Study on ATP Concentration Changes in Cytosol of Individual Cultured Neurons during Glutamate-induced Deregulation of Calcium Homeostasis

摘要

For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]_C), pH (pH_c), and intracellular free Ca~(2+) concentration ([Ca~(2+)]_i) of the individual neurons challenged with toxic glutamate (Glu) concentrations was performed. To this end, the ATP-sensor AT1.03, which binds to ATP and therefore enhances the efficiency of resonance energy transfer between blue fluorescent protein (energy donor) and yellow-green fluorescent protein (energy acceptor), was expressed in cultured hippocampal neurons isolated from 1-2-day-old rat pups. Excitation of fluorescence in the acceptor protein allowed monitoring changes in pH_c. Cells were loaded with fluorescent low-affinity Ca~(2+) indicators Fura-FF or X-rhod-FF to register [Ca~(2+)]_i. It was shown that Glu (20 uM, glycine 10 μM, Mg~(2+)-free) produced a rapid acidification of the cytosol and decrease in [ATP]_C. An approximately linear relationship (r~2 = 0.56) between the rate of [ATP]_C decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]_C decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]_C of as much as 15.9%. In the phase of high [Ca~(2+)]_i, the plateau of [ATP]_C dropped to 10.4% compared to [ATP]_C in resting neurons (100%). In the presence of the Na~+/K~+-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]_C in the phase of the high [Ca~(2+)]_i plateau was only 36.6%. Changes in [ATP]_C, [Ca~(2+)]_i, mitochondrial potential, and pH_c in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na~+/K~+-ATPase ouabain, led us to suggest that in addition to increase in proton conductivity and decline in [ATP]_C, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na~+/Ca~(2+) exchange.