Protective effects of Potassium transPort in mitochondria from rat myometrium under activation of mitochondrial Permeability transition

We demonstrated using PBFI k+-sensitive fluorescent probe an enhancement of both components of k+-cycle – the aTP-sensitive k+-uptake and quinine-sensitive K+/H+-exchange – under the Ca2+ induced opening of mitochondrial permeability transition pore (MPTP) in rat myometrium mitochondria. Addition of CaCl2 (100 μM) to K +-free medium results in the enhancement of reactive oxygen species (ROS) production, which was eliminated by cyclosporine A. Addition of CaCl2 to k +-rich medium did not increase the rate of ROS production, but blocking of mitoKaTP-channels with glybenclamide (10 mcM) increased production of ROS. We conclude that k+-cycle exerts a protective influence in mitochondria from rat myometrium by regulation of matrix volume and rate of ROS production under the condition of Ca2+-induced MPTP.


M
itochondria play an important role in regulation of cell life and death as they are involved in realization of the most important functions, such as providing of energy substrates and control over cell death [1].The inner membrane of mitochondria is encrusted with respira tory chain complex and ATPsynthase, which pro duces ATP, using energy of Δμ H+ in the process.The inner membrane of mitochondria is thus impermea ble to ions, and their matrix homeostasis is regulated via functioning of ion transporting systems of chan nels and exchangers [1,2].The mitochondrial ion homeostasis is a finely tuned process, imbalances in it may adversely affect mitochondrial functions and may provoke cell death.Particularly, matrix overload with Ca 2+ is known to induce cyclosporine A (CsA)sensitive mitochondrial permeabili ty transition pore (MPTP), that exists in either high or low conductan ce state.The highconductance state causes inner membrane rupture followed by cell death.Imbalan ced Ca 2+ homeostasis and MPTP induction caused by it are associated with a number of pathologies such as neurodegenerative diseases and disorders of heart, smooth and skeletal muscles [3,4].
Mitochondria are capable of maintaining nor mal functioning under adverse conditions [5].K + transport across the inner membrane may be one of the factors that help to sustain the mitochondrial function within physiological margins [6].K ions homeostasis is maintained by potassium channels, which provide for accumulation of the ion in mito chondria, and by K + /H + exchanger, which releases K + in cytosol in exchange for H + .The latter's function servers to maintain stable matrix volume and mi tochondrial membranes' integrity.Potassium chan nels, i.e. mitochondrial K + ATP channels (mitoK + ATP channels) regulate matrix volume, function of electrontransport chain, as well as indirectly af fect F 0 F 1 ATPase and generation of reactive oxygen species (ROS) [2,7].For instance, Costa et al. [8] demonstrated, that cooperative activity of mitoK + ATP channel and K + /H + exchanger in muscle mitochon dria is responsible for creating and supporting a new equilibrium matrix volume.K + influx through the mitoK + ATP channel causes alkalinization of matrix that mediates increased ROS production [9].The last effect, in particular, is perceived as key to cy toprotective mitoK + ATP channel effects under MPTP activation.For example, Costa et al. [8] showed, that mitoK + ATP channel activation indirectly prevents MPTP opening through increased ROS production and consequent protein kinase C activation, which is one of the key enzymes protecting cells from death [10].These effects are probably involved in the phe nomenon of preconditioning -the protective influ ence of series of short ischemic periods prior to pro longed ischemia [7,10].Cell damage under adverse conditions, namely oxidative stress, has been proven експериментальні роботи doi: http://dx.doi.org/10.15407/ubj87.06.086 to include the disruption of Ca 2+ homeostasis, which in turn causes necrotic or apoptotic cell death [11].There is also data supporting activation and cytopro tective effects of mitoK + ATP channels under increased cytosol Ca 2+ concentration and induction of MPTP opening [12].
Nevertheless, the mechanism underlying cell protection by mitoK + ATP channel has not been studied thoroughly.The mitoK + ATP channels' priori ty role over K + ATP channels of plasma membrane in preconditioning remains a speculative subject as well.The influence of activation of K + transport in smooth muscle mitochondria on ROS generation has not been investigated.Also, there is no data as to specifics of functioning of mitoK + ATP channels from myometrium under stress conditions, such as Ca 2+ overload.Therefore, we pursued the aim to investigate, using probes and selective blockers of K + ATP channels and MPTP, the effect of Ca 2+ on K + transport, dynamics of changes in matrix volume and ROS gene ration in isolated rat myometrium mi tochondria.

materials and methods
Isolation of mitochondrial fraction from myometrium.Mature female white rats (150200 g body mass) were anesthetized with ethyl ether and decapitated.Uterus tissue was cleaned from blood and fat, minced and homogenized on ice in 8 ml of isolation buffer solution of the following composi tion: 250 mM sucrose, 1 mM EDTA, 10 mM HEPES (pH 7.2 titred by 2 M Tris).The homogenate was centrifuged for 7 min at 1000 g and 4 °C.The su pernatant was centrifuged for 7 min.at 12 000 g and 4 °C.The sediment of mitochondria was resuspended in isolation buffer without EDTA and stored on ice.Protein content was determined using Bradford assay.
Concentrated glibenclamide and CsA solu tions in dimethyl sulfoxide were applied introduced into incubation medium in cuvette at 1 μl per 1.8 ml of working volume (final concentration of 10 μM).CaCl 2 solution was introduced in the cuvette to final concentration of 100 μM.
The suspension was incubated with 40 μM acetoxy methyl ether of PBFI (PBFI-AM) and 0.5 μl of 20% solution of F127 nonionic surfactant for 10 min at room temperature.The suspension was then mixed for 2 min with 500 μl of buffer for K ions substitu tion in mitochondrial matrix, consisting of 175 mM sucrose, 10 mM HEPES (pH 7.2 at 25 °C), 5 mM succinate, 5 mM Na 2 HPO 4 1 mM MgCl 2 , and 50 mM tetraethyl ammonium.7 ml of mitochondria isola tion buffer was then added to the mix, followed by centrifugation for 10 min at 12000 g and 4 °C.The sediment was resuspended in buffer with 250 mM sucrose and 10 mM HEPES (pH 7.2 at 4 °C and no EDTA added) and kept on ice.Probe fluorescence was registered at 340 nm and 380 nm wavelength of excitation and 480 nm wavelength of emission.Final protein concentration in a sample was 5560 µg/ml.
The measurements were performed with PTI Quanta Master 40 spectrofluorometer (Canada) in a standard fluorometric cuvette in thermostatic sam ple holder with magnetic stirrer at 28 °C in standard incubation medium consisting of 10 mM HEPES (pH 7.2 at 28 °C titred by 2 M Tris), 125 mM KCl, 5 mM Na 2 HPO 4 , 1 mM MgCl 2 , 5 mM succinate, 5 µM rotenone, 2 µg/ml oligomycin.KCl was re placed with NaCl on equimolar basis in certain sam ples.Final protein concentration in a sample was 5560 µg/ml.
Mitochondria swelling was measured by regis tering side lateral light scattering at 520 nm in standard incubation medium (see above) as de scribed [8].The signal registration begun 1 s after the added mitochondria suspension had been intro duced in the incubation medium.
ROS generation was assayed with ROSsensi tive 2′,7′-dichlorofluorescein diacetate probe (DCF-DA).Probe solution was introduced into incubation medium, and the readings were taken immediately.Final probe concentration was 4 µM.Fluorescence excitation wavelength was 540 nm, and emission was registered at 520 nm 1 s after mitochondria suspension had been introduced into the incubation medium.

results and discussion
Mitochondria isolation in sucroserich K + free medium leads to a markedly decreased content of these cations in matrix.Afterwards, as the mitochon dria are introduced into incubation medium contain ing KCl, P i anions, and respiratory chain substrates, they begin to uptake K + intensively, which has been demonstrated by various methods, including fluo rescent spectroscopy with potas siumsensitive PBFI probe [8].In order to ensure the adequacy of the probe's response to K + in isolated mitochondria from the rat myometrium, we studied intensity of fluores cence PBFI loaded in mitochondria depending on KCl concentration in incubation medium.Introduc tion of mitochondria loaded with PBFI into the in cubation medium with various KCl concentrations (see Materials and Methods) led to dosedependent increase in PBFI fluorescence, which testifies to the adequacy of response of the probe to K + accu mulation in mitochondrial matrix (Fig. 1, a).The maximum probe's response was registered at KCl concentrations close to physiologic (125 mM), and consequently this concentration was used in the standard incubation medium.K + influx into mito chondrial matrix in these experimental conditions is by diffusion due to high membrane potential, and by K + transport channels (ATPsensitive, in particular) on the inner mitochondrial membrane [13].As has been demonstrated in numerous publications, the functioning of mitoK + ATP channels is blocked by ATP [8].ATP in concentration of 200 µM in the standard incubation medium inhibited K + accumulation in myometrium mitochondria as well (Fig. 1, B, 8).The inhibiting effect of ATP was totally eliminated by mitoK + ATP channels activator diazoxide (Fig. 1, B, 9), which is also in accordance with data from experi ments performed on mitochondria from other tissues [2,8,14].Thus, our results corroborate the existence of K + ATP channels in the rat myometrium mitochon dria.
As has been mentioned by various authors, the pathological conditions inhibiting normal muscle tis sue and cell functioning are mediated by disruptions in Ca 2+ homeostasis, increase in its cytosol concen trations, sometimes by orders of magnitude above the physiological values, and may lead to nega tive consequences for the myocyte and the muscle in general [11].Hence, the understanding of possible mecha nisms underlying myocytes protection under damaging conditions with increased cytosol Ca 2+ is an important research task.The possibility of regula tion of K + ATP channels by Ca 2+ has been demonstra ted [15].Since the activation of these channels ex hibits a cytoprotective effect in conditions associated with imbalanced Ca 2+ homeostasis, it is interesting to investigate their functional properties in smooth muscle mitochondria under high Ca 2+ concentration.K + accumulation in matrix was found to decrease in incubation medium with 100 µM of CaCl 2 (Fig. 1,  B, 2).Ca 2+ induced decrease in K + accumulation was eliminated by 0.5 mM EGTA, a Ca 2+ chelator (Fig. 1, B, 4).The data presented on Fig. 1, B, sup port the assumption that this influence of Ca 2+ on K + accumulation in myometrium mitochondria is media ted via Ca 2+ uniporter.The inhibiting effect of Ca 2+ on K + accumulation was eliminated by 10 µM of ruthenium red, an inhibitor of Ca 2+ uniporter (Fig. 1,  B, 3).
The blockade of total accumulation of K + in mi tochondria may be caused by either partial inhibition of K + uptake into the matrix or by activation of K + release.Diazoxide, an activator of K + ATP channels, in concentration of 50 µM did not exert any effect on K + accumulation in the presence of 100 µM of CaCl 2 (Fig. 1, B, 6).Lack of activation of K + accumulation by diazoxide in our conditions may have at least two explanations: a) K + ATP channels are already in active state in the presence of Ca 2+ , or b) K + ATP channels are inhibited, and the activator cannot affect their func tional state.Since introduction of 10 µM glibencla mide, an inhibitor of K + ATP channels, in the presence of Ca 2+ restores fluorescent signal to control levels, it evidences in favor of our first assumption (Fig. 1,  B, 5).We have demonstrated the specificity of in hibiting effect of glibenclamide on K + transport in myometrium mitochondria in particular in our previ ous work [16].We have also demonstrated inhibition by glibenclamide of ATPsensitive K + transport in myometrium mitochondria with PBFI probe (Fig. 1, B, 10).This tendency of increase in PBFI signal in the presence of K + ATP channels inhibitors allowed us to formulate a hypothesis of simultaneous acti vation of mitoK + ATP channels and K + /H + exchanger, which was confirmed in further experiments with quinine, an inhibitor of K + /H + exchanger.Namely, quinine, when introduced in 0.5 mM concentration, restored K + to values higher than that of the control (Fig. 1, B, 7).We can thus assume that activation of mitoK + ATP channels is coupled with activation of K + / H + exchanger.Therefore, we can assume from these results, that in our conditions we observe potassium cycle activation in the presence of calcium in mito chondria of myometrium, particularly K + influx into matrix through the K + ATP channels and release the K + /H + -exchanger , which is eliminated by the specific inhibitors -gliben clamide, ATP and quinine.Garlid et al. argue that the regulation of mitochondrial ma trix volume is the main function of potassium cycle under physiological conditions, which may also pro tect mitochondria under stress [2,14].It is known that increased mitochondrial volume is a condition for activation of the K + /H + exchanger as a participant in potassium cycle.The activation of the potassium cycle helps to establish new matrix volume equilib rium, yet it is the K + /H + exchanger that maintains integrity of mitochondrial membranes [2,8].Basing on our data we assume that potassium cycle activa tion, and the K + /H + exchanger in particular, may be involved in maintenance of integrity of myometri um's mitochondrial membranes under Ca 2+ overload and induction of MPTP.

Fig. 1. K ions accumulation in isolated mitochondria from myometrium under various conditions. A, fluorescence of PBFI probe loaded in isolated mitochondria depending on KCl concentration in incubation medium; 1 -125 mM KCl, 2 -50 mM KCl, 3 -10 mM KCl, 4 -50 mM choline chloride (in cases 2, 3 and 4 the incubation medium contained also sucrose, the final osmolarity was 250 mOsm/l). B, effect of Ca 2+ , inhibitors of K + transport and Ca 2+ uniporter on total accumulation of potassium ions in isolated mitochondria; 1 -K + accumulation in standard incubation medium, 2 -K + accumulation in the presence of 100 µM CaCl
It has been demonstrated that opening of Ca 2+ induced MPTP may result in uncontrolled increase in matrix volume followed by outer membrane rup ture and induction of apoptosis or necrosis [11].The change in mitochondrial volume resulting from MPTP opening may be observed by registering of side light scattering at 520 nm [17].Introduction of mitochondrial suspension in the standard incuba tion medium with 100 µM CaCl 2 (see Material and Methods section) caused a noticeable drop in lateral light scattering in comparison to control (Fig. 2, a).According to our results, ruthenium red (10 µM), CsA (10 µM) and EGTA inhibited Ca 2+ induced swelling of mitochondria (Fig. 2, a).The Ca 2+ sen sitivity of changes in lateral light scattering of mito chondria suspension leads to the following conclu sions: a. Ca 2+ induced swelling of mitochondria is mediated by transport of Ca 2+ into matrix through mitochondrial uniporter as it is inhibited by ruthe nium red and EGTA, a Ca 2+ chelator; b. this process is media ted by MPTP induction as it is inhibited by CsA, which is an MPTP inhibitor.
One of the functions of mitochondrial potas sium cycle and the mitoK + ATP channels has been presumed to be matrix volume regulation [2].We hence investigated the possible involvement of the mitoK + ATP channels in Ca 2+ induced swelling of mi tochondria from myometrium.We found that gliben clamide, an inhibitor of mitoK + ATP channels, in con centrations of 10 µM partially inhibited the changes in lateral light scattering of mitochondrial suspen sion induced by Ca 2+ (Fig. 2, B).It must be noted that the inhibitor itself did not affect the process in any way in the absence of Ca ions (data not shown).Our data are in accordance with the results of Jaburek et al. [18], who propose the existence of various func tional states for mitoK + ATP channels with differing sensitivity to inhibitors.Also, glibenclamide did not affect the change of mitochondrial volume in K + free medium (replaced equimolarly with Na + ) (Fig. 2, B).These results may indicate that changes in light scat tering of mitochondria suspension in the presence of 100 µM CaCl 2 are caused by two processes: the induction of CsAsensitive MPTP and the swelling of mitochondria due to K + influx through potassium channels, i.e. glibenclamidesensitive mitoK + ATP channels.Costa et al. suppose that one of the main physiological effects of mitoK + ATP channels activa tion on mitochondrial function is the increase in sta ble matrix volume, and the protective effect against damage to the cell that is ascribed to these channels results directly from regulation of mitochondrial volu me [8].
ROS are byproducts of cellular metabolism and are produced as a result of functioning of mito chondrial electrontransport chain, among other processes .They participate in intracellular signal pathways, regulating numerous processes under physiological conditions, including gene expres sion and muscle contraction.Nevertheless, under pathological conditions associated with imbalance in intracellular Ca 2+ homeostasis the increased ROS gene ration leads to MPTP opening followed by ne crosis or apoptosis [11].Taking into account the cy toprotective effect of mitoK + ATP channels activation under conditions arising from increased concentra tion of Ca 2+ [3,17], we also aimed to investigate the effect of K + transport in mitochondria of myometri um on the rate of ROS generation in the presen ce of Ca 2+ .Under standard conditions (Ca 2+ free) ROS generation does not result in MPTP opening, as in troduction of CsA, an inhibitor of MPTP, does not affect the process in any way (data not shown).It is worth mentioning that in the incubation medium containing K + the rate of ROS generation was higher than in the potassiumfree medium (replaced by equimolar Na + ) (Fig. 3), which is in accordance with data by others [19] that activation of K + influx in mitochondria potentiates ROS generation in heart mitochondria.While addition of 100 µM CaCl 2 did not cause further increase in ROS generation in the medium containing K + , it did result in higher ROS generation in the potas siumfree medium, and these changes were eliminated by CsA (data not shown).Glibenclamide (10 µM) potentiates rate of ROS gene ration in the standard medium with KCl and did not affect the process in the potassiumfree medium (Fig. 3), which proves yet again the specificity of its експериментальні роботи ATP channels of cardiomyocytes decreases ROS generation in the mitochondria in re sponse to increased local oxidant levels [14].
Therefore, the obtained results lead to con clusion that Ca 2+ induces opening of CsAsensitive MPTP in mitochondria from the myometrium, followed by matrix swelling and increased rate of ROS gene ration.We suppose that both components of potassium cycle in the myometrium mitochon dria -accumulation in matrix, including influx through mitoK + ATP channels, and release through K + / H + exchanger -are activated under the induction of MPTP.Activation of K + influx into the matrix in tensifies ROS generation under normal conditions and inhibits it under induction of MPTP.While the induction of MPTP is associated with swelling of mitochondria and accumulation of cytotoxic ROS, the activation of potassium cycle, on the other hand, regu lates the rate of ROS generation and matrix volu me, thus preventing membrane rupture and overproduction of damaging ROS.к л ю ч о в і с л о в а: іони са, циклоспорин чутлива мітохондріальна пора, глібенкламід, K + атр -канали, K + /н + -обмін, мітохондрії.