Superoxide Contributes to Development of Salt Sensitivity and Hypertension Induced by Nitric Oxide Deficiency
Department of Physiology, Tulane Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center, New Orleans, La.
Abstract
This study was performed to examine the role of superoxide (O2eC) in the development of salt sensitivity and hypertension induced by inhibition of nitric oxide (NO) generation. Male Sprague-Dawley rats were fed with diet containing either normal salt (NS) (0.4% NaCl) or high salt (HS) (4% NaCl). These rats were treated with or without an NO synthase inhibitor, nitro-L-arginine methylester (L-NAME) (15 mg/kg/d) and O2eC scavenger, tempol (30 mg/kg per day) in the drinking water for 4 weeks. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and urine collection was performed during the course of experimental periods. At the end of 4 weeks, L-NAME treatment resulted in greater increases in SBP in HS rats (127±2 to 172±3 mm Hg; n=8) than in NS rats (130±2 to 156±2 mm Hg; n=9). Co-administration of tempol with L-NAME markedly attenuated these SBP responses to a similar level in both HS (128±3 to 147±2 mm Hg; n=8) and NS rats (126±2 to 142±3 mm Hg; n=8). Urinary 8-isoprostane excretion (UIsoV) increased in response to L-NAME treatment that was higher in HS (10.6±0.5 to 21.5±0.8 ng/d) than in NS rats (10.8±0.7 to 16.9±0.6 ng/d). Co-treatment with tempol completely abolished these UIsoV responses to L-NAME in both HS and NS rats but did not alter urinary H2O2 excretion rate. The decreases in urinary nitrate/nitrite excretion in response to L-NAME treatment were not altered by co-administration of tempol in both HS and NS rats. These data suggest that enhancement of O2eC activity during NO inhibition contributes to the development of salt sensitivity that is associated with NO-deficient hypertension.
Key Words: hypertension kidney nitric oxide
Introduction
Superoxide (O2eC) and other reactive oxygen species are constant products of cellular metabolism. However, the development of oxidative stress is dependent on the balance between their production and degradation.1,2 O2eC is usually instantly reduced by the enzyme superoxide dismutase (SOD) normally present in living tissues.3 Nitric oxide (NO), another free radical, is also known to act as an antioxidative agent by constant elimination of O2eC from the tissue, thus helping to maintain minimal level of O2eC in normal condition and provides a protective function against the action of O2eC in many organs including the kidney.4eC6 Thus, it is possible that in the condition of NO deficiency, there is an increasing accumulation of O2eC in biological tissues.5 In fact, we have observed in an earlier study that acute NOS inhibition leads to enhanced O2eC activity that exerts vasoconstriction, antidiuresis, and antinatriuresis effects in the kidney.6,7 The results of these studies indicate that NO deficiency can lead to the development of oxidative stress in the body.
Oxidative stress has been suggested to be involved in the pathophysiology of many forms of hypertension;8,9 however, the exact mechanism is not yet fully understood. Scavenging of O2eC significantly reduces blood pressure in different models of hypertension,10,11 especially those associated with salt-sensitivity.12,13 Previous studies have indicated that inhibition of NO generation during high-salt intake leads to the development of salt-sensitive hypertension and the impairment of kidney function.14eC16 Thus, the increases in O2eC level caused by NO deficiency may significantly contribute to the development of salt-sensitive forms of hypertension.
The specific aim of this study was to examine the role of O2eC generation in the development of salt-sensitive hypertension during chronic nitric oxide synthase (NOS) inhibition in rats. To induce NO deficiency, chronic treatment with NOS inhibitor, nitro-L-arginine methyl ester (L-NAME) was given to rats during normal and high-salt intake.14,17 Blood pressure and excretory responses were evaluated with or without co-treatment of O2eC scavenger, tempol (4-hydroxy-tetramethylpiperidime-1-oxyl), during the course of 4-week treatment with L-NAME in these rats.10,18,19
Materials and Methods
The study was performed in male Sprague-Dawley rats (Charles River Laboratories; Wilmington, Mass) in accordance with the guidelines and practices established by the Tulane University Animal Care and Use Committee. After 3 days of acclimatization, animals (220 to 250 grams body weight) were randomly divided into 6 experimental groups based on diet (normal salt, NS, 0.4% NaCl; or high-salt, HS, 4% NaCl; Harlan-Teklad, Madison, Wis) and drug treatment (NOS inhibitor, L-NAME, and O2eC scavenger, tempol; Sigma, St. Louis, Mo): (1) NS, nontreated; (2) NS, L-NAME; (3) NS, L-NAME + tempol; (4) HS, nontreated; (5) HS, L-NAME; (6) HS, L-NAME + tempol. As in our pilot study, tempol treatment alone (n=2) was without any effect on blood pressure and, also, similar observation with chronic treatment of tempol was previously reported in normotensive rats;10,11 we did not feel the necessity to repeat these experiments in the present study. Because control rats with HS intake also remained normotensive, we also did not conduct experiments with tempol treatment alone in these rats.
L-NAME at a dose of 15 mg/kg per day was given in drinking water for a 4-week experimental period. This dose of L-NAME was previously used by other investigators14,17 as an adequate dose to achieve near-maximal inhibition of NOS in the body. Tempol was also added with L-NAME to the drinking water at a dose of 30 mg/kg per day as previously reported.10,18,19 This dose of tempol was adequate to decrease blood pressure and plasma or urine 8-isoprostane levels in hypertensive rats.10,18,19 Systolic blood pressure (SBP) was measured by tail-cuff plethysmography. Basal blood pressure was measured for 3 consecutive days before the starting the protocol and then at 3- to 4-day intervals during the 4-week study period. The 24-hour urine collection was performed in metabolic cages on the day before the start of treatment to establish basal excretory parameters and then on days 7, 14, 21, and 28 of experiment. Body weight and water intake were also recorded on each day of urine collections. The status of glomerular filtration rate (GFR) was assessed by calculating creatinine clearance to assess the differences in overall kidney function in these groups of experimental animals. To determine creatinine concentration in plasma samples, arterial blood samples were collected from a carotid arterial cannula placed in rats under anesthesia (pentobarbital, 50 mg/kg intraperitoneal).
Analytical Methods and Statistics
Urinary excretion of sodium and potassium were assessed by flame photometry. Concentration of 8-isoprostane in urine samples was determined by enzyme immunoassay (Cayman Chemical, Ann Arbor, Mich)10,18 and H2O2 concentration was measured by colorimetric assay (Cayman Chemical).20 Nitrate/nitrite (NOx) concentration was also measured colorimetrically (Assay Design, Ann Arbor, Mich).6,20 To estimate glomerular filtration rate (GFR), creatinine clearance was calculated from the plasma and urine concentration determined colorimetrically. Results are expressed as mean±SEM. Statistical comparisons within groups were conducted by the use of ANOVA for repeated measurements, followed by Newman-Keuls test. Unpaired Student t test was used for comparisons between groups. Statistical significance is defined at a value of P=0.05.
Results
Blood Pressure Responses
As shown in Figure 1, there were no significant differences in SBP at the end of 4-week experimental period in nontreated control groups with NS (127±2 to 133±2 mm Hg) or HS intake (129±2 to 136±3 mm Hg). These Sprague Dawley rats are normally not salt-sensitive as indicated by the fact that HS intake alone for 4 weeks did not significantly alter SBP. Chronic L-NAME treatment resulted in greater increases in SBP in HS rats (127±2 to 172±3 mm Hg; P<0.05) than in NS rats (130±2 to 156±2 mm Hg; P<0.05). Higher dose of L-NAME used in other studies in rats15 did not cause much difference in blood pressure responses compared with what we had observed in the present investigation indicating that this dose was sufficient to achieve a near maximal NOS inhibition. These data confirmed the previous observation14eC17 that NOS inhibition leads to a salt-sensitive model of hypertension. However, co-administration of tempol with L-NAME markedly attenuated SBP in both HS (128±3 to 147±2 mm Hg; P<0.05) as well as NS rats (126±2 to 142±3 mm Hg; P<0.05).
Excretory Responses
Urine volumes collected for 24 hours are given in the Table. In addition, recorded body weight and 24-hour water intake are also given in the Table. It was observed that an age-related weight gains in these animals were slightly less in L-NAMEeCtreated animals compared with untreated group in both NS- and HS-fed rats. Such a slight decrease in weight gain was not seen in groups of rats co-treated with tempol. Thus, the retention of sodium caused by L-NAME administration did not cause any weight gains in these groups of animals. We also observed that the water intake was mostly remained unchanged during L-NAME or L-NAME plus tempol treatment though generally HS intake groups showed higher water intake compared with NS groups as expected (Table).
Body Weight, Water Intake, and Urine Volume During 4-Week Experimental Period
The responses in urinary excretion rate of NO metabolites, nitrate/nitrite (UNOxV), to treatment with L-NAME and tempol in these experimental animals are shown in Figure 2. As expected, UNOxV was significantly lower in L-NAMEeCtreated animals in both NS (21.1±1.2 to 14.8±0.9 eol/d) and HS intake groups (21.8±1.6 to 11.7±0.7 eol/d). Co-treatment with tempol did not alter the observed reduction in UNOxV induced by inhibition of NO generation either in the NS (15.6±1.3 eol/d) or in the HS group (13.5±0.9 eol/d). As shown in Figure 3, urinary 8-isoprostane excretion (UISOV) caused by chronic L-NAME treatment for 4 weeks was significantly higher in both NS (16.9±0.3 ng/d) and HS (21.5±0.8 ng/d) groups compared with corresponding NS (12.5±0.7 ng/d) and HS (13.7±0.9 ng/d) control group, indicating that NO deficiency leads to higher O2eC activity. However, co-administration of tempol completely abolished UISOV responses to L-NAME in both NS (13.8±0.7 ng/d) and HS (15.8±0.9 ng/d) groups, indicating that the dose of tempol effectively reduces O2eC activity in these rats during NOS inhibition. In NS rats, L-NAME treatment caused slightly but significantly lower sodium excretion on days 21 and 28 compared with the NS nontreated rats (Figure 4A). In the NS L-NAME plus tempol-treated group, sodium excretion was higher compared with the NS L-NAMEeCtreated group. HS intake caused expected increases in sodium excretion compared with NS groups (Figure 4B). However, in the HS L-NAME group, sodium excretion was significantly lower on days 7, 14, and 21 compared with the HS nontreated group. In the HS L-NAME plus tempol-treated group, sodium excretion was significantly higher than that in the HS L-NAME group.
The urinary excretion rate of H2O2 (UH2O2V) was determined in the samples collected from the experimental animals at the end of week 4 of treatment period. The results are illustrated in Figure 5. We observed that administration of L-NAME did not alter UH2O2V significantly in these rats fed NS or HS diets. Co-administration of tempol with L-NAME did not cause any significant increase in UH2O2V in both HS and NS groups. However, it was observed that UH2O2V was higher in nontreated HS group of rats compared with that in nontreated NS groups as reported earlier in another study.21
At the end of 4 weeks, creatinine clearance was calculated to determine the changes in estimated GFR (Figure 6). Attenuated creatinine clearances caused by chronic L-NAME treatment in both NS and HS rats compared with nontreated groups were partially restored in groups co-treated with tempol, suggesting that the decrease in GFR during NOS blockade is partly caused by enhancement of O2eC activity in the kidney.
Discussion
In the present investigation, it was observed that scavenging of O2eC by chronic tempol administration attenuated the SBP response to chronic L-NAME administration in rats during NS as well as HS intake. However, SBP during co-treatment of L-NAME and tempol remained significantly higher than that in the nontreated control groups (Figure 1). Urinary excretion rate of sodium was seen lower in L-NAMEeCtreated rats but not in tempol co-treated rats in both NS and HS intake groups (Figure 4). The decreases in creatinine clearance (GFR) in L-NAMEeCtreated rats were seen partially restored by co-treatment of tempol (Figure 6). Such decreases in GFR in L-NAMEeCtreated groups were caused presumably caused by increases in pre-glomerular arteriolar resistances by the lack of NO production.2 However, partial restoration of GFR during co-treatment with tempol indicated that an enhancement of O2eC caused by NOS inhibition also played a role in such increases in pre-glomerular resistances as suggested in earlier studies.2,22 Thus, the findings in the present study have indicated that an enhanced O2eC activity modulates both renal hemodynamics and excretory function in the condition of NO deficiency that may be involved in the development of hypertension induced by NOS inhibition.
It has been demonstrated that NOS inhibition enhances vascular O2eC release both in rats23,24 and in humans,25 and such enhanced O2eC production was abolished by the use of a O2eC scavenger.25 Although we did not measure directly the O2eC level in the present study, we observed that UISOV (a marker for endogenous O2eC activity) increased in L-NAMEeCtreated rats and the response was greater in the HS group of rats compared with that in the NS group (Figure 3). These UISOV responses to L-NAME were completely prevented in rats co-treated with tempol indicating an increase in O2eC activity during NOS inhibition. In our previous studies in dogs,6,20 we also observed an increase in UISOV during acute NOS inhibition in the kidney that was ameliorated by co-administration of tempol.
Although L-NAMEeCinduced SBP response was much greater in the HS intake group compared with the NS intake group, tempol treatment caused attenuation of SBP to similar levels in both groups that are not significantly different from each other (Figure 1). Because tempol administration abolished the differences in hypertensive responses to L-NAME during varying salt intake, these findings indicate that the development of salt-sensitivity induced by chronic NOS inhibition is mainly attributed to increases in endogenous O2eC activity. Tempol treatment did not alter UNOxV in the NOS inhibited rats, indicating that the blood pressure lowering effect of tempol in the present study was not caused by reversal of NO bioavailability but rather was caused by decreases in O2eC activity. It has also been shown that tempol treatment significantly attenuates blood pressure in several hypertensive models that are particularly associated with salt-sensitivity.12,13,18,26 Thus, the results of present investigation further support an important role of O2eC in the development of salt-sensitivity in NO-deficient hypertension.
It may be argued that tempol as a SOD mimetic lead to increases in H2O2 levels that influence the SBP responses in rats in the present investigation. However, it was observed that tempol administration in L-NAMEeCtreated rats did not cause any significant increase in UH2O2V in both HS and NS groups (Figure 5). Thus, it would argue against any significant contribution of H2O2 in the observed marked attenuation of the SBP responses to tempol in L-NAMEeCtreated rats. Although the effects of catalase administration after tempol treatment had not been examined in the present study, we have reported earlier that acute administration of tempol did not alter UH2O2V either in dogs20 or in normotensive, as well as angiotensin IIeCinduced hypertensive rats.27 We also observed that there was no significant difference between the renal responses to intra-arterial administration of tempol with or without catalase in rats.27 As reported previously,21 UH2O2V was seen higher in nontreated HS group of rats compared with that in nontreated NS groups in our present study. It is to be noted here that such differences in UH2O2V was observed, although blood pressure levels are similar in both nontreated NS and HS groups. However, the effects of a possible change in vascular level of H2O2 during tempol administration are yet to be determined conclusively.21,24,28eC30 It has been shown that H2O2 acts as a vasodilator,24,30 implicating that enhancement of its vascular level would cause a decrease in blood pressure. However, some studies have implicated that increases in renal medullary tissue H2O2 level causes hypertension in rats,28,29 an effect that is opposite to the present finding of tempol-induced reduction in SBP.
Oxidative stress and hypertension are closely associated with higher sympathetic activity.31,32 Thus, it could be argued that tempol-induced changes in blood pressure observed in the present study may be influenced by its inhibitory effects on sympathetic activity32 or antioxidant-induced changes in the release of norepinephrine from the nerve terminals.33 However, it was also demonstrated that enhanced O2eC activity by SOD inhibition can cause stimulation of sympathetic activity that was shown to be inhibited by tempol.34 Thus, reduction of endogenous O2eC activity by tempol administration can indirectly be associated with a possible reduction in sympathetic activity in hypertensive rats in the present study.
The exact mechanism that produces increases in the endogenous level of O2eC during NOS inhibition is not yet clear. Both NO and O2eC are constant products of cellular metabolism, and both of these molecules are constantly interacting with each other in biological tissues.5 Normally, O2eC in the tissue is kept to a minimal level by the antioxidative function of SOD as well as NO. However, when NO production is diminished in the tissue, it is expected that this balance may be altered allowing O2eC accumulation in the tissue because of its inadequate removal by NO.6 It is also possible that the activity of enzymes responsible for endogenous production of O2eC may be upregulated during NOS inhibition.35 Further experiments are required to determine the activity of these oxidative enzymes during NO synthase inhibition.
In conclusion, these data demonstrate that the enhanced O2eC activity caused by chronic NOS inhibition contributes to the development of salt sensitivity that is involved in the pathophysiology of the NO-deficient form of hypertension.
Perspectives
The findings of this present study further support our previous observations indicating an important role of the interaction between O2eC and NO in the regulation of renal function and blood pressure.4eC9 NO provides a protective role against the actions of O2eC by acting as an important antioxidative agent in the body. The development of any imbalance between oxidative and antioxidative processes in living tissues would lead to derangements in organ function including the kidney. The results of the present study, which demonstrate a close relation between enhancement of O2eC activity and the development of salt sensitivity during NOS inhibition, provide an important clue in our quest in understanding the pathophysiology of salt-sensitive hypertension. Thus, it is imperative that further emphasis should be focused on complete elucidation of the interactive role of O2eC and NO in the regulation of many organ functions to increase our knowledge on physiology as well as pathophysiologic processes of many diseases that are linked to NO metabolism and oxidative stress.
Acknowledgments
We gratefully acknowledge the technical help provided by Alexander Castillo and Kevin Wellen.
This study was supported by National Heart, Lung, and Blood Institute grant HL-51306.
References
Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840eC844.
Wilcox CS. Redox regulation of the afferent arteriole and tubuloglomerular feedback. Acta Physiol Scand. 2003; 179: 217eC223.
Kitiyakara C, Chabrashvili T, Chen Y, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. J Am Soc Nephrol. 2003; 14: 2775eC2782.
Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension:what is the clinical significance Hypertension. 2004; 44: 248eC252.
Modlinger PS, Wilcox CS, Aslam S. Nitric oxide, oxidative stress, and progression of chronic renal failure. Semin Nephrol. 2004; 24: 354eC365.
Majid DSA, Nishiyama A, Jackson KE, Castillo A. Inhibition of nitric oxide synthase enhances superoxide activity in canine kidney. Am J Physiol Regul Integr Comp Physiol. 2004; 287: R27eCR32.
Majid DSA, Nishiyama A. Nitric oxide blockade enhances renal responses to superoxide dismutase inhibition in dogs. Hypertension. 2002; 39: 293eC297.
Romero JC, Reckelhoff JF. Role of angiotensin and oxidative stress in essential hypertension. Hypertension. 1999; 34: 943eC949.
Wilcox CS. Reactive oxygen species: roles in blood pressure and kidney function. Curr Hypertens Rep. 2002; 4: 160eC166.
Schnackenberg CG, Wilcox CS. Two-week administration of tempol attenuates both hypertension and renal excretion of 8-iso prostaglandin f2alpha. Hypertension. 1999; 33: 424eC428.
Welch WJ, Blau J, Xie H, Chabrashvili T, Wilcox CS. Angiotensin-induced defects in renal oxygenation: role of oxidative stress. Am J Physiol Heart Circ Physiol. 2005; 288: H22eCH28.
Manning RD Jr, Meng S, Tian N. Renal and vascular oxidative stress and salt-sensitivity of arterial pressure. Acta Physiol Scand. 2003; 179: 243eC250.
Howard LL, Patterson ME, Mullins JJ, Mitchell KD. Salt-sensitive hypertension develops after transient induction of ANG II-dependent hypertension in Cyp1a1-Ren2 transgenic rats. Am J Physiol Renal Physiol. 2005; 288: F810eCF815.
Tolins JP, Shultz PJ. Endogenous nitric oxide synthesis determines sensitivity to the pressor effect of salt. Kidney Int. 1994; 46: 230eC236.
Yamada SS, Sassaki AL, Fujihara CK, Malheiros DM, De Nucci G, Zatz R. Effect of salt intake and inhibitor dose on arterial hypertension and renal injury induced by chronic nitric oxide blockade. Hypertension. 1996; 27: 1165eC1172.
Nakanishi K, Hara N, Nagai Y. Salt-sensitive hypertension in conscious rats induced by chronic nitric oxide blockade. Am J Hypertens. 2002; 15: 150eC156.
Scrogin KE, Hatton DC, Chi Y, Luft FC. Chronic nitric oxide inhibition with L-NAME: effects on autonomic control of the cardiovascular system. Am J Physiol. 1998; 274: R367eCR374.
Hoagland KM, Maier KG, Roman RJ. Contributions of 20-HETE to the antihypertensive effects of tempol in Dahl salt-sensitive rats. Hypertension. 2003; 41: 697eC702.
Zhang Y, Croft KD, Mori TA, Schyvens CG, McKenzie KU, Whitworth JA. The antioxidant tempol prevents and partially reverses dexamethasone-induced hypertension in the rat. Am J Hypertens. 2004; 17: 260eC265.
Majid DS, Nishiyama A, Jackson KE, Castillo A. Superoxide scavenging attenuates renal responses to ANG II during nitric oxide synthase inhibition in anesthetized dogs. Am J Physiol Renal Physiol. 2005; 288: F412eCF419.
Williams JM, Pollock JS, Pollock DM. Arterial pressure response to the antioxidant tempol and ETB receptor blockade in rats on a high-salt diet. Hypertension. 2004; 44: 770eC775.
Haque MZ, Majid DS. Assessment of renal functional phenotype in mice lacking gp91PHOX subunit of NAD(P)H oxidase. Hypertension. 2004; 43: 335eC340.
Usui M, Egashira K, Kitamoto S, Koyanagi M, Katoh M, Kataoka C, Shimokawa H, Takeshita A. Pathogenic role of oxidative stress in vascular angiotensin-converting enzyme activation in long-term blockade of nitric oxide synthesis in rats. Hypertension. 1999; 34: 546eC551.
Cosentino F, Barker JE, Brand MP, Heales SJ, Werner ER, Tippins JR, West N, Channon KM, Volpe M, Luscher TF. Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice. Arterioscler Thromb Vasc Biol. 2001; 21: 496eC502.
Guzik TJ, West NE, Pillai R, Taggart DP, Channon KM. Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension. 2002; 39: 1088eC1094.
Park JB, Touyz RM, Chen X, Schiffrin EL. Chronic treatment with a superoxide dismutase mimetic prevents vascular remodeling and progression of hypertension in salt-loaded stroke-prone spontaneously hypertensive rats. Am J Hypertens. 2002; 15: 78eC84.
Kopkan L, Castillo A, Navar LG, Majid DS. Renal hemodynamic and excretory response to superoxide anion scavenging in angiotensin II-induced hypertensive rats. J Am Soc Nephrol. 2004; 15: 207A. Abstract.
Makino A, Skelton MM, Zou AP, Cowley AW Jr. Increased renal medullary H2O2 leads to hypertension. Hypertension. 2003; 42: 25eC30.
Chen YF, Cowley AW Jr, Zou AP. Increased H2O2 counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla. Am J Physiol Regul Integr Comp Physiol. 2003; 285: R827eCR833.
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201eC1209.
Campese VM, Ye S, Zhong H, Yanamadala V, Ye Z, Chiu J. Reactive oxygen species stimulate central and peripheral sympathetic nervous system activity. Am J Physiol Heart Circ Physiol. 2004; 287: H695eCH703.
Xu H, Fink GD, Galligan JJ. Tempol lowers blood pressure and sympathetic nerve activity but not vascular O2eC in DOCA-salt rats. Hypertension. 2004; 43: 329eC334.
Jou SB, Cheng JT. The role of free radicals in the release of noradrenaline from myenteric nerve terminals of guinea-pig ileum. J Auton Nerv Syst. 1997; 66: 126eC130.
Shokoji T, Fujisawa Y, Kimura S, Rahman M, Kiyomoto H, Matsubara K, Moriwaki K, Aki Y, Miyatake A, Kohno M, Abe Y, Nishiyama A. Effects of local administrations of tempol and diethyldithio-carbamic on peripheral nerve activity. Hypertension. 2004; 44: 236eC243.
Husain K, Hazelrigg SR. Oxidative injury due to chronic nitric oxide synthase inhibition in rat: effect of regular exercise on the heart. Biochim Biophys Acta. 2002; 1587: 75eC82., 百拇医药(Libor Kopkan; Dewan S. A.)
Abstract
This study was performed to examine the role of superoxide (O2eC) in the development of salt sensitivity and hypertension induced by inhibition of nitric oxide (NO) generation. Male Sprague-Dawley rats were fed with diet containing either normal salt (NS) (0.4% NaCl) or high salt (HS) (4% NaCl). These rats were treated with or without an NO synthase inhibitor, nitro-L-arginine methylester (L-NAME) (15 mg/kg/d) and O2eC scavenger, tempol (30 mg/kg per day) in the drinking water for 4 weeks. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and urine collection was performed during the course of experimental periods. At the end of 4 weeks, L-NAME treatment resulted in greater increases in SBP in HS rats (127±2 to 172±3 mm Hg; n=8) than in NS rats (130±2 to 156±2 mm Hg; n=9). Co-administration of tempol with L-NAME markedly attenuated these SBP responses to a similar level in both HS (128±3 to 147±2 mm Hg; n=8) and NS rats (126±2 to 142±3 mm Hg; n=8). Urinary 8-isoprostane excretion (UIsoV) increased in response to L-NAME treatment that was higher in HS (10.6±0.5 to 21.5±0.8 ng/d) than in NS rats (10.8±0.7 to 16.9±0.6 ng/d). Co-treatment with tempol completely abolished these UIsoV responses to L-NAME in both HS and NS rats but did not alter urinary H2O2 excretion rate. The decreases in urinary nitrate/nitrite excretion in response to L-NAME treatment were not altered by co-administration of tempol in both HS and NS rats. These data suggest that enhancement of O2eC activity during NO inhibition contributes to the development of salt sensitivity that is associated with NO-deficient hypertension.
Key Words: hypertension kidney nitric oxide
Introduction
Superoxide (O2eC) and other reactive oxygen species are constant products of cellular metabolism. However, the development of oxidative stress is dependent on the balance between their production and degradation.1,2 O2eC is usually instantly reduced by the enzyme superoxide dismutase (SOD) normally present in living tissues.3 Nitric oxide (NO), another free radical, is also known to act as an antioxidative agent by constant elimination of O2eC from the tissue, thus helping to maintain minimal level of O2eC in normal condition and provides a protective function against the action of O2eC in many organs including the kidney.4eC6 Thus, it is possible that in the condition of NO deficiency, there is an increasing accumulation of O2eC in biological tissues.5 In fact, we have observed in an earlier study that acute NOS inhibition leads to enhanced O2eC activity that exerts vasoconstriction, antidiuresis, and antinatriuresis effects in the kidney.6,7 The results of these studies indicate that NO deficiency can lead to the development of oxidative stress in the body.
Oxidative stress has been suggested to be involved in the pathophysiology of many forms of hypertension;8,9 however, the exact mechanism is not yet fully understood. Scavenging of O2eC significantly reduces blood pressure in different models of hypertension,10,11 especially those associated with salt-sensitivity.12,13 Previous studies have indicated that inhibition of NO generation during high-salt intake leads to the development of salt-sensitive hypertension and the impairment of kidney function.14eC16 Thus, the increases in O2eC level caused by NO deficiency may significantly contribute to the development of salt-sensitive forms of hypertension.
The specific aim of this study was to examine the role of O2eC generation in the development of salt-sensitive hypertension during chronic nitric oxide synthase (NOS) inhibition in rats. To induce NO deficiency, chronic treatment with NOS inhibitor, nitro-L-arginine methyl ester (L-NAME) was given to rats during normal and high-salt intake.14,17 Blood pressure and excretory responses were evaluated with or without co-treatment of O2eC scavenger, tempol (4-hydroxy-tetramethylpiperidime-1-oxyl), during the course of 4-week treatment with L-NAME in these rats.10,18,19
Materials and Methods
The study was performed in male Sprague-Dawley rats (Charles River Laboratories; Wilmington, Mass) in accordance with the guidelines and practices established by the Tulane University Animal Care and Use Committee. After 3 days of acclimatization, animals (220 to 250 grams body weight) were randomly divided into 6 experimental groups based on diet (normal salt, NS, 0.4% NaCl; or high-salt, HS, 4% NaCl; Harlan-Teklad, Madison, Wis) and drug treatment (NOS inhibitor, L-NAME, and O2eC scavenger, tempol; Sigma, St. Louis, Mo): (1) NS, nontreated; (2) NS, L-NAME; (3) NS, L-NAME + tempol; (4) HS, nontreated; (5) HS, L-NAME; (6) HS, L-NAME + tempol. As in our pilot study, tempol treatment alone (n=2) was without any effect on blood pressure and, also, similar observation with chronic treatment of tempol was previously reported in normotensive rats;10,11 we did not feel the necessity to repeat these experiments in the present study. Because control rats with HS intake also remained normotensive, we also did not conduct experiments with tempol treatment alone in these rats.
L-NAME at a dose of 15 mg/kg per day was given in drinking water for a 4-week experimental period. This dose of L-NAME was previously used by other investigators14,17 as an adequate dose to achieve near-maximal inhibition of NOS in the body. Tempol was also added with L-NAME to the drinking water at a dose of 30 mg/kg per day as previously reported.10,18,19 This dose of tempol was adequate to decrease blood pressure and plasma or urine 8-isoprostane levels in hypertensive rats.10,18,19 Systolic blood pressure (SBP) was measured by tail-cuff plethysmography. Basal blood pressure was measured for 3 consecutive days before the starting the protocol and then at 3- to 4-day intervals during the 4-week study period. The 24-hour urine collection was performed in metabolic cages on the day before the start of treatment to establish basal excretory parameters and then on days 7, 14, 21, and 28 of experiment. Body weight and water intake were also recorded on each day of urine collections. The status of glomerular filtration rate (GFR) was assessed by calculating creatinine clearance to assess the differences in overall kidney function in these groups of experimental animals. To determine creatinine concentration in plasma samples, arterial blood samples were collected from a carotid arterial cannula placed in rats under anesthesia (pentobarbital, 50 mg/kg intraperitoneal).
Analytical Methods and Statistics
Urinary excretion of sodium and potassium were assessed by flame photometry. Concentration of 8-isoprostane in urine samples was determined by enzyme immunoassay (Cayman Chemical, Ann Arbor, Mich)10,18 and H2O2 concentration was measured by colorimetric assay (Cayman Chemical).20 Nitrate/nitrite (NOx) concentration was also measured colorimetrically (Assay Design, Ann Arbor, Mich).6,20 To estimate glomerular filtration rate (GFR), creatinine clearance was calculated from the plasma and urine concentration determined colorimetrically. Results are expressed as mean±SEM. Statistical comparisons within groups were conducted by the use of ANOVA for repeated measurements, followed by Newman-Keuls test. Unpaired Student t test was used for comparisons between groups. Statistical significance is defined at a value of P=0.05.
Results
Blood Pressure Responses
As shown in Figure 1, there were no significant differences in SBP at the end of 4-week experimental period in nontreated control groups with NS (127±2 to 133±2 mm Hg) or HS intake (129±2 to 136±3 mm Hg). These Sprague Dawley rats are normally not salt-sensitive as indicated by the fact that HS intake alone for 4 weeks did not significantly alter SBP. Chronic L-NAME treatment resulted in greater increases in SBP in HS rats (127±2 to 172±3 mm Hg; P<0.05) than in NS rats (130±2 to 156±2 mm Hg; P<0.05). Higher dose of L-NAME used in other studies in rats15 did not cause much difference in blood pressure responses compared with what we had observed in the present investigation indicating that this dose was sufficient to achieve a near maximal NOS inhibition. These data confirmed the previous observation14eC17 that NOS inhibition leads to a salt-sensitive model of hypertension. However, co-administration of tempol with L-NAME markedly attenuated SBP in both HS (128±3 to 147±2 mm Hg; P<0.05) as well as NS rats (126±2 to 142±3 mm Hg; P<0.05).
Excretory Responses
Urine volumes collected for 24 hours are given in the Table. In addition, recorded body weight and 24-hour water intake are also given in the Table. It was observed that an age-related weight gains in these animals were slightly less in L-NAMEeCtreated animals compared with untreated group in both NS- and HS-fed rats. Such a slight decrease in weight gain was not seen in groups of rats co-treated with tempol. Thus, the retention of sodium caused by L-NAME administration did not cause any weight gains in these groups of animals. We also observed that the water intake was mostly remained unchanged during L-NAME or L-NAME plus tempol treatment though generally HS intake groups showed higher water intake compared with NS groups as expected (Table).
Body Weight, Water Intake, and Urine Volume During 4-Week Experimental Period
The responses in urinary excretion rate of NO metabolites, nitrate/nitrite (UNOxV), to treatment with L-NAME and tempol in these experimental animals are shown in Figure 2. As expected, UNOxV was significantly lower in L-NAMEeCtreated animals in both NS (21.1±1.2 to 14.8±0.9 eol/d) and HS intake groups (21.8±1.6 to 11.7±0.7 eol/d). Co-treatment with tempol did not alter the observed reduction in UNOxV induced by inhibition of NO generation either in the NS (15.6±1.3 eol/d) or in the HS group (13.5±0.9 eol/d). As shown in Figure 3, urinary 8-isoprostane excretion (UISOV) caused by chronic L-NAME treatment for 4 weeks was significantly higher in both NS (16.9±0.3 ng/d) and HS (21.5±0.8 ng/d) groups compared with corresponding NS (12.5±0.7 ng/d) and HS (13.7±0.9 ng/d) control group, indicating that NO deficiency leads to higher O2eC activity. However, co-administration of tempol completely abolished UISOV responses to L-NAME in both NS (13.8±0.7 ng/d) and HS (15.8±0.9 ng/d) groups, indicating that the dose of tempol effectively reduces O2eC activity in these rats during NOS inhibition. In NS rats, L-NAME treatment caused slightly but significantly lower sodium excretion on days 21 and 28 compared with the NS nontreated rats (Figure 4A). In the NS L-NAME plus tempol-treated group, sodium excretion was higher compared with the NS L-NAMEeCtreated group. HS intake caused expected increases in sodium excretion compared with NS groups (Figure 4B). However, in the HS L-NAME group, sodium excretion was significantly lower on days 7, 14, and 21 compared with the HS nontreated group. In the HS L-NAME plus tempol-treated group, sodium excretion was significantly higher than that in the HS L-NAME group.
The urinary excretion rate of H2O2 (UH2O2V) was determined in the samples collected from the experimental animals at the end of week 4 of treatment period. The results are illustrated in Figure 5. We observed that administration of L-NAME did not alter UH2O2V significantly in these rats fed NS or HS diets. Co-administration of tempol with L-NAME did not cause any significant increase in UH2O2V in both HS and NS groups. However, it was observed that UH2O2V was higher in nontreated HS group of rats compared with that in nontreated NS groups as reported earlier in another study.21
At the end of 4 weeks, creatinine clearance was calculated to determine the changes in estimated GFR (Figure 6). Attenuated creatinine clearances caused by chronic L-NAME treatment in both NS and HS rats compared with nontreated groups were partially restored in groups co-treated with tempol, suggesting that the decrease in GFR during NOS blockade is partly caused by enhancement of O2eC activity in the kidney.
Discussion
In the present investigation, it was observed that scavenging of O2eC by chronic tempol administration attenuated the SBP response to chronic L-NAME administration in rats during NS as well as HS intake. However, SBP during co-treatment of L-NAME and tempol remained significantly higher than that in the nontreated control groups (Figure 1). Urinary excretion rate of sodium was seen lower in L-NAMEeCtreated rats but not in tempol co-treated rats in both NS and HS intake groups (Figure 4). The decreases in creatinine clearance (GFR) in L-NAMEeCtreated rats were seen partially restored by co-treatment of tempol (Figure 6). Such decreases in GFR in L-NAMEeCtreated groups were caused presumably caused by increases in pre-glomerular arteriolar resistances by the lack of NO production.2 However, partial restoration of GFR during co-treatment with tempol indicated that an enhancement of O2eC caused by NOS inhibition also played a role in such increases in pre-glomerular resistances as suggested in earlier studies.2,22 Thus, the findings in the present study have indicated that an enhanced O2eC activity modulates both renal hemodynamics and excretory function in the condition of NO deficiency that may be involved in the development of hypertension induced by NOS inhibition.
It has been demonstrated that NOS inhibition enhances vascular O2eC release both in rats23,24 and in humans,25 and such enhanced O2eC production was abolished by the use of a O2eC scavenger.25 Although we did not measure directly the O2eC level in the present study, we observed that UISOV (a marker for endogenous O2eC activity) increased in L-NAMEeCtreated rats and the response was greater in the HS group of rats compared with that in the NS group (Figure 3). These UISOV responses to L-NAME were completely prevented in rats co-treated with tempol indicating an increase in O2eC activity during NOS inhibition. In our previous studies in dogs,6,20 we also observed an increase in UISOV during acute NOS inhibition in the kidney that was ameliorated by co-administration of tempol.
Although L-NAMEeCinduced SBP response was much greater in the HS intake group compared with the NS intake group, tempol treatment caused attenuation of SBP to similar levels in both groups that are not significantly different from each other (Figure 1). Because tempol administration abolished the differences in hypertensive responses to L-NAME during varying salt intake, these findings indicate that the development of salt-sensitivity induced by chronic NOS inhibition is mainly attributed to increases in endogenous O2eC activity. Tempol treatment did not alter UNOxV in the NOS inhibited rats, indicating that the blood pressure lowering effect of tempol in the present study was not caused by reversal of NO bioavailability but rather was caused by decreases in O2eC activity. It has also been shown that tempol treatment significantly attenuates blood pressure in several hypertensive models that are particularly associated with salt-sensitivity.12,13,18,26 Thus, the results of present investigation further support an important role of O2eC in the development of salt-sensitivity in NO-deficient hypertension.
It may be argued that tempol as a SOD mimetic lead to increases in H2O2 levels that influence the SBP responses in rats in the present investigation. However, it was observed that tempol administration in L-NAMEeCtreated rats did not cause any significant increase in UH2O2V in both HS and NS groups (Figure 5). Thus, it would argue against any significant contribution of H2O2 in the observed marked attenuation of the SBP responses to tempol in L-NAMEeCtreated rats. Although the effects of catalase administration after tempol treatment had not been examined in the present study, we have reported earlier that acute administration of tempol did not alter UH2O2V either in dogs20 or in normotensive, as well as angiotensin IIeCinduced hypertensive rats.27 We also observed that there was no significant difference between the renal responses to intra-arterial administration of tempol with or without catalase in rats.27 As reported previously,21 UH2O2V was seen higher in nontreated HS group of rats compared with that in nontreated NS groups in our present study. It is to be noted here that such differences in UH2O2V was observed, although blood pressure levels are similar in both nontreated NS and HS groups. However, the effects of a possible change in vascular level of H2O2 during tempol administration are yet to be determined conclusively.21,24,28eC30 It has been shown that H2O2 acts as a vasodilator,24,30 implicating that enhancement of its vascular level would cause a decrease in blood pressure. However, some studies have implicated that increases in renal medullary tissue H2O2 level causes hypertension in rats,28,29 an effect that is opposite to the present finding of tempol-induced reduction in SBP.
Oxidative stress and hypertension are closely associated with higher sympathetic activity.31,32 Thus, it could be argued that tempol-induced changes in blood pressure observed in the present study may be influenced by its inhibitory effects on sympathetic activity32 or antioxidant-induced changes in the release of norepinephrine from the nerve terminals.33 However, it was also demonstrated that enhanced O2eC activity by SOD inhibition can cause stimulation of sympathetic activity that was shown to be inhibited by tempol.34 Thus, reduction of endogenous O2eC activity by tempol administration can indirectly be associated with a possible reduction in sympathetic activity in hypertensive rats in the present study.
The exact mechanism that produces increases in the endogenous level of O2eC during NOS inhibition is not yet clear. Both NO and O2eC are constant products of cellular metabolism, and both of these molecules are constantly interacting with each other in biological tissues.5 Normally, O2eC in the tissue is kept to a minimal level by the antioxidative function of SOD as well as NO. However, when NO production is diminished in the tissue, it is expected that this balance may be altered allowing O2eC accumulation in the tissue because of its inadequate removal by NO.6 It is also possible that the activity of enzymes responsible for endogenous production of O2eC may be upregulated during NOS inhibition.35 Further experiments are required to determine the activity of these oxidative enzymes during NO synthase inhibition.
In conclusion, these data demonstrate that the enhanced O2eC activity caused by chronic NOS inhibition contributes to the development of salt sensitivity that is involved in the pathophysiology of the NO-deficient form of hypertension.
Perspectives
The findings of this present study further support our previous observations indicating an important role of the interaction between O2eC and NO in the regulation of renal function and blood pressure.4eC9 NO provides a protective role against the actions of O2eC by acting as an important antioxidative agent in the body. The development of any imbalance between oxidative and antioxidative processes in living tissues would lead to derangements in organ function including the kidney. The results of the present study, which demonstrate a close relation between enhancement of O2eC activity and the development of salt sensitivity during NOS inhibition, provide an important clue in our quest in understanding the pathophysiology of salt-sensitive hypertension. Thus, it is imperative that further emphasis should be focused on complete elucidation of the interactive role of O2eC and NO in the regulation of many organ functions to increase our knowledge on physiology as well as pathophysiologic processes of many diseases that are linked to NO metabolism and oxidative stress.
Acknowledgments
We gratefully acknowledge the technical help provided by Alexander Castillo and Kevin Wellen.
This study was supported by National Heart, Lung, and Blood Institute grant HL-51306.
References
Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840eC844.
Wilcox CS. Redox regulation of the afferent arteriole and tubuloglomerular feedback. Acta Physiol Scand. 2003; 179: 217eC223.
Kitiyakara C, Chabrashvili T, Chen Y, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. J Am Soc Nephrol. 2003; 14: 2775eC2782.
Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension:what is the clinical significance Hypertension. 2004; 44: 248eC252.
Modlinger PS, Wilcox CS, Aslam S. Nitric oxide, oxidative stress, and progression of chronic renal failure. Semin Nephrol. 2004; 24: 354eC365.
Majid DSA, Nishiyama A, Jackson KE, Castillo A. Inhibition of nitric oxide synthase enhances superoxide activity in canine kidney. Am J Physiol Regul Integr Comp Physiol. 2004; 287: R27eCR32.
Majid DSA, Nishiyama A. Nitric oxide blockade enhances renal responses to superoxide dismutase inhibition in dogs. Hypertension. 2002; 39: 293eC297.
Romero JC, Reckelhoff JF. Role of angiotensin and oxidative stress in essential hypertension. Hypertension. 1999; 34: 943eC949.
Wilcox CS. Reactive oxygen species: roles in blood pressure and kidney function. Curr Hypertens Rep. 2002; 4: 160eC166.
Schnackenberg CG, Wilcox CS. Two-week administration of tempol attenuates both hypertension and renal excretion of 8-iso prostaglandin f2alpha. Hypertension. 1999; 33: 424eC428.
Welch WJ, Blau J, Xie H, Chabrashvili T, Wilcox CS. Angiotensin-induced defects in renal oxygenation: role of oxidative stress. Am J Physiol Heart Circ Physiol. 2005; 288: H22eCH28.
Manning RD Jr, Meng S, Tian N. Renal and vascular oxidative stress and salt-sensitivity of arterial pressure. Acta Physiol Scand. 2003; 179: 243eC250.
Howard LL, Patterson ME, Mullins JJ, Mitchell KD. Salt-sensitive hypertension develops after transient induction of ANG II-dependent hypertension in Cyp1a1-Ren2 transgenic rats. Am J Physiol Renal Physiol. 2005; 288: F810eCF815.
Tolins JP, Shultz PJ. Endogenous nitric oxide synthesis determines sensitivity to the pressor effect of salt. Kidney Int. 1994; 46: 230eC236.
Yamada SS, Sassaki AL, Fujihara CK, Malheiros DM, De Nucci G, Zatz R. Effect of salt intake and inhibitor dose on arterial hypertension and renal injury induced by chronic nitric oxide blockade. Hypertension. 1996; 27: 1165eC1172.
Nakanishi K, Hara N, Nagai Y. Salt-sensitive hypertension in conscious rats induced by chronic nitric oxide blockade. Am J Hypertens. 2002; 15: 150eC156.
Scrogin KE, Hatton DC, Chi Y, Luft FC. Chronic nitric oxide inhibition with L-NAME: effects on autonomic control of the cardiovascular system. Am J Physiol. 1998; 274: R367eCR374.
Hoagland KM, Maier KG, Roman RJ. Contributions of 20-HETE to the antihypertensive effects of tempol in Dahl salt-sensitive rats. Hypertension. 2003; 41: 697eC702.
Zhang Y, Croft KD, Mori TA, Schyvens CG, McKenzie KU, Whitworth JA. The antioxidant tempol prevents and partially reverses dexamethasone-induced hypertension in the rat. Am J Hypertens. 2004; 17: 260eC265.
Majid DS, Nishiyama A, Jackson KE, Castillo A. Superoxide scavenging attenuates renal responses to ANG II during nitric oxide synthase inhibition in anesthetized dogs. Am J Physiol Renal Physiol. 2005; 288: F412eCF419.
Williams JM, Pollock JS, Pollock DM. Arterial pressure response to the antioxidant tempol and ETB receptor blockade in rats on a high-salt diet. Hypertension. 2004; 44: 770eC775.
Haque MZ, Majid DS. Assessment of renal functional phenotype in mice lacking gp91PHOX subunit of NAD(P)H oxidase. Hypertension. 2004; 43: 335eC340.
Usui M, Egashira K, Kitamoto S, Koyanagi M, Katoh M, Kataoka C, Shimokawa H, Takeshita A. Pathogenic role of oxidative stress in vascular angiotensin-converting enzyme activation in long-term blockade of nitric oxide synthesis in rats. Hypertension. 1999; 34: 546eC551.
Cosentino F, Barker JE, Brand MP, Heales SJ, Werner ER, Tippins JR, West N, Channon KM, Volpe M, Luscher TF. Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice. Arterioscler Thromb Vasc Biol. 2001; 21: 496eC502.
Guzik TJ, West NE, Pillai R, Taggart DP, Channon KM. Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension. 2002; 39: 1088eC1094.
Park JB, Touyz RM, Chen X, Schiffrin EL. Chronic treatment with a superoxide dismutase mimetic prevents vascular remodeling and progression of hypertension in salt-loaded stroke-prone spontaneously hypertensive rats. Am J Hypertens. 2002; 15: 78eC84.
Kopkan L, Castillo A, Navar LG, Majid DS. Renal hemodynamic and excretory response to superoxide anion scavenging in angiotensin II-induced hypertensive rats. J Am Soc Nephrol. 2004; 15: 207A. Abstract.
Makino A, Skelton MM, Zou AP, Cowley AW Jr. Increased renal medullary H2O2 leads to hypertension. Hypertension. 2003; 42: 25eC30.
Chen YF, Cowley AW Jr, Zou AP. Increased H2O2 counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla. Am J Physiol Regul Integr Comp Physiol. 2003; 285: R827eCR833.
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201eC1209.
Campese VM, Ye S, Zhong H, Yanamadala V, Ye Z, Chiu J. Reactive oxygen species stimulate central and peripheral sympathetic nervous system activity. Am J Physiol Heart Circ Physiol. 2004; 287: H695eCH703.
Xu H, Fink GD, Galligan JJ. Tempol lowers blood pressure and sympathetic nerve activity but not vascular O2eC in DOCA-salt rats. Hypertension. 2004; 43: 329eC334.
Jou SB, Cheng JT. The role of free radicals in the release of noradrenaline from myenteric nerve terminals of guinea-pig ileum. J Auton Nerv Syst. 1997; 66: 126eC130.
Shokoji T, Fujisawa Y, Kimura S, Rahman M, Kiyomoto H, Matsubara K, Moriwaki K, Aki Y, Miyatake A, Kohno M, Abe Y, Nishiyama A. Effects of local administrations of tempol and diethyldithio-carbamic on peripheral nerve activity. Hypertension. 2004; 44: 236eC243.
Husain K, Hazelrigg SR. Oxidative injury due to chronic nitric oxide synthase inhibition in rat: effect of regular exercise on the heart. Biochim Biophys Acta. 2002; 1587: 75eC82., 百拇医药(Libor Kopkan; Dewan S. A.)