Quantitative analysis of sanshool compounds in Japanese pepper (Xanthoxylum piperitum DC.) and their pungent characteristics

The distributions of each sanshool in the Japanese pepper plant grown in various regions and the change in composition of sanshools during maturation of the fruit were investigated. The degree of pungency, defined as the amount of a sanshool/the threshold value, was calculated, and the pungent qualities of the products were evaluated and compared. The degree of pungency and amount of a sanshool showed a positive correlation. In young leaves and flowers, the degree of pungency was less than that in the fruits, the main compound being alpha-sanshool, while the two hydroxy sanshools were detected only in trace amounts. The main compound in fruits was hydroxy alpha-sanshool, whose threshold value was higher than that of alpha-sanshool. It is concluded that the pungency of Japanese pepper should be evaluated not only by the threshold values, but also by the pungent qualities, the composition of sanshools, and the usage of each product of Japanese pepper.     

Psychophysical Evaluation of a Sanshool Derivative (Alkylamide) and the Elucidation of Mechanisms Subserving Tingle

Previous studies investigated the neural and molecular underpinnings of the tingle sensation evoked by sanshool and other natural or synthetic alkylamides. Currently, we sought to characterize the psychophysical properties associated with administration of these compounds. Like other chemesthetic stimuli, the synthetic tingle analog isobutylalkylamide (IBA) evoked a sensation that was temporally dynamic. Repeated IBA application at short (30 sec) interstimulus intervals (ISI) resulted in a tingle sensation that increased across trials. Application at longer ISIs (∼30 min) resulted in a sensation of decreased intensity consistent with self-desensitization. Prior treatment with the TRPV1 or TRPA1 agonists, capsaicin and mustard oil did not cross-desensitize the tingle sensation evoked by IBA suggesting that neither TRPV1 nor TRPA1 participate in the transduction mechanism sub-serving tingle. When evaluated over 30-min time period, lingual IBA evoked a sensation that was described initially as tingling and pungent but after approximately 15 min, as a cooling sensation. Further, we found that the sensation evoked by lingual IBA was potentiated by simultaneous application of cold (0°C) and cool (21°C) thermal stimuli but was unaffected by warm (33°C) and hot (41°C) temperatures. Finally, to test the hypothesis that the tingling sensation is subserved by the activation of mechanosensitve fibers, we evaluated lingual tactile thresholds in the presence and absence of lingual IBA. The presence of IBA significantly raised lingual tactile thresholds, whereas capsaicin did not, identifying a role for mechanosensitive fibers in conveying the tingle sensation evoked by sanshool-like compounds. Collectively, these results show that lingual alkylamide evokes a complex sensation that is temporally dynamic and consistent with in vitro and in vivo experiments suggesting these compounds activate mechanosensitve neurons via blockade of KCNK two-pore potassium channels to induce the novel tingling sensation.     

DESCRIPTIVE ANALYSIS OF ORAL PUNGENCY1

Pungent spices are well-recognized for their aromatic and pungent nature; however, relatively little is known about their pungent or ‘burning’ responses. Therefore, this research was undertaken to characterize oral pungency of the principal irritants or red pepper (capsaicin), black pepper (piperine), cinnamon (cinnamaldehyde), cumin (cuminaldehyde), cloves (eugenol), ginger (ginger oleoresin), and alcohol (ethanol). These compounds were evaluated for four pungent qualities (burning, tingling, numbing, overall), two temporal qualities (lag time, overall duration), and three spatial qualities (longitudinal location, lateral location, localized/diffuse). The pungency of cinnamaldehyde was primarily burning and tingling. It had a quick onset and rapid decay. The pungency of eugenol had a long-lasting, predominantly numbing effect. The pungency piperine, capsaicin and ginger were primarily burning, but had different temporal and spatial responses. The pungency of ethanol was most diffuse in nature, with some burning and tingling sensations. It had the shortest perceived onset and overall duration. The pungency of cuminaldehyde was equally burning, tingling and numbing.     

Touch inhibits touch: sanshool-induced paradoxical tingling reveals perceptual interaction between somatosensory submodalities

Human perception of touch is mediated by inputs from multiple channels. Classical theories postulate independent contributions of each channel to each tactile feature, with little or no interaction between channels. In contrast to this view, we show that inputs from two sub-modalities of mechanical input channels interact to determine tactile perception. The flutter-range vibration channel was activated anomalously using hydroxy-α-sanshool, a bioactive compound of Szechuan pepper, which chemically induces vibration-like tingling sensations. We tested whether this tingling sensation on the lips was modulated by sustained mechanical pressure. Across four experiments, we show that sustained touch inhibits sanshool tingling sensations in a location-specific, pressure-level and time-dependent manner. Additional experiments ruled out the mediation of this interaction by nociceptive or affective (C-tactile) channels. These results reveal novel inhibitory influence from steady pressure onto flutter-range tactile perceptual channels, consistent with early-stage interactions between mechanoreceptor inputs within the somatosensory pathway.     

Galangal Pungent Component, 1′-Acetoxychavicol Acetate,Activates TRPA1

We investigated the activation of transient receptor potential cation channel (TRP) subfamily V, member 1 (TRPV1) and TRP subfamily A, member 1 (TRPA1) by 1′-acetoxychavicol acetate (ACA), the main pungent component in galangal. ACA did not activate TRPV1-expressing human embryonic kidney (HEK) cells, but strongly activated TRPA1-expressing HEK cells. ACA was more potent than allyl isothiocyanate, the typical TRPA1 agonist.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

The complete amino-acid sequence of the alpha and beta subunits of B-phycoerythrin from the rhodophytan alga Porphyridium cruentum

Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.     

The effects of vanilloid analogues structurally related to capsaicin on the transient receptor potential vanilloid 1 channel

Transient receptor potential vanilloid 1 (TRPV1) is known as a receptor of capsaicin, a spicy ingredient of chili peppers. It is also sensitive to a variety of pungent compounds and is involved in nociception. Here, we focused on the structural characteristics of capsaicin, and investigated whether vanillylmanderic acid (VMA), vanillic acid (VAcid), vanillyl alcohol (VAlc), vanillyl butyl ether (VBE), and vanillin, containing a vanillyl skeleton similar to capsaicin, affected the TRPV1 activities. For detection of TRPV1 activity, intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in HEK 293 cells heterologously expressing mouse TRPV1 (mTRPV1-HEK) and in mouse sensory neurons. Except for vanillin, four vanilloid analogues dose-dependently increased [Ca²⁺]_i in mTRPV1-HEK. The solutions that dissolved VMA, VAcid and vanillin at high concentrations were acidic, whereas those of VAlc and VBE were neutral. Neutralized VAcid evoked [Ca²⁺]_i increases but neutralized VMA did not. Mutation of capsaicin-sensing sites diminished [Ca²⁺]_i responses to VAcid, VAlc and VBE. VAcid, VMA, and vanillin suppressed the activation of TRPV1 induced by capsaicin. VAcid and VMA also inhibited the acid-induced TRPV1 activation. In sensory neurons, VMA diminished TRPV1 activation by capsaicin or acids. The present data indicate that these structural characteristics of chemical compounds on TRPV1 may provide strategies for the development of novel analgesic drugs targeting nociceptive TRPV1.     

Regulation of TRPV1 channel activities by intracellular ATP in the absence of capsaicin

Transient receptor potential vanilloid 1 (TRPV1) is a voltage-dependent non-selective cation channel activated by capsaicin, the main pungent ingredient of chili peppers, and noxious heat. Although TRPV1 channels produce outwardly rectifying currents even in the absence of capsaicin, little is known about the regulation mechanism of the TRPV1 currents. In the present study, we found that intracellular ATP regulates the basal activities of TRPV1 channels in a concentration-dependent manner. The ATP-dependent regulation of TRPV1 channels was mediated by phosphoinositides. Moreover, an increase in intracellular ATP concentration negatively shifted voltage-dependent activation of TRPV1 channels. These results suggest that the ATP-dependent production of phosphoinositides regulates the voltage-dependent gating of the basal TRPV1 channel activities in the absence of capsaicin.     

Endocannabinoid activation of the TRPV1 ion channel is distinct from activation by capsaicin

Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the “tail-up, head-down” configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.     

Oleuropein aglycone enhances UCP1 expression in brown adipose tissue in high-fat-diet-induced obese rats by activating β-adrenergic signaling

Oleuropein is the pungent principle of raw olives. Oleuropein aglycone (OA) is a major phenolic compound in extra virgin olive oil and the absorbed form of oleuropein. We aimed to determine the mechanism underlying the nutritional effects of oleuropein and OA on interscapular brown adipose tissue (IBAT) in rats with high-fat (HF) diet-induced obesity by examining the agonistic activity of oleuropein and OA toward the transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). Four-week-old male Sprague–Dawley rats were fed an HF (palm oil 30% wt:wt) diet alone or with oleuropein (HF-O, 1 g/kg diet) for 28 days. In rats fed HF-O compared to HF, urinary noradrenaline, adrenaline and UCP1 levels in IBAT were significantly higher, whereas plasma leptin levels and the total weight of the abdominal cavity adipose tissue were significantly lower. In anaesthetized 7-week-old male Sprague–Dawley rats, the OA (3.8 mg of intravenous injection)-induced increase in plasma noradrenaline secretion was suppressed by TRPA1 or TRPV1 antagonist and by a β2- or β3-adrenoceptor antagonist. Furthermore, OA-activated rat and human TRPV1s expressed on HEK293 cells at the same level as zingerone (pungent component in ginger). OA also activated humanTRPA1, and its potency was approximately 10-fold stronger than that for TRPV1. These findings suggest that OA is the agonist of both TRPA1 and TRPV1 and that OA enhances UCP1 expression in IBAT with a concomitant decrease in the visceral fat mass of HF-diet-induced obese rats through enhanced noradrenaline secretion via β-adrenergic action following TRPA1 and TRPV1 activation.     

Structural Determinants of the Transient Receptor Potential 1 (TRPV1) Channel Activation by Phospholipid Analogs

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1.     

Chiroptical properties of binuclear rhodium complexes of lanostane alcohols

The circular dichroism (CD) of the in situ-formed Rh2(OCOCF3)4 complexes of sterically hindered, secondary lanostane alcohols was investigated. The main object of the present studies are derivatives of 3beta-, 7alpha-, 7beta-, and 11beta-hydroxylanostanes with or without an additional functional group, e.g., double bond, oxo-, hydroxy-, or acetoxy groups. Up to five Cotton effects (CEs) can be found in the CD spectra of Rh-complexes of these alcohols in the spectral range between 650-300 nm. Correlation of the CEs signs with the absolute stereochemistry at the carbon atom bearing the hydroxy group was investigated. The Rh-complex with the 3beta-acetoxylanostan-11beta-ol ligand was isolated in the crystalline form. Its polymeric structure, determined by the X-ray method, shows the di-Rh-units linked by the axially ligating oxygen atoms of the hydroxy and acetoxy groups. In the latter case, the coordination takes place through the carbonyl oxygen.     

Molecular determinants of vanilloid sensitivity in TRPV1

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.     

Isolation and identification of a humanTRPV1 activating compound from soy sauce

Transient receptor potential vanilloid 1 (TRPV1) was identified as a receptor of capsaicin, which is a pungent ingredient in hot red peppers. Due to its relevance for nociception, a physiological and pharmacological study of TRPV1 has also been developed. Therefore, it is important to enrich scientific knowledge regarding the TRPV1 activating or inhibiting compounds. In this study, we fractionated soy sauce based on the human TRPV1 (hTRPV1) activity using column chromatography and purified 5-(9H-pyrido[3,4-b]indol-1-yl)-2-furanmethanol (perlolyrine) as an hTRPV1-activating compound. Additionally, perlolyrine activates the human transient receptor potential ankyrin 1 (hTRPA1). The EC₅₀ of hTRPV1 and hTRPA1 were 2.87 and 1.67 μmol L^?1, respectively. HPLC quantification of soy sauces showed that they contain 2.22–12.13 μmol L^?1 of perlolyrine. The sensory evaluation revealed that perlolyrine has taste modification effect. The results of this study, for the first time, suggest that perlolyrine induces the activation of hTRPV1 and hTRPA1.     

Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations

The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate.     

TRPA1 Agonists—Allyl Isothiocyanate and Cinnamaldehyde—Induce Adrenaline Secretion

Thermosensitive transient receptor potential (TRP) channels, especially TRPV1 and TRPA1, are activated by the pungent compounds present in spices. TRPV1 activation by the intake of capsaicin, the irritant in hot pepper, induces adrenaline secretion and increases energy consumption. TRPV1 is mainly expressed in the sensory neurons and coexpressed with TRPA1 at a high frequency. However, the mechanism underlying adrenaline secretion by TRPA1 agonists such as allyl isothiocyanate (AITC) and cinnamaldehyde (CNA), the pungent ingredients in mustard and cinnamon, is not known. We examined whether AITC and CNA could induce adrenaline secretion in anesthetized rats. An intravenous injection of AITC or CNA (10 mg/kg) increased adrenaline secretion. These responses disappeared completely in capsaicin-treated rats with an impaired sensory nerve function. Moreover, pretreatment with cholinergic blockers (hexamethonium and atropine) attenuated the AITC- or CNA-induced adrenaline secretion. These results suggest that TRPA1 agonists activate the sensory nerves and induce adrenaline secretion via the central nervous system.     

TRPA1 agonists--allyl isothiocyanate and cinnamaldehyde--induce adrenaline secretion

Thermosensitive transient receptor potential (TRP) channels, especially TRPV1 and TRPA1, are activated by the pungent compounds present in spices. TRPV1 activation by the intake of capsaicin, the irritant in hot pepper, induces adrenaline secretion and increases energy consumption. TRPV1 is mainly expressed in the sensory neurons and coexpressed with TRPA1 at a high frequency. However, the mechanism underlying adrenaline secretion by TRPA1 agonists such as allyl isothiocyanate (AITC) and cinnamaldehyde (CNA), the pungent ingredients in mustard and cinnamon, is not known. We examined whether AITC and CNA could induce adrenaline secretion in anesthetized rats. An intravenous injection of AITC or CNA (10 mg/kg) increased adrenaline secretion. These responses disappeared completely in capsaicin-treated rats with an impaired sensory nerve function. Moreover, pretreatment with cholinergic blockers (hexamethonium and atropine) attenuated the AITC- or CNA-induced adrenaline secretion. These results suggest that TRPA1 agonists activate the sensory nerves and induce adrenaline secretion via the central nervous system.