The Cellular Uptake of Anandamide Is Coupled to
Its Breakdown by Fatty-acid Amide Hydrolase*
Dale G.
Deutsch
§,
Sherrye T.
Glaser
,
Judy M.
Howell
,
Jeffrey S.
Kunz
,
Robyn A.
Puffenbarger
,
Cecilia J.
Hillard¶, and
Nada
Abumrad
From the
Departments of Biochemistry and Cell Biology
and
Physiology and Biophysics, State University of New York at
Stony Brook, Stony Brook, New York 11974-5215 and the ¶ Department
of Pharmacology and Toxicology, Medical College of Wisconsin,
Milwaukee, Wisconsin 53226-0509
Received for publication, April 13, 2000, and in revised form, December 14, 2000
 |
ABSTRACT |
Anandamide is an endogenous compound
that acts as an agonist at cannabinoid receptors. It is inactivated via
intracellular degradation after its uptake into cells by a
carrier-mediated process that depends upon a concentration gradient.
The fate of anandamide in those cells containing an amidase called
fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and
ethanolamine. The active site nucleophilic serine of FAAH is
inactivated by a variety of inhibitors including
methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl
fluoride. In the current report, the net uptake of anandamide in
cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH,
was decreased by nearly 50% after 6 min of incubation in the presence
of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH,
is not inhibited by MAFP. Free anandamide was found in all
MAFP-treated cells and in control Hep2 cells, whereas phospholipid was
the main product in N18 and C6 control cells when analyzed by
TLC. The intracellular concentration of anandamide in N18, C6,
and Hep2 cells was up to 18-fold greater than the extracellular
concentration of 100 nM, which strongly suggests that
it is sequestered within the cell by binding to membranes or proteins.
The accumulation of anandamide and/or its breakdown products was found
to vary among the different cell types, and this correlated
approximately with the amount of FAAH activity, suggesting that the
breakdown of anandamide is in part a driving force for uptake. This was
shown most clearly in Hep2 cells transfected with FAAH. The uptake in
these cells was 2-fold greater than in vector-transfected or
untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors
reduce anandamide uptake by cells by shifting the anandamide
concentration gradient in a direction that favors equilibrium. Because
inhibition of FAAH increases the levels of extracellular
anandamide, it may be a useful target for the design of therapeutic agents.
 |
INTRODUCTION |
Endocannabinoids, such as anandamide (arachidonyl ethanolamide)
and 2-arachidonyl glycerol, are endogenous ligands that bind to the
cannabinoid receptors (1-3). Emerging evidence suggests that they are
involved in many physiological phenomena such as pain, locomotion,
memory, learning, blood pressure, immunity, sleep, reproduction, mood,
perception, response to stress, and so forth (for review see Ref. 4).
9-Tetrahydrocannabinol, the active component of
marijuana, appears to mimic many of the physiological and
pharmacological effects of the endogenous cannabinoids, in some cases
to an extreme degree (e.g. a marijuana "high").
Anandamide is transported into the neuroblastoma, glioma, brain neuron,
brain astrocyte, cerebellar granule cells, leukocyte, macrophage,
leukemia, and lymphoma cells in culture (5-10). The driving force for
uptake is substrate concentration (facilitated diffusion) rather than
an active cotransport system (11, 12). The transport appears to be
carrier-mediated, and specific transport inhibitors have been described
(9, 11, 13-16). After being transported into the cell, anandamide is
subsequently broken down into arachidonic acid and ethanolamine by an
endoplasmic reticular integral membrane-bound enzyme called fatty-acid
amide hydrolase (FAAH),1
anandamide amidase, or anandamide amidohydrolase (for review see Ref.
17). Interestingly, the catalytic site contains at least two important
serines and a lysine (not the histidine, serine, and aspartate triad
found in many hydrolytic serine active site enzymes) with Ser-241
acting as the nucleophile involved in the bond breaking of substrates
(18-20). It has been shown that FAAH is inhibited by a variety of
compounds, such as methylarachidonylfluorophosphonate (MAFP) and
palmitylsulfonyl fluoride (PSF) (21-23). Employing these inhibitors,
we show that the net movement of anandamide into the cells is coupled
to the activity of intracellular FAAH. We propose that the inhibition
of anandamide breakdown results in its intracellular build-up and the
attainment of equilibrium between free intracellular and extracellular
anandamide, and this disfavors further net uptake.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
N18TG2 neuroblastoma, C6 glioma (kindly
provided by Allyn Howlett and Joel Levine, respectively), and human
laryngeal carcinoma cells (Hep2), provided by our in-house cell culture
facility, were grown in 35 × 10-mm dishes in 2 ml of Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 1%
penicillin/streptomycin, and L-glutamine (Life
Technologies, Inc.). All cells were grown at 37 °C with 5%
CO2. For all experiments, the number of cells plated for
Hep2 and C6 was ~1.7 × 106, and the number of cells
plated for N18 was ~7 × 105. The exact cell numbers
were determined for comparison of the uptake rates in N18
neuroblastoma, C6 glioma, and Hep2 carcinoma (see below and Fig.
3).
Time Course of Anandamide Uptake--
The effects of MAFP and
PSF on anandamide uptake were measured using
arachidonyl-[5,6,8,9,11,12,14,15-3H]ethanolamide
called [3H]anandamide (172 Ci/mmol, 62.2 nCi/µl)
from PerkinElmer Life Sciences as substrate. Growth medium was
removed from the cells and replaced with 750 µl of supplemented
Dulbecco's modified Eagle's medium containing 40 nCi[3H]anandamide (100 nM anandamide).
The cells were incubated for 1-6 min at 37 °C with 5%
CO2. To account for nonspecific binding to cellular
membrane, parallel incubations were carried out at 4 °C. The counts
from the nonspecific binding were subtracted from the total amount of
anandamide taken up after each incubation so only the carrier-mediated
transport uptake was represented in the data. To study whether MAFP
affected the cellular uptake of anandamide, incubations were carried
out after a 10-min preincubation of the cells with 1 µM
MAFP (Cayman Chemical, Ann Arbor, MI) or 100 nM
pamitylsulfonyl fluoride (provided by Alex Makriyannis, University of
Connecticut) in the medium at 37 °C. After the incubation, the
medium was immediately removed, and the cells were washed with ice-cold
supplemented Dulbecco's modified Eagle's medium. The cells were
scraped from the plate after the addition of 0.4 ml of 2 mM
EDTA in phosphate-buffered saline. This procedure was repeated two
additional times to maximize yield. The labeled compounds were then
extracted from both the medium and the cells by the addition of 2 volumes of chloroform:methanol (1:1) (Fisher) mixed thoroughly and spun
down in a clinical centrifuge for 5 min. Finally, 100 µl of the
organic layer from both the cell and medium extractions were placed in
scintillation vials with 3.0 ml of ScintiVerse II scintillation fluid
(Fisher). The samples were counted in an LKB Rack beta scintillation counter.
Determination of Km and
Vmax--
Saturation kinetics were determined in N18, C6,
and Hep2 cells plated at a minimal density of 1 × 106
cells. They were incubated for either 3 s (to determine
nonspecific binding) or 60 s at 37 °C. For N18 and C6 cells,
these experiments were performed with
[lsqb]3H]anandamide (0.09-3.0 nM) and
unlabeled anandamide to yield total concentrations ranging from 0.03 to
1.0 µM anandamide. For Hep2 cells,
[lsqb]3H]anandamide (0.01-5.5
nM) was added to unlabeled anandamide over a concentration
range of 0.25-100 µM anandamide. The same procedure was
used for these experiments as for the time course of anandamide uptake
with the exception that the experiment was terminated by the addition
of 5 ml of ice-cold supplemented Dulbecco's modified Eagle's medium,
which was then immediately removed. Experiments were conducted in
duplicate or triplicate and repeated two or three separate times.
Calculations were performed by subtracting the nonspecific binding at
3 s from the 60-s incubation, and the picomoles taken up were
corrected for the number of cells. To determine the
Km and Vmax, the data were
analyzed employing a Lineweaver-Burk plot using the linear
regression program of Sigma Plot (SPSS, Chicago, IL).
5-Min Uptake Experiments and TLC Analysis--
The same
procedure described above for the time course experiments was employed
for these 5-min incubations, with the exception that the level of
radioactivity was increased to 600 nCi/dish to have enough counts for
thin layer chromatography analysis. After the 5-min incubation, the
labeled anandamide was extracted from both the medium and the cells by
the addition of 2 volumes of chloroform:methanol (1:1) mixed thoroughly
and spun down in a clinical centrifuge for 5 min. 100 µl of the
organic layer were removed for counting from both the cellular (1.2 ml
total) and medium (750 µl) extracts. The remaining portions of the
organic extracts from the cells and medium were dried down. The residue was resuspended in 40 µl of chloroform:methanol (1:1), and this was
spotted on silica-based thin layer chromatography plates provided by
Analtech (Newark, DE). The solvent consisted of a 6:3:1 mixture of
ethyl acetate, hexane, and acetic acid (Fisher). Arachidonic acid and
anandamide (PerkinElmer Life Sciences) standards were run alongside the
experimental samples. The plate was then treated with
EN3HANCE autoradiography spray provided by PerkinElmer Life
Sciences and exposed on Kodak X-Omat AR film. After film development,
the areas corresponding to the bands on the x-ray film were scraped off
the plates and placed in scintillation vials with scintillation fluid
to quantify the products of the TLC analysis (anandamide, phospholipids, and arachidonic acid). Each experiment was performed in
triplicate. Following the time course experiments and TLC analysis, the
raw results were corrected to account for the percentages of the
organic layer, which was taken from each sample. The inhibition of
uptake values in Table I was obtained by averaging the triplicates and
assuming that the uptake of controls for each cell line was 100%.
Comparison of Anandamide Uptake in N18 Neuroblastoma, C6 Glioma,
and Hep2 Carcinoma--
To statistically quantify the uptake rates in
these three cell lines, the percentage of the total anandamide taken up
by each cell line was compared after 5 min of incubation under
identical conditions at the same time. The amount of uptake was then
related to the total number of cells, thus allowing the comparison of uptake rates among different cell lines. Each experiment was performed in triplicate on 35-mm plates, which were ~50% confluent. A fourth plate was run in parallel to determine the number of cells/plate.
Enzyme Assay for FAAH--
The enzyme assay for FAAH activity in
N18, C6, and Hep2 cells was conducted as described previously (24).
Cells were washed with ice cold PBS and scraped in ice-cold Tris-EDTA,
pH 7.6. The cells were then disrupted by sonication. Incubations were
performed in triplicate at 37 °C in a water bath with shaking. Each
incubation contained 10 µl of 50 mg/ml defatted bovine serum albumin
in H2O, 50 µl of the cellular extract, 30 µM anandamide (Cayman Chemical Co., Ann Arbor, MI), and
0.01 mCi of 120 mCi/mmol
arachidonyl[ethanolamine-1,2-14C]ethanolamide
(PerkinElmer Life Sciences). The control tubes contained everything
except the cell extract. The reactions were terminated after 30 min by
the addition of 2 volumes of chloroform:methanol (1:1). The
radioactivity in the aqueous phase was measured by liquid scintillation
counting. The number of cells/plate was determined with a
hemacytometer. The specific activity was expressed as nanomoles of
anandamide hydrolyzed/106 cells/h. This was more meaningful
than activity based on the amount of protein because protein levels
varied widely among the different cell types.
Transfection of Hep2 Cells with Rat FAAH cDNA--
Cells
were seeded at 3 × 105 cells/35-mm dish on day 1. On
day 2, cells were transfected with 2.5 µg of DNA using 2.5 µl of LipofectAMINE according to the manufacturer's instructions (Life Technologies, Inc.). On day 4, cells were either harvested in 150 µl
of Tris-EDTA, pH 7.4, for FAAH assays or used for uptake experiments. The control Hep2 cells were either untransfected and
serum-starved or were transfected with the pcDNA3 vector
(Invitrogen, Carlsbad, CA), and the experimental Hep2 cells were
transfected with rat pcDNA3-FAAH cDNA (30).
Comparison of Anandamide Uptake in Hep2, Hep2 Vector-transfected,
and Hep2 FAAH-transfected Cells--
To quantify the anandamide uptake
rates of untransfected, vector-transfected, and FAAH-transfected cells,
the total anandamide taken up was compared after 5 min of incubation
under identical conditions. The cells were used for uptake 48 h
after transfection. The same procedure that was used for the time
course experiments was used for these incubations, with the exception
that 200 nCi/dish was used, nonspecific binding to the cellular
membrane was determined by incubating parallel plates in 37 °C
uptake medium for 3 s, and all incubations were stopped by adding
4 °C medium.
 |
RESULTS |
Anandamide uptake in N18, C6, and Hep2 cells was saturable and
temperature- and time-dependent. The apparent
Km and Vmax values were 1.8 µM and 17.4 pmol/min/106 cells for N18 cells,
0.7 µM and 3.9 pmol/min/106 cells for C6
cells, and 20.9 µM and 5.9 pmol/min/106 cells
for Hep2 cells, respectively.
Time course experiments with N18, C6, and Hep2 cells were conducted to
qualitatively determine the effects of two FAAH inhibitors, MAFP and
PSF, on anandamide uptake. Fig.
1A shows that after
preincubating C6 cells with MAFP for 10 min, the amount of anandamide
that enters the cell is reduced at each time point from 2 to 6 min. At
6 min, cellular net uptake of labeled anandamide in the control without inhibitor is about double the net cellular uptake of labeled anandamide in the presence of MAFP. The results of the N18 cell time course experiment in Fig. 1C demonstrate that the effect of MAFP on
cellular uptake is not restricted to C6 cells. After 6 min, the control cells have taken up approximately twice as much labeled anandamide as
did the MAFP-treated cells. To study the effects of PSF, the other
hydrolase inhibitor used in these studies, on anandamide uptake,
identical experiments were conducted in N18 and C6 cell lines. Fig. 1,
B and D, shows that preincubating C6 and N18
cells with 100 nM PSF for 10 min also reduces the amount of
anandamide entering the cell at each time point from 2 up to 6 min. In
experiments similar to those described above for C6 and N18 cells,
uptake in Hep2 cells was characterized in the presence of MAFP and PSF. In contrast to the results found with N18 and C6 cells, net uptake was
not decreased in the presence of either inhibitor (Fig. 1, E
and F). The shapes of the curves resulting from the time
course experiments are interesting. First, anandamide is accumulated at
a greater rate during the first min than during the rest of the
experiment, and the amount taken up is approximately the same with or
without the inhibitor. Second, the cells with uninhibited FAAH show a
linear uptake after 1 min, whereas those that are inhibited or lack
FAAH appear to level slowly to a plateau. The results in Fig. 1 are
qualitatively representative of at least three experiments conducted
with N18, C6, and Hep2 cells.

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Fig. 1.
Time course illustrating the effect of MAFP
or PSF on anandamide uptake in C6 glioma cells, N18 neuroblastoma, and
Hep2 laryngeal carcinoma cells. represents uptake at 37 °C
(corrected for nonspecific binding at 4 °C), and represents
uptake at 37 °C in the presence of 1 µM MAFP or 100 nM PSF (corrected for nonspecific binding at 4 °C).
Uptake is measured as picomoles of anandamide present inside the cell
at a particular time (1-6 min). The points were generated
using Sigma Plot, and the curves were fitted using
regression analysis (Hyperbola III).
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To more accurately characterize the effect of FAAH inhibition on
anandamide uptake, the total cellular radioactivity resulting from
anandamide incorporation was quantified in N18, C6, and Hep2 cells
after an incubation of 5 min in the absence or presence of MAFP (Table
I). MAFP inhibited the incorporation by
~40% in both N18 and C6, and this effect was highly significant for
both of these cell lines (p = 0.0001). However, MAFP
did not inhibit anandamide incorporation in Hep2. In fact, it seemed to
cause a slight stimulation, although this effect was not quite
significant (p = 0.0553). Whereas 1 µM
MAFP was chosen for these experiments, similar results were observed at
100 nM (data not shown). The observed inhibition in N18 and
C6 cells in the presence of MAFP was not due to toxicity because cells
exposed to MAFP do not exhibit decreased viability as observed after
trypan blue staining (data not shown).
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Table I
The effect of MAFP upon the cellular uptake of anandamide in C6 glioma
cells, N18 neuroblastoma, and Hep2 laryngeal carcinoma cells
Quantification of label in cells after 5-min incubation with
[3H]anandamide. The total number of counts was determined in
the absence ( ) and presence (+) of 1 µM MAFP. These
values were corrected for nonspecific binding by the subtraction of
counts from a 4 °C incubation. The average of the controls for each
cell line was normalized to 100%. A Student's t test (two
sided) was used to compare the differences between those cells in the
presence (+) and absence ( ) of MAFP and determine the p
value. The percent inhibition of uptake was calculated by (dpm in
control cells dpm in MAFP-treated cells) × 100/dpm in
control cells. The remainder of the sample was used for thin layer
chromatography analysis as shown in Fig. 2.
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To determine the fate of [lsqb]3H]anandamide
(arachidonyl[5, 6, 8, 9, 11, 12, 14, 15-3H]ethanolamide)
after being taken up by the cells, thin layer chromatography was
performed on those control and experimental C6, N18, and Hep2 cells,
which were quantified in Table I. After incubation of C6 and N18 with
anandamide in the absence of inhibitor (
), the main radioactive
product formed inside the cell was phospholipids (83% for C6, 93% for
N18), which remain at the origin in this TLC development system (Fig.
2). For the C6 and N18 cells, only 14 and
3% of the radioactivity in the cell was free anandamide, respectively,
and only 3% of the radioactivity was detected as arachidonic acid in
the C6 cells and none in the N18 cells. Because of overexposure, the
TLC chromatogram for N18 cells (Fig. 2) gives the impression of quite
high anandamide levels in the samples without inhibitor (
), although
counting of the samples scraped from the plate actually shows only 3%
free anandamide. In the presence of MAFP (+), the entire radioactivity
in the C6 and N18 cells is accounted for by anandamide (Fig. 2).
Anandamide accumulated in these cells because the breakdown reaction by
FAAH was rendered inactive. These experiments raise the possibility
that MAFP exerts its effect by inhibiting the transporter. However, it
seems unlikely that MAFP exerts its effect in this way as shown by the
TLC experiments with Hep2 cells (Fig. 2). The anandamide transport
mechanism is operational in these cells in the absence of MAFP (
).
However, unlike the situation with control C6 and N18 cells, anandamide is not broken down (>90% of the radioactivity in the cell is
anandamide) after being transported into the cells because these cells
lack FAAH (5). Significantly, in the presence of MAFP (+) transport is
not inhibited because there is no significant difference in the amount
of anandamide on the TLC, and as mentioned above (Table I), there seems
to be a slight stimulation. The TLC pattern for Hep2 cells with (+) or
without (
) MAFP treatment is the same as that observed in N18 and
C6 cells that have been treated with MAFP.

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Fig. 2.
TLC autoradiogram of C6 glioma cells, N18
neuroblastoma cells, and Hep2 human laryngeal carcinoma cells incubated
for 5 min. As indicated, experiments were conducted with or
without 1 µM MAFP. Arrows indicated the
position of anandamide (AEA), arachidonic acid
(AA), and phospholipids (PL) as determined by
standards. The contrast in the N18 autoradiograph was adjusted to make
it comparable with the C6 and Hep2 panels. The apparent large amount of
anandamide in the control N18 cells is an artifact due to overexposure
of the TLC radiogram that caused saturation of the film even at low
levels of radioactivity. Exposure times for these autoradiographs are 4 days for C6, 7 days for N18, and 2 days for Hep2. Replicate
chromatograms for each experiment were essentially identical.
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An experiment was performed to make a side by side comparison of N18,
C6, and Hep2 in terms of their uptake and enzyme activity. Interestingly, the absolute rate of uptake was found to vary among the
different cell types when the cell numbers were carefully quantified,
and this correlated approximately with the amount of FAAH activity
(Fig. 3). The N18 cells had the greatest
total uptake with the C6 and Hep2 cells having ~64 and 51% of the
uptake of the N18, respectively. The FAAH activity in C6 was found to be approximately 60% of that found in N18, although there was no
measurable FAAH activity in Hep2 cells as mentioned above.

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Fig. 3.
Comparison of uptake and FAAH activity among
N18, C6, and Hep2 cell lines. Each experiment was performed in
triplicate (repeated twice for the N18 and C6 and three times for the
Hep2 cells), and the graph indicates the mean ± S.E. of each
group. Each experiment was standardized for percentage
uptake/106 cells. The graph was generated using Sigma Plot.
In N18 cells, the uptake was 29 ± 1.64 pmol/106
cells, and the specific activity of FAAH was 0.87 ± 0.24 nmol/h/106 cells, and these values were normalized to 100%
on the bar graph.
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FAAH transfection experiments were undertaken to demonstrate that
uptake is dependent upon FAAH activity in Hep2 cells. The Hep2 cells,
which have no measurable FAAH activity, were transfected with a
pcDNA3 vector as a control or with pcDNA3-FAAH cDNA (Fig. 4). The vector-transfected Hep2 cells had
no measurable FAAH activity, whereas the Hep2 cells transfected with
pcDNA3-FAAH cDNA were very active (2.92 nmol ± 0.19/h/106 cells), demonstrating the expression of FAAH
(p < 0.0001). Significantly, anandamide uptake in the
transfected cells doubled (8.27 ± 1.0 pmol for transfected
versus 4.28 ± 0.21 pmol for untransfected cells and
4.29 ± 0.34 pmol for vector-transfected cells) (Fig. 4).
This finding demonstrates that uptake is dependent upon FAAH activity.

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Fig. 4.
Comparison of anandamide uptake and FAAH
enzymatic activity in pcDNA vector-transfected and
pcDNA3- FAAH-transfected Hep2 cells. The values shown are the
mean ± S.E. from two experiments, each experiment was performed
in triplicate. The uptake of anandamide in Hep2 cells transfected with
pcDNA3-FAAH was 8.2 ± 0.37 pmol/106 cells, the
specific activity of FAAH was 2.92 ± 0.19 nmol/h/106
cells, and these values were normalized to 100%.
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The intracellular concentration of anandamide was calculated for each
of the cell types (Table II). The
intracellular concentration of anandamide in N18, C6, and Hep2 cells
was determined to be ~5-, 7-, and 18-fold greater, respectively, than
the extracellular concentration of 100 nM that was used in
these experiments. These data strongly suggest some sequestering
mechanisms for accumulating anandamide inside cells without FAAH or
when FAAH is inhibited with MAFP.
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Table II
Intracellular concentrations of anandamide in the presence of inhibitor
The intracellular concentrations of anandamide were calculated in N18,
C6, and Hep2 cells after a preincubation with 1 µM MAFP
for 10 min followed by a 5-min incubation at 37 °C in supplemented
Dulbecco's modified Eagle's medium containing 100 nM
anandamide and 1 µM MAFP. Cell volumes of N18 and C6
cells were described previously (37). The intracellular volume of Hep2
cells was kindly provided by Janikke Ludt and Kirsten Sandvig (personal
communication) from calculations related to prior experiments (38).
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DISCUSSION |
It is known that anandamide uptake is an energy-independent,
reversible process and is governed by a concentration gradient of
unbound anandamide, which exists across the cellular membrane, i.e. a facilitated diffusion-mediated transporter (8, 12, 25). The cells employed in the current study also demonstrate the
characteristics of facilitated diffusion in that they exhibit saturation kinetics as well as temperature and time dependence. Using
1-min incubation periods to obviate any effects due to metabolism, the
uptake of anandamide as a function of concentration exhibited Michaelis-Menten kinetics. The Km of ~1
µM that we determined for the N18 and C6 cells was
similar to that reported for cerebral cortical neurons and astrocytes,
astrocytoma, and CHP100 neuroblastoma (13, 14, 25). The
Km that we determined for the Hep2 (20 µM) was similar to reports for cerebellar granule neurons (8). In addition to a facilitated diffusion component of the uptake, a
nonsaturable proportion of the uptake, particularly with lipophilic
molecules such as anandamide, may occur via simple diffusion across the
membrane (for review see Ref. 26).
FAAH has been shown by immunohistochemistry to be localized to the
endoplasmic reticulum (27, 28). It is the enzyme that is responsible
for the inactivation of anandamide in brain, liver, other tissues, and
in many cell lines (20, 29, 30). In our experiments we found a
correlation between the rate of uptake in cell lines and the amount of
FAAH, giving further credence to the hypothesis that anandamide
hydrolysis drives uptake (Fig. 3). However, the most unequivocal
demonstration that anandamide uptake is coupled to its metabolism by
FAAH was shown in Hep2 cells transfected with FAAH. Strikingly, the
total amount of anandamide uptake doubled in Hep2 cells transfected
with FAAH (p < 0.0003). Although this experiment
definitively demonstrates that uptake is dependent upon FAAH activity,
the nature of the transporter in Hep2 may be structurally different
from those of N18 and C6 and other systems reported to transport
anandamide. A putative transporter(s) for anandamide has not been
characterized. The large variation in Km values (for
review see Ref. 32) for cells that take up anandamide suggests that
there may be more than one transporter. Recently, another enzyme was
described that hydrolyzes anandamide (31). However, it occurs in a
human megakaryoblastic cell line, has a pH optimum of around 5, and is
less sensitive to phenylmethylsulfonyl fluoride (PMSF) and MAFP.
A model that accounts for our observations is shown in Fig.
5. The first step is anandamide uptake
into the cell (5-10). In the C6 and N18 control cells, the observed
linear increase in disintegrations/min inside the cell (Fig.
5A, Control) reflects the continued cellular
accumulation of anandamide and its products. The driving force for the
continued movement of anandamide inside the cell is its degradation. A
breakdown results in low anandamide concentration inside the cell, and
this disfavors the establishment of equilibrium between anandamide
inside and outside the cell. The linear uptake rates indicate that
there are no changes in substrate supply (less than 20% of medium
anandamide was taken up in 5 min) or cell viability (enzymatic activity
was constant) during the assay. In the control cells, there is only a
small amount of anandamide accumulation in the cells after 6 min (Fig. 2), and the same results were observed with longer incubation periods
up to 2.5 h (5, 6). The increase of disintegrations/min inside the
cell reflects the fact that the bulk of [3H]anandamide
taken up into the cell is rapidly degraded to other radioactive
products containing arachidonic acid (the labeled portion of
anandamide) due to the presence of intracellular FAAH. Arachidonic acid
does not accumulate because it is rapidly transformed to phospholipid
(see Fig. 2). During the incubation period used in these experiments,
insignificant amounts of labeled phospholipids and arachidonic acid
that are formed inside the cells are exported back into the medium as
observed by TLC analysis of lipids in the medium (data not shown).
Similarly, only small amounts of anandamide would be expected to be
exported into the medium, although the transport is reversible (6, 8).
When anandamide is incubated with N18 and C6 cells for longer periods
(30 min-2.5 h), anandamide uptake continues to be unabated, and the
label from the phospholipid is subsequently incorporated into other
compounds, such as triglycerides and cholesterol esters (5, 6).

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Fig. 5.
Proposed mechanism for the inhibition of
cellular anandamide uptake by MAFP. A, in the control
cells (Control), the degradation of anandamide
([3H]anandamide, ) by FAAH, after being taken up by
the carrier from the medium, is illustrated. The presence of FAAH in
the endoplasmic reticulum ensures a low concentration of free
anandamide in the cell relative to the medium, and this concentration
gradient drives the uptake. Large amounts of labeled metabolites
([3H]anandamide metabolites, ) accumulate in the cell.
B, the MAFP-treated cells illustrate the effect of MAFP on
anandamide uptake (FAAH Inhibited). As the cell takes up
anandamide ([3H]anandamide, ), the presence of MAFP
prevents its breakdown by FAAH. The intracellular concentration
increases, and the attainment of equilibrium between free intracellular
and extracellular anandamide prevents further accumulation.
Intracellular membranes or a putative anandamide-binding protein act as
a "sink" for anandamide.
|
|
In C6 and N18 in the presence of inhibitors (Fig. 5, FAAH
Inhibited), the shape of the uptake curves (Fig. 1) reflects the situation when cellular degradation of anandamide is blocked. The
inhibition of anandamide breakdown results in its intracellular build-up and the attainment of equilibrium between free intracellular and extracellular anandamide. The shifting of the anandamide
concentration gradient in a direction that favors equilibrium prevents
further net movement of anandamide from the outside to the inside of
the cell. The shape of the experimental curves in Fig. 1 supports this
interpretation of the data because uptake reached a plateau quickly
after the 1-min time point, reflecting equilibration of free
nonmetabolized anandamide across the membrane. The nearly identical
uptake seen after a 1-min incubation in control and FAAH-inhibited
cells suggests that the uptake during this time period is entirely due
to movement of anandamide across the membrane via the carrier.
Furthermore, these data indicate that the inhibitor is specific for
FAAH and does not affect the transporter directly. The complete
leveling off of anandamide accumulation in FAAH-inhibited cells
indicates that the catabolism of anandamide by FAAH is a major driving
force for its accumulation in these particular cells. For example, in
the case where uptake would have reflected metabolism, rates during the
first minute would have to be much higher in control cells because
anandamide would be siphoned away and not allowed to equilibrate with
extracellular anandamide. This observation suggests that the inhibitor
could be very useful in cases where complete suppression of anandamide
utilization is desired. However, even in the presence of this
inhibitor, because membrane transport is not inhibited, anandamide
becomes highly concentrated in the cells (Table II). One possible
explanation for this is that after uptake, anandamide was dispersed
throughout intracellular membranes and/or to an anandamide-binding
protein. (12, 32). It has been observed that long chain fatty acid
diffusion within the cytoplasm is slow and lacks selective targeting
toward specific organelles. However, fatty acids bound to a cytoplasmic
binding protein have increased solubility and targeting (26, 33). It is
possible that a binding protein exists that binds anandamide and speeds
up the process of anandamide intracellular transport.
Hep2 cells, which lack FAAH, have the same time course and TLC profile
as the cells incubated with FAAH inhibitor. MAFP does not inhibit
uptake in Hep2, demonstrating that MAFP does not exert its effect on
uptake at the level of the putative transporter in these cells.
Beltramo et al.(14), using relatively weak FAAH inhibitors
with cortical astrocytes in culture, concluded that there was no effect
upon uptake of anandamide after a 4-min incubation. Likewise, they did
not observe any inhibition of FAAH by
N-(4-hydroxyphenyl)arachidonylamide, a selective uptake
inhibitor. We observed a 15% inhibition of uptake in Hep2 cells in the
presence of 10 µM
N-(4-hydroxyphenyl)arachidonylamide (data not shown).
Bisogno et al. (34) reported that PMSF, which strongly
inhibited FAAH, gave nearly 50% inhibition of uptake during a 20-min
incubation in RBL-2H3 basophils, and this was postulated to
occur as a result of alkylating the transporter. Likewise, Maccarrone
et al. (25) reported that PMSF inhibited uptake 50% in
RBL-2H3 cells. The mechanism they postulated was that the transporter
may contain a cystine that was inactivated. In view of our current
study, it is possible that the effect of PMSF on uptake may be
secondary to its effect on FAAH. This has recently been proposed for
PMSF, arachidonyltrifluoromethylketone, and MAFP in RBL-2H3 cells (12).
The approach in our study distinguishes between the two possible
mechanisms (i.e. directly blocking the carrier for transport
or the indirect effect of FAAH inhibition). These studies establish a
theoretical basis for the use of FAAH inhibitors as therapeutic agents
to increase the levels of extracellular anandamide. For example, PSF
has been shown to raise exogenous levels of anandamide in brain
hippocampal slice (35). Also, analogs of MAFP have recently been shown
to be very potent and long acting antinociceptive agents
in vivo (36).
 |
ACKNOWLEDGEMENTS |
We thank Rebecca Rowehl and Lee Ann Silver
for the help with cell culture and Lou Charnon-Deutsch and Karen
Henrickson for the artwork. We also thank Eric Barker for providing a
preprint of manuscripts for us to read.
 |
FOOTNOTES |
*
This work was supported by National Institute of Health
Grants DA09374 (to D. G. D.), DA09155 (to C. J. H.), and NIDDK33301 (to N. A.). A preliminary report was presented at the International Cannabinoid Research Society, Acapulco, Mexico (Deutsch, D. G., Kunz, J. S., Abumrad, N., and Hillard, C. C. (1999)
Symposium on the Cannabinoids, p. 28, International
Cannabinoid Research Society, Burlington, VT June 18-20, 1999. These studies were presented in Jeffrey S. Kunz's Honors Thesis at
SUNY at Stony Brook.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 631-632-8595;
Fax: 631-632-8575; E-mail: ddeutsch@notes.cc.sunysb.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M003161200
 |
ABBREVIATIONS |
The abbreviations used are:
FAAH, fatty-acid
amide hydrolase;
MAFP, methylarachidonylfluorophosphonate;
PSF, palmitylsulfonyl fluoride;
PMSF, phenylmethylsulfonyl fluoride;
N18, N18TG2 neuroblastoma cells;
C6, C6 glioma cells;
Hep2, human laryngeal
carcinoma cells.
 |
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