Replacement of {alpha}1-Na-K-ATPase of Dahl rats by Milan rats lowers blood pressure but does not affect its activity

SERGEI N. ORLOV1,2, JULIE DUTIL1, PAVEL HAMET1 and ALAN Y. DENG1

1 Research Centre, Centre Hospitalier de l’Université de Montreal, Hôtel Dieu, Montreal, Quebec, H2W 1T8, Canada
2 Department of Biomembranes, Faculty of Biology, Moscow State University, 119899 Moscow, Russia


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both linkage and use of congenic strains have shown that a chromosome region near the gene for the Na-K-ATPase {alpha}1-subunit (Atp1a1) contained a quantitative trait locus (QTL) for blood pressure (BP). Currently, two congenic strains, designated S.M5 and S.M6, were made by replacing a segment of the Dahl salt-sensitive SS/Jr (S) rat by the homologous region of the Milan normotensive rat (MNS). In S.M5, the gene for Atp1a1 is from the MNS strain; whereas in S.M6, Atp1a1 is from the S strain. The baseline activity of the {alpha}1-Na-K-ATPase and its stoichiometry were evaluated by an assay of ouabain-sensitive inwardly and outwardly directed 86Rb and 22Na fluxes in erythrocytes. The two congenic strains showed a similar BP, but both had a BP lower than that of S rats (P < 0.0001). Neither the {alpha}1-Na-K-ATPase activity nor its stoichiometry was affected by the substitution of the Atp1a1 alleles of S by those of MNS. Thus the BP-lowering effects observed in S.M5 and S.M6 could not be attributed to the {alpha}1-Na-K-ATPase activity or its stoichiometry. Atp1a1 is not supported as a candidate to be a BP QTL.

quantitative trait loci; genetic hypertension; telemetry


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN OUR PREVIOUS WORK, regions on rat chromosome 2 (Chr 2) were shown to carry quantitative trait loci (QTL) for blood pressure (BP) (36). To fine map the QTL, we made two congenic strains by replacing a segment of the Dahl salt-sensitive SS/Jr (S) strain with that of the Milan normotensive (MNS) strain (8). We specifically targeted the Na-K-ATPase {alpha}1-subunit locus (Atp1a1) for functional assays.

Atp1a1 encodes the {alpha}1-isoform of the Na-K-ATPase subunit ubiquitously expressed in all tissues studied so far (10, 18). It is implicated in the BP regulation, because the {alpha}1-Na-K-ATPase is the sole active Na+ transporter located on the basolateral membrane of renal epithelial cells (12), which plays a pivotal role in the extracellular fluid volume adjustment (9, 23). An elevated Na+:K+ stoichiometry of this enzyme could be sufficient to maintain an augmented transcellular salt and fluid transport via a membrane hyperpolarization and an activation of electrogenic ion carriers (16). In addition to its implication in the kidney function, it was also suggested that an inhibition of the {alpha}1-Na-K-ATPase by endogenous ouabain or ouabain-like factors could maintain the elevated vascular smooth muscle tone and peripheral resistance (1).

The role of the {alpha}1-Na-K-ATPase in genetic hypertension of the S rat remains controversial. Herrera and Ruiz-Opazo (13) sequenced cDNA clones from kidney libraries and found an adenine (A) to thymidine (T) transversion at the nucleotide 1079 in the Atp1a1 coding region of the S rat compared with the Dahl salt-resistant rat (R). This mutation led to the G276L substitution in the enzyme. However, Kurtz and coworkers (21) could not reproduce this result. Later on, Ruiz-Opazo and coworkers (20) argued that the negative result obtained by Kurtz and coworkers might be caused by a consistent Tag PCR error that selectively substituted A to T-1079. Subsequently, Herrera and coworkers (14) developed transgenic S rats bearing the {alpha}1-Na-K-ATPase cDNAs from the R rat. These rats exhibited smaller salt-sensitive hypertension. In the same study, they also found a linkage between the alleles of the Atp1a1 locus and BP variations in an F2(S x R) population. In contrary, Rapp and Dene (19) detected no such linkage while studying another independently derived F2(S x R) population. Zicha and coworkers (28) found no linkage between Atp1a1 polymorphisms and BP in an F2(S x R) population, although BP of these F2 hybrids correlated positively with ouabain-sensitive Na+ extrusion and Na+ content in their erythrocytes. Recently, Harris and coworkers (11) carried out a site-directed mutagenesis to artificially introduce a mutation of the A-to-T base change in the Atp1a1 coding region. They found that the S rat does not have the mutation at all compared with the positive control that does (11). Therefore, the mutation detected by Herrera and coworkers (13) remains to be duplicated by other investigators.

Conflicting results were also obtained in the studies of the {alpha}1-Na-K-ATPase activity in salt-sensitive hypertension. No systematic differences in the rate of ouabain-sensitive Na+ and K+ fluxes were observed by Zicha and Duhm (27) while comparing the erythrocytes from S and R rats fed on the low-salt diet. A high-salt diet led to a rise in the ouabain-sensitive Na+ and K+ fluxes in S rats only (26). In the Xenopus laevis oocyte expression system, cells carrying the total RNA from the S kidneys or cDNAs derived from the in vitro transcribed Atp1a1-mRNA from S exhibited a decreased ouabain-sensitive 86Rb influx and non-altered ouabain-sensitive 22Na efflux compared with R rats (13). The same differences in the ouabain-sensitive ion fluxes in erythrocytes were observed comparing the S and R rats (2). Based on these results, Herrera et al. (14) speculated that G276L substitution could lead to an elevated stoichiometry of the {alpha}1-Na-K-ATPase (3Na+:1K+ in S vs. 3Na+:2K+ in R).

Regardless of the controversy over the base difference between S and R, Atp1a1 was a candidate for a BP QTL potentially determining an allelic difference between S and MNS, because a substitution of the S chromosome region containing Atp1a1 by that of the MNS lowered BP (4). To further examine the candidacy of Atp1a1, we constructed two congenic strains, each by replacing a fragment of the S chromosome by that of MNS (8). The obvious difference between them is one congenic strain is homozygous SS, whereas the other is homozygous MM, at the Atp1a1 locus. We then compared the effect of the salt diet on BP and the activity of the erythrocyte Na/K pump in these congenic strains with those of the S strain. Since the {alpha}1-Na-K-ATPase is the only isoform expressed in mammalian erythrocytes (10, 18) we used these cells to examine the effect of the Atp1a1 substitution on the {alpha}1-Na-K-ATPase activity and its stoichiometry. Other cell types such as vascular muscle cells would have other isoforms of the Na-K-ATPase in addition to the {alpha}1-isoform (15).


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Animal studies.
The animals used, breeding scheme for generating congenic strains, and their BP measurements were the same as published previously (8). The congenic strains made and the chromosome regions involved are outlined in Fig. 1. For BP studies, the mating pairs of the S and congenic strains to be studied were bred simultaneously. Male rats were weaned at 21 days of age, maintained on a low-salt diet (0.2% NaCl, Harlan Teklad 7034), and then fed a high-salt diet (2% NaCl, Harlan Teklad 94217) starting from 35 days of age until the end of the experiment. The age of the animals at death was 14 wk from the date of birth. The protocols for handling as well as maintaining animals were approved by our institutional animal committee. All the procedures for the experiment were in accordance with the guidelines of local, provincial, and federal regulations.



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Fig. 1. Chromosome substitutions and quantitative trait locus (QTL) mapping. The linkage map is essentially the same as published previously (8), which is based on an F2(S x MNS) population. Numbers to the left of the linkage map are units in cM. The order for most of the loci on the map has been initially determined by linkage using the MAPMAKER program, then verified by scoring crossovers during the construction of congenic substrains. Solid bars under congenic strains symbolize the S chromosome fragments that have been replaced by that of the Milan normotensive (MNS) rat. The entire region indicated by solid bars and junctions between the solid and open bars are homozygous MM on the map for all the markers listed in the corresponding positions. Open bars on ends of solid bars indicate the ambiguities of crossover breakpoints between markers. Junctions between solid and open bars as well as ends of chromosome regions of interest in each strain are connected by dotted lines to the marker positions on the map. Atp1a1 (in bold and indicated by a left-pointing arrow), Na-K-ATPase {alpha}1-subunit; Fgg, fibrinogen-{gamma}; Gca, guanylyl cyclase A/atrial natriuretic peptide receptor; Hsd3b, 3-hydroxysteroid dehydrogenase/{delta}-5 isomerase; and Nep, neutral endopeptidase. The rest of the markers are anonymous (http://www.genome.wi.mit.edu/rat/public/). S.M5 and S.M6 are S.MNS-Nep/D2Mit14 and S.MNS-Nep/Gca, respectively (8). S, the Dahl salt-sensitive strain; n = 11, 5, and 7 for S, S.M5, and S.M6, respectively. The chromosome markers are SS for S.M6, but MM homozygous for S.M5 at Atp1a1 and flanking markers Hsd3b, D2Got121, D2Rat47, and D2Mit14 (4, 5). All the other markers not in the region indicated by the solid bar are SS in both S.M5 and S.M6. The marker for Atp1a1 is for the 5'-untranslated region (4, 5). Bottom: mean arterial pressure (MAP) values were 183.0 ± 5.7, 135.7 ± 5.5, and 126.4 ± 3.7 mmHg for S, S.M5, and S.M6, respectively.

 
Na/K pump activity and stoichiometry.
Blood samples, 5–10 ml, were collected from the heart in a syringe rinsed with 150 mM NaCl containing ~20 U of heparin per ml of blood and stored on ice no more than 2 h before the ion flux measurement. Then, samples were centrifuged (~1,000 g, 10 min), plasma and white cells were aspirated, and erythrocytes were washed twice with 5 ml of medium A containing 150 mM NaCl and 10 mM HEPES-Tris (pH 7.4, room temperature). Packed erythrocytes, 50 µl, were taken out and mixed with 1 ml of medium B containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 2.5 mM NaH2PO4, 10 mM glucose, and 20 mM HEPES-Tris (pH 7.4, 37°C). The erythrocytes were centrifuged, and the supernatant was aspirated. For influx studies, 0.2 ml of medium B without radioactivity was added to the cells; but to the efflux studies, cells were mixed with 0.2 ml medium B containing 4 µCi/ml 22Na or 0.5 µCi of 86Rb.

To study the isotope influx rate, after 3 h of preincubation, the cells were centrifuged, and the supernatant was aspirated. Then, 0.2 ml of medium B containing 4 µCi/ml 22Na or 0.5 µCi/ml of 86Rb with or without ouabain was added. It is well-documented that the {alpha}1-isoform of Na-K-ATPase from rodents possesses an extremely low affinity for ouabain compared with other species (24). Indeed, in preliminary experiments, we found that an elevation in the ouabain concentration from 2.5 to 10 mM increased the ouabain-sensitive 86Rb influx by 10–15% (data not shown). However, because of possible side effects of ouabain in a high concentration on the membrane integrity and on ion transporters other than the Na/K pump (see below), 2.5 mM ouabain was used in our study. In the absence of ouabain, the kinetics of isotope influx and efflux is linear up to 20–30 min (data not shown). Based on these results, the incubation time was limited to 15 min, and the isotope uptake was terminated by an addition of 1 ml ice-cold medium C containing 100 mM MgCl2 and 10 mM HEPES-Tris buffer (pH 7.4). The erythrocytes were centrifuged, washed twice with the ice-cold medium C, and lysed by a mixture of 0.5 ml of 0.5% Triton X-100 and 0.5 ml of 10% trichloroacetic acid (TCA). These samples were centrifuged (2,000 g, 10 min), and the protein-free lysate was transferred into 10 ml of scintillation cocktail.

To measure the isotope efflux, 50 µl of cells preincubated during 5 h in 0.2 ml of medium B containing 22Na or 86Rb were mixed with 1 ml of ice-cold medium B, centrifuged, and washed twice with the same ice-cold medium. Then, 1 ml of medium B with or without 2.5 mM ouabain was then added. After an incubation of 15 min at 37°C, these samples were centrifuged, and 0.8 ml of the supernatant was measured for radioactivity with a liquid scintillation analyzer (model TR-1600; Canberra-Packard Canada, Mississauga, Ontario, Canada).

To measure the total content of the intracellular Na+ and K+, after 3 h of incubation in medium B, erythrocytes were sedimented, washed three times with ice-cold medium C, and lysed by Triton-TCA as indicated above. Aliquots of the protein-free supernatant were diluted with water and subjected to flame photometry (Flapho-4, Carl Zeiss, Germany).

The stoichiometry of the Na/K pump (S) was calculated as S = (VOSNaE - VOSNaI) x (VOSKI - VOSKE)-1, where VOSNaE and VOSKI are values of ouabain-sensitive (subscript "OS") components of Na+ efflux (efflux indicated by subscript "E") and of K+ influx (influx indicated by subscript "I"), respectively. These parameters were also used as a measure of the Na/K pump activity. VOSNaI and VOSKE are values of ouabain-sensitive components of Na+ influx and K+ efflux, respectively; these values were used to evaluate the Na+/Na+ (VOSNaI) and K+/K+ (VOSKE) mode of operation of the Na/K pump (7). To determine VOSNaI and VOSKI, the rate of K+ (86Rb) and Na+ (22Na) influx in the absence and presence of ouabain was measured and calculated as V = A x (amt)-1, where A is the radioactivity of cell lysate (in cpm), a is the specific radioactivity of 86Rb or 22Na in the incubation medium (in cpm/nmol), m is the content of erythrocytes in the samples (in liters), and t is the time of incubation (0.25 h).

The calculation of absolute values for the rate of isotope efflux is complicated because of a lack of information on the content of intracellular exchangeable K+ and Na+, which makes it difficult to determine the values of the intracellular specific radioactivity of 86Rb and 22Na. To overcome this problem, we assumed that under steady-state conditions the total values of inward and outward ion fluxes are the same, i.e., VNaE = VNaI and VKE = VKI, where V is the rate of Na and K efflux and influx in the absence of ouabain. This assumption is applicable for our study because the same conditions were used to measure the rates of isotope influx and efflux (17). Based on the above assumption, VOSNaE and VOSKE were calculated as VOSNaE = VNaI x (ANat - ANao) x ANat-1, and VOSKE = VKI x (ARbt - ARbo) x ANat-1, where ANat, ARbt, ANao, and ARbo are the radioactivity of 22Na (subscript Na) or 86Rb (subscript "Rb") in the incubation medium after 15 min of incubation without (subscript "t") and with ouabain (subscript "o"), respectively. The values of the ouabain-resistant (subscript "OR") Na+ and K+ efflux were calculated as VORNaE = VNaI x ANao x ANat-1, and VORKE = VKI x ARbo x ARbt-1. All measurements of ion fluxes were done in triplicates. Isotopes and ouabain were purchased from Amersham (Mississauga, Ontario, Canada) and Sigma (St. Louis, MO), respectively. The remaining chemicals were obtained from Sigma, GIBCO-BRL (Life Technologies, Gaithersburg, MD) and Anachemia (Montreal, Quebec, Canada).

Statistical analysis.
Repeated measures ANOVA followed by Dunnett in the SYSTAT 9 program (SPSS Sci., Chicago, IL) was used to compare the significance level for a difference or a lack of it in BP between a congenic strain and the S strain. To analyze ion flux data, means and standard errors were calculated and statistical significance was assessed by a Student’s t-test for unpaired data.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Proof of the Atp1a1 allelic substitutions in a congenic strain.
As shown in Fig. 1, the regions homozygous for MNS in the congenic strains S.M5 and S.M6 are indicated by solid bars. The regions where the exact extent of the MNS replacements was uncertain are indicated by open bars on ends of the solid bars. The rest of Chr 2 and the rest of the genome are essentially SS homozygous in both S.M5 and S.M6 (data not shown). The microsatellite marker for Atp1a1 is located in the 5'-untranslated region of the gene (4, 5). D2Mit14 located between Atp1a1 and D2Wox37 (5) is MM in S.M5 but SS in S.M6 (Fig. 1). Additional markers surrounding Atp1a1 were also MM in S.M5 but SS in S.M6. They include D2Rat47, D2Got121, and Hsd3b (Fig. 1).

BP measurements and comparisons.
For the simplicity of presentation, only the averaged values of 24 h for the day when the pump activity was analyzed were included. For a more detailed presentation of BPs for the congenic strains, please see Ref. 8. The mean arterial pressures (MAPs) for the strains are given to show the BP effect in a congenic strain. The variations in both systolic and diastolic pressures were the same (data not shown). MAPs were significantly lowered (P < 0.0001) in the S.M5 (n = 5) and S.M6 (n = 7) congenic strains compared with that of the S (n = 9) strain.

Effect of salt diet on ion transport in erythrocytes from S.
Neither the intracellular Na+ nor K+ content measured in erythrocytes incubated for 3 h in medium B was different in rats fed on high- and low-salt diet (data not shown). Table 1 shows that none of the components of ion fluxes in erythrocytes from the S rats was affected by the salt diet. These negative results are consistent with previous data of other investigators (27). Also in line with the work of Canessa and coworkers (2), a low-salt diet was used in the further studies on ion transports in erythrocytes from congenic rats.


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Table 1. Comparison of the ion transport in erythrocytes from S rats kept on low- and high-salt diets

 
Ouabain-sensitive ion fluxes in erythrocytes.
The substitution of the Atp1a1 alleles of the S rat with those of the MNS rat in S.M5 did not affect the ouabain-sensitive K+ influx and ouabain-sensitive Na+ efflux, which were used as a measure of the baseline Na-K-ATPase activity (Fig. 2). A comparison of the values of the ouabain-sensitive K+ influx and efflux (~3,000 and 1,300 nmol per liter of cells per hour) shows that ~45% of the total ouabain-sensitive K+ fluxes was mediated by the K+/K+ mode of operation of the Na/K pump. This mode of operation was not altered in S.M5 congenic rats.



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Fig. 2. Ouabain-sensitive K+ and Na+ fluxes in the erythrocytes of S and congenic S.M5 and S.M6 rats. Values are means ± SE obtained for 11 S, 8 S.M5, and 7 S.M6 rats.

 
In contrast to S.M5, the S.M6 strain has the same Atp1a1 alleles as those of S and was used as a control strain for S.M5. As predicted, neither the ouabain-sensitive Na+ influx nor the ouabain-sensitive K+ influx and efflux were significantly altered in S.M6 compared with S rats (Fig. 2). An elevation of ouabain-sensitive Na+ efflux observed in S.M6 is probably caused by a slight increase in the intracellular Na+ content. This is evident from the studies by flame photometry (6.2 ± 0.4, 6.2 ± 0.3, and 8.1 ± 0.5 mmol per liter of packed erythrocytes of S, S.M5, and S.M6, respectively). We did not see significant differences in the content of the intracellular potassium among these strains (data not shown).

Ouabain-resistant ion fluxes in erythrocytes.
While measuring ion fluxes in the presence of the Na-K-ATPase inhibitor ouabain, we did not notice any differences between S.M5 and S rats (Fig. 3). However, the rates of ouabain-resistant Na+ influx and efflux were both increased by 50–80% in S.M6 rats compared with S and S.M5 rats.



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Fig. 3. Ouabain-resistant K+ and Na+ fluxes pump in the erythrocytes of S and congenic S.M5 and S.M6 rats. Values are means ± SE obtained for 11 S, 8 S.M5, and 7 S.M6 rats.

 
Na/K pump stoichiometry.
Table 1 shows that the salt diet did not significantly affect the stoichiometry of the Na-K-ATPase in erythrocytes from the S rats. There were no significant differences in this parameter between S and congenic rats (Fig. 4).



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Fig. 4. Stoichiometry of the Na/K pump in erythrocytes of S and congenic S.M5 and S.M6 rats. Values are means ± SE obtained for 11 S, 8 S.M5, and 7 S.M6 rats. The values corresponding to 3Na+:2K+ and 3Na+:1K+ stoichiometry are indicated horizontal lines.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of our study contain two major findings concerning a potential role of Atp1a1 in hypertension. First, S.M6 carried same alleles as S, whereas S.M5 has the MNS alleles, at Atp1a1. However, both S.M5 and S.M6 have similar BP-attenuating effects (Fig. 1). Because of this Atp1a1-independent outcome, Atp1a1 itself is an unlikely candidate for the BP QTL. Second, replacing the Atp1a1 alleles of the S rats by those of the MNS rats did not affect the activity and stoichiometry of the {alpha}1-Na-K-ATPase. These results indicate that either the {alpha}1-Na-K-ATPase in S rats is not mutated or a mutation does not alter the pump function. A congenic strain containing a minimum flanking region around Atp1a1 would prove that there would be no BP effect.

Although MNS rats per se were not directly analyzed for either BP or the pump activity, S.M6 served as an effective control because it contained a small segment from the MNS rat not including Atp1a1. If any gene from the MNS genome adjacent to, but excluding Atp1a1, affected the pump activity, then S.M6 should have provided the adequate information. S.M6 as a control is important in another respect. As S.M6 was derived from the same progenitor congenic strain S.M (8) as S.M5, any BP effects of the residual MNS background as well as potential position effects resulting from making the congenic strains had to be ruled out (3). The MNS rat would not be a good control, because it differs greatly from S in the genome on the rest of the chromosomes and because it gives no information on the effects of genome changes during the congenic construction. If one could observe a difference in the {alpha}1-Na-K-ATPase activity between MNS and S, then this difference could not be a test for the Atp1a1 gene alone. It could be due to many other genes from MNS modifying it such as the adducin gene (22). Of course, if there were no differences, then the MNS genome other than Atp1a1 might be influencing the outcome.

The rates of ouabain-resistant Na+ influx and efflux mediated by ion transporters distinct from the Na-K-ATPase were both increased in S.M6 compared with S and S.M5 (Fig. 3). As an explanation, an elevated ouabain-resistant Na+ flux could be considered as a mechanism for the increase in an intracellular Na+ content in S.M6, which in turn could lead to an augmented ouabain-sensitive Na+ efflux (Fig. 2) by a feedback activation of the Na-K-ATPase. Because the values of the ouabain-resistant K+ influx and efflux in congenic and S rats were the same, it appeared unlikely that the Na-K-Cl cotransporter and a nonselective monovalent ion leakage across the erythrocyte membrane contribute to the elevated ouabain-resistant Na+ fluxes in S.M6 rats. Because our experiments were conducted in the bicarbonate-free medium, the impact of NaCO3-/Cl- exchanger seemed also unlikely. One aspect is certain: this increase in the ouabain-resistant Na+ fluxes in S.M6 is not due directly to the Atp1a1 gene, because S.M6 has the same Atp1a1 alleles as S.

The intriguing fact is that S.M6 has a shorter MNS chromosome segment than S.M5. When the nonoverlapping region was removed, which contained the Atp1a1 gene and was unique to S.M5, the rates of ouabain-resistant Na+ influx and efflux went up. It seemed that some gene(s) in this unique segment from the MNS rat actually modified the permeability of erythrocyte membranes to Na+. This phenomenon might involve gene-gene interactions/modifications between the Atp1a1 alleles from the S rat and another unknown modifying gene(s) of the MNS rat. Alternatively, the unidentified residual MNS genetic background might be different between S.M5 and S.M6 and consequently might have influenced the Na-K-ATPase activity differently. In our genomic scan, we found 88 markers to be SS homozygous for both S.M5 and S.M6 strains (data not shown). Thus a new congenic strain by replacing the MNS Atp1a1 alleles with those of the S rat might have to be constructed to examine this Atp1a1-independent phenomenon.

It is noted that possible altered properties of the {alpha}1-isoform of the Na/K pump of the S rat were based on two observations from the same research team, which showed decreased values of the ouabain-sensitive K+ influx in the Xenopus laevis oocytes transfected with RNAs from the S kidneys (13) and in the erythrocytes from the S rat compared with the R rat (2). Because the values of the ouabain-sensitive Na+ influx were not altered, it was speculated that the stoichiometry of the {alpha}1-isoform of the Na/K pump in the S rat could be 3Na+:1K+ compared with a "normal" 3Na+:2K+ ratio of this transporter in the R rat (14). Nevertheless, the data obtained in our study indicated that the stoichiometry of the Na-K-ATPase was not affected by the substitution of the Atp1a1 alleles from the S rats by these of the MNS rats (Fig. 4), despite a decrease in BP as the result of it. In addition, the values of stoichiometry in both congenic strains as well as in S rats were in the range from 1.4 to 2.0, which is closer to the 1.5 value of the "normal" ratio rather than to the 3.0 value predicted from the 3Na+:1K+ stoichiometry. These observations are not consistent with the notion of a functional mutation in the {alpha}1-Na-K-ATPase of the S rat.

Because both intracellular Na+ and extracellular phosphate affect the stoichiometry of the erythrocyte Na-K-ATPase (7), different conditions used in separate experiments could be a factor for the differing outcomes. Canessa and coworkers (2) measured a unidirectional 22Na and 86Rb influx and efflux. Afterwards, the total content of intracellular monovalent cations was used to calculate the specific intracellular activity of isotopes (2), which might not allow a precise measurement of the Na+:K+ stoichiometry (see METHODS). Moreover, they used the phosphate-free medium and Na+-loaded K+-depleted cells to study the isotope efflux. In our studies, we used cells with physiologically low intracellular Na+/K+ ratio, and the medium contained inorganic phosphate at the concentration corresponding to its level in rat plasma (2.5 mM).

It is noteworthy that an addition of 2.5 mM ouabain led to an elevation of the Na+ influx by 2–5%, which resulted in the negative values of ouabain-sensitive component of Na+ influx used to evaluate the Na+/Na+ mode of operation of the Na-K-ATPase (Table 1, Fig. 2). Presumably, the addition of ouabain could activate other Na+ transport pathways via an interaction with the inactivated Na-K-ATPase. This hypothesis is supported by the recent data on the production of oxygen reactive species in ouabain-treated cardiac myocytes independently of the intracellular Na+/K+ ratio (25). However, the side effect of a high concentration of ouabain on Na+ transporters other than the Na-K-ATPase cannot be excluded. An inhibition of the ouabain-sensitive Na+ efflux extruding a small portion of the intracellular 22Na could also lead to this phenomenon.

In conclusion, our data showed that neither the {alpha}1-Na-K-ATPase activity nor its stoichiometry was affected by the substitution of the Atp1a1 alleles of S by those of MNS. Thus the BP-lowering effects observed in S.M5 and S.M6 could not be attributed to the {alpha} Na-K-ATPase activity or its stoichiometry. Atp1a1 is not supported as a candidate to be a BP QTL.


    ACKNOWLEDGMENTS
 
We appreciate the assistance of Nathalie Bourcier, Annie Ménard, Julie Roy, and Marie-Claude Guertin.

This work was supported by grants from American Heart Association (AHA) National Center, from the Heart and Stroke Foundation of Canada (Quebec), and from Canadian Institutes for Health Research (CIHR) (to A. Y. Deng), from the Kidney Foundation of Canada (to S. N. Orlov), and by a CIHR grant (to P. Hamet). A. Y. Deng is an Established Investigator of the AHA. J. Dutil is a holder of the CIHR studentship.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. Y. Deng, Research Centre, Centre Hospitalier de l’Université de Montreal, 7-132 Pavillon Jeanne Mance, 3840, rue St. Urbain, Montreal, Quebec, Canada H2W 1T8 (E-mail: alan.deng{at}umontreal.ca).

10.1152/physiolgenomics.00059.2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Blaustein MP. Endogenous ouabain: role in the pathogenesis of hypertension. Kidney Int 49: 1748–1753, 1996.[ISI][Medline]
  2. Canessa M, Romero JR, Ruiz-Opazo N, and Herrera VLM. The {alpha}1 Na+-K+ pump of Dahl salt-sensitive rats exhibits altered Na+ modulation of K+ transport in red blood cells. J Membr Biol 134: 107–122, 1993.[ISI][Medline]
  3. Deng AY. In search of hypertension genes in Dahl salt-sensitive rats. J Hypertens 16: 1707–1717, 1998.[ISI][Medline]
  4. Deng AY, Dene H, and Rapp JP. Congenic strains for the blood pressure quantitative trait locus on rat chromosome 2. Hypertension 30: 199–202, 1997.[Abstract/Free Full Text]
  5. Deng AY, Jackson CM, Hoebee B, and Rapp JP. Mapping of rat chromosome 2 markers generated from chromosome-sorted DNA. Mamm Genome 8: 731–735, 1997.[ISI][Medline]
  6. Deng AY and Rapp JP. Cosegregation of blood pressure with angiotensin converting enzyme and atrial natriuretic peptide receptor genes using Dahl salt-sensitive rats. Nat Genet 1: 267–272, 1992.[ISI][Medline]
  7. Duhm J. Na+ and K+ transport in human and rat erythrocytes: features complicating the interpretation of data. In: Salt and Hypertension. Dietary Minerals, Volume Homeostasis and Cardiovascular Regulation, edited by Retting R, Ganten D, and Luft FC. New York: Springer-Verlag, 1989, p. 31–51.
  8. Dutil J and Deng AY. Further chromosomal mapping of a blood pressure QTL in Dahl rats on chromosome 2 using congenic strains. Physiol Genomics 6: 3–9, 2001.[Abstract/Free Full Text]
  9. Gagnon F, Hamet P, and Orlov SN. Na+,K+ pump and Na+-coupled ion carriers in isolated mammalian kidney epithelial cells: regulation by protein kinase C. Can J Physiol Pharmacol 77: 305–319, 1999.[ISI][Medline]
  10. Geering K. Na,K-ATPase. Curr Opin Nephrol Hypertens 6: 434–439, 1997.[ISI][Medline]
  11. Harris EL, Kelly G, and Barnard R. Neither GH nor SS/Jr rats have an A1079T transition in the {alpha}1 Na+,K+-ATPase gene. Hypertension 38: 786–792, 2001.[Abstract/Free Full Text]
  12. Herrera VLM, Cova T, Sasson D, and Ruiz-Opazo N. Developmental cell-specific regulation of {alpha}1, {alpha}2, and {alpha}3 Na-K-ATPase gene expression. Am J Physiol Cell Physiol 266: C1301–C1312, 1994.[Abstract/Free Full Text]
  13. Herrera VLM and Ruiz-Opazo N. Alteration of Na+,K+-ATPase 86Rb influx by a single amino acid substitution. Science 249: 1023–1026, 1990.[ISI][Medline]
  14. Herrera VLM, Xie H, Lopez LV, Schork NJ, and Ruiz-Opazo N. The {alpha}1 Na,K-ATPase gene is a susceptibility hypertension gene in the Dahl salt-sensitiveHSD rat. J Clin Invest 102: 1102–1111, 1998.[Abstract/Free Full Text]
  15. Juhaszova M and Blaustein MP. Na+ pump low and high ouabain affinity {alpha} subunit isoforms are differently distributed in cells. Proc Natl Acad Sci USA 94: 1800–1805, 1997.[Abstract/Free Full Text]
  16. Orosz DE and Hopfer U. Pathophysiological consequences of changes in the coupling ratio of Na,K-ATPase for renal sodium reabsorption and its implications for hypertension. Hypertension 27: 219–227, 1996.[Abstract/Free Full Text]
  17. Postnov YV, Orlov SN, Gulak PV, and Shevchenko AS. Altered permeability of the erythrocyte membrane for sodium and potassium in spontaneously hypertensive rats. Pflügers Arch 365: 257–263, 1976.
  18. Pressley TA. Structure and function of the Na,K pump: ten years of molecular biology. Miner Electrolyte Metab 22: 264–271, 1996.[ISI][Medline]
  19. Rapp JP and Dene H. Failure of alleles at the Na+,K+-ATPase {alpha}1 locus to cosegregate with blood pressure in Dahl rats. J Hypertens 8: 457–462, 1990.[ISI][Medline]
  20. Ruiz-Opazo N, Barany F, Hirayama K, and Herrera VLM. Confirmation of mutant {alpha}1 Na,K-ATPase gene and transcript in Dahl salt-sensitive/JR rats. Hypertension 24: 260–270, 1994.[Abstract]
  21. Simonet L, Lezin ES, and Kurtz TW. Sequence analysis of the {alpha}1 Na+,K+-ATPase gene in the Dahl salt-sensitive rat. Hypertension 18: 689–693, 1991.[Abstract]
  22. Tripodi G, Valtorta F, Torielli L, Chieregatti E, Salardi S, Trusolino L, Menegon A, Ferrari P, Marchisio P, and Bianchi G. Hypertension-associated point mutation in the adducin {alpha} and ß subunits affect actin cytoskeleton and ion transport. J Clin Invest 97: 2815–2822, 1996.[Abstract/Free Full Text]
  23. Vander AJ. Renal Physiology (5th ed.). New York: McGraw-Hill, 1991.
  24. Willis JS and Ellory JC. Ouabain-sensitivity: diversities and disparities. In: Current Topics in Membranes and Transport. New York: Academic, 1983, vol. 19, p. 227–280.
  25. Xie Z, Kometiani P, Liu J, Li J, Shapiro JI, and Askari A. Intracellular reactive oxygen species mediate the linkage of Na+/K+-ATPase to hypertrophy and its marker genes in cardiac myocytes. J Biol Chem 274: 19323–19328, 1999.[Abstract/Free Full Text]
  26. Zicha J, Dobesova Z, Vokurkova M, and Kunes J. Abnormal Na,K-pump activity cosegregates with blood pressure in Dahl SS/Jr x SR/Jr F2 hybrids fed a high-salt diet since weaning (Abstract). Hypertension 34: 708, 1999.
  27. Zicha J and Duhm J. Kinetics of Na+ and K+ transport in red blood cells of Dahl rats. Effect of age and salt. Hypertension 15: 612–27, 1990.[Abstract]
  28. Zicha J, Negrin CD, Dobesová Z, Carr F, Vokurková M, McBride MW, Kunes J, and Dominiczak AF. Altered Na+-K+ pump activity and plasma lipids in salt-sensitive Dahl rats: relationship to Atp1a1 gene. Physiol Genomics 6: 99–104, 2001.[Abstract/Free Full Text]