Long-term High Osmolality Activates Na+-H+ Exchange and Protein Kinase C in Aortic Smooth Muscle Cells
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 pHi 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.
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
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
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).
pHi 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 pHi 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 pHi 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.
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 5× Denhardt’s solution, 5× 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 30×106 cpm [32P]DNA probe for NHE-1 or β-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 2× 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 β-glycerophosphate, 5.5 mmol/L d-glucose, 5 mmol/L EGTA, 1 mmol/L CaCl2, 100 μmol/L [γ-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.
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.
The data are expressed as mean±SEM. Statistical analysis was performed by ANOVA, with P<.05 considered statistically significant.
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).
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.
We then sought to determine the effect of long-term exposure to hypertonic medium on pHi 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 pHi recovery from an acid load by using BCECF. Fig 3⇓ shows tracings from representative experiments performed according to this protocol. As illustrated, Na+-dependent pHi 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 pHi recovery from an acid load (dpHi/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 pHi 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, pHi 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 pHi.
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 (β-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).
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.
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 pHi 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.
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 pHi 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 pHi. 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.
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.
- © 1995 American Heart Association, Inc.
Grinstein S, Cohen S, Goetz JD, Rothstein A, Mellors A, Gelfand EW. Activation of the Na+/H+ antiport by changes in cell volume and by phorbol esters: possible role of protein kinase. Curr Top Membr Trans. 1986;26:115-134.
Grinstein S, Woodside M, Sardet C, Pouyssegur JD, Rotin D. Activation of the Na+/H+ antiporter during cell volume regulation. J Biol Chem. 1992;267:23823-23828.
Soleimani M, Bookstein C, McAteer JA, Hattabaugh YJ, Musch M, Rao MC, Howard RL, Chang EB. Effect of high osmolality on Na+/H+ exchange in renal proximal tubule cells. J Biol Chem. 1994;269:15613-15618.
Soleimani M, Howard RL. Presence of chloride-formate exchange in vascular smooth muscle and cardiac cells. Circ Res. 1994; 74:48-55.
Chaillet JR, Boron WF. Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. J Gen Physiol. 1985;86:765-794.
Bergman JA, McAteer JA, Evan AP, Soleimani M. Use of the pH sensitive dye BCECF to study pH regulation in cultured human kidney proximal tubule cells. J Tissue Cult Methods. 1991;13:205-210.
Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980;77:520-525.
Williams BR, Schrier W. Characterization of glucose-induced in situ protein kinase C activation in cultured vascular smooth muscle cells: role of protein kinase C. Diabetes. 1992;41:1464-1472.
Heasley LE, Johnson GL. Regulation of protein kinase C by nerve growth factor and epidermal growth factor and phorbol esters in PC12 pheochromocytoma cells. J Biol Chem. 1989;264:8646-8652.
Heasley LE, Johnson GL. Detection of nerve growth factor and epidermal growth factor-regulated protein kinases in PC12 cells with synthetic peptide substrates. Mol Pharmacol. 1989;35:331-338.
Grinstein S, Cohen S, Goetz JD, Rothstein AE, Gelfand W. Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbol esters. Proc Natl Acad Sci U S A. 1985;82:1429-1433.
Weinman ED, Shenolikar S. Protein kinase C activates the renal apical membrane Na+-H+ exchanger. J Membr Biol. 1989;93:133-139.
Horie S, Moe O, Yamaji Y, Cano A, Miller RT, Alpern RJ. Role of protein kinase C and transcription factor AP-1 in the acid-induced increase in Na/H antiporter activity. Proc Natl Acad Sci U S A. 1991;89:5236-5240.
Horie S, Moe O, Miller RT, Alpern RJ. Long-term activation of protein kinase C causes chronic Na/H antiporter stimulation in cultured proximal tubule cells. J Clin Invest. 1991;89:365-372.
Williams B, Howard RL. Glucose-induced changes in Na+/H+ antiport activity and gene expression in cultured vascular smooth muscle cells: role of protein kinase C. J Clin Invest. 1994;93:2623-2631.
Williams B, Tsai P, Schrier RW. Glucose-induced down regulation of angiotensin II and arginine vasopressin receptors in cultured aortic vascular smooth muscle cells: role of protein kinase C. J Clin Invest. 1992;90:1992-1999.
Soleimani M, Singh G, Bizal GL, Gullans S, McAteer J. Na+/H+ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells: expression, functional localization, and differential regulation. J Biol Chem. 1994;269:27973-27978.
Horie S, Moe O, Tejedor A, Alpern RJ. Preincubation in acid medium increases Na/H antiporter activity in cultured renal proximal tubule cells. Proc Natl Acad Sci U S A. 1990;87:1703-1708.
Moe O, Miller RT, Horie S, Cano A, Presig PA, Alpern RJ. Differential regulation of Na/H antiporter by acid in renal epithelial cells and fibroblasts. J Clin Invest. 1991;88:1703-1708.
Itoh T, Yamauchi A, Miyai A, Yokoyama K, Fugiwara Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells. J Clin Invest. 1994;93:2387-2392.