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.
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Abstract |
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Methods. [Ca2+]i was measured in Fura-2-loaded HEK293hTRPV1 and HEK293rTRPV1 cells. To assess native CB1 receptor activity, [35S]GTPS 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
9-tetrahydrocannabinol failed to increase [Ca2+]i, although its solvent ethanol was an effective TRPV1 activator. In the [35S]GTP
S binding assay using rat cerebellar membranes, anandamide (pEC50
5.8) and
9-tetrahydrocannabinol (pEC50
7.1), but not capsaicin, stimulated binding.
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 9-tetrahydrocannabinol activated CB1, anandamide is an endogenous agonist for both receptor systems.
Keywords: agonist, capsaicin ; analgesia ; cannabinoids, anandamide ; receptors, nociception ; receptors, vanilloid
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Introduction |
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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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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.
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Materials and methods |
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Drug stocks
For fluorimetry, 10 mM stocks of capsaicin and anandamide were prepared in DMSO and further diluted in KrebsHEPES buffer for the former and a mix of KrebsHEPES 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 KrebsHEPES buffer. A 10 mM stock of THC was prepared in ethanol and further diluted in ethanol on ice when required.
For the [35S]GTPS assay, capsaicin, anandamide and
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 1830) were maintained in minimum essential medium (MEM) supplemented with 10% fetal calf serum, L-glutamine 0.2 mM, fungizone 2.5 µg ml1, penicillin 100 IU ml1 and streptomycin 100 µg ml1 at 37°C in 5% carbon dioxideair mixture. Cells were passaged with trypsinEDTA 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 KrebsHEPES 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 KrebsHEPES 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 KrebsHEPES buffer and stored on ice until required.
Aliquots (1.8 ml) of warmed KrebsHEPES buffer and 200 µl cells were pipetted into a quartz cuvette containing a small magnetic stirrer that was finally placed ino a PerkinElmer 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, PerkinElmer) 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]GTPS binding assay
[35S]GTPS 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
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 TrisHCl 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
S was reconstituted in TrisHCl 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
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
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 [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
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 GaddumSchild equation assuming a slope of unity:
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Results |
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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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Discussion |
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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-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]GTPS 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.
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Acknowledgments |
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Footnotes |
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References |
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