Identi fi cation of nitric oxide in mitochondria of myometrium cell

Aim. To demonstrate the possibility of NO synthesis in intact myocytes of uterus. Methods. Confocal scanning microscopy method, NO-sensitive fl uorescent probe DAF-FM, MitoTracker Orange CM-H2TMRos. Results. The basal production of NO in intact myocytes was shown using DAF-FM. Incubation of myocytes with NO donor – sodium nitroprusside (SNP) – led to an increase of the DAF-FM-T fl uorescent signal. On the contrary, the addition of NO-synthase inhibitor – N-nitro-L-arginine (NA) – results in the reduction of fl uorescent intensity. It was demonstrated colocalizition of specifi c probe for mitochondria MitoTracker Orange CM-H2TMRos and NO-sensitive dye DAF-FM. Conclusions. For the fi rst time it has been demonstrated the presence of NO in smooth muscle cell mitochondria using laser confocal microscopy, NO-sensitive probe DAF-FM and specifi c marker of the functionally active mitochondria MitoTracker Orange CM-H2TMRos. K e y w o r d s: nitric oxide, myometrium, DAF-FM, confocal microscopy.


Introduction
Nitric oxide (NO) plays an essential role in many physiological processes, particulary in vasodilation, neurotransmission, immune responses, etc. [1,2].The re are convincing proofs of the functional role of NO as a tocolytic agent.The ability of nitrocompounds to modulate activity of the Ca 2+ -transport system in myometrium cells was established [3].At the same time, the identifi cation of sources of its biosynthesis in the uterus myocytes is still a problem, because of a short time of existence and a high reactivity of nitric oxide.
The presence of the Ca 2+ -dependent isoforms of NO-synthase in mitochondria (mtNOS) was proved by immunohistochemical methods for single tissues [4][5][6].On the one hand, nitric oxide can regulate the activity of electron transport chain in mitochondria through a reverse decrease in the cytochrome c-oxidase activity, and control of mitochondria pH.On the other hand, an excess production of NO together with intensifi cation of the superoxide anion formation in mitochondria is accompanied by the peroxynitrite generation, damage of the respiratory chain components, mitochondria depolarization and the apoptosis development [4][5][6].So in general, the mitochondria and cells life and destruction depend on the level of NO production.This indicates the importance of identifi cation of NO biosynthesis in the intact cells mitochondria.
Modern fl uorescent probe DAF-FM allows the recording of NO production, even the registration of the low NO concentrations (2-5 nM) in intact cells; the laser confocal microscopy method allows the visualization of the NO formation and the confi rmation of the link of NO biosynthesis by these organelles using specifi c probe for mitochondria [4,7].
So, the purpose of this work was to demonstrate the possibility of NO synthesis in the intact myocytes of uterus by energized mitochondria using the sensitive fl uorescent dye DAF-FM, the selective Identifi cation of nitric oxide in mitochondria of myometrium cell mar ker for mitochondria MitoTracker Orange CM-H 2 TMRos and confocal laser scanning microscopy.

Materials and Methods
Myocytes were isolated from the uteruses of white nonlinear nonpregnant rats, by the method of Mollard using collagenase and soybean trypsin inhibitor [8].The animals were narcotized by inhalation of diethyl ether, after that they were decapitated.All regulations for the work with laboratory animals were maintained (International Convention, Strasbourg, 1986).
For the visualization of mitochondria and cell nucleus the fl uorescent dyes MitoTracker Orange CM-H 2 TMRos (200 nM) and Hoechst 33342 (50 nM) were used respectively [10].The loading of immobilized myocytes with NO-sensitive fl uorescent pro be DAF-FM (C 21 H 14 F 2 N 2 O 5 , 4-amino-5-me thy la mi no-2′,7′-difluo rescein, diaminofl uoresce in-FM) in concentration 10 μM was carried out for 15 min at 24 C.The experiments with confocal microscopy were performed in Multi Track mode.The Multi Track function permits several tracks to be defi ned as one confi guration for the scan procedure.Each track is a separate unit and can be confi gurated independently of the other tracks with regard to channels.Fluorescence of Hoechst 33342 was excited using laser wavelength 405 nm, and registered with the fi lter BP 420-480.For MitoTracker Orange CM-H 2 TMRos, the fl uorescence laser at the wavelength of 543 nm was used, and fl uorescence was registered in the range of 560-615 nm (fi lter BP 560-615); DAF-FM fl uorescence was excited at the wavelength 488 nm, and the emission was collected in the 505-530 nm signal range (fi lter BP 505-530) [http://www.lifetechnologies.com/order/catalog/product/D23844,9].The study of fl uorescent dye distribution in a cell was performed in Time Series mode.For the quantitative analysis of the time-dependent fl uorescence intensity of the cell, the ROI (Region Of Interest) mode was used.
The data are presented as mean ± SEM [10].

Results and Discussion
In the experiment an active acid form of DAF (DAF-FM) was used.It interacts directly with NO in the presence of O 2 , forming triasole-fl uorescein (DAF-FM-T) derivative, that has high quantum yield of fl uorescence [7,11].The effi ciency of DAF-FM entering into myoplasm was increased by permeabilization of the plasma membrane by 0.1 % digitonin, although the dye can penetrate partially through the membrane into a cell by diffusion [12].The treatment of cells by the detergent signifi cantly reduces the contribution of NO-synthase associated with the plasma membrane in a fl uorescent signal.On the other hand, the dye entering the mitochondria was increased.These studies revealed (Fig. 1), that there was green fl uorescent signal after pre-incubation with DAF-FM.The result refl ects a basal NO production [4].The signal was not associated with the cells autofl uorescence in this area.A high sensitivity of the used dye permits to register a basal level of NO in myocytes, that is formed as a result of the constitutive NO-synthase functioning.
The signal is detected in the cell myoplasm, mapping its contours and forming the heterogeneous dyed areas.There were the NO-positively dyed areas also in the nuclei, and sometimes outside the cells, as a result of the diffusion.
The incubation of myocytes with 0.1 mM sodium nitroprusside (SNP) -NO donor -led to a substantial increase of fl uorescent signal (Fig. 2).The fl uorescence increased in myocytes and outside the cells, because NO was generated by SNP outside the cells too.
The incubation of cells with increased concentration (0.1 and 0.2 mM) of N-nitro-L-arginine (NA)a nonselective inhibitor of the Ca 2+ -dependent isoforms of NO-synthase [13] -was accompanied by the dose-dependent reduction of DAF-FM-T fl uorescent intensity on average by 20 % (0.1 mM inhibitor) and 40 % (0.2 mM inhibitor).The growth of fl uorescent response with adding SNP occurred in the presence of inhibitor (Fig. 3, 4).
These results show that DAF-FM responds to NO. DAF-FM does not test a level of a wide range of the active nitrogen and oxygen metabolites, for example NO 2 -, NO 3 -, ONOO -, • O 2 -, H 2 O 2 , etc. [4].There are good reasons to use DAF-FM for detection of nitric oxide in intact cell in our further investigations.
Colocalization of the NO-sensitive dye DAF-FM and the specifi c probe for mitochondria MitoTracker Orange CM-H 2 TMRos, accumulated only in the intact energized organells [14], was demonstrated by the next studies (Fig. 5).
The computer analysis the image outside the cell nucleus has revealed an identical distribution of both fl uorescent dyes (Fig. 6).
We have shown the potential presence of NO in mitochondria of uterus myocytes.Nonetheless, the nitric oxide biosynthesis in cells is linked not only with mitochondria.Constitutive NO-synthase is associated with the plasma membrane, sarcoplasmic reticulum and other intracellular structures.NO, synthesized by these structures, can diffuse to mitochondria and vice versa.Further researches are necessary to clarify the subcellular distribution of NOsynthases in the intact cells.

Conclusions
In this work for the fi rst time the presence of NO in smooth muscle cell mitochondria has been demonstrated using the laser confocal microscopy, NOsensitive probe DAF-FM and specifi c marker for the functionally active mitochondria MitoTracker Orange CM-H 2 TMRos.It has been also shown that the amount of NO changes in the presence of the nitric oxide donor and NO-synthase inhibitor.