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Differential ontogenesis of D1 and D2 dopaminergic receptors in the chick embryo retina
Developmental Brain Research, 12 (1984) 217-223 Elsevier
217
Differential Ontogenesis of D 1 and D 2 Dopaminergic Receptors in the Chick Embryo Retina ANA LUCIA M. VENTURA l, WILLIAM L. KLEIN2 and FERNANDO G. DE MELLO 1 l lnstituto de Biofisica, Universidade Federal do Rio de Janeiro, Centro de Ci~ncias da Satide, Cidade Universitdria, llha do Fundao, Rio de Janeiro 21941 (Brazil)and 2Department of Neurobiology and Physiology, Northwestern University, Evanston, 1L 60201 (U.S.A.)
(Accepted September 20th, 1983) Key words: retina ontogeny-- dopamine receptors - - receptor regulation
The differentiation of D 1 and D2 dopamine receptors was investigated during the ontogenesis of the chick embryo retina. Our resuits reveal an interesting complexity in dopaminergic differentiation, with one major receptor system developing before synapses and another one developing after. The dopamine-dependent increase of chick retina cAMP level differentiates early during retina ontogeny. By the embryonic day 10-11 10-4 M dopamine and ADTN elicit a 13-fold increase in cAMP content of the retina. However, [3H]spiperone (D 2 ligand) binds very little to crude membrane preparation of retinas from embryos in the same developmental stage (12-13 fmol/mg protein). High specific binding of [3H]spiperone is only detected after the embryonic day 17-18, attaining 80 to 100 fmol of specific spiperone binding sites in the retinas from post-hatched animals. Apomorphine also promotes the accumulation of cAMP of retinas from early embryonic stages. However, it is only 20-30% as effective as ADTN or dopamine. In addition, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine effect remains practically constant throughout this period. Both dopamine and apomorphine are equally effective in eliciting cAMP accumulation of retinas from post-hatched animals. Moreover, apomorphine is a potent inhibitor of dopamine-induced cAMP level of the embryonic tissue. The results presented here indicate that D 1 and D2 receptors differentiate independently from each other, and that apomorphine elevates retina cAMP levels via a subclass of D 1receptors that does not desensitize significantlyduring retina development. INTRODUCTION At least two types of dopamine receptors have been established in the CNS 15. O n e is coupled to adenylate cyclase and its activation by specific agonists leads to an increase in the cellular cyclic A M P level. This receptor has been designated D 1. Specific ligands to D~ receptors have not been entirely characterized 2J. The other class is designated D 2. These receptors have high affinity for b u t y r o p h e n o n e s and do not mediate dopamine activation of the adenylate cyclase systems. In a few cases D 2 activation in fact inhibits the enzyme s . The presence of D 1 receptors in the retina is well documented. D o p a m i n e sensitive adenylate cyclase systems have been characterized in a n u m b e r of species, including rat and bovine 1, rabbit 2, guinea pig23, carp 24, and chicken retinas 4.
The observation that [3H]spiperone binds to a population of dopamine receptors in the retina of several species 17 supports the idea that D2 receptors are also present in this tissue, although recent studies with 3H-domperidone, (supposedly a D 2 specific ligand)23 showed that this ligand did not bind specifically to retina homogenates. However, b o u n d [3H]spiperone was clearly displaced by cold domperidone and by other b u t y r o p h e n o n e derivatives as well 25. The chick retina is a particularly useful model for neurochemical studies of the embryonic CNS, because it can be easily obtained throughout most of the embryo lifetime. In 1978, we showed that dopaminedependent adenylate cyclase of the chick retina differentiated early during the ontogenesis of the tissue 4. Retinas obtained from 7-day-old embryos already responded to dopamine with increased cyclic A M P level. By the eighth embryonic day, full differ-
Correspondence: F. G. de Mello, Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Centro de Cifncias da Safide, Cidade Universit~iria,Ilha do Fund~o, Rio de Janeiro 21941, Brazil.
0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.
218 entiation of the dopamine coupled cyclase system of the retina was observed. In this report we show that the ontogenesis of dopamine displaceable [3H]spiperone binding sites in chick retina does not correlate with the appearance of dopamine dependent adenylate cyclase system of the tissue. In addition, our data also suggest that apomorphine-dependent cyclic AMP accumulation of the retina is mediated by a subclass of D l receptor. MATERIAL AND METHODS
Reagents Cyclic AMP, protein kinase, bovine serum albumin, serotonin creatine sulphate (Sigma); Basal Medium of Eagle (BME), (GIBCO); 3-isobutyl-l-methylxanthine (IBMX), (Aldrich); dopamine, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes), (Calbiochem); apomorphine (Merck); 2amino-6,7-dihydroxy- 1,2,3,4-tetrahydronaphthalene (ADTN), (Burroughs-Wellcome); spiperone (Jansen); [3H]cyclic AMP (42.5Ci/mmol) and [3H]spiperone (35.9 Ci/mmol) (New England Nuclear) were used throughout this study. (+) and (--)Butaclamol was kindly supplied to us by Dr. D. J. Marshall from Ayerst Laboratories. All other reagents were of analytical grade. Fertilized white Leghorn eggs were obtained from a local hatchery. Retina dissection and incubation In all experiments, unless stated otherwise, the procedures were the same as described before 4,20. The embryos were staged according to Hamburger and Hamilton H, the eyes removed, transferred to cold Ca 2+, Mg 2+ free media (CMF) and the retinal tissue dissected free of the pigmented epithelium. After a brief (30 s) centrifugation at 400 g the tissue was resuspended in 2 ml of BME with 25 mM Hepes, pH 7.4 (adjusted with NaOH), 0.5 mM IBMX, 0 . 5 m M pargyline, 0.l mM sodium-ascorbate at 37 °C. The tissue was then incubated at 37 °C for 10 min, then each test compound was added to the final concentration required. The tissue was further incubated for 5 min. The reaction was stopped by the addition of trichloroacetic acid (TCA) to 5% final concentration. The cyclic AMP was purified according to Matsuzawa and Nirenberg is and assayed by the method of Gilman 9. Protein was estimated by the
method of Lowry et al. ~' using bovine serum albunlin as standard.
FH]spiperone binding assay Retinas from embryos in different stages of differentiation were dissected free of the pigmented epithelium, as above, and immediately transferred to ice-cold 50 mM Tris, pH 7.4 containing 120 mM NaCI, 5 mM KCI, 2 mM MgCI> 1 mM CaC/, (assay buffer). The tissue was disrupted in a polytron homogenizer (Brinkman Instruments) using setting 6 for 25 s. The homogenates were centrifuged at 45,000 g for 45 rain and the pellet was rinsed and resuspended in the assay buffer (crude membrane). No [3H]spiperone specific binding was detected m the 45,000 g supernatant in any of the embryonic stages studied. The amount of protein present in the pellet represented approximately 65% of the total protein of the retina homogenates from embryos until the fifteenth day of development and approximately 50ofof the total protein of retinal homogenates of older embryos and post-hatch chicken. [3H]spiperone binding was carried out essentially as described by Creese et al.~. The assay was conducted at 37 °C in a final volume of 0.2 ml containing 50 mM Tris, pH 7.4, 120 mM NaCI, 5 mM KCI, 2 mM MgCI-. 1 mM CaCI2, 0.1 mM pargyline, each one of the test compounds and 0.1-0.2 mg of protein. The reaction was interrupted with 10 ml of ice-cold 50 mM Tris, pH 7.7, immediately filtered through Whatman GF/B glass fiber filters and further washed with another 10 ml of the same buffer. All the filtration procedure took less than 10 s. The specific [3H]spiperone binding was estimated as that displaceable bv 0.1 mM dopamine which corresponded to the stereospecific binding of [3H]spiperone as estimated by the difference of binding in the presence of 1 a M (+) and (--)butaclamol respectively. RESULTS Fig. 1. shows the effect of dopaminergic agonists upon the cAMP level of retinas obtained from 12- to 13-day-old chick embryos. Dopamine and ADTN were equally effective in increasing the cAMP level of the tissue, with an EDs~ of approximately 2-3 ,uM. Apomorphine also promoted the accumulation of cAMP in the embryonic retina. Although the EDs,~
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-LO60RU6 MOLARITY Fig. 1. Effect of dopaminergic agonists upon the level of cAMP of chick embryo retina. Retinas from 12- to 13-day-old embryos were dissected and incubated for 10 min at 37 °C in BME containing: 0.1 mM pargiline, 0.1 mM sodium ascorbate, 25 mM Hepes and 0.5 mM IBMX. Then ADTN (triangles), dopamine (circles), or apomorphine (squares), were added and the retinas were further incubated for 5 rain. The reaction was stopped with 5% TCA final concentration. The points represent the mean of 2-3 experiments + S.E.M.
for apomorphine was similar to that of the other two agonists (approximately 2 ktM) the maximal increase in cAMP accumulation observed with this compound was only 20-30% of that observed with dopamine or ADTN. Moreover, when apomorphine was added in the incubation medium together with dopamine, it had a potent inhibitory effect upon the dopamine stimulated cAMP level of the embryonic tissue (Fig. 2). The K i for apomorphine calculated from these experiments was approximately 0.1 ~M. At the maximal apomorphine inhibitory effect, (100/~M of this compound), the cAMP level never went below the level observed with apomorphine alone. The inhibitory effect of apomorphine was reversible by increasing dopamine concentration (Fig. 2, inset). The EDs0 for dopamine to increase cAMP level of the tissue in the presence of 10/~M apomorphine was over an order of magnitude (40 #M) higher than that found when dopamine was added alone to the medium (approximately 2/~M). The inhibition was fully
-LOG APOBBORPHINEMOLARIT¥ Fig. 2. Effect of apomorphine upon the dopamine-dependent cAMP accumulation of chick embryo retina. Retinas from 12to 13-day-old embryos were used. The incubation procedure was the same as that described in the legend of Fig. 1. Dopamine (0.1 mM) was added together with apomorphine in the concentrations indicated. The reaction was stopped 5 min after the addition of the compounds, with TCA 5% final concentration. The inset shows the reversibility of apomorphine inhibitory effect by increasing dopamine concentration. The concentration of apomorphine used in this experiment was 10/~M. Each point represents the mean value of 2-3 experiments + S.E.M. Triangles represent the effect of two concentrations of apomorphine added alone in the incubation medium. The square indicates the level of cAMP with no drug added.
overcome at a dopamine concentration equal to 1 mM. The stimulatory effect of dopamine and apomorphine upon the cyclase system of the retina was compared at different stages of the embryonic development and also in retinas from animals of the posthatch period. A remarkable desensitization of the dopamine-dependent cAMP accumulation was observed during the course of tissue differentiation, attaining a very low responsiveness to dopamine in retinas obtained from 2- to 3-day-old chicken (Table I and Fig. 3). The apomorphine-dependent cAMP accumulation of the retina did not desensitize nearly as much as the dopamine response, remaining relatively the same throughout the embryonic lifetime. Moreover, retina from 2- to 3-day-old chicken was equally responsive to both dopamine and apomorphine (Fig. 3 and Table I).
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D E V E L O P M E N T A L S T A G E (days) Fig. 3. Ratios between stimulated and basal level of cAMP of the retina in different stages of embryonic development. The incubation procedure was the same as that described in the legend of Fig. 1. The curve represented by circles refers to the ratio between dopamine-stimulated versus non-stimulated levels of retina cAMP, and the triangles represent the apomorphinestimulated versus non-stimulated cAMP levels. The inset shows the cAMP basal level of retinas in different stages of embryonic development.
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DEVELOPMENTAL STAGE, (days)
While dopamine- and apomorphine-dependent c u m u l a t i o n of c A M P
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of t h e c h i c k r e t i n a d i f f e r e n -
t i a t e d e a r l y in t h e o n t o g e n e s i s of t h e t i s s u e (Fig. 3 a n d T a b l e I), t h e n u m b e r of [ 3 H ] s p i p e r o n e specific b i n d i n g sites in t h e e m b r y o n i c r e t i n a i n c r e a s e d signifi c a n t l y o n l y a f t e r e m b r y o n i c d a y 17-18. Fig. 4 s h o w s t h a t t h e n u m b e r of d o p a m i n e d i s p l a c e a b l e [3H]spipe-
Fig. 4. Developmental profile of specific [3HJspiperone binding sites of chick retina. The assay procedure is described in Material and Methods. The concentration of spiperone used in each assay was 3 riM. The specific binding was estimated as that displaceable by 0.1 mM dopamine. Each point is the mean value of 3-7 experiments + S.E.M. In all experiments quintuplicate samples were run in parallel to estimate the number of specific sites shown. The inset shows the Scatchard analysis of 2 independent experiments with crude membrane preparations of retinas from 2- to 3-day-old chicken.
TABLE I
Effect of dopamine and apomorphine upon the cA MP level of retinas from embryos in different stages of development and from posthatched chicken The incubation procedure was the same as that described in the legend of Fig. 1. The data of the embryonic period represent the mean _+ S.E.M. of at least 3 independent experiments. For the post-hatched period the data is the mean _+ S.E.M. of 7 independent experiments. Dopamine and apomorphine were used at a concentration of 10-~ M.
Developmental stage (days) Embryonic 10 11 12 13 16 Post-hatched 3
Basal
Dopamine Apomorphine (pmol cA MP/mg protein)
Apomorphine + dopamine
18.0 20.0 19.0 20.8 66.2
-201.0 228.6 + 26.0 246.2 + 10.0 372.2 + 34.0
--112.0 _+ 14.0 57.6 + 4.0 167.0 + 27.0
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49.9 -83.9 55.2 113.0
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Fig. 5. Inhibition of specific [3H]spiperone binding in crude membrane preparation of chicken retina by antagonist and agonist drugs. The specific binding was defined as the amount of [3H]spiperone displaced by 1 ~M (+)butaclamol. Each point is the mean value of 2-3 experiments with quadruplicate determinations in each experiment. S.E.M. values were less than 15% of themean. The concentration of [3H]spiperone used was in the range of 0.85-1.6 nM.
in the pre-hatching period and remained equally high in retinas of post-hatched chicken. The K d of spiperone for these sites as estimated by Scatchard analysis in retinas from post-hatch animals was approximately 1 nM, with an estimated Bma x of 100 fmol/mg protein (Fig. 4, inset). The displacement of [3H]spiperone binding by antagonist and agonist drugs is shown in Fig. 5. With a Ki value of 1.6 nM, spiperone was the most effective compound to compete with [3H]spiperone. In addition, while (+)butaclamol ( K i = 31 nM) displaced bound [3H]spiperone, (--)butaclamol did not compete for spiperone binding sites up to 1000 nM concentration. Among the agonist drugs tested, apomorphine was the most potent compound with a K i value equal to 8 nM, followed by dopamine, with a K i value of 156 nM. Serotonin was not a good displacer of bound [3H]spiperone, showing a K i greater than 26,000 nM. These experiments indicate that [3H]spiperone binds to dopaminergic receptor sites of the D 2 type. Moreover, in contrast to the high affinity shown by spiperone for the D 2 receptors, this compound was a poor inhibitor of the D 1receptor coupled to the cyclase system. Inhibition of 50% of the cAMP accumulation promoted by 1 I~M dopamine was achieved only with 1/xM spiperone (data not shown). DISCUSSION
Our data show that the embryonic chick retina is
capable of responding to dopamine with increased cAMP level in a very early stage of embryonic development. All the agonists tested were effective in promoting cAMP accumulation in the tissue. However, [3H]spiperone bound very little to crude membrane preparation of retina from early embryonic stages. High stereospecific binding of [3H]spiperone was only detected after embryonic day 18, with kinetic characteristics of interaction with D 2 receptors. These data put together show that D1 and D 2 receptors of the chick retina follow a different pattern of differentiation, suggesting that they may be in different cells. Since cyclase response of chick retina to dopamine can be detected already in embryos on embryonic day 7, the embryonic D 1 receptors could be important in decoding differentiation signals triggered by dopamine, when functional dopaminergic synapses are formed in the tissue. The occurrence of dopamine in the embryonic chick retina was first detected on day 15 of development 14, time coincident with appearance of the first synaptic structures in the tissue 12,19,22. An endogenous increase in cAMP level of the embryonic chick retina is also observed during this period 4, probably due to functional dopaminergic synapses that are formed after day 15 of development 5. While D 2 receptors increase after embryonic day 18, cyclase response to dopamine decreases considerably during the same period. The low cyclase responsiveness of the mature tissue to dopamine is possibly due to a desensitization of the D I dopaminergic system. We have recently shown that dopamine-dependent cAMP accumulation desensitizes in cultured retina cells chronically exposed to dopamine. It was also shown that light deprivation associated with decreased dopamine release 13 could induce a dopamine hyperresponsive state in retinas of young chicken 6. Our data are therefore consistent with the idea that endogenous dopamine output of dopaminergic synapses leads to the observed desensitization of the D 1 system of the tissue and that this system is regulated 'in vivo' during embryonic development. Although apomorphine promotes an increase in the cAMP level of the retina, it is only 20-30% as effective as ADTN or dopamine itself. Moreover, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine-dependent cAMP accumulation decreases
222 only slightly during the same period. In addition, as opposed to what is observed in the early embryonic stages of retina differentiation, both dopamine and apomorphine are equally effective in increasing cAMP accumulation of retinas from post-hatched animals. These data suggest that apomorphine me-
er subclass of D 1 receptor. The differential desensitization of responses to dopaminc and apomorphine does not seem consistent with apomorphine being a simple partial agonist for a uniform population of D 1 receptors during embryonic development. Also, if apomorphine was acting as a partial agonist, the dose
diates its effect via a subclass of D 1 receptors whose sensitivity to apomorphine remains practically con-
needed to inhibit stimulation by 100/~M dopamine should be about two orders of magnitude greater
stant during retina ontogeny. This idea is strength-
than observed.
ened by the fact that the time course of dopaminergic desensitization of cultured embryonic retina cells exposed to saturating concentrations of dopamine, reveals two decaying kinetic components with half lives of 6 and 22 h respectively~. Our data also show that apomorphine is a potent
ACKNOWLEDGEMENTS
competitive inhibitor of the highly dopamine respon-
This work was supported by grants to F . G . d . M . from CNPq, F I N E P and C P E G U F R J ; and partly by the Brazilian American Institute of Rio de Janeiro
sive D1 system present in the embryonic tissue, indicating that this compound also interacts with this oth-
(IBEU). A . L . M . V . is the recipient of a fellowship from CAPES.
REFERENCES
10 Haggendal, J. and Malmfors, T., Identification and cellular localization of the catecholamines in the retina and the choroid of the rabbit, Actaphysiol. scand., 64 (1965) 58-66. 11 Hamburger, V. and Hamilton, H. L., A series of normal stages in the development of the chick embryo, J. Morph.. 88 (1951) 49-92. 12 Hughes, W. F. and La Velle, A., On the synaptogenic sequence in the chick retina, Anat. Rec., 179 (1974) 297-302. 13 Iuvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons, Science, 202 (1978) 901-902. 14 Kato, S., Nakamura, T. and Negishi, K., Postnatal development of dopaminergic cells in the rat retina, J. comp. Neurol., 191 (1980) 227-236. 15 Kebabian, J. W. and Calne, D. B., Multiple receptors for dopamine, Nature (Lond.), 277 (1979) 93-96. 16 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951)265-275. 17 Makman, M. H., Dvorkin, B., Horowitz, S. G. and Thai. L. J., Properties of dopamine agonist and antagonist binding sites in the mammalian retina, Brain Res., 194 (1980) 403-418. 18 Matsuzawa, H. and Nirenberg, M. W., Receptor mediated shifts in cGMP and cAMP levels in neuroblastoma cells, Proc. nat. Acad. Sci. U.S.A., 72 (1975) 3472-3476. 19 Meller, K., Elektronenmikroskopische befunde zur differenzierung der rezeptorzellen und bipolarzellen der retina und ihrer synaptischen verbindungen, Z. Zellforsch., 64 (1964) 733-750. 20 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 (1982) 493-500. 21 Seeman, P., Brain dopamine receptors, Pharmacol. Rev., 32 (1980) 229-313.
1 Brown. J. H. and Makman, M. H., Stimulation by dopamine of adenylate cyclase in retinal homogenates and adenosine-Y-5'-cyclic monophosphate formation in intact retina, Proc. nat. Acad. Sci. U.S.A., 69 (1972)53%543. 2 Bucher, M B. and Schorderet, M., Dopamine and apomorphine sensitive adenylate cyclase in homogenates of rabbit retina, Naunyn-Schmiedeberg's Arch. exp. Pharmacol., 288 (1975) 103-107. 3 Creese, L., Burt, D. R. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science, 197 (1977) 596-598. 4 De Mello, F. G., The ontogeny of dopamine-dependent increase of adenosine-3'-5'-cyclic monophosphate in the chick retina, J. Neurochem., 31 (1978) 1049-1053. 5 De Mello, F. G. and de Mello, M. C. F., Dopamine dependent modulation of cAMP level in the chick retina. In R. Levi-Montalcini (Eds.), Nerve Cells, Transmitters and Behaviour, Pontificiae Academiae Scientiarum Scripta Varia, Vaticano, 1980, pp. 343-355. 6 De Mello, M. C. F., Ventura, A. L. M., Paes de Carvalho, R., Klein, W. L. and de Mello, F. G., Regulation of dopamine- and adenosine-dependent adenylate cyclase systems of chicken embryo retina cells in culture, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 5708-5712. 7 Ehinger, B. and Flack, B., Adrenergic retinal neurons of some new world monkeys, Z. Zellforsch., 100 (1969) 364-375. 8 Frey, E. A., Cote, T. E., Grewe, C. W. and Kebabian, J. W., 3H-spiroperidol identifies a D2 dopamine receptor inhibiting adenylate cyclase activity in the intermediary lobe of the rat pituitary gland, Endocrinology, 110 (1982) 1897-1904. 9 Gilman, A. G., Protein binding assay for adenosine-3'-5'cyclic monophosphate, Proc. nat. Acad. Sci. U.S.A., 67 ( 197013/)5-312.
223 22 Sheffield, J. B. and Fischman, D, A., Intercellular junctions in the developing neural retina of the chick embryo, Z. Zellforsch., 104 (1970) 405-418. 23 Watling, K. J., Dowling, J. E. and Iversen, L. L., Dopamine receptors in the retina may all be linked to adenylate cyclase, Nature (Lond.), 281 (1979) 578-580. 24 Watling, K. J., Dowling, J. E. and Iversen, L. L., Dopaminergic mechanisms in the carp retina: effects of dopa-
mine, K ÷ and light on cyclic AMP synthesis. In Neurochemistry of the Retina, Pergamon Press, Oxford, 1980, pp. 519-537. 25 Watling, K. J. and Iversen, L. L., Comparison of the binding of 3H-spiperone and 3H-domperidone in homogenates of mammalian retina and caudate nucleus, J. Neurochem., 37 (1981) 113/)-1143.
217
Differential Ontogenesis of D 1 and D 2 Dopaminergic Receptors in the Chick Embryo Retina ANA LUCIA M. VENTURA l, WILLIAM L. KLEIN2 and FERNANDO G. DE MELLO 1 l lnstituto de Biofisica, Universidade Federal do Rio de Janeiro, Centro de Ci~ncias da Satide, Cidade Universitdria, llha do Fundao, Rio de Janeiro 21941 (Brazil)and 2Department of Neurobiology and Physiology, Northwestern University, Evanston, 1L 60201 (U.S.A.)
(Accepted September 20th, 1983) Key words: retina ontogeny-- dopamine receptors - - receptor regulation
The differentiation of D 1 and D2 dopamine receptors was investigated during the ontogenesis of the chick embryo retina. Our resuits reveal an interesting complexity in dopaminergic differentiation, with one major receptor system developing before synapses and another one developing after. The dopamine-dependent increase of chick retina cAMP level differentiates early during retina ontogeny. By the embryonic day 10-11 10-4 M dopamine and ADTN elicit a 13-fold increase in cAMP content of the retina. However, [3H]spiperone (D 2 ligand) binds very little to crude membrane preparation of retinas from embryos in the same developmental stage (12-13 fmol/mg protein). High specific binding of [3H]spiperone is only detected after the embryonic day 17-18, attaining 80 to 100 fmol of specific spiperone binding sites in the retinas from post-hatched animals. Apomorphine also promotes the accumulation of cAMP of retinas from early embryonic stages. However, it is only 20-30% as effective as ADTN or dopamine. In addition, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine effect remains practically constant throughout this period. Both dopamine and apomorphine are equally effective in eliciting cAMP accumulation of retinas from post-hatched animals. Moreover, apomorphine is a potent inhibitor of dopamine-induced cAMP level of the embryonic tissue. The results presented here indicate that D 1 and D2 receptors differentiate independently from each other, and that apomorphine elevates retina cAMP levels via a subclass of D 1receptors that does not desensitize significantlyduring retina development. INTRODUCTION At least two types of dopamine receptors have been established in the CNS 15. O n e is coupled to adenylate cyclase and its activation by specific agonists leads to an increase in the cellular cyclic A M P level. This receptor has been designated D 1. Specific ligands to D~ receptors have not been entirely characterized 2J. The other class is designated D 2. These receptors have high affinity for b u t y r o p h e n o n e s and do not mediate dopamine activation of the adenylate cyclase systems. In a few cases D 2 activation in fact inhibits the enzyme s . The presence of D 1 receptors in the retina is well documented. D o p a m i n e sensitive adenylate cyclase systems have been characterized in a n u m b e r of species, including rat and bovine 1, rabbit 2, guinea pig23, carp 24, and chicken retinas 4.
The observation that [3H]spiperone binds to a population of dopamine receptors in the retina of several species 17 supports the idea that D2 receptors are also present in this tissue, although recent studies with 3H-domperidone, (supposedly a D 2 specific ligand)23 showed that this ligand did not bind specifically to retina homogenates. However, b o u n d [3H]spiperone was clearly displaced by cold domperidone and by other b u t y r o p h e n o n e derivatives as well 25. The chick retina is a particularly useful model for neurochemical studies of the embryonic CNS, because it can be easily obtained throughout most of the embryo lifetime. In 1978, we showed that dopaminedependent adenylate cyclase of the chick retina differentiated early during the ontogenesis of the tissue 4. Retinas obtained from 7-day-old embryos already responded to dopamine with increased cyclic A M P level. By the eighth embryonic day, full differ-
Correspondence: F. G. de Mello, Instituto de Biofisica, Universidade Federal do Rio de Janeiro, Centro de Cifncias da Safide, Cidade Universit~iria,Ilha do Fund~o, Rio de Janeiro 21941, Brazil.
0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.
218 entiation of the dopamine coupled cyclase system of the retina was observed. In this report we show that the ontogenesis of dopamine displaceable [3H]spiperone binding sites in chick retina does not correlate with the appearance of dopamine dependent adenylate cyclase system of the tissue. In addition, our data also suggest that apomorphine-dependent cyclic AMP accumulation of the retina is mediated by a subclass of D l receptor. MATERIAL AND METHODS
Reagents Cyclic AMP, protein kinase, bovine serum albumin, serotonin creatine sulphate (Sigma); Basal Medium of Eagle (BME), (GIBCO); 3-isobutyl-l-methylxanthine (IBMX), (Aldrich); dopamine, N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes), (Calbiochem); apomorphine (Merck); 2amino-6,7-dihydroxy- 1,2,3,4-tetrahydronaphthalene (ADTN), (Burroughs-Wellcome); spiperone (Jansen); [3H]cyclic AMP (42.5Ci/mmol) and [3H]spiperone (35.9 Ci/mmol) (New England Nuclear) were used throughout this study. (+) and (--)Butaclamol was kindly supplied to us by Dr. D. J. Marshall from Ayerst Laboratories. All other reagents were of analytical grade. Fertilized white Leghorn eggs were obtained from a local hatchery. Retina dissection and incubation In all experiments, unless stated otherwise, the procedures were the same as described before 4,20. The embryos were staged according to Hamburger and Hamilton H, the eyes removed, transferred to cold Ca 2+, Mg 2+ free media (CMF) and the retinal tissue dissected free of the pigmented epithelium. After a brief (30 s) centrifugation at 400 g the tissue was resuspended in 2 ml of BME with 25 mM Hepes, pH 7.4 (adjusted with NaOH), 0.5 mM IBMX, 0 . 5 m M pargyline, 0.l mM sodium-ascorbate at 37 °C. The tissue was then incubated at 37 °C for 10 min, then each test compound was added to the final concentration required. The tissue was further incubated for 5 min. The reaction was stopped by the addition of trichloroacetic acid (TCA) to 5% final concentration. The cyclic AMP was purified according to Matsuzawa and Nirenberg is and assayed by the method of Gilman 9. Protein was estimated by the
method of Lowry et al. ~' using bovine serum albunlin as standard.
FH]spiperone binding assay Retinas from embryos in different stages of differentiation were dissected free of the pigmented epithelium, as above, and immediately transferred to ice-cold 50 mM Tris, pH 7.4 containing 120 mM NaCI, 5 mM KCI, 2 mM MgCI> 1 mM CaC/, (assay buffer). The tissue was disrupted in a polytron homogenizer (Brinkman Instruments) using setting 6 for 25 s. The homogenates were centrifuged at 45,000 g for 45 rain and the pellet was rinsed and resuspended in the assay buffer (crude membrane). No [3H]spiperone specific binding was detected m the 45,000 g supernatant in any of the embryonic stages studied. The amount of protein present in the pellet represented approximately 65% of the total protein of the retina homogenates from embryos until the fifteenth day of development and approximately 50ofof the total protein of retinal homogenates of older embryos and post-hatch chicken. [3H]spiperone binding was carried out essentially as described by Creese et al.~. The assay was conducted at 37 °C in a final volume of 0.2 ml containing 50 mM Tris, pH 7.4, 120 mM NaCI, 5 mM KCI, 2 mM MgCI-. 1 mM CaCI2, 0.1 mM pargyline, each one of the test compounds and 0.1-0.2 mg of protein. The reaction was interrupted with 10 ml of ice-cold 50 mM Tris, pH 7.7, immediately filtered through Whatman GF/B glass fiber filters and further washed with another 10 ml of the same buffer. All the filtration procedure took less than 10 s. The specific [3H]spiperone binding was estimated as that displaceable bv 0.1 mM dopamine which corresponded to the stereospecific binding of [3H]spiperone as estimated by the difference of binding in the presence of 1 a M (+) and (--)butaclamol respectively. RESULTS Fig. 1. shows the effect of dopaminergic agonists upon the cAMP level of retinas obtained from 12- to 13-day-old chick embryos. Dopamine and ADTN were equally effective in increasing the cAMP level of the tissue, with an EDs~ of approximately 2-3 ,uM. Apomorphine also promoted the accumulation of cAMP in the embryonic retina. Although the EDs,~
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-LO60RU6 MOLARITY Fig. 1. Effect of dopaminergic agonists upon the level of cAMP of chick embryo retina. Retinas from 12- to 13-day-old embryos were dissected and incubated for 10 min at 37 °C in BME containing: 0.1 mM pargiline, 0.1 mM sodium ascorbate, 25 mM Hepes and 0.5 mM IBMX. Then ADTN (triangles), dopamine (circles), or apomorphine (squares), were added and the retinas were further incubated for 5 rain. The reaction was stopped with 5% TCA final concentration. The points represent the mean of 2-3 experiments + S.E.M.
for apomorphine was similar to that of the other two agonists (approximately 2 ktM) the maximal increase in cAMP accumulation observed with this compound was only 20-30% of that observed with dopamine or ADTN. Moreover, when apomorphine was added in the incubation medium together with dopamine, it had a potent inhibitory effect upon the dopamine stimulated cAMP level of the embryonic tissue (Fig. 2). The K i for apomorphine calculated from these experiments was approximately 0.1 ~M. At the maximal apomorphine inhibitory effect, (100/~M of this compound), the cAMP level never went below the level observed with apomorphine alone. The inhibitory effect of apomorphine was reversible by increasing dopamine concentration (Fig. 2, inset). The EDs0 for dopamine to increase cAMP level of the tissue in the presence of 10/~M apomorphine was over an order of magnitude (40 #M) higher than that found when dopamine was added alone to the medium (approximately 2/~M). The inhibition was fully
-LOG APOBBORPHINEMOLARIT¥ Fig. 2. Effect of apomorphine upon the dopamine-dependent cAMP accumulation of chick embryo retina. Retinas from 12to 13-day-old embryos were used. The incubation procedure was the same as that described in the legend of Fig. 1. Dopamine (0.1 mM) was added together with apomorphine in the concentrations indicated. The reaction was stopped 5 min after the addition of the compounds, with TCA 5% final concentration. The inset shows the reversibility of apomorphine inhibitory effect by increasing dopamine concentration. The concentration of apomorphine used in this experiment was 10/~M. Each point represents the mean value of 2-3 experiments + S.E.M. Triangles represent the effect of two concentrations of apomorphine added alone in the incubation medium. The square indicates the level of cAMP with no drug added.
overcome at a dopamine concentration equal to 1 mM. The stimulatory effect of dopamine and apomorphine upon the cyclase system of the retina was compared at different stages of the embryonic development and also in retinas from animals of the posthatch period. A remarkable desensitization of the dopamine-dependent cAMP accumulation was observed during the course of tissue differentiation, attaining a very low responsiveness to dopamine in retinas obtained from 2- to 3-day-old chicken (Table I and Fig. 3). The apomorphine-dependent cAMP accumulation of the retina did not desensitize nearly as much as the dopamine response, remaining relatively the same throughout the embryonic lifetime. Moreover, retina from 2- to 3-day-old chicken was equally responsive to both dopamine and apomorphine (Fig. 3 and Table I).
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D E V E L O P M E N T A L S T A G E (days) Fig. 3. Ratios between stimulated and basal level of cAMP of the retina in different stages of embryonic development. The incubation procedure was the same as that described in the legend of Fig. 1. The curve represented by circles refers to the ratio between dopamine-stimulated versus non-stimulated levels of retina cAMP, and the triangles represent the apomorphinestimulated versus non-stimulated cAMP levels. The inset shows the cAMP basal level of retinas in different stages of embryonic development.
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DEVELOPMENTAL STAGE, (days)
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ac-
of t h e c h i c k r e t i n a d i f f e r e n -
t i a t e d e a r l y in t h e o n t o g e n e s i s of t h e t i s s u e (Fig. 3 a n d T a b l e I), t h e n u m b e r of [ 3 H ] s p i p e r o n e specific b i n d i n g sites in t h e e m b r y o n i c r e t i n a i n c r e a s e d signifi c a n t l y o n l y a f t e r e m b r y o n i c d a y 17-18. Fig. 4 s h o w s t h a t t h e n u m b e r of d o p a m i n e d i s p l a c e a b l e [3H]spipe-
Fig. 4. Developmental profile of specific [3HJspiperone binding sites of chick retina. The assay procedure is described in Material and Methods. The concentration of spiperone used in each assay was 3 riM. The specific binding was estimated as that displaceable by 0.1 mM dopamine. Each point is the mean value of 3-7 experiments + S.E.M. In all experiments quintuplicate samples were run in parallel to estimate the number of specific sites shown. The inset shows the Scatchard analysis of 2 independent experiments with crude membrane preparations of retinas from 2- to 3-day-old chicken.
TABLE I
Effect of dopamine and apomorphine upon the cA MP level of retinas from embryos in different stages of development and from posthatched chicken The incubation procedure was the same as that described in the legend of Fig. 1. The data of the embryonic period represent the mean _+ S.E.M. of at least 3 independent experiments. For the post-hatched period the data is the mean _+ S.E.M. of 7 independent experiments. Dopamine and apomorphine were used at a concentration of 10-~ M.
Developmental stage (days) Embryonic 10 11 12 13 16 Post-hatched 3
Basal
Dopamine Apomorphine (pmol cA MP/mg protein)
Apomorphine + dopamine
18.0 20.0 19.0 20.8 66.2
-201.0 228.6 + 26.0 246.2 + 10.0 372.2 + 34.0
--112.0 _+ 14.0 57.6 + 4.0 167.0 + 27.0
_+ 4.2 _+ 1.8 + 4.2 _+ 14.0
44.1 _.+4.6
89.6 + 8.0
49.9 -83.9 55.2 113.0
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65.0 _+ 7.0
68.0 _+ 6.0
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Fig. 5. Inhibition of specific [3H]spiperone binding in crude membrane preparation of chicken retina by antagonist and agonist drugs. The specific binding was defined as the amount of [3H]spiperone displaced by 1 ~M (+)butaclamol. Each point is the mean value of 2-3 experiments with quadruplicate determinations in each experiment. S.E.M. values were less than 15% of themean. The concentration of [3H]spiperone used was in the range of 0.85-1.6 nM.
in the pre-hatching period and remained equally high in retinas of post-hatched chicken. The K d of spiperone for these sites as estimated by Scatchard analysis in retinas from post-hatch animals was approximately 1 nM, with an estimated Bma x of 100 fmol/mg protein (Fig. 4, inset). The displacement of [3H]spiperone binding by antagonist and agonist drugs is shown in Fig. 5. With a Ki value of 1.6 nM, spiperone was the most effective compound to compete with [3H]spiperone. In addition, while (+)butaclamol ( K i = 31 nM) displaced bound [3H]spiperone, (--)butaclamol did not compete for spiperone binding sites up to 1000 nM concentration. Among the agonist drugs tested, apomorphine was the most potent compound with a K i value equal to 8 nM, followed by dopamine, with a K i value of 156 nM. Serotonin was not a good displacer of bound [3H]spiperone, showing a K i greater than 26,000 nM. These experiments indicate that [3H]spiperone binds to dopaminergic receptor sites of the D 2 type. Moreover, in contrast to the high affinity shown by spiperone for the D 2 receptors, this compound was a poor inhibitor of the D 1receptor coupled to the cyclase system. Inhibition of 50% of the cAMP accumulation promoted by 1 I~M dopamine was achieved only with 1/xM spiperone (data not shown). DISCUSSION
Our data show that the embryonic chick retina is
capable of responding to dopamine with increased cAMP level in a very early stage of embryonic development. All the agonists tested were effective in promoting cAMP accumulation in the tissue. However, [3H]spiperone bound very little to crude membrane preparation of retina from early embryonic stages. High stereospecific binding of [3H]spiperone was only detected after embryonic day 18, with kinetic characteristics of interaction with D 2 receptors. These data put together show that D1 and D 2 receptors of the chick retina follow a different pattern of differentiation, suggesting that they may be in different cells. Since cyclase response of chick retina to dopamine can be detected already in embryos on embryonic day 7, the embryonic D 1 receptors could be important in decoding differentiation signals triggered by dopamine, when functional dopaminergic synapses are formed in the tissue. The occurrence of dopamine in the embryonic chick retina was first detected on day 15 of development 14, time coincident with appearance of the first synaptic structures in the tissue 12,19,22. An endogenous increase in cAMP level of the embryonic chick retina is also observed during this period 4, probably due to functional dopaminergic synapses that are formed after day 15 of development 5. While D 2 receptors increase after embryonic day 18, cyclase response to dopamine decreases considerably during the same period. The low cyclase responsiveness of the mature tissue to dopamine is possibly due to a desensitization of the D I dopaminergic system. We have recently shown that dopamine-dependent cAMP accumulation desensitizes in cultured retina cells chronically exposed to dopamine. It was also shown that light deprivation associated with decreased dopamine release 13 could induce a dopamine hyperresponsive state in retinas of young chicken 6. Our data are therefore consistent with the idea that endogenous dopamine output of dopaminergic synapses leads to the observed desensitization of the D 1 system of the tissue and that this system is regulated 'in vivo' during embryonic development. Although apomorphine promotes an increase in the cAMP level of the retina, it is only 20-30% as effective as ADTN or dopamine itself. Moreover, while the dopamine responsiveness of the tissue decreases sharply during its ontogeny, the apomorphine-dependent cAMP accumulation decreases
222 only slightly during the same period. In addition, as opposed to what is observed in the early embryonic stages of retina differentiation, both dopamine and apomorphine are equally effective in increasing cAMP accumulation of retinas from post-hatched animals. These data suggest that apomorphine me-
er subclass of D 1 receptor. The differential desensitization of responses to dopaminc and apomorphine does not seem consistent with apomorphine being a simple partial agonist for a uniform population of D 1 receptors during embryonic development. Also, if apomorphine was acting as a partial agonist, the dose
diates its effect via a subclass of D 1 receptors whose sensitivity to apomorphine remains practically con-
needed to inhibit stimulation by 100/~M dopamine should be about two orders of magnitude greater
stant during retina ontogeny. This idea is strength-
than observed.
ened by the fact that the time course of dopaminergic desensitization of cultured embryonic retina cells exposed to saturating concentrations of dopamine, reveals two decaying kinetic components with half lives of 6 and 22 h respectively~. Our data also show that apomorphine is a potent
ACKNOWLEDGEMENTS
competitive inhibitor of the highly dopamine respon-
This work was supported by grants to F . G . d . M . from CNPq, F I N E P and C P E G U F R J ; and partly by the Brazilian American Institute of Rio de Janeiro
sive D1 system present in the embryonic tissue, indicating that this compound also interacts with this oth-
(IBEU). A . L . M . V . is the recipient of a fellowship from CAPES.
REFERENCES
10 Haggendal, J. and Malmfors, T., Identification and cellular localization of the catecholamines in the retina and the choroid of the rabbit, Actaphysiol. scand., 64 (1965) 58-66. 11 Hamburger, V. and Hamilton, H. L., A series of normal stages in the development of the chick embryo, J. Morph.. 88 (1951) 49-92. 12 Hughes, W. F. and La Velle, A., On the synaptogenic sequence in the chick retina, Anat. Rec., 179 (1974) 297-302. 13 Iuvone, P. M., Galli, C. L., Garrison-Gund, C. K. and Neff, N. H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons, Science, 202 (1978) 901-902. 14 Kato, S., Nakamura, T. and Negishi, K., Postnatal development of dopaminergic cells in the rat retina, J. comp. Neurol., 191 (1980) 227-236. 15 Kebabian, J. W. and Calne, D. B., Multiple receptors for dopamine, Nature (Lond.), 277 (1979) 93-96. 16 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951)265-275. 17 Makman, M. H., Dvorkin, B., Horowitz, S. G. and Thai. L. J., Properties of dopamine agonist and antagonist binding sites in the mammalian retina, Brain Res., 194 (1980) 403-418. 18 Matsuzawa, H. and Nirenberg, M. W., Receptor mediated shifts in cGMP and cAMP levels in neuroblastoma cells, Proc. nat. Acad. Sci. U.S.A., 72 (1975) 3472-3476. 19 Meller, K., Elektronenmikroskopische befunde zur differenzierung der rezeptorzellen und bipolarzellen der retina und ihrer synaptischen verbindungen, Z. Zellforsch., 64 (1964) 733-750. 20 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 (1982) 493-500. 21 Seeman, P., Brain dopamine receptors, Pharmacol. Rev., 32 (1980) 229-313.
1 Brown. J. H. and Makman, M. H., Stimulation by dopamine of adenylate cyclase in retinal homogenates and adenosine-Y-5'-cyclic monophosphate formation in intact retina, Proc. nat. Acad. Sci. U.S.A., 69 (1972)53%543. 2 Bucher, M B. and Schorderet, M., Dopamine and apomorphine sensitive adenylate cyclase in homogenates of rabbit retina, Naunyn-Schmiedeberg's Arch. exp. Pharmacol., 288 (1975) 103-107. 3 Creese, L., Burt, D. R. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity, Science, 197 (1977) 596-598. 4 De Mello, F. G., The ontogeny of dopamine-dependent increase of adenosine-3'-5'-cyclic monophosphate in the chick retina, J. Neurochem., 31 (1978) 1049-1053. 5 De Mello, F. G. and de Mello, M. C. F., Dopamine dependent modulation of cAMP level in the chick retina. In R. Levi-Montalcini (Eds.), Nerve Cells, Transmitters and Behaviour, Pontificiae Academiae Scientiarum Scripta Varia, Vaticano, 1980, pp. 343-355. 6 De Mello, M. C. F., Ventura, A. L. M., Paes de Carvalho, R., Klein, W. L. and de Mello, F. G., Regulation of dopamine- and adenosine-dependent adenylate cyclase systems of chicken embryo retina cells in culture, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 5708-5712. 7 Ehinger, B. and Flack, B., Adrenergic retinal neurons of some new world monkeys, Z. Zellforsch., 100 (1969) 364-375. 8 Frey, E. A., Cote, T. E., Grewe, C. W. and Kebabian, J. W., 3H-spiroperidol identifies a D2 dopamine receptor inhibiting adenylate cyclase activity in the intermediary lobe of the rat pituitary gland, Endocrinology, 110 (1982) 1897-1904. 9 Gilman, A. G., Protein binding assay for adenosine-3'-5'cyclic monophosphate, Proc. nat. Acad. Sci. U.S.A., 67 ( 197013/)5-312.
223 22 Sheffield, J. B. and Fischman, D, A., Intercellular junctions in the developing neural retina of the chick embryo, Z. Zellforsch., 104 (1970) 405-418. 23 Watling, K. J., Dowling, J. E. and Iversen, L. L., Dopamine receptors in the retina may all be linked to adenylate cyclase, Nature (Lond.), 281 (1979) 578-580. 24 Watling, K. J., Dowling, J. E. and Iversen, L. L., Dopaminergic mechanisms in the carp retina: effects of dopa-
mine, K ÷ and light on cyclic AMP synthesis. In Neurochemistry of the Retina, Pergamon Press, Oxford, 1980, pp. 519-537. 25 Watling, K. J. and Iversen, L. L., Comparison of the binding of 3H-spiperone and 3H-domperidone in homogenates of mammalian retina and caudate nucleus, J. Neurochem., 37 (1981) 113/)-1143.