Neurobiologyof Aging, Vol. 17, No. 4, pp. 557-563, 1996 Copyright © 1996 ElsevierScienceInc. Printed in the USA. All rights reserved 0197-4580/96 $15.00 + .00 ELSEVIER
Region-Specific Downregulation of Free Intrace,llular Calcium in the Aged Rat Brain H E N R I K E H A R T M A N N , K A R S T E N V E L B I N G E R , A N N E E C K E R T A N D W A L T E R E. M L I L L E R 1
Department of Psychopharmacology, Central Institute of Mental Health, J5, 68159 Mannheim, Germany R e c e i v e d 9 M a r c h 1995; R e v i s e d 5 July 1995; A c c e p t e d 29 S e p t e m b e r 1995 HARTMANN, H., K. VELBINGER, A. ECKERT AND W.E. MI]LLER. Region-specific downregulation of free intracellular calcium in the aged rat brain. NEUROBIOL AGING 17(4) 557-563, 1996.--Age-related changes in resting levels of the free intracellular calciu~a concentration ([Ca2+]i) as well as alterations of the rise in [Ca2+]i following depolarization have been investigated in acutely isolated brain cells of various regions of the rat brain. Characterization of the Ca2+ responses following KC1 depolarization in the hippocampus, cortex, striatum, and cerebellum of young rats revealed significant regional differences in the basal [Ca2+]~level as well as in the KCl-induced rise in [Ca2+]i. However, there was no correlation between both parameters. Resting [Ca2+]i as well as Ca2÷ responses after depolarization were lower in the hippocampus and cortex of the aged animals, but not in the striatum or cerebellum. It is concluded that the Ca2÷ homeostasis in the first two regions is specially susceptible to the aging process, resulting in a downregulation of [Ca2÷]/, probably as a consequence of an enhanced sensitivity of mechanisms regulating transmembraneous Ca2÷ fluxes. The cellular Ca2÷ homeostasis was altered in a comparable way in rat spleenocytes. The rise in [Ca2+]i in the aged animals following stimulation of lymphocytes with the mitogen phytohemagglutinin (PHA) was significantly reduced in the plateau phase, which is maintained by Ca2÷ influx mechanisms. The data indicate that age-related disturbances of the cellular Caa+ homeostasis may be present in different cell types and seem to affect mainly transmembraneous Ca2÷ flux much more than intracellular Ca2+ release. Aging
Free inlxacellular calcium
ALTERATIONS of the free intracellular calcium concentration ([Ca2*]~) play a key role for neuronal function and are of major importance for activating and inhibiting mechanisms regulating neuronal signal transduction. The central role of [Ca2+] i in cellular signalling led to the hypothesis that disturbances of the neuronal Ca 2+ homeostasis may represent a basic mechanism of brain aging and of age-related deficits of brain function (3,10). To prove this hypothesis, a variety of studies have been performed characterizing alterations of Ca 2+ regulating mechanisms with aging. These investigations included studies on altered activity or sensitivity of mechanisms like the Na÷/Ca 2÷ exchanger (19,23) or the Ca 2÷ ATPase (11,12,21,23). Moreover, by the use of electrophysiological methods, altered characteristics of Ca 2÷ currents (34) and of Ca2+dependent afterhyperpolarization (16,27) have been reported. In many cases, these data have been interpreted as indication for an age-related enhancement of [Ca2+]~. However, comparable few data are available on age-related alterations of the [Ca2+]~ itself and most have been performed in synaptosomal preparations. These studies revealed conflicting results indicating increased or unaltered basal [Ca2+]i or depolarization-induced rises in [Ca2+]i (4, 18,21,22). Additional support that [Ca2*]i is not generally enhanced with aging comes from recent studies where significant age-related reductions of basal [Ca2+]~ (6,37) and depolarizationinduced rise in [Ca2+]i in acutely dissociated mouse brain cells (6) or rat brain synaptoneurosomes (37) have been shown. Considering the different susceptibility of distinct brain regions for aging
and for age-related neurodegenerative processes (2) it may be assumed that some brain regions are more affected than others by alterations of Ca 2÷ regulating mechanisms. To test this hypothesis, we evaluated the basal [Ca2+] i and the depolarization-induced rise in [Ca2+]i in the cortex, hippocampus, striatum, and cerebellum of young and aged rats. To investigate the hypothesis that age-related alterations of the Ca 2÷ homeostasis are possibly not restricted to brain cells but can also be found in other cell systems, we included rat spleenocytes in our present study. METHOD
Materials Young (4--6 months) and aged (21-23 months) male Wistar rats were kindly donated by Tropon (Kt~ln, Germany). Fura-2 AM was purchased from Molecular Probes (USA). All other chemicals were obtained from commercial suppliers.
Method Rats were killed by decapitation and the brains immediatedly removed on ice. At the same time, spleens were also removed and further processed as described below.
Preparation of Mechanically Dissociated Brain Cells The cerebellum, hippocampus, striatum, and cortex were dissected and each tissue fraction transfered into 2 ml of medium I
To whom requests for reprints should be addressed. 557
(NaCl 138, KCI 5.4, Na2HPO 4 0.17, KH2PO4 0.22, glucose 5.5, and sucrose 58.4, all in mmol/1, pH 7.35). Mechanically dissociated brain cells were prepared basically following the method of Stoll et al. (36). The tissue was minced with a scalpel and further dissociated by trituration through a nylon mesh (pore diameter 102 ixm) with a pasteur pipette. The resulting suspension was filtered by gravity through a fresh nylon mesh with the same pore diameter and the dissociated cell aggregates were washed twice with medium II (NaC1 110, KC1 5.3, CaCI 2 1.8, MgC12 1, glucose 25, sucrose 70, and HEPES ( 4 - 2 - h y d r o x y e t h y l ) - l - p i p e r a z i n e ethanesulfonic acid) 20, all in mmol/1, pH 7.4) by centrifugation (400 x g for 3 rain at 4°C). To account for the varying amount of tissue of the different regions all suspensions were initially adjusted to 20 mg wet weight/ml, resulting in similar protein concentrations for all samples. Vitality of the cells in the different brain regions was tested by Trypan blue exclusion test. It was > 90% for the cortex, hippocampus, and striatum. Vitality in the cerebellum was somewhat lower (75-80%), however, vitality in all regions remained constant for at least 3 h.
Determination of [Ca2+]i in Brain Cells After the last centrifugation step the pellet was resuspended in medium III (like medium II but sucrose 40 mmol/1) and incubated in 3 ml for 20 min in a shaking water bath (37°C). After this equilibration the suspension was washed again by centrifugation (400 x g, 3 min, room temperature) and the pellet resuspended in 1 ml Hank's Balanced Salt Solution (HBSS) (glucose 6, MgSO 4 1, CaC12 1, KC1 5, NaC1 137, Na2nPO 4 0.3, HEPES 10, all mmol/1, pH 7.4) and the cells incubated with fura-2 AM (10 txmol/1) for 45 min in a shaking water bath. After dye loading, cells were washed three times (400 x g, 3 min, room temperature) to remove extracellular fura-2 AM. The final pellets were resuspended in HBSS and divided into aliquots of 1 ml, which were kept at 37°C prior to measurement. To ensure that the fluorescence is only due to intracellulary incorporated fura-2 cells were washed by centrifugation immediately before fluorescence measurement to remove leaked dye and finally resuspended in lml HBSS. Fluorescence was measured with a SLM Aminco 4800 spectrofluorometer. Samples were kept at 37°C under magnetic stirring. After equilibration (90 s) to get the basal [Ca2+]~, KC1 (25 t*1) was added in the appropriate concentration. [Ca2+]~ was calculated from the ratio of fluorescence intensities at excitation wavelenghts of 340 and 380 nm (emission at 510 nm) in intact cells, after lysis at saturating (Rmax, SDS 0.2%) and at very low (Rmin, EGTA 6 mmol/1, TRIS 30 mmol/1) free Ca 2+ concentrations according to Grynkiewicz et al. (5).
Isolation of Rat Spleen T-Lymphocytes After removal, the spleen was washed in RPMI 1640/5% fetal calf serum (FCS) and the cells collected by squeezing the tissue with two curved needles. The spleen cells were depleted of Blymphocytes by passage over nylon wool columns (14) and further depleted of erythrocytes by NH4C1 lysis. In a previous study (9), the percentage of T-cells in this preparation was found to be approximately 88%.
HARTMANN ET AL. 15 or 100 ~g/ml. Ca z+ measurements and calculations were performed as described above. R . . . . was determined by the use of Triton 0.5%.
Statistics Differences in responses were assessed for significance by ANOVA (SAS Institute, Cary, NC). Differences were considered statistically significant for p < 0.05. RESULTS
Figure 1 shows typical time courses of the KC1 (60 mmol/1)induced rise in [Ca2÷]i in the hippocampus and cerebellum of the adult rat. Depolarization of the cells induces a rapid rise in [Ca2÷]i, which returns to a lower plateau level at about 15-20 s after addition of KC1. This rise is solely due to Ca 2÷ influx, as no response can be found in the presence of EGTA (data not shown). The basal [Ca2÷]i and the plateau level after depolarization are stable for the whole time period investigated (Fig. 1). As previously shown in mouse brain cells, the [Ca2+],. levels remain almost unchanged for up to 60 min under these conditions (6) indicating a preserved cellular integrity with intact mechanisms regulating the neuronal Ca 2÷ homeostasis. Interestingly, the resting [Ca2÷]i levels differ significantly between the various regions investigated (Figs. 1 and 2 left y-axis, open bars). Lowest basal levels can be found in the cerebellum whereas highest levels are found in the cortical preparation. In all regions, depolarization with KC1 induces a concentration-dependent rise in [Ca2+]i with a half maximal response at about 20 mmol/1 and a maximal response at about 60 mmol/l KC1 (Fig. 2, inset). At higher concentrations (80 mmol/ 1) Ca 2÷ levels tend to decline (Fig. 2, inset). The maximal rise over baseline induced with 60 mmol/1 KC1 differs considerably between the brain regions, with the highest response in the hippocampus (Fig. 2, right y-axis, hatched bars). The rise in [Ca2÷]i in percent of baseline is highest (about 190%) in the hippocampus, followed by 150% in the cerebellum, 120% in the cortex and 80% in the striatum (Fig. 2, inset). A comparison of the regional-dependent differences in basal [Ca2+]i with the depolarization induced rise in [Ca2+]i does not indicate any correlation between both parameters. To investigate if the Ca 2÷ regulating mechanisms are differently affected by aging in the four regions, we evaluated resting [Ca2÷]~ and the depolarization-induced rise in [Ca2÷],. in brain cells
E 4. ,N
1200 900 600
Measurement of [Ca2+]i in Spleen T-Lymphocytes For measurement of [Ca2+]i lymphocytes (107 cells/ml) were loaded with fura-2 AM (3 I.~mol/1) for 40 min at 37°C. After washing, the samples were equilibrated in a cuvette at 37°C for 3 rain (cell density 2.5 x 10 6 cells/ml). Lymphocytes were stimulated with phytohemagglutinin (PHA) at a final concentration of
oeHippocampus Cerebellum I
time (see) FIG. 1. Time course of [Ca2+]i after depolarization with KC1 (60 mmol/l) in acutely dissociated brain cells from the hippocampus (closed circles) and cerebellum (open circles) of adult rats. Data are means + SEM, n = 5, each representing an individual animal.
FREE INTRACELLULAR CALCIUM IN THE A G I N G BRAIN
1000 [ ]
i / i
600 /// /// ///
I11 I I I I I I iii i i i i i i / / / iii i i i / / /
Cortex Hippocampus Striatum Cerebellum FIG. 2. Basal (open bars, left y-axis, n = 11) [Ca2÷]i and KCI (60 mmoVl) (hatched bars, right y-axis, n = 7) induced rise in [Ca2÷]~ in acutely dissociated brain cells from the cortex, hippocampus, striatum, and cerebellum of adult rats. Data are means _+ SEM each value representing an individual animal. Basal [Ca2+]i differs significantly between the regions (ANOVA p < 0.0001, F = 35.15) as well as rise in [Ca2+]i after depolarization with KC1 (60 mmoVl). (ANOVA p < 0.0001, F = 67.63). Inset: percental increase of [Ca2+]i above basal levels in the hippocampus (H, n = 5-7), cerebellum (Ce, n = 9-10), Cortex (Co, n = 7-9), and striatum (S, n = 5-6) after stimulation with increasing KC1 concentrations. Response to depolarization in the regions differs significantly. (ANOVA region p < 0.0001, F = 58.94, region x concentration p < 0.0002, F = 3.52).
no significant changes of the basal [Ca2+] i can be found (Fig. 3), there is only a small tendency to an age-related reduced Ca 2÷ response which does not reach statistical significance (Tables 1 and 2). To study if age-related alterations of Ca 2÷ regulation can also be found in other cell systems, T-lymphocytes were isolated from spleens of young and aged rats. Stimulation of the lymphocytes with the mitogen PHA (15 ixg/ml) results in a concentration-dependent rise in [Ca2+] i. Again, this response is significantly reduced in cells from aged animals (Fig. 5). The PHA-induced rise in [Ca2+]i is mediated by two mechanisms, initially by IP 3mediated Ca 2÷ release from intracellular stores, whereas the plateau phase is dominated by Ca 2÷ influx from the extracellular space (24). Comparison of the time course of the PHA (15 txg/ml) mediated response in cells from young and aged rats reveals a significant age x time interaction, suggesting that age-related disturbances are mainly restricted to the plateau phase dominated by Ca 2÷ influx. The same characteristic age-related difference can be seen after stimulation with a high dose of PHA (100 ixg/ml) (Fig. 5, inset) where a significant age x time interaction indicates again a specific reduction of the rise in [Ca2+]i in the Ca 2÷ influx phase.
Cortex lOOO [ ] Young 800
of adult and aged Wistar rats in parallel experiments. Again, basal [Ca2+]i differs significantly between the four regions of adult rats and differs in neurons front aged rats in a similar pattern (Fig. 3). However, [Ca2+]i resting ]evels are always lower in brain cells from aged animals, mostly pronounced in the cortex and the hippocampus (Fig. 3). Interestingly, in the same two brain regions, the depolarizationinduced increase in [Ca2+L is also significantly reduced with aging (Fig. 4A and B). This is the case for the initial maximal rise in [Ca2+]~ after addition of KC1 as well as for the plateau phase (Table 2). However, in the striatal and cerebellar cell preparations, where
E 60O ¢:
200 100 0
///, ///i ///i ///1
Hippocampus [ ] Young
E 600 c
i/i //I /ii
[ ] Aged m O
~ ] Aged
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FIG. 3. Basal [Ca2+]i in acutely dissociated brain cells of the cortex, hippocampus, striatum, and cerebellum of young (3 months) and aged (22 months) rats. Values represent means + SEM, n = 11-14, each representing an individual animal. ANOVA p < 0.0001, F = 35.15 for regiondependent differences, ANOVA p < 0.05, F = 5.3 for age-related differences, post hoc test revealed a significant reduction of the basal [Ca2÷]i in the cortex and hippocampus (*p < 0.05).
FIG. 4. Concentration-dependent increase in [Ca2+]i above basal [Ca2+]i after stimulation with increasing KC1 concentrations in acutely dissociated neurons from the cortex (A) and hippocampus (B) from young (3 months) and aged (22 months) rats. Data represent means ± SEM of seven to eight experiments each representing an individual animal. ANOVA revealed significant age-related differences. (A) p < 0.001, F = 13.03, (B) p < 0.0005, F = 14.59. *p < 0.05, **p < 0.01, t-test.
HARTMANN ET AL.
TABLE 1 DEPOLARIZATION-INDUCEDMAXIMALRISE IN [Ca2+]iIN DIFFERENT BRAIN REGIONSOF YOUNGAND AGED RATS [CaZ+]i (nmol/l) (Peak) Brain Region Cortex Young Aged Hippocampus Young Aged Striatum Young Aged Cerebellum Young Aged
KC1 10 mmol/1 KC120mmol/l KC160mmol/1
374.4 _ 22.3 261.7 _ 18.3
560.3 _+25.0 442.7 _+29.1
593.9 -+31.1 557.2 + 38.7
p < 0.001 F = 13.03
380.7_+ 24.6 267.5 _+23.1
768.9_+46.0 906.9-- 52.8 583.2___35.1 800.0 __49.9
p <0.001 F = 14.59
218.1 _+26.7 414.6_+41.9 194.9_+ 19.7 349.5 _+35.8
424.4__.34.1 430.4_+ 56.5
231.9_+23.8 448.5__32.9 211.3 _+19.1 435.1 _+34.3
510.3_+32.9 554.6 _+34.5
Maximal initial rise in [Ca2+]~in mechanically dissociated brain cells of various brain regions of young and aged rats after depolarization with increasing KC1 concentrations. Data are means + SEM, n = 7-8. ANOVA revealed a significant age effect over all regions, p < 0.002, F = 12.00. DISCUSSION We investigated the resting levels of [Ca2÷]i and the depolarization-induced rise in [Ca2+]~ in mechanically dissociated brain cells from various rat brain regions. This preparation has been shown to represent an excellent model to study a variety of signal transduction mechanisms like the properties of various receptor systems (36), the activity of the phosphatidylinositolphospholipase C (PI-PLC) (6,8) or the adenylatecyclase systems (1), and also to measure central [Ca2+]~ (6-8) in individual animals. In a previous study (40) using a similar preparation of acutely dissociated rat brain cells the proportion of neuronal to glia cells has been shown to be about 75 to 25% in the rat cortex and hippocampus. Comparable to our previous studies in mouse brain cells (6) as well as to other studies using similar rat brain preparations (37,40), basal [Ca2+]i levels are increased compared to basal [Ca2+]i in neuronal tissue culture. However, slightly enhanced basal [Ca2+] i levels do not seem to be specific for the mechanical dissociation of brain cells as resting [Ca2+]i levels in brain slices, which might contain many more preserved cells, have been shown to be 200-300 nmol/1 (30), which is comparable to [Ca2+]i resting levels in our cell system. More important, somewhat enhanced basal [Ca2÷]~ levels do not affect cellular intergrity, as the resting [Ca2+]i levels remains stable for at least 60 rain (6) at a [Ca2+]ex of 1 mmol/1, indicating that the Ca 2+ extrusion mechanisms are capable to maintain a Ca 2+ gradient of about 3000 between the extracellular and the intracellular space. Moreover, during the whole time period the cells remain responsive to depolarization with KC1 indicating that no irreversible inactivation of Ca 2+ regulating mechanisms has occured (6). Interestingly, we found different resting levels as well as different maximal responses upon depolarization with KC1 in the four different rat brain regions investigated. If the levels of the maximal response were only dependent on the driving force resulting from the Ca z+ gradient between extracellular and intracellular space, the highest maximal response should be in the region with the lowest basal [Ca2÷]i. However, this correlation has not been found. This indicates different activities and sensitivities of mechanisms involved in maintenance of basal [Ca2+]~ and in the regulation of [Ca2+] i elevation after cell activation in the different brain regions.
Comparison of [Ca2+]i levels in brain cells of hippocampus, cortex, striatum, and cerebellum from young and aged rats revealed reduced basal [Ca2+] i and a reduced Ca 2÷ response only in the first two areas in aging, whereas in the latter two areas only minor and not significant changes of the Ca 2+ regulation could be found. These results confirm and further extend our previous findings in mouse brain cells, indicating an age-related reduced resting [Ca2+]i and a reduced Ca 2÷ response following depolarization (6, 9). To our knowledge, these are the first data describing agerelated region-specific disturbances of the central [Ca2+]i. Interestingly, a recent study by Villa et al. (39) indicated a specific loss of calbindin and calretinin in the hippocampus of aged SpragueDawley rats with no change in the cerebellum. Because the preparation used contains different cells and not only neurons (40), we cannot completely refuse the possibility that changes of cellular composition during aging might influence our data to some extent. However, the preliminary findings of Verkhratsky et al. (38) on a single cell preparation strongly support the assumption of decreased [Ca2+]i after stimulation in rat cortical neurons in aging. So far, only few and rather conflicting data on age-related alterations of the resting [Ca2+]~ are available. In synaptosomes prepared from the whole rat brain or from the rat cortex increased (20) or unchanged (4,18,23) basal [Ca2+]i levels have been reported although most studies used the fura-2 method. Increased basal [Ca2+]i levels have also been observed in freshly isolated central neurons and primarily cultured neurons from the lumbar dorsal root ganglia of rats (38). A recent study by Strosznajder et al. (37) performed in rat cortex synaptoneurosomes confirmed our findings of reduced resting [Ca2÷]i levels with aging. Data about age-dependent alterations of the activation-induced Ca 2+ response are more consistent. Besides our previous (6) and present findings of a reduced Ca 2÷ response in rat and mouse brain, a reduced Ca 2÷ response upon depolarization has also been shown by Verldaratsky et al. in freshly isolated cortical neurons of aged rats (38). Moreover, Reynolds and Carlen (34) showed a reduction in Ca 2+ currents in aged hippocampal dentate gyms granule neurons. Further support for a reduced Ca 2+ response with aging c o m e s f r o m studies in s y n a p t o s o m e s w h e r e a r e d u c e d 45CaZ+uptake after depolarization has been reported (41). Pitler and Landfield reported a prolongation of Ca z+ spike duration in hippocampal slices of aged rats (32) probably causing a prolonged Ca2+-dependent afterhyperpolarization (AHP) (16). Prolonged AHP in hippocampal slices from aged rabbits has also been shown by Moyer et al. (27). Both findings have been interpreted as indices of elevated [Ca2+]i in aging. However, investigations by Potier et al. (33) in Sprague-Dawley rats also revealed an age-related prolongation in spike duration, but changes in AHP were inconsistent and were very much dependent on the experimental conditions like the holding potential of the neuron and on the number of action potentials used to trigger the AHP. Interestingly, studies on the effects of CaZ+-dependent currents in cat neocortical neurons by Schwindt et al. (35) revealed an enhanced AHP induced by a small reduction of [Ca2+] i with low concentrations of the Ca 2÷ chelator BAPTA. These data indicate that the relationship between alterations of [Ca2+]i and mechanisms of AHP might be more complex and reduced [Ca2+]i levels and prolonged AHP might not be necesserily contradictory. We interprete our findings of a reduced basal [Ca2+] i as well as of a reduced Ca 2+ response after depolarization as indices of an age-related downregulation of the Ca z+ homeostasis. A specific impairment of the energy metabolism in the aged neuron is not a very convincing explanation for the described age-related alterations of [Ca2+]~, as this would presumedly result in a reduced gradient between [Ca2+]ex and [Ca2+]~. However, the opposite has been shown in this and previous studies (6). Furthermore, it does
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TABLE 2 PLATEAU LEVEL OF [Ca2% IN DIFFERENT RAT BRAIN REGIONS OF YOUNG AND AGED RATS AFI'ER DEPOLARIZATION W I T H KC1
[Ca/*]e (nmol/1) (Plateau) Brain Region
Cortex Young Aged Hippocampus Young Aged Stfiatum Young Aged Cerebellum Young Aged
KCI 10 mmol/l
KCI 20 mmol/1
KCI 60 mmol/l
210.8 _+14.6 170.9_+ 13.0
389.7 _+15.0 336.4_+20.1
430.9 _ 16.7 414.1 _+29.6
p < 0.01 F = 7.92
240.2 _+8.22 177.1 _+9.7
507.8 _+19.6 435.3 _+17.9
623.9 _+20.4 611.2 -+28.1
p < 0.005 F = 9.94
143.6 -+ 12.3 130.9 -+ 11.4
298.3 _+20.8 253.1 _+28.6
325.8 _+26.7 316.6 _+33.1
181.4 _+ 16.5 127.5 _+10.8
298.4 -+ 9.9 284.1 +_ 19.5
407.3 -+ 34.9 412.1 _+14.6
Plateau level of [Ca2+]i in mechanically dissociated brain cells of various brain regions of young and aged rats after depolarization with increasing KC1 concentrations. Data are means -+ SEM, n = 7-8. ANOVA revealed a significant age effect over all regions, p < 0.005, F = 9.62.
not seem very likely that a global age-enhanced vulnerability of aged brain cells to the preparation procedure is reflected in our findings, as other complex ~ignal transduction mechanisms like the receptor-coupled PLC activation has been shown to be unaltered with aging (8). Therefore, the ability of the aged neurons to keep Ca 2+ homeostasis on a lower level might suggest an enhanced
time (sec) FIG. 5. Time course of PHA (15 ~g/ml) induced rise in [Ca2+]~ in lymphocytes from young (3 months, open circles) and aged (22 months, closed circles) rats. Data are means _ SEM of eight to nine experiments, each representing an individual animal. ANOVA revealed a significant age effect, p < 0.05, F = 5.4, time :.: age interaction p < 0.0001, F = 8.61. Inset: time course of PHA (100 i~ghnl) induced rise in [Ca2+]i. Data are means -+ SEM of eight to nine experiments, each representing an individual animal. The responses in in the lymphocytes from the aged animals are significantly reduced (age effect p < 0.05, F = 6.05, tine x agep < 0.0001, F = 5.61).
sensitivity of Ca2+-dependent mechanisms terminating Ca 2+ influx, like Ca2+-dependent Ca 2+ channel inactivation or the Na+/ Ca 2÷ exchanger. The assumption of an enhanced Ca 2+ sensitivity is supported by previous studies in mouse brain cells where the Ca 2÷ stimulated PLC activity was increased and could be activated by smaller changes of [Ca2+] i (6). Comparable results in the cerebral cortex of the rat have been recently reported by Strosznajder et al. (37), where the activity of membrane bound PLC and PLA 2 in the presence of endogenous Ca 2+ was increased with aging, whereas [Ca2+]i in synaptoneurosomes of this brain area was decreased. The assumption of an age-related increased Ca 2÷ sensitivity of central neurons and an adaptive downregulation of [Ca2+]i may also integrate the finding of a prolonged AHP (16) where the underlying mechanism of the Ca2+-dependent K ÷ conductance might be activated by lower [Ca2+]~. Presently, we do not know the reason for the enhanced Ca 2+ sensitivity, but it seems conceivable that these alterations develop slowly over time and may be compensated under normal conditions. However, in situations of additional stress like ischemia or hypoglycemia, the capacity of mechanisms regulating [Ca2+] i may reach its limit and may, therefore, contribute to the enhanced vulnerability of the aging brain for neurodegenerative processes resulting in neuronal death. It should be noted that age-related changes of Ca 2÷ regulation were mainly seen in the cortex and hippocampus, two brain regions especially susceptible to neurodegeneration during aging. Interestingly, the reduced Ca 2÷ reponse was not restricted to brain cells, but Ca 2÷ responses in rat lymphocytes following stimulation with a mitogen were also reduced. Similar findings of a reduced Ca 2÷ response in aged murine lymphocytes after stimulation with ionomycin (27) or concanavalin A (17,31) have been shown previously. Comparable to our hypothesis it has been suggested that one mechanism underlying the reduced reponse in aged lymphocytes is an increased sensitivity of a transmembraneous Ca 2÷ plasma pump (26). Further support for the assumption of a rather general age-related reduced Ca 2÷ responsiveness comes from findings of Kirischuk et al. (15), where a reduced Ca 2÷ reponse was found in the rat dorsal root ganglion after depolarization. Moreover, Ca 2÷ uptake (28) as well as [Ca2+]i (28) have been
H A R T M A N N ET AL.
shown to be decreased in aged fibroblasts. However, no alteration of [Ca2+] i in fibroblasts were found by Huang et al. (13). On the other hand, reduced [caa+]i in aging is not just a general phenomenon of all cell systems, since even in the brain [Ca2+]i in striatal and cerebellar cells is not changed. An explanation for the regional selectivity cannot yet be given. In conclusion, our findings indicate a region-specific reduction of the Ca a÷ responsiveness in aged brain cells. These alterations may contribute to age-related alterations of signal transduction mechanisms and may contribute to age-related neurodegeneration
due to disturbances of the adaptive capacity of Ca 2+ regulating mechanisms. Moreover, our present data as well as previous findings in lymphocytes (9,25,26) together with investigations in other cell systems may indicate that impairment of the Ca 2+ responsiveness is not restricted to cells of some brain regions but can also be found in some peripheral neuronal and nonneuronal cell types. The common characteristics of these age-related alterations seem to be disturbances of Ca 2+ regulating mechanisms mainly affecting the transmembraneous Ca 2+ flux with less pronounced alterations of the intracellular Ca 2+ release.
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