Hyperbaric Oxygen in vivo Accelerates the Loss of Cytoskeletal Proteins and MIP26 in Guinea Pig Lens Nucleus

Exp. Eye Res. (1999), 68, 493–504Article No. exer.1998.0630, available online at http :}}www.idealibrary.com on

Hyperbaric Oxygen in vivo Accelerates the Loss of Cytoskeletal

Proteins and MIP26 in Guinea Pig Lens Nucleus†

VANITA A. PADGAONKAR, LI-REN LIN‡, VICTOR R. LEVERENZ, ALLAN RINKE,

VENKAT N. REDDY‡ FRANK J. GIBLIN*

Eye Research Institute of Oakland University, Rochester, MI 48309-4480, U.S.A.

(Received Oxford 1 October 1998 and accepted in revised form 10 December 1998)

Previous studies have shown that treatment of guinea pigs with hyperbaric oxygen (HBO) producescertain changes in the lens nuclei of the animals which are typical of those occurring during aging. Theseinclude an increase in nuclear light scattering (NLS), elevation in levels of oxidized thiols, loss of water-soluble protein and damage to nuclear membranes. The present study investigated the effect of HBO-treatment in vivo on lens cytoskeletal proteins and MIP26 which are also known to undergo alterationwith age. Young (2-month-old) and old (18-month-old) guinea pigs were treated 15 and 30 times withHBO (3 times per week with 2±5 atmospheres of 100% oxygen for 2±5 hr periods). SDS-PAGE and Westernblotting showed that HBO-treatment of the older animals accelerated the age-related loss of five nuclearcytoskeletal proteins including actin, vimentin, ankyrin, α-actinin and tubulin, compared to levelspresent in age-matched controls (effects on spectrin and the beaded filaments were not investigated in thisstudy). Treatment of the young animals with HBO produced losses which were primarily associated withconcentrations of the nuclear α- and β-tubulins ; these cytoskeletal proteins were observed to be mostsensitive to the induced oxidative stress, and were affected earliest in the study. Disulfide-crosslinking,rather than proteolysis, appeared to be the main cause of the HBO-induced cytoskeletal protein loss(elevated levels of calcium, which might have induced proteolysis, were not found in the experimentalnuclei). Loss of MIP26 was observed only in the older guinea pigs treated 30 times with HBO; bothdisulfide-crosslinking and degradation to MIP22 were associated with the disappearance. Thus, nuclearMIP26 was susceptible to oxidative stress, but less so than the cytoskeletal proteins, particularly thetubulins. No cortical effects on either MIP26 or the cytoskeletal proteins were observed under any of thetreatment protocols. No direct link was observed between an HBO-induced increase in NLS (observed inboth the young and old animals using slit-lamp biomicroscopy) and losses of either MIP26 or thecytoskeletal proteins. The appearance of HBO-induced nuclear opacity without any change in the levelsof nuclear sodium, potassium or calcium is similar to that observed previously for human senile purenuclear cataracts. The results provide additional evidence that molecular oxygen can enter the nucleusof the lens and promote age-related events. The observed effects on MIP26 and the cytoskeletal proteinsare indicative of an increased level of lens nuclear oxidative stress in the HBO model, possibly a precursorto nuclear cataract. # 1999 Academic Press

Key words : lens ; nuclear light scattering; hyperbaric oxygen; guinea pig; cytoskeletal proteins ; MIP26;MIP22; aging; disulfide crosslinking; tubulin; oxidative stress.

1. Introduction

The treatment of human patients and experimental

animals with hyperbaric oxygen (HBO) has been

shown to produce a loss of transparency in the nuclear

region of the lens (Giblin et al., 1995; Palmquist,

Philipson and Barr, 1984; Schocket et al., 1972).

Studies conducted on HBO-treated guinea pigs (Giblin

et al., 1995) have demonstrated that a state of

oxidative stress exists in the lens nuclei of the animals,

as evidenced by damage to lens nuclear membranes,

loss of water-soluble protein, increase in urea-insoluble

protein, elevation in the levels of oxidized thiols and

* Address correspondence to: Frank J. Giblin, Eye ResearchInstitute, Oakland University, Rochester, MI 48309-4480, U.S.A.

† Preliminary results of this work were presented at the 1996and 1998 annual meetings of the Association for Research in Visionand Ophthalmology.

‡ Current address : Kellogg Eye Center, University of Michigan,1000 Wall St., Ann Arbor, MI 48105, U.S.A.

increased lens nuclear light scattering (NLS). Each of

these events is associated with changes which take

place in the aging human lens and during the

formation of cataract. Morphological effects taking

place in the lens nuclear membranes of HBO-treated

guinea pigs were similar to those reported to be

present in human immature nuclear cataract, a

disease which has been linked with oxidative damage

(Giblin et al., 1995; Spector, 1982). For these reasons,

experimental HBO treatment may be a useful method

for investigating mechanisms of aging and cataract

development in the central region of the lens.

The lens contains a variety of cytoskeletal proteins

including intermediate filaments, microfilaments and

microtubules (Alcala and Maisel, 1985; Jaffe and

Horwitz, 1991; Zigler, 1994). Although the major

proteins of the lens cytoskeleton are actin and

vimentin, the tissue is known to contain a number of

similar proteins including tubulin, ankyrin, α-actinin,

spectrin, myosin, tropomyosin and beaded filaments.

0014–4835}99}040493­12 $30.00}0 # 1999 Academic Press

494 V. A. PADGAONKAR ET AL.

All of the cytoskeletal proteins present in the eryth-

rocyte membrane are represented in the lens (Allen et

al., 1987). The majority of lens cytoskeletal proteins

are at their highest concentration in the epithelium;

however, the lens-specific beaded filaments are present

only in the fibers (Prescott et al., 1996). Precise roles

for cytoskeletal proteins in the lens are not known. It

is generally believed that they are associated with

maintenance of epithelial and fiber cell structure,

organization of cytoplasmic elements of the epithelium

and superficial fibers, maintenance of stability during

elongation of epithelial cells into fibers and provision of

flexibility during lens accommodation. Typically, the

content of cytoskeletal proteins in the lens decreases

with age, particularly in the nucleus. Loss of these

proteins, including vimentin and α-actinin, has been

observed to accelerate in the human lens during the

development of senile cataract (Ozaki, Jap and

Bloemendal, 1985).

MIP26 is the most abundant lens membrane

protein, constituting 50–60% of the total protein

present in the membrane fraction (Alcala and Maisel,

1985; Jaffe and Horwitz, 1991; Zigler, 1994). It is

specific for the lens and lens fibers, but its exact

function in the tissue is not known. Recent hypotheses

have proposed that MIP26 may have a number of

different roles including function as a weak water

channel (Chandy et al., 1997; Kushmerick et al.,

1995; Mulders et al., 1995), a fiber cell adhesion

protein (Costello, McIntosh and Robertson, 1989;

Michea, de la Fuente and Lagos, 1994) and a

nonselective ion channel (Ehring et al., 1990; Shen et

al., 1991; Zampighi, Hall and Kreman, 1985). It has

been known for some time that another lens mem-

brane protein, MIP22; increases in abundance with

age (Alcala, Valentine and Maisel, 1980; Horwitz et

al., 1979; Roy, 1979; Roy, Spector and Farnsworth,

1979) and in a number of different experimental

models for cataract.

The purpose of the present study was to investigate

the possible loss of cytoskeletal proteins and MIP26 in

the lens nuclei of HBO-treated guinea pigs as additional

evidence that this treatment can accelerate the effect

of aging in the central region of the lens.

2. Materials and Methods

Male Hartley guinea pigs of two different ages were

used in the study. ‘Retired breeder ’ animals, initially

17–18 months old, were purchased from Kuiper

Rabbit Ranch (Indianapolis, IN, U.S.A.) and 1-month-

old animals were from Kingstar (Kingston, NH,

U.S.A.). The lenses of the animals were examined

carefully by slit-lamp biomicroscopy prior to the

experiments, and animals with cortical or nuclear

opacities were excluded from the study.

Hyperbaric O#

Treatment

Procedures for treating guinea pigs with HBO in a

45 in long, 18 in diameter pressure vessel (Amron

International, Escondido, CA, U.S.A.) have been

described previously (Giblin et al., 1995). The animals

were exposed to 2±5 atmospheres absolute (22±3 psig)

of 100% O#(USP Grade Medical Gas, Liquid Carbonic,

Chicago, IL, U.S.A.) for 2±5 hr periods, three times

per week, on alternate days. Soda lime (Sodasorb,

W. R. Grace and Co., Lexington, MA, U.S.A.) was

placed in the chamber to absorb CO#and ice was used

to maintain the temperature in the vessel below 25°C.

Up to 16 guinea pigs at one time were treated in the

chamber in divided lucite boxes ; the location of each

animal was rotated in the chamber between treat-

ments. The total number of HBO-treatments was

either 15 (5 weeks) or 30 (10 weeks), with a few

animals treated 51 times with oxygen over a 17 week

period. Age-matched controls were included with each

group of O#-treated animals. The transparency of

lenses of control and HBO-treated guinea pigs was

assessed using a Zeiss slit-lamp photomicroscope

following induction of full mydriasis with tropicamide

(1%) and phenylephrine (10%). The results were

documented by photography. Following killing of the

animals by CO#asphyxiation, the eyes were enucleated

and the lenses removed by posterior approach.

SDS-PAGE and Western Blotting

Isolated lenses were frozen immediately in crushed

dry ice and stored in liquid nitrogen until required.

Frozen lenses were divided with the use of a cork borer

into a ‘doughnut’, which was used as the cortical

sample, and a cylinder. Ten percent of each end of the

cylinder was trimmed off and discarded; the rest,

accounting for approximately 25% of the total lens

weight, was taken as the nucleus. The lens cortex or

nucleus was homogenized (100 mg wet weight per ml

buffer) at 0–3°C in 5 m Tris–HCl buffer, pH 7±9containing 50 m iodoacetamide (IAA), 0±1 m

phenylmethylsulfonylfluoride (PMSF) and 2 m EDTA

(all from Sigma Chemical Co., St. Louis, MO, U.S.A.).

In addition to the use of IAA and EDTA, the tissues

were also homogenized in a N#atmosphere to protect

against artifactual oxidation of protein -SH groups.

The homogenate was centrifuged at 12000 g for

30 min and the supernatant, containing water-soluble

proteins, was sampled for analysis of protein con-

centration and then frozen for future study. The

water-insoluble pellet was washed 3 times with the

homogenizing buffer, and then resuspended in 10

volumes of 8 urea (ACS Reagent Grade, Sigma)

prepared in homogenizing buffer without PMSF or

IAA. Following centrifugation at 12000 g for 30 min,

the supernatant was collected as the urea-soluble (US)

fraction. The pellet was washed twice with 8 urea,

once with Tris–HCl buffer containing EDTA, once with

0±1 NaOH and finally, again with Tris–HCl buffer.

The urea insoluble (UI) pellet was suspended in sodium

dodecyl sulfate (SDS) sample buffer (50 m Tris–HCl

O2 AFFECTS LENS CYTOSKELETON AND MIP26 495

F. 1. Slit-lamp biomicroscopy photographs of guinea pigeyes. (A) 3±25-month-old control. (B) 3±25-month-old after15 treatments with hyperbaric oxygen over a 5-week period.(C) 4±5-month-old control. (D) 4±5-month-old after 30treatments with HBO over a 10-week period. (E) 19±5-month-old control (F) 19±5-month-old after 15 treatmentswith HBO (G) 20±5-month-old control (H) 20±5-month-oldafter 30 treatments with HBO (I) 22±25-month-old control(J) 22±25-month-old after 51 treatments with HBO.

buffer, pH 6±8 containing 1% SDS and 10% glycerol)

and then either held at room temperature for 2 hr or

heated in a boiling water bath for 3 min [the two

procedures yielded identical results ; mercaptoethanol

was not included in the boiling mixture since this

combination has been reported to cause aggregation

of MIP26 (Alcala and Maisel, 1985)], prior to analysis

by SDS-PAGE (Laemmli, 1970). US protein samples

were mixed with one-tenth volume of 10¬-concen-

trated SDS sample buffer for analysis by SDS-PAGE.

Protein concentrations were determined with Pierce

BCA Protein Assay Reagent (Pierce Chemical Co.,

Rockford, IL U.S.A.) using bovine serum albumin

as a standard. Electrophoresis, either with or without

treatment with 5% mercaptoethanol, was conducted

with 10% gels using a Hoefer mini-gel system (Hoefer

Scientific Instruments, San Francisco, CA, U.S.A.).

The prestained molecular weight standards (Bio-Rad

Laboratories, Hercules, CA, U.S.A.) included phos-

phorylase B (104 kD), bovine serum albumin (81 kD),

ovalbumin (52 kD), carbonic anhydrase (34 kD),

soybean trypsin inhibitor (28 kD) and lysozyme

(20 kD). Quantitation of the densities of protein bands

on gels or stains on immunoblots was accomplished

using a Scanmaster 3­ scanner (Howtek, Hudson,

NH, U.S.A.).

Immunoblot procedures were conducted using

mouse monoclonal antibodies to vimentin, α-tubulin,

β-tubulin and spectrin, and rabbit polyclonal anti-

bodies to actin and α-actinin (all from Sigma). A

mouse monoclonal antibody to ankyrin (Research

Plus, Inc., Bayonne, NJ, U.S.A.) was also used. A 1:40

dilution was employed for the vimentin antibody,

whereas a 1:100 dilution was used for each of the

others. The transblot was developed using an Ampli-

fied Alkaline Phosphatase Immuno-blot assay (Bio-

Rad). The immunoblotting procedures have been

described in detail previously (Padgaonkar et al.,

1997).

Protease Treatment

Partially purified MIP26 was degraded to a lower

molecular weight species by incubation with staphy-

lococcus aureus protease (Type XVII-B, Sigma). The

MIP26 was isolated from 18-month-old guinea pig

lens cortical and nuclear UI proteins. The protein

(20 µg) was incubated for 20 min at 37°C with 0, 12

or 36 µg of protease in 2 m Tris–HCl buffer, pH 7±9.

The final concentration of SDS in the incubation

mixture was 0±2%. Proteolysis was stopped by in-

creasing the SDS concentration to 1% prior to analysis

by SDS-PAGE.

Cation Analysis

Measurement of levels of sodium and potassium in

guinea pig lens cortex and nucleus were carried out in

10% trichloroacetic acid (TCA) solutions (1 ml per

tissue) following isolation of the tissues with a cork

borer. Tissue homogenizers were soaked in 1% nitric

acid and rinsed with deionized water prior to use. For

determination of total calcium concentration, cortex

was teased away from the nucleus using stainless steel

forceps which had been soaked in 50 m EDTA for a

few hours. The isolated intact nucleus was 45% of the

total weight. The tissues were homogenized in 0±5 ml

of 10% TCA containing 0±2% lanthanum chloride to

minimize phosphate interference. Measurements of

496 V. A. PADGAONKAR ET AL.

the concentrations of each of the three cations were

made using a GBC double beam atomic absorption

spectrophotometer with an air}acetylene flame (Model

902, GBC Scientific Equipment, Arlington Heights, IL,

U.S.A.).

3. Results

The effect of HBO-treatment of guinea pigs on lens

NLS was evaluated in young (initially 2-month-old)

and old (initially 18-month-old) animals using slit-

lamp biomicroscopy. For young guinea pigs, a slight

increase in scattering was evident after 15 treatments

of the animals with HBO, compared to age-matched

controls [Fig. 1(A) and (B)]. The level of NLS increased

in the lenses of the animals following 30 treatments

with O#

[Fig. 1(C) and (D)]. Similar slit-lamp results

were obtained when 18-month-old guinea pigs were

treated 15 and 30 times with HBO [Fig. 1(E)–(H)]. A

few of the older animals received additional treatments

with HBO for a total of 51 times. Lens NLS in these

animals was observed to become increasingly more

dense [Fig. 1(I) and (J)].

We investigated the effect of aging and in vivo HBO-

treatment on US proteins present in the lens nucleus of

guinea pigs, first studying normal, untreated 1-month-

old animals, as well as 18-month-old animals with

and without HBO treatment. SDS-PAGE analysis of the

F. 2. SDS-PAGE and Western blotting for guinea pig lens nuclear urea-soluble proteins, with and without hyperbaricoxygen treatment in vivo. Left : SDS-PAGE; from left to right : normal 1-month-old, control 19±25-month-old, 19±25-month-old after 15 treatments with HBO, control 20±5-month-old, 20±5-month-old after 30 treatments with HBO. Arrowheads indicatechanges in band densities for five cytoskeletal proteins. Note the increased aggregate formation at the top of the experimentalgels (arrows). For each analysis, 40 µg of protein was applied to the gel. Center : Western blot, anti-actin. Right : Western blot,anti-vimentin. Far right : MW markers. The data represent typical results for 3 experiments.

US nuclear proteins from a 1-month-old untreated

animal showed prominent staining for bands having

molecular weights of 43, 51, 58, 90 and 235 kD

(Fig. 2). As the guinea pigs aged from 1 month to

19±25 months of age (the age of the 15 HBO-treatment

control animal), there was a 50–85% decrease in the

intensity of staining of the bands for the five proteins

(as determined by density scanning of the gels).

Treatment of 18-month-old animals 15 times with

HBO, further decreased the intensity of staining of the

cytoskeletal protein bands by approximately 50%

compared to the age-matched controls. As the

untreated control animals aged from 19±25 to 20±5months, the intensity of staining of the 5 protein bands

also decreased by about 50% (Fig. 2). Treatment of the

guinea pigs 30 times with HBO produced a near

complete loss of any remaining cytoskeletal proteins

in the lens nucleus. Following both 15 and 30 HBO

treatments, a significant increase in the amount of

high molecular weight protein aggregate was detected

at the top of the gel, compared to the age-matched

controls. This protein was found to disappear upon

treatment with mercaptoethanol (results not shown).

Western blot analysis using anti-actin showed a 70%

decrease of this cytoskeletal protein (43 kD) in the lens

nucleus as the normal animal aged from 1 to 19±25

months (Fig. 2). An additional 70% decrease occurred

following 15 HBO-treatments of the 18-month-old

O2 AFFECTS LENS CYTOSKELETON AND MIP26 497

guinea pig compared to the age-matched control, and

there was nearly complete loss of actin in the nucleus

after 30 HBO treatments. Western blot analysis using

anti-vimentin showed that 15 HBO-treatments caused

a nearly complete loss of vimentin (58 kD) in the lens

nucleus. Additional blotting experiments indicated a

similar age- and HBO-induced decrease of nuclear

cytoskeletal proteins including β-tubulin (51 kD), α-

actinin (90 kD) and ankyrin (235 kD) (results not

shown). α-tubulin was not detectable in the nucleus of

the normal 18-month-old guinea pig lens.

The cytoskeletal protein spectrin with a MW of

240 kD would have migrated at the approximate

position of ankyrin in Fig. 2. However, we were

unsuccessful in obtaining significant immunostaining

in either old or young guinea pig lens nuclei using a

monoclonal antibody to spectrin. The beaded filaments

filensin (115 kD) and CP49 (49 kD) would have

migrated at the approximate positions of α-actinin and

β-tubulin, respectively, in Fig. 2. However, we did not

have access to appropriate antibodies to investigate

the beaded filament proteins in this study.

SDS-PAGE was also used to investigate possible

effects of the HBO treatment on cortical US proteins of

18-month-old guinea pig lenses. After 30 treatments

of the animals with HBO, in contrast to results for the

lens nucleus, no significant changes in the levels of

cytoskeletal proteins in the experimental cortex were

observed compared to the 20±5-month-old controls

(Fig. 3).

Next, looking at younger animals, we investigated

possible effects on cytoskeletal proteins in the lens

nucleus of 2-month-old guinea pigs after 30 HBO-

treatments. Results of SDS-PAGE indicated signifi-

cantly less effects of the O#-treatment on cytoskeletal

proteins present in the younger lens nucleus (Fig. 4)

compared to those which had been observed for the

older animals. Staining patterns for ankyrin (235 kD)

and actin (43 kD) were minimally affected by the

HBO-treatment (!10% decrease in band intensities)

while staining for α-actinin (90 kD) was decreased by

about 25%. However, HBO-induced decreases in

staining of approximately 60% were observed in

regions corresponding to the α- and β-tubulins at

58 and 51 kD, respectively. A slight increase in the

amount of high molecular weight protein aggregate at

the top of the experimental gel was found to disappear

upon treatment with mercaptoethanol. Western blot

results showed no effect of 30 HBO-treatments of the

young animals on lens nuclear vimentin (Fig. 4) and

actin (data not shown). However, staining for the α-

and β-tubulins was significantly decreased in the

experimental samples ; compared to the age-matched

controls, the band for α-tubulin at 58 kD was

decreased by nearly 70% and four bands for β-tubulin

at 51 kD and lower were decreased overall by 60%

(Fig. 4, without mercaptoethanol). Similar effects on

the nuclear tubulins were obtained after 15 treatments

of the young animals with HBO (data not shown);

however, after 15 treatments, no corresponding effects

were observed on nuclear α-actinin, compared to the

25% loss of the protein observed after 30 treatments.

Although treatment of the tubulin samples with

mercaptoethanol produced no significant recovery for

the α-tubulin immunostain at 58 kD, almost complete

recovery was observed for the four β-tubulin immuno-

stain bands at 51 and lower kD. Possible HBO-induced

loss of cytoskeletal proteins in the lens cortex of the

young animals was also investigated with SDS-PAGE,

and identical to the results for the older animals, no

effects of O#on the cortical cytoskeletal proteins were

observed (results not shown).

We next investigated possible effects of the HBO-

treatment on the levels of MIP26 in the guinea pig

lens. When 18-month-old animals were treated 30

times with HBO, a 42% loss was observed in the

amount of MIP26 present in the lens nucleus

compared to the age-matched control (Fig 5, without

mercaptoethanol). In addition, degraded MIP at

approximately 22 kD MW was apparent in the gel,

and there was evidence for a larger amount of

aggregated protein located at the top of the ex-

perimental gel compared to the control (Fig. 5, without

mercaptoethanol). The relationship of MIP26 to

MIP22 observed in Fig. 5 is similar to that obtained

following the treatment of isolated guinea pig lens

nuclear MIP26 with protease (Fig. 6). Treatment of

the nuclear samples with mercaptoethanol produced a

12% increase in the amount of MIP26, a 2-fold

increase in the level of MIP22 and disappearance of

the increased amount of aggregated protein at the top

of the experimental gel (Fig. 5, with mercaptoethanol).

The sum of the densities of the bands for MIP26 and

MIP22 for the reduced nuclear samples accounted

for about 94% of the corresponding control MIP26

density.

In contrast to the results for 30 HBO-treatments, no

loss of MIP26 was observed in the experimental

nucleus of the older animals after 15 treatments (data

not shown). In addition, we investigated younger

animals and studied MIP26 in lenses of 2-month-old

guinea pigs after 30 treatments with HBO. In contrast

to the results for the older animals, no loss of MIP26

was observed in the experimental nucleus after

treatment of the young animals with O#(Fig. 7). With

regard to cortical MIP26, the results were consistent

in that the HBO-treatment produced no effects on the

cortical membrane protein under any of the conditions

used to treat the old or young animals (results not

shown).

Since other studies (Mitton et al., 1996; Yoshida,

Murachi and Tsukahara, 1984) have implicated

calcium-activated proteolysis in the degradation of

lens cytoskeletal proteins and MIP26, we measured

levels of total calcium, as well as the concentrations of

sodium and potassium, in the lenses of 18-month-old

guinea pigs treated 30 times with HBO. For the lens

nucleus, no significant change was observed in the

498 V. A. PADGAONKAR ET AL.

F. 3. SDS-PAGE for old guinea pig lens cortical urea-soluble proteins after 30 hyperbaric oxygen treatments invivo. Left : 20±5-month-old control ; right : 20±5-month-oldanimal treated 30 times with HBO; samples were treatedwithout and with mercaptoethanol (ME). For each analysis20 µg of protein was applied to the gel. MW markers areon the far right. The data represent typical results for 3experiments.

experimental levels of sodium, potassium or calcium

compared to controls (Table I, P"0±6). For the lens

cortex, slight changes were observed for the ex-

perimental samples including a 7% increase in sodium,

a 5% loss in potassium and a 25% increase in

calcium; however, none of the values were highly

significant (Table I, P¯0±07–0±08).

4. Discussion

This study showed that treatment of 18-month-old

guinea pigs with HBO accelerated the age-related loss

of cytoskeletal proteins in the lens nucleus of the

animals. As the control guinea pigs aged from 1 to 19

months, a "50% loss of actin, vimentin, tubulin,

α-actinin and ankyrin occurred in the lens nucleus

(Fig. 2). Treatment of the animals 15 times with HBO

increased this loss by an additional 50%, and 30

treatments produced a nearly complete loss of the

proteins (Fig. 2). It has been known for some time that

the content of cytoskeletal proteins in the lens

decreases dramatically with age, particularly during

the period in which cortical fibers become compacted

into the nucleus (Alcala and Maisel, 1985; Maisel and

Ellis, 1984; Mousa and Trevithick, 1979; Ramaekers,

Boomkens and Bloemendal, 1981). A major cause of

this age-related loss of cytoskeletal proteins is believed

to be proteolytic degradation by calcium-activated

enzymes (Roy, Chiesa and Spector, 1983; Truscott,

Marcantonio and Duncan, 1989; Yoshida, Murachi

and Tsukahara, 1984). However, the HBO-induced

disappearance of the proteins observed in the lens

nucleus in this study, appeared to be more the result

of disulfide-crosslinked protein aggregation (Fig. 2).

Since levels of total calcium in the experimental lens

nucleus remained unchanged compared to controls

(Table I), it is less likely that proteolysis was the cause

of the cytoskeletal protein loss. While it is true that the

concentration of free calcium determines the rate of

proteolysis ; it has been reported for a study of rabbit

lenses that an increase in free calcium was always

accompanied by a corresponding increase in total

calcium; i.e., free and bound calcium did not re-

distribute such that levels of total calcium remained

unchanged (Hightower, Duncan and Harrison, 1985).

The absence of any change in the levels of sodium,

potassium and calcium in the experimental lens nuclei

of the present study (Table I) matches that which

has been found previously for human pure nuclear

cataracts (Duncan and Bushell, 1975).

It was not surprising that lens nuclear cytoskeletal

proteins became oxidized as a result of exposure to

HBO in vivo. Previous studies have shown that a state

of oxidative stress exists in the lens nucleus of guinea

pigs treated with HBO, and that oxidation and

insolubilization of lens crystallin proteins occur (Giblin

et al., 1995). Cytoskeletal proteins, especially tubulin,

actin and vimentin, are highly susceptible to oxidative

damage as a result of a high content of accessible

sulfhydryl (-SH) groups that are essential for their

function (DalleDonne, Milzani and Colombo, 1995;

Luduena and Roach, 1991; Mellon and Rebhun,

1976; Milzani, DalleDonne and Colombo, 1997;

Mirabelli et al., 1988; Rogers, Morris and Blake,

1991). The formation of disulfide-crosslinked high

molecular weight aggregates, as observed in this

study, is a common feature of oxidatively damaged

cytoskeletal proteins (Ellis et al., 1984; Graceffa,

Adam and Lehman, 1993; Schwartz et al., 1987;

Zaremba et al., 1984). Lens epithelial cells which have

been exposed to sulfhydryl oxidants such as H#O#

or

diamide suffer a severe disruption of cytoskeletal

protein organization (Ikebe et al., 1989; Prescott et al.,

1991), and cytoskeletal components have been

reported to be present in the high molecular weight

aggregated protein of human senile cataracts (Roy et

al., 1984). Two recent studies using oxidative models

for cataract in vivo (buthionine sulfoxamine cataract

in the mouse and selenite cataract in the rat) have also

demonstrated a heightened loss of lens cytoskeletal

proteins in the early stages of cataract (Calvin et al.,

1992; Matsushima et al., 1997). However, in these

particular models, unlike the present study, calcium-

O2 AFFECTS LENS CYTOSKELETON AND MIP26 499

F. 4. SDS-PAGE and Western blotting for lens nuclear, urea-soluble proteins from young guinea pigs treated 30 times withhyperbaric O

#. Samples were treated without (®ME) or with (­ME) mercaptoethanol. From left to right : SDS-PAGE: 4±5-

month-old control, 4±5-month-old animals treated 30 times with HBO. For each analysis, 40 µg of protein was applied to thegel. Note the increased amount of high molecular weight protein aggregate located at the top of the experimental gel, and itsdisappearance upon treatment with mercaptoethanol (arrows). Western blots, control and experimental, without and with ME,for anti-vimentin, anti-α-tubulin and anti-β-tubulin. Note the recovery in β-tubulin immunostaining upon treatment of theexperimental sample with mercaptoethanol. Far right : MW markers. The data represent typical results for 3 experiments.

activated proteolysis by calpain was believed to be the

major cause of the protein loss.

Our finding that HBO-induced loss of cytoskeletal

proteins took place only in the lens nucleus, not the

cortex, is consistent with our previous in vivo and in

vitro studies indicating that oxidative changes

associated with this model are confined to the

central region of the lens (Giblin et al., 1988, 1995;

Padgaonkar, Giblin and Reddy, 1989). One possible

reason for this observation may be the relatively low

level of GSH (3 µmol g−") which exists in the nuclear

region of the 20-month-old guinea pig, compared to

that present in the cortex (15 µmol g−") (Giblin et al.,

1995). Thus, the lens nucleus is more susceptible to

oxidative damage since its lower level of GSH coupled

with a relatively inactive glutathione redox cycle

(Giblin et al., 1988) would make the tissue less able to

prevent the formation of protein disulfide. The im-

portance of GSH and an active glutathione redox cycle

in maintaining cytoskeletal protein sulfhydryls and a

normal cytoskeletal protein architecture has been

demonstrated previously (Ikebe et al., 1989; Li, Zhao

and Chou, 1993). A similar disparity in the levels of

GSH present in the nucleus and cortex exists in the

human lens (Lou and Dickerson, 1992), and may be

an initiating factor in the formation of human senile

nuclear cataract (Sweeney and Truscott, 1998). A

recent study has shown that GSH may possess an

essential antioxidant function that is independent of

its ability to detoxify H#O#

(Reddan et al., 1999).

The lens cytoskeletal proteins of young (2-month-

old) guinea pigs were considerably less susceptible to

HBO-treatment in vivo, compared to the older animals.

In contrast to results for the older guinea pigs, no

significant changes were observed in the levels of

actin, vimentin and ankyrin in the lens nucleus of the

2-month-old animals after 30 O#-treatments (Fig. 4).

One reason for this finding may be the presence of a

higher level of antioxidant activity in the younger

lens nucleus; for example, the concentration of GSH

in the nuclear region for a 2-month-old animal is

5³0±5 µmol g−" (unpublished data, n¯4) compared

to 3 µmol g−" for the older animal.

The tubulins were the first cytoskeletal proteins to

be affected by oxidative stress in the lens nucleus.

Unlike other cytoskeletal proteins, the α- and β-

tubulins suffered a 60–70% loss in the lens nucleus

of the younger guinea pigs after 15 and 30 HBO-

treatments. The SDS-PAGE results without and with

mercaptoethanol indicated that disulfide-crosslinked

aggregation of the tubulins had taken place (Fig. 4) ;

however, we are unable to explain why only β-

500 V. A. PADGAONKAR ET AL.

F. 5. SDS-PAGE for lens nuclear urea-insoluble proteinsfrom old guinea pigs treated 30 times with hyperbaric O

#.

20±5-month-old controls and 20±5-month-old animals after30 treatments with HBO. Data are without (®ME) and with(­ME) treatment of the samples with mercaptoethanol.For each analysis, 15 µg of protein was applied to the gel.Note the higher level of aggregated protein at the topof the experimental gel compared to the control andits disappearance upon treatment with mercaptoethanol(arrowheads). Far right : MW markers. The data are typicalresults for 3 experiments.

tubulin, and not also α-tubulin, recovered following

mercaptoethanol treatment. The greater susceptibility

of the nuclear tubulins to oxidative damage compared

to other cytoskeletal proteins present in the nucleus

may be associated with the known hypersensitivity of

the tubulins to sulfhydryl oxidation (Luduena and

Roach, 1991; Steiner, 1985) and the need for these

proteins to co-exist with high levels of GSH for normal

function (Zuelke, Jones and Perreault, 1997). The

tubulins, with 8 and 12 free -SH groups per β- and α-

subunit (Krauhs et al., 1981; Ponstingl et al., 1981),

have a substantially higher sulfhydryl content than

other cytoskeletal proteins ; vimentin, for example,

which is also considered to be oxidation-sensitive,

contains only a single cysteine residue (Rogers, Morris

F. 6. Degradation of isolated guinea pig lens cortical(left) and nuclear (right) MIP26 by staphylococcus aureusprotease. 20 µg of MIP26 was incubated at 37°C with 0,12 µg (1¬) or 36 µg (3¬) of protease for 20 min. ‘P’indicates the protease band. Far right : MW markers.

F. 7. SDS-PAGE for lens nuclear urea-insoluble proteinsfrom young guinea pigs treated 30 times with hyperbaric O

#,

4±5-month-old controls and 4±5-month-old animals after 30treatments with HBO. Data correspond to without (®ME)and with (­ME) treatment of the samples with mercapto-ethanol. For each analysis, 15 µg of protein was applied tothe gel. Far right : MW markers. The data are typical resultsfor 3 experiments.

O2 AFFECTS LENS CYTOSKELETON AND MIP26 501

T I

Effect of 30 treatments of 18-month-old guinea pigs with

hyperbaric oxygen on the levels of sodium, potassium and

calcium in the lens cortex and nucleus

Cation concentration (m)

Cortex Nucleus

SodiumControl (6) 17±6³1±0 11±8³1±0Oxygen (6) 18±8³1±2 12±2³1±6

P¯0±07 P¯0±63Potassium

Control (6) 133±3³6±6 95±9³7±4Oxygen (6) 127±0³4±3 98±9³11±3

P¯0±08 P¯0±61Calcium

Control (4) 0±44³0±05 0±46³0±10Oxygen (4) 0±55³0±09 0±45³0±07

P¯0±08 P¯0±87

Each result is expressed as the mean³.. The number ofexperiments is in parentheses (lenses in each group were fromseparate animals ; sodium and potassium values were determined inthe same lens). Values used for the water content of cortex andnucleus were 70% and 55%, respectively.

and Blake, 1991). The inherent instability of tubulin

and its tendency to aggregate have been suggested as

reasons why the protein requires protection by αB-

crystallin chaperone activity (Arai and Atomi, 1997).

The HBO-induced loss of cytoskeletal proteins

occurring in the lens nucleus was most likely not

related to the observed increase in NLS. Although, for

the older animals, accelerated cytoskeletal protein loss

coincided with increased nuclear scatter, a substantial

age-related decrease in the level of the proteins was

also seen in the control lens nuclei which maintained

low scatter. For the young animals, HBO-treatment

produced increased NLS without any significant effects

on the nuclear levels of actin, vimentin, α-actinin or

ankyrin, indicating that the cause for increased scatter

was independent of changes to these cytoskeletal

proteins. Although young experimental lenses with

increased NLS did show significant loss of the nuclear

tubulins (Fig 4), old control lenses with low scatter

also had severely diminished nuclear tubulin content.

These results are in accordance with previous con-

clusions (Maisel and Ellis, 1984; Ringens, Hoenders

and Bloemendal, 1982) that the age-related loss of

vimentin, tubulin and other cytoskeletal proteins in

the human lens nucleus is not a direct initiator of

nuclear cataract since the same changes are evident in

old clear lenses (Bradley, Ireland and Maisel, 1979;

Kuwabara, 1968). With regard to actin, other

investigators have suggested that this cytoskeletal

protein may not be required for any specific function

in the compacted nuclear fiber cells (Mousa and

Trevithick, 1979). Although an accelerated loss of

vimentin has been reported to occur in the nucleus of

human nuclear cataracts (Ozaki, Jap and Bloemendal,

1985), a finding similar to the results of this study

with HBO, vimentin-knockout mice, surprisingly, have

been found to possess a normal lens cortex and

nucleus (Colucci-Guyon et al., 1994). Regarding

lens cortical changes, HBO-treatment produced no

increased scatter or loss of cytoskeletal proteins in that

region. It is possible, as discussed above, that the high

level of cortical GSH in the guinea pig lens prevented

such effects. It has been proposed, however, that the

loss of certain cortical cytoskeletal proteins, including

actin and the beaded filaments, may be associated

with the development of cortical cataracts (Maisel and

Ellis, 1984; Mousa, Creighton and Trevithick, 1979;

Tagliavini, Gandolfi and Maraini, 1986).

Significant loss of the membrane protein MIP26 was

evident in the nucleus of lenses of 18-month-old

guinea pigs treated 30 (Fig. 5), but not 15, times with

HBO. No effects on the protein were observed following

exposure of young animals to elevated O#. Thus,

although nuclear MIP26 was susceptible to oxidative

stress, it was less so compared to the nuclear

cytoskeletal proteins, particularly, the nuclear α- and

β-tubulins.

The HBO-induced loss of nuclear MIP26 included

degradation of the protein to MIP22 (Fig. 5), a result

which further confirms the ability of molecular oxygen

to accelerate a known age-induced effect in the lens

center (Alcala, Valentine and Maisel, 1980; Horwitz et

al., 1979; Roy, 1979; Roy, Spector and Farnsworth,

1979). The main mechanism for cleavage of MIP26 in

the aging lens is believed to be enzymatic proteolysis ;

however, a specific endogenous proteolytic enzyme for

this function has never been identified (Joseph Horwitz,

personal communication). An alternative mechanism

which has been suggested by Takemoto and Takehana

(1986) is that oxygen free radical-induced protein

fragmentation (Kim, Rhee and Stadtman, 1985; Wolff

and Dean, 1986) might also play a role in the

degradation process. The shift from MIP26 to MIP22

in the aging lens has been observed to be more active

in the nucleus; at ages 55–60 years in the human,

although the lens cortex contains equal amounts of

both proteins, the nucleus is significantly richer in

MIP22 (Horwitz et al., 1979; Roy, Spector and

Farnsworth, 1979). It is possible that some of the

nuclear MIP22 formed in the aging lens may be the

result of oxidative stress. Studies are ongoing in our

laboratory to investigate possible oxygen free radical-

induced degradation of MIP26.

Similar to the results of this study, an accelerated

degradation of MIP26 in the lens has been shown to

take place in a number of different experimental

models for cataract including the X-rayed rabbit

(Garadi, Giblin and Reddy, 1982), the Nakano mouse

(Roy, Garner and Spector, 1982), U18666A cataract

in the rat (Alcala, Cenedella and Katar, 1985),

galactose cataract in the rat (Alcala et al., 1986),

selenite cataract in the rat (David et al., 1988), the

502 V. A. PADGAONKAR ET AL.

Emory mouse (Lo and Kuck, 1990) and the HIV-1

protease transgenic mouse (Mitton et al., 1996). The

mechanism of cleavage of MIP26 in a number of the

above models appears to be activation of calpain by an

elevated calcium level ; for example, MIP26 degra-

dation was prevented in the HIV-1 protease model by

culturing the lenses with a calpain inhibitor (Mitton et

al., 1996). However, as mentioned previously, levels of

calcium did not rise in the nucleus of the lenses of

the HBO-treated animals. The present study did not

investigate whether the C- or N-terminal ends of the

nuclear MIP26 were cleaved. In the selenite and HIV-

1 protease experimental models, the C-terminal of the

protein was proteolyzed (David et al., 1988; Mitton et

al., 1996), while both ends of MIP26 may be cleaved

in the aging human lens (Takemoto and Takehana,

1986).

In addition to degradation of MIP26, HBO-induced

loss of the protein included the formation of disulfide-

crosslinked aggregates (Fig. 5). Although the amino

acid sequence of guinea pig MIP26 has not been

determined, bovine and human MIP26 contain three

and two cysteine residues, respectively (Gorin et al.,

1984; Pisano and Chepelinsky, 1991). The -SH groups

of MIP26 have been shown to be freely accessible to

the in vitro binding of compounds such as palmitic

acid (Manenti, Dunia and Benedetti, 1990). Whether

MIP26 or MIP22 were involved in the crosslinking

observed in the present study is not clear ; both

proteins appeared to increase in the SDS-PAGE gel

upon treatment with mercaptoethanol (Fig. 5). In

addition, possible crosslinking of crystallins to the

membrane protein cannot be ruled out. Alpha

crystallin has been shown to crosslink in vitro to both

MIP26 and MIP22 by a glycation-mediated process

(Prabhakaram and Ortwerth, 1992).

Similar to the data for the cytoskeletal proteins, loss

of nuclear MIP26 also did not appear to be linked with

the observed increase in NLS. Although no changes in

the levels of MIP26 were observed for the HBO-treated

younger guinea pigs or the older animals treated 15

times with HBO, each of these protocols produced

some degree of increased NLS compared to controls. It

is possible that the loss of MIP26, as well as the

aggregation of cytoskeletal proteins as discussed

above, reflect a higher level of oxidative stress existing

in the lens nuclear membranes of the HBO-treated

animals. There is also little likelihood that degradation

of MIP26 to MIP22 in the aging human lens may

affect transparency since changes in the relative

abundance of the two proteins appear to be complete

by the age of forty (Roy, 1979). However, loss of the

C-terminal end of MIP26 may be detrimental to the

lens since this region in the bovine protein has been

linked with control of the closing of the channel gate

(Ehring et al., 1991) via its sites for phosphorylation

(Garland and Russell, 1985; Lampe and Johnson,

1990; Schey et al., 1997) and its interaction with

calmodulin (Girsch and Peracchia, 1991) [contrasting

results, however, have been obtained for chicken lens

MIP26 (Modesto et al., 1996)].

In summary, this study has shown that molecular

oxygen is able to enter the nucleus of the lens in vivo

and accelerate age-related losses of membrane cyto-

skeletal proteins and MIP26. Oxidative stress and

disulfide-crosslinking were implicated as causes of the

protein losses, and the nuclear tubulins were found to

be particularly vulnerable to oxidative damage. No

direct link was found between HBO-induced loss of

transparency in the lens nucleus and the disap-

pearance of the membrane proteins. However, the

observed oxidative effects on lens cytoskeletal proteins

and MIP26 are indicative of a general state of nuclear

oxidative stress in the HBO model, possibly a precursor

to nuclear cataract.

Acknowledgements

The authors thank Ann Dunlop for the long-term careof the guinea pigs, Alice Carleton for the typing of themanuscript and Ken Hightower for helpful discussionsconcerning free and bound lens calcium. We appreciate theassistance of the following students in treating the animalswith hyperbaric oxygen: Vikas Agarwal, Navneet Brar,Michael Faw, Aaron Gibson, Valerie Heller, Tom Ickes,Amy Komendera, Thiti Napawan, Lauren O’Toole, VaideheePadgaonkar, Cory Renauer, Katherine Rylien, MatthewStevens and Chris Supina. The work was supported byNational Institutes of Health Research Grants EY02027(F. J. Giblin), EY00484 (V. N. Reddy) and EY05230 (CoreGrant for Vision Research). The study was part of theCooperative Cataract Research Group (CCRG) program.Allan Rinke was the recipient of a student fellowship inhonor of Neil and Donna Weisman by Fight for Sight, Inc.,NYC, a division of Prevent Blindness America, and anOakland University Undergraduate Research Grant.

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