DoBC:The Main Achievements of the Department

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In recent years, efforts of the scientific staff have been aimed at studying the role of endogenous hydrogen sulfide in various elements of the cardiovascular system (heart, blood vessels, cardiovascular neurons, mitochondria), and interaction between hydrogen sulfide and nitric oxide, another powerful transmitter in the body.

It has been revealed that hydrogen sulfide is actively involved in the heterometric regulation of cardiac function. Modulation of hydrogen sulfide synthesis affects the level of mitochondrial membrane potential and the respiration rate. Following blockade of enzymes for hydrogen sulfide synthesis, either cystathionine-γ-lyase or 3-mercaptopyruvate sulfur transferase, or simultaneous inhibition of both enzymes, the mitochondrial membrane potential in the heart is reduced significantly. The maximum reduction of the latter is observed after inhibiting 3-mercaptopyruvate sulfur transferase by aspartate. The mitochondrial respiration rate increases significantly after combined blockade of both cytosolic and mitochondrial enzymes. Changes in mitochondrial membrane potential coincide with the physiological response of the heart to the volume load. Blockade of hydrogen sulfide synthesis in the mitochondria by aspartate, accompanied by a significant decrease in membrane potential, results in the inhibition of cardiac response to volume load compared with that in control conditions. The latter reflects a reduction in the functional reserves of the heart under inhibited hydrogen sulfide synthesis.

It has been shown that hydrogen sulfide can carry neuroprotective effect by reducing 6-hydroxydopamine-induced degeneration of dopaminergic neurons in the midbrain (model of Parkinson's disease). Moreover, hydrogen sulfide exerts positive effect on the contractile function of the heart in rats, reducing the sensitivity of mitochondrial permeability transition pores to calcium, normalizing mitochondrial respiration and increasing the degree of coupling of oxidation and phosphorylation.

In the medulla oblongata of rats, a significant content of endogenous hydrogen sulfide has been detected. In hypertension, the level of hydrogen sulfide in the rat medulla oblongata is much lower that that in the normotensive rats. After preliminary inhibition of cystathionine -β-synthase (which is expressed primarily in neurons of the brain), or an inhibition of 3-mercaptopyruvate sulfur transferase (which is thought to be the mitochondrial enzyme), a significant weakening of the hemodynamic effects is observed. Changes in hemodynamics after inhibiting cystathionine-γ-lyase are neglegible. These data suggest that the effects of L-cysteine in the medulla oblongata are implemented mainly through the activation of cystathionine -β-synthase and 3-mercaptopyruvate sulfur transferase. It has been also shown that an inhibition of neuronal NO-synthase reduces the effects of L-cysteine introduced in the medullary nuclei, testifying to the interaction between hydrogen sulfide and nitric oxide systems in the cardiovascular control. We were the first to show that an administration of single-walled carbon nanotubes in the medullary nuclei results in hypotensive responses in both normotensive and spontaneously hypertensive rats. Single-walled carbon nanotubes influence the neural medullary cardiovascular control in rats following their intramedullary or peripheral (intravenous, intraperitoneal, subcutaneous) administration. Endogenous hydrogen sulfide and nitric oxide contribute to the implementation of the hypotensive effect of single-walled carbon nanotubes.

In the study on adult and old rats, it has been revealed that blockade of cystathionine-γ-lyase (CSE), enzyme for de novo synthesis of hydrogen sulfide, restores acetylcholine-induced relaxation of the aorta smooth muscles, suppressed in old rats. This effect is removed by inhibiting of nitric oxide (NO) synthesis. The levels of intramitochondrial pools of H2S and NO2-, as well as cNOS activity in the heart decrease with age. An introduction of propargyl glycine largely restores the pools of H2S and NO2- and stimulates reduced activity of cNOS. Thus, stimulation of H2S and NO synthesis by propargyl glycine results in recovering of suppressed endothelium-dependent relaxation of the aorta smooth muscles in old rats and improving the cardiac function via the stimulation of H2S and NO synthesis. NaHS, introduced in vivo, inhibits oxidative and nitrosative stress, restores cNOS coupling and increases the constitutive synthesis of NO.

It has been found that an activation of free radical processes, particularly during aging, ischemia-reperfusion of the myocardium, and ischemic pre-conditioning results in enhanced expression of UCP2 and UCP3 genes in the heart tissues. Blockade these uncoupling proteins reduces negative effects of oxidative stress, indicating the participation of these proteins in the formation of cardioprotective myocardial response.

The features of calcium accumulation by isolated heart mitochondria from adult rats under physiological conditions and in case of mitochondrial dysfunction due to their load by high concentrations of calcium ions have been determined. The possibility of using flow cytometry to study mitochondrial calcium homeostasis in isolated cardiomyocytes was demonstrated. The dependence of calcium accumulation on incubation temperature, availability of Mg2+ -ATP complex substrate and succinate has been shown. It was found that mitochondria from isolated heart have limited ability to store calcium. The data on the impact of exogenous hydrogen sulfide and inhibitors of its synthesis on the accumulation of Ca2+ by the mitochondria in cardiomyocytes of adult rats have been obtained. It was found that an accumulation of calcium in isolated mitochondria depends on the concentration of NaHS. Inhibition of 3-mercaptopyruvate sulfur transferase, mitochondrial enzyme, does not affect the accumulation of calcium, which may testify to mitochondrial regulation of calcium-transporting system by cytosolic hydrogen sulfide, not mitochondrial one.

It has been found that endogenous hydrogen sulfide pools are reduced in the heart mitochondria in aging. This process is normalized under the action of pyridoxal-5-phosphate, in other words, hydrogen sulfide content increases probably due to an activation of H2S-synthesizing enzymes. It has been revealed that degree of swelling of the heart mitochondria from old rats after the introduction of Ca2+ inductor pyridoxal-5-phosphate lowers significantly compared to the heart mitochondria without inductor, which may indicate an increase in resistance due to reduced mitochondrial membrane permeability in the presence of Ca2+ inductor. The activation of hydrogen sulfide biosynthesis results in reduced sensitivity of mitochondrial permeability transition pore to its inducer calcium in the heart mitochondria from old rats due to increased threshold concentration, which causes swelling of organelles. The experimental results suggest that an increase in the content of hydrogen sulfide through the activation of its biosynthesis in the body is effective in inhibiting mitochondrial permeability transition pore, and it can be useful to correct mitochondrial dysfunction in various pathological conditions of the cardiovascular system in aging.

In biochemical studies, it has been shown that hydrogen sulfide donor NaHS at physiological concentrations inhibits calcium-induced mitochondrial permeability transition pore opening, indicating the protective effect on the pore formation in rat heart. An increase in the activity of arginase, nitrate reductase, and enzymes for constitutive nitric oxide synthesis has been detected in the mitochondria from rat heart after either the introduction of L-cysteine, substrate of hydrogen sulfide synthesis, or its antagonist propargyl glycine, and their joint actions that may be a manifestation of hormesis. Temporal correlation between increased pools of two powerful vasodilators, namely sulfur- containing and nitrogen- containing, was found in the blood plasma and the heart of rats. With aging, endogenous pools of hydrogen sulfide are reduced in the blood plasma and the heart mitochondria, at that oxidative and nitrosative stress is considerably weakened not only following a single injection of L-cysteine, but also propargyl glycine, an inhibitor of H2S synthesis. Reducing of endogenous pools of hydrogen sulfide in the heart mitochondria from old rats leads to an increase in peroxynitrite generation that is metabolized to generate *OH and * NO2-. In the heart mitochondria of old rats, propargyl glycine stimulates the activity of constitutive NO-synthase (cNOS), while the activity of inducible isoform (iNOS) is inhibited by propargyl glycine and L-cysteine, and their combined administration has an additive effect.

It has been shown that omega 3 polyunsaturated fatty acids (linoleic acid) cause hyperpolarization of endothelial cells due to the stimulation of calcium-dependent potassium channels of large conductance. This effect is receptor-independent and preserved in the membrane patch when you add omega 3 fatty acids to the inner surface of the membrane. Reduction of cholesterol level in the membrane of endothelial cells almost completely inhibits this effect, indicating the regulatory role of cholesterol in the effect of omega 3 fatty acids on calcium-dependent potassium channels of large conductivity. A similar effect is observed when adding anandamide, an agonist of cannabinoid receptors types I and II. Results can extend modern understanding of the mechanisms of action of omega 3 polyunsaturated fatty acids and agonists of cannabinoid receptors.

The effects of Ca2+ -induced mitochondrial permeability transition pore (MPTP) opening in the states of submaximal conductivity on the formation of ROS in the mitochondria of the rat brain and the liver were shown, as well as a linear dependence of ROS generation (JR) on the rate of Ca2+ -stimulated respiration (JO2) and exponential dependence of JR on membrane potential (ΔΨm). Based on the obtained results, it was concluded that in the conditions of submaximal conductivity, the role of MPTP in ROS generation is determined by its contribution to both the respiration rate and the modulation of ΔΨm. The data obtained indicate potential-dependent mechanism of regulating ROS generation in mitochondria of the liver and the brain of rats by MPTP opening in states of submaximal conductivity.

The levels of expression of mRNA cystathionine-γ-lyase, cystathionine-β-synthase and 3-mercaptopyruvate sulfur transferase were studied by PCR in real time method in the heart from adult and old rats in control and after the modulation of endogenous production of hydrogen sulfide. It has been revealed that the level of mRNA 3-MPST is significantly higher than mRNA CSE in the heart of adult and old animals. An increase in the expression of mRNA of two enzymes was found in the heart of old rats compared to adult animals. Moderate short-term inhibition of CSE together with a single injection of the amino acid L-cysteine is accompanied by increased expression of CSE mRNA in the heart of only adult rats, while in old animals substantial decline was observed. 3- MPST mRNA expression significantly increases (6.3-fold (p <0.05) in adults and 2.6-fold (p <0.05) in old animals), compared to control rats. Thus, under conditions of moderate short-term inhibition of CSE with following administration of amino acid L-cysteine, changes in expression of H2S-synthesizing enzymes occur, which are aimed at activating the endogenous synthesis of hydrogen sulfide as an important regulatory factor in the cardiovascular system under physiological and pathological conditions.