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Gintonin stimulates gliotransmitter release in cortical primary astrocytes
Neuroscience Letters 603 (2015) 19–24
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research paper
Gintonin stimulates gliotransmitter release in cortical primary astrocytes Hyunsook Kim a , Byung-Hwan Lee a , Sun-Hye Choi a , Hyeon-Joong Kim a , Suk-Won Jung a , Sung-Hee Hwang b , Hyewon Rhim c , Hyung-Chun Kim d , Ik-Hyun Cho e , Seung-Yeol Nah a,∗ a
Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine Konkuk University, Seoul 143-701, South Korea Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University, Wonju 220-702, South Korea c Center for Neuroscience, Korea Institute of Science and Technology, Seoul 139-791,South Korea d Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chuncheon 200-701, South Korea e Department of Convergence Medical Science, College of Oriental Korean Medicine, Kyung Hee University, Seoul 130-701, South Korea b
h i g h l i g h t s • • • •
G␣q /11 protein-coupled receptor-mediated [Ca2+ ]i transients of astrocytes are coupled to release of gliotransmitters. Gintonin treatment to astrocytes activates [Ca2+ ]i transients pathway via LPA receptor activation. Gintonin-mediated [Ca2+ ]i transients are coupled to release of ATP and glutamate. Gintonin regulates gliotransmitter release via LPA receptor activation in primary astrocytes.
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Article history: Received 15 May 2015 Received in revised form 6 July 2015 Accepted 7 July 2015 Available online 17 July 2015 Keywords: Ginseng Gintonin Astrocytes Lysophosphatidic acid receptor [Ca2+ ]i transients Gliotransmitter release
a b s t r a c t Lysophosphatidic acid (LPA) is a simple and minor phospholipid, but serves as a lipid-derived neurotransmitter via activation of G protein-coupled LPA receptors. Astrocytes abundantly express LPA receptors and contain gliotransmitters that modulate astrocyte-neuron interactions. Gintonin is a novel ginseng-derived G protein-coupled LPA receptor ligand. Gintonin induces [Ca2+ ]i transients in neuronal and non-neuronal cells via activation of LPA receptors, which regulate calcium-dependent ion channels and receptors. A line of evidence shows that neurotransmitter-mediated [Ca2+ ]i elevations in astrocytes are coupled with gliotransmitter release. However, little is known about whether gintonin-mediated [Ca2+ ]i transients are coupled to gliotransmitter release in astrocytes. In the present study, we examined the effects of gintonin on adenosine triphosphate (ATP) and glutamate release in mouse cortical primary astrocytes. Application of gintonin to astrocytes induced [Ca2+ ]i transients in a concentrationdependent and reversible manner. However, ginsenosides, other active ingredients in ginseng, had no effect on [Ca2+ ]i transients. The induction of gintonin-mediated [Ca2+ ]i transients was attenuated/blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca2+ chelator. Gintonin treatment on astrocytes increased ATP and glutamate release in a concentration- and time-dependent manner. BAPTA and Ki16425 attenuated gintonin-mediated ATP and glutamate release in astrocytes. The present study shows that gintoninmediated [Ca2+ ]i transients are coupled to gliotransmitter release via LPA receptor activation. Finally, gintonin-mediated [Ca2+ ]i transients and gliotransmitter release from astrocytes via LPA receptor activation might explain one mechanism of gintonin-mediated neuromodulation in the central nervous system. © 2015 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: GT, gintonin; LPA, lysophosphatidic acid; [Ca2+]i, intracellular calcium concentration; ATP, adenosine triphosphate; GLP, ginseng major latex-like protein; FBS, fetal bovine serum; HEPES, N-2-hydroxyethyl-piperazine-N -2-ethanesulfonic acid. ∗ Corresponding author. Fax: +82 2 450 3037. E-mail address: [email protected] (S.-Y. Nah). http://dx.doi.org/10.1016/j.neulet.2015.07.012 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction Lysophosphatidic acid (LPA) is a simple phospholipid, but the actions of LPA are diverse with a myriad effects on animal nervous systems [1]. Endogenous LPAs are involved in various events through lipid-derived growth factor-like behaviors. The primary cellular actions of LPA are to elicit transient increases in the intracellular calcium concentration [Ca2+ ]i and induce cell proliferation, differentiation, morphological changes, migration, and survival through the activation of LPA receptors. The activations of G protein-coupled LPA receptors are coupled to diverse activities such as brain development, angiogenesis, embryo implantation, spermatogenesis, and wound healing. On the other hand, astrocytes closely modulate neuronal activities by forming a tripartite synapse and releasing gliotransmitters in addition to metabolic support and protection of neurons in the brain [2,3]. Recent studies have shown that astrocytes express LPA receptors abundantly [4,5], although the role of LPA receptors in astrocyte-neuron interactions is not well understood. There are reports that LPA treatment to astrocytes induces neuronal differentiation and axonal outgrowth of neurons, supporting evidence of LPA-mediated release of growth factors via LPA receptor signaling pathways indirectly affecting neuronal activity [6]. However, relatively little is known about LPA effects on gliotransmitter release in astrocytes. Ginseng is a traditional herbal medicine with diverse biological effects. Recent study shows ginseng contains a novel ginsengderived LPA receptor ligand, gintonin [7]. Gintonin consists of LPAs such as LPA C18:2 , LPA C18:1 , and LPA C16:0 , as well as ginseng major latex-like protein (GLP) 151 and ginseng ribonuclease-like storage protein [7]. In particular, gintonin induced [Ca2+ ]i transients with a low EC50 values in cells expressing LPA1, LPA2, LPA3, or LPA5 receptor subtypes, indicating that gintonin is a novel high affinity ligand to these LPA receptors [7]. The primary action of gintonin is to induce [Ca2+ ]i transients through activation of G protein-coupled LPA receptor signaling pathways [7]. In addition, gintonin-mediated LPA receptor activation, and the following [Ca2+ ]i transients, are linked to the regulation of intracellular Ca2+ -dependent ion channels and receptors [8]. Further studies showed that gintonin mediated-[Ca2+ ]i transients are also coupled to interneuron communication [9]. Although previous reports raise the possibility that gintonin-mediated [Ca2+ ]i transients via LPA receptors play a key role in intra- and inter-neuronal communication, it has not been demonstrated that gintonin-mediated activation of G protein-coupled LPA receptors in primary astrocytes is coupled to the regulation of gliotransmitter release. In the present study, we examined the effects of gintonin on [Ca2+ ]i transients and the release of adenosine triphosphate (ATP) and glutamate in cultured mouse cortical astrocytes. We report that gintonin induces [Ca2+ ]i transients via membrane signaling transduction pathways of LPA receptors, and that gintonin-mediated [Ca2+ ]i transients are coupled to the stimulation of ATP and glutamate release. Additionally, we discuss the pharmacological roles of gintonin-mediated neuromodulation via the release of gliotransmitters from astrocytes.
sn-Glycero-3-phosphate, 857130P) was purchased from Avanti Polar Lipids, Inc. (Alabama, USA). All other reagents, including ATP and N-2-hydroxyethyl-piperazine-N -2-ethanesulfonic acid (HEPES), were purchased from Sigma–Aldrich (St Louis, MO, USA). 2.2. Mouse cortical primary astrocytes culture Primary astrocyte cultures were prepared from the cerebral cortices of postnatal day 1 ICR (CD-1® ) mice according to the method of Shano et al. [5]. Briefly, primary astrocyte cultures were prepared from the cerebral cortices of 1-day-old neonatal ICR mice. Cells were seeded in culture plates coated with poly-l-lysine hydrobromide (100 g/mL; Sigma–Aldrich) and grown in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 g/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 ◦ C. At the time of primary cell confluence (5–7 days), cells were harvested with a 0.05% trypsin/EDTA solution (Life technologies, Carlsbad, CA), seeded in culture plates previously treated with poly-l-lysine hydrobromide (100 g/mL; Sigma–Aldrich), and grown in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 g/mL streptomycin for further experiments. 2.3. Measurement of intracellular calcium concentration The free [Ca2+ ]i was measured by dual excitation spectrofluorometric analysis of cell suspensions loaded with Fura-2 AM, as previously described [7]. Briefly, astrocytes were harvested with a trypsin/EDTA solution and re-suspended in a HBS. The cells were incubated with Fura-2 AM (final concentration 2.5 M) in HBS at 37 ◦ C for 40 min. Extracellular Fura-2 AM was removed by centrifugation. Each aliquot of 3 × 106 cells was loaded into a cuvette and free calcium mobilization was measured using a RF-5301PC spectrofluorophotometer and Supercap software (Ex: 340 nm and 380 nm; Em: 520 nm) (Shimadzu, Tokyo, Japan). 2.4. Measurement of ATP and glutamate release For ATP assay experiments, cells were seeded on 24-well plates at a density of 4 × 104 cells per well. For measurement of ATP release, astrocytes were treated with DMEM in the presence or absence of gintonin at 37 ◦ C. For glutamate assay experiments, cells were seeded on 6-well plates at a density of 2 × 105 cells per well. To determine glutamate release, astrocytes were treated with HEPES-buffered saline solution (HBS: 120 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.5 mM CaCl2 , 10 mM glucose, 25 mM HEPES, pH 7.4) in the presence or absence of gintonin at 37 ◦ C. The supernatants from control and treated astrocytes were collected, centrifuged at 12,000 rpm for 5 min, and the concentrations of ATP and glutamate in the supernatant were determined according to the manufacturer’s instruction—ATP Bioluminescent Assay Kit (Sigma–Aldrich) and Glutamate Assay Kit, fluorometric (Abcam, Cambridge, MA), respectively. 2.5. Data analysis
2. Materials and methods 2.1. Materials Gintonin, devoid of ginseng saponins, was prepared from Panax ginseng according to previously described methods [10]. Gintonin was dissolved in deionized water and diluted with medium before use. Dulbecco’s Minimum Essential Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen (Camarillo, CA, USA). LPA (1-oleoyl-2-hydroxy-
To obtain concentration-response curves for the effects of gintonin on [Ca2+ ]i transients, the peak increase of [Ca2+ ]i transient amplitudes at different concentrations of gintonin were plotted. Origin software (OriginLab, Northampton, MA, USA) was used to fit the data to the Hill equation: y/ymax = [A]nH /([A]nH + [EC50 ]nH ), where y is the peak at a given concentration of gintonin, ymax is the maximal peak in the absence of gintonin, EC50 is the concentration of gintonin producing a half-maximal effect, [A] is the concentration of gintonin, and nH is the Hill coefficient. All values are presented as the mean ± the standard error of the mean (S.E.M).
H. Kim et al. / Neuroscience Letters 603 (2015) 19–24
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Fig. 1. Effects of gintonin or ginsenosides on intracellular calcium transients in primary astrocytes. (A) A representative trace obtained after gintonin (GT, 1 g/mL), ginsenosides (30 M, each), or LPA (LPA C18:1 , 1 M) treatment in primary astrocytes. (B) Histograms representing net increases of gintonin-mediated [Ca2+ ]i transients calculated from traces obtained in the presence of ginsenosides, gintonin (GT), or LPA. (C) A representative trace obtained after gintonin treatment (0.01–1 g/mL) showing induction of a [Ca2+ ]i transient. (D) Gintonin treatment (0.01–1 g/mL) induces a [Ca2+ ]i transient. Concentration-response relationship curve for gintonin-induced [Ca2+ ]i transients in astrocytes. Each point represents the mean ± S.E.M. (n = 3–4).
3. Results 3.1. Effect of gintonin, ginsenosides, and LPA on [Ca2+ ]i transients in cultured mouse cortical primary astrocytes Since it is known that cultured cortical astrocytes express LPA15 receptor subtypes [5], we first examined the effects of gintonin, ginsenosides, and LPA on [Ca2+ ]i transients in cultured mouse cortical astrocytes. As shown in Fig. 1A, gintonin and LPA C18:1 treatment induced a transient rise of [Ca2+ ]i in astrocytes in a reversible manner. Gintonin- and LPA-induced [Ca2+ ]i transients occurred without a detectable lag, reached peak values within a few seconds, and then gradually decreased. Ginsenosides, such as ginsenoside Rg1 , Rb1 , and Rg3 , had no effect on [Ca2+ ]i transients in cultured mouse cortical astrocytes. Gintonin also induced [Ca2+ ]i transients in a concentration-dependent manner (Fig. 1C). The EC50 was 0.03 ± 0.003 g/mL (Fig. 1D). These results indicate that gintonin, but not ginsenosides, are the main active ingredient in ginseng inducing intracellular Ca2+ mobilization in astrocytes, and that gintonin, but not ginsenosides, use membrane signaling transduction pathways for [Ca2+ ]i transients. 3.2. Signal transduction pathway of gintonin-mediated [Ca2+ ]i transients in primary astrocytes We first examined the effects of gintonin on [Ca2+ ]i transients in the absence or presence of the LPA1/3 receptor antagonist
Ki16425. As shown in Fig. 2A and B, the presence of Ki16425 significantly attenuated gintonin-mediated [Ca2+ ]i transients. However, the phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca2+ chelator (BAPTAAM) all blocked gintonin-mediated [Ca2+ ]i transients in astrocytes (Fig. 2C and D). These results show that gintonin elicits the release of Ca2+ from intracellular stores to induce [Ca2+ ]i transients in astrocytes via activation of the LPA receptor-phospholipase Cintracellular IP3 receptor signaling transduction pathway. 3.3. Effects of gintonin on gliotransmitter release in primary astrocytes Since astrocytes are the main glial cells containing gliotransmitters, which they release in response to [Ca2+ ]i transients from neurotransmitters or hormones [11], we next examined the effects of gintonin on gliotransmitter release in astrocytes. As shown in Fig. 3A, gintonin treatment for 5 min on astrocytes induced ATP release in a concentration-dependent manner in the range of 0.1–10 g/mL. LPA C18:1 (1 M) treatment also induced ATP release (Fig. 3A). The EC50 for ATP release by gintonin was 0.31 ± 0.06 g/mL (Fig. 3A). The intracellular calcium chelator BAPTA-AM and LPA1/3 receptor antagonist Ki16425 blocked gintonin-mediated ATP release (Fig. 3A). In addition, we also examined the effect of gintonin on glutamate release. In time-dependent experiments, gintonin-mediated ATP release was maximal at 5 min after gintonin treatment and decreased to near basal levels after
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Fig. 2. Effect of signal transduction pathway inhibitors on gintonin-induced [Ca2+ ]i transients in primary astrocytes. (A and C) Representative traces of gintonin-mediated [Ca2+ ]i transients in the absence or presence of various antagonists or blockers. Arrows indicate application of gintonin (1 g/mL). An LPA1/3 receptor antagonist (Ki16425, 10 M), PLC inhibitor (U73122, 5 M), IP3 receptor antagonist (2-APB, 100 M), or intracellular Ca2+ chelator (BAPTA-AM, 50 M) were added before gintonin application. (B and D) Histograms representing net increases of gintonin-mediated [Ca2+ ]i transients calculated from traces obtained in the absence or presence of various antagonists or blockers. *p < 0.05, compared to gintonin only treatment. Data are means ± S.E.M. (n = 3–4).
30 min (Fig. 3B). As shown in Fig. 4A, gintonin also increased glutamate release in a concentration-dependent manner two-fold at 3 g/mL after gintonin treatment compared to control saline treatment, respectively. The EC50 for glutamate release by gintonin was 0.18 ± 3.43 g/mL (Fig. 4A). LPA C18:1 (1 M) treatment also induced glutamate release (Fig. 4A). The intracellular calcium chelator BAPTA-AM and LPA1/3 receptor antagonist Ki16425 also blocked gintonin-mediated glutamate release (Fig. 3A). In timedependent experiments, gintonin-mediated glutamate release was maximal 15 min after gintonin treatment and showed a decreasing tendency after 30 min (Fig. 4B). These results indicate gintonin induces ATP and glutamate release in primary astrocytes in an intracellular calcium- and LPA receptor-dependent manner. 4. Discussion Although astrocytes are the most numerous cell type in the mammalian brain cortex including humans [12], they have been regarded as having a simple structural function in the central nervous system. However, accumulating evidence shows that astrocytes play many important roles in brain development, physiology, and pathophysiology [13]. For playing their role in metabolic and homeostatic functions of the nervous system, astrocytes contain gliotransmitters, which are used for intercellular communication between astrocytes and neurons [11]. In previous reports, we have shown gintonin enhanced synaptic transmission by stimulating the release of glutamate in hippocampal slices [9]. Gintonin
also enhances NMDA receptor channel currents [14]. In addition, we demonstrated that gintonin-mediated [Ca2+ ]i transients in PC12 cells are coupled to dopamine release [15]. However, the relationship between gintonin and astrocytes, and whether gintonin can regulate gliotransmitter release in astrocytes, was previously unknown, even though primary astrocytes abundantly express LPA receptor subtypes [4,5]. First, we showed that gintonin, but not ginsenosides, induced [Ca2+ ]i transients in astrocytes. This demonstrates gintonin, not ginsenosides, is the main active ingredient of ginseng inducing transient intracellular calcium mobilization in primary astrocytes. Secondly, gintonin-induced [Ca2+ ]i transients are achieved through an LPA receptor-mediated signal transduction pathway in primary astrocytes. Third, gintonin-induced [Ca2+ ]i transients are coupled to gliotransmitter release. Thus, this is the first report showing gintonin regulates gliotransmitter release (ATP and glutamate) in primary astrocytes. ATP and glutamate are the prototypic gliotransmitters in astrocytes and Ca2+ signaling plays a key role in the release of gliotransmitters [11]. Astrocytes release ATP and glutamate when they are stimulated with neurotransmitters, such as G␣q/11 protein-coupled receptor ligands linked to intracellular calcium elevation [16]. Released gliotransmitters interact with receptors located on presynaptic or postsynaptic neurons to form tripartite synapse, and can regulate synaptic transmission in the central nervous system [3]. Although LPA is a neurolipid known to have mitogen-like growth factor effects on cell proliferation, migration, and anti-apoptosis
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Fig. 3. Effect of gintonin on ATP release in primary astrocytes. (A) ATP release from primary astrocytes by treatment with gintonin and effect of various concentrations of gintonin (GT) on ATP release in astrocytes. ATP release was also examined in astrocytes treated with gintonin or LPA (1 M) at the indicated concentrations for 5 min, or with 1 g/mL gintonin in the presence of BAPTA-AM (BA, 10 M, for 30 min) or Ki16425 (10 M). Data are means ± S.E.M. (n = 3–4). (B) Time course of gintoninmediated ATP release. ATP concentrations in each sample were determined by an ATP assay kit, as described in the Materials and Methods. * p < 0.05, compared to control (Con), # p < 0.05, compared to GT (1 g/mL) alone treatment.
in the nervous system, recent studies showed LPA also plays a role as a neurotransmitter, or regulator of neurotransmitter release. For example, Dubin et al. [17] showed that LPA exhibits neurotransmitter-like behavior by increasing both Cl− and nonselective cation currents in early embryonic cortical neuroblastic cells. In addition, Trimbuch et al. [18] showed that LPA is released from astrocytes and that LPA released in the mammalian hippocampus interacts with LPA receptors at presynaptic sites to induce hippocampal excitation by stimulating glutamate release. The present study provides additional evidence that an exogenous LPA receptor ligand, gintonin, via LPA receptors, can induce gliotransmitter release from astrocytes. Since it is known that ATP, and glutamate regulate synaptic transmission and brain plasticity [19], gintonin-mediated release of ATP, and glutamate raises the possibility that exogenous LPA could modulate interactions between astrocytes and neurons through gliotransmission. Gintonin is a unique form of herbal-medicine LPA, as it consists of LPA-ginseng protein complexes. The recent elucidation of the three-dimensional structure of GLP151, a protein component of gintonin, revealed how it binds to LPA. The phosphate head group of LPA binds to imidazole ring histidine residues at the C-terminal of GLP151 with hydrogen bonds and acyl-chains of LPA bind to another portion of GLP151 [20]. In addition, they
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Fig. 4. Effect of gintonin on glutamate release in primary astrocytes. (A) Glutamate release from primary astrocytes by treatment with gintonin or LPA (1 M). Effect of various concentration of gintonin (GT) on glutamate release in astrocytes. Glutamate release was also examined in astrocytes treated with gintonin at the indicated concentrations for 5 min, or with 1 g/mL gintonin in the presence of BAPTA-AM (BA, 10 M, for 30 min), or Ki16425 (10 M). Data are means ± S.E.M. (n = 3–4). (B) Time course of gintonin-mediated glutamate release. Glutamate concentrations in each sample were determined by a glutamate assay kit, as described in the Materials and Methods. * p < 0.05, compared to control (Con), # p < 0.05, compared to GT (1 g/mL) alone treatment.
showed GLP151 protein could function as a carrier/transporter of LPA and deliver it to LPA receptors [20]. The present study provides additional information showing that gintonin might be a main regulator of ginseng-induced gliotransmitter release by acting on astrocytes. Since gliotransmitters play an important role in learning and memory through interactions with neurons [21], the present study suggests that gintonin-mediated stimulation of gliotransmitter release, via G protein-coupled LPA receptors, might also contribute to in vivo amelioration of learning and memory deficits induced by amyloid- or in animal models of Alzheimer’s disease [22]. In conclusion, we have demonstrated that gintonin-mediated activation of LPA receptors is linked to ATP and glutamate release in cultured mouse cortical astrocytes. Finally, we suggest that gintonin-mediated gliotransmitter release might be a molecular basis for the pharmacological effects of ginseng in the nervous system.
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Acknowledgments This work was supported by the Basic Science Research Program (NRF-2014R1A1A2054538) which is funded by the Ministry of Education, Science, and Technology and by the BK21 plus project fund to S.-Y. Nah. Hyunsook Kim was supported by the KU-Research Professor Program of Konkuk University. References [1] Y.C. Yung, N.C. Stoddard, H. Mirendil, J. Chun, Lysophosphatidic acid signaling in the nervous system, Neuron 85 (2015) 669–682. [2] M.M. Halassa, T. Fellin, P.G. Haydon, The tripartite synapse: roles for gliotransmission in health and disease, Trends Mol. Med. 13 (2007) 54–63. [3] A. Araque, G. Carmignoto, P.G. Haydon, S.H. Oliet, R. Robitaille, A. Volterra, Gliotransmitters travel in time and space, Neuron 81 (2014) 728–739. [4] S. Tabuchi, K. Kume, M. Aihara, T. Shimizu, Expression of lysophosphatidic acid receptor in rat astrocytes: mitogenic effect and expression of neurotrophic genes, Neurochem. Res. 25 (2000) 573–582. [5] S. Shano, R. Moriyama, J. Chun, N. Fukushima, Lysophosphatidic acid stimulates astrocyte proliferation through LPA1, Neurochem. Int. 52 (2008) 216–220. [6] T.C. Spohr, R.S. Dezonne, S.K. Rehen, F.C. Gomes, Astrocytes treated by lysophosphatidic acid induce axonal outgrowth of cortical progenitors through extracellular matrix protein and epidermal growth factor signaling pathway, J. Neurochem. 119 (2011) 113–123. [7] S.H. Hwang, T.J. Shin, S.H. Choi, H.J. Cho, B.H. Lee, M.K. Pyo, J.H. Lee, J. Kang, H.J. Kim, C.W. Park, H.C. Shin, S.Y. Nah, Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids–protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity, Mol. Cells 33 (2012) 151–162. [8] J.H. Lee, S.H. Choi, B.H. Lee, S.H. Hwang, H.J. Kim, J. Rhee, C. Chung, S.Y. Nah, Activation of lysophosphatidic acid receptor by gintonin inhibits Kv1 2 channel activity: involvement of tyrosine kinase and receptor protein tyrosine phosphatase alpha, Neurosci. Lett. 548 (2013) 143–148. [9] H. Park, S. Kim, J. Rhee, H.J. Kim, J.S. Han, S.Y. Nah, C. Chung, Synaptic enhancement induced by gintonin via lysophosphatidic acid receptor activation in central synapses, J. Neurophysiology 113 (2015) 1493–1500. [10] M.K. Pyo, S.H. Choi, S.H. Hwang, T.J. Shin, B.H. Lee, S.M. Lee, Y. Lim, D. Kim, S.Y. Nah, Novel glycoproteins from ginseng, J. Ginseng Res. 35 (2011) 92–103.
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Research paper
Gintonin stimulates gliotransmitter release in cortical primary astrocytes Hyunsook Kim a , Byung-Hwan Lee a , Sun-Hye Choi a , Hyeon-Joong Kim a , Suk-Won Jung a , Sung-Hee Hwang b , Hyewon Rhim c , Hyung-Chun Kim d , Ik-Hyun Cho e , Seung-Yeol Nah a,∗ a
Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine Konkuk University, Seoul 143-701, South Korea Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University, Wonju 220-702, South Korea c Center for Neuroscience, Korea Institute of Science and Technology, Seoul 139-791,South Korea d Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chuncheon 200-701, South Korea e Department of Convergence Medical Science, College of Oriental Korean Medicine, Kyung Hee University, Seoul 130-701, South Korea b
h i g h l i g h t s • • • •
G␣q /11 protein-coupled receptor-mediated [Ca2+ ]i transients of astrocytes are coupled to release of gliotransmitters. Gintonin treatment to astrocytes activates [Ca2+ ]i transients pathway via LPA receptor activation. Gintonin-mediated [Ca2+ ]i transients are coupled to release of ATP and glutamate. Gintonin regulates gliotransmitter release via LPA receptor activation in primary astrocytes.
a r t i c l e
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Article history: Received 15 May 2015 Received in revised form 6 July 2015 Accepted 7 July 2015 Available online 17 July 2015 Keywords: Ginseng Gintonin Astrocytes Lysophosphatidic acid receptor [Ca2+ ]i transients Gliotransmitter release
a b s t r a c t Lysophosphatidic acid (LPA) is a simple and minor phospholipid, but serves as a lipid-derived neurotransmitter via activation of G protein-coupled LPA receptors. Astrocytes abundantly express LPA receptors and contain gliotransmitters that modulate astrocyte-neuron interactions. Gintonin is a novel ginseng-derived G protein-coupled LPA receptor ligand. Gintonin induces [Ca2+ ]i transients in neuronal and non-neuronal cells via activation of LPA receptors, which regulate calcium-dependent ion channels and receptors. A line of evidence shows that neurotransmitter-mediated [Ca2+ ]i elevations in astrocytes are coupled with gliotransmitter release. However, little is known about whether gintonin-mediated [Ca2+ ]i transients are coupled to gliotransmitter release in astrocytes. In the present study, we examined the effects of gintonin on adenosine triphosphate (ATP) and glutamate release in mouse cortical primary astrocytes. Application of gintonin to astrocytes induced [Ca2+ ]i transients in a concentrationdependent and reversible manner. However, ginsenosides, other active ingredients in ginseng, had no effect on [Ca2+ ]i transients. The induction of gintonin-mediated [Ca2+ ]i transients was attenuated/blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca2+ chelator. Gintonin treatment on astrocytes increased ATP and glutamate release in a concentration- and time-dependent manner. BAPTA and Ki16425 attenuated gintonin-mediated ATP and glutamate release in astrocytes. The present study shows that gintoninmediated [Ca2+ ]i transients are coupled to gliotransmitter release via LPA receptor activation. Finally, gintonin-mediated [Ca2+ ]i transients and gliotransmitter release from astrocytes via LPA receptor activation might explain one mechanism of gintonin-mediated neuromodulation in the central nervous system. © 2015 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: GT, gintonin; LPA, lysophosphatidic acid; [Ca2+]i, intracellular calcium concentration; ATP, adenosine triphosphate; GLP, ginseng major latex-like protein; FBS, fetal bovine serum; HEPES, N-2-hydroxyethyl-piperazine-N -2-ethanesulfonic acid. ∗ Corresponding author. Fax: +82 2 450 3037. E-mail address: [email protected] (S.-Y. Nah). http://dx.doi.org/10.1016/j.neulet.2015.07.012 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction Lysophosphatidic acid (LPA) is a simple phospholipid, but the actions of LPA are diverse with a myriad effects on animal nervous systems [1]. Endogenous LPAs are involved in various events through lipid-derived growth factor-like behaviors. The primary cellular actions of LPA are to elicit transient increases in the intracellular calcium concentration [Ca2+ ]i and induce cell proliferation, differentiation, morphological changes, migration, and survival through the activation of LPA receptors. The activations of G protein-coupled LPA receptors are coupled to diverse activities such as brain development, angiogenesis, embryo implantation, spermatogenesis, and wound healing. On the other hand, astrocytes closely modulate neuronal activities by forming a tripartite synapse and releasing gliotransmitters in addition to metabolic support and protection of neurons in the brain [2,3]. Recent studies have shown that astrocytes express LPA receptors abundantly [4,5], although the role of LPA receptors in astrocyte-neuron interactions is not well understood. There are reports that LPA treatment to astrocytes induces neuronal differentiation and axonal outgrowth of neurons, supporting evidence of LPA-mediated release of growth factors via LPA receptor signaling pathways indirectly affecting neuronal activity [6]. However, relatively little is known about LPA effects on gliotransmitter release in astrocytes. Ginseng is a traditional herbal medicine with diverse biological effects. Recent study shows ginseng contains a novel ginsengderived LPA receptor ligand, gintonin [7]. Gintonin consists of LPAs such as LPA C18:2 , LPA C18:1 , and LPA C16:0 , as well as ginseng major latex-like protein (GLP) 151 and ginseng ribonuclease-like storage protein [7]. In particular, gintonin induced [Ca2+ ]i transients with a low EC50 values in cells expressing LPA1, LPA2, LPA3, or LPA5 receptor subtypes, indicating that gintonin is a novel high affinity ligand to these LPA receptors [7]. The primary action of gintonin is to induce [Ca2+ ]i transients through activation of G protein-coupled LPA receptor signaling pathways [7]. In addition, gintonin-mediated LPA receptor activation, and the following [Ca2+ ]i transients, are linked to the regulation of intracellular Ca2+ -dependent ion channels and receptors [8]. Further studies showed that gintonin mediated-[Ca2+ ]i transients are also coupled to interneuron communication [9]. Although previous reports raise the possibility that gintonin-mediated [Ca2+ ]i transients via LPA receptors play a key role in intra- and inter-neuronal communication, it has not been demonstrated that gintonin-mediated activation of G protein-coupled LPA receptors in primary astrocytes is coupled to the regulation of gliotransmitter release. In the present study, we examined the effects of gintonin on [Ca2+ ]i transients and the release of adenosine triphosphate (ATP) and glutamate in cultured mouse cortical astrocytes. We report that gintonin induces [Ca2+ ]i transients via membrane signaling transduction pathways of LPA receptors, and that gintonin-mediated [Ca2+ ]i transients are coupled to the stimulation of ATP and glutamate release. Additionally, we discuss the pharmacological roles of gintonin-mediated neuromodulation via the release of gliotransmitters from astrocytes.
sn-Glycero-3-phosphate, 857130P) was purchased from Avanti Polar Lipids, Inc. (Alabama, USA). All other reagents, including ATP and N-2-hydroxyethyl-piperazine-N -2-ethanesulfonic acid (HEPES), were purchased from Sigma–Aldrich (St Louis, MO, USA). 2.2. Mouse cortical primary astrocytes culture Primary astrocyte cultures were prepared from the cerebral cortices of postnatal day 1 ICR (CD-1® ) mice according to the method of Shano et al. [5]. Briefly, primary astrocyte cultures were prepared from the cerebral cortices of 1-day-old neonatal ICR mice. Cells were seeded in culture plates coated with poly-l-lysine hydrobromide (100 g/mL; Sigma–Aldrich) and grown in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 g/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 ◦ C. At the time of primary cell confluence (5–7 days), cells were harvested with a 0.05% trypsin/EDTA solution (Life technologies, Carlsbad, CA), seeded in culture plates previously treated with poly-l-lysine hydrobromide (100 g/mL; Sigma–Aldrich), and grown in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 g/mL streptomycin for further experiments. 2.3. Measurement of intracellular calcium concentration The free [Ca2+ ]i was measured by dual excitation spectrofluorometric analysis of cell suspensions loaded with Fura-2 AM, as previously described [7]. Briefly, astrocytes were harvested with a trypsin/EDTA solution and re-suspended in a HBS. The cells were incubated with Fura-2 AM (final concentration 2.5 M) in HBS at 37 ◦ C for 40 min. Extracellular Fura-2 AM was removed by centrifugation. Each aliquot of 3 × 106 cells was loaded into a cuvette and free calcium mobilization was measured using a RF-5301PC spectrofluorophotometer and Supercap software (Ex: 340 nm and 380 nm; Em: 520 nm) (Shimadzu, Tokyo, Japan). 2.4. Measurement of ATP and glutamate release For ATP assay experiments, cells were seeded on 24-well plates at a density of 4 × 104 cells per well. For measurement of ATP release, astrocytes were treated with DMEM in the presence or absence of gintonin at 37 ◦ C. For glutamate assay experiments, cells were seeded on 6-well plates at a density of 2 × 105 cells per well. To determine glutamate release, astrocytes were treated with HEPES-buffered saline solution (HBS: 120 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.5 mM CaCl2 , 10 mM glucose, 25 mM HEPES, pH 7.4) in the presence or absence of gintonin at 37 ◦ C. The supernatants from control and treated astrocytes were collected, centrifuged at 12,000 rpm for 5 min, and the concentrations of ATP and glutamate in the supernatant were determined according to the manufacturer’s instruction—ATP Bioluminescent Assay Kit (Sigma–Aldrich) and Glutamate Assay Kit, fluorometric (Abcam, Cambridge, MA), respectively. 2.5. Data analysis
2. Materials and methods 2.1. Materials Gintonin, devoid of ginseng saponins, was prepared from Panax ginseng according to previously described methods [10]. Gintonin was dissolved in deionized water and diluted with medium before use. Dulbecco’s Minimum Essential Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen (Camarillo, CA, USA). LPA (1-oleoyl-2-hydroxy-
To obtain concentration-response curves for the effects of gintonin on [Ca2+ ]i transients, the peak increase of [Ca2+ ]i transient amplitudes at different concentrations of gintonin were plotted. Origin software (OriginLab, Northampton, MA, USA) was used to fit the data to the Hill equation: y/ymax = [A]nH /([A]nH + [EC50 ]nH ), where y is the peak at a given concentration of gintonin, ymax is the maximal peak in the absence of gintonin, EC50 is the concentration of gintonin producing a half-maximal effect, [A] is the concentration of gintonin, and nH is the Hill coefficient. All values are presented as the mean ± the standard error of the mean (S.E.M).
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Fig. 1. Effects of gintonin or ginsenosides on intracellular calcium transients in primary astrocytes. (A) A representative trace obtained after gintonin (GT, 1 g/mL), ginsenosides (30 M, each), or LPA (LPA C18:1 , 1 M) treatment in primary astrocytes. (B) Histograms representing net increases of gintonin-mediated [Ca2+ ]i transients calculated from traces obtained in the presence of ginsenosides, gintonin (GT), or LPA. (C) A representative trace obtained after gintonin treatment (0.01–1 g/mL) showing induction of a [Ca2+ ]i transient. (D) Gintonin treatment (0.01–1 g/mL) induces a [Ca2+ ]i transient. Concentration-response relationship curve for gintonin-induced [Ca2+ ]i transients in astrocytes. Each point represents the mean ± S.E.M. (n = 3–4).
3. Results 3.1. Effect of gintonin, ginsenosides, and LPA on [Ca2+ ]i transients in cultured mouse cortical primary astrocytes Since it is known that cultured cortical astrocytes express LPA15 receptor subtypes [5], we first examined the effects of gintonin, ginsenosides, and LPA on [Ca2+ ]i transients in cultured mouse cortical astrocytes. As shown in Fig. 1A, gintonin and LPA C18:1 treatment induced a transient rise of [Ca2+ ]i in astrocytes in a reversible manner. Gintonin- and LPA-induced [Ca2+ ]i transients occurred without a detectable lag, reached peak values within a few seconds, and then gradually decreased. Ginsenosides, such as ginsenoside Rg1 , Rb1 , and Rg3 , had no effect on [Ca2+ ]i transients in cultured mouse cortical astrocytes. Gintonin also induced [Ca2+ ]i transients in a concentration-dependent manner (Fig. 1C). The EC50 was 0.03 ± 0.003 g/mL (Fig. 1D). These results indicate that gintonin, but not ginsenosides, are the main active ingredient in ginseng inducing intracellular Ca2+ mobilization in astrocytes, and that gintonin, but not ginsenosides, use membrane signaling transduction pathways for [Ca2+ ]i transients. 3.2. Signal transduction pathway of gintonin-mediated [Ca2+ ]i transients in primary astrocytes We first examined the effects of gintonin on [Ca2+ ]i transients in the absence or presence of the LPA1/3 receptor antagonist
Ki16425. As shown in Fig. 2A and B, the presence of Ki16425 significantly attenuated gintonin-mediated [Ca2+ ]i transients. However, the phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca2+ chelator (BAPTAAM) all blocked gintonin-mediated [Ca2+ ]i transients in astrocytes (Fig. 2C and D). These results show that gintonin elicits the release of Ca2+ from intracellular stores to induce [Ca2+ ]i transients in astrocytes via activation of the LPA receptor-phospholipase Cintracellular IP3 receptor signaling transduction pathway. 3.3. Effects of gintonin on gliotransmitter release in primary astrocytes Since astrocytes are the main glial cells containing gliotransmitters, which they release in response to [Ca2+ ]i transients from neurotransmitters or hormones [11], we next examined the effects of gintonin on gliotransmitter release in astrocytes. As shown in Fig. 3A, gintonin treatment for 5 min on astrocytes induced ATP release in a concentration-dependent manner in the range of 0.1–10 g/mL. LPA C18:1 (1 M) treatment also induced ATP release (Fig. 3A). The EC50 for ATP release by gintonin was 0.31 ± 0.06 g/mL (Fig. 3A). The intracellular calcium chelator BAPTA-AM and LPA1/3 receptor antagonist Ki16425 blocked gintonin-mediated ATP release (Fig. 3A). In addition, we also examined the effect of gintonin on glutamate release. In time-dependent experiments, gintonin-mediated ATP release was maximal at 5 min after gintonin treatment and decreased to near basal levels after
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Fig. 2. Effect of signal transduction pathway inhibitors on gintonin-induced [Ca2+ ]i transients in primary astrocytes. (A and C) Representative traces of gintonin-mediated [Ca2+ ]i transients in the absence or presence of various antagonists or blockers. Arrows indicate application of gintonin (1 g/mL). An LPA1/3 receptor antagonist (Ki16425, 10 M), PLC inhibitor (U73122, 5 M), IP3 receptor antagonist (2-APB, 100 M), or intracellular Ca2+ chelator (BAPTA-AM, 50 M) were added before gintonin application. (B and D) Histograms representing net increases of gintonin-mediated [Ca2+ ]i transients calculated from traces obtained in the absence or presence of various antagonists or blockers. *p < 0.05, compared to gintonin only treatment. Data are means ± S.E.M. (n = 3–4).
30 min (Fig. 3B). As shown in Fig. 4A, gintonin also increased glutamate release in a concentration-dependent manner two-fold at 3 g/mL after gintonin treatment compared to control saline treatment, respectively. The EC50 for glutamate release by gintonin was 0.18 ± 3.43 g/mL (Fig. 4A). LPA C18:1 (1 M) treatment also induced glutamate release (Fig. 4A). The intracellular calcium chelator BAPTA-AM and LPA1/3 receptor antagonist Ki16425 also blocked gintonin-mediated glutamate release (Fig. 3A). In timedependent experiments, gintonin-mediated glutamate release was maximal 15 min after gintonin treatment and showed a decreasing tendency after 30 min (Fig. 4B). These results indicate gintonin induces ATP and glutamate release in primary astrocytes in an intracellular calcium- and LPA receptor-dependent manner. 4. Discussion Although astrocytes are the most numerous cell type in the mammalian brain cortex including humans [12], they have been regarded as having a simple structural function in the central nervous system. However, accumulating evidence shows that astrocytes play many important roles in brain development, physiology, and pathophysiology [13]. For playing their role in metabolic and homeostatic functions of the nervous system, astrocytes contain gliotransmitters, which are used for intercellular communication between astrocytes and neurons [11]. In previous reports, we have shown gintonin enhanced synaptic transmission by stimulating the release of glutamate in hippocampal slices [9]. Gintonin
also enhances NMDA receptor channel currents [14]. In addition, we demonstrated that gintonin-mediated [Ca2+ ]i transients in PC12 cells are coupled to dopamine release [15]. However, the relationship between gintonin and astrocytes, and whether gintonin can regulate gliotransmitter release in astrocytes, was previously unknown, even though primary astrocytes abundantly express LPA receptor subtypes [4,5]. First, we showed that gintonin, but not ginsenosides, induced [Ca2+ ]i transients in astrocytes. This demonstrates gintonin, not ginsenosides, is the main active ingredient of ginseng inducing transient intracellular calcium mobilization in primary astrocytes. Secondly, gintonin-induced [Ca2+ ]i transients are achieved through an LPA receptor-mediated signal transduction pathway in primary astrocytes. Third, gintonin-induced [Ca2+ ]i transients are coupled to gliotransmitter release. Thus, this is the first report showing gintonin regulates gliotransmitter release (ATP and glutamate) in primary astrocytes. ATP and glutamate are the prototypic gliotransmitters in astrocytes and Ca2+ signaling plays a key role in the release of gliotransmitters [11]. Astrocytes release ATP and glutamate when they are stimulated with neurotransmitters, such as G␣q/11 protein-coupled receptor ligands linked to intracellular calcium elevation [16]. Released gliotransmitters interact with receptors located on presynaptic or postsynaptic neurons to form tripartite synapse, and can regulate synaptic transmission in the central nervous system [3]. Although LPA is a neurolipid known to have mitogen-like growth factor effects on cell proliferation, migration, and anti-apoptosis
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Fig. 3. Effect of gintonin on ATP release in primary astrocytes. (A) ATP release from primary astrocytes by treatment with gintonin and effect of various concentrations of gintonin (GT) on ATP release in astrocytes. ATP release was also examined in astrocytes treated with gintonin or LPA (1 M) at the indicated concentrations for 5 min, or with 1 g/mL gintonin in the presence of BAPTA-AM (BA, 10 M, for 30 min) or Ki16425 (10 M). Data are means ± S.E.M. (n = 3–4). (B) Time course of gintoninmediated ATP release. ATP concentrations in each sample were determined by an ATP assay kit, as described in the Materials and Methods. * p < 0.05, compared to control (Con), # p < 0.05, compared to GT (1 g/mL) alone treatment.
in the nervous system, recent studies showed LPA also plays a role as a neurotransmitter, or regulator of neurotransmitter release. For example, Dubin et al. [17] showed that LPA exhibits neurotransmitter-like behavior by increasing both Cl− and nonselective cation currents in early embryonic cortical neuroblastic cells. In addition, Trimbuch et al. [18] showed that LPA is released from astrocytes and that LPA released in the mammalian hippocampus interacts with LPA receptors at presynaptic sites to induce hippocampal excitation by stimulating glutamate release. The present study provides additional evidence that an exogenous LPA receptor ligand, gintonin, via LPA receptors, can induce gliotransmitter release from astrocytes. Since it is known that ATP, and glutamate regulate synaptic transmission and brain plasticity [19], gintonin-mediated release of ATP, and glutamate raises the possibility that exogenous LPA could modulate interactions between astrocytes and neurons through gliotransmission. Gintonin is a unique form of herbal-medicine LPA, as it consists of LPA-ginseng protein complexes. The recent elucidation of the three-dimensional structure of GLP151, a protein component of gintonin, revealed how it binds to LPA. The phosphate head group of LPA binds to imidazole ring histidine residues at the C-terminal of GLP151 with hydrogen bonds and acyl-chains of LPA bind to another portion of GLP151 [20]. In addition, they
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Fig. 4. Effect of gintonin on glutamate release in primary astrocytes. (A) Glutamate release from primary astrocytes by treatment with gintonin or LPA (1 M). Effect of various concentration of gintonin (GT) on glutamate release in astrocytes. Glutamate release was also examined in astrocytes treated with gintonin at the indicated concentrations for 5 min, or with 1 g/mL gintonin in the presence of BAPTA-AM (BA, 10 M, for 30 min), or Ki16425 (10 M). Data are means ± S.E.M. (n = 3–4). (B) Time course of gintonin-mediated glutamate release. Glutamate concentrations in each sample were determined by a glutamate assay kit, as described in the Materials and Methods. * p < 0.05, compared to control (Con), # p < 0.05, compared to GT (1 g/mL) alone treatment.
showed GLP151 protein could function as a carrier/transporter of LPA and deliver it to LPA receptors [20]. The present study provides additional information showing that gintonin might be a main regulator of ginseng-induced gliotransmitter release by acting on astrocytes. Since gliotransmitters play an important role in learning and memory through interactions with neurons [21], the present study suggests that gintonin-mediated stimulation of gliotransmitter release, via G protein-coupled LPA receptors, might also contribute to in vivo amelioration of learning and memory deficits induced by amyloid- or in animal models of Alzheimer’s disease [22]. In conclusion, we have demonstrated that gintonin-mediated activation of LPA receptors is linked to ATP and glutamate release in cultured mouse cortical astrocytes. Finally, we suggest that gintonin-mediated gliotransmitter release might be a molecular basis for the pharmacological effects of ginseng in the nervous system.
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Acknowledgments This work was supported by the Basic Science Research Program (NRF-2014R1A1A2054538) which is funded by the Ministry of Education, Science, and Technology and by the BK21 plus project fund to S.-Y. Nah. Hyunsook Kim was supported by the KU-Research Professor Program of Konkuk University. References [1] Y.C. Yung, N.C. Stoddard, H. Mirendil, J. Chun, Lysophosphatidic acid signaling in the nervous system, Neuron 85 (2015) 669–682. [2] M.M. Halassa, T. Fellin, P.G. Haydon, The tripartite synapse: roles for gliotransmission in health and disease, Trends Mol. Med. 13 (2007) 54–63. [3] A. Araque, G. Carmignoto, P.G. Haydon, S.H. Oliet, R. Robitaille, A. Volterra, Gliotransmitters travel in time and space, Neuron 81 (2014) 728–739. [4] S. Tabuchi, K. Kume, M. Aihara, T. Shimizu, Expression of lysophosphatidic acid receptor in rat astrocytes: mitogenic effect and expression of neurotrophic genes, Neurochem. Res. 25 (2000) 573–582. [5] S. Shano, R. Moriyama, J. Chun, N. Fukushima, Lysophosphatidic acid stimulates astrocyte proliferation through LPA1, Neurochem. Int. 52 (2008) 216–220. [6] T.C. Spohr, R.S. Dezonne, S.K. Rehen, F.C. Gomes, Astrocytes treated by lysophosphatidic acid induce axonal outgrowth of cortical progenitors through extracellular matrix protein and epidermal growth factor signaling pathway, J. Neurochem. 119 (2011) 113–123. [7] S.H. Hwang, T.J. Shin, S.H. Choi, H.J. Cho, B.H. Lee, M.K. Pyo, J.H. Lee, J. Kang, H.J. Kim, C.W. Park, H.C. Shin, S.Y. Nah, Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids–protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity, Mol. Cells 33 (2012) 151–162. [8] J.H. Lee, S.H. Choi, B.H. Lee, S.H. Hwang, H.J. Kim, J. Rhee, C. Chung, S.Y. Nah, Activation of lysophosphatidic acid receptor by gintonin inhibits Kv1 2 channel activity: involvement of tyrosine kinase and receptor protein tyrosine phosphatase alpha, Neurosci. Lett. 548 (2013) 143–148. [9] H. Park, S. Kim, J. Rhee, H.J. Kim, J.S. Han, S.Y. Nah, C. Chung, Synaptic enhancement induced by gintonin via lysophosphatidic acid receptor activation in central synapses, J. Neurophysiology 113 (2015) 1493–1500. [10] M.K. Pyo, S.H. Choi, S.H. Hwang, T.J. Shin, B.H. Lee, S.M. Lee, Y. Lim, D. Kim, S.Y. Nah, Novel glycoproteins from ginseng, J. Ginseng Res. 35 (2011) 92–103.
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