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Keywords:

  • chick;
  • desensitization;
  • in ovo injection;
  • neurotransmitter;
  • receptor up- and down-regulation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N6-cyclohexyladenosine (CHA) or antagonist 8-Cyclopentyl-1, 3-dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [3H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A2A agonist 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [3H]DPCPX binding, and reduced A2A receptors. The A2A antagonists 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-trizolo[1,5-c] pyrimidine (SCH58261) and 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S-(4-nitrobenzyl)-6-thioinosine (NBMPR) also reduced [3H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [3H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long-term treatment with A1 and A2A receptors modulators.

Abbreviations used
CGS21680

3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino] ethyl]phenyl] propanoic acid

CHA

N6-cyclohexyladenosine

DMSO

dimethyl sulphoxide

DPCPX

8-Cyclopentyl-1, 3-dipropylxanthine

NBMPR

S-(4-nitrobenzyl)-6-thioinosine

SCH58261

7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-trizolo[1,5-c] pyrimidine

ZM241385

4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol

Neurotransmitters and different chemical mediators regulate several important events during CNS development, such as neurite outgrowth, growth cone motility (Lankford et al. 1987; Kater and Lipton 1995), cell morphology (Cepko et al. 1996), synapse formation, and cell death (Cook et al. 1998; Calaza and Gardino 2010). Interestingly, the expression onset of several neurotransmitter receptors is an early event during embryogenesis and in many cases precedes synapse formation (Hokoç et al. 1990; Gardino et al. 1993), suggesting that these early events are fundamental for the correct formation and functioning of CNS.

The retina is an essential part of the visual system as it transduces the light stimulus and sends a pre-processed signal to brain areas. The chick retina is a useful model to study retinal development and specially the role played by chemical mediators during development. This model is suitable for in vitro studies as the embryonic retinal cells can be dissociated and grown in culture for relatively long periods (de Mello et al. 1982; Adler et al. 1984; Lankford et al. 1987). The developing chick embryo is also a very interesting model for in vivo studies, in particular by using the injection of different substances in the eggs at specific developmental periods (Linser and Moscona 1979; Magalhães et al. 2006; Pohl-Guimarães et al. 2010; Socodato et al. 2011). The knowledge about the development of different neurotransmitter systems and signaling pathways, as well as the interference of distinct drugs at specific developmental periods could also be important to study disease mechanisms and to evaluate possible side effects of drugs eventually chosen to treat some diseases during pregnancy or in infants.

Adenosine is an important neuromodulator in both central and peripheral nervous system. The main function of adenosine in the adult CNS appears to be the regulation of excitatory neurotransmitter release (Sebastião and Ribeiro 2000), affecting sleep (Porkka-Heiskanen and Kalinchuk 2010), synaptic plasticity (de Mendonça and Ribeiro 2001), and cell death induced by glutamate (Ferreira and Paes de Carvalho 2001). The nucleoside activates four types of G protein-coupled receptors named A1 and A3 receptors, which inhibit adenylyl cyclase (AC) by activating Gi protein, and A2A and A2B receptors, which classically increase AC activity through Gs/Golf (Fredholm et al. 2001).

The adenosine system has been shown to be present since early stages of chicken retinal development. Immunostaining for adenosine was found in an early stage of development (embryonic day 12, E12) and after hatching (Paes-de-Carvalho et al. 1992). Adenosine uptake and release occur mainly through equilibrative transporters (Thorn and Jarvis 1996 for review; Visser et al. 2002). In the retina, equilibrative transporters are found since E8 at all retina extensions and in later stages restricted to plexiform layers (Paes-de-Carvalho et al. 1992). A1 receptors are expressed in the outer plexiform layer and inner retina since E10 (Paes-de-Carvalho 1990; Paes-de-Carvalho et al. 1992) and promote the inhibition of cyclic AMP accumulation induced by dopamine (Paes-de-Carvalho and Mello 1985). It has also been previously shown that adenosine has the ability to induce cyclic AMP accumulation in retinas of chick embryos at E14, reaching a peak at E17 and a large decrease is observed in post-hatched animals (Paes-de-Carvalho and Mello 1982). The presence of the adenosine system throughout development suggests it might have a major role in retina functionality. Indeed, it has been shown that activation of A2A receptors inhibits calcium influx (Stella et al. 2002) and induces opsin expression in salamander (Alfinito et al. 2002). Moreover, activation of A1 receptors in cultured retinal cells also reduces calcium influx induced by depolarization (Santos et al. 2000) or glutamate (Hartwick et al. 2004). Considering these studies and the reports showing that many aspects of CNS development are regulated by calcium and cyclic AMP, it seems important to understand the mechanisms that regulate the expression of adenosine receptors.

Prolonged activation of G protein-coupled receptors leads to a decrease in receptor activity and/or expression, a phenomenon called homologous desensitization. It has been shown in different species, both in culture and in vivo models, that chronic exposure of A1 receptors to selective agonists decrease receptor protein and mRNA levels as well as Gi protein (Fernandez et al. 1996; Ciruela et al. 1997; Vendite et al. 1998; Roman et al. 2008; León et al. 2009). Recent evidence (Pereira et al. 2010) showed an in vitro and ex vivo modulation of A1 receptor expression in chick retinal cell monolayer and explant cultures incubated for 3–5 days with A2A receptor agonists. However, it is important to use an in vivo approach to verify the existence of these mechanisms in a more preserved microenvironment. Therefore, the main goal of this study was to evaluate the regulation of A1 receptor expression in retinas of chicken embryos treated in ovo with A1 or A2A receptor agonists and antagonists for 48 h.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

Adenosine deaminase, N6-cyclohexyladenosine (CHA), 2-p-(carboxyethyl) phenethylamino-5′-N-ethylcarboxyaminoadenosine hydrochloride (CGS21680), S-(4-nitrobenzyl)-6-thioinosine (NBMPR), glutamine, bovine serum albumin, Triton X-100, and dimethyl sulphoxide (DMSO) were obtained from Sigma/RBI (St Louis, MO, USA); anti-A1 receptor and anti-A2A receptor antibodies were from Chemicon International (Billerica, MA, USA). [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, 130 Ci/mmol), anti-rabbit secondary antibody, glycine, sodium dodecyl sulfate, Tris, acrylamide polyacrylamide gel electrophoresis, N-methylene-bis-acrylamide, mercaptoethanol, hyperfilm, ECL kit, first-strand cDNA synthesis kit, DNase I, DEPC-treated water were purchased from Amersham Pharmacia Biotech UK Limited (Buckinghamshire, UK). 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385, Tocris, Ellisville, MO, USA) was a gift from Dr. Rodrigo A. Cunha (Coimbra University, Coimbra, Portugal). Trizol reagent was supplied by Invitrogen Corporation (Carlsbad, CA, USA) .

Injection of adenosine agonists or antagonists in the egg

Fertilized White Leghorn eggs were obtained from a local hatchery and incubated at 37.8°C and a relative humidity of 80–90%. The procedures for the use of animals were in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ and approved by the commission of animal care CEPA/PROPPi from Federal Fluminense University, protocol #112.

In sterile condition, eggs at E14 were injected in their air chambers with 10 μL of CHA, DPCPX, CGS21680, SCH58261, ZM241385 (600 μM diluted in DMSO), or 6 μL of NBMPR (10 μM diluted in DMSO). At this stage, the approximate volume of the egg is around 60 mL and so we estimated that the final concentrations of CHA, DPCPX, CGS21680, SCH58261, and ZM241385 in the retina would be around 100 nM, whereas the concentration of NBMPR would be around 1 μM. For the time curve of CGS21680, eggs were injected at E14 with CGS21680 and killed 6, 18, 24, and 48 h later. Control animals were injected with 10 μL (or 6 μL for NBPMR experiment) of DMSO.

The eggs with the injected embryos returned to incubator until being killed 48 h later (at E16). The staging of the embryos was systematically determined based on Hamburger and Hamilton (1951). The retinas were immediately dissected and processed for RT-PCR, western blot, or radioligand assays.

Binding assays in homogenates

E16 retinas were dissected from other ocular tissues and retinal pigmented epithelium in calcium and magnesium-free balanced salt solution, and homogenized in Tris-HCl (50 mM, pH 7.4). Homogenates were centrifuged and the pellet was incubated with adenosine deaminase (0.5 U/mL) for 30 min, centrifuged, and stored at – 70°C until use. Samples containing 0.1 mg protein were incubated for 1 h with 5 nM [3H]DPCPX. Specific binding was calculated as the total binding minus the non-specific binding achieved in the presence of 100 μM non-radioactive CHA.

For kinetic studies of A1 receptors, homogenates were incubated for 1 h with 5 nM [3H]DPCPX in the presence of increasing concentrations of non-radioactive DPCPX (0.1–100 nM). For saturation analyses, samples were incubated in the presence of increasing concentrations of [3H]DPCPX (0.2–15 nM). The radioactivity was determined by scintillation spectroscopy. Protein was determined by the method of Lowry et al. (1951). The data were analyzed by curve fitting (one site –specific binding equation) using the Graphpad Prism program.

Western blot

Retinas were dissected in calcium and magnesium-free balanced salt solution, homogenized in sample buffer, and boiled for 5 min. Total protein amount in each sample was determined using Bradford reagent, with bovine serum albumin as standard. Samples containing 60 μg protein were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the proteins transferred to polyvinylidene fluoride membranes, which were incubated overnight with the antibodies anti-A1 adenosine receptor (1 : 100) or anti-A2A adenosine receptor (1 : 1000). Then, membranes were washed, incubated with peroxidase-conjugated anti-rabbit secondary antibody 1 : 500 (A1 receptor) and 1 : 3000 (A2A receptor), and revealed by ECL chemiluminescence. To obtain the loading control, after revelation, membranes were washed in Tris-buffered saline (20 mM Tris, 200 mM NaCl, pH 7.6) and incubated for 30 min with glycine 0.2 M (pH 2.2) to remove primary and secondary antibodies. Then, membranes were incubated overnight with α-tubulin antibody (1 : 70 000), washed, incubated with peroxidase-conjugated anti-mouse secondary antibody 1 : 5000, and revealed by the ECL chemiluminescence. Quantitative analysis of blots was performed by scanning images and using the computer program Scion Image (Scion Corporation, Frederick, MD, USA).

Real-time quantitative PCR

Total retinal RNA was extracted using Trizol reagent, according to the manufacturer's protocol. Extracted RNA samples were treated for 10 min at 37°C with RNase-free DNase I 0.5 U/μg of RNA. First-strand synthesis of cDNA was performed in a solution containing 1 μg RNA, reverse transcriptase, random hexamers, and dithiothreitol for 1 h at 37°C using First-Strand cDNA synthesis kit (GE Healthcare Life Sciences, Buckinghamshire, UK) . The quantitative real-time PCR amplification was performed using GoTaq qPCR Master Mix (Promega, Sunnyvale, CA, USA) and carried out on a Real-Time PCR System (StepOne; Applied Biosystems, Carlsbad, CA, USA) under the following conditions: an initial holding stage at 95°C for 2 min was followed by 40 cycles: denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min. Melt curve was performed (slow ramp of 0.3°C/20 s to 95°C). Specific primer sequences, corresponding base sites, and size of PCR products to A1 adenosine receptor were as follows: (GenBank sequence no S78192), 5′-CTTCTTCGTCTGGGTCCTG-3′ (base position 1032) and 5′-ATCTGCAGGAAGGCTGTCC-3′ (base position 1367). Specific primer sequences to L27, ribosomal protein used in all PCR experiments as an internal control (GenBank sequence no X56852) were as follows: 5′-AAGCCGGGGAAGGTGGTG-3′ (base position 42) and 5′- GGGTGGGCATCAGGTGGT-3′ (base position 276). Data were analyzed and quantified using Step-One software (Applied Biosystems). Standard curves were produced by amplification of DNA dilution series and the amount of samples was calculated from standard curve.

Statistics

Data were analyzed using one-way anova (three or more experimental groups) followed by the Bonferroni post-test or t-test (two experimental groups) using the software Graphpad Prism (GraphPad Software Inc., San Diego, CA, USA). Data are shown as mean ± standard error of the mean (mean ± SEM).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In vivo modulation of A1 receptor activity regulates its own expression

To study the regulation of A1 receptors in vivo, E14 chicken eggs were injected with A1 receptor agonists and/or antagonists, and, 48 h later, E16 retinas were dissected and processed for western blot or binding of [3H]DPCPX. This particular time window (E14–E16) was chosen mainly because of the developmental features of the adenosine system. Adenosine shows the ability to induce cyclic AMP accumulation in retinas of chick embryos from E14 and this effect is maximal at E16/E17 (Paes-de-Carvalho and Mello 1982). Moreover, the expression of A1 receptors also peaks around E16 (Paes-de-Carvalho 1990) making this period appropriate to study the regulation of A1 receptor expression and its modulation by A2A receptors. As shown in Fig. 1a and b, injection of N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, decreased the level of A1 receptor protein by 29.9 ± 5.0% when compared with control. This result suggests that CHA injected in ovo is able to activate A1 receptor and stimulate its down-regulation. On the other hand, treatment with DPCPX, a selective A1 receptor antagonist, induced an increase of 27 ± 17.6% in the levels of this protein. These results are in agreement with previous published data showing that chronic inhibition or stimulation of A1 receptor leads to a respective up-regulation (Fastbom and Fredhom 1990; Hettinger-Smith et al. 1996) or down-regulation (Stille and Stiles 1991; Hettinger-Smith et al. 1996) of this receptor. In addition, when CHA and DPCPX were simultaneously injected, the level of A1 receptor protein remained similar to control retinas.

image

Figure 1. Analysis of A1 receptor expression in E16 retinas treated for 48 h with A1 receptor agonists and/or antagonists. (a, d, and f) Representative immunoblots of A1 receptor and α-tubulin in retinas treated with: CHA, DCPCX, CHA+DPCPX (a); CGS21680 6 h, 18 h, 24 h, and 48 h (d); CGS21680, ZM241385, CGS21680 + ZM241385 (f). (b, e, and g) Quantification of A1 receptor levels using α-tubulin as a reference protein of experiments showed in a (Control = 100.0% n = 10; CHA = 70.1 ± 5.0% n = 5; DPCPX= 127.4 ± 17.6% n = 4; CHA+DPCPX= 88.3 ± 3.6% n = 4), d (Control = 100.0%; CGS21680 6 h = 94.4 ± 3.7% n = 3; CGS21680 18 h = 108.6 ± 5.5% n = 3; CGS21680 24 h = 133.2 ± 8.7% n = 4; CGS21680 48 h = 132.9 ± 9.4% n = 5) and f (Control = 100.0% n = 13, CGS21680 = 132.1 ± 7.1% n = 8; ZM241385 = 71.0 ± 7.6% n = 7; CGS21680 + ZM241385 = 91.1 ± 8.8% n = 8). (c and h) Binding sites for [3H]DPCPX in retinal homogenates from animal whose eggs were injected with adenosine receptor agonists and/or antagonists. The homogenates were incubated with 5nM [3H]DPCPX in presence or absence of 100μM CHA. (c) CHA, DPCPX, CHA+DPCPX (Control = 100.0% n = 8; CHA= 72.5 ± 3.7% n = 5; DPCPX= 144.9 ± 4.0% n = 3; CHA+DPCPX= 107.0 ± 13.7% n = 3); (h) CGS21680, SCH58261, ZM241385, SCH58261 + CGS21680, CGS21680 + ZM241385 (Control = 100.0% n = 18, CGS21680 = 130.7 ± 10.4% n = 8, SCH58261 = 66.6 ± 6.1% n = 7, CGS21680 + SCH58261 = 106.6 ± 6.6% n = 5 ZM241385 = 69.4 ± 4.5% n = 6, CGS21680 + ZM241385 = 91.3 ± 15.3% n = 5) The results obtained as fmoles/mg protein in retinal homogenates were normalized to 100% (76 ± 3.4 fmoles/mg protein in c; 66.9 ± 3.4 fmoles/mg protein in h). Control animals were injected with DMSO, the vehicle used to dilute all drugs. The asterisks indicate that the differences are significant (*< 0.05, **< 0.01, and ***< 0.001) when compared with control retina.

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Another method performed to evaluate the regulation of A1 receptor expression was the analysis of the binding sites for [3H]DPCPX. In this way, retinal samples exposed to specific drugs were challenged with 5nM of [3H]DPCPX. As shown in Fig. 1c, the treatment with CHA reduced by 27.5% whereas DPCPX increased by 44.9%, the number of binding sites for [3H]DPCPX. On the other hand, simultaneous injection with both drugs did not affect the [3H]DPCPX binding as compared to control (Fig. 1c). Together, the binding results corroborate the western blot data and also indicate that drug injections in ovo constitute a good method for the study of drug modulation of retinal neurochemistry during development.

Is A1 receptor expression regulated by the activation of A2A receptors?

As previous studies have demonstrated a modulation of receptor expression in cultured retinal cells (Pereira et al. 2010), we have also investigated the possible modulation of A1 receptor by in vivo activation/inhibition of A2A receptors. To this purpose, E14 eggs were injected with selective A2A receptor agonist CGS21680 and killed 6, 18, 24, and 48 h after treatment. As shown in Fig. 1d and e, treatment for 6 h did not change the A1 receptor levels. However, from 18 h on, there was an increase of 18.6 ± 5.5%, reaching maximum effect in 48 h (32.9 ± 9.4%). Therefore, all subsequent experiments were done using 48 h. To discard non-specific effect of CGS21680, animals were injected simultaneously with CGS21680 and ZM241385, a selective antagonist of A2A receptor. As expected, ZM241385 was able to block the increase of A1 receptor expression induced by CGS21680 (Fig. 1f and g), suggesting that the increase in A1 receptor expression is mediated by A2A receptor activation. Interestingly, injection of ZM241385 alone reduced by 29 ± 7.6% the amount of A1 receptor protein (Fig. 1f and g). Then, we evaluated the effects of A2A agonist and antagonists in the [3H]DPCPX binding. The A2A agonist CGS21680 induced an increase of [3H]DPCPX-binding sites (130.7 ± 10.4%) and this effect was blocked by the A2A antagonists SCH58261 or ZM241385 (Fig. 1h). On the other hand, either SCH58261 or ZM241385 were able to reduce [3H]DPCPX binding, respectively, in 66.6 ± 6.1% and 69.4 ± 4.5% (Fig. 1h). These results demonstrate that endogenous adenosine, through activation of A2A receptors, contributes to the regulation of A1 receptor expression during retinal development.

The results showed that exposure to CGS21680 increased the expression of A1 receptor, whereas CHA induced a reduction, probably, by down-regulation. Thus, we investigated how the levels of A1 receptor would be when both A1 and A2A receptors were stimulated. Exposure to CGS21680 plus CHA did not affect A1 receptor levels (91.9 ± 17.3%) when compared to control (Fig. 2a and b).

image

Figure 2. Analysis of A1 receptor expression in E16 retinas treated for 48 h with A1 receptor agonists and/or antagonists. (a, c, and e) Representative immunoblots of A1 receptor and α-tubulin in retinas treated with: CHA, CGS21680, CHA+CGS21680 (a); CGS21680, DPCX, CGS21680 + DPCPX (c); CHA, ZM241385, CHA+ZM241385 (e). (b, d, and f) Quantification of A1 receptor levels using α-tubulin as a reference protein of experiments showed in b (Control = 100.0% n=; CGS21680 = 136.7 ± 9.7% n = 4; CHA= 74.4 ± 3.0% n = 4; CGS21680 + CHA= 91.9 ± 17.3% n = 4), d (Control = 100.0% n = 15; CGS21680 = 134.9 ± 7.5% n = 9; DPCPX= 132.9 ± 9.2% n = 6; CGS21680 + DPCPX= 140.0 ± 19.6% n = 4) and f (Control = 100.0% n = 8, CHA= 75.9 ± 3.5% n = 7; ZM241385 = 63.2 ± 9.5% n = 4; CHA+ZM 241385 = 46.5 ± 20.4% n = 3). (g) Binding sites for [3H]DPCPX in homogenates of retinas from animal whose eggs were injected with CHA, SCH58261, and CHA+SCH58261. The homogenates were incubated with 5nM [3H]DPCPX in presence or absence of 100μM CHA. (Control = 100.00% n = 7; CHA= 72.48 ± 3.67% n = 5; SCH58261 = 65.52 ± 10.5%, n = 4; CHA+SCH58261 = 60.40 ± 7.0% n = 5). Eggs from control animals were injected with DMSO, the vehicle used to dilute all drugs. The results obtained as fmoles/mg protein in retinal homogenates were normalized to 100% (82.8 ± 6.0) and represent the mean ± SEM of at least three separate experiments performed in duplicate. Asterisks indicate that the differences are significant (*< 0.05, **< 0.01, and ***< 0.001) when compared with control retina.

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As shown in Fig. 1a and c, as CGS21680, DPCPX was able to increase the levels of A1 receptor. To evaluate if the simultaneous activation of A2A and inhibition of A1 receptors would be synergic, embryos were exposed to CGS plus DPCPX. Although CGS21680 and DPCPX alone induced an increase in A1 receptor (134.9 ± 7.4% and 132.9 ± 9.2%, respectively), CGS21680 plus DPCPX was not able to enhance this effect (140 ± 19.6%) (Fig. 2c and d).

On the other hand, stimulation of A1 receptor or inhibition of A2A receptor induced a reduction in A1 receptor expression. Therefore, we investigated whether a simultaneous injection of CHA and ZM241385 would promote a synergic effect. As it can be noticed in Fig. 2e and f, this effect was not observed. The same result was found in retinas exposed to CHA and SCH58261 (Figure S1). The combination of CHA and SCH58261 did not show an additive reduction in [3H]DPCPX binding (Fig. 2g).

Down-regulation and up-regulation of the A2A receptors

As mentioned above, the long-term treatment with CHA or DPCPX was able to reduce or increase, respectively, the amount of A1 receptor protein. We then evaluated whether the same down-regulation and up-regulation phenomenon occurs with A2A receptors in retinas exposed to CGS21680 and/or ZM241385. Treatment with CGS21680 reduced the expression of A2A receptor protein by 21.1 ± 4.8% (Fig. 3a and b), whereas ZM241385 increased the expression (125.6 ± 7.3%). Treatment with both drugs did not change the A2A receptor levels when compared to control (108.5 ± 1%).

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Figure 3. Analysis of A2A receptor protein levels in E16 retinas after 48 h of treatment with CGS21680, ZM241385, and CGS21680 + ZM241385. (a) Representative immunoblot of A2A receptor and α-tubulin. (b) Quantification of A2A receptor level using the α-tubulin as a reference protein (Control = 100.0% n = 7; CGS21680 = 78.9 ± 4.8% n = 6; ZM241385 = 125.6 ± 7.3%; CGS21680 + ZM211385 = 108.5 ± 1.0% n = 3). Control retinas were obtained from animals whose eggs were injected with DMSO, the vehicle used to dilute all drugs. The results represent the mean ± SEM of three separate experiments. The asterisks indicate that the differences are significant (**< 0.01) when compared with control retina.

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Binding studies of A1 receptor

It was also interesting to investigate whether pharmacological properties of A1 receptors were affected by the long-term treatment. To address this question, kinetic studies of [3H]DPCPX binding were performed using retina homogenates from control animals or animals injected with CHA or CGS21680. As shown in Fig. 4a, [3H]DPCPX binding was homogeneous in control or treated retinas with similar Kd values but different Bmax values (Fig. 4c), with CGS 21680 injection promoting an increase of approximately 27% and CHA decreasing approximately 32% the Bmax values in relation to control. We have also performed displacement experiments in the presence of increasing concentrations of unlabeled DPCPX (Fig. 4b), which also showed no significant variation of EC50 values between the different conditions and the control (Fig. 4c). The results clearly demonstrate that the treatment with the A1 agonist CHA promotes a decrease of Bmax whereas treatment with the A2A agonist CGS 21680 promotes an increase in Bmax, and that both treatments do not promote any change in Kd values.

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Figure 4. Kinetics of [3H]DPCPX binding displacement and saturation. (a) Retinal homogenates from embryos whose eggs were treated for 48 h with CHA or CGS21680 were incubated in the presence of increasing concentrations of [3H]DPCPX (0.2nM–15nM) or (b) incubated in the presence of increasing concentrations of unlabeled DPCPX (0.1nM–100nM). (c) Table representative with values of Bmax, Kd, and EC50. The values of Bmax are represented as fmoles/mg protein, whereas Kd and EC50 as nanomolar. Control animals were injected with DMSO. The results obtained as fmoles/mg protein were normalized to 100% (74 ± 3.4 fmoles/mg protein) and represent the mean ± SEM of three separate experiments performed in duplicate. The non-linear regression (F test with Kd and Bmax as chosen parameters) was used for the statistical analysis of saturation curves (a). The asterisks indicate that the differences are significant (***< 0.0001) when compared with control retina.

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Role of the adenosine transporter (ENT-1) in the expression of A1 receptor

The results found with A2A receptor antagonists indicate that the basal expression of A1 receptors would be controlled, at least in part, by the activation of A2A receptors by endogenous adenosine. To confirm this hypothesis, fertilized eggs were injected with NBMPR, a blocker of type-1 equilibrative adenosine transporter. Interestingly, NBMPR induced a reduction of 36.6 ± 4.4% of [3H]DPCPX-binding sites after 48 h (Fig. 5a). This reduction could be because of accumulation of adenosine in the extracellular space, and chronic activation of A1 receptors. To address this issue, NBMPR was injected with DPCPX. Interestingly, [3H]DPCPX binding was similar to control when ENT-1 and A1 receptor were simultaneously blocked, suggesting that the reduction induced by ENT-1 inhibition would be because of A1 receptor activation by endogenous adenosine accumulated in the extracellular environment (Fig. 5a).

image

Figure 5. Analysis of [3H]DPCPX-binding sites in homogenates of E16 retinas treated for 48 h with NBMPR alone or in the presence of DPCPX and ZM241385. Homogenates were incubated with 5nM [3H]DPCPX in absence or presence of 100μM CHA. (a) DPCPX, NBMPR, DPCPX+NBMPR (Control = 100.0% n = 7; DPCPX=126.4 ± 5.5% n = 5; NBMPR=63.44 ± 4.4% n = 5; DPCPX+NBMPR=104.7 ± 7.4% n = 4). (b) NBMPR, ZM241385, NBMPR+ZM241385 (Control = 100.0% n = 7; NBMPR=67.8 ± 4.4% n = 7; ZM241385 = 71.9 ± 3.6% n = 9; NBMPR+ZM241385 = 66.5 ± 4.5% n = 3) Control animals were injected with DMSO. The results obtained as fmoles/mg protein were normalized to 100% (76 ± 3.4 fmoles/mg protein for a and 86.9 ± 11.5 for b) and represent the mean ± SEM of three separate experiments performed in duplicate. Asterisks indicate that the differences are significant (***< 0.001) when compared with control retina.

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Then, we evaluated the possible synergistic effect of NBMPR and ZM241385, as both drugs reduced the binding sites for [3H]DPCPX. Fig. 5b shows a reduction in [3H]DPCPX-binding sites in retinas exposed to NBMPR and ZM241385; however, this reduction was similar to that found with these drugs alone. Thus, the reduction induced by NBMPR and ZM241385 alone appears to be maximum, which is in agreement with the results found in retinas simultaneously treated with CHA and ZM241385.

Modulation of A1 receptor mRNA

We have also investigated the effect of the injection of adenosine receptor agonists and/or antagonists on A1 receptor mRNA levels in retinas of E14–E16 embryos by real-time RT-PCR. Interestingly, treatment with CHA, DPCPX, CGS21680, and ZM241385 did not change the amount of mRNA compared to control (Control = 1.00 ± 0.1 n = 3; CHA = 0.99 ± 0.1 n = 3; DPCPX = 0.89 ± 0.2 n = 3; CGS21680 = 1.11 ± 0.1 n = 3; ZM241385 = 0.85 ± 0.1 n = 3).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The expression of receptors for chemical signals represents a key regulatory event during CNS development, as these receptors appear to be involved in signaling related to neuronal and glial morphogenesis, synapse formation, activation of different signaling pathways, and gene expression (Martins and Pearson 2007 for review). Previous work has shown an important role for the nucleoside adenosine as a signaling molecule during CNS development, especially in the retina (Paes-de-Carvalho 2002). We have shown recently (Pereira et al. 2010) that a chronic activation of A2A adenosine receptors is able to increase the expression of A1 receptors in a cyclic AMP/PKA-dependent way, and that this increase is accompanied by a decrease of A2A receptor expression. This study extends those previous findings and shows that the long-term in vivo pharmacological activation or blockade of adenosine receptors regulates their expression in the developing chick retina. Interestingly, our evidence also shows that endogenous adenosine plays a crucial role in the regulation of adenosine receptor levels during development.

Down-regulation of adenosine A1 receptors

We found that 48-h exposure to CHA caused the down-regulation of A1 receptors, promoting a decrease in Bmax with no changes in Kd. These data are in accordance to many studies reporting the same effect using other A1 receptor agonists (Abbracchio et al. 1992; Hettinger-Smith et al. 1996; Coelho et al. 2006). Our results have also shown that the reduction of retinal [3H]DPCPX-binding sites and A1 receptor protein caused by the long-term treatment with CHA was inhibited by DPCPX, demonstrating that these drugs are reaching the retina at the estimated concentrations to stimulate or block the A1 receptor subtype (Jacobson and Gao 2006 for review).

The treatment with A2A antagonists (SCH58261 or ZM241385) alone reduced A1 receptor levels in a way similar to the treatment with CHA. On the other hand, treatment with these drugs in combination did not show a synergistic effect. The lack of synergy observed with CHA and SCH58261 or ZM241385 could suggest a common target site for these two agents, with each one providing maximal activation when added alone. Alternatively, the decrease in A1 receptor levels induced by CHA, SCH58261, or ZM241385 could occur through two different modulatory events, perhaps one by homologous desensitization of A1 receptor and another through a signaling pathway coupled to A2A receptor inhibition, which converge somewhere in the intracellular pathway

Up-regulation of adenosine A1 receptors

This study shows that injection of DPCPX in E14 chick eggs induces an increase of A1 receptor-binding sites in E16 retinas, characterizing a receptor up-regulation. Some studies have shown a similar effect in other CNS regions after chronic treatment with adenosine antagonists (Hettinger-Smith et al. 1996). Moreover, DDT1 MF-2 cells treated for 18 h with IBMX or xanthine (non-selective adenosine receptor antagonists) showed a reduction in adenylyl cyclase activity associated with an increase in the amount of A1 receptors (Stille and Stiles 1991). These data indicate that drugs injected in ovo reach the retina in concentrations selective to their targets.

A1 receptor modulation by A2A receptor activation

Our results also show that stimulation with CGS21680 promoted a significant increase in the amount of A1 receptors in a time-dependent manner, an effect blocked by SCH58261 or ZM241385. These results corroborate the findings of Pereira et al. (2010) showing the modulation of A1 receptor expression when cultured retinal cells were chronically treated with A2A receptor agonists. We now extend those findings from the in vitro and ex vivo to the in vivo situation, corroborating the idea that the developing chick embryo is an excellent model for neurochemical development studies. The data suggest the existence of a pathway in the intact retina that mediates the up-regulation of A1 receptors when A2A receptors are chronically activated. Moreover, a long-term treatment with the A2A receptor agonist CGS21680 also decreases A2A receptor expression, whereas the antagonist ZM241385 reduced protein expression. One question that arises is whether the effects on A1 receptor expression actually depend on the concomitant changes found in the A2A receptor expression. In cultured retinal neurons, the up-regulation of A1 receptors induced by chronic [N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl] adenosine), an A2A receptor agonist, depends on the cAMP/PKA pathway (Pereira et al. 2010). Likely, the same process is present in the intact retina, but we cannot exclude that down-regulation of A2A receptors participates in this phenomenon. Also, it cannot be ruled out that the chronic activation of A2A receptor induces homologous desensitization via PKA pathway and this event decreases the inhibition of A1 receptor expression. It is suitable to postulate that, at this embryonic stage, endogenous activation of A2A receptor is inhibiting A1 receptor expression. There is already evidence in the literature of a complementary developmental pattern of A1 and A2A receptors in the superior colliculus (Tavares-Gomes et al. 2009).

The administration of CGS21680 and DPCPX together did not show a synergistic effect in increasing the amount of A1 adenosine receptor. This finding raises again the assumption that the activation of A2A receptor or inhibition of A1 receptor per se has a maximum effect in regulating A1 receptor. Interestingly, the simultaneous activation of A1 receptor with CHA and A2A receptor with CGS21680 did not change the amount of A1 receptor when compared to control, indicating a compensatory effect between the receptors.

The results with A2A antagonists decreasing A1 receptor expression indicate that a basal release of endogenous adenosine regulates the expression of A1 receptors during retinal development. Indeed, NBMPR, an adenosine transporter blocker, reduced A1 receptor expression. Interestingly, this reduction was inhibited by blocking A1 receptor with DPCPX, but blocking A2A receptor with ZM241385 had no effect. These data indicate that an increase of extracellular adenosine would lead to activation of A1 receptor and consequently their reduction by down-regulation.

Other studies indicate a modulation of A1 receptor expression by A2A (Lopes et al. 1999; Castillo et al. 2008) or A3 receptors. Dunwiddie et al. (1997) have shown that the selective activation of A3 receptors promotes a heterologous desensitization of A1 receptors in the CA1 area of rat hippocampus. Similarly, A2A receptor activation also promotes a reduction of A1 receptor agonist affinity in A1/A2A receptor heteromers (Ciruela et al. 2006). Another study shows that C6 glioma cells subjected to different periods of hypoxia present a down-regulation of A1 receptors and an up-regulation of A2A receptors in a manner dependent on extracellular adenosine. Interestingly, the effect of hypoxia on A2A receptor expression is inhibited by an A1 receptor antagonist (Castillo et al. 2008). These data together with the results found in cultures of retinal neurons (Pereira et al. 2010) and the data presented herein suggest that the expression of A1 and A2A receptors could be mutually interdependent.

Changes in A1 receptor mRNA

Exposure to CGS21680 and DPCPX increased whereas CHA and ZM241385 reduced the amount of A1 receptor protein levels. However, the real-time RT-PCR analysis revealed no changes in mRNA levels. These results indicate that changes in A1 receptor expression do not involve transcriptional regulation. Modifications in A1 receptor levels could occur by alteration in the translational rate of pre-existent A1 receptor mRNA and/or in the degradation of the A1 receptor itself.

In contrast to these data, some studies show an increase of mRNA levels (Vendite et al. 1998; Jajoo et al. 2009; Ruiz et al. 2011) whereas others demonstrate a reduction (León et al. 2009; Lorenzo et al. 2010) of A1 receptor. These conflicting data appear to be because of different experimental conditions and/or developmental stages studied.

Adenosine receptor expression and chick retina development

Recently, Socodato et al. (2011) have shown that purified neuronal cultures from E6 retinas treated with A2A agonists [N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl] adenosine) or CGS21680 for 4 days present a robust cell death. The same effect is observed in retinas from animals injected in ovo at E6 and analyzed at E9 (Socodato et al. 2011). In contrast, purified neuronal cultures from E8 retinas pre-incubated with A2A agonists for 24 h are protected from glutamate or refeeding-induced cell death (Ferreira and Paes de Carvalho 2001; Mejía-García and Paes-de-Carvalho 2007). Thus, death or survival of retinal neurons in culture or in intact tissue induced by a chronic treatment with A2A agonists depends on a specific window of development. One possibility is that these phenomena are related to the induction of A1 receptor expression mediated by the long-term treatment with A2A agonists.

The adenosine system is present early in the chick retinal development. A2A receptors can be detected since E6 (Pereira M., Vardiero E. and Paes-de-Carvalho R., unpublished results) and A1 receptors are expressed in low amounts in E10 retinas, reaching a peak of expression at E16 (Paes-de-Carvalho 1990). The data presented here together with previous works suggest a possible mechanism to explain the regulation of A1 receptor expression by A2A receptors in the developing chick retina. It is possible that the regulation of adenosine concentration in the extracellular space would activate A2A receptor and induce A1 receptor expression in the retina of older embryos. Thus, endogenous adenosine has a role in retinal development and on the expression of its receptors. Moreover, the peak of A1 receptor expression coincides with the period of cAMP production induced by the A2A receptor pathway. Perhaps during chick retina development, the peak of A1 receptor expression depends on cAMP formation and PKA activation induced by A2A receptor, the same pathways involved in the increase of A1 receptor expression in culture (Pereira et al. 2010).

In conclusion, this study adds some important information about the mechanisms controlling the expression of adenosine receptors in the developing retina and places endogenous adenosine as a key agent in the expression of their receptors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We greatly acknowledge Ms. Luzeli R. de Assis and Sarah de Alencar Rodrigues for the technical assistance. We are also grateful to Dr Karen de Jesus Oliveira for her assistance in real-time RT-PCR experiments and to Renato Esteves da Silva Socodato for the help with figures design. This work was supported by grants from CNPq, PROPPi-UFF, FAPERJ, INNT/INCT/CNPq, CNPq, and PRONEX/MCT. RBS was the recipient of a graduate student fellowship from CAPES. The authors declare that they have no competing financial interests.

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  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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