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

---

A. M. Surin^1,2*, 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: +7 (495) 151-1726; E-mail: surin­_am@yahoo.com

²Scientific Center for Children’s 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, Vorob’evy Gory 1/12, 119234 Moscow, Russia

^* To whom correspondence should be addressed.

Received August 30, 2013; Revision received October 28, 2013

---

For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]_c), pH (pH_c), 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 pH_c. 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 (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 pH_c 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.

---

KEY WORDS: neurons, glutamate, calcium deregulation, ATP, mitochondria

DOI: 10.1134/S0006297914020084

---