'Entourage' effects of N-acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism

Article abstract of DOI:10.1038/sj.bjp.0704732

1. The abilities of a series of saturated N-acyl ethanolamines and related compounds to affect the ability of anandamide (AEA) to produce a Ca²⁺ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) has been investigated. 2. The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither affected basal Ca²⁺-influx, nor the influx in response to a submaximal concentration of AEA (1 μM). In contrast, the C12:0, C17:0, C18:0 ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an augmentation of the response to AEA. 3. Lauroylethanolamide (C12:0) produced a leftward shift in the dose-response curve for AEA. EC₅₀ values for AEA to produce Ca²⁺ influx into hVR1-HEK293 cells were 1.8, 1.5, 1.1 and 0.22 μM in the presence of 0, 1, 3 and 10 μM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose-response curves to capsaicin. 4. Palmitoylethylamide was synthesized and found to be a mixed-type inhibitor (K_i(slope) 4.1 μM, K_i(intercept) 66 μM) of [³H]-AEA metabolism by rat brain membranes. 5. The -amide, -ethylamide, -isopropylamide, -butylamide, -cyclohexamide and -trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca²⁺ influx response to 1 μM AEA. 6. There was no obvious relation between the abilities of the compounds to enhance the Ca²⁺ influx response to 1 μM AEA into hVR1-HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes. 7. It is concluded that although palmitoylethanolamide has entourage-like effects at VR1 receptors expressed on hVR1-HEK293 cells, other N-acyl ethanolamines have even more dramatic potentiating effects. It is possible that they may play an important role under conditions where their synthesis is increased, such as in severe inflammation.

Full text of DOI:10.1038/sj.bjp.0704732

British Journal of Pharmacology (2002) 136, 452 ± 458
ã
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`Entourage' e€ects of N-acyl ethanolamines at human vanilloid
receptors. Comparison of e€ects upon anandamide-induced
vanilloid receptor activation and upon anandamide metabolism
2
3
3
1Darren Smart, Kent-Olov Jonsson, Severine Vandevoorde, Didier M. Lambert &
*,2Christopher J. Fowler
1Neuroscience Research, Glaxo SmithKline Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19
2
3
5AW; Department of Pharmacology and Clinical Neuroscience, Umea University, SE-901 87 Umea, Sweden and Unite de
Chimie pharmaceutique et de Radiopharmacie, Universite catholique de Louvain, Avenue Mounier, 73, UCL-CMFA 73.40,
B-1200 Brussels, Belgium
1
The abilities of a series of saturated N-acyl ethanolamines and related compounds to a€ect the
ability of anandamide (AEA) to produce a Ca2+ in¯ux into human embryonic kidney cells
expressing the human vanilloid receptor (hVR1-HEK293 cells) has been investigated.
2
The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither a€ected basal Ca2+-in¯ux, nor the in¯ux
in response to a submaximal concentration of AEA (1 mM). In contrast, the C12:0, C17:0, C18:0
ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated
the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an
augmentation of the response to AEA.
3
Lauroylethanolamide (C12:0) produced a leftward shift in the dose-response curve for AEA.
EC50 values for AEA to produce Ca2+ in¯ux into hVR1-HEK293 cells were 1.8, 1.5, 1.1 and
0.22 mM in the presence of 0, 1, 3 and 10 mM lauroylethanolamide, respectively. Lauroylethanolamide
did not a€ect the dose ± response curves to capsaicin.
4
Palmitoylethylamide was synthesized and found to be a mixed-type inhibitor (Ki(slope) 4.1 mM,
Ki(intercept) 66 mM) of [3H]-AEA metabolism by rat brain membranes.
5
The -amide, -ethylamide, -isopropylamide, -butylamide, -cyclohexamide and -tri¯uoromethyl
ketone analogues of palmitoylethanolamide had little or no e€ect on the Ca2+ in¯ux response to
1 mM AEA.
6
There was no obvious relation between the abilities of the compounds to enhance the Ca2+ in¯ux
response to 1 mM AEA into hVR1-HEK293 cells and to prevent the hydrolysis of AEA by rat brain
membranes.
7
It is concluded that although palmitoylethanolamide has entourage-like e€ects at VR1 receptors
expressed on hVR1-HEK293 cells, other N-acyl ethanolamines have even more dramatic
potentiating e€ects. It is possible that they may play an important role under conditions where
their synthesis is increased, such as in severe in¯ammation.
British Journal of Pharmacology (2002) 136, 452 ± 458
Keywords: Vanilloid receptors; anandamide; fatty acid amidohydrolase; N-acyl ethanolamines
Abbreviations: AEA, anandamide; FAAH, fatty acid amidohydrolase; MEM, minimal essential medium; OEA, oleoyletha-
nolamide; PA-Et, palmitoylethylamide; PA-NH2, palmitamide; PBA, palmitoylbutylamide; PCA, palmitoylcy-
clohexamide; PEA, palmitoylethanolamide; PIA, palmitoylisopropylamide; PTMK, palmitoyl tri¯uoromethyl
ketone; VR, vanilloid receptors
Introduction
It is now well established that the N-acyl ethanolamine
anandamide (arachidonoyl ethanolamide C20:4, AEA) not
only acts as an endogenous ligand at cannabinoid receptors
(Devane et al., 1992), but is also capable of activating
vanilloid receptors (VR) (Zygmunt et al., 1999; Smart et al.,
2000). In contrast to the activation produced by hydrogen
ions (Caterina et al., 1997; McLatchie & Bevan, 2001), the
VR agonist capsaicin is believed to activate VR1 receptors
intracellularly (Jung et al., 1999; Welch et al., 2000), and a
similar mechanism may exist for AEA, since blockade of
AEA facilitated transport into hVR1-HEK cells transfected
with VR1 receptors inhibits AEA-induced in¯ux of calcium
into the cell (De Petrocellis et al., 2001a). Intracellular AEA
is metabolized to arachidonic acid by the enzyme fatty acid
amide hydrolase (FAAH) (Deutsch & Chin, 1993), and
inhibition of the enzyme potentiates the e€ects of AEA upon
VR1 receptors (De Petrocellis et al., 2001a; Ross et al., 2001).
Whilst it is clear that AEA activates VR1 receptors, it is
not as yet proven beyond doubt that AEA acts as an
`endovanilloid' in the same way as it acts as an endocanna-
binoid (see Di Marzo et al., 2001). The main obstacle to the
acceptance of AEA as an endovanilloid has been its relatively
low potency (Zygmunt et al., 1999; Smart et al., 2000), even
*Author for correspondence; E-mail: cf@pharm.umu.se

D. Smart et al
Palmitoylethanolamide analogues and VR1 receptors
453
after FAAH inhibition (Ross et al., 2001). However, there is
evidence that the potency of AEA towards VR1 receptors can
be regulated (Di Marzo et al., 2001). Thus, the naturally-
occurring N-acyl ethanolamine palmitoylethanolamide (PEA,
C16:0) potentiates the e€ects of AEA and resiniferatoxin at
VR1 receptors (De Petrocellis et al., 2001b). It is not known,
however, whether these e€ects of PEA are shared by other N-
acyl ethanolamines. In consequence, in the present study, the
e€ects of a series of PEA analogues and homologues have
been investigated with respect to their ability to potentiate the
actions of AEA at VR1 receptors.
amino acids, 10% foetal calf serum, and 0.2 mM L-glutamine,
and maintained under 95%/5% O2/CO2 at 378C. Cells were
passaged every 3 ± 4 days and the highest passage number
used was 27.
Measurement of [Ca2+]i using the FLIPR technology
The method of Smart et al. (2000) was used. hVR1-HEK293
cells were seeded (density 256103 cells well71) into black
walled clear-base 96-well plates (Costar, U.K.) and cultured
overnight in MEM, supplemented as described above. The
cells were then incubated at 258C for 120 min with MEM
containing the cytoplasmic calcium indicator, Fluo-3AM
(4 mM; Te¯abs, Austin, TX, U.S.A.). After washing the plates
four times with Tyrode's medium containing 0.1% bovine
serum albumin, they were placed into a FLIPR (Molecular
Devices, U.K.) to monitor cell ¯uorescence (lEX=488 nm,
Methods
Compounds
Anandamide [ethanolamine-1-3H] ([3H]-AEA; speci®c activity
60 Ci mmol71) was obtained from American Radiolabeled
Chemicals, Inc. (St. Louis, MO, U.S.A.). The syntheses of
propanoylethanolamide (C3:0), butanoylethanolamide (C4:0),
hexanoylethanolamide (C6:0), octanoylethanolamide (C8:0),
decanoylethanolamide (C10:0), lauroylethanolamide (C12:0),
lEM=540 nm) before and after the addition of various
compounds in the presence or absence of 1 mM AEA.
Incubations were routinely run over 3 min, and the responses
generally appeared within a few seconds of injection of the
anandamide, peaking at about 30 s. Responses were
measured as peak ¯uorescence intensity (FI) minus basal
FI, and are expressed as a percentage of the response
obtained with either anandamide or capsaicin, as appro-
priate.
myristoylethanolamide
(C14:0),
palmitoylethanolamide
(C16:0), palmitamide (C16:0), palmitoylbutylamide (C16:0),
palmitoylisopropylamide (C16:0), palmitoylcyclohexamide
(C16:0) and oleoylethanolamide (C18:1) have been described
earlier (Lambert et al., 1999; 2001; Jonsson et al., 2001).
Capsaicin and non-radioactive AEA (for the VR1 experi-
ments) was purchased from Tocris (Bristol, U.K.). Heptade-
canoylethanolamide (C17:0), stearoylethanolamide (C18:0),
palmitoyl tri¯uoromethyl ketone and non-radioactive AEA
(for the FAAH experiments) were purchased from Cayman
Chemical Company (Ann Arbor, MI, U.S.A.). Fatty acid free
bovine serum albumin was obtained from the Sigma
Chemical Co. (St Louis, MO, U.S.A.). Cell culture media
were obtained from Gibco/Life Technologies.
Assay of FAAH activity
The assay used was that of Omeir et al. (1995) adapted to the
tritiated substrate (Fowler et al., 1997). Brie¯y, frozen brains
(minus cerebellum) from adult rats that had been stored at
7708C were thawed and homogenized at 48C in 20 mM
HEPES bu€er, pH 7.0, with 1 mM MgCl2. The homogenates
were centrifuged twice at 36,0006g for 20 min at 48C. Tissue
pellets were resuspended in homogenization bu€er and
incubated at 378C for 15 min, after which they were
centrifuged at 36,0006g for 20 min at 48C. Membranes were
re-suspended in 50 mM Tris-HCl bu€er, pH 7.4, containing
1 mM EDTA and 3 mM MgCl2, and stored at 7708C until
used for assay. Upon assay, membranes [12.5 mg protein
assay71], palmitoylethylamide (PA-Et) or ethanol carrier,
[3H]-AEA and assay bu€er (10 mM Tris-HCl, 1 mM EDTA,
1% (w v71) fatty acid-free bovine serum albumin, pH 7.6)
(®nal assay volume of 200 ml) were incubated for 10 min at
378C. Reactions were stopped by placing the tubes in ice and
thereafter adding 400 ml of chloroform : methanol (1 : 1 v
v71). Phases were separated by centrifugation in a bench
centrifuge after the samples had been thoroughly mixed.
Aliquots (200 ml) of the methanol/bu€er phase containing the
[3H]-ethanolamine product were removed and analysed for
radioactivity by liquid scintillation spectroscopy with quench
correction. Blanks contained distilled water instead of the
membranes.
Synthesis of palmitoylethylamide (PA-Et)
In a two-neck ¯ask, 4.17 g of ethylamine (92.6 mmoles) were
poured into 20 ml of dry methylene chloride. The solution
was cooled in an ice bath and magnetically stirred. Palmitoyl
chloride (2.74 g, 10 mmoles) was added dropwise. The
reaction mixture was stirred for 12 h at room temperature
and then washed with 5% sodium bicarbonate solution, HCl
1 M and brine. The organic layer was dried (MgSO4) and the
solvent evaporated under reduced pressure to give 2.34 g
(83%) of a white solid.
m.p.: 62 ± 638C (uncorrected); TLC (chloroform : methanol
6 : 4 v v71) Rf=0.86; spectroscopic data: 1H NMR (d-CDCl3) d
(p.p.m.) 0.86 (t, J=7 Hz, 3 H); 1.14 (t, J=7 Hz, 3 H); 1.16 ±
1.24 (m, 26 H); 2.1 (t, J=7 Hz, 2 H); 3.2 ± 3.33 (m, 2 H); 5.4
(NH); 13C NMR (d-CDCl3) d (p.p.m.) 14.1; 14.95 (CH3); 22.7;
25.8; 29.26; 29.37; 29.5; 29.63; 29.64; 29.65; 29.66; 29.68; 29.69 ;
29.8; 31.94; 34.3; 36.94 (CH2); 172.97 (C=O); mass spectro-
metry [M+]=283; IR v (cm71): 3298 (NH); 1633 (C=O).
Calculation of EC50, Ki(slope) and Ki(intercept) values
EC50 values were calculated from the mean values at each
AEA or capsaicin concentration using the built-in equation
`sigmoid dose-response (variable slope)' of the GraphPad
Prism computer programme (GraphPad Software Inc., San
Diego, CA, U.S.A.). Top and bottom values were set to 100
Cell culture
hVR1-HEK293 cells were grown as monolayers in minimum
essential medium (MEM) supplemented with non-essential
British Journal of Pharmacology vol 136 (3)

454
D. Smart et al
Palmitoylethanolamide analogues and VR1 receptors
and 0, respectively. Apparent KM and Vmax values for
inhibition of [3H]-AEA metabolism by PA-Et were calculated
from the mean data using the Direct Linear Plot analysis
(Eisenthal
& Cornish-Bowden, 1974) and the Enzyme
Kinetics v 1.4 software package (Trinity Software, Campton,
NH, U.S.A.). These values were used in secondary replots of
[PA-Et] vs KMapp/Vmax
Ki(slope) and Ki(intercept) values, respectively.
and 1/Vmax
to determine
app
app
Results
Effect of N-acylethanolamines upon AEA-induced
activation of VR1 receptors in hVR1-HEK293 cells
A series of N-acylethanolamines ranging from 3 carbons
(propanoylethanolamide) up to 18 carbons (stearoylethano-
lamide [C18:0], oleoylethanolamide (OEA, [C18:1]) were
investigated (Figure 1). The compounds were tested at a
concentration of 10 mM for e€ects per se, and for their ability
to a€ect the Ca2+-in¯ux response produced by a submaximal
concentration of AEA (1 mM). Two series of experiments
were undertaken, with AEA, PEA and OEA being tested in
both experiments. Very similar values were seen for these
compounds in both sets of experiments. For simplicity, the
second of these values are shown in the ®gures.
With the exception of PEA, none of the N-acyl
a
Ca2+-response per se in the
ethanolamines produced
hVR1-HEK293 cells (Figure 1a). The C3:0, C4:0, C6:0 and
C10:0 compounds were also without e€ect upon the response
to AEA. In contrast, the response to AEA was greatly
potentiated by the C12:0, C17:0, C18:0 and C18:1 com-
pounds, so that in the case of the C12:0 compound
(lauroylethanolamine), the response to 1 mM AEA became
maximal (Figure 1a). In Figure 1b, the ability of the
compounds to inhibit the FAAH-catalyzed metabolism of
AEA is shown. There was no obvious relation between the
abilities of the compounds to inhibit AEA metabolism and to
potentiate the VR1-mediated Ca2+ in¯ux in the hVR1-
HEK293 cells. Thus, for example, the C18:0 and C18:1
compounds potentiate the Ca2+ in¯ux response to AEA to
about the same extent, but have greatly di€ering abilities to
inhibit [3H]-AEA metabolism. Conversely, the C12:0 and
C14:0 compounds produce rather similar inhibitions of [3H]-
AEA metabolism, but di€er greatly in their ability to
potentiate the Ca2+ in¯ux response to AEA (Figure 1a, b).
Figure 1 (a) E€ects of a series of N-acyl ethanolamines upon the
Ca2+-in¯ux in hVR1-HEK293 cells in the absence and presence of
1 mM AEA. The compounds were used at a concentration of 10 mM.
Shown are means+s.e.mean of three experiments. The `C' numbers
given in the abscissa indicate the acyl chain length and degree of
saturation, respectively. Thus, for example, C20:4 indicates a 20
carbon length side chain with four double bonds, i.e. the
arachidonoyl group in AEA. (b) Inhibition of the metabolism of
2 mM [3H]-AEA by rat brain membranes by the N-acyl ethanolamines
at concentrations of 10 mM (unless otherwise shown). The data are
means+s.e.mean and are taken from the raw data of Figure 3 of
Jonsson et al. (2001).
the range of 10 ± 13 nM being found for the four curves
(Figure 2b).
Effects of PEA analogues upon FAAH activity and the
Ca2+ influx response to AEA and capsaicin in
hVR1-HEK293 cells
Comparison of the effects of lauroylethanolamide
(C12:0) upon the Ca2+ influx response to AEA and
capsaicin in hVR1-HEK293 cells
Six analogues to PEA where the ethanolamine group
was replaced by -amide (PA-NH2), -ethylamide (PA-Et),
-isopropylamide (PIA), -butylamide (PBA), -cyclohexamide
(PCA) and -tri¯uoromethyl ketone (PTMK) groups were
investigated. Of these, no data is available on the ability of
PA-Et to interact with FAAH, so the e€ects of this
compound upon [3H]-AEA by rat brain membranes was
determined. PA-Et was found to be a mixed-type inhibitor,
with a Ki(slope) value of 4.1 mM and a Ki(intercept) value of
66 mM (Figure 3).
Dose-response curves for the stimulation of Ca2+ in¯ux in
hVR1-HEK293 cells by AEA and capsaicin are shown in
Figure 2. Lauroylethanolamide produced a leftward shift in
the AEA dose-response curve, so that the potency of AEA
was increased approximately 10 fold at the highest
lauroylethanolamide concentration tested (10 mM) (Figure
2a). Thus, the EC50 values for AEA calculated from the
curves were 1.8, 1.5, 1.1 and 0.22 mM in the presence of 0, 1,
3 and 10 mM lauroylethanolamide, respectively. In contrast
to the e€ects on AEA, lauroylethanolamide did not a€ect
the dose ± response curves to capsaicin, with EC50 values in
In Figure 4, the e€ects of the six PEA analogues upon
basal and AEA-induced Ca2+ in¯ux in hVR1-HEK293 cells
British Journal of Pharmacology vol 136 (3)

D. Smart et al
Palmitoylethanolamide analogues and VR1 receptors
455
Figure 3 Mode of inhibition of [3H]-AEA metabolism in rat brain
membranes by palmitoylethylamide (PA-Et). Means+s.e.mean of
three experiments are shown in the main plot.
A secondary
Lineweaver ± Burk replot of the mean data is shown in the insert to
illustrate the mixed-type nature of the inhibition. The Ki(slope) and
Ki(intercept) values were calculated from the mean data as described in
Methods.
Figure 2 E€ects of lauroylethanolamide (C12:0) upon the the Ca2+
-
In
a
recent study, it was reported that the N-
in¯ux in hVR1-HEK293 cells produced by (a) AEA and (b)
capsaicin. Data are means of three experiments. In (a) the s.e.means
are shown (when not enclosed by the symbols) for the sake of clarity
only for the curves for 0, 3 and 10 mM lauroylethanolamide. In (b)
the s.e.means are shown only for the curves for 0 and 10 mM
lauroylethanolamide.
acylethanolamine palmitoylethanolamide (C16:0) was able
to increase the VR1-mediated Ca2+ in¯ux in response to
AEA in hVR1-HEK293 cells (De Petrocellis et al., 2001b).
These authors found that PEA produced a 2 fold decrease in
the EC50 value for AEA and was also able to potentiate the
responses to the VR1 stimulators capsaicin and resinifer-
atoxin, although the stimulation of the response to
resiniferatoxin was more marked than for capsaicin. At a
concentration of 1 mM AEA (the concentration used in the
present study), PEA was without e€ect upon the response to
AEA, whereas a potentiation was seen at AEA concentra-
tions 5250 nM (De Petrocellis et al., 2001b). The apparent
potency of AEA is highly dependent upon the assay
conditions, in particular upon the concentration of bovine
serum albumin (De Petrocellis et al., 2001b) (which is
required in the FLIPR assay to prevent binding of the AEA
to the plastic wells). However, the AEA concentration used
here (1 mM) relative to its EC50 value (1.8 mM) corresponds
to a concentration of *240 nM under the conditions used
by De Petrocellis et al. (2001b) relative to their EC50 value
are shown together with data on the abilities of the
compounds to a€ect [3H]-AEA metabolism. The experiments
were conducted concomitantly with the experiments shown in
Figure 1, so the data for AEA is the same in both ®gures.
None of the compounds produced marked e€ects either per
se, or upon the Ca2+ in¯ux response to 1 mM AEA (Figure
4a), despite a large variation in the abilities of the compounds
to prevent [3H]-AEA metabolism (Figure 4b).
Discussion
In the short time since the publication by Zygmunt et al.
(1999) that AEA was able to activate VR1 receptors, a
number of studies, not the least in this journal, have
demonstrated the involvement of these sensory nerve
receptors in the pharmacological e€ects of exogenously
applied AEA in nociceptive (Gauldie et al., 2001; Tognetto
et al., 2001) and pulmonary (Craib et al., 2001; Smith &
McQueen, 2001) function as a consequence of its e€ects on
sensory neurotransmission (Zygmunt et al., 1999; Ralevic et
al., 2001; Ross et al., 2001; Mang et al., 2001). However, it is
still a matter of debate as to whether AEA acts as an
endogenous vanilloid (see Di Marzo et al., 2001).
of 0.44 mM. In addition to its e€ects on AEA-induced Ca2+
-
in¯ux, the authors found that PEA produced a 5 fold
decrease in the Ki for the inhibition of [3H]-resinaferatoxin
binding by AEA (De Petrocellis et al., 2001b). The authors
suggested that this action of PEA could be described as an
`entourage' e€ect, similar to that seen for the ability of the
2-arachidonoyl glycerol homologues 2-palmitoylglycerol and
2-linoleoyl-glycerol to potentiate the cannabinoid receptor-
mediated e€ects of 2-arachidonoyl glycerol without them-
selves having anities for cannabinoid receptors (Ben-
Shabat et al., 1998).
British Journal of Pharmacology vol 136 (3)

456
D. Smart et al
Palmitoylethanolamide analogues and VR1 receptors
Figure 4 (a) E€ects of a series of palmitoylethanolamide analogues upon the Ca2+-in¯ux in hVR1-HEK293 cells in the absence
and presence of 1 mM AEA. Shown are means+s.e.mean of three experiments. The concentrations of the palmitoylethanolamide
analogues were in all cases 10 mM, except for PTMK where a concentration of 2 mM was used. (b) Inhibition of the metabolism of
2 mM [3H]-AEA by rat brain membranes by the palmitoylethanolamide analogues at concentrations of 10 mM (unless otherwise
shown). The data are means+s.e.mean and are taken from the raw data of Figure 4 of Jonsson et al. (2001), with the exception of
PA-Et and PA-NH2, which are from unpublished data.
In the present study, we have investigated a series of
response to capsaicin reported by De Petrocellis et al.
(2001b) was rather modest.
palmitoylethanolamide homologues and analogues in order
to investigate whether such an entourage e€ect is unique for
PEA, or whether other N-acylethanolamines produce even
more dramatic e€ects. In addition, in view of the ®nding that
PEA acts as a partial agonist at VR1-receptors overexpressed
in hVR1-HEK293 cells (Smart et al., 2000), e€ects of the
compounds per se upon VR1-receptors were investigated.
PEA was found to produce e€ects both per se and upon the
response to AEA, essentially in agreement with the study of
De Petrocellis et al. (2001b). These e€ects are lost when the
-ethanolamine group was replaced by -ethylamine (PA-Et),
-isopropylamine (PIA), -butylamine (PBA), -cyclohexamine
(PCA) and -tri¯uoromethyl ketone (PTMK) groups, suggest-
ing a degree of speci®city in the entourage actions of PEA,
although it should be pointed out as a caveat that the modest
inhibitory e€ects of PIA and PCA on the transport of AEA,
at least in rodent cell lines (Jonsson et al., 2001), may mask
small augmentations of the response to AEA, given that in
It has long been known that N-acyl ethanolamines are
substrates for amidohydrolase activities that are presumed to
be identical to fatty acid amide hydrolase (FAAH, Epps et
al., 1982; Schmid et al., 1985, Deutsch & Chin, 1993), and
that they will thus compete with AEA for this enzyme
(Jonsson et al., 2001). In theory, such a potentiation of the
response to AEA, but not capsaicin, could be a result of
competition for FAAH, so that more AEA is available for
interaction with the VR1 receptor. Certainly, the FAAH
inhibitors phenylsulphonyl methyl ¯uoride and methyl
arachidonoyl ¯uorophosphonate potentiate the Ca2+-re-
sponse to a low concentration (100 nM) of AEA but not
capsaicin (10 nM) in hVR1-HEK cells (De Petrocellis et al.,
2001a). However, in that study, the FAAH inhibitors were
preincubated with the cells for 10 min prior to addition of the
agonists, whereas in the present study, they were added
concomitantly. Given the very short incubation times used in
the FLIPR methodology (the peak response to AEA occurs
at about 30 s after administration, Smart et al., 2000),
together with the ®nding by De Petrocellis et al. (2001b), that
PEA at the concentrations used did not a€ect [14C]-AEA
hydrolysis by hVR1-HEK293 cell membranes, it is unlikely
that FAAH inhibition is involved in the actions of the NAEs.
Consistent with this conclusion, the present data show no
relationship between the ability of the compounds tested to
inhibit [3H]-AEA metabolism and their ability to potentiate
the VR1-mediated actions of AEA. The best illustrations of
this are PTMK, stearoylethanolamide and OEA. PTMK
produces an almost complete inhibition of FAAH but does
not a€ect the VR1 response to AEA; Stearoylethanolamide
and OEA produce similar robust augmentations of the VR1
response to AEA despite large di€erences in their abilities to
a€ect AEA metabolism. Of course, it should be mentioned as
a caveat that the FAAH data was obtained using rat brain
membranes, whereas the HEK cells are of human origin.
However, rat and human FAAH have very similar sequences
(82% amino acid identity and only one divergent amino acid
in the amidase signature sequence) and are able to hydrolyze
hVR1-HEK cells AEA uptake inhibitors block the Ca2+
-
response to this compound (De Petrocellis et al., 2001a). The
®nding that palmitamide (PA-NH2) has at best modest e€ects
in this assay is important. This compound is a good substrate
of FAAH (Boger et al., 2000), and its low activity would
suggest that the e€ects of PEA are not a result of its
metabolism to the free fatty acid.
PEA, however, was not the best entourage compound at
the assay concentration of AEA used, since lauroylethano-
lamide (C12:0), heptadecanoylethanolamide (C17:0), stear-
oylethanolamide (C18:0) and oleoylethanolamide (C18:1)
produced much more robust potentiations of the e€ect of
AEA without producing Ca2+-in¯uxes in the cells in its
absence. The C12:0 compound potentiated the e€ects of
AEA but not of capsaicin, suggesting that in some way it
increases the anity of the VR1 receptor for the
endogenous compound but not the exogenous compound.
At ®rst sight, this di€ers from the observations with PEA
(De Petrocellis et al., 2001b), and might suggest that the
C12:0 and C16:0 compounds may exert their e€ects in
di€erent ways. However, the e€ect of PEA upon the
British Journal of Pharmacology vol 136 (3)

D. Smart et al
Palmitoylethanolamide analogues and VR1 receptors
457
a
series of N-acyl amides with rather similar relative
levels of the C16:0 and C18:0 N-acyl ethanolamines were
increased 39 and 21 fold (Kondo et al., 1998). Although there
is no apparent increased sensitivity of peripheral nociceptors
to exogenously applied AEA in arthritic rat knee joints
(Gauldie et al., 2001), it is nevertheless tempting to speculate
that N-acyl ethanolamines may be an important regulator of
the actions of AEA upon vanilloid receptors under severe
in¯ammatory conditions.
speci®cities (Cravatt et al., 1996; Giang & Cravatt, 1997).
Thus, in agreement with the conclusion of De Petrocellis et
al. (2001b), FAAH inhibition does not account for the
potentiation of AEA e€ects upon Ca2+-in¯ux in hVR1-
HEK293 cells produced by the N-acyl ethanolamines.
In conclusion, the present study has con®rmed that PEA
has entourage-like e€ects at VR1 receptors expressed on
hVR1-HEK293 cells and demonstrated that other N-acyl
ethanolamines have even more dramatic potentiating e€ects.
It is of course hazardous to extrapolate data obtained from
transfected cells to the situation in vivo. However, one
important property of N-acyl ethanolamines in the body is
that their synthesis is dramatically increased under certain
conditions of cellular stress, ischaemia and in¯ammation (see
e.g. Epps et al., 1979; Kondo et al., 1998; Berdyshev et al.,
2000; Hansen et al., 2001). Thus, for example, 9 h following
cadmium chloride-induced in¯ammation in the rat testis, the
This study was supported by grants from the Swedish Medical
Research Foundation (Grant no. 12158), the Swedish Asthma- and
Allergy Association's Research Foundation, Konung Gustav V's
and Drottning Victorias Foundation, the Swedish Psoriasis
Association, and the Research Funds of the Medical Odontologi-
cal Faculty, Umea University. The chemical work was ®nancially
supported by the Belgian National Fund for Scienti®c Research
and a FSR grant from the Universite catholique de Louvain.
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(Received February 14, 2002
Revised March 14, 2002
Accepted March 22, 2002)
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