EFFECT OF LOW DOSE L-NAME PRETREATMENT ON NITRIC OXIDE/REACTIVE OXYGEN SPECIES BALANCE AND VASOACTIVITY IN L-NAME/SALT-INDUCED HYPERTENSIVE RATS

High salt levels have been associated with cardiovascular diseases and inflammatory injury in arteries (1). There is now compelling evidence to indicate that high sodium intake exerts detrimental effects on cardiovascular structure via blood pressure-dependent and blood pressure-independent mechanisms (2). In animal models, high sodium chloride levels have been demonstrated to cause excessive inflammatory activation, triggering ischemia injury and end-organ stress mediated by reactive oxygen species (ROS) and pro-inflammatory cytokine secretion, leading to irreversible cardiac cell damage (3, 4). It has been shown that enhanced ROS formation is an early hallmark of NaCl-induced endothelial dysfunction and that high salt intake increased vascular superoxide formation (5, 6).

NG-nitro-L-arginine methyl ester (L-NAME) is nitric oxide (NO) synthase inhibitor, commonly used for the induction of NO-deficient hypertension. Chronic L-NAME treatment resulted in hypertension, decreased NO synthase (NOS) activity, myocardial hypertrophy and fibrosis (7, 8), reduced vasorelaxation (9, 10) as well as vascular wall thickening (11, 12). Administration of L-NAME is also associated with increased production of ROS and depletion of endogenous antioxidants (13, 14).

Previously we have shown the dual effect of L-NAME in peripheral tissues and the brain. We found that prolonging the L-NAME treatment from 4 to 7 weeks increased NOS activity in the peripheral tissues, while in the brain regions it was highly significantly decreased (15). NO synthase inhibitor was paradoxically able to increase NO production in the left ventricle and aorta of Wistar rats when administered in a low dose and for a long time. Chronic low-dose L-NAME-treatment also increased the endothelium-dependent vasorelaxation and reduced vasoconstriction of the femoral artery. Moreover, no negative effects of low-dose L-NAME on the morphological parameters of the heart, aorta and femoral artery were observed (16, 17). Results of Andelova et al. (18) also indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts.

In summary, high sodium chloride level, similarly like high L-NAME level lead to inflammation and ischemic injury by ROS and pro-inflammatory cytokine secretion (3, 4, 11-14). On the other hand, low-dose L-NAME-treatment increased cardioprotection, lead to the endothelium-dependent vasorelaxation and reduced vasoconstriction (16-18). Therefore, we hypothesize that a low dose of L-NAME may act as an adaptation to the stress that leads to oxidative damage and cardiovascular risks including hypertension due to high salt dose.

Specifically, in this study we have investigated whether low dose of L-NAME could adapt the animals and prevent them to develop hypertension due to salt diet or high dose of L-NAME. Therefore, the aim of the present work was to find out the effect of L-NAME as well as salt diet on oxidative damage and lipid peroxidation, nitric oxide synthase activity, protein expressions of eNOS and Nox-2 in the heart left ventricle and kidney, blood pressure and arterial reactivity in rats pretreated with low dose of L-NAME.

MATERIALS AND METHODS

Animals and treatment

Procedures were performed in accordance with institutional guidelines and were approved by the State Veterinary and food Administration of the Slovak Republic and by an Ethical committee according to the European Convention for the protection of Vertebrate Animals used for Experimental and other Scientific purposes, Directive 2010/63/EU of the European Parliament.

The experiments were carried out in male normotensive Wistar Kyoto (WKY) rats housed in a room with a maintained temperature (22 ± 2°C), relative humidity (55 ± 10%), and 12-h light/dark cycle. The animals had free access to standard lab chow (pelleted ST-1 diet) and water ad libitum. Rats were divided into five groups: (1) control group, (2) 12-week-old male WKY rats orally treated with NG-nitro-L-arginine methyl ester (L-NAME, 2 mg/kg/day) for 7 weeks (n = 6 in each group). From the fourth week of treatment, (3) higher dose of L-NAME (40 mg/kg/day), or (4) salt diet (8% NaCl), or (5) both higher dose of L-NAME and salt diet were applied to the low dose of L-NAME (2 mg/kg/day). The food with salt diet (8% NaCl) was prepared from ST-1 diet in the service department of our Centre (Dobra Voda) and certified by The Central Controlling and Testing Institute in Agriculture - SK 100089.

Systolic blood pressure was measured by tail-cuff plethysmography every week. At the end of experiment the animals were sacrificed. Samples of the heart left ventricle (LV), aorta and kidney were used for the determination of NOS activity, conjugated dienes (CD) concentration and Western blot analysis. Superior mesenteric artery was removed and prepared for isometric tension recording.

Functional studies on isolated arteries

Superior mesenteric artery was carefully excised and cut into segments which were used as ring preparations for measurement of isometric tension in arterial smooth muscle. The arterial rings were suspended in 20 ml organ baths filled with oxygenated (95% O2 + 5% CO2) modified Krebs solution maintained at 37°C. The Krebs solution had the following composition (in mmol/l): NaCl 118, KCl 5, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11, and CaNa2 EDTA 0.03. Isometric tension in arterial wall was recorded using a force-displacement transducer Sanborn FT 10 (Sanborn, Baltimore, USA). The preparations were first equilibrated under a resting tension of 10 mN for 60 – 90 min, and the solution was changed every 15 min.

To examine the endothelium-dependent vasorelaxation, the arterial preparations were first precontracted by phenylephrine (10–6 mol/l). When the contraction reached a plateau, increasing concentrations of acetylcholine were applied in a cumulative manner (10–9 – 10–5 mol/l). The magnitude of relaxation was assessed as the decrease in arterial tension in response to particular acetylcholine concentration.

Neurogenic contractions were induced by electrical stimulation of periarterial sympathetic nerves. The arterial rings were stimulated by two parallel platinum plate electrodes placed on either side of the preparation and connected to an electrostimulator ST-3 (Hungary). Contractile responses to electrical stimuli were obtained using square pulses of 0.5 ms in duration, at supramaximal voltage (> 30 V), applied at 2 Hz, for a period of 20 s. The obtained neurogenic contractions were blocked by phentolamine (10–5 mol/l) or tetrodotoxin (10–6 mol/l), indicating that they are induced mainly by nerve-released (endogenous) noradrenaline.

Nitric oxide synthase activity

Total NO synthase activity was determined in crude homogenates of left ventricle (LV) and kidney by measuring (3H)-L-citrulline formation from (3H)-L-arginine (MP Biochemicals, California, USA) as described elsewhere (19, 20). (3H)-L-citrulline was measured with the Quanta Smart triCarb Liquid Scintillation Analyzer (Packard Instrument Company, Meriden, Ct, USA). NOS activity was expressed as pkat/min per gram of protein.

Endothelial nitric oxide synthase and nicotinamide adenine dinucleotide phosphate hydrogen oxidase subunit (Nox-2) expressions

For Western blot analysis, samples of the tissues (LV and kidney) were probed with a polyclonal rabbit anti-endothelial NOS, anti-Nox2 (anti-gp 91 phox) and anti-GAPDH (as control) antibodies (Abcam, UK; Bio-Rad, USA) as described elsewhere (22). Antibodies were detected using a secondary peroxidase-conjugated antirabbit antibody (Abcam, UK). The bands were visualized using the enhanced chemiluminescence system (ECL, Amersham, UK), quantified by using ChemiDoc™ Touch Imagine System (Image Lab™ Touch software, Bio-Rad, USA), and normalized to GAPDH bands.

Concentration of conjugated dienes

Concentration of conjugated dienes (CD) was determined as a marker of oxidative damage and lipid peroxidation. CD concentration was measured in lipid extracts of LV and kidney homogenates (21). After chloroform evaporation under inert atmosphere and addition of cyclohexane, conjugated diene concentrations were determined spectrophotometrically (l = 233 nm, GBC UV/VIS 911 A).

Statistical analysis

Results are expressed as means ± S.E.M. One-way ANOVA and Duncan‘s post-hoc test were used for statistical analysis. P < 0.05 value was considered statistically significant. Isometric responses are expressed as the active wall tension in mN and normalized to the length (in mm) of the particular preparation.

RESULTS

Blood pressure

Systolic blood pressure was monitored every week and the time course of systolic blood pressure development is shown in Fig. 1. Chronic low dose L-NAME-treatment did not change systolic blood pressure. A significant increase in the blood pressure was seen already after the first week of administration of SD, LN40, and LN40 + SD. This increase persisted till the end of the experiment. At the end of experiment, low-dose L-NAME administration did not change systolic blood pressure in comparison with control WKY rats (123.3 ± 2.0 mmHg versus 120.1 ± 1.3 mm Hg). After administration of higher dose of L-NAME, blood pressure was significantly increased comparing to LN2 (145.1 ± 2.1 mm Hg versus 123.3 ± 2.0 mm Hg, P < 0.05). Salt diet and combination of higher dose of L-NAME and salt diet led to a significant elevation of systolic blood pressure in comparison with LN2 group (151.6 ± 3.0 mm Hg and 153.2 ± 2.3 versus123.3 ± 2.0 mm Hg, P < 0.05).

Fig. 1. Time course of systolic blood pressure development during treatment. C, control WKY rats; LN, NG-nitro-L-arginine methyl ester; LN2, rats treated with low dose of LN (2 mg/kg/day); LN 40, rats treated with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD) were applied. Data are means ± SEM (n = 6); +P < 0.05 as compared to LN2 group; ►P < 0.05 as compared to LN 40 group.

Arterial reactivity in vitro

Treatment of WKY rats with low dose of L-NAME significantly decreased endothelium-dependent relaxations in their superior mesenteric arteries. Salt diet induced further impairment of arterial relaxant responses in low dose L-NAME - treated rats. Application of high dose of L-NAME almost completely eliminated relaxations to acetylcholine and salt diet did not further modify these responses (Fig. 2).

L-NAME administered in low doses, alone or in combination with salt diet, did not significantly alter contractile responses of superior mesenteric arteries to electrical stimulation of periarterial sympathetic nerves, comparing to control WKY rats. However, high dose of L-NAME caused increase in neurogenic contractions and salt diet did not produce any additional changes in these responses in high dose L-NAME - treated rats (Fig. 3).

Fig. 2. Endothelium-dependent relaxant responses to acetylcholine in superior mesenteric arteries. C, control WKY rats; LN, NG-nitro-L-arginine methyl ester; LN2, rats treated with low dose of LN (2 mg/kg/day); LN 40, rats treated with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD) were applied. Data are means ± SEM (n = 6); *P < 0.05 as compared to controls; +P < 0.05 as compared to LN2 group.

Fig. 3. Contractile responses to electrical stimulation (2 Hz) of periarterial sympathetic nerves in superior mesenteric arteries. C, control WKY rats; LN, NG-nitro-L-arginine methyl ester; LN2, rats treated with low dose of LN (2 mg/kg/day); LN 40, rats treated with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD) were applied. Data are means ± SEM (n = 6); +P < 0.05 as compared to LN2 group.

Nitric oxide synthase activity

There was no difference in NOS activity in the left ventricle between control group and LN2 group. However, NOS activity in the kidney was significantly decreased approximately by 53%. Higher dose of L-NAME, salt diet and combination of higher dose of L-NAME and salt diet induced significant decrease in NOS activity in the left ventricle compared with LN2 group (Fig. 4a). In the kidney, NOS activity was significantly lower only after salt diet and combination of higher dose of L-NAME and salt diet (Fig. 4b).

Fig. 4. Total NO synthase activity (a) in the left heart ventricle (b) in the kidney. C, control WKY rats; LN, NG-nitro-L-arginine methyl ester; LN2, rats treated with low dose of LN (2 mg/kg/day); LN 40, rats treated with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD) were applied. Data are means ± SEM (n = 6); *P < 0.05 as compared to controls; +P < 0.05 as compared to LN2 group.

Protein expressions of endothelial nitric oxide oxide synthase and NADPH oxidase 2

Endothelial NOS (eNOS) protein expression was upregulated only in the left ventricle of LN2 group. Administration of higher dose of L-NAME, salt diet as well as combination of both factors decreased eNOS expression in LV (Fig. 5a). On the other hand, eNOS expression was downregulated in LN2 group in the kidney. Salt diet, LN40, and LN40 + SD increased eNOS expression in comparison to LN2 (Fig. 5b).

Fig. 5. eNOS protein expression (a) in the left heart ventricle (b) in the kidney. C, control WKY rats; LN, NG-nitro-L-arginine methyl ester; LN2, rats treated with low dose of LN (2 mg/kg/day); LN 40, rats treated with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD) were applied. Data are means ± SEM (n = 6); *P < 0.05 as compared to controls; +P < 0.05 as compared to LN2 group.

No significant changes in Nox-2 (subunit of NADPH oxidase) expression were seen in the left ventricle (Fig. 6a). Quantitative analysis showed significant differences in Nox-2 expression in the kidney. Nox-2 expression was increased in WKY rats treated with LN2. We did not observe any changes of Nox-2 expression after salt diet administration, but it was markedly upregulated after higher dose of L-NAME and combination (Fig. 6b).

Fig. 6. NOX2 protein expression (a) in the left heart ventricle (b) in the kidney. C, control WKY rats; LN, NG-nitro-L-arginine methyl ester; LN2, rats treated with low dose of LN (2 mg/kg/day); LN 40, rats treated with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD) were applied. Data are means ± SEM (n = 6); *P < 0.05 as compared to controls; +P < 0.05 as compared to LN2 group.

Conjugated dienes concentration

No significant changes in conjugated dienes (CD) concentration were seen in the left ventricle (Fig. 7a). The levels of CD in the kidney were increased significantly in LN2 group as compared control group. Salt diet did not change CD concentration in comparison to the LN2 group. Administration of higher dose of L-NAME increased the concentration of conjugated dienes versus LN2 group. Additional elevation of CD concentration was observed after combination of higher dose of L-NAME and salt diet (Fig. 7b).

Fig. 7. Conjugated dienes concentration (a) in the left heart ventricle (b) in the kidney. C, control WKY rats; LN, NG-nitro-L-arginine methyl ester; LN2, rats treated with low dose of LN (2 mg/kg/day); LN 40, rats treated with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD) were applied. Data are means ± SEM (n = 6); *P < 0.05 as compared to controls; +P < 0.05 as compared to LN2 group; ►P < 0.05 as compared to LN 40 group.

DISCUSSION

In the present study, we tested the hypothesis whether low dose of L-NAME could adapt the animals and attenuate or prevent the development of hypertension induced by salt diet or high dose of L-NAME. Chronic low dose L-NAME-treatment resulted in increase of CD concentration in the kidney, but there were no significant changes in the blood pressure and NOS activity in the heart. In isolated mesenteric arteries, this treatment partially reduced the endothelium-dependent relaxation, but it did not affect the sympathoadrenergic contractile responses which remained unchanged also after application of salt diet. Interestingly, after pretreatment with low dose of L-NAME, high salt intake did not change CD concentration as well as Nox-2 protein expression. Combination of salt diet with high dose L-NAME-treatment did not lead to the additive decrease of NOS activity, but it increased the level of ROS followed by increased blood pressure.

Previously, Bernatova et al. (16) documented similarly to our results that administration of low doses of L-NAME (1.5 mg/kg/day) resulted only in a transient mild elevation of BP after three and six weeks of treatment and an extension of treatment to eight weeks resulted in normalization of BP. This was associated with elevation of NOS activity in the left ventricle and aorta. In our study, low dose L-NAME-treatment did not change NOS activity, but cardiac eNOS protein expression was upregulated. On the other hand, NOS activity was decreased in the kidney as well as eNOS protein expression. Many studies have suggested that the effect of L-NAME administration in vivo on NOS expression may differ depending on the treatment period and the tissue investigated (15, 23). The mechanism of eNOS induction after L-NAME treatment, precisely described by Grumbach et al. (24), includes activation of transcriptional regulatory protein - nuclear factor - κB (NF-κB), usually associated with inducible NOS (iNOS) induction (25). In the model of L-NAME-induced hypertension, both a decreased level of NO and increased ROS generation may participate in NF-κB activation and may evoke higher eNOS or iNOS expression (26, 27). Moreover, under conditions of intracellular ROS overproduction, cofactors needed for NO synthesis, especially tetrahydrobiopterin, may be oxidized which leads to uncoupling of the NOS dimer resulted in decreased NOS activity. In addition, in condition of oxidative stress, NOS produces rather superoxide radical than NO (28). Regarding tissue oxidative damage, in our study no significant changes in CD concentration, marker of lipid peroxidation, were seen in the left ventricle after low dose of L-NAME. The level of ROS in the left ventricle was not changed, which was associated with unchanged NOS activity. On the other hand, the levels of CD in the kidney were significantly increased and NOS activity was decreased after low dose of L-NAME treatment indicating a greater sensitivity of the kidney to L-NAME. Similarly, a salt diet also affected mainly the kidney reflecting the individual response of the tissue to the load.

Administration of higher dose of L-NAME from the fourth week of treatment decreased NOS activity and resulted in additional elevation of CD concentration as well as systolic blood pressure. It means that low dose L-NAME-treatment did not adapt the animals and was not able to prevent the changes developed by higher dose of L-NAME. The results are in agreement with other studies using higher L-NAME doses (7, 29). Regarding protein expressions, higher dose of L-NAME administered for 4 weeks reduced eNOS expression in the LV and markedly upregulated the expression of NADPH oxidase subunit, Nox-2, in the kidney. Our findings support the previous reports which suggest that the L-NAME BP-raising mechanism might not solely depend on NOS inhibition but may involve oxidative stress (30).

It is known that high dose of L-NAME effectively blocks NO-dependent vascular relaxation (31) and severely impairs vascular functions (9). The incapability of low dose L-NAME-treatment to reduce the adverse effects of subsequent high dose L-NAME-treatment or salt diet was evident also in endothelium-dependent relaxant response of mesenteric artery, in which both interventions further impaired this reaction. Moreover, treatment with only low dose of L-NAME caused significant diminution of mesenteric arterial relaxation which indicates that the boundary dose of L-NAME at which it could still have adaptive/preventive effect is not definite and might be highly dependent on the sensitivity of the regulatory systems in the particular organism.

Although increase in blood pressure induced by pharmacological blockade of NO production has been attributed to inhibition of NOS in cardiovascular system, it was documented that there is a strong sympathetic component to L-NAME-induced hypertension (32). This is also in agreement with the decrease of NOS expression and activity within the brain regulatory centres during chronic L-NAME treatment observed in our previous work (15). In this study, however, low dose of L-NAME did not evoke the increase in arterial sympathoadrenergic contraction, not even when the anticontractile influence of endothelium was reduced. The presented results suggest that in low dose L-NAME-treated rats, despite the impaired endothelium-dependent relaxant response, the possible down-regulation of arterial adrenergic receptor system or contractile signalling pathways could be one of the mechanisms of adaptation which was able to maintain the blood pressure at the unchanged level. On the other hand, in group with high dose of L-NAME, the neurogenic contractions were enhanced probably due to substantial NO deficiency and smooth muscle hypertrophy (11, 12).

A high sodium chloride intake accelerates hypertension development and aggravates its complications in humans as well as in animal models. Many reports indicate that oxidative stress is an important contributing factor in hypertension induced by salt diet (33, 34). A high salt diet increased lipid peroxidation, NADPH oxidase activity and reduced the expression of Cu/Zn- and Mn-SOD in rat kidneys (35). It has been suggested that expression of eNOS was significantly decreased in the renal cortex of chronically salt-loaded animals (36). In our study we reported for the first time that after pretreatment with low dose of L-NAME, high sodium intake did not induce lipid peroxidation in the kidney, because there is an evidence showing that high salt per se increases lipid peroxidation (35, 37). This fact furthermore corresponded with protein expression of NADPH oxidase subunit which expression did not change as well. Although in our experimental study, we don’t have a group that would receive only a salt diet, we assume that BP increase in SD group was developed by high salt-dependent mechanisms. We based this statement also on the fact, that the SD and LN 40 + SD groups had the same BP development, while the LN 40 group had a significantly lower BP compared to them.

In the present study a combination of high-L-NAME with high-sodium diet produced a marked induction of ROS production measured as increased CD concentration. This combination accelerated the development of hypertension accompanied by decreased NOS activity as well as impaired endothelium-dependent relaxation in WKY rats, despite pretreatment with low dose of L-NAME. We also demonstrated that oxidative stress was probably derived from NADPH oxidase, because we found out upregulated Nox-2 protein expression after the combination. Our findings are in good accordance with previous study of Kopkan and Majid (38) demonstrating that high sodium intake, when given in combination with L-NAME, promotes oxidative damage. In the study superoxide scavenger, tempol, was able to abolish the differences in hypertensive responses to L-NAME during varying salt intake; therefore, the development of salt-sensitivity induced by chronic NOS inhibition could be mainly attributed to increase in endogenous ROS. The study indicated that the blood pressure lowering effect of tempol was not caused by reversal of NO bioavailability but rather by decreasing level of ROS (38).

Many studies have shown that high salt intake leads to endothelial dysfunction and impaired vascular reactivity due to increased oxidative stress in different vascular beds in both animals and humans. Zheng et al. found out that vascular eNOS was uncoupled from NO formation by oxidation of tetrahydrobiopterin in blood vessels from deoxycorticosterone acetate (DOCA)-salt rats (39). A recent research article reported that arteries from rats subjected to high-salt intake were unable to properly regulate intracellular calcium levels and showed augmented activity of the calcium sensitization pathway RhoA/ROCK. This pathway also regulates eNOS, which is well-known as a pivotal mediator for endothelial function. These changes may precede the development of vascular diseases induced by high-salt intake (40).

Moreover, it was documented that high dietary sodium intake can reduce the vascular smooth muscle sensitivity to NO due to decreased cyclic guanosine monophosphate production in thoracic aorta smooth muscle of spontaneously hypertensive rats (41). Therefore, the observed decrease in arterial endothelium-dependent responses after high salt intake could be caused partially by altered dilatory responsiveness of arterial smooth muscle to NO itself.

As mentioned above, overproduction of ROS and decreased NO bioavailability in arteries manifest not only at the level of endothelial vasorelaxation (9, 10, 42, 43) but also in sympathoadrenergic contractile responses which might reflect the altered mass and architectonics of tunica media as well as the changes in activity of central and peripheral autonomic nervous structures during these pathological conditions (12, 32). It was documented that increased dietary salt augments the activity of central sympathetic nervous system (44) and that it can both enhance and inhibit the function of sympathetic postganglionic neurons (45). From the presented results it seems that in the group of rats with salt diet, arterial adrenergic mechanisms were capable to adapt to higher central sympathetic stimulation by dampening the sensitivity which caused that neurogenic contractions were not increased. Moreover, the potential adapting influence of low dose L-NAME pretreatment could also contribute to this effect.

In conclusion, the results of the present study showed that chronic mild inhibition of NOS in rats by treating with low dose of L-NAME could not sufficiently adapt their regulatory systems to prevent the impairments produced by subsequent application of high dose of L-NAME and salt diet. On the other hand, rats administered with low dose of L-NAME showed enhanced expression of eNOS in the heart left ventricle and were resistant to salt diet-induced increase of reactive oxygen species in the kidney and to augmentation of sympathoadrenergic system in the mesenteric artery (see the summarization of the results in Fig. 8). These findings indicate that chronic low dose of L-NAME treatment has a potential to trigger at least some adapting mechanisms deserving further investigation.

Fig. 8. Schematic summarization of the results obtained in this study. Effect of treatment with LN2, SD, and LN40/LN40 + SD on hemodynamics (arterial reactivity), NOS activity and eNOS expression, and oxidative status (CD concentration and Nox-2 expression) in the heart and in the kidney. LN, NG-nitro-L-arginine methyl ester; LN2, treatment with low dose of LN (2 mg/kg/day); LN 40, treatment with low dose of LN and from the fourth week of treatment, higher dose of LN (40 mg/kg/day), or salt diet (SD), or both higher dose of L-NAME and salt diet (LN 40 + SD); CD, conjugated dienes.
(–) no change, ↑ increase, ↓ decrease in the respective parameter.

Abbreviations: BP, blood pressure; CD, conjugated dienes; COX-2, cyclooxygenase 2; DOCA, deoxycorticosterone acetate; L-NAME, NG-nitro-L-arginine methyl ester; LV, left ventricle; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; NF-κB, nuclear factor kappaB; NO, nitric oxide; NOS, nitric oxide synthase; Nox-2, isoform of NADPH oxidase; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; SD, salt diet; SOD, superoxide dismutase; WKY, Wistar-Kyoto.

Acknowledgements: This study was partially supported by research grants: APVV-14-0932, VEGA 2/0151/18, and VEGA 2/0147/18.

Conflict of interests: None declared.

REFERENCES

1. Zhang F, Tang Z, Hou X, et al. VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis. Proc Natl Acad Sci USA 2009; 106: 6152-6157.
2. Meneton P, Jeunemaitre X, de Wardener HE, MacGregor GA. Links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases. Physiol Rev 2005; 85: 679-715.
3. Mozaffari MS, Patel C, Ballas C, Schaffer SW. Effects of excess salt and fat intake on myocardial function and infarct size in rat. Life Sci 2006; 78: 1808-1813.
4. Ketonen J, Mervaala E. Effects of dietary sodium on reactive oxygen species formation and endothelial dysfunction in low-density lipoprotein receptor-deficient mice on high-fat diet. Heart Vessels 2008; 23: 420-429.
5. Lenda DM, Sauls BA, Boegehold MA. Reactive oxygen species may contribute to reduced endothelium-dependent dilation in rats fed high salt. Am J Physiol Heart Circ Physiol 2000; 279: H7-H14.
6. Zhu J, Mori T, Huang T, Lombard JH. Effect of high-salt diet on NO release and superoxide production in rat aorta. Am J Physiol Heart Circ Physiol 2004; 286: H575-H583.
7. Pechanova O, Bernatova I, Pelouch V, Babal P. L-NAME-induced protein remodeling and fibrosis in the rat heart. Physiol Res 1999; 48: 353-362.
8. Simko F, Simko J. The potential role of nitric oxide in the hypertrophic growth of the left ventricle. Physiol Res 2000; 49: 37-46.
9. Bernatova I, Pechanova O, Babal P, Kysela S, Stvrtina S, Andriantsitohaina R. Wine polyphenols improve cardiovascular remodeling and vascular function in NO-deficient hypertension. Am J Physiol Heart Circ Physiol 2002; 282: H942-H948.
10. Gerova M, Torok J, Pechanova O, Matuskova J. Rilmenidine prevents blood pressure increase in rats with compromised nitric oxide production. Acta Pharmacol Sin 2004; 25: 1640-1646.
11. Gerova M, Kristek F. Efficiency of NO donors in substituting impaired endogenous NO production: a functional and morphological study. Physiol Res 2001; 50: 165-173.
12. Simko F, Matuskova J, Luptak I, et al. Spironolactone differently influences remodeling of the left ventricle and aorta in L-NAME-induced hypertension. Physiol Res 2007; 56 (Suppl. 2): S25-S32.
13. Abdel-Rahman RF, Hessin AF, Abdelbaset M, Ogaly HA, Abd-Elsalam RM, Hassan SM. Antihypertensive effects of roselle-olive combination in L-NAME-induced hypertensive rats. Oxid Med Cell Longev 2017; 2017: 9460653. doi: 10.1155/2017/9460653
14. Cebova M, Klimentova J, Janega P, Pechanova O. Effect of bioactive compound of Aronia melanocarpa on cardiovascular system in experimental hypertension. Oxid Med Cell Longev 2017; 2017: 8156594. doi: 10.1155/2017/8156594
15. Vrankova S, Barta A, Parohova J, Pechanova O. Could NF-kappa B play a role in blood pressure regulation? Focused on L-NAME-induced hypertension. J Hypertens 2009; 27 (Suppl. 4): S140.
16. Bernatova I, Kopincova J, Puzserova A, Janega P, Babal P. Chronic low-dose L-NAME treatment increases nitric oxide production and vasorelaxation in normotensive rats. Physiol Res 2007; 56 (Suppl. 2): S17-S24.
17. Kopincova J, Puzserova A, Bernatova I. L-NAME in the cardiovascular system - nitric oxide synthase activator? Pharmacol Rep 2012; 64: 511-520.
18. Andelova E, Bartekova M, Pancza D, Styk J, Ravingerova T. The role of NO in ischemia/reperfusion injury in isolated rat heart. Gen Physiol Biophys 2005; 24: 411-426.
19. Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990; 87: 682-685.
20. Pechanova O, Bernatova I, Pelouch V, Simko F. Protein remodeling in the heart of NO-deficient hypertension: the effect of captopril. J Mol Cell Cardiol 1997; 29: 3365-3374.
21. Kogure K, Watson BD, Busto R, Abe K. Potentiation of lipid peroxides by ischemia in rat brain. Neurochem Res 1982; 7: 437-454.
22. Bourcier T, Sukhova G, Libby P. The nuclear factor kappa-B signaling pathway participates in dysregulation of vascular smooth muscle cells in vitro and in human atherosclerosis. J Biol Chem 1997; 272: 15817-15824.
23. Simko F, Pechanova O, Repova K, et al. Lactacystin-induced model of hypertension in rats: effects of melatonin and captopril. Int J Mol Sci 2017; 18: E1612. doi: 10.3390/ijms18081612
24. Grumbach IM, Chen W, Mertens SA, Harrison DG. A negative feedback mechanism involving nitric oxide and nuclear factor kappa-B modulates endothelial nitric oxide synthase transcription. J Mol Cell Cardiol 2005; 39: 595-603.
25. Sun Y, Carretero OA, Xu J, et al. Deletion of inducible nitric oxide synthase provides cardioprotection in mice with 2-kidney, 1-clip hypertension. Hypertension 2009; 53: 49-56.
26. Pechanova O, Simko F. Chronic antioxidant therapy fails to ameliorate hypertension: potential mechanisms behind. J Hypertens 2009; 27 (Suppl. 6): S32-S36.
27. Zhen J, Lu H, Wang XQ, Vaziri ND, Zhou XJ. Upregulation of endothelial and inducible nitric oxide synthase expression by reactive oxygen species. Am J Hypertens 2008; 21: 28-34.
28. Xu H, Pritchard KA. Targeted increases in endothelial cell superoxide anion production stimulate eNOS-dependent nitric oxide production, not uncoupled eNOS activity. Arterioscler Thromb Vasc Biol 2008; 28:1580-1581.
29. Wang Y, Zhang F, Liu Y, et al. Nebivolol alleviates aortic remodeling through eNOS upregulation and inhibition of oxidative stress in L-NAME-induced hypertensive rats. Clin Exp Hypertens 2017; 39: 628-639.
30. Jaarin K, Foong WD, Yeoh MH, et al. Mechanisms of the antihypertensive effects of Nigella sativa oil in L-NAME-induced hypertensive rats. Clinics (Sao Paulo) 2015; 70: 751-757.
31. Kim HY, Lee YJ, Han BH, et al. Mantidis ootheca induces vascular relaxation through PI3K/AKT-mediated nitric oxide-cyclic GMP-protein kinase G signaling in endothelial cells. J Physiol Pharmacol 2017; 68: 215-221.
32. Sander M, Hansen PG, Victor RG. Sympathetically mediated hypertension caused by chronic inhibition of nitric oxide. Hypertension 1995; 26: 691-695.
33. Greaney JL, DuPont JJ, Lennon-Edwards SL, Sanders PW, Edwards DG, Farquhar WB. Dietary sodium loading impairs microvascular function independent of blood pressure in humans: role of oxidative stress. J Physiol 2012; 590: 5519-5528.
34. Feng D, Yang C, Geurts AM, et al. Increased expression of NAD(P)H oxidase subunit p67(phox) in the renal medulla contributes to excess oxidative stress and salt-sensitive hypertension. Cell Metab 2012; 15: 201-208.
35. Kitiyakara C, Chabrashvili T, Chen Y, et al. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. J Am Soc Nephrol 2003; 14: 2775-2782.
36. Ni Z, Vaziri ND. Effect of salt loading on nitric oxide synthase expression in normotensive rats. Am J Hypertens 2001; 14: 155-163.
37. Cosic A, Jukic I, Stupin A, et al. Attenuated flow-induced dilatation of middle cerebral arteries is related to increased vascular oxidative stress in rats on a short-term high salt diet. J Physiol 2016; 594: 4917-4931.
38. Kopkan L, Majid DS. Superoxide contributes to development of salt sensitivity and hypertension induced by nitric oxide deficiency. Hypertension 2005; 46: 1026-1031.
39. Zheng JS, Yang XQ, Lookingland KJ, et al. Gene transfer of human guanosine 5’-triphosphate cyclohydrolase I restores vascular tetrahydrobiopterin level and endothelial function in low renin hypertension. Circulation 2003; 108: 1238-1245.
40. Crestani S, Webb RC, da Silva-Santos JE. High-salt intake augments the activity of the RhoA/ROCK pathway and reduces intracellular calcium in arteries from rats. Am J Hypertens 2017; 30: 389-399.
41. Kagota S, Tamashiro A, Yamaguchi Y, et al. Downregulation of vascular soluble guanylate cyclase induced by high salt intake in spontaneously hypertensive rats. Br J Pharmacol 2001; 134: 737-744.
42. Berenyiova A, Drobna M, Cebova M, Kristek F, Cacanyiova S. Changes in the vasoactive effects of nitric oxide, hydrogen sulfide and the structure of the rat thoracic aorta: the role of age and essential hypertension. J Physiol Pharmacol 2018; 69: 535-546.
43. Sun YY, Su XH, Jin JY, et al. Rumex acetosa L. induces vasorelaxation in rat aorta via activation of PI3-kinase/Akt- AND Ca(2+)-eNOS-NO signaling in endothelial cells. J Physiol Pharmacol 2015; 66: 907-915.
44. Stocker SD, Madden CJ, Sved AF. Excess dietary salt intake alters the excitability of central sympathetic networks. Physiol Behav 2010; 100: 519-524.
45. Habecker BA, Grygielko ET, Huhtala TA, Foote B, Brooks VL. Ganglionic tyrosine hydroxylase and norepinephrine transporter are decreased by increased sodium chloride in vivo and in vitro. Auton Neurosci 2003; 107: 85-98.