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Signal transduction in aging
Arch. Gerontol. Geriatr. suppl. 4 (1994) 235-246 9 1994 Elsevier Science Ireland Ltd. All rights reserved. 0 1 6 7 - 4 9 4 3 / 9 4 / $ 0 7 . 0 0
235
SIGNAL TRANSDUCTION IN AGING
M. SUGAWA and T. MAY Deptartment of Neuropsychopharmacology, Ulmenallee 30, D-14050 Berlin, FRG
Freie Universit~t Berlin,
UKRV-C,
SU~.,~MARY The possible age-related involvement of two d i f f e r e n t signal transduction pathways in the rat CNS was investigated. In the p h o s p h y t i d y l inositol (PI) response, higher phospholipase-C (PL-C) a c t i v i t y and drastically higher (almost 2.5-fold) i n o s i t o l ( 1 , 4 , 5 ) t r i s p h o s p h a t e ( I n s ( 1 , 4 , 5 ) P 3) concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to young adult (approximately 3.5 months) rats were observed. In the adenosine 3 " : 5 " - c y c l i c monophosphate (cAhIP) cascade, a significantly higher endogenus cAMP level and a significant decline of the adenylate cyclase ( A C , ATP pyrophosphate-lyase ( c y c l i z i n g ) , EC 4 . 6 . 1 . 1 . ) a c t i v i t y were observed in striatal tissue from Y3oung rats in comparison with aged rats. Binding saturation exl~eriments with [ H]SCH 23390 at the dopamine (DA) D I receptor (D I) and [ J H ] s p i p e r o n e at the D A D 2 receptor (D 2) revealed no change in the a f f i n i t y (Kd) but a significant decrease in the density (13max) of D I ( - 3 1 % , p < 0.005) and of D 2 (-22 %, p < 0.05), respectively, in the aged versus young striata. DA seems to s l i g h t l y inhibit total inositol phosphate formation and this effect was antagonized by ( - ) - s u l p i r i d e . A significant decrease (p < 0.05) in the AC a c t i v i t y stimulated by 10 ~M DA in the senescent compared to the young animals was monitored. A p p a r e n t l y , the age-related decline of the AC a c t i v i t y was independent of changes of G s and G i a c t i v i t y . Keywords:
inositol phosphate, adenylate cyclase, dopamine, striatum
INTRODUCTION In the last decade studies on the interactions
between
and abnormalities of various n e u r o t r a n s m i t t e r - r e c e p t o r s revealed interesting of guanine
results.
nucleotide-binding
functional
defects
in aging processes have
In contrast to the receptor level, the involvement and several
second messen-
gers has recently taken a shape in aging studies.
Age-related
changes in the
phosphytidyl
months)
inositol
mately 24 months) (Nalepa et a l . , (Surichamorn aged
rats.
response
(G-protein)
in young
rats were observed.
1989; Guiramand et a l . , et
al.,
1989)
Furthermore,
(Crew et a l . , et a l . ,
(PI)
protein
the
1986; Miyamoto et a l . ,
show either
1989; Mundy et a l . , receptor-mediated
which
and old
are mediated
differences
PI signal transductions.
(approxi-
a reduction
1991) or no change Pi via
breakdown
in
~l-adrenergic
1990} and glutamate receptors
1989) depend on strain-specific
receptor-regulated
The findings
in muscarinic responses
(2-5
(Guiramand
in the responses of various
However,
there has been a lack of
studies about alterations in PI responses in v e r y old Wistar rats.
236
In the cAMP cascade, a g e - r e l a t e d and T h o r n b e r r y ,
1978; Nomura et a l . ,
were observed (Puri and V o l i c e r ,
alterations
of the AC a c t i v i t y
(Schmidt
1985) and of the c o n c e n t r a t i o n
of cAMP
1977) in the aging process.
Is t h e r e any alteration of AC catalytic unit a c t i v i t y and of the endogenous cAMP concentration in e x t r e m e l y aged rats? I f the catalyc unit a c t i v i t y would be reduced, Is this
is this d e p e n d e n t on the presence or absence of activated G-proteins? due to an a g e - d e p e n d e n t r e d u c t i o n
either
in G - p r o t e i n
function
or
in
coupling of the activated Gs/G i with the catalyc unit complex? It is well described that alterations such as sensorimotoric, Sabol et a l . ,
in the regulation of v a r i o u s f u n c t i o n s ,
occur in the striatum of senescence (Finch et a l . ,
1985; Joseph et a l . ,
1990).
This means,
in t u r n ,
an area for o b s e r v i n g how these f u n c t i o n s are controlled A decline
of age-related
(Joseph et a l . ,
striatal
dopamine
receptor
the striatum
in the aging
activity
1981; is
process.
seems established
1990). Also in the corpora striata of old rats, a decrease in the
level of dopamine (DA) D 2 r e c e p t o r mRNA was observed (Mesco et a l . , The p r e s e n t work was c a r r i e d out to gain i n s i g h t fect of D A D I and D 2 r e c e p t o r s , y o u n g adult vs.
into the a g e - r e l a t e d e f -
of PI b r e a k d o w n and cAMP cascades in the
the aged rat b r a i n .
volvement of these signal
1991).
It is the f i r s t attempt to elucidate the i n -
transduction
pathways
in the e x t r e m e l y aged Wistar
rats. MATERIALS AND METHODS Animals. tertal,
F.R.G.)
light/dark All
Female Wistar
animals
cycle
rats
(Hagemann,
were housed in g r o u p s (light:
had
06.00 a.m.
a standard
diet
Lippische V e r s u c h s t i e r z u c h t ,
Ex-
of 4-5 animals per cage with a 12 h r s
- 06.00 p . m . ) (Altromin
1324)
at 21~ and
and 50 % h u m i d i t y .
water
ad
libitum.
The
y o u n g ( w e i g h i n g 200-220 g) and old ( w e i g h i n g 285 + 22 g) rats were sacrificed by decapitation at an age of about 3.5 and 40 months of age, r e s p e c t i v e l y .
The
50 % s u r v i v a l level of these rats at our housing conditions is about 29 months. DA b i n d i n g studies.
The two b i n d i n g assays for d e t e r m i n i n g
the c h a r a c t e -
r i s t i c s of D I and D 2 and the AC assay were performed with common homogenate pools of striatal membranes of single animals. The assays of saturation
and of DA displacement of
Pont, New England Nuclear) b i n d i n g D I and binding of 1.0
D 2 were c a r r i e d out.
Non-specific
[3H]SCH
23390 ( D u -
[ 3 H ] s p i p e r o n e (Amersham B u c h l e r )
b i n d i n g was defined in the presence
]~M (+)-butaclamol ( D I ) and 30 uM ( - ) - s u l p i r i d e ( D 2 ) . Incubation times 3 [ H ] s p i p e r o n e and 120 rain for [ 3 H ] S C H 23390 b i n d i n g at 23~
were 90 min for
The b i n d i n g was terminated by rapid f i l t r a t i o n with a cell h a r v e s t e r . [3H]forskolin
(FSK) b i n d i n g assay. Striatal tissue was homogenized at 4~
in 3 ml of 0.32 M sucrose.
Each r e s u l t i n g
striatal
pellet was rehomogenized in
237
3.2 ml of 50 mM T r i s - H C l ,
pH 7.4,
10 mM NaF, and 5 mM MgCI 2.
b i n d i n g was performed at six c o n c e n t r a t i o n s (3.5 - 70 nM). at 23~
[3H]FSK-
Incubation (60 rain
was terminated by f i l t r a t i o n .
Assay of phospholipase C ( P L - C ) a c t i v i t y using exogenous prelabelled s u b strates. with
Phosphoinositide h y d r o l y s i s
exogenous
(reflecting
PL-C a c t i v i t y )
[ 3 H ] P t d l n s ( 4 , 5 ) P 2 (Amersham
lease of water soluble [ 3 H ] i n o s i t o l
Duchler)
(pll
7.5,
50 mM T r i s - H C l ,
t r y p s i n i n h i b i t o r ( t y p e I I , Sigma), oxidase, EC 1 . 4 . 3 . 4 ) .
by measuring
the re-
phosphates. Tissues obtained from the fore-
brain and the striatum were homogenized at 0~ genization b u f f e r
was determined
in 10 volumes ( w t / v o l ) 5 mM MgCI2,
of homo-
60 12g/ml soybean
10 12M p a r g y l i n e (an i n h i b i t o r of monoamine
The homogenate was c e n t r i f u g e d at 500 g for 10 rain and
the pellet was discarded.
The s u p e r n a t a n t was then c e n t r i f u g e d at 40000 g for
60 rain. The pellet was resuspended. Tissue obtained from the f o r e b r a i n and the striatum ( e i t h e r a single b r a i n was used or two brains were pooled) were homogenized.
For measuring the PL-C a c t i v i t y ,
assay tube) was incubated at 37~ ( 4 , 5 ) P 2 vesicles.
with reaction b u f f e r c o n t a i n i n g
(I
The PL-C a c t i v i t y
12M, RBI,
BIOTREND)
121
[3H]Ptdlns-
To regulate the PL-C a c t i v i t y via DA r e c e p t o r s ,
Sigma) a n d ( - ) - s u l p i r i d e solvent.
the homogenate (5 12g p r o t e i n / 6 0 DA (I
were d e l i v e r e d
12M,
in aqueous
is expressed in dpm induced by the enzyme (total
dpm - blank dpm) d i v i d e d by dpm of the sample in the absence of the enzyme. AC assay. The AC assay volume was 100 121 and included 30 121 membrane homogenate, 50 mM T r i s - H C I ,
pH 7.4, 2 mM MgCI, 0.3 mM EGTA, 10 I~M p a r g y -
line, I mM IBMX, 100 pM cAMP, 100 pM A T P , 5 mM c r e a t i n e - p h o s p h a t e , creatine-kinase,
I mg/ml
bovine serum albumin
New England
Nuclear).
(BSA)
152 U/I
and about 400,000 cpm
(~-32P)ATP
(DuPont,
min at 30~
in the absence of presence of d i f f e r e n t stimulators: 10 12M GppNHp,
10 pM FSK, 10 121ki FSK plus 100 nM GppNHp,
The samples were
incubated
10
10 ~,I FSK plus 10 mM NaF, 10
p~1 DA, 10 ~M DA plus 200 nM (+)SCH 23390. Determination of the endogenous inositol 1 , 4 , 5 - t r i s p h o s p h a t e ( I n s ( 1 , 4 , 5 ) P 3 ) concentration.
Tissues form v a r i o u s regions of rat b r a i n ,
i.e.,
cortex,
stria-
turn, and hippocampus were homogenized in water and 20 % p e r c h l o r i c acid ( 4 : 1 , v o l / v o l ) and c e n t r i f u g e d at 2000 g for 20 min at 4~ and the s u p e r n a t a n t was t i t r a t e d contained: T r i s - b u f f e r , 12g p r o t e i n / m l ) , protein/ml),
and
and
[3H]Ins(1,4,5)P3
Ins(1,4,5)P 3 binding
10 M KOH.
Each assay tube
(3000 cpm), homogenate ( c o n t a i n i n g 1.5
I n s ( 1 , q , 5 ) P 3 (3000 cpm),
t e r 15 min incubation at 0~ at 4~
to pH 8.0 with
The pellet was discarded
protein
homogenate ( c o n t a i n i n g in a p o l y p r o p y l e n e
1.5
tube.
12g Af-
the tubes were c e n t r i f u g e d at 2000 g for 15 rain
The pellet was dissolved in 100 pl H20. Detrmination of the endogenous cAMP c o n c e n t r a t i o n .
The tissue was homo-
genized in 667 1~I (2 striata pooled) aqueous 4 mM EDTA solution at 4~
fol-
238
lowed by heating
for 3 min in boiling
17,800 g at 4~
Fifty
water,
and centrifugation
for
5 rain at
~I of the supernatant was assayed for cAMP applying a
radioimmunoassay kit (Amersham B u c h l e r ) . Protein
determination
and data analysis.
mined with BSA as the standard. Significance
of differences
(p
The protein
content
was d e t e r -
All results are presented as mean + S.E.M.
< 0.05)
between
young
and aged animals were
calculated by S t u d e n t ' s t - t e s t s . For f u r t h e r
details
see the references:
Sugawa
(1993),
Sugawa and May
(1993), May and Sugawa (1993). RESULTS AND DISCUSSION DA receptor analysis in aging.
Binding saturation experiments with tritated
SCH 23390 at the D I receptor and [ 3 H ] s p i p e r o n e at the D 2 receptor revealed no change in the affinity DI
(-31
IB),
(K d) but a significant decrease in the density (Bmax) of
~o, p < 0.005,
respectively,
data not shown)
in the aged versus
and of D 2 (-22 ~o, p < 0.05,
young
striata.
The specific
Figure
binding
ir,
fmol/mg protein of a single concentration of tritated SCH 23390 (Figure I A ) and t r i t a t e d spiperone (data not shown) are significantly reduced at most of the DA concentrations
tested
in the senescent compared
the young
according
to
the differences in the B The proportion
values. max of the h i g h - a f f i n i t y
with
agonist
binding
state
(high)
signifi-
cantly decreased (p < 0.025) in the older (20.9 + 3 . 2 ~) in comparison with the young
(30.6 + 2.9 ~o) in D I ,
but increased n o n - s i g n i f i c a n t l y
in D 2 (47.9 + 2.6
versus 40.5 + 5.1 ~o). Age-related PL-C a c t i v i t y observed
second
messenger
in forebrain
tissue
(data not shown).
generating
isolated
However,
enzyme
from young
higher
activity. and old
PL-C a c t i v i t y
No d i f f e r e n t rats
has been
is observed in the
striatum of aged than the young (Figure 2). Variation of the incubation time of PL-C
demonstrates
pronounced PL-C
that
the difference
at incubations
activity
between
exceeding 5 minutes.
in the aged striatum
old and young
tissue
is most
A f t e r a 5 min incubation,
is 20 % higher,
after
the
20 rain about 45
higher (p < 0.05). The basal AC a c t i v i t y ficantly
in the striatal
(p < 0.01) reduced,
i.e.,
membrane of aged animals is signi-
approximately 35 % less than in young (data
not shown). DA receptor-mediated
signal transmission
pathways.
The young adult and
old rat striat'um tend to have a D A D 2 receptor mediated i n h i b i t o r y the phosphoinositol cascade. 20 ~o) which
is antagonized
1 IJM DA inhibits the PL-C a c t i v i t y by I
IJM ( - ) - s u l p i r i d e
in each case.
response in
(approximately However,
effect was significant only in the young striatum (p < 0.05, Figure 3).
the
239
A t_ o.
600 -
500
400
-
-
§
I-
*
o E 300 -
~ o
200 -
.
+
§
t~
0Y n
.
+ Aged 100
-
m
0-
9
8 -].og
7 [dopamine]
6
5
(M)
B 400 350
300
I
2so 200 \ ~
150 100 50
Y:totll
R:totll
Y:high
R:high
Figure I . A. Displacement of the specific [3H]SCH 23390 b i n d i n g . ( D I) by DA of 6 young (O} and 6 aged (+) rat striata. B. The binding of [aH]spiperone (D 2) if, 6 young (Y) and 6 aged (A) rat striata. Left two columns show total Bma x values determined by Scatchard anylyses of the 3H-antagonist binding saturation experiments. The two right columns show Bmax values of the highaffinity agonist binding state calculated by Scatchard and DA displacement analyses (means + S . E . M . ) . Asterisks indicate significant differences at p < 0.05. (May and Sugawa, 1993).
The D I mediated AC activation was investigated with 10 IJM DA. The DA stimulating effect on the AC was fully reversible in both the aged and young rats by the addition of 200 nM of SCH 23390, i . e . , diated (Figure q).
it is probably only D I me-
In the aged compared with the young rats, 10 pM DA dis-
plays a significant reduction (p < 0.05) from 36.5 w+ 6.9 96 to 23.6 + 3.9 ~ in AC activation calculated as ~o stimulation above basal activity (Figure 4). Coupling between G-protein and AC catalyc subunit in aging. The absolute AC a c t i v i t y upon stimulation by various pharmacological agents is in each case significantly lower in the aged striatum as compared to the young (p < 0.01 or
240
5.5 5.0 4.5 4.0
3.5
9~
>-, '-~ 3,0
2.5 2.0 1.5 1.0 0.5 0.0( 0
5
I
I
I
I
I
I
i
10
15
20
25
30
35
40
Time
Figure 2. Incubation time-dependence rat striatum. Results are the means each performed in triplicate. At 5 and than the symbol. Asterisks indicate 0 . 0 5 ) . (Sugawa, 1993}. 120
100
[min]
of PI hydrolysis in young (V) and old (O) + S . E . M . of 3 independent experiments, a t 20 min ( y ) , the e r r o r bars are smaller statistically significant differences (p <
-
-
T
T
8O u}
so
~J
4O
20 f-
oi A
B
C
young
D
A
B
C
D
old
F i g u r e 3. Effect of DA and D 2 antagonists on tile h y d r o l y s i s of exogenous PI in y o u n g and old rat s t r i a t u m , measured as the appearance of w a t e r - s o l u b l e inositol phosphates. Pargyline was always p r e s e n t . The following ligands were added in both age g r o u p s . A: no a d d i t i o n , c o n t r o l , set as 100 %; on an absolute scale, h o w e v e r , the e x t e n t of PI h y d r o l y s i s is a p p r o x i m a t e l y 40 % l a r g e r in the old tissue; B: I ~M DA; C: I pM DA + 10 nM haloperidol; D: I ~I~A DA + I laM ( - ) - s u l p i r i d e . Mean + S . E . M . of t h r e e i n d e p e n d e n t e x p e r i m e n t s each performed in t r i p l i c a t e . A s t e r i s k denotes s i g n i f i c a n t d i f f e r e n c e s between y o u n g and old g r o u p (p < 0 . 0 5 } .
241
0.05,
data not shown).
The observed
age-related
decline
in the AC a c t i v i t y
might be caused by effects of the coupling of G-proteins with the AC catalytic s u b u n i t , therefore,
the percentage stimulation of the control (basal) AC a c t i v i t y
by various reagents (defined as relative stimualation), solely, lated.
and the combinations of GppNHp plus FSK, No significant differences,
however,
i.e.,
GppNHp, NaF, FSK
NaF plus FSK, was calcu-
in the relative stimulation of the AC
a c t i v i t y by any pharmacological substances used in the striatum were observed. 50
r "6 o~ 40
E
~
p
]
30
20
10
~
l
0 DA
l
SCH23390
DA SCH23390
Y
A
Figure 4. The stimulation by 10 ~M DA of the AC (in ~ above the basal activ i t y ) and the inhibition of the AC stimulation by the addition of 200 nM SCH 23390 in 6 young ( Y ) and 6 aged (A) rat striata. Means + S.E.M. Asterisk denotes significant difference between young and old g r o u p s - ( p < 0.05). To answer the question,
whether
the G s-AC catalyc subunit
involved in the aging process, a [3H]FSK to the specific differences binding
[3H]FSK
were
binding
observed.
binding
test was applied.
in young and aged striatum,
The
calculated
affinity
(Kd)
between these two g r o u p s ,
i.e.,
_+ 0.27 pmollmg protein (aged)
According
no age-related
values
of
[3H]FSK
(Bmax) values display as well no difference
2.00 + 0.48 pmol/mg protein
In the young rat brain,
pocampus,
(young)
the highest concentration
of I n s ( 1 , 4 , 5 ) P 3 and is found
the striatum and the cortex contain two- and f o u r - f o l d
respectively.
A similar
I n s ( 1 , 4 , 5 ) P 3 concentration
campus and cortex for young adult and aged tissue. the young tissue,
and 2.19
(data not shown).
Age-dependent difference of endogenous concentration
4,5)P 3,
is
were 14.6 + 1.9 nM for the young (n = 6) and 15.6 + 2.4 nM for the
aged tissues (n = 7). The density
cAMP.
pathway
in the old rat striatal
in the hipless I n s ( 1 , -
is found
However,
in hippo-
in contrast
I n s ( 1 , 4 , 5 ) P 3 concentration
to
is as high
242
as in the hippocampus.
In fact,
the aged striatum contains approximately 250
more I n s ( 1 , 4 , 5 ] P 3 than the young striatum (P < 0.005, data not shown). The cAMP level in the aged striatum is approximately 40 % higher as compared to young striatum (p < 0.05, data not shown). On the way to a better transduction
pathways
understanding
in aging:
Working
of the involvement
hypotheses.
The
of the signal
question
remains,
why is there such a drastically higher endogenous I n s ( 1 , 4 , 5 ) P 3 concentration in the old compared with the young rat striatum,
even though at the receptor le-
vel there is only a slight reduction [ b y about 20 $} in the D A D 2 receptor density
in the 40-month-old
I n s ( 1 , 4 , 5 ) P 3 to
rat striatum.
I n s ( 1 , 3 , 4 , 5 ) P 4 or
phosphate-5-phosphatase)
which
One possible explanation
I n s ( 1 , 4 , 5 ) P 3 5-phosphatase
hydrolyzes
is that either (inositol
I n s ( 1 , 4 , 5 ) P 3 to I n s ( I , 4 ) 2 ,
polyare ei-
ther less active or are present in lower amounts in old tissue (Figure 5, r i g h t side).
r
i
-
w4
PKA
Figure 5. Schematic drawing of the possible age-related involvement in the cAMP (left side) and PI ( r i g h t side) signal transduction pathways, a: 5-phosphatase, b: 3-kinase. In the present s t u d y a much higher
(about 40 ~) cAMP concentration
the striatum of extremely aged animals was detected.
in
This significantly d i f f e r e n t
concentration of the endogenous cAMP in the striatal tissue between young and aged cannot be explained less basal AC a c t i v i t y membranes.
by differences
(about 35 % reduction)
A conceivable
explanation
activity
(Stancheva
and Alova,
1991)
activity
(Govoni
al.o
in
et
in the AC a c t i v i t y ,
1988)
the
was found
is an age-related or a different older.
Another
since significant
in the aged striatal decline
protein
of cAMP-PDE
kinase A
possibility
could
(PKA) be a
changed ratio of AC and cAMP-PDE, either of the a c t i v i t y of of the amount, the extremely aged animals (Figure 5, left side).
in
243
A number of biochemical changes with senescence in the DA pathway, cluding
DA regulated
(Nomura,
1985;
signal transmissions,
Sabol et a l . ,
1985;
have been observed
Morelli
et a l . ,
1990).
in-
in the rat CNS
Our
recent
study
showed a significant decrease in the density of DA D I and D 2 receptors in the aged-vs,
young striata (May and Sugawa, 1993). It could be explained that the
higher D A D 2 receptor density and higher DA concentration tum lead to the more pronounced
in the young s t r i a -
inhibition of PL-C a c t i v i t y ,
lower endogenous I n s ( 1 , 4 , 5 ) P 3 concentration
and therefore,
to
in the young rat in comparison to
the aged. It has been postulated that D A D 2 receptors regulate inhibition of the activity
of various
and Meldolesi,
cell types
1989),
including
evidence of age-related at the receptor 1989).
(Morra et a l . ,
level
1991; Vallar et a l . ,
brain cells (Pizzi et a l . ,
D A D 2 receptor dysfunction (Joyce et a l . ,
In our present s t u d y ,
1986;
1988).
1988; Vallar
There is some
in the rat striatum system
Norman et a l . ,
1987;
there is no significant difference
Han et a l . ,
of the effector
mechanisms mediated by D A D 2 receptor sites between the young adult and the aged striatum. In
senescence,
interactions
volved in signal transduction Johnson,
1989).
However,
between
receptors
are probably altered
in the striatum,
and
their
(Severson,
all applied
G-proteins
in-
1987; Green and
pharmacological
agents
stimulated the AC in the young animals much s t r o n g e r than in aged correlating well with the results of the basal AC a c t i v i t y . tein-AC coupling on aging was investigated.
The involvement of the G - p r o -
FSK appears to have two d i f f e r e n t
interactions with AC. One requires Gs and another does not (Seamon and Daly, 1981; Schneyer et a l . ,
1983; Battaglia et a l . ,
1986). The age-related activation
of AC by FSK in the presence and absence of the guanine GppNHp (which stimulates at low concentration
nucleotide
(100 nM) preferentially
analog, G i ) , and
in combination with 10 mM NaF (which activates Gs) was focussed in a series of experiments.
In striatal tissue, stimulation by FSK was inhibited by the additon
of GppNHp and was superadditive by the addition of NaF in respect to activation of the AC.
It is likely that the changes in AC catalytic unit a c t i v i t y with
age occur
independently
(O'Conner
et a l . ,
the
[311]FSK
by a filtration binding
in
the absence
1983) and G i.
binding
test,
or
in
These findings
since
the
the
presence
of activated
G
s are consistent with data from
high-affinity
FSK
binding
determined
binding assay probably reflects only the G s - p r o t e i n coupled AC
site as previously discussed
no change in the specific
(Laurenza and Seamon,
FSK binding
1991).
There was
between young and aged animals in the
striatum. Taken together,
the age-related
of alterations in Gs and G i a c t i v i t y . aged rats,
decline of the AC a c t i v i t y
is independent
In the striatal membranes of our extremely
it seems that the reduced AC a c t i v i t y is due to either the reduction
244
in the number of the AC molecules, the AC phosphorylation state (desensitizat i o n ) , or even an altered AC gene expression d u r i n g aging. T h e r e f o r e , a f u n c tional down-regulation of AC a c t i v i t y occurs in aging. Our recent investigation revealed an imbalance in DA D I / D 2 receptor mediated cAMP signal transduction in v e r y old rats. We observed the decline of the AC a c t i v i t y by DA D I receptor in aged animals in comparison to the young. We could prove that the age-related alterations in AC catalytic u n i t a c t i v i t y occur independently of G - p r o t e i n s , i . e . , side}.
Gas - AC coupling (see Figure 5, left
It is possible that the coupling of receptors, e . g . ,
DA D I , and G - p r o -
tein a n d / o r the feedback mechanisms between PKA and those receptors might be altered in the striatum of aged animals ( F i g u r e 5, left side). It is of interest in the f u t u r e to examine how the c r o s s - t a l k between these two signal transduction pathways is altered in aging processes. ACKNOWLEDGEMENTS The authors thar~k Sharra for encouragement, This paper is dedicated to prof. Dr. H. Coper. REFERENCES Battaglia, G., Norman, A . B . , Hess, E.J. and Creese, I. (1986): Forskolin potentiates the stimulation of rat striatal adenylate cyclase mediated by D-I dopamine receptors, guanine nucleotides, and sodium fluoride. J. Neurochem., 46, 1180-1185. Crew, F . T . , Gonzales, R . A . , Polavcik, R., Phillips, M . I . , Theiss, C. and Raizada, M. (1986): Changes in receptor stimulated phosphoinositide h y d r o lysis in brain d u r i n g ethanol administration, aging, and other pathological conditions. Psychopharmacol. B u l l . , 22, 775-780. Finch, C . E . , Rand~ll, P.K. and Marshall, J . F . (1981): Aging and basal gangliar functions. Ann. Rev. Gerontol, G e r i a t r . , 2, 49-87. Govoni, S., Rius, R . A . , Battaini, F. and T r a b u c c h i , M. (1988): Reduced cAMP-dependent phosphorylation in striatum and nucleus accumbens of aged rats: Evidence of an altered functioning of D I dopaminoceptive neurons. J. Gerontol., 43, B93-B97. Green, A. and Johnson, J . L . (1989): Evidence for altered expression of the GTP-dependent r e g u l a t o r y proteins G and Gi, in adipocytes from aged rats. Biochem. J . , 258, 607-610. s Guiramand, J . , Sassetti, I. and Recasens, M. (1989): Developmental changes in the chemosensitivity of rat brain synaptoneurosomes to e x c i t a t o r y amino acids, estimated by inositol phosphate formation. I n t . J. Dev. Neurosci., 7, 257-266. Han, Z . , K u y a t t , B . L . , Kochman, K . A . , DeSouza, E.B. and Roth, G.S.(1989): Effect of aging on concentrations of D~-receptor-containing neurons in the rat striatum. Brain Res., 498, 299-307.z Joseph, J . A . , Roth, G.S. and Strong, R. (1990): The striatum, a microcosm for the examination of age-related alterations in the CNS: a selected review. Rev. Biol. Res. A g i n g . , 4, 181-199. Joyce, J . N . , Loeschen, S . K . , Sapp, D.W. and Marshall, J.F. (1986): A g e - r e l a ted regional loss of caudate-putamen dopamine receptors revealed by quantitative autoradiography. Brain. Res., 378, 158-163. Laurenza, A. and Seamon, K . B . (1991): H i g h - a f f i n i t y binding sites for (3H)Forskolin. Methods in Enzymol., 195, 52-65. May, T . and Sugawa, M. (1993): Altered dopamine receptor mediated signal transmission in the striatum of aged rats. Brain. Res., 604, 106-111.
245
Mesco, E . R . , Joseph, J . A . , Blake, M.J. and Roth, G.S. (1991): Loss of D^ receptors d u r i n g aging in partially due to decreased levels of mRNA. Braif~ Res., 545, 355-357. Miyamoto, A . , Araiso, T . , Koyama, T. and Ohshika, H. (1990): Membrane v i s cosity correlates with ~1-adrenergic signal transduction of the aged rat cerebral c o r t e x . J. Neurochem., 55, 70-75. Morra, M., Leboulenger, F . , Desrues, L . , Tonon, M.Ch. and V a u d r y , H. (1991): Dopamine i n h i b i t s inositol phosphate production, arachidonic acid formation, and corticosteroid release by frog adrenal gland t h r o u g h a pertussis t o x i n - s e n s i t i v e G-protein. Endocrinol., 128, 2625-2632. Morelli, M., Mennini, T . , Cagnotto, A . , Toffano, G. and Di Chiara, G. (1990): Quantitative autoradiographical analysis of the age-related modulation of central dopamine D I and D~ receptors. Neurosci., 36, 403-410. Mundy, W., Tandon, P., Ali, . and Tilson, H. (1991): Age-related changes in receptor-mediated phosphoinositide h y d r o l y s i s in various regions of rat brain. Life Sci., 49, PL97-PL102. Nalepa, I . , Pintor, A . , Fortuna, S., Vetulani, J. and Michalek, H. (1989): Increased responsiveness of the cerebral cortical phosphatidylinositol system to noradrenaline and carbachol in senescent rats. Neurosci, L e t t . , 107, 195-199. Nomura, Y . , Makihata, J. and Segawa, T . (1985): Activation of adenylate cyclase by dopamine, GTP, NaF and forskolin in striatal membranes of neonatal and senescent rats. Eur. J. Pharmacol., 106, 437-440. Norman, A . B . , Battaglia, G. and Creese, I. (1987): Differential recovery rates of rat D~ dopamine receptors as a function of aging and chronic reserpine treatmen~ following i r r e v e r s i b l e modification: A key to receptor r e g u l a t o r y mechanisms. J. Neurosci., 7, 1484-1491. O ' C o n n e r , S.W., Scarpace, P.J. and Abrass, I . B . (1983): Age-associated decrease in the catalytic u n i t a c t i v i t y of rat myocardial adenylate cyclase. Mech. Ageing D e v . , 21, 357-363. Pizzi, M., Da Prada, M., Valerio, A . , Memo, M., Spano, P.F. and Haefely, W.E. (1988): Dopamine D~ receptor stimulation inhibits inositol phosphate generating system in rat s-triatal slices. Brain Res., 456, 235-240. Puri, S.K. and Volicer, L. (1977): Effect of aging on cyclic AMP levels and adenylate cyclase and phosphodiesterase activities in the rat corpus striatum. Mech. Ageing and D e v . , 6, 53-58. Sabol, K . E . , Naill, D . B . , Wages, S . A . , C h u r c h , W.H. and Justice, J . B . ( 1 9 8 5 ) : Dopamine depletion in a striatal subregion d i s r u p t s performance of a skilled motor task in the rat. Brain Res., 335, 33-43. Schmidt, M.J. and T h o r n b e r r y , J . F . (1978): Cyclic AMP and cyclic GMP accumulation in v i t r o in brain regions of young, old and aged rats. Brain Res., 139, 169-177. Schneyer, C . R . , Pineyro, M.A. and Gregerman, R . I . (1983): Mechanism of action of forskolin on adenylate cyclase: effect on bovine sperm complemented with e r y t h r o c y t e membranes. Life Sci., 33, 275-279. Seamon, K.B. and Daly, J.W. (1981): Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulator protein. J. Biol. Chem., 256, 9799-9801. Severson, J . A . (1987): Synaptic regulation of neurotransmitter function in aging. In: Review of Biological research in A g i n g , Vol. 3, pp. 191-206. Editor: M. Rothstein. A . R . Liss I n c . , New York. Stancheva, S.L. and Alova, L.G. (1991): Age-related changes of cyclic AMP phosphodiesterase a c t i v i t y in rat brain regions and a new phosphodiesterase i n h i b i t o r - n o o t r o p i c agent adafenoxate. Gen. Pharmac., 22, 955-958. Sugawa, M. (1993): Alterations of inositol phosphate t u r n o v e r in striatum of aged rats. Eur. J. Pharmacol. Mol. Pharmacol. Sect., 247, 39-44. Sugawa, M. and May, T. (1993): Age-related alteration in signal t r a n s d u c t i o n : Involvement of the cAMP cascade. Brain. Res., 618, 57-62. Surichamorn, W., Abdallah, E . A . M . and EI-Fakahany, E. (1989): Aging does not alter brain muscarinic receptor-mediated phosphoinositide h y d r o l y s i s
246
and its inhibition by phosrbol esters, tetrodotoxin and receptor desensitization. J. Pharmacol. Exp. T h e r . , 251, 543-549. Vallar, L. and Meldolesi, J. (1989); Mechanisms of signal transduction at the dopamine D 2 receptor. Trends Pharmacol. Sci., 101, 75-77. Vallar, L . , V i n c e n t i n i , L.M. and Meldgl+esi, J. (1988): I n h i b i t i o n of inositol phosphate production is a late, Ca- -dependent effect of D 2 dopaminergic receptor activation in rat lactotroph cells. J. Biol. Chem., 263, 1012710134. Walker, J . B . and Walker, J.P. (1973): Properties of adenylate cyclase from senescent rat brain. Brain Res., 54, 391-396.
235
SIGNAL TRANSDUCTION IN AGING
M. SUGAWA and T. MAY Deptartment of Neuropsychopharmacology, Ulmenallee 30, D-14050 Berlin, FRG
Freie Universit~t Berlin,
UKRV-C,
SU~.,~MARY The possible age-related involvement of two d i f f e r e n t signal transduction pathways in the rat CNS was investigated. In the p h o s p h y t i d y l inositol (PI) response, higher phospholipase-C (PL-C) a c t i v i t y and drastically higher (almost 2.5-fold) i n o s i t o l ( 1 , 4 , 5 ) t r i s p h o s p h a t e ( I n s ( 1 , 4 , 5 ) P 3) concentration in the corpus striatum (caudate-putamen) of extremely old (approximately 40 months) female Wistar rats in comparison to young adult (approximately 3.5 months) rats were observed. In the adenosine 3 " : 5 " - c y c l i c monophosphate (cAhIP) cascade, a significantly higher endogenus cAMP level and a significant decline of the adenylate cyclase ( A C , ATP pyrophosphate-lyase ( c y c l i z i n g ) , EC 4 . 6 . 1 . 1 . ) a c t i v i t y were observed in striatal tissue from Y3oung rats in comparison with aged rats. Binding saturation exl~eriments with [ H]SCH 23390 at the dopamine (DA) D I receptor (D I) and [ J H ] s p i p e r o n e at the D A D 2 receptor (D 2) revealed no change in the a f f i n i t y (Kd) but a significant decrease in the density (13max) of D I ( - 3 1 % , p < 0.005) and of D 2 (-22 %, p < 0.05), respectively, in the aged versus young striata. DA seems to s l i g h t l y inhibit total inositol phosphate formation and this effect was antagonized by ( - ) - s u l p i r i d e . A significant decrease (p < 0.05) in the AC a c t i v i t y stimulated by 10 ~M DA in the senescent compared to the young animals was monitored. A p p a r e n t l y , the age-related decline of the AC a c t i v i t y was independent of changes of G s and G i a c t i v i t y . Keywords:
inositol phosphate, adenylate cyclase, dopamine, striatum
INTRODUCTION In the last decade studies on the interactions
between
and abnormalities of various n e u r o t r a n s m i t t e r - r e c e p t o r s revealed interesting of guanine
results.
nucleotide-binding
functional
defects
in aging processes have
In contrast to the receptor level, the involvement and several
second messen-
gers has recently taken a shape in aging studies.
Age-related
changes in the
phosphytidyl
months)
inositol
mately 24 months) (Nalepa et a l . , (Surichamorn aged
rats.
response
(G-protein)
in young
rats were observed.
1989; Guiramand et a l . , et
al.,
1989)
Furthermore,
(Crew et a l . , et a l . ,
(PI)
protein
the
1986; Miyamoto et a l . ,
show either
1989; Mundy et a l . , receptor-mediated
which
and old
are mediated
differences
PI signal transductions.
(approxi-
a reduction
1991) or no change Pi via
breakdown
in
~l-adrenergic
1990} and glutamate receptors
1989) depend on strain-specific
receptor-regulated
The findings
in muscarinic responses
(2-5
(Guiramand
in the responses of various
However,
there has been a lack of
studies about alterations in PI responses in v e r y old Wistar rats.
236
In the cAMP cascade, a g e - r e l a t e d and T h o r n b e r r y ,
1978; Nomura et a l . ,
were observed (Puri and V o l i c e r ,
alterations
of the AC a c t i v i t y
(Schmidt
1985) and of the c o n c e n t r a t i o n
of cAMP
1977) in the aging process.
Is t h e r e any alteration of AC catalytic unit a c t i v i t y and of the endogenous cAMP concentration in e x t r e m e l y aged rats? I f the catalyc unit a c t i v i t y would be reduced, Is this
is this d e p e n d e n t on the presence or absence of activated G-proteins? due to an a g e - d e p e n d e n t r e d u c t i o n
either
in G - p r o t e i n
function
or
in
coupling of the activated Gs/G i with the catalyc unit complex? It is well described that alterations such as sensorimotoric, Sabol et a l . ,
in the regulation of v a r i o u s f u n c t i o n s ,
occur in the striatum of senescence (Finch et a l . ,
1985; Joseph et a l . ,
1990).
This means,
in t u r n ,
an area for o b s e r v i n g how these f u n c t i o n s are controlled A decline
of age-related
(Joseph et a l . ,
striatal
dopamine
receptor
the striatum
in the aging
activity
1981; is
process.
seems established
1990). Also in the corpora striata of old rats, a decrease in the
level of dopamine (DA) D 2 r e c e p t o r mRNA was observed (Mesco et a l . , The p r e s e n t work was c a r r i e d out to gain i n s i g h t fect of D A D I and D 2 r e c e p t o r s , y o u n g adult vs.
into the a g e - r e l a t e d e f -
of PI b r e a k d o w n and cAMP cascades in the
the aged rat b r a i n .
volvement of these signal
1991).
It is the f i r s t attempt to elucidate the i n -
transduction
pathways
in the e x t r e m e l y aged Wistar
rats. MATERIALS AND METHODS Animals. tertal,
F.R.G.)
light/dark All
Female Wistar
animals
cycle
rats
(Hagemann,
were housed in g r o u p s (light:
had
06.00 a.m.
a standard
diet
Lippische V e r s u c h s t i e r z u c h t ,
Ex-
of 4-5 animals per cage with a 12 h r s
- 06.00 p . m . ) (Altromin
1324)
at 21~ and
and 50 % h u m i d i t y .
water
ad
libitum.
The
y o u n g ( w e i g h i n g 200-220 g) and old ( w e i g h i n g 285 + 22 g) rats were sacrificed by decapitation at an age of about 3.5 and 40 months of age, r e s p e c t i v e l y .
The
50 % s u r v i v a l level of these rats at our housing conditions is about 29 months. DA b i n d i n g studies.
The two b i n d i n g assays for d e t e r m i n i n g
the c h a r a c t e -
r i s t i c s of D I and D 2 and the AC assay were performed with common homogenate pools of striatal membranes of single animals. The assays of saturation
and of DA displacement of
Pont, New England Nuclear) b i n d i n g D I and binding of 1.0
D 2 were c a r r i e d out.
Non-specific
[3H]SCH
23390 ( D u -
[ 3 H ] s p i p e r o n e (Amersham B u c h l e r )
b i n d i n g was defined in the presence
]~M (+)-butaclamol ( D I ) and 30 uM ( - ) - s u l p i r i d e ( D 2 ) . Incubation times 3 [ H ] s p i p e r o n e and 120 rain for [ 3 H ] S C H 23390 b i n d i n g at 23~
were 90 min for
The b i n d i n g was terminated by rapid f i l t r a t i o n with a cell h a r v e s t e r . [3H]forskolin
(FSK) b i n d i n g assay. Striatal tissue was homogenized at 4~
in 3 ml of 0.32 M sucrose.
Each r e s u l t i n g
striatal
pellet was rehomogenized in
237
3.2 ml of 50 mM T r i s - H C l ,
pH 7.4,
10 mM NaF, and 5 mM MgCI 2.
b i n d i n g was performed at six c o n c e n t r a t i o n s (3.5 - 70 nM). at 23~
[3H]FSK-
Incubation (60 rain
was terminated by f i l t r a t i o n .
Assay of phospholipase C ( P L - C ) a c t i v i t y using exogenous prelabelled s u b strates. with
Phosphoinositide h y d r o l y s i s
exogenous
(reflecting
PL-C a c t i v i t y )
[ 3 H ] P t d l n s ( 4 , 5 ) P 2 (Amersham
lease of water soluble [ 3 H ] i n o s i t o l
Duchler)
(pll
7.5,
50 mM T r i s - H C l ,
t r y p s i n i n h i b i t o r ( t y p e I I , Sigma), oxidase, EC 1 . 4 . 3 . 4 ) .
by measuring
the re-
phosphates. Tissues obtained from the fore-
brain and the striatum were homogenized at 0~ genization b u f f e r
was determined
in 10 volumes ( w t / v o l ) 5 mM MgCI2,
of homo-
60 12g/ml soybean
10 12M p a r g y l i n e (an i n h i b i t o r of monoamine
The homogenate was c e n t r i f u g e d at 500 g for 10 rain and
the pellet was discarded.
The s u p e r n a t a n t was then c e n t r i f u g e d at 40000 g for
60 rain. The pellet was resuspended. Tissue obtained from the f o r e b r a i n and the striatum ( e i t h e r a single b r a i n was used or two brains were pooled) were homogenized.
For measuring the PL-C a c t i v i t y ,
assay tube) was incubated at 37~ ( 4 , 5 ) P 2 vesicles.
with reaction b u f f e r c o n t a i n i n g
(I
The PL-C a c t i v i t y
12M, RBI,
BIOTREND)
121
[3H]Ptdlns-
To regulate the PL-C a c t i v i t y via DA r e c e p t o r s ,
Sigma) a n d ( - ) - s u l p i r i d e solvent.
the homogenate (5 12g p r o t e i n / 6 0 DA (I
were d e l i v e r e d
12M,
in aqueous
is expressed in dpm induced by the enzyme (total
dpm - blank dpm) d i v i d e d by dpm of the sample in the absence of the enzyme. AC assay. The AC assay volume was 100 121 and included 30 121 membrane homogenate, 50 mM T r i s - H C I ,
pH 7.4, 2 mM MgCI, 0.3 mM EGTA, 10 I~M p a r g y -
line, I mM IBMX, 100 pM cAMP, 100 pM A T P , 5 mM c r e a t i n e - p h o s p h a t e , creatine-kinase,
I mg/ml
bovine serum albumin
New England
Nuclear).
(BSA)
152 U/I
and about 400,000 cpm
(~-32P)ATP
(DuPont,
min at 30~
in the absence of presence of d i f f e r e n t stimulators: 10 12M GppNHp,
10 pM FSK, 10 121ki FSK plus 100 nM GppNHp,
The samples were
incubated
10
10 ~,I FSK plus 10 mM NaF, 10
p~1 DA, 10 ~M DA plus 200 nM (+)SCH 23390. Determination of the endogenous inositol 1 , 4 , 5 - t r i s p h o s p h a t e ( I n s ( 1 , 4 , 5 ) P 3 ) concentration.
Tissues form v a r i o u s regions of rat b r a i n ,
i.e.,
cortex,
stria-
turn, and hippocampus were homogenized in water and 20 % p e r c h l o r i c acid ( 4 : 1 , v o l / v o l ) and c e n t r i f u g e d at 2000 g for 20 min at 4~ and the s u p e r n a t a n t was t i t r a t e d contained: T r i s - b u f f e r , 12g p r o t e i n / m l ) , protein/ml),
and
and
[3H]Ins(1,4,5)P3
Ins(1,4,5)P 3 binding
10 M KOH.
Each assay tube
(3000 cpm), homogenate ( c o n t a i n i n g 1.5
I n s ( 1 , q , 5 ) P 3 (3000 cpm),
t e r 15 min incubation at 0~ at 4~
to pH 8.0 with
The pellet was discarded
protein
homogenate ( c o n t a i n i n g in a p o l y p r o p y l e n e
1.5
tube.
12g Af-
the tubes were c e n t r i f u g e d at 2000 g for 15 rain
The pellet was dissolved in 100 pl H20. Detrmination of the endogenous cAMP c o n c e n t r a t i o n .
The tissue was homo-
genized in 667 1~I (2 striata pooled) aqueous 4 mM EDTA solution at 4~
fol-
238
lowed by heating
for 3 min in boiling
17,800 g at 4~
Fifty
water,
and centrifugation
for
5 rain at
~I of the supernatant was assayed for cAMP applying a
radioimmunoassay kit (Amersham B u c h l e r ) . Protein
determination
and data analysis.
mined with BSA as the standard. Significance
of differences
(p
The protein
content
was d e t e r -
All results are presented as mean + S.E.M.
< 0.05)
between
young
and aged animals were
calculated by S t u d e n t ' s t - t e s t s . For f u r t h e r
details
see the references:
Sugawa
(1993),
Sugawa and May
(1993), May and Sugawa (1993). RESULTS AND DISCUSSION DA receptor analysis in aging.
Binding saturation experiments with tritated
SCH 23390 at the D I receptor and [ 3 H ] s p i p e r o n e at the D 2 receptor revealed no change in the affinity DI
(-31
IB),
(K d) but a significant decrease in the density (Bmax) of
~o, p < 0.005,
respectively,
data not shown)
in the aged versus
and of D 2 (-22 ~o, p < 0.05,
young
striata.
The specific
Figure
binding
ir,
fmol/mg protein of a single concentration of tritated SCH 23390 (Figure I A ) and t r i t a t e d spiperone (data not shown) are significantly reduced at most of the DA concentrations
tested
in the senescent compared
the young
according
to
the differences in the B The proportion
values. max of the h i g h - a f f i n i t y
with
agonist
binding
state
(high)
signifi-
cantly decreased (p < 0.025) in the older (20.9 + 3 . 2 ~) in comparison with the young
(30.6 + 2.9 ~o) in D I ,
but increased n o n - s i g n i f i c a n t l y
in D 2 (47.9 + 2.6
versus 40.5 + 5.1 ~o). Age-related PL-C a c t i v i t y observed
second
messenger
in forebrain
tissue
(data not shown).
generating
isolated
However,
enzyme
from young
higher
activity. and old
PL-C a c t i v i t y
No d i f f e r e n t rats
has been
is observed in the
striatum of aged than the young (Figure 2). Variation of the incubation time of PL-C
demonstrates
pronounced PL-C
that
the difference
at incubations
activity
between
exceeding 5 minutes.
in the aged striatum
old and young
tissue
is most
A f t e r a 5 min incubation,
is 20 % higher,
after
the
20 rain about 45
higher (p < 0.05). The basal AC a c t i v i t y ficantly
in the striatal
(p < 0.01) reduced,
i.e.,
membrane of aged animals is signi-
approximately 35 % less than in young (data
not shown). DA receptor-mediated
signal transmission
pathways.
The young adult and
old rat striat'um tend to have a D A D 2 receptor mediated i n h i b i t o r y the phosphoinositol cascade. 20 ~o) which
is antagonized
1 IJM DA inhibits the PL-C a c t i v i t y by I
IJM ( - ) - s u l p i r i d e
in each case.
response in
(approximately However,
effect was significant only in the young striatum (p < 0.05, Figure 3).
the
239
A t_ o.
600 -
500
400
-
-
§
I-
*
o E 300 -
~ o
200 -
.
+
§
t~
0Y n
.
+ Aged 100
-
m
0-
9
8 -].og
7 [dopamine]
6
5
(M)
B 400 350
300
I
2so 200 \ ~
150 100 50
Y:totll
R:totll
Y:high
R:high
Figure I . A. Displacement of the specific [3H]SCH 23390 b i n d i n g . ( D I) by DA of 6 young (O} and 6 aged (+) rat striata. B. The binding of [aH]spiperone (D 2) if, 6 young (Y) and 6 aged (A) rat striata. Left two columns show total Bma x values determined by Scatchard anylyses of the 3H-antagonist binding saturation experiments. The two right columns show Bmax values of the highaffinity agonist binding state calculated by Scatchard and DA displacement analyses (means + S . E . M . ) . Asterisks indicate significant differences at p < 0.05. (May and Sugawa, 1993).
The D I mediated AC activation was investigated with 10 IJM DA. The DA stimulating effect on the AC was fully reversible in both the aged and young rats by the addition of 200 nM of SCH 23390, i . e . , diated (Figure q).
it is probably only D I me-
In the aged compared with the young rats, 10 pM DA dis-
plays a significant reduction (p < 0.05) from 36.5 w+ 6.9 96 to 23.6 + 3.9 ~ in AC activation calculated as ~o stimulation above basal activity (Figure 4). Coupling between G-protein and AC catalyc subunit in aging. The absolute AC a c t i v i t y upon stimulation by various pharmacological agents is in each case significantly lower in the aged striatum as compared to the young (p < 0.01 or
240
5.5 5.0 4.5 4.0
3.5
9~
>-, '-~ 3,0
2.5 2.0 1.5 1.0 0.5 0.0( 0
5
I
I
I
I
I
I
i
10
15
20
25
30
35
40
Time
Figure 2. Incubation time-dependence rat striatum. Results are the means each performed in triplicate. At 5 and than the symbol. Asterisks indicate 0 . 0 5 ) . (Sugawa, 1993}. 120
100
[min]
of PI hydrolysis in young (V) and old (O) + S . E . M . of 3 independent experiments, a t 20 min ( y ) , the e r r o r bars are smaller statistically significant differences (p <
-
-
T
T
8O u}
so
~J
4O
20 f-
oi A
B
C
young
D
A
B
C
D
old
F i g u r e 3. Effect of DA and D 2 antagonists on tile h y d r o l y s i s of exogenous PI in y o u n g and old rat s t r i a t u m , measured as the appearance of w a t e r - s o l u b l e inositol phosphates. Pargyline was always p r e s e n t . The following ligands were added in both age g r o u p s . A: no a d d i t i o n , c o n t r o l , set as 100 %; on an absolute scale, h o w e v e r , the e x t e n t of PI h y d r o l y s i s is a p p r o x i m a t e l y 40 % l a r g e r in the old tissue; B: I ~M DA; C: I pM DA + 10 nM haloperidol; D: I ~I~A DA + I laM ( - ) - s u l p i r i d e . Mean + S . E . M . of t h r e e i n d e p e n d e n t e x p e r i m e n t s each performed in t r i p l i c a t e . A s t e r i s k denotes s i g n i f i c a n t d i f f e r e n c e s between y o u n g and old g r o u p (p < 0 . 0 5 } .
241
0.05,
data not shown).
The observed
age-related
decline
in the AC a c t i v i t y
might be caused by effects of the coupling of G-proteins with the AC catalytic s u b u n i t , therefore,
the percentage stimulation of the control (basal) AC a c t i v i t y
by various reagents (defined as relative stimualation), solely, lated.
and the combinations of GppNHp plus FSK, No significant differences,
however,
i.e.,
GppNHp, NaF, FSK
NaF plus FSK, was calcu-
in the relative stimulation of the AC
a c t i v i t y by any pharmacological substances used in the striatum were observed. 50
r "6 o~ 40
E
~
p
]
30
20
10
~
l
0 DA
l
SCH23390
DA SCH23390
Y
A
Figure 4. The stimulation by 10 ~M DA of the AC (in ~ above the basal activ i t y ) and the inhibition of the AC stimulation by the addition of 200 nM SCH 23390 in 6 young ( Y ) and 6 aged (A) rat striata. Means + S.E.M. Asterisk denotes significant difference between young and old g r o u p s - ( p < 0.05). To answer the question,
whether
the G s-AC catalyc subunit
involved in the aging process, a [3H]FSK to the specific differences binding
[3H]FSK
were
binding
observed.
binding
test was applied.
in young and aged striatum,
The
calculated
affinity
(Kd)
between these two g r o u p s ,
i.e.,
_+ 0.27 pmollmg protein (aged)
According
no age-related
values
of
[3H]FSK
(Bmax) values display as well no difference
2.00 + 0.48 pmol/mg protein
In the young rat brain,
pocampus,
(young)
the highest concentration
of I n s ( 1 , 4 , 5 ) P 3 and is found
the striatum and the cortex contain two- and f o u r - f o l d
respectively.
A similar
I n s ( 1 , 4 , 5 ) P 3 concentration
campus and cortex for young adult and aged tissue. the young tissue,
and 2.19
(data not shown).
Age-dependent difference of endogenous concentration
4,5)P 3,
is
were 14.6 + 1.9 nM for the young (n = 6) and 15.6 + 2.4 nM for the
aged tissues (n = 7). The density
cAMP.
pathway
in the old rat striatal
in the hipless I n s ( 1 , -
is found
However,
in hippo-
in contrast
I n s ( 1 , 4 , 5 ) P 3 concentration
to
is as high
242
as in the hippocampus.
In fact,
the aged striatum contains approximately 250
more I n s ( 1 , 4 , 5 ] P 3 than the young striatum (P < 0.005, data not shown). The cAMP level in the aged striatum is approximately 40 % higher as compared to young striatum (p < 0.05, data not shown). On the way to a better transduction
pathways
understanding
in aging:
Working
of the involvement
hypotheses.
The
of the signal
question
remains,
why is there such a drastically higher endogenous I n s ( 1 , 4 , 5 ) P 3 concentration in the old compared with the young rat striatum,
even though at the receptor le-
vel there is only a slight reduction [ b y about 20 $} in the D A D 2 receptor density
in the 40-month-old
I n s ( 1 , 4 , 5 ) P 3 to
rat striatum.
I n s ( 1 , 3 , 4 , 5 ) P 4 or
phosphate-5-phosphatase)
which
One possible explanation
I n s ( 1 , 4 , 5 ) P 3 5-phosphatase
hydrolyzes
is that either (inositol
I n s ( 1 , 4 , 5 ) P 3 to I n s ( I , 4 ) 2 ,
polyare ei-
ther less active or are present in lower amounts in old tissue (Figure 5, r i g h t side).
r
i
-
w4
PKA
Figure 5. Schematic drawing of the possible age-related involvement in the cAMP (left side) and PI ( r i g h t side) signal transduction pathways, a: 5-phosphatase, b: 3-kinase. In the present s t u d y a much higher
(about 40 ~) cAMP concentration
the striatum of extremely aged animals was detected.
in
This significantly d i f f e r e n t
concentration of the endogenous cAMP in the striatal tissue between young and aged cannot be explained less basal AC a c t i v i t y membranes.
by differences
(about 35 % reduction)
A conceivable
explanation
activity
(Stancheva
and Alova,
1991)
activity
(Govoni
al.o
in
et
in the AC a c t i v i t y ,
1988)
the
was found
is an age-related or a different older.
Another
since significant
in the aged striatal decline
protein
of cAMP-PDE
kinase A
possibility
could
(PKA) be a
changed ratio of AC and cAMP-PDE, either of the a c t i v i t y of of the amount, the extremely aged animals (Figure 5, left side).
in
243
A number of biochemical changes with senescence in the DA pathway, cluding
DA regulated
(Nomura,
1985;
signal transmissions,
Sabol et a l . ,
1985;
have been observed
Morelli
et a l . ,
1990).
in-
in the rat CNS
Our
recent
study
showed a significant decrease in the density of DA D I and D 2 receptors in the aged-vs,
young striata (May and Sugawa, 1993). It could be explained that the
higher D A D 2 receptor density and higher DA concentration tum lead to the more pronounced
in the young s t r i a -
inhibition of PL-C a c t i v i t y ,
lower endogenous I n s ( 1 , 4 , 5 ) P 3 concentration
and therefore,
to
in the young rat in comparison to
the aged. It has been postulated that D A D 2 receptors regulate inhibition of the activity
of various
and Meldolesi,
cell types
1989),
including
evidence of age-related at the receptor 1989).
(Morra et a l . ,
level
1991; Vallar et a l . ,
brain cells (Pizzi et a l . ,
D A D 2 receptor dysfunction (Joyce et a l . ,
In our present s t u d y ,
1986;
1988).
1988; Vallar
There is some
in the rat striatum system
Norman et a l . ,
1987;
there is no significant difference
Han et a l . ,
of the effector
mechanisms mediated by D A D 2 receptor sites between the young adult and the aged striatum. In
senescence,
interactions
volved in signal transduction Johnson,
1989).
However,
between
receptors
are probably altered
in the striatum,
and
their
(Severson,
all applied
G-proteins
in-
1987; Green and
pharmacological
agents
stimulated the AC in the young animals much s t r o n g e r than in aged correlating well with the results of the basal AC a c t i v i t y . tein-AC coupling on aging was investigated.
The involvement of the G - p r o -
FSK appears to have two d i f f e r e n t
interactions with AC. One requires Gs and another does not (Seamon and Daly, 1981; Schneyer et a l . ,
1983; Battaglia et a l . ,
1986). The age-related activation
of AC by FSK in the presence and absence of the guanine GppNHp (which stimulates at low concentration
nucleotide
(100 nM) preferentially
analog, G i ) , and
in combination with 10 mM NaF (which activates Gs) was focussed in a series of experiments.
In striatal tissue, stimulation by FSK was inhibited by the additon
of GppNHp and was superadditive by the addition of NaF in respect to activation of the AC.
It is likely that the changes in AC catalytic unit a c t i v i t y with
age occur
independently
(O'Conner
et a l . ,
the
[311]FSK
by a filtration binding
in
the absence
1983) and G i.
binding
test,
or
in
These findings
since
the
the
presence
of activated
G
s are consistent with data from
high-affinity
FSK
binding
determined
binding assay probably reflects only the G s - p r o t e i n coupled AC
site as previously discussed
no change in the specific
(Laurenza and Seamon,
FSK binding
1991).
There was
between young and aged animals in the
striatum. Taken together,
the age-related
of alterations in Gs and G i a c t i v i t y . aged rats,
decline of the AC a c t i v i t y
is independent
In the striatal membranes of our extremely
it seems that the reduced AC a c t i v i t y is due to either the reduction
244
in the number of the AC molecules, the AC phosphorylation state (desensitizat i o n ) , or even an altered AC gene expression d u r i n g aging. T h e r e f o r e , a f u n c tional down-regulation of AC a c t i v i t y occurs in aging. Our recent investigation revealed an imbalance in DA D I / D 2 receptor mediated cAMP signal transduction in v e r y old rats. We observed the decline of the AC a c t i v i t y by DA D I receptor in aged animals in comparison to the young. We could prove that the age-related alterations in AC catalytic u n i t a c t i v i t y occur independently of G - p r o t e i n s , i . e . , side}.
Gas - AC coupling (see Figure 5, left
It is possible that the coupling of receptors, e . g . ,
DA D I , and G - p r o -
tein a n d / o r the feedback mechanisms between PKA and those receptors might be altered in the striatum of aged animals ( F i g u r e 5, left side). It is of interest in the f u t u r e to examine how the c r o s s - t a l k between these two signal transduction pathways is altered in aging processes. ACKNOWLEDGEMENTS The authors thar~k Sharra for encouragement, This paper is dedicated to prof. Dr. H. Coper. REFERENCES Battaglia, G., Norman, A . B . , Hess, E.J. and Creese, I. (1986): Forskolin potentiates the stimulation of rat striatal adenylate cyclase mediated by D-I dopamine receptors, guanine nucleotides, and sodium fluoride. J. Neurochem., 46, 1180-1185. Crew, F . T . , Gonzales, R . A . , Polavcik, R., Phillips, M . I . , Theiss, C. and Raizada, M. (1986): Changes in receptor stimulated phosphoinositide h y d r o lysis in brain d u r i n g ethanol administration, aging, and other pathological conditions. Psychopharmacol. B u l l . , 22, 775-780. Finch, C . E . , Rand~ll, P.K. and Marshall, J . F . (1981): Aging and basal gangliar functions. Ann. Rev. Gerontol, G e r i a t r . , 2, 49-87. Govoni, S., Rius, R . A . , Battaini, F. and T r a b u c c h i , M. (1988): Reduced cAMP-dependent phosphorylation in striatum and nucleus accumbens of aged rats: Evidence of an altered functioning of D I dopaminoceptive neurons. J. Gerontol., 43, B93-B97. Green, A. and Johnson, J . L . (1989): Evidence for altered expression of the GTP-dependent r e g u l a t o r y proteins G and Gi, in adipocytes from aged rats. Biochem. J . , 258, 607-610. s Guiramand, J . , Sassetti, I. and Recasens, M. (1989): Developmental changes in the chemosensitivity of rat brain synaptoneurosomes to e x c i t a t o r y amino acids, estimated by inositol phosphate formation. I n t . J. Dev. Neurosci., 7, 257-266. Han, Z . , K u y a t t , B . L . , Kochman, K . A . , DeSouza, E.B. and Roth, G.S.(1989): Effect of aging on concentrations of D~-receptor-containing neurons in the rat striatum. Brain Res., 498, 299-307.z Joseph, J . A . , Roth, G.S. and Strong, R. (1990): The striatum, a microcosm for the examination of age-related alterations in the CNS: a selected review. Rev. Biol. Res. A g i n g . , 4, 181-199. Joyce, J . N . , Loeschen, S . K . , Sapp, D.W. and Marshall, J.F. (1986): A g e - r e l a ted regional loss of caudate-putamen dopamine receptors revealed by quantitative autoradiography. Brain. Res., 378, 158-163. Laurenza, A. and Seamon, K . B . (1991): H i g h - a f f i n i t y binding sites for (3H)Forskolin. Methods in Enzymol., 195, 52-65. May, T . and Sugawa, M. (1993): Altered dopamine receptor mediated signal transmission in the striatum of aged rats. Brain. Res., 604, 106-111.
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