MODULATION OF SPONTANEOUS MOTILITY AND GABA-DEPENDENT REGULATION OF FUNCTIONS OF STOMACH SMOOTH MUSCLES IN VIVO

Electron scanning microscopy was used to obtain the image and identify the size of TiO 2 nanoparticles. Using the tenzometric method in the isometric mode, it was estab­ lished that chronic effect of TiO 2 on stomach smooth muscles led to redistribution of the amplitudes of spontaneous contractions in terms of their frequencies. An increase in frequency of their contractions, decrease in the duration of contraction–relaxation cycle, the disturbance of the asymmetry of the duration of contraction–relaxation development, a reduction in Montevideo index of contractions and Alexandria index of contractions were demonstrated. It was also shown the existance of the divergence in numerical va­ lues of frequency­amplitude complexes, TiO 2 -modified spontaneous contractive activi ty of smooth muscles of stomach and caecum . In the conditions of long-term chronic influ­ ence (100 days), TiO 2 removes the regulatory mechanisms of depressing a release of inhibition neuromediators from neurons of the intramural nervous interlacement, medi­ ated by GABA А ­ and GABA С ­receptors, in smooth musc les of stomach and caecum dioxide nanoparticles, mechanoki­ netics, GABA­dependent regulation, spontaneous and induced con­


INTRODUCTION
Current development of high technologies, became the foundation for extensive production of nanosized materials, in particular, titanium dioxide (ТіО 2 ), whose applica tion takes a large sector of different kinds of industry, including pharmacological indus try, the sphere of food production and medicine. This means that using ТіО 2 in technolo gies, pertaining to microelectronics, biosensorics, production of plastic masses, creation of dyes, biocompatible implant materials, medicinal preparations, food dyes (Е171), food products, others. Nanosized ТіО 2 material is present in the form of three crystal structures: rutile, anatase, and brookite, but the first two forms are predominantly used in the industrial production (usually in the form of their mixture with different stoichio metry of components) [18,24]. ТіО 2 used in the form of anatase is much more chemically active, compared to rutile; when exposed to ultraviolet, it generates reactive oxygen intermediaries [10,15]. Similar effects occur inside the tissue cells due to the interaction between TiO 2 nanoparticles and macromolecules [7,17]. The intake of TiO 2 to human organism takes place with food, water, and air, and the main ways of its transportation are gastrointestinal tract (average daily amount of consumed ТіО 2 (0.002-5) mg and respiratory tracts (daily amount of consumed ТіО 2 -0.0007 mg) [14,16,22], which in the global practice may already be referred to the nanohazard problems in the sphere of Environmental and Health Safety (protection of environment and human health from harmful impact of some objects, including nanoscaled). Our studies, conducted in vitro using stomach smooth muscles (SM) [19], demonstrated that nanosized ТіО 2 material changed the energetic and calcium homeostasis of smooth muscle mitochondria, the conductivity of their plasmatic membrane for Са 2+ and cholinergic excitation. At the same time, the mechanisms of releasing these cations from rianodine and IP 3 sensitive stores of sarcoplasmic reticulum were not sensitive to the impact of TiO 2 . It was demon strated [21] that in vitro ТіО 2 in smooth muscles of large intestines modulated histamine and nicotineinduced mechanisms of regulating its functional activity. It was stated that the intake route of nanosized TiO 2 material to human organism via the gastrointestinal tract is the most probable, and food impact in the gastric cavity is the longest compared to other gastrointestinal tract parts. The aim of this work was to study changes in vivo in spontaneous motility and GABAdependent regulation of functions of stomach smooth muscles, caused by chronic effect of nanosized TiO 2 material.

MATERIALS AND METHODS
8weekold Wistar rats of both genders were used for in vivo experiments. The rats were kept in standard conditions of the vivarium (room temperature of 20±2 °C, relative humidity -50-70 %, lightdarkness cycle -12:12 h). All manipulations with animals were carried out in accordance with the International Convention of animals and the Law of Ukraine "On protection of animals from cruelty". Protocol N 2 (October 20, 2016) of the meeting of Bioethics Committee of Educational and Scientific Centre "Institute of Biology and Medi cine" Taras Shevchenko KNU. Killing of animals carried out by the in jection of lethal dose of anesthetic protocol (Sigma).
TiO 2 nanoparticles were dispersed in the distilled water using ultrasound for 15 min; to obtain the homogenic suspension the latter was additionally stirred with the mecha nical stirrer prior to each use. The intragastric dose was selected according to the data about the intake of TiO 2 with food products in Great Britain: 5 mg per person daily, that is equivalent to 0.1 mg/kg of weight per day [22]. Due to this, every day the rats were intragastrically administrated the suspension of TiO 2 with the consideration of 0.1 mg/kg in the first experimental group for 30 days and in the second experimental group for 100 days. The bodyweight of rats was estimated every 4-6 days.
The experiments were conducted using the isolated preparations of circular smooth muscles of rats' antrum, loaded with TiO 2 . The registration of spontaneous contractibi lity of smooth muscle stripes (SMS) of caecum and myometrium was performed by the tenzometric method in the isometric mode with the following calculations: frequencies of preparation contractions for 10 min; averaged value of contractionrelaxation cycle; duration of some contraction fragments: contraction phase, relaxation phase; asymme try coefficient; Montevideo index of contractions (MU); Alexandria index (AU) of contrac tions. The method [4,11] was also used to conduct the kinetic analysis of spontaneous contractionsrelaxations of muscle preparations and the ones, induced by high potas sium Krebs solution, with the estimation of normalized maximal velocities of contrac tions (V nc ) -relaxations (V nr ).
Statistical analysis of the experiment results was performed using OriginPro 8 program. The unpaired version of Student's ttest was used to determine the reliable differences between the mean values of two samples. Multiple comparisons were per formed using the parametric one-way ANOVA. The results were considered significant on condition of the probability value of р under 5 % (р < 0.05). The results were pre sented as the arithmetic mean ± standard error of the mean value, n -number of ex periments.
The nanoparticles of TiO 2 (PlasmaChem GmbH, D12489 Berlin, Germany) were used in the form of nanopowder (mixture of rutile and anatase) with the average size of particles (21±5) nm (Fig. 1). The mea surements were conducted using the scanning electron microscope Tes can Mira 3 LMU, kindly provided for our work by NanoMedTech LLC) ( Fig. 1), specific area (50±10) sq.m./g; purity > 99.5 %, content of Al 2 O 3 < 0.3 wt; SiO 2 < 0.2 wt. The nanopow der of TiO 2 was previously resus pended in dimethylsulfoxide (DMSO) assuming the presence of 0.25 % DMSO in the final volume. Likewise, all the control solutions contained 0.25 % DMSO. The destruction of the aggregates of ТіО 2 nanoparticles in the suspension was performed us ing the ultrasound processing for 2 min at the frequency of 37 kHz. The zetapotential of TiO 2 nanoparti cles, estimated using the Zetasizer nano device (kindly provided by NanoMedTech LLC), was (7.93) mV.

RESULTS AND DISCUSSION
Our previous іn vivo studies [20] demonstrated that TiO 2 used in nanosized form is an efficient modulator of the activity related to the functioning of pace-maker cells [6,9,23], spontaneous contractive activity of stomach smooth muscles of rats. The elemental ana lysis of the content of micro and macroelements in rat tissues [20] demonstrated that ТіО 2 is bioavailable for cells of different organs, with the highest nonuniform accu mulation in smooth muscles of gastrointestinal tract: in саесum and stomach. In this work, the tenzometric method in the isometric mode was used to study the spontaneous contractive activity of isolated smooth muscle stripes of circular smooth muscles of an trum of three groups of rats: control group (n = 6) and two groups of animals (6 per group), loaded with the suspension of nanosized TiO 2 material in the abovementioned concentration for 30 and 100 days. Similarly to the previous experiments, the duration of regist ration of spontaneous contractions in both groups of rats was 60 min. During this time period, the level of basal tone of muscle preparations remained stable. Fig. 2, А pre sents the results of tenzometric measurements in control and experimental groups of rats loaded with TiO 2 . There was the distribution of amplitudes of spontaneous contrac tions of smooth muscles by frequencies with three significant maxima in the control. The contractions with the amplitude of (14.8±1.0) mN, n = 12.З were registered with the highest frequency (13±0.8) %. The frequency of muscle preparation contractions, calcu lated in the control (Fig. 3), for 10 min was (12±0.6) %; the averaged value for the dura tion of the contractionrelaxation cycle (contraction act) was (48±3.4) s, the duration of some contraction fragments was as follows: contraction phase -(16.2±0.9) s, relaxation phase -(31.8±1.7) s; assymetry coefficient -(0.53±0.03); MU index of contractions -(143.8±7.2); AU index of contractions -(6900±483).
The following series of experiments involved the study of the changes in spontane ous contractive activity of stomach smooth muscles of rats, loaded with TiO 2 for 30 days (Fig. 2, B). In these conditions, compared to the control, in the dependency chart, "the amplitude of contractions -the frequency of the incidence of contractions with this am plitude", there is a shift towards their smaller amplitudes with three significant peaks. The highest incidence (10.5±0.4) %, n = 12 was noted for contractions with the following amplitudes: (6.5±0.4) mN (two maxima) and (4.6±0.24) mN. The calculations of parame ters of these spontaneous contractions (Fig. 3) demonstrated that in these conditions their frequency for 10 min, compared to the control, increases and amounts to (16±0.8) s; the duration of the contractionrelaxation cycle decreases almost 1.5 times and is (26.4±1.3) s. There was also a decrease in the duration of some contraction fragments: contraction phase -(11.2±0.7) s, relaxation phase -(15.2±0.9) s; asymmetry coeffi cient -(0.82±0.05); MU index of contractions -(93.6±6.5); AU index of contractions -(2470±148).
The study of spontaneous contractions of smooth muscle stripes of rats, loaded with TiO 2 suspension for 100 days, demonstrated (Fig. 2, C) that, compared to the con trol, there is further transformation of their amplitudefrequency characteristics. The am plitudes of such contractions cover the range from 3 to 6.5 mN, n = 12: the highest inci dence (13.2±0.7) % was registered for contraction with the amplitude of (4.4±0.31) mN, as stated above, at the same frequency in the control its value was (14. of which was (16.8±0.9) s continued decreasing, whereas the duration of the contrac tion phase had no statistically significant differences from the control. There is also noteworthy impairment of such an important index of spontaneous contractive activity, as asymmetry coefficient, at the effect of TiO 2 suspension in the rats of group two (dura tion of load with ТіО 2 -30 days) and three (duration of load with ТіО 2 -100 days): while in the control its value was (0.53±0.03), in group two -(0.82±0.05), in group three its value approximated 1 (1.07±0.06), which is likely related to the fact that this nanosized material modulates mutually coordinated mechanisms of increasing and decreasing the intracellular concentration of calcium ions in the interstitial cells of stomach smooth muscles, the pacemaker activity of which is known [23] to be determined by cyclic pro cesses of releasing these cations from sarcoplasmic reticulum and their being absorbed  Рис. 3. Гістограми параметрів кінетичного аналізу спонтанної скорочувальної активності кільцевих гладеньких м'язів шлункa (C -контроль; 30 та 100 -термін (діб) навантаження щурів суспензією нанорозмірного матеріалу діоксиду титану): 1 -частота скорочення препаратів за 10 хв; 2 -усе реднене значення тривалості циклу скорочення-розслаблення (с); 3 -показник скоротливого індексу в одиницях Монтевідео; 4 -показник скоротливого індексу в Олександрійських одини цях. * р<0,05 -зміни вірогідні щодо контролю Taking the abovementioned results into consideration, it was interesting to analyze the divergence in numerical values between some parameters of frequencyamplitude complexes of spontaneous contractive activity of circular smooth muscles of stomach and smooth muscles of caecum, modified by TiO 2 in vivo (the results of experimental studies in vivo using smooth muscles of caecum are presented in our work [20]). The calculations of the parameter value ratio (PVR) for the frequencyamplitude complexes of spontaneous activity of smooth muscles of stomach and caecum demonstrated consi derable differences in the mechanisms of their formation. In control, these values for such parameters as contraction frequency, duration of contractionrelaxation cycle, AU index of contractions differed considerably for different muscles. The comparative analy sis demonstrated that in the control the frequency of contractions of caecum preparations С 30 100 С 30 Test modes 100 С 30 100 С 30 100 1 exceeded the same parameter for stomach smooth muscles considerably, and their ratio was far from 1, amounting to (0.27±0.01), n = 12. The value of this ratio was the same in conditions of loading the rats with ТіО 2 for 30 and 100 days. As for PVR for such parameters as duration of contractionrelaxation cycle and AU index of contractions for the mentioned muscles, their difference in the values was also noted, especially for smooth muscles of rats, loaded with TiO 2 for 30 days. For instance, in control, PVR by the index of the duration of contractionrelaxation cycle was (3.8±0.2), n = 12, whereas at the effect of ТіО 2 for 30 days it was twice higher. There was also a double increase in the PVR value for AU index of contractions compared to the control. Taking the above mentioned into consideration, it is possible to assume some differences in the mecha nisms of the formation of spontaneous contractive activity of smooth muscles of sto mach and intestines in the control and at the effect of TiO 2 . GABA is a neuromediator of both central and peripheral nervous systems [1,2,13]. Our studies [12] using the method of molecular docking of nanosized TiO 2 material to the extracellular part of GABAreceptor we have determined potentially probable sites of binding nanosized TiO 2 material of different affinity and analyzed the character of bonds, stabilizing them according to their amino acid composition. It is known [2,8] that GABAreceptors (GABA А and GABA С ) of neurons of intramural nervous system are involved in NOdependent regulation of functions of smooth muscles of gastrointestinal tract. It was interesting to study the participation of this neuromediator of nonadrenergic noncholinergic neurons of stomach smooth muscles in the formation of the tonic com ponent of high potassium contraction and its modulation with TiO 2 . It was demonstrated (Fig. 4, А) that in response to the application of high potassium (80 mmol/l) solution, smooth muscle stripes developed contractionsrelaxations with the phase component of (23±1.5) mN, n = 12; here, the ratio of the phase component of contraction to the tonic component was (1.3±0.08). The kinetic analysis of the curves for high potassium con traction and the calculations of normalized maximal velocities for separate contraction and relaxation phases demonstrated that such parameter as V nc acquires the value of (7.7±0.4) min 1 , where as V nr -(1.8±0.31) min 1 . Then, the plateau of the tonic compo nent of high potassium contraction was consecutively applied high potassium solution in the abovementioned concentration with the content of GABA in the concentrations of (10 8 -10 5 ) mol/l. It was determined (Fig. 4, B) that the application of GABA in these conditions was accompanied with fluctuations in the basic level of the tonic component of high potassium contraction according to acid concentration. The substitution of the abovementioned composition with normal Krebs solution was accompanied with the res toration of the initial level of the basal tone of muscle preparations. The study of changes in K + induced (80 mmol/l) contractionrelaxation of smooth muscle stripes of rats, loaded with the suspension of nanosized ТіО 2 material for 30 days in the abovementioned concentration, demonstrated (Fig. 4, C) that its amplitude remained in the range of the control, whereas the velocity of relaxation increases (V nr -(2.98±0.03) min 1 , n = 12), compared to the control. At the same time, the value of V nc (6.16±0.61) min 1 remained unchanged. In these conditions, there was a decrease in the tonic component of high potassium contraction, accompanied with the decrease in the ratio of its components, which was (1.05±0.05), compared to the control. The application of GABA (10 8 -10 5 ) mol/l on the tonic component of high potassium contraction was accompanied with insignificant increase in the level of its plateau (Fig. 4, D). It is known [3,5] that acetylcholine, an agonist of muscarinic cholinoreceptors, par ticipates in the parasympathetic control of contractive activity of smooth muscles along with inhibition neuromediators. Taking the abovementioned into consideration, the con traction of preparations of circular smooth muscles of antrum, activated by acetylcholine in the concentration of 10 -5 mol/l, was registered in the work. It was determined (Fig. 5, А) that the averaged value of the phase component of such contraction was (26.3±1.8) mN, n=12, and its ratio to the tonic component -(1.25±0.07). Here the estimated maxi mal velocity of the contraction phase was (7.25±1.17) min 1 , whereas the normalized maximal velocity of the relaxation phase -(1.47±0.33) min 1 . Then, the plateau of tonic component of acetylcholine contraction was consecutively applied Krebs solution with the GABA in the concentrations of (10 8 -10 5 ) mol/l (Fig. 5, B). It was determined that in these conditions there was an increase in the basic level of the tonic component, the plateau of which witnessed the increase in spontaneous contractions of smooth mus cles. We also studied of acetylcholineinduced contractions of circular smooth muscles of antrum (in the abovementioned concentration) of the group of rats (n = 6), which were loaded with the suspension of nanosized TiO 2 material for 100 days (Fig. 5, C). It was determined that, compared to the control group of animals, the value of the phase com ponent of acetylcholine contraction of smooth muscles had no changes. The ratio of the phase component of acetylcholine contraction to its tonic component and the values of maximal velocities of contraction and relaxation for smooth muscle stripes also remained in the range of the control. The application of GABA in the abovementioned concentra tions on the tonic component of acetylcholine of contractions caused no significant chang es. For comparison, similar studies were conducted in vivo using smooth muscles of caecum. It was determined (Fig. 6, A, B) that in the control GABA (10 5 ) mol/l, applied to the plateau of the tonic component of high potassium contraction, causes a considerable increase in its level, which exceeds the values of the similar parameter in stomach smooth muscles. Similarly to our previous studies [20], smooth muscles of caecum, loaded with the increase in the normalized maximal velocity of the contraction phase and relaxation phase compared to the control group of rats, and the increase in the ratio of the phase component and the tonic component (Fig. 6, C). The data of the cumulative effect of GABA in the concentrations of (10 8 -10 5 ) mol/l on the tonic component of high potassium contraction of caecum in the control group of rats, presented in Fig. 6, D, demonstrate a considerable increase in the level of its plateau. Thus, the obtained data of experimental studies testify to the relevant role of the mediator of intramural nervous system, nitrogen oxide, in the mechanisms of relaxation of smooth muscles of caecum and stomach, acti vated with high potassium solution or acetylcholine. Similar to stomach smooth muscles of rats, loaded with ТіО 2 for 100 days, in smooth muscles of саесum this nanosized mate rial removes the mechanisms of inhibiting the release of NO from neurons of intramural nervous system, mediated via GABA А and GABA С receptors.