Tag Archives: nitric oxide

Nitric oxide as the regulator of intracellular homeostasis in the uterus myocytes

Yu. V. Danylovych

Palladin Institute of Biochemistry, National Academy of Sciences, Kyiv, Ukraine;
e-mail: danylovych@biochem.kiev.ua

The published data on the mechanisms and regulation of active and passive Ca2+ transport in the myometrium have been analyzed. Particular attention is paid to the cGMP-dependent and independent pathways of action of nitric oxide or its derivatives on intracellular Ca2+ homeostasis of uterine smooth muscle and its contractile activi­ty. Information on the effect of nitric oxide on Ca2+-transport systems of other types of smooth muscles is provided in a comparative aspect. Based on own experimental results and literature data a scheme of NO action in the myometrium is suggested in which nitric oxide or its derivatives cause­ Ca2+-dependent polarization of the sarcolemma. In accordance with our results, this effect may be based on the increase of sarcolemma Ca2+ permeability under the influence of NO or its derivatives and the stimulation of at least the initial passive transport of the cation in the myocytes mediated by dihydropyridine-sensitive channels. Additional factors that contribute to the polarization of the membrane are the increase of protons transport from the muscle cells and stimulation of Na+, K+-ATPase. Acting on the sarcoplasmic reticulum, nitrosactive compounds activate the inclusion of calcium in this compartment and inhibit Ca2+-induced release of the cation. The latter effects are able to provide compensation for NO-induced Ca2+ increase in myocytes and supress the electro-mechanical coupling at Ca2+ release from the reticulum. NO-derivates also inhibit a key link in the smooth muscle contractile act – the formation of the Ca2+-calmodulin complex.

Signal mediators at induction of heat resistance of wheat plantlets by short-term heating

Yu. V. Karpets, Yu. E. Kolupaev, T. O. Yastreb

V. V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

The effects of functional interplay of calcium ions, reactive oxygen species (ROS) and nitric oxide (NO) in the cells of wheat plantlets roots (Triticum aestivum L.) at the induction of their heat resistance by a short-term influence of hyperthermia (heating at the temperature of 42 °С during 1 minute) have been investigated. The transitional increase of NO and H2O2 content, invoked by heating, was suppressed by the treatment of plantlets with the antagonists of calcium EGTA (chelator of exocellular calcium), lanthanum chloride (blocker of calcium channels of various types) and neomycin (inhibitor of phosphatidylinositol-dependent phospholipase C). The rise of hydrogen peroxide content, caused by hardening, was partially suppressed by the action of inhibitors of nitrate reductase (sodium wolframate) and NO-synthase (NG-nitro-L-arginine methyl ester – L-NAME), and the increasing of nitric oxide content was suppressed by the treatment of plants with the antioxidant ionol and with the scavenger of hydrogen peroxide (dimethylthiourea). These compounds and antagonists of calcium also partially removed the effect of the rise of plantlets’ heat resistance, invoked by hardening heating. The conclusion on calcium’s role in the activation of enzymatic systems, generating reactive oxygen species and nitric oxide, and on the functional interplay of these signal mediators at the induction of heat resistance of plantlets by hardening heating is made.

The effect of nitric oxide on synaptic vesicle proton gradient and mitochondrial potential of brain nerve terminals

A. S. Tarasenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: tas@biochem.kiev.ua

The effect of nitric oxide on synaptic vesicle proton gradient and membrane potential of rat brain nerve terminals was studied. It has been shown that nitric oxide in the form of S-nitrosothiols at nanomolar concentrations had no effect on the studied parameters, but caused a rapid dissipation of synaptic vesicle proton gradient and depolarization of mitochondrial membrane in the presence of a SH-reducing compound such as dithiothreitol. Both processes were reversible and the rate of H+-gradient restoration depended on the redox potential of nerve terminals, namely the molar ratio of reductant/oxidant. This facts, as well as insensitivity of the studied processes to the inhibitor of NO-sensitive guanylate cyclase such as ODQ, allow suggesting that post-translational modification of thiol residues of the mitochondrial and synaptic vesicle proteins underlies the effect of nitric oxide on the key functional parameters of presynaptic nerve terminals.

Peculiarities of arginase and NO-synthase pathways of L-arginine metabolism in peripheral blood lymphocytes of patients with ovarian cancer

O. I. Yakubets, R. V. Fafula, D. Z. Vorobets, Z. D. Vorobets

Danylo Halytski Lviv National Medical University, Ukraine;
е-mail: vorobets@meduniv.lviv.ua

The peculiarities of arginase and NO-synthase pathways of L-arginine metabolism in peripheral blood lymphocytes of patients with ovarian cancer were studied. It was shown that the development of cancer pathology is associated with an imbalance in the NO synthesis in blood lymphocytes. The reason for such imbalance is the activation of arginase and inducible isoform of NO-synthase (iNOS) and significant inhibition of its constitutive isoform. The analysis of the kinetic properties of NOS of blood lymphocytes of patients with ovarian cancer was carried out. It was shown that the affinity constant of iNOS affinity for L-arginine is 5.4-fold lower than for eNOS of blood lymphocytes of persons in the control group. The inhibition of eNOS occurs via non-competitive type and is related to the reduction of maximum reaction rate.

The effect of N-stearoylethanolamine on the activity of antioxidant enzymes, content of lipid peroxidation products and nitric oxide in the blood plasma and liver of rats with induced insulin-resistance

O. V. Onopchenko, G. V. Kosiakova, T. M. Goridko,
A. G. Berdyschev, O. F. Meged, N. M. Hula

Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv;
e-mail: onop.89.av@mail.ru

The influence of N-stearoylethanolamine (NSE) on the content of lipid peroxidation products­, activity of antioxidant enzymes and the nitric oxide level in the liver and blood plasma of rats with insulin-resistance(IR) state was investigated. IR state was induced in rats by prolonged high-fat diet (58% of energy derived from fat) for 6 months combined with one injection of streptozotocin (15 mg/kg of body weight). The existence of IR state was estimated by results of glucosotolerance test and blood plasma insulin content.  The level of lipid peroxides products was shown to be higher in the liver of insulin resistant animals as a result of reduced superoxide dismutase and catalase activity, however, glutathione peroxidase activity was increased. The increase of nitric-oxide content in the liver and blood plasma of high-fat diet rats compared with healthy control animals was also observed. The administration of the NSE suspension per os in a dose of 50 mg/kg during 2 weeks to the rats with induced insulin-resistance state contributed to the increase of superoxide dismutase, catalase and glutathione peroxidase activi­ty. In consequence of antioxidant enzymes activation the intensity of POL process was decreased. The NSE administration caused normalization of nitric oxide level, restoring pro-/antioxidant balan­ce in the liver and blood plasma of rats with IR state.
In conclusion, the NSE administration to the rats with insulin-resistance state restored pro-/antioxidant balance and enhanced the content of nitric oxide, therefore, improving insulin sensitivity.

Interconnection between nitric oxide formation and hypersensitivity parameters under guinea pig model of acute asthma with multiple challenges

O. O. Parilova, S. G. Shandrenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: elenparil@gmail.com

An immunoregulatory role of nitric oxide (NO) in the development of adaptive immune responses associated with allergic diseases is very important. The present study extended these observations by the examination of the reciprocal changes in characteristic immunologic parameters of the disease and NO level of bronchoalveolar lavage (BAL) cells under guinea pig model of acute asthma with multiple challenges. Development of guinea pig Th2 mediated asthma was accompanied by increasing the level of allergic markers: ovalbumin (OVA) specific IgG and IL-4. We demonstrated that the infiltrate of airway cells contributes to NO synthesis in the respiratory tract during allergic inflammation. The level of intracellular NO formation significantly correlated with plasma allergen specific IgG value in OVA-induced asthma. The presented data evidence that the elevated intracellular NO level in BAL fluid may reflect a nitrosative stress in respiratory tract in general, when allergic asthma exacerbation is present.

Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells

Yu. V. Danylovych, S. A. Karakhim, H. V. Danylovych, O. V. Kolomiets, S. O. Kosterin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua

We demonstrated using Ca2+-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes’ inner mitochondrial membrane by a protonophore carbonyl cyanide m-chlorophenyl hуdrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3′-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane’s electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hуdrazone causes substantial increase in fluorescence of Ca2+-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca2+-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.

Effect of anticonvulsants on the nitric oxide system

L. A. Gromov1, I. F. Belenichev2, L. G. Gonchar-Cherdakli1, G. A. Zhernovaja2

1SI Іnstitute of Pharmacology and Toxicology, National Academy of Medical Sciences of Ukraine, Kyiv;
2Zaporizhia State Medical University, Ukraine;
e-mail: llrr@ukr.net

The effect of phenobarbital, carbamazepine, valproate sodium, depakine, topiramate and lamotrigine on the content of NO and NO-synthase activity in white rat brain tissues has been studied. It was established that the action of carbamazepine, valproate sodium, topiramate and lamotrigine decreases the activity of NO-synthase and the level of NO in the brain tissues. The amount of NO does not change while NO-synthase activity increases with the introduction of phenobarbital. The involvement of nitric oxide in the mechanism of action of the studied anticonvulsant drugs is discussed.

Reactive oxygen species and stress signaling in plants

Yu. E. Kolupaev, Yu. V. Karpets

V. V. Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@mail.ru

Data on the basic processes and the compartments, involved in formation of reactive oxygen species (ROS) in plant cells, are generalised. The features of structure and regulation of NADPH-oxidase as the one of main enzymatic producers of ROS are characterized. The two-component histidine kinases, ROS-sensitive transcript-factors, ROS-sensitive protein kinase and redox-regulated ionic channels are discussed as the possible sensors of redox-signals in plant cells. The interaction between ROS and other signal mediators, in particular nitric oxide and calcium ions, is discussed. The ROS role as the signal mediators in the development of plant resistance to hyperthermia, osmotic shock and other abiotic stressors is analyzed.

Investigation of nitrosactive compounds influence on polarization of the mitochondrial inner membrane in the rat uterus myocytes using potential sensitive fluorescent probe DіOC(6)(3)

Yu. V. Danylovych, H. V. Danylovych, O. V. Kolomiets, S. O. Kosterin, S. A. Karakhim, A. Yu. Chunikhin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua

The effect of nitrosactive compounds (sodium nitroprusside and sodium nitrite) on the polarization level of the uterus myocytes inner mitochondrial membrane using the confocal laser microscopy and fluorescent probe potentialsensitive DiOC6(3) (3,3′-dihexyloxacarbocyanine) was ivestigated. Colocalisation of mitochondrial membranes specific fluorescent probes (MitoTracker Orange CM H2TMRos­, 10 – nonyl acridine orange and DiOC6(3)) was demon­strated. It was shown that sodium nitroprusside at 0.1 mM concentration caused a moderate decrease in mitochondrial transmembrane potential. That observation was confirmed by flow cytometry. Action efficiency of sodium nitrite in a similar concentration was significantly lower than that of sodium nitroprusside. It is shown that it was sodium nitroprusside which caused a slight swelling of the mitochondria. A possible protecting role of nitric oxi­de as to mitochondria was discussed.