Tag Archives: nitric oxide

The effect of thymic mesenchymal stromal cells on arginase activity and nitric oxide produced by mouse macrophages

R. S. Dovgiy1,2, I. S. Nikolsky3, L. M. Skivka1

1Taras Shevchenko National University of Kyiv, Ukraine;
2Institute of Gerontology, NAMS of Ukraine, Kyiv;
3State Institute of Genetic and Regenerative
Medicine NAMS of Ukraine, Kyiv;
e-mail: romandovgiy@gmail.com

Mesenchymal stromal cells (MSC) gained much attention due to their therapeutic properties, media­ted largely by anti-inflammatory action. We aimed to investigate the capacity of MSC obtained from young mice to modulate arginine metabolism of macrophages from old animals. Bone marrow cells obtained from young and aged mice were cocultivated with MSC in the presence of M-CSF. Nitric oxide production was analyzed in supernatants by Griess reaction, and arginase activity was measured in cell lysates. We have found that arginase activity was significantly lower in macrophages isolated from old mice as compared to young animals (P ˂ 0.05). Syngeneic MSC addition markedly stimulated arginase activity in macrophages from both young and aged mice (P ˂ 0.001), with greater effect in old animals. There were no significant differences in nitric oxide level between groups. In summary, there was more pronounced anti-inflammatory shift in macrophage metabolism in aged animals upon cocultivation with MSC.

Functioning of nitric oxide cycle in gastric mucosa of rats under excessive combined intake of sodium nitrate and fluoride

O. Ye. Akimov, V. O. Kostenko

Ukrainian Medical Stomatological Academy, Poltava, Ukraine;
e-mail: riseofrevan@mail.ru

In the article the function of nitric oxide (·NO) cycle in rat’s gastric mucosa was assessed under excessive combined chronic fluoride and nitrate intake during 30 days. It was estimated that general nitric oxide synthase activity (NOS) was increased during excessive sodium fluoride intake meanwhile influence on gene­ral nitrate reduction was not statistically significant, but general nitrite reduction was increased. General arginase activity decreased. Excessive sodium nitrate intake decreased NOS activity, but increased nitrate, nitrite reduction and general arginase activity. Combined sodium nitrate and fluoride intake increased NOS by 18.9%, nitrate reduction by 71.7%, nitrite by 161.5%, arginase activity increase by 61.4%. The highest amounts of peroxynitrite were obtained from excessive sodium fluoride intake group, excessive sodium nitrate intake showed the lowest levels and combined excessive sodium nitrate and fluoride intake levels had intermediate results. Summarizing the data obtained, the authors have made a conclusion that combined excessive sodium nitrate and fluoride intake creates optimal conditions for functioning of nitrate-nitrite reductases.

Effect of nitric oxide donor SNAP on GABA release from rat brain nerve terminals

A. S. Tarasenko

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

In this work we investigated the effect of nanomolar concentrations of nitric oxide on the release of gamma-aminobutyric acid (GABA) from rat brain nerve terminals using a radioisotope method with [3H]GABA and a spectrofluorimetric method with Ca2+-sensitive probe Fluo-4 AM. It was shown that in the presen­ce of dithiothreitol (DTT), nitric oxide donor SNAP at concentration, in which it produces NO in the nanomolar range, caused Ca2+-independent [3H]GABA release from nerve terminals. The applications of 4-aminopyridine (4-AP) and nipecotic acid (NA), as the inducers of GABA release from vesicular and cytoplasmic pools, showed that the maximum of SNAP/+DTT-induced [3H]GABA release was registered at 10th min of incubation and coincided in time with significant increase (almost double) in NA-induced [3H]GABA release. At this time point, 4-AP-induced release of [3H]GABA was drastically reduced. At the 15th min of incubation of nerve terminals with SNAP/+DTT, the opposite picture was observed: the decrease in NA- and increase in 4-AP-induced [3H]GABA release. Thus, nitric oxide in the form of S-nitrosothiols at nanomolar concentrations causes Ca2+-independent GABA leakage from synaptic vesicles into cytosol with subsequent release from nerve terminals. The reuptake of the neurotransmitter and its re-accumulation in synaptic vesicles occur later.

Prednisolone and vitamin D(3) modulate oxidative metabolism and cell death pathways in blood and bone marrow mononuclear cells

I. O. Shymanskyy, O. O. Lisakovska, A. O. Mazanova,
D. O. Labudzynskyi, A. V. Khomenko, M. M. Veliky

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: ishymansk@inbox.ru

The study was designed to evaluate reactive oxygen species (ROS)/nitric oxide (NO) formation and apoptotic/necrotic cell death elicited by prednisolone in peripheral blood and bone marrow mononuclear cells and to define the efficacy of vitamin D3 to counter glucocorticoid (GC)-induced changes. It was shown that prednisolone (5 mg per kg of female Wistar rat’s body weight for 30 days) evoked ROS and NO overproduction by blood mononuclear cells (monocytes and lymphocytes) that correlated with increased cell apoptosis and necrosis. In contrast, prednisolone did not affect ROS/NO levels in bone marrow mononuclear cells that corresponded to lower level of cell death than in the control. Alterations of prooxidant processes revealed in mononuclear cells and associated with GC action were accompanied by vitamin D3 deficiency in animals, which was assessed by the decreased level of blood serum 25-hydroxivitamin D3 (25OHD3). Vitamin D3 administration (100 IU per rat daily for 30 days, concurrently with prednisolone administration) completely restored 25OHD3 content to the control values and significantly reversed ROS and NO formation in blood mononuclear cells, thus leading to decreased apoptosis. In bone marrow, vitamin D3 activated ROS/NO production and protein nitration that may play a role in prevention of prednisolone-elicited increase in bone resorption. We conclude that vitamin D3 shows a profound protection against GC-associated cellular damage through regulating intracellular ROS/NO formation and cell death pathways.

The mechanism of VEGF-mediated endothelial cells survival and proliferation in conditions of unfed-culture

T. V. Nikolaienko, V. V. Nikulina, D. V. Shelest, L. V. Garmanchuk

Educational and Scientific Centre “Institute of Biology”,
Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: nikolaenkotetiana@yandex.ua

The mechanisms of VEGF-mediated effects on endothelial cells during cancer development and progression is not clear. In present study the biological effects of VEGF, VEGF-rich culture medium of peritoneal macrophages from mice with Lewis lung carcinoma were studied on MAEC cell line under conditions of unfed culture. We have shown that VEGF increased cell proliferation by the 5th day of culturing vs control and anti-VEGF-treated cells. This effect was associated with increased consumption of glucose and NO production by the 2nd day while decreased – on the 5th day of cell culturing. VEGF-mediated NO production was dependent on Ca2+ ions. Block of Ca2+-channels (LaCl3) had more pronounced inhibitory effect vs chelator of Ca2+ ions (EDTA).  It was shown that peritoneal macrophages are the main suppliers of VEGF at tumor angiogenesis, as evidenced by the data obtained on model system of endothelial cells synchronized in G0/G1 phase.

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.