Age-related changes of dopamine sensitivie cyclic amp generation in the rat frontal cortex

Meachanisms of Ageing and Development, 54 (1990) 63--73

63

Elsevier Scientific Publishers Ireland Ltd.

A G E - R E L A T E D C H A N G E S OF D O P A M I N E S E N S I T I V E C Y C L I C A M P G E N E R A T I O N IN T H E R A T F R O N T A L C O R T E X

F R A N C E S C O A M E N T A a,b," C A R L O C A V A L L O T T I a, W A D E L. COLLIER% M A N U E L A DE M I C H E L E a and A L B E R T O R I C C P RDipartimento di Scienze Neurologiche, University "'La Sapienza'; ODipartimento di Sanita Pubblica e Biologia Cellulare, University "'Tor Vergata'" Rome (Italy) and CManatee Community College, South Campus, Venice, FL (U.S.A.)

(ReceivedAugust 2nd, 1989) (Revision receivedNovember 15th, 1989) SUMMARY The dopamine (DA) D-1 and D-2 receptors coupled to 3',5'-cyclic adenosine monophosphate (cAMP) generation were studied in m e m b r a n e particles of the frontal cortex in young (3-month-old), adult (12-month-old) and aged (24-month-old) male Sprague--Dawley rats. Activation of D-1 receptors with DA, apomorphine or fenoldopam enhanced accumulation of c A M P in the frontal cortex of young rats. The stimulatory effect elicited by DA on c A M P generation declined by about 20070 in adult rats. No further decline in c A M P accumulation was noticeable in aged animals. The response to dopaminergic agonists was blocked by the D-1 receptor antagonist SCH 29390 in the three age groups examined. The presence of D-2 receptors, negatively coupled to c A M P generation, was demonstrated by incubating frontal cortex membrane particles with SCH 23390 and then with DA. This inhibitory response, was also elicited with D-2 receptor agonists quinpirole or bromocriptine in the absence of SCH 23390 in which these compounds produced a decrease in cAMP. The decrease in c A M P caused following D-2 receptor stimulation was shown to be enhanced with age. No difference was observed between the three age groups of animals in the activation of c A M P production by forskolin. The present data suggest a selective decrease in the coupling between the D-1 receptor and c A M P generation in the frontal cortex of adult and aged rats and of an age-dependent increase in the coupling between the D-2 receptor and c A M P inhibition. The functional consequences of these biochemical changes may have important implications in the aging of the rat frontal cortex.

*To whom all correspondence should be addressed at: Dipartimento di Scienze Neurologiche Via A. Bor-

eiU, 50, 100161, Rome, Italy. 0047-6374/90/$03.50 Printed and Published in Ireland

© 1990Elsevier Scientific Publishers Ireland Ltd.

64

Key words: Aging; Frontal cortex; Dopamine receptors; cAMP; Rat INTRODUCTION

The central dopaminergic system undergoes a variety of age-dependent changes (for a review see Ref. 1). They consist in the progressive loss of dopaminergic nerve cell bodies in the substantia nigra [2]; in the reduction of dopamine (DA) and its metabolite homovanillic acid in extrapyramidal nuclei, in limbic cortex and in frontal and temporal areas [3--5]; and in the reduction of the DA synthesizing enzymes tyrosine hydroxylase and DOPA-decarboxylase in the striatum and in the substantia nigra [7--10]. Moreover, reduced density of DA receptor sites (D-I and D-2) has been described in extrapyramidal nuclei using either radioreceptor binding techniques and DA-sensitive adenylate cyclase assay [ 11--21]. Although the existence of a mesocortical dopaminergic system supplying frontal, cingular and entorinal cortices was demonstrated almost 15 years ago [22--25], the topic of the characterization of DA receptors populations in these cortical areas received less attention than in the case of extrapyramidal nuclei. On the other hand, the presence of D-1 and D-2 receptor sites in the frontal cortex has been rather recently demonstrated using adenylate cyclase assay and radioreceptor binding associated with autoradiography [26--30]. Since no information is so far available as to the age-related changes of DA receptors in the rat frontal cortex, we characterized the DA receptors that are coupled to 3',5'-cyclic adenosine monophospate (cAMP) generating system in the rat frontal cortex as a function of age. MATERIALS AND M E T H O D S

Male Sprague--Dawley rats (Charles River, Italy) of three (considered to be young, n = 8), 12 (considered to be adult, n = 10) and 24 (considered to be old, n = 10) months of age were used. The animals were kept under a constant light/dark cycle (light period from 0700 h to 1900 h) at an ambient temperature of 22 __. 1 °C. They had free access to water and laboratory chow (4 RF 18, ltalmangimi, Italy). Rats were weighed, anaesthetized with ether and killed by decapitation. The cranium was quickly opened, the brain was removed and either the left as the right frontal cortex were dissected out and homogenized in 2 mM T r i s - - H C l (pH 7.5) containing 2 mM EGTA. Membrane particles of the frontal cortex were prepared according to the procedure described in earlier papers [31,32]. Briefly, the homogenates were centrifuged at 1000 g for 15 min and pellets were discarded. The supernatants were further centrifuged at 50 000 g for 25 min at 4°C. The resulting pellets were washed with the incubation buffer, resuspended and centrifuged. The final pellets were used for adenylate cyclase assay. Adenylate cyclase was assayed in a 500/al reaction mixture containing Tris--HC1

65 (80 mM), EGTA (0.6 raM), 3-isobuthyl-l-methyl-xantine (0.5 mM), ATP (2 mM) MgSO 4 (16 raM), phosphocreatine (5 mM), creatine phosphokinase (50 units/ml), ascorbic acid (0.02%). The tubes containing membrane particles were preincubated 2 min. Then appropiate concentrations of agonists and/or antagonists were added (unless otherwise specified) and the incubation was continued for 10 min at 30°C. After incubation, the samples were placed in boiling water bath for 3 min, and cAMP was isolated by chromatography on Dowex 50W-X4 followed by neutral alumina [33]. cAMP levels were then measured by radioimmunoassay using the Amersham Radiochemical Centre (U.K.) kit. Protein was measured by the procedure by Lowry e t al. [34] against a standard of bovine serum albumin. In a series of preliminary experiments the DA-sensitive cAMP generation was assayed in samples of the pellets discarded after homogenization. However, in this preparation DA or dopaminergic agonists and antagonists did not cause any change in cAMP levels (data not shown). Statistical significance was assessed by analysis of variance (ANOVA) followed by the Duncan's test. The chemicals used to prepare reagents were purchased from Sigma Chemical Co. (U.S.A.) unless otherwise specified. Fenoldopam and SCH 23390 were obtained from SK&F Laboratories (U.S.A.) and Schering Plough (U.S.A.), respectively. Quinpirole and bromocriptine were products of Lilly Research Labs. (U.S.A.) and Sandoz (Switzerland), respectively. Isomers of sulpiride were obtained from Ravizza (Italy). RESULTS Increasing concentrations of DA stimulated the production of cAMP by frontal cortex membrane particles (Fig. 1). Maximal activation was achieved with about 100 /aM DA. There was a linear increase of cAMP during the first 5 min incubation period in the presence of 100/aM DA and no significant changes in cAMP levels were observed during an additional 10 min of incubation (data not shown). The other D-1 agonists apomorphine and fenoldopam also increased the production of cAMP (Fig. 1). Apomorphine and fenoldopam had a lower potency than DA (Fig. 1). The stimulatory effects elicited by DA and dopaminergic agonists on cAMP generation significantly declined (P < 0.01) in adult rats (Fig. I). No further decline was noticeable in old rats (Fig. 1). The calculated ECs0 values for dopamine, apomorphine and fenoldopam were 1.9/aM, 3.2/aM and 0.9/aM in young rats, respectively; 4.6/aM (P < 0.01 vs. young), 4.7/aM ( P < 0.01 vs. young) and 3.0/aM ( P < 0.001 v s . young) in adult rats; 4.5/aM ( P < 0.01 vs. young; not significant v s . adult), 4.8 laM ( P < 0.01 vs. young; not significant vs. adult), 3.1/aM ( P < 0.001 v s . young; not significant v s . adult). When membrane particles were incubated with DA or dopaminergic agonists in the presence of the D-1 receptor antagonist SCH 23390, a significant blockade of cAMP production was observed as well as a decrease of basal activity (Table I). The

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Fig. 1. Accumulation of c A M P by m e m b r a n e particles of frontal cortex of y o u n g (n = 8, panel A), adult (n = 10, panel B) and old (n = 10, panel C) rats. Values are expressed as percentage of increase over basal activity, induced by increasing concentrations of D A ( • ), a p o m o r p h i n e (11) or fenoldopam (A). M e m b r a n e particles were preincubated for 2 min and then agonists added and the incubation was continued for 10 min. For this figure and the following figures data are presented as the mean _+ S.E.M. of 3 independent determinations per animal. For basal activity values of each age group see Table I. *P < 0.01 v s . young (starting from this concentration).

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67 TABLE I EFFECT OF FORSKOLIN ON cAMP GENERATING SYSTEM IN THE FRONTAL CORTEX OF YOUNG, ADULT AND OLD RATS Drug

None (basal activity) Forskolin(10) DA (100) Forskolin (10) ± SCH 23390 (10) DA(100) + SCH23390 DA (100) + S-sulpiride (54)

c A M P ( p m o l / m g protein/lO min ± S.E.M.)

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When present, forskolin SCH 23390 or S-sulpiride were preincubated with membrane particles of rat frontal cortex for 2 rain. DA was then added after preincubation and the reaction continued for a further 10 rain. The reaction was stopped by placing the samples in a boiling water bath for 3 rain. Data are presented as the mean of three independent determinations per animal. •P < 0.001 when compared with basal activity. bp< 0.01 when compared with young. cp< 0.001 when compared with DA alone. dp< 0.001 when compared with young.

loss in c A M P c o n c e n t r a t i o n s i n d u c e d b y d o p a m i n e r g i c a g o n i s t s plus S C H 23390 was significantly higher in a d u l t ( P < 0.001) a n d o l d ( P < 0.001) t h a n in y o u n g rats ( T a b l e I). N o significant differences in this i n h i b i t o r y effect were o b s e r v e d b e t w e e n a d u l t a n d o l d rats ( T a b l e I). T h e ICs0 value f o r S C H 23390 in inhibiting D A - s e n s i t i v e c A M P a c c u m u l a t i o n to b a s a l levels was a b o u t 0.1/~M in the three age g r o u p s ( d a t a n o t shown). T h e selective D-2 agonists q u i n p i r o l e a n d b r o m o c r i p t i n e i n d u c e d a significant decrease o f c A M P p r o d u c t i o n even in the a b s e n c e o f S C H 23390 (Fig. 2). T h e inhibit o r y effects o f q u i n p i r o l e a n d b r o m o c r i p t i n e o n c A M P g e n e r a t i n g system were sign i f i c a n t l y higher in a d u l t ( P < 0.01) a n d o l d ( P < 0.01) t h a n in y o u n g rats (Fig. 2). N o changes were n o t i c e a b l e b e t w e e n a d u l t a n d o l d a n i m a l s (Fig. 2). T h e ICs0 values for b r o m o c r i p t i n e a n d q u i n p i r o l e were 1.8/~M a n d 6.4/~M, respectively in y o u n g rats; 1.0 ~ ( P < 0.01 v s . y o u n g ) a n d 5.6/~M ( P < 0.05 v s . y o u n g ) , respectively in a d u l t rats; 1.1/~M ( P < 0.01 v s . y o u n g ; n o t significant v s . a d u l t ) a n d 5.5/~M ( P < 0.05 v s . y o u n g ; n o t significant v s . adult). T h e i n h i b i t i o n o f c A M P p r o d u c t i o n seen with D A + S C H 23390 o r with D-2 agonists was a n t a g o n i z e d b y i n c u b a t i n g f r o n t a l c o r t e x m e m b r a n e particles with S-sulpiride, b u t n o t with R - s u l p i r i d e (Fig. 3). S- o r

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Fig. 2. Incubation of basal c A M P accumulation by quinpirole ( • ) or bromocriptine (/x). Membrane particles of frontal cortex of young (n = 8, panel A), adult (n = 10, panel B) and old (n = 10, panel C) rats were preincubated for 2 min and then the D-2 agonist added and the incubation continued for 10 min. For basal activity of each age group, see Table 1. The values were significantly different from basal activity (P < 0.001) starting from quinpirole concentration of 1/aM and from bromocriptine concentration of 0.1/aM in the three age groups. *P < 0.01 v s . young (starting from this concentration).

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Fig. 3. Effect of R- or S-sulpiride on the DA-induced inhibition o f c A M P accumulation in the m e m b r a n e particles o f frontal cortex of y o u n g (n = 8, panel A), adult (n = 10, panel B) and old (n = 10, panel C) rats. S-sulpiride ( o ) was more active than R-sulpiride ( 1 ) i n reversing the D A + SCH 23390 inhibition o f c A M P accumulation. R- (D) or S-sulpiride (O) alone had no action of their own. O n the left top are shown c A M P levels in the presence o f D A (100/~M); on the left bottom are shown basal c A M P levels. Membrane particles were incubated with SCH 23390 (10/aM) for 2 m i n before adding DA and sulpiride. The incubation was then continued for an additional l0 min.

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70 R-sulpiride alone had no effect on cAMP concentrations in the three age groups (Fig. 3). The incubation o f membrane particles o f frontal cortex with DA in the presence of the D-2 receptor antagonist S-sulpiride caused a higher increase of cAMP levels than DA alone in the three age groups (Table I). However, this effect was not statistically significant (Table I). Basal cAMP levels and cyclic nucleotide concentrations elicited by forskolin were not significantly different in the three age groups investigated (Table I). DISCUSSION For many years, the dopaminergic innervation o f the cerebral cortex has been rather neglected. There is now striking evidence based on histofluorescence observations, biochemical analysis of the distribution of DA or [3H]DA uptake activity, DA-sensitive adenylate cyclase and electrophysiological studies suggesting that dopaminergic projections supply the cerebral cortex (for a review see Ref. 35). Dopaminergic fibres to the frontal cortex originate primarily from mesencephalic neurons (A9 and A10 groups) [36]. The rat frontal cortex represents also the terminal area of neurons originating in the thalamic mediodorsal nucleus and receives fibres from the amygdala [37,38]. Moreover, it has been suggested that the mesocortical frontal dopaminergic system is involved in the regulation of emotional states and of complex behaviours [35]. More recent studies attempted to characterize the DA receptor subtypes present demonstrated the existence of both D-l and D-2 receptors in the frontal cortex linked with the activation and the inhibition of adenylate cyclase activity respectively [26--30]. The present study was designed to analyze whether the frontal cortex DA receptor system linked to the stimulation or inhibition of the cAMP generating system undergoes age-dependent changes. Our findings suggest that similarly to studies reported for rabbit frontal cortex DA-sensitive adenylate cyclase [16], for rat striatal DAdependent adenylate cyclase [11,12] and for D-1 receptors characterized using binding techniques [ 19--21 ], rat frontal cortex DA-sensitive cAMP generation is reduced with age. The loss of the post-receptor activation of cAMP generation, likely corresponding to D-1 receptors [26,30,39], occurs between the young age and the maturity. No further changes were noticeable in senescence similarly as described for DAsensitive striatal adenylate cyclase [40]. The same is true for DA-sensitive cAMP inhibition, likely corresponding to D-2 receptors [30,41,42], that is increased between 3 and 12 months of age. Forskolin, an activator of the catalytic unit of the adenylate cyclase complex amplifying receptor-mediated responses [43], increased to the same extent cAMP generating system in the three age groups examined. These findings suggest that frontal cortex DA receptors rather than the catalytic unit of the adenylate cyclase undergo age-dependent changes. In contrast to the D-2 striatal receptors which decreased with age [44--48], our

71

findings suggest an age-related increase in the coupling between the D-2 receptor and cAMP generation starting from 12 months of age. Supporting this hypothesis are the findings of lower IC50 values for bromocriptine and quinpirole in the frontal cortex of adult and old rats in comparison with young animals (see Fig. 2). Whether this enhanced D-2 mediated inhibition of cAMP generation in the frontal cortex of adult or old animals, in comparison with young ones, may be related to a compensation for the reduction in D-1 receptor responses or represents the expression of other functional phenomena cannot be established on the basis of the above data. However, the functional consequences of these biochemical changes may have important implications in the aging of the rat frontal cortex. ACKNOWLEDGEMENTS

The present study was supported by a grant of the University "La Sapienza" (Progetti di Facolt/0. The authors are greatly indebted with Sigma Tau Industrie Farmaceutiche Riunite S.p.A. (Pomezia, Italy) for the gift of experimental animals. REFERENCES 1 2 3 4 5 6

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