L. G. Babich¹, S. G. Shlykov¹, A. M. Kushnarova-Vakal¹, N. I. Kupynyak², V. V. Manko², V. P. Fomin³, S. O. Kosterin¹

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: babich@biochem.kiev.ua;
²Ivan Franko National University of Lviv, Ukraine;
³University of Delaware, Newark, USA

The aim of the study was to show the relationships between ionized Ca concentration ([Ca²⁺]_m) in the mitochondria matrix and functional activity of this organelle. [Ca²⁺]_m was determined using the fluorescent probe Fluo-4, AM. Total level of Ca²⁺ accumulation in mitochondria was monitored using ⁴⁵Ca²⁺ as radioactive tracer. It was shown that incubation of myometrium mitochondria with 3 mM Mg²⁺ resulted in the low level of [Ca²⁺]_m. Subsequent addition of 100 µM Ca²⁺ resulted in 8 times increase of [Ca²⁺]_m but in low level of total calcium accumulation. Normalized fluorescence of Ca²⁺-sensitive probe Fluo-4 in response to the Ca²⁺ addition was higher than 2.5. At the same time, [Ca²⁺]_m was considerably higher in the medium containing­ 3 mМ АТР and 3 mМ Mg²⁺. Subsequent addition of 100 µM Ca²⁺ to the incubation medium resulted in only 2.4 times increase of [Ca²⁺]_m but considerably higher level of total calcium accumulation was observed. Normali­zed fluorescence of Fluo-4 in response to the Ca²⁺ addition was lower than 1.3. In liver mitochondria higher rate of oxygen consumption was detected in the presence of an oxidative substrate succinate than of pyruvate or α-ketoglutarate. At the presence of an oxidative substrate succinate normalized fluorescence of Fluo-4 in liver mitochondria in response to the Ca²⁺ addition was lower than 1.3. It was concluded that low level of [Ca²⁺]_m was correlated with low functional activity of this organelle and, vise versa, high level of [Ca²⁺]_m was correlated with high functional activity. It was suggested that normalized fluorescence changes in response to the Са²⁺ addition could be used as a test of the mitochondrial functional activity: lower normalized fluorescence values − higher functional activity.

Keywords: ionized Са concentration, liver, mitochondria, myometrium, total Са(2+) accumulation