Glutathione metabolism in the lung: Inhibition of its synthesis leads

Loading....

Loading....

Loading....

Loading....

Loading....

Full text

(1)

Biochemistry

Glutathione metabolism

in

the lung: Inhibition of its synthesis leads

to

lamellar

body and mitochondrial defects

[lymphocytes/lungtype2cells/buthioninesulfoximine/glutathionemono(glycyl)ester/intestine]

JOHANNES

MARTENSSON*, AJEY JAINt, WILLIAM FRAYERt,

AND

ALTON

MEISTER*

*Departments of Biochemistry andtPediatrics,CornellUniversity Medical College, 1300 York Avenue, New York, NY 10021

ContributedbyAlton Meister,April20,1989

ABSTRACT Mice treated with buthioninesulfoximine, an inhibitor ofglutathione synthesis, showedstriking alterations ofmorphology of lungtype2celllameilar bodies (swelling and disintegration) and mitochondria (degeneration) and of lung capillary endothelial cells (mitochondrial swelling). These ef-fects probably may be ascribed toglutathione deficiency; ad-ministration of glutathione monoester protects

against

them. Measurements of arteriovenous plasma glutathione levels across thelung indicate that thenet uptake ofglutathione by thisorgan is substantial. Thus,glutathione exported from the liver to theblood plasma is utilized by the lung which, like liver, kidney, andlymphocytes (and unlike skeletal muscle), exhibits a high overall rate ofglutathione turnover. Intraperitoneal Hijection of glutathione into buthionine sulfo treated mice leads to very high levels of plasma glutathione without significant increase in theglutathiolelevelsofliver,lung, and lymphocytes; on the other hand, admirationof glutathione monoester leads to markedlyincreased tissue and mitochon-drial levels ofglutathione. Admirationofglutathione mo-noester (in contrast to glutathione) to control mice also in-creasesmitochondrialglutathione levels. Thefindings indicate that glutathione is required for mitochondrial integrity and that itprobably also functionsinthe processing and storage of surfactant in lamellar bodies. The morphological changes observed aftertreatmentwithbuthioninesulfoximine and their prevention by glutathione monoester as well as finding on glutathione metabolism indicate that this tripeptide plays an important role in the lung. Thepreviously observed failure of buthioninesulfoximine-treated mice to gain weightismainly due to glutathione deficiencyintheintestinal mucosa.

Previousstudies (1) on the effectsof inhibition of glutathione

(GSH) synthesisonskeletalmuscleprovided evidencethat

(i)

GSH functions in muscle under normalconditions, (it) the levels of GSH in muscle are greatly in excess of those

required, and (iib) GSH isrequired for mitochondrial

integ-rity.It wasalso found thatadministration of GSHmonoester

(incontrast tothatofGSH) led to maintenance of a significant level of GSH inmusclemitochondria duringarelatively long

periodof administration ofbuthioninesulfoximine(BSO),an

inhibitor

of

GSH synthesis.

Inthepresentworkwehave extended thisapproachtothe

lung,which likekidney and liver (and in contrast to skeletal muscle) exhibits ahighrate of turnoverof GSH. Wefound evidenceofsignificantcellular damage to lung and to a lesser extent to lymphocytes after prolonged inhibition of GSH

synthesis produced byadministrationofBSO. Interestingly,

wefound striking evidence of lamellar body damage in type 2cells anddegenerationofmitochondriain these cells and in

capillaryendothelial cells. Thesechangeswerepreventedby administration ofGSH monoester. Administration of GSH

monoester prevented the marked decline in tissue GSH levels

found after administration of BSO and led to levels of

mitochondrialGSH in thelung and liver that were higher than thosefound in untreated controls.

EXPERIMENTAL PROCEDURES

Materials. Mice (male, 28-32 g, Swiss-Webster; Taconic

Farms) were maintained on Purina Chow. L-Buthionine-[S,R]-sulfoximine (BSO)(2-4), L-buthionine sulfone(4), and GSH monoisopropyl (glycyl) ester (5) wereprepared as de-scribed. Hypaque

(50%16)

waspurchased fromWinthrop-Breon Labs (New York). An aqueous solution of GSH

monoisopro-pylesterwaspreparedin the 1/2(H2SO4) form andadjustedto

pH6.5-6.8 by cautiousaddition of NaOHimmediatelyprior to use. Instudies inwhich this compound wasgiven,control experiments were carried out in which mice were given equimolaramountsofGSH,isopropanol, andNa2SO4.

Methods. BSO wasadministered tomiceas stated below

(see Figs. 1 and 2 and Table 1). L-Buthionine sulfone was given to mice in equimolar amounts in parallelstudies.The tissues were obtained as follows. Mice wereanesthetized by intraperitoneal injection of a mixture of xylazine (5 or 10

mg/kgof body weight) and ketamine (100mg/kg);within 3

min, the thoracicandperitoneal cavities wereopened. The lung andliver were perfused from

the

right ventricle with 10 ml of cold saline after

clamping

of the venous heart input. Perfusion was completed within 2 min as the lung turned

white and theliver became light brown. Theleft inferiorlobe

ofthe lung was excised, rinsed with cold saline, blotted, weighed,andhomogenized in 5 vol of5%sulfosalicylicacid. Apieceof the left lobe ofthe liver wasexcisedandprocessed

as described for the lung. The tissue homogenates were

centrifuged for5minat10,000 x gat

40C,

and the supernatant solutions wereanalyzedfor GSH.

Lymphocytes were obtained from whole blood (0.7 ml)

drawn from the right ventricle into a heparinized syringe containing0.7,umol ofEDTAand 14 amoleach of L-serine and sodium borate (6). Samples obtained from two mice were

pooledand mixed

with

2.6mlof coldsaline and then

layered

ontopofaHypaque/Ficoll gradientinaplastictube(6,7).The

gradientconsistedoftwophases:anupperphasecontaining3 mlofamixtureof10partsof33.9%Hypaque and 24 parts of

9% Ficoll,andalowerphasecontainingamixtureof3mlof

10 parts of

50%o

Hypaque and 30 parts of

9%o

Ficoll. The

lymphocyteswereseparated fromgranulocytes by differential centrifugation; theywerecentrifugedat300x gfor 30minat

40C

rather than 800x gasrecommended(7)so as todecrease

contamination by platelets. The lymphocyte

suspensions

(which contained about

10%

monocytes) were washed by centrifugationwith cold salinecontaining1mMEDTA,20 mM

L-serine,and 20 mMsodium borate. Contaminating

erythro-cytes were lysed by suspension of the cells in 0.15 mM ammonium chlorideat

370C

for 5 min(6,8).Thelymphocytes

Abbreviations:GSH, glutathione; BSO, buthionine sulfoximine.

5296 Thepublicationcostsof this article weredefrayedin partbypagecharge payment.Thisarticlemustthereforebehereby marked "advertisement" inaccordance with 18U.S.C.§1734 solelytoindicatethisfact.

(2)

Proc. Natl.Acad. Sci. USA86 (1989) 5297 werewashed twice with saline (containing EDTA, serine, and

sodiumborate),counted, and lysed bytwocycles of

freezing/

thawing in 0.1-0.2 mlof 4.3%sulfosalicylic acidasdescribed (6, 9). After centrifugation, the supernatant solutions were immediatelyanalyzedfor total GSH.

Determinations of total GSH were also carried out on plasma obtained by immediate centrifugation of 0.2 ml of

wholeblood[from the right ventricle (see above) or by direct aspiration (0.05 ml) from the left ventricle] at10,000 x gat

40C for 1 min (10, 11). A portion of the clear supernatant

solutionwasimmediatelymixedwithsulfosalicylicacid(final concentration, 4.3%) and then centrifuged for 5 minat 40C. The supernatant solution obtainedwas usedfor determina-tion of total GSH. The presence of hemoglobin in thesamples

was examined (12, 13). The minute amount ofhemoglobin

found indicated that the presence of erythrocytes could accountfor <0.3%of the observed GSH content(14).

GSH determinations carried out as described above on

control samples oflung,liver,andlymphocyteswere,within

experimental error, identical to values obtained on mice

killed byspinal cord transection.Theanesthesiaprocedureis advantageous in that significantly larger amounts of blood maybeobtainedthanareobtained afterspinalcord transec-tion. However, the perfusion was completed within 5 min; afterlongerperiods ofanesthesia,evenwithone-thirdof the doses statedabove, lower GSH levelswerefound.

Total GSH was determined by the glutathione disulfide reductase-5,5-dithiobis(2-nitrobenzoate)

recycling.

method

(11). For plasma, values that include mixed disulfides be-tween GSH and cysteine or proteins were alsodetermined

afterreduction with5 mMdithiothreitol(9). BSOwas

deter-minedbyuseofaDurrum(model 500) aminoacidanalyzer.

The activities of -glutamylcysteine synthetase, y-glutamyl transpeptidase,andcitratesynthase (15,16)weredetermined

as stated (1). Protein was determined (17), and electron

microscopy(1)was performed as previouslystated. The mitochondrial fractionsofliver and lungwere sepa-ratedbytheprocedurepreviously given (1, 16, 18, 19)except that heparin was omitted in the homogenization step. The

fractions obtained by thisprocedurewerechecked by elec-tronmicroscopy. The fractions from livercontainedvirtually uncontaminated mitochondria; those from lung contained predominantly mitochondriaand about 5% lamellarbodies.

RESULTS

Effect ofAdministration of BSOonGSH Levels.

Adminis-tration of BSO led torapid decline ofthelevels of GSH in the lung, lymphocytes (Fig. 1), liver, and plasma (Fig. 2). The initialrapid rates ofdecline,expressedasthetimerequiredfor a50% decrease in the level, were about 25 and 45 min for lymphocytes andlung,respectively.Thedeclineintheplasma

GSHlevel,asexpected (20, 21),wasclosely

parallel

tothat

of

the liver

(ti,2

= about 65min). Thedeclineof the GSH levels in the liver wasbiphasic;thesecond slower rate,asdiscussed

previously (1, 18), isthoughttoreflect loss of GSH from the

mitochondria. The slower laterratesof GSHdisappearance

were0.01%, 0.02%, 0.05%,and0.05% ofthecorresponding

initialratesforlymphocytes, lung, liver, andplasma,

respec-tively.Thedisappearance of GSHfrom

lymphocytes

andlung

exhibited bothrapidand slowphasesbutseemedtobe more

complexthanin liver. The dataobtainedonGSH levels in the lungareprobablyafunction of severalfactors, includingthe cellularheterogeneity ofthis organ, theratesof cellularexport ofGSH, and the intracellular levels of BSO. Although

rela-tively high overall levels ofBSO were maintained in these

experiments,theintracellularlevelofBSO in the variouslung

celltypesisnotknown.BSOwasrapidly cleared fromplasma

(til2

= 30-40min),andthiscompoundwas

rapidly

excreted in

theurine. Preliminarystudies inwhich single doses of BSO

were administeredindicate that the time requiredfora

50%

loo -J 0 z 0 (-IL 0 0I~ (I)

oD

EI

6~~~~~~~~~~~~

0 100 200 300 400 500 HOURS

FIG. 1. Effect oftreatmentwith BSOontheGSHleveloflungand lymphocytes. Mice were given BSO twicedaily by

intraperitoneal

injection(4mmol/kg)at9a.m.andat6p.m.for21days.BSOwasalso givenin thedrinkingwater(20mM).Controlswereinjectedwithan

equivalentvolumeof saline andweregiventapwater todrink. Control valuesof GSHwere1.69±0.12,umol/g(lung)and 1.39 + 0.15nmol per106cells

(lymphocytes).

After0.25hr, 0.5 hr, 1 hr, 2 hr, 1

day,

2 days,4days,7days,15days,and 21daysof BSO treatment, the GSH values(mean ±SD;n=3-5)forlungwere,respectively,1.31±0.16,

1.04±0.15,0.92± 0.05,

0..91

±0.10,0.54 ±0.06,0.47±0.05,0.43 ± 0.05, 0.42 ± 0.04, 0.39 ± 0.04, and 0.38 ± 0.04

tumol/g.

The

correspondingvaluesfor

lymphocytes

were0.97 ±0.11,0.51±0.08, 0.42±0.05,0.36±0.05,0.27+ 0.04,0.25 ±0.04,0.24± 0.04,0.18 ±0.02,0.16±0.02,and 0.14±0.03nmol per106cells.

(Inset)

Effect

onlung (o)and

lymphocyte

(e)

GSH levels ofasingle

injection

(-)

andoftwoinjections(---)ofBSO(at0and 1hr;eachdose,8

mmol/kg).

decrease in tissue BSO level is about

40,

50,

and 120 min for

liver, lung,

and

lymphocytes,

respectively.

After

discontinu-ation oftreatmentwith

BSO,

the tissuelevelsofBSOdeclined

rapidly,

and the levels of GSH increased

significantly

(see

Fig.

1 Insetand

Fig.

2

Inset).

Therates atwhichnormallevelsof

GSH return werelower than the initialrates of GSH

disap-pearance, and theGSH values"overshot"thecontrolvalues asobserved in the heart

(see

figure

1 inref.

1).

The

rapid

rates at which GSH levels increased after

discontinuation of BSOtreatment

precluded

studies on the effects of amino acid

supplementation

onBSO-treated

mice,

aswasdone in studiesonmuscle

(1). However,

studiesonfed and fastedmice showed thatadministrationeitherof

cysteine

orofamixture of

cysteine,

glycine,

and

glutamate

increased

the GSH levelsof

lung,

liver,

and

lymphocytes

toabout the same extent. Administration of

glutamate (or

of

a-keto-glutarate)

alone alsoincreased liver and

lung

GSH levels in fed mice.Administration of

glycine

or

glutamine

didnotincrease GSH levels in these tissues orin

lymphocytes.

The

'y-glutamylcysteine

synthetase

activities of

lympho-cytes and

lung

werefoundtobe 87.0+4.8

(mean

± SD;n=

5)

and 10.3±0.1

nmol/hr

per mgof

protein,

respectively;

the valuefor liver is 82.3 + 1.5

nmol/hr

per mgof

protein

(18).

(3)

100 -J 0 z 0 LL 0 0R I-n,. 80 60 40 20 E (n

ad

1.0 0.5 0 100 200 300 400 500 HOURS

FIG. 2. Effect ofasingledose(Inset)ormultipledoses of BSO at9 a.m.and 6 p.m.(each dose,2mmol/kg)onGSHlevelsofliver (A)andplasma (A). ControlvaluesofGSHwere8.26±0.06

srnol/g

(liver)and 58.3±4.8AM(venousplasmafromrightventricle).After 0.25hr,0.5hr,1hr,2hr.1day,4days (only liver),9days,15days, and 21daysofBSO treatment, the GSH values(mean±SD;n=3-5) for liverwere,respectively, 6.61 ±0.28,5.78 ± 0.25,4.54 ± 0.21, 2.48±0.09,1.10±0.08,0.83±0.05,0.66±0.04,0.59±0.03,and 0.52±0.02

Amol/g.

ThecorrespondingGSHvalues forplasmawere

50.7 ±2.9,46.6± 3.1,33.2 ± 3.2,12.9±1.5, 3.50± 0.50,2.92±

0.3,1.80±0.3,and 1.20±0.2

AsM.

AplasmaBSO value ofcloseto

zero wasfoundat2 hr.

The

y-rglutamyl

transpeptidase activities of lymphocytes, lung,kidney, andliver are,respectively,41.2 ± 1.2(mean ±

SD;n = 5), 25.0 + 0.3 26,300 ± 3700(22), and1.92 ±0.05

nmol/hr

per mgofprotein (18); seealso refs. 20and 23-27. Utilization of GSH by the Lung. Previous studies on

ne-phrectomized mice led to the conclusion that about two-thirdsofthearterialbloodplasmaGSHisused

by

the

kidney

and that about one-third of the plasma GSH is used by extrarenal

-t-glutamyl

transpeptidase activity (21). On the basis of studiesontheisolatedperfusedratlung, Berggrenet al.(24) concludedthat thelungin additiontothekidneymay utilizeplasmaGSH and thatutilization of GSHisprobably mediatedby extracellular breakdown andresynthesisrather than by direct

uptake.

The present

studies,

which lead to similarconclusions,make itpossibletoconsidera quantita-tive estimate of the amount of GSH used by the lung; interestingly, thelungsuse moreGSHthando thekidneys. Plasma from blood drawn fromtheright ventriclehasGSH

levelsof58.3 ±4.8

,M

(Fig. 1),and levels that include GSH

mixed disulfidesof 62.1 ± 5.0

AM.

Incontrast, GSH levels

ofplasmataken from theleft ventriclewerefoundtobe 22.1 ± 2.3

pAM,

and levels thatinclude GSH mixed disulfides of 29.9 ± 7.5

,uM.

The arteriovenous

difference,

about 32

nmol/ml,

whenmultiplied bythecardiacoutput[=6.3ml of

plasma permin

(28)],

indicates that the loss of GSH from plasma during transit through the lungisabout 200 nmolper min. This value is greater than has been estimated for

utilization ofGSH

by

mouse

kidney

(about

50

nmol/min)

(29)J

Morphological Changes Associated with Administration of

BSO. Electron

microscopic

studies ofthe liver and

kidney

after 3 weeks oftreatmentwith BSO showednoabnormalities.

Incontrast,

striking

changeswerenotedin the

lung

type2cells

and

lung capillary

endothelial cells

(Fig. 3).

Intype 2cellsthere was a

significant

decreaseinthenumberof mitochondria and

evidence ofmitochondrial

swelling

and

degeneration.

After treatmentwithBSO the lamellar bodiesseemedtodominate thetype 2

cells;

thelamellar bodies became

larger,

less

dense,

and

appeared

to occupy more space than in the controls.

Swelling

of thelamellar bodieswasassociatedwith

disruption

ofthecharacteristic latticestructureofthese

organelles.

There was a decrease in the numberofmicrovilli on the alveolar

surfaceofthe type 2

cells,

swelling

and

thickening

ofthebasal

membrane,

and

swelling

ofmitochondriainthe

lung

capillary

endothelial cells.

Blunting

of themicrovilli of the

lymphocytes

wasalsoseen.These

changes

in

morphology

were not seenin

mice

given

buthionine sulfonenor were

they

found inmice

treated with BSO and GSHmonoester.

They

werefoundin

mice treated with BSO and GSH. Determinations of citrate

synthase activity

werecarriedout onthemitochondrial

frac-tionobtained from

lung;

after9

days

oftreatmentwith

BSO,

this

activity

was0.025±0.003

,umol/min

per mgof

protein

as

compared

with values of0.094 + 0.007 for controls. Mice treated with BSOfor3weeks and then allowedto recoverfor 8 weeks didnot show the

morphological

changes

described

above;

thecitrate

synthase activity

of themitochondrial frac-tion of

lung

wasaboutthesame asthatof untreatedcontrols.

Effect ofAdministrationof GSH Monoester and of GSHon GSHLevels.When miceweretreated with BSOfor 9

days,

there was as

expected

a marked decline in the levels of cellular GSHandof

plasma

GSH

(Table

1,

experiment

2vs.

experiment 1).

When GSH monoester was

given

together

withBSO

(Table

1,

experiment 3),

theliver GSHlevelswere about thesame as

the

levels in

controls,

andthe GSHlevel oflivermitochondriawassomewhat greaterthan thelevelsin controls. Administration of GSH monoester led to

higher

levelsofGSH in the

lung (experiment

3vs.

experiment

2);

the

mitochondrial fraction obtained from

lung

had GSH levels

thatwere

higher

thanthose in thecontrols

(experiment

3vs.

experiment

1).

TheGSH valuesfor

lymphocytes

treated with

BSO and GSHmonoester were

higher

than thosefound for the BSO-treated mice

(experiment

3vs.

experiment 2).

When GSHwasgiven

together

withBSO

(experiment 4),

very

high

plasma

levelsofGSHwerefound

(about

80 timesgreaterthan the control

values),

but there was

only

a

slight

increase

(perhaps

within thelimitsof

error)

oftheGSH levels of liver

and

lung,

ofthemitochondrial fractions obtainedfrom these

tissues,

and of the

lymphocytes.

As shown

previously,

ad-ministrationofGSHtountreatedmice has

only

a

slight

effect or no effect on liver and

kidney

GSH

levels

(32).

Similar

findings

were made in the present studies on

lung;

for

example,

untreated mice

injected

with GSH

(5

mmol/kg)

exhibited

lung

GSH levels thatwere

only

30% greater than those of controlsafter2 hrand wereabout thesame as the

controls after 4 hr. Administration of GSH monoester to untreated mice ledtolevels of livermitochondrial GSHthat were

significantly higher

(120%)

than thosefoundin

con-trols,

whereasadministrationof

equimolar

amountsof GSH didnotaffect livermitochondrial GSHlevels.

tThearteriovenous difference inplasmaGSH level acrosstherat

lungis smaller thanwasfound hereforthemousebecause there is

alowerplasmalevel of GSH in theratrightventriclethan in thatof themouse.EstimatesofGSHutilizationbyratlungand

kidney

(30)

basedon arteriovenous differences inplasmaGSH levels across

these organs andcardiacoutput(31)indicatethatkidneyand

lung

(4)

Proc. Natl. Acad. Sci. USA86(1989) 5299 f TZ

..

li tt- .*!

a.

*.

,,

FIG. 3. Representativeelectronmicrographsof thelungsof control mice(aandb)andmice treated with BSO for 21days (candd).cand d show lamellarbody(lb)swellinganddisintegration (one arrow),mitochondrial (m) degeneration (2 arrows),endothelial cell(ec)mitochondrial swelling,andthickeningof the basal membrane(bm). [aandc=x5400(horizontalbar in the lowerrightcorner=0.60

/Am);

b and d= x17,800

(horizontal bar in the lowerrightcorner=0.18Am).] DISCUSSION

Theproductionof cellular GSHdeficiency bytreatmentwith BSO hasadvantagesovervarious methodsofGSHdepletion

(compare ref.20). Thus, decreased GSH levelscanbe main-tained for relatively long periods, facilitating detection of structural and other abnormalities. Itseemssignificantthat the

effects of BSO on tissue and mitochondrial GSH levels,

skeletalmuscledegeneration (1),morphological changesin the

lung, anddepression ofweight gain (1, §)arevirtually

com-pletely preventedby giving GSHmonoesterbutnotby

giving

GSH. Although BSOishighly effective as aninhibitor,

rela-tively highdosesmustbegiventomaintain effective cellular

levels,andBSOisonly slowlytransportedintosome organ-elles[e.g.,mitochondria(18)]andtissues[e.g.,brain(20,

21)].

led us to examine theeffect of BSO administration onintestinal GSH levels. After 14 days of treatment with BSO (oral and intraperitoneal),thelevels of GSH inmousejejunalmucosa,colon mucosa, and pancreas were about4% of the controls. Electron microscopy showed microvillus and mitochondrial degeneration andvacuolization of theepithelialcellsofjejunalmucosa.Previous studies showed thatratjejunal mucosalcells have high levels of GSH(33).Itisprobablethat thenormalabsorptivefunctions of the gutrequireGSH.

§Theeffect of BSO on weight gain notedpreviously (1)was

con-firmed in the presentstudies; this effect is largely prevented by administration ofGSHmonoesterbut isnotaffectedby adminis-tration of GSH. ThefindingthatmicegivenBSOdevelopdiarrhea

(5)

Table 1. Effect of administration of GSH monoester and of GSH on GSH levels GSH levels

Liver Lung

Mitochondrial, Mitochondrial, Lymphocyte

Total, nmol/mgof Total, nmol/mg of total, nmol Plasma Exp. Treatment* ,Umol/g protein Jtmol/g protein per106cells total,

1LM

1 None 8.26 6.4 1.69 4.7 1.39 58.3

2 BSO 0.98 3.8 0.36 0.86 0.20 0.90

3 BSO + GSH monoester 8.29 10.8 0.71 10.0 0.45 43.0

4 BSO + GSH 1.10 3.9 0.42 0.87 0.24 5080t

*Mice were treated with saline (experiment 1) and with BSO (experiments 2-4) for 9 days. The BSO was given intraperitoneally(4mmol/kg)at10 a.m.and6 p.m. Inexperiment 3, GSHmonoisopropylester (5mmol/kg;1-1.3ml) was injectedintraperitoneallyas anisosmolarsolution (pH 6.5-6.8) twice daily (8 a.m. and 4 p.m.) for 9 days. In experiment 4, mice wereinjectedwithGSH,isopropanol,andNa2SO4in amountsequimolartotheester given inexperiment3.Mice (4-5 perexperiment) weresacrificedat 11 a.m. on day 9,and GSHdeterminations were done as described. Data are given asmeans;SDs were + 0.04-0.08fortissues and 0.2-480 for plasma.

tTheGSH levels in plasma (from rightventricle)were25,100 and 12,080

,.M

at 50min and 120 min, respectively, after GSH administration.

Themorphological changes observed here after treatment with BSO and their prevention by GSH monoester strongly suggestthat GSH isrequired forlung mitochondrialintegrity and for otheraspects of lung function. GSHprobably plays arole in theprocessingand storageoflung surfactant. The

highlevel of GSH in the alveolarepithelial liningfluid and related studies on this interesting finding (34) suggest that surfactant and GSH areofmajorimportancein the protection of thelungagainsttoxiccompoundsin theinhaledair.Export of GSHfrom such cellsasphagocytes,type 2cells,

lympho-cytes, and fibroblasts may contribute to the GSH of the alveolarliningfluid. The presentstudies revealanadditional aspectof theinterorgantransport of GSH in which plasma GSH(whosemajorsourceis theliver)isutilizedby the lung. Administrationof GSH ledtoextraordinarily highplasma GSHlevels(Table 1) but not to significantincreases in the GSH levels oflung,liver,orlymphocytes.Thesefindingsare in accord with the view that there is normally little or no transport of intact GSH into cells.AdministrationofGSHto control mice doesnotleadtoappreciableincreases in tissue GSH levels,andthe small increases thatareobserved most

likelymaybe ascribedtoextracellulardegradation,transport of theproducts,andintracellularsynthesis ofGSH(compare

ref. 20). Underphysiological conditions many cells export

GSH; the reverse of this process has not been observed. Theseconsiderations donotexcludethelikely possibilitythat administered GSH mightprotectagainst extracellularly ap-plied toxicity and that it can serve as a good source of cysteinefor GSHsynthesis.The substantialincrease in GSH levelsfound afteradministrationof GSHmonoesterconfirms

that thiscompound iswelltransportedand converted

intra-cellularlytoGSH(5, 32).Thehighlevelsof GSH foundinthe

mitochondrial fractions oflungandliver afteradministration ofGSH monoestersuggest thatthisagentmaybeusefulfor

protectionagainst various typesoftoxicity.

Wethank Dr. Donald A.Fischman,Mrs. LeeCohen-Gould,and Mr. James Dennis for valuable advice and help in the electron microscopystudies and Dr.Carl G. Becker forproviding equipment forleukocyte counting.This workwassupportedin partbyfunds from the National Institutes of Health(Grant2 R37DK-12034).J.M.,

on leave from the Department ofClinical Chemistry, University Hospital,Link6ping,Sweden, acknowledgesgenerous supportfrom the Swedish MedicalResearchCouncil (05644-06B),ToreNilsson Research Fund, Throne-Holst Foundation, Sweden-American Foundation,SwedishSocietyofMedicine,Medical Research Coun-cil of theSwedishLife InsuranceCompanies, S-O Liljedahl Foun-dation, Ollie and Elof Ericssons Foundation, andthe Albert and NannaSkantze Foundation.

1. Mfrtensson,J. &Meister, A. (1989) Proc.Natl.Acad. Sci. USA86, 471-475.

2. Griffith, 0. W., Anderson, M. E. & Meister, A. (1979) J. Biol. Chem. 254, 1205-1210.

3. Griffith, 0. W. & Meister, A. (1979) Proc. Natl. Acad. Sci. USA 76, 2715-2719.

4. Griffith, 0. W. (1982) J. Biol. Chem. 257, 13704-13712.

5. Anderson, M.E., Powrie, F., Puri, R. N. &Meister, A.(1985)

Arch.Biochem.Biophys. 239, 538-548.

6. MArtensson,J. (1986)Metabolism35,118-121.

7. English, D. & Andersen, B.R. (1974)J. Immunol. Methods 5, 249-252.

8. Boyum,A.(1968) Scand.J.Clin.Lab. Invest. 21,97-102.

9. Mirtensson, J. (1987)J.Chromatogr. 420, 152-157.

10. Anderson,M.E. &Meister,A.(1980)J.Biol. Chem.255,9530-9533. 11. Anderson, M. E. (1985) MethodsEnzymol.113,550-551.

12. Cross, C. E., Watanabe,T. T.,Hasegawa,G.K.,Goralnik,G.N., Roertgen,K.E.,Kaizu, T., Reiser,K.M.,Gorin,A. B.&Last, J.A.(1979) Toxicol.Appl.Pharmacol. 48,99-109.

13. Marklund, S. (1979)Clin. Chim.Acta92,229-234.

14. Smith,J. R.(1974)J.Lab.Clin. Med.83, 444-450. 15. Srere,P. A. (1969)MethodsEnzymol. 13, 3-11.

16. Robinson, J.B., Jr, & Srere, P. A. (1985)J. Biol. Chem. 260,

10800-10805.

17. Bradford, M.M.(1976) Anal. Biochem.72,248-254.

18. Griffith,0.W.& Meister,A.(1985) Proc.Natl. Acad.Sci. USA82,

4668-4672.

19. Nedergaard, J. & Cannon,B.(1979)MethodsEnzymol.55, 3-28.

20. Meister,A.& Anderson, M. E. (1983)Annu. Rev.Biochem. 52, 711-760.

21. Griffith, 0.W.&Meister,A.(1979)Proc.Nat!.Acad.Sci.USA 76, 5606-5610.

22. Orlowski,M.&Wilk,S.(1975)Eur.J. Biochem.53, 581-590. 23. Tate, S. S.&Meister,A.(1985)MethodsEnzymol. 113,400-404. 24. Berggren,M.,Dawson,J.& Moldeus,P.(1984) FEBS Lett. 176,

189-192.

25. Dethmers,J. K.&Meister,A.(1981)Proc.Nat!. Acad.Sci.USA 78, 7792-7796.

26. Novogrodsky, A., Tate, S. S. & Meister, A. (1976)Proc. Nat!.

Acad. Sci. USA 73, 2414-2418.

27. Dawson, J.R., Vahikangas, K., Jernstr6m, B. & Moldeus, P.

(1984)Eur.J.Biochem. 138,439-443.

28. Foster,H.L.,Small, J. D.&Fox,J.G., eds. (1983) TheMouse in BiomedicalResearch, NormativeBiology, Immunology,and

Hus-bandry (Academic,NewYork), Vol.III.

29. Meister,A.(1983)in FunctionsofGlutathione-Biochemical, Phys-iologicalandToxicologicalAspects, eds. Larsson, A.,Orrenius,S., Holmgren,A.&Mannervik,B.(Raven,NewYork),pp. 1-22.

30. Anderson, M.E., Bridges, R.J. & Meister, A. (1980) Biochem. Biophys.Res.Commun.96,848-853.

31. Altman, P. L. & Dittmer,D.S., eds. (1974) BiologyDataBook (Fed.Am.Soc. Exp.Biol., Bethesda, MD), 2ndEd., Vol.3. 32. Puri,R.N. &Meister,A.(1983)Proc.Nat!.Acad. Sci. USA80,

5258-5260.

33. Cornell,J.S. &Meister,A.(1976)Proc.Nat!.Acad.Sci.USA73, 420-422.

34. Cantin, A.M., North, S.L., Hubbard, R.L. & Crystal, R.G. (1987)J.Appl.Physiol. 63,152-157.

Figure

Updating...

References

Updating...

Related subjects :