Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis

ISSN 0006 2979, Biochemistry (Moscow), 2014, Vol. 79, No. 2, pp. 146 157. Pleiades Publishing, Ltd., 2014. Published in Russian in Biokhimiya, 2014, Vol. 79, No. 2, pp. 196 208.

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

A. M. Surin1,2*, L. R. Gorbacheva3, I. G. Savinkova3,R. R. Sharipov1, B. I. Khodorov1, and V. G. Pinelis2

1Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Baltiiskaya ul. 8, 125315 Moscow, Russia; fax: [phone]; E mail: [email protected]

2Scientific Center for Childrens Health, Russian Academy of Medical Sciences, Lomonosovsky pr. 2/1, 119991 Moscow, Russia

3Department of Human and Animal Physiology, Faculty of Biology, Lomonosov Moscow State University, Vorobevy Gory 1/12, 119234 Moscow, Russia

Received August 30, 2013 Revision received October 28, 2013

AbstractFor 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) concen

trations 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 M, glycine 10 M, Mg2+ free) produced a rapid acid ification of the cytosol and decrease in [ATP]c. An approximately linear relationship (r2 = 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...