Effects of magnesium, ruthenium red and the antibiotic ionophore A-23187 on initial rates of calcium uptake and release by heart mitochondria

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Effects of Magnesium, A-231 87 on Initial

167,104-115 (1974)

Ruthenium

Red and the Antibiotic

Rates of Calcium

Uptake

Heart Mitochondria LOUIS Division

of Biochemistry,

University

lonophore

and Release by

l

A. SORDAHL of Texas Medical

Branch,

Galveston,

Texas 77550

Received August 9, 1974 Initial rates of calcium uptake by rabbit heart mitochondria were measured by dual-beam spectroscopy. Mg’+ produced a marked reduction in the rate of Ca*+ uptake. In the absence of Mg’+, Cal+-stimulated mitochondrial respiration could not be further increased by addition of ADP. After Ca*+ addition with Mgz+ present, ADP produced apparent coupled respiratory stimulation. Both the Ca *+ transport inhibitor, ruthenium red, and the divalent cation-specific, ionophoretic antibiotic, A-23187, inhibited Caz+ uptake in the presence or absence of Mg*+. Ruthenium red added to mitochondria after Cal+ accumulation caused a slow efflux of Caz+ from mitochondria; A-23187 caused a rapid discharge of accumulated Ca I+ from mitochondria in the presence or absence of ruthenium red. The results suggest that Mgz+ exerts a “protective” effect on the mitochondrial phosphorylating mechanism and may modulate the competitive effects of Ca*+ and ADP for electron transport chain-generated energy. Further, the effects of ruthenium red and A-23187 suggest the possibility of two pathways or “channels” for mitochondrial Cal+ uptake and release.

Most studies of in uitro mitochondrial calcium transport have involved incubation of mitochondria with isotopically labeled calcium and subsequent analysis of calcium uptake after centrifugation or filtration (1). These studies have provided a substantial amount of information on the energy-linked accumulation of calcium by mitochondria (1, 2). However, the determination of initial velocities of mitochondrial calcium uptake, particularly at low cation concentrations, has been by extrapolation or indirect means (l-6). Since a number of investigators (1, 2, 4, 7-9) have suggested that mitochondrial calcium transport may play a role in maintaining ionic homeostasis within the cell, it is important to know the rate at which calcium can be transported into mitochondria at physiological concentrations of the cation. Further, the ‘This work was supported by grants from American Heart Association, Texas Affiliate, Eli Lilly and Company, and DHEW-5-Sol-RR-05427-12.

factors which can modify or regulate this uptake and release of calcium need to be elucidated. This is particularly significant in a tissue such as heart where the movements of calcium are in a constant and dynamic state of flux (10). The use of calcium-sensitive agents (aequorin or murexide) has permitted the direct measurement of mitochondrial calcium uptake by spectrophotometric means (11-13). These techniques allow rapid monitoring of uptake and release of small amounts of calcium (-lo-’ M) by isolated mitochondria. In two recent reports (14, 15) the initial velocities of calcium uptake by liver (15) and cardiac (14) mitochondria have been measured using stopped-flow and dual-wavelength spectrophotometric techniques with the chelometric dye, murexide (13). On the basis of these studies, Vinogradov and Scarpa (15) have proposed a model of mitochondrial cation transport involving the binding of two calcium ions

104 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.

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to a transport complex in the membrane in order to achieve maximal active transport of calcium. In the case of liver mitochondria, the binding and subsequent transport of calcium by this complex can be competitively slowed by the presence of manganese (15). Two compounds have recently been shown to have differential effects on mitochondrial calcium transport (16, 17). Moore (16) has demonstrated that the muco- and glycoprotein-specific reagent ruthenium red, used to stain membranes, produces selective inhibition of active calcium uptake into mitochondria while exhibiting no inhibitory effects on respiratory activity or potassium ion transport. Vasington et al. (18) confirmed Moore’s studies (16) and further observed that ruthenium red did not prevent the efflux of calcium from mitochondria. These investigators (18) suggested that different paths for calcium uptake and release may exist in mitochondria. Rossi et al. (19) reported that ruthenium red can promote the release of calcium from calcium-loaded rat liver mitochondria. It has been suggested that ruthemum red specifically acts to compete with calcium for transport and/or binding sites in the inner mitochondrial membrane (X9). Reed and Lardy (17) have reported that a new ionophoretic antibiotic (A-23187, Eli Lilly and Company), which is specific for the divalent cations magnesium and calcium, produces a rapid, energy-dissipating efflux of calcium from mitochondria. This effect on calcium discharge from mitochondria can be attenuated by the presence of magnesium (17). Wong et al. (20) have essentially confirmed these results. Mitochondria are capable of actively transporting a number of other cations (1). In particular, heart mitochondria have been shown to accumulate magnesium under specific conditions (1, 21). It is generally accepted that calcium accumulation and oxidative phosphorylation are noncompeting processes in mitochondria (1). That is, mitochondria will preferentially accumulate calcium in lieu of phosphorylation of ADP, as shown with rat liver mitochondria (22). This report extends

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these studies to heart mitochondria with somewhat differing results and confirms some of the studies on initial velocities of calcium uptake by isolated heart mitochondria (14). Further, the effects of ruthenium red (16, 18, 19) and A-23187 (17, 20) on heart mitochondrial calcium transport are reported. A modulatory effect of magnesium on rapid calcium uptake by heart mitochondria will be demonstrated and the results suggest a possible “protective” effect of magnesium on the phosphorylating capacity of heart mitochondria during active calcium accumulation. Utilizing ruthenium red and A-23187 as differential inhibitors, the possible existence of two different “channels” for calcium movement through the mitochondrial membrane will be discussed. EXPERIMENTAL

PROCEDURES

Rabbit heart mitochondria were isolated by a modification of previously described techniques (12). This essentially involved the isolation of mitochondria in a medium consisting of 0.18 M KCl, 10 mM EGTA, 0.5% bovine serum albumin, pH 7.2. The mitochondria were first washed in a medium containing 1 mM EGTA and then in 0.1 mM EGTA. The final suspension of mitochondria was in the above medium minus EGTA or in 0.25 M sucrose-10 mM Tris-HCl, pH 7.2. Mitochondrial respiratory activity was measured by polarographic means (12). Rapid uptake of calcium was monitored using the chelometric dye murexide in a DW-2 dual-wavelength spectrophotometer (American Instrument Company) at t,he wavelength pair 541-507 nm by modification (23) of previously described methods (12). Mitochondrial respiratory activity during rapid calcium uptake was measured with a vibrating platinum electrode inserted into the reaction cuvette. Additions to the reaction medium were made by injection through a rubber diaphragm in the electrode assembly positioned over the cuvette in the spectrophotometer. The vibrating platinum electrode served as a mixing device, but to ensure complete mixing the syringe was back-aspirated twice with each addition. This created a marked addition artifact that has been eliminated from the figures. Mixing was complete in 3-5 s. The reaction medium for calcium uptake consisted of: 0.25 M sucrose, 5 mM Tris-HCl (pH 7.2), 70 mM NaCl or KCl, 8 mM inorganic phosphate, 50 pM murexide, 5 pg rotenone, and 3 mg mitochondrial protein in a total volume of 3.0 ml. Succinate was added at a final concentration of 5 mM and the temperature of the reaction was 30°C. This medium has been found to give optimal activities for both respiration and cal-

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cium uptake by heart mitochondrial preparations in this laboratory. Parallel assays of mitochondrial calcium uptake and release were also done for all experiments described by a Millipore filtration method (12) utilizing Wa in essentially the same assay medium outlined above. Changes in the redox state of cytochrome b were measured by dualwavelength spectrophotometry at the wavelength pair 430-410 nm (3). Mitochondrial protein was determined by a biuret method (24). Ruthenium red (tetraaminorutheniumhydrochlorochloride) was obtained from Alfa Laboratories, Sigma Chemical Company and K and K Laboratories, with all preparations giving comparable results. Spectrophotometric analysis of the “crude” aqueous solutions of ruthenium red revealed a c, M of 14.7 at 533 nm. This indicates that solutions were only approximately 22% pure with respect to ruthenium red. A similar observation has recently been reported by Reed and Bygrave (32). Purification of the ruthenium red according to the method of Luft (33) resulted in a c,,,L of 68 at 533 nm for ruthenium red solutions in 0.1 M ammonium acetate. This is identical to the c,,,~ value reported by Luft (33). Since most of the experiments reported here using ruthenium red were done prior to

determining its purity, additions where indicated are designated “crude ruthenium red” and the actual amount of dye is only about 20% of the value stated. However, it should be pointed out that these “crude” amounts used are the same as those reported by other investigators (16, 18, 19) and with similar effects, i.e., complete inhibition of mitochondrial calcium uptake with no effect on oxidative phosphorylation. Murexide (ammonium purpurate) was obtained from both Sigma and Fisher Chemical Companies and a freshly prepared stock solution (1 mM) used each day. The ionophoretic antibiotic A-23187 (dissolved in 70% ethanol at 1 mg/ml) was the generous gift of Dr. Robert L. Hamill, Eli Lilly and Company. RESULTS

Respiration-supported calcium uptake by isolated rabbit heart mitochondria measured by spectrophotometric means is shown in Fig. 1. The upward deflection of the traces indicate an increase in calcium concentration in the medium as measured by the murexide dye. A downward deflection of the traces is the uptake of calcium

3-

m

I’

FIG. 1. Representative traces of mitochondrial calcium uptake measured by dual-beam spectroscopy. The chelometric dye murexide exhibits linear changes in absorbance at the wavelength pair 541-507 nm, when complexing with calcium. The left side of A demonstrates these changes with three calcium additions of 100 nmoles each. A single addition of 300 nmoles (100 PM) calcium has been made in B and C. Subsequent addition of succinate (Succ) produces respiration-supported calcium uptake (downward deflection). The breaks in the traces represent artifacts at the point of addition. The reaction medium consists of 0.25 M sucrose, 10 mM Tris-HCl (pH 7.2) 70 mM NaCl, 8 mM phosphate, 50 @M murexide, 5 pg rotenone, and 3 mg mitochondrial protein in a total volume of 3.0 ml. Succinate is added at a final concentration of 5 mM; temperature = 30°C. The figures to the right of the traces indicate initial rates of calcium uptake in nmoles/min/mg. A: Uptake in the absence of magnesium. B: 1.0 mM magnesium present. C: Composite trace in the absence of magnesium to indicate calcium uptake proceeds normally with oligomycin (3 rg) present (solid line), but is inhibited (dotted lines) by addition of respiratory chain inhibitors (antimycin A, 5 pg; malonate, 5 mM, or KCN, 2 msr) before or after succinate addition.

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by mitochondria. The linearity of the reaction is demonstrated in Fig. lA, where sequential amounts of calcium (100 nmoles) were added giving equal changes in absorbance. It should be noted that the change in absorbance is slightly greater in Fig. 1B with the same amount of calcium added than in Fig. 1A. This is probably due to magnesium preventing calcium phosphate formation. Since phosphate and succinate are known to complex calcium, spectrophotometric calibration of the murexide response to added amounts of calcium in the absence of these anions was routinely done. The differences in murexide response ,to free calcium in the presence and absence of these anions were then taken into account so that in all experiments reported here the figures represent true free calcium concentrations and rates of uptake. After calcium addition, subsequent addition of succinate (Succ) produced rapid uptake of calcium from the medium by mitochondria. The fact that the uptake of calcium was totally respiratory substrate dependent is demonstrated in Fig. 1C. The presence of oligomycin did not alter the succinate-driven uptake of calcium in these preparations while electron-transport chain inhibitors such as antimycin A, malonate, or KCN totally prevented the uptake of calcium (Fig. lC, dotted lines). With the addition of these metabolic inhibitors, or when the system became anaerobic, the release of 70% of the accumulated calcium from the mitochondria was generally observed (data not shown). Reversing the order of addition of calcium and succinate had no effect on the velocities of the reactions. Analysis of the initial velocities of calcium uptake by rabbit heart mitochondria at varying concentrations of calcium indicated an apparent K, of approximately 55-65 pM (Fig. 2). Since stopped-flow techniques were not used, the data do not extend to zero time or calcium concentration; however, the results appear similar to those reported by Scarpa and Graziotti (14) for cardiac mitochondria. Under the conditions of the experiments reported here, the rates of uptake remained linear for all concentrations of calcium used (Fig. 2) until approxi-

mately 80% of the calcium had been removed from the medium (Fig. 1A). In the presence of magnesium the initial rates of calcium uptake by rabbit heart mitochondria are markedly decreased (Fig. 1B). Table I indicates the effects of increasing concentrations of magnesium on the initial velocities of calcium uptake by rabbit heart mitochondria. The possibility that magnesium may have been affecting the release of calcium from the murexide dye is excluded by the fact that removal of calcium with EGTA from the dye, in the presence of totally inhibited mitochondria, was the same in the presence or absence of magnesium (data not shown). It has also 300 275 250 >F t 225 > 200 d 175 k 150 ; 125 2 100 * 75 +o 0 50

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40

60

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100 120 140 [Co++] pM

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FIG. 2. Initial rates of rabbit heart mitochondrial calcium uptake at varying concentrations of calcium. Experimental conditions same as Fig. 1 minus magnesium. The rates of calcium uptake were determined by measuring the initial linear portion of uptake trace after the addition of succinate. This curve is based on 10 separate experiments. The dotted line indicates that no determinations were made between 0 and 15 pM calcium concentration. TABLE I EFFECTS OF INCREASING @Ag*+] ON INITIAL RATES OF Cal+ UPTAKE BY HEART MITOCHONDRIA~

Initial rates of uptakeb (nmoles/min/mg protein) 0 0.33 1.0 1.65 3.3

230 + 21 171 jz 16 loo+12 60 f 10 50 f 10

a The initial concentration of Ca*+ in these experiments was 100 MM. bThese figures represent the average of five experiments. All other conditions as in Fig. 1.

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the cytochromes. This also implies the direct involvement of the energy-transducing mechanism in the active transport processes at thg inner mitochondrial membrane (30). Changes in the redox states of cytochrome b, concomitant with calcium uptake and followed by addition of ADP, reveal distinct differences in the presence and absence of exogenous magnesium (Fig. 4). In the absence of magnesium, cytochrome b did not reach a reduced (upward deflection) steady state until all of the calcium had been accumulated (Fig. 4A). Subsequent addition of ADP produced a transient oxidation of cytochrome b and a small efflux of calcium from the mitochondria (Fig. 4A). As cytochrome b returned to the original reduced steady state, the calcium that had been released from the mitochondria was reaccumulated (Fig. 4A). In the presence of magnesium (Fig. 4B), cytochrome b rapidly reached a reduced steady state before all of the calcium had been removed from the medium. Subsequent addition of ADP produced a marked, transient oxidation (downward

been established that magnesium does not affect murexide response to calcium (13). Measurement of mitochondrial respiratory activity revealed a marked stimulation of State 3 respiration by exogenous magnesium (Fig. 3A, +Mg’+). Under conditions identical to those employed for calcium uptake, a distinct stimulation in mitochondrial respiration was observed by the addition of calcium in the absence of exogenous magnesium (Fig. 3B). Subsequent addition of ADP failed to produce any significant increase in mitochondrial oxygen consumption. In the presence of magnesium, calcium also produced a distinct but lesser stimulation of respiration and the subsequent addition of ADP produced a marked increase in respiratory activity, characterized by an apparent State 3 to State 4 respiratory transition (Fig. 3C). Since the outer mitochondrial membrane is readily permeable to most substances, it is generally assumed that active transport through the inner membrane is reflected by changes in the redox states of 960

Yw ADP

R -2 130 ADP /

135 M

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FIG. 3. Oxygen electrode traces of mitochondrial succinate-linked respiration during calcium uptake, + magnesium. Assay conditions are identical to those in Fig, 1. Magnesium when present, 1.0 mM. The numbers next to the traces indicate the rates of oxygen consumption in natoms/min/mg mitochondrial protein. A: Coupled respiratory activity * magnesium. After addition of mitochondria (M,), ADP (500 nmoles) was added to produce a State 3 respiratory burst. B: Oxygen consumption during calcium uptake minus magnesium. Calcium (300 nmoles) is added followed by succinate (Succ). Subsequent addition of ADP (500 nmoles) produces no further stimulation in respiration. C: Oxygen consumption during calcium uptake plus magnesium; additions same as B.

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A -McJ”

FIG. 4. Changes in cytochrome b redox states during calcium uptake and subsequent ADP addition, * magnesium. The upper traces in each panel represent the independent measurement of calcium uptake + magnesium, as in Fig. 1. The lower traces represent cytochrome b reduction (upward deflection) measured at the wavelength pair 430-410 nm by dual-beam spectroscopy. Assay conditions are identical to Fig. 1 except that no murexide is present during assay of cytochrome b. Additions: Ca2+ = 300 nmoles, Succ (succinate) = 5 mM, and ADP = 500 nmoles. A: Minus magnesium. B: Plus magnesium, 1.0 mM.

deflection) of’ cytochrome b and eventually it returned to a lower reduced steady state than the original (Fig. 4B). This is consistent with the observed increase in the rates of oxygen consumption after ADP addition (Fig. 3C), at which point a substantial amount of calcium (upward deflection) was observed to come out of the mitochondria and was then reaccumulated as cytochrome b once again approached a new reduced steady state (Fig. 4B). Studies of the competitive effects of calcium and ADP on mitochondrial calcium uptake and cytochrome b reduction are shown in Fig. 5. In the absence of magnesium, a slight decrease in the rate of calcium uptake was observed with the simultaneous addition of both calcium and ADP (Fig. 5B). Cytochrome b exhibited prolongation of a more oxidized steady state, indicating a possible phosphorylation of the added ADP after calcium had been accumulated (Fig. 5B). In the presence of magnesium (Fig. 5D), a marked decrease in the rate of calcium uptake was observed with the simultaneous addition of calcium and ADP. Cytochrome b reached an initial reduced steady state that coincided with a marked change or slowing in

the rate of calcium uptake (Fig. 5D). Finally, when all the calcium had been accumulated, cytochrome b further increased its reduced steady-state level. To further define the effects of magnesium on mitochondrial calcium uptake, an inhibitor of calcium transport, ruthenium red (16, 18), and the ionophoretic antibiotic A-23187 (17) were used in studies of both the uptake and release of calcium by these mitochondrial preparations. Figure 6 is a composite set of traces showing the effects of ruthenium red and the ionophoretie antibiotic A-23187 on the uptake and release of calcium by mitochondria in the absence of magnesium. The unlabeled solid heavy line is the control calcium uptake curve. The beginning of calcium release (upward deflection) is shown at the point when the system normally becomes anaerobic. If ruthenium red (Fig. 6; +RR, solid line) or the antibiotic A-23187 (Fig. 6; +A-23, heavy solid line) are present at the initiation of calcium uptake, little or no uptake will occur. Addition of ruthenium red after calcium uptake is completed produces a rapid efflux of approximately 40-50% of the accumulated calcium (Fig 6; +RR, dashed line). Addition of A-23 alone

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OXA

FIG. 5. Calcium uptake and changes in cytochrome b redox states during simultaneous additions of calcium and ADP f magnesium. Assay conditions are identical to Fig. 1 and measurements of cytochrome b as in Fig. 4. The numbers to the right of the upper traces are the rates of calcium uptake expressed in nmoles/min/mg. Calcium and ADP, when added, 300 and 500 nmoles, respectively. A: Control, calcium uptake and cytochrome b reduction minus magnesium. B: Calcium uptake and cytochrome b reduction with simultaneous addition of calcium and ADP minus magnesium. C: Control, calcium uptake and cytochrome b reduction plus magnesium (1.0 mM). D: Calcium uptake and cytochrome b reduction with simultaneous addition of calcium and ADP plus magnesium (1.0 mhr).

or with ruthenium red resulted in a complete, instantaneous discharge of the accumulated calcium (Fig. 6; A-23 +RR, dashed line). Figure 7 shows the effects of ruthenium red and A-23187 on the uptake and release of calcium in the presence of exogenous magnesium. The unlabeled heavy, solid line is the control uptake of calcium. Although the release of calcium when the system becomes anaerobic is not shown in Fig. 7, this also occurred in the presence of magnesium. With magnesium present, ruthenium red still produces total inhibition of calcium uptake (Fig. 7; +RR, solid line), however, a significant amount of calcium is taken up in the presence of A-23187 (Fig. 7; +A-23, heavy solid line). This lack of total inhibition of calcium uptake by A-23187 (Fig. 7) is probably due to some binding of the antibiotic by magnesium (17). Increasing the concentration of A-23187 from 1 &mg protein to 3 pg/mg protein effectively and completely inhibited calcium uptake in the presence of

magnesium (data not shown). Addition of either ruthenium red or A-23187, after calcium has been accumulated with magnesium present, produces a slow efflux of approximately 30% of the accumulated calcium (Fig. 7; +RR or A-23, lower dashed lines). However, when both ruthenium red and A-23187 are added, after calcium uptake, a rapid and complete discharge of the accumulated calcium is observed (Fig. 7; A-23 +RR, upper dashed line). All of the above results were duplicated qualitatively utilizing ‘Va and Millipore filtration techniques at higher concentrations of calcium

(300 PM). DISCUSSION

These data are essentially in agreement with those of Scarpa and Graziotti (14) with respect to the apparent K, (-55-65 PM) for half-maximal rates of calcium uptake by heart mitochondria, as well as the concentration of calcium necessary to reach a maximum velocity of uptake (-200

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100 -‘RR ,-.--------h-23. A-23+ RR +1-z, ; -Mg[Q”]PM I 50 rl [

I,,,,,,,,,,,. +--------= ,

“3 FIG. 6. Composite curves of mitochondrial calcium uptake and release in the absence of Mg*+ with addition of “crude” ruthenium red (+RR) and the antibiotic A-23187 (A-23). Assay conditions as in Fig. 1. Although ruthenium red exhibits absorbance at 541 nm, because of the nature of dual-beam spectroscopy this absorbance was cancelled by optical attenuation when the dye was present at the beginning of the experiment. When the dye was added at later points in an experiment, prior determinations of absorbance change were made and corrected for by optical attenuation. The heavy solid line (unlabeled) is the control uptake (downward deflection) of calcium and the beginning of release (system anaerobic) at 7 min (upward deflection). Eventually 60-70s of this accumulated calcium would be released. The heavy solid line, +A-23, indicates inhibition of calcium uptake by the antibiotic ,4-23187. The light solid line, +RR, indicates the inhibition of calcium uptake by ruthenium red. The lower dashed line, +RR, indicates addition of ruthenium red after calcium uptake and the efflux of calcium from the mitochondria (upward deflection). The upper dashed line, A-23; A-23 +RR, indicates the discharge of accumulated calcium from mitochondria by the addition of the antibiotic A-23187 alone or by addition of A-23187 and ruthenium red together. Additions where indicated: A-23187 (A-23), 1 &mg mitochondrial protein; “crude” ruthenium red (RR), -3.0 nmoles/mg mitochondrial protein.

The data also appear to support the concept that mammalian mitochondria, obtained from normal hearts, would not be capable of sequestering calcium at rates and in amounts sufficient to participate directly in the relaxation phase of the cardiac cycle (14). The low in vitro rates of calcium uptake by heart mitochondria at calcium concentrations that are considered to exist intracellularly (0.1 PM-~ pM) during the cardiac cycle (10,25) may represent a physiological mechanism that prevents cardiac mitochondria in viuo from respond-

PM).

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ing to the normal fluxes of intracellular calcium, thereby maintaining mitochondrial respiratory activity primarily for ATP production. However, extrapolation of in vitro results to in viuo conditions should be approached with caution. One discrepancy between the data reported here and those of Scarpa and Graziotti (14) was the presence of 5 mM magnesium in their assay medium during the determination of the initial velocities of calcium uptake. However, this might be explained by the differences in measurement techniques and the character of the calcium uptake curve in the presence of magnesium. The calcium uptake trace is considerably more hyperbolic in the presence of magnesium (Fig. lB), that is, a continuing decrease in the velocity of uptake occurs as the external calcium concentration decreases. Scarpa and Graziotti (14) used a stopped-flow technique in

[b”]YM I

FIG. 7. Composite curves of mitochondrial calcium uptake and release in the presence of Mg*+ (1.0 mM) with additions of ruthenium red (+RR) and the antibiotic A-23187 (+A-23). Assay conditions as in Fig. 1. The heavy solid line (unlabeled) is the control uptake (downward deflection) of calcium. Although not shown, 5060% of the accumulated calcium would be released when the system becomes anaerobic. The heavy solid line, +A-23, is partial inhibition of calcium uptake by the antibiotic A-23187. The light, solid line, +RR, is the inhibition of calcium uptake by ruthenium red. The two lower dashed lines, +RR and A-23, indicate the release of accumulated calcium by the addition of ruthenium red or the antibiotic A-23187. The upper dashed line, A-23 +RR, indicates the discharge of accumulated calcium upon addition of both A-23187 and ruthenium red. Additions where indicated: A-23187 (A-23), 1 pg/mg mitochondrial protein; “crude” ruthenium red (RR), -3.0 nmoles/mg mitochondrial protein.

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which the initial velocities of their reactions were measured in the first 500 ms. In the experiments reported here, an artifact of mixing during initiation of the reaction occurs during the first 3-5 s (Fig. 1) and the initial velocity of the reaction is sunsequently determined by measuring the linear portion of the slope of the uptake curve during the next lo-20 s (Fig. 1). The results of Scarpa and Graziotti (14) indicate that perhaps no differences exist in the initial velocities of calcium uptake in the presence or absence of magnesium when measured by very rapid means. However, this does not obviate the fact that subsequent mitochondrial calcium accumulation (lo-20 s after initiation of the reaction) is markedly decreased by the presence of magnesium (Figs. lB, 5B; Table I). Magnesium is also facilitating some “protective” effect on mitochondrial oxidative phosphorylation during calcium accumulation (Figs. 3 and 4) and altering the response of heart mitochondria to the simultaneous addition of calcium and ADP (Fig. 5). The data indicate a regulatory effect of magnesium on the respiration-supported uptake of calcium by heart mitochondria. The decreased rates of uptake in the presence of magnesium (Fig. 1 and Table I) are not due to inhibitory effects of magnesium on respiration (Fig. 3). In fact, magnesium produces a marked stimulation of mitochondrial respiratory activity (Fig. 3A). It should be noted that magnesium also apparently activates mitochondrial ATPase resulting in somewhat lower ADP:O ratios (Fig. 3A and C). However, calciumstimulated respiration is less in the presence of magnesium (Fig. 3C) than in its absence (Fig. 3B). In the presence of magnesium, mitochondria apparently have retained the capability to phosphorylate ADP after calcium uptake (Fig. 3C). An effect on electron transport chain activity is further evidenced by the differential changes in the redox state of cytochrome b in the presence and absence of magnesium (Fig. 4). The small, transient oxidation of cytochrome b and the efflux of calcium from mitochondria (Fig. 44, -MgZ+) with no significant change in respiratory rate

(Fig. 3B) is at present unresolved. However, the differences in the mitochondrial response to calcium and ADP when magnesium is present or absent appear to be quite different. It is attractive to speculate that magnesium is acting to “protect” the highenergy intermediate state by reducing the energy load on the redox pressure of mitochondria in such a manner that not all of the energy generated from electron transport can be directed exclusively to the uptake of calcium. The distinct slowing of the calcium uptake curve in Fig. 5D, when calcium and ADP were simultaneously present, and the apparent ability of mitochondria to undergo a marked stimulation of respiration after calcium accumulation followed by addition of ADP (Fig. 3C) would tend to support this view. These data could also be interpreted as a competitive effect of magnesium for divalent cation transport at the inner mitochondrial membrane. By competitively slowing the rate of binding and subsequent transport of calcium in the mitochondrial membrane, magnesium would, in effect, slow the response of the electron transport chain to a given quantity of calcium. The ability of heart mitochondria to phosphorylate ADP after accumulation of small amounts of calcium (100 pM) and in the presence of magnesium is similar to the results reported by Rossi and Lehninger (22) for rat liver mitochondria. Rossi and Lehninger (22) also concluded from their experiments that simultaneous addition of calcium and ADP to liver mitochondria, with magnesium present, resulted first in the preferential accumulation of calcium and secondly in the phosphorylation of ADP. The results shown here in Fig. 5 would suggest that, in the presence of magnesium, heart mitochondria initially begin to accumulate calcium with added ADP (Fig. 5D) but, after approximately half the calcium is taken up, some phosphorylation of ADP may also be occurring. In the absence of magnesium, heart mitochondria appear to first accumulate the added calcium and subsequently phosphorylate the added ADP (Fig. 5A and B). Since direct measurements of ADP phosphorylation were not done under these _. conditions (Fig. 5), this conclusion still

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remains speculative. It should also be pointed out that in their experiments with rat liver mitochondria, Rossi and Lehninger (22) did not make direct determination of calcium uptake but measured the respiratory responses of mitochondria to added calcium and ADP by polarographic means. An alternative explanation would involve the existence of two active calcium “channels” or sites through transport which calcium may be actively moved through the mitochondrial membrane in the absence of magnesium. However, in the presence of magnesium one channel would, in effect, be blocked and thereby the rate of total accumulation of calcium into mitochondria slowed. This, in turn, would have the effect of reducing the amount of energy demand placed on the electron transport chain and permit mitochondria to retain their phosphorylative capacity. Additional evidence that magnesium may not be effectively competing with calcium for active transport is that the conditions for active uptake of magnesium that have been observed in heart mitochondria were at far higher concentrations of magnesium than were used in these expeiments (21). It is also generally accepted that magnesium is not a particularly effective competitor of calcium for active transport into mitochondria (1). One other possibility would be that the “apparent” uptake of calcium in these studies is actually energized binding of the cation to the mitochondrial membrane and not transport per se. A-23 could then be conceived as more readily dislocating calcium from the membrane binding sites than ruthenium red. Magnesium would simply be interfering with calcium binding. This latter possibility seems unlikely, however, since Mela (29) has shown under conditions very similar to those employed here that a distinct increase in intramitochondrial pH occurs, indicating actual transport of calcium into the intramitochondrial compartment. Ruthenium red, in the presence or absence of magnesium, totally inhibits the active uptake of calcium into mitochondria (Figs. 6 and 7) and is consistent with previously reported results (16, 18, 19). The

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antibiotic A-23187 (A-23) more effectively blocks calcium uptake in the absence of magnesium (Fig. 6) than in its presence (Fig. 7). It appears that the effect of A-23 in only slowing the rate of uptake in the presence of magnesium represents a competition between calcium and magnesium for the antibiotic since total blockade of calcium uptake into mitochondria in the presence of magnesium can be effected by increasing the concentration of A-23 (data not shown). The effects of ruthenium red and A-23 on the release of accumulated calcium from mitochondria are somewhat more complex, because unlike other inhibitors of mitochondrial membrane translocases (26-28), ruthenium red permits the leakage of the accumulated calcium from mitochondria. It would appear that the binding of ruthenium red to the mitochondrial membrane alters the active transport process so that energy from electron transport can no longer he transduced to maintain the ionic gradient and, therefore, the calcium begins to leak out (Figs. 6 and 7). At the concentrations of “crude” ruthenium red used in these experiments (-3.0 nmoles per mg mitochondrial protein) mitochondrial respiration is neither inhibited nor uncoupled (data not shown). This is in agreement with the results of Moore (16) and Vasington et al. (18). Vasington et al. (18) also observed the loss of accumulated calcium from rat liver mitochondria upon addition of ruthenium red and suggested that the pathways for calcium uptake and discharge from mitochondria may be different. In a recent report. Rossi et al. (19) concluded that ruthenium red can promote the discharge of calcium from liver mitochondria only if added before all the calcium is taken up. This is not the case for the experiments reported here, the differences between the results reported here and those of Rossi et al. (19) may be due to differences in assay conditions. In the absence of magnesium, A-23 will promote the almost instantaneous discharge of calcium out of the mitochondria and will do so in the presence or absence of ruthenium red (Fig. 6). The slower rate of calcium discharge in the presence of magnesium (Fig. 7) and at low

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A-23 concentration (1 pg/mg mitochondrial protein) probably represents a competition of magnesium for the antibiotic, since the rates of calcium discharge from mitochondria can be increased by increasing the amounts of A-23 added. Reed and Lardy (17) have suggested that A-23 interacts with a mitochondrial divalent cation carrier to produce a discharge of calcium from mitochondria. They further suggested that magnesium would slow this rate of efflux by competitively combining with antibiotic (17). This explanation would suffice for the action of A-23 alone, however, when ruthenium red has completely inhibited the divalent cation carrier after calcium accumulation, A-23 still produces a rapid efflux of calcium from mitochondria (Figs. 6 and 7). For this reason, it is suggested that A-23 acts at another “channel” in the mitochondrial membrane to produce calcium efflux. Recently, Leblanc and Clauser (31) have demonstrated differential effects of ADP and magnesium on the accumulation of calcium into hog heart mitochondria. On the basis of their experiments, Leblanc and Clauser (31) have suggested that ADP and magnesium act to protect the structural integrity of the mitochondrial membrane from displacement by calcium, which, in turn, maintains mitochondrial respiratory control during active calcium uptake. The physiological significance of mitochondrial calcium transport in uiuo is not yet understood. A number of hypotheses have been suggested, particularly for the role of mitochondrial calcium transport in the maintenance of ionic homeostasis within the cell (1, 2, 4, 7-9). However, the concise conditions under which calcium transport does occur in viva have not been elucidated. For in vitro conditions, the data presented here suggest that magnesium “protects” the phosphorylating mechanism from complete uncoupling or inhibition during active calcium uptake, regulating to some extent the utilization of “high-energy intermediates.” Further, two “channels” or sites may exist in the mitochondrial membrane for calcium uptake and release, one modulated by magnesium. Kinetic analysis of the interactions of cal-

cium and magnesium at the mitochondrial membrane are currently in progress. ACKNOWLEDGMENTS The technical assistance of Mr. Michael these studies is gratefully appreciated.

Stewart in

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