Abstract Enhanced production of oxygen free radicals may play a role in hypertension by affecting vascular smooth muscle contraction, resistance to blood flow, and organ damage. The aim of this study was to determine whether oxygen free radicals are involved in the development of salt-induced hypertension. Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed either a high salt (6.0% NaCl) or low salt (0.3% NaCl) diet for 4 weeks. The high salt diet caused the development of severe hypertension in Dahl-S animals and had no effect on blood pressure in Dahl-R animals. A tetranitroblue tetrazolium dye was used to detect superoxide radicals in microvessels of the mesentery. Light absorption measurements revealed enhanced staining along the endothelium of arterioles and venules in hypertensive Dahl-S animals, with significantly lower values in normotensive animals. In addition, a Clark electrochemical electrode was used to measure hydrogen peroxide levels in fresh plasma. Hypertensive Dahl-S animals had a higher plasma hydrogen peroxide concentration compared with their normotensive counterparts (2.81±0.43 versus 2.10±0.41 μmol/L), while no difference was detected between high- and low salt–treated Dahl-R animals (1.70±0.35 versus 1.56±0.51 μmol/L). The plasma hydrogen peroxide levels of all groups correlated with mean arterial pressure (r=.77). These findings demonstrate an enhanced production of oxygen free radicals in the microvasculature of hypertensive Dahl-S rats.
- hypertension, salt-sensitive
- free radicals
- nitroblue tetrazolium
- hydrogen peroxide
- rats, Dahl
An important unresolved issue in hypertension research is the mechanism for organ damage during the development of the syndrome. Reactive oxygen species such as superoxide radical (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (·OH), may play a critical role in the pathogenesis of hypertension, as well as in other conditions such as atherosclerosis, stroke, and myocardial infarction.1 Oxygen free radicals may play a dual role. On one hand, they may affect vascular resistance by inactivating NO, thereby causing arteriolar vasoconstriction and elevation of peripheral hemodynamic resistance;2 on the other hand, they may serve as trigger mechanisms for lesion formation.
Recent studies by Prabha et al3 and Kumar and Das4 indicate that essential hypertension is associated with increased superoxide anion and hydrogen peroxide production by circulating leukocytes as well as decreased NO and antioxidant levels. They also showed that free radical production and NO levels in hypertensives reverted to normal values when treated with antihypertensive medication. In the genetic salt-sensitive Dahl hypertensive rat (Dahl-S), there is increasing evidence for a suppression of NO release, presumably from endothelial cells, which leads to constriction of peripheral arterioles and elevation of peripheral vascular resistance.5 There is also evidence that the Dahl-S has an elevated number of circulating leukocytes that produce superoxide compared with its normotensive control, the Dahl salt-resistant rat (Dahl-R).6 But no direct measurements of free radical production in the Dahl hypertensive model are available.
Thus, the purpose of this study was to study the involvement of oxygen radicals in the Dahl model of hypertension. In addition to hemodynamic and arteriolar tone measurements, we used tetranitroblue tetrazolium reduction as an index of spontaneous in vivo superoxide radical production in microvessels of the rat mesentery. Furthermore, we used a modification of the Clark electrode method to determine plasma hydrogen peroxide levels in Dahl-R and Dahl-S animals.
All animal procedures were previously reviewed and approved by the University of California at San Diego Animal Subject Committee. Male Dahl-S and Dahl-R rats were obtained at 6 weeks of age from Harlan Sprague-Dawley (Indianapolis, Ind) and maintained on a diet of standard rat chow (0.3% NaCl) and water ad libitum for a 1-week observation period. The animals were then divided into four different diet groups as follows: Dahl-S, 0.3% NaCl control diet (n=9); Dahl-S, 6.0% NaCl enriched diet (n=12); Dahl-R, 0.3% NaCl control diet (n=7); and Dahl-R, 6.0% NaCl enriched diet (n=7). All animals were maintained on either the 0.3% NaCl control diet or the 6.0% NaCl enriched diet (ICN Nutritional Biochemicals) for 4 weeks. A femoral catheter (Clay Adams PE-50), was inserted for blood pressure measurements and blood sample withdrawal using 4% xylocaine (Astra USA Inc) as a local anesthetic. After the animals were allowed to recover from the surgical procedure, mean, systolic, and diastolic blood pressures and heart rate were continuously monitored in conscious animals via a transducer (Statham) for 30 minutes.
After administration of general anesthesia with pentobarbital (50 mg/kg IV, Abbot Laboratories), rats were placed on a heated stage. The abdomen was opened by a small midline incision, and the ileocecal portion of the mesentery was gently exteriorized for intravital microscopy as previously described.7 The preparation was kept at 37°C and continuously superfused (1.0 mL/min) with a Krebs-Henseleit bicarbonate-buffered solution saturated with a 95% N2 5% CO2 gas mixture. Special precautions were taken to avoid interruption of the suffusate on the tissue because even superficial drying causes rapid cell injury.8
The mesenteric microcirculation was visualized through an intravital microscope (Technical Instruments) with the use of a digital color coupled charge device (CCD) camera (Optronics Engineering) and a 40× water immersion objective (Zeiss). Single unbranched vessels with diameter between 20 and 40 μm and length of approximately 150 μm were selected. The images were recorded with a videocassette recorder (Panasonic) for playback analysis.
In addition to steady state values, arterioles were studied after local application of the vasodilator papaverine (1.0 mg/mL, American Regent Laboratories). Measurements before and after application of papaverine provided steady state lumen diameters (Dss) and maximal diameters (Dmax). All diameter measurements were measured off-line using NIH Image 1.35 at constant central blood pressures native to each animal. The tone (T) was computed as T=(Dmax−Dss/Dmax) and is a nondimensional parameter that specifies the degree of active smooth muscle constriction such that T=0% in dilated vessels and T=100% in fully constricted vessels with an occluded lumen. No correction for the irregular shape of the lumen cross section of contracting arterioles was made.9
In Vivo Superoxide Production
Following the method of DeLano et al,10 TNBT (Vector Laboratories) was used to detect superoxide radicals in vivo. TNBT is reduced by superoxide radicals into the insoluble blue/black product formazan. This reaction is specific for superoxide radicals, since the reduction can be inhibited by superoxide dismutase.11 TNBT was topically superfused over the mesentery preparation for 1 hour. The preparation was then rinsed with the superfusion buffer, fixed with formalin (Sigma Chemical), and excised.
Whole-mount specimens were placed on a glass slide and viewed using a laboratory microscope (Leitz) and 40× water immersion objective. Images were digitized using a CCD camera and NIH Image 1.35. Using a video photometric window (1×5 μm), a series of average gray-value measurements were made along the endothelium of mesenteric microvessels. As an index of superoxide formation, measurements were expressed in terms of light absorption (A) as where It is the gray value along the endothelium and Io is the gray value in an avascular region without formazan deposits. Measurements were carried out on arterioles as well as venules.
Plasma Hydrogen Peroxide Production Levels
Blood samples were taken from the femoral arterial catheter with 10 U/mL heparin as an anticoagulant (Pharmacia & Upjohn Co), and immediately placed on ice (4°C) until measurements were performed. All measurements were completed on samples within 2 to 3 hours, a period of time that did not significantly affect the peroxide levels in these samples.
To measure hydrogen peroxide production levels, a 0.75-mL aliquot of anticoagulated blood was placed in a 2-mL centrifuge tube, incubated at 37°C for 10 minutes, and then centrifuged at 500g for 10 minutes. Thereafter, either sodium azide (NaN3; 10 μL of a 2-mol/L stock solution; Sigma Chemical) or catalase (10 μL containing 0.25 mg of the enzyme; Calbiochem) was added to the plasma layer and gently stirred. The Clark electrode was placed in the plasma of the aliquot (about 3 mm above the buffy coat), and its output was recorded for 10 minutes, at which point a steady electrode current was reached. Measurements were performed in duplicate, and the electrode was cleaned between measurements with a potent aqueous oxidizing solution (3% hydrogen peroxide; Fisher Scientific).12
Hydrogen peroxide was measured continuously with the electrode’s platinum anode, which was biased at 0.6 V with respect to the silver/silver chloride cathode. Hydrogen peroxide is oxidized at the surface of the anode according to the reaction H2O2→O2+2H++2e−.
Because the output voltage generated by the electrode is not specific for hydrogen peroxide, a differential measurement was carried out using catalase and sodium azide. Catalase is an enzyme that which catalyzes the reaction of hydrogen peroxide to oxygen and water (2H2O2→O2+2H2O) and serves to eliminate hydrogen peroxide. Thus, the addition of catalase provides a baseline or background current. In contrast, sodium azide inhibits enzymes such as catalase and myeloperoxidase, which break down hydrogen peroxide. Thus, the reading in a sample with azide gives the current caused by hydrogen peroxide as well as the background. The difference between these two currents provides a signal that is caused by hydrogen peroxide alone. Calibration of the system was performed by adding known concentrations of hydrogen peroxide to a buffered saline solution and to plasma. A linear response was found in the range between 0 and 10 μmol/L.
Measurements are presented as the mean value±SD. Unpaired comparisons between the 6.0% NaCl and 0.3% NaCl groups were carried out by Student’s t test. Analyses were performed on Statworks 1.2. Values of P<.01 were considered significant.
High salt–treated Dahl-S animals had substantially elevated mean, systolic, and diastolic blood pressures when compared with low salt–treated Dahl-S animals (P<.001; Table 1⇓). In contrast, the high salt diet had no effect on blood pressure in Dahl-R animals. Hypertensive Dahl-S animals also displayed a lower body weight and hematocrit compared to its normotensive counterpart. Basal heart rates remained unchanged among all groups.
In Vivo Superoxide
Light micrographs of TNBT-stained mesenteric arterioles and venules are shown in Fig 1⇓. Reduced TNBT, in the form of blue/black formazan crystals, is seen along the endothelium of vessels and not in the surrounding interstitial space. Based on light absorption measurements, high salt–treated Dahl-S arterioles stain 18% darker than low salt–treated Dahl-S arterioles (Fig 2⇓). In contrast, only a 6% difference in staining level was detected between high and low salt–treated Dahl-R arterioles. Similarly, high salt–treated Dahl-S venules stained 15% darker than low salt–treated Dahl-S venules, and no difference was detected between high and low salt–treated Dahl-R venules. Invariably, TNBT staining in venules was stronger than in arterioles.
Plasma Hydrogen Peroxide Production
Hypertensive high salt–treated Dahl-S animals had a significantly higher plasma hydrogen peroxide concentration when compared with their normotensive low salt controls (2.81±0.43 versus 2.10±0.41 μmol/L; P<.01; Fig 3⇓). In contrast, no plasma peroxide difference was detected between high and low salt–treated Dahl-R animals (1.70±0.35 versus 1.56±0.51 μmol/L), and both of these animals had lower peroxide values than either Dahl-S animal. The plasma hydrogen peroxide concentration showed a linear correlation with mean arterial blood pressure in the four experimental groups (r=.77, n=35; Fig 4⇓).
No difference in resting luminal diameter among the groups was found. The resting diameters for high and low salt–treated Dahl-R and high and low salt–treated Dahl-S animals were 17.6±4.0, 17.6±2.2, 18.6±3.5, and 18.5±4.6 μm, respectively. The arterioles of the high salt–treated Dahl-S animals exhibited significantly higher tone values than the low salt–treated Dahl-S animals (10.9±4.4 versus 3.0±0.9%; P<.001). In contrast, no difference was detected between high and low salt–treated Dahl-R animals (3.0±1.2% versus 3.6±0.5%). There was a positive linear correlation between arteriolar tone and plasma peroxide concentration (r=.86, n=35; Fig 5⇓).
The question of whether elevated blood pressure alone constitutes a risk for cardiovascular complications in hypertensive subjects is unresolved. Few studies have provided a direct link between high blood pressure per se and the complications that accompany the hypertensive state, suggesting that direct treatment of elevated blood pressure may not necessarily address other factors in the development of the disease. The data in the literature provide no indication of the mechanisms that predispose hypertensives to organ injury.
Recently, we obtained direct evidence for a significant oxidative stress in the microvascular wall of spontaneously hypertensive rats (SHR) when compared with their normotensive control, the Wistar-Kyoto rat (WKY).13 Oxygen radicals are also produced spontaneously by circulating leukocytes of Dahl-S hypertensive rats but significantly less in leukocytes of their normotensive control, the Dahl-R rat.6 The present observations suggest that the progression of the hypertensive syndrome in the Dahl rat is also accompanied by overproduction of oxygen free radicals in the circulation.
It has been demonstrated in vivo that the establishment of salt-induced hypertension in the Dahl model is mediated by a suppression of basal NO synthesis/release5 and that flow-dependent vasodilation is modulated by salt intake even in Dahl-R rats.14 Moreover, hypertension in the Dahl model is preventable by administration of l-arginine, an NO donor.15 However, increasing evidence suggests that hypertension may be associated with increased decomposition of NO by superoxide and less by altered release of NO.16
In the SHR, the elevated production of oxygen free radicals involves an adrenal component, since adrenalectomy leads to a reduction of oxidative stress as well as arterial blood pressure.13 Administration of glucocorticoids to normotensive adrenalectomized SHR leads to return of the hypertensive state and elevated oxidative stress. There is also evidence to suggest that the Dahl hypertensive rat model involves an adrenal component.17 In fact, the involvement of mineralocorticoids in the development of salt-induced hypertension has been well characterized.18 19 20 However, the underlying mechanism that leads to the oxidative stress remains largely unexplored.
To understand the mechanism for oxygen free radical formation in hypertension, the cellular source must be identified. The endothelial cell, which is recognized as a source of NO, has also been identified as a potential source of oxygen free radical generation.21 22 Superoxide radicals in and around the vascular endothelial cells were found to play a critical role in the pathogenesis of hypertension. The present observations on the location of tetranitroblue tetrazolium reaction products, or formazan crystals, in the rat mesentery support the hypothesis that a major source of oxygen free radicals may be the endothelium of both the arterioles and venules. In addition, the increased staining in high salt–treated Dahl-S animals suggests that hypertension in the Dahl rat is associated with increased superoxide generation. Interestingly, the staining level in venules was higher than arterioles, the significance of which remains unknown. Other sources of oxygen free radicals could be circulating leukocytes6 and mitochondria.
The increase in plasma hydrogen peroxide concentration is most likely the result of the increase in superoxide. Because breakdown of superoxide, either spontaneously or via superoxide dismutase, leads to hydrogen peroxide, hydrogen peroxide levels may be used as a secondary, more stable indicator of oxidative stress. Not all superoxide radicals, however, are converted into hydrogen peroxide, since NO has been shown to be highly reactive with superoxide to form the stable product peroxynitrite (ONOO−).23 A higher arteriolar tone is associated with the increase in plasma hydrogen peroxide level in the high salt Dahl-S when compared with the low salt Dahl-S. The correlation between arteriolar tone and plasma hydrogen peroxide suggests that animals that have the highest plasma peroxide levels may also have arterioles in the most constricted state. At the same time, such animals may be subject to the largest oxidative stress, a condition that promotes the actual disease process by expression of inflammatory products, such as platelet activating factor, cytokines, and leukocyte adhesion molecules. The direct action of hydrogen peroxide on arteriolar tone is complex. Hydrogen peroxide has been shown to produce vasodilation in coronary arteries as well as cerebral and skeletal muscle arterioles.24 25 26 However, hydrogen peroxide has also been shown to produce vasoconstriction in carotid and pulmonary arteries.27 28 The response observed is often biphasic contraction after relaxation or vice versa and may be concentration dependent.
The in vivo TNBT assay is advantageous because it gives a visual display of oxidative stress within the microvascular network. However, superoxide is an unstable and short-lived radical, so detection errors may arise from reduction of TNBT independent of superoxide. For that reason, we also developed the assay for plasma hydrogen peroxide, a more stable oxygen species. A differential measurement, using catalase and sodium azide, was carried out because the output voltage generated by the electrode may also detect other species in plasma. The accuracy and stability of the hydrogen peroxide readings were confirmed by repeated measurements on a single sample. The second measurement differed from the first by only 5%, and the readings were stable over a 5-hour time period.
The relationship between oxygen free radicals and essential hypertension has received relatively limited attention. However, initial results suggest the trend that essential hypertension is associated with increased oxygen free radical production and decreased antioxidant capacity. Prabha et al3 demonstrated increased superoxide anion and hydrogen peroxide production by polymorphonuclear leukocytes of essential hypertensives; Kumar and Das4 showed decreased vitamin E and superoxide dismutase levels, and Tse et al29 showed decreased vitamin C and thiol levels in hypertensives. Thus, treatment directed at the oxygen free radical formation in addition to reduction of the central blood pressure may yield a benefit that remains to be explored.
The present observations may have other implications. The oxidative stress in hypertensives suggests not only that enhanced levels of oxygen radicals and deficiency of NO production may be responsible for the elevated tone in the arterioles and the elevated peripheral resistance, but that the oxidative stress also may be responsible for impaired endothelial cell function30 31 and enhanced vascular reactivity to different stimuli, such as oxygen.32 33
In summary, the present results suggest an enhanced production of superoxide radicals within the arterioles and venules of hypertensive Dahl-S animals. The endothelial cell appears to be one of the sources of this overproduction. Concomitant with the increase of superoxide production, there is an elevated level in plasma hydrogen peroxide and arteriolar tone.
Selected Abbreviations and Acronyms
|Dahl-R||=||salt-resistant Dahl rat|
|Dahl-S||=||salt-sensitive Dahl rat|
|TNBT||=||tetranitroblue tetrazolium solution|
This research was supported by National Institutes of Health grant HL-10881. The authors would also like to thank Dr Benjamin W. Zweifach for his advice and guidance.
- Received October 7, 1996.
- Revision received November 21, 1996.
- Accepted June 16, 1997.
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