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Anxiolytic agent, dihydrohonokiol-B, recovers amyloid β protein-induced neurotoxicity in cultured rat hippocampal neurons
Neuroscience Letters 384 (2005) 44–47
Anxiolytic agent, dihydrohonokiol-B, recovers amyloid  protein-induced neurotoxicity in cultured rat hippocampal neurons Bing Liu, Naoki Hattori, Nan-Yan Zhang, Bo Wu, Li Yang, Kaori Kitagawa, Zheng-Mei Xiong, Takao Irie, Chiyoko Inagaki ∗ Department of Pharmacology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan Received 5 February 2005; received in revised form 14 April 2005; accepted 15 April 2005
Abstract The effects of anxiolytic honokiol derivative, dihydrohonokiol-B (DHH-B), on amyloid  protein (A25–35 , 10 nM)-induced changes in Cl− -ATPase activity, intracellular Cl− concentration ([Cl− ]i ) and glutamate neurotoxicity were examined in cultured rat hippocampal neurons. DHH-B (10 ng/ml) recovered A-induced decrease in neuronal Cl− -ATPase activity without any changes in the activities of Na+ /K+ ATPase and anion-insensitive Mg2+ -ATPase. A GABAC receptor antagonist (1,2,5,6,-tetrahydropyridin-4-yl) methyl-phosphinic acid (TPMPA, 15 M), inhibited the protective effects of DHH-B on Cl− -ATPase activity. DHH-B reduced A-induced elevation of [Cl− ]i as assayed using a Cl− -sensitive fluorescent dye, and prevented A-induced aggravation of glutamate neurotoxicity. These data suggest that DHH-B exerts the neuroprotective action against A through GABAC receptor stimulation. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Amyloid  protein; Dihydrohonokiol-B; Cl− -ATPase; GABAC receptor; Intracellular Cl− concentration
Dihydrohonokiol (DHH-B; 3 -(2 propenyl)-5-(1,1 -biphenyl)-2,-4 -diol), a partially reduced derivative of honokiol isolated from magnolia bark [9], exhibits a potent anxiolytic activity in mice [6] and has been used in traditional Chinese medicine [10]. We previously showed that an ammoniainduced increase in intracellular chloride concentration (a model for ammonia neurotoxicity in hepatic coma) was recovered by DHH-B through GABAC receptor stimulation in primary cultured rat hippocampal neurons [5]. We also demonstrated that a GABAC receptor agonist suppressed ammonia-induced apoptosis in a GABAC receptor antagonist (TPMPA)-sensitive manner [12] and that GABAC receptor subunits were expressed in neurons in cultures and slices of rat brain hippocampi [7]. Thus, DHH-B may exert GABAC stimulation activity to protect neuronal death. We previously showed another model of pathophysiological neuronal death using amyloid  (A) proteins, i.e. pathogenic peptides of Alzheimer’s disease (AD) [11]. In ∗
Corresponding author. Tel.: +81 6 6993 9427; fax: +81 6 6992 2940. E-mail address: [email protected] (C. Inagaki).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.081
this system, pathophysiological concentration (≤ 10 nM) of A proteins, reduced neuronal Cl− -ATPase activity in cultured rat hippocampal neurons leading to the increases in the intraneuronal Cl− concentration ([Cl− ]i ) as well as glutamate-induced neurotoxicity, the relative potency being A25–35 > A1–42 > A1–40 . Since the activity of Cl− ATPase, which is a candidate for outwardly directed active Cl− -transporter [3], was found to be reduced in the brains of AD [2], this in vitro model seems to be a relevant one for neuronal death in AD. In the present study, we examined whether DHH-B had a protective effect against A-induced neurotoxicity via GABAC receptor stimulation. Hippocampal tissues of 19-day-old Wistar rat embryos were triturated in Ca2+ - and Mg2+ -free Hank’s solution as described previously [4]. The cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM l-glutamine, 100 IU/ml penicillin G sulfate, 10% fetal calf serum and 10% horse serum. The cells were seeded in poly-l-lysine-coated plastic dishes at a density of 2.55 × 105 cells/cm2 . After incubation for 2 days, the
B. Liu et al. / Neuroscience Letters 384 (2005) 44–47
cells were exposed to 5 M adenine-9-arabinofuranoside (Ara-A) in modified Eagle’s medium (MEM) supplemented with 2 mM l-glutamine and 5% horse serum for 4 days [1]. A25–35 and other reagents were applied for 2 days from the 8th day of culture. For monitoring glutamate excitotoxicity, the cells were exposed to glutamate (10 M, 10 min) on the 10th day of culture and assayed for cell viability after another 2-day culture in the media with or without A and other reagents. Plasma membrane-rich fractions were prepared on the 10th day of culture, as described previously [11]. Briefly, the cultured cells were homogenized in ice-cold buffer solution containing 0.25 M sucrose, 1 mM EDTA–Tris (pH 7.4), 12.5 mM Tris–2-(N-morpholino)-ethanesulfonic acid (Tris–Mes, pH 7.4), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 50 units/ml aprotinin, and centrifuged (10,000 × g, 15 min; 100,000 × g, 20 min). The pellets were suspended in 2 mM EDTA–Tris (pH 7.4), stirred for 30 min, and centrifuged (100,000 × g, 20 min). The resulting pellets were resuspended in 2 mM EDTA–Tris (pH 7.4) and used as plasma membrane-rich fractions. The protein concentration was determined by the method of Lowry et al. [8]. ATPase activities were determined by spectrophotometric measurement of the inorganic phosphate liberated. The incubation was carried out for 15 min at 37 ◦ C in 200 l reaction buffer containing 100 mM Tris–Mes (pH 7.4), 1 mM EDTA–Tris, 100 mM NaCl, 10 mM KCl, 6 mM magnesium acetate, 6 mM ATP–Tris (pH 7.4), 2 mM NaN3 and 3–6 g membrane protein with or without 1 mM ouabain and/or 0.3 mM ethacrynic acid (EA). The reaction was terminated by the addition of 10% trichloroacetic acid. Na+ /K+ -ATPase was calculated by subtracting the ATPase activity in the presence of 1 mM ouabain from the total ATPase activity. The activity in the presence of 1 mM ouabain and 0.3 mM EA was designated as anion-insensitive Mg2+ -ATPase. The difference
45
Fig. 2. Effects of DHH-B on the A25–35 -induced changes in [Cl− ]i in cultured rat hippocampal neurons. Neurons on the 8th day of culture were exposed to 10 nM A25–35 with or without 10 ng/ml DHH-B for 2 days. [Cl− ]i of pyramidal cell-like neurons was assayed fluorometrically using a Cl− -sensitive fluorescent dye (MQAE) on the 10th day of culture (n = 8–14, P < 0.05).
between ATPase activities in the presence and absence of 0.3 mM EA was designated as Cl− -ATPase activity. For the measurement of [Cl− ]i , dissociated hippocampal cells were cultured on poly-l-lysine-coated coverslips in plastic dishes, and treated as described above. The cells were washed with modified Krebs–HEPES buffer solution (pH 7.3) containing 128 mM NaCl, 2.5 mM KCl, 2.7 mM CaCl2 , 1 mM MgSO4 , 20 mM HEPES and 16 mM glucose, exposed to 5 mM N-(6-methoxyquinolyl)-acetoethyl ester (MQAE), a Cl− -sensitive fluorescent dye, in the same buffer solution for 1 h at 37 ◦ C, and then washed with a dye-free buffer solution. Fluorescence intensity of a single pyramidal cell-like
Fig. 1. Effects of dihydrohonokiol-B (DHH-B) on the A25–35 -induced changes in Cl− -ATPase in cultured rat hippocampal neurons. Neurons on the 8th day of culture were exposed to 10 nM A25–35 with or without 10 ng/ml DHH-B and/or 15 M TPMPA for 2 days. Cell membranes were prepared and assayed for ATPase activities of Cl− -ATPase, Na+ /K+ -ATPase and anion-insensitive Mg2+ -ATPase on the 10th day of culture (n = 5–14, P < 0.05, P < 0.01). Mean ± S.E. values of each control ATPase activity (mol Pi/mg protein/h) were: Cl− -ATPase, 4.8 ± 0.48; Na+ /K+ -ATPase, 5.3 ± 0.3; anion-insensitive Mg2+ ATPase, 18.2 ± 1.0, respectively.
46
B. Liu et al. / Neuroscience Letters 384 (2005) 44–47
Fig. 3. Effects of DHH-B on the A25–35 -induced increase in glutamate neurotoxicity. Neurons on the 8th day of culture were treated with or without 10 nM A25–35 and/or 10 ng/ml DHH-B for 2 days, and then exposed to 10 M glutamate for 10 min. WST-8 reducing activity (A) and release of LDH (B) were assayed 2 days after the glutamate exposure (n = 8–14, P < 0.05).
neuron was measured in a cell-chamber at room temperature using an inverted fluorescence microscope system. Excitation and emission wavelengths were collected at 360 and 510 nm, respectively. The fluorescence intensity of MQAE in each neuron was calibrated for Cl− concentration as described previously [4] and neuronal [Cl− ]i was estimated to be a value corresponding to the initial fluorescence intensity. Cell viability was assessed by measuring the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium monosodium salt (WST-8) reduction (Dojindo, Tokyo, Japan) reflecting mitochondrial activity, and lactate dehydrogenase (LDH) release (Wako, Osaka, Japan) from damaged plasma membranes. Data were presented as mean ± S.E. Differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by Fisher PLSD test. When only two groups were compared, Student’s t-test was used. The differences between means with P < 0.05 were considered to be significant. The sources of the materials used were as follows: DMEM and MEM were purchased from Nissui, Tokyo, Japan; Ara-A, A25–35 , PMSF, EA, ATP and ouabain were from Sigma, St. Louis, MO, USA; fetal calf serum, horse serum and l-glutamine were from ICN biomedicals, Aurora, OH, USA; (1,2,5,6,-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) was from Tocris (Avonmouth, UK). When the cultured rat hippocampal neurons were treated with 10 nM A25–35 for 2 days, Cl− -ATPase activity in plasma membrane fractions was significantly reduced to the level of 63% of control (Fig. 1A) without significant changes in the activities of plasma membrane Na+ /K+ -ATPase and anion-insensitive Mg2+ -ATPase (Fig. 1B and C). DHH-B prevented the A-induced inhibition of Cl− -ATPase activity at a concentration of 10 ng/ml, without any effects on the other two plasma membrane ATPase activities (Fig. 1). Treatment with DHH-B alone did not affect the Cl− -ATPase activity, suggesting that DHH-B recovered A-induced inhibition of Cl− -ATPase activity not through its own stimula-
tory effect. TPMPA, a GABAC receptor antagonist, did not affect the Cl− -ATPase activity by itself, but blocked the protective effect of DHH-B, suggesting that GABAC receptor stimulation may be involved in the protective mechanism of DHH-B against A-induced inhibition of Cl− -ATPase activity. Treatment with A25–35 and DHH-B did not change the morphology of cultured neurons and total amounts of membrane proteins (data not shown). When the cultured neurons were treated with 10 nM A25–35 for 2 days, [Cl− ]i increased to a level three times higher than that of control, and 10 ng/ml DHH-B completely blocked this effect (Fig. 2). Glutamate (10 M, 10 min), when applied to the Atreated cells, but not to non-treated cells, decreased the cell viability as observed in a decrease in WST-8 reducing activity (Fig. 3A) and an increase in LDH release (Fig. 3B). Co-application of DHH-B with A25–35 (10 nM) prevented the A plus glutamate-induced effects on the neuronal viability (Fig. 3A and B), suggesting that DHH-B has protective effects on A-induced enhancement of glutamate neurotoxicity, which may reflect a preapoptotic condition in early pathophysiological profiles of AD. Thus, DHH-B attenuated A-induced inhibition of neuronal Cl− -ATPase activity and resulting elevation of neuronal [Cl− ]i , as well as the A-induced aggravation of glutamate neurotoxicity. Since such a protective effect of DHH-B on Cl− -ATPase was reversed by GABAC receptor antagonist, TPMPA, DHH-B may exert the neuroprotective action through GABAC receptor stimulation. Collectively, DHH-B and GABAC receptor agonists can be one of the therapeutic and/or preventive strategies in Alzheimer’s disease patients.
Acknowledgments DHH-B was a kind gift from Dr. Yuji Maruyama at Research Division for Assessment of Complementary Medicine (NPO), Gunma 370-0867, Japan. This work was supported by grants from the Japanese Ministry of Education, Culture,
B. Liu et al. / Neuroscience Letters 384 (2005) 44–47
Sports, Science, and Technology; Japanese Private School Promotion Foundation; Salt Science Research Foundation, Japan; and a grant-in-aid for the 21st Century of Excellence (COE) program.
[6]
[7]
References [1] R.W. Burry, D.A. Kniss, R.H. Ho, Enhanced survival of apparent presynaptic elements on polylysine-coated beads by inhibition of non-neuronal cell proliferation, Brain Res. 346 (1985) 42–50. [2] N. Hattori, K. Kitagawa, T. Higashida, K. Yagyu, S. Shimohama, T. Wataya, G. Perry, M.A. Smith, C. Inagaki, Cl− -ATPase and Na+ /K+ ATPase activities in Alzheimer’s disease brains, Neurosci. Lett. 254 (1998) 141–144. [3] C. Inagaki, N. Hattori, K. Kitagawa, X.T. Zeng, K. Yagyu, Cl− ATPase in rat brain and kidney, J. Exp. Zool. 289 (2001) 224– 231. [4] M. Inoue, M. Hara, X.T. Zeng, T. Hirose, S. Ohnishi, T. Yasukura, T. Uriu, K. Omori, A. Minato, C. Inagaki, An ATP-driven Cl− pump regulates Cl− concentrations in rat hippocampal neurons, Neurosci. Lett. 134 (1991) 75–78. [5] T. Irie, E. Miyamoto, K. Kitagawa, Y. Maruyama, K. Inoue, C. Inagaki, An anxiolytic agent, dihydrohonokiol-B, inhibits ammonia-
[8]
[9] [10]
[11]
[12]
47
induced increases in the intracellular Cl− of cultured rat hippocampal neurons via GABAC receptors, Neurosci. Lett. 312 (2001) 121–123. H. Kuribara, E. Kishi, M. Kimura, S.T. Weintraub, Y. Maruyama, Comparative assessment of the anxiolytic-like activities of honokiol and derivatives, Pharmacol. Biochem. Behav. 67 (2000) 597–601. B. Liu, N. Hattori, B. Jiang, Y. Nakayama, N.Y. Zhang, B. Wu, K. Kitagawa, M. Taketo, H. Matsuda, C. Inagaki, Single cell RTPCR demonstrates differential expression of GABAC receptor rho subunits in rat hippocampal pyramidal and granule cells, Brain Res. Mol. Brain Res. 123 (2004) 1–6. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein Measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. Y. Maruyama, H. Kuribara, Overview of the pharmacological features of honokiol, CNS Drug Rev. 6 (2000) 35–44. Y. Maruyama, H. Kuribara, M. Morita, M. Yuzurihara, S.T. Weintraub, Identification of magnolol and honokiol as anxiolytic agents in extracts of saiboku-to, an oriental herbal medicine, J. Nat. Prod. 61 (1998) 135–138. K. Yagyu, K. Kitagawa, T. Irie, B. Wu, X.T. Zeng, N. Hattori, C. Inagaki, Amyloid beta proteins inhibit Cl− -ATPase activity in cultured rat hippocampal neurons, J. Neurochem. 78 (2001) 569–576. L. Yang, K. Omori, K. Omori, H. Otani, J. Suzukawa, C. Inagaki, GABAC receptor agonist suppressed ammonia-induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level, J. Neurochem. 87 (2003) 791–800.
Anxiolytic agent, dihydrohonokiol-B, recovers amyloid  protein-induced neurotoxicity in cultured rat hippocampal neurons Bing Liu, Naoki Hattori, Nan-Yan Zhang, Bo Wu, Li Yang, Kaori Kitagawa, Zheng-Mei Xiong, Takao Irie, Chiyoko Inagaki ∗ Department of Pharmacology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan Received 5 February 2005; received in revised form 14 April 2005; accepted 15 April 2005
Abstract The effects of anxiolytic honokiol derivative, dihydrohonokiol-B (DHH-B), on amyloid  protein (A25–35 , 10 nM)-induced changes in Cl− -ATPase activity, intracellular Cl− concentration ([Cl− ]i ) and glutamate neurotoxicity were examined in cultured rat hippocampal neurons. DHH-B (10 ng/ml) recovered A-induced decrease in neuronal Cl− -ATPase activity without any changes in the activities of Na+ /K+ ATPase and anion-insensitive Mg2+ -ATPase. A GABAC receptor antagonist (1,2,5,6,-tetrahydropyridin-4-yl) methyl-phosphinic acid (TPMPA, 15 M), inhibited the protective effects of DHH-B on Cl− -ATPase activity. DHH-B reduced A-induced elevation of [Cl− ]i as assayed using a Cl− -sensitive fluorescent dye, and prevented A-induced aggravation of glutamate neurotoxicity. These data suggest that DHH-B exerts the neuroprotective action against A through GABAC receptor stimulation. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Amyloid  protein; Dihydrohonokiol-B; Cl− -ATPase; GABAC receptor; Intracellular Cl− concentration
Dihydrohonokiol (DHH-B; 3 -(2 propenyl)-5-(1,1 -biphenyl)-2,-4 -diol), a partially reduced derivative of honokiol isolated from magnolia bark [9], exhibits a potent anxiolytic activity in mice [6] and has been used in traditional Chinese medicine [10]. We previously showed that an ammoniainduced increase in intracellular chloride concentration (a model for ammonia neurotoxicity in hepatic coma) was recovered by DHH-B through GABAC receptor stimulation in primary cultured rat hippocampal neurons [5]. We also demonstrated that a GABAC receptor agonist suppressed ammonia-induced apoptosis in a GABAC receptor antagonist (TPMPA)-sensitive manner [12] and that GABAC receptor subunits were expressed in neurons in cultures and slices of rat brain hippocampi [7]. Thus, DHH-B may exert GABAC stimulation activity to protect neuronal death. We previously showed another model of pathophysiological neuronal death using amyloid  (A) proteins, i.e. pathogenic peptides of Alzheimer’s disease (AD) [11]. In ∗
Corresponding author. Tel.: +81 6 6993 9427; fax: +81 6 6992 2940. E-mail address: [email protected] (C. Inagaki).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.081
this system, pathophysiological concentration (≤ 10 nM) of A proteins, reduced neuronal Cl− -ATPase activity in cultured rat hippocampal neurons leading to the increases in the intraneuronal Cl− concentration ([Cl− ]i ) as well as glutamate-induced neurotoxicity, the relative potency being A25–35 > A1–42 > A1–40 . Since the activity of Cl− ATPase, which is a candidate for outwardly directed active Cl− -transporter [3], was found to be reduced in the brains of AD [2], this in vitro model seems to be a relevant one for neuronal death in AD. In the present study, we examined whether DHH-B had a protective effect against A-induced neurotoxicity via GABAC receptor stimulation. Hippocampal tissues of 19-day-old Wistar rat embryos were triturated in Ca2+ - and Mg2+ -free Hank’s solution as described previously [4]. The cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM l-glutamine, 100 IU/ml penicillin G sulfate, 10% fetal calf serum and 10% horse serum. The cells were seeded in poly-l-lysine-coated plastic dishes at a density of 2.55 × 105 cells/cm2 . After incubation for 2 days, the
B. Liu et al. / Neuroscience Letters 384 (2005) 44–47
cells were exposed to 5 M adenine-9-arabinofuranoside (Ara-A) in modified Eagle’s medium (MEM) supplemented with 2 mM l-glutamine and 5% horse serum for 4 days [1]. A25–35 and other reagents were applied for 2 days from the 8th day of culture. For monitoring glutamate excitotoxicity, the cells were exposed to glutamate (10 M, 10 min) on the 10th day of culture and assayed for cell viability after another 2-day culture in the media with or without A and other reagents. Plasma membrane-rich fractions were prepared on the 10th day of culture, as described previously [11]. Briefly, the cultured cells were homogenized in ice-cold buffer solution containing 0.25 M sucrose, 1 mM EDTA–Tris (pH 7.4), 12.5 mM Tris–2-(N-morpholino)-ethanesulfonic acid (Tris–Mes, pH 7.4), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 50 units/ml aprotinin, and centrifuged (10,000 × g, 15 min; 100,000 × g, 20 min). The pellets were suspended in 2 mM EDTA–Tris (pH 7.4), stirred for 30 min, and centrifuged (100,000 × g, 20 min). The resulting pellets were resuspended in 2 mM EDTA–Tris (pH 7.4) and used as plasma membrane-rich fractions. The protein concentration was determined by the method of Lowry et al. [8]. ATPase activities were determined by spectrophotometric measurement of the inorganic phosphate liberated. The incubation was carried out for 15 min at 37 ◦ C in 200 l reaction buffer containing 100 mM Tris–Mes (pH 7.4), 1 mM EDTA–Tris, 100 mM NaCl, 10 mM KCl, 6 mM magnesium acetate, 6 mM ATP–Tris (pH 7.4), 2 mM NaN3 and 3–6 g membrane protein with or without 1 mM ouabain and/or 0.3 mM ethacrynic acid (EA). The reaction was terminated by the addition of 10% trichloroacetic acid. Na+ /K+ -ATPase was calculated by subtracting the ATPase activity in the presence of 1 mM ouabain from the total ATPase activity. The activity in the presence of 1 mM ouabain and 0.3 mM EA was designated as anion-insensitive Mg2+ -ATPase. The difference
45
Fig. 2. Effects of DHH-B on the A25–35 -induced changes in [Cl− ]i in cultured rat hippocampal neurons. Neurons on the 8th day of culture were exposed to 10 nM A25–35 with or without 10 ng/ml DHH-B for 2 days. [Cl− ]i of pyramidal cell-like neurons was assayed fluorometrically using a Cl− -sensitive fluorescent dye (MQAE) on the 10th day of culture (n = 8–14, P < 0.05).
between ATPase activities in the presence and absence of 0.3 mM EA was designated as Cl− -ATPase activity. For the measurement of [Cl− ]i , dissociated hippocampal cells were cultured on poly-l-lysine-coated coverslips in plastic dishes, and treated as described above. The cells were washed with modified Krebs–HEPES buffer solution (pH 7.3) containing 128 mM NaCl, 2.5 mM KCl, 2.7 mM CaCl2 , 1 mM MgSO4 , 20 mM HEPES and 16 mM glucose, exposed to 5 mM N-(6-methoxyquinolyl)-acetoethyl ester (MQAE), a Cl− -sensitive fluorescent dye, in the same buffer solution for 1 h at 37 ◦ C, and then washed with a dye-free buffer solution. Fluorescence intensity of a single pyramidal cell-like
Fig. 1. Effects of dihydrohonokiol-B (DHH-B) on the A25–35 -induced changes in Cl− -ATPase in cultured rat hippocampal neurons. Neurons on the 8th day of culture were exposed to 10 nM A25–35 with or without 10 ng/ml DHH-B and/or 15 M TPMPA for 2 days. Cell membranes were prepared and assayed for ATPase activities of Cl− -ATPase, Na+ /K+ -ATPase and anion-insensitive Mg2+ -ATPase on the 10th day of culture (n = 5–14, P < 0.05, P < 0.01). Mean ± S.E. values of each control ATPase activity (mol Pi/mg protein/h) were: Cl− -ATPase, 4.8 ± 0.48; Na+ /K+ -ATPase, 5.3 ± 0.3; anion-insensitive Mg2+ ATPase, 18.2 ± 1.0, respectively.
46
B. Liu et al. / Neuroscience Letters 384 (2005) 44–47
Fig. 3. Effects of DHH-B on the A25–35 -induced increase in glutamate neurotoxicity. Neurons on the 8th day of culture were treated with or without 10 nM A25–35 and/or 10 ng/ml DHH-B for 2 days, and then exposed to 10 M glutamate for 10 min. WST-8 reducing activity (A) and release of LDH (B) were assayed 2 days after the glutamate exposure (n = 8–14, P < 0.05).
neuron was measured in a cell-chamber at room temperature using an inverted fluorescence microscope system. Excitation and emission wavelengths were collected at 360 and 510 nm, respectively. The fluorescence intensity of MQAE in each neuron was calibrated for Cl− concentration as described previously [4] and neuronal [Cl− ]i was estimated to be a value corresponding to the initial fluorescence intensity. Cell viability was assessed by measuring the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)2H-tetrazolium monosodium salt (WST-8) reduction (Dojindo, Tokyo, Japan) reflecting mitochondrial activity, and lactate dehydrogenase (LDH) release (Wako, Osaka, Japan) from damaged plasma membranes. Data were presented as mean ± S.E. Differences between groups were evaluated by one-way analysis of variance (ANOVA) followed by Fisher PLSD test. When only two groups were compared, Student’s t-test was used. The differences between means with P < 0.05 were considered to be significant. The sources of the materials used were as follows: DMEM and MEM were purchased from Nissui, Tokyo, Japan; Ara-A, A25–35 , PMSF, EA, ATP and ouabain were from Sigma, St. Louis, MO, USA; fetal calf serum, horse serum and l-glutamine were from ICN biomedicals, Aurora, OH, USA; (1,2,5,6,-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) was from Tocris (Avonmouth, UK). When the cultured rat hippocampal neurons were treated with 10 nM A25–35 for 2 days, Cl− -ATPase activity in plasma membrane fractions was significantly reduced to the level of 63% of control (Fig. 1A) without significant changes in the activities of plasma membrane Na+ /K+ -ATPase and anion-insensitive Mg2+ -ATPase (Fig. 1B and C). DHH-B prevented the A-induced inhibition of Cl− -ATPase activity at a concentration of 10 ng/ml, without any effects on the other two plasma membrane ATPase activities (Fig. 1). Treatment with DHH-B alone did not affect the Cl− -ATPase activity, suggesting that DHH-B recovered A-induced inhibition of Cl− -ATPase activity not through its own stimula-
tory effect. TPMPA, a GABAC receptor antagonist, did not affect the Cl− -ATPase activity by itself, but blocked the protective effect of DHH-B, suggesting that GABAC receptor stimulation may be involved in the protective mechanism of DHH-B against A-induced inhibition of Cl− -ATPase activity. Treatment with A25–35 and DHH-B did not change the morphology of cultured neurons and total amounts of membrane proteins (data not shown). When the cultured neurons were treated with 10 nM A25–35 for 2 days, [Cl− ]i increased to a level three times higher than that of control, and 10 ng/ml DHH-B completely blocked this effect (Fig. 2). Glutamate (10 M, 10 min), when applied to the Atreated cells, but not to non-treated cells, decreased the cell viability as observed in a decrease in WST-8 reducing activity (Fig. 3A) and an increase in LDH release (Fig. 3B). Co-application of DHH-B with A25–35 (10 nM) prevented the A plus glutamate-induced effects on the neuronal viability (Fig. 3A and B), suggesting that DHH-B has protective effects on A-induced enhancement of glutamate neurotoxicity, which may reflect a preapoptotic condition in early pathophysiological profiles of AD. Thus, DHH-B attenuated A-induced inhibition of neuronal Cl− -ATPase activity and resulting elevation of neuronal [Cl− ]i , as well as the A-induced aggravation of glutamate neurotoxicity. Since such a protective effect of DHH-B on Cl− -ATPase was reversed by GABAC receptor antagonist, TPMPA, DHH-B may exert the neuroprotective action through GABAC receptor stimulation. Collectively, DHH-B and GABAC receptor agonists can be one of the therapeutic and/or preventive strategies in Alzheimer’s disease patients.
Acknowledgments DHH-B was a kind gift from Dr. Yuji Maruyama at Research Division for Assessment of Complementary Medicine (NPO), Gunma 370-0867, Japan. This work was supported by grants from the Japanese Ministry of Education, Culture,
B. Liu et al. / Neuroscience Letters 384 (2005) 44–47
Sports, Science, and Technology; Japanese Private School Promotion Foundation; Salt Science Research Foundation, Japan; and a grant-in-aid for the 21st Century of Excellence (COE) program.
[6]
[7]
References [1] R.W. Burry, D.A. Kniss, R.H. Ho, Enhanced survival of apparent presynaptic elements on polylysine-coated beads by inhibition of non-neuronal cell proliferation, Brain Res. 346 (1985) 42–50. [2] N. Hattori, K. Kitagawa, T. Higashida, K. Yagyu, S. Shimohama, T. Wataya, G. Perry, M.A. Smith, C. Inagaki, Cl− -ATPase and Na+ /K+ ATPase activities in Alzheimer’s disease brains, Neurosci. Lett. 254 (1998) 141–144. [3] C. Inagaki, N. Hattori, K. Kitagawa, X.T. Zeng, K. Yagyu, Cl− ATPase in rat brain and kidney, J. Exp. Zool. 289 (2001) 224– 231. [4] M. Inoue, M. Hara, X.T. Zeng, T. Hirose, S. Ohnishi, T. Yasukura, T. Uriu, K. Omori, A. Minato, C. Inagaki, An ATP-driven Cl− pump regulates Cl− concentrations in rat hippocampal neurons, Neurosci. Lett. 134 (1991) 75–78. [5] T. Irie, E. Miyamoto, K. Kitagawa, Y. Maruyama, K. Inoue, C. Inagaki, An anxiolytic agent, dihydrohonokiol-B, inhibits ammonia-
[8]
[9] [10]
[11]
[12]
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induced increases in the intracellular Cl− of cultured rat hippocampal neurons via GABAC receptors, Neurosci. Lett. 312 (2001) 121–123. H. Kuribara, E. Kishi, M. Kimura, S.T. Weintraub, Y. Maruyama, Comparative assessment of the anxiolytic-like activities of honokiol and derivatives, Pharmacol. Biochem. Behav. 67 (2000) 597–601. B. Liu, N. Hattori, B. Jiang, Y. Nakayama, N.Y. Zhang, B. Wu, K. Kitagawa, M. Taketo, H. Matsuda, C. Inagaki, Single cell RTPCR demonstrates differential expression of GABAC receptor rho subunits in rat hippocampal pyramidal and granule cells, Brain Res. Mol. Brain Res. 123 (2004) 1–6. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein Measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. Y. Maruyama, H. Kuribara, Overview of the pharmacological features of honokiol, CNS Drug Rev. 6 (2000) 35–44. Y. Maruyama, H. Kuribara, M. Morita, M. Yuzurihara, S.T. Weintraub, Identification of magnolol and honokiol as anxiolytic agents in extracts of saiboku-to, an oriental herbal medicine, J. Nat. Prod. 61 (1998) 135–138. K. Yagyu, K. Kitagawa, T. Irie, B. Wu, X.T. Zeng, N. Hattori, C. Inagaki, Amyloid beta proteins inhibit Cl− -ATPase activity in cultured rat hippocampal neurons, J. Neurochem. 78 (2001) 569–576. L. Yang, K. Omori, K. Omori, H. Otani, J. Suzukawa, C. Inagaki, GABAC receptor agonist suppressed ammonia-induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level, J. Neurochem. 87 (2003) 791–800.