From the ¶ Endocannabinoid Research Group, Istituto per la
Chimica di Molecole di Interesse Biologico and the
Istituto di Cibernetica, C. N. R., Via Toiano 6, 80072, Arco Felice, Napoli, Italy, the
Department of Experimental
Medicine and Biochemical Sciences, University of Rome Tor Vergata,
Italy, and ** Neurology, GlaxoSmithKline, New Frontiers Science Park,
Third Avenue, Harlow, Essex CM19 5AW, United Kingdom
Received for publication, September 19, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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The endogenous ligand of CB1
cannabinoid receptors, anandamide, is also a full agonist at
vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby
causing an increase in cytosolic Ca2+ concentration in
human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of
anandamide facilitated transport into cells, VDM11 and VDM13, and two
inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl
fluoride and methylarachidonoyl fluorophosphonate, inhibited and
enhanced, respectively, the VR1-mediated effect of anandamide, but not
of resiniferatoxin or capsaicin. The nitric oxide donor, sodium
nitroprusside, known to stimulate anandamide transport, enhanced
anandamide effect on the cytosolic Ca2+ concentration.
Accordingly, hVR1-HEK cells contain an anandamide membrane transporter
inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and
an anandamide hydrolase activity sensitive to phenylmethylsulfonyl
fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid
amide hydrolase transcript. These findings suggest the following. (i)
Anandamide activates VR1 receptors by acting at an intracellular site.
(ii) Degradation by fatty acid amide hydrolase limits anandamide
activity on VR1; and (iii) the anandamide membrane transporter
inhibitors can be used to distinguish between CB1 or VR1
receptor-mediated actions of anandamide. By contrast, the
CB1 receptor antagonist SR141716A inhibited also the
VR1-mediated effect of anandamide and capsaicin on cytosolic
Ca2+ concentration, although at concentrations higher than
those required for CB1 antagonism.
Anandamide (N-arachidonoyl ethanolamine,
AEA,1 Ref. 1) was isolated
from porcine brain and proposed as an endogenous agonist of cannabinoid
CB1 receptors, which are present in the nervous system as
well as in cardiovascular, reproductive, and gastrointestinal tissues
(2). Since its discovery, several observations have supported the
hypothesis that AEA is an endocannabinoid, i.e. a functional
activator of CB1 receptors (see Refs. 3-6 for recent reviews). However, there have been numerous reports showing that AEA is
only a partial agonist at these receptors (see Refs. 7, 8 for
examples), whereas other observations have suggested that there may be
additional molecular targets for this substance (see Refs. 9, 10 for
examples, and Ref. 6 for review). Several investigators (11-14) have
provided pharmacological and molecular evidence that AEA is a full
agonist at the receptor for capsaicin, the substance responsible for
the pungent taste of hot chili peppers (15). This receptor is a
ligand-gated, nonselective cation channel and was recently cloned and
named VR1 (16). It is most abundant in sensory neurons but is also found in select areas of the central nervous system of rats and men
(17). AEA, at concentrations similar or higher than those necessary to
activate CB1 cannabinoid receptors, elicits typical VR1-mediated functional responses, such as endothelium-independent vasodilation of small arteries (11), cation inward currents in sensory
neurons (12), apoptosis (13), and inhibition of electrically stimulated
mouse vas deferens (14). The selective antagonist of vanilloid
receptors, capsazepine (18) strongly reduces these effects.
Furthermore, AEA activates VR1-mediated cation currents in human
embryonic kidney (HEK) 293 cells or Xenopus oocytes
overexpressing either rat or human VR1 (11, 12), and displaces the high
affinity VR1 ligand, [3H]resiniferatoxin, from binding
sites in membranes of CHO cells overexpressing rat VR1 (14). These data
indicate that, along with its proposed role as an endocannabinoid, AEA
might also function as an endovanilloid. However, whether or not AEA
behaves as a physiological ligand for VR1 is still being debated (19,
20), and further studies need to be performed to give an answer to this question.
The possible regulation of the activity of AEA at VR1 receptors by
biochemical events leading to the physiological inactivation of AEA and
by agents that pharmacologically or physiologically modulate these
events, could be taken as evidence in favor of a possible role for this
compound also as an endovanilloid. Furthermore, the use of
antagonists for either CB1 or VR1 receptors, such as SR1417161 (21) and capsazepine (18), respectively, should help in
discriminating between those effects of AEA that are mediated by either
receptor. Therefore, in the present study we have addressed the
question of whether the mechanisms previously shown to cause the
inactivation of AEA, i.e. uptake by cells facilitated by a selective AEA membrane transporter (AMT), followed by either hydrolysis catalyzed by fatty acid amide hydrolase (FAAH) or oxidation by enzymes
of the arachidonate cascade such as lipoxygenases (LOXs) (see Refs. 4,
22, 23 for specific reviews), also modulate the effect of AEA on VR1
receptors. Furthermore, based on previous reports showing that
SR141716A, particularly at high concentrations, is not selective for
the CB1 receptor (24, 25), we have investigated the effects
of this compound on a typical vanilloid-like, and non-CB1-mediated, effect of AEA, to assess whether it can
be used to discriminate between VR1- and CB1-mediated AEA
actions. Our results suggest that AEA interacts with VR1 at an
intracellular site, and, hence, its activity at either VR1 or
CB1 receptors can be regulated via the AMT and is
significantly limited by intracellular enzymatic hydrolysis. Moreover,
our data show that SR141716A can act as an inhibitor of VR1-mediated
signaling, albeit at concentrations higher than those required for
CB1 receptor antagonism and as such should be used with
some caution to distinguish between AEA as an endocannabinoid or endovanilloid.
Transfected Cells--
Expression of hVR1 cDNA into HEK 293 cells was carried out as described previously (26). Cells were grown as
monolayers in minimum essential medium supplemented with nonessential
amino acids, 10% fetal calf serum, and 0.2 mM glutamine
and maintained under 95:5% O2/CO2 at
37 °C.
Compounds--
Capsaicin, AEA, and methylarachidonoyl
fluorophosphonate (MAFP) were purchased from Cayman Chemicals (Ann
Arbor, MI). VDM11 and VDM13 (Fig. 1; Ref. 27) were synthesized from the
corresponding amines and arachidonoyl chloride (all from Sigma) in
dimethylformamide, in the presence of 1.1 equivalents of triethylamine
for 18 h at 4 °C. The reaction was stopped by adding water and
by extracting the products with diethyl ether. The compounds were
purified by direct phase-high pressure liquid chromatography, and
chemical structures were confirmed by means of proton nuclear magnetic resonance and infrared spectroscopy. [14C]AEA (5 mCi/mmol) was synthesized from [14C]ethanolamine and
arachidonoyl chloride as described (1). SR141716A was kindly donated by
Sanofi Recherche, Montpellier, France. Sodium nitroprusside (SNP),
phenylmethylsulfonyl fluoride (PMSF), arachidonic acid,
5,8,11,14-eicosatetraynoic acid (ETYA) and caffeic acid (CA) were
purchased from Sigma. Capsazepine and resiniferatoxin were purchased
from Alexis Biochemicals. The unselective AMT inhibitor AM404 was
purchased from Cayman Chemicals (Ann Arbour, MI). The 5(S)-,
11(S)-, and 15(S)- hydroperoxy derivatives of AEA
(Fig. 1) were generated by incubating 40 µM AEA with
soybean lipoxygenase-1 (sLOX) or barley lipoxygenase-1 (bLOX), purified as reported (sLOX, Ref. 28; bLOX, Ref. 29). AEA was incubated with
lipoxygenase (1 unit per 3 µmol of substrate) in 100 mM
sodium borate buffer (pH 9.0 for sLOX and pH 7.0 for bLOX), following the reaction spectrophotometrically at 236 nm. After completion (15 min), the pH was lowered to 4, and the products were purified with SPE
columns (Bakerbond 500 mg, J.T. Baker) and then with reverse phase-HPLC
as described (30). Reverse phase-HPLC was carried out on a Cosmosil
5C18 AR column (5 µm, 250 × 4.6 mm, Nacalai Tesque, Japan)
using tetrahydrofuran/methanol/water/acetic acid (25:40:35:0.1,
v/v/v/v) as eluent, at a flow rate of 1 ml/min. 15-hydroperoxy-eicosatetraenoylethanolamide (HETEE) was the major product (~95%) of sLOX, whereas 11- and 5-HEA were the major
products (~70% and ~15% respectively) of bLOX. The enantiomeric
ratio (S/R) of each hydroperoxide was found to be
95:5 by chiral separations and CD spectroscopy (30).
Cytosolic Ca2+ Concentration (CCC) Assays--
The
effect of the substances on CCC (27) was determined by using Fluo-3, a
selective intracellular fluorescent probe for Ca2+. One day
prior to experiments, hVR1-HEK cells were transferred into 6-well
dishes coated with poly-L-lysine (Sigma) and grown in the
culture medium mentioned above. On the day of the experiment, the cells
(50,000-60,000 per well) were loaded for 2 h at 25 °C with 4 µM fluo-3 methylester (Molecular Probes) in
Me2SO containing 0.04% pluoronic. After the loading,
hVR1-HEK cells were washed with Tyrode pH = 7.4, trypsinized,
resuspended in Tyrode, and transferred to the cuvette of the
fluorescence detector (Perkin-Elmer LS50B) under continuous stirring.
Experiments were carried out by measuring cell fluorescence at 25 °C
( AEA Hydrolase Activity Assays--
hVR1-HEK cells were cultured
as described above. The effect of VDM11, VDM13, PMSF, and MAFP on the
enzymatic hydrolysis of AEA was studied as described previously (31) by
using cell membranes incubated with either of the two compounds at
different concentrations and [14C]AEA (9 µM) in 50 mM Tris-HCl, pH 9, for 30 min at
37 °C. [14C]Ethanolamine produced from
[14C]AEA hydrolysis was measured by scintillation
counting of the aqueous phase after extraction of the incubation
mixture with 2 volumes of CHCl3/CH3OH 2:1
(v/v).
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for
FAAH--
Analysis of the RNA from hVR1-HEK cells for the presence of
a FAAH transcript was carried out by means of RT-PCR. Total RNA was
extracted from cells (10 × 106/test) as described
(32). cDNA synthesis was performed in a 20-µl reaction mixture
containing 75 mM KCl, 3 mM MgCl2,
10 mM dithiothreitol, 1 mM dNTPs, 50 mM Tris-HCl pH 8.3, 5 µg of total RNA, 0.125 A260 units of hexanucleotide mixture (Roche
Molecular Biochemicals) for random priming and 200 units of Superscript II RNase H AEA Transporter Assays--
The time- and
temperature-dependent uptake of [14C]AEA by
intact hVR1-HEK cells was studied as described previously for RBL-2H3 cells (31). The effect of compounds on the uptake of AEA was studied by
using 3.6 µM (10,000 cpm) [14C]AEA. Cells
were incubated with [14C]AEA for 5 min at 37 °C, with
or without varying concentrations of the inhibitors. Residual
[14C]AEA in the incubation medium after extraction with
CHCl3/CH3OH 2:1 (v/v), determined by
scintillation counting of the lyophilized organic phase, was used as a
measure of the AEA that was taken up by cells. Data are expressed as
the concentration exerting 50% inhibition of AEA uptake
(IC50).
Receptor Binding Assays--
Displacement assays for
CB1 receptors were carried out by using
[3H]SR141716A (0.4 nM, 55 Ci/mmol, Amersham
Pharmacia Biotech) as the high affinity ligand and the filtration
technique previously described (33) on membrane preparations (0.4 mg/tube) from male CD rat brains (Charles River, Italia) and in the
presence of 100 µM PMSF. Specific binding was calculated
with 1 µM SR141716A (a gift from Sanofi Recherche,
France) and was 84.0%. The Ki value for capsazepine
was calculated by applying the Cheng-Prusoff equation to the
IC50 value (obtained by GraphPad) for the displacement of
the bound [3H]SR141716A by increasing concentrations of
the test compounds.
Effect of AMT Modulators on AEA Action at VR1 Receptors--
In
agreement with previous studies (11, 12, 27), we found that AEA,
capsaicin, and AM404 are full agonists at human VR1 receptors
overexpressed in HEK cells in as much as they elicited a typical VR1
response, i.e. the increase of CCC. The EC50 for AEA, capsaicin, and AM404 were 0.52 ± 0.12, 0.019 ± 0.009, and 0.035 ± 0.011 µM, respectively. These effects
were blocked by EGTA (4 mM, not shown) and by increasing
concentrations of the VR1 antagonist capsazepine (see below) and were
not observed in nontransfected HEK cells (data not shown).
Two selective and recently developed inhibitors of the AMT, VDM11, and
VDM13 (Ref. 27, Fig. 1) strongly
inhibited the AEA effect on CCC in hVR1-HEK cells (Fig.
2, Table I). The IC50
for VDM11 with a 5-min preincubation was
3.9 ± 1.1 µM, a value similar to that for VDM11
inhibition of the AMT in rat C6 glioma and basophilic leukemia
(RBL-2H3) cells (IC50 ~10 µM, Ref. 27).
VDM11 (4 µM) did not affect the efficacy and potency of
capsaicin or resiniferatoxin on CCC (Table I). This compound, at a
10-µM concentration, did inhibit the capsaicin effect if
preincubated 5 min prior to the VR1 agonist, but it was inactive when
preincubated 30 min before capsaicin (data not shown). It is noteworthy
that VDM11 and VDM13 alone (10 µM) (i) exhibited a
negligible stimulatory effect on CCC (14.1 ± 5.9 and <5% of the
effect of ionomycin, respectively); (ii) had little effect on
[3H]SR141716A specific binding from rat brain membranes
(11.6 ± 1.8 and 29.1 ± 2.3% displacement, respectively;
Ki>20 µM in both cases); and (iii)
did not significantly inhibit [14C]AEA hydrolysis by
hVR1-HEK cells (<5% and 15.3 ± 4.5% inhibition, respectively)
(see also Ref. 27).
Finally, the NO donor SNP enhanced dose-dependently the
effect of AEA, but not capsaicin or resiniferatoxin, on CCC (Table I).
SNP alone did not produce any significant effect on CCC (<5% of the
effect of ionomycin) or [14C]AEA hydrolysis (<5% inhibition).
AEA Uptake by hVR1-HEK Cells--
Intact hVR1-HEK cells were shown
to uptake [14C]AEA in a time- and
temperature-dependent manner (Fig.
3A). This process was inhibited dose-dependently by coincubation with VDM11 and
AM404 (estimated IC50 = 4.0 ± 0.6 and 3.6 ± 0.7 µM, respectively) and VDM13, and was enhanced by SNP (5 mM) (Fig. 3B and data not shown).
Effect of FAAH Inhibitors on AEA Action at VR1 Receptors--
The
general serine protease inhibitor PMSF and the more selective FAAH
inhibitor MAFP (34, 35) were used. We found that both compounds,
preincubated with cells for 10 min and at concentrations previously
found to be fully effective against AEA hydrolysis and FAAH in other
cell lines, dose-dependently enhanced the effect of
submaximal doses of AEA, but not capsaicin, on VR1-mediated CCC
increase (Fig. 2, Table I). Neither PMSF (250 µM) nor
MAFP (75 nM) alone exhibited any significant effect on CCC
(<5% of ionomycin effect, data not shown).
Effect of FAAH Inhibitors on AEA Hydrolysis by hVR1-HEK Cell
Membranes--
PMSF and MAFP dose-dependently inhibited
the hydrolysis of AEA by hVR1-HEK cell membranes. Basal activity at pH
9 was 27.0 ± 5.8 pmol min hVR1-HEK Cells Express a FAAH Transcript--
cDNA, obtained
by retrotranscription of total RNA from hVR1-HEK cells, was amplified
by RT-PCR using probes specific for human FAAH. The amplified fragment
was analyzed by agarose gel electrophoresis and showed a single band of
the molecular size (202 bp) expected from the human FAAH-encoding
cDNA fragment (Fig. 3C, lane 1). This band
could not be attributed to the presence of contaminating DNA, because
in this case the expected molecular weight should have been higher (425 bp, Fig. 3C, lane 2). No band was present in an
RNA sample that had not undergone retrotranscription (Fig. 3C, lane 3).
Effect of Hydroperoxy-eicosatetraenoylethanolamides on VR1
Receptors--
5(S)-, 11(S)-, and
15(S)-hydroperoxy-eicosatetraenoylethanolamide were prepared
and purified as described in "Experimental Procedures" and were
tested on CCC in hVR1-HEK cells. Only the latter compound was found to
induce an increase in CCC (Fig. 4), and
only at concentrations significantly higher than those necessary to AEA
to exert the same effect. We also found that nonselective inhibition of
LOX by CA (50 µM) and ETYA (10 µM) did not
influence the effect on CCC of either sub- or near-maximal
concentrations of AEA, whereas arachidonic acid (10 µM)
exerted only a very little effect per se (Table I and data
not shown).
Effect of SR141716A and Capsazepine on VR1 Receptors--
We
tested several concentrations of SR141716A (with 30-min preincubation)
on the effects of several concentrations of capsaicin, AEA, and AM404
on hVR1-mediated CCC increase in hVR1-HEK cells (Figs. 5 and
6). SR141716A inhibited
the effect of the agonists with
Ki values of 1.1 ± 0.2, 1.3 ± 0.1, and
1.0 ± 0.1 µM for AEA (0.5 µM), AM404
(32 nM), and capsaicin (20 nM), respectively (means ± S.D., n = 3). SR141716A per
se also exerted a weak enhancement of CCC but only at high doses
(17.9 ± 2.1 and 36.0 ± 3.2% stimulation at 5 and 10 µM, respectively) and in a manner partly sensitive to 5 µM capsazepine (7.1 ± 1.2 and 19.5 ± 1.6%
stimulation, respectively, means ± S.D., n = 3, p < 0.05 by the t test). The dose-response curve of capsaicin in the presence of the antagonist was shifted to the
right to parallel curves (Fig. 5C). The dose-response curves for AEA and AM404 were also shifted to the right when they were administered after increasing concentrations of SR141716A (Fig. 5,
A and B), although the maximal effects of these
two agonists in the presence of the two highest concentrations of the
antagonist did not reach the values observed in its absence.
Furthermore, the inhibitory effect of SR141716A on capsaicin-induced
CCC increase reached a maximum with a 2.5-µM
concentration, whereas with AEA and AM404, the effect was maximal at 5 µM (Figs. 5 and 6). With 2.5 µM SR141716A,
however, it was possible to calculate Kc values for inhibition of CCC increase induced by the three VR1 agonists, and
these values were similar for AEA, AM404, and capsaicin (0.13 ± 0.04, 0.20 ± 0.06, and 0.28 ± 0.03 µM,
respectively, means ± S.D., n = 3). Capsazepine
was ineffective as a CB1 receptor ligand (Ki>10 µM) in CB1
receptor binding assays but inhibited AEA effect on CCC with a potency
~30-fold higher than SR141716A (Ki = 35.5 ± 3.2 nM, Fig. 6C).
We confirmed that AEA acts as a full agonist on VR1 receptors by
enhancing CCC in HEK cells transfected with human VR1 receptor cDNA. This effect was due uniquely to activation of VR1, and not CB1, receptors because it was: 1) observed, at lower doses,
also with capsaicin, which does not activate CB1 receptors
(36); 2) completely blocked by the selective VR1 receptor antagonist, capsazepine; and 3) not observed in nontransfected HEK cells. Furthermore, HEK cells do not express CB1 receptors (see
below). One of the aims of this study was to understand whether AEA
actions at VR1 receptors are regulated by the mechanisms previously
described to lead to AEA metabolism in intact living cells. This issue
is not all obvious because, for example, recent studies suggest that the binding site for capsaicin on VR1 receptors is intracellular (37),
and not extracellular as for most membrane receptors described to date.
Therefore, to activate VR1 receptors, capsaicin must first cross the
cell membrane, and it is possible that the transport of AEA into cells
via the AMT facilitates rather than terminate AEA action at VR1.
Furthermore, a recent report showed that LOX products of arachidonic
acid, the hydroperoxyeicosatetraenoic acids (HPETEs), are more potent
than AEA as agonists of rat VR1 receptors (38). Therefore, hydrolysis
of AEA to arachidonic acid might mediate AEA effect on VR1 receptors.
The data presented here demonstrate that AEA-facilitated transport into
cells through the AMT is necessary for VR1 activation by this compound;
and AEA enzymatic hydrolysis plays an important role in terminating, as
opposed to mediating, the vanilloid-like activity of AEA.
We found that two selective and recently developed inhibitors of the
AMT, VDM11, and VDM13 (Ref. 27, Fig. 1), strongly inhibit the AEA
effect on CCC in hVR1-HEK cells. This is exactly the opposite of what
was previously found for the effect of AMT inhibitors on AEA actions
that are mediated by CB1 receptors (39, 40). For example,
VDM11 (10 µM) enhances 2-fold the potency of a typical CB1-mediated effect of AEA (41) that is the mobilization of Ca2+ from intracellular stores in N18TG2
cells.2 Indeed, intact
hVR1-HEK cells were shown here to take up AEA via a time- and
temperature-dependent mechanism that could be inhibited by
VDM11 with exactly the same potency as that observed for the inhibition
of AEA-induced increase of CCC. VR1 receptors are readily desensitized
following treatment with vanilloid receptor agonists (15). This is the
reason why we did not use the previously developed AMT inhibitors,
linvanil (40) and AM404 (39), which potently activate per se
both rat and human VR1 (14, 27, 42-44). With these compounds we might
have observed either desensitization or an AMT-independent potentiation
of AEA effect on VR1. By contrast, VDM11 and VDM13, whereas being
equipotent to the most widely used AMT inhibitor, AM404 (39) in both
hVR1-HEK (this study) and other cells (27), are almost inactive
per se on VR1 receptors (27), thus making it unlikely that
their inhibitory action on AEA-induced CCC increase was because of
desensitization of these receptors. Furthermore, a half-maximal
concentration of VDM11 did not inhibit the effect on CCC produced by
either resiniferatoxin or capsaicin, thus arguing against a possible
action of this AMT inhibitor through VR1 desensitization or, for
example, as a VR1 antagonist. An inhibitory effect on capsaicin-induced
CCC increase was observed only with a maximal concentration of VDM11
and with a 5 min pre-incubation. This might be explained with the
observation that capsaicin is also recognized by the AMT, although to a
smaller extent than AEA (44). In fact, when administered to cells 30 min before capsaicin, VDM11 did not inhibit its effect (27), thus
conclusively ruling out desensitization of VR1 receptors, a phenomenon
that would last for several hours. Finally, VDM11 and VDM13 are also
almost inactive on proteins that might have interfered with the action
of AEA on CCC, i.e. CB1 cannabinoid receptors
(which, however, are not expressed in the cell model under study) and
FAAH. Therefore, to the best of our knowledge, the effect of these two
compounds on AEA-induced and VR1-mediated rise in CCC could be due only
to interference with the AMT. In further support of the facilitating
role of the AMT in the action of AEA on VR1 receptors we found that the
NO-donor, SNP, previously shown to activate the AMT in several cell
types (40, 45), significantly enhanced both AEA uptake by hVR1-HEK
cells and the stimulatory effect of AEA (but not capsaicin or
resiniferatoxin) on VR1-mediated CCC increase in these cells. It is
noteworthy that NO donors were previously shown to inhibit a
CB1 receptor-mediated effect of AEA, i.e. the
inhibition of adenylate cyclase (40).
These data indicate that AEA uptake by cells via the AMT plays a
permissive role in the action of exogenous AEA on VR1 receptors, and
suggest that AEA site of action on VR1 receptors, like for capsaicin
(37), is intracellular. This finding was confirmed by preliminary data
obtained in dorsal root
ganglia3 indicating that
VDM11 (3 µM) also suppresses another VR1-mediated effect
of AEA (1 µM), i.e. the release of calcitonin
gene-related peptide (46) and has numerous important implications.
First, AEA is not stored in and released from preformed vescicles, and the biochemical pathway most likely to generate this compound in
stimulated neurons is the phospholipase D-catalyzed hydrolysis of a
membrane phospholipid precursor (see Ref. 47 and Ref. 3 for review).
Therefore, it is possible that in certain cells AEA synthesized
de novo acts first as an intracellular endovanilloid and
then as an extracellular endocannabinoid, once it is released from the
cells, most likely through the AMT itself (22). It is possible that
cells have the means of shifting the target (and the subsequent
biological effect) of extracellular AEA from CB1 to VR1
receptors, or of intracellular AEA from VR1 to CB1
receptors, by regulating the activity of the AMT. This strategy could
be also exploited pharmacologically to select the pharmacological action of exogenous AEA or, preferably, of its more stable synthetic analogs such as the CB1/VR1 hybrid ligand, arvanil (33,
44). This would be important, for example, for the therapeutic
treatment of pain, as it has been shown that both CB1 and
VR1 receptor agonists can produce analgesia, although through different
mechanisms and under different conditions. On the other hand, AEA was
suggested to exert opposite effects on cancer cell apoptosis depending
on whether it acts via VR1 or CB1 receptors (13). Thus,
coadministration of AEA and arvanil with either inhibitors or
activators of AMT may lead the different effects on pain or cancer cell
growth in vivo. Finally, the capability of some synthetic
VR1 agonists, such as olvanil and arvanil (44), to activate hVR1 more
potently than capsaicin (14, 27), may be due in part to their being recognized by the AMT (44, 48) and, hence, penetrating more rapidly
into the cell and acting on an intracellular site on VR1. Therefore,
one possible strategy for the development of ultrapotent VR1 agonists
to be used as therapeutic agents could be the design of compounds that
at once activate VR1 and are recognized by the AMT with high efficacy.
The next step in our study was to understand the role of AEA enzymatic
hydrolysis in the modulation of AEA activity at VR1 receptors. We
studied the effects of two inhibitors of AEA hydrolysis, the general
serine protease inhibitor PMSF, and the more selective FAAH inhibitor
MAFP (34, 35). We found that both compounds, although being inactive
per se on CCC, significantly enhanced the activity of
submaximal doses of AEA, but not capsaicin, on VR1-mediated CCC
increase. At exactly the same concentrations required to enhance AEA
activity at VR1, PMSF and MAFP also blocked the hydrolysis of AEA by
hVR1-HEK cells, which, as shown here by using the RT-PCR methodology,
express a FAAH-like transcript. We cannot rule out the possibility that
PMSF enhances the effect of AEA on CCC by directly interacting with VR1
receptors. However, this compound usually behaves as an alkylating
agent for Ser and Cys residues, and would be more likely to inactivate
VR1 receptors, rather than facilitate their activation by AEA.
Therefore, these findings suggest that the effect of PMSF and,
particularly, the FAAH-selective MAFP on AEA-induced CCC increase is
due to their inhibition of FAAH-catalyzed AEA hydrolysis. Hence, it can
be proposed that, as in the case of endocannabinoid AEA (2), also the
biological activity of endovanilloid AEA is limited by enzymatic hydrolysis. Furthermore, we could estimate an EC50 ~100
nM for AEA effect on VR1-mediated CCC increase in the
presence of either 250 µM PMSF or 75 nM MAFP
(Table I). This value is not very different from the
Ki = 30-50 nM of AEA in CB1
receptor binding assays carried out in the presence of PMSF (2). Thus,
it is possible that AEA potency at VR1 receptors is comparable with that at CB1 cannabinoid receptors.
Based on the finding that HPETEs behave as full agonists at VR1
receptors (38), it could be possible that also the hydroperoxy derivatives of AEA, the HETEEs, activate these receptors and mediate the vanilloid-like actions of AEA. On the other hand, it is possible that LOX-catalyzed oxidation of AEA, which leads to compounds that are
still active on CB1 receptors (49), is used instead to
inactivate AEA as an endovanilloid. Indeed 5(S)-,
11(S)-, and 15(S)-HETEE were found here much less
active than AEA on VR1-mediated CCC increase in HVR1-HEK cells.
However, it is possible that these compounds could not penetrate the
cell membrane and activate the putative intracellular VR1 site for AEA
proposed here. In fact, the HETEEs tested here and are not recognized
as substrates by the AMT.4
Nevertheless, because of their lipophilic nature, they might still be
capable of diffusing through the cell membrane, which would explain why
the 15(S)-HETEE did exhibit measurable VR1 activity. Our
additional finding that nonselective inhibitors of LOX do not influence
the effect on CCC of either sub- or near-maximal concentrations of AEA
(whereas arachidonic acid exerted only a very little effect) was not
surprising because constitutive LOX activity seems to be low in HEK
cells (50, 51). Further studies are needed to fully assess the role of
LOXs in AEA activity at VR1 receptors.
A corollary to the findings discussed so far is that truly selective
inhibitors of AMT, but not FAAH, can be used to distinguish between the
actions of anandamide at VR1 and CB1 receptors. In principle, also the CB1 receptor antagonist SR141716A (21)
could be employed to discriminate between these two types of AEA
pharmacological activity. However, this antagonist, albeit generally
selective for CB1 receptors, acts also on other molecular
targets at concentrations higher than 1 µM (24, 25, 52).
Furthermore, preliminary experiments revealed that a single high dose
of SR141716A can antagonize capsaicin-induced and vanilloid
receptor-mediated vasodilation of rat mesenteric arteries (11). Indeed,
we found here that SR141716A, at concentrations of >1
µM, inhibits the effects of capsaicin, AEA, and AM404 on
hVR1-mediated CCC increase in hVR1-HEK cells. Several lines of evidence
suggest that this inhibitory effect of SR141716A is due, at least in
part, to an interaction with VR1 but not CB1 receptors.
First, in the cell model used in this study, i.e. HEK 293 cells transfected with VR1 receptor cDNA, CB1 receptors
are not expressed, as assessed by
RT-PCR,5 nor is any specific
binding for [3H]SR141716A found in nontransfected HEK 293 cells, according to Tao and Abood (53). Second, the effects of AEA,
capsaicin, and AM404 on CCC are clearly mediated by VR1 and not
CB1 receptors (see beginning of "Discussion"). Indeed,
if AEA were increasing CCC via CB1 receptors AMT inhibitors
should have enhanced, and not inhibited, this effect (see above).
Third, the inhibitory effect of SR141716A on AEA-induced CCC increase
was observed at concentrations that were three orders of magnitude
higher than those necessary to antagonize CB1
receptor-mediated effects of AEA (Kc ~0.3
nM, Ref. 33; Ki for human
CB1 = 5.6 nM, Ref. 21; Kd
for CB1 receptors 0.19-1.24 nM, Ref. 2). Finally, SR141716A per se exerted a weak agonist effect on
CCC in a manner sensitive to the VR1 antagonist capsazepine (shown here
to be highly selective for VR1 versus CB1
receptors). This latter observation suggests that SR141716A may act as
a partial agonist and, subsequently, competitive antagonist for VR1
receptors. It is unlikely, however, that SR141716A inhibitory action
was because of desensitization of VR1 receptors, because the
dose-response curves of capsaicin in the presence of the antagonist
were shifted to the right to parallel curves. On the other hand, an
unusual dose-response curve was observed for the inhibition of
capsaicin effect on CCC, with a narrow range of active doses and a
ceiling effect at 2.5 µM SR141716A particularly
noticeable with concentrations of capsaicin > 30 nM.
A similar ceiling effect was also previously observed for another
non-CB1-mediated inhibitory effect of SR141716A (52), and
may be due either to the fact that this compound becomes active as a
VR1 agonist at In conclusion, the present study supports a role of AEA as an
endovanilloid acting upon the VR1 receptor at an intracellular site.
Our data also suggest that the activity of AEA at the vanilloid receptor may be controlled by the endocannabinoid pathway components that regulate its internalization and degradation. Hence,
targeting of AMT and FAAH may offer indirect means of influencing
VR1-mediated sensory signaling and pain. Further studies will be
required to determine which of the several pharmacological actions
described so far for AEA are exerted through vanilloid receptors; we
have shown here that selective AMT inhibitors, but not necessarily the
CB1 receptor antagonist SR141716A, can be used as
biochemical tools to distinguish between the vanilloid-like and
cannabimimetic actions of AEA.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EX = 488 nm,
EM = 540 nm) before and
after the addition of the test compounds at various concentrations.
Capsazepine and SR141716A or EGTA (4 mM), MAFP, PMSF, SNP,
ETYA, and CA were added 30 or 10 min, respectively, before AEA or
capsaicin or resiniferatoxin, whereas the AMT inhibitors were added 5 min before. Data are expressed as the concentration exerting a
half-maximal effect (EC50). The efficacy of the effect was
determined by comparing it to the analogous effect observed with 4 µM ionomycin. The inhibitory effects of AMT inhibitors and of antagonists were expressed as IC50 calculated by
GraphPad software. For the antagonists, Ki values at
approximately half-saturating concentrations of agonists were
calculated by means of the Cheng-Prusoff equation.
Kc values for competitive antagonism were
calculated by means of the following equation: Kc = [ant]/{(EC
1}, where [ant] is the concentration of SR141716A (2.5 µM) and EC
reverse transcriptase (Life Technologies,
Inc.). The cDNA reaction mixture was incubated at 25 °C for 10 min and then at 42 °C for 50 min, and the reaction was stopped by
heating at 95 °C for 5 min followed by lowering the temperature at
4 °C. RT-PCR amplification was performed by means of a combination
of hot-start and touch-down PCR, and using 2 µl of the cDNA and
1.25 units of Taq GOLD (PerkinElmer Life Sciences) in 50 µl of its buffer containing 3 mM MgCl2, 250 µM of each dNTPs, and 0.5 µM each of 5' and
3' primers. Reactions were performed in a Gene Amp PCR System 9600 thermocycler (PerkinElmer Life Sciences). The amplification profile
consisted of an initial denaturation of 10 min at 92 °C and 15 cycles of 30 s at 95 °C, 1 min at 66 °C (annealing) and 1 min at 68 °C (with an annealing temperature stepping down of 1 °C
every 3 cycles, from 66 to 61 °C), followed by 25 cycles of 30 s at 95 °C, 1 min at 61 °C, and 1 min at 72 °C. A final
extension of 15 min was carried out at 72 °C. The primers used were:
FAAH sense primer, 5'-GCCTGGGAAGTGAACAAAGGGACC-3' and FAAH antisense
primer, 5'-CCACTACGCTGTCGCACTCCGCCG-3'. The expected sizes of the
amplicon was 202 bp.
2-microglobulin was used as the housekeeping
gene (32). PCR primers for FAAH were selected on the basis of the
sequence of the FAAH human gene (GenBankTM/EBI
accession number AF098012) by including the intron 497-722. In the
presence of contaminant genomic DNA, the expected size of the amplicon
would be 425 bp. PCR products (15 µl) were electrophoresed on 2%
agarose gel (MS agarose, Roche Molecular Biochemicals) in 1× TAE
buffer at 4 V/cm for 4 h. Ethidium bromide (0.1 µg/ml) was
included both in the gel and electrophoresis buffer, and PCR products
were detected by UV visualization. No PCR product was detected in the
absence of cDNA, primers, or Red-hot DNA polymerase. DNA ladder
(100-bp molecular ruler, Bio-Rad) was run as a marker.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (6K):
[in a new window]
Fig. 1.
Chemical structures of the two new AMT
inhibitors, VDM11 and VDM13 that have little or no activity at VR1
receptors (27).
View larger version (11K):
[in a new window]
Fig. 2.
Dose-related effect of the AMT inhibitor,
VDM11, and the FAAH inhibitor, MAFP, on AEA-induced increase of
cytosolic Ca2+ concentration in hVR1-HEK
cells. The effects are reported as percent of the effects observed
with AEA + vehicle (see Table I). The concentration of AEA used was 0.1 µM with MAFP and 1 µM with VDM11. Data are
means ± S.D. of n = 3 experiments. VDM11 was
added 5 min before AEA; MAFP 10 min before AEA.
Effect of various substances on anandamide (AEA)-, capsaicin (Caps)-,
and resiniferatoxin (RTX)-induced increase in cytosolic Ca2+
concentration (CCC) in hVR1-HEK cells
View larger version (58K):
[in a new window]
Fig. 3.
Uptake of anandamide and FAAH expression in
intact hVR1-HEK cells. A, time-dependent
uptake of [14C]anandamide and effect of low temperature.
B, effect of various substances on the amounts of
[14C]anandamide taken up after 5-min incubation with
cells. VDM11 5, VDM11 5 µM; VDM11 10, VDM11 10 µM; VDM11 50, VDM11 50 µM; VDM13 10, VDM13
10 µM; VDM13 50, VDM13 50 µM; SNP 5, sodium
nitroprusside 5 mM. Data are means ± S.D. of
n = 3 experiments. *, p < 0.05; **,
p < 0.01 versus control, as assessed by
analysis of variance. Inhibitors were added 5 min before
[14C]anandamide. C, agarose gel
electrophoresis of RT-PCR transcripts obtained by using cDNA
(lane 2), DNA (lane 3), and RNA (lane
4) from hVR1-HEK cells. Amplification was carried out by using
oligoprobes for human FAAH. A DNA ladder as bp-molecular ruler is also
shown. This figure is representative of three separate
experiments.
1 mg
protein
1, which was decreased to 11.1 ± 3.9 and
2.9 ± 0.4 pmol min
1 mg protein
1 with
100 and 250 µM PMSF, and to 21.1 ± 3.5, 8.8 ± 1.5 and 3.0 ± 0.7 pmol min
1 mg
protein
1, with 10, 50, and 75 nM MAFP,
respectively (n = 3 ± S.E., p < 0.01 by analysis of variance with the two highest concentrations of inhibitors).
View larger version (10K):
[in a new window]
Fig. 4.
Effect of various HETEE on cytosolic
Ca2+ concentration into hVR1-HEK cells. The effects
are reported as percent of the effects observed with 4 µM
ionomycin. Data are means ± S.D. of n = 3 experiments.
View larger version (15K):
[in a new window]
Fig. 5.
Effect of various concentrations of SR141716A
on the cytosolic Ca2+ concentration in hVR1-HEK cells
induced by anandamide (AEA, A), AM404
(AM, B), and capsaicin
(caps, C). The effects are
reported as percent of the effects observed with 4 µM
ionomycin. SR1, SR141716A 1 µM, SR2.5, SR141716A 2.5 µM, SR5, SR141716A 5 µM. The dose-response
curves obtained with 10 µM SR141716A are not shown
because they were almost superimposable to those obtained with 5 µM. In all cases, SR141716A was added 30 min before the
agonists. Data are means ± S.D. of n = 3 experiments.
View larger version (11K):
[in a new window]
Fig. 6.
Effect of various doses of SR141716A
(A, B) and capsazepine
(C) on the increase of cytosolic Ca2+
concentration in hVR1-HEK cells induced by anandamide (0.5 µM; A and
C) or capsaicin (20 nM;
B). The effects are reported as percent of
the effects observed with agonist + vehicle (see Table I). Both
antagonists were added 30 min before AEA. Data are means ± S.D.
of n = 3 experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 µM concentrations, or to nonselective effects at high doses. Also with AEA the inhibitory effect of SR141716A
was observed in a narrow range of concentrations, and, moreover, could
not be overridden by high concentrations of the compound. This may be
because of effects of the antagonist also on non-VR1 targets,
i.e. inhibition of the AMT (39). Although its mechanism of
action needs further investigation, the VR1 antagonistic effect of
SR141716A, particularly when the antagonist is systemically administered and its local concentrations in tissues cannot be established with accuracy, implies that this compound should be used
with some caution when discriminating between CB1- and
VR1-mediated actions of AEA.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to P. Hayes and W. Cairns for the cloning and expression of human VR1, to A. Schiano Moriello and Ines Brandi for technical assistance, and to S. Piantedosi for some of the artwork.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Ministero dell Università e della Ricerca Scientifica e Tecnologica Grant 3933 (to V. D. M.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
39-081-853-4156; Fax: 39-081-804-1770; E-mail:
vdimarzo@icmib.na.cnr.it.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M008555200
2 V. Di Marzo and L. De Petrocellis, unpublished observations.
3 P. Geppetti and V. Di Marzo, unpublished observations.
4 M. Maccarrone, unpublished data.
5 J.B. Davis, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: AEA, arachidonoylethanolamide, anandamide; HEK, human embryonic kidney; CCC, cytosolic Ca2+ concentration; PMSF, phenylmethylsulfonyl fluoride; MAFP, methylarachidonoyl fluorophosphonate; SNP, sodium nitroprusside; LOX, lipoxygenase; ETYA, 5,8,11,14-eicosatetraynoic acid; CA, caffeic acid; RP-HPLC, reverse phase-high pressure liquid chromatography; FAAH, fatty acid amide hydrolase; AMT, anandamide membrane transporter; HPETEs, hydroperoxy-eicosatetraenoic acids; VDM11, N-(2-methyl-4-hydroxy-phenyl)-arachidonamide; VDM13, N-arachidonoyl-5-methoxytryptamine; HETEE, hydroperoxy-eicosatetraenoylethanolamide; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pairs.
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