Sodium Transport in the Choroid Plexus and Salt-Sensitive Hypertension
To elucidate the role of epithelial sodium channels (ENaCs) and Na+-K+-ATPase in Na+ transport by the choroid plexus, we studied ENaC expression and Na+ transport in the choroid plexus. Lateral ventricle choroid plexuses were obtained from young male Wistar, Dahl salt–resistant (SS.BN13), and Dahl salt–sensitive (SS/MCW) rats on a regular (0.3%) or high- (8.0%) salt diet. The effects of ENaC blocker benzamil and Na+-K+-ATPase blocker ouabain on sodium transport were evaluated by measuring the amounts of retained 22Na+ and by evaluating intracellular [Na+] with Sodium Green fluorescence. In Wistar rats, ENaC distribution was as follows: microvilli, 10% to 30%; cytoplasm, 60% to 80%; and basolateral membrane, 5% to 10%. Benzamil (10−8 m) decreased 22Na+ retention by 20% and ouabain (10−3 m) increased retention by 40%, whereas ouabain and benzamil combined caused no change. Similar changes were noted in intracellular [Na+]. In Dahl rats on a regular salt diet, intracellular [Na+] was similar, but the amount of retained 22Na+ was less in sensitive versus resistant rats. High salt did not affect ENaC mRNA or protein, nor the benzamil induced decreases in retained 22Na+ or intracellular [Na+] in either strain. However, high salt increased intracellular [Na+] and attenuated the increase in uptake of 22Na+ by ouabain in resistant but not sensitive rats, suggesting a decrease in Na+-K+-ATPase activity only in resistant rats. These findings suggest that both ENaC and Na+-K+-ATPase regulate Na+ transport in the choroid plexus. Aberrant regulation of Na+ transport and of Na+-K+-ATPase activity, but not of ENaCs, might contribute to the increase in cerebrospinal fluid [Na+] in Dahl salt-sensitive rats on a high-salt diet.
Strict regulation of [Na+] in the cerebrospinal fluid (CSF) and, thus, the central nervous system is crucial for normal functioning of neurons. An increase in CSF [Na+] by as little as 2 mmol/L can increase the firing rate of neurons.1,2⇓ A chronic 5-mmol/L increase in CSF [Na+] causes sympathetic hyperactivity and hypertension.3 Such a subtle increase in CSF [Na+] might be a primary abnormality in salt-induced hypertension. CSF [Na+] increases by ≤5 mmol/L in Dahl salt-sensitive (S) rats and spontaneously hypertensive rats on a high-salt diet, and this increase appears to precede the increase in blood pressure, whereas CSF [Na+] shows minimal changes in “salt-resistant” strains, such as Wistar-Kyoto and Dahl salt-resistant (R) rats.3,4⇓
The epithelium of the choroid plexus (CP) is the major site for production of CSF, which is later absorbed by arachnoid granulations.5,6⇓ Net transport of Na+ between plasma and CSF is a balance of Na+ influx into and efflux out of the CP cells, which is accomplished by selective distribution and activity of a variety of enzymes and transporters.7,8⇓ An increase in CSF [Na+] in Dahl S rats on a high-salt diet may, thus, be attributed to an increased efflux of Na+ into the CSF, decreased reuptake of Na+ from the CSF, or both. However, the actual mechanisms contributing to the increase in CSF [Na+] in Dahl S rats or spontaneously hypertensive rats on a high-salt diet have not yet been studied.
A number of Na+ transporters mediate Na+ influx into the CP. On the basis of their distribution either on the apical or the basolateral surface, they contribute to the transport of Na+ from CSF or plasma into the CP cells along a prevailing electrochemical gradient. The Na+ channel blocker, amiloride, decreases Na+ transport into the CP and CSF.9–11⇓⇓ Blockade of the sodium hydrogen exchanger (NHE) was first thought to be responsible for this, but NHE proteins are almost undetectable in the CP of rats.5,12⇓ Amiloride also blocks epithelial sodium channels (ENaCs), which we reported recently to be present in the CP of rats.13 ENaCs are members of the amiloride-sensitive Na+ channels with 3 subunits, α, β, and γ, forming a heteromultimeric Na+ channel in many transporting epithelia.14–16⇓⇓
Na+-K+-ATPase forms the major pathway for active Na+ efflux from the CP. The predominant isoform in the choroid cells is the α1 Na+-K+-ATPase, whereas all 3 α isoforms are abundant in the brain microvessels.17–19⇓⇓ Its predominant location on the ventricular surface of the epithelium provides the driving force for directional transport of Na+ into the CSF against an electrochemical gradient.18–20⇓⇓ The Na+-K+-ATPase inhibitor ouabain, at 6.7×10−5 m, has no effect on the flux of 22Na+ from ventricle to blood but reduces the flux from blood to ventricle by 33% in the CP of frogs.11 Similarly, 10−6 m ouabain added to the artificial CSF used for ventriculo-cisternal perfusion of rabbits reduces the transport of 22Na+ from blood to CSF by 50% to 60%.21 ICV infusion of ouabain at 10 μg/d decreases CSF [Na+] by 6 to 8 mmol/L in Wistar rats on a high-salt diet.3
Considering that the role of ENaCs in Na+ transport by the CP has not yet been studied at all, the first objective of the current study was to assess in Wistar rats the expression and subcellular distribution of ENaCs in the CP and the effects of blockers of the ENaC and Na+-K+-ATPase on parameters of Na+ transport by the CP in vitro. This first set of experiments demonstrated that both the ENaC and Na+-K+-ATPase contribute to Na+ transport by the CP. As a second objective, we then evaluated whether aberrant regulation of the ENaC or Na+-K+-ATPase may contribute to the salt-induced increase in CSF [Na+] in Dahl S rats and assessed the above parameters in Dahl S versus Dahl R rats on a regular or high-salt diet.
Animals and Protocols
Young 5- to 6-week–old male Wistar, Dahl S (SS/MCW), and Dahl R (SS.BN13) rats received a regular (0.3%, 120 μmol of Na+ per gram) or high- (8.0%, 1370 μmol of Na+ per gram) salt diet for 7 to 14 days. For details of methods, see the online Data Supplement at http://hyper.ahajournals.org.
The CP of the lateral ventricles was micropunched for total RNA isolation as described previously.13 Genomic DNA was removed by DNAse I (Ambion), and cDNAs were synthesized by Superscript II RNase H− Reverse Transcriptase (Invitrogen). The mRNA abundance of α, β, and γ ENaCs was measured by quantitative real-time PCR. Phospho-glycerate kinase 1 was used as the housekeeping gene. The PCR conditions and external standard for phospho-glycerate kinase 1 and α, β, and γ ENaCs were the same as those described previously.13
CP protein extracts (5 μg) were run on a 4% to 12% gradient gel (Bio-Rad) by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane was probed with the primary antibodies to α-ENaC (1:500), β-ENaC, or γ-ENaC (1:1000)22 overnight at 4°C, followed by goat antirabbit secondary antibody (1:5000; Santa Cruz). β-Actin (1:10000) was used as an internal standard to compare band density.
Immunohistochemistry was performed as described previously13 using the primary antibodies22 diluted in blocking solution (1:250 for α- and γ-ENaC; 1:500 for β-ENaC) and was processed using the Vectastain Elite ABC kit (Vector Laboratories). Antigen-antibody reactions were developed with Vector diaminobenzidine kit with nickel enhancement. Intensity and percentage area of the staining in CP cells were assessed using Image Pro (version 6.2; Media Cybernetics, Inc). In addition, distribution of the staining in the apical membranes and cytoplasm was assessed blindly by 2 individuals and scored 1 (<30%), 2 (30% to 70%), and 3 (>70%) on the basis of the area stained.
Immunoelectron microscopy was done following standard protocols with the primary antibodies diluted in 0.1% BSA in PBS (1:100). The number of gold particles (per 100 μm2) in the cytoplasm, apical, or basal membrane was counted in cells having intact morphology (4 to 8 per rat). Approximately 4000 immunoelectron microscopy fields were quantified.
Studies With Isolated CP
The CP of lateral ventricles was quickly removed after decapitation and stored on individual snap-well inserts (Corning) of 6-well culture plates. The insert and well were filled with 500 and 2000 μL, respectively, of artificial cerebrospinal fluid (aCSF) or aCSF+drug.
Uptake of 22Na+
From each rat, 1 CP was bathed in aCSF as a control and the contralateral CP was bathed in aCSF containing drugs: benzamil (1, 10, or 100 nM), ouabain (1 mmol/L or 1 μmol/L), ethyl isopropyl amiloride (EIPA; 1 mmol/L), or both benzamil and ouabain for 2 hours. Each CP was then put in 0.5-mL Eppendorf tubes containing 250 μL of the same incubation solution with 1 μCi/mL of 22Na+ and incubated for 30 seconds. Activity of 22Na+ was assessed on a liquid scintillation counter (1219 RackBeta) and is expressed as counts per minute per 100 μg of CP. Retention of 22Na+ was negligible in CP briefly dipped in aCSF containing 22Na+. The amount of retained 22Na+ increased with increasing duration of incubation and approached a steady-state volume of distribution (Vd) within 1 minute (Figure S1 in the online Data Supplement), with no further uptake beyond 1 minute.
Measurement of Intracellular [Na+]
The CPs were first loaded with 10 μmol/L of Sodium Green (Molecular Probes). After 3 washes in aCSF, the CPs were divided into 3 to 5 pieces and put in aCSF or aCSF containing drugs as above for 2 hours. After the incubation period, each piece was transferred to glass coverslips and viewed in an inverted epifluorescence microscope. Sodium Green was excited by an Argon Laser at 488 nm, and emissions between 500 to 560 nm were assessed. Fluorescence intensity of Sodium Green was calibrated to the [Na+] by incubation in the presence of gramicidin (5 μmol/L) and by varying the [Na+] in aCSF by replacing Na+ with N-methyl-d-glucamine. Intensity of Sodium Green fluorescence increased when the CSF [Na+] was increased. The change in fluorescence intensity with varying [Na+] was used to generate a calibration curve for the measurement of intracellular [Na+] ([Na+]i; Figure S2).
Data are expressed as mean±SEM. Effects of individual drugs on Na+ transport in Wistar rats were assessed by paired t test. The effects of diet and strain on gene expression and parameters of Na+ transport were evaluated by 2-way ANOVA. Effects were considered statistically significant at P<0.05.
Expression and Subcellular Distribution of ENaCs in the CP
Both mRNA and protein of all 3 of the ENaC subunits were expressed in the CP of all 3 of the rat strains studied. Figure 1A shows the major bands detected by the antibodies in the kidney (as positive control) and the CP of Wistar rats: ≈80 and 37 kDa for α-ENaC, ≈85 kDa for β-ENaC, and ≈90 and 70 kDa for γ-ENaC. Immunoreactivity was present in >70% of CP cells and was most prominent in the apical membranes and cytoplasm (Figure 1B). Similarly, at the subcellular level, ≈35% of α-ENaC and ≈15% to 20% of β- and γ-ENaCs were in the apical microvilli, only ≈5% to 10% in the basolateral membranes and the remainder in the cytoplasm (Figure 1B).
Na+ Transport in the CP
Figure 2 shows the effect of various drugs on the retention of 22Na+ by the CP in Wistar rats on a regular salt diet. Ouabain (1 μmol/L), which blocks α2 and α3 Na+-K-ATPase, did not affect the amount of 22Na+ retained, whereas 1 mmol/L of ouabain, which blocks all of the α isoforms, increased retention by ≈40%. Benzamil (1 nM) did not have a significant effect, but there was a dose-dependent decrease in the amount of retained 22Na+ with 10 and 100 nM benzamil. To minimize the possible involvement of other channels, further experiments were continued using only the lowest effective dose of benzamil (10 nM). This dose of benzamil decreased retained 22Na+ in the CP at 30, 45, and 60 seconds by 18%, 20%, and 25%, respectively (Figure S1). Combining 1 mmol/L of ouabain and 10 nM benzamil together caused no overall change in the amount of retained 22Na+ (Figure 2). The NHE blocker EIPA at 1 μmol/L had minimal effect (≈10% decrease) on retained 22Na+.
Intensity of the Sodium Green (Figure S3) was low under basal conditions, with a calculated [Na+]i of 35±3 mmol/L. Fluorescence intensity was lower in the presence of 10 nM benzamil with [Na+]i at 16±2 mmol and higher in the presence of 1 mmol/L of ouabain with [Na+]i at 60±3 mmol/L. Fluorescence intensity was similar to controls in the presence of both ouabain and benzamil, with a calculated [Na+]i of 39±2 mmol/L.
Effect of High-Salt Diet on ENaC Expression in Dahl Rats
Abundance of α-ENaC mRNA was similar in Dahl R and S rats, but the abundance of both 80- and 37-kDa protein fractions of α-ENaC was higher in Dahl S than in Dahl R rats. Relative abundance of the 37- and 80-kDa fractions of α-ENaC was similar in S and R rats (Table and Figure 3). Immunoreactivity to α-ENaC showed low staining intensity and was similar in R and S rats. High-salt diet did not affect α-ENaC mRNA abundance, protein quantity, or immunodensity in either strain (Table and Figure 3).
β-ENaC mRNA was ≈2-fold less, whereas β-ENaC protein abundance was similar and immunoreactivity greater in the apical membrane in the CP of Dahl S versus R rats (Table and Figure 3). High-salt diet did not affect β-ENaC mRNA or protein abundance in Dahl R and S rats, and the higher immunoreactivity in the Dahl S rats persisted on a high-salt diet (Table and Figure 3).
Abundance of the mRNA and the 90-kDa and 70-kDa bands of γ-ENaC was similar, whereas immunoreactivity to γ-ENaC was higher in both apical membranes and cytoplasm of CPs of Dahl S versus R rats. A high-salt diet tended to lower (P=0.08) γ-ENaC mRNA but did not affect protein abundance or distribution (Table and Figure 3). Figure S4 shows the actual Western blots in Dahl rats on regular and high salt.
Effect of High-Salt Diet on Na+ Transport in the CP
On regular salt, the amount of retained 22Na+ (Figure 4) was lower in the CP of Dahl S versus Dahl R rats, whereas [Na+]i (Figure 5) was similar. Benzamil decreased retention of 22Na+ and [Na+]i to the same extent in both strains (Figures 4 and 5⇓). Ouabain increased the amount of retained 22Na+ by ≈40% (Figure 4) in rats on a regular salt diet but increased [Na+]i only in Dahl R rats (Figure 5). A high-salt diet significantly increased [Na+]i (Figure 5) and lowered the amount of retained 22Na+ in R rats only (Figure 4). The decrease in retention of 22Na+ and in [Na+]i by benzamil was not affected by a high-salt diet in either strain. However, high salt attenuated the ouabain-induced increase in 22Na+ retention by ≈50% in Dahl R rats and, if anything, enhanced the effect in Dahl S rats (Figure 4). The ouabain-induced increase in [Na+]i was also less in Dahl R rats on high salt, whereas in Dahl S rats this response remained absent (Figure 5). Dahl R and Wistar rats showed the same pattern of responses to high salt (Figure S5).
The major findings of the current study are that, in the epithelial cells of the CP, ENaC is prominent on the apical microvilli and contributes to Na+ transport. Both benzamil-blockable Na+ influx and ouabain-blockable efflux contribute to Na+ transport across the CP. On regular salt, the amounts of retained 22Na+ and ouabain-induced increase in [Na+]i are both lower in Dahl S rats. High-salt diet increases [Na+]i and decreases the amount of retained 22Na+ and ouabain-blockable efflux only in R rats. The effects of benzamil were not dependent on strain or dietary salt. Figure 6 provides a schematic outline of the main locations of ENaC and Na+-K+-ATPase in CP cells and their possible roles in Na+ transport across the CP in Dahl S versus R rats, as will be discussed in detail.
Sodium Transport in the CP
Benzamil dose-dependently blocks a number of channels, including ENaC, acid-sensing ion channel, NHE, and Na+–Ca2+ exchanger channel.23 However, it has the highest potency for ENaCs.23 At the nanomolar concentration used in the current experiments, the benzamil blockade can be assumed to be limited to ENaCs.23 The specific NHE blocker EIPA at a high concentration of 1 μmol/L decreased retained 22Na+ only by ≈10%, whereas benzamil at 10 nM caused a 25% decrease, suggesting that a significant portion of the previously9–11⇓⇓ reported inhibition of Na+ influx by amiloride is ENaC mediated. Inhibition of this ENaC-mediated influx of Na+, while efflux continues, caused a marked decrease in [Na+]i, as measured by Sodium Green fluorescence.
The choroid cells in CP express the α1, β1 and β3 subunits of the Na+-K+-ATPase, whereas the blood vessels express all of the α1 to α3 and β1 to β3 subunits.24,25⇓ In the current study, 1 μmol/L of ouabain, which blocks the α2 and α3 subunits, did not have a significant effect on retained 22Na+, but 1 mmol/L of ouabain increased retention by 40% and increased [Na+]i by ≈50%. This finding indicates that ouabain inhibits the efflux by blockade of the α1 subunit of Na+-K+-ATPase predominantly in the CP cells. Blockade of the enzyme by ouabain, together with blockade of ENaC by benzamil, resulted in unchanged 22Na+ retention and [Na+]i, indicating that, similar to renal epithelia,26 ENaC-mediated influx and Na+-K+-ATPase-dependent efflux jointly contribute to Na+ transport across the choroid epithelial cells.
ENaC Localization and Expression in the CP
In absorbing epithelia of the kidneys and colon, ENaC is expressed on the luminal surface, where it mediates the final steps of Na+ reabsorption.16 The basolateral Na+-K+-ATPase generates the electrochemical gradient required for ENaC-mediated Na+ transport.26 In the CP, Na+-K+-ATPase is localized mainly on the microvilli and contributes to Na+ secretion into the CSF, and its blockade clearly lowers CSF [Na+].3 However, in contrast to the pattern in renal epithelia, we demonstrate by electron microscopy that, in Wistar rats, only 5% to 10% of the ENaC is localized on the basolateral membrane and 15% to 30% is expressed on the microvilli. This pattern is similar to that in the ciliary body of the eye, where ENaC appears to contribute to Na+ reabsorption from the aqueous humor.27,28⇓ Therefore, ENaC might contribute to Na+ influx into choroid cells primarily from the CSF and, to a lesser extent, from the blood. Additional studies are clearly required to elucidate the mechanisms that regulate cellular distribution and function of ENaC in the CP, which may involve functions29–31⇓⇓ other than Na+-transport.
Dahl and Wistar rats showed a similar pattern for ENaC localization. On a regular salt diet, components of ENaC expression appeared to be higher in S versus R rats, that is, higher protein abundance for α and higher staining intensity for β and γ subunits. This pattern was not influenced by high-salt diet and may indicate a higher ENaC activity in S versus R rats.
Na+ Transport in the CP of Dahl R and S Rats
Most interestingly, on regular salt, retention of 22Na+ was lower but [Na+]i was similar in the CP of S versus R rats. Considering also that CSF uptake of IV 22Na+ is enhanced in vivo in S versus R rats,4 these findings may reflect enhanced Na+ transport across the CP. Such enhanced transport may be because of increased activity of Na+-K+-ATPase associated with increased influx through Na+ channels, such as the ENaC in S rats. However, the benzamil-induced decreases in 22Na+ uptake and in [Na+]i were similar between the 2 strains. It appears that the higher abundance of ENaCs at the apical membranes in S versus R rats does not lead to significantly higher activity and Na+ influx. Alternatively, blockade by benzamil of higher ENaC-dependent influx at the apical membrane may be offset by a larger non–ENaC-dependent influx at the basolateral membranes of S rats. Ouabain also similarly increased 22Na+ retention in S and R rats, consistent with a similar role for Na+-K+-ATPase in efflux in the 2 strains. However, ouabain increased [Na+]i only in R rats and not at all in S rats. The increase in 22Na+ retention but absence of an increase in [Na+]i by ouabain in S rats may, therefore, reflect enhanced turnover of Na+i, that is, larger influx and efflux, with the latter not dependent on Na+-K+-ATPase. Additional studies are required to elucidate the actual mechanisms for this enhanced influx and efflux in S versus R rats.
High Salt Lowers Na+-K+-ATPase-Mediated Efflux in R But Not S Rats
A high-salt diet increased [Na+]i by ≈30% and decreased retained 22Na+ by ≈20% in R rats but caused no changes in S rats. In renal epithelia, an increase in [Na+]i would be compensated by greater activity of Na+-K+-ATPase to maintain [Na+]i, cell volume, and osmolarity. In the CP of Dahl R rats on high salt, the opposite appears to occur. High-salt diet markedly decreased the ouabain-sensitive component of efflux and the further increase in [Na+]i. Such a decrease in Na+-K+-ATPase activity in the CP of R rats on a high-salt diet might be a mechanism to prevent increases in CSF [Na+] and resultant sympathoexcitation and hypertension. The mechanism(s) responsible for this decrease in activity still need to be explored, and these studies may lead to new insights into the salt resistance of, for example, R rats, and, by extension, humans. In the current experimental design, any endogenous inhibitor of enzyme activity should have dissociated by the ≈2 hours preincubation. Mechanisms causing a decrease in expression appear to be more likely.
In contrast to Dahl R, in S rats, high salt failed to inhibit ouabain-dependent Na+ transport and, if anything, caused a moderate increase. This enhanced activity or increased expression of the pump may reflect a primary abnormality of mechanisms regulating the pump in Dahl S rats as compared with R or Wistar rats.32,33⇓ A persistent high activity of the pump would contribute to an increase in CSF [Na+], resulting in increased release of ouabain-like compounds (“ouabain”)34 to inhibit this increase in pump activity32 and to blunt the increase in CSF [Na+].3 Alternatively, CSF [Na+] may increase through other mechanisms, and the resultant increase in concentration of ouabain would also inhibit the pump but also enhance Na+-K+-ATPase expression in the CP through negative feedback.35,36⇓ In the current experimental design, any endogenous ouabain should have dissociated by ≈2 hours preincubation,36 thereby exposing the uninhibited pump activity. In support of this second possibility, despite the somewhat larger inhibition of efflux by ouabain in S rats on high salt, ouabain still did not increase [Na+]i. This pattern of changes is consistent with this second possibility and the above-stated conclusion of enhanced efflux of Na+ (into the CSF) in S rats not dependent on Na+-K+-ATPase. Additional studies, including molecular biology approaches, will provide further insights in this regard.
The present studies on Na+-transport in the CP of Dahl rats appear to point to mechanisms contributing to an increase in CSF [Na+] and thereby possibly to hypertension in Dahl S rats on a high-salt diet. Dahl SS/MCW clearly differed from Dahl SS.BN1337 rats, whereas the latter responded similar to Wistar rats as another control strain. Additional studies in congenic or consomic strains are needed to assess whether these CP mechanisms indeed track the CSF [Na+] and hypertension.
Being the prime source of CSF, Na+ transport mechanisms in the CP are important in the regulation of CSF [Na+]. The current study suggests aberrant regulation of Na+ transport and of Na+-K-ATPase in the CP of S rat, that may contribute to the increase in CSF [Na+] in this strain on a high-salt diet. Future studies using microarrays and tissue arrays in the early phase of a high-salt diet might elucidate the possible mediators of this abnormal Na+ transport and identify new candidate genes causing the increase in CSF [Na+] and hypertension in S rats on a high-salt diet, as well the salt resistance of Dahl R rats.
We are thankful to Dr Lawrence G. Palmer (Cornell University) for the generous gift of ENaC antibodies. We also thank Dr Bing Huang, Peter Rippstein, Roselyn White, Li Bi, and Junhui Tan for technical assistance and Danielle Oja for formatting assistance.
Sources of Funding
This work was supported by Canadian Institutes of Health Research Operating Grant FRN-MOP:74432. M.S.A. was supported by a Pfizer/Canadian Institutes of Health Research/Canadian Hypertension Society doctoral research award and an Ontario Graduate Scholarship in Science and Technology.
F.H.H.L. holds the Pfizer Chair in Hypertension Research, an endowed chair supported by Pfizer Canada, the University of Ottawa Heart Institute Foundation, and the Canadian Institutes of Health Research.
- Received October 29, 2008.
- Revision received November 18, 2008.
- Accepted June 30, 2009.
- ↵Huang BS, Van Vliet BN, Leenen FH. Increases in CSF [Na+] precede the increases in blood pressure in Dahl S rats and SHR on a high-salt diet. Am J Physiol. 2004; 287: H1160–H1166.
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- ↵Jernigan NL, LaMarca B, Speed J, Galmiche L, Granger JP, Drummond HA. Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries. Am J Physiol. 2008; 294: H409–H420.
- ↵Zicha J, Negrin CD, Dobesova Z, Carr F, Vokurkova M, McBride MW, Kunes J, Dominiczak AF. Altered Na+-K+ pump activity and plasma lipids in salt-hypertensive Dahl rats: relationship to Atp1a1 gene. Physiol Genomics. 2001; 6: 99–104.
- ↵Wang H, Leenen FH. Brain sodium channels mediate increases in brain “ouabain” and blood pressure in Dahl S rats. Hypertension. 2002; 40: 96–100.
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