Effect of senescence on Ca2+—ion transport by heart mitochondria

Mechanisms of Ageing and Development, 19 (1982) 5-13

EFFECT OF SENESCENCE MITOCHONDRIA

O N Ca2+-.-ION T R A N S P O R T

5

BY H E A R T

RICHARD G. HANSFORD and FRANCES CASTRO Laboratory o f Molecular Aging, National Institute on Aging, National Institutes o f Health, Gerontology Research Center, Baltimore Oty Hospitals, Baltimore, MD 21224 (U.S.A.)

(Received December 11, 1981)

SUMMARY Both the rate of uptake of Ca 2÷ into isolated rat heart mitochondria and the rate of release of Ca 2÷ by a separate, Na+-dependent, pathway were shown to be diminished in senescence (24-month relative to 6-month animal). These processes were studied at lower concentrations of Ca 2÷ and loads of Ca 2+ per mg of mitochondrial protein than those generally employed, and it is argued that these are more appropriate physiologically/'In addition, Ca 2÷ release was characterized in terms of the sum of the added Ca 2÷ and the endogenous Ca2÷ of the mitochondrial preparation; the endogenous Ca 2÷ content was found to be unchanged with age. The decrements in rates of transport are not caused by altered rates of substrate oxidation and are inferred to reflect decreased carrier-protein content or activity. The buffering of extramitochondrial free Ca 2÷ concentration by heart mitochondria was studied and found to be less than complete at mitochondrial Ca 2÷ loads inferred to be physiological. No change was shown in senescence, in keeping with the essentially equal age-linked decrements in the activity of mitochondrial Ca 2÷ uptake and release.

INTRODUCTION Previous work from this laboratory [ 1] established that the rate of inactivation of 2-oxoglutarate dehydrogenase on adding the chelating agent EGTA to a mitochondrial incubation was 40% lower when the mitochondria were derived from the hearts of 24month-old rats, as opposed to 6-month-old animals. This dehydrogenase is activated by Ca 2+ [2] and is located within the permeability barrier of the mitochondrial inner membrane [3]; the inactivation by the chelating agent thus presumably involves the withdrawal of Ca 2+ from the inner (matrix) compartment of the mitochondrion, involving the participation of a membrane transport system. The latter conclusion was strengthened by the finding that inactivation requires Na÷ [1], which is known to be transported into heart mitochondria during Ca 2÷ release, in an electrically neutral exchange process [4, 5]. 0047-6374/82/0000-0000/$02.75

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The clear inference was that this transport process was less active in senescence, and this is examined directly in the present paper by using the metallochromic indicator Arsenazo III and a dual-wavelength spectrophotometer to measure extramitochondrial free Ca 2÷ concentration (see ref. 6). In addition, the rate of Ca 2÷ uptake into the mitochondria, which is an electrophoretic process involving an entirely separate carrier from that catalysing the release (see ref. 7), is measured as a function of age. The demonstration that both Ca 2÷ uptake and release activities are altered in senescence (see below) raised the question of whether heart mitochondria from the senescent animal would buffer extramitochondrial free Ca 2÷ concentration to the same value as mitochondria from young adult animals. This arises because the extramitochondrial Ca 2÷ concentration is understood to be a steady-state value reflecting the opposition of influx and efflux pathways [8]. Any change in this balance may be physiologically important, as the role of mitochondria in the homeostasis of cytosolic free Ca 2÷ concentrations has been emphasized [9], and this is discussed in more detail below (see Discussion section). For these reasons, the extramitochondrial free Ca 2÷ concentration was measured directly with a Ca2÷-selective electrode, and its response to changes in mitochondrial Ca 2÷ load and to the presence of Mg2÷ investigated in mitochondria from young adult and senescent animals.

MATERIALS AND METHODS

Preparation of mitochondria Heart mitoch0ndria were prepared from male, Wistar-derived rats, maintained at the Gerontology Research Center, by a procedure described previously [10]. Care was taken to subject hearts from animals of each age group to the same length of time of proteolytic digestion. Mitochondria were given a final wash in EGTA-free preparation medium. Uptake and release of Ca e÷by mitochondria Mitochondrial Ca 2+ uptake was studied at 25 °C using an Aminco-Chance DW2A dual-wavelength spectrophotometer, at the wavelengths 650 and 575 nm, and the metallochromic indicator Arsenazo III [6]. Mitochondria (2 mg of protein) were added to a cuvette containing 2 ml of 0.13 M KC1, 20 mM HEPES (potassium salt), 5 mM potassium phosphate, 5 mM L-glutamate, 5 mM L-malate and 30/IM Arsenazo III, pH 7.2. Five minutes later, CaC12 was added to give 10 or 20/aM and the rate of Ca 2+ disappearance from the medium was recorded. When a steady-state had been reached (9 min after the addition ofmitochondria) the release of Ca 2+ was initiated by the addition of 1'.5 nmol of Ruthenium Red (to block re-uptake) and 40/2mol of NaC1. A slower chart-speed was used for recording release than for recording uptake. For the experiments of Table IC, the protocol was similar, except that the Ca 2+uptake phase was not studied, and incubations contained CaCI2 ( 5 - 4 0 nmol) or EGTA ( 4 - 1 0 nmol) initially. The release of mitochondrial Ca 2÷ was elicited by the addition of Ruthenium Red and NaC1 (to 20 mM) 5 rain after the mitochondria, and this rate was

recorded. The release process was allowed to go to completion, at 11 min, and the total absorbance change subsequent to the addition of Ruthenium Red and NaC1 was taken to be a measure of the mitochondrial Ca 2+ content at the moment of initiating release. This quantity was compared to the concentration of the substrate of an enzyme reaction, designated "S", and used in plots of 1Iv against I/S, where v is the velocity of Ca 2+ release. The Vmax and Km values derived from such plots are presented in Table I; it is to be noted that the use of the term "Km" is not rigorous, as the parameter is actually the Ca2+ content at which a half-maximal velocity is achieved, rather than a concentration. For these studies, the wavelength pair 575 and 585 nm was used. Within each mitochondrial preparation, each experiment was duplicated.

The buffering o f extramitochondrial free Ca2÷ concentration by heart mitochondria Mitochondria (2.5 mg of protein) were added to 5 ml of medium comprising 0.13 M KC1, 20 mM HEPES (potassium salt), 5 mM KPi, 10 mM NaC1, 5 mM L-malate and 5 mM L-glutamate, at pH 7.2 and 25 °C. In addition, either CaC12 (0-7.5/aM) or EGTA (0-2/IM) was present in each incubation; MgC12 was present at 1 mM in half of the experiments, and this is indicated where applicable (Fig. 2). The mitochondria either accumulated Ca 2÷ from the medium or partially released endogenous Ca 2÷, depending on the starting pCa (-log Ca 2÷ activity) of the surrounding fluid. These movements were recorded using a Ca2+-sensitive electrode (Radiometer F2002). Ten minutes after the addition of the mitochondria, the pCa of the incubation medium had attained a steady-state, and this was determined by adding Ca2÷-EGTA buffers of known pCa at pH 7.2 [11], on the basis of trial and error, until the voltage given by the electrode at the end of the mitochondrial uptake (or release) phase was exactly regained. An association constant of 1.204 × 10 7 w a s used in calculating the pCa values of the buffers, at pH 7.2. R eagen ts Arsenazo III was type 1, from Sigma Chemical Co. Ruthenium Red was a gift from Dr. J. Wehrle, Department of Physiological Chemistry, Johns Hopkins University, and had been recrystallized. All other reagents were of the highest grade available commercially.

RESULTS Figure 1 illustrates the design of the experiments used to collect data on the activity of both mitochondrial Ca r" uptake and release. The same amount of mitochoridrial protein was used on each occasion, so that the mitochondrial Ca 2÷ load was always the same (i.e. per mg of protein), at the same extramitochondrial Ca2÷ concentration. As seen in Fig. 1, essentially complete uptake of the added Ca 2+ occurs, energized by the transmembrane electrical potential generated by the oxidation of glutamate plus malate (see refs. 12 and 13). Release is then initiated by the addition of NaC1, to potentiate efflux by Ca2+/2 Na+ exchange, and of Ruthenium Red, to block any re-uptake [4].

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Fig. 1. The uptake and release of Ca 2÷ by rat heart mitochondria, as measured with the indicator Arsenzo III. CaCI2 was added at the point indicated to give 10/aM and 11 nmol/mg of mitochondrial protein. At the completion of the uptake phase, the release of Ca 2+ was initiated by the addition of 1.5 nmol of Ruthenium Red plus 40 #mol of NaCI (RR + NaC1). Details are given in the Materials and Methods section. The mitochondria used in this particular experiment were derived from a 6-monthold rat.

Rates of uptake are significantly decreased in senescence, at both 10/aM and 30/aM Ca 2+, as given in Table IA. It should be noted that Vmax and Km cannot be accurately determined for Ca 2÷ uptake, as the v versus S relationship is sigmoidal for heart mitochondria [14] and Vmax may be limited by the proton-pumping capacity of the respiratory chain rather than by the turnover of the transport protein [8]. However, a similar percentage decremer/t with old age at 10 and 30/aM Ca 2÷ would make a change in Vmax more likely than a change in Km. Since Ca 2+ uptake is secondary to the generation of a membrane potential by respiration [12, 13], it was important to establish whether the ability to oxidize glutamate plus malate was impaired in old age. Measurements of ADPstimulated 02 uptake with 5 mM L-glutamate and 5 mM L-malate as substrate gave values of 278 + 8 (n = 5) and 262 -+ 9 (n = 5) ng • atoms of 02 per min per mg of protein, at 25 °C, for mitochondria from 6-month-old and 24-month-old rats, respectively. The comparable figures for the oxidation of 2.5 mM pyruvate and 1 mM L-malate were 360 + 30 (n = 5) and 326 +- 10 (n = 5) ng • atoms of O2 per min per mg o f protein. Thus, there was no evidence for the impaired oxidation with old age of the substrate combination used in the transport studies, nor of pyruvate. In addition, respiratory control ratios (see ref. 15) were unchanged, so that it is unlikely that the membrane potential available for driving Ca 2* transport was less in the mitochondria from the senescent animals. Table IB presents results for the rate of Ca 2÷ release from the mitochondria, obtained in experiments o f the type shown in Fig. 1. It is seen that there is a highly significant decrease in activity with senescence, this being most apparent at the smallest load of Ca 2÷ (11 nmol/mg of mitochondrial protein). In these experiments, no measurement was made of the endogenous content of mitochondrial Ca 2÷, which could begin to be significant in comparison with the amount added when the latter is as low as 11 nmol/mg protein. For this reason, a separate set o f experiments was carried out, using different preparations of

TABLE I EFFECT OF SENESCENCE ON Ca 2÷ UPTAKE AND RELEASE BY RAT HEART MITOCHONDRIA (IA)

OB)

Ca2+ concentration (gM)

Rate of Ca2+ uptake (nmol per rain per mg protein) 6 months

24 months

Difference (%) p

10 30

24.0 -+0.8(9) 94.5 -+4.9(5)

16.4 -+ 1.5(9) 68.6 -+5.1(9)

-32 -27

Ca2+ load (nmol/mg protein)

Rate o f Ca2+ release (nmol per min per mg protein) 6 months

11 33 42

6.95 ± 0.02(9) 10.5 ~ 0.7(5) 11.0 -+0.6(5) 6 months

Oc)

24 months 4.59 -+0.38(8) 7.46 -+0.47(8) 8.25 + 0.56(6) 24 months

<0.001 <0.01

Difference (%) p -34 -29 -25

<0.001 <0.005 <0.01

Difference (%) p

Km for Cae+ release (nmol Ca2+permgprotein}

6.05 -+0.46(5)

6.64 -+ 1.33(5)

+10

N.S.a

Vmax for Ca2+ release (nmol per min per mg protein)

9.95 ± 0.69(5)

7.28 -+0.42(5)

-27

<0.01

Uptake and release of Ca2÷ were measured using the metallochromic indicator Arsenazo III and a dualwavelength spectrophotometer, as described in the Materials and Methods section. Results are presented as the mean ± S.E.M., with the number of mitochondrial preparations in parentheses. The preparations used in (C) were different from those used in (A) and (B). aN.S. = not significant (p > 0.05).

mitochondria, and allowing Ca 2* release to go to completion in every case (unlike Fig. 1). Thus, the total amount o f Ca 2* released from the mitochondria on the addition o f NaC1 and Ruthenium Red could be taken as the amount within the mitochondrion and available for release at that point. The validity of this assumption was shown by the finding that the ionophore A23187 gave rise to an identical amount o f Ca 2÷ release, when added in place o f NaCI and Ruthenium Red (results now shown). Release of mitochondrial Mg ~÷ by the ionophore was no problem, provided that the Ca2+-specific wavelength pair for Arsenazo III o f 675 and 685 nm [6] was used. This intramitochondrial Ca 2÷ content was manipulated in these experiments b y the initial addition o f small amounts o f CaC12 or EGTA, resulting in a range o f 1 . 0 - 2 0 nmol/mg o f protein. When velocity o f release is expressed as a function o f mitochondrial Ca 2÷ content, a rectangular hyperbola is obtained, yielding linear plots o f 1Iv versus I[S, where S is mitochondrial Ca 2÷ content, at the moment of addition o f NaC1 and Ruthenium Red. One such plot is shown in Fig. 2. Analysis o f the combined results o f these experiments (Table IC) gives a highly significant decrease with senescence in the Vmax o f Ca 2÷ release, but no change in the " K m " - - t h i s term, as mentioned above, being used loosely and applied to content, rather than to concentration. Thus, studies using in all 14 preparations from 6-month-old animals and 13

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Fig. 2. Double reciprocal plot of rate of Ca 2+egress v e r s u s the mitochondrial content of Ca~+. The data were derived from a series of experiments of the type shown in the second portion of Fig. 1. Release was allowed to proceed to completion and the Ca 2÷ content which is referred to is the sum of the endogenous Ca 2. and that added, as described in the text.

preparations from 24-month-old animals (Table I A - C ) corroborate an age-linked decrease in Ca 2÷ release, regardless of whether endogenous Ca 2÷ content is considered. Mitochondria have been shown to act as effective buffers of extramitochondrial free Ca 2÷ concentration, when the mitochondrial load of Ca 2÷ is in the range 1 5 - 1 0 0 nmol/mg of protein [8, 16, 17]. The experiments of Fig. 3 were designed to see whether that is true at lower Ca 2÷ loads, which we believe to be more physiological (see Discussion) and whether the buffering function is impaired in old age. It was found that mitochondria functioned as excellent buffers of additions of CaC12 of 15 nmol/mg protein (Fig. 3). The pCa value achieved in the steady-state was lower in the presence of Mg2÷, which is consistent with the reported inhibition of the electrogenic uptake pathway by this cation in heart [18]. When endogenous Ca 2÷ was withdrawn by the addition of small amounts of EGTA, the pCa began to rise above the buffered value (Fig. 3). No significant difference could be established between mitochondria from 6-month-old and 24-month-old animals at any value of added CaC12 or EGTA, in studies using 5 preparations and 4 preparations, respectively. As the measured pCa values are expressed as a function of CaC12 (or EGTA) added to the mitochondrial incubation in Fig. 3, knowledge of the endogenous Ca 2÷ content of these mitochondria is crucial. This was measured using Arsenazo III as described above, and was 2.84 + 0.23 (n = 8) nmol/mg for the 6-month-old animals and 2.82 + 0.21 (n = 6) nmol/mg for the 24-month-old animals. Thus, at any given addition, the mitochondrial content of Ca 2÷ may be taken as being the same for both age groups in Fig. 3, and they can be fairly compared.

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nm01EGTA/mgprotan nm01CaCIdmgprotein Fig. 3. The buffering of extzamitochondr~l free Ca 2. concentration as a function of mitochondrial

Ca 2. load, in heart mitochondria from young adult and senescent rats. In separate experiments, respiring mitochondria were either loaded with small amounts of Ca 2. (up to 15 nmol/mg protein) or were partially depleted of their endogenous Ca =* content by the addition of EGTA (4 nmol/mg protein). When a steady-state had been achieved, the free Ca 2. concentration of the mitochondrial incubation (expressed here as pCa, or -log Ca 2. activity) was measured using an ion-selective electrode and by reference to Ca2+-EGTA buffers. Full details are given in the Materials and Methods section. Each point represents the mean (-+ S.E.M.) of experiments performed with three mitochondrial preparations. Solid symbols indicate results from 6-month-old animals; open symbols are results from 24month-old animals. Circles indicate the incubation medium given in Materials and Methods; squares indicate the additional presence of 1 mM MgC12. DISCUSSION This paper presents decrements with senescence in the rates o f both uptake and release o f mitochondrial Ca 2. (Table I). These are not part o f a generalized deterioration in structure o f the mitochondria, nor the reflection of an increased contamination o f the preparations from the older animals with non-mitochondrial protein. This can be asserted because the specific activities o f other oxidative processes are undiminished in these preparations, as shown for the oxidations o f glutamate plus malate and pyruvate plus malate in the Results section and in other work from this laboratory [10]. Equally, the finding that sparked these investigations, that the rate o f EGTA-induced inactivation o f 2oxoglutarate dehydrogenase was lower in preparations from senescent animals [1], cannot be explained on the grounds o f increased contamination o f the preparations, in that the t i n for enzyme inactivation was found to be greater in old age. Combined with the finding reported here o f an unchanged endogenous Ca 2. content, this defines a decreased activity o f Ca 2÷ release in the functional mitochondria o f the preparation. Great emphasis was placed in the uptake and release experiments (Fig. 1, Table I) on the use o f smaller loads o f Ca 2. per mg of mitochondrial protein than those generally used in in vitro studies o f mitochondria (for reviews, see refs. 9 and 19). The reason for this is that we have identified the range 0 . 5 - 3 nmol o f Ca 2+ per mg o f mitochondrial protein as allowing the modulation o f 2-oxoglutarate dehydrogenase activity in the mitochondrion [1], and presume that this is the range which obtains in vivo. Indeed, one direct study o f mitochondria in situ in arterial smooth muscle [20] using electron-probe

12 analysis does pinpoint such a low mitochondrial Ca 2÷ content. At such levels, the egress pathway for Ca 2÷ is not saturated by available Ca 2÷ within the mitochondrion (Fig. 2), and any age-linked change in affinity, or Vr,~a~,would affect the activity of transport. In fact, we found that affinity (Ca 2÷ c o n t e n t for half-maximal velocity) is not changed in senescence, but that the Vmax of the process is diminished (Table IC). A consequence of the lack of saturation of the Ca 2+ release pathway by Ca 2+ contents which we presume to be physiological is that heart mitochondria do not act as perfect buffers of extramitochondrial pCa (Fig. 3). At higher loads ( 1 5 - 6 0 nmol/mg) we have confirmed (results not shown) the near-perfect buffering emphasized by other investigators [8, 17]. We would like to suggest, in keeping with the proposal by Denton and McCormack [21] that the mitochondrial Ca 2÷ transport processes exist to regulate the intramitochondrial Ca 2÷ activity, rather than that of the cytosol, which is under the primary control of plasma membrane and reticulum transport activities. The need to regulate the intramitochondrial Ca 2÷ activity arises because of the Ca 2÷ sensitivity of three flux-determining dehydrogenases involved in pyruvate oxidation and the operation of the tricarboxylate cycle [2, 2 2 - 2 4 ] . The similar degree of loss of activity of both Ca 2+ uptake and release which is seen in senescence (Table I) may be the reason why no age-linked change in extramitochondrial pCa could be established (Fig. 3). The rationale is that the pCa value reflects the resultant of the opposed activities of uptake and release. For the same reason, the intramitochondrial pCa may not be changed in senescence. Unfortunately, the latter parameter cannot yet be measured directly at the low Ca 2÷ loads employed in this study, though some progress has been made recently at higher loads [25]. If a beat-to-beat oscillation of matrix pCa occurs in response to variations in cytosolic pCa, then this may be damped in the aging heart. It should be noted that an altered contractile response to catecholamines has already been described for the aging heart, and this is thought to involve changes in Ca 2÷ sequestration and release by the sarcoplasmic reticulum [26, 27]. It is striking that significant and extensive [30-40%] decreases in the specific activity of three transport systems have now been demonstrated in heart mitochondria in old age [10, 28] (see also this paper). However, it may be premature to infer a general mechanism for these changes, in that acylcarnitine transport is less active as a consequence of a decreased intramitochondrial substrate pool [10], whereas adenine nucleotide transport is apparently decreased in response to changes in membrane composition [28] and, presumably, fluidity [29]. The changes in membrane composition could well underly the decreased Vmax for Ca 2+ transport identified in the present paper, and the impact of these changes on other permeability properties, notably the transport of respiratory substrates, in heart mitochondria would seem a fruitful field for further study. REFERENCES 1 R. G. Hansford and F. Castro, Effect of micromolar concentrations of free calcium ions on the reduction of heart mitochondrial NAD(P) by 2-oxoglutarate. Biochem. J., 198 (1981) 525. 2 J. G. McCormack and R. M. Denton, The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex. Biochem. J., 180 (1979) 533. 3 J. B. Chappell, Mitochondrial transporting systems. Br. Med. Bull., 24 (1968) 150.

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