Abstract
The vanilloid receptor transient receptor potential type V1 (TRPV1) integrates responses to multiple stimuli, such as capsaicin, acid, heat, and endovanilloids and plays an important role in the transmission of inflammatory pain. Here, we report the identification and in vitro characterization of A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel, potent, and selective TRPV1 antagonist. A-425619 was found to potently block capsaicin-evoked increases in intracellular calcium concentrations in HEK293 cells expressing recombinant human TRPV1 receptors (IC50 = 5 nM). A-425619 showed similar potency (IC50 = 3–4 nM) to block TRPV1 receptor activation by anandamide and N-arachidonoyl-dopamine. Electrophysiological experiments showed that A-425619 also potently blocked the activation of native TRPV1 channels in rat dorsal root ganglion neurons (IC50 = 9 nM). When compared with other known TRPV1 antagonists, A-425619 exhibited superior potency in blocking both naive and phorbol estersensitized TRPV1 receptors. Like capsazepine, A-425619 demonstrated competitive antagonism (pA2 = 2.5 nM) of capsaicinevoked calcium flux. Moreover, A-425619 was 25- to 50-fold more potent than capsazepine in blocking TRPV1 activation. A-425619 showed no significant interaction with a wide range of receptors, enzymes, and ion channels, indicating a high degree of selectivity for TRPV1 receptors. These data show that A-425619 is a structurally novel, potent, and selective TRPV1 antagonist.
The vanilloid receptor VR1, recently termed TRPV1, is a nonselective cation channel predominantly expressed by peripheral nociceptors. TRPV1 receptors are readily activated by noxious chemicals, such as capsaicin and resiniferatoxin, protons (pH < 6.0), and noxious heat (>43°C) (Caterina et al., 1997; Tominaga et al., 1998). TRPV1 is also activated or potentiated by endovanilloids, such as anandamide, N-arachidonoyl-dopamine (NADA) (Huang et al., 2002), and N-oleoyl-dopamine (Chu et al., 2003) as well as eicosanoids, such as 12-hydroper-oxyeicosatetraenoic acid (Hwang et al., 2000).
An interesting property of this channel is that these diverse stimuli not only directly activate TRPV1 but also sensitize and reduce the activation thresholds of the channel to other stimuli (Di Marzo et al., 2002). For example, exposure of TRPV1-expressing cells to acidic conditions sensitizes the channel to activation by heat or by capsaicin (Tominaga et al., 1998). In addition, several studies have shown that TRPV1 receptors can be sensitized by inflammatory agents, including bradykinin, NGF, and ATP, acting via second messengers downstream of receptors for these agents (Cortright and Szallasi, 2004). Direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) or phorbol-12,13-dibutyrate (PDBu) leads to similar sensitization of TRPV1 responses to other stimuli (Di Marzo et al., 2002; El Kouhen et al., 2003). These data suggest that TRPV1 plays a key role in the integration of noxious signals after inflammation or tissue injury.
The prototypic TRPV1 receptor antagonist capsazepine has been extensively studied and shown to inhibit nocifensive and hyperalgesic responses not only to capsaicin but also to inflammatory agents (Nagy et al., 2004). However, capsazepine has only modest potency and low specificity, also antagonizing voltage-activated calcium channels (Docherty et al., 1997), acetylcholine receptors (Liu and Simon, 1997), and hyperpolarizing-activated cyclic nucleotide-gated channels such as HCN1 (Gill et al., 2004). Efforts from several groups have been directed to the development of novel TRPV1 receptor antagonists with improved potency and/or selectivity compared with capsazepine. For example, arginine-rich peptides have been reported as TRPV1 blockers with analgesic activities (Planells-Cases et al., 2000). Other TRPV1 antagonists have been described recently, including N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyr-azine-1(2H)-carbox-amide (Valenzano et al., 2003), JYL1421 and KJM429 (Wang et al., 2002), SB-366791 (Gunthorpe et al., 2004), N-(4-chlorobenzyl)-N′-(4-hydroxy-3-iodo-5-methoxybenzyl)thiourea (Toth et al., 2004), compound 41 (Swanson et al., 2005), AMG9810 (Doherty et al., 2005), and 5-iodo-resiniferatoxin (Wahl et al., 2001). The present studies were carried out to characterize a novel TRPV1 receptor antagonist A-425619 that was optimized from hits identified by high-throughput screening of chemical libraries (Gomtsyan et al., 2005).
Materials and Methods
Materials. Cell culture media and fetal bovine serum were obtained from Sigma-Aldrich (St. Louis, MO). G-418 sulfate was obtained from Calbiochem (San Diego, CA). Dulbecco's phosphate-buffered saline, pH 7.4 (D-PBS) (with calcium, magnesium, and 1 mg/ml d-glucose) was obtained from Invitrogen (Carlsbad, CA). Fluo-4 AM was purchased from Tef Labs (Austin, TX). NADA was purchased from Tocris Cookson Inc. (Ellisville, MO). A-425619 was synthesized at Abbott Laboratories (Abbott Park, IL). All other chemicals were obtained from Sigma-Aldrich, unless otherwise indicated.
Ca2+ Flux Assay. Cloning and stable expression of the human TRPV1 receptor in HEK293 cells have been previously detailed (Witte et al., 2002). TRPV1-mediated elevation of intracellular calcium levels was measured using the fluorescent calcium chelating dye fluo-4, as described previously (Smart et al., 2001). Briefly, cells were grown as a monolayer in black-walled clear bottom 96-well Biocoat plates (precoated with poly-d-lysine) (BD Biosciences, San Jose, CA). Growth medium was comprised of Dulbecco's modified Eagle's medium (DMEM) (with 4.5 mg/ml d-glucose), 4 mM l-glutamine, 300 μg/ml G-418 sulfate, and 10% (v/v) fetal bovine serum. Prior to the start of the assay, the cells were incubated with 2 μM (acetyloxy)methyl ester form of fluo-4 (fluo-4 AM) in D-PBS for 2 h at 25°C. Subsequently, cells were washed with D-PBS to remove extra-cellular fluo-4 AM, and 100 to 150 μl of D-PBS was added to each well. In some experiments, cells were pretreated with 100 nM PDBu in D-PBS for 20 min at 25°C to sensitize TRPV1 to capsaicin, NADA, or heat. All test compounds were dissolved in DMSO (10 mM stocks), except NADA (5 mg/ml in ethanol) and ruthenium red (10 mM in dH20), and then diluted in D-PBS to obtain (4×) solutions. Test compounds (50 μl of the 4× solutions) were added to the cells at a delivery rate of 50 μl/s. Antagonists were added to the cells 5 min before addition of agonist, and final assay volume was 200 μl. Acid activation studies of the TRPV1 receptor were performed in a similar manner, except ambient pH was lowered to pH 6.7 to facilitate detection of a pure TRPV1-mediated increased intracellular Ca2+. Antagonist solutions were prepared in the ambient pH buffer. Acidic pH solutions were prepared by titration of D-PBS with 1 M HCl, and then 50 μl was added to the cells at a delivery rate of 50 μl/s. For heat activation studies, the liquid contents of the wells were aspirated and replaced with 50 μl of D-PBS or test compound solution at ambient room temperature (25°C). A 96-well assay plate of D-PBS (250 μl/well) was preheated on a hot orbital shaker (Daigger, Vernon Hills, IL) to 50°C, and then 150 μl of heated solution was added to the cells at a delivery rate of 50 μl/s to attain a peak temperature of 38°C. Peak temperature was determined using a TH-1 Therm probe (Cell MicroControls, Norfolk, VA). Changes in fluorescence were recorded over time in a fluorometric imaging plate reader (FLIPR) (Molecular Devices, Sunnyvale, CA) (λEX = 488 nm, λEM = 540 nm). Antagonists were tested at 11 concentrations (indicated on each graph). The peak increase in fluorescence over baseline [relative fluorescence units (RFU)] was calculated and expressed as a percentage of the maximal agonist response (in absence of antagonist). EC50 and IC50 values were calculated from curve fits of the concentration-effect data using a four-parameter logistic Hill equation (GraphPad Prism; GraphPad Software Inc., San Diego, CA). Significant differences were calculated by unpaired, two-tailed Student's t tests.
Dorsal Root Ganglion (DRG) Neuronal Cultures. Adult male Sprague-Dawley rats (∼8 weeks old, 250–300 g) were deeply anesthetized with CO2 and sacrificed. Lumbar (L4–L6) DRG were dissected from the vertebral column and placed in DMEM (Hyclone Laboratories, Logan, UT) containing 0.3% collagenase B (Roche Diagnostics, Indianapolis, IN) for 60 min at 37°C. The collagenase was replaced with 0.25% trypsin (Invitrogen) in Ca2+/Mg2+-free D-PBS, and further digested for 30 min at 37°C. After washing in fresh DMEM, ganglia were dissociated by trituration using a fire-polished Pasteur pipette. Cells were washed in fresh DMEM and triturated again using a smaller bore fire-polished pipette to obtain a single-cell suspension. DRG cells were then plated on polyethylenimine-treated 12-mm glass coverslips. Cells were plated at a density of one DRG per coverslip in 1 ml of DMEM supplemented with 10% fetal bovine serum, NGF (50 ng/ml), and 100 U/ml Pen/Strep. Neurons were used for electrophysiological recording within 12 to 24 h. Experimental procedures involving rats were conducted under a protocol approved by an Institutional Animal Care and Use Committee.
Electrophysiology. Rat DRGs were maintained at room temperature in an extracellular recording solution (pH 7.4, 325 mOsm) consisting of 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,10 mM HEPES, and 12 mM glucose. Patch pipettes composed of borosilicate glass (1B150F-3; World Precision Instruments, Inc., Sara-sota, FL), were pulled and fire-polished using a DMZ-Universal micropipette puller (Zeitz, Augsburg, Germany). Pipettes (2–6 MΩ) were filled with an internal solution (pH 7.3, 295 mOsm) consisting of 122.5 mM K-aspartate, 20 mM KCl, 1 mM MgCl2, 10 mM EGTA, 5 mM HEPES, and 2 mM ATP·Mg. Standard whole-cell recording techniques were utilized for voltage-clamp studies using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA) (Hamill et al., 1981).
Cells were continuously perfused with extracellular solution at a rate of 0.5 ml/min. Capsaicin was applied to small- to medium-sized neurons (25- to 35-μm diameter) for 5 s using a piezoelectric-driven theta-tube application device (Burleigh Instruments, Fishers, NY) controlled by Axon Instruments pClamp 9 software (Axon Instruments Inc.). Control responses typically ran down for the first 5 to 10 min following whole-cell configuration. Therefore, capsaicin was applied alone at 2-min intervals until successive applications produced currents of similar amplitude. At this point, increasing concentrations of A-425619 were preapplied to the neuron for ∼60 s, followed by coapplication of capsaicin and A-425619. Peak current amplitudes were measured and plotted as a function of antagonist concentration. In a subset of neurons, the washout of the inhibition by A-425619 was monitored by continuing application of capsaicin at 2-min intervals while applying external solution to the cell. Current amplitudes were typically recovered to >80% of control responses within 4 to 6 min.
For electrophysiological studies, a 10 mM stock solution of A-425619 dissolved in DMSO was serially diluted 1:10 in DMSO. On the day of recording, the resulting stock solutions were diluted 1:1000 or 1:333 into external solution for the final concentrations. DMSO alone at these concentrations had no effect on capsaicin-activated currents.
Results
Activation of Human TRPV1 Receptors Stably Expressed in HEK293 Cells
Characterization of recombinant human TRPV1 receptors stably expressed in HEK293 cells was carried out using a Ca2+ flux-based assay (FLIPR) as previously reported (Smart et al., 2001; Witte et al., 2002). Figure 1 shows representative FLIPR tracings following activation of TRPV1 receptors by different stimuli. Addition of 50 nM capsaicin, a concentration equivalent to EC50 (Fig. 2A), elicited a rapid and a robust increase in intracellular calcium concentrations with a maximum response obtained within 10 to 20 s (Fig. 1A). Untransfected HEK293 cells (null) showed no response to capsaicin. Concentrations equivalent to EC50 values of NADA (3 μM) and anandamide (10 μM) (Fig. 2A) evoked a response similar to that evoked by capsaicin (Fig. 1, B and C). Both anandamide and NADA behaved as less potent agonists at the TRPV1 receptor with maximum calcium signals occurring at 60 to 120 s. Note that NADA and anandamide also produced small calcium signals (∼10%) in null HEK293 cells, indicating that these endogenous ligands evoked non-TRPV1 receptor-mediated responses in addition to activating TRPV1 (Fig. 1, B and C). Acid activation of TRPV1 receptors was performed by reducing the extracellular pH, as described under Materials and Methods. As shown in Fig. 1D, the acid (pH 5.5)-evoked increased intracellular calcium response was immediate and transient. This effect was specific to the TRPV1 receptor since no acid response was observed with null HEK293 cells (Figs. 1D and 2B).
In Vitro Pharmacological Characterization of A-425619
A-425619 Is a Highly Potent Antagonist at the Recombinant Human TRPV1 Receptor. The abilities of A-425619 and other TRPV1 antagonists (Fig. 3) to inhibit receptor activation were investigated using HEK293 cells stably expressing human TRPV1 receptors. As shown in Fig. 4A, A-425619 blocked TRPV1 activation by 50 nM capsaicin in a concentration-dependent manner. A-425619 was a more potent antagonist (IC50 = 5 nM) than capsazepine (IC50 = 149 nM) or ruthenium red (IC50 = 512 nM) (Fig. 4A). Under the same conditions, A-425619 was 15-fold more potent than I-RTX (IC50 = 75 nM) in blocking TRPV1 receptor activation by capsaicin (Fig. 4A; Table 1).
A-425619 was also very potent in blocking TRPV1 activation by 3 μM NADA (IC50 = 4 nM) or 10 μM anandamide (IC50 = 3 nM), with a rank order of potencies of A-425619 > I-RTX > capsazepine > ruthenium red (Fig. 4, B and C). However, the activation of TRPV1 receptor-mediated Ca2+ flux evoked by either NADA or anandamide was not completely inhibited by any of the TRPV1 receptor antagonists (Fig. 4, B and C). These data are consistent with results obtained in null HEK293 cells (Fig. 1), which indicate that these ligands induced a small, non-TRPV1 receptor-mediated increased intracellular calcium in addition to activating TRPV1 receptors. This nonspecific effect was more evident in the case of anandamide, where ∼30% of the total response was unaffected by TRPV1 receptor antagonists (Fig. 4C).
A-425619 was highly potent in blocking activation of TRPV1 receptors by acid (pH 5.5) (Fig. 4D). The rank order of potencies was A-425619 (2 nM) > capsazepine (50 nM) > I-RTX (88 nM) > ruthenium red (386 nM) (Fig. 4D). The pIC50 and Hill slope values for A-425619 and other antagonists in blocking TRPV1 activation by different stimuli are summarized in Table 1.
A-425619 Is a Potent Antagonist at the Native Rat TRPV1 Receptor. The ability of A-425619 to block native TRPV1 receptor activation was examined electrophysiologically in cultured rat DRG neurons, as described under Materials and Methods. Application of 1 μM capsaicin to small-diameter neurons clamped at -70 mV elicited large inward currents. Capsaicin-evoked currents were reduced in the presence of A-425619 in a concentration-dependent manner (IC50 = 9 nM) and were completely blocked at 100 nM (Fig. 5). TRPV1 receptor block by A-425619 was reversible since capsaicin-evoked currents were recovered following antagonist washout (Fig. 5A). Additionally, A-425619 was able to potently block the activation of TRPV1 receptors by endogenous ligands. Anandamide (10 μM) elicited a large current that was fully blocked by 100 nM A-425619 (data not shown).
A-425619 Is a Highly Selective TRPV1 Antagonist. To determine the specificity of A-425619, the compound was profiled in a large panel of in vitro binding assays (CEREP, Poitiers, France). These assays included G protein-coupled receptors, enzymes, transporters, and ion channels, which are listed in Table 2. A-425619 was found to be inactive (IC50 > 10 μM) at most of the tested targets. Additionally, A-425619 was weak or inactive in functional FLIPR-based assays against other targets, including P2X3 (IC50 > 10 μM), P2X2/3 (IC50 > 10 μM), TRPM8 (IC50 = 8 μM), and TRPA1 (IC50 > 10 μM) receptors. Thus, the present data demonstrate that A-425619 is a highly selective antagonist of TRPV1 receptors.
A-425619 Is a Competitive Antagonist at the TRPV1 Receptor. To determine the nature of A-425619 antagonism at the TRPV1 receptor, capsaicin concentration-effect curves were determined in the presence of increasing concentrations of A-425619. Figure 6A shows that capsaicin concentration-effect curves were shifted to the right with increasing A-425619 concentrations, without affecting the maximal capsaicin response. This indicates that A-425619 acts as a competitive antagonist at the TRPV1 receptor capsaicin-binding site. A Schild plot analysis yielded a pA2 of 2.5 nM and a slope factor of 1.06 ± 0.05 (Fig. 6B). Under the same conditions, concentration response curves for capsaicin were generated with increasing concentrations of capsazepine and ruthenium red. Consistent with competitive antagonism by capsazepine, capsaicin dose-response curves were shifted to the right as a function of increasing concentrations of capsazepine, without change in the maximal responses (slope = 1.16 ± 0.08) (Fig. 6C). In contrast, increasing concentrations of the antagonist ruthenium red induced a large decrease in the efficacy of capsaicin to stimulate calcium flux with a small rightward shift of the capsaicin dose-response curves (Fig. 6D), consistent with noncompetitive antagonism.
A-425619 Is a Potent Antagonist at the Sensitized TRPV1 Receptor. Agents such as heat, acid, and endovanilloids not only activate TRPV1 receptors but also sensitize the channel responses to other stimuli (Cortright and Szallasi, 2004). We recently reported that sensitization of TRPV1 under acidic conditions involves receptor phosphorylation and that PKC plays an important role in this acid-induced sensitization (El Kouhen et al., 2003). The ability of A-425619 to block the activation of sensitized TRPV1 receptors was investigated under different conditions. In the presence of 100 nM PDBu or pH 6.0, the potency of capsaicin to stimulate increased intracellular calcium was enhanced 2- to 4-fold (control capsaicin concentration-effect curves in the absence of antagonist; Fig. 7, A and B). EC50 values for capsaicin were 8.5 and 15 nM, after PDBu and acid pretreatment, respectively (Fig. 7, A and B). The EC50 value for capsaicin to activate the naive TRPV1 receptor was ∼50 nM. In the presence of increasing concentrations of A-425619, concentration-effect curves of capsaicin were shifted to the right (Fig. 7, A and B). Schild plot analysis yielded pA2 values of 0.8 and 4.3 nM after sensitization by PDBu and acid, respectively (Fig. 7C). These pA2 values were not significantly different (p > 0.05) from that generated in naive conditions (2.5 nM) (Fig. 6B) and showed that A-425619 remained a potent antagonist at sensitized TRPV1 receptors. The rank order of potencies for antagonist block of sensitized TRPV1 was the same as that in blocking activation of naive TRPV1, A-425619 > I-RTX > capsazepine > ruthenium red (Fig. 8; Table 3).
The ability of heat to activate TRPV1 receptors was also assessed in the calcium flux assay on both naive and PDBu-sensitized channels, as described under Materials and Methods. A small but significant response was observed in TRPV1 receptor-expressing HEK293 cells in response to heat (38°C). This signal was dramatically increased when the cells were pretreated for 20 min with the PKC activator PDBu (100 nM) (Fig. 9A). However, the increased intracellular calcium evoked by heat in PDBu-treated cells (see Materials and Methods) was transient and much shorter in duration than that induced by other stimuli (Fig. 1). The transient nature of this response may be due, at least in part, to the methodology used as well as to the response of the channel to heat. The small heat-evoked signal obtained in null cells was unaffected by the PDBu pretreatment (Fig. 9A). The ability of A-425619 and other antagonists to block the response of the sensitized TRPV1 receptor to heat was also examined. A-425619 effectively attenuated this response and was approximately 25- and 50-fold more potent than I-RTX and capsazepine, respectively (Fig. 9B; Table 3).
Discussion
The present data demonstrate that the structurally novel compound A-425619 is a highly potent TRPV1 receptor antagonist. A-425619 is a competitive antagonist of capsaicin-evoked receptor activation and can potently block both naive and sensitized TRPV1 receptor responses to a variety of stimuli. A-425619 is a potent antagonist at both recombinant human and native rat TRPV1 receptors and shows a high degree of specificity compared with its activity at other cell surface receptors and ion channels.
It is now generally accepted that TRPV1 is an integrator of multiple and diverse stimuli, such as vanilloids, acid (pH < 6.0), heat (>43°C), and endogenous arachidonic acid derivatives (Caterina et al., 1997; Tominaga et al., 1998). These agents can directly activate TRPV1 receptors as well as sensitize channel responses to other noxious stimuli (Di Marzo et al., 2002). Moreover, inflammatory agents, including ATP, NGF, and bradykinin, can potentiate TRPV1 responses by activating specific kinases, which phosphorylate the TRPV1 channel (Di Marzo et al., 2002). Interestingly, both NGF and bradykinin also induce the hydrolysis of phosphatidylinositol (4,5)-biphosphate, which leads to the release of TRPV1 receptor from an inhibited state (Chuang et al., 2001). These studies indicate that the TRPV1 channel is regulated by multiple mechanisms and support the importance of this channel in pain transmission during inflammation or tissue injury.
The present work demonstrates that the structurally novel TRPV1 receptor antagonist A-425619 is 25- to 50-fold more potent than capsazepine in blocking activation of TRPV1 receptors by a variety of stimuli, including capsaicin, acid, heat, NADA, and anandamide. A-425619 completely blocked TRPV1 activation by capsaicin, acid, and heat. However, the calcium flux induced by NADA and anandamide was not fully blocked by A-425619, consistent with previous reports, that NADA and anandamide have additional activities, including effects on fatty acid amide hydrolase and cannabinoid CB1 receptors (Huang et al., 2002; Chu et al., 2003). Moreover, in vitro and in vivo studies with capsazepine have been difficult to interpret due to the low selectivity of this antagonist for blocking TRPV1 receptors (Nagy et al., 2004). Thus, the availability of highly potent and selective antagonists of TRPV1 receptors not only will help to elucidate the complex pharmacology of this interesting channel but also may have therapeutic potential as novel analgesics.
Consistent with other reports, both acid and PDBu pre-treatments reduced activation thresholds of TRPV1 receptors by capsaicin, heat, anandamide, or NADA (El Kouhen et al., 2003; Cortright and Szallasi, 2004). Interestingly, here we provide evidence that A-425619 effectively blocked the activation of both naive and sensitized TRPV1 channels. Sensi-tization or phosphorylation of TRPV1 seems to increase the affinity of the channel to agonist e.g., capsaicin (Premkumar and Ahern, 2000; Vellani et al., 2001). However, a Schild plot analysis indicated that the affinity of A-425619 to block sensitized TRPV1 receptors remained comparable with that blocking naive TRPV1 receptors. The present data suggest that TRPV1 antagonists such as A-425619 may serve as an effective tool in blocking TRPV1 activation during inflammation or tissue injury.
Like capsazepine, A-425619 competitively blocked the ability of capsaicin to stimulate calcium flux through TRPV1 receptors. The capsaicin recognition site has been proposed to be predominantly localized on intracellular domains of TRPV1 (Jung et al., 1999; Jordt and Julius, 2002), although there is evidence of an extracellular domain as well (Vyklicky et al., 2003). In contrast, the proton interaction site is proposed to be extracellular (Jordt et al., 2000). Since A-425619 blocks activation of TRPV1 receptors by multiple stimuli, it is possible that this compound blocks or modulates the gating mechanism of the channel. It has been reported that the noncompetitive antagonist ruthenium red blocks the activation of TRPV1 channels by different stimuli through blocking of the channel gating (Czirjak and Enyedi, 2003). Alternatively, protons and heat could act as TRPV1 modulators by sensitizing the channel to activation by endogenous vanilloids. In this case, A-425619 would inhibit proton, heat, or PDBu activation of TRPV1 by competing for recognition site(s) for capsaicin or endogenous vanilloids. Evidence supporting this latter interpretation comes from demonstrations that the potency and efficacy of anandamide to activate TRPV1 receptors is greatly enhanced under acidic conditions (Premkumar and Ahern, 2000; Vellani et al., 2001). Although the exact mode by which capsaicin-competitive antagonists block TRPV1 receptor activation in response to other stimuli remains unclear, the present data demonstrate that A-425619 can effectively block TRPV1 receptor activation by a variety of pronociceptive stimuli and that the potency of this antagonist for TRPV1 receptors is largely unaffected by the state of channel activation. The present data show that A-425619 serves as a useful tool to enhance the understanding of the complex TRPV1 pharmacology. In addition, the structurally novel TRPV1 antagonist A-425619 will be useful in defining the analgesic potential of blockade of TRPV1 in vivo (Honore et al., 2005).
Footnotes
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.105.084103.
-
ABBREVIATIONS: TRPV1, transient receptor potential type V1; NADA, N-arachidonoyl-dopamine; NGF, nerve growth factor; PKC, protein kinase C; PDBu, phorbol-12,13-dibutyrate; SB-366791, SN-(3-methoxyphenyl)-4-chlorocinnamide; compound 41, 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-carboxylic acid (5-trifluoromethyl pyridin-2-yl)amide; AMG9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide; A-425619, 1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea; D-PBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; AM, acetoxymethyl ester; DMSO, dimethyl sulfoxide; FLIPR, fluorometric imaging plate reader; RFU, relative fluorescence unit(s); DRG, dorsal root ganglion; I-RTX, 5-iodo-resiniferatoxin; JYL1421, N-(4-tert-butylbenzyl)-N′-[3-fluoro-4-(methylsulfonylamino)benzyl]thiourea; KJM429, N-(4-tert-butylbenzyl)-N′-[4-(methylsulfonylamino)benzyl]thiourea.
- Received January 21, 2005.
- Accepted April 14, 2005.
- The American Society for Pharmacology and Experimental Therapeutics