Changes in the profile of NO synthases affect coronary blood flow autoregulation and myocardial contractile activity during restraint stress in rats.

The efficiency of autoregulation of the coronary blood flow and contractile activity of the myocardium in the presence of inhibitors of constitutive and inducible NO synthases was studied in rats exposed to 6-h restraint stress. Intracoronary administration of S-methylisothiourea (10 µmol/liter), but not L-NAME (60 µmol/liter) fully prevented post-stress increase in the volume coronary blood flow rate, intensity of heart perfusion, and reduction of ventricular developed pressure at all levels of perfusion pressure. Real-time PCR showed 6-fold increased expression of inducible NO-synthase mRNA in the heart tissue against the background of unchanged expression of neuronal and endothelial NO synthases and 2-3-fold elevated content of transcripts of stress-inducible genes Hspa1a and Hspbp1. It was shown that the hypotension of coronary vessels and reduced contractile function of the myocardium are related to NO production by inducible NO synthase in endotheliocytes of coronary vessels and cardiomyocytes.

[The special role of inducible NO-synthase in the mechanism of rat coronary vessels tone regulation during immobilization stress, combined with diabetes mellitus].

Persistent hyperglycemia after intraperitoneal injection of streptozocin (50 mg/kg) to the rats prevents decrease of coronary vessels myogenic tone and myocardial contractility during subsequent 6-hr immobilization stress. The intensity of these abnormalities in all experimental groups is significantly reduced with the supplement of the perfusate with inducible NO-synthase (iNOS) selective blocker S-methylisothiourea. Similar rise of the NO2-/NO3- concentration in the rats' blood after "stress", "diabetes mellitus" and "diabetes mellitus + stress" combines with an increase of iNOS transcripts in the myocardium to 6, 5.8 and 51 times compared with control. These data testify to a substantial modification of the cells from coronary vessels in the presence of marked and persistent hyperglycemia. Inducible NO-synthase uncoupling caused by excessive formation of the reactive oxygen and nitrogen species in the myocardium may be one of number mechanisms responsible for such phenotypic vascular alteration in the "stress + diabetes mellitus" group.

Functional activity of BK(Ca) channels in coronary vascular smooth muscle cells during combined exposure to hyperglycemia and stress.

The effect of tetraethylammonium in a dose of 1 mM (inhibiting functional activity and preventing opening of BKCa channels) was least pronounced during restraint stress. The influence of this agent was more significant in animals exposed to 14-day hyperglycemia alone or in combination with restraint stress. Therefore, hyperglycemia and stress (individual or combined exposure) significantly inhibit functional activity of BKCa channels in smooth muscles of the coronary vessels. Our results suggest that the development of hyperglycemia is realized via pathogenetic mechanisms of vascular injury in the heart (similarly to stress conditions). Permanent increase in blood glucose level and 6-h immobilization probably induces nonspecific post-stress abnormalities in channel function.

[Changes in endothelium-dependent dilation and α1-adrenoreactivity of rat aorta caused by inducible NO-synthase inhibition after motor activity restrictions].

The aim of work was to study the influence of the highly selective blocker of the inducible NO-synthase (iNOS) of S-methylthiourea on the alteration of the endothelium-dependent vasodilation and α1-adrenoreactivity of the isolated rat aortic rings which underwent a short-term restriction of physical activity. The experiments were carried out on rat aortic rings preparations from female-rats bathed in Krebs-Henseleit solution, bubbled with 95% O2 and 5% CO2 and contracting in isometric mode. Endothelium-dependent dilation was caused by cumulative addition of acetylcholine (10-(10)-10(-4) M) after phenylephrine precontraction(10(-6) M). Adrenoreactivity was assessed through the response to increasing concentrations of α1-adrenergic receptor agonist. The 60-minute immobilization stress, characterized by the increase of the relative weight of the adrenal glands by 19.5%, the concentration of glucocorticoids (twice as much), of NO2/NO3 (stable NO degradation products) by 35%, the reduction in the level of thyroxine (by 16%), triiodothyronine (by 10%) and the increase in thyrotropic hormone by 45%, interleukin-1b (twice as much) and the appearance of tumour necrosis factor alpha in the blood serum, was accompanied by the two types of reaction of isolated aortic rings to acetylcholine and phenylephrine. The first one was expressed in the enhancing of acetylcholine-induced dilation of isolated aortic rings and the reduction of its response to α1-adrenergic stimulant phenylephrine. The second one showed a decrease in the response of isolated aortic rings to acetylcholine and enhancing the response to phenylephrine. But both of these reaction types were eliminated by using highly selective inducible NO-synthase inhibitor with S-methylisothiourea. However, it was differently directed with a different type of reaction. Taken together, these results suggest that the iNOS is formed in the cells of rat aorta under short-term stress. In some cases it can be a source of a large number of NO (coupling state of iNOS), and in another contribute reduce its bioavailability (uncoupling state of iNOS).

Adaptation to short-term stress exposures prevents poststress dysfunction of calcium-activated potassium channels in coronary vessels.

Adaptation to short-term stress exposures prevents immobilization stress-induced decrease in functional activity of BK(Ca)channels in smooth muscle cells of coronary vessels developing against the background of NO overproduction and increase in the relative content of oxidized glutathione (change in redox state of the glutathione system). The protective antistress mechanisms of adaptation probably include prevention of NO overproduction by inducible NO synthase and maintenance of dependent glutathione redox state functional activity of BK(Ca)channels in smooth muscle cells of coronary vessels.

[Nitrogen monoxide and functional activity of large conductance Ca-activated potassium channels in coronary vessels in rats with limited motor activity].

The study was aimed at the detection of endothelial factors contribution and nitric monoxide produced by constitutional and inducible NO-synthase in regulation of Ca-activated potassium channels activity of high conductivity in immobilization stress. The experiments were conducted in isolated rat-female hearts after retrograde perfusion with Krebs-Henseleit solution bubbled with 95% O2, 5% CO2 at 37 degrees C. To study BK(Ca)-channels role in the regulation of coronary vessels tone and contractile myocardial function BK(Ca)-channel blocker tetraethylammonium (ImM/l concentration) (Sigma, USA) was added in perfused solution. The obtained results justify: firstly, the non-selective blockade of constitutional and inducible NO-synthase L-NAME accompanied by the reduction of coronary flow volume velocity to control values level in the group of animals underwent to stress; second, selective blocker of inducible NO-synthase S-methylisotiourea completely eliminates evoked by stress hypotonia of cardiac vessels and reduction of contractile activity of left ventricle in stressed rats; thirdly, stress reduces the functional activity of Ca-activated potassium channels and the removal of endothelium or blockade of nitric monoxide completely it restores.

[Adaptation to short-term stress exposure increases the activity ATP-sensitive potassium channels in the smooth muscle cells of coronary blood vessels].

The study was aimed at the effect of prior adaptation to short-term stress exposure on changes in K(ATP)-channel activity induced by severe stress and the dependence of the changes on the state of endothelium which plays important role in autoregulation of the coronary flow and myocardial contractility. Experiments were conducted on isolation hearts of female rats. At the first step of experiment, the heart was perfused by Krebs-Henseleit solution; at the second step, the heart was perfused with the same solution in which glibenclamide (1 microM), glibenclamide with saponin or N(omega)-nitro-L-arginine (60 microM) methyl ether was added. During the experiment, the perfusion pressure was stepwise elevated from 40 to 120 mm Hg with 20 mm intervals (coronary autoregulation). Adaptation to short-term stress prevented development of stress-specific myocardial hyperperfusion (increased volumetric velocity of coronary flow against the background of decreased myocardial contractility) and the reduction of coronary dilation reserve. In coronary vessels of adapted rats, as distinct from control rats, basal glibenclamide-sensitive functional activity of K(ATP)-channels depended on presence and functional activity of endotheliocytes; it was reduced in presence of endothelium and increased after de-endothelization or NO synthase inhibition. In all experimental groups, the increase in glibenclamide-sensitive functional activity of K(ATP)-channels induced by NO synthase inhibition more than twice as great as after the endothelium denudation. In adapted animals, stress did not decrease the functional activity of K(ATP)-channels and their activity slightly depended on presence of endotheliocytes. In addition, the elevation of their functional activity characteristic of adaptation and evident after endothelium removal has vanished. Therefore adaptation to short-term stress exposure is associated with a potential increase in basal activity of K(ATP)-channels which enhances the potency of vascular dilation system and may apparently reduce the risk of high vascular tone when such important local regulatory system as the NO system is damaged.

[Participation of ATP-sensitive potassium channel in autoregulation of coronary blood flow under the condition of limited motor activity].

The aim of study involved detection of the effect of the K(ATP)-channel blocker glibenclamide on autoregulation of coronary flow, the expression of reactive hyperemia, the value of coronary dilatation reserve, and the myocardial contractile function in isolated rat hearts after a 6-hour immobilization stress. The experiments have been performed on 64 isolated rat hearts (female): into the cavity of left ventricle, a latex balloon connected with electromanometer has been introduced. Every experiment consisted of 2 stages. The heart has been perfused by Krebs-Henseleite solution in the first stage, and in the second stage--by the same solution with glibenclamide (1 mkM) or its combination with verapamile (10(-)6 M) or saponin (44 mcg/ml of coronary flow within 2 minutes) added to it. During the experiment, the perfusion pressure has been elevated step by step from 40 to 120 mm Hg with 20 mm Hg steps (coronary autoregulation). In rats after immobilization, the glibenclamide effect on cardiac vessel tone and expression of maximal hyperemic coronary flow (in contrast to its influence on myocardial contractile function) is lower than in control and depends on endotheliocyte presence which suggests an important role of endothelium in maintaining cardiac vessel smooth cell activity of K(ATP)-channels inhibited under the stress condition. After immobilization stress, the role of endothelium in the reactive hyperemia origin was enhanced, that of the K(ATP)-channels was reduced. The general activity of both mechanisms of tone regulation of cardiac vessels remains the same as in control. This suggests that the K(ATP)-channels as nitric monoxide and eicozanoids are the local system of myogenic tone regulation of the rat cardiac vessels; that the rat immobilization inhibits the activity KATp-channel's smooth cells of coronary vessels and creates a marked dependence of their activity on endothelium presence.

[The role of superoxide anions in regulation of coronary vessel tone and myocardial contractile function in changed redox-state of the myocardium].

The study relates to the character of tirone effect (chemical trap of superoxide--anions) on regulation of coronary vessel tone and myocardial contractile function in normal and changed cell redox-state of coronary and cardiac vessels. The experiments were performed in 64 female Wistar rats (180-320 g). The coronary blood flow and myocardial contractile junction were studied in isolated heart preparations. To determine the role of superoxide-anions in regulation of coronary vessel tone, tirone was added to the perfusion solution (4,5-dihydroxy-1,3-benzene disulfonic acid, 10 mm, Sigma USA). Preliminary injection of N-acetyl-L-cysteine evoked a 16 % increase, whereas injection of L-buthionine-(S,R)-sulfoximine reduced concentration of nonprotein thiol group in the myocardium and erythrocytes of experimental animals by 37%. The influence of superoxide anions on the cardiac vessel tone and myocardial contractile function was due to nitric monoxide participation the concentration of which increased in binding of superoxide anions and was directly dependent on concentration of sulfhydrilis groups in the cardiac cells. The oxygen active forms and cellular redox-state seem to play an important role in the regulation mechanisms of the coronary vessel tone and myocardial contractile function.

[Indices of cholecystokinin secretion, vegetative regulation of heart rate and anxiety level in patients with a motor dysfunction of the gallbladder]. / Pokazateli sekretsii kholetsistokinina, vegetativnoi reguliatsii serdechnogo ritma i uroven' trevozhnosti u bol'nykh s motornoi disfunktsiei zhelchnogo nuzyria.

We examined 36 patients with biliary diseases (14 men and 22 women) at the age of 19-62 (19 patients with chronic non-calculous cholecystitis, 11 patients with cholelithiasis, and 6 patients with gallbladder dysfunctions) as well as 14 practically healthy people from the control group. Methods of the study included dynamic sorbite echography of the gallbladder, determination of serum cholecystokinin by the radioimmunoassay (RIA) as well as examination of variability of the cardiac rate and anxiety level. Patients with a motor dysfunction of the gallbladder suffer from disorders of the correlation between cholecystokinin secretion, the functional state of the vegetative nervous system and the nature of the motor and evacuation function of the gallbladder.

[Indicators of cholecystokinin secretion, vegetative regulation of cardiac rhythm, and level of anxiety in patients with motor dysfunction of the gall bladder]. / Pokazateli sekretsii kholetsistokinina, vegetativnoi reguliatsii serdechnogo ritma i uroven' trevozhnosti u bol'nykh s motornoi disfunktsiei zhelchnogo puzyria.

We examined 36 patients with biliary diseases (14 males and 22 females) at the age of 19-62 (19 with chronic acalculous cholecystitis, 11 with cholelithiasis, 6 with gall-bladder dysfunction) and 14 practically healthy people as a control group. The examination methods comprised dynamic gall-bladder ultrasonography with Sorbitol, serum cholecystokinin detection with the help of radioimmunoassay, cardiac rate variability and anxiety level tests. Patients with motor gall-bladder dysfunction have derangements of interrelations between cholecystokinin secretion, the functional state of the vegetative nervous system, and the nature of the gall-bladder motor evacuation function.

[Nitric oxide storage in the cardiovascular system]. / Deponirovanie oksida azota v serdechno-sosudistoi sisteme.

Nitric oxide (NO) is a highly reactive substance with short lifetime. In conditions of a living organism NO can be bound by the complexes used for transport and intracellular storage of NO. The main biological forms of NO store include S-nitrosothiols and dinitrosyl iron complexes capable of interconversion. The NO store formed by these complexes in the vascular wall, on the one hand, provides for protection from excessive free NO after its overproduction and, on the other hand, can be an additional NO source when it is deficient. Apparently, the efficiency of NO storage is genetically determined and corresponds to the inherited level of NO production in the organism. Controlled modulation of formation and dissociation of the NO store is a promising trend for further investigation.

[Changes in autoregulation of coronary blood flow in rats with various sensitivity to stress]. / K mekhanizmu razvitiia izmenenii autoreguliatsii koronarnogo krovotoka u krys s razlichnoi chuvstvitel'nost'iu k stressu.

The purpose of work: study of a role of endothelial nitric oxide in development of stress-induced changes in autoregulation of coronary blood flow in rats with various types of behaviour. The experiments were performed on isolated hearts of female-rats, in the "open field" test, depending on the type of impelling and searching activity of animals subdivided into two groups: "active" and "passive". After a 6-hour immobilization stress only in "passive" rats an increase of volumetric velocity of coronary flow; a decrease of an autoregulation index, coronary reserve against the background of intravascular pressure reduction, were found out. The blockade of nitric oxide synthesis in this group completely eliminated the stress-induced decrease of coronary vascular tone and essentially limited the caused by stress dissociation of coronary flow and the contractility function of the myocardium. In blood plasma of "passive" animals the nitrite/nitrate contents was by 55% more than of the "active" rats. After the transferred stress, in "passive" animals the nitrite/nitrate concentration in blood plasma increased by 29% and in "active" rats--by 136%; the absolute values, however, did not differ between the groups. Thus the autoregulation of coronary flow seems to be subject to action of stress in the rats showing a "passive" type of behaviour in the test "open field", and practically does not change in "active" animals; secondly, in spite of the fact that the stress-induced amplification of NO-producing function andothelium of coronary blood vessels is stereotyped in different rats, in "passive" rats, apparently, sensitivity of coronary vessels to nitric oxide is higher than at "active" those.

[Autoregulation of coronary vessels following the acute blood loss and its combination with preexisting immobilization stress]. / Autoreguliatsiia koronarnykh sosudov posle ostroi krovopoteri i ee sochetanii s predvaritel'no perenesennym stressom.

A 6-hour immobilization stress and a 2-hour posthaemorrhagic hypotension caused elevation of the coronary flow, deterioration of coronary autoregulation, decreasing of the coronary dilation reserve, and diminishing of the left ventricular pressure. In the "stress + haemorrhage" group, the changes of the coronary vessels autoregulation ability were less obvious. Concentration of NO3-/NO2- in the blood serum was elevated after stress. Inhibition of the NO-synthase decreased the coronary flow. Thereupon, a pre-existing immobilization stress alters the coronary vessels tone.

[The mechanism of stressor suppression of the contractile function and reactivity of aortic smooth muscle]. / K mekhanizmu stressornogo ugneteniia sokratitel'noi funktsii i reaktivnosti gladkoi myshtsy aorty.

Blockade of the NO synthesis did not change the stressor augmentation of the acetylcholine-induced endothelium-dependent relaxation. The immobilisation stress seems to suppress the contractile function and the reactivity of the aorta smooth muscle reactivity as the result of augmentation of the NO basal production in endotheliocytes.

[The mechanism of the action of low doses of thyroid hormones on the stressor-induced changes in rat vascular tonus]. / K mekhanizmu vliianiia malykh doz tireoidnykh gormonov na stressornye izmeneniia tonusa sosudov krys.

Small doses of thyroidine were found to diminish a 6-hr immobilisation-induced decrease of the mean AP and duration of a hypertension response to blockade of NO-synthasa. The thyroidine seems to act on the system of endothelial nitric oxide synthasa.

[Changes in the endotheliocyte activity of the coronary vessels under the influence of stress]. / Izmenenie aktivnosti éndoteliotsitov koronarnykh sosudov pod vliianiem stressa.

The effect of saponin and blockade of the nitrogen oxide upon the coronary endothelium was found in rats subjected to a 6-hrs stress. The damage of the endothelium and the blockade abolished the stressor-induced increase in the coronary blood flow velocity. The index of autoregulation and the latter's efficiency, however, remained the same. Preliminary administration of L-arginine prevented the effect of NG-monomethyl-L-arginine upon the coronary flow in the isolated hearts of rats subjected to stress. The post-stressor increase in the coronary blood flow seems to be due to an increase in releasing the nitrogen oxide from the coronary vessels' endotheliocytes.

[The autoregulation of the coronary flow of the isolated heart in rats after saponin damage to the endothelium]. / Autoreguliatsiia koronarnogo potoka izolirovannogo serdtsa krys posle povrezhdeniia éndoteliia saponinom.

The damage of the endothelium with saponin completely abolished the autoregulation of the coronary vessels in the Langendorff isolated heart and reduced the volume velocity of the coronary blood flow by 34% at the perfusion pressure 40 mmHg. Indomethacin and verapamil augmented the coronary flow in intact rats. All these agents decreased the distensibility of the coronary vessels by 43-49% on the average. The damage of the endothelium of coronary vessels reduced the maximum reactive hyperemia and, respectively, the coronary vasodilator reserve.

[The autoregulation of the coronary flow of the isolated rat heart after NO-synthase blockade]. / Autoreguliatsiia koronarnogo toka izolirovannogo serdtsa krys posle blokady NO-sintazy.

The experiments were performed on 67 rat hearts isolated by the Langendorff method. Perfusion pressure (PP) was stepwise increased from 40 to 120 mm Hg. It was found that NO-synthase blockade by NG-monomethyl-L-arginine (NG-MMLA) decreased volume velocity coronary flow (VVCF) at PP 40 and 60 mm Hg, autoregulation index and shifted the onset of effective autoregulation to the right. In cases of high coronary tone, caused by introduction of pituitrine, the autoregulation and its effectiveness was unchanged. The combination of NG-MMLA and pituitrine decreased the autoregulation index again. In cardiac perfusion under constant pressure, the value of maximal hyperemic coronary flow after introduction of NG-MMLA was decreased by 30-57%, and as a result the coronary reserve was decreased by 28.3%. Thus, NO, which released from endothelium of coronary vessels plays a significant role in mechanism of coronary autoregulation.