Resveratrol enhances 5-hydroxytryptamine type 3A receptor-mediated ion currents: the role of arginine 222 residue in pre-transmembrane domain I.

Resveratrol, which is found in grapes, red wine, and berries, has many beneficial health effects, such as anti-cancer, neuro-protective, anti-inflammatory, and life-prolonging effects. However, the cellular mechanisms by which resveratrol acts are relatively unknown, especially in terms of possible regulation of receptors involved in synaptic transmission. 5-Hydroxytryptamine type 3A (5-HT(3A)) receptor is one of several ligand-gated ion channels involved in fast synaptic transmission. In the present study, we investigated the effect of resveratrol on mouse 5-HT(3A) receptor channel activity. 5-HT(3A) receptor was expressed in Xenopus oocytes, and the current was measured using a two-electrode voltage clamp technique. Treatment of resveratrol itself had no effect on the oocytes injected with H(2)O as well as on the oocytes injected with 5-HT(3A) receptor cRNA. In the oocytes injected with 5-HT(3A) receptor cRNA, co- or pre-treatment of resveratrol with 5-HT potentiated 5-HT-induced inward peak current (I(5-HT)) with concentration-, reversible, and voltage-independent manners. The EC(50) of resveratrol was 28.0±2.4 µM. The presence of resveratrol caused a leftward shift of 5-HT concentration-response curve. Protein kinase C (PKC) activator or inhibitor had no effect on resveratrol action on I(5-HT). Site-directed mutations of pre-transmembrane domain 1 (pre-TM1) such as R222A, R222D, R222E, R222K, and R222T abolished or attenuated resveratrol-induced enhancement of I(5-HT), indicating that resveratrol might interact with pre-TM1 of 5-HT(3A) receptor. These results indicate that resveratrol might regulate 5-HT(3A) receptor channel activity via interaction with the N-terminal domain and these results further show that resveratrol-mediated regulation of 5-HT(3A) receptor channel activity might be one of cellular mechanisms of resveratrol action.

Effects of quercetin on human α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated ion currents.

Quercetin is a low molecular weight flavonoid found in dietary fruits and vegetables. Quercetin, like other flavonoids, has demonstrated neuroprotective effects in vitro and in vivo. However, relatively little is known about how quercetin achieves its neuroprotective abilities. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is one of several excitatory receptors, which play an important role in postsynaptic neurotransmission. Over-stimulation of ionotropic glutamate receptor including AMPA receptors is closely associated with excitatory neurotoxicities. In the present study, we investigated the effects of quercetin on the glutamate-induced inward current (IGlu) in Xenopus oocytes that heterologously express human AMPA receptor and stargazin, an auxiliary subunit of AMPA receptor. IGlu was measured using the two-electrode voltage clamp technique. In oocytes injected with cRNAs coding AMPA receptor (GluR1) and stargazin, quercetin inhibited IGlu in a reversible and concentration-dependent manner. The IC50 was 84.9+/-15.0 microM. Quercetin action on IGlu was attenuated by increasing glutamate concentration, and was membrane holding potential-dependent. These results show a possibility that quercetin interacts with AMPA receptor, which was heterologously expressed in Xenopus oocytes and that quercetin action on IGlu of AMPA receptor could be one of contributions of quercetin-mediated neuroprotections.

Ginsenoside Rg3 inhibits human Kv1.4 channel currents by interacting with the Lys531 residue.

We have demonstrated previously that the 20(S) but not the 20(R) form of ginsenoside Rg(3) inhibited K(+) currents flowing through Kv1.4 (hKv1.4) channels expressed in Xenopus laevis oocytes, pointing to the presence of specific interaction site(s) for Rg(3) in the hKv1.4 channel. In the current study, we sought to identify this site(s). To this end, we first assessed how point mutations of various amino acid residues of the hKv1.4 channel affected inhibition by 20(S)-ginsenoside Rg(3) (Rg(3)). Lys531 residue is known to be a key site for K(+) activation and to be part of the extracellular tetraethylammonium (TEA) binding site; the mutation K531Y abolished the Rg(3) effect and made the Kv1.4 channel sensitive to TEA applied to the extracellular side of the membrane. Mutations of many other residues, including the pH sensitive-site (H507Q), were without any significant effect. We next examined whether K(+) and TEA could alter the effect of Rg(3) and vice versa. We found that 1) raising [K(+)](o) reduced the inhibitory effect of Rg(3) on hKv1.4 channel currents, whereas Rg(3) shifted the K(+) activation curve to the right, and 2) TEA caused a rightward shift of the Rg(3) concentration-response curve of wild-type hKv1.4 channel currents, whereas Rg(3) caused a rightward shift of the TEA concentration-response curve of K531Y mutant channel currents. The docked modeling revealed that Lys531 plays a key role in forming hydrogen bonds between Rg(3) and hKv1.4 channels. These results indicate that Rg(3) inhibits the hKv1.4 channel current by interacting with residue Lys531.

Involvement of batrachotoxin binding sites in ginsenoside-mediated voltage-gated Na+ channel regulation.

Recently, we showed that the 20(S)-ginsenoside Rg3 (Rg3), an active ingredient of Panax ginseng, inhibits rat brain NaV1.2 channel peak currents (INa). Batrachotoxin (BTX) is a steroidal alkaloid neurotoxin and activates NaV channels through interacting with transmembrane domain-I-segment 6 (IS6) of channels. Recent report shows that ginsenoside inhibits BTX binding in rat brain membrane fractions. However, it needs to be confirmed whether biochemical mechanism is relevant physiologically and which residues of the BTX binding sites are important for ginsenoside regulations. Here, we demonstrate that mutations of BTX binding sites such as N418K and L421K of rat brain NaV1.2 and L437K of mouse skeletal muscle NaV1.4 channel reduce or abolish Rg3 inhibition of I(Na) and attenuate Rg3-mediated depolarizing shift of the activation voltage and use-dependent inhibition. These results indicate that BTX binding sites play an important role in modifying Rg3-mediated Na+ channel properties.

Human glycine alpha1 receptor inhibition by quercetin is abolished or inversed by alpha267 mutations in transmembrane domain 2.

Quercetin, one of the flavonoids, is a compound of low molecular weight found in fruits and vegetables. Besides its antioxidative effect, quercetin also shows a wide range of diverse neuropharmacological actions. However, the cellular mechanisms of quercetin's actions, especially on ligand-gated ion channels and synaptic transmissions, are not well studied. We investigated the effect of quercetin on the human glycine alpha1 receptor channel expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Application of quercetin reversibly inhibited glycine-induced current (I(Gly)). Quercetin's inhibition depends on its dose, with an IC(50) of 21.5+/-.2 microM. The inhibition was sensitive to membrane voltages. Site-directed mutations of S267 to S267Y but not S267A, S267F, S267G, S267K, S267L and S267T at transmembrane domain 2 (TM2) nearly abolished quercetin-induced inhibition of I(Gly). In contrast, in site-directed mutant receptors such as S267 to S267I, S267R and S267V, quercetin enhanced I(Gly) compared to the wild-type receptor. The EC(50) was 22.6+/-1.4, 25.5+/-4.2, and 14.5+/-3.1 microM for S267I, S267R and S267V, respectively. These results indicate that quercetin might regulate the human glycine alpha(1) receptor via interaction with amino acid residue alpha267 and that alpha267 plays a key role in determining the regulatory consequences of the human glycine alpha1 receptor by quercetin.

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents.

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X1-P2X7) have been identified. Most of the P2X1 receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X1 receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X1 receptors. In the present study, we examined the effect of gintonin on P2X1 receptor channel activity. P2X1 receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X1 receptors induced large inward currents (I ATP ), but repetitive ATP treatments induced a rapid desensitization of I ATP . Gintonin treatment after P2X1 receptor desensitization potentiated I ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I ATP . Gintoninmediated I ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X1 receptor greatly attenuated the gintonin-mediated I ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X1 receptor through a phosphoinositide-dependent pathway.

Suppression of metastasis of intravenously-inoculated B16/F10 melanoma cells by the novel ginseng-derived ingredient, gintonin: involvement of autotaxin inhibition.

Ginseng has been used for cancer prevention. However, little is known about its active components and the molecular mechanisms underlying its effects. Recently, we isolated a unique lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin contains approximately 9.5% LPA, mainly LPA C18:2. Autotaxin (ATX) is responsible for metastasis by overproducing LPA in cancers. However, LPA, particularly LPA C18:2, is a strong negative feedback ATX inhibitor. It is unknown whether gintonin inhibits ATX activity and whether gintonin­induced ATX inhibition is coupled with antimetastatic activity. In this study, we examined whether gintonin and LPA C18:2 inhibit ATX activity and metastasis­related cellular activities in melanoma cells. We found that gintonin and LPA C18:2 inhibited the purified and secreted ATX activity from melanoma cells in a concentration­dependent manner. Gintonin also inhibited cell migration with a minimal inhibition of cell growth. The oral administration of gintonin or LPA C18:2 inhibited lung metastasis induced by tail­vein inoculations of melanoma cells. Moreover, the oral administration of gintonin significantly suppressed the tumor growth induced by subcutaneous grafts of melanoma cells. A histological analysis showed that the oral administration of gintonin reduced tumor necrosis, the pleomorphism of tumor cells, tumor cell mitosis and angiogenesis. The present study demonstrates that the gintonin­induced inhibition of ATX activity may be the molecular basis of ginseng­induced antimetastatic and antitumor activities.

Effects of Ginsenoside Metabolites on GABAA Receptor-Mediated Ion Currents.

In a previous report, we demonstrated that ginsenoside Rc, one of major ginsenosides from Panax ginseng, enhances γ-aminobutyric acid (GABA) receptorA (GABAA)-mediated ion channel currents. However, little is known about the effects of ginsenoside metabolites on GABAA receptor channel activity. The present study investigated the effects of ginsenoside metabolites on human recombinant GABAA receptor (α1ß1γ2s) channel activity expressed in Xenopus oocytes using a two-electrode voltage clamp technique. M4, a metabolite of protopanaxatriol ginsenosides, more potently inhibited the GABA-induced inward peak current (IGABA ) than protopanaxadiol (PPD), a metabolite of PPD ginsenosides. The effect of M4 and PPD on IGABA was both concentration-dependent and reversible. The half-inhibitory concentration (IC50) values of M4 and PPD were 17.1±2.2 and 23.1±8.6 µM, respectively. The inhibition of IGABA by M4 and PPD was voltage-independent and non-competitive. This study implies that the regulation of GABAA receptor channel activity by ginsenoside metabolites differs from that of ginsenosides.

Inhibitory effects of dextrorotatory morphinans on the human 5-HT(3A) receptor expressed in Xenopus oocytes: Involvement of the N-terminal domain of the 5-HT(3A) receptor.

We previously developed a series of dextromethorphan (DM, 3-methoxy-17-methylmorphinan) analogs modified at positions 3 and 17 of the morphinan ring system. Recent reports have shown that DM attenuates abdominal pain caused by irritable bowel syndrome, and multidrug regimens that include DM prevent nausea/vomiting following cancer surgery. However, little is known regarding the molecular mechanisms underlying the beneficial effects of DM. Here, we investigated the effects of DM, 3 of its analogs (AM, 3-allyloxy-17-methoxymorphian; CM, 3-cyclopropyl-17-methoxymorphinan; and DF, 3-methyl-17-methylmorphinan), and 1 of its metabolites (HM, 3-methoxymorphinan) on the activity of the human 5-HT(3A) receptor channel expressed in Xenopus laevis oocytes, using the 2-microelectrode voltage clamp technique. We found that intra-oocyte injection of human 5-HT(3A) receptor cRNAs elicited an inward current (I(5-HT)) in the presence of 5-HT. Cotreatment with AM, CM, DF, DM, or HM inhibited I(5-HT) in a dose-dependent, voltage-independent, and reversible manner. The IC(50) values for AM, CM, DF, DM, and HM were 24.5±1.4, 21.5±4.2, 132.6±35.8, 181.3±23.5, and 191.3±31.5µM, respectively. The IC(50) values of AM and CM were 7-fold lower than that of DM, and mechanistic analysis revealed that DM, DF, HM, AM, and CM were competitive inhibitors of I(5-HT). Point mutations of Arg241 in the N-terminal, but not amino acids in the pore region, to other amino acid residues attenuated or abolished DM- and DM-analog-induced inhibition of I(5-HT). Together, these results demonstrated that dextrorotatory morphinans might regulate 5-HT(3A) receptor channel activity via interaction with its N-terminal domain.

Effects of protopanaxatriol-ginsenoside metabolites on rat N-methyl-d-aspartic Acid receptor-mediated ion currents.

Ginsenosides are low molecular weight glycosides found in ginseng that exhibit neuroprotective effects through inhibition of N-methyl-D-aspartic acid (NMDA) receptor channel activity. Ginsenosides, like other natural compounds, are metabolized by gastric juices and intestinal microorganisms to produce ginsenoside metabolites. However, little is known about how ginsenoside metabolites regulate NMDA receptor channel activity. In the present study, we investigated the effects of ginsenoside metabolites, such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT), on oocytes that heterologously express the rat NMDA receptor. NMDA receptor-mediated ion current (I(NMDA)) was measured using the 2-electrode voltage clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, PPT, but not CK or PPD, reversibly inhibited I(NMDA) in a concentration-dependent manner. The IC(50) for PPT on I(NMDA) was 48.1±4.6 µM, was non-competitive with NMDA, and was independent of the membrane holding potential. These results demonstrate the possibility that PPT interacts with the NMDA receptor, although not at the NMDA binding site, and that the inhibitory effects of PPT on I(NMDA) could be related to ginseng-mediated neuroprotection.

Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids-protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity.

Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demonstrated that it induced [Ca2+]i transients in cells via G protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobilization and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s)were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i transients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 >LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells expressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G protein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)-Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morphological changes, which were blocked by Rho kinase inhibitorY-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, attenuates Alzheimer's disease-related neuropathies: involvement of non-amyloidogenic processing.

Ginseng extracts show cognition-enhancing effects in Alzheimer's disease (AD) patients. However, little is known about the active components and molecular mechanisms of how ginseng exerts its effects. Recently, we isolated a novel lysophosphatidic acid (LPA) receptor-activating ligand from ginseng, gintonin. AD is caused by amyloid-ß protein (Aß) accumulation. Aß is derived from amyloid-ß protein precursors (AßPPs) through the amyloidogenic pathway. In contrast, non-amyloidogenic pathways produce beneficial, soluble AßPPα (sAßPPα). Here, we describe our investigations of the effect of gintonin on sAßPPα release, Aß formation, Swedish-AßPP transfection-mediated neurotoxicity in SH-SY5Y neuroblastoma cells, and Aß-induced neuropathy in mice. Gintonin promoted sAßPPα release in a concentration- and time-dependent manner. Gintonin action was also blocked by the Ca2+ chelator BAPTA, α-secretase inhibitor TAPI-2, and protein-trafficking inhibitor brefeldin. Gintonin decreased Aß1-42 release and attenuated Aß1-40-induced cytotoxicity in SH-SY5Y cells. Gintonin also rescued Aß1-40-induced cognitive dysfunction in mice. Moreover, in a transgenic mouse AD model, long-term oral administration of gintonin attenuated amyloid plaque deposition as well as short- and long-term memory impairment. In the present study, we demonstrated that gintonin mediated the promotion of non-amyloidogenic processing to stimulate sAßPPα release to restore brain function in mice with AD. Gintonin could be a useful agent for AD prevention or therapy.

Gintonin, a ginseng-derived novel ingredient, evokes long-term potentiation through N-methyl-D-aspartic acid receptor activation: involvement of LPA receptors.

Ginseng has been shown to have memory-improving effects in human. However, little is known about the active components and the molecular mechanisms underlying its effects. Recently, we isolated novel lysophosphatidic acids (LPAs)-ginseng protein complex derived from ginseng, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity. Gintonin activated Ca²âº-activated Clchannels in Xenopus oocytes through the activation of endogenous LPA receptor. In the present study, we investigated whether the activation of LPA receptor by gintonin is coupled to the regulation of N-methyl-D-aspartic acid (NMDA) receptor channel activity in Xenopus oocytes expressing rat NMDA receptors. The NMDA receptor-mediated ion current (I ( NMDA )) was measured using the two-electrode voltage-clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, gintonin enhanced I ( NMDA ) in a concentration-dependent manner. Gintonin-mediated I ( NMDA ) enhancement was blocked by Ki16425, an LPA1/3 receptor antagonist. Gintonin action was blocked by a PLC inhibitor, IP3 receptor antagonist, Ca²âº chelator, and a tyrosine kinase inhibitor. The site-directed mutation of Ser1308 of the NMDA receptor, which is phosphorylated by protein kinase C (PKC), to an Ala residue, or co-expression of receptor protein tyrosine phosphatase with the NMDA receptor attenuated gintonin action. Moreover, gintonin treatment elicited a transient elevation of [Ca²âº](i) in cultured hippocampal neurons and elevated longterm potentiation (LTP) in both concentration-dependent manners in rat hippocampal slices. Gintonin-mediated LTP induction was abolished by Ki16425. These results indicate that gintonin-mediated I ( NMDA ) potentiation and LTP induction in the hippocampus via the activation of LPA receptor might be responsible for ginseng-mediated improvement of memory-related brain functions.

Effect of dextromethorphan on human K(v)1.3 channel activity: involvement of C-type inactivation.

Dextromethorphan exhibits neuroprotective effects against inflammation-mediated neurodegeneration. However, relatively little is known regarding the molecular mechanism for this inflammation-mediated neuroprotection. Human K(v)1.3 channels, one of the voltage-gated potassium channels, are widely expressed in the immune and nervous systems. Activation of human K(v)1.3 channels causes neuroglia-mediated neurodegeneration. Agents that inhibit human K(v)1.3 channel activity have been developed as novel drugs for immunosuppression. In the present study, we investigated the effects of dextromethorphan on human K(v)1.3 or K(v)1.2 channel activity heterologously expressed in Xenopus laevis oocytes. The channel currents were measured with the two-electrode voltage clamp technique. Activation of both channels induced outward peak and steady-state currents. Dextromethorphan treatment induced a slight inhibition of peak currents in human K(v)1.2 and K(v)1.3 channels, whereas dextromethorphan profoundly inhibited the steady-state currents of human K(v)1.3 channels compared to K(v)1.2 channel currents. Dextromethorphan's action on steady-state currents of human K(v)1.3 channels was in a concentration-dependent manner. The half-maximal inhibitory concentration (IC(50)) on steady-state currents of human K(v)1.3 channels was 12.8±1.6µM. Dextromethorphan also accelerated the C-type inactivation rate, increased the current decay rate, and inhibited currents in a use-dependent manner. These results indicate that dextromethorphan accelerates C-type inactivation of human K(v)1.3 channels and acts as an open-channel blocker. These results further suggest the possibility that dextromethorphan-mediated acceleration of C-type inactivation of human K(v)1.3 channels might be one of the cellular bases of dextromethorphan-mediated protection against inflammation-mediated neurodegeneration.

Quercetin Inhibits α3ß4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes.

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-HT(3A) and α7 nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal α3ß4 nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal α3 and ß4 subunits. Treatment with acetylcholine elicited an inward peak current (I(ACh)) in oocytes expressing α3ß4 nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited I(ACh) in oocytes expressing α3ß4 nicotinic acetylcholine receptors. The inhibition of I(ACh) by quercetin was reversible and concentration-dependent. The half-inhibitory concentration (IC(50)) of quercetin was 14.9±0.8 µM in oocytes expressing α3ß4 nicotinic acetylcholine receptor. The inhibition of I(ACh) by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate α3ß4 nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.

Effects of quercetin on α9α10 nicotinic acetylcholine receptor-mediated ion currents.

Quercetin, one of the flavonoids, is a low molecular weight substance found in fruits and vegetables. Quercetin, like other flavonoids, has a wide range of neuropharmacological actions and antioxidant effects. The α9α10 nicotinic acetylcholine receptor is one of the numerous nicotinic acetylcholine receptors that exist as a heteropentameric form between efferent olivocochlear fibers and hair cells of the cochlea. In this study, we report the effects of quercetin on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage-clamp technique. Treatment with acetylcholine evoked inward currents (I(ACh)) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Quercetin blocked I(ACh) in concentration-dependent and reversible manners, and the blocking effect on I(ACh) was stronger with pre-application than co-application of quercetin. The half maximal inhibitory concentration (IC(50)) of quercetin was 45.4±10.1µM. Quercetin-mediated I(ACh) inhibition was not affected by acetylcholine concentration and was independent of membrane-holding potential. Although the inhibitory effect of quercetin was significantly attenuated in the absence of extracellular Ca(2+), the action of quercetin was independent of extracellular Ca(2+) concentration, indicating that the presence of extracellular Ca(2+) might be needed for quercetin-related effects and might play an important role in quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor. These results indicate that quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor could provide a molecular basis for quercetin actions at the cellular level.

Ginsenoside Rg(3) decelerates hERG K(+) channel deactivation through Ser631 residue interaction.

The human ether-a-go-go-related gene (hERG) cardiac K(+) channels are one of the representative pharmacological targets for development of drugs against cardiovascular diseases such as arrhythmia. Panax ginseng has been known to have cardio-protective effects. However, little is known about the molecular mechanisms of how ginsenosides, the active ingredients in Panax ginseng, interact with hERG K(+) channel proteins. In the present study, we first examined the effects of various ginsenosides on hERG K(+) channel activity by expressing human α subunits in Xenopus oocytes. Among them ginsenoside Rg(3) (Rg(3)) most potently enhanced outward I(hERG) and peak I(tail). Rg(3) induced a large persistent deactivating-tail current (I(deactivating-tail)) and profoundly decelerated deactivating current decay in both concentration- and voltage-dependent manners. The EC(50) for steady-state I(hERG), peak I(tail), and persistent I(deactivating-tail) was 0.41±0.05, 0.61±0.11, and 0.36±0.04µM, respectively. Rg(3) actions were blocked by bepridil, a hERG K(+) channel antagonist. Site-directed mutation of S631, which is located at the channel pore entryway, to S631C in hERG K(+) channel abolished Rg(3) actions on hERG K(+) channels. These results indicate that S631 residue of hERG K(+) channel plays an important role in Rg(3)-mediated induction of a persistent I(deactivating-tail) and in a deceleration of hERG K(+) channel deactivation.

Inhibitory Effects of Quercetin on Muscle-type of Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes.

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as α3ß4, α7 and α9α10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (I(ACh)) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited I(ACh). Pre-treatment of quercetin further inhibited I(ACh) in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of I(ACh) by quercetin was reversible and concentration-dependent. The IC(50) of quercetin was 18.9±1.2 µM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of I(ACh) by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.

A simple method for the preparation of crude gintonin from ginseng root, stem, and leaf.

Ginseng has been used as a general tonic agent to invigorate the human body as an adaptogenic agent. In a previous report, we have shown that ginseng contains a novel glycolipoprotein called gintonin. The main function of gintonin is to transiently enhance intracellular free Ca(2+) [Ca(2+)]i levels in animal cells. The previous method for gintonin isolation included multiple steps using organic solvents. In the present report, we developed a simple method for the preparation of crude gintonin from ginseng root as well as stem and leaf, which produced a higher yield of gintonin than the previous one. The yield of gintonin was 0.20%, 0.29%, and 0.81% from ginseng root, stem, and leaf, respectively. The apparent molecular weight of gintonin isolated from stem and leaf through sodium dodecyl sulfate polyacrylamide gel electrophoresis was almost same as that from root but the compositions of amino acids, carbohydrates or lipids differed slightly between them. We also examined the effects of crude gintonin from ginseng root, stem, and leaf on endogenous Ca(2+)-activated Cl- channel (CaCC) activity of Xenopus oocytes through mobilization of [Ca(2+)]i. We found that the order of potency for the activation of CaCC was ginseng root > stem > leaf. The ED50 was 1.4±1.4, 4.5±5.9, and 3.9±1.1 µg/mL for root, stem and leaf, respectively. In the present study, we demonstrated for the first time that in addition to ginseng root, ginseng stem and leaf also contain gintonin. Gintonin can be prepared from a simple method with higher yield of gintonin from ginseng root, stem, and leaf. Finally, these results demonstrate the possibility that ginseng stem and leaf could also be utilized for ginstonin preparation after a simple procedure, rather than being discarded.

An edible gintonin preparation from ginseng.

Ginseng, the root of Panax ginseng, is one of the oldest herbal medicines. It has a variety of physiological and pharmacological effects. Recently, we isolated a subset of glycolipoproteins that we designated gintonin, and demonstrated that it induced transient change in intracellular calcium concentration ([Ca(2+)]i) in cells via G-protein-coupled receptor signaling pathway(s). The previous method for gintonin isolation included multiple steps using methanol, butanol, and other organic solvents. In the present study, we developed a much simple method for the preparation of gintonin from ginseng root using 80% ethanol extraction. The extracted fraction was designated edible gintonin. This method produced a high yield of gintonin (0.20%). The chemical characteristics of gintonin such as molecular weight and the composition of the extract product were almost identical as the gintonin prepared using the previous extraction regimen involving various organic solvents. We also examined the physiological effects of edible gintonin on endogenous Ca(2+)-activated Cl(-) channel activity of Xenopus oocytes. The 50% effective dose was 1.03±0.3 µg/mL. Finally, since gintonin preparation through ethanol extraction is easily reproducible, gintonin could be commercially applied for ginseng-derived functional health food and/or drug following the confirmations of in vitro and in vivo physiological and pharmacological effects of gintonin.