INVOLVEMENT OF KАТР-CHANNELS OF PLASMA AND MITOCHONDRIAL MEMBRANES IN MAINTAINING THE CONTRACTIVE..

© 2020 O.V. Tsymbalyuk et al.; Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia Biologica. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://www.budapestopenaccessinitiative.org/ and Creative Commons Attribution 4.0 License), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. UDC: 577.353

K + -channels, in particular, ATP-sensitive K + -channels of plasma and inner mitochondrial membranes, play a significant role in regulating the contraction-relaxation processes in visceral smooth muscles. The specificities of these channels and their role in maintaining excitation have been well investigated for the cardiac muscle and smooth vascular muscles. However, scarce data exist about the involvement of these channels, especially the mitochondrial ones, in regulating myometrium contractions. The tenzometric methods and mechanokinetic analysis were used to study the regularities in oxytocin-induced contractions of smooth muscles of non-pregnant rats myometrium under the conditions of modulating ATP-sensitive K + -channels of plasma and inner mitochondrial membranes. It was determined that prior incubation of myometrium in the presence of the blocking agents of K ATP -channels of plasma membrane and mitochondria (glibenclamide and 5-hydroxydecanoate, respectively) was generally associated with the increase in the phase and, in some cases, tonic components of oxytocin-induced contractions. There was an increase in the amplitude of oxytocin-induced contractions at the background of the impact of glibenclamide (the range of concentrations used was 1-10 µM) without significant changes in the area under these mechanograms; the velocity of relaxation increased considerably as well. The blocking agent of mitochondrial ATP-sensitive K + -channels, 5-hydroxydecanoate (in the concentrations of 50 µM and 200 µM) caused considerable changes in the kinetics of the processes of intensification (the increase in velocity) and decline (more than 2-fold decrease in the velocity) in comparison with the force of oxytocin-induced contractions, but it did not impact their amplitude and the area under mechanograms. The most significant effect, demonstrated by

INTRODUCTION
Smooth muscles (SM) play a vital role in the functioning of internal organs and systems of the organism, forming the structural-functional basis for the gastrointestinal tract, the vascular bed, the genito-urinary system, and respiratory pathways. In general, the regulation of the capability to maintain basal strain and active contraction of SM is ensured by changes in the intracellular concentration of Ca 2+ ions, which is conditioned by the combination of diverse processes: the work of energy-dependent systems of Ca 2+ -extrusion from the cell, the penetration of these ions via calcium channels from the extracellular space and the release from the intracellular depots (sarcoplasmatic reticulum and mitochondria) [4,5,39,30]. The regulation of Ca 2+ -dependent processes of SM contraction-relaxation is considerably performed by K + -channels, including ATP-sensiti ve K + -channels (K ATP -channels). It was established that the activation of these channels reduced the duration of the action potential, thus decreasing the release of Ca 2+ into the cytosol that conditioned muscle relaxation [1,31,36].
K ATP -channels of two types are expressed in the uterus tissues: the inwardly rectifying K ATP -channel of plasma membrane and mitochondrial ATP-sensitive K + -transporter (mitoK ATP -channel) [23,25,27]. K ATP -channels of plasma membrane play one of the key roles in regulating the membrane potential of the myocyte and the contractive activity of myometrium tissue. For instance, it was demonstrated that during pregnancy and up to delivery there were changes in the properties of these channels, including the sensiti vity to pharmacological and physiological regulators (Ca 2+ ions and membrane potential) along with the decrease in the expression of its subunits [10,23]. According to other researchers [11,26], the expression rate of these channels depends on the age and the functional state of myometrium: on the one hand, it decreases at late stages of pregnancy, and on the other -it increases in non-pregnant uterus of a woman with age. It indicates the relevance of ATP-sensitive K + -channels as regulators of the functional state and excitation of the uterus.
MitoK ATP -channels are capable of regulating the bioenergetic state of mitochondria, affecting such relevant indices as the intensity of breathing and membrane potential of the inner membrane, which, in its turn, impacts the physiological state of smooth muscle cells (SMC) and SM tissues in general [32,34]. They also regulate the velocity of forming reactive oxygen intermediates and demonstrate their cytoprotective action under pathological conditions, under the influence of pro-oxidant factors and high concentration of Ca 2+ ions [19].
Both plasma and mitochondrial K ATP -channels in muscles are essential for maintaining normal functioning of myocytes and SM in general. The activation of K ATP -chan nels leads to the decrease in muscle excitation and preservation of the energy balance in all the types of muscles, without exceptions, under both normal and pathological conditions. It was concluded that the cells of myometrium of both non-pregnant and pregnant women and female rats contain diazoxide-sensitive K ATP -chan nels, whose activation results in inhibiting the contractions of the uterus [15,25,27]. The scientific data demonstrate good prospects for applying the effectors of K + -chan nels (including the activators of K ATP -channels) as tocolytics during the therapy of premature delivery [2,13,14].
The aim of our study was to investigate the involvement of K ATP -channels of plasma and mitochondrial membranes in maintaining the coordinated contractive activity and their role in modulating oxytocin-induced contractions of myometrium of intact fragment of the uterus wall with preserved mucosa.

MATERIALS AND METHODS
The experiments were conducted using Wistar line rats (the average weight of animals was 200-250 g). All the manipulations with animals were conducted according to the International convention for the protection of animals and the Law of Ukraine "On Protection of Animals from Cruelty" (the Minutes of the meeting of bioethics commission of SSC Institute of Biology and Medicine No. 3 dated May 2, 2019). The animals were killed by the introduction of a lethal dose of propofol narcosis (Sigma).
The tenzometric experiments were conducted using the preparations of longitudinal smooth muscles of uterine horns. Muscle stripes (the average size -2×10 mm) were placed into the working chamber of 2 mL with the flowing Krebs solution (the flow rate of 5 mL/min), thermostated at 37 °С. The Krebs solution contained (in mM): 20.4 NaCl; 5.9 KCl; 15.5 NaHCO 3 ; 1.2 NaH 2 PO 4 ; 1.2 MgCl 2 ; 2.5 CaCl 2 ; 11.5 glucose; рН of solution was 7.4. The preparations were provided with passive tension at the rate of 10 mN and left for 1 h (until achieving stable reproduction of contractions). The contractive activity was studied in the isometric mode using the force sensing device. The signals were registered with an analogue-to-digital transformer.
The contractions were induced by the application of uterotonic hormone oxytocin (0.1 i.u., the average concentration of protein with hormonal activity is about 0.033 µM; Gideon Richter, Hungary). The activator of ATP-sensitive K + -channels of plasma membrane, diazoxide (concentrations of 50, 100, 150 and 200 µM; Sigma, USA), the blocking agent for these channels, glibenclamide (concentrations of 1, 2, 4, 6, 8 and 10 µM; Sigma, USA) and the blocking agent for ATP-sensitive K + -channels of mitochondria, 5-hydroxydecanoate (concentrations of 50 µM and 200 µM; Sigma, USA) were used in the experiment.
Stock solutions of diazoxide and glibenclamide were prepared by preliminary dissolution of the substances in the organic solvent dimethylsulfoxide (DMSO) and added to Krebs solution to obtain the final aliquot of DMSO of 0.1% from the total volume of Krebs solution. All the other contractions, including the control ones, were studied in the solutions, containing 0.1% DMSO. The stock solution of 5-hydroxydecanoate was prepared by preliminary dissolution of this substance in distilled water.
Non-selective blocking of ATP-sensitive K + -channels was done by preliminary incubation for 20 min of SM preparations with glibenclamide, and their activation -with diazoxide. The blocking of ATP-sensitive K + -channels of mitochondrial membranes was done by preliminary incubation for 20 min of SM with 5-hydroxydecanoate (5-HD). After the preliminary incubation of preparations with the above-mentioned substances, the effect of the latter on spontaneous and acetyl choline-induced contractions of SM was tested.
The mechanokinetics of the process of contractions-relaxations of muscle preparations (the calculations of normalized maximal velocities for contraction and relaxation phases, V nс and V nr , respectively) was studied according to the methods described in [3]. The contraction phase was defined as a fragment of contractive response from the beginning of force variation up to its maximal value (amplitude of phase contraction). The relaxation phase started with the maximum of phase contraction and lasted till achieving the stable level of the tonic contraction. The introduction of the tonic contraction into the contractive responses of smooth muscle preparations was calculated as a percentage of stationary strain relative to the maximal force of contractive responses.
The experimental data was processed by the variation statistics methods using OriginPro 8 program. The samples were checked in terms of belonging to normally distributed general populations according to Shapiro-Wilk criterion. The paired version of Student's t-test was used to determine the reliable differences between the mean values of samplings. In all cases the results were considered reliable on condition of the probability value р, under 5 % (р < 0.05). The validation analysis of data approximation by the linear function was performed using Fisher's F-criterion; determination coefficients (R 2 ) were at least 0.9. The results were presented as the arithmetic mean ± standard error of the mean value, n -number of experiments.

RESULTS AND DISCUSSION
The mechanokinetic parameters of contractions of rat myometrium under activation of oxytocin receptors with uterotonic oxytocin were investigated (Fig. 1A). This agonist is known to activate receptors, associated with G q/11 -proteins, inducing contractions via inositol triphosphate-dependent pathway [4]. The contractions of myometrium preparations, induced by oxytocin (0.1 i.u.), can be characterized by the following average kinetic parameters: the velocity of contraction phase V nc (3.31 ± 0.59) min -1 (n = 9); the velocity of relaxation phase V nr (0.23 ± 0.05) min -1 (n = 9).
It was determined that the prior incubation of myometrium in the presence of the blocking agents of K ATP -channels of plasma membrane and mitochondria was generally accompanied by the increase in the phase and, in some cases, tonic components of oxytocin-induced contractions. For instance, under blocking of K ATP -channels of SMC with glibenclamide at the cumulative increase in its concentration in the washing solution from 1 µM to 10 µM, there was a dose-dependent increase in the amplitude of oxytocin-induced contractions (Fig. 2). In the presence of glibenclamide in the concentration of 1 µM, the amplitude of contraction was 110.7 % on average, and under the increased concentration of this blocking agent up to 10 µM, it was 131.6 % as compared to the control. It should be noted that the glibenclamide concentrations used were capable of blocking effect on K ATP -channels of the plasma membrane, but they were not sufficient to induce significant changes in the conductance of these channels in mitochondria [22]. preparations considerably increased, depending on the dose (Fig. 2). For instance, at the cumulative increase in glibenclamide concentration in the washing solution up to 10 µM, the indices of V nc and V nr were 89.9 % on average (n = 6, p<0.05) and 185.7 % (n = 6, p<0.01) (as compared to the control values taken as 100 %). At the background of the cumulative increase in the concentrations of the activator for ATP of K ATP -channels of diazoxide (50, 100, 150 and 200 µM), there was a decrease in the amplitude of oxytocin-induced contractions (Fig. 3). For instance, in the presence of 200 µM of diazoxide, the average amplitude was 84.3 % as compared to the control. It should be noted that this substance caused a considerable dose-dependent decrease in the area under curves for oxytocin-induced contractions; for instance, under the influence of 200 µM of diazoxide this index decreased almost twice as compared to the control (Fig. 3).
At the background of the activation of K ATP -channels with diazoxide, there were considerable changes (an increase) observed in the kinetic parameters of oxytocininduced contractions of myometrium (Fig. 3). For instance, in the presence of 200 µM of diazoxide in the washing solution, the indices of normalized maximal velocities for the phases of contraction and relaxation, V nc and V nr , respectively, were as follows: 238.5 % (n = 6, p<0.01) and 183.8 % (n = 6, p<0.01) (as compared to the control values taken as 100 %). In general, the blocking of K ATP -channels of the inner mitochondrial membrane with 5-HD (50 µM and 200 µM) caused no changes in the amplitude or the area under the tenzometric curve for oxytocin-induced contractions (Fig. 4). However, in these conditions there were considerable transformations in the kinetics of these curves: the contraction phase accelerated greatly, whereas the relaxation phase slowed down significantly. For instance, under the influence of 50 µM and 200 µM of 5-HD the average parameter of V nc was 173.2 % (n = 6, p<0.05) and 203.9 % (n = 6, p<0.05), respectively, and the normalized maximal velocities of relaxation V nr were as follows: 73.8 % (n = 6, p<0.05) and 42.9 % (n = 6, p<0.05) (as compared to the control values taken as 100 %).
The combined effect of the blocking agents of K ATP -channels of plasma and inner mitochondrial membranes, glibenclamide (10 µM) and 5-HD (200 µM), was accompanied with the increase in the amplitude of oxytocin-induced contractions up to 121.0 % on average (n = 6, p<0.05), but the area under the curves for contractions showed no significant changes (Fig. 5А). However, in these conditions, there were significant changes in the normalized maximal velocities of the contraction and relaxation phases, V nc and V nr : these indices were 83.6 % (n = 6, p<0.05) and 163.9 % (n = 6, p<0.05) respectively (the data presented was re-calculated in comparison to the control taken as 100 %) (Fig. 5А).
We also studied oxytocin-induced (0. [Diazoxide] (µM) V nr * * * * * * * * * * * * * * the blocking agent of mitoK ATP -channels, 5-HD (200 µM) (Fig. 1F). In these conditions, there were significant changes in the kinetic indices of V nc and V nr -up to 235.9 % (n = 6, p<0.01) and 63.7 % (n = 6, p<0.05) respectively (the data presented was re-calculated in comparison to the control taken as 100 %) (Fig. 5B). Uterine tubes contain the areas with sources of spontaneous activity in the cervical and ovarial sections. The research group of K.V. Kazarian et al. [20,21] determined that the main source of rhythm in the uterine tubes is the ovarial section. The main uterotonic hormone, oxytocin, promotes the dominance of the ovarial section and provides coordinated functioning of all the rhythmogenic parts of myometrium [20]. The control of excitation and contractive activity of SM in uterine ensures the preservation of fertility and the ability to bear the fetus and endure the delivery [35] since at least half of the cases of newborns' deaths are related to premature delivery, the cellular basis of which is the disrupted regulation of SM strain and the processes of contractions and relaxations of myometrium.
Potassium channels is an important factor of myometrium excitation regulation including K ATP -channels of inward rectification. V nr * * * * * * * K ATP -channel of the plasma membrane. Two isoforms of K ATP -channels of the plasma membrane are expressed in the myometrium of non-pregnant rats and humans: prevailing domination is noted for isoform Kir6.1/SUR2B, whereas Kir6.2/SUR1 is detected in the insignificant amount [11,23,25]. During the pregnancy of women, there is a decrease in the expression of Kir6.1/SUR2B, thus, the tissues of uterus get ready to the delivery activity [11]; this regularity was not observed in the myometrium tissue of female rats. За 100 % прийнято відповідні показники у контролі; (M±m, n = 6); * -p<0,05; ** -p<0,01 -різниця достовірна щодо контролю ATP-sensitive K + -channels of plasma and mitochondrial membranes of smooth muscle cells have a similar heterotetrameric structure and similar mechanisms of pharmacological and physiological regulation. The activation of both types of K ATP -channels demonstrates cytoprotective effect and improves the bioenergetic state of the cell. For instance, the investigations of the processes of ischemia-reperfusion of myocardium and other tissues showed that the activation of both plasma and mitochondrial K ATPchannels underlies the phenomenon of ischemic preconditioning, which resulted in the protection of cells from destruction and, in case of myocardium, the decrease in the area damaged by necrosis [5,6,9,24]. Under normoxia, the activation of plasma K ATPchannels in the myocardium reduces the potential of the effect and ensures relaxation of the cardiac muscle. In smooth muscles, the plasma K ATP -channels also fulfill relevant functions depending on their localization. For instance, in SMC of vascular muscles they are involved in the regulation of the vascular tone [8]. Besides vessels, K ATP -channels were also identified in the plasma membrane of SMC of other muscles, including gastrointestinal and urinary tracts. The obtained results are in agreement with the data of K. Sawada et al. [28] who demonstrated that the activation of K ATP -channels of the plasma membrane led to inhibiting the amplitude of oxytocin-induced contractions, and the combined use of activators and blocking agents of these channels eliminated the effect of the former. Similar results were obtained using non-pregnant mice myometrium [17]. It should be noted that, in addition to impacting oxytocin-induced contractions, K ATP -channels are involved in the regulation of other agonist-induced contractive responses (including the ones to the activity of acetylcholine and prostaglandin F2α) [16][17][18].
The scientific data related to the expression and role of mitochondrial K ATP -channels in the functioning of SM, is very fragmented and mostly limited to the vascular wall [15]. MitoK ATP -channels are localized in the inner mitochondrial membrane; they transport K + ions to the matrix, ensuring the driving force of K + /Н + -antiporter and thus participating in the regulation of the volume and energetic homeostasis of mitochondria.
While analyzing the obtained results, one may envisage that K ATP -channels of the plasma membrane and mitoK ATP -channels ensure the modulation of oxytocin-induced contractions of myometrium. Their cumulative blocking leads to the increase in the amplitude of these contractions. However, this effect exert considerable impact on the total efficiency of the contractive function of myometrium, as the parameter of the area under mechanogram remains at the level of control values.
The normalized maximal velocity of relaxation, V nr , is the parameter, most sensitive to the activity of the effectors of K ATP -channels; in general, it increases both under nonselective blocking and activation of these channels. It is noteworthy that in case of selective blocking of mitoK ATP -channels this parameter decreases considerably, and this slowing-down of the muscle relaxation process is not eliminated in the presence of the non-selective activator of K ATP -channels, diazoxide. Therefore, it may be predicted that mitoK ATP -channels fulfil a vital function in the myometrium tissue, defining the velocity of relaxation, thus regulating the duration of the oxytocin-induced spasm of the uterus. This process can be mediated by the regulatory effect of mitoK ATP -channels on the processes of accumulation of Ca 2+ ions by myocyte mitochondria [13].

CONCLUSIONS
Our research demonstrated that K ATP -channels of plasma membrane and mitoK ATPchannels in non-pregnant rats' myometrium play a modulating role in forming the contractive response to the uterotonic hormone oxytocin. These channels are involved in the regulation of the contractive function of myometrium, modulating the amplitude of contractions, the ability of long-term strain support, and the kinetics of contraction and relaxation processes.