NHE-1 Protein in Vascular Smooth Muscle and Lymphocytes From the Spontaneously Hypertensive Rat
Abstract The present study examined the abundance of NHE-1 protein in cultured vascular smooth muscle cells (VSMCs), freshly isolated thymocytes, and fresh aortic tissue from spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. Two sets of affinity-purified antibodies (Ab(765-778) and Ab(698-711)) against different epitopes of the NHE-1 isoform of the Na+-H+ antiporter were used. Each set of antibodies recognized a major protein band at 105 to 110 kD that was more abundant in protein lysates prepared from cultured VSMCs from the SHR than those from WKY rats (Ab(765-778) 0.047±0.011 vs 0.010±0.002 O.D. units/10 μg protein, P<.001 for SHR and WKY, respectively; and Ab(698-711) 0.173±0.026 vs 0.087±0.028 O.D. units/10 μg protein, P<.05, for SHR and WKY, respectively). The increase in NHE-1 protein abundance in cultured VSMCs from the SHR was associated with a greater Vmax of the Na+-H+ antiporter as compared to those from WKY rats (17.93±2.07 vs 8.16±1.05 mmol H+/min, P<.001, respectively). In contrast to cultured VSMCs, there was no difference in the relative abundance of NHE-1 protein in fresh aortic tissue (0.075±0.018 vs 0.083±0.017 O.D. units/10 μg protein, from SHR and WKY, respectively) or in freshly isolated thymocytes (0.158±0.046 vs 0.226±0.054 O.D. units/10 μg protein, from SHR and WKY, respectively). We conclude that the increase in the Vmax of the Na+-H+ antiporter in cultured VSMCs from the SHR, compared to those from WKY rats, is due, at least in part, to increased levels of NHE-1 protein.
Cultured vascular smooth muscle cells (VSMCs) from the spontaneously hypertensive rat (SHR) exhibit increased activity of the Na+-H+ antiporter as compared to normotensive Wistar-Kyoto (WKY) rats.1 2 3 Analysis of the kinetic properties of the Na+-H+ antiporter in cultured VSMCs from the SHR has revealed that the Vmax of the Na+-H+ antiporter is increased as compared to VSMCs from WKY rats.1 2 3 In contrast, thymic lymphocytes from the SHR display similar kinetic properties for the Na+-H+ antiporter as compared to those from WKY rats.4 These discordant findings may reflect cell-specific differences in the properties of the Na+-H+ antiporter or, more likely, differences in the phenotype expressed in cultured VSMCs as compared to freshly isolated lymphocytes. Cultured VSMCs from the SHR exhibit a growth phenotype characterized by enhanced proliferation, a feature observed only in early passaged VSMCs.1 2 Increased activity of the Na+-H+ antiporter in cultured VSMCs from the SHR likewise is observed in early passaged cells (passages 2 through 6) but not in cells grown in late subpassages (passages 7 through 10). Because the NHE-1 isoform of the antiporter is involved in the regulation of cell growth, the alterations in Na+-H+ antiporter activity and cell proliferation observed in cultured VSMCs from the SHR may be interrelated.1
The molecular basis for the observed increase in Vmax of the Na+-H+ antiporter in cultured VSMCs has not been defined. VSMCs, like lymphocytes, possess the NHE-1 isoform of the Na+-H+ antiporter. Other isoforms of the Na+-H+ antiporter (NHE-2, NHE-3, and NHE-4) are not expressed in cultured VSMCs.5 Studies by Lucchesi et al5 and LaPointe et al1 showed that NHE-1 mRNA levels are not different between VSMCs derived from SHR and WKY rats. This finding suggests that the greater level of Na+-H+ antiporter activity in cultured VSMCs from the SHR is due to a posttranscriptional alteration.1 An increase in the abundance of NHE-1 protein in cultured VSMCs from the SHR seems a likely mechanism for such an alteration and one that could also explain the elevated Vmax of the Na+-H+ antiporter. In a recent study by Siczkowski et al,6 however, differences in the level of NHE-1 protein between cultured VSMCs from SHR and WKY rats could not be found.
In the present study, we addressed these issues in cultured VSMCs from the SHR and further examined NHE-1 abundance in freshly isolated VSMCs and thymocytes. We show that cultured VSMCs from the SHR have increased levels of NHE-1 protein, whereas freshly isolated VSMCs and thymocytes do not.
Animals and Cells
Rats were purchased from Taconic Farms (Germantown, NY) and used at 12 to 18 weeks of age. The SHRs had significantly higher blood pressures and were significantly smaller than the WKY rats as determined by body weight prior to death (data not shown).
In total, 12 SHR and 9 WKY rats were used for establishment of VSMC cultures. Cell lines were established from individual donor animals as described previously to avoid mixed populations of cells that would result from pooling of cells derived from different donor animals.1 7 The aorta was cleaned of blood and connective tissue and then incubated in phosphate buffered saline (PBS) containing collagenase IV and elastase (Sigma Chemical Co). The endothelium and adventitia were removed, and the intact muscularis was reincubated with collagenase and elastase for 30 to 60 minutes. It was cut into 1 to 2 mm2 pieces, and the VSMCs were dispersed by vigorously pipetting through a 5-ml serological pipette. The dispersed cells were washed once by centrifugation with PBS and resuspended in culture media. The cells were grown in Dulbecco’s modified Eagle’s medium plus Ham’s F-12 nutrient mixture (1:1, Sigma), 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (250 μg/mL) at 37°C in a humidified atmosphere of 5% CO2. Cultures were fed or passaged twice weekly. Experiments were performed on cells grown in passages 2 through 8.
VSMCs used for immunoblots were grown to confluence in 100-mm-diameter dishes and were placed in serum-free media for 24 hours prior to use according to protocols used previously to examine Na+-H+ antiporter activity in cultured VSMCs from the SHR.1 6 The following day, the cells were washed twice with PBS and then scraped off the dish into 1 mL of PBS containing 10 μg/mL each DNase, RNase, chymostatin, leupeptin, aprotinin, and pepstatin (CLAP) and 0.5 mmol/L PMSF.
Fresh aortas were obtained from five SHR and five WKY rats. Thoracic aortas were incubated with collagenase and elastase for 15 minutes followed by the removal of the endothelium and adventitia by microdissection. The muscularis was then cut into small pieces and homogenized by hand with a Teflon pestle in PBS containing DNase, RNase, and protease inhibitors as described above.
Thymuses were obtained from nine SHR and nine WKY rats. Thymic lymphocytes were isolated by teasing apart the thymus in RPMI 1640.4 The cell suspension was collected and washed three times by centrifugation in RPMI. The final pellet was resuspended in the homogenization buffer and homogenized with a Kinematica homogenizer.
Cell homogenates were centrifuged at 14 000 rpm in a desktop microcentrifuge, and the protein was extracted from the pellets by incubation at 37°C in 0.5 mL of a 2% Triton X-100 solution containing 15 mmol/L Tris, pH 8.0, 4 mmol/L EGTA, 120 mmol/L NaCl, 2 mmol/L MgCl2, 10 μg/mL DNase, 10 μg/mL RNase, 10 μg/mL CLAP, and 0.5 mmol/L PMSF. Preliminary studies showed that no detectable NHE-1 protein remained in the pellet after the Triton X extraction. The lysates were used immediately or stored at −85°C until used.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were used to perform an analysis of the molecular weights and quantification of the relative amount of NHE-1 proteins.8 Equal amounts of protein from cell lysates isolated on the same day from SHR and WKY rats were subjected to SDS-PAGE electrophoresis (10% polyacrylamide) under reducing conditions. After electrophoresis, the proteins were transferred overnight onto nitrocellulose paper by electrophoresis.9 The complete transfer of protein from the gel was confirmed in some experiments by silver staining of the gel after electrophoretic transfer.8
Detection of the NHE-1 proteins was performed using double antibody staining. Prior to probing with the primary antibody, the nitrocellulose was blocked for nonspecific protein binding by incubation for at least 1 hour with 5% powdered milk in PBS containing 0.1% Tween 20 (PBS-T). After blocking, the nitrocellulose was washed once in 0.3% PBS-T and then twice in 0.1% PBS-T. The nitrocellulose was then probed with one of the rabbit polyclonal antibodies against NHE-1 diluted in 0.1% PBS-T plus 0.2% fish skin gelatin. After probing, the nitrocellulose was then subjected to multiple washes with PBS-T and then incubated with horseradish peroxidase–labeled donkey anti-rabbit IgG in PBS-T containing fish skin gelatin (Amersham Corp). The excess secondary IgG was removed by a series of washes with PBS-T, and the bound antibody was visualized using enhanced chemoluminescence (ECL) detection (Amersham) for 5 to 30 minutes.
Affinity-purified antibodies against the cytoplasmic tail of NHE-1 were a generous gift from Drs. O. Moe and R. Alpern (Dallas, Tex). Two separate sets of polyclonal antibodies, generated against different epitopes of NHE-1, were used. The first antibody (Ab(765-778)) was raised against a peptide sequence corresponding to amino acids 765 to 778, near the carboxy terminal end of the cytosolic portion of the published sequence of the human NHE-1 protein,10 whereas the other antibody preparation (Ab(698-711)) was generated against a peptide sequence corresponding to amino acids 698 to 711 of the published NHE-1 sequence. There is only one amino acid difference between the human and rat sequences for each peptide used to make these antibodies. Glutamic acid at position 710 in the human sequence is replaced with alanine in the rat, and threonine at position 769 in the human sequence is replaced with proline in the rat. Both sets of antibodies recognized a protein band at about 110 kD (Fig 1⇓), the reported molecular weight of NHE-1.10 Recognition of the 110 kD protein was blocked by preincubation of each anti-NHE-1 antibody with excess of their respective immunizing peptides (Fig 1⇓), indicating that this protein band was specifically recognized by the antibodies used.
Na+-H+ Antiporter Kinetics
The pH-sensitive fluorescent probe, 2′,7′-bis(carboxyethyl)-5,6-carboxy-fluorescein (Molecular Probes) was used to measure pHi in a superfused system as described previously.1 The standard assay solution had the following composition (mmol/L): NaCl 136.8, KCl 4.7, CaCl2 1.25, MgCl2 1.25, Na2HPO4 0.97, NaH2PO4 0.23, glucose 5, HEPES 5, pH 7.4. Na+ kinetics were determined as the initial pHi recovery rates in acidified cells assayed in the presence of different Na+ concentrations (0-140 mmol/L). Choline was iso-osmotically substituted for sodium. We have shown previously that the pHi recovery under these conditions is Na+ dependent and inhibitable by ethyl-isopropylamiloride.1 The pHi recovery data was transformed to net rates of H+ efflux by multiplying the change in pHi by the buffering power of the cells.1 Determination of kinetic parameters for the Na+-H+ antiporter was done using a double reciprocal plot.
Miscellaneous Measurements and Statistical Analysis
To determine the effect of serum starvation on VSMC proliferation, the proportion of cells in the G0-G1 phase of the cell cycle was determined by fluorescence-activated cell sorting (FACS) analysis. After 24 to 36 hours of serum deprivation, the cells were trypsinized and resuspended in Dulbecco’s PBS. The cells were fixed in 50% ethanol and kept at 4°C. Before cell cycle analysis, the cells were washed twice with PBS and stained with propidium iodide. RNase (180 μ/mL) was added to each sample. After a 30-minute incubation at 37°C, the level of propidium iodide staining was determined using a flow cytometer (Epics-XL, Coulter Corp).
Protein concentrations of cell lysate preparations were measured using a BCA protein assay (Amersham). Densitometry was performed using a Bio-Rad model GS 670 Imaging Densitometer and Molecular Analyst version 1.1.1 software (Bio-Rad).
All data are reported as mean±SEM. Differences between SHR and WKY cells were determined using Student’s t test. Differences were considered statistically significant when P<.05.
Cultured VSMCs from SHR and WKY rats were acutely acidified to a pHi of about 6.4, and the pHi recovery was determined in the presence of varying concentrations of external Na+. For every concentration of Na+ examined, the rates of pHi recovery from cell acidification were faster in cells derived from the SHR than in those derived from WKY rats (Fig 2⇓). The Vmax of the Na+-H+ antiporter was greater in VSMCs derived from the SHR than those derived from WKY rats, whereas the Km and Hill coefficients were not significantly different between the two strains (Table⇓). These kinetic studies are consistent with the greater Vmax for internal H+ that we have reported previously in VSMCs derived from the SHR.1
To determine if the greater Vmax of the antiporter in VSMCs derived from the SHR was due to an increase in the abundance of NHE-1 protein, the relative abundances of NHE-1 protein in cell lysates from cultured VSMCs from SHR and WKY rats were determined using Western blot analysis. A representative Western blot using (Ab(698-711)) is shown in Fig 3⇓. Densitometric analysis of lysate preparations derived from eight SHR cell lines and seven WKY cell lines and probed with Ab(765-778) indicated that the relative abundance of NHE-1 protein was increased in cultured VSMCs derived from SHR as compared to those from WKY rats (Fig 4A⇓).
It is possible, although not likely, that the increase in density of the 110-kD band was due to a greater affinity of Ab(765-778) for NHE-1 protein in lysates from cells from SHR than in those from cells from WKY rats. This possibility was examined by using a different set of polyclonal antibodies raised against a different region of the NHE-1 isoform of the Na+-H+ antiporter (Ab(698-711)). Densitometric analysis of lysate preparations derived from eight SHR cell lines and six WKY cell lines and probed with Ab(698-711) also revealed that the relative abundance of NHE-1 protein was increased in cultured VSMCs derived from SHR as compared to those derived from WKY rats (Fig 4B⇑). Thus, directionally similar results were obtained using both sets of antibodies. Although both antibodies gave qualitatively similar results, Ab(698-711) gave a greater signal than Ab(765-778) and was therefore used in additional studies to examine the relative abundance of NHE-1 protein in fresh aortic tissue and thymus. Correlations between the Vmax of the Na+-H+ antiporter and NHE-1 protein abundance could not be determined because these two parameters were measured in cells from different rats. Moreover, correlations between the Vmax of the Na+-H+ antiporter and NHE-1 protein abundance would require precise quantification of the amount of NHE-1 protein that is present in each preparation.
To ensure that the cells from both the SHR and WKY rats had reached the same level of quiescence after serum deprivation, FACS analysis was performed to determine the percentage of cells in the G0-G1 phase of the cell cycle. After serum deprivation, the majority of the cells were in G0-G1, and there was no difference between cultured VSMCs from SHR and WKY rats (82±2%, n=10 and 81±3%, n=9, respectively) as determined by FACS analysis.
Fresh Aortic Tissue
To determine if the increased abundance of NHE-1 protein observed in cultured VSMCs from the SHR was also observed in freshly isolated aortic tissue, Western blots were performed on lysates from fresh aortic tissue using Ab(698-711). In a total of 10 assays from each group (duplicate experiments were performed), there was no significant difference in the relative abundance of NHE-1 protein in aortic tissue obtained from five SHR as compared to tissue obtained from five age-matched WKY rats (Fig 5⇓).
Western blots were also performed on freshly obtained thymic lymphocytes using Ab(698-711) (in a total of 12 assays per group). A representative Western blot of thymocytes is shown in Fig 6⇓. The relative abundance of NHE-1 protein was not significantly different between thymic lymphocytes from nine SHR and nine WKY rats (Fig 7⇓).
This study shows that the relative abundance of NHE-1 protein in cultured aortic VSMCs from the SHR is increased as compared to those from WKY rats. Furthermore, this study confirms previous work showing that the Vmax of the Na+-H+ antiporter is increased in cultured aortic VSMCs from the SHR.1 2 3 Our finding that NHE-1 protein is increased in cultured VSMCs from the SHR provides an explanation for the observed increase in the Vmax of the Na+-H+ antiporter. A direct correlation between NHE-1 protein abundance and the Vmax of the Na+-H+ antiporter, however, could not be demonstrated in the present study, because these two parameters were not measured in the same cells.
Our findings in cultured VSMCs from the SHR differ from the work of Siczkowski et al.6 These authors were unable to detect differences in the relative abundance of NHE-1 protein in cultured VSMCs derived from SHR and WKY rats and concluded that differences in functional Na+-H+ antiporter activity was due to an increased turnover number per NHE-1 molecule in the SHR cells.6 In the present study, by contrast, cultured VSMCs derived from the SHR displayed an increase in the relative abundance of NHE-1 protein compared to those derived from WKY rats. This finding was confirmed using two different sets of polyclonal antibodies generated against different epitopes of the NHE-1 protein. A number of possibilities could account for the disparity between the two studies. In our study, the protein content of each extraction was measured, and the gels were loaded with equal amounts of protein. Siczkowski et al,6 on the other hand, normalized their data based upon DNA content, which could have obscured a difference in the relative concentration of NHE-1 protein between SHR and WKY cells, which may display differences in cell size (ie, total protein per cell) and in cell ploidy.11 The contrasting results of these two studies, in terms of NHE-1 protein expression, could also be due to differences in the methods used to extract the NHE-1 protein or may reflect different subpopulations of SHR and WKY rat strains due to outbreeding and/or genetic drift.12 Another important difference between these two studies is that we used cultured cells grown in early passages (most of the data were obtained from cells assayed in the second through fourth passages), whereas Siczkowski et al6 used cells grown in later subpassages (5 through 10). Studies by Berk et al2 and by us1 reported that differences in Na+-H+ exchange activity between cultured VSMCs derived from SHR and WKY rats are no longer present when cells are grown in later subpassages.
VSMCs in culture predominantly express a proliferative phenotype, whereas freshly isolated VSMCs predominantly express a contractile (nonproliferative) phenotype.13 The increase in both Na+-H+ antiporter activity and NHE-1 protein abundance observed in cultured VSMCs from the SHR may be related to the increase in cell proliferation that characterizes their growth phenotype.1 2 14 Indeed, several studies have shown that cultured VSMCs from the SHR exhibit hyperplasia as compared to those derived from WKY rats.1 2 14 The faster growth rate in cultured VSMCs from the SHR may necessitate an increase in the activity of the Na+-H+ antiporter as a way of preventing the intracellular pH from falling during periods of enhanced cellular metabolic activity. In the chronic state, this increase in Na+-H+ antiporter activity may be provided by an increase in the abundance of NHE-1 protein. Fresh aortic tissue from adult SHR rats, by contrast, does not appear to have an increase in NHE-1 protein abundance, possibly because this tissue shows little or no evidence of hyperplasia.15 16
The exact mechanism by which NHE-1 protein is increased in cultured VSMCs from the SHR is, as of yet, unknown. The previous finding that mRNA levels of the NHE-1 isoform are similar between cultured VSMCs from SHR and WKY rats argues against altered NHE-1 gene transcription in the SHR.1 5 The amount of functional NHE-1 protein, however, is not only dependent on the amount of NHE-1 mRNA but also depends on translation rate, stability of the protein products, delivery of NHE-1 to the cell membrane, and the turnover rate of the membrane-bound proteins. From our data, it cannot be determined if the increase in NHE-1 protein in cultured VSMCs from the cells is due to increased NHE-1 translation, a prolonged half-life of the NHE-1 protein in the plasma membrane, or some other alteration.
The finding that NHE-1 protein was similarly expressed in thymic lymphocytes from SHR and WKY rats is in agreement with our previous work showing that the kinetic properties of the Na+-H+ antiporter are indistinguishable from each other in freshly isolated thymocytes from SHR and WKY rats.4 The potential significance of an overactive Na+-H+ antiporter in terms of blood pressure regulation may differ in vascular from nonvascular tissues. Some of the features displayed in cultured VSMCs mimic the ultrastructure and biochemical and immunochemical features that occur in VSMCs in vivo in some forms of hypertension, atherosclerosis, and vascular balloon injury.17 Cultured VSMCs may thus be used as an in vitro model for the vascular cell proliferation that occurs in vivo under certain pathological conditions. In other tissues, differences in the activity of the Na+-H+ antiporter between SHR and WKY cells may depend on the prevailing intracellular pH (ie, H+ substrate availability) independently from the amount of NHE-1 protein present. We have reported that the intracellular pH is reduced in lymphocytes from the SHR, which leads to a secondary activation of the Na+-H+ antiporter.18 19 20 In response to acid loading or metabolic acidosis, the activity of the Na+-H+ antiporter in renal proximal cells21 22 and lymphocytes20 increases. An increase in Na+-H+ antiporter activity, either primary or secondary to reduced pHi, would promote cell Na+ influx and intracellular Na+ accumulation.23 This increase in cell Na+ could contribute to an increase in intracellular Ca2+ via a plasma membrane Na+-Ca2 exchanger and thus increased reactivity to contractile stimuli.
In summary, this study shows that there is an increase in the relative abundance of NHE-1 protein in cultured VSMCs from the SHR as compared to cultured VSMCs from WKY rats. In contrast, NHE-1 protein abundance was not different between lymphocytes or in fresh aortic tissue from SHR and WKY rats. We suggest that in cultured VSMCs from the SHR, an increase in NHE-1 protein abundance is a feature associated with the growth phenotype that is characterized by augmented vascular cell proliferation.
This study was supported by an American Heart Association grant, Chicago Chapter, to M.S.L. and a Veterans Affairs Merit Review grant to D.B. Parts of this work were presented at the annual meeting of the American Heart Association High Blood Pressure Council, Chicago, 1996. The authors would like to thank Drs Orson Moe and Robert Alpern from Southwestern University Medical School (Dallas, Tex) for their generous gift of the polyclonal antibodies against NHE-1 protein used in this study.
- Received November 19, 1996.
- Revision received December 10, 1996.
- Accepted March 26, 1997.
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