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Originally published In Press as doi:10.1074/jbc.M406260200 on June 8, 2004

J. Biol. Chem., Vol. 279, Issue 34, 35133-35138, August 20, 2004
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Warm Temperature-sensitive Transient Receptor Potential Vanilloid 4 (TRPV4) Plays an Essential Role in Thermal Hyperalgesia*

Hiroshi Todaka, Junichi Taniguchi, Jun-ichi Satoh, Atsuko Mizuno, and Makoto Suzuki{ddagger}

From the Department of Pharmacology, Division of Molecular Pharmacology, Jichi Medical School 3311-1, Yakushiji, Minamikawachi, Tochigi 329-0498, Japan

Received for publication, June 4, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals sense various ranges of temperatures by cutaneous thermal stimuli. Transient receptor potential vanilloid 4 (TRPV4) is a cation channel activated at a warm temperature (over 30 °C) in exogenously expressed cells. We found in the present study that TRPV4 is essential in thermal hyperalgesia at a warm temperature in vivo. TRPV4–/– and TRPV4+/+ mice exhibited the same latency of escape from 35–50 °C hotplates. Neuronal activity in the femoral nerve, however, revealed that the number and activity level of neurons decreased in response to a warm temperature in TRPV4–/– mice. TRPV4–/– mice displayed a significantly longer latency to escape from the plates at 35– 45 °C when hyperalgesia was induced by carrageenan without changes in foot volumes. TRPV4 therefore determines the sensitivity rather than the threshold of painful heat detection and plays an essential role in thermal hyperalgesia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sensation of warm to hot temperatures is an essential afferent neural activity in animals. An insight into the molecular basis of this sensation has been obtained by discovery of a capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1)1 (1). TRPV1 encodes a nonselective cation channel protein originally identified as the receptor for capsaicin, the principal pungent constituent in hot peppers. In addition to capsaicin and related vanilloid compounds, this channel is activated by acidic pH or by painfully hot temperatures. Responses to noxious heat are diminished in mice lacking TRPV1 (2). Detectable responses to hot temperatures (42–52 °C) are absent in sensory neurons cultured from these animals. Therefore, TRPV1 plays an essential role in the detection of hot temperature. However, a significant component of heat responsiveness remains in TRPV1-knock-out mice and in skin-nerve preparations explanted from them (2, 3). These findings indicate the existence of TRPV1-independent mechanisms of heat detection.

The mechanisms by which mammals detect innocuous warm temperatures are even less well understood than the mechanism underlying noxious thermosensation. A subset of sensory nerve fibers responsive to heat ranging from 30 to 42 °C has been identified in vivo in a number of mammalian species (4, 5), although the heat transduction mechanisms accounting for such responsiveness have not been clarified. Recently, it has been reported that two TRPV1-related ion channel proteins, TRPV3 and TRPV4, can be activated by mild temperature elevations exceeding ~30–35 °C (6, 7). TRPV3 and TRPV4 are located in cultured keratinocytes and play an essential role in the detection of warm temperatures and hypoosmolarity in vitro (8).

We previously found that TRPV4 might be a mediator of sensory neuron responsiveness to hypoosmolarity as well as a contributor to mechanical nociception (9–11) in vivo by using mice lacking TRPV4. However, the role of TRPV4 in the detection of warm temperatures in vivo remains obscure.

The mice lacking TRPV1 showed impairment in detection of chemically induced inflammatory pain when tested on hot-plates over 50 °C (3). Carrageenan is a widely used reagent known for the ability to induce an acute inflammation. Carrageenan is a sulfated polysaccharide extracted mainly from an alga. When injected into the hind paw of a rat, foot volume had increased at 3 h after injection, the gain of which was used as an indicator of the inflammation or as an assay to test anti-inflammatory drugs (12). In the first 1.5 h of the early phase of inflammation, thermal hyperalgesia is observed without remarkable edema. Thus use of such inflammatory agents may amplify the role of TRPV4 in thermal sensation in vivo.

We thus examined the contribution of TRPV4 to the detection of warm temperatures and to chemically induced hyperalgesia using TRPV4-knock-out mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—Mice were maintained on ad libitum diet chow and tap water for drinking. TRPV4-knock-out mice were backcrossed into a C57BL/6J inbred strain (Clea Japan Inc., Tokyo) to 4–5 generations. The male TRPV4 null (TRPV4–/–) mice and the wild type (TRPV4+/+) mice were used at 8–12 weeks of age for hotplate tests and at 10–24 weeks of age for recordings of neuronal discharges. Foot volumes before and after injection of capsaicin, carrageenan, or a vehicle were measured by immersion of the foot in water. The Animal Experiment Committee at the Jichi Medical School approved all of the experimental procedures used in this study.

Hotplate Test—The hotplate test was performed by a conventional method. A mouse placed on a thermal plate jumped over a fence (13 cm in height) to escape from the temperature. The duration (s) until escape was measured. The test was performed in triplicate for each mouse.

Acute Inflammatory Responses—Injections of chemical reagents were performed under brief anesthesia with ether inhalation. For carrageenan hypersensitivity, carrageenan (20 mg/ml, Sigma) was suspended in an isotonic saline solution and injected into the plantar surfaces of both hind paws in a volume of 20 µl by using a 26-gauge needle under brief anesthesia with ether. After 20 min, mice were tested for thermal sensitivity by the hotplate test. For capsaicin hypersensitivity, capsaicin (20 mg/ml, Sigma) in an isotonic saline solution containing 10% ethanol and 0.5% Tween 20 was injected into both hind paws in a volume of 20 µl. After 20 min, thermal sensitivity was tested as described above.

Recording and Analysis—Recordings were performed under anesthesia with urethane (1 g/kg, intraperitoneal). To apply thermal stress immediately, an aluminum pipe perfused with temperature-controlled water was attached to the hind paw of the mouse. The temperature on the surface of the hind paw was measured with a thermometer. Frequency of discharges in vivo from the hind limbs was electrically measured with a DAM-8 amplifier (World Precision Instruments, Inc., Sarasota, FL) using a method described previously (11). Femoral neural signals recorded over a period of 15 s were stored on a computer and analyzed. Signal-to-noise ratios greater than 3 were stored, and the rates of discharges were determined using the burst analysis program in pSTAT (Axon Instruments, Inc., Union City, CA). This method enables simultaneous detection of a few neuronal activities. We measured activations of three neurons at most in the same recording when they were discriminated by magnitudes of amplitude. The data were analyzed using Student's t test or two-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
With a rise in hotplate temperature over 35 °C, a significant decrease in the latency of response was observed compared with the latency of response observed at room temperature. An increase in the temperature of the hotplate decreased the latency of response regardless of the genotype (Fig. 1).



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  		FIG. 1.
Results of hotplate tests on TRPV4+/+ and TRPV4–/– mice. TRPV4+/+ (open columns) and TRPV4–/– mice (filled columns) (n = 12) were subjected to hotplate escape tests. The duration until escape from the hotplate at a given temperature was measured in triplicate in each experiment.

 
We next examined responses of neurons in the hind paw to warm temperature by recording femoral nerve activities. We measured neuronal discharges of several levels through a microelectrode located in the extracellular space. Two representative levels of neuronal discharge are shown in Fig. 2a. In TRPV4+/+ mice, we found that the activity level with higher amplitude increased when the temperature of the hind paw was increased to 40 °C within 15 s. Mean count of discharges in the following 15 s were expressed as frequency (Hz). Neurons with discharges that increased by at least 1 Hz in response to the high temperature were regarded as sensitive neurons. About 80% (46 of the 56 trials) of the neurons in TRPV4+/+ mice were sensitive to a temperature of 40 °C. The number of sensitive neurons was much less (19 of the 129 trials) in TRPV4–/– mice, the difference between the numbers of sensitive neurons in TRPV4–/– and TRPV4+/+ mice being statistically significant (Fig. 2b, chi-square test).



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  		FIG. 2.
Neuronal responses to high temperatures in TRPV4+/+ and TRPV4–/– mice. a, a representative 15-s discharge of a warmth-sensitive neuron in a TRPV4+/+ mouse is shown. The discharge was recorded in the femoral nerve with the mouse's hind paw on the thermal device. After recording (at 25 °C), the device was heated to 40 °C within 15 s, and then recording was performed (at 40 °C). Two levels of activities (arrows) can be discriminated from the basal level (line). The higher one is regarded as a warmth-sensitive discharge. b, rates of detection of warmth-sensitive (40 °C) and heat-sensitive (50 °C) neurons are shown. Neurons with discharges that increased at least 1 Hz in response to high temperature were counted as sensitive neurons. The populations of neurons sensitive to 40 °C and to 50 °C are shown. A significant (*, p < 0.01) difference was obtained by the {chi}2 test. n.s, not significant. c, the mean discharges in 10 TRPV4+/+ and TRPV4–/– mice were recorded at temperatures from 25 to 50 °Cby5 °C increments (each n = 10). These discharges were initiated at the same frequency at 25 °C regardless of the genotype. A thermometer on the hind paw measured the temperatures. d, rundown of discharge in warmth-sensitive neurons. The temperature (40 °C) was maintained for 1 min, and the mean discharges in 10 TRPV4+/+ and TRPV4–/– mice were recorded. The fate of discharge was calculated every 15 s, and activities are expressed as a percentage of the initial activities.

 
With a rise in foot temperature, warmth-sensitive neuronal activity began at about 33 °C in TRPV4+/+ mice. Neuronal activity in TRPV4–/– mice hardly began until over 40 °C. Although the threshold of temperature was not exactly calculated, we observed that temperature-sensitive neuronal activity began at about 33 °C in TRPV4+/+ mice and at about 40 °C in TRPV4–/– mice.

Most of the neuronal activities in TRPV4–/– mice were high threshold-type (Fig. 2c). We observed another type of activity in TRPV4–/– mice. This activity was greatest around 30 °C and then decreased during the rise in temperature.

We also observed a time-dependent rundown when the foot was maintained at 40 °C in TRPV4–/– mice. The activity levels of neurons in TRPV4–/– mice and in TRPV4+/+ mice at 40 °C were defined as the base-line activity. The rundown in activity could be observed for the next 1 min in which the activity level decreased similarly in TRPV4+/+ mice and in TRPV4–/– mice (Fig. 2d).

Reproduction of the temperature-evoked discharge was examined in the same nerve. It took at least 30 min for recovery of the discharge in response to the high temperature. We therefore did not obtain an exact magnitude of discharge at 50 °C when we measured it after recording at 40 °C. We therefore independently examined the response of femoral nerve activity to heat (50 °C) on the hind paw. Surprisingly, the populations of heat-sensitive neurons in TRPV4+/+ mice (13 of 32 trials) and TRPV4–/– mice (22 of 42 trials) were not different.

The frequencies of discharge by the warmth (to 40 °C) and heat (to 50 °C) are summarized in Fig. 3a. Discharge was obtained 3–5 times in each mouse. At temperatures from 25 to 40 °C, the mean frequency of discharge in TRPV4+/+ mice increased significantly (from 4.3 ± 0.7 to 7.2 ± 1.0 Hz, n = 56, p < 0.01, paired t test), whereas the mean frequency of discharge in TRPV4–/– mice was not altered (from 3.6 ± 0.5 to 3.1 ± 0.4 Hz, n = 129). At temperatures from 25 to 50 °C, the mean frequencies of discharge in TRPV4+/+ and TRPV4–/– mice increased significantly. The frequency of heat-responsive discharge in TRPV4+/+ mice increased from 4.1 ± 0.7 to 10.0 ± 1.2 Hz (n = 32), and the mean frequencies of discharge in TRPV4–/– mice increased from 3.4 ± 0.4 to 8.1 ± 1.3 Hz (n = 42). The magnitudes of basal levels were significantly different.



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  		FIG. 3.
Frequencies of discharge in femoral nerves in response to high temperatures in TRPV4+/+ and TRPV4–/– mice. a, neuronal discharges were recorded 3–5 times in TRPV4+/+ and TRPV4–/– mice. The frequencies of discharge over periods of 15 s were plotted before and after application of a high temperature (40 °C in the upper panels and 50 °C in the lower panels) to the hind paws of TRPV4+/+ (left panels) and TRPV4–/– (right panels) mice. The sensitive neurons are indicated by open circles for TRPV4+/+ mice and by closed circles for TRPV4–/– mice. Means ± S.E. of frequencies are plotted by a circle with a bar. Significant differences were obtained by Student's t test (*, p < 0.05; **, p < 0.001). b, the activities of the sensitive neurons are summarized. Means ± S.E. of frequencies are plotted by a circle with a bar. The number of observations is shown in parentheses. Significant differences in the responses were obtained by two-way ANOVA (*), and the gains in activity by temperature were compared by Student's t test (**, p < 0.01).

 
Based on the data above, temperature-sensitive activities were analyzed separately (Fig. 3b). There were 19 nerves capable of response to warm temperatures (from 2.9 ± 1.0 to 5.5 ± 1.0 Hz) in TRPV4–/– mice. The warmth-sensitive nerves in TRPV4+/+ mice were activated from 3.3 ± 0.6 to 8.8 ± 1.0 Hz (n = 46). The increments of discharges were analyzed by two-way ANOVA, and the result indicated a significant difference (p < 0.001). The magnitude (5.6 ± 0.8 Hz) of evoked activity in TRPV4+/+ mice was significantly different from the magnitude (2.6 ± 0.5 Hz) of evoked activity in TRPV4–/– mice. Therefore, both the numbers and magnitudes of evoked activity of 40 °C-sensitive neurons in TRPV4–/– mice were decreased in comparison with the numbers and magnitudes of evoked activity in TRPV4+/+ mice.

At temperatures from 25 to 50 °C, the mean frequencies of discharge in TRPV4+/+ and TRPV4–/– mice increased significantly. The frequency of heat-responsive discharge in TRPV4+/+ mice increased from 7.0 ± 0.9 to 11.9 ± 1.24 Hz (n = 13), and the frequency of heat-responsive discharge in TRPV4–/– mice increased from 1.7 ± 0.4 to 7.4 ± 1.3 Hz (n = 22). The basal levels were significantly different. In contrast to the warmth-sensitive activity, neuronal discharge evoked by heat in TRPV4–/– mice was not significantly different from neuronal discharge evoked in TRPV4+/+ mice (two-way ANOVA). The magnitude (5.0 ± 1.4 Hz) of evoked activity in TRPV4+/+ mice was not significantly different from the magnitude (5.6 ± 1.1 Hz) of evoked activity in TRPV4–/– mice. Therefore, the number of heat-sensitive neurons in TRPV4–/– mice was the same as that in TRPV4+/+ mice, and the magnitude of evoked activity of the neurons in TRPV4–/– mice was not significantly different from the magnitude of evoked activity of the neurons in TRPV4+/+ mice.

Hyperalgesic response to thermal stimuli associated with inflammation was tested by injecting carrageenan into the hind paw and subsequent stimulation by a hotplate. We examined the recovery time of escape latency because brief anesthesia with ether was used for the injection. Twenty min was required to reproduce the same latency. The hind paw did not appear to be swollen during this procedure. Analysis of the carrageenan-induced hind paw inflammation showed that there were no significant differences between the volumes of the lower legs in TRPV4–/– and TRPV4+/+ mice either before or after injection of the irritants (0.26 ± 0.03 g before injection, 0.27 ± 0.03gat20 min after injection, 0.25 ± 0.03 g at 4 h after injection, n = 12, p > 0.1, ANOVA). At 20 min after carrageenan injection, TRPV4+/+ mice at 40 °C exhibited a significant decrease in latencies compared with base-line pre-inflammation responses (n = 10, paired t test, p < 0.001), whereas TRPV4–/– mice did not exhibit a difference in latencies compared with base-line pre-inflammation responses (n = 10) (Fig. 4a). Significant differences between carrageenan-injected TRPV4+/+ and TRPV4–/– mice were found at all temperatures tested except 50 °C (Fig. 4b).



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  		FIG. 4.
Hyperalgesic tests on TRPV4+/+ and TRPV4–/– mice by carrageenan injection. TRPV4+/+ (open columns) and TRPV4–/– mice (filled columns) (n = 10) were subjected to hotplate escape tests. The duration until escape from the hotplate at a given temperature was measured in triplicate in each experiment. a, carrageenan was injected into the hind paws of the mice, and the tests were performed before injection and at 20 min and 4 h after injection. b, under brief anesthesia, carrageenan was injected into the hind paws of mice, and the test was performed 20 min after the injection. Significant differences were obtained by Student's t test (**, p < 0.001). c, the mean discharges in 10 TRPV4+/+ and TRPV4–/– mice were recorded at 25 to 50 °C. The mean discharges are plotted for TRPV4+/+ (n = 10, open circles) and TRPV4–/– mice (n = 10, closed circles). Three types of neuronal activities are independently plotted in TRPV4–/– mice. The number of mice is shown in parentheses.

 
To investigate the neuronal response to carrageenan, we identified warmth-sensitive neurons and then injected carrageenan. We observed that carrageenan-induced discharges were quite different in magnitude from the discharges of the warmth-sensing neurons (n = 20). Nonetheless, the threshold of warmth-sensitive neuronal activity was lowered at 20 min after injection of carrageenan in TRPV4+/+ mice. On the other hand, warmth-sensitive neurons of the high threshold-type (n = 7) or of the low threshold-type, possessing peak activity in 30 °C (n = 2), remained unchanged in TRPV4–/– mice. We could find one neuron in TRPV4–/– activated at 29 °C like that in TRPV4+/+ after injection of carrageenan. Thus, knockout of TRPV4 eliminated the generation of the carrageenan-induced thermal hyperalgesic response of the femoral neurons.

We also injected capsaicin and found no significant swelling in our experimental setting (leg volumes, 0.26 ± 0.03 before injection, 0.27 ± 0.02 at 20 min after injection, and 0.27 ± 0.02 at 4 h after injection). In contrast to carrageenan injection, 2 of the 20 warmth-sensitive neurons showed sensitivity to capsaicin. Hotplate tests revealed significant differences between latencies at all temperatures tested, including 50 °C, in capsaicin-injected TRPV4+/+ and TRPV4–/– mice (Fig. 5).



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  		FIG. 5.
Hyperalgesic tests on TRPV4+/+ and TRPV4–/– mice by capsaicin injection. TRPV4+/+ (open columns) and TRPV4–/– (filled columns) mice (n = 10) were subjected to hotplate escape tests. The duration until escape from the hotplate at a given temperature was measured in triplicate in each experiment. a, capsaicin was injected into the hind paws of the mice, and the tests were performed before injection and at 20 min and 4 h after injection. b, under brief anesthesia, capsaicin was injected into the hind paws of the mice, and the test was performed 20 min after the injection. Significant differences were obtained by Student's t test (*, p < 0.05; **, p < 0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TRPVs are multisensitive cation channels that are potentiated by temperature (TRPV1, > 42 °C (1); TRPV2, > 52 °C (13), TRPV3, > 31 °C (6), TRPV4 > 25 °C (7)) in heterogeneously expressed cells. The present results suggested that TRPV4 plays a major role in the detection of a warm temperature rather than a hot temperature in vivo. TRPV4 plays a major role in determining temperature sensitivity. The range of temperatures activating TRPV4 in vitro is compatible with the present findings. The number and discharge frequency of warmth-sensitive neurons in TRPV4–/– mice were clearly decreased, whereas the number and discharge frequency of heat-sensitive neurons in TRPV4–/– mice, presumably possessing TRPV1, were not different in activity and in number (Fig. 3). In TRPV1-knock-out mice, escape latency in the hotplate test (over 50 °C) after injection of carrageenan was not changed, whereas escape latency at warm temperatures has not been tested (2). It is therefore reasonable to assume that TRPV1-containing neurons play a major role in heat detection by determining the threshold of pain when exposed to harmful heat. TRPV3 is another warm temperature-sensitive channel that has been proposed to form a heterogeneous multimer with TRPV1 in sensory neurons (14). Thus, whether TRPV3 plays a role in the detection of hot or warm sensations in vivo will remain unclear until experiments using TRPV3-knock-out mice are performed.

The sigmoid relation of neuronal activity against temperature was shifted to the left in TRPV4+/+ mice (Fig. 4) by injection of carrageenan, whereas neurons with high and low threshold-type activities were still observed in TRPV4–/– mice. Neurons with high threshold-type activities exhibited the sigmoid relation and may contain TRPV1/TRPV3. The activity of this type at warm temperature is not influenced by carrageenan-induced inflammation. Neurons with low threshold-type activities exhibited a decrease in activity at over 35 °C and may detect temperatures lower than 30 °C because maximum activity was observed at around 30 °C. Molecules that detect this range of temperatures have not been identified in vitro or in vivo. Such molecules might be detected in the future by using TRPV4–/– mice.

Warm temperature-sensitive neurons, which are apparently different from pain-sensitive neurons, are found in various species, including humans and monkeys (15, 16). Warmth-sensitive neurons were not sensitive to carrageenan and only 10% of them were sensitive to capsaicin in the present study. Warm temperature-sensitive neurons have been shown to possess mechanosensitive and chemosensitive units (16, 17). These findings are in accordance with the characteristics of TRPV4–/– mice, which exhibit impaired responses to pressure and acetic acid irritation (11).

In TRPV4–/– mice, inflammatory and thermal hyperalgesia induced by capsaicin and carrageenan was remarkably suppressed at a warm temperature. The contribution of TRPV4 to hyperalgesia has been suggested in a situation in which hypotonicity increases the TRPV4-mediated current in primary afferent nociceptive nerve fibers through prostaglandin E2 (9). We injected isotonic solution as a vehicle and did not find significant swelling of the hind paw at 20 min after the injection of the chemical irritants. Mouse foot swelling does not occur even 4 h after the injection (3). Thus, we examined the direct influence of chemical irritants rather than the indirect effect of swelling that caused mechanical stress to induce neural discharges. In this situation, many factors in addition to prostaglandin E2 play roles in hyperalgesia. Mechanisms of hyperalgesia have been investigated with reagents and various knock-out mice lacking receptors (18, 19), enzymes (20), mediators (21), and ion channels (22, 3). It is interesting that carrageenan-induced hyperalgesia is blocked by activation of cannabinoid receptors (23, 24). On the other hand, anandamide, a second messenger of cannabinoid receptors, activates TRPV4 in vascular endothelial cells (25). Mechanisms other than the cannabinoid pathway should be considered for the activation of TRPV4 under the condition of carrageenan-induced hyperalgesia.

It was recently suggested from the results of a study using antisense oligonucleotides that TRPV4 may play a role in the detection of taxol-induced inflammation at room temperature (26). The results suggesting the importance of TRPV4 in chemically induced inflammation are compatible with the present findings. Furthermore, they have suggested that activation of TRPV4 by taxol depends on the Src tyrosine kinase. Thus there are at least two pathways to activate TRPV4, the anandamide-arachidonic and the Src tyrosine kinase pathways. Although mediators and second messengers of chemical-induced inflammation may communicate by a complex mechanism, TRPV4 is one of the final pathways for sensing inflammation at a warm temperature.

There are several sites and cells that sense warm temperature. In addition to neurons, keratinocytes possess TRPV4 and TRPV3 and outward cation currents are evoked by a temperature of 42 °C. Most keratinocytes from a TRPV4–/– mouse lacked the ability to respond to a warm temperature, although the current based on TRPV3 partially remained (8). Thus, TRPV4 in keratinocytes also plays an important role in the mechanism of thermal hyperalgesia.

Thermosensitive channels can be targets of drugs to relieve pain. Acid-sensing cation channel 3 (22) and TRPV1 (3) have been reported to play important roles in thermal hyperalgesia. The importance of these channels has been indicated by results of an experiment using carrageenan-injected hind paws subjected to a temperature of > 50 °C by a highly intensified beam. However, the contribution of these channels in thermal hyperalgesia from moderate temperatures (35–45 °C) is negligible in vivo.

The absence of warmth-sensitive neurons in TRPV4–/– mice was caused from knock-out of this gene. We have made mice lacking TRPV4 by insertion of a neomycin-resistant gene into the 4th exon. Thus, full-length mRNA was not detected in our TRPV4–/– mice. However, a part of the product of TRPV4 could be detected in our TRPV4–/– mice because an N-terminal spliced form of TRPV4 (amino acids 314 to end) might be produced in wild mice. This spliced form, if present, was not functionally active when exogenously expressed in Chinese hamster ovary cells (27) or when endogenously detected in cultured keratinocytes (8). Interestingly, others have independently constructed mice lacking TRPV4 in which the resistant gene disrupted an exon near the C-terminal region (28). If their phenotype differs from ours, the difference may be because of the spliced form of TRPV4.

We conclude from the results of this study that TRPV4 plays a major role in the detection of warmth in the sensory system and will become a novel target for treatment of inflammatory and thermal hyperalgesia.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology, Division of Molecular Pharmacology, Jichi Medical School 3311-1, Yakushiji, Minamikawachi, Tochigi 329-0498, Japan. Tel.: 81-28-558-7326; Fax: 81-28-544-5541.

1 The abbreviations used are: TRPV, transient receptor potential vanilloid; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank Yuki Oyama for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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