Ni2+, a new inhibitor of mitochondrial calcium transport

177

Biochimica et Biophysica Acta, 656 (1981) 177-182

Elsevier/North-Holland Biomedical Press BBA 99966 Ni 2+, A NEW INHIBITOR OF MITOCHONDRIAL CALCIUM TRANSPORT ERZSEBET LIGETI a JUDIT BODNAR a EVA KAROLY a and ERNO LINDNER b a Experimental Research Department and 2nd PhysiologicalInstitute, Semmelweis University Medical School, Lrll6~ut 78/.4, 1082 Budapest, and b Department for A naly tical Chemistry, Budapest Technical University, Budapest {Hungary)

(Received April 16th, 1981)

Key words: Ni 2+effect; Ca2+ transport; Respiration; Volume change; (Rat liver mitochondria)

1. The effect of Ni 2÷ on respiration, volume changes and Ca2+ movements was investigated in rat liver mitochon. dria. 2. Ni 2+ inhibited Ca2+ uptake into respiring mitochondria, Ca2+-stimulated respiration and swelling in Ca2+ salts, whereas it did not inhibit either state 4 and DNP-stimulated respiration, or swelling in K÷ salt in the presence of valinomycin. 3. The inhibitory concentration of Ni 2+ depended strongly on the applied Ca2+ concentration. As revealed by direct methods, 50% inhibition of Ca 2+ influx Was achieved by approx. 2-fold excess of Ni 2+. 4. If added to Ca2÷-loaded mitochondria, Ni 2+ gave rise to slow Ca2+ release and inhibited uncoupler-induced efflux slightly. 5. It is concluded that N i 2+ is a potent inhibitor of mitochondrial Ca 2+ transport. Ca 2+ influx is far more sensitive to inh~ition than Ca2÷ efflux.

Introduction Mitochondria are effective sinks for cytoplasmic Ca2÷ in various cells and assist in keeping the cytoplasmic free Ca 2÷ concentration low [ 1,2]. This function is served by two distinct transport pathways characterized in the last few years [3]. Ca2÷ influx down the electrochemical gradient proceeds via a uniport mechanism. The reversal of this pathway is probably responsible for Ca2÷ efflux in the case of decreased membrane potential (Ark), e.g., upon addition of uncouplers or respiratory inhibitors. A separate efflux pathway is supposed to operate when Ca2÷ efflux occurs under conditions of high A~ [4]. This efflux process is activated by Na ÷ in heart and brain, whereas it is Na÷-independent in liver mitochondria [5]. The characterization of the distinct transport pathways was made partially possible by the use of inhib-

Abbreviation: CCCP, carbonylcyanide m-chlorophenylhydrazone.

itors: ruthenium red and La a÷. Both of them inhibit Ca2÷ uptake via the uniport [6-8] and decrease the rate of Ca2÷ efflux due to uncoupling [9-11]. On the other hand, the separate efflux pathway is ruthenium red-insensitive and can be:blocked only by La a÷ [12, 13]. In the present paper a novel inhibitor of Ca2÷ transport is described which resembles ruthenium red in its inhibitory properties. Methods Rat liver mitochondria were prepared according to Johnson and Lardy [14]. Protein content was measured by the biuret method with bovine serum albumin as standard. Respiration was monitored by a Clark-type electrode attached to an O2-meter and a potentiometric recorder, the whole set being produced by Radelkis, Hungary (OP.925). The measurements were carried out in a glass cell maintained at 25°C. All other experiments were done at room temperature Mitochondrial volume changes were followed at 546 nm by a Beckman DK-2A spectrophotometer

0005-2787/81/0000-0000/$02.50 © 1981 Elsevier/North-HollandBiomedical Press

178 with a high-absorbance cell in the reference pathway. Ca 2÷ movements were followed either electrochemically or by the 4SCa-technique. For the electrochemical measurements a neutral carrier based Ca2+-selective membrane [15] was used in a Philips IS-560 electrode body. The selectivity coefficients (log KMz+,ca2+) of the Ca2+-selective electrode for interfering ions determined with the separate solution method were the following: Na ÷, 3.4; K ÷, 3.7; Mg2+, 4.3; Ni 2÷, 3.3; Sr 2+, 2.1. The Ca2+-electrode was attached to a Radelkis pH meter (OP-205) and recorder (OH-814). Calibrations were done with known amounts of CaCI2 as described by Crompton et al. [16]. For determination of 4SCa uptake and release mitochondria were suspended in a medium of low buffer capacity and changes of the extramitochondrial H÷-concentration occurring during Ca 2÷ movements were followed by a Radiometer pH meter type 26, using a glass electrode (Radiometer G 222C) and a calomel reference electrode (Radiometer K 401). Sampling was timed on the basis of the H÷-electrode trace. At the time given 1 ml suspension was withdrawn by an Eppendorf sampler and spun down for 30 s in a table centrifuge (Mechanika Precyzyjna, Warsawa, Type 320a). A 200 /21 aliquot of the supernatant was pipetted into scintillation vial and counted in a Packard Tri-carb 2101 liquid scintillation spectrometer. Parallel incubations agreed within 5%. Compositions of the incubation media are given individually in the legends to figures and tables. Materials. CCCP was produced by Calbiochem, rotenone by K and K Laboratories, Inc. (Plainview, New York), oligomycin by Serva. All the other reagents were of analytical grade purity. Results

Effect o f Ni 2+on respiration The rate of oxygen consumption with succinate as substrate was determined in the unstimulated state and after addition of Ca 2+ or CCCP, respectively (Fig. 1A). The presence of Ni 2+ strongly inhibited Ca 2÷stimulated respiration, whereas uncoupled oxidation was reduced o~aly by approx. 10%. Unstimulated respiration was not influenced at all. Similar results were obtained when different H ÷ donating anions

A

B

100='q

~o E ~

80-

~ a >'6, x c o

20 i

0 °'2 0'4 01G 0'8 ,o Ni 2+

0

( rnM )

0!2 0', 0'G 0'8

Fig. 1. Effect of Ni2+ on the respiration of mitochondria. The medium contained: 245 mM sucrose, 5 mM KCI, 5 mM MgC12, 5 mM Tris-HCl, 1.7 mM Tris-phosphate, 3.4 mM Trissuccinate, 1/aM rotenone and 3 mg mitochondrial protein in a final volume of 2.3 ml; the pH being 7.0. Respiration was stimulated in A by the addition of either 170 /aM CaC12 (=133 nmol. mg-t) or 1.3 /aM CCCP, and in B by 110 /aM ADP. NiCI2 was always added before succinate. Respiration: • =, unstimulated; X X, Ca~'-stimulated; o o, CCCP-stimulated; • A, ADP-stimulated.

(phosphate, acetate), different respiratory substrates (succinate, 13-hydroxybutyrate or glutamate plus malate) or different divalent cations (Ca 2+, Sr 2÷) were used. Thus, the main effect of Ni 2÷ is inhibition of Ca2+-stimulated respiration. However, Ni 2+ also diminished the rate of ADP-stimulated respiration (Fig. 1B). It is likely that Ni 2÷ ions form a tight complex with ADP, thus decreasing the concentration of the free nucleotide. Effect o f Ni 2+ on volume changes in respiring mitoehondria Addition of permeant cations (e.g., Ca 2+, Sr 2+ or K + together with valinomycin) to respiring mitochondria in the presence of acetate results in rapid swelling in consequence of penetration of the cation plus acetate ions into the matrix space followed by osmotic movement of water. The presence of Ni z+ inhibited swelling due to strontium-acetate uptake (Fig. 2A). If swelling was induced by 166/aM Sr 2÷, then 330/aM Ni 2+ was sufficient to block the process almost completely (Fig. 2A curve d). Increasing the concentration of Sr 2÷ decreased the inhibitory effect of Ni 2÷, and only much higher Ni 2+ levels could prevent swelling (data not shown). The same results were obtained if Sr 2+ was replaced by Ca 2+. On the other hand Ni 2+ did not influence the volume-increase due

179 to penetration of K* in the presence of valinomycin (Fig. 2B curve e). Addition of Ni 2+ alone did not induce swelling. These observations point to the specificity of the Ni2+-effect on Ca 2+ and Sr 2. transport. The optical technique is suitable for following volume changes accompanying Sr 2. (or Ca 2*) efflux, too. After an initial aerobic swelling release o f the divalent cation was induced by the uncoupler CCCP and the process was visualized as a rapid shrinkage phase (Fig. 2C). 330 ~M Ni 2., which prevented swelling, had only minor influence on shrinkage (compare curves d and g of Fig. 2). At higher concentrations Ni 2. had a pronounced effect on CCCP-induced shrinkage (curve h of Fig. 2), but complete inhibition was achieved only by extreme doses (over 30 mM).

Effect o f Ni 2+ on volume changes in non-respiring mitochondria Swelling of non-respiring mitochondria in the isosmotic solution of a freely permeable anion (e.g., NO~, C10~, SCN-, etc) depends only on the penetration of the cation present. Selwyn et al. [17] applied this technique to measure passive influx of divalent

A

B Ni2~" Sr 2+

¢b

I

Sr 2+

22:

/--,.-4 Imin

Fig. 2. Effect of Ni2+ on volume changes in respiring mitochondria. The medium contained: 227 mM sucrose, 6.6 mM KCI, 3.3 mM MgC12, 3 mM Tris-succinate, 20 mM Tris-HCI, 16 mM Tris-acetate, 1 ~aM rotenone and 3.6 mg mitochondrial protein in a final volume of 3 ml, the pH being 7,0. Swelling was induced in A by the addition of 166 /~I~!

Sr(NO3)2 (=140 nmol .rag -t) in B by 166 /aM Sr(NO3)~ then by 166 ng valinomycin/mg, and shrinkage was triggered in C by 0.66 ttM CCCP. Applied Ni2+ concentrations (~uM): a, 0; b, 33 ; c, 100; d, 330; e, 660; f, 0; g, 330; h, 1650.

I

0.1 A

I lmin

Fig. 3. Effect of Ni2+ on volume changes in non-respiring mitochondria. The medium contained 80 mM St(NO3)2, 10 mM Tris-HCl and 1 IzM rotenone in a f'mal volume of 3 ml; pH being 7.0. Swelling was started by addition of 4.2 mg mitochondrial protein to the medium. Applied Ni2+ concent_rations: a, 0; b, 3.3 raM; c, 8.3 mM; d, 20 mM.

cations into respiration-inhibited mitochondria. Following the above principle we recorded swelling in 80 mM Sr(NO3)2. Fig. 3 demonstrates that also passive Sr 2÷ uptake was effectively inhibited by Ni 2÷. Because of the high Sr 2÷ concentration higher concentrations of Ni 2÷ were needed than in the experiments on respiring mitochondria. The possibility that 20 mM Ni 2÷ acted simply by increasing the osmolarity of the medium could be ruled out as addition of 30 mM Tris-HC1 had no effect. Swelling in 120 mM KNO3 in the presence of valinomyein, was not inhibited by Ni 2÷ (not shown).

Effect o f Ni ~+on calcium transport All the previous methods reflected the movements of Ca ~+ (or Sr ~+) across the mitochondrial membrane indirectly. In the following experiments Ca 2+ transport was measured directly.

180 The original traces obtained with the Ca2+-selec tive electrode are represented in Fig. 4. On the addition of succinate, a sharp drop of extramitochondrial free Ca 2÷ concentration was seen due to chelation of the divalent cation by the dicarboxylic acid. After this abrupt change rapid Ca 2+ uptake into mitochondria proceeded for approx. 1 min, followed by a phase of slow influx. Addition of CCCP after completion of Ca 2÷ uptake resulted in a fast Ca 2÷ release. Ni 2+ added before succinate inhibited Ca 2÷ influx (brokep lines). On the other hand, if Ni 2÷ were added after the rapid uptake phase it induced a slow Ca 2+ efflux, the rate of which increased as the inhibition of Ca 2÷ uptake by higher Ni 2+ concentrations became more and more pronounced. Thus, the spontaneous Ca 2÷ efflux from mitochondria could be made apparent after blocking Ca 2÷ influx via the uniport mechanism. At relatively high concentrations of Ni 2+ injected, a sharp change in the electrode potential could be observed (Fig. 4D) according to the Ni 2÷ interference at diluted Ca 2÷ solutions. Uncoupling by CCCP produced fast Ca 2+ release, however, this process was slower in the presence of Ni 2÷ than in its absence. The results of a similar experiment carried out by the 4SCa-technique are summarized in Table I.

Olt

B

C

Succ

Succ

D Succ

100o E

200-

300

~

CCCP

" 350

37o]

CCCP Ni 2~

5rain

TABLE I EFFECT OF Ni2+ ON Ca2+ MOVEMENTS FOLLOWED BY 4 s Ca-TECHNIQUE

A

Succ

Increasing concentrations of Ni 2÷ gradually decreased Ca2+ uptake, but increased the rate of spontaneous Ca 2+ release. The rate of Ca 2+ efflux in the case of inhibited influx pathway (11.9 n m o l ' mg -1) is in agreement with data published earlier [5,18[. CCCPinduced efflux was inhibited by approx. 20% in the presence of 5 mM Ni 2+. Thus, direct methods for measuring Ca z+ transport showed that Ni 2+ inhibited Ca 2÷ influx completely, and CCCP-induced Ca 2÷ efflux partially. Spontaneous Ca 2+ release became apparent after blocking the uptake mechanism, showing that the separate efflux pathway was not completely inhibited by Ni 2÷. The possibility of partial inhibition of this latter process can not be ruled out on the basis of our experiments. The possibility of competition between Ni 2+ and Ca 2+ was also investigated with the 4SCa-technique (Fig. 5). A 10-fold increase in the Ca 2÷ concentration drastically decreased the inhibitory effect of 400 #M Ni 2÷ (from 98% to 46%). However, the present experimental technique does not allow kinetic analysis of the inhibition.

Ni 2+

Ni 2+

CCCP

Fig. 4. Effect of Ni2+ on Ca2+ movements followed by the ion-selective electrode. Composition of the medium: 247 mM sucrose, 5 mM KC1, 2.5 mM Tris-HCl, 3 mM MgC12, 1/sM rotenone, 0.8 mM Tris-phosphate, 57 #M CaC12 (122 nmol - mg-t), 3 mg mitochondrial protein and 1.7 /~g oligomycin per mg (pH 7.0). Final volume: 6.5 ml. Ca2÷ uptake was initiated by 3.8 mM sodium-succinate and Ca2÷ release by 0.6 ztM CCCP. Applied Ni2+ concentrations: A, 0; b, 15.4 /IM; C, 154 xtM; D, 616 #M. - . . . . . , Ni2+ added before succinate.

Composition of the medium: 246 mM sucrose, 5 mM KC1, 3 mM MgC12, 1 mM Tris-phosphate, 0.5 mM Tris-HC1, 1/~M rotenone, 118 ~tM 45CAC12 (83 nmol. mg-1; 2.5 - 10-8 Ci), 3 mg mitochondrial protein, 1.7 ~g oligomycin/mg. Final volume: 2.1 ml, pH 7.0. Ca2+ uptake was initiated by 4.7 mM sodium succinate and release by 0.5 ~tM CCCP. Ni2÷ was added before succinate if Ca2+ uptake were measured, or 60 s after succinate, if Ca2+ release were investigated. CCCP was added 3 min after succinate. Samples were taken 60 s after the last addition. Ni2+ concn. (raM)

Ca2+ uptake (nmol •mg-1 )

Ca2+ release (nmol -mg -1 ) Spontaneous

0 0.2 0.5

81.9 51.9 30.3

0 2.6 3.1

1.0

13.5

3.7

2.0 5.0

7.3 2.5

5.9 11.9

CCCPinduced 38.3 38.8 41.5 34.1 31.6 30.5

181 160140120E

-6 100E 8060-

E

40-

(_)

200 0

~ 100

, 2O0

, 300

Ca =+ (IJM) Fig. 5. Effect of Ni2÷ on Ca 2+ uptake in the presence of various concentrations of Ca2+. Composition of the medium: 246 mM sucrose, 5 mM KC1, 3 mM MgC12, 0.5 mM Tris-HC1, 1 mM Tris-phosphate, 1/zM rotenone, 3 mg mitochondrial protein, 1.7 /zg/mg oligomycin a n d various amounts of 10 mM 4SCaC12 (activity: 10 -6 Ci • m r i ) . Final volume: 2.1 ml; pH 7.0. Ca 2+ uptake was initiated by 4.7 mM sodium succinate. If present, NiC12 was added in a concentration of 400 #M before succinate, k X, in the absence of Ni~; o o, in the presence of Ni 2+.

effects was observed upon addition of Ni2+ (Figs. 1. and 2B). Thus, the effects of Ni 2÷ can be ascribed to a direct action on the Ca2÷-transporting system. This view is supported by the observation that Ni 2+ inhibits passive Sr 2+ influx but not K ÷ influx into non-respiring mitochondria (Fig. 3). Apparently Ni 2+ is not rapidly transported by the Ca2+-uniporter as it neither stimulates respiration (Fig. 1A) nor induces aerobic swelling (Fig. 2A). However, slow penetration of Ni 2+ into the mitochondrial matrix cannnot be ruled out. This has to be examined by direct uptake measurements. The slow release observed after addition of Ni 2+ to Ca2÷-loaded mitochondria (Fig. 4 and Table I) is the consequence of the function of the separate efflux route in the presence of inhibited influx pathway. Thus, Ni 2+ - at a concentration of approx. 1 mM completely inhibits Ca2+ uptake and the related processes, partially inhibits uncoupler-induced Ca2÷ release and the accompanying phenomena, b u t - in contrast with the effect of La 3÷ - leaves the separate efflux pathway either totally or partially intact. All the above effects are similar to the known effects of ruthenium red. We conclude that Ni 2÷ represents a new and potentially useful tool in experimental studies on Ca 2÷ transport in mitochondria.

Acknowledgements Discussion The results obtained with different techniques accord in the finding that Ni 2÷ inhibits Ca2÷ uptake into respiring mitochondria. On the basis of the described control experiments a primary action on the respiratory chain, or on the carriers of respiratory substrates or phosphate, respectively, were excluded. The possibility of a decrease of the membrane potential has been considered. Ni 2+, as a heavy metal might have various side effects rendering the membrane leaky and diminishing A~b. In this way both the inhibition of Ca2+ uptake and the induction of efflux could be explained. Although we do not have direct data on the membrane potential a decrease of the A~k seems highly improbable. In the case of lower Aft the rate of state 4 respiration ought to increase and that of K+ uptake (in the presence of valinomycin) ought to decrease. Neither of these

The authors are indebted to Professor A. Fony6 for continuous support and for careful revision of the manuscript, to Dr. Kl~ra T6th for helpful discussions, to Dr. Z. Marcsek for the use of the liquid scintillation spectrometer and to Mrs. Juliana Szilassy for her excellent and devoted technical assistance. The work is part of the research project 'Carrier mechanism of biological membranes' and was supported by grant No. 1-07-0306-01-1/F of the Hungarian Ministry of Health.

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