The present work shows the existence of adenosine-dependent cyclic adenosine monophosphate (AMP) accumulation in the chick optic tectum. When tecta ...
Developmental Brain Research, 35 (1987) 141-147 Elsevier
141
BRD 50592
Development of adenosine-dependent cyclic AMP accumulation in the avian optic tectum Ana Lucia Marques Ventura and Roberto Paes de Carvalho Departamento de Neurobiologia, lnstituto de Biologia, Universidade Federal Fluminense, Niteroi (Brazil) (Accepted 3 February 1987) Key words: Adenosine; Cyclic adenosine monophosphate; Optic tectum; Neurotransmitter; Chick embryo
The present work shows the existence of adenosine-dependent cyclic adenosine monophosphate (AMP) accumulation in the chick optic tectum. When tecta from 18-day-old embryos were incubated with the phosphodiesterase inhibitor IBMX and RO 20-1724, the cyclic AMP level increased from 39.2 to 73.3 and 285.5 pmol/mg protein, respectively. The high level obtained with RO 20-1724 could be inhibited by increasing concentrations of IBMX or by adenosine deaminase, but not by dipyridamole. 2-Chloroadenosine promoted a dose-dependent cyclic AMP accumulation in tecta incubated with RO 20-1724 and adenosine deaminase. This effect was blocked by IBMX and varied substantially during the development of the tissue. The degree of stimulation increased after day 11 of incubation, attaining maximal levels on day 14. The effect of 2-chloroadenosine remained constant until day 18, a period when both the protein content and the basal cyclic AMP levels are increasing in the developing tectum. The cyclic AMP increase elicited by 2-chloroadenosine was greatly reduced in tecta from 20-day-old embryos and 2-day-old chicks. The putative transmitters glutamate and glycine and the neurotransmitter analogs isoproterenol and carbachol had no stimulatory effect on the cyclic AMP accumulation of tecta from 10- and 17-day-old embryos.
INTRODUCTION The optic tectum is an area of the CNS which receives the m a j o r i t y of the ganglion cell axons from the retina. The n e u r o t r a n s m i t t e r s released by retinal ganglion cell terminals are not known at present. T h e excitatory amino acids g l u t a m a t e and aspartate, as well as G A B A and serotonin, have b e e n d e t e c t e d in the pigeon optic tectum 1s'33, and in its h o m o l o g o u s structure, the superior colliculus of the rat 26. High affinity receptors for G A B A and acetylcholine 29 and the cholinergic enzymes choline acetyltransferase (CHAT) and acetylcholinesterase (ACHE) have been detected in the pigeon and chick tectum 17'31. Recently, n e u r o p e p t i d e - l i k e immunoreactivity, organized in a laminar pattern, has been shown to occur in the anuran tectum ~9. A d e n o s i n e is considered to be a n e u r o t r a n s m i t t e r or n e u r o m o d u l a t o r in the CNS 32. This nucleoside can
be released by electrical stimulation of neural tissues as well as by depolarizing agents 2.15. It has a potent inhibitory effect in a variety of electrophysiological p h e n o m e n a . These effects are believed to be due to the inhibition of excitatory transmitter release 7,s. In addition, hyperpolarizing postsynaptic effects of adenosine have also been r e p o r t e d 1. A d e n o s i n e regulates adenylate cyclase by interacting with two types of m e m b r a n e receptors and with an intracellular inhibitory site. The m e m b r a n e receptors are n a m e d A 1 and A 2 and their activation by adenosine leads to inhibition and stimulation of adenylate cyclase, respectively 3a. The presence of A 1 adenosine receptors in several areas of the CNS has been detected by ligand binding studies 3'1°. The distribution of these receptors 1°'2°, as well as of adenosine transport sites 12, is not ubiquitous in the CNS. Previous studies have d e m o n s t r a t e d the existence of both A 1 and A2 adenosine receptors in the chick
Correspondence: A.L. Marques Ventura, Departamento de Neurobiologia, Universidade Federal Fluminense, Caixa Postal 229, Niteroi 24000, Rio de Janeiro, Brazil. 0165-3806/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
142 embryonic retina 27'2s. The nucleoside promotes the accumulation of cyclic AMP in the intact retina as well as in cultured cells from the chick embryo. These effects are mediated by the activation of A 2 receptors whose expression begins on day 14 of development 27. Adenosine A 1 receptors are also present in the chick embryo retina and their activation promotes the inhibition of the dopamine-dependent cyclic AMP accumulation of this tissue 28. Adenosine A] receptors 14 as well as adenosine uptake sites are co-localized with the enzyme adenosine deaminase in the rat superior colliculus 25. The present work shows that the non-metabolizable adenosine analog 2-chloroadenosine promotes the accumulation of cyclic AMP in the chick optic tectum. This effect is probably mediated by the activation of adenosine receptors of the A 2 subtype. The sensitivity of the rectum to this nucleoside varies significantly during the development of the chick, suggesting a neuromodulatory role for adenosine in this tissue. MATERIAL AND METHODS Cyclic AMP, protein kinase from bovine heart, bovine serum albumin, adenosine deaminase (E.C. 3.5.4.4) type II, 2-chloroadenosine, dipyridamole, dopamine (Sigma, St. Louis, MO); IBMX (Aldrich, Milwaukee, WI); RO 20-1724 (Hoffmann-La Roche, Nutley, NJ); [3H]cyclic AMP (New England Nuclear, Boston, MA); basal medium of Eagle (BME) (Gibco, Grand Island, NY); all other reagents were of analytical grade. Fertilized white leghorn eggs were obtained from a local hatchery.
containing 25 mM HEPES. pH 7.4 (adjusted with NaOH 1 N) and 0.5 mM RO 20-1724 at 37 °C for 10 min. Then, each test compound, at the required concentration, was added and the tissue was incubated for an additional 10 rain, except when otherwise indicated. The reactions were interrupted by the addition of TCA to a 5% final concentration. The cyclic AMP was purified according to Matsuzawa and Niremberg 23 and assayed by the method of Gilman I3. The protein content was determined by the method of Lowry et al. 22, using bovine serum albumin as standard. RESULTS Fig. 1A shows that a significant increase of tectal protein was observed during the course of tissue differentiation. The protein concentration was low (approximately 1.5 mg/tectum) in early developmental stages (10- to 12-day-old embryos) and gradually increased after this period. In tecta from 17-day-old embryos, a protein content of 4.5-5.0 mg/tectum was detected. This relatively high value remained unchanged in tecta from post-hatched chicks. The basal cyclic AMP content of the chick tectum also increased gradually during the development (Fig. 1B). Freshly dissected tecta were immediately
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Dissection and incubation procedures The embryos were staged according to Hamburger and Hamilton 16 and immediately decapitated. After removal of the tissues surrounding the embryo encephalon, the optic lobes were obtained by cutting the pedunculi and immediately transferred to cold Hank's balanced saline solution without calcium and magnesium (CMF). Then, the involving membranes were removed and the tecta divided into 6 or 7 pieces which were quickly washed in the same saline and transferred to the incubation medium. All incubations followed the procedure previously described 5. Briefly, each tectum was incubated in 2 mi of BME
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Fig. 1. A: protein content of the chick optic tectum as a function of the developmental stage. Points represent the mean of at least 3 tissue samples determined in duplicates. Bars represent the S.E.M. B: developmental profile of the basal cyclic AMP levels in the chick embryo optic tectum, Data represent the cyclicAMP content measured in two separate experiments assayed in duplicate. Bars represent the deviation from the mean.
143 transferred to a 5% T C A solution and the cyclic A M P was purified and d e t e r m i n e d . The cyclic A M P level of tecta from 12-day-old e m b r y o s was approximately 18.5 p m o l / m g protein, a value that r e m a i n e d constant until day 14 of d e v e l o p m e n t . In later stages the cyclic A M P concentration increased, attaining the level of 48.0 + 2.4 p m o l / m g p r o t e i n at hatching. In tecta from 2-day-old chicks, a lower cyclic A M P level was o b s e r v e d (33.7 + 2.4 pmol/mg protein). The cyclic A M P levels in freshly dissected tissues usually reflects the steady-state concentration of the nucleotide. The rise in cyclic A M P concentration observed during the e m b r y o n i c d e v e l o p m e n t of tectum could be due, at least in part, to a change in cyclic A M P p h o s p h o d i e s t e r a s e activity or to the a p p e a r ance of a stimulation of a d e n y l a t e cyclase by an endogenous m o d u l a t o r . R O 20-1724 is a p o t e n t phosphodiesterase inhibitor in m a n y tissues and I B M X , besides being an inhibitor of this enzyme, is also an adenosine antagonist 3'32. Previous work showed that retina cells r e s p o n d distinctly to these two compounds because of the stimulation of adenylate cyclase by e n d o g e n o u s adenosine 27. Optic tecta from 16-day-old e m b r y o s were incubated for 20 min at 37 °C in m e d i u m containing R O 20-1724 and increasing concentrations of I B M X (Fig. 2). In the presence of R O 20-1724 the cyclic A M P concentration increased from 26.8 + 2.2 to 133.0 + 24.5 pmol/mg protein. The incubation of the tissues with R O 201724 plus increasing concentrations of I B M X produced a d o s e - d e p e n d e n t reduction in the cyclic A M P levels o b s e r v e d with R O 20-1724 alone. A 50% reduction of tectal cyclic A M P content from 16-day-old e m b r y o s was o b t a i n e d with a p p r o x i m a t e l y 200 g M IBMX. The inhibition of cyclic A M P accumulation induced by I B M X suggested the existence of an adenosine-sensitive adenylate cyclase system in this area of the visual system, stimulated by e n d o g e n o u s nucleoside released during the incubation of the tissue. This idea was c o r r o b o r a t e d by the e x p e r i m e n t s shown in Table I. Tecta from 18-day-old e m b r y o s were incub a t e d for 20 min at 37 °C with 0.5 m M R O 20-1724 plus the drugs indicated. In some cases, 100/xM 2chloroadenosine was a d d e d to the m e d i u m 10 min after the beginning of the incubation period. W h e n tecta were incubated in the presence of R O 20-1724 alone, a high cyclic A M P content was o b s e r v e d
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Fig. 2. Effect of increasing concentrations of IBMX on the cyclic AMP level of tecta from 19-day-old embryos incubated with RO 20-1724. The tissues were dissected in CMF and incubated for 20 min at 37 °C in BME buffered with HEPES, pH 7.4, containing 0.5 mM RO 20-1724 and the indicated concentrations of IBMX. Results represent the mean of two separate experiments assayed in duplicate. Bars represent the deviation from the mean.
TABLE I Effect of adenosine deaminase, phosphodiesterase inhibitors and dipyridamole on the cyclic A M P levels of tecta from 17-18day-old chick embryos
Tecta were incubated for 20 min at 37 °C in BME buffered with HEPES, pH 7.4, containing the indicated drugs. 2-Chloroadenosine was added to the medium 10 min after the beginning of incubation. Numbers in parentheses represent the number of experiments performed in each case. Additions to the medium
pmol/mg protein +_deviation from the mean
None IBMX RO 20-1724 RO 20-1724 + adenosine deaminase RO 20-1724 + IBMX RO 20-1724 + 2-chloroadenosine RO 20-1724 + adenosine deaminase + 2-chloroadenosine RO 20-1724 + adenosine deaminase + 2-chloroadenosine + IBMX RO 20-1724 + dipyridamole
39.273.3 + 285.5 + 63.2 + 68.4 + 274.8 +
1.3 (2) 21.1 (2) 20.1 (4) 2.8 (2) 11.1 (2) 29.5 (2)
224.9 + 15.0 (2) 52.5 _+5.0 (2) 292.8 _+80.1 (4)
144 (285.5 + 20.1 pmol/mg protein). In contrast, when the tissue was incubated with R O 20-1724 plus 0.5 U/ml adenosine d e a m i n a s e or 0.5 m M I B M X , the cyclic A M P levels decreased to 63.2 + 2.8 and 68.4 + 11.1 pmol/mg protein, respectively. The addition of 100 ~tM 2-chloroadenosine to the incubation m e d i u m containing only R O 20-1724 did not p r o m o t e a significant increase of the cyclic A M P level. On the other hand, 100/~M 2-chloroadenosine induced a 3-fold increase in the cyclic A M P content when tectum was incubated with R O 20-1724 and adenosine deaminase (from 63.2 + 2.8 to 224.9 + 15.0 pmol/mg protein). The cyclic A M P accumulation induced by 2chloroadenosine was c o m p l e t e l y abolished when the CI
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Fig. 3. Effect of increasing concentrations of 2-chloroadenosine on the cyclic AMP level of tecta from 19-day-old chick embryos incubated with RO 20-1724 plus adenosine deaminase. Tecta were incubated for 10 min at 37 °C in BME buffered with HEPES pH 7.4, containing 0.5 mM RO 20-1724 plus 0.5 U/ml adenosine deaminase. At the end of this period the indicated concentrations of 2-chloroadenosine were added and the tissues were further incubated for another 10 min. The reaction was stopped by TCA and the cyclic AMP was purified and determined as described in Materials and Methods. Points are the mean of two separate experiments in which the cyclic AMP content was assayed in duplicate. Bars represent the deviation from the mean. Inset: effect of 2-chloroadenosine on the cyclic AMP levels of tecta from 14-day-old chick embryos as a function of incubation time. The incubation procedure was the same as described above except that the reaction was stopped at the indicated times after the addition of 100 gM 2chloroadenosine. Open circles represent the basal cyclic AMP and closed circles the stimulated levels. Points are the mean of two separate experiments assayed in duplicate and the bars represent the deviation from the mean.
tissue was pre-incubated with I B M X (Table I). Dipyridamole, a c o m p o u n d known to block adenosine uptake 32'34, did not reduce cyclic A M P accumulation o b s e r v e d when the tissue was incubated with R O 201724 alone (Table I). The effect of increasing concentrations of 2-chloroadenosine, an adenosine analog resistant to adenosine deaminase, upon the cyclic A M P content of tecta from 19-day-old e m b r y o s incubated in the presence of R O 20-1724 and adenosine deaminase, is illustrated in Fig. 3. The nucleoside p r o m o t e d a dosed e p e n d e n t accumulation of cyclic A M P and a 3-fold increase was o b s e r v e d with 2-chloroadenosine concentrations equal to or higher than 10/~M. The EDs0 estimated from these experiments was approximatel y 3/~M. M o r e o v e r , 2-chloroadenosine-elicited cyclic A M P accumulation is t i m e - d e p e n d e n t (Fig. 3, inset). The cyclic A M P level increased during the first period of incubation, attaining equilibrium after 10 mln. In o r d e r to study the d e v e l o p m e n t of the adenosine-dependent cyclic A M P accumulation in the chick optic tectum, tissues from e m b r y o s at several ages were incubated for 10 rain in B M E containing R O 20-1724 plus adenosine deaminase. A f t e r this period, 100/tM 2-chloroadenosine was a d d e d and the tecta were further incubated for 10 min. The results of these experiments are shown in Fig. 4. A t . 5 - 2 . 0 fold stimulation was o b t a i n e d when the tecta from 10- to l l - d a y - o l d e m b r y o s were used. A gradual increase in the response to 2-chloroadenosine was observed in the subsequent days and a maximal effect (3-fold stimulation) was observed in tecta from 14day-old embryos. This level of cyclic A M P accumulation r e m a i n e d the same until e m b r y o n i c day 18. In tecta from 20-day-old e m b r y o s and 2-day-old posthatched chicks, a low level of response to the adenosine analog was o b s e r v e d ( a p p r o x i m a t e l y 1.5-fold). The cyclic A M P increase p r o m o t e d by 2-chloroadenosine in the optic tectum could be m e d i a t e d by the release of other putative neurotransmitters that could activate adenylate cyclase systems eventually present in the tissue. To investigate this possibility, we studied the effect of some c o m p o u n d s upon tectum cyclic A M P content. Table II shows that when tecta from 10- and 17-day-old e m b r y o s were incubated with 2-chloroadenosine, their cyclic A M P content increases from 34.9 to 71.7 and from 72.0 to
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Effect of some putative neurotransmitters and agonists on the cyclic A M P level of tectum from developing chick embryos
The incubation procedure was the same as described in the legend to Fig. 3. Data are expressed in pmol/mg protein + deviation from the mean of two separate experiments assayed in duplicate; n.t., means not tested.
2-chl~roadenosin
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