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, 25 (39), 8924-37

Camphor Activates and Strongly Desensitizes the Transient Receptor Potential Vanilloid Subtype 1 Channel in a Vanilloid-Independent Mechanism

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Camphor Activates and Strongly Desensitizes the Transient Receptor Potential Vanilloid Subtype 1 Channel in a Vanilloid-Independent Mechanism

Haoxing Xu et al. J Neurosci.

Abstract

Camphor is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite its long history of common medical use, the underlying molecular mechanism of camphor action is not understood. Capsaicin and menthol, two other topically applied agents widely used for similar purposes, are known to excite and desensitize sensory nerves by acting on two members of transient receptor potential (TRP) channel superfamily: heat-sensitive TRP vanilloid subtype 1 (TRPV1) and cold-sensitive TRP channel M8, respectively. Camphor has recently been shown to activate TRPV3, and here we show that camphor also activates heterologously expressed TRPV1, requiring higher concentrations than capsaicin. Activation was enhanced by phospholipase C-coupled receptor stimulation mimicking inflamed conditions. Similar camphor-activated TRPV1-like currents were observed in isolated rat DRG neurons and were strongly potentiated after activation of protein kinase C with phorbol-12-myristate-13-acetate. Camphor activation of rat TRPV1 was mediated by distinct channel regions from capsaicin, as indicated by camphor activation in the presence of the competitive inhibitor capsazepine and in a capsaicin-insensitive point mutant. Camphor did not activate the capsaicin-insensitive chicken TRPV1. TRPV1 desensitization is believed to contribute to the analgesic actions of capsaicin. We found that, although camphor activates TRPV1 less effectively, camphor application desensitized TRPV1 more rapidly and completely than capsaicin. Conversely, TRPV3 current sensitized after repeated camphor applications, which is inconsistent with the analgesic role of camphor. We also found that camphor inhibited several other related TRP channels, including ankyrin-repeat TRP 1 (TRPA1). The camphor-induced desensitization of TRPV1 and block of TRPA1 may underlie the analgesic effects of camphor.

Figures

Figure 1.
Figure 1.
Camphor activates heterologously expressed rat TRPV1. A, Structure of the (+) and (–) stereoisomers of camphor. B, Both camphor stereoisomers activated rTRPV1. The weaker action of (–)-camphor in comparison with (+)-camphor was likely the result of tachyphylaxis. Ramp currents were evoked by 400 ms voltage ramps from –100 to +100 mV applied every 4 s. Each symbol represents the current amplitude at +60 mV (red triangles) and –60 mV (black circles). Note the zero current level (indicated by blue dashed line). C, rTRPV1 was activated by camphor, anandamide, and capsaicin. Capsazepine (10 μm) strongly inhibited the anandamide-induced currents. D, Effect of camphor on rTRPV1-mediated current elicited by voltage steps (protocol shown in the top panel).
Figure 2.
Figure 2.
Camphor activates TRPV1 at lower concentrations than TRPV3. A, Concentration dependence of the activation of TRPV1 by camphor (1, 3, and 10 mm). Representative ramp currents shown in B correspond to the filled circles with the same color in A. Note the negative slope in the inward current during the ramp, characteristic of ITRPV1. C, Average TRPV1 current densities (current amplitude normalized to the capacitance of each cell; pF) elicited by 1–10 mm camphor. Data are shown as mean ± SEM. D–F, Concentration dependence of the activation of TRPV3 by 1–10 mm camphor. The representative ramp currents are shown in E. Note the change of rectification during the development of ITRPV3 after camphor application, as the peak current became linear. F, Average ITRPV3 density induced by different concentrations of camphor.
Figure 3.
Figure 3.
Camphor induces [Ca2+]i rise in TRPV1- and TRPV3-expressing cells but not in TRPV2-expressing cells. A, Camphor and capsaicin increased fura-2 ratios (F340/F380) of HEK cells transfected with the rTRPV1-EGFP construct. EGFP-positive cells were identified by measuring F440. TRPV1-positive cells had higher fura-2 ratios under resting states, indicative of higher basal [Ca2+]i level. Camphor (5 mm) increased fura-2 ratios in most of these TRPV1-positive (expressing) cells with no, or only very minimal, effects on TRPV1-negative cells. Capsaicin (1 μm) induced increases of fura-2 ratios in almost all of the TRPV1-positive cells. All cells responded to Ca2+ ionophore ionomycin (1 μm). B, Average changes (mean ± SEM; solid line) in the fura-2 ratio induced by camphor and capsaicin in TRPV1-positive cells (blue) and TRPV1-negative cells (black). Responses from individual cells were plotted in dotted lines of various colors. C, D, Camphor (5 mm) failed to increase fura-2 ratios in TRPV2-positive cells. The same cells responded with 200–500 μm DPBA, an agonist of TRPV1–3 channels. E, F, TRPV3-expressing cells responded strongly to camphor (5 mm) but not to capsaicin (1 μm).
Figure 4.
Figure 4.
Camphor does not activate currents in cells expressing several related TRP channels. A, B, Camphor (10 mm) did not activate current from a TRPV2-expressing cell. The expression of TRPV2 was confirmed by the response induced by 300 μm DPBA. C, D, Camphor (10 mm) failed to increase the basal current from a TRPV4-transfected cell. Indeed, the small constitutive activity was completely inhibited. 4-αPDD (1 μm) readily activated a doubly rectifying current characteristic of ITRPV4. E, F, TRPM8 was not activated by 10 mm camphor but by 500 μm menthol. A small outward-rectifying basal current was seen after break in (data not shown) but decayed almost completely. G, H, Application of 10 mm camphor to a cell cotransfected with mTRPC5 and the M1 receptor did not activate TRPC5 currents (ITRPC5). Subsequent application of 100 μm carbachol activated large inward and outward currents with the characteristic I–V of ITRPC5.
Figure 5.
Figure 5.
Camphor inhibits TRPA1-mediated basal currents. A, B, The basal TRPA1 activity was reversibly inhibited by camphor. ITRPA1 was stimulated by 200 μm mustard oil (allyl isothiocyanate). C, Dose dependence of camphor-induced inhibition of basal ITRPA1 (measured at –80 mV; n = 6). Data were fitted with a sigmoid curve with an IC50 value of 0.66 mm and a Hill coefficient of 0.94.
Figure 6.
Figure 6.
Camphor does not activate TRPV1 by a vanilloid-dependent mechanism. A, B, Chicken TRPV1 was not activated by camphor or capsaicin. Application of low pH (pH 4.2) solution did activate an outwardly rectifying cTRPV1-mediated current. C, D, A rat-chicken chimeric TRPV1 (schematic structure inset) was activated by capsaicin but not by camphor. E, F, A capsaicin-insensitive point mutant of rat TRPV1 (Y511A_rTRPV1) was only very weakly activated by capsaicin but retained apparently normal camphor sensitivity. G, Camphor activation of TRPV1 in the presence of 10 μm capsazepine, a competitive inhibitor at the vanilloid binding site. A low concentration of camphor (3 mm) was used here to minimize desensitization. H, Camphor-induced inward IrTRPV1 in the presence of capsazepine was completely inhibited by the pore blocker ruthenium red (10 μm). Low Ca2+ solution was used here to minimize desensitization.
Figure 7.
Figure 7.
Camphor-activated IrTRPV1 exhibits fast acute desensitization and strong tachyphylaxis. A, B, IrTRPV1 activated by 10 mm camphor quickly and completely desensitized during an ∼60 s application. Capsaicin-activated currents desensitized with much slower kinetics and less completely. C, Camphor-activated inward IrTRPV1 underwent very strong tachyphylaxis, giving much smaller response on repeated applications of 10 mm camphor. D, In contrast, ITRPV3 was slightly sensitized after repeated stimulation with 4 mm camphor.
Figure 8.
Figure 8.
Camphor dramatically enhances tachyphylaxis of proton and capsaicin-activated TRPV1 current. A, B, Proton-activated IrTRPV1 recovered nearly completely during 5 min separating two exposures to low pH solution. A 5 min exposure to 10 mm camphor, applied between low-pH solutions, strongly reduced the TRPV1 current during the subsequent low-pH activation. Even prolonged washout did not result in substantial recovery. Note the transient camphor-induced current. C, Proton-induced current from a cTRPV1-expressing cell was barely reduced by 5 min of camphor exposure. D, Capsaicin-induced IrTRPV1 was also attenuated by camphor application. E, Summary of the effects of camphor on tachyphylaxis of proton and capsaicin-activated currents from chicken or rat TRPV1-expressing cells. The recovery ratio for two consecutive stimulations was defined as the percentage of the latter response relative to the initial. Recovery ratios for both proton-induced IrTRPV1 (red) and capsaicin-induced IrTRPV1 (blue) exhibited significant difference with (+) or without (–) 5 min of camphor exposure (*p < 0.001). There was no significant difference on the recovery of proton-induced IcTRPV1 (black; #p > 0.05).
Figure 9.
Figure 9.
Camphor activation of rTRPV1 is enhanced at elevated temperatures and sensitized by receptor stimulation in HM1 cells. A, The effect of 3 mm camphor on IrTRPV1 at elevated temperatures. Temperature protocol is shown below. Camphor-induced IrTRPV1 quickly desensitized after reaching its peak. B, Average current densities elicited by elevated temperature and 3 mm camphor at warm temperatures (∼33–35°C) are shown. The “3 camphor” shows the average current density elicited by 3 mm camphor in a separate group of cells (Fig. 2C). C, In an rTRPV1-expressing HM1 cell, the small IrTRPV1 induced by 1.5 mm camphor was dramatically potentiated by 20 μm carbachol application. The representative ramp current is shown in D. E, Summary of the effects of carbachol on camphor-activated IrTRPV1 at both –60 and +60 mV.
Figure 10.
Figure 10.
Camphor activates a TRPV1-like current in DRG neurons. A, Currents evoked by voltage ramps (400 ms; from –100 to +100 mV) are shown in basal conditions (dashed line), in the presence of 3 mm (red line) and 10 mm (blue line) camphor, and during application of 1 μm capsaicin (solid black line). The holding potential between ramps was –60 mV. B, Increasing concentrations of camphor (applied during bars) elicited increasing amounts of current in a DRG neuron. The currentat +80 mV(triangles) and –80 mV (squares) during the ramps are shown. The positions of the ramp currents plotted in A are indicated by the green symbols; the dotted line indicates zero current level. C, Currents in the presence of 10 mm camphor recorded from a camphor-unresponsive neuron (green line) and from the same camphor-responsive neuron shown in A for comparison. The entire voltage protocol is shown, including the 40 ms at –100 and +100 mV flanking the ramp. The black dashed line shows the basal current recorded in the camphor-unresponsive neuron, which happened to have similar amplitude to the camphor-responsive cell.
Figure 11.
Figure 11.
PMA treatment enhances camphor-activated currents in DRG neurons. A, Ramp currents from –100 to +100 mV at baseline before PMA addition (dashed line) and after a 16 s application of 1 μm PMA application (red line). The application of 10 mm camphor (blue line) and 1 μm capsaicin (solid black line) after PMA treatment elicited large ramp currents similar to heterologously expressed TRPV1. B, The current at +80 mV (triangles) and –80 mV (squares) during voltage ramps in the cell in A is plotted. The positions of the ramp currents displayed in A are indicated by the green symbols. The dotted line indicates zero current level.

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