Effects of Catechin on Activity of Angiotensin-Converting Enzyme and Generation of Reactive Oxygen Species in Rat Aorta.

Activity of angiotensin-converting enzyme (ACE) and intensity of ROS generation in the aorta were studied in male Wistar rats intraperitoneally injected with a mixture of (+) and (-) stereoisomers of catechin. ACE activity in aortal segments was evaluated by hydrolysis of hippuryl-histidine-leucine; ROS generation was measured by oxidation of dichlorodihydrofluorescein. The dynamics of ACE activity and intensity of ROS generation in the aorta after catechin administration and their dependence on the catechin dose were studied. The effects of dihydroquercetin and fucoidin on the studied parameters were analyzed. Catechin increased ACE activity; the maximum increase was achieved in 3 h after administration. Catechin dose producing a half-maximum increase in ACE activity was 0.04 µg/kg. The effects of catechin on ACE activity was attenuated by dihydroquercetin and completely abolished by fucoidin. Catechin did not enhance ROS production in the aorta, and in a dose of 0.1 µg/kg even inhibited this process. It is hypothesized that isomers of catechin produce opposite effects on ROS generation in the aorta.

Blood Pressure Changes After Exposures Increasing Angiotensin-Converting Enzyme Activity and After Its Normalization with Dihydroquercetin in Male Wistar Rats.

Changes in BP and HR were assessed after exposures increasing activity of angiotensin-converting enzyme: ionizing radiation, NO synthase inhibitor (L-NAME), and dexamethasone. Effects of dihydroquercetin and angiotensin-converting enzyme inhibitor enalapril on activity of this enzyme, BP, and HR were also evaluated under these exposures. Wistar male rats were subjected to X-ray irradiation in a dose of 2.5 Gy. Angiotensin-converting enzyme activity in the aorta sections was determined by Hip-His-Leu hydrolysis. BP and HR were recorded using a non-invasive tail-cuff method and PowerLab 8/35 software. BP and HR were not altered with the increase in activity of angiotensin-converting enzyme after irradiation. In case of prolonged (7 days) treatment with NO synthase inhibitor and dexamethasone, the increase in enzyme activity was accompanied by elevation of BP and, in the case of NO synthase inhibitor, HR reduction. Dihydroquercetin normalized the enzyme activity and lowered BP, but not to the normal level. Enalapril normalized BP, increased by NO synthase inhibitor solution intake; at the same time, the angiotensinconverting enzyme activity decreased more than 2-fold in comparison with the normal.

Dihydroquercetin and Fucoidin Inhibit the Increase of Angiotensin-Converting Enzyme Activity in the Rat Aorta after Irradiation.

The time course of angiotensin-converting enzyme activity in the rat aorta after fractionated exposure to ionizing radiation and the effects of dihydroquercetin and fucoidin on this parameter were studied. Male Wistar rats were exposed to single or repeated (fractionated) X-ray radiation in a dose of 2.5 Gy at 200 kV. Activity of angiotensin-converting enzyme in aorta segments was evaluated 2 h after the last exposure by hydrolysis of hippuryl-histidineleucin substrate. Enzyme activity in the rat aorta was higher than normally after all the studied doses of fractionated exposure (2.5 Gy per fraction) with the maximum effect after the total dose of 7.5 Gy (3 fractions). Fucoidin, a blocker of endothelium receptors realizing the leukocyte adhesion to the endothelium, and flavonoid dihydroquercetin inhibiting expression of adhesion molecules in the endothelium abolished the increase in activity of angiotensinconverting enzyme in the rat aorta after single exposure; moreover, dihydroquercetin reduced significantly the effect of fractionated exposure. These data indicate that leukocyte adhesion to the endothelium is an important factor contributing to the increase of angiotensin-converting enzyme activity in the aorta.

Ionizing Radiation Enhances Activity of Angiotensin-Converting Enzyme in Rat Aorta.

We analyzed changes in angiotensin-converting enzyme activity in rat aorta at the early terms after irradiation in doses equal to one fraction dose used in tumor radiotherapy. Male Wistar rats were exposed to whole body or local (chest) X-ray irradiation (200 kV, 1-7.5 Gy). The activity of the enzyme in aorta segments was measured in 1-48 h after irradiation by hydrolysis of hippuryl-histidine-leucine. Activity of angiotensin-converting enzyme in rat aorta was increased 1-24 h after whole body irradiation in a dose of 2.5 Gy with a peak in 2 h after exposure. After local exposure, enzyme activity also increased in 2 h, but returned to the control level in 24 h. In 2 h after whole-body irradiation in doses >2.5 Gy, the increase in enzyme activity was less pronounced and after exposure to 7.5 Gy, it did not differ from the control. During local exposure, the effect did not decrease with increasing the irradiation dose. The fraction of blood monocytes adherent to plastic in rats subjected to whole body irradiation decreases with increasing the dose. In rats subjected to local irradiation in a dose of 7.5 Gy, monocyte adhesion to plastic did not differ from the control. These data suggest that the increase in activity of angiotensin-converting enzyme in the aorta after irradiation is determined by monocyte adhesion to the endothelium; the decrease in this effects with increasing the dose can be explained by radiation damage of monocytes.

Lipoxygenase Inhibitors Nordihydroguaiaretic Acid and Fungus Lecanicillum lecanii Extract Induce Death of Lymphoid Leukemia Cells.

We studied the effect of lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and fungus Lecanicillum lecanii extract on lymphatic leukemia P388 cells. The cells grown in the abdominal cavity of DBA2 mice for 7 days were transferred into a nutrient medium. The effect of lipoxygenase inhibitors was evaluated by changes in cell number, trypan blue staining, nucleus damage, and changes in cell distribution by DNA content after 22-h incubation. NDGA and fungus extract induced apoptotic death of lymphatic leukemia cells, which was seen from nucleus damage and reduced DNA content in cells. IC50 for NDGA and fungus extract was 0.66 and 5.5 µg/ml, respectively.

Dihydroquercetin Does Not Affect Age-Dependent Increase in Blood Pressure and Angiotensin-Converting Enzyme Activity in the Aorta of Hypertensive Rats.

We analyzed changes in angiotensin-converting enzyme activity in the aorta of hypertensive SHR rats against the background of age-related BP increase (from week 7 to 14) and the effect of dihydroquercetin on BP rise and angiotensin-converting enzyme activity. Normotensive WKY rats of the same age were used as the control. BP and activity of angiotensin-converting enzyme in the aorta of SHR rats increased with age. Dihydroquercetin in doses of 100 and 300 µg/kg per day had no effect on the increase of these parameters; dihydroquercetin administered to 14-week-old WKY rats in a dose of 300 µg/kg reduced activity of the angiotensin-converting enzyme. Thus, the early (7-14 weeks) increase in BP and angiotensin-converting enzyme activity in the aorta of SHR rats was not modified by flavonoids (dihydroquercetin) in contrast to other rat strains and humans, which is indicative of specificity of hypertension mechanism in SHR rats.

Taxifolin and Fucoidin Abolish the Irradiation-Induced Increase in the Production of Reactive Oxygen Species in Rat Aorta.

We studied changes in ROS content in the aorta of Wistar rats at early terms after irradiation in doses equal to single fraction used in tumor radiotherapy and the effects of taxifolin and fucoidin, blockers of leukocyte adhesion to endothelium, on ROS content. Male rats were exposed to X-rays (200 kW) in doses of 1-7.5 Gy. ROS production in aorta segments was measured in 1-48 h after irradiation by dichlorodihydrofluorescein oxidation. The content of ROS in the aorta of rats exposed to radiation in doses of 1-2.5 Gy increased in 1-24 h after irradiation, the peak ROS content was found in 2 h after irradiation. Taxifolin (100 µg/kg dihydroquercetin once a day with drinking water) and fucoidin (10 mg/kg, i.v.) abolished ROS accumulation. The content of ROS in rat aorta increased in 1-24 h after irradiation in doses used for tumor radiotherapy and this increase can be determined by leukocyte adhesion to the endothelium.

[Screening of Organisms Producing Inhibitors of 15-Lipoxygenase Among Micromycetes.]

The effects of extracts from the mycelium of Lecanicilium lecaniiNo.169, Beauveria fellina No.7 and Beauveria bassianaNo.15 on the activity of 15-lpoxygenase (15-LO) recovered from rat reticulocytes was investigated. The activity of 15-LO was determined by oxidation of linolic acid. The extract from the mycelium of the fungal complex was shown to inhibit 15-LO (IC50 of 12 mcg/ml). The inhibitory effect of the combined extract on 15-LO was due to the substances recovered from Lecanicilium lecanii No.169. The extract fractions responsible for the activity were determined and the compounds containing the fractions were identified. They proved to be 10 - 4-hydroxybenzoic acid and 4-hydroxybenzyl alcohol and genistein, a flavonoid from fraction 11. The possible role of the inhibitory effect of the compounds on 15-LO in the antiatherosclerotic activity of the fungal extract is discussed.

[Change of Tumor Cell Multiple Drug Resistance Inhibitory Activity of Oligomycins in Complexes with Lithium and Zinc].

Oligomycins and their complexes with lithium and zinc were shown to be less active vs. cyclosporin A in inhibition of transport proteins responsible for multiple drug resistance of lymphoid leukosis P388VR cells, while certain oligomycin complexes were tens or hundreds times more active than cyclosporin A by inhibition of transport proteins in another type of tumor cells, i.e. human larynx cancer Hep-2, that makes possible the use of the oligomycins complexes with lithium and zinc for inhibition of multiple drug resistance of certain tumor types.

Camel thorn extract reduces activity of angiotensin-converting enzyme in rat aorta increased during aging and treatment with NO-synthase inhibitor.

We studied the effect of camel thorn extract on activity of angiotensin-converting enzyme in rat aorta increased in animals during aging or treatment with NO-synthase inhibitor. Intake of camel thorn extract with drinking water reduced activity of angiotensin-converting enzyme; the effect increased with increasing the dose of the extract. Angiotensin-converting enzyme activity in older rats and animals treated with NO-synthase inhibitor decreased to the values observed in young control rats at extract concentration of 0.2%. Comparison of the effects of camel thorn extract with those of flavonoid taxifolin showed that the extract was not inferior to taxifolin in preventing the early stages of aortic atherosclerosis caused by increased activity of angiotensin-converting enzyme.

Effects of high-salt diet on the activity of angiotensin-converting enzyme and generation of reactive oxygen species in rat aorta.

The dynamics of angiotensin-converting enzyme activity in the aorta and blood plasma and generation of ROS in the aorta were studied in rats subjected to two high-salt diets (0.4% and 1% NaCl solutions). During high-salt diets, activity of angiotensin-converting enzyme in the aorta progressively decreased to the minimal during week 1 and remained lower than control level for 1 month. Both diets were followed by a decrease in ROS concentration in the aorta, and this effect was more pronounced when the dosage of salt increased. ROS level in the aorta varied similarly during both diets, but the amplitude of the response was significantly higher in rats receiving 0.4% salt solution. ROS amount in the aorta during high-salt diets differed from the control level and depended from salt dosage and the duration of administration.

[Multidrug resistance increase of tumor cells at the effect of radiation and phorbol ether depends on protein kinase C and reactive oxygen species].

The effects of phorbol ether (PMA) and ionizing radiation on multidrug resistance (MDR) of human larynx cancer cells HEp-2 and the dependences of these effects on protein kinase C (PKC) activity and reactive oxygen species (ROS) production were studied. MDR was determined by transport rate of rhodamine 123 off cells and production of ROS in cells was measured by means of 2'7'-dichlorodigidrofuorescein oxidation to fluorescent 2',7'-dichlorofluorescein. ROS production was increased in cells at PMA treatment. This increase was caused by PKC dependent activation of NADPH-oxidase because the ROS increase suppressed completely with PKC and oxidase inhibitors. It was shown that tumor cell MDR was increased 16 h after PMA (100 nM) and radiation (1 Gy) treatment. The MDR increase depends on PKC activity and ROS increase in cells at both influences since PKC inhibitor and antioxidant, lipoic acid suppressed MDR increase. The obtained data are in accordance with hypothesis about the necessity of activation both signalization and stress reaction for initiation of transcription of transport protein genes which responsible for MDR of tumor cells.

[Ionic mechanisms of cardiotropic action of a new class III antiarrhythmic drug RG-2]. / Ionnye mekhanizmy kardiotropnogo deistviia preparata III klassa RG-2.

A perforated patch-clamp analysis of the effect of a novel class III antiarrhythmic agent RG-2, on voltage-dependent currents was made in rat ventricular myocytes. In these cells, RG-2 decreased delayed rectifier outward K(+) current, I(k), in concentration dependent manner with threshold concentration 0.1 microM/l. In contrast, the drug did not have significant effects on the transient outward and inward rectifier K(+) current. RG-2 in concentration dependent manner decreased Ca(2+) current (I(Ca,L)) with threshold concentration 1 microM/l, tenfold higher than threshold concentration for I(k). We can conclude that decreasing of I(k) may explain prolongation of cardiac repolarization induced by RG-2, and contribute to its antiarrhythmic action.

[Selective cytostatic and cytotoxic effects of avermectins]. / Izbiratel'noe tsitostaticheskoe i tsitotoksicheskoe deistvie avermektinov.

A natural avermectin complex, aversectin C, was shown to be capable of exerting selective cytostatic effect. It killed proliferating neuroblastoma B 103 cells but was non-toxic for differentiated cells of this culture. The activity of aversectin C was related neither to activation of the GABA alpha-receptors nor to their blocking and was at a large extent due to the action of avermectin A1, a component of aversectin C.

The regulation of L-type Ca2+ currents in cardiac myocytes of hibernating animals.

The action of isoproterenol and BAY K 8644 on voltage-dependent Ca2+ currents in isolated ground squirrel cardiac myocytes was studied in two (active and hibernating) states of the animal. In cardiac myocytes of active animals the effect of both drugs was shown to depend on the holding potential. At Vh of about -50 mV both isoproterenol and BAY K 8644 increased the Ca2+ current and their action was additive. At Vh of about -20 mV, both drugs inhibited the Ca2+ current. In cardiac myocytes from hibernating animals, isoproterenol increased the Ca2+ current at any holding potentials, while the effect of BAY K 8644 did not differ significantly from its effect on active animals. The combined action of the two drugs caused the inhibition of the Ca2+ current at high holding potentials. In terms of the two-site Ca2+ channel model, this means that one of the two pathways of channel phosphorylation is blocked in hibernating animal cardiac cells, and BAY K 8644 restores this pathway.

The regulation of L-type Ca2+ currents in rat cardiac myocytes.

The regulation of L-type Ca2+ current in isolated rat cardiac cells was studied using the perforated patch-clamp technique. A dual effect of the cAMP-dependent phosphorylation activator, isoproterenol, at different holding potentials (V(h)) was shown. The currents increased at V(h) = -50 mV and decreased at V(h) = -30 mV. A dihydropyridine agonist, BAY K 8644, and isoproterenol had an additive effect on the activation of Ca2+ channels at holding potentials close to the resting potential. The additivity was disturbed at more positive V(h). The activating effect of BAY K 8644 did not virtually change in the presence of a protein kinase blocker, H8, and a phosphatase activator, acetylcholine. The results were interpreted within the framework of a two-site phosphorylation model with two independent pathways of Ca2+ current regulation.

Effect of isoproterenol on the L-type Ca2+ current in cardiac cells from rats and hybernating ground squirrels.

The perforated patch clamp method was used to study the effect of the agonist of beta-adrenoreceptors isoproterenol on L-type Ca2+ current in cardiocytes of rats and ground squirrels in two states: active and hibernating. It is shown that isoproterenol exerts a dual effect on Ca2+ currents of rats and ground squirrels in the active state: at Vh = -50 mV, the current increases, whereas at Vh = -30 mV, it decreases. In hibernating ground squirrels, the dual effect of isoproterenol is not observed: isoproterenol increases Ca2+ current at any Vh values. The hypothesis is put forward that, during the entrance of ground squirrels into hibernation, the phosphorylation of one of the sites (not cAMP-dependent) of L-type Ca2+ channels is blocked.

Selective cytostatic and neurotoxic effects of avermectins and activation of the GABAalpha receptors.

A natural avermectin complex, aversectin C, was shown to be capable of exerting selective cytostatic and neurotoxic effects on mammalian cells. Specifically, it killed proliferating neuroblastoma B103 cells but was non-toxic for differentiated cells of this culture. The antiproliferation action of aversectin C was not inhibited by bicuculline or picrotoxin, antagonists of the GABAalpha receptors, and was partly due to the action of avermectin A1, a component of aversectin C. Aversectin C irreversibly suppressed activity of 60% neurons in medial septal slices of the rat brain. More than 55% of them were the GABAalpha- and B1-sensitive neurons whereas the rest, about 45% neurons, were the GABAalpha-insensitive and the neurotoxic effect of aversectin C was caused mainly by the B2 component.

The effect of some peptides from the hibernating brain on Ca2+ current in cardiac cells and on the activity of septal neurons.

The effects of the peptides TSKYR and DY isolated from the brain of hibernating ground squirrels on Ca2+ current were studied. TSKYR activated Ca2+ current in frog auricle fibers and in single cells from frog ventricle whereas DY blocked Ca2+ current in both preparations. In isolated rat and ground squirrel cardiocytes, TSKYR had no effect on Ca2+ current, and DY increased it. In brain slices of rat, DY blocked the activity of medial septal neurons. TSKYR increased activity of septal neurons at the initial phase, which was followed by decrease of neuronal activity.