Journal of the American College of Nutrition, Vol. 23, No. 6, 742S-744S (2004)
Published by the American College of Nutrition
Investigation of Intracellular Magnesium Mobilization Pathways I Pc12 Cells B Simultaneous Mg-Ca Fluorescent Imaging
Takeshi Kubota, MS,
Yutaka Shindo, BT,
Kentaro Tokuno, BT,
Hirokazu Komatsu, MT,
Hiroto Ogawa, PhD,
Yoshiichiro Kitamura, PhD,
Koji Suzuki, PhD and
Kotaro Oka, PhD
School of Fundamental Science and Technology (T.K., Y.K., K.O.)
Department of System Design Engineering (Y.S., K.T., K.S.)
Department of Applied Chemistry (H.K.) Keio University
Department of Biology, Saitama Medical School (H.O.)
Department of Biosciences and Informatics (Y.K., K.O.), Keio University, Yokohama, JAPAN
Address reprint requests to: Kotaro Oka, PhD, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, JAPAN. E-mail: oka{at}bio.keio.ac.jp
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ABSTRACT
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Objective and Methods: PC12 cells were loaded with a novel Mg indicator KMG-104 and Ca indicator fura-2, and intracellular Mg was studied in the endoplasmic reticulums (ERs), mitochondria, and Mg-ATP. Under coexistence of the two indicators, fluorescent signals of Mg and Ca can be measured separately. Mg release from the ER was investigated by photolysis of caged compounds.
Results: Transient [Ca] i increase by uncaging of caged Ca or caged IP 3 or bath-application of caffeine (10 mM) induced no [Mg] i increase. These results suggest that there is no mechanism for Mg release from the ER through ryanodine receptors or IP 3 receptors. In order to investigate the possibility of Mg release from Mg-ATP by energy consumption, we depleted ATP by oligomycin, an inhibitor of mitochondrial ATP synthase. Treating with oligomycin (4 µM) for several minutes showed no change of [Mg] i and [Ca] i.
Conclusions: This result shows that Mg-ATP is not a Mg store. Since, when cells were treated by an uncoupler FCCP (3 µM), [Mg] i and [Ca] i increased, we concluded that mitochondria participate in maintenance of intracellular Mg stores.
Key words: intracellular Mg store, FCCP, photolysis, ATP, mitochondria
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INTRODUCTION
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Magnesium, an abundant cation in cells, is involved in many biological functions. There are over 300 enzymatic, Mg dependent reactions [1]. Moreover, it has been known that intracellular Mg concentration ([Mg] i) changes in response to extracellular stimulus, and in some cases, [Mg] i change is accompanied by [Ca] i change [2,3]. The Mg stores in cells have not been identified yet, but there are three candidates; endoplasmic reticulums (ERs), mitochondria, and Mg-ATP. Ca is usually accumulated in ER, and released through ryanodine receptors and/or IP 3 receptors by ligand gated manner. We investigated whether Mg is also released from ER by using a receptor’s agonist (caffeine) or caged compounds of Ca and IP 3. We also checked whether Ca induces Mg release from ER with the same methods. To investigate the Mg accumulation in mitochondria, an uncoupler, FCCP was used to inhibit mitochondrial activity. ATP strongly binds to Mg, and exists as a Mg-ATP complex in cells. When ATP decomposes to ADP by energy consumption, free Mg will be released to the cytosol. To check this Mg release, we depleted ATP by inhibiting ATP synthase. We investigated these three possibilities by simultaneous Mg-Ca measurement with a novel high-selective Mg indicator and a Ca indicator.
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MATERIALS AND METHODS
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Selectivity of Mg and Ca indicators was confirmed in vitro in HEPES buffered solution with 2 µM fura-2 and 10 µM KMG-104. PC12 cells were incubated with 10 µM KMG-104-AM in the culture medium for 30 min at 37°C, and then washed twice, and further incubated for 15 min for complete hydrolysis of the acetoxymethyl ester form. Loading of other AM reagents, 2 µM fura-2-AM, or 10 µM NP-EGTA-AM, was carried out at the same time as a mixture with 0.02% Pluronic F-127. Excitation wavelengths for KMG-104 were at 490 nm, and fura-2 was 340 and 380 nm, respectively. Fluorescent images were acquired with an inverted microscope (ECLIPSE TE300, Nikon) equipped with a x20 (S Fluor, Nikon) or a x40 (S Fluor, Nikon) objective, a 505 nm dichroic mirror and a 535/55 nm barrier filter. A 150 W Xe lamp with a monochrometer unit was used for multiple excitations, and fluorescence was measured with a CCD camera (HiSCA, Hamamatsu Photonics).
Caged IP 3 was microinjected with indicators into PC12 cells. The concentrations of caged IP 3, KMG-104, and fura-2 in the pipette were 0.5, 10, and 2 mM, respectively. Caged compounds were photoreleased by the UV light with 355 nm wavelength from a YAG laser.
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RESULTS
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Properties of KMG-104
MG-104 was designed to have high specificity for Mg. KMG-104 is useful for confocal laser scanning microscopy because it is excited at 490 nm, and emitted fluorescence around 510 nm. Fluorescent intensity of KMG-104 increases with [Mg] increase, and it shows no response to Na, K, and Ca; the dissociation constant (K d) for Mg is 3 mM. From these characteristics, KMG-104 is able to trace only [Mg] change in physiological conditions.
Double Staining of Mg and Ca
Before double staining experiments, we investigated in vitro whether Mg and Ca could be measured selectively in the presence of KMG-104 and fura-2. Fluorescence from the mixture in solution of the two indicators excited at 340, 380, and 490 nm wavelength were measured when [Mg] was changed in the range of 0–100 mM, and [Ca] is constant (100 nM). Same measurement was performed when [Ca] was changed (0–10 µM) in the presence of 1 mM Mg. From these experiments, we demonstrated that KMG-104 and fura-2 were specific for each cation, in the physiological range of concentrations.
Determination of the Intracellular Mg Store
Mg release from ER through two types of receptors was investigated with an agonist and caged compounds. To measure [Mg] i and [Ca] i changes simultaneously, PC12 cells were double stained with KMG-104 and fura-2. Ryanodine receptors were activated by uncaging of caged Ca or bath-application of caffeine (10 mM). These stimuli induced [Ca] i increase and this indicates Ca release from ER through ryanodine receptors, but [Mg] i did not changed (Fig. 1). Uncaging of caged IP 3 induced [Ca] i increase from ER through IP 3 receptors, but [Mg] i also did not change. These results suggest that there is no mechanism of Mg release from ER through ryanodine receptors or IP 3 receptors during Ca mobilization.
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Fig. 1. Mg is not released from ER through ryanodine receptors. Caged Ca was uncaged in both KMG-104 and fura-2 loaded PC12 cells. KMG-104 was excited at 490 nm, and fura-2 was at 380 nm wavelength. Laser flashes induced [Ca] i increase (triangles), but [Mg] i did not change (circles). Decrease of fura-2 fluorescence excited at 380 nm wavelength implies [Ca] i increase. Arrowheads indicate photolysis flashes.
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In order to investigate the possibility of Mg release from Mg-ATP by energy consumption, we depleted ATP by oligomycin, an inhibitor of mitochondrial ATP synthase. We expected [ATP] i decrease to cause free Mg increase. Treating with oligomycin (4 µM) for several minutes showed no change of [Mg] i and [Ca] i. This indicates that Mg-ATP is not a candidate of Mg store.
Only when cells were treated by FCCP (3 µM), did both [Mg] i and [Ca] i increase (Fig. 2). In our previous study, we observed FCCP induced [Mg] i increase [4,5]. FCCP is a mitochondrial uncoupler, and it eliminates mitochondrial electrochemical potentials across the inner membrane.
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Fig. 2. [Mg] i increased when mitochondrial activity was inhibited. Double stained PC12 cells were treated by mitochondrial inhibitor, FCCP. Bath application of 3 µM FCCP induced both [Mg] i (triangles) and [Ca] i (circles) increase. Excitation wavelengths are 490 nm for KMG-104 and 380 nm for fura-2. Decrease of fura-2 fluorescence implies [Ca] i increase. Bar indicates FCCP application.
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DISCUSSION
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We demonstrated that mitochondria play a role as intracellular Mg stores, by simultaneous measurement of Mg and Ca. For applying the double staining method described here, it is necessary to choose the suitable indicators that have high selectivity to each ion and are clearly distinguishable by their optical characteristics. KMG-104 and fura-2 is an appropriate combination that satisfies such requirements. This double staining method and laser photolysis technique revealed that there are no Mg release mechanisms induced by Ca increase from ER, and also that Mg-ATP is not a Mg source. Because the binding constant of ADP to Mg is low, Mg is released from the Mg-ATP complex during energy consumption. It is known that Ca is accumulated in mitochondria through a Ca uniporter using electrochemical gradient of inner membrane [6]. If mitochondria have a similar mechanism for accumulation of Mg, FCCP collapses the electrochemical gradient and induces Mg release to the cytosol.
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ACKNOWLEDGMENTS
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This research was partially supported by the Ministry of Education, Culture, Sports, Science and Technology, Grant-in-Aid for the 21st Century Center of Excellence (COE) Program entitled "Understanding and Control of Life’s via Systems Biology, Keio University," and by Special Coordination Funds for Promoting Science and Technology.
Received August 5, 2004.
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ABSTRACT
INTRODUCTION
