127
Clinical Science (1983) 65,127-135
Calcium transport in synaptosomes and subcellular
membrane fractions of brain tissue in spontaneously
hypertensive rats
G. M. KRAVTSOV, S. N. ORLOV, N. I. POKUDIN A N D Yu. V. POSTNOV
Central Research Luboratoty of the Ministry ofPublic Health of the U.S.S.R.,Moscow, U.S.S.R.
(Received 22 July118 October 1982; accepted 7 February 1983)
summary
1. The uptake of Na+ and CaZ+by synaptosomes and uptake of CaZ+by the mitochondria
and microsomes of brain tissue of rats with spontaneous hypertension (SH rats) and normotensive
Kyoto-Wistar rats (WKY rats) were studied with
an isotope-exchange method.
2. By means of inhibitor analysis it has been
shown that calcium influx into the synaptosomes
during depolarization of their plasma membrane
takes place only through the potential-dependent
channels in both groups of animals.
3. Basal CaZ+uptake by the synaptosomes of
hypertensive rats was increased, apparently by
partial depolarization of the synaptosome membrane caused by the increased membrane permeability to Na+ (basal Na+ uptake by synaptosomes
was found to be increased in hypertensive rats).
4. CaZ+ uptake by mitochondria of hypertensive rats was increased, and the Ca" uptake
by microsomes was decreased in these rats compared with controls.
5. The increment of the maximal CaZ+transport rate in microsomes after the addition of
calmodulin was decreased in spontaneously hypertensive rats compared with normotensive animals.
Thus alterations in the interaction of calmodulin
with the Caz+-transporting systems of the plasma
membrane are an important part of the widespread membrane defect observed in spontaneous
hypertension.
Correspondence: Professor Yu. V. Postnov,
Central Research Laboratory, Ministry of Public
Health, Timoshenko av. 21, Moscow 121359,
U.S.S.R.
6. The changes in the Ca'+-transporting and
Caz+-regulating systems of the synaptosomes of
brain tissue in spontaneously hypertensive rats
may be the basis for the increase of the intrasynaptosomal CaZ+ concentration and, in turn,
for the alteration in the rate of neurotransmitter
release.
Key words: brain, calcium transport, membranes,
spontaneous hypertension, synaptosomes.
Abbreviations: TTX, tetrodotoxin.
Introduction
Recent studies have demonstrated abnormalities
in the plasma membrane of several types of cells
in primary hypertension (both in spontaneous
hypertension of rats and in essential hypertension
of humans). Altered membrane control over intracellular calcium distribution and increased membrane permeability for univalent cations were
observed, particularly, in myocardiocytes . p d
vascular smooth muscle cells of spontaneously
hypertensive Kyoto-Wistar rats [l-41 and in
adipocytes and erythrocytes in both forms of
primary hypertension [5-8]. Thus it has become
evident that membrane alterations are widespread
in the tissues and that primary hypertension might
be considered as a special kind of membrane
pathology. Recent findings of decreased CaZ+binding ability in plasma membranes'of synaptosomes in spontaneously hypertensive (SH) rats [9]
supports this point of view.
It is known that the free calcium concentration
in nerve endings plays the key role in regulation
G.M.Kravtsov et al.
128
of neurotransmitter release [ 101. Ca2+ concentration in cytoplasm is determined both by the rate
of Ca2+ influx during membrane depolarization
and by the activity of Ca2+-transportingsystems.
At present there is little information concerning
the mechanism of Ca2+ influx into nerve endings;
in particular, the relative role of Ca2+- and Na+channels in the total Ca2+ uptake has not been
established. Among the systems responsible for
CaZ+transport against the electrochemical gradient
the leading role of Na+-Ca2+ exchange has been
considered [ 1I]. However, it has been shown that
the significance of this system in the maintenance
of free Ca2+ concentration of the nerve terminal
during its depolarization is rather limited [12].
ATP-dependent Caz+-pumps in the plasma membrane and in intrasynaptosomal vesicles, as well as
the Caz+-transport system of mitochondria, were
considered t o be more likely Ca2+-buffering
systems of nerve terminals [ 13-15]. The relative
roles of these systems in the handling of low
concentrations of CaZ+have not been established.
In the present work the features of Ca2+ uptake
by synaptosomes during their depolarization, as
well as Ca2+ accumulation by subcellular membrane fractions of the brain, have been studied
in spontaneously hypertensive rats.
Material and methods
Rats
Sixteen-week-old male Okamoto-Aoki spontaneously hypertensive (SH) rats weighing 230260 g (blood pressure 170-200 mmHg) and
normotensive Kyoto-Wistar rats (WKY rats) of
the same age and sex (blood pressure 120-130
mmHg) were used.
Preparation of membrane fractions
Synaptosomes were prepared by a modification of Cotman's method [16]. Rat brain (without
cerebellum) was homogenized in 20% (w/v)
medium A (0.32 mol/l sucrose, 5 mmol/l HEPES,
pH 7.4) at 0-2'C, with Teflon/glass (clearance 0.10.08mm), once or twice at 400 rev./min, and
diluted twice with the same medium. The homogenate was centrifuged at 3000g for 10 min.
The supernatant was centrifuged (20 OOOg, 15 min)
and the pellet was washed with medium A under
the same conditions. The sediment was resuspended
in 30 ml of medium A and transferred into a discontinuous Ficoll-sucrose density gradient, consisting of 10 ml each of 7.5%, 12%and 17% Ficoll
(w/v in medium A). The fractions were separated
by centrifugation (25 000 rev./min for 60 min;
rotor type SW-27, Beckman, U.S.A.). The fractions between sucrose (0.32 mol/l)-7.5% Ficoll,
-7.5-1276 Ficoll and -12-17% Ficoll, and the
fraction at the bottom (the fractions F1, F2, F3
and F4 respectively), were washed in 80 ml of
0.32 mol/l sucrose (57 OOOg for 30 min). The
supernatant obtained after 20 000 g centrifugation was sedimented (30 000 g for 30 min),
yielding a pellet (PI) and supernatant (Sl). The
P, pellet was resuspended and washed in 0.32
mol/l sucrose (57 OOOg for 40 min): fraction F5.
The S, supernatant was centrifuged at 120 000 g
for 90 min and washed in 0.32 mol/l sucrose
under the same conditions: fraction F6.
Identification o f membrane fractions
Among the fractions F1-F6 of WKY rat brain
the highest activity of plasma membrane markers
(acetylcholinesterase [ 171, S'mcleotidase [ 181)
was observed in fraction Fz. The activity of mito-
TABLE 1. Protein content and relative enzyme activities in membrane fractions of
WK Y rat brain
The protein content is given as percentage of the total protein of the homogenate.
The total enzyme activity in fractions F1-F, was taken as 100%.
Fractions
Protein
Relative activities (%)
(% of total)
F,
F2
F3
F,
F,
F6
4.59
2.1
2.3
1.4
1.5
2.9
Acetylcholinesterase
5'-Nucleo-
tidase
Succinate
dehydrogenase
2.1
8.9
6.3
3.4
11.5
61.2
24
25
10
2
11
28
6.6
7.2
24.2
48.4
12.1
1.5
Glucose 6-
phosphatase
0.5
0.5
0
0.5
0.5
98
Calcium transport in synaptosomes in hypertension
chrondrial enzyme markers (succinate dehydrogenase [19]) was negligible in fraction F2 and the
highest activity was in fraction F4 (Table 1). Data
obtained in this study for identification of fractions F2and F4 are in accordance with the results
of other authors [16]. It should be noted that the
maximum glucose 6-phosphatase activity [20] was
observed in fraction F6, together with h g h activity
of both S'hucleotidase and acetylcholinesterase
(Table 1). Taking into consideration the data on
the distribution of glucose 6-phosphatase activity
in membrane fractions of liver and kidney, for
which this enzyme was found to be a marker of
endoplasmic reticulum [20], it may be suggested
that our fraction F6 consisted mostly of endoplasmic reticulum, which contains the enzymes
commonly acting as markers for plasma membranes, or non-synaptosomal plasma membranes
enriched by glucose 6-phosphatase.
There were no significant differences in the
activities of membrane marker enzymes between
WKY and SH rats.
On the basis of the data mentioned above,
membrane fractions Fz (synaptosomes), F4 (mitochrondria) and F6 (microsomes) were used for
study of Ca2+transport.
129
After the incubation, 100-150 pg of synaptosome protein was transferred to Millipore HA
filters, which had been stored in KC1 solution
(1 mol/l) and washed just before the experiment
with 5 ml of the following medium (mmol/l):
NaCl, 150; EGTA, 1; Tris-HC1, 10 (pH 7.1;
0-2°C). The samples on the filters were washed
three times with 5 ml of the same medium.
Sodium uptake by synaptosomes
To determine Na+ uptake synaptosomes were
put in medium B containing 0.3 mmol of oubainll,
where 45CaC12was substituted for "NaCl(20pCi/
ml). After 0.5, 10 and 30min of incubation an
aliquot of synaptosome suspension containing
2 mg of protein was placed on filters (Whatman,
GF/c, U.K.) and washed four times with 5 ml of
the same wash medium.
Gz2' accumulation by mitochondria and micro-
somes
Membrane fraction protein (30-40 pg) was
added to 50ml of medium and incubated at
37°C with periodic shaking. The composition of
the incubation medium was (mmol/l): imidazoleHCl, 40 (pH 6.9); H a , 120; NaCl, 6.5; MgCl2,3;
K2HP04, 3; ATP-Tris, 4 ; EGTA, 0.1; CaCl,,
Calcium uptake by synaptosomes
0.01-0.3; 45CaC12, 1.5 pCi/ml. Accumulation of
To study the kinetics of Ca2+uptake, synapto- CaZ+by the mitochondria was determined in the
somes were preincubated for 10 min (37°C) in the presence of 4 mmol of Tris-succinatell. Free
following medium (mmol/l): HEPES-Tris, 20 (pH calcium concentration in the incubation medium
7.4); MgCl,, 135; K2HP04, 0.5; glucose, 10. Pro- was calculated from the value K, [Ca-EGTA]*- =
tein concentration was 1-2 mg/ml. After pre- 1.3568 x lo6mol/l [21]. After the incubation,
incubation, aliquots of synaptosomes were put in the suspension (200 pl) was filtered through Millimedium B [composition (mmol/l): HEPES-Tris,
pore filters (HA) and the filters were washed
20 (pH 7.4,37"C); MgCl,, 1; KC1, 5; NaC1, 135; three times with 5 ml of wash medium. RadioK2HP04, 0.5; CaC1, 1;45CaC1z,5 pCi/ml; glucose, activity of the filters and in the incubation
101 or in medium C (the same as medium B, but medium was measured in Bray solution [22]. The
with NaCl at 50 mmol/l and KCl at 80 mmol/l). kinetics of Ca2+ accumulation by the membrane
In a number of experiments the Na+/K+concen- fraction was linear up to 10 min. To determine the
rate of Ca2+ accumulation the incubation time
tration ratio was varied.
Except for synaptosomal membrane depolariza- was limited to 5 min. The dependence of calcium
tion due to the elimination of K+-diffusionpoten- accumulation rate on the Ca2+concentrations was
tial (C media), synaptosomal depolarization was expressed in a Lineweaver-Burk plot. A HP-9815A
caused either by the addition of veratrine to computer was used to calculate the affinity (K,)
medium B, leading to the opening of Na+-channels, and the maximal rate (Vmax.)of Ca2+accumulaor by ouabain, which depolarizes the membrane tion.
by Na',K+-dependent ATPase inhibition. Specific
Na+-channel(tetrodotoxin, TTX) and Ca"channe1
Calmodulin concentration
(verapamil) blockers were used in the experiments.
Inhibitors of electron transport and ATPase of
Concentration of calmodulin in the nerve tissue
mitochrondria (rotenone and oligomycin) were cytosol (supernatant obtained after the microsome
added 2min before the end of preincubation. sedimentation) was determined by the increase of
Inhibitor concentrations are presented in the ATPdependent Ca2+ accumulation by inside-out
vesicles of erythrocyte membranes. Both the
Tables and Figures.
G. M. Kravtsov et al.
130
method of preparation of rat erythrocyte ghosts
enriched by inside-out vesicles and the method of
determination of CaZ+ accumulation by these
vesicles have been described before [23].
Reagents
TTX, rotenone and oligomycin (Bohringer,
F.R.G.), veratrine (active principle of veratridine)
(Sigma, U.S.A.) and verapamil (LEX,Yugoslavia)
were used in this study. Calmodulin was kindly
provided by Dr V. A. Tkachuk (Biochemistry
Department of Moscow University).
Results
Effect of depolarizing agents on Ca2+accumulation b y synaptosomes
Addition of veratrine (70 pg/ml) t o medium B
or changes in the K+/Na+ concentration ratio
(medium C) increased CaZ+ uptake by synaptosomes 2-2.5-fold (Fig. 1). The maximum effect
of potassium depolarization was observed at
3-5 min and, for veratrine depolarization, at
15 min of incubation (Fig. 1). Ouabain, a Na+,K+ATPase inhibitor, caused an increase in Ca2+
uptake. The addition of TTX t o medium B par-
6 -
4.
2 -
0-
I
0
10
20
30
L
0
10
20
30
Time (mm)
Time (min)
FIG. 1. Kinetics of calcium uptake by synaptosomes of control rats. 1, Control
(medium B); 2, medium B + ATP (1 mmol/l); 3, medium B -!- veratrine (70 pg/ml);
4, medium B -I-ouabain (0.3 mmol/l); 5 , medium B -!- veratrine (70 pg/ml) -!- ouabain
(0.3 mmol/l); 6, medium C. In ( b ) the media contained oligomycin (2 pg/ml) -!rotenone (2 pg/ml).
TABLE2. Effect of blocking agents on calcium uptake by synaptosomes
Means f SE are given. The time of incubation was limited t o 5 and 15 min in media B
and C respectively. TTX, Tetrodotoxin.
Medium
Blocking agent
Ca’+ (nmol/mg of protein)
WKY rats
(n=5)
B (low
potassium)
B + ouabain
(0.3 mm041)
B + veratrine
(7O~dml)
C (high
potassium
TTX (500 nmol/l)
Verapamil(100pmol/l)
Oligomycin (2pglml) + rotenone (2 pg/ml)
TTX (500 nmol/l)
Verapamil(lOOpmol/l)
TTX (500 nmol/l)
Verapamil(lOOpmoI/l)
TTX (500 nmol/l)
Verapamil(100 pmol/l)
1.72 i 0.16
1.68 i 0.14
1.55 i 0.10
0.60 i 0.08
2.68 i 0.20
2.03 i 0.25
4.26 i 0.31
1.68 i 0.16
1.58 f 0.09
4.81 i 0.40
4.10 i 0.35
1.53 i 0.10
* Difference between SH and WKY rats with P < 0.01.
SH rats
(n =5)
2.93 i 0.15*
2.22i 0.1*
1.53 i 0.08
0.98 i 0.05
4.24 i 0.29*
2.96 i 0.17*
1.63 i 0.07
4.31 i 0.40
1.72 i 0.16
1.50 i 0.10
4.78 i 0.45
4.81 i 0.37
1.60 i 0.08
Gdcium transport in synaptosomes in hypertension
tially decreased the Ca2+uptake by synaptosomes
caused by ouabain and completely blocked veratrine-induced Ca2+ uptake. There was no effect
of TTX on K+-induced Ca2+ uptake (Table 2).
Verapamil not only abolished the effect of all
depolarizing agents on Ca2+ uptake by synaptosomes, but also decreased basal Ca2+ uptake in
medium B by 10-15%. Oligomycin and rotenone,
depolarizing the mitochondria1 membrane, led
to a drastic decrease both in basal and in veratrine- or K+-induced Ca2+uptake (Fig. lb).
Basal Ca2+ uptake by synaptosomes of hypertensive rats was 70% higher than that of normotensive control rats (Table 2). The differences
disappeared only after verapamil treatment.
The dependence of Ca2+ uptake by synaptosomes on the K+/Na+ concentration ratio is
shown in Fig. 2. Differences of Ca2+ uptake between synaptosomes from hypertensive and
normotensive rats were revealed at moderate (up
to 60 mmol/l) potassium concentrations, where
Ca" uptake by synaptosomes of SH rats was
considerably higher.
The effect of ouabain on Ca" uptake by the
SH rat synaptosomes was greater than that observed in synaptosomes of normotensive rats
(Table 2). In SH rats the magnitudes of ouabain-,
veratrine- and potassium-induced Ca" uptake
were practically the same. The differences in the
ouabain effect on synaptosomes of both groups
of animals remained after addition of TTX,
whereas they were not observed after treatment
with verapamil.
6r
B
2
't
0
11
0
20
40
60
80
100 K*
120
100
80
60
40Na+
I
1
140
13.1
t
~
0
~
~~~~~
2
1
3
Ca'+ bmol/l)
FIG. 3. Dependence of the rate of calcium accumulation by mitochondria on the free calcium
concentration in the incubation medium. The
number of observations is 4 for each group. The
brain tissue from eight rats was used for each
observation.
Ca2+ accumulation by mitochondria and microsomes
The affinity of the Ca'+-transporting system
of mitochondria for Ca2+in hypertensive rats was
approximately twice that in normotensive rats
(1.03 k0.08 pmol/l and 0.58 f 0.60 pmol/l respectively; Fig. 3). The maximal rate of Ca2+accumulation by mitochondria of both groups did not
differ (Table 3). The affinity of the Ca2+-transporting system of microsomes for Ca2+was thrice
that of mitochondria (Fig. 4, Table 4). On the
other hand, the maximal rate of Ca" accumulation by microsomes was about one-twentieth of
that of mitochondria. The addition of calmodulin
led to an increase both in the maximal rate of
Ca2+ accumulation by microsomes of normotensive rats and in the affinity for Ca". The effect
of calmodulin on the Ca2+pump of SH rat microsomes was negligible (Fig. 4, Table 4).
Concn. (mmol/l)
FIG.2. Dependence of calcium uptake by synapto-
Gdmodulin content
somes on the potassium/sodium concentration
ratio in the incubation medium. The number of
observations is 5 for each group. The brain tissue
from eight rats was used for each observation.
The content of calmodulin in cytosol of brain
tissue (supernatant obtained after sedimentation
of microsomes) did not differ in the two groups of
G.M.Kravtsov et al.
132
o l , ,
,
,
,
,
0
2
3
4
5
1
0
1
2
3
4
5
Ca" bmol/l)
Calf bmol/l)
FIG. 4. Dependence of the rate of calcium accumulation by microsomes on the free
calcium concentration in the incubation medium: ( 1 ) without calmodulin; (2) with
calmodulin (0.8 pmol/l). The number of observations is 4 for each group. The brain
tissue from eight rats was used for each observation.
TABLE3 . Affinity for Caz+ ( K , ) and the maximal
rate (Vmax.) o f calcium accumulation by rat brain
mitochondria
n is the number of observations. The brain tissue
from eight rats was used for each observation.
Means f SE are given. N.S., Not significant.
Group
1 WKY rats
2 SH rats
P (1 vs 2)
11
K, bmol/l)
4
4
1.03 i0.08
0.58 f 0.06
9.93t 1.20
10.73 f 1.41
< 0.001
N.S.
"ma,.
(nmol min-'
rng-' of protein)
animals. In both the addition of 3 p l of cytosol
to the 5 0 0 ~ 1of the incubation medium led t o a
5.0-5.2-fold increase in the rate of CaZ+accumulation by the inside-out vesicles of the erythrocyte
membranes. The same increase in the rate of Ca2+
accumulation could be produced by the addition
of 1.5 nmol of bovine brain calmodulin (Fig. 5).
Discussion
Fraction F z , which has been characterized as
synaptosomes, was enriched by enzyme markers
of plasma membranes and contained the minimal
admixtures of mitochondria and endoplasmic
reticulum (Table 1). This conclusion is in an agreement with the practical absence of effect of ATP
(1 mmol/l) on 45Ca uptake b y fraction Fz (data are
not presented). Fraction F1,which was similar to
fraction F z in 5'-nucleotidase activity, consists
mostly of myelin [ 161.
The increase in CaZ+ uptake caused by potassium depolarization was completely inhibited by
verapamil (Table 2). The Na+-channel inhibitor
TTX did not affect potassium-stimulated CaZ'
uptake; this agrees with previously published
results [24]. Ouabain, the Na+,K'-ATPase inhibitor, is capable of lowering the membrane potential
(by 10-20 mV) by abolishing its electrogenic
component dependent on the Na' pump [25].
Such a decrease of potential apparently leads to
the opening of a part of the potential-dependent
Na+-channels and to still greater membrane
depolarization, which in turn causes the opening
of Caz+-channels in the plasmalemma and leads
to Ca2+ influx into the nerve endings (synaptosomes). The partial decrease of CaZ+accumulation
by synaptosomes under the effect of TTX, as well
as its complete disappearance due t o verapamil,
support this explanation (Table 2).
Increases in Na+ permeability, induced by
veratrine, give rise to synaptosome depolarization,
which in its turn leads to the opening of potentialdependent Caz+-channels in the membrane and, as
a result, t o an increase in Ca2+uptake. The alternative pathway is CaZ' entry into synaptosomes
through potential-dependent Na+ channels. TTX
prevented the effect of veratrine (Table 2). However, verapamil also completely abolished the
veratrine-induced Ca" uptake (Table 2). If Caz+
influx occurred (if only in part) along the TTXsensitive Na+-channels, verapamil would not be
able to abolish the veratrine effect completely.
Thus analysis with several inhibitors has shown
that CaZ+ influx into synaptosomes in all types
of membrane depolarization used in the study
occurs only through potential-dependent CaZ+channels.
The differences in CaZ+ uptake by synaptosomes in SH rats and control rats were observed
in the absence of depolarizing agents or during
133
Cirlcium transport in synaptosomes in hypertension
TABLE 4. Effect of calmodulin on the affinity for Caz+ ( K , ) and the maximal rate
( Vmax,) o f calcium accumulation by rat brain microsomes
n is the number of observations. The brain tissue from eight rats was used for each
observation. Means? SE are given. N.S., Not significant.
With calmodulin
(0.8 pmol/l)
Without calmodulin
Group
1 WKY rats
2 SHrats
P ( l vs2)
n
Km~mol/l)
4
4
Vmax:
Km OcmoW)
Vmax:
(nmol mm-l
mg-I of protein)
0.334 i 0.034
0.289i0.061
0.582 i 0.036
0.397i0.05
0.267 i 0.026
0.253 i 0.028
0.723 i 0.036
0.459 i 0.031
N.S.
< 0.05
N.S.
<0.01
(nmol mm-I
mg-' of protein)
0.0015 0.05 0.15
0.5
1.5
5
15
Calmodulin (nmol)
0
1
2
3
4
5
Supernatant &I)
FIG. 5 . Dependence of the rate of Ca2+uptake by inside-out vesicles of the rat erythrocyte membrane on calmodulin (1) or supernatant of WKY (2) and SH (3) rat brain
tissue content in 0.5 ml of the incubation medium. The incubation medium also contained imidazole-HC1 (50 mmol/l) (pH 6.9), KC1 (100 mmol/l), MgClz ( 5 mmol/l),
EGTA (0.1 mmol/l), ATP (disodium; 4 mmol/l) and 4sCaC12 (0.04 mmol/l) (for
details see [23]). The number of observations is 4 for each group. The brain tissue
from eight rats was used for each observation.
moderate plasma membrane depolarization (by
means of Na+,K+-ATPase inhibition and by increases in K+ concentration 'in the incubation
media up to 50-60mmol/l). Since these differences disappeared after verapamil action, it could
be suggested that they were determined by the
increase in SH rat synaptosomes of Caw influx
through Ca"-channels owing to partial membrane
depolarization.
Partial synaptolemma depolarization might
result from the decrease of K+ diffusion potential
(due to the decreased intrasynaptosomal K+concentration) or from the increased permeability of
the synaptolemma to Na'. To examine the latter
suggestion, the kinetics of "Na influx into synaptosomes were studied. The results given in Fig. 6
show that Na+ uptake rate by synaptosomes in the
absence of depolarizing agents (medium B) is
considerably hgher in hypertensive rats than in
control animals.
Thus there are grounds to believe that altered
permeability of the synaptolemma for Na+ in
SH rats leads to its depolarization and, as a result,
to an increased CaZ+influx into nerve endings.
The high affinity constant for Ca" (Km = 1.03
pmolll) and the high maximal Ca2+accumulation
rate (VmaX.= 9.2 nmol min-' mg-' of protein)
enable mitochondria to compete effectively with
MgZ+,CaZ+-ATPase
of the plasma membrane for
cytosolic ionized calcium, during the early stages
of depolarization. Additional data concerning the
leading contribution of mitochondria to the
regulation of intrasynaptosomal Ca2+ concentration during depolarization were obtained in
G. M.Kravtsov et ai.
134
0.3
h
0.2
-
0.1
-
.d
c
2a
CI
2
=r
g
3
0
10
20
30
Time (min)
FIG. 6 . Kinetics of sodium uptake by synaptosomes. The number of observations is 5 for each
group. The brain tissue from eight rats was used
for each observation.
experiments with rotenone and oligomycin. The
addition of these electron-transport inhibitors of
mitochondria reduced both basal Ca2+ accumulation by mitochondria and the effect of all depolarizing agents used in the study (Fig. 1). These
results are in good agreement with the data of
Akerman & Nickolls [13].
The other membrane fraction of nerve tissue
cells in our study possessing Ca" accumulating
ability was the microsome fraction. Besides the
high activity of marker enzymes of the plasma
membrane (S'-nucleotidase and acetylcholinesterase), high activity of the endoplasmic reticulum
marker (i.e. glucose 6-phosphatase) was observed
in this fraction (Table 2). A membrane fraction
similar in enzyme marker composition was obtained b y Schellenberg & Swanson [26]. It should
be noted that the affinity of the Ca'+-transporting
system of microsomes for Ca'L+( K , = 0.33 pmol/
1) (Fig. 4, Table 4) is similar to that found by
Blaustein et al. for the fraction of lysed synaptosomes [27]. We suggest that the latter fraction may
contain a microsome admixture. It should also be
noted that the increase of Ca" accumulation by
the microsome fraction was observed during K+induced depolarization and the existence of the
Na+-Ca'+ exchange system was shown to be
identical with that described for synaptosomes
[26]. In our study there was increased Ca" uptake
by microsomes after the addition of calmodulin
(Table 4). Previously the calmodulin effect on
Ca'+ uptake has been demonstrated in inside-out
vesicles of the presynaptic membranes [ 151.
Hence we assume that the properties of the Ca'+transporting system of both the synaptolemma
and microsomes are similar (if not identical).
Therefore we can evaluate the contribution of
plasma membrane and of mitochondria in Ca2+transport regulation in depolarized synaptosomes.
The values of K, and V,.
for Ca'+-transporting
systems of mitochondria and microsomes suggest
that mitochondria play the leading role in the
maintenance of low intrasynaptosomal CaZ+
concentration.
Together with increased calcium influx in
synaptosomes, defects of both mitochondria1 and
microsomal Caz+-transporting systems were observed in the brain tissue of spontaneously hypertensive rats. Whereas Ca2+uptake by mitochondria
of hypertensive rats was higher than that of
normotensive animals, in microsomes of SH rats
it was, on the contrary, reduced. Because of this
the part played by changes in subcellular calcium
handling in determining the rate of the neurotransmitter release by nerve terminals of spontaneously hypertensive rats may be obtained only
by direct investigations. In hypertension the
increment of the maximal Caz+-transportrate after
calmodulin action was lowered both in microsomes of brain tissue (Table 4)and in erythrocytes
[28]. This suggests that the altered interaction
between calmodulin and membrane Ca'+-transporting systems is an important part of a widespread
membrane defect in spontaneous hypertension.
The molecular mechanism of this defect, which
also reveals itself by the increase of plasma membrane permeability for univalent cations [7,8, 14,
181, the decrease of Ca'+-binding ability [6,9],
has been investigated. The results of the present
study, as well as the investigations carried out on
the adipose tissue of SH rats [5], indicate, however, that the membrane defect in primary hypertension is not limited to plasma membranes but
appears also in mitochondria.
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