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Ca2+ exchange activity is increased in Alzheimer's disease brain tissues
Brain Research, 543 (1991) 139-147
139
Elsevier BRES 16406
Na+/Ca 2 + exchange activity is increased in Alzheimer's disease brain tissues Robert A. Colvin 1, Jonathan W. Bennett 1, Sharon L. Colvin 1, Richard A. Allen 1, John Martinez I and Gary D. Miner 2 1Department of Zoological and Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, OH 45701 (U.S.A.) and 2The Alzheimer's Foundation, Tulsa, OK 74137 (U.S.A.) (Accepted 2 October 1990)
Key words: Sodium/calcium exchange; Ion transport; Plasma membrane; Alzheimer's disease; Neurodegenerative disease; Human post-mortem study
These studies were performed to determine the changes that occur in Na+/Ca2÷ exchange activity in Alzheimer's disease (AD) brain tissues. Cerebral plasma membrane vesicles were purified by sucrose density gradient centrifugation from frozen postmortem hippocampal/temporal cortex tissue slices derived from age matched brains of normal, AD and non-Alzheimer dementia (NAD) origin (autopsy confirmed). Membrane marker assays (Na/K ATPase, muscarinic receptor, cytochrome c oxidase) revealed no change in membrane purity across different preparations. Thin-section electron microscopy revealed predominantly intact unilamellar vesicles. Vesicles were preincubated for 15 min (37 °C) in buffer containing 132 mM NaCI, 5 mM KCI, 1.3 mM MgCI2, 10 mM glucose and 10 mM HEPES (pH 7.4). Ca2÷ uptake was initiated by diluting vesicles 20-fold with buffer containing either 132 mM NaC1 or 132 mM choline chloride and 4SCaCl2 then terminated by addition of 200/zM LaC!3 and rapid filtration. Ca2+ content increased rapidly at first and then maintained a steady plateau for up to 5 min. When the Ca2÷ ionophore A23187 (10 ~M) with 100/.tM EGTA was added after 4 min, Ca2÷ content was reduced to 10% of its original value. Ruthenium red (10/~M) had no effect on Ca2÷ content. Na+-dependent Ca2÷ uptake (Ca2+ content measured in choline chloride minus that measured in NaCI) was increased in AD brains as evidenced by both an increase in the initial rise in Ca2÷ content and in elevated values of peak plateau Ca2+ content. The Kmand Vr,ax for Na+/Ca2+ exchange was estimated from Lineweaver-Burk analysis of the effect of increasing extravesicular concentrations of Ca2÷ on Nat-dependent Ca2÷ uptake after 30 s. The values obtained for the Km (uM) and VmaX (nmol/mg/min) were (respectively): normal (n = 6) 57.9 + 28.1 and 4.45 _+ 0.58; AD (n = 6) 71.2 + 29.3 and 6.68 + 1.58; NAD (n = 4) 66.1 + 3.3 and 3.76 + 1.50 (mean + S.D.). The Vm~x for Na+/Ca2÷ exchange in AD brain tissues was significantly (P < 0.05) elevated when compared with normal or NAD brains. There was no correlation evident in either normal, AD, NAD or the total sample population between Na+/Ca2+ exchange Vm~xor Kr, and autolysis time or age at death. The results suggest that in brain regions suffering the greatest degeneration due to AD the surviving neurons have increased Na+/Ca2+ exchange activity. This increase in Na+/Ca2÷ exchange activity may provide clues to a better understanding of the pathogenesis of nerve cell degeneration in AD. INTRODUCTION The mechanisms of neuronal cell dysfunction and death that underlie normal aging and the h u m a n neurodegenerative diseases (e.g. Parkinson's, H u n t i n g t o n ' s and Alzheimer's diseases) are still largely unknown. Similarly, very little is understood concerning the cellular defense mechanisms that act to prevent or reverse potentially lethal changes in n e u r o n a l structure and function occurring in aging and degenerative disease states. The 'calcium hypothesis of brain aging '13 proposes that cellular mechanisms that act to modulate the concentration of free intracellular calcium ([Cai]) play a critical role in brain aging and that changes in [Ca d of short or long duration contribute to the causative factors accounting for cell dysfunction and degeneration.
A n extensive body of literature suggests that neuronal Ca 2÷ metabolism is affected by aging (for review see ref. 9) and it is now well established that increases in [Cai] caused by overstimulation by excitatory amino acids are a direct causative factor in ischemia-induced n e u r o n a l death 6"12'18'29'30. The evidence relating excitotoxicity and changes in [Cai] to the neurodegenerative processes occurring in Alzheimer's disease ( A D ) , is still indirect and inconclusive. Although the large pyramidal neurons of the cortex and hippocampus, which are k n o w n to degenerate in A_D, are thought to have a high density of excitatory amino acid receptors 7, when tissues are assayed for excitatory amino acid receptor binding site density the results are conflicting24. Some studies show no change 8 while others observe decreases in selective regions of the A D brain 2A6,26,3°-33 or a loss of glycine-
Correspondence: R.A. Colvin, Department of Zoological and Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, OH 45701, U.S.A. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
140 dependent binding 29,35. Peterson and c o - w o r k e r s 27"2~, using skin fibroblasts obtained from AD patients, have shown that these cells demonstrate higher levels of bound calcium but lower levels of [Cai] when compared with young and normal aged donors. It is clear that any mechanisms underlying changes in cellular Ca 2+ metabolism associated with A D must involve complex interactions between Ca 2+ binding proteins, Ca 2+ sequestering organelles and Ca 2+ transport proteins such as the Na+/Ca 2+ exchanger. Na+/Ca 2+ exchange is found in high density in the plasma membranes of excitable cells in such tissues as heart and nerve. This protein generally catalyzes the transport of sodium ions across the plasma membrane in exchange for calcium ions and the exchange process is reversible. Na+/Ca 2+ exchange is electrogenic as 3 sodium ions are exchanged for each calcium ion 5. The presumed role of Na+/Ca 2+ exchange is to maintain the resting concentration of [Ca d at submicromolar levels by pumping excess Ca 2+ out of the cell. Hence, the theory that Na+/Ca z+ exchange plays an important role in protecting neurons from CaZ+-induced damages after excitatory amino acid overstimulation has been proposed TM. Na+/Ca 2÷ exchange in the nervous system was first demonstrated in the squid giant axon 3. The direct measurement of Na+/Ca 2+ exchange in the mammalian nervous system is most often accomplished by the preparation of synaptosomes (pinched-off nerve terminals) and the analysis of the effects of sodium ions on calcium ion influx and effiux using radioactive Ca 2+4. Na+-dependent Ca 2÷ uptake has also been studied in purified synaptic plasma membranes 1°'11'2°'21. Studies of the changes in Na+/Ca 2+ exchange activity in synaptic plasma membranes from the aging rat brain have been reported by Michaelis and co-workers 22'23. They found lower Na+/Ca 2+ exchange activity in aged rats, which could be explained by an increase in the Kact for C a 2+ with little or no effect on Vmax. An increase in resting [Cai] and increased responses to agents that elevate [Cai] (e.g. KCI depolarization) were also observed in intact synaptosomes from aged rat brain 23. In the present study we tested the hypothesis that differences exist in the Na+/Ca 2+ exchange activity measured in cerebral plasma membrane vesicles purified from human post-mortem brain tissues of normal, AD and non-Alzheimer's dementia origin. We report that Na+/Ca 2+ exchange activity is significantly elevated in the plasma membranes purified from AD brain tissue. The results suggest that the plasma membranes of surviving neurons in brain areas suffering the greatest neural degeneration due to AD have elevated Na+/Ca 2+ exchange activity. This increase in Na+/Ca 2+ exchange activity may provide clues to a better understanding of
the pathogenesis of nerve cell degeneration in AD. MATERIALS AND METHODS
Preparation of cerebral plasma membrane vesicles Tissue sections from the frontal cortex and hippocampus/temporal cortex regions of human brain were obtained from the National Neurological Research Bank, VAMC Wadsworth, Los Angeles, CA 90083. The clinical diagnosis of Alzheimer's disease was confirmed by pathological diagnosis at the time of autopsy. It was not determined whether Alzheimer's disease patients were of familial or non-familial types. The non-Alzheimer's dementia patients were individuals that clinically presented with progressive dementia or chronic dementia of undetermined origin and upon neuropathological investigation showed no changes characteristic of Alzheimer's disease. Normals were individuals without clinical presentation of dementia at the time of death. Neuropathological investigation at autopsy showed in every case grossly normal brain and spinal cord and microscopic sections of the hippocampus and cortex were unremarkable. The cause of death of the normal patients was non-neurological. Frozen brain sections (-70 °C) were thawed, and the tissue was rapidly dissected in an ice bath. Homogenization of the tissue was performed in 20 vols. of buffer A containing 0.32 M sucrose, 10 mM HEPES (pH 7.4) and the protease inhibitors: soybean trypsin inhibitor (50/~g/ml), leupeptin (0.5/~g/ml) and pepstatin (0.7/~g/ml) utilizing a Brinkmann Polytron at a setting of 4 (10 s, repeated 3-4 times). The homogenate was then centrifuged at 1000 g for 10 min. The resulting supernatant fluid was centrifuged at 17,000 g for 20 min. The pellet was resuspended in buffer A and carefully layered over a sucrose step gradient in a SW-41 centrifuge tube. The step gradient contained 4 ml each of 0.8 M and 0.6 M sucrose containing the protease inhibitors described above. The tubes were spun in a SW-41 rotor at 25,000 rpm for 2 h. The membrane material at the 0.6 M-0.8 M interface was recovered using a Pasteur pipette and resuspended in 10 vols. of buffer A. The resulting suspension was centrifuged at 100,000 g for 40 rain. The pellet was resuspended in buffer A to a concentration of 10-20 mg/ml and frozen in aliquots in liquid N 2. Membrane preparations were stored at -70 °C until used. Membrane protein determinations were performed by the method of Lowry ~5 using bovine serum albumin as a standard.
Assay of Na/Ca exchange activity Cerebral plasma membrane vesicles were thawed, diluted to 2 mg/ml in buffer B containing 132 mM NaC1, 5 mM KCI, 1.3 mM MgCI z, 10 mM glucose, 10 mM HEPES (pH 7.4) and then kept on ice until use. In order to load the vesicles with Na +, 25/A of the membrane suspension was added to test tubes incubating in a 37 °C water bath. After 15 min at 37 °C, the membrane suspension was diluted to 0.5 ml by the addition of either buffer B or buffer C in which choline chloride was substituted for NaCI. Each buffer contained various concentrations of CaCO 3 with the addition of 45CAC12 (0.01 mCi/ml). Reactions were stopped by the addition of 200/~M LaCI 3 and placing the tubes on ice. Zero time points were determined by addition of 200 ~M LaCI 3 to Na+-loaded vesicles before the addition of either buffer B or buffer C. The membrane suspensions on ice were then filtered on a Brandel M-24RP Cell Harvester using No. 30 glass fiber filters (Schleicher and Schuell). The filters were washed 3 x with 5 ml of buffer D containing 132 mM NaCI, 5 mM KCI, 200~M LaCI 3 and 10 mM Hepes (pH 7.4), placed in 3 ml of Biofluor and radioactivity determined in a liquid scintillation counter. Na+-dependent Ca 2+ uptake was defined as the change in Ca 2÷ content in buffer C minus that measured in buffer B.
Electron microscopy Cerebral plasma membrane vesicles in buffer B were diluted to approximately 0.05 mg/ml and then centrifuged in a SW-27 rotor for 30 min. The buffer was decanted and the primary fixative (4%
141 glutaraldehyde and 1% tannic acid in 0.1 M sodium cacodylate buffer) was gently pipetted over the pellet. Following fixation for 2 h at 4 °C the pellets were rinsed with the same buffer, then detached from the centrifuge tube using a spatula and cut into 1 mm square blocks with a razor blade. The blocks were further fixed and stained with 2% osmium tetroxide (1.5 h at 4 °C) and 1% aqueous uranylacetate (0.5 h at 22 °C), with an intervening buffer rinse. The blocks were then dehydrated with a graded alcohol series and carried through propylene oxide to embedment in epon resin (Polysciences Poly/Bed 812). Sections with silver to gold interference colors were cut with a diamond knife on an AO Ultracut microtome, collected on uncoated 200 mesh copper grids, and examined with a Philips EM 400 electron microscope with or without additional staining with lead citrate.
SDS polyacrylamide gel electrophoresis Protein profiles of cerebral plasma membrane vesicles were obtained according to the method of Laemmli 14 on 8-18% linear gradient gels stained with Coomassie blue.
Membrane marker assays Equilibrium binding assays for [3H]ouabain (14.6 Ci/mmol) and [3H]quinuclidinylbenzilate (QNB) (32.9 Ci/mmol) were carded out at 37 °C in 5 ml vols. [3H]ouabain binding was assayed after incubation of vesicles in 120 mM NaCl, 1 mM MgATP and 20 mM Tris-HC! (pH 7.4) for 60 min. [3H]ouabain was diluted to 5.0 Ci/mmol with non-radioactive ouabain (Sigma) before use. [3H]QNB binding was assayed after incubation in 5 mM MgCi 2, 50 mM Tris-HCi (pH 7.4) for 90 min. Specific binding of [3H]ouabain and [3H]QNB was the amount of binding displaced by 2.10-4 M ouabain and 1.10-6 M atropine sulfate, respectively. For Scatchard analysis, concentration ranges for [3H]ouabain were 1.34-66.7 nM and for [3H]QNB were 0.01-0.5 nM. Protein present in each assay tube was 50 /~g. Binding reactions were terminated by rapid filtration on the Brandel M-24RP Cell Harvester, the filters (No. 30 glass, Schleicher and Schuell) being rinsed twice with 5 ml of 50 mM Tris-HCl (pH 7.4) and radioactivity determined as described above. Computer-generated linear least-squares analyses of Scatchard plots were obtained to determine the maximal number of binding sites (Bmax) as well as the equilibrium dissociation constant (Ko). Cytochrome c oxidase activity was assayed by the method of Sottocasa et al. 34 using Sigma type III cytochrome c.
TABLE I
Properties of the membrane preparations used in this study Values are means + standard deviation.
Normal brain
AD brain
NAD brain
Number Age(years) Autolysis time (h)
6 71.8+10.0 15.7+6.76
6 74.3+5.28 16.1+7.47
4
QNBbindingK a(pM) Bmax(pmol/mg)
43.5+23.3 31.3+4.08 28.3+5.87 1.33+0.437 1.33+0.340 0.804+0.530
75.5+9.98 8.38+3.82
OuabainbindingKd(nM ) 11.2+1.95 Bmax (pmol/mg) 26.3+4.40
10.0+4.05 22.8+9.53
11.1+2.24 18.9+11.4
Cytochrome c oxidase (,umol/min/mg x 103)
5.17+1.00
3.40+0.490*
6.70+1.70
* Significantly different from normal brain (P < 0.05).
Methods). The biochemical properties of membrane preparations recovered from the 0.6 M/0.8 M sucrose interface are shown in Table I. Membrane markers of plasma membrane origin (binding of [3H]QNB and [3H]ouabain) and mitochondrial membrane (cytochrome c oxidase) were analyzed. The brain tissues studied were age-matched and had post-mortem delays (autolysis
kD
AD
NORMAL
220--
Materials [3H]ouabain, [3H]QNB and 45CAC12 were purchased from New England Nuclear (Boston, MA). The Ca 2÷ ionophore A23187 was purchased from Calbiochem. All other reagents were of the highest purity available.
Statistics Nonlinear regression was used to fit a sigmoid curve to the calcium dose response data using a 4 parameter logistic equation. Goodness of fit was quantitated by the least-squares method. Lineweaver-Burk plots were analyzed by linear least-squares analysis. Means were obtained from the data collected from normal, Alzheimer's disease and non-Alzheimer's dementia tissues and were compared using one-way Anova with Scheffe's method and judged significantly different when P < 0.05.
6760-
36-
RESULTS
Characterization o f the membrane preparations used in
this study
18.5--
In order to study Na+/Ca 2+ exchange activity in human brain, cerebral plasma membranes were purified from frozen post-mortem tissues by homogenization and sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (see M a t e r i a l s a n d
Fig. 1. Electrophoretic separation of normal and A D cerebral plasma membrane vesicle proteins on polyacrylamide gel after solubilization in SDS. Twenty/~g protein was applied to each lane. The standards used were: ferritin 220,000; albumin 67,000; eatalase 60,000; lactate dehydrogenase 36,000; and ferritin 18,500.
142 times) ranging from 5 to 26 h; however, only the N A D group had a mean post-mortem delay that appeared different from the other two groups (although not significant P = 0.175). The membrane fraction obtained was high in plasma membrane content as evidenced by the high density for [3H]QNB and [3H]ouabain binding
sites. Mitochondrial membranes appeared to represent only a minor contaminant. N A D membrane preparations were significantly lower in cytochrome coxidase activity when compared with normal. Importantly, the plasma membrane marker activities obtained from membrane preparations of different origin were similar, indicating
Fig. 2. A: thin section electron mlcrograph showing typical profiles of cerebral plasma membrane vesicles derived from AD brain tissue. B: thin section electron micrograph of cerebral plasma membrane vesicles derived from normal brain. Both normal and AD brain tissues were from frontal cortex. Original magnification x62,500.
143 4
that no significant differences in the degree of purity existed. Fig. 1 shows the electrophoretic profile of normal and A D p l a s m a m e m b r a n e proteins stained with Coomassie blue. N o r m a l and A D p r e p a r a t i o n s showed nearly identical patterns of protein staining. In both p r e p a r a tions proteins ranged in molecular weight from 20 to 220 kDa. The morphological properties of normal and A D p l a s m a m e m b r a n e p r e p a r a t i o n s were examined by thinsection electron microscopy (see Fig. 2A,B). In both p r e p a r a t i o n s , thin section profiles revealed p r e d o m i nantly intact unilamellar vesicles. Morphological findings d e m o n s t r a t e d few detectable differences i n the two preparations. This finding was consistent with the biochemical evidence (Table I and Fig. 1) and demonstrates that the m o r p h o l o g y of purified cerebral plasma membranes was not affected by the origin of the tissue used.
Characterization of Na+lCa2+ exchange C e r e b r a l p l a s m a m e m b r a n e vesicles from human brain catalyzed N a + - d e p e n d e n t Ca 2÷ u p t a k e as shown by the time course o f Fig. 3. Fig. 3 A shows that Ca 2+ content of N a ÷ - l o a d e d vesicles placed in a choline chloride m e d i u m increased rapidly at first and then maintained a steady plateau for up to 5 min. N a + - d e p e n d e n t Ca 2+ u p t a k e could be easily distinguished from Ca 2+ uptake in the absence of a Na ÷ gradient (i.e. N a ÷ loaded vesicles placed in NaCI media). N a + / C a 2÷ exchange, shown in Fig. 3B, was defined as the change in Ca 2+ content in vesicles a d d e d to a choline chloride m e d i u m minus that
B
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i
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Fig. 4. Kinetic analysis of Na+/Ca2÷ exchange activity in cerebral plasma membrane vesicles derived from normal tissue-hippocampus/temporal cortex region. A: aliquots (25 /~1) of membrane vesicles equilibrated with buffer B were diluted 20-fold into either buffer B or buffer C containing increasing concentrations of *SCaCO3 at 37 °C. Na+-dependent Ca 2+ uptake was terminated after 30 s and is plotted as a function of added 4SCaCO3. Results were analyzed by computer assisted non-linear regression analysis. K m = 102 #M, Vmax = 3.66 nmoi/mg/min. B: the same data as presented in Fig. 4A except that results are presented in a Lineweaver-Burk representation and analyzed by linear regression. Km = 74.7/~M, Vmax = 3.11 nmol/mg/min.
m e a s u r e d after addition to a NaCI m e d i u m . R u t h e n i u m red, which has been shown to be an effective inhibitor of the mitochondrial Ca 2÷ u n i p o r t e r 23, had no effect on Ca 2+ content at a concentration of 10 p M (data not shown). W h e n vesicles were first l o a d e d with 4SCa by N a + / C a 2+ exchange and then e x p o s e d to the Ca2+-ionophore A23187 (Fig. 3B arrow) with 100/~M E G T A present to maintain low extravesicular Ca 2+, Ca 2+ content was reduced to a p p r o x i m a t e l y 10% of its original value within 1 min. Only a small a m o u n t of vesicle-associated Ca 2÷ was lost when E G T A was a d d e d alone. These data confirm that Ca 2+ that becomes associated with the
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TIME (seconds) Fig. 3. Na+/Ca2+ exchange in cerebral plasma membrane vesicles derived from normal tissue-hippocampus/temporal cortex region. A: aliquots (25 /~1) of vesicles equilibrated with buffer B were diluted 20-fold with either buffer B (O) or buffer C (©) containing 100/~M 45CACO3 at 37 °C. B: data points represent Na+-dependent Ca 2+ uptake (Ca 2÷ content in buffer C minus that measured in buffer B shown in Fig. 3A). At the time indicated by the arrow the incubation media were made to contain 100 gM EGTA or 10 pM A23187 and 100 pM EGTA. Reactions were terminated at indicated times as described under Materials and Methods.
2 NAO 0
i
i
60
t20
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i
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Fig. 5. Representative experiments showing the overall effect of tissue origin on the time course of Na+/Ca2+ exchange. Na +dependent Ca 2+ uptake was measured as described in Fig. 3. Tissue origin: normal (O), AD ( i ) and NAD (O)-hippocampus/temporal cortex region.
144 vesicles during a transport reaction is, in fact, internalized. Fig. 4 shows data attempting to quantitate the relationship between Na+/Ca 2+ exchange activity and the concentration of extravesicular Ca 2+. Ca 2+ content was measured 30 s after the start of the reaction and plotted as a function of a d d e d Ca 2+. It should be noted that because of the curvature of the line that described Ca 2÷ content, m e a s u r e m e n t s made at 30 s underestimated the true initial velocity of increasing Ca 2+ content. Nevertheless, as can be seen in Fig. 4 A , B , measurements of Ca 2+ content at 30 s provided a good first approximation of the true initial velocity enabling an estimate of the K m and Vmax to be made. Estimates of K m and Vm~x could be o b t a i n e d from either a computer-assisted non-linear regression analysis of log [Ca] vs. V (Fig. 4A) or linear regression analysis of the L i n e w e a v e r - B u r k plot (Fig. 4B). A l t h o u g h similar estimates were obtained by either m e t h o d (see legend to Fig. 4), all the results described below were the result of L i n e w e a v e r - B u r k analysis. Effect o f tissue origin on Na+/Ca 2+ exchange Fig. 5 demonstrates the effect of tissue origin on the time course of N a + / C a 2÷ exchange in human cerebral
plasma m e m b r a n e s . It can be seen that m e m b r a n e s derived from A D brain had increased Na÷/Ca 2+ exchange activity as evidenced by both an increase in the initial rise in Ca 2÷ content and in elevated values of peak plateau Ca 2+ content. N A D tissues yielded m e m b r a n e s that gave values for Ca 2+ content that were similar or slightly less than that of normal origin. It is important to note that Ca 2÷ content m e a s u r e d in the absence of a Na ÷ gradient (see Fig. 3A) was not affected by tissue origin
4-
•
B
A
(data not shown). This observation further confirmed the similar purity of m e m b r a n e preparations from different tissue origins. As Na÷/Ca 2+ exchange was d e t e r m i n e d as the difference between N a ÷ - d e p e n d e n t and Na+-inde p e n d e n t Ca 2+ uptake, the increases seen in m e m b r a n e s derived from A D tissues were clearly the result of increased N a ÷ - g r a d i e n t - d e p e n d e n t Ca 2+ uptake. Next, we p e r f o r m e d a kinetic analysis of the extravesicular Ca 2+ d e p e n d e n c e of N a + / C a > exchange in each of the 3 tissue types. Fig. 6 shows both a non-linear fit (Fig. 6A) and L i n e w e a v e r - B u r k analysis (Fig. 6B) of the relationship between the concentration of a d d e d extravesicular Ca 2+ and the velocity of the reaction measured at 30 s (see also Fig. 4). It can be seen with either plot that the dominant effect observed in A D tissue was an increase in the estimated Vmax for N a + / C a 2+ exchange. A summary of the kinetic data o b t a i n e d for Na+/Ca 2÷ exchange in all the tissue samples studied is shown in Table II. The larger degree of uncertainty in the estimation of K m for the normal and A D tissues may be explained, in part, by a greater uncertainty in the estimate of initial velocities at low Ca 2+ concentrations. W h e n using the L i n e w e a v e r - B u r k analysis, this uncertainty is more likely to affect the K m d e t e r m i n a t i o n than the Vmax determination. The mean values obtained for the g m of Na+/Ca 2+ exchange were not significantly different when c o m p a r e d b e t w e e n the 3 groups: normal, A D and N A D . On the o t h e r hand, the mean Vmax of Na÷/Ca 2÷ exchange was significantly increased for A D tissue ( P < 0.05) when c o m p a r e d with either normal or N A D tissue. Figs. 7 and 8 show a composite view of the Vmax and K m d e t e r m i n e d for each tissue sample plotted vs the age at death or autolysis time for that particular sample. There was no correlation evident in either normal, A D , N A D or the total sample population between Na+/Ca 2÷ exchange Vmax o r K m and autolysis time or age at death.
3TABLE II 2-
• •
Summary of the kinetic constants obtained from Na+/Ca2+ exchange initial velocities estimated at various concentrations of extravesicular calcium
Q
i-
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.
.
,
.
.
,
50 t00 t/Ca 2+ X l0-a M
log [Ca2+] added Fig. 6. Representative experiments showing kinetic analysis of the effect of tissue origin on Na+/Ca2+ exchange estimated initial velocity. The effect of increasing added 45CaCO~ on Na +-dependent Ca~+ uptake after 30 s was measured as described in Fig. 4. A: computer assisted non-linear regression analysis. B: LineweaverBurk analysis with linear regression. Tissue origin: normal (O), AD (11) and NAD (O)-hippocampus/temporal cortex region.
Initial velocities (30 s after the start of the reaction) were estimated over a range of added Ca 2÷ concentrations of 0.01-0.3 mM. K m and Vmax were estimated by Lineweaver-Burk analysis. Values are means _+standard deviation. Origin
n
K,, (I~M)
V , ~ (nmol/mg/min)
Normal AD NAD
6 6 4
57.9 + 28.1 71.2 + 29.3 66.1 + 3.3
4.45 + 0.58 6.68 + 1.58* 3.76 + 1.50
*
Significantly different from either normal or NAD brain 0.05).
(P <
145
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AGE AT DEATH (years) Fig. 7. Na+/Ca 2+ exchange V,,,aX(A) or Km (B) plotted against age at death (years) for either normal, AD, NAD or the total sample population. There was no correlation (P > 0.05) in either normal, A D , NAD, or the total sample population• The r-values for the regression lines were: 0.256, 0.004, 0.734 and 0.020 for Vmax;0.370, 0.010, 0.615 and 0.041 for Kin, respectively.
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AUTOLYSIS TIME (hours) Fig. 8. Na+/Ca 2+ exchange Vm~ (A) or Km (B) plotted against autolysis time (h) for either normal, AD, NAD or the total sample population. There was no correlation (P > 0.05) in either normal, AD, NAD, or the total sample population. The r-values for the regression lines were: 0.010, 0.113, 0.385 and 0.158 for Vmax;and 0.350, 0.007, 0.001 and 0.064 for Km respectively.
DISCUSSION The major finding of this study was that Na+/Ca 2+ exchange activity measured in cerebral plasma membrane vesicles derived from A D post-mortem brain tissues was significantly greater than that measured in vesicles from normal or N A D brain tissues. What is the possible role of Na+/Ca 2÷ exchange in the pathogenesis of nerve cell degeneration in A D ? The recent study of Mattson et a1.18 has clearly shown cell-specific differences in calcium homeostatic systems and that cells with superior Na+/ Ca 2÷ exchange capacity are more likely to survive an insult of increased [Cai]. It is attractive to speculate that the increased Na+/Ca 2÷ exchange activity observed in the plasma membranes of neurons from A D brain (present study) reflects the properties of surviving neurons with increased capacity for Na+/Ca 2÷ exchange. These neurons may have survived the neurodegenerative process of A D by virtue of their increased Na÷/Ca 2+ exchange activity. If this were true, it is probable that increased intracellular Ca 2÷ played a key role in the death of the non-survivors. In support of this, it has been found that
the antigenic changes in neuronal cytoarchitecture seen in cultured hippocampal cells after treatment with glutamate and Ca 2÷ influx are similar to that seen in the neurofibrillary tangles of A D 17. The conclusions of the present study must be discussed in terms of the considerable difficulty that exists in interpreting human post-mortem studies. In fact, the study of A D in post-mortem tissues presents unique factors that can adversely affect clear interpretation of the data. In post-mortem studies in general it is difficult if not impossible to make a direct correlation between a neurochemical measurement and a pathological observation. Even worse for the A D researcher is the c o m m o n practice to reserve one hemisphere for pathology while the other is frozen for neurochemical studies. Several factors (epiphenomena) are known to affect the results of post-mortem studies 25. These include: patient age, sex, drug history, immediate pre-mortem status (sudden death or prolonged coma) and postmortem delay. In the present study brain tissues were age matched with similar post-mortem delays (see Table I).
146 The effect of post-mortem delay on Na+/Ca 2+ exchange activity in h u m a n brain tissues has not been characterized. Unfortunately, in this retrospective study, patient records of the medication administered and the duration of disease were not readily available. The study of A D in post-mortem tissues presents yet two more complicating issues of the severity of the disease and tissue atrophy. In this study we are considering that all brain samples from A D patients reflect end-stage disease, although this clearly may not be the case as older patients may die at more early stages of the disease. It is known that changes occur in the relative proportion of different n e u r o n types and glial cells during the tissue atrophy of A D . It is unlikely that the proliferation of astrocytes that occurs in A D would account for the increased Na+/Ca 2+ exchange activity, as Na+/Ca 2÷ exchange is found in such high density in the ABBREVIATIONS AD EGTA
Alzheimer's disease ethyleneglycol bis(fl-aminoethylether)-N,N'-tetraacetic acid
REFERENCES 1 Akerman, K.E.O. and Nichoils, D.G., Intrasynaptosomal compartmentation of calcium during depolarization-induced calcium uptake across the plasma membrane, Biochim. Biophys. Acta, 645 (1981) 41-48. 2 Akiyama, H., McGeer, P.L., Itagaki, S., McGeer, E.G. and Kaneko, T., Loss of glutaminase-positive cortical neurons in Alzheimer's disease, Neurochem. Res., 14 (1989) 353-358. 3 Blaustein, M.P. and Hodgkin, A.L., The effect of cyanide on the efflux of calcium from squid axons, J. Physiol., 200 (1969) 497-527. 4 Blaustein, M.P. and Oborn, C.J., The influence of sodium on calcium fluxes in pinched-off nerve terminals in vitro, J. Physiol., 247 (1975) 657-686. 5 Carafoli, E., Intracellular calcium homeostasis, Ann. Rev. Biochem., 56 (1987) 395-433. 6 Choi, D.W., Ionic dependence of glutamate neurotoxicity, J. Neurosci., 7 (1987) 369-379. 7 Cotman, C.W., Monaghan, D.T., Ottersen, O.P. and StormMathisen, J., Anatomical organization of excitatory amino acid receptors and their pathways, Trends Neurosci., 10 (1987) 273-280. 8 Geddes, J.W., Chang-Chui, H., Cooper, S.M., Lott, I.T. and Cotman, C.W., Density and distribution of NMDA receptors in the human hippocampus in Alzheimer's disease, Brain Research, 399 (1986) 156-161. 9 Gibson, G.E. and Peterson, C., Calcium and the aging nervous system, Neurobiol. Aging, 8 (1987) 329-343. 10 Gill, D.L., Grollman, E.F. and Kohn, L.D., Calcium transport mechanisms in membrane vesicles from guinea pig brain synaptosomes, J. Biol. Chem., 256 (1981) 184-192. 11 Gill, D.L., Sodium channel, sodium pump and sodium-calcium exchange activities in synaptosomal plasma membrane vesicles, J. Biol. Chem., 257 (1982) 10986-10990. 12 Gill, R., Foster, A.C. and Woodruff, G.N., MK-801 is neuroprotective in gerbils when administered during the post-ischaemic period, Neuroscience, 25 (1988) 847-855. 13 Khachaturian, Z.S., Introduction and overview. Ann. New York
plasma m e m b r a n e s of neurons. In fact, glial cell proliferation would be expected to cause an underestimation of the increase in Na+/Ca 2÷ exchange activity. Notwithstanding the possibility of post-mortem artifact, the observations of this study are consistent with the view that increased Na+/Ca 2÷ exchange activity in A D brain tissues is a result of the disease state. A better understanding of the role of Na÷/Ca 2÷ exchange and increased intraceilular Ca 2÷ in the pathogenesis of the A D must await further knowledge of the temporal sequence of events that underlie the formation of senile plaques, neurofibrillary tangles and cell death. Acknowledgements. We would like to thank David Henderson, Department of Anatomy, Oral Roberts University School of Medicine, who performed the electron microscopy. This work was supported in part by a grant from the Founders of Doctors' Hospital, Tulsa, OK, U.S.A.
HEPES NAD SDS QNB
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid non-Alzheimer dementia sodium dodecylsulfate quinuclidinylbenzilate
Acad. Sci., 568 (1989) 1-4. 14 Laemli, V.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. 15 Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, A.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 254-275. 16 Maragos, W.E, Chu, D.C.M., Young, A.B., D'Amato, C.J. and Penny, J.B., Loss of hippocampal [aH]TCP binding in Alzheimer's disease, Neurosci. Lett., 74 (1987) 371-376. 17 Mattson, M.P., Antigenic changes similar to those seen in neuroflbrillary tangles are elicited by glutamate and Ca2+ influx in cultured hippocampal neurons, Neuron, 4 (1990) 105-117. 18 Mattson, M.E, Guthrie, P.B. and Kater, S.B., A role for N a + - d e p e n d e n t Ca 2+ extrusion in protection against neuronal excitotoxicity, FASEB J., 3 (1989) 2519- 2526. 19 Mattson, M.P. and Kater, S.B., Excitatory and inhibitory neurotransmitters in the generation and degeneration of hippocampal neuroarchitecture, Brain Research, 278 (1989) 337-348. 20 Michaelis, M.L. and Michaelis, E.K., Ca2+ fluxes in resealed synaptic plasma membrane vesicles, Life Sci., 28 (1981) 37-45. 21 Michaelis, M.L. and Michaelis, E.K., Alcohol and local anesthetic effects on Na~--dependent Ca 2+ flUXeSin brain synaptic membrane vesicles, Biochem. Pharmacol., 32 (1983) 963-969. 22 Michaelis, M.L., Johe, K. and Kitos, T.E., Age-dependent alterations in synaptic membrane systems for Ca2+ regulation, Mech. Ageing Dev., 25 (1984) 215-225. 23 Michaelis, M.L., Ca2÷ handling systems and neuronal aging, Ann. New York Acad. Sci., 568 (1989) 89-94. 24 Palmer, A.M. and Gershon, S., Is the neuronal basis of Alzheimer's disease cholinergic or glutamatergic, FASEB J., 4 (1990) 2745-2752. 25 Palmer, A.M., Lowe, S.L., Francis, ET. and Bowen, D.M., Are post-mortem biochemical studies of human brain worthwhile?, Biochem. Soc. Trans., 16 (1988) 472-475. 26 Penney, J.B., Maragos, W.E, Greenamyre, J.T., Debowey, D.L., Holungsworth, Z. and Young, A.B., Excitatory amino acid binding sites in the hippocampal region of Alzheimer's disease and other dementias, J. Neurol. Neurosur. Psych., 53 (1990) 314-320.
147 27 Peterson, C. and Goldman, J.E., Alterations in calcium content and biochemical processes in cultured skin fibroblasts from aged and Alzheimer donors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 2758-2762. 28 Peterson, C., Ratan, R., Shelanski, M.L. and Goldman, J.E., Cytosolic free calcium and cell spreading decrease in fibroblasts from aged and Alzheimer donors, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7999-8001. 29 Procter, A.W., Stirling, J.M., Stratmann, G.C., Cross, A.J. and Bowen.D.M., Loss of glycine-dependent radioligand binding to the N-methyI-D-aspartatephenylcudine receptor complex in patients with Alzheimer's disease, Neurosci. Lett., 101 (1989) 62-66. 30 Procter, A.W., Palmer, A.M., Francis, P.T., Lowe, S.L., Neary, D., Murphy, E., Doshi, R. and Bowen, D.M., Evidence of glutamatergic denervation and possible abnormal metabolism in Alzheimer's disease, J. Neurochem., 50 (1988) 790-802. 31 Siesjo, B.K. and Bengtsson, E, Calcium fluxes, calcium antagonists and calcium related pathology in brain ischemia, hypogly-
32
33
34
35
cemia, and spreading depression: a unifying hypothesis, J. Cereb. Blood Flow Metab., 9 (1989) 127-140. Simon, R.P., Griffiths, T., Evans, M.C., Swan, J.H. and Meldrum, B.S., Calcium overload in selectively vulnerable neurons of the hippocampus during and after ischaemia: an electron microscopy study in the rat, J. Cereb. Blood Flow Metab., 4 (1984) 350-361. Simpson, M.D.C., Roysston, M.C., Deakin, J.F.W., Cross, A.J., Mann, D.M.A. and Slater, P., Regional changes in [3H] D-aspartate and [3HI TCP binding sites in Alzheimer's disease brains, Brain Research, 462 (1988) 76-82. Sottocasa, G.L., Kuytenstierna, L., Ernster, A. and Bergtrand, A., An electron- transport system associated with the outer membrane of liver mitochondria, J. Cell. Biol., 32 (1967) 415-438. Steele, J.E., Palmer, A.M., Stratmann, G.C. and Bowen, D.M., The N-methyl-D-aspartate receptor complex in Alzheimer's disease: reduced regulation by glycine but not zinc, Brain Research, 500 (1989) 369-373.
139
Elsevier BRES 16406
Na+/Ca 2 + exchange activity is increased in Alzheimer's disease brain tissues Robert A. Colvin 1, Jonathan W. Bennett 1, Sharon L. Colvin 1, Richard A. Allen 1, John Martinez I and Gary D. Miner 2 1Department of Zoological and Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, OH 45701 (U.S.A.) and 2The Alzheimer's Foundation, Tulsa, OK 74137 (U.S.A.) (Accepted 2 October 1990)
Key words: Sodium/calcium exchange; Ion transport; Plasma membrane; Alzheimer's disease; Neurodegenerative disease; Human post-mortem study
These studies were performed to determine the changes that occur in Na+/Ca2÷ exchange activity in Alzheimer's disease (AD) brain tissues. Cerebral plasma membrane vesicles were purified by sucrose density gradient centrifugation from frozen postmortem hippocampal/temporal cortex tissue slices derived from age matched brains of normal, AD and non-Alzheimer dementia (NAD) origin (autopsy confirmed). Membrane marker assays (Na/K ATPase, muscarinic receptor, cytochrome c oxidase) revealed no change in membrane purity across different preparations. Thin-section electron microscopy revealed predominantly intact unilamellar vesicles. Vesicles were preincubated for 15 min (37 °C) in buffer containing 132 mM NaCI, 5 mM KCI, 1.3 mM MgCI2, 10 mM glucose and 10 mM HEPES (pH 7.4). Ca2÷ uptake was initiated by diluting vesicles 20-fold with buffer containing either 132 mM NaC1 or 132 mM choline chloride and 4SCaCl2 then terminated by addition of 200/zM LaC!3 and rapid filtration. Ca2+ content increased rapidly at first and then maintained a steady plateau for up to 5 min. When the Ca2÷ ionophore A23187 (10 ~M) with 100/.tM EGTA was added after 4 min, Ca2÷ content was reduced to 10% of its original value. Ruthenium red (10/~M) had no effect on Ca2÷ content. Na+-dependent Ca2÷ uptake (Ca2+ content measured in choline chloride minus that measured in NaCI) was increased in AD brains as evidenced by both an increase in the initial rise in Ca2÷ content and in elevated values of peak plateau Ca2+ content. The Kmand Vr,ax for Na+/Ca2+ exchange was estimated from Lineweaver-Burk analysis of the effect of increasing extravesicular concentrations of Ca2÷ on Nat-dependent Ca2÷ uptake after 30 s. The values obtained for the Km (uM) and VmaX (nmol/mg/min) were (respectively): normal (n = 6) 57.9 + 28.1 and 4.45 _+ 0.58; AD (n = 6) 71.2 + 29.3 and 6.68 + 1.58; NAD (n = 4) 66.1 + 3.3 and 3.76 + 1.50 (mean + S.D.). The Vm~x for Na+/Ca2÷ exchange in AD brain tissues was significantly (P < 0.05) elevated when compared with normal or NAD brains. There was no correlation evident in either normal, AD, NAD or the total sample population between Na+/Ca2+ exchange Vm~xor Kr, and autolysis time or age at death. The results suggest that in brain regions suffering the greatest degeneration due to AD the surviving neurons have increased Na+/Ca2+ exchange activity. This increase in Na+/Ca2÷ exchange activity may provide clues to a better understanding of the pathogenesis of nerve cell degeneration in AD. INTRODUCTION The mechanisms of neuronal cell dysfunction and death that underlie normal aging and the h u m a n neurodegenerative diseases (e.g. Parkinson's, H u n t i n g t o n ' s and Alzheimer's diseases) are still largely unknown. Similarly, very little is understood concerning the cellular defense mechanisms that act to prevent or reverse potentially lethal changes in n e u r o n a l structure and function occurring in aging and degenerative disease states. The 'calcium hypothesis of brain aging '13 proposes that cellular mechanisms that act to modulate the concentration of free intracellular calcium ([Cai]) play a critical role in brain aging and that changes in [Ca d of short or long duration contribute to the causative factors accounting for cell dysfunction and degeneration.
A n extensive body of literature suggests that neuronal Ca 2÷ metabolism is affected by aging (for review see ref. 9) and it is now well established that increases in [Cai] caused by overstimulation by excitatory amino acids are a direct causative factor in ischemia-induced n e u r o n a l death 6"12'18'29'30. The evidence relating excitotoxicity and changes in [Cai] to the neurodegenerative processes occurring in Alzheimer's disease ( A D ) , is still indirect and inconclusive. Although the large pyramidal neurons of the cortex and hippocampus, which are k n o w n to degenerate in A_D, are thought to have a high density of excitatory amino acid receptors 7, when tissues are assayed for excitatory amino acid receptor binding site density the results are conflicting24. Some studies show no change 8 while others observe decreases in selective regions of the A D brain 2A6,26,3°-33 or a loss of glycine-
Correspondence: R.A. Colvin, Department of Zoological and Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, OH 45701, U.S.A. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
140 dependent binding 29,35. Peterson and c o - w o r k e r s 27"2~, using skin fibroblasts obtained from AD patients, have shown that these cells demonstrate higher levels of bound calcium but lower levels of [Cai] when compared with young and normal aged donors. It is clear that any mechanisms underlying changes in cellular Ca 2+ metabolism associated with A D must involve complex interactions between Ca 2+ binding proteins, Ca 2+ sequestering organelles and Ca 2+ transport proteins such as the Na+/Ca 2+ exchanger. Na+/Ca 2+ exchange is found in high density in the plasma membranes of excitable cells in such tissues as heart and nerve. This protein generally catalyzes the transport of sodium ions across the plasma membrane in exchange for calcium ions and the exchange process is reversible. Na+/Ca 2+ exchange is electrogenic as 3 sodium ions are exchanged for each calcium ion 5. The presumed role of Na+/Ca 2+ exchange is to maintain the resting concentration of [Ca d at submicromolar levels by pumping excess Ca 2+ out of the cell. Hence, the theory that Na+/Ca z+ exchange plays an important role in protecting neurons from CaZ+-induced damages after excitatory amino acid overstimulation has been proposed TM. Na+/Ca 2÷ exchange in the nervous system was first demonstrated in the squid giant axon 3. The direct measurement of Na+/Ca 2+ exchange in the mammalian nervous system is most often accomplished by the preparation of synaptosomes (pinched-off nerve terminals) and the analysis of the effects of sodium ions on calcium ion influx and effiux using radioactive Ca 2+4. Na+-dependent Ca 2÷ uptake has also been studied in purified synaptic plasma membranes 1°'11'2°'21. Studies of the changes in Na+/Ca 2+ exchange activity in synaptic plasma membranes from the aging rat brain have been reported by Michaelis and co-workers 22'23. They found lower Na+/Ca 2+ exchange activity in aged rats, which could be explained by an increase in the Kact for C a 2+ with little or no effect on Vmax. An increase in resting [Cai] and increased responses to agents that elevate [Cai] (e.g. KCI depolarization) were also observed in intact synaptosomes from aged rat brain 23. In the present study we tested the hypothesis that differences exist in the Na+/Ca 2+ exchange activity measured in cerebral plasma membrane vesicles purified from human post-mortem brain tissues of normal, AD and non-Alzheimer's dementia origin. We report that Na+/Ca 2+ exchange activity is significantly elevated in the plasma membranes purified from AD brain tissue. The results suggest that the plasma membranes of surviving neurons in brain areas suffering the greatest neural degeneration due to AD have elevated Na+/Ca 2+ exchange activity. This increase in Na+/Ca 2+ exchange activity may provide clues to a better understanding of
the pathogenesis of nerve cell degeneration in AD. MATERIALS AND METHODS
Preparation of cerebral plasma membrane vesicles Tissue sections from the frontal cortex and hippocampus/temporal cortex regions of human brain were obtained from the National Neurological Research Bank, VAMC Wadsworth, Los Angeles, CA 90083. The clinical diagnosis of Alzheimer's disease was confirmed by pathological diagnosis at the time of autopsy. It was not determined whether Alzheimer's disease patients were of familial or non-familial types. The non-Alzheimer's dementia patients were individuals that clinically presented with progressive dementia or chronic dementia of undetermined origin and upon neuropathological investigation showed no changes characteristic of Alzheimer's disease. Normals were individuals without clinical presentation of dementia at the time of death. Neuropathological investigation at autopsy showed in every case grossly normal brain and spinal cord and microscopic sections of the hippocampus and cortex were unremarkable. The cause of death of the normal patients was non-neurological. Frozen brain sections (-70 °C) were thawed, and the tissue was rapidly dissected in an ice bath. Homogenization of the tissue was performed in 20 vols. of buffer A containing 0.32 M sucrose, 10 mM HEPES (pH 7.4) and the protease inhibitors: soybean trypsin inhibitor (50/~g/ml), leupeptin (0.5/~g/ml) and pepstatin (0.7/~g/ml) utilizing a Brinkmann Polytron at a setting of 4 (10 s, repeated 3-4 times). The homogenate was then centrifuged at 1000 g for 10 min. The resulting supernatant fluid was centrifuged at 17,000 g for 20 min. The pellet was resuspended in buffer A and carefully layered over a sucrose step gradient in a SW-41 centrifuge tube. The step gradient contained 4 ml each of 0.8 M and 0.6 M sucrose containing the protease inhibitors described above. The tubes were spun in a SW-41 rotor at 25,000 rpm for 2 h. The membrane material at the 0.6 M-0.8 M interface was recovered using a Pasteur pipette and resuspended in 10 vols. of buffer A. The resulting suspension was centrifuged at 100,000 g for 40 rain. The pellet was resuspended in buffer A to a concentration of 10-20 mg/ml and frozen in aliquots in liquid N 2. Membrane preparations were stored at -70 °C until used. Membrane protein determinations were performed by the method of Lowry ~5 using bovine serum albumin as a standard.
Assay of Na/Ca exchange activity Cerebral plasma membrane vesicles were thawed, diluted to 2 mg/ml in buffer B containing 132 mM NaC1, 5 mM KCI, 1.3 mM MgCI z, 10 mM glucose, 10 mM HEPES (pH 7.4) and then kept on ice until use. In order to load the vesicles with Na +, 25/A of the membrane suspension was added to test tubes incubating in a 37 °C water bath. After 15 min at 37 °C, the membrane suspension was diluted to 0.5 ml by the addition of either buffer B or buffer C in which choline chloride was substituted for NaCI. Each buffer contained various concentrations of CaCO 3 with the addition of 45CAC12 (0.01 mCi/ml). Reactions were stopped by the addition of 200/~M LaCI 3 and placing the tubes on ice. Zero time points were determined by addition of 200 ~M LaCI 3 to Na+-loaded vesicles before the addition of either buffer B or buffer C. The membrane suspensions on ice were then filtered on a Brandel M-24RP Cell Harvester using No. 30 glass fiber filters (Schleicher and Schuell). The filters were washed 3 x with 5 ml of buffer D containing 132 mM NaCI, 5 mM KCI, 200~M LaCI 3 and 10 mM Hepes (pH 7.4), placed in 3 ml of Biofluor and radioactivity determined in a liquid scintillation counter. Na+-dependent Ca 2+ uptake was defined as the change in Ca 2÷ content in buffer C minus that measured in buffer B.
Electron microscopy Cerebral plasma membrane vesicles in buffer B were diluted to approximately 0.05 mg/ml and then centrifuged in a SW-27 rotor for 30 min. The buffer was decanted and the primary fixative (4%
141 glutaraldehyde and 1% tannic acid in 0.1 M sodium cacodylate buffer) was gently pipetted over the pellet. Following fixation for 2 h at 4 °C the pellets were rinsed with the same buffer, then detached from the centrifuge tube using a spatula and cut into 1 mm square blocks with a razor blade. The blocks were further fixed and stained with 2% osmium tetroxide (1.5 h at 4 °C) and 1% aqueous uranylacetate (0.5 h at 22 °C), with an intervening buffer rinse. The blocks were then dehydrated with a graded alcohol series and carried through propylene oxide to embedment in epon resin (Polysciences Poly/Bed 812). Sections with silver to gold interference colors were cut with a diamond knife on an AO Ultracut microtome, collected on uncoated 200 mesh copper grids, and examined with a Philips EM 400 electron microscope with or without additional staining with lead citrate.
SDS polyacrylamide gel electrophoresis Protein profiles of cerebral plasma membrane vesicles were obtained according to the method of Laemmli 14 on 8-18% linear gradient gels stained with Coomassie blue.
Membrane marker assays Equilibrium binding assays for [3H]ouabain (14.6 Ci/mmol) and [3H]quinuclidinylbenzilate (QNB) (32.9 Ci/mmol) were carded out at 37 °C in 5 ml vols. [3H]ouabain binding was assayed after incubation of vesicles in 120 mM NaCl, 1 mM MgATP and 20 mM Tris-HC! (pH 7.4) for 60 min. [3H]ouabain was diluted to 5.0 Ci/mmol with non-radioactive ouabain (Sigma) before use. [3H]QNB binding was assayed after incubation in 5 mM MgCi 2, 50 mM Tris-HCi (pH 7.4) for 90 min. Specific binding of [3H]ouabain and [3H]QNB was the amount of binding displaced by 2.10-4 M ouabain and 1.10-6 M atropine sulfate, respectively. For Scatchard analysis, concentration ranges for [3H]ouabain were 1.34-66.7 nM and for [3H]QNB were 0.01-0.5 nM. Protein present in each assay tube was 50 /~g. Binding reactions were terminated by rapid filtration on the Brandel M-24RP Cell Harvester, the filters (No. 30 glass, Schleicher and Schuell) being rinsed twice with 5 ml of 50 mM Tris-HCl (pH 7.4) and radioactivity determined as described above. Computer-generated linear least-squares analyses of Scatchard plots were obtained to determine the maximal number of binding sites (Bmax) as well as the equilibrium dissociation constant (Ko). Cytochrome c oxidase activity was assayed by the method of Sottocasa et al. 34 using Sigma type III cytochrome c.
TABLE I
Properties of the membrane preparations used in this study Values are means + standard deviation.
Normal brain
AD brain
NAD brain
Number Age(years) Autolysis time (h)
6 71.8+10.0 15.7+6.76
6 74.3+5.28 16.1+7.47
4
QNBbindingK a(pM) Bmax(pmol/mg)
43.5+23.3 31.3+4.08 28.3+5.87 1.33+0.437 1.33+0.340 0.804+0.530
75.5+9.98 8.38+3.82
OuabainbindingKd(nM ) 11.2+1.95 Bmax (pmol/mg) 26.3+4.40
10.0+4.05 22.8+9.53
11.1+2.24 18.9+11.4
Cytochrome c oxidase (,umol/min/mg x 103)
5.17+1.00
3.40+0.490*
6.70+1.70
* Significantly different from normal brain (P < 0.05).
Methods). The biochemical properties of membrane preparations recovered from the 0.6 M/0.8 M sucrose interface are shown in Table I. Membrane markers of plasma membrane origin (binding of [3H]QNB and [3H]ouabain) and mitochondrial membrane (cytochrome c oxidase) were analyzed. The brain tissues studied were age-matched and had post-mortem delays (autolysis
kD
AD
NORMAL
220--
Materials [3H]ouabain, [3H]QNB and 45CAC12 were purchased from New England Nuclear (Boston, MA). The Ca 2÷ ionophore A23187 was purchased from Calbiochem. All other reagents were of the highest purity available.
Statistics Nonlinear regression was used to fit a sigmoid curve to the calcium dose response data using a 4 parameter logistic equation. Goodness of fit was quantitated by the least-squares method. Lineweaver-Burk plots were analyzed by linear least-squares analysis. Means were obtained from the data collected from normal, Alzheimer's disease and non-Alzheimer's dementia tissues and were compared using one-way Anova with Scheffe's method and judged significantly different when P < 0.05.
6760-
36-
RESULTS
Characterization o f the membrane preparations used in
this study
18.5--
In order to study Na+/Ca 2+ exchange activity in human brain, cerebral plasma membranes were purified from frozen post-mortem tissues by homogenization and sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (see M a t e r i a l s a n d
Fig. 1. Electrophoretic separation of normal and A D cerebral plasma membrane vesicle proteins on polyacrylamide gel after solubilization in SDS. Twenty/~g protein was applied to each lane. The standards used were: ferritin 220,000; albumin 67,000; eatalase 60,000; lactate dehydrogenase 36,000; and ferritin 18,500.
142 times) ranging from 5 to 26 h; however, only the N A D group had a mean post-mortem delay that appeared different from the other two groups (although not significant P = 0.175). The membrane fraction obtained was high in plasma membrane content as evidenced by the high density for [3H]QNB and [3H]ouabain binding
sites. Mitochondrial membranes appeared to represent only a minor contaminant. N A D membrane preparations were significantly lower in cytochrome coxidase activity when compared with normal. Importantly, the plasma membrane marker activities obtained from membrane preparations of different origin were similar, indicating
Fig. 2. A: thin section electron mlcrograph showing typical profiles of cerebral plasma membrane vesicles derived from AD brain tissue. B: thin section electron micrograph of cerebral plasma membrane vesicles derived from normal brain. Both normal and AD brain tissues were from frontal cortex. Original magnification x62,500.
143 4
that no significant differences in the degree of purity existed. Fig. 1 shows the electrophoretic profile of normal and A D p l a s m a m e m b r a n e proteins stained with Coomassie blue. N o r m a l and A D p r e p a r a t i o n s showed nearly identical patterns of protein staining. In both p r e p a r a tions proteins ranged in molecular weight from 20 to 220 kDa. The morphological properties of normal and A D p l a s m a m e m b r a n e p r e p a r a t i o n s were examined by thinsection electron microscopy (see Fig. 2A,B). In both p r e p a r a t i o n s , thin section profiles revealed p r e d o m i nantly intact unilamellar vesicles. Morphological findings d e m o n s t r a t e d few detectable differences i n the two preparations. This finding was consistent with the biochemical evidence (Table I and Fig. 1) and demonstrates that the m o r p h o l o g y of purified cerebral plasma membranes was not affected by the origin of the tissue used.
Characterization of Na+lCa2+ exchange C e r e b r a l p l a s m a m e m b r a n e vesicles from human brain catalyzed N a + - d e p e n d e n t Ca 2÷ u p t a k e as shown by the time course o f Fig. 3. Fig. 3 A shows that Ca 2+ content of N a ÷ - l o a d e d vesicles placed in a choline chloride m e d i u m increased rapidly at first and then maintained a steady plateau for up to 5 min. N a + - d e p e n d e n t Ca 2+ u p t a k e could be easily distinguished from Ca 2+ uptake in the absence of a Na ÷ gradient (i.e. N a ÷ loaded vesicles placed in NaCI media). N a + / C a 2÷ exchange, shown in Fig. 3B, was defined as the change in Ca 2+ content in vesicles a d d e d to a choline chloride m e d i u m minus that
B
•21 .
A
B 2
,3
0
|
i
-5
-4
i
,
,
I
50
-3
log [Caz+] added
J
' t00
l/Ca a+ x 10-a M
Fig. 4. Kinetic analysis of Na+/Ca2÷ exchange activity in cerebral plasma membrane vesicles derived from normal tissue-hippocampus/temporal cortex region. A: aliquots (25 /~1) of membrane vesicles equilibrated with buffer B were diluted 20-fold into either buffer B or buffer C containing increasing concentrations of *SCaCO3 at 37 °C. Na+-dependent Ca 2+ uptake was terminated after 30 s and is plotted as a function of added 4SCaCO3. Results were analyzed by computer assisted non-linear regression analysis. K m = 102 #M, Vmax = 3.66 nmoi/mg/min. B: the same data as presented in Fig. 4A except that results are presented in a Lineweaver-Burk representation and analyzed by linear regression. Km = 74.7/~M, Vmax = 3.11 nmol/mg/min.
m e a s u r e d after addition to a NaCI m e d i u m . R u t h e n i u m red, which has been shown to be an effective inhibitor of the mitochondrial Ca 2÷ u n i p o r t e r 23, had no effect on Ca 2+ content at a concentration of 10 p M (data not shown). W h e n vesicles were first l o a d e d with 4SCa by N a + / C a 2+ exchange and then e x p o s e d to the Ca2+-ionophore A23187 (Fig. 3B arrow) with 100/~M E G T A present to maintain low extravesicular Ca 2+, Ca 2+ content was reduced to a p p r o x i m a t e l y 10% of its original value within 1 min. Only a small a m o u n t of vesicle-associated Ca 2÷ was lost when E G T A was a d d e d alone. These data confirm that Ca 2+ that becomes associated with the
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TIME (seconds) Fig. 3. Na+/Ca2+ exchange in cerebral plasma membrane vesicles derived from normal tissue-hippocampus/temporal cortex region. A: aliquots (25 /~1) of vesicles equilibrated with buffer B were diluted 20-fold with either buffer B (O) or buffer C (©) containing 100/~M 45CACO3 at 37 °C. B: data points represent Na+-dependent Ca 2+ uptake (Ca 2÷ content in buffer C minus that measured in buffer B shown in Fig. 3A). At the time indicated by the arrow the incubation media were made to contain 100 gM EGTA or 10 pM A23187 and 100 pM EGTA. Reactions were terminated at indicated times as described under Materials and Methods.
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Fig. 5. Representative experiments showing the overall effect of tissue origin on the time course of Na+/Ca2+ exchange. Na +dependent Ca 2+ uptake was measured as described in Fig. 3. Tissue origin: normal (O), AD ( i ) and NAD (O)-hippocampus/temporal cortex region.
144 vesicles during a transport reaction is, in fact, internalized. Fig. 4 shows data attempting to quantitate the relationship between Na+/Ca 2+ exchange activity and the concentration of extravesicular Ca 2+. Ca 2+ content was measured 30 s after the start of the reaction and plotted as a function of a d d e d Ca 2+. It should be noted that because of the curvature of the line that described Ca 2÷ content, m e a s u r e m e n t s made at 30 s underestimated the true initial velocity of increasing Ca 2+ content. Nevertheless, as can be seen in Fig. 4 A , B , measurements of Ca 2+ content at 30 s provided a good first approximation of the true initial velocity enabling an estimate of the K m and Vmax to be made. Estimates of K m and Vm~x could be o b t a i n e d from either a computer-assisted non-linear regression analysis of log [Ca] vs. V (Fig. 4A) or linear regression analysis of the L i n e w e a v e r - B u r k plot (Fig. 4B). A l t h o u g h similar estimates were obtained by either m e t h o d (see legend to Fig. 4), all the results described below were the result of L i n e w e a v e r - B u r k analysis. Effect o f tissue origin on Na+/Ca 2+ exchange Fig. 5 demonstrates the effect of tissue origin on the time course of N a + / C a 2÷ exchange in human cerebral
plasma m e m b r a n e s . It can be seen that m e m b r a n e s derived from A D brain had increased Na÷/Ca 2+ exchange activity as evidenced by both an increase in the initial rise in Ca 2÷ content and in elevated values of peak plateau Ca 2+ content. N A D tissues yielded m e m b r a n e s that gave values for Ca 2+ content that were similar or slightly less than that of normal origin. It is important to note that Ca 2÷ content m e a s u r e d in the absence of a Na ÷ gradient (see Fig. 3A) was not affected by tissue origin
4-
•
B
A
(data not shown). This observation further confirmed the similar purity of m e m b r a n e preparations from different tissue origins. As Na÷/Ca 2+ exchange was d e t e r m i n e d as the difference between N a ÷ - d e p e n d e n t and Na+-inde p e n d e n t Ca 2+ uptake, the increases seen in m e m b r a n e s derived from A D tissues were clearly the result of increased N a ÷ - g r a d i e n t - d e p e n d e n t Ca 2+ uptake. Next, we p e r f o r m e d a kinetic analysis of the extravesicular Ca 2+ d e p e n d e n c e of N a + / C a > exchange in each of the 3 tissue types. Fig. 6 shows both a non-linear fit (Fig. 6A) and L i n e w e a v e r - B u r k analysis (Fig. 6B) of the relationship between the concentration of a d d e d extravesicular Ca 2+ and the velocity of the reaction measured at 30 s (see also Fig. 4). It can be seen with either plot that the dominant effect observed in A D tissue was an increase in the estimated Vmax for N a + / C a 2+ exchange. A summary of the kinetic data o b t a i n e d for Na+/Ca 2÷ exchange in all the tissue samples studied is shown in Table II. The larger degree of uncertainty in the estimation of K m for the normal and A D tissues may be explained, in part, by a greater uncertainty in the estimate of initial velocities at low Ca 2+ concentrations. W h e n using the L i n e w e a v e r - B u r k analysis, this uncertainty is more likely to affect the K m d e t e r m i n a t i o n than the Vmax determination. The mean values obtained for the g m of Na+/Ca 2+ exchange were not significantly different when c o m p a r e d b e t w e e n the 3 groups: normal, A D and N A D . On the o t h e r hand, the mean Vmax of Na÷/Ca 2÷ exchange was significantly increased for A D tissue ( P < 0.05) when c o m p a r e d with either normal or N A D tissue. Figs. 7 and 8 show a composite view of the Vmax and K m d e t e r m i n e d for each tissue sample plotted vs the age at death or autolysis time for that particular sample. There was no correlation evident in either normal, A D , N A D or the total sample population between Na+/Ca 2÷ exchange Vmax o r K m and autolysis time or age at death.
3TABLE II 2-
• •
Summary of the kinetic constants obtained from Na+/Ca2+ exchange initial velocities estimated at various concentrations of extravesicular calcium
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log [Ca2+] added Fig. 6. Representative experiments showing kinetic analysis of the effect of tissue origin on Na+/Ca2+ exchange estimated initial velocity. The effect of increasing added 45CaCO~ on Na +-dependent Ca~+ uptake after 30 s was measured as described in Fig. 4. A: computer assisted non-linear regression analysis. B: LineweaverBurk analysis with linear regression. Tissue origin: normal (O), AD (11) and NAD (O)-hippocampus/temporal cortex region.
Initial velocities (30 s after the start of the reaction) were estimated over a range of added Ca 2÷ concentrations of 0.01-0.3 mM. K m and Vmax were estimated by Lineweaver-Burk analysis. Values are means _+standard deviation. Origin
n
K,, (I~M)
V , ~ (nmol/mg/min)
Normal AD NAD
6 6 4
57.9 + 28.1 71.2 + 29.3 66.1 + 3.3
4.45 + 0.58 6.68 + 1.58* 3.76 + 1.50
*
Significantly different from either normal or NAD brain 0.05).
(P <
145
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AGE AT DEATH (years) Fig. 7. Na+/Ca 2+ exchange V,,,aX(A) or Km (B) plotted against age at death (years) for either normal, AD, NAD or the total sample population. There was no correlation (P > 0.05) in either normal, A D , NAD, or the total sample population• The r-values for the regression lines were: 0.256, 0.004, 0.734 and 0.020 for Vmax;0.370, 0.010, 0.615 and 0.041 for Kin, respectively.
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AUTOLYSIS TIME (hours) Fig. 8. Na+/Ca 2+ exchange Vm~ (A) or Km (B) plotted against autolysis time (h) for either normal, AD, NAD or the total sample population. There was no correlation (P > 0.05) in either normal, AD, NAD, or the total sample population. The r-values for the regression lines were: 0.010, 0.113, 0.385 and 0.158 for Vmax;and 0.350, 0.007, 0.001 and 0.064 for Km respectively.
DISCUSSION The major finding of this study was that Na+/Ca 2+ exchange activity measured in cerebral plasma membrane vesicles derived from A D post-mortem brain tissues was significantly greater than that measured in vesicles from normal or N A D brain tissues. What is the possible role of Na+/Ca 2÷ exchange in the pathogenesis of nerve cell degeneration in A D ? The recent study of Mattson et a1.18 has clearly shown cell-specific differences in calcium homeostatic systems and that cells with superior Na+/ Ca 2÷ exchange capacity are more likely to survive an insult of increased [Cai]. It is attractive to speculate that the increased Na+/Ca 2÷ exchange activity observed in the plasma membranes of neurons from A D brain (present study) reflects the properties of surviving neurons with increased capacity for Na+/Ca 2÷ exchange. These neurons may have survived the neurodegenerative process of A D by virtue of their increased Na÷/Ca 2+ exchange activity. If this were true, it is probable that increased intracellular Ca 2÷ played a key role in the death of the non-survivors. In support of this, it has been found that
the antigenic changes in neuronal cytoarchitecture seen in cultured hippocampal cells after treatment with glutamate and Ca 2÷ influx are similar to that seen in the neurofibrillary tangles of A D 17. The conclusions of the present study must be discussed in terms of the considerable difficulty that exists in interpreting human post-mortem studies. In fact, the study of A D in post-mortem tissues presents unique factors that can adversely affect clear interpretation of the data. In post-mortem studies in general it is difficult if not impossible to make a direct correlation between a neurochemical measurement and a pathological observation. Even worse for the A D researcher is the c o m m o n practice to reserve one hemisphere for pathology while the other is frozen for neurochemical studies. Several factors (epiphenomena) are known to affect the results of post-mortem studies 25. These include: patient age, sex, drug history, immediate pre-mortem status (sudden death or prolonged coma) and postmortem delay. In the present study brain tissues were age matched with similar post-mortem delays (see Table I).
146 The effect of post-mortem delay on Na+/Ca 2+ exchange activity in h u m a n brain tissues has not been characterized. Unfortunately, in this retrospective study, patient records of the medication administered and the duration of disease were not readily available. The study of A D in post-mortem tissues presents yet two more complicating issues of the severity of the disease and tissue atrophy. In this study we are considering that all brain samples from A D patients reflect end-stage disease, although this clearly may not be the case as older patients may die at more early stages of the disease. It is known that changes occur in the relative proportion of different n e u r o n types and glial cells during the tissue atrophy of A D . It is unlikely that the proliferation of astrocytes that occurs in A D would account for the increased Na+/Ca 2+ exchange activity, as Na+/Ca 2÷ exchange is found in such high density in the ABBREVIATIONS AD EGTA
Alzheimer's disease ethyleneglycol bis(fl-aminoethylether)-N,N'-tetraacetic acid
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plasma m e m b r a n e s of neurons. In fact, glial cell proliferation would be expected to cause an underestimation of the increase in Na+/Ca 2÷ exchange activity. Notwithstanding the possibility of post-mortem artifact, the observations of this study are consistent with the view that increased Na+/Ca 2÷ exchange activity in A D brain tissues is a result of the disease state. A better understanding of the role of Na÷/Ca 2÷ exchange and increased intraceilular Ca 2÷ in the pathogenesis of the A D must await further knowledge of the temporal sequence of events that underlie the formation of senile plaques, neurofibrillary tangles and cell death. Acknowledgements. We would like to thank David Henderson, Department of Anatomy, Oral Roberts University School of Medicine, who performed the electron microscopy. This work was supported in part by a grant from the Founders of Doctors' Hospital, Tulsa, OK, U.S.A.
HEPES NAD SDS QNB
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid non-Alzheimer dementia sodium dodecylsulfate quinuclidinylbenzilate
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