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Obovatol isolated from Magnolia obovata enhances pentobarbital-induced sleeping time: possible involvement of [GABA.sub.A] receptors/chloride channel activation.

Abstract

This study aimed to investigate the effects of obovatol isolated from Magnolia obovata on pentobarbital-induced sleeping behaviors and to determine whether these effects were mediated by GABAa receptors/chloride channel activation, using a western blot technique and [Cl.sup.-] sensitive fluorescence probe. [GABA.sub.A] receptors subunits expression and chloride influx were investigated in cultured cerebellar granule cells. Obovatol (0.05, 0.1, and 0.2 mg/kg) prolonged the sleeping time induced by pentobarbital (42 mg/kg). In addition, obovatol (20 and 50 [micro]M) significantly increased CI- influx in the primary cultured cerebellar granule cells. Moreover, obovatol increased the expression of [GABA.sub.A] receptor [alpha]-, [beta]-, and [gamma]-subunits. However, it had no effect on the abundance of the expression of glutamic acid decarboxylase (GAD), suggesting that obovatol might not activate GAD. These results suggest that obovatol potentiates pentobarbital-induced sleeping time through the [GABA.sub.A] receptors/chloride channel activation.

[C]2008 Elsevier GmbH. All rights reserved.

Keywords: Obovatol; Magnolia obovata; Pentobarbital; Sleep; [GABA.sub.A] receptors; Chloride influx; Glutamic acid decarboxylase (GAD)

Introduction

Magnolia obovata has been traditionally used for the treatment of thrombotic stroke, depression, anxiety, and inflammatory and neuronal diseases in oriental countries for a long time (Watanabe et al. 1983; Hirano et al. 1991; Lo et al. 1994). Biophenolic compounds such as magnolol, honokiol, and obovatol isolated from Magnolia obovata have been found to be anxiolytic and muscle relaxant (Maruyama et al. 1998; Seo et al. 2007). From the previous experiment, it was reported that obovatol has anxiolytic-like effects in animal models paradigm, suggesting that these effects are involved in GABA/benzodiazepine receptor complex (Seo et al. 2007).

GABA is released from the terminal of specific inhibitory neurons. GABA binds to its receptors and produces an increase in membrane permeability to [Cl.sup.-] ions, which elicit a hyperpolarization of the postsynaptic membrane. Benzodiazepines and barbiturates show agonistic effects on the [GABA.sub.A], receptors. Their binding to the allosteric site of the receptors enhances the affinity of the GABA-binding site for GABA, which results in an enhancement of chloride influx and facilitates the GABAergic transmission (Olsen 1981; Ticku and Maksay 1983). On the other hand, the [GABA.sub.A] receptors consist of different subunits. To date, four different types of subunits ([alpha], [beta], and [gamma]) have been described, each of which encloses different membranes. In addition, the critical step in GABA biosynthesis is the decarboxylation of glutamate by glutamic acid decarboxylase (GAD). GAD exists in two different isoforms, GAD65 and GAD67. The level of GAD65 and GAD67 is reported to be up-regulated in the GABAergic interneurons.

GABA receptors have been known to play an important role in the modulation of barbiturate-induced sleeping through interaction with GABAergic systems (Doghramji 2006). Therefore, we were interested in whether the enhancement of pentobarbital-induced sleeping behaviors by obovatol is mediated by [GABA.sub.A] receptors/chloride channel activation.

Materials and methods

Animals

The animals used were ICR male mice (purchased from Samtako, Korea), weighing 22-25 g, in groups of 10-12 heads. The mice were housed in acrylic cages with water and food available ad libitum under an artificial 12-h light/dark cycle (light on at 7:00 am) and at a constant temperature (22 [+ or -] 2 [degrees]C). To ensure adaptation to the new environment, the mice were kept in the departmental holding room for 1 week before testing. The mice were fasted for 24 h before the injection of pentobarbital. All the experiments were conducted in accordance with the National Institute of Toxicological Research on the Korea Food and Drug Administration guidelines for the care and use of laboratory animals.

Drugs

Obovatol (Fig. 1, purity, [greater than or equal to] 95%) was isolated from the leaves of Magnolia obovata. The leaves of Magnolia obovata were harvested in the fall in Daejeon, Korea, and identified as described elsewhere (Kwon et al. 1997). The dried and milled sample (I kg) was soaked in chloroform-acetone (1:1, v/v, 51) at room temperature for 5 days. The extract was concentrated under reduced pressure, and purified by slica gel, CI8 column chromatography, and preparative thin layer chromatography (TLC). Finally, high-performance liquid chromatography (HPLC) (Phenomenex, Ultracarb 10 ODS; 250 x 21.2 mm at 285 nm, flow rate 3.5 ml/min), using a linear gradient rising from 80% to 90% methanol for 50 min was used in purifying and collecting the obovatol (120 mg); the [t.sub.R] in HPLC of obovatol was 35 min (Hwang et al. 2002).

[FIGURE 1 OMITTED]

Pentobarbital sodium was obtained from Hanlim Pharm. Co., Ltd. (Korea). Fetal bovine serum (FBS) and DMEM were purchased from GIBCO (Grand Island, NY, USA). The [Cl.sup.-] -sensitive fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was obtained from Invitrogen (USA). The specific rabbit polyclonal antibodies against [GABA.sub.A] receptor subunits or GAD65/67 and the corresponding conjugated anti-rabbit immunoglobulin G-horseradish peroxidase were obtained from Santa Cruz Biotechnology Inc. (USA). The ECL Western blotting detection system was obtained from Roche Diagnostics (USA). All the other chemicals used in these experiments were obtained from Sigma (St Louis, MO, USA).

Pentobarbital-induced sleeping

Pentobarbital sodium was diluted in 0.9% physiological saline and administered to each mouse intraper-itoneally (i.p.) to induce sleep. Test samples suspended in 1% CMC in physiological saline were administered orally (p.o.) to the mice (0.l ml/l0 g). Around 10-12 mice were used for each treatment group. All experiments were carried out between 1:00 and 5:00 pm. Animals were fasted for 24 h, prior to the experiment. Pentobarbital was given to animals placed in a box 30 min after the oral administration of test drugs. Those animals that stopped moving in the box within 15 min after pentobarbital injection were immediately transferred to another box. Those individuals that stayed immobile for more than 3 min were judged to be asleep. The time that elapsed from receiving pentobarbital until an animal, positioned delicately on its back, lost its righting reflex represented latency to onset of sleeping. The animals were observed constantly, and the time of awakening, characterized by righting of the animal, was noted. The sleeping time was defined as the time taken for the animal to regain spontaneous movements after being transferred to the second box. Animals that failed to fall asleep within 15 min after pentobarbital administration were excluded from the experiments (Wolfman et al. 1996; Darias et al. 1998).

Primary culture of cerebellar neurons

Primary cultures of cerebellar neurons enriched in granule cells were prepared from cerebella of 8-d-old SD rats as previously reported (Han et al. 2007). After cell culture for 8-d, cells express functional [GABA.sub.A] receptors, with an expression pattern similar to that apparent observed in the cerebellum during postnatal development, but different from that observed in the adult rat cerebellum. Briefly, cells were plated (1 x [10.sup.6] cells per 0.2 ml) in 96 microplates or (2 x [10.sub.6] per 2.0 ml) in 60-mm dishes that had been coated with poly-o-lysine (10 mg/ml) (Sigma, USA). The cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, gentamicin (100 mg/ml), antibiotic-antimycotic solution (100 unit/ml) and 25 mM KC1. Cytosine arabinofurano-side (10 mM) was added to cultures 18-24h after plating, to inhibit the proliferation of nonneuronal cells.

Measurement of intracellular chloride influx

The intracellular chloride ion ([Cl.sup.-]) concentration of cerebellar granule cells was estimated using the [Cl.sup.-] sensitive fluorescence probe MQAE, according to the method of West and Molloy (1996) with a slight modification. The buffer (pH 7.4) used contained the following: 2.4 mM [HPO.sub.4.sup.2-], 0.6 mM [H.sub.2][PO.sub.4.sup.-], 10 mM HEPES, 10mM D-glucose, and 1 mM [MgSO.sub.4]. A variety of MQAE loading conditions were assessed. The cells were incubated overnight in a medium containing 10 mM MQAE. After loading, the cells were washed three times in the relevant [Cl.sub.-] -containing buffer. Then, the wash buffer was replaced with the buffer with or without the compounds or control. Repetitive fluorescence measurements were initiated immediately using a FLUOstar (excitation wavelength: 320 nm; emission wavelength: 460 nm; BMG LabTechnol-ogy, Germany). The data were presented as the relative fluorescence [F.sub.0]/F, where [F.sub.0] was the fluorescence without [Cl.sup.-] and F was the fluorescence as a function of time. The [F.sub.O]/F values were directly proportional to [[Cl.sup.-]],.

Expression of [GABA.sub.A] receptor subunits and GAD65/67

At the eighth day of culture, the treatment of obovatol to the primary cultured cerebellar neurons was initiated as our previous report (Ma et al. 2007). Obovatol was dissolved in ethanol and diluted sequentially in culture medium to final concentrations of 20 [micro]M. Control group was treated with solvent alone at the same dilution as that used for drug treatment (0.1% v/v). The culture medium was completely replaced every day with the fresh medium containing the appropriate drug. After treatment of obovatol, cells were harvested and treated with lysis buffer. The extracts were centrifuged at 20000g for 20min. Equal amounts of proteins were separated on a SDS 12% polyacrylamide gel, and transferred to a nitrocellulose membrane (Hyboud ECL, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The blots were blocked for 2 h at room temperature with 5% (w/v) non-fat dried milk in Tris-buffered saline solution (10 mM Tris, pH 8.0 and 150mM NaCl) containing 0.05% Tween-20. The membrane was incubated with the specific rabbit polyclonal antibodies against [GABA.sub.A] receptor subunits (1:500) or GAD65/67, for 6h at room temperature. The blot was then incubated with the corresponding conjugated anti-rabbit immunoglobulin G-horseradish peroxidase. The immunoreactive proteins were detected using the ECL Western blotting detection system.

Statistical analysis

Data were presented as the mean [+ or -] SEM. For statistical comparison, the results were analyzed using analysis of variance (ANOVA). A P-value <0.05 was considered a statistically significant difference. In case of significant variation, the individual values were compared with Dunnett's test.

Results

Effects of obovatol on pentobarbital-induced sleeping behaviors in mice

Thirty minutes after the oral administration of obovatol (0.05, 0.1, and 0.2 mg/kg), the ICR mice received sodium pentobarbital (42 mg/kg, i.p.). The time elapsed from pentobarbital injection to the loss of the righting reflex was taken as sleeping latency. The time elapsed between the loss and the voluntary recovery of the righting reflex was considered as the total sleeping time. Although all these three dosages of obovatol had no effect on the latency of pentobarbital-induced sleep (data not shown), all dosages of obovatol (0.05, 0.1, and 0.2mg/kg) significantly increased sleeping time induced by pentobarbital from 75.9 [+ or -] 2.3 min to 106.5, 132.2, and 152.0 min respectively (Fig. 2).

[FIGURE 2 OMITTED]

Effects of obovatol on [Cl.sup.-] influx in the primary cultured cerebellar neurons

Resting intracellular [Cl.sup.-] concentrations ([[Cl.sup.-]],-) were calibrated using standard [Cl.sup.-] solutions of 0, 10, 20, and 40 mM, each containing 140mM [K.sup.+]. Appropriate amounts of methylsulfate were used to replace [Cl.sup.-] in these solutions. Tributyltin chloride (5mM) and niger-icin (5 mM) were present to artificially facilitate the balance between intracellular [Cl.sup.-] and extracellular [Cl.sup.-] concentrations. Resting [[CI.sup.-]]/ in cultured cerebellar granule cells was 20.3 [ +or -] 1.3 mM. and treatment of granule cells with obovatol (20 and 50 [micro]M) increased [[Cl.sup.-]], to 30.3 and 36.9 mM, respectively. Pentobarbital (10 [micro]M) also increased the influx of [Cl.sup.-] in primary cultured cerebellar granule cells (Fig. 3).

[FIGURE 3 OMITTED]

Effects of obovatol on the expression of [GABA.sub.A] receptors subunits and GAD 65/67

Treatment of primary cultured cerebellar granule cells with 20 [micro]M obovatol could significantly increase the [GABA.sub.A] receptors [alpha]-. [beta]-, and [gamma]-subunits (Figs. 4 A-C). At the same time, to make sure whether obovatol affects pentobarbital-induced sleeping behaviors through the synthesis of GABA, the effects of obovatol on the expression of GAD65/67 was detected. However, the result showed that chronic treatment of obovatol did not affect the abundance of GAD expression (Fig. 4D).

[FIGURE 4 OMITTED]

Discussion

A variety of modulators of GABA transmission, including neurosteroids, benzodiazepines, and barbiturates, were investigated in in vitro and in in vivo models (Darias et al. 1998). Enhancement of GABAergic neuronal inhibition underlies the therapeutic action in the treatment of generalized anxiety disorders, panic anxiety, sleep disturbances, and epilepsy including status epilepticus. These properties of GABA also take a growing interest in attractive targets for pharmacological research (Ma et al. 2007, 2008). Structural and physiological heterogeneity of the pentameric composition of the [GABA.sub.A] receptors, as well as the different distribution of its receptor subtypes in specific brain areas, provide an important basis for the development of therapeutic drugs (Sieghart and Sperk 2002). The modulators of [GABA.sub.A] receptors have been the focus in the therapeutics to insomnia (Zammit 2007). GABA as a major inhibitory amino acid transmitter in CNS involves in the counterpoise between excitatory and inhibitory regulations in CNS. Therefore, we are interested in investigating whether the prolongation of obovatol is due to an effect on the CNS via the GABAergic systems.

[GABA.sub.A] receptors are a major member of the ligand-gated ion channel family. They are also the target for a wide range of therapeutic agents such as benzodiazepines, barbiturates, and anesthetics. Barbiturates, such as pentobarbital, have three distinct effects on the [GABA.sub.A] receptor activity. At low concentrations, pentobarbital modulates GABA-mediated [Cl.sup.-] current. At higher concentrations, pentobarbital directly activates the [GABA.sub.A] receptor in the absence of GABA. To investigate the detailed mechanisms involved in the prolongation to the pentobarbital-induced sleeping time caused by obovatol, the effect of obovatol on the chloride influx in primary cultured cerebellar neurons was evaluated. Our results showed that both obovatol and pentobarbital increased the influx of chloride in the primary cultured cerebellar granule cells. The results indicate that obovatol has a similar action of pentobarbital on the GABA-gated chloride- channel. So, it is proposed that obovatol might act on [GABA.sub.A] receptors to induce chloride channel opening, and modulate pentobarbital-induced pharmacological properties like a GABA receptors agonist.

Therefore, we tried to find out the typical GABA responding subunits, by which obovatol acts on [GABA.sub.A] receptors and potentiates pentobarbital-induced sleeping. Our results showed that treatment of primary cultured cerebellar granule cells with 20 [micro]M obovatol significantly increased the [GABA.sub.A] receptors [alpha]-, [beta]-, and, [gamma]-subunits. These receptor subtypes have discrete distributions in the brain, suggesting that they fulfill different functional roles (Pirker et al. 2000). Expression studies of different subunit combinations have clearly defined some of the pharmacological differences between specific receptor combinations that are thought to be expressed in vivo (Korpi et al. 2002). It has been confirmed that removal of either [[alpha].sub.1] or [[beta].sub.2] subunits of [GABA.sub.A] receptors produces strong and selective decreases in hypnotic effects of different drugs (Blednov et al. 2003). That may be of the explanation for our in vivo results. From an additional experiment, we investigated the effects of obovatol on the expression of GAD65/67. GAD65/67, which is necessary for GABA synthesis, plays a major role in GABA transmission in normal physiological condition. Obovatol showed no effect on the GAD65/67 expression. So, it means that obovatol did not increase GABA level by direct activation of GAD.

In this study, obovatol, a biophenolic component isolated from Magnolia obovata potentiated pentobar-bital-induced sleeping behaviors in mice. Obovatol (20 and 50 [micro]M) also significantly increased the chloride influx and the expression of [GABA.sub.A] receptors [alpha]-, [beta] and [gamma]-subunits in the primary cultured cerebellar granule cells. However, obovatol did not activate GAD. Therefore, we suggest obovatol enhances pento-barbital-induced sleeping behaviors, the influence on GABAergic system might be involved in its mechanisms. Further investigation is needed for pharmacological actions of obovatol.

Acknowledgement

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2005-005-J15002).

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Hong Ma (a), Young-Jun Jo (a), Yuan Ma (b), Jin-Tae Hong (a), Byoung-Mog Kwon (c), Ki-Wan Oh (a), *

(a) College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea

(b) Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, South Korea

(c) Korea Research Institute of Biosciences (KRIBB), Deajeon 305-764, South Korea

* Corrcsponding author. Tel./fax: +8243 261 2827.

E-mail address: kiwan@chungbuk.ac.kr (K.-W. Oh).

doi: 10.1016/j.phymed.2008.12.007
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Author:Ma, Hong; Jo, Young-Jun; Ma, Yuan; Hong, Jin-Tae; Kwon, Byoung-Mog; Oh, Ki-Wan
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9SOUT
Date:Apr 1, 2009
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