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Ca2+ exchange activity in human brain: The effect of normal aging
Neurobiologyof Aging, Vol. 14, pp. 373-381, 1993
0197-4580/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
Analysis of Na +/Ca 2+ Exchange Activity in Human Brain: The Effect of Normal Aging ROBERT
A. COLVIN, 1 ANFAN
WU, NANCY
DAVIS AND CATHY
A. MURPHY
Department of Biological Sciences, Ohio University College of Osteopathic Medicine, Athens, OH 45701 R e c e i v e d 23 N o v e m b e r 1992; R e v i s e d 8 F e b r u a r y 1993; A c c e p t e d 8 M a r c h 1993 COLVIN, R. A., A. WU, N. DAVIS AND C. A. MURPHY. Analysis of Na +/Cae+ exchange activity in human brain: The effect of normal aging. NEUROBIOL AGING 14(4) 373-381, 1993.--Na+/Ca 2+ exchange activity and passive permeability to Ca2+ were analyzed in plasma membrane vesicles (PMV) purified from whole rat brain and three regions of human brain: frontal cortex, temporal cortex, and cerebellum. Accumulation of Ca2+ due to Na+/Ca 2+ exchange activity showed a characteristic pattern of an initial rapid rise in Ca 2+ content followed by a stable plateau in both rat and human brain. Total Ca2+ accumulation in rat brain PMV was on average three-fold higher than in human brain. Passive permeability to Ca2+ was measured as the rate of Ca2 ÷ release from PMV first loaded with 4SCa by Na+/Ca 2+ exchange and then exposed to 1 mM EGTA. The Ca2+ permeabilities of human and rat brain PMV were similar. Ca 2+ release from rat brain PMV was faster overall and was resolved into fast and slow components while in human brain a single slow component was found. Post mortem delay up to 4 h had no effect on Na+/Ca 2+ exchange Km for Ca 2+ , Vm~,, and peak uptake and Ca2+ release rate in rat brain PMV. Human frontal cortex was shown to have a greater Na+/Ca 2+ exchange activity than that found in the cerebellum. The frontal cortex, temporal cortex and cerebellum had similar Ca 2+ permeabilities. Age-related effects on Na+/Ca 2+ exchange activity and Ca 2+ permeability were determined in 15 tissues from human frontal cortex (age at death 21 to 93 years). No significant age related effects were seen. These data do not rule out the loss of Ca2+ homeostasis as a component of normal brain aging but do indicate that significant changes in Na+/Ca 2÷ exchange activity and Ca 2+ permeability do not accompany normal human brain aging. Ca2 +
Transport
Brain aging
Plasma membrane vesicles
Ca 2+ permeability
Rat brain
Ca2 + efflux
man tissue (3,8). In these studies, the human brain N a + / C a 2+ exchanger was shown to have many properties similar to that seen in animal tissues. Furthermore, Na +/Ca 2 ÷ exchange activity was increased in brain tissues from Alzheimer disease patients when compared with aged matched controls (3). The ability to measure N a + / C a 2÷ exchange activity in human postmortem tissues now allows a more direct test of the Ca 2 ÷ hypothesis of human brain aging to be performed. In the present studies, N a + / C a 2+ exchange activity and the passive permeability to Ca 2 + were determined in PMV isolated from postmortem human brain tissues derived from individuals having various ages at death.
THE " c a l c i u m hypothesis of brain a g i n g " (9) proposes that changes in the cellular mechanisms that act to modulate the concentration of free intracellular calcium ([Cad) within neurons and glia contribute to the causative factors leading to the cognitive decline associated with normal aging. The mechanisms of homeostatic control of [Ca~] in the human central nervous (CNS) system must involve complex interactions between Ca 2÷ binding proteins, Ca 2+ sequestering organelles, and Ca 2 + transport proteins. The Na +/Ca 2 + exchanger is an important Ca 2 + transporting protein in the plasma membranes of neurons and glial cells. This protein generally catalyzes the transport of Na + ions across the plasma membrane in exchange for Ca 2 + ions. Studies of rat hippocampal neurons (15), striatal neurons, and cortical astrocytes in culture (1) have clearly demonstrated the importance of Na + graclient dependent Ca 2+ extrusion for maintaining low [Ca~]. In these studies it was also shown that Na +/Ca 2 + exchange plays an even more important role in Ca 2 + extrusion after cell activation. Comparing rats of different chronological ages, Michaelis and coworkers demonstrated age-related deficits in Ca 2 + homeostatic function in brain synaptosomes (13), which could be explained by a decrease in N a + / C a 2+ exchange activity (14). The decrease in exchange activity was the result of an increase of Kact for Ca 2 + with little or no effect on V,,,,=. Recently, it has been demonstrated that N a + / C a 2+ exchange activity can be measured in purified plasma membrane vesicles (PMV) isolated from postmortem hu-
METHOD
Preparation of Plasma Membrane Vesicles Frozen tissue sections from human brain were obtained from the National Neurological Research Bank (VAMC Wadsworth, Los Angeles, CA). The characteristics of the brain tissues used in this study are listed in Table 1. Neuropathological investigation at autopsy showed in every case grossly normal brain and spinal cord and microscopic sections of the hippocampus and cortex were unremarkable. Frozen ( - 8 0 ° C ) human brain sections and whole rat brains were used for the preparation of P M V as described previously (3).
1 To whom requests for reprints should be addressed. 373
374
COLVIN ET AL, TABLE 1 CHARACTERISTICSOF BRAINTISSUESUSED
Age (years)
Post Mortem Delay (h)
Cause of Death
21 22 22 30 40 41 44 44 45 47 48 50 57 59 65 66 68 71 72 78 93
36.5 15 18 I1 18 16 12 9.5 18.5 18.5 9 13.5 11.5 11 16 15.5 16.5 12.7 11.5 31 10 15.8 -+ 6.61"
multiple injuries multiple injuries multiple injuries drug overdose malignant melanoma auto accident renal carcinoma metastatic adenocarcinoma bronchopneumonia myocardial infarction bronchopneumonia aspiration sepsis myocardial infarction acute respiratory failure pancreatic carcinoma bronchopneumonia bronchopneumonia lung carcinoma empyema myocardial infarction lung carcinoma
* Mean Post-Mortem Delay +-- SD. The crude microsomal fraction was resuspended in Buffer A conmining 0.32 M sucrose, 10 mM HEPES (pH 7.4), and the protease inhibitors- soybean trypsin inhibitor (50 Ixg/ml), leupeptin (0.5 I~g/ml), and pepstatin (0.7 I~g/ml). For human tissues the crude microsomal fraction was layered over an equal volume of 0.6 M sucrose containing protease inhibitors and centrifuged in an S W 41 rotor at 25,000 rpm for 30 min. The pellet obtained was resuspended in Buffer A and layered over a sucrose gradient conmining 0.6 M and 0.8 M sucrose as the steps. The crude microsomal fraction from rat brain was loaded directly on the sucrose step gradient. The gradients were centrifuged in an SW-41 rotor at 25,000 rpm for 2 h. The PMV were recovered from the 0.6--0.8 M interface and resuspended in 10 vol of Buffer A. The resulting suspension was centrifuged at × 100,000 g for 40 min. The pellet was resuspended in Buffer A to a concentration of 10-20 mg/ml and frozen in aliquots in liquid nitrogen. PMV preparations were stored at - 8 0 ° C until used. Membrane protein determinations were performed by the method of Lowry (12) using bovine serum albumin as a standard. Assay of Ca 2 ÷ Accumulation and Release Due to Na ÷~Caz ÷ Exchange Activity
To preload PMV with Na + , PMV were thawed and diluted to 3 mg/ml (human brain) or 2 mg/ml (rat brain) in Buffer B conmining 137 mM NaC1 and 10 mM HEPES (pH 7.4). These concentrations of membrane protein were found to be optimal for measuring Ca / ÷ transport. The PMV were loaded with Na n by incubating 25 i~1 of the membrane suspension at 37°C for 5 min, after which the test tubes were again placed on ice. To initiate a Ca 2+ transport reaction, Na +-loaded PMV were diluted to 0.5 ml by the addition of either Buffer B or Buffer C in which choline chloride was substituted for NaC1. Each buffer contained various concentrations of CaCO3 and 100 t~M EGTA with the addition of either 0.19 mM (initial velocity) or 0.019 mM (time course)
~5CaC12 (NEN). Free Ca ~ ~ ion concentrations in the assay solutions were determined from an iterative computer program using published dissociation constants (4). Mock solutions were prepared and free Ca 2 + ion concentrations were validated using a Ca 2 ÷ ion selective electrode (Philips). Reactions were stopped at various times by the addition of l mM LaCI 3 and placing the tubes on ice. The membrane suspensions on ice were then filtered on a Brandel M-24RP cell harvester using No. 30 glass fiber filters (Schleicher & Schuell). The filters were washed three times with 5 ml of ice-cold wash buffer containing 137 mM choline CI, 10 mM HEPES (pH 7.4), and 1 mM EGTA, placed in 3 ml of Ultima Gold (Packard) and 45Ca trapped on the filter determined in a scintillation counter with nearly 100% counting efficiency. As total Ca 2+ added to the buffers was known (45Ca plus carrier Ca 2 + ), aliquots were counted to determine the specific activity of 4~Ca. Using this value, the counts on the filter were converted to nmol Ca 2 +/mg protein. Na +-dependent Ca 2 ÷ accumulation was determined as the change in Ca 2 + content in Buffer C minus that measured in Buffer B. For determination of Km for Ca 2+ and V . . . . Na+/Ca-' + exchange was initiated and Ca 2 + accumulation allowed to proceed for either 5 or 10 s before being stopped by addition of 1 mM LaCI 3. The free Ca 2 + ion concentrations ranged between 0.54 ~xM and 2571xM. The initial velocity of Na ÷ dependent Ca 2÷ accumulation was expressed as nmol Ca 2 +/mg/s and plotted as a function of free [CaS+]. The data obtained were fit by nonlinear regression to a rectangular hyperbola to obtain Km and V..... . Assay of Na n Dependent Ca 2+ Efflux
PMV were first loaded with 45Ca via Na ÷ dependent Ca 2+ uptake as described above. After 5 min at 37°C a second dilution buffer was added to reach a final volume of 1 ml and initiate Ca / ÷ efflux. The second dilution buffer contained concentrations of Na ÷ and choline such that when added to the reaction mixture the desired concentrations of Na n and choline were obtained. After various times, efflux was stopped by addition of 1 mM LaCI3, placing the tubes on ice, and rapid filtration (see above). The same combinations of Na + and choline were also added to Na + loaded PMV initially diluted into Buffer B. The values obtained for Ca / ÷ content were then subtracted from the Ca / ÷ content measured in PMV initially diluted into Buffer C. Assay o f Passive Ca 2 + Permeability
PMV were first loaded with 4~Ca exactly as described above. After 5 min at 37°C, 1 mM EGTA was added to inhibit Na + ~Caz + exchange and start the measurement of Ca 2 ÷ release. Ca 2 ÷ release was stopped at various times by the addition of 100 I~M LaC13, placing the tubes on ice and rapid filtration (see above). Na ÷ loaded PMV diluted into Buffer B with addition of 1 mM EGTA served as a control (as described above). The data obtained were fit by nonlinear regression to either a single or a double exponential decay. SDS Polyacrylamide Gel Electrophoresis
Protein profiles of PMV from human or rat brain were obtained according to the method of Laemmli (11) on 8% to 18% linear gradient gels (Pharmacia) and stained with silver stain (Bio Rad). Statistics
Computer assisted nonlinear regression (GraphPad Inplot, San Diego, CA) was used for curve fitting. Goodness of fit was quantitated by the least-squares method. Data presented in graphical
Na+/Ca 2+ EXCHANGE ACTIVITY AND AGING
STD.
A
B
C
375 squares analysis was used to estimate age dependent relationships in the data and judged significant when p < 0.05.
D
RESULTS
Comparison of Na +/Ca 2+ Exchange Activity and Passive Ca 2+ Permeability Measured in Rat and Human Brain
212,000 170,000 116,000 53,000
20,000 6,500
FIG. 1. Electrophoretic separation of human and rat brain plasma membrane vesicle proteins on polyacrylamide gel after solubilization in SDS. Twenty p.g protein was applied to each lane. (A) human frontal cortex, (B) human temporal cortex, (C) human cerebellum, and (D) whole rat brain.
format are single experiments representative of data obtained in at least three different preparations of PMV. When appropriate, means were obtained from the data collected and compared using one-way analysis of variance (ANOVA) with Scheff6's method and judged significantly different when p < 0.05. Linear least
Previous studies (3,7) have shown the suitability of postmortem human brain tissues for analysis of Na+/Ca 2÷ exchange activity. In the present studies, initial experiments were aimed at comparing Na ÷/Ca 2 ÷ exchange activity of rat and human brain PMV that were prepared in a similar manner. Figure 1 shows the electrophoretic profile of human and rat brain PMV proteins. The human and rat brain membranes showed nearly identical patterns of protein staining. The most notable difference was a greater abundance of low molecular weight proteins (<20 kDa) in each region of human brain tested (A: frontal cortex, B: temporal cortex, C: cerebellum). Plasma membrane vesicles prepared from either rat brain or human brain showed a characteristic pattern of Ca 2 + accumulation due to Na+/Ca 2+ exchange activity. Initially, there was a rapid rate of Ca 2 ÷ accumulation that began to plateau within 2 to 3 rain. This plateau level of Ca 2÷ accumulation (termed "peak uptake") was stable for up to 10 min after the start of the reaction. Figure 2A shows the time course of Ca 2 ÷ accumulation seen for PMV prepared from either whole rat brain or human frontal cortex. The appearance of the curves were very similar, the major difference being a three-fold higher peak uptake seen with rat brain. Depending on the Ca 2 + accumulating activity of the preparation, the shortest time points that could be reliably measured were 5-10 s. During the first 10 s of the reaction, Ca 2+ accumulation was nearly linear. Therefore, measuring Ca 2 ÷ accumulation at 5 or 10 s was a good estimate of the initial velocity of the reaction. The K m and Vm~ for Ca 2 ÷ accumulation were estimated by measuring the initial velocity at various free Ca 2÷. Consistent with the measurements of peak uptake, Vm,= estimates for rat brain were threefold higher than the estimates for human frontal cortex. On the other hand, Km estimates for rat brain and human frontal cortex did not differ significantly (compare Tables 2 and 3). The plateau phase of Ca 2 + accumulation is a steady state composed of several Ca 2 ÷ fluxes. The Na ÷/Ca 2 ÷ exchanger mediates a small component of Na + dependent Ca 2 ÷ influx and a larger
3
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FIG. 2. Time course of Ca 2+ accumulation and passive Ca 2+ release. (A) Aliquots (25 ixl) of ( I ) rat brain or (©) human frontal cortex
PMV were preincubated in buffer containing 137 mM NaC1 and then diluted twenty-fold with either the same buffer or buffer containing 137 mM choline chloride; the difference in Ca2+ content representing Na + dependent Ca2+ accumulation. (B) After preloading PMV with 45Ca via Na÷/Ca 2+ exchange, 1 mM EGTA was added to inhibit the exchanger and initiate Ca2+ release. Computer assisted nonlinear regression was used to fit Ca2+ release data to either 2 component (rat brain) or 1 component (human brain) exponential decay.
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COLVIN ET A L
TABLE 2 THE EFFECT OF SHORT-TERM POST MORTEM DELAY ON PARAMETERSOF Na * Ca2* EXCHANGE Time of Delay Na ÷/Caz+ Parameter Vm~ nmol/ mg/s K m I~M Peak nmol/ mg/5min
Control
30 Min
60 Min
90 Min
2H
4H
2 H (b~ 25°
0.394 -- 0.14 54.8 +_ 42.2
0.345 +-- .081 54.3 - 12.9
0.306 ± 0.025 37.7 ± 8.7
0.308 ±- 0.0760 38.7 +- 3.29
0.298 ± .086 32.9 ± 14.1
0.313 +-- .019 38.3 ±- 14.2
0.262 ± 0.041 32.6 ± 6.29
3.76 +-0.246
4.17 +- 0.305
3.61
±
0.75
3.14 ±
0.159
3.71
---1.19
4.45 ±
1.83
4.35 +-0.936
All values are the mean +- SD, for three separate determinations from three different tissues. Vmax and Kn~ values were determined by measuring Na+-dependent Ca2+ accumulation 5 s after the start of the reaction in the presence of free [Ca2÷ ] ranging from 0.54 to 257 IxM. Peak values were determined by measuring Na ÷-dependent Ca2÷ accumulation 5 min after the start of the reaction in the presence of free [Ca2+ ] equaling 55.2 ~M. component of Ca2+/Ca 2+ exchange during the plateau phase. Ca 2 ÷ release, representing the passive permeability of the PMV to Ca 2÷ also occurs during the plateau phase. This passive Ca 2÷ release is shown in Fig. 2B. PMV were first loaded with Ca 2 ÷ by N a ÷ / C a 2+ exchange, then the N a + / C a 2÷ exchanger was inhibited by addition of 1 mM EGTA. The Ca 2 ÷ release that resulted was due to the passive permeability of the PMV to Ca 2 ÷. In contrast to Ca 2+ accumulation, comparison of Ca 2 + release data for rat and human brain P M V revealed important differences (see Fig. 2B). Ca 2 ÷ release from rat brain PMV in this representative experiment was resolved into a fast (T 1/2 = 0.212 m i n - ~) and a slow (T 1/2 = 1.13 min - 1 ) component by nonlinear regression. However, Ca 2 ÷ release from human brain PMV demonstrated only the slow component of release (T 1/2 = 8.87 m i n - ~ ) . The computer generated lines of Fig. 2B were extrapolated beyond the actual data points to demonstrate that both curves approached zero. The time course of Ca 2÷ release was different when increasing extravesicular Na ÷ was used to initiate Ca 2 + efflux. In Fig. 3 is shown the effect of diluting P M V preloaded with Ca 2÷ by N a + / Ca 2 + exchange into solutions containing either zero, 20 mM, or 60 mM Na ÷ . Under these conditions, reversal of N a ÷ / C a 2+ exchange mediates a rapid but transient Ca 2 ÷ release whose magnitude was dependent on the concentration of Na ÷ used. One minute after the reversal of N a + / C a 2÷ exchange was initiated, Ca 2 + content reached an apparent steady state. This property of Na ÷ induced Ca 2 ÷ release differed markedly from passive Ca 2 ÷ release, which showed a steady decline in Ca 2 ÷ content with time. Effect o f Postmortem Delay on N a +/Ca e + Exchange
The differences observed in N a + / C a 2+ exchange activity between rat and human brain, particularly the large difference in
peak uptakes, might be the result of postmortem delay in the human brain tissues. No effect of long term (i.e., 5-26 h) postmortem delay on Na ÷/Ca 2 ÷ exchange activity was seen in previous studies (3,8). However, the period immediately following death (0-4 h) may be the most critical for producing membrane proteolysis, lipolysis, and artifactual changes in N a + / C a 2+ exchange activity and Ca 2÷ permeability. Animal models can be used to analyze the effects of postmortem delay on biochemical processes. Human brain cooling curves have been generated and it has been shown that the cooling curves generated can be reproduced in animals by immersion of the head in a cooled incubator following decapitation (20,21). Cadavers are normally placed in a refrigerator at 4°C within 4 h of death. To mimic these conditions, rat brains also were incubated for 2 h at 25°C. Rats (aged 6 months) were decapitated and the intact skulls were incubated for various times (0, 30, 60, 90 min; 2 and 4 h) at 0-4°C and for 2 h at 25°C. After the incubation was complete, the brain tissue was carefully removed from the skull, quick frozen in liquid N 2 and then stored frozen ( - 80°C) for later use. The frozen rat brain tissues were then processed into PMV as described in the Method section. To determine the effect of postmortem delay on the electrophoretic profile of plasma membrane proteins, S D S - P A G E was performed on each membrane preparation (see Fig. 4). Each lane showed nearly identical patterns of protein staining. There was no evidence of postmortem effects on the molecular weight of the major protein species associated with purified PMV. Table 2 lists means +-- SD obtained for N a + / C a 2÷ exchange K m for Ca 2÷ , Vm~x, and peak uptake after different times and conditions of postmortem delay. There was no significant effect of postmortem delay on these measurements of Na +/Ca 2 ÷ exchange activity. The lack of an effect of postmortem delay also was reflected in the time course of Ca 2 + accumulation as shown in Fig.
TABLE 3 COMPARISON OF Ca2÷ TRANSPORT IN PMV PURIFIED FROM DIFFERENT REGIONS OF HUMAN BRAIN Brain Regions
Age (years)
Vmax (nmoldmg/s)
K m (~M)
Peak nmol/mg/5 min
T 1/2 rain
Frontal cortex Temporal cortex Cerebellum
48.3 --- 21.0 (15) 47.6 ±- 8.16 (8) 57.7 --- 18.9 (12)
0.123 --- 0.042 (15) 0.088 - 0.054 (8) 0.058 --- 0.029 (12)*
41.7 ±- 27.2 (15) 28.2 --- 16.5 (8) 27.5 +- 27.6 (12)
1.28 ±- 0.494 (12) 1.39 +- 0.295 (4) 0.997 --- 0.525 (12)
7.93 - 5.10 (14) 5.45 +- 2.83 (4) 3.64 -+ 4.13 (12)
All values are the mean +- SD. Vmax and I ~ values were determined by measuring Na+-dependent Ca 2+ accumulation at either 5 s (frontal cortex, temporal cortex) or 10 s (cerebellum) after the start of the reaction in the presence of free [Caz+] ranging from 0.54 to 257 ~M. Peak values were determined by measuring Na + -dependent Caz + accumulation 5 min after the start of the reaction in the presence of free [Ca2 + ] equaling 55.2 izM. Release data fit well to a single component fit and were not analyzed for a two component fit. * Significantly different from frontal cortex p < 0.05.
N a + / C a 2+ E X C H A N G E ACTIVITY AND AGING
377
mortem delays resulted in a loss of the fast component of release (T 1/2 = 4.31 - 1.39 r a i n - l ; mean - SD). Additionally, the T 1/2 for the slow component showed a small gradual decrease indicating an increase in release rate.
7 6¸ Z
Lrd
5
Characterization of Na +~Ca2 + Exchange Activity in Different Areas of Human Brain
t.-
Next, N a + / C a 2+ exchange activity was assayed using P M V purified from temporal cortex and cerebellar regions of human brain and compared with frontal cortex. For each brain region, the time course of Ca 2 + accumulation is shown in Fig. 6A and calcium release in Fig. 6B. Table 3 lists the average cumulative data for all tissues assayed. The pattern of Ca 2 + accumulation resulting from N a + / C a 2 + exchange activity was very similar in all three brain regions. Table 3 shows that the mean V,n,~ values obtained for temporal cortex and cerebellum were less than those obtained for frontal cortex. The mean value for cerebellum was significantly (p < 0.05) less than that in frontal cortex. There were no significant differences seen in the mean values of K m for Ca 2 + and peak uptake obtained for each brain region. These findings indicated that PMV from cerebellum had a slower rate of Ca 2 + accumulation but still could accumulate nearly the same total amount of Ca 2 + after 5 min. Figure 6B shows that similar patterns of passive Ca 2 + release were seen for each brain region. In all three brain regions, only the slow component of Ca 2 + release was seen. Cumulative data are shown in Table 3, which shows that the mean release rate of cerebellar PMV was approximately one half that seen in frontal cortex. This difference was not significant (p = 0.068). These data provided evidence to suggest that P M V from different brain regions may differ in their Ca 2 + permeability as well.
rj
,~ ,-" 3f PEAK: 5 0
0
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Ca CONTENT
FIG. 3. Time course of Na+dependent Ca 2+ efflux. Rat brain PMV were first loaded with 45Ca by Na+/Ca 2÷ exchange. After 5 minutes, various final concentrations of Na + (O zero Na, • 20 mM Na, • 60 mM Na) were used to initiate Na+-dependent Ca2+ effiux. Data represent Ca2+ content measured after the start of efflux. Ca2+ content in Na + loaded PMV diluted into buffer containing 137 mM NaCI and treated similarly to Ca2÷ loaded PMV was subtracted from each determination. 5A, which compares no delay with 4 h of delay. Figure 5B shows the effect of postmortem delay on Ca 2 ÷ release. The rate of release for P M V prepared from control tissues (no delay) appeared to fit well to two components of release (T 1/2fast = 0.443 ----0.542 r a i n - 1, T 1/2slow = 5.78 - 5.61 m i n - 1; mean --+ SD). This was also true of 30- and 60-min delays. However, longer post-
A
B
C
D
E
F
G
STD.
212,000 170,000 116,000 53,000
20,000 6,500 FIG. 4. Electrophoretic separation of rat brain plasma membrane vesicle proteins after various periods of postmortem delay. Twenty p,g protein was applied to each lane. (A) no delay, (B) 30 rain, (C) 60 rain, (D) 90 min, (E) 2 h, (F) 4 h (G) 2 h at 25°C.
378
COLVIN ET AL.
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FIG. 5. Effect of postmortem delay on the time course of Ca2 + accumulation, (A) and passive Ca2 + release (B). Ca 2 ÷ content was determined as described in Fig. 2. • no delay, • 4 h delay. Age-Related Effects on Human Brain Na +/Ca 2 + Exchange Activity and Ca 2 ÷ Permeability To analyze the effects of normal aging, we choose to study brain tissues from individuals who had died at different ages from non-neurological causes and whose brains were unremarkable upon autopsy. These experiments were performed on 15 different tissues from human frontal cortex ranging in age at death from 21 to 93 years. Figure 7 shows the time course of Ca 2 ÷ accumulation due to N a ÷ / C a 2÷ exchange for three frontal cortex tissues with ages at death 22, 44, and 72 years. Figure 7 serves to demonstrate the typical variation shown between different tissues. Notwithstanding such differences in the extent of Ca 2 ÷ accumulation in different tissues, there was no discernible age-related effect on the pattern of Ca 2 ÷ accumulation or the peak uptake. Figure 8A and B show scattergrams of age-related variations in N a ÷ / C a 2÷ exchange V,,~, and K,, for Ca 2 ÷. No significant effect of age was seen on either the K,, or V,,,~,. The mean --- SD values for K,, and V,,,= obtained for all the tissues studied were 41.7 --+ 27.2 IxM and 0.123 --- 0.042 nmol/mg/s, respectively (n = 15). Peak uptake data are plotted against age in Fig. 8C. As expected, from the initial velocity data, there was no significant effect of age on the peak uptake. The mean +-- SD value for peak uptake for all tissues tested was 1.28 --- 0.494 nmol/mg/5 min (n = 12). The time course of Ca 2 + release after addition of 1 mM EGTA was fit to a single exponential decay in all the tissues tested. As Fig. 8D illustrates, there were no significant age related
effects on PMV Ca 2 ÷ permeability. The mean + SD value for T 1/2 for all the tissues tested was 7.93 +- 5.1 min (n = 14). To determine if age-related effects exist on Na n dependent Ca 2 + efflux, 60 mM Na ÷ was added to PMV preloaded with 45Ca2 + and the Ca 2 + content determined 1 min later. This value was then expressed as a percentage of the peak Ca 2÷ content measured before addition of Na ÷. Figure 9 shows Ca 2 ÷ efflux data plotted against age, from 11 tissues with ages at death ranging from 22 to 93. There were no significant age related effects on Na n dependent Ca 2 + efflux, which would be consistent with no change in the Na ÷ affinity of the exchanger with aging. DISCUSSION Perhaps the most important functions of the N a + / C a 2+ exchanger, in terms of cell survival, are Ca 2 ÷ extrusion and the ability to return [Cai] to low resting values after cell activation (1,14,15). The important kinetic characteristics of the N a + / C a 2÷ exchanger that in large p a n determine its ability to maintain low [CaJ are its affinity for Ca 2+ and Na n and its maximum capacity for transporting Ca 2 +. In the present studies, the Vmax and K m for Ca 2 ÷ activation of Ca 2 + accumulation were easily determined by analyzing the effect of increasing Ca 2 ÷ concentrations on the initial velocity of Ca 2+ transport. The affinity for Na n was estimated from the ability of added Na + to promote Ca 2 + efflux from Ca 2 ÷ loaded PMV. Plasma membrane vesicles were isolated from three regions of human brain: frontal cortex, temporal cortex, and
is[ 1 05
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0
~
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FIG. 6. The time course of Ca2 + accumulation (A) and passive Ca 2 ÷ release (B) in different regions of human brain, determined as described in Fig. 2. • frontal cortex, O temporal cortex, • cerebellum.
N a + / C a 2+ E X C H A N G E ACTIVITY AND AGING
379
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FIG. 7. Time course of C a 2 + accumulation in human frontal cortex. Tissues were from individuals whose ages at death were: 0 22 years, • 44 years, and • 72 years.
cerebellum; and Na÷-dependent Ca 2+ accumulation was measured. It was discovered that frontal cortex tissues had larger V,,,~ values than either the temporal cortex or cerebellum. This finding predicts a nonuniform distribution of the Na +/Ca 2 + exchanger in human brain and that different brain regions have different capacities for Ca 2+ extrusion.
Although rat brain PMV and human brain PMV had N a + / Ca 2 + exchange activities that were very similar in terms of their time course of accumulation, rat brain P M V had a three-fold greater capacity for accumulating Ca 2 ÷. The greater capacity for Ca 2+ accumulation in rat brain was not due to lower Ca 2+ permeability as this was shown to be smaller in human brain. The intactness of purified vesicle preparations can have an affect on V , ~ and/or peak uptake values. Vesicle intactness can be determined biochemically by measuring ouabain binding in the presence and absence of alamethicin (2). When this experiment was performed the results showed similar levels of stimulation of ouabain binding by alamethicin in rat brain and human brain. This indicates that vesicle intactness could not explain the difference between rat brain and human brain Na ÷/Ca 2 + exchange activity. The Ca 2÷ accumulating capacity of PMV was apparently directly related to the estimated V,,~ for N a + / C a 2+ exchange, as this value was also three-fold greater in rat brain, whereas the K m for Ca z + activation of Na ÷/Ca 2 ÷ exchange activity was shown to be similar in rat brain and human brain (compare Tables 2 and 3). These findings lead to the interesting conclusion that rat brain has a much greater capacity for Ca 2+ extrusion than human brain. These results suggest that human brain may be less efficient at lowering [Call and be more susceptible to Ca2+-induced neurodegeneration. An analysis of the passive permeability to Ca 2 + of PMV also showed differences between rat brain and human brain. Ca 2+ release from rat brain PMV could be fit to two components of release (fast and slow) whereas human brain PMV appeared to be
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FIG. 8. N a + / C a 2+ exchange (A) V,,,~, (B) K m for C a 2+ , (C) p e a k uptake, and (D) C a 2+ release rate plotted against age at death. There was no correlation (p > 0.05) between age at death and each parameter tested. The R-square for the regression lines were V,,~: 0.0065, Kin: 0.012, Peak uptake: 0.003, and T 1/2: 0.041.
380
COLVIN ET AL,
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AGE ( y e a r s ) FIG. 9. Na + dependent Caz + efflux plotted against age at death. PMV were first loaded with 4SCa as described in Fig. 3. Sixty mM Na + was added to initiate Ca2+ efflux and efflux was stopped 1 min later by addition of 100 ~,M LaC13. The amount of Ca~+ remaining after addition of Na ÷ was expressed as a percentage of Ca2+ remaining after addition of choline. The R-square for the regression line was 0.093.
adequately described by a single exponential decay (slow). In addition, Ca 2+ release (expressed in terms of T 1/2) was faster from rat brain PMV. Using the data obtained in the present studies, it is impossible to define precisely the nature and physiological significance of the fluxes associated with the passive release of Ca 2 ÷. Ca 2 ÷ release could be mediated by any number of ion channel proteins that are known to exist in the plasma membrane. Furthermore, these ion channels may exhibit Ca 2 ÷ permeability only under the unphysiological and depolarized conditions that these assays were performed. It is interesting to note, however, that rat brain PMV with their greater Ca / ÷ permeability also have greater Na÷/Ca 2 ÷ exchange activity and presumably, greater capacity for Ca 2 ÷ extrusion. One artifactual explanation for the greater Na+/Ca 2÷ exchange activity seen in rat brain would be loss of activity due to the postmortem delay of human tissues• Previous studies (3) have shown that Na÷/Ca 2+ exchange activity of human brain PMV was unaffected by long term (5 to 26 h) postmortem delay. However, of greater interest is the period of time immediately after death when changes in brain chemistry are probably occurring most rapidly. To study the effects of short-term postmortem delay on Na ÷/Ca 2 ÷ exchange activity an animal model was used (see Results section). The molecular weights of the major protein species of rat brain PMV were not affected by various lengths of postmortem delay, showing that the integrity and composition of the membrane were maintained. Na+/Ca 2÷ exchange activity showed a remarkable stability during periods of postmortem delay up to 4 h. It has been known for many years that the exchanger is able to function normally even after extensive proteolysis (18). These findings are supported by molecular studies showing that large deletions of portions of the exchanger protein do not affect its transport activity (16,17). Considering these findings, it is not surprising that Na +/Ca 2 + exchange activity is well preserved during postmortem delay. The only measurement of Ca 2 + ion flux across PMV that was affected by short-term postmortem delay was • pass,ve Ca 2 ÷ release. The observed effects were limited to small increases in Ca 2 ÷ permeability and a loss of the fast component of release. These effects were probably not large enough to have significantly affected the analysis of Ca 2 ÷ accumulation due to Na+/Ca 2÷ exchange. It is tempting to speculate that human tis-
sues undergo a similar change in release rate during the first few hours of postmortem delay. However, without data from human tissues with no postmortem delay, such a conclusion is unwarranted. In conclusion, it is important to note that as long as the brain is kept in situ during postmortem delay, surprisingly long postmortem delays can be experienced without compromising the integrity of purified plasma membranes or the ability to measure ion fluxes. The ultimate objective of these studies was to understand how normal aging affects Na +/Ca 2 + exchange activity and Ca 2 + permeability in human brain. To accomplish this goal, brain tissues from individuals with different chronological ages of death were analyzed. The major difficulties in performing these studies were the limited availability of well characterized control tissues with ages at death less than 60 and the large degree of variability seen in human postmortem tissues. These factors make it possible that, when comparing group means, differences might exist but the differences would not reach statistical significance. This may be the case for the release rate in cerebellum or the peak uptake in temporal cortex (see Table 3). Notwithstanding the limited number of tissues that were available, the lack of age-related effects on Na +/Ca 2 ÷ exchange activity and Ca 2 + permeability was convincing after viewing the scattergrams of Figs. 8 and 9. Previous studies using animal models have demonstrated age-related reductions in brain (14) and heart (7,10) Na ÷/Ca 2 ÷ exchange activity. It was suggested by these studies that the efficiency of Ca 2 ÷ extrusion was lower in aged animals and that the reduction in Na÷/Ca 2 + exchange activity therefore had important functional consequences. The present finding that such changes do not occur in human brain are important because they point out that significant differences do exist between human and rodent models used for exploring the neurobiology of aging (6). The results of the present studies must be interpreted with caution. No correlation between Ca 2 + transport and chronological age was found, however, the extent of biological aging of the tissues was unknown. The chronological ages of the brain tissues used did cover the range when cognitive decline is normally seen in human subjects, but the level of cognitive function at the time of death was not determined. Therefore, there was no way to relate chronological with biological age. When an attempt is made to correlate a variable such as Na÷/Ca 2+ exchange activity o r C a 2 + permeability with longevity it can be argued that the study is biased toward a negative result (19). It follows that the more important a variable is to survival and longevity the more likely it is that aged survivors will have maintained this characteristic when compared to their younger counterparts. Therefore, one is justified in interpreting the results of the present study as clearly identifying Na+/Ca 2+ exchange activity and maintenance of low plasma membrane permeability to Ca 2 ÷ as important for longevity. This interpretation has important implications for the " C a 2 + hypothesis of aging". Many studies have suggested that alterations in Ca 2 ÷ homeostasis occur in the aging nervous system (for review see ref. 5) and this has led to the proposal that changes in cellular mechanisms that act to modulate [Ca 2÷ ~] within neurons and glia contribute to the causative factors leading to age-related nervous system dysfunction. Although most researchers would agree that a loss of Ca / ÷ homeostasis is the final common pathway of cell death, the causative role of changes in Ca 2÷ homeostasis for age-related nervous system dysfunction is much more contentious. The results of the present studies do suggest that changes in Na+/Ca 2+ exchange activity and C a 2 + permeability are not good candidates to be causative factors in Ca / ÷ homeostatic changes associated with aging. Rather, we would predict that maintenance of normal levels of Na +/Ca 2 ÷ exchange activity and low plasma membrane permeability to Ca/+ would be associated with longevity. What still
N a + / C a 2+ E X C H A N G E ACTIVITY AND AGING
381
eludes our grasp is evidence of a direct causative nature that would relate the cognitive decline associated with aging with a detriment in a specific aspect of Ca 2+ homeostasis.
ACKNOWLEDGEMENTS This work was supported by a grant from the National Institute of Neurological Disorders and Stroke No. NS30384.
REFERENCES 1. Blaustein, M. P.; Goldman W. F.; Fontana G.; Kruegler, B. K.; Steele, T. D.; Weiss, D. N.; Yarowsky, P. J. Physiological roles of the sodium-calcium exchanger in nerve and muscle. Annal. N.Y. Acad. Sci. 639:254-274; 1991. 2. Colvin, R. A.; Ashavaid, T. F.; Herbette, L. Structure-function studies of canine cardiac sarcolemmal membranes I. Estimation of receptor site densities. Biochem. Biophys. ACTA 812:601-608; 1985. 3. Colvin, R. A.; Bennett, J. W.; Colvin, S. L.; Allen, R. A.; Martinez, J. A.; Miner, G. D. Na+/Ca 2+ exchange activity is increased in Alzheimer's disease brain tissues. Brain Research 543:139-147; 1991. 4. Fabiato, A. Computer programs for calculating total from specific free or free from specific total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 157:378416; 1988. 5. Gibson, G. E.; Peterson, C. Calcium and the aging nervous system. Neurobiol. Aging 8:329-343; 1987. 6. Hazzard, D. G. Relevance of the rodent model to human aging studies. Neurobiol. Aging 12:645--649; 1991. 7. Heyliger, C. E.; Prakash, A. R.; McNeil, J. H. Alterations in membrane Na+-Ca 2+ exchange in the aging myocardium. Age 11:1-6; 1988. 8. Hoel, G.; Michaelis, M. L.; Freed, W. J.; Klienman, J. E. Characterization of Na÷-Ca 2+ exchange activity in plasma membrane vesicles from postmortem human brain. Neurochem. Research 15:881887; 1990. 9. Khachaturian, Z. S. Introduqtion and overview. Annal. N.Y. Acad. Sci. 568:1--4; 1989. 10. Khatter, J. C.; Navarmam, S.; Agbanyo, M. Mechanisms of reduced digitalis tolerance with advancing age in the guinea pig. Biochem. Pharmacol. 40:997-1003; 1990. 11. Laemmli, V. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685; 1970.
12. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, A. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 254-275; 1951. 13. Michaelis, M. L.; Foster, C. T.; Jayawickreme, C. Regulation of calcium levels in brain tissue from adult and aged rats. Mech. Aging Develop. 62:291-306; 1992. 14. Michaelis, M. L.; Johe, E.; Kitos, T. E. Age-dependent alterations in synaptic membrane systems for Ca2 + regulation. Mech. Aging Develop. 25:215-225; 1984. 15. Mattson, M. P.; Guthrie, P. B.; Kater S. B. A role for Na +- dependent Ca2÷ extrusion in protection against neuronal excitotoxicity. FASEB J, 3:2519-2526; 1989. 16. Nicoll, D. A.; Hilgemann, D. W.; Philipson, K. D. A role for the hydrophilic region encompassing amino acid residues 218-737 (Loop F) of the cardiac sarcolemmal Na+-Ca 2+ exchanger. Biophys. J. 61:A387; 1992. 17. Nicoll, D. A.; Longoni, S.; Philipson, K. D. Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca 2+ exchanger. Science 250:562-565; 1990. 18. Philipson, K. D.; Nishimoto, A. Y. Stimulation of Na+/Ca 2+ exchange in cardiac sarcolemmal vesicles by proteinase pretreatment. Am. J. Physiol. 243:C191-C195; 1982. 19. Rogers, J. Efficient experimental design in aging studies. Neurobiol. Aging 12:695-697; 1991. 20. Spokes, E. G. S.; Kock, D. J. Postmortem stability of dopamine, glutamine decarboxylase and acetyltransferase in the mouse brain under conditions simulating the handling of human autopsy material. J, Neurochem. 31:381-383; 1978. 21. Stopa, E. G.; Uhl, G. R.; O'Hara, B. F.; Chorsky, R. L.; King, J. C.; Bird, E. D.; Wolfe, H. Somatostatin-gene expression in the postmortem adult and fetal human brain. Peptide Res. 5:201-205; 1992.
0197-4580/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
Analysis of Na +/Ca 2+ Exchange Activity in Human Brain: The Effect of Normal Aging ROBERT
A. COLVIN, 1 ANFAN
WU, NANCY
DAVIS AND CATHY
A. MURPHY
Department of Biological Sciences, Ohio University College of Osteopathic Medicine, Athens, OH 45701 R e c e i v e d 23 N o v e m b e r 1992; R e v i s e d 8 F e b r u a r y 1993; A c c e p t e d 8 M a r c h 1993 COLVIN, R. A., A. WU, N. DAVIS AND C. A. MURPHY. Analysis of Na +/Cae+ exchange activity in human brain: The effect of normal aging. NEUROBIOL AGING 14(4) 373-381, 1993.--Na+/Ca 2+ exchange activity and passive permeability to Ca2+ were analyzed in plasma membrane vesicles (PMV) purified from whole rat brain and three regions of human brain: frontal cortex, temporal cortex, and cerebellum. Accumulation of Ca2+ due to Na+/Ca 2+ exchange activity showed a characteristic pattern of an initial rapid rise in Ca 2+ content followed by a stable plateau in both rat and human brain. Total Ca2+ accumulation in rat brain PMV was on average three-fold higher than in human brain. Passive permeability to Ca2+ was measured as the rate of Ca2 ÷ release from PMV first loaded with 4SCa by Na+/Ca 2+ exchange and then exposed to 1 mM EGTA. The Ca2+ permeabilities of human and rat brain PMV were similar. Ca 2+ release from rat brain PMV was faster overall and was resolved into fast and slow components while in human brain a single slow component was found. Post mortem delay up to 4 h had no effect on Na+/Ca 2+ exchange Km for Ca 2+ , Vm~,, and peak uptake and Ca2+ release rate in rat brain PMV. Human frontal cortex was shown to have a greater Na+/Ca 2+ exchange activity than that found in the cerebellum. The frontal cortex, temporal cortex and cerebellum had similar Ca 2+ permeabilities. Age-related effects on Na+/Ca 2+ exchange activity and Ca 2+ permeability were determined in 15 tissues from human frontal cortex (age at death 21 to 93 years). No significant age related effects were seen. These data do not rule out the loss of Ca2+ homeostasis as a component of normal brain aging but do indicate that significant changes in Na+/Ca 2÷ exchange activity and Ca 2+ permeability do not accompany normal human brain aging. Ca2 +
Transport
Brain aging
Plasma membrane vesicles
Ca 2+ permeability
Rat brain
Ca2 + efflux
man tissue (3,8). In these studies, the human brain N a + / C a 2+ exchanger was shown to have many properties similar to that seen in animal tissues. Furthermore, Na +/Ca 2 ÷ exchange activity was increased in brain tissues from Alzheimer disease patients when compared with aged matched controls (3). The ability to measure N a + / C a 2÷ exchange activity in human postmortem tissues now allows a more direct test of the Ca 2 ÷ hypothesis of human brain aging to be performed. In the present studies, N a + / C a 2+ exchange activity and the passive permeability to Ca 2 + were determined in PMV isolated from postmortem human brain tissues derived from individuals having various ages at death.
THE " c a l c i u m hypothesis of brain a g i n g " (9) proposes that changes in the cellular mechanisms that act to modulate the concentration of free intracellular calcium ([Cad) within neurons and glia contribute to the causative factors leading to the cognitive decline associated with normal aging. The mechanisms of homeostatic control of [Ca~] in the human central nervous (CNS) system must involve complex interactions between Ca 2÷ binding proteins, Ca 2+ sequestering organelles, and Ca 2 + transport proteins. The Na +/Ca 2 + exchanger is an important Ca 2 + transporting protein in the plasma membranes of neurons and glial cells. This protein generally catalyzes the transport of Na + ions across the plasma membrane in exchange for Ca 2 + ions. Studies of rat hippocampal neurons (15), striatal neurons, and cortical astrocytes in culture (1) have clearly demonstrated the importance of Na + graclient dependent Ca 2+ extrusion for maintaining low [Ca~]. In these studies it was also shown that Na +/Ca 2 + exchange plays an even more important role in Ca 2 + extrusion after cell activation. Comparing rats of different chronological ages, Michaelis and coworkers demonstrated age-related deficits in Ca 2 + homeostatic function in brain synaptosomes (13), which could be explained by a decrease in N a + / C a 2+ exchange activity (14). The decrease in exchange activity was the result of an increase of Kact for Ca 2 + with little or no effect on V,,,,=. Recently, it has been demonstrated that N a + / C a 2+ exchange activity can be measured in purified plasma membrane vesicles (PMV) isolated from postmortem hu-
METHOD
Preparation of Plasma Membrane Vesicles Frozen tissue sections from human brain were obtained from the National Neurological Research Bank (VAMC Wadsworth, Los Angeles, CA). The characteristics of the brain tissues used in this study are listed in Table 1. Neuropathological investigation at autopsy showed in every case grossly normal brain and spinal cord and microscopic sections of the hippocampus and cortex were unremarkable. Frozen ( - 8 0 ° C ) human brain sections and whole rat brains were used for the preparation of P M V as described previously (3).
1 To whom requests for reprints should be addressed. 373
374
COLVIN ET AL, TABLE 1 CHARACTERISTICSOF BRAINTISSUESUSED
Age (years)
Post Mortem Delay (h)
Cause of Death
21 22 22 30 40 41 44 44 45 47 48 50 57 59 65 66 68 71 72 78 93
36.5 15 18 I1 18 16 12 9.5 18.5 18.5 9 13.5 11.5 11 16 15.5 16.5 12.7 11.5 31 10 15.8 -+ 6.61"
multiple injuries multiple injuries multiple injuries drug overdose malignant melanoma auto accident renal carcinoma metastatic adenocarcinoma bronchopneumonia myocardial infarction bronchopneumonia aspiration sepsis myocardial infarction acute respiratory failure pancreatic carcinoma bronchopneumonia bronchopneumonia lung carcinoma empyema myocardial infarction lung carcinoma
* Mean Post-Mortem Delay +-- SD. The crude microsomal fraction was resuspended in Buffer A conmining 0.32 M sucrose, 10 mM HEPES (pH 7.4), and the protease inhibitors- soybean trypsin inhibitor (50 Ixg/ml), leupeptin (0.5 I~g/ml), and pepstatin (0.7 I~g/ml). For human tissues the crude microsomal fraction was layered over an equal volume of 0.6 M sucrose containing protease inhibitors and centrifuged in an S W 41 rotor at 25,000 rpm for 30 min. The pellet obtained was resuspended in Buffer A and layered over a sucrose gradient conmining 0.6 M and 0.8 M sucrose as the steps. The crude microsomal fraction from rat brain was loaded directly on the sucrose step gradient. The gradients were centrifuged in an SW-41 rotor at 25,000 rpm for 2 h. The PMV were recovered from the 0.6--0.8 M interface and resuspended in 10 vol of Buffer A. The resulting suspension was centrifuged at × 100,000 g for 40 min. The pellet was resuspended in Buffer A to a concentration of 10-20 mg/ml and frozen in aliquots in liquid nitrogen. PMV preparations were stored at - 8 0 ° C until used. Membrane protein determinations were performed by the method of Lowry (12) using bovine serum albumin as a standard. Assay of Ca 2 ÷ Accumulation and Release Due to Na ÷~Caz ÷ Exchange Activity
To preload PMV with Na + , PMV were thawed and diluted to 3 mg/ml (human brain) or 2 mg/ml (rat brain) in Buffer B conmining 137 mM NaC1 and 10 mM HEPES (pH 7.4). These concentrations of membrane protein were found to be optimal for measuring Ca / ÷ transport. The PMV were loaded with Na n by incubating 25 i~1 of the membrane suspension at 37°C for 5 min, after which the test tubes were again placed on ice. To initiate a Ca 2+ transport reaction, Na +-loaded PMV were diluted to 0.5 ml by the addition of either Buffer B or Buffer C in which choline chloride was substituted for NaC1. Each buffer contained various concentrations of CaCO3 and 100 t~M EGTA with the addition of either 0.19 mM (initial velocity) or 0.019 mM (time course)
~5CaC12 (NEN). Free Ca ~ ~ ion concentrations in the assay solutions were determined from an iterative computer program using published dissociation constants (4). Mock solutions were prepared and free Ca 2 + ion concentrations were validated using a Ca 2 ÷ ion selective electrode (Philips). Reactions were stopped at various times by the addition of l mM LaCI 3 and placing the tubes on ice. The membrane suspensions on ice were then filtered on a Brandel M-24RP cell harvester using No. 30 glass fiber filters (Schleicher & Schuell). The filters were washed three times with 5 ml of ice-cold wash buffer containing 137 mM choline CI, 10 mM HEPES (pH 7.4), and 1 mM EGTA, placed in 3 ml of Ultima Gold (Packard) and 45Ca trapped on the filter determined in a scintillation counter with nearly 100% counting efficiency. As total Ca 2+ added to the buffers was known (45Ca plus carrier Ca 2 + ), aliquots were counted to determine the specific activity of 4~Ca. Using this value, the counts on the filter were converted to nmol Ca 2 +/mg protein. Na +-dependent Ca 2 ÷ accumulation was determined as the change in Ca 2 + content in Buffer C minus that measured in Buffer B. For determination of Km for Ca 2+ and V . . . . Na+/Ca-' + exchange was initiated and Ca 2 + accumulation allowed to proceed for either 5 or 10 s before being stopped by addition of 1 mM LaCI 3. The free Ca 2 + ion concentrations ranged between 0.54 ~xM and 2571xM. The initial velocity of Na ÷ dependent Ca 2÷ accumulation was expressed as nmol Ca 2 +/mg/s and plotted as a function of free [CaS+]. The data obtained were fit by nonlinear regression to a rectangular hyperbola to obtain Km and V..... . Assay of Na n Dependent Ca 2+ Efflux
PMV were first loaded with 45Ca via Na ÷ dependent Ca 2+ uptake as described above. After 5 min at 37°C a second dilution buffer was added to reach a final volume of 1 ml and initiate Ca / ÷ efflux. The second dilution buffer contained concentrations of Na ÷ and choline such that when added to the reaction mixture the desired concentrations of Na n and choline were obtained. After various times, efflux was stopped by addition of 1 mM LaCI3, placing the tubes on ice, and rapid filtration (see above). The same combinations of Na + and choline were also added to Na + loaded PMV initially diluted into Buffer B. The values obtained for Ca / ÷ content were then subtracted from the Ca / ÷ content measured in PMV initially diluted into Buffer C. Assay o f Passive Ca 2 + Permeability
PMV were first loaded with 4~Ca exactly as described above. After 5 min at 37°C, 1 mM EGTA was added to inhibit Na + ~Caz + exchange and start the measurement of Ca 2 ÷ release. Ca 2 ÷ release was stopped at various times by the addition of 100 I~M LaC13, placing the tubes on ice and rapid filtration (see above). Na ÷ loaded PMV diluted into Buffer B with addition of 1 mM EGTA served as a control (as described above). The data obtained were fit by nonlinear regression to either a single or a double exponential decay. SDS Polyacrylamide Gel Electrophoresis
Protein profiles of PMV from human or rat brain were obtained according to the method of Laemmli (11) on 8% to 18% linear gradient gels (Pharmacia) and stained with silver stain (Bio Rad). Statistics
Computer assisted nonlinear regression (GraphPad Inplot, San Diego, CA) was used for curve fitting. Goodness of fit was quantitated by the least-squares method. Data presented in graphical
Na+/Ca 2+ EXCHANGE ACTIVITY AND AGING
STD.
A
B
C
375 squares analysis was used to estimate age dependent relationships in the data and judged significant when p < 0.05.
D
RESULTS
Comparison of Na +/Ca 2+ Exchange Activity and Passive Ca 2+ Permeability Measured in Rat and Human Brain
212,000 170,000 116,000 53,000
20,000 6,500
FIG. 1. Electrophoretic separation of human and rat brain plasma membrane vesicle proteins on polyacrylamide gel after solubilization in SDS. Twenty p.g protein was applied to each lane. (A) human frontal cortex, (B) human temporal cortex, (C) human cerebellum, and (D) whole rat brain.
format are single experiments representative of data obtained in at least three different preparations of PMV. When appropriate, means were obtained from the data collected and compared using one-way analysis of variance (ANOVA) with Scheff6's method and judged significantly different when p < 0.05. Linear least
Previous studies (3,7) have shown the suitability of postmortem human brain tissues for analysis of Na+/Ca 2÷ exchange activity. In the present studies, initial experiments were aimed at comparing Na ÷/Ca 2 ÷ exchange activity of rat and human brain PMV that were prepared in a similar manner. Figure 1 shows the electrophoretic profile of human and rat brain PMV proteins. The human and rat brain membranes showed nearly identical patterns of protein staining. The most notable difference was a greater abundance of low molecular weight proteins (<20 kDa) in each region of human brain tested (A: frontal cortex, B: temporal cortex, C: cerebellum). Plasma membrane vesicles prepared from either rat brain or human brain showed a characteristic pattern of Ca 2 + accumulation due to Na+/Ca 2+ exchange activity. Initially, there was a rapid rate of Ca 2 ÷ accumulation that began to plateau within 2 to 3 rain. This plateau level of Ca 2÷ accumulation (termed "peak uptake") was stable for up to 10 min after the start of the reaction. Figure 2A shows the time course of Ca 2 ÷ accumulation seen for PMV prepared from either whole rat brain or human frontal cortex. The appearance of the curves were very similar, the major difference being a three-fold higher peak uptake seen with rat brain. Depending on the Ca 2 + accumulating activity of the preparation, the shortest time points that could be reliably measured were 5-10 s. During the first 10 s of the reaction, Ca 2+ accumulation was nearly linear. Therefore, measuring Ca 2 ÷ accumulation at 5 or 10 s was a good estimate of the initial velocity of the reaction. The K m and Vm~ for Ca 2 ÷ accumulation were estimated by measuring the initial velocity at various free Ca 2÷. Consistent with the measurements of peak uptake, Vm,= estimates for rat brain were threefold higher than the estimates for human frontal cortex. On the other hand, Km estimates for rat brain and human frontal cortex did not differ significantly (compare Tables 2 and 3). The plateau phase of Ca 2 + accumulation is a steady state composed of several Ca 2 ÷ fluxes. The Na ÷/Ca 2 ÷ exchanger mediates a small component of Na + dependent Ca 2 ÷ influx and a larger
3
1
f
I
I
100
200
I
I
300 400 SECONDS
I
I
500
600
0
t
0
500 SECONDS
I
]000
FIG. 2. Time course of Ca 2+ accumulation and passive Ca 2+ release. (A) Aliquots (25 ixl) of ( I ) rat brain or (©) human frontal cortex
PMV were preincubated in buffer containing 137 mM NaC1 and then diluted twenty-fold with either the same buffer or buffer containing 137 mM choline chloride; the difference in Ca2+ content representing Na + dependent Ca2+ accumulation. (B) After preloading PMV with 45Ca via Na÷/Ca 2+ exchange, 1 mM EGTA was added to inhibit the exchanger and initiate Ca2+ release. Computer assisted nonlinear regression was used to fit Ca2+ release data to either 2 component (rat brain) or 1 component (human brain) exponential decay.
376
COLVIN ET A L
TABLE 2 THE EFFECT OF SHORT-TERM POST MORTEM DELAY ON PARAMETERSOF Na * Ca2* EXCHANGE Time of Delay Na ÷/Caz+ Parameter Vm~ nmol/ mg/s K m I~M Peak nmol/ mg/5min
Control
30 Min
60 Min
90 Min
2H
4H
2 H (b~ 25°
0.394 -- 0.14 54.8 +_ 42.2
0.345 +-- .081 54.3 - 12.9
0.306 ± 0.025 37.7 ± 8.7
0.308 ±- 0.0760 38.7 +- 3.29
0.298 ± .086 32.9 ± 14.1
0.313 +-- .019 38.3 ±- 14.2
0.262 ± 0.041 32.6 ± 6.29
3.76 +-0.246
4.17 +- 0.305
3.61
±
0.75
3.14 ±
0.159
3.71
---1.19
4.45 ±
1.83
4.35 +-0.936
All values are the mean +- SD, for three separate determinations from three different tissues. Vmax and Kn~ values were determined by measuring Na+-dependent Ca2+ accumulation 5 s after the start of the reaction in the presence of free [Ca2÷ ] ranging from 0.54 to 257 IxM. Peak values were determined by measuring Na ÷-dependent Ca2÷ accumulation 5 min after the start of the reaction in the presence of free [Ca2+ ] equaling 55.2 ~M. component of Ca2+/Ca 2+ exchange during the plateau phase. Ca 2 ÷ release, representing the passive permeability of the PMV to Ca 2÷ also occurs during the plateau phase. This passive Ca 2÷ release is shown in Fig. 2B. PMV were first loaded with Ca 2 ÷ by N a ÷ / C a 2+ exchange, then the N a + / C a 2÷ exchanger was inhibited by addition of 1 mM EGTA. The Ca 2 ÷ release that resulted was due to the passive permeability of the PMV to Ca 2 ÷. In contrast to Ca 2+ accumulation, comparison of Ca 2 + release data for rat and human brain P M V revealed important differences (see Fig. 2B). Ca 2 ÷ release from rat brain PMV in this representative experiment was resolved into a fast (T 1/2 = 0.212 m i n - ~) and a slow (T 1/2 = 1.13 min - 1 ) component by nonlinear regression. However, Ca 2 ÷ release from human brain PMV demonstrated only the slow component of release (T 1/2 = 8.87 m i n - ~ ) . The computer generated lines of Fig. 2B were extrapolated beyond the actual data points to demonstrate that both curves approached zero. The time course of Ca 2÷ release was different when increasing extravesicular Na ÷ was used to initiate Ca 2 + efflux. In Fig. 3 is shown the effect of diluting P M V preloaded with Ca 2÷ by N a + / Ca 2 + exchange into solutions containing either zero, 20 mM, or 60 mM Na ÷ . Under these conditions, reversal of N a ÷ / C a 2+ exchange mediates a rapid but transient Ca 2 ÷ release whose magnitude was dependent on the concentration of Na ÷ used. One minute after the reversal of N a + / C a 2÷ exchange was initiated, Ca 2 + content reached an apparent steady state. This property of Na ÷ induced Ca 2 ÷ release differed markedly from passive Ca 2 ÷ release, which showed a steady decline in Ca 2 ÷ content with time. Effect o f Postmortem Delay on N a +/Ca e + Exchange
The differences observed in N a + / C a 2+ exchange activity between rat and human brain, particularly the large difference in
peak uptakes, might be the result of postmortem delay in the human brain tissues. No effect of long term (i.e., 5-26 h) postmortem delay on Na ÷/Ca 2 ÷ exchange activity was seen in previous studies (3,8). However, the period immediately following death (0-4 h) may be the most critical for producing membrane proteolysis, lipolysis, and artifactual changes in N a + / C a 2+ exchange activity and Ca 2÷ permeability. Animal models can be used to analyze the effects of postmortem delay on biochemical processes. Human brain cooling curves have been generated and it has been shown that the cooling curves generated can be reproduced in animals by immersion of the head in a cooled incubator following decapitation (20,21). Cadavers are normally placed in a refrigerator at 4°C within 4 h of death. To mimic these conditions, rat brains also were incubated for 2 h at 25°C. Rats (aged 6 months) were decapitated and the intact skulls were incubated for various times (0, 30, 60, 90 min; 2 and 4 h) at 0-4°C and for 2 h at 25°C. After the incubation was complete, the brain tissue was carefully removed from the skull, quick frozen in liquid N 2 and then stored frozen ( - 80°C) for later use. The frozen rat brain tissues were then processed into PMV as described in the Method section. To determine the effect of postmortem delay on the electrophoretic profile of plasma membrane proteins, S D S - P A G E was performed on each membrane preparation (see Fig. 4). Each lane showed nearly identical patterns of protein staining. There was no evidence of postmortem effects on the molecular weight of the major protein species associated with purified PMV. Table 2 lists means +-- SD obtained for N a + / C a 2÷ exchange K m for Ca 2÷ , Vm~x, and peak uptake after different times and conditions of postmortem delay. There was no significant effect of postmortem delay on these measurements of Na +/Ca 2 ÷ exchange activity. The lack of an effect of postmortem delay also was reflected in the time course of Ca 2 + accumulation as shown in Fig.
TABLE 3 COMPARISON OF Ca2÷ TRANSPORT IN PMV PURIFIED FROM DIFFERENT REGIONS OF HUMAN BRAIN Brain Regions
Age (years)
Vmax (nmoldmg/s)
K m (~M)
Peak nmol/mg/5 min
T 1/2 rain
Frontal cortex Temporal cortex Cerebellum
48.3 --- 21.0 (15) 47.6 ±- 8.16 (8) 57.7 --- 18.9 (12)
0.123 --- 0.042 (15) 0.088 - 0.054 (8) 0.058 --- 0.029 (12)*
41.7 ±- 27.2 (15) 28.2 --- 16.5 (8) 27.5 +- 27.6 (12)
1.28 ±- 0.494 (12) 1.39 +- 0.295 (4) 0.997 --- 0.525 (12)
7.93 - 5.10 (14) 5.45 +- 2.83 (4) 3.64 -+ 4.13 (12)
All values are the mean +- SD. Vmax and I ~ values were determined by measuring Na+-dependent Ca 2+ accumulation at either 5 s (frontal cortex, temporal cortex) or 10 s (cerebellum) after the start of the reaction in the presence of free [Caz+] ranging from 0.54 to 257 ~M. Peak values were determined by measuring Na + -dependent Caz + accumulation 5 min after the start of the reaction in the presence of free [Ca2 + ] equaling 55.2 izM. Release data fit well to a single component fit and were not analyzed for a two component fit. * Significantly different from frontal cortex p < 0.05.
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mortem delays resulted in a loss of the fast component of release (T 1/2 = 4.31 - 1.39 r a i n - l ; mean - SD). Additionally, the T 1/2 for the slow component showed a small gradual decrease indicating an increase in release rate.
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Next, N a + / C a 2+ exchange activity was assayed using P M V purified from temporal cortex and cerebellar regions of human brain and compared with frontal cortex. For each brain region, the time course of Ca 2 + accumulation is shown in Fig. 6A and calcium release in Fig. 6B. Table 3 lists the average cumulative data for all tissues assayed. The pattern of Ca 2 + accumulation resulting from N a + / C a 2 + exchange activity was very similar in all three brain regions. Table 3 shows that the mean V,n,~ values obtained for temporal cortex and cerebellum were less than those obtained for frontal cortex. The mean value for cerebellum was significantly (p < 0.05) less than that in frontal cortex. There were no significant differences seen in the mean values of K m for Ca 2 + and peak uptake obtained for each brain region. These findings indicated that PMV from cerebellum had a slower rate of Ca 2 + accumulation but still could accumulate nearly the same total amount of Ca 2 + after 5 min. Figure 6B shows that similar patterns of passive Ca 2 + release were seen for each brain region. In all three brain regions, only the slow component of Ca 2 + release was seen. Cumulative data are shown in Table 3, which shows that the mean release rate of cerebellar PMV was approximately one half that seen in frontal cortex. This difference was not significant (p = 0.068). These data provided evidence to suggest that P M V from different brain regions may differ in their Ca 2 + permeability as well.
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FIG. 3. Time course of Na+dependent Ca 2+ efflux. Rat brain PMV were first loaded with 45Ca by Na+/Ca 2÷ exchange. After 5 minutes, various final concentrations of Na + (O zero Na, • 20 mM Na, • 60 mM Na) were used to initiate Na+-dependent Ca2+ effiux. Data represent Ca2+ content measured after the start of efflux. Ca2+ content in Na + loaded PMV diluted into buffer containing 137 mM NaCI and treated similarly to Ca2÷ loaded PMV was subtracted from each determination. 5A, which compares no delay with 4 h of delay. Figure 5B shows the effect of postmortem delay on Ca 2 ÷ release. The rate of release for P M V prepared from control tissues (no delay) appeared to fit well to two components of release (T 1/2fast = 0.443 ----0.542 r a i n - 1, T 1/2slow = 5.78 - 5.61 m i n - 1; mean --+ SD). This was also true of 30- and 60-min delays. However, longer post-
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20,000 6,500 FIG. 4. Electrophoretic separation of rat brain plasma membrane vesicle proteins after various periods of postmortem delay. Twenty p,g protein was applied to each lane. (A) no delay, (B) 30 rain, (C) 60 rain, (D) 90 min, (E) 2 h, (F) 4 h (G) 2 h at 25°C.
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FIG. 5. Effect of postmortem delay on the time course of Ca2 + accumulation, (A) and passive Ca2 + release (B). Ca 2 ÷ content was determined as described in Fig. 2. • no delay, • 4 h delay. Age-Related Effects on Human Brain Na +/Ca 2 + Exchange Activity and Ca 2 ÷ Permeability To analyze the effects of normal aging, we choose to study brain tissues from individuals who had died at different ages from non-neurological causes and whose brains were unremarkable upon autopsy. These experiments were performed on 15 different tissues from human frontal cortex ranging in age at death from 21 to 93 years. Figure 7 shows the time course of Ca 2 ÷ accumulation due to N a ÷ / C a 2÷ exchange for three frontal cortex tissues with ages at death 22, 44, and 72 years. Figure 7 serves to demonstrate the typical variation shown between different tissues. Notwithstanding such differences in the extent of Ca 2 ÷ accumulation in different tissues, there was no discernible age-related effect on the pattern of Ca 2 ÷ accumulation or the peak uptake. Figure 8A and B show scattergrams of age-related variations in N a ÷ / C a 2÷ exchange V,,~, and K,, for Ca 2 ÷. No significant effect of age was seen on either the K,, or V,,,~,. The mean --- SD values for K,, and V,,,= obtained for all the tissues studied were 41.7 --+ 27.2 IxM and 0.123 --- 0.042 nmol/mg/s, respectively (n = 15). Peak uptake data are plotted against age in Fig. 8C. As expected, from the initial velocity data, there was no significant effect of age on the peak uptake. The mean +-- SD value for peak uptake for all tissues tested was 1.28 --- 0.494 nmol/mg/5 min (n = 12). The time course of Ca 2 + release after addition of 1 mM EGTA was fit to a single exponential decay in all the tissues tested. As Fig. 8D illustrates, there were no significant age related
effects on PMV Ca 2 ÷ permeability. The mean + SD value for T 1/2 for all the tissues tested was 7.93 +- 5.1 min (n = 14). To determine if age-related effects exist on Na n dependent Ca 2 + efflux, 60 mM Na ÷ was added to PMV preloaded with 45Ca2 + and the Ca 2 + content determined 1 min later. This value was then expressed as a percentage of the peak Ca 2÷ content measured before addition of Na ÷. Figure 9 shows Ca 2 ÷ efflux data plotted against age, from 11 tissues with ages at death ranging from 22 to 93. There were no significant age related effects on Na n dependent Ca 2 + efflux, which would be consistent with no change in the Na ÷ affinity of the exchanger with aging. DISCUSSION Perhaps the most important functions of the N a + / C a 2+ exchanger, in terms of cell survival, are Ca 2 ÷ extrusion and the ability to return [Cai] to low resting values after cell activation (1,14,15). The important kinetic characteristics of the N a + / C a 2÷ exchanger that in large p a n determine its ability to maintain low [CaJ are its affinity for Ca 2+ and Na n and its maximum capacity for transporting Ca 2 +. In the present studies, the Vmax and K m for Ca 2 ÷ activation of Ca 2 + accumulation were easily determined by analyzing the effect of increasing Ca 2 ÷ concentrations on the initial velocity of Ca 2+ transport. The affinity for Na n was estimated from the ability of added Na + to promote Ca 2 + efflux from Ca 2 ÷ loaded PMV. Plasma membrane vesicles were isolated from three regions of human brain: frontal cortex, temporal cortex, and
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N a + / C a 2+ E X C H A N G E ACTIVITY AND AGING
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cerebellum; and Na÷-dependent Ca 2+ accumulation was measured. It was discovered that frontal cortex tissues had larger V,,,~ values than either the temporal cortex or cerebellum. This finding predicts a nonuniform distribution of the Na +/Ca 2 + exchanger in human brain and that different brain regions have different capacities for Ca 2+ extrusion.
Although rat brain PMV and human brain PMV had N a + / Ca 2 + exchange activities that were very similar in terms of their time course of accumulation, rat brain P M V had a three-fold greater capacity for accumulating Ca 2 ÷. The greater capacity for Ca 2+ accumulation in rat brain was not due to lower Ca 2+ permeability as this was shown to be smaller in human brain. The intactness of purified vesicle preparations can have an affect on V , ~ and/or peak uptake values. Vesicle intactness can be determined biochemically by measuring ouabain binding in the presence and absence of alamethicin (2). When this experiment was performed the results showed similar levels of stimulation of ouabain binding by alamethicin in rat brain and human brain. This indicates that vesicle intactness could not explain the difference between rat brain and human brain Na ÷/Ca 2 + exchange activity. The Ca 2÷ accumulating capacity of PMV was apparently directly related to the estimated V,,~ for N a + / C a 2+ exchange, as this value was also three-fold greater in rat brain, whereas the K m for Ca z + activation of Na ÷/Ca 2 ÷ exchange activity was shown to be similar in rat brain and human brain (compare Tables 2 and 3). These findings lead to the interesting conclusion that rat brain has a much greater capacity for Ca 2+ extrusion than human brain. These results suggest that human brain may be less efficient at lowering [Call and be more susceptible to Ca2+-induced neurodegeneration. An analysis of the passive permeability to Ca 2 + of PMV also showed differences between rat brain and human brain. Ca 2+ release from rat brain PMV could be fit to two components of release (fast and slow) whereas human brain PMV appeared to be
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adequately described by a single exponential decay (slow). In addition, Ca 2+ release (expressed in terms of T 1/2) was faster from rat brain PMV. Using the data obtained in the present studies, it is impossible to define precisely the nature and physiological significance of the fluxes associated with the passive release of Ca 2 ÷. Ca 2 ÷ release could be mediated by any number of ion channel proteins that are known to exist in the plasma membrane. Furthermore, these ion channels may exhibit Ca 2 ÷ permeability only under the unphysiological and depolarized conditions that these assays were performed. It is interesting to note, however, that rat brain PMV with their greater Ca / ÷ permeability also have greater Na÷/Ca 2 ÷ exchange activity and presumably, greater capacity for Ca 2 ÷ extrusion. One artifactual explanation for the greater Na+/Ca 2÷ exchange activity seen in rat brain would be loss of activity due to the postmortem delay of human tissues• Previous studies (3) have shown that Na÷/Ca 2+ exchange activity of human brain PMV was unaffected by long term (5 to 26 h) postmortem delay. However, of greater interest is the period of time immediately after death when changes in brain chemistry are probably occurring most rapidly. To study the effects of short-term postmortem delay on Na ÷/Ca 2 ÷ exchange activity an animal model was used (see Results section). The molecular weights of the major protein species of rat brain PMV were not affected by various lengths of postmortem delay, showing that the integrity and composition of the membrane were maintained. Na+/Ca 2÷ exchange activity showed a remarkable stability during periods of postmortem delay up to 4 h. It has been known for many years that the exchanger is able to function normally even after extensive proteolysis (18). These findings are supported by molecular studies showing that large deletions of portions of the exchanger protein do not affect its transport activity (16,17). Considering these findings, it is not surprising that Na +/Ca 2 + exchange activity is well preserved during postmortem delay. The only measurement of Ca 2 + ion flux across PMV that was affected by short-term postmortem delay was • pass,ve Ca 2 ÷ release. The observed effects were limited to small increases in Ca 2 ÷ permeability and a loss of the fast component of release. These effects were probably not large enough to have significantly affected the analysis of Ca 2 ÷ accumulation due to Na+/Ca 2÷ exchange. It is tempting to speculate that human tis-
sues undergo a similar change in release rate during the first few hours of postmortem delay. However, without data from human tissues with no postmortem delay, such a conclusion is unwarranted. In conclusion, it is important to note that as long as the brain is kept in situ during postmortem delay, surprisingly long postmortem delays can be experienced without compromising the integrity of purified plasma membranes or the ability to measure ion fluxes. The ultimate objective of these studies was to understand how normal aging affects Na +/Ca 2 + exchange activity and Ca 2 + permeability in human brain. To accomplish this goal, brain tissues from individuals with different chronological ages of death were analyzed. The major difficulties in performing these studies were the limited availability of well characterized control tissues with ages at death less than 60 and the large degree of variability seen in human postmortem tissues. These factors make it possible that, when comparing group means, differences might exist but the differences would not reach statistical significance. This may be the case for the release rate in cerebellum or the peak uptake in temporal cortex (see Table 3). Notwithstanding the limited number of tissues that were available, the lack of age-related effects on Na +/Ca 2 ÷ exchange activity and Ca 2 + permeability was convincing after viewing the scattergrams of Figs. 8 and 9. Previous studies using animal models have demonstrated age-related reductions in brain (14) and heart (7,10) Na ÷/Ca 2 ÷ exchange activity. It was suggested by these studies that the efficiency of Ca 2 ÷ extrusion was lower in aged animals and that the reduction in Na÷/Ca 2 + exchange activity therefore had important functional consequences. The present finding that such changes do not occur in human brain are important because they point out that significant differences do exist between human and rodent models used for exploring the neurobiology of aging (6). The results of the present studies must be interpreted with caution. No correlation between Ca 2 + transport and chronological age was found, however, the extent of biological aging of the tissues was unknown. The chronological ages of the brain tissues used did cover the range when cognitive decline is normally seen in human subjects, but the level of cognitive function at the time of death was not determined. Therefore, there was no way to relate chronological with biological age. When an attempt is made to correlate a variable such as Na÷/Ca 2+ exchange activity o r C a 2 + permeability with longevity it can be argued that the study is biased toward a negative result (19). It follows that the more important a variable is to survival and longevity the more likely it is that aged survivors will have maintained this characteristic when compared to their younger counterparts. Therefore, one is justified in interpreting the results of the present study as clearly identifying Na+/Ca 2+ exchange activity and maintenance of low plasma membrane permeability to Ca 2 ÷ as important for longevity. This interpretation has important implications for the " C a 2 + hypothesis of aging". Many studies have suggested that alterations in Ca 2 ÷ homeostasis occur in the aging nervous system (for review see ref. 5) and this has led to the proposal that changes in cellular mechanisms that act to modulate [Ca 2÷ ~] within neurons and glia contribute to the causative factors leading to age-related nervous system dysfunction. Although most researchers would agree that a loss of Ca / ÷ homeostasis is the final common pathway of cell death, the causative role of changes in Ca 2÷ homeostasis for age-related nervous system dysfunction is much more contentious. The results of the present studies do suggest that changes in Na+/Ca 2+ exchange activity and C a 2 + permeability are not good candidates to be causative factors in Ca / ÷ homeostatic changes associated with aging. Rather, we would predict that maintenance of normal levels of Na +/Ca 2 ÷ exchange activity and low plasma membrane permeability to Ca/+ would be associated with longevity. What still
N a + / C a 2+ E X C H A N G E ACTIVITY AND AGING
381
eludes our grasp is evidence of a direct causative nature that would relate the cognitive decline associated with aging with a detriment in a specific aspect of Ca 2+ homeostasis.
ACKNOWLEDGEMENTS This work was supported by a grant from the National Institute of Neurological Disorders and Stroke No. NS30384.
REFERENCES 1. Blaustein, M. P.; Goldman W. F.; Fontana G.; Kruegler, B. K.; Steele, T. D.; Weiss, D. N.; Yarowsky, P. J. Physiological roles of the sodium-calcium exchanger in nerve and muscle. Annal. N.Y. Acad. Sci. 639:254-274; 1991. 2. Colvin, R. A.; Ashavaid, T. F.; Herbette, L. Structure-function studies of canine cardiac sarcolemmal membranes I. Estimation of receptor site densities. Biochem. Biophys. ACTA 812:601-608; 1985. 3. Colvin, R. A.; Bennett, J. W.; Colvin, S. L.; Allen, R. A.; Martinez, J. A.; Miner, G. D. Na+/Ca 2+ exchange activity is increased in Alzheimer's disease brain tissues. Brain Research 543:139-147; 1991. 4. Fabiato, A. Computer programs for calculating total from specific free or free from specific total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 157:378416; 1988. 5. Gibson, G. E.; Peterson, C. Calcium and the aging nervous system. Neurobiol. Aging 8:329-343; 1987. 6. Hazzard, D. G. Relevance of the rodent model to human aging studies. Neurobiol. Aging 12:645--649; 1991. 7. Heyliger, C. E.; Prakash, A. R.; McNeil, J. H. Alterations in membrane Na+-Ca 2+ exchange in the aging myocardium. Age 11:1-6; 1988. 8. Hoel, G.; Michaelis, M. L.; Freed, W. J.; Klienman, J. E. Characterization of Na÷-Ca 2+ exchange activity in plasma membrane vesicles from postmortem human brain. Neurochem. Research 15:881887; 1990. 9. Khachaturian, Z. S. Introduqtion and overview. Annal. N.Y. Acad. Sci. 568:1--4; 1989. 10. Khatter, J. C.; Navarmam, S.; Agbanyo, M. Mechanisms of reduced digitalis tolerance with advancing age in the guinea pig. Biochem. Pharmacol. 40:997-1003; 1990. 11. Laemmli, V. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685; 1970.
12. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, A. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 254-275; 1951. 13. Michaelis, M. L.; Foster, C. T.; Jayawickreme, C. Regulation of calcium levels in brain tissue from adult and aged rats. Mech. Aging Develop. 62:291-306; 1992. 14. Michaelis, M. L.; Johe, E.; Kitos, T. E. Age-dependent alterations in synaptic membrane systems for Ca2 + regulation. Mech. Aging Develop. 25:215-225; 1984. 15. Mattson, M. P.; Guthrie, P. B.; Kater S. B. A role for Na +- dependent Ca2÷ extrusion in protection against neuronal excitotoxicity. FASEB J, 3:2519-2526; 1989. 16. Nicoll, D. A.; Hilgemann, D. W.; Philipson, K. D. A role for the hydrophilic region encompassing amino acid residues 218-737 (Loop F) of the cardiac sarcolemmal Na+-Ca 2+ exchanger. Biophys. J. 61:A387; 1992. 17. Nicoll, D. A.; Longoni, S.; Philipson, K. D. Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca 2+ exchanger. Science 250:562-565; 1990. 18. Philipson, K. D.; Nishimoto, A. Y. Stimulation of Na+/Ca 2+ exchange in cardiac sarcolemmal vesicles by proteinase pretreatment. Am. J. Physiol. 243:C191-C195; 1982. 19. Rogers, J. Efficient experimental design in aging studies. Neurobiol. Aging 12:695-697; 1991. 20. Spokes, E. G. S.; Kock, D. J. Postmortem stability of dopamine, glutamine decarboxylase and acetyltransferase in the mouse brain under conditions simulating the handling of human autopsy material. J, Neurochem. 31:381-383; 1978. 21. Stopa, E. G.; Uhl, G. R.; O'Hara, B. F.; Chorsky, R. L.; King, J. C.; Bird, E. D.; Wolfe, H. Somatostatin-gene expression in the postmortem adult and fetal human brain. Peptide Res. 5:201-205; 1992.