A Novel Effect of Bifemelane, a Nootropic Drug, on Intracellular
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J Pharmacol Sci 100, 126 – 132 (2006) Journal of Pharmacological Sciences ©2006 The Japanese Pharmacological Society Full Paper A Novel Effect of Bifemelane, a Nootropic Drug, on Intracellular Ca2+ Levels in Rat Cerebral Astrocytes Yoshitoku Yoshida1, Akira Nakane1, Mitsuhiro Morita1, and Yoshihisa Kudo1,* 1School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Received November 10, 2005; Accepted December 8, 2005 Abstract. We investigated the effects of bifemelane, a nootropic drug, on the intracellular 2+ 2+ calcium concentration ([Ca ]i) in rat cerebral astrocytes using a Ca imaging device. At concentrations of 10 – 30 µM, bifemelane induced a slow onset and small increase in the 2+ [Ca ]i, while at higher concentrations (100 – 300 µM), it induced a rapid transient increase in the 2+ [Ca ]i during administration and a second large increase was seen during drug washout. The first peak was observed in Ca2+-free medium, but its onset was significantly delayed, and no second peak was seen. Neither of these effects was seen in cells treated with thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, in Ca2+-free medium. When thapsigargin- 2+ 2+ treated astrocytes were returned to normal medium containing Ca (1.8 mM), the [Ca ]i increased significantly, and this effect was reversely inhibited by bifemelane. We conclude that bifemelane causes the first peak by stimulating release from intracellular Ca2+ stores and the second by capacitive entry through store–operated Ca2+ channels. Although the detail mecha- nisms of action of the drug are still unknown, bifemelane will be provided as a pharmacological tool for basic studies on astrocytes. Keywords: bifemelane, astrocyte, Ca2+, capacitive entry, nootropic drug Introduction melane on cultured rat hippocampal astrocytes, we found that the drug increased the intracellular calcium Bifemelane was prescribed in Japan as a therapeutic concentration with a very slow time-course. Since recent drug for early stage dementia due to senescence, studies on astrocytes have revealed the dynamic roles of ischemic brain damage, or Alzheimer’s disease during these cells in regulating synaptic activities by releasing the period of 1996 – 1998. Its effectiveness was neurotransmitters, such as glutamate and ATP (6 – 10), 2+ evaluated in basic and clinical studies which showed a the effect of bifemelane on the [Ca ]i may explain positive improvement of age-related cognitive disorders its therapeutic effects. In the present study, we examined in patients and in experimental dementia models, its effects on astrocytes cultured in astrocyte defined showing, for example, improvement in long-term poten- medium which have stable Ca2+ responses to neuro- tiation in the hippocampus after ischemic injury and transmitters (11). protection against glutamate-induced neurotoxicity (1 – 5). However, its effectiveness as a therapeutic agent Materials and Methods for dementia and psychological disorders due to brain ischemia was doubted following double-blindfolded Animal experiments were performed in accordance tests on patients and it was deleted from the list of with The Japanese Pharmacological Society “Guiding therapeutic medicines in 1998. Principles for the Care and Use of Laboratory Animals” In preliminary experiments on the effects of bife- and were approved by the Animal Care Committee of Tokyo University of Pharmacy and Life Science. *Corresponding author. [email protected] Published online in J-STAGE: February 11, 2006 DOI: 10.1254/jphs.FP0050926 126 Effect of Bifemelane on Astrocytes 127 Primary rat cerebral cortex cultures Intracellular Ca2+ measurement by an imaging method Cerebral cells were cultured according to a previously Three types of extracellular medium were used in the described method (12). Briefly, the cerebral cortex was physiological experiments; these were i) basal salt dissected from 18-day-old embryonic Wistar rats solution (BSS; 130 mM NaCl, 5.4 mM KCl, 5.5 mM (purchased as pregnant rats from Japan SLC, Inc., glucose, 2 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, Shizuoka) and incubated for 20 min at 37°C with 0.25% pH 7.4); ii) nominally Ca2+-free medium (BSS without 2+ trypsin (Difco & BD, Franklin Lakes, NJ, USA) and the CaCl2); and iii) completely Ca -free medium 0.02% DNase I (Sigma-Aldrich, Tokyo). The cells were (nominally Ca2+-free medium containing 5 mM EGTA). then dissociated by repeated pipetting and plated on To load the calcium indicator, the cells were incubated glass cover slips (Matsunami #1; Matsunami Glass, Ind., for 45 min at 30°C in BSS containing fura-2/AM Ltd., Kishiwada) with four wells (15 mm in diameter) (7.5 µM), washed three times, and incubated for 20 min formed by an adhesive silicon rubber template at room temperature (23°C–26°C). (Flexiperm; Heraeus, Hanau, Germany) at a density of The cells placed in an observation bath (working 5 × 105 cells per well. The cultures were maintained volume: about 0.3 ml) are perfused by BSS continuously for one week in an incubator in Dulbecco’s modified at a rate of 2 ml/min. The chemicals to be tested are Eagle’s medium (DMEM; cat. No. 430-2100, containing applied by perfusion at the same rate which will replace no L-glutamate or L-aspartate; Gibco-BRL (Life the bathing medium into the test condition within 10 s. Technologies, Inc., Rockville, MD, USA), 5% v/v Fluorescence images were obtained using an inverted precolostrum new born calf serum (Mitsubishi Chemical microscope (IX-70; Olympus, Tokyo), a filter exchang- Co., Tokyo), containing 5% v/v heat-inactivated horse ing device (OSP-EXA, Olympus), and a cooled CCD serum (Gibco-BRL). camera (C6790; Hamamatsu Photonics, Hamamatsu); and the images were analyzed using an image analyzer Astrocyte specific cultures from rat cerebral cortex designed for Ca2+ imaging with software for Ca2+ Astrocytes were isolated from the cerebral cortex of measurement (AQUACOSMOS, Hamamatsu Photonics). postnatal day 1 Wistar rats, using a modification of a previously described method (11). Briefly, brain cells Drugs used were prepared from the cortices of 10 – 15 brains by Bifemelane hydrochloride (4-(O-benzylphenoxy)-N- trypsinization, trituration, and filtration and then seeded methylbutylamine hydrochloride) was a gift from at a concentration of 1.3 × 104 cells/cm2 in 75 cm2 Mitsubishi Welpharma Co. (Tokyo). trans-ACPD plastic flasks (Sumitomo Bakelite, Tokyo). The cells [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t- were cultured at 37°C in 5% CO2 humidified air in basal ACPD)], N-methyl-D-aspartate (NMDA), and ethylene Eagle’s medium containing 10% fetal calf serum (FCS; glycol bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic Iquitech-Bio, Ingram, TX, USA), with a medium change acid (EGTA) were obtained from Sigma Chemicals Co. every three days. After 12 days in culture, the resulting (St. Louis, MO, USA). Fura-2/AM was purchased from mixed glial culture was shaken at 260 rpm for 18 h at Dojindo Co. (Kumamoto). Thapsigargin was purchased 37°C and then rinsed with medium to remove non- from Tocris Bioscience (Avonmouth, UK). astrocytic cells. The adherent cells were sub-cultured by trypsinization and seeded at the indicated densities in Results DMEM (Gibco-BRL) containing 25 mM HEPES, pH 7.4, and 10% FCS onto 12 mm diameter coverslips Effects of bifemelane on primary cerebral cultures (Matsunami) coated with poly-D-lysine (10 µg/ml) in To examine the effect of bifemelane on the Ca2+ 24-well plates (Sumitomo Bakelite). After 48 h, the dynamics of brain cells, we first applied the agent to medium was changed to astrocyte-defined medium fura-2 loaded primary cultures from the rat cerebral (ADM) (Gibco-BRL), a modified version of that cortex, which included both neuronal and glial cells, previously reported (11), consisting of DMEM contain- and compared the effect with those of agonists for two ing 25 mM HEPES, pH 7.4, 1 mM pyruvate, 2 mM different glutamate receptors, NMDA (an ionotropic glutamine, 50 mg/ml of human apotransferrin (Gibco- glutamate receptor agonist) and t-ACPD (a metabotropic BRL), 10 ng/ml of D-biotin, 5.2 ng/ml of sodium glutamate receptor agonist). Bifemelane at a concentra- 2+ selenite, 1.5 mg/ml of bovine fibronectin (Gibco-BRL), tion of 300 µM caused an obvious increase in the [Ca ]i 0.5 mg/ml of heparan sulfate, 5 mg/ml of insulin in about 20% of these cells, the majority of which (Gibco-BRL), 10 ng/ml of epidermal growth factor also responded to t-ACPD and were different from those (Gibco-BRL), and 5 ng/ml of basic fibroblast growth that responded to NMDA (Fig. 1). Since our previous factor (Gibco-BRL). studies on primary cultured brain cells showed that the 128 Y Yoshida et al Fig. 1. Imaging analysis of the effects of glutamate agonists and bifemelane on rat cerebral cells in primary culture. The cells were loaded with fura-2/AM for 45 min and then examined under a fluores- cence microscope equipped with an image analysis system (see Materials and Methods). The same cells were then sequentially exposed for 30 s to NMDA (100 µM), t-ACPD (100 µM), or bifemelane (300 µM) with 10-min washes between treatments. The cells were examined immedi- ately after the 30-s treatment. As shown in color scale, cells that respond to the drugs are reddish and those that do not are bluish (F340/F380 ratio). 2+ NMDA-positive cells in these cultures are neuronal cells Source of the increased [Ca ]i induced by bifemelane and that the t-ACPD-positive cells are mainly astrocytes To determine the source of the calcium producing 2+ (13, 14), these results indicate that the bifemelane- the increased [Ca ]i, we pretreated the cells with sensitive cells are astrocytes. nominally Ca2+-free BSS for 90 s before adding bife- melane.