Báo cáo khoa học: Uptake and metabolism of [3H]anandamide by rabbit platelets Lack of transporter? ppt

Uptake and metabolism of [
3
H]anandamide by rabbit platelets
Lack of transporter?
Lambrini Fasia, Vivi Karava and Athanassia Siafaka-Kapadai
Department of Chemistry (Biochemistry), University of Athens, Greece
Anandamide is an endogenous ligand for cannabinoid
receptor and its protein-mediated transport across cellular
membranes has been demonstrated in cells derived from
brain as well as in cells of the immune system. This lipid
is inactivated via intracellular degradation by a fatty acid
amidohydrolase (FAAH). In the present study, we report
that rabbit platelets, in contrast to human platelets, do not
possess a carrier-mediated mechanism for the transport of
[
3
H]anandamide into the cell, i.e. cellular uptake was not
temperature dependent and its accumulation was not satu-
rable. This endocannabinoid appears to enter the cell by
simple diffusion. Once taken up by rabbit platelets,
[
3
H]anandamide was rapidly metabolized into compounds
which were secreted into the medium. Small amounts of free
arachidonic acid as well as phospholipids were amongst the
metabolic products. FAAH inhibitors did not decrease
anandamide uptake, whereas these compounds inhibited
anandamide metabolism. In conclusion, anandamide is
rapidly taken up by rabbit platelets and metabolized mainly
into water-soluble metabolites. Interestingly, the present
study also suggests the absence of a transporter for anand-

amide in these cells.
Keywords: anandamide; anandamide transporter; fatty acid
amidohydrolase; rabbit platelets.
Anandamide (N-arachidonoylethanolamine) is the most
important member of a class of endogenous lipids called
N-acylethanolamines that have been proposed as the
physiological ligands for the cannabinoid (CB) receptors
[1,2]. In addition to anandamide, there is another endo-
cannabinoid namely 2-arachidonoylglycerol that has been
isolated from rat brain and canine gut [3,4].
For anandamide signaling via CB receptors, an active
uptake mechanism to transport anandamide into the cell
has been reported. The properties of this transport process
together with the transport mechanisms of the related
endogenous compounds 2-arachidonoylglycerol and palmi-
toylethanolamide have recently been reviewed [5]. Cellular
uptake is followed by the rapid degradation of anandamide
by an endoplasmic reticular integral membrane-bound
enzyme called fatty-acid amide hydrolase (FAAH) [6,7].
Anandamide uptake has been demonstrated in neuro-
blastoma and glioma cells [8], cortical neurons [9], cerebellar
granule neurons [10], cerebral cortical neurons and
astrocytes [11], macrophages and basophils [12], human
platelets [13], human mast cells [14] and human endothelial
cells [15]. In these cells, anandamide transport has the
characteristics of facilitated diffusion rather than an active
cotransport system or passive diffusion, as it follows
saturation kinetics, is temperature- and time-dependent,
shows structural specificity among N-acylethanolamines, is
bidirectional and lacks the requirement of ATP or extracel-

lular sodium [9–11,16]. Several specific inhibitors of anand-
amide transport have been described including various
structural analogs of anandamide [11,17–22]. Ligand struc-
tural requirements of anandamide transporter are very
different from those for the CB1 receptor. However the
transporter and the FAAH do share some of them [21].
The kinetic parameters of anandamide accumulation
among different cell types is varied suggesting the existence
of different subtypes of anandamide carrier [16]. For
example, in the cerebellar granule neurons, K
m
¼ 41 ±
15 l
M
[10] while in the human umbelical vein endothelial
cells K
m
¼ 190 ± 10 n
M
[15]. It should be noted that
Bisogno et al. demonstrated that different kinetics might
depend on the experimental protocol [22].
Studies in several cell types have shown that the net
movement of anandamide into the cells is coupled to the
activity of intracellular FAAH [23,24]. FAAH is responsible
for the catabolism of anandamide and it contributes to
anandamide uptake by making and maintaining a concen-
tration gradient between the extracellular space and the
interior of the cell. Therefore, in the presence of various
inhibitors of FAAH (e.g. phenylmethylsulfonyl fluoride),

the uptake is limited by the shifting of anandamide
concentration gradient in a direction that leads to equilib-
rium [23,24].
FAAH is the main catabolic enzyme in the conversion of
anandamide into free arachidonic acid and ethanolamine
Correspondence to A. Siafaka-Kapadai, Department of Chemistry
(Biochemistry), University of Athens, Panepistimioupolis,
157 71 Athens, Greece.
Fax: +30 210 7274476, Tel.: +30 210 7274493,
E-mail:
Abbreviations: CB, cannabinoid receptors; FAAH, fatty acid
amidohydrolase; COX, cyclooxygenase; POPOP, 1,4-di[2-(5-phenyl-
oxazole)] benzene; PPO, 2,5-diphenyloxazole; ACD, acid-citrate-
dextrose; Tg/Ca, Tyrodes/gelatin/Ca
2+
; PL, phospholipids;
LOX, lipoxygenase.
Enzymes: fatty acid amide hydrolase (arachidonoylethanolamide
amidohydrolase; EC 3.5.1.4).
(Received 4 December 2002, revised 6 May 2003,
accepted 19 June 2003)
Eur. J. Biochem. 270, 3498–3506 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03724.x
[25,26]. FAAH is also capable of metabolizing several other
fatty acid amides [7] and esters such as oleamide [27] and
2-arachidonoylglycerol [28] as well as catalyzing the reverse
condensation of free arachidonic acid and ethanolamine to
the formation of anandamide [6,29,30]. FAAH is a mem-
brane-bound protein localized mainly in the microsomal
and mitochondrial fractions [6,31–33]. The K
m

of the
enzyme for anandamide has been reported to be 2–67 l
M
depending on the enzyme preparations and assay conditions
and the optimum pH is 8.5–10 [25,26]. The existence and
secretion of a FAAH from the unicellular eucaryote
Tetrahymena pyriformis, has been recently reported by our
laboratory [34]. Other enzymes also directly metabolize
anandamide. Various purified lipoxygenases as well as
lipoxygenases derived from tissues convert anandamide to
polar metabolites (hydroperoxides and hydroperoxy-deriv-
atives) [35–37]. Recombinant human cyclooxygenase-2
(hCOX-2) but not hCOX-1 has the ability to directly
oxidize anandamide to yield prostaglandin E
2
ethanolamide
[38].
Here, we report the results of a study aimed at assessing
the possibility that anandamide is taken up by rabbit
platelets via a carrier-mediated transport system as it has
been suggested for a number of cells including human
platelets, and subsequently exploring anandamide meta-
bolic fate.
Materials and methods
Materials
[
3
H]Anandamide (200 CiÆmmol
)1
), radiolabelled at the

arachidonic moiety, was purchased from American Radio-
labelled Chemicals Inc. (St Louis, MO, USA). Anandamide,
arachidonic acid, phosphatidylethanolamine, phenyl-
methylsulfonyl fluoride, caffeic acid, indomethacin, bovine
serum albumin, BSA, 1,4-di[2-(5-phenyloxazole)] benzene
(POPOP), 2,5-diphenyloxazole (PPO) and naphthalene
were from Sigma Chemicals Co. VDM11 was from Tocris
Cookson Ltd (UK). Other chemicals were of the highest
purity available.
Buffers
The anticoagulant solution, acid/citrate/dextrose (ACD),
contained 1.36% citric acid, 2.5% trisodium citrate and
2.0% dextrose (w/v). The resuspension buffers for the
washed rabbit platelets were: (a) Tyrodes/gelatin/Ca
2+
pH 7.2 (Tg/Ca) which contained 0.8% NaCl, 0.02% KCl,
0.02% MgCl
2
, 0.1% dextrose, 0.25% gelatin and 0.02%
CaCl
2
(w/v) and (b) 10 m
M
NaCl/P
i
pH 7.4 which con-
tained 0.14% Na
2
HPO
4

,0.12%NaH
2
PO
4
and 0.82%
NaCl (w/v).
Preparation of washed rabbit platelets
Blood was drawn from the central vein of the ear of male
rabbits and was collected into an ACD anticoagulant
solution. Platelets were washed as described previously
[39,40]. Platelets were finally resuspended in Tg/Ca pH 7.2
or a 10 m
M
NaCl/P
i
pH 7.4, at a concentration of 3 · 10
8
plateletsÆmL
)1
.
Measurement of [
3
H]anandamide accumulation
by rabbit platelets
Platelet suspension (3 · 10
8
plateletsÆmL
)1
)inTg/Cawas
incubated with 1.25 n

M
[
3
H]anandamide at 37 °Cfor
various time intervals. The incubation was stopped by
the addition of 4% v/v ice-cold formol and the
suspension was placed on ice. Platelets were separated
from the medium by centrifugation at 13 000 g for
2 min, washed with 0.9% v/v NaCl and extracted
according to Bligh and Dyer [41]. [
3
H]Anandamide
(100 n
M
) uptake was also studied in platelet suspension
in 10 m
M
NaCl/P
i
. In this case, after the incubation with
[
3
H]anandamide, platelets were washed with NaCl/P
i
containing 1% w/v BSA and the study was repeated at
various temperatures (0–4, 25 and 37 °C) as well as in the
presence of VDM11 (20 l
M
)at37°C. For the kinetic
studies, [

3
H]anandamide at concentrations between 100
and 2000 n
M
was used and the incubation took place at
37 °C. The concentration of phenylmethylsulfonyl fluor-
ide used was 2 m
M
and the preincubation time was
15 min.
Metabolism of [
3
H] anandamide by intact rabbit platelets
Washed rabbit platelets were resuspended in Tg/Ca
pH 7.2 or in 10 m
M
NaCl/P
i
resulting in a final concen-
tration of 3 · 10
8
plateletsÆmL
)1
. The platelet suspension
was incubated with [
3
H]anandamide 1.25 n
M
(specific
activity 200 CiÆmmoL

)1
)or450n
M
in certain experiments,
at 37 °C for different time intervals. Lipids from 0.5-mL
aliquots of the platelet suspension were extracted accord-
ing to Bligh and Dyer [41] and were separated by TLC on
heat-activated silica-gel G-plates using CHCl
3
/CH
3
OH/
NH
4
OH 80 : 20 : 2 (v/v). After visualization by exposure
to iodine vapors, lipids corresponding to free arachidonic
acid, phosphatidylethanolamine and anandamide were
scraped off the plates and their radioactivity was meas-
ured by liquid scintillation counting using a toluene base
(5 g PPO and 0.3 g POPOP in 1 L toluene) as the
scintillation fluid. The radioactivity of the methanol/water
phase was also measured using dioxan/water base (100 g
napthalene, 7 g PPO, 0.3 g POPOP, 200 mL water in 1 L
dioxan) as the scintillation fluid. The liquid scintillation
counter used was a Wallac 1209 Rackbeta, Pharmacia.
Inthesamemanner,for[
3
H]anandamide accumulation
experiments, after incubation of platelets with radiolabeled
anandamide, the cells were separated from the medium by

centrifugation at 13 000 g for 2 min, washed and the lipids
from platelets and media were extracted and separated
as described above. The experiments were repeated after
preincubation of platelet suspension with either 2 m
M
phenylmethylsulfonyl fluoride or 100 l
M
caffeic acid, or
0.5 and 75 l
M
indomethacin.
Results
[
3
H]Anandamide uptake and metabolism
[
3
H]Anandamide was rapidly taken up by rabbit platelets
as shown in Fig. 1. At 2 min, there was a high percentage
of radioactivity incorporated into platelets (32.6 ± 2.7%).
Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J. Biochem. 270) 3499
Surprisingly, the amount of radioactivity associated with
the platelets was reduced over time in parallel with an
increase in extracellular radioactivity. This uptake and
metabolism was completed within 20 min and then the
amount of radioactivity remained constant in both the
platelets and the extracellular space. Our studies with intact
rabbit platelets showed that these cells were capable of
rapidly metabolizing [
3

H]anandamide (Fig. 2). The main
metabolic products in the control cells were found in the
water/methanol phase of the Bligh–Dyer extraction and
referred throughout this study as Ôwater-solubleÕ com-
pounds, and the metabolism was completed at 20 min
when a plateau was reached. The metabolism occurred
rapidly, since at 5 min almost 50% of the exogenously
added [
3
H]anandamide had been catabolized. Only a small
percentage of [
3
H]anandamide metabolic products were
free arachidonic acid and phospholipids ( 2% and
 10% at 20 min, respectively, Fig. 2).
Very similar results were obtained when the metabolism
experiment was performed in the presence of 100 l
M
caffeic acid (a LOX inhibitor). The main metabolic
products were water-soluble compounds and the metabo-
lism occurred rapidly ( 50% at 5 min) and reached a
plateau at 20 min (Fig. 3A). In the presence of 0.5 l
M
indomethacin (a COX inhibitor) a small inhibition was
observed ( 10% at 5, 10 and 20 min) and reached a
plateau at 40 min. The extent of the metabolism at
40 min was the same compared to the control cells (not
shown). In the presence of 75 l
M
indomethacin, a

significant inhibition of anandamide metabolism was
observed. At 5 min, the inhibition was  65% and the
metabolism reached a plateau at 20 min when the
inhibition was approximately 50% (Fig. 3A). This inhibi-
tion was almost totally attributed to water-soluble
metabolites (Fig. 3B).
In subsequent studies, attempts were made to further
identify the metabolic products of [
3
H]anandamide in
intact platelets and in the extracellular space. For this
purpose, lipids were extracted from platelets as well as
from the medium and were separated by TLC. The
results are presented in Fig. 4. After a 2-min incubation,
the radioactivity incorporated in platelets corresponded
mainly to [
3
H]anandamide. Thereafter, the amount of
[
3
H]anandamide in platelets was reduced rapidly with
time while the amount of other radiolabeled products,
phospholipids, free arachidonic acid or water-soluble
compounds remained constant. The radioactivity profile
of the medium was completely different. At 2 min, the
total amount of radioactivity found in the media was
low and corresponded mainly to [
3
H]anandamide, which
had not been bound to platelets. This is consistent with

the observation that this amount was unchangeable with
time. On the other hand, the total amount of radio-
activity in the medium increased rapidly with time and
this increase was attributed totally to water-soluble
compounds.
In order to test the possible involvement of FAAH on
[
3
H]anandamide uptake and metabolism by rabbit platelets,
the effect of phenylmethylsulfonyl fluoride on [
3
H]ananda-
mide uptake was determined (Fig. 5). Preincubation of the
platelet suspension with phenylmethylsulfonyl fluoride had
no effect on the rapid uptake of [
3
H]anandamide by
Fig. 1. Distribution of radioactivity in platelets and medium. Platelet
suspension in Tg/Ca (3 · 10
8
plateletsÆmL
)1
) was incubated with
[
3
H]anandamide (1.25 n
M
)at37°C. At the time intervals indicated,
0.5 mL of platelet suspension was centrifuged, the supernatant (Y-1)
was removed and the cells were washed with 0.9% NaCl (w/v). The

supernatant (Y-2) was removed, lipids were extracted from platelets
and their radioactivity was measured. The sum of radioactivity in Y-1
and Y-2 is the radioactivity of extracellular space. Values are the
means ± SD of duplicate samples of three independent experiments.
Total c.p.m., 15 000–45 000.
Fig. 2. [
3
H]Anandamide metabolism by intact rabbit platelets. Platelet
suspension in Tg/Ca (3 · 10
8
plateletsÆmL
)1
) was incubated with
[
3
H]anandamide (1.25 n
M
)at37°C. At the time intervals indicated,
0.5 mL of platelet suspension were extracted according to Bligh and
Dyer [41]. Lipids were subjected to TLC and radioactivity was meas-
ured. The radioactivity of water-methanol phase was also measured.
(j) Water-soluble compounds (h) phospholipids (m) free arachidonic
acid (d) anandamide. Values are the means ± SD of duplicate sam-
ples of three independent experiments. Total c.p.m., 15000–45 000.
3500 L. Fasia et al. (Eur. J. Biochem. 270) Ó FEBS 2003
platelets. The amount of radioactivity in both platelets and
extracellular space remained constant with time. Phenyl-
methylsulfonyl fluoride is a well-known, strong inhibitor of
FAAH. The effect of phenylmethylsulfonyl fluoride on
[

3
H]anandamide accumulation may be due to the inhibition
of the anandamide metabolism. Similar results were
obtained using a more specific FAAH inhibitor, namely
arachidonoyltrifluoromethyl-ketone (data not shown).
After preincubation of platelets with phenylmethylsulfonyl
fluoride, the distribution of radioactivity in platelets corres-
ponded to nonmetabolized [
3
H]anandamide (Fig. 6). Based
on these results, we assumed at the time that the uptake of
anandamide was carrier-mediated, coupled to its metabo-
lism and reached a plateau in 2 min when the metabolism
was inhibited by phenylmethylsulfonyl fluoride. In that case,
the uptake should be also temperature- and concentration-
dependent, well known characteristics of a facilitated
diffusion.
Effect of temperature on [
3
H]anandamide uptake
and metabolism
Studies were then undertaken to determine if anandamide is
transported across the cellular membrane via facilitated
diffusion as has been shown for a number of cell types. Since
this type of transport is temperature dependent, ananda-
mide uptake at 37 °C, 25 °C and 0–4 °C was examined. In
these experiments, 100 n
M
[
3

H]anandamide in 10 m
M
NaCl/P
i
was used in order to have experimental conditions
comparable to those previously reported in human platelets
[13] as well as other cells [12,15]. The profile of [
3
H]anand-
amide uptake was the same (Fig. 7) although the percentage
of radioactivity of platelet fraction was lower in NaCl/P
i
compared to that in Tg/Ca (Fig. 1). This difference could be
attributed to the higher [
3
H]anandamide concentration used
(100 n
M
instead of 1.25 n
M
) as well as to the presence of 1%
w/v BSA in the washing buffer. BSA apparently removed
Fig. 3. Effect of caffeic acid and indomethacin on [
3
H]anandamide metabolism by intact rabbit platelets. (A) Platelet suspension in 10 m
M
NaCl/P
i
(3 · 10
8

plateletsÆmL
)1
) was incubated with [
3
H]anandamide (450 n
M
)at37 °C. At the time intervals indicated, the medium (extracellular space) of
0.5 mL of the platelet suspension was removed by centrifugation. The platelets were extracted twice according to Bligh and Dyer [41]. Lipids were
subjected to TLC and radioactivity corresponding to nonmetabolized anandamide was measured. (B) Distribution of the radioactivity in water-
soluble compounds and in lipids extracted from platelets. Incubation time with [
3
H]anandamide: 40 min. W: water-soluble compounds, PL:
phospholipids, FFA: free arachidonic acid. Values are the means ± SD of duplicate samples of three independent experiments. Total c.p.m.,
6000–25 000.
Fig. 4. Distribution of the radioactivity of the platelets (A) and the extracellular space (B) into various lipids. Platelet suspension in Tg/Ca (3 · 10
8
plateletsÆmL
)1
) was incubated with [
3
H]anandamide (1.25 n
M
)at37°C. At the time intervals indicated, the medium (extracellular space) of 0.5 mL
of the platelet suspension was removed by centrifugation. The platelets as well as the extracellular medium were extracted twice according to Bligh-
Dyer [41] method. Lipids were subjected to TLC and radioactivity was measured. (j) Water-soluble compounds (m) phospholipids ())free
arachidonic acid (s) anandamide. Values are the means ± SEM of duplicate samples of one representative experiment.
Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J. Biochem. 270) 3501
[
3
H]anandamide that had not entered the platelets and

could have been nonspecifically bound to the plasma
membrane. In order to further test this hypothesis, platelets
were incubated with [
3
H]anandamide for 1–2 min (low
metabolism) and were then separated from the medium
(M
1
) by centrifugation and washed with NaCl/P
i
containing
BSA (M
2
). Interestingly, the radioactivity found in M
2
(corresponded mainly to [
3
H]anandamide) was higher than
that found in M
1
which corresponded mainly to water-
soluble compounds produced after [
3
H]anandamide meta-
bolism (data not shown). These results suggest that
[
3
H]anandamide, at least in part, may not be transferred
into the cells but is possibly nonspecifically bound to the
plasma membranes from which it was removed after

treatment with BSA.
As shown in Fig. 7, the profile of [
3
H]anandamide uptake
was identical at 37 °Cand25°C. At these two tempera-
tures, the metabolism of [
3
H]anandamide took place to
the same extent and resulted in the same products. When the
uptake of [
3
H]anandamide at 0–4 °C was studied, the
amount of radioactivity found in platelets was larger than
that at 37 °C (Fig. 7). This observation can be explained by
thesmallerextentof[
3
H]anandamide metabolism by intact
rabbit platelets at this low temperature. [
3
H]Anandamide
that was not metabolized remained bound to the platelets,
and resulted in a smaller amount of radioactivity being
released into the extracellular space at 0–4 °C compared to
37 °C.Inthecasethat[
3
H]anandamide had been trans-
ferred via facilitated diffusion, the uptake at 4 °C should
have been much lower.
Fig. 5. Effect of phenylmethylsulfonyl fluoride on [
3

H]anandamide
uptake by rabbit platelets. Platelet suspension in Tg/Ca (3 · 10
8
plateletsÆmL
)1
) was incubated with [
3
H]anandamide (1.25 n
M
)at
37 °C in the absence or presence of 2 m
M
phenylmethylsulfonyl
fluoride. At the time intervals indicated, the medium (extracellular
space) of 0.5 mL of the platelet suspension was removed by centrifu-
gation. The platelets as well as the extracellular medium were extracted
twice according to Bligh and Dyer [41]. The radioactivity of platelets
and extracellular space was measured. Values are the means ± SD of
duplicate samples of three independent experiments. Total c.p.m.,
15 000–45 000.
Fig. 6. Distribution of the radioactivity into various lipids in the presence
of phenylmethylsulfonyl fluoride. Platelet suspension in Tg/Ca (3 · 10
8
plateletsÆmL
)1
) preincubated with phenylmethylsulfonyl fluoride
(2 m
M
), was then incubated with [
3

H]anandamide (1.25 n
M
)at37°C.
At the time intervals indicated, the medium (extracellular space) of
0.5 mL of the platelet suspension was removed by centrifugation. The
platelets were extracted twice according to Bligh and Dyer [41]. Lipids
weresubjectedtoTLCandradioactivitywasmeasured.(j)Water
soluble compounds (m) phospholipids (h) free arachidonic acid ())
anandamide. Values are the means ± SD of duplicate samples of
three independent experiments. Total c.p.m., 15 000–45 000.
Fig. 7. Effect of temperature and VDM11 on [
3
H]anandamide uptake
by rabbit platelets. Platelet suspension in NaCl/P
i
(3 · 10
8
plate-
letsÆmL
)1
)wasincubatedwith[
3
H]anandamide (100 n
M
)atvarious
temperatures or after preincubation with 20 l
ı
`
VDM11 (for 10 min).
At the time intervals indicated, the medium (extracellular space) of

0.5 mL of the platelet suspension was removed by centrifugation. After
centrifugation, the supernatant was decanted and the platelets were
washed with NaCl/P
i
containing 1% w/v BSA. The platelets as well as
the extracellular medium were extracted twice according to Bligh and
Dyer [41]. The radioactivity of platelets and extracellular space was
measured. Values are the means ± SD of duplicate samples of two to
five independent experiments. Total c.p.m., 8000–45 000.
3502 L. Fasia et al. (Eur. J. Biochem. 270) Ó FEBS 2003
To explore this hypothesis further, the experiments at
37 °C were repeated in the presence of VDM11, a specific
anandamide membrane transporter inhibitor. Platelets
were preincubated for 10 min with 20 l
M
VDM11 as the
50% inhibitory concentration (IC
50
) reported for other
cells was 10–11 l
M
[19]. As shown in Fig. 7, although a
small inhibition ( 20%) of uptake was observed at 2 min,
the profile of [
3
H]anandamide uptake was almost identical
with or without VDM11 at 37 °C. Thus, these results
indicate the absence of a membrane transporter in rabbit
platelets.
Effect of concentration on [

3
H]anandamide uptake
and metabolism
Among the criteria for the characterization of a transport
process across cellular membranes as carrier-mediated, is its
saturation at high ligand concentrations. Therefore studies
were undertaken at [
3
H]anandamide concentrations of
100–2000 n
M
. Similar concentrations were used for human
platelets [13]. Cellular uptake was estimated from the total
radioactivity in platelets and the extracellular space after
1–2 min of incubation with [
3
H]anandamide. The catabol-
ism of [
3
H]anandamide was low during this time (Fig. 2).
The amount of radioactivity found in platelets was a linear
function of [
3
H]anandamide concentration (Fig. 8). These
results indicate that anandamide crossed the platelet plasma
membrane by simple diffusion and not by a carrier-
mediated transport. Again, the accumulation of [
3
H]anand-
amide in platelets was higher at 0–4 °Cthanat37°C due to

decreased metabolism. Interestingly, the uptake was also
linear but higher both in the presence of phenylmethylsul-
fonyl fluoride, and 0–4 °C apparently due to the inhibition
of FAAH activity, or decreased metabolism, respectively
(Fig. 8A and B).
Discussion
Initially, the purpose of these experiments was to investigate
the existence of a transporter in rabbit platelets, as it has
been suggested for human platelets [13] as well as for a
number of cells [8–12,14,15]. Additionally, it has been
reported by Braud et al. [42] and by our laboratory [43], that
aggregation of rabbit platelets caused by anandamide is
accomplished through its conversion to arachidonic acid by
the action of a FAAH; this is in contrast to human platelets
in which the process is independent of the arachidonate
cascade. The effect on rabbit platelets was blocked by the
FAAH inhibitor, phenylmethylsulfonyl fluoride. Having in
mind that in almost every cell type tested, FAAH is
localized in the membrane of microsomes (endoplasmic
reticulum) or mitochondria [6,31–33], it was assumed at
thetimethat[
3
H]anandamide should be taken up by a
facilitated diffusion process. Subsequently, [
3
H]anandamide
would be hydrolyzed to arachidonic acid, which in turn
would induce platelet aggregation through a well-known
process [44,45]. It should be noted that enzymes involved in
arachidonic acid metabolism, such as LOX and COX are

also localized inside the cell [44,46]. Surprisingly, the results
presented here indicate that rabbit platelets do not possess a
carrier-mediated mechanism for the transport of [
3
H]anand-
amide into the cell, i.e. the process was not temperature
dependent (Fig. 7) and was not saturable (Fig. 8) in
contrast to the results reported for human platelets [13]
and for other cells [8–12,14,15].
The uptake of [
3
H]anandamide was exactly the same at 37
and 25 °C (Fig. 7) but was higher at 0–4 °C, apparently due
to the lower degree of metabolism at 0–4 °Ccomparedto
that at 37 °Corat25°C. At these temperatures, even after
only 1–2-min incubation there was some anandamide
metabolism (Fig. 2). Therefore, the higher degree of uptake
was probably due to the diminished anandamide meta-
bolism.
Furthermore, very interesting results came from experi-
ments in which FAAH activity was inhibited; inhibition
resulted in an increase of the uptake (Figs 5, 6 and 8). If a
transporter is present, inhibition of anandamide hydrolysis
decreases rather than enhances the uptake as it has been
shown in previous studies [23]. On the contrary, if a
membrane transporter is really absent, as we suggest here,
Fig. 8. Concentration dependence of [
3
H]anandamide uptake by rabbit platelets. Platelet suspension in NaCl/P
i

(3 · 10
8
plateletsÆmL
)1
)wasincu-
bated with various concentrations of [
3
H]anandamide for 1 min at 0–4 °Cor37°C (A) or in the presence of 2 m
M
phenylmethylsulfonyl fluoride
(B). At the time intervals indicated, the medium (extracellular space) of 0.5 mL of the platelet suspension was removed by centrifugation. After
centrifugation, the supernatant was decanted and the platelets were washed with NaCl/P
i
containing 1% w/v BSA. The platelets were extracted
twice according to Bligh and Dyer. The radioactivity of platelets was measured. Values are the means ± SD of duplicate samples of three
independent experiments.
Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J. Biochem. 270) 3503
the inhibition of FAAH should increase the extent of the
apparent uptake. Hence, these results demonstrate the
absence of a membrane transporter in rabbit platelets.
Time dependence experiments (Fig. 1) revealed that
radioactivity was rapidly associated with platelets and then
gradually decreased within the cell and increased in the
extracellular space, suggesting the existence of a metabolic
process. Preincubation of platelets with phenylmethylsulfo-
nyl fluoride resulted in the rapid uptake of anandamide
( 90% of the total [
3
H]anandamide at 2 min) which
remained almost unchanged up to 40 min (Fig. 5). In the

presence of phenylmethylsulfonyl fluoride, anandamide
was neither metabolized nor released into the medium
(Fig. 6). The phenomenon seems to be rather a passive
diffusion and/or a nonspecific binding to the membrane of
platelets according to the results presented in Fig. 8. The
concentration dependence curve was linear up to 2000 n
M
anandamide, while in human platelets, the uptake was
saturable (K
m
¼ 200 ± 20 n
M
at 37 °C). In the presence
of phenylmethylsulfonyl fluoride, the concentration
dependence curve was also linear but higher (e.g. at
2000 n
M
[
3
H]anandamide the uptake in the presence of
phenylmethylsulfonyl fluoride was threefold higher than in
control cells). On the contrary, for human platelets it has
been reported that 100 l
M
phenylmethylsulfonyl fluoride
reduced anandamide uptake by  40% of the untreated
control [13] as it should be expected if a membrane
transporter is present [23]. Additionally, the uptake was
also linear but higher at 0–4 °C compared to 37 °C
(Fig. 8A) also suggesting the absence of a carrier-mediated

process, since this kind of transport could not take place at
low temperature.
Although, the possibility of the existence of a hidden
carrier-mediated transport along with the passive diffusion
could not be totally excluded, since the uptake was almost
the same at 1–2 min in the presence of phenylmethylsulfo-
nyl fluoride (Figs 5 and 8) and there is a small inhibition
at 2 min by VDM11 (Fig. 7), our data do not provide
any other evidence to support this hypothesis. The
coexistence of passive diffusion and a facilitated transport
has been reported for palmitoylethanolamide in Neuro-2a
neuroblastoma and rat RBL-2H3 basophilic leukaemia
cells, but the uptake was temperature sensitive in these
cells [47].
Rabbit platelets could play a role in rapidly removing
anandamide from the extracellular space and in metaboli-
zing it as it has been suggested for other cells [16], and/or
anandamide could be a precursor for the strong agonist
arachidonic acid and its metabolic products.
The [
3
H]anandamide metabolism in intact cells was
investigated in order to determine the assumed main
metabolic products, e.g. arachidonic acid and phospho-
lipids (PL) [13,23] in both cells and extracellular space,
using a TLC separation of total lipids extracted by the
Bligh–Dyer method. Our results showed that most of
radioactivity found in platelets after 2 min, was non-
metabolized [
3

H]anandamide, which was subsequently
metabolized mainly to methanol/water-soluble products
that increased dramatically and reached a plateau after
10–20 min. Interestingly, no initial increase in free fatty
acid was detected ( 2% after 2 min, and reached a
plateau ( 5%) after 20 min) (Fig. 4).
Results obtained in the presence of caffeic acid (a LOX
inhibitor) suggested that LOX was not involved in the
metabolism since no inhibition was observed. When indo-
methacin (a COX inhibitor) was used at 0.5 l
M
,a
concentration that inhibited platelet aggregation induced
by 14 l
M
anandamide [43], only a small inhibition was
observed. On the contrary, a significant inhibition ( 50%)
of [
3
H]anandamide metabolism by 75 l
M
indomethacin was
observed, suggesting the involvement of COX. This inhibi-
tion could be also attributed to the inhibition of FAAH as it
has been reported that indomethacin is a competitive
inhibitor of rat brain FAAH enzyme (K
i
¼ 120 l
M
)[48].

On the other hand, it is well known that platelets possess
two major enzymatic routes for arachidonic acid metabo-
lism, the cyclooxygenase (COX) and the lipoxygenase
(LOX) pathways. In the COX pathway, the main product
is thromboxane A
2
and other prostaglandins while in the
LOX pathway the main product is 12-monohydroxyeicosa-
tetraenoic acid. Another minor pathway for arachidonic
acid metabolism in platelets has also been reported: a
nonenzymatic free-radical catalyzed peroxidation to iso-
prostanes such as 8-Epi-PGF
2a
[49,50]. The products of
arachidonic acid peroxidation are water soluble, as reported
in the literature in experiments with rabbit platelets labeled
with [
3
H]arachidonic acid [51]. The identification of meth-
anol/water-soluble products of [
3
H]anandamide metabolism
was not addressed in the present study but it could be
speculated that these were oxidation products of ananda-
mide and/or arachidonic acid produced by the action of
FAAH activity. Additionally, data from studies performed
in our laboratory with rabbit platelet homogenate showed
the presence of FAAH activity which is localized mainly in
the plasma membrane-rich fraction of rabbit platelets and is
much higher than that in human platelets (L. Fasia and

A. Siafaka, unpublished data). Moreover, the localization of
endogenous FAAH in the plasma membrane has been also
reported for the rat liver [7].
In conclusion, the above results revealed a major
difference between human and rabbit platelets, since
[
3
H]anandamide is not taken up by a carrier-mediated
process in rabbit platelets in contrast to human platelets.
Anandamide is taken up by rabbit platelets through
passive diffusion, and subsequently rapidly metabolized
apparentlybytheactionofaFAAH,incontrasttorat
platelets where no FAAH expression was found [52].
Rabbit platelets could act as modulators to control
anandamide concentration and keep it at physiological
levels. Alternatively, anandamide could be a precursor for
arachidonic acid and its metabolic products. Further
studies are required to conclusively prove this suggestion
and clarify the possibility of the involvement of other
enzyme(s) (besides FAAH) in the metabolism of anand-
amide for the production of water-soluble metabolites.
These metabolites could be products of arachidonic acid
produced by the action of FAAH, but the direct action of
other enzyme(s) on anandamide could not be excluded.
Acknowledgements
This work was supported in part by University of Athens Special
Account for Research Grants (70/4/3351). The authors would like to
thank L. McManus (UTHSCSA, USA) for reading the manuscript.
3504 L. Fasia et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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