Investigation of intracellular magnesium mobilization pathways I PC12 cells B simultaneous Mg-Ca fluorescent imaging

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 IP3 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 IP3 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 microM) 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 microM), [Mg]i and [CA]i increased, we concluded that mitochondria participate in maintenance of intracellular Mg stores.