intraterminal mitochondria; it is blocked by ruthenium red, by FCCP, and by azide. + dinitrophenol + oligomycin. There is, however, a residual ATP-dependent ...
Calcium Buffering in Presynaptic Nerve Terminals I. Evidence for Involvement of a Nonmitochondrial A TP-Dependent Sequestration Mechanism M O R D E C A I P. B L A U S T E I N , R O N A L D W. R A T Z L A F F , C. K E N D R I C K , and E R I K S. S C H W E I T Z E R
NANCY
From the Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110. Dr. Kendrick's present address is Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
A B $ T R AC T A latent A T P - d e p e n d e n t Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. T h e data indicate that a fraction of the Ca uptake (measured with ~Ca) is associated with the intraterminal mitochondria; it is blocked by r u t h e n i u m red, by FCCP, and by azide + dinitrophenol + oligomycin. T h e r e is, however, a residual A T P - d e p e n d e n t Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondriai) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. T h e proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the A T P - d e p e n d e n t Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca 2+ concentration (buffered with EGTA) is 80% of the 45Ca was lost in 30 s, and >90% was lost in 3 min; in contrast, only -20% of the 4SCa was lost after 5 min when the efflux solution contained 250 tzM EGTA (which should have reduced the free Ca ~+ to below 10-T M). The EGTA may be expected to compete effectively for superficially bound Ca, whereas A-23187 may simply make membranes permeable to Ca. Thus, it seems reasonable to conclude that most of the Ca is stored in membrane-bound organelles which survive the osmotic shock, and that the Ca is not simply bound to exposed surfaces (membranes). Net Ca Sequestration
The experiments described in the preceding section indicate that ATP promotes the transport of ~Ca into compartments which are not in direct communication with the bathing medium. One possibility is that this transport simply involves the exchange of ~Ca for the endogenous 4~ in the tissue fragments (cf. 7); alternatively, of course, the ATP may promote net Ca sequestration. Measurements by atomic absorption spectrophotometry of net Ca concentrations in incubation fluids and tissue pellets provide strong support for the latter alternative. Fig. 8, which presents data from three experiments, shows that ATP promotes the net accumulation of Ca by the tissue pellets; although not illustrated here, there was a concomitant decrease in the Ca concentration of the ATP-containing incubation fluids, relative to the ATP-free controls (cf. 6). As reported previously (6), the net transfer of Ca from medium to tissue, in the presence of ATP, was enhanced somewhat when oxalate was present in the medium; this is consistent with earlier observations (35) that oxalate enhances ATP-stimulated ~Ca uptake by the tissue fragments. As in the case of sarcoplasmic reticulum (43), the results with oxalate can be readily explained if the organelles into which the Ca is transported are permeable to the oxalate so that, as the Ca accumulates inside the vesicles, the Ca-oxalate solubility product is
32
THE JOURNAL
OF GENERAL PHYSIOLOGY
9 VOLUME
72 9 1978
exceeded and Ca-oxalate precipitates; the intravesicular ionized Ca 2+ concentration is thereby r e d u c e d , so that more Ca can be t r a n s p o r t e d in. This may indicate that the Ca is t r a n s p o r t e d against a concentration gradient. T h e effects observed with oxalate may be r a t h e r small (6) because p h o s p h a t e ions can p r o d u c e the same effect (cf. 63): p h o s p h a t e ions are normally present in r a t h e r I00
i-T. 50 ,E
c
lo
5
I
0
I
I
I
2 5 Time (min)
I
I
4
5
FIGURE 7. Effects of EGTA - ADP and ionophore A-23187 on Ca efflux from *~Ca-loaded disrupted terminals. Previously lysed synaptosomes were loaded with ~Ca by incubating them for 10 rain at 30~ C in KCI medium (Table I) containing 10 /zM CaCI2 (labeled with tracer ~Ca) and 1 mM ATP. The suspensions were centrifuged at 15,000 g (max) for 6 min at 4~ C. The supernatant solutions were discarded, and the pellets were resuspended in Ca-free KCI medium (Table I). Aliquots were removed and filtered (zero-time samples), and additional Ca-free KC1 medium was added to each sample; this solution also contained (final concentration after dilution) 250 /zM EGTA (@), or 10 /zM A-23187 (&). The samples were incubated at 30~ C; portions of the suspensions were removed at the indicated times, and filtered through Millipore filters. The protein concentration in the efflux suspensions was 1.0 mg/ml; the fully loaded tissue fragments (i.e., at t = 0) contained approximately 900 pmol Ca (as ~Ca) per mg protein. Each point represents the mean of three determinations normalized to the fully loaded (100% Ca remaining at t = 0) condition; the bars indicate -+SEM.
high concentration, even if no e x o g e n o u s inorganic p h o s p h a t e is a d d e d , because o f A T P hydrolysis (7).
Distribution of Ca-Accumulating Particles in a Discontinuous Sucrose Density Gradient In an effort to obtain i n f o r m a t i o n about the identity o f the organelles responsible for the A T P - d e p e n d e n t Ca uptake, a series o f subfractionation studies were u n d e r t a k e n with lysed synaptosomes (see Methods). T h e various centrifugation fractions (Fig. 1) were assayed for protein content and A T P - d e p e n d e n t Ca uptake activity; data f r o m a representative e x p e r i m e n t are shown in Table V.
Bta~USTZIN ZT AL.
33
Calcium Sequestration in Nerve Terminals
O n e o f the main observations was that only a tiny fraction ( < 1 % ) o f the n o n m i t o c h o n d r i a l A T P - d e p e n d e n t u p t a k e activity f r o m the lysate was f o u n d in the synaptic vesicle pellet (1)4); m o r e o v e r , the relative specific activity o f the Ca u p t a k e was r a t h e r low ( - 0 . 5 ) . Nearly all o f the activity s e d i m e n t e d in the m o r e dense fraction (P~). A similar result was o b t a i n e d w h e n Pa was pelleted at 11,500 []
16
9 ATP-free
I S = +ATP
14
BE=,,
-I"-
~o
j Ii A
o ~ o
Z
0 A
B
C
I
FIGURE 8. Effects of ATP on net Ca uptake by disrupted synaptosomes. Data from three experiments (A, B and C, respectively) are shown. Equilibrated synaptosome pellets were resuspended in 1.0 ml lysis solution; after 1 min, 1.0-ml aliquots of buffered solutions containing KCI, NaCI, and CaC12 were added to give a final Ca concentration of 6-8 /~M, and a solution composition similar to "KCI medium" (Table I). All solutions contained 0.1 mM DNP, 0.! mM NaNa, and 0.7 #g/ml oligomycin; in some instances (hatched bars) the solutions contained ATP (final concentration = 1 mM). The protein concentration in the suspensions was 0.4-0.6 mg/ml. The suspensions were incubated at 30~ C for 7 (A), 15 (B), or 21 min (C), and then centrifuged at 15,000g (max) for 6 min at 4~ C. Both supernatant solutions and pellets were assayed for Ca content (see Methods). The data shown represent the means of Ca determinations on three (B) or four (A and C) tissue samples; error bars indicate -SEM. g for 20 min, even t h o u g h , in this e x p e r i m e n t , 1)4 (the synaptic vesicle fraction) c o n t a i n e d nearly 2% o f the total lysate protein (data not shown). U p o n sucrose g r a d i e n t fractionation o f Pa, the Ca u p t a k e was m a r k e d l y e n r i c h e d (specific activity = 4-6) at the 0.6-1.0 M sucrose interface (see T a b l e V). Very little activity r e m a i n e d at or above the 0.32-0.6 M sucrose interface, a n d little activity was o b s e r v e d in the m i t o c h o n d r i a l pellet (P'a; see Refs. 14, 15, 67). A b o u t 80% or m o r e o f the n o n m i t o c h o n d r i a l Ca u p t a k e activity was r e c o v e r e d routinely, a f t e r sucrose g r a d i e n t fractionation. In s u m , a l t h o u g h these data do not p r o v i d e positive identification o f the Ca sequestration sites, they do indicate that n e i t h e r the "synaptic vesicles," p r e s u m ably, those associated with n e u r o t r a n s m i t t e r storage (cf. 15, 67), n o r the m i t o c h o n d r i a are responsible for this activity.
34
THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 72 9 1 9 7 8 DISCUSSION
Methodology: Use of Osmotically Disrupted Synaptosomes Before evaluating the data obtained in this study, it is imperative to consider the validity o f the osmotically disrupted s y n a p t o s o m e p r e p a r a t i o n that was employed in most o f the experiments. As noted in Results, the use o f osmotic shock seems to be justified by the evidence that organelles such as m i t o c h o n d r i a and sarcoplasmic reticulum survive this type o f treatment, and by the fact that c o m p a r a b l e results are obtained when saponin is used to disrupt the terminals. TABLE
V
DISTRIBUTION OF ~Ca UPTAKE ACTIVITY IN DISRUPTED SYNAPTOSOMES SUBJECTED TO DIFFERENTIAL AND DISCONTINUOUS SUCROSE GRADIENT CENTRIFUGATION ATP-delmndent r uptake
Fraction
RSAt
Total activity
Total protein
pmoll5 rain
rag
pmollmg protein per 5 rain*
Whole lysate Pa Light band (0.6-1.0 M interface) Heavy band (1.0-1.2 M interface) Pellet (P'a) Residual sucrose Gradient totals 83 P4
S,j
652 893 3,873
( 1.0) 1.37 5.94
33,056 31,656 7,281
50.7 35.4 1.88
1,198
1.84
5,726
4.78
265 1,076
0.41 (2 30)
89 341
0.14 0.52
-
Overall totals
-
3,678 ~ 26,132 (83%)w 1,066 136 -
27,334 (83%)w
13.9 8.78 29.3 (83%)[I 11.3 0.4 10.5 51.5 (102%)[[
* Incubated at 30~ in the presence of 0.1 mM NAN3,0.1 mM DNP, and 0.7 p.g/ml oligomycin. Relative specific activity (=ATP-dependent uptake in fraction + ATP-dependent Ca uptake in whole lysate). wPercent of activity recovered. ]1Percent of protein recovered. In addition to osmotic shock and saponin treatment, we also a t t e m p t e d to expose intracellular organelles by e m p l o y i n g very vigorous h o m o g e n i z a t i o n conditions. T h e latter p r o c e d u r e yielded preparations with relatively little, and poorly reproducible A T P - d e p e n d e n t Ca uptake activity, in contrast to the osmotic shock m e t h o d . Several protocols for osmotic shock disruption were tested. W h e n the hypotonic b u f f e r ("lysis') solution contained 36 mM KCI, in addition to the constituents listed in Table I, the yield o f A T P - d e p e n d e n t Ca uptake activity was r e d u c e d by - 2 0 - 3 0 % (data not shown). O n the o t h e r h a n d , m a r k e d variation in the d u r a t i o n o f e x p o s u r e to the standard lysis solution had but little effect: t r e a t m e n t for as short a period as 12 s or as long as 7 min (cf. Fig. 3) resulted in preparations with essentially identical A T P - d e p e n d e n t Ca uptake activity. T h u s , the critical step a p p e a r e d to be the initial e x p o s u r e to the
Bt.AUSTEI~ ET AL. CalciumSequestration in Nerve Terminals
35
very hypotonic solution. A 1-min exposure to the lysis solution followed by return to normotonic solution was arbitrarily chosen for use in most experiments. Another important caveat, in terms o f transmitter specificity, concerns the heterogeneity of the nerve terminal population in our synaptosome preparations. This will be a particularly crucial concern in evaluating the quantitative aspects o f Ca storage (7); however, for present purposes it seems adequate to assume that most nerve terminals handle Ca in a uniform fashion, and that the Ca-storing systems unde r discussion are probably present in most types o f terminals. Nevertheless, it should be clearly recognized that our data merely reflect the average o f the activity in the heterogeneous population o f terminals. Finally, the question o f whether or not the Ca-storing organeUes are actually intraterminal structures must be reconsidered. Several observations are consistent with the hypothesis that they are derived from nerve terminals. In the first place, as will be illustrated in the subsequent article (7; see Figs. 9 and 10), nerve terminals apparently do have nonmitochondrial Ca sequestration sites with a high affinity for Ca. T h e present report shows that synaptosome preparations, in which a large fraction of the terminals are structurally and functionally intact (5), have a latent nonmitochondrial Ca uptake activity; this activity is manifest by procedures known to disrupt plasma membranes, thereby permitting the Ca sequestering organelles direct access to the A T P and (or) Ca in the media. T h e n , because the nonmitochondrial Ca uptake activity and the nerve terminals distribute in parallel fashion on preparative sucrose gradients (Table II and related text), we conclude that intraterminal organelles are probably responsible for most of this Ca uptake activity. However, we cannot eliminate the possibility that structures derived from other types of cells (e.g., glia) may also be involved. The Sites of Ca Sequestration in Presynaptic Nerve Terminals
T h e evidence presented in this report clearly shows that there are at least two types of sites, presumably situated within presynaptic terminals, that can store Ca in the presence o f A T P probably as a consequence of A T P hydrolysis (cf. 7). One type of Ca storage site has all the properties normally associated with mitochondrial Ca accumulation: Ca storage is promoted by ATP, and is blocked by a variety o f mitochondrial poisons (DNP, oligomycin, NaNa, FCCP and ruthenium red). In preliminary studies (not shown), we also found that the mitochondrial poison-sensitive Ca uptake is promoted by a normal Krebs cycle intermediate, succinate. This Ca uptake is unaffected by saponin, presumably because the organelles involved have little cholesterol in their membranes; it is minimally affected by trypsin, which can be explained if the trypsin is prevented by the outer membrane (56) from reaching the active sites o f Ca translocation at the inner membrane (39). Taken together, these observations indicate that one site of Ca sequestration is the intraterminal mitochondrion, as suggested by previous workers (59). O ur data also show that there is a second site o f Ca sequestration, with somewhat different properties: the Ca uptake is supported by ATP, but not by succinate (data not shown); it is unaffected by mitochondrial poisons, but is
36
T H E JOURNAL OF GENERAL P H Y S I O L O G Y ' V O L U M E
7 2 . 1978
abolished by trypsin. This Ca storage system has a somewhat higher affinity for Ca than does the mitochondrial system (Table IV; and see 7). The nonmitochondrial Ca storage system apparently involves membranebound organelles: the accumulated Ca is not removed by the Ca chelator, EGTA, but Ca is rapidly released and uptake is prevented by A-23187, an agent known to increase markedly the Ca permeability of natural membranes and lipid bilayers. What is the Nature of the Second (Nonmitochondrial) Ca Storage Site? A variety of organelles must be considered as possible candidates for sites of Ca sequestration: (a) plasma membrane vesicles; (b) synaptic (transmitter-storing) vesicles; (c) endoplasmic reticulum; and (d) mitochondria (which might, for example, have two different types of Ca storage mechanisms). To begin with, mitochondria may be ruled out by the fractionation studies (Tables II and V), because most of the poison-insensitive Ca uptake activity is found in the intermediate-density fractions, and relatively little activity appears in the mitochondrial pellets. The possibility that the ATP-stimulated transport we observed may be a manifestation of Ca extrusion, i.e., Ca transport into everted (inside-out) plasma membrane vesicles formed at the time of lysis, must also be considered. However, two observations appear to eliminate this possibility. One is that the nonmitochondrial ATP-stimulated Ca uptake is not affected by low concentrations of saponin or digitonin. Saponin exposes both the mitochondrial and nonmitochondrial Ca uptake mechanisms (Table III), probably by disrupting the cholesterol-rich (9) plasma membrane. The second relevant observation is that the Ca sequestration proceeds in the virtual absence of Na, either inside or outside the vesicles (see Fig. 3). Net Ca extrusion from resealed presynaptic terminals requires external Na (4); this would be equivalent to Na inside the hypothetical everted plasma membrane vesicles, but the lysis solution contained no Na (Table I). Recently, Blitz et al. (8) suggested that "coated vesicles" from brain may take up Ca by an ATP-dependent mechanism. Their conclusion rests primarily on the assumption that other organelles did not significantly contaminate their preparations. A variety of observations and considerations have led us to question the conclusion of Blitz et al., and to propose, instead, that the nonmitochondrial sites of Ca sequestration may be the intraterminal smooth endoplasmic reticulum. Synaptic vesicle membranes have a cholesterol content comparable to that of synaptic plasma membranes (10, 53); thus, the fact that the Ca uptake activity is resistant to saponin (Table III) may imply that synaptic vesicles are not involved in Ca sequestration. If the "coated vesicles" of Blitz et al. are, as they state, involved in synaptic vesicle membrane recycling, they presumably include components of the smooth ("uncoated") synaptic vesicle membranes. A second critical observation is that synaptic vesicle fractions prepared by the method of DeRobertis et al. (14) contain little ATP-dependent Ca uptake
BLAUSTgIN ET AL. CalciumSequestration in Nerve Terminals
37
activity; m o s t o f t h e activity is f o u n d i n m o r e d e n s e f r a c t i o n s ( T a b l e V). 4 T h e s e d a t a , too, a r e s u g g e s t i v e , b u t n o t c o n c l u s i v e . F i n a l l y , as n o t e d in t h e I n t r o d u c t i o n , t h e r e is c o n s i d e r a b l e b i o c h e m i c a l a n d m o r p h o l o g i c a l e v i d e n c e t h a t , in m a n y types o f cells i n c l u d i n g s e c r e t o r y cells, C a is s e q u e s t e r e d in s m o o t h e n d o p l a s m i c r e t i c u l u m . O n e o f t h e m o s t c o n v i n c i n g d e m o n s t r a t i o n s is t h a t o f H e n k a r t et al. (29). T h e y f o u n d t h a t t h e e n d o p l a s m i c r e t i c u l u m in t h e a x o p l a s m o f s q u i d g i a n t a x o n s swelled w h e n t h e a x o n s g a i n e d Ca as a r e s u l t o f i n c u b a t i o n in C a - r i c h o r N a - f r e e sea w a t e r s (cf. 28); m o r e o v e r , w h e n o x a l a t e was i n j e c t e d i n t o t h e a x o p l a s m , e l e c t r o n - d e n s e p r e c i p i t a t e s , p r e s u m a b l y , Ca o x a l a t e , w e r e f o r m e d in t h e e n d o p l a s m i c r e t i c u l u m o f Cal o a d e d a x o n s (29). I n s u m , t h e s e c o n s i d e r a t i o n s a r e c o n s i s t e n t with t h e view t h a t e n d o p l a s m i c r e t i c u l u m , w h i c h is also s e e n in p r e s y n a p t i c t e r m i n a l s , 5 a n d n o t s y n a p t i c vesicles, m a y be t h e site o f n o n m i t o c h o n d r i a l A T P - d e p e n d e n t Ca s e q u e s t r a t i o n i n neuronal (including nerve terminal) cytoplasm. We thank Dr. B. K. Krueger for his constructive critique of the manuscript, Mr. V. Creasy for fabricating the filter banks, Ms. J. Jones for preparing the typescript, and Mrs. S. McConnell and Mr. W. DiPalma for the illustrations. This research was supported by grant NS-08442 from the National Institute of Neurological and Communicative Disorders and Stroke. Mr. Schweitzer is a National Science Foundation predoctoral fellow. Received for publication 27 December 1977. REFERENCES 1. BAKER, P. F. 1972. T r a n s p o r t and metabolism of calcium ions in nerve. Prog. Biophys. Mol. Biol. 24:177-223. 2. BLAUSTEIN, M. P. 1974. T h e interrelationship between sodium and calcium fluxes across cell membranes. Rev. Physiol. Biochem. Pharmacol. 70:33-82. 3. BLAUSTEIN, M. P. 1975. Effects of potassium, veratridine and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro. J. Physiol. (Lond. ) . 247:617-655. 4. BI~USTEIN, M. P., and A. C. ECTOR. 1976. Carrier-mediated s o d i u m - d e p e n d e n t and calcium-dependent calcium efflux from pinched-off presynaptic nerve terminals (synaptosomes) in vitro. Biochim. Biophys. Acta. 419:295-308. 5. BLAUSTEIN, M. P., N. C. KENDRICK, R. C. FRIED, and R. W. RATZLAFF. 1977. Calcium metabolism at the mammalian presynaptic nerve terminal: lessons from the synaptosome. In Neuroscience Symposia. VoI. II. Approaches to the Cell 4 In most cell fractionation studies the smooth endoplasmic reticulum is recovered in a "microsome" fraction which does not generally sediment at 15,000 g. However, the homogenization conditions employed in these experiments are usually quite harsh, often involving, for example, tissue disruption in a Polytron homogenizer or Waring Blender. In contrast, when osmotic shock is used to disrupt cells, the endoplasmic reticulum may not fragment into microsomes; the endoplasmic reticulum may then sediment along with most other cell membranes, including the plasmalemma, as appears to be the case in our preparations. 6 McGraw, C. F., A. V. Somlyo, and M. P. Blaustein. 1978. Ultrastructural localization of calcium by electron probe analysis in presynaptic nerve terminals. Soc. Neurosci. Syrup. In press.
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THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 72 9 1978
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23. 24.
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