Download AGE-RELATED CHANGES OF Na+, K+ - ATPase, Ca+2

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2004, 55, 2, 457–465
www.jpp.krakow.pl
T. TORLIÑSKA, A. GROCHOWALSKA.
AGE-RELATED CHANGES OF Na , K
+
AND Mg
+2
- ATPase, Ca
+
+2
- ATPase
-ATPase ACTIVITIES IN RAT BRAIN SYNAPTOSOMES.
Department of Physiology, University of Medical Sciences, Poznañ, Poland
Cerebral metabolism of glucose, one of the determinants of tissue ATP level, is
crucial for central nervous system function. The activity of P-type pumps, namely
Na ,
+
K
-
+
ATPase,
Ca
+2
-
ATPase
and
Mg
+2
-
ATPase
were
examined
in
brain
synaptosomes of 5 - day, 3 - month and 18 - month - old rats to determine if changes
in enzyme activity related to aging are potentially associated with alterations in
glucose homeostasis.
Activities of all the ATPases studied in isolated brain synaptosomes were expressed
in µmol of Pi liberated from ATP by 1 mg of synaptosome protein during one hour.
Serum glucose concentration was measured by the glucose oxidase method and
insulin level was estimated by the RIA. Our results demonstrate that 18 - month - old
rats are characterized by hyperglycemia and hyperinsulinemia. Their serum glucose
concentration was significantly increased approx. 62.3% and 135.8 % as compared
to 3 - month - old rats and 5 - day, newborn rats, respectively. An enormous increase
in
serum
insulin
concentration
in
the
old,
hyperglycemic
rats
was
observed
concomitantly. As a result of these changes the insulin - to - glucose ratio in the old
rats was greatly increased approx. (270% and 230%) compared to young, mature and
newborn rats. Hyperglycemia and hyperinsulinemia occurring in the old rats, had a
different impact on activities of the ATPases tested. Our results have revealed that
Na , K
+
+
- ATPase activity remains almost unchanged with age, the activity of Ca
ATPase decreases, whereas that of Mg
+2
+2
-
- ATPase increases significantly in old,
insulin resistant rats. In conclusion it seems that changes in activity of different P -
type pumps may differ with aging and that adaptation of specific ATPases to internal
environment alterations is not identical.
Key
w o r d s : Na ,K -ATPase,
+
+
Ca
+2
-ATPase,
Mg
+2
hyperglycemia, hyperinsulinemia, aging.
-ATPase,
brain
synaptosomes,
458
INTRODUCTION
It is generally accepted that cerebral metabolism of glucose, which appears
one of the determinants of tissue ATP level, is crucial for central nervous system
(CNS)
activity.
Na ,
+
K
+
-
stimulated
ATPase
(E.C.
3.6.1.3.)
is
known
to
be
involved in the maintenance of sodium and potassium gradients across plasma
membranes
at
the
expense
of
ATP
hydrolysis,
with
very
high
activity
in
electrically excitable tissues. The activity of this enzyme has been found to
increase in the developing rat brain and to decrease during aging (1).
A
study
on
synaptosomal
the
effect
fractions
of
from
aging
the
on
rat
Na ,
brain
+
K
-
+
ATPase
parietal
cortex,
activity
in
crude
hippocampus
and
striatum revealed a progressive decline in enzyme activity from 12 months to 24
months of age (2). Zaidi and co-workers (3) found that also Ca
-ATPase activity
2+
in F344/BFN1 rat brain synaptic plasma membranes exhibited a progressive agedependent decrease which might be accounted for some loss of PMCA from the
membranes and for age-related structural changes of calmodulin.
By contrast, Kennedy et al., (4) showed that aging-related changes in energy
utilization demonstrated in brain slices and homogenates were not associated with
alterations
in
the
Na ,
+
K -ATPase
+
enzyme
system.
Moreover
as
shown
by
Mooradian et al., (5) sodium - potassium or magnesium ATPase activity was not
altered with aging or diabetes but exhibited reduced inhibition by NEM ( Nethylmaleimide ), the known (H ) - ATPase inhibitor.
+
Insulin resistance in old, compared with young, humans and animals has been
well documented to lead finally to hyperglycemia and hyperinsulinemia.
Insulin cannot be synthesized by the central nervous system and is transported
from the blood to the brain acting as a multiple functioning molecule, i.e. growth
factor
during
development
and
modulator
of
electrical
activity
via
phosphorylation of voltage-gated ion channels (6). According to Baura et al., (7),
the delivery of plasma insulin into the CNS is saturable and is facilitated by an
insulin - receptor mediated transport process. The results of Zaia and Piantanelli
(8) showed a significant decrease in the number and increase in affinity of insulin
binding sites in brains of old animals that may be responsible for alterations in
central insulin action, among them neuromodulation.
As shown by Aragno et al., the hyperglycemia-induced damage of central
nervous system is brought about by a detrimental effect of chronic hyperglycemia
on the integrity of synaptic membranes due to overproduction of free radicals (9).
The aim of our study was to elucidate if there is any relationship between
hyperglycemia and hyperinsulinemia found in old animals and changes in activity
of several ATPases, including Na , K -ATPase, Ca
+
+
-ATPase and Mg
+2
-ATPase in
+2
synaptosomes isolated from rat brain during aging. Our review of the literature
revealed no study on the activity of ATPases in the CNS along with blood glucose
and insulin concentrations during aging, performed under identical conditions at
the same time.
459
MATERIAL AND METHODS
The experiments were performed on male Wistar rats and conducted following the experimental
protocol approved by the Committee for Research and Animal Ethics of the University of Medical
Sciences in Poznañ. The animals were housed in cages at normal room temperature and maintained
on standard laboratory chow (LSM) with free access to food and water. All the experiments were
carried
out
between
9-11
a.m.
The
rats
were
sacrificed
by
decapitation
and
after
complete
exsanguination brains were removed immediately and used for isolation of synaptosomes. In the
present study brain synaptosomes were isolated from three groups of rats: newborn (5-day-old),
young (3-month-old) and old (18-month) animals.
The tissue was homogenized in 20 vol of 0.32 M sucrose at 4°C.
Preparation of synaptosomes
Synaptosomes were isolated from rat brains by the method of Lin and Way (10, 11). Briefly, a
whole-brain homogenates in 0.32 M sucrose - 5 mM HEPES buffer, pH 7.5, were centrifuged at
1000 x g for 10 min. The resultant supernatant was centrifuged for 30 minutes at 17 000 x g to
obtain the crude synaptosomal fraction. The fraction, washed twice and suspended in 0.32 M
sucrose HEPES - buffer was subjected to Ficoll gradient centrifugation (63 000 x g for 1 hour).
Synaptosomes collected at the interfaces between Ficoll layers (7.5% and 12% in sucrose - HEPES
solution) were pelleted and suspended in 0.32 M sucrose solution for enzyme assays within 2 hours.
All the procedures described above were carried out at 4°C.
ATPases Assay
The Na , K -ATPase activity was estimated by the method of Muszbek et al., (12). The enzyme
+
+
activity was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP
during the incubation of synaptosomal fraction. The reaction mixture contained 100 mM NaCl, 20
mM KCl, 2mM ATP (disodium salt), 30mM Tris-HCl buffer (pH 7.4) and the synaptosomes (50 µg
of protein) in a final volume of 1 ml. After a 10-min. preincubation at 37°C to allow the ouabain
(1.5 mM) to react with the ATP-ase, the reaction was initiated by addition of ATP, and terminated
after 15 min. incubation by addition of 500 µl of 15% (w/v) trichloroacetic acid. The released
inorganic phosphate was assayed by the spectrophotometric method of Goldberg (13). Na , K +
+
ATPase activity was calculated from the difference between amounts of inorganic phosphate found
after incubation in the absence and presence of 1.5 M ouabain.
The Mg
+2
-ATPase activity and Ca
+2
-ATPase activity were measured by the method of Lin and
Way (11). The assay medium (1 ml) contained 50 mM imidazole-HCl buffer (pH 7.5), 0.4 mM
MgCl2 or 0.4 mM CaCl2, 2 mM ATP and the synaptosomal fraction (50 µg protein) suspended in
0.32 M sucrose. After 15 minutes of incubation at 37°C the reaction was stopped by adding 1.2 ml
of ice cold 10% TCA. The Ca
+2
- ATPase (or Mg
+2
- ATPase) activity represented the difference
between the enzyme activity in the presence and absence of Ca
+2
(or Mg
) cations.
+2
Activities of all the ATPases tested, were expressed in µmol Pi liberated from ATP by 1 mg of
synaptosome protein during one hour (µmol Pi/mg protein/hour). Protein concentration was assayed
by the method of Lowry et al., (14) using bovine serum albumin as a standard.
Other analyses
Serum insulin was measured by a standard radioimmunoassay (RIA) using kits for rat insulin
(Linco, Research Inc. USA). Glucose was determined by the glucose oxidase method (Sigma).
460
Student's
t-test
was
used
for
statistical
evaluation
and
differences
were
considered
to
be
significant at a level of p<0.05.
RESULTS
The
results
summarized
in
Tab.
1
demonstrate
that
18-month-old
rats,
weighing 595 - 630 g, can be regarded as hyperglycemic and hyperinsulinemic
animals. Their serum glucose concentration was significantly increased by about
62.3% and 135.8% as compared to 3-month-old rats (weighing 220 - 250 g) and
5-day-newborn rats (5.8 - 6.0 g), respectively. As shown in the same table, an
enormous
increase
in
serum
insulin
concentration
was
observed
in
the
old,
hyperglycemic rats in comparison to insulin levels measured in serum of newborn
and young, mature rats. As a result of these changes the insulin-to-glucose ratio
in the old rats was greatly increased, by about 270% as compared to young rats,
and by about 230% in comparison to the newborns. These findings confirm that
the 18-month-old rats, used in our experiments, are characterized by an agedependent insulin resistance.
Tab. 1. Serum glucose and insulin concentrations and
the insulin/glucose ratio in newborn (5 days),
young (3 months) and old (18 months) rats.
NEWBORN RATS
YOUNG RATS
OLD RATS
Glucose ( mmol/l )
4.35 ± 0.50
6.32 ± 0.30
10.26 ± 0.30*
Insulin (IRI) µU/ml
14.88 ± 3.71
19.00 ± 1.91
114.06 ± 5.59*
IRI/glucose µU/mol
3.35 ± 0.66
3.01 ± 0.98
11.16 ± 0.56*
Results are presented as mean ± SEM
( n = 10
)
!"#$%
*Significantly
different from newborn rats ( p < 0.01 )
&
''''
(%%
Tab. 2. Activities of Na , K , Mg
in
+
synaptosomes
(18 months) rats.
+
isolated
-
)*+', .
-
#
-)
+2
and Ca
+2
from
/
-)
- ATPases (expressed in µmol Pi x mg of protein
brains
of
0*12
newborn
(5
days),
31
2'
young
4
(3
44
2
months)
''
NEWBORN RATS
NEWBORN RATS
+
+
x hour )
-1
YOUNG RATS
YOUNG RATS
and
OLD RATS
OLD RATS
15.80 ± 1.30 *
13.50 ± 1.20**
Ca - ATPase
6.67 ± 2.04
*
10.40 ± 0.80 *
7.40 ± 0.60****
Mg+2 - ATPase
2.31 ± 0.90
8.30 ± 0.90*
+2
Ca+2 - ATPase
Mg+2 - ATPase
6.88 ± 2.08
15.80 ± 1.30
6.67 ± 2.04
10.40 ± 0.80
8.30 ± 0.90*
2.31 ± 0.90
13.50 ± 1.20
7.40 ± 0.60
*
*
11.60 ± 0.80**
11.60 ± 0.80**
Results are presented as mean ± SEM
!"#$%
!"#$%
( n = 10 )
*Significantly
different from newborn rats ( p < 0.001 )
&
''''
(%%%
''''
(%%%
**Significantly
different from young rats ( p < 0.001 )
&
&&
''''
(%%%
''''
(%%%
&&
old
6.88 ± 2.08
Na , K - ATPase
Na+, K+ - ATPase
*
-1
461
Fig. 1. Relative differences in activities of Na , K
+
+
- ATPase , Ca
2+
- ATPase and Mg
2+
- ATPase in
newborn ( 5-day-old ) rats (A) and 18-month-old rats (B) expressed in % of control activity. Values
obtained from young mature rats ( 3-month-old) were accepted as control ( 100% )
As depicted in Tab. 2 activities of Na , K , Ca
+
in
µmol
Pi
x
mg
protein
x
hour)
in
+
+2
and Mg
synaptosomes
+2
-ATPases (expressed
isolated
from
brains
of
newborn, young and old rats differ among the groups tested. The activities of
Na ,
+
K -ATPase
+
synaptosomes
and
Ca
isolated
+2
-ATPase
from
surprisingly, the activity of Mg
were
young,
+2
found
mature
to
rats
be
the
highest
(3-month-old)
in
brain
whereas
-ATPase was significantly elevated in the group
of 18-month-old rats.
When values of ATPase activities estimated in young, mature rats, commonly
used as experimental animals, were accepted as 100% (Fig.1) it was found that
greater relative differences can be observed in the group of newborn rats than in
the old rat group. These results may also suggest that changes in activities of all
synaptosomal ATPases tested, could provide an index of brain maturity not only
in the developing rat brain, as shown previously, but also in the postnatal period.
On the contrary, relative differences in activities of Ca
-ATPase and Na , K -
+2
+
+
ATPase in synaptosomes isolated from brains of the old, 18-month animals were
smaller or even insignificant, except of those concerning Mg
-ATPase activity
+2
that appeared significantly elevated.
462
DISCUSSION
It has been proved that either glycemia per se or glucose metabolites are
responsible for the blood-brain barrier abnormality which occurs in diabetes.
Knudsen
and
Jacobsen
(15)
suggest
that
the
specific
decrease
of
sodium
permeability could be the result of glucose-mediated inhibition of the Na , K +
ATPase
localized
at
the
blood-brain
barrier.
However
in
some
+
conditions
hyperglycemia can be beneficial for the organism. For example data of Ekholm
et al., (16) indicate that hyperglycemia retards the loss of ion homeostasis by
leading
to
production
of
additional
ATP
supporting
Na ,
+
K -driven
ATPase
+
activity during ischemia.
In the present study we have shown that serum glucose concentration was
significantly increased in old, 18-month rats as compared with the newborn
animals. These changes were accompanied by an increase of immunoreactive
serum insulin concentration in old rats. As a result of these changes IRI/glucose
ratio in the old rats was greatly elevated obviously showing insulin resistance.
Hyperglycemia
and
hyperinsulinemia
observed
in
our
study
had
a
different
impact on activities of the ATPases tested. There are striking differences among
Na , K -ATPase, Ca
+
+
-ATPase and Mg
+2
-ATPase in the synaptosomal fraction
+2
isolated from brains of old hyperglycemic animals. Our results revealed that Na ,
+
K -ATPase activity remains almost unchanged with age, the activity of Mg
+
-
+2
ATPase increases significantly whereas activity of Ca
-ATPase decreases. The
+2
changes in the latter enzyme can be accounted for the hyperglycemia found in old
rats, that in turn may lead to the enzyme protein glycation as shown by Janicki et
al., (17).
Another pathway, in addition to generation of free radicals that is involved in
age-related cellular degeneration is the accumulation of advanced glycosylation
end-products (AGE). Under in vivo conditions AGE formation depends mainly
on the blood glucose concentration that has been proved to be elevated with age
(18). There is a growing number of data showing that hyperglycemia may inhibit
the activity of several enzymes due to enzyme protein glycation (19,20).
It has been shown that also experimental diabetes is associated with reduction
of activity of P-type pumps, e.g. Na , K -ATPase in the central nervous system
+
+
(21). In contrast, the marked hyperglycemia caused by the administration of 2DG to satiated rats was associated with significant increase in Na , K -ATPase
+
+
activity and in [3 H]-ouabain and [3 H]-mazindol binding to hypothalamic sites
which are functionally coupled in their response to circulating glucose (22).
Plasma membrane Ca
-ATPase (PMCA), a regulator of intracellular calcium,
2+
is known to be an enzyme, the activity of which undergoes early developmental
changes in rat brain as a function of its maturity (23).
According to Zaidi and Michaelis, (24) PMCA appears to be very sensitive to
the inhibitory effect of reactive oxygen species ( ROS ) due to the age dependent
oxidative modification of PMCA and the related chronic oxidative stress.
463
Alterations in the capacity to maintain normal calcium homeostasis have
been suggested to underlie the reduced cellular function bound with the aging
process.
It
seems
likely
that
in
the
brain,
multiple
methionines
within
the
calmodulin molecule become oxidized to methionine sulfoxides, resulting in an
inability to activate a range of target proteins, including plasma membrane Ca
2+
-
ATPase. (25).
Recent evidence indicates, that chronic hyperglycemia may inhibit plasma
membrane Ca
-ATPase (PMCA) in cells of various tissues. The P-type plasma
2+
membrane PMCA, a regulator of cytosolic Ca
has a diminished activity in
2+
erythrocyte and renal cortical cell membranes in uncontrolled diabetes.
As shown by Janicki et al., (17) PMCA pumping activity in SPM vesicles
prepared from the cerebra of hyperglycemic rats was depressed by about 8.4%,
compared to control normoglycemic rats. The brain synaptic PCMA inhibition
was accompanied by glycation of hemoglobin in rats with streptozocin-induced
diabetes.
In
addition,
PCMA
activity
in
synaptic
plasma
membranes
from
normoglycemic rats was inhibited by prior incubation with glucose. This may
indicate that hyperglycemia inhibits brain Ca
-ATPase by glycation, particularly
2+
because brain glucose concentration has been shown to be increased ten-fold in
diabetic rats.
Our present study reveals that activity of Ca
2+
- ATPase in synaptosomes
prepared from the brains of 18 - month - old rats decreased by about 28.8% as
compared to the activity of the enzyme in brains of young, mature rats (3-monthold). These changes in PCMA activity can be accounted for the hyperglycemia
found in old rats, that in turn may generate formation of reactive oxygen species
and/or lead to glycation, causing damage of the enzyme protein.
ATPase
ATPase,
is
II,
a
a
vanadate-sensitive
member
of
a
and
subfamily
phosphatidylserine-dependent
of
P-type
ATPases
and
is
Mg
-
2+
presumably
responsible for aminophospholipid translocation activity in eukaryotic cells. (26).
The aminophospholipid translocation activity plays an important physiological
role in the maintenance of membrane phospholipid asymmetry that is observed
not only in plasma membranes but also in membranes of other cellular organelles.
As shown by Nedeljkovic and co-workers (27) Mg
-ATPase is not uniformly
2+
distributed and differs in respect to affinity for ATP in rat brain regions. It has
been confirmed that the enzyme is activated by milimolar concentrations of Mg
2+
and that it cannot be effectively inhibited by known ATPase inhibitors.
Comparison of Na , K -ATPase and Mg
+
+
-ATPase activities in the synaptic
2+
plasma membrane from various regions of rat brain reveals that moderate hypoxia
increases the activity of synaptosomal Mg
-ATPase whereas activities of both
2+
Ca
- ATPase and Na , K -ATPase are decreased (28).
2+
+
+
In our present study it was only the Mg
+2
-ATPase, the activity of which has
been shown to become gradually higher with age. As compared to newborn rats
the activity of this enzyme was 5-fold higher in old animals, and this may confirm
464
its crucial role in maintaining the membrane phospholipid asymmetry as well as
in the process of signal transduction via controlling ATP concentration.
It thus may be concluded that changes in activity of different P-type ATPases,
including Na , K -ATPase, Ca
-ATPase and Mg
same
their
+
+
2+
conditions
and
that
-ATPase, may differ even in the
2+
adaptation
to
internal
and/or
external
environmental changes is not identical.
Acknowledgements: This work was supported by the research grant N
o
501-1-11-0 from The
University of Medical Sciences in Poznañ
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Received:
September 8, 2003
Accepted:
May 7, 2004
Authors address: Teresa Torliñska, Department of Physiology, University of Medical Sciences,
Œwiêcicki str. 6, 60-781 Poznañ, Poland, tel/fax: + 48 61 8 65 91 60
E-mail: [email protected]