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Abstract
For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]^sub c^), pH (pH^sub c^), and intracellular free Ca^sup 2+^ concentration ([Ca^sup 2+^]^sub 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^sub c^. Cells were loaded with fluorescent low-affinity Ca^sup 2+^ indicators Fura-FF or X-rhod-FF to register [Ca^sup 2+^]^sub i^. It was shown that Glu (20 [mu]M, glycine 10 [mu]M, Mg^sup 2+^-free) produced a rapid acidification of the cytosol and decrease in [ATP]^sub c^. An approximately linear relationship (r ^sup 2^ = 0.56) between the rate of [ATP]^sub c^ decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]^sub c^ decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]^sub c^ of as much as 15.9%. In the phase of high [Ca^sup 2+^]^sub i^, the plateau of [ATP]^sub c^ dropped to 10.4% compared to [ATP]^sub c^ in resting neurons (100%). In the presence of the Na^sup +^/K^sup +^-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]^sub c^ in the phase of the high [Ca^sup 2+^]^sub i^ plateau was only 36.6%. Changes in [ATP]^sub c^, [Ca^sup 2+^]^sub i^, mitochondrial potential, and pH^sub c^ in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na^sup +^/K^sup +^-ATPase ouabain, led us to suggest that in addition to increase in proton conductivity and decline in [ATP]^sub c^, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na^sup +^/Ca^sup 2+^ exchange.[PUBLICATION ABSTRACT]