The first application of sensory structures based on photoelectric transducer for the study of enzymatic reactions

Background. The development of highly sensitive sensor equipment with the possibility of registering analytes in real time is a fast growing research area and a promising diagnostic biomedical technology. Currently, the standard laboratory method for determining the activities of ATPses is an indirect spectroscopic study of the concentration of inorganic phosphate formed as a result of ATP hydrolysis by these enzymes. However, there is no commercially available phosphate sensor with satisfactory parameters of sensitivity, selectivity and stability over time. The purpose of our research was the deve­lopment of a photoelectric recombination sensor system for the real-time detection of biochemical markers and its testing on the example of screening ATPase activity of rat erythrocyte plasma membrane suspension preparations. Materials and Methods. Experiments were performed on suspension preparations of plasma membranes of erythrocytes of Wistar rats. Preparations of plasma membrane suspensions obtained by Dodge method from each animal were divided into aliquots and used for the simultaneous study of ATPase activity by the reference method of Rathbun & Betlach, as well as the registration of photocurrents induced during the passage of the ATPase reaction using the photoelectric recombination multisensor system of our own design. Results. The application of silicon sensory structures based on photoelectrical transducer principle for detecting the activity of adenosine triphosphate hydrolases on the example of total Mg2+,Na+,K+-ATPases preparations of plasma membranes of rat erythro­cytes has been experimentally tested. The directly measured analytic parameter is the photocurrent of the deep silicon barrier structure under illumination with high absorption coefficient. The physical features of the device operation have been examined. Detection of such metabolites becomes possible due to reactions intermediates with their own dipole moment (inorganic phosphate, which is one of the products of ATP hydrolysis). The drastic change of photocurrent that characterizes the course of biochemical reaction was observed in real time. The effect is explained by local electrostatic influence on the parameters of recombination centers at the silicon surface that results in surface recombination velocity change. The sensor operation is qualitatively explained in the frame of Stevenson-Keyes’s theory. Conclusions. Our approach can be regarded as a promising way to elaborate technically simple and highly sensitive method for detection of quantitative behavior of enzymatic reactions. Moreover, the local modification of silicon surface allows obtaining time depending scenarios of the adsorption and thus improving the sensitivity of the sensor. These circumstances open up the possibility of elaborating the complex sensory structures with optimized parameters for certain enzymatic reactions

Inhibition of Na(+),K(+)-ATPase and activation of myosin ATPase by calix[4]arene C-107 cause stimulation of isolated smooth muscle contractile activity

The discovery of compounds that might modify myometrial contractility is an important area of researches. In our previous experiments, we found that some representatives of macrocyclic compounds fami­ly – calix[4]arenes – can modify the enzymatic and transport activity of membrane-bound cation-transport ATP hydrolases. The aim of this work was to study and compare the effect of calix[4]arene C-107 on the enzymatic activities of Mg2+-dependent ATPases of the uterine smooth muscle, namely: ouabain-sensitive Na+,K+-ATPase, plasma membrane Ca2+-independent “basal” Mg2+-ATPase, ATPase of the actomyosin complex and myosin subfragment-1, with effect on the contractile activity of the myometrium. It was shown that calix[4]arene C-107 efficiently inhibited myometrium Na+,K+-ATPase (I50 = 54 ± 6 nM) selectively to other ATP-hydrolases of the plasma membrane and simultaneously activated the enzymatic activity of the myosin ATPase of smooth muscles (A50 = 9.6 ± 0.7 μM). Such reciprocal biochemical effects led to the stimulation of the smooth muscle contractile activity that was demonstrated by the tensometric method using different isolated smooth muscles. Calix[4]arene С-107 was shown to stimulate the increase of the tonic component of myometrium contractions induced by oxytocin, as well as contractions of the caecum muscles induced by high-potassium solution or acetylcholine, and to maintain increased tension for a long time. Thus, calix[4]arene C-107 is a prospective compound for enhancing the smooth muscle basal tone and/or contraction in case of hypotonic dysfunctions

Diazepinone effect on liver tissue respiration and serum lipid content in rats with a rotenone model of Parkinson’s disease

Parkinson’s disease (PD) is a chronic and progressive age-related neurodegenerative disorder. Accumulation of α-synuclein aggregates, oxidative stress, mitochondrial dysfunction and lipid metabolism disturbance are thought to be the key violations at PD pathogenesis. Despite long-time research the causes of PD occurrence are not yet clear. We investigated the influence of diazepinon, a new derivative of benzodiazepine, on liver tissue respiration (LTR), serum lipid content and behavioral parameters of rats with modeled PD induced by intraperitoneal injections of 2.0 mg/kg rotenone (ROT) within 28 days. LTR was assessed using the polarograph LP-9. Blood samples for biochemical analysis were collected from the inferior vena cava. The behavioral parameters of rats were studied by the open field test. We showed that in rats with ROT – induced PD the coefficient of liver oxygen consumption was decreased by 33.5% (P < 0.001), the serum content of phospholipids, cholesterol, cholesterol esters, free fatty acids and triglycerides was reduced by 21.4% (P < 0.001), 28.8% (P < 0.001), 26.8% (P < 0.001), 30.3% (P < 0.01) and 41.5% (P < 0.001) respectively and the motor disorders were detected. Diazepinone application resulted in a full recovery of LTR, serum concentration of phospholipids, partial recovery of serum free fatty acids and triglycerides content and significant improvement of motor behavior. However diazepinone did not affect the reduced concentration of cholesterol and cholesterol esters in the serum of rats with simulated PD

Ethanol reforming in non-equilibrium plasma of glow discharge

The results of a detailed kinetic study of the main plasma chemical processes in non-equilibrium ethanol/argon plasma are presented. It is shown that at the beginning of the discharge the molecular hydrogen is mainly generated in the reaction of ethanol H-abstraction. Later hydrogen is formed from active H, CH2OH and CH3CHOH and formaldehyde. Comparison with experimental data has shown that the used kinetic mechanism predicts well the concentrations of main species at the reactor outlet.Comment: 16 pages, 8 figure

Influence of calixarene C-99 on contractile activity of rat large intestine smooth muscles

The regularities of influence of new high-affinity inhibitor of plasma membrane sodium pump calixarene C-99 on the contractile activity of rat’s large intestine circular smooth muscle were studied. It was found that under preliminary complete blockage of the sodium pump using ouabain (100 µM) the calixarene C-99 (100 µM) causes activation of smooth muscles spontaneous contractions. It has been also found that under ouabain preliminary action the calixarene changes mechanokinetical parameters of contractile responses to depolarization of smooth muscle cells plasma membranes via potassium solution (80 mM), as well as per application of the muscarinic acetylcholine agonist (10 µM). It has been shown that calixarene C-99 has no effect on the Ca2+ mobilization from the sarcoplasmic reticulum. Thus, the calixarene C-99 action on intestinal smooth muscles is not limited to the plasma membrane’s sodium pump blocking. Possible mechanisms of action calixarenes C-99 on smooth muscle’s contractile activity are discussed

Kinetics of relaxation of rat myometrium in conditions of inhibition of plasma membrane calcium pump and systems of active Ca2+ transport of intracellular Ca2+-depot

The processes of regulation of smooth muscle contractile function depend greatly on the efficiency of maintaining Са2+-homeostasis by systems of active ionic transport of the plasma membrane (calcium pump and Na+,Ca2+-exchanger) and intracellular stores (sarcoplasmic reticulum and mitochondria). In this work, the effect of calixarene C-716 (macrocyclic compound capable of blocking functioning of the plasma membrane calcium pump) on the kinetics of relaxing K+-induced contraction of rat myomet­rium smooth muscles, while inhibiting the systems of active transport of calcium ions of intracellular Са2+-stores, were investigated. It was established that the application of 100 M calixarene at the maximum of the phase component of the K+-induced contractile response did not change the kinetic parameters of the relaxation process (the normalized maximal velocity of relaxation phase Vnr and the half-maximal time of relaxation τ1/2). The tonic component of K+-induced contraction remained without statistically significant changes. Blocking of Са2+-accumulating function of mitochondria by the protonophore СССР caused a significant increase in both the parameter Vnr and the tonic component of K+-induced contraction, while the τ1/2 index remained at the control level. Blocking calcium pump of sarcoplasmatic reticulum with thapsigargin (TG) caused a conside­rable increase in the τ1/2 ,Vnr parameters and the tonic component of the K+-induced contraction. Combination of TG and СССР induce further increase in the Vnr parameter and the tonic component of K+-induced contraction. The action of calixarene С-716 (100 M) at the background of the impact of TG and СССР was accompanied by further increase in the tonic contraction, Vnr remained at the increased level, and the τ1/2 index decreased. Possible mechanisms for changing the mechanokinetic parameters of relaxing K+-induced contraction of myometrium is discussed. Analysis of the results of our experimental data and the data of other researchers allow predicting that an increase in the rate of relaxation of myometrium smooth muscles at the action of calixarene C-716 under the conditions of blockage of intracellular Ca2+ depots is associated with a significant activation of NO synthesis by constitutive forms of NO-synthases, whereas an increase in the tonic component of K+-contraction under these conditions is probably due to an elevated concentration of Ca2+ ions in the myocytes

Involvement of KАТР-channels of plasma and mitochondrial membranes in maintaining the contractive function of myometrium of non-pregnant rat uterus

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 KATP-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 the activator of ATP-sensitive K+-channels of diazoxide (the range of concentrations was 50–200 µM), was the reduction in the area under oxytocin-induced contractions by half. This compound also caused a decrease in the amplitude of contractions and a considerable increase in the maximal velocities of the intensification and decline phases for the force of oxytocin-induced contractions. The combined effect of the blo­cking agents of KATP-channels of plasma and inner mitochondrial membranes, glibenclamide (10 µM) and 5-hydroxydecanoate (200 µM), was accompanied by the increase in the amplitude of oxytocin-induced contractions and the changes in the kinetics of contraction and relaxation processes. Thus, the data obtained demonstrate that KATP-channels of plasma membrane and mitoKATP-channels 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

Thermomechanokinetics of viscoelastic deformation of smooth muscles in the rat gastrointestinal tract. II. Thermomechanokinetics of hysteresis of stomach and large intestine smooth muscles

The thermomechanokinetical properties of smooth muscle to a certain extent determine the functional properties of the gastrointestinal tract. We has previously investigated the changes induced by temperature on the kinetics of elastic deformation of rats gastric smooth muscle. In the present study, the thermomechanokinetical properties of stress-induced deformation and relaxation (hysteresis curves) of smooth muscle strips of the stomach and the large intestine (colon and caecum) were analyzed. Proposed and tested mathematical phenomenological description of the hysteresis curves of muscles using quantitative parameters: force constants k1 and k2 (for the application and removal of the load, respectively); indexes P1/2,c (the load value, which causes the demimaximal strip deformation) and P1/2,r (the residual load, which is half the strip length recovery). An area of full cycle hysteresis is characterized hysteresis loop quantitatively, and serves as the energy characteristic of temporary irreversibility of the length return of deformed smooth muscle strips to the unladen condition. Using these quantitative parameters shows that the deformation of the stomach smooth muscle has a relatively low temperature sensitivity and physical sense is a passive process. Discusses possible mechanisms that cause thermoinduced mechanokinetical changes of hysteresis parameters in smooth muscles

Thermomechanokinetics of viscoelastic deformation of smooth muscles in the rat gastrointestinal tract. I. Dynamic properties of the stretch in stomach smooth muscles

Functioning of the digestive system strongly depends on mechanical properties of smooth muscles that form the walls of the gastrointestinal tract organs. a quantitative analysis of temperature effects on the mechanokinetical curves „load-time” (at fixed loads) and „load-deformation” (with constant and gradual-increasing load) of rat stomach circular smooth muscles, in the work was carried out. Shows that the value of relative deformation [(L–Lp)/Lp] of muscle preparations – the parameter that depends on temperature; their high values are observed with a physiologically-relevant temperatures, whereas temperature shifts towards moderate cooling or heating it reduces. Increases in temperature up to 48°C had the opposite effect, it accompanied by decrea­sing of [(L–Lp)/Lp] at an average of 27% compared with the value that occurred in 37°C. by calculation of the relative change in stiffness coefficient it is shown that a moderate cooling (22°C) and heating (48°C) gastric smooth muscles causes it to increase by an average of 34% compared with their value in 37°C. However, the relatively low thermosensitivity of viscoelastic deformation of stomach smooth muscles indicates that in the physical sense in this process is predominate a passive component

Influence of calixarene C-90 on contractile activity of rat myometrium smooth muscles

It is known that calix[4]arene with cipher C-90 selectively and with high affinity inhibits Ca2+,Mg2+-ATPase of smooth muscle cells plasma membrane preparations. The work wis devoted to investigation of the influence of calixarene C-90 (10 µM) on spontaneous and induced (high-potassium solution and oxytocin) contractions of rat uterus longitudinal smooth muscles. Contractile activity was studied tensometrically in the isometric mode, analysis of the kinetic properties of contractions was performed by the calculation of the normalized maximal velocity of contraction (Vnс) and relaxation (Vnr) phases. Calixarene C-90 changed the spontaneous contractile activity, causing a decrease in amplitude and has no significant effect on the frequency, while slowing down of the relaxation phase of individual contractions (decreasing parameter Vnr) occurred. In the presence of non-selective NO-synthase inhibitor L-NAME (100 µM), calixarene C-90 did not cause a reduction of the amplitude of spontaneous contractions and the speed of relaxation phase returned to the control level. Furthermore, calixarene C-90 was equally contributing factor to reduced force of both oxytocin-induced (0.1 IU) and K+-induced (80 mM) contractions without affecting the nature of the increase in contractile force responses (normalized maximal velocity of contraction phase stayed at control level). The relaxation velocity of caused contractions recieved opposite changes depending on the nature of the contractile stimulation: in case of oxytocin-evoked contractions – decreased, while for K+-induced contractions – increased. In the presence of L-NAME calixarene C-90 did not cause inhibition of the maximal force K+- and oxytocin-induced contractions, but evoked changes in the kinetical para­meters of contractile responses (decrease Vnr). Thus, blocking of NO synthesis resulted in the removal of inhibiting both spontaneous and evoked contractions of smooth muscle myometrium under the influence of calixarene C-90. These results suggest that inhibition force of uterus smooth muscle contractions under the influence of calixarene C-90 is by NO-dependent way, whereas slow relaxation (decrease in normalized maximal velocity Vnr) is caused by the inhibition of Ca2+-transport function of the plasma membrane calcium pump