Influence of aging and thymus on the beta-adrenergic dependent adenylyl cyclase activity in mouse brain cortex

Arch. Gerontol. Geriatr. suppl. 3 (1992) 359-366 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-4932/92/$05.00

359

INFLUENCE OF AGING AND THYMUS ON THE BETA-ADRENERGIC DEPENDENT ADENYLYL CYCLASE A C T I V I T Y IN MOUSE BRAIN CORTEX

C. VlTICCHI and L. PIANTANELLI Center of Biochemistry, Gerontologic Research Department, I . N . R . C . A . , Via B i r a r e l l i , 8, 1-60121 Ancona, Italy SUMMARY 13-adrenoceptor (13AR) density has previously been found altered in brain cortex of aging mice. In the present paper the question has been addressed whether adenylyl cyclase (AC) also presents similar age-related changes. Due to the fact that age-related receptor impairments have p r e v i o u s l y been corrected by thymic g r a f t s , the influence of thymus was also studied. Therefore, e x p e r i ments were performed on y o u n g , athymic nude y o u n g , old and thymus-grafted old mice. BAR characteristics and basal and isoproterenol-stimulated AC a c t i v i t y were assayed in the same membrane preparations. Our results confirm a decrease of receptor density of 131 subtype in both old and athymic nude young mice, paralleled by a decreased isoproterenol-stimulated AC a c t i v i t y . A neonatal thymus grafted into old recipients is able to correct the changes in receptor density and the height of the peak of AC response. The location of the peak, however, remains shifted to the r i g h t , as it occurs in nude and in untreated old mice. It can be concluded that alterations may occur at d i f f e r e n t steps along the stimulus transducing chain, as suggested by the differential effect induced by thymus on receptor changes, which are completely recovered, and AC activity alterations, only partially corrected. Keywords: nude mice

adenylyl

cyclase,

13-adrenergic

receptors,

brain

cortex,

thymus,

INTRODUCTION A consistent number of observations show that the catecholaminergic system is deeply impaired d u r i n g aging (Roth, 1979; Scarpace, 1986; Weiss, 1988) and reports of experiments performed on various tissues of mice of different ages demonstrate an age-related decrease of the 13-adrenergic receptor

(13AR)

density (Greenberg and Weiss, 1978; Piantanelli et a l . , 1980; Fattoretti et a l . , 1982).

In p a r t i c u l a r ,

13AR density was found altered in brain cortex of aging

mice (Piantanelli et a l . ,

1985)

where the receptor decrease is completely in

charge of the BI subtype (Viticchi et a l . , 1989) probably located on the neuronal cells of the brain cortex (Minneman et a l . , 1979). The age-dependent receptor density decrease is not d e f i n i t i v e as it was completely recovered by a neonatal thymus grafted into old recipients (Fattoretti et a l . , et a l . , 1985; Viticchi et a l . ,

1989).

1982; Piantanelli

In addition it has been demonstrated that

the thymic corrective action is exerted only on 131 subtype, the same subpopulation showing changes with aging. Receptor density may be considered a'good aging correlate and may represent a possible " p r i m a r y "

mechanism involved in neuroendocrine aging

(Roth

360 and Hess,

1982).

However,

the characteristics

a f f i n i t y and subtype d i s t r i b u t i o n nergic system efficiency mediated

such as d e n s i t y ,

since the action of hormones on their target cells are

by a complex mechanism that

(Narayanan and Derby,

of receptors

give us only partial information about 13-adreinvolves

several other

1982; Sibley and Lefkowitz,

important

steps

1985). cAMP is one of the

second messengers through which catecholamines act within the cell; then, aden y l y l cyclase (AC) certainly

is a key point in the pathway of the B-adrenergic

stimulation and may play a fundamental

role also in the age-related

alterations

observed in the system (Dillon et a l . , 1980). The aim of this work was to s t u d y the influence of the age and thymus on BARs and AC a c t i v i t y

parallelly.

Results of these experiments could give f u r -

t h e r insights on the understanding alterations

of the

13-adrenergic

of the mechanisms of both the age-related

responsiveness

(Piantanelli

et a l . ,

1978;

Giu-

dicelli and Pecquery, 1978; Feldman, 1986) and the corrective action of the t h y mus (Fabris

and

Piantanelli,

1982;

Fattoretti

et a l . ,

1982;

1985). Receptor characteristics and basal and isoproterenol

Piantanelli

et a l . ,

(IPR) stimulated AC

a c t i v i t y were measured in the same membrane preparations from y o u n g , athymic nude young, old and t h y m u s - g r a f t e d old mice. MATERIALS AND METHODS Materials.

(-)-(3H)-dihydroalprenolol

Amersham (Great B r i t a i n ) ,

(DHA) and cAMP assay kit were from

(dl)-isoproterenol

was from Sigma (St.

Louis, USA),

while A T P , creatine kinase and creatine phosphate were from Boehringer many).

Practolol

was a kind

gift

of the

ICI-Pharma.

(Ger-

Other chemicals were at

level of the laboratory standard. Animals.

3 and 21 month old male B a l b l c - n u and 2 month old male B a l b / c -

nu nude mice of our own colony were used.

When t h y m u s - g r a f t e d

old animals

were used, the g r a f t of neonatal thymus was performed into the recipients u n der

the

kidney

capsule j u s t

one

month

before

their

sacrifice.

Animals

were

killed by cervical dislocation and the brain cortex was drawn immediately. Membrane already

preparation

described

and

(Piantanelli

receptor et a l . ,

washed in ice-cold 10 mM T r i s - H C l buffer,

then

it was homogenized

buffer,

centrifuged

protein concentration buffer

and finally

(pH 7 . 4 ) ,

Membranes

Briefly,

suspended

Incubation

prepared

was minced

as and

0.82 mM EDTA

(pH 7 . 4 ) ,

in the same buffer

concentrations

ranging from 0.3 to 4 nM and run at 37~

were

tissue

0.25 M sucrose,

in 50 mM T r i s - H C l

of about I mglml.

in presence of increasing

assay.

1985).

10 mM MgCI 2 to get a final

was performed

in the same

of the labeled antagonist

DHA

for 10 min.

When 131 and 132 determination was made, the specific 131-antagonist practoIoi was used and the estimation of the two subtypes was obtained according to

361 the mathematical reaction

procedure previously

was stopped by adding

described

7 ml of ice-cold

f i l t e r e d t h r o u g h Whatman GF/c f i l t e r s . Packard

scintillator

concentration

using

buffer

et a l . ,

1982).

The

and the m i x t u r e

was

Filters were then d r i e d and counted in a

a Toluene-Triton

was determined

(Fattoretti

according

X-100

to the

scintillation

Lowry

method

liquid.

Protein

(Lowry

et a l . ,

1951) using BSA as s t a n d a r d . AC a c t i v i t y

assay.

r e c e p t o r determination 80 mM T r i s - H C l 1.5 mM A T P .

Membranes

from

the

and enzyme a c t i v i t y

(pH 7 . 4 ) ,

same p r e p a r a t i o n

assay.

were

The incubation

used

for

system was:

10 mM MgCI2, 0.75 mM EGTA, 4.2 mM teophylline and

The protein concentration was about 0.05 mg/ml.

A T P was p r o v i -

ded by the presence of 0.25 mg/ml of creatine kinase and 5 mM creatine phosphate (ATP r e g e n e r a t i n g system).

When it was p r e s e n t IPR ranged from 10-8 to

10 -4 M. Incubation started by adding ATP and the A T P r e g e n e r a t i n g system to the membrane suspension and ran for AC a c t i v i t y

was proportional

10 min at 37~

to the incubation

U n d e r these conditions

time and p r o t e i n

concentration.

The enzyme a c t i v i t y was stopped by boiling the samples for 2.5 min, and cAMP was measured using the Amersham cAMP assay kit.

AC a c t i v i t y

was expressed

as pmole cAMPlmg p r o t e i n l m i n . RESULTS 13AR c h a r a c t e r i s t i c s preparations periments

are

and AC a c t i v i t y

reported

dealing

f e r e n t animal models used. not show s i g n i f i c a n t contrary,

were measured in the same membrane

in o r d e r to compare them d i r e c t l y . with

membranes from brain

Data on receptor a f f i n i t y

changes when the d i f f e r e n t

BAR d e n s i t y

In Table I the results of e x cortex

of the d i f -

are not given

as they do

models are compared.

shows a decrease in the brain

On the

c o r t e x of old and nude

mice when t h e y are compared to the young ones. Such a decrease is recovered when a neonatal thymus is g r a f t e d into old recipients one month before e x p e r i ments were performed.

Furthermore

both the receptor decrease and the reco-

v e r i n g effect of the thymus are completely in charge of the 131 r e c e p t o r s u b t y p e since the 82 remains nearly constant in the d i f f e r e n t animal models. Age and thymus do not influence basal AC a c t i v i t y

significantly.

In fact,

results r e p o r t e d in Figure I show that in unstimulated conditions AC a c t i v i t y is similar in the animal models s t u d i e d ,

i r r e s p e c t i v e of the d i f f e r e n t levels of 13AR

density. In the presence of the 8-agonist

IPR the a c t i v i t y

of AC was increased in

all models, b u t the degree of this increase over the basal level varies according to the animal model i n v e s t i g a t e d .

In Figure 2 results are r e p o r t e d from e x p e r i -

ments

in y o u n g ,

on AC a c t i v i t y

mice at increasing

measured

concentrations

of

IPR.

old,

old

thymus-grafted

In membranes of young

and nude animals the

362 Table I BAR DENSITY OF MOUSE BRAIN CORTEX (mean fmole/mg + S . E . M . ) Animal

BAR d e n s i t y

models

Total

BI

B2

YOUNG

82.8 + 7.2

55.0 + 6.8

27.7 + 4.4

p

0.01

0.01

49.7 + 1.6

16.5 + 2.5

<

OLD p < OLD + THYMUS p

0.001 56.6 + 3.9

0.001

0.001

54.5 + 1.7

18.0 + 1.5

0.01

0.01

<

NUDE p

0,001 86.3 + 4.0

<

33.2 + 3.0 29.7 + 2.7 35.9 + 2.0

Notes: p indicates the results of statistical analysis (t t e s t ) , always r e f e r r i n g to comparison of the u p p e r and lower line, e x c e p t the last row of the table which is r e f e r r e d to nude vs. y o u n g comparisons. Missing p values indicate no statistically significant differences.

100

200

T

Z

g~

"1"

& 0

o

Y

0

O+T

N

F i g u r e I . Results are p r e s e n t e d on brain c o r t e x total BAR d e n s i t y (I-I) and basal AC a c t i v i t y (ll~) from young ( Y ) , y o u n g athymic nude ( N ) , old (O) and t h y m u s - g r a f t e d old ( O + T) mice. Data d e r i v e from e x p e r i m e n t s performed on 6 animals. Bars r e p r e s e n t SEM. No statistically s i g n i f i c a n t changes have been o b s e r v e d in AC a c t i v i t y . Results of statistical analysis of BAR d e n s i t y are r e p o r t e d in Table I, which also show the d i f f e r e n t i a l changes of r e c e p t o r subpopulation s.

363

18~I 160

~2

140

12o

<

0

8

7

- log

[IPR, mol/1]

Figure 2. Dose dependence of AC a c t i v i t y in mouse brain cortex stimulated by increasing concentrations of IPR. Data derive from experiments performed on young { O ) , young athymic nude [ Z ~ ) , old ( I ' I ) and t h y m u s - g r a f t e d old [mR) mice. Each group consisted of 6 animals. _l~ars represent SEM. The peak of AC a c t i v i t y in young mice is obtained with 10 M IPR. The peaks of the other animal models are shifted to the r i g h t . The following differences between the peak heights are statistically significant: y o u n g - o l d , p < 0.05, "thymus-grafted old-old, p < 0.02, t h y m u s - g r a f t e d old-nude, p < 0.05, y o u n g - n u d e , n . s . 100

100

r/]

o

50

50 o

r~

I=u o o

~

< 0

0

Y

O

O+T

N

Figure 3 . Brain cortex 13AR density and IPR-stimulated AC a c t i v i t y are compared in young ( Y ) , young athymic nude ( N ) , old [O) and t h y m u s - g r a f t e d old {O + T) mice. Each group consisted of 6 animals. Bars represent SEM.

364 stimulation

reaches its maximum value when I uM of the agonist is present.

membranes derived from old mice the peak is significantly to that from the young ones. in old mice,

though

Nude mice show a trend similar to that observed

the height of the peak is not statistically

that of young animals.

In addition,

right.

different

from

in old and nude mice the peak of response

is shifted

to the

stimulation

The age-dependent

is completely recovered in t h y m u s - g r a f t e d

peak of response occurs at a higher young mice.

In

lower when compared

decrease of the

AC

old animals, although the

IPR concentration

Figure 3 compares brain cortex

IPR-induced

than that observed

BAR density and the percent

in in-

crease of AC stimulated a c t i v i t y at the peak in the same membrane preparations from the different animal models.

Data show that there is a s t r i c t

relationship

between the capability of the tissue to respond to the stimulation and the availability of BARs. DISCUSSION The capability of a system to respond to a hormonal

stimulation

is r e g u -

lated by several factors each of them representing a potential limiting point for altered

efficiency

seph,

of the responsiveness

1988; Scarpace,

1990).

During

(Roth and Hess,

aging

several

lated by the 13-adrenergic system are impaired man,

1986) and

brain

cortex

it was reported

(Piantanelli

et a l . ,

that 1985,

biological

(Piantanelli

the density

1982;

of

Kohno et a l . ,

Roth and Jo-

functions

et a l . ,

modu-

1978;

Feld-

BARs decreases both

in

1986) and submandibular

glands of aging mice (Fattoretti et a l . , 1982), while their affinity does not show significant

changes.

The

receptor

decrease

may e x e r t

different

effects

upon

various steps of the transmission chain of the hormonal message leading to the stimulation of AC a c t i v i t y .

Results reported in this paper suggest that the res-

ponsiveness of AC a c t i v i t y to adrenergic stimulation is linked to the availability of receptors,

in agreement

(Giudicelli and Pecquery,

with

results

1978, Dolphin et a l . ,

ponsiveness to pharmacological mulated conditions,

previous

treatments

on the c o n t r a r y ,

on impairment

1979; Scarpace,

(Piantanelli

et a l . ,

of

I~AR,

AC

1986) and res1978).

In u n s t i -

there are no changes in AC a c t i v i t y from

the various animal models, probably because only a v e r y small amount of receptors is involved in basal a c t i v i t y (Severne et a l . , 1984). Young nude mice present alterations in I~AR density and I PR-stimulated AC activity

similar

to those found

in old animals.

These findings,

those previously observed on the alteration of I PR-induced

together

stimulation

with

of sub-

mandibular glands' DNA synthesis (Piantanelli et a l . , 1978), confirm the already suggested

role of the thymus

in the correct

13-adrenergic system (Fattoretti et a l . , al.,

1989).

This

role is f u r t h e r

development

and maintenance

of

1982, Piantanelli et a l . , 1985; Viticchi et

supported

by

results

obtained

with

thymus-

365 grafted old mice. In fact, one month after the neonatal gland is implanted, impairments in both BARs and AC are corrected.

It is worth noting that the reco-

v e r i n g effect of the thymus is exerted when grafted into old animals, that is, in animals showing considerable environmental alterations (Roth, 1979; Roth and Hess, 1982) AC a c t i v i t y

impairment does not seem completely due to the age-related

changes in BARs. In fact, the peak of stimulated AC a c t i v i t y remains shifted to the r i g h t even in t h y m u s - g r a f t e d mice, as happens in nude and untreated old animals. T h u s , the higher concentration of IPR needed to reach the same height of the peak suggests that additional alterations do occur, other than receptor changes (Feldman,

1986).

Moreover,

it is worth

noting that

impaired mechanisms are not recovered by thymic action.

such eventually

In conclusion, though

age-related modifications in 6ARs can be considered responsible for the majority of AC a c t i v i t y changes, other alterations may occur at d i f f e r e n t steps along the 6-adrenergic message transducing chain (O'Connor et alo, 1983). F u r t h e r studies on this aspect are needed, as well as on the differential role played by 61 and 62 receptor subtypes on the modulation of AC a c t i v i t y . ACKNOWLEDGEMENTS We thank Mr. Flavio Marchegiani and Ms. Giovanna Pennesi for their technical assistance and Ms. Monica Glebocki for k i n d l y reading the manuscript. REFERENCES Dillon, N., C h u n g , S., Kelly, J. and O'Malley, K. (1980): Age and beta adrenoceptor-mediated function. Clin. Pharmacol. T h e r . , 27, 769-772. Dolphin, A . , A d r i e n , J . , Hamon, M. and Bockaert, J. (1979): I d e n t i t y of (3H)dihydroalprenolol binding sites and 6-adrenergic receptors coupled with adenylate cyclase in the central nervous system: pharmacological properties, d i s t r i b u t i o n and adaptive responsiveness. Mol. Pharmacol., 15, 1-15. Fabris, N. and Piantanelli, L. (1982): T h y m u s - n e u r o e n d o c r i n e interaction d u r i n g development and aging. In: Endocrine and Neuroendocrine Mechanisms of A g i n g . CRC Series in A g i n g , pp. 161-181. Editors: R.C. Adelman and G.S. Roth. CRC Press, Boca Raton, USA. Fattoretti, P., V i t i c c h i , C. and Piantanelli, L. (1982): Age-dependent decrease of beta-adrenoceptor density in the submandibular glands of mice and its modulation by the thymus. A r c h . Gerontol. G e r i a t r . , 1, 229-240. Feldman, R.D. (1986}: Physiological and molecular correlates of age-related changes in the human 6-adrenergic receptor system. Faseb. J . , 45, 48-50. Giudicelli, Y. and Pecquery, R. (1978): 6-adrenergic receptors and catecholamine-sensitive adenylate cyclase in rat fat-cell membranes: influence of g r o w t h , cell size and aging. Eur. J. Biochem., 90, 413-419. Greenberg, L.H. and Weiss, B. (1978): 6-adrenergic receptors in aged rat brain reduced number and capacity of pineal gland to develop supersensit i v i t y . Science, 201, 61-63. Kohno, A . , Seeman, P. and Cinader, B. (1986): Age-related changes of betaadrenoceptors in aging inbred mice. J. Gerontol., 41, 439-444. Lowry, O . H . , Rosebrough, N . J . , Farr, A . L . and Randall, R.J. (1951): Protein measurement with the folin reagent. J. Biol. Chem., 193, 265-275. Minneman, K . P . , Dibner, M . D . , Wolfe, B . B : and Molinoff, P.B. (1979): Beta-1 and beta-2 adrenergic receptors in rat cerebral cortex are independently regulated. Science, 204, 866-868.

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