Inhibitory Effect of Jujuboside A on Glutamate-Mediated Excitatory Signal Pathway in Hippocampus

• Abstract
• Introduction
• Materials and Methods
• Animals and drugs
• Microdialysis and high-performance liquid chromatography procedures
• Measurement of intracellular calcium in cultured hippocampal neurons
• Results
• Effect of JuA on Glu levels in hippocampus:
• Calcium changes in cultured hippocampal neurons
• Discussion
• References

Abstract

Jujuboside A (JuA) is a main component of jujubogenin extracted from the seed of Ziziphus jujuba Mill var spinosa (Bunge) Hu ex H F Chou (Ziziphus), which is widely used in Chinese traditional medicine for the treatment of insomnia and anxiety. Previously, we reported the inhibitory effects of JuA on hippocampal formation in vivo and in vitro, the present study was carried out to examine the effects of JuA on glutamate (Glu)-mediated excitatory signal pathway in hippocampus. Microdialysis coupled with high-performance liquid chromatography (HPLC) was used to monitor the changes of Glu levels in the hippocampus induced by penicillin sodium, or a mixture of penicillin sodium and JuA. The results showed that penicillin increased the hippocampal Glu concentration (p < 0.01) and a high dose of JuA (0.1 g/L) significantly blocked penicillin-induced Glu release (p < 0.05). Moreover, the effect of JuA on intracellular Ca²⁺ changes after the stimulation by Glu was studied in cultured hippocampal neurons with confocal laser scanning microscope (CLSM). It was found that Glu (0.5 mM) induced an intracellular [Ca²⁺]_i increase (p < 0.01), and JuA significantly inhibited the Glu-induced Ca²⁺ increase. The calmodulin (CaM) antagonist trifluoperazine (TFP) showed a similar inhibitory effect as JuA. These observations suggested that JuA has inhibitory effects on Glu-mediated excitatory signal pathway in hippocampus and probably acts through its anti-calmodulin action.

Introduction

Ziziphus jujuba Mill var. spinosa (Bunge) Hu ex H F Chou, is widely used in Chinese traditional medicine for the treatment of insomnia and anxiety [1]. Experimental studies validated that it could inhibit the spontaneous activity, facilitate the hypnotic action of pentobarbital, and antagonize the excitatory action of morphine and pentylenetetrazole [1], [2]. JuA is a main component of jujubogenin. Some reports showed that JuA is a non-competitive inhibitor of CaM [3] and has an inhibitory effect on the activities of mice [4]. In our previous studies, we found that JuA has inhibitory effects on hippocampal formation in vivo and in vitro. JuA significantly decreased the slopes of excitatory postsynaptic potential (EPSP) and the amplitudes of population spike (PS) in the responses of hippocampal cells [5]. However, the mechanisms of JuA’s inhibitory effect have not yet been fully elucidated.

In the present study, in order to understand the underlying mechanisms, we investigated the effect of JuA on Glu-mediated excitatory signal pathway in hippocampus. Glu is a major excitatory neurotransmitter in hippocampus. It is released in response to presynaptic neuronal membrane depolarization and actives postsynaptic Glu receptors. The activation of Glu receptor then opens the ion channel coupled to the receptor, allowing the passage of extracellular calcium into the intracellular cytosol, which in turn triggers a series of biochemical events [6]. Ca²⁺ plays a key role in the transduction of extracellular signals and in the control of neuronal functions. It is involved in control of neuronal membrane excitability, neurosecretion, synaptic plasticity, gene expression and programmed cell death [7]. It was known that elevated level of extracellular Glu is responsible for neuronal hyperactivity and degeneration in brain disorders, including stroke, epilepsy and Parkinson's disease [8]. Although the mechanisms underlying the neuronal damage induced by Glu are not yet fully understood, Glu-mediated overload of intracellular free calcium appears to be a prominent contributor [9]. For the importance of glutamate and calcium in neuronal excitatory pathway, we studied the effects of JuA on changes of extracellular Glu and intracellular calcium. Since JuA is an inhibitor of CaM, the effect of TFP, a kind of calmodulin antagonist, was also studied in the experiment.

Materials and Methods

Animals and drugs

Adult Sprague-Dawley rats of both sexes (weight 240 - 260 g) and pups (3 - 5 days old) were obtained from Zhejiang Center of Laboratory Animals (Grade II, Certificate No 2 001 001). JuA (purity 99 %) was provided by National Institute for the Control of Pharmaceutical and Biological Products. Penicillin sodium and urethane were obtained from China Medical Bioproduct Co. Fluo-3/AM was purchased from Molecular Probe. Other drugs were purchased from Sigma.

Microdialysis and high-performance liquid chromatography procedures

Animal preparation: After being anesthetized with urethane (1.25 g/kg, s. c.), the adult rat was placed in a stereotaxic frame. Guide cannulas were implanted chronically through the skull and aimed at the lateral ventricle (AP = -0.8, L = 1.5, H = -3.5) and hippocampus (AP = -5.8, L = 5.0, H = -3.0). The guide cannulas were fixed to the skull with stainless metal screws and dental cement. After surgery, the animals were allowed to recover for one week.

Microdialysis procedure: According to the different injections during the experiments, rats were randomly assigned to five group: ACSF group, penicillin sodium group (1000 kIU/L penicillin sodium), low dose JuA group (0.05 g/L JuA + 1000 kIU/L penicillin sodium), high dose JuA group (0.1 g/L JuA + 1000 kIU/L penicillin sodium) and TFP group (50 μM TFP + 1000 kIU/L penicillin sodium). In each group, six rats were studied. The rat was placed in a freely moving system (BAS, MD-1575). Microdialysis probes (BAS, MD-2204, 4 mm membrane) were perfused with artificial cerebrospinal fluid (ACSF: 126 mM NaCl, 27.5 mM NaHCO₃, 2.4 mM KCl, 5 mM KH₂PO₄, 5 mM Na₂HPO₄, 0.5 mM Na₂SO₄, 0.82 mM MgCl₂·6H₂O, 1.1 mM CaCl₂·2 H₂O, 5 mM glucose, and pH 7.4) at a flow rate of 1 μL/min. Perfusate collected for the first 120 min was not analyzed, to allow equilibration between the brain tissue and perfusion solution before sampling. Subsequently, samples were collected at 30 min intervals and the concentration of Glu was determined by HPLC. The next two samples that were collected during the first hour served as Glu baseline levels. At the beginning of the second hour, through the probe placed in the lateral ventricle, each rat was injected with 3 μL relevant drugs according to its group. Samples were collected every 30 min thereafter, and the amount of Glu was measured to evaluate the effects of the various drugs.

Measurement of Glu by HPLC: Glu levels were measured by OPA/ß-mercaptoethanol precolumn derivatization, reversed-phase gradient elution and fluorescence detection. Glu in the dialysate was first derivatized to the fluorescent isoindoles. 20 μL dialysate sample and 10 μL OPA (Sigma) derivating fluid were allowed to react for 1 min at room temperature. The HPLC employed buffer A: 0.1 M KH₂PO₄ buffer (adjusted to pH 6.60): methanol = 65 : 35 v/v, and buffer B: 0.1 M KH₂PO₄ buffer (adjusted to pH6.60): methanol = 10 : 90 v/v. Buffer A was ultrasonically degassed while buffer B was filtered and degassed through a 0.2-μm nitrocellulose membrane under vacuum. The two-buffer HPLC system (Shimadzu-10AVP, Japan) was coupled to a fluorescent detector (RF-10AXL, Shimadzu, Japan). Separation was achieved on a C18 column (Hypersil, BDS, 5μm). 20 μL of the reaction mixture were injected into the column and separated with a gradient from A:B (100 : 0) to A:B (60 : 40) within 12 min, then eluted with 100 % B for 5 min to elute other components. The flow rate was 1 mL/min; excitation wavelength: 357 nm; emission wavelength: 455 nm. The areas under the peaks of the Glu were used for calculating the concentrations. The recovery after derivatizaton was 106 ± 8 %.

Histology: At the end of experiments, the rats were euthanized with pentobarbital overdose and decapitated. Brains were removed and the positions of probes were verified by the freezing-microtome technique. Probe placements were verified for all data presented in this study and are demonstrated in Fig. [1].

Measurement of intracellular calcium in cultured hippocampal neurons

Hippocampal cell cultures: Primary hippocampal cell cultures from 3 - 5 day-old Sprague Dawley rats were prepared as described previously [3]. Briefly, hippocampi were rapidly dissected, dissociated by trypsinization and gentle trituration, and plated onto precoated (poly-D-lysine, 10 μg/mL) glass coverslips in DMEM containing 10 % FCS. The culture media was refreshed every 3 days. At the third day in culture, the media was supplemented with 5-fluoro-2′-deoxyuridine to block glial proliferation. Cells between 10 and 12 days in culture were used for experiments.

Calcium measurements: A calcium indicator Fluo-3 was used to evaluate the intensity of intracelluar Ca²⁺. Cells were incubated at 37 °C for 30 min with the acetoxymethyl ester of fluo-3 (fluo-3-AM, 5 μM; Molecular Probes). The fluorescence was measured with a confocal laser scanning microscopy (Zeiss LSM510, Germany). Fluo-3 was excited by the 488 nm laser and emissions between 515 and 560 nm were obtained. Images of 512 × 512 pixels were taken with a 20 × objective. The percentage of the change of fluorescence intensity was calculated as: (F/F₀) × 100 %. F represents the maximal fluorescence intensity monitored after stimulation and F₀ is the fluorescence intensity in resting conditions. Cells were randomly assigned to five group: ACSF group, Glu groups (0.5 mM), low dose JuA group (0.05 g/L JuA + 0.5 mM Glu), high dose JuA group (0.1 g/L JuA + 0.5 mM Glu), and TFP group (50μM TFP + 0.5 mM Glu). In JuA group and TFP group, JuA or TFP were added 10 min prior to Glu.

Statistical analysis: Experiments were repeated on more than five coverslips. Data are presented as mean ± SEM. Statistical significance was evaluated with either Student’s t test or one-way ANOVA. Significance was accepted at the P < 0.05 level.

Results

Effect of JuA on Glu levels in hippocampus:

As illustrated in Fig. [1], the microdialysis probe was located within the hippocampal formation, the infusion probe in lateral ventricle. In ACSF group, the mean concentration of Glu was 6.14 ± 1.03 μM. Direct microinjection of penicillin (1000 kIU/L) into lateral ventricle could greatly elevate the Glu level in hippocampus to 298 ± 49 % compared with its normal level (p < 0.01). When JuA was co-injected into lateral ventricle with penicillin (1000 kIU/L), the increase of Glu caused by penicillin was reduced by JuA with a dose dependent manner. The Glu concentrations were 2.38 ± 0.76 and 1.67 ± 0.37 times baseline with 0.05 and 0.1 g/L JuA, respectively. There was a significant difference (p < 0.05) between penicillin group and high dose JuaA group. 50 μM TFP completely inhibited the effect of penicillin, the Glu concentration was 1.01 ± 0.17 times baseline (Fig. [2]).

Calcium changes in cultured hippocampal neurons

Glu (0.5 mM) significantly increased the intracellular [Ca²⁺]_i in cultured hippocampal neurons and the relative increase in fluorescence of fluo-3 was 287 ± 70 % (p < 0.01). JuA significantly inhibited Glu-induced [Ca²⁺]_i rise, the relative increases in fluorescence were 211 ± 37 % and 148 ± 36 % with 0.05 and 0.1 g/L JuA, respectively (p < 0.01). Two typical fluorescence changes in the Glu group and the high-dose JuA group are shown in Fig. [3]. Likewise, TFP also significantly reduced the [Ca²⁺]_i increase induced by Glu (Fig. [4]).

Discussion

The major finding in this experiment was that JuA, a component extracted from a Chinese herb, has an inhibitory effect on Glu-mediated excitatory signal pathway in the hippocampus. Our result revealed that JuA showed a similar effect with TFP.

In this study, we observed that JuA reduced the elevation of Glu level induced by penicillin sodium. Penicillin is a GABA_A receptor blocker and is often used to induce epilepsy [10], [11]. It could promote the release of Glu indirectly [12], as confirmed by our data (Fig. [2]). When co-injected with penicillin sodium and JuA, the increase in extracellular levels of Glu was dose-dependently inhibited (Fig. [2]). It was reported that Glu is released from vesicles in presynaptic terminals by a Ca²⁺-dependent mechanism [13]. Evidence demonstrated that CaM regulates synaptic protein phosphorylation and plays a role in regulating neurotransmitter release [14], [15]. It is, therefore, likely that the JuA reduced the extracellular level of Glu by suppressing Glu release from presynaptic terminals through inhibiting the activity of CaM. In addition, as shown in Fig. [4], JuA attenuated the elevation of intracellular Ca²⁺. This also contributed to the inhibition of Glu release.

It is well known that Ca²⁺ regulates numerous physiological cellular phenomena as a second messenger. Glu activates two principal classes of ionotropic receptors, NMDA receptors and AMPA/KA receptors, then induces an increase in the intracellular concentration of Ca²⁺. Our data showed that JuA not only inhibited the release of Glu, but also depressed the elevation of intracellular Ca²⁺ induced by Glu. This could facilitate the inhibitory effect of JuA on Glu-mediated excitatory signal pathway. There is evidence that CaM antagonists inhibit the elevation of cytosolic calcium induced by high K⁺ depolarization in synaptosomes [16] and block Ca²⁺ channels in smooth muscle from rat vas deferens [17]. It is suggested that CaM governs two important parameters: membrane potential and [Ca²⁺]_i, by regulating ion pumps and channels [18]. In our study, both JuA and TFP inhibited the Glu-induced Ca²⁺ increase. This result suggested that JuA could reduce Ca²⁺ increase by inhibiting CaM activity.

TFP is often used as a CaM inhibitor and is a useful pharmacological tool for determining whether calmodulin is involved in a cell response. We found that TFP and JuA had similar effects, which indicated the effect of JuA is correlated with its anti-calmodulin action. A notable thing was that TFP fully inhibited the elevation of Glu induced by penicillin, which may not be totally attributed to its blocking CaM function. In clinic, TFP is a widely used phenothiazine antipsychotic. It is known that benzodiazepine receptor and GABA_A receptor compose a benzodiazepine-GABA receptor-ionophore complex. A variety of centrally acting anxiolytic, depressant, anticonvulsant and convulsant drugs bind to one of the sites in the complex and modulate the binding of ligands at the other sites [19]. Phenothiazines were reported to alter GABA_A receptor kinetics in hippocampal cells [20]. Thus, it seems that TFP may impact penicillin binding to the benzodiazepine site at the GABA_A ionophore and then directly inhibit the effect of penicillin.

In summary, our data indicated that JuA inhibited the release of Glu and the elevation of intracellular Ca²⁺, which may be one of the underlying mechanisms of its clinical effects. The present results also suggested that JuA’s effects perhaps were mediated through its anti-calmodulin action. To verify it, further study is still needed to be done.

References

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Prof Xiaoxiang Zheng

Department of Biomedical Engineering

Zhejiang University (Yuquan campus)

Hangzhou 310027

P. R. China

Fax: +86-571-87951676

Email: zxx@mail.hz.zj.cn

References

• 1 Hong G X. Advances in research on the seed of Suanzaoren.  J Tradit Chin Med. 1987;  12 51-3
  PubMedSearch in Google Scholar
• 2 Yang H Y. Central pharmacological effects of five Chinese herbs.  J Pharm Chin (Taipei). 1987;  40 193-200
  PubMedSearch in Google Scholar
• 3 Zhou Y, Li Y, Wang Z, Ou Y, Zhou X. ¹H NMR and spin-labeled EPR studies on the interaction of calmodulin with jujuboside A.  Biochem Biophys Res Commun. 1994;  202 148-54
  CrossrefPubMedSearch in Google Scholar
• 4 Wu S X, Zhang J X, Xu T, Li L F, Zhao S Y, Lan M Y. Effects of seeds, leaves and fruits of Ziziphus spinosa and JuA on central nervous system function.  J Trad Chin Med. 1993;  18 685-7
  PubMedSearch in Google Scholar
• 5 Shou C, Feng Z, Wang J, Zheng X. The effects of jujuboside A on rat hippocampus in vivo and in vitro .  Planta Med. 2002;  68 799-803
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Muir K W, Lees K R. Clinical experience with excitatory amino acid antagonist drugs.  Stroke. 1995;  26 503-13
  PubMedSearch in Google Scholar
• 7 Ghosh A, Greenberg M E. Calcium signaling in neurons: molecular mechanisms and cellular consequences.  Science. 1995;  268 239-47
  CrossrefPubMedSearch in Google Scholar
• 8 Coyle J T, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders.  Science. 1993;  262 689-95
  CrossrefPubMedSearch in Google Scholar
• 9 Sapolsky R M. Cellular defenses against excitotoxic insults.  J Neurochem. 2001;  76 1601-11
  CrossrefPubMedSearch in Google Scholar
• 10 Ostojic Z S, Ruzdijic S, Car M, Rakic L, Veskov R. The connection between absence-like seizures and hypothermia induced by penicillin: possible implication on other animal models of petit mal epilepsy.  Brain Res. 1997;  777 86-94
  CrossrefPubMedSearch in Google Scholar
• 11 Sugimoto M, Fukami S, Kayakiri H, Yamazaki S, Matsuoka N, Uchida I. et al . The beta-lactam antibiotics, penicillin-G and cefoselis have different mechanisms and sites of action at GABA(A) receptors.  Br J Pharmacol. 2002;  135 427-32
  CrossrefPubMedSearch in Google Scholar
• 12 Chen X, Sheng C, Zheng X. Direct nitric oxide imaging in cultured hippocampal neurons with diaminoanthraquinone and confocal microscopy.  Cell Biol Int. 2001;  25 593-8
  CrossrefPubMedSearch in Google Scholar
• 13 Birnbaumer L, Campbell K P, Catterall W A, Harpold M M, Hofmann F, Horne W A. et al . The naming of voltage-gated calcium channels.  Neuron. 1994;  13 505-6
  CrossrefPubMedSearch in Google Scholar
• 14 DeLorenzo R J. Calmodulin in neurotransmitter release and synaptic function.  Fed Proc. 1982;  41 2265-72
  PubMedSearch in Google Scholar
• 15 Sandoval M E, Aquino G, Chavez J L. Sodium-dependent, calmodulin-dependent transmitter release from synaptosomes.  Neurosci Lett. 1985;  56 271-7
  CrossrefPubMedSearch in Google Scholar
• 16 Sitges M, Talamo B R. Sphingosine, W-7, and trifluoperazine inhibit the elevation in cytosolic calcium induced by high K⁺ depolarization in synaptosomes.  J Neurochem. 1993;  61 443-50
  PubMedSearch in Google Scholar
• 17 Nakazawa K, Higo K, Abe K, Tanaka Y, Saito H, Matsuki N. Blockade by calmodulin inhibitors of Ca²⁺ channels in smooth muscle from rat vas deferens.  Br J Pharmacol. 1993;  109 37-41
  PubMedSearch in Google Scholar
• 18 Saimi Y, Kung C. Calmodulin as an ion channel subunit.  Annu Rev Physiol. 2002;  64 289-311
  CrossrefPubMedSearch in Google Scholar
• 19 Ticku M K. Benzodiazepine-GABA receptor-ionophore complex. Current concepts.  Neuropharmacol. 1983;  22 1459-70
  CrossrefPubMedSearch in Google Scholar
• 20 Mozrzymas J W, Barberis A, Cherubini E. Facilitation of miniature GABAergic currents by chlorpromazine in cultured rat hippocampal cells.  Neuroreport. 1999;  10 2251-2254
  PubMedSearch in Google Scholar

Prof Xiaoxiang Zheng

Department of Biomedical Engineering

Zhejiang University (Yuquan campus)

Hangzhou 310027

P. R. China

Fax: +86-571-87951676

Email: zxx@mail.hz.zj.cn