Exp. Eye Res. (2000) 71, 405±413doi:10.1006/exer.2000.0896, available online at http://www.idealibrary.com on

Fatty Acid Cytotoxicity to Bovine Lens Epithelial Cells: Investigationson Cell Viability, ecto-ATPase, Na�, K�-ATPase and Intracellular

Sodium Concentrations{

NGA NGUYEN, DAGOBERT GLANZ AND DIETMAR GLAESSER*

Institut fuÈ r Physiologische Chemie, Medizinische FakultaÈt, Martin-Luther-UniversitaÈt Halle-Wittenberg,Halle (Saale), Germany

(Rece 000)

cytotoxic(Glaesser

0014-483

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* AddressInstitut fuÈ rLuther-UnivGermany.

ived 18 February 2000, accepted in revised form 11 July 2000 and published electronically 29 August 2

Unsaturated non-esteri®ed fatty acids have been shown to be cytotoxic in micromolar concentrations tobovine lens epithelial cells, in the following order: arachidonic acid 4 linoleic acid 4 oleic acid � lino-lenic acid. As unsaturated free fatty acids are known to be Na�, K�-ATPase inhibitors, the aim of the studywas to investigate whether or not the fatty acid cytotoxicity is correlated with effects on Na�, K�-ATPaseactivity and function in bovine lens epithelial cells. Furthermore, we also examined the effects of linoleicacid on an ecto-ATPase activity which could be demonstrated on the outside of primarily cultured bovinelens epithelial cells. It has already been shown that 10 mmol lÿ1 linoleic acid was cytotoxic but did notimpair the ecto-ATPase activity of intact cells nor the Na�, K�-ATPase in enriched membrane fractions.Na�, K�-ATPase activity was slightly activated with 10 mmol lÿ1 linoleic acid and inhibited by about 50 %with 100 mmol lÿ1. Using the sodium-binding benzofuran isophthalate, measurements of intracellularsodium concentrations were carried out. In serum-starved bovine lens epithelial cells the basal [Na�]inwas clearly lower than 5 mmol lÿ1. When the function of the Na�, K�-ATPase was interrupted byomitting K�-ions from the medium, [Na�]in increased at a rate of 0.318 mmol lÿ1 minÿ1. Linoleic acidintensi®ed that increase strongly in a concentration dependent manner. However, in K�-containingmedium the linoleic acid-induced increase of [Na�]in was completely prevented. Therefore, the highlinoleioc acid cytotoxicity cannot be mediated by linoleic acid effects on Na�, K�-ATPase activity and

#

function in bovine lens epithelial cells. 2000 Academic PressKey words: lens epithelial cells; linoleic acid cytotoxicity; intracellular sodium concentration; Na�,

K�-ATPase; ecto-ATPase.

1. Introduction

From the early studies of Bonting, Simon andHawkins (1961) up to now (Delamere et al., 1997;Garner and Kong, 1999), much attention has beenpaid to the Na�, K�-ATPase of eye lens epithelial cellsas a possible target for cataractogenic risk factors.Cortical opacities in human lenses are regularlyaccompanied by elevated contents of sodium anddecreased contents of potassium (Duncan andBushell, 1975). H2O2 produces both opaci®cationand inhibition of Na�, K�-ATPase activity in organcultured lenses from cattle (Garner, Garner andSpector, 1983), rabbits (Giblin, McCredy and Reddy,1982) and rats (Fukui, 1976). Cation transport andNa�, K�-ATPase activity progressively decline inselenite-treated rabbit lenses (Hightower andMcCredy, 1994). cis-Linoleic acid as well as othercis-unsaturated free fatty acids have been shown to be

to bovine lens epithelial cells in cultureet al., 1996; Glanz et al., 1997). So far the

5/00/100405�09 $35.00/0

rk was supported by Grant IIIGK-GRK 134/1 from therschungsgemeinschaft.

correspondence to: Professor Dr Dietmar Glaesser,Physiologische Chemie, Medizinische FakultaÈt, Martin-ersitaÈt Halle-Wittenberg, D-06097 Halle (Saale),

mechanism is unknown, but from lipidperoxidationexperiments we have concluded that lipidperoxida-tion products are not involved. First, lipidper-oxidation products formed by autoxidation of linoleicacid were less cytotoxic than linoleic acid itself.Second, the content of protein bound sulphydrylgroups and the level of reduced glutathione were notaffected, indicating that the antioxidative system ofthe lens is not occupied by cytotoxic concentrationsof linoleic acid (Glaesser et al., 1996; Fass andGlaesser, 1997). On the other hand, cell damage iscorrelated with cellular uptake rates of the fatty acidmolecules themselves (Trimborn et al., 2000). Swarts,Schuurmanns Stekhoven and De Pont (1990) havereported on the binding of unsaturated free fatty acidsto Na�, K�-ATPase preparations leading to theinactivation of the enzyme. Furthermore, linoleicacid and oleic acid have been identi®ed as endogenousNa�, K�-ATPase inhibitors in volume-expanded hogplasma (Tamura et al., 1985), and in bovine blood(Tal et al., 1989) as well as in patients suffering fromessential hypertension (Kelly et al., 1986). Na�, K�-ATPase inhibitors were found which proved to be

non-esteri®ed fatty acids. Therefore, our investigationswere primarily designed to examine whether or not a

# 2000 Academic Press

counted. The whole procedure was carried out in a

correlation exists between linoleic acid cytotoxicityand linoleic acid effects on Na�, K�-ATPase activityand function in bovine lens epithelial cells. Further-more, we also examined the in¯uence of linoleic acidon an ecto-ATPase activity of bovine lens epithelial

406

cells which had been characterized earlier (Bergner

complete description of the assay has been given in

density was measured at 635 nm (Shimadzu Spectro-

and Glaesser, 1979; Bergner, 1984).

2. Materials and Methods

Chemicals and Culture Media

Sodium-binding benzofuran isophthalate acetoxy-methyl ester (SBFI-AM) and Pluronic F127 were fromMolecular Probes (Eugene, Oregon, U.S.A.), fetal calfserum was purchased from C.C.Pro (Karlsruhe,Germany). Neutral red solution (3.3 g lÿ1 D-PBS),fatty acids, ATP, ouabain, EDTA, Gramicidin D,Amphotericin B and minimal essential mediumaccording to Eagle (MEM) were from Sigma(Deisenhofen, Germany). The HEPES-buffered glucosecontaining salt solution (HBS) was prepared asdescribed (Glaesser et al., 1984). It contained5 mmol lÿ1 glucose, 140 mmol lÿ1 NaCl, 5 mmollÿ1 KCl, 0.2 mmol lÿ1 EDTA, 1 mmol lÿ1 free Ca2�,1 mmol lÿ1 free Mg2�, 5 mmol lÿ1 HEPES, and200 000 IU lÿ1 Penicillin G. The pH was 7.4. Fattyacids were dissolved in acetone, the ®nal concen-

tration of acetone in the test-tubes, including the controls, was 0.5 %.

Cell Culture

Primary cultures of epithelial-capsule-preparationswere performed as described (Glaesser et al., 1984).Brie¯y, bovine eyes from 1±2 year old animals werecollected at the abattoir and immediately transportedin warm water at 358C to the laboratory. The centralzone of the anterior lens capsule was cut around, theepithelial-capsule-preparation was attached to a coverslide by capillary forcesÐepithelial cells facingupwardsÐand transferred to a Petri dish containing2 ml MEM plus 10 % fetal calf serum. The prep-arations were ®xed with small pieces of glass at theiredges. All manipulations were carried out in a roomat 358C. Subculturing has been done according to

Iwig et al. (1995) twice a week with a splitting rate of

released ATPase activity or inorganic phosphate into

1 : 2 or 1 : 4.

Cell Detachment Test

One hundred thousand subcultured bovine lensepithelial cells were seeded into Petri dishes, 3.5 cm indiameter, and precultured for 24 hr in MEM plus10 % fetal calf serum. After extensive washing withHBS, further culturing was done with 2 ml HBS pluseffector for 5 hr. After this the cultures were washed

with HBS and detached cells were sucked off. Theadherent cells were loosened by trypsination and

N. NGUYEN ET AL.

shaker with 50 rotations per minute at 358C.

Neutral Red Cytotoxicity Assay

The neutral red test was performed according toSkehan (1995) with some minor modi®cations. A

Trimborn et al. (2000).

Determination of Protein and Inorganic Phosphate

Protein was determined according to a micromodi-®cation of Lowry's method (Langner et al., 1971).Inorganic phosphate was assayed spectrophotometri-cally using the reagent of Hess and Derr (1975). Thereagent consisted of 0.03375 % malachitgreen,1.05 % ammonium molybdate, 0.04 % Triton-X100,and 1 mol lÿ1 HCl, freshly made. Twenty ml aliquotsof the probes were added to 200 ml of reagent, optical

photometer) after exactly 3 min.

Determination of Ecto-ATPase Activity of Bovine LensEpithelial Cells in Primary Culture

Epithelial-capsule-preparations were cultured inMEM plus 10 % fetal calf serum for 1 hr. Thereafterthey were thoroughly washed twice with HBS,removing damaged epithelial cells which are easilyloosened from the capsule. The determination of ecto-ATPase activity was performed by incubating onepreparation in 3 ml HBS at 358C. The reaction wasstarted by the addition of ATP, ®nal concentration0.5 mmol lÿ1, and followed over a period of 28 minby measuring inorganic phosphate every 7 min. Afterthat the preparation was thoroughly washed andincubated for 3 hr in HBS in the presence of 10 mmollÿ1 linoleic acid. Thereafter ecto-ATPase activity wasexamined again. To make sure that the increase ofmedium phosphate is directly a result of ecto-ATPaseactivity of the epithelial cells, various controlexperiments have to be done. Epithelium-capsule-preparations freed from damaged cells did not releaseany ATPase activity into the medium but releasedsmall amounts of inorganic phosphate. The phos-phate release amounted to less than 3 % of the ecto-ATPase activity and could be neglected. Lens capsuleswithout epithelial cells neither hydrolysed ATP nor

the medium.

Determination of Na�, K�-ATPase Activity in MembraneEnriched Fractions of Subcultured Bovine Lens EpithelialCells

Membrane enriched fractions were prepared accord-ing to Garner et al. (1983). Con¯uent subcultured

Student's t-test with unpaired data was used to detect

bovine lens epithelial cells from 10 culture ¯asks(250 ml ¯asks) were thoroughly washed with physio-logical NaCl, mechanically loosened with the help of arubber and placed in 10 ml buffer (250 mmol lÿ1

saccharose; 2 mmol lÿ1 Tris±HCl pH 7.0; 1 mmol lÿ1

EDTA). The cell suspension was homogenized using aPotter-Elvehjem glass±glass homogenizer and centri-fuged at 8000 g for 15 min (Biofuge RS). The pelletwas rehomogenized in fresh buffer and recentrifuged.The supernatants were combined and centrifuged at100 000 g for 1 hr at 48C (Beckman Spinco L2-65B).The resulting pellet was suspended in 1.3 ml ofcentrifugation buffer. For the determination of theNa�, K�-ATPase activity 50 ml aliquots of themembrane enriched fraction were preincubated for20 min at 378C with a reaction mixture consisting of100 mmol lÿ1 NaCl, 20 mmol lÿ1 KCl, 4 mmol lÿ1

MgCl2 , 0.5 mmol lÿ1 EDTA and 50 mmol lÿ1 Tris±HCl pH 7.0. The reaction was started by the additionof 2 mmol lÿ1 ATP, the total volume was 500 ml.After a 30 min incubation at 378C, 100 ml aliquotswere withdrawn and pipetted to 50 ml of 3 % TCA.Inorganic phosphate was assayed as described above.The ouabain-sensitive Na�, K�-ATPase activity wascalculated as the difference between inorganic phos-

FATTY ACID CYTOTOXICITY TO LENS CELLS

phate released in the absence and presence of0.1 mmol lÿ1 ouabain. provable at the concentrations used (Table I and

10 mmol lÿ1 linoleic acid for 3 hr at 358C retained99 % of the ecto-ATPase activity (n � 3).

TABLE I

Cytotoxicity of non-esteri®ed fatty acids to subculturedbovine lens epithelial cells evaluated with the neutral red

cytotoxicity assay

Fatty acid Carbon chain NR50

Myristic acid 14 : 0 4160Stearic acid 18 : 0 4160Oleic acid 18 : 1 cis 24.1 + 1.65Linoleic acid 18 : 2 cis 15.6 + 0.34Linolelaidic acid 18 : 2 trans 75.9 + 3.50Linolenic acid 18 : 3 cis 25.3 + 2.90Arachidonic acid 20 : 4 cis 7.4 + 0.59

The NR50 values were determined in MEM and are given in mmolÿ1

Intracellular Na� Measurements

Subcultured bovine lens epithelial cells wereprecultured on coverslips in MEM plus 10 % fetalcalf serum for 24 hr at 378C. Cell loading wasperformed by incubating 1 coverslip in 1 ml HBScontaining 10 mmol lÿ1 SBFI-AM and 10 ml Pluronic(20 % in DMSO) for 1 hr at 378C. Then the coverslipswere incubated for another 0.5 hr at 378C in dye-freeHBS and placed in POC-chambers (Bachofer,Reutlingen). Measurements were made at 228Cusing an Axiovert1 100 inverted microscope withTV attachment. The objective was an Achrostigmat40x/1.30 oil. The light source was a Xenon XBO75 W short-arc lamp. The Atto¯uor1 Ratio VisionImaging System was used to record the images. SBFIwas alternately excited at 334 and 380 nm, theemission was measured at 520 nm. Calibration of theexcitation ratio in terms of [Na�]in was accomplishedin situ by application of 10 mmol lÿ1 Gramicidin Dand 2.5 mmol lÿ1 Amphotericin B (Harootunian et al.,1989). Calibration buffers were prepared from twosolutions of equal ionic strength. One contained130 mmol lÿ1 sodium gluconate and 30 mmol lÿ1

NaCl, the other contained 130 mmol lÿ1 potassiumgluconate and 30 mmol lÿ1 KCl. Both solutionscontained additionally 1 mmol lÿ1 CaCl2 , 1 mmollÿ1 MgSO4 , and 10 mmol lÿ1 HEPES and were

titrated with N-methyl-D-glucamine to pH 7.1 (Negu-lescu and Machen, 1990).

Calculations

The results are expressed as average values withstandard deviation of the mean. NR50 is de®ned as thevalue giving 50 % inhibition of neutral red uptake.

407

signi®cant differences.

3. Results

Fatty Acid Cytotoxicity

Cell retraction, bleb formation, pycnotic cell nucleiand eventually cell detachment are characteristicmorphological signs of fatty acid cytotoxicity tobovine lens epithelial cells (Fig. 1). Data obtainedwith the cell detachment test revealed cytotoxicity forthe cis-con®gurated fatty acids 16 : 1, 18 : 1, 18 : 2,18 : 3 and 18 : 4 with the highest cytotoxicity forarachidonic acid. The trans-con®gurated as well asthe esteri®ed counterparts proved to be nontoxic atthe concentration used. Furthermore, no cytotoxicitycould be found with saturated fatty acids [Fig. 2(A)and (B)]. The results were con®rmed with the neutralred viability assay. The trans-con®gurated counter-part of linoleic acid was clearly less cytotoxic and withmyristic acid and stearic acid no cytotoxicity was

Fig. 3).

In¯uence of Cytotoxic Concentrations of Linoleic Acid onEcto-ATPase and Na�, K�-ATPase Activities

Using epithelial-capsule-preparations an ecto-ATPase activity of primarily cultured bovine lensepithelial cells could be demonstrated (Fig. 4), whichhas been characterized previously (Bergner andGlaesser, 1979; Bergner, 1984). No in¯uence oflinoleic acid on the ecto-ATPase activity could befound. Preparations which had been incubated with

l fatty acid (n � 6). There is no signi®cant difference incytotoxicity between oleic acid and linolenic acid. All otherdifferences are signi®cant with P 5 0.02.

inhibited the ouabain insensitive activity by 32.8 %

FIG. 1. Morphological signs of cell damage caused bylinoleic acid. The cells were precultured in MEM plus 10 %fetal calf serum for 24 hr. (A) Cells after 24 hr in HBSwithout linoleic acid. (B) Cells after 5 hr in HBS plus10 mmol lÿ1 linoleic acid. The cells begin to retract and cellnuclei become prominent. (C) Cells after 24 hr in HBS plus25 mmol lÿ1 linoleic acid. The cells are contracted andseveral have already detached from the substratum.Granulation, which can be identi®ed as bleb formation at

FIG. 2. In¯uence of fatty acids on subcultured bovine lensepithelial cells, evaluated with the cell detachment test. Thecells were treated with 10 mmol lÿ1 effector for 5 hr in HBS.(A) Physiologically occurring fatty acids. (B) cis-Unsaturatedfatty acids as well as their trans-con®gurated and esteri®edcounterparts. Signi®cant differences were found for all cis-con®gurated fatty acids vs the control P 5 0.01, for eachcis-con®gurated fatty acid vs the others P 5 0.01 as well asvs their trans-con®gurated and esteri®ed counterpartsP 5 0.01. Saturated fatty acids, trans-con®gurated andesteri®ed fatty acids were not signi®cantly different vs the

408 N. NGUYEN ET AL.

Na�, K�-ATPase activity in crude homogenatesfrom subcultured bovine lens epithelial cells was3.2 nmol Pi per mg protein per min amounting to18.5 % of the total ATPase activity. Na�, K�-ATPaseactivity of the partially puri®ed membrane fractionwas 9.7 nmol Pi per mg protein per min and

higher magni®cation, as well as pycnotic nuclei arediscernible. 150�.

amounted to 42.1 % of the total ATPase activity inthis fraction. Ten mmol lÿ1 linoleic acid caused an

40.7 % activation but 100 mmol lÿ1 linoleic acidproduced an 51.7 % inhibition of the Na�, K�-ATPaseactivity. One hundred mmol lÿ1 linoleic acid also

control.

(Fig. 5).

Multipoint SBFI In Situ Calibration

Subcultured bovine lens epithelial cells, preloadedwith SBFI-AM, were examined in HBS pH 7.1.Alternately exciting at 334 and 380 nm, whilemonitoring the emission at 520 nm, we determinedthe response of the ¯uorescence±excitation ratio.Calibration curves were established using GramicidinD and Amphotericin B to equilibrate [Na�]in with

external concentrations. Results from ten multipointcalibration experiments, carried out with single cells,

FIG. 3. Sensitivity of subcultured bovine lens epithelialcells to linoleic acid, evaluated with the neutral redcytotoxicity assay in MEM. NR50 � 15.6 mmol lÿ1 linoleicacid (n � 3).

FIG. 4. Epithelium-capsule-preparations offer a well-suitedand stable system for demonstrating the ecto-ATPaseactivity of primarily cultured bovine lens epithelial cells.The enzyme activity was determined with 1 epithelium-

FIG. 5. Effect of linoleic acid on ATPase activities ofpartially puri®ed membrane fractions of bovine lensepithelial cells. ATPase activities were determined withoutouabain (overall activity) and in the presence of 0.1 mmollÿ1 ouabain (ouabain insensitive activity). The Na�, K�-ATPase activity was calculated as the difference of bothassays. 10 mmol lÿ1 linoleic acid signi®cantly activated theNa�, K�-ATPase activity (P 5 0.01), 100 mmol lÿ1 linoleicacid signi®cantly inhibited the Na�, K�-ATPase activity(P 5 0.01) as well as the ouabaine insensitive (P 5 0.05)and the overall activity (P 5 0.01).

FATTY ACID CYTOTOXICITY TO LENS CELLS 409

are shown in Fig. 6(A). To construct the calibrationcurve of the ¯uorescence intensity ratios in terms of[Na�]in , the data from Fig. 6(A) were ®tted to theequation

y � Rmin � �Rmax ÿ Rmin�=�1 � Kd=�Na���;

where Rmin is the ratio at zero sodium, Rmax is theratio at saturating sodium concentration, and Kd isthe sodium concentration at 50 % of the total change.The resulting calibration curve is shown in Fig. 6(B).

SBFI-AM molecules easily penetrate cell mem-branes. During the cleavage of SBFI-AM by intra-cellularly occurring esterases four molecules offormaldehyde and acetic acid are formed for eachtrapped molecule of SBFI (Negulescu and Machen,1990. Therefore, in our dye loading protocol the 1 hrincubation period with SBFI-AM was followed by a30 min incubation period in dye-free medium, which

capsule-preparation in 3 ml HBS at 358C (n � 7).

facilitates both the SBFI-AM hydrolization to becomecomplete and the leakage of formaldehyde and acetic

acid, which otherwise might damage the cells. Cells,pretreated in such a way, were uniformly ¯uorescentwith no detectable differences between nucleic andcytoplasmic cell regions and the ¯uorescence-exci-tation ratios of the baseline values remained suf®-ciently constant throughout the experiments. Theplateau formation shortly after changing the cali-bration buffers [Fig. 6(A)] hint at a rapid equilibrationbetween intra- and extracellular sodium concen-trations in the presence of the two ionophores. Atthe end of the experiment the 160 mmol lÿ1 Na�

buffer was replaced by HBS without ionophores. Thecells reached the baseline very quickly, indicating anintact and ef®ciently working sodium pumpingmachinery. Furthermore, it also means that the effectsof the ionophores are fully reversible.

It can be taken from Fig. 6(A) that the basal [Na�]in

of bovine lens epithelial cells in HBS is less than5 mmol lÿ1. However, exact data could not beestablished as the corresponding ¯uorescence ratioswere to close to Rmin . It is well known that theresolution of the method varies from cell type to celltype as the cytoplasma of the cells differently in¯uencethe excitation spectra as well as the sodium af®nity ofSBFI (Negulescu and Machen, 1990). While in freesolution the Kd is about 17 mmol lÿ1 Na�, we foundfor lens cells 42.3 mmol lÿ1 Na� [Fig. 6(B)].Harootunian et al. (1989) reported the basal [Na�]in

in serum-starved ®broblats to be 4.2 mmol lÿ1, whichcould be increased two-fold by 5 % serum. Fig. 6(A)reveals that the basal [Na�]in in serum-starved bovine

lens epithelial cells is clearly less than 5 mmol lÿ1 andmay presumably range between 1 and 3 mmol lÿ1.

FIG. 6. Multipoint SBFI calibration in subcultured bovinelens epithelial cells. SBFI-loaded cells were superfused withcalibration buffers of varying [Na�], but a constant[Na�] � [K�] of 160 mM. The calibration buffers containedtwo ionophores to equilibrate intra- and extracellular Na�.SBFI was alternately excited at 334 and 380 nm, the¯uorescence intensities were measured at 520 nm. (A) Theexperiment demonstrates the effects of step changes inextracellular [Na�]. Measurements from single cells,n � 10. (B) Calibration curves of the SBFI-¯uorescenceintensity ratio in terms of [Na�]in , calculated with the datafrom (A). Rmin is the ratio at 0 mmol lÿ1 Na�, Rmax is theratio at saturating [Na�], and Kd is the [Na�] at 50 % of thetotal change.

FIG. 7. Effects of cytotoxic concentrations of linoleic acidon [Na�]in and the function of [Na�]in , K�-ATPase. (A) Thecells were precultured in HBS. At the time zero HBS wasreplaced by K�-free HBS. After switching off the Na�,K�-ATPase activity by removal of extracellular potassiumions [Na�]in increased at a rate of 0.318 mmol lÿ1 minÿ1.Linoleic acid intensi®ed the increase of [Na�]in in aconcentration dependent manner (results from three toten experiments with measurements from single cells each).Linoleic acid concentrations: (s) without; (h) 25 mmol lÿ1;(n) 50 mmol lÿ1; (,) 100 mmol lÿ1. (B) The linoleic acid-induced increase in [Na�]in is prevented in K�-containingHBS which allows the Na�, K�-ATPase to remain inoperation. (h) at time zero HBS was replaced by K�-freeHBS containing 100 mmol lÿ1 linoleic acid; (s) at time zeroHBS was replaced by HBS containing 100 mmol lÿ1 linoleicacid (results from two experiments with measurements from

410 N. NGUYEN ET AL.

In¯uence of Cytotoxic Concentrations of Linoleic Acid on[Na�]in

In culture media lacking potassium ions, Na�, K�-ATPase is stopped functioning and the resultingincrease in [Na�]in is due to Na�-in¯ux (Negulescuand Machen, 1990). Using K�-free HBS, [Na�]in

increased in lens cells at a rate of 0.318 mmol lÿ1

minÿ1. Linoleic acid intensi®ed the increase of [Na�]in

in a concentration dependent manner. With 25, 50and 100 mmol lÿ1 linoleic acid the rates increased to0.651, 1.442 and 5.588 mmol Na� lÿ1 minÿ1,respectively [Fig. 7(A)]. However, this strong linoleicacid-caused increase in [Na�]in could be prevented in

K�-containing medium which enables the Na�, K�-ATPase to remain in operation [Fig. 7(B)].

4. Discussion

The mechanism by which unsaturated free fattyacids exert toxic effects on bovine lens epithelial cellsis unknown so far. Results obtained with the celldetachment test reveal that both the free carboxylgroup and the cis-con®gurated double bond areimportant for the fatty acid cytotoxicity. Saturated,esteri®ed or trans-con®gurated counterparts were noteffective. These results could be con®rmed with themore sensitive neutral red viability assay which not

®ve single cells each).

only records dead or living cells but also slight cellulardysfunction. This led us to assume that unsaturated

free fatty acids do not directly injure cell-substratumcontacts or the plasma membrane but rather impairenzymatic activities and metabolic functions of thecells ®rst. The capacity of free fatty acids as inhibitorsof Na�, K�-ATPase activity in partially puri®edpreparations has been repeatedly reported (Tamuraet al., 1985; Kelly et al., 1986). Working withpreparations from rabbit kidney outer medulla Swartset al. (1990) showed that the enzyme was inhibited bypalmitoleic acid, oleic acid, linoleic acid and arachi-donic acid, while saturated and esteri®ed fatty acidshad little or no effect. These ®ndings correspondexactly to the fatty acid cytotoxicity to bovine lensepithelial cells. Therefore, in this paper we havestudied the effects of linoleic acid, one of the mostcytotoxic fatty acid to bovine lens epithelial cells inculture, in more detail. The results reveal that thehighly cytotoxic concentration of 100 mmol lÿ1

linoleic acid inhibited the Na�, K�-ATPase activityof partially puri®ed membrane preparations by about50 % and in addition it strongly elevated the passivein¯ux of sodium ions in intact cells. However, in spiteof these effects, the intracellular concentration ofsodium ions remained nearly unchanged. We assumethat the residual Na�, K�-ATPase activity wassuf®cient to compensate the linoleic acid-inducedincrease of the Na�-in¯ux. In addition, there isevidence from the literature to suggest that the Na�,K�-ATPase behaves differently in response toexogenous agents in intact membranes when com-pared with isolated enzyme preparations. Ascorbicacid inhibits the puri®ed but not the cellular ATPase(Ng, Akera and Haw, 1985). Cholesterol, on the otherhand, inhibits the sodium pump in intact membranesbut stimulates the soluble preparation of the enzyme(Yeagle, 1985). A ®ve- to ten-fold increase in themembrane concentrations of palmitic acid, stearicacid, oleic acid, and linoleic acid, respectively, did nothave marked effects on the sodium pump of erythro-cytes (Dwight, Mendes Ribeiro and Hendry, 1992).The demonstration of normal sodium pump functionin the presence of the highly cytotoxic concentrationof 100 mmol lÿ1 linoleic acid makes it extremelyunlikely that the fatty acid cytotoxicity to bovine lensepithelial cells is mediated by an impairment of thesodium pump activity. This conclusion is corroboratedby the fact, that 10 mmol lÿ1 linoleic acid, whichproved already to be cytotoxic in both viability assays,did not inhibit but activated the Na�, K�-ATPaseactivity slightly.

ecto-ATPases are ATP-splitting activities located atthe outer surface of a large variety of cell types. Theyusually require Ca2� or Mg2� for activity andhydrolyse a broader spectrum of nucleotides. Theyare supposed to be part of an enzyme cascade whichcatalyses the metabolism of extracellularly occurringnucleotides. Extracellularly occurring ATP, ADP, UTP

FATTY ACID CYTOTOXICITY TO LENS CELLS

and UDP are signalling molecules which exert theirfunctions via surface-located receptors, so called

purinoceptors, which are involved in various physio-logical functions, including calcium-homeostasis ofthe cells (Burnstock, 1997). In the nervous systemecto-ATPase activity is detectable at sites where ATP issupposed to act as a synaptic transmitter (Zinchuk,Okada and Kobayashi, 1999a). In heart tissue ecto-ATPase activity has been observed at sites at whichextracellular ATP can exert its actions on the cardiacmuscle cells (Zinchuk, Okada and Kobayashi, 1999b).It has been shown that micromolar concentrations ofthe lipid peroxidation product 4-hydroxynonenalstrongly inhibit the ecto-ATPase activity from ratbrain (Foley, 1999).

A high ecto-ATPase activity has also been demon-strated on the plasma membrane of primarily culturedbovine lens epithelial cells. The enzyme, whichhydrolyses ATP 4 ITP 4 UTP 4ADP, could beclearly differentiated from other ATPases of the cells.Interestingly, the lens ecto-ATPase is positivelycorrelated with cell age. It increases three-fold duringageing of G0-epithelial cells in vivo (from 4 months to70 months old animals) and drops down to very lowlevels in mitotically growing cells in culture (Bergnerand Glaesser, 1979; Bergner, 1984). Though thephysiological role of the lens ecto-ATPase is not yetclear, the enzyme may be part of a regulatory systemincluding extracellularly occurring nucleotides andpurinoceptors as it has been suggested for othertissues, Purinoceptors have been demonstrated onhuman (Riach et al., 1995) as well as bovine(Lennarz, Glanz and Glaesser, 1997) lens epithelialcells. Micromolar concentrations of C6-substitutedpurines and purine nucleotids interfere with severalcalcium-dependent phenomena in primarily culturedbovine lens epithelial cells. Added at the beginning ofprimary culture, they cause the epithelium to contractvery strongly, inhibit the calcium-dependent ¯atten-ing and retard the calcium-dependent G04G1transition of the cells (Glaesser and Iwig, 1975;Glaesser, Rattke and Iwig, 1979).

As shown herein, 10 mmol lÿ1 linoleic acid, whichis already cytotoxic, neither inhibited the plasmamembrane-anchored Na�, K�-ATPase nor the plasmamembrane-bound ecto-ATPase. These ®ndings lead usto suggest that fatty acid cytotoxicity to bovine lensepithelial cells is not mediated via the plasmamembrane as the primary target. We assume thatcytotoxic fatty acid molecules pass the plasmamembrane and exert their effects intracellularly ®rst.This assumption is also in accordance with earlier®ndings showing that a linoleic acid caused release oflactate dehydrogenase is preceded by an inhibition ofmitochondrial dehydrogenase activities (Fass andGlaesser, 1997)

Cellular uptake as well as cytotoxicity of fatty acidsare primarily dependent on the fatty acid to albuminmolar ratio in the medium, and in in vitro experi-

411

ments, cell damage appeared only when the ratioexceeded 1 : 1 (Glaesser et al., 1996; Trimborn et al.,

2000). Due to very low albumin concentrations, thebuffering capacity for non-esteri®ed fatty acids inaqueous humor is extremely low. Detailed measure-ments of albumin and non-esteri®ed fatty acidconcentrations in bovine aqueous humor supportthe assumption that free fatty acids might becomecataractogenic risk factors, especially in pathologicalsituations (John and Glaesser, 2000). Providedhuman lens epithelial cells prove to be also sensitive,fatty acids might be a cataractogenic risk factors inhumans, particularly in pathological situations likediabetes, when in serum the concentration of non-esteri®ed fatty acids considerably rises. Experiments

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with cultured human lens epithelial cells are nowunder way.

Acknowledgements

This work was supported by a grant from the DeutscheForschungsgemeinschaft. The authors are grateful toProfessor Dr M. Iwig and Mrs B. Michel for providing uswith subcultured bovine lens epithelial cells and wegratefully acknowledge the skilful technical assistance of

Mrs Gudrun Zschau as well as the careful preparation of the manuscript by Mrs Gabriele Liebert-Hoang.

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