CALCIUM INFLUX BUT NOT PH OR ATP LEVEL MEDIATES GLUTAMATE-INDUCED CHANGES IN INTRACELLULAR MAGNESIUM IN CORTICAL-NEURONS

1. We have recently shown that glutamate increases [Mg2+](i) in cultured rat cortical neurons. However, the mechanism of this increase in [Mg2+](i) is not well understood. We used fluorescence microscopic methods to measure [Mg2+](i), [Ca2+](i), and pH(i) in single neurons. Intracellular ATP analysis was performed by high-performance liquid chromatography (HPLC). 2. A 25-mM NH4Cl pulse followed by Na+-free wash rapidly acidified the cytosol. In 2',7'-bis(2-carboxyethyl)-5 (6)-carboxyfluorescein (BCECF)-loaded neurons, the pH(i) was reduced by 2.46 units, and in magfura-2-loaded neurons the [Mg2+](i) was increased by 0.62 mM. Five-minute treatment with 100 mu M glutamate, on the other hand, reduced the cytosolic pH by 0.73 units and increased the [Mg2+](i) by 7.24 mM in rat cortical neurons. These results indicate that change in pH(i) does not play a significant role in the glutamate-induced [Mg2+](i) elevation. 3. The metabolic inhibition (5 mM KCN and 1 mM iodoacetate) for 30 min significantly reduced the intracellular ATP levels. However, 5-min treatment with 100 mu M glutamate did not significantly deplete intracellular ATP in cultured cortical neurons. When tested under similar conditions in magfura-2-loaded neurons, glutamate increased [Mg2+](i) to a significantly larger extent than metabolic inhibition. This suggests that ATP depletion and subsequent release of Mg2+ from Mg2+-ATP complex is not the primary source of [Mg](i) elevation observed during glutamate stimulation. 4. To further study the role of glutamate-induced Ca2+ influx in subsequent [Mg2+](i) elevation, extracellular Ca2+ was elevated from 1.4 to 3.0 mM during glutamate application in magfura-2-loaded neurons. Increasing extracellular Ca2+ significantly increased the [Mg2+](i) response to glutamate in these cells. This effect could not be attributed to very large [Ca2+](i) increases interfering with the magfura-2 signal. 5. The results of this study are consistent with the hypothesis that magnitude of Ca2+ entry is the primary determinant of the size of glutamate-induced [Mg2+](i) change in rat cortical neurons.