Abstract
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-[3H]Dipropyl-8-cyclopentylxanthine ([3H]DPCPX), a selective adenosine A1receptor antagonist, and [3H]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 [3H]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 [3H]DPCPX binding in sham-operated rats, adrenalectomized rats and rats adrenalectomized and treated with dexamethasone. Moreover, no differences in displacement of [3H]DPCPX by the adenosine receptor agonistN 6-(R-phenylisopropyl)adenosine were found among these groups. Adrenalectomy did not significantly affect the number of [3H]CGS 21680 binding sites in striatum or the mRNA encoding adenosine A2A receptors. No changes in the affinity of [3H]CGS 21680 for adenosine A2A receptors or in the potency of the adenosine receptor agonist 2-chloroadenosine to displace [3H]CGS 21680 were found. Dexamethasone treatment decreased cAMP formation induced by the nonselective adenosine agonist 5′-N-ethylcarboxamidoadenosine 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 γ-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 (Rudolphiet 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 (Goodman and Snyder, 1982; Fastbomet 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 γ-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 A1receptor-mediated 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 A1and 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 5:00p.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 −80°C. Sagittal sections (10- or 14-μm thick) were made and thaw-mounted on poly-l-lysine (50 mg/ml)- or gelatin-coated slides.
In situ hybridization.
The 48-mer A1adenosine 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 (Johanssonet al., 1993, 1994). The oligodeoxyribonucleotides (Scandinavian Gene Synthesis, Köping, Sweden) were radiolabeled, using terminal deoxyribonucleotidyl transferase (Amersham, Solna, Sweden) and α-35S-dATP (DuPont-NEN, Stockholm, Sweden), to a specific activity of about 109 cpm/μg. Slide-mounted, 14-μm sections were hybridized in a cocktail containing 50% formamide (Fluka, Buchs, Switzerland), 4× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate), 1× 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 107 cpm/ml probe. After hybridization for 16 hr at 42°C, the sections were washed four times for 15 min each in 1× 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 β-max film (Amersham) for 1 week (preproenkephalin probe) or 3 weeks (A1 and A2A probe).
Ligand-binding autoradiography.
For receptor autoradiography, 10-μm 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 mM MgCl2 with or without 100 μM GTP for A1 or 10 mM MgCl2 with or without 1000 μM GTP for A2 receptors. The ligand used for A1receptors was [3H]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 [3H]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 μM (R)-PIA (Boehringer, Mannheim, Germany) (for A1 receptors) or 20 μM 2-chloroadenosine (Sigma, LabKemi) (for A2A receptors). Displacement studies were performed with (R)-PIA (10−10, 10−9, 10−8 and 10−7 M) for A1 receptors (0.5 nM DPCPX) and with 2-chloroadenosine (10−9, 10−8, 10−7 and 10−6 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 3Hyperfilm (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 × 104 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 × 105 cells, 0.35 ml) were transferred to test tubes. NECA (10−8 to 10−3 M; Sigma, LabKemi), a potent nonselective adenosine receptor agonist, was added, together with 30 μM 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 μM 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 [3H]cAMP was separated from free by rapid filtration over glass fiber filters (Skatron AS, Tranby, Norway).
Data analysis.
The films from the in situhybridization and autoradiographic studies were analyzed with a microcomputer imaging device system (Imaging Research, St. Catharine’s, Canada). The system was calibrated with a Kodak density wedge when films from in situ hybridization experiments were analyzed, and the results are presented as optical density values. For films from ligand-binding autoradiographic studies, the optical density values were converted to binding density (femtomoles per milligram of gray matter) by using calibrated plastic standards (Amersham) and the specific activity of the respective radioligand.
Statistics.
When measurements were done in several regions of the rat brain, an overall analysis was performed with two-way ANOVA (treatment × region) (GraphPad PRISM, San Diego, CA). Additionally, for individual regions a one-way ANOVA followed by Bonferroni’s correction for pairwise comparisons was used (GraphPad InStat; ISI Software, San Diego, CA). A two-tailed, unpaired, Student’s t test was used for analysis of the in vitro studies (GraphPad InStat; ISI Software). P values of <.05 were considered significant.
Results
Effect of adrenalectomy on preproenkephalin mRNA.
To verify that the adrenalectomy and the treatment with dexamethasone did influence the central nervous system, we examined the expression of preproenkephalin mRNA in caudate putamen; previous reports (e.g., Chao and McEwen, 1990; Mailleux and Vanderhaeghen, 1993) demonstrated clear-cut effects on this neuropeptide. As seen in figure 1, the expected decrease in preproenkephalin mRNA was observed after adrenalectomy, and the effect of adrenalectomy was reversed by the chosen dose of dexamethasone.
Effects of adrenalectomy on the expression of adenosine A1 receptors and corresponding mRNA in rat brain.
In agreement with previous studies (e.g.,Johansson et al., 1993), there was a widespread distribution of both [3H]DPCPX binding and mRNA encoding adenosine A1 receptors (fig. 2). Also in agreement with previous data (Johansson et al., 1993), we found that there was no exact correspondence between the distribution of A1 receptors, as determined by [3H]DPCPX binding, and the distribution of A1 receptor mRNA. As discussed elsewhere (e.g., Johansson et al., 1993), this difference may be accounted for by the fact that A1 receptors are present in many nerve terminals, where they regulate transmitter release (Fredholm and Dunwiddie, 1988), whereas mRNA is found mainly in cell bodies.
Overall analyses with two-way ANOVAs showed that adrenalectomized rats had a significantly lower number of binding sites for [3H]DPCPX and a decreased amount of mRNA encoding adenosine A1 receptors, compared with sham-operated and adrenalectomized rats treated with dexamethasone as glucocorticoid replacement (fig. 3, A and B; tables 1and 2). However, there was some variability among different brain areas, and significant differences inB max values between adrenalectomized and sham-operated rats were found only in caudate putamen (fig. 3C) and frontoparietal cortex (table 1). In addition, in caudate putamen, frontal parietal cortex, stratum radiatum of CA3, ventrolateral thalamus and the molecular layer of cerebellar cortex there were significant differences in receptor numbers in adrenalectomized rats that received no replacement therapy and those that were given dexamethasone (table 1).
No specific region showed a significant difference in mRNA expression of adenosine A1 receptors between sham-operated and adrenalectomized rats (table 2). However, in both the granular layer of gyrus dentatus and the pyramidal layer of CA1, dexamethasone caused a significant increase in A1 mRNA expression in adrenalectomized rats.
When all of the areas were analyzed together, no significant alterations of the Kd values for [3H]DPCPX binding were found. However, there was a significant difference in stratum radiatum of CA1 between sham-operated and adrenalectomized rats, which was absent in adrenalectomized rats treated with dexamethasone. In addition, theKd values were significantly different in the frontoparietal part of the cerebral cortex between adrenalectomized rats and those treated with dexamethasone (table 1). There is no obvious explanation for these effects, but they might be secondary to effects of glucocorticoids on other receptors that can influence the affinity of adenosine A1 receptors for adenosine. There are several examples of such receptor-receptor interactions (Agnati et al., 1995).
It was previously found that drug treatment can influence not only total receptor number, as assessed by antagonist binding, but also the coupling to G proteins (e.g., Green and Stiles, 1986;Fastbom and Fredholm, 1990; Hoppe and Lohse, 1995). We therefore examined whether adrenalectomy altered the ability of GTP to influence [3H]DPCPX binding or the affinity of an agonist for the binding sites. However, as illustrated in figure 4A, none of the groups differed in the potency with which (R)-PIA, an agonist at adenosine receptors, displaced [3H]DPCPX binding. Moreover, addition of 100 μM GTP to the incubation solution increased the binding to similar extents in the three different treatment groups (fig. 4B).
Effects of adrenalectomy on the expression of adenosine A2A receptors and corresponding mRNA in rat brain.
In agreement with previous results (e.g.,Johansson et al., 1993), expression of adenosine A2A receptors and their corresponding mRNA was much higher in caudate putamen than in other brain regions (fig. 5). The respective B max andKd values for [3H]CGS 21680 binding were 403 ± 13.4 fmol/mg gray matter and 2.74 ± 0.28 nM for adrenalectomized rats, 392 ± 11.8 fmol/mg gray matter and 2.99 ± 0.27 nM for sham-operated rats and 377 ± 15.5 fmol/mg gray matter and 3.09 ± 0.38 nM for adrenalectomized rats treated with dexamethasone, respectively. These values were not significantly different, nor were there any significant differences in A2A receptor mRNA expression among the three groups. A similar decrease in [3H]CGS 21680 binding was found in all three groups when 1000 μM GTP was added to the incubation solution; the decreases were 47%, 46% and 57% in adrenalectomized, sham-operated and adrenalectomized/dexamethasone-treated rats, respectively. The adenosine agonist 2-chloroadenosine displaced [3H]CGS 21680 binding with similar potencies in all three groups. The IC50 values were 59.9 ± 24.8 nM for adrenalectomized rats, 71.8 ± 28.3 nM for sham-operated rats and 78.6 ± 25.5 nM for adrenalectomized rats treated with dexamethasone.
Effects of dexamethasone on NECA-induced cAMP formation in PC-12 and Jurkat cells.
The lack of significant effect of adrenalectomy or dexamethasone on A2A receptors in the striatum is in apparent contrast to the previous finding that A2receptor-mediated effects in DDT1 MF-2 cells are down-regulated by dexamethasone (Gerwins and Fredholm, 1991). To ensure that the discrepancy is not caused by a difference in the type of assay used (binding vs. cAMP measurements), we examined whether dexamethasone could affect cAMP responses mediated by A2Areceptors in PC-12 cells. However, dexamethasone did not have any significant effect on NECA-induced cAMP responses in PC-12 cells (fig.6A). The cAMP response in DDT1 MF-2 cells may be due to stimulation of A2B rather than A2A receptors. A2B receptors are found in Jurkat cells (van der Ploeg et al., 1996). In these cells, treatment with dexamethasone (100 nM) for 24 hr significantly decreased the cAMP formation after stimulation with NECA at 10−4, 10−5 or 10−7 M (fig. 6B).
Discussion
Our main finding is that adrenal steroids regulate the expression of adenosine A1 receptors not only in vitro but also in vivo. The number of adenosine A1 receptors was decreased after adrenalectomy. The effect of adrenalectomy was counteracted by daily dexamethasone injections, indicating that it is due to loss of endogenous glucocorticoids. Although decreases in adenosine A1 receptors after adrenalectomy were observed in most brain regions, the magnitude of the effect differed among brain areas. Significant decreases ofB max values were found only in caudate putamen and frontoparietal cortex. However, when adrenalectomized rats that received no glucocorticoid replacement were compared with rats that were given dexamethasone, significant differences in receptor number were found in caudate putamen, frontoparietal cortex, stratum radiatum of CA3, ventrolateral thalamus and the molecular layer of cerebellar cortex. There was no direct correspondence between the number of glucocorticoid receptors and the alterations in adenosine A1 receptor density in the examined regions.
A complicating factor for the interpretation of these region-specific differences is that many adenosine A1 receptors are located on nerve terminals in regions distinct from those where the cell bodies are located. Glucocorticoids might therefore regulate the transcription and translation of adenosine A1 receptors in the cell bodies but the consequent alterations in receptor number would be found mostly in nerve terminal regions. In fact, results concerning adenosine A1 receptor mRNA are consistent with this view (see below).
In agreement with several previous studies, GTP markedly increased the binding of the receptor antagonist [3H]DPCPX (Fastbom and Fredholm, 1990; Parkinson and Fredholm, 1992). The reason is that adenosine receptor agonist ligands, including the endogenous ligand adenosine, bind in a pseudoirreversible manner to the receptors in the presence of magnesium when GTP levels are low. This tightly bound adenosine cannot be removed by addition of the enzyme adenosine deaminase. When GTP (or GDP) is added in concentrations approaching physiological levels, the bound adenosine rapidly dissociates and the antagonist radioligand can gain access to the receptor (see Parkinson and Fredholm, 1992). The difference in [3H]DPCPX binding in the presence and absence of GTP therefore reflects the number of A1 receptors tightly associated with the holotrimeric G protein. The present data thus indicate that this proportion is not altered by adrenalectomy or by dexamethasone.
There were significant overall effects of adrenalectomy and of dexamethasone not only on the number of A1 receptors but also on the expression of A1 receptor mRNA. The effect of dexamethasone was most pronounced in the granular layer of gyrus dentatus and the pyramidal layer of CA1. These two hippocampal regions, along with the granular layer in cerebellum, express the highest levels of glucocorticoid receptors in the brain (Cintra et al., 1994). It therefore seems reasonable to assume that altered transcription of the A1 receptor gene can account for at least part of the change in the amount of receptor protein.
There are several reasons for believing that corticosteroids regulate adenosine A1 receptors mainly via glucocorticoid receptors and not via mineralocorticoid receptors. First, dexamethasone treatment reversed the decrease in adenosine A1 receptors induced by adrenalectomy and, because dexamethasone binds to glucocorticoid receptors with much higher affinity than to mineralocorticoid receptors (Chao et al., 1989), this compound mainly reveals effects mediated via the glucocorticoid receptor. Second, adrenalectomy down-regulated adenosine A1 receptors in several brain regions that are known to express high levels of glucocorticoid receptors but only low levels of mineralocorticoid receptors. Third, Gerwins and Fredholm (1991) found up-regulation of adenosine A1 receptors in vitroafter stimulation of glucocorticoid, but not mineralocorticoid, receptors. In fact, the changes in adenosine receptors were smallest in the hippocampal area, where mineralocorticoid receptors are most abundant. This might indicate that mineralocorticoid receptors and glucocorticoid receptors play opposing roles in the regulation of adenosine A1 receptors. Another possibility is that heterodimeric mineralocorticoid and glucocorticoid receptors have different effects, compared with the homodimers (see Trapp and Holsboer, 1996).
The adenosine A1 receptor gene has been shown to contain at least four exons (Ren and Stiles, 1995), which can generate two different transcripts that are expressed in a tissue-specific manner (Ren and Stiles, 1995). It is not known how these are regulated, but it is interesting to note that there are several AP-1 consensus sites. It has been shown in vitro that activated glucocorticoid receptors can interact with AP-1 (Schule et al., 1990;Yang-Yen et al., 1990; Beato et al., 1995).
Whereas glucocorticoid altered A1 receptors, it had no apparent effect on A2A receptors or on A2Areceptor mRNA. This could not be ascribed to a lack of effect on gene transcription in striatal adenosine A2A receptor-containing neurons, because a significant alteration was found in the level of mRNA coding for the coexisting neuropeptide preproenkephalin. Indeed, preproenkephalin mRNA and adenosine A2A receptor mRNA are expressed in the same subpopulation of striatal neurons (Schiffmannet al., 1991).
This lack of effect of dexamethasone on A2A receptors is in apparent contrast to the findings of Yingling et al. (1994)on cAMP responses to adenosine analogs in PC-18 cells. We therefore examined the effect of dexamethasone treatment on cAMP responses to an adenosine analog in PC-12 cells. We previously showed that, in the clone used, the response can be accounted for by stimulation of A2A receptors (van der Ploeg et al., 1996). Our findings confirmed the results on striatal A2A receptors but differed from those reported for PC-18 cells. We have no good explanation for this apparent discrepancy, but it should be noted that Yingling and coworkers used (R)-PIA as an agonist despite the fact that it is more potent on A1 receptors than on A2A receptors. Furthermore, it was found that the enhancement of (R)-PIA-stimulated cAMP responses was markedly reduced by inclusion of a phosphodiesterase inhibitor, suggesting that part of the effect was due to down-regulation of this enzyme (Yingling et al., 1994). In our experiments, the phosphodiesterase inhibitor rolipram was used.
Our experiments in PC-12 cells were run in parallel with experiments in Jurkat cells, which express mainly adenosine A2B receptors, rather than A2A receptors (van der Ploeg et al., 1996). In contrast to the results in PC-12 cells, the functional responses in Jurkat cells were decreased after dexamethasone. These findings provide further evidence for a down-regulation of A2B receptors. They also suggest that the functional A2 effects reported previously in DDT1 MF-2 cells (Gerwins and Fredholm, 1991) may reflect alterations in A2B receptors. Although further studies on A2Breceptors are needed, the data thus indicate that glucocorticoid effects on adenosine receptors can range from up-regulation (A1 receptor) or no effect (A2A receptor) to down-regulation (A2B receptor).
The present data thus show that endogenous and exogenous glucocorticoids regulate the number of adenosine A1receptors at least in part by altering the expression of the corresponding mRNA in rat brain. In contrast, no effects on adenosine A2A receptors or their mRNA were found. It is tempting to speculate that the alterations in adenosine A1 receptors can have functional consequences, because these receptors are known to modulate many brain functions. For example, adenosine A1receptors modulate seizure activity (Dragunow, 1988), and it is possibly relevant that dexamethasone treatment increases the threshold of the adenosine receptor antagonist theophylline to cause seizures (Hoffman et al., 1994). If adenosine A1receptors are important there, then it can be predicted that adrenalectomy would render animals more susceptible to these and other effects of adenosine A1 receptor antagonists.
Acknowledgments
We thank Dr. A. Cintra for help with the adrenalectomy and Susanne Ahlberg for help with the cAMP measurements in PC-12 and Jurkat cells.
Footnotes
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Send reprint requests to: Per Svenningsson, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
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↵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]-5′-N-ethylcarboxamidoadenosine
- DPCPX
- 1,3-dipropyl-8-cyclopentylxanthine
- NECA
- 5′-N-ethylcarboxamidoadenosine
- (R)-PIA
- N 6-(R-phenylisopropyl)adenosine
- SSC
- standard saline citrate
- Received August 13, 1996.
- Accepted October 21, 1996.
- The American Society for Pharmacology and Experimental Therapeutics