CC BY
10
Mamatova Irodakhon Yusupovna, Phd., student, of the Andijan State University,
of Uzbekistan
Almatov Karim Tajibaevich,
Professor, of the National University of Uzbekistan E-mail: [email protected]
THE EFFECT TEMPERATURE OF DIFFERENT INTENSITY OF ENVIRONMENTS FOR THE ACTIVATION NADH OXIDASE OF THE RATS LIVER MITOCHONDRION
Abstract: Study of the determination of the negative and positive effect of temperature of various intensity (22-41 o C) on the processes in the cell and tissues on the functions and structures of mitochondria, will give a chance to understand the mechanism of more effective treatment. Keywords: liver, mitochondria, NADH2, rotenone high temperature.
Actuality of the topic: It is known that climatic and meteorological factors exert a great influence on the general state of the organism and the processes of its vital activity. Among them, special attention is paid to the thermal factor (hyperthermia), which is one of the specific elements of the hot climate of Central Asia. It is known that hyperthermia refers to dangerous stressing states. However, heat therapy was applied in antiquity, and artificial hyperthermia is likely to become one of the medical technologies of the 21st century. One of the directions of the problem of hyperthermia is the study of physiological and biochemical reactions of the organism to the effect of high temperature.
There are many organelles in the cell of living organisms with individual functions that support the vital activity of the cell. Among these organelles mitochondria is half the cell, producing the energy necessary for the functioning of the cell. Substrates are oxidized in the mitochondria, ATP is produced and it has all the properties inherent in the cell such as: contraction, ion transport, synthesis of proteins and lipids, and the transfer of genetic information by inheritance [1, 3, 5, 24].
In recent years, information has been obtained about the favorable effect in the treatment of diseases under the influence of controlled ambient temperature [8;11; 13; 14; 15]. But at the same time, the structures and functions of the mitochondria of various organs are almost not studied in the treatment of humans. Study of
the destruction of the synthetic processes of the central nervous system, heart, liver, kidneys and other organs, a decrease in the function of cells associated with energy by 75-80%, with a 15-20% decrease in the formation of ATP in mitochondria [3]. The study of the determination of the negative and positive effect of temperature on the processes in the cell and tissues on the functions and structures of mitochondria will give a chance to understand the mechanism of more effective treatment.
Objective: Determination of the amount of NADH oxidizes of rat liver mitochondria under the influence of temperature of different intensity (22-24, 28-30, 35-36 and 41-42 o C).
Materials and methods of research: The studies were carried out on male rats of the Westar line, weighing 160-180 g. Experimental animals were on a standard diet and a feeding regimen.
For the study, the animals were divided into 4 groups. The first group (22-24 0 C) was left for monitoring. The second, third and fourth groups of rats were left at 2830; 35-36 and 41-42 o C. After 30 and 60 minutes, the rats were sacrificed, the liver was removed and immersed in a beaker with a release medium. The liver was cleaned of extraneous tissue (fat, connective tissue), then its mass was determined by weighing, cut by scissors and placed in a tenfold volume compared to the organs of a pre-cooled isolation medium and homogenized for 30-40 seconds in a homogenizer with Teflon pestle. Mitochondria were
isolated from the liver of animals according to the conventional method of differential centrifugation [7] with some modifications [19].The mitochondria were then washed twice with isolation medium without ethylene-diaminetetraacetate.
The activity of oxidize systems of liver mitochondria was determined after freezing and thawing of the mitochondria. NADH2- and succinate oxidase activities were evaluated by adding 3 ^mol NADH2, 10 ^mol succinate to a 1 ml cell. The N2-oxidase activity was also determined in the presence of 2 ^g of rotenone. Change medium: 0.25 M sucrose containing 50 mM Tris-HCl, pH 7.4 and 5 mM histidine [20]. The activity of oxidase systems in the mitochondria was registered polarograph-ically with a rotating platinum electrode under standard conditions in a 1-ml polarograph cell at 25 0 C.
Results and discussion:
It is known that liver mitochondria have two oxidation systems - the internal phosphorylating pathway for oxidation of succinate and substrates oxidized via NAD,
and the external pathway of free oxidation of added NADH; the initial part of the respiratory chain of this path is NADH2-cytochrome b5-reductase [12]. One of these oxidation pathways is inhibited by rotenone (internal oxidation pathway). In Table 1 shows the results of the separate determination of the oxidation rates of NADH on the inner and outer paths at 25 0 C. It can be seen that moderate and high ambient temperatures affect the NAD oxidase systems of rat liver mitochondria differently. Holding the animals at 28-30 o C for 30 and 60 minutes increases the rate of oxidation of NADH through the respiratory chain by 6.7 and 12.9%, respectively, from the control (at 22-24 o C), along the external - the rotenone-sensitive oxidation pathway NADH increased by 4.6 and 12.3%. With an increase in the temperature of the medium to 35-36 o C the increase in the activity of oxidase systems is markedly enhanced. In this case, the rotenone of sensitive NADH oxidase is increased by 22.8 and 32.8%, and the rotenone of insensitive NADH oxidase is increased by 24.8 and 38.4%.
Table 1.- Influence of different intensity of ambient temperature on NADN oxidase systems of liver mitochondria membranes (M ± m; n = 5-6)
Time, min Calcium-accumulation capacity of mitochondria,nmol / mg protein
Temperature intensity, ° C
22-24 28-30 35-36 41-42
the rotenone sensitive NADH oxidase
30 53.44 ± 3.09 57.02 ± 3.00 65.62 ± 4.12** 49.64 ± 2.13
% 100 106.7 122.8 92.9
60 55.32 ± 3.55 62.45 ± 3.67** 73.46 ± 4.42*** 45.75 ± 3.09**
% 100 112.9 132.8 82.7
the rotenone insensitive NADH oxidase
30 5.05 ± 0.32 5.28 ± 0.40 6.30 ± 0.49*** 7.69 ± 0.54****
% 100 104.6 124.8 152.4
60 5.11 ± 0.49 5.74 ± 0.41* 7.07 ± 0.54** 9.33 ± 0.66***
% 100 112.3 138.4 192.5
Thus, an increase in the temperature of the medium accelerates the activity both along the internal and external oxidation pathways of NADN. With increasing intensity of temperature, these processes are markedly enhanced. In our opinion, an increase in temperature to 35-36 o C is associated with an increase in the access of substrates to the active center of NADH-dehydrogenase of liver mitochondria. At the same time, there is no elimination of cytochrome c from the inner membrane of the mitochondria. This means that the controlled high tem-
perature does not disrupt the compactness of the mitochondrial membranes.
However, at a temperature of41-42 0 C, another pattern is observed: the internal oxidation pathway of NADH is inhibited, and the external pathway rises NADH is markedly increased. Under these experimental conditions, the activity of the rotenone of sensitive NADH oxidase decreases by 7.1 and 17.3%, respectively, from the control level (22-24 o C), while the rotenone of insensitive NADH oxidase, by contrast, increases by 52.4 and 92.5%.
In our opinion, the conditions for activation ofthe external pathway of oxidation of NADH and inhibition of the internal pathway of oxidation ofsubstrates under conditions of high temperature of the medium are associated with a violation of the function of coenzyme Qand desorption of cytochrome c from the inner mitochondrial membrane into the inter membrane space, as a result of which the flavin 5- cytochrome c 5 to cytochrome oxidase. These results indicate a profound disruption of conjugation between protein phospholipid bonds of the inner mitochondrial membrane during swelling [17]. Disturbances in membranes associated with changes in phospholipids significantly change the ability of the inner mitochondrial membrane to accept cytochrome c [17]. These characteristics are very sensitive to the formation of membrane disturbances and change even at small degrees of "hidden" damage [17] In our opinion, the cleavage of the phospholipids of the internal mitochondrial membranes by endogenous phospholipases upon swelling leads to a disruption of the function of the free-floating carrier of the reducing equivalents from dehydrogenases to cytochrome chains, coenzyme Q[9] In the respiratory chain, there are three binding centers for coenzyme Q 1) investigated and characterized by LS Yaguzhinsky and coworkers [23] hydro-phobic site in succinate dehydrogenase; 2) the region of the respiratory chain between the cytochromes b and c1, where antimycin A, 2-hydroxy-3-alkylbenzo and naphthoquinones bind; 3) the site of binding of the rotenone to NADH-dehydrogenase [24]. At these binding points, the coenzyme Qinteraction ofthe coenzyme nucleus with the corresponding enzyme is realized at the expense of different functional groups of the coenzyme Qmolecule [23].
Damaged mitochondria are the trigger for the release of cytochrome c through mitochondrial pores [22]. Released by cytochrome c is the "death sentence" of the cell [6]. Cytochrome c entails the transmission of an apop-tosis signal, the result of which is often various diseases. Mitochondrial DNA mutations (mtDNA) can also be the cause of many diseases [6]. Compared to nuclear DNA, mtDNA is round, small and simple [10; 16]. An interesting detail about mutations and mtDNA diseases is that the more nuclear mutations occur, the more severe symptoms are encountered [19]. Generation of ATP may decrease with mtDNA mutations; therefore, cells that depend on DNA produced in mitochondria are considered to be the highest risk of disease [11]. Many
patients in resuscitation departments have a clinical state of mitochondrial dysfunction caused by active oxygen species and mtDNA damage [10].
The most important antioxidant of mitochondria is coenzyme Q10, which is contained in virtually all tissues of the body. As is known, coenzyme Q10 is a carrier of electrons in the respiratory chain, while at the same time effectively protects the lipids of biological membranes and lipoproteins of blood from peroxidation, protects DNA and proteins from oxidative modification [22].
Here, first of all, we are not interested in what further processes will be and the role of cytochrome c, which is released, is, but the fact of its release from the mitochondria. From our point of view, this simple, at first glance, act is very important, because as a positive feedback supports the violation of the transport of electrons in the respiratory chain, reduces the utilization of molecular oxygen by mitochondria and, therefore, promotes its accumulation in the cell and the stability necessary for apoptosis states of oxidative stress first in the mitochondria, then in the cytoplasm and throughout the entire cell. In addition, positive feedback on the maintenance of oxidative stress in cells is realized, obviously, through other channels. One of them is the active form of oxygen-dependent damage to mitochondrial DNA (mtDNA)
with the formation and accumulation, in particular, of
8 hydroxy 2' deoxyguanosine. This oxidative damage of mtDNA is repaired to some extent [2]. Another channel of such feedback may act by oxidative damage to the mtDNA polymerase, which should result in a decrease in mtDNA replication and, accordingly, mitochondrial base attenuation. Thus, in mitochondria, there are different ways of keeping the oxidative stress that arises in them no lower than a certain level.
Discussion.When rats are kept at28-30 and35-36 o C, the permeability of membranes for substrates is slightly exceeded, but stability will not change. The cell passed from one physiologically metabolic state to the second active state, improves the delivery of substrates, oxygens and energy to cells and tissues of the body. When the rats are kept at a temperature of 41-42 o C, the mitochondria of the liver are inflated, the outer membranes are cracked and, in connection with the injury of the inner membrane, cytochrome c and other propooptical proteins from the membrane will escape and reach the nucleus of the cells to injure it.
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