Expression of A1 Adenosine Receptors Modulating Dopamine ...

NCIIIYI( h n i l ~ f n Raven P re s, New York C‘ 1985 International Society for Neurochemistry

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Expression of A, Adenosine Receptors Modulating Dopamine-Dependent Cyclic AMP Accumulation in the Chick Embryo Retina R. Paes de Carvalho and F. G. de Mello Laboratdrio de Neuroqrtirnica, lnstituto de Biofisica, Cidade Universifciria. Rio de Juneiro, Brusil

Abstract: Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-oldchick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopaminedependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-dayold chick embryo retinas, with an IC,, of approximately I k M . This effect was not blocked by dipyridamole. N6(I-Phenylisopropy1)adenosine.(1-PIA) was the most potent adenosine analog tested, showing an IC,, of 0. l p M which was two orders of magnitude lower than its stereoisomer d-PIA (10 FM).The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-lmethylxanthine (IBMX) or by adenosine deaminase. Ad-

enine was not effective, whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5day-old posthatched chickens doparnine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP. In this case, 2-chloroadenosine did not interfere with the response elicited by either one of the compounds. The data suggest the existence of two populations of D, dopamine receptors in the chick embryo retina-one that is negatively modulated by adenosine and another that is not under purinergic control. Key Words: Adenosine receptors-Doparnine-Cyclic AMP-Chick retina-Development. Paes de Carvalho R. and de Mello F. G. Expression of A, adenosine receptors modulating dopamine-dependent cyclic AMP accumulation in the chick embryo retina. J . Neurochetn. 44,845-851 (1985).

Adenosine is a putative neurotransmitter o r neuromodulator in the C N S . This purine has potent electrophysiological effects, blocking postsynaptic potentials a n d d e p r e s s i n g n e u r o n a l firing (Dunwiddie and Hoffer, 1980; Lee e t al., 1983). It is postulated that these effects are mediated thruugh t h e inhibition of transmitter release by the purine (Sawynok and Jhamandas, 1976; Michaelis e t al., 1979; Motley and Collins, 1983). Specific receptor binding sites for adenosine were detected in several areas of the C N S (Bruns e t al., 1980; Schwabe and Trost.

1980; L e w i s et al., 1981; Barnes and Thampy. 1982; Marangos e t al., 1982). Moreover, adenosine regulates adenylate cyclase activity by interacting with specific extracellular receptor sites, whose activation d e p e n d s on the integrity of the ribose moiety of the nucleoside (R site). This effect is blocked by m e t h y l x a n t h i n e s ( F a i n a n d Malbon, 1979; Van Calker e t al., 1979; L o n d o s e t al., 1980). An intracellular site of adenosine interaction was also described (P site). T h e activation of these sites by adenosine leads t o the inhibition of adenylate cy-

Received June 1 I . 1984: accepted August 29. 1984. Address correspondence and reprint requests t o F. G. d e Mello, Laboratorio de Neuroquimica, lnstituto de Biofisica. U.F.R.J., C.C.S., Bloco G, Cidade Universitaria. llhado Fundao 21941, Rio d e Janeiro, Brasil. The present address of R. F’aes de Carvalho is Departamento de Neurobiologia, lnstituto de Biologia, U.F.F.. Morro S. Joao Batista sino.. CP 229, Niteroi. 24000, Rio de Janeiro. Brasil.

Abbrei.in?ions used; B M E . Basal medium of Eagle: CMF. C a 2 + , Mg2--Free medium: HEPES. N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid: IBMX. 3-lsobutyl- I-methylxanthine: NECA. 5’-N-Ethylcarboxamideadenosine: Nh-MA. N6Methyladenosine: PIA. N“-Phenylisopropyladenosine:R0201724. 4-(3-Butoxy-4-methoxybenzyl-2-imidorolidinone~: TCA. Trichloroacetic acid.

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clase activity. The efficacy of adenosine to promote its effect through intracellular receptor sites depends o n the integrity of the purine moiety of the nucleoside and is blocked by dipyridamole, a potent inhibitor of adenosine uptake into the cells (Van Calker et al.. 1979). Methylxanthines d o not compete with adenosine for t h e s e sites (Fain and Malbon. 1979). At least t w o classes of adenosine receptors with R-site characteristics have been proposed. They have been designated A , and A? receptors, whose activation by specific agonists leads to the inhibition o r stimulation respectively, of adenylate cyclase (Van Calker et al., 1979). Previous work from our laboratory showed that adenosine promotes the accumulation of cyclic AMP in the chick retina. This effect varies substantially during the ontogeny of the tissue. appearing by day 14 of embryonic development and attaining maximal sensitivity by embryonic day 17. Retinas obtained from chicken of the posthatch period are characterized by a reduced responsiveness to adenosine (Paes d e Carvalho and de Mello, 1982). Dopamine also promotes the accumulation of cyclic A M P in the chick retina. This effect is expressed early in the embryonic life of the animal and is present in retinas from 7-day-old embryos. Maximal tissue responsiveness to dopamine is observed after embryonic day 8 (de Mello. 1978). Also a reduced sensitivity to dopamine was detected in retinas from posthatched chickens (de Mello and d e Mello, 1980). The present report shows that adenosine and related analogs inhibit, in a dose-dependent fashion, the accumulation of cyclic A M P elicited by dopamine in the chick embryo retina. This effect is mediated by A, receptors which are already detected in retinas from 10-day-old embryos. MATERIALS AND METHODS Materials Cyclic AMP, AMP, ATP, adenine, protein kinase from bovine heart, bovine serum albumin, 6-amino-2-chloropurine riboside (2-chloroadenosine), A@-methyladenosine (@-MA), adenosine deaminase (EC 3.5.4.4) type I1 (Sigma Chemical Co., St. Louis, MO); pargyline (Regis Chem. Co., Chicago, IL); basal medium of Eagle (BME) (GIBCO Grand Island, NY); 3-isobutyl-I-methylxanthine (IBMX) (Aldrich Chem. Co., Milwaukee, WI); dopamine, adenosine (Calbiochem., Los Angeles, CA): 4-(3-butoxy4-methoxybenzyl-2-imidozolidinone) (R020-1724) (Hoffmann LaRoche Inc., Nutley, NJ); dipyridamole (Persantin) (Boehringer Ingelhein, S . Paulo, Brasil); and [3H]cyclic AMP (New England Nuclear Inc., Boston, MA) were used throughout this study. The d and 1 stereoisomers of A@-phenylisopropyladenosine (PIA) were kindly supplied to us by Dr. John Daly (National Institutes of Health). 5'-N-ethylcarboxamideadenosine (NECA) was a generous gift from Dr. J.

J . Neurorhem., Vol. 44.N o . 3, 1985

Wolff (National Institutes of Health). All other reagents were of analytical grade. Fertilized white Leghorn eggs were obtained from a local hatchery. Methods Rerim dissection. The embryos were staged according to Hamburger and Hamilton (1951)and retina dissections performed according to a previously published procedure (Piddington and Moscona, 1965).The eyes were removed and transferred to cold Ca2+,Mg'+-free medium (CMF), and the retinal tissue was dissected free of the pigmented epithelium. After a brief (30 s) centrifugation at 400 fi'. the tissue was resuspended in 2 ml of BME at 37°C containing 25 mM HEPES (pH 7.4) (adjusted with NaOH). 0.5 mM RO20-1724 or IBMX, 0. I mM pargyline, and 0. I mM sodium ascorbate. Incrrhariorz cind ussay procedures. All incubations followed the procedure described previously (de Mello. 1978). After transferring the dissected retinas to BME containing the compounds listed above. the tissues were incubated at 37°C for 10 min; then each test compound was added to the final concentration required. The retinas were further incubated at 37°C for 10 min and the reactions were interrupted by the addition of trichloroacetic acid (TCA) to a 5% final concentration. The cyclic AMP was purified according to Matsuzawa and Nirenberg (1975) and assayed by the method of Gilman (1970). Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.

RESULTS The cyclic AMP levels of retinas from 16-day-old chick embryos incubated in the presence of 0.5 m M R020-1724 plus 0. I unit/ml of adenosine deaminase increased from 40 to 144 and 306 p m o l h g protein when the tissue was exposed for 10 min either to 100 p.M 2-choloroadenosine o r 100 p.M dopamine, respectively (Table 1). When both compounds were added together t o the incubation medium, the retina cyclic AMP content increased from 40 to approximately 250 pmol/mg protein, showing that the activation of the two adenylate cyclase systems were not additive with respect to cyclic AMP accumulation (Table 1). One possible explanation for these results is that the adenosine- and dopamine-dependent adenylate cyclase systems present in the tissue were located on the same cell type and that the addition of 2-chloroadenosine o r dopamine, in saturating concentrations, would stimulate maximally the same pool of enzyme molecules present in the cell membrane. This is an unlikely possibility, since the simultaneous addition of lower concentrations of 2-chloroadenosine and dopamine still were not additive with respect t o cyclic AMP accumulation (Table 1). Alternatively one could imagine the existence of an adenosine receptor in the retina whose activation would lead to the inhibition of the dopamine-promoted cyclic AMP accumulation of the tissue. To test this hypothesis, retinas from 12-day-

A , ADENOSINE RECEPTORS IN THE CHICK RETINA TABLE 1. Effect of dopamine and 2-chloroadenosine on the cyclic AMP level of retinas from 16-day-old chick embryos Additions to the incubation medium

Concentration used (pM)

pmol cyclic AMP/mg protein f SEM

None 2-Chloroadenosine 2-Chloroadenosine Dopamine Dopamine 2-Chloroadenosine + Dopamine 2-Chloroadenosine + Dopamine

-

40.1 2 11.5 129.7 2 1.1

~~

5 100

I 100 5 1 100

I00

144.0 f 12.2 121.8 2 16.2 306.7 5 21.2

121.1

2

5.9

250.2

2

6.6

Retinas were preincubated for 10 min in BME containing 25

mM HEPES (pH 7.4) (adjusted with NaOH), 0.1 mM pargyline, 0.1 mM sodium ascorbate, 0.5 mM R020-1724. and 0.1 unit/ml of adenosine deaminase. Dopamine and 2-chloroadenosine were added at the concentrations indicated and the retinas were further incubated for 10 min at 37°C. The reaction was stopped by TCA to 5% final concentration. The data represent the average of two experiments 2 the deviation of the individual values from the values shown.

old chick embryos, which do not respond to adenosine (Paes de Carvalho and de Mello, 1982; and Fig. 1 legend) and 2-chloroadenosine (see Table 5) in terms of shifting its cyclic AMP content, were preincubated with 0.5 mM R020-1724 for 10 min at 37°C. Then 100 pM dopamine and various concentrations of adenosine were added and the retinas were further incubated for another 10 min. The reaction was interrupted by the addition of TCA

idamole. At the end of this period 100 pM dopamine and adenosine at the concentra-

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(5% final concentration). The result of this experiment is shown in Fig. 1. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine, with an IC,, of approximately 1 p M (circles). Dipyridamole, a potent inhibitor of adenosine uptake into cells (Van Calker et al., 1979), did not interfere with the inhibition (triangles), indicating that the adenosine effect was mediated via membrane receptors oriented towards the extracellular environment. Among the adenosine analogs tested 1-PIA was the most potent compound to exert the inhibitory effect, with a n IC,, of approximately 0.1 pM. The stereoisomer d-PIA was much less effective than IPIA, showing a n IC,, two orders of magnitude lower than its active isomer (Fig. 2). Other adenosine analogs had intermediate potencies. Their inhibition constants (IC,,) are listed in Table 2. The resulting rank order of potency I-PIA > 2-chloroadenosine > adenosine > N6-methyladenosine > dPIA = NECA is consistent with the order of potency for the activation of A, receptors in other systems (Van Calker et al., 1979; Londos et al., 1980). For comparison we show the EC,, values for cyclic AMP accumulation induced by adenosine analogs in retinas from 16- to 17-day-old chick embryos. The rank order of potency, NECA = 2-chloroadenosine > I-PlA > d-PIA, strengthens the idea that these effects are mediated by A, receptors. The specificity with which these receptors respond to the different purine compounds can be analysed in Table 3, which shows the effect of several nucleosides and nucleotides on the dopamine-dependent cyclic AMP accumulation of retinas from 10- to 12-day-old embryos, incubated in the presence of 0.5 m M R020-1724 plus 0.5 unit/ml adenosine deaminase. Under these conditions only 2-chloroadenosine significantly inhibited the dopamine-dependent cyclic AMP accumulation. Adenine was completely ineffective, whereas ATP and AMP promoted a partial inhibition only at con-

FIG. 2. Effect of increasing concentrations of I-PIA and dPIA upon the dopamine-elicited cyclic AMP accumulation in retinas from 11-day-old chick embryos. The procedure was the same as that described in the legend to Fig. 1 except that the retinas were also incubated in the presence of 0.2 unit'rnl of adenosine deaminase. The data represent t h e mean 2 SEM from three to four experiments. The cyclic AMP levels were: basal. 14.7 f 2.8 Dmollma Drotein (n = 5); dopamine-stimulated, 222.5 t 20.7 prnol/mg protein (n = 8).

-.

i

1 E

2

I

-Log PIA (M)

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TABLE 2. Rank order of potency ojdi‘rrrnt pttrinrrgic rigonisis itpun itrhihitiori and stimtclurion ctf’ udmylate tyke systems of the chick embryo retinti

Compound I-PIA 2-Chloroadenosine Adenosine N,-MA d-PIA NECA

Inhibition (10- to 12-day old) IC,, crM)

Stimulation (16- to 17-day old) ECq, ( F A # )

0.1

0.3 I .o 5.0 10.0 10.0

The lCsovalues were estimated on the basis of dose-effect curves for each compound. All the incubation procedures were described in the legend to Table I . The range of concentrations used for each compound vaned from to lo-’ M . The EC,,, values were also estimated using dose-effect curves obtained for each compound in the range of IO-’to lo-’ M concentrations.

centrations as high as 100 p M (not shown). The effects of these nucleotides are not due to their conversion to adenosine, since the retinas were incubated in the presence of adenosine deaminase. Further, the presence of this enzyme in the incubation medium abolished the effect of 100 p,M adenosine. These data further suggest that products originating from adenosine metabolism are also ineffective upon the A, receptors. IBMX, at the concentration of 0.5 mM. completely blocked the 2-chloroadenosine-elicitedinhibition of dopamine-dependent cyclic AMP accumulation in the retina (Table 4). In addition. the inhibitory effect of purinergic agonist upon the dopamine-elicited cyclic AMP accumulation was not reversed by increasing dopamine concentration up to 1 or 10 mM, respectively (Table 4). The maximal inhibition by adenosine upon the dopamine-promoted cyclic AMP accumulation was 70%; 30% of the dopamine-elicited increase i n cyclic AMP level was insensitive to the nucleoside treatment at the highest concentration of adenosine used (Fig. I). All other adenosine analogs tested TABLE 3. Effect of nucleosides und nrrclroiides upon dopumine-dependent cyclic A M P incv-euse in retincis from 10- to 12-day-old chick embryos Additions to the incubation medium

Concentration used (pM)

pmol cyclic AMPlmg protein -c SEM

Control 2-Chloroadenosine Adenosine Adenine AMP ATP

-

276.3 f 22.2 (6) 83.3 2 5.8 (4) 304.4 f 51.1 (2) 378.5 2 37.0 (2) 297.2 f 89.3 (21 226.8 f 7.8 (4)

10 100

100 10

10

The assay procedure was the same as that described in the legend to Table I except that the adenosine deaminase concentration was 0.5 unit/ml. The numbers in parentheses represent the number of experiments performed in each case.

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None Dopamine Dopamine + I-chloroddenome Dopamine + 2-chloroadenosine + IBMX Dopamine + 2-chloroadeno\ine Dopamine + 2-chloroadenosine

The assay procedure was the same as that decribed in the legend to Table I . IBMX was present in the medium during the preincubation time and thereafter. The numbers in parenthe\e\ represent the number of experiment5 performed in each case. The basal cyclic AMP level shown ic the result of an individual experiment.

also showed the same pattern of 70% maximal inhibition at high concentrations (Fig. 2 and Table 4). Moreover, preincubation of the tissue with 100 p M 2-chloroadenosine for 10 min before the addition of dopamine, or the incubation of the retinas in the presence of dopamine and 2-chloroadenosine simultaneously, for more than 10 min. did not increase the inhibitory effect of the purinergic agonist above the 70% inhibition observed with the normal procedure (data not shown). These facts suggested the existence of two populations of D, dopamine receptors modulating adenylate cyclaw activity in the chick embryo retina: One whose activation is negatively modulated by adenosine agonists and another that is not subjected to purinergic control. Previous work from our laboratory rcvcaled that apomorphine, a dopamine agonist. also increnwd the cyclic AMP content of the chick embryo retina via a subclass of D, receptors whose sensitivity t o apomorphine was practically constant throughout the embryonic and postcmbryonic periods. This compound was only 20-30% as effective as dopamine in activating the cyclasc system of the embryonic retina in the early stages of development ( 10to 12-day-old embryos). In addition, it was shown that the highly dopamine-sensitive D, system of the embryonic tissue constituted another subclass of D, receptors that was only present in the embryonic retina and was considerably reduced in the retina of posthatched animals. At this stage. both apomorphine and dopamine were equally effective in eliciting the cyclic AMP accumulation of the retinal tissue (Ventura et al.. 1984). Table 5 shows the results of experiments that were conducted to test the hypothesis that only the

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A , ADENOSINE RECEPTORS IN THE CHICK RETINA TABLE 5 . Effect of 2-chloroadenosine upon dopamine and apomorphine-promoted cyclic AMP accumulation in retinas from 11-day-old embryos and 5-day-old posthatched animals Additions to the incubation medium

1 I-Day-old embryos"

5-Da y-old post-hatched chicken" -~

~

Basal 14.5 5 0.6 (3) 19.3 2 0.09 (3) 2-Chloroadenosine Dopamine 254.8 5 20.1 (8) Dopamine + 2-chloroadenosine 83.3 2 5.8 (4) Apomorphine 51.5 2 8.6 (3) Apomorphine + 2-chloroadenosine 41.5 2 5.1 (4)

30.6 t 1.9 (6) 48.8 ? 2.9 (9) 49.0 t 2.4 (8) 91.1 t 5 . 5 (9) 49.0 t 1.7 (8) 67.7

? ~~

4.7 (8) ~

~

The assay procedure was the same as that described in the legend to Table I except that the adenosine deaminase concentration was 0.5 unithl. The numbers in parentheses indicate the number of experiments performed in each case. The concentrations of 2-chloroadenosine, dopamine, and apomorphine were 100 p M . The differences between the basal cyclic AMP level and the dopamine-, apomorphine-, and 2-chloroadenosine-stimulated levels of 5-day-old posthatched chicken are significant at p < 0.0s. '' pmol cyclic AMPimg protein -+ SEM.

embryonic D, subclass of dopamine receptors in the retina was under the control of purinergic agonists. As shown before, 100 p M dopamine increased the cyclic AMP content of 1 1-day-old chick embryo retinas by a factor of 18-fold. Apomorphine stimulated only 3.6-fold the cyclic nucleotide level of retinas obtained from embryos at the same developmental stage. As opposed to what is observed with respect to the effect of purinergic agonists upon the dopamine-promoted cyclic AMP accumulation, 2chloroadenosine did not affect significantly the increase in the cyclic nucleotide content elicited by apomorphine. Moreover, the stimulation of 5-dayold posthatched chicken retinas by dopamine or 2chloroadenosine (100 ~ h 4 )increased the cyclic AMP content of the tissue from 30 to approximately 49 and 48 pmol/mg protein, respectively. When the two compounds were added together to the incubation medium the retina cyclic AMP content increased from 30 to 91 pmolimg protein. Therefore, dopamine and 2-chloroadenosine added together onto retinas from posthatched chicken caused an effect upon cyclic AMP content that was approximately additive. In addition, the effect of saturating concentrations of apomorphine and 2-chloradenosine upon retinas from 5-day-old chicken were also additive with respect to cyclic AMP accumulation. Table 5 also confirms our previous data (Ventura et al., 1984) which show that the apomorphine effect upon the cyclic AMP content of retinas from 11day-old embryos and 5-day-old chicken is approximately the same. This is not the case when retinas from both developmental stages are stimulated by dopamine.

DISCUSSION The data presented in this work together with previous reports from our laboratory (de Mello, 1978; Paes d e Carvalho and de Mello, 1982; Ventura et al., 1984) reveal an interesting complexity of the mechanisms that regulate the adenylate cyclase activity of the embryonic chick retina (see summary of events in Table 6). Although dopamine and 2chloroadenosine independently promote the accumulation of cyclic AMP in retinas from 16-day-old chick embryos (Table l), in retinas obtained from 12-day-old embryos only dopamine is capable of increasing the cyclic AMP content of the tissue (Paes de Carvalho and de Mello, 1982). However purinergic agonists interfere with the dopamine-elicited cyclic AMP accumulation of retinas from the early embryonic stages, promoting a 70% inhibition of the dopamine-dependent increase of cyclic AMP levels; 30% of the cyclic AMP increase elicited by dopamine is insensitive to purinergic agonists. Recently our laboratory proposed the existence of two subtypes of D, receptor systems in the chick retina (Ventura et al., 1984). One is highly sensitive to dopamine or ADTN and insensitive to apomorphine, as measured by changes in the cyclic AMP content of the tissue. This cyclase system of the embryonic tissue declines to almost undetectable levels in retinas obtained from posthatched animals. On the other hand, the other subtype of D, receptor is responsive to both dopamine and apomorphine. This system is expressed in the retina throughout the embryo development and does not disappear as the tissue differentiates towards the posthatched stage. T h e n , apomorphine and dopamine are equally effective in activating this D, receptor-cyclase system. In the early stages of differentiation of the embryonic tissue the apomorphine-responsive system accounts for only 30% of the total do-

TABLE 6. Summary of the changes displayed by dopamine and adenosine receptors in the developing chick retina Levels of dopamine and adenosine receptors in the retinal tissue Developmental stage

D,

DIE

D:

A,

A:

Embryos, Not 9- to 12 day-old Low High Low High detected Embryos, 16- to 18 day-old Low Decreasing Increasing High High Posthatched chicken Not Not 5-day-old Low detected High detected Low The levels of responsiveness of D, (de Mello, 1978). A , (this report). and A, (F'aes de Carvalho and de Mello. 1982) receptors were assessed by measuring the effects of specific dopaminergic and purinergic agonista upon the cyclic AMP levels of the retina. The D, receptor was stimulated by ['H]spiroperidol binding studies (Ventura et al.. 1984). D,E refers to the embryonic subclass of D, receptors (Ventura et al., 1984: and thia report).

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pamine-dependent cyclic AMP accumulation of the tissue. Our data suggest that the dopaminergic D, receptor population present in the embryonic tissue, which is not under purinergic control. is the same as that which is stimulated by apomorphine. In fact, this concept is supported by the finding that 2-chloroadenosine does not interfere with t h e apomorphine-promoted increase of the cyclic AMP level of the retina from the early embryonic stages (Table 5 ) . Moreover, retinas obtained from posthatched animals, which are characterized by the presence of only the D, system that is equally sensitive to dopamine and apomorphine, is not subjected to the inhibitory response caused by 2-chloroadenosine. Instead, when dopamine or apomorphine are added together with 2-chloroadenosine onto the mature tissue, the level of cyclic AMP attained at the end of 10 min of stimulation is approximately the sum of the levels observed with either dopamine, apomorphine or 2-chloroadenosine alone (Table 5). In this case the cyclase stimulation by 2-chloroadenosine is probably mediated by A, receptors (Paes de Carvalho and de Mello, 1982). In the embryonic tissue, as expected, the inhibitory effect of purinergic agonists upon dopaminedependent cyclic AMP accumulation cannot be reversed by increasing the concentrations of dopamine. This fact indicates that the negative modulation of the dopamine-activated cyclase system promoted by the purine is not via changes in the affinity of dopamine receptor for its ligand. It rather suggests the occurrence of changes in the coupling mechanism between dopamine receptor and adenylate cyclase molecules. caused by the activation of an independent class of adenosine receptors of the A, type. The inhibition of dopamine-dependent cyclic AMP accumulation by adenosine and related compounds is already present in retinas from 10-dayold chick embryos while the adenosine-elicited cyclic AMP accumulation, mediated by A2 receptors, appears only after embryonic day 14 (Paes de Carvalho and de Mello, 1982). Therefore A , receptors are expressed before A, receptors in the developing retina, and both classes of receptors transiently coexist in the tissue after day 14 of development. After hatching, however, we can detect only the response mediated by A, receptors. Whether the A, or the embryonic D, receptors or both disappear as the tissue differentiates remains to be determined. The transitory expression of an adenylate cyclase system subjected to such complex control mediated by dopamine and adenosine receptors raises intriguing questions, as to what would be their functional role. The complexity of this cyclase system is not only restricted to the modulatory role exerted by doparnine and adenosine upon its activity. In 1.Neurorhem., Yo/.44,No. 3, 1985

fact, the cyclase system whose activity is modulated by the embryonic subtype of D, receptor is not evenly distributed in the tissue. It is topographically organized. so that the tissue responsiveness to dopamine is predominantly observed in the dorsoposterior region of the embryonic retina (dc Mello and de Mello. 1984). One can speculate t h a t the fine control of the cyclase activity by purinergic and dopaminergic receptors of a certain type of cell may be important to modulate embryological functions occurring in a precise region of the tissue within a specific time of development. Evidence that hormones o r neurotransmitters that affect cyclic AMP levels in the retina or other neuronal cells may serve as signals to control the differentiation of specific synaptic functions has recently been proposed (Nirenberg et al., 1983: Puro, 1983: Yeh et al., 1983). Whether these mechanisms constitute a general feature of the embryonic CNS remains to be investigated. Acknowledgments: This work was supported by grants awarded to F. G . de Mello by CNPq. FINEP, and CEPG (U.F.R.J.). R. Paes de Carvalho was the recipient of a fellowship from CNPq. We thank Miss Neila Soares and Miss Aurizete Gomes for technical assistance and D r s . M . C. F. de Mello. J. L. M. do Nascimento, and A . L. M. Ventura for helpful discussions.

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J . Nerirochem.. Vol. 44. N o . 3 . 1985