Angelika Puzserova

Endothelial dysfunction in spontaneously hypertensive rats: focus on methodological aspects

Despite the apparent consensus on the existence of endothelial dysfunction in conduit and resistance arteries of spontaneously hypertensive rats (SHR), a commonly employed experimental model of hypertension, there are a number of reports showing that endothelium-dependent vasodilatory responses are similar, or even increased, in SHR compared with their normotensive counterparts. The present paper aims to discuss the rationale for these apparent discrepancies, including the effect of age, type of artery and methodological aspects. Data from the literature indicate that the age of the animal is a contributing factor and that endothelial dysfunction is likely to be a consequence of hypertension. In addition, the use of antioxidant additives, such as ascorbic acid or ethylene diaminetetraacetic acid, and differences in the level of initial arterial stretch, might also be of importance because they may modify the oxidative status of the artery and the levels of vasoactive factors released by the endothelium.

L-NAME in the cardiovascular system – nitric oxide synthase activator?

L-arginine analogues are widely used inhibitors of nitric oxide synthase (NOS) activity both in vitro and in vivo, with N(ω)-nitro-L-arginine methyl ester (L-NAME) being at the head. On the one hand, acute and chronic L-NAME treatment leads to changes in blood pressure and vascular reactivity due to decreased nitric oxide (NO) bioavailability. However, lower doses of L-NAME may also activate NO production via feedback regulatory mechanisms if administered for longer time. Such L-NAME-induced activation has been observed in both NOS expression and activity and revealed considerable differences in regulatory mechanisms of NO production between particular tissues depending on the amount of L-NAME. Moreover, feedback activation of NO production by L-NAME seems to be regulated diversely under conditions of hypertension. This review summarizes the mechanisms of NOS regulation in order to better understand the apparent discrepancies found in the current literature.

(–)‐Epicatechin reduces blood pressure and improves vasorelaxation in spontaneously hypertensive rats by NO‐mediated mechanism

Studies in humans have found consumption of certain flavanoid‐containing foods to be associated with improvement in endothelial function and with reduction of blood pressure (BP). (–)‐Epicatechin is a compound representative of the flavanols (a subfamily of flavonoids), abundant in cocoa seeds, which is preserved during the industrialization process to chocolate. The antihypertensive effect of dietary (–)‐epicatechin was investigated on spontaneously hypertensive rats (SHRs). Consumption of (–)‐epicatechin‐supplemented diet (3 g (–)‐epicatechin/kg diet) decreased BP in SHR by 27 and 23 mm Hg on days 2 and 6, respectively. On day 6, a 173% increase of nitric oxide synthase (NOS) activity was observed in the aorta of EPI‐SHR as compared to nonsupplemented SHR (P < 0.05). Responses to acetylcholine (ACh) were then examined in femoral arteries in the absence and the presence of L‐NAME, a nonselective NOS inhibitor, to assess the ACh‐mediated relaxation ascribed to NO‐dependent and ‐independent mechanisms. Acetylcholine‐induced endothelium‐dependent relaxation in the femoral artery was significantly higher in EPI‐SHR than in SHR, with a predominance of the NO‐dependent component of this relaxation. The endothelium‐independent relaxation, assayed by using the NO donor sodium nitroprusside, resulted in nonsignificant difference in the three experimental groups, demonstrating an unaffected function of vascular smooth muscle cells. These results give further support to the concept that (–)‐epicatechin can modulate BP in hypertension by increasing NO levels in the vasculature.

Determination of motor activity and anxiety-related behaviour in rodents: methodological aspects and role of nitric oxide

ABSTRACT In various areas of the bio-medical, pharmacological and psychological research a multitude of behavioural tests have been used to investigate the effects of environmental, genetic and epi-genetic factors as well as pharmacological substances or diseased states on behaviour and thus on the physiological and psycho-social status of experimental subjects. This article is reviewing the most frequently used behavioural tests in animal research (open field, elevated plus maze, zero maze, and black and white box). It provides a summary of common characteristics as well as differences in the methods used in various studies to determine motor activity, anxiety and emotionality. Additionally to methodological aspects, strain, sex and stress-related differences as well as the involvement of nitric oxide in modulation of motor activity and anxiety of rodents were briefly reviewed.

Biochemical aspects of nitric oxide synthase feedback regulation by nitric oxide

Biochemical aspects of nitric oxide synthase feedback regulation by nitric oxide Nitric oxide (NO) is a small gas molecule derived from at least three isoforms of the enzyme termed nitric oxide synthase (NOS). More than 15 years ago, the question of feedback regulation of NOS activity and expression by its own product was raised. Since then, a number of trials have verified the existence of negative feedback loop both in vitro and in vivo. NO, whether released from exogenous donors or applied in authentic NO solution, is able to inhibit NOS activity and also intervenes in NOS expression processes by its effect on transcriptional nuclear factor NF-κB. The existence of negative feedback regulation of NOS may provide a powerful tool for experimental and clinical use, especially in inflammation, when massive NOS expression may be detrimental.

Long-term social stress induces nitric oxide-independent endothelial dysfunction in normotensive rats

As chronic stress is a significant risk factor for several cardiovascular disorders, this study investigated the hypothesis that long-term stress produced by crowding may lead to alterations in nitric oxide (NO) production and NO-dependent relaxation in the course of stress, resulting in endothelial dysfunction and hypertension in Wistar–Kyoto (WKY) rats. For this purpose, male WKY rats were divided into control (480 cm2/rat, four rats/cage, n = 8) and crowded (200 cm2/rat, five rats/cage, n = 10) groups for 8 or 12 weeks. Vasorelaxation was evaluated in vitro as a response to acetylcholine (ACh) of femoral arteries pre-contracted by serotonin, before and after NO synthase inhibition (N G-nitro-l-arginine methyl ester, 300 μmol/l). Crowding increased plasma corticosterone concentration but failed to affect blood pressure (determined by tail-cuff plethysmography) of rats. NO production was unchanged in the hypothalamus and left ventricle of both stressed groups; however it was significantly elevated in the aorta. Maximal ACh-induced relaxation was elevated significantly after 8-week stress, but reduced after 12 weeks. Stress elevated the NO-dependent component and reduced the NO-independent component of ACh-induced relaxation in both crowded groups. However, a reduction in the NO-independent component was more pronounced after 12-week versus 8-week stress. In conclusion, elevated endothelium-dependent relaxation was observed after 8-week stress, while the extension of stress exposure resulted in a reduction in arterial relaxation associated with a more pronounced decrease of its NO-independent component. Thus, elevation of the NO-dependent component of relaxation can be considered as an adaptation mechanism, and impairment of NO-independent relaxation might be the initial step in chronic stress-induced cardiovascular disorders.

Chronic social stress increases nitric oxide-dependent vasorelaxation in normotensive rats

Chronic social stress increases nitric oxide-dependent vasorelaxation in normotensive rats The aim of this study was to examine oxidative load and endothelium-dependent vasorelaxation in the serotonin pre-constricted femoral artery (FA) of Wistar-Kyoto (WKY) rats exposed to chronic social stress produced by crowding in the presence or absence of ascorbic acid (AsA) in working solution. Adult male rats were randomly divided into control (living space: 480 cm2/rat) or stressed (living space: 200 cm2/rat) groups for 8 weeks. Blood pressure and heart rate, determined using tail-cuff plethysmography, were not influenced by stress vs. control. Conjugated dienes (CD) and concentrations of thiobarbituric acid-reactive substances (TBARS) were measured in the left ventricle and liver (for assessment of oxidative load) and were found unchanged by chronic crowding. The nitric oxide (NO)-dependent component of endothelium-dependent relaxation was investigated in the FA using a wire myograph. In both the presence and absence of AsA, acetylcholine-induced relaxation of the FA of stressed rats significantly exceeded that of the controls, which was associated with an increase of the NO-dependent component. In conclusion, the data showed that chronic crowding did not produce oxidative stress in the organs investigated and indicate that elevation of NO production during chronic stress is an important way of adaptation, which may prevent normotensive rats from the development of stress-induced hypertension.

Epicatechin Prevents Blood Pressure Increase and Reduces Locomotor Hyperactivity in Young Spontaneously Hypertensive Rats

This study investigated the effects of subchronic (−)-epicatechin (Epi) treatment on locomotor activity and hypertension development in young spontaneously hypertensive rats (SHR). Epi was administered in drinking water (100 mg/kg/day) for 2 weeks. Epi significantly prevented the development of hypertension (138 ± 2 versus 169 ± 5 mmHg, p < 0.001) and reduced total distance traveled in the open-field test (22 ± 2 versus 35 ± 4 m, p < 0.01). In blood, Epi significantly enhanced erythrocyte deformability, increased total antioxidant capacity, and decreased nitrotyrosine concentration. In the aorta, Epi significantly increased nitric oxide (NO) synthase (NOS) activity and elevated the NO-dependent vasorelaxation. In the left heart ventricle, Epi increased NOS activity without altering gene expressions of nNOS, iNOS, and eNOS. Moreover, Epi reduced superoxide production in the left heart ventricle and the aorta. In the brain, Epi increased nNOS gene expression (in the brainstem and cerebellum) and eNOS expression (in the cerebellum) but had no effect on overall NOS activity. In conclusion, Epi prevented the development of hypertension and reduced locomotor hyperactivity in young SHR. These effects resulted from improved cardiovascular NO bioavailability concurrently with increased erythrocyte deformability, without changes in NO production in the brain.

Genotype-Related Effect of Crowding Stress on Blood Pressure and Vascular Function in Young Female Rats

This study investigated the influence of chronic crowding stress on nitric oxide (NO) production, vascular function and oxidative status in young Wistar-Kyoto (WKY), borderline hypertensive (BHR) and spontaneously hypertensive (SHR) female rats. Five-week old rats were exposed to crowding for two weeks. Crowding elevated plasma corticosterone (P < 0.05) and accelerated BP (P < 0.01 versus basal) only in BHR. NO production and superoxide concentration were significantly higher in the aortas of control BHR and SHR versus WKY. Total acetylcholine (ACh)-induced relaxation in the femoral artery was reduced in control SHR versus WKY and BHR, and stress did not affect it significantly in any genotype. The attenuation of ACh-induced relaxation in SHR versus WKY was associated with reduction of its NO-independent component. Crowding elevated NO production in all strains investigated but superoxide concentration was increased only in WKY, which resulted in reduced NO-dependent relaxation in WKY. In crowded BHR and SHR, superoxide concentration was either unchanged or reduced, respectively, but NO-dependent relaxation was unchanged in both BHR and SHR versus their respective control group. This study points to genotype-related differences in stress vulnerability in young female rats. The most pronounced negative influence of stress was observed in BHR despite preserved endothelial function.

Sex differences in the blood antioxidant defense system in juvenile rats with various genetic predispositions to hypertension

This study investigated the contribution of blood oxidative stress (OS) to the development of hypertension, as well as sex differences in the antioxidant defense system (ADS) in genetic models of hypertension. Nine-week-old normotensive Wistar-Kyoto (WKY) rats, borderline hypertensive rats (BHR) and spontaneously hypertensive rats (SHR) of both sexes were used. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography, the trolox equivalent antioxidant capacity (TEAC) and the concentration of lipid peroxides (LP) were determined in plasma. The activity of the antioxidant enzymes Cu/Zn–superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) was determined in erythrocytes. SBP was significantly elevated in BHR and SHR in both sexes. BHR and SHR males had a higher SBP than the respective females. Sex-dependent differences in the ADS were found only in SHR, in which TEAC, SOD and CAT were significantly higher in males than in females. No differences in TEAC, SOD, CAT and GPx were observed between BHR (males and females) and WKY controls. LP levels were similar in all the groups investigated. Significant positive correlations were observed between SBP and both SOD and CAT. TEAC correlated positively with SOD and LP. As no signs of oxidative damage to lipids were found in young BHR and SHR of either sex, OS in the blood does not seem to be causatively related to the development of hypertension in these rats. However, despite activated antioxidant defenses, the positive correlation between plasma TEAC and LP suggests that oxidative damage is progressing slowly and therefore it seems to be a consequence rather than the cause of hypertension.