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    Role of bradykinin in acid-induced mesenteric hyperemia and duodenal villous damage. Leung Felix W,Iwata Fumihiro,Kao John,Seno Kyoji,Itoh Makoto,Leung Joseph W C Life sciences Intestinal mucosal capsaicin-sensitive afferent nerves mediate, in part, the mesenteric hyperemia after intraduodenal acidification. The hyperemia plays a role in protecting the duodenal mucosa against acid damage. We tested the hypothesis that bradykinin contributes to this protective hyperemia. A specific antagonist of bradykinin will attenuate the hyperemia and exacerbate duodenal villous damage induced by acid. Study 1: Intravenous vehicle, or the specific bradykinin B2 receptor antagonist (HOE 140) was administered to anesthetized rats. This was followed by intraduodenal bolus administration of 160 microM capsaicin or 0.1 N HCl, and then intravenous bradykinin. Study 2: Intravenous administration of vehicle or HOE 140 was followed by duodenal perfusion with 0.1 N HCl. Superior mesenteric artery blood flow (pulsed Doppler flowmetry) (Study 1) and duodenal villous damage (histology) (Study 2) were recorded. HOE 140 significantly reduced the hyperemia induced by bradykinin and intraduodenal capsaicin or acid. Deep villous injury was significantly increased after treatment with HOE 140. These findings support the hypothesis that acid-induced and afferent nerve-mediated mesenteric hyperemia is modulated by a mechanism that involves bradykinin B2 receptor. Antagonism of bradykinin B2 receptor also increased acid-induced deep duodenal villous damage. Thus, maintenance of bradykinin-mediated mesenteric hyperemia, is a previous unrecognized mechanism associated with protection of the rat duodenal mucosa against acid-induced damage. 10.1016/s0024-3205(01)01455-2
    Distribution and characterization of vanilloid receptors in the rat stomach. Nozawa Y,Nishihara K,Yamamoto A,Nakano M,Ajioka H,Matsuura N Neuroscience letters The cloned vanilloid receptor-1 (VR1) is recognized as a common molecular target for protons, noxious heat, and vanilloids. The presence of VR1 in the dorsal root, trigeminal, and nodose ganglia has been firmly established, but it is unclear in the gut, despite this VR1 may be important for gastric mucosal homeostasis. In this study we used an antibody and a radioligand to show the distribution of vanilloid receptors (VRs) in rat stomach and to characterize it. The deafferentiation of capsaicin-sensitive nerves in rats was induced by consecutive injections of capsaicin. VR1-immunopositive nerve endings were predominantly found in the mucous neck cells of the proliferation zone, and around blood vessels in the submucosa. Radioreceptor assay using [3H]-resiniferatoxin (RTX) revealed the existence of high affinity and single-class binding site in the membrane fractions of the mucosa. Capsaicin completely inhibited the specific binding of [3H]-RTX. Both the VR1 immunoreactivity and the receptor density of [3H]-RTX binding sites significantly reduced by the application of capsaicin for prolonged periods of time in the mucosa of rats. Our results indicate that VRs are expressed in the rat stomach, and suggest that they may be involved in mucosal protection by increasing cell proliferation and blood flow. 10.1016/s0304-3940(01)02021-3
    Direct evidence for ATP release from non-adrenergic, non-cholinergic ("purinergic") nerves in the guinea-pig taenia coli and bladder. Burnstock G,Cocks T,Kasakov L,Wong H K European journal of pharmacology Demonstration of release of ATP from smooth muscle preparations during stimulation of purinergic nerves is complicated by the difficulty in showing whether it comes from nerve or muscle. ATP released during relaxation of the guinea-pig taenia coli and contraction of bladder strips in response to purinergic nerve stimulation was measured in the superfusate using the luciferin-luciferase ATP assay method. The amount of ATP increased 2-6 fold during isometric responses to purinergic nerve stimulation. This release was blocked by tetrodotoxin but not by adrenergic nerve destruction with 6-hydroxydopamine. No significant release of ATP was detected during comparable responses elicited by direct muscle stimulation. These results provide further support for the purinergic nerve hypothesis. 10.1016/0014-2999(78)90070-5
    The contribution of cholinergic postganglionic neurotransmission to contractions of rabbit detrusor. Downie J W,Dean D M The Journal of pharmacology and experimental therapeutics Field stimulation was used to elicit a contractile response in muscle strips from rabbit detrusor. The blockade of this response by tetrodotoxin (1 X 10(-7) M) ranged from 100% at 1 Hz to 86% at 40 Hz. At concentrations which produced strictly muscarinic antagonism (up to 4 X 10(-7) M) atropine depressed the frequency-response curve by about 42% at maximum but was much less effective at frequencies below 10 Hz. Similarly, treatment of the strips with hemicholinium-3 (5.2 X 10(-4) M) for 90 minutes in the presence of field stimulation at 60 Hz, depressed the frequency-response curve by 52% at maximum but produced less depression below 10 Hz. The hemicholinium-3-resistant response was neither depressed further by atropine (4 X 10(-7) M) nor potentiated by physostigmine (2 X 10(-6) M). Although hemicholinium-3 has antimuscarinic and anticholinesterase properties, these were found not to interfere with the tests for residual cholinergic transmission. Therefore, it was concluded that only part of the motor neurotransmission in rabbit detrusor is cholinergic. The remaining portion is predominant at frequencies below 10 Hz and is postulated to involve a chemical mediator other than acetylcholine.
    Identification of an aspartic residue in the P-loop of the vanilloid receptor that modulates pore properties. García-Martínez C,Morenilla-Palao C,Planells-Cases R,Merino J M,Ferrer-Montiel A The Journal of biological chemistry Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway. 10.1074/jbc.M002391200
    Inhibition of calcineurin inhibits the desensitization of capsaicin-evoked currents in cultured dorsal root ganglion neurones from adult rats. Docherty R J,Yeats J C,Bevan S,Boddeke H W Pflugers Archiv : European journal of physiology Capsaicin activates a non-specific cation conductance in mammalian sensory neurones. If capsaicin is applied continuously or repeatedly then there is a progressive decline in responsiveness. We have studied the mechanism of this desensitization using electrophysiological methods in cultured dorsal root ganglion neurones from adult rats. The rate of desensitization of capsaicin-induced responses is partly dependent on the extracellular calcium concentration and is slower when extracellular calcium is reduced. Desensitization is strongly inhibited by intracellular application of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid (BAPTA). These data suggest that desensitization is due to a rapid rise in intracellular calcium levels which occurs when capsaicin-sensitive ion channels are activated. Desensitization is not reduced by the non-specific protein kinase inhibitors H7 or staurosporine or by okadaic acid, a selective inhibitor of protein phosphatases 1 and 2A. Desensitization is greatly reduced by cyclosporin A complexed to cyclophilin, which is a specific inhibitor of protein phoshatase 2B (calcineurin). A mechanism for desensitization of capsaicin responsiveness is proposed whereby the evoked rise in calcium activates calcineurin leading to dephosphorylation and desensitization of the capsaicin-sensitive ion channels.
    Evidence for angiotensin II receptors in the urinary bladder of the rabbit. Anderson G F,Barraco R A,Normile H J,Rosen T N Canadian journal of physiology and pharmacology Dose-response (DR) curves for several angiotensin analogs were examined on isolated rabbit detrusor strips with washout and rest between each addition. The order of potency was [Val5]-angiotensin II greater than [Ile5]-angiotensin II greater than [Ile5]-angiotensin I greater than [Val4]-angiotensin III. Repeated cumulative DR to [Val5]-AII resulted in a gradual increase in potency and intrinsic activity for four DR. However, the maximum force generated occurred at lower agonist concentrations and was less than that of the single methods, suggesting tachyphylaxis. Atropine (1.0 microM) shifted the cumulative DR curve downward, suggesting some cholinergic component possibly involving a presynaptic site of action. The magnitude of field-stimulated atropine-resistant contractions was reduced by both 1.0 and 10 microM saralasin as well as 10 microM naloxone. Tissue binding with 125I-labelled angiotensin II on isolated detrusor smooth muscle membranes indicated specific binding saturation occurred at 14.3 fmol/mg with a KD of 0.72 nM in EDTA-Tris buffered saline. Thus our results show that angiotensin II (AII) receptors can be demonstrated in destrusor muscle by ligand binding experiments on cell membranes and that saralasin and naloxone partially block atropine-resistant contractions. However, it seems unlikely that AII serves as a neurotransmitter because of the delay in onset of action of exogenous AII in isolated bath experiments and the apparent inability of saralasin to totally abolish the atropine-resistant field-stimulated preparation. If AII serves a role in neurotransmission it most probably is as a neuromodulator. 10.1139/y84-062
    Evidence for functional tachykinin NK1 receptors on human isolated small bronchi. Naline E,Molimard M,Regoli D,Emonds-Alt X,Bellamy J F,Advenier C The American journal of physiology On human small isolated bronchi (diameter < 1 mm), but not on larger bronchi (diameter 3-5 mm), substance P (SP) and specific tachykinin SP-preferring neurokinin (NK1) receptor agonists {[beta Ala4, Sar9, Met(O2)11]SP-(4-11), [Sar9, Met(O2)11]SP, [Arg6,Sar9,Met(O2)11]SP-(6-11), and septide; 10(-10) to 10(-6) M} produced a concentration-dependent contraction that occurred at low concentrations (pD2 values of 7.79-8.33) and was characterized by a low intrinsic activity [maximal effect (Emax) of 38-45% of Emax induced by 3 mM acetylcholine, in a noncumulative manner]. Comparison of cumulative and noncumulative concentration-response curves to SP and NK1 receptor agonists suggest rapid receptor desensitization. The SP (10(-8) M)-induced contraction was inhibited by tachykinin NK1 receptor antagonists (rank order of potency: SR-140333 > CP-96,345 > RP-67580) but not by the tachykinin NK2 receptor antagonist SR-48968. Indomethacin (10(-6) M) abolished the SP-induced contraction. Our results suggest that tachykinin NK1 receptors are present on human small bronchi and that their stimulation induces a prostanoid-dependent contraction. The small isolated bronchus is an interesting model of human tissue to test NK1 receptor antagonists. 10.1152/ajplung.1996.271.5.L763
    Capsaicin-like activity of some natural pungent substances on peripheral endings of visceral primary afferents. Patacchini R,Maggi C A,Meli A Naunyn-Schmiedeberg's archives of pharmacology 1. The effects of some naturally occurring pungent substances, piperine, mustard oil, eugenol and curcumin, were compared to those of capsaicin in the rat isolated urinary bladder. 2. All test compounds dose-dependently contracted the rat bladder and produced desensitization toward capsaicin (1 mumol/l). Development of cross-tachyphylaxis among the natural pungent substances on one hand and capsaicin on the other, suggested a common site of action on visceral primary afferents. 3. Contractile responses to piperine, mustard oil and eugenol were partially tetrodotoxin and ruthenium red-sensitive, suggesting that activation of sensory terminals by these agents takes place indirectly, as well as by a direct action on sensory receptors. 4. The presence of the secondary acrylamide linkage (present in the backbone of capsaicin, but not in that of test compounds) does not appear to be essential to produce desensitization of sensory nerve terminals.
    Pharmacological characterisation of the plant sesquiterpenes polygodial and drimanial as vanilloid receptor agonists. André Eunice,Campi Barbara,Trevisani Marcello,Ferreira Juliano,Malheiros Angela,Yunes Rosendo A,Calixto João B,Geppetti Pierangelo Biochemical pharmacology This study was designed to assess the participation of transient receptor potential vanilloid 1 (TRPV1) in the biological effects induced by the plant-derived sesquiterpenes polygodial and drimanial. In rat isolated urinary bladder, polygodial and drimanial produced a tachykinin-mediated contraction that was inhibited by combination of NK(1) and NK(2) tachykinin receptor antagonists, SR 140333 and SR 48968. Furthermore, two different TRPV1 antagonists, capsazepine and ruthenium red prevented the contraction induced by both compounds. In addition, capsaicin, polygodial and drimanial displaced in a concentration-dependent manner the specific binding sites of [(3)H]-resiniferatoxin to rat spinal cord membranes, with a IC(50) values of 0.48, 4.2 and 3.2 microM, respectively. Likewise, capsaicin, polygodial and drimanial promoted an increase of [(45)Ca(2+)] uptake in rat spinal cord synaptosomes. In cultured rat trigeminal neurons, polygodial, drimanial and capsaicin were also able to significantly increase the intracellular Ca(2+) levels, effect that was significantly prevented by capsazepine. Together, the present results strongly suggest that the pharmacological actions of plant-derived sesquiterpenes polygodial and drimanial, seem to be partially mediated by activation of TRPV1. Additional investigations are needed to completely define the pharmacodynamic properties of these sesquiterpenes. 10.1016/j.bcp.2005.12.030
    New evidence on the mechanisms underlying bradykinin-mediated contraction of the pig iris sphincter in vitro. El Sayah Mariem,Calixto João B Peptides We have reported previously that bradykinin (BK) induces potent and reproducible concentration-dependent contractions of the pig iris sphincter (PIS) muscle in vitro through the activation of BK B(2) receptors. Here we attempted to investigate additional mechanisms by which BK induces contraction of the PIS in vitro. BK-mediated contraction of the PIS relied largely on the external Ca2+ influx by a mechanism sensitive to the L-, N- and P-type of Ca2+ channel selective blockers. Likewise, BK-induced contraction of the PIS was greatly inhibited by the CGRP-(8-37), NK(2) or NK(3) receptor antagonists (SR 48968, SR 142801), and to a lesser extent by the NK(1) antagonist (FK 888). Capsaicin desensitization of PIS or capsazepine pre-incubation also significantly reduced BK-mediated contraction in the PIS. Furthermore, KT 5720 or GF 109203X (the protein kinase A and C inhibitors, respectively) also significantly inhibited BK-mediated contraction. Taken together, these results indicate that BK-mediated contraction of the PIS seems to be mediated primarily by the release of CGRP and tachykinins from sensory nerve fibers, and relies largely on extracellular Ca2+ influx via activation of L-, N- and P-type of Ca2+ channels. Finally, these responses are mediated by activation of both protein kinase A- and C-dependent mechanisms. 10.1016/s0196-9781(03)00182-7
    Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and alpha, beta-methylene ATP in guinea-pig urinary bladder. Usune S,Katsuragi T,Furukawa T British journal of pharmacology 1. The contraction and intracellular Ca2+ change evoked by diadenosine tetraphosphate (AP4A) were studied in the outer longitudinal muscle of the guinea-pig urinary bladder and compared with those evoked by ATP and alpha, beta-methylene ATP (a P2-purinoceptor agonist). 2. AP4A, ATP and alpha, beta-methylene ATP produced concentration-dependent transient contractions. These contractions were inhibited by PPADS (pyridoralphosphate-6-azophenyl- 2'-4'-disulphonic acid), 0.3- 30 microM, a P2x-purinoceptor antagonist, and suramin, 1-300 microM, a P2-purinoceptor antagonist in a concentration-dependent manner. From Schild plot analysis, the apparent pA2 values for PPADS for contractions evoked by AP4A, ATP and alpha, beta-methylene ATP were 6.86, 6.56, 6.74, and those for suramin were 6.01, 4.59 and 5.12, respectively; the Schild slopes for PPADS were 1.07, 1.14 and 1.06, and, those for suramin 0.75, 1.05 and 1.16, respectively. 3. AP4A (10 microM) and ATP (100 microM) failed to elicit any contraction of the tissue after a desensitization produced by repeated application of alpha, beta-methylene ATP (1 microM). 4. In fluorescence experiments with fura-2, the increases in [Ca2+]i and contraction evoked by AP4A were suppressed by suramin and nifedipine, an L-type Ca2+ channel blocker. 5. These findings suggest that P2x-purinoceptors, which are more sensitive to PPADS than suramin, exist on the outer longitudinal muscles of guinea-pig urinary bladder, and that the AP4A-evoked contraction results from Ca2+ influx. 10.1111/j.1476-5381.1996.tb15246.x
    Pharmacological investigation of hydrogen sulfide (H2S) contractile activity in rat detrusor muscle. Patacchini Riccardo,Santicioli Paolo,Giuliani Sandro,Maggi Carlo Alberto European journal of pharmacology We have investigated the mechanism through which hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat isolated urinary bladder. Sodium hydrogen sulfide (NaHS), a donor of H2S, produced concentration-dependent contractile responses (pEC50=3.5+/-0.1) that were unaffected by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine (30 microM) and SB 366791 (10 microM) and by the N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CTX; 100 nM). In contrast, the unselective transient receptor potential (TRP) cation channels blocker ruthenium red (30 microM) almost abolished NaHS-induced contractions. Ruthenium red (30 microM) greatly reduced capsaicin-induced contractions, whereas it did not attenuate the contractile response to neurokinin A. The putative TRPV1 receptor antagonist iodo-resiniferatoxin, from 100 nM upward, produced agonist responses per se, and could not be tested against NaHS. We conclude that H2S either acts at TRPV1 receptorial sites unblocked by capsazepine or SB 366791, or stimulates a still unidentified transient receptor potential-like channel co-expressed with TRPV1 on sensory neurons. 10.1016/j.ejphar.2005.01.005
    Study of the mechanisms involved in the bradykinin-induced contraction of the pig iris sphincter muscle in vitro. El Sayah Mariem,Calixto João B European journal of pharmacology This study was designed to investigate the mechanisms by which bradykinin induces contraction of the pig iris sphincter muscle in vitro. Addition of bradykinin, Lys-bradykinin and Met-Lys-bradykinin to the pig iris sphincter resulted in a graded contraction with a mean EC(50s) of 21, 11 and 5 nM, respectively. The bradykinin B(1) receptor agonist des-Arg(9)-bradykinin only caused a slight contraction, measured 6 h after the tissue was set up. The B(2) receptor antagonists FR 173657 ((E)-3-(6-acetamido-3-pyridyl)-N [N-2-4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylamino-carbonyl-ethyl] acrylamide) and Hoe 140 (D-Arg(0)-[Hyp(3), Thi(5), D-Tic(7), Oic(8)]-bradykinin produced a graded shift to the right associated with marked inhibition of the bradykinin-induced contraction. Atropine, guanethidine or tetrodotoxin significantly reduced the bradykinin-induced contraction. Dazoxiben, an inhibitor of thromboxane A(2), and MK-571 (3-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3oxo-propyl) thio) methyl) propanoic acid, a leukotriene D(4) receptor-selective antagonist, also caused inhibition of the bradykinin-mediated contraction. Cyclooxygenase-1 and -2 inhibitors, indomethacin, ibuprofen, valeryl salicylate and NS 398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methanosulfonamide) all significantly inhibited the bradykinin-mediated contraction without affecting the carbachol-induced contraction of the pig iris sphincter. Taken together, these results indicate that the bradykinin-mediated contraction of the pig iris sphincter muscle seems to be mediated primarily by the activation of the B(2) receptor release of acetylcholine, noradrenaline and both cyclooxygenase-1 and -2 metabolites besides the release of leukotriene D(4) and tromboxane A(2) from the arachidonic acid pathway. 10.1016/s0014-2999(02)02702-4
    Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. Nagata Keiichi,Duggan Anne,Kumar Gagan,García-Añoveros Jaime The Journal of neuroscience : the official journal of the Society for Neuroscience Mechanosensory channels of sensory cells mediate the sensations of hearing, touch, and some forms of pain. The TRPA1 (a member of the TRP family of ion channel proteins) channel is activated by pain-producing chemicals, and its inhibition impairs hair cell mechanotransduction. As shown here and previously, TRPA1 is expressed by hair cells as well as by most nociceptors (small neurons of dorsal root, trigeminal, and nodose ganglia) and localizes to their sensory terminals (mechanosensory stereocilia and peripheral free nerves, respectively). Thus, TRPA1 channels are proposed to mediate transduction in both hair cells and nociceptors. Accordingly, we find that heterologously expressed TRPA1 display channel behaviors expected for both auditory and nociceptive transducers. First, TRPA1 and the hair cell transducer share a unique set of pore properties not described for any other channel (block by gadolinium, amiloride, gentamicin, and ruthenium red, a ranging conductance of approximately 100 pS that is reduced to 54% by calcium, permeating calcium-induced potentiation followed by closure, and reopening by depolarization), supporting a direct role of TRPA1 as a pore-forming subunit of the hair cell transducer. Second, TRPA1 channels inactivate in hyperpolarized cells but remain open in depolarized cells. This property provides a mechanism for the lack of desensitization, coincidence detection, and allodynia that characterize pain by allowing a sensory neuron to respond constantly to sustained stimulation that is suprathreshold (i.e., noxious) and yet permitting the same cell to ignore sustained stimulation that is subthreshold (i.e., innocuous). Our results support a TRPA1 role in both nociceptor and hair cell transduction. 10.1523/JNEUROSCI.0013-05.2005
    The pungency of garlic: activation of TRPA1 and TRPV1 in response to allicin. Macpherson Lindsey J,Geierstanger Bernhard H,Viswanath Veena,Bandell Michael,Eid Samer R,Hwang SunWook,Patapoutian Ardem Current biology : CB Garlic's pungent flavor has made it a popular ingredient in cuisines around the world and throughout history. Garlic's health benefits have been elevated from folklore to clinical study. Although there is some controversy as to the efficacy of garlic, garlic products are one of the most popular herbal supplements in the U.S. Chemically complex, garlic contains different assortments of sulfur compounds depending on whether the cloves are intact, crushed, cooked, or raw. Raw garlic, when cut and placed on the tongue or lips, elicits painful burning and prickling sensations through unknown mechanisms. Here, we show that raw but not baked garlic activates TRPA1 and TRPV1, two temperature-activated ion channels that belong to the transient receptor potential (TRP) family. These thermoTRPs are present in the pain-sensing neurons that innervate the mouth. We further show that allicin, an unstable component of fresh garlic, is the chemical responsible for TRPA1 and TRPV1 activation and is therefore likely to cause garlic's pungency. 10.1016/j.cub.2005.04.018
    Pungent products from garlic activate the sensory ion channel TRPA1. Bautista Diana M,Movahed Pouya,Hinman Andrew,Axelsson Helena E,Sterner Olov,Högestätt Edward D,Julius David,Jordt Sven-Eric,Zygmunt Peter M Proceedings of the National Academy of Sciences of the United States of America Garlic belongs to the Allium family of plants that produce organosulfur compounds, such as allicin and diallyl disulfide (DADS), which account for their pungency and spicy aroma. Many health benefits have been ascribed to Allium extracts, including hypotensive and vasorelaxant activities. However, the molecular mechanisms underlying these effects remain unknown. Intriguingly, allicin and DADS share structural similarities with allyl isothiocyanate, the pungent ingredient in wasabi and other mustard plants that induces pain and inflammation by activating TRPA1, an excitatory ion channel on primary sensory neurons of the pain pathway. Here we show that allicin and DADS excite an allyl isothiocyanate-sensitive subpopulation of sensory neurons and induce vasodilation by activating capsaicin-sensitive perivascular sensory nerve endings. Moreover, allicin and DADS activate the cloned TRPA1 channel when expressed in heterologous systems. These and other results suggest that garlic excites sensory neurons primarily through activation of TRPA1. Thus different plant genera, including Allium and Brassica, have developed evolutionary convergent strategies that target TRPA1 channels on sensory nerve endings to achieve chemical deterrence. 10.1073/pnas.0505356102
    An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts. Jaquemar D,Schenker T,Trueb B The Journal of biological chemistry We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control. 10.1074/jbc.274.11.7325
    Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Birder L A,Kanai A J,de Groat W C,Kiss S,Nealen M L,Burke N E,Dineley K E,Watkins S,Reynolds I J,Caterina M J Proceedings of the National Academy of Sciences of the United States of America 10.1073/pnas.231243698
    The basis for drug treatment of the overactive bladder. Andersson K E,Chapple C,Wein A World journal of urology The normal bladder functions, storage and elimination of urine, are dependent on neural circuits in the brain and spinal cord that coordinate the activity of the detrusor and that of the smooth and striated muscles of the outflow region. Disturbances at different levels may cause the overactive bladder (OAB) syndrome, characterized by urge, frequency and urge incontinence. Knowledge about the mechanisms controlling both normal and abnormal micturition is mandatory for the detection of targets for pharmacological intervention. Such targets may be found in the central nervous system (CNS) or peripherally. Several CNS transmitters can modulate voiding, but few drugs with a defined CNS site of action have been demonstrated to be clinically useful. Traditionally, drugs for treatment o 10.1007/pl00007101
    Direct activation of capsaicin receptors by products of lipoxygenases: endogenous capsaicin-like substances. Hwang S W,Cho H,Kwak J,Lee S Y,Kang C J,Jung J,Cho S,Min K H,Suh Y G,Kim D,Oh U Proceedings of the National Academy of Sciences of the United States of America Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction. 10.1073/pnas.97.11.6155
    Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Zygmunt P M,Petersson J,Andersson D A,Chuang H,Sørgård M,Di Marzo V,Julius D,Högestätt E D Nature The endogenous cannabinoid receptor agonist anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. Here we show that the vasodilator response to anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP, but not the cannabinoid CB1 receptor blocker SR141716A, inhibited the vasodilator effect of anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (HU 210, WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic the action of anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibited anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Our results indicate that anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems. 10.1038/22761
    The cloned capsaicin receptor integrates multiple pain-producing stimuli. Tominaga M,Caterina M J,Malmberg A B,Rosen T A,Gilbert H,Skinner K,Raumann B E,Basbaum A I,Julius D Neuron Capsaicin, the main pungent ingredient in "hot" chili peppers, elicits buming pain by activating specific (vanilloid) receptors on sensory nerve endings. The cloned vanilloid receptor (VR1) is a cation channel that is also activated by noxious heat. Here, analysis of heat-evoked single channel currents in excised membrane patches suggests that heat gates VR1 directly. We also show that protons decrease the temperature threshold for VR1 activation such that even moderately acidic conditions (pH < or = 5.9) activate VR1 at room temperature. VR1 can therefore be viewed as a molecular integrator of chemical and physical stimuli that elicit pain. Immunocytochemical analysis indicates that the receptor is located in a neurochemically heterogeneous population of small diameter primary afferent fibers. A role for VR1 in injury-induced hypersensitivity at the level of the sensory neuron is presented.
    The capsaicin receptor: a heat-activated ion channel in the pain pathway. Caterina M J,Schumacher M A,Tominaga M,Rosen T A,Levine J D,Julius D Nature Capsaicin, the main pungent ingredient in 'hot' chilli peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. We have used an expression cloning strategy based on calcium influx to isolate a functional cDNA encoding a capsaicin receptor from sensory neurons. This receptor is a non-selective cation channel that is structurally related to members of the TRP family of ion channels. The cloned capsaicin receptor is also activated by increases in temperature in the noxious range, suggesting that it functions as a transducer of painful thermal stimuli in vivo. 10.1038/39807
    Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder. Andrade Edinéia Lemos,Ferreira Juliano,André Eunice,Calixto João B Biochemical pharmacology TRPA1 is a member of the transient receptor potential (TRP) channel family present in sensory neurons. Here we show that vanilloid receptor (TRPV1) stimulation with capsaicin and activation of TRPA1 with allyl isothiocyanate or cinnamaldehyde cause a graded contraction of the rat urinary bladder in vitro. Repeated applications of maximal concentrations of the agonists produce desensitization to their contractile effects. Moreover, contraction caused by TRPA1 agonists generates cross-desensitization with capsaicin. The TRP receptor antagonist ruthenium red (10-100 microM) inhibits capsaicin (0.03 microM), allyl isothiocyanate (100 microM) and cinnamaldehyde (300 microM)-induced contractions in the rat urinary bladder. The selective TRPV1 receptor antagonist SB 366791 (10 microM) blocks capsaicin-induced contraction, but partially reduces allyl isothiocyanate- or cinnamaldehyde-mediated contraction. However, allyl isothiocyanate and cinnamaldehyde (10-1000 microM) completely fail to interfere with the specific binding sites for the TRPV1 agonist [(3)H]-resiniferatoxin. Allyl isothiocyanate or cinnamaldehyde-mediated contractions of rat urinary bladder, which rely on external Ca(2+) influx, are significantly inhibited by tachykinin receptor antagonists as well as by tetrodotoxin (1 microM) or indomethacin (1 microM). Allyl isothiocyanate-induced contraction is not changed by atropine (1 microM) or suramin (300 microM). The exposure of urinary bladders to allyl isothiocyanate (100 microM) causes an increase in the prostaglandin E(2) and substance P levels. Taken together, these results indicate that TRPA1 agonists contract rat urinary bladder through sensory fibre stimulation, depending on extracellular Ca(2+) influx and release of tachykinins and cyclooxygenase metabolites, probably prostaglandin E(2). Thus, TRPA1 appears to exert an important role in urinary bladder function. 10.1016/j.bcp.2006.04.003
    Isolation and receptor profiling of ileal enterochromaffin cells. Schäfermeyer A,Gratzl M,Rad R,Dossumbekova A,Sachs G,Prinz C Acta physiologica Scandinavica AIM AND METHODS:Enterochromaffin (EC) cells, interspersed throughout the gastrointestinal mucosa, provide most of the serotonin of the body and control intestinal motility, secretion and absorption. We purified EC cells from the rat ileum by a combination of elutriation and density gradient centrifugation in order to characterize the function of this important cell type. RESULTS:Immunostaining showed that there were 84% serotonin-positive cells in the highly enriched EC fraction as compared with 12% in unfractionated cells, yielding a approximately sevenfold enrichment. Serotonin measurements in the cell suspensions indicated a seven to 14-fold enrichment. Presence of alpha- and beta-adrenoreceptor isoforms, muscarinic M3 and gama-aminobutyric acid (GABA)-A receptors was confirmed by RT-PCR and cytochemistry. Increased expression of VMAT-1 and GABA-A mRNA was also shown by quantitative TaqMan PCR using EC cell RNA. Serotonin release in isolated EC cells was stimulated by noradrenaline, and to a smaller extent, by carbachol, while GABA addition was without effect. CONCLUSION:Our data provide a basis for a new approach to characterize receptors on this unique cell type. 10.1111/j.1365-201X.2004.01299.x
    Distinct 5-HT receptors mediate the peristaltic reflex induced by mucosal stimuli in human and guinea pig intestine. Foxx-Orenstein A E,Kuemmerle J F,Grider J R Gastroenterology BACKGROUND & AIMS:The role of 5-hydroxytryptamine (5-HT) in regulating the peristaltic reflex in humans is unknown. The neural pathways subserving peristalsis induced by mucosal stimulation were characterized in human jejunum and guinea pig colon. METHODS:Compartmented flat-sheet preparations that enable measurement of 5-HT and sensory transmitter release into one compartment and mechanical responses in adjacent compartments were used. RESULTS:Mucosal stimuli (2-8 brush strokes) caused concomitant release of 5-HT and calcitonin gene-related peptide (CGRP) into the compartment where stimulation was applied in both species; in contrast, muscle stretch caused release of CGRP only. CGRP release as well as ascending contraction and descending relaxation of circular muscle induced by mucosal stimulation were inhibited by a selective 5-HT4 antagonist in human jejunum and by selective 5-HT4 and 5-HT3 antagonists in guinea pig colon. The effects of the 5-HT3 and 5-HT4 antagonists in guinea pig colon were additive. A selective 5-HT1P antagonist mimicked the effect of the 5-HT4 antagonist. The CGRP antagonist human CGRP8-37 inhibited ascending and descending responses in both species. CONCLUSIONS:5-HT released by mucosal stimulation initiates a peristaltic reflex by activating 5-HT4/5-HT1P receptors on sensory CGRP neurons in human intestine and 5-HT4/5-HT1P and 5-HT3 receptors in guinea pig colon. 10.1053/gast.1996.v111.pm8898642
    An examination of the 5-HT3 receptor mediating contraction and evoked [3H]-acetylcholine release in the guinea-pig ileum. Fox A,Morton I K British journal of pharmacology 1. The relative contributions of two classes of 5-hydroxytryptamine (5-HT) receptor (5-HT2 and 5-HT3) to the contractile action of 5-HT, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT) and alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) were studied in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. Contractility studies were combined with an analysis of the effects of the three agonists on [3H]-acetylcholine ([3H]-ACh) release from preparations preincubated with [3H]-choline. 2. In contracting the LMMP, 5-HT was approximately one order of magnitude more active than 2-methyl-5-HT and alpha-methyl-5-HT, with relative activities for 5-HT: 2-methyl-5-HT: alpha-methyl-5-HT of 1.00: 0.13: 0.10. 3. Ketanserin (1 microM) was without effect on the concentration-response curves for concentration to 5-HT. 2-methyl-5-HT or alpha-methyl-5-HT, whilst ondansetron (GR38032F: 1 microM) produced a parallel rightward displacement of the upper part of the concentration-response curves to 5-HT and alpha-methyl-5-HT and of the entire curve to 2-methyl-5-HT. 4. In increasing the spontaneous release of [3H]-ACh from the LMMP, 5-HT was again approximately one order of magnitude more active than 2-methyl-5-HT and alpha-methyl-5-HT with relative activities for 5-HT: 2-methyl-5-HT: alpha-methyl-5-HT of 1.00: 0.19: 0.11. 5. Ondansetron (1 microM) greatly attenuated the increase in spontaneous [3H]-ACh release evoked by all three agonists. pKB estimates of 7.62 + 0.12 and 7.64 + 0.09 were obtained for ondansetron antagonism of 5-HT and 2-methyl-5-HT-evoked increases respectively. 6. These data suggest that the contractile action of 5-HT, 2-methyl-5-HT and a-methyl-5-HT in the guinea-pig ileum can, under these conditions, be accounted for largely in terms of 5-HT3 receptor activation. Estimates for pKB obtained with ondansetron are in accordance with those previously obtained from contractility studies in this preparation and these findings are discussed in terms of the postulated existence of subtypes of 5-HT3 receptors. 10.1111/j.1476-5381.1990.tb14119.x
    Serotonin as a useful parameter for cold and warm ischemic injury in small bowel transplantation. Kaihara S,Egawa H,Inomata Y,Uemoto S,Asonuma K,Tanaka K Transplantation We investigated serotonin as a parameter of cold and warm ischemic injury prior to transplantation. Lewis rats were used as both donors and recipients, and the proximal 20 cm of jejunum served as the graft. The grafts were preserved in 4 degrees C lactated Ringer's solution for 0, 6, 12, 18, and 24 hr after harvest for cold ischemia (n=7/group). The superior mesenteric artery was clamped for 0, 15, 30, 60, and 120 min before harvest for warm ischemia (n=7/group). The serotonin concentration was measured in the luminal effluent and the preservation solution before transplantation, and total serotonin was calculated as the sum of these amounts. Finally, transplantation was performed heterotopically. Total serotonin increased significantly with both cold and warm ischemic time (P<0.01 by analysis of variance, Fisher's PLSD); however, between 18 hr and 24 hr of cold ischemic time only, there were no significant changes. Total serotonin levels correlated well with cold and warm ischemic time, as shown by linear regression analysis (cold ischemia: R2=80.2%, P<0.01; warm ischemia: R2=92.8%, P<0.01). We established the cutoff level of total serotonin to predict the graft survival at 2200 ng, and using this critical level, graft survival was predicted by total serotonin with a sensitivity of 71.4% and a specificity of 89.8%. Immunohistochemical staining with the serotonin antibody revealed that the number of serotonin-positive cells decreased with both cold and warm ischemic time. In conclusion, serotonin is a useful parameter of cold and warm ischemic injury before transplantation and can assist in predicting graft survival. 10.1097/00007890-199708150-00005
    Serotonin levels in the small bowel mucosa as a marker of ischemic injury during small bowel preservation. Matia Ivan,Baláz Peter,Jackanin Stanislav,Rybárová Emília,Kron Ivan,Pomfy Mikulás,Fronek Jirí,Ryska Miroslav Annals of transplantation Of the serotonin occurring in the small bowel mucosa, 95% is present in enterochromaffine cells. The cold ischemia during small bowel transplantation results in mucosal injury and releasing of serotonin into the lumen. Because of it, the mucosal concentration of serotonin is decreasing. The aim of our study was to establish the correlation between changes in serotonin levels in small bowel mucosa during grafts preservation and cold ischemic time. Wistar rats (n= 35) weighing 322+/-18g, divided into five main groups (n= 7/group) according to the time of small bowel grafts preservation (0, 1, 6, 9, and 12 hours), were used as experimental animals. The grafts were preserved in 4 degrees C histidine-tryptophane-ketoglutarate (HTK) solution. Tissue samples for mucosal serotonin concentration measurement and for light microscopic evaluation were taken after predefined cold ischemic times. Quantitative histological assessment was made using the Park's small bowel wall injury grading scheme. The t-test for dependent samples was used for statistical analysis. The mean serotonin mucosal concentrations after 0, 1, 6, 9, and 12 hours of cold ischemic injury were 433.09+/-160.33, 402.6+/-120.53, 412.5+/-47.57 ng/mL, 190.8+/-45.88 and 145.2+/-16.78 ng/mL Statistically significant differences (p<0.05) were between 6, 9, and 12 hours of cold ischemia. Morphological changes of small bowel mucosa graded by Park's scheme after the same ischemic intervals were 0, 0.5+/-0.47, 0.97+/-0.41, 1.74+/-0.69, and 1.84+/-0.64. Statistically significant differences (p<0.05) were demonstrated between all preservation times except between 9 and 12 hours of cold ischemia. Morphological changes in small bowel mucosa correlated with cold ischemic time, as well as with serotonin mucosal concentration. These data indicate the possibility of use a serotonin concentration in small bowel mucosa as a parameter of small bowel grafts ischemic injury.
    Mechanical stimulation activates Galphaq signaling pathways and 5-hydroxytryptamine release from human carcinoid BON cells. Kim M,Javed N H,Yu J G,Christofi F,Cooke H J The Journal of clinical investigation 5-Hydroxytryptamine (5-HT) released from enterochromaffin cells activates secretory and peristaltic reflexes necessary for lubrication and propulsion of intestinal luminal contents. The aim of this study was to identify mechanosensitive intracellular signaling pathways that regulate 5-HT release. Human carcinoid BON cells displayed 5-HT immunoreactivity associated with granules dispersed throughout the cells or at the borders. Mechanical stimulation by rotational shaking released 5-HT from BON cells or from guinea pig jejunum during neural blockade with tetrodotoxin. In streptolysin O-permeabilized cells, guanosine 5'-O- (2-thiodiphosphate) (GDP-beta-S) and a synthetic peptide derived from the COOH terminus of Galphaq abolished mechanically evoked 5-HT release, while the NH(2)-terminal peptide did not. An antisense phosphorothioated oligonucleotide targeted to a unique sequence of Galphaq abolished mechanically evoked 5-HT release and reduced Galphaq protein levels without affecting the expression of Galpha(11). Depletion and chelation of extracellular calcium did not alter mechanically evoked 5-HT release, whereas depletion of intracellular calcium stores by thapsigargin and chelation of intracellular calcium by 1,2-bis (o-Aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) reduced 5-HT release. Mechanically evoked 5-HT release was inhibited by somatostatin-14 in a concentration-dependent manner. The results suggest that mechanical stimulation of enterochromaffin-derived BON cells directly or indirectly stimulates a G protein-coupled receptor that activates Galphaq, mobilizes intracellular calcium, and causes 5-HT release. 10.1172/JCI12467
    Short-chain fatty acids stimulate colonic transit via intraluminal 5-HT release in rats. Fukumoto Satoshi,Tatewaki Makoto,Yamada Tadanori,Fujimiya Mineko,Mantyh Chris,Voss Miranda,Eubanks Steve,Harris Mary,Pappas Theodore N,Takahashi Toku American journal of physiology. Regulatory, integrative and comparative physiology We studied whether physiological concentration of short-chain fatty acids (SCFAs) affects colonic transit and colonic motility in conscious rats. Intraluminal administration of SCFAs (100-200 mM) into the proximal colon significantly accelerated colonic transit. The stimulatory effect of SCFAs on colonic transit was abolished by perivagal capsaicin treatment, atropine, hexamethonium, and vagotomy, but not by guanethidine. The stimulatory effect of SCFAs on colonic transit was also abolished by intraluminal pretreatment with lidocaine and a 5-hydroxytryptamine (HT)(3) receptor antagonist. Intraluminal administration of SCFAs provoked contractions at the proximal colon, which migrated to the mid- and distal colon. SCFAs caused a significant increase in the luminal concentration of 5-HT of the vascularly isolated and luminally perfused rat colon ex vivo. It is suggested that the release of 5-HT from enterochromaffin cells in response to SCFAs stimulates 5-HT(3) receptors located on the vagal sensory fibers. The sensory information is transferred to the vagal efferent and stimulates the release of acetylcholine from the colonic myenteric plexus, resulting in muscle contraction. 10.1152/ajpregu.00442.2002
    Characterization of YM060, a potent and selective 5-hydroxytryptamine3 receptor antagonist, in rabbit nodose ganglion and N1E-115 neuroblastoma cells. Ito H,Hidaka K,Miyata K,Kamato T,Nishida A,Honda K The Journal of pharmacology and experimental therapeutics The 5-hydroxytryptamine (5-HT)3 receptor blocking properties of YM060, [(R)-5-[(1-methyl-3-indolyl)carbonyl]-4,5,6,7-tetrahydro-1H- benzimidazole hydrochloride], were examined by electrophysiological and radioligand binding studies. Results were compared with those for ondansetron, granisetron and the enantiomer (S-form) of YM060. 5-HT and 2-methyl-5-HT, a selective 5-HT3 receptor agonist, induced dose-dependent depolarizations of rabbit nodose ganglion with ED50 values of 24.0 (19.9-29.1) and 40.1 (30.9-52.1) nmol, respectively (geometric mean, 95% CL). YM060, ondansetron, granisetron and the S-form dose-dependently inhibited 5-HT-induced depolarizations with IC50 values of 3.85 (2.47-5.98), 1.55 (1.26-1.91), 1.45 (1.18-1.79) and 13.5 (11.2-16.2) nM, respectively. Methysergide, a 5-HT1-like and 5-HT2 receptor antagonist, at a concentration of 10(-5) M had no effect on responses to 5-HT. YM060 up to 10(-5) M produced no significant depression of depolarizing responses to 1,1-dimethyl-4-phenylpiperazinium iodide and gamma-aminobutyric acid. YM060, ondansetron, granisetron and the S-form displaced specific binding of [3H]GR65630 to N1E-115 neuroblastoma cell membranes with Ki values of 0.091 (0.086-0.097), 7.03 (5.96-8.01), 2.02 (1.74-2.30) and 10.3 (9.96-10.6) nM, respectively. These results show that YM060, compared with ondansetron and granisetron, has considerably higher affinity for 5-HT3 receptors in N1E-115 cells and slightly less potent 5-HT3 receptor antagonistic activity in rabbit nodose ganglion. Moreover, the isomeric activity ratio (R-form/S-form) was approximately 112 in N1E-115 cells and no greater than 4 in the ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)
    Block by ruthenium red of cloned neuronal voltage-gated calcium channels. Cibulsky S M,Sather W A The Journal of pharmacology and experimental therapeutics The dye ruthenium red (RuR) has diverse experimental uses, including block of ion channels. RuR is a well described antagonist of one class of intracellular Ca2+ release channels, the ryanodine receptors, but recently this compound has also been identified as a putative blocker of voltage-gated calcium channels of the surface membrane involved in neurotransmitter release. Using electrophysiological methods, we have studied the action of RuR upon pure populations of neuronal voltage-gated ion channels heterologously expressed in Xenopus laevis oocytes. All four channel types studied, including class A (P/Q-type), class B (N-type), class C (L-type), and class E channels, are sensitive to RuR, with IC50 values ranging from 0.7 to 67.1 microM. Block of class C and class E channels most likely results from 1:1 binding of ruthenium red at a site in the extracellular entrance to the pore, resulting in obstruction of permeant ion flux through these channels. The mechanism of block of class A and class B channels is more complex, requiring binding of more than one molecule of RuR per channel.
    Contractile effect of TRPA1 receptor agonists in the isolated mouse intestine. Penuelas Angelica,Tashima Kimihito,Tsuchiya Shizuko,Matsumoto Kenjiro,Nakamura Tomonori,Horie Syunji,Yano Shingo European journal of pharmacology TRPA1 is a member of the transient receptor potential (TRP) channel family expressed in sensory neurons. The present study focused on the effects of TRPA1 activation on contractile responses in isolated mouse intestine preparations. The jejunum, ileum, and proximal and distal colon were surgically isolated from male ddY mice. Intestinal motility was recorded as changes in isotonic tension. TRPA1, TRPM8, and TRPV1 expressions were examined by reverse transcription-polymerase chain reaction (RT-PCR). A TRPA1 agonist allyl isothiocyanate (AITC) dose-dependently induced contractions in the proximal and distal colon, whereas in the jejunum and ileum, even 100 muM AITC caused very little contraction. Likewise, a TRPA1 and TRPM8 agonist icilin, a TRPA1 agonist allicin, and a TRPV1 agonist capsaicin induced contractions in the colon. However, a TRPM8 agonist menthol induced long-lasting relaxation in the colon. Repeated exposure to AITC produced desensitization of its own contraction in the colon. Moreover, contractions induced by AITC generate cross-desensitization with icilin and capsaicin. Tetrodotoxin completely abolished AITC-induced contractions in the colon, whereas atropine significantly attenuated AITC-induced contractions in the distal colon, but not in the proximal colon. Menthol-induced relaxation in the colon was not inhibited by tetrodotoxin and atropine. RT-PCR analysis revealed the expression of TRPA1 and TRPV1, but not TRPM8, throughout the mouse intestine. These results suggest that TRPA1, but not TRPM8, are functionally expressed in the enteric nervous system throughout the mouse intestine on neurons that may also co-express TRPV1, yet the contractile responses to TRPA1 activation differ depending on their location along the intestine. 10.1016/j.ejphar.2007.08.015
    Protective effects of Delta(9)-tetrahydrocannabinol against N-methyl-d-aspartate-induced AF5 cell death. Chen Jia,Lee Chun-Ting,Errico Stacie,Deng Xiaolin,Cadet Jean L,Freed William J Brain research. Molecular brain research The neuroprotective effects of Delta(9)-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl-d-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant alpha-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation. 10.1016/j.molbrainres.2004.10.044
    ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Story Gina M,Peier Andrea M,Reeve Alison J,Eid Samer R,Mosbacher Johannes,Hricik Todd R,Earley Taryn J,Hergarden Anne C,Andersson David A,Hwang Sun Wook,McIntyre Peter,Jegla Tim,Bevan Stuart,Patapoutian Ardem Cell Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol. 10.1016/s0092-8674(03)00158-2
    Plasticity in serotonin control mechanisms in the gut. Gershon Michael D Current opinion in pharmacology 5-hydroxytryptamine (5-HT or serotonin) is a charged molecule and must be transported across biological membranes. Enzymes that catabolize 5-HT are all intracellular; therefore, 5-HT inactivation requires a high affinity transporter, known as serotonin transporter (SERT or 5-HTT). In the central and enteric nervous systems, SERT is located in serotonergic neurons; however, these neurons are not present in the gastrointestinal mucosa, where 5-HT initiates peristaltic and secretory reflexes. Instead, SERT is expressed by enterocytes. The severity of gastrointestinal effects caused by drugs that inhibit SERT, such as tricyclic antidepressants, selective serotonin reuptake inhibitors and cocaine, does not usually prevent their therapeutic or recreational use because backup transporters and alterations in receptor gene expression allow the gut to adapt, albeit imperfectly, to their toxicity. 10.1016/j.coph.2003.07.005
    The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gershon Michael D,Tack Jan Gastroenterology Serotonin is an important gastrointestinal signaling molecule. It is a paracrine messenger utilized by enterochromaffin (EC) cells, which function as sensory transducers. Serotonin activates intrinsic and extrinsic primary afferent neurons to, respectively, initiate peristaltic and secretory reflexes and to transmit information to the central nervous system. Serotonin is also a neurotransmitter utilized by a system of long descending myenteric interneurons. Serotonin is synthesized through the actions of 2 different tryptophan hydroxylases, TpH1 and TpH2, which are found, respectively, in EC cells and neurons. Serotonin is inactivated by the serotonin reuptake transporter (SERT)-mediated uptake into enterocytes or neurons. The presence of many serotonin receptor subtypes enables selective drugs to be designed to therapeutically modulate gastrointestinal motility, secretion, and sensation. Current examples include tegaserod, a 5-HT(4) partial agonist, which has been approved for treatment of irritable bowel syndrome (IBS) with constipation in women and for chronic constipation in men and women. The 5-HT(3) antagonists, granisetron and ondansetron, are useful in combating the nausea associated with cancer chemotherapy, and alosetron is employed in the treatment of IBS with diarrhea. Serotonergic signaling abnormalities have also been putatively implicated in the pathogenesis of functional bowel diseases. Other compounds, for which efficacy has not been rigorously established, but which may have value, include tricyclic antidepressants and serotonin selective reuptake inhibitors to combat IBS, and 5-HT(1) agonists, which enhance gastric accommodation, to treat functional dyspepsia. The initial success encountered with serotonergic agents holds promise for newer and more potent insights and therapies of brain-gut disorders. 10.1053/j.gastro.2006.11.002
    Inhalational anesthetics activate two-pore-domain background K+ channels. Patel A J,Honoré E,Lesage F,Fink M,Romey G,Lazdunski M Nature neuroscience Volatile anesthetics produce safe, reversible unconsciousness, amnesia and analgesia via hyperpolarization of mammalian neurons. In molluscan pacemaker neurons, they activate an inhibitory synaptic K+ current (IKAn), proposed to be important in general anesthesia. Here we show that TASK and TREK-1, two recently cloned mammalian two-P-domain K+ channels similar to IKAn in biophysical properties, are activated by volatile general anesthetics. Chloroform, diethyl ether, halothane and isoflurane activated TREK-1, whereas only halothane and isoflurane activated TASK. Carboxy (C)-terminal regions were critical for anesthetic activation in both channels. Thus both TREK-1 and TASK are possibly important target sites for these agents. 10.1038/8084
    TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells. Nozawa Katsura,Kawabata-Shoda Eri,Doihara Hitoshi,Kojima Ryosuke,Okada Hidetsugu,Mochizuki Shinobu,Sano Yorikata,Inamura Kohei,Matsushime Hitoshi,Koizumi Tomonobu,Yokoyama Toshihide,Ito Hiroyuki Proceedings of the National Academy of Sciences of the United States of America Serotonin (5-hydroxytryptamine; 5-HT) is abundantly present throughout the gastrointestinal tract and stored mostly in enterochromaffin (EC) cells, which are located on the mucosal surface. 5-HT released from EC cells stimulate both intrinsic and extrinsic nerves, which results in various physiological and pathophysiological responses, such as gastrointestinal contractions. EC cells are believed to have the ability to respond to the chemical composition of the luminal contents of the gut; however, the underlying molecular and cellular mechanisms have not been identified. Here, we demonstrate that the transient receptor potential (TRP) cation channel TRPA1, which is activated by pungent compounds or cold temperature, is highly expressed in EC cells. We also found that TRPA1 agonists, including allyl isothiocyanate and cinnamaldehyde, stimulate EC cell functions, such as increasing intracellular Ca(2+) levels and 5-HT release, by using highly concentrated EC cell fractions and a model of EC cell function, the RIN14B cell line. Furthermore, we showed that allyl isothiocyanate promotes the contraction of isolated guinea pig ileum via the 5-HT(3) receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function. 10.1073/pnas.0805323106
    Pharmacokinetics of daikenchuto, a traditional Japanese medicine (kampo) after single oral administration to healthy Japanese volunteers. Munekage Masaya,Kitagawa Hiroyuki,Ichikawa Kengo,Watanabe Junko,Aoki Katsuyuki,Kono Toru,Hanazaki Kazuhiro Drug metabolism and disposition: the biological fate of chemicals The pharmacokinetics of daikenchuto (TJ-100), a pharmaceutical-grade traditional Japanese medicine, were investigated in healthy Japanese volunteers after a single oral administration of 2.5-, 5-, and 10-g doses. Six ingredients [hydroxy-α-sanshool (HAS), hydroxy-β-sanshool (HBS), [6]-shogaol (6S), [10]-shogaol (10S), ginsenoside Rb₁(GRB1), and ginsenoside Rg₁(GRG1)] of TJ-100 were determined by using liquid chromatography-tandem mass spectrometry. The results indicated that HAS, an ingredient derived from Zanthoxylum piperitum fruit, exhibited the highest plasma concentration among the six ingredients investigated. The plasma concentrations of HAS, HBS, 6S, and 10S reached the maximum concentration (approximately 400, 80, 0.14, and 0.6 ng/ml, respectively, after a 5-g administration of TJ-100) within 30 min after administration, and the mean half-life was approximately 2 h. Thus, these compounds were rapidly absorbed and eliminated. The plasma concentration of GRB1 reached the maximum concentration (2 ng/ml after a 5-g administration of TJ-100) at approximately 4 h after administration and the half-life of GRB1 was approximately 40 h. The plasma concentration of GRG1 was extremely low (<0.023 ng/ml). The pharmacokinetics of HAS, HBS, 6S, and 10S, were linear within the range of 2.5 to 10 g/day of TJ-100. On the other hand, the kinetics of GRB1 and GRG1 were not proportional to dosage, and plateauing was observed. 10.1124/dmd.111.040097
    A functional role for the two-pore domain potassium channel TASK-1 in cerebellar granule neurons. Millar J A,Barratt L,Southan A P,Page K M,Fyffe R E,Robertson B,Mathie A Proceedings of the National Academy of Sciences of the United States of America Cerebellar granule neurons (CGNs) are one of the most populous cells in the mammalian brain. They express an outwardly rectifying potassium current, termed a "standing-outward" K(+) current, or IK(SO), which does not inactivate. It is active at the resting potential of CGNs, and blocking IK(SO) leads to cell depolarization. IK(SO) is blocked by Ba(2+) ions and is regulated by activation of muscarinic M(3) receptors, but it is insensitive to the classical broad-spectrum potassium channel blocking drugs 4-aminopyridine and tetraethylammonium ions. The molecular nature of this important current has yet to be established, but in this study, we provide strong evidence to suggest that IK(SO) is the functional correlate of the recently identified two-pore domain potassium channel TASK-1. We show that IK(SO) has no threshold for activation by voltage and that it is blocked by small extracellular acidifications. Both of these are properties that are diagnostic of TASK-1 channels. In addition, we show that TASK-1 currents expressed in Xenopus oocytes are inhibited after activation of endogenous M(3) muscarinic receptors. Finally, we demonstrate that mRNA for TASK-1 is found in CGNs and that TASK-1 protein is expressed in CGN membranes. This description of a functional two-pore domain potassium channel in the mammalian central nervous system indicates its physiological importance in controlling cell excitability and how agents that modify its activity, such as agonists at G protein-coupled receptors and hydrogen ions, can profoundly alter both the neuron's resting potential and its excitability. 10.1073/pnas.97.7.3614
    TASK-3 immunoreactivity shows differential distribution in the human gastrointestinal tract. Kovács Ilona,Pocsai Krisztina,Czifra Gabriella,Sarkadi László,Szucs Géza,Nemes Zoltán,Rusznák Zoltán Virchows Archiv : an international journal of pathology The presence and distribution of TASK-3 immunopositivity (a channel with potential oncogenic significance) was investigated in the human gastrointestinal system. The immunohistochemical reactions were performed with two commercially available polyclonal antibodies, targeting different epitopes of the channel protein. Experiments conducted on frozen and formalin-fixed samples indicated that the application of a suitable antigen retrieval (AR) technique was essential to produce consistent, strong and reproducible TASK-3-specific immunolabelling of the formalin-fixed tissue. The lack of or inappropriate selection of the AR resulted in false-negative reactions. As for the distribution of the TASK-3 channels, strong immunolabelling was observed in the gastric and large intestinal mucosa, with particularly prominent immunoreactivity of the epithelial cells. In contrast, the smooth-muscle layers demonstrated weak TASK-3 positivity. Intense TASK-3 expression was noted in both the exocrine and endocrine pancreas, but the islets of Langerhans exhibited more powerful reactions. The ductal apparatus of the submandibular gland and lymphocytes situated in pericolonic lymph nodes were also TASK-3 positive. Strong TASK-3 positivity could also be observed in malignant gastrointestinal tumours, with intense nuclear-perinuclear labelling of some of the tumour cells. The present findings suggest that TASK-3 channels may have roles in the gastrointestinal functions, including insular hormone secretion. 10.1007/s00428-005-1205-7
    Hydroxy-alpha-sanshool activates TRPV1 and TRPA1 in sensory neurons. Koo Jae Yeon,Jang Yongwoo,Cho Hawon,Lee Chang-Hun,Jang Kyoung Hwa,Chang Yong Ha,Shin Jongheon,Oh Uhtaek The European journal of neuroscience Sanshools are major active ingredients of Zanthoxylum piperitum and are used as food additives in East Asia. Sanshools cause irritant, tingling and sometimes paresthetic sensations on the tongue. However, the molecular mechanism underlying the pungent or tingling sensation induced by sanshools is not known. Because many transient receptor potential (TRP) channels are responsible for the sensations induced by various spices and food additives, we expressed 17 TRP channels in human embryonic kidney (HEK) cells and investigated their activation by hydroxy-alpha-sanshool (HalphaSS) or hydroxy-beta-sanshool (HbetaSS) isolated from Zanthoxylum piperitum. It was found that HalphaSS, but not HbetaSS, depolarized sensory neurons with concomitant firing of action potentials and evoked inward currents. Among 17 TRP channels expressed in HEK cells, HalphaSS caused Ca(2+) influx in cells transfected with TRPV1 or TRPA1, and evoked robust inward currents in cells transfected with TRPV1 or TRPA1. In primary cultured sensory neurons, HalphaSS induced inward currents and Ca(2+) influx in a capsazepine-dependent manner. Moreover, HalphaSS-induced currents and Ca(2+) influx were greatly diminished in TRPV1(-/-) mice. HalphaSS evoked licking behavior when injected into a single hind paw of wild-type mice, but this was much reduced in TRPV1-deficient mice. These results indicate that TRPV1 and TRPA1 are molecular targets of HalphaSS in sensory neurons. We conclude that the activations of TRPV1 and TRPA1 by HalphaSS explain its unique pungent, tingling sensation. 10.1111/j.1460-9568.2007.05743.x
    Localized release of serotonin (5-hydroxytryptamine) by a fecal pellet regulates migrating motor complexes in murine colon. Heredia Dante J,Dickson Eamonn J,Bayguinov Peter O,Hennig Grant W,Smith Terence K Gastroenterology BACKGROUND & AIMS:The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity. METHODS:Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa. RESULTS:Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation. CONCLUSIONS:The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion. 10.1053/j.gastro.2008.12.010
    Potassium leak channels and the KCNK family of two-P-domain subunits. Goldstein S A,Bockenhauer D,O'Kelly I,Zilberberg N Nature reviews. Neuroscience With a bang, a new family of potassium channels has exploded into view. Although KCNK channels were discovered only five years ago, they already outnumber other channel types. KCNK channels are easy to identify because of their unique structure--they possess two preforming domains in each subunit. The new channels function in a most remarkable fashion: they are highly regulated, potassium-selective leak channels. Although leak currents are fundamental to the function of nerves and muscles, the molecular basis for this type of conductance had been a mystery. Here we review the discovery of KCNK channels, what has been learned about them and what lies ahead. Even though two-P-domain channels are widespread and essential, they were hidden from sight in plain view--our most basic questions remain to be answered. 10.1038/35058574
    TASK Channel Deletion Reduces Sensitivity to Local Anesthetic-induced Seizures. Du Guizhi,Chen Xiangdong,Todorovic Marko S,Shu Shaofang,Kapur Jaideep,Bayliss Douglas A Anesthesiology BACKGROUND:Local anesthetics (LAs) are typically used for regional anesthesia but can be given systemically to mitigate postoperative pain, supplement general anesthesia, or prevent cardiac arrhythmias. However, systemic application or inadvertent intravenous injection can be associated with substantial toxicity, including seizure induction. The molecular basis for this toxic action remains unclear. METHODS:We characterized inhibition by different LAs of homomeric and heteromeric K channels containing TASK-1 (K2P3.1, KCNK3) and TASK-3 (K2P9.1, KCNK9) subunits in a mammalian expression system. In addition, we used TASK-1/TASK-3 knockout mice to test the possibility that TASK channels contribute to LA-evoked seizures. RESULTS:LAs inhibited homomeric and heteromeric TASK channels in a range relevant for seizure induction; channels containing TASK-1 subunits were most sensitive and IC₅₀ values indicated a rank order potency of bupivacaine > ropivacaine >> lidocaine. LAs induced tonic-clonic seizures in mice with the same rank order potency, but higher LA doses were required to evoke seizures in TASK knockout mice. For bupivacaine, which produced the longest seizure times, seizure duration was significantly shorter in TASK knockout mice; bupivacaine-induced seizures were associated with an increase in electroencephalogram power at frequencies less than 5 Hz in both wild-type and TASK knockout mice. CONCLUSIONS:These data suggest that increased neuronal excitability associated with TASK channel inhibition by LAs contributes to seizure induction. Because all LAs were capable of evoking seizures in TASK channel deleted mice, albeit at higher doses, the results imply that other molecular targets must also be involved in this toxic action. 10.1097/ALN.0b013e3182343660
    Neurogenic and myogenic properties of pan-colonic motor patterns and their spatiotemporal organization in rats. Chen Ji-Hong,Zhang Qian,Yu Yuanjie,Li Kongling,Liao Hong,Jiang Longying,Hong Lu,Du Xiaohui,Hu Xinghai,Chen Sifeng,Yin Sheng,Gao Qingmin,Yin Xiangdong,Luo Hesheng,Huizinga Jan D PloS one BACKGROUND AND AIMS:Better understanding of intrinsic control mechanisms of colonic motility will lead to better treatment options for colonic dysmotility. The aim was to investigate neurogenic and myogenic control mechanisms underlying pan-colonic motor patterns. METHODS:Analysis of in vitro video recordings of whole rat colon motility was used to explore motor patterns and their spatiotemporal organizations and to identify mechanisms of neurogenic and myogenic control using pharmacological tools. RESULTS:Study of the pan-colonic spatiotemporal organization of motor patterns revealed: fluid-induced or spontaneous rhythmic propulsive long distance contractions (LDCs, 0.4-1.5/min, involving the whole colon), rhythmic propulsive motor complexes (RPMCs) (0.8-2.5/min, dominant in distal colon), ripples (10-14/min, dominant in proximal colon), segmentation and retrograde contractions (0.1-0.8/min, prominent in distal and mid colon). Spontaneous rhythmic LDCs were the dominant pattern, blocked by tetrodotoxin, lidocaine or blockers of cholinergic, nitrergic or serotonergic pathways. Change from propulsion to segmentation and distal retrograde contractions was most prominent after blocking 5-HT3 receptors. In the presence of all neural blockers, bethanechol consistently evoked rhythmic LDC-like propulsive contractions in the same frequency range as the LDCs, indicating the existence of myogenic mechanisms of initiation and propulsion. CONCLUSIONS:Neurogenic and myogenic control systems orchestrate distinct and variable motor patterns at different regions of the pan-colon. Cholinergic, nitrergic and serotonergic pathways are essential for rhythmic LDCs to develop. Rhythmic motor patterns in presence of neural blockade indicate the involvement of myogenic control systems and suggest a role for the networks of interstitial cells of Cajal as pacemakers. 10.1371/journal.pone.0060474
    Emerging roles for two-pore-domain potassium channels and their potential therapeutic impact. Bayliss Douglas A,Barrett Paula Q Trends in pharmacological sciences A distinct gene family of widely distributed and well-modulated two-pore-domain background potassium (K(2P)) channels establish resting membrane potential and cell excitability. By using new mouse models in which K(2P)-channel genes are deleted, the contributions of these channels to important physiological functions are now being revealed. Here, we highlight results of recent studies using mice deleted for K(2P)-channel subunits that uncover physiological functions of these channels, mostly those of the TASK and TREK subgroup. Consistent with activation of these K(2P) channels by volatile anesthetics, TASK-1, TASK-3 and TREK-1 contribute to anesthetic-induced hypnosis and immobilization. The acid-sensitive TASK channels are not required for brainstem control of breathing by CO(2) or pH, despite widespread expression in respiratory-related neurons. TASK channels are necessary, however, for homeostatic regulation of adrenal aldosterone secretion. The heat-, stretch- and lipid-activated TREK-1 channels contribute to temperature and mechanical pain sensation, neuroprotection by polyunsaturated fatty acids and, unexpectedly, mood regulation. The alkaline-activated TASK-2 channel is necessary for HCO(3)(-) reabsorption and osmotic volume regulation in kidney proximal tubule cells. Development of compounds that selectively modulate K(2P) channels is crucial for verifying these results and assessing the efficacy of therapies targeting these interesting channels. 10.1016/j.tips.2008.07.013
    Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels. Bautista Diana M,Sigal Yaron M,Milstein Aaron D,Garrison Jennifer L,Zorn Julie A,Tsuruda Pamela R,Nicoll Roger A,Julius David Nature neuroscience In traditional folk medicine, Xanthoxylum plants are referred to as 'toothache trees' because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-alpha-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-alpha-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-alpha-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience. 10.1038/nn.2143
    Hydroxy-α sanshool induces colonic motor activity in rat proximal colon: a possible involvement of KCNK9. Kubota Kunitsugu,Ohtake Nobuhiro,Ohbuchi Katsuya,Mase Akihito,Imamura Sachiko,Sudo Yuka,Miyano Kanako,Yamamoto Masahiro,Kono Toru,Uezono Yasuhito American journal of physiology. Gastrointestinal and liver physiology Various colonic motor activities are thought to mediate propulsion and mixing/absorption of colonic content. The Japanese traditional medicine daikenchuto (TU-100), which is widely used for postoperative ileus in Japan, accelerates colonic emptying in healthy humans. Hydroxy-α sanshool (HAS), a readily absorbable active ingredient of TU-100 and a KCNK3/KCNK9/KCNK18 blocker as well as TRPV1/TRPA1 agonist, has been investigated for its effects on colonic motility. Motility was evaluated by intraluminal pressure and video imaging of rat proximal colons in an organ bath. Distribution of KCNKs was investigated by RT-PCR, in situ hybridization, and immunohistochemistry. Current and membrane potential were evaluated with use of recombinant KCNK3- or KCNK9-expressing Xenopus oocytes and Chinese hamster ovary cells. Defecation frequency in rats was measured. HAS dose dependently induced strong propulsive "squeezing" motility, presumably as long-distance contraction (LDC). TRPV1/TRPA1 agonists induced different motility patterns. The effect of HAS was unaltered by TRPV1/TRPA1 antagonists and desensitization. Lidocaine (a nonselective KCNK blocker) and hydroxy-β sanshool (a geometrical isomer of HAS and KCNK3 blocker) also induced colonic motility as a rhythmic propagating ripple (RPR) and a LDC-like motion, respectively. HAS-induced "LDC," but not lidocaine-induced "RPR," was abrogated by a neuroleptic agent tetrodotoxin. KCNK3 and KCNK9 were located mainly in longitudinal smooth muscle cells and in neural cells in the myenteric plexus, respectively. Administration of HAS or TU-100 increased defecation frequency in normal and laparotomy rats. HAS may evoke strong LDC possibly via blockage of the neural KCNK9 channel in the colonic myenteric plexus. 10.1152/ajpgi.00114.2014
    Effect of dietary resistant starch and protein on colonic fermentation and intestinal tumourigenesis in rats. Le Leu Richard K,Brown Ian L,Hu Ying,Morita Tatsuya,Esterman Adrian,Young Graeme P Carcinogenesis Protein as well as starch is fermented in the colon, but the interaction between protein and starch fermentation and the impact on colonic oncogenesis is unknown. High-protein diets increase delivery of protein to the colon and might promote oncogenesis through generation of toxic products. We investigated the interaction of resistant starch (RS) with digestion-resistant potato protein (PP) on colonic fermentation events and their relationship to intestinal tumourigenesis. Male Sprague-Dawley rats were fed an AIN-76A-based diet for 4 weeks and intestinal neoplasms were induced by azoxymethane. Experimental diets included the following: no added RS or PP, 10% high amylose maize starch (source of RS) replacing digestible starch, 15% PP replacing casein and 10% high amylose maize starch+15% PP. Rats were maintained on diets until killed at 30 weeks. Feeding RS significantly increased short-chain fatty acid (SCFA) levels (P<0.001) in the caecum and colon. Importantly, butyrate concentration was significantly increased in the distal colon with RS (P<0.001). Feeding PP increased protein fermentation products, but this effect was reduced by adding RS to the diet. Intestinal neoplasms and colorectal adenocarcinomas were reduced by feeding RS (P<0.01) regardless of whether PP was fed, whereas PP alone increased the incidence and number of small intestinal neoplasms including the adenocarcinomas (P<0.01). In conclusion, RS altered the colonic luminal environment by increasing the concentration of SCFAs including butyrate and lowering production of potentially toxic protein fermentation products. These effects of RS not only protected against intestinal tumourigenesis but also ameliorated the tumour-enhancing effects of feeding indigestible protein. 10.1093/carcin/bgl245
    Inhibition of liver microsomal cytochrome P450 activity and metabolism of the tobacco-specific nitrosamine NNK by capsaicin and ellagic acid. Zhang Z,Hamilton S M,Stewart C,Strother A,Teel R W Anticancer research The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK), present in tobacco and tobacco smoke, is metabolically activated by microsomal enzymes. In this study, we examined the effect of capsaicin and ellagic acid on the in vitro metabolism of NNK by hamster and rat liver microsomes. Capsaicin is the principal component of Capsicum fruits used widely throughout the world as a food additive. Ellagic acid, with reported anticarcinogenic properties, is found in various soft fruits and nuts. Both capsaicin and ellagic acid inhibited the major pathways of NNK-reduction, N-pyridine oxidation and a-hydroxylation by hamster liver microsomes. Capsaicin inhibited NNK-reduction and a-hydroxylation and ellagic acid inhibited N-oxidation and a-hydroxylation by rat liver microsomes. The effects of capsaicin and ellagic acid on isozymes of cytochrome P450 were observed in the hydroxylation reactions of the metabolism of the steroid hormone testosterone. Results of these experiments indicated that both capsaicin and ellagic acid strongly inhibited the constitutive enzymes CYP 2A2, 3A1, 2C11, 2B1, 2B2 and 2C6. This study suggests that capsaicin and ellagic acid, as naturally occurring dietary constituents, possess antimutagenic and anticarcinogenic properties through the inhibition of xenobiotic metabolizing enzymes.
    Beneficial influence of dietary curcumin, capsaicin and garlic on erythrocyte integrity in high-fat fed rats. Kempaiah Rayavara K,Srinivasan Krishnapura The Journal of nutritional biochemistry In rats rendered hyperlipidemic by maintaining them on a high-fat diet (30%) for 8 weeks, inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet produced significant hypotriglyceridemic effect. Plasma cholesterol remained unaffected in high-fat treatment. Hepatic triglyceride content was significantly higher in high-fat fed rats, and this increase was effectively countered by inclusion of the hypolipidemic spice agents -- curcumin, capsaicin or garlic in the diet. The lipid profile of erythrocyte membranes of hyperlipidemic rats was similar to basal controls. An examination of the osmotic fragility of erythrocytes in various groups indicated that the red blood cells of hyperlipidemic rats display a slight resistance to osmotic lysis. Inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet, which produced the hypotriglyceridemic effect, appeared to beneficially correct this altered osmotic fragility of erythrocytes. Activities of ouabain-sensitive Na(+),K(+)-ATPase as well as acetylcholinesterase of erythrocyte membranes in high-fat fed rats remained unaltered. Activity of Ca(2+),Mg(2+)-ATPase in erythrocyte membrane was significantly decreased in high-fat fed animals, whereas dietary spice principles and garlic countered this reduction in enzyme activity. In the absence of any change in the cholesterol/phospholipid molar ratio in the erythrocyte membrane, a decreased activity of membrane-bound Ca(2+),Mg(2+)-ATPase could have probably contributed to the accumulation of intracellular calcium leading to the diminished deformability of the erythrocytes in high-fat fed rats. 10.1016/j.jnutbio.2005.09.005
    Effect of capsaicin on substrate oxidation and weight maintenance after modest body-weight loss in human subjects. Lejeune Manuela P G M,Kovacs Eva M R,Westerterp-Plantenga Margriet S The British journal of nutrition The aim of the present study was to investigate whether capsaicin assists weight maintenance by limiting weight regain after weight loss of 5 to 10 %. In this randomized double-blind placebo-controlled study, ninety-one moderately overweight subjects were randomly assigned to an intensive group that underwent all the measurements, and an extensive group that underwent the same measurements except the metabolism measurements. After a 4-week very-low-energy diet (VLED) intervention, a 3-month weight-maintenance period followed. During weight maintenance, subjects were divided into a capsaicin (135 mg capsaicin/d) and a placebo group. Body mass was measured before and after the VLED and after 1, 2 and 3 months of weight maintenance. The mean body-mass loss during the VLED was 6.6 (SD 2.0) kg (7.8 (SD 1.8) % initial body mass), and was not different between the subsequent treatment and placebo group. During weight maintenance, mean % regain during treatment was not significantly different compared with placebo (33.3 (SD 35.7) v. 19.2 (SD 41.8) %, P=0.09). RQ was significantly less increased during weight maintenance in the treatment group compared with placebo (0.04 (SD 0.06) v. 0.07 (SD 0.05), P<0.05), indicating a relatively more sustained fat oxidation. Fat oxidation (g/h) after weight maintenance was higher in the capsaicin group compared with placebo (4.2 (SD 1.1) v. 3.5 (SD 0.9), P<0.05). These results indicate that capsaicin treatment caused sustained fat oxidation during weight maintenance compared with placebo. However, capsaicin treatment has no limiting effect on 3-month weight regain after modest weight loss. 10.1079/bjn2003938
    Enhanced energy expenditure and fat oxidation in humans with high BMI scores by the ingestion of novel and non-pungent capsaicin analogues (capsinoids). Inoue Naohiko,Matsunaga Yoshiko,Satoh Hitoshi,Takahashi Michio Bioscience, biotechnology, and biochemistry The biochemical and physiological indices were monitored in 44 subjects after 4-week capsinoids (capsaicin analogues with low pungency) intake. The subjects were randomly assigned to 3 groups: CSNs3 (3 mg/kg of capsinoids), CSNs10 (10 mg/kg of capsinoids) and the control (placebo). Measurements were performed in the morning on overnight-fasted subjects. The oxygen consumption (VO(2)), resting energy expenditure (REE) and fat oxidation increased slightly compared to pre-administration values without any adverse effects, although the increase was not significant. The increase in fat oxidation was positively and significantly correlated with the body mass index (BMI). A meta-analysis was therefore conducted on a subgroup consisting of subjects with BMI >or= 25 (n=28). As a result, not only VO(2) increased significantly (p<0.05) in the CSNs10 group, but also REE in the CSNs10 group and fat oxidation in the CSNs3 and CSNs10 groups tended to increase (p<0.1). Consequently, a capsinoids intake would be able to enhance the energy expenditure and fat burning in humans, particularly those with high BMI. 10.1271/bbb.60341
    Synthesis, cannabinoid receptor affinity, molecular modeling studies and in vivo pharmacological evaluation of new substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides. 2. Effect of the 3-carboxamide substituent on the affinity and selectivity profile. Silvestri Romano,Ligresti Alessia,La Regina Giuseppe,Piscitelli Francesco,Gatti Valerio,Brizzi Antonella,Pasquini Serena,Lavecchia Antonio,Allarà Marco,Fantini Noemi,Carai Mauro Antonio Maria,Novellino Ettore,Colombo Giancarlo,Di Marzo Vincenzo,Corelli Federico Bioorganic & medicinal chemistry New substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized by replacing the 2,4-dichlorobenzyl and cyclohexyl moieties at the 3-carboxamide nitrogen of the previously reported CB(1) receptor antagonists/inverse agonists 4 and 5. Several ligands showed potent affinity for the hCB(1) receptor, with K(i) concentrations comparable to the reference compounds 1, 4 and 5, and exhibited CB(1) selectivity comparable to 1 and 2. Docking experiments and molecular dynamics (MD) simulations explained the potent hCB(1) binding affinity of compounds 31 and 37. According to our previous studies, 31 and 37 formed a H-bond with K3.28(192), which accounted for the high affinity for the receptor inactive state and the inverse agonist activity. The finding of inhibition of food intake following their acute administration to rats, supported the concept that the CB(1) selective compounds 4 and 52 act as antagonists/inverse agonists. 10.1016/j.bmc.2009.06.027
    Synthesis and alpha-D-glucosidase inhibitory activity of N-substituted valiolamine derivatives as potential oral antidiabetic agents. Horii S,Fukase H,Matsuo T,Kameda Y,Asano N,Matsui K Journal of medicinal chemistry Various kinds of N-substituted valiolamine derivatives, including compounds 23a, 24a, and 34a, which are structurally analogous to the key pseudodisaccharides (25a and 26a) of naturally occurring oligosaccharide alpha-D-glucosidase inhibitors, have been synthesized and estimated by the measure of inhibitory activity against porcine sucrase and maltase. The N-substituted valiolamine derivatives evaluated in this study have been found to be more potent than the corresponding N-substituted valienamine derivatives as well as the parent valiolamine. It is noteworthy that even simple N-substituted valiolamine derivatives such as N-[2-hydroxy-1-(hydroxymethyl)ethyl]-, N-[(1R,2R)-2-hydroxycyclohexyl]-, and N-[(R)-(-)-beta-hydroxyphenethyl]valiolamine (6, 8a, and 9a) have the stronger alpha-D-glucosidase inhibitory activity against porcine intestinal maltase and sucrase than naturally occurring oligosaccharide alpha-D-glucosidase inhibitors. 10.1021/jm00156a023
    Intrinsic primary afferent neurons of the intestine. Furness J B,Kunze W A,Bertrand P P,Clerc N,Bornstein J C Progress in neurobiology After a long period of inconclusive observations, the intrinsic primary afferent neurons of the intestine have been identified. The intestine is thus equipped with two groups of afferent neurons, those with cell bodies in cranial and dorsal root ganglia, and these recently identified afferent neurons with cell bodies in the wall of the intestine. The first, tentative, identification of intrinsic primary afferent neurons was by their morphology, which is type II in the terminology of Dogiel. These are multipolar neurons, with some axons that project to other nerve cells in the intestine and other axons that project to the mucosa. Definitive identification came only recently when action potentials were recorded intracellularly from Dogiel type II neurons in response to chemicals applied to the lumenal surface of the intestine and in response to tension in the muscle. These action potentials persisted after all synaptic transmission was blocked, proving the Dogiel type II neurons to be primary afferent neurons. Less direct evidence indicates that intrinsic primary afferent neurons that respond to mechanical stimulation of the mucosal lining are also Dogiel type II neurons. Electrophysiologically, the Dogiel type II neurons are referred to as AH neurons. They exhibit broad action potentials that are followed by early and late afterhyperpolarizing potentials. The intrinsic primary afferent neurons connect with each other at synapses where they transmit via slow excitatory postsynaptic potentials, that last for tens of seconds. Thus the intrinsic primary afferent neurons form self-reinforcing networks. The slow excitatory postsynaptic potentials counteract the late afterhyperpolarizing potentials, thereby increasing the period during which the cells can fire action potentials at high rates. Intrinsic primary afferent neurons transmit to second order neurons (interneurons and motor neurons) via both slow and fast excitatory postsynaptic potentials. Excitation of the intrinsic primary afferent neurons by lumenal chemicals or mechanical stimulation of the mucosa appears to be indirect, via the release of active compounds from endocrine cells in the epithelium. Stretch-induced activation of the intrinsic primary afferent neurons is at least partly dependent on tension generation in smooth muscle, that is itself sensitive to stretch. The intrinsic primary afferent neurons of the intestine are the only vertebrate primary afferent neurons so far identified with cell bodies in a peripheral organ. They are multipolar and receive synapses on their cell bodies, unlike cranial and spinal primary afferent neurons. They communicate with each other via slow excitatory synaptic potentials in self reinforcing networks and with interneurons and motor neurons via both fast and slow EPSPs. 10.1016/s0301-0082(97)00051-8
    TLC and HPLC Analysis of Alkamides in Echinacea Drugs1,2. Bauer R,Remiger P Planta medica HPLC and TLC methods are presented for the analysis of the alkamides in ECHINACEA PURPUREA, E. ANGUSTIFOLIA, and E. PALLIDA. The roots of E. PURPUREA and E. ANGUSTIFOLIA contain different structural types of alkamides, while the roots of E. PALLIDA were almost void of amides. In contrast, the aerial parts of the three ECHINACEA species yielded very similar alkamide patterns. The methods are suitable for the analytical characterization and standardization of the three species. 10.1055/s-2006-962030
    Medicinal properties of Echinacea: a critical review. Barrett B Phytomedicine : international journal of phytotherapy and phytopharmacology Preparations from Echinacea purpurea are among the most widely used herbal medicines. Most uses of E. purpurea are based on the reported immunological properties. A series of experiments have demonstrated that E. purpurea extracts do indeed demonstrate significant immunomodulatory activities. Among the many pharmacological properties reported, macrophage activation has been demonstrated most convincingly. Phagocytotic indices and macrophage-derived cytokine concentrations have been shown to be Echinacea-responsive in a variety of assays. Activation of polymorphonuclear leukocytes and natural killer cells has also been reasonably demonstrated. Changes in the numbers and activities of T- and B-cell leukocytes have been reported, but are less certain. Despite this cellular evidence of immunostimulation, pathways leading to enhanced resistance to infectious disease have not been described adequately. Several dozen human experiments--including a number of blind randomized trials--have reported health benefits. The most robust data come from trials testing E. purpurea extracts in the treatment for acute upper respiratory infection. Although suggestive of modest benefit, these trials are limited both in size and in methodological quality. Hence, while there is a great deal of moderately good-quality scientific data regarding E. purpurea, effectiveness in treating illness or in enhancing human health has not yet been proven beyond a reasonable doubt. 10.1078/094471103321648692