1Department of Biochemistry and Molecular Biology, 2Department of Ophthalmology and Visual Sciences, and 3Department of Pharmacology and Toxicology, University of Louisville, School of Medicine, Louisville, Kentucky 40202
Submitted 1 May 2003 ; accepted in final form 2 September 2003
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ABSTRACT |
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lens; Na,K-ATPase; tyrosine phosphorylation; Lyn
Regulation of Na,K-ATPase function can occur through several different protein kinase-mediated mechanisms. Studies on the effects of phosphorylation by PKA and PKC on Na,K-ATPase function reveal a wide range of response patterns in different tissue types, some stimulatory, some inhibitory (42). The activation of tyrosine kinases appears to stimulate Na,K-ATPase in the intact kidney proximal tubule, as judged by an increase of ouabain-sensitive 86Rb uptake (22, 36). The proximal tubule response may involve recruitment of tyrosine-phosphorylated Na,K-ATPase protein from the cytosol to the plasma membrane (36). In rat skeletal muscle, insulin causes tyrosine phosphorylation of Na,K-ATPase 1 and
2 protein (8) and stimulates Na,K-ATPase
1 and
2 translocation to the plasma membrane (2). In rat astrocytes, tyrosine kinase activation by insulin causes an increase of Na,K-ATPase activity associated with a selective increase in the synthesis of Na,K-ATPase
1 protein (34). In other tissues, however, tyrosine kinase activation appears to inhibit Na,K-ATPase function. In cultured rabbit nonpigmented ciliary epithelium, tyrosine kinase inhibitors were found to prevent the inhibitory action of dopamine and D-1 agonists on Na,K-ATPase-mediated ion transport (35). In the intact, organ-cultured lens, previous studies show genistein reduces the inhibitory effects of endothelin (ET-1) on ouabain-sensitive 86Rb uptake (38). Also in the lens, thrombin-induced inhibition of Na,K-ATPase-mediated active ion transport is suppressed by herbimycin A, an inhibitor of Src-family tyrosine kinases (37). Inhibition of Na,K-ATPase function occurs in parallel with increased tyrosine phosphorylation of multiple membrane proteins in the epithelium of thrombin-treated lenses.
Nonreceptor tyrosine kinases, including Src-family kinases, are activated in thrombin-treated tissues (23). In platelets, the Src-family kinase Lyn plays a key role in the response to thrombin (26). When platelets from Lyn-deficient mice are challenged with thrombin, normal platelet aggregation, thromboxane A2 production, and ADP secretion fail to occur (10). In the present study, experiments were conducted to test whether a change of intrinsic Na,K-ATPase activity is detectable after Lyn kinase-dependent tyrosine phosphorylation of isolated, partially purified lens epithelium membrane material. Lyn treatment caused a partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase 1-catalytic subunit.
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EXPERIMENTAL PROCEDURES |
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Membrane preparation. Membrane preparations containing plasma membranes as well as intracellular membranes were obtained following methodology described by Okafor et al. (37). Previously frozen samples of lens capsule epithelium and kidney outer medulla were homogenized in ice-cold homogenization buffer A [150 mM sucrose, 4 mM EGTA, 5 mM HEPES, 800 µM dithiothreitol (DTT), pH 7.4] in the presence of protease inhibitors [100 µM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml antipain, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 2 µg/ml aprotinin] using a glass homogenizer. The homogenate was centrifuged at 115,000 g for 60 min at 4°C. To remove extrinsic proteins, the membrane pellet was then resuspended in homogenization buffer A containing 600 mM KCl and subjected to centrifugation once again at 115,000 g for 60 min at 4°C (16). The membrane pellet was resuspended in homogenization buffer A and subjected to centrifugation a final time at 115,000 g for 60 min at 4°C. The final pellet containing epithelium or kidney membrane material was resuspended in buffer A and the protein content was measured using the BCA protein assay kit (Pierce, Rockford, IL).
Lyn-dependent phosphorylation and Na,K-ATPase activity measurement. Lens epithelium or kidney membrane material was incubated in kinase buffer containing 1 mM EGTA, 10 mM Tris, pH 7.2, 20 mM MgCl2, 1 mM ATP, 0.2 mM sodium orthovanadate, 10 µg/ml pepstatin A, 10 µg/ml antipain, 10 µg/ml leupeptin, 1 mM PMSF, 5 mM DTT, and Lyn kinase (0.08 units/µg protein) (Upstate Biotechnology, Lake Placid, NY) for 2030 min at 30°C. Treated material was then used for Western blot analysis, immunoprecipitation, or Na,K-ATPase activity measurements. Sodium orthovanadate, an inhibitor of Na,K-ATPase activity, was removed before Na,K-ATPase activity measurements. To remove sodium orthovanadate, the membrane material was centrifuged at 100,000 g for 3 min. The membrane pellet was resuspended two times in 100 µl of centrifugation buffer [10 mM Tris, pH 7.2, 5 mM DTT, 10% glycerol (wt/vol)] and centrifuged at 100,000 g for 3 min. The final pellet was resuspended in 100 µl of Na,K-ATPase buffer and assayed immediately for Na,K-ATPase activity.
In some experiments, Lyn kinase-treated membrane material was subsequently incubated with protein tyrosine phosphatase 1B (PTP-1B) (0.5 U/µl) (Upstate Biotechnology, Lake Placid, NY) for 30 min at 37°C in PTP-1B assay buffer (25 mM HEPES, 50 mM NaCl, 5 mM DTT, 2.5 mM EDTA, 100 µg/ml bovine serum albumin, 10 µg/ml pepstatin, 10 µg/ml antipain, 10 µg/ml leupeptin, and 1 mM PMSF, pH 7.2). To remove buffer constituents, the PTP-1B-treated membrane material was resuspended once in 200 µl of centrifugation buffer and centrifuged at 100,000 g for 3 min. The final pellet was resuspended in Na,K-ATPase assay buffer and assayed immediately for Na,K-ATPase activity.
Na,K-ATPase activity was determined as described by Okafor et al. (37). Aliquots of Lyn kinase-treated and untreated epithelium membrane material (100 µg) or kidney membrane material (
25 µg) were added to Na,K-ATPase buffer (100 mM NaCl, 10 mM KCl, 3 mM MgCl2, 1 mM EGTA, pH 7.4). Ouabain, a specific inhibitor of Na,K-ATPase (46), was added to half the sample aliquots to a final concentration of 1 mM. Samples were preincubated for 15 min at 37°C with gentle agitation. ATP hydrolysis was initiated by the addition of ATP to a final concentration of 1 mM. The ATP hydrolysis reaction was carried out for 45 min at 37°C with gentle agitation. The reaction was stopped with the addition of 15% ice-cold trichloroacetic acid. ATP hydrolysis was quantified using a colorimetric method that measured the amount of inorganic phosphate released in each reaction sample (37). Less than 20% of the available substrate ATP was hydrolyzed. The difference in ATP hydrolysis in the presence and absence of ouabain was a measurement of Na,K-ATPase activity. The data are presented as nanomoles phosphate released per milligram protein per minute.
Because Na,K-ATPase activity was measured in samples of lens epithelium membrane material that had been treated with buffer containing 0.2 mM sodium orthovanadate and then washed, separate studies were conducted to confirm that Na,K-ATPase activity was not inhibited by residual vanadate. Na,K-ATPase activity was 9.7 ± 0.4 nmoles Pi·mg protein1·min1 (mean ± SE; n = 5) in vanadate-treated samples, which was not significantly different from the activity of 10.2 ± 0.6 measured in control samples.
86Rb uptake. Ouabain-sensitive 86Rb uptake by the intact lens was used as an index of Na,K-ATPase-mediated active sodium-potassium transport. Intact lenses were bathed at 37°C in Krebs solution with the following composition (in mM): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, 1 MgCl2, and 5.5 glucose at pH 7.4. It was assumed that the Na,K-ATPase mechanism transports 86Rb similarly to potassium. Lenses were preincubated for 10 min in Krebs solution containing test agents, and then 86Rb (0.1 µCi/ml) was added. Half of the lenses in each group also received 1 mM ouabain, added simultaneously with the 86Rb. The 86Rb uptake period was 30 min. Previous studies show that during this time, 86Rb uptake is linear. After the 30-min 86Rb uptake period, each lens was placed in a large volume of ice-cold nonradioactive Krebs solution for 2 min to wash 86Rb from extracellular space. After this, the lenses were weighed, lyophilized, and reweighed to determine water content. The dried tissue was digested in 30% nitric acid, and radioactivity in the acid digest was measured by scintillation counting. On the basis of the specific activity of 86Rb in the medium, the data were calculated as nanomoles potassium (86Rb) accumulated per gram of lens water per minute.
Western blot analysis. Membrane material was solubilized with Laemmli sample dilution buffer, and proteins were separated on a 7.5% gel by SDS-PAGE at 40 mA for 2 h using the Laemmli buffer system (31). Proteins were electrophoretically transferred to nitrocellulose sheets at 30 V for 16 h. The nitrocellulose membranes were blocked for 1 h with 5% dry milk in TTBS (30 mM Tris, 150 mM NaCl, 0.5% Tween-20, pH 7.4). For immunodetection of Na,K-ATPase 1, Lyn kinase, or tyrosine phosphoproteins, the nitrocellulose membranes were incubated at room temperature for 60 min with either a monoclonal antibody directed against Na,K-ATPase
1 (Sigma, St. Louis, MO), Lyn kinase (Upstate Biotechnology), or anti-phosphotyrosine antibody PY20 (Transduction Lab, Lexington, KY) conjugated to horseradish peroxidase. Nitrocellulose membranes probed for Na,K-ATPase
1 and Lyn kinase were washed with TTBS twice for 15 min and then three times for 5 min before being incubated for 60 min with a horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA). Nitrocellulose membranes probed for PY20 were washed with TTBS twice for 15 min and then four times for 5 min at room temperature and then visualized with chemiluminescence substrate (Pierce, Rockford, IL). Nitrocellulose membranes were exposed to X-ray film.
Immunoprecipitation. Following a methodology modified from a technique described by Khundmiri and Lederer (29), lens epithelium (500 µg) and kidney membrane material (200 µg) were solubilized in sufficient immunoprecipitation buffer (10 µM deoxycholate, 100 mM D-mannitol, 5 mM Tris, pH 7.6, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml antipain, and 10 µg/ml pepstatin A) to bring the final protein concentration to 2 or 0.8 µg/µl, respectively. The membrane material was mixed on a rotating wheel at 4°C for 3 h. The insoluble material was then pelleted by centrifugation at 8,000 g for 15 min at 4°C. The supernatant (250 µl) was removed then precleared with 20 µg of mouse IgG and 50 µl of immobilized protein G (ImmunoPure, Pierce, Rockford, IL) for 15 h on a rotating wheel at 4°C. The membrane material mixture was then centrifuged at 1,000 g for 3 min at 4°C. The supernatant was removed and precleared once again with rabbit IgG and 50 µl of immobilized protein A (ImmunoPure) for 3 h on a rotating wheel at 4°C. The supernatant was transferred to a fresh microcentrifuge tube, and 10 µg of polyclonal antibody directed against Na,K-ATPase 1 polypeptide (RDI, Flanders, NJ) was added. The membrane material mixture was incubated on a rotating wheel for 15 h at 4°C. After this, 50 µl of immobilized protein A (ImmunoPure) were added and mixed for an additional 3 h on a rotating wheel at 4°C. The mixture was then washed with 200 µl of PBS, pH 7.4, and then centrifuged at 1,000 g for 3 min at 4°C. The wash procedure was repeated two more times, and then immunoprecipitated Na,K-ATPase
1 polypeptide was dissociated from the protein A and antibody mixture by being incubated in 45 µl of Laemmli sample dilution buffer for 20 min at 65°C. The samples were centrifuged at 4,000 g for 5 min. The supernatant was then subjected to SDS-PAGE, followed by Western blot analysis. In some experiments, a different immunoprecipitating antibody was used. The immunoprecipitation was carried out using 2 µg of monoclonal antibody directed against tyrosine phosphoproteins (PY99) (Santa Cruz Biotechnology, Santa Cruz, CA).
In some experiments, Lyn kinase-treated membrane material was incubated with PTP-1B (100 mU/µl) for 30 min at 37°C in PTP-1B assay buffer. The Lyn-PTP-1B-treated membrane material was subjected to immunoprecipitation, followed by SDS-PAGE. Resolved proteins were analyzed for tyrosine phosphoproteins by Western blot.
Statistical analysis. Student's t-test was used for statistical analysis.
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RESULTS |
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Thrombin induces the activation of nonreceptor tyrosine kinases (11, 30, 32), including Lyn, a Src tyrosine kinase family member (10, 26). Lyn kinase is expressed in lens tissue. When lens epithelium membrane material was isolated and used for Western blot analysis, two immunopositive bands corresponding to the two known isoforms of Lyn kinase were detected (Fig. 2), although the results do not signify the degree to which Lyn is activated. To determine the effects of tyrosine kinase-mediated phosphorylation on lens epithelium membrane proteins in vitro, isolated lens epithelium membrane material was incubated with active, partially-purified Lyn kinase (0.08 units Lyn/µg membrane material) in ATP-containing kinase reaction buffer. After this, tyrosine phosphorylation and Na,K-ATPase activity were examined. Western blot analysis revealed a marked increase in several phosphotyrosine protein bands (Fig. 3). A smaller increase of phosphotyrosine band density observed in the presence of ATP but absence of added Lyn may signify the activity of endogenous Lyn and other tyrosine kinases. No increase was observed in the absence of both ATP and Lyn (data not shown). Na,K-ATPase activity was reduced significantly in lens epithelium material subjected to Lyn pretreatment (Table 1). To compare the effects of Lyn on a different tissue, Na,K-ATPase activity was also measured in membrane material isolated from kidney medulla. Lyn pretreatment was found to cause a 20% reduction of Na,K-ATPase activity measured in kidney membrane material (Table 1).
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Studies were conducted to determine whether tyrosine phosphorylation of the Na,K-ATPase 1 polypeptide occurs. Na,K-ATPase
1 protein was first isolated from lens epithelium membrane samples by immunoprecipitation using a polyclonal antibody directed against the Na,K-ATPase
1-subunit, and then the immunoprecipitated Na,K-ATPase
1 protein was subjected to treatment with Lyn kinase. The Lyn-treated immunoprecipitates were resolved by SDS-PAGE and subjected to Western blot analysis (Fig. 4). A dense, 100-kDa tyrosine phosphoprotein band was observed in Lyn-treated Na,K-ATPase
1 immunoprecipitates. The 100-kDa tyrosine phosphoprotein band was undetectable in Na,K-ATPase
1 immunoprecipitates that were not treated with Lyn. For technical reasons, Na,-K-ATPase activity could not be determined reliably in the immunoprecipitates.
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To examine the effects of Lyn on Na,K-ATPase in the nonsolubilized membrane, lens epithelium membrane material was treated first with Lyn before Na,K-ATPase 1 protein was isolated by immunoprecipitation. Western blot analysis revealed a 100-kDa phosphotyrosine band in immunoprecipitates isolated from lens epithelium membrane material that had been subjected to Lyn treatment (Fig. 5A). A similar result was observed in membrane material isolated from porcine kidney medulla, a non-lens tissue in which the major Na,K-ATPase isoform is
1 (Fig. 5B). Phosphotyrosine bands were not detectable in immunoprecipitates isolated from either lens or kidney membrane material that was not treated with Lyn.
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The results suggest Na,K-ATPase 1 polypeptide is subject to tyrosine phosphorylation by Lyn. To confirm this idea, studies were conducted to test whether Na,K-ATPase
1 protein could be immunoprecipitated from Lyn-treated lens membrane material using a monoclonal antibody directed against tyrosine phosphoproteins. A 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase
1 protein was observed in immunoprecipitates isolated from Lyn-treated lens membrane material (Fig. 6). Neither Na,K-ATPase
1 nor phosphotyrosine bands were detected in immunoprecipitates isolated from non-Lyn treated lens epithelium membrane material or control samples in which the immunoprecipitating antibody was substituted for mouse IgG.
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In some studies, lens epithelium membrane material was first treated with Lyn kinase and then subjected to tyrosine phosphatase treatment with PTP-1B. Treated membrane material was then immunoprecipitated with a polyclonal antibody directed against Na,K-ATPase 1. A 100-kDa phosphotyrosine band was observed in the Na,K-ATPase
1 immunoprecipitate obtained from Lyn kinase-treated lens epithelium membrane material but not from membrane material that had subsequently been treated with PTP-1B (Fig. 7). Na,K-ATPase activity measured in Lyn-treated lens epithelium membrane material increased from 7.1 ± 1.7 nmoles Pi·mg protein1·min1 to 12.3 ± 2.4 by subsequent PTP-1B treatment. This represents a 39.5 ± 14% increase in Na,K-ATPase activity (data as mean ± SE; n = 5; P < 0.05). In comparison, PTP-1B failed to change Na,K-ATPase activity in lens epithelium membrane material that was not pretreated with Lyn (data not shown).
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DISCUSSION |
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Na,K-ATPase 1 is the main isoform expressed in porcine lens cells, and neither Na,K-ATPase
2 or
3 isoforms are detectable by Western blot (19). Immunoprecipitation using antibodies directed against Na,K-ATPase
1 and against phosphotyrosine residues confirmed tyrosine phosphorylation of Na,K-ATPase
1 polypeptide in Lyn-treated membrane material. It was also demonstrated that Na,K-ATPase
1 polypeptide could be isolated from lens epithelium first by immunoprecipitation and then subjected to Lyn treatment to elicit tyrosine phosphorylation detectable by Western blot. Furthermore, recombinant protein tyrosine phosphatase PTP-1B was found to cause reversal of Lyn-induced tyrosine phosphorylation. Taken together, the results suggest Lyn causes tyrosine phosphorylation of Na,K-ATPase
1 polypeptide.
Exogenous Lyn was used in this study as a means of causing tyrosine phosphorylation in isolated lens epithelium membrane material. Endogenous Lyn kinase was detected in lens epithelium, but the Western blot results do not provide information on its activation state. Lyn kinase, a member of the Src-family of tyrosine kinases, is a membrane-associated nonreceptor tyrosine kinase that is also expressed in myeloid and B lymphoid hematopoietic cells (17). Lyn has also been detected in human endometrium and brain endothelium, where it is thought to play an important role in human reproduction and blood-brain barrier development, respectively (1, 13). It is also expressed in intestinal crypt cells (39). Alternative splicing of the Lyn gene results in the expression of Lyn A (56 kDa) and Lyn B (53 kDa) forms of Lyn (40). Except for a 20-amino acid deletion, Lyn B is identical to Lyn A (47). Lyn kinase is thought to be involved in signal transduction mechanisms after activation of B cell antigen receptors, FcE high-affinity receptors (25), and interleukin-3 receptors (44). Lyn kinase phosphorylates many substrates, including phosphoinositol-3 kinase (PI3-K), ras GTPase-activating protein (GAP), and mitogen-activating protein kinase (MAPK) (12).
Several studies have shown that the Na,K-ATPase 1-subunit is phosphorylated by PKC and PKA on serine-threonine residues (5, 9). However, the existence of additional phosphorylation sites was suspected because neither the deletion of the known Na,K-ATPase
1-subunit serine-threonine sites nor treatment with PKC or PKC inhibitors was able to fully suppress residual levels of Na,K-ATPase
1 phosphorylation (4). This fits with the notion that Na,K-ATPase
1 can be subject to tyrosine phosphorylation. To examine a possible tyrosine phosphorylation site, Feraille et al. (22) analyzed Na,K-ATPase pump activity in opossum kidney (OK) cells transfected with mutant Na,K-ATPase
1 in which tyrosine-10 was substituted either by alanine or glutamate. Insulin-induced stimulation of Na,K-ATPase function was suppressed in cells expressing the Tyr-10 substitutions. Consistent with the idea of phosphorylation of the Na,K-ATPase
1-subunit at Tyr-10 in the presence of a Src-family tyrosine kinase, this region of the Na,K-ATPase
1 protein exhibits a Src kinase consensus phosphorylation sequence composed of multiple acidic residues (50). Interestingly, the gastric H+,K+-ATPase proton pump is also subject to phosphorylation at Tyr-10 (43).
Lyn kinase-induced inhibition of Na,K-ATPase activity in isolated, partially purified lens epithelium and kidney medulla membrane material observed in the present study differs from the response to insulin and other agonists in intact astrocytes, proximal tubule, and skeletal muscle in which tyrosine phosphorylation is associated with stimulation of Na,K-ATPase function (8, 21, 22, 34, 36). This may reflect differences in the cascade of events triggered by insulin and partially purified Lyn, differences in the response of intact cells in which changes in Na,K-ATPase synthesis or recruitment to the plasma membrane may occur (2, 21, 34, 36), differences in cell-specific regulatory mechanisms, or differences in Na,K-ATPase 1 isoform characteristics. In the porcine lens, the Na,K-ATPase
1 isoform is predominant, although long-term changes in Na,K-ATPase activity might occur through the upregulation of the
2-subunit in response to alteration of cytoplasmic ion balance (19).
The lens epithelium is specialized for active sodium-potassium transport. Na,K-ATPase-mediated ion transport by the epithelial monolayer is essential for maintenance of electrolyte homeostasis in the mass fiber cells that constitute the bulk of the lens (33). The results of the present study suggest that changes in the activity of Lyn or other tyrosine kinases could lead to modulation of Na,K-ATPase function in lens epithelium. Modulation of Na,K-ATPase activity as the result of Lyn kinase activation has not previously been reported, and although there is strong evidence from several different cell types indicating the susceptibility of Na,K-ATPase to tyrosine phosphorylation, the identity of the tyrosine kinases that influence Na,K-ATPase in intact tissues is not known (21, 38, 48). In gastric mucosa, there is evidence suggesting that plasma membrane H+,K+-ATPase is subject to tyrosine phosphorylation (27), and chromatographic separation of detergent-solubilized membrane material revealed an endogenous Src-family kinase at 60 kDa (28). Src family kinases are known to influence other ion transporters. In platelets, for example, phosphorylation of plasma membranes by pp60Src kinase resulted in significant inhibition of calcium ATPase activity that correlated with the degree of PMCA tyrosine phosphorylation (15). In mouse erythrocytes, activation of Src family tyrosine kinases appears to modify K+-Cl cotransporter function (14). In the lens, Lyn kinase is likely to be just one of several nonreceptor tyrosine kinases, and it is possible that other tyrosine kinases also influence Na,K-ATPase activity. The present experiments did not permit us to identify the tyrosine kinases activated by thrombin.
Several studies have identified Src family tyrosine kinases in the lens. It has been suggested that nonreceptor tyrosine kinases play an essential role in differentiation with inhibition of Src family tyrosine kinases acting as one of the events required for lens epithelial cells to withdraw from the cell cycle and commence differentiation toward the lens fiber cell phenotype (45). When the intact lens is maintained in organ culture, inhibition of Src family tyrosine kinases with PP1 appears to prevent opacification (49).
In summary, the results of the present study suggest that Na,K-ATPase activity in lens epithelium is susceptible to modulation by tyrosine phosphorylation. The significance of modulating Na,K-ATPase activity in lens epithelium remains to be determined. It has been proposed that spatial localization of high-Na,K-ATPase activity to specific regions of the lens surface is essential to support circulation of electrical currents that work via electroosmosis to speed solute movement through the tortuous extracellular space between the tightly packed lens cells (33). Although Na,K-ATPase protein is abundant in all lens cells (16, 18), Na,K-ATPase activity is higher at the epithelium than the fibers and highest in epithelium at the equator of the lens (7, 24, 41). To establish the circulating currents, there may be a need for mechanisms that modulate Na,K-ATPase activity to produce unequal activity in different parts of the lens.
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ACKNOWLEDGMENTS |
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This work was supported by National Eye Institute Grant EY-09532, a Senior Scientific Award from Research to Prevent Blindness (NAD), and the Ky Lions Eye Foundation.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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