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Information about the catalytic and kinetic properties of mitochondria NO-synthase from uterus smooth muscle is missing currently. According to the data on MitoTracker Orange CM-H2TMRos and 4-аmino-5-methylamino-2',7'-difluorescein, diaminofluorescein-FM (DAF-FM) dye co-localization in uterine smooth muscle cells, presented in this paper, NO can be synthesized in their mitochondria. High activity of NO synthase requires the presence of substrates of respiration, L-arginine, Ca 2+ and NADPH. It is established that the dependence of NO production on the concentration of L-arginine has a bell-shaped character with a maximum of 75 μM, and the apparent affinity constant for L-arginine is 28.9 ± 9.1 μM. The dependence of NO production on Ca 2+ concentration has a maximum at 100-250 μM; the activation constant for Ca 2+ is 44.4 ± 14.5 μM. The inhibitor of Ca 2+ transport in mitochondria ruthenium red (RuR), as well as the inhibitor of NO-synthase N G -nitro-L-arginine (NA), reduces NO production. The biosynthesis of NO by mitochondria depends on its energized level: it is stimulated by the addition of respiration substrates, suppressed with specific inhibitors of the electron transport chain (rotenone and antimycin A) and carbonyl-cyanide 3-chlorophenylhydrazone (CCCP) protonophore.

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

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In this paper, a Petri net-based model of the fluorescence of the nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH 2). NADH and FAD nucleotides from the electron transport chain and the swelling of isolated mitochondria is presented. The model describes selected aspects of these processes under the influence of the chosen electron transport chain chemical modificators. The model expressed in the language of the Petri net theory has an intuitive graphical interpretation and can be analyzed using rigorous mathematical methods. For the simulation experiment, we chose the Cell Illustrator v.3 software (Human Genome Center, University of Tokyo, Japan). An analysis of the fluorescent response of NADH/FAD in the isolated mitochondria to specific electron transport chain inhibitors (rotenone, antimycin A, and sodium cyanide (NaCN)) and protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP) revealed a correlation between the changes in the NADH/FAD fluorescence (their redox state) and the functions of particular complexes in the inner mitochondrial membrane. The inhibition of the electron transport chain resulted in the organelle's swelling; we obtained mathematical equations through modeling to formalize the process of mitochondria swelling and NADH/FAD fluorescence changes in a medium with sodium azide. In particular, these equations adequately and simultaneously described the time characteristics of the reduction of the fluorescence of nucleotides and the swelling of mitochondria. Our model enabled us to predict the changes in the organelle NADH/FAD fluorescence and their hydrodynamic diameters in time. Furthermore, it helped us to optimize the experimental procedures, allowing us to analyze the process dynamics and to compare the modeling results with actual observations under changing compositions of the incubation media and the presence of activators/inhibitors.

It was proved that No synthesis in isolated mitochondria of rat uterus smooth muscle depended on the entry of exogenous ca 2+ to mitochondria (inhibited by 1-10 mM Mg 2+ in the absence of ATP and by 10 μM ruthenium red) and was suppressed by calmodulin antagonists (0.1-10 μM calmidazolium and 1-100 μM trifluoperazine). It was blocked by N G -nitro-l-arginine, a known antagonist of the constitutive No-synthase, with a half-maximal inhibition effect at about 25 μM. Moderate deholesterinization of the plasma membrane of myocytes after processing with 0.01% digitonin was followed by increased nitric oxide biosynthesis by cells. The data obtained suggested that mitochondria and plasmalemma is a possible source of No synthesis in uterine myocytes. k e y w o r d s: nitric oxide, mtNoS, mitochondria, calcium, uterus smooth muscle.

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A mathematical model is developed using Petri net methodology that is able to predict simultaneous changes in bio(physical)chemical parameters of mitochondria, namely hydrodynamic diameter, fluorescence of NADH, DCF fluorescence signal depending on the time of action and concentration of sodium azide and predict these changes for unknown NaN 3 concentration. The model is created for real experimental conditions, it combines functional changes of mitochondria with a structural representation of learned processes.