J Physiol Biochem DOI 10.

1007/s13105-010-0070-2

ORIGINAL PAPER

Effects of antenatal, postpartum and post-weaning melatonin supplementation on blood pressure and renal antioxidant enzyme activities in spontaneously hypertensive rats
S. K. Lee & K. N. S. Sirajudeen & Arunkumar Sundaram & Rahimah Zakaria & H. J. Singh

Received: 7 October 2010 / Accepted: 17 December 2010 # University of Navarra 2011

Abstract Although melatonin lowers blood pressure in spontaneously hypertensive rats (SHR), its effect following antenatal and postpartum supplementation on the subsequent development of hypertension in SHR pups remains unknown. To investigate this, SHR dams were given melatonin in drinking water (10 mg/kg body weight/day) from day 1 of pregnancy until day 21 postpartum. After weaning, a group of male pups continued to receive melatonin till the age of 16 weeks (Mel-SHR), while no further melatonin was given to another group of male pups (Maternal-Mel-SHR). Controls received plain drinking water. Systolic blood pressure (SBP) was measured at 4, 6, 8, 12 and 16 weeks of age, after which the kidneys were collected for analysis of antioxidant enzyme profiles. SBP was
S. K. Lee : A. Sundaram : R. Zakaria Department of Physiology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia K. N. S. Sirajudeen (*) Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia e-mail: sirajuden@kb.usm.my H. J. Singh Faculty of Medicine, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia e-mail: hjsingh@salam.uitm.edu.my

significantly lower till the age of 8 weeks in MaternalMel-SHR and Mel-SHR than that in the controls, after which no significant difference was evident in SBP between the controls and Maternal-Mel-SHR. SBP in Mel-SHR was lower than that in controls and MaternalMel-SHR at 12 and 16 weeks of age. Renal glutathione peroxidase (GPx) and glutathione s-transferase (GST) activities, levels of total glutathione and relative GPx-1 protein were significantly higher in Mel-SHR. GPx protein was however significantly higher in Mel-SHR. No significant differences were evident between the three groups in the activities of superoxide dismutase, catalase and glutathione reductase. In conclusion, it appears that while antenatal and postpartum melatonin supplementation decreases the rate of rise in blood pressure in SHR offspring, it however does not alter the tendency of offspring of SHR to develop hypertension. Keywords Antenatal . Postpartum melatonin . GPx-1 . Blood pressure . SHR

Introduction Epidemiologic data and experimental studies have shown that pre- and postnatal environmental factors contribute to the adult blood pressure profile [6, 22, 29, 49], and positive association between adult hypertension and intrauterine growth restriction, low

S.K. Lee et al.

birth weight, and early postnatal malnutrition has been reported [5, 7, 23, 29]. For example, pups born to SpragueDawley dams given a low protein diet from the 12th day of pregnancy had low birth weight, and their systolic blood pressures at the age of 8 weeks were 2025 mmHg higher than that in controls [49]. Elevated blood pressure in offspring has also been reported following antenatal administration of glucocorticoids, hypoxia, impaired placenta perfusion or diabetes during gestation [1, 2, 31], as well as neonatal hyperoxia and modifications of nutritional regimens [51]. Oxidative stress during critical growth and development period has been proposed to contribute to the perinatal origins of high blood pressure [11]. In this regard, renal oxidative stress has been suggested to play a pathogenetic role in prenatally programmed hypertension in the rat [47]. The kidneys, which are involved in blood pressure regulation, are extremely sensitive to the effects of an adverse environment during early development [4]. A lack of coordinated upregulation of the antioxidant enzymes and discordance between their protein abundance and enzymatic activity has been reported in the kidneys of spontaneously hypertensive rats [52]. Although several perinatal environmental modifications have been attempted to modify adult blood pressure profile in hypertension susceptible models, including dietary supplements and psychosocial stimuli [39, 50, 53], studies examining the impact of melatonin supplementation during the pre- and postnatal periods on the development of hypertension are extremely limited particularly when melatonin, a pineal hormone, is known for its potent antioxidant properties [40, 41]. Most studies examining the effect of melatonin on hypertension have involved humans or animals with established hypertension, where blood pressure has been found to decrease following melatonin supplementation [15, 19, 30, 45]. It is however unclear if melatonin supplementation from as early as the gestation period could reprogramme the blood pressure profile in genetically hypertensive SHR offspring. This study therefore examined the effects of antenatal, postpartum (from birth to day 21 of age), and post-weaning melatonin supplementation for up to 16 weeks of age, on the development of raised blood pressure, as well as in the renal enzymatic antioxidant activities, their protein, and mRNA expressions in male offspring of SHR.

Materials and methods Experimental design Breeding colonies of SHR were obtained from the Laboratory Animals Research Unit of Universiti Sains Malaysia, Health Campus, Kubang Kerian, Kelantan. The rats were housed at room temperature, with 12:12-h light:dark cycle, and had access ad libitum to rat chow and tap water throughout the period of study. Prior to mating, breeder males (>24 weeks old) and virgin females (12 weeks old) were screened for resting systolic blood pressures (SBP) using indirect tail cuff plethysmography (Life Science, Model 179, USA). SHR with SBP of <150 mmHg were removed from the breeding colony. After confirmation of proestrus on a vaginal smear, each female was cohabitated individually overnight with a fertile male. Mating was confirmed by the presence of sperms on the vaginal smear the next morning, and this was then considered as pregnancy day 1. Pregnant rats were housed individually throughout the pregnancy (the number of dams used was 24). Pregnant females were randomised into either a control group (8 dams) or a melatoninsupplemented group (16 dams). Melatonin was administered to the dams from day 1 of pregnancy until postnatal day 21. At birth, each litter was adjusted to eight pups per dam, and from the litter, only male offspring were included in this study. This was to avoid any unexpected variation between male and female rats in their response to melatonin supplementation. The pups were weaned at postnatal day 21 (3 weeks of age). From the pool of male offspring of melatonin-supplemented dams, six of them continued to receive melatonin in drinking water till the age of 16 weeks (Mel-SHR), and another six received plain drinking water (Maternal-Mel-SHR). Similarly, six male offspring from the nonsupplemented dams were randomised as SHR controls. The study design was approved by the Animal Ethics and Welfare Committee of Universiti Sains Malaysia. Melatonin administration Melatonin was given in drinking water at a dose of 10 mg/kg body weight per day. The concentration of melatonin in drinking water was calculated based on the average daily water consumption of the rats. The

Antenatal, postpartum and post-weaning melatonin supplementation

animals were placed individually in metabolic cages to monitor their daily water intake and to ensure that the prescribed dose of melatonin was delivered. During the experiment, concentration of melatonin in the drinking water was adjusted to match the agerelated increase in body weight and water consumption. Drinking bottles containing melatonin were wrapped in aluminium foil to prevent degradation of melatonin by exposure to light. Blood pressure measurement Blood pressures were measured using tail cuff plethysmography between 0800 and 1100 hours, and each time the mean of three consecutive measurements was recorded for each rat. SBP of the dams was measured before and after pregnancy and that of the offspring at 4, 6, 8, 12 and 16 weeks of age. Sample collection At 16 weeks of age, the animals were sacrificed under diethyl ether anaesthesia. Their kidneys were harvested, cleaned with sterile normal saline and snapfrozen in liquid nitrogen and stored at 80C until further analysis. Estimation of enzymatic antioxidants activities, levels of thiobarbituric acid reactive substances and hydrogen peroxide Whole kidney homogenates (10% w/v) were prepared in 0.1 M TrisHCl buffer, pH 7.4 at 04C using an ice-chilled glass homogenising vessel in a homogenizer fitted with Teflon pestle and homogenised at 900 rpm. Homogenates were then centrifuged at 3,000g, at 4C for 20 min to remove nuclear fragments and tissue debris without membrane fragment precipitation. Aliquots of the supernatant fractions were then stored at 80C in microcentrifuge tubes until further analysis. Superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione s-transferase (GST) activities, levels of total glutathione and hydrogen peroxide (H2O2) level were measured using commercially available kits (Calbiochem, Germany for SOD and GPx activity; Cayman, USA for GR and GST activity; Sigma, USA for total glutathione level; Invitrogen, USA for H2O2 level). Catalase (CAT)

activity was measured according to Goth [17]. The levels of thiobarbituric acid reactive substance (TBARS), a lipid peroxidation marker, were measured according to Ohkawa et al. [33]. Total protein was determined using a protein assay kit (Biorad, USA). Determination of GPx-1 and GST-M1 protein levels Enzyme protein and relative mRNA levels were only determined when significant difference in the enzymatic activity was evident between melatonin-treated and untreated SHR. Kidney homogenates (10% w/v) were prepared in 0.1 M TrisHCl buffer, pH 7.4 containing protease inhibitor cocktail (Calbiochem, Germany) at 04C using a Teflon-pestle-fitted homogenizer. Homogenates were then centrifuged at 9,000g at 4C for 10 min. A portion of the supernatant was used for the determination of total protein concentration using a Bio-Rad Protein Assay kit. Total cellular protein (50 g each) was electrophoresed in 12% Trisglycine SDS polyacrylamide gel. The isolated proteins were transferred onto 0.45-m nitrocellulose membrane (Amersham, Germany), blocked in 5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) at room temperature for 30 min, and then washed three times with PBS-T and incubated with the primary antibody to gluthathione peroxidase-1 (GPx-1, 1:10,000; Novus Biologicals, USA), glutathione s-transferase M1 (GSTM1, 1:1,000; Calbiochem, Germany) or beta actin (1:10,000; Calbiochem, Germany) for 2 h at room temperature on an orbital shaker. After incubation, it was washed three times with PBS-T, and the blots were incubated with secondary HRP-conjugated antibody (anti-sheep for GPx-1, anti-rabbit for GST-M1 and beta actin) at room temperature for 1 h. Following three washes with PBS-T, the membrane was developed using enhanced chemiluminescence (ECL) reagent (Amersham, UK) and exposed to a CCD camera (Alpha Innotech Image Analyzer, USA). Band intensity was measured using Alpha Innotech Image Analyzer software. Band intensity of the target protein was normalised with the band intensity of beta actin. Determination of GPx-1 and GST-M1 mRNA levels After extraction of total RNA using Rneasy Mini Kit (Qiagen, Germany), cDNA was synthesised using Revert Aid H Minus First Strand cDNA Synthesis Kit (Fermentas, USA). Primer sequences for beta actin,

S.K. Lee et al.

GPx-1 and GST-M1 were obtained from previous literatures [8, 28]. Real-time PCR assays were performed using SYBR Green I PCR kit (Stratagene, USA) via Mx 3000PTM instrument (Stratagene, USA). The amplification was started with initial PCR activation at 95C for 10 min, which was then followed by three-step cycling consisting of 36 cycles, and involving the denaturation stage at 95C for 30 s; annealing step at 60C for 1 min and then ending with the extension step at 72C for 1 min. After completion of the 36 cycles of amplification, the PCR products were subjected to the dissociation phase. Dissociation curve was checked for a single peak appearance before data analysis. Relative expression of mRNA was determined by the comparative 2TC method [26]. Statistical analysis Normal distribution and the homogeneity of variance of all the measured parameters were checked with normality test and Levene's test. The data in this study met the assumptions for parametric statistical analysis, i.e. data of each parameter were normally distributed, and the variances were equal. Data were analysed using oneway analysis of variance test (ANOVA) for multiple comparisons and followed by post hoc Tukey studies. Pearson correlation test was used to test the correlation between two variables. Data were analysed using Statistical Package for the Social Science (SPSS) software version 15. A p value of <0.05 was considered statistically significant. Data are expressed as mean and standard error of the mean (meanSEM).

difference between the mean SBP of the three groups (Fig. 1). Mean SBP in Maternal-Mel-SHR at 6 and 8 weeks of age were significantly lower than those in age-matched SHR controls (p<0.001; Fig. 1). This difference however was no longer evident at 12 and 16 weeks of age. Mean SBP in Mel-SHR at weeks 6, 8, 12 and 16 were significantly lower than those in SHR controls over the corresponding weeks, and at weeks 12 and 16 in Maternal-Mel-SHR (p<0.001; Fig. 1). SBP in all the three groups at 16 weeks of age however was consistently higher than their SBP at 4, 6, 8 and 12 weeks of age. AOE activities, relative mRNA and protein levels of renal antioxidant enzymes, TBARS, and H2O2 levels GPx activity (Fig. 2) and relative GPx-1 protein level (Table 3) were significantly higher in Mel-SHR when compared to those in SHR controls or those in Maternal-Mel-SHR (p<0.05). However, there was no significant difference in the relative GPx-1 mRNA levels between the three groups. GST activity was significantly higher in Mel-SHR when compared to that in SHR or Maternal-Mel-SHR (p<0.01 and p< 0.05, respectively, Fig. 3). Levels of GST-M1 protein and its mRNA were not different between the three groups. Total glutathione was significantly higher in Mel-SHR when compared to SHR controls (p<0.05; Fig. 4). Levels of TBARS, H2O2, and SOD, CAT and GR activities were also not different between the three groups (Table 3). No significant correlation was evident between SBP and GPx activity, or GST activity or total glutathione either in Mel-SHR (r=0.220, p=0.675; r=0.285, p=0.585 and r=0.457, p=0.362, respectively) or in SHR (r=0.567, p=0.241; r=0.451, p= 0.369 and 0.037, p=0.944, respectively).

Results Maternal SBP, pregnancy duration and outcome SBP was significantly lower in SHR dams receiving melatonin supplementation during pregnancy (p<0.001, Table 1). Melatonin supplementation at a dose of 10 mg/kg body weight per day did not affect the duration of pregnancy, litter size, body weight of pups at birth or the growth of the pups until weaning (Table 2). SBP of offspring In the offspring, SBP at 4 weeks of age were within the normotensive range, and there was no significant

Discussion Although melatonin administration to SHR dams during pregnancy and 21 days postpartum lowered the SBP (Table 1), and delayed the rise in SBP in the offspring, it however did not completely ameliorate the hypertension in the offspring. Continuation of melatonin supplementation till the age of 16 weeks also did not prevent the rise in blood pressure,

Antenatal, postpartum and post-weaning melatonin supplementation Table 1 Maternal systolic blood pressure (SBP, mmHg) in melatonin-treated SHR (Mel-SHR) and untreated controls Groups SHR dams(n=8) Mel-SHR dams(n=8) Values are expressed as meanSEM *p<0.001, compared to SHR dams Maternal SBP (mmHg) before mating 162.592.43 163.022.50 Maternal SBP (mmHg) at day 21 postpartum 168.332.17 148.002.59*

although the rate of rise was lower (Fig. 1). These findings confirm that while daily oral administration of melatonin at a dose of 10 mg/kg bodyweight during the antenatal and 3 weeks postnatal period results in a slightly lower rise in blood pressure in SHR, it however does not cause any epigenetic modifications or in any way alter the tendency of the SHR offspring towards hypertension. In addition, this study also confirmed that oral melatonin (10 mg/ kg body weight per day) given from day 1 of pregnancy does not affect the pregnancy outcome or the growth of the offspring in SHR (Table 2) [18]. There is little information in the literature on the impact of maternal melatonin supplementation on the development of hypertension in the SHR offspring, except for one isolated study investigating the effect of maternal melatonin supplementation on open-field behaviour, where slightly lower SBP was reported between 11 and 27 weeks of age [20], but not before in SHR offspring that had received melatonin during gestation and up to the weaning period. This appears to be the first study of its kind examining the effect of melatonin during the antenatal and postpartum periods on the development of blood pressure in the rat. While it was not the objective of this study to examine the exact mechanism of action of melatonin, some parameters relating to the renal antioxidant system were nevertheless measured, given reports on the antioxidant properties of melatonin [16, 46], and

evidence implicating an altered renal antioxidant status in the pathogenesis of hypertension [47, 52]. With regard to the antioxidant status, renal GPx activity, its relative GPx-1 protein level, GST activity and total glutathione levels were significantly higher in Mel-SHR when compared to SHR and MaternalMel-SHR (Figs. 2, 3 and 4). As Maternal-Mel-SHR ceased to receive further melatonin supplementation from the age of 3 weeks onwards, the effect of melatonin might have dissipated between the age of 3 and 16 weeks. No significant difference was evident in SOD or CAT activities between the three groups (Table 3). Interestingly, relative GPx-1 mRNA levels were not different between the three groups (Table 3). Melatonin has been shown to increase GPx-1 mRNA level in other tissues like those from human chorion, rat liver and rat brain cortex [21, 34, 35], but this effect was however not evident in this study. The reason for this is not clear, but it might be dependent on the type of tissue, dose and duration of melatonin administration [37]. The higher relative GPx-1 protein and activity in Mel-SHR (Fig. 2, Table 3) suggests the presence of higher relative functional protein levels following melatonin supplementation. It has been reported that GPx protein is much more vulnerable to peroxide-induced and hydroxyl radical-mediated inactivation and degradation as compared to other enzymatic antioxidants like SOD and CAT protein

Table 2 Duration of pregnancy, litter size, birth weight and body weight of pups at postnatal day 21 of melatonin-treated (Mel-SHR) and untreated SHR (SHR) Groups Gestation duration Number of pups (days) per litter 22.50.10 22.30.13 8.80.36 9.00.30 Mean birth weight (g); n Mean body weight of male pups at day 21 litter=8 postnatal (g); n litter=8 5.400.04 5.340.03 26.450.09 27.780.08

SHR(n=8) Mel-SHR (n=8)

Values are expressed as meanSEM

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Systolic blood pressure (mmHg)
200
*** ^^^ *** ### *** ###

150

*** ^^^

SHR controls Maternal-Mel-SHR Mel-SHR

100 4 6 8 12 16

Age (weeks)

Fig. 1 Systolic blood pressure (mmHg) in controls and melatonin-treated SHR offspring. Values are expressed as meanSEM (n=6 per group). ***p<0.001, Mel-SHR compared to SHR controls; ^^^p<0.001, Maternal-Mel-SHR compared to SHR controls; ###p<0.001, Mel-SHR compared to Maternal-Mel SHR

[38]. The higher GPx-1 protein level in melatonin treated group might therefore be due to a protective effect of melatonin in preventing inactivation or breakdown of GPx protein. No significant differences were evident in the relative GST-M1 protein or its mRNA levels between the three groups. Whether the slight hypotensive effect evident in rats given melatonin is due to its effects on renal GPx and GST activities is uncertain, although their higher levels following melatonin supplementation seem to suggest so. Similar increases might have also occurred in other tissues, which might have collectively contributed to the lower blood pressure. In this regard, increased blood GPx activity has been reported in the sheep following melatonin implants [3]. GPx catalyses the detoxification of harmful peroxides, including hydrogen peroxide (H2O2), to water, and a lower renal GPx activity in SHR has been reported before [24]. It might be hypothesised that GPx deficiency or its
400 GPx activity (U/mg protein) 380 360 340 320 300 280 SHR controls MaternalMel-SHR Mel-SHR

* ^

Fig. 2 Renal GPx activity in controls and melatonin-treated SHR offspring. Values are expressed as meanSEM (n=6 per group). *p<0.05, compared to SHR controls; ^p<0.05, compared to Maternal-Mel-SHR

decreased activity could lead to increased accumulation of peroxides like H2O2, which, after its conversion to hydroxyl radical, could inactivate the vasodilator, nitric oxide, through formation of peroxynitrite and impair endothelium-dependent relaxation [32, 44]. GPx deficiency has also been linked to endothelial dysfunction [10, 14] and atherosclerosis [25], both of which are believed to contribute to the pathogenesis of hypertension. However, while GPx activity was increased in the melatonin treated group, H2O2 levels were not different between the three groups (Table 3). In addition, the levels of TBARS, a marker of lipid peroxidation, were also not significantly different between the three groups (Table 3). It has been reported that oral melatonin given to SHR, aged 2325 weeks for 6 weeks, attenuated the raised blood pressure and also lowered the levels of renal TBARS compared to the untreated SHR controls [30]. Melatonin supplementation has been found to reduce the levels of markers of lipid peroxidation in subjects exposed to oxidative stress [12, 13, 36] but not in normal control rats [12]. The reason for the difference in the results of this study and the others is not clearly apparent. Measurement of other markers of oxidative stress would also be required to confirm if the hypotensive action of melatonin was through the maintenance of redox homeostasis. From the data so far, it seems unlikely that the hypotensive effect of melatonin evident in this study is related to its direct or indirect H2O2 scavenging activity, but might be related to either the upregulation of other antioxidants or scavenging of other peroxides that might be responsible for the raised blood pressure. Alternatively, the hypotensive actions of melatonin might be due to a number of other hypotensive mechanisms attributed to melatonin, some of which include its vasodilating effect through MT2 receptors [27], and reduction in sympathetic activity [9]. GSH is an essential cosubstrate for GPx enzymatic activity, and GR converts the oxidised glutathione to its reduced state. Although GR activity was not significantly different between Mel-SHR and controls, total glutathione level was significantly higher in melatonin-treated rats. Stimulation of intracellular synthesis of GSH by exogenous melatonin has been reported before [48], and lower erythrocyte GSH level has been found in subjects with essential hypertension when compared to normotensive volunteers [43]. The lower ratio of reduced/oxidised glutathione in hyper-

Antenatal, postpartum and post-weaning melatonin supplementation Table 3 Activities, relative mRNA and protein levels of renal antioxidant enzymes, and H2O2 levels in control and melatonin-treated SHR offspring Parameter/group SOD activity (U/mg protein) CAT activity (U/mg protein) Relative GPx-1 protein level Relative GPx-1 mRNA level (fold change) Relative GST-M1 protein level Relative GST-M1 mRNA level (fold change) GR activity (U/mg protein) TBARS level (nmol/mg protein) H2O2 level (M) Values are expressed as meanSEM *p<0.05, compared to SHR controls; **p<0.05, compared to Maternal-Mel-SHR SHR controls(n=6) 15.931.00 554.1318.72 1.000.04 1.000.13 1.000.07 1.000.07 70.610.86 2.130.05 40.513.45 Maternal-Mel-SHR(n=6) 14.680.73 545.8312.77 1.010.05 1.110.09 0.980.06 1.030.08 71.691.16 2.190.07 41.092.80 Mel-SHR(n=6) 14.560.95 574.5316.65 1.170.06*, ** 1.160.08 1.060.06 1.060.05 73.431.38 2.250.10 42.242.62

tensives is reportedly restored to normal following antioxidant (vitamin C and E) supplementation [42]. It is therefore possible that the higher GPx activity evident following melatonin supplementation in MelSHR might be due to a simultaneous stimulation of GSH, which is an important cofactor in GPx activity, but its contribution to the lower blood pressure however remains unclear. Analysis of the correlations between SBP and activities of GPx and GST and total glutathione did not reveal any significant correlations either in untreated SHR or in melatonin-supplemented SHR, indicating that melatonin does not significantly alter the relationships between SBP and GPx activity or GST activity or total glutathione. In conclusion, it appears that although melatonin supplementation during antenatal, postpartum and post-weaning periods delays or reduces the rate of
60
GST activity (U/mg protein)

rise in blood pressure in SHR offspring, it however has little influence on the underlying genetic abnormality in this strain of rat as SBP returns to its hypertensive levels soon after cessation of melatonin supplementation. Antenatal and 3 weeks of postnatal melatonin supplementation did not reprogramme the blood pressure profile in SHR offspring. We are however unsure if higher doses of melatonin might have suppressed or delayed the rise in blood pressure further. Although the hypotensive effect of melatonin appears concomitant to changes in GPx, GST and total glutathione level, there was however no significant correlation between SBP and the activities of GPx or GST or total glutathione. Given that the levels of TBARS and H2O2 were not different between the three groups, their involvement in the pathogenesis of
Total glutathione level (nmol/mg protein)

** ^

50 40 30 20 10 0 SHR controls Maternal-MelSHRv Mel-SHR

1.2

0.8

0.6

0.4 SHR controls MaternalMel-SHR Mel-SHR

Fig. 3 Renal GST activity in controls and melatonin-treated SHR offspring. Values are expressed as meanSEM (n=6 per group). **p< 0.01, compared to SHR controls; ^p < 0.05, compared to Maternal-Mel-SHR

Fig. 4 Renal total glutathione level in controls and melatonintreated SHR offspring. Values are expressed as meanSEM (n=6 per group). *p<0.05, compared to SHR controls

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hypertension in SHR or their role in the hypotensive action of melatonin still remains unclear and needs further investigation.
Acknowledgements This study was supported by Research University Grant, Universiti Sains Malaysia (RU 1001/PPSP/ 811018) and Fundamental Research Grant Scheme (203/PPSP/ 6170021), Malaysia. Lee SK is a scholar under the USM Fellowship Scheme.

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