Involvement of mitochondria in the age-dependent decrease in calcium uptake of rat brain synaptosomes

36

Brain Research. 37811986) 36-48

Elsevier BRE 11841

Involvement of Mitochondria in the Age-Dependent Decrease in Calcium Uptake of Rat Brain Synaptosomes JAVIER VITORICA and JORGINA SATRUSTEGUI Departamento de Bioquimica y Biologia Molecular, Centro de Biologia Molecular, C. S. 1. C. Universidad A ut6noma de Madrid, Cantoblanco, Madrid 28049 (Spain)

(Accepted November 26th, 1985) Key words: calcium uptake - - calcium distribution - - brain mitochondrion - - synaptosome - - ageing - - membrane lipid fluidity

Calcium uptake in rat brain synaptosomes decreases during ageing 4°. The possible involvement of mitochondria in altered calcium homeostasis has been investigated. Mitochondria isolated from old rat brain showed decreased calcium uptake rates. Since neither the mitochondrial membrane potential nor the ApCa decreases with age, it was concluded that variations in the driving force for calcium uptake were not the cause for impaired calcium transport in mitochondria from aged rat brain. The steady state calcium distribution in isolated aged rat brain mitochondria was achieved at higher extramitochondrial calcium concentrations than that of adults. Studying the effects of the selective release of calcium from the mitochondrial pool by the addition of an uncoupler to 45Ca loaded synaptosomes incubated in high-potassium media, it was found that the intrasynaptic mitochondrial pool and the intra/extramitochondria145Ca distribution also decreased considerably in 24-month-old rats. Steady state fluorescence anisotropy (rs) of diphenylhexatriene-labelledmitoplasts from 'free' brain mitochondria increased with ageing. However, since no changes in rs from synaptosomal mitochondria were found in 24-month-old rats, it is suggested that alterations in lipid dynamics are not involved in the impaired calcium uptake observed in brain mitochondria from aged rats. The implications of these findings in the calcium homeostasis of brain endings are discussed.

INTRODUCTION

by blocking outward, voltage-activated K+-chan nels 40,59.

The involvement of Ca 2+ m o v e m e n t s in the coupling between stimulus and secretion is firmly established. A n u m b e r of age-related deficiencies in secretion or n e u r o t r a n s m i t t e r release a p p e a r to be due to impaired calcium fluxes or distribution. F o r example, the physiological response to a - a d r e n e r g i c stimulation of parotid gland cells decreases in old rats and this involves changes in phospholipid turnover and calcium fluxes 19. The c a l c i u m - d e p e n d e n t release of acetylcholine is m a r k e d l y depressed in brain slices from aged mice ~5, a p h e n o m e n o n that is accompanied by a decrease in calcium u p t a k e by synaptosomes derived from aged rats 4°. These alterations are abolished by the use of agents that p r o m o t e an increase in cellular calcium content, such as the Ca 2÷ ionophore A-23187 (ref. 19) or 3,4-aminopyridine, a c o m p o u n d that a p p a r e n t l y prolongs depolarization

In addition to the plasma m e m b r a n e systems involved in active Ca2+-extrusion, mitochondria and microsomes are among the intracellular organelles that could be involved in altered calcium homeostasis. Brain m i t o c h o n d r i a possess an electrophoretic uniporter for the uptake of Ca z÷ and a ruthenium red insensitive pathway for electroneutral Ca 2÷ effiux which is activated by Na ÷ (refs. 10, 35). In the presence of A D P and phosphate, brain m i t o c h o n d r i a can take up and retain high calcium loads without a loss in m e m b r a n e potential 38'62. A primary p u r p o s e of this study was to search for variations in the properties of the calcium transport systems of brain m i t o c h o n d r i a as a possible cause of the observed decrease in calcium u p t a k e shown by synaptosomes of aged rats. O u r results show that the activity of the calcium u n i p o r t e r of isolated brain mi-

Correspondence: J. Satrt~stegui, Departamento de Bioquimica y Biologia Molecular, Centro de Biologia Molecular, C.S.I.C. Universidad Aut6noma de Madrid, Cantoblanco, Madrid 28049, Spain. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

37 tochondria decreases with age and that this results in an altered calcium distribution across the mitochondrial membrane. A second aspect of this study deals with the investigation of the subcellular distribution of the transported calcium in synaptosomes of adult and aged rats. With the use of an uncoupler added to calciumloaded synaptosomes 1'2°'49, we have found that the intramitochondrial calcium pool decreases with age resulting in a change in calcium distribution. From these results it may be suggested that the altered calcium homeostasis observed in nerve endings from old rats can be accounted for by the drop in activity in the calcium uniporter of mitochondria. Changes in the fluidity of membrane lipids are known to occur during ageing 64 and during lipid peroxidation 13. There is evidence that the fluidity state of the lipid phase in a membrane is important for the activity of intrinsic membrane proteins 29'39'57. To evaluate the influence of an altered lipid phase in the decreased calcium uptake in old rat brain mitochondria, we have investigated the lipid mobility of mitochondrial membranes from animals of various ages. No correlation was found between changes in lipid fluidity and calcium uniporter activity in rat brain mitochondria of synaptic origin, suggesting that changes in membrane lipid dynamics are not responsible for the reduced calcium uptake in old rat brain mitochondria. A preliminary account of this work has been presented elsewhere 46. MATERIALS AND METHODS Male adult (2-3 months) and 12- or 24-month-old Wistar rats, fed 'ad libitum' on a standard laboratory diet, were used throughout this study.

Isolation of brain mitochondria A fraction containing synaptosomal and non-synaptosomal brain mitochondria was prepared according to the method of Nicholls 35 with the modifications specified earlier 45,62. The separate synaptic and nonsynaptic brain mitochondrial fractions were prepared as described previously 61.

Isolation of synaptosomes Synaptosomes were prepared according to the

method of Booth and Clark 3 using a discontinuous Ficoil gradient. The final pellets were resuspended in 0.32 M sucrose, 10 mM Tris-HCl pH 7.4, and stored at 0 °C for no more than 2 h in 30 mg of protein/ml.

Preparation of rat brain mitoplasts Mitoplasts of both mitochondrial fractions (synaptic and non-synaptic) were obtained according to the method of Schmaitman and Greenawalt 47 by treatment with digitonin, as described earlier 61.

Measurement of the efflux of Ca2+ from brain mitochondria The effiux of Ca 2+ from CaZ+-loaded mitochondria was estimated with the use of the metallochromic indicator Arsenazo III and an Aminco DW2a, a dualwavelength spectrophotometer, at the wavelength pair 675-685 nm as described previously 45,62. Briefly, mitochondria (2 mg of protein) were incubated in 0.32 M mannitol, 10 mM Tris-HC1, 20 mM KCI, 150 oo o

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Fig. 1. Age-dependent variations in the rates of C a 2+ uptake of rat brain mitochondria. 3- (O) and 24- (O) month-old rat brain mitochondria (0.6 mg protein) were incubated in 0.32 M manitol, 10 mM Tris/HCl, 20 mM KCi, 0.1% BSA (fatty acid free) pH 7.4, 2.5 mM succinate (K salt), 2.4/~M rotenone, 0.8 mM Pi, 0.2 mM ADP, 0.4 nmoi oligomycin/mgprotein in a nitriloacetate C a 2+ buffer and different calcium loads. Calcium movements were followed with a C a 2+ sensitive electrode. Calcium uptake was estimated after the addition of a calibrated calcium-dose through the difference in calcium concentration observed following each calcium addition. The inset shows the logarithmic representation of the C a 2+ uptake rate with respect to pCa in old (O) and adult (O) rat brain mitochondria. The slopes and intercepts of those lines were significantly different (V < 0.0001).

38 0.1% BSA (fatty acid flee) pH 7.4, 0.2 mM Arsenazo III, 2.5 mM succinate (K salt), 2.4/aM rotenone, and different CaCI: additions to provide the desired loading. Ca 2+ efflux was estimated after the addition of 1.2/aM ruthenium red in order to stop the active influx. The amount of mitochondrial Ca 2+ taken up was measured by noting the initial Ca ~+ in the medium and the final Ca 2+ in the medium when a new equilibrium was reached after CaCI: addition.

Measurement of Ca2+ uptake and pCa e+ of the extramitochondrial medium The Ca 2+ activity of the incubation medium was measured using a Ca 2+ ion-selective electrode 44 as described before 62 in the presence of 2 mM potassium nitriloacetate in order to buffer extramitochondrial Ca 2+ ' For the Ca 2+ uptake experiments, mitochondria (0.8-1 mg/ml) were incubated as described above in the presence of different Ca 2+ concentrations. When an initial steady-state Ca 2+ distribution was obtained, different amounts of Ca 2+ were added. The rate of Ca 2+ uptake was calculated from the difference between the total initial extramitochondrial Ca 2+ and the total extramitochondrial Ca 2+ after 12 s following Ca 2+ addition. Measurement of 45Ca2+ uptake by synaptosomes Calcium uptake was determined by a filtration technique. Only plastic equipment was used since calcium binds to glass, Synaptosomes (2 mg protein/ml) were preincubated for 5 min at 30 °C in 'low potassium media' (in mM): NaC1 145, KC1 5, MgC12 1, KH204 P 0.4, glucose 10, Tris HCI 10, pH 7.4. After this preincubation, an aliquot was transferred to 'low potassium media' or 'high potassium media' to give a final concentration of 5 or 65 mM potassium, containing 2.3 mM CaC12 (0.75/aCi 45Ca;+/ml). The composition of the high potassium media was the same of that of the low potassium media, except that NaC1 was isosmotically replaced by KC1. 45Ca2+ accumulation was measured at 30 °C and appropriate incubation times as indicated in the figures. Uptake of 45Ca2+ was terminated by the addition of E G T A and ruthenium red to give a final concentration of 3 mM and 7.5/aM respectively. When incubations were carried out during short periods of time, (30 s or less) 10/aM ruthenium red was present

from the start of the incubation. After the uptake reaction was stopped, the synaptosomes were filtered through Whatman glass microfiber filters (GF/B). Each filter was washed with 10 ml of 0.32 M sucrose, 10 mM Tris-HC1, pH 7.4 (ref. 9) and counted for radioactivity.

Membrane potential measurements Membrane potential determinations were based on the equilibrium distribution of [3H]tetraphenyiphosphonium ([3H]TPP+) between the intrasynaptosomal and extrasynaptosomal space. Synaptosomes (2 mg of protein/ml) were preincubated for 5 min at 30 °C as described for calcium uptake experiments. After preincubation, aliquots of the synaptosomal preparation were transferred to a medium containing 1/aM [3H]TPP+, 10/~M FCCP, 10/aM oligomycin, 2, 3 mM CaCI2, 0.4 mM KHzO4P, 1 mM MgCI 2, 10 mM glucose, 10 mM Tris-HC1 pH 7.4, and KC1 to give a final concentration of 65 mM potassium (the osmolarity was maintained constant at 150 mM with NaC1). After 30 s the reaction was stopped by filtration as described above. The contribution of the mitochondriai membrane potential to TPP + accumulation was prevented by the addition of FCCP. Inclusion of oligomycin in the incubation medium prevented ATP hydrolysis by mitochondrial ATPases. To determine the intrasynaptosomal TPP + concentrations, the intrasynaptosomai water space was determined from the distribution of [14C]inulin and 3H20. Membrane potentials (Em) were calculated from measurements of [3H]TPP + accumulation using the Nernst equation assuming that Em in 120 mM K + was 0 mV 5~. Steady-state fluorescence polarization The steady-state fluorescence polarization and fluorescence anisotropy were determined as described by Vorbeck et al. 64 by using all-trans-l,6-diphenyl1,3,5-hexatriene (DPH). Mitoplasts were labelled at a protein concentration of 50-150/ag per ml in a 1000-fold dilution of 2 mM DPH stock solution (tetrahydrofuran as solvent) containing 0.25 mM sucrose, and 10 mM Tris-HCi pH 7.4, and incubated for 20 min or other lengths of time at 25 °C. Steady-state fluorescence polarization was measured with an RRS 1000 Schoeffel spectrofluorimeter

39 connected to a HP 9815 microcomputer and equipped with a temperature-controlled cell block. The excitation and emission wavelengths were 365 and 430 nm respectively. The corrected steady-state fluorescence polarization (P) was calculated as: p = (I w - Ivh/Ivv+ Ivh) where Ivv and Ivh are observed intensities measured with polarizers parallel and perpendicular to the vertically oriental polarizer exciting beam respectively. Light transmission was not affected by differences in polarization. The steady-state fluorescence anisotropy (rs) was calculated from the relation: r~ = 2P/(3 - P) where P is the corrected fluorescence polarization. To evaluate whether ageing affects the incorporation of D P H into mitoplasts, we have tested the effect of different incubation times on fluorescence polarization of mitochondrial membranes from various ages. Since steady-state polarization reached a plateau value after about 6 min incubation with D P H in mitochondria from both 3- and 24-month-old animals, all measurements were carried out after a 20min incubation. Light scattering effects were non-detectable in our experimental conditions since it was observed that dilutions of the samples from adult and aged animals did not affect steady-state fluorescence polarization values.

data and between plateau values of calcium efflux (Fig. 3) were evaluated by Student's t-test. RESULTS

Calcium uptake in aged rat brain mitochondria Mitochondria incubated in the presence of A D P and phosphate can take up and retain high calcium loads without a loss of their membrane potentials 38. Therefore, we have used mitochondria incubated in the presence of 0.2 mM ADP and 0.8 mM Pi to determine calcium uptake rates in mitochondria from adult and old animals. The rate of Ca 2+ uptake was estimated with a Ca 2+ sensitive electrode in mitochondria incubated with different calcium loads and the results were corrected for the initial efflux rates which were determined separately. Fig. 1 shows that the dependency between the rate of calcium uptake and the extramitochondrial free calcium concentration is exponential; the logarithmic representation of the uptake rate with respect to pCa 2+ gives a straight line with a correlation coefficient of 0.958 for adults and 0.960 for older rats. The rates of Ca 2+ uptake in old rat brain mitochondria fall consistently below those of adult animals and this difference is enhanced

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Enzymatic assays Citrate synthase, acetylcholinesterase and N A D H cytochrome C reductase were assayed as described in refs. 14, 23 and 56, respectively. Other methods Mitochondrial membrane potential was measured by monitoring the movements of TPP + with a tetraphenylphosphonium-selective electrode 62. Proteins were determined by the biuret method. The comparison of the slopes of the concentrationdependent phases of calcium efflux and uptake (Figs. 1 and 3) was carried out by the F-test for the differences between two regression coefficients 53. The comparison between the means of different groups of

ADP

150 2 min Fig. 2. Effect of calcium in tetraphenylphosphonium distribution in rat brain mitochondria. Adult ( - - ) and 24-month-old (---) mitochondria (0.8 mg protein) were incubated with succinate (K+ salt) 0.4 nml of oligomycin/mgof protein, 0.8 mM Pi and 2.4 ~M rotenone in the presence of 1.25 #M tetraphenylphosphonium. Subsequent additions were Ca2+ (10 nmol Ca2+/mgprotein -1) and ADP (0.2 mM).

40 at higher Ca 2+ concentrations. A decrease in Ca 2+ uptake has been also observed in heart mitochondria from old rats 17.

Effect of age on the calcium electrochemical gradient In order to define the influence of age on the uniporter reaction, it is necessary to distinguish between effects brought about by changes in the driving force for Ca 2+ uptake (A/~Ca2+) and those on the uniporter activity. Of the components of A/2Ca 2+, A ~p is not affected by age in rat brain mitochondria incubated in the absence of calcium 63. The effect of the different calcium loads on A~p of brain mitochondria from adult and old rats is shown in Fig. 2. Each calcium addition results in a small decrease and subsequent recovery of TPP ÷ accumulation in mitochondria. These transitions in TPP + accumulation are essentially the same irrespective of the age of the animal. To study the variations of,dpCa, the other component of AfiCa, we have analyzed whether old and adult mitochondria incubated in a range of calcium concentrations differed in the concentration of free

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calcium present in the matrix. We incubated mitochondria in a range of calcium concentrations and determined the rate of calcium efflux promoted by the addition of the calcium ionophore A23187. The ionophore suppresses carrier-mediated effects on the rate of calcium effiux, thus allowing the determination of relative free calcium concentrations of different mitochondrial preparations ~6"62. Fig. 3 shows the result of such experiment. The rates of calcium effiux appear to be concentration-dependent below 25-30 nmol mitochondrial Ca/mg protein and level off above those values. The slope of the concentrationdependent phase and the total effiux at the plateau level are reduced in older animals, indicating that the internal free calcium concentration decreases and the calcium-buffering power of the mitochondriai matrix increases with age. These results suggest that mitochondrial preparations from aged rat brain may have a larger ApCa than those from adult rats. Taken together, the results suggest that there is no decrease of A/~Ca during ageing and consequently, differences in the uptake rate cannot be attributed to changes in the driving force for calcium uptake but rather to changes in the activity of the uniporter.

J 50

Fig. 3. Differences between the Ca 2+ buffering power of the mitochondrial matrix in old and adult rats. Adult (A) and 24month-old rat brain mitochondria (&) were incubated with succinate (K salt), 0.2 mM ADP, 0.8mM Pi, 0.4 nmol oligomycin/mg protein, rotenone and increasing amounts of calcium. The rate of calcium efflux initiated by the simultaneous addition of ruthenium red, 10 mM NaCI and 0.2 MM A23187, is plotted against the amount of Ca2+ taken up by mitochondria prior to ruthenium red addition. The data correspond to individual results obtained in 3 different experiments. The slopes of the rates of Ca2+ efflux vs Ca2+ load showed significant differences, when compared by the F-test, between adult and old rat brain mitochondria (P < 0.05). The plateau values were also significantlydifferent (P < 0.005).

The regulation of calcium distribution across the mitochondrial membrane results from the balance of the calcium uptake and efflux rates 5"36. Therefore, any variation in the calcium uptake process ought to lead to a resetting of the calcium steady-state distribution. To study the consequences of the decreased calcium uniporter activity, we have determined the steady-state calcium distribution in mitochondria incubated at different calcium concentrations. Fig. 4 shows that the Ca 2+ concentration at which influx and effiux balance is significantly higher in old than in adult mitochondria and this difference is more pronounced over the higher range of calcium concentrations. These results are consistent with the prediction that a lowering of the calcium influx rate in mitochondria from old rats would increase the extramitochondrial calcium concentration at steady-state. It could be argued that the decrease in calcium uptake rate could result from an increased contamination of the mitochondrial preparations from older rats with other organelles or from increased amounts of broken mitochondria. However, two lines of evi-

41

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Fig. 4. Steady-state Ca 2+ distribution in brain mitochondria incubated with 0.2 mM ADP and 0.8 mM Pi. Adult (Lk) and 27month-old rat brain mitochondria (0.6 mg protein (&)) were incubated with 0.8 mM Pi, 0.2 mM ADP, 0.4 nmol oligomycin/mg protein in a nitrilo-acetate Ca 2÷ buffer in the presence of succinate (K salt). The steady-state pCa after addition of different calcium loads was estimated with a calcium-sensitive electrode. The results are mean + S.E.M. for 7 experiments.

The difference in steady-state between mitochondria from adult and old animals was tested for significance by Student's t-test. The following degrees of significance are noted in the fig.: * P < 0.05; ** P < 0.025.

dence suggest that there is no increase in contamination in preparations of old animals. (1) The internal volume (i.e. sucrose impermeable space) and the accumulations of tetraphenylphosphonium and [14C]acetic acid per milligram of mitochondrial protein measured in the same mitochondrial preparations are identical in energized old and adult brain mitochondria 63 and (2) the total activity and recovery of the matrix and of the outer mitochondrial membrane marker enzymes, citrate synthase, and NADH-cytochrome, c reductase, in synaptosomal and free mitochondrial fractions do not vary with ageing 61. Consequently, the variations in the Ca 2÷ transport activities that we are now reporting cannot be accounted for by a leakiness of the mitochondria or redistribution of protein between the fractions, and are, therefore, truly age-related.

Calcium distribution in synaptosomes The brain mitochondrial preparation used in these studies is heterogenous 26. Mitochondria belonging to this fraction can arise from neuron or glial cell bodies or from nerve endings 7,24. Since it is mitochondria

arising from the latter that could be involved in synaptosomal calcium uptake, it was necessary to show that the altered calcium transport properties observed in isolated mitochondria were also present in synaptosomes. To this end, rat brain synaptosomes were loaded with 45Ca by incubation in 2.3 mM Ca2CI at low and high potassium concentrations, and the intrasynaptosomal location of 45Ca was then investigated using a non-disruptive technique based on the selective release of calcium from the mitochondrial pool by collapsing the mitochondrial membrane potential 2°. This was accomplished by the addition of FCCP and oligomycin to prevent ATP hydrolysis by mitochondrial ATPase 1'49'52. Under those conditions we have tested that the resting synaptosomal membrane potential is maintained over a period of around 4 min, and over periods longer than 10 min in K+-depola rized synaptosomes where the main source of ATP utilization, i.e. Na+,K+-ATPase activity, is reduced (results not shown). Fig. 5 shows the results of such experiment. At all ages, the time course and total uptake values in high K ÷ media are much higher than those found in low K ÷ media as has been observed by other authors 49. However, a progressive decrease in calcium uptake in 12- and 24-month-old rats can be observed both under resting (low K ÷) and depolarizing (high K ÷) conditions, in agreement with the results of Peterson and Gibson 4°. The age-dependent differences in calcium accumulation values increase with longer incubation times and are absent between 12- and 24month-old rats at 30 s. These differences in calcium uptake c a n n o t b e attributed to age-dependent changes in synaptosomal volume or membrane potential since these were found to be unchanged in 24month-old rats (results not shown). The addition of FCCP and oligomycin to K+-depo larized synaptosomes after 10 min incubation results in a rapid release of 45Ca. This is followed by a slower release that levels off after about 12 min when the initial non-mitochondrial pool size is regained and net Ca2÷-extrusion by plasma membrane Ca2+-ATPase is arrested ~. The residual synaptosoma145Ca contents observed in rats of all ages are very similar and their values are close to those found in synaptosomes incubated in low K ÷ media, a condition in which the FCCP-promoted Ca 2+ release was found to be barely

42

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Fig. 5. Difference in calcium uptake by synaptosomes in rats of increasing age. Calcium uptake in synaptosomes for 3- ((3, Q), 12- ([2, II) and 24-month-old rats (~, • ) was measured in low (closed symbols) or high (open symbols) potassium concentration in the presence of 2.3 mM CI2Ca (0.75/~Ci 45Ca2+/ml). 10 ktM FCCP and 10/~M oligomycin were added at t = 10 min. The results are means S.E.M. of 5 experiments. * and ** indicate that significant degrees of difference (P < 0.05 or P < 0.025) exist between adults and 12(a) or 24- (b) month-old rats, or between 12- and 24-month-old rats (c).

detectable (results not shown). This suggests that the extra 4SCa taken up by depolarization is located mainly within mitochondria, in agreement with the results of A k e r m a n and Nicholls z. Under our incubation conditions this represents 6.91 _+ 0.593 nmol Ca2+/mg protein or 38.1 + 1.75% of the total 4SCa taken up after 10 min in depolarizing medium for synaptosomes from 3-month-old animals and 5.95 + 0.43 nmol Ca2+/mg protein (35 + 2.2% of total 4SCa taken up) in synaptosomes from 12-month-old rats. However, the corresponding values for 24-month-old rats are significantly lower (P < 0.0025): 3.74 + 0.30 nmol Ca2+/mg protein or 27.94 + 1.99% of total calcium. By dividing mitochondrial (FCCP-releasable) by extramitochondrial (total minus FCCP-releasable) 45Ca it can be observed that the calcium distribution within synaptosomes is 0.625 + 0.048, 0.599 + 0.033 and 0.393 + 0.033 at 3, 12 and 24 months of age re-

spectively. Whereas the values in 3- and 12-monthold rats are again similar, the calcium distribution drops significantly (P < 0.0025) in 24-month-old rats, in agreement with the observations on isolated mitochondria. Since significant differences in calcium distribution do not appear until 24 months of age, it is suggested that a process other than impaired mitochondrial calcium uptake accounts for the decrease in calcium uptake in synaptosomes observed at 12 months.

Influence of mitochondria in calcium uptake by synaptosomes If the large differences in calcium uptake by synaptosomes of old animals are due to impaired Ca 2÷ uptake in mitochondria, then, it follows that those differences could disappear if the mitochondrial contribution to calcium accumulation is abolished. To test this possibility 4SCa uptake experiments were con-

43 ducted in synaptosomes in which the intrasynaptic mitochondrial membrane potential was abolished by the addition of FCCP at time zero of the calcium uptake assays. As observed in Fig. 6, this results in a marked decrease in total calcium uptake. Similar results are observed when F C C P is substituted by 10 a M antimycin A (results not shown). U n d e r those conditions, the differences between the two age groups studied (3- and 24-month-old rats) were substantially decreased (compare uptake values in Figs. 5 and 6). Therefore, this result indicates that decreased calcium influx in mitochondria is one of the main causes contributing to impaired calcium uptake in synaptosomes from old rats. However, since differences in total calcium accumulation can still be observed (Fig. 6), it is suggested that the calcium uniporter of mitochondria is not the only system involved in impaired calcium influx in synaptosomes from 24-month-old rats. M e m b r a n e fluidity studies

The decrease in calcium uptake observed in 24month-old rat brain mitochondria could be due to

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changes in the carrier protein itself, or to changes in the fluidity of the membrane lipids. To study the changes in lipid dynamics in the inner mitochondrial membrane, mitoplasts were labelled with D P H and tested for fluorescence depolarization. Fig. 7 shows the temperature-dependence of steady-state fluorescence anisotropy in mitoplasts from free and synaptosomal mitochondria of rats of different ages. The Arrhenius plots of r s for free mitochondria of adult and old animals are largely parallel. However, the rs values are high'er over the whole range of temperatures in old than in adult mitochondrial preparations, a finding that has been often encountered when the cholesterol proportion of the membrane increases 8'51. It is conceivable that this could be the cause for the age-dependent increase in steady-state fluorescence anisotropy since, Vorbeck et al. 64 and Lewin and Timiras 2s have reported that the cholesterol proportion of hepatic and cardiac mitochondria increases markedly in 29- and 33-month-old rats. Fig. 7B shows that unlike the results obtained on free mitochondria, steady-state fluorescence anisotropy does not vary with age in synaptosomal mitochondria. As observed, in 3-month-old animals, r s values of synaptosomal mitochondria are higher than those of free mitochondria, whereas in older animals

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Fig. 6. Effect of FCCP in calcium uptake by synaptosomes from 3- and 24-month-old rats. Calcium uptake was determined in synaptosomes from adult (O) and 24-month-old rats (0) incubated in high potassium media in the presence of 10 /~M FCCP and 10/~M oligomycin. The results are means + S.E.M. of 5 experiments. The following degrees of significance are marked: * P < 0.05; ** P < 0.025.

Fig. 7. Influence of age on temperature dependence of fluorescence anisotropy. Fluorescence anisotropy was measured, as described in Materials and Methods, in mitoplasts from nonsynaptic (A) or synaptic (B) mitochondria obtained from 3-(0) or 24-month-old rats (0) respectively. The results are mean + S.E.M. of 3 experiments. The numbers associated with the graphs indicate the temperature at which breaking points are observed (means + S.E.M.).

44 the differences in r~ between both mitochondria types tend to disappear. We have used different criteria in order to rule out that age-dependent differences in contaminating proteins are the cause of the results observed. Thus, the purity of the mitoplast preparations used in these assays was investigated with the use of acetylcholine esterase as a marker enzyme of microsomes, because the fluidity of this membrane fraction is markedly lower than that of mitochondria. In contrast to the results of Leong et al. 26, we have found that the synaptic mitochondrial preparation is more contaminated with microsomal protein than that of free mitochondria. However, the degree of contamination was unchanged with age: 4% and 2.6% of microsomal protein were present in mitoplasts from free mitochondria of 3- and 24-month-old rats respectively, whereas in synaptosomal mitochondria, contamination of mitoplasts with microsoreal protein was 13% or 12%, in 3- or 24-month-old rats. DISCUSSION Calcium uptake by synaptosomes is severely reduced during ageing (see ref. 40 and this paper) and the results presented here suggest that the decrease in calcium uptake by mitochondria could be involved in impaired calcium homeostasis in synaptosomes isolated from aged animals. We have found that calcium uptake in mitochondria is markedly decreased in 24-month-old rats (Fig. 1) and that this does not result from changes in either component of the driving force for calcium uptake (,d~, Fig. 2, or ApCa, Fig. 3), but rather to a decrease in the activity of the uniporter itself. The decrease in calcium influx results in a change in steady-state calcium distribution across the mitochondrial membrane (Fig. 4), a result that could be expected if the steady-state was the result of an equilibrium between influx and efflux rates as has been previously reported 5'36. We have assessed that the altered calcium uptake properties observed in isolated mitochondria from 24-month-old rats are also present in intrasynaptosomal mitochondria. Fig. 5 shows that the decrease in calcium uptake in synaptosomes from 24-month-old animals is accompanied by a change in calcium distribution. The mitochondrial pool of calcium decreases and the extramitochondrial calcium pool increases

resulting in (Ca)in/(Ca)out values of 0.62 and 0.59 in 3- and 12-month-old animals and as low as 0.39 in 24month-old rats. It could be argued that impaired calcium distribution in synaptosomes could result from a decrease in mitochondrial protein within the nerve endings during ageing. We have tested this possibility with the use of citrate synthase as a marker enzyme for mitochondria in our synaptosomal preparations. The finding of no variations with age in citrate synthase activity from synaptosomes excludes that a loss of mitochondria rather than a decrease in the calcium uniporter activity is the cause of the results observed. The decrease in calcium uptake in mitochondria could be due to changes in the enzyme protein itself or to changes in the membrane lipid domain. As shown in Fig. 7, we have observed a decrease in fuidity of the lipid phase of the inner mitochondrial membrane in preparations of 'free' mitochondria from old rat brain, a finding that could be involved in the altered calcium uptake activity observed in the total mitochondrial population (free and synaptosomal). However, since both the total and free mitochondrial preparations are heterogenous 26 it is possible that the change in calcium transport properties and in lipid dynamics that have been observed in these fractions may not belong to one and the same mitochondrial type. This ambiguity is eliminated when calcium uptake and membrane fluidity are compared within the same mitochondrial population, i.e. that of synaptic origin. Since in this population we did not find any changes in lipid mobility that would follow the decrease in mitochondrial calcium contents with age, it is suggested that a change in the carrier protein itself rather than alterations in membrane lipid mobility might be the cause of the decrease in the calcium uniporter activity in brain mitochondria from 24-monthold animals. The role of mitochondria in the regulation of calcium homeostasis is complex. On the one hand, Denton and McCormarck n, in studies on Ca 2+ sensitivity of intramitochondrial dehydrogenases, suggested that the physiological function of the C a 2+ transporting systems in the inner mitochondrial membrane is to control the intramitochondrial Ca 2+ concentration and thereby regulate a variety of Ca2+-dependent enzymes. To function in this way, the concentration of free calcium within mitochondria should be below

45 the levels saturating the regulated enzymes, i.e. in the range of 10-6 M. Since estimates of the calcium content of mitochondria of various origins have yielded conflicting results 20'21'32'34'43'50'54'55, it is still not clear whether interaction of calcium with mitochondria in vivo functions in the Ca2÷-activation of Krebs cycle activity. Among other factors, free matrix calcium concentrations depend on Pi and ADP levels 62'65, which probably act by altering the calcium activity coefficient within mitochondria. To our knowledge, the combination of Pi, ADP and Ca 2+ as possible source of variation has not been considered in this context, and therefore, conclusions as to the influence of a decrease in the calcium uniporter activity in Ca2÷-activation of Krebs cycle enzymes do not seem warranted. On the other hand, attention has been focused for some time on the role of mitochondria in the regulation of intracellular calcium concentration. Provided that the mitochondrial matrix free calcium concentration is sufficiently high (around 30 nmol/mg protein at 0.2 mM Pi and ADP with rat brain mitochondria but lower values with increasing Pi), the velocity of calcium efflux will be independent of changes of the mitochondrial calcium content 62. Then, any change in cytosolic calcium concentration would lead to an increase in calcium uptake by rat brain mitochondria and a rapid lowering of cytosolic calcium 37'65. If the FCCP-releaseable calcium represents a minimal estimate of the calcium concentration in intrasynaptosomal mitochondria, it can be assumed that the mitochondrial calcium pool is low under resting conditions: in fact, we did not find any detectable CaE÷-release when FCCP was added to synaptosomes incubated under low K ÷. This indicates that the mitochondrial calcium pool present under resting conditions probably does not exceed the lower detection limit of our experimental conditions, that is around 0.2 nmol Ca/mg synaptosomal protein. In our hands, and from the comparison of citrate synthase activities in synaptosomes and mitochondria, 10% of synaptosomal protein corresponds to mitochondria. Therefore, the intrasynaptosomal mitochondrial pool would be no larger than 2 nmol Ca2÷/mg mitochondrial protein in undepolarized synaptosomes, a value close to those reported by other authors for liver or brain 1'43. At these low calcium concentrations and with relatively normal Pi levels (below 0.8 mM)

mitochondria could not function as buffer of cytosolic calcium. However, when synaptosomes are depolarized with high K ÷, and possibly under prolonged stimulation in vivo, net calcium inflow takes place. Thus, intracellular calcium was found to increase 1.28-2.5fold in pre- and postsynaptic endings of hippocampal slices during long-term potentiation 2'22. The capacity of microsomes as intracellular calcium sinks is very small, and, due to its high affinity for calcium 33'48, this compartment is probably more saturated than mitochondria under resting conditions 4,6,41,42. Therefore, calcium accumulation will take place largely in mitochondria even though the calcium affinity of this organelle is relatively low 37. Thus, when synaptosomes are preloaded with 45Ca at low K + and subsequently depolarized, the addition of FCCP at the onset of depolarization prevents all calcium uptake (results not shown). The local overshoots of calcium concentration within the cytosol of nerve cells could facilitate mitochondrial calcium uptake 18,6°. From the experiments of Fig. 5 it can be assumed that mitochondria from 3-month-old rats accumulate around 69 nmol Ca2+/mg protein after 10 min incubation of synaptosomes in high K ÷ medium, which is well above the calcium concentration required to buffer cytosolic calcium. Under similar conditions mitochondria from 24-month-old rats accumulate 37 nmol/mg protein which is again within the range of concentrations required to buffer cytosolic calcium. However, this buffering is probably exerted at much higher cytosolic calcium levels since synaptic intra/ extramitochondrial calcium distributions are shifted to much lower values in aged rat brain (see above). From the previous considerations it would be expected that under sustained or repeated depolarization a decrease in the calcium uniporter activity of mitochondria from old animals could result in an increase in cytosolic calcium concentration. In turn, this could lead to a resetting of different processes linked to calcium homeostasis in the aged rat brain, for example, to changes in calcium uptake or efflux through the plasma membrane inasmuch as those processes are dependent on cytosolic calcium levels, or to shifts in the Ca2+-regulation of cytosolic proteins. Support to these possibilities stems from the following observations.

46 (1) After-hyperpolarizations induced by spike bursts in C A I neurons of hippocampal slices result from Ca2+-dependent K + conductances and are considerably prolonged in cells of aged rats, indicating that cytosolic calcium levels remain higher in old animals after a current-induced burst in action potentials 25. (2) By abolishing the mitochondrial membrane potential at the time of depolarization, the differences in Ca 2÷ uptake between 24- and 3-month-old rat brain synaptosomes decrease considerably (Fig. 6). This suggests that the differences in calcium handling between adult and old mitochondria are among the main causes of the age-dependent decrease in calcium uptake in synaptosomes. To summarize, the findings of these studies seem to provide clear evidence that altered calcium homeostasis in aged mammalian nerve endings is due, at least in part, to changes in the activity of the calcium uniporter of mitochondria. Experiments are currently under way to explore the influence of altered calcium transport by aged brain mitochondria in cytosolic calcium levels and in Ca e+ transport through the different systems involved in calcium entry in nerve cells (Ca2+-channels, Na+/Ca 2+ exchange). Additional studies are required to analyze other possible causes for the decrease in calcium uptake by synapto-

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ACKNOWLEDGEMENTS This work was supported by grants from the Fondo de Investigaciones Sanitarias and Comisi6n Asesora de Investigaci6n Cientffica y T6cnica and by a grant from the Caja de Ahorros y Monte de Piedad to Javier Vitorica. We are deeply indebted to Dr. Pedro Aparicio for permission to use the Aminco D W 2 a dual-wavelength spectrophotometer at the Centro de Investigaciones Biol6gicas C.S.I.C. We thank Miss Maria Victoria Mora Gil for her technical assistance.

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