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The symptoms of patients suffering from persistent trigeminalneuralgia may be alleviated by the induction of peripheral nerveblock with perineural injections of alcohol. This procedure is ini-tially painful due to an intense burning sensation1. Similarly,patients with esophagitis provide anecdotal evidence that the con-sumption of alcoholic beverages causes a burning pain propor-tional to the alcoholic strength of the drink. In addition, theburning pain produced by the application of alcoholic tincturesto skin wounds is familiar to all. Common to all these phenome-na is a “burning” sensation, which raises the possibility thatethanol may produce activity in the nociceptors responsible forsensing noxious heat. Consequently, we have studied the effect ofethanol upon vanilloid receptor-1 (VR1)2,3, which has been iden-tified previously as a receptor for noxious heat2. VR1—which ispredominantly expressed in afferent Aδ- and C-fibers2—is a poly-modal receptor4 that is activated by noxious heat (above ∼ 42ºC),extracellular acidic pH and some lipids—for example, anan-damide5,6—or 12- or 15-HPETE7. It is plausible that irritants thatelicit a “burning” sensation may do so by activating VR1 or anoth-er thermoreceptor. The studies we describe here, which used avariety of established methods for studying sensory neurons—including the measurement of neuropeptide release, elevation ofcytoplasmic Ca2+ concentrations in recombinant and native cellsystems, and electrophysiological recording—were done in order

Ethanol elicits and potentiatesnociceptor responses via thevanilloid receptor-1

M. Trevisani1,*, D. Smart2,*, M. J. Gunthorpe2,*, M. Tognetto3, M. Barbieri1, B. Campi3, S. Amadesi1, J. Gray2, J. C. Jerman4, S. J. Brough4, D. Owen2, G. D. Smith2, A. D. Randall2, S. Harrison1, A. Bianchi3, J. B. Davis2 and P. Geppetti1

1 Department of Experimental and Clinical Medicine, Headache Center, University of Ferrara, Via Fossato di Mortara 19, 44100 Ferrara, Italy2 Neurology-CEDD, GlaxoSmithKline, Third Avenue, Harlow CM19 5AW, UK3 Department of Pharmacology, University of Catania, Viale Andrea Doria 86, 95123 Catania, Italy4 Discovery Research, GlaxoSmithKline, Third Avenue, Harlow CM19 5AW, UK* These authors contributed equally to this work.

Correspondence should be addressed to J.B.D. (John_B_Davis@gsk.com)

Published online: 29 April 2002 DOI: 10.1038/nn852

The vanilloid receptor-1 (VR1) is a heat-gated ion channel that is responsible for the burningsensation elicited by capsaicin. A similar sensation is reported by patients with esophagitis whenthey consume alcoholic beverages or are administered alcohol by injection as a medical treatment.We report here that ethanol activates primary sensory neurons, resulting in neuropeptide release orplasma extravasation in the esophagus, spinal cord or skin. Sensory neurons from trigeminal or dor-sal root ganglia as well as VR1-expressing HEK293 cells responded to ethanol in a concentration-dependent and capsazepine-sensitive fashion. Ethanol potentiated the response of VR1 to capsaicin,protons and heat and lowered the threshold for heat activation of VR1 from ∼ 42ºC to ∼ 34ºC. Thisprovides a likely mechanistic explanation for the ethanol-induced sensory responses that occur atbody temperature and for the sensitivity of inflamed tissues to ethanol, such as might be found inesophagitis, neuralgia or wounds.

to establish whether a VR1-mediated mechanism may provide anexplanation for the painful burning sensations described above.

RESULTSNeuropeptide release and plasma extravasationVR1 is expressed on a subset of peptidergic nociceptors that areable to signal via the release of neuropeptides. Consequently, theactivation of VR1 leads to release of the neuropeptides substance-P (SP) and calcitonin gene–related peptide (CGRP)8. To inves-tigate the events underlying the generation of “burning”responses to ethanol, we first measured the release of SP fromdorsal spinal cord (Fig. 1a and b), esophagus (Fig. 1c and d) andskin (Fig. 1e and f), where central and peripheral endings of pri-mary afferents terminate. Ethanol (0.1–3%, equivalent to0.017–0.51 M) caused a concentration-dependent release of SP-like immunoreactivity (SP-LI) from the tissues, including skin(where exposure to similar concentrations of ethanol can beexpected during various treatments). Pretreatment with cap-saicin or removal of extracellular Ca2+ ions practically abolishedthe responses.

The inhibitory effect of capsaicin pretreatment indicated thatethanol causes neuropeptide release from the central (dorsalspinal cord) and peripheral (esophagus and skin) terminals ofcapsaicin-sensitive nociceptors in C- and Aδ-fibers, which are

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the major known source of SP in sensory neurons. The roleplayed by VR1 in this response was supported by the require-ment for extracellular Ca2+ ions, which suggested the involve-ment of a Ca2+-permeable ion channel, and by inhibition of theresponse by the VR1 antagonist capsazepine9 (10 µM). As wellas antagonizing VR110, capsazepine is reported to inhibit volt-age-gated calcium channels. However, capsazepine had no effecton KCl (80 mM)–mediated SP-LI release from the dorsal spinalcord (75 ± 7 fmol/g/20 min for capsazepine and 78 ± 8 fmol/g/20min for vehicle; in both cases, n = 5). These data indicated it wasunlikely that inhibition of voltage-gated channels contributed tothe inhibitory effect of capsazepine observed in our experiments.In addition, ethanol did not appear to be neurotoxic: capsaicin(1 µM) caused the release of SP-LI after pretreatment of dorsalspinal cord with 3% (0.51 M) ethanol (124 ± 10 fmol/g/20 min,n = 5) or vehicle (145 ± 12 fmol/g/20 min, n = 5). Similar resultswere obtained for ethanol-induced release of a second sensoryneuropeptide, CGRP (data not shown).

In various rat tissues, including esophagus11, stimulationof primary afferents and the subsequent release of SP causesan increase in plasma extravasation via the activation of

endothelial NK1 receptors. In the rat esophagus, a substantialincrease in Evans blue extravasation was caused by both ethanol(100 µl, 50% ethanol intra-esophageal instillation) and SP (0.1nmol/kg, intravenously). The NK1 receptor antagonistSR140333 significantly inhibited both responses, whereas cap-sazepine blocked only the effect of ethanol (Fig. 1g and h). Plas-ma extravasation (6.3 ± 1.1 ng/mg, n = 5) induced byintra-esophageal capsaicin (100 µl, 1 mM) was also inhibited bycapsazepine (2.3 ± 0.4 ng/mg, n = 5, P < 0.01). These data indi-cate that ethanol stimulated the release of tachykinins, and sub-sequent plasma extravasation, via VR1 activation.

Responses of primary sensory neurons The release of neuropeptides and the desensitizing effect of cap-saicin strongly suggested that the effect of ethanol upon esopha-gus, skin and dorsal spinal cord was mediated by peptidergicnociceptors that express VR1. To further dissect these sensoryresponses to ethanol, we studied primary neurons isolated fromtrigeminal and dorsal root ganglia (DRG), cultured at 37°C, usingCa2+ imaging (Fig. 2). In trigeminal ganglion neurons (TGNs),ethanol caused a concentration-dependent increase in cytosolicCa2+ ion concentration ([Ca2+]i) (Fig. 2a), which was inhibited bycapsazepine (10 µM) (Fig. 2b and c). A similar but less intenseresponse to ethanol was observed in DRG neurons; this was alsoinhibited by capsazepine (Table 1). Ninety-eight per cent of theTGN and DRG cells grown in culture responded to capsaicin(0.1–1 µM), which indicated that these were C- or Aδ-fiber noci-ceptors8. All the cells that responded to ethanol also respondedto capsaicin (data not shown). In contrast, a non-VR1-expressingcell type, human hepatoma (Hep G2) cells, showed a smallincrease in [Ca2+]i in response to ethanol; however, this minorresponse was not affected by capsazepine (Table 1). A propor-tion of the neuronal response to ethanol was capsazepine-insen-sitive, which indicated that ethanol may exert some effects viaadditional, VR1-independent mechanisms. Ethanol also facili-tated VR1 responses to other agonists. Ethanol 1% (0.17 M)increased the efficacy (Emax relative to control of 1.15 ± 0.03,n = 4, P < 0.01), but not the potency (pEC50 values of 7.15 ± 0.06versus 7.13 ± 0.05), of capsaicin-induced Ca2+ responses in DRGcells (Fig. 2d).

Recombinant VR1 responsesThe responses observed in isolated neurons confirmed that theeffect of ethanol upon ex vivo tissue preparations was mediatedvia the neurons in those tissues. However, both the native sys-tems express a diverse complement of neuronal receptors thatmight contribute to an ethanol-evoked response. If VR1 plays aprimary role in the ethanol-mediated response, as is suggestedby inhibition of the response by low extracellular Ca2+ concen-

Fig. 1. Ethanol-induced neurotransmitter release and plasma extravasa-tion. Release of SP-LI from (a) rat dorsal spinal cord, (c) esophagus and(e) skin was induced by increasing concentrations of ethanol. Theeffects of capsaicin (CAP) pretreatment (10 µM for 60 min beforeethanol addition), Ca2+-free medium plus 1 mM EGTA (–Ca2+) and 10µM capsazepine (CZP) on the SP-LI release that was induced by 3%ethanol from (b) rat dorsal spinal cord, (d) esophagus and (f) skin werealso assessed. Increases in plasma extravasation that were induced by(g) intra-esophageal instillation of 50% ethanol or (h) intravenous SP(0.1 nmol/kg) as well as the effect of 1 mg/kg of intravenous tachykininNK1 receptor antagonist SR140333 (SR) or intra-esophageal instillationof 100 µM capsazepine were also analyzed. Data are mean ± s.e.m. of atleast five experiments; *, P < 0.05.

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ethanol—were seen in VR1-expressing cells. The highest con-centration tested, 3% ethanol, caused a 165 ± 21% (n = 10)increase in the capsaicin-gated current (Fig. 3b). Under theseconditions, in which cells were maintained at room temperature,ethanol caused no stimulation when applied alone (Fig. 3c). Incontrast, when the fluorimetric imaging plate reader (FLIPR)platform was used to follow intracellular Ca2+ fluxes, we foundthat 1% (0.17 M) ethanol increased baseline [Ca2+]i slightly(Fig. 2f) and that significant responses to ethanol occurred at37°C (Fig. 2a and e).

These observations may be explained by the suggestion thatethanol is able to potentiate a wide range of VR1 activators,including heat. Varying ambient temperature between experi-mental systems might thus explain the changing degree ofresponse seen to ethanol alone. Thus, we next establishedwhether ethanol had similar effects upon activation of VR1 byprotons4, the endo-cannabinoid anandamide5,6 and heat (seebelow). Ethanol potentiated the activation of VR1 by protonsin a concentration-dependent manner analogous to that seenwith capsaicin and ethanol (Fig. 2g). Ethanol 1% increased the

pEC50 for protons from 6.26 ± 0.02 to 6.50 ± 0.03 (n = 5, P < 0.01) and the efficacy to 1.18 ± 0.02 (P < 0.01). The potentiating effect of ethanol wasobserved in whole-cell recordings. The application ofa pH 6 solution had little effect alone, but did activateVR1 when 3% (0.51 M) ethanol was coadministered(Fig. 3e). Similarly, 0.3% ethanol also enhanced theanandamide (1 µM)-induced activation of VR1, asmeasured by either FLIPR (12,801 ± 1,021 versus 4,625± 857 fluorescence intensity units (FIU), n = 3, P < 0.01) or electrophysiology (Fig. 3f). VR1-dependenteffects should be antagonized by recognized VR1 antag-onists. Both the ethanol-induced Ca2+ responses(Fig. 2e) and current responses to ethanol and capsaicin(Fig. 3a and b) were antagonized by capsazepine (pIC506.67 ± 0.02, n = 6) (Figs. 2b, c and 3g) and also by the

tration or capsazepine, then a VR1-dependent response toethanol should be obtained in a recombinant system that express-es VR1. At 37°C, 0.1–3% (0.017–0.51 M) ethanol caused amarked concentration-dependent [Ca2+]i increase in human VR1(hVR1)-expressing HEK293 cells that was practically absent inwild-type HEK293 cells (Fig. 2e). This showed that VR1 can beactivated by ethanol. The effect appeared to be relatively specificto VR1, as ethanol failed to enhance the endogenous carbachol-induced muscarinic Ca2+ response in the same cells (data notshown). Ethanol also enhanced the Ca2+ response to capsaicin (0.1nM to 10 µM) in a concentration-dependent manner (Fig. 2f); itincreased both the potency and efficacy of capsaicin. Ethanol0.3% (0.051 M) increased the potency of capsaicin from pEC508.22 ± 0.03 to 8.76 ± 0.05 (n = 4, P < 0.01) and the efficacy to1.18 ± 0.02 (P < 0.01). The modulatory effect of ethanol on hVR1responses was similarly observed in whole cell patch-clamprecordings done at 22–24°C (Fig. 3). In control experiments, cap-saicin alone and capsaicin that was applied with ethanol had noeffect on wild-type HEK293 cells (n = 7; Fig. 3a), whereas cap-saicin-gated currents—which were markedly potentiated by

Fig. 2. Ethanol stimulates and potentiates native andrecombinant VR1 responses. (a) Typical traces thatvisualize [Ca2+]i during exposure to ethanol and cap-saicin, at 37ºC, in cultured TGNs that were pretreatedwith (a) vehicle or (b) capsazepine. (c) Pooled data fromexperiments similar to those shown in (a). Eighty neuronswere analyzed; *P < 0.01 versus vehicle. (d) Cytoplasmic[Ca2+]i was monitored in DRG cells before and after theaddition of capsaicin (0.1 nM to 10 µM) in the absence() or presence () of 1% ethanol. Responses were mea-sured as peak increases in fluorescence minus basalamounts; they are expressed relative to a 1 µM capsaicincontrol (n = 4). (e) Ethanol induced a concentration-dependent Ca2+ response. [Ca2+]i was monitored at37ºC with Fura-2 in hVR1-expressing HEK293 (n = 86)and wild-type (Wt) HEK293 (n = 148) cells. Responseswere measured relative to the peak response to 5 µMionomycin. Ethanol enhancement of Ca2+ response to(f) capsaicin or (g) protons. [Ca2+]i was monitored withFluo-3 in hVR1-expressing HEK293 cells before and afterthe addition of capsaicin (0.1 nM to 10 µM) or HCl(0.4–1.5 mM) in the absence () or presence of 0.1% (), 0.3% () or 1% () ethanol. Responses were measured as peak increases in fluorescenceminus basal amounts and were expressed relative to 100 nM capsaicin (n = 6). (h) The ethanol-induced Ca2+ response is VR1-mediated. Fluo-3–loaded hVR1-expressing HEK293 cells were preincubated for 30 min with capsazepine (CZP, 1 µM), ruthenium red (RR, 0.3 µM), Rp-cAMP (Ch.Cl,10 µM), H89 (10 µM), BIM (1 µM), Ro318220 (10 µM) or chelerythrine chloride (10 µM). Rp-cAMP, H89, BIM, Ro318220 and Ch.Cl were added toblock kinase signaling pathways. Changes in [Ca2+]i were monitored before and after the addition of 3% ethanol. Responses were measured as peakincreases in fluorescence minus basal amounts and were expressed relative to a 100 nM capsaicin control (n = 3–8).

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Table 1. TGN and DRG neuron responses to ethanol are inhibitedby capsazepine.

Ethanol DRG DRG TGN TGN Hep G2 Hep G2concentration vehicle CPZ vehicle CPZ vehicle CPZ

0.05 M 5 ± 1 2 ± 1a 8 ± 2 1 ± 1a 7 ± 2 5 ± 2(0.3%)

0.17 M 17 ± 2 7 ± 2a 23 ± 2 9 ± 2a 12 ± 2 13 ± 2(1%)

0.51 M 32 ± 2 18 ± 2a 38 ± 3 21 ± 3a 17 ± 2 15 ± 3(3%)

Capsaicin 56 ± 4 9 ± 2a 71 ± 4 2 ± 2a ND ND

Data are shown as a percentage of the control ionomycin response. aP < 0.05 betweencells treated with capsazepine (CPZ, 10 µM) and vehicle (0.01% ethanol) . ND, no data.

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non-competitive, but less selective, pore-blocker rutheniumred (Figs. 2h and 3h). In addition, current–voltage relation-ships for currents elicited by capsaicin alone or capsaicin andethanol (Fig. 3d) showed pronounced outward rectificationand reversal potentials that were close to 0 mV (–3.7 ± 1.1 mV,n = 5), which are characteristics of VR1-mediated conductance.

VR1 accounts for the majority of the effect of ethanol onhVR1-expressing HEK293 cells and at least part of ethanol-evoked responses in primary neurons. Effects of alcohols on theactivity of other ion channels, including nicotinic, GABA andglycine receptor channels, have been described12. However, notall membrane proteins are affected; for example, no effect ofethanol upon the activity of endogenous muscarinic receptorswas found, as might have been expected if the effect were medi-ated via non-specific effects upon membrane biophysics. There isevidence for the existence of specific alcohol-binding sites13.Ethanol has also been linked to protein kinase C (PKC) translo-cation14 and, as PKC activation leads to an increased probabilityof VR1 opening15, we tested the effect of PKC inhibitors on theethanol-induced signal (Fig. 2h). We found that these inhibitorshad no effect, which suggests that PKC is not involved.

Effect upon thermal response threshold VR1VR1 is widely believed to be a physiological thermosensor capa-ble of converting noxious heat into the depolarization and firingof sensory neurons. The effect of ambient temperature upon thebasal response to ethanol alone is noted above. Thus, we deter-mined the effects of ethanol on the temperature responsiveness ofVR1 (Fig. 4). We found that ethanol had no effect on the small,heat-induced increases in leak currents in wild-type HEK293(Fig. 4a and c). In hVR1-expressing HEK293 cells, a well defined

heat-activated current was recruited at athreshold of ∼ 42°C; this was accompa-nied by a large increase in current noise,which was attributable to the activationof a channel of high single channel con-ductance (Fig. 4b). This basal thresholdfor VR1 activation at ∼ 42°C1,3 was con-sistent with the activity of the endoge-nous heat-receptor in DRG neurons16,17.Ethanol 3% caused a large shift in thethreshold of VR1 activation, so thatchannel activity attributable to VR1was observed at temperatures as lowas 34°C. At higher temperatures,much greater activation of VR1 wasalways seen in the presence of ethanol(Fig. 4b and c).

DISCUSSIONOur data demonstrate the ethanol-mediated modulation of VR1 function.Ethanol (0.1–3%) caused the potentia-tion of VR1 activity produced by vanil-loids, anandamide, protons or heat.These concentrations were lower thanthose found in the alcoholic beveragesor medications (up to 30% ethanol) towhich wounds or damaged tissues maybe exposed18. Shifts in the heat-medi-ated VR1-gating range were consistentwith the induction of VR1 activity atnormal body temperature. This pro-

vides further evidence that modulatory factors—for exampleethanol or bradykinin19—are capable of sensitizing VR1 so thatbody temperature is sufficient to activate VR1. The ability ofethanol to affect C- and Aδ-fiber nociceptors is emphasized byits capacity to release SP from the central and peripheral terminalsof these neurons and to cause capsazepine-sensitive excitatoryeffects in isolated neurons from sensory ganglia. Such a sensiti-zation mechanism may contribute to the persistent pain thatresults from inflammatory or neuropathic injury. A further impli-cation of these results is that interpretations of the effects ofethanol on somatic and visceral tissues now require considera-tion of the potential contributions of VR1. In addition, becauseof the polymodal nature of VR1, these considerations must alsotake into account the local temperature, pH and presence of anyendogenous activators of VR1. These factors may vary greatlybetween normal and damaged or inflamed tissues, whosepathologies engage VR1 function20,21.

Our data on the VR1-dependent release of neuropeptidesfrom esophagus—together with reports of the upregulation ofVR1 expression in inflamed bowel22 and enhanced sensitivity tocapsaicin in modeled esophagitis23—highlight a possible role ofVR1 in visceral pain, where tissue injury has increased exposureof sensory nerve endings. Our findings provide the followingmechanistic explanation for the burning pain, described bypatients, that ethanol evokes18: exposure to ethanol may suffi-ciently lower the threshold of VR1 receptors, which are recruitedby inflammation, so that they become activated at body temper-ature or via the activators mentioned above19. These parallelsbetween in vitro experiments and clinical symptoms encouragethe further study of VR1 for the development of treatments forpainful wounds and inflamed tissues.

Fig. 3. Ethanol modulates capsaicin-, anandamide- or proton-gated inward currents recorded fromhVR1-expressing HEK293 cells. (a) Sample traces that resulted from the application of 500 nM cap-saicin alone (filled bar) or with 3% ethanol (open bar). (b) Percentage potentiation of the 500 nM cap-saicin response induced by 0.3–3% ethanol at 22–24ºC (n = 4–10). (c) At 22–24°C, hVR1-expressingHEK293 cells responded to 1 µM capsaicin but not to 3% ethanol (n = 5). (d) Current-voltage rela-tionship for capsaicin-gated hVR1 currents and ethanol-potentiated, capsaicin-gated currents thatwere assessed with a voltage-ramp protocol (–70 to +70 mV in 1 s). The effects of ethanol on the cur-rent elicited by endogenous VR1 ligands were also examined. (e) Acid (pH 6) was applied alone orwith 3% ethanol and then blocked by 10 µM capsazepine (CZP); data from three samples were pooled.(f) Anandamide (AEA, 1 µM) was applied alone or with 3% ethanol; data from five samples werepooled. The antagonistic effect of (g) 10 µM capsazepine (hatched bar, n = 4) and (h) 10 µM ruthe-nium red (RR, shaded bar, n = 4) on 3% ethanol potentiated 500 nM capsaicin (filled bar) currents.(a–h) The vertical current calibration bar corresponds to 200 pA.

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METHODSPeptide-release assays. Thick slices (∼ 0.4 mm) of the dorsal horn of lum-bar enlargements of the dorsal spinal cord, esophagus or skin from theshaved dorsum of rats were stabilized for 60 min in Kreb’s solution at37°C. Fractions (4 ml) were collected at 10-min intervals into ethanoicacid (final concentration 2 N) before, during and after administrationof ethanol. Fractions were freeze-dried, reconstituted with assay bufferand analyzed for CGRP and SP immunoreactivities as described24.

Plasma extravasation. Male Sprague-Dawley rats were anesthetized(pentobarbital 60 mg/kg, intraperitoneally). A cannula was insertedinto the oral part of the esophagus and the aboral ending of the esoph-agus was ligatured. One minute after injection of the dye Evans blue(30 mg/kg) into the jugular vein, 50% ethanol (100 µl), its vehicle (100µl of 0.9% saline) or 1 mM capsaicin (100 µl in 5% ethanol) were giventhrough the esophageal cannula; alternatively, SP was administeredintravenously. Vehicles did not cause any measurable plasma extrava-sation (data not shown). Capsazepine, administered via the esophagealcannula, intravenous SR140333 or a combination of their vehicles (1%intra-esophageal ethanol and 10% intravenous DMSO, respectively)were administered 15 min before the dye was injected. Rats were killed5 min after SP or ethanol were administered and the esophagus wasremoved, weighed and incubated in formamide for 24 h at 37°C.Extravasated Evans blue was measured spectrophotometrically at 620nm. Ethical approval for animal work was obtained from the local Uni-versity of Ferrara ethics committee.

Cell culture. DRG and TGNs were taken from 1–3-day-old rats and cul-tured as described20,24. The human hepatoma cell line (a gift of F. Bernar-di, University of Ferrara) was grown in Dulbecco’s modified Eagle’smedium/F12.

Measurement of changes in [Ca2+]i. [Ca2+]i was determined with theCa2+ -sensitive fluorescent dye Fura-2, which was loaded into Hep G2cells or sensory neurons as its acetoxymethylester form (40 min, 37°C).The bath solution consisted of 1.4 mM CaCl2, 5.4 mM KCl, 0.4 mMMgSO4, 135 mM NaCl, 5 mM D-glucose, 10 mM HEPES and 0.1% bovineserum albumin at pH 7.4. Alternate excitation at 340 nM and 380 nM wassupplied and the F340/F380 emission ratio recorded with a dynamic imageanalysis system (Laboratory Automation 2.0, RCS, Florence, Italy). Ratiochanges were expressed as a percentage of the peak response to iono-mycin (5 µM)24. After 10 min of stabilization, responses to increasingconcentrations of ethanol (0.1–3% or 0.017 – 0.51 M) and capsaicin (0.1µM) were recorded in the presence of capsazepine (10 µM) or its vehicle.In some experiments, plated neurons were pre-exposed to capsaicin (10µM) for 60 min to desensitize them. [Ca2+]i was also monitored withFLIPR (Molecular Devices, Wokingham, UK), a high-throughput plat-form for the fluorimetric measurement of intracellular Ca2+ concentra-tions, as described6. Briefly, hVR1-expressing HEK293 cells loaded withFluo-3 (4 µM; Teflabs, Austin, USA) were incubated at 25°C for 2 h, whiledissociated rat DRG cells that had been seeded into 384 plates (10,000cells per well) were incubated with Fluo-4 (2 µM) at 37°C for 1 h. Mod-ulating agents were preincubated with the cells for 30 min at 25°C in

Tyrode buffer. A FLIPR was used to monitor cell fluorescence (λex = 488nm, λem = 540 nm) before and after the addition of agonists. Responseswere measured as peak minus basal FIU and, where appropriate, wereexpressed as a fraction of the maximum capsaicin-induced response.Curve-fitting and parameter estimation were done with Graph Pad Prism3.00 (GraphPad Software, San Diego, California).

Whole cell patch-clamp electrophysiology. All recordings were madewith standard whole-cell patch-clamp methods, as described3; theywere done at room temperature (22–24ºC) unless otherwise stated. Theextracellular solution consisted of 130 mM NaCl, 5 mM KCl, 2 mMCaCl2, 1 mM MgCl2, 30 mM glucose and 25 mM HEPES-NaOH at pH7.3. Patch pipettes (resistance 2–5 MΩ) were filled with 140 mM CsCl,4 mM MgCl2, 10 mM EGTA and 10 mM HEPES-CsOH at pH 7.3.Drugs were applied with an automated perfusion device (time for solu-tion exchange ∼ 30 ms). Temperature jump experiments were done asdescribed3. Data are presented as mean ± s.e.m.

Ethanol (0.01%), at a concentration that did not produce anydetectable release of neuropeptides or mobilization of calcium (datanot shown), or DMSO (0.1%) vehicles were used for the delivery ofdrugs in in vitro studies. Statistical comparisons were made whereappropriate with Student’s t-tests or the ANOVA and Dunnett’s test.

AcknowledgmentsWe thank C. Farrant and S. Lomax for preparation of artwork. This work was

supported in part by ARCA (Padua) and Cofin (MIUIR, Rome).

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 26 NOVEMBER 2001; ACCEPTED 25 MARCH 2002.

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Fig. 4. Ethanol shifts the threshold for VR1 heat activation. Whole cellinward currents were induced by a shift (gray bar) in temperature from24ºC to the indicated temperatures in (a) wild-type HEK293 cells and(b) hVR1-expressing HEK293 cells in the absence or presence of 3%(0.51 M) ethanol (open bar). (c) Pooled data from experiments similarto those shown in (a) and (b) were used to define the temperatureresponse profile for heat-evoked currents in wild-type (squares) orVR1-expressing (circles) cells either in the presence (filled symbols) orabsence (open symbols) of 3% ethanol. Asterisks indicate the lowesttemperature at which the VR1 heat-activated current—in the absence(*) or presence (**) of ethanol—were significantly different (n = 7, P < 0.05 by Student’s unpaired t-tests) to control recordings forHEK293 cells.

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