Thyroid hormone-divalent cation interactions

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 188, No. 1, May, pp. 220-225, 1978

Thyroid Effect

Hormone-Divalent

of Thyroid

Hormone

Cation

on Mitochondrial

Interactions Calcium

Metabolism

P. A. HERD Department

of Medicine,

The Miriam Hospital, Division of Biology Providence, Rhode Island 029X

and Medicine,

Brown

University,

Received May 2, 1977; revised December 20, 1977 The effect of L-triiodotbyronine (Ta) on calcium metabolism was studied in mitochondria isolated from thyroidectomized rats. The maximal rate of low-level calcium uptake ( V) was enhanced by TZ in the absence or the presence of added acetate; the K,,, value was unchanged. Further, the rate constant (K,) derived from measurement of the efflux of calcium was stimulated IO-fold upon the addition of Ts. This stimulating effert was not seen in the presence of acetate. In studies of adenine nucleotide translocation, calcium stimulated the translocation of ATP out of these mitochondria, but only in the presence of Ts. These results suggest that Ta alters calcium ion mobility within the mitochondrial membrane and hence modulates calcium-dependent metabolic processes. The precise locus of this affect remains to be identified.

take (lo), calcium efflux, and the calciumdependent translocation of ATP out of the mitochondrion were found to be sensitive to L-triiodothyronine added in vitro.

The mechanism(s) by which thyroid hormones act is largely unknown, although the metabolic consequences of altered thyroid state have been well described (1, 2). Indeed, although the rapid binding of hormone to nuclear protein [e.g., Ref. (3)] has been suggested to play a primary role resulting in the initiation of cytoribosomal protein synthesis (4), concomitant direct, hormonally mediated alterations in cell metabolism [e.g., heart rate (5) and mitochondrial metabolism (6-8)] have also been described. The relationship between these early physiological changes and later events secondary to cytoribosomal protein synthesis has not been explored. We recently described the formation of a spectrally distinct complex between L-triiodothyronine and calcium in nonpolar environments (9). These observations suggest that direct, hormone-mediated alterations in membrane calcium metabolism could provide a basis for the in vitro (8) and in uiuo (5-7) effects of thyroid hormone, particularly those observed prior to the initiation of cytoribosomal protein synthesis. In this report, the effect of thyroid hormone on mitochondrial calcium metabolism is reported. “Low-level” calcium up-

METHODS Analytical grade inorganic reagents and biochemical reagents were purchased from Sigma Chemical Co., St. Louis. [W]ATP and %a’+ were obtained from New England Nuclear Corp., Boston. Male albino rats, 50-60 g (CD strain, Charles River Laboratories), were thyroidectomized and maintained for at least 4 weeks prior to study. Previous studies (P. Herd, unpublished observation) have shown that during this period serum thyroxine levels drop to undetectable levels and body weight increases are markedly reduced (t150-g final weight) vs controls (>250-g final weight). Mitochondria were prepared from livers of two to three animals as previously described (7). The initial homogenization mixture contained 0.2 mM EDTA added to the standard homogenization medium [MSH’ = 210 mM mannitol/70 mM sucrose/2 mM Hepes (N’ Abbreviations used: MSH, 210 mM mannitol/70 mM sucrose/2 mM Hepes buffer; KHS, the standard reaction mixture (see Methods); Ca/O, nanomoles of calcium sequestered per nanogram. atom oxygen consumed; ADP/O, nanomoles of ADP phosphorylated per nanogram-atom oxygen consumed; K1/2, calcium concentration at half-maximal velocity; V, maximum rate of calcium uptake; T3, L-triiodothyronine. 220

0003-9861/78/1881-0220$02.00/O Copyright Q 1978by Academic Press, Inc. All rights of reproduction in any form reserved.

THYROID

HORMONE

AND

MITOCHONDRIAL

2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), pH 7.2, buffer] and was deleted from the last two rewashing steps. Mitochondrial protein was determined by the biuret method (ll), with bovine serum albumin as a standard. Oxygen consumption studies were carried out at 37°C with a Clark-type oxygen electrode. The standard reaction mixture (designated KHS) contained 105 mM KCl, 10 mM Hepes (pH 7.2), 5.7 mM Tris-succinate, 0.1 pM rotenone, 30 mM MSH, and 1 mg/ml of mitochondrial protein. Other additions are noted in the text. The kinetics of calcium uptake and efflux were measured according to the protocol described by Reed and Bygrave (12) except that the standard reaction mixture contained KHS and 1 mg/ml of mitochondrial protein. Other additions are as noted in the text. Values of K, and V for calcium ion influx were calculated by linear regression analysis of the plots of l/V vs l/(Ca)’ (13). In all cases, the zero-order correlation coefficient was 0.98-1.00 and the standard error of each estimate was t2.570. An efflux rate constant (K,) was calculated assuming that: (i) all sequestered calcium is mobile, after nonspecific binding is taken into account (12); and (ii) the rate of calcium efflux is directly proportional to the intramitochondrial mobile pool (12). The possibility that short-term (15-s) or long-term (20.min) incubation of these mitochondria with thyroid hormone and/or calcium caused mitochondrial swelling (8) was ruled out in preliminary experiments (N = 4) in which r,-triiodothyronine (0.1, 1.0, and 10 pM) and/or calcium (0.1 mM) was incubated with mitochondria in KHS for 20 min at O’C. Mitochondrial volumes were then determined (14). In no case was swelling observed; the total mitochondrial volume was 1.20 f 0.12 &mg of mitochondrial protein, the sucrose-permeant space was 0.70 + 0.09 pl/mg, and the sucrose-impermeant space was 0.50 f 0.11 pl/mg. These values are identical to those previously obtained with this method (13) for untreated mitochondria. Adenine nucleotide translocation was measured by the “back-exchange method” of Pfaff and Klingenberg (15). Mitochondria (30 mg) were preincubated for 90 min at 0°C in KHS medium containing 0.2 gCi of [‘?]ATP (0.26 mM). The mitochondria were centrifuged at 14,500g for 10 min and washed twice, all at 0°C. The pellet was resuspended in KHS at 10 mg/ml. Aliquots (0.1 ml) were preincubated in KHS (final volume, 1.0 ml) for 1 min at 0°C during which time additional reactants were added. Nucleotide exchange was initiated by the addition of 266 nmol of nonradioactive ATP. The reaction was stopped at +20 s by the addition of 0.5 pM Atractyloside (final concentration) and the mitochondria were pelleted through silicone (General Electric Co. F-50) at 12,000g for 5 min in an Eppendorf microcentrifuge. Aliquots of the supernatant were

CALCIUM

METABOLISM

221

counted for radioactivity and data were calculated as percentage efflux per 20 s (14) using appropriate controls. Radioactivity measurements of ‘“Caz+ and [‘“ClATP were carried out in Aquasol (New England Nuclear Corp., Boston) to >99.9% reliability in a Packard liquid scintillation counter. The data represent the results of at least two experiments, each from at least three separate preparations of mitochondria. Where applicable, Student’s t test is used to compare experimental and control groups. RESULTS

Oxygen Consumption Associated with Calcium Uptake The respiratory cycle associated with low-level calcium uptake (10) is altered by exogenously added L-triiodothyronine (Fig. 1). There is a dose-dependent increase in the State 3 (Ca) respiration rate, and no changes occur in the steady-state or State 4 (Ca) rate or in the Ca/O ratio. Concomitant measurements of ADP-induced respiratory cycles requiring the addition of phosphate (0.5 mu) showed no effect of the added hormone on the State 3 (ADP) rate, the State 4 (ADP) rate, or the ADP/O ratio (Data not shown). These results are interpreted to suggest that the kinetics of calcium uptake as measured by State 3 (Ca) may be altered by added thyroid hormone. Kinetics The rates of calcium uptake vs exogenous calcium concentration in the presence and L-triioabsence of 1 PM (0.5 nmol/mg/ml) dothyronine are plotted in Fig. 2a. The sigmoidicities of the two curves, derived from Hill plots (not shown), are identical, 1.8, in agreement with those obtained from mitochondria isolated from euthyroid animals (13, 16). A reciprocal plot analysis of these data (Fig. 2b) shows that added hormone enhances V; the Kl,z value is unchanged. In Table I, additional uptake data are presented. The stimulatory effect of added L-triiodothyronine on V is significantly elevated at 0.1 PM L-triiodothyronine and maximal at 1.0 PM L-triiodothyronine. In the presence of added acetate, similar results are obtained: V is enhanced and the Kl,z value is unaffected by added hormone. The rate constant derived from measure-

P. A. HERD

222

RCR or Co++/0

oo.bo, log

T3 (pm)

1. Effect of L-triiodothyronine (Tz) on oxygen consumption rates, respiratory control ratios (RCR), and Ca*+/O ratios in mitochondria obtained from thyroidectomized rats. (See Methods for experimental protocol.) L-Triiodothyronine was incubated with the mitochondria for 1 min before the addition of calcium (80 nmol/mg). Data are derived from duplicate experiments obtained from eight separate preparations of mitochondria. The range of values in each case was +5% of the mean value plotted. P < 0.05 for State 3 (Ca’+ at 0.01 pM Ta) vs control. FIG.

i,co nmoles mg .15sec at 0°C

I (Ca+2 1g FIG. 2. Effect of L-triiodothyronine (T3) on the kinetics of calcium uptake. (a) Calcium uptake ( Vc.) is plotted as a function of exogenous calcium concentration obtained with the calcium buffer NTA (see Methods) in the presence and absence of 1.0 pM Ts. (b) Reciprocal plots of these data. V and Kllz values were computed from a linear regression analysis of these data, as described under Methods and listed in Table I. Co*

(@M)

ments of the passive efflux of calcium (Table I) is increased upon the addition of thyroid hormone. The maximal effect is obtained at 1.0 PM. In contrast, in the presence of acetate, this stimulatory effect is not obtained.

Calcium location

and Adenine

Nucleotide

Trans-

Babior and co-workers (17) were unable to demonstrate a direct effect of thryoid hormone on the uptake of ADP by liver

THYROID

HORMONE

AND

MITOCHONDRIAL TABLE

CALCIUM

223

METABOLISM

I

EFFECT OF L-TRIIODOTHYRONINF, ON THE KINETIC PARAMETERS ASSOCIATED WITH CALCIUM UPTAKE AND EFFILJX FROM MITOCHONDRIA OBTAINED FHOM THYROIDECTOMIZED RATS~ N

Control +T;j +T:j +Ta Control +Ta +T, +T,

14 4 10 4 6 2 6 2

(0.1 PM) (1.0 PM) (10.0 PM) + 10 mM acetate (0.1 PM) (1.0 PM) (10.0 /tM)

” See Methods for experimental protocol. Ca’+ sequestered per milligram minute. * P < 0.05. **P
Uptake

P, (0.5 mM) Ta (1.0 PM) Ca2+ (0.1 mM) I’, + Ta Ca2+ + T:, Ca + P, +T:, (0.01 PM) +T:, (0.10 PM) +Ta (1.0 PM) +Ta (10.0 PM)

II

6 6 6 6 6 6 6 2 4 6 2

V

K1

5.16 f 0.2 5.84 + 0.2* 6.05 + 0.2* * 6.00+0.1** 14.1 f 0.55

8.07 8.07 8.07 8.07 4.66

15.8 + 0.61: -

4.66

N = number of duplicates.

EFFECT OF L-TRIIODOTHYHONINE AND CALCIUM ON ADENINE NLJCLEOTIDF: TRANSLOCATION RATES IN MITOCHONDHIA OBTAINED FROM THYROIDECTOMIZED RATS” N Percentage exAddition change in 20 s6 1.20 1.30 1.27 1.50 1.40 2.57 1.40 19.60 22.80 20.70 12.50

” See Methods for experimental protocol. h Data were obtained from three separate preparations of mitochondria. Basal adenine nucleotide translocation was 0.032 + 0.003 nmol of labeled nucleotide exchanged/mg 20 s at 0°C. In each case, the range of data was less than +lO% of the recorded value.

mitochondria, but noted a significant increase in nucleotide translocation within 24 h after in vivo administration of the hormone. In studies on ATP translocation, added triiodothyronine had no effect on the magnitude of ATP efflux and neither calcium nor phosphate had an observable effect (Table II). The combination of calcium plus L-triiodothryonine, however, produced a moderate, twofold increase in exchange activity (vs control, in the absence of T3, P

Efflux

K, (min” ) 0.34 + 0.018 1.75 * 0.04** 4.07 f 0.086** 4.00 * 0.110” 0.522 + 0.022 0.535 0.500 -c 0.05 0.521

Uptake data presented as nanomoles of

c 0.10). Most strikingly, the further addition of phosphate (0.5 mM) produced an lofold increase in the rate of nucleotide efflux, and this response was maximal at the lowest concentration of L-triiodothyronine tested (0.01 PM). DISCUSSION

Calcium Transport These results demonstrate that L-triiodothyronine directly modulates calcium ion metabolism in mitochondria isolated from thyroidectomized rats. Recently, Moyle and Mitchell (18) reported that calcium uptake may be mediated by a phosphatedependent transport mechanism. In rat liver mitochondria, low levels of added phosphate (co.5 mM) have no effect on the kinetics of calcium uptake (10, 19), hence endogenous phosphate levels are not limiting under these conditions. In contrast, the addition of L-triiodothyronine (Table I) increased V without altering Kllz, suggesting that the affinity of calcium for the carrier mechanism was not altered; rather, the kinetics of transfer across the membrane appear to be affected. Potential sites for hormone action include alteration of the numbers of ions transported per transfer cycle, altered mobility of the calcium in the mitochondrial membrane, and/or altered rate of release of the transport calcium. Since Hill plots of the uptake data, in the presence or absence of L-triiodothyronine, are identical (sig-

224

P. A. HERD

moidicity, n = 1.8), the number of binding sites per transfer step is unaltered by added hormone. Acetate decreases the matrix pH and thereby enhances calcium transport by increasing the rate of release of the sequestered calcium from the carrier mechanism on the inner surface of the inner mitochondrial membrane (20). Acetate did not alter the hormone-induced increase in V (Table I), hence the effect of added hormone would appear to influence events preceding this site. In contrast, acetate inhibited the hormone-dependent stimulation of calcium efflux (Table I), suggesting that the increased intramitochondrial hydrogen ion concentration was the limiting factor (20, 21) decreasing calcium binding to the carrier mechanism. Although the mechanism of calcium efflux remains unclear (20, 23), Azzone et al. (21) recently emphasized the role of hydrogen ion permeability as well as kinetic factors as determinants of calcium flux. In the present experiments, hydrogen ion leak would not appear to play a significant role in the observed changes in K, (Table I) since State 4 (Ca) respiration is unaltered (Fig. 1); hence added hormone did not alter the steady-state energy requirements to maintain the sequestered calcium, and these mitochondria did not undergo volume changes. Calcium mobility within the membrane cannot be readily evaluated directly. However, since calcium uptake and efflux are affected by added hormone (Table I), a common locus may be altered. In support of this view, we have shown (9) that thyroid hormone forms a stable complex with calcium only in nonpolar environments. An intramembranous cation-hormone interaction is consistent with these observations, and attempts to detect this complex in biological membranes are currently in progress (P. Herd, studies in progress). Adenine Nucleotide Translocation The effect of thyroid hormone on mitochondrial adenine nucleotide translocation has been studied previously (17, 24, 25). These investigations observed decreased activity with respect to ADP uptake and

efflux in mitochondria obtained from hypothyroid rata (17, 24). In addition, Hoch (25) reported increased levels of ATP and ADP in hypothyroid mitochondria and suggested that this increase nucleotide level could, in part, maintain an adequate extramitochondrial nucleotide supply even at lowered transport rates. Further, these transfer rates and intramitrochondrial nucleotide levels were reversed toward normal at 1 day (17) and 3 days (24) (the earliest time points tested) following in uiuo administration of the hormone. Spencer and Bygrave (26) noted that potassium and calcium independently enhance the translocation of ATP and, to a smaller extent, ADP out of the mitochondrion. This effect of added cation is independent of energy conservation by these mitochondria and is not mimicked by manganese, magnesium, or sodium. The loci of these cationic effects are not known. Our data (Table II) confirm the observation that nucleotide efflux is low in mitochondria from hypothyroid rats (17). The effects of exogenous calcium becomes evident only upon the addition of L-triiodothyronine (Table II), suggesting a requirement for the presence of thyroid hormone in manifesting calcium’s effect. The nature of the stimulatory effect of added phosphate in the presence of calcium plus triiodothyronine (Table II) is unclear (10, 15, 19). The phosphate level used in our experiments does not uncouple these mitochondria (7, 10,19). Since endogenous phosphate is also required for calcium movement in the membrane (18), the possibility that the endogenous phosphate pool was depleted by the incubation procedure cannot be ruled out (28,29). Importantly, the stimulation of nucleotide translocation required the addition of thyroid hormone (Ca + Pi vs Ca + Pi + T3). Since the stimulation of nucleotide translocation occurs at very low levels of added hormone (<0.0005 nmol/mg of mitochondria/ml), it is conceivable that nucleotide translocation may be a primary locus for thyroid hormone action on cellular metabolism. In the steady state, phosphate-coupled respiration is limited by the activity of the adenine nucleotide translocase (27).

THYROID

HORMONE

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MITOCHONDRIAL

Hence we may hypothesize that this stimulation of Ca2’-dependent nucleotide translocation by thyroid hormone may modulate the altered cytoplasmic energy (ATP) requirements [e.g., for nucleoprotein phosphorylation (30) and/or cytoribosomal protein synthesis] observed following hormone administration (4). The mechanism of this action is unclear, but these data are consistent with the hypothesis that a thyroid hormone-calcium ion complex (9) may mediate membranous, calcium-dependent metabolic processes. ACKNOWLEDGMENTS This project was supported by The Miriam Hospital and the Rhode Island Heart Association. We wish to thank Dr. R. P. Davis for his advice and comments. REFERENCES 1. PITT-RIVERS, R., AND TATA, J. R. (1959) The Thyroid Hormones, Pergamon Press, Oxford. 2. FHEF,DBEHG, A. S., AND HAMOLSKY, M. (1974) in Handbook of Physiology, Section 7: Endocrinology, (Greep, R. O., and Astwood, E. R., eds.), Vol. 3, pp. 435-468, Williams and Wilkins, Baltimore. 3. SPINDI.ER, B., MACLEOD, K., RING, J., AND BAXTER, J. (1975) J. BioZ. Chem. 250, 4113-4119. 4. TATA, R., JH., ERNSTER, L., LINDBERG, O., ARRHENIUS, E., PETERSON, S., AND HEDMAN, R. (1963) Biochem. J. 86.408-428. 5. Guz, A., KLJHI,AND, G. S., AND FEEEDBF,RG, A. S. (1961) Amer. J. Physiol. 200,58-60. 6. HOCII, F. L. (1967) Proc. Nut. Acad. Sci. USA 58, 506-511.

7. HERD, P. A., KAPLAY, S. S., AND SANADI, D. R. (1974) Endocrinology 94,464-474. 8. CASH, W., GRADY, M., CARLSON, H., AND KONG, E. E. (1969) J. Biol. Chem. 241, 1945-1950. 9. HERD, P., AND DAVIS, R. P. (1978) Submitted for publication. 10. ROSSI, C. S., AND LEHNINGEH, A. L. (1964) J. Biol.

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Chem. 239,3971-3980. 11. GORNALL, A. G., BORDAWIIJ,, C. S., AND DAVID, M. (1949) J. Biol. Chem. 177,751-766. 12. REED, K., AND BYGRAVE, F. (1975) Anal. Biothem. 67,44-54. 13. VINOGRADOV, A., AND SCARPA, A. (1973) J. Biol. Chem. 248,5527-5531. 14. HERD, P. A., AND MARTIN, H. (1975) Biochem. Pharmacol. 24,1179-1186. 15. PFAFF, E., AND KLINGENRERG, M. (1968) Eur. J. Biochem. 6,68-79. 16. BYGHAVE, F. L., REED, K. C., ANI) SPENCF,II, T. (1971) Nature New Biol. 230,89-91. 17. BARIOR, B. M., CREAGAN, S., INGRAR, S. H., ANI) KIPNES, R. (1973) Proc. Nut. Acad. Sci. USA 70,98-102.

18. MOYI,E, J., AND MITCHEI,L, P. (1977) FEBS Lett. 77,136-140.

19. DRAHOTA, Z., CAHNAFOLI, E., ROSSI, C. S., GAMBLE;, R., AND LFXNINGER, A. L. (1965) J. Biol. Chem.240,2712-2720. 20. RE:EI), K., AND BYGRAVF., F. (1975) Eur. J. Biothem. 55,497-504. 21. AZZONF,, G. F., POZZAN, T., MASSAHI, S., BHLJGADIN, M., AND DELL’ANTONE, P. (1977) FEBS Lett. 78, 21-24. 22. CHOMPTON, M., CAPANO, M., AND CAHAFOI.I, E. (1976) Eur. J. Biochem. 69,453-462. 23. HARRIS, E. J., AND ZAHN, B. (1977) FEBS Lett. 79, 284-291. 24. PORTNAY, G., MCCLENDON, F., B~JSH, J. E., BHAVERMAN, L., AND BABIOR, B. M. (1973) Biochem. Biophys. Res. Commun. 55, 17-21. 25. HOCH, F. L. (1973) Fed. Proc. 34, 541 (abstract). 26. SPENCER, T., AND BYGHAVE, F. (1972) Biochem. J. 129, 355-365. 27. CARAFOLI, E., AND LEHNINOER, A. (1971) Biothem. J. 122.681-690. 28. BRAND, M. D., REYNAFAHJF,, B., AND LEHNINGER, A. L. (1976) J. Biol. Chem. 251.5670-567s. 29. KLINGENBEHG, M. (1970) Essays Biochem. 6, 119-15s. 30. TANINGHER, M., MOLINAHI, M. P. AND CESAHONE, C. F. (1977) Endocrinology 101, 1221-1227.