Full text of DOI:10.1038/sj.bjp.0703918

British Journal of Pharmacology (2001) 132, 1084 ± 1094
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Pharmacological di€erences between the human and rat vanilloid
receptor 1 (VR1)
1
1
1
*,1Peter McIntyre, 1,2Linda M. McLatchie, Anne Chambers, Elsa Phillips, Melanie Clarke,
1Jonathan Savidge, Christy Toms, Marcus Peacock, Kirti Shah, Janet Winter,
1Natasha Weerasakera, 1,3Mike Webb, Humphrey P. Rang, Stuart Bevan & Iain F. James
1
1
1
1
1
1
1
1Novartis Institute for Medical Sciences, 5 Gower Place London WC1E 6BN
1
Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and
expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells.
Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with
similar EC50 values. Capsaicin had lower potency on both channels, when measured
2
a
electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+0.20 mM; hu-
man=1.90+0.30 mM: CHO cells: rat=0.20+0.06 mM; human=0.19+0.08 mM).
3 In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent
protein, aequorin, the EC50 values for capsaicin-induced responses were similar in both cell lines
(rat=0.35+0.06 mM, human=0.53+0.03 mM).
4
The threshold for activation by acidic solutions was lower for human VR1 channels than that for
rat VR1 (EC50 pH 5.49+0.04 and pH 5.78+0.09, respectively).
5
6
The threshold for heat activation was identical (428C) for rat and human VR1.
PPAHV was an agonist at rat VR1 (EC50 between 3 and 10 mM) but was virtually inactive at the
human VR1 (EC50410 mM).
Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of
human VR1 than rat VR1.
Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also
7
8
more e€ective at inhibiting the noxious heat response of human than of rat VR1.
British Journal of Pharmacology (2001) 132, 1084 ± 1094
Keywords: Capsaicin; capsazepine; VR1; pain response; proton activation; heat activation; intracellular calcium
Abbreviations: CGRP, calcitonin gene-related peptide; CPZ, capsazepine; CHO, chinese hamster ovary; cDNA, copy DNA;
DRG, dorsal root ganglion; NGF, nerve growth factor; PPAHV, Phorbol 12-phenylacetate 13 acetate 20-
homovanillate; RR, ruthenium red; VR1, Vanilloid receptor 1; VRL, Vanilloid receptor like
Introduction
Capsaicin, the pungent vanilloid agent in chilli peppers,
activates a subset of somatic and visceral primary a€erent
sensory neurones, notably the polymodal nociceptors (see
Bevan & Szolcsanyi, 1990). These have conduction velocities in
the C- and Ad-®bre range (see Szolcsanyi, 1993), are responsive
to noxious mechanical, thermal, and chemical stimuli and
convey nociceptive signals from the periphery to the spinal
cord. A vanilloid receptor (VR1) that is activated by capsaicin,
low pH solutions and temperatures greater than 428C has been
cloned from rat dorsal root ganglia (Caterina et al., 1997;
Tominaga et al., 1998). VR1 is a non-selective cation channel,
with high permeability for divalent cations, and is localized to a
subset of primary a€erent neurones (Caterina et al., 1997;
Helliwell et al., 1998). A second VR1 homologue termed the
Vanilloid Receptor-like protein-1 (VRL-1) (Caterina et al.,
1999) does not respond to capsaicin or protons but is sensitive
to heat at high temperatures with a threshold of about 528C and
it is possible that this channel may play a role in the
physiological response to noxious heat in vivo.
Studies in transgenic mice lacking the VR1 receptor
provide strong evidence for a role of VR1 in the sensation
of noxious heat in vivo. As well as losing vanilloid-evoked
pain behaviour, these mice showed reduced thermal hyper-
algesia in models of in¯ammatory pain, whereas responses to
mechanical stimuli were normal (Caterina et al., 2000; Davis
et al., 2000).
In addition to its sensitivity to capsaicin, protons and heat,
rat VR1 is activated by endogenous lipoxygenase products
that are likely to be expressed in in¯amed tissues (Hwang et
al., 2000) and high concentrations of anandamide, an
endogenous activator of the cannabinoid receptor, also
activate rat and human VR1 (Zygmunt et al., 1999; Smart
et al., 2000). Other in¯ammatory agents such as PGE2 which
do not directly activate VR1, sensitize the vanilloid receptor
to other noxious stimuli (Kress et al., 1997; Vyklicky et al.,
*Author for correspondence;
E-mail: Peter.McIntyre@pharma.novartis.com
Current addresses: 2Cardiovascular Research, The Rayne Institute,
St. Thomas' Hospital, Lambeth Palace Road, London, SE1 7EH,
U.K.; 3MRL San Diego, 5050 Coast Boulevard South, La Jolla, CA
92037, U.S.A.

P. McIntyre et al
Human and rat VR1 pharmacology
1085
1998). Furthermore, the hyperalgesic neurotrophin, nerve
growth factor (NGF), also acutely sensitizes the response of
adult rat sensory neurons to capsaicin, as well as having the
longer term e€ect of upregulating the expression of the
receptor itself (Bevan & Winter, 1995; Shu & Mendell, 1999).
VR1 is therefore able to detect and integrate multiple pain
stimuli, especially in in¯amed conditions when products of
lipoxygenase and cyclo-oxygenase pathways as well as NGF
are present at elevated concentrations. It is likely that drugs
that interfere with VR1 function will be analgesic.
at 458C) and the rat library was screened at high stringency
(0.26SSC at 658C). Positive cDNA clones were excised as
pBKCMV plasmid clones, in E. coli and sequenced on an
ABI 377 DNA sequencer (PE Applied Biosystems). Only the
longest clone was selected from the human library and
sequenced, but several clones were isolated and sequenced
from the rat library. A rat VR1 clone, which contained the
entire protein coding domain was used for further studies.
The sequence of the human VR1 has been deposited with
Genbank with the accession number: AJ 272063.
A number of other exogenous activators of vanilloid
receptors have been identi®ed. These include resiniferatoxin
(RTX) the ultrapotent agonist isolated from the plant
Euphorbia resinifera and its non pungent analogue, Phorbol
12-phenylacetate 13 acetate 20-homovanillate (PPAHV).
Studies with these compounds have suggested that their
interaction with the receptor may di€er signi®cantly from
that of capsaicin (Walpole et al., 1996; Liu, et al., 1998;
Szallasi et al., 1999; Jerman et al., 2000).
In vitro transcription
Copy RNA was prepared from Not1 linearized pBKCMV
DNA, with T3 RNA polymerase with a Stratagene mRNA
capping kit. The RNA was precipitated with ethanol and
rinsed before being resuspended in 10 ml of distilled water at
1 mg ml71
.
Here, we describe the cloning of a functional human VR1
homologue from human dorsal root ganglion (DRG) and a
comparison of its pharmacology with that of the rat VR1.
Both receptors were expressed in Xenopus oocytes and in
Chinese Hamster ovary (CHO) cells and characterized
electrophysiologically and by monitoring intracellular calcium
concentration changes either in cell populations with
aequorin luminescence or in individual cells by ratiometric
imaging of fura 2 ¯uorescence. Both receptors responded to
capsaicin, protons and heat. Although capsaicin had similar
potency at the two receptors, signi®cant pharmacological
di€erences were found between the rat and human VR1.
Oocyte preparation, injection and recording
Female Xenopus laevis were anaesthetized with tricaine by a
Home Oce approved procedure and their ovaries removed.
Following defolliculation with collagenase (type 1, Sigma) in
divalent cation free media (mM: NaCl 82.5, KCl 2.5,
Na2HPO4 1.2, HEPES 5, adjusted to pH 7.5 with NaOH)
mature stage
V and VI oocytes were injected with
approximately 50 nl of RNA (*1 mg ml71) and maintained
at 188C in ND96 solution (mM: NaCl 96, KCl 2, MgCl2 1,
CaCl2 1.8, HEPES 5, sodium pyruvate 2.5, adjusted to
pH 7.5 with NaOH), supplemented with 50 mg ml71 genta-
mycin, until required.
Recordings were made from oocytes bathed in ND96
solution pH 7.4 under two-electrode voltage clamp, 3 ± 5 days
following RNA injection, using a Geneclamp 500 ampli®er
and pClamp software (Axon Instuments). Electrodes were
pulled from standard borosilicate glass (Clark Electromedical
Instruments) and had resistance of about 0.2 MO when ®lled
with 3 M KCl. Capsaicin-induced currents were elicited at
room temperature, at a holding potential of 760 mV unless
otherwise stated. Oocytes were microperfused with ND96 and
drug-containing solutions.
Methods
Hank's balanced salt solution (HBSS), phosphate bu€ered
saline (PBS) and all cell culture reagents were obtained from
Gibco BRL. Geneticin (G418), ruthenium red, and capsaicin
were obtained from Sigma. All restriction enzymes were
obtained from New England Biolabs. Viewplates were
obtained from Packard Instruments Ltd. Capsazepine and
coelenterazine h were synthesized at Novartis. Phorbol 12-
phenylacetate 13-acetate 20-homovanillate (PPHAV) was
obtained from Alexis.
Generation of human and rat VR1 expressing CHO cell
lines
Cloning
Cells lines expressing aequorin have been used previously to
measure calcium ¯uxes (see Brini et al., 1995 for a description
of this technique).
Human DRG RNA was purchased from Analytical
Biochemical Services (MA, U.S.A.). Rat DRG RNA was
prepared from dorsal root ganglia that were isolated from
adult male Sprague-Dawley rats which had been killed by
CO2 asphyxiation using a Home Oce approved procedure
and were frozen on dry-ice. RNA was extracted by the
method of Chomczynski & Sacchi (1987). Poly A+ RNA was
puri®ed by oligo dT chromatography (Aviv & Leder, 1972).
Lambda ZAP express cDNA libraries were made with a
cDNA synthesis kit (Stratagene) according to the manufac-
turers instructions. The rat and human lambda ZAP
expressing DRG cDNA libraries were screened with a cDNA
989 bp probe which hybridized to part of the coding domain
of rat VR1 (Helliwell et al., 1998). The human library was
screened at low stringency (26 standard saline citrate (SSC)
An aequorin-expressing host cell line was generated. The
aequorin gene was subcloned into pXMT3 mammalian
expression plasmid which encodes the dihydrofolate reductase
gene. The aequorin cDNA and the pXMT3 plasmid were gifts
from Dr K. Stoekli and Dr S. Geisse, respectively, at Novartis,
Basle. CHO-DUKX cells were co-transfected with the
aequorin-pXMT3 construct and pCEP4 plasmid (Invitrogen)
which contains a hygromycin resistance gene. The transfected
cells were selected by growth in MEM medium containing
200 mg ml71 hygromycin (Gibco BRL) followed by further
selection in MEM alpha medium without nucleosides supple-
mented with dialysed foetal calf serum (Gibco BRL). Cell lines
were cloned from the selected population by limiting dilution.
British Journal of Pharmacology vol 132 (5)

1086
P. McIntyre et al
Human and rat VR1 pharmacology
The 3.3 kb ratVR1 cDNA insert was prepared for
expression in mammalian cells by removing it from the
pBK-CMV plasmid with EcoR1 and Not1 digestion and
ligating it into pIRESneo (Clontech). The human VR1 cDNA
insert of 3.5 kb was similarily subcloned into the same
expression vector. The VR1-pIRESneo constructs were
transfected into the CHO-aequorin host cell line by means
of Lipofectamine Plus (Gibco BRL). Following selecion in
medium containing 700 mg ml71 G418, the cell populations
were cloned by limiting dilution. Resulting clones were
screened in the aequorin assay and the highest expressing
clones of each species were chosen for further study.
NaCl, 5 KCl, 10 glucose, 10 HEPES, 2 CaCl2, 1 MgCl2)
adjusted to pH 7.4 with NaOH, to which capsaicin was
added. All solutions contained 0.1% dimethyl sulphoxide
(DMSO) as a solvent for capsaicin. Recordings were made
with borosilicate glass patch pipettes (2 ± 6 MO) ®lled with;
(mM) 130 CsCl, 1 CaCl2, 10 EGTA, 1 MgCl2, 10 HEPES)
adjusted to pH 7.4 with CsOH with an Axoclamp 200A
ampli®er and pClamp6 software (Axon instruments). Solu-
tion changes, including capsaicin application were made using
a U-tube system (Bevan & Winter, 1995). Experiments were
carried out at room temperature and only one cell was used
from each coverslip to minimize desensitization.
Aequorin assay
Imaging of intracellular calcium levels [Ca2+
]
i
Aequorin- and VR1- expressing cells were grown in 96-well
viewplates (Packard) and incubated at 378C with 20 mM
coelenterazine h and 30 mM reduced glutathione in 50 ml of
medium per well. At the start of the assay, the medium
containing coelenterazine h was removed and cells were
washed once with 100 ml of HBSS containing 75 mM CaCl2
and bu€ered to pH 7.4 with 10 mM HEPES. This was
replaced with 100 ml of the same bu€er containing test
compounds where appropriate. Cells were incubated for
10 min at room temperature. They were then placed in the
measuring chamber of a luminometer (Wallac Microbeta Jet).
Agonists, with the exception of PPAHV, were injected in a
volume of 20 ml HBSS at 6 fold ®nal concentration. Because
of its low solubility, PPAHV was added at twice the ®nal
concentration to 50 ml of cell-containing solution giving a
®nal volume of 100 ml. Unless otherwise stated in the text,
luminescence signals were integrated over 10 s for the rat
VR1 expressing cells and 20 s for the human VR1 expressing
cells, because of the di€erent kinetics of response at the two
receptors. Control experiments were carried out on the host
CHO-aequorin cell line which did not express VR1.
Measurements were made in quadruplicate. Results were
expressed as mean+s.e.mean and curve ®tting was performed
with Microsoft Origin software.
Cells grown on Poly-D-Lysine coated glass coverslips were
loaded with fura-2 AM (5 mM, 45 min), washed and
incubated at room temperature for at least a further 30 mins.
Coverslips were placed in a laminar ¯ow perfusion chamber
(Warner Instrument Corp.) and constantly perfused with
HEPES-bu€ered saline (2 mM Ca2+) via a local perfusion
pipette through which drugs and heated solutions were also
applied. Heat stimulations consisted of a linear increase (18C
s71) in perfusate temperature from 25 to 508C. Perfusate
temperature was controlled by a regulated Peltier device and
was monitored at the tip of the perfusion pipe by a miniature
thermocouple. Images of fura-2 loaded cells with the
excitation wavelength alternating between 340 and 380 nm
were captured with a cooled CCD camera (Photometrics).
Following subtraction of background ¯uorescence, the ratio
of ¯uorescence intensity at the two wavelengths was
calculated. Ratio levels in groups of 20 ± 40 individual cells
were analysed using ImageMaster sofware (PTI, NJ, U.S.A.).
Over the range of calcium concentrations measured, the ratio
values increased linearly with [Ca2+]. The magnitude of
responses is quoted as the maximum ratio increase over the
basal ratio levels immediately prior to stimulation, represent-
ing mean data from at least three separate experiments on
three di€erent passages of CHO cells.
In initial experiments with solutions containing 1.25 mM
calcium, the magnitude of the calcium signal evoked by
capsaicin was sucient to deplete most of the aequorin. In
order to avoid such saturation e€ects, aequorin measure-
ments with capsaicin as an agonist were carried out in bu€er
containing 75 mM calcium. The presence of unused aequorin
was con®rmed at the end of the assay by measuring the signal
produced by addition of 1% Triton X-100. In low pH
experiments, assay solutions with 75 mM calcium gave a very
small response to a pH 5 stimulus compared to the signal
evoked by 3 mM capsaicin. A proton-induced signal of a
similar magnitude to capsaicin was obtained when solution
containing 1.25 mM calcium was used so all pH experiments
were conducted in this higher calcium solution.
Statistical analysis
Experimental results are expressed as mean+s.e.mean and
the statistical signi®cance of any di€erences between pairs
were determined using two-tailed Student's t-test, unless
otherwise stated. Di€erences between multiple samples were
analysed using ANOVA.
Results
Cloning
Full-length cDNA clones were identi®ed from human and rat
DRG libraries by screening with part of the coding domain
of the rat VR1 (Helliwell et al., 1998). The human clone had
a 3463 base pair insert which encoded a novel 839 amino acid
protein that gave a predicted protein sequence with 85%
identity to the rat VR1 (Figure 1), and 47 and 48% identity
to the rat and human VRL-1 proteins respectively. Compar-
ison of the human and rat VR1 amino acid sequences (Figure
1) revealed that the ankyrin repeats and transmembrane
Electrophysiological recording
Human and rat VR1-expressing CHO cells grown on Poly-D-
Lysine coated glass coverslips were studied under whole-cell
voltage clamp. Steady state current was recorded with
membrane potential held at +30 mV to minimise the e€ects
of desensitisation (Yeats et al., 1992; Bevan & Docherty,
1993; Liu & Simon, 1996) in a standard solution (mM, 140
British Journal of Pharmacology vol 132 (5)

P. McIntyre et al
Human and rat VR1 pharmacology
1087
Figure 1 Alignment of the putative protein sequences of human and rat VR1. The predicted sequences of the human (above, this
paper) and rat (below, Caterina et al., 1997, Accession number AF029310) were aligned using the GCG GAP program (University
of Wisconsin, Genetics Computer Group). Solid vertical bars indicate identity, a colon indicates strong similarity, a period indicates
weak similarity and a gap indicates no similarity. Putative ankyrin repeats are underlined and predicted transmembrane domains are
in bold and underlined. The human VR1 sequence has been deposited in the EMBL database and been assigned accession number
AJ272063.
domains are conserved and that the C-terminus is more
highly conserved than the N-terminus.
tively. Di€erences in capsazepine potency were not related to
di€erent expression levels for human and rat VR1, since
capsaicin elicited currents of similar amplitude in oocytes
expressing either type of receptors (Figure 2d).
Properties of receptors expressed in oocytes
To determine whether the human cDNA clone encodes a
vanilloid receptor, RNA was transcribed and expressed in
Xenopus oocytes. Under two-electrode voltage clamp at a
holding potential of 760 mV, oocytes expressing the human
VR1 clone showed a capsaicin-evoked inward current. A
log(concentration)-response curve was constructed for both
the rat and human VR1 clones (Figure 2a) by sequential
application of increasing concentrations of capsaicin. The
curves were essentially superimposable with EC50 values of
1.9 mM (rat 1.9+0.2 mM, n=6; human 1.9+0.3 mM, n=10).
Current-voltage relationships of the capsaicin-evoked re-
sponses were generated by stepping to di€erent membrane
potentials from the holding value of 760 mV. The current
elicited at each potential was normalized to that recorded at
+80 mV. As shown in Figure 2b, the currents evoked by
capsaicin acting on both human and rat VR1 receptors showed
similar outwardly rectifying current-voltage relationships.
The competitive antagonist capsazepine inhibited the
capsaicin-evoked current at both receptors but, as shown in
Figure 2c, capsazepine was more potent at the human than
the rat receptor. To compare this di€erence in more detail
capsazepine concentration-inhibition curves were constructed
for the response to 1 mM capsaicin (Figure 2d). Although
there are not enough data points on the curves to calculate
precise IC50 values, capsazepine is clearly more potent at the
human than the rat VR1. Fitting curves to the data points
allowed us to estimate approximate IC50 values of 0.24+0.03
and 1.70+0.20 mM (n=9) for human and rat VR1 respec-
Properties of receptors expressed in CHO cells
For a more extensive pharmacological analysis, human and
rat VR1 were expressed in mammalian CHO cells and their
properties examined either electrophysiologically or by
measuring the evoked changes in [Ca2+]i.
Agonists
When studied electrophysiologically under whole cell voltage
clamp (Figure 3a) log(concentration)-response curves to
capsaicin (measured at +30 mV to minimize desensitization),
had similar EC50 values for the human (0.19+0.08 mM, n=4)
and rat (0.20+0.06 mM, n=4) VR1 (P40.5).
Figure 3b shows log(concentration)-response curves to
capsaicin for both rat and human VR1 in the CHO-aequorin
assay. The calculated EC50 values of 0.35+0.06 mM for the
rat and 0.53+0.03 mM for the human were not signi®cantly
di€erent (P40.1).
In the aequorin assay, PPAHV showed concentration-
dependent agonist activity on the rat VR1 with an EC50 value
between 3 and 10 mM but essentially no activity at the human
VR1 receptor at 10 mM under the assay condition used
(Figure 3c). In electrophysiological studies, a response of the
human VR1 to 3 mM PPAHV was detected at 760 mV but
this was about 20 fold smaller in magnitude than that of the
rat clone when scaled to the response to 300 nM capsaicin
(data not shown).
British Journal of Pharmacology vol 132 (5)

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P. McIntyre et al
Human and rat VR1 pharmacology
Figure 2 The human VR1 clone codes for a functional capsaicin receptor. Human and rat VR1 were expressed in oocytes. (a)
Capsaicin log (concentration)-response curve for the human clone (n=10) compared to that for the rat (n=6). Currents recorded at
760 mV by sequential application of increasing concentrations of capsaicin and normalized to the response elicited by 30 mM
capsaicin. (b) Current voltage relationships produced using voltage steps from the holding value of 760 mV in the presence of 1 mM
capsaicin, normalized to the current at +80 mV. Leak current in the absence of capsaicin subtracted (n=8, human; n=3, rat). (c)
Reversible antagonism of the response to 1 mM capsaicin by 300 nM or 10 mM capsazepine as indicated. Wash period of 5 min
indicated by gap in trace. (d) Percentage inhibition of the response to 1 mM capsaicin by increasing concentration of capsazepine,
expressed as a percentage of the response in the absence of antagonist (n=12 human; n=9 rat). The inset in (d) shows mean
currents with 1 mM capsaicin (values, 136+27 nA (n=12), and 112+23 nA (n=9)). This shows that the di€erences seen with
capsazepine are not due to di€erences in expression level.
Antagonists
individual pH-response curves showed that the EC50 value
for the rat VR1 (pH 5.78+0.09, n=11) was signi®cantly
higher than the value for the human VR1 (pH 5.49+0.04,
n=11, P50.001).
The competitive VR1 inhibitor capsazepine (CPZ) showed a 6
fold higher potency at the human receptor than at the rat
receptor when tested against EC50 concentrations of capsaicin
in the aequorin assay (Figure 4). The IC50 values of
0.039+0.004 mM and 0.22+0.02 mM were calculated for
human and rat expressing VR1 cells respectively. These
values were signi®cantly di€erent (P50.01). The e€ects of
another capsaicin antagonist ruthenium red (RR) were also
studied in the aequorin assay where ruthenium red showed a
2.3 fold greater potency at human VR1 (IC50 0.90+0.02 mM)
than at the rat VR1 (0.21+0.03 mM, P50.05).
When a maximally e€ective acidic pH (pH 5.0) was used
to stimulate VR1, capsazepine blocked the calcium response
to pH stimulation of human VR1 with an IC50 of
0.32+0.02 mM. In contrast capsazepine did not block the
pH5 activation of rat VR1 at concentrations of up to 30 mM
(Figure 5b). We tested the e€ect of increasing the activating
proton concentration on the inhibition by capsazepine, to
see if the inhibition of the low pH response was
surmountable, which would be indicative of competitive
inhibition, or non-surmountable, which would be indicative
of non-competitive inhibition. Capsazepine showed equal
ability to inhibit the responses of human VR1 to pH 4,
pH 4.5 or pH 5.0 solutions (see Figure 5c). The IC50 values
Protons
Low pH solutions activated both rat and human VR1. A
small di€erence in pH sensitivity was noted between species
(Figure 5a). In aequorin-expressing CHO cells, the threshold
for activation of the rat receptor was consistently higher than
the threshold for the human receptor. Analysis of the
of
0.22+0.02 mM
pH 4;
0.21+0.02 mM
pH 4.5;
0.19+0.02 mM pH 5 were not signi®cantly di€erent
(P40.05) which implies that capsazepine was not
competitive inhibitor of the low pH response.
a
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P. McIntyre et al
Human and rat VR1 pharmacology
1089
Figure 4 Capsazepine and ruthenium red inhibit capsaicin responses
in rat- and human-VR1 expressing CHO cells. (a) Capsazepine
inhibited the capsaicin response in CHO cells expressing aequorin
and either rat or human VR1. Various concentrations of capsazepine
were added to the cells for 10 min prior to the assay, then the
response was measured over 10 s (rat VR1) or 20 s (human VR1)
after the addition of an EC50 concentration of capsaicin. Capsazepine
e€ectively inhibited both rat VR1 and human VR1 responses to
capsazepine (IC50 values of 0.22+0.02 and 0.04+ 0.00 mM respec-
tively). Signal was normalized to that produced by the EC50
concentration of capsaicin with no antagonist as 100%, to allow
comparison between rat and human VR1 expressing cell lines. (b)
Ruthenium red inhibition of capsaicin responses was measured as in
(a). Ruthenium red was an e€ective blocker of capsaicin responses in
rat VR1 and human VR1 (IC50 values of 0.22+0.03 and
0.09+0.02 mM respectively).
Figure 3 Agonism of capsaicin and PPAHV in rat- and human-
VR1 expressing CHO cells measures using aequorin. (a) Capsaicin
log(concentration)-response curves for CHO cells stably expressing
human and rat VR1 clones. The current elicited by increasing
concentrations of capsaicin was measured at +30 mV and normal-
ized to that elicited by 10 mM capsaicin for each cell (n=4 for each).
EC50 values, 0.20+0.06 mM rat and 0.19+0.08 mM human. (b) CHO
cells expressing aequorin and either human or rat VR1 were
challenged with a range of capsaicin concentrations and the calcium
signal averaged over the ®rst 10 s for rat VR1 and the ®rst 20 s for
human VR1 cells. Graphs represent mean of three separate log
(concentration) response experiments. EC50 values were similar using
this protocol, 0.53+0.03 mM for human VR1 and 0.35+0.06 mM for
rat VR1. Results are expressed as the percentage of maximal
response. (c) CHO cells expressing aequorin and rat VR1 were
challenged with 1, 3 and 10 mM of PPAHV and the luminescence was
averaged over the ®rst 10 s. CHO cells expressing aequorin and
human VR1 were challenged with 10 mM PPAHV and the
luminescence was averaged over the ®rst 20 s. Results are expressed
as the percentage of maximal response to 3 mM capsaicin of cells
assayed in parallel.
Ruthenium red blocked the low pH activation of cells
expressing either rat or human VR1 (Figure 5d) with similar
IC50 values (rat 0.34+0.05; human 0.24+0.04, n=4).
Heat
Rat VR1 is activated by heat in the noxious range, from
42 ± 508C (Tominaga et al., 1998). We investigated the heat
response of CHO cells transfected with rat and human VR1
by measuring intracellular calcium concentrations in
individual cells using ratiometric imaging of fura-2 (Figure
6). An increase in perfusate temperature from 25 to 508C
(18 s71) stimulated substantial increases in [Ca2+]i in both
human and rat VR1-CHO cells, with similar average
magnitudes (average peak ratio increases of 3.8+0.2
(n=318) and 3.7+0.1 (n=239), respectively). These re-
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P. McIntyre et al
Human and rat VR1 pharmacology
Figure 5 Ability of Ruthenium Red and Capsazepine to block low pH-induced responses in rat- and human-VR1 expressing CHO
cells. (a) Low pH solutions stimulate uptake of Ca2+ in CHO cells that express aequorin and either rat or human VR1. Rat VR1 is
more sensitive to low pH with a signi®cant stimulation seen at pH 6.0 whereas human VR1 is not activated until pH is dropped to
5.5. CHO cells that express aequorin but not receptor show no calcium signal at low pH. (b) CHO cells that express aequorin and
either rat or human VR1were preincubated with a range of capsazepine concentrations and challenged with pH 5 bu€er as in Figure
4a. Capsazepine completely inhibited the human VR1 proton induced calcium signal (IC50=0.31+0.02 mM), but was ine€ective
against the rat VR1. Signal was normalized to pH5 signal with no antagonist as 100% to allow comparison between rat and human
VR1 expressing cell lines. (c) CHO cells that express aequorin and human VR1were preincubated with a range of capsazepine
concentrations and challenged with pH 4.0, pH 4.5 or pH 5 bu€er. Capsazepine was equally e€ective at blocking the proton-
induced response at each pH used. (d) Ruthenium red was e€ective at blocking pH 5-induced calcium signal in both rat and human
VR1 expressing cells.
sponses were non-linear with temperature. and showed a
clear threshold temperature at which the rate of [Ca2+]i rise
increased dramatically (Figure 6a,b). This was estimated to
be 42.4+0.38C for human VR1, similar to the estimate of
42.6+0.38C for rat VR1 (Savidge et al., 2001). These
speci®c heat responses were not seen in non-transfected
CHO cells.
As previously reported (Savidge et al., 2001), 1 and 10 mM
capsazepine produced almost complete blockade of responses
in rat VR1-CHO stimulated by 100 nM capsaicin
(99.8+0.4% inhibition with 10 mM capsazepine and 88+2%
with 1 mM capsazepine), but responses to heat were inhibited
by only 37+2% (n=119) by 10 mM capsazepine and 28+2%
(n=105) by 1 mM capsazepine. In contrast, heat responses in
Discussion
We have described the cloning of a novel cDNA sequence
which, from sequence homology and functional evaluation
is the human orthologue of the rat VR1. The protein
encoded by this sequence is a vanilloid receptor sensitive to
capsaicin and protons, which is consistent with a previous
study on cultured human DRG neurones (Baumann et al.,
1996) and it is also sensitive to heat. We have generated
CHO cell lines stably expressing rat or human VR1,
allowing us to carry out a detailed comparison of the
pharmacology of these species orthologues for the ®rst
time. After submission of this manuscript, Hayes et al.
(2000) reported cloning of a human VR1 orthologue. The
pharmacology of this receptor expressed in HEK293 cells is
consistent with our results.
cells expressing human VR1 showed a much greater
sensitivity to capsazepine blockade (Figure 6c). At a
concentration of 1 mM, capsazepine produced a reversible
blockade of heat responses (93+0.8%, n=75), which was
signi®cantly greater than the inhibition seen with the same
concentration of capsazepine on rat VR1 (Figure 6d,
P50.0001).
In the Xenopus oocyte and CHO cell expression systems,
the EC50 for capsaicin was the same for both the rat and
human VR1 although the potency was lower in the Xenopus
oocyte system than in CHO cells. A lower potency for
capsaicin at rat VR1 expressed in the Xenopus oocyte system
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P. McIntyre et al
Human and rat VR1 pharmacology
1091
Figure 6 E€ect of heat on intracellular calcium concentration ([Ca2+]i- expressed as ratio values) in VR1-expressing CHO cells. (a)
human and (b) rat VR1 CHO cells show a rapid increase in [Ca2+]i when the temperature rises above around 408C. Each trace on
graphs (a) and (b) represent a single cell in the same representative experiment. The estimated average threshold temperature at
which [Ca2+]i began to increase rapidly showed no di€erence between rat- and human- VR1. (c) Average [Ca2+]i increases in a
group of around 30 human VR1 transfected- CHO cells from a single representative experiment in response to heating to 508C (as
in a)) in the absence and presence of capsazepine (1 mM). Responses to heat in human VR1-CHO showed virtually complete and
reversible inhibition by capsazepine (1 mM). (d) Comparison of the e€ect of 1 mM capsazepine (CPZ) on the heat response in human
(®lled bars) and rat (open bars)- VR1 CHO cells. Heat responses in the presence of capsazepine, and those following capsazepine
washo€ for approximately 5 min (`Recovery') were normalized to an initial response to heat in the same cells (`Control'). Inhibition
of the heat response by capsazepine in rat VR1-CHO is markedly weaker than in human VR1-CHO.
compared to the mammalian cells was also seen by Caterina
and colleagues (1997) and this may represent some secondary
modi®cation of the receptors that di€ers in the two
expression systems.
are di€erences between the agonist binding site in the two
species.
Studies on the in vivo activity of capsaicin and PPAHV in
rat suggest that both compounds are capable of acting as
agonists of VR1, although some di€erences in their action
have been noted. PPAHV is less pungent than capsaicin and
although both compounds are capable of causing e€ective
desensitization against neurogenic in¯ammation in adult rats,
administration of PPAHV at a dose sucient to result in
desensitization of the neurogenic response does not result in a
hypothermia response that is normally seen with capsaicin
(Appendino et al., 1996).
Ruthenium red inhibited the responses of both rat and
human VR1 to capsaicin and low pH, and has also been
shown to inhibit the heat response in rat-VR1 CHO cells
(Savidge et al., 2001). In contrast, capsazepine showed
species-dependent inhibitory e€ects. We observed that
capsazepine could inhibit capsaicin activation of both human
Although human and rat VR1 expressing cells had
similar EC50 values for capsaicin, they responded di€er-
ently to PPAHV, which had an EC50 of between 3 and
10 mM at the rat VR1 but was essentially inactive up to a
concentration of 10 mM at the human VR1. This
compound is probably a full agonist at the rat VR1 as,
at 10 mM, it produced approximately 80% of the maximal
response seen with capsaicin at the rat receptor; however,
the lack of solubility of the compound prevented us from
acquiring reliable data at higher concentrations. The failure
to evoke robust responses with PPAHV at the human
VR1 in either electrophysiological or calcium assay
experiments implies a di€erence in either anity or ecacy
at rat and human VR1. This, in turn, suggests that there
British Journal of Pharmacology vol 132 (5)

1092
P. McIntyre et al
Human and rat VR1 pharmacology
and rat VR1 but that its potency on the human VR1 was
about 6 fold greater than on the rat VR1. Furthermore,
whilst both the human and rat VR1 channels could be
activated by protons, capsazepine blocked the low pH-evoked
responses of human but not rat VR1. Capsazepine was only a
weak inhibitor of the heat response mediated by rat-VR1, but
was highly e€ective at blocking the heat response mediated
by human-VR1.
region between transmembrane domains 5 and 6, towards the
outer side of the membrane, in rat VR1 a€ects the proton-
potentiation of the capsaicin response. Further evidence for
di€erent sites of interaction comes from mutation of
glutamate 648 in rat VR1 which abolishes the ability of
protons to activate the channel while leaving the responses to
capsaicin and heat una€ected.
The estimated IC50 value for capsazepine blockade of
capsaicin at the rat VR1 receptor was 0.22+0.02 mM which is
in good agreement with the value of 0.28 mM reported by
Caterina et al. (1997) for rat VR1 expressed in Xenopus
oocytes and is similar to the value of 0.42+0.05 mM obtained
using a calcium uptake assay of native receptors on cultured
rat DRG neurones (Bevan et al., 1992a). The value we
obtained is approximately 10 times higher than the pKB value
of 7.52+0.08 (28 nM) reported by Jerman et al. (2000). The
reason for this di€erence is not clear. In Xenopus oocytes
capsazepine was approximately six times more potent at
blocking capsaicin activation of the human than the rat
receptor, which is the same order of di€erence seen in the
mammalian expression system. The absolute values that we
see in the oocyte system are higher than observed in the
mammalian expression system and are also higher for rat
VR1 than the value observed by Caterina et al. (1997).
Di€erent experimental conditions for oocyte recordings could
account for the di€erence between the two groups.
Effect of capsazepine on the heat response of rat- and
human- VR1
In our hands 1 mM capsazepine completely inhibited the heat
response in CHO cells expressing human VR1, in contrast to
rat VR1 expressing cells where 10 mM capsazepine reduced
the response by only a third. The weak inhibitory e€ects of
capsazepine on heat responses on cells expressing rat VR1 are
similar to the ®ndings of Rang and colleagues that
capsazepine could inhibit heat responses only slightly, even
at 10 mM, in rat cultured dorsal root ganglion cells (Nagy &
Rang, 1999; Savidge et al., 2001). Treede and colleagues
(Kirschstein et al., 1999) found a similar degree of blockade
in cultured rat dorsal root ganglion cells using the same
(10 mM) capsazepine concentration. Thus, the pharmacology
of the heat reponse of recombinant rat VR1 appears to re¯ect
that of the native receptor, at least for capsazepine.
Tominaga et al. (1998) showed that 10 mM capsazepine
produced a more substantial blockade of the heat response
of rat VR1 expressed in HEK 293 cells. However, they also
saw a much more substantial rundown of the heat response
than we observed.
Effect of capsazepine on pH responses of rat- and
human- VR1 CHO cells
Capsazepine blocks voltage gated calcium channels
(Docherty et al., 1997) and also blocks activation of nicotinic
acetylcholine receptors (Liu & Simon, 1997). The concentra-
tions at which these e€ects are seen above 10 mM and
therefore do not explain the species selectivity that we
describe. Further work will be necessary to determine if the
e€ects of capsazepine on agonist activation and the e€ects of
this compound against protons are due to interactions at
separate sites.
Taken together, our data suggest that the site and
mechanism of activation of VR1 by capsaicin is di€erent
from that for protons and heat. Capsazepine, as well as being
a competitive antagonist at the capsaicin binding site, can
inhibit the proton activation of human but not rat VR1 in a
non-competitive manner. The identi®cation of a species-
speci®c e€ect of capsazepine will allow us to map the binding
site of capsazepine in the human VR1 using a chimeric
receptor approach, which in turn will provide important
information about the way in which the channel operates and
the mechanisms of capsazepine block.
Capsazepine failed to inhibit the responses of rat VR1 to low
pH at concentrations 100 times greater than the IC50 value at
the human VR1. This is in contrast to the results of Jerman
et al. (2000) who stated that capsazepine blocked low pH
activation of rat VR1 expressed in human embryonic kidney
cells, although these authors provided no data, and
Tominaga et al. (1998) who showed that 10 mM capsazepine
reversibly blocked the low pH response of rat VR1 in HEK
cells by 79% in calcium-free conditions. Our results are
consistent with the ®ndings of earlier studies on rat cultured
dorsal root ganglion cells which showed that capsazepine did
not inhibit the sustained inward current induced by acidic
(pH 6.1 or 5.5) extracellular solutions (Bevan et al., 1992b;
Vyklicky et al., 1998). In the current study, the block of
proton responses by capsazepine at human VR1 could not be
overcome by further lowering the pH (Figure 5c) suggesting
that capsazepine does not compete with protons for the same
binding site. This implies, in turn, that capsaicin and protons
bind to di€erent sites. Oh and colleagues have published
evidence that capsaicin binds to an intracellular site on the
native rat VR1 (Jung et al., 1999) whereas protons probably
exert their e€ect from outside the cell. A recent study by
Julius and colleagues (Jordt et al., 2000) has shown that
mutation of glutamate 600 in the putative pore forming
We thank Armand Wanner (Novartis, Switzerland) for help with
DNA sequencing.
British Journal of Pharmacology vol 132 (5)

P. McIntyre et al
Human and rat VR1 pharmacology
1093
References
APPENDINO, G., CRAVOTTO, G., PALMISANO, G., ANNUNZIATA,
R. & SZALLASI, A. (1996). Synthesis and evaluation of phorboid
20-homovanillates: discovery of a class of ligands binding to the
vanilloid (capsaicin) receptor with di€erent degrees of coopera-
tivity. J. Med. Chem., 39, 3123 ± 3131.
HWANG, S.W., KWAK, J.Y., LEE, S.-L., KANG, C.-J., JUNG, J., CHO, S.,
MIN, K.H., SUH, Y.G., KIM, D. & OH, U. (2000). Direct activation
of capsaicin receptors by products of lipoxygenases: Endogenous
capsaicin-like substances. Proc. Natl. Acad. Sci. U.S.A., 97,
6155 ± 6160.
AVIV, H. & LEDER, P. (1972). Puri®cation of biologically active
globin messenger RNA by chromatography on oligothymidylic
acid-cellulose. Proc. Natl. Acad. Sci. U.S.A., 69, 1408 ± 1412.
BAUMANN, T.K., BURCHIEL, K.J., INGRAM, S.L. & MARTENSON,
M.E. (1996). Responses of adult dorsal root ganglion neurones in
culture to capsaicin and low pH. Pain, 65, 31 ± 38.
BEVAN, S. & DOCHERTY, R.J. (1993). Cellular mechanisms of the
action of capsaicin. In Capsaicin in the study of pain. ed. Wood,
J.N., pp. 27 ± 44, London: Academic Press.
JERMAN, J.C., BROUGH, S.J., PRINJHA, R., HARRIES, M.H., DAVIS,
J.B. & SMART, D. (2000). Characterisation using FLIPR of rat
vanilloid recepor (rVR1) pharmacology. Br. J. Pharmacol., 130,
916 ± 922.
JORDT, S.-E., TOMINAGA, M. & JULIUS, D. (2000). Acid potentiation
of the capsaicin receptor determined by a key extracellular site.
Proc. Natl. Acad. Sci. U.S.A., 97, 8134 ± 8139.
JUNG, J., HWANG, S.W., KWAK, J., LEE, S.Y., KANG, C.J., KIM, W.B.,
KIM, D. & OH, U. (1999). Capsaicin binds to the intracellular
domain of the capsaicin-activated ion channel. J. Neurosci., 19,
529 ± 538.
KIRSCHSTEIN, T., GREFFRATH, W., BUSSELBERG, D. & TREEDE,
R.D. (1999). Inhibition of rapid heat responses in nociceptive
primary sensory neurons of rats by vanilloid receptor antago-
nists. J. Neurophysiol., 82, 2853 ± 2860.
KRESS, M., REEH, P.W. & VYKLICKY, L. (1997). An interaction of
in¯ammatory mediators and protons in small diameter dorsal
root ganglion neurons of the rat. Neurosci. Lett., 224, 1 ± 4.
LIU, L. & SIMON, S.A. (1996). Similarities and di€erences in the
currents activated by capsaicin, piperine and zingerone in rat
trigeminal ganglion cells. J. Neurophysiol., 76, 1858 ± 1869.
LIU, L. & SIMON, S.A. (1997). Capsazepine, a vanilloid receptor
antagonist, inhibits nicotinic acetylcholine receptors in rat
trigeminal ganglia. Neurosci. Lett., 228, 29 ± 32.
BEVAN, S., HOTHI, S., HUGHES, G., JAMES, I.F., RANG, H.P., SHAH,
& YEATS, J. (1992a). Capsazepine: a
K., WALPOLE, C.S.J.
competitive antagonist of the sensory neurone excitant capsaicin.
Br. J. Pharmacol., 107, 544 ± 552.
BEVAN, S., RANG, H.P. & SHAH, K. (1992b). Capsazepine does not
block the proton-induced activation of rat sensory neurones. Br.
J. Pharmacol., 107, 235P.
BEVAN, S. & SZOLCSANYI, J. (1990). Sensory neuron-speci®c actions
of capsaicin: Mechanisms and applications. Trends Pharmacol.
Sci., 11, 330 ± 333.
BEVAN, S.
& WINTER, J. (1995). Nerve growth factor (NGF)
di€erentially regulates the chemosensitivity of adult rat cultured
sensory neurons. J. Neurosci., 15, 4918 ± 4926.
BRINI, M., MARSAULT, R., BASTIANUTTO, C., ALVAREZ, J.,
POZZAN, T. & RIZZUTO, R. (1995). Transfected aequorin in the
measurement of cytosolic Ca2+ concentration ([Ca2+]i). J. Biol.
Chem., 270, 9896 ± 9903.
CATERINA, M.J., LEFFLER, A., MALMBERG, A.B., MARTIN, W. J.,
TRAFTON, J., PETERSEN-ZEITZ, K.R., KOLTZENBURG, M.,
BASBAUM, A.I. & JULIUS, D. (2000). Impaired nociception and
pain sensation in mice lacking the capsaicin receptor. Science,
288, 306 ± 313.
LIU, L., SZALLASI, A.& SIMON, S.A. (1998).
A non-pungent
resiniferatoxin analogue, phorbol 12-phenylacetate 13 acetate
20-homovanillate, reveals vanilloid receptor subtypes on rat
trigeminal ganglion neurons. Neurosci, 84, 569 ± 581.
NAGY, I. & RANG, H.P. (1999). Similarities and di€erences between
the responses of rat sensory neurons to noxious heat and
capsaicin. J. Neurosci., 19, 10647 ± 10655.
CATERINA, M.J., ROSEN, T.A., TOMINAGA, M., BRAKE, A.J.
&
SAVIDGE, J.R., RANASINGHE, S.P. & RANG, H.P. (2001). Compar-
ison of intracellular calcium signals evoked by heat and capsaicin
in cultured dorsal root ganglion neurons and in a cell line
expressing the rat vanilloid receptor, VR1. Neuroscience., 102
177 ± 184.
SHU, X. & MENDELL, L.M. (1999). Nerve growth factor acutely
sensitizes the response of adult rat sensory neurons to capsaicin.
Neurosci. Lett., 274, 159 ± 162.
SMART, D., GUNTHORPE, M.J., JERMAN, J.C., NASIR, S., GRAY, J.,
MUIR, A.I., CHAMBERS, J.K., RANDALL, A.D. & DAVIS, J.B.
(2000). The endogenous lipid anandamide is a full agonist at the
human vanilloid receptor (hVR1). Br. J. Pharmacol., 129, 227 ±
230.
SZALLASI, A., SZABO, T., BIRO, T., MODARRES, S., BLUMBERG,
P.M., KRAUSE, J.E., CORTRIGHT, D.N. & APPENDINO, G. (1999).
Resiniferatoxin-type phorboid vanilloids display capsaicin-like
selectivity at native vanilloid receptors on rat DRG neurons and
at the cloned vanilloid receptor VR1. Br. J. Pharmacol., 128,
428 ± 434.
JULIUS, D. (1999). A capsaicin receptor homologue with a high
threshold for noxious heat. Nature, 398, 436 ± 441.
CATERINA, M.J., SCHUMACHER, M.A., TOMINAGA, M., ROSEN,
T.A., LEVINE, J.D. & JULIUS, D. (1997). The capsaicin receptor: A
heat-activated ion channel in the pain pathway. Nature, 389,
816 ± 824.
CHOMCZYNSKI, P. & SATCCHI, . (1987). Single step method of RNA
isolation by acid guanidinium-phenol-chloroform extraction.
Anal. Biochem., 162, 156 ± 159.
DAVIS, J.B., GRAY, J., GUNTHORPE, M.J., HATCHER, J.P., DAVEY,
P.T., OVEREND, P., HARRIES, M.H., LATCHAM, J., CLAPHAM,
C., ATKINSON, K., HUGHES, S.A., RANCE, K., GRAU, E.,
HARPER, A.J., PUGH, P.L., ROGERS, D.C., BINGHAM, S.,
RANDALL, A. & SHEARDOWN, S.A. (2000). Vanilloid receptor-
1 is essential for in¯ammatory thermal hyperalgesia. Nature, 405,
183 ± 187.
DOCHERTY, R.J., YEATS, J.C. & PIPER, A.S. (1997). Capsazepine
block of voltage-activated calcium channels in adult rat dorsal
root ganglion neurons in culture. Br. J. Pharmacol., 121, 1461 ±
1467.
HAYES, P., MEADOWS, H.J., HARRIES, M.H., DUCKWORTH, M.D.,
CAIRNS, W., HARRISON, D.C., CLARKE, C., GUNTHORPE, M.,
ELLINGTON, K., PRINJHA, R.K., BARTON, A.J., MEDHURST,
A.D., SMITH, G.D., TOPP, S., MURDOCK, P., SANGER, G.J.,
TERRETT, J., JENKINS, O., RANDALL, A., BENHAM, C.D.,
SZOLCSANYI, J. (1993). Actions of capsaicin on sensory receptors. In
Capsaicin in the study of pain. ed. Wood, J.N., pp. 1 ± 26, London:
Academic Press.
TOMINAGA, M., CATERINA, M.J., MALMBERG, A.B., ROSEN, T.A.,
GILBERT, H., SKINNER, K., RAUMANN, B.E., BASBAUM, A.I. &
JULIUS, D. (1998). The cloned capsaicin receptor integrates
multiple pain-producing stimuli. Neuron, 21, 531 ± 543.
VYKLICKY, L., KNOTKOVA-URBANCOVA, H., VITASKOVA, Z.,
VLACHOVA, V., KRESS, M. & REEH, P.W. (1998). In¯ammatory
mediators at acidic pH activate capsaicin receptors in cultured
sensory neurons from newborn rats. J. Neurophysiol., 79, 670 ±
676.
GLOGER, I.S.
& DAVIS, J.B. (2000). Cloning and functional
expression of a human orthologue of rat vanilloid receptor-1.
Pain, 88, 207 ± 217.
HELLIWELL, R.J.A., MCLATCHIE, L.M., CLARKE, M., WINTER, J.,
BEVAN, S.
& MCINTYRE, P. (1998). Capsaicin sensitivity is
associated with the expression of the vanilloid (capsaicin)
receptor (VR1) mRNA in adult rat sensory ganglia. Neurosci.
Lett., 250, 177 ± 180.
British Journal of Pharmacology vol 132 (5)

1094
P. McIntyre et al
Human and rat VR1 pharmacology
WALPOLE, C.S.J., BEVAN, S., BLOOMFEILD, G., BRECKENRIDGE,
R., JAMES, I.F., RITCHIE, T., SZALLASI, A., WINTER, J.
ZYGMUNT, P.M., PETERSSON, J., ANDERSSON, D.A., CHUANG, H.,
SORGARD, M., DI, M.V., JULIUS, D. & HOGESTATT, E.D. (1999).
Vanilloid receptors on sensory nerves mediate the vasodilator
action of anandamide. Nature, 400, 452 ± 457.
&
WRIGGLESWORTH, R. (1996). Similarities and di€erences in
the structure activity relationships of capsaicin and resinifer-
atoxin analogues. J. Med. Chem., 39, 2939 ± 2952.
YEATS, J., BEVAN, S.J.
&
DOCHERTY, R.J. (1992). Calcium-
(Received August 24, 2000
Revised December 6, 2000
Accepted January 2, 2001)
dependent and independent desensitization of capsaicin-evoked
responses in voltage-clamped adult rat dorsal root ganglion
(DRG) neurones in culture. J. Physiol., 446, 390P.
British Journal of Pharmacology vol 132 (5)