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NEUROPHARMACOLOGY
Departments of Biological Chemistry and Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland (I.S., T.I., M.J.C.); and Dainippon Pharmaceutical Company, Limited, Suita/Osaka, Japan (I.S., N.H.)
Received January 28, 2005; accepted June 6, 2005.
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Abstract |
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; Caterina and Julius, 2001). Mice lacking TRPV1 are deficient in responsiveness to vanilloids and noxious heat, as well as in inflammation-induced thermal hyperalgesia (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 systemically or directly into the preoptic/anterior hypothalamus (Jancso-Gabor et al., 1970a,b; 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 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, although tantalizing, remains unclear. 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.
Resiniferatoxin (RTX) is an ultrapotent vanilloid receptor agonist produced 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 [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 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 on the presence of TRPV1. We further found that, in vitro, I-RTX exhibits partial agonist activity at recombinant rat TRPV1.
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). Mice were housed at 24–25°C on a 12-h light/dark (8:00 AM to 8:00 PM) photocycle and provided with food and water ad libitum. All experiments were conducted on age-matched male mice 2 to 4 months old between the hours of 10:00 AM and 7:30 PM. Cell culture reagents were obtained from Invitrogen (Carlsbad, CA), and other chemicals, including I-RTX, were from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. For body temperature measurements, I-RTX, RTX, capsaicin, and capsazepine were dissolved in a solution of 10% ethanol and 10% Tween 80 in pyrogen-free saline and were injected s.c. at 0.1 ml/10 g of 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. For rectal temperature measurements, mice were placed in individual cages at room temperature (22–25°C). Rectal temperatures were recorded from awake mice by repeated insertion of a lubricated rectal thermocouple probe (model RET-3; Physitemp Instruments, Inc., Clifton, NJ) prior to and following s.c. injection of capsaicin, RTX, or I-RTX. In the experiment shown in Fig. 1C, after the final rectal temperature recording, mice injected with RTX were given food and water. The following day, temperature measurements were resumed prior to I-RTX administration.
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6 days prior to experiments. During the experiment, mice in individual cages were placed on telemetric receiver platforms (MiniMitter), and their body temperatures were monitored continuously using Vital View software (MiniMitter).
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 (Guler et al., 2002), were subjected to Ca2+ imaging and whole-cell voltage-clamp recording. Fluorometric Ca2+ imaging was performed as described (Guler et al., 2002) using an inverted microscope (Nikon, Melville, NY), fluorescent light source (DG4; Sutter Instrument Company, Novato, CA), and charge-coupled device camera (Roper Scientific, Trenton, NJ). Bath solution for imaging contained 130 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0.6 mM MgCl2, 10 mM HEPES, 1.2 mM NaHCO3, and 10 mM 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) at 37°C for 40 min and 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 140 mM NaCl, 0.1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.4 (adjusted with NaOH). Cells were superfused with bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM 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 (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 Inc., Union City, CA) were used. Borosilicate glass electrodes had tip resistances of 1.5 to 2.0 M
. Series resistance was typically less than 10 M, 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.
High-Performance Liquid Chromatography. High-performance liquid chromatography (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; 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 was 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 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 + (B50/x)H], 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.
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), 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 Student's t test) and reversed nearly to baseline by 240 min (Fig. 1, A and D). We next sought to determine whether I-RTX would inhibit this response. Unexpectedly, however, we observed that s.c. 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. 1, B and 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 toward 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. 1, A and B). As previously reported (Szallasi and Blumberg, 1989), noniodinated RTX at 0.001 and 0.01 µmol/kg also produced a significant and robust decrease in rectal temperature (Fig. 1, C and 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 to 300 min and showed no signs of reversal during the 6-h observation period. By the following day, however, body temperature returned to normal levels in these mice (Fig. 1C).
We also observed that I-RTX (1 µmol/kg) failed to produce hypothermia 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 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 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 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. To address this apparent paradox, we performed microscopic fluorescent 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 relative intracellular Ca2+ concentration ([Ca2+]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 and C). During persistent I-RTX stimulation, upon reaching a plateau level, relative [Ca2+]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 to 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 TRPV1 agonist. The [Ca2+]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 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.
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I-RTX Evokes Whole-Cell Currents in TRPV1-Expressing Human Embryonic Kidney Cells. To further confirm the ability of I-RTX to activate TRPV1, we conducted whole-cell voltage-clamp recordings in TRPV1-expressing HEK293 cells. 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. 3, A and 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 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 +80 mV, 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 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 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 is a weak partial agonist of TRPV1, as suggested by our Ca2+ imaging experiments. I-RTX 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.
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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 Student's t test). Superimposition of I-RTX onto cells pretreated for 1 min 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 Student's t test), similar to that induced by the same concentration of I-RTX 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 ultrapotent 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, although we used several 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 with 3 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 Student's t test; Fig. 3B). Furthermore, when the kinetics of current 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 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.
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). The doses of I-RTX required to produce a significant decrease in body temperature were much higher than might have been predicted 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 capsaicin-evoked responses. A similar situation was recently reported for two other TRPV1 ligands, JYL1511 and JYL827. These compounds inhibit TRPV1 binding of [3H]RTX and 45Ca2+-uptake induced by capsaicin with similar potencies (Ki, 29–50 nM; IC50, 34–67 nM), although either compound alone also causes increases in intracellular Ca2+ in TRPV1-expressing 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 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 pathways. An alternative explanation, and one that we cannot exclude, is that some fraction of the administered I-RTX is deiodinated 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, although they recorded only at negative potentials and may, therefore, have missed weak responses. Other groups have confirmed these general results using whole-cell voltage-clamp in Chinese hamster ovary cells expressing human TRPV1 (Seabrook et al., 2002), Ca2+ imaging in rat trigeminal neurons and HEK293 cells expressing human TRPV1 (Rigoni et al., 2003), calcitonin gene-related peptide (CGRP) release from spinal cord slices (Rigoni 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 (45°C)-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 because of its tendency to reduce body temperature (Dogan 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 wild-type and TRPV1-/- mice (Seabrook et al., 2002).
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 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 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 agonists (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 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 rapid in onset but more transient than those evoked by RTX. Another iodinated form of RTX, 2-iodoresiniferatoxin, has been reported to exhibit partial agonist activity at TRPV1 (McDonnell et al., 2002). However, since the potency of this compound is reported to be comparable with that of the I-RTX used in our experiments, trace contamination by this compound is also unlikely to explain our results. Together, these findings argue that I-RTX, rather than RTX or 2-iodoresiniferatoxin, 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.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: TRPV1, transient receptor potential vanilloid 1; RTX, resiniferatoxin, 6,7-deepoxy-6,7-didehydro-5-deoxy-21-dephenyl-21-(phenylmethyl)-daphnetoxin,20-(4-hydroxy-3-ethoxybenzeneacetate); I-RTX, 5-iodoresiniferatoxin, 6,7-deepoxy-6,7-didehydro-5-deoxy-21-dephenyl-21-(phenylmethyl)-daphnetoxin,20-(4-hydroxy-5-iodo-3-ethoxybenzeneacetate); DMSO, dimethylsulfoxide; HEK, human embryonic kidney; HPLC, high-performance liquid chromatography; JYL1511, N-(4-tert-butylbenzyl)-N'-[3-methoxy-4-(methylsulfonylamino)benzyl]thiourea; JYL827, N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N'-[4-(methylsulfonylamino)benzyl]thiourea; CGRP, calcitonin gene-related peptide.
1 Both authors contributed equally to this work. 
Address correspondence to: Dr. Michael J. Caterina, Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. E-mail: caterina{at}jhmi.edu
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Fura ratio was calculated by subtracting baseline value (period a in A) from peak I-RTX-stimulated value (period b in A). D, cumulative Ca2+ response evoked by I-RTX plus capsaicin (maximum during time c minus maximum during time a in A). Dashed line, fit from C. E, I-RTX inhibition of subsequent response to 1 µM capsaicin (maximum during time c minus maximum during time b in A). Data represent mean ± S.E.M. (n = 5).
