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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

The endocannabinoid anandamide and cannabinoid (CB) receptors have been implicated in the hypotension in various forms of shock and in advanced liver cirrhosis. Anandamide also activates vanilloid TRPV1 receptors on sensory nerve terminals, triggering the release of calcitonin gene-related peptide which elicits vasorelaxation in isolated blood vessels in vitro. However, the contribution of TRPV1 receptors to the in vivo hypotensive effect of anandamide is equivocal. We compared the cardiac performance of anaesthetized TRPV1 knockout (TRPV1−/−) mice and their wild-type (TRPV1+/+) littermates and analysed in detail the haemodynamic effects of anandamide using the Millar pressure–volume conductance catheter system. Baseline cardiovascular parameters and systolic and diastolic function at different preloads were similar in TRPV1−/− and TRPV1+/+ mice. The predominant hypotensive response to bolus intravenous injections of anandamide and the associated decrease in cardiac contractility and total peripheral resistance (TPR) were similar in TRPV1+/+ and TRPV1−/− mice, as was the ability of the CB1 receptor antagonist SR141716 to completely block these effects. In TRPV1+/+ mice, this hypotensive response was preceded by a transient, profound drop in cardiac contractility and heart rate and an increase in TPR, followed by a brief pressor response, effects which were unaffected by SR141716 and were absent in TRPV1−/− mice. These results indicate that mice lacking TRPV1 receptors have a normal cardiovascular profile and their predominant cardiovascular depressor response to anandamide is mediated through CB1 receptors. The role of TRPV1 receptors is limited to the transient activation of the Bezold-Jarisch reflex by very high initial plasma concentrations of anandamide.

The biological effects of marijuana and its main psychoactive ingredient, Δ9-tetrahydrocannabinol (THC), are mediated by specific receptors. To date, two cannabinoid (CB) receptors have been identified by molecular cloning: the CB1 receptor, which is highly expressed in the brain (Matsuda et al. 1990), but is also present in peripheral tissues including the heart and vascular tissues (Gebremedhin et al. 1999; Liu et al. 2000; Bonz et al. 2003), and the CB2 receptor, expressed primarily by immune and haematopoietic cells (Munro et al. 1993). The natural ligands of these receptors are lipid-like substances called endocannabinoids, which include arachidonoyl ethanolamide or anandamide and 2-arachidonoylglycerol (reviewed by Mechoulam et al. 1998). Cannabinoids elicit not only neurobehavioural and immunological effects, but also cardiovascular effects such as profound hypotension (Lake et al. 1997a; Hillard, 2000; Kunos et al. 2002; Randall et al. 2002; Ralevic et al. 2002). Anandamide has been implicated in the pathomechanism of hypotension associated with various forms of shock, including haemorrhagic (Wagner et al. 1997), endotoxic (Varga et al. 1998) and cardiogenic shock (Wagner et al. 2001a), and advanced liver cirrhosis (Bátkai et al. 2001). Increased sensitivity of hypertensive rats to the hypotensive action of anandamide (Lake et al. 1997b) could also suggest a role for endocannabinoids in hypertension.

In anaesthetized rats, intravenous administration of anandamide causes a triphasic blood pressure response, in which the major, prolonged hypotensive effect (phase III) is preceded by a transient, vagally mediated fall in heart rate and blood pressure (phase I) followed by a brief, non-sympathetically mediated pressor response of unknown mechanism (phase II) (Varga et al. 1995). Also in anaesthetized rats it has been observed (Malinowska et al. 2001) that the phase I bradycardic response was dose-dependently inhibited by the vanilloid TRPV1 receptor antagonist capsazepine and the non-selective inhibitor ruthenium red. These two inhibitors had no effect on the phase III hypotension, which was abolished by the cannabinoid CB1 receptor antagonist SR141716 (Malinowska et al. 2001) and was also absent in CB1 receptor knockout mice (Ledent et al. 1999; Járai et al. 1999).

At micromolar concentrations, anandamide binds to vanilloid TRPV1 receptors (Zygmunt et al. 1999), and there is evidence that the in vitro vasodilator effect of anandamide in certain vascular beds involves activation of TRPV1 receptors on sensory nerve terminals, causing the release of calcitonin gene-related peptide (CGRP) and the activation of CGRP receptors (Zygmunt et al. 1999). Interplay between the vanilloid and endocannabinoid systems has recently been implicated in blood pressure regulation in hypertension (Li et al. 2003). However, the involvement of TRPV1 receptors in the in vivo hypotensive response to anandamide is uncertain (Szolcsányi, 2000; Ralevic et al. 2002; Kunos et al. 2002) and only based on pharmacological inhibitors whose specificity has been questioned (Ray et al. 2003). Therefore, the aim of this study was to characterize the cardiovascular profile of anaesthetized TRPV1 knockout mice (TRPV1−/−) and their wild-type littermates (TRPV1+/+), and to use them for a detailed analysis of the haemodynamic effects of anandamide, including its effect on myocardial function, using the Millar pressure–volume conductance catheter system (Pacher et al. 2003). The results indicate that the predominant hypotensive effect of anandamide involves a profound decrease in cardiac contractility and is mediated exclusively by cannabinoid CB1 receptors in both TRPV1+/+ and TRPV1−/− mice, but the transient activation of the cardiogenic sympathetic reflex by very high initial concentration of anandamide involves TRPV1 receptors.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

All protocols were approved by the NIAAA Animal Care and Use Committee and were performed in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Haemodynamic measurements

Male TRPV1−/− and TRPV1+/+ mice weighing 25–30 g were used for the study. Heterozygote breeding pairs were kindly provided by Dr David Julius, University of California at San Francisco, and additional mice were purchased from the Jackson Laboratory, Bar Harbour, ME, USA. Mice were anaesthetized with pentobarbital sodium (80 mg kg−1i.p.) and tracheotomized to facilitate breathing. Animals were placed on controlled heating pads, and core temperature measured via a rectal probe was maintained at 37°C. A microtip pressure–volume catheter (SPR-839; Millar Instruments, Houston, TX, USA) was inserted into the right carotid artery and advanced into the left ventricle (LV) as described (Pacher et al. 2002a,b,c, 2003). Polyethylene cannulae (P10) were inserted into the right femoral artery and vein for measurement of mean arterial pressure (MAP) and administration of drugs, respectively. After stabilization for 20 min, the signals were continuously recorded at a sampling rate of 1000 s−1 using an ARIA pressure–volume conductance system (Millar Instruments) coupled to a Powerlab/4SP A/D converter (AD Instruments, Mountain View, CA, USA), and then stored and displayed on a computer. All pressure–volume loop data were analysed with a cardiac pressure–volume analysis program (PVAN3.2; Millar Instruments), and the heart rate (HR), maximal left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), MAP, maximal slope of systolic pressure increment (+dP/dt) and diastolic decrement (−dP/dt), ejection fraction (EF), stroke volume (SV), arterial elastance (Ea; end-systolic pressure/stroke volume), cardiac output (CO) and stroke work (SW) were computed. The relaxation time constant (τ), an index of diastolic function, was also calculated by two different methods (Weiss method: regression of log[pressure]versus time; Glantz method: regression of dP/dtversus pressure) using PVAN3.2. Total peripheral resistance (TPR) was calculated by the equation: TPR = MAP/CO.

In six additional TRPV1+/+ and six TRPV1−/− mice, haemodynamic parameters were determined under conditions of changing preload, elicited by transiently compressing the inferior vena cava (IVC) using a cotton swab, inserted through a small, transverse, upper abdominal incision. This technique yields very reproducible occlusions in mice without opening the chest cavity. Since +dP/dt may be preload-dependent (Kass et al. 1987), in these animals pressure–volume (PV) loops recorded at different preloads were used to derive other useful systolic function indices that may be less influenced by loading conditions and cardiac mass. These measures include the dP/dt–end diastolic volume (EDV) relation (dP/d –EDV) (Kass et al. 1987), the preload-recruitable stroke work (PRSW), which represents the slope of the relation between stroke work and EDV and is independent of chamber size and mass (Kass et al. 1987), and the end-systolic pressure–volume relation (ESPVR, Emax) (Nakano et al. 1990). The slope of the end diastolic pressure–volume relation (EDPVR), an index of LV stiffness, was also calculated from PV relations using PVAN 3.2. (see Table 1 and Fig. 1). At the end of the experiments, animals were killed by an overdose of anaesthetic (sodium pentobarbital).

Table 1.  Baseline haemodynamic parameters in TRPV1−/− and TRPV1+/+ mice measured by Millar pressure–volume conductance catheter system
 TRPV1+/+TRPV1−/−
  1. Values are means ±s.e.m. from 8–20 experiments.

HR (min−1)448.8 ± 19.8 492 ± 25
MAP (mmHg)81.6 ± 3.1 79.5 ± 2.2
LVESP (mmHg)100.8 ± 3.8  98.1 ± 2.7
LVEDP (mmHg) 6.0 ± 0.6  4.5 ± 0.7
CO (μl min−1)9610 ± 67210220 ± 974
EF (%)53.9 ± 2.2 49.6 ± 2.8
SW (mmHg μl)1697 ± 1181592 ± 99
+dP/dt (mmHg s−1)9174 ± 582 9110 ± 409
−dP/dt (mmHg s−1)7706 ± 549 7885 ± 345
τ (Weiss) (ms) 7.3 ± 0.5  6.3 ± 0.3
τ (Glantz) (ms) 12.2 ± 1   10.3 ± 0.6
TPR (mmHg ml−1 min) 9.5 ± 0.6  9.6 ± 1.2
SV (μl)21.4 ± 0.8 20.7 ± 1.3
Ea (mmHg μl−1) 4.6 ± 0.2  5.1 ± 0.5
Emax (mmHg μl−1) 7.4 ± 0.6  7.4 ± 0.4
PRSW (mmHg)67.6 ± 6.4 69.9 ± 6.6
(+dP/dt)/EDV (mmHg s−1μl) 250 ± 47  267 ± 86
EDPVR slope (mmHg μl−1) 0.19 ± 0.05  0.21 ± 0.04
image

Figure 1. Representative pressure–volume relations following vena cava inferior occlusions in TRPV1+/+ and TRPV1−/− mice Note that the slope of systolic and diastolic pressure–volume relations (ESPVR and EDPVR) are similar in both mouse strains.

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Calibrations

The volume calibration of this conductance system was performed as previously described (Pacher et al. 2003). Briefly, seven cylindrical holes in a block 1 cm deep and with known diameters ranging from 1.4 to 5 mm were filled with fresh heparinized whole murine blood. An interelectrode distance of 4.5 mm was used to calculate the absolute volume in each cylinder. In this calibration, the linear regression between the absolute volume in each cylinder versus the raw signal acquired by the conductance catheter was used as the volume calibration formula. At the end of each experiment, 10 μl of 15% saline was injected i.v. and from the shift of pressure–volume relation, parallel conductance volume (Vp) was calculated by PVAN 3.2 and used for correction for the cardiac mass volume as previously described (Pacher et al. 2003).

Drugs

Anandamide was from Tocris (Baldwin, MO, USA); SR141716 was from the National Institute on Drug Abuse Drug Supply Program (Research Triangle Park, NC, USA). Capsaicin was from Sigma Chemicals (St Louis, MO, USA). Anandamide and SR141716 were emulsified in corn oil: water (1: 4); capsaicin was dissolved in ethanol: Tween80: saline 1: 1: 8, as described (Malinowska et al. 2001).

Statistical analyses

In each phase, at least 10 PV loops were examined to generate an average. Time-dependent variables were analysed by ANOVA followed by Dunnett's post hoc test. In other cases, Student's t test was used, as appropriate. Values with P < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Characterization of cardiac function in TRPV1−/− and TRPV1+/+ mice

Baseline cardiovascular characteristics (MAP, LVSP, LVEDP, Ea, +dP/dt, −dP/dt, HR, EF, τ, SV, SW, CO and TPR) were not significantly different in TRPV1−/− and TRPV1+/+ mice (Table 1). Figure 1 illustrates typical PV loops obtained after inferior vena cava occlusions in both strains. Note that the slopes of systolic and diastolic pressure volume relations (ESPVR and EDPVR) are similar in TRPV1−/− and TRPV1+/+ mice (Fig. 1). The load-independent indices of contractility and left ventricular stiffness (Emax, dP/dt–EDV, PRSW, EDPVR) were also similar in both strains and are summarized in Table 1.

Haemodynamic effects of anandamide in TRPV1+/+ mice

Bolus injections of anandamide (20 mg kg−1i.v.) caused a triphasic effect in TRPV1+/+ mice (Figs 2, 3 and 5). The transient first phase, which lasted a few seconds, was characterized by profound decreases in cardiac contractility and heart rate and an increase in TPR, followed by a brief pressor response (phase II) associated with increased cardiac contractility. The third, prolonged hypotensive phase was characterized by decreased cardiac contractility and TPR and it lasted up to 10 min (Figs 2, 3 and 5). Pretreatment of the mice with a CB1 receptor antagonist, SR141716 (3 mg kg−1i.v.), had no effect on the first and second phases of the response to anandamide, but completely prevented the subsequent hypotension and the associated decreases in TPR and cardiac contractility (Figs 4 and 5).

image

Figure 2. Haemodynamic effects of anandamide in anaesthetized TRPV1+/+ (A) and TRPV1-/- (B) Representative recordings of the effect of intravenous injection of anandamide (20 mg kg−1, AEA) on mean arterial pressure (MAP, top panel) and cardiac contractility (LVSP and dP/dt; middle panel) and pressure–volume relations (bottom panel) in anaesthetized TRPV1+/+ (A) and TRPV1−/− (B) mice. The five parts of the middle and bottom panels represent baseline conditions (Bl), phase I, phase II, and phase III of the anandamide response and conditions 10 min after injection (10 min). The arrows indicate the injection of the drug.

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image

Figure 3. Haemodynamic effects of anandamide (AEA) in TRPV1+/+ (•) and in TRPV1−/− mice (bsl00043) Values are mean ±s.e.m. (n= 6 for each condition). *P < 0.05, versus baseline of TRPV1+/+; #P < 0.05, versus baseline of TRPV1−/−; †P < 0.05, TRPV1+/+versus TRPV1−/−. Arrow indicates anandamide injection (0 min).

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image

Figure 5. Peak haemodynamic changes in phases I, II and III of the anandamide response (20 mg kg−1, i.v.) in TRPV1+/+ (black and dark grey columns) and TRPV1−/− mice (open and light grey columns) pretreated with vehicle or SR141716 (grey) Values are means ±s.e.m. and are expressed as percentage change from baseline; *P < 0.05, versus baseline values; #P < 0.05, TRPV1+/+versus TRPV1−/− (n= 6 for each condition).

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image

Figure 4. Inhibition of CB1 receptor prevents major haemodynamic effects of anandamide (phase III) in both TRPV1+/+ (A) and TRPV1-/- (B) mice Representative recordings of the effects of anandamide (20 mg kg−1i.v., AEA) after pretreatment with SR141716 (3 mg kg−1, i.v.) on mean arterial pressure (MAP, top panel) and cardiac contractility (LVSP and dP/dt; middle panel) and pressure–volume relations (bottom panel) in anaesthetized TRPV1+/+ (A) and TRPV1−/− mice (B). The five parts of the middle and bottom panels represent the same five stages as described in the legend for Fig. 2. The arrows indicate the injection of the drugs.

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Haemodynamic effects of anandamide in TRPV1−/− mice

In TRPV1−/− mice, the anandamide-induced initial component (phase I), present in TRPV1+/+ littermates, was absent, and the phase II pressor response was also markedly reduced (Figs 4 and 5). In contrast, the subsequent prolonged hypotensive response accompanied by decreased cardiac contractility and TPR were similar to the responses observed in TRPV1+/+ mice, and were similarly completely antagonized by pretreatment with SR141716 (Figs 4 and 5).

Effect of capsaicin on MAP in TRPV1−/− and TRPV1+/+ mice

In TRPV1+/+ mice, capsaicin (10 and 100 μg kg−1i.v.) evoked a brief blood pressure response similar to phase I and II responses to anandamide (Fig. 6). This response was completely absent in TRPV1−/− mice.

image

Figure 6. Representative tracings of MAP illustrate the effect of i.v. capsaicin injections in TRPV1+/+ (top) and TRPV1−/− mice (bottom) Capsaicin doses: left, 10 μg kg−1; right, 100 μg kg−1. Similar response was seen in 4 experiments. The arrows indicate the injection of the drug.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

This is the first study to characterize the cardiovascular profile of TRPV1−/− mice and to describe the haemodynamic effects of anandamide using the Millar pressure–volume conductance catheter system. The results indicate that the predominant hypotensive effect of anandamide (phase III) involves a profound decrease in cardiac contractility in addition to decreased TPR, and is mediated by CB1 but not TRPV1 receptors. We also show that TRPV1−/− mice under pentobarbital anaesthesia have normal cardiac function and blood pressure.

The in vivo cardiovascular effects of cannabinoids are complex and may involve modulation of the autonomic outflow in both the central (Niederhoffer & Szabo, 2000) and the peripheral nervous systems (Ishac et al. 1996), as well as direct effects on the myocardium (Bonz et al. 2003) and vasculature (Gebremedhin et al. 1999; Járai et al. 1999; Wagner et al. 2001b). CB1 receptors are present in both the vasculature (Gebremedhin et al. 1999; Liu et al. 2000), where they mediate vasodilatation (Gebremedhin et al. 1999), and in the myocardium where they mediate negative inotropy (Bonz et al. 2003), and both of these sites may be implicated in the hypotensive effect of anandamide (Wagner et al. 2001). Although some synthetic cannabinoids can cause centrally mediated sympathoexcitatory effects (Niederhoffer & Szabo, 2000), the cardiovascular depressor effects of anandamide do not have a centrally mediated component (Varga et al. 1996). Additionally, presynaptic CB1 are present in sympathetic nerve terminals where their stimulation inhibits noradrenaline (norepinephrine) release (Ishac et al. 1996), and such receptors are likely to mediate the bradycardic effects of anandamide (Wagner et al. 2001b).

Structural similarities between anandamide and vanilloid compounds such as capsaicin (Di Marzo et al. 1998) raised the possibility of interplay between these two systems. Indeed, Zygmunt et al. (1999) demonstrated that in rat mesenteric arteries, the endothelium-independent vasodilator effect of anandamide is inhibited by the TRPV1 receptor antagonist capsazepine or by a calcitonin gene-related peptide (CGRP) receptor antagonist. They further demonstrated that anandamide binds to the cloned TRPV1 receptor with micromolar affinity, and at nanomolar concentrations it releases immunoreactive CGRP from sensory nerve terminals located in the vascular adventitia (Zygmunt et al. 1999). A similar involvement of TRPV1 receptors in the mesenteric vasodilator action of methanandamide has also been proposed (Ralevic et al. 2000). These observations support the hypothesis that anandamide acts at TRPV1 receptors in sensory nerves to release the potent vasodilator peptide CGRP.

The above results do not implicate the endothelium in the vasodilator effect of anandamide. In two other studies in which the vasodilatation induced by anandamide was found to have both endothelium-dependent and -independent components, the role of TRPV1 receptors was confirmed for the endothelium-independent component only (Járai et al. 1999; Mukhopadhyay et al. 2002). The endothelium-dependent vasodilator effect of anandamide is unaffected by capsazepine in rabbit aortic rings (Mukhopadhyay et al. 2002). Interestingly, sensory nerve terminals also appear to have CB1 receptors, stimulation of which by very low doses of anandamide or by the synthetic cannabinoid HU-210, neither of which interacts with TRPV1 receptors, inhibits sensory neurotransmission (reviewed in Ralevic et al. 2002). Furthermore, a recent study by Zygmunt et al. (2002) indicates that THC and cannabinol, but not other psychotropic cannabinoids, can elicit CGRP release from periarterial sensory nerves by a mechanism that is independent of not only CB1 and CB2 receptors, but also of vanilloid TRPV1 receptors. Thus, the sensory nerve-dependent effects of cannabinoids are complex, as interactions with CB1 and TRPV1 receptors appear to have opposite functional consequences and there may be additional actions independent of both of these receptors. TRPV1 receptors do not appear to be involved in the dilatation of isolated coronary arteries by anandamide either in the sheep, where the effect is endothelium dependent (Grainger & Boachie-Ansah, 2001), or in the rat, where it is endothelium independent (White et al. 2001). Furthermore, in the rat mesenteric arterial bed, the role of sensory nerves and vanilloid receptors in the dilator effect of anandamide was found to be conditional on the presence of nitric oxide (Harris et al. 2002).

TRPV1-containing afferent nerve fibres are present on the epicardial surface of the heart and the activation of these receptors by epicardially injected capsaicin evokes a sympathoexcitatory response with a brief increase in blood pressure (Zahner et al. 2003). Capsaicin infusion also induces a moderate pressor effect in pigs (Kapoor et al. 2003). In the present experiments, i.v. injection of 10 μg kg−1 capsaicin evoked only a brief pressor response, while at the much higher dose of 100 μg kg−1 its effect had both a depressor and a pressor component (Fig. 6). The finding that capsaicin elicited no change in blood pressure in TRPV1−/− mice (Fig. 6) suggests that TRPV1 receptors mediate the cardiogenic sympathetic or Bezold-Jarisch reflex in mice, which is in agreement with recent findings in rats (Zahner et al. 2003).

In the absence of anandamide-induced hypotension in CB1 knockout mice, the physiological relevance of the interaction of anandamide with TRPV1 receptors has been questioned (Szolcsányi, 2000). In the present study we have attempted to resolve this issue. We previously reported that anandamide causes a triphasic blood pressure response where the predominant hypotensive effect is preceded by a transient, vagally mediated drop in heart rate and blood pressure followed by a brief pressor response (Varga et al. 1995). Here we show that this initial component is missing in TRPV1−/− mice and is unaffected by the CB1 antagonist SR141716 in wild-type mice. Bolus intravenous injections of anandamide may reach high enough plasma concentrations for a few seconds to activate TRPV1 receptors, which may explain the above findings. Indeed, the phase I transient hypotension and bradycardia do not appear when anandamide is injected slowly to limit its peak plasma concentration (Kunos G, Bátkai S. & Pacher P, unpublished observations). In contrast, the prolonged hypotensive phase of the anandamide response is characterized by decreased cardiac contractility and TPR, which were similar in TRPV1+/+ and TRPV1−/− mice and were completely antagonized by SR141716, implicating CB1 receptors. In agreement with this observation, the sustained hypotensive and bradycardic effects of cannabinoids are totally absent in mice lacking the CB1 receptor (Ledent et al. 1999; Járai et al. 1999). Thus, TRPV1 receptors are not involved in the sustained cardiovascular response to anandamide, but may become transiently activated in response to pharmacological concentrations achieved after bolus i.v. injections. These findings are in agreement with a recent report by Malinowska et al. (2001), who found that in anaesthetized rats the transient vagal activation to a bolus injection of anandamide was partially blocked by the TRPV1 antagonists capsazepine or ruthenium red, whereas the CB1-mediated prolonged hypotension remained unaffected.

In conclusion, our results demonstrate that the sustained hypotensive effect of anandamide involves a marked cardiodepressor component in addition to a decrease in TPR, and these effects are mediated by CB1 but not TRPV1 receptors. We also demonstrate that TRPV1−/− mice have normal cardiac performance and blood pressure, and that TRPV1 receptors mediate the cardiogenic sympathetic reflex.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

Authors are indebted to Dr David Julius, University of California at San Francisco for kindly providing us with TRPV1−/− mice.