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

Abstract

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 Ca²⁺-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, Ca²⁺ 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 [Ca²⁺](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.

Fig. 1.

Effects of gintonin on I_NMDA. (A) A representative trace of gintonin (1 μg/ml) (GT)-mediated potentiation of I_NMDA in oocytes expressing the rat NMDA receptor. The trace represents 6 separate oocytes. (B) Concentration-dependent effect of gintonin on I_NMDA potentiation. The trace represents 6 separate oocytes. Application of NMDA (300 μM) and glycine (10 μM) elicited I_NMDA. Gintonin application after NMDA (300 μM) and glycine (10 μM) potentiated I_NMDA. (C) Concentration-dependent potentiating effects of gintonin on I_NMDA. Oocytes were voltage-clamped at a holding potential of −60 mV. Each point represents the mean ± SEM (n = 4–5/group).

Fig. 2.

Effects of an LPA 1/3 receptor antagonist on gintonin-mediated potentiation of I_NMDA. (A) Representative traces of gintonin (1 μg/ml) (GT)-mediated potentiation of I_NMDA in the absence (upper) or presence (lower) of Ki16425. Treatment with Ki16425 attenuated gintonin-mediated potentiation of I_NMDA as well as the Ca²⁺-activated Cl⁻ current. (B) Summary of the percent potentiation induced by gintonin in the absence or presence of Ki16425. Oocytes were voltage-clamped at a holding potential of −60 mV. Data represent the mean ± SEM. (n = 6–7/group).

Fig. 3.

Effects of a PLC inhibitor, IP₃ receptor antagonist, or BAPTA on gintonin-mediated potentiation of I_NMDA. (A–C) Representative traces of gintonin (1 μg/ml) (GT)-mediated potentiation of I_NMDA in the presence of an active PLC inhibitor U-73122, IP₃ receptor antagonist 2-APB, or intracellular Ca²⁺ chelator BAPTA. (D) Summary of the percent potentiation induced by gintonin in the absence (Con) or presence of various inhibitors or calcium chelator (U-73122, 2-APB, stauroporine, or BAPTA). Oocytes were voltage-clamped at a holding potential of −60 mV. Data represent the mean ± SEM (n = 6–7/group).

Fig. 4.

Effects of mutation of the PKC phosphorylation site, use of a tyrosine kinase inhibitor, or co-expression of receptor protein tyrosine phosphatase on gintonin-mediated potentiation of I_NMDA. A summary of the percent potentiation induced by gintonin in site-directed mutation of the PKC phosphorylation site (A), or co-expression of receptor protein tyrosine phosphatase (B), or in the absence (Con) or in the presence of tyrosine kinase (C). Oocytes were voltage-clamped at a holding potential of −60 mV. A, ^*p < 0.05, compared to control; #p < 0.05, compared to S1312A mutant. B, ^*p < 0.05, compared to control. C, ^*p < 0.05, compared to control; Data represent the mean ± SEM (n = 4–5/group).

Fig. 5.

Effects of gintonin on [Ca²⁺]ⁱ transients in cultured rat hippocampal neurons. (A) Representative traces in the absence (Con) or presence of various concentrations of gintonin. (B) Concentration-dependent effects of gintonin on [Ca²⁺]ⁱ transients. Each point represents the mean ± SEM (n = 7–10).

Fig. 6.

Procedure for LTP induction and activity calculation in rat hippocampal slices. (A) Upper panel: The protocol for theta burst stimulation (TBS) for acute hippocampal slices (a) or tetanic train stimulation for organotypic hippocampal slices (b). (B) Activity was calculated using area measuring of field potential trajectory. The signal was divided into 2 parts: activation and non-activation. The circle area within the line was calculated as the actual activity response. The artifact as indicated by the arrow was not calculated.

Fig. 7.

Effects of gintonin and an LPA1/3 receptor antagonist on LTP following tetanic stimulation of the Schaffer collateral (SC) area in rat hippocampal slices. LTP could be induced and maintained at CA3/CA1 (A) The LTP was increased by gintonin treatment in a concentration-dependent manner in acute hippocampal slices. Pooled data obtained from 4 different experiments. Data are expressed as percentages of control fEPSP amplitude. (B) Co-application of gintonin (1 μg/ml) with Ki16425 (1 nM), an LPA1/3 receptor antagonist, abolished gintonin-mediated LTP induction in organotypic hippocampal slices. The vertical arrow indicates the TBS addition point. Each point of the bars corresponds to the SEM (n = 4 each). (C) The summary histograms for normalized fEPSP values that show LTP levels at 40 min after TBS (^*p < 0.05, compared to control; ^#p < 0.01, compared to control, n = 4). (D) The summary histograms for normalized fEPSP values that show LTP levels at 40 min after TBS (^*p < 0.05, compared to control; ^#p < 0.05, compared to only gintonin treatment, n = 4).

Fig. 8.

Representative traces from control, gintonin alone, or gintonin + Ki16425 in rat hippocampal slices. (A) Traces were obtained 50 min after TBS in control or with different concentrations of gintonin. (B) Traces were obtained 50 min after TBS in control, gintonin, gintonin + Ki16425, or Ki16425 alone. Sample traces show fEPSP evoked by test pulse stimulation.