Mi Kyung Pyo

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.

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 inhibitor Y-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.

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.

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 Rg3 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 Rg3 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 Rg3 (Rg3). 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 Rg3 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 Rg3 and vice versa. We found that 1) raising [K+]o reduced the inhibitory effect of Rg3 on hKv1.4 channel currents, whereas Rg3 shifted the K+ activation curve to the right, and 2) TEA caused a rightward shift of the Rg3 concentration-response curve of wild-type hKv1.4 channel currents, whereas Rg3 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 Rg3 and hKv1.4 channels. These results indicate that Rg3 inhibits the hKv1.4 channel current by interacting with residue Lys531.

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 Ca2+ [Ca2+]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 Ca2+-activated Cl- channel (CaCC) activity of Xenopus oocytes through mobilization of [Ca2+]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.

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.

Quercetin enhances human α7 nicotinic acetylcholine receptor-mediated ion current through interactions with Ca2+ binding sites

The flavonoid quercetin is a low molecular weight substance found in fruits and vegetables. Aside from its anti-oxidative effect, quercetin, like other flavonoids, has a wide range of neuropharmacological actions. The α7 nicotinic acetylcholine receptor (α7 nAChR) has a Ca2+-binding site, is highly permeable to the Ca2+ ion, and plays important roles in Ca2+-related normal brain functions. Dysfunctions of α7 nAChR are associated with a variety of neurological disorders. In the present study, we investigated the effects of quercetin on the ACh-induced inward peak current (IACh) in Xenopus oocytes that heterologously express human α7 nAChR. IACh was measured with the two-electrode voltage clamp technique. In oocytes injected with α7 nAChR cRNA, the effects of the co-application of quercetin on IACh were concentration-dependent and reversible. The ED50 was 36.1 + 6.1 μM. Quercetin-mediated enhancement of IACh caused more potentiation when quercetin was pre-applied. The degree of IACh potentiation by quercetin pre-application was time-dependent and saturated after 1 min. Quercetin-mediated IACh enhancement was not affected by ACh concentration and was voltage-independent. However, quercetin-mediated IACh enhancement was dependent on extracellular Ca2+ concentrations and was specific to the Ca2+ ion, since the removal of extracellular Ca2+ or the addition of Ba2+ instead of Ca2+ greatly diminished quercetin enhancement of IACh. The mutation of Glu195 to Gln195, in the Ca2+-binding site, almost completely diminished quercetin-mediated IACh enhancement. These results indicate that quercetin-mediated IACh enhancement human α7 nAChR heterologously expressed in Xenopus oocytes could be achieved through interactions with the Ca2+-binding site of the receptor.

Human glycine α1 receptor inhibition by quercetin is abolished or inversed by α267 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 quercetins 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)). Quercetins 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.

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.

A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated α7 nicotinic acetylcholine receptor regulation

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not α7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of α7 nAChR induces alterations in channel gating properties and converts α7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg3 (Rg3) activity against the α7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg3. We further characterized Rg3 regulation of L247T receptors. We found that Rg3 inhibition of mutant α7 nAChR channel currents was reversible and concentration-dependent. Rg3 inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg3 and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg3 interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg3 forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg3 localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg3 at the channel pore.