Characterization and comparison of recombinant human and rat TRPV1 receptors: effects of exo- and endocannabinoids{dagger}

P. M. W. Lam, J. McDonald and D. G. Lambert*

Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, LE1 5WW, UK

* Corresponding author. E-mail: DGL3{at}le.ac.uk

Accepted for publication December 21, 2004.


    Abstract
 Top
 Footnotes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. TRPV1 is a ligand-gated ion channel whose activation by capsaicin increases intracellular Ca2+ ([Ca2+]i). TRPV1 and cannabinoid CB1 receptor activation are capable of eliciting analgesia. In this study, using recombinant human (h) and rat (r) TRPV1 receptors expressed in HEK293 cells, we have performed a comparison of both TRPV1 species at 22 and 37°C and compared endo- and exocannabinoid activity at both receptors.

Methods. [Ca2+]i was measured in Fura-2-loaded HEK293hTRPV1 and HEK293rTRPV1 cells. To assess native CB1 receptor activity, [35S]GTP{gamma}S binding to membranes prepared from rat cerebellum was measured.

Results. Both capsaicin (pEC50 rat ~6.9 and pEC50 human ~6.8 at 37°C) and anandamide (pEC50 rat ~5.3 and pEC50 human ~5.8 at 37°C) produced a concentration-dependent increase in [Ca2+]i in rat and human systems and at 22 and 37°C. In HEK293rTRPV1 cells, anandamide appeared to be a partial agonist. Capsazepine demonstrated competitive antagonism at both human and rat TRPV1 receptors and at both temperatures studied. Capsazepine effects were not temperature dependent: pKB at rTRPV1 was 5.98 at 22°C and 6.02 at 37°C, and pKB at hTRPV1 was 6.76 at 22°C and 6.75 at 37°C. However, there was a consistent 6-fold increase in capsazepine potency for hTRPV1 relative to rTRPV1. The exocannabinoid {Delta}9-tetrahydrocannabinol failed to increase [Ca2+]i, although its solvent ethanol was an effective TRPV1 activator. In the [35S]GTP{gamma}S binding assay using rat cerebellar membranes, anandamide (pEC50 ~5.8) and {Delta}9-tetrahydrocannabinol (pEC50 ~7.1), but not capsaicin, stimulated binding. {Delta}9-tetrahydrocannabinol was a partial agonist. pEC50 values for anandamide at rTRPV1 and rCB1 were similar.

Conclusions. There were small differences in the pharmacology of rat and human TRPV1 receptors. Whilst capsaicin activated TRPV1 and {Delta}9-tetrahydrocannabinol activated CB1, anandamide is an endogenous agonist for both receptor systems.

Keywords: agonist, capsaicin ; analgesia ; cannabinoids, anandamide ; receptors, nociception ; receptors, vanilloid


    Introduction
 Top
 Footnotes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
TRPV1 (previously known as VR1) is a non-selective ligand-gated ion channel and is a member of the transient receptor potential (TRP) superfamily. Caterina and colleagues1 cloned TRPV1 from a rat dorsal root cDNA library encoding a protein of 838 amino acids with a molecular mass of 95 kDa. Cloning studies by other groups revealed close sequence homology across a range of species.24 A series of related TRPV channels have been described which display differences in activation and are proposed to function in a range of sensory roles.5 TRPV1 is relatively highly expressed in sensory neurones, with lower expression in brain, kidney, lung and spleen.1 3 Capsaicin, the pungent vanilloid component in chilli peppers, is the classic agonist at TRPV1 on C-fibres and a small population of A{delta}-fibres. Depolarization of the sensory nerves involves an influx of sodium and calcium ions with the generated action potential perceived as pain. Paradoxically, sustained application of capsaicin induces analgesia, which is likely to result from a chemical denervation.6

{Delta}9-Tetrahydrocannabinol (THC), the major psychoactive compound from maurijuana (Cannabis sativa), binds to CB1 located in mammalian brain.7 There is much current interest and controversy regarding the use of cannabis and cannabinoids for the treatment of pain.79

Identification of an endogenous ligand at TRPV1 is controversial. However, anandamide (AEA) has been proposed as an ideal candidate because of its structural similarity to capsaicin (Fig. 1) and lipids are thought to regulate TRP channel function.10 Moreover, this suggests some overlap between TRPV1 and CB1 receptor systems. In addition, AEA was found to produce vasodilation in arterial preparations, although this action was only sensitive to the TRPV1 antagonist capsazepine and not to the CB1 antagonist SR141716A.11 Also, AEA activates TRPV1 in recombinant and endogenous systems, which reinforces the possibility that it functions as an endovanilloid.12



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Fig 1 Structures of capsaicin and anandamide.

 
TRPV1 can also be described as polymodal as it can be activated by noxious heat (>43°C), protons and various lipids including the endocannabinoid AEA. Activation of recombinant TRPV1 by capsaicin, acid, heat and AEA is blocked by the competitive antagonist capsazepine,13 although there are marked differences in its antagonist potency against these different TRPV1 activators.

Mice lacking TRPV1 receptors displayed decreased capsaicin sensitivity and thermal hyperalgesia, implicating TRPV1 function in nociceptive processing and inflammation.14 Capsaicin is used in the pain clinic as a topical cream for chronic pain of neuropathic or musculoskeletal origin. A recent review concluded that capsaicin had moderate to poor efficacy in treatment of moderate or severe chronic pain. Also, local adverse effects limited continual use of capsaicin for some patients. However, capsaicin is beneficial to patients who are unresponsive to, or intolerant of, other treatments.15 Clearly, there is a need for non-pungent TRPV1 agonists and model systems in which to evaluate their activity. Measurements of intracellular Ca2+ in cells expressing recombinant TRPV1 receptors is of value as the TRPV1 receptor is located on nociceptors whose activation produces a Ca2+-dependent release of excitatory transmitters.16 Desensitization following capsaicin application is preceded by a rise in Ca2+;17 however, there was no capsaicin-mediated rise in Ca2+ in cultured dorsal root ganglion (DRG) neurones from TRPV1 knockout animals and no paw licking/shaking behaviour was observed in these animals.14

There have been few studies characterizing and comparing recombinant rat (r) and human (h) TRPV1 receptors. Therefore in this study we have made a detailed comparison of rat and human TRPV1 receptors heterologously expressed in human embryonic kidney (HEK293) cells (HEK293rTRPV1, HEK293hTRPV1). As an index of receptor activation, we have measured intracellular Ca2+ ([Ca2+]i) in Fura-2-loaded cells and examined the effects of a range of agonists and antagonists at the two most common experimental temperatures (22 and 37°C) to facilitate comparison with the literature. In addition, we have examined the effects of AEA (endocannabinoid) and THC (exocannabinoid) at both TRPV1 and CB1 (endogenously expressed in rat cerebellum) to compare exo- and endocannabinoids and any dual activation of TRPV1 and CB1 receptors.


    Materials and methods
 Top
 Footnotes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sources of materials
Triton X-100 was obtained from BDH Laboratory Supplies (UK); Folin's reagent, D-glucose, sodium chloride, sodium hydroxide and scintillation fluid were obtained from Fisher Scientific (UK); ethanol was obtained from Hayman Ltd (UK); cell culture media and reagents were obtained from Invitrogen (UK); [35S]GTP{gamma}S (1250 Ci/mmol) was obtained from Perkin–Elmer Life Sciences, Inc. (USA); anandamide, bacitracin, bovine serum albumin (BSA), capsaicin, capsazepine, dimethyl sulphoxide (DMSO), dithiothreitol (DTT), ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), Fura-2 acetoxymethyl ester (Fura-2AM), GTP{gamma}S, Sigmacote and THC were obtained from the Sigma Chemical Co. (UK); Whatman G/F B-filters were obtained from VWR International Ltd (UK). All other reagents were of the highest purity available.

Drug stocks
For fluorimetry, 10 mM stocks of capsaicin and anandamide were prepared in DMSO and further diluted in Krebs–HEPES buffer for the former and a mix of Krebs–HEPES buffer and DMSO for the latter (owing to the high insolubility of anandamide) when required. A 10 mM stock of capsazepine was prepared in DMSO and further diluted in Krebs–HEPES buffer. A 10 mM stock of THC was prepared in ethanol and further diluted in ethanol on ice when required.

For the [35S]GTP{gamma}S assay, capsaicin, anandamide and {Delta}9-THC stocks were diluted in DMSO because of buffer incompatibility.

Solvent controls were used as appropriate. All stocks were stored at –20°C and dilutions of the stocks were prepared fresh on the day of an experiment.

Cell culture
HEK293hTRPV1 and HEK293rTRPV1 cells (passages 18–30) were maintained in minimum essential medium (MEM) supplemented with 10% fetal calf serum, L-glutamine 0.2 mM, fungizone 2.5 µg ml–1, penicillin 100 IU ml–1 and streptomycin 100 µg ml–1 at 37°C in 5% carbon dioxide–air mixture. Cells were passaged with trypsin–EDTA when required and used when confluent.

Fluorimetric measurement of intracellular free calcium [Ca2+]i
Cells were detached, and were washed twice with and suspended in Krebs–HEPES buffer of the following composition: NaCl 143 mM, glucose 11.7 mM, HEPES 10 mM, KCl 4.7 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM and CaCl2 2.6 mM. Suspensions were incubated with a final concentration of 5 µM Fura-2AM for 30 min at 37°C. The cells were resuspended again in Krebs–HEPES buffer and incubated for a further 20 min at room temperature in the dark to allow for dye de-esterification. The cells were then resuspended in Krebs–HEPES buffer and stored on ice until required.

Aliquots (1.8 ml) of warmed Krebs–HEPES buffer and 200 µl cells were pipetted into a quartz cuvette containing a small magnetic stirrer that was finally placed ino a Perkin–Elmer LS50B fluorimeter. The temperature of the cuvette was maintained by external water tubes connected to a water bath that was heated 2°C above the desired temperature to compensate for intermediate cooling. The temperature was confirmed using a thermocouple thermometer. The cells were allowed to warm to the required temperature for 2 min prior to the addition of different agonists at varying concentrations. The antagonist capsazepine was pre-incubated for 10 min prior to the addition of agonist. As this was a cuvette-based assay, single concentrations of agonists were used in different cuvettes, i.e. concentration response curves were not cumulative. Fluorescence emission was measured at 510 nm with excitation at 340 nm and 380 nm. [Ca2+]i was estimated using built-in software (FLDM, Perkin–Elmer) and the Grynkiewicz equation.18 Rmax and Rmin were obtained with Triton-X100 and EGTA respectively. The Kd values for Fura-2 were 145 nM and 225 nM at 22°C and 37°C, respectively.18 19

[35S]GTP{gamma}S binding assay
[35S]GTP{gamma}S binding was performed according to methodology described by Berger and colleagues20 with modifications.21 All test tubes were treated with Sigmacote prior to experimenting to reduce adherence of the ligands (especially THC). All [35S]GTP{gamma}S buffers were adjusted to pH 7.4 with sodium hydroxide. Cerebella dissected from the brains of female Wistar rats were homogenized in buffer containing Tris–HCl 50 mM and EGTA 0.2 mM. Membrane fractions were collected by centrifugation for 10 min at 20 375g and 4°C. The homogenization and centrifugation was performed a total of three times. Stock [35S]GTP{gamma}S was reconstituted in Tris–HCl 50 mM and DTT 10 mM. The membrane fraction protein concentration was determined using the method of Lowry and colleagues.22 Then, 20 µg of membranes were incubated in 0.5 ml volumes of buffer containing Tris 50 mM, EGTA 0.2 mM, magnesium chloride 1 mM, sodium chloride 100 mM, bacitracin 0.15 mM, GDP 5 µM, [35S]GTP{gamma}S ~150 pM and different ligands at various concentrations. The reaction was incubated for 1 h at 30°C with gentle shaking and terminated by filtration through Whatman GF/B filters using a Brandel harvester. Non-specific binding was determined in the presence of GTP{gamma}S 10 µM.

Data analysis
Data are presented as mean (SEM) of n experiments or as single representative fluorimetric traces (from n experiments). [Ca2+]i concentration response curves were converted to {Delta}[Ca2+]i (maximum [Ca2+]i – basal [Ca2+]i) and analysed using GRAPHPAD PRISM (V3.0) to derive potency pEC50 (concentration producing 50% of the maximum response Emax) and efficacy Emax. [35S]GTP{gamma}S data were expressed as stimulation factor (ratio of agonist-stimulated specific binding to basal specific binding) and analysed using GRAPHPAD PRISM (V3.0). Fits were ‘sigmoid concentration response curves’. In capsazepine inhibition studies data were normalized to the maximum response produced by capsaicin alone and apparent pKB values (antagonist potency) were calculated using the Gaddum–Schild equation assuming a slope of unity:

Where appropriate, data were analysed using unpaired t-tests or analysis of variance (ANOVA) with the Bonferroni correction. Differences were considered significant when P≤0.05.


    Results
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 Footnotes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Typical temporal profiles
Capsaicin produced a time- and concentration-dependent increase in [Ca2+]i (Figs 2 and 3, and Table 1). Figure 2 shows typical traces in response to varying concentrations of capsaicin in HEK293rTRPV1 and HEK293hTRPV1 cells at 22 and 37°C. Addition of capsaicin at 60 s caused a rapid (~40 s) elevation in [Ca2+]i, and this reached a maximum at ~10 µM capsaicin. The [Ca2+]i response in HEK293hTRPV1 was greater than in HEK293rTRPV1, and we believe that this is suggestive of higher expression in the former cell line. In the HEK293hTRPV1 cells the response was biphasic in nature, with this profile being less pronounced at the lower temperature (22°C). Once the maximum [Ca2+]i response was achieved, there was a gradual decrease. Overall, there was some variability in [Ca2+]i responses between receptor species and temperatures; in HEK293rTRPV1, [Ca2+]i with 10 µM capsaicin ranged from 79 to 90 nM at 22°C and from 146 to 198 nM at 37°C. In HEK293hTRPV1, [Ca2+]i ranged from 188 to 475 nM at 22°C and from 488 to 1046 nM at 37°C.



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Fig 2 Typical traces obtained from fluorimetric measurements in a capsaicin concentration–response curve: (A) hTRPV1, 22°C; (B) rTRPV1, 22°C; (C) hTRPV1, 37°C; (D) rTRPV1, 37°C. Arrows indicate the addition of capsaicin at 60 s. Data are from single representative experiments as shown in Table 1.

 


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Fig 3 Concentration response curves for capsaicin (Cap) and AEA: (A) hTRPV1, 22°C; (B) rTRPV1, 22°C; (C) hTRPV1, 37°C; (D) rTRPV1, 37°C. Data are mean (SEM) (n=3–7).

 

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Table 1 Potency (pEC50) and efficacy (Emax) of capsaicin and anandamide at rat and human TRPV1 receptors. Data are mean (SEM).

 
Comparison of capsaicin and the endocannabinoid anandamide at TRPV1
Figure 3 shows capsaicin concentration response curves in HEK293hTRPV1 and HEK293rTRPV1 cells at 22 and 37°C and the corresponding pEC50 values are given in Table 1. Generally, there was no difference in the potency, which ranged from 132 to 275 nM, between receptor species and temperatures. However, the potency of capsaicin in HEK293rTRPV1 cells increased significantly at 37°C relative to 22°C (P=0.05). There was a temperature-dependent increase in Emax at both receptor species. AEA also produced a concentration-dependent increase in [Ca2+]i in HEK293hTRPV1 and HEK293rTRPV1 cells at 22 and 37°C (Figure 3 and Table 1). Overall, EC50 ranged from 1.5 to 4.8 µM. Similar to capsaicin, there was generally no difference in response to AEA. However, the efficacy at 37°C increased relative to that at 22°C at both receptors. At rat TRPV1 receptors (with lower expression), AEA acted as a partial agonist relative to capsaicin at both temperatures. The relative intrinsic activity values {alpha} of capsaicin relative to AEA at rTRPV1 were 0.36 and 0.29 at 22°C and 37°C, respectively, and at hTRPV1 were 0.96 and 0.75 at 22°C and 37°C, respectively.

Capsazepine antagonism
Capsazepine 30 µM competitively antagonized the capsaicin-mediated increase in [Ca2+]i in HEK293hTRPV1 and HEK293rTRPV1 at 22 and 37°C (Figure 4 and Table 2). Capsazepine was ~6-fold more potent at hTRPV1 than at rTRPV1. pKB analysis with AEA as the agonist could not be performed because of its low potency and relative insolubility.



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Fig 4 Capsazepine (Cpz) 30 µM produced a rightward shift in the concentration–response curve to capsaicin indicative of competitive antagonism. Data (mean [SEM], n=3–4) are normalized to the maximum capsaicin response in each individual experiment. (A) hTRPV1, 22°C; (B) rTRPV1, 22°C; (C) hTRPV1, 37°C; (D) rTRPV1, 37°C.

 

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Table 2 Effect of capsazepine on capsaicin-mediated increase in [Ca2+]i at TRPV1 at 22 and 37°C. Data are mean (SEM) (n=4 except at hTRPV1 22°C where n=3) and rTRPV1/hTRPV1 calculated from the antilog pKB values.

 
Effects of the exocannabinoid THC at hTRPV1
THC 100 µM (in 170 mM ethanol) produced a time-dependent increase in [Ca2+]i in HEK293hTRPV1 at 37°C (Fig. 5A). However, this could be mimicked by the addition of 170 mM ethanol alone and in a capsazepine (30 µM) sensitive manner (Fig. 5B). The ethanol effect was probed further and appeared to be concentration dependent. However, the response failed to saturate despite using concentrations up to 1.5 M (Fig. 5C).



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Fig 5 Effect of ethanol and THC on [Ca2+]i at hTRPV1 at 37°C. Addition of (A) THC 100 µM and (B) ethanol 170 mM (indicated by the initial arrows) produced a capsazepine-sensitive increase in [Ca2+]i. Capsazepine (Cpz) or DMSO addition is shown by the later arrow (capsazepine 30 µM in DMSO to 0.3% v/v final). (C) Ethanol concentration response at hTRPV1 at 22°C (n=4) and 37°C (n=5). Data are mean (SEM).

 
[35S]GTP{gamma}S assay
AEA and THC produced a concentration-dependent and saturable increase in [35S]GTP{gamma}S binding with pEC50 and Emax values (stimulation factor) of 5.79 (SEM 0.09) and 7.10 (0.13) (P<0.05) and 2.29 (0.06) and 1.54 (0.04) (P<0.05), respectively. THC was a partial agonist relative to AEA in this system and the relative intrinsic activity {alpha} was 0.41. Capsaicin failed to stimulate [35S]GTP{gamma}S binding (Fig. 6). The pEC50 value of AEA at CB1 is in agreement with that at hTRPV1 and rTRPV1 at 22 and 37°C (P>0.05).



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Fig 6 Effects of capsaicin (Cap), AEA and THC on [35S]GTP{gamma}S binding to CB1 receptors in rat cerebellar membranes. Data are mean (SEM) (n=3) and are expressed as stimulation factor (ratio of agonist-stimulated specific binding to basal specific binding).

 

    Discussion
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 Footnotes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Capsaicin and anandamide
TRPV1, a ligand-gated Ca2+-permeable ion channel, is involved in nociceptive signalling and this receptor is a therapeutic target in pain management.23 Capsaicin and AEA are agonists at recombinant rat and human TRPV1 receptors4 13 24 25 and native TRPV1 in sensory neurons.1 26 The capsaicin response at recombinant rat and human TRPV1 receptors was examined at 22 and 37°C (two commonly used experimental temperatures) and there was a temperature-dependent increase in maximum response (with no major changes in potency). However, we have previously reported for recombinant HEK293rTRPV1 cells that capsaicin potency decreased with increasing temperature, although in our original paper we went up to 50°C, a temperature that would activate TRPV1.24 We have no explanation for this discrepancy. At rTRPV1 the pEC50 value reported here of 6.56 for capsaicin (22°C) was lower than recently reported by us and others4 24 in transfected HEK293 cells. However, the pEC50 values at 37°C were in good agreement.24 At hTRPV1, our pEC50 values for capsaicin were 6.88 and 6.77 at 22°C and 37°C, respectively, and these were in reasonable agreement with those determined from FLIPR-based calcium assays in HEK293hTRPV1 at 25°C.13 25 Species differences for capsaicin have not been reported in other studies.4 13 25 At the higher capsaicin concentrations (100 µM) there was an apparent decline in Ca2+ responses which we feel may result from an acute desensitization.

The endocannabinoid AEA activates not only the CB1 receptor27 but also TRPV1.28 AEA response at hTRPV1 at 22°C (pEC50=5.82) is consistent with reported values of 5.6,13 5.9525 and 5.69.24 No difference in potency was observed for the AEA response at hTRPV1 or rTRPV1 with increasing temperature but there was an increase in maximum response, consistent with changes observed for capsaicin. Reports differ in classifying AEA as either a partial28 or full agonist at recombinant TRPV1,25 and this may be due to species or expression differences. We have shown that AEA is a partial agonist in rat, whilst in human a full agonist profile is observed. As more receptors are present in cells expressing the human clone, we suspect that higher expression coupled with the possible introduction of a receptor reserve increases relative intrinsic activity.

Capsazepine
Capsazepine is a competitive antagonist at TRPV1.29 We have clearly shown competitive antagonism at both rTRPV1 and hTRPV1 at 22 and 37°C with this molecule. Capsazepine potency was not temperature-dependent at TRPV1, as confirmed by others,30 but there was an ~6-fold increase in capsazepine potency for hTRPV1 compared with rTRPV1. This difference is consistent with that reported by McIntyre and colleagues.31 pKB values for this antagonist have proved highly variable; for example, in FLIPR-based assays, this varied from 6.58 to 7.31 in HEK293hTRPV1 cells13 25 and to 7.52 in HEK293rTRPV1.4 Other reports include values of 6.04 in guinea-pig trachea, 5.12 in guinea-pig bronchi (sensory neurones) and 6.65 at rTRPV1 transfected Chinese hamster ovary (CHO) cells.3133 The reason for these differences is unclear.

Investigation of exocannabinoid activity at TRPV1
The exocannabinoid THC activates CB1 receptors; however, activity at TRPV1 has not been extensively examined. We have attempted to determine whether THC was able to activate TRPV1 in a similar fashion to that of the endocannabinoid AEA. THC produced elevated [Ca2+]i levels but subsequent investigations with an ethanol control attributed this effect to ethanol only. These results are supported by ethanol activity at TRPV1 reported by Trevisani and colleagues.34 Capsaicin, AEA, protons and heat can potentiate the modulation of TRPV1 activity by ethanol. Also, the threshold for heat activation of TRPV1 dropped from 42 to 34°C. Ethanol was found to increase the potency and efficacy of capsaicin and caused the release of substance P from central and peripheral terminals of capsaicin-sensitive nociceptors in C- and A{delta}-fibres. Release of substance P is related to the pain sensation, and ethanol can lower the threshold of TRPV1 to body temperature; thus the data provided an explanation for the burning pain sensed by patients with oesophagitis after consuming alcoholic beverages.34 These data indicate that endo- rather than exocannabinoids are TRPV1 activators.

The [35S]GTP{gamma}S binding assay was employed to act as a positive control for THC activity and to allow a comparison of AEA potency at CB1 and TRPV1 to be made. Both AEA and THC activated the CB1 receptor. AEA displayed a lower potency than a previously reported pEC50 value of 6.56, while THC was consistent with the literature.35 Our data demonstrated that THC was a partial agonist relative to AEA. As expected, capsaicin had no effect at the CB1 receptor.

Speculation exists over the possibility that AEA and other similar unidentified lipid compounds are endogenous ligands at TRPV1. We have clearly demonstrated that AEA is active at both TRPV1 and CB1. It has been known for some time that TRPV1 receptors mediate the vasodilation produced in response to AEA.28 In summary, capsaicin activates TRPV1, THC activates CB1 and AEA activates both receptors. Therefore it is tempting to suggest that CB1 and TRPV1 may be metabotrophic and ionotrophic members of a family of endocannabinoid receptors.


    Acknowledgments
 
We thank GlaxoSmithKline (Essex, UK) for providing the HEK293TRPV1 clones and appreciate their continued advice and support, especially from Dr D. Smart. We are grateful to the British Journal of Anaesthesia and the Royal College of Anaesthetists for project grant funding (PhD studentship for PMWL).


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 Footnotes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
{dagger} Presented in abstract form in the following publications: Lam PMW, Smart D, Lambert DG. Anandamide but not {Delta}9-tetrahydrocannabinol activates recombinant human vanilloid receptors. Br J Anaesth 2003; 90: 418P; Lam PMW, Smart D, Lambert DG. Differences in the affinity of capsazepine at recombinant rat and human VR1 receptors. Br J Pharmacol 2003; 138: 220P. Back


    References
 Top
 Footnotes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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