Katsuya Ohbuchi

Epithelial transient receptor potential ankyrin 1 (TRPA1)-dependent adrenomedullin upregulates blood flow in rat small intestine

The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.

Multitargeted effects of hangeshashinto for treatment of chemotherapy-induced oral mucositis on inducible prostaglandin E2 production in human oral keratinocytes.

Objective. Chemotherapy-induced oral mucositis (COM) is characterized by painful inflammation with prolonged damage that involves the pathological pain-evoking prostaglandin E2 (PGE2). We previously found that gargling with hangeshashinto (HST), a traditional Japanese medicine, was effective for the treatment of COM. However, little is known regarding the mechanisms. Our aim was to identify the active ingredients and clarify the characteristic effects of HST on the PGE2 system. Methods. Prostanoids produced by human oral keratinocytes (HOK) stimulated with IL-1β were measured by enzyme immunoassay. Active ingredients that regulate PGE2 production were identified and quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and a culture system of HOK cells. Results. Inducible PGE2, PGD2, and PGF2α, metabolites of cyclooxygenase (COX) pathways, were reduced by HST (10-300 µg/mL) without inducing cytotoxicity. The active ingredients of HST were quantified by LC-MS/MS, and [6]-shogaol, [6]-gingerol, wogonin, baicalein, baicalin, and berberine were shown to reduce PGE2 production. A mixture of these 6 ingredients at concentrations equal to 300 µg/mL of HST strongly suppressed PGE2 production to the same level as HST. [6]-Shogaol and [6]-gingerol did not decrease COX-2 mRNA expression and mostly inhibited PGE2 metabolic activity in an assay using intact HOK cells, suggesting that they regulate PGE2 synthesis at the posttranscriptional level. Wogonin, baicalin, and berberine inhibited expression of COX-2 mRNA without affecting PGE2 metabolic activity. Moreover, wogonin, but not [6]-shogaol, suppressed phosphorylation of mitogen-activated protein kinases (p38s and JNKs). Conclusions. These lines show that HST includes several PGE2-regulating ingredients that have different mechanisms and can function as a multicomponent and multitarget agent for treatment of COM, indicating that HST may be beneficial in a new medical strategy for COM treatment.

Hydroxy-α sanshool induces colonic motor activity in rat proximal colon: a possible involvement of KCNK9

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.

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.

Ignavine: a novel allosteric modulator of the μ opioid receptor

Processed Aconiti tuber (PAT) is used to treat pain associated with various disorders. Although it has been demonstrated that the κ opioid receptor (KOR) signaling pathway is a mediator of the analgesic effect of PAT, active components affecting opioid signaling have not yet been identified. In this study, we explored candidate components of PAT by pharmacokinetic analysis and identified ignavine, which is a different structure from aconitine alkaloids. A receptor binding assay of opioid receptors showed that ignavine specifically binds the μ opioid receptor (MOR), not the KOR. Receptor internalization assay in MOR-expressing cell lines revealed that ignavine augmented the responses produced by D-Ala(2)-N-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), a representative MOR agonist, at a low concentration and inhibited it at a higher concentration. Ignavine also exerted positive modulatory activity for DAMGO, endomorphin-1 and morphine in cAMP assay. Additionally, ignavine alone showed an analgesic effect in vivo. In silico simulation analysis suggested that ignavine would induce a unique structural change distinguished from those induced by a representative MOR agonist and antagonist. These data collectively suggest the possibility that ignavine could be a novel allosteric modulator of the MOR. The present results may open the way for the development of a novel pain management strategy.

Transient receptor potential ankyrin 1 agonists improve intestinal transit in a murine model of postoperative ileus

Stimulation of transient receptor potential ankyrin 1 (TRPA1), which abundantly expressed in enterochromaffin cells (ECC), has been reported to exert apparently contradictory results in in vitro contractility and in vivo gastrointestinal (GI) transit evaluations. The pharmaceutical‐grade Japanese traditional medicine daikenchuto (TU‐100) has been reported to be beneficial for postoperative ileus (POI) and accelerate GI transit in animals and humans. TU‐100 was recently shown to increase intestinal blood flow via stimulation of TRPA1 in the epithelial cells of the small intestine (SI).

Tramadol and its metabolite m1 selectively suppress transient receptor potential ankyrin 1 activity, but not transient receptor potential vanilloid 1 activity.

BACKGROUND:The transient receptor potential vanilloid 1 (TRPV1) and the transient receptor potential ankyrin 1 (TRPA1), which are expressed in sensory neurons, are polymodal nonselective cation channels that sense noxious stimuli. Recent reports showed that these channels play important roles in inflammatory, neuropathic, or cancer pain, suggesting that they may serve as attractive analgesic pharmacological targets. Tramadol is an effective analgesic that is widely used in clinical practice. Reportedly, tramadol and its metabolite (M1) bind to &mgr;-opioid receptors and/or inhibit reuptake of monoamines in the central nervous system, resulting in the activation of the descending inhibitory system. However, the fundamental mechanisms of tramadol in pain control remain unclear. TRPV1 and TRPA1 may be targets of tramadol; however, they have not been studied extensively. METHODS:We examined whether and how tramadol and M1 act on human embryonic kidney 293 (HEK293) cells expressing human TRPV1 (hTRPV1) or hTRPA1 by using a Ca2+ imaging assay and whole-cell patch-clamp recording. RESULTS:Tramadol and M1 (0.01–10 &mgr;M) alone did not increase in intracellular Ca2+ concentration ([Ca2+]i) in HEK293 cells expressing hTRPV1 or hTRPA1 compared with capsaicin (a TRPV1 agonist) or the allyl isothiocyanate (AITC, a TRPA1 agonist), respectively. Furthermore, in HEK293 cells expressing hTRPV1, pretreatment with tramadol or M1 for 5 minutes did not change the increase in [Ca2+]i induced by capsaicin. Conversely, pretreatment with tramadol (0.1–10 &mgr;M) and M1 (1–10 &mgr;M) significantly suppressed the AITC-induced [Ca2+]i increases in HEK293 cells expressing hTRPA1. In addition, the patch-clamp study showed that pretreatment with tramadol and M1 (10 &mgr;M) decreased the inward currents induced by AITC. CONCLUSIONS:These data indicate that tramadol and M1 selectively inhibit the function of hTRPA1, but not that of hTRPV1, and that hTRPA1 may play a role in the analgesic effects of these compounds.

Involvement of Interleukin-17A-Induced Hypercontractility of Intestinal Smooth Muscle Cells in Persistent Gut Motor Dysfunction

Background and Aim The etiology of post-inflammatory gastrointestinal (GI) motility dysfunction, after resolution of acute symptoms of inflammatory bowel diseases (IBD) and intestinal infection, is largely unknown, however, a possible involvement of T cells is suggested. Methods Using the mouse model of T cell activation-induced enteritis, we investigated whether enhancement of smooth muscle cell (SMC) contraction by interleukin (IL)-17A is involved in postinflammatory GI hypermotility. Results Activation of CD3 induces temporal enteritis with GI hypomotility in the midst of, and hypermotility after resolution of, intestinal inflammation. Prolonged upregulation of IL-17A was prominent and IL-17A injection directly enhanced GI transit and contractility of intestinal strips. Postinflammatory hypermotility was not observed in IL-17A-deficient mice. Incubation of a muscle strip and SMCs with IL-17A in vitro resulted in enhanced contractility with increased phosphorylation of Ser19 in myosin light chain 2 (p-MLC), a surrogate marker as well as a critical mechanistic factor of SMC contractility. Using primary cultured murine and human intestinal SMCs, IκBζ- and p38 mitogen-activated protein kinase (p38MAPK)-mediated downregulation of the regulator of G protein signaling 4 (RGS4), which suppresses muscarinic signaling of contraction by promoting inactivation/desensitization of Gαq/11 protein, has been suggested to be involved in IL-17A-induced hypercontractility. The opposite effect of L-1β was mediated by IκBζ and c-jun N-terminal kinase (JNK) activation. Conclusions We propose and discuss the possible involvement of IL-17A and its downstream signaling cascade in SMCs in diarrheal hypermotility in various GI disorders.

Wogonin Attenuates Ovalbumin Antigen-Induced Neutrophilic Airway Inflammation by Inhibiting Th17 Differentiation

Allergic airway inflammation is generally considered to be a Th2-type immune response. Recent studies, however, have demonstrated that Th17-type immune responses also play important roles in this process, particularly in the pathogenesis of neutrophilic airway inflammation, a hallmark of severe asthma. We scrutinized several Kampo extracts that reportedly exhibit anti-inflammatory activity by using in vitro differentiation system of human and mouse naïve T cells. We found that hange-shashin-to (HST) and oren-gedoku-to (OGT) possess inhibitory activity for Th17 responses in vitro. Indeed, wogonin and berberine, major components common to HST and OGT, exhibit Th17-inhibitory activities in both murine and human systems in vitro. We therefore evaluated whether wogonin suppresses OVA-induced neutrophilic airway inflammation in OVA TCR-transgenic DO11.10 mice. Consequently, oral administration of wogonin significantly improved OVA-induced neutrophilic airway inflammation. Wogonin suppressed the differentiation of naïve T cells to Th17 cells, while showing no effects on activated Th17 cells.

Arf6 mediates Schwann cell differentiation and myelination.

During development of the peripheral nervous system (PNS), Schwann cells wrap neuronal axons, becoming the myelin sheaths that help axonal functions. While the intercellular signals controlling the myelination process between Schwann cells and peripheral neurons are well studied, the transduction of these signals in Schwann cells still remains elusive. Here, we show that Arf6, an Arf protein of the small GTPase family, is involved in promoting the myelination process. Knockdown of Arf6 with the small-interfering (si)RNA in primary Schwann cells markedly decreases dibutyl-cyclic AMP-induced myelin marker protein expression, indicating that Arf6 plays a role in differentiation-like phenotypic changes. To obtain in vivo evidence, we generated small-hairpin (sh)RNA transgenic mice targeting Arf6 for Schwann cells. Transgenic mice exhibited reduced myelin thickness compared to littermate controls, consistent with the defective myelin formation observed in the transgenic mouse-derived Schwann cell and neuronal culture system. Transgenic mice also exhibited decreased phosphorylation of myelination-related signaling molecules such as Akt kinase cascade proteins as well as downregulation of myelin marker proteins. These results suggest that signaling through Arf6 is required for Schwann cell myelination, adding Arf6 to the list of intracellular signaling molecules involved in the myelination process.