Influence of Social Isolation Stress on Age-Related Changes in Functional Activity and Expression of Receptors of Endogenous Vasoconstrictors in Rat Aorta.

Social isolation stress was modeled by long-term isolation of 12-month-old rats in individual cages over 28 weeks. It was found that sensitization of blood vessels to the vasoconstrictor action of serotonin due to overexpression of 5HT2A-type receptor genes, as well as an imbalance in the expression level of endothelin ETA- and ETB-receptors (55 and 153%, respectively) are the early signs of vascular aging. A significant contribution to the development of age-related changes in the contractile properties of blood vessels is made by the stress component, which is manifested at the level of glucocorticoid-dependent mechanisms of regulation of gene expression. The decrease in the expression of glucocorticoid receptors caused by isolation stress leads to a decrease in the expression of the genes responsible for the synthesis of V1A-R and ATII-R and to the development of vascular hyporeactivity to the vasoconstrictor action of ATII and AVP. In the aorta of stressed rats, the α1-AR mRNA level increases by 3 times. At the same time, stress did not affect the dynamics of age-related changes in the expression of genes encoding 5HT2A-R and ETA/ETB-R.

On the Mechanism of the Cardioprotective Action of σ1 Receptor Agonist Anxiolytic Fabomotizole Hydrochloride (Afobazole).

Original translational rat model of chronic heart failure provoked by experimental anterior transmural myocardium infarction was employed to examine the preventive action of anxiolytic Afobazole (15 mg/kg/day administered intraperitoneally during the first 15 days after coronary occlusion) on the development of the heart failure assessed in 3 months after infarction. Afobazole prevented the development of pathologic remodeling of the myocardium, maintained its inotropic function, and decreased the plasma level of brain natriuretic peptide known as a biochemical marker of chronic heart failure. In the myocardium, Afobazole down-regulated overexpression of the genes induced in chronic heart failure and assessed by corresponding RNA levels, which code angiotensin (AT1A-R), vasopressin (V1A-R), and glucocorticoid (GR) receptors as well as Epac2 protein. The revealed biochemical changes are consistent with the data on cardioprotective action of Afobazole.

Epac Proteins and Calmodulin as Possible Arrhythmogenesis Trigger in Alcoholic Cardiomyopathy.

The expression of Epac proteins (exchange protein directly activated by cAMP) and calmodulin (CaM) was assessed by the content of the corresponding mRNA in biopsy specimens of cardiac atrium, left ventricle, and thoracic aorta of rats with alcoholic cardiomyopathy. In the myocardium, overexpression of Еpac1, Ерас2, and СаМ mRNA was found. The content of Epac2 mRNA in the left ventricle was elevated by 2.9 times (p=0.000001), in the left atrium by 3.2 times (p=0.00001), in the right atrium by 3 times (p=0.00001). In contrast to the myocardial tissue, the content of CaM mRNA in the thoracic aorta was not increased, but showed a tendency to decrease, when compared to the control values, while the level of Epac1 and Epac2 mRNA was increased. The assumption is made that regulatory proteins Epac and CaM can play a key role in arrhythmogenesis development under conditions of alcoholic cardiomyopathy.

[Vascular reactivity and receptor expression of endogenous vasoconstrictor in rats with alcoholic cardiomyopathy and insulation stress].

On the model of alcohol cardiomyopathy studied the effect of chronic ethanol consumption and the insulation stress on the reactivity of isolated rat aorta and the expression of the endogenous vasoconstrictor receptors in the aorta. Pushing alcoholization outbred rats was carried out for 24-28 weeks, using as the sole source of liquid 10% ethanol solution. In assessing the results of the study took into account the age of the animals. It is found that the reactivity of isolated aortic rings dissected from the body of old (40-45 weeks) nonstressed rats in response to endothelin-1 (ET1), noradrenaline (NA), arginine vasopressin (AVP) or angiotensin II (ATII) is not different from such reactivity for young animals. However, with the increase in life expectancy increases the sensitivity of vessels to vasoconstrictor action of serotonin (5HT). Prolonged stress insulation and the consumption of high doses of ethanol the stress lead to increased ET1- and NA-induced contraction of the aortic rings and a significant decrease in contractile response of the aorta to the impact ATII and AVP. Stress and alco- hol in combination with stress causing reduction mRNA ETA-R, AT1A-R. and V1A-R and increased mRNA α1-AR in rat aorta. It is found that in the vessels of stressed and alcoholized animals reduced level of expression of cytosolic glucocorticoid receptors (GR), which is a transcription factor for genes ETA-R, AT1A-R V1A-R. It is propoused that the development of vascular hyporesponsiveness of stressed and alcoholized rats to action ATII and AVP is the result of reducing the expression of their receptors on the GR-dependent mechanism. It is shown that under the influence of ethanol vessels become hyporeactivity selectively with respect to the action of 5HT. The mechanism of this process is unclear. Importantly, the changes in the contractile properties vessels recovered from the rat at 1 month after the abolition of the reception of ethanol (step abstinence) were similar to changes found at the alcohohzed animals. Thus, the importance of breaking the neuroendocrine regulation of vascular tone during long-term consumption of ethanol has a stressor components. Furthermore, in this experimental model we not received data in favor ethanol direct impact on the development of hypertension.

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes.

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 µM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 µM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 µM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.

[Activation of "silent" vasoconstrictive 5-HT1A receptors as a possible mechanism of synergism in angiotensin II and serotonin effect on vascular tone].

It has been shown that the agonist of 5HT1A-receptors 8-OH-DPAT induces contraction of aortic rings in the presence of angiotensin II. This effect is not associated with activation of alpha1-adrenoceptors by 8-OH-DPAT as it is reproduced in the presence of prazosin which completely suppresses the nonspecific vasoconstrictive effect of 8-OH-DPAT via alpha1-adrenoceptors on the aorta incubated without angiotensin II. Synergism in the action of angiotensin II and 8-OH-DPAT is completely preserved after partial desensitization of the receptors of angiotensin II. It has been found that 8-OH-DPAT increases the free cytoplasmic calcium concentration in cultured smooth muscle cells from the rat aorta. The data obtained support the hypothesis about the existence of "silent" vasoconstrictive 5HT1A-receptors. It has been suggested that activation of these receptors underlies synergism in vasoconstrictive action of serotonin and angiotensin II.

Inversion of the response to serotonin in rats with traumatic shock.

Normally serotonin reduced blood pressure. It was shown that in rats with traumatic shock its hypotensive effect was transformed into hypertensive one. In vitro serotonin exhibited a slight vasoconstrictor effect on isolated rat aorta, while 24 h after injury, the strength of aortic contractions in response to serotonin increased 2.2 times. Desensitization of glucocorticoid receptors caused by injection of high doses of dexamethasone (3 mg/kg) to rats for 5 days led to similar changes in serotonin effect. We hypothesized that inversion of the response to serotonin in shock was caused by increased activity and/or expression of vasoconstrictor serotonin receptors in blood vessels.

[Activators of protein kinase A and Epac proteins enhance the contractile response of the isolated snail (Helix pomatia) heart to serotonin].

The mechanisms of cAMP action on the contractility of the isolated heart were studied in the snail Helix pomatia. Serotonin is a powerful activator of heart contractility in this animal. Preincubation of the isolated heart ventricle with the activator of protein kinase A (PKA) Sp-8-bromoadenosine-3',5'-cyclic monophosphothioate (200 microM) or the activator of Epac proteins 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (100 microM) proved to enhance the amplitude of contractions induced by serotonin. Two types of changes in the contractile response were observed: each agent caused either a uniform increase in the amplitude of heart contractions at all concentrations of serotonin or an abrupt increase in the response to the first minimum dose of serotonin. These results indicate that Epac proteins along with PKA are involved in the transmission of cAMP effect on heart contractility.

[The role of desensitization of glucocorticoid receptors in the development of vascular resistance to endogenous vasoconstrictors in traumatic shock].

The fact that the activity of cytosol glucocorticoid receptors decreases in shock have been shown before [Golikov P. P. et al., 2001]. The connection between the development of vascular hyporeactivity to endogenous vasoconstrictors and desensitization of glucocorticoid receptors was studied in this investigation. On Kenton traumatic model in a rat experiment, it was shown that the strength of the isometric constriction of the isolated aorta in response to angiotensin II, endothelin-1, phenylephrine, noradrenaline, and vasopressin falls on the second day after a severe mechanical injury (3.3, 2.1, 1.7, 1.6, and 1.5 times, respectively; p < 0.01). On the contrary, the strength of the constriction in response to serotonin increases more then twice. Artificial desensitization of glucocorticoid receptors by long-term administration of dexamethasone (3 mg per kg during five days) results in similar changes of vascular reactivity i.e. a 2.5, 2, 7, and 1.4-fold decrease in the strength of aortal constriction in response to angiotensin II, vasopressin, and endothelin-1, respectively. The strength of the constriction in response to serotonin tended to increase as well. Carbahol-induced relaxation of the aorta pre-constricted with noradrenaline did not change compared with control, being 70 to 80%, both in shock and after desensitization of glucocorticoid receptors with dexamethasone. Presumably, the pathogenetic mechanism of pressor reaction suppression, connected with a decrease in cytosol glucocorticoid receptor activity and thus with inhibition of glucocorticoid-induced expression of the membrane receptors of endogenous vasoconstrictors, is realized in traumatic shock together with other mechanisms.

[Activation of cardiac contractivity of the edible snail H. pomatia under effect of thrombin. Study of role of cAMP].

Thrombin acts on mammalian cells through specific, the so-called protease-activated receptors (PARs). The thrombin action is mediated via three out of four known types of these receptors PAR(1, 3, 4). Mammalian thrombin receptors, apart from performance of other functions, control cardiac and vascular contractility. It is not known whether receptors of such kind exist in invertebrate animals. In the present work we have showed for the first time that thrombin in the concentration range of 0.01-1 units/ml increases amplitude of contractions of the isolated heart ventricle of the edible snail Helix pomatia. Its effect is reproduced by peptide ligands of receptors PAR1 and PAR4 that have sequences Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and Glu-Tyr-Pro-Gly-Lys-Phe (QYPGKF), respectively. A potent activati of cardiac contractivity of H. pomatia is serotonin. A comparative study of mechanisms of action of serotonin and thrombin on the edible snail heart was carried out. cAMP participates in transduction of signal from serotonin receptors. On the membrane preparation from the H. pomatia heart, it was shown that thrombin and peptide ligands PAR(1, 4), unlike serotonin, did not increase adenylyl cyclase activity. Thus, mechanism of activation of cardiac contractivity of H. pomatia by thrombin differs from the action mechanism of serotonin. It is suggested that molluscs have receptors homologous to protease activated mammalian receptors.