Lipophilicity of capsaicinoids and capsinoids influences the multiple activation process of rat TRPV1

Article abstract of DOI:10.1016/j.lfs.2006.07.024

Analogs of capsaicin, such as capsaicinoids and capsinoids, activate a cation channel, transient receptor potential cation channel vanilloid subfamily 1 (TRPV1), and then increase the intracellular calcium concentration ([Ca²⁺]_i). Th

Full text of DOI:10.1016/j.lfs.2006.07.024

Life Sciences 79 (2006) 2303–2310
www.elsevier.com/locate/lifescie
Lipophilicity of capsaicinoids and capsinoids influences the multiple
activation process of rat TRPV1
a
a
b
b
Akihito Morita a, , Yusaku Iwasaki , Kenji Kobata , Tohko Iida , Tomohiro Higashi ,
Kyoko Oda a, Asami Suzuki a, Masataka Narukawa a, Shiho Sasakuma a, Hidehiko Yokogoshi a,
Susumu Yazawa c, Makoto Tominaga b, Tatsuo Watanabe a
⁎
a
School of Food and Nutritional Sciences and COE Program in the 21st Century, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
b
Department Section of Cell Signaling, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji, Okazaki,
Aichi 444-8787, Japan
c
Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received 29 September 2005; accepted 28 July 2006
Abstract
Analogs of capsaicin, such as capsaicinoids and capsinoids, activate a cation channel, transient receptor potential cation channel vanilloid
subfamily 1 (TRPV1), and then increase the intracellular calcium concentration ([Ca2+]i). These compounds would be expected to activate
TRPV1 via different mechanism(s), depending on their properties. We synthesized several capsaicinoids and capsinoids that have variable
lengths of acyl moiety. The activities of these compounds towards TRPV1 heterologously expressed in HEK293 cells were determined by
measuring [Ca2+]i. When an extracellular or intracellular Ca2+ source was removed, some agonists such as capsaicin could increase [Ca2+]i.
However, a highly lipophilic capsaicinoid containing C18:0 and capsinoids containing C14:0, C18:0, or C18:1 (the latter was named
olvanilate) could not elicit a large increase in [Ca2+]i in the absence of an extracellular or intracellular Ca2+ source. These results suggest that
highly lipophilic compounds cause only a slight Ca2+ influx, via TRPV1 in the plasma membrane, and are not able to activate TRPV1 in the
endoplasmic reticulum.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Capsaicinoids; Capsinoids; Intracellular calcium concentration; Lipophilicity; TRPV1
Introduction
1999). CST and related compounds are capsaicin analogs called
capsinoids that have an ester bond instead of the amide bond
between the vanillyl moiety and the fatty acid chain (Kobata
et al., 1998, 1999).
Capsaicin (CAP; isoC10:1VA, Fig. 1), which is the major
pungent principle of red peppers (Capsicum spp.), has diverse
biological activities (Szallasi and Blumberg, 1999). There are
natural and synthetic analogs, which are generally known as
capsaicinoids. Their common structure is a group of acid amides
of vanillylamine and fatty acids. We have also isolated a novel
compound, capsiate (CST; isoC10:1VOH, Fig. 1), from ‘CH-19
Sweet’, a non-pungent red pepper cultivar (Kobata et al., 1998,
Several capsaicinoids and CST activate a specific receptor,
transient receptor potential vanilloid subtype 1 (TRPV1) (Cate-
rina et al., 1997; Tominaga et al., 1998; Appendino et al., 2002;
Iida et al., 2003). Activation of TRPV1 causes a rapid rise in the
intracellular free Ca2+ concentration ([Ca2+]i) (Caterina et al.,
1997; Tominaga et al., 1998). There are two pools of TRPV1 in
cells: one in the plasma membrane (TRPV1PM) and the other in
the endoplasmic reticulum (TRPV1ER) (Olah et al., 2001; Karai
et al., 2004). Thus, TRPV1 regulates two calcium compartments:
extracellular Ca2+ and the intracellular Ca2+ store (Eun et al.,
2001; Marshall et al., 2003). It had been thought that all
⁎
Corresponding author. Tel.: +81 54 264 5558; fax: +81 54 264 5586.
E-mail address: moritaa@u-shizuoka-ken.ac.jp (A. Morita).
0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2006.07.024

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A. Morita et al. / Life Sciences 79 (2006) 2303–2310
Fig. 1. Structures of capsaicinoids and capsinoids. Six capsaicinoids and six capsinoids used in this study are shown. Three compounds were previously named as
capsaicin (CAP), olvanil (OLV), and capsiate (CST). One capsinoid containing oleic acid was newly synthesized and named as olvanilate (OLT).
capsaicinoids and capsinoids activate both pools of TRPV1 and
mobilize both Ca2+ compartments. However, a recent report
indicated that different vanilloid agonists cause different patterns
of Ca2+ response in CHO cells heterologously expressing rat
TRPV1 (Toth et al., 2005). The capsaicinoids and capsinoids
appear to increase [Ca2+]i via different mechanism(s), depending
on their different properties.
suggest that the high lipophilicity of the compounds influences
the activation process of TRPV1.
Materials and methods
Materials
For this investigation, we synthesized four capsaicinoids and
five capsinoids containing varying lengths of fatty acids, as in
Fig. 1. The activity of these compounds towards TRPV1 hetero-
logously expressed in HEK293 cells was determined by
measuring [Ca2+]i. Drugs such as N,N,N′,N′-tetrakis(2-pyridyl-
methyl)ethylenediamine (TPEN) (Hofer et al., 1998) or tapsi-
gargin (Bian et al., 1991; Lytton et al., 1991) were used to reduce
the amount of Ca2+ in the intracellular Ca2+ store. To minimize
the amount of extracellular Ca2+, we used Ca2+-free assay buffer
containing 0.125 mM EGTA. In both cases, certain capsaicinoids
and capsinoids containing long acyl moieties, such as C18:0VA,
C14:0VOH, C18:1VOH (OLT), and C18:0VOH (see Fig. 1),
elicited an extremely slight increase in [Ca2+]i. These results
Capsaicin and capsazepine were purchased from Sigma.
Capsaicinoids were synthesized by the condensation of vanil-
lylamine hydrochloride with the corresponding acyl chlorides in
dry pyridine (Rangoonwala and Seitz, 1970). Capsinoids were
synthesized enzymatically (Kobata et al., 2002). The purity of all
compounds was confirmed to be over 95% by reversed phase
HPLC. Capsaicinoids and capsinoids were dissolved in dimethyl
sulfoxide (DMSO) and stored at −20 °C. The DMSO stock was
diluted with aqueous medium just before each experiment
because diluted capsinoids are unstable in aqueous solution
(Sutoh et al., 2001; Iida et al., 2003). All other chemicals were of
guaranteed reagent grade. All cell culture media were obtained
from Invitrogen. All culture dishes and plates were from Falcon.

A. Morita et al. / Life Sciences 79 (2006) 2303–2310
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Measurement of lipophilicity
2-AM (5 μM; Molecular Probes), and chromophore EL (0.15%;
Sigma) at 37 °C for 30 min. After washing twice with loading
buffer, the cells were stocked in the loading buffer at below 4 °C
(b4 h).
We determined the lipophilicity of the compounds as log P,
as described in a previous report (Iida et al., 2003). Table 1
shows that the log P values of the capsaicinoids and capsinoids
are proportional to the acyl chain length. Compounds with
unsaturated acyl chains have lower log P values.
A cuvette containing Fura 2-loaded cells (6×105 cells/2 ml)
was placed in a fluorospectrophotometer (CAF-110; Jasco Inc.,
Tokyo, Japan). After incubation with stirring at 37 °C for at least
1 min, the test compound was added by microsyringe. Time
dependent changes of the fluorescence (excitation wavelength
set at 340 or 380 nm and emission wavelength at 500 nm) were
recorded and analyzed using PowerLab system MacLab/4e and
Chart 4 (AD Instruments). The fluorescence ratio (340/380)
was converted to [Ca2+]i according to the equation published by
Grynkiewicz et al. (1985), where Rmax and Rmin were deter-
mined using 0.1% Triton X-100 and 5 mM EGTA (pH 8.0),
respectively. The effective dissociation constant for Fura 2 at
37 °C was 224 nM.
Cloning and expression of rTRPV1
Total RNA of C6 glioma cells was isolated using RNeasy
Mini Kit (Qiagen). The first strand cDNA was transcribed by
SuperScript II (Invitrogen) using oligo dT primer. A rat TRPV1
cDNAwas amplified by Expand HiFi (Roche Diagnostics) using
forward and reverse primers incorporating the restriction sites
shown, RVR1F1-HindIII (5′-atccaagcttgaaaggatggaacaacggg)
and RVR1R2-XbaI (5′-tatctctagattatttctcccctgggacc). Reaction
products were cloned into the HindIII-XbaI sites of pcDNA3
(Invitrogen), and the sequence was confirmed using a BigDye
Terminator cycle sequence kit and ABI PRISM 310 genetic
analyzer (Applied Biosystems). HEK293 cells were transfected
with rTRPV1-pcDNA3 construct using SuperFect Transfection
Reagent (Qiagen), according to the manufacturer's instructions.
Stable clones were selected in 600 μg/ml G418 (Sigma).
Northern blot and Western blot analyses using antibody against
rTRPV1 (Alpha Diagnostic) confirmed the rTRPV1 expression.
One clone, expressing TRPV1, was named HEK293VR11.
In some experiments, capsazepine (100 μM), an antagonist
of TRPV1, was added at least 1 min prior to the addition of the
test compounds. To analyze the effect of BSA, we treated cells
with loading buffer in the presence or absence of 0.1% BSA. To
exclude the contribution of the intracellular Ca2+ store, we used
two drugs, TPEN and tapsigargin. We added 10 μM TPEN to
the loading buffer during all periods. In the case of tapsigargin,
we added it (50 μM) 3 min before the addition of the
capsaicinoids or capsinoids. To exclude the contribution of
extracellular Ca2+, we suspended Fura 2-loaded cells with Ca2+-
free buffer, and added EGTA (0.125 mM) 1 min before the
addition of the test compounds.
Electrophysiology
Whole-cell patch-clamp recordings were carried out 1 or
2 days after seeding of the HEK293VR11 cells to new dishes.
The standard bath solution contained 140 mM NaCl, 5 mM
KCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, and 10 mM
glucose; it was adjusted to pH 7.4 with NaOH. The pipette
solution contained 140 mM KCl, 5 mM EGTA, and 10 mM
HEPES; it was adjusted to pH 7.4 using KOH. All patch-
clamp experiments were performed at room temperature
(22 °C).
Data analysis
The basal level of [Ca2+]i was calculated from the average
value of the fluorescence ratio (340/380) during the 10 s imme-
diately preceding the addition of test chemical. The maximum
level of [Ca2+]i was calculated from the maximum increase in
the ratio (340/380) during the first minute after the addition of
the test chemical. The change (Δ[Ca2+]i) was calculated by
subtraction of the former from the latter. For C18:0VA,
C14:0VOH and C18:0VOH, maximum values were obtained
during the first 5 min after addition. The increase in Δ[Ca2+]i
induced by 10 μM CAP in the absence of TPEN was 586 nM.
Using this value, we normalized the Δ[Ca2+]i values induced by
the capsaicinoids or capsinoids, except for the experiments in
which the extracellular Ca2+ was removed. Curve fitting and
parameter estimation were carried out using Prism 4 (Graph
Pad Software).
Measurement of [Ca2+
]
i
HEK293VR11 cells were maintained in Dulbecco's modi-
fied Eagle's medium containing 10% fetal bovine serum,
100 units/ml of penicillin, 100 μg/ml of streptomycin, and
250 ng/ml of amphotericin B under 5% CO2/air at 37 °C. Cells
were sub-cultured every week and the highest passage number
used was 50.
HEK293VR11 cells were plated in 150 mm dishes at a
density of 1.8×107 cells. After 3–4 days, cells were detached,
using 10 ml of Ca2+-free PBS containing 0.5 mM EDTA, and
then collected by centrifugation. The collected cells were
washed twice with loading buffer [5.37 mM KCl, 0.441 mM
KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4–7H2O,
5.56 mM glucose, 20 mM HEPES, 1 mM CaCl2, and 0.1%
bovine serum albumin (BSA), pH 7.4] and resuspended in the
same buffer containing the cytoplasmic calcium indicator, Fura
Results
The rTRPV1 cDNA was cloned from rat C6 glioma cells
because these cells had been shown to respond to capsaicin
(Bíró et al., 1998). The nucleotide sequence of this clone was
almost identical to rat TRPV1 cDNA (Caterina et al., 1997),
except that it produced one amino acid substitution: Thr at
position 239 was replaced by Ala. After transfecting with this
clone and selecting by the presence of G418 (0.6 μg/ml), we

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A. Morita et al. / Life Sciences 79 (2006) 2303–2310
Table 1
These values were very similar to those obtained in the cells
transiently expressing TRPV1 (99 nM and 290 nM for CAP and
CST, respectively) (Iida et al., 2003). In addition to the
electrophysiological properties, we measured the change in
[Ca2+]i (Fig. 2B). CAP increased [Ca2+]i concentration-
dependently. The EC50 value was 81.9 nM, which was a little
smaller than that found by measuring the membrane current (Fig.
2A) and similar to the values (15 to 190 nM) previously reported
by others (Jerman et al., 2000; Appendino et al., 2002). CSTalso
increased [Ca2+]i, and its EC50 value was 606 nM, which was a
little greater than that found by measuring the membrane current
(Fig. 2A). These results indicate that CST opens the TRPV1
channel and then increases [Ca2+]i, similarly to CAP.
Lipophilicity and pharmacokinetic parameters of capsaicinoids and capsinoids
Compounds
Log TPEN (−)
TPEN (+)
P
EC50
(μM)
Max EC50
(%) (μM)
Max
(%)
Capsaicinoids isoC10:1VA
C10:0VA
(CAP) 3.81 0.0819 99.0
4.01 0.235
4.38 62.6
98.8 11.6
63.8
7.32 63.1
1.79 39.8
0.216 10.5
58.7
68.7
5.46 15.9
C14:0VA
C18:1VA
C18:0VA
7.37 0.190 100.1
(OLV) 9.12 0.00686 97.1
10.16 0.0609 82.6
Capsinoids
isoC10:1VOH (CST) 5.80 0.606
91.3 16.8
84.7 18.8
75.5
C10:0VOH
C14:0VOH
6.77 1.27
9.86 1.44
C18:1VOH (OLT) 11.47 0.198
C18:0VOH 12.93 NC
66.2 10.7
NC 4.93 10.9
17.5
All five capsaicinoids (Fig. 1) increased [Ca2+]i concentra-
tion-dependently (Table 1). These changes in [Ca2+]i response
were not observed in the parent HEK293 cells and were
inhibited by capsazepine, a specific antagonist for TRPV1
(data not shown). Therefore, these effects appeared via
activation of TRPV1. The potency (EC50 values) for four of
the capsaicinoids (CAP, OLV, C10:0VA, C14:0VA) were
comparable to those reported by others (Jerman et al., 2000;
Appendino et al., 2002). Their efficiency (maximum response)
was almost the same.
The log octanol/water partition coefficient (log P) and pharmacokinetic
parameter was calculated as described in Materials and methods. The partition
coefficient of CAP was measured directly using an octanol–water partitioning
system. Other log P values were calculated from the retention time in HPLC.
The increase of Δ[Ca2+]i induced by 10 μM CAP in the absence of TPEN was
586 nM. Using this value, we normalized the Δ[Ca2+]i values induced by other
capsaicinoids or capsinoids in the presence or absence of TPEN.
NC; not calculated.
obtained HEK293VR11 cells, which expressed TRPV1 mRNA
and protein (data not shown).
In our experiments, 0.1% BSA was added into the loading
buffer to promote dissolution of the lipophilic compounds in
aqueous solution. BSA binds a variety of lipophilic ligands, such
as fatty acids and lysolecithin (Peters, 1996). It has been reported
that BSA decreases the effect of anandamide on TRPV1 (De
Petrocellis et al., 2001). However, our data (Fig. 3A) show that
BSA had no effect on the action of CAP and C18:0VA.
We examined the electrophysiological properties of the
cloned cells to confirm the functional expression of the rTRPV1
receptor. As shown in Fig. 2A, CAP and CST induced a
concentration-dependent current in this cell line. The EC50
values for CAP and CSTwere 114 nM and 477 nM, respectively.
Fig. 2. CAP- and CST-induced changes in membrane potential and [Ca2+]i. (A) Membrane currents were normalized to the response maximally activated by 1 μM
capsaicin or 3 μM capsiate and expressed as a percent of the maximal response. Each point represents a mean value SEM from three independent cells. Averaged data
were fitted with the Hill equation. EC50 values for capsaicin and capsiate concentration–response curves were 112 nM and 477 nM, respectively. (B) Δ[Ca2+]i values
were normalized to the response maximally activated by 10 μM capsaicin and expressed as a percentage of the maximal response. Each point represents the mean
values SEM from six to seven independent experiments. Data were fitted with the Hill equation. EC50 values for capsaicin and capsiate concentration–response
curves were 81.9 nM and 606 nM, respectively.

A. Morita et al. / Life Sciences 79 (2006) 2303–2310
2307
Fig. 3. The different responses to Δ[Ca2+]i induced by C18:0VA or CAP. (A) Δ[Ca2+]i is indicated after the addition of 1 μM each of CAP or C18:0VA in the absence
(open bar) or presence (dotted bar) of 0.1% BSA. Δ[Ca2+]i values were normalized to the maximal response activated by 10 μM capsaicin in the presence of BSA and
expressed as a percent of the maximal response. Each point represents the mean values SEM from three independent experiments. (B) The fluorescence ratio
(excitation 340 nm/excitation 380 nm) is indicated after the addition of 1 μM each of CAP or C18:0VA. Arrows indicate the time of the sample addition. (C)
Concentration–response curves for CAP (circles) and C18:0VA (squares) are indicated. Open symbols indicate maximum values during 1 min after sample addition
(short periods). Closed symbols indicate the maximum value during 5 min after sample addition (long periods). Each point represents the mean values SEM from
three and seven independent experiments for C18:0VA and CAP, respectively.
The potency of C18:0VA (60.9 nM) was similar to that of
CAP (81.9 nM), and the efficiency of C18:0VA (maximum
response) was 82.6% of that of CAP (Table 1). This result
differs from that of a previous report (Appendino et al., 2002).
The reason for this discrepancy remains obscure. However, we
observed that C18:0VA gradually increases the fluorescence
ratio (340/380) (Fig. 3B). When [Ca2+]i was measured during
the first minute, the EC50 value for C18:0VA was not mea-
surable and its maximum response was 50% (Fig. 3C), similar
to the previous results (Appendino et al., 2002). On the contrary,
when we measured [Ca2+]i over 5 min, C18:0VA increased the
fluorescence ratio to a maximal level (Fig. 3B). The effect of
C18:0VA was almost the same as that of CAP (Fig. 3C).
Therefore, C18:0VA is one of the TRPV1 agonists, but it has a
somewhat unique property.
CST is known to activate TRPV1 (Iida et al., 2003), but the
activity of the other capsinoids has not been reported. We found
that all the newly synthesized capsinoids increased [Ca2+]i via
Fig. 4. Tapsigargin inhibits the increase of Δ[Ca2+]i induced by highly lipophilic agonists. Δ[Ca2+]i values were normalized to the maximal response activated by
10 μM capsaicin and expressed as a percent of the maximal response. Vehicle (open bar) or 50 μM of tapsigargin (dotted bar) were added to a loading buffer at 1 min
prior to the addition of the indicated capsaicinoids (A) or capsinoids (B). Each bar represents the mean values SEM from three experiments.

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A. Morita et al. / Life Sciences 79 (2006) 2303–2310
Fig. 5. The absence of extracellular Ca2+ inhibits the increase of Δ[Ca2+]i induced by highly lipophilic agonists. The Fura 2-loaded cells were resuspended with Ca2+
-
free loading buffer. EGTA (0.125 mM) was added at 1 min prior to the addition of 10 μM of the capsaicinoids (A) or 30 μM of the capsinoids (B). Each bar represents
the mean values SEM from three experiments.
the activation of TRPV1, as these effects were not observed in
the parent HEK293 cells and were inhibited by capsazepine
(data not shown). The EC50 value of C10:0VOH was two-fold
larger than that of CAP, and its maximum response was almost
identical to that of CAP (Table 1). C14:0VOH and OLT in-
creased [Ca2+]i, but the maximum responses were lower at
75.5% and 66.2%, respectively. The properties of the latter two
capsinoids are similar to those of C18:0VA. The addition of
more than 100 μM C18:0VOH made the assay solution turbid
because of its low solubility. Therefore, we could not obtain the
correct value for the fluorescence. The response of 30 μM
C18:0VOH was about 60% of that of 10 μM capsaicin, but did
not reach the maximum level. Thus, the EC50 value and the
maximum response of C18:0VOH could not be calculated.
CAP regulates [Ca2+]i via TRPV1 with multiple mechanisms.
TRPV1 distributes in the plasma and the ER membranes (Olah
et al., 2001; Karai et al., 2004). Thus, there are two Ca2+ sources,
extracellular and intracellular (Eun et al., 2001; Marshall et al.,
2003).
the presence of TPEN) (Table 1). This inhibition suggests that
C18:0VA elicits a very slight influx of calcium via TRPV1PM in
the absence of an intracellular Ca2+ store.
Capsinoids containing medium chain acyl groups (C10:0VOH
and CST) increased [Ca2+]i even in the presence of TPEN
(Table 1). As expected from the effect of C18:0VA, capsinoids
containing long fatty acids (C14:0VOH, C18:0VOH, and OLT)
could not elicit a large increase in [Ca2+]i (Table 1). Because the
log P values of the latter group are higher than that of C18:0VA
(Table 1), these compounds are extremely lipophilic. It is possible
that these highly lipophilic capsaicinoids and capsinoids elicit
only a slight calcium influx via TRPV1PM in the absence of an
intracellular Ca2+ store.
In addition to TPEN, tapsigargin also depletes the intracellular
Ca2+ store (Bian et al., 1991; Lytton et al., 1991). Fig. 4 shows the
effects of tapsigargin on the increase in [Ca2+]i induced by
capsaicinoids and capsinoids. As expected from the results with
TPEN, tapsigargin also decreased the efficiencies of the highly
lipophilic capsaicinoid and capsinoids to less than 10%. There-
fore, we believe that the extremely lipophilic capsaicinoid
(C18:0VA) and capsinoids (C14:0VOH, C18:0VOH and OLT)
elicit only a slight calcium influx via TRPV1PM in the absence of
an intracellular Ca2+ store.
To eliminate contributions to [Ca2+]i from the intracellular
Ca2+ store, we treated the cells with 10 μM TPEN, a membrane-
permeable multivalent cation chelator. Because TPEN has only
moderate affinity for Ca2+ (Kd =130 mM), it should chelate Ca2+
in the intracellular store (Hofer et al., 1998). Four capsaicinoids
(CAP, C10:0VA, C14:0VA and OLV) elicited a [Ca2+]i increase
even in the presence of TPEN, although the EC50 values
increased and the efficiencies (maximum responses) decreased
(Table 1). These capsaicinoids are able to increase [Ca2+]i via
TRPV1PM in the absence of an intracellular Ca2+ store.
Interestingly, the effect of C18:0VA was inhibited by the
presence of TPEN (Table 1). The efficiency of C18:0VA was
decreased from 82.6% (in the absence of TPEN) to 10.5% (in
To eliminate the influence of extracellular Ca2+, we sus-
pended Fura 2-loaded cells with Ca2+-free assay buffer, and
added EGTA (0.125 mM) 1 min prior to the addition of the test
compounds. CAP increased [Ca2+]i in the absence of extracel-
lular Ca2+ (Fig. 5). This increase is due to the release of Ca2+
from the intracellular Ca2+ store via TRPV1 in the ER mem-
brane, as previously reported by others (Olah et al., 2001; Karai
et al., 2004). Three capsaicinoids (C10:0VA, C14:0VA and
OLV) and two capsinoids (CST and C10:0VOH) also increased

A. Morita et al. / Life Sciences 79 (2006) 2303–2310
2309
[Ca2+]i, but to a lesser extent than CAP (Fig. 5). These six
ligands, which are of low- or mid-level lipophilicity, can acti-
vate TRPV1ER. However, one capsaicinoid (C18:0VA) and
three capsinoids (C14:0VOH, OLT and C18:0VOH) elicited
only a slight release of calcium from the intracellular Ca2+ store
(Fig. 5). It is possible that highly lipophilic capsaicinoids and
that TRPV1 has multiple binding sites, with at least five to seven
closed conformations, and that partial binding would lead to
partial channel opening (Hui et al., 2003). Further experiments
are necessary to confirm this possibility.
Furthermore, we must consider several amplification proces-
ses, including intracellular Ca2+ stores, because highly lipo-
philic TRPV1 agonists can increase [Ca2+]i under normal
conditions. It had been postulated that a Ca2+ influx via TRPV1
was amplified by several mechanisms, such as CICR (calcium-
induced calcium release) from the intracellular Ca2+ store or
SOCE (store operated calcium entry) from an extracellular site
(Karai et al., 2004). Recently, van der Stelt et al. (2005) dem-
onstrated that anandamide acted as an intracellular messenger
amplifying Ca2+ influx via TRPV1 channels. They hypothesize
that anandamide may function as a store-operated messenger
signaling to TRPV1 to gate extracellular Ca2+. It is possible that
highly lipophilic TRPV1 agonists stimulate metabolically the
capsinoids are not able to activate TRPV1ER
.
Discussion
All capsaicinoids and capsinoids increased [Ca2+]i under
normal conditions. However, one capsaicinoid (C18:0VA) and
three capsinoids (C14:0VOH, C18:0VOH and OLT) that have
high lipophilicity (log PN9.5) exhibited different effects in the
absence of extracellular Ca2+ or intracellular Ca2+ sources.
When the extracellular Ca2+ was removed from the medium,
agonists such as CAP and CST increased [Ca2+]i (Fig. 5). These
agonists activated the TRPV1ER, as described by others (Olah
et al., 2001; Karai et al., 2004). However, highly lipophilic
agonists did not elicit large increases in [Ca2+]i (Fig. 5). It has
been reported that the high lipophilicity of fatty acids decreases
the dissociation of these compounds from the plasma membrane
(Hamilton, 1998; Zhang et al., 1996). Electrophysiological
evidence indicated that the lipophilic agonists such as OLV
were not washed out from the membrane but CAP was easily
(Iida et al., 2003). Therefore, highly lipophilic agonists do not
penetrate the plasma membrane and are not able to activate
production of anandamide, which then activates TRPV1PM
.
Therefore, the response to C18:0VA would be slower than that
to CAP (Fig. 3). Further experiments are necessary to elucidate
the precise mechanism(s) by which highly lipophilic agonists
increase [Ca2+]i under normal conditions.
Our results indicate that TRPV1 agonist potency is partially
dependent on its lipophilicity, which influences its transport
across the cell membrane. Indeed, some endogenous compounds,
called endovanilloids (Chu et al., 2003), need to be carried inside
the cell via a selective transporter to interact with TRPV1 (Huang
et al., 2002). Using several agonists with different lipophilicities,
we would be able to distinguish the multiple mechanisms of the
activation process of TRPV1.
TRPV1ER
.
When the intracellular Ca2+ store was depleted by the ad-
dition of TPEN or tapsigargin, agonists such as CAP and CST
increased [Ca2+]i (Table 1 and Fig. 4). It is remarkable that the
highly lipophilic agonists increased [Ca2+]i only very slightly in
the presence of TPEN and tapsigargin (Table 1 and Fig. 4).
These results suggest that highly lipophilic agonists elicit little
Ca2+ influx via TRPV1PM. This suggestion is supported by
evidence that C18:0VA did not increase [45Ca2+] uptake into
DRG neurons (Walpole et al., 1993).
Conclusion
A highly lipophilic capsaicinoid containing C18:0 and cap-
sinoids containing C14:0, C18:0, or C18:1 (the latter was named
olvanilate) could not elicit a large increase in [Ca2+]i in the
absence of an extracellular or intracellular Ca2+ source. These
results suggest that highly lipophilic compounds cause only a
slight Ca2+ influx via TRPV1 in the plasma membrane and are
not able to activate TRPV1 in the endoplasmic reticulum. The
capsaicinoids and capsinoids increase [Ca2+]i through different
mechanism(s), depending on their properties.
It is necessary to consider why highly lipophilic agonists only
slightly open the TRPV1PM channels. We postulate that highly
lipophilic agonists would partially bind to TRPV1PM. Recently,
three laboratories have reported that Met 547 of TRPV1, near the
extracellular side of transmembrane domain 4, was functioning
as a binding site of TRPV1 to CAP (Chou et al., 2004; Gavva
et al., 2004; Phillips et al., 2004). The other candidates for
binding sites are located at the intracellular N-terminal cytosolic
domain (Jordt and Julius, 2002) or the C-terminal cytosolic
domain (Jung et al., 2002). Agonists such as CAP may have
adequate lipophilicity to penetrate the plasma membrane. They
can bind to both transmembrane and intracellular sites; they then
elicit prolonged channel openings. On the other hand, highly
lipophilic compounds do not easily dissociate from the plasma
membrane (Hamilton, 1998; Zhang et al., 1996). Thus, highly
lipophilic agonists would stay in the plasma membrane and act
only at Met 547 in the transmembrane domain. They would bind
to TRPV1 partially and open the TRPV1 channel partially, so
that only small amounts of Ca2+ penetrate into the cells. Recent
results from measuring single channel activity have also shown
Acknowledgements
This work was supported in part by Morinaga and Co., Ltd.,
and a Grant-in-aid for Scientific Research from the Ministry of
Education, Sports, Culture, Science and Technology of Japan. We
are grateful to Dr. A. Utani, School ofMedicine, Kyoto University,
and Dr. S. Sugiyama, Medical School of Saitama University, for
providing HEK293 cells and C6 glioma cells, respectively.
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