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¹ Institute for Heart Research, Department of Biochemistry, Slovak Academy of Sciences, PO Box 104, 840 05 Bratislava 104, Slovak Republic² Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, 813 71 Bratislava, Slovak Republic³ Pharmacologie et Physico–Chimie des Interactions Cellulaires et Moléculaires, Université Louis Pasteur de Strasbourg, Unité Mixte de Recherche Centre National de la Recherche Scientifique 7034, Faculté Pharmacie, Illkirch, France

	Abstract

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It has been suggested that polyphenolic substances provide protection against the risk factors of cardiovascular diseases. The present study was designed to investigate whether application of red wine polyphenols influences the kinetic properties of the renal Na⁺,K⁺-ATPase in rats with hypertension (164 ± 8 mmHg) that was experimentally induced by the NO synthase inhibitor N^G.-nitro-Larginine methyl ester (L-NAME). Polyphenols in a dose of 40 mg kg^–1 day^–1 in drinking fluid induced different effects on the properties of the renal Na⁺,K⁺-ATPase depending on the mode of their administration. Preventive application of polyphenols during the development of hypertension (144 ± 5 mmHg) partially protected the Na⁺,K⁺-ATPase molecule against hypertension-induced deterioration via increased capability of the enzyme to bind ATP and/or Na⁺ as suggested by decrease of K_m and K_Na, respectively, even to values lower than in controls. However, polyphenols did not prevent the hypertension-induced reduction of the number of active Na⁺,K⁺-ATPase molecules as shown by similar V_max values as compared to the hypertensive L-NAME group. The above protection is probably secured by a NO-dependent mechanism as suggested by 150% increase of the NO synthesis. Additional treatment of already hypertensive animals with polyphenols (153 ± 8 mmHg) resulted in partial restoration of the Na⁺,K⁺-ATPase affinities especially for sodium as indicated by significant diminution of K_Na. However, polyphenols in this mode of application did not slow down the L-NAME-induced decrease in the number of Na⁺,K⁺-ATPase molecules in the kidney as suggested by additional significant decrease in V_max values when comparing this group with the control group and also the hypertensive L-NAME group. In this case the polyphenols affected the Na,K-ATPase molecule in a NO-independent way as indicated by the fact that polyphenols failed to restore normal NO synthesis.

(Received 17 June 2003; accepted after revision 10 October 2003)
Corresponding author Norbert Vrbjar: Institute for Heart Research, Department of Biochemistry, Slovak Academy of Sciences, Dúbravská cesta 9, PO Box 104, 840 05 Bratislava 45, Slovak Republic. Email: usrdnorb{at}savba.sk

	Introduction

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Epidemiological studies have shown that consumption of foods and beverages rich in polyphenols, including those contained in fruit, vegetables, tea or red wine, is associated with lower incidence of cardiovascular diseases (Hertog et al. 1993; Keli et al. 1996; St Leger et al. 1979) as a consequence of their protective role in vivo (Whitehead et al. 1995; Serafiny et al. 1996). After terminating the application of NO synthase inhibitor in experimental NO-deficient hypertension in rats, administration of polyphenols from red wine, resulted in accelerated and larger decreases of blood pressure, improvement of structural and functional properties of the cardiovascular system (Bernátováet al. 2002) and ameliorated functional properties of the Na⁺,K⁺-ATPase in kidney (Javorkováet al. 2003). At rest, the Na⁺,K⁺-ATPase converts 20–30% of the ATP production in mammals to active transport of Na⁺ and K⁺ in kidney, central nervous system and other cells of the body where Na⁺ and K⁺ gradients are required for maintaining the membrane potential and cell volume (Jorgensen & Pedersen, 2001). The alterations of Na⁺,K⁺-ATPase properties and the resultant changes in control of Na⁺ homeostasis and membrane potential, both key factors in regulation of vascular tone and blood pressure, were suggested as an underlying factor in hypertension (Hermsmeyer, 1982; Blaustein et al. 1986; Herrera et al. 1988; Sweadner et al. 1994; Magyar et al. 1995; Trouve et al. 2000). Application of polyphenols from red wine during the recovery period after the induction of NO-deficient hypertension helped to restore the affinities of the binding sites for ATP and Na⁺ as well as inducing an additional positive effect of fully restoring the number of Na⁺,K⁺-ATPase molecules in renal tissue (Javorkováet al. 2003). Because it was shown that polyphenols from red wine partially prevented the increase in blood pressure when they were applied simultaneously with the NO synthase inhibitor (Pechánová & Bernátová, 2000), the present study was designed to investigate whether polyphenols applied simultaneously with the NO synthase inhibitor N^G.-nitro-L-arginine methyl ester (L-NAME) affect the functional properties of the Na⁺,K⁺-ATPase in kidney. To study the properties of the Na⁺,K⁺-ATPase we focused our attention on two main issues concerning the response to blood pressure variations, that is, the energy supply provided by ATP, and the affinity changes of the Na⁺ binding site in the enzyme molecule using kinetic evaluation as a tool.

	Methods

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Male Wistar-Kyoto rats (15 weeks old) weighing approximately 350 g, were divided into four groups. The first group (n= 12) served as a control group (C). The second group (n= 12) received N^G.-nitro-L-arginine methyl ester (L-NAME) in a dose of 40 mg kg^–1 day^–1 in drinking water for 4 weeks (hypertensive L-NAME group, LN). The third group (n= 12) received the same dose of L-NAME together with an alcohol-free extract of polyphenolic compounds from red wine for 4 weeks (LNPF4). The fourth group (n= 9) was treated with L-NAME for 7 weeks but during the last 3 weeks the treatment was supplemented with polyphenols (LNPF7/3). Red wine polyphenols were provided by D. Ageron (Société Francaise de Distillerie, Vallont Pont d'Arc, France) and their composition has been described previously (Andriambeloson et al. 1999) as follows in mg (g dry powder)^–1: proanthocyanidins 480, total anthocyanins 61, free anthocyanins 19, catechin 38, hydroxycinnamic acid 18, flavonols 14. Red wine polyphenols were administered in the drinking water after testing the amount of daily water consumption individually for each animal 1 week before the experiment. During the experiment water consumption was controlled and adjusted to receive a total dose of 40 mg kg^–1 day^–1. During the experiment all animals were kept in individual cages at a temperature of 22–24 °C and were fed with a regular pellet diet. The systolic blood pressure (SBP) was measured weekly by the non-invasive method of tail cuff plethysmography. All procedures and experimental protocols were approved by the Veterinary Council of the Slovak Republic and conform to the European Convention on Animal Protection. At the end of the experiments, rats were killed under diethyl ether anaesthesia, the kidneys were quickly excised, aliquots of samples of renal tissue were used for determination of NO synthase activity and the remaining parts were immediately frozen in liquid nitrogen and stored for further investigations of Na⁺,K⁺-ATPase properties.

The calcium- and calmodulin-dependent NO synthase activity was determined in crude homogenates of fresh renal tissue by measuring the formation of L-[³H]citrulline (L-Cit) from L-[³H]arginine (Amersham, UK) according to the method of Bredt & Snyder (1990) with a modification as described by Bernátováet al. (1996).

The plasma membrane fraction from kidney was isolated according to the method of Jorgensen (1974). The amount of protein was determined by the procedure of Lowry et al. (1951) using bovine serum albumin as a standard.

ATP kinetics of Na⁺,K⁺-ATPase was estimated at a temperature of 37 °C by measuring the hydrolysis of ATP by 10 µg plasma membrane proteins in the presence of increasing concentrations of substrate ATP in the range of 0.16–16.0 mmol l^–1. The total volume of medium was 0.5 ml containing (mmol l^–1): MgCl₂ 4, KCl 10, NaCl 100 and imidazole 50 (pH 7.4). After 20 min of preincubation in substrate-free medium, the reaction was started by addition of ATP and after 20 min the reaction was stopped by addition of 0.3 ml 12 % ice-cold solution of trichloroacetic acid. The liberated inorganic phosphorus was determined according to the method of Taussky & Shorr (1953). In order to establish the Na⁺,K⁺-ATPase activity, the ATP hydrolysis that occurred in the presence of Mg²⁺ alone was subtracted.

The Na⁺,K⁺-ATPase kinetics for cofactor Na⁺ were determined by the same method, in the presence of increasing concentrations of NaCl in the range of 1.0–100.0 mmol l^–1. The amount of ATP was constant (8 mmol l^–1).

The kinetic parameters were evaluated from obtained data by direct non-linear regression. All results were expressed as mean ±S.E.M. The significance of differences between the individual groups was determined using ANOVA and the Bonferroni test. A value of P P 0.05 was regarded as significant.

	Results

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Systolic blood pressure

Systolic blood pressure (SBP) was similar in all groups (124 ± 5 mmHg) at the beginning of the experiment. In the control group, this value did not change significantly during the experiment (49 days). Daily application of L-NAME induced a significant elevation of SBP to 164 ± 8 mmHg. Simultaneous treatment with L-NAME and polyphenols during the first 4 weeks of the experiment partially protected the rats against the increase in blood pressure as shown by a value of 144 ± 5 mmHg in the LNPF4 group. Administration of polyphenols for 3 weeks to rats with already developed hypertension and continuing treatment with L-NAME (LNPF7/3) did not decrease significantly the SBP (153 ± 8 mmHg) as compared to the LN group (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Figure 1.  Systolic blood pressure in the four groups of rats Systolic blood pressure in control rats (C), in rats with L-NAME-induced NO-deficient hypertension (LN), in rats treated simultaneously with L-NAME and polyphenols from red wine during the first 4 weeks (LNPF4) and in rats treated with L-NAME for 7 weeks and supplemented with polyphenols during the last 3 weeks of treatment (LNPF7/3). Data represent means ±S.E.M.aP P 0.001 as compared to group C; bP P 0.001 as compared to group LN.

The body weight of the animals at the beginning of our experiment was 350 ± 10 g. During the experiment the gain in weight was similar in all investigated groups (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Figure 2.  Body weight in the four groups of rats Body weight development in control rats (C), in rats with L-NAME-induced NO-deficient hypertension (LN), in rats treated simultaneously with L-NAME and polyphenols from red wine during the first 4 weeks (LNPF4) and in rats treated with L-NAME for 7 weeks and supplemented with polyphenols during the last 3 weeks of treatment (LNPF7/3). Data represent means ±S.E.M.

Administration of L-NAME for 4 weeks induced 40% inhibition of the NO synthase activity. Simultaneous treatment with L-NAME and polyphenols in the LNPF4 group induced a significant increase in NO synthase activity which exceeded its value in the control group by 25%, and was 110% greater than in the LN group. On the other hand in the LNPF7/3 group with the treatment by polyphenols following the development of hypertension, the NO synthase activity remained similar to that of the LN group (Table 1).

Activation of the renal Na⁺,K⁺-ATPase with increasing concentrations of the substrate (ATP), resulted in V_max (maximum velocity) of 25.44 ± 0.66 µmol inorganic of inorganic phosphate (P_i) mg^–1 h^–1 and the K_m (concentration ATP necessary for half-maximal activation of the enzyme) was 1.49 ± 0.09 mmol l^–1 ATP in the control group.

In the hypertensive LN group we observed a significant decrease of the enzyme activity (by 43%–28%) throughout the investigated concentration range of ATP with greater decrease in the presence of lower concentrations of substrate (Fig. 3). Data evaluation by non-linear regression revealed significant changes of K_m and V_max values as shown by 34% increase in K_m and 26% decrease in V_max as compared to the control group (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Figure 3.  Activation of renal Na+,K+-ATPase in the four groups of rats Activation of renal Na+,K+-ATPase by substrate ATP in control rats (C), in rats with L-NAME-induced NO-deficient hypertension (LN), in rats treated simultaneously with L-NAME and polyphenols from red wine during the first 4 weeks (LNPF4) and in rats treated with L-NAME for 7 weeks and supplemented with polyphenols during the last 3 weeks of treatment (LNPF7/3). Inset, activation of the enzyme in the whole range of ATP concentrations.

View larger version (30K): [in this window] [in a new window]   Figure 4.  Kinetic parameters of Na+,K+-ATPase activation by substrate ATP in the four groups of rats Kinetic parameters of Na+,K+-ATPase activation by substrate ATP from kidneys in control rats (C), in rats with L-NAME-induced NO-deficient hypertension (LN), in rats treated simultaneously with L-NAME and polyphenols from red wine during the first 4 weeks (LNPF4) and in rats treated with L-NAME for 7 weeks and supplemented with polyphenols during the last 3 weeks of treatment (LNPF7/3). Left panel shows the maximal velocities (Vmax) of enzyme activities and right panel shows Michaelis-Menten constants (Km). Data are means ±S.E.M.aP P 0.001 as compared to group C; bP P 0.001 as compared to group LN.

Comparing the LNPF4 group with controls we observed a biphasic effect of increasing ATP concentrations on the enzyme activity. Below 1 mmol l^–1 ATP the enzyme activity was increased with the highest increase of 19%, and above this concentration the enzyme activity decreased with the highest inhibitory effect reaching 21% (Fig. 3). Both investigated kinetic parameters were altered as shown by a significant decrease of V_max by 23% as well as a 39% decrease in the K_m value (Fig. 4).

When comparing the LNPF7/3 group with the hypertensive LN group we observed a biphasic effect, as shown by an increase (maximum 11%) of the enzyme activity below 1.4 mmol l^–1 ATP and its decrease (maximum 14%) above this concentration (Fig. 3). There were also statistically significant decreases in K_m (27%) as well V_max (17%) value (Fig. 4).

The application of polyphenols in the LNPF7/3 group did not restore the hypertension-induced effect on the Na⁺,K⁺-ATPase as shown by comparison with controls. The enzyme activity in the LNPF7/3 group was significantly lower (by 37%) throughout the investigated range of ATP (Fig. 3) resulting in unchanged value of K_m while the V_max was depressed by 38% (Fig. 4).

Na⁺,K⁺-ATPase: Na⁺ kinetics

Activation of the enzyme with increasing concentrations of NaCl, resulted in a value of V_max of 26.99 ± 0.91 µmol P_i mg^–1 h^–1 and the K_Na (concentration of Na⁺ necessary for half-maximal activation of the enzyme) value was 5.74 ± 0.34 mmol (l NaCl)^–1 in the control group.

In the hypertensive LN group we observed a significant decrease (by 58–23%) of the enzyme activity throughout the applied concentration range of NaCl as compared to control group (Fig. 5). This inhibition was reflected also in significant changes of both investigated kinetic parameters. The V_max value decreased by 18% and the K_Na value increased by 121% (Fig. 6).

View larger version (18K): [in this window] [in a new window]   Figure 5.  Activation of renal Na+,K+-ATPase by cofactor Na+ in the four groups of rats Activation of renal Na+,K+-ATPase by cofactor Na+ in control rats (C), in rats with L-NAME-induced NO-deficient hypertension (LN), in rats treated simultaneously with L-NAME and polyphenols from red wine during the first 4 weeks (LNPF4) and in rats treated with L-NAME for 7 weeks and supplemented with polyphenols during the last 3 weeks of treatment (LNPF7/3). Inset, activation of the enzyme in the whole range of NaCl concentrations investigated.

View larger version (27K): [in this window] [in a new window]   Figure 6.  Kinetic parameters of Na+,K+-ATPase activation by cofactor Na+ in the four groups of rats Kinetic parameters of Na+,K+-ATPase activation by cofactor Na+ from kidneys in control rats (C), in rats with L-NAME-induced NO-deficient hypertension (LN), in rats treated simultaneously with L-NAME and polyphenols from red wine during the first 4 weeks (LNPF4) and in rats treated with L-NAME for 7 weeks and supplemented with polyphenols during the last 3 weeks of treatment (LNPF7/3). Left panel shows the maximal velocities (Vmax) of enzyme activity and the right panel shows the KNa values representing the concentrations of NaCl necessary for half-maximal activation of the enzyme. Data are means ±S.E.M.aP P 0.001 as compared to group C; bP P 0.001 as compared to group LN.

Comparing the LNPF4 group with controls we observed an inhibition (by 5–19%) of the enzyme in the whole concentration range of NaCl (Fig. 5) resulting in a significant decrease of V_max by 20% as well as the K_Na value by 19% (Fig. 6).

In the LNPF7/3 group we observed a biphasic effect, depending on the NaCl concentration, as compared to the hypertensive LN group (Fig. 5). Below 8 mmol l^–1 NaCl the enzyme activity was stimulated (maximum 153%) and above this concentration the enzyme activity was decreased (maximum 36%). Evaluating the above data we observed a significant decrease of K_Na by 85% with a statistically significant 42% decrease in V_max (Fig. 6).

When comparing the LNPF7/3 and the control groups, we observed again a biphasic effect on the Na⁺,K⁺-ATPase activity. In the presence of the lowest cofactor concentration the stimulatory effect was 6% (Fig. 5). The inhibitory effect on the enzyme started above 1 mmol l^–1 (maximum 51%). These changes resulted in statistically significant 53% decrease of V_max accompanied by a significant 66% decrease in the K_Na value (Fig. 6).

	Discussion

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In the present study we have attempted to answer two main questions concerning the functional properties of the renal Na,K-ATPase in various stages of experimentally induced hypertension. The first question was whether an alcohol-free extract of polyphenolic compounds from red wine has a preventive effect against the development of hypertension. The second question was whether polyphenols might counteract pre-existing hypertension in the persisting presence of hypertension-evoking factors.

In this study we used the experimental model of NO-deficient hypertension by inhibiting NO synthesis using an L-arginine analogue L-NAME. In this model various alterations in the cardiovascular system have been observed previously, for example sustained hypertension, left ventricular hypertrophy (Bernátováet al. 1996), metabolic disturbances and ultrastructural alterations of the heart (Tribulováet al. 2000) and enhanced triggering of angiogenesis (Okruhlicováet al. 2000). NO-deficient hypertension also induces glomerular sclerosis/hypertrophy and tubular remodelling in the renal cortical region (Mandarim-de-Lacerda & Pereira, 2001). In addition to morphological alterations the NO-deficient hypertension induced a profound deterioration in the functional properties of the renal Na⁺,K⁺-ATPase as also documented in our previous study (Vrbjar et al. 2002). Hypertension induced a decrease in the number of active enzyme molecules in renal plasma membranes as indicated by the decrease in V_max. The functional alterations of the enzyme resulted in qualitative changes with depressed affinities to Na⁺ as well as to the substrate (ATP), as shown by an increase in the K_Na and K_m values.

Previous studies of the effect of red wine revealed its participation in the prevention of renal damage mediated by oxidative stress (Orellana et al. 2002; Rodrigo et al. 2002; Rodrigo & Rivera, 2002). In our study we used an alcohol-free extract from red wine in a dose of 40 mg kg^–1 day^–1. This amount of polyphenols was higher than used in previous studies utilizing wine, because of the absence of alcohol which might influence the absorption and distribution of polyphenolic compounds in the organism. However, this dose is even lower than the amount used usually for polyphenolic compounds from green tea (50–100 mg kg^–1 day^–1) which also revealed protective effects on the kidney (Sabu et al. 2002; Yokozawa et al. 2003).

The present study for the first time provides evidence that polyphenols from red wine in the absence of alcohol may cause different effects depending on the time of their application during the development of hypertension. Polyphenols when applied simultaneously with the NO synthase inhibitor L-NAME during the development of hypertension completely prevented the inhibition of NO synthesis in kidneys, together with partial prevention of the increase in blood pressure. However, simultaneous application of polyphenols together with L-NAME during sustained hypertension failed to eliminate either the decrease of NO synthesis or the increase of blood pressure.

As far as the Na⁺,K⁺-ATPase is concerned, polyphenols protected the qualitative properties of the enzyme molecule during the development of hypertension. Application of polyphenols increased the capability of renal Na⁺,K⁺-ATPase to utilize substrate as a source of energy necessary for the transport of excessive Na⁺ out of the intracellular space as suggested by improved enzyme affinity for ATP in the LNPF4 group as shown by significant decrease of K_m value when compared to the hypertensive LN group. When comparing the activation of the enzyme by substrate in the LNPF4 group with controls our data indicated that polyphenols protected the ATP-binding site of the enzyme molecule against hypertension-induced deterioration to a limited extent. In the presence of higher concentrations of substrate the ATP-binding site in the enzyme molecule remains impaired. The protection of qualitative properties of the Na⁺,K⁺-ATPase especially in the vicinity of ATP-binding site might be a consequence of elevated NO synthesis, as documented previously in cardiac and renal tissue after cessation of L-NAME treatment (Vrbjar et al. 1999b; Vrbjar et al. 2002) or in hearts of spontaneously hypertensive rats with increased synthesis of NO (Vrbjar & Pechánová, 2002). The suggested involvement of the NO pathway is supported also by the previous observation that absence of NO synthase was followed by a decrease in Na⁺,K⁺-ATPase activity in cardiac tissue (Zhou et al. 2002). The above hypothesis is in agreement with recent studies showing that polyphenols from red wine and green tea may act in kidney via a NO-dependent mechanism (Giovannini et al. 2001; Wakabayashi, 2002; Yokozawa et al. 2003). Previous studies using various models of hypertension have suggested that preservation of lower blood pressure is associated with an increase in the affinities of the Na⁺-binding site (Vrbjar et al. 1999a, b, 2002; Javorkováet al. 2003). Our present finding of a protective effect of preventive application of polyphenols on the maintenance of SBP as well as NO synthesis are supported also by improved affinity of the Na⁺-binding site in the enzyme molecule as shown by a profound decrease of the K_Na value as compared to the hypertensive LN group. Since V_max is independent of substrate concentration it provides information about the changes in number of active enzyme molecules in the tissue. Our present data, in agreement with previous observations, showed a hypertension-induced decrease in the number of active Na⁺,K⁺-ATPase molecules in renal tissue as suggested by the decrease in V_max in both types of enzyme activation with ATP and also Na⁺. Preventive application of polyphenols did not eliminate the L-NAME-induced decrease in the number of enzyme molecules in the renal tissue as suggested by similarities in V_max values when comparing the LNPF4 group with the hypertensive LN group. This fact was confirmed in both types of enzyme activation by ATP and/or Na⁺.

In the part of the study designed to investigate the effect of polyphenols after development of NO-deficient hypertension (LNPF7/3 group), polyphenols partially suppressed the negative influence of L-NAME-induced hypertension on Na⁺,K⁺-ATPase affinities to ATP and Na⁺. Concerning the activation of the enzyme by ATP, our results indicate improved affinity of the ATP-binding site as shown by a significant decrease in K_m, suggesting a better utilization of ATP particularly in the presence of lower concentrations of substrate. Although the addition of polyphenols in this part of the experiment caused K_m to return to the control level, the activity of the Na⁺,K⁺-ATPase in the presence of higher ATP concentrations was still markedly lower than in controls. The effect of polyphenols in suppressing the damage to the Na⁺-binding site observed in the hypertensive LN group was also apparent from the significant decrease of K_Na in the LNPF7/3 group. The above increase of the enzyme affinity to Na⁺ was effectively reflected in increased Na⁺,K⁺-ATPase activity especially in the presence of lower concentrations of Na⁺. This protective effect of polyphenols in the LNPF7/3 group was not observed in the presence of higher concentrations of Na⁺. It seems that additional administration of polyphenols to already hypertensive animals affected the Na,K-ATPase molecule in a NO-independent way as indicated by the fact that polyphenols failed to restore normal NO synthesis.

Concerning the quantity of Na⁺,K⁺-ATPase molecules, additional application of polyphenols did not slow down the L-NAME-induced decrease in the number of active enzyme molecules in kidney as suggested by the additional significant decrease in V_max when comparing the LNPF7/3 group with the control group and also the LN group. This fact was confirmed in both types of enzyme activation, by ATP and/or Na⁺.

It should be mentioned that the observed effects of polyphenopls from red wine may be important especially in pathophysiological situations such as development and persistence of hypertension, while administration of red wine to control animals did not change the activity of renal Na⁺,K⁺-ATPase (Rodrigo et al. 2002).

In conclusion, application of polyphenols induced different effects on properties of the renal Na⁺,K⁺-ATPase depending on the time of their administration. Preventive application of polyphenols during the development of hypertension induced a partial protection of the Na⁺,K⁺-ATPase probably by inhibiting the increase of SBP via a NO-dependent mechanism. In contrast the additional treatment of already hypertensive animals with polyphenols failed to counteract hypertension and depression of NO synthesis but induced a smaller effect on the Na⁺,K⁺-ATPase molecule via an unknown mechanism.

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	Acknowledgements

 
This research was supported by Slovak Grant Agencies (grants No. VEGA-2/, 2064/22 and APVT-51/013802). The authors thank Mrs M. Hybelová and Mrs Z. Hradecká for their technical assistance.

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