Tag Archives: nitric oxide

Indexes of nitric oxide system in experimental antiphospholipid syndrome

O. Z. Yaremchuk, K. A. Posokhova, І. P. Kuzmak,
M. I. Kulitska, I. М. Klishch, M. M. Korda

I. Horbachevsky Ternopil National Medical University, Ukraine;
e-mail: yaremchuk@tdmu.edu.ua

Received: 11 November 2019; Accepted: 21 January 2020

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antibo­dies to negatively charged membrane phospholipids (aPL).  Endothelial dysfunction is one of the most dangerous­ APS manifestations followed by thrombosis, placental insufficiency and often foetal death due to circulatory disorders in placenta blood vessels. It is established that synthesis and bioavailability of nitric oxide (NO) in the endothelium are impaired at APS, but the role of NO system in pregnancy failure at this pathology remains ambiguous. The aim of this research was to estimate the indexes of the nitric oxide system in animals with an experimental antiphospholipid syndrome before pregnancy and on the 18th day of pregnancy, without­ treatment and under treatment with nitric oxide synthesis modulators (L-arginine and aminoguanidine). In the blood serum and liver of the BALB/c mice with experimental APS, the content of eNOS and iNOS by ELISA and the level of NO2 and NO3 with the use of Gris reagent were determined before pregnancy and on the 18th day of pregnancy. The data obtained indicate the relative inefficient NO production by eNOS and NO hyperproduction by iNOS in the blood serum and liver of mice in the pathogenesis of experimental APS. Thus, in mice with APS before pregnancy and on the 18th day of the pregnancy, the eNOS content and NO2 level were decreased while the iNOS content and NO3 level were increased compared to the indexes in the control animal group. L-arginine administration to the animals with APS at the follow-up periods resulted in an increased eNOS content and NO2, NO3 levels in blood serum and liver with the simultaneous decrease in iNOS content in the liver as compared to indexes in untreated mice with APS. The combined use of L-arginine and selective iNOS inhibitor aminoguanidine caused a significant increase in eNOS content and a decrease in iNOS content followed by normalization of NO2 and NO3 levels in blood and liver of mice with  experimental APS before pregnancy and on the 18th day of pregnancy compared to untreated mice with APS.

Sources and regulation of nitric oxide synthesis in uterus smooth muscle cells

H. V. Danylovych, Yu. V. Danylovych, T. V. Bohach,
V. T. Hurska, S. O. Kosterin

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

Received: 28 February 2019; Accepted: 17 May 2019

It was proved that NO synthesis in isolated mitochondria of rat uterus smooth muscle depended on the entry of exogenous Ca2+ to mitochondria (inhibited by 1-10 mM Mg2+ in the absence of ATP and by 10 μM ruthenium red) and was suppressed by calmodulin antagonists (0.1-10 μM calmidazolium and 1-100 μM trifluoperazine). It was blocked by NG-nitro-L-arginine, a known antagonist of the constitutive NO-synthase, with a half-maximal inhibition effect at about 25 μM. Moderate deholesterinization of the plasma membrane of myocytes after processing with 0.01% digitonin was followed by increased nitric oxide biosynthesis by cells. The data obtained suggested that mitochondria and plasmalemma is a possible source of NO synthesis in uterine myocytes.

Effect of sodium nitroprusside and S-nitrosoglutathione on pigment content and antioxidant system of tocopherol-deficient plants of Arabidopsis thaliana

N. M. Semchuk, Yu. V. Vasylyk, O. I. Kubrak, V. I. Lushchak

Vassyl Stefanyk Precarpathian National University, Ivano-Frankivsk, Ukraine;
e-mail: lushchak@pu.if.ua

Sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) were used as a source of exogenous nitric oxide (NO) to investigate their effects on biochemical parameters and antioxidant enzyme response in leaves of wild type Columbia and tocopherol-deficient vte4 and vte1 mutant lines of Arabidopsis thaliana plants and possible tocopherol involvement in regulation of antioxidant response under NO-induced stress. SNP enhanced the activity of the enzymes, that scavenge hydrogen peroxide in leaves of all studied lines, and increased glutathione reductase and glutathione-S-transferase activity there. In addition, it decreased the intensity of lipid peroxidation in vte1 mutant line leaves. At the same time, GSNO increased the levels of protein carbonyls and inactivated enzymes ascorbate peroxidase, guaiacol peroxidase and dehydroascorbate reductase in almost all investigated plant lines. In contrast to wild type, GSNO increased superoxide dismutase activity and decreased catalase activity and chlorophyll a/b ratio in the leaves of two mutant lines. It can be assumed that tocopherols in some way are responsible for plant protection against NO-induced stress. However the mechanisms of this protection remain unknown.

The biosynthesis of nitric oxide from L-arginine. Nitric oxide formation features and its functional role in mitochondria

G. V. Danylovych, T. V. Bohach, Yu. V. Danylovych

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

Modern data on biochemical patterns of nitric oxide biosynthesis in mammal cells from L-arginine in normoxic conditions is described.  The attention of the authors is given to the results of the recent years on the structure and regulation features isoforms of NO-synthase. The emphasis is put on the latest conception of the compartmentalization of certain isoforms of these enzymes in cells and on the possibility of the directed transport of nitric oxide in the vascular wall. The central place in the review is devoted to issues on the endogenous formation of NO in mitochondria and its potential physiological significance. Our own results on the identification of NO in mitochondria of the uterine smooth muscle, biochemical characteristics of this process and NO possible role in Са2+ transport regulation by organelles are presented and discussed.

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.