title: Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis.
abstract: For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]c), pH (pHc), and intracellular free Ca2+ concentration ([Ca2+]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 pHc. Cells were loaded with fluorescent low-affinity Ca2+ indicators Fura-FF or X-rhod-FF to register [Ca2+]i. It was shown that Glu (20 µM, glycine 10 µM, Mg2+-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 [Ca2+]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 [Ca2+]i plateau was only 36.6%. Changes in [ATP]c, [Ca2+]i, mitochondrial potential, and pHc in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na+/K+-ATPase ouabain (0.5 mM), 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+/Ca2+ exchange.
authors.affiliation: Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, 125315, Russia. surin-_am@yahoo.com.
authors.forename: A M
authors.initials: AM
authors.lastname: Surin
authors.forename: L R
authors.initials: LR
authors.lastname: Gorbacheva
authors.forename: I G
authors.initials: IG
authors.lastname: Savinkova
authors.forename: R R
authors.initials: RR
authors.lastname: Sharipov
authors.forename: B I
authors.initials: BI
authors.lastname: Khodorov
authors.forename: V G
authors.initials: VG
authors.lastname: Pinelis
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology*; D006706:Homeostasis / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D051381:Rats
chemical_list: D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D002118:Calcium