Exp. Eye Res. (1992) 54, 321-328

Na,K-ATPase MARGARET

of Cultured Bovine H,O, Effects H. GARNER”“, REN-RONG

AFSHIN WANG6~~~

Lens Epithelial

BAHADOR”, ABRAHAM

BAO-THU THI SPECTOR*

Cells: NGUYEN”,

aDepartment of Ophthalmology, University of California, Irvine, CA 92715 and b Biochemistry and Molecular Biology Laboratory, Department of Ophthalamolog y, Co//ege of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A. (Received

Bethesda

31 July

7990 and accepted

in revised

form 18 April

1991)

Na,K-ATPase function was studied in cultured bovine lens epithelial cells under confluent and nonconfluent conditions. The afEn@ of the Na,K-ATPase for the cardiac glycoside, ouabain, ditrers between the confluent and non-confluent cultures. The confluent cells have a higher ffiity for ouabain than do the non-confluent cells. The ouabain affinity of the confluent cells is similar to that for the Na,K-ATPase isolated from the bovine axolemma and the bovine lens cortex. The ouabain atsnity of the non-confluent cells is similar to that for the Na,K-ATPase of the renal medulla and bovine lens epithelium. Similar results are not found with confluent and non-confluent h4DCK cells, H,O, treatment of confluent and nonconfluent lens epithelial cell cultures has differing effects on the Na,K-ATPase function. In the confluent cell preparations, H,O, affects K+-dependent dephosphorylation of the intermediate phosphoenzyme. In the non-confluent preparations, H,O, appears to inhibit K+-occlusion. Key words : lens ; Na,K-ATPase ; hydrogen peroxide ; ouabain afIlnity.

1. Introduction Altered Na+ and K+ concentrations are observed in many human cataract lenses and have been correlated with increasing lens color and with cortical opaciflcation (Mercantonio et al., 1980; Marlat, Baracchi and Maraini, 1981; Pasino and Maraini, 1982 ; Maraini and Pasino, 1983). The change in monovalent cation concentrations may in part be attributed to decreased efficiency of the sodium pump (Na,K-ATPase). Na,KATPase is an intrinsic membrane enzyme which transports 2K+ into the cell and 3Na+ out of the cell, both against steep gradients; the energy for the process is supplied by the hydrolysis of ATP (for review see Skou, 1988). The steady-state hydrolysis of near saturating concentrations of ATP by lens Na,K-ATPase (V&J decreases with increasing severity of human cataract (Kobayashi, Roy and Spector, 1983). The K, for ATP appears to be altered in 2 7% of the senile cataracts studied when compared to clear lenses (Garner and Spector, 1986). K+ influx, measured using ssRb+ as a tracer, also decreases (Maraini and Pasino, 1983). However, the decreased K+ transport does not always correlate directly with decreasing V,, for ATP hydrolysis. Since there is increased oxidation of lens methionine and cysteine (Garner and Spector, 1980) with the development of cataract, and since there appear to be higher than normal concentrations of H,O, (Spector and Garner, 1981) in the aqueous * Current address: Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas. 5323 Harry Hines Blvd, Dallas, TX 75235-8592, 0014-4835/92/030321+08 21

U.S.A. $03.00/O

humor of some cataract patients, many studies have been initiated to examine the effect of H,O, on Na,KATPase function. Results using lens organ culture models suggest that H,O, can alter Na,K-ATPase function. In bovine lenses treated for 1 hr with 1 mM H,O,, K+ transport appears to be completely uncoupled from ATP hydrolysis (Garner, Garner and Spector, 1983). At lower concentrations of H,O,, partial uncoupling is observed. Although V,,, for ATP hydrolysis is the same as the control, the steady-state kinetics of ATP hydrolysis shift from negative co-operativity to positive co-operativity (Garner, Garner and Spector, 1984). Positively co-operative ATP hydrolysis has been observed in 10% of a senile cataract population (Garner and Spector, 1986). The change in pump efficiency in the bovine lens model system would appear to be linked to specific K+ stimulatory sites on the enzyme (Garner, Garner and Spector, 1986). Incubation of rabbit lenses (Delamere et al., 1988) in 0.5 mu H,O, for 1 hr leads to a 50% decrease in K+ transport. Once the H,O, is removed the K+ transport continues to decline over the next 20 hr. There is a concomitant increase in intracellular Na+ from 15 to 35m~andadecreaseinK+from118to105m~.V,, (ATP hydrolysis at near saturating ATP concentrations) is normal, a result similar to that reported for the bovine lens. There is conflicting evidence as to the ability of lens glutathione in the rabbit model system to prevent the oxidative damage to Na,K-ATPase (Delamere et al., 1988; Reddy et al., 1988). Studies using cultured lens epithelial cells yield results diametrically opposed to those reported for lens 0 1992 AcademicPressLimited EER 54

M. Ii. GARNER

322

organ culture. Although H,O, treatment causes a loss of glutathione (Spector, Huang and Wang, 1985) and DNA damage (Kleinman, Wang and Spector, 1990), K+ transport, as measured using *6Rb+ as a tracer is actually stimulated (Spector et al., 1987). A similar result was reported when rabbit lenses were treated with 0.080 mM H,O, (Delamere et al., 1988). Increasing glutathione levels (above normal), in the cultured bovine lens epithelium model, causes increased pump inhibition (Spector et al., 1987). Since the results using the lens organ culture model differ from those using the cultured epithelial cell model, experiments were designed to define more clearly the effects of H,O, on Na,K-ATPase function in cultured epithelial cells. Ouabain binding, phosphoenzyme formation, and K+-occlusion are reported for confluent and non-confluent cells. The effects of H,O, on these functions of Na,K-ATPase differ depending upon whether or not the cells are confluent. 2. Materials and Methods

Bovine lenses were provided by the Great American Veal Company, Newark, NJ and Marx Brothers Meats, Inc., Shrewsbury, NJ, within 2 hr of death. ATP (disodium salt) was obtained from Boehringer Mannheim, ATP (tris salt) was obtained from Sigma (St Louis, MO). 86RbC1 (484 mCi mmol-l), [3H]ouabain (18 Ci mmol-‘), and Y~~P-ATP (6000 Ci mmol-‘) were obtained from Amersham. Madin-Darby canine kidney (MDCK) cells were obtained from American Type Culture Collection. Cell Culture and Treatment with H,O, MDCK cells were propagated in minimum essential medium (Eagle) with Earle’s BSS containing 10% fetal bovine serum. Lens epithelial cells were obtained from first- and second-passage monolayer cultures derived from bovine lens epithelium explants. These cultures were prepared from young calf lenses and maintained as described previously (Spector et al., 1985, 1987) in loo-mm culture plates, with 5 ml of media per plate. The 2 x lo6 lens epithelial cells were initially obtained from confluent plates and consequently plated out so as to be confluent or non-confluent. These cells were passaged by a brief treatment with trypsin to detach the cells from the plate. Then, soybean trypsin inhibitor was immediately added to prevent proteolytic cleavage by trypsin. The cells were given 24 hr to recover before treatment with H,O,. The lens epithelial cells, 2 x lo6 cells, either confluent or non-confluent (24 hr after plating) were treated in EMEM medium containing O-2 mM H,O, in the absence of fetal bovine serum for 5 min. The ratio of H,O,: cells was the same in confluent and nonconfluent plates. After 5 min, catalase was added to destroy the H,O,. Cells were directly lysed (after removal of the culture

ET AL.

medium) in situ in the culture plates in 2.5-3.0 ml of 30 mM imidazole buffer, pH 7.4, containing 1 mM EDTA, 0.05 mM PMSF and 0.0625 % sodium deoxycholate. Consequently, the cells were removed from the plates first by gentle agitation, and then, by scraping with a rubber policeman. Detachment was complete in 10 min ; complete lysis was accomplished by two freeze/thaw cycles or by homogenization using a Dounce homogenizer. The homogenate was incubated an additional 20 min to ensure adequate detergent activation of the Na,K-ATPase (Jorgensen, 1988). Isolation of Na,K-ATPase Rich Fractions from Tissue A microsomal fraction was isolated from lens epithelium, lens cortex, renal medulla and brain axolemma by differential centrifugation of tissue homogenized in 25 mM imidazole buffer, pH 7.2, containing 250 mM sucrose, 1 mM EDTA, 0.5 mM phenylmethanesulfonyi fluoride (PMSF), 0.8 mM benzamidine, 2 ,u~ leupeptin (Jorgensen, 1988). Where sufficient tissue was available the microsomes were further purified by sucrose density gradient centrifugation following detergent activation using SDS or sodium deoxycholate (Garner et al., 1983 ; Jorgensen, 1988). Ouabain Binding Ouabain binding (Wallick and Schwartz, 1988) was measured in 50 mM Tris (TRIZMA base) buffer, pH 7.2 (pH adjusted with phosphoric acid), containing 5 mM MgCl,. Ouabain concentrations were varied from lOms to 10m3 M with [3H]ouabain (1 x 10s dpm) as a tracer. For a typical assay, 100 ~1 of the detergent activated cell or tissue preparation was added to 900 ~1 of the appropriate ouabain solution containing the radiolabel and incubated at room temperature for 30 min. After removing 100 ~1 of the mixture for total medium cpm, 800 ,ul were loaded on a millipore fllter (type HA, 0.45 PM) ; the filter was rinsed with 12 ml of cold 50 mM Tris buffer. Each filter was placed in a scintillation vial, clarified in 10 ml of liquid scintillation cocktail (Readi-protein, Beckman or Liquistint, National Diagnostics, Mannville, NJ) and counted. Counts were obtained using a LKB 1217 Rackbeta counter or Beckman LS5000TD counter. Moles ouabain bound were obtained by multiplying one-eighth of the filter :medium ratio by the appropriate ouabain concentration per ml. Ouabain Inhibition of Na,K-ATPase Activity ATP hydrolysis was measured in 30 mM histidine buffer, pH 7.4, containing 130 mM NaCl, 20 mM KCl, 6 mM MgCl, and 5 mM ATP in the presence of varying concentrations of ouabain. Phosphate release was measured directly (Garner and Spector, 1987)) or

H,D,

323

indirectly using the coupled assay described previously (Garner, Garner and Spector, 1984).

1Om8 to 10m4 molar. In all cases, saturation was achieved by 10m5 M ouabain. Each preparation appeared to bind comparable amounts of ouabain at saturation (Fig. l), 54 + 11 pmol per 2 million cells, which is the mean+s.~. of all the different values in Fig. 1. This indicates that there are roughly 18 x lo6 pump molecules per cell in the confluent and nonconfluent cultures, a value which is an order of magnitude greater than the value obtained for the fresh bovine lens epithelium. Fresh bovine lens epithelium binds 3.7 & 02 pmol ouabain per 2 million cells. In culture there is an apparent increase in the number of pump molecules per cell to a value similar to that observed in dispersed cells of the duck salt gland (Hootman and Ernst, 1988). The cultured lens epithelial cell preparations differed in the values for SO% saturation. Plotted in Fig. 2 is fractional saturation (bound/bound,,,,) vs. ouabain concentration for the control preparations of confluent second-passage cells (m-m), non-confluent firstpassage cells (0-O) and for the non-confluent second-passage cells (@.s..@). The constants obtained

CULTURED

LENS

EPITHELIUM

Na-PUMP

AND

Phosphoenzyme Formation Phosphoenzyme formation from ATP (Post, Toda and Rogers, 1975) was measured in 2 5 mM imidazole buffer, pH 74, containing 5 mM MgCl,, 1 SO mM NaCl and 100 PM [32P]ATP (specific activity, 2 yCi ml-l). Measurements in the same buffer containing 150 mM KC1 instead of NaCl were used as blanks. In a typical assay, 100 ,ul of the lysed cell preparation were added to 9 10 ,~l of the appropriate assay buffer and incubated for 15 set at room temperature. Then 50 ~1 of a solution of SO% TCA in 1 M phosphoric acid were added to each tube. After removing lOO$ for the determination of medium counts, 800 ~1 aliquots were placed on Millipore filters (0.45 pm, type HA), and the filters were rinsed with 10 ml of a cold solution of 5 % TCA in 0.1 M phosphoric acid. The filters were transferred to vials: 5 ml of Liquiscint were added to clarify the filters before counting. To calculate the amount of phosphate bound, one-eighth the filter: medium ratio was multiplied by the concentration of ATP per ml.

from fits of the data to a single site ligand binding equation are collected in Table I. The I&,, for the nonconfluent cells is three to four times greater than the

constant for the confluent cells. Hill plots of the data K+-occlusion

for the three preparations are linear with slopesnear 1. This indicates the appropriateness of the single site

K+ occlusion was determined as described previously (Shani-Sekler et al., 1988 ; Garner, Bahador and Sachs, 1990). For a typical assay, 100 ~1 of the lysed cell preparation were mixed with 200 ,ul of Tris buffer, pH 7.2, containing 4 mM KC1 and 6 mM MgCl, with 1 ,&i of s6RbC1 as a tracer in the presence or absence of 6 mM ATP. Bound K+ was separated from free K+ by cation exchange chromatography. Some of the sample (2 SO ~1) was placed on a 2-cm3 column of Dowex 50 x 2 (Tris form, pre-equilibrated with albumin and 0.200 M sucrose). The column was eluted with 2 ml of 0.200 M sucrose. The eluent was collected in scintillation vials and counted using Cerenkov counting. Ten microliters of the original mixture were placed in vials with water for determination of the total counts. The amount bound was computed by multiplying onetwenty-fifth of the effluent: total ratio by the concentration of K+ ml-’ assay. To calculate K+ occluded, the mol K+ bound per mol Na,K-ATPase in presence of ATP is subtracted from the moles bound in the absence of ATP. This definition assumes that ATPdependent deocclusion of the K+-occluded state is normal. ,I 9

ligand binding model. While the Hill plots clearly demonstrate a difference between the confluent cells

3. Results

3L

To describe adequately changes in function, a method was needed to determine total Na,K-ATPase in the cell preparation. For this purpose, ouabain binding was measured for each control and H,O,-treated preparation. Ouabain concentrations were varied from

and the two non-confluent cell preparations, there is no significant difference in ouabain binding between the non-confluent first-passage and non-confluent second-passagepreparations.

I

I

Confluent

FIG.

2 x lo6

First possoge non-confluent

Second possoge non-confluent

1. Plotsshowingthe picomolesof ouabainboundper cells for control

(C). and H,O,-treated

(E) in

contluent, non-confluent first-passage and non-confluent second-passage cultures of bovine lens epitbelium. Since ouabain binds to Na,K-ATPasein a 1: 1 ratio, the plots indicatethat eachpreparationhad roughly the samenumber of pump moleculesper cell. The resultsare means+ S.D. of three cWerentexperiments(n = 3). 21-2

324

M. H. GARNER

ET AL

Log hobainl

0.0 x IO”

I

I

I

I

I

2.0 Y lo-

4.0 x o6

6-O x 1O-6

6.0 x OS

I.0 x lo-5

IOuabainlChn

A

1

FIG. 2. Plots showing fractional saturation (bound/bound,,,,) vs. ouabain concentration for the control preparationsof confluent second-passage (a-m), non-confluentfirst-passage (O-O), and non-confluentsecond-passage (@....a) culturesof bovine lensepithelium.Eachpreparationcontainedapproximately2 x lo8 cells.The plotsshowthe relative afkity’ (K,,) of the Na pump for ouabainin each cell preparation.The valuesare listedin TableI. The resultsare means+s.~.of three different experiments(n = 3). The insert is a Hill plot of the samedata. Y-representsfractional saturation. In all cases,the slopeof the Hill plot is 1: the R values are greaterthan 095, indicating a reasonablefit.

TABLE I

K,, valuesfor ouabain binding Cell type/tissue source

KS0value @M)

Co&rent monolayer, flrst passage (bovine lens) Confluentmonolayer, fourth passage, (bovine lens) Non-con&rent cells,ilrst passage (bovine lens) Non-confluent cells,secondpassage (bovine lens) Bovine axolemma (tissuepreparation) Bovine renal medulla (tissuepreparation) Bovine lensepithelium (tissuepreparation central epithelium) Superikial bovine lenscortex (tissuepreparation) Confluentmonolayer MDCK, passage55 and 57 (dog) Non-confluentMDCK. passage55 and 57

0.3 f 0.1 0.2 f0.1 (0.2 fo.l)* 1.2fO.l

(l.o+Ol)*

1.1kO.l (l-2+01)* 04kO.l 0.9 f0.1 l.Of0.2 0.4kO.2 38.0 + 5.0 0.08 + 0.01

* Numbers in parentheses represent those values obtained for cells treated with H,O,. For the bovine lens, the results are means + S.D. of three different experiments (n = 3).

The apparent difference in ouabain affinity between preparations is similar to that observed (Table I) between the Na,K-ATPase of the bovine renal medulla and the bovine brain (axolennna) or between the lens epithelium and superficial lens cortex. H,O, had no apparent effect on the K,, values for ouabain binding (values in parentheses, Table I). To determine the possibility that the difference in KhO values for ouabain binding to confluent and non-confluent cultures was unique to lens cells, ouabain binding to confluent and nonconfluent and non-confluent

confluent cultures of MDCK cells was measured. The results, listed in Table I, differ significantly from those obtained for the cultured bovine lens epithelium. The non-confluent MDCK cells display a very high affinity for ouabain ; the confluent cells display a very low aikity for ouabain. These differences may reflect the different origins of the cultured cells, i.e. species and tissue. The remaining experiments were designed to define the differential effects of H,O, on Na,K-ATPase function in confluent and non-confluent cultured

CULTURED

LENS

EPITHELIUM

Na-PUMP

ADP

AND

325

H,O,

K

Mg2+ ATP No-occlusion

FIG. 3. Schematicrepresentationof the catalytic cycle of Na,K-ATPasewhere El and E2 representthe Na+ and K+ conformations, respectively: ElP and E2P are the two phosphoenzymeintermediates.

bovine lens epithelial cell preparations. To facilitate

further description of the results of these studies, normal Na,K-ATPase function will be reviewed. In order to transport successfully 2K+ ions into the cell and 3Na+ ions out of the cell, there are three requirements: (1) energy for the transport must be supplied by ATP hydrolysis ; (2) the monovalent cation sites must change from facing the intacellular side of the membrane to facing the extracellular side of the membrane and return to the intracellular side during one cycle ; and (3) the atsnity of the monovalent cation sites must change as the sidedness changes. These requirements are demonstrated schematically in Fig. 3. Na,K-ATPase exists ln two conformations. In the El conformation, the monovalent cation sites face the intracellular side of the membrane and have a higher tinity for Na+ than K+ even though K+ concentrations are higher in the intracellular compartment. Mg2+ATP binds to the El conformation and is hydrolysed (step 1). During the hydrolysis, the y TABLE

phosphate of ATP is transferred to an enzyme aspartic acid to form the ElP phosphoenzyme (Post et al., 1975). As this occurs, the conformation begins to change ; Na+ is occluded and cannot be released from either side of the membrane. Once the enzyme completes the conformational change to the E2 state, the monovalent cation sites face the extracellular side of the membrane and have a higher aillnity for K+ than Na+ even though Na+ concentrations are higher than K+ concentrations on this side of the membrane. K+ replaces Na+, and, as the aspartyl phosphate bond is hydrolysed, the conformation again begins to change. K+ is occluded and cannot be released from either side of the membrane. The binding of ATP to a second low afllnlty site on the 82 conformation causes the occluded state to convert to the El conformation. K’ is replaced by Na+ and the system is poised for another cycle. First, the effect of H,O, on steps 1 and 2 of the cycle, the formation of the phosphoenzyme in the presence of Na+ and K+, was determined. Under normal conditions, phosphorylation to ElP occurs in the presence of a high concentration of Na+ and dephosphorylation occurs in the presence of a high concentration of K+. Listed in Table II are the values obtained for control and H,O,-treated preparations. In all cases, except the H,O,-treated con&rent cells, the picomoles of phosphate bound in NaCl medium are greater than the picomoles bound in the KC1 medium. The difference represents the phosphorylated Na,K-ATPase and approximately 1 mol of Pi is bound mol-’ Na,KATPase (as determined by ouabain binding). In the case of the H,O,-treated confluent cells, similar amounts of phosphate bind in both KC1 and NaCl, and the amount bound is similar to that for the confluent control cells in the presence of NaCl. These data suggest that in no instance with non-confluent cells does H,O, prevent the formation of the phosphoenzyme intermediate, that is ATP can still be hydrolysed. However, treatment of confluent cells with H,O, yields an enzyme that forms a stable phosphoenzyme II

Effects of H,O, on phosphoenzyme formation

Confluent ceils secondpassage

NaCl (150 mM) KC1 (150 mhi) Na,K-ATPase (NaCl-KCl)

Control

H202

4.2 f0.3*

4.8 f 0.3*

1.0*0.1*

4.5 f0.51

3.2 f 0.3’ (1*03)-t

-0.2+0.8* m

Dividing cells

Dividing cells

secondpassage Control

fkst passage

H, 0,

Control

JW,

2.8 f02*

3.2+ 0.4*

2.9 f 0.2

2.2 + 0.3

0*8kO.l*

1.0+0.1*

0.9 kO.1

1.1+0.1

1.9 f 0,4* (0.9)$

2.2 +05* uvt

1.0+0.3* (1~2Yt

1.0 f 0.4

The results are means f S.D. of three different experiments. * Units are pmol of phosphoenzyme per 100pl of lysedcellsuspension. t Values in parentheses are the moles of phosphoenzyme formed per mol Na,K-ATPase. by ouabain binding.

The Na.K-ATPase

(1.3H

concentration

was determined

M. H. GARNER

Confluent

First possage non-conf bent

Second passage non-mfluent

FIG. 4. Plots showing mol K+ occluded mol-’ Na,K-ATPase for control and experimental (H,O,-treated) in confluent, first-passage and second-passagepreparations of bovine lens epithelium. Occluded K+ (,,Rb+ used as a tracer) was separated from free K+ by the column method. The results are means & S.D. of three different experiments (n = 3).

Intermediate even in the presence of KCl, MgCl, and ATP. This means that step 2 is either inhibited or that the conditions for dephosphorylation are dramatically different for the Na,K-ATPase of the H,O,-treated confluent cells. Next, the effect of H,O, on steps 3 and 4 of the cycle, the ability of the enzyme to occlude K+ and the ability of ATP to unload the K+-occluded state, was determined. For Na,K-ATPase, in the presence of low concentrations of ATP, step 4 becomes rate limiting. Therefore, in the absence of ATP, K+ (*,Rb+ as a tracer) binds tightly and the K+-occluded Na,K-ATPase complex can be isolated, while similar experiments in the presence of mM concentrations of ATP result in an enzyme free of bound K+. The results of the occlusion assays are shown graphically in Fig. 4. In the nonconfluent first- and second-passage control preparations, approximately 2 mol K+ are occluded mol-’ Na,K-ATPase (as determined by ouabain binding). H,O, caused a 92 and 50% inhibition of occlusion in the non-confluent first- and second-passage cell preparations, respectively. There was little evidence of K+occlusion for either the control or H,O,-treated confluent cell preparations. Therefore, in the coniluent monolayer, either ATP dependent deocclusion (step 4) is no longer rate limiting under the conditions used for the assay, or ATP no longer causes deocclusion of the K+-occluded state (step 4 is inhibited). In either case K+ transport would be expected to be altered. 4. Discussion H,O, would appear to alter Na,K-ATPase function in both confluent and non-confluent (actively growing) cultured bovine lens epithelial cells. In nonconfluent cells, the major effect is the apparent

ET AL.

inhibition of Na,K-ATPase-dependent K’ occlusion. What the effects are on K+ transport in non-confluent cells is unclear at the present time. For a related pump, the H,K-ATPase of the parietal cell, K+-occlusion cannot be measured because there is an increase in the rate of conversion of E2K to ElK in the absence of ATP (Lorentzon, Sachs and Wallmark, 1988). The H,K-ATPase still pumps a K+ gradient (Wallmark et al. (1987). In a previous study (Spector et al., 1987) treatment of bovine lens cells (24 hr after replating from a confluent culture) in culture under similar conditions to those used for the present study led to an apparent stimulation of Na,K-ATPase-dependent K+ transport. Further study is needed to determine whether H,O, inhibition of K+-occlusion indicates an inhibition of Na,K-ATPase-dependent K+ transport. It is interesting to note that there is no apparent K+occlusion in either the control or H,O,-treated preparations from confluent cells. In confluent cells, the major effect of H,O, is on the stability of the phosphoenzyme in the presence of KCl. This change would be expected to cause partial or complete inhibition of K+ transport. Step 2 of the mechanism is rate limiting under normal conditions where ATP concentrations are in the millimolar range ; K+ dependent dephosphorylation step 3 is usually rapid. After H,O, treatment of the confluent lens epithelial cells, the phosphoenzyme is stable even in the presence of K+, indicating either that step 3 is inhibited completely or rate limiting. It is interesting to note that K+-occlusion cannot be detected in preparations from either control or H,O,-treated confluent lens epithelial cell cultures. These differences in H,O, modification of Na,KATPase function between confluent and nonconfluent cultured lens epithelial cells may be related to the observed differences in ouabain affinity. Those cells with low affinity for ouabain, the non-confluent cells, are those where the most apparent H,O, effect is inhibition of K+-occlusion. Those cells with higher ouabain affinity, the confluent cells, show inhibition of K+-dependent dephosphorylation of the phosphoenzyme intermediate. The observed difference in ouabain affinity (for review see Sweadner, 1989) may indicate that the two cell types, confluent and nonconfluent, express different catalytic subunits. Two catalytic subunits have been observed by SDS-PAGE separation of chick lens preparations (Takemoto, Hansen and Hokm, 1981, 1982). In mammals there are at least three genes for the catalytic subunit of Na,K-ATPase, a-1~~2, and a3 (Shull, Schwartz and Lingrel, 1985 ; Kawakami et al., 1986 ; Ovchinnikov et al., 1986, 1987, 1988; Shull andLingre1, 1987). In rat, a2 and a3 (Hsu and Guidotti, 1989) are found in high concentration in the axolemma ; the axolemma preparation has a high affinity for ouabain (Sweadner, 1979). In rat renal medulla, the al (Orlowski and Lingrel, 19 8 8) subunit is found in high concentration ; the renal medulla Na,K-ATPase has a lower ouabain

CULTURED

LENS

EPITHELIUM

Na-PUMP

AND

327

H,O,

affinity (Urayama and Nakao, 1979). Similar difierences in ouabain al%nity have been reported for rabbit brain and kidney (Skou, 1962). In the studies reported here, bovine brain Na,K-ATPase has a higher ouabain affinity than bovine kidney Na,K-ATPase. The difference between brain and kidney ouabain aflinity is similar to the difference observed between lens cortex and lens epithelium, or between confluent and nonconfluent cultured lens epithelium. However, differences in ouabain affinity have also been attributed to EDTA treatment of the microsomes as in MOPC plasmacytoma cells (Lelievre et al., 1985) oncogene transformation (Tagliaferri et al., 1987) or mutation of a cell line to express a small protein which alters the ouabain affinity of Na,K-ATPase (Schultz and Cantley, 1988). Biphasic ouabain aiilnity was reported previously for rabbit lens preparations (Neville, Paterson and Hamilton, 1978). In guinea-pig (McDonough, 1985) and pig (Sen and Pfeiffer, 1982) lenses the low al%nity component appears to be predominant. Obviously, further studies are needed to determine which catalytic subunits of Na,K-ATPase are expressed in the lens and in cultured lens epithelial cells. The apparent conflicting results on the effects of H,O, on Na,K-ATPase function may actually reflect differences in the Na,K-ATPase population in the model system of choice. Acknowledgements We wish to thank Drs H. Enomoto, W. H. Garner, and R. Chiesa for advice. We wish to thank J. Yamamoto, J. Davis and’ R. Chiesa for technical assistance. This project was funded by grants from the National Institutes of Health, EYO7010 (M.H.G.) and other National Eye Institute grants (A.S.).

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