serum (Flow Laboratories, Rockville, MD) were used throughout the study. Fluphenazine HCl was a gift from. Squibb Ind., S. Paulo, Brasil. Butaclamol was kindly.
0022-3042/82/0102-O493/$02.75/0
Journal of Neurochemistry Raven Press, New York @ 1982 International Society for Neurochemistry
Adenosine-Elicited Accumulation of Adenosine 3’, 5‘-Cyclic Monophosphate in the Chick Embryo Retina R. Paes de Carvalho and F. G. de Mello Instituto de Biojlsicu du Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
Abstract: The cyclic AMP level of 17-day-old chick embryo retina increased from 20 to 33 I pmoVmg protein when the tissue was incubated for 20 min in the (RO 20-1724). presence of 4-(3-butoxy-4-methoxybenzyl-2-imidozolinone) The addition of 0.5 m~-3-isobutyl-I-methylxanthine (IBMX) or 0.5 unitsiml of adenosine deaminase (EC 3.5.4.4) to the medium reduced the increase of cyclic AMP content from 20 to 100 pmoVmg protein. Dipyridamole did not interfere with the rise of the retina cyclic AMP level observed with RO 20-1724. The EC,, of 6-amino-2-chloropurine riboside (2-chloroadenosine)-elicited accumulation of cylic AMP of retinas incubated in the presence of RO 20-1724 plus adenosine deaminase was approximately 1 W M . When retina incubation was carried out in the presence of 0.5 mM-IBMX, the 2-chloroadenosine dose-
response curve was shifted to the right two orders of magnitude. Maximal stimulation of the cyclic AMP level of 17-day-old chick embryo retina incubated in the presence of 0.5 mM-IBMX was observed at 1 mwadenosine concentration. This effect was not blocked by dopamine antagonists. Guanosine and adenine did not affect the retinal cyclic AMP level. AMP and ATP had a slight stimulatory effect. Adenosine response of embryonic retina increased sharply from the 14th to the 17th embryonic day. A similar, but not identical, adenosine effect was observed in cultured retina cells. Key Words: Adenosine-Cyclic AMP-Chick retina-Ontogeny-Culture. Paes de Carvalho R. and de Mello F. G. Adenosine-elicited accumulation of adenosine 3‘, 5’-cyclic monophosphate in the chick embryo retina. J . Neurochem. 38, 493-500 (1982). The original demonstration by Sattin and Rall (1970) that adenosine was a potent activator of cyclic AMP accumulation in brain slices suggested a possible role of this purine a s a neurotransmitter in the nervous system. Since then, a n increasing amount of information has appeared in the literature reinforcing this concept and confirming the existence of membrane purinergic receptors capable of modulating adenylate cylase (EC 4.6.1.1) activity in various CNS areas and in peripheral tissues (Huang and Daly, 1974; Burnstock, 1978; Fain and Malbon, 1979). The retina, a part of the CNS, is organized to receive and modulate partly the visual information. This is accomplished with relatively few cell types
(Stell, 1972). Many compounds that have been identified in the brain as putative neurotransmitters have also been found in the retinas of different species (Curtis and Johnston, 1974). The presence of GABA (Pasantes-Morales e t al., 1972; Lam, 1972a; Macaione, 1972; Kennedy and Voaden, 1974; Yates and Keen, 1976), acetylcholine (Lam, 1972b; Puro et al., 1977), and dopamine (Boycott et al., 1975), as well a s their respective receptors (Enna and Snyder, 1976; Vogel e t al., 1976; Vogel and Nirenberg, 1976; Vogel e t al., 1977; Sugiyama e t al., 1977; Redburn e t al., 1980; Makman et al., 1980), suggests that these compounds play a n important role in the neurotransmission of the retinal tissues. In addition, evidence shows that neuropep-
Received May 11, 1981;accepted August 1 1 , 1981. Address correspondence and reprint requests to F. G . de Mello, Instituto de Bioflsica, Centro de Cikncias da Salide, Cidade UniversitBria, Ilha do FundBo, Rio de Janeiro, RJ 21941, Brasil.
Abbreviations used: B M E , Basal medium of Eagle; 2Chloroadenosine, 6-Amino-2-chloropurine riboside; HEPES, N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid; IBMX, 3-Isobutyl-1-methylxanthine;RO 20 1724, 4-(3-Butoxy-4methoxybenzyl-2-imidozolidinone); TCA, Trichloroacetic acid.
493
R . PAES DE CARVALHO A N D F. G . D E MELLO
494
tides such as substance P (Winder and Patsalos, 1974; Karten and Brecha, 1980) and endorphins (Brecha et al., 1979) also are present in bovine and avian retina. In the last few years our attention has been focused on the embryonic differentiation of the chick retina, especially neurochemical aspects (de Mello et al., 1976; Puro et al., 1977; de Mello, 1978). In earlier communications we reported that dopamine-dependent accumulation of cyclic AMP in chick retina differentiates early in the embryonic life of the animal. Dopamine sensitivity was present in retinas from 7-day-old embryos, and b y the 8th embryonic d a y maximal c yc l a se re sponse t o dopamine was observed (de Mello, 1978). In this work we demonstrate that adenosine promotes the accumulation of cyclic AMP in the intact embryonic chick retina and in cultured retina cells. Our evidence suggests that this purine is released spontaneously in this tissue and that adenosine sensitivity of the chick retina varies dramatically during the ontogeny of the tissue. Further, we show that retina from the posthatch period is characterized by a reduced sensitivity of the tissue to adenosine.
MATERIALS AND METHODS Cyclic AMP, AMP, ATP, guanosine, adenine, protein kinase from bovine heart, bovine serum albumin, 6amino-2-chloropurine riboside (2-chloroadenosine), 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, (IBMX) Grand Island, NY); 3-isobutyl-I-methylxanthine (Aldrich Chem. Co., Milwaukee, WI); dopamine, adenosine (Calbiochem., Los Angeles, CA); 4-(3butoxy-4-methoxybenzyl-2-i~idozolidinone) (RO 20 1724) (Hoffman La Roche Inc., Nutley, NJ); dipyridamole (Persantin) (Boehringer Ingelheim, S. Paulo, Brasil); [3H]cyclic AMP (New England Nuclear Inc., Boston, MA); three times crystallized trypsin (Worthington Biochemical Co., Freehold, NJ); and fetal calf serum (Flow Laboratories, Rockville, MD) were used throughout the study. Fluphenazine HCl was a gift from Squibb Ind., S. Paulo, Brasil. Butaclamol was kindly supplied to u s by Dr. D. J. Marshall from Ayerst Laboratories, Montreal, Canada. All other reagents were of analytical grade. Fertilized white Leghorn eggs were obtained from a local hatchery. Retina dissections were performed according to a previously published procedure (Piddington and Moscona, 1965) and the embryos staged according to Hamburger and Hamilton (1951).
Incubation and assay procedures All incubations followed the procedure described previously (de Mello, 1978), except that the incubation time in the presence of each test compound was 10 min. Cyclic AMP was extracted with 5% trichloroacetic acid (TCA), purified according to Matsuzawa and Nirenberg (1979, and assayed by the method of Gilman (1970). Protein was determined by the method of Lowry et al. (1951).
J . Neurochem., Vol. 38. N o . 2 , 1982
Retina cell cultures Primary cultures of retina cells were prepared by a modification of a method described previously (Vogel et al., 1976). Usually, retinas from 9-day-old embryos were dissected under sterile conditions, transferred to 3.0 ml of CA+-, Mg2+-free medium containing 0.05% trypsin, and incubated in a water bath at 37°C for 12-13 min. The trypsinized tissue was then centrifuged at 500 x g for 30-60 s. The supernatant was discarded and the retinas were resuspended in BME (Earle’s salts) containing 5% fetal calf serum and 2 mwglutamine. The cells were dissociated mechanically by pipetting the tissue 10 times with a large-bore 5-ml pipette. The yield was approximately 70 x 1oG cellshetina with a viability >95%, estimated by trypan blue exclusion. The cells were plated in 35-mm plastic Petri dishes (approximately 35 x 106 cells/ dish) and transferred to an incubator with a humidified atmosphere of 95% air-5% COz. RESULTS The level of cyclic AMP in retinas isolated from 17-day-old embryonic chick and incubated for 20 min at 37°C in assay medium without phosphodiesterase inhibitor was 20 pmoVmg protein, which was approximately the same level observed in freshly dissected retinas at this stage of development (de Mello, 1978). When 0.5 mM of the phosphodiesterase inhibitor IBMX was present in the medium, the level of cyclic nucleotide increased to 100 pmol of cyclic AMP/mg protein. In contrast, when retinas were incubated for the same period in the presence of RO 20 1724, another potent phosphodiesterase inhibitor, the level of cyclic AMP of the tissue increased to 331 pmol of cyclic AMP/mg protein (Table 1). At least two hypotheses could account for this discrepancy. One possibility is that retina phosphodiesterases are differentially sensitive to these TABLE 1. Effrct of phosphodiesteruse inhibitors on the cyclic AMP level of retinas from 17-day-old chick embryo Treatment of retinas during incubation period None IBMX RO 20 1724 RO 20 1724 + IBMX RO 20 1724 + Adenosine deaminase RO 20 1724 + Dipyridamole
Concentration pmol cyclic AMPimg used (mM) protein ? S.E.M.
0.5 0.5 0.5 0.5 0.5 0.1
20 ? 2 (4) 100? 3 (17) 331 2 14 ( 5 ) 99 ? 2 ( 6 ) 60
-C
4 (5)
412
?
3 0 (4)
The retinas were incubated for 20 min at 37°C in BME containing 25 mM-HEPES, pH 7.4 (adjusted with NaOH), plus one of the compounds listed above. Adenosine deaminase concentration was 0.5 units/ml. The reaction was then interrupted by the addition of TCA (5% final concentration). Numbers in parentheses indicate the number of experiments performed in each case.
ADENOSINE-ELICITED CYCLIC AMP INCREASE IN RETINA
two inhibitors. The alternative is that IBMX can prevent an endogeneous stimulation of a cyclase system of the retina that is not blocked by RO 20 1724. To distinguish between these two possibilities, we incubated retinas in medium containing both IBMX and RO 20 1724 (0.5 mM each). Under this condition, the level of cyclic AMP observed after 20 min incubation was the same as that of retinas exposed to IBMX alone, i.e., 99 pmol cyclic AMP/mg protein. This result indicates that IBMX is inhibiting endogenous stimulation of cyclase activity. Since IBMX is a potent adenosine receptor antagonist (Van Calker et al., 1978; Smellie et al., 1979), the high level of cyclic AMP observed in the tissue in the presence of RO 20 1724 alone could be explained by an endogenous source of adenosine that could stimulate adenylate cyclase molecules present in the retinal tissue. This explanation is consistent with our observations that in retinas incubated in medium containing RO 20 1724 plus adenosine deaminase (to eliminate the endogenous source of adenosine) the cyclic AMP content was reduced to the level observed in the presence of IBMX. Further, the high retinal cyclic AMP content with RO 20 1724 could be reduced by IBMX in a dose-dependent manner. A reduction of 50% in the content of cyclic AMP was obtained with approximately 0.1 mM-IBMX (Fig. 1). These results strongly suggest the existence of an adenosinedependent cyclase system in embryonic retinal
!I&@-*-
i
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5 4 3 -LOG IBMX MOLARITY FIG. 1. Effect of increasing concentrations of IBMX on the level of cyclic AMP of 17-day-old chick embryo retinas incubated in the presence of 0.5 mM-RO 20 1724. The incubation was carried out in assay medium as described in the footnote to Table 1. Each point is the average of two experiments
*
S.E.M.
Figure 2 shows the results of experiments designed to test the effect of added adenosine or 2chloroadenosine on the level of cyclic AMP of isolated 17-day-old embryonic retinas. Retinas were incubated for 10 min at 37°C in medium containing either IBMX o r RO 20 1724 plus adenosine deaminase, and then adenosine or 2-chloroadenosine was added to the medium at the concentrations indicated in Fig. 2. After the incubation continued for an additional 10 min, the reaction was stopped by the addition of TCA (5% final concentration). The curve represented by filled triangles shows the effect of 2-chloroadenosine on the cyclic AMP level of retinal tissue incubated in the presence of 0.5 mM-RO 20 1724 plus adenosine deaminase. Maximal accumulation of retina cyclic AMP promoted by 2-chloroadenosine was achieved at a concentration of 0.01 mM. When the tissue was preincubated with 0.5 mM-IBMX plus adenosine deaminase (open triangles), 2-chloroadenosine also increased the level of cyclic AMP. However, the concentration required to obtain maximal stimulation was two or1
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8
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-LOG NUCLEOSIDE MOLARITY FIG. 2. Effect of adenosine and 2-chloroadenosine on the level of retina cyclic AMP from 17-day-old chick embryos. The tissue was preincubated for 10 min at 3 7 T in BME containing 25 mM-HEPES, pH 7.4 (adjusted with NaOH), and (a) 0.5 mM-RO 20 1724.0.5 unitsiml of adenosine deaminase (A); (b) 0.5 mM-IBMX, 0.5 units/ml of adenosine deaminase (A); (c) 0.5 mM-IBMX (0).Then either 2-chloroadenosine (triangles) or adenosine (circles) was added at the concentrations indicated and the incubation allowed to proceed for another 10 min. The reaction was stopped by the addition of TCA (5% final concentration). Each point is the average of duplicate experiments in which the individual values were within 15% of the values shown.
J . Neurochem., Vol. 38, N o . 2, 1982
R . PAES DE CARVALHO A N D F. G . DE MELLO
496
a slight increase in cyclic AMP content of the tissue. Further, the presence of adenosine deaminase in the incubation medium abolished retinal response to 1 mM-adenosine. The maturation of the adenosine-dependent increase of retina cyclic AMP occurs after the 14th embryonic day (Fig. 3). Maximal differentiation of adenosine responsiveness was attained in retinas from 17- to 18-day-old embryos. Further, the levels of retinal cyclic AMP from 12-day-old chick embryos in the absence and presence of 2-chloroadenosine were 50 p m o l h g protein and 33 pmoVmg protein, respectively. After hatching, however, retinal responses to adenosine were greatly reduced. Diminished responses to dopamine also have been observed in chick retinas from the posthatch period (de Mello and de Mello, 1980). The chick embryo retina can be easily dissociated into single cells, and, under appropriate conditions, the cells will develop many properties of the intact tissue (Vogel e t al., 1976; Puro e t al., 1977; Sugiyama et al., 1977). Figure 4 shows that retinal cells obtained from 9-day-old chick embryos and maintained for as long as 8 days in culture respond to adenosine with higher cyclic AMP levels. In contrast with intact retinas, which increase cyclic AMP in response to adenosine only after the 14th embryonic day, cells in culture responded to this purine as soon as 1 day after cells from 9-day-old retinas were plated. However, an increase in the response of retina cells to adenosine was observed after the fourth day of culture. From this stage on, the level of the cyclic nucleotide increased from a basal level of approximately 50 pmoVmg protein to approximately 350 pmoVmg protein when 0.1 mMadenosine was used. The same pattern of response was obtained with 2-chloroadenosine (Table 4). The basal level of cyclic AMP of cultures incubated for 20 min in the presence of RO 20 1724 throughout this experiment was approximately 50 pmol/mg protein, i.e., seven times lower than that found in the intact retina.
ders of magnitude higher than that observed in the presence of 0.5 mM-RO 20 1724 (filled triangles). Adenosine also enhanced the cyclic AMP level of retinas maintained in IBMX without adenosine deaminase in the medium. In this case the concentration of adenosine required for maximal stimulation was 1 mM (filled circles). Since dopamine also elevates the cyclic AMP content of chick embryo retinas (de Mello, 1978), the effect of adenosine and 2-chloroadenosine described above could reflect an indirect action of these compounds mediated by an enhanced output of endogenous dopamine present in dopaminergic circuits of the tissue. This possibility was ruled out by the data shown in Table 2. Seventeen-day-old embryonic retinas were incubated for 10 min in the presence of 0.5 mM-IBMX together with either 10 p M - ( +)-butaclamol or 100 pM-fluphenazine, potent dopamine antagonists (Clement-Cormier et al., 1974; Iversen, 1975; Seeman et al., 1975). Adenosine then was added to a final concentration of I mM and the tissue further incubated for 10 min. The reaction was stopped by addition of TCA. Retinas in the presence of IBMX alone showed a fourfold increase of their cyclic AMP concentration when either 0.01 mM-dopamine or 1 mM-adenosine was added to the medium. The presence of 10 p M - ( +)-butaclamol or 100 pM-fluphenazine in the incubation medium fully prevented the rise of retinal cyclic AMP content caused by dopamine. However, the dopamine antagonists did not affect the cyclic AMP accumulation of retinas stimulated by adenosine. Table 3 shows the effect of several nucleosides and nucleotides on the level of cyclic AMP of embryonic chick retina. These experiments were carried out in the presence of 0.5 mM-RO 20 1724 and 0.5 unit/ml of adenosine deaminase. Under these conditions only 2-chloroadenosine significantly increased cyclic AMP content of the tissue. Guanosine and adenine did not change the level of the cyclic nucleotide, whereas AMP and ATP elicited
TABLE 2. Effect of dopumine antagonists on adenosine-stimulated level of retina cyclic AMP from 17-day-old chick embryos ~~
~~
~
~
Additions Retina treatment during incubation period
Concentration used (mM)
Control Fluphenazine (+)-Butaclamol
0.1 0.01
None
*
*
Adenosine (1 mM)
(pmol cyclic AMPimg protein) 374 t 36 3 7 6 2 14 9 88 5 369 2 77 5 86 8 340 ? 30
9 5 ? 13
77 71
Dopamine (0.1 mM)
* *
The retinas were preincubated for 10 min at 37°C in BME containing 25 mM-HEPES pH 7.4 (adjusted with NaOH), 0.5 mM-IBMX, 0.1 mM-pargyline, and 0.1 mM-sodium ascorbate and either fluphenazine or (+)-butaclamol. Then dopamine or adenosine was added to the medium and the incubation allowed to proceed for an additional 10 min. The reaction was stopped by TCA (5% final concentration). The data shown are the average of two separate experiments S.E.M.
*
J . Neurochem.. Vul. 38, N o . 2 , 1982
497
ADENOSINE-ELICITED CYCLIC A M P INCREASE I N RETINA TABLE 3. Effect of nucleosides and nucleotides on the level of retina cyclic A M P from 16-day-old chick embryos ~
r
I
I
I
~~
Additions to the incubation medium
Concentration used (mM)
pmol cyclic AMPimg protein ? S.E.M.
None 2-Chloroadenosine Adenosine Guanosine AMP ATP Adenine
0.01
60 ? 8 214? I1 73 5 7 61 t 2 92 ? 3 89 ? 1 70 ? 7
0.01
0.01 0.01 0.01 0.01
The retinas were incubated for 10 min at 37°C in BME containing 25 mM-HEPES, pH 7.4 (adjusted with NaOH), 0.5 mMRO 20 1724, and 0.5 unitiml of adenosine deaminase. Then each test compound was added and the tissue was further incubated for 10 min. The reaction was stopped by the addition of TCA (5% final concentration). The numbers indicate the average value of two separate experiments.
Figure 5 shows that the adenosine concentration required for maximal increase of the cyclic nucleotide level in the cultures was 0.1 mM, one order of magnitude higher than that found with intact tissue under the same incubation procedure. Equilibrium is reached after 10 min exposure to adenosine, as shown in the inset of Fig. 5 .
DAYS OF CELLS IN CULTURE FIG. 4. Effect of adenosine on the level of cyclic AMP of cultured retina cells from chick embryos. The cultures were washed twice at 37°C with 2 ml of serum-free BME buffered with 25 mM-HEPES, pH 7.4 (adjusted with NaOH). Then, the dishes were preincubated for 10 min at 37°C in serum-free BME containing 25 mM-HEPES, pH 7.4 (adjusted with NaOH), and 0.5 mM-RO 20 1724. Adenosine was added to a final concentration of 0.1 mM and the cultures were further incubated for 10 min. The reaction was stopped by the addition of TCA (5% final concentration). Each point represents the mean value of two to four experiments ? S.E.M. The points without bars are the results of individual determinations.
DISCUSSION
odenosine
f
The chick embryo retina is easily accessible and can be obtained free from surrounding structures during most of the embryonic life span. Its structure and morphological differentiation have been well documented; this tissue is thus an excellent model for the study of CNS differentiation (Coulombre, 1955). Moreover, the embryonic retina is suitable for tissue culture technique, which has been routinely used in the study of morphological and
tI II
TABLE 4. Effect of 2-chloroadenosine on the cyclic A M P level of cultured retina cells from chick embryo
A
Ib
Days of cell in 2-Chloroadenosine culture Basal (0.1 m M )
%-
I I 1 14 I I'e ' I ' DEVELOPMENTAL STAGE (DAYS) FIG. 3. Embryonic developmental profile of chick retina responsiveness to adenosine. Retinas were incubated for 10 min at 37°C in BME containing 25 mM-HEPES, pH 7.4 (adjusted with NaOH), and 0.5 mM-IBMX. Then adenosine was added to a final concentration of 1mM and the retinas were incubated for 10 min. The reaction was stopped by the addition of TCA (5% final Concentration). Each point represents the value of individual determinations.
04/
1
4 6 8
61 43 48 54
130 L 9 34s ? 34 251 ? 26 327 2 56
The assay procedure was the same as described in the legend to Fig. 4. The data are measured in pmol cyclic A M P h g protein and represent the average value oftwo cultures ~ s . E . M .
J . Neurochem., Vol. 38, N o . 2, 1982
R . PAES DE CARVALHO AND F. G. DE MELLO
498 r-fl
4t
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FIG. 5. Effect of increasing concentrations of adenosine on the level of cyclic AMP of cultured retina cells from chick embryos. The assay procedure is described in the legend to Fig. 4. Each point represents the mean value of two experiments. The values found in each individual experiments were within 15% of the values shown.
neurochemical aspects of the retina ontogeny (Sheffield and Moscona, 1970; Vogel and Nirenberg, 1976; Vogel et al., 1976; Puro et al., 1977). The present work shows the existence of an adenosine-dependent accumulation of cyclic AMP in the chick embryo retina. The comparison between the level of cyclic AMP of 17-day-old chick embryo retinas incubated in the presence of either IBMX or RO 20 1724 but without added adenosine showed a significant threefold difference, with higher cyclic nucleotide content in retinas exposed to RO 20 1724. The presence of adenosine deaminase in the incubation medium during tissue treatment by RO 20 1724 reduced the cyclic AMP level to that observed in IBMX-treated retinas. Ample evidence indicates that IBMX is a potent adenosine antagonist (PrCrnont et al., 1977; Van Calker et al., 1978, 1979; Smellie et al., 1979). Therefore, we interpret our findings as due to an antagonist effect of IBMX, which prevents the activation of retina adenylate cyclase molecules mediated by an endogenous adenosine output in the embryonic tissue. The lower level of cyclic AMP content of retina exposed either to IBMX or adenosine deaminase was enhanced by the addition of 2-chloroadenosine in the medium in a dose-dependent manner. This effect was also observed when adenosine was added to IBMX-treated tissue. However, adenosine deaminase abolished the adenosine effect, further suggesting that inosine was ineffective in stimulatJ . Neurochem.. Vol. 38, N o . 2 , 1982
ing adenosine receptors responsible for activating adenylate cyclase molecules of the tissue. When dipyridamole, an adenosine uptake inhibitor (Huang and Daly, 1974; Van Calker et al., 1979; Bender et al., 1980), was added to the medium, it did not interfere significantly with cyclic AMP accumulation observed with RO 20 1724 treatment. This fact strongly suggests that the effect of adenosine or adenosine analogs on the cyclic AMP content of the retina is mediated by cell membrane receptors, possibly of the R type (Fain and Malbon, 1979; Londos et al., 1980), rather than through changes in the intracellular pool of the nucleoside. The course of maturation of adenosine-elicited accumulation of retina cyclic AMP during chick embryo development differs significantly from what has been reported for the dopamine-dependent adenylate cyclase system of the same tissue (de Mello, 1978). Retinas obtained from embryos between the 8th and 14th embryonic days were unresponsive to adenosine. From the 15th embryonic day on, the retinal tissue showed a substantial cyclic AMP accumulation mediated by adenosine. It is unlikely that the lack of retinal responsiveness to adenosine in the early stages of embryonic development is due to high tissue adenosine deaminase activity, as 2-chloroadenosine was unable to elicit cyclic AMP accumulation of chick retinas from 12day-old embryos. The chick neural retina possess its definitive number of cells by the 15th embryonic day. Complete differentiation of rods and cones is observed in the fundic region of the retina by the 19th day of embryo development (Coulombre, 1955). According to Lindeman (1947) it is also at this stage that the retina becomes capable of visual function, as revealed by the pupillary constrictor reflex. Fully differentiated rods and cones, however, are observed in the periphery of the neural retina by 1 or 2 days after hatching. An increase in the thickness of the inner plexiform layer of the chick retina is observed after the 12th embryonic day. Thus the inner plexiform layer of the retina from 17-day-old embryos is about 17 pm thick as compared with 5 1 pm in 15-day-old chicken (Coulombre, 1955). The low adenosine response of the chick retina from the posthatch period is consistent with the idea that the adenosine-cyclase system of retinal tissue is desensitized due to synaptic activity possibly modulated by visual function. Preliminary experiments in our laboratory show that adenosine N-,oxide increased only slightly the cyclic AMP level of retinas from posthatch animals, but was fully active in stimulating retinas from 17-day-old embryos (data not shown). Since this adenosine analog reportedly is not broken down by adenosine deaminase (AnandSrivastava and Johnson, 1980), the lower response of the postembryonic tissue to adenosine may not be due to high deaminase activity. In addition, we
ADENOSINE-ELICITED CYCLIC AMP INCREASE IN RETINA have shown (de Mello and de Mello, 1980) that the dopamine-dependent cyclase system of the posthatch chick retina reduced its response t o the amine. However, the retinas became hyperresponsive to dopamine after the chicks were chronically treated by the dopamine antagonist fluphenazine, or kept in a dark environment for relatively long periods of time (de Mello and de Mello, 1980). The regulation of cell susceptibility to respond to neurotransmitters and hormones has been the subject of a recent review (Klein and Wolf, 1981). Accounts from other laboratories have shown that adenine nucleotides also activate brain adenosine-dependent adenylate cyclase (Sattin and Rall, 1970; Saito, 1977; Fain and Malbon, 1979). However, many effects ascribed to adenine nucleotides may be caused by sample contamination with adenosine. In our case, AMP and ATP slightly increased the retinal cyclic AMP level from 16-dayold embryos. Since these experiments were carried out in the presence of adenosine deaminase, sample contamination with adenosine can be ruled out. These nucleotides were less effective than adenosine in promoting cyclic AMP accumulation in the retinal tissue, supporting the concept that the interaction between adenosine and its receptor is highly specific. During morphological differentiation of the chick retina, synaptic contacts are not seen in the tissue until the 13th day of development (Meller, 1964; Sheffeld and Fischman, 1970; Hughes and L a Velle, 1974). Our findings concerning the differentiation of adenosine-elicited accumulation of retinal cyclic AMP shows good correlation between the appearance of synaptic contacts in the chick embryo retina and the onset of response of the tissue to adenosine. Dopamine antagonists did not prevent the adenosine-elicited cyclic AMP accumulation of the tissue. Therefore, adenosine nucleoside effects are not mediated by increased dopamine output due to enhanced synaptic activity. The results suggest instead a direct stimulation of adenylate cyclase molecules coupled to membrane adenosine receptors. The accumulation of cyclic AMP due to adenosine was also observed in retina cell culture. However, the time course of maturation of the adenosine response of these cells was anticipated by at least 4 days. Thus, a stage of retina cell differentiation equivalent to intact retinas from 10-day-old embryos exhibited significant responses to both adenosine and 2-chloroadenosine. Maximal retina cell susceptibility to adenosine stimulation was attained in a differentiation stage of cells equivalent to that of retinas from 13-day-old embryos. Although the maximal change in the level of retina cyclic AMP caused by adenosine was very similar to that of retinas obtained from embryos “in ovo,” the basal cyclic AMP level of cultures submitted to RO
499
20 1724 treatment was low compared with the level in intact tissue. These events may indicate that the adenosine release mechanism is impaired or diminished in cell culture. Alternatively, a cell population responsible for synthesizing and releasing the nucleoside could have been selected out during the procedure used for tissue dissociation. Acknowledgments: The authors are grateful to Dr. W. L.
Klein for his critical review of the manuscript. This w or k was supported by grants from CNPq, FINEP and CEPG UFRJ. Dr. Paes de Carvalho is the recipient of a fellowship from the Brasilian National Research Council (CNPq).
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