ę 1995 American Heart Association, Inc.
From the Vascular Pathophysiology Unit, Department of Internal Medicine, Center for Biomedical Research, School of Medicine, University of Navarra, Pamplona (A.G., R.L., A.A., A.F., J.D.), and the Department of Medicine, University Hospital, School of Medicine, University of Zaragoza, Zaragoza, Spain (J.T., J.B., J.D.).
Correspondence to Javier Diez, MD, PhD, Unidad de FisiopatologÝa Vascular, Departamento de Medicina Interna, Centro de Investigaciones BiomÚdicas, Facultad de Medicina, C/ Irunlarrea s/n, 31080 Pamplona, Spain.
Key Words: hypertension, essential • lymphocytes • Na+-H+ exchanger • NHE-1 isoform
Several isoforms of the Na+-H+ exchanger protein have been cloned in mammalian cells and termed NHE-1, NHE-2, NHE-3, and NHE-4.5 6 7 8 9 Recent studies indicate that human lymphocytes express the mRNA encoding for the NHE-1 isoform of the Na+-H+ exchanger.10 Several observations suggest that NHE-1 participates in intracellular pH regulation11 and cellular volume homeostasis.12 Furthermore, NHE-1 appears to play a role in the initiation of cell growth.11
We designed the present study to investigate whether the enhanced Na+-H+ exchange activity detectable in lymphocytes from essential hypertensive patients could be explained by a parallel increase in NHE-1 mRNA levels. We investigated the steady-state levels of NHE-1 mRNA using a quantitative polymerase chain reaction (PCR) assay.
The control group consisted of 21 white normotensive subjects. These control subjects were normotensive patients of the outpatient clinics of the University Hospital at the University of Zaragoza.
The study was approved by the institutional Ethics Committee, and all subjects gave informed consent before inclusion. All subjects were studied under the conditions of an unlimited Na+ diet.
Medical examination consisted of a complete medical history, physical examination, and radiological, biochemical, and hormonal evaluations.13 The biochemical evaluation included determination of plasma glucose and plasma renin activity; measurement of serum cholesterol, potassium, bicarbonate, and creatinine; and determination of urinary sodium and creatinine. Blood samples were drawn with subjects in a fasting condition at 8 AM. Biochemical parameters were measured by routine laboratory methods. Plasma renin activity was estimated by radioimmunoassay for angiotensin I (Sorin, Sallugia).14 Creatinine clearance was calculated according to the standard formula: Creatinine Clearance=Urinary CreatininexUrinary Output (per minute)/Serum Creatinine.
Isolation of Lymphocytes
Early in the morning, venous blood (90 mL) was drawn from each subject maintained in fasting conditions. Blood was collected in heparinized tubes and diluted with an equal quantity of 0.9% NaCl. This solution was layered onto a sterile sodium metrizoate/Ficoll mixture (Lymphoprep, Nycomed, Pharma AS) and centrifuged at 800g for 15 minutes at 20░C. The thin lymphocyte layer resting on the density interface was collected and divided into two portions. One portion (40x106 cells) was washed three times in 0.9% NaCl, and the cells were homogenized in 4 mL guanidium isothiocyanate lysis buffer for the preparation of RNA and stored at -80░C until further manipulations. The remaining cells were used for the measurement of intracellular pH and the determination of Na+-H+ exchange activity. These cells were washed twice with a solution containing (mmol/L) NaCl 118, NaHCO3 20, KCl 2, MgCl2 1, CaCl2 1, glucose 10, and Tris-3-(N-morpholino)propanesulfonic acid (MOPS) 15, pH 7.4, at 37░C. The cells were resuspended in the same solution and kept at room temperature ready for use.
Measurement of Intracellular pH
Intracellular pH was measured fluorometrically using the pH-sensitive carboxyfluorescein derivative 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (Molecular Probes).15 Lymphocytes were incubated at a concentration of 20x106/mL with BCECF-AM (5 Ámol/L) for 30 minutes at 37░C in the above-mentioned medium. After incubation, the cells were washed twice and resuspended in medium that lacked the dye. The dye-loaded cells were kept at 20░C until ready for use.
Fluorescence determinations were carried out at 37░C with a flow cytometer (FACScan, Becton Dickinson) with excitation and emission wavelengths set at 488 and 530 nm, respectively. Intracellular pH was calculated from the BCECF fluorescence signals using the slope and intercept of a calibration line generated for each experiment. The calibration curve was performed using the nigericin technique described by Thomas et al.16 The calibration solutions contained (mmol/L) KCl 140, MgCl2 1, CaCl2 1, glucose 10, nigericin 0.020, and Tris-2-(N-morpholino)ethanesulfonic acid (MES) 20, for pH lower than 7.0, and Tris-MOPS 20, for pH higher than 7.0. The solutions were titrated to an external pH between 6.0 and 7.6 (at least five points) using dilute acid (HCl) or base (Tris base). Over this pH range, the fluorescence increased linearly with pH (r.998).
Measurement of Buffering Power
Buffering power was determined as described by Roos and Boron.17 The resting intracellular pH of BCECF-loaded lymphocytes was first determined. Then 5 mmol/L NH4Cl was added to the cell suspension, and the new intracellular pH was recorded. The buffering power was calculated as the ratio of intracellular NH4+ to intracellular pH, where intracellular NH4+ equals the rise in NH4+ produced by the alkaline pulse and intracellular pH is the difference between resting intracellular pH and intracellular pH after the NH4Cl pulse. The intracellular NH4+ concentration was calculated using a pKa for NH3 at 37░C of 8.89 and assuming that NH3 is in equilibrium across the cell membrane.17
Measurement of Na+-H+ Exchange Activity
For the analysis of Na+-H+ exchange activity, intracellular pH was acidified in a portion of the BCECF-loaded cells. Acidification of lymphocyte pH was performed as follows: Cells were incubated in an acid-loading solution with 2 Ámol/L nigericin for 5 minutes in a shaking water bath at 37░C. The acid-loading solution contained (mmol/L) KCl 150, MgCl2 1, CaCl2 1, glucose 10, and Tris-MES 20, pH 6.0, at 37░C. The cells were then washed and resuspended in an identical medium at the same pH but which lacked the ionophore and contained in addition fatty acid–free albumin (1 mg/mL) to scavenge residual nigericin.
Na+-H+ exchange activity was assayed as the initial rate of change in intracellular pH after an acid load. To measure the activity of the Na+-H+ exchanger, we compared the rate of intracellular pH recovery in acid-loaded lymphocytes in the presence and absence of Na+ (Na+ had been replaced with an equimolar amount of Ch+) as previously reported.18 The composition of the medium was (mmol/L) NaCl (or choline chloride) 140, KCl 2, MgCl2 1, CaCl2 1, glucose 10, and Tris-MOPS 20, pH 8.0, at 37░C. Both media were nominally HCO3- free. These were also the optimal conditions for promoting maximal exchange activity.18
Na+-dependent intracellular pH recovery was analyzed by fitting the intracellular pH versus time record to a single-exponential function (Fig 1),19 and its value (V) was estimated from the equation
where K is the slope of the linear regression line, intracellular pHmax is the maximal difference of intracellular pH recovery between cells incubated in media with and without Na+, and t is the time (in hours).
The initial rate of Na+-dependent H+ efflux was calculated multiplying V by the buffering power measured for cells at that particular intracellular pH and was expressed in millimoles per liter of cells per hour. In control experiments (n=3), 100 Ámol/L amiloride inhibited more than 97% of the Na+-dependent H+ efflux. We also examined the kinetics of amiloride inhibition of Na+-dependent H+ efflux. The data indicated half-maximal inhibition of Na+-dependent H+ efflux at an amiloride concentration of 5 Ámol/L. Therefore, this Na+-dependent H+ efflux was considered to represent the maximal activity (or Vmax) of the Na+-H+ exchanger in lymphocytes.
Total cell RNA was obtained using the guanidinium isothiocyanate–phenol–chloroform method.20 Briefly, 400 ÁL of 2 mol/L sodium acetate (pH 4.3), 4 mL of water-saturated phenol, and 1 mL of chloroform/isoamilic alcohol (49:1) were added to homogenized cells. RNA was precipitated with 2.5 vol ethanol, and the pellet was washed with ethanol at 75%. The resultant pellet was vacuum dried and resuspended in diethyl pirocarbonate–treated water. The RNA concentration was spectrophotometrically determined, and RNA was stored at -80░C.
For reverse transcription (RT) reaction, we used 1 Ág total RNA in a final volume of 20 ÁL. The following reagents were added to the mix reaction: 2 ÁL RT buffer (GIBCO BRL), 5 mmol/L each deoxy-nucleotide-triphosphate (Boehringer Mannheim Biochemica GmbH), 100 ng/ÁL random hexamers (Boehringer Mannheim), 200 U Moloney murine leukemia virus RT (GIBCO BRL), 5 mmol/L dithiothreitol, and 20 U RNAsin (Pharmacia Biotech Inc). cDNA synthesis proceeded for 60 minutes at 37░C and was stopped by heat (5 minutes at 95░C) and finally quick chilled in ice.
Before the quantitation assays, PCR optimal conditions were determined for three parameters: Mg2+ concentration, primer concentration, and primer annealing temperature for both NHE-1 and ▀-actin cDNA amplification. The conditions chosen were as follows: for NHE-1, 1.5 mmol/L, 100 ng/ÁL each primer, and 57░C; for ▀-actin, 1.5 mmol/L, 40 ng/ÁL each primer, and 57░C. Oligonucleotides (5'-3') d(CTTCCTCTACAGCTACATGG) and d(CATAGGCGATGATGAACTGG) were upstream and downstream primers, respectively, used for amplification of a 342 bp fragment (nucleotides 1409-1751) from human NHE-1 cDNA.21 Oligonucleotides (5'-3') d(TCTACAATGAGCTGCGTGTG) and d(GGTGAGGATCTTCATGAGGT) were used to amplify a 314 bp fragment from human ▀-actin cDNA located between nucleotides 1319 and 2079 in the reported human ▀-actin gene sequence.22 The final volume of the PCR reaction was 50 ÁL, with 2 U of Taq polymerase (Promega) and 3 ÁCi of [32P]dCTP (Amersham). The mixture was overlaid with 50 ÁL mineral oil and amplified in a GeneAtaq Thermocicler (Pharmacia Biotech AB). NHE-1 cDNA fragments were amplified by 25 cycles and ▀-actin fragments by 20 cycles with the following amplification profile: 94░C, 57░C, and 72░C at 1 minute for each step.
Although we performed the RT reaction in the same reaction tube, we carried out PCR reactions for NHE-1 and ▀-actin in separate tubes because variable amplification efficiency was observed when PCR was performed in the same tube.
Electrophoresis and Radioactivity Determinations
After amplification, 19-ÁL aliquots of the PCR reactions were electrophoresed on a 2% agarose gel, and bands were visualized by ethidium bromide. Bands of equal size were excised from the gel and radioactivity measured in a ▀ liquid scintillation counter (LKB Wallec). The obtained values were corrected with background radioactivity from blank reactions without RNA input. Finally, values corresponding to NHE-1 amplification were normalized with those for ▀-actin amplification.
Values are expressed as mean and range or mean▒SEM. Student's t test for single comparisons of quantitative parameters was used to assess statistical significance between normotensive subjects and hypertensive patients. The correlation between continuously distributed variables was tested by univariate regression analysis.
Intracellular pH and Buffering Power
Intracellular pH values obtained in lymphocytes from 21 normotensive subjects ranged from 7.26 to 7.48 (mean, 7.39▒0.01) (Fig 2). The reproducibility of the pH measurement was assessed by calculating the index of intra-assay variation in three samples (<0.20%) and the mean coefficient of interassay variation in four subjects (<0.20%). Intracellular pH was diminished (P<.001) in hypertensive (7.34▒0.01) compared with normotensive (Fig 2) individuals.
Buffering power was lower (P<.01) in lymphocytes from hypertensive patients compared with lymphocytes from normotensive subjects (8.66▒0.45 versus 12.80▒1.30 mmol H+/L cells per pH unit).
Na+-H+ Exchange Activity
The Na+-dependent H+ efflux measured in lymphocytes from normotensive and hypertensive individuals is presented in Fig 3. Maximal exchange activity in normotensive subjects ranged from 485 to 1367 mmol H+/L cells per hour (mean, 881▒56 mmol/L cells per hour). Measurement of the exchanger had an index of intra-assay variation and a mean coefficient of interassay variation of 1% (n=3) and 2% (n=4), respectively. Maximal Na+-H+ exchange activity was increased (P<.01) in lymphocytes from hypertensive patients (1262▒100 mmol H+/L cells per hour) compared with lymphocytes from normotensive subjects.
Maximal Na+-H+ exchange activity was directly correlated with systolic pressure (r=.371, P<.05, y=121+0.013x), diastolic pressure (r=.442, P<.01, y=73.2+0.014x), and mean arterial pressure (r=.448, P<.01, y=89+0.013x) (Fig 4) in all subjects. No correlation was found between exchange activity and intracellular pH.
PCR Amplification of Sequences Encoding for NHE-1 and ▀-Actin
The main problem of PCR-based quantitative methods arises from their great sensitivity. DNA amplification by PCR represents an exponential reaction, reaching a plateau effect at higher cycle numbers. The kinetic behavior of the process implies that small changes in amplification efficiency may significantly influence the final amounts of PCR product. However, quantitation requires accurate measurement in the phase of exponential amplification. Thus, we made amplification profiles for NHE-1 and ▀-actin to test the amount of RNA input and the cycle number needed to find the exponential range in the RT-PCR reaction. These experiments were performed using different amounts of RNA input in the RT reaction. Then, in the PCR process, the reaction was stopped each five cycles, and 7-ÁL aliquots were loaded onto an electrophoresis agarose gel. Kinetic behavior was assessed by addition of a 32P-labeled nucleotide (Fig 5). The arrows in Fig 5 indicate the amount of RNA and the number of cycles chosen for each cDNA amplification in further quantitative assays (500 ng RNA and 25 cycles for NHE-1; 300 ng RNA and 20 cycles for ▀-actin).
To assess whether the levels of ▀-actin remain unchanged,23 we carried out an RT-PCR reaction to amplify the ▀-actin cDNA fragment in six normotensive subjects and five hypertensive patients randomly selected. As shown in Fig 6, the values measured in the two groups of subjects were not significantly different (68 553▒7416 cpm in hypertensive patients and 66 251▒4165 cpm in normotensive subjects).
Analysis of NHE-1– and ▀-Actin–Specific PCR Products
The identity of the PCR products from NHE-1 and ▀-actin cDNA amplification was verified. The ▀-actin amplified fragment was digested with BstEII and Bgl II. BstEII yielded the predicted restriction fragments (205 and 109 bp), and Bgl II did not digest the amplified PCR product (no restriction site for this endonuclease is present in the amplified region) (Fig 8). The NHE-1 amplified fragment was purified and cloned in a p-GEM-t plasmid (Promega) and sequenced following a conventional dideoxy method (Fig 9).24
Changes in NHE-1 Expression in Human Lymphocytes in
The results of NHE-1 expression obtained in lymphocytes in the two groups of subjects studied are presented in Fig 10. The steady-state level of lymphocyte NHE-1 mRNA in normotensive subjects ranged from 0.01 to 0.26 (mean, 0.12▒0.02). To assess the reproducibility of our assay, we calculated the index of intra-assay variation in nine samples (13%) and the mean coefficient of interassay variation in nine subjects (31%). NHE-1 transcript levels were elevated (P<.05) in hypertensive patients (0.16▒0.01) with respect to normotensive subjects. No correlations were found between NHE-1 mRNA levels and either intracellular pH or Na+-H+ exchange activity.
The mechanisms by which the Na+-H+ exchanger is activated in essential hypertensive patients could include changes in the intracellular buffer capacity for H+, modifications of intracellular pH, and variations of cellular volume.26 An enhanced Na+-H+ exchange activity might be caused by a reduced buffer capacity for H+ in hypertensive patients. Although a reduced buffer capacity was found in cells from hypertensive patients in the present study, previous investigations have excluded any relation between the two parameters in hypertension.27 28
We have found that resting intracellular pH is diminished in lymphocytes from hypertensive patients compared with those from normotensive subjects. This is in agreement with the previous observation by Batlle et al29 of a reduced intracellular pH in lymphocytes from the spontaneously hypertensive rat. It has been shown that the human lymphocyte Na+-H+ exchanger is activated by an intracellular pH lower than 7.0, with a pK of 6.57.18 Thus, the lymphocyte Na+-H+ exchanger appears not to be involved in the regulation of resting intracellular pH. Accordingly, we believe that the activation of the exchanger seen in hypertensive patients cannot be attributed to the diminished resting intracellular pH present in their lymphocytes.
On the other hand, the involvement of the Na+-H+ exchanger in cellular volume regulation occurs under specific circumstances, namely, the acute response to cellular volume shrinkage associated with increased osmolality of the extracellular medium.30 Although we did not measure lymphocyte volume in the present study, other authors have found that the cell volume of lymphocytes is increased in hypertensive compared with normotensive individuals.4 Thus, it appears that changes in lymphocyte volume are not involved in the stimulation of Na+-H+ exchange activity in hypertension.
It has been previously demonstrated in lymphocytes from healthy subjects that Na+-H+ exchange activity is related to both the fluidity of the membrane and serum cholesterol.25 However, no associations were found in the present study between serum cholesterol and exchange activity, suggesting that changes in membrane fluidity do not account for the increased Na+-H+ exchange activity in hypertensive patients.
Conflicting results have been reported in the literature concerning the effects of several hormones, namely, aldosterone and insulin, on Na+-H+ exchange activity. Although Wehling et al31 reported a stimulatory effect of aldosterone on the lymphocyte exchanger, Quednau et al32 found no such effect. On the other hand, whereas some in vitro studies have demonstrated that erythrocyte Na+-H+ exchange activity may be stimulated by incubation with insulin,33 34 other studies failed to observe a similar effect in lymphocytes.25 Neither aldosterone nor insulin was measured in plasma from subjects in the present study. Nevertheless, none of the hypertensive patients exhibited biochemical features suggesting the existence of hyperaldosteronism or hyperinsulinemia.
It has been postulated that changes in cytosolic Ca2+ and Na+-H+ exchange activity in essential hypertension are closely linked, the rise in cytosolic Ca2+ being the cause of enhanced exchange activity.35 However, this theory is weakened when one considers that normal values for cytosolic Ca2+ fall in the nanomolar range and that cytosolic Ca2+ must be elevated to the micromolar range until an activation of the exchanger is observable,36 which is hardly likely.
Protein kinase C enhances the activity of the Na+-H+ exchanger.36 Thus, it could be proposed that an increased protein kinase C activity in essential hypertension should activate the exchanger via increased phosphorylation. However, no evidence has yet been provided that protein kinase C activity is increased in essential hypertension. On the other hand, phosphorylation of the Na+-H+ exchanger should result in kinetic changes opposite to those reported in the literature in hypertensive patients.2 37
Since there is no apparent explanation for the increased maximal activity of the Na+-H+ exchanger in lymphocytes from essential hypertensive patients, we established an RT-PCR assay to investigate whether an increase in NHE-1 mRNA levels would occur, thereby explaining the increase in exchanger activity.
Conventional specific techniques for gene expression measurement include Northern blot and RNase protection assay. However, these methods are not sensitive enough to detect mRNA in samples limited by either low copy number per cell or low cell number. Although the NHE-1 gene is ubiquitously expressed in all cell types so far studied, it is still unknown whether a low number of copies is present in the human lymphocyte. On the other hand, according to the protocol approved by the institutional Ethics Committee of our hospital, only 90 mL of blood was drawn from each subject to measure transport activity and mRNA expression. This meant that a maximal number of 40x106 cells was available to measure lymphocyte NHE-1 mRNA in each subject.
We therefore chose the RT-PCR method, which after amplification, allows for the identification of specific cDNA copies with low levels of mRNA, far below the detection limit of the above-mentioned techniques.38 39 In addition, recent studies in which RT-PCR was validated with respect to the above-mentioned techniques have demonstrated that the amount of mRNA estimated by the RT-PCR method correlated closely with the results of Northern blot analysis40 and RNase protection assay.41
Amplification of the target sequence with a constitutively expressed reference gene has been frequently applied to measure relative changes in mRNA levels.42 43 44 Thus, we chose to amplify the ▀-actin gene as a standard. Our results indicate that lymphocyte ▀-actin mRNA abundance remained unchanged in lymphocytes from patients with essential hypertension.
The main finding of the present study is that the steady-state level of lymphocyte NHE-1 mRNA was significantly increased in hypertensive patients with respect to normotensive subjects. This observation is in disagreement with recent results by Rosskopf et al.45 These authors were unable to find increased steady-state NHE-1 mRNA transcript levels in immortalized lymphoblasts from essential hypertensive patients. However, several reasons may account for the apparent discrepancy. First, Rosskopf et al measured NHE-1 mRNA in cultures of lymphoblasts grown from Epstein-Barr virus–infected lymphocytes. Thus, the specific characteristics of each cellular model may explain the different results. Second, Rosskopf et al analyzed NHE-1 mRNA expression by Northern blot. Therefore, differences in methodology may also influence the diversity of findings. Finally, Rosskopf et al measured NHE-1 mRNA levels in a small group of nine male normotensive subjects and a small group of 10 male hypertensive patients. In addition, numerical data relative to results of the Northern blot analysis suggest that a great interindividual variation occurs in the hypertensive group. It is likely that with the use of a larger group of hypertensive and normotensive individuals, the differences could reach statistical significance, as has occurred in the present study.
Studies on rats and cultured cells of renal origin have demonstrated that metabolic acidosis enhances both Na+-H+ exchange activity and NHE-1 mRNA transcript levels.46 47 48 Very recently, Quednau et al32 have found enhanced Na+-H+ exchange activity and NHE-1 mRNA levels in human lymphocytes during metabolic acidosis. Therefore, it appears that long-term metabolic acidosis influences the regulation of the Na+-H+ exchanger at the transcriptional level. Since none of the patients included in the present study exhibited metabolic acidosis, this cause can be excluded as being responsible for the enhanced expression of the NHE-1 gene seen in hypertensive patients.
Intracellular acidosis has been hypothesized as a possible mechanism of induction of expression of the Na+-H+ exchanger.49 Although an association was found in the present study between diminished intracellular pH and enhanced NHE-1 mRNA levels in lymphocytes from hypertensive patients, additional studies are needed to ascertain whether a causal relation exists between the two alterations.
Several other factors have been shown to be involved in the regulation of NHE-1 mRNA levels in a variety of cell types. For instance, in vascular smooth muscle, serum and platelet-derived growth factor increase NHE-1 mRNA levels.50 51 Further experiments are required to elucidate the possible role of these factors in the overexpression of NHE-1 mRNA in essential hypertension.
Our results show that the enhancement of maximal Na+-H+ exchange activity in essential hypertension is accompanied by a corresponding increase in NHE-1 mRNA abundance. The level of NHE-1 mRNA was increased by a factor of approximately 1.33 in hypertensive patients compared with normotensive subjects. This value is similar to the value by which the maximal activity was enhanced in lymphocytes from hypertensive patients compared with normotensive subjects. Nevertheless, no correlation was found in this study between the two parameters. Therefore, our findings do not prove a causal relation between increased transport activity and the rise in NHE-1 mRNA. Further investigations are required to confirm whether the number of exchangers is increased in lymphocyte membranes from patients with essential hypertension.
It is unknown whether lymphocytes express other Na+-H+ exchanger isoforms (ie, NHE-2, NHE-3, or NHE-4). Whether essential hypertension can stimulate the expression of these additional isoforms of the Na+-H+ exchanger in lymphocytes remains unknown. Thus, our study does not exclude the possibility that the transcription of additional isoforms of the exchanger is enhanced in hypertension, thereby contributing to the observed enhancement of Na+-H+ exchange activity. Subsequent studies should be performed to investigate this issue.
In summary, our finding of increased expression of the NHE-1 isoform of the Na+-H+ exchanger in lymphocytes from patients with essential hypertension might be indicative of an increased number of transporters in this condition. In turn, this could explain the increased maximal transport activity of the exchanger seen in lymphocytes from the same patients. The mechanism underlying this genetic change in hypertension remains to be defined.
Received August 8, 1994; first decision September 2, 1994; accepted November 7, 1994.
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