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(Circulation Research. 1995;76:530-535.)
© 1995 American Heart Association, Inc.

Articles

Long-term High Osmolality Activates Na⁺-H⁺ Exchange and Protein Kinase C in Aortic Smooth Muscle Cells

Manoocher Soleimani, Gurinder Singh, Jesus H. Dominguez, Randy L. Howard

From the Department of Medicine, Indiana University School of Medicine, and Veterans Affairs Medical Center, Indianapolis, Ind.

Correspondence to Manoocher Soleimani, MD, Nephrology Section, Department of Medicine, Fesler Hall 108, 1120 South Dr, Indiana University School of Medicine, Indianapolis, IN 46202-5116.

	Abstract

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Abstract The effect of long-term exposure to hypertonic medium on Na⁺-H⁺ exchange activity was studied in cultured vascular smooth muscle (VSM) cells by using a combination of ²²Na⁺ influx and pH measurement with the pH-sensitive dye BCECF. Incubation of VSM cells in high-osmolality medium (510 mOsm/L) for 48 hours significantly increased the acid-stimulated ²²Na⁺ influx (control, 3.16±0.41 nmol/mg protein per minute; high osmolality, 6.40±0.66 nmol/mg protein per minute; P<.01) and Na⁺-dependent pH_i recovery (control, 0.29±0.06 pH/min; high osmolality, 0.65±0.13 pH/min; P<.03). Activation of Na⁺-H⁺ exchange was osmolality dependent and reached maximal stimulation at 700 mOsm/L. Na⁺-H⁺ exchanger stimulation was independent of serum in the culture media. Na⁺-H⁺ exchanger isoform (NHE-1) mRNA in VSM cells cultured in high-osmolality medium was unchanged from that in VSM cells cultured in control medium, indicating an absence of transcriptional regulation by high osmolality. Long-term high osmolality significantly increased protein kinase C (PKC) activity in cultured VSM cells, as assessed by phosphorylation of a PKC-specific substrate (control, 20.9±2.1 pmol phosphorylation/mg protein per minute; high osmolality, 33.6±2.9 pmol phosphorylation/mg protein per minute; P<.01). Downregulation of PKC by preincubation of VSM cells with 0.1 µmol/L phorbol 12-myristate 13-acetate (PMA) prevented osmolality-induced stimulation of the Na⁺-H⁺ exchanger (control plus PMA, 0.27±0.05 pH/min; high osmolality plus PMA, 0.33±0.08 pH/min; P>.05). These results indicate that long-term exposure to hypertonic medium stimulates Na⁺-H⁺ exchange activity in cultured VSM cells and that this effect is independent of antiporter gene expression regulation. The results further demonstrate that the stimulatory effect of osmolality on Na⁺-H⁺ exchanger is mediated via posttranslational modification of the Na⁺-H⁺ exchanger by chronic PKC activation. The Na⁺-H⁺ exchanger may be involved in VSM cell volume regulation in long-term high osmolality.

Key Words: Na⁺-H⁺ exchanger • high osmolality • vascular smooth muscle cells

	Introduction

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Exposure to high extracellular osmolality is associated with acute increases of Na⁺-H⁺ exchanger activity in nonepithelial cells.¹ This adaptive response increases Na⁺ influx and intracellular osmolality, therefore preventing cell dehydration.¹ The stimulation of Na⁺-H⁺ exchange is an early response and occurs in a matter of several minutes. The molecular mechanism of osmotic-induced Na⁺-H⁺ exchange activation remains unknown. Several studies have suggested possible roles for protein kinase A,² protein kinase C (PKC),¹ or calmodulin-calcium–dependent protein kinase³ phosphorylation in mediating Na⁺-H⁺ exchange stimulation in high osmolality, although evidence for a phosphorylation-independent mechanism has also been reported.⁴ Although the effect of osmotic shrinkage on Na⁺-H⁺ exchange activation has been studied after acute exposure of the cells to hypertonic medium, the role of Na⁺-H⁺ exchanger activity during longer term osmolar stress is unknown. Furthermore, the cellular mechanism(s) mediating such possible adaptive regulation remains speculative.

Vascular smooth muscle (VSM) cells expressing the Na⁺-H⁺ exchanger isoform (NHE-1) stimulate Na⁺-H⁺ exchanger activity in response to acute elevation of osmolality.⁵ This effect is independent of PKC and calcium-calmodulin–dependent protein kinase.⁵ We now have studied the effect of long-term high osmolality on Na⁺-H⁺ exchanger and PKC activity in cultured VSM cells. The results illustrate that long-term hyperosmolality increases Na⁺-H⁺ exchanger activity via chronic PKC activation and posttranslational modification. The stimulatory effect of high osmolality on Na⁺-H⁺ exchange activity in VSM cells might be of clinical importance. Specifically, modulation of Na⁺-H⁺ exchanger activity may contribute to cell volume regulation of VSM in patients with elevated blood sugar, advanced renal failure, or volume depletion, which are conditions manifested by increased plasma osmolality.

	Materials and Methods

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Cell Culture Procedures
Cultured VSM cells from rat aorta were grown in DMEM (400 mg/dL glucose) supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin as previously described.⁶ The cells were maintained and grown at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cell viability was checked by the exclusion of trypan blue (0.3%) and always exceeded 95%. The cultures reached confluence after 7 to 10 days and were subcultured by using trypsin-EDTA (0.25% to 1.0%) treatment. Cells were positively identified as smooth muscle by indirect immunofluorescent staining for myosin by using rabbit anti-myosin (smooth and skeletal) antibody and anti-rabbit IgG fluorescein isothiocyanate conjugate. Cells from passages 3 to 8 were used for experiments.

Measurement of the Na⁺-H⁺ Exchanger Activity
²²Na⁺ Influx
Uptake of radiolabeled Na⁺ by cultured VSM cells grown in 24-well plastic plates was measured as previously described.⁵ VSM cells were washed three times with a Na⁺-free buffer consisting of (mmol/L) chloride salt of N-methyl-D-glucamine (NMDG) 140, KCl 4, MgCl₂ 2, CaCl₂ 1, and HEPES 7, pH 7.4 (solution A). The cells were then incubated in the presence of an ammonium-containing solution consisting of (mmol/L) chloride salt of NMDG 110, NH₄Cl 30, KCl 4, MgCl₂ 2, CaCl₂ 1, and HEPES 7, pH 7.4, (solution B) for 10 minutes. Thereafter, the ammonium-containing solution was replaced with uptake solution (solution A) containing 2 mmol/L ²²NaCl. The ²²Na uptake reaction was stopped after 4 minutes by using four rapid washes with ice-cold saline. Cell-associated radioactivity was extracted with 1 mL of 1N sodium hydroxide and counted by scintillation spectroscopy. Protein concentration was determined by the bicinchoninic acid method according to the manufacturer's protocol (Pierce Co).

pH_i Measurement
pH_i in VSM cells grown on coverslips was measured by the use of the pH-sensitive dye BCECF as previously described⁷ and used.⁸ VSM cells were incubated in the presence of 5 µmol/L BCECF for 15 minutes in a solution consisting of (mmol/L) NaCl 140, KCl 4, MgCl₂ 2, CaCl₂ 1, and HEPES 7, pH 7.4 (solution C). The fluorescence of BCECF was monitored in a thermoregulated (37°C) double–excitation beam spectrofluorometer (PTI double-beam fluorometer, Delta Scan I, Photon Technologies Inc). A calibration curve was generated by use of the KCl/nigericin technique and solutions of varying pH. The fluorescence ratio at excitation wavelengths of 500 and 450 nm was used to determine pH_i values in the experimental groups by comparison with the calibration curve. Acid loading of VSM cells grown on coverslips was achieved by use of an NH₄ pulse with an NH₄-containing solution (solution B). Cell acidification was induced by replacing NH₄Cl solution with a Na⁺-free solution (solution A). After intracellular acidosis, the initial rate of pH_i recovery was monitored in the presence of a Na⁺-containing solution (solution C).

VSM cells were grown to near confluence on plastic dishes or coverslips, deprived of serum for 12 hours, and then incubated in normal (310 mOsm/L) or hypertonic (410 mOsm/L) medium for 24 hours. In the hypertonic groups, osmolality of the medium was increased to 510 mOsm/L (up to 710 mOsm/L in some experiments) for an additional 24 hours. Increments in medium osmolality were achieved by the addition of NaCl.

Isolation of Total and Poly(A⁺) RNA
Total cellular RNA was extracted from confluent cultured VSM cells in multiple 100-mm dishes by the method of Chomczynski and Sacchi.⁹ In brief, cells were scraped and homogenized in 10 to 12 vol of 4 mol/L guanidinium thiocyanate, 25 mmol/L sodium citrate (pH 7), 0.5% sarcosyl, and 0.1 mol/L 2-mercaptoethanol. RNA was extracted by phenol/chloroform/isoamyl alcohol and precipitated by isopropanol.⁹ Poly(A⁺) RNA was prepared by using oligo dT-cellulose spin columns (5 Prime 3 Prime, Inc). Total and poly(A⁺) RNA were quantified by spectrophotometry.

Northern Hybridization
Poly(A⁺) RNA samples (10 to 15 µg per lane) were fractionated on a 1% agarose-formaldehyde gel and transferred to nylon membranes by capillary diffusion.¹⁰ The membranes were prehybridized for 15 hours at 42°C with 5x Denhardt's solution, 5x standard saline citrate (SSC), 50% formamide, 0.5% sodium dodecyl sulfate (SDS), and 0.5 mg/mL sheared salmon sperm DNA. The membranes were hybridized overnight in the above solution with 20 to 30x10⁶ cpm [³²P]DNA probe for NHE-1 or ß-actin. The cDNAs were labeled with [³²P]deoxynucleotides by use of a random-primed DNA labeling kit (5 Prime 3 Prime, Inc). The membranes were washed in 2x SSC/0.5% SDS solution for 30 minutes at room temperature, for 30 minutes at 60°C, and for 45 minutes at 65°C and exposed to Kodak X-OMAT film at -70°C with intensifying screens for 24 to 72 hours. The Pst I–Pst I fragment (nucleotides 478 to 1850) from the rat NHE-1 cDNA was used as specific probe in the Northern blot analysis. The NHE-1 cDNA was a generous gift from Dr Gary E. Shull, University of Cincinnati (Ohio).

Assay of PKC Activity
PKC activity was measured as phosphorylation of a PKC-specific substrate according to established methods.^{11 12 13} Briefly, VSM cells were grown in 96-well microtiter plates to near confluence and then exposed to control or hypertonic medium for 48 hours in a manner similar to ²²Na⁺ influx experiments. A PKC assay buffer consisting of 140 mmol/L NaCl, 5.4 mmol/L KCl, 10 mmol/L MgCl₂, 0.3 mmol/L sodium phosphate, 0.4 mmol/L potassium phosphate, 25 mmol/L ß-glycerophosphate, 5.5 mmol/L D-glucose, 5 mmol/L EGTA, 1 mmol/L CaCl₂, 100 µmol/L [-³²P]ATP, 50 µg/mL digitonin, and 20 mmol/L HEPES (pH 7.2, 30°C) was prepared. The culture medium was aspirated and replaced with 40 µL of the PKC assay buffer containing 100 µmol/L of a highly specific PKC substrate (VRKRTLRRL).^{12 13} The phosphorylation reaction was terminated after 10 minutes by the addition of 10 µL of 25% trichloroacetic acid. Aliquots (45 µL) of the reaction mixture were then blotted onto phosphocellulose, washed, and assayed by liquid scintillation spectroscopy.

Materials
²²Na, [³²P]dCTP, and [³²P]ATP were purchased from New England Nuclear. PKC-specific peptide (VRKRTLRRL), amiloride, dimethylamiloride (DMA), and nitrocellulose filters were obtained from Sigma Chemical Co. BCECF and nigericin were obtained from Molecular Probes Inc. Oligo dT-cellulose spin columns and random-primed DNA labeling kit were purchased from 5 Prime 3 Prime, Inc. Ninety-six–well microtiter plates were purchased from Becton Dickinson Labware.

Data Analysis
The data are expressed as mean±SEM. Statistical analysis was performed by ANOVA, with P<.05 considered statistically significant.

	Results

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In the first series of experiments, we examined the effect of hypertonicity on Na⁺-H⁺ exchanger in VSM cells. Cultured VSM cells were grown to near confluence in 24-well plates, deprived of serum for 12 hours, and exposed to either isotonic (310 mOsm/L) or hypertonic (410 mOsm/L) medium for 24 hours. The osmolality of the hypertonic medium was increased to 510 mOsm/L for another 24 hours. The medium was removed after 48 hours, and Na⁺-H⁺ exchanger activity was assayed after acid loading by ²²Na⁺ influx in the presence of isotonic medium in both groups. As illustrated in Fig 1, acid-stimulated, DMA-sensitive ²²Na⁺ uptake was significantly increased in VSM cells grown in hypertonic medium (P<.01). We also examined the effect of various increments in medium osmolality on the acid-stimulated ²²Na⁺ influx. Cultured VSM cells were grown to near confluence in 24-well plates and exposed to either isotonic (310 mOsm/L) or hypertonic (410 mOsm/L) medium for 24 hours. Thereafter, the osmolality of the hypertonic medium was increased to 610, 710, or 810 mOsm/L by addition of NaCl for another 24 hours. Acid-stimulated ²²Na⁺ uptake was maximal at 710 mOsm/L (7.22±0.54, 8.22±0.52, and 5.57±0.49 nmol/mg protein in 610, 710, and 810 mOsm/L, respectively; n=4 for each group).

View larger version (48K): [in this window] [in a new window]   Figure 1. Bar graph showing the effect of prolonged high osmolality (high Osm) on dimethylamiloride (DMA)–sensitive 22Na+ influx in vascular smooth muscle cells. Cells were grown in 24-well plastic plates and exposed to control (regular osmolality) medium (310 mOsm/L) or high Osm medium (410 mOsm/L) for 24 hours. Thereafter, the osmolality of the hypertonic media was increased to 510 mOsm/L for another 24 hours. The media were aspirated, the cells were acidified, and 22Na influx was assayed at 4 minutes in regular (310 mOsm/L) medium with or without 0.1 mmol/L DMA. Values shown for uptake represent mean±SEM for four separate experiments performed in quadruplicate.

The results of the experiments in Fig 1 suggest that VSM cells are osmotically tolerant cells and activate Na⁺-H⁺ exchange in response to long-term exposure to hypertonic medium. In the next series of experiments, we examined the effect of serum on high-osmolality–induced Na⁺-H⁺ exchange activation. VSM cells were grown to near confluence and incubated in normal or high-osmolality medium in the absence of serum. The increments in osmolality were achieved in a manner similar to that described for Fig 1. As illustrated in Fig 2, the stimulatory effect of high osmolality on Na⁺-H⁺ exchange in VSM cells was independent of serum in the culture medium.

View larger version (25K): [in this window] [in a new window]   Figure 2. Bar graph showing the effect of serum on high-osmolality (high Osm)–induced 22Na+ influx in vascular smooth muscle cells. Cells were incubated in regular (control) or high Osm medium without serum for 48 hours. The increments in osmolality were achieved in a manner similar to that described in Fig 1. The media were aspirated, and the cells were assayed for 22Na influx as described in Fig 1. Values shown for uptake represent mean±SEM for three separate experiments performed in quadruplicate. DMA indicates dimethylamiloride.

We then sought to determine the effect of long-term exposure to hypertonic medium on pH_i and Na⁺-dependent hydrogen efflux by using the pH-sensitive dye BCECF. VSM cells were grown on coverslips, incubated in normal or hypertonic medium for 48 hours as described in Fig 1, and assayed for Na⁺-dependent pH_i recovery from an acid load by using BCECF. Fig 3 shows tracings from representative experiments performed according to this protocol. As illustrated, Na⁺-dependent pH_i recovery was significantly increased in VSM cells incubated in high osmolality (510 mOsm/L) compared with control cells (310 mOsm/L). The results of seven separate experiments showed that initial pH_i recovery from an acid load (dpH_i/dt) was as follows: control, 0.29±0.06 pH/min; high osmolality, 0.65±0.13 pH/min (P<.03, n=7 coverslips for each group). Steady state pH_i was 7.21±0.05 in the control cells and 7.25±0.06 in the high-osmolality cells (P>.05, n=7 coverslips for each group). After acid loading with NH₄Cl, pH_i decreased to 6.31±0.07 in control cells and 6.36±0.08 in high-osmolality cells (P>.05, n=7 coverslips for each group). These results suggest that the increased Na⁺-H⁺ exchange activity in hypertonic medium (Figs 1 through 3) represents a primary adaptive stimulation of the exchanger and is not secondary to alterations in pH_i.

View larger version (9K): [in this window] [in a new window]   Figure 3. Tracings showing the effect of prolonged high osmolality (high Osm) on Na+-dependent pHi recovery in vascular smooth muscle cells by use of the pH-sensitive dye BCECF (representative experiments). Cells were grown on coverslips and exposed to control (regular osmolality) medium (310 mOsm/L) or high Osm medium in a manner similar to that described in Fig 1. Na+-H+ exchanger was assayed as Na+-dependent pHi recovery from an acid load. NMDG indicates N-methyl-D-glucamine.

The data in Figs 1 through 3 indicate that Na⁺-H⁺ exchanger activity in VSM cells is increased by prolonged exposure to hypertonic medium. To determine if this effect is regulated at the transcription level, we examined the effect of elevated osmolality on Na⁺-H⁺ exchanger (NHE-1) mRNA steady state levels in VSM cells. Cells were grown in 100-mm dishes and exposed to either isotonic or hypertonic medium by using the same protocol used to test the effect of high osmolality on Na⁺-H⁺ exchanger activity (Fig 1). Poly(A⁺) RNA was isolated from each group, size-fractionated, transferred to a nylon membrane, and probed with radiolabeled NHE-1 cDNA. A representative experiment demonstrates that the levels of NHE-1 mRNA and the constitutive control (ß-actin mRNA) were not affected by high osmolality (Fig 4). Three Northern blots were performed on three separate Poly(A⁺) RNA samples, which were pooled from ten 100-mm plates of VSM cells incubated in control or high osmolality. Densitometric scanning of the blots from three separate experiments did not show any significant difference between control and high-osmolality groups (high osmolality, 96±6.3%; control, 100%; P>.05).

View larger version (28K): [in this window] [in a new window]   Figure 4. Northern blot analysis of Na+-H+ exchanger isoform (NHE-1) mRNA. Vascular smooth muscle cells were grown in 100-mm dishes and exposed to control or high-osmolality (high Osm) medium in a manner similar to that described in Fig 1. Poly(A+) RNA was extracted from both groups, size-fractionated, transferred, and probed for NHE-1 mRNA. ß-Actin mRNA levels are measured as controls.

The results of the above experiments illustrate that NHE-1 mRNA does not increase in response to long-term exposure to hypertonic medium. As such, transcription of the antiporter is unlikely to be involved in the increase in Na⁺-H⁺ exchange activity. Next, we examined a potential posttranslational modification of the exchanger in response to prolonged high osmolality. We focused on the long-term effect of PKC activation, because several studies have shown the important role of PKC in regulation of the Na⁺-H⁺ exchanger.^{10 14 15 16 17 18 19 20 21 22} To determine whether PKC activity is increased with long-term exposure to hypertonic medium, cells were grown to confluence in 96-well plates and exposed to control or high-osmolality medium for 48 hours. PKC activity was assayed by phosphorylation of the highly specific PKC substrate (VRKRTLRRL).^{11 12 13} Fig 5 shows that a 48-hour exposure to high osmolality significantly increased PKC activity in VSM cells (P<.01). These results indicate sustained stimulation of PKC in response to high osmolality.

View larger version (42K): [in this window] [in a new window]   Figure 5. Bar graph showing the effect of chronic high osmolality (high Osm) on protein kinase C (PKC) activity in vascular smooth muscle (VSM) cells as assessed by PKC-specific substrate phosphorylation. Left and right bars show PKC activity in VSM cells after prolonged exposure to control or high Osm medium, respectively. Values shown for activities represent mean±SEM for three separate experiments performed in quadruplicate.

The results of the experiments in Figs 1 and 5 indicate that long-term high osmolality increases Na⁺-H⁺ exchanger and PKC activity in VSM cells. To examine the role of PKC activation in mediating the effect of prolonged hypertonicity on Na⁺-H⁺ exchanger, PKC activity was downregulated by incubating the VSM cells with phorbol 12-myristate 13-acetate (PMA). Na⁺-H⁺ exchanger activity was assayed by the ²²Na⁺ influx or pH-sensitive dye BCECF measurements. Cells were grown to confluence and exposed to control or high-osmolality medium in the presence or absence of 0.1 µmol/L PMA for 48 hours. Thereafter, PKC activity was assayed by phosphorylation of the PKC-specific substrate (VRKRTLRRL) in a manner similar to that performed for Fig 5. The results showed that PKC activity was downregulated after preincubation with 0.1 µmol/L PMA, and there was no significant difference between control and high-osmolality groups (control, 12.1±1.3 pmol phosphorylation per milligram protein per minute; high osmolality, 13.8±1.6 pmol phosphorylation per milligram protein per minute; P>.05; n=3). This confirms previous reports on PKC downregulation in VSM cells with 0.1 µmol/L PMA.^{11 12 13}

Fig 6 (top) shows that downregulation of PKC with 0.1 µmol/L PMA nearly abolished the stimulation of acid-stimulated DMA-sensitive ²²Na⁺ influx by high osmolality (P>.05 for control plus PMA vs high osmolality plus PMA, n=4). Fig 6 (bottom) demonstrates the initial rate of pH_i recovery from an acid load in cells that were exposed to control or high-osmolality medium in the presence and absence of PMA. As illustrated, Na⁺-dependent acid efflux was significantly higher in the high-osmolality group (lane 3) compared with the control group (lane 1). Downregulation of PKC activity significantly prevented the effect of high osmolality (lane 4) on Na⁺-H⁺ exchanger compared with the control condition (lane 2). Taken together, these results suggest that long-term high osmolality induces Na⁺-H⁺ exchanger stimulation via PKC activation.

View larger version (29K): [in this window] [in a new window]   Figure 6. Top, Bar graph showing the effect of protein kinase C (PKC) downregulation on dimethylamiloride (DMA)–sensitive 22Na+ influx in control and prolonged high-osmolality (high Osm) groups (control with and without DMA and high Osm with and without DMA). All groups were exposed to phorbol 12-myristate 13-acetate (PMA). Values shown for uptake represent mean±SEM for four separate experiments performed in quadruplicate. Bottom, Bar graph showing the effect of PKC downregulation on Na+-dependent pHi recovery from an acid load in control and prolonged high Osm groups by using the pH-sensitive dye BCECF (control with and without PMA and high Osm with and without PMA). Values shown for dpHi/dt represent mean±SEM for seven coverslips.

	Discussion

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Acute increments in extracellular osmolality have been shown to activate the Na⁺-H⁺ exchanger in several nonepithelial cells.¹ The molecular mechanism(s) of this activation remains unknown. Several studies have suggested possible roles for protein kinases in mediating this response.^{1 2 3} One recent report indicated that a phosphorylation-independent mechanism might be responsible for osmotic activation of the Na⁺-H⁺ exchanger.⁴ Although the effect of high osmolality on Na⁺-H⁺ exchanger activity has been examined after short-term (acute) exposure of the cells to hypertonic media, there is little information on the effect of long-term high osmolality on Na⁺-H⁺ exchanger.

The results of the experiments shown in Figs 1 through 3 support the view that prolonged high osmolality increases Na⁺-H⁺ exchange activity in VSM cells. Furthermore, the demonstration of comparable steady-state and acid load pH_i at all levels of osmolality tested indicates that high-osmolality–induced Na⁺-H⁺ exchanger stimulation is a primary adaptive process and is not secondary to alterations in pH_i. In additional studies not shown, we found that a comparable increase in the osmolality of the medium by sucrose induced similar Na⁺-H⁺ exchanger activation, indicating that the stimulatory effect of high osmolality on Na⁺-H⁺ exchanger activity is likely due to hypertonicity rather than an increased concentration of sodium or chloride. Our results also indicate that transcription of the Na⁺-H⁺ exchanger is not involved in mediating high-osmolality–induced antiporter activation, since NHE-1 mRNA levels remained unchanged in control and high-osmolality groups (Fig 4). The experiments in Fig 5 illustrate that long-term high osmolality induces chronic stimulation of PKC. The role of chronic PKC stimulation in Na⁺-H⁺ exchange activation was studied. Incubation of VSM cells for 48 hours with PMA, a maneuver that downregulates PKC in VSM cells,^{11 23} almost abolished the effect of long-term hypertonicity on Na⁺-H⁺ exchange activity (Fig 6). These results indicate that long-term high osmolality, via sustained PKC stimulation, activates the Na⁺-H⁺ exchanger. These are the first studies demonstrating sustained PKC stimulation in long-term hypertonicity. The results further demonstrate that the mechanisms of osmotic activation of the Na⁺-H⁺ exchanger in VSM cells in acute⁵ and chronic (Figs 1 through 6) states are different. Na⁺-H⁺ exchanger activation in response to hypertonicity was inhibited by PKC downregulation in chronic (Fig 6) but not acute states.⁵ Recent studies by others have suggested that short-term PKC stimulation of renal epithelial cells chronically increases the Na⁺-H⁺ exchanger (NHE-1) activity and mRNA expression.²⁰ The authors, to achieve PKC stimulation, exposed the renal epithelial cells to PMA for 2 hours.²⁰ The Na⁺-H⁺ exchanger activity and mRNA levels were assayed 24 hours later, but the PKC activity was not measured in those experiments.²⁰ As such, the relation of PKC activity and Na⁺-H⁺ exchanger activity remains unknown. Recent studies of VSM cells showed that long-term high glucose (20 mmol/L) activated Na⁺-H⁺ exchanger²² and NHE-1 mRNA levels and induced sustained stimulation of PKC.^{11 22 23} Downregulation of PKC prevented the high-glucose stimulation of Na⁺-H⁺ exchanger in VSM cells.²² The stimulation of Na⁺-H⁺ exchanger in high-glucose medium was independent of the osmolality of the medium, since comparable increases in the osmolality of the medium by mannitol did not affect the Na⁺-H⁺ exchanger.²²

Whether sustained stimulation of PKC in long-term high osmolality is unique to VSM cells is poorly understood. Recent studies in our laboratory show that long-term high osmolality induces sustained PKC activation in LLC-PK₁ cells. The results further demonstrate that prolonged high osmolality, via chronic PKC activation, decreases Na⁺-H⁺ exchanger (NHE-3) activity in LLC-PK₁ cells with no significant effect on NHE-3 mRNA expression (M. Soleimani et al, unpublished data, 1994). Taken together, these results suggest that adaptive regulation of the Na⁺-H⁺ exchanger in long-term high osmolality may be mediated via chronic PKC activation. However, one should be cautious with generalizations concerning this mechanism. Chronic exposure of inner medullary collecting duct (mIMCD-3) cells (which express NHE-1 and NHE-2) to hypertonic medium increased Na⁺-H⁺ exchanger activity via increased NHE-2 mRNA expression.²⁴ NHE-1 mRNA levels in mIMCD-3 cells were decreased with long-term exposure to hypertonic medium.²⁴ Lack of changes in NHE-1 mRNA in VSM cells (Fig 4) and decreased levels in mIMCD-3 cells in prolonged high osmolality²⁴ suggest that NHE-1 is differentially regulated in these two cell lines. Recent experiments examining regulation of the Na⁺-H⁺ exchanger in chronic acidosis have shown differential regulation of NHE-1 in cultured renal and fibroblast cells.^{25 26} NHE-1 mRNA was upregulated in renal proximal tubule cells and downregulated in fibroblast cells in response to long-term acidosis.²⁶ Taken together, these results illustrate that regulation of the Na⁺-H⁺ exchanger in response to pathophysiological states is dependent on the cell and experimental conditions under study. Whether this is secondary to differences in cell types (epithelial versus nonepithelial) or reflects differences in intracellular signaling pathways remains unknown. The pathways mediating the PKC-induced phosphorylation of Na⁺-H⁺ exchanger isoform NHE-1 in high osmolality are not clear. Whether PKC directly phosphorylates NHE-1 or activates a phosphorylation cascade in long-term hypertonicity remains unknown. Recent studies in Madin-Darby canine kidney cells show that acute increment in osmolality of the medium activates mitogen-activated protein (MAP) kinase and that this effect is PKC dependent.²⁷ MAP kinase activity declined rapidly in response to high osmolality.²⁷ As such, MAP kinase would be an unlikely candidate to mediate PKC effect in long-term osmolality. The presence of another phosphorylation cascade involving PKC that is activated by high osmolality cannot be ruled out at this time.

In conclusion, the results of the above studies demonstrate that long-term exposure to hypertonic medium increases Na⁺-H⁺ exchanger activity in VSM cells. This effect may be related to posttranslational modification of the Na⁺-H⁺ exchanger and is mediated via chronic PKC activation. We suggest that chronic upregulation of the Na⁺-H⁺ exchanger, by increased Na⁺ influx, may help prevent VSM cell dehydration in long-term high osmolality.

	Acknowledgments

 
This study was supported by a merit review grant from the Department of Veterans Affairs; a Grant-in-Aid from the American Heart Association, Indiana Affiliate, Inc (Dr Soleimani); and National Institute of Diabetes and Digestive and Kidney Diseases grant DK-43640 (Dr Dominguez). Dr Howard was the recipient of a Physician Scientist Award from the National Institutes of Health (DK-02116). The excellent technical assistance of Yolanda J. Hattabaugh and Daniel J. Homco is gratefully acknowledged.

Received September 13, 1994; accepted December 21, 1994.

	References

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