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(Investigative Ophthalmology and Visual Science. 2002;43:2714-2720.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

The Influence of Cycloheximide on Na,K-ATPase Activity in Cultured Human Lens Epithelial Cells

Guangming Cui1, William L. Dean2 and Nicholas A. Delamere1,3

1 From the Departments of Ophthalmology and Visual Sciences, 2 Biochemistry and Molecular Biology, and 3 Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Earlier studies from this laboratory demonstrated the ability of lens epithelium to synthesize new Na,K-adenosine triphosphatase (Na,K-ATPase) catalytic subunit ({alpha}) polypeptide under conditions of increased ion permeability. In the present study, the authors considered whether continuous synthesis of Na,K-ATPase protein is necessary for maintenance of Na,K-ATPase activity in lens cells.

METHODS. Na,K-ATPase activity was measured by quantifying the ouabain-sensitive rate of ATP hydrolysis in cultured human lens epithelial cells (HLE-B3) permeabilized with digitonin. The abundance of Na,K-ATPase {alpha} subunit was determined by Western blot analysis. Synthesis of Na,K-ATPase {alpha}1 polypeptide was investigated by measuring 35S-methionine incorporation.

RESULTS. Na,K-ATPase activity was reduced to less than 20% of the control level in HLE-B3 cells exposed to 100 µM cycloheximide for 24 hours. However, as judged by Western blot density, the abundance of Na,K-ATPase {alpha}1 and {alpha}3 subunit in cycloheximide-treated cells was 90% and 84% of the control level, respectively. 35S-methionine incorporation experiments revealed detectable labeling of Na,K-ATPase {alpha}1 subunit polypeptide within 30 minutes, consistent with {alpha}1 polypeptide synthesis. Na,K-ATPase {alpha}1 polypeptide labeling was also detected in the epithelium of intact rat lenses that had been allowed to incorporate 35S-methionine. Cycloheximide abolished 35S-methionine incorporation into Na,K-ATPase {alpha}1 subunit polypeptide of HLE-B3 cells. When added during the chase phase of the experiment, cycloheximide was found to slow the disappearance of labeled {alpha}1 polypeptide, consistent with a reduced rate of polypeptide degradation.

CONCLUSIONS. The results suggest that a continuous cycle of Na,K-ATPase {alpha}1 synthesis and degradation may occur in lens epithelial cells. Cycloheximide appeared to inhibit Na,K-ATPase protein synthesis and degradation. The observed reduction of Na,K-ATPase activity after treatment with cycloheximide indicates that even though Na,K-ATPase remains abundant, Na,K-ATPase becomes inactivated when protein synthesis is inhibited.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the lens, as in other tissues, a low concentration of intracellular sodium and a high concentration of intracellular potassium is maintained by Na,K-adenosine triphosphatase (Na,K-ATPase).1 Na,K-ATPase is a plasma membrane–bound transporter that uses the energy from adenosine triphosphate (ATP) hydrolysis to export sodium ions and import potassium.2 Although it represents only a small fraction of the total number of lens cells, the epithelial monolayer on the anterior surface of the lens is believed to conduct much of the Na,K-ATPase mediated ion transport for the entire cell mass. The activity of Na,K-ATPase in the lens epithelial monolayer is maintained at a higher level than elsewhere in the lens.3 4

In an earlier study, the synthesis of new Na,K-ATPase catalytic subunit polypeptide in lens epithelial cells was observed when the ion permeability of intact cultured porcine lens was increased by exposing the lens to amphotericin B.5 This suggests that synthesis of additional Na,K-ATPase protein molecules is one way for the lens epithelium to increase Na,K-ATPase activity. Recognizing the apparent ability of lens epithelium to synthesize new Na,K-ATPase catalytic subunit protein under conditions of increased ion permeability led us to consider whether continuous synthesis of Na,K-ATPase protein is necessary for the maintenance of Na,K-ATPase activity in resting lens cells. In the present study, cultured human lens epithelial cells (HLE-B3) were exposed to cycloheximide, a protein synthesis inhibitor, and the consequent changes in Na,K-ATPase activity were examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
A human lens epithelial cell line (HLE-B3) transformed with adenovirus 12-SV40 was used in this study.6 7 This cell line was the kind gift of Usha Andley (Washington University, St. Louis, MO). Cells were cultured at 37°C in minimum essential medium (MEM; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% gentamicin in humidified air containing 5% CO2.

Rat Lens
Sprague-Dawley rat eyes were obtained from Harlan Bioproducts (Indianapolis, IN) and were shipped to the laboratory overnight on ice. To obtain the lens, the globe was dissected, the suspensory ligaments were cut, and the lens was gently removed and placed in medium 199 (M199; Gibco, Rockville, MD) at 37°C.

Na,K-ATPase Activity Assay
Na,K-ATPase activity was determined in confluent cell monolayers immediately after a period of incubation in the presence or absence of cycloheximide (100 µM). The assay technique has been used earlier to measure Na,K-ATPase activity in nonpigmented ciliary epithelium.8 The methodology is based on a technique developed for analysis of receptor-mediated modulation of Na,K-ATPase activity in kidney tubule.9 The MEM was removed, and digitonin permeabilization was performed by adding 15 µM digitonin in ATPase assay buffer (40 mM histidine, 100 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM EGTA [pH 7.4]) for 10 minutes on ice. After this, the digitonin buffer was removed and replaced with ATPase assay buffer. Ouabain was applied to half of the cells at a final concentration of 1 mM, which is sufficient to inhibit Na,K-ATPase activity by approximately 100%.10 ATP hydrolysis was initiated by adding ATP containing a trace amount of {gamma}-32P labeled ATP. The final concentration of ATP in the reaction was 1 mM. After 30 minutes at 37°C, the reaction was stopped by adding 10% ice-cold trichloroacetic acid and chilling the cells on ice. An aliquot of the reaction mixture was removed and mixed with 4% ammonium molybdate in 2 N H2SO4. Inorganic phosphate forms a complex with ammonium molybdate11 and is then extracted by mixing with benzene/isobutanol (1:1). Radioactivity of the inorganic phosphate was measured in an aliquot of the organic layer, by scintillation spectrophotometer (Beckman, Fullerton, CA). Specific activity was determined by measuring the radioactivity in an aliquot of the {gamma}-32P labeled ATP. A sham control was used to determine nonspecific ATP hydrolysis by a parallel assay on blank culture wells without cells. Nonspecific ATP hydrolysis was found to generate 2.80 ± 0.05 nM inorganic phosphate (Pi) per well compared with 10.00 ± 0.35 nM per well determined in wells containing control cells without 1 mM ouabain (mean ± SE; n = 12). Protein content in each well was determined by lysing cells with 0.5 N NaOH and taking an aliquot for protein assay using a kit (Bio-Rad, Hercules, CA) based on an assay described by Bradford.12

Potassium (86Rb) Uptake
Because Na,K-ATPase transports rubidium in a manner similar to potassium,13 ouabain-sensitive 86Rb uptake was measured as an index of Na,K-ATPase-mediated potassium transport in confluent monolayers of intact HLE-B3 cells grown in 24-well plates. Some cells were incubated with cycloheximide (100 µM) for 24 hours before measurement of 86Rb uptake. Control cells were not treated. At the end of the 24-hour pretreatment period, the MEM was removed and replaced with Krebs solution with or without cycloheximide (100 µM) for 1 hour. The composition of the Krebs solution (in mM) was: 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, 1 MgCl2, and 5.5 glucose at pH 7.4. Then ouabain (1 mM) was added to half of the cells. 86Rb uptake was initiated by adding a trace amount of 86RbCl (0.1 µCi/mL) to all the cells. Throughout the experiment, the temperature was maintained at 37°C, and the atmosphere contained 5% CO2. After 15 minutes, the radioactive solution was removed, and the cells were washed twice with ice-cold Krebs buffer. Cells were then lysed by adding 0.5 N NaOH for 12 hours at room temperature. The amount of 86Rb in the cell lysate was quantified by scintillation counter. The protein content in the lysate was measured using the protein assay kit (Bio-Rad).

Isolation of Membrane Material
Membrane material was isolated from confluent HLE-B3 cells grown in 25-cm2 flasks, by a method based on the technique described by Moseley et al.14 The MEM was removed and replaced with ice-cold buffer A (150 mM sucrose, 5 mM HEPES, 875 µM dithiothreitol [DTT], pH 7.4) that contained 4 mM EGTA and a mixture of protease inhibitors (100 µM phenylmethylsulfonyl fluoride [PMSF], 10 µg/mL antipain, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 2 µg/mL aprotinin). The culture flasks were chilled on ice, and cells were lifted off with a scraper. In some experiments the capsule-epithelium was removed from rat lenses and used as a source of membrane material. The capsule-epithelium was homogenized in ice-cold buffer A containing protease inhibitors, using a Dounce glass homogenizer (Bellco Glass Co., Vineland, NJ). Cells suspensions were then transferred to centrifuge tubes and briefly sonicated (three times for 5 seconds each). Membrane material was pelleted by centrifugation at 100,000g at 4°C. The pellet was resuspended in buffer A without EGTA or protease inhibitors. Protein concentration was determined using the protein assay kit (Bio-Rad). By comparing the cell count with the protein level, the amount of membrane protein per cell was determined as 0.126 ± 0.004 ng/cell (mean ± SE; n = 3). This amount was not significantly different from the 0.118 ng/cell determined in cells exposed to 100 µM cycloheximide for 24 hours.

Western Blot Analysis
Western blot experiments were conducted using a methodology based on the technique of Moseley et al.14 Membrane material was solubilized in Laemmli buffer15 and separated on 10% SDS-PAGE at 20 mA per gel for 120 minutes. A 50-µg protein aliquot of membrane material (equivalent to ~4 x 105 cells) was applied to each lane. After electrophoresis, proteins were transferred from the gel to a nitrocellulose membrane by applying 30 V for 12 hours. The nitrocellulose membrane was blocked in 5% milk in TTBS (30 mM Tris, 150 mM NaCl, 0.5% [vol/vol] Tween-20 [pH7.4]) for 1 hour. For immunodetection of Na,K-ATPase {alpha} subunits, the nitrocellulose membrane was incubated with a monoclonal antibody directed against either the Na,K-ATPase {alpha}1 isoform (Upstate Biotechnology, Lake Placid, NY) or the Na,K-ATPase {alpha}3 isoform (Affinity Bioreagents, Golden, CO) for 1 hour. After one wash in TTBS for 15 minutes and then four washes for 5 minutes each, the nitrocellulose membrane was incubated for 1 hour with HRP-conjugated secondary antibody and washed again as just described. Na,K-ATPase polypeptide was visualized by developing the nitrocellulose in enhanced chemiluminescence Western blot detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 minute and exposed to x-ray film for 0.5 to 2 minutes. Immunoblot band density was quantified using 1D image-analysis software (Kodak, Rochester, NY).

35S-Methionine Metabolic Labeling
HLE-B3 cells grown in 25-cm2 flasks were used 1 day after reaching confluence. Adult rat lenses were used within 1 hour of dissection from the eye. The MEM or M199 culture medium was removed, and cells or the intact lenses were washed several times with Hanks’ balanced salt solution. After the technique detailed earlier by Lescale-Matys et al.,16 the cells were starved of methionine before the addition of 35S-methionine. Methionine-free MEM without fetal bovine serum or gentamicin was then added for 1 hour. After this, 100 µCi/mL 35S-methionine was added to initiate labeling. After incubation at 37°C for a specified time, the radioactive medium was removed and replaced with 2 mL ice-cold buffer A. The HLE-B3 cells were then detached by freezing the flask to -80°C and thawing on ice. In rat lens experiments, the capsule-epithelium was removed from each intact lens and pooled. After this, membrane material was prepared as described earlier.

For pulse-chase experiments, 35S-methionine pulse labeling was performed for 2 hours as has been described. Then a chase phase was initiated by replacing the 35S-methionine–containing medium with nonradioactive MEM containing a normal concentration of nonradiolabeled methionine. After a specified period at 37°C, the MEM was removed and replaced with 2 mL ice-cold buffer A. The cells were detached from the flasks, and membrane material was prepared as for Western blot experiments earlier.

Immunoprecipitation
The immunoprecipitation strategy was based on a methodology described earlier for isolation of plasma membrane Ca-ATPase.17 Membrane material (400 µg of protein) was solubilized in sufficient immunoprecipitation buffer (30 mM Tris, 150 mM NaCl, 10 mM EGTA, 1% Triton X-100, 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL antipain, 10 µg/mL pepstatin A, and 1 mM DTT [pH 7.4]) for a final protein concentration of 1 µg/µL. The membrane material mixture was agitated gently on a rotating mixer for 1 hour at 4°C. Insoluble material was then pelleted by centrifugation at 8000g at 4°C for 10 minutes. The supernatant was transferred to a fresh 1.5-mL microfuge tube, and 10 µg monoclonal antibody directed against Na,K-ATPase {alpha}1 polypeptide (Upstate Biotechnology) was added. The mixture was incubated at 4°C overnight with gentle agitation on a rotating mixer. After this, 50 µL immobilized protein A (ImmunoPure; Pierce, Rockford, IL) was added for an additional incubation of 1.5 hours at 4°C on the rotating mixer. The immobilized protein A had been washed two times previously with immunoprecipitation buffer. The reaction mixture was then centrifuged at 3000g for 3 minutes at 4°C. The pellet was washed twice with 200 µL immunoprecipitation buffer and then washed twice with 200 µL Tris-NaCl (30 mM Tris and 150 mM NaCl [pH 7.4]). The immunoprecipitated Na,K-ATPase {alpha}1 polypeptide was dissociated from the antibody and immobilized protein A by incubating in 45 µL of Laemmli buffer for 20 minutes at 40°C, followed by centrifugation at 4000g for 5 minutes. The supernatant was subjected to electrophoresis by SDS-PAGE. After electrophoresis, the gel was fixed in solution containing 50% methanol and 10% acetic acid and then vacuum dried on a gel drier. 35S-labeled Na,K-ATPase {alpha}1 polypeptide was visualized by exposing the dried gel to x-ray film for 3 to 6 days.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cycloheximide is a well-recognized inhibitor of protein synthesis at the ribosome level.18 Confluent monolayers of human lens epithelial cells (HLE-B3) were exposed to medium containing 100 µM cycloheximide for specified periods of time up to 24 hours. The cells were then permeabilized with 15 µM digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was determined. In control HLE-B3 cells that were not treated with cycloheximide, Na,K-ATPase activity was 48.3 ± 2.6 nmol Pi/mg protein per 45 minutes (mean ± SE, n = 12). Cycloheximide pretreatment caused a marked decrease in Na,K-ATPase activity. After a 24-hours incubation in the presence of cycloheximide, Na,K-ATPase activity in HLE-B3 cells was reduced to 9.6% of the control value (Fig. 1 ; significantly different from control; P < 0.001). In separate experiments, potassium (86Rb) uptake was measured as an index of Na,K-ATPase-mediated ion transport in confluent monolayers of intact HLE-B3 cells that had been pretreated with 100 µM cycloheximide for 24 hours (Fig. 1) . In cells that had been subjected to 24 hours of cycloheximide treatment, the rate of ouabain-sensitive potassium (86Rb) uptake was 8.57 ± 0.57 nmol potassium/mg of protein per minute (mean ± SE, n =12). This was significantly (P < 0.001) less than the rate of 18.15 ± 0.57 nmol potassium/mg of protein per minute measured in control cells that were not treated with cycloheximide.



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  		Figure 1. Top: Na,K-ATPase activity measured in HLE-B3 cells subjected to a 4- or 24-hour preincubation period in the presence of 100 µM cycloheximide (Cx). Immediately after the preincubation period, the culture medium containing cycloheximide was removed, and the cells were permeabilized with digitonin. ATP hydrolysis was then measured in the presence or absence of ouabain. The results are shown as mean ± SE (vertical bar), n = 12. Bottom: Ouabain-sensitive potassium (86Rb) uptake rate measured in control cells (no cycloheximide) and in cells exposed to 100 µM cycloheximide added 24 hours before the initiation of 86Rb uptake. The results are shown as the mean ± SE (vertical bar), n = 12. *Significant difference from control (P < 0.001).

 
To determine the influence of cycloheximide on Na,K-ATPase {alpha}1 protein abundance, Western blot studies were conducted with a monoclonal antibody directed against the Na,K-ATPase {alpha}1 subunit. Cell monolayers were incubated in the presence or absence of cycloheximide, membrane microsomes were prepared, and 50 µg of membrane microsome material (equivalent to ~4 x 105 cells) was resolved on SDS-PAGE for the immunoblot. A single band was recognized at an apparent molecular weight of 102 kDa (Fig. 2) . The band density of Na,K-ATPase {alpha}1 polypeptide determined in cells treated with cycloheximide for 24 hours was 90.3% ± 1.2% (mean ± SE; n = 5) of the band density determined in control cells (significantly different from control; P < 0.001).



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  		Figure 2. Top: Western blot of Na,K-ATPase {alpha}1 and {alpha}3 polypeptide determined in membrane material isolated from HLE-B3 cells that were subjected to a 24-hour preincubation period in the presence or absence (control) of 100 µM cycloheximide (Cx). Membrane material isolated from rat brain (left lane) was used as a positive control. Fifty micrograms protein (equivalent to 4 x 105 cells) was applied to each lane. Molecular size markers (in kilodaltons) are shown at left. The relative immunoblot density (Cx-treated versus control) was measured in several different experiments. The data shown in the bottom panel are the mean ± SE (vertical bar); n = 5 for {alpha}1, n = 3 for {alpha}3. *Significant difference from control (P < 0.0001).

 
Two different isoforms of Na,K-ATPase, {alpha}1 and {alpha}3, were detected in the HLE-B3 cells. Na,K-ATPase {alpha}2 was not detected (data not shown). The influence of cycloheximide on the abundance of Na,K-ATPase {alpha}3 polypeptide was also examined. Western blot studies were conducted with a monoclonal antibody directed against the {alpha}3 polypeptide. A single band was detected at an apparent molecular weight of 119 kDa. In cells treated with cycloheximide for 24 hours, the Na,K-ATPase {alpha}3 band density was 84.4% ± 2.0% (mean ± SE; n = 3) of the band density determined in control cells (significantly different from control; P < 0.001).

To examine the basis for the inhibitory influence of cycloheximide on Na,K-ATPase activity, experiments were first conducted to determine whether detectable synthesis of the {alpha}1 catalytic Na,K-ATPase polypeptide occurs in HLE-B3 cells. Pulse labeling of HLE-B3 cells was performed by adding 35S-methionine to the culture medium. Cell membrane material was prepared from the cells that had incorporated 35S-methionine and was then subjected to immunoprecipitation with a monoclonal antibody directed against the {alpha}1 subunit of Na,K-ATPase. The immunoprecipitated proteins were subsequently eluted and resolved by SDS-PAGE. Newly synthesized protein labeled with 35S-methionine was visualized by autoradiography. A single band at the apparent molecular weight of 102 kDa was detected (Fig. 3) , consistent with isolation of 35S-labeled Na,K-ATPase {alpha}1 polypeptide from the membrane protein mixture. 35S-methionine detectable in the 102-kDa protein was detectable as early as 30 minutes after the start of 35S-methionine metabolic labelling.



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  		Figure 3. Top: Autoradiograph of 35S-methionine incorporation into protein isolated from HLE-B3 cells by immunoprecipitation with an antibody directed against Na,K-ATPase {alpha}1. The autoradiograph shows the result of a typical experiment in which the cells were allowed to incorporate 35S-methionine for periods up to 240 minutes. Bottom: densitometric analysis of the autoradiograph.

 
When 100 µM cycloheximide was added to the 35S-methionine–containing medium, no detectable incorporation of 35S into the immunoprecipitated 102-kDa polypeptide was observed (Fig. 4) . This suggests that cycloheximide inhibits the synthesis of new Na,K-ATPase {alpha}1 polypeptide in HLE-B3 cells.



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  		Figure 4. The influence of 100 µM cycloheximide on 35S-methionine incorporation into protein isolated from HLE-B3 cells by immunoprecipitation using an antibody directed against Na,K-ATPase {alpha}1. The autoradiograph shows the result of a typical experiment in which cells were cultured in 35S-methionine–containing medium for 4 hours in the presence (+) or absence (-) of cycloheximide (100 µM). A radiolabeled band was detected in membrane material isolated from control cells but not in that from cycloheximide-treated cells.

 
To maintain a stable abundance of polypeptide, the rate of synthesis must be matched by an equivalent rate of polypeptide degradation. Pulse-chase labeling experiments were conducted to examine the degradation of Na,K-ATPase {alpha}1 polypeptide in the presence or absence of cycloheximide. Cells were first allowed to incorporate 35S-methionine under normal conditions (no cycloheximide) and then were incubated in the presence or absence of cycloheximide (100 µM) in medium containing a normal concentration of nonradioactive methionine. Cell membrane material was prepared from this source and then subjected to immunoprecipitation with an antibody directed against the {alpha}1 subunit of Na,K-ATPase. The immunoprecipitated material was then subjected to separation by SDS-PAGE. As judged by autoradiography, the 35S labeling intensity of the 102-kDa Na,K-ATPase {alpha}1 band diminished as a function of the incubation time in nonradioactive methionine (Fig. 5) . After 2 hours in nonradioactive methionine, the labeling of the 102-kDa band had decreased significantly. Based on analysis of the time course in five independent experiments, the half-life for the decrease in labeling was 32 ± 3 minutes (mean ± SE, n = 5). In the presence of cycloheximide the 35S labeling intensity of the 102-kDa band was observed to decrease less rapidly. The slow decay of labeling in the cycloheximide-treated group did not enable calculation of the half-life in that group.



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  		Figure 5. Autoradiograph of typical 35S-methionine pulse-chase experiments in which HLE-B3 cells were cultured in the (A) absence (control) or (B) presence of 100 µM cycloheximide during the chase phase. The cells were initially allowed to incorporate 35S-methionine for 2 hours under control conditions. Then the cells were incubated in medium containing nonradioactive methionine, with or without cycloheximide for periods up to 240 minutes. Protein was isolated from the cells by immunoprecipitation with an antibody directed against Na,K-ATPase {alpha}1. Left: labeling of the immunoprecipitated 102-kDa band. Right: relative labeling intensity of the102-kDa band, quantified and plotted versus time.

 
To determine whether Na,K-ATPase {alpha}1 polypeptide synthesis occurs in the epithelial cell layer of the intact lens, 35S-methionine labeling was examined in rat lenses. Intact adult rat lenses were incubated for 4 hours in 35S-methionine–containing culture medium, and the capsule-epithelium was removed from each lens and used as a source of membrane material that was then subjected to immunoprecipitation with a monoclonal antibody directed against the {alpha}1 subunit of Na,K-ATPase. The immunoprecipitated protein was resolved by SDS-PAGE. A single radiolabeled band at 110 kDa was observed by autoradiography, consistent with 35S-methionine labeling of newly synthesized Na,K-ATPase {alpha}1 polypeptide (Fig. 6) .



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  		Figure 6. Autoradiograph of 35S-methionine incorporation into protein isolated from rat lens epithelium by immunoprecipitation, with an antibody directed against Na,K-ATPase {alpha}1. The autoradiograph shows the result of a typical experiment in which intact adult rat lenses were allowed to incorporate 35S-methionine for 4 hours.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Na,K-ATPase activity in the human lens epithelial cell line HLE-B3 was significantly inhibited in cells that had been pretreated with cycloheximide. One possible interpretation of this finding is that during the 24-hour time span of these experiments, synthesis of Na,K-ATPase protein was required for maintenance of the amount of Na,K-ATPase protein in the cells. However, this was not the case. As judged by immunoblot density, the abundance of Na,K-ATPase {alpha}1 and {alpha}3 catalytic subunit protein was relatively similar (within 10%–15%) in control and cycloheximide-treated cells. There was no detectable change of electrophoretic mobility of either the Na,K-ATPase {alpha}1 or {alpha}3 immunoreactive bands detected by Western blot. The Na,K-ATPase {alpha}1 and {alpha}2 immunoblots offered no evidence to explain why Na,K-ATPase activity was reduced by approximately 90% in cells pretreated with cycloheximide.

It was reported earlier19 that exposure to cycloheximide for 12 hours did not change the Western blot band intensity for Na,K-ATPase {alpha}1 polypeptide in HL60 cells, but in that study the abundance of Na,K-ATPase {alpha}3 protein was reduced with a half-life of approximately 9 hours after treatment with cycloheximide. The response to cycloheximide may be different for different enzymes, because tryptophan hydroxylase activity in rat basophilic leukemia cells and mouse mastocytoma cells is inhibited by cycloheximide20 but in those cells, cycloheximide caused a rapid decrease in the abundance of the enzyme polypeptide.

It has been suggested that synthesis of protein by lens cells is slow.21 A slow rate of protein synthesis would fit with the absence of a pronounced effect of cycloheximide on the abundance of Na,K-ATPase {alpha} polypeptide, but would not explain the inhibitory effect of cycloheximide pretreatment on Na,K-ATPase activity. Cycloheximide does not appear to have a direct inhibitory effect on Na,K-ATPase activity, because it was found that 500 µM cycloheximide did not suppress the cAMP-induced stimulation of Na,K-ATPase activity in the avian nasal salt gland.22 Another possible interpretation of the relatively stable amount of Na,K-ATPase {alpha} polypeptide in cycloheximide-treated cells is that cycloheximide simply fails to inhibit Na,K-ATPase synthesis. However, direct examination of Na,K-ATPase {alpha}1 polypeptide synthesis using 35S-methionine incorporation revealed significant label incorporation into immunoprecipitated Na,K-ATPase {alpha}1 polypeptide, and this was suppressed by cycloheximide.

Results from 35S-methionine pulse-chase experiments were consistent with a cycle of Na,K-ATPase {alpha}1 protein synthesis and degradation. For technical reasons, it is difficult to use the 35S-methionine labeling data to calculate an accurate half-life for protein synthesis. In part, this is because 35S-methionine labeling experiments are conducted in medium with a very low substrate methionine concentration that can partially inhibit the rate of protein synthesis. In addition, during 35S-methionine labeling, the two processes of protein synthesis and protein degradation occur simultaneously, so that some of the labeled protein could be degraded before detection. These factors tend to produce an overestimate of the half-life of protein synthesis. This could explain why the half-life of 35S-methionine labeling was approximately 60 minutes, which is greater than the half-life of 32 ± 3 minutes calculated for protein degradation by using the pulse–chase data. Even so, the correlation between synthesis and degradation rates is reasonable. This is to be expected in a normal cell, where synthesis and degradation are in equilibrium and the abundance of Na,K-ATPase protein remains constant.

The 35S-methionine pulse–chase time course for Na,K-ATPase {alpha}1 labeling in HLE-B3 cells appeared to occur on a time scale similar to that reported for Na,K-ATPase ß subunit in Madin-Darby canine kidney (MDCK) cells,23 but a slower time course has been reported for the Na,K-ATPase {alpha} subunit in HeLa cells where the half-life is 5 hours24 and the LLC-PK1 pig kidney cell line16 where the half-life is 10 to 12 hours. In general, the turnover of Na,K-ATPase protein is reported to occur at a faster rate than the turnover of other membrane proteins.25 In 35S-methionine pulse-chase studies, cycloheximide was found to slow the rate at which 35S was lost from immunoprecipitated Na,K-ATPase {alpha}1 protein. This finding suggests that the rate of Na,K-ATPase polypeptide degradation is reduced in the presence of cycloheximide. A similar effect of cycloheximide has been reported in smooth muscle and in neurons, where the rate of both protein synthesis as well as degradation is reduced in the presence of cycloheximide.26 27 The ability of cycloheximide to slow HLE-B3 cell Na,K-ATPase {alpha}1 protein degradation is consistent with the observed maintenance of {alpha}1 abundance when {alpha}1 synthesis is inhibited by cycloheximide.

The results of studies in HLE-B3 cells suggest that a continuous cycle of Na,K-ATPase {alpha}1 synthesis and degradation may occur in lens epithelial cells. For technical reasons, we have not yet been able to measure Na,K-ATPase {alpha}3 synthesis. We cannot rule out the possibility that cells in culture may behave in a different way from cells in the intact lens. However, 35S-methionine incorporation studies were conducted using the intact adult rat lens, and we were able to detect label incorporation into immunoprecipitated Na,K-ATPase {alpha} subunit polypeptide within 4 hours. This suggests that Na,K-ATPase synthesis also occurs in the intact tissue.

The findings presented in this study suggest that Na,K-ATPase protein synthesis could be important for maintenance of Na,K-ATPase activity in lens epithelial cells. After inhibition of protein synthesis, the abundance of Na,K-ATPase protein remains close to normal, but activity of the enzyme is reduced. We speculate that under normal circumstances, the pool of Na,K-ATPase protein is continually being replenished as new proteins are synthesized and old proteins are degraded. Apparently, cycloheximide inhibits both protein synthesis and degradation. Na,K-ATPase activity may become inactivated as the result of modification of protein that is retained longer than normal. There could be multiple causes of such putative inactivation. The Na,K-ATPase protein may be subjected to phosphorylation, oxidative damage, or glycation. The mechanism is a topic for further study.


   Footnotes
 
Supported by National Eye Institute Grant EY09532, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness, Inc. NAD is the recipient of a Senior Scientific Investigator Award from Research to Prevent Blindness, Inc.

Submitted for publication January 22, 2002; revised April 1, 2002; accepted April 9, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Nicholas A. Delamere, Department of Ophthalmology, University of Louisville School of Medicine, 301 E. Muhammad Ali Boulevard, Louisville, KY 40202; delamere{at}louisville.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S. Tamiya, W. L. Dean, C. A. Paterson, and N. A. Delamere
Regional Distribution of Na,K-ATPase Activity in Porcine Lens Epithelium
Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4395 - 4399.
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