Andrej Barta

Melatonin interactions with blood pressure and vascular function during l-NAME-induced hypertension

Abstract:  The mechanisms responsible for the antihypertensive effect of melatonin are not completely understood. To elucidate the possible role of the nitric oxide (NO) pathway in the hemodynamic actions of melatonin, the effects of this indolamine on vascular function during hypertension induced by the NO‐synthase (NOS) inhibitor, Nω‐nitro‐l‐arginine‐methyl ester (l‐NAME) were investigated. Four groups of male adult Wistar rats were employed: control, L‐NAME (40 mg/kg), melatonin (10 mg/kg) and l‐NAME + melatonin for 5 wks. Systolic and diastolic blood pressure were measured invasively in the carotid artery. Conjugated dienes concentration (an oxidative load marker), NOS RNA expression and its activity and RNA expression of cyclooxygenase‐(COX)‐1 and COX‐2 were determined in the aorta. Acetylcholine‐induced responses and their NO‐mediated component were evaluated in femoral and mesenteric artery. Moreover, endothelium‐derived constricting factor (EDCF)‐dependent vasoconstriction and inner diameter were determined in the femoral artery. Chronic l‐NAME treatment induced hypertension, elevated the oxidative load and inhibited NOS activity. Moreover, impaired NO‐dependent relaxation, augmented EDCF‐constriction, increased COX‐2 expression and reduced arterial inner diameter were observed. Melatonin added to l‐NAME treatment completely prevented elevation of the oxidative load in the aorta. However, melatonin was not able to prevent NOS activity decline, elevation of COX‐2 expression or the impairment of vascular responses (except moderate improvement in relaxation of small mesenteric arteries) and it exerted only slight antihypertensive effect. In conclusion, in addition to the reduction of the oxidative load, the restoration of the NO pathway seems to play an important role in the antihypertensive effect of melatonin.

Melatonin prevents fibrosis but not hypertrophy development in the left ventricle of NG-nitro-L-arginine-methyl ester hypertensive rats

Objective Melatonin was shown to reduce blood pressure, enhance nitric oxide availability and scavenge free radicals. There is, however, a shortage of data with respect to the effect of melatonin on pathological left ventricular remodelling associated with haemodynamic overload. Design We investigated whether melatonin was able to prevent left ventricular hypertrophy (LVH) and fibrosis associated with NG-nitro-L-arginine-methyl ester (L-NAME)-induced hypertension. Methods Four groups of male Wistar rats were investigated: control, L-NAME (50 mg/kg per day), melatonin (10 mg/kg per day) and L-NAME plus melatonin. Blood pressure was measured non-invasively each week. After 5 weeks of treatment the animals were killed and nitric oxide synthase (NOS) activity, endothelial and inducible NOS expression, the level of collagenous proteins, hydroxyproline and conjugated dienes in the left ventricle were determined. Results The administration of L-NAME inhibited NOS activity, increased conjugated dienes concentration, elevated blood pressure and induced LVH and fibrosis (indicated by increased collagenous proteins and hydroxyproline levels). The addition of melatonin to L-NAME treatment failed to prevent the attenuation of NOS activity and the development of LVH and prevented hypertension only partly. The administration of melatonin, however, completely prevented the increase in conjugated dienes concentration and the development of left ventricular fibrosis. NOS expression was not different among experimental groups. Conclusion Melatonin prevented the development of left ventricular fibrosis and the increase in oxidative load in rats with L-NAME-induced hypertension. The antifibrotic effect of melatonin seems to be independent of its effects on NOS activity and might be linked to its antioxidant properties.

The effects of new Alibernet red wine extract on nitric oxide and reactive oxygen species production in spontaneously hypertensive rats.

We aimed to perform a chemical analysis of both Alibernet red wine and an alcohol-free Alibernet red wine extract (AWE) and to investigate the effects of AWE on nitric oxide and reactive oxygen species production as well as blood pressure development in normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs). Total antioxidant capacity together with total phenolic and selected mineral content was measured in wine and AWE. Young 6-week-old male WKY and SHR were treated with AWE (24,2 mg/kg/day) for 3 weeks. Total NOS and SOD activities, eNOS and SOD1 protein expressions, and superoxide production were determined in the tissues. Both antioxidant capacity and phenolic content were significantly higher in AWE compared to wine. The AWE increased NOS activity in the left ventricle, aorta, and kidney of SHR, while it did not change NOS activity in WKY rats. Similarly, increased SOD activity in the plasma and left ventricle was observed in SHR only. There were no changes in eNOS and SOD1 expressions. In conclusion, phenolics and minerals included in AWE may contribute directly to increased NOS and SOD activities of SHR. Nevertheless, 3 weeks of AWE treatment failed to affect blood pressure of SHR.

Effect of nuclear factor kappa B inhibition on L-NAME-induced hypertension and cardiovascular remodelling.

Objective We aimed to analyze effects of nuclear factor-kappa B (NF-κB) inhibition on blood pressure (BP) regulation and cardiovascular remodelling. Design Adult 12-week-old male Wistar Kyoto rats (WKY) were treated with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg/day) for seven weeks. From the fourth week of L-NAME treatment, the NF-κB inhibitor lactacystin (1 μg/kg) was applied once a week. Furthermore, age-matched WKY received L-NAME or lactacystin alone for 7 or 3 weeks, respectively. Methods Total NOS activity was determined in the left ventricle (LV) and aorta. The concentration of conjugated dienes, fibrosis, and collagen I and III levels were determined in the LV. The cross-sectional area (CSA) and wall thickness to internal diameter ratio (WT/ID) were measured in the aorta. Results L-NAME treatment increased BP significantly (145 ± 2 mmHg vs. 110 ± 1 mmHg in controls). The addition of lactacystin resulted in further significant increase in BP (161 ± 3 mmHg). Similarly, lactacystin potentiated the increased conjugated dienes concentration induced by L-NAME. Whereas L-NAME alone did not affect NOS activity, the addition of lactacystin decreased it in both tissues investigated. The addition of lactacystin did not affect LV hypertrophy, fibrosis, and collagen I and III, already increased by L-NAME; however, it further amplified CSA in the aorta increased by L-NAME alone. WT/ID increased significantly only after the addition of lactacystin. Conclusion Decreased NOS activity along with increased oxidative load may be responsible for decreased NO bio-availability and further BP increase after NF-κB inhibition in L-NAME-induced hypertension. Increased CSA and WT/ID could contribute to this hypertensive process.

The Regulatory Role of Nuclear Factor Kappa B in the Heart of Hereditary Hypertriglyceridemic Rat

Activation of nuclear factor-κB (NF-κB) by increased production of reactive oxygen species (ROS) might induce transcription and expression of different antioxidant enzymes and also of nitric oxide synthase (NOS) isoforms. Thus, we aimed at studying the effect of NF-κB inhibition, caused by JSH-23 (4-methyl-N 1-(3-phenyl-propyl)-benzene-1,2-diamine) injection, on ROS and NO generation in hereditary hypertriglyceridemic (HTG) rats. 12-week-old, male Wistar and HTG rats were treated with JSH-23 (bolus, 10 μmol, i.v.). After one week, blood pressure (BP), superoxide dismutase (SOD) activity, SOD1, endothelial NOS (eNOS), and NF-κB (p65) protein expressions were higher in the heart of HTG rats compared to control rats. On the other hand, NOS activity was decreased. In HTG rats, JSH-23 treatment increased BP and heart conjugated dienes (CD) concentration (measured as the marker of tissue oxidative damage). Concomitantly, SOD activity together with SOD1 expression was decreased, while NOS activity and eNOS protein expression were increased significantly. In conclusion, NF-κB inhibition in HTG rats led to decreased ROS degradation by SOD followed by increased oxidative damage in the heart and BP elevation. In these conditions, increased NO generation may represent rather a counterregulatory mechanism activated by ROS. Nevertheless, this mechanism was not sufficient enough to compensate BP increase in HTG rats.

604 EFFECT OF MELATONIN ON BLOOD PRESSURE AND FIBROSIS ENLARGEMENT IN THE HEART AND AORTA IN EXPERIMENTAL METABOLIC SYNDROME

Objectives: Melatonin is involved in many signaling processes via receptor-mediated or receptor-independent mechanisms. We aimed to find out whether melatonin can modify blood pressure (BP) and morphological parameters of heart and aorta in rats with metabolic syndrome. Methods: Males, 6-week-old rats were divided into four groups: Wistar Kyoto rats; spontaneously hypertensive rats (SHR); rats with metabolic syndrome (SHR-cp); and SHR-cp treated with melatonin (10 mg/kg/day) for 3 weeks. BP was measured by telemetry. Tissue sections embedded in paraffin were stained with hematoxylin–eosin and picrosirius red. Heart and aorta collagen levels, cross section area and wall thickness of the aorta were analyzed. Melatonin MT(1) and MT(2) receptors were determined by immunohistochemical and Western blot methods. Results: BP increase in SHR-cp was comparable to that of SHR. Melatonin treatment reduced BP increase of SHR-cp by 12%. Both heart and aorta collagen levels in SHR-cp were increased significantly comparing to SHR. Melatonin failed to affect fibrosis enlargement in the heart. On the other hand, it was able to reduce fibrosis enlargement in the aorta. Comparing to SHR, expression of MT(1) receptors was elevated in SHR-cp with higher density in the aorta than in the heart. Thus, melatonin reduced fibrosis enlargement in the aorta – the tissue where expression of MT(1) receptors was shown to be higher. In Conclusion: Decrease of fibrosis enlargement in the aorta after melatonin treatment may contribute to BP reduction in rats with metabolic syndrome. Moreover, contribution of MT(1) receptor – mediated effect of melatonin is suggested. (APVV-0538-07, APVV-0742-10, VEGA-2/0190/11, VEGA-2/0178/09).

EFFECTS OF STANDARD AND ALTERNATIVE THERAPY IN EXPERIMENTAL METABOLIC SYNDROME

while vasodilation increased in the presence of PVAT at 30 weeks of age. Serum TBARS levels increased with age, and there were no significant differences in waist length/body length, as an index of abdominal obesity, and systolic blood pressure. Conclusion. This study indicates that compensatory effects of PVAT on vasodilation differ by vascular site and age in SHRSP.ZF rats with metabolic syndrome. These findings suggest that PVAT has an important role in retaining blood circulation, especially in the arteries supplying blood to organs such as the kidneys and intestines. Furthermore, oxidative stress may be involved in the development or dysfunction of PVAT effects. Acknowledgments. This work was partly supported by MEXT KAKENHI (grant number 16K08563 to SK).

Effects Of The Direct Renin Inhibitor Aliskiren On Oxidative Stress In Isolated Rat Heart

Abstract Increased activity of the renin-angiotensin-aldosterone system (RAAS) plays a significant role in the development and progression of various cardio-metabolic diseases, such as hypertension, atherosclerosis and heart failure. Aliskiren is the newest antihypertensive drug and the first orally active direct renin inhibitor to become available for clinical use. This study investigated the acute and direct effects of Aliskiren on different parameters of oxidative stress on isolated rat heart. The hearts of male Wistar albino rats (n = 24, 8 per experimental group, age 8 weeks, body mass 180–200 g), were excised and retrogradely perfused according to the Langendorfftechnique at a gradually increasing perfusion pressure (40-120 cmH2O). Markers of oxidative stress (NO2−, TBARS, H2O2 and O2−) were measured spectrophotometrically after perfusion with three different concentrations of Aliskiren (0.1 μM, 1 μM, and 10 μM). The results demonstrated possible dose-dependent cardioprotective properties of Aliskiren, particularly with higher CPP. Lipid peroxidation (TBARS) levels decreased with the highest dose of Aliskiren and higher CPP, and the same trend was observed in nitrite (NO2−) and hydrogen peroxide (H2O2) levels. These findings indicate that the acute effects of Aliskiren do not likely promote the production of reactive oxygen species upon higher pressure with the highest dose. Aliskiren may exert beneficial effects on oxidative stress biomarkers.

884 Effect of nuclear factor-kappa B inhibition on heart remodelling in experimental hypertension

Objectives: We aimed to analyze effects of NF-&kgr;B inhibition on blood pressure (BP) and heart remodelling in NG-nitro-L-arginine methyl ester (L-NAME)-hypertensive animals. Methods: Males, 12-week-old Wistar Kyoto rats (WKY) were treated with nitric oxide synthase (NOS) inhibitor L-NAME (40 mg/kg/day) for seven weeks. From the fourth week of treatment, NF-&kgr;B inhibitor lactacystin (1 mg/kg) was applied once a week. Furthermore, age-matched WKY received L-NAME or lactacystin alone for 7 or 3 weeks, respectively. NOS activity, expressions of eNOS mRNA and protein, concentration of conjugated dienes and collagen I and III levels were determined in the heart. NF-&kgr;B (p65) protein expression was measured by immunohistochemical and Western blot methods. Results: L-NAME treatment increased BP significantly (145 ± 2 vs. 110 ± 3 mmHg in controls). Addition of lactacystin resulted in further significant BP increase (161 ± 3 mmHg). L-NAME led to increased NF-&kgr;B (p65) expression followed by elevation of both eNOS mRNA and protein expressions. Addition of lactacystin blocked, however, elevated eNOS expression followed by decreased NOS activity. Furthermore, lactacystin potentiated increased conjugated dienes concentration and fibrotic process induced by L-NAME treatment. Addition of lactacystin, however, did not affect heart hypertrophy and collagen I and III already increased by L-NAME. Conclusion: Decreased NOS activity along with increased oxidative load may be responsible for decreased NO bioavailability and further BP increase after NF-&kgr;B inhibition in L-NAME-hypertension. Decreased level of NO may also contribute to fibrosis enlargement in the heart after lactacystin treatment. Thus, NO could represent a regulatory factor responsible for different NF-&kgr;B-dependent growth responses. (APVV-0538-07, APVV-0742-10, VEGA-2/0190/11, VEGA-2/0178/09).

THE ROLE OF NUCLEAR FACTOR KAPPA B IN L-NAME-INDUCED HYPERTENSION: PP.29.139

Objective: Recently we have demonstrated involvement of NF-κB in the upregulation of endothelial nitric oxide synthase (eNOS) in hypertension induced by NG-nitro-L-arginine methyl ester (L-NAME). Thus, the goal of our study was to analyze an effect of NF-κB inhibitor, lactacystin, in L-NAME-induced hypertension. Methods and Design: Adult 12-week-old male Wistar rats were subjected to treatment with L-NAME (40 mg/kg/day) for seven weeks (n = 14). Half of the rats received lactacystin together with L-NAME for last three weeks. Next 16-week-old male Wistar rats received lactacystin only for 3 weeks (n = 7). Blood pressure was measured by tail-cuff plethysmography every week. Total NOS activity was determined by measuring the formation of L-[3H] citrulline from L-[3H] arginine. Endothelial NOS and NF-κB (p65) protein expressions were determined immunohistochemically and by Western blot analysis. Results: Lactacystin treatment did not affect the blood pressure (103 ± 6 mmHg), while 7-week-L-NAME treatment increased blood pressure (141 ± 3 mmHg) by 38% comparing the age-matched untreated animals (104 ± 5 mmHg). Addition of lactacystin to the L-NAME increased blood pressure significantly (159 ± 4 mmHg) by 54% comparing the untreated control group and by 12% comparing the L-NAME group. L-NAME treatment led to increased NF-κB expression followed by elevation of both eNOS protein expression and total NOS activity in the aorta, heart and kidney. Addition of lactacystin blocked, however, elevated eNOS protein expression in all tissues investigated. Conclusion: We hypothesized that NF-κB is responsible for upregulation of eNOS protein expression which may represent one of the counterregulatory mechanisms activated to compensate decreased NO production and increased blood pressure after long-term L-NAME treatment.