THE Jourmar, OF BIOLOGICAL CHE~~~STRY Vol. 241, No. 1, Issue of January 10, 1966

Printed in U.S.A.

Binding of Calcium by Liver Mitochondria of Rats Treated with

Steroid Hormones*

(Received for publication, May 18, 1965)

DANIEL V. KINIBERG AND SARA A. GOLDSTEIN From the Department of Medicine, Columbia University College oj Physicians and Surgeons, New York, New York 10032

SUMMARY

1. Prior treatment of rats with certain steroid hormones affects the binding of calcium to subsequently isolated liver mitochondria in vifro. Mitochondria isolated from animals treated with cortisol bind less calcium than do mitochondria from control animals under conditions in which binding is supported either by adenosine triphosphate alone or primarily by a respiratory substrate. Treatment with other glucocorti- coids (prednisone and cortisone) likewise diminishes sub- strate-dependent calcium binding to mitochondria in vitro.

2. Prior treatment with deoxycorticosterone enhances the binding of calcium to subsequently isolated mitochondria in both ATP- and substrate-dependent systems. Treatment with aldosterone and 17&estradiol also increases calcium binding under respiratory substrate-dependent conditions in vifro. Treatment with testosterone or progesterone is inef- fective in altering mitochondrial calcium binding in vifro.

3. The results suggest that treatment of rats with cortisol or deoxycorticosterone affects the ATPase activity of subse- quently isolated mitochondria and that this effect may be responsible for alterations in the ATP-dependent binding of calcium in vitro. The effect of cortisol treatment on dimin- ishing ATP-dependent calcium binding is potentiated by the addition of oligomycin in vifro.

4. Antimycin inhibits the substrate-dependent binding of calcium to mitochondria in vitro. This effect is potentiated by cortisol treatment of the animal, probably by virtue of a steroid-induced block in the electron transport sequence at or near the antimycin-sensitive step.

5. It is suggested that cortisol treatment interferes with the utilization of ATP or respiratory substrate necessary to support mitochondrial calcium binding in vifro.

The effects of steroid hormones on calcium metabolism are poorly understood. Estrogens and testosterone are capable of enhancing calcium absorption in individuals with osteoporosis

* Presented in part to the American Society of. Biological Chemists, Atlantic City, New Jersey, April 13, 1965. This work was supported by Grants AM-07625 and HE-05741 from the Na- t.ional Institutes of Health, and by American Cancer Society Institutional Grant IN-4E.

(I), but the mechanism of this hormonal action remains unex plained. Glucocorticoids may decrease calcium absorption and, in addition, may effect a return towards normal of the serum calcium levels in certain hypercalcemic states (24). Further, glucocorticoid administration or adrenalectomy may alter the transport of calcium by segments of isolated rat intestine in vitro, but the mechanism of t’hese effects is likewise unexplained (5-7).

Calcium binding by mitochondria was initially reported in 1953 by Slater and Cleland (8) ; these observations have been confirmed and extended (9-14). The accumulation of calcium by isolated liver and kidney mitochondria is an active process which can be supported by either mitochondrial respiration or adenosine triphosphate (11, 14, 15). The maximal binding of large quantities of calcium, however, requires the presence of an oxidizable substrate, ATP, magnesium ions, and inorganic phos- phate (10, 11, 14).

The present investigation was undertaken in an effort to define the basis for the steroid hormone-induced effects on cal- cium metabolism. These studies show an effect of prior treat- ment of rats with certain steroid hormones upon the binding of calcium by isolated rat liver mitochondria in vitro. Furthermore, the present findings suggest the locus in the respiratory chain which is sensitive to glucocorticoids. This system in vitro appears to be a useful model for the study of the mechanism of hormonal influences on calcium and phosphorous metabolism.

EXPERIMENTAL PROCEDURE

Materials-The chemicals used in the incubation mixture and in the protein and inorganic phosphate determinations were of analytical grade and were purchased from the Fisher Scientific Company. Tris was procured from Sigma. Dipotassium ATP, succinic acid, n-glutamic acid, and a-ketoglutaric acid, all of which were neutralized with KOH, were obtained from Mann. 4SCa P-3 (specific activity > 10,000 mC per g) was purchased from Oak Ridge. Oligomycin B and antimycin A were procured from the Wisconsin Alumni Research Foundation. Ouabain was purchased from Mann, and Amytal was a gift of Eli Lilly and Company.

Cortisone was obtained from Merck (Cortone acetate, 25 mg per ml in NaCl suspension). Testosterone (Perandren pro- pionate, 25 mg per ml in sesame oil), deoxycorticosterone (Percorten acetate, 5 mg per ml in sesame oil), and crystalline d-aldosterone acetate were obtained from Ciba. The d-aldos- terone acetate was dissolved (0.33 mg per ml) in sesame oil.

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96 Binding of Calcium by Rat Liver Mitochondria Vol. 241, No. 1

Progesterone and 17@-estradiol were obtained from Schering as Proluton, 25 mg per ml in sesame oil, and Progynon benzoate, 1 mg per ml in sesame oil, respectively. Cortisol and prednisone (17a!, 21.dihydroxy-l , 4-pregnadiene-3,11,20-trione) were pur- chased in crystalline form from Steraloids, Inc., and dissolved in propylene glycol (2.5 and 1.0 mg per ml, respectively).

Animal Treatment-Albino male rats of the Sherman strain were obtained at a weight of 125 g and fed a diet adequate in vitamin D and containing 1.44 y0 calcium and 1.17 y0 phosphorus. The animals were given injections subcutaneously with either 2.5 mg of cortisol, 2.5 mg of deoxycorticosterone acetate, 5.0 mg of cortisone acetate, 1.0 mg of prednisone, 2.5 mg of testosterone propionate, 2.5 mg of progesterone, 1.0 mg of 17/%estradiol benzoate, or 0.66 mg of d-aldosterone acetate daily for 7 days with control animals receiving injections of the appropriate vehicle.

Preparation of Mitochondria-After 7 days of treatment, two or more control and an equal number of steroid-treated animals were killed by a sharp blow on the head followed by decapitation. The livers were removed quickly and chilled in ice-cold isotonic sucrose. A 40% homogenate of the pooled liver from control or treated animals was prepared at 0” with a Potter-Elvehjem homogenizer fitted with a Teflon pestle. Subsequent steps were carried out at a 0” in an International model PR-2 centrifuge with the No. 269 and 295 rotors. The homogenate (10 ml) was added to 30 ml of cold 0.25 M sucrose and centrifuged at 600 x g for 10 min to remove nuclei, cell debris, and unbroken cells. The upper layer was removed and centrifuged at 8000 x g for 10 min to sediment the mitochondria. The pellet was washed once with cold 0.25 M sucrose, sedimented at 14,000 X g, and subsequently resuspended in cold isotonic sucrose with the aid of a homogenizer and Teflon pestle at a slow speed. The final suspensions of mitochondria from control and treated animals were diluted with 0.25 M sucrose to contain between 2.0 and 4.0 mg of mitochondrial protein per ml as determined by a modifica- tion of the biuret procedure (16).

Reaction Mixtures-For those experiments in which the mito- chondria were not incubated prior to the addition of calcium, the basic reaction mixture contained, in a final volume of 3.0 ml, 168 pmoles of Tris-HCl buffer (pH 7.4), 120 pmoles of KCl, 15 pmoles of MgC12, 125 pmoles of sucrose, 3.0 to 22.5 pmoles of dipotassium ATP, 0.75 pmole of CaClz containing 45Ca, and 0.5 ml of mitochondrial suspension equivalent to 1.0 to 2.0 mg of protein.

Unless otherwise indicated, in those experiments in which the mitochondria were incubated before the addition of calcium, 0.5 ml of the mitochondrial suspension was added to 1.0 ml of me- dium containing 168 pmoles of Tris-HCl buffer (pH 7.4), 120 pmoles of KCl, 15 pmoles of MgC12, and 125 pmoles of sucrose. After 15 min of incubation at 30”, 0.75 pmole of CaClz labeled with 45Ca, 0 to 15 pmoles of dipotassium ATP, 0 to 30 pmoles of potassium phosphate buffer (pH 7.4), and 0 to 30 pmoles of potassium succinate were added in 1.5 ml, thus bringing the total volume to 3.0 ml. In larger experiments multiples of the basic 3.0.ml reaction mixture were used.

The mixtures were incubated in a Dubnoff-type shaker at 30” in air. Aliquots were removed at varying times, chilled at 0” in polypropylene tubes, and centrifuged at 28,700 x g at 0” for 10 min. The supernatant fluid was decanted and 0.25-ml aliquots were counted as infinitely thin samples on aluminum planchets in the Geiger region with a thin window detector.

Results are expressed as micromoles of calcium bound per mg of mitochondrial protein and represent the mean values obtained from three or more separate experiments performed in an identi- cal manner. In preliminary experiments, the supernatant solution and 1 y0 sodium dodecyl sulfate extracts of mitochondria were both counted and subjected to calcium determinations with a titrimetric procedure (17). This revealed a good correlation between calcium disappearance from the medium and mito- chondrial calcium content as determined by both procedures. Inorganic phosphate was determined by the procedure of Fiske and SubbaRow (18) after removal of the protein by the addition of an equal volume of cold 10% trichloroacetic acid and cen- trifugation at 4”.

RESULTS

Mitochondria not Incubated Prior to Addition of Calcium- Previous studies employing low calcium concentrations in the medium have indicated that the binding of calcium by mito- chondria may be supported either by ATP alone or by oxidative phosphorylation (11, 14, 15). Fig. 1 illustrates the results of initial studies in which calcium binding was measured in mito- chondria isolated from the livers of control and cortisol-treated animals. The mitochondria were not subjected to prior incuba- tion in the absence of calcium, ATP, inorganic phosphate, or substrate. Varying concentrations of ATP were present in a medium devoid of added succinate and inorganic phosphate. With increasing ATP concentrations (1.0 to 7.5 mM) increasing amounts of calcium were bound to the mitochondria derived from both the control and cortisol-treated animals. Differences in binding between the two preparations of mitochondria were evident at ATP concentrations between 1.0 and 3.0 mM. With 2.0 mM ATP in the medium, the mitochondria from the control animals bound twice as much calcium as those from the cortisol- treated animals (0.26 Fmole of Ca++ per mg as opposed to 0.13 pmole of Ca++ per mg at 30 min). The diminished binding by liver mitochondria obtained from cortisol-treated animals per- sisted throughout the 70.min incubation. Of note is the fact that at 1 mM ATP, where binding was not appreciable, the differ- ences between the two mitochondrial preparations were small. Likewise, at 7.5 111~ ATP, where binding was near maximal, there were no demonstrable differences between mitochondria from control and cortisol-treated animals.

The effects of treatment with deoxycorticosterone, a mineralo- corticoid, were examined under similar conditions. As shown in Fig. 2, mitochondria from the deoxycorticosterone-treated animals bound more calcium at all points in time than did those from the control animals. The effect of the mineralocorticoid, which was opposite in direction to that of cortisol, was also minimized in the presence of increasing concentrations of ATP, where binding was nearly complete in both the control and steroid-treated groups.

Inorganic phosphate was measured in the trichloroacetic acid supernatant fluids from the experiments in which mitochondria not subjected to prior incubation were studied with 0.25 mM Ca+f and 3 mM ATP in the medium. At 30 min the mito- chondria from control animals bound 0.33 pmole of Ca++ per mg of protein as opposed to 0.30 pmole per mg bound to the mitochondria from cortisol-treated animals. At the same time there were 1.41 pmoles of Pi per mg of protein in the control flasks, as opposed to 2.34 pmoles of Pi per mg in the flasks con- taining mitochondria from the cortisol-treated animals. Under

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Issue of January 10, 1966 D. V. Kimberg and X. A. Goldstein 97

0.4

- ‘I

CONTROL 7.5 mM ATP

mM ATP

CONTROL I ml,, ATP

CORTISOL I mM ATP

\ \tCORTISOL 2mM ATP

IO 20 30 40 50 60 70

TIME (MINUTES)

FIG. 1 (left). Effect of cortisol treatment on calcium binding to mitochondria in the absence of substrat’e and with varying ATP concentrations. The mit.ochondria were not subjected to prior incubation and the experiments were performed as described in the text. The medium contained 56 mM Tris-HCI buffer (pH 7.4), 40 rnM KCl, 5 mM MgClt, 41.7 mM sucrose, 0.25 mM CaClz (labeled with Wa), concent’rations of ATP from 1.0 to 7.5 mM, and liver mitochondria from control and cortisol-treated rat,s (3.0 to 6.0 mg of protein) in a volume of 9.0 ml.

the same assay conditions, calcium binding at 30 min to mito- chondria from control and deoxycorticosterone-treated animals was 0.32 and 0.34 pmole per mg of protein, respectively. Pi in the media from control and deoxycorticosterone flasks was 2.22 and 1.50 pmoles per mg of protein, respectively. Inorganic phosphate is indeed the major anion accompanying Ca++ ac- cumulation (ll), and in these experiments it was probably derived from ATP. In view of the relatively small difference in calcium binding under these experimental conditions, the striking disparity in the amount of inorganic phosphate in the media must be related to other factors.

Similar experiments were conducted in which mitochondria from control and from cortisol- and deoxycorticosterone-treated animals were added to a more complete medium without prior ‘incubation. This medium contained varying amounts of ATP (0.25 to 3.0 mM), 10 mM potassium succinate, and 10 mM potas- sium phosphate. At 1.0 mM ATP the results were similar to those obtained with ATP (1.5 to 3.0 mM) in the absence of sub- strate. The calcium binding to the mitochondria from cortisol- treated animals was less than control, and that to the mito- chondria from deoxycorticosterone-treated animals was greater than the control. Once again, with increasing ATP concentra- tions in the complete medium, the effects of prior treatment with steroids became less evident.

Incubation of Mitochondria Prior to Addition of Calcium-In an attempt to define the effects of steroid hormone treatment more clearly, studies lvere undertaken in which the mitochondria

,oOCA 3 mM

ROL 3 mM

CONTROL I.5 m

CONTROL I mM

ATP

ATP

10 20 30 40 50 60 70

TIME (MINUTES)

FIG. 2 (right). Effect of deoxycorticosterone (DOCK) treat- ment on calcium binding to mitochondria in the absence of sub- strate and with varying ATP concentrations. The mitochondria were not subject,ed to prior incubation and the experiments were performed as described in the t,ext. The medium was identical with that in Fig. 1 except for the ATP concentrations, which varied between 1.0 and 3.0 mM.

from control and treated animals were incubated at 30” for 15 min before the addition of calcium, ATP, inorganic phosphate, or substrate. Both prior incubation and suboptimal ATP concentrations have been shown to increase the requirement for substrate in providing for optimal calcium binding (10). Thus, the depletion of endogenous substrate by prior incubation should make calcium binding more directly dependent upon ATP in experiments in which subsequently added ATP is the sole energy source. Contrary to the results of others (15), significant and consistent binding of calcium could not be supported with succi- nate and inorganic phosphate alone in experiments in which the mitochondria were incubated before the addition of calcium, substrate, and inorganic phosphate. It was necessary to add small, suboptimal amounts of ATP (0.75 to 1.5 mM) along with the succinate and inorganic phosphate. Under these circum- stances calcium binding is largely dependent upon the added respiratory substrate (10, 19). The method by which these experiments T+eere conducted is detailed under “Experimental Procedure.”

ATP-dependent Calcium Binding-Experiments were per- formed in which mitochondria from control and cortisol-treated animals were incubated for 15 min before the addition of CaClz labeled with 45Ca and varying amounts of ATP. Cortisol treat- ment diminishes the ATP-dependent binding of calcium. This effect is not marked at low (1.0 mM) ATP concentrations, where binding is not great, or at high (5.0 mM) ATP concentrations, where binding is nearly complete in both preparations of mito-

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98 Binding oyf Calcium by Rat Liver Mitochondria Vol. 241, No. 1

CONTROL + SUCCINATE

IO 20 30 40 50 60 70

TIME (MINUTES)

0.6

0.5

z

w

L LT 0.4 a

”

2 w

;: zz 0.3

P

w

::

L 23

: 0.2

cl?

0. I

FIG. 3 (left). Effect of succinate (in the presence of 2.0 rn~ ATP) on calcium binding to mitochondria from control and corti- sol-treated animals. The incubations were performed as de- scribed in the text. Liver mitochondria from control and corti- sol-treated rats (1.5 ml containing 3.0 to 6.0 mg of protein) were incubated for 15 min at 30” by addition to 3.0 ml of a medium containing 168 mM Tris-HCl buffer (pH 7.4), 120 rnM KCl, 15 rnM MgC12, and 125 rnM sucrose. At the end of the 15min incubation, 4.5 ml of a medium containing 0.5 mM CaClz (labeled with 45Ca),

chondria. With 2.0 mM ATP the mitochondria from the control animals bound 0.22 pmole of Ca+f per mg of protein at 30 min as opposed to 0.15 pmole per mg bound to mitochondria from the cortisol-treated animals.

Similar experiments were conducted with mitochondria from control and deoxycorticosterone-treated animals. The ATP- dependent binding of calcium was greater in the mitochondria from the deoxycorticosterone-treated animals than in mito- chondria from the controls. This effect, which is opposite in direction to that of cortisol, is best seen at intermediate ATP concentrations (1.5 and 3.0 mM), and was negligible with 0.75 and 5.0 mM ATP. With 1.5 n?M ATP the mitochondria from the deoxycorticosterone-treated animals bound 0.12 pmole of Ca++ per mg of protein at 30 min as opposed to 0.01 pmole per mg bound to mitochondria from the control animals.

Subsfrate-dependent Calcium Binding-Further studies were performed to define the effects of steroid hormone treatment on mitochondria incubated for 15 min before the addition of CaClz

CONTROL + SUCCINATE

IO 20 30 40 50 60 70

TIME (MINUTES)

4.0 mM ATP, and either water or 20 mM succinate were added, bringing the final volume to 9.0 ml, wit.h a final concentration of 0.25 mM CaC12.

FIG. 4 (right). Effect of succinate (in the presence of 5.0 rnM ATP) on calcium binding to mit’ochondria from control and cor- tisol-treated animals. The mitochondria were subjected to prior incubat,ion and the experiments were performed as described in the text. The media were identical with t’hose in Fig. 3 except, for final ATP concentration.

along with a more complete medium. Figs. 3 and 4 illustrat,e the effects of adding 10 mM succinate to the medium. At the lower (2.0 IIIM) ATP concentration (Fig. 3) the addition of succi- nate not only increased the binding of calcium by mitochondria from both control and cortisol-treated animals, but markedly enhanced the difference in uptake between the two. At 5.0 mM ATP (Fig. 4) the addition of succinate only slightly increased the already great binding of calcium to bot’h groups of mito- chondria.

As shown in Fig. 5, the addition of 10 lllM phosphate t’o the system containing 10 mM succinate and little (0.75 mM) ATP caused increased binding to mitochondria from both control and cortisol-treated animals and accentuated the differences between them. At higher ATP concentrations the effects of phosphate on augmenting calcium binding were less apparent and the corti- sol effect was likewise less marked.

Wit,h previously incubated mitochondria from control and deoxycorticosterone-treated animals, the addition of 10 mM

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Issue of January lo,1966 D. V. Kin&erg and X. A. Goldstein 99

succinate and 10 mM phosphate along with 0.75 mM ATP and 0.25 mM CaClz augmented the binding of calcium and accen- tuated the increased binding to the mitochondria from the deoxycorticosterone-treated animals.

EJects of Various Steroid Hormones on Substrate-dependent Calcium Binding-In view of the differences in the binding of

calcium to mitochondria isolated from control as opposed to cortisol- and deoxycorticosterone-treated animals, the effects of treatment with other steroid hormones were explored. A complete system (ATP, succinate, and inorganic phosphate) with varying ATP concentrations was used for this purpose. The mitochondria were subjected to incubation before the addition of calcium, ATP, succinate, and inorganic phosphate.

Treatment with cortisol, cortisone, and prednisone, all gluco- corticoids, resulted in diminished binding of calcium to subse-

--

IO 20 30 40 50 60 70

TIME (MINUTES)

FIG. 5. Effect of phosphate (in the presence of 0.75 rn~ ATP and 10 rn~ succinate) on calcium binding to mitochondria from control and cortisol-treated animals. The incubations were per- formed as described in the text. The incubation conditions w’ere identical with those in Fig. 3. At the end of 15 min of prior incu- bation, 4.5 ml of a medium containing 0.5 mM CaC12 (labeled wit.h ‘Wa), 1.5 mM ATP, 20 rnM succinate, and cit’hcr water or 20 rnM potassium phosphate buffer (pH 7.4) were added, bringing t,he final volume to 9.0 ml, wit,h a final concentrat’ion of 0.25 rnM CaC12.

TABLE I

E$ect of cortisol treatment on substrate-dependent calcilrm binding

The mitochondria were subjected to prior incubation. The incubations were performed as described in the text. The condi- tions were identical with those in Fig. G.

I Ca++ bound

0.26 0.2F 0.21 0.15

fimole/mg mitochondrial protein

0.12 0.35 0.14 0.40 0.12 0.38 0.22 0.40 0.12 0.40 0.26 0.42 0.08 0.40 0.21 0.41 O.OB 0.39 0.15 0.40

0.29 0.36 0.39 0.39 0.39

TABLE II

E$ect of cortisone treatment on substrate-dependent calcium binding The mitochondria were subject.ed to prior incubation. The

incubations were performed as described in the text. The condi- tions were identical with those in Fig. G.

Ca++ bound

Time

min

10 20 30 50 70

C.:,P:::H., ~ 1.5 my ATP ~ 2.0 IIIM ATP

Control Cortisone Control ( Cortisone

/.mrole/ntg nzitochondriel @&2in

0.13 0.02 0.29 0.08 0.29 0.10 0.20 0.04 0.39 0.14 0.37 0.17 0.15 0.03 0.30 0.12 0.3B 0.19 0.11 0.02 0.36 0.12 0.36 0.18 0.02 0.00 0.33 0.08 0.34 0.14

ATF

ATF

ATF

PREDNISONE 1.5 mM ATF

CONTROL 075 mM AT

I

IO 20 30 40 50 60 70

TIME (MINUTES)

FIG. (i. Effect of prednisone treatment on the substrate-de- pendent calcium binding to mitochondria subject.ed to prior incu- bation. The incubations were performed as described in the text. Liver mitochondria from corkol and cortisol-treat,ed rats (1.5 ml containing 3.0 to 8.0 mg of protein) were incubated for 15 min at, 30” by addition tSo 3.0 ml of a medium containing lG8 rnM Tris- I-ICI buffer (pH 7.4), 120 mM KCl, 15 mM MgC12, and 125 rnM sucrose. At the end of the 15.min incubation, 4.5 ml of a medium containing 0.5 mM CaClz (labeled with %a), 20 mM succina.te, 20 mM potassium phosphate buffer (pH 7.4), and concentrations of ATP, from 1.5 to 4.0 mM, were added, bringing the final volume to 9.0 ml. The final concentration of CaC12 was 0.25 mM, along with 10 mM succinate, 10 mM potassium phosphate, and 0.75 to 2.0 mM ATP.

quently isolated mitochondria at all points in time at ATP concentrations from 0.75 to 2.0 111~ (Tables I and II, and Fig. 6). The effects were maximal with 1.5 mM ATP in the presence of 10 IILM succinate and 10 mM inorganic phosphate. Rapid sampling techniques clearly revealed differences in the initial rates of

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100 Binding oj Calcium by Rat Liver Mitochondria Vol. 241, No. 1

ROL 1.5 mM ATP

LDOSTERONE 0.75 m

CONTROL 0.75

IO 20 30 40 50 60 70

TIME (MINUTES)

IO 20 30 40 50 60 70

TIME (MINUTES)

FIG. 7 (left). Effect of aldosterone treatment on the substrate- FIG. 8 (right). Effect of estradiol treatment on the substrate- dependent calcium binding to mitochondris. The incubations dependent calcium binding to mitochondria. The incubations were performed as described in the text. The conditions were were performed as described in the text. The conditions were dentical with those in Fig. 6. identical u-ith those in Fig. G.

calcium uptake within the first 3 min, before maximum binding had occurred. Treatment with deoxycorticosterone and aldos- terone acetate, both mineralocorticoids, resulted in the opposite effect; that is, one of enhanced binding to t.he mitochondria from t’he steroid-treated animals (Fig. 7).

dependent binding of calcium to mitochondria from either the control or cortisol-treated animals.

Estradiol treatment likewise caused increased binding of calcium to subsequently isolated mitochondria (Fig. 8). Treat- ment with testosterone and progesterone was totally ineffective in altering mitochondrial calcium binding in this system.

In experiments with glucocorticoid and estrogen treatment, an effect on mitochondrial calcium uptake was demonstrable as early as 18 hours following t,he injection of a single dose of hor- mone. The steroid effects were near maximal and quite con- sist.ent after 2 days of treatment.

Oligomycin B has been shown to inhibit calcium binding to mitochondria in ATP-dependent systems (15). As seen in Table III, oligomycin (0.02 and 0.04 pg per ml) preferentially inhibited binding to t.he mitochondria from cortisol-treated animals in a medium where the binding was dependent upon added ATP. Concent,rations of oligomycin between 0.08 and 0.20 pg per ml caused nearly complete inhibition of binding in both groups of mitochondria. Thus, oligomycin potentiated the decrease in binding induced by cortisol treatment. In the presence of respiratory subst’rate as well as ATP, oligomycin at concentrations as high as 1.0 pg per ml had no effect on binding to mitochondria from either control or cortisol-treated animals.

Effects of Various Substrates and Inhibitors on Calcium Bind- ing-The diminished binding of calcium to mitochondria isolated from cortisol-treated animals was also noted with 10 mM glu- tamate or 10 mM cr-ketoglutarate as the substrate. Although the binding of calcium was greater in mitochondria from both cont.rol and steroid-treated animals with succinate as the sub- strate, the relative magnitude of t’he cortisol effect on binding was unchanged in the presence of the other substrates. Ouabain, at a concentration of 5 x 1O-5 M, failed to affect the ATP-

Antimycin A, which inhibit,s the electron transport sequence between cytochrome b or coenzyme & and cytochrome cl (20), similarly potentiated the decrease in binding induced by cortisol (Table IV). These studies were carried out in the complete system with succinate as the substrate. At an antimycin con- centration of 9.0 X 10-g M there was no effect on binding to mitochondria from either control or cortisol-treated animals. At a concentration of 3.6 X lo-* M there was only minimal in- hibition of binding to the mitochondria from t’he control animals,

, LESTRADIOL 1.5 mM ATF

-CONTROL -CONTROL 1.5 mM 1.5 mM ATF ATF

m,..

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Issue of January lo,1966 D. V. Kimberg and S. A. Goldstein 101

but a striking effect on calcium binding to the mitochondria from the cortisol-treated animals. Higher concentrations of antimycin (9.0 X lo-8 and 3.6 X 10-T M) were associated with progressive inhibition of uptake in both the control and cortisol- treated animals. Similar but less striking findings of the same nature were apparent with glutamate or cY-ketoglutarate instead of succinate as the substrate.

The effect of Amytal, which has been considered to be an inhibitor of electron transport in the region of the NAD-linked flavoprotein (21), was also investigated. Low concentrations of Amytal (1.6 X 1O-4 M) were ineffective in altering calcium binding to mitochondria from either control or cortisol-treated animals in the complete system with glutamate or a-keto- glutarate as substrates. At slightly higher Amytal concentra- tions (3.1 x lop4 and 6.2 x 10e4 M) there was a small, incon- sistent preferential inhibition of binding to the mitochondria from control animals. In a number of experiments Amytal caused 10% inhibition of calcium binding to the mitochondria from the controls with no effect on binding to the mitochondria from cortisol-treated animals.

DISCUSSION

The present study has shown that calcium binding by isolated rat liver mitochondria may be affected by prior treatment of the animals with certain steroid hormones. Glucocorticoid treatment diminishes the binding of calcium to subsequently isolated mitochondria in vitro under conditions in which the binding is supported either by ATP alone or primarily by a respiratory substrate. These effects are dependent upon the presence of suboptimal concentrations of ATP or substrate, such that the complete binding of the calcium present in the medium does not occur with the mitochondria from the control animals. These findings suggest that mitochondria from glucocorticoid- treated animals fail to utilize either ATP or respiratory substrate as efficiently as the mitochondria from control animals.

It is conceivable that treatment with cortisol, deoxycorticos- terone, and certain other steroid hormones may lead to changes in mitochondrial ATPase activity, mitochondrial morphology, and respiratory efficiency. Such changes could be responsible for or related to the hormonal effects on calcium binding noted in this study. The glucocorticoid effect upon the ATP-de- pendent binding of calcium may be mediated by alterations in the activity of a mitochondrial ATPase or by interference with the reactions concerned with ATP synthesis. The marked increase in inorganic phosphate in the media containing mito- chondria from cortisol-treated animals as opposed to mito- chondria from control animals tends to support such an inter- pretation. The addition in vitro of a variety of steroid hormones (22, 23) or the administration of deoxycorticosterone to intact rats (23) has been reported to activate a latent mitochondrial ATPase. The solubilized mitochondrial ATPase, however, may be‘ inhibited by deoxycorticosterone (24, 25). Furthermore, the addition in vitro of deoxycorticosterone may cause reversible mitochondrial swelling (21). Electron micrographs of the livers from cortisol-treated animals do reveal striking increases in mitochondrial size and changes in structure.l Preliminary observations2 also suggest that cortisol treatment releases a latent mitochondrial ATPase in subsequently isolated rat liver

1 J. Wiener, personal communication. 2 D. V. Kimberg and S. A. Goldstein, unpublished observations.

TABLE III

Effect of oligomycin B on ATP-dependent binding of calcium to mitochondria from control and co&sol-treated animals

The incubations were carried out as described in the text. Liver mitochondria from control and cortisol-treated rats (1.5 ml containing 3.0 to 6.0 mg protein) were incubated in 3.0 ml of a medium identical with that described in Fig. 3, aside from the inclusion of 0.18 ml of either 100% ethanol or oligomycin in 100% ethanol. Following 15 min of incubation, 4.5 ml of a medium con- taining 0.5 mM CaC& (labeled with 45Ca) and 4.0 mM ATP were added, bringing the final volume to 9.0 ml with final concentra- tions of 0.25 mM CaCle and 2.0 mM ATP. In the absence of oligo- mycin the mitochondria from control and cortisol-treated animals bound 0.26 and 0.15 Fmole of Ca++ per mg of protein, respectively. The data in the table represent the oligomycin related per cent inhibition of binding to mitochondria from control and cortisol- treated animals at each concentration of oligomycin.

Oligomycin B

P&?/ml 0.004 0.008 0.012 0.020 0.040 0.080 0.160 0.200

Inhibition of binding to mitochondria

Control animals Cortisol-treated animals

YO % 0 0 0 0 0 0 0 40

15 40 85 87

100 100 100 100

TABLE IV

Efect of antimycin A on substrate-dependent binding of calcilLm to mitochondria from control and cortisol-treated animals

The incubations were carried out as described in the text. Liver mitochondria from control and cortisol-treated rats (1.5 ml containing 3.0 to 6.0 mg of protein) were incubated in 3.0 ml of a medium identical with that described in Fig. 3, aside from the inclusion of 0.18 ml of either 100% ethanol or antimycin in 100% ethanol. Following 15 min of incubation, 4.5 ml of a medium con- taining 0.5 mM CaC12 (labeled with 45Ca), 20 mM succinate, 20 mM potassium phosphate buffer, pH 7.4, and 3.0 mM ATP were added, bringing the final volume to 9.0 ml. Samples were taken 30 min following the addition of the calcium-containing medium. In the absence of antimycin, the mitochondria from control and cortisol-treated animals bound 0.46 and 0.34 pmole of Ca++ per mg of protein, respectively. The data in the table represent the antimycin-related per cent inhibition of binding to mitochondria from control and cortisol-treated animals at each concentration of antimycin

Antimycin A

‘3f

1.8 x 10-s 3.6 X 10-E 9.0 x 10-S 3.6 X 10-7

Inhibition of binding to mitochondria

Control animals Cortisol-treated animals

% % 0 12 7 56

60 70 65 70

mitochondria, whereas deoxycorticosterone treatment may inhibit this enzyme. Thus, cortisol treatment may interfere with the ATP-dependent binding of calcium in vitro by means

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102 Binding ~1 Cal&m by Rat Liver Mitochondria Vol. 241, Ko. 1

of an activation of a latent mitochondrial ATPase and depletion of the ATP in the medium with failure to form high energy intermediates necessary for calcium binding. Oligomycin B, probably by virtue of the fact that it prevents the format,ion of a necessary high energy intermediate from ATP, will inhibit calcium binding to mitochondria in ATP-dependent systems (15). The potentiation by oligomycin of the cortisol effect on ATP- dependent binding observed in the experiments reported here may reflect further impairment of ATP utilization in a system already possessing a defect. I f indeed a steroid-sensitive ATPase is of importance in causing the diminished ATP-dependent binding to mitochondria from cortisol-treated animals, we can assume from the data presented that the enzyme is insensitive to ouabain.

more precise localization of the various steroid hormone effects, the possible interactions with parathyroid hormone and vitamin D at the mitochondrial level, and the physiological significance, if any, of this model system all require further investigation. The present findings do, however, broaden the potential useful- ness of this model in vitro for the study of factors concerned with calcium and phosphorous metabolism.

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Daniel V. Kimberg and Sara A. GoldsteinHormones

Binding of Calcium by Liver Mitochondria of Rats Treated with Steroid

1966, 241:95-103.J. Biol. Chem. 

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