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Effect of magnesium on calcium-dependent brain function that prolongs ethanol-induced sleeping time in mice
Neuroscience Letters 294 (2000) 5±8
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Effect of magnesium on calcium-dependent brain function that prolongs ethanol-induced sleeping time in mice Den'etsu Sutoo*, Kayo Akiyama Institute of Medical Science, University of Tsukuba, Tsukuba 305-8575, Japan Received 23 August 2000; received in revised form 12 September 2000; accepted 12 September 2000
Abstract The effect of intracerebroventricular (i.c.v.) administration of magnesium on calcium- and dopamine-dependent brain function was investigated behaviorally and biochemically. The duration of ethanol-induced sleeping time in mice was prolonged following i.c.v. administration of calcium chloride (10 mmol/kg) or dopamine (30 nmol/mouse); however, it was not affected by magnesium chloride (10 or 40 mmol/kg). The ability of calcium to prolong ethanol-induced sleeping time was inhibited by the administration of magnesium chloride. The brain dopamine level in mice was signi®cantly increased following i.c.v. administration of calcium chloride. Taking into consideration these results and those from previous studies, it is suggested that calcium enhances dopamine synthesis in the brain through a calmodulin-dependent system, and the increase in dopamine level prolongs ethanol-induced sleeping time. However, magnesium inhibits dopamine release. Therefore, magnesium may inhibit calcium-dependent brain function through dopaminergic neurons, and consequently reduce the effect of calcium on ethanol activity. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcium/calmodulin; Dopamine release; Dopamine synthesis; Ethanol-induced sleep; Magnesium; Mice brain; Tyrosine hydroxylase
We have investigated pharmacologically and behaviorally the central dopamine-synthesizing mechanism through a calcium/calmodulin-dependent system. When ethanol is injected i.p. at a dose of 4.5 g/kg, mice lose their righting re¯ex and lapse into a state resembling sleep for approximately 1 h. The duration of ethanol-induced sleeplike state (ethanol-induced sleeping time) was prolonged following the intracerebroventricular (i.c.v.) administration of dopamine, norepinephrine or monoamine oxidase inhibitors. The administration of calcium chloride also prolonged the ethanol-induced sleeping time. This effect of calcium was abolished by the administration of calmodulin antagonists, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) and tri¯uoperazine, or a tyrosine hydroxylase inhibitor, a-methyltyrosine (aMPT). These results suggest that calcium may activate tyrosine hydroxylase in the brain via a calmodulin-dependent pathway, leading to increased levels of catecholamines in the brain that may subsequently prolong the ethanol-induced sleeping time [8,9]. This * Corresponding author. Tel.: 181-298-53-3113; fax: 181-29854-9817. E-mail address: [email protected] (D. Sutoo).
conclusion was con®rmed by biochemical and quantitative immunohistochemical techniques. Although the norepinephrine level in the mouse brains did not change signi®cantly, dopamine levels in the lateral part of the neostriatum and nucleus accumbens were increased following the i.c.v. administration of calcium. Moreover, the effect of calcium to enhance dopamine synthesis was abolished by W-7 [8,13]. The brain regions in which the amount of dopamine was increased by calcium were those areas with high levels of calmodulin and tyrosine hydroxylase [14]. These results suggest that the calcium/calmodulin-dependent system activates dopamine synthesis in speci®c brain regions such as the neostriatum and nucleus accumbens. We have applied these fundamental studies concerning calcium-dependent dopamine synthesizing system to elucidate a few brain functions and mechanisms for understanding of some diseases [15±18]. To elucidate the mechanism of metal-toxicosis, the effect of various metal ions on the ethanol-induced sleeping time in mice was investigated [10]. The ethanol-induced sleeping time was prolonged following the i.c.v. injection of cadmium chloride, zinc chloride, manganese chloride, or mercury chloride, as well as calcium chloride. The ability of these cations to prolong
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 53 7- 8
6
D. Sutoo, K. Akiyama / Neuroscience Letters 294 (2000) 5±8
ethanol-induced sleeping time was inhibited by the administration of W-7 or aMPT. These ®ndings show that cadmium, zinc, manganese, or mercury activates cerebral calmodulin which increases the activity of tyrosine hydroxylase, and that the subsequent increase in dopamine levels in the brain prolongs the ethanol-induced sleeping time. Moreover, it was con®rmed by quantitative immunohistochemical [16] and 1H-nuclear magnetic resonance ( 1H-NMR) studies [12] that cadmium, zinc, manganese and mercury bind to calmodulin and activate it, as calcium does. However, magnesium chloride is different from the other divalent cations in that it does not signi®cantly activate calmodulin [11,12] and does not affect the duration of the ethanol-induced sleeping time [10]. In the present study, to elucidate the relationships between calcium and magnesium with the respect to brain functions, the effect of magnesium on the calcium- and dopamine-dependent brain function was investigated. Male mice of the ddY strain were provided by Japan SLC, Inc. (Shizuoka, Japan). The mice weighed approximately 25 g (5 weeks old) at the start of the experiment and all animals were used only once. They were housed in groups of 8±10 in stainless steel cages at room temperature (22 ^ 28C) for more than one week before use in the experiments, and were exposed to a 12-h light±dark cycle. Food and water were provided ad libitum. All animals were handled in compliance with the `Guiding Principles for the Care and Use of Laboratory Animals' formulated by the Japanese Pharmacological Society. Experiments were carried out between 1000 and 1600 h. Ethanol (30% in saline, v/v) was injected i.p. at a dose of 4.5 g/kg, and the duration of ethanol-induced sleeping time was measured. Sleeping time duration is de®ned as the time from the loss of the righting re¯ex to the time when the righting re¯ex is restored, occurring twice within 30 s. Room temperature, extraneous sounds, and ethanol concentrations were carefully controlled. First, the sleeping times in mice treated with calcium, magnesium, dopamine, aMPT and saline were compared. Next, the effects of administration of calcium together with magnesium or with aMPT on the ethanol-induced sleeping time were analyzed. Mice were injected into the lateral ventricle with calcium chloride (10 mmol/kg), magnesium chloride (10 or 40 mmol/kg) or dopamine (30 nmol/mouse), 30 min, 30 min or 15 min, respectively, before the injection of ethanol. a-Methyltyrosine (100 mg/kg) was injected i.p. 24 h before the injection of ethanol. All chemicals and drugs were dissolved in physiological saline. The doses and other conditions for administration were based on the results of our previous tests [8±10], and i.c.v. injections were performed in conscious mice, as described by Clark et al. [2] using an injection volume of 5 ml/mouse. The data were analyzed using ANOVA and Newman-Keuls t-test for subsequent comparisons among groups. The effects of magnesium or calcium on the brain dopamine level in mice were analyzed. High-performance liquid
chromatography (HPLC) technique was employed to determine the dopamine content in the brain. Four animal groups (9±11 mice/group), i.e. mice given an i.c.v. injection of calcium chloride (10 mmol/kg), magnesium chloride (40 mmol/kg), calcium chloride plus magnesium chloride, or saline were prepared. Thirty minutes after injection, the brains were taken out quickly and divided into two parts, the cerebrum and rhombencephalon, as described in our previous report [8]. Only the cerebrum was analyzed in the present study. The brain tissue was homogenized with 0.1 M perchloric acid (4:1, v/w) in a glass homogenizer. After centrifugation for 10 min at 25 000 £ g (48C), the supernatant ¯uid was diluted (1:400) with distilled deionized water and analyzed using an automatic catecholamine analyzer (Tosoh HLC-725CA, Tokyo, Japan). As shown in Fig. 1, injection of calcium chloride (10 mmol/kg) or dopamine (30 nmol/mouse) prolonged ethanol-induced sleeping time in mice by 56% (P , 0:01) or 51% (P , 0:01), respectively, when compared to the saline-treated control group. The ethanol-induced sleeping time did not change following the administration of magnesium chloride (10 or 40 mmol/kg) or aMPT alone. Moreover, the ability of calcium to enhance ethanol-induced sleeping time was inhibited by administration of magnesium chloride or aMPT. Namely, ethanol-induced sleeping time in mice treated with magnesium chloride (40 mmol/kg) together with calcium chloride, or with aMPT (100 mg/kg) 24 h before
Fig. 1. Effects of calcium, magnesium, dopamine and aMPT on the duration of ethanol-induced sleeping time in mice. Mice were injected i.c.v. with calcium chloride, magnesium chloride or dopamine, 30, 30 or 15 min, respectively, before the injection of ethanol. a-Methyltyrosine was injected i.p. 24 h before the injection of ethanol. The administration of calcium chloride (10 mmol/kg) or dopamine (30 nmol/mouse) prolonged the duration of ethanol (4.5 g/kg)-induced sleeping time. However, it was not prolonged following the administration of magnesium chloride (10 or 40 mmol/kg). Moreover, the ability of calcium to prolong ethanol-induced sleeping time was inhibited by the administration of magnesium chloride (40 mmol/kg) or aMPT (100 mg/kg). Each value represents the mean ^ SEM of 11±14 mice. *P , 0:01 compared to saline-treated mice group; d P , 0:01 compared to calcium chloride-treated mice group, using Newman±Keuls t-test.
D. Sutoo, K. Akiyama / Neuroscience Letters 294 (2000) 5±8
Fig. 2. Effects of i.c.v. administration of magnesium and calcium on the cerebral dopamine content in mice. The administration of calcium chloride (10 mmol/kg) or magnesium chloride (40 mmol/kg) increased the cerebral dopamine level. Each value represents the mean ^ SEM of 9±11 mice. *P , 0:05 compared to saline-treated mice group, using Newman±Keuls t-test.
the injection of calcium chloride was signi®cantly shorter by 36% (P , 0:01) or 33% (P , 0:01), respectively, when compared to mice treated with calcium chloride alone. The effects of calcium or magnesium on the cerebral dopamine level in mice are shown in Fig. 2. The dopamine level signi®cantly increased following the administration of calcium chloride (10 mmol/kg) or magnesium chloride (40 mmol/kg) by 9% (P , 0:05) and 8% (P , 0:05), respectively, as compared with the control. The dopamine level slightly increased following the administration of mixture solution of calcium and magnesium, but it did not signi®cantly change. In this study, the duration of ethanol-induced sleeping time was prolonged following the i.c.v. administration of calcium chloride, but it was not affected by magnesium chloride. These ®ndings agreed with our previous observations [10]. Here, the ability of calcium to prolong ethanolinduced sleeping time was signi®cantly inhibited by the i.c.v. administration of magnesium chloride. In the biochemical test, on the other hand, the brain dopamine level increased following the i.c.v. administration of magnesium, as well as the administration of calcium. These ®ndings suggest that dopamine synthesis in the brain is enhanced by not only calcium but also magnesium, and that the function of increased dopamine is inactivated by magnesium. These ®ndings are supported by previous reports. Tyrosine hydroxylase was activated and dopamine synthesis was enhanced by magnesium via cAMP-dependent protein kinase [3,6]. The dopamine release stimulated by N-methyl-d-aspartate [4,5], nitric oxide [20], or potassium chloride [20] was inhibited by magnesium. Moreover, the inhibition of dopamine release by adenosine was mediated by the adenosine A1 receptor, and this inhibition was enhanced by magnesium [7]. It must be investigated in the future whether or not the sites of dopamine synthesis activated by calcium, dopamine synthesis activated by magnesium, and dopamine release inhibited by magnesium are the same. The relationships between calcium and magnesium in the dopaminergic system and the calmodulin system are
7
summarized as follows: (a) calcium binds to calmodulin and activates it, however magnesium does not completely bind to calmodulin therefore it does not activate it [12]; (b) magnesium affects the conformation of interface sites for target enzymes of calcium-bound calmodulin, as the calmodulin antagonist does [11]. Thus, magnesium inhibits the calmodulin-dependent activation of certain enzymes [1,19]; (c) calcium activates dopamine synthesis through calmodulin-dependent protein kinase [9,17], and magnesium activates dopamine synthesis through cAMP-dependent protein kinase [3,6]; and, (d) calcium enhances the dopamine release in synapse, whereas magnesium inhibits it [4,5,7,20]. In conclusion, it is suggested that calcium enhances dopamine synthesis in the brain through a calmodulin-dependent system, and the increase in dopamine level prolongs ethanol-induced sleeping time. However, magnesium inhibits dopamine release. Therefore, magnesium may inhibit calcium-dependent brain function through dopaminergic neurons, and consequently reduce the effect of calcium on ethanol activity. The authors would like to thank Mr Yoshimasa Itoh and Mr Jiro Ishii, Tosoh Corporation, Tokyo, Japan, for technical assistance in HPLC. [1] Brostrom, C.O. and Wolff, D.J., Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3 0 ,5 0 -monophosphate and guanosine 3 0 ,5 0 monophosphate as substrates, Arch. Biochem. Biophys., 172 (1976) 301±311. [2] Clark, W.G., Vivonia, C.A. and Baxter, C.F., Accurate freehand injection into the lateral brain ventricle of the conscious mouse, J. Appl. Physiol., 25 (1968) 319±321. [3] Edelman, A.M., Raese, J.D., Lazar, M.A. and Barchas, J.D., Tyrosine hydroxylase: studies on the phosphorylation of a puri®ed preparation of the brain enzyme by the cyclic AMPdependent protein kinase, J. Pharmacol. Exp. Ther., 216 (1981) 647±653. [4] Johnson, K.M. and Jeng, Y.J., Pharmacological evidence for N-methyl-d-aspartate receptors on nigrostriatal dopaminergic nerve terminals, Can. J. Physiol. Pharmacol., 69 (1991) 1416±1421. [5] MartIÂnez-Fong, D., Rosales, M.G., GoÂngora-Alfaro, J.L., HernaÂndez, S. and Aceves, J., NMDA receptor mediates dopamine release in the striatum of unanesthetized rats as measured by brain microdialysis, Brain Res., 595 (1992) 309±315. [6] Morgenroth III, V.H., Hegstrand, L.R., Roth, R.H. and Greengard, P., Evidence for involvement of protein kinase in the activation by adenosine 3 0 ,5 0 -monophosphate of brain tyrosine 3-monooxygenase, J. Biol. Chem., 250 (1975) 1946± 1948. [7] Okada, M., Mizuno, K., Okuyama, M. and Kaneko, S., Magnesium ion augmentation of inhibitory effects of adenosine on dopamine release in the rat striatum, Psychiatry Clin. Neurosci., 50 (1996) 147±156. [8] Sutoo, D. and Sano, K., Modulating effects of biogenic amines on calcium and ethanol-induced sleeping time, Alcohol, 1 (1984) 141±144. [9] Sutoo, D., Akiyama, K. and Iimura, K., Effect of calmodulin antagonists on calcium and ethanol-induced sleeping time in mice, Pharmacol. Biochem. Behav., 23 (1985) 627±631.
8
D. Sutoo, K. Akiyama / Neuroscience Letters 294 (2000) 5±8
[10] Sutoo, D., Akiyama, K. and Iimura, K., The ability of divalent cations to enhance ethanol-induced sleeping time, Alcohol, 3 (1986) 69±72. [11] Sutoo, D., Akiyama, K., Fujii, N. and Matsushita, K., 1H-NMR studies of calmodulin: various divalent cation-induced conformational changes, Kitasato Arch. Exp. Med., 61 (1988) 149±160. [12] Sutoo, D., Akiyama, K., Fujii, N. and Matsushita, K., 1H-NMR studies of calmodulin: the character of the calcium binding sites, Jpn. J. Pharmacol., 50 (1989) 217±227. [13] Sutoo, D., Akiyama, K. and Geffard, M., Central dopaminesynthesis regulation by the calcium-calmodulin-dependent system, Brain Res. Bull., 22 (1989) 565±569. [14] Sutoo, D., Akiyama, K., Yabe, K. and Kohno, K., Multiple analysis of tyrosine hydroxylase and calmodulin distributions in the forebrain of the rat using a microphotometry system, Brain Res. Bull., 26 (1991) 973±982. [15] Sutoo, D., Akiyama, K. and Takita, H., The effect of convulsions on the recti®cation of central nervous system disorders in epileptic mice, Physiol. Behav., 52 (1992) 865±872.
[16] Sutoo, D., Disturbances of brain function by exogenous cadmium, In R.L. Isaacson and K.F. Jensen (Eds.), The Vulnerable Brain and Environmental Risks, Vol. 3, Toxins in Air and Water, Plenum Press, New York, 1994, pp. 281± 300. [17] Sutoo, D. and Akiyama, K., Regulation of blood pressure with calcium-dependent dopamine synthesizing system in the brain and its related phenomena, Brain Res. Rev., 25 (1997) 1±26. [18] Sutoo, D., Akiyama, K. and Matsui, A., Gastric ulcer formation in cold-stressed mice related to a central calciumdependent-dopamine synthesizing system, Neurosci. Lett., 249 (1998) 9±12. [19] Weiss, B., Differential activation and inhibition of the multiple forms of cyclic nucleotide phosphodiesterase, Adv. Cyclic Nucleotide Res., 5 (1975) 195±211. [20] West, A.R. and Galloway, M.P., Nitric oxide and potassium chloride-facilitated striatal dopamine ef¯ux in vivo: role of calcium-dependent release mechanisms, Neurochem. Int., 33 (1999) 493±501.
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Effect of magnesium on calcium-dependent brain function that prolongs ethanol-induced sleeping time in mice Den'etsu Sutoo*, Kayo Akiyama Institute of Medical Science, University of Tsukuba, Tsukuba 305-8575, Japan Received 23 August 2000; received in revised form 12 September 2000; accepted 12 September 2000
Abstract The effect of intracerebroventricular (i.c.v.) administration of magnesium on calcium- and dopamine-dependent brain function was investigated behaviorally and biochemically. The duration of ethanol-induced sleeping time in mice was prolonged following i.c.v. administration of calcium chloride (10 mmol/kg) or dopamine (30 nmol/mouse); however, it was not affected by magnesium chloride (10 or 40 mmol/kg). The ability of calcium to prolong ethanol-induced sleeping time was inhibited by the administration of magnesium chloride. The brain dopamine level in mice was signi®cantly increased following i.c.v. administration of calcium chloride. Taking into consideration these results and those from previous studies, it is suggested that calcium enhances dopamine synthesis in the brain through a calmodulin-dependent system, and the increase in dopamine level prolongs ethanol-induced sleeping time. However, magnesium inhibits dopamine release. Therefore, magnesium may inhibit calcium-dependent brain function through dopaminergic neurons, and consequently reduce the effect of calcium on ethanol activity. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcium/calmodulin; Dopamine release; Dopamine synthesis; Ethanol-induced sleep; Magnesium; Mice brain; Tyrosine hydroxylase
We have investigated pharmacologically and behaviorally the central dopamine-synthesizing mechanism through a calcium/calmodulin-dependent system. When ethanol is injected i.p. at a dose of 4.5 g/kg, mice lose their righting re¯ex and lapse into a state resembling sleep for approximately 1 h. The duration of ethanol-induced sleeplike state (ethanol-induced sleeping time) was prolonged following the intracerebroventricular (i.c.v.) administration of dopamine, norepinephrine or monoamine oxidase inhibitors. The administration of calcium chloride also prolonged the ethanol-induced sleeping time. This effect of calcium was abolished by the administration of calmodulin antagonists, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) and tri¯uoperazine, or a tyrosine hydroxylase inhibitor, a-methyltyrosine (aMPT). These results suggest that calcium may activate tyrosine hydroxylase in the brain via a calmodulin-dependent pathway, leading to increased levels of catecholamines in the brain that may subsequently prolong the ethanol-induced sleeping time [8,9]. This * Corresponding author. Tel.: 181-298-53-3113; fax: 181-29854-9817. E-mail address: [email protected] (D. Sutoo).
conclusion was con®rmed by biochemical and quantitative immunohistochemical techniques. Although the norepinephrine level in the mouse brains did not change signi®cantly, dopamine levels in the lateral part of the neostriatum and nucleus accumbens were increased following the i.c.v. administration of calcium. Moreover, the effect of calcium to enhance dopamine synthesis was abolished by W-7 [8,13]. The brain regions in which the amount of dopamine was increased by calcium were those areas with high levels of calmodulin and tyrosine hydroxylase [14]. These results suggest that the calcium/calmodulin-dependent system activates dopamine synthesis in speci®c brain regions such as the neostriatum and nucleus accumbens. We have applied these fundamental studies concerning calcium-dependent dopamine synthesizing system to elucidate a few brain functions and mechanisms for understanding of some diseases [15±18]. To elucidate the mechanism of metal-toxicosis, the effect of various metal ions on the ethanol-induced sleeping time in mice was investigated [10]. The ethanol-induced sleeping time was prolonged following the i.c.v. injection of cadmium chloride, zinc chloride, manganese chloride, or mercury chloride, as well as calcium chloride. The ability of these cations to prolong
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 53 7- 8
6
D. Sutoo, K. Akiyama / Neuroscience Letters 294 (2000) 5±8
ethanol-induced sleeping time was inhibited by the administration of W-7 or aMPT. These ®ndings show that cadmium, zinc, manganese, or mercury activates cerebral calmodulin which increases the activity of tyrosine hydroxylase, and that the subsequent increase in dopamine levels in the brain prolongs the ethanol-induced sleeping time. Moreover, it was con®rmed by quantitative immunohistochemical [16] and 1H-nuclear magnetic resonance ( 1H-NMR) studies [12] that cadmium, zinc, manganese and mercury bind to calmodulin and activate it, as calcium does. However, magnesium chloride is different from the other divalent cations in that it does not signi®cantly activate calmodulin [11,12] and does not affect the duration of the ethanol-induced sleeping time [10]. In the present study, to elucidate the relationships between calcium and magnesium with the respect to brain functions, the effect of magnesium on the calcium- and dopamine-dependent brain function was investigated. Male mice of the ddY strain were provided by Japan SLC, Inc. (Shizuoka, Japan). The mice weighed approximately 25 g (5 weeks old) at the start of the experiment and all animals were used only once. They were housed in groups of 8±10 in stainless steel cages at room temperature (22 ^ 28C) for more than one week before use in the experiments, and were exposed to a 12-h light±dark cycle. Food and water were provided ad libitum. All animals were handled in compliance with the `Guiding Principles for the Care and Use of Laboratory Animals' formulated by the Japanese Pharmacological Society. Experiments were carried out between 1000 and 1600 h. Ethanol (30% in saline, v/v) was injected i.p. at a dose of 4.5 g/kg, and the duration of ethanol-induced sleeping time was measured. Sleeping time duration is de®ned as the time from the loss of the righting re¯ex to the time when the righting re¯ex is restored, occurring twice within 30 s. Room temperature, extraneous sounds, and ethanol concentrations were carefully controlled. First, the sleeping times in mice treated with calcium, magnesium, dopamine, aMPT and saline were compared. Next, the effects of administration of calcium together with magnesium or with aMPT on the ethanol-induced sleeping time were analyzed. Mice were injected into the lateral ventricle with calcium chloride (10 mmol/kg), magnesium chloride (10 or 40 mmol/kg) or dopamine (30 nmol/mouse), 30 min, 30 min or 15 min, respectively, before the injection of ethanol. a-Methyltyrosine (100 mg/kg) was injected i.p. 24 h before the injection of ethanol. All chemicals and drugs were dissolved in physiological saline. The doses and other conditions for administration were based on the results of our previous tests [8±10], and i.c.v. injections were performed in conscious mice, as described by Clark et al. [2] using an injection volume of 5 ml/mouse. The data were analyzed using ANOVA and Newman-Keuls t-test for subsequent comparisons among groups. The effects of magnesium or calcium on the brain dopamine level in mice were analyzed. High-performance liquid
chromatography (HPLC) technique was employed to determine the dopamine content in the brain. Four animal groups (9±11 mice/group), i.e. mice given an i.c.v. injection of calcium chloride (10 mmol/kg), magnesium chloride (40 mmol/kg), calcium chloride plus magnesium chloride, or saline were prepared. Thirty minutes after injection, the brains were taken out quickly and divided into two parts, the cerebrum and rhombencephalon, as described in our previous report [8]. Only the cerebrum was analyzed in the present study. The brain tissue was homogenized with 0.1 M perchloric acid (4:1, v/w) in a glass homogenizer. After centrifugation for 10 min at 25 000 £ g (48C), the supernatant ¯uid was diluted (1:400) with distilled deionized water and analyzed using an automatic catecholamine analyzer (Tosoh HLC-725CA, Tokyo, Japan). As shown in Fig. 1, injection of calcium chloride (10 mmol/kg) or dopamine (30 nmol/mouse) prolonged ethanol-induced sleeping time in mice by 56% (P , 0:01) or 51% (P , 0:01), respectively, when compared to the saline-treated control group. The ethanol-induced sleeping time did not change following the administration of magnesium chloride (10 or 40 mmol/kg) or aMPT alone. Moreover, the ability of calcium to enhance ethanol-induced sleeping time was inhibited by administration of magnesium chloride or aMPT. Namely, ethanol-induced sleeping time in mice treated with magnesium chloride (40 mmol/kg) together with calcium chloride, or with aMPT (100 mg/kg) 24 h before
Fig. 1. Effects of calcium, magnesium, dopamine and aMPT on the duration of ethanol-induced sleeping time in mice. Mice were injected i.c.v. with calcium chloride, magnesium chloride or dopamine, 30, 30 or 15 min, respectively, before the injection of ethanol. a-Methyltyrosine was injected i.p. 24 h before the injection of ethanol. The administration of calcium chloride (10 mmol/kg) or dopamine (30 nmol/mouse) prolonged the duration of ethanol (4.5 g/kg)-induced sleeping time. However, it was not prolonged following the administration of magnesium chloride (10 or 40 mmol/kg). Moreover, the ability of calcium to prolong ethanol-induced sleeping time was inhibited by the administration of magnesium chloride (40 mmol/kg) or aMPT (100 mg/kg). Each value represents the mean ^ SEM of 11±14 mice. *P , 0:01 compared to saline-treated mice group; d P , 0:01 compared to calcium chloride-treated mice group, using Newman±Keuls t-test.
D. Sutoo, K. Akiyama / Neuroscience Letters 294 (2000) 5±8
Fig. 2. Effects of i.c.v. administration of magnesium and calcium on the cerebral dopamine content in mice. The administration of calcium chloride (10 mmol/kg) or magnesium chloride (40 mmol/kg) increased the cerebral dopamine level. Each value represents the mean ^ SEM of 9±11 mice. *P , 0:05 compared to saline-treated mice group, using Newman±Keuls t-test.
the injection of calcium chloride was signi®cantly shorter by 36% (P , 0:01) or 33% (P , 0:01), respectively, when compared to mice treated with calcium chloride alone. The effects of calcium or magnesium on the cerebral dopamine level in mice are shown in Fig. 2. The dopamine level signi®cantly increased following the administration of calcium chloride (10 mmol/kg) or magnesium chloride (40 mmol/kg) by 9% (P , 0:05) and 8% (P , 0:05), respectively, as compared with the control. The dopamine level slightly increased following the administration of mixture solution of calcium and magnesium, but it did not signi®cantly change. In this study, the duration of ethanol-induced sleeping time was prolonged following the i.c.v. administration of calcium chloride, but it was not affected by magnesium chloride. These ®ndings agreed with our previous observations [10]. Here, the ability of calcium to prolong ethanolinduced sleeping time was signi®cantly inhibited by the i.c.v. administration of magnesium chloride. In the biochemical test, on the other hand, the brain dopamine level increased following the i.c.v. administration of magnesium, as well as the administration of calcium. These ®ndings suggest that dopamine synthesis in the brain is enhanced by not only calcium but also magnesium, and that the function of increased dopamine is inactivated by magnesium. These ®ndings are supported by previous reports. Tyrosine hydroxylase was activated and dopamine synthesis was enhanced by magnesium via cAMP-dependent protein kinase [3,6]. The dopamine release stimulated by N-methyl-d-aspartate [4,5], nitric oxide [20], or potassium chloride [20] was inhibited by magnesium. Moreover, the inhibition of dopamine release by adenosine was mediated by the adenosine A1 receptor, and this inhibition was enhanced by magnesium [7]. It must be investigated in the future whether or not the sites of dopamine synthesis activated by calcium, dopamine synthesis activated by magnesium, and dopamine release inhibited by magnesium are the same. The relationships between calcium and magnesium in the dopaminergic system and the calmodulin system are
7
summarized as follows: (a) calcium binds to calmodulin and activates it, however magnesium does not completely bind to calmodulin therefore it does not activate it [12]; (b) magnesium affects the conformation of interface sites for target enzymes of calcium-bound calmodulin, as the calmodulin antagonist does [11]. Thus, magnesium inhibits the calmodulin-dependent activation of certain enzymes [1,19]; (c) calcium activates dopamine synthesis through calmodulin-dependent protein kinase [9,17], and magnesium activates dopamine synthesis through cAMP-dependent protein kinase [3,6]; and, (d) calcium enhances the dopamine release in synapse, whereas magnesium inhibits it [4,5,7,20]. In conclusion, it is suggested that calcium enhances dopamine synthesis in the brain through a calmodulin-dependent system, and the increase in dopamine level prolongs ethanol-induced sleeping time. However, magnesium inhibits dopamine release. Therefore, magnesium may inhibit calcium-dependent brain function through dopaminergic neurons, and consequently reduce the effect of calcium on ethanol activity. The authors would like to thank Mr Yoshimasa Itoh and Mr Jiro Ishii, Tosoh Corporation, Tokyo, Japan, for technical assistance in HPLC. [1] Brostrom, C.O. and Wolff, D.J., Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3 0 ,5 0 -monophosphate and guanosine 3 0 ,5 0 monophosphate as substrates, Arch. Biochem. Biophys., 172 (1976) 301±311. [2] Clark, W.G., Vivonia, C.A. and Baxter, C.F., Accurate freehand injection into the lateral brain ventricle of the conscious mouse, J. Appl. Physiol., 25 (1968) 319±321. [3] Edelman, A.M., Raese, J.D., Lazar, M.A. and Barchas, J.D., Tyrosine hydroxylase: studies on the phosphorylation of a puri®ed preparation of the brain enzyme by the cyclic AMPdependent protein kinase, J. Pharmacol. Exp. Ther., 216 (1981) 647±653. [4] Johnson, K.M. and Jeng, Y.J., Pharmacological evidence for N-methyl-d-aspartate receptors on nigrostriatal dopaminergic nerve terminals, Can. J. Physiol. Pharmacol., 69 (1991) 1416±1421. [5] MartIÂnez-Fong, D., Rosales, M.G., GoÂngora-Alfaro, J.L., HernaÂndez, S. and Aceves, J., NMDA receptor mediates dopamine release in the striatum of unanesthetized rats as measured by brain microdialysis, Brain Res., 595 (1992) 309±315. [6] Morgenroth III, V.H., Hegstrand, L.R., Roth, R.H. and Greengard, P., Evidence for involvement of protein kinase in the activation by adenosine 3 0 ,5 0 -monophosphate of brain tyrosine 3-monooxygenase, J. Biol. Chem., 250 (1975) 1946± 1948. [7] Okada, M., Mizuno, K., Okuyama, M. and Kaneko, S., Magnesium ion augmentation of inhibitory effects of adenosine on dopamine release in the rat striatum, Psychiatry Clin. Neurosci., 50 (1996) 147±156. [8] Sutoo, D. and Sano, K., Modulating effects of biogenic amines on calcium and ethanol-induced sleeping time, Alcohol, 1 (1984) 141±144. [9] Sutoo, D., Akiyama, K. and Iimura, K., Effect of calmodulin antagonists on calcium and ethanol-induced sleeping time in mice, Pharmacol. Biochem. Behav., 23 (1985) 627±631.
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D. Sutoo, K. Akiyama / Neuroscience Letters 294 (2000) 5±8
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