Effects of active oxygen generated by DTTFe2+ on cardiac Na+Ca2+ exchange and membrane permeability to Ca2+

J Mol

Cell

Cardiol21,

1009-1016

(1989)

Effects of Active Oxygen Generated by DTT/Fe2+ Permeability Na + /Cal + Exchange and Membrane Zhong Q. ShP, Allan J. Davison*

on Cardiac to Ca* +

and Glen F. Tibbitsl*

Cardiac Membrane Research Lab.’ and Bioenergetics Lab.2, Kinesiology, Simon Fraser University, Burnaby, BC, Canada V5A IS6 (Received 9 November 1988, accepted in revisedform 31 May 1989) SHI, A. J. DAVISON AND G. F. TIBBITS. Effects of Active Oxygen Generated by DTT/Fe’+ on Cardiac Na+/Ca 2+ Exchange and Membrane Permeability to Ca2+ Journal of Molecular and Cellular Cardiology (1989) 21, 1009~-1016. Sarcolemmal vesicles isolated from bovine heart were preincubated at 37°C with an oxygen radical generating system consisting of 1 mM dithiothreitol (DTT) and 50 PM FeS04. Exposure of the vesicles for 1 to 40 treatment decreased the apparent K,,, for mins stimulated Na’/Ca2’ exchange about 2.5-fold. The DTT/Fe2+ Ca*+ ofNa;-dependent Ca’+ uptake by 80?, (from 63 to 13 PM). The effect on V,,,,,was much smaller however. The resulting stimulation of exchange activity was diminished by the presence of desferrioxamine (957,;) or catalase (SOY/,). In contrast, superoxide dismutase and sodium formate did not prevent the effects ofDTT/Fe*+ on the exchanger. Neither Zn2+ nor Gas+ could replace Fe2+ in the stimulation ofNa+/Ca2+ exchange. Passit v exchange to continue to plateau values and then Ca2+ efflux was determined by first allowing Na+/Ca*+ diluting the loaded vesicles in the presence of EGTA. Ca ” leakage from the vesicles was slightly but significantly (P < 0.05) increased by the action of DTT/Fe’+, the rate constants for the passive Ca2+ efflux being 0.22 and 0.26/min in control and treated groups, respectively. The calcium loading observed in myocytrs in exchange by active oxygen ma) ischemia/reperfusion injury suggests that the stimulation of Na+/Ca*+ moderate the myocardial response to oxygen mediated injuries including ischemia/reperfusion injury. However, the clinical relevance of these phenomena is far from clear as thr stimulation depends in part on the K,,, for Ca2 ’ prior to treatment.

2. Q

KEY

Na+/Ca*+

WORDS:

exchange;

sarcolemma;

oxygen

free radicals;

myocardium:

Ca’+

permeability.

Introduction

Active oxygen has been suggestedto mediate myocardial ischemia/reperfusion injury [ZO,II]. Speciessuch asH202, . O,, and . OH [I]. At least three distinct sarcolemmal pro- accelerate membrane lipid peroxidation. teins control this flux: voltage-dependent causing impaired membrane function and inCa2+ channels, Na+/Ca2+ exchange, and an hibiting some integral membrane-bound enATP-dependent Ca2+ pump [Z]. Sarco- zymes [12-141. lemma1 Na+/Ca*’ exchange is an antiport Recently, Reeveset al. [15] found that prosystem with a coupling ratio of 3 : 1 [3, 41, In oxidants stimulate Na+/Ca’+ exchange b>the heart, Na’/Ca2’ exchange is a primary decreasingK,,, for Ca’+. This important findmeansof efflux of Ca*+ [3, 41. However, the ing prompted the current investigation of a dependence of the antiporter on membrane number of questionswhich were not answered potential, ENa and Ec,, suggests that in the initial study. These include: (a) whether Na+/Ca2+ exchange also promotes the net or not the treatment changed the V,,,,,; (b) entry of Ca2+ into the cell during the pro- whether the effect would be seen in preparlonged depolarization of the cardiac action ations in which the K,,, (Ca*+) (untreated1 potential [.5J. In cardiac sarcolemmal vesicles wasmore typical than in previous studies,and Na+/Ca*+ exchange is very sensitive to mem- (c) whether there were effectson passiveCa*+ brane lipid composition [4, G-91. permeability in cardiac sarcolemmal vesicles. Transsarcolemmal to the regulation

*To

whom

Ca* + transport of myocardial

all correspondence

0022.2828/89/101009

should

+ 08 $03.00/O

is crucial contractility

bc addressed. cl 1989 Academic

Press Limitrd

1010

Z. Q

Shi

Because of the potential clinical importance of this observation, the current experiments were designed to investigate the above issues, as well as the universality of the phenomena. The oxygen free radical generating system studied increased both the initial rate of Naf/Ca2+ exchange and passive Cazf permeability in sarcolemma from both bovine and rat ventricles. Materials

and Methods

All experiments were performed on cardiac sarcolemmalvesiclesisolated from bovine ventricles unless noted otherwise in which rat ventricles were used. The rat sarcolemmal vesicleswere isolated by the procedure of Bers [16] as modified by Tibbits et al. [17]. Bovine hearts were obtained from a local slaughterhouse and approximately 60 g of ventricles trimmed of fat and connective tissue were used.The ventricular tissuewas homogenized with a Waring commercial blender at high speedfor one burst of 30 s. The solutions and the remainder of the isolation procedure were identical to that described for the rat [17j. Sarcolemmal vesicles for both specieswere harvested from the interface of 28 and 30% (w/w) sucrose from a discontinuous sucrose gradient. This fraction was diluted, spun down ( 177000 x g; 60 min), resuspendedin 140 mM NaCl and 12.5 mM 3-(N-morpholino) propanesulfonic acid (MOPS) (pH 7.4 at 37”C), and then stored in liquid nitrogen at - 190°C in small aliquots. Protein was determined by the method of Bradford [18] and the sarcolemmal marker K+-stimulated pnitrophenylphosphatase (K+ pNPPase) was assayedaswe have described previously [19]. Na+/Ca*+ exchange was determined as Nat+-dependent Ca*+ uptake using a technique that has been described [ZU]. A 5~1 aliquot of Na+-loaded sarcolemmal vesicles (N 3 mg protein/ml) was suspended on the wall of a polystyrene tube containing 245 ~1of uptake medium maintained at 37°C. The medium contained either 140 mM KC1 or NaCl, 12.5 mM MOPS (pH 7.4 @ 37”C), 0.4 pM valinomycin, 2.58 @i 45Ca2’ and a concentration of 40Ca2+ ranging from 10 to 160 PM. The uptake was initiated by vortex mixing and quenched at a preset time by a custombuilt device which quenchesthe exchange by

et al.

the automatic addition of 30 ~1 of a solution containing 140 mM KC1 and 1 mM LaCls. An aliquot of 220 ~1 was then filtered (Millipore0.45 PM) and the filters were washed twice with 3 ml of a rinsing solution (containing 140 mM KC1 and 1 mM LaCls). The filters were then dried and counted by standard liquid scintillation procedures in a Beckman LS7000 counter. The uptake by vesiclesdiluted into NaCl represented blanks (absence of a Na+ transsarcolemmal gradient) and was subtracted from that obtained with K+ dilution. This corrects for any 45Ca2+ counted that was bound superficially to the sarcolemma or that which permeated the vesicle by some pathway other than Na+/Ca*+ exchange. Sarcolemmal vesicles were incubated with an oxygen free radical generator, which consistedof 1 mMdithiothreitol (DTT) and 50 pM FeS04 unlessstated otherwise. Control sarcolemmal vesicles were incubated at 37°C with 50 pM ZnS04 or Ga2(S04)s, or with no additives for 40 mins (unlessstated otherwise). Any scavengerswere added to the incubation mixture prior to addition of the test reagents. The time course of Na+/Ca*+ exchange was determined as described above but the extravesicular Ca*+ concentration was 10 PM, and the uptake was quenched at times ranging from 2 to 120 s. The characteristics of Na+/Ca*+ exchange in both treated and control groups were invesvarious tigated in media containing [40Ca2+]e by quenching the uptake after 2 s. The rate of uptake during this period of time represented the initial rate. The effect of the oxygen free radical generator on the apparent X, for Ca*+ and the V,,,,, was then determined using an Eadie-Hofstee plot of the data. PassiveCa*+ eIIlux from sarcolemmal vesicles was measured as described previously [20]. The vesicles were first loaded with 45Ca2+ by Na+/Ca’+ exchange (with [Ca2+lo being 10 PM) for 120 s. Efflux was initiated by a ten-fold dilution with a solution containing 140 mM KC1 and 1 mM EGTA. Aliquots (400 ~1) of this dilution were taken at 15, 60, 120, 180 and 240 s and then filtered, rinsed and counted as described above. The Ca*+ content of the vesiclesafter theseperiods of efflux was expressedas a percentage of that at time zero.

Active

0,

and

Na+/Ca*

l&L

The 45CaZ+ was purchased from DuPont (New England Nuclear). DTT, catalase, and EGTA were purchased from Sigma Chemical Company. Superoxide dismutase was purchased from Boehringer Mannheim GmbH, Penzberg Control Laboratory; Gaz(S04)s was purchased from Fluka. Stock solutions of FeS04, Gal(S04)s, ZnS04 and DTT were prepared immediately before the experiment each day. Results

Unless stated otherwise, all results were obtained from bovine cardiac sarcolemmal vesicles. The time course of sarcolemmal Na+dependent Ca’ + uptake in responseto treatment with 1mM DTT and 50~~ FeS04 is shown in Figure 1. Uptake was linear in both groups for at least 3 s (Fig. 1 inset). During this period of linear uptake, the treated group exhibited velocities about 250% higher than the control group although the steady-state vesicular Ca2+ content was not significantly different between the two groups. Pretreatment with DTT (1 mM) and FeS04 (50 PM) resulted in a significant stimulation of exchange at all extravesicular Na+/Ca’+ Ca2+ concentrations lessthan 80 ,UM,asshown in Figure 2(a). Stimulation at low Ca2+ concentrations was more dramatic than at higher

2.5-

0

Tl

I

I x1

I 40

I 60 t(s)

I 80

1 100

1 J 120

FIGURE 1. Time course of Na’-dependent Ca2* uptake after incubating sarcolemma with DTT (1 mM) and FeS04 (50 1(M) for 40 min at 37°C. The uptake was determined with [Ca’+]o= 10 PM. Open circle (-G-) represents treated group; closed circle (-•-) represents control. Four different sarcolemmal preparations were used in five separate experiments and vertical bars denote SAM. Inset: Expanded time scale ofNa+/Ca’+ exchange. Uptake was significantly (P < 0.05) higher in the DTT/Fe’+ treated group for all points shown in the inset (2-8

s).

Ca2+ levels. Eadie-Hofstee plots [Fig. 2(b) ] revealed an increase in the affinity of the exchanger for Ca2+ with apparent K,,, values of 13 and 63 PM in the treated versus control membranes, respectively. The corresponding V,,,,, values [from Fig. 2(b)] were 2.64 and 3.21 nmol/mg prot./s in the treated and control groups, respectively. Although the relatively small decreasein V,,, observed at 50 ,UM

1

__-----

(a)

1011

+ Exchange

3.0 lb) 2.5 \

2 2.0 i F‘0 1.5E c ’

l.O-

0.5-

I 0

20

40

60

60

[Co’+]

100 /LM

120

140

150

0

Y) \ \

\

\ \

tl

\

\

I

,

0.05

0.1

0.15

\ ‘1 ( 2

v Ka2’)

FIGURE 2. Dependence of Na+/Ca’+ exchange on [Ca2+lo after DTT (1 mM) and F&O4 (50 w) treatment. (a) Na,‘-dependent Ca2+ influx, measured at 2 s, as a function of [Ca* ‘10. Uptake was significantly (P < 0.05) higher in the treated group for all points with [Ca*+]o < 40 p. (b) Eadie-Hofstee plots of the data. Lines were drawn by leastsquares linear regression analysis. (Open circle (-0-) represents treated group; closed circle (-O-) represents control. Vertical bars denote S.E.M. for 6 separate determinations in four different sarcolemmal preparations.

1012

2. Q Shi et al.

FeS04 was not significant (P > 0.05), there was a large (50%) and significant decrease in ~‘lnax in response to 10 PM FeS04 and 1 mM DTT treatment (Fig. 3). Catalase, superoxide dismutase, sodium formate and desferrioxamine had differing effects on the stimulation of exchanger activity by DTT/Fe ” (Fig. 3). Stimulation was almost completely blocked by 100 pM desferrioxamine. Catalase (ranging from 1300 to 6500 U/ml) inhibited the increase in exchange activity by 60% as shown in Table 1. Boiling the catalase not only eliminated the inhibition but enhanced the ability of DTT/Fe’+ to decrease the A^,. In contrast, superoxide dismutase (ranging from 357 to 1785 U/ml) (Table 2) and sodium formate ( 15 mM) had no effect (Fig. 3). Incubation with FeS04 (50 pMj alone at 37°C for 40 mins resulted in a response similar to DTT/Fe ‘+ . However, pretreatment with 50 PM of either ZnS04 or Ga2(S04)s under the same experimental conditions (Fig. 4) had no effect on the exchange activity. The time course of desferrioxamine treatin Figure 5. The ment is shown DTT/Fe’+ treatment required no more than 1 min for a complete effect on both the apparent A”,,, and V,,, of the exchanger. The effects of DTT/Fe’+ on the passive Ca2+ permeability of sarcolemmal vesicles is shown in Table 3. Ca2+ leakage from the

but consistently vesicles was slightly (P < 0.05) increased by the action of the free radical generating system. The overall rate constants of passive Ca2+ efflux calculated from the relevant equation were 0.22 and 0.26 min- ’ for the control and treated groups, respectively. In the rat heart (in which the pretreatment of sarKm for Ca2 + was 21 PM) treatment colemmal vesicles resulted in 7791, stimulation of initial rates measured at 10 pM Ca2+. This stimulation was due to a decrease in the K,,, (Ca2+) to 15 FM as well as an increase in the ~‘nlax from 1.14 to 1.55 nmol/mg prot./s. Discussion Pretreatment of sarcolemmal vesicles with DTT/Fe2 + stimulates Nat/Ca2+ exchange and increases passive Ca2+ permeability. The approximate 2.5-fold stimulation of the initial rate of Na+/Ca2+ exchange by DTT/Fe2+ observed at low Ca2+ concentrations results from the dramatic effect on the apparent K,,, for Ca2+. There was no significant effect of DTT/Fe’+ on the I’,,,,, between treated and control groups ( Fe2 + being 50 ,LLM). This suggests that the altered exchange mechanism is due primarily to an increase in affinity for Ca2+. Catalase decreased the stimulation of exchange activity by 601,;,. This Na+/Ca2+

CTL DTT’Fe CAT B-CAT SOD

SF DESF

Agents present FIGURE 3. Summary of the scavenger effects [3900 U/ml cat&se (CAT), 3900 U/ml boiled catalase (B-CAT), 107 1 U/ml superoxide dismutase (SOD), 15 rnM sodium formate (SF)] and the metal chelator [100/.~ desferrioxamine (DESF)] on the kinetic parameten of Na+/Ca*+ exchange stimulated by Irn~ DTT and 10 PM Fe’+. CTL represents the control group (no radical generator or scavenger). Data are expressed as percent of control. Experimental conditions were as described in Tables 1, 2, and Figure 5.

Active TABLE

0, and Na+/Ca*

+ Exchange

1. Effect of catalase on the kinetic parameters alterations of Na’/Ca’+ exchange”

Active catalase (kdmli

1013 of DTT/Fe’+-induced 0 0 of Control

Boiled catalase Wdml)

DTT (mM)

FeSOh (PM) 10 10 10 10 10 10 10 10 10 10

100 200 300 500 100 200 300 500

i’ msx

x-Ill 32.7 46.9 56.9 60.0 58.7 30.9 33.3 26.9 21.1 24.6

100 f + k + + rfr + f f k

4.5 5.8 6.2 6.9 6.5 4.9 4.6 4.3 3.0 3.0

45.4 42.9 47.1 55.5 57.1 54.5 61.4 62.1 61.4 70.6

100 * + f f f + + f k +

2.8 2.5 2.3 3.1 3.1 4.0 4.2 4.4 3.8 3.8

“Sarcolemmal vesicles were preincubated for 40 min at 37°C in the presence or absence of 1 rnM DTT, 10 PM FeS04, and either active or boiled catalase, then immediately assayed for Na’/Ca” exchange as described in Methods. [Ca*+] of the extravcsicular mrdium was varied between lo-160 PM. Uptake was quenched at 2 s. Data represent mean _+ SE. of experiments with 3 different sarcolemmal preparations and arc expressed as a prrcrnt of control. In these experiments the absolute values of 100°/, were as follows: A-,,, 55.7 & 3.1 PM, lmax 3.6 + 0.1 nmol/mg pro+ for experiments with active catalasr and X, 68.4 + 3.5 PM, L,,, 4.7 i 0. I nmol/mg pro+ for experiments with boiled catalasr..

shows that H202, at least in part, mediates the stimulation of exchange activity by DTT/Fe ‘+ . This is consistent with autoxidation of DTT [ZZ, 221, producing Hz02 in the presence of Fe ‘+. The accumulation ofH202, in turn, results in formation of. OH and .02-

TABLE

by Fenton-type reactions including the ironcatalyzed Haber-Weiss reaction [23]. Catalase inactivated by boiling did not prevent this stimulatory effect, which excludes metal binding effects. Superoxide dismutase and sodium formate had little or no effect on stimulation of

2. Effect of superoxide dismutasc (SOD) on the kinetic parameters of DTT/Fe2+ -induced alteration of Na+/Ca2+ exchangea ‘I,, of Control DTT (rnMl

100 200 300 500

F&O4

--_____

(PM!

1 1 1 1 1

10 10 10 10 10

fi-nl

19.1 22.5 22.0 24.8 24.7

100 + + * k 2

i ‘Illax

4.6 7.8 5.5 6.1 5.9

25.0 26.3 25.8 26.7 27.5

100 + + + + k

2.6 3.9 2.7 2.7 2.7

aSarcolemmal vesicles were preincubated for 40 min at 37°C in thr presence or absence of 1 rn~ DTT, 10 P’M FeSO., and superoxide dismutasr. then immediately assayed for Naf/Caz+ exchange as described in Methods. [Ca’+] of the extravesicular medium was varied between 10 to 160 PM. Uptake was quenched at 2 s. Data represent mean & S.E. of three separate experiments with two different sarcolemmal preparations and are expressed as a percent of control where 1000!, equals 72.6 k 9.1 PM for the A*,,, and 2.4 i 0.1 nmol/mg prot/s for the r,,,.

Z. Q

0

CTL

Fez+ Agents

Shi

et al.

i!n2+ present

Delay before adding desferrb%amln

(mln)

FIGURE 4. Effect of Fe*+ alone, Zn’+, and Ga’+ on the kinetic parameters of Na+/Ca2+ exchange. Sarcolemmal vesicles were preincubated for 40 min at 37°C in the absence [control (CTL)] or presence of 50 PM FeS04, 50 PM Z&04, or 50 PM Gal(SOb)s individually, then immediately assayed for Na+/Ca” exchange as described in Methods. Data are expressed as percent of control.

FIGURE 5. Time course of desferrioxamine effects on the kinetic parameters of Na+ /Ca2 + exchange stimulated by DTT/Fe ‘+ Sarcolemmal vesicles were incubated with 1 rnM DTT and 50 PM FeS04. Desferrioxamine (100 FM) was added either before (n = 1) or 1, 5, 40, and 80 min (n = 3) after the addition of DTT/Fe2+, then immediately assayed for Na+/Ca” exchange as described in Methods. Data are expressed as a percent of that at time = 0 min.

exchange activity, suggesting that ’ Os- and . OH are not involved. Desferrioxamine, an iron chelator with an affinity for iron several orders ofmagnitude greater than for Ca’+ [24], completely blocked the effect of DTT/Fe’+ on the exchanger. Moreover, Fe’+ alone could stimulate Na+ /Ca2 + exchange activity (Fig. 4). Direct iron binding to the exchanger protein could be responsible for the effects of DTT/Fe 2+ . However, this is negated by the following observations: ( 1) adding an equivalent concentration of Fe2+ to the reaction medium increased K,,, for Ca2+ in a manner consistent with competitive inhibition (data not shown); (2) incubation of sarcolemmal vesicles with Zn2 + (Z&04) had no effect on

the exchanger (Zn2+ is a stable divalent metal with the same crystal ionic radius (0.74 A) as ferrous ions); (3) similarly, incubation of sarcolemmal vesicles with Ga3 + [Gaz (SOa) s] also had no effect on the exchanger (Ga3+ is a stable trivalent metal with a crystal ionic radius similar (0.62 vs. 0.64 A) to ferric ions). Taken together with the inhibition by catalase, these data strongly support a mechanism in which redox reactions mediate the response. In agreement with this is the observation that anaerobiosis markedly reduced the degree of stimulation produced by DTT/Fe’ + [151. There are several other candidates for the mechanism by which DTT/Fe’+ stimulates Na+/Ca’+ exchange activity. First, it is

TABLE

3. Rates of passive Ca*+ treatment. Data represent sarcolemmal preparations.

efflux mean

from sarcolemmal +_ S.E. of six separate

vesicles after determinations

Ca’+ content (% of content at DTT

@Ml 1 “P < 0.05

FeS04

(PM)

Ca2+ content (t = (nmol/mg prot) 8.0 6.9

50 (treated

vs control).

DTT and FeS04 in three different

t = 0)

0)

ke 120s 64.5 + 0.7 59.5 &- 1.3a

360 s 41.5 &- 0.7 35.5 + 1.1”

(min-‘) 0.22 0.26”

Active

0, and Na+/CazC

known that DTT/Fe 2+, by generation of active oxygen species can induce lipid peroxidation [251. The activity of Na+/Ca2 + exchange in cardiac sarcolemmal vesicles is very sensitive to the membrane lipid environment [&9, 26, 271. If lipid peroxidation occurs under these conditions, the end-products and lipid bilayer disorder may modify the exchange protein to provide greater accessibility for Naf and Ca 2 + . This is consistent with the observation that passive CaZt permeability is increased by DTT/Fe” pretreatment, indicating that the membrane lipid environment may be altered. However, the incubation of membrane vesicles with DTT/Fe2’ requires less than one minute to stimulate Na+/Ca2’ exchange. This argues against membrane lipid peroxidation which usually entails a latent period one or two orders of magnitude longer. A more plausible explanation is oxidation of SH groups and/or methionine residues of the exchanger protein. Sulfur-containing residues reportedly interact with a Na+-binding site of the Na+/Ca2+ antiport in cardiac sarcolemmal vesicles [28]. This proximity renders the Na+/Ca2+ exchanger susceptible to SH reagents. The current data confirm and extend the finding of Reeves et al. [IS], that the exchanger is stimulated by DTT/Fe’+. Howelrer, in contrast to their observation that the exchanger is stimulated at 1 PM iron, we found no effect of DTT/Fe2 + on the K,,, for Ca2+ until the Fe’+ concentration was increased to 10 PM, and the most effective range was 50 to 100 ,UM (data not shown). Ten PM FeS04 in the presence of 1 mM DTT decreased both the apparent K,,, for Ca2 + (albeit to a lesser extent than 50 PM) and I’,,,,, (Table 1). These changes reflect more than simply increased affinity for Ca2+. Indeed they represent a decrease in the efficiency of‘ Ca2+ translocation across the sarcolemmal membrane. These discrepant results may bc due to differences in sarcolemmal preparations used (although the species is the same) or difherences in contaminating levels of Fe’+ in the solutions. Although the A-m for Ca’+ in the untreated vesicles is not reported in that study, it appears to be greater than 200 ,UM well outside the normal reported range of 1-40 PM 141.

Exchange

1015

The observed differences in the degree of stimulation may depend on the K, (Ca2+) of the untreated sarcolemmal vesicles. Reeves et N IO-fold stimulation in al. [E] observed bovine heart sarcolemmal vesicle preparations in which the untreated K,,, (Ca”) exceeded 200 PM. The current data show a 2.5-fold stimulation of initial rates in bovine hearts with an initial k^, (Ca2+) of 63 PM. Using rat sarcolemmal vesicles with a X, (Ca2+) of 21 PM the stimulation was 1.7 fold. Both the pretreatment A-m and the degree of stimulation are very similar in the rat to that found in canine sarcolemmal vesicles (K. D. Philipson personal communication). The apparent Km (Ca2+) values are for some reason very variable in ho. The elevated values found in the bovine heart could be a consequence of the prolonged period ( - 20 min) of ischemia prior to isolation. In order to resolve more definitively the mechanism of this stimulation it may be important for future investigations to characterize the potential effects of such treatment on the lipid bilayer and the redox state of thiol groups on the Na+/Ca’+ exchanger. It is tempting to propose that the stimulated Na+/Ca’+ exchange activity is a mechanism for protection against the Ca2+ overload during cell threatening insults, including myocardial ischemia/reperfusion injury. However, the stimulation entails a decrease in the AAm ( Ca2 ’ ) to the range of 13-24 ,UM. The degree of stimulation would depend on the X, (Ca2’) prior to treatment. Whether the phenomena described in this study could moderate the increased Ca2+ permeability which contributes to the Ca2+ overload frequently observed during ischemia remains to be investigated. Acknowledgements The authors thank Drs K. D. Philipson and J. P. Reeves for insightful discussions. This work was supported by a grant from the BC Heart Foundation to GFT and a Presidential Rcsearch Grant from Simon Fraser University to GFT. We gratefully acknowledge the kind gift of desferrioxamine from the Ciba Foundation and the expert technical assistance of MS Haruyo Kashihara.

1016

2. Q. Shi et al.

References 1 2 3 4 5 6 7 8 9 10 I1

12

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

LANCER, G. A. The role of calcium in the control of myocardial contractility: An update. J Mol Cell Cardiol 12, 231-239 (1980). CARAFOLI, E. The homeostasis of calcium in heart cells. J Mol Cell Cardiol 17, 203-2 12 (1985). REEVES, J. P. The sarcolemmal sodium-calcium exchange system. In: Current Topics in Membrunes and Transport, F. Bronner, A. E. Shamoo (Eds) pp. 77-127. London, Academic Press, Inc. vol. 25, (1985). PHILIPSON, K. D. Sodium-calcium exchange in plasma membrane vesicles. Ann Rev Physiol47, 561-57 1 (1985). MULLINS, L. J. The generation of electric current in cardiac fibers by Na/Ca exchange. Am J Physiol 236, ClO3-Cl10 (1979). PHILIPSON, K. D., FRANK, J. S., NISHIMOTO, A. Y. Effects of phospholipase C on the Na+-Gas+ exchanger and Ca2+ permeability of cardiac sarcolemmal vesicles. J Biol Chem 258, 5905-5910 (1983). PHILIPSON, K. D., NISHIMOTO, A. Y. Interaction of charged amphiphiles with Na+/Ca’+ exchange in cardiac sarcolemmal vesicles. J Biol Chem 259, 13999-l 4002 ( 1984). PHILIPSON, K. D., NISHIMOTO, A. Y. Stimulation of Na+-Cazf exchange in cardiac sarcolemmal vesicles by phospholipase D. J Biol Chem 259, 1619 (1984). PHILIPSON, K. D., WARD, R. Effects of fatty acids on Na+-Ca*+ exchange and Ca2+ permeability of cardiac sarcolemmal vesicles. J Biol Chem 260,9666-967 1. HESS, M. L., MANSON, N. H. Molecular oxygen: friend and foe. J Mol Cell Cardiol 16,969985 (1984). MCCORD, J. M., ROY, R. S., SCHAFFER, S. W. Free radicals and myocardial ischemia: the role of xanthine oxidase. In: Advances In Myocardiology, P. Harris, P. A. Poole-Wilson (Eds) vol. 5, pp. 183-189. New York and London: Plenum Medical Book Company (1983). KONG, S., DAVISON, A. J. The relative effectiveness of ‘OH, HaOs, Oa-, and reducing free radicals in causing damage to biomembranes. A study of radiation damage to erythrocyte ghosts using selective free radical scavengers. Biochim Biophys Acta 640, 3 13-325 ( 1981). KAKO, K. J. Free radical effects on membrane protein in myocardial ischemia/reperfusion injury. J Mol Cell Cardiol 19, 209211 (1987). KRAMER, J. H., MAK, I. T., WEGLICKI, W. B. Differential sensitivity of canine cardiac sarcolemmal and microsomal enzymes to inhibition by free radical-induced lipid peroxidation. Circ Res 55, 120-124 (1984). REEVES, J. P., BAILEY, C. A., HALE, C. C. Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles. J Biol Chem 261, 494884955 (1986). BERS, D. M. Isolation and characterization of cardiac sarcolemma. Biochim Biophys Acta 555, 131-146 (1979). TIBBITS, G. F., KASHIHARA, H., O’REILLY, K. Na+-Ca*+ exchange in cardiac sarcolemma: modulation of Ca2+ affinity by exercise. Am J Physiol 256, C638-C642 (1989). BRADFORD, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 249-254 (1976). TIBBITS, G. F., SASAKI, M., IKEDA, M., SHIMADA, K., TSURUHARA, T., NAGATOMO, Characterization of rat myocardial sarcolemma. J Mol Cell Cardiol 13, 105 I-1061 (1981). TIBBITS, G. F., PHILIPSON, K.D. Na-dependent alkaline earth metal uptake in cardiac sarcolemmal vesicles. Biochim Biophys Acta 817, 327-332 (1985). TIEN, M., BUCHER, J, R., AUST, S. D. Thiol-dependent lipid peroxidation. Biochem Biophys Res Commun 107, 279-285 ( 1982). TROTTA, P. P., PINKUS, L. M., MEISTER, A. Inhibition by dithiothreitol of the utilization of glutamine by carbamyl phosphate synthase. J Biol Chem 249, 1915-1921 (1974). HABER, F., WEISS, J. The catalytic decomposition of hydrogen peroxide by ion salts. Proc R Sot 147, 3322351 (1934). KEBERLE, H. (1964) The biochemistry ofdesferrioxaminc and its relation to iron metabolism. Annals NY Acad Sci 119, 758-768 (1964). HALLIWELL, B., GUTTERIDGE, J. M. C. Free Radicals In Biology And Medicine, pp. 139-189. Oxford, Clarendon Press. (1985). PHILIPSON, K. D., WARD, R. Modulation of Na+-Ca2+ exchange and Ca’+ permeability in cardiac sarcolemmal vesicles by doxylstearic acids, Biochim Biophys Acta 897, 152-158 (1987). YAMASAKI, Y., ITO, K., ENOMOTO, Y., SUTKO, J. L. Alterations by saponins ofpassive Ca2+ permeability and Na’Gas’ exchange activity of canine cardiac sarcolemmal vesicles. Biochim Biophys Acta 897, 481-487 (1987). PIERCE, G. N., WARD, R., PHILIPSON, K. D. Role for sulfur-containing groups in the Na+-Gas+ exchange of cardiac sarcolemmal vesicles. Membr Biol94, 2 17-225 (1986).