R-00461-2001.R1

Renal Medullary Nitric Oxide Deficit of

Dahl S Rats Enhances Hypertensive Actions of Angiotensin II

Mátyás Szentiványi Jr.*, Ai-Ping Zou, David L. Mattson, Paulo Soares,

Carol Moreno, Richard J. Roman, and Allen W. Cowley Jr.

*Clinical Research Department-2nd Institute of Physiology, Semmelweis University of Medicine, Budapest, Hungary; Department of Physiology, Medical College of Wisconsin, Milwaukee,

Wisconsin

Szentiványi Jr.: Impaired renal medullary NO in Dahl S rats

Correspondence:Allen W. Cowley Jr., Ph.D. Department of Physiology, Medical College of Wisconsin 8701 Watertown Plank Rd. P.O.B. 26509 Milwaukee WI 53226-0509 Fax: (414) 456-6546 Phone: (414) 456-8266 e-mail: cowley@mcw.edu

Copyright 2002 by the American Physiological Society.

AJP-Regu Articles in PresS. Published on April 4, 2002 as DOI 10.1152/ajpregu.00461.2001

2

ABSTRACT

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 angiotensin II

(AngII). Nitric oxide 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 down regulation 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 i.v. infusion of AngII (5 ng/kg/min) did not increase the

concentration of NO in the renal medulla as measured by a microdialysis with oxyhemoglobin

trapping technique. In contrast, AngII produced a 38 % increase in the concentration of NO

(87+8 to 117+8 nmol/L) in the outer medulla of Brown Norway rats. The same i.v. dose of

AngII reduced renal medullary blood flow as determined by laser-Doppler flowmetry in Dahl S,

but not in BN rats. A 7-day i.v. AngII infusion at a dose of 3 ng/kg/min 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). AngII failed to increase MAP after NO substrate was provided by infusion of

L-arginine (300 g/kg/min) into the renal medulla of Dahl S rats. Intravenous infusion of L-

arginine at the same dose had no effect on the AngII-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 AngII.

Key words: Dahl S rats, Brown-Norway rats, nitric oxide, nitric oxide synthase, renal medullary

blood flow, angiotensin II

3

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 i.v. 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 with 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 (8,22,26). Several vasoconstrictors, such as angiotensin

II (AngII), 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 AngII infusion in captopril treated rats was shown to increase 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 L-NAME

into the renal medullary interstitial space, was shown to enhance the medullary vasoconstrictor

effects to small subpressor elevations of plasma AngII (46). Chronic i.v. administration of

AngII 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).

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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 to 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 AngII administration; 3) exhibit an exaggerated reduction of MBF in

the face of small subpressor elevations of circulating AngII; and 4) would respond to AngII

chronically like Brown Norway (BN) rats if L-arg was administered into the renal medullary

interstitial space (e.g. Would AngII-induced hypertension be prevented?).

In these studies, inbred strains of Dahl S rats were compared to 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 (7,34). Although comparisons between

Dahl S and 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 AngII resulted in a clear elevation of medullary NO concentrations.

Both rat strains were maintained on a low salt (0.4 %) diet throughout all studies. AngII was infused

intravenously for a week to Dahl S and BN rats at 3 ng/kg/min, a dose that was shown to be non-

pressor in Sprague Dawley rats (36). The ability of systemic administration of ANGII 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

5

the hypertensive actions of small elevations of circulating AngII (3ng/kg/min infused i.v. for one

week) and whether a continuous administration of L-arg into the renal medulla of Dahl S rats would

return AngII hyper-responsiveness to normal.

METHODS

Twelve week old male inbred Dahl S(SS/JrHsdMcw) and BN(BN/SsMcw) rats obtained from

colonies maintained at the Medical College of Wisconsin (7) were studied. Rats were given free

access to tap water and maintained throughout the study on low salt rat chow (0.4% NaCl; Dyets,

Inc., Bethlehem, PA).

NOS enzyme activity in the renal cortex, 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]-arginine to citrulline using a modification of the radioactive HPLC method originally described

by Carlberg (1) and modified by Wu et al. (39). The kidneys from Dahl S and BN rats were rapidly

removed from pentobarbital anesthetized rats, immersed in ice-cold saline, and the cortex, outer

medulla and inner medulla separated. The tissue was homogenized in 3 volumes of a 20 mM HEPES

buffer containing 1 mM EDTA, 1 mM DTT, 2 mM pepstatin, 1 mM leupeptin and 0.1 mM PMSF.

The homogenate was centrifuged at 3,000 g for 5 minutes and 11,000 g for 15 minutes. 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 Corporation, St Louis, MO) for 10 minutes followed by a brief

centrifugation (3000 g) to remove endogenous L-arginine from the homogenate (25). This procedure

removed >97 % of 3H-arginine counts added to the samples to monitor the efficiency of the arginine

removal. It is important to note that the Dowex-50W resin had to be extensively washed with water

to remove residual acidity prior to use. Aliquots of the Dowex-treated homogenates (100 g protein

for inner medulla, 250 g for outer medulla and cortex) were incubated with L-[3H]-arginine (0.2

Ci, 20 M) in 100 l of 20 mM HEPES buffer containing 2 mM, CaCl2, 1 mM NADPH, 1.5 g/ml

6

calmodulin, 2.5 M FAD, 1 M FMN, 20 M tetrahydrobiopterin and 0.4 mM L-proline to inhibit

arginase activity (2). Inner medullary samples were incubated for 30 minutes at 37o C, outer medulla

for 60 minutes and cortical tissue incubated for 90 minutes. The reactions were stopped by the

addition of 50 l of 20 mM EGTA. The conversion of L-arginine to L-citrulline after derivatization

of the sample with 2 volumes of o-pthalaldehyde reagent (P-0532) was determined by RP-HPLC

using a 4.6 X 25cm 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 determined from the ratio of counts in the citrulline peak to

the sum of the counts in the arginine 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 g of the renal outer medullary protein, and 25 g 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, 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 hours at a 1:1000 dilution

as we have previously described (21). Bound primary antibodies were detected with a horseradish

peroxidase labeled secondary antibody (goat anti-mouse IgG; 1:1000; 2 hr) and enhanced

chemiluminescence (SuperSignal, Pierce Chemical). The band intensities were quantitated using

densitometry (Scion Image, Scion Corporation).

Comparison of renal medullary [NO], MAP, and MBF responses to AngII (5 ng/kg/min) in

anesthetized Dahl S (N=6) and BN (N=6) rats. Rats were anesthetized with Inactin (50 mg/kg i.p.)

and ketamine (33 mg/kg i.m.) and surgically prepared for measurement of renal medullary NO

7

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 l/min during a 2 hour

equilibration period followed by two 30-minute control collection periods (70 l/30 min). Then

AngII was infused i.v. at a rate of 5 ng/kg/min. for a one hour 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, Inc.). MBF was measured using an optical

fiber (500 micron; Edmund Scientific) implanted into the renal medulla to a depth of 5.5 millimeters.

MBF changes were determined using a Perimed PeriFlux PF3 Flowmeter (Perimed, Sweden) as

described in previous studies (20,24,26).

Chronic measurement of MAP in Dahl S (N=6) and BN rats (N=6). Rats were anesthetized with

xylazine (2 mg/kg i.p.) and ketamine (30 mg/kg i.p.) for implantation of indwelling arterial and

venous catheters which 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 (7,23).

Rats received a continuous saline i.v. infusion at a rate of 0.25 ml/hour, the rate of delivery

subsequently used for the AngII. One week following surgery, daily 2 hr. measurements of MAP

were begun using an on-line data collection and analysis system (7). After 3 days of stable and

reproducible MAP measurements, AngII (Sigma Chemical) was infused i.v. at a dose of 3 ng/kg/min.

Infusion of AngII was continued for 7 days with daily MAP measurements made throughout and 2

days following the cessation of AngII. Previous studies in our laboratory have demonstrated that this

dose does not elevate arterial pressure when infused i.v. for one week in uninephrectomized Sprague

Dawley rats (36).

Effect of medullary versus intravenous L-arginine (L-arg) administration on AngII-induced

hypertension in Dahl S (N=5) rats. To assess whether a deficiency in the formation of NO in the

8

renal medulla sensitizes Dahl S rats to the chronic hypertensive effects of AngII, 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 (22,23,24). Following another week of recovery, MAP was measured for 2 hours

daily. Following three stable control days, the renal medullary interstitial infusion of L-arg was

begun at a dose of 300 g/kg/min in a volume of 8 l/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 i.v. AngII infusion (3 ng/kg/min.) was started and

continued for 7 days followed by 2 post-control 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 anti-

hypertensive 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 g/kg/min) while

isotonic saline was administered into the medullary interstitial space. On the third day of i.v. L-arg

infusion, AngII (3 ng/kg/min) was added to the i.v. infusate for a 7 day period followed by 2 post

control days.

Statistics. Data are presented as mean values standard error of the mean (SEM). For statistical

comparisons of the chronic experiments, one-way analysis of variance (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 as statistically significant.

RESULTS

NOS enzyme activity in the renal cortex, outer and inner medulla of Dahl S and BN rats. As

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seen in Figure 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 pmoles citrulline/min-1.mg protein). No differences between

the two strains were found in NOS activity in inner medullary homogenates (60.0+5.6 in BN vs.

49.0+3.0 in Dahl S, pmoles citrulline/min-1.mg protein). NOS activity in aliquots of renal cortex

homogenates was similar in the two strains (0.9+0.1. vs. 0.9+0.1 pmoles citrulline/min-1.mg protein

in Dahl S and BN respectively).

NOS protein expression in the outer and inner medulla of Dahl S and BN rats. As can be

seen in Figure 2, the levels of NOS I, NOS II and NOS III protein expressed was 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).

Comparison of renal medullary [NO], MAP, and MBF responses to AngII in

anesthetized Dahl S and BN rats. As seen in Figure 3, i.v. AngII infusion (5 ng/kg/min) 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 Figure 3 (lower panel) indicating that AngII 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

AngII stimulation. The low dose of AngII used in this study did not change MAP in either strain of

rat in these acute experiments (MAP: 131 3 vs.134 4 in Dahl S; 114 3 vs. 118 4 mmHg in BN).

Chronic measurement of MAP in i.v. AngII-infused Dahl S and BN rats. A comparison

of the pressor effects of a chronic i.v. infusion of AngII (3 ng/kg/min) in Dahl S and BN rats is

presented in Figure 4. Ang II had no effect on MAP over 7 days in BN rats but increased MAP

10

significantly in Dahl S rats. The pressure increase was sustained throughout the AngII 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 following cessation of the infusion of

AngII.

Effect of medullary versus intravenous L-arg administration on AngII-induced

hypertension in Dahl S rats. As seen on Figure 5, a renal medullary interstitial infusion of L-arg to

increase NO levels completely blocked the pressor response to AngII in Dahl S rats. The same dose

of L-arg infused intravenously did not prevent this AngII-induced hypertension in this strain of rats.

DISCUSSION

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 AngII. 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 AngII 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 AngII 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 AngII (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

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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

AngII.

A blunted ability of the renal medulla of Dahl S rats to generate NO in response to AngII

stimulation is of particular interest because this would be expected to reduce the excretion of sodium

and thereby shift the chronic pressure relationship (21) through two mechanisms. First, by reducing

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 (18,32,46). This, in turn, may be related to the

production of 20-HETE in these epithelial cells since this cytochrome P450 product of arachadonic

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). Since NO binds to heme in P4504A 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 AngII increases oxygen free radical production in the outer medulla that would, in part,

buffer the actions of AngII stimulated NO production (12). NADH-oxidase levels are higher in the

medulla of Sprague Dawley rats compared to the cortex (45) and it is possible that Dahl S rats exhibit

greater levels of oxidative stress than BN rats. Thus, there appears 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 AngII.

The second mechanism leading to decreased sodium excretion could be by the reduction of

medullary blood flow via enhanced vasoconstrictor effects of AngII in the presence of reduced levels

of NO. The rich density of contractile pericytes surrounding the vasa recta vessels in the outer

12

medulla (29) provide an ideal site for NO regulation of blood flow to both outer and the inner

medulla. We have previously shown that a normally non-hypertensive dose of AngII produced

sustained hypertension when medullary NOS activity was blunted by a chronic medullary infusion of

a non-hypertensive dose of L-NAME (36). In this same study, it was also found that medullary blood

flow was chronically reduced by AngII 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 found that kidneys of Dahl S rats fed

a high salt diet exhibited lower Ca++-dependent NOS activity levels than Dahl salt-resistant rats (17).

Castrop and Kurtz reported a decrease in NOS I gene expression in whole kidney homogenates of

Dahl S rats when compared to Dahl R rats (3). 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 when

compared to 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 co-segregation analysis by Deng and Rapp 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 (9). In other studies (10,11), Deng et al. 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 co-segregation 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; Chr12-NOS I). It was found that a positive correlation

existed between AngII-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

13

those homozygous for the BN allele. These data indicated that at least one NOS loci (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 FMN 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 NOSII compared to the wildtype. Accelerated

degradation of the mutant gene also appeared to contribute to the diminished NO production in cells

expressing the Dahl S NOSII gene (40).

Restoration of medullary [NO] by L-arginine prevents subpressor AngII-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 AngII-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 AngII hypersensitivity. The

antihypertensive effects of the L-arg administration were almost certainly due to the intrarenal effects

of L-arg since intravenous administration of the same dose of L-arg failed to reduce the hypertensive

effects of AngII. 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

14

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 AngII in Dahl S rats. As a

consequence, hypertension occurred in Dahl S rats with small elevations of circulating AngII that had

no effect in the Brown Norway strain.

ACKNOWLEDGMENTS

This work was supported by the National Institute of Health grant HL-29587. The authors thank

Meredith M. Skelton for careful review of the manuscript and Ryan Hagemeier and Robert Klum for

their careful analyses of NOS by HPLC. Mátyás Szentiványi, Jr. was a visiting postdoctoral fellow

from Semmelweis University of Medicine, Budapest, Hungary with support from OKTA T030245.

Paulo Soares was a visiting fellow from the Dept. Internal Medicine, State Univ. of Campenas, Brasil.

Carol Moreno is a visiting postdoctoral fellow supported by Fundacion Seneca in Spain.

15

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20

FIGURE LEGENDS

Figure 1. Comparison of nitric oxide synthase (NOS) activity in the inner and medulla and the cortex

of the kidney of Dahl S ( DS; black bars; N=6) and Brown Norway (BN;N=6) rats. * significant

difference from BN (P<0.05).

Figure 2. Densitometric analysis of the Western blots of the three isoforms: NOS I, NOS II, and NOS

III, using proteins isolated from homogenates of the outer medulla (lower panel) and the inner medulla

(upper panel) for the Dahl S (white bar; N=5) and Brown Norway (black bar; BN; N=5) rats.

*significant difference from BN (P<0.05).

Figure 3. Comparison of changes in medullary blood flow (MBF) and changes in medullary nitric

oxide concentration ([NO]) following one hour i.v. administration of Angiotensin II (AngII) in Dahl S

(N=6) and Brown Norway (BN; N=6) rats. * significant difference from control (P<0.05).

Figure 4: Average changes in mean arterial pressure (MAP) with i.v. infusion of Angiotensin II (AngII;

3.0 ng/kg/min) in Brown Norway rats (BN; closed circles) and in Dahl S rats (DS; open circles). *

indicates significant difference from the control days; P<0.05.

Figure 5: Average daily changes in mean arterial pressure (MAP) with i.v. infusion of angiotensin II

(AngII: 3.0 ng/kg/min) in Dahl S rats infused with L-arginine (L-Arg) administered intravenously (i.v.;

closed circles) or into the renal interstitium (r.i.; open circles). * significant difference from control;

P<0.05)

21

Figure 1:

0

20

40

60

80

100

0

2

4

6

8

10

12

NO

S ac

tivity

(pm

oles

citr

ullin

e fo

rmed

/m

in. m

g pr

otei

n)

Outer MedullaInner Medulla

BN BNDS DS

*0

2

4

6

8

10

12

BNDS

Cortex

22

Figure 2:

Outer MedullaNOS I NOS II NOS III

DS DSDSBN BNBN

Inner MedullaNOS I NOS II NOS III

DS DSDSBN BNBNDen

sito

met

ic U

nits

Den

sito

met

ic U

nits

*

* **

2500

2000

1500

1000

500

0

2500

2000

1500

1000

500

0

Outer MedullaNOS I NOS II NOS III

DS DSDSBN BNBNDS DSDSBN BNBN

Inner MedullaNOS I NOS II NOS III

DS DSDSBN BNBNDen

sito

met

ic U

nits

Den

sito

met

ic U

nits

*

* **

2500

2000

1500

1000

500

0

2500

2000

1500

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500

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23

Figure 3:

Dahl-S BNM

BF

(%)

*

60

70

80

90

100

60

70

80

90

100

Med

ulla

ry[N

O]

(nm

ol/L

)

40

60

80

100

120

140

Control AngII40

60

80

100

120

140

Control AngII

A.

*B.

Dahl-S BNDahl-S BNM

BF

(%)

*

60

70

80

90

100

60

70

80

90

100

Med

ulla

ry[N

O]

(nm

ol/L

)

40

60

80

100

120

140

Control AngII40

60

80

100

120

140

Control AngII

A.

*B.

24

Figure 4:

Time (days)

MAP(mmHg)

1 2 3 4 5 6 7 8 9 10 11 12

90100110120130140150

AngII 3 ng/kg/min i.v.

* * * * ** *

25

Figure 5:

Time (days)

MAP(mmHg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

110120130140150160170

AngII 3 ng/kg/min i.v.

* * * * *

L-Arg 300 ug/kg/min.