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Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, stimulates neurotransmitter release
Neuroscience Letters 584 (2015) 356–361
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Short communication
Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, stimulates neurotransmitter release Sung-Hee Hwang a,1 , Byung-Hwan Lee b,1 , Sun-Hye Choi b , Hyeon-Joong Kim b , Seok-Won Jung b , Hyun-Sook Kim b , Ho-Chul Shin c , Hyun Jin Park d , Keun Hong Park d , Myung Koo Lee d , Seung-Yeol Nah b,∗ a
Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University, Wonju 220-702, South Korea Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea c Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, South Korea d College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea b
h i g h l i g h t s • • • • •
Application of gintonin to PC12 cells induced intracellular calcium transients. Gintonin treatment in PC12 cells increased the release of dopamine. Gintonin-induced [Ca2+ ]i transients are coupled to dopamine release. Intraperitoneal administration of gintonin to mice increased serum dopamine level. Gintonin may regulate neurotransmitter release via LPA receptor activation.
a r t i c l e
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Article history: Received 29 September 2014 Received in revised form 30 October 2014 Accepted 6 November 2014 Available online 11 November 2014 Keywords: Ginseng Gintonin LPA receptor [Ca2+ ]i transient Dopamine Neurotransmitter release
a b s t r a c t Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an intracellular calcium concentration [Ca2+ ]i transient via activation of LPA receptors and regulates calcium-dependent ion channels and receptors. [Ca2+ ]i elevation by neurotransmitters or depolarization is usually coupled to neurotransmitter release in neuronal cells. Little is known about whether gintonin-mediated [Ca2+ ]i transients are also coupled to neurotransmitter release. The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and is widely used as a model for catecholamine release. In the present study, we examined the effects of gintonin on dopamine release in PC12 cells. Application of gintonin to PC12 cells induced [Ca2+ ]i transients in concentration-dependent and reversible manners. However, ginsenoside Rg3 , another active ingredient of ginseng, induced a lagged and irreversible [Ca2+ ]i increase. The induction of gintonin-mediated [Ca2+ ]i transients was attenuated or blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5triphosphate receptor antagonist, and an intracellular Ca2+ chelator. Repeated treatment with gintonin induced homologous desensitization of [Ca2+ ]i transients. Gintonin treatment in PC12 cells increased the release of dopamine in a concentration-dependent manner. Intraperitoneal administration of gintonin to mice also increased serum dopamine concentrations. The present study shows that gintonin-mediated [Ca2+ ]i transients are coupled to dopamine release via LPA receptor activation. Finally, gintonin-mediated [Ca2+ ]i transients and dopamine release via LPA receptor activation might explain one mechanism of gintonin-mediated inter-neuronal modulation in the nervous system. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: GT, gintonin; LPA receptor, lysophosphatidic acid receptor; GPCR, G protein-coupled receptor; PC12, pheochromocytoma cell 12; [Ca2+ ]i , intracellular calcium concentration. ∗ Corresponding author. Tel.: +82 2 450 4154; fax: +82 2 450 3037. E-mail address: [email protected] (S.-Y. Nah). 1 Both authors contributed equally. http://dx.doi.org/10.1016/j.neulet.2014.11.007 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Ginseng is a traditional herbal medicine that possesses a variety of physiological and pharmacological effects as a tonic [12]. Recent studies have shown that ginseng contains a novel G protein-coupled lysophosphatidic acid (LPA) receptor ligand, known as gintonin [10,13,15]. Gintonin induces a [Ca2+ ]i transient through LPA receptor activation but not S1P and other fatty
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acid receptors with high affinity in cells expressing LPA receptors endogenously or heterologously. Gintonin-mediated LPA receptor activation is also accountable for diverse downstream events, including stimulation of phospholipase C, protein kinase C (PKC), mitogen-activated protein kinases, and phosphoinositide 4-kinase (PI4 kinase) through multiple G proteins, such as G␣i/o , G␣12/13 , and G␣q/11 [2,10,13,16]. In addition, gintonin has been reported to regulate various ion channels (e.g., Ca2+ -activated Cl− , Ca2+ activated K+ , and voltage-gated Kv1.2) and receptors (e.g., NMDA and P2X1 ) through LPA receptor activation [9,10]. In addition, gintonin can induce long-term potentiation (LTP) in hippocampal slices [16]. Although, previous reports raise the possibility that gintonin-mediated [Ca2+ ]i transients and ensuing NMDA receptor activation leading to LTP induction might be closely related to the regulation of neurotransmitter release in the nervous system, it has not been demonstrated that the activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of neurotransmitter release. PC12 cells are derived from the pheochromocytoma of the rat adrenal chromaffin. These cells are widely used to study the modulation of neurotransmitter release by a variety of neurotransmitters or drugs because they possess vesicles containing catecholaminergic neurotransmitters, primarily dopamine [7,11], and the release of these catecholamines is associated with elevation of cytosolic calcium, similar to adrenal chromaffin cells [8]. For example, stimulation of PC12 cells by a cholinergic agonist or depolarization by high extracellular K+ induces a [Ca2+ ]i transient and results in the release of dopamine [8]. Currently, PC12 pheochromocytoma cells are utilized to obtain information about the role of bioactive compounds that could affect in vitro neurotransmitter release [6,20]. In the present study, we examined the effects of gintonin on a [Ca2+ ]i transients and the release of dopamine in PC12 cells. We report here that gintonin induces a [Ca2+ ]i transient via membrane signaling transduction pathways of LPA receptors, and that gintonin-mediated [Ca2+ ]i transients are coupled to the stimulation of dopamine release. Additionally, we discuss the physiological and pharmacological role of gintonin-mediated dopamine release in the nervous system.
2. Materials and methods 2.1. Materials Gintonin, devoid of ginseng saponins, was prepared from Panax ginseng according to previously described methods [15]. Gintonin was dissolved in deionized water and then diluted with medium before use. RPMI1640 medium, DMEM, fetal bovine serum (FBS), horse serum (HS), penicillin, and streptomycin were purchased from Invitrogen (Camarillo, CA, USA). Lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate, 857130P) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). All other reagents used, including dopamine and N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), were purchased from Sigma–Aldrich (St. Louis, MO, USA).
2.2. Cell culture PC12 cells, a rat pheochromocytoma cell line, were obtained from Korean Cell Line Bank (KCLB, Seoul, Korea) and were grown in RPMI 1640 supplemented with 10% heat-inactivated HS, 5% heat-inactivated FBS, 100 units/mL penicillin, and 100 g/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 ◦ C.
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2.3. Measurement of intracellular calcium concentration The intracellular free calcium concentration was measured by dual excitation spectrofluorometric analysis of cell suspensions loaded with Fura-2 AM as previously described [10]. Briefly, PC12 cells (either untreated or treated with NGF) were harvested with trypsin/EDTA solution and resuspended in HEPES-buffered saline solution (HBS: 120 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.5 mM CaCl2 , 10 mM glucose, 25 mM HEPES, and pH 7.4). The cells were incubated with Fura-2 AM (final concentration 2.5 M) in HBS at 37 ◦ C for 30 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 dopamine release PC12 cells were treated with Lock’s buffer [145 mM NaCl, 5.6 mM KCl, 12 mM MgCl2 , 22 mM CaCl2 , 10 mM glucose, and 10 mM HEPES (pH adjusted to 7.4 with NaOH)] in the presence or absence of gintonin at 37 ◦ C. The samples were centrifuged at 12,000 rpm for 5 min and the levels of released dopamine in the supernatant were determined by an HPLC system, as previously described [19]. Six-week-old male balb/c mice (Koatech Technology Corporation, Seoul, Korea) were housed under specific pathogen-free conditions. Mice were intraperitoneally administered saline solution (Sal) or gintonin (100 mg/kg). Mice were anesthetized with Zoletil 50 and Rompun, sacrificed, and blood samples were collected. Dopamine levels in serum were determined by HPLC [19]. Animal experiments were conducted in strict accordance with the recommendations in the guide for the care and use of Laboratory Animals of the National Institutes of Health. 2.5. Data analysis To obtain concentration-response curves of the effects of gintonin on [Ca2+ ]i transients, the peak increase of [Ca2+ ]i transient amplitudes at different concentrations of gintonin were plotted, and 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 ± S.E.M. The significance of differences between control and treatment values was determined using Student’s t-test. Values of p < 0.05 were considered statistically significant. 3. Results 3.1. Effect of gintonin on [Ca2+ ]i transients in PC12 pheochromocytoma cells In the present study, we first examined the effects of gintonin on [Ca2+ ]i transients in PC12 cells. As shown in Fig. 1A and B, gintonin treatment induced a transient rise of [Ca2+ ]i in PC12 cells in a reversible and concentration-dependent manner. The EC50 was 0.06 ± 0.01 g/mL. Gintonin-induced [Ca2+ ]i transients initiated without a detectable lag and reached peak values within a few seconds, and [Ca2+ ]i gradually decreased but did not return to its basal level until the end of time point (100 s). We observed that treatment of PC12 cells with LPA C18:1 also induced a [Ca2+ ]i transient, similar to gintonin (Fig. 3A and B). When we examined the effect of acetylcholine on [Ca2+ ]i transients, we found that acetylcholine application also induced a [Ca2+ ]i transient, similar to gintonin
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Fig. 1. Effects of gintonin (GT) on intracellular calcium transients in PC12 cells. (A) A representative trace obtained after gintonin treatment in PC12 cells. Gintonin treatment (0.03–1 g/mL) induces a [Ca2+ ]i transient. (B) Concentration-response relationship curve for gintonin-induced [Ca2+ ]i transients in PC12 cells. Each point represents the mean ± S.E.M. (n = 3–4). (C–F) Representative [Ca2+ ]i transient traces obtained under the indicated treatment. The arrows indicate application of gintonin (GT; gintonin 1 g/mL) or acetylcholine (ACh; acetylcholine 50 M). Gintonin shows homologous desensitization, but not heterologous desensitization with acetylcholine (ACh).
(Fig. 1D). Repeated treatment of PC12 cells with gintonin induced a remarkable attenuation of [Ca2+ ]i transients, indicating homologous desensitization (Fig. 1C). Homologous desensitization was also observed after repeated treatment with acetylcholine (Fig. 1D). When we treated cells with gintonin followed by acetylcholine, or vice versa (Fig. 1E and F), we did not observe heterologous desensitization to the two ligands. In addition, we could observe that co-treatment of acetylcholine and gintonin induced additive increases of [Ca2+ ]i transients (Fig. S1). These results indicate that the membrane signaling transduction pathway for gintonin might be distinct from that of acetylcholine.
3.2. Signal transduction pathway of gintonin-mediated [Ca2+ ]i transients in PC12 pheochromocytoma cells We 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. 2, the presence of Ki16425 significantly attenuated the gintonin-mediated [Ca2+ ]i transient, but the S1P receptor antagonist JTE-013 had almost no effect (Fig. 2A and B). However, the active phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca2+ chelator (BAPTA-AM) all blocked
gintonin-mediated [Ca2+ ]i transients in PC12 cells (Fig. 2C and D). These results show that gintonin, via activation of the LPA receptorphospholipase C-intracellular IP3 receptor signaling transduction pathway, elicits the release of Ca2+ from intracellular stores to increase [Ca2+ ]i .
3.3. Gintonin’s effects differ from those of ginsenosides on [Ca2+ ]i transient induction in PC12 cells We next compared the effect of representative ginsenosides, such as ginsenoside Rb1 , Rg1 , and Rg3 , which are ginseng glycosides known to regulate various ion channels and receptors [14], with gintonin in the induction of [Ca2+ ]i transients in PC12 cells. As shown in Fig. 3, the ginsenosides Rb1 and Rg1 had no effect on [Ca2+ ]i transients even at the highest concentration of 100 M. Interestingly, the ginsenoside Rg3 showed increased [Ca2+ ]i , however the pattern of the [Ca2+ ]i transient induced by ginsenoside Rg3 was different from that of gintonin. Unlike gintonin, ginsenoside Rg3 induced a slow and lagging rise of [Ca2+ ]i and did not show a sharp peak or return to resting levels. These results suggest that the gintonin-mediated [Ca2+ ]i transient are distinct from that of ginsenoside Rg3 .
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Fig. 2. Effect of signal transduction pathway inhibitors on gintonin-induced [Ca2+ ]i transients in PC12 cells. (A and C) Representative traces of gintonin-mediated [Ca2+ ]i transients in the absence or presence of various antagonist. The arrows indicate application of gintonin (1 g/mL). An LPA1/3 receptor antagonist (Ki16425, 10 M), S1P receptor antagonist (JTE-013, 10 M), PLC inhibitor (U73122, 5 M), IP3 receptor antagonist (2-APB, 100 M), or 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 pharmacological agents. *p < 0.05, compared to gintonin only treatment. Data are means ± S.E.M. (n = 3–4).
Fig. 3. Effects of ginsenosides and gintonin on intracellular calcium transients in PC12 cells. (A) Representative traces obtained after ginsenoside Rg3 (Rg3 , 10–100 M), gintonin (GT, 1 g/mL), or LPA (1 M) treatment of PC12 cells. (B) Histograms representing net increases of ginsenoside Rg3 -, gintonin- or LPA-mediated [Ca2+ ]i increases calculated from traces. (C) Representative traces obtained after ginsenoside Rb1 (Rb1 , 10–100 M) or gintonin (GT, 1 g/mL) treatment of PC12 cells. Inset: net increases of ginsenoside Rb1 - or gintonin-mediated [Ca2+ ]i transients. (D) Representative traces obtained after ginsenoside Rg1 (Rg1 , 10–100 M) or gintonin (GT, 1 g/mL) treatment in PC12 cells. Inset: net increases of ginsenoside Rg1 - or gintonin-mediated [Ca2+ ]i transients. *p < 0.05, compared to gintonin treatment. Data are means ± S.E.M. (n = 3–4).
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that stimulate dopamine release did not affect cell viability (Fig. S2). In addition, we further examined acute effect of gintonin (100 mg/kg) on dopamine concentrations in serum after intraperitoneal administration. As shown in Fig. 4B, gintonin increased serum dopamine concentrations by 1.6-, 2.2-, and 2.3-fold 10, 20, and 30 min after gintonin administration when compared to saline treatment, respectively, but at lower than 100 mg/kg did not show a slight but not significant effect on dopamine in serum (Fig. 3). These results indicate that gintonin induces dopamine release in vitro as well as in vivo.
4. Discussion
Fig. 4. Effect of gintonin (GT) on dopamine release in PC12 cells (A) and in mice (B). (A) Dopamine release from PC12 cells by treatment with gintonin. Dopamine release was examined in PC12 cells treated with gintonin at indicated concentrations for 10 min or with 1 g/mL gintonin in the presence of BAPTA-AM (BA, 50 M, for 30 min). Data are means ± S.E.M. (n = 3–4). (B) Effect of various concentration of gintonin (GT) on dopamine release in mice. The acute effect of the indicated concentration of gintonin (GT) on dopamine concentrations in 30 min after intraperitoneal administration in mice (n = 5). (C) The acute effect of gintonin (GT, 100 mg/kg) on dopamine concentrations in serum 10, 20, and 30 min after intraperitoneal administration in mice (n = 5). (Sal saline; gintonin, 100 mg/kg). Dopamine concentration in each sample was determined by HPLC, as described in Section 2. *p < 0.05, compared to control (Con).
3.4. Effects of gintonin on dopamine release in PC12 cells and mice Since the induction of depolarization by elevation of extracellular K+ or receptor ligands that induce a [Ca2+ ]i transient is coupled to dopamine release, we first examined the effects of gintonin on PC12 cells. As shown in Fig. 4A, gintonin treatment for 10 min in PC12 cells induced dopamine release in the range of 0.1–3 g/mL, but at more than 10 g/mL had no effect on dopamine release. The intracellular calcium chelator BAPTA-AM blocked gintoninmediated dopamine release (Fig. 4A), and gintonin concentrations
In previous reports, we have shown that gintonin increases the release of soluble amyloid precursor protein ␣ (sAPP␣), but not A. Gintonin-mediated stimulation of sAPP␣ release is dependent on [Ca2+ ]i transients via LPA receptor activation [9]. Gintonin also enhances NMDA receptor channel currents via phosphorylation of the Tyr132 residue of NMDA receptors via calcium-dependent tyrosine kinase and induces LTP in rat hippocampal slices [16]. In addition, gintonin not only attenuates A-induced learning and memory deficits, but also ameliorates Alzheimer’s disease-related memory deficits in a transgenic animal model of Alzheimer’s disease [9]. Thus, gintonin-mediated effects seen in vitro and in vivo suggest the possibility that the gintonin-mediated transient elevation of [Ca2+ ]i could not only be closely associated with the regulation of intracellular Ca2+ -dependent ion channels and receptors, but could also be coupled to the regulation of neurotransmitter release and ultimately exert intercellular effects in the nervous system. However, it was previously unknown whether gintonin could stimulate neurotransmitter release in the nervous system. Here, we showed that gintonin-mediated [Ca2+ ]i transients are coupled to the stimulation of dopamine release in PC12 cells and mice. We first showed that gintonin application induced [Ca2+ ]i transients and stimulated dopamine release through a LPA receptor-mediated signal transduction pathway in PC12 cells without affecting cell viability. Secondly, gintonin-induced [Ca2+ ]i transients showed homologous, but not heterologous, desensitization to acetylcholine. Third, in vivo intraperitoneal administration of gintonin in mice increased serum dopamine concentration. Thus, the present study shows that gintonin regulates neurotransmitter release in neuronal cells. Because gintonin-induced [Ca2+ ]i transients showed homologous desensitization, but not heterologous desensitization to acetylcholine, the possibility that the signal transduction pathway of gintonin-mediated [Ca2+ ]i transients is different from that of acetylcholine exists. Previous reports have shown that acetylcholine induces [Ca2+ ]i transients through activation of nicotinic acetylcholine receptors, which are ligand-gated ion channels [8], whereas gintonin activates the G protein-coupled LPA receptor signaling transduction pathway to induce [Ca2+ ]i transients (Fig. 2A and B) [10]. Supporting this notion is that co-treatment of acetylcholine and gintonin caused additive effects on [Ca2+ ]i transients (Fig. S1). Thus, it appears that gintonin utilizes a different signaling transduction pathway than acetylcholine. Interestingly, although, ginsenoside Rb1 and Rg1 had no effect on [Ca2+ ]i transients, we observed that ginsenoside Rg3 also caused an elevation of intracellular calcium levels. However, the pattern of ginsenoside Rg3 -induced increase in cytosolic calcium levels is a quite different from that of gintonin, since ginsenoside Rg3 -induced changes did not show the typical pattern of receptormediated [Ca2+ ]i transients. Rather, ginsenoside Rg3 showed a lagged increase in cytosolic calcium that did not reach a peak and did not return to resting levels, whereas gintonin showed typical reversible receptor-mediated [Ca2+ ]i transients. Previous reports
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have shown that ginsenosides, including Rg3 , inhibited Ca2+ , and Na+ influx induced by nicotinic acetylcholine receptors or high extracellular K+ , and attenuated catecholamine release from cultured bovine adrenal medullary cells [17,18]. These results suggest that ginsenosides inhibit catecholamine release when cells are activated or excited by other excitatory ligands or depolarization, whereas gintonin induces [Ca2+ ]i transients and catecholamine release via LPA receptor activation. Thus, gintonin-mediated regulation of [Ca2+ ]i transients and catecholamine release differs from that of ginsenosides. Future studies will be required to elucidate what the pharmacological roles of these two components are in their differential regulation of [Ca2+ ]i transients and catecholamine release. LPA is a simple phospholipid derivative that mediates a variety of cellular effects as a mitogen in most cell types and organs [3]. Recent studies have shown that there are at least six LPA receptor subtypes (LPA1–6) expressed in many cell types across different tissues, including the adrenal medulla [1,3]. Gintonin is a unique form of plant LPA, as it consists of LPA-ginseng protein complexes. The protein components of gintonin may function as a carrier and/or stabilizer of LPAs, and help LPAs act as functional high affinity ligands for G protein-coupled LPA receptors [10]. Although, LPA is a known neurolipid in the nervous system, our understanding of its role as a neurotransmitter or regulator of neurotransmitter release is still rudimentary. Recent studies have shown that LPA can evoke [Ca2+ ]i transients and exhibit neurotransmitter-like activities in embryonic cortical neuroblasts [4,5]. The present study provides additional evidence that gintonin could act as regulator of neurotransmitter release by stimulating dopamine release. Taken together with previous research, the present study provides further evidence that gintonin-mediated stimulation of neurotransmitter release in the hippocampus might be responsible for LTP induction and amelioration of learning and memory deficits induced by A, or in animal models of Alzheimer’s disease. In conclusion, we have demonstrated that LPA receptor activation by gintonin is coupled to dopamine release in PC12 cells. Finally, we suggest that gintonin-mediated neurotransmitter release via LPA receptor activation might be one of the molecular basis for the pharmacological effects of ginseng. Acknowledgments This work was supported by the Basic Science Research Program (2011-0021144) and the Priority Research Centers Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science, and Technology (2012-0006686) and by the BK21 plus project fund to S.-Y. Nah. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2014.11.007.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Short communication
Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, stimulates neurotransmitter release Sung-Hee Hwang a,1 , Byung-Hwan Lee b,1 , Sun-Hye Choi b , Hyeon-Joong Kim b , Seok-Won Jung b , Hyun-Sook Kim b , Ho-Chul Shin c , Hyun Jin Park d , Keun Hong Park d , Myung Koo Lee d , Seung-Yeol Nah b,∗ a
Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University, Wonju 220-702, South Korea Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, South Korea c Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, South Korea d College of Pharmacy and Research Center for Bioresource and Health, Chungbuk National University, Cheongju 361-763, South Korea b
h i g h l i g h t s • • • • •
Application of gintonin to PC12 cells induced intracellular calcium transients. Gintonin treatment in PC12 cells increased the release of dopamine. Gintonin-induced [Ca2+ ]i transients are coupled to dopamine release. Intraperitoneal administration of gintonin to mice increased serum dopamine level. Gintonin may regulate neurotransmitter release via LPA receptor activation.
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Article history: Received 29 September 2014 Received in revised form 30 October 2014 Accepted 6 November 2014 Available online 11 November 2014 Keywords: Ginseng Gintonin LPA receptor [Ca2+ ]i transient Dopamine Neurotransmitter release
a b s t r a c t Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an intracellular calcium concentration [Ca2+ ]i transient via activation of LPA receptors and regulates calcium-dependent ion channels and receptors. [Ca2+ ]i elevation by neurotransmitters or depolarization is usually coupled to neurotransmitter release in neuronal cells. Little is known about whether gintonin-mediated [Ca2+ ]i transients are also coupled to neurotransmitter release. The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and is widely used as a model for catecholamine release. In the present study, we examined the effects of gintonin on dopamine release in PC12 cells. Application of gintonin to PC12 cells induced [Ca2+ ]i transients in concentration-dependent and reversible manners. However, ginsenoside Rg3 , another active ingredient of ginseng, induced a lagged and irreversible [Ca2+ ]i increase. The induction of gintonin-mediated [Ca2+ ]i transients was attenuated or blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5triphosphate receptor antagonist, and an intracellular Ca2+ chelator. Repeated treatment with gintonin induced homologous desensitization of [Ca2+ ]i transients. Gintonin treatment in PC12 cells increased the release of dopamine in a concentration-dependent manner. Intraperitoneal administration of gintonin to mice also increased serum dopamine concentrations. The present study shows that gintonin-mediated [Ca2+ ]i transients are coupled to dopamine release via LPA receptor activation. Finally, gintonin-mediated [Ca2+ ]i transients and dopamine release via LPA receptor activation might explain one mechanism of gintonin-mediated inter-neuronal modulation in the nervous system. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: GT, gintonin; LPA receptor, lysophosphatidic acid receptor; GPCR, G protein-coupled receptor; PC12, pheochromocytoma cell 12; [Ca2+ ]i , intracellular calcium concentration. ∗ Corresponding author. Tel.: +82 2 450 4154; fax: +82 2 450 3037. E-mail address: [email protected] (S.-Y. Nah). 1 Both authors contributed equally. http://dx.doi.org/10.1016/j.neulet.2014.11.007 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Ginseng is a traditional herbal medicine that possesses a variety of physiological and pharmacological effects as a tonic [12]. Recent studies have shown that ginseng contains a novel G protein-coupled lysophosphatidic acid (LPA) receptor ligand, known as gintonin [10,13,15]. Gintonin induces a [Ca2+ ]i transient through LPA receptor activation but not S1P and other fatty
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acid receptors with high affinity in cells expressing LPA receptors endogenously or heterologously. Gintonin-mediated LPA receptor activation is also accountable for diverse downstream events, including stimulation of phospholipase C, protein kinase C (PKC), mitogen-activated protein kinases, and phosphoinositide 4-kinase (PI4 kinase) through multiple G proteins, such as G␣i/o , G␣12/13 , and G␣q/11 [2,10,13,16]. In addition, gintonin has been reported to regulate various ion channels (e.g., Ca2+ -activated Cl− , Ca2+ activated K+ , and voltage-gated Kv1.2) and receptors (e.g., NMDA and P2X1 ) through LPA receptor activation [9,10]. In addition, gintonin can induce long-term potentiation (LTP) in hippocampal slices [16]. Although, previous reports raise the possibility that gintonin-mediated [Ca2+ ]i transients and ensuing NMDA receptor activation leading to LTP induction might be closely related to the regulation of neurotransmitter release in the nervous system, it has not been demonstrated that the activation of G protein-coupled LPA receptors by gintonin is coupled to the regulation of neurotransmitter release. PC12 cells are derived from the pheochromocytoma of the rat adrenal chromaffin. These cells are widely used to study the modulation of neurotransmitter release by a variety of neurotransmitters or drugs because they possess vesicles containing catecholaminergic neurotransmitters, primarily dopamine [7,11], and the release of these catecholamines is associated with elevation of cytosolic calcium, similar to adrenal chromaffin cells [8]. For example, stimulation of PC12 cells by a cholinergic agonist or depolarization by high extracellular K+ induces a [Ca2+ ]i transient and results in the release of dopamine [8]. Currently, PC12 pheochromocytoma cells are utilized to obtain information about the role of bioactive compounds that could affect in vitro neurotransmitter release [6,20]. In the present study, we examined the effects of gintonin on a [Ca2+ ]i transients and the release of dopamine in PC12 cells. We report here that gintonin induces a [Ca2+ ]i transient via membrane signaling transduction pathways of LPA receptors, and that gintonin-mediated [Ca2+ ]i transients are coupled to the stimulation of dopamine release. Additionally, we discuss the physiological and pharmacological role of gintonin-mediated dopamine release in the nervous system.
2. Materials and methods 2.1. Materials Gintonin, devoid of ginseng saponins, was prepared from Panax ginseng according to previously described methods [15]. Gintonin was dissolved in deionized water and then diluted with medium before use. RPMI1640 medium, DMEM, fetal bovine serum (FBS), horse serum (HS), penicillin, and streptomycin were purchased from Invitrogen (Camarillo, CA, USA). Lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate, 857130P) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). All other reagents used, including dopamine and N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), were purchased from Sigma–Aldrich (St. Louis, MO, USA).
2.2. Cell culture PC12 cells, a rat pheochromocytoma cell line, were obtained from Korean Cell Line Bank (KCLB, Seoul, Korea) and were grown in RPMI 1640 supplemented with 10% heat-inactivated HS, 5% heat-inactivated FBS, 100 units/mL penicillin, and 100 g/mL streptomycin in a humidified atmosphere with 5% CO2 at 37 ◦ C.
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2.3. Measurement of intracellular calcium concentration The intracellular free calcium concentration was measured by dual excitation spectrofluorometric analysis of cell suspensions loaded with Fura-2 AM as previously described [10]. Briefly, PC12 cells (either untreated or treated with NGF) were harvested with trypsin/EDTA solution and resuspended in HEPES-buffered saline solution (HBS: 120 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.5 mM CaCl2 , 10 mM glucose, 25 mM HEPES, and pH 7.4). The cells were incubated with Fura-2 AM (final concentration 2.5 M) in HBS at 37 ◦ C for 30 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 dopamine release PC12 cells were treated with Lock’s buffer [145 mM NaCl, 5.6 mM KCl, 12 mM MgCl2 , 22 mM CaCl2 , 10 mM glucose, and 10 mM HEPES (pH adjusted to 7.4 with NaOH)] in the presence or absence of gintonin at 37 ◦ C. The samples were centrifuged at 12,000 rpm for 5 min and the levels of released dopamine in the supernatant were determined by an HPLC system, as previously described [19]. Six-week-old male balb/c mice (Koatech Technology Corporation, Seoul, Korea) were housed under specific pathogen-free conditions. Mice were intraperitoneally administered saline solution (Sal) or gintonin (100 mg/kg). Mice were anesthetized with Zoletil 50 and Rompun, sacrificed, and blood samples were collected. Dopamine levels in serum were determined by HPLC [19]. Animal experiments were conducted in strict accordance with the recommendations in the guide for the care and use of Laboratory Animals of the National Institutes of Health. 2.5. Data analysis To obtain concentration-response curves of the effects of gintonin on [Ca2+ ]i transients, the peak increase of [Ca2+ ]i transient amplitudes at different concentrations of gintonin were plotted, and 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 ± S.E.M. The significance of differences between control and treatment values was determined using Student’s t-test. Values of p < 0.05 were considered statistically significant. 3. Results 3.1. Effect of gintonin on [Ca2+ ]i transients in PC12 pheochromocytoma cells In the present study, we first examined the effects of gintonin on [Ca2+ ]i transients in PC12 cells. As shown in Fig. 1A and B, gintonin treatment induced a transient rise of [Ca2+ ]i in PC12 cells in a reversible and concentration-dependent manner. The EC50 was 0.06 ± 0.01 g/mL. Gintonin-induced [Ca2+ ]i transients initiated without a detectable lag and reached peak values within a few seconds, and [Ca2+ ]i gradually decreased but did not return to its basal level until the end of time point (100 s). We observed that treatment of PC12 cells with LPA C18:1 also induced a [Ca2+ ]i transient, similar to gintonin (Fig. 3A and B). When we examined the effect of acetylcholine on [Ca2+ ]i transients, we found that acetylcholine application also induced a [Ca2+ ]i transient, similar to gintonin
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Fig. 1. Effects of gintonin (GT) on intracellular calcium transients in PC12 cells. (A) A representative trace obtained after gintonin treatment in PC12 cells. Gintonin treatment (0.03–1 g/mL) induces a [Ca2+ ]i transient. (B) Concentration-response relationship curve for gintonin-induced [Ca2+ ]i transients in PC12 cells. Each point represents the mean ± S.E.M. (n = 3–4). (C–F) Representative [Ca2+ ]i transient traces obtained under the indicated treatment. The arrows indicate application of gintonin (GT; gintonin 1 g/mL) or acetylcholine (ACh; acetylcholine 50 M). Gintonin shows homologous desensitization, but not heterologous desensitization with acetylcholine (ACh).
(Fig. 1D). Repeated treatment of PC12 cells with gintonin induced a remarkable attenuation of [Ca2+ ]i transients, indicating homologous desensitization (Fig. 1C). Homologous desensitization was also observed after repeated treatment with acetylcholine (Fig. 1D). When we treated cells with gintonin followed by acetylcholine, or vice versa (Fig. 1E and F), we did not observe heterologous desensitization to the two ligands. In addition, we could observe that co-treatment of acetylcholine and gintonin induced additive increases of [Ca2+ ]i transients (Fig. S1). These results indicate that the membrane signaling transduction pathway for gintonin might be distinct from that of acetylcholine.
3.2. Signal transduction pathway of gintonin-mediated [Ca2+ ]i transients in PC12 pheochromocytoma cells We 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. 2, the presence of Ki16425 significantly attenuated the gintonin-mediated [Ca2+ ]i transient, but the S1P receptor antagonist JTE-013 had almost no effect (Fig. 2A and B). However, the active phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca2+ chelator (BAPTA-AM) all blocked
gintonin-mediated [Ca2+ ]i transients in PC12 cells (Fig. 2C and D). These results show that gintonin, via activation of the LPA receptorphospholipase C-intracellular IP3 receptor signaling transduction pathway, elicits the release of Ca2+ from intracellular stores to increase [Ca2+ ]i .
3.3. Gintonin’s effects differ from those of ginsenosides on [Ca2+ ]i transient induction in PC12 cells We next compared the effect of representative ginsenosides, such as ginsenoside Rb1 , Rg1 , and Rg3 , which are ginseng glycosides known to regulate various ion channels and receptors [14], with gintonin in the induction of [Ca2+ ]i transients in PC12 cells. As shown in Fig. 3, the ginsenosides Rb1 and Rg1 had no effect on [Ca2+ ]i transients even at the highest concentration of 100 M. Interestingly, the ginsenoside Rg3 showed increased [Ca2+ ]i , however the pattern of the [Ca2+ ]i transient induced by ginsenoside Rg3 was different from that of gintonin. Unlike gintonin, ginsenoside Rg3 induced a slow and lagging rise of [Ca2+ ]i and did not show a sharp peak or return to resting levels. These results suggest that the gintonin-mediated [Ca2+ ]i transient are distinct from that of ginsenoside Rg3 .
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Fig. 2. Effect of signal transduction pathway inhibitors on gintonin-induced [Ca2+ ]i transients in PC12 cells. (A and C) Representative traces of gintonin-mediated [Ca2+ ]i transients in the absence or presence of various antagonist. The arrows indicate application of gintonin (1 g/mL). An LPA1/3 receptor antagonist (Ki16425, 10 M), S1P receptor antagonist (JTE-013, 10 M), PLC inhibitor (U73122, 5 M), IP3 receptor antagonist (2-APB, 100 M), or 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 pharmacological agents. *p < 0.05, compared to gintonin only treatment. Data are means ± S.E.M. (n = 3–4).
Fig. 3. Effects of ginsenosides and gintonin on intracellular calcium transients in PC12 cells. (A) Representative traces obtained after ginsenoside Rg3 (Rg3 , 10–100 M), gintonin (GT, 1 g/mL), or LPA (1 M) treatment of PC12 cells. (B) Histograms representing net increases of ginsenoside Rg3 -, gintonin- or LPA-mediated [Ca2+ ]i increases calculated from traces. (C) Representative traces obtained after ginsenoside Rb1 (Rb1 , 10–100 M) or gintonin (GT, 1 g/mL) treatment of PC12 cells. Inset: net increases of ginsenoside Rb1 - or gintonin-mediated [Ca2+ ]i transients. (D) Representative traces obtained after ginsenoside Rg1 (Rg1 , 10–100 M) or gintonin (GT, 1 g/mL) treatment in PC12 cells. Inset: net increases of ginsenoside Rg1 - or gintonin-mediated [Ca2+ ]i transients. *p < 0.05, compared to gintonin treatment. Data are means ± S.E.M. (n = 3–4).
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that stimulate dopamine release did not affect cell viability (Fig. S2). In addition, we further examined acute effect of gintonin (100 mg/kg) on dopamine concentrations in serum after intraperitoneal administration. As shown in Fig. 4B, gintonin increased serum dopamine concentrations by 1.6-, 2.2-, and 2.3-fold 10, 20, and 30 min after gintonin administration when compared to saline treatment, respectively, but at lower than 100 mg/kg did not show a slight but not significant effect on dopamine in serum (Fig. 3). These results indicate that gintonin induces dopamine release in vitro as well as in vivo.
4. Discussion
Fig. 4. Effect of gintonin (GT) on dopamine release in PC12 cells (A) and in mice (B). (A) Dopamine release from PC12 cells by treatment with gintonin. Dopamine release was examined in PC12 cells treated with gintonin at indicated concentrations for 10 min or with 1 g/mL gintonin in the presence of BAPTA-AM (BA, 50 M, for 30 min). Data are means ± S.E.M. (n = 3–4). (B) Effect of various concentration of gintonin (GT) on dopamine release in mice. The acute effect of the indicated concentration of gintonin (GT) on dopamine concentrations in 30 min after intraperitoneal administration in mice (n = 5). (C) The acute effect of gintonin (GT, 100 mg/kg) on dopamine concentrations in serum 10, 20, and 30 min after intraperitoneal administration in mice (n = 5). (Sal saline; gintonin, 100 mg/kg). Dopamine concentration in each sample was determined by HPLC, as described in Section 2. *p < 0.05, compared to control (Con).
3.4. Effects of gintonin on dopamine release in PC12 cells and mice Since the induction of depolarization by elevation of extracellular K+ or receptor ligands that induce a [Ca2+ ]i transient is coupled to dopamine release, we first examined the effects of gintonin on PC12 cells. As shown in Fig. 4A, gintonin treatment for 10 min in PC12 cells induced dopamine release in the range of 0.1–3 g/mL, but at more than 10 g/mL had no effect on dopamine release. The intracellular calcium chelator BAPTA-AM blocked gintoninmediated dopamine release (Fig. 4A), and gintonin concentrations
In previous reports, we have shown that gintonin increases the release of soluble amyloid precursor protein ␣ (sAPP␣), but not A. Gintonin-mediated stimulation of sAPP␣ release is dependent on [Ca2+ ]i transients via LPA receptor activation [9]. Gintonin also enhances NMDA receptor channel currents via phosphorylation of the Tyr132 residue of NMDA receptors via calcium-dependent tyrosine kinase and induces LTP in rat hippocampal slices [16]. In addition, gintonin not only attenuates A-induced learning and memory deficits, but also ameliorates Alzheimer’s disease-related memory deficits in a transgenic animal model of Alzheimer’s disease [9]. Thus, gintonin-mediated effects seen in vitro and in vivo suggest the possibility that the gintonin-mediated transient elevation of [Ca2+ ]i could not only be closely associated with the regulation of intracellular Ca2+ -dependent ion channels and receptors, but could also be coupled to the regulation of neurotransmitter release and ultimately exert intercellular effects in the nervous system. However, it was previously unknown whether gintonin could stimulate neurotransmitter release in the nervous system. Here, we showed that gintonin-mediated [Ca2+ ]i transients are coupled to the stimulation of dopamine release in PC12 cells and mice. We first showed that gintonin application induced [Ca2+ ]i transients and stimulated dopamine release through a LPA receptor-mediated signal transduction pathway in PC12 cells without affecting cell viability. Secondly, gintonin-induced [Ca2+ ]i transients showed homologous, but not heterologous, desensitization to acetylcholine. Third, in vivo intraperitoneal administration of gintonin in mice increased serum dopamine concentration. Thus, the present study shows that gintonin regulates neurotransmitter release in neuronal cells. Because gintonin-induced [Ca2+ ]i transients showed homologous desensitization, but not heterologous desensitization to acetylcholine, the possibility that the signal transduction pathway of gintonin-mediated [Ca2+ ]i transients is different from that of acetylcholine exists. Previous reports have shown that acetylcholine induces [Ca2+ ]i transients through activation of nicotinic acetylcholine receptors, which are ligand-gated ion channels [8], whereas gintonin activates the G protein-coupled LPA receptor signaling transduction pathway to induce [Ca2+ ]i transients (Fig. 2A and B) [10]. Supporting this notion is that co-treatment of acetylcholine and gintonin caused additive effects on [Ca2+ ]i transients (Fig. S1). Thus, it appears that gintonin utilizes a different signaling transduction pathway than acetylcholine. Interestingly, although, ginsenoside Rb1 and Rg1 had no effect on [Ca2+ ]i transients, we observed that ginsenoside Rg3 also caused an elevation of intracellular calcium levels. However, the pattern of ginsenoside Rg3 -induced increase in cytosolic calcium levels is a quite different from that of gintonin, since ginsenoside Rg3 -induced changes did not show the typical pattern of receptormediated [Ca2+ ]i transients. Rather, ginsenoside Rg3 showed a lagged increase in cytosolic calcium that did not reach a peak and did not return to resting levels, whereas gintonin showed typical reversible receptor-mediated [Ca2+ ]i transients. Previous reports
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have shown that ginsenosides, including Rg3 , inhibited Ca2+ , and Na+ influx induced by nicotinic acetylcholine receptors or high extracellular K+ , and attenuated catecholamine release from cultured bovine adrenal medullary cells [17,18]. These results suggest that ginsenosides inhibit catecholamine release when cells are activated or excited by other excitatory ligands or depolarization, whereas gintonin induces [Ca2+ ]i transients and catecholamine release via LPA receptor activation. Thus, gintonin-mediated regulation of [Ca2+ ]i transients and catecholamine release differs from that of ginsenosides. Future studies will be required to elucidate what the pharmacological roles of these two components are in their differential regulation of [Ca2+ ]i transients and catecholamine release. LPA is a simple phospholipid derivative that mediates a variety of cellular effects as a mitogen in most cell types and organs [3]. Recent studies have shown that there are at least six LPA receptor subtypes (LPA1–6) expressed in many cell types across different tissues, including the adrenal medulla [1,3]. Gintonin is a unique form of plant LPA, as it consists of LPA-ginseng protein complexes. The protein components of gintonin may function as a carrier and/or stabilizer of LPAs, and help LPAs act as functional high affinity ligands for G protein-coupled LPA receptors [10]. Although, LPA is a known neurolipid in the nervous system, our understanding of its role as a neurotransmitter or regulator of neurotransmitter release is still rudimentary. Recent studies have shown that LPA can evoke [Ca2+ ]i transients and exhibit neurotransmitter-like activities in embryonic cortical neuroblasts [4,5]. The present study provides additional evidence that gintonin could act as regulator of neurotransmitter release by stimulating dopamine release. Taken together with previous research, the present study provides further evidence that gintonin-mediated stimulation of neurotransmitter release in the hippocampus might be responsible for LTP induction and amelioration of learning and memory deficits induced by A, or in animal models of Alzheimer’s disease. In conclusion, we have demonstrated that LPA receptor activation by gintonin is coupled to dopamine release in PC12 cells. Finally, we suggest that gintonin-mediated neurotransmitter release via LPA receptor activation might be one of the molecular basis for the pharmacological effects of ginseng. Acknowledgments This work was supported by the Basic Science Research Program (2011-0021144) and the Priority Research Centers Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science, and Technology (2012-0006686) and by the BK21 plus project fund to S.-Y. Nah. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2014.11.007.
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