaser et al. (2003) suggested that the uptake of AEA at short incubation times was not a saturable process. Current thinking seems to be moving towards a consensus that although the uptake can in some cells be driven to a certain extent by fatty acid amide hydrolase, an accumulation process, possibly involving endocytosis and possibly into distinct intracellular compartments or alternatively by proteins shuttling AEA from the cell membrane to the endoplasmic reticulum, does concentrate this endocannabinoid against its gradient (see Hillard and Jarrahian, 2003, Ligresti et al., 2004, Fowler et al., 2004, Ortega-Gutiérrez et al., 2004, Fegley et al., 2004, McFarland et al., 2004, McFarland and Barker, 2004, for recent discussions).

In many respects, the field of AEA uptake is rather reminiscent of that of long chain fatty acid uptake, where both passive and active processes, the latter involving lipid rafts, have been identified (see e.g. Ibrahimi et al., 1996, Stump et al., 2001, Hamilton et al., 2002, Pohl et al., 2004). However, one major difference between the two fields is the use in the assays of fatty acid-free bovine serum albumin (BSA). Long chain fatty acids in the blood exist bound to albumins (review, see Spector, 1975), and fatty acid-free BSA is routinely used in uptake assays. In contrast, most studies of AEA uptake have not used fatty acid-free BSA, possibly in view of the initial study demonstrating that the uptake of AEA into neuronal cultures was completely blocked by 0.5% BSA (Di Marzo et al., 1994). Indeed, one criticism of the study of Glaser et al. (2003) was their use of a “high” (0.4%) fatty acid-free BSA concentration, the contention being that the specific (i.e. BSA sensitive) process was blocked and that the non-specific process remained (see Hillard and Jarrahian, 2003). Nevertheless, the fact remains that AEA binds with a high (nanomolar) affinity to BSA (Bojesen and Hansen, 2003), and that in a physiological setting, it is likely that AEA binding proteins will be present in the extracellular medium. In this respect, lower concentrations of BSA might be appropriate in AEA uptake assays.

In a recent study, Ligresti et al. (2004) reported that the uptake of AEA into RBL-2H3 cells was reduced, but not obliterated, when the assays were undertaken in the presence of 0.4% fatty acid-free BSA, and that the remaining activity could still partially be inhibited by the uptake inhibitor OMDM-2 ((9Z)-N-[1-((R)-4-hydroxbenzyl)-2-hydroxyethyl]-9-octadecenamide). Another recent study found that the sensitivity of inhibition of AEA uptake into RBL2H3 cells by UCM707 (N-(Fur-3-ylmethyl)arachidonamide) was similar when assays were run in the absence or presence of 0.15% fatty acid-free BSA, a concentration which had a modest effect on the uptake per se, but essentially prevented non-specific adsorption of the AEA to the cell walls (Fowler et al., 2004). Such findings motivate an investigation as to whether saturable transport of AEA can be identified for assays run in the presence of fatty acid-free BSA.

Section snippets

Materials

Radioactive anandamide [arachidononyl 5, 6, 8, 9, 11, 12, 14, 15-3H] ethanolamide (spec. act., 60 Ci mmol−1), was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). Unlabeled anandamide and URB 597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) was obtained from Cayman Chemical company (Ann Arbor, MI, USA). AM404 (N-(4-hydroxyphenyl)arachidonylamide) was purchased from Tocris Cookson (Bristol, UK). All cell culture media, sera and supplements were purchased from

Characteristics of [3H]AEA uptake in the presence of fatty acid-free BSA

In an initial experiment using an incubation time of 4 min, the effects of different concentrations of fatty acid-free BSA in the incubation medium during the AEA uptake was investigated (Fig. 1A). Parallel wells were run in the absence of cells, since AEA is known to bind to, and be released from, cell culture plates (see Karlsson et al., 2004). As expected, fatty acid-free BSA reduced the observed uptake. The presence of 0.1% fatty acid-free BSA in the buffer considerably reduced the level of

Discussion

The aim of the present study has been very simple, namely to determine whether a saturable uptake of AEA can be seen in assays run in the presence of a low concentration of fatty acid-free BSA. Although it has been argued that the use of BSA is inadvisable since it will essentially remove all the “specific” uptake of AEA (Hillard and Jarrahian, 2003), our view is that the presence of an AEA binding protein in the assay medium reflects better the situation in vivo, where there are likely to be a

Acknowledgements

The technical expertise of Britt Jacobsson and Ingrid Persson are greatly appreciated. The authors are also grateful to Dr. Harvey Motulsky for useful discussions concerning the analysis of the kinetic data. This study was supported by grants from the Swedish Research Council (Grant no. 12158, medicine), Konung Gustav V's and Drottning Victorias Foundation, Gun and Bertil Stohne's Foundation, Stiftelsen för Gamla Tjänarinnor, and the Research Funds of the Medical Faculty, Umeå University.

References (34)

  • M. Ramakrishnan et al.

    N-Myristoylamine-cholesterol (1:1) complex: first evidence from differential scanning calorimetry, fast-atom-bombardment mass spectrometry and computational modelling

    FEBS Lett.

    (2002)
  • P.C. Schmid et al.

    Properties of rat liver N-acylethanolamine amidohydrolase

    J. Biol. Chem.

    (1985)
  • A.A. Spector

    Fatty acid binding to plasma albumin

    J. Lipid Res.

    (1975)
  • D.D. Stump et al.

    Oleic acid uptake and binding by rat adipocytes define dual pathways for cellular fatty acid uptake

    J. Lipid Res.

    (2001)
  • T.A. Day et al.

    Role of fatty acid amide hydrolase in the transport of the endogenous cannabinoid anandamide

    Mol. Pharmacol.

    (2001)
  • L. De Petrocellis et al.

    The endocannabinoid system: a general view and latest additions

    Br. J. Pharmacol.

    (2004)
  • D.G. Deutsch et al.

    Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist

    Biochem. Pharmacol.

    (1993)
  • Cited by (28)

    • Inhibition of fatty acid amide hydrolase and cyclooxygenase by the N-(3-methylpyridin-2-yl)amide derivatives of flurbiprofen and naproxen

      2013, European Journal of Pharmacology
      Citation Excerpt :

      Non-specific binding was determined in the presence of 10 μM CP55,940. The method of Rakhshan et al. (2000) as modified by Sandberg and Fowler (2005) was used. RBL2H3 cells were cultured in minimum essential medium with Earl's salts, 15% foetal bovine serum and 100 U/mL penicillin+100 mg/mL streptomycin (“medium”).

    • Chapter 2 Organized Trafficking of Anandamide and Related Lipids

      2009, Vitamins and Hormones
      Citation Excerpt :

      Recently, a few reports have shown that initial AEA uptake kinetics are linear, and thus, suggest passive diffusion of AEA across the plasma membrane (Glaser et al., 2003; Kaczocha et al., 2006; Sandberg and Fowler, 2005). In these studies, researchers measured AEA uptake at time points less than 1 min (Glaser et al., 2003; Kaczocha et al., 2006; Sandberg and Fowler, 2005), as opposed to experiments determining steady‐state uptake kinetics at time points ranging anywhere from 5 min to 40 h (Dickason‐Chesterfield et al., 2006; Fegley et al., 2004; Maccarrone et al., 2000; McFarland et al., 2008; Moore et al., 2005; Rakhshan et al., 2000). It was argued that at early time points under 1 min, FAAH has not yet begun to metabolize AEA and therefore cannot influence transport or interfere with the analysis of AEA uptake kinetics (Glaser et al., 2003; Kaczocha et al., 2006).

    • Anandamide uptake is consistent with rate-limited diffusion and is regulated by the degree of its hydrolysis by fatty acid amide hydrolase

      2006, Journal of Biological Chemistry
      Citation Excerpt :

      For other cell types, it has been shown that incubation times longer than 1 min cannot isolate AEA uptake occurring at the plasma membrane from downstream processes, including FAAH activity and intracellular sequestration (11, 12, 19, 20, 49). Short incubation times have been used extensively to determine transport of fatty acids (50) (for review, see Ref. 51), and only recently extended to include AEA (18, 19, 22). In our hands, at 37 °C the rate of AEA accumulation in RBL-2H3 cells displayed linear uptake kinetics at 25 s, when plotted as a function of total [AEA] in the presence of BSA (Fig. 2, A and C), consistent with previous work implicating simple diffusion.

    View all citing articles on Scopus
    View full text