JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 JPETThis Fast article Forward. has not been Published copyedited and on formatted. June 9, The 2005 final asversion DOI:10.1124/jpet.105.084277 may differ from this version.

JPET #84277

TITLE:

5-Iodoresiniferatoxin evokes hypothermia in mice and is a partial TRPV1 in vitro

Isao Shimizu1, Tohko Iida1, Nobuhiko Horiuchi, and Michael J. Caterina Downloaded from

Departments of Biological Chemistry and Neuroscience

Johns Hopkins School of Medicine, Baltimore, MD 21205 USA (I.S., T.I., and M.J.C.) jpet.aspetjournals.org and Dainippon Pharmaceutical Company, Limited, Suita/Osaka 564-0053 Japan (I.S. and

N.H.)

at ASPET Journals on September 27, 2021

1

Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

RUNNING TITLE: IRTX activates TRPV1 in vivo and in vitro

Address correspondence to: Michael J. Caterina, M.D., Ph.D., Associate Professor,

Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe

Street, Baltimore, MD 21205

email: [email protected]. Downloaded from

Phone: (410) 502-5457

FAX: (410) 955-5759 jpet.aspetjournals.org

Number of text pages: 31 (including title pages and figure legends)

Number of tables: 0 at ASPET Journals on September 27, 2021

Number of figures: 4

Number of References: 33

Words in Abstract: 149

Words in Introduction: 607

Words in Discussion: 1289

Nonstandard Abbreviations: TRPV1, transient receptor potential vanilloid 1; I-RTX,

5-iodoresiniferatoxin,

(6,7-deepoxy-6,7-didehydro-5-deoxy-21-dephenyl-21-(phenylmethyl)-daphnetoxin,20-(4-h ydroxy-5-iodo-3-ethoxybenzeneacetate,; RTX, ,

6,7-deepoxy-6,7-didehydro-5-deoxy-21-dephenyl-21-(phenylmethyl)-daphnetoxin,20-(4-hy

2 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

droxy-3-ethoxybenzeneacetate; DMSO, dimethylsulfoxide; HEK, human embryonic kidney; ANOVA, analysis of variance; CGRP, calcitonin gene-related peptide; 2I-RTX,

2-iodoresiniferatoxin; CAP, ; CPZ, capsazepine.

Recommended Section Assignment: Neuropharmacology Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

3 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

ABSTRACT

Transient receptor potential vanilloid 1 (TRPV1) is a capsaicin- and heat-gated ion channel required for normal in vivo responses to these painful stimuli. However growing evidence suggests that TRPV1 also participates in thermoregulation. We therefore examined the effects of a selective TRPV1 antagonist, 5-Iodoresiniferatoxin (I-RTX), on mouse body

temperature. Surprisingly, subcutaneous administration of I-RTX (0.1 - 1 µmol/kg) Downloaded from evoked a hypothermic response similar to that evoked by capsaicin (9.8 µmol/kg) in naïve wild-type mice, but not in mice pretreated with resiniferatoxin, a potent TRPV1 agonist, or jpet.aspetjournals.org in naïve TRPV1 null mice. In response to I-RTX in vitro, HEK293 cells expressing rat

2+ TRPV1 exhibited increases in intracellular Ca (biphasic, EC50 = 56.7 nM and 9.9 µM)

2+ that depended on Ca influx and outwardly rectifying, capsazepine-sensitive, currents that at ASPET Journals on September 27, 2021 were smaller than those evoked by 1µM capsaicin. Thus, I-RTX induces

TRPV1-dependent hypothermia in vivo and is a partial TRPV1 agonist in vitro.

4 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

INTRODUCTION

Transient receptor potential receptor vanilloid 1 (TRPV1) is a non-selective cation channel whose expression is most prominent in a subset of small- to medium-diameter sensory neurons and that can be activated by pungent vanilloid compounds such as capsaicin. Alternatively, TRPV1 can be gated by noxious heat (> 42°C), extracellular

acidity (pH < 6), or certain endogenous lipids such as endocannabinoids (Caterina et al., Downloaded from

1997; Caterina and Julius, 2001). Mice lacking TRPV1 are deficient in responsiveness to vanilloids and noxious heat, as well as in inflammation-induced thermal hyperalgesia jpet.aspetjournals.org (Caterina et al., 2000; Davis et al., 2000). A number of sometimes-conflicting observations have led to speculation that TRPV1 also participates in thermoregulation.

For example, capsaicin evokes robust hypothermia in rodents when administered either at ASPET Journals on September 27, 2021 systemically or directly into the preoptic/anterior hypothalamus (Jancso-Gabor et al.,

1970a; Jancso-Gabor et al., 1970b; deVries and Blumberg, 1989), but evokes no change in body temperature in mice lacking TRPV1 (Caterina et al., 2000). Basal body temperature in mice lacking TRPV1 has been reported by some to be normal (Caterina et al., 2000; Iida et al., 2005) but by others to exhibit exaggerated circadian fluctuations (Szelenyi et al.,

2004). Furthermore, rodents that have been systemically desensitized with capsaicin exhibit impaired tolerance to elevated ambient temperature (Jancso-Gabor et al., 1970a;

Szolcsanyi, 1983; Szelenyi et al., 2004). There is also growing evidence that TRPV1 is important for normal fever generation in response to endotoxin (Szekely and Szolcsanyi,

1979; Dogan et al., 2004; Iida et al., 2005). Capsazepine, the most commonly used

TRPV1 antagonist, has not been reported to produce changes in either basal body

5 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

temperature or thermal tolerance, but does appear to block the initial phase of endotoxin-induced fever (Dogan et al., 2004). However, interpretation of these results is made difficult by capsazepine’s relatively low in vivo potency at rodent TRPV1 (McIntyre et al., 2001; Phillips et al., 2004) and the fact that this compound can inhibit other ion channels, besides TRPV1 (Liu and Simon, 1997; Ray et al., 2003). Thus, the precise

relationship between TRPV1 and thermoregulation, while tantalizing, remains unclear. Downloaded from

One approach that might facilitate our understanding of this relationship would be to examine the effects on thermoregulation of more potent and selective TRPV1 antagonists. jpet.aspetjournals.org Resiniferatoxin

(6,7-deepoxy-6,7-didehydro-5-deoxy-21-dephenyl-21-(phenylmethyl)-daphnetoxin,20-(4-h ydroxy-3-ethoxybenzeneacetate; RTX) is an ultrapotent vanilloid receptor agonist produced at ASPET Journals on September 27, 2021 by plant species of the genus Euphorbia. This compound binds to and activates both native and recombinant TRPV1 with high potency (Kd 20 - 100 pM, EC50 1 - 50 nM)

(Szallasi and Blumberg, 1990; Caterina et al., 1997; Szallasi et al., 1999). Recently, a form of RTX iodinated at the 5’ position of the vanilloid moiety

(6,7-deepoxy-6,7-didehydro-5-deoxy-21-dephenyl-21-(phenylmethyl)-daphnetoxin,20-(4-h ydroxy-5-iodo-3-ethoxybenzeneacetate ; 5-iodoresiniferatoxin; I-RTX) was shown to be a

TRPV1 ligand and modulator with extremely interesting properties (Wahl et al., 2001).

Like RTX, I-RTX potently binds TRPV1 and displaces other vanilloid ligands from this channel (Ki = 4.8 ± 0.6 nM at native rat TRPV1, 5.8 ± 1.1 nM at recombinant rat TRPV1).

In contrast to RTX, however, I-RTX was found to inhibit capsaicin-evoked current responses in Xenopus oocytes expressing TRPV1 (Wahl et al., 2001). Independent

6 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

studies revealed that I-RTX could block not only vanilloid-evoked TRPV1 responses, but also those evoked by protons or heat, both in cells transfected with recombinant TRPV1 and in sensory neurons expressing native TRPV1 (Seabrook et al., 2002; Rigoni et al.,

2003; Correll et al., 2004). Given these properties, we sought to determine the effects of

I-RTX on mouse body temperature regulation. Unexpectedly, we found that I-RTX, like

RTX and capsaicin, could evoke robust, dose-dependent hypothermia that was dependent Downloaded from on the presence of TRPV1. We further found that, in vitro, I-RTX exhibits partial agonist activity at recombinant rat TRPV1. jpet.aspetjournals.org

METHODS

at ASPET Journals on September 27, 2021

Experimental Animals and Chemical regents

All procedures were approved by The Johns Hopkins Animal Care and Use

Committee. Wild-type C57BL/6 mice were obtained from the Jackson Laboratory (Bar

Harbor, ME). TRPV1-/- mice, backcrossed against a C57BL/6 background were described previously (Caterina et al., 2000). Mice were housed at 24-25°C on a 12 hr light (08:00 -

20:00)/ 12 hr dark photocycle and provided with food and water ad libitum. All experiments were conducted on age-matched male mice 2 - 4 months old, between the hours of 10:00 and 19:30.

Cell culture reagents were obtained from Invitrogen (Carlsbad, CA) and other chemicals, including I-RTX, were from Sigma (St. Louis, MO) unless otherwise noted.

For body temperature measurements, I-RTX, RTX, capsaicin, and capsazepine were

7 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

dissolved in a solution of 10% , 10% Tween 80 in pyrogen-free saline, and were injected subcutaneously at 0.1 ml/ 10 g body weight. For in vitro studies, RTX, I-RTX, capsaicin and capsazepine stocks were dissolved in ethanol or dimethylsulfoxide (DMSO) and diluted daily to their final working concentrations.

Body temperature measurements Downloaded from

For rectal temperature measurements, mice were placed in individual cages at room temperature (22 - 25°C). Rectal temperatures were recorded from awake mice by jpet.aspetjournals.org repeated insertion of a lubricated rectal thermocouple probe (model RET-3, Physitemp,

Clifton, NJ) prior to and following subcutaneous (s.c.) injection of capsaicin, RTX or

I-RTX. In the experiment shown in Fig. 1C, after the final rectal temperature recording, at ASPET Journals on September 27, 2021 mice injected with RTX were given food and water. The following day, temperature measurements were resumed prior to I-RTX administration.

Peritoneally-implanted telemetric temperature probes (MiniMitter, Bend OR) were used for the core body temperature measurements shown in Fig. 1E. Mice were deeply anesthetized with ketamine (80 mg/kg) and xylazine (8 mg/kg), administered intraperitoneally (i.p.). A small incision was made under aseptic conditions in the lower peritoneal cavity and a sterilized probe inserted into the cavity and sutured to the abdominal wall. Abdominal musculature and skin were closed separately with 4-0 silk (U.S. Surgical,

Norwalk, CT), and antibiotic ointment applied. Mice were rested ≥ 6 days prior to experiments. During the experiment, mice in individual cages were placed on telemetric receiver platforms (Minimitter, Bend OR) and their body temperatures were monitored

8 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

continuously using Vital View software (Minimitter, Bend OR).

Calcium imaging and electrophysiology

HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, penicillin, streptomycin, and L-glutamine. HEK293

cells stably transfected with cDNA encoding rat TRPV1 or control vector, pCDNA3 Downloaded from

(Guler et al., 2002), were subjected to Ca2+ imaging and whole-cell voltage-clamp recording. Fluorimetric Ca2+ imaging was performed as described (Guler et al., 2002) using jpet.aspetjournals.org an inverted microscope (Nikon, Melville, NY), fluorescent light source (DG4, Sutter

Instruments, Santa Rosa, CA), and CCD camera (Roper, Tuscon, AZ). Bath solution for imaging contained (in mM) 130 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 10 HEPES, 1.2 at ASPET Journals on September 27, 2021

NaHCO3, and 10 glucose, adjusted to pH 7.45 with NaOH. Cells were loaded with 10 µM fura 2-acetomethoxy ester (Molecular Probes, Eugene, OR) in bath solution containing

0.02% pleuronic acid (Molecular Probes, Eugene, OR) at 37°C for 40 min, then rinsed.

The ratio of 510 nm emission at 340 nm/380 nm excitation was measured using Ratiotool software (Isee Imaging, Raleigh NC). For whole-cell voltage clamp, recording pipettes were filled with internal solution containing (in mM) 140 NaCl, 0.1 MgCl2, 5 EGTA, 10

HEPES, pH 7.4 (adjusted with NaOH). Cells were superfused with bath solution containing (in mM) 140 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, pH 7.4

(adjusted with NaOH). After establishment of the whole-cell configuration, bath solution was switched to a calcium-free bath solution, in which CaCl2 was replaced with 5 mM

EGTA. I-RTX in Ca2+-free bath solution was prepared from stocks prepared in ethanol

9 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

(6.63 mM) or DMSO (13.25 mM) for Ca2+ imaging and electrophysiology, respectively, and added to a fixed-volume recording chamber by pipet. An Axopatch200B amplifier and pCLAMP 9.0 software (Axon Instruments, Union City, CA) were used. Borosilicate glass electrodes had tip resistances of 1.5 – 2.0 MΩ. Series resistance was typically less than 10

MΩ, without compensation. A KCl-agar bridge was used. No correction was made for

liquid junction potential, given that it was less than 1.0 mV. Downloaded from

High Performance Liquid Chromatography jpet.aspetjournals.org HPLC analysis was performed using a SCL-10A system controller, LC-10AS liquid chromatograph, C-R7A plus chromatopac, and SPD-10A UV-VIS detector (Shimadzu,

Kyoto, Japan) together with a Capcell PAK C18 SG120 separation column (4.6 X 150 mm; at ASPET Journals on September 27, 2021

Shiseido, Tokyo, Japan). The mobile phase consisted of H2O:CH3CN:MeOH (15:40:45%), and was delivered at 0.75 ml/min. UV absorbance of I-RTX and RTX was monitored at

254 nm. I-RTX and RTX were dissolved in ethanol, and 4 to 6 µl injected for HPLC analyses.

Statistical Analysis

Data were expressed as mean ± S.E.M. Statistical analysis was performed using unpaired or paired Student’s t test for comparison between two groups, Dunnett’s multiple range test for multiple comparisons, or two-way analysis of variance (ANOVA) followed by Bonferroni correction, as indicated in figure legends. Curve fitting for calcium imaging analysis was performed using the following equation: y = Amin + (Amax-Amin)/[1 +

10 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

H (B50/x) ], where Amax and Amin are the maximum and minimum ratio changes, B is the drug concentration producing a half-maximal response, x is the agonist concentration, and H is the Hill coefficient.

Results

I-RTX produces hypothermia in wild-type C57BL/6 mice, but not in RTX-pretreated Downloaded from or TRPV1 null mutant mice

We first confirmed the hypothermic effect of capsaicin using awake mice (Fig. 1A). jpet.aspetjournals.org Consistent with previous reports (deVries and Blumberg, 1989), capsaicin (9.8 µmol/kg, s.c.) evoked a significant reduction in rectal temperature that reached a nadir within 60 min

(-3.9 ± 0.5 °C versus –0.5 ± 0.2 °C for vehicle, n = 4, p < 0.001, unpaired t-test) and at ASPET Journals on September 27, 2021 reversed nearly to baseline by 240 min (Fig. 1A, D). We next sought to determine whether I-RTX would inhibit this response. Unexpectedly, however, we observed that subcutaneous injection of I-RTX, alone, produced a robust and transient hypothermia in wild-type mice that was similar in maximal amplitude (-5.0 ± 0.1°C at 1 µmol/kg, n = 4, p

< 0.001 versus vehicle, Dunnett’s multiple range test, Fig. 1B, D) to that evoked by capsaicin. As shown in Fig. 1B, this effect was dose-dependent, with a significant reduction in body temperature at 0.1 and 1 µmol/kg I-RTX, but only a tendency towards hypothermia at 0.01 µmol/kg. Similar to capsaicin, the maximum hypothermic effect of

I-RTX at 1 µmol/kg was observed from 60 to 100 min after the injection, with a nearly complete recovery by 360 min (Fig. 1A, B). As previously reported (Szallasi and

Blumberg, 1989), non-iodinated RTX, at 0.001 and 0.01 µmol/kg, also produced a

11 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

significant and robust decrease in rectal temperature (Fig. 1C, D). However, this effect persisted much longer than that evoked by either I-RTX or capsaicin. In fact, mice injected with 0.01 µmol/kg RTX exhibited a hypothermic response that reached a peak

(-8.1 ± 0.2 °C, n = 4, p < 0.001 versus vehicle, Dunnett’s multiple range test) at approximately 150-300 min and showed no signs of reversal during the 6-hour observation

period. By the following day, however, body temperature returned to normal levels in Downloaded from these mice (Fig. 1C).

We also observed that I-RTX (1 µmol/kg) failed to produce hypothermia jpet.aspetjournals.org when injected into mice that had been treated the previous day with 0.01

µmol/kg RTX (Fig. 1C). These results suggest that RTX and I-RTX may be influencing body temperature via a common mechanism. To further establish at ASPET Journals on September 27, 2021 whether the hypothermic effect of I-RTX was being mediated by TRPV1, we administered this agent to mice in which the TRPV1 gene had been disrupted. We previously demonstrated that TRPV1-/- mice fail to exhibit capsaicin-evoked hypothermia (Caterina et al., 2000), despite normal hypothermic responses to another chemical agent, ethanol (Iida et al., 2005). As shown in Fig. 1E, I-RTX (1 µmol/kg, s.c.) produced no detectable hypothermia in TRPV1-/- mice. Therefore, the hypothermic effect of I-RTX is most likely the result of its interactions with TRPV1. We further examined whether another TRPV1 antagonist, capsazepine, would alter mouse body temperature when administered systemically. Subcutaneous injection of this compound at either of two doses (30 µmol/kg and 100 µmol/kg) failed to produce any detectable change in body temperature (Fig. 1F), confirming that the I-RTX evoked hypothermia is not a common consequence of TRPV1

12 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

antagonism

I-RTX increases intracellular Ca2+ in HEK293 cells expressing TRPV1

The results outlined above demonstrate that I-RTX, which is recognized as a

TRPV1 antagonist, exhibits TRPV1 agonist-like effects on the mouse thermoregulatory

system. In order to address this apparent paradox, we performed microscopic fluorescent Downloaded from

Ca2+ imaging on HEK293 cells stably expressing rat TRPV1. As shown in Fig. 2A, in accordance with our in vivo findings, I-RTX alone concentration-dependently increased jpet.aspetjournals.org 2+ 2+ relative intracellular Ca concentration ([Ca ]i) (reflected by an increase in fura 2 fluorescence ratio) in TRPV1-expressing cells, but not in cells stably transfected with control vector (pCDNA3) (Fig. 2 A, C). During persistent I-RTX stimulation, upon at ASPET Journals on September 27, 2021

2+ reaching a plateau level, relative [Ca ]i remained stable, with no evidence of desensitization. The concentration-dependence of the I-RTX-evoked response appeared to be biphasic, with one plateau at 1 to 3 µM and another at 15 to 30 µM (EC50 = 56.7 nM at

30 nM-1 µM and 9.9 µM and at 1-30 µM) (Fig. 2C). Solubility precluded us from applying higher I-RTX concentrations. The existence of two components in our data suggests that I-RTX might have two mechanisms and/or sites of action on TRPV1.

Furthermore, the maximum response induced by I-RTX (20 µM) was 77.1 ± 4.3% (n = 5) of that induced by 1 µM capsaicin (Fig. 2C), indicating that this compound is not a full

2+ TRPV1 agonist. The [Ca ]i increase evoked by I-RTX application was reversibly inhibited by elimination of extracellular Ca2+ (Fig. 2B), suggesting that this increase requires Ca2+ influx through TRPV1 channels on the plasma membrane, rather than release

13 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

from internal Ca2+ stores. Recovery of the response upon Ca2+ restoration was achieved even after I-RTX had been removed from the bath solution, suggesting that I-RTX, like

RTX (Caterina et al., 1997), either remains tightly bound to TRPV1 or persists in the plasma membrane to allow continued TRPV1 activation.

In addition to partial agonist behavior, we also observed evidence of I-RTX

antagonism at TRPV1. I-RTX concentration-dependently reduced the response to Downloaded from subsequent application of 1 µM capsaicin in TRPV1-expressing cells (Fig. 2D). Up to 1

µM I-RTX, the ratio change upon capsaicin treatment decreased, consistent with jpet.aspetjournals.org antagonism. However, at higher concentrations (1 - 30 µM), the agonistic effects of I-RTX became more obvious and increased the overall Ca2+ influx. When this confounding

agonism was subtracted from the overall response, the IC50 of the inhibition was calculated at ASPET Journals on September 27, 2021 to be 54.4 nM, similar to the EC50 of the first phase of I-RTX agonism (Fig. 2E).

I-RTX evokes whole-cell currents in TRPV1-expressing HEK cells.

To further confirm the ability of I-RTX to activate TRPV1, we conducted whole-cell voltage-clamp recordings in TRPV1-expressing HEK293 cells. In order to avoid Ca2+-dependent TRPV1 desensitization (Tominaga et al., 1998), we conducted these experiments in the absence of extracellular Ca2+. Consistent with our Ca2+ imaging studies,

I-RTX alone (30 µM) evoked currents similar in their outwardly rectifying current-voltage relationship to those induced by capsaicin (10 nM, Fig. 3A, B). Activation by I-RTX, however, was apparently slower than that evoked by capsaicin, with a longer time required to reach a maximal response. The effect of I-RTX was sustained during several minutes of

14 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

application, and persisted even after washout (data not shown). As illustrated in Fig. 3B,

I-RTX increased the current amplitude in a concentration-dependent manner, whereas no such response was observed in pcDNA3 control cells (-0.2 ± 0.1 pA/pF at +80mV, 30 µM

I-RTX, n = 4). Although we did not examine the concentration-dependence of this current response in detail, its overall profile was consistent with that observed in the Ca2+ imaging analysis. I-RTX-evoked currents appeared to be confined to cells expressing a high level Downloaded from of TRPV1; when stimulated with 10 nM capsaicin, these cells invariably exhibited responses of > 40 pA/pF at +80 mV (n = 20, data not shown). Moreover, the maximum jpet.aspetjournals.org response at +80 mV evoked by 30 µM I-RTX (46.5 ± 5.4 pA/pF, n = 4) was only 14.1% of that evoked by 1 µM capsaicin (330.5 ± 58.2 pA/pF, n = 4, Fig. 3B), indicating that I-RTX

2+ is a weak partial agonist of TRPV1, as suggested by our Ca imaging experiments. I-RTX at ASPET Journals on September 27, 2021 induced only a small increase in inward currents (Fig. 3B inset). Even at the highest

I-RTX concentration (30 µM), the current density was 1.3 ± 0.2 pA/pF (n = 4), which was only 0.4% of that evoked by 1 µM capsaicin (366.6 ± 44.1 pA/pF, n = 4) at –80 mV, reflecting the strong outward rectification of I-RTX-evoked TRPV1 currents.

We next examined the effects of capsazepine on the I-RTX-evoked current response (Fig. 3C). After the response to I-RTX (300 nM) reached a plateau, capsazepine

(10 µM) reversibly reduced the outward currents at +80 mV by 64.3 ± 5.0% (n = 4, p <

0.05, paired t-test). Superimposition of I-RTX onto cells pretreated for one minute with capsazepine induced only a slight increase in outward currents (2.3 ± 0.7 pA/pF, n = 4).

During washout, however, the current amplitude gradually increased to 15.2 ± 3.5 pA/pF (n

= 4, p < 0.05, paired t-test), similar to that induced by the same concentration of I-RTX

15 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

without capsazepine (Fig. 3C). These findings demonstrate that capsazepine can antagonize the effects of I-RTX and further suggest that, whereas capsazepine is cleared rapidly from cells, I-RTX persists to evoke its agonist effects.

Because I-RTX is structurally similar to RTX, an ultra potent TRPV1 agonist, we were concerned that the agonistic activity observed in the present study might be due to

contaminating RTX in the I-RTX preparation, even though we used several Downloaded from independently-purchased vials of the drug that were nominally more than 99.9% pure. To address this remote possibility, we compared the response of TRPV1-expressing cells to 3 jpet.aspetjournals.org pM RTX with that to 3 nM I-RTX. The current response evoked by 3 nM I-RTX (8.0 ±

2.2 pA/pF, n = 8) was significantly greater than that evoked by 3 pM RTX (2.8 ± 0.8 pA/pF, n = 7, p < 0.05, unpaired t-test, Fig. 3B). Furthermore, when the kinetics of current at ASPET Journals on September 27, 2021 development were compared following application of I-RTX (300 nM) versus RTX (300 pM), I-RTX induced more rapid responses than RTX (Fig. 3D). Finally, we analyzed the

I-RTX used in our studies by high performance liquid chromatography (HPLC). As reported by others (Wahl et al., 2001), I-RTX and RTX could be well-resolved using this method (Fig. 4). No peak corresponding to RTX was observed in the I-RTX lot used in our experiments. Based on mixing experiments in which we artificially introduced contaminating RTX at levels of 0.1% to 1%, we were able to confirm that any RTX in our initial I-RTX stock must be present at less than 0.1% (Fig. 4C). Together, these findings argue against contaminating RTX as the cause of the agonist behavior we observed.

Discussion

16 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

In this study, we have demonstrated that I-RTX induces TRPV1-mediated hypothermia when administered systemically in the mouse. This hypothesis is strongly supported by the fact that decreases in body temperature following subcutaneous I-RTX injection were not observed in either wild-type mice pretreated with RTX or TRPV1 null mutant mice. It also allows one to reconcile our I-RTX body temperature results with the

lack of apparent effect of capsazepine on mouse body temperature and the recent report that Downloaded from another, more selective TRPV1 antagonist,

4-(3-Trifluoromethylpyridin-2-yl)piperazine-1-carboxylic Acid jpet.aspetjournals.org (5-Trifluoromethylpyridin-2-yl)amide, actually produces a small but significant degree of hyperthermia in rats (Swanson et al., 2005). The doses of I-RTX required to produce a significant decrease in body temperature were much higher than might have been predicted at ASPET Journals on September 27, 2021 from the in vitro affinity of I-RTX for TRPV1 (Wahl et al., 2001), but are well within the range of I-RTX concentrations required to inhibit capsaicin-induced Evans blue extravasation and acetic acid-induced writhing in mice (EC50 = 0.41 and 0.42 µmol/kg, i.p., respectively) (Rigoni et al., 2003).

Our in vitro studies support the notion that I-RTX can activate TRPV1, and suggest that it does so as a partial agonist. I-RTX concentration-dependently evoked Ca2+ influx in

TRPV1-expressing cells. This effect appeared to have two kinetic components. The mechanistic basis for the existence of two components is unclear, but they may reflect different sites or modes of action on TRPV1. In either case, both Ca2+ influx and current responses could be observed over the entire dynamic range of concentrations. Moreover, the higher-affinity component exhibited a potency similar to that of I-RTX inhibition of

17 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

capsaicin-evoked responses. A similar situation was recently reported for two other

TRPV1 ligands,

N-(4-tert-butylbenzyl)-N'-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea (JYL1511) and

N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N'-[4-(methylsulfonylamino)benzyl]thio

3 45 2+ urea (JYL827). These compounds inhibit TRPV1 binding of [ H]RTX and Ca -uptake Downloaded from induced by capsaicin with similar potencies (Ki = 29 - 50 nM, IC50 = 34-67 nM), although either compound, alone, also cause increases in intracellular Ca2+ in TRPV1-expressing jpet.aspetjournals.org cells (Wang et al., 2003). The degree of I-RTX partial agonism estimated from our Ca2+ imaging experiments was greater than that estimated from our voltage clamp experiments.

In addition, the Ca2+ influx observed in I-RTX-treated TRPV1 cells appeared to be out of at ASPET Journals on September 27, 2021 proportion to the relatively small I-RTX-evoked inward currents observed in these cells.

These differences might reflect amplification of Ca2+ responses by mechanisms downstream of TRPV1 activation, such as Ca2+ release from intracellular stores or activation of other cell-surface Ca2+ influx pathways. Regardless, their dependence on extracellular Ca2+ and their absence from cells stably transfected with control vector demonstrate the dependence of both the influx and current responses on TRPV1. The apparent discrepancy between in vitro and in vivo efficacy of I-RTX further suggests that the mouse thermoregulatory system may be unusually sensitive to I-RTX agonism. Such sensitivity could arise from species differences (rat versus mouse) in I-RTX agonism, from the expression of a variant of TRPV1 with exceptional I-RTX sensitivity, or from amplification of small TRPV1-evoked current responses in thermoregulatory signaling

18 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

pathways. An alternative explanation, and one that we cannot exclude, is that some fraction of the administered I-RTX is de-iodinated to RTX in vivo and that the latter compound augments the otherwise weak agonist effects of I-RTX.

Partial agonist activity is an unexpected property of I-RTX, given previous in vitro and in vivo studies of this compound. Wahl et al. (2001) reported that I-RTX at up to 3 µM

did not affect the basal current in voltage-clamped Xenopus oocytes expressing TRPV1, Downloaded from though they recorded only at negative potentials, and may therefore have missed weak responses. Other groups have confirmed these general results using whole-cell jpet.aspetjournals.org voltage-clamp in CHO cells expressing human TRPV1 (Seabrook et al., 2002), Ca2+ imaging in rat trigeminal neurons and HEK 293 cells expressing human TRPV1 (Rigoni et al., 2003), calcitonin gene –related peptide (CGRP) release from spinal cord slices (Rigoni at ASPET Journals on September 27, 2021 et al., 2003) and peripheral nerve recordings from a guinea pig airway smooth muscle preparation (Undem and Kollarik, 2002). However, several reports have provided evidence consistent with agonist activity of I-RTX. For example, topical application of 10

µM I-RTX has been reported to acutely increase the frequency of urinary bladder contraction and reduce the number of CGRP-immunoreactive fibers in the rat, suggesting that, at least in the bladder, I-RTX acts preferentially to activate and consequently cause regression of TRPV1-expressing neurons (Charrua et al., 2004). Similarly, I-RTX (10

µM) has been reported to facilitate, rather than inhibit, noxious heat (45ºC)-evoked release of CGRP from isolated rat skin (Petho et al., 2004). In accordance with our findings, another group recently reported that they could not use I-RTX as a TRPV1 antagonist to study thermoregulation in the rat, owing to its tendency to reduce body temperature (Dogan

19 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

et al., 2004). However, no thermoregulatory data were shown for I-RTX in that study and the dependence of the effect on TRPV1 was not evaluated. It has also been reported that intraplantar administration of I-RTX (0.1 to 1 nmol/paw) causes paw flinching behavior in the rat, although this effect might not be mediated by TRPV1, since at a higher dose (10 nmol/paw) I-RTX-evoked equivalent flinching behavior in wildtype and TRPV1-/- mice

(Seabrook et al., 2002). Downloaded from

The reasons for these apparent discrepancies between studies are not clear. They might, in some cases, relate to the specific functional assays employed. For example, the jpet.aspetjournals.org effects of I-RTX on body temperature might be more readily appreciated than nociceptive or other in vivo effects. In vitro, differences between studies might be related to the species of TRPV1 used. For instance, although Seabrook et al. (2002) did examine the at ASPET Journals on September 27, 2021 effects of I-RTX alone at strongly positive potentials, their use of human TRPV1 may have prevented them from observing I-RTX-evoked currents, given the 8-fold lower affinity of this compound for human, as opposed to rat TRPV1. TRPV1 expression level also influences the likelihood of observing I-RTX responses, since in our experiments, I-RTX agonism was confined to cells that exhibited strong current responses to a low dose of capsaicin. Finally, we cannot exclude the possibility that some “antagonistic” effects reported for I-RTX are due to terminal regression or other toxic effects on

TRPV1-expressing neurons similar to those produced by other TRPV1 (Jancso et al., 1977; Simone et al., 1998).

For several reasons, we can be confident that the effects we observed are unlikely to be the result of trace contamination of our drug preparations with RTX. First, we

20 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

demonstrated that the level of contaminating RTX was substantially less than 0.1%. In our control experiments, the effect of RTX at a dose equivalent to 0.1% contamination produced a current response significantly smaller than that evoked by I-RTX. Second, the kinetics of RTX and I-RTX-evoked current responses are different, with I-RTX-evoked currents reaching a maximum sooner than those evoked by RTX. Third, the kinetics of

RTX and I-RTX-evoked hypothermia are different, with the response to I-RTX being more Downloaded from rapid in onset but more transient than those evoked by RTX. Another iodinated form of

RTX, 2-iodoresiniferatoxin (2I-RTX) has been reported to exhibit partial agonist activity at jpet.aspetjournals.org TRPV1 (McDonnell et al., 2002). However, since the potency of this compound is reported to be comparable to that of the I-RTX used in our experiments, trace contamination by this compound is also unlikely to explain our results. Together, these at ASPET Journals on September 27, 2021 findings argue that I-RTX, rather than RTX or 2I-RTX, is responsible for the effects reported in the present study.

In conclusion, we have found that I-RTX induces robust hypothermia through a

TRPV1-dependent mechanism, and exerts weak, partial agonism on recombinant TRPV1 in vitro. These effects demonstrate that I-RTX should be used with caution, and may not be appropriate for use as an in vivo TRPV1 antagonist. However, they also indicate that

I-RTX might serve as a useful pharmacological tool to probe the contributions of TRPV1 to thermoregulatory processes.

Acknowledgements: The authors thank Juan Wang for expert technical assistance, Man

Kyo Chung for critical reading of the manuscript, Naoyuki Yoshida and Katsuyoshi

21 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

Kawashima, Christopher Gross, Phillip Cole and the Caterina lab for helpful suggestions. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

22 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

REFERENCES

Caterina MJ and Julius D (2001) The vanilloid receptor: a molecular gateway to the pain

pathway. Ann. Rev. Neurosci. 24:487-517.

Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR,

Koltzenburg M, Basbaum AI and Julius D (2000) Impaired nociception and pain Downloaded from

sensation in mice lacking the capsaicin receptor. Science 288:306-313.

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD and Julius D (1997) jpet.aspetjournals.org The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature

389:816-824.

Charrua A, Avelino A, Cruz C and Cruz F (2004) Iodoresiniferatoxin (IRTX) applied at ASPET Journals on September 27, 2021

topically to the rat bladder acts as a potent TRPV1 agonist. Society For

Neuroscience Annual Meeting Abstracts 743.7.

Correll CC, Phelps PT, Anthes JC, Umland S and Greenfeder S (2004) Cloning and

pharmacological characterization of mouse TRPV1. Neurosci Lett 370:55-60.

Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham

J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL,

Rogers DC, Bingham S, Randall A and Sheardown SA (2000) Vanilloid receptor-1

is essential for inflammatory thermal hyperalgesia. Nature 405:183-187. deVries DJ and Blumberg PM (1989) Thermoregulatory effects of resiniferatoxin in the

mouse: comparison with capsaicin. Life Sci. 44:711-715.

Dogan MD, Patel S, Rudaya AY, Steiner AA, Szekely M and Romanovsky AA (2004)

23 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

Lipopolysaccharide fever is initiated via a capsaicin-sensitive mechanism

independent of the subtype-1 vanilloid receptor. Br J Pharmacol 143:1023-1032.

Guler AD, Lee H, Iida T, Shimizu I, Tominaga M and Caterina M (2002) Heat-evoked

activation of the ion channel, TRPV4. J Neurosci 22:6408-6414.

Iida T, Shimizu I, Nealen M, Campbell A and Caterina M (2005) Attenuated fever response

in mice lacking TRPV1. Neurosci Lett 378:28-33. Downloaded from

Jancso G, Kiraly E and Jancso-Gabor A (1977) Pharmacologically induced selective

degeneration of chemosensitive primary sensory neurons. Nature 270:741-743. jpet.aspetjournals.org Jancso-Gabor A, Szolcsanyi J and Jancso N (1970a) Irreversible impairment of

thermoregulation induced by capsaicin and similar pungent substances in rats and

guinea pigs. J. Physiol. (London) 206:495-507. at ASPET Journals on September 27, 2021

Jancso-Gabor A, Szolcsanyi J and Jancso N (1970b) Stimulation and desensitization of the

hypothalamic heat-sensitive structures by capsaicin in rats. J. Physiol., London

208:449-459.

Liu L and Simon SA (1997) Capsazepine, a vanilloid , inhibits nicotinic

acetylcholine receptors in rat trigeminal ganglia. Neurosci. Lett. 228:29-32.

McDonnell ME, Zhang SP, Dubin AE and Dax SL (2002) Synthesis and in vitro evaluation

of a novel iodinated resiniferatoxin derivative that is an agonist at the human

vanilloid VR1 receptor. Bioorg Med Chem Lett 12:1189-1192.

McIntyre P, McLatchie LM, Chambers A, Phillips E, Clarke M, Savidge J, Toms C,

Peacock M, Shah K, Winter J, Weerasakera N, Webb M, Rang HP, Bevan S and

James IF (2001) Pharmacological differences between the human and rat vanilloid

24 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

receptor 1 (VR1). Br J Pharmacol 132:1084-1094.

Petho G, Izydorczyk I and Reeh PW (2004) Effects of TRPV1 receptor antagonists on

stimulated iCGRP release from isolated skin of rats and TRPV1 mutant mice. Pain

109:284-290.

Phillips E, Reeve A, Bevan S and McIntyre P (2004) Identification of species-specific

determinants of the action of the antagonist capsazepine and the agonist PPAHV on Downloaded from

TRPV1. J Biol Chem 279:17165-17172.

Ray AM, Benham CD, Roberts JC, Gill CH, Lanneau C, Gitterman DP, Harries M, Davis jpet.aspetjournals.org JB and Davies CH (2003) Capsazepine protects against neuronal injury caused by

oxygen glucose deprivation by inhibiting I(h). J Neurosci 23:10146-10153.

Rigoni M, Trevisani M, Gazzieri D, Nadaletto R, Tognetto M, Creminon C, Davis JB, at ASPET Journals on September 27, 2021

Campi B, Amadesi S, Geppetti P and Harrison S (2003) Neurogenic responses

mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity

antagonist, iodo-resiniferatoxin. Br J Pharmacol 138:977-985.

Seabrook GR, Sutton KG, Jarolimek W, Hollingworth GJ, Teague S, Webb J, Clark N,

Boyce S, Kerby J, Ali Z, Chou M, Middleton R, Kaczorowski G and Jones AB

(2002) Functional properties of the high-affinity TRPV1 (VR1) vanilloid receptor

antagonist (4-hydroxy-5-iodo-3-methoxyphenylacetate ester) iodo-resiniferatoxin. J

Pharmacol Exp Ther 303:1052-1060.

Simone DA, Nolano M, Johnson T, Wendelschafer-Crabb G and Kennedy WR (1998)

Intradermal injection of capsaicin in humans produces degeneration and subsequent

reinnervation of epidermal nerve fibers: correlation with sensory function. J

25 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

Neurosci 18:8947-8959.

Swanson DM, Dubin AE, Shah C, Nasser N, Chang L, Dax SL, Jetter M, Breitenbucher JG,

Liu C, Mazur C, Lord B, Gonzales L, Hoey K, Rizzolio M, Bogenstaetter M, Codd

EE, Lee DH, Zhang SP, Chaplan SR, Carruthers NI. (2005) Identification and

biological evaluation of 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-carboxylic acid

(5-trifluoromethylpyridin-2-yl)amide, a high affinity TRPV1 (VR1) vanilloid receptor Downloaded from

antagonist. J Med Chem. 48:1857-72.

Szallasi A and Blumberg PM (1989) Resiniferatoxin, a phorbol-related diterpene, acts as an jpet.aspetjournals.org ultrapotent analog of capsaicin, the irritant constituent in red pepper. Neuroscience

30:515-520.

Szallasi A and Blumberg PM (1990) Specific binding of resiniferatoxin, an ultrapotent at ASPET Journals on September 27, 2021

capsaicin analog, by dorsal root ganglion membranes. Brain Res. 524:106-111.

Szallasi A, Blumberg PM, Annicelli LL, Krause JE and Cortright DN (1999) The cloned rat

vanilloid receptor VR1 mediates both R-type binding and C-type calcium response

in dorsal root ganglion neurons. Mol Pharmacol 56:581-587.

Szekely M and Szolcsanyi J (1979) Endotoxin fever in capsaicin treated rats. Acta Physiol.

Acad. Scien. Hung. 53:469-477.

Szelenyi Z, Hummel Z, Szolcsanyi J and Davis JB (2004) Daily body temperature rhythm

and heat tolerance in TRPV1 knockout and capsaicin pretreated mice. Eur J

Neurosci 19:1421-1424.

Szolcsanyi J (1983) Disturbances of thermoregulation induced by capsaicin. J. Therm. Biol.

8:207-212.

26 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE,

Basbaum AI and Julius D (1998) The cloned capsaicin receptor integrates multiple

pain-producing stimuli. Neuron 21:1-20.

Undem BJ and Kollarik M (2002) Characterization of the vanilloid receptor 1 antagonist

iodo-resiniferatoxin on the afferent and efferent function of vagal sensory C-fibers.

J Pharmacol Exp Ther 303:716-722. Downloaded from

Wahl P, Foged C, Tullin S and Thomsen C (2001) Iodo-resiniferatoxin, a new potent

vanilloid receptor antagonist. Mol Pharmacol 59:9-15. jpet.aspetjournals.org Wang Y, Toth A, Tran R, Szabo T, Welter JD, Blumberg PM, Lee J, Kang SU and Lim JO

(2003) High-affinity partial agonists of the vanilloid receptor. Mol Pharmacol

64:325-333. at ASPET Journals on September 27, 2021

27 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

FOOTNOTES

1These authors contributed equally to this study

This work was supported by grants from The W.M. Keck Foundation, The Searle Scholars

Program, The Arnold and Mabel Beckman Foundation and Dainippon Pharmaceuticals to

M.J.C. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021

28 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

FIGURE LEGENDS

Figure 1. Systemic administration of I-RTX, capsaicin or RTX causes hypothermia in wild-type but not in TRPV1-/- mice. (A), Changes in rectal temperature following s.c. administration of vehicle (10% ethanol/10% Tween 80 in saline; closed circles; n = 5) or

capsaicin (CAP, 9.8 µmol/kg, open circles; n = 5). (B), Changes in rectal temperature Downloaded from following s.c. administration of vehicle (closed circles; n = 7), or I-RTX at 0.01 µmol/kg

(open squares; n = 5), 0.1 µmol/kg (open triangles; n = 5) or 1 µmol/kg (open circles; n = 4). jpet.aspetjournals.org (C), Changes in rectal temperature following s.c. administration of vehicle (closed circles; n

= 7), or RTX at 0.001 µmol/kg (open squares; n = 4) or 0.01 µmol/kg (open triangles; n =

4). The following day, I-RTX (1 µmol/kg) was injected s.c to the group that had received at ASPET Journals on September 27, 2021

0.01 µmol/kg RTX. Data represent mean ± SEM. *p < 0.05, ** p < 0.01,

*** p < 0.001; compared with the corresponding vehicle control point (unpaired t-test with

Bonferroni correction for capsaicin and two-way ANOVA with Bonferroni correction for

I-RTX and RTX). (D), Maximum changes in body temperature (from preinjection value at time 0) following s.c. administration of vehicle (open bar; n = 12), I-RTX (filled bars; n = 4

- 5), capsaicin (hatched bar; n = 5) or RTX (dotted bars; n = 4). *** p < 0.001; compared with vehicle (Dunnett’s multiple range test for I-RTX and RTX, and unpaired t-test for capsaicin). Doses are in µmol/kg. V, vehicle. (E) Changes in peritoneal temperature following s.c. administration of I-RTX (1 µmol/kg) in wild-type mice (dashed lines; n = 2) or TRPV1 null mutant mice (solid lines; n = 3). (F) Lack of changes in rectal temperature following s.c. administration of capsazepine (CPZ, 30 µmol/kg, open triangles; 100

29 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

µmol/kg, open squares) or vehicle (filled circles) (n = 4 per group).

Figure 2. I-RTX increases free intracellular [Ca2+] in HEK293 cells expressing rat

TRPV1. (A) Population-averaged fura ratios in the presence of bath solution (a), I-RTX

(b) and I-RTX plus 1 µM capsaicin (c) in HEK293 cells stably transfected with TRPV1

(closed symbols) or control vector pCDNA3 (open squares). During period (b), Downloaded from

TRPV1-expressing cells were treated with vehicle (0.45% ethanol, filled squares, trace 2) or I-RTX (30 nM, circles, trace 3; 1 µM, triangles, trace 4; 20 µM, diamonds, trace 5), and jpet.aspetjournals.org pCDNA3 cells were treated with I-RTX (30 µM, open squares, trace 1). (B)

2+ Representative changes in relative [Ca ]i in TRPV1-expressing cells upon application of 3

2+ µM I-RTX and transient removal of extracellular Ca . The variability in response at ASPET Journals on September 27, 2021 amplitude evident from this figure is typical of responses in our TRPV1 stable cell line, regardless of the agonist used. (C-E) Summary of concentration-dependent agonistic and antagonistic effects of I-RTX on TRPV1. Closed circles, I-RTX in TRPV1-expressing cells; open square, 1 µM capsaicin only in naïve TRPV1-expressing cells; open triangle,

I-RTX in stable pCDNA3-transfected cells. Panel C shows agonistic effect of I-RTX on

2+ the change in relative [Ca ]i. ∆Fura ratio was calculated by subtracting baseline value

(period (a) in panel A) from peak I-RTX-stimulated value (period (b) in panel A). Panel D shows cumulative Ca2+ response evoked by I-RTX plus capsaicin (maximum during time

(c) minus maximum during time (a) in panel A). Dashed line indicates fit from panel C.

Panel E shows I-RTX inhibition of subsequent response to 1 µM capsaicin (maximum

30 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

during time (c) minus maximum during time (b) in panel A). Data represent mean ± SEM

(n = 5).

Figure 3. I-RTX evokes TRPV1-mediated currents in HEK293 cells. (A) Top,

Whole-cell current responses of TRPV1-expressing cells during sequential applications of

capsaicin (CAP, 10 nM) and I-RTX (30 µM). In this and other panels, currents were Downloaded from recorded during repetitive (0.5 Hz) 200 ms voltage ramps from –100 to +100 mV and the values at +80 mV (upward trace) and -80 mV (downward trace) plotted and connected. jpet.aspetjournals.org Bottom, Current-voltage traces obtained during voltage-ramps at times a-c (defined at top)

(B) Changes in current density (current amplitude divided by membrane capacitance) at ±

80 mV in stable TRPV1 -transfected cells evoked by vehicle (0.23% DMSO), I-RTX, RTX at ASPET Journals on September 27, 2021 or capsaicin (+80 mV, filled bars; -80 mV, open bars, n = 4-8). Inset, magnification of current responses at -80mV in cells treated with vehicle (DMSO) or I-RTX. (C) Changes in current response at ±80 mV in TRPV1-expressing cells during application of I-RTX (300 nM) with capsazepine (CPZ, 10 µM) present during the indicated time period. Bar graphs show changes in current density at +80 mV at time points a and b. (n = 4, * p < 0.05, paired t-test). (D) Current responses at +80 mV in TRPV1-expressing cells during application of

I-RTX (300 nM, black traces, n = 4) or RTX (300 pM, gray traces, n = 4). Arrow indicates the time of drug application. The dashed line indicates zero current or potential level.

Figure 4. Lack of detectable RTX contamination in I-RTX. High performance liquid chromatography was performed on solutions of I-RTX, RTX, and mixtures of the two. (A

31 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #84277

and B) Chromatographic separation of the I-RTX (A) and RTX (B) preparations used in this study on a C18 column. Numbers indicate retention time, in minutes. (C) Expansion of chromatograms derived from region indicated by shaded bar in panel (A). The indicated mixtures of I-RTX and RTX were resolved as in panels (A) and (B). Black arrow, RTX peak. Gray arrows: I-RTX peak (truncated).

Downloaded from

jpet.aspetjournals.org at ASPET Journals on September 27, 2021

32 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021 JPET Fast Forward. Published on June 9, 2005 as DOI: 10.1124/jpet.105.084277 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 27, 2021