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Phosphorylation-Dependent Desensitization by Anandamide of Vanilloid Receptor-1 (TRPV1) Function in Rat Skeletal Muscle Arterioles and in Chinese Hamster Ovary Cells Expressing TRPV1

Erzsébet Lizanecz, Zsolt Bagi, Enik T. Pásztor, Zoltán Papp, István Édes, Noémi Kedei, Peter M. Blumberg, and Attila Tóth

Division of Clinical Physiology, Institute of Cardiology, University of Debrecen, Debrecen, Hungary (E.L., Z.B., E.T.P., Z.P., I.É., A.T.); and Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (N.K., P.M.B., A.T.)

Received for publication June 10, 2005.

Accepted for publication December 6, 2005.

	Abstract

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It has been proposed that activation of vanilloid receptor-1 (TRPV1) affects the vasotone of resistance arteries. One of the endogenous activators of TRPV1 is anandamide. The effects of anandamide on TRPV1 responsiveness were tested on isolated, pressurized (80 mm Hg) skeletal muscle (m. gracilis) arterioles (179 ± 33 µm in diameter). We found that the TRPV1 agonist capsaicin (1 µM) elicited a substantial constriction in isolated arterioles (51 ± 12%). In contrast, anandamide (0-100 µM) did not affect arteriolar diameter significantly (3 ± 5%). Isolated vessels were also preincubated with anandamide (30 µM for 20 min). This anandamide pretreatment completely blocked capsaicin-induced arteriolar constriction (response decreased to 1 ± 0.6%), and this inhibition was reversed by a protein phosphatase-2B inhibitor (cyclosporin-A; 100 nM, 5 min) treatment (constriction, 31 ± 1%). An exogenous TRPV1-expressing cell line [Chinese hamster ovary (CHO)-TRPV1] was used to specifically evaluate TRPV1-mediated effects of anandamide. The efficacy of anandamide in this system, as determined by ⁴⁵Ca²⁺ uptake, was 65 ± 8% of that of capsaicin. Upon treatment of the cells with cyclosporin-A or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), anandamide was transformed to a full agonist. Anandamide treatment caused an acute desensitization in these cells as measured by intracellular Ca²⁺ imaging. Application of cyclosporin-A or PMA reversed this desensitization. Our data suggest that anandamide may cause a complete (albeit phosphorylation-dependent) desensitization of TRPV1 in skeletal muscle arterioles and in CHO-TRPV1 cells, which apparently transforms the ligand-gated TRPV1 into a phosphorylationgated channel. This property of anandamide may provide a new therapeutic strategy to manipulate TRPV1 activity.

Compared with the well characterized effects of TRPV1 upon stimulation by exogenous substances (Szallasi and Blumberg, 1999), relatively little is known about its endogenous ligands. So far, anandamide (N-arachidonoyl-ethanolamide) (Zygmunt et al., 1999), N-arachidonoyl-dopamine (NADA) (Huang et al., 2002), and different lipoxygenase products (Hwang et al., 2000) have been identified as endogenous agonists of TRPV1. Among them, anandamide was originally identified as an endocannabinoid activating the G-protein-coupled metabotropic cannabinoid CB1 receptor (Devane et al., 1992).

The reported potencies of anandamide to activate TRPV1 and CB1 receptors are in the same range (from 10 nM to 10 µM) (Di Marzo et al., 2002b; Ross, 2003). It is noteworthy that the potency and efficacy of anandamide is affected by its metabolism. Ross et al. (2001) reported only low efficacy of anandamide at TRPV1 receptors overexpressed in CHO cells, which was greatly enhanced by inhibition of fatty acid amide hydrolase.

As has been pointed out previously (Ross, 2003), the partial activation of TRPV1 may evoke partial inhibition for other endogenous ligands. Such ligands could be NADA (Huang et al., 2002) or other as-yet-unidentified endogenous ethanolamides (Szolcsanyi, 2000). It has also been suggested that stimulation of TRPV1 by anandamide leads to desensitization to subsequent agonist challenge (tachyphylaxis) (Smart et al., 2001; Tognetto et al., 2001).

Both anandamide and capsaicin cause vasodilation of mesenteric, hepatic, basilar, and meningeal arterioles (Zygmunt et al., 1999; Ralevic et al., 2001; Harris et al., 2002; Akerman et al., 2004; O'Sullivan et al., 2004) through the stimulation of cannabinoid and TRPV1 receptors. In these studies, anandamide-mediated vasorelaxation was mainly (Zygmunt et al., 1999; Ralevic et al., 2001; Harris et al., 2002; Akerman et al., 2004) or partly (O'Sullivan et al., 2004) attributed to the activation of TRPV1. In mesenteric arteries, TRPV1 activation produces vasodilation as a result of calcitonin gene-related peptide release from the sensory nerve terminals innervating the blood vessels (Zygmunt et al., 1999). However, it has also been reported that activation of TRPV1 can cause vasoconstriction in rat (Scotland et al., 2004) and canine (Porszasz et al., 2002) mesenteric arteries or in coronary vessels (Szolcsanyi et al., 2001). In these latter cases, TRPV1-mediated substance P (Scotland et al., 2004) or endothelin (Szolcsanyi et al., 2001) release was suggested as possible mechanisms. Moreover, it was also reported that in vivo anandamide treatment (0.075-3 mg/kg) caused a transient vasoconstriction in renal, mesenteric, and hindquarters vessels in conscious rats (Gardiner et al., 2002). This latter constriction lasted less than 60 s and was not mediated by cannabinoid receptors. In contrast, little is known about the functional role of TRPV1 in skeletal muscle arterioles. In addition, the vascular and cellular effects of agonists (if any) at high concentrations for a longer period of time are not addressed.

In this study, we present evidence that functional TRPV1 is expressed in skeletal muscle arterioles and its stimulation by capsaicin leads to robust vasoconstriction in isolated vessels possessing spontaneous myogenic tone. In contrast, anandamide stimulation was without short-term effects on the same vessels but caused complete desensitization of TRPV1 receptors to capsaicin. This anandamide-mediated desensitization of TRPV1 was reversed by calcineurin (protein phosphatase 2B) inhibition. To investigate the underlying mechanisms, a TRPV1-overexpressing cell line was used. In this system, anandamide caused a desensitization of TRPV1 that was modulated by PKC and protein phosphatase 2B in the continuous presence of anandamide.

Our data suggest that anandamide can be an endogenous desensitizer of TRPV1, in addition to being a partial agonist of this receptor. We propose a mechanism for the indirect gating of TRPV1 by reversible phosphorylation in the continuous presence of anandamide and suggest that this behavior, in general, represents a potential therapeutic approach for modulating TRPV1 activity by partial agonists.

	Materials and Methods

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Isolation of Skeletal Muscle Arterioles and Measurement of Vessel Diameter. Wistar rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Using microsurgical instruments and an operating microscope, a gracilis muscle arteriole (1.5 mm long) running intramuscularly was isolated and transferred into an organ chamber containing two glass micropipettes filled with physiological salt solution composed of 110 mM NaCl, 5.0 mM KCl, 2.5 mM CaCl₂, 1.0 mM MgSO₄, 1.0 mM KH₂PO₄, 5.0 mM glucose, and 24.0 mM NaHCO₃ equilibrated with a gas mixture of 10% O₂ and 5% CO₂, balanced with nitrogen, at pH 7.4. Vessels were cannulated on both ends and micropipettes were connected with silicone tubing to an adjustable physiological salt solution reservoir. Inflow and outflow pressures were set to 80 mm Hg. Temperature was set at 37°C by a temperature controller. The internal arteriolar diameter at the midpoint of the arteriolar segment was measured by videomicroscopy (Bagi et al., 2005). Animal experiments were approved by the University of Debrecen Medical and Health Science Center and were in accordance with the standards established by the National Institutes of Health.

CHO-TRPV1 Cell Culturing. The selected stable CHO cell clone expressing TRPV1 (Tet-Off-induced CHO-TRPV1 cells) (Szallasi et al., 1999) was cultured in maintaining media (Ham's F12 medium supplemented with 10% bovine serum albumin (BSA), 25 mM HEPES, pH 7.5, 250 µg/ml G418 (Geneticin) (all from Life Technologies Inc., Rockville, MD) and 1 mg/l tetracycline (Calbiochem, La Jolla, CA). For ⁴⁵Ca²⁺ uptake, CHO-TRPV1 cells were plated in 24-well plates to yield a cell density of 20 to 40% confluence. The next day, the medium was changed to remove the tetracycline to induce TRPV1 expression. Experiments were performed approximately 48 h after induction. For intracellular Ca²⁺ concentration measurements, CHO-TRPV1 cells were plated on 25-mm, round, glass coverslips in maintaining media (Ham's F12 supplemented with 10% fetal bovine serum, 25 mM HEPES, pH 7.5, 250 µg/ml G418, and 1 mg/liter tetracycline). The next day, the medium was changed to inducing medium (maintaining medium without tetracycline but containing 1 mM sodium butyrate) to induce TRPV1 expression. Experiments were performed approximately 24 h after induction.

⁴⁵Ca²⁺ Uptake Experiments. Experiments were performed as described previously (Toth et al., 2004). In brief, CHO-TRPV1 cells were used immediately upon removal from the CO₂ incubator or were preincubated with 100 nM PMA, 100 nM cyclosporin-A (CY-A), or with 100 nM PMA and 100 nM CY-A applied together. For the ⁴⁵Ca²⁺ uptake assay, cells were incubated for 5 min at 37°C in a total volume of 500 µl of serum-free Dulbecco's modified Eagle's medium (Life Technologies Inc.) containing 1.8 mM CaCl₂ in the presence of 0.25 mg/ml BSA (Sigma, St. Louis, MO), 1 µCi/ml ⁴⁵Ca²⁺ (MP Bio-medicals, Irvine, CA), and increasing concentrations of anandamide. Maximal response was determined by the response caused by 300 nM capsaicin on the same plate. Immediately after the incubation, extracellular ⁴⁵Ca²⁺ was removed by washing the cells three times with ice-cold DPBS (Life Technologies Inc.) containing 1.8 mM CaCl₂. Then, 400 µl of radioimmunoprecipitation assay buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) was added to each well to lyse the cells. Plates were shaken slowly for 20 min. Then, 300 µl of cell lysate was transferred from each well into a scintillation vial and radioactivity was determined by scintillation counting. For each data point in each experiment, four wells were assayed. Data from these experiments were analyzed by computer fit to the Hill equation. Each experiment was performed at least three times.

Ca²⁺ Imaging. Experiments were performed as described previously (Toth et al., 2003). In brief, CHO-TRPV1 cells were transferred to DPBS containing 1 mg/ml BSA and 5 µM fura2-acetoxymethyl ester (Molecular Probes, Eugene, OR) for 2 h at room temperature. The cells were then kept in maintaining media at room temperature until the measurements, which were carried out in DPBS. The fluorescence of individual cells was measured with an InCyt Im2 fluorescence imaging system (Intracellular Imaging Inc., Cincinnati, OH). The cells within a field were illuminated alternately at 340 and 380 nm. Emitted light >510 nm was measured. Data were analyzed with the InCyt 4.5 software and further processed with Excel (Microsoft Corp, Redmond, WA) and Prism 2.0 (Graphpad Software, Inc., San Diego, CA) software.

Immunohistochemistry. Frozen skeletal muscle (m. gracilis) tissue samples were embedded in Tissue-Tek O.C.T compound (Electron Microscopy Sciences, Hatfield, PA). Cryostat sections (thickness, 10 µm) were placed on adhesive slides and fixed in acetone for 10 min. The slices were blocked with normal goat sera (1.5% in phosphate-buffered saline; Sigma) for 20 min and stained with anti-capsaicin receptor antibody (raised in rabbit; Calbiochem) at 1:100 dilution in the blocking buffer. Then, the slices were incubated with a biotin-labeled anti-rabbit antibody (1:200, Vector Laboratories, Burlingame, CA) and the immunocomplexes were visualized by a Vector VIP substrate (Vector Laboratories) according to the manufacturer's instructions. Finally, the slices were also stained with methyl green to visualize the nuclei.

	Results

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The vasoactive effects of anandamide and capsaicin were measured in resistance arterioles (132-223 µm in diameter at 80 mm Hg) of skeletal muscle (m. gracilis) of the rat. The isolated arteries were mounted in a perfusion myograph system and developed spontaneous myogenic tone without the use of any vasoactive agent. The functionality of the endothelium was tested at the beginning of each experiment by administration of acetylcholine, whereas smooth muscle functions were checked by addition of norepinephrine (Table 1).

First, the effects of TRPV1 stimulation were tested on the isolated vessels. In this series of experiments, 1 µM capsaicin caused 58 ± 8% constriction (n = 5), which was blocked by the competitive TRPV1 antagonist capsazepine (10 µM, constriction was 2 ± 5%; n = 5, p < 0.01) suggesting that the capsaicin effects were TRPV1-specific (Fig. 1A). To investigate the desensitization of TRPV1 in this system in separate experiments, isolated vessels were incubated with 1 µM capsaicin for 20 min, followed by 40-min regeneration, and the vessels were tested again with 1 µM capsaicin. According to the results, the maximal constriction was 51 ± 12% (n = 5, Fig. 1B, capsaicin before), and it did not decrease significantly after the 20-min incubation with capsaicin, followed by a 40-min regeneration period (maximal response is 35 ± 7%, n = 5; Fig. 1B, capsaicin after, p = 0.29). To further pursue the specificity of TRPV1-specific constriction in this system, immunohistochemistry was also performed (Fig. 1C) and revealed a strong TRPV1-like immunostaining in the smooth muscle layer of skeletal muscle vessels.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Specificity of capsaicin-mediated vasoconstriction. Skeletal muscle arteries were isolated and tested with capsaicin. Response to 1 µM capsaicin was measured in the absence (A, first bar, n = 5) or in the presence of the TRPV1-specific antagonist capsazepine (10 µM, second bar, n = 5). To measure the capsaicin-mediated desensitization of TRPV1 in a separate series of experiments, vessels were treated with capsaicin (1 µM, 20 min, maximal response is shown on B, first bar), and the responsiveness of the same vessels were determined after 40 min regeneration (1 µM capsaicin, second bar, n = 5). Finally, the expression of TRPV1 was detected in the skeletal muscle arteries by immunohistochemistry (C). Consecutive slides of m. gracilis were stained with a polyclonal anti TRPV1 antibody (dilution 1:100; Calbiochem) and TRPV1 immunostaining was visualized with Vector VIP substrate. Slides with the omission of the primary antibody served as controls for background staining. The lumen (V) and the smooth muscle layer (arrows) of the vessels are shown.

Effects of anandamide on the skeletal muscle arteriolar diameter were tested by cumulative dose-response measurements. After 1 min of incubation, no significant changes in vessel diameter were detected over a range of 1 nM to 100 µM anandamide compared with the control (response to 100 µM is shown in Fig. 2; dilation, 3 ± 5%). It was also suggested previously that anandamide can desensitize the TRPV1 (Di Marzo et al., 2001a; Smart et al., 2001; Tognetto et al., 2001; Helyes et al., 2003; Toth et al., 2005). We therefore tested the effects of 30 µM anandamide (for 20 min followed by a 40-min regeneration period) on the responsiveness of TRPV1 in this system (Fig. 2). Capsaicin (1 µM) alone significantly constricted these vessels (constriction is 51 ± 12%; n = 5, p = 0.018). Capsaicin-induced constriction was completely abolished by anandamide (30 µM) pretreatment (constriction decreased to 1 ± 0.6%; n = 5). The inhibitory effect of anandamide on TRPV1 was reversed by the calcineurin (protein phosphatase 2B) inhibitor CY-A (100 nM; response to 1 µM capsaicin was 31 ± 1%; n = 4, p = 0.014 for constriction, but no significant difference from capsaicin alone, p = 0.12). Furthermore, anandamide caused a significant vasoconstriction (maximal constriction is 7 ± 2%; n = 4, p = 0.01) when applied after 100 nM CY-A (5-min pretreatment). CY-A alone had no effects on vessel diameter (1-1000 nM; n = 5, data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effects of anandamide on the diameter of isolated skeletal muscle arterioles. Skeletal muscle arteries were isolated and tested with anandamide and capsaicin. Response to anandamide (100 µM, n = 5) is shown on the first bar. In case of the second bar, vessels were preincubated with 100 nM CY-A for 5 min before anandamide stimulation (n = 4). Capsaicin response (1 µM, n = 5) is shown on the third bar. The fourth bar represents the capsaicin response after anandamide treatment (30 µM, 15 min, n = 5). The fifth bar shows the response to capsaicin after incubation with anandamide (30 µM for 15 min) and CY-A (100 nM, for 5 min at the end of anandamide treatment, n = 5). Bars denote mean ± S.E.M. Significant differences from control (p < 0.05) are labeled by asterisks. Ana, anandamide; Caps, capsaicin.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Characterization of the agonist effect of anandamide on CHO-TRPV1 cells. CHO-TRPV1 cells were treated with different concentrations of anandamide (1-200 µM) for 5 min and the 45Ca2+ uptake was measured by scintillation counting. Dose-response relations for anandamide were determined in the absence of effectors (control, n = 7) or in the presence of the fatty acid amide hydrolase inhibitor PMSF (0.5 mM, n = 4), the calcineurin inhibitor CY-A (100 nM, n = 5), the PKC activator PMA (100 nM, n = 5), or in the simultaneous presence of CY-A and PMA (100 nM CY-A + 100 nM PMA, n = 3). Four parallel determinations were done in each separate experiment and a representative experiment is shown in A. The data from the replicates are summarized in B [maximal effects (Bmax) compared with the effect of 300 nM capsaicin on the same plate; bars are mean ± S.E.M.] and C (apparent Kd values; bars are mean ± S.E.M.). Significant differences versus control (p < 0.05) are labeled by asterisks.

To test the anandamide-mediated desensitization specifically on TRPV1 receptors, CHO-TRPV1 cells, which do not express cannabinoid receptors, were preincubated with anandamide (1-100 µM for 15 min at 37°C), then ⁴⁵Ca²⁺ uptake was initiated by 50 nM capsaicin (the half-maximally effective dose of capsaicin in this system). Anandamide inhibited the capsaicin-induced ⁴⁵Ca²⁺ uptake in a dose-dependent manner (Fig. 4) with an apparent IC₅₀ value of 21 ± 2 µM (n = 3). This IC₅₀ is near the K_d determined in the previous assays (Fig. 3, K_d = 30 ± 6 µM, n = 7).

View larger version (11K): [in this window] [in a new window]   Fig. 4. Antagonist effect of anandamide on CHO-TRPV1 cells. CHO-TRPV1 cells were treated with anandamide (0-100 µM) for 15 min, then 45Ca2+ and 50 nM capsaicin (elicits half-maximal effects in this system) was added for an additional 5 min. The 45Ca2+ uptake was detected by scintillation counting. Four parallel determinations were performed at each point in each experiment. A representative result is shown of the three independent separate experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Sensitization of TRPV1 to anandamide by enhanced phosphorylation. CHO-TRPV1 cells were loaded with the Ca2+ indicator fura-2. Cells were pretreated with buffer alone (A, control), with 100 nM PMA (B) or with 100 nM CY-A (C) for 15 min. Then, intracellular Ca2+ elevations were evoked by addition of anandamide (30 µM) at 1 min. Responses were recorded for an additional 9 min. Responses of 27 to 49 individual cells were recorded and averaged in each separate experiment. A representative experiment is shown of the three independent experiments performed.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Resensitization of TRPV1 to anandamide by enhanced phosphorylation. CHO-TRPV1 cells were loaded with the Ca2+ indicator fura-2. Intracellular Ca2+ elevations were evoked by anandamide (30 µM) at 1 min. When the cells desensitized and responses reached a plateau (usually in 3-5 min), buffer alone (A, control), 100 nM PMA (B), or 100 nM CY-A (C) was added in the continued presence of anandamide and responses were recorded for an additional 5 min. Responses of 32 to 49 individual cells were detected and averaged in each separate experiment. A representative experiment is shown from the three experiments performed.

	Discussion

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The observed efficacy (65 ± 8% relative to that of capsaicin) and potency (30 ± 6 µM) of anandamide on TRPV1 is somewhat lower than that reported by others. In particular, Di Marzo et al. (2001b) determined a potency of anandamide of 800 ± 200 nM at CB1 receptors from rat brain and 350 ± 91 nM at TRPV1 receptors overexpressed in HEK cells. One explanation of these differences may be that fatty acid amide hydrolase activities are different in these systems. Indeed, Ross et al. (2001) reported efficacy of anandamide very similar to that reported here (10 µM anandamide had 17.8 ± 3.9% efficacy in their system, compared with 21.6 ± 2.6% determined here) using TRPV1 expressed in CHO cells. In addition, we confirmed that application of PMSF was able to sensitize TRPV1-expressing cells to anandamide, as described by Ross et al. (2001), although we found only a moderate sensitization compared with their measurements. Further possibilities may also contribute to the differences in the potency and efficacy of anandamide, including different levels of TRPV1 expression, differences in protein phosphorylation (Vellani et al., 2001), anandamide transporters (De Petrocellis et al., 2001), and other factors (Di Marzo et al., 2001a; Ross, 2003). Some of these other factors (PKC activation and protein phosphatase 2B inhibition) were also confirmed to be effective in this work.

The tissue concentration of anandamide usually ranges between 4 and 200 nM, but there are also data suggesting that much higher concentrations (more than 2 µM in the supernatant) could be synthesized by stimulated cultured primary neurons (Ahluwalia et al., 2003). In this study, we did not attempt to determine what concentration of anandamide is present physiologically. Instead, we accepted that anandamide is a physiological regulator of TRPV1 and tested our system to make sure we were using a sufficient concentration of anandamide to have effects on TRPV1. In addition, we have also shown that in our CHO-TRPV1 system the potency of anandamide is most likely to be underestimated (because of effects of PKC activation, calcineurin, and fatty acid amide hydrolase inhibition).

Our data suggest that the observed low efficacy of anandamide compared with capsaicin on TRPV1 can possibly be related to the desensitization of TRPV1. Indeed, it has been published that stimulation of TRPV1 leads to desensitization to subsequent agonist challenge (well known as tachyphylaxis) in the case of anandamide (Di Marzo et al., 2001b; Smart et al., 2001; Tognetto et al., 2001; Helyes et al., 2003). However, in contrast with the others, we used a model of acute desensitization (decrease in the responsiveness in the continuous presence of the agonist) instead of tachyphylaxis. The physiological difference between the two models is that in the case of tachyphylaxis, we expect repeated bursts of release of anandamide, whereas in case of acute desensitization, we expect the continuous presence of the anandamide. This behavior (acute desensitization) is not limited to anandamide because other endogenous TRPV1 ligands, such as NADA (Toth et al., 2003) or N-oleoyldopamine (Szolcsanyi et al., 2004) were also reported to cause acute desensitization, although without data about the role of phosphorylation.

One of the prominent mechanisms of TRPV1 desensitization is receptor dephosphorylation. It was suggested that PKC is one of the kinases involved in the phosphorylation of TRPV1 (Vellani et al., 2001; Bhave et al., 2003; Mandadi et al., 2004), whereas calcineurin was suggested as the phosphatase (Docherty et al., 1996). It has also been shown that changes in the phosphorylation state of TRPV1 could reverse the desensitization (tachyphylaxis) caused by repeated applications of 100 nM capsaicin (Mandadi et al., 2004). Considering that some of the PKC isozymes and the calcineurin are calcium-dependent enzymes, an intriguing possibility is that the actual activities of these kinases and phosphatase are most probable depends on the level of TRPV1 activation (intracellular Ca²⁺ concentration) and their Ca²⁺ sensitivity. In arterioles, activation of PKC led to TRPV1-independent vascular effects; therefore, we used the calcineurin inhibitor CY-A, which was without vasoactive effects when applied alone. Treatment with CY-A, indeed, reversed the desensitization of TRPV1 as evaluated with capsaicin.

To further investigate possible cellular mechanisms, we used a heterologous TRPV1 expression system in which TRPV1 of the rat was expressed in CHO cells, and we tested the effects of phosphorylation on the acute desensitization of TRPV1. The advantage of this system is that TRPV1-mediated effects could be specifically observed, because there is no endogenous expression of cannabinoid or TRPV1 receptors in the CHO cells. Both the potency and the efficacy of anandamide in this system were found to be similar to that reported previously (Ross, 2003). Moreover, the partial efficacy allowed the investigation of phosphorylation in regulating the responsiveness of TRPV1 to anandamide. Our data support the idea that both the potency and efficacy of anandamide are controlled by phosphorylation. The acute desensitization of TRPV1 by anandamide was strong enough to cause reduced capsaicin-mediated responses. Results of intracellular Ca²⁺ imaging experiments suggested that the partial efficacy of anandamide was the reason for the acute TRPV1 desensitization. This desensitization was blocked (Fig. 5) and reversed (Fig. 6) by PKC activation and calcineurin inhibition. Moreover, a higher ⁴⁵Ca²⁺ uptake was found in the case of CY-A compared with control or PMA treatments. This observation suggests that there might be multiple physiologically relevant phosphorylation sites in TRPV1, in addition to the sensitivity of the PKC sites to dephosphorylation catalyzed by calcineurin.

Based on our observations and those of others, here we propose a mechanism for the regulation of TRPV1 responsiveness upon stimulation with anandamide (Fig. 7). According to this model, we suggest three different phases: resting, activated, and desensitized. In the resting phase, there are no ligands present, and TRPV1 is inactive. The steady-state level of phosphorylation determines the sensitivity of TRPV1 to anandamide. The phosphorylation of TRPV1 is a function of the apparent phosphatase (e.g., protein phosphatase 2B, calcineurin) and kinase (e.g., PKC) activities. If TRPV1 phosphorylation is enhanced (e.g., by activation of PKC or inhibition of calcineurin) TRPV1 sensitivity to anandamide is higher. The second phase is the activation of the TRPV1 by anandamide. At the appearance of anandamide, the channel activity increases and the intracellular Ca²⁺ concentration rises. This activation, however, is followed by a rapid desensitization (third phase). During this acute desensitization period (in the continuous presence of the ligand), the activity of the receptor and the intracellular Ca²⁺ concentrations are apparently regulated by reversible phosphorylation: dephosphorylation keeps the TRPV1 desensitized (and the intracellular Ca²⁺ concentration low), whereas phosphorylation sensitizes the TRPV1. In this desensitized period, the TRPV1 behaves as a phosphorylation gated channel until anandamide is present in sufficient concentration. However, when anandamide concentration decreases, the channel will be closed independently from its phosphorylation state.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Proposed mechanism of TRPV1 responsiveness to anandamide in CHO-TRPV1 cells. Under resting conditions (bottom left) the phosphorylation state of TRPV1 (phosphorylated receptor is represented by -Pon the top of the cells, whereas dephosphorylated is without P on the bottom) is regulated by kinases (like protein kinase C, PKC) and protein phosphatase 2B (calcineurin, PP2B). This determines its sensitivity to ligands, like anandamide (). The Ca2+ concentration rises upon anandamide binding to TRPV1 (receptor is activated) as a result of the Ca2+ influx through the activated channel. It results in the higher activity of PP2B compared with PKC and the consequent dephosphorylation of the receptor. The dephosphorylated receptor remains in its desensitized state as far as anandamide is present. However, if the level of receptor phosphorylation increased (as a result of kinase activation or phosphatase inhibition) the receptor could be reactivated (resensitized). Finally, when anandamide dissociates, the receptor returns to its resting (inactivated) state irrespectively to its phosphorylation level.

Finally, these data add to the complexity of the physiological effects of anandamide. The role of anandamide as an endogenous activator of TRPV1 is under discussion (Smart and Jerman, 2000; Szolcsanyi, 2000; Zygmunt et al., 2000; Di Marzo et al., 2001a; Ross, 2003; Wang et al., 2003). Our data presented here add to the range of possibilities, suggesting that anandamide is able to desensitize TRPV1 in vivo. This desensitization might be modulated by CB1 receptors, when TRPV1 and CB1 receptors are expressed in the same cells (Helyes et al., 2003), or might be mediated by the stimulation of TRPV1 itself (CHO-TRPV1 data, presented here).

In summary, the physiological effects of anandamide on the vasculature are most probably mediated by both TRPV1 and CB1 receptors in general. It is therefore possible that anandamide has dual actions at CB1 and TRPV1 receptors: in the short term, anandamide activates the CB1 and TRPV1 receptors simultaneously, whereas at longer times, it can desensitize the TRPV1 receptors and convert them to a metabotropic (phosphorylation dependent) receptor. In most cases, both TRPV1 and CB1 stimulation lead to vasodilation, which makes it complicated to determine the relative contributions of these receptors to the vasodilation. However, it is shown here that TRPV1 stimulation leads to vasoconstriction in the case of skeletal muscle arterioles. Investigating the effects of anandamide on these vessels, we found a complete desensitization of TRPV1. Our data suggest that anandamide is an endogenous partial activator of TRPV1, but at least in some cases (as in the case of the skeletal muscle arterioles), it can function as a desensitizer of TRPV1, which apparently transforms the ligand gated TRPV1 to a phosphorylation gated channel.

	Footnotes

 
This research was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. In addition, A.T. and Z.B. were supported by the János Bolyai Research Fellowship and by the Hungarian Research Fund (OTKA F/15 048873 and 048837, respectively).

ABBREVIATIONS: TRPV1 or VR1, vanilloid (capsaicin) receptor 1; NADA, N-arachidonoyl-dopamine; CHO, Chinese hamster ovary; PKC, protein kinase C; CHO-TRPV1, Chinese hamster ovary cells expressing the rat vanilloid receptor 1; PMA, phorbol 12-myristate 13-acetate; CY-A, cyclosporin-A; DPBS, Dulbecco's phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride.

Address correspondence to: Dr. Attila Tóth, Division of Clinical Physiology, Institute of Cardiology, University of Debrecen, P.O. Box 1, 4004, Debrecen, Hungary. E-mail: atitoth{at}jaguar.unideb.hu

	References

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