Article pubs.acs.org/jnp
Partial Activation and Inhibition of TRPV1 Channels by Evodiamine and Rutaecarpine, Two Major Components of the Fruits of Evodia rutaecarpa Shenglan Wang,†,‡ Satoshi Yamamoto,† Yoko Kogure,† Wensheng Zhang,⊥ Koichi Noguchi,‡,§ and Yi Dai*,†,‡,§ †
Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe, Hyogo 6508530, Japan Traditional Medicine Research Center, Chinese Medicine Confucius Institute at Hyogo College of Medicine, Kobe, Hyogo 6508530, Japan § Department of Anatomy and Neuroscience, Hyogo College of Medicine, Nishinomiya, Hyogo 6638501, Japan ⊥ State Key Laboratory of Earth Surface Processes and Resource Ecology, Beijing Normal University, Beijing 100875, People’s Republic of China ‡
ABSTRACT: Evodiamine (1) and rutaecarpine (2) are the two major components of Evodia rutaecarpa, which has long been used in traditional medicine for the treatment of many diseases. Using transient receptor potential vanilloid 1 (TRPV1)expressing HEK293 cells and patch-clamp recording, the inhibitory actions of 1 and 2 against TRPV1 channels were investigated. The effects of these compounds against capsaicin- or proton-activated TRPV1 activities were evaluated. The results showed that, although 1 and 2 can activate TRPV1, the maximum response was 3.5- or 9-fold lower than that of capsaicin, respectively, suggesting partial agonism. In comparison to capsaicin, coadministration of 1 and capsaicin increased the half-maximal effective concentration (EC50) of capsaicin-activated TRPV1 currents as shown by a right shift in the dose−response curve, whereas coadministration of 1 with protons failed to inhibit the proton-induced current. Moreover, preadministration of 1, but not 2, inhibited both capsaicin- and proton-induced TRPV1 currents, which might involve channel desensitization. Taken together, 1 and 2 may share the same binding site with capsaicin and act as partial agonists (antagonists) of TRPV1. Evodiamine (1), but not rutaecarpine (2), can desensitize or competitively inhibit the activity of TRPV1.
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efficiency of E. rutaecarpa fruits are attributed mainly to the presence of 1 and 2.3,4 These compounds have been reported to possess multiple biological effects, such as antinociceptive, anti-inflammatory, antineoplastic, antidiabetic, and thermoregulatory effects,5−9 of which some are related to TRPV1 activity. In fact, 1 has been characterized as a putative agonist for TRPV1 both in vitro10 and in vivo.11−13 However, as the main components of E. rutaecarpa fruit, which is used in current clinics in Asian countries, the effects of 1 and 2 on exogenous (capsaicin) and endogenous agonist (proton)-induced TRPV1 activation remain poorly understand. In the present study, it is demonstrated for the first time that 1 and 2 partially activate TRPV1 and inhibit capsaicin (or proton)-induced TRPV1 activation through competitive partial antagonism and/or channel desensitization.
ransient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on either sensory neuronal systems (especially on primary afferents) or nonneuronal sites, such as the epithelium of the bladder, the lungs, and hair cells of the cochlea. Capsaicin, protons, heat, and a variety of endogenous lipids activate TRPV1. Activation of TRPV1 has been implicated in the pathophysiology of many neuronal (especially pain-related) and non-neuronal diseases, such as cystitis, asthma, and hearing loss.1 Since TRPV1 is a critical therapeutic target for the treatment of pain, as well as other conditions or diseases, many TRPV1 antagonists have been developed for clinical trials. However, these antagonists show adverse effects such as hyperthermia.1,2 Evodia rutaecarpa (Juss.) Benth. (Rutaceae fruits) have been used as a traditional Chinese medicine (named “Wu Zhu Yu”) for thousands of years for the treatment of headache, abdominal pain, dysentery, and cardiovascular diseases. Evodiamine (1) and rutaecarpine (2) are the two major components of E. rutaecarpa fruits. The pharmacological action and therapeutic © XXXX American Chemical Society and American Society of Pharmacognosy
Received: July 6, 2015
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DOI: 10.1021/acs.jnatprod.5b00599 J. Nat. Prod. XXXX, XXX, XXX−XXX
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RESULTS AND DISCUSSION Evodiamine (1) and Rutaecarpine (2) Activate TRPV1 as Partial Agonists. To determine the agonistic action of 1 or 2 on TRP channels, activated currents in TRPV1- or TRPA1transfected HEK293 cells by these compounds were examined. Compounds 1 (10 μM) and 2 (50 μM) did not induce any current in untransfected or TRPA1-transfected HEK293 cells as measured by voltage clamp recordings (data not shown). In contrast, the application of 1 and 2 induced significant inward currents in TRPV1-transfected HEK293 cells and displayed outward rectification properties with a reversal potential of 0 mV, similar to the reversal potential of capsaicin-induced TRPV1 currents (Figure 1A). The activated currents of both 1 and 2 were blocked completely and reversibly by capsazepine (10 μM) (Figure 1B). In comparison to capsaicin, 1 or 2 did not elicit the maximum TRPV1 response (−142.8 ± 58.9 pA/ pF, n = 7 for 1, −55.0 ± 15.7 pA/pF, n = 7 for 2, and −497.0 ± 89.6 pA/pF, n = 7 for capsaicin). The maximum currents induced by 1 or 2 were 3.5- or 9.0-fold lower than that induced by capsaicin, respectively. The half-maximal effective concentration (EC50) of 1 or 2 on TRPV1 was 0.60 or 2.06 μM, which was 10- or 30-fold higher than that of capsaicin, respectively (Figure 2A). These values for 1 are supported by the findings of previous studies, which reported that 1 was 3-fold less active than capsaicin in inducing contractions of the guinea pig atrium and bronchi.11,12 TRPV1 is an oligomeric protein with a homotetramer as the predominant form that is arranged symmetrically in the membrane, which provides multiple binding sites for its agonists.14 The Hill slope is often used to assess cooperativity in channel-gating transitions (ligand binding or activation). To analyze the dose curve data in Figure 2A, the Hill plot data were determined using a previously reported method by plotting the logarithm of the ratio of responsive to unresponsive levels of agonist-induced current against the logarithm of the given concentration of agonists (see Experimental Section).15 A Hill value “ >1” represents the positive, whereas “ 0.05 vs control; −200.3 ± 41.5 pA/pF, n = 6, for 2 cotreatment, p > 0.05 vs control) (Figure 6A, C, D). Unlike capsaicin, protons bind to the outer pore region to open the channel.21,23,24 The present data showed that pretreatment, but not cotreatment, with 1 inhibited protonactivated TRPV1 activation and suggested that 1 does not share the same binding site as protons. Thus, the inhibition was due to channel desensitization rather than competitive partial agonist binding. The lack of effect of 2 may be due to its low efficiency against TRPV1, which could not result in desensitization of TRPV1 (Figure 3D). Taken together, the present study has demonstrated that 1 and 2 can partially activate TRPV1 and evodiamine (1) can inhibit the different agonist-induced TRPV1 channel activations through distinct mechanisms. As mentioned previously, 1 and 2 are known to possess multiple biological effects.13,25−27 Given that TRPV1 is expressed in various organs (tissues) and has various physiological roles other than that of a pain sensor,28−32 the present observations may provide a common mechanism encompassing the broader biological effects of 1 and 2.
Table 3. IC50 Values of Evodiamine (1) on Capsaicin Current Density
a
1 (μM)
CAP (μM)
IC50 (μM)
0 0.1 0.5 1 10
0.1
0.44
CAP current density (pA/pF) −327.4 −242.6 −143.4 −132.5 −60.3
± ± ± ± ±
52.3 25.2 28.5a 16.2a 12.7b
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EXPERIMENTAL SECTION
Test Compounds. Compound 1 was obtained from Alexis Corporation (Lausen, Switzerland), and compound 2 and capsazepine were from Sigma-Aldrich (St Louis, MO, USA). Allyl isothiocyanate and capsaicin were from Nacalai Tesque (Kyoto, Japan). The purity of each standard compound was no less than 98% by HPLC analysis.
p < 0.05. bp < 0.01 versus 0.1 μM capsaicin without 1 pretreatment.
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DOI: 10.1021/acs.jnatprod.5b00599 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Mammalian Cell Culture. Human embryonic kidney-derived (HEK) 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), glutamax, penicillin, and streptomycin (Invitrogen, Carlsbad, CA, USA). HEK293 cells were transfected with 1 μg of human TRPA1 (hTRPA1) cDNA or rat TRPV1 (rTRPV1) cDNA using Lipofectamine LTX and PLUS Reagent (Invitrogen). An enhanced green fluorescence protein reporter plasmid (BD Biosciences, San Jose, CA, USA) was cotransfected with the TRP channels. The hTRPA1 and rTRPV1 cDNAs were generous gifts from Prof. Makoto Tominaga (Okazaki Institute for Integrative Bioscience, Okazaki, Japan). Electrophysiology. Whole-cell patch-clamp recordings were performed 2 days after transfection of HEK293 cells with hTRPA1 cDNA or 1 day after transfection with rTRPV1 cDNA. Voltage-clamp experiments were performed at a −60 mV holding potential, and cell capacitance and series resistance were compensated by 80% in all tests. Recordings were sampled at 5 kHz and filtered at 2 kHz. Voltage steps ranging from −100 to +100 mV (100 ms) were applied to generate an I−V curve. The bath solution was used as the steady-state leak current shown in Figure 1A. Agonists were applied until the evoked currents underwent desensitization. In all experiments, the current magnitude was quantified by the peak current amplitude. Patch-clamp experiments were performed at room temperature (∼25 °C). The standard bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4 (adjusted with NaOH). The calcium-free bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, and 10 mM glucose, pH 7.4 (adjusted with NaOH). The pipet solution contained 140 mM KCl, 2 mM MgCl2, 0.5 mM CaCl2, 5 mM MgATP, 5 mM EGTA, and 5 mM HEPES, pH 7.2 (adjusted with KOH). The bath solution was buffered to pH 6 with 10 mM 2morpholinoethanesulfonic acid monohydrate (Dojindo, Osaka, Japan) for examining the proton-induced current responses. Acidsensing ion channels (ASIC channels) have been reported to be expressed in HEK cells. The proton-produced ASIC currents showed a sharp increase followed by an acute decrease (desensitization) within 5 s.33 To avoid ASIC currents in recording proton-induced TRPV1 currents, the peak currents were just taken into account outside the application period of an initial 5 s. The solutions containing each compound were applied to the chamber (1 mL) through a gravity system at a flow rate of 3−4 mL/min. Statistical Analysis. All results are expressed as the means ± SEM. An unpaired t-test was used to compare the electrophysiological data between the two groups. A difference was accepted as significant if the probability was less than 5% (p < 0.05). Sigmoidal curve fitting for the EC50 or IC50 was performed using Origin 9 (Originlab, Northampton, MA, USA). The Hill plots shown in Figure 2B were generated using the equation previously described:15 log10 X/(Xmax − X) vs log[A], where Xmax was the maximum response in current density induced by capsaicin at 10 μM, and X is the agonist-induced current density at a given concentration ([A]). Schild regressions are plotted with log[(A′/ A) − 1] and log[B] to estimate the slope. A′ is the EC50 value of 1, A is the EC50 value of capsaicin, and B is the concentration of 1.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81-78-304-3147. Fax: +81-78-304-2847. E-mail: ydai@ huhs.ac.jp. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research Kakenhi 26460713 (Y.D.). REFERENCES
(1) Brederson, J. D.; Kym, P. R.; Szallasi, A. Eur. J. Pharmacol. 2013, 716, 61−76. F
DOI: 10.1021/acs.jnatprod.5b00599 J. Nat. Prod. XXXX, XXX, XXX−XXX
