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Am J Physiol Regul Integr Comp Physiol 283: R266-R272, 2002. First published April 4, 2002; doi:10.1152/ajpregu.00461.2001
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Vol. 283, Issue 1, R266-R272, July 2002

Renal medullary nitric oxide deficit of Dahl S rats enhances hypertensive actions of angiotensin II

Mátyás Szentiványi Jr.1, Ai-Ping Zou2, David L. Mattson2, Paulo Soares2, Carol Moreno2, Richard J. Roman2, and Allen W. Cowley Jr.2

1 Clinical Research Department, 2nd Institute of Physiology, Semmelweis University of Medicine, H-1088 Budapest, Hungary; and 2 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-0509


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies were designed to examine the hypothesis that the renal medulla of Dahl salt-sensitive (Dahl S) rats has a reduced capacity to generate nitric oxide (NO), which diminishes the ability to buffer against the chronic hypertensive effects of small elevations of circulating ANG II. NO synthase (NOS) activity in the outer medulla of Dahl S rats (arginine-citrulline conversion assay) was significantly reduced. This decrease in NOS activity was associated with the downregulation of protein expression of NOS I, NOS II, and NOS III isoforms in this region as determined by Western blot analysis. In anesthetized Dahl S rats, we observed that a low subpressor intravenous infusion of ANG II (5 ng · kg-1 · min-1) did not increase the concentration of NO in the renal medulla as measured by a microdialysis with oxyhemoglobin trapping technique. In contrast, ANG II produced a 38% increase in the concentration of NO (87 ± 8 to 117 ± 8 nmol/l) in the outer medulla of Brown-Norway (BN) rats. The same intravenous dose of ANG II reduced renal medullary blood flow as determined by laser-Doppler flowmetry in Dahl S, but not in BN rats. A 7-day intravenous ANG II infusion at a dose of 3 ng · kg-1 · min-1 did not change mean arterial pressure (MAP) in the BN rats but increased MAP in Dahl S rats from 120 ± 2 to 138 ± 2 mmHg (P < 0.05). ANG II failed to increase MAP after NO substrate was provided by infusion of L-arginine (300 µg · kg-1 · min-1) into the renal medulla of Dahl S rats. Intravenous infusion of L-arginine at the same dose had no effect on the ANG II-induced hypertension. These results indicate that an impaired NO counterregulatory system in the outer medulla of Dahl S rats makes them more susceptible to the hypertensive actions of small elevations of ANG II.

Brown-Norway rats; nitric oxide synthase; renal medullary blood flow


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THERE HAS BEEN persistent evidence, albeit indirect, that Dahl salt-sensitive (Dahl S) rats may have a reduced ability to produce nitric oxide (NO) in response to different stimuli such as a high-salt diet and vasoconstrictors (5, 6, 14, 16, 27, 33). These findings are consistent with observations that chronic oral or intravenous administration of L-arginine (L-Arg) prevents salt-induced hypertension in Dahl S rats and normalizes the pressure-natriuresis relationship (31). There is evidence of a reduced ability of NO to block chloride reabsorption in the thick ascending limb in Dahl S rats, which could account in part for the salt sensitivity of blood pressure in this strain (13). Consistent with these findings, it was observed that chronic infusion of L-Arg into the renal medullary interstitial space alone was able to prevent salt-induced hypertension in Dahl S rats at doses that have no effect when infused intravenously (23, 24). Taken together, these data suggest that the renal medulla of Dahl S rats may have both a reduced ability to produce NO in response to various stimuli as well as a reduced responsiveness to NO, but this has not been directly ascertained.

NO production is known to play an important role in the regulation of medullary blood flow (MBF) and arterial blood pressure (7, 22, 26). Several vasoconstrictors, such as ANG II, arginine vasopressin (AVP), and norepinephrine, can stimulate the release of medullary NO, which in turn buffers the vascular actions of these agonists (30, 37, 42, 46). Chronic ANG II infusion in captopril-treated rats was shown to increase NO synthase (NOS) III protein levels in homogenates of whole kidney of Sprague-Dawley rats (15). A moderate reduction of NOS activity within the renal medulla resulting from the infusion of a subpressor dose of NG-nitro-L-arginine methyl ester (L-NAME) into the renal medullary interstitial space was shown to enhance the medullary vasoconstrictor effects to small subpressor elevations of plasma ANG II (46). Chronic intravenous administration of ANG II or AVP administered at a dose that normally does not raise arterial pressure produced a sustained hypertension in rats with blunted medullary NOS activity (36, 37).

Recently, we found that mRNA expression of NOS I and NOS III in the outer medulla of the kidney was significantly less in Dahl S rats compared with Brown-Norway (BN) rats (41). Furthermore, these Dahl S rats exhibited sustained hypertension in response to small, normally subpressor elevations of plasma AVP. It was these observations that motivated the design of the present study to more completely characterize the production of NO.

Therefore, studies were carried out to determine whether Dahl S rats 1) exhibit a reduced medullary expression of NOS protein and NOS enzyme activity, 2) exhibit a reduced elevation of medullary NO in response to ANG II administration, 3) exhibit an exaggerated reduction of MBF in the face of small subpressor elevations of circulating ANG II, and 4) would respond to ANG II chronically like BN rats if L-Arg was administered into the renal medullary interstitial space (e.g., would ANG II-induced hypertension be prevented?).

In these studies, inbred strains of Dahl S rats were compared with an inbred strain of normotensive, salt-insensitive BN rats. These two strains were chosen because extensive genetic and functional data have now been obtained from genetic linkage studies using an intercross of these two strains of rats, both of which were inbred in our department (8, 34). Although comparisons between Dahl S and Dahl salt-resistant (Dahl R) strains could have served the same purpose, the aim of the present study was to compare a rat strain in which the renal medulla had a reduced ability to produce NO to a strain in which administration of ANG II resulted in a clear elevation of medullary NO concentrations.

Both rat strains were maintained on a low-salt (0.4%) diet throughout all studies. ANG II was infused intravenously for 1 wk to Dahl S and BN rats at 3 ng · kg-1 · min-1, a dose that was shown to be nonpressor in Sprague-Dawley rats (36). The ability of systemic administration of ANG II administered intravenously at a subpressor dose to change MBF and stimulate medullary NO production was determined first in anesthetized rats (46). Chronically instrumented rats were then studied to determine whether blunted medullary NO production in Dahl S rats would sensitize these animals to the hypertensive actions of small elevations of circulating ANG II (3 ng · kg-1 · min-1 infused intravenously for 1 wk) and whether a continuous administration of L-Arg into the renal medulla of Dahl S rats would return ANG II hyperresponsiveness to normal.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Twelve-week-old male inbred Dahl S (SS/JrHsdMcw) and BN (BN/SsMcw) rats obtained from colonies maintained at the Medical College of Wisconsin (8) were studied. Rats were given free access to tap water and maintained throughout the study on low-salt rat chow (0.4% NaCl; Dyets, Bethlehem, PA).

NOS enzyme activity in the renal cortex and outer and inner medulla of Dahl S (n = 7) and BN (n = 7) rats. NOS activity in renal homogenates was determined by measuring the conversion of L-[3H]Arg to citrulline using a modification of the radioactive HPLC method originally described by Carlberg (2) and modified by Wu et al. (39). The kidneys from Dahl S and BN rats were rapidly removed from pentobarbital sodium-anesthetized rats and were immersed in ice-cold saline, and the cortex, outer medulla, and inner medulla were separated. The tissue was homogenized in 3 vol of a 20 mM HEPES buffer containing 1 mM EDTA, 1 mM dithiothreitol, 2 mM pepstatin, 1 mM leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 3,000 g for 5 min and 11,000 g for 15 min. The supernatant was mixed (2:1) with a suspension of Dowex-50W resin in water at pH 5.5 (HCR-W2, ionic form: Sigma Chemical, St Louis, MO) for 10 min followed by a brief centrifugation (3,000 g) to remove endogenous L-Arg from the homogenate (25). This procedure removed >97% of [3H]Arg counts added to the samples to monitor the efficiency of the Arg removal. It is important to note that the Dowex-50W resin had to be extensively washed with water to remove residual acidity before use. Aliquots of the Dowex-treated homogenates (100 µg protein for inner medulla, 250 µg for outer medulla and cortex) were incubated with L-[3H]Arg (0.2 µCi, 20 µM) in 100 µl of 20 mM HEPES buffer containing 2 mM CaCl2, 1 mM NADPH, 1.5 µg/ml calmodulin, 2.5 µM FAD, 1 µM FMN, 20 µM tetrahydrobiopterin, and 0.4 mM L-proline to inhibit arginase activity (1). Inner medullary samples were incubated for 30 min at 37°C, outer medulla for 60 min, and cortical tissue for 90 min. The reactions were stopped by the addition of 50 µl of 20 mM EGTA. The conversion of L-Arg to L-citrulline after derivatization of the sample with 2 vol of o-phthalaldehyde reagent (P-0532) was determined by reverse phase-HPLC using a 4.6 × 25-cm LC-18-DB column (Supelco), which was eluted isocratically at a rate of 1.5 ml/min using 11.5% methanol, 11.5% acetonitrile, 1% tetrahydrofuran, and 0.1 M KH2PO4 (pH 5.9) as the mobile phase. Products were monitored using a radioactive flow detector (Radiomatic Instruments, Tampa, FL), and the activity was determined from the ratio of counts in the citrulline peak to the sum of the counts in the Arg and citrulline peaks.

NOS protein expression in the outer and inner medulla of Dahl S (n = 5) and BN rats (n = 5). Expression of NOS protein was measured in homogenates of renal inner and outer medulla. Aliquots of 50 µg of the renal outer medullary protein and 25 µg of inner medullary homogenate protein were loaded into each lane and size separated by electrophoresis through an 8% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad), blocked overnight at 4°C, and then incubated with a monoclonal anti-NOS I antibody (which cross-reacts with NOS II; Transduction Labs) and a monoclonal anti-NOS III antibody (Transduction Labs) for 2 h at a 1:1,000 dilution as we have previously described (21). Bound primary antibodies were detected with a horseradish peroxidase-labeled secondary antibody (goat anti-mouse IgG; 1:1,000; 2 h) and enhanced chemiluminescence (SuperSignal, Pierce Chemical). The band intensities were quantitated using densitometry (Scion Image, Scion).

Comparison of renal medullary NO concentration ([NO]), mean arterial pressure, and MBF responses to ANG II (5 ng · kg-1 · min-1) in anesthetized Dahl S (n = 6) and BN (n = 6) rats. Rats were anesthetized with Inactin (50 mg/kg ip) and ketamine (33 mg/kg im) and surgically prepared for measurement of renal medullary NO concentration using the microdialysis/hemoglobin trapping technique described in our previous studies (43, 46). The microdialysis probe was perfused at a rate of 2.3 µl/min during a 2-h equilibration period followed by two 30-min control collection periods (70 µl/30 min). Then ANG II was infused intravenously at a rate of 5 ng · kg-1 · min-1 for a 1-h equilibration period, and dialysate fluid was collected during experimental periods. NO-dependent conversion of oxyhemoglobin to methemoglobin in the dialysate samples was determined using a wavelength scanning mode of a DU-640 Beckman spectrophotometer (Beckman Instruments). MBF was measured using an optical fiber (500 µm; Edmund Scientific) implanted into the renal medulla to a depth of 5.5 mm. MBF changes were determined using a Perimed PeriFlux PF3 Flowmeter (Perimed) as described in previous studies (20, 24, 26).

Chronic measurement of mean arterial pressure in Dahl S (n = 6) and BN rats (n = 6). Rats were anesthetized with xylazine (2 mg/kg ip) and ketamine (30 mg/kg ip) for implantation of indwelling arterial and venous catheters that were tunneled subcutaneously to the back of the neck and exited through a flexible spring for attachment to a swivel over the home cage as described in detail previously (8, 23). Rats received a continuous saline intravenous infusion at a rate of 0.25 ml/h, the rate of delivery subsequently used for the ANG II. One week after surgery, daily 2-h measurements of mean arterial pressure (MAP) were begun using an on-line data collection and analysis system (8). After 3 days of stable and reproducible MAP measurements, ANG II (Sigma Chemical) was infused intravenously at a dose of 3 ng · kg-1 · min-1. Infusion of ANG II was continued for 7 days with daily MAP measurements made throughout and 2 days after the cessation of ANG II. Previous studies in our laboratory have demonstrated that this dose does not elevate arterial pressure when infused intravenously for 1 wk in uninephrectomized Sprague-Dawley rats (36).

Effect of medullary vs. intravenous L-Arg administration on ANG II-induced hypertension in Dahl S (n = 5) rats. To assess whether a deficiency in the formation of NO in the renal medulla sensitizes Dahl S rats to the chronic hypertensive effects of ANG II, a protocol was designed in which medullary NO production was enhanced by local renal medullary infusion of L-Arg. Rats were uninephrectomized to avoid interactions with the contralateral kidney. One week later, femoral arterial and venous catheters as well as a renal medullary interstitial catheter were implanted under anesthesia (22-24). After another week of recovery, MAP was measured for 2 h daily. After 3 stable control days, the renal medullary interstitial infusion of L-Arg was begun at a dose of 300 µg · kg-1 · min-1 in a volume of 8 µl/min. This dose of L-Arg administered into the renal medulla was shown previously to increase medullary [NO] (43) while having no effect on basal MBF (23, 24). After 3 days of L-Arg infusion, the intravenous ANG II infusion (3 ng · kg-1 · min-1) was started and continued for 7 days followed by 2 postcontrol days.

Another instrumented group of uninephrectomized Dahl S (n = 5) rats was studied as a control group to determine whether the renal medullary infusion of L-Arg alone could be exerting antihypertensive effects by escape from the kidney and recirculation in the systemic vasculature. In this group of Dahl S rats, L-Arg was infused intravenously at the same rate (300 µg · kg-1 · min-1) while isotonic saline was administered into the medullary interstitial space. On the 3rd day of intravenous L-Arg infusion, ANG II (3 ng · kg-1 · min-1) was added to the intravenous infusate for a 7-day period followed by 2 postcontrol days.

Statistics. Data are presented as means ± SE. For statistical comparisons of the chronic experiments, one-way ANOVA for repeated measures was used and Tukey's multiple range test as a post hoc analysis was carried out. One-way ANOVA with Tukey's multiple range test was used for all other comparisons. All statistical analyses were performed on the raw data. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NOS enzyme activity in the renal cortex and outer and inner medulla of Dahl S and BN rats. As seen in Fig. 1, the NOS enzyme activity of the Dahl S rats was nearly one-third that of the BN rats in the outer medulla (1.8 ± 0.4 and 11.6 ± 1 pmol citrulline · min-1 · mg protein-1). No differences between the two strains were found in NOS activity in inner medullary homogenates (60.0 ± 5.6 vs. 49.0 ± 3.0 pmol citrulline · min-1 · mg protein-1 in BN and Dahl S, respectively). NOS activity in aliquots of renal cortex homogenates was similar in the two strains (0.9 ± 0.1 vs. 0.9 ± 0.1 pmol citrulline · min-1 · mg protein-1 in Dahl S and BN, respectively).


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  		Fig. 1.   Comparison of nitric oxide (NO) synthase (NOS) activity in the inner medulla (A), outer medulla (B), and cortex of the kidney of Dahl salt-sensitive (DS; filled bars; n = 6; C) and Brown-Norway (BN; n = 6) rats. * Significantly different from BN (P < 0.05).

NOS protein expression in the outer and inner medulla of Dahl S and BN rats. As can be seen in Fig. 2, the levels of NOS I, NOS II, and NOS III protein expressed were significantly lower in the outer medulla of Dahl S than in BN rats, while no significant differences between the strains were observed in the inner medulla. Previous studies have indicated little or no expression of any of the NOS isoforms in homogenates of cortex (21).


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  		Fig. 2.   Densitometric analysis of the Western blots of the 3 isoforms of NOS (NOS I, NOS II, and NOS III), using proteins isolated from homogenates of the inner medulla (A) and the outer medulla (B) for Dahl S (filled bars; n = 5) and BN (open bars; n = 5) rats. * Significantly different from BN (P < 0.05).

Comparison of renal medullary [NO], MAP, and MBF responses to ANG II in anesthetized Dahl S and BN rats. As seen in Fig. 3A, intravenous ANG II infusion (5 ng · kg-1 · min-1) given acutely produced small but consistent reductions of the MBF signal (~10%) in anesthetized Dahl S rats (P < 0.05), but not in BN rats. These changes were consistent with the results of the microdialysis studies presented in Fig. 3B, indicating that ANG II had no effect on medullary [NO] in Dahl S rats, while BN rats exhibited a 38% [NO] increase (P < 0.05). There was no difference in the resting medullary [NO] levels of Dahl S and BN rats (89 ± 3 vs. 87 ± 8 nmol/l, respectively), suggesting that the deficit was due to the inability of Dahl S rats to increase [NO] with ANG II stimulation. The low dose of ANG II used in this study did not change MAP in either strain of rat in these acute experiments (MAP: 131 ± 3 vs. 134 ± 4 mmHg in Dahl S; 114 ± 3 vs. 118 ± 4 mmHg in BN).


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  		Fig. 3.   Comparison of changes in medullary blood flow (MBF; A) and changes in medullary NO concentration ([NO]; B) after 1-h intravenous administration of ANG II in Dahl S (n = 6) and BN (n = 6) rats. * Significantly different from control (P < 0.05).

Chronic measurement of MAP in intravenous ANG II-infused Dahl S and BN rats. A comparison of the pressor effects of a chronic intravenous infusion of ANG II (3 ng · kg-1 · min-1) in Dahl S and BN rats is presented in Fig. 4. ANG II had no effect on MAP over 7 days in BN rats but increased MAP significantly in Dahl S rats. The pressure increase was sustained throughout the ANG II infusion, reaching its highest level after 7 days, at which time MAP was 18 mmHg higher than during the control days. MAP returned to within control level 2 days after cessation of the infusion of ANG II.


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  		Fig. 4.   Average changes in mean arterial pressure (MAP) with intravenous infusion of ANG II (3.0 ng · kg-1 · min-1) in BN rats and in Dahl S rats. * Significantly different from control days (P < 0.05).

Effect of medullary vs. intravenous L-Arg administration on ANG II-induced hypertension in Dahl S rats. As seen in Fig. 5, a renal medullary interstitial infusion of L-Arg to increase NO levels completely blocked the pressor response to ANG II in Dahl S rats. The same dose of L-Arg infused intravenously did not prevent this ANG II-induced hypertension in this strain of rats.


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  		Fig. 5.   Average daily changes in MAP with intravenous infusion of ANG II (3.0 ng · kg-1 · min-1) in Dahl S rats infused with L-arginine (L-Arg) administered intravenously (iv; filled circles) or into the renal interstitium (ri; open circles). * Significantly different from control days (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study advances our understanding of the role of renal medullary NO in the development of salt-induced hypertension in the Dahl S rat in several ways. First, it provides direct evidence that in a naturally occurring animal model, the reduced ability of the renal medulla to produce NO can lead to the enhancement of the vasoconstrictor actions of ANG II. Second, this study has demonstrated that if this NO deficit was corrected in the Dahl S rat by the local administration of L-Arg, the hypertensive actions of subpressor amounts of ANG II converted the Dahl S phenotype to that of the BN rat strain.

Evidence and consequences of reduced medullary NOS activity and reduced ability to generate NO responses to ANG II stimulation in Dahl S rats. We have shown previously that lowering of medullary NOS activity with a pharmacological inhibitor (L-NAME) in Sprague-Dawley rats led to hypersensitivity of the medullary vasculature to ANG II (46) and to a salt-sensitive form of hypertension (21). It was also shown that salt-induced hypertension in Dahl S rats could be prevented by medullary L-Arg administration, suggesting indirectly that NO production may be reduced in the Dahl S rat (24). The present study, however, provides direct evidence that, with NOS mRNA reduced in the Dahl S rat (41), there is also a reduction in the protein expression of all three NOS isoforms, most notably in the renal outer medulla. This is turn was accompanied by a significant reduction in NOS enzyme activity and medullary NO tissue concentrations in response to ANG II.

A blunted ability of the renal medulla of Dahl S rats to generate NO in response to ANG II stimulation is of particular interest because this would be expected to reduce the excretion of sodium and thereby shift the chronic pressure relationship (21) by increasing sodium reabsorption in the medullary thick ascending limb (mTAL) as shown by Oritz and Garvin (28). NO reduction may indeed account in part for observations that sodium reabsorption by the thick ascending limb is elevated in Dahl S rats (18, 32, 44). This, in turn, may be related to the production of 20-hydroxyeicosatetraenoic acid (20-HETE) in these epithelial cells because this cytochrome P-450 product of arachidonic acid metabolism has vasoconstrictor actions and reduces sodium reabsorption in the medullary thick ascending limb of Henle (44). This same study showed that Dahl S rats have a reduced capacity to produce 20-HETE in the outer medulla (44). Because NO binds to heme in P-4504A enzymes and inhibits the formation of 20-HETE (35), some of the observed response differences between Dahl S and BN could be influenced by these complex interactions. Preliminary studies in our laboratory also suggest that ANG II increases oxygen free radical production in the outer medulla that would, in part, buffer the actions of ANG II-stimulated NO production (12). NADH oxidase levels are higher in the medulla of Sprague-Dawley rats compared with the cortex (45), and it is possible that Dahl S rats exhibit greater levels of oxidative stress than BN rats. Thus there appear to exist several important pathways whereby the reduction of NOS activity in the outer medulla of Dahl S rats could modify the vasoconstrictor effects of plasma ANG II.

A second mechanism leading to decreased sodium excretion could be by the reduction of MBF via enhanced vasoconstrictor effects of ANG II in the presence of reduced levels of NO. The rich density of contractile pericytes surrounding the vasa recta vessels in the outer medulla (29) provides an ideal site for NO regulation of blood flow to both the outer and the inner medulla. We have previously shown that a normally nonhypertensive dose of ANG II produced sustained hypertension when medullary NOS activity was blunted by a chronic medullary infusion of a nonhypertensive dose of L-NAME (36). In this same study, it was also found that MBF was chronically reduced by ANG II in these L-NAME-infused rats (36).

Studies by others have found reduced renal excretion of cGMP and nitrate/nitrite (33) in Dahl S rats, suggesting impaired NO synthesis. Ikeda and colleagues (17) found that kidneys of Dahl S rats fed a high-salt diet exhibited lower Ca2+-dependent NOS activity levels than Dahl R rats. Castrop and Kurtz (3) reported a decrease in NOS I gene expression in whole kidney homogenates of Dahl S rats compared with Dahl R rats. Although these previous studies did not distinguish cortical from medullary NO production, it was recently reported that immunohistochemical expression of NOS III was lower in both the renal cortical and medullary tissue of Dahl S rats compared with Dahl R rats (19).

The impaired NO synthesis that we have found in the outer medulla of the Dahl S rat may be genetically determined. A genetic cosegregation analysis by Deng and Rapp (11) using an intercross between the Dahl S and Dahl R strains found that it was unlikely that NOS III was segregating with the trait of blood pressure salt sensitivity. In other studies, Deng et al. (9, 10) found that although NOS I or NOS III did not segregate with blood pressure, a blood pressure quantitative trait loci (QTL) for NOS II was suggested. A cosegregation analysis between the Dahl S and BN strains carried out in our laboratory recently identified QTLs for arterial pressure and renal blood flow responses to acetylcholine on chromosomes 4, 10, and 12 (34). Interestingly, all three regions harbor NOS genes (Chr 4-NOS III; Chr 10-NOS II; Chr 12-NOS I). It was found that a positive correlation existed between ANG II-induced changes in blood pressure in rats homozygous for the NOS II allele on chromosome 10 in the F2 generation rats that were homozygous for Dahl S alleles, but not for those homozygous for the BN allele. These data indicated that at least one NOS locus (NOS II) could confer a distinct pressure phenotype to these rats (34). Chen et al. (4) recently identified a single nucleotide transversion in the NOS II gene that produced an amino acid substitution (S714P) between the FAD and flavine mononucleotide binding sites and a restriction fragment length polymorphism, which was present only in Dahl S rats. In subsequent studies from this group, expression of this mutation in COS-7 cells showed a decrease in the Vmax but no change in Km of the enzyme for L-Arg of the mutant NOS II gene. Within the physiological concentration range of L-Arg, the amount of NO produced was reduced in cells transfected with the mutant NOS II compared with the wild type. Accelerated degradation of the mutant gene also appeared to contribute to the diminished NO production in cells expressing the Dahl S NOS II gene (40).

Restoration of medullary [NO] by L-Arg prevents subpressor ANG II-induced hypertension in Dahl S rats. It is interesting that the basal NO concentrations within the renal medullary tissue did not differ between the Dahl S and BN rat strains as determined by the oxyhemoglobin microdialysis method. This would suggest that there exists sufficient L-Arg substrate and NOS enzyme activity to maintain resting vascular tone in the renal medulla.

However, administration of L-Arg into the renal medulla prevented ANG II-induced hypertension in the Dahl S rats. These results are consistent with the conclusion that reduced medullary NO production in Dahl S rats contributed importantly to the ANG II hypersensitivity. The antihypertensive effects of the L-Arg administration were almost certainly due to the intrarenal effects of L-Arg because intravenous administration of the same dose of L-Arg failed to reduce the hypertensive effects of ANG II. The dose of L-Arg administered in the present study has been shown to produce an elevation of medullary NO concentration in Sprague-Dawley rats (44).

Studies by others have found that salt-induced hypertension in Dahl S rats could be prevented by high-dose oral or systemic administration of L-Arg (5, 6, 16, 31). Miyata et al. (23, 24) demonstrated the importance of the renal medullary impairment of NO production in salt-induced hypertension of Dahl S rats. Specifically, renal medullary interstitial infusion of L-Arg in the same low doses used in the present study protected Dahl S rats from high salt-induced hypertension, whereas D-Arg did not, nor did intravenous administration of the same dose of L-Arg.

In summary, the present results indicate that there exists an inherited defect in the ability of the Dahl S rat to produce NO within the outer medulla of the kidney. This defect was manifested by reduced protein expression of all three NOS isoforms, a reduction of NOS enzyme activity, and a failure of medullary NO concentrations to increase in response to ANG II in Dahl S rats. As a consequence, hypertension occurred in Dahl S rats with small elevations of circulating ANG II that had no effect in the BN strain.


    ACKNOWLEDGEMENTS

The authors thank M. M. Skelton for careful review of the manuscript and R. Hagemeier and R. Klum for careful analyses of NOS by HPLC.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-29587. M. Szentiványi, Jr., was a visiting postdoctoral fellow from Semmelweis Univ. of Medicine, Budapest, Hungary, with support from OKTA T030245. P. Soares was a visiting fellow from the Dept. of Internal Medicine, State Univ. of Campinas, Campinas, Brazil. C. Moreno is a visiting postdoctoral fellow supported by Fundacion Seneca in Spain.

Address for reprint requests and other correspondence: A. W. Cowley, Jr., Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., P.O. Box 26509, Milwaukee, WI 53226-0509 (E-mail: cowley{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published April 4, 2002;10.1152/ajpregu.00461.2001

Received 2 August 2001; accepted in final form 29 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 283(1):R266-R272
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