аrene С-956 is effeCtive inhibitor of Н +-С a 2 +-exChanger in smooth musCle mitoChondria

it was shown that calix[4]arene c-956 exhibited a pronounced concentration-dependent (10-100 μm) inhibitory effect on the h+-ca2+-exchanger of the inner mitochondrial membrane of rat uterine myocytes (Ki 35.1 ± 7.9 μm). the inhibitory effect of calix[4]arene c-956 was accompanied by a decrease in the initial rate (V0 ) and an increase in the magnitude of the characteristic time (τ1/2) of the ΔрН-induced Са 2+ release. At the same time, it did not affect the potential-dependent accumulation of ca2+ in mitochondria. thus, the action of calix[4]arene c-956 might be directed on increasing the concentration of ca ions in the mitochondrial matrix. the calculation of basic kinetic parameters of the ca2+ transport from isolated organelles (in the case of its non-zero stationary level), based on changes in fluorescence of ca2+-sensitive dye Fluo-4 Am in mitochondria was performed. the proposed approach can be used for the kinetic analysis of the exponential decrease of the fluorescence response of any probes under the same experimental conditions.


e x p e r i m e n t a l w o r k s e x p e r i m e n t a l w o r k s
it was shown that calix [4]arene c-956 exhibited a pronounced concentration-dependent (10-100 μm) inhibitory effect on the h + -ca 2+ -exchanger of the inner mitochondrial membrane of rat uterine myocytes (K i 35.1 ± 7.9 μm). the inhibitory effect of calix [4]arene c-956 was accompanied by a decrease in the initial rate (V 0 ) and an increase in the magnitude of the characteristic time (τ 1/2 ) of the ΔрН-induced Са 2+ release.At the same time, it did not affect the potential-dependent accumulation of ca 2+ in mitochondria.thus, the action of calix [4]arene c-956 might be directed on increasing the concentration of ca ions in the mitochondrial matrix.the calculation of basic kinetic parameters of the ca 2+ transport from isolated organelles (in the case of its non-zero stationary level), based on changes in fluorescence of ca 2+ -sensitive dye Fluo-4 Am in mitochondria was performed.the proposed approach can be used for the kinetic analysis of the exponential decrease of the fluorescence response of any probes under the same experimental conditions.K e y w o r d s: calix [4]arenes, h + -ca 2+ -exchanger, mitochondria, smooth muscle, uterus.
M itochondria play a central role in many fundamental biological processes, such as energy supply of cellular functions, oxidative metabolism, steroidogenesis, programmed cell death, etc. [1][2][3][4].Changes in the Ca 2+ concentration in mitochondria are major factors in the regulation of biochemical processes in the matrix and the functioning of the electron transport chain.An increase in Ca 2+ concentration within mitochondrial matrix activates the ATP synthesis and enzymes of the tricarboxylic acid cycle while overloading the organelles with a cation induces cell death.It has been established that mitochondria play an essential role in the intracellular Ca 2+ signaling in smooth muscle cells owing to their ability to accumulate and release significant amounts of Ca ions [1,[5][6][7].The vital task of modern membranology is the search for exogenous non-toxic compounds that could efficiently and selectively regulate Ca 2+ transport in the inner mitochondrial membrane and, thus, modulate Ca 2+ homeostasis.
In recent years, much attention has been paid to the supramolecular macrocyclic polyphenol compounds calix [4]arenes, particularly their ability to effectively alter the metabolic, energy and transport processes in subcellular organelles [8].Modern research is also focused on studying of calix [4]arenes derivatives that specifically affect the cation-transport systems in subcellular structures of smooth muscle.In particular, it has been established that the calix [4]arenes C-97, C-99 and C-107 alter the polarization of the inner membrane, the redox state of purine and flavin nucleotides of the electron transport chain, as well as mitochondrial Ca 2+ -homeostasis [9,10].
The mitochondria of uterine smooth muscle cells are high capacious Ca 2+ -storage units, and able, along with the Ca 2+ -pumps of the plasma membrane doi: https://doi.org/10.15407/ubj90.01.025 and sarcoplasmic reticulum, to terminate the Ca 2+ signal and maintain a low physiological cation concentration in the cytosol [11].The regulation of the mitochondrial Ca 2+ concentration is based on the coordinated functioning of systems that support potential-dependent calcium accumulation and cation release from the matrix into the cytosol.Two systems that regulate the Ca 2+ concentration in the matrix have been identified in the myometrial mitochondria: a Ca 2+ -uniporter and an H + -Ca 2+ -exchanger [11].The main mechanism for the Ca 2+ uptake into the mitochondrial matrix is the functioning of the Ca 2+uniporter, whose activity is optimal at micromolar Ca 2+ concentrations out of mitochondria and which support a potential-dependent (electrophoretic), highly capacious, ruthenium red-sensitive cation accumulation pathway [12].In smooth muscles, unlike other electroexcitable tissues, Na + -dependent Ca 2+ transport from mitochondria is less significant compared to the H + -Ca 2+ -exchanger [11], since the myocytes excitation and, hence, the development of the action potential occurs due to not only sodium but also calcium inward currents [13,14].It was found that in the sarcolemma of the uterine myocytes Ca 2+independent Mg 2+ -ATPase, whose function may be a production of H + in the myoplasm, exhibited a high activity [15].These data indicate the primary role of the H + -Ca 2+ -exchanger in the mitochondria of the uterus smooth muscle cells.A similar transport system operates in liver and kidney cells [16].The H + -Ca 2+ -exchanger ensure a ΔpH-dependent decrease in Ca 2+ concentration in the mitochondria.The molecular basis of this process is the protein Mdm38/Letm1 (leucine-zipper-EF hand-containing transmembrane region) of the mitochondrial inner membrane [17].Overloading with Ca 2+ and de-energization of mitochondria are accompanied by the opening of a nonspecific permeability transition pore through which cation may also be transported [12,17,18].
Due to the importance of biochemical processes within mitochondria for the functioning of the whole cell, as well as the direct relationship between mitochondrial dysfunction and the development of smooth muscle pathologies, the search for non-toxic and selective modifiers of the mitochondrial Catransport systems is a focus of research interest.The ability of some supramolecular macrocyclic calix [4]arenes, in particular, C-90 and C-956, to inhibit the activity of the plasma membrane and sarcoplasmic reticulum Ca 2+ -pumps in smooth muscle draws interest to these compounds as possible regulators of Ca 2+ homeostasis in myocytes.

Synthesis of calix
Isolation of myometrium mitochondria.The rats were anesthetized by inhalation of diethyl ether and then decapitated.All animal experiments were performed in accordance with the rules and regulations of The European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986).
Mitochondria were isolated from rat myometrium using differential centrifugation [19].According to electron microscopy, the isolated mitochondria had round shape of 500 nm in size with cristae, i.e., they had morphological features of intact organelles.Their hydrodynamic diameter measured by photon correlation spectroscopy was 549 ± 20 nm.This value increased significantly to 804 ± 35 nm at mitochondrial inner membrane rupture by antibiotic alamethicin indicating that mitochondria remained intact during isolation.Isolated mitochondria were functionally active and supported energy-dependent Ca 2+ accumulation in the presence of succinate and Mg-ATP 2- [19].
The protein concentration in the mitochondrial fraction was determined by the Bradford assay [20].Its average value was 2 mg/ml, and in the sample -25 μg/ml.
Procedure for loading fluorescent probe Fluo-4 Am into mitochondria.The loading fluorescent probe Fluo-4 AM at a final concentration of 2 μM into mitochondria was carried out in a medium containing 10 mM Hepes (pH 7.4; 37 °C), 250 mM sucrose, 0.1% bovine serum albumin for 30 min at 37 °C.To facilitate the loading, the dye was mixed with Pluronic F-127 (0.02%) [19].
Studying ionized ca 2+ content in mitochondria by spectrofluorimetry.The assessment of the relative values of the Ca 2+ level in the myometrium mitochondrial matrix loaded with Fluo-4 AM (λ ex = 490 nm, λ fl = 520 nm) was performed using Quanta Master 40 PRTI (Canada) and FelixGX 4.1.0.3096 software.
The Fluo-4 fluorescent signal was presented in relative units as (F-F 0 )/F 0 , where F 0 is the fluorescence intensity at the start of the experiment.F is the fluorescence intensity recorded as the experiment runs.
The apparent inhibition constant K i and Hill coefficient were calculated by the Hill method in [4]arene]}.K -control in the absence of calix [4]arene, F 0 -initial fluorescence, F -fluorescence at appropriate intervals.
Statistical data analysis was performed with common statistical methods [21] and Student's t-test using the IBM standard software.
The following reagents were used in the experiments: Hepes, DMSO, cyclosporin A, sodium succinate, sucrose, ATP, bovine serum albumin (Sigma, USA); Fluo-4 AM, Pluronic F-127 (Invitrogen, USA); mineral salts of domestic production.The solutions were prepared with double distilled water (conductivity under 1.5 μS/cm.The water conductivity was measured by a conductivity meter type OK-102/1 (Hungary).

results and discussion
Calix [4]arenes C-90 and C-956 are structural analogs (Fig. 1) that differ in existence of two elongated aliphatic chains in the C-956 structure that facilitates interaction of C-956 with hydrophobic parts of subcellular membranes and penetration into a lipid bilayer.
It was found that calix [4]arene C-956 at a concentration of 100 μM almost completely blocked the ΔpH-induced Ca 2+ release from isolated rat myometrium mitochondria and did not affect the potentialdependent accumulation of this cation.Its structural precursor, C-90, at the same concentration exhibited slight inhibitory effect (P < 0.1) on both Ca 2+ exchange systems in the inner mitochondrial membrane (Fig. 2).
(20 μM) led to an increase in Ca 2+ concentration in myoplasm (according to the changes in the fluorescence intensity of the Ca 2+ -sensitive Fluo-4 AM dye and confocal microscopy), however, the Ca 2+ concentration decreased over time to basal level indicating a trigger for cellular mechanisms for reducing the level of ionized Ca 2+ in myoplasm.This phenomenon might be account for the function of mitochondria as a potent Ca 2+ store and/or Na + -Ca 2+ -exchanger of the plasma membrane [23].These authors showed that calix [4]arene C-956 inhibited the plasma membrane Ca 2+ , Mg 2+ -ATPase even more effectively, and caused a transient increase in Ca 2+ concentration in myoplasm.In view of the presented in this study, the inhibition of H + -Ca 2+ -exchanger by C-956 compound may lead to a shift in the equilibrium in the inner mitochondrial membrane Ca 2+ transport toward the calcium accumulation and a corresponding Thus, the results obtained indicate that calix [4]arene C-956 affects the H + -Ca 2+ -exchanger of myometrium mitochondria, but not the system of potential-dependent Ca 2+ accumulation in these subcellular structures.
The unique property of the calix [4]arenes is their specific structure-dependent effect on the cation-transport systems of subcellular organelles.In particular, we showed that the high-affinity inhibitors of Na + , K + -ATPase namely, C-97, C-99 and C-107 depolarized the plasma membrane of rat uterine myocytes and transiently hyperpolarized the internal mitochondrial membrane [9].The latter effect testifies to the penetration of calix [4]arenes into the cell and their direct interaction with mitochondria.The indicated calix [4]arenes were shown to effec-tively block the potential-dependent Ca 2+ accumulation and reduce the cation concentration in the myometrium mitochondria, probably, stimulating the H + -Ca 2+ -exchanger [10].In this respect, the effects of mentioned calix [4]arenes, as well as C-956, on the Ca 2+ transport in mitochondria differ significantly, that may be due to different chemical nature, size, hydrophobicity, charge and relative position of the substituents on the calix [4]arene cup.
We studied the concentration and time dependences of the calix [4]arene C-956 inhibitory effect on the ΔpH-induced Ca 2+ release from isolated mitochondria in concentration range of 10-100 μM (Fig. 3, in 3 a -data are given only for three concentrations) The calculated apparent inhibition constant K i for calix [4]arene C-956 effect on the mitochondrial H + -Ca 2+ -exchanger was 35.1 ± 7.9 μM, and the Hill coefficient (n H ) was close to 1 (Fig. 3, B).
The time dependence of the cation release for both the control and in the presence of calix [4]arene is an exponential curve, and the level of the cation in the matrix, according to the changes in Fluo-4 fluorescence, reaches a constant level and, hence, does not reach zero values (Fig. 4).In the case, when the cation release from the matrix becomes constant over time and does not tend to zero, the calculation of the kinetic parameters of the transport process is not trivial.Therefore, we proposed a methodology for calculating the initial rate (V 0 ) and the characteristic time (τ 1/2 ) of ΔpH-induced Ca 2+ release from isolated mitochondria.It is based on the assumption that a decrease in the fluorescence of the Ca 2+ -sen-Fig.3. concentration and time dependences of calix [4]arene С-956 effect on ΔрН-induced Са 2+ release from rat myometrium isolated mitochondria.A -the time dependences of Са 2+ release at different concentrations of calix [4] Fluorescence Fluo-4, relative units sitive Fluo-4 probe loaded into mitochondria adequately reflects a decrease in the Ca 2+ concentration in the matrix.

arene С-956, the data of a typical experiment (n = 5). B -concentration dependence of the inhibitory effect of calix[4]arene С-956, (m ± m, n = 5). C -control in the absence of calix[4]arene
Assuming that F 0 is the initial fluorescence, F is the fluorescence at the appropriate time interval, and F′ is the constant level of fluorescence reached over time as a result of the release of a certain amount of Ca 2+ from the matrix, we obtain the equation: where t -time, k -rate constant.Particular cases: According to the equation ( 5), we obtain a typical linear dependence in the coordinates {ln ((F 0 -F′)/(F-F′)); t} (Fig. 5).
From the resulting equation, we calculated the characteristic time (τ 1/2 ) and the initial rate V 0 of the ΔpH-induced Ca 2+ release (equations ( 6) and ( 7)): We performed calculations with the use of equations ( 6) and (7), and obtained the corresponding dependences (Fig. 6).
Our previous results [19] demonstrated that the H + -Ca 2+ -exchanger of the inner mitochondrial membrane of myometrium cells is an electrogenic system involved in calcium ions transport in the stoichiometric ratio 1H + : 1Ca 2+ .Plasma membrane Ca 2+ , Mg 2+ -ATPase -another subcellular transport system of uterine smooth muscles -also functions in the electrogenic mode and can transport one Ca ion from the cell in exchange for H + [24].In each case, H + can be the counter-ion upon Ca 2+ transport across the membrane.Taking into account that both systems are highly sensitive to the inhibitory effect of C-956 and have similar inhibitory constants (tens of µM) [23], it can be suggested that the calix [4]arene C-956 targets specifically at the H + -transport component of these systems.More detailed studies of the transporter structure along with further research of Thus, the results obtained indicate the following : calix [4]arene C-956 (derivative of calix [4]arene C-90) in the range of micromolar concentrations exhibited a pronounced inhibitory effect on the H + -Ca 2+ -exchanger, which was a decrease in the initial rate V 0 and, as a result, an increase in the characteristic time τ 1/2 of ΔpH-induced Ca 2+ release from mitochondria.Though, C-956 did not affect the potential-dependent accumulation of the calcium ions by these subcellular organelles.The methodology for calculating the basic kinetic parameters of the Ca 2+ transport from isolated mitochondria, in the case when the transport process reaches a nonzero constant level, was proposed.This approach can be used for the kinetic analysis of the exponential decrease in the fluorescent response of any probe.This work was financed by the target complex multidisciplinary research program of the NAS of Ukraine "Molecular and cellular biotechnologies for the needs of medicine, industry and agriculture" (State Registration No. 0115U003639).

Fig. 5 .
Fig. 5. the linear dependence of changes in the logarithmic value of the relative fluorescence signal Fluo-4 in mitochondria on time was subsequently used to calculate the constant of the rate of ΔрНinduced Са 2+ release from mitochondria.the data of a typical experiment