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 cultur ed rat cortical neurons. However, the mechanism of this increase in [M g2+](i) is not well understood. We used fluorescence microscopic metho ds to measure [Mg2+](i), [Ca2+](i), and pH(i) in single neurons. Intra cellular ATP analysis was performed by high-performance liquid chromat ography (HPLC). 2. A 25-mM NH4Cl pulse followed by Na+-free wash rapid ly acidified the cytosol. In 2',7'-bis(2-carboxyethyl)-5 (6)-carboxyfl uorescein (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 m M. Five-minute treatment with 100 mu M glutamate, on the other hand, r educed 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 iodoacet ate) for 30 min significantly reduced the intracellular ATP levels. Ho wever, 5-min treatment with 100 mu M glutamate did not significantly d eplete intracellular ATP in cultured cortical neurons. When tested und er 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 observ ed during glutamate stimulation. 4. To further study the role of gluta mate-induced Ca2+ influx in subsequent [Mg2+](i) elevation, extracellu lar Ca2+ was elevated from 1.4 to 3.0 mM during glutamate application in magfura-2-loaded neurons. Increasing extracellular Ca2+ significant ly increased the [Mg2+](i) response to glutamate in these cells. This effect could not be attributed to very large [Ca2+](i) increases inter fering with the magfura-2 signal. 5. The results of this study are con sistent with the hypothesis that magnitude of Ca2+ entry is the primar y determinant of the size of glutamate-induced [Mg2+](i) change in rat cortical neurons.