10th International Magnesium Symposium Schedule of Events

19
Investigation of intracellular magnesium mobilization pathways in PC12 cells
by simultaneous Mg-Ca fluorescent imaging
Takeshi Kubota 1 , Hirokazu Komatsu 4 , Yutaka Shindo 2 , Kentaro Tokuno 2 , Yoshiichiro
Kitamura 1 , Hiroto Ogawa 3 , Koji Suzuki 4 , and Kotaro Oka 1 , 1 Center for Life Science
and Technology, Keio University, Yokohama, Japan, 2 Department of System Design
Engineering, Keio University, Yokohama, Japan, 3 Department of Biology, Saitama
Medical School, Saitama, Japan, 4 Department of Applied Chemistry, Keio University,
Yokohama, Japan. kubota@bpni.bio.keio.ac.jp
Intracellular Mg2+ concentration shows dynamic change in response to various
extracellular stimuli in excitable cells, and it suggests that the concentration change
plays important roles in intracellular signal transduction. To clarify the role of
intracellular Mg2+ and Ca2+ during stimulation, we examined the correlation of these
divalent cations in PC12 cells by fluorescent imaging techniques. At first, we
developed a novel fluorescent Mg indicator KMG-104 (Ex = 504 nm, Em = 523 nm).
The dissociation constant (Kd) of KMG-104 is 3 mM, near to the intracellular Mg2+
concentration (about 1 mM), and not affected by Ca2+ disturbance. PC12 cells were
double-stained by KMG-104 and a Ca2+ indicator, Fura-Red, and the fluorescences
exited at 480 nm from these dyes were acquired simultaneously with a W-View
optical system with a 535/55 BP (KMG-104) and a 600 LP (Fura-Red) filters,
respectively. When the cells were stimulated with a mitochondrial uncoupler, FCCP
(3 µM), both Mg2+ and Ca2+ increased. After washing the FCCP, Mg2+
concentration returned to the basal level, but Ca2+ concentration did not recover. To
investigate the correlation of Ca2+ and Mg2+ mobilization directly, intracellular Ca2+
concentration was increased by photolysis of caged-Ca2+. Transient increase of
Ca2+ caused no Mg2+ concentration change. From these results, we presume Mg2+
mobilization pathway does not correspond with Ca2+ cascades. A plausible source
of Mg2+ mobilization is Mg2+ release from intracellular Mg-ATP complex by ATP
consumptions.