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 [3H]anandamide into the cell, i.e.
Trang 1Uptake and metabolism of [3H]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
[3H]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,
[3H]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, Km¼ 41 ±
15 lM [10] while in the human umbelical vein endothelial cells Km¼ 190 ± 10 nM [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: siafaka@chem.uoa.gr
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/Ca2+; 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)
Trang 2atives) [35–37] Recombinant human cyclooxygenase-2
(hCOX-2) but not hCOX-1 has the ability to directly
oxidize anandamide to yield prostaglandin E2ethanolamide
[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
[3H]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/Ca2+
pH 7.2 (Tg/Ca) which contained 0.8% NaCl, 0.02% KCl,
0.02% MgCl2, 0.1% dextrose, 0.25% gelatin and 0.02%
CaCl2 (w/v) and (b) 10 mM NaCl/Pi pH 7.4 which
con-tained 0.14% Na2HPO4, 0.12% NaH2PO4 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 mMNaCl/PipH 7.4, at a concentration of 3· 108
plateletsÆmL)1
presence of VDM11 (20 lM) at 37C For the kinetic studies, [3H]anandamide at concentrations between 100 and 2000 nM was used and the incubation took place at
37C The concentration of phenylmethylsulfonyl fluor-ide used was 2 mM and the preincubation time was
15 min
Metabolism of [3H] anandamide by intact rabbit platelets Washed rabbit platelets were resuspended in Tg/Ca
pH 7.2 or in 10 mM NaCl/Piresulting in a final concen-tration of 3· 108 plateletsÆmL)1 The platelet suspension was incubated with [3H]anandamide 1.25 nM (specific activity 200 CiÆmmoL)1) or 450 nMin certain experiments,
at 37C 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 CHCl3/CH3OH/
NH4OH 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
In the same manner, for [3H]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 mM
phenylmethylsulfonyl fluoride or 100 lM caffeic acid, or 0.5 and 75 lMindomethacin
Results [3H]Anandamide uptake and metabolism [3H]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%)
Trang 3Surprisingly, 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 [3H]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 water-soluble
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 [3H]anandamide had been catabolized Only a small
percentage of [3H]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 lM
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 lM
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 lM 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 [3H]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 [3H]anandamide Thereafter, the amount of [3H]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 [3H]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 [3H]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 [3H]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 ) at 37 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 [3H]Anandamide metabolism by intact rabbit platelets Platelet suspension in Tg/Ca (3 · 10 8 plateletsÆmL)1) was incubated with [3H]anandamide (1.25 n M ) at 37 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 measmeas-ured (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.
Trang 4platelets 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
[3H]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 [3H]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 metabometabo-lism
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 [3H]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 37C, 25 C and 0–4 C was examined In these experiments, 100 nM [3H]anandamide in 10 mM
NaCl/Piwas 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/Pi compared to that in Tg/Ca (Fig 1) This difference could be attributed to the higher [3H]anandamide concentration used (100 nMinstead of 1.25 nM) as well as to the presence of 1% w/v BSA in the washing buffer BSA apparently removed
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 ) at 37 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.
Trang 5[3H]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 [3H]anandamide for 1–2 min (low metabolism) and were then separated from the medium (M1) by centrifugation and washed with NaCl/Picontaining BSA (M2) Interestingly, the radioactivity found in M2 (corresponded mainly to [3H]anandamide) was higher than that found in M1 which corresponded mainly to water-soluble compounds produced after [3H]anandamide meta-bolism (data not shown) These results suggest that [3H]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 [3H]anandamide uptake was identical at 37C and 25 C At these two tempera-tures, the metabolism of [3H]anandamide took place to the same extent and resulted in the same products When the uptake of [3H]anandamide at 0–4C was studied, the amount of radioactivity found in platelets was larger than that at 37C (Fig 7) This observation can be explained by the smaller extent of [3H]anandamide metabolism by intact rabbit platelets at this low temperature [3H]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 0–4C compared to
37C In the case that [3H]anandamide had been trans-ferred via facilitated diffusion, the uptake at 4C should have been much lower
Fig 5 Effect of phenylmethylsulfonyl fluoride on [3H]anandamide
uptake by rabbit platelets Platelet suspension in Tg/Ca (3 · 10 8
plateletsÆmL)1) was incubated with [3H]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 [3H]anandamide (1.25 n M ) at 37 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 was measured (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 [3H]anandamide uptake
by rabbit platelets Platelet suspension in NaCl/P i (3 · 10 8 plate-letsÆmL)1) was incubated with [3H]anandamide (100 n M ) at various 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.
Trang 6To explore this hypothesis further, the experiments at
37C were repeated in the presence of VDM11, a specific
anandamide membrane transporter inhibitor Platelets
were preincubated for 10 min with 20 lMVDM11 as the
50% inhibitory concentration (IC50) reported for other
cells was 10–11 lM [19] As shown in Fig 7, although a
small inhibition ( 20%) of uptake was observed at 2 min,
the profile of [3H]anandamide uptake was almost identical
with or without VDM11 at 37C Thus, these results
indicate the absence of a membrane transporter in rabbit
platelets
Effect of concentration on [3H]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 [3H]anandamide concentrations of
100–2000 nM 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 [3H]anandamide The
catabol-ism of [3H]anandamide was low during this time (Fig 2)
The amount of radioactivity found in platelets was a linear
function of [3H]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 0–4C than at 37 C due to
decreased metabolism Interestingly, the uptake was also
linear but higher both in the presence of
phenylmethylsul-fonyl fluoride, and 0–4C 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 the time that [3H]anandamide should be taken up by a facilitated diffusion process Subsequently, [3H]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 [3H]anandamide was exactly the same at 37 and 25C (Fig 7) but was higher at 0–4 C, apparently due
to the lower degree of metabolism at 0–4C compared to that at 37C or at 25 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,
(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.
Trang 7the 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 [3H]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 nM
anandamide, while in human platelets, the uptake was
saturable (Km¼ 200 ± 20 nM at 37C) In the presence
of phenylmethylsulfonyl fluoride, the concentration
dependence curve was also linear but higher (e.g at
2000 nM [3H]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 lM 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 0–4C 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 [3H]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 [3H]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 lM, a concentration that inhibited platelet aggregation induced
by 14 lM anandamide [43], only a small inhibition was observed On the contrary, a significant inhibition ( 50%)
of [3H]anandamide metabolism by 75 lMindomethacin was observed, suggesting the involvement of COX This inhibi-tion could be also attributed to the inhibiinhibi-tion of FAAH as it has been reported that indomethacin is a competitive inhibitor of rat brain FAAH enzyme (Ki¼ 120 lM) [ 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 A2 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-PGF2a [49,50] The products of arachidonic acid peroxidation are water soluble, as reported
in the literature in experiments with rabbit platelets labeled with [3H]arachidonic acid [51] The identification of meth-anol/water-soluble products of [3H]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 [3H]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 apparently by the action of a FAAH, in contrast to rat 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.
Trang 8Itoh, K., Yamashita, A & Waku, K (1995)
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