Different effects of metformin and phenformin on hypoxia-induced Ca²⁺ fluxes in cultured primary neurons

Recent evidence suggests that metformin and phenformin may exert beneficial effects against neuronal injury in the ischemic brain, however, the difference of action between these two drugs and the molecular mechanism of such protection is not clear. In this study, we investigated whether mild hypoxia-affected neurons exhibit changes in cytosolic calcium handling and whether metformin and phenformin exert any effect on calcium homeostasis in hypoxia-affected neurons. Cultured primary rat cortical cells were stained with calcium sensitive dye Oregon Green 488 BAPTA-1 AM and spontaneous calcium dependent changes of fluorescence were recorded. Using obtained fluorescence traces we estimated changes in relative amplitude of recorded spontaneous signals, changes in frequency of spontaneous activity, and changes in decay of fluorescence traces. We found that hypoxia caused reduction of the relative signal amplitude, increased the spontaneous activity, and slowed the decay of calcium concentration. After pre-treatment of cells with 0.1-0.5 mM metformin, the relative signal amplitude increased and the frequency of spontaneous signals decreased in hypoxia-affected neurons. However, pre-treatment with 1-25 mu M phenformin neither increased the relative signal amplitude nor reduced the frequency of spontaneous signals. The decay of fluorescence traces became faster after application of metformin or phenformin comparing to neurons under hypoxic conditions. These results suggest different action of metformin and phenformin in improvement of Ca2+ homeostasis in hypoxia-affected neurons, which may have different effects on neuronal survival and functions after hypoxia/ischemia.