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,1
* Vascular Biology Center, Department of Pharmacology and Toxicology, and Department of Surgery, Medical College of Georgia, Augusta, Georgia 30912
1To whom requests for reprints should be addressed at Vascular Biology Center, Medical College of Georgia, 1459 Laney-Walker Boulevard, Augusta, GA 30912. E-mail: dpollock{at}mail.mcg.edu
Abstract
Evidence for endothelin (ET) involvement in the control of fluid volume balance and arterial pressure has been derived in part from the observations that rats lacking the ETB receptor develop hypertension when placed on a high-salt (HS) diet. The present study was designed to determine the effect of superoxide on salt-induced hypertension in male and female ETB-deficient (sl/sl) and wild-type control (wt) rats. After 14 days on a HS (8% NaCl) diet, female sl/sl rats had significantly elevated arterial pressure (183 ± 2 mm Hg, tail cuff) compared with female wt rats (134 ± 2 mm Hg). The response to a HS diet was lower in male sl/sl rats (166 ± 6 mm Hg) yet was significantly greater than that in male wt controls (135 ± 3 mm Hg). Separate groups of male and female sl/sl and wt rats were given tempol (1 mM in drinking water) during HS treatment. Arterial pressures were 149 ± 5 mm Hg in male and 143 ± 3 mm Hg in female sl/sl rats treated with tempol, values that were similar to those of controls on a normal salt diet. After 14 days, however, male and female sl/ sl rats recovered from the blood pressure–lowering effects of tempol. On Day 15, arterial pressures in female sl/sl rats on a HS diet were 160 ± 6 mm Hg and 177 ± 6 mm Hg in tempol-treated and untreated groups, respectively. In male sl/sl rats, arterial pressures were 155 ± 3 mm Hg and 165 ± 5 mm Hg in tempol-treated and untreated groups, respectively. On Day 15, no differences among groups with or without tempol were observed in plasma thiobarbituric acid–reactive substance (TBARS) concentrations or in urinary excretion of TBARS. Plasma ET-1 concentrations were significantly higher in female vs. male sl/sl rats. These results indicate that the early stages of salt-dependent hypertension produced by ETB receptor deficiency are dependent on superoxide and that the elevated pressure in the female rats may be due to elevated circulating levels of ET-1.
Key Words: gender • endothelin • reactive oxygen species • salt-sensitive hypertension • ET receptor
Introduction
The endothelin (ET) system is an important regulator of blood pressure. Binding of vascular smooth muscle cell ETA receptors by ET-1 results in vasoconstriction, while ET-1 activation of ETB receptors on endothelial and renal tubular cells results in vasodilation and natriuresis (1–3). It has been previously demonstrated that treatment with a high-salt (HS) diet in rats following chronic blockade of the ETB receptor or in ETB receptor–deficient rats results in the development of hypertension and elevated plasma and urinary ET-1 (4–8). From these studies, it has been concluded that the lack of ETB receptor function results in a salt-sensitive form of hypertension.
Other results have suggested that salt-dependent hypertension in ETB receptor–deficient rats may be linked with an ETA receptor–dependent increase in reactive oxygen species (5). Elmarakby et al. (5) observed that ETA receptor blockade prevented the hypertension and increase in oxidative stress in ETB receptor–deficient rats placed on a HS diet. ET-1 has been shown to stimulate superoxide production in the vasculature; therefore, increased ET-1 in salt-sensitive hypertension may contribute to an increase in oxidative stress and an elevation in blood pressure (9–11). This is supported by the finding that the elevated blood pressure with ETB receptor blockade or long-term infusion of exogenous ET-1 may be attenuated by antioxidant treatment (8, 12). It has recently been reported that treatment with the superoxide dismutase (SOD) mimetic tempol attenuates the development of hypertension in the initial days of treatment in rats on a HS diet given an ETB receptor antagonist, supporting the hypothesis that superoxide contributes to the maintenance of arterial pressure in rats on a HS diet (8).
A gender difference exists in the development of salt-sensitive hypertension in ETB receptor–deficient rats, with females having a greater rise in blood pressure and a larger increase in plasma ET levels in response to a HS diet compared with male ETB receptor–deficient rats (6). Therefore, we hypothesize that the greater rise in blood pressure in female ETB receptor–deficient rats on a HS diet is due to the stimulation of superoxide. This hypothesis was tested by treating male and female ETB receptor–deficient rats with tempol in conjunction with a HS diet.
Materials and Methods
Tail-Cuff Measurements and Metabolic Cage Studies.
Male and female wild-type (wt) and homozygous (sl/sl) rats deficient of ETB receptors on a Wistar-Kyoto genetic background (13) were obtained from our local breeding colony. Experiments were conducted (at a mean age of 13 weeks) in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved and monitored by the Medical College of Georgia Institutional Animal Care and Use Committee. Rats were housed in temperature and humidity–controlled light-cycled quarters. All rats were placed on a HS (8% NaCl) diet, and a subset of male and female rats of both strains received tempol (1 mM) in the drinking water. This concentration of tempol has been shown to reduce super-oxide levels in the kidney and vascular tissue (14, 15). Blood pressures were confirmed using the tail-cuff method (16). Before beginning treatment and at 7 and 14 days after starting treatment, rats were placed in metabolic cages (Nalgene Co., Rochester, NY) to allow quantitative measurement of food and water intake and to facilitate 24-hr urine collection. Under sodium pentobarbital anesthesia (65 mg/kg ip), a terminal blood sample was obtained and centrifuged, and plasma was frozen at –80°C.
Assays and Chemicals.
Urinary ET levels were determined using radioimmunoassay (GE Healthcare BioSciences, Piscataway, NJ). Plasma ET concentrations were measured using enzyme-linked immunosorbent assay (ELI-SA) (QuantiGlo; R&D Systems, Minneapolis, MN). Plasma and urinary thiobarbituric acid–reactive substance (TBARS) concentrations were determined by measuring the reaction of malondialdehyde with thiobarbituric acid (OXItek; ZeptoMetrix Corporation, Buffalo, NY). Urinary H2O2 excretion was determined using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, Eugene, OR). Plasma 8-isoprostane levels were determined using enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). The urinary microalbumin level was measured using a competitive ELISA (Nephrat; Exocell, Philadelphia, PA).
Statistical Analysis.
Two-factor analysis of variance with repeated measures was used to evaluate tail-cuff pressure and excretion data. Post hoc contrasts were used to compare individual means using Bonferroni correction. One-way analysis of variance was used to compare data obtained from analysis of plasma taken at the end of the experiment. Values are reported as means ± SEM. P < 0.05 was considered statistically significant.
Results
Before the start of the HS diet, male and female rats had similar water intake (data not shown). Water intake was increased in male and female rats in response to a HS diet, although the increase in water intake was greater in male rats. Tempol treatment did not affect water or food intake in male or female rats.
As previously shown (6), a HS diet resulted in an increase in systolic arterial pressure in male and female sl/sl rats but not in wt rats (Fig. 1
). The increase in pressure was greater in female sl/sl rats compared with male sl/sl rats. In both genders, treatment with tempol delayed the rise in arterial pressure in response to a HS diet and abolished the gender difference. Tempol did not, however, block a rise in pressure. Following 14 days of a HS diet, male and female sl/sl rats were recovering from the blood pressure–lowering effects of tempol.
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). Treatment with tempol had no effect on plasma ET levels in male rats but lowered ET levels in female sl/sl rats. A HS diet increased urinary ET excretion irrespective of genotype and gender (Table). The increase was more pronounced in wt males compared with sl/sl males. Tempol did not alter ET excretion in any group.
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) were comparable with respect to genotype and gender; tempol had no effect on these measurements. H2O2 excretion was increased in response to a HS diet in wt and sl/sl male and female rats. The increase in H2O2 excretion was greater in sl/sl males compared with wt males. Treatment with tempol decreased H2O2 excretion in sl/sl rats only. Among females, there was no difference with regard to genotype in the increase in H2O2 excretion; however, similar to males, tempol reduced H2O2 in female sl/sl rats only. Discussion
The ETB receptor–deficient rat has elevated plasma levels of ET-1 and is a model of salt-sensitive hypertension that can be prevented by ETA receptor blockade (4, 5). In the past few years, accumulating evidence has indicated that ET-1 can stimulate increases in oxidative stress, particularly superoxide (11, 12, 17, 18). In contrast to most models of hypertension, female ETB receptor–deficient rats develop more severe hypertension in response to a HS diet compared with male rats (6). Therefore, the present study addressed two primary questions: first, whether salt-dependent hypertension in the ETB receptor–deficient rat was dependent on superoxide and, second, whether the gender difference in the hypertension in this model could be due to differences in superoxide activity. Treatment with tempol blunted the development of salt-dependent hypertension in male and female ETB receptor–deficient rats. This finding supports a role for superoxide to contribute to the development of salt-dependent hypertension in ETB receptor–deficient rats. Furthermore, these data support the hypothesis that female sl/sl rats produce more superoxide or are more sensitive to the blood pressure–raising effects of superoxide compared with males.
In male and female sl/sl rats, tempol severely blunted the development of salt-induced hypertension, abolishing the gender difference in blood pressure. In both sexes, there appeared to be an "escape" from the blood pressure–lowering effects of tempol toward the end of the second week of treatment. This phenomenon was similar to previous observations in Sprague-Dawley rats in which hypertension was produced by chronic ETB receptor blockade and a HS diet (8). In contrast to the present study, it was previously observed that tempol increased H2O2 levels following chronic ETB receptor blockade and a HS diet (8). Tempol is an SOD mimetic and as such can increase the generation of H2O2 derived from superoxide. Antioxidants such as catalase and glutathione peroxidase can then convert H2O2 to water. With short-term ETB receptor blockade, this antioxidant capacity may be overwhelmed, resulting in increased H2O2 levels. In contrast, a decrease in H2O2 in sl/sl rats may be the result of an increase in antioxidant potential to catalyze the breakdown of H2O2 that may be established as the ETB receptor–deficient rats grow and develop. Future studies will need to investigate the level of antioxidant capacity during genetic and pharmacologic disruption of ETB receptor function.
Measures of oxidative stress in the present study, however, did not reveal a clear gender difference in the degree of oxidative stress. It was somewhat surprising that sl/sl rats of either sex did not have any greater degree of oxidative stress compared with wt control rats, but this could be due to the indirect nature of these measurements or, as already mentioned, an upregulation of antioxidant mechanisms in this strain. Moreover, there was no direct evidence of tempol lowering measures of oxidative stress; yet, tempol treatment delayed the rise in blood pressure in male and female sl/sl rats. It is possible that the blood pressure–lowering effects of tempol are occurring independent of lowering superoxide levels. However, we find a more likely explanation is that tempol lowers local super-oxide levels in the kidney or in peripheral nerves that would not result in changes in plasma reactive oxygen species, allowing for better blood pressure control. These findings unfortunately reflect the reality that all of the current means of assessing superoxide in vivo have severe limitations.
The lack of a gender difference in blood pressure during tempol treatment in ETB receptor–deficient rats on a HS diet is consistent with the hypothesis that superoxide contributes to the salt-induced hypertension in this model and that the higher pressures seen in the female rats can be accounted for by superoxide. As previously reported, female ETB receptor–deficient rats not only have an exaggerated salt-dependent hypertension compared with male rats, but this is associated with higher levels of circulating ET-1 compared with males (6). Given that ETA receptor blockade can normalize blood pressure in this model (5), it is reasonable to postulate that the elevated ET-1 in female rats accounts for the elevated arterial pressure. ETA receptor blockade can prevent or reverse the hypertension produced by chronic ETB receptor blockade (7, 19). The mechanism for why female rats on a HS diet have higher ET-1 levels is not clear but may be related to increased oxidative stress. For the most part, ET-1 levels appear to be highly sensitive to the activity of ETB receptors, which have even been referred to as clearance receptors. Because neither female or male sl/sl rats have functional ETB receptors within the circulation, differences in ETB receptor function cannot account for these differences. It would appear that a HS diet stimulates more ET-1 production in females compared with males, although the mechanism of how a HS diet increases ET-1 production or activity is not known. Following tempol treatment, plasma ET-1 levels in female sl/sl rats are decreased to the level seen in male sl/sl rats, regardless of tempol treatment. Although oxidative stress is implicated in the rise in plasma ET-1 in female sl/sl rats, the mechanism by which this is occurring is unknown.
Finally, male and female sl/sl rats, but not wt rats, had elevated microalbumin excretion when placed on a HS diet, indicating some degree of hypertension-induced renal injury. Tempol prevented microalbuminuria only in the male rats, indicating that the injury in female rats may not be dependent on superoxide. However, it is possible that a more severe degree of oxidative stress–induced injury is more difficult to overcome with tempol treatment in the female rats. A more thorough investigation of renal injury in this model is warranted.
In conclusion, results from our study indicate that the early stages of salt-dependent hypertension produced by ETB receptor deficiency are dependent on superoxide. The elevated pressure in the female rats may be due to elevated circulating levels of ET-1.
Acknowledgments
We thank Lindsay Hammett, Amy Dukes, Heather Walker, and Carolyn Rhoden for expert technical assistance.
Footnotes
This study was supported by National Heart Lung and Blood Institute grants HL64776, HL69999, and HL74167 and by American Heart Association Established Investigator Awards (J.S.P. and D.M.P.).
Received for publication September 30, 2005. Accepted for publication December 21, 2005.
References
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