Glucocorticoids Regulate the Expression of Adenosine A1 but not A2A Receptors in Rat Brain PER SVENNINGSSON and BERTIL B. FREDHOLM

The effect of adrenalectomy on the expression of adenosine receptors and their mRNA in rat brain was examined using quantitative autoradiography and in situ hybridization. 1,3[H]Dipropyl-8-cyclopentylxanthine ([H]DPCPX), a selective adenosine A1 receptor antagonist, and [ H]CGS 21680, a selective adenosine A2A receptor agonist, were used as radioligands. One week after adrenalectomy, the expression of mRNA for adenosine A1 receptors was significantly decreased, as was the number of binding sites for [H]DPCPX. These effects were significantly counteracted by replacement treatment with dexamethasone (1.5 mg/kg i.p., twice daily). Addition of GTP caused a similar increase of [H]DPCPX binding in sham-operated rats, adrenalectomized rats and rats adrenalectomized and treated with dexamethasone. Moreover, no differences in displacement of [H]DPCPX by the adenosine receptor agonist N-(R-phenylisopropyl)adenosine were found among these groups. Adrenalectomy did not significantly affect the number of [H]CGS 21680 binding sites in striatum or the mRNA encoding adenosine A2A receptors. No changes in the affinity of [ H]CGS 21680 for adenosine A2A receptors or in the potency of the adenosine receptor agonist 2-chloroadenosine to displace [H]CGS 21680 were found. Dexamethasone treatment decreased cAMP formation induced by the nonselective adenosine agonist 59-Nethylcarboxamidoadenosine in Jurkat cells, which express adenosine A2B receptors, but did not alter it in PC-12 cells, which express mostly A2A receptors. The results suggest that endogenous corticosteroids positively regulate the expression of adenosine A1 receptors, at least partly at the transcriptional level. In contrast, corticosteroids do not regulate the expression of adenosine A2A receptors. Corticosteroids acting on mineralocorticoid and glucocorticoid receptors, both of which bind to DNA (Evans and Arriza, 1989), regulate the transcription of many genes, including those for receptors. Thus, it has been shown that corticosteroids, via glucocorticoid receptors, can increase the mRNA expression and responsiveness of beta-2 adrenergic receptors in DDT1 MF-2 cells (Collins et al., 1988), and it is well documented that corticosteroids can regulate mRNA and receptor expression in vivo. Removal of endogenous corticosteroids by adrenalectomy has, for example, been shown to significantly affect both mRNA and receptor density for 5-hydroxytryptamine1A and g-aminobutyric acidA receptors in the rat hippocampus (Chalmers et al., 1993; Orchinik et al., 1994), a region in the central nervous system where both mineralocorticoid and glucocorticoid receptors are expressed (McEwen et al., 1968; Chao et al., 1989; Cintra et al., 1994). Whereas mineralocorticoid receptors are expressed at high levels only in hippocampus, glucocorticoid receptors are widely distributed, with high to moderate levels in, for example, hippocampus and cerebral and cerebellar cortex and striatum (Cintra et al., 1991, 1994). In the latter region glucocorticoid receptors are involved in the transcriptional regulation of cannabinoid receptors and the neuropeptides proenkephalin and protachykinin (for review, see Chao and McEwen, 1990; Mailleux and Vanderhaeghen, 1993; Angulo and McEwen, 1994). Adenosine is a potent endogenous neuromodulator that has been suggested to act as an endogenous neuroprotective agent (Rudolphi et al., 1992), as a regulator of seizure susceptibility (Dragunow, 1988), as an endogenous analgetic (Sawynok, 1995) and in the regulation of sensorimotor control (for review, see Ferré et al., 1992; Fredholm, 1995). Adenosine in physiological concentrations exerts its action in the brain mainly via the G protein-coupled adenosine A1 and A2A receptors (Rudolphi et al., 1992). Adenosine A1 receptors are widely distributed in the central nervous system, with high levels of expression in glucocorticoid receptor-rich areas like hippocampus and cerebral and cerebellar cortex (GoodReceived for publication August 13, 1996. 1 Recipient of a doctoral fellowship from the Knut and Alice Wallenberg Foundation. ABBREVIATIONS: ANOVA, analysis of variance; cAMP, cyclic AMP; CGS 21680, 2-[p-(2-carbonylethyl)phenylethylamino]-59-N-ethylcarboxamidoadenosine; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; NECA, 59-N-ethylcarboxamidoadenosine; (R)-PIA, N-(R-phenylisopropyl)adenosine; SSC, standard saline citrate. 0022-3565/97/2802-1094$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 280, No. 2 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 280:1094–1101, 1997 1094 at A PE T Jornals on July 9, 2017 jpet.asjournals.org D ow nladed from man and Snyder, 1982; Fastbom et al., 1987). Adenosine A2A receptors and their corresponding mRNAs have a more restricted distribution and are mostly found in striatum. A2A receptors are colocalized with dopamine D2 receptors in g-aminobutyric acid-ergic medium-sized neurons that also contain enkephalin (Schiffmann et al., 1991; Fink et al., 1992). These striatal neurons also show high levels of immunoreactivity for glucocorticoid receptors (Cintra et al., 1991). These data suggest that glucocorticoids may regulate adenosine receptors in the brain. Indeed, there is some evidence that stressful stimuli, which are known to affect glucocorticoid levels, can influence A1 receptors (Boulenger et al., 1984, 1986) in the central nervous system. Gerwins and Fredholm (1991) showed that in vitro treatment with the synthetic steroid dexamethasone increases the number of adenosine A1 receptors and enhances A1 receptormediated responses in smooth muscle cells. However, it is not known whether such a regulation of adenosine A1 receptors occurs at the transcriptional level or whether it occurs in the central nervous system in vivo. There is likewise no information about the role that corticosteroids have in the regulation of adenosine A2A receptors. In the present study, we investigated the effects of adrenalectomy on the expression of adenosine A1 and A2A receptors and their corresponding mRNA in rat brain. Materials and Methods Animals and treatment. The experiments were approved by the regional animal ethics committee. Male Sprague-Dawley rats (ALAB, Stockholm, Sweden) weighing 200 to 230 g were used. The rats were housed two per cage and maintained on a 12/12-hr light/ dark cycle. All rats had free access to food and drinking water. Bilateral adrenalectomy was performed via a lumbar approach under chloral hydrate (400 mg/kg) anesthesia. Sham operations were identical to adrenalectomy, but the adrenal glands were left intact. The adrenalectomized rats received 0.9% NaCl for daily drinking and were injected i.p. twice daily (10:00 A.M. and 5:00 P.M.) with either 1 ml of dexamethasone (1.5 mg/kg; Sigma, LabKemi, Stockholm, Sweden), dissolved in saline and a few drops of Tween 80, or vehicle. At 10:00 A.M. on day 7, rats were briefly anesthetized with CO2 and killed by decapitation. The brains were rapidly dissected out and frozen at 280°C. Sagittal sections (10or 14-mm thick) were made and thaw-mounted on poly-L-lysine (50 mg/ml)or gelatincoated slides. In situ hybridization. The 48-mer A1 adenosine receptor probe was complementary to nucleotides 985 to 1032 of the rat A1 receptor (Mahan et al., 1991). The 44-mer A2A probe was complementary to nucleotides 916 to 959 of the dog RDC8 cDNA (Schiffmann et al., 1990). The 48-mer preproenkephalin probe was complementary to nucleotides 388 to 435 of the rat preproenkephalin gene (Yoshikawa et al., 1984). The specificity of each probe was tested earlier (Johansson et al., 1993, 1994). The oligodeoxyribonucleotides (Scandinavian Gene Synthesis, Köping, Sweden) were radiolabeled, using terminal deoxyribonucleotidyl transferase (Amersham, Solna, Sweden) and a-S-dATP (DuPont-NEN, Stockholm, Sweden), to a specific activity of about 10 cpm/mg. Slide-mounted, 14-mm sections were hybridized in a cocktail containing 50% formamide (Fluka, Buchs, Switzerland), 43 SSC (13 SSC is 0.15 M NaCl, 0.015 M sodium citrate), 13 Denhardt’s solution, 1% sarcosyl, 0.02 M sodium phosphate (pH 7.0), 10% dextran sulfate, 0.5 mg/ml yeast tRNA (Sigma, LabKemi), 0.06 M dithiothreitol, 0.1 mg/ml sheared salmon sperm DNA and 10 cpm/ml probe. After hybridization for 16 hr at 42°C, the sections were washed four times for 15 min each in 13 SSC at 55°C (A1 probe and preproenkephalin probe) or 45°C (A2A probe), dipped briefly in water, 70% ethanol, 95% ethanol and 99.5% ethanol and air-dried. Finally the sections were apposed to Hyperfilm b-max film (Amersham) for 1 week (preproenkephalin probe) or 3 weeks (A1 and A2A probe). Ligand-binding autoradiography. For receptor autoradiography, 10-mm sections were preincubated in 170 mM Tris-HCl buffer containing 1 mM EDTA and 2 U/ml adenosine deaminase (calf intestine; Boehringer, Mannheim, Germany) at 37°C for 30 min. Sections were then washed twice for 10 min at 23°C in 170 mM Tris-HCl buffer with 10 mM MgCl2 for A2 receptors or 1 mM MgCl2 for A1 receptors. Incubations were performed for 2 hr at 23°C in Tris-HCl buffer containing the radioligand at the appropriate concentration, 2 U/ml adenosine deaminase and 1 mMMgCl2 with or without 100 mM GTP for A1 or 10 mM MgCl2 with or without 1000 mM GTP for A2 receptors. The ligand used for A1 receptors was [ H]DPCPX (0.125, 0.25, 0.5, 1, 2.5 and 5 nM, 60–80 Ci/mmol; DuPont-NEN), and the ligand for the study of A2A receptors was [ H]CGS 21680 (0.5, 1.25, 2.5, 5, 10 and 20 nM, 48.1 Ci/mmol; DuPont-NEN). Nonspecific binding was defined with 20 mM (R)-PIA (Boehringer, Mannheim, Germany) (for A1 receptors) or 20 mM 2-chloroadenosine (Sigma, LabKemi) (for A2A receptors). Displacement studies were performed with (R)-PIA (10, 10, 10 and 10 M) for A1 receptors (0.5 nM DPCPX) and with 2-chloroadenosine (10, 10, 10 and 10 M) for A2 receptors (2.5 nM CGS 21680). Sections were then washed twice for 5 min each in ice-cold Tris-HCl, dipped quickly three times in ice-cold distilled water and dried at 4°C over a strong fan. The dried sections, together with plastic tritium standards (Amersham), were apposed to Hyperfilm (Amersham) for 5 weeks. In vitro assays. PC-12 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with penicillin, streptomycin, L-glutamine and 5% fetal calf serum/10% horse serum at 37°C in 5% CO2/95% air. Jurkat cells were maintained in RPMI 1640 medium supplemented with penicillin, streptomycin, L-glutamine and 7.5% fetal calf serum. Cells were subcultured (5 3 10 cells/ml) for 12 hr before addition of dexamethasone (100 nM). Cells were then incubated for 24 hr. After being washed twice with assay medium, aliquots (0.5 3 10 cells, 0.35 ml) were transferred to test tubes. NECA (10 to 10 M; Sigma, LabKemi), a potent nonselective adenosine receptor agonist, was added, together with 30 mM levels of the phosphodiesterase inhibitor rolipram (Research Biochemicals Inc., Natick, MA), to a final volume of 0.5 ml. To amplify the response, Jurkat cells were studied in the presence of 10 mM forskolin (van der Ploeg et al., 1996). Reactions were terminated, after 10 min of incubation at 37°C, by addition of perchloric acid to a final concentration of 0.4 M. Samples were neutralized with KOH, and the cAMP content in the supernatants was determined with a protein-binding assay (Nordstedt and Fredholm, 1990), where bound [H]cAMP was separated from free by rapid filtration over glass fiber filters (Skatron AS,