Phorbol Ester Activation of the Rat Vascular Myocyte Na⁺-H⁺ Exchanger Isoform 1

A well-recognized membrane transport abnormality in hypertension is elevation of NHE activity.¹² This has been demonstrated in skeletal muscle of SHR compared with normotensive WKY in vivo³ and persists despite culture in vitro of striated⁴ and vascular⁴⁵⁶ myocytes. This increased NHE activity of SHR vascular myocytes is associated with an increased cell proliferation rate⁵⁶ that may be due to hyperresponsiveness of these cells to agonists and growth factors.⁷⁸⁹¹⁰ The increased NHE activity of SHR vascular myocytes in vitro is associated with an increased V_max of the exchanger,⁴ with no change in the pH_0.5. This increase in V_max of the SHR exchanger is not due to an increased expression of NHE isoform 1 (NHE-1) mRNA¹¹ ; the presence of other NHE isoforms such as NHE-2, -3, and -4¹¹ ; or increased cellular content of NHE-1 protein.¹² NHE-1 is thus the sole isoform present in vascular myocytes, and it is likely that a posttranslational processing mechanism acting on this isoform may be responsible for the increased exchanger activity in SHR cells.

One process that could lead to activation of NHE activity is phosphorylation.¹³¹⁴¹⁵ Stimulation of cells with α-thrombin, growth factors, okadaic acid, and phorbol esters causes a dramatic increase in NHE-1 phosphorylation accompanied by enhancement of exchanger activity. Many agonists stimulate phosphoinositide hydrolysis to generate inositol 1,4,5-trisphosphate and diacylglycerol, the latter messenger then stimulating PKC. The C-terminal of rat NHE-1 possesses a putative PKC phosphorylation site.¹⁶ Furthermore, some previous reports have suggested an increased contractile response to phorbol esters (which substitute for diacylglycerol in stimulating PKC) in isolated aortas and renal artery of SHR,¹⁷¹⁸ with increased PKC activity in the soluble fraction of renal artery.¹⁸ Antagonists of PKC could relax aortic tone in vitro and lower blood pressure of SHR in vivo.¹⁹ These findings have prompted us to investigate further the mechanism of NHE-1 activation by phorbol esters in vascular myocytes of SHR compared with WKY and whether differences in NHE-1 activation between these rat strains could be attributed to alterations in the degree of NHE-1 phosphorylation. Our studies demonstrate that the response to phorbol ester stimulation of NHE-1 in vascular myocytes of both strains involved a substantial increase in the V_max of the exchanger. This was not associated with any significant alteration in the subcellular distribution of NHE-1 in either cell type. However, stimulation by phorbol ester was accompanied by an increased affinity for intracellular H⁺ in WKY cells (increased pH_0.5), whereas SHR cells demonstrated a reduced cooperativity for intracellular H⁺ (lowered Hill coefficient), perhaps indicating an anomalous interaction with the intracellular H⁺ modifier site. These very different kinetic responses to phorbol esters between the rat strains were accompanied by a reduction in NHE-1 phosphorylation in WKY cells (and to a lesser extent in SHR cells), thus dissociating phorbol ester stimulation of NHE-1 from an increase in its phosphorylation.

Methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F-12 medium, chick embryo extract, and [³²P]orthophosphate were from ICN Flow. Fetal calf serum was from Advanced Protein Products. Ham’s F-12 growth medium was buffered with 14 mmol/L NaHCO₃ (pH 7.1, with 5% CO₂ in air) and contained 15% fetal calf serum, 0.5% (wt/vol) chick embryo extract, 2 mmol/L glutamine, 10⁵ IU penicillin per liter, and 100 mg streptomycin per liter. Protein A–Sepharose-CL4B beads were obtained from Pharmacia LKB Biotechnology. ¹⁴C molecular weight markers, the enhanced chemiluminescence kit, and Aurodye were purchased from Amersham International. 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was from Cambridge Bioscience. All other chemicals, including TPA, H-7, and Percoll, were from Sigma Chemical Co. The specific PKC inhibitor Ro 318220,²⁰ which inhibits both the calcium-sensitive and -insensitive atypical isoforms of PKC, was obtained from Dr J. Lawton, Roche Products Ltd.

Culture of Vascular Myocytes and Measurement of NHE Activity

WKY and SHR were maintained at a colony established at the Biomedical Services Unit, Leicester University. Blood pressures of 12-week-old rats by the tail-cuff method confirmed values in SHR exceeding 170 mm Hg, with values less than 120 mm Hg in WKY. Vascular myocytes were obtained from thoracic aortas by enzymatic dissociation as previously described⁴ and maintained in Ham’s F-12 growth medium. Studies were performed on cultures between passages 3 and 8 because previous work suggested that NHE abnormalities were found predominantly in early-passage SHR cells.⁵⁶ NHE activity was measured in SHR and WKY confluent vascular myocytes seeded onto glass coverslips as described previously.⁴¹² Cells were rendered quiescent by serum deprivation for 24 hours in DMEM containing 1 g/L bovine serum albumin, 2 mmol/L glutamine, and antibiotics. After loading with 5 μmol/L BCECF-AM for 0.5 hour in serum-free DMEM at 37°C and deesterification for a further 0.5 hour, pH_i was measured after incubation of coverslips in HBSS composed of (mmol/L) NaCl 130, KCl 5, CaCl₂ 1.8, MgSO₄ 1, glucose 5, and HEPES 20 as well as 1 g/L bovine serum albumin, pH 7.4. All measurements were performed at 37°C with a thermostatted sample compartment holder within a dual grating fluorometer (Deltascan, Photon Technology International Inc). NHE activity was determined at a range of different pH_i from 6.0 to 7.2 after clamping of pH_i with nigericin and monensin (5 μmol/L of each) in isotonic KCl buffers (replacing the NaCl of HBSS with KCl and omitting the bovine serum albumin) by measuring the difference between the total rate of change of pH_i and that measured in Na⁺-free medium (replacing the NaCl in HBSS with N-methyl-d-glucamine chloride, pH 7.4), as described.⁴ Values of Na⁺-dependent rate of pH_i change were converted to efflux rates by multiplying them by the buffering capacities measured at these various clamped pH_i values. Measurements were then repeated on cells that had been preincubated with 100 nmol/L TPA in HBSS at 37°C. Studies with Ro 318220 were performed by incubation of cells with 10 μmol/L of the inhibitor for 20 minutes at 37°C and then measurement of pH_i and NHE activity or by addition of 100 nmol/L TPA after preincubation with the inhibitor. These studies were repeated with another PKC inhibitor, H-7 (50 μmol/L). An additional technique of downregulating PKC by prolonged (24 hours) incubation of cells in 100 nmol/L TPA was also used, and fluxes were then investigated in cells that were subsequently stimulated with 100 nmol/L TPA.

NHE fluxes were fitted to the logistic (Hill) equation to obtain values for V_max, pH_0.5, and the apparent Hill coefficient for the internal H⁺ binding site of the antiport. The computer program used to derive these parameters was a recursive nonlinear least-squares algorithm (P-fit, Biosoft Corp). These parameters were also checked with another nonlinear optimization algorithm that we have used previously²¹ and that uses the pattern search method to minimize the sum of errors squared between calculated and observed NHE flux values. Both algorithms produced very similar results for the different parameters V_max, pH_0.5, and the Hill coefficient.

Determination of NHE-1 Abundance by Western Blotting

We have previously described the use of NHE-1–specific polyclonal antibodies to the last 157 amino acids of the regulatory C-terminal of NHE-1 for detection of NHE-1 protein in vascular myocytes.¹²²² G252 and G253 are protein A Sepharose–purified immunoglobulin fractions from these antisera.

We measured NHE-1 abundance in cultures from SHR and WKY myocytes by Western blots of these cell extracts using a previously described method employing G252 antibody.¹²²² This ensured that the subsequent phosphorylation studies used similar numbers of NHE-1 transporters in the two different strains. Serum-deprived (24 hours) quiescent cultures were studied before and after 20 minutes of stimulation with 100 nmol/L TPA at 37°C. Briefly, after cells were snap-frozen with liquid nitrogen, they were scraped off the tissue culture flasks into a buffer containing (mmol/L) Tris 50 (pH 7.4), NaCl 140, EDTA 5, phenylmethylsulfonyl fluoride 1, o-phenanthroline 1, and iodoacetamide 1. An equal volume of buffer containing 125 mmol/L Tris (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 0.004% bromophenol blue was added, and the extracts were boiled for 3 minutes. Proteins were resolved on 7.5% SDS-PAGE gels followed by electrotransfer onto supported nitrocellulose. Membranes were blocked overnight with 10% low-fat milk powder in TBS-Tween (composed of 20 mmol/L Tris [pH 7.4], 137 mmol/L NaCl, and 1 g/L Tween 20). The first antibody incubation was with 1 μg/mL of G252 antibody in 5% low-fat milk powder in TBS-Tween for 2.5 hours. After extensive washes in TBS-Tween, horseradish peroxidase–linked donkey anti-rabbit second antibody (1:1500 dilution) was incubated with the membranes for 1 hour. Detection was achieved with the enhanced chemiluminescence kit from Amersham. The bands corresponding to NHE-1 (molecular weight around 95 kD) were measured on a Bio-Rad densitometer.¹²²²

Subcellular Fractionation of Vascular Myocyte Homogenates

For investigation of whether there was any intracellular compartmentalization of NHE-1 or an alteration of this subcellular distribution after 20 minutes of stimulation with 100 nmol/L TPA at 37°C, monolayers of SHR or WKY myocytes were detached from flasks with EDTA after two brief washes in HBSS.²¹²² Cells were then disrupted by nitrogen cavitation in 3 mL of homogenization buffer composed of 250 mmol/L sucrose, 1 mmol/L EDTA, and 1 mmol/L phenylmethylsulfonyl fluoride, pH 7.2, as described.²¹²² After a 10-minute centrifugation (600g), the postnuclear supernatant was layered onto a self-generated 30% Percoll gradient preformed on an ultracentrifuge at 27 000g for 60 minutes at 4°C. Membranes and cellular organelles were recovered at the following densities (in grams per milliliter, as indicated by beads): 1.042, 1.05, 1.056, 1.069 to 1.083, and 1.102 to 1.128. Proteins were determined by the fluorescamine method,²¹²² and 7.5% SDS-PAGE gels were then loaded with equal amounts of protein per track. Western blots were performed as described above. Enzyme markers were used for determination of the location of membrane and organelle fractions as described.²¹²² The plasma membrane marker 5′-nucleotidase (EC 3.1.3.5) was enriched in the fraction with a density of 1.042 g/mL in all extracts.

Determination of NHE-1 Phosphorylation

In the present studies, G253 was used for immunoprecipitation of labeled NHE-1, which is visualized as a 95-kD phosphoprotein in rat vascular myocyte extracts, as illustrated previously.²³ Nonspecific immunoglobulin did not immunoprecipitate any labeled protein in this region (data not shown). The method for immunoprecipitation of NHE-1²³ was adapted from that of Sardet et al.¹³ Quiescent vascular myocyte cultures were loaded with [³²P]orthophosphate (50 μCi/mL) for 3 hours in phosphate-free HBSS composed of (mmol/L) NaCl 130, KCl 5, CaCl₂ 1.8, MgSO₄ 1, glucose 5, HEPES 20, and glutamine 2 as well as 1 g/L bovine serum albumin, pH 7.4. After brief washes with cold HBSS, cells were snap-frozen with liquid nitrogen. One milliliter of cold (4°C) extraction buffer containing 10 g/L polyoxyethylene-8-lauryl ether and (mmol/L) Tris 30, NaCl 130, EDTA 5, phenylmethylsulfonyl fluoride 1, o-phenanthroline 1, iodoacetamide 1, sodium fluoride 100, sodium orthovanadate 5, ATP 10, and sodium pyrophosphate 10 as well as 1 mg/L pepstatin A and 2 mg/L leupeptin was then added to the frozen cells, and the monolayers were scraped off the plastic flasks. Extracts were sonicated and then clarified by centrifugation at 14 000g. The supernatant was preabsorbed with protein A–Sepharose-CL4B beads followed by centrifugation. The antibody G253 was then added to the supernatant (final concentration, 100 μg/mL), and the samples were rotated end on end for 2 hours at 4°C. Immunoprecipitates were recovered after a 1-hour incubation with protein A–Sepharose-CL4B beads (previously treated with unlabeled vascular myocyte cell extracts to reduce nonspecific binding). After the beads had been washed extensively, Laemmli sample buffer was added and extracts were boiled. Phosphoproteins were resolved on 7.5% SDS-PAGE gels and quantified by autoradiography on preflashed x-ray films.²³ All values have been normalized to an arbitrary value of 1 for the quiescent WKY cell extract. With these techniques, our preliminary studies had indicated that SHR myocytes showed a larger increment in pH_i and NHE-1 phosphorylation than WKY myocytes when stimulated with 100 nmol/L arginine vasopressin for 20 minutes (results not shown).

We investigated the effect of TPA on NHE-1 phosphorylation by incubating quiescent ³²P-labeled myocyte cultures in HBSS containing 100 nmol/L TPA for 20 minutes at 37°C and then snap-freezing the monolayer with liquid nitrogen. Incubations with 10 μmol/L Ro 318220 were performed for 40 minutes at 37°C followed by snap-freezing. Alternatively, 100 nmol/L TPA was added to cells preincubated with Ro 318220 for 20 minutes, and after a further 20 minutes, the reaction was terminated by freezing with liquid nitrogen. We determined the effect of H-7 by incubating cells for 20 minutes at 37°C with the inhibitor (50 μmol/L) and then snap-freezing the cells. Downregulation of PKC was also achieved by incubation of cells for 24 hours with 100 nmol/L TPA (the last 3 hours of the incubation was performed in phosphate-free HBSS containing [³²P]orthophosphate).

Statistics

Results are expressed as mean±SE, and comparisons were made by ANOVA and Student’s t test, performed on an Oxstat statistics package (Microsoft Corp). Two-tailed probability values less than .05 were considered significant.

Results

Fig 1 shows the effect of 100 nmol/L TPA on the pH_i of serum-deprived SHR and WKY vascular myocytes. TPA led to significant alkalinization in both cell types, a peak effect being achieved between 10 and 20 minutes. Although the pH_i at rest was higher in SHR than WKY myocytes (P<.05), the difference became more marked after stimulation with TPA (P<.005 at 20 minutes after TPA). In the presence of 100 nmol/L ethylisopropyl amiloride, no increase in pH_i was demonstrable, indicating that NHE-1 was stimulated by TPA (data not shown). All subsequent experiments were therefore performed on cells after stimulation with TPA for 20 minutes.

Intracellular pH and NHE activity at pH_i 6.0 were measured in both cell types before and after stimulation with TPA as well as in the absence and presence of the PKC inhibitors Ro 318220 and H-7. ANOVA revealed very significant differences between the quiescent and TPA-stimulated cells in the absence and presence of Ro 318220 and H-7 for both intracellular pH and NHE activity measurements (P<.005 for both parameters in WKY and SHR cells). The NHE activity of SHR cells was significantly higher than that in WKY cells before stimulation (P<.001, Fig 2). On stimulation with TPA, NHE activity was increased in both cell types (P<.001). The NHE activity of SHR cells after TPA stimulation remained significantly elevated compared with that in WKY cells (P<.001, Fig 2). The PKC inhibitor Ro 318220 was incubated with these cells for assessment of the dependence of unstimulated NHE activity and pH_i on PKC. Ro 318220 had no effect on either pH_i or NHE activity of both WKY and SHR cells, suggesting that neither parameter depended on PKC activity. However, this inhibitor abolished the TPA-induced alkalinization and increase in NHE activity of both cell types (Fig 2), thus indicating that PKC may be involved in the TPA stimulation of NHE activity in both cell types. We confirmed this effect of Ro 318220 by incubating cells with another PKC inhibitor (H-7) and also by downregulating PKC with prolonged 24-hour exposure to 100 nmol/L TPA. Both maneuvers did not alter pH_i or NHE activity of the WKY and SHR cells but did abolish the subsequent stimulation by 20-minute incubations with TPA (Fig 2).

We examined the kinetics of NHE activity in greater detail by clamping pH_i to different levels between 6 and 7.2 and investigating the Na⁺-dependent H⁺ efflux in both cell types before and after TPA stimulation. It was not possible to clamp pH_i below 6.0 because the dye BCECF is not very sensitive to changes in pH below this value. In unstimulated cells, the V_max of SHR cells was significantly increased compared with that in WKY cells (P<.001), with similar values for the Hill coefficient, although SHR cells also exhibited an increased affinity for intracellular H⁺ (an increased pH_0.5, P<.005, Fig 3 and Table). The increased V_max in quiescent SHR cells confirms our previous report,⁴ although in those serum-stimulated cells⁴ we could not demonstrate any differences in the Hill coefficient or pH_0.5. Incubation with TPA led to very significant increases in the V_max of NHE-1 in both WKY and SHR cells (Fig 3 and Table, P<.001 for both strains). However, in addition, TPA-stimulated WKY cells demonstrated an increased affinity for intracellular H⁺ (a rise in pH_0.5, P<.001), with no change in the Hill coefficient. In contrast, when stimulated with TPA, SHR cells showed no change in the affinity for intracellular H⁺ but rather a reduced Hill coefficient (P<.01), indicating a reduced cooperativity of H⁺ at the internal modifier site of NHE-1. Thus, after stimulation with TPA, the V_max in SHR cells was higher (P<.001), with a lowered Hill coefficient (P<.01) and lower pH_0.5 (P<.001) than these parameters in WKY cells.

The similar abundance of NHE-1 in SHR and WKY vascular myocytes has previously been described.¹² In the current investigation, Western blots with the NHE-1–specific antibody G252 revealed that the ratio of NHE-1 protein in SHR and WKY vascular myocyte extracts was 0.99±0.11 (n=3). Incubation with 100 nmol/L TPA for 20 minutes did not significantly alter the amount of NHE-1 extracted from the WKY cell cultures using the buffer containing polyoxyethylene-8-lauryl ether (Fig 4). The ratio of NHE-1 densities after TPA and in quiescent WKY cells was 1.01±0.11 (n=3). In contrast, the ratio of NHE-1 densities in extracts after 20 minutes of TPA stimulation and in quiescent SHR cells was 0.80±0.11 (n=3), indicating a slight reduction in extractable NHE-1 from SHR myocytes (Fig 4). Thus, the stimulation of NHE-1 activity measured fluorometrically was not accompanied by any increase in extractable NHE-1 in either cell type.

Because of the increased V_max of both cell types stimulated by TPA, we examined whether this phenomenon could be attributed to an altered subcellular localization of NHE-1. Using isopycnic centrifugation through a Percoll gradient,²² we found that the NHE-1 of quiescent SHR and WKY cells was predominantly located in the plasma membrane fraction in three separate experiments (as indicated by the location of the marker enzyme 5′-nucleotidase at a density of 1.042 g/mL, which was enriched by 3.2- to 3.8-fold compared with the postnuclear supernatant). In this fraction, immunoreactive NHE-1 was enriched approximately 2.2- to 2.4-fold in extracts of both cell types compared with the postnuclear supernatant. After 20 minutes of TPA stimulation, the subcellular distribution of NHE-1 did not change, the majority of the exchanger being recovered in the plasma membrane fraction (in which NHE-1 was enriched 2.1- to 2.2-fold compared with the postnuclear supernatant). Thus, the increase in V_max with TPA stimulation of both cell types was not associated with any redistribution of NHE-1 from an intracellular compartment to the cell surface.

Since phosphorylation of NHE-1 may be one mechanism leading to its activation,¹³¹⁴¹⁵ we examined this posttranslational event in SHR and WKY vascular myocytes stimulated with TPA. In unstimulated cells, we had previously shown that NHE-1 phosphorylation in SHR cells was approximately double that of WKY cells²³ although the NHE-1 protein content was very similar between strains.¹² All NHE-1 phosphorylation results reported here have been corrected to an arbitrary value of 1 in the quiescent WKY cultures. When WKY vascular myocytes were stimulated with TPA, NHE-1 phosphorylation was significantly reduced, as detected by densitometric traces of the region around 95 kD of the autoradiographs (Table). ANOVA revealed significant differences between the quiescent and TPA-stimulated cells and the effect of Ro 318220 incubation with and without TPA (P<.05 by ANOVA in WKY cells). In six experiments, the NHE-1 phosphorylation in TPA-stimulated cells fell significantly compared with that in quiescent WKY cells (Table, P<.001). Incubation of WKY cells with the PKC inhibitors Ro 318220 and H-7 or downregulation of PKC with prolonged TPA treatment (24 hours) did not significantly alter NHE-1 phosphorylation (Table). In the presence of Ro 318220, TPA did not significantly lower NHE-1 phosphorylation (Table).

In contrast, the NHE-1 phosphorylation of SHR cells exhibited a different response to TPA stimulation. Significant differences between SHR cells before and after TPA stimulation in the presence and absence of Ro 318220 or H-7 were revealed by ANOVA (P<.005). The NHE-1 phosphorylation in quiescent SHR cells was elevated compared with that in quiescent WKY cells (Table, P<.001). NHE-1 phosphorylation was reduced by TPA stimulation in SHR cells (P<.03), but this reduction was to a level of about 70% of the NHE-1 phosphorylation values in quiescent SHR cells. This reduction was significantly different from the response in WKY cells (P<.02), in which TPA reduced NHE-1 phosphorylation to about 33% of values in untreated WKY cells. However, the PKC inhibitors Ro 318220 and H-7 led to a substantial increase in NHE-1 phosphorylation in SHR cells (Table, P<.002 compared with quiescent SHR cultures), even though there were no associated changes in NHE activity and pH_i. Coincubation of SHR cells with TPA and Ro 318220 led to intermediate levels of NHE-1 phosphorylation. Prolonged incubation with TPA to downregulate PKC activity had no effect on NHE-1 phosphorylation, but this procedure may not have achieved complete downregulation of all the PKC isoforms.

This response to Ro 318220 in SHR cells appeared to be unique to this hypertensive model because lymphoblasts derived from patients with hypertension²⁴ in whom we had shown an increased NHE-1 phosphorylation compared with normotensive subjects do not demonstrate an enhancement of NHE-1 phosphorylation on incubation with Ro 318220 (10 μmol/L of the inhibitor for 40 minutes at 37°C, data not shown).

Discussion

SHR vascular myocytes cultured in vitro exhibit a number of differences compared with WKY cells. These include an increased proliferation rate⁵⁶⁷ and altered responsiveness to growth factors and vascular agonists.⁵⁸⁹¹⁰ The generation of diacylglycerol as a second messenger during stimulation leads to activation of PKC, and increased responsiveness of SHR aortas to phorbol esters has been demonstrated.¹⁷¹⁸¹⁹ Previous findings of increased NHE activity have been associated with the increased proliferation rate of SHR vascular myocytes.⁵⁶

In the present study, we have confirmed the findings of others⁵⁶ and ourselves⁴ that NHE activity is elevated in unstimulated SHR vascular myocytes compared with WKY cells and that this was mainly attributed to an increased V_max of NHE-1. However, in our previous study on SHR vascular myocytes that had not been serum deprived,⁴ we did not observe any differences in the Hill coefficient or pH_0.5 of NHE-1. In contrast, the present study on serum-withdrawn quiescent cells demonstrated a higher value for the pH_0.5 of NHE-1 in SHR cells (indicating a higher H⁺ affinity) than WKY cells. Nevertheless, the elevated V_max of SHR cells compared with that of WKY cells is present in both quiescent and serum-fed cells.

TPA incubation led to a more substantial alkalinization in SHR cells, which confirmed findings in a previous study.¹⁰ TPA also led to an increase in V_max of both cell types that was not due to an increased cellular NHE-1 protein content. This increased V_max was also not associated with any redistribution of NHE-1 from intracellular compartments to the cell surface. The effect of TPA on NHE activity has previously been documented to be solely an effect on altered pH_0.5 of NHE²⁵ rather than on V_max, but this may be due to cell-specific differences in response. Furthermore, in some cell types, TPA has been reported either to have no effect on NHE-1 activity or to suppress the V_max in addition to lowering the Hill coefficient.²⁶ Thus, the response of NHE-1 to TPA may depend on the intracellular milieu in which the exchanger is expressed, different kinetic responses being reported in different cell types. In the WKY cells, we have demonstrated that the pH_0.5 was increased by TPA, indicating an increased affinity for intracellular H⁺ resembling the previously reported effect of TPA.²⁵ This contrasts with the effect of TPA on SHR cells, in which no change in affinity for intracellular H⁺ was demonstrated and stimulation was associated with a fall in the cooperativity of H⁺ acting at the internal proton modifier site of NHE-1.²⁷ This accounts for the less sigmoidal activation curve of NHE-1 in TPA-stimulated SHR cells, so that activity falls more gradually as pH increases. Thus, in the stimulated SHR cells, pH_i could rise further before NHE activity is switched off, explaining the greater alkalinization response of these cells. It is uncertain whether this accounts for a proportion of the increased cell proliferation rate and the hyperresponsiveness to different growth factors and agonists reported in SHR vascular myocytes.^5678910 Parallels can be drawn between our study and that of Rosskopf et al,²⁸ who have studied the kinetics of NHE-1 in TPA-stimulated lymphoblasts from hypertensive humans. They demonstrated that these TPA-stimulated hypertensive cells showed a lower Hill coefficient for intracellular H⁺ and an increased V_max, findings very similar to those we have presented for TPA-stimulated SHR vascular myocytes. In addition, pH_0.5 for intracellular H⁺ was lower in the hypertensive compared with normotensive lymphoblasts, identical to the comparison of TPA-stimulated SHR and WKY cells. Thus, these similarities between studies on humans and the rat model suggest that TPA stimulation of NHE-1 is fundamentally different in cells from hypertensive compared with normotensive individuals.

We have attempted to examine the effect of TPA stimulation of these cells on one mechanism previously described to be associated with NHE-1 activation, ie, NHE-1 phosphorylation.¹³¹⁴¹⁵ We had previously suggested that the increased NHE activity in SHR vascular myocytes may be associated with the increased phosphorylation of NHE-1.²³ However, stimulation of SHR cells with TPA was not associated with a further increase in phosphorylation but a small decrease in NHE-1 phosphorylation of about 30%. In contrast, stimulation of WKY cells actually led to a greater dephosphorylation of NHE-1 of more than 67%. These findings, taken together with the kinetic changes of NHE activity after TPA stimulation, illustrate the very different responses that SHR and WKY cells exhibit, although it is uncertain to what extent dephosphorylation of NHE-1 contributes to the changes in the Hill coefficient or pH_0.5 induced by TPA in these cells. In addition, our results contrast with those reported earlier¹³ in which phorbol esters were reported to enhance NHE-1 phosphorylation. It is possible that the cell lines used for these experiments possess a different balance in the signaling mechanisms leading to NHE-1 activation compared with our primary cultures of vascular myocytes, since these continuous lines also overexpress growth factor (tyrosine kinase–linked) receptors.¹³ Moreover, other researchers have demonstrated that the NHE-1 activity of HL60 cells was stimulated with phorbol esters and that this led to dephosphorylation of NHE-1.²⁹ Furthermore, several reports have suggested that activation of NHE-1 may not be associated with its phosphorylation. For example, the increase in NHE activity with an osmotic stimulus is not associated with an increased phosphorylation.³⁰ Deletion mutants of NHE-1 revealed that stimulation of the exchanger without an increased phosphorylation was still possible when sections of the regulatory C-terminal domain were removed, although the response to growth factors was halved,³¹ suggesting that other regulatory proteins may interact with and activate NHE-1.³¹³² Thus, one possibility for the differences in the response of SHR and WKY cells to TPA may be an altered interaction between such regulatory proteins and NHE-1 that is phosphorylated to varying degrees in these cells.

Inhibition of PKC in the present studies led to no significant changes in NHE activity or pH_i in either the WKY or SHR cells, although the effect of TPA on stimulating NHE-1 activity was abolished. Furthermore, downregulation of PKC with prolonged TPA incubation led to no significant changes in NHE activity. This meant that the NHE activity of quiescent vascular myocytes was PKC independent, as was the difference in NHE activity between quiescent SHR and WKY cells. The relationship of NHE-1 phosphorylation to PKC activity was also documented to be inverse rather than direct (as demonstrated by TPA-induced dephosphorylation of NHE-1, especially in WKY cells, and the elevation of NHE-1 phosphorylation by PKC inhibitors, especially in SHR cells). This concurred with the data reported for HL60 cells²⁹ as opposed to ER22 fibroblasts¹³ and presents further evidence that non–phosphorylation-dependent mechanisms exist for the PKC-mediated activation of NHE-1.³⁰³¹³²

In summary, we have demonstrated that the kinetics of NHE-1 in SHR and WKY cells differ in the quiescent state and this is manifested as an increased V_max and increased pH_0.5 in SHR cells. TPA led to increases in V_max in both cell types but to an increased internal H⁺ affinity in WKY cells and a reduced cooperativity at the internal H⁺ modifier site of NHE-1 in SHR cells. These changes in V_max were not associated with any alteration in the subcellular localization of NHE-1 in both cell types. The kinetics differences between SHR and WKY cells resemble those described for TPA-stimulated cells from hypertensive and normotensive humans. The combined effect of increased V_max and reduced cooperativity for H⁺ at the internal modifier site of NHE-1 in SHR cells may account for the greater alkalinization response of these cells. NHE-1 phosphorylation was increased in SHR compared with WKY cells in the quiescent state, and studies with TPA and PKC inhibitors indicate an inverse relationship between PKC activity and NHE-1 phosphorylation and an upregulated NHE-1 phosphorylation mechanism in SHR cells. Non–phosphorylation-dependent mechanisms may be involved in the TPA-induced stimulation of NHE-1, a possibility being the interaction of putative regulatory proteins with the C-terminal control region of NHE-1 (although the degree of this interaction may be altered by NHE-1 phosphorylation). These possibilities remain to be explored in this rat model of hypertension.

Selected Abbreviations and Acronyms

• Download figure
• Download PowerPoint

• Download figure
• Download PowerPoint

• Download figure
• Download PowerPoint

• Download figure
• Download PowerPoint

Footnotes