Manganese suppresses ATP-dependent intercellular calcium waves in astrocyte networks through alteration of mitochondrial and endoplasmic reticulum calcium dynamics

The neurotoxicity of manganese [Mn] is due in part to glutamate excitotoxicity. Release of ATP by astrocytes is a critical modulator of glutamatergic neurotransmission, which is regulated by calcium (Ca2+) waves that propagate through astrocytic networks in response to synaptic activity. It was postulated that Mn alters ATP-dependent intracellular Ca2+ dynamics in astrocytes, thereby suppressing Ca2+ wave activity. Confluent primary cultures of cortical astrocytes were loaded with the Ca2+-sensitive dye fluo-4 and examined by fluorescence microscopy for Ca2+ wave activity following micropipet mechanical stimulation of a single cell. Mitochondrial Ca2+ was evaluated by fluorescence microscopy following addition of ATP using the mitochondrial-specific Ca2+ dye rhod-2-AM. Imaging studies revealed that pretreatment of astrocytes with 1–10 μM Mn significantly reduced the rate, area, and amplitude of mechanically induced Ca2+ waves. This attenuation was not a result of inhibited mitochondrial calcium uptake because robust calcium waves were still observed following pretreatment of astrocytes with Ru360, an inhibitor of mitochondrial Ca2+ uptake, either in coupling or uncoupling conditions. However, determination of endoplasmic reticulum (ER) Ca2+ levels in cells using the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin indicated that Mn reduced the available pool of releasable ER Ca2+ at concentrations as low as 1 μM. Examination of ATP-stimulated changes in mitochondrial Ca2+ indicated that, in cells pretreated with Mn, mitochondria retained high levels of Ca2+. It is concluded that exposure of astrocytes to low concentrations of Mn2+ results in sequestration of Ca2+ within the mitochondria that reduces the available pool of releasable Ca2+ within the ER, thereby inhibiting calcium wave activity.