[Vascular reactivity and metabolism of the reactive oxygen species and nitrogen in effects of low doses of radiation].

The disturbance of endothelium-dependent and endothelium-independent vascular reactions of relaxation was registered in the preparations of aorta of radiosensitive BALB/c mice, exposed to chronic external gamma-irradiation (cumulative dose of 0.43 Sv). Low doses of radiation induced an intensive hydrolysis of membrane phospholipids by phospholipase A2, displayed by an increase in the level of eukosanoisds--LTC4 and TxB2, formed under effects of lipid oxidases (lipoxygenase and cyclooxygenase) at the same time with O2 generation. High doses of O2- can also be formed under the effect of low doses of radiation along xanthine oxidase pathway simultaneously with uric acid. In these conditions *OH-radical is formed not only at the expense of water radiolysis, which is observed under the effect of high doses, as well as along the two--NO-dependent and NO-independent--pathways. Significant increase in the content of lipid peroxidation products--dienic conjugates and valonic dialdehyde--in the organs of cardiovascular system is a confirmation of active generation of *OH and *NO2 under the effect of low doses of radiation. The latter induce significant changes in the pools of NO stable metabolites, which can cause disturbance of NO-dependent physiological functions of both heart and aorta. Significant decrease in the levels of nitrite and nitrozothiols in these conditions may result in an oxidative stress. In increased simultaneous generation of *O2- and NO they may bind and thus form a toxic substance peroxynitrite. This notion can be confirmed by the low doses of nitrite, which are formed spontaneously in the presence of molecular oxygen against the background of increased or control levels of nitrate, which is formed mainly at the degradation of peroxynitrite, i.e. at high levels of superoxide anion.

MITOCHONDRIAL DYSFUNCTION IN THE AGING HEART IS ACCOMPANIED BY CONSTITUTIVE NO-SYNTHASES UNCOUPLING ON THE BACKGROUND OF OXIDATIVE AND NITROSATIVE STRESS.

We investigated the sensitivity of mitochondrial permeability transition pore (MPTP) opening to its inductor calcium, as well as indicators of oxidative and nitrosative stress in adult and old rat heart mitochondria and heart tissues. The coupling index of constitutive Ca2+/calmodulin-dependent NO-synthase (сNOS) was calculated based on experimentally found parameters. The aging is characterized by oxidative and nitrosative stress, which accompanied by a decrease of the cNOS coupling index and an increased sensitivity of MPTP to calcium. We found that in the heart mitochondria of old rats such oxidative stress indicators as the rate of generation of superoxide (• O2 - ) and hydroxyl (• OH) anion radicals were significantly increased (in 4 and 2,7 times respectively). Also, increased levels of urea and products of early lipid peroxidation ­ conjugated dienes display the intensification of oxidative stress. Such indicators of nitrosative stress as nitrate reductase and iNOS activity were also enhanced. At the same time under aging the NO2 - pools, generated when the heart oxygenation is normal, nitrosothiols pools and the activity of cNOS were decreased. It is due to the enhanced level of cNOS uncoupling, resulting in increased oxidative stress. It was also shown the lower level of hydrogen sulfide (H2S) in old rat heart mitochondria. Thus, we observed the increased sensitivity of MPTP to calcium, due to decreased concentration of its inhibitors nitric oxide and hydrogen sulfide and increased levels of its inducers ROS and RNS in old rat heart mitochondria.

[Dependence of calcium absorption in the chicken small intestine on its Ca2+-binding protein content and the effect of exogenous nucleotide precursors on this process]. / Zavisimost' absorbtsii kal'tsiia v tonkom kishechnike tsypliat ot soderzhaniia ca2+-sviazyvaiushchego gelka i vlianie na etot protsess ekzogennykh predshestvennikov nukleotidov.

A dependence was studied between the level of calcium absorption and the content of calcium-binding protein in the small intestine of D-hypovitaminous chickens to whom different exogenic precursors of nucleotides (potassium orotate or guanine) were administered at different stages of the response (0-72 h) to a single administration of vitamin D3 in a dose of 500 MU. It is established that under the effect of potassium orotate or guanine the degree of discrepancy between calcium adsorption and the content of Ca2+-binding protein increases at all stages under investigation. The results of the mathematical analysis of the experimental data which is based on the supposition of the linear dependence between the level of absorbed calcium and Ca2+-binding protein evidence for a considerable rise in the excess amount of the latter, which possibly does not take part in cation absorption, in the chickens who were administered nucleotide exogenic precursors. The data obtained show that the amount of calcium-binding protein is not a limiting factor in the mechanism of vitamin D3 stimulation of calcium absorption in the chicken small intestine.

[Mechanism of action of ecdysterone in chickens with D-hypovitaminosis. Early phase of effect]. / Izuchenie mekhanizmov deistviia ékdisterona u tsypliat s D-gipovitaminozom. Ranniaia faza deistviia.

Early action of ecdysterone in enterocytes of thin intestine of chickens with experimental D-hypovitaminosis has been studied with the aim to find out molecular mechanisms of its action. Changes in biosynthesis of nucleic acids accompanied with normalization of Ca translocation, biosynthesis of proteins, lipids and nucleic acid precursors (purine and pyrimidine nucleotides) as well as oxypurines were observed over the time interval (0-2 h) after ecdysterone administration. Multifunction character of the early action of ecdysterone indicates to the intermediary mechanism of gene activation by ecdysterone and provides for the availability of the secondary mediator for realization of its biological action in enterocytes.

[Effect of ecdysterone on the metabolism of purine and pyrimidine nucleotides in chicken tissues]. / Vliianie ékdisterona na obmen purinovykh i pirimidinovykh nukleotidov v tkaniiakh tsypliat.

Hormonal amounts (10(-7) M) of ecdysterone phytoecdysteroid have been studied for their effect on the metabolic processes in the chicken tissues. Changes in the blood serum parameters which characterize the state of the integral systems of mineral, lipid, protein and nucleotide metabolism as well as the dynamics of metabolism of nucleic acids and their precursors--purine and pyrimidine nucleotides in the tissues with actively functioning protein-synthesizing system prove that the biological effect of ecdysterone after its introduction to the bird organism manifests both in the non-genome early phase of the action (0-1 h) and in the late genome phase (24-72 h), i.e. in analogy to the time of response to the introduction of classical steroid hormones. It is supposed that polyfunctional early effect of ecdysterone is mediated by its action on one of universal transmembrane signal systems of the cells--adenylate cyclase or polyphosphoinosite one, while the presence of the expressed changes in metabolism of nucleic acids in the late phase unambiguously indicate to its action in the bird organism as that of a typical steroid hormone.

[Modulation of intracellular pools of cyclic purine nucleotides by biologically active oxysterol-ecdysterone and vitamin D3]. / Moduliatsiia vnutrishn'oklitynnykh puliv tsyklichnykh purynovykh nukleotydiv biolohichno aktyvnymy oksysterynamy-ekdysteronom ta vitaminom D3.

The dynamics of changes in the level of cyclic purine nucleotides was studied in the tissues of rats with experimental D-hypovitaminosis in the range of 5 min-24 h after administration of hormonal doses of polyhydroxylated ecdysterone hydroxysterolne and predecessor of 1.25-dihydroxycholecalciferol (calcitriol)--vitamin D3. The early sharp increase of intracellular pools cEMP in the liver tissue, small intestine, brain and heart mucosa was observed. It precedes the modulation of nucleic acid biosynthesis and supposes activation of the effect of oxysterenes of G-signal (phospholipid) system in the early phase at the expense of interaction with receptors exposed on plasmatic membrane of the cells.

[Mechanisms of the early effect of biologically active hydroxysterols: calcitriol and ecdysterone. Modulation of intracellular pools of arachidonic acid and products of its oxidative metabolism]. / Mekhanizmy rannoï diï biolohichno aktyvnykh oksysteryniv: kal'tsitryolu ta ekdysteronu. Moduliatsiia vnutrishn'oklitynnykh puliv arakhidonovoï kysloty ta produktiv ïï okysniuval'noho metabolizmu.

It is established that ecdysterone (20-hydroxyecdysone) in concentration of 10(-8) M and calcitriol (1 alpha, 25-dihydroxyvitamin D3) in concentration 10(-12) M evoke in the studied tissues-targets of rats (brain, heart, liver, intestine mucosa) rapid (0.5-2 min) transit (2.5-4 times) increase of the intracellular pools of arachidonic acid, eicosanoid--total peptidoleukotrienes C4/D4/E4 (2.5-6 times), and, especially prostaglandins--stable metabolite prostacyclin 6-ketoprostaglandin F1 alpha (10-400 times). Ecdysterone in concentration 10(-11)-10(-9) inhibits arachidonic cascade in the brain and heart. Activation of arachidonic cascade and further increase of biosynthesis of cGMP are considered as a single negative link of autoregulation of activity of phospholipid-dependent signal system of cells and ecdysterone as a possible efficient modulator intracellular pools of eicosanoid in the tissues of warm-blooded animals.

[Purine nucleoside cycles in chicken tissues]. / Purinnukleozidnye tsikly v tkaniakh tsypliat.

The activity of enzymes catalyzing the reactions of successive degradation to the IMP and GMP bases as well as reactions of the reutilization and degradation of the hypoxanthine and guanine bases in the chicken liver and spleen is determined. The passage rate of [8-14C]hypoxanthine label through IMP and [8-3H]guanine label through GMP is studied together with the metabolism intensity of adenine-hypoxanthine-, xanthine- and guanine-containing components and labelled acid of the acid-soluble fraction of the test tissues in experiments in vivo. The results obtained evidence for functioning of conjugated ways of hypoxanthine- and guanine-derivatives in the so-called nucleoside cycles in the chicken tissues, the activity of the guanosine cycle (GMP----guanosine----guanine----CMP) in the liver being higher than that in inosine one (IMP----inosine----hypoxanthine----IMP), whereas in the spleen, vice versa, the activity of the metabolism of hypoxanthine derivatives is higher than that of guanine derivatives.

[Characteristics of purine nucleotide metabolism and GMP-reductase activity in chicken tissues]. / Osobennosti obmena purinovykh nukleotidov i aktivnost' GMP-reduktazy v tkaniakh ptits.

The [3H]guanosine and [3H]guanine label is shown to be distributed unevenly in the purine components of chicken tissues. 60 min after isotope administration about 80% of radioactivity is localized in xanthine and uric acid in the liver and duodenum, that agrees with high activity of purine nucleoside phosphorylase (EC 2.4.2.1) and guanine deaminase (EC 3.5.4.3). At the same time over 50% of label is found in the spleen in adenine nucleotides of the pool, RNA as well as in hypoxanthine and only 20% in oxypurines. Such a distribution of the label is in direct correlation with the activity of GMP-reductase (EC 1.6.6.8) catalyzing the reduction deamination of GMP in IMP.

[Effect of potassium orotate on metabolism in chickens under different provision with vitamin D3]. / Vliianie orotate kaliia na obmen veshchestv u tsypliat pri pazlichnoi obespechennosti ikh vitaminom D3.

Vitamin D3 induction of the vitamin-D3-dependent proteins biosynthesis and certain indexes characterizing the rachitic state in animals were studied as affected by potassium orotate. Introduction of potassium orotate to the rachitogenic diet in combination with vitamin D3 is shown to normalize the processess of bone mineralization and to increase intensity of chicken growth. In duodenum mucosa with rachitis orotate normalizes the activity of alkaline phosphatase, raises the content of calcium-binding protein and stimulates the latter formation after administration of vitamin D3; orotate favours redistribution of the purine and pyrimidine nucleotides content. Radioactive orotic acid is subjected to intensive metabolism in mucosa and is converted to nucleotides of pool and RNA.

C(27)-steroid hormones calcitriol and ecdysterone activates hydrolysis of neutral lipids--cholesterol esters and triacylglycerols--in its early pregenomic phase of action. / C(27)-steroïdni hormony kal'tsytriol ta ekdysteron aktyvuiut' hidroliz neitral'nykh lipidiv--efiriv kholesterolu ta tryatsylhlitseroliv--u rannii dohenomnii fazi diï.

We have examined in vivo capacities of two C27-steroid hormones-phytoecdysteroid ecdysterone (20-hydroxyecdysone, 10(-8)M) and calcitriol (1 alpha, 25-dihydroxyvitamin D3, 10(-12)M), as a modulators of neutral lipids hydrolysis in the brain and heart cells. Severe lines of evidence indicate that both hormones may acts as positive regulators of cholesterol esters and triacylglycerol hydrolysis in early (0.5-30 min) pregenomic phase of its actions, yielding known (DAG, free polyunsaturated fatty acids) and possible (free cholesterol) lipid second messengers.

The C(27)-steroid hormones calcitriol and ecdysterone in the early phase of its action activates hydrolysis of phosphatidylcholines in target animal tissues. / C(27)-steroïdni hormony kal'tsytriol ta ekdysteron u rannii fazi diï aktyvuiut' u tkaninakh-misheniakh tvaryn hidroliz fosfatydylkholiniv.

We have examined in vivo the capacities of two C27-steroid hormones--phytoecdysteroid ecdysterone (20-hydroxyecdysone, 10(-8)M) and calcitriol (1 alpha, 25-dihydroxyvitamin D3, 10(-12)M), as a modulators of phosphatidylcholine hydrolysis in the brain and heart cells of rats. Sever lines of evidence indicate that both hormones may acts as positive regulators of membrane phosphatidylcholine hydrolysis by phospholipases A2, C and D in early (0.5-30 min) membrane phase of its actions, yielding known lipid second messengers (diacylglycerol, phosphatidic acid) and soluble products, that may acts as second messengers (choline,choline phosphate, glycerophosphocholine).

[In vitro study of the early membrane effects of C27-steroid hormone ecdysterone, immobilized on the nanodispersed magnetite surface]. / Doslidzhennia in vitro rannikh membrannikh efektiv C27-steroïdnoho hormonu ekdysteronu, immobilizovannoho na poverkhni nanodyspersnoho mahnetytu.

The object of the study was to reveal the existence of receptor interactions of C27-steroid hormone ecdysterone with the surface of various cells (liver and spleen macrophages, thymus and spleen lymphocytes, erythrocytes and hepatocytes from rat organs) in experiments in vitro using ecdysterone immobilized on the nanodispersed iron surface (which did not penetrate the cell during observation) and the model of fast (during 15 s) activation of phospholipid signal system with ecdysterone. The parameters, which are characteristic of cell phospholipid signal system activation, were evaluated (the concentrations of free arachidonic acid, diene conjugates, tromboxane B2 and leukotriene C4) and its alteration in response to free and immobilized ecdysterone introduction in suspensins of intact cells and cells with the surface modified in vivo by pre-injection of antigen (human erythrocytes). Concurrent binding of immbilized ecdysterone with intact rat cells in the presence of free ecdysterone was also investigated. It was established that there are high-affinity ecdysterone-specific binding sites on the plasma membranes of all cells investigated, Kd estimated according to two different methods varied in the region of 10(-8)-10(-7) M. The characteristic features of the changes in parameters characteristic of phospholipid system activation were revealed to be queite similar in intact cells, as well as in cells with modified surfaces, under introduction of free ecdysterone and immobilized ecdysterone, which did not penetrate into cytosole. The data obtained point to the existence of ecdysterone receptors on the surface of cells plasma membranes and are indicative of the membrane effects as mechanisms of the early pregenomic phase of cells activation by ecdysterone.

[Endothelial dysfunction in obese hypertensive patients]. / Dysfunktsiia endoteliiu u khvorykh na hipertonichnu khvorobu iz suputnim ozhyrinniam.

A total of 28 patients with stage II hypertensive disease (HD) were examined, their age ranging between 32 to 64 years. Of these, 17 subjects were diagnosed as having had alimentary obesity. The control group comprised 11 subjects with normal level of arterial pressure and body mass. Studied in the above patients were blood serum content of nitrogen oxide by determining levels of its stable metabolites NO2- and NO3- anions. HD patients presenting with normal body weight and those with obesity demonstrated significantly lower blood serum levels of anions NO2- and NO3- as compared to healthy subjects. In HD patients, obesity is accompanied by a more significant reduction in blood serum levels of NO2- and NO3-, which fact might be caused by inhibition of nitratereductase.

[Propargylglycine restores endothelium-dependent relaxation of aortic smooth muscles in old rats].

In the study we investigated the effect of blockade cystathionine-gamma -lyase (CSE), an enzyme of hydrogen sulfide (H2S) (de novo) synthesis on the endothelium-dependent relaxation of aortic smooth muscle (SM) in old rats. It has been shown that an inhibition of CSE by propargylglycine (PAG) results in restoration of a decreased ACh-induced relaxation of aorta in old rats. This effect of PAG was removed by blocking nitric oxide (NO) synthesis in the endothelial cells. Age-related changes in the levels of H2S, NO2- and enzyme activity of the constitutive synthesis of NO (cNOS) in the heart, were determined. It has been shown that PAG introduction elevates a decreased levels of HzS, NO2- and stimulates the suppressed activity of cNOS in old rats. These results suggest that PAG activates alternative ways of H2S synthesis and stimulates the constitutive synthesis of NO. These actions of PAG restore endothelial function in old rats.

[Induction of nitrosative stress in mitochondria of rats hearts in experimental ischemia-reperfusion of the brain and its correction by ecdysterone].

On the model of focal ischemia-reperfusion of the brain investigated the induction of nitrosative stress in mitochondria of rats hearts and possible mechanisms of protective action of ecdysterone. It is shown that focal ischemia-reperfusion of the brain induced in myocardial mitochondria the activation of constitutive and inducible de novo synthesis of NO by oxidation of L-arginine and not oxidative synthesis of NO through the recovery of oxidized stable metabolites of NO. Strong evidence of induction of nitrosative stress in heart mitochondria by focal ischemia-reperfusion of the brain, was a significant increase in mitochondrial pool of nitrate- and nitrite-anions and pools of nitrosothiols, that is proof of the formation and decay of peroxynitrite--a key marker of nitrosative stress. Also was observed increase in heart mitochondria by focal ischemia-reperfusion of the brain, content key regulator of de novo synthesis of NO-hydrogen sulfide and activity of inducible arginase II and, as a result, the pool of carbamide, which is also a regulator of the synthesis of NO. Previous introduction for animals herbal extract Serratsula coronata, enriched ecdysterone, reduces induction nitrosative stress in mitochondria of rats hearts under conditions of focal ischemia-reperfusion of the brain.

[Induction of oxidative stress in heart mitochondria in brain focal ischemia-reperfusion and protective effect of ecdysterone].

Based on the fact that the acute phase of ischemic stroke is accompanied by the development of heart damage, manifestations of which are oxidative stress, morphological changes in the myocardium, in the model of brain focal ischemia-reperfusion, we investigated the oxidative stress in rat heart mitochondria and possible mechanisms of cardioprotective effect of ecdysterone. Under the conditions of brain focal ischemia-reperfusion, there is an increase rate of the generation of reactive oxygen species: superoxide (*O2-) and hydroxyl radicals (*OH), pools of stable hydrogen peroxide (H2O2), accumulate products of lipid peroxidation (diene conjugates and malonic dialdehyde), as a result of activation xanthine oxidase (marker uric acid), lipooxygenase (marker leukotriene C4) and cyclooxygenase (marker tromboksane B2) ways of *O2-(generating). In animals that received ecdysterone for 18 days, under conditions of brain focal ischemia-reperfusion, the rate of reactive oxygen species generation and the pools of lipid peroxidation products were decreased, and the survival of animals was increased. The obtained results support the development of oxidative stress in heart mitochondria of rats, powerful antiradical properties ofecdysterone, its cardioprotective effect, in conditions of brain focal ischemia-reperfusion.

[Biochemical mechanisms of the cardioprotective effect of the K(ATP) channels opener flocalin (medicinal form) in ischemia-reperfusion of myocardium].

In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) of myocardium it was investigated changes of biochemical processes in arterial blood at intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible mechanisms of cardioprotective action offlocalin, which prevented the opening of a mitochondrial permeability transition pore (MPTP) and inhibition of apoptosis induced by it. They consist, from one side, in activating of the constitutive de novo biosynthesis of nitric oxide by cNOS, from other side, in suppression of inducible nitric oxide de novo synthesis by iNOS in such way to prevent the formation of toxic peroxynitrite by co-operation of surplus nitric oxide with superoxide anion, thereby limits the generation of toxic active forms of nitrogen (*NO2) and oxygen (*OH). The first effect of flocalin takes place due to limitation the degradation of L-arginine by arginase which keeps substrat for cNOS, second--due to the inhibition of superoxide generation, in particular, by xanthine oxidase (marker uric acid), lipoxigenase (marker LTC4) and cyclooxygenase (marker TxB2). Because LTC4 have coronaroconstrictory, arrhythmogenic and chemoattractory properties in the conditions of myocardial ischemia, inhibition of its production both with superoxide generation (markers H2O2 and diene conjugates) may be the another mechanisms of flocalin's cardioprotection. Powerful antiischemic action of flocalin (marker nitrite anion) as the mechanisms of cardioprotection is possible as well as inhibition of ATP and GTP degradation (marker hypoxanthine+xanthine+inosine levels in the blood) and, possibly, stimulation ofhaem degradation by haem oxygenase (markers total bilirubin and Fe in the blood). Diminishing content of free arachidonic acid in arterial blood can testify inhibition of cellular membranes phospholipides degradation by phospholipase A2 as a result of flocalin cardioprotection.

[Magnetic-laser influence on the system of nitric oxide and contractile activity of smooth muscles of rat aorta under hypertension].

The study was conducted on three groups of rats: Group I included Wistar rats with normal blood pressure (first control group); group II - rats with genetically determined hypertension (second control group); group Ill - rats with genetically determined hypertension under the influence ofmagnetic-laser power (study group). For the low-intensively magnetic-laser influence (MLI) we have used device MIT-MT, Ukraine, which was designed for the treatment of low-frequency magnetic field using optical flow blue and red ranges of spectrum. The MLI duration was 15 minutes for the blue range, and 25 minutes for the red one. Biochemical studies included the determination of the activity of isoenzymes of NO-synthase: constitutive (cNOS) and inducible (iNOS), the content of free hemoglobin, stable metabolites of NO, namely nitrite - (NO2(-)) and nitrate - (NO3(-)) anions, resistance to acid hemolysis of red blood cells. The contractile activity of smooth muscles of the aorta was measured. We found that magnetic-laser exposure of rats with genetically determined hypertension in the red (630 nm) and blue (470 nm wavelength) optical range even after a single session leads to an increased synthesis of nitric oxide in the blood plasma. Our data sindicate that the most effective in the intensification of endogenous nitric oxide (increase of NO2(-) and reduction of NO3(-)) and endothelium-dependent responses of aorta in rats with genetically determined hypertension was a ten-day course of the magnetic-laser exposure in the optical flow of the blue spectral range. Also, after 10 sessions of magnetic-laser exposure in rats from the above specified spectrum a stabilization of erythrocyte membranes was observed.

[The changes of metabolism in myocardium at ischemia-reperfusion and activating of the ATP-sensitive potassium channels].

In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) it was investigated the changes of biochemical processes in the different areas of heart (intact, risk and necrotic zone) during intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible cardioprotective mechanisms of flocalin action at ischemia-reperfusion conditions: the preservation of sufficient levels of de novo (by cNOS) NO synthesis, an inhibition of de novo (by iNOS) and salvage (by NADH-dependent nitratreductase) NO synthesis, an inhibition of L-arginine degradation by arginase, an inhibition of oxidizing metabolism due to limitation of ROS and RNS generation, inhibition of free arachidonic acid and eicosanoids synthesis, inhibition of ATP and GTP degradations and, possibly, stimulation of protective haem degradation. These changes may prevent formation of toxic peroxynitrite and suggest the possibility of participating in flocalin-mediated cardioprotective effects of warning a mitochondrial permeability transition pore (MPTP) opening and inhibition of apoptosis and/or necrosis of cardiomyocytes induced by it.