Sang Min Jeong

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.

Protective effects of ginseng saponins on 3-nitropropionic acid-induced striatal degeneration in rats.

The precise cause of neuronal cell death in Huntingtons disease (HD) is not known. Systemic administration of 3-nitropropionic acid (3-NP), an irreversible succinate dehydrogenase inhibitor, not only induces a cellular ATP depletions but also causes a selective striatal degeneration similar to that seen in HD. Recent accumulating reports have shown that ginseng saponins (GTS), the major active ingredients of Panax ginseng, have protective effects against neurotoxin insults. In the present study, we examined in vitro and in vivo effects of GTS on striatal neurotoxicity induced by repeated treatment of 3-NP in rats. Here, we report that systemic administration of GTS produced significant protections against systemic 3-NP- and intrastriatal malonate-induced lesions in rat striatum with dose-dependent manner. GTS also improved significantly 3-NP-caused behavioral impairment and extended survival. However, GTS itself had no effect on 3-NP-induced inhibition of succinate dehydrogenase activity. To explain the mechanisms underlying in vivo protective effects of GTS against 3-NP-induced striatal degeneration, we examined in vitro effect of GTS against 3-NP-caused cytotoxicity using cultured rat striatal neurons. We found that GTS inhibited 3-NP-induced intracellular Ca(2+) elevations. GTS restored 3-NP-caused mitochondrial transmembrane potential reduction in cultured rat striatal neurons. GTS also prevented 3-NP-induced striatal neuronal cell deaths with dose-dependent manner. The EC(50) was 12.6 +/- 0. 7microg/ml. These results suggest that in vivo protective effects of GTS against 3-NP-induced rat striatal degeneration might be achieved via in vitro inhibition of 3-NP-induced intracellular Ca(2+) elevations and cytotoxicity of striatal neurons.

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.

Ginseng saponins induce store-operated calcium entry in Xenopus oocytes

We investigated the effect of the active ingredients of Panax ginseng, ginsenosides, on store‐operated Ca2+ entry (SOCE) using a two‐electrode voltage clamp technique in Xenopus oocytes in which SOCE is monitored through Ca2+‐activated Cl− currents. Under hyperpolarizing voltage clamp conditions, treatment with ginsenosides produced a biphasic Ca2+‐activated Cl− current consisting of a rapid transient inward current and a slowly developing secondary sustained inward current. The transient inward current was inactivated rapidly, whereas the sustained inward current persisted for nearly 10 min. The effect of ginsenosides on the biphasic current was dose‐dependent and reversible. The EC50 was 42.8±11.6 and 46.6±7.1 μg ml−1 for the transient and sustained inward current, respectively. In the absence of extracellular Ca2+ ginsenosides induced only a transient inward current but in the presence of extracellular Ca2+ ginsenosides induced the biphasic current. Magnitudes of the sustained currents were dependent on extracellular Ca2+ concentration. Sustained inward current induced by ginsenosides, but not transient inward current, and ginsenoside‐induced store‐operated Ca2+ (SOC) currents (ISOC) were blocked by La3+, a Ca2+ channel blocker, suggesting that the sustained inward current and ISOC was derived from an influx of extracellular Ca2+. Treatment with 2‐APB and heparin, which are IP3 receptor antagonists, inhibited the ginsenoside‐induced biphasic current. Treatment with the PLC inhibitor, U73122, also inhibited the ginsenoside‐induced biphasic current. Intraoocyte injection of ATP‐γS, but not adenylyl AMP‐PCP, induced a persistent activation of ginsenoside‐induced sustained current but did not affect the transient current. In rat hippocampal neurons, ginsenosides inhibited both carbachol‐stimulated intracellular Ca2+ release and intracellular Ca2+ depletion‐activated SOCE. These results indicate that ginsenoside might act as a differential regulator of intracellular Ca2+ levels in neurons and Xenopus oocytes.

Effects of Korean red ginseng extract on cisplatin-induced nausea and vomiting

Ginseng, the root ofPanax ginseng C. A. Meyer, is well known as a tonic medicine for restoring and enhancing human health. In traditional medicine, ginseng is utilized for the alleviation of emesis, which includes nausea and vomiting. However, it has not yet been demonstrated whether ginseng exhibitsin vivo anti-nausea and anti-vomiting properties. In this study, we examined the anti-emetic effect of Korean red ginseng total extract (KRGE) on cisplatin-induced nausea and vomiting using ferrets. Intraperitoneal administration (i.p.) of cisplatin (7.5 mg/kg) induced both nausea and vomiting with one-hour latency. The episodes of nausea and vomiting reached a peak after 1.5 h and persisted for 3 h. Treatment with KRGEvia oral route significantly reduced the cisplatin-induced nausea and vomiting in a dose-dependent manner. The anti-emetic effect was 12.7±8.6, 31.8±6.9, and 67.6±4.0% with doses of 0.3, 1.0, and 3.0 g/kg of KRGE, respectively. Pretreatment with KRGEvia oral route 1 and 2 h before cisplatin administration also significantly attenuated the cisplatin-induced nausea and vomiting. However this did not occur with a pretreatment 4 h before cisplatin administration. These results are supportive of KRGE being utilized as an anti-emetic agent against nausea and vomiting caused by chemotherapy (i.e. cisplatin).

Involvement of 5‐HT3 receptors in the development and expression of methamphetamine‐induced behavioral sensitization: 5‐HT3A receptor channel and binding study

Methamphetamine (MAP) is one of the most commonly abused drugs in Asia, and previous studies suggest that serotonin 3 receptors (5‐HT3) are involved in MAP‐induced locomotion and reward. However, little is known about the role of 5‐HT3 receptors in MAP‐induced behavioral sensitization. Here, we measured the effects of MDL 72222, a 5‐HT3 antagonist, and SR 57227 A, a 5‐HT3 agonist, on the development and expression of MAP‐induced behavioral sensitization, and alternations of 5‐HT3 receptor binding labeled with the 5‐HT3‐selective antagonist, [3H]GR65630, in mice. In addition, we investigated the effects of MAP on 5‐HT3A receptor channel activity in Xenopus laevis oocytes expressing 5‐HT3A receptors. We found that MDL 72222 attenuated both the development and expression of behavioral sensitization to MAP (1.0 mg/kg, i.p.), and that this attenuating effect of MDL 72222 was reversed by pre‐treatment with SR 57227 A. In oocytes expressing 5‐HT3A receptor, MAP exhibited a dual modulation of 5‐HT3A receptor channel activity, i.e. pre‐treatment with a low dose of MAP (0.1 µm) enhanced 5‐HT‐induced inward peak current (I5‐HT) but a high dose of MAP (100 µm) inhibited I5‐HT. The acute administration of MDL 72222 with MAP decreased [3H]GR65630 binding versus MAP alone in the mouse striatum. Our results suggest that MDL 72222 attenuates MAP‐induced behavioral sensitization via 5‐HT3 receptors in the caudate putamen, and that 5‐HT3 receptor antagonists like MDL 72222 have potential as novel anti‐psychotic agents for the treatment of MAP dependence and psychosis.

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.

Ginseng and ion channels : Are ginsenosides, active component of Panax ginseng, differential modulator of ion channels?

The last two decades have shown a marked expansion in publications of diverse effects of Panax ginseng. Ginsenosides, as active ingredients of Panax ginseng, are saponins found in only ginseng. Recently, a line of evidences shows that ginsenosides regulate various types of ion channel activity such as Ca²^+, K^+, Na^+, Cl^-, or ligand gated ion channels (i.e. 5-HT₃, nicotinic acetylcholine, or NMDA receptor) in neuronal, non-neuronal cells, and heterologously expressed cells. Ginsenosides inhibit voltage-dependent Ca²^+, K^+, and Na^+ channels, whereas ginsenosides activate Ca²^+-activated Cl^- and Ca²^+-activated K^+ channels. Ginsenosides also inhibit excitatory ligand-gated ion channels such as 5-HT₃. nicotinic acetylcholine, and NMDA receptors. This review will introduce recent findings on the ginsenoside-induced differential regulations of ion channel activities and will further expand the possibilities how these ginsenoside-induced ion channel regulations are coupled to biological effects of Panax ginseng.

Differential effect of bovine serum albumin on ginsenoside metabolite-induced inhibition of α3β4 nicotinic acetylcholine receptor expressed inXenopus oocytes

Ginsenosides, major active ingredients ofPanax ginseng, that exhibit various pharmacological and physiological actions are transformed into compound K (CK) or M4 by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. Recent reports shows that ginsenosides might play a role as pro-drugs for these metabolites. In present study, we investigated the effect of bovine serum albumin (BSA), which is one of major binding proteins on various neurotransmitters, hormones, and other pharmacological agents, on ginsenoside Rg2-, CK-, or M4-induced regulation of α3β4 nicotinic acetylcholine (ACh) receptor channel activity expressed inXenopus oocytes. In the absence of BSA, treatment of ACh elicited inward peak current (lACh) in oocytes expressing α3β4 nicotinic ACh receptor. Co-treatment of ginsenoside Rg2, CK, or M4 with ACh inhibitedlACh in oocytes expressing α3β4 nicotinic ACh receptor with reversible and dose-dependent manner. In the presence of 1% BSA, treatment of ACh still elicitedlACh in oocytes expressing α3β4 nicotinic ACh receptor and co-treatment of ginsenoside Rg2 or M4 but not CK with ACh inhibitedlACh in oocytes expressing α3β4 nicotinic ACh receptor with reversible and dose-dependent manner. These results show that BSA interferes the action of CK rather than M4 on the inhibitory effect oflACh in oocytes expressing α3β4 nicotinic ACh receptor and further suggest that BSA exhibits a differential interaction on ginsenoside metabolites.