The effect of N-acetylcysteine and melatonin in adult spontaneously hypertensive rats with established hypertension

European Journal of Pharmacology 561 (2007) 129 – 136 www.elsevier.com/locate/ejphar

The effect of N-acetylcysteine and melatonin in adult spontaneously hypertensive rats with established hypertension Olga Pechánová a,d,⁎, Josef Zicha d , Ludovít Paulis a,b,d , Woineshet Zenebe a , Zdenka Dobesová d , Stanislava Kojsová a , Lýdia Jendeková a , Martina Sládková a , Ima Dovinová a,c , Fedor Simko b , Jaroslav Kunes d a

Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak Republic b Department of Pathophysiology, Medical Faculty, Comenius University, Bratislava, Slovak Republic c Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic d CRC and Institute of Physiology, AS CR, Prague, Czech Republic Received 25 May 2006; received in revised form 10 January 2007; accepted 11 January 2007 Available online 1 February 2007

Abstract The attenuated nitric oxide (NO) formation and/or elevated production of reactive oxygen species are often found in experimental and human hypertension. We aimed to determine possible effects of N-acetylcysteine (1.5 g/kg/day) and N-acetyl-5-methoxytryptamine (melatonin, 10 mg/ kg/day) in adult spontaneously hypertensive rats (SHR) with established hypertension. After a six-week-treatment, blood pressure was measured and NO synthase (NOS) activity, concentration of conjugated dienes, protein expression of endothelial NOS, inducible NOS and nuclear factor-κB (NF-κB) in the left ventricle were determined. Both treatments improved the NO pathway by means of enhanced NOS activity and reduced reactive oxygen species level as indicated by decreased conjugated diene concentrations and lowered NF-κB expression. N-acetylcysteine (but not melatonin) also increased the endothelial NOS protein expression. However, only melatonin was able to reduce blood pressure significantly. Subsequent in vitro study revealed that both N-acetylcysteine and melatonin lowered the tone of phenylephrine-precontracted femoral artery via NO-dependent relaxation. Nevertheless, melatonin-induced relaxation also involved NO-independent component which was preserved even after the blockade of soluble guanylate cyclase by oxadiazolo[4,3-a]quinoxalin-1-one. In conclusion, both N-acetylcysteine and melatonin were able to improve the NO/reactive oxygen species balance in adult SHR, but blood pressure was significantly lowered by melatonin only. This implies that a partial restoration of NO/reactive oxygen species balance achieved by the antioxidants such as N-acetylcysteine has no therapeutic effect in adult rats with established hypertension. The observed antihypertensive effect of melatonin is thus mediated by additional mechanisms independent of NO pathway. © 2007 Elsevier B.V. All rights reserved. Keywords: Spontaneous hypertension; NO synthase; Oxidative load; N-acetylcysteine; Melatonin

1. Introduction Nitric oxide (NO) plays a key role in the maintenance of vascular tone affecting the level of blood pressure (Berry et al., 2001; Vanhoutte, 2001; Wilcox, 2002). Relative NO deficiency (namely compared to existing sympathetic hyperactivity) was demonstrated in different forms of hypertension (Zicha et al., ⁎ Corresponding author. Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Sienkiewiczova 1, 813 71 Bratislava, Slovak Republic. Tel.: +421 2 52926271; fax: +421 2 52968516. E-mail address: [email protected] (O. Pechánová). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.01.035

2001; Manning et al., 2003; Payne et al., 2003). However, several studies have indicated that in spontaneous hypertension the basal NO production is not impaired (Briones et al., 2000; Radaelli et al., 1998) but may be even enhanced (Vaziri et al., 1998; Nava et al., 1998). This means that despite its increased formation, NO was not able to stimulate sufficient formation of cyclic guanosine monophosphate (cGMP) and to maintain an adequate NO-dependent vasodilation tone (Nava et al., 1998). The fact that the endogenously produced NO in spontaneously hypertensive rats (SHR) is not able to raise appropriately cGMP levels could be explained by thickened fibrotic intimal layer forming a physical barrier to NO or by the increased oxidative

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load counterbalancing the enhanced NO formation (Noll et al., 1997). In addition, NO and superoxides react to the powerful oxidants peroxynitrites, which can form hydroxyl radicals and nitrate protein tyrosine residues, resulting in an impairment of cellular signaling (Bouloumie et al., 1997). Thus, it can be assumed that chronic antioxidant treatment could exert beneficial effects in hypertension which is characterized by increased oxidative load. The sulfhydryl donor and antioxidant, N-acetylcysteine, enhances biological effects of nitric oxide by decreasing the level of reactive oxygen species (preventing thus peroxynitrate formation) and by protecting NO (formation of S-nitrosothiol compounds) from free radical destruction (Lahera et al., 1993). Chronic administration of N-acetylcysteine protected against vascular dysfunction (Cabassi et al., 2001) and reduced blood pressure in spontaneous hypertension (Girouard et al., 2003). Moreover, this antihypertensive effect of N-acetylcysteine was probably mediated by increased NO-dependent vasodilation (Girouard et al., 2003). The pineal hormone, N-acetyl-5-methoxytryptamine (melatonin), was reported to have potent direct (Allegra et al., 2003) and indirect (Reiter, 2000) antioxidant properties, which could participate in the mechanism of its antihypertensive effect demonstrated in experimental (Kawashima et al., 1987) and clinical hypertension (Scheer et al., 2004). Similarly to Nacetylcysteine, the reduction of blood pressure after melatonin treatment was associated with improved antioxidant reserve (Girouard et al., 2004). The vascular relaxation observed after acute melatonin administration was related to decreased oxidative load, increased NO concentration and increased cGMP levels in smooth muscle cells (Anwar et al., 2001). However, the receptor-mediated pathways may also contribute to the melatonin-induced enhancement of NO formation or may directly influence cGMP levels in smooth muscle cells (Brydon et al., 1999). In the present study we aimed to investigate whether chronic N-acetylcysteine or melatonin treatment is able to decrease blood pressure in SHR with established hypertension, by means of modulating the NO pathway and oxidative load. Furthermore, we tried to analyze whether this beneficial effect of antioxidant treatment is related to direct relaxation of blood vessels isolated from SHR. 2. Methods 2.1. Animals and treatment All procedures and experimental protocols were approved by the Ethical Committee of the Institute of Physiology AS CR, and conform to the European Convention on Animal Protection and Guidelines on Research Animal Use. All the chemicals used were purchased from Sigma Chemicals Co. (Germany) except of [3H]-L-arginine (Amersham, UK). Male adult SHR (the colony of Institute of Physiology AS CR, Prague established from breeding pairs purchased at Charles River) aged 12 weeks were randomly divided into three groups (n = 8 in each group): control SHR group, N-acetylcysteine-

treated SHR (1.5 g/kg/day, 20 g/l) and melatonin-treated SHR (10 mg/kg/day, 100 mg/l). Both drugs were given in the drinking fluid. The drinking bottles containing melatonin solution were protected from light by aluminum foil. During the experiment, drinking fluid consumption was controlled and adjusted, if necessary. In addition, age-matched Wistar-Kyoto rats were divided into 3 groups (n = 8 in each group) with the same treatment as the SHR to serve as control. All animals were housed in the room with a stable temperature of 23 ± 1 °C and fed with a regular pellet diet ad libitum. After six weeks of treatment, the blood pressure was determined by direct puncture of the carotid artery under a light ether anesthesia. Thereafter the animals were sacrificed and body weight and heart weight were determined. Relative weight was calculated as heart weight/body weight ratio. Samples of the left ventricle were used for the determination of NO synthase activity, conjugated dienes as well as for Western blot analysis. Femoral arteries for wire myography were isolated from adult 12-week-old SHR (n = 8) males. 2.2. Total NO synthase activity Left ventricles were excised, weighed and homogenized for determination of NO synthase activity. Total NO synthase activity was determined in crude homogenates by measuring the formation of L-[ 3 H]citrulline (L-Cit) from L-[ 3 H]arginine (Amersham, UK) as previously described by Bredt and Snyder (1990) with minor modifications (Pechánová et al., 1997). 2.3. Western blot analysis Samples of the left ventricle were homogenized in 25 mmol/ L Tris–HCl, pH 7.4, containing 5 mmol/l EDTA, 50 mmol/ l NaCl, 1 μmol/l leupeptin, 0.3 μmol/l aprotinin, 0.1 mmol/ l PMSF, 1 mmol/l pepstatin and 1% sodium dodecyl sulfate. After the centrifugation (15,000 ×g, 20 min, twice) supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels. Following the electrophoresis, proteins were transferred to nitrocellulose membranes and were probed with a polyclonal rabbit antiendothelial NO synthase and anti-inducible NO synthase antibodies (Alexis Biochemicals, Germany) and a polyclonal rabbit anti-nuclear factor-κB (NF-κB) antibody which recognizes the 65 kDa RelA (p65) protein (Santa Cruz Biotechnology, CA). Bound antibodies were detected using a secondary peroxidase-conjugated anti-rabbit antibody (Alexis Biochemicals, Germany). The bands were visualized using the enhanced chemiluminescence system (ECL, Amersham, UK) and analyzed densitometrically using Photo-Capt V.99 software. 2.4. Conjugated diene concentration The concentration of conjugated dienes was measured in lipid extracts of the left ventricle homogenates according to Kogure et al. (1982). Briefly, after chloroform evaporation under the inert atmosphere and addition of cyclohexane, conjugated diene concentrations were determined spectrophotometrically (λ = 233 nm, Bio-Rad, GBC 911A).

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Table 1 Body weight (BW), relative heart weight (HW/BW), systolic blood pressure (SBP) in untreated adult Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) or in rats subjected to chronic treatment with N-acetylcysteine (NAC, 1.5 g/kg/day) or melatonin (mel, 10 mg/kg/day) for 6 weeks WKY BW [g] HW/BW [mg/g] SBP [mm Hg]

WKY + NAC a

354.0 ± 9.2 2.31 ± 0.06a 120.5 ± 3.6a

a

340.8 ± 9.1 2.44 ± 0.08a 121.8 ± 4.3a

WKY + Mel a

345.6 ± 8.7 2.29 ± 0.06a 125.1 ± 5.2a

SHR

SHR + NAC

SHR + Mel

294.0 ± 6.2 3.41 ± 0.04 191.4 ± 4.6

270.4 ± 5.7 3.34 ± 0.06 181.1 ± 6.4

295.4 ± 5.5 3.28 ± 0.06 165.6 ± 5.5b

Data are means ± SEM, Significant differences (P b 0.05): acompared to corresponding SHR, bcompared to untreated SHR.

2.5. Determination of antioxidant activity by TEAC assay

2.7. Statistical analysis

The antioxidant activity was measured by the TEAC (trolox equivalent antioxidant capacity) assay (Re et al., 1999). 2,2′azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) monocation solution was prepared by mixing equal volumes of 2.45 mmol/l potassium persulfate and 7 mmol/l ABTS and kept in the dark at room temperature for 6 h. The ABTS + radical cation solution was diluted with PBS to give an absorbance of 0.70 ± 0.03 at 734 nm. N-acetylcysteine, melatonin and standard Trolox were added as mmol/l solutions and diluted to the final concentrations of 1, 2.5, 5, 10 μmol/l in PBS, pH 7.4. The decrease in absorbance caused by antioxidants, measured at 0, 3 and 6 min reflected the ABTS + radical cation scavenging capacity and was plotted against the concentration of the antioxidant. The TEAC value represents the ratio between the linear plot slope for scavenging of ABTS + radical cation by Nacetylcysteine and melatonin compared to the Trolox. Multiple comparison testing of slopes was carried out using the simtest function from the MULTCOMP package.

The results are expressed as mean ± SEM. Values were considered to differ significantly if the two-tailed probability value (P) was less than 0.05 (one-way ANOVA with Bonferroni post-test and Spearman test for correlation). The Tukey-type test with implementation according to Westfall (1997) and Bretz et al. (2001) was used for calculation of multiplicity-adjusted Pvalues for comparisons of antioxidant activity by TEAC assay. 3. Results 3.1. Cardiovascular parameters After six weeks of experiment, systolic blood pressure was 120.5 ± 3.6 mm Hg in the Wistar-Kyoto rats. Both Nacetylcysteine and melatonin treatment had no effect on the

2.6. Wire myography The femoral artery was removed, carefully cleaned of adhering fat and connective tissue and then cut into rings (1.5–2 mm length). Two stainless-steel wires were passed through the lumen taking care not to damage the endothelium and mounted in a Mulvany–Halpern myograph. The internal diameter of the vessels was 400–550 μm. The chamber was filled with physiological salt solution (composition in mmol/l: NaCl 118, KCl 5, NaHCO3 25, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, EDTA 1, ascorbic acid 1.1, glucose 11), maintained at 37 °C and continuously bubbled with a 95% O2 and 5% CO2 mixture. After the equilibration period of 45 min, the rings were precontracted with phenylephrine (10− 5 mol/l) and tested for the presence of functional endothelium by determining the ability of acetylcholine (10− 6 mol/l) to induce relaxation greater than 60%. The extent of relaxation was expressed as the percentage of phenylephrine-induced contraction. Thereafter, the rings of rat femoral artery were precontracted with phenylephrine (10− 5 mol/l) and after the contraction reached a steady state, N-acetylcysteine or melatonin was added cumulatively in a concentration range of 10- 5–10− 2 mol/l and 10− 7–10− 3 mol/l, respectively. Relaxation responses were repeated after 10− 4 mol/l ω-nitro-L-arginine methylester (LNAME) and L-NAME + 10− 5 mol/l 1H-[1,2,4] oxadiazolo[4,3a]quinoxalin-1-one (ODQ) preincubation.

Fig. 1. Effect of chronic N-acetylcysteine (NAC) and melatonin (mel) treatment on NOS activity in the left ventricle of WKY and SHR. Significant differences: ⁎P b 0.05 treated vs. untreated rats of the same genotype, +P b 0.05 WKY vs. corresponding SHR.

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Fig. 3. Effect of chronic N-acetylcysteine (NAC) and melatonin (mel) treatment on concentration of conjugated dienes (CD) in the left ventricle of WKY and SHR. Significant differences: ⁎P b 0.05 treated vs. untreated rats of the same genotype, +P b 0.05 WKY vs. corresponding SHR.

Fig. 2. Effect of chronic N-acetylcysteine (NAC) and melatonin (mel) treatment on eNOS and NF-κB protein expression in the left ventricle of SHR. Significant differences: ⁎P b 0.05 treated vs. untreated rats of the same genotype.

systolic blood pressure. Systolic blood pressure in the SHR group was increased significantly compared to Wistar-Kyoto rats to 191.4 ± 4.6 mm Hg (P b 0.05). The chronic Nacetylcysteine treatment caused only a non-significant reduction of the systolic blood pressure, whereas melatonin reduced systolic blood pressure by 13.5% (P b 0.05) (Table 1). The heart weight/body weight ratio in the Wistar-Kyoto rats was 2.31 ± 0.06 mg/g and was not affected by N-acetylcysteine or melatonin treatment. The spontaneously hypertensive rats developed cardiac hypertrophy as evidenced by increased heart weight/body weight ratio to 3.41 ± 0.04 mg/g as compared to Wistar-Kyoto rats (P b 0.05). Both N-acetylcysteine and melatonin treatment did not decrease heart weight/body weight ratio in SHR significantly (Table 1).

treatment enhanced the NOS activity by 19.2% (P b 0.05) and melatonin treatment by 14.9% (P b 0.05) (Fig. 1). In the left ventricle of SHR the NOS activity was 5.04 ± 0.12 pkat/g proteins. The NOS activity was enhanced by 14.3% (P b 0.05) in the SHR + N-acetylcysteine group and by 19.3% (P b 0.05) in the SHR + melatonin group (Fig. 1). The N-acetylcysteine treatment increased endothelial NOS protein expression in the left ventricle of the SHR by 28% (P b 0.05), while melatonin did not change endothelial NOS protein expression significantly (Fig. 2). The same was true for Wistar-Kyoto rats (data not shown). Neither Nacetylcysteine nor melatonin administration caused significant changes in the expression of inducible NOS protein.

3.2. NO pathway The NOS activity was 4.07 ± 0.12 pkat/g proteins in the left ventricle of the control Wistar-Kyoto rats. The N-acetylcysteine

▪

Fig. 4. Relationship between conjugated diene (CD) concentration and NF-κB protein expression in the left ventricle of SHR. Control SHR ( ), Nacetylcysteine (NAC)-treated SHR (♦) and melatonin (mel)-treated SHR (▴).

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▪

Fig. 5. The antioxidant activity of Trolox ( ), N-acetylcysteine (NAC) (♦) and melatonin (mel) (▴) measured by the TEAC (trolox equivalent antioxidant capacity) assay. ⁎P b 0.01, Tukey multiple comparisons NAC vs. melatonin.

3.3. Oxidative load The conjugated diene concentration was 68 ± 5 nmol/g tissue in the left ventricle of control Wistar-Kyoto rats. Neither Nacetylcysteine treatment, nor the melatonin treatment changed the conjugated diene concentration (Fig. 3). In the left ventricle of the SHR the conjugated diene concentration was elevated to 93 ± 5 nmol/g tissue (P b 0.05 vs. Wistar-Kyoto rats). In the SHR + N-acetylcysteine group the conjugated diene concentration was decreased by 16% (P b 0.05) and in the SHR + melatonin group by 23% (P b 0.05) (Fig. 3). N-acetylcysteine and melatonin treatment decreased NF-κB protein expression in the left ventricle of the SHR by 20% (P b 0.05) and 24% (P b 0.05), respectively (Fig. 2). Similar results were also obtained in Wistar-Kyoto rats (data not shown). Moreover, a positive correlation between NF-κB protein expression and conjugated diene concentration in the left ventricle of control SHR (r = 0.7619, P b 0.05) and even stronger correlations were present in SHR treated with N-acetylcysteine (r = 0.8735, P b 0.01) or melatonin (r = 0.8743, P b 0.01). Fig. 4 shows the correlation within all groups (r = 0.8464, P b 0.0001).

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Fig. 7. The effect of acute melatonin administration on femoral artery from untreated SHR precontracted with 10− 5 mol/l phenylephrine. The influence of 10− 4 mol/l ω-nitro-L-arginine methylester (L-NAME) and L-NAME + oxadiazolo quinoxaline (ODQ, 10− 5 mol/l) preincubation. #Indicates significant (P b 0.05) melatonin-induced relaxation, ⁎ significantly different (P b 0.05) compared to melatonin-induced relaxation, +significantly different (P b 0.05) compared to melatonin-induced relaxation of L-NAME preincubated vessel.

3.4. TEAC assay The antioxidant activity of N-acetylcysteine was comparable with the activity of Trolox. However, the antioxidant activity of melatonin was 2.27 times higher than that of N-acetylcysteine. The difference between slopes for melatonin (11.752) and N-acetylcysteine (5.179) was significant (melatonin/N-acetylcysteine =2.269± 0.463, P b 0.01, Tukey multiple comparisons of slopes, Fig. 5). 3.5. Vascular relaxation Both N-acetylcysteine (10− 5–10− 2 mol/l) and melatonin (10 − 7 –10 − 3 mol/l) elicited dose-dependent relaxation of phenylephrine-precontracted femoral artery with intact endothelium isolated from SHR. The relaxing responses to Nacetylcysteine were completely abolished in the presence of LNAME (Fig. 6). The relaxing responses to melatonin were only reduced by the preincubation with L-NAME (10− 4 mol/l) and a significant NO-independent component of melatonin-induced relaxation was preserved even after ODQ preincubation (10− 5 mol/l), (Fig. 7). 4. Discussion

Fig. 6. The effect of acute N-acetylcysteine (NAC) administration on femoral artery from untreated SHR precontracted with 10− 5 mol/l phenylephrine. The influence of 10− 4 mol/l ω-nitro-L-arginine methylester (L-NAME) and L-NAME + oxadiazolo quinoxaline (ODQ, 10− 5 mol/l) preincubation. #Indicates significant (P b 0.05) NAC-induced relaxation, ⁎ significantly different (P b 0.05) compared to NAC-induced relaxation.

In the present study, we have evaluated the effects of chronic antioxidant treatment by N-acetylcysteine and melatonin in adult spontaneously hypertensive rats. Only melatonin (but not N-acetylcysteine) was able to reduce blood pressure significantly. Both treatments enhanced NO synthase activity and reduced oxidative load as was demonstrated by reduced concentrations of conjugated dienes and decreased nuclear factor-κB expression. Subsequent in vitro study revealed that both N-acetylcysteine and melatonin lowered the tone of phenylephrine-precontracted femoral artery via NO-dependent relaxation. Nevertheless, melatonin-induced relaxation also involved an NO-independent component which was preserved even after the blockade of soluble guanylate cyclase by ODQ.

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Several authors documented the increased NO production in young SHR both before and after the onset of hypertension (Radaelli et al., 1998; Vaziri et al., 1998). Nava et al. (1998) suggested that despite its increased formation, nitric oxide is not able to stimulate sufficient formation of cyclic GMP and to maintain an adequate NO-dependent vasodilatation. The decreased biological NO effects could be explained by increased reactive oxygen species generation in SHR. Indeed, increased activity and/or enhanced expression of xanthine oxidase and NADPH oxidase, the enzymes responsible for reactive oxygen species generation, were found in different tissues of SHR (Yang et al., 2002; Chabrashvili et al., 2002). Since the increased expression of NADPH oxidase in kidney preceded the development of hypertension, it could be one of the causes rather than a consequence of the hypertension (Chabrashvili et al., 2002). It was therefore hypothesized that the antioxidant treatment might be efficient in achieving the regression of hypertension in the adult SHR. Both substances used in our study have been reported to posses potent antioxidant effects which can improve the efficacy of NO pathway in SHR. N-acetylcysteine not only decreases the level of reactive oxygen species but it may also protect NO by forming S-nitrosothiols (Lahera et al., 1993). Melatonin, especially in higher doses, also shows potent antioxidant effects (Allegra et al., 2003; Reiter, 2000). We have also demonstrated that antioxidant activity of melatonin measured by TEAC assay was 2.27 times higher than that of Nacetylcysteine. This finding suggests further antioxidant effects of melatonin — not revealed by the determination of conjugated diene concentration or nuclear factor-κB protein expression. However melatonin may additionally influence cardiovascular system through melatonin receptor-dependent pathways, which may involve a decrease in cyclic adenosine monophosphate level (Capsoni et al., 1994), increase in intracellular Ca2+ levels (Brydon et al., 1999) or act through other alternative pathways (Mei et al., 2001; Vacas et al., 1981; Nelson et al., 1996). Melatonin receptors have been found on vascular smooth muscle cells (Capsoni et al., 1994) as well as on endothelial cells (Masana et al., 2002). In our study the antioxidant properties of both N-acetylcysteine and melatonin were confirmed by decreased conjugated diene concentration and reduced NF-κB expression. These results are in agreement with those of Cabassi et al. (2001) who observed a reduction of aortic lipid peroxidation indicated by lower concentration of malondialdehyde after N-acetylcysteine treatment. Furthermore, N-acetylcysteine treatment also reduced NF-κB protein expression in aldosterone–salt hypertensive rats (Sun et al., 2002). In agreement with our study, it has been reported that N-acetylcysteine can prevent NF-κB activation by suppressing the generation of reactive oxygen species (for review see Das and Maulik, 2004) and that not only intracellular superoxide and renal malondialdehyde content but also the immunohistological expression of the 65-kDA DNAbinding subunit of NF-κB were reduced by melatonin treatment (Nava et al., 2003). Moreover, we have shown a positive correlation between NF-κB protein expression and conjugated diene concentration in the left ventricle of control SHR and even

stronger correlation was present in SHR treated with Nacetylcysteine or melatonin. In addition, Girouard et al. (2003) have demonstrated that the antioxidant properties of melatonin account for its antihypertensive effect similarly as in the case of N-acetylcysteine. However, in our study, only melatonin was able to reduce blood pressure significantly. The observed antihypertensive effect of melatonin confirms previous reports about its blood pressure lowering effect in SHR (Kawashima et al., 1987), in essential hypertensives (Scheer et al., 2004) and also in healthy subjects (Arangino et al., 1999). Using the same dose of melatonin and same duration of treatment, Nava et al. (2003) obtained slightly greater decrease of blood pressure in SHR (by 20%) than we did (by 14%). On the other hand, the inability of N-acetylcysteine to reduce blood pressure is partly controversial to findings about the preventive effect of N-acetylcysteine on the development of L-NAME-induced hypertension (Rauchová et al., 2005), salt hypertension in Dahl rats (Kuneš et al., 2004) or spontaneous hypertension in young SHR (Pechánová et al., 2004). It should be noted that this study was performed in adult SHR with established form of hypertension which seem to be less susceptible to possible therapeutic effect of chronic Nacetylcysteine administration. The enhanced NOS activity in both N-acetylcysteine- and melatonin-treated animals, indicates an improved efficacy of NO pathway. Moreover, the effects of NO formed are further increased due to decreased reactive oxygen species level. This may explain the enhanced endothelium-dependent relaxation in SHR after N-acetylcysteine (Cabassi et al., 2001; Girouard et al., 2003) or melatonin treatment (Girouard et al., 2001). Furthermore, N-acetylcysteine also increased endothelial NOS protein expression. A similar N-acetylcysteine-induced activation of endothelial NOS protein expression was observed in cultured bovine aortic endothelial cells (Ramasamy et al., 1999). However, the fact, that the enhancement of NOS activity after melatonin treatment was not associated with increased NOS protein expression, suggests that the increase in protein expression is not essential for improving the enzyme activity. A possible explanation would be a direct effect of melatonin on endothelial intracellular Ca2+ concentrations which could further enhance NOS activity. Indeed increased intracellular Ca2+ levels in endothelial cells were observed after melatonin treatment (Pogan et al., 2002). However, the reduction of reactive oxygen species level, which results in stabilizing the NOS enzyme in homodimerized form, may also increase NOS activity (Maxwell, 2002). Our comparison of the two antioxidants has shown for the first time that the reduction of oxidative load and improvement of NO pathway are not sufficient to decrease blood pressure in SHR. This suggests that beside enhanced NOS activity and reduced reactive oxygen species level other mechanisms may be responsible for blood pressure lowering effects of melatonin. This hypothesis was further analyzed by means of in vitro experiments on isolated femoral arteries. We have observed an endothelium-dependent relaxation after acute melatonin administration. This result confirms the data of Anwar et al. (2001) who have found, that the vasorelaxation after melatonin

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administration is associated with increased NO level and increased cGMP concentration in vascular smooth muscle cells. In our experiment, the melatonin-induced relaxation was only partially inhibited by NOS inhibitor L-NAME administration with an additive effect of soluble guanylate cyclase inhibitor ODQ, suggesting the presence of a significant NO-dependent component. Nevertheless, the melatonin-induced relaxation was still preserved after a combined L-NAME plus ODQ administration. This in vitro finding supports our results from in vivo experiment which indicate that additional mechanisms different from the improvement of NO pathway (e.g. melatonin receptormediated vasodilation) may be involved in the blood pressure lowering and vasorelaxant effects of melatonin. In conclusion, both N-acetylcysteine and melatonin treatment were able to improve the altered NO/reactive oxygen species balance in adult SHR, but only melatonin was able to lower blood pressure significantly. This implies that a partial restoration of NO/reactive oxygen species balance achieved by the treatment with antioxidants such as N-acetylcysteine has no therapeutic effect in adult rats with established hypertension. The antihypertensive effect of melatonin is thus mediated by additional mechanisms independent of NO pathway. 4.1. Limitation of the study It is a general problem in experiments comparing the biological effects of different substances to choose the appropriate dose(s) of the tested drugs. The different antioxidant effect of melatonin and N-acetylcysteine might be potentially determined by different biological availability of these substances. The biological availability could be influenced by differences in digestion, resorption or elimination by kidney or liver. These factors can determine different concentration of these antioxidants in the blood. However, it is very probable that the plasmatic concentration of both N-acetylcysteine and melatonin were sufficient enough to induce well expressed protective effect, since both drugs were given in high pharmacological doses, several times exceeding the common clinical dose. Acknowledgements The study was supported by the research grants VEGA 2/ 6148/26, 1/3429/06, 1/3442/26 and APVT 51-027404, 51017902 as well as by the Grant Agency of the Czech Republic (305/03/0769), Ministry of Health CR (NR 7786/2004) and the research project AV0Z 5011922. Technical assistance of A. Petrová and I. Nahodilová is highly appreciated. References Allegra, M., Reiter, R.J., Tan, D.X., Gentile, C., Tesoriere, L., Livrea, M.A., 2003. The chemistry of melatonin's interaction with reactive species. J. Pineal Res. 34, 1–10. Anwar, M.M., Meki, A.R.M.A., Rahma, H.H.A., 2001. Inhibitory effects of melatonin on vascular reactivity: possible role of vasoactive mediators. Comp. Biochem. Physiol., C. Toxicol. Pharmacol. 130, 357–367.

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