Vol. 182, No.

2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 31, 1992 Pages 906-912

REGULATION OF INTRACELLULAR Mg= BY SUPEROXIDE IN AMNION CELLS

NobuyukiMasnMoto, * Jirou Mizuki, AkiraMiyakeandOsaluTanizawa

Heiichi Tasaka,

Department
of ObstetricsandGynecology,
OsakaUniversityMedical
School,l-l-50, Fuknshiwa,
Fukushimakn,
Osaka553, Japan

Received December 16, 1991

Changesof intracellular free Mg concentration([Mg]i) in humanarnion cells inducedby superoxideanion

were determined using a highly v*-sensitive fluorescent dyeMga-fnra2or M$*-indol. Superoxideanion,
producedby addition of xanthine oxidaseto hypoxanthine,induceddecreaseof [Meli. The decreasewas
significantly inhibited by an anion channelblocker, 4,4diisothiocyano-2,2disulfonic acid stilbene (DIDS).
Superoxidedisnutase(SOD),injected into cells by cell fusion, also inhibited the changeof [MgZli, but
catalasedid not. Superoxideanion inducedprompt increaseof intracellular pll (pili) as well as decreaseof
[Mg]i and subsequentlyactivated the increaseof intracellular free Ca ([Cali) and the release of ara-
chidonate. In contrast to superoxideanion, f&Cl whichinduces increaseof pHi in annion cells increased
[M$]i. The elevation of basal level of [H<li by Mg-ionophoreinhibited the changeof [Ca*]i and the
releaseof arachidonateinducedby superoxideanion. Theseresults suggestthat superoxideanion, transported
throughanionchannelsinto cells, decreasesIMgali directly, not due to a pH-effect and that the decrease
of [@Ii mayregnlate biological functionsof the cells via increaseof [Ca]i. 0 1992 Academic Press, Inc

Recently, the intracellular free Mg concentration ([MgZ]i) has been considered to play an

important role in regulation of cell functions. White and Hartzell showed by the whole cell

patch clamp technique that change in (Mgli fron 0.3 to 3.0 11 caused 50% decrease in the

voltage-gated CaZ current in isolated cardiac ayocytes (1).

CAMP-dependent Moreover Meissner

and Hendersonfound that [Mg]i nodulates calcium release from the sarcoplasnic reticulum (2).

Various compounds Modulating Mg influx and efflux have been reported (3,4). But the

significance of change in [Mgli is not yet clear, and little is known about biological

materials that change IIg2li. Recently, the developnents of a Mg-sensitive fluorescent dye

Mg-fura (5-7) or Mg-indol, which is not affected by the intracellular free Ca

concentration ([Ca]i), and a digital imaging aicroscopic system have allowed direct neasurewent

of [MgZ]i in single cells. Therefore we used this procedure to measure [Mg2]i in cultured

hunan annioncells.

Superoxideanion is consideredto have variouseffects in various tissues including brain, heart and

kidney, althoughits nodeof action is not yet clearly understood. Wehave reported that superoxideanion

*
To whom reprint requests should be addressed.

0006-291X/92 $1.50
Copyright 0 1992 by Academic Press, Inc.
All rights of reproduction in any form reserved. 906
Vol. 182, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

increases[Cali in humanvoletrial cells without causingcell injury (8) and that it also induces in-

creasesof intracellular pll (plli), ]Ca]i and releaseof arachidonatein humana1nioncells (9). In this

study, we deterlined the effects of superoxideanion on ]M$*]i and clarify the roles of [M$+]i as an intra-

cellular modulator.

MATERIALS AND METHODS

Materials NgZ*-fura2,MgZ-fura2-AH,Mga-indol and I$-indol-AM were obtained fro1 MolecularProbe.

Trypsin, hypoxanthine(BX),xanthine oxidase(X0) and 4,4diisothiocyano-2,2disulfonic acid stilbene @IDS)
were obtained fro1 Sigma. Indol, indol-AM,superoxidedisiutase (SOD),catalase and NB&l were obtained
from WakoPureChe1icalIndustries (Tokyo,Japan). Mg2+-ionophore (N,N-Diheptyl-N,N-dilethyl-1,4-butane-
diaaide) was obtained fro1 Fluka Cheiical Industries (Tokyo,Japan). BVJ (Sendaivirus) was gifted from
Dr.Haneda(OsakaUniversity).

Cell preparation Amnioncells werepreparedas follows : Theieibrane wasrinsed with Hanksbalanced

salt solution and choppedinto 5-10 11 pieces. Thepiecesweredigestedtwice with 0.25%trypsin supplelent-
ed with penicillin (200 units/Ill, streptonycin (200.~g/11), 0.1%bovine serumalbuminand 20 1H HEPES in
aediua 199 at 37 C for 40 1in. Cell aggregateswere dissociatedby gentle pipetting in phosphate-buffered
saline (PBS). Non-dispersed fragmentswereseparatedby filtration throughnylon resh. For removalof red
blood cells, cell suspensionscontainingapproxilately107cells in 2 11 werelayeredonto 10 11 of 90%Percoll
and centrifuged at 500x g for 10 iin. The isolated cells werewashedand counted. Saiplesof 0.5 x 10
cells wereplacedon 0.14 11 glass coverslipswhichweresealedundera 1.0 c1 diameterhole in the bottoa of
speciallydesigned35 II culture dishes. Thecells were1aintainedat 37Cin an atmosphere of 95%air-5%COZ
in 1ediu1199with 10%fetal bovineseru1andwereusedfor experiientsafter 2-3 days.

Measurelentof (NgZ]i -~
and ]Ca*li b video-recordingor laser-scanning(10) Cells wereloadedwith a Hague-
sium-sensitivefluorescent dye, MgZ-fura2-AN (5 fi M), in aediur 199 at 31C for 60 min. The [Mgli of
a1nioncells was1easuredat 100isec intervals with a digital i1aging microscopicsystemH-1000(Inter deck.
Inc. Japan).Theratio of the intensities of fluorescenteiission at 510 nmwith excitation at 335 nmand 370
nr was1easured. The fluorescentexcitation bea wasled to drop on the cells, and euission imageswere re-
cordedwith videofilm and analyzedwith a coiputer.
In anotherexperiient, cells wereloadedwith a magnesium-sensitivefluorescentdye, Mg-indol-AN(5,~M),
in 1ediu1199 at 31Cfor 60 1in. The[M~*]i of cells loadedwith If-indol-AM wascalculatedfro1 data ob-
tained with a digital scan-iiagingliicroscopicsyste1, ACAS470workstation. Cells werescannedby a 1 .UI
spot of an argonion laser bea at 345n1. hissions at 405n1and485n1fro1 the illulinated spot on the cells
weredirected to a sensitive photo1ultiplier tube, and the photo-currentwasmeasured with a high-speeddata
acquisition interface. The [w]i in variousportions of single cells was calculatedfrom the ratio of the
fluorescencesat the two e1issionwavelengths using a standardcalibration curve of Ng concentration.
[Ca*]i wasdetenined from the ratio of eiission fluorescencesat 405 nmand 485n1 on excitation at 355 nm
with indol-AMby the sa1e1ethod as for deteriination of [Mg2li. Solution in whichNa wasreplacedby
cholinewaspreparedas Na* free solution.

Releaseof arachidonate For aeasureaentof the releaseof arachidonate, the cells werepreincubated with
--
[HI arachidonate( 0.25 .UCi/11, 100 Ci/1101 ; NewEnglandNuclear)for 2h to allow incorporationof [HI
arachidonateinto esterified lipids. Thecells werethen washedfour tiles with PBSand incubatedwith vehi-
cle or test agents. The1ediu1 wasthen extractedwith ethyl acetateand arachidonatewasanalyzedby thin-
layer chroiatography.

Erythrocyteghosts---
and cell fusion As describedpreviously (ll), Erythrocyteghosts loadedwith SODwere
prepared. A 1ixture of 0.211 of packederythrocytesand 1.811 of 20 units SODor 200 ,Ug catalasein reverse
PBSwasdialyzed against 48011of g-fold diluted reversePBScontaining4mNMgCIZfor 301in at 4C. After
further dialysis against 50011of isotonic PBSfor 301in at roo1 telperature, the erythrocyte ghostswere
washed4 times. 0.511 of 20%erythrocyteghosts and0.511 of the inactivated HVJ(POOOHAU/al) wereaddedto
cultured cells on a glass coverslip. The1ixture waskept at 4C for 201in for cell agglutination and then

907
Vol. 182, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

shakengently at 31Cfor 20minfor cell fusion betweenghostsand cells (12,13). Theamnioncells werewashed
4 times to eliminate non-fusedghosts. Thesetreatmentsallowedinjection of SODor catalaseinto cells. It
wasdeterminedby the cytochrome C method(14) for SODand a spectophotoaetricmethod(15) for catalase that
the treated cells contained20-fold SODand la-fold catalase activity respectivelyof non-treatedcells 2 h
after cell fusion. Thus,we examined[Mf]i studies within 2h. Thesetreatmentsdid not affect the basal
levels of [Ca*]i, [Mg*li and pHi.

Analysisof data Data are shownas meanvalues plus or minusthe standarderror of the mean of multiple
deterli&iG in at least three replicate experiments. Homoscedasticity of data was analyzed
by Bartletts test. The significance of differences was assessed by analysis of variance or
Kruskal Wallis test, followed by multiple comparisonsby the method of Dunnett or scheffe and a
P value of less than 0.05 wereconsideredsignificant.

RESULTS

Snperoxide
wasproducedby addition of xanthine oxidase to hypoxanthine@X-X0]. The basal level of

[rag*]i in annioncells was0.76 + 0.02 (n=20]. HX(lmM)-X0(10

munits/al] induced decreaseof ]Vli within

10 set after the addition of xanthineoxidaseand [MgIi then slowly returned to a steadylevel of nearly

the basal level after 3-4 min but did not completelyrecoverthe pre-level (Fig.lA). The distribution of

[Mgli in the resting cells wasshownin Fig.%A. Decrease

of [MgZli wasobservedin almostarea 1Osecafter

addition of HX-X0(Fig.%B]. 4minafter addition HX-X0,]Mgli partially returnedto the basal level (Fig.ZC)

This decreasein [Mgzli was significantly inhibited by the pretreatmentof a scavengerof superoxideanion,

SOD(10 units/ml] or an anion channelblocker, DIDS(100~M] (Fig.3). Furthermore,using cell fusion tech-

nique, we determinedthe effects of superoxideanion, transportedinto cells, on [Mg*li and the release of

arachidonate. SODinjected into cells reducedthe changeof [Mg*]i and the releaseof arachidonateinducedby

HX-X0,but intracellular catalase ( a scavengerof hydrogenperoxide) did not affect them(Fig.4). As Lynch

and Fridovich reported that superoxide crossesthe membrane

through anion channels(16,171, our results

suggestthat superoxide induces decreaseof ]MgZli after its transport into the cytosol. To determine

whethersuperoxideactivates Na/Mg exchanger, weexaminedits effect on [Mgli of amnioncells in Na

A 1.1
B
1.5
x0 NH&I
1 0.9 1 1.3 1
-5
.- 1.1
+ 0.7
b
E
0.5 lisl 0.7 LTSI
0.9
0 100 200 0 100 200
Time kec) Time (sac)
Fig. 1 Tile courses of change of [Mg*li in single cells of cultured human annion. (A) Addition of
-1
xanthlne oxidase (XO:lOmits/ml) to hypoxanthine(InM) induced decreaseof [Mg]i in an amion cell. This
changeoccurred within 10 set after stimulation. The recovery to almost the basal level wasslow taking 3-4
min. (B) NIL&l induced increase of lMg*li in an amnioncell.

908
Vol. 182, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

C g) 0.1
E,

02 3
7
0.0

0 =20mM =OmM

F&2. Distributions of [Mg2]i in various portions of single cells were shownby computerimage graphics.
[Mg**]i is codedin ranging through the spectrua from white to black, as calibrated in the right hand scale.
Changesof [Mg*]i in annion cells was shown:(A) Before stionlation, (B) 1Osecafter addition of xanthine
oxidase and (C) 4minafter addition of xanthine oxidase.
F&. Changesof [Mg*]i induced by hypoxanthin (BX:laM) - xanthine oxidase (XO:lOmunits/al) in various
treatments. The cells were pretreated with superoxide dismutase(SOD:lOU/ml)or the anion channel blocker,
4,4vdiisothiocyano-2,2, disulfonic acid stilbene (DIDS:lOOpM) for lOoin. The changeof [Mg]i induced by
addition of xanthine oxidase to hypoxanthine(BX-X0)wassignificantly inhibited by SODor DIDS, respectively.
The extracellular mediumwas replaced by minimal Na medinnor nediumwith a high Mg concentration (20~)~
and the cells were stimulated by BX-X0 2Oseclater. Thesemedia, in which [Mg*]i increases slowly, but
not appreciably within lOOsec, did not affect the changesinduced by BX-X0. Dataare means + S.E (n=lO).

free-solution. Omission of Na fro1 the Bedim resulted in gradual increase of [Mg*]i, indicating the

existence of Na/Mg exchanger in alnion cells. But the absence of extracellular Na did not inhibit the

decrease of [M$]i induced by superoxide (Fig.3). A high extracellular concentrationof Mg2(20aM)

also did

not affect the changeof [Mg*]i inducedby superoxide(Fig.3). Theseresults suggest that superoxide did

not activate [Mgli efflux. Both snperoxideand&Cl increasepHi and subsequently[Ca*li in amion

cells (9). But unlike superoxideNH.&1increase[Mg2li (Fig.lB), whichindicates that the changeof [Mfji

A B
3000
0.:3-

t
z

0.:z-

f
!G
.-
r
Or
$ 0.l-

0.cI- 0
control SOD i CAT i control SOD i CAT i

FiJJ. Changes of [Mg*]i (A) and the release of arachidonate (8) induced by hypoxanthine- xanthine oxidase
in the injection of scavengersinto cells. Intracellular SOD(SODI ) and catalase (CATL ) were injected by
cell fusion technique. Data are mean+ SE (n=lO).

909
Vol. 182, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mg ionophore

m. Effects of pretreatment with Mg**ionophore on changein [Ca*li and arachidonate release induced by
superoxide. The basal level of (Mg*]i increased ou treataent with Kg ionophore (l-100~ M) in 5rWMgZ
solution for 1Oain (lower). 1-10~ M Mp* ionophore did not affect the basal level of [Ca*]i but 1OObM mg*
ionophore increased [Ca*li (upper). After treatment with these solutions, the media were replaced with
solution containing 1aM I$, resulting in a high [Kg*]i and low extracellular Kg*. The cells were then
proaptly stimulated by hypoxanthine (1N) and xanthine oxidase (lOuunits/al) @X-X0), and the increase of
[Ca*li (peak value) and the release of arachidonate for 30 nin after stiaulation were measured. The increase
of [Ca*]i and the release of arachidonate were inhibited by pretreataent with Mg2* ionophore (upper and
niddle). Thebasal release of arachidonate of control cells did not change with changeof [Mg]i. Data are
leans * S.E.

induced by superoxide is not dependent on pHi. The influence of pH (6.8-8.2) on the fluorescence of Me-

indol was ninilal with Mg concentrations of below 0.02 IM in cells. Wepreviously suggested that the

increase of [Ca+li induced by superoxide way be partially dependent on change of pHi (9). MgYelective

Idga-ionophore (18) (below 10~ M) alone, which did not affect [Ca*]i, increasedthe basal level of [Mg]i.

To deteruinewhetherthe changeof [Mga]i affects [Ca]i, we pretreated aunioncells with w-ionophore

(l-100~ M] for 10 uin in 5 ul Mg* solution, washedthemand then prouptly addedHX(luM]-X0(10

uunits/ul) in

control solution (np,=lti). The increaseof [Ca*li and the releaseof arachidonateinducedby HX-X0were

significantly inhibited in high [Ng?]i cells as comparedwith valuesfor control cells of low [Ig2]i in the

sale solution (Fig.5). Theseresults suggestthat the decreaseof (&?+]i inducedby superoxideuay activate

the increaseof ]Cali.

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Vol. 182, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION

Intracellular MC+hasbeenconsideredto play importantroles in energywetabolisaand as an intracellular

signal. In this study we showedthat superoxideinduces decreaseof ]Mg2li, whichresults in increaseof

]Cali. Wehavereported that the increaseof [Ca]i inducedby superoxideinduces releaseof arachidonate

(9). Thuswe suggestthat decreaseof [Mg2li nay be an initial processin the biological function of annion

cells. In heart cells, [Hg Ii is reported to increasemarkedlyduring ischeaia and to decreaseagain on

reperfusion (19,20). Injury of the central nervoussystemor spinal cord causes decreaseof [Mg]i in re-

gionsof injury (21-23). Thesedecreasesof [Wli on reperfusionof heart cells or injury of nervoustissue

lay also be causedby productionof superoxide. Possibleaechanisasof the changeof [Mg]i maybe as fol-

lows ; (i) changein influx or efflux through the aeabrane,(ii) changeof intracellular binding sites to

aewbranes(24), (iii) mobilizationof Mg* to the endoplasaicreticulum (ER)(25), and (iv) a chemicalre-

sponseof Mg to superoxide. In this study, we found that the response of [Mg2li to superoxidewas not

dependenton either an M$/Na exchangeror a I$ gradient acrossthe cell membrane,

whichindicates that

superoxidedid not affect MgZ+efflux. With regard to the secondwechanisa

mentionedabove, Corkeyet al.

showedthat nest intracellular Hg is in a boundfora and that a small changeof binding sites inducesa

large changeof [Mg*]i (26). In connectionwith the third wechanisa

it is notedworthythat, Baunann
et al.

reported that Mg* can balance changeof Ca* aoveuentin bee photo receptors (25). Theyshowed that
release of Ca from the ERis associatedwith a significant increase in the Mg content of the ER.

However,it is not yet clear whetherthe decreaseof IMg*li inducedby superoxidewascausedby changeof

its binding to newbranes,its uptakeby the ERor a cheaicalresponseof superoxide.

Increaseof [MgZ+]iis reported to inhibit CaZinflux throughthe aeabrane(1,27,28) or CaZ*release from

Ca2*stores (2). But there are no reports on whether decreaseof [lg2li belowthe physiologicallevel

affects [Ca*li. Our data showthat superoxideinduced decreaseof ]M$]i, subsequently activating

[Ca*]i increase. Thesefindings suggestthe aechanisaof the effect of superoxideon cellular functions.

REFERENCES
1. White,R.E.andHartzel1,C.S.(1988)Science239, 778-780
2. Meissner,G.andHenderson,J.S. (1987)J.Biol.Cheu.262, 3065-3073
3. Grubbs,R.D.andkguire,M.E, (1987)Magnesium6, 113-127
4. Flataan,P.W.(1984)J.Meabr.Biol.80, 1-14
5. Ra,ju,B.,Murphy,E.,Levy,L.A., Hall,R.D., andLondon,R.E.(1989)h.J.Physiol. 256, C540-548
6. Murphy,E.,Freudenrich,
C.C., Levy,L.A.,London,R.E.,andLieberuan,M.(1989)
Proc.Natl.Acad.Sci.USA.
86, 2981-2984
7. Quawwe,G.Aand Rabkin,S.W.(1990)Biochew.Biopbys.Res.Coaaun.167, 1406-1412
8. Masuaoto,N.,Tasaka,H.,Miyake,A.,andTanizawa,O. (1990)J.Biol.Chew.265, 22533-22534

911
Vol. 182, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

9. Ikebuchi,Y.,Masuwoto,N., Tasaka,H.,Hoike,K.,Kasahara,K.,Miyake,A.,andTanizawa,O.
(1991)J.Biol.Chew.266, 13233-13237
10. Masumoto,N., Tasaka,K.,Basahara,B., Miyake,A.,andTanizawa,O. (1991)
J.Biol.Chew.266, 6485-6488
11. Furusawa,M., Yamaizuwi,M., Nishirra,T., Uchida,T.,andOkada,Y.(1976)
Methodsin Cell Biology14, 73-80
12. FnrusawaM.,Nishinra,T., YawaizuwiM.,andOkada,Y.(1974)Nature 249, 449-450
13. YasaizuziM.,Furusawa,M., Uchida,T.,Nishirura,T., andOkada,Y.(1978)
Cell.Struct.Funct.3, 293-304
14. Bulter,J., Jayson,G.G.,andSwallow,A.J.(1975)BiocheM.Biophys.Acta 408, 215-222
15. Beers,R.F.Jr.and Sizer,I.W. (1952)J.Biol.CheM.195, 133-140
16. Lyuch,R.E.andFridovich,I. (1978)J.Biol.Chew.253, 1838-1845
17. Lynch,R.E.andFridovich,I. (1978)J.Biol.Chew.253, 4697-4699
18. Lanter,F. Erne,D.,Awwann,D., andSiwon,W.(1980)Anal.Chea.52, 2400-2402
19. Hirkels,J.H., Echteld,C.J.A., andRuigrok,T.J.C. (1989)J.Mol.Cell.Cardiol. 21, 1209-1218
20. Murphy,E.,Steenbergen,C., Levy,L.A.,Raju,B., and London,R.E.(1989)
J.Biol.Chea.264, 5622-5627
21. Vink,R., Mclntosh,T.B.,Dewediuk,P.,andFaden,A.I.(1987)
Biochew.Biophys.Res.Comun. 149, 594-599
22. Vink,R., Mclntosh,T.K.,Dewediuk,P., Weiner,M.W., andFaden,A.I. (1988)
J.Biol.Chem.263, 757-761
23. Vink,R., Yul,S.W.,Lewke,M.,Dewediuk,P.,andFaden,A.I.(1989)Brain.Res.490, 144-147
24. Veloso,D.,Guyun,R.W., OskarssonM.,andVeech,R.L.(1973)J.BioI.CheM.248, 4811-4819
25. Bauaann,O., Walz,B.,Sowlyo,A.V.,andSow1y0,A.P. (1991)
Proc.Natl.Acad.Sci.USA.88, 741-744
26. Corkey,B.E.,Duszynski,J., Rich,T.L., Matschins$,B., andWillianson,J.R.
(1988)J.Biol.Chew.261, 2567-2574
27. Agus,Z.S.,Kelepouris,E.,Dukes,I., andMorad,M.(1989)Aw.J.Physiol.256, C452-455
28. Hartzel1,H.C.andWhite,R.E.(1989)J.Gen.P$siol. 94, 745-767

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