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Mg2+ ATPase and ATP-dependent Ca2+ transport in synaptic membranes
Brain Research, 329 (1985) 39-47 Elsevier
39
BRE 10578
Activation of Central Muscarinic Receptors Inhibit Ca2+/Mg 2+ ATPase and ATP-Dependent Ca 2÷ Transport in Synaptic Membranes DAVID H. ROSS, S. MARTIN SHREEVE and M. G. HAMILTON Division of Molecular Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284 (U.S.A.)
(Accepted June 12th, 1984) Key words: Ca2+/Mg2+ ATPase - - ATP-dependent Ca2÷ uptake - - cholinergic agonists - - transmitter release
Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg 2÷ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ = 0.5/xM, close to the cytosolic concentration of Ca2÷. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20/~M), both Ca2+/Mg2÷ ATPase and ATP-dependent Ca2÷ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 #M) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2÷ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.
INTRODUCTION It is believed that cholinergic nerve terminals contain receptors which, when activated, regulate the release of acetylcholine (ACh) by a feedback mechanism. Electrically evoked release of A C h from brain slices was reduced following administration of cholinesterase inhibitorsS,24. Muscarinic antagonists block or potentiate this response in a concentration-dependent fashion. Hadhazy and Szerb 12 have reported that potassium-dependent release of A C h from hippocampus, striatum and cortex slices was reduced by physiostigmine, oxotremorine and carbamylcholine, while atropine potentiated the release. These findings were confirmed by Nordstrom and Bartfai 21, who demonstrated that [3H]ACh release from the hippocampus was reduced by muscarinic agonists (carbachol) and increased by antagonists (atropine). This evidence suggests that presynaptic muscarinic receptors, which are responsive to agonists and antagonists, may modulate the release of ACh. Trans-
mitter release mechanisms are known to be Ca2+-dependent t4 and probably involve phosphorylation of proteins 7. Krueger et al. 13 have shown that agents which induce or enhance depolarization of synaptosomes increase the level of intracellular Ca 2÷ and Ca2+-dependent protein phosphorylation. Recent studies by D e L o r e n z o and colleagues7, s have also demonstrated that depolarization of synaptic protein produces Ca2+-dependent phosphorylation of synaptic vesicles which is calmodulin-dependent and resuits in neurotransmitter release. Since changes in intracellular Ca 2÷ levels provide the signal for release, muscarinic agonists and antagonists may alter A C h release by interfering with buffering mechanisms for the optimal levels of intracellular Ca 2÷. Michaelson et al. 17 reported that muscarinic agonists (oxotremorine) block the K+-dependent release of acetylcholine from cholinergic Torpedo synaptosomes and effect antagonized by atropine. While calcium-dependent K+-stimulated release of acetylcholine was seen to occur simultaneously with 45Ca2+ in-
Correspondence: D. H. Ross, Division of Molecular Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A.
40 flux; no effect of oxotremorin~ or atropine on K +stimulated 45Ca2+ influx could be demonstrated. The release of ACh was also seen to parallel an increase in Ca2+-dependent phosphorylation of a 100,000 dalton protein. Muscarine inhibited this Ca2+-depend ent phosphorylation, a response which was antagonized by atropine, these studies prompted us to evaluate two mechanisms which buffer cytosolic Ca 2+ and which may be subject to cholinergic receptor regulation. Ca2+/Mg2+ ATPase and ATP-dependent Ca 2+ sequestration in synaptic membranes are believed to play a major role in regulating cytosolic Ca 2+ (refs. 13, 20, 22). This paper reports the effects of cholinergic receptor activation on two Ca 2÷ buffering mechanisms, Ca2+/Mg 2÷ ATPase and ATP-dependent Ca 2+ uptake in synaptosomal membrane preparations.
band of tissue at the 7.5-12% interphase was removed and slowly diluted with an equal volume of D-glucose and HEPES. The pH was adjusted to 7.4 with HCI. This suspension was centrifuged at 25,000 g for 20 min to concentrate synaptosomal protein. The resultant pellet was slowly resuspended in resting buffer to a protein concentration of 1.1)-1.5 mg/ml and used as outlined below.
Preparation of lysed synaptosomal membranes
MATERIALS AND METHODS
Synaptosomes, isolated as described above, were resuspended in 20 mM Tris - 0.5 mM DTE, pH 8.1, in an ice bath for 60 min, to hypotonically lyse them. This membrane suspension was mixed every 20 min to facilitate the lysing procedure. At the end of 60 min, the suspension was pelleted at 40,000 g for 20 min and resuspended to a protein concentration of 0.5-1.0 mg/ml. This fraction was used for enzyme and ATP-dependent Ca 2+ uptake assays.
Materials
Measurement of Ca2+accumulation by synaptosomes
HEPES, sucrose, Na2-ATP and dithioerythritol (DTE) were obtained from Sigma (St. Louis, MO). All buffer salts were used as their chlorides and were purchased from Fisher Scientific (Pittsburgh, PA). Sodium dodecyi sulfate (Lauryl sulfate) was purchased from Pierce Chemicals (Rockford, IL). [y-32p]ATP (10-40 Ci/mmol), [45CaZ+]chloride, PPO and POPOP were obtained from New England Nuclear (Boston, MA), Millipore filters (0.45/~m, Type HAMP), were purchased from Millipore (Bedford, MA). Ammonium molybdate and isobutanol were purchased from Aldrich and Fisher Chemicals.
Methods Isolation of synaptosomes Male rats (150-200 g, Sprague-Dawley) were sacrificed by decapitation, brains rapidly removed and washed with ice-cold saline. A 10% homogenate was made of the cortex in 0.32 M sucrose and a crude P2 pellet fraction containing nerve endings isolated by differential centrifugation, according to the method outlined by Cotman and Matthews 6. This P2 pellet was resuspended in 0.32 M sucrose and layered over a 7.5-12% Ficoil (in 0.32 M sucrose) gradient and centrifuged at 65,000 g for 60 min. The resultant
Synaptosomes were suspended in resting buffer (RB) containing, in mM, NaCI (132), KC1 (5), MgC12 (1.3), D-glucose (10) and Tris (25), and the pH was adjusted to 7.4 with HCI. Synaptosomes were generally used within 30 min of final resuspension and kept cold (4 °C) until temperature equilibration, One-mt aliquots of this suspension were pre-equilibrated with appropriate [Ca2+]o at 37 °C for 5 min. in control samples, synaptosomes were pre-equilibrated at 37 °C for 10 rain. To initiate the uptake process, 100 ~1 of synaptosome suspension was added to each incubation tube (also preincubated at 37 °C). Each incubation tube then contained 100 #1 of synaptosome suspension, 100 pl ~5Ca2+ (0.45 ~Ci) and 800/~1 Ca2+C12 at various concentrations prepared in RB. Depolarization of synaptosomes was effected by substituting KCI for NaC1 in the buffer, achieving a final concentration of 25 mM K +. The combined Na +- and K + concentration was constant at 137 raM. The tubes were incubated in Dubnoff metabolic shaker at 37 °C, for 5 s (fast phase) or 30 s (slow phase). Termination of the reaction was performed by rapid addition of 10 ml ice-cold quench buffer (RB containing 3 mM EGTA). In some experiments, the synaptosomes were lysed at this point by using a hypotonic quench buffer (containing in mM, E G T A (3) and Tris (25), pH 7.4). The contents of each tube were
41 immediately filtered over Gelman (GA-6, Metricel, 0.45/~m, 25 mm) filters. The filters were washed (4 × 5 ml) with ice-cold RB containing 500/~M Ca 2÷ (high affinity) or 4 mM Ca 2÷ (low affinity). Filtration time under these conditions was about 10 s. Filters were allowed to dry and counted by liquid scintillation spectrometry. Ca 2+ accumulation was expressed as nmol of Ca 2÷ accumulation/mg of protein. Voltage-' dependent Ca 2÷ accumulation was calculated as the difference between the accumulation at 5 mM K ÷ and that at 25 mM K+.
Measurement of Ca2+/Mg2+ A TPase activity in lysed synaptosomes CaZ+-stimulated ATP hydrolysis was measured as outlined by Javors et al. 13 with the following modifications: lysed synaptosomes were preincubated at 37 °C in the presence or absence of drugs at different concentrations. The total final volume (2 ml) of the reaction medium contained KC1 (100 mM), H E P E S (20 mM, pH 7.4), MgCI 2 (250/~m), E G T A (100/~M), ouabain (1 mM), 50-150 ktg membrane protein and ATP (100 ~M). Ca2+ concentrations in the media were regulated from 0.1 to 5/~M with E G T A buffers, as described by Bartfai 1. The enzyme reaction was initiated by the addition of the ATP solution containing [3zp]ATP, following pre-equilibration with substrates. The reaction was terminated 60 s later, with a 2-ml volume of 5% T C A - 5 % SDS stopping solution. Ammonium molybdate (2 ml) was added, followed by isobutanol (1.5 ml) to complex and extract the released 32p i into the organic phase. The mixture was either shaken or vortexed; then the two phases were allowed to stand 10 min to separate. The organic layer (500/A) was removed by Eppendorf pipette, transferred to liquid scintillation vials and counted in a toluene POPOPPPO fluor by an Isocap 300. Ca2+-stimulated ATP hydrolysis (Ca 2÷ ATPase) is expressed in nmol Pi released/mg/min by subtracting Mg2+-stimulated ATPase activity from Ca2+Mg 2÷ activity. A tissue blank containing no Ca 2÷ or Mg 2÷ was included to determine non-specific ATPase activity.
A TP-dependent Ca2+ uptake by lysed synaptosomes ATP-dependent and -independent transport by lysed synaptosomal membranes was measured in the presence and absence of 100/~M ATP, as described
by Javors et al. 13. Lysed synaptosomai membranes were prepared and preincubated in the same manner as for Ca2+ stimulated ATPase activity, using identical buffer components and reaction times. 45Ca2+ (0.6/~Ci) was added two min prior to the addition of ATP (100/~M) to reaction media containing varying concentrations of Catree, 2+ as determined by E G T A buffers 2z. Following the addition of 45Ca2+, ATP was added to initiate the transport reaction for 60 s. The reactions were terminated by addition of ice-cold stopping solution (3 ml) containing'KC1 (100 mM), E G T A (100/~M), CaCl 2 (500/tm), HEPES (20 mM) and MgC12 (250/tM), at pH 7.4, 25 °C, followed by rapid filtration (5-7 s) over MiUipore filters (0.45 HAMP). The filters were washed 4 times with 3 ml stopping solution, allowed to dry and then counted in a toluene-based POPOP-PPO scintillation solution. Ca 2+ uptake was expressed as ATP-stimulated uptake in nmol/mg/min, uncorrected for ATP-independent Ca 2+ binding.
Assay of Na+-dependent Cae+ efflux Na+-dependent Ca 2+ effiux was performed according to the method outlined by Blaustein and Ector 2. Synaptosomes were prepared as outlined above. Synaptosomes were resuspended in a physiological medium (0.6-0.85 mg protein in 0.5 ml of Na + + 5 K + buffer) and equilibrated for 12 min, at 30 °C. Calcium loading of the synaptosomes was initiated by addition of 0.5 ml of 137 mM potassium chloride-5 mM NaC! containing 45Ca2+ (--- 2 ~Ci//~mol Ca 2+) in a final volume of 1.0 ml. This preparation was incubated for 2 min at 30 °C with 45Ca2+; 0.5 ml of the suspension was then transferred to a tube containing 10 ml of the effiux solution (containing 132 mM NaC1 and 5 mM KC1). In some experiments, the Na + was replaced by equimolar chloride to check for the Na + dependency. When choline was substituted, the solution did not contain Ca 2+. The solutions were mixed and then filtered at various time intervals. The suspensions were vacuum filtered on pre-washed 0.3/~M Millipore filters. Each filter was then washed with 10 ml ice-cold choline + 5 K +. Filters were dried, placed in liquid scintillation vials and counted in a toluene/ETOH-based fluor with PPO and POPOP. Protein content was determined by Lowry et al. 16. Ca 2+ effiux was calculated as the difference between 45Ca2+ remaining on the filter af-
42 ter immediate loading and the a m o u n t remaining at different intervals and expressed as % of control.
150 ¸ ,_>
Miscellaneous procedures Protein m e a s u r e m e n t s on synaptosomal suspensions were m a d e by the m e t h o d of Lowry et al.16 using bovine serum albumin as a standard, and an autoanalyzer. The water used in the experiments was double-deionized and was shown by atomic absorption spectroscopy to contain negligible amounts of Ca 2+. In addition, the buffers containing Ca 2+ were analyzed by atomic absorption spectroscopy and found to be accurate ( + 0.05 ¢tM). Standard errors for mean stimulated uptake were calculated as described by Nachshen and Blaustein x9. RESULTS
Effects of cholinergic agonists on Ca2+/Mg2+ A TPase activity in lysed synaptosomal membranes Changes in the release pattern of the cholinergic transmitter acetylcholine ( A C h ) may result from altered intracellular Ca 2÷ buffering mechanisms. It was of interest, therefore, to evaluate the effects of two cholinergic agonists, muscarine and o x o t r e m o rine, on Ca2+/Mg2+ A T P a s e activity. These studies are presented in Fig. 1. E n z y m e activity was evaluated in lysed synaptosomes in the presence of muscarine ( 1 - 2 0 / ~ M ) following a drug incubation p e r i o d of 15 min. E n z y m e activity was evaluated as a function of various Ca 2÷ concentrations, from 0.1 to 5/~M. Ca 2÷ concentrations used were designed to approximate those cytosolic concentrations believed reached during the depolarization cycle 3.4. A s seen in Fig. 1, increasing the concentration of [Ca2+]o p r o d u c e d an activation of Cae+/Mg 2+ A T P a s e , as m e a s u r e d by an increase in A T P hydrolysis. This response to increasing [Ca2+]o reached a maximum at 5 # M , a concentration which is believed to be maximal for free cytosolic Ca 2÷ in the nerve terminal9. Muscarine, in increasing concentrations, p r o d u c e d a progressive decrease in Ca2+/Mg 2+ A T P a s e activity. Similar changes were seen when the agonist o x o t r e m o r i n e was used. As illustrated in Fig. 2, o x o t r e m o r i n e ( 1 - 2 0 / ~ M ) also decreased Ca2+/Mg2+ A T P a s e activity by reducing the Vmax. O x o t r e m o r i n e a p p e a r e d to be less potent than muscarine when comparing the d e g r e e e of inhibition of enzyme activity seen at 2.5 and 5.0/~M Ca:*.
Conlrol E
arine
"-. I00(1)
20,~M
_
i
w(/'/ ÷o (_.9
Ca'}ree [#M]
Fig. 1. Effect of muscarine on Ca2+-stimulated ATPase as a function of [Ca2+}o. Lysed synaptosomes were preincubated with muscarine (15 min) at 1, 10 and 20~M. Ca2+/Mg2+ ATPase was 2+ assayed by measuring Pi release at different Catr~e concentrations, from 0.1-5.0#M. Each point on the curve represents the mean of 6 individual experiments, each performed in triplicate. ABMDP computer program package, from the University of California, was used to compute analysis of variance. A threeway analysis of variance was used to determine the differences between curves at each drug concentration. Values at 2.5 and 5.0 #M Ca 2+ were significantly different from controls at 1, 10 and 20 mM muscarine. P < 0.05.
Influence of atropine on muscarine-dependent decrease in Ca2+/Mg2 +A TPase activity in lysed synaptosomes The inhibition of Ca2+/Mg2+ A T P a s e by muscarine (10 # M ) was evaluated for r e c e p t o r specificity by atropine protection experiments. W h e n muscarine (10 150Control
~c ,::[ g ,m ~
~ine
I00,
io~. M
~
20/~M
E .U') "O ¢..)
0
i
2 Co "'free
3
4
5
[/~M}
Fig. 2. The effect of oxotremorine on Ca2+-stimulated ATPase as a function of [Ca2+}o. Lysed synaptosomes were preincubated as outlined in the legend to Fig. 1. Each point on the curve represents the mean of 6 individual experiments, each performed in triplicate. The data were statistically treated as outlined for Fig. 1. Values at 5.0 #M Ca 2. were significantly different from controls at 1, 10 and 20ktM T oxotremorine. P < 0.05.
43 fects on Ca2+/Mg 2+ ATPase at any of the Ca 2+ con-
150-
Control
oJ
E
"-.
,u.M Atropine Muscorine
I00"
p.M Muscorine ~
c
50
u) ;o (..1
CO'free i/~M] Fig. 3. Interactions of atropine and muscarine on Ca2+-stimulated ATPase activity. Lysed synaptosomes were preincubated with atropine (1/~M) 5 min prior to the addition of muscarine (1/~M). Fifteen min later, the reaction was initiated by the addition of ATP. Each point on the curve represents 4 separate experiments, each performed in triplicate. Statistics were used as outlined in Fig. 1. Muscarine (1/~M) was significantly different from control and atropine + muscarine at P < 0.05. /~M) was p r e i n c u b a t e d with lysed s y n a p t o s o m e s , the resulting Ca2+/Mg 2+ A T P a s e activity was inhibited a p p r o x i m a t e l y 40%, at 5.0/~M Ca 2+. W h e n atropine (1/~M) was p r e i n c u b a t e d prior to the addition of muscarine, no significant inhibition of enzyme activity could be detected. A t r o p i n e , when incubated with the m e m b r a n e s alone, p r o d u c e d no significant ef-
4
Control Muscorine °j'M
~
8 e~/,/~"~
~
Effects of muscarine on A TP-dependent Ca2+ uptake in lysed synaptosomal membranes Ca 2÷ m a y be t a k e n up by an A T P - d e p e n d e n t mechanism into s m o o t h endoplasmic reticulum 3,4. F o r this reason, it was of interest to evaluate the effects of the cholinergic agonist muscarine on A T P d e p e n d e n t Ca 2+ uptake. Ca 2+ u p t a k e was m e a s u r e d in the presence of 100 p M A T P as a function of Ca2+ concentrations ranging from 0.1 to 5 p M . These studies are p r e s e n t e d in Fig. 4. A T P - d e p e n d e n t Ca 2÷ uptake increased as the concentration of Ca 2+ increased, until saturation of Ca 2+ uptake was reached at 5.0 ktM Ca 2+. A T P - d e p e n d e n t Ca 2+ u p t a k e was evaluated in the presence of various concentrations of muscarine, ranging from 0.1 to 10/~M. Muscarine p r o d u c e d a c o n c e n t r a t i o n - d e p e n d e n t decrease in Ca 2+ uptake seen at 2.5 and 5.0/~M Ca 2+. Decreases in Ca 2+ uptake were significantly different from control (P < 0.01) following 1 and 1 0 ~ M muscarine. The inhibition of A T P - d e p e n d e n t Ca 2+ uptake following muscarine a p p e a r e d due to a reduction in Vmax for Ca2+-stimulated A T P - d e p e n d e n t uptake with no significant change in affinity (Kin) for Ca 2+. The a p p a r e n t activation of muscarinic receptors in reducing A T P - d e p e n d e n t Ca 2+ uptake could be completely abolished by p r e t r e a t m e n t with 0.5/~M atro-
Control **oE 4'
~
p~
/~ I . O ~ M
•lo m e-~L~ Q. "~ 2 o) a e-
IO.OM-M
~
o
p
i
n
e
+ Muscorine
m
C,O ,M
/
ge2. c'-, c
Muscorlne
Q.
Q_
o
i
;' i 4 CO*~ree [~M]
g
Fig. 4. The effect of muscarine on ATP-dependent Ca 2+ uptake. Lysed synaptosomes were preincubated with muscarine (0.1-10.0/~M) for 15 min prior to initiation of the reaction with ATP. Each point on the curve represents the mean of 4 separate experiments, each performed in triplicate. Statistics were applied as outlined in Fig. 1. Muscarine (1.0 and 10 k~M) was' significantly different from control (P < 0.05) at 2.5 and 5.0/~M
Ca2+.
centrations tested.
o
i
2 CO*free [,~M]
Fig. 5. Effects of atropine and muscarine on ATP-dependent Ca2+ uptake. Atropine was preincubated alone and in combination with muscarine with lysed synaptosomes, as outlined in Fig. 3. Reactions were initiated by the addition of ATP. Each point on the curve represents the mean of 4 separate experiments, each performed in triplicate. Statistics were applied as outlined in Fig. 1. Muscarine (10/~M) was significantly different from control (P < 0.05).
44 TABLE 1
Effects of cholinergic agents on voltage-dependent Ca2* influx Voltage-dependent Ca 2+ influx was measured in synaptosomes from rat brain cortex, as described in Methods. Aliquots of synaptosomes were preincubated with the drugs listed above for 15 rain in resting buffer, then either depolarized or left in resting buffer to measure asCa2+ influx. Values are expressed as nmol/mg protein/5 s and are the means ± S.D. for 6 separate experiments, each one in triplicate.
Drug
Dose
5 s K+-stimulated (25 mM)
30 s K" -stimulated (25 mM)
M
Influx (nmol/mg)
Influx (nmol/mg)
Muscarine
0 5 x 10-4 5x10-5 5 x 1 0 -6
4.76 ± 0.19 4.51 ± 0.20 4.92±0.23 4.75±0.19
9.70 _+ 0.46 9.04 ± 0.66 9.88±0.68 9.70±0.46
Oxotremorine
0 lxl0 3 l x l 0 -4 1xl0 5
4.76±0.19 4.21±0.62 3.98±0.94 4.73±0.05
9.70±0.46 9.41±1.20 9.89±1.20 9.65±0.35
Atropine
0 l x l 0 -3 1 x l 0 -4 1 x l 0 -5
6.45±0.54 5.25±0.18 8.17±0.80 6.78±0.26
12.65±2.60 8.65±1.70 9.19±0.5(/ 9.74±1.20
Scopolamine
0 1x10-3 lx10-4 1x10-5
3.09 2.52 3.19 5.83
+ 0.50 + 0.60 + 0.14 _+ 2.80
5.86 5.62 4.59 6.49
___1.11 + 0.78 ± 0.73 ± 0.28
p r e i n c u b a t e d w i t h lysed s y n a p t o s o m e s p r i o r to t h e
Fast and slow phase voltage-dependent Ca 2+ influx [ollowing pretreatment with cholinergic drum
addition of muscarine (10/~M), significant blockade
A c e t y l c h o l i n e r e l e a s e is t r i g g e r e d b y r a i s i n g t h e
of t h e effects o f m u s c a r i n e c o u l d b e s e e n at 1.1, 2.5
levels of f r e e c y t o s o l i c C a 2+ in t h e n e r v e t e r m i n a l s
p i n e (Fig. 5). W h e n this c h o l i n e r g i c a n t a g o n i s t was
a n d 5 , 0 / ~ M C a 2+. N o c h a n g e s w e r e s e e n w h e n a t r o -
t h r o u g h v o l t a g e - d e p e n d e n t d e p o l a r i z a t i o n a n d CaZ+
pine was preincubated with lysed synaptosomes.
influx. T h e t i m e - c o u r s e o f v o l t a g e - d e p e n d e n t
Ca2+
influx i n t o s y n a p t o s o m e s f r o m r o d e n t b r a i n h a s rec e n t l y b e e n d e s c r i b e d in t e r m s o f fast a n d slow p h a s e s TABLE II
Na*-stimulated Ca2+ efflux from rat brain synaptosomes Na+-Ca z+ exchange was measured by loading synaptosomes with 45Ca2+ under depolarizing conditions, then reversing the Na+/K + ratio to produce Na+-dependent Ca 2+ effiux. Drugs were preincubated with synaptosomes for 15 rain. The values shown are the mean of 6 experiments and are expressed as a ratio of 45Ca2+ effiuxed as a function of time where 0 time = 100% of control.
Time (s)
Control
% of Control (mean + S.D.) Muscarine (~M)
25 50 100 200 600 1200
71 67 49 41 23 18
Atropine (~M)
10
100
1000
10
100
1000
73 64 48 37 21 22
74 63 38 36 t8 17
72 63 56 39 19 19
72 60 48 37 24 18
70 67 47 43 24 18
76 71 511 40 19 21
45 for Ca 2+ entry 19. It was of interest to evaluate muscarinic agonists on Ca 2÷ influx through voltage-sensitive Ca 2+ channels. The possibility that presynaptic muscarinic receptors may be coupled to Ca 2÷ channels and limiting Ca2+-dependent ACh release, was tested, using muscarine and oxotremorine. These agonists, together with two cholinergic antagonists, were evaluated for their efficacy on voltage-dependent Ca 2÷ influx during fast phase and slow phase calcium entry. These results are presented in Table I. Muscarine and oxotremorine were tested at concentrations ranging from 50 to 1000/~M. Ca 2÷ influx was examined in the presence of 25 mM K ÷ at 5 s and 30 s, representing fast and slow phase Ca 2+ entry, respectively. As seen from these experiments, no significant responses were seen when either drug was preincubated with synaptosomes under physiological buffer conditions. Resting Ca 2÷ influx (5 mM K ÷) was also unaffected by preincubation with either drug (data not included). Two cholinergic antagonists were preincubated for 15 min under identical conditions, to evaluate their action on fast and slow phase Ca 2÷ entry. As seen in Table I, neither atropine nor scopolamine (10-1000/~M) produced any significant effect on either fast or slow phase Ca 2+ entry, nor did these drugs alter Ca 2÷ entry during resting membrane conditions (5 mM K+; data not included).
Effects of rnuscarinic drugs on Na+-Ca 2÷ exchange in synaptosomes Reduction in cytosolic Ca 2÷ by increased efflux from synaptosomes represents one mechanism by which muscarinic agonists may alter intracellular Ca 2+ levels to decrease transmitter release. Since Na+-induced Ca 2÷ efflux has been implicated as a buffering mechanism for cytosolic Ca 2÷ (refs. 2, 11) we have evaluated the effects of muscarine and atropine on Na+-induced Ca 2÷ efflux from intact synaptosomes. Table II outlines these experiments, demonstrating the effects of muscarine and atropine (10-1000gM) on Ca 2+ efflux, from 5 s to 1200 s. As seen from these experiments, Na+-stimulated Ca 2+ effiux occurs almost to completion by 1200 s. Rate constants for Ca 2÷ efflux were not significantly affected by the wide ranging concentrations of muscarine or atropine.
DISCUSSION The results presented in this paper demonstrate that two of the buffer mechanisms necessary for the regulation of synaptosomal Ca 2÷ levels may be modulated by activation of muscarinic receptors. Both muscarine (1-20/~M) and oxotremorine (1-20 #M) produced significant inhibition of Ca2+/Mg 2+ ATPase activity in synaptosomal membranes (Figs. 1 and 2). This enzyme has recently been implicated in Ca 2÷ buffering mechanisms in synaptosomes by a number of laboratories13,~8,22, 23. Both muscarine and oxotremorine inhibition of Ca2+/Mg 2+ ATPase could be reversed by preincubation with atropine. ATP-dependent Ca 2÷ uptake, stimulated by ATP hydrolysis, was also inhibited by muscarine over a similar concentration range. This effect was completely blocked by atropine preincubation. Transmitter release occurs via depolarization, based on a rise in cytosolic Ca 2+ in the nerve terminal 14. In order for release to terminate, however, the rise in cytosolic Ca 2+ must be terminated by some one or more intracellular Ca 2÷ buffering mechanisms 9. Of these mechanisms evaluated here, Ca2+/Mg 2÷ ATPase is believed to play a major role in regulation of nerve terminal Ca 2÷ levels e0. Our studies here demonstrate that neither muscarinic agonists nor antagonists altered the voltage-dependent Ca2+ entry by the fast or slow phase, nor did they affect Na+-Ca 2+ exchange. In contrast, our studies indicate that intracellular mechanisms for (1) maintaining optimal intracellular Ca 2+ levels and (2) enzyme-dependent Ca 2÷ extrusion from the synaptosome are significantly altered by activation of muscarinic receptors. It may be therefore concluded that this alteration may reduce the coupling between Ca 2÷ and transmitter release. The transient rise in cytosolic Ca e+ that would be expected following inhibition of intracellular Ca 2÷ buffering mechanism may be overcome by ATP Ca2+ uptake into synaptic mitochondria 20. Alternatively, increased cytosolic Ca 2÷ may activate and then limit Ca2+/Mg 2+ ATPase 9, thereby prolonging the recovery rate of the enzyme. The increases in Ca 2+ in the cytosol occurring following inhibition of enzyme activity may be expected to partially increase transmitter release. However, a prolonged rise in cytosolic Ca 2+ would be expected to activate Ca2+-dependent
46 K + conductance. Thus, Ca2+-mediated K ÷ conduc-
tion of Ca2+/Mg 2+ ATPase and/or A T P - d e p e n d e n t
tance may account for the reduced rate of transmitter release observed following muscarinic agonist treat-
Ca 2+ uptake may reduce the coupling between optimal cytosolic Ca 2+ and transmitter release. The
ment.
mechanism by which reduced ATPase activity is in-
The studies presented here are supported by the recent studies of Michaeison et al. 17, who demon-
volved with the release process remains to be elucidated; however, Ca2+/Mg2+ ATPase may be nec-
strated that muscarine blocked the Ca2+-dependent
essary to regulate critical Ca 2+ levels on the mem-
phosphorylation of a 100,000 dalton protein in synap-
brane for synaptic vesicle attachment or regulation of K + conductance14.
tosomes, in parallel with the blockade of transmitter release. A t r o p i n e treatment reversed this inhibition of phosphorylation and transmitter release. W h e t h e r or not CaZ+/Mg2+ ATPase was directly affected in
ACKNOWLEDGEMENTS
their study was not determined; however, it is known
The authors wish to thank the expert editorial assistance of Marilyn Wilson for preparation of this manuscript. The authors also wish to thank N. Monis
that CaZ+/Mg 2+ ATPase in synaptic m e m b r a n e s undergoes Ca2+-dependent phosphorylation 13. If activation of muscarinic receptors leads to reduced transmitter release through a mechanism involving altered intracellular Ca 2+ movements, then reduced Ca2+/Mg 2÷ A T P a s e activity may be directly implicated. In the present studies, and those reported by Michaelson et al. 17, voltage-dependent Ca 2÷ influx
and H. L. Cardenas for their expert technical assistance on this project. This research was supported by US A r m y Research and Development Contract D A M D 17-81-C1206 and U S A F Program Project in Neurosciences F33615-83-C-0624 to D . H . R .
was unaffected, leading to the conclusion that altera-
REFERENCES l Bartfai, T., Preparation of metal-chelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes, Cyclic Nucleotide Res., 10 (1979) 219-242. 2 Blaustein, M. P. and Ector, A. C., Carrier-mediated sodium-dependent and calcium-dependent calcium efflux from pinched-off presynaptic nerve terminals (synaptosomes) in vitro, Biochim. Biophys. Acta, 419 (1976) 295-308. 3 Blaustein, M. P., Ratzlaff, R. W., Kendrick, N. C. and Schweitzer, E. S., Calcium buffering in Presynaptic Nerve Terminals. I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism, J. gen. Physiol., 72 (1978) 15-41. 4 Blaustein, M. P., Ratzlaff, R. W. and Schweitzer, E. S., Calcium buffering in presynaptic nerve terminals. II. Kinetic properties of the nonmitochondrial Ca ÷+ sequestration mechanism, J. gen. Physiol., 72 (1978) 43-66. 5 Bourdois, P. S., Mitchell, J. F., Somogzi, G. T. and Szerb, J. C., The output per stimulus of acetylcholine from cerebral cortex slices in the presence or absence of cholinesterase inhibition, Brit. J. PharmacoL, 52 (1974) 509-517. 6 Cotman, C. W. and Matthews, D. A., Synaptic plasma membranes from rat brain synaptosomes. Isolation and partial characterization, Biochim. Biophys. Acta, 249 (1971) 380-394. 7 DeLorenzo, R. J., Freedman, S. D., Yohe, W. B. and Maurer, S. C., Stimulation of Ca++-dependent neurotransmitter release and synaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles. Proc. nat. Acad. Sci.
(U.S.A.), 76 (1979) 1838-1842. 8 DeLorenzo, R. J., Calmodulin in neurotransminer release and synaptic function, Fed. Proc., 41 (1982) 2265-2272. 9 Duncan, C. J., Properties of a Ca ++ ATPase activity of mammalian synaptic membrane preparations, J. Neurochem., 27 (1976) 1277-1279. 10 Garrett, K. M. and Ross, D. H., Effects of in vivo ethanol administration on Ca++/Mg÷* ATPase and ATP-dependent Ca ++ uptake activity in synaptosomal membranes, Neurochem. Res., 8 (1983) 1013-1028. 11 Gill, D. L,, Grollman, E. P. and Kohn, L. D., Calcium transport mechanisms in membrane vesicles from guinea pig brain synaptosomes, J. biol. Chem., 256 (1981) 184-192. 12 Hadhazy, P. and Szerb, J. C., The effect of cholinergic drugs on [3H]acetylcholine release from slices of rat hippocampus, striatum and cortex, Brain Research, 123 (1977) 311-322. 13 Javors, M. A., Bowden, C. L. and Ross, D. H., Kinetic characterization of Ca +÷ transport in synaptic membranes, J. Neurochem., 37 (1981) 381-387. 14 Kelly, R. B., Deutsch, J. W., Carlson, S. S. and Wagner, J. A., Biochemistry of neurotransmitter release. Ann. Rev. Neurosci., 2 (1979) 399-446. 15 Krueger, B. K., Forn, J. and Greengard, P., Depolarization-induced phosphorylation of specific proteins mediated by calcium ion influx in rat brain synaptosomes, J. biol. Chem., 252 (1977) 2764-2773. 16 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall~ R. J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 17 Michaelson, D. M.. Avissar, S., Kloog, Y. and Sokolovsky,
47 M., Mechanism of acetylcholine release: possible involvement of presynaptic muscarinic receptors in regulation of acetylcholine release and protein phosphorylation, Proc. nat. Acad. Sci. (U.S.A.), 76 (1979) 6336-6340. 18 Michaelis, E. K., Michaelis, M. L., Chang, H. H. and Kitos, T. E., High affinity Ca÷+-stimulated Mg÷÷-dependent ATPase in rat brain synaptosomes, synaptic membranes and microsomes, J. biol. Chem., 258 (1983) 6101-6108. 19 Nachshen, D. A. and Blaustein, M. P., Some properties of potassium-stimulated calcium influx in presynaptic nerve endings, J. Gen. Physiol., 76 (1980) 709-728. 20 Nicholls, D. G. and Akerman, K. E. O., Biochemical approaches to the study of cytosolic calcium regulation in nerve endings, Phil. Trans. B., (1981) B296, pp. 115-122.
21 Nordstrom, P. and Bartfai, T., Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence, Acta Physiol., 108 (1980) 347-353. 22 Ross, D. H. and Cardenas, H. L., Calmodulin stimulation of Ca÷+-dependent ATP hydrolysis and ATP-dependent Ca ÷÷ transport in synaptic membranes, J. Neurochem., 41 (1983) 161-171. 23 Sorenson, R. G. and Mahler, H., Calcium-stimulated adenosine triphosphates in synaptic membranes, J. Neurochem., 37 (1981) 1407-1418. 24 Szerb, J. C. and Somogyi, C. T., Depression of acetylcholine release in cerebral cortical slices by cholinesterase inhibition and by oxotremorine, Nature (Lond.), 241 (1973) 121-122.
39
BRE 10578
Activation of Central Muscarinic Receptors Inhibit Ca2+/Mg 2+ ATPase and ATP-Dependent Ca 2÷ Transport in Synaptic Membranes DAVID H. ROSS, S. MARTIN SHREEVE and M. G. HAMILTON Division of Molecular Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284 (U.S.A.)
(Accepted June 12th, 1984) Key words: Ca2+/Mg2+ ATPase - - ATP-dependent Ca2÷ uptake - - cholinergic agonists - - transmitter release
Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg 2÷ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ = 0.5/xM, close to the cytosolic concentration of Ca2÷. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20/~M), both Ca2+/Mg2÷ ATPase and ATP-dependent Ca2÷ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 #M) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2÷ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.
INTRODUCTION It is believed that cholinergic nerve terminals contain receptors which, when activated, regulate the release of acetylcholine (ACh) by a feedback mechanism. Electrically evoked release of A C h from brain slices was reduced following administration of cholinesterase inhibitorsS,24. Muscarinic antagonists block or potentiate this response in a concentration-dependent fashion. Hadhazy and Szerb 12 have reported that potassium-dependent release of A C h from hippocampus, striatum and cortex slices was reduced by physiostigmine, oxotremorine and carbamylcholine, while atropine potentiated the release. These findings were confirmed by Nordstrom and Bartfai 21, who demonstrated that [3H]ACh release from the hippocampus was reduced by muscarinic agonists (carbachol) and increased by antagonists (atropine). This evidence suggests that presynaptic muscarinic receptors, which are responsive to agonists and antagonists, may modulate the release of ACh. Trans-
mitter release mechanisms are known to be Ca2+-dependent t4 and probably involve phosphorylation of proteins 7. Krueger et al. 13 have shown that agents which induce or enhance depolarization of synaptosomes increase the level of intracellular Ca 2÷ and Ca2+-dependent protein phosphorylation. Recent studies by D e L o r e n z o and colleagues7, s have also demonstrated that depolarization of synaptic protein produces Ca2+-dependent phosphorylation of synaptic vesicles which is calmodulin-dependent and resuits in neurotransmitter release. Since changes in intracellular Ca 2÷ levels provide the signal for release, muscarinic agonists and antagonists may alter A C h release by interfering with buffering mechanisms for the optimal levels of intracellular Ca 2÷. Michaelson et al. 17 reported that muscarinic agonists (oxotremorine) block the K+-dependent release of acetylcholine from cholinergic Torpedo synaptosomes and effect antagonized by atropine. While calcium-dependent K+-stimulated release of acetylcholine was seen to occur simultaneously with 45Ca2+ in-
Correspondence: D. H. Ross, Division of Molecular Pharmacology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A.
40 flux; no effect of oxotremorin~ or atropine on K +stimulated 45Ca2+ influx could be demonstrated. The release of ACh was also seen to parallel an increase in Ca2+-dependent phosphorylation of a 100,000 dalton protein. Muscarine inhibited this Ca2+-depend ent phosphorylation, a response which was antagonized by atropine, these studies prompted us to evaluate two mechanisms which buffer cytosolic Ca 2+ and which may be subject to cholinergic receptor regulation. Ca2+/Mg2+ ATPase and ATP-dependent Ca 2+ sequestration in synaptic membranes are believed to play a major role in regulating cytosolic Ca 2+ (refs. 13, 20, 22). This paper reports the effects of cholinergic receptor activation on two Ca 2÷ buffering mechanisms, Ca2+/Mg 2÷ ATPase and ATP-dependent Ca 2+ uptake in synaptosomal membrane preparations.
band of tissue at the 7.5-12% interphase was removed and slowly diluted with an equal volume of D-glucose and HEPES. The pH was adjusted to 7.4 with HCI. This suspension was centrifuged at 25,000 g for 20 min to concentrate synaptosomal protein. The resultant pellet was slowly resuspended in resting buffer to a protein concentration of 1.1)-1.5 mg/ml and used as outlined below.
Preparation of lysed synaptosomal membranes
MATERIALS AND METHODS
Synaptosomes, isolated as described above, were resuspended in 20 mM Tris - 0.5 mM DTE, pH 8.1, in an ice bath for 60 min, to hypotonically lyse them. This membrane suspension was mixed every 20 min to facilitate the lysing procedure. At the end of 60 min, the suspension was pelleted at 40,000 g for 20 min and resuspended to a protein concentration of 0.5-1.0 mg/ml. This fraction was used for enzyme and ATP-dependent Ca 2+ uptake assays.
Materials
Measurement of Ca2+accumulation by synaptosomes
HEPES, sucrose, Na2-ATP and dithioerythritol (DTE) were obtained from Sigma (St. Louis, MO). All buffer salts were used as their chlorides and were purchased from Fisher Scientific (Pittsburgh, PA). Sodium dodecyi sulfate (Lauryl sulfate) was purchased from Pierce Chemicals (Rockford, IL). [y-32p]ATP (10-40 Ci/mmol), [45CaZ+]chloride, PPO and POPOP were obtained from New England Nuclear (Boston, MA), Millipore filters (0.45/~m, Type HAMP), were purchased from Millipore (Bedford, MA). Ammonium molybdate and isobutanol were purchased from Aldrich and Fisher Chemicals.
Methods Isolation of synaptosomes Male rats (150-200 g, Sprague-Dawley) were sacrificed by decapitation, brains rapidly removed and washed with ice-cold saline. A 10% homogenate was made of the cortex in 0.32 M sucrose and a crude P2 pellet fraction containing nerve endings isolated by differential centrifugation, according to the method outlined by Cotman and Matthews 6. This P2 pellet was resuspended in 0.32 M sucrose and layered over a 7.5-12% Ficoil (in 0.32 M sucrose) gradient and centrifuged at 65,000 g for 60 min. The resultant
Synaptosomes were suspended in resting buffer (RB) containing, in mM, NaCI (132), KC1 (5), MgC12 (1.3), D-glucose (10) and Tris (25), and the pH was adjusted to 7.4 with HCI. Synaptosomes were generally used within 30 min of final resuspension and kept cold (4 °C) until temperature equilibration, One-mt aliquots of this suspension were pre-equilibrated with appropriate [Ca2+]o at 37 °C for 5 min. in control samples, synaptosomes were pre-equilibrated at 37 °C for 10 rain. To initiate the uptake process, 100 ~1 of synaptosome suspension was added to each incubation tube (also preincubated at 37 °C). Each incubation tube then contained 100 #1 of synaptosome suspension, 100 pl ~5Ca2+ (0.45 ~Ci) and 800/~1 Ca2+C12 at various concentrations prepared in RB. Depolarization of synaptosomes was effected by substituting KCI for NaC1 in the buffer, achieving a final concentration of 25 mM K +. The combined Na +- and K + concentration was constant at 137 raM. The tubes were incubated in Dubnoff metabolic shaker at 37 °C, for 5 s (fast phase) or 30 s (slow phase). Termination of the reaction was performed by rapid addition of 10 ml ice-cold quench buffer (RB containing 3 mM EGTA). In some experiments, the synaptosomes were lysed at this point by using a hypotonic quench buffer (containing in mM, E G T A (3) and Tris (25), pH 7.4). The contents of each tube were
41 immediately filtered over Gelman (GA-6, Metricel, 0.45/~m, 25 mm) filters. The filters were washed (4 × 5 ml) with ice-cold RB containing 500/~M Ca 2÷ (high affinity) or 4 mM Ca 2÷ (low affinity). Filtration time under these conditions was about 10 s. Filters were allowed to dry and counted by liquid scintillation spectrometry. Ca 2+ accumulation was expressed as nmol of Ca 2÷ accumulation/mg of protein. Voltage-' dependent Ca 2÷ accumulation was calculated as the difference between the accumulation at 5 mM K ÷ and that at 25 mM K+.
Measurement of Ca2+/Mg2+ A TPase activity in lysed synaptosomes CaZ+-stimulated ATP hydrolysis was measured as outlined by Javors et al. 13 with the following modifications: lysed synaptosomes were preincubated at 37 °C in the presence or absence of drugs at different concentrations. The total final volume (2 ml) of the reaction medium contained KC1 (100 mM), H E P E S (20 mM, pH 7.4), MgCI 2 (250/~m), E G T A (100/~M), ouabain (1 mM), 50-150 ktg membrane protein and ATP (100 ~M). Ca2+ concentrations in the media were regulated from 0.1 to 5/~M with E G T A buffers, as described by Bartfai 1. The enzyme reaction was initiated by the addition of the ATP solution containing [3zp]ATP, following pre-equilibration with substrates. The reaction was terminated 60 s later, with a 2-ml volume of 5% T C A - 5 % SDS stopping solution. Ammonium molybdate (2 ml) was added, followed by isobutanol (1.5 ml) to complex and extract the released 32p i into the organic phase. The mixture was either shaken or vortexed; then the two phases were allowed to stand 10 min to separate. The organic layer (500/A) was removed by Eppendorf pipette, transferred to liquid scintillation vials and counted in a toluene POPOPPPO fluor by an Isocap 300. Ca2+-stimulated ATP hydrolysis (Ca 2÷ ATPase) is expressed in nmol Pi released/mg/min by subtracting Mg2+-stimulated ATPase activity from Ca2+Mg 2÷ activity. A tissue blank containing no Ca 2÷ or Mg 2÷ was included to determine non-specific ATPase activity.
A TP-dependent Ca2+ uptake by lysed synaptosomes ATP-dependent and -independent transport by lysed synaptosomal membranes was measured in the presence and absence of 100/~M ATP, as described
by Javors et al. 13. Lysed synaptosomai membranes were prepared and preincubated in the same manner as for Ca2+ stimulated ATPase activity, using identical buffer components and reaction times. 45Ca2+ (0.6/~Ci) was added two min prior to the addition of ATP (100/~M) to reaction media containing varying concentrations of Catree, 2+ as determined by E G T A buffers 2z. Following the addition of 45Ca2+, ATP was added to initiate the transport reaction for 60 s. The reactions were terminated by addition of ice-cold stopping solution (3 ml) containing'KC1 (100 mM), E G T A (100/~M), CaCl 2 (500/tm), HEPES (20 mM) and MgC12 (250/tM), at pH 7.4, 25 °C, followed by rapid filtration (5-7 s) over MiUipore filters (0.45 HAMP). The filters were washed 4 times with 3 ml stopping solution, allowed to dry and then counted in a toluene-based POPOP-PPO scintillation solution. Ca 2+ uptake was expressed as ATP-stimulated uptake in nmol/mg/min, uncorrected for ATP-independent Ca 2+ binding.
Assay of Na+-dependent Cae+ efflux Na+-dependent Ca 2+ effiux was performed according to the method outlined by Blaustein and Ector 2. Synaptosomes were prepared as outlined above. Synaptosomes were resuspended in a physiological medium (0.6-0.85 mg protein in 0.5 ml of Na + + 5 K + buffer) and equilibrated for 12 min, at 30 °C. Calcium loading of the synaptosomes was initiated by addition of 0.5 ml of 137 mM potassium chloride-5 mM NaC! containing 45Ca2+ (--- 2 ~Ci//~mol Ca 2+) in a final volume of 1.0 ml. This preparation was incubated for 2 min at 30 °C with 45Ca2+; 0.5 ml of the suspension was then transferred to a tube containing 10 ml of the effiux solution (containing 132 mM NaC1 and 5 mM KC1). In some experiments, the Na + was replaced by equimolar chloride to check for the Na + dependency. When choline was substituted, the solution did not contain Ca 2+. The solutions were mixed and then filtered at various time intervals. The suspensions were vacuum filtered on pre-washed 0.3/~M Millipore filters. Each filter was then washed with 10 ml ice-cold choline + 5 K +. Filters were dried, placed in liquid scintillation vials and counted in a toluene/ETOH-based fluor with PPO and POPOP. Protein content was determined by Lowry et al. 16. Ca 2+ effiux was calculated as the difference between 45Ca2+ remaining on the filter af-
42 ter immediate loading and the a m o u n t remaining at different intervals and expressed as % of control.
150 ¸ ,_>
Miscellaneous procedures Protein m e a s u r e m e n t s on synaptosomal suspensions were m a d e by the m e t h o d of Lowry et al.16 using bovine serum albumin as a standard, and an autoanalyzer. The water used in the experiments was double-deionized and was shown by atomic absorption spectroscopy to contain negligible amounts of Ca 2+. In addition, the buffers containing Ca 2+ were analyzed by atomic absorption spectroscopy and found to be accurate ( + 0.05 ¢tM). Standard errors for mean stimulated uptake were calculated as described by Nachshen and Blaustein x9. RESULTS
Effects of cholinergic agonists on Ca2+/Mg2+ A TPase activity in lysed synaptosomal membranes Changes in the release pattern of the cholinergic transmitter acetylcholine ( A C h ) may result from altered intracellular Ca 2÷ buffering mechanisms. It was of interest, therefore, to evaluate the effects of two cholinergic agonists, muscarine and o x o t r e m o rine, on Ca2+/Mg2+ A T P a s e activity. These studies are presented in Fig. 1. E n z y m e activity was evaluated in lysed synaptosomes in the presence of muscarine ( 1 - 2 0 / ~ M ) following a drug incubation p e r i o d of 15 min. E n z y m e activity was evaluated as a function of various Ca 2÷ concentrations, from 0.1 to 5/~M. Ca 2÷ concentrations used were designed to approximate those cytosolic concentrations believed reached during the depolarization cycle 3.4. A s seen in Fig. 1, increasing the concentration of [Ca2+]o p r o d u c e d an activation of Cae+/Mg 2+ A T P a s e , as m e a s u r e d by an increase in A T P hydrolysis. This response to increasing [Ca2+]o reached a maximum at 5 # M , a concentration which is believed to be maximal for free cytosolic Ca 2÷ in the nerve terminal9. Muscarine, in increasing concentrations, p r o d u c e d a progressive decrease in Ca2+/Mg 2+ A T P a s e activity. Similar changes were seen when the agonist o x o t r e m o r i n e was used. As illustrated in Fig. 2, o x o t r e m o r i n e ( 1 - 2 0 / ~ M ) also decreased Ca2+/Mg2+ A T P a s e activity by reducing the Vmax. O x o t r e m o r i n e a p p e a r e d to be less potent than muscarine when comparing the d e g r e e e of inhibition of enzyme activity seen at 2.5 and 5.0/~M Ca:*.
Conlrol E
arine
"-. I00(1)
20,~M
_
i
w(/'/ ÷o (_.9
Ca'}ree [#M]
Fig. 1. Effect of muscarine on Ca2+-stimulated ATPase as a function of [Ca2+}o. Lysed synaptosomes were preincubated with muscarine (15 min) at 1, 10 and 20~M. Ca2+/Mg2+ ATPase was 2+ assayed by measuring Pi release at different Catr~e concentrations, from 0.1-5.0#M. Each point on the curve represents the mean of 6 individual experiments, each performed in triplicate. ABMDP computer program package, from the University of California, was used to compute analysis of variance. A threeway analysis of variance was used to determine the differences between curves at each drug concentration. Values at 2.5 and 5.0 #M Ca 2+ were significantly different from controls at 1, 10 and 20 mM muscarine. P < 0.05.
Influence of atropine on muscarine-dependent decrease in Ca2+/Mg2 +A TPase activity in lysed synaptosomes The inhibition of Ca2+/Mg2+ A T P a s e by muscarine (10 # M ) was evaluated for r e c e p t o r specificity by atropine protection experiments. W h e n muscarine (10 150Control
~c ,::[ g ,m ~
~ine
I00,
io~. M
~
20/~M
E .U') "O ¢..)
0
i
2 Co "'free
3
4
5
[/~M}
Fig. 2. The effect of oxotremorine on Ca2+-stimulated ATPase as a function of [Ca2+}o. Lysed synaptosomes were preincubated as outlined in the legend to Fig. 1. Each point on the curve represents the mean of 6 individual experiments, each performed in triplicate. The data were statistically treated as outlined for Fig. 1. Values at 5.0 #M Ca 2. were significantly different from controls at 1, 10 and 20ktM T oxotremorine. P < 0.05.
43 fects on Ca2+/Mg 2+ ATPase at any of the Ca 2+ con-
150-
Control
oJ
E
"-.
,u.M Atropine Muscorine
I00"
p.M Muscorine ~
c
50
u) ;o (..1
CO'free i/~M] Fig. 3. Interactions of atropine and muscarine on Ca2+-stimulated ATPase activity. Lysed synaptosomes were preincubated with atropine (1/~M) 5 min prior to the addition of muscarine (1/~M). Fifteen min later, the reaction was initiated by the addition of ATP. Each point on the curve represents 4 separate experiments, each performed in triplicate. Statistics were used as outlined in Fig. 1. Muscarine (1/~M) was significantly different from control and atropine + muscarine at P < 0.05. /~M) was p r e i n c u b a t e d with lysed s y n a p t o s o m e s , the resulting Ca2+/Mg 2+ A T P a s e activity was inhibited a p p r o x i m a t e l y 40%, at 5.0/~M Ca 2+. W h e n atropine (1/~M) was p r e i n c u b a t e d prior to the addition of muscarine, no significant inhibition of enzyme activity could be detected. A t r o p i n e , when incubated with the m e m b r a n e s alone, p r o d u c e d no significant ef-
4
Control Muscorine °j'M
~
8 e~/,/~"~
~
Effects of muscarine on A TP-dependent Ca2+ uptake in lysed synaptosomal membranes Ca 2÷ m a y be t a k e n up by an A T P - d e p e n d e n t mechanism into s m o o t h endoplasmic reticulum 3,4. F o r this reason, it was of interest to evaluate the effects of the cholinergic agonist muscarine on A T P d e p e n d e n t Ca 2+ uptake. Ca 2+ u p t a k e was m e a s u r e d in the presence of 100 p M A T P as a function of Ca2+ concentrations ranging from 0.1 to 5 p M . These studies are p r e s e n t e d in Fig. 4. A T P - d e p e n d e n t Ca 2÷ uptake increased as the concentration of Ca 2+ increased, until saturation of Ca 2+ uptake was reached at 5.0 ktM Ca 2+. A T P - d e p e n d e n t Ca 2+ u p t a k e was evaluated in the presence of various concentrations of muscarine, ranging from 0.1 to 10/~M. Muscarine p r o d u c e d a c o n c e n t r a t i o n - d e p e n d e n t decrease in Ca 2+ uptake seen at 2.5 and 5.0/~M Ca 2+. Decreases in Ca 2+ uptake were significantly different from control (P < 0.01) following 1 and 1 0 ~ M muscarine. The inhibition of A T P - d e p e n d e n t Ca 2+ uptake following muscarine a p p e a r e d due to a reduction in Vmax for Ca2+-stimulated A T P - d e p e n d e n t uptake with no significant change in affinity (Kin) for Ca 2+. The a p p a r e n t activation of muscarinic receptors in reducing A T P - d e p e n d e n t Ca 2+ uptake could be completely abolished by p r e t r e a t m e n t with 0.5/~M atro-
Control **oE 4'
~
p~
/~ I . O ~ M
•lo m e-~L~ Q. "~ 2 o) a e-
IO.OM-M
~
o
p
i
n
e
+ Muscorine
m
C,O ,M
/
ge2. c'-, c
Muscorlne
Q.
Q_
o
i
;' i 4 CO*~ree [~M]
g
Fig. 4. The effect of muscarine on ATP-dependent Ca 2+ uptake. Lysed synaptosomes were preincubated with muscarine (0.1-10.0/~M) for 15 min prior to initiation of the reaction with ATP. Each point on the curve represents the mean of 4 separate experiments, each performed in triplicate. Statistics were applied as outlined in Fig. 1. Muscarine (1.0 and 10 k~M) was' significantly different from control (P < 0.05) at 2.5 and 5.0/~M
Ca2+.
centrations tested.
o
i
2 CO*free [,~M]
Fig. 5. Effects of atropine and muscarine on ATP-dependent Ca2+ uptake. Atropine was preincubated alone and in combination with muscarine with lysed synaptosomes, as outlined in Fig. 3. Reactions were initiated by the addition of ATP. Each point on the curve represents the mean of 4 separate experiments, each performed in triplicate. Statistics were applied as outlined in Fig. 1. Muscarine (10/~M) was significantly different from control (P < 0.05).
44 TABLE 1
Effects of cholinergic agents on voltage-dependent Ca2* influx Voltage-dependent Ca 2+ influx was measured in synaptosomes from rat brain cortex, as described in Methods. Aliquots of synaptosomes were preincubated with the drugs listed above for 15 rain in resting buffer, then either depolarized or left in resting buffer to measure asCa2+ influx. Values are expressed as nmol/mg protein/5 s and are the means ± S.D. for 6 separate experiments, each one in triplicate.
Drug
Dose
5 s K+-stimulated (25 mM)
30 s K" -stimulated (25 mM)
M
Influx (nmol/mg)
Influx (nmol/mg)
Muscarine
0 5 x 10-4 5x10-5 5 x 1 0 -6
4.76 ± 0.19 4.51 ± 0.20 4.92±0.23 4.75±0.19
9.70 _+ 0.46 9.04 ± 0.66 9.88±0.68 9.70±0.46
Oxotremorine
0 lxl0 3 l x l 0 -4 1xl0 5
4.76±0.19 4.21±0.62 3.98±0.94 4.73±0.05
9.70±0.46 9.41±1.20 9.89±1.20 9.65±0.35
Atropine
0 l x l 0 -3 1 x l 0 -4 1 x l 0 -5
6.45±0.54 5.25±0.18 8.17±0.80 6.78±0.26
12.65±2.60 8.65±1.70 9.19±0.5(/ 9.74±1.20
Scopolamine
0 1x10-3 lx10-4 1x10-5
3.09 2.52 3.19 5.83
+ 0.50 + 0.60 + 0.14 _+ 2.80
5.86 5.62 4.59 6.49
___1.11 + 0.78 ± 0.73 ± 0.28
p r e i n c u b a t e d w i t h lysed s y n a p t o s o m e s p r i o r to t h e
Fast and slow phase voltage-dependent Ca 2+ influx [ollowing pretreatment with cholinergic drum
addition of muscarine (10/~M), significant blockade
A c e t y l c h o l i n e r e l e a s e is t r i g g e r e d b y r a i s i n g t h e
of t h e effects o f m u s c a r i n e c o u l d b e s e e n at 1.1, 2.5
levels of f r e e c y t o s o l i c C a 2+ in t h e n e r v e t e r m i n a l s
p i n e (Fig. 5). W h e n this c h o l i n e r g i c a n t a g o n i s t was
a n d 5 , 0 / ~ M C a 2+. N o c h a n g e s w e r e s e e n w h e n a t r o -
t h r o u g h v o l t a g e - d e p e n d e n t d e p o l a r i z a t i o n a n d CaZ+
pine was preincubated with lysed synaptosomes.
influx. T h e t i m e - c o u r s e o f v o l t a g e - d e p e n d e n t
Ca2+
influx i n t o s y n a p t o s o m e s f r o m r o d e n t b r a i n h a s rec e n t l y b e e n d e s c r i b e d in t e r m s o f fast a n d slow p h a s e s TABLE II
Na*-stimulated Ca2+ efflux from rat brain synaptosomes Na+-Ca z+ exchange was measured by loading synaptosomes with 45Ca2+ under depolarizing conditions, then reversing the Na+/K + ratio to produce Na+-dependent Ca 2+ effiux. Drugs were preincubated with synaptosomes for 15 rain. The values shown are the mean of 6 experiments and are expressed as a ratio of 45Ca2+ effiuxed as a function of time where 0 time = 100% of control.
Time (s)
Control
% of Control (mean + S.D.) Muscarine (~M)
25 50 100 200 600 1200
71 67 49 41 23 18
Atropine (~M)
10
100
1000
10
100
1000
73 64 48 37 21 22
74 63 38 36 t8 17
72 63 56 39 19 19
72 60 48 37 24 18
70 67 47 43 24 18
76 71 511 40 19 21
45 for Ca 2+ entry 19. It was of interest to evaluate muscarinic agonists on Ca 2÷ influx through voltage-sensitive Ca 2+ channels. The possibility that presynaptic muscarinic receptors may be coupled to Ca 2÷ channels and limiting Ca2+-dependent ACh release, was tested, using muscarine and oxotremorine. These agonists, together with two cholinergic antagonists, were evaluated for their efficacy on voltage-dependent Ca 2÷ influx during fast phase and slow phase calcium entry. These results are presented in Table I. Muscarine and oxotremorine were tested at concentrations ranging from 50 to 1000/~M. Ca 2÷ influx was examined in the presence of 25 mM K ÷ at 5 s and 30 s, representing fast and slow phase Ca 2+ entry, respectively. As seen from these experiments, no significant responses were seen when either drug was preincubated with synaptosomes under physiological buffer conditions. Resting Ca 2÷ influx (5 mM K ÷) was also unaffected by preincubation with either drug (data not included). Two cholinergic antagonists were preincubated for 15 min under identical conditions, to evaluate their action on fast and slow phase Ca 2÷ entry. As seen in Table I, neither atropine nor scopolamine (10-1000/~M) produced any significant effect on either fast or slow phase Ca 2+ entry, nor did these drugs alter Ca 2÷ entry during resting membrane conditions (5 mM K+; data not included).
Effects of rnuscarinic drugs on Na+-Ca 2÷ exchange in synaptosomes Reduction in cytosolic Ca 2÷ by increased efflux from synaptosomes represents one mechanism by which muscarinic agonists may alter intracellular Ca 2+ levels to decrease transmitter release. Since Na+-induced Ca 2÷ efflux has been implicated as a buffering mechanism for cytosolic Ca 2÷ (refs. 2, 11) we have evaluated the effects of muscarine and atropine on Na+-induced Ca 2÷ efflux from intact synaptosomes. Table II outlines these experiments, demonstrating the effects of muscarine and atropine (10-1000gM) on Ca 2+ efflux, from 5 s to 1200 s. As seen from these experiments, Na+-stimulated Ca 2+ effiux occurs almost to completion by 1200 s. Rate constants for Ca 2÷ efflux were not significantly affected by the wide ranging concentrations of muscarine or atropine.
DISCUSSION The results presented in this paper demonstrate that two of the buffer mechanisms necessary for the regulation of synaptosomal Ca 2÷ levels may be modulated by activation of muscarinic receptors. Both muscarine (1-20/~M) and oxotremorine (1-20 #M) produced significant inhibition of Ca2+/Mg 2+ ATPase activity in synaptosomal membranes (Figs. 1 and 2). This enzyme has recently been implicated in Ca 2÷ buffering mechanisms in synaptosomes by a number of laboratories13,~8,22, 23. Both muscarine and oxotremorine inhibition of Ca2+/Mg 2+ ATPase could be reversed by preincubation with atropine. ATP-dependent Ca 2÷ uptake, stimulated by ATP hydrolysis, was also inhibited by muscarine over a similar concentration range. This effect was completely blocked by atropine preincubation. Transmitter release occurs via depolarization, based on a rise in cytosolic Ca 2+ in the nerve terminal 14. In order for release to terminate, however, the rise in cytosolic Ca 2+ must be terminated by some one or more intracellular Ca 2÷ buffering mechanisms 9. Of these mechanisms evaluated here, Ca2+/Mg 2÷ ATPase is believed to play a major role in regulation of nerve terminal Ca 2÷ levels e0. Our studies here demonstrate that neither muscarinic agonists nor antagonists altered the voltage-dependent Ca2+ entry by the fast or slow phase, nor did they affect Na+-Ca 2+ exchange. In contrast, our studies indicate that intracellular mechanisms for (1) maintaining optimal intracellular Ca 2+ levels and (2) enzyme-dependent Ca 2÷ extrusion from the synaptosome are significantly altered by activation of muscarinic receptors. It may be therefore concluded that this alteration may reduce the coupling between Ca 2÷ and transmitter release. The transient rise in cytosolic Ca e+ that would be expected following inhibition of intracellular Ca 2÷ buffering mechanism may be overcome by ATP Ca2+ uptake into synaptic mitochondria 20. Alternatively, increased cytosolic Ca 2÷ may activate and then limit Ca2+/Mg 2+ ATPase 9, thereby prolonging the recovery rate of the enzyme. The increases in Ca 2+ in the cytosol occurring following inhibition of enzyme activity may be expected to partially increase transmitter release. However, a prolonged rise in cytosolic Ca 2+ would be expected to activate Ca2+-dependent
46 K + conductance. Thus, Ca2+-mediated K ÷ conduc-
tion of Ca2+/Mg 2+ ATPase and/or A T P - d e p e n d e n t
tance may account for the reduced rate of transmitter release observed following muscarinic agonist treat-
Ca 2+ uptake may reduce the coupling between optimal cytosolic Ca 2+ and transmitter release. The
ment.
mechanism by which reduced ATPase activity is in-
The studies presented here are supported by the recent studies of Michaeison et al. 17, who demon-
volved with the release process remains to be elucidated; however, Ca2+/Mg2+ ATPase may be nec-
strated that muscarine blocked the Ca2+-dependent
essary to regulate critical Ca 2+ levels on the mem-
phosphorylation of a 100,000 dalton protein in synap-
brane for synaptic vesicle attachment or regulation of K + conductance14.
tosomes, in parallel with the blockade of transmitter release. A t r o p i n e treatment reversed this inhibition of phosphorylation and transmitter release. W h e t h e r or not CaZ+/Mg2+ ATPase was directly affected in
ACKNOWLEDGEMENTS
their study was not determined; however, it is known
The authors wish to thank the expert editorial assistance of Marilyn Wilson for preparation of this manuscript. The authors also wish to thank N. Monis
that CaZ+/Mg 2+ ATPase in synaptic m e m b r a n e s undergoes Ca2+-dependent phosphorylation 13. If activation of muscarinic receptors leads to reduced transmitter release through a mechanism involving altered intracellular Ca 2+ movements, then reduced Ca2+/Mg 2÷ A T P a s e activity may be directly implicated. In the present studies, and those reported by Michaelson et al. 17, voltage-dependent Ca 2÷ influx
and H. L. Cardenas for their expert technical assistance on this project. This research was supported by US A r m y Research and Development Contract D A M D 17-81-C1206 and U S A F Program Project in Neurosciences F33615-83-C-0624 to D . H . R .
was unaffected, leading to the conclusion that altera-
REFERENCES l Bartfai, T., Preparation of metal-chelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes, Cyclic Nucleotide Res., 10 (1979) 219-242. 2 Blaustein, M. P. and Ector, A. C., Carrier-mediated sodium-dependent and calcium-dependent calcium efflux from pinched-off presynaptic nerve terminals (synaptosomes) in vitro, Biochim. Biophys. Acta, 419 (1976) 295-308. 3 Blaustein, M. P., Ratzlaff, R. W., Kendrick, N. C. and Schweitzer, E. S., Calcium buffering in Presynaptic Nerve Terminals. I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism, J. gen. Physiol., 72 (1978) 15-41. 4 Blaustein, M. P., Ratzlaff, R. W. and Schweitzer, E. S., Calcium buffering in presynaptic nerve terminals. II. Kinetic properties of the nonmitochondrial Ca ÷+ sequestration mechanism, J. gen. Physiol., 72 (1978) 43-66. 5 Bourdois, P. S., Mitchell, J. F., Somogzi, G. T. and Szerb, J. C., The output per stimulus of acetylcholine from cerebral cortex slices in the presence or absence of cholinesterase inhibition, Brit. J. PharmacoL, 52 (1974) 509-517. 6 Cotman, C. W. and Matthews, D. A., Synaptic plasma membranes from rat brain synaptosomes. Isolation and partial characterization, Biochim. Biophys. Acta, 249 (1971) 380-394. 7 DeLorenzo, R. J., Freedman, S. D., Yohe, W. B. and Maurer, S. C., Stimulation of Ca++-dependent neurotransmitter release and synaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles. Proc. nat. Acad. Sci.
(U.S.A.), 76 (1979) 1838-1842. 8 DeLorenzo, R. J., Calmodulin in neurotransminer release and synaptic function, Fed. Proc., 41 (1982) 2265-2272. 9 Duncan, C. J., Properties of a Ca ++ ATPase activity of mammalian synaptic membrane preparations, J. Neurochem., 27 (1976) 1277-1279. 10 Garrett, K. M. and Ross, D. H., Effects of in vivo ethanol administration on Ca++/Mg÷* ATPase and ATP-dependent Ca ++ uptake activity in synaptosomal membranes, Neurochem. Res., 8 (1983) 1013-1028. 11 Gill, D. L,, Grollman, E. P. and Kohn, L. D., Calcium transport mechanisms in membrane vesicles from guinea pig brain synaptosomes, J. biol. Chem., 256 (1981) 184-192. 12 Hadhazy, P. and Szerb, J. C., The effect of cholinergic drugs on [3H]acetylcholine release from slices of rat hippocampus, striatum and cortex, Brain Research, 123 (1977) 311-322. 13 Javors, M. A., Bowden, C. L. and Ross, D. H., Kinetic characterization of Ca +÷ transport in synaptic membranes, J. Neurochem., 37 (1981) 381-387. 14 Kelly, R. B., Deutsch, J. W., Carlson, S. S. and Wagner, J. A., Biochemistry of neurotransmitter release. Ann. Rev. Neurosci., 2 (1979) 399-446. 15 Krueger, B. K., Forn, J. and Greengard, P., Depolarization-induced phosphorylation of specific proteins mediated by calcium ion influx in rat brain synaptosomes, J. biol. Chem., 252 (1977) 2764-2773. 16 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall~ R. J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 17 Michaelson, D. M.. Avissar, S., Kloog, Y. and Sokolovsky,
47 M., Mechanism of acetylcholine release: possible involvement of presynaptic muscarinic receptors in regulation of acetylcholine release and protein phosphorylation, Proc. nat. Acad. Sci. (U.S.A.), 76 (1979) 6336-6340. 18 Michaelis, E. K., Michaelis, M. L., Chang, H. H. and Kitos, T. E., High affinity Ca÷+-stimulated Mg÷÷-dependent ATPase in rat brain synaptosomes, synaptic membranes and microsomes, J. biol. Chem., 258 (1983) 6101-6108. 19 Nachshen, D. A. and Blaustein, M. P., Some properties of potassium-stimulated calcium influx in presynaptic nerve endings, J. Gen. Physiol., 76 (1980) 709-728. 20 Nicholls, D. G. and Akerman, K. E. O., Biochemical approaches to the study of cytosolic calcium regulation in nerve endings, Phil. Trans. B., (1981) B296, pp. 115-122.
21 Nordstrom, P. and Bartfai, T., Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence, Acta Physiol., 108 (1980) 347-353. 22 Ross, D. H. and Cardenas, H. L., Calmodulin stimulation of Ca÷+-dependent ATP hydrolysis and ATP-dependent Ca ÷÷ transport in synaptic membranes, J. Neurochem., 41 (1983) 161-171. 23 Sorenson, R. G. and Mahler, H., Calcium-stimulated adenosine triphosphates in synaptic membranes, J. Neurochem., 37 (1981) 1407-1418. 24 Szerb, J. C. and Somogyi, C. T., Depression of acetylcholine release in cerebral cortical slices by cholinesterase inhibition and by oxotremorine, Nature (Lond.), 241 (1973) 121-122.