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 pHi 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 pHi 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 microM 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 pHi 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 microM 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 Mg(2+)-ATP complex is not the primary source of [Mg2+]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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D016548:Prosencephalon / Q000187:drug effects; D051381:Rats; D013997:Time Factors
D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium