Effects of Gintonin-Enriched Fraction in an Atopic Dermatitis Animal Model: Involvement of Autotaxin Regulation.

Ginseng extract has been used for prevention of atopic dermatitis (AD) in experimental animal models. However, little is known about its active ingredients and the molecular mechanisms underlying its anti-AD effects. Recently, we isolated a unique lysophosphatidic acid (LPA) receptor ligand, gintonin, from ginseng. Gintonin, the glycolipoprotein fraction of ginseng, contains LPAs, mainly LPA C18 : 2 with other minor lysophospholipid components. A line of evidence showed that serum autotaxin (ATX) activity and level are significantly elevated in human AD patients compared to those in normal controls, which indicates that ATX may be involved in human AD. In a previous study, we demonstrated that gintonin exerted anti-inflammatory effects via inhibition of microglial activation and proinflammatory cytokine production by immune cells and that it strongly inhibited ATX activity. In this study, we investigated whether oral administration of the gintonin-enriched fraction (GEF) could ameliorate the symptoms of 2,4-dinitrofluorobenzene (DNFB)-induced AD in NC/Nga mice. We found that oral administration of GEF to DNFB-induced AD mice for 2 weeks reduced ear swelling and AD skin index. In addition, oral administration of GEF reduced the serum levels of immunoglobulin E, histamine, interleukin-4, and interferon-γ. Histological examination showed that oral administration of GEF attenuated skin inflammation and significantly reduced eosinophil and mast cell infiltration into the skin. Moreover, oral administration of GEF not only decreased serum ATX level but also reduced serum ATX activity. The present study shows that the anti-AD effects of ginseng might be attributed to GEF-induced anti-inflammatory activity and ATX regulation.

Plant Lysophosphatidic Acids: A Rich Source for Bioactive Lysophosphatidic Acids and Their Pharmacological Applications.

Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphatidic acid; LPA) is a simple and minor phospholipid in plants. Plant LPAs are merely metabolic intermediates in de novo lipid synthesis in plant cell membranes or for glycerophospholipid storage. The production and metabolisms of LPAs in animals are also well characterized and LPAs have diverse cellular effects in animal systems; i.e., from brain development to wound healing through the activation of G protein-coupled LPA receptors. Recent studies show that various foodstuffs such as soybean, cabbage and seeds such as sesame and sunflower contain bioactive LPAs. Some LPAs are produced from phosphatidic acid during the digestion of foodstuff. In addition, herbal medicines such as corydalis tuber, and especially ginseng, contain large amounts of LPAs compared to foodstuffs. Herbal LPAs bind to cell surface LPA receptors in animal cells and exert their biological effects. Herbal LPAs elicit [Ca(2+)]i transient and are coupled to various Ca(2+)-dependent ion channels and receptor regulations via the activation of LPA receptors. They also showed beneficial effects of in vitro wound healing, in vivo anti-gastric ulcer, anti-Alzheimer's disease, autotaxin inhibition and anti-metastasis activity. Thus, herbal LPAs can be useful agents for human health. Humans can utilize exogenous plant-derived LPAs for preventive or therapeutic purposes if plant-derived LPAs are developed as functional foods or natural medicine targeting LPA receptors. This brief review article introduces the known rich sources of herbal LPAs and herbal LPA binding protein, describes their biological effects, and further addresses possible clinical applications.

Preparation of a Monoclonal Antibody against Gintonin and Its Use in an Enzyme Immunoassay.

Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an [Ca(2+)]i transient in animal cells via activation of LPA receptors. In vitro studies have shown that gintonin regulates various calcium-dependent ion channels and receptors. In in vivo studies, gintonin elicits anti-Alzheimer's disease activity through the activation of the non-amyloidogenic pathway and anti-metastatic effects through the inhibition of autotaxin. However, a method for gintonin quantitation in ginseng has not been developed. In the present study, we developed an enzyme immunoassay (EIA) to measure gintonin. A monoclonal antibody was raised in a mouse using gintonin as the immunogen, and an indirect competitive EIA was used to measure gintonin. The working range was 0.01-10 µg per assay. The anti-gintonin monoclonal antibody did not cross-react with the ginsenosides Ra, Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, and Rg3 or with LPAs such as LPA C16:0, LPA C18:0, LPA C18:1, and LPA C18:2. Using a standard curve, we measured the amount of gintonin in various ginseng extract fractions. Interestingly, we only detected a little amount of gintonin in conventional hot water extracts of Korean red ginseng. However, we can measure gintonin after ethanol extraction of Korean red ginseng marc. Thus, gintonin can be extracted from ginseng with ethanol but not water, and the remaining Korean red ginseng marc can be used to obtain gintonin. These results indicate that the EIA with the anti-gintonin monoclonal antibody can be used to quantify gintonin in various ginseng preparations, including commercial ginseng products.

Resveratrol inhibits glycine receptor-mediated ion currents.

Resveratrol is found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-nociceptive, and life-prolonging effects. However, the single cellular mechanisms by which resveratrol acts are relatively unknown, especially in terms of possible regulation of receptors involved in synaptic transmission. The glycine receptor is an inhibitory ligand-gated ion channel involved in fast synaptic transmission in spinal cord. In the present study, we investigated the effect of resveratrol on human glycine receptor channel activity. Glycine α1 receptors were expressed in Xenopus oocytes and glycine receptor channel activity was measured using a two-electrode voltage clamp technique. Treatment with resveratrol alone had no effect on oocytes injected with H2O or on oocytes injected with glycine α1 receptor cRNA. In the oocytes injected with glycine α1 receptor cRNA, co- or pre-treatment of resveratrol with glycine inhibited the glycine-induced inward peak current (IGly) in a reversible manner. The inhibitory effect of resveratrol on IGly was also concentration dependent, voltage independent, and non-competitive. These results indicate that resveratrol regulates glycine receptor channel activity and that resveratrol-mediated regulation of glycine receptor channel activity is one of several cellular action mechanisms of resveratrol for pain regulation.

Effects of ginsenoside Rg3 on α9α10 nicotinic acetylcholine receptor-mediated ion currents.

Ginsenosides is a low molecular weight substance found in ginseng as one of the active ingredients. Ginsenosides, like other herbal medicines, has a wide range of neuropharmacological actions including neuroprotective effects. The α9α10 nicotinic acetylcholine receptor is one of numerous nicotinic acetylcholine receptors that exists as a heteropentameric form in auditory hair cells of the cochlea. In this study, we report the effects of ginsenosides on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage clamp technique. Treatment with acetylcholine evoked inward currents (IACh) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Ginsenosides blocked IACh in order of potency of Rg3> Rb2> CK>Re = Rg2> Rf>Rc> Rb1> Rg1 with reversible manners, and the blocking effect of Rg3 on IACh was same after pre-application than co-application of Rg3. The half maximal inhibitory concentration (IC50) of Rg3 was 39.6 ± 4.9 µm. Rg3-induced IACh inhibition was not affected by acetylcholine concentration and was independent of membrane holding potential. Although the inhibitory effect of Rg3 on IACh was abolished in oocytes expressing α9 subunit alone, indicating that the presence of α10 subunit might be required for Rg3-induced regulations of α9α10 nicotinic acetylcholine receptor channel activity. These results indicate that α10 subunit of α9α10 nicotinic acetylcholine receptor might play an important role in Rg3-induced regulation of the α9α10 nicotinic acetylcholine receptor.

Inhibitory Effects of Ginsenoside Metabolites, Compound K and Protopanaxatriol, on GABAC Receptor-Mediated Ion Currents.

Ginsenosides, one of the active ingredients of Panax ginseng, show various pharmacological and physiological effects, and they are converted into compound K (CK) or protopanaxatriol (M4) by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. The γ-aminobutyric acid receptorC (GABAC) is primarily expressed in retinal bipolar cells and several regions of the brain. However, little is known of the effects of ginsenoside metabolites on GABAC receptor channel activity. In the present study, we examined the effects of CK and M4 on the activity of human recombinant GABAC receptor (ρ1) channels expressed in Xenopus oocytes by using a 2-electrode voltage clamp technique. In oocytes expressing GABAC receptor cRNA, we found that CK or M4 alone had no effect in oocytes. However, co-application of either CK or M4 with GABA inhibited the GABA-induced inward peak current (IGABA ). Interestingly, pre-application of M4 inhibited IGABA more potently than CK in a dose-dependent and reversible manner. The half-inhibitory concentration (IC50) values of CK and M4 were 52.1±2.3 and 45.7±3.9 µM, respectively. Inhibition of IGABA by CK and M4 was voltage-independent and non-competitive. This study implies that ginsenoside metabolites may regulate GABAC receptor channel activity in the brain, including in the eyes.

Resveratrol Inhibits GABAC ρ Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes.

Resveratrol is a phytoalexin found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-inflammatory, and life-prolonging effects. However, relatively little is known about the effects of resveratrol on the regulation of ligand-gated ion channels. We have previously reported that resveratrol regulates subsets of homomeric ligand-gated ion channels such as those of 5-HT3A receptors. The γ-aminobutyric acidC (GABAC) receptor is mainly expressed in retinal bipolar cells and plays an important role in visual processing. In the present study, we examined the effects of resveratrol on the channel activity of homomeric GABAC receptor expressed in Xenopus oocytes injected with cRNA encoding human GABAC ρ subunits. Our data show that the application of GABA elicits an inward peak current (IGABA ) in oocytes that express the GABAC receptor. Resveratrol treatment had no effect on oocytes injected with H2O or with GABAC receptor cRNA. Co-treatment with resveratrol and GABA inhibited IGABA in oocytes with GABAC receptors. The inhibition of IGABA by resveratrol was in a reversible and concentration-dependent manner. The IC50 of resveratrol was 28.9±2.8 µM in oocytes expressing GABAC receptor. The inhibition of IGABA by resveratrol was in voltage-independent and non-competitive manner. These results indicate that resveratrol might regulate GABAC receptor expression and that this regulation might be one of the pharmacological actions of resveratrol on the nervous system.

Activation of lysophosphatidic Acid receptor is coupled to enhancement of ca(2+)-activated potassium channel currents.

The calcium-activated K(+) (BKCa) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca(2+) is the main regulator of BKCa channel activation. The BKCa channel contains two high affinity Ca(2+) binding sites, namely, regulators of K(+) conductance, RCK1 and the Ca(2+) bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca(2+) levels through diverse G proteins such as Gαq/11, Gαi, Gα12/13, and Gαs and the related signal transduction pathway. In the present study, we examined LPA effects on BKCa channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BKCa channel activation was also attenuated by the PLC inhibitor U-73122, IP3 inhibitor 2-APB, Ca(2+) chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BKCa channel activation. The present study indicates that LPA-mediated activation of the BKCa channel is achieved through the PLC, IP3, Ca(2+), and PKC pathway and that LPA-mediated activation of the BKCa channel could be one of the biological effects of LPA in the nervous and vascular systems.

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 Gintonin-enriched fraction on the gene expression of six lysophosphatidic receptor subtypes.

BACKGROUND: Gintonin, isolated from ginseng, acts as a ginseng-derived lysophosphatidic acid (LPA) receptor ligand and elicits the [Ca2+]i transient through six LPA receptor subtypes (LPARSs). However, the long-term effects of gintonin-enriched fraction (GEF) on the gene expression of six LPARSs remain unknown. We examined changes in the gene expression of six LPA receptors in the mouse whole brain, heart, lungs, liver, kidneys, spleen, small intestine, colon, and testis after long-term oral GEF administration. METHODS: C57BL/6 mice were divided into two groups: control vehicle and GEF (100 mg/kg, p.o.). After 21-day saline or GEF treatment, total RNA was extracted from nine mouse organs. Quantitative-real-time PCR (qRT-PCR) and western blot were performed to quantify changes in the gene and protein expression of the six LPARSs, respectively. RESULTS: qRT-PCR analysis before GEF treatment revealed that the LPA6 RS was predominant in all organs except the small intestine. The LPA2 RS was most abundant in the small intestine. Long-term GEF administration differentially regulated the six LPARSs. Upon GEF treatment, the LPA6 RS significantly increased in the liver, small intestine, colon, and testis but decreased in the whole brain, heart, lungs, and kidneys. Western blot analysis of the LPA6 RS confirmed the differential effects of GEF on LPA6 receptor protein levels in the whole brain, liver, small intestine, and testis. CONCLUSION: The LPA6 receptor was predominantly expressed in all nine organs examined; long-term oral GEF administration differentially regulated LPA3, LPA4, and LPA6 receptors in the whole brain, heart, lungs, liver, kidneys, small intestine, and testis.

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.

Quinazolindione derivatives as potent 5-HT3A receptor antagonists.

5-HT(3A) receptor antagonists have been used mainly for the treatment of nausea and vomiting. These days, the antagonists are of special interest due to their therapeutic potential to treat other diseases such as depression, psychotic disorder, drug abuse, and irritable bowel syndrome. To discover novel 5-HT(3A) receptor antagonists, we screened our in-house small molecule library, resulting in identifying the quinazolindione derivatives as potent 5-HT(3A) receptor antagonists. For the purpose of structure-activity relationship study, 24 quinazolindione analogues were biologically evaluated against 5-HT(3A) receptor. Among those, KKHT10612 shows the best antagonistic effect against 5-HT(3A) receptor with an IC(50) value of 0.8 microM which is comparable with that of the reference compound, MDL72222, and selectivity over T-type calcium channel as well.

Pr-lynx1, a modulator of nicotinic acetylcholine receptors in the insect.

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the gene itself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface as a GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by alpha3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing alpha3beta4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.

Bioactive lipids in gintonin-enriched fraction from ginseng.

BACKGROUND: Ginseng is a traditional herbal medicine for human health. Ginseng contains a bioactive ligand named gintonin. The active ingredient of gintonin is lysophosphatidic acid C18:2 (LPA C18:2). We previously developed a method for gintonin-enriched fraction (GEF) preparation to mass-produce gintonin from ginseng. However, previous studies did not show the presence of other bioactive lipids besides LPAs. The aim of this study was to quantify the fatty acids, lysophospholipids (LPLs), and phospholipids (PLs) besides LPAs in GEF. METHODS: We prepared GEF from white ginseng. We used gas chromatography-mass spectrometry for fatty acid analysis and liquid chromatography-tandem mass spectrometry for PL analysis, and quantified the fatty acids, LPLs, and PLs in GEF using respective standards. We examined the effect of GEF on insulin secretion in INS-1 cells. RESULTS: GEF contains about 7.5% linoleic (C18:2), 2.8% palmitic (C16:0), and 1.5% oleic acids (C18:1). GEF contains about 0.2% LPA C18:2, 0.06% LPA C16:0, and 0.02% LPA C18:1. GEF contains 0.08% lysophosphatidylcholine, 0.03% lysophosphatidylethanolamine, and 0.13% lysophosphatidylinositols. GEF also contains about 1% phosphatidic acid (PA) 16:0-18:2, 0.5% PA 18:2-18:2, and 0.2% PA 16:0-18:1. GEF-mediated insulin secretion was not blocked by LPA receptor antagonist. CONCLUSION: We determined four characteristics of GEF through lipid analysis and insulin secretion. First, GEF contains a large amount of linoleic acid (C18:2), PA 16:0-18:2, and LPA C18:2 compared with other lipids. Second, the main fatty acid component of LPLs and PLs is linoleic acid (C18:2). Third, GEF stimulates insulin secretion not through LPA receptors. Finally, GEF contains bioactive lipids besides LPAs.

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.

Gintonin absorption in intestinal model systems.

BACKGROUND: Recently, we identified a novel ginseng-derived lysophosphatidic acid receptor ligand, called gintonin. We showed that gintonin induces [Ca2+]i transient-mediated morphological changes, proliferation, and migration in cells expressing lysophosphatidic acid receptors and that oral administration of gintonin exhibits anti-Alzheimer disease effects in model mice. However, little is known about the intestinal absorption of gintonin. The aim of this study was to investigate gintonin absorption using two model systems. METHODS: Gintonin membrane permeation was examined using a parallel artificial membrane permeation assay, and gintonin absorption was evaluated in a mouse everted intestinal sac model. RESULTS: The parallel artificial membrane permeation assay showed that gintonin could permeate an artificial membrane in a dose-dependent manner. In the everted sac model, gintonin absorption increased with incubation time (from 0 min to 60 min), followed by a decrease in absorption. Gintonin absorption into everted sacs was also dose dependent, with a nonlinear correlation between gintonin absorption and concentration at 0.1-3 mg/mL and saturation at 3-5 mg/mL. Gintonin absorption was inhibited by the Rho kinase inhibitor Y-27632 and the sodium-glucose transporter inhibitor phloridzin. Moreover, lipid extraction with methanol also attenuated gintonin absorption, suggesting the importance of the lipid portion of gintonin in absorption. This result shows that gintonin might be absorbed through passive diffusion, paracellular, and active transport pathways. CONCLUSION: The present study shows that gintonin could be absorbed in the intestine through transcellular and paracellular diffusion, and active transport. In addition, the lipid component of gintonin might play a key role in its intestinal absorption.

Identification of ginsenoside interaction sites in 5-HT3A receptors.

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.

Neuroprotective effects of ginsenoside Rg3 against homocysteine-induced excitotoxicity in rat hippocampus.

We previously demonstrated that ginsenoside Rg(3) (Rg(3)), one of the active ingredients in Panax ginseng, attenuates NMDA receptor-mediated currents and NMDA-induced neurotoxicity (Kim, S., Kim, T., Ahn, K., Park, W.K., Nah, S.Y., Rhim, H., 2004. Ginsenoside Rg(3) antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons. Biochem. Biophys. Res. Commun. 323, 416-424). Accumulating evidence suggests that homocysteine (HC), a metabolite of methionine, exerts its excitotoxicity through NMDA receptor activation. In the present study, we examined the neuroprotective effects of Rg(3) on HC-induced hippocampal excitotoxicity in vitro and in vivo. Our in vitro studies using rat cultured hippocampal neurons revealed that Rg(3) treatment significantly and dose-dependently inhibited HC-induced hippocampal cell death, with an EC(50) value of 28.7+/-7.5 muM. Rg(3) treatment not only significantly reduced HC-induced DNA damage, but also dose-dependently attenuated HC-induced caspase-3 activity in vitro. Our in vivo studies revealed that intracerebroventricular (i.c.v.) pre-administration of Rg(3) significantly and dose-dependently reduced i.c.v. HC-induced hippocampal damage in rats. To examine the mechanisms underlying the in vitro and in vivo neuroprotective effects of Rg(3) against HC-induced hippocampal excitotoxicity, we examined the effect of Rg(3) on HC-induced intracellular Ca(2+) elevations in cultured hippocampal cells and found that Rg(3) treatment dose-dependently inhibited HC-induced intracellular Ca(2+) elevation, with an IC(50) value of 41.5+/-17.5 muM. In addition, Rg(3) treatment dose-dependently inhibited HC-induced currents in Xenopus oocytes expressing the NMDA receptor, with an IC(50) of 47.3+/-14.2 muM. These results collectively indicate that Rg(3)-induced neuroprotection against HC in rat hippocampus might be achieved via inhibition of HC-mediated NMDA receptor activation.

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.