- Email: [email protected]
Adaptive changes in adenosine receptors following long-term treatment with the adenosine receptor agonist R-phenylisopropyl adenosine
Life !kknces, Vol. 58, No. 9, pp. ?69-?76, 1996 Comnieht 0 19% Elsevier Science Inc. Print-ii ii the USA. All rights ~crved o&24-3205/% $15.00 t .oo
0024-3205(95)02355-O
ELSEVIER
ADAPTIVE CHANGES IN ADENOSINE RECEPTORS FOLLOWING LONG-TERM TREATMENT WITH THE ADENOSINE RECEPTOR AGONIST R-PHENYLISOPROPYL ADENOSINE Mercedes Fernandez’,
Per Svenningsson
and Bertil B. Fredholm*
Department of Physiology and Pharmacology, Division of Molecular Neuropharmacology, Karolinska Institute& S-171 77 Stockholm, Sweden (Received in final form December 12, 19%)
Summaw in brain A, and AlA receptors and in the corresponding mRNA were studied using quantitative receptor autoradiography and in sifu hybridisation. [3H]DPCPX was used as an antagonist ligand at Ai receptors and [3H]-CGS 21680 as an agonist ligand at AzA receptors. Treatment of rats with the relatively Ai receptor selective adenosine analogue R-PIA (0.3 mg/kg) for 7 days in the presence of the peripherally acting antagonist 8-p-sulfophenyltheophylline (S-PST; 10 mg/kg) caused a decrease in the binding of the Ai receptor ligand, but not in that of the AZA receptor ligand. The effect on Ai receptors was also seen in the presence of 100 PM GTP that decreases agonist binding to insignificant levels, There was no change in either Ai or AZ* receptor mRNA. No significant changes were detected following administration of either R-PIA or S-PST alone. These results thus demonstrate an effect on brain A, receptors after systemic administration of R-PIA in the presence of a peripherally acting adenosine antagonist, demonstrating that, under these conditions, the agonist reaches receptors in significant amounts. Changes
Key Words: adenosine receptors, R-phenykopropyl
adenosine, mRNA
It is known that long-term treatment with adenosine receptor antagonists leads to an increase in the number of adenosine Ai receptors (1, 2) which is not accompanied by changes in the amount of Ar receptor n-RNA (3). By contrast, there are only small, and generally insignificant changes in A*.,, receptors and mRNA (3). Long-term treatment with antagonists also causes a number of adaptive changes that may or may not be related to these changes in adenosine receptors (4, 5). There is also some evidence that long-term intracerebroventricular treatment with adenosine receptor agonists can produce adaptive changes in the number of Ai receptors (6). However, little is known about the adaptive changes in central adenosine receptors following systemic administration of an adenosine
’ Present address: Dipartimento Quimica, Inorgzinica, Orgknica y Bioquimica, Universidad Castilla-La Mancha, Ciudad Real, Spain. * To whom all correspondence should be sent at the above address.
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receptor agonist. In the present series of experiments we administered the relatively Al receptor selective agonist R-N6-phenylisopropyl adenosine (R-PIA) (7) once daily by i.p. injection for 7 days. The number of Al and AZAreceptors was examined by quantitative receptor autoradiography and their corresponding mRNAs using in situ hybridisation. When adenosine receptor agonists are given systemically they produce marked effects on e.g. the cardiovascular system. Even though the magnitude of these effects may decrease upon repeated administration (8) we wanted to minimise the peripheral cardiovascular effects by combining the adenosine receptor agonist R-PIA with the peripherally acting antagonist S-p-sulfophenyltheophylline (S-PST). The results suggest that an A1 receptor agonist can, via a direct action at central sites, decrease binding to Al receptors, and that this is not due to any effect at the transcriptional level.
Materials and Methods A total of six male Sprague-Dawley rats per treatment was used. One group of animals received saline injections, the second group injections of R-PIA (0.3 mg/kg, i.p ), the third group S-PST (10 mg/kg, i.p.) and the fourth group both R-PIA and S-PST. The treatment lasted for seven days. After anaesthesia with CO1 the rats were decapitated, the whole brain removed and frozen on dry ice. Saggital sections of each brain were cut with a Leitz cryostat. Fourteen-pm-thick sections were thaw-mounted on poly-L-lysine (50 &ml) coated slides for it2 silu hybridisation. Ten pm thick sections were thaw-mounted on gelatine coated slides for quantitative receptor autoradiography. The 48-mer Al adenosine receptor probe was complementary to nucleotides 985- 1032 of the rat Al receptor (3, 9). The 44-mer AzA probe was complementary to nucleotides 916-959 of the dog RDCS cDNA (10). The oligodeoxyribonucleotides were radiolabelled using terminal deoxyribonucleotidyl transferase (Amersham) and a-35S-dATP (Amersham) to a specific activity of about 1O9 CPM/pg. Slide mounted sections were hybridised in a cocktail containing 50% formamide (Baker), 4 x SSC, 1 x Denhardt’s solution, 1% sarcosyl, 0.02 M NaP04 (pH 7.0), 10% dextran sulfate, 0.5 mg/ml yeast tRNA (Sigma), 0.06 M dithiothreitol, 0.1 mg/ml sheared salmon sperm DNA and lo7 CPM/ml of probe. After hybridisation for 16 h at 42°C the sections were washed four times for 15 min each in 1 x SSC at 55°C (Al probe) or 45°C (A** probe), then dipped briefly in water, 70%, 95% and 99.5% ethanol and air-dried. Finally the sections were apposed to Hyperfilm @-max film (Amersham) for 4 weeks (Al-probe) or 3 weeks (Az~ probe). For receptor autoradiography 10 pm sections were pre-incubated in 170 mM Tris-HCl buffer containing 1 mM EDTA and 2 U/ml adenosine deaminase 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 AZ* receptors or 1 mM MgClz for A1 receptors. Incubations were performed for 2 h at 23°C in Tris-HCl buffer containing the radioligand at the appropriate concentration, 2 U/ml adenosine deaminase, 1 mM MgC& with or without 100 pM GTP for A1 and 10 mM MgC12 for AZAreceptors. The ligand used for A, receptors was [3H]-1,3-dipropyl-8-cyclopentyl xanthine (DPCPX; 0.5 nM; 60-80 Ciimmol; DuPont-NEN) and the ligand for the study of A1A receptors was [“HI-CGS 2 I680 (2-[p-(2-carboxyethyl)-phenethylamino]-5’-N-ethylcarboxamidoadenosine; 2 nM, 48.1 Ci/mmol DuPont-NEN, Stockholm, Sweden). Non-specific binding was defined by 20 pM R-PIA (for A, receptors) or 20 pM 2-chloroadenosine (2-CADO; for A2~ receptors) (11). Sections were then washed twice for 5 min each in ice-cold Tris-HCI, 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 Hyperfdm-3H (Amersham) for 5 weeks. The autoradiograms were analysed with a Microcomputer Imaging Device (Imaging Research). Optical densities were converted to binding
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density (fmol/mg grey matter) using the plastic standards and the specific activity of the radioligands. In some experiments a high concentration of GTP was added to the incubation mixture. It is well known that GTP shifts virtually all agonist binding to G protein coupled receptors to the low affinity state (see 12). This facilitates the dissociation of agonist ligands, including endogenous adenosine from the A1 receptor (13, 14). It has been shown that such treatment with GTP (or a GTP analogue) can significantly increase antagonist binding and that this is due to the removal of endogenous adenosine formed in the slices and cryptically bound to the receptors (14). In order to measure binding, without having to take into account possible changes in the amount of endogenous agonist, GTP is added. The results from the quantitative receptor autoradiography were analysed by ANOVA procedures in the SYSTAT program. Statistical hypotheses were considered significant if pcO.05.
Statistics.
Results In agreement with previous results the Ai receptor ligand was enriched in hippocampus (CAl, CA2, CA3), cerebellum and some cortical layers (15, 16). Since the autoradiograms looked virtually identical to those already published they are not shown. The mentioned regions as well as the medial and lateral geniculate nucleus, globus pallidus and the dorsolateral part of the caudate-putamen were used to quantitate Ai receptor binding. Binding was considerably higher in sections treated with 100 pM GTP (Fig I), probably indicating the removal of endogenous adenosine cryptically bound to the receptors (14, 16). Whereas neither R-PIA nor 8 PST given alone produced any significant changes in Ai ligand binding, their combination produced a significant decrease in binding sites in all regions studied except the cortex in the absence of GTP (Fig la). The magnitude of the change was similar in all regions and quite modest (IO-20% decrease). In the presence of GTP significant effects were observed only in the CA3 region of the hippocampus, in the medial geniculate nucleus and in the caudate-putamen. The magnitude of the change was at least as great as in the absence of GTP and the apparent difference between results in the two assay conditions is probably mainly due to the fact that twelve measurements were made in the absence of GTP and only six in its presence. Since GTP dramatically decreases the affinity of agonists to the receptor, the results show that the effect of systemic R-PIA-treatment on the number of DPCPX binding sites is not due to the exogenous agonist competing for the binding sites. Whereas the binding of DPCPX was no significant changes in the amount lc). These results can be compared receptor antagonist, caffeine, which receptors without any accompanying
reduced, indicating a reduced number of receptors, there were of Ai receptor mRNA as judged by in situ hybridisation (Fig with the effects of long-term treatment with an adenosine demonstrated a significant up-regulation of the number of changes at the mRNA level (3).
R-PIA is an agonist not only at Ai adenosine receptors but it can also weakly activate adenosine Azn receptors (see 7) which are enriched in the dopamine-rich regions of the brain such as the striatum (17). However, R-PIA treatment was unable to alter the binding of the selective agonist ligand CGS 21680 or the amount of A 2~ receptor mRNA (Fig 2). Under the incubation conditions used (2 nM ligand and development of film for 5 weeks) there is detectable binding only in the caudate-putamen, nucleus accumbens and tuberculum olfactorium. The detection of binding sites in other regions requires either higher ligand concentrations or much longer exposure times (18).
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Fig. 1. Histograms showing the effect of R-PIA (0.3 mgkg). S-PST (10 mgkg ip) and their combination on A, receptors in selected brain regions. First bar (untilled) untreated controls: Second bar (striped) animals treated with R-PIA: Third bar (cross-hatched) animals treated with g-PST; Fourth bar (filled) animals treated with both drugs. The following regions were analysed: Cere - cerebellum; CAl, CA2 and CA3 regions of the hippocampus: Cort - cortex (all three panels); GP - globus pallidus; CP - caudate putamen (only for binding). In addition the medial and lateral geniculate nuclei were analysed and statistically evaluated, but the results from these regions are not shown. Panel a. shows the binding of the A, receptor antagonist DPCPX in the absence of added GTP. Mean f s.e.m. of 11 to 12 sections from 6 animals. There were highly significant differences (p
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Fig. 2. Histograms showing the effect of R-PIA (0.3 mgkg), S-PST (10 mg/kg i.p.) and their combination on A?* receptor binding. measured as CGS 21680 binding, and AlrZ receptor mRNA in the caudateputamen. The former is given as fmol bound/mg tissue, the latter in arbitrary density units scaled similarly. Mean i s.c.m. of 10 to 11 sections from 6 animals. There were no significant differences between treatments.
Discusssion
The present results demonstrate that systemic long-term administration of the somewhat adenosine A1 receptor selective agonist R-PIA can cause a down-regulation of adenosine A, receptor binding sites in several regions of the brain. This effect cannot be ascribed to changes in the expression of the corresponding mRNA, and is therefore mediated at the protein level. The effect also cannot be ascribed to peripheral actions of the agonist since the magnitude of the changes was larger if these peripheral effects were blocked by the peripheral receptor antagonist S-PST (19) than if they were not. There are several possible explanations for the finding that R-PIA treatment alone had no significant effect whereas the drug given together with S-PST was effective. The most likely explanation is that this dose of R-PIA causes a major decrease in blood pressure and heart rate and thus in cardiac output (19, 20). Therefore it is expected that a much smaller amount of the drug reaches the brain. In the same doses, R-PIA, given without a peripheral antagonist, also causes a marked reduction of body temperature, while the shivering activity is suppressed (I 9, 20). It is easy to imagine that these profound peripheral effects will also have central counterparts. When all the results from all the examined brain regions were pooled, nificant change in the number of A1 receptors was found in the group
a small and statistically sigof animals treated only with
R-PIA Down-regulates Brain At Receptors
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the peripherally acting antagonist S-PST. It is important that this was seen examined in the absence of GTP. Under these circumstances changes in the agonist bound to the receptor can significantly affect the apparent receptor no significant effect of S-PST alone was seen in the slices treated with GTP does not alter receptor number.
only in the brain slices amount of endogenous number. The fact that suggests that the drug
Vol. 58, No. 9, 1996
The absence of any effect of R-PIA on ATAreceptors could be ascribed either to its being a weak agonist at these receptors or to the fact that down-regulation of A2r, receptor mediated signalling following agonist treatment is due not to changes at the receptor level but to subsequent steps in the signalling cascade (21). In favour of the latter possibility speaks the fact that long-term treatment with antagonists that are as active on A 2Aas on Ar receptors only affect the latter receptors (3). There are functional adaptations in adenosine A, but not in adenosine Axn receptor mediated responses (22). Furthermore, in brain slices treated with adenosine analogues adenosine Ai, but not adenosine A*n, receptor responses are down-regulated (23). Earlier studies have shown that the amount of R-PIA that reaches the brain immediately after injection is very low and has insignificant functional consequences (24) The present results suggest that after repeated administration the compound can reach central adenosine Ai receptors in sufficient concentrations to affect the receptors. This conclusion is also substantiated by recent findings that long-term treatment with another adenosine Ai receptor agonist can lead to adaptive changes in centrally evoked epileptic seizures (25). The results also suggest that long-term treatment with adenosine receptor agonists could lead to a tolerance to the central actions just as it has been reported to do for the peripheral actions (see 8).
Acknowlednments These studies were supported by grants from the Swedish Medical Research Council (proj. no 2553) by Karolinska Institutet, by Ostermans Foundation and by the 1987 Fund for Stroke Research. References 1. B.B. FREDHOLM, Acta Physiol. Stand. 115 283-286 (1982). 2. V. RAMKUMAR, J.R. BUMGARNER, K.A. JACOBSON and G.L. STILES, J. Clin. Invest. g 242-247 (1988). 3. B. JOHANSSON, S. AHLBERG, I. VAN DER PLOEG, S. BRENE, N. LINDEFORS, H. PERSSON and B.B. FREDHOLM, Naunyn-Schmiedeberg’s Arch. Pharmacol. 347 407-414 (1993). 4. R.C. SANDERS and T.F. MURRAY, Neurosci. Lett. 101325-330 (1989). 5. V. GEORGIEV, B. JOHANSSON and B.B. FREDHOLM, Brain Res. 612 271-277 (1993). 6. N.M. PORTER, M. RADULOVACKI and R.D. GREEN, J. Pharmacol. Exp. Ther. 244 218-225 (1988). 7. B.B. FREDHOLM, M.P. ABBRACCHIO, G. BURNSTOCK, J.W. DALY, T.K. HARDEN, K.A. JACOBSON, P. LEFF and M. WILLIAMS, Pharmacol. Rev. 46 143-156 (1994). 8. C. CASATI, S. MONOMOLI, S. DIONISOTTI, C. ZOCCHI, E. BONIZZONI and E. ONGINI, J. Pharmacol. Exp. Ther. 268 1506-1511 (1994). 9. L.C. MAHAN, L.D. MCVITTIE, E.M. SMYK-RANDALL, H. NAKATA, F.J. MONSMA JR, C.R. GERFEN and D.R. SIBLEY, Mol. Pharmacol. 40 l-7 (1991). 10. S.N. SCHIFFMANN, F. LIBERT, G. VASSART, J.E. DUMONT and J-J. VANDERHAEGHEN, Brain Res. 519 333-337 (1990).
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11. F.E. PARKINSON and B..B. FREDHOLM, Mol. Neuropharmacol. 1 179- 186 (1992). 12. A.G. GILMAN, Ann. Rev. Biochem. 56 615-649 (I 987). 13. I. VAN DER PLOEG, F.E. PARKINSON and B.B. FREDHOLM, J. Neurochem. 58 12211229 (1992). 14. F.E. PARKINSON and B.B. FREDHOLM, J. Neurochem. r 94 I-950 (I 992). 15. J. FASTBOM, A. PAZOS and J.M. PALACIOS, Neuroscience 22 8 13-826 (1987). 16. J. FASTBOM and B.B. FREDHOLM, Neuroscience 34 759-769 (1990). 17. F.E. PARKINSON and B.B. FREDHOLM, Naunyn-Schmiedeberg’s Arch Pharmacol. 342 8589 (1990). 18. B. JOHANSSON, V. GEORGIEV, F.E PARKINSON and B.B. FREDHOLM, Eur. J. Pharmacol., Mol. Pharmacol. Sect. 247 103-I 10 (1993). 19. B. JONZON, A. BERGQUIST, Y-O. LI and B.B. FREDHOLM, Acta Physiol Stand. 126 491-498 (1986). 20. H. VAPAATALO, D. ONKEN, P.J. NEUVONEN and E. WESTERMANN, Arzneimittelforschung 25 407-410 (1975). 21. Y. CHERN, H-L. LAI, J.C. FONG and Y LIANG, Mol. Pharmacol. 44 950-958 (1993). 22. B.B. FREDHOLM, B. JONZON and E LINDGREN, Acta Physiol. Stand. 122 55-59 (1984). 23. M.P. ABBRACCHIO, G. FOGLIATTO, A.M. PAOLETTI, G.E ROVATI and F CATTABENI, Eur. J. Pharmacol., Mol. Pharmacol. Sect 258 3 17-324 (1992) 24. M.S. BRODIE, K. LEE, B.B. FREDHOLM, L. STkILE and T.V. DUNWIDDIE, Brain Res. 415 323-330 (1987). 25. D.K.J.E. VON LUBITZ, I.A. PAUL, X-D. JI, M. CARTER and K.A. JACOBSON, Eur. J. Pharmacol. 253 95-99 (1994).
0024-3205(95)02355-O
ELSEVIER
ADAPTIVE CHANGES IN ADENOSINE RECEPTORS FOLLOWING LONG-TERM TREATMENT WITH THE ADENOSINE RECEPTOR AGONIST R-PHENYLISOPROPYL ADENOSINE Mercedes Fernandez’,
Per Svenningsson
and Bertil B. Fredholm*
Department of Physiology and Pharmacology, Division of Molecular Neuropharmacology, Karolinska Institute& S-171 77 Stockholm, Sweden (Received in final form December 12, 19%)
Summaw in brain A, and AlA receptors and in the corresponding mRNA were studied using quantitative receptor autoradiography and in sifu hybridisation. [3H]DPCPX was used as an antagonist ligand at Ai receptors and [3H]-CGS 21680 as an agonist ligand at AzA receptors. Treatment of rats with the relatively Ai receptor selective adenosine analogue R-PIA (0.3 mg/kg) for 7 days in the presence of the peripherally acting antagonist 8-p-sulfophenyltheophylline (S-PST; 10 mg/kg) caused a decrease in the binding of the Ai receptor ligand, but not in that of the AZA receptor ligand. The effect on Ai receptors was also seen in the presence of 100 PM GTP that decreases agonist binding to insignificant levels, There was no change in either Ai or AZ* receptor mRNA. No significant changes were detected following administration of either R-PIA or S-PST alone. These results thus demonstrate an effect on brain A, receptors after systemic administration of R-PIA in the presence of a peripherally acting adenosine antagonist, demonstrating that, under these conditions, the agonist reaches receptors in significant amounts. Changes
Key Words: adenosine receptors, R-phenykopropyl
adenosine, mRNA
It is known that long-term treatment with adenosine receptor antagonists leads to an increase in the number of adenosine Ai receptors (1, 2) which is not accompanied by changes in the amount of Ar receptor n-RNA (3). By contrast, there are only small, and generally insignificant changes in A*.,, receptors and mRNA (3). Long-term treatment with antagonists also causes a number of adaptive changes that may or may not be related to these changes in adenosine receptors (4, 5). There is also some evidence that long-term intracerebroventricular treatment with adenosine receptor agonists can produce adaptive changes in the number of Ai receptors (6). However, little is known about the adaptive changes in central adenosine receptors following systemic administration of an adenosine
’ Present address: Dipartimento Quimica, Inorgzinica, Orgknica y Bioquimica, Universidad Castilla-La Mancha, Ciudad Real, Spain. * To whom all correspondence should be sent at the above address.
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receptor agonist. In the present series of experiments we administered the relatively Al receptor selective agonist R-N6-phenylisopropyl adenosine (R-PIA) (7) once daily by i.p. injection for 7 days. The number of Al and AZAreceptors was examined by quantitative receptor autoradiography and their corresponding mRNAs using in situ hybridisation. When adenosine receptor agonists are given systemically they produce marked effects on e.g. the cardiovascular system. Even though the magnitude of these effects may decrease upon repeated administration (8) we wanted to minimise the peripheral cardiovascular effects by combining the adenosine receptor agonist R-PIA with the peripherally acting antagonist S-p-sulfophenyltheophylline (S-PST). The results suggest that an A1 receptor agonist can, via a direct action at central sites, decrease binding to Al receptors, and that this is not due to any effect at the transcriptional level.
Materials and Methods A total of six male Sprague-Dawley rats per treatment was used. One group of animals received saline injections, the second group injections of R-PIA (0.3 mg/kg, i.p ), the third group S-PST (10 mg/kg, i.p.) and the fourth group both R-PIA and S-PST. The treatment lasted for seven days. After anaesthesia with CO1 the rats were decapitated, the whole brain removed and frozen on dry ice. Saggital sections of each brain were cut with a Leitz cryostat. Fourteen-pm-thick sections were thaw-mounted on poly-L-lysine (50 &ml) coated slides for it2 silu hybridisation. Ten pm thick sections were thaw-mounted on gelatine coated slides for quantitative receptor autoradiography. The 48-mer Al adenosine receptor probe was complementary to nucleotides 985- 1032 of the rat Al receptor (3, 9). The 44-mer AzA probe was complementary to nucleotides 916-959 of the dog RDCS cDNA (10). The oligodeoxyribonucleotides were radiolabelled using terminal deoxyribonucleotidyl transferase (Amersham) and a-35S-dATP (Amersham) to a specific activity of about 1O9 CPM/pg. Slide mounted sections were hybridised in a cocktail containing 50% formamide (Baker), 4 x SSC, 1 x Denhardt’s solution, 1% sarcosyl, 0.02 M NaP04 (pH 7.0), 10% dextran sulfate, 0.5 mg/ml yeast tRNA (Sigma), 0.06 M dithiothreitol, 0.1 mg/ml sheared salmon sperm DNA and lo7 CPM/ml of probe. After hybridisation for 16 h at 42°C the sections were washed four times for 15 min each in 1 x SSC at 55°C (Al probe) or 45°C (A** probe), then dipped briefly in water, 70%, 95% and 99.5% ethanol and air-dried. Finally the sections were apposed to Hyperfilm @-max film (Amersham) for 4 weeks (Al-probe) or 3 weeks (Az~ probe). For receptor autoradiography 10 pm sections were pre-incubated in 170 mM Tris-HCl buffer containing 1 mM EDTA and 2 U/ml adenosine deaminase 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 AZ* receptors or 1 mM MgClz for A1 receptors. Incubations were performed for 2 h at 23°C in Tris-HCl buffer containing the radioligand at the appropriate concentration, 2 U/ml adenosine deaminase, 1 mM MgC& with or without 100 pM GTP for A1 and 10 mM MgC12 for AZAreceptors. The ligand used for A, receptors was [3H]-1,3-dipropyl-8-cyclopentyl xanthine (DPCPX; 0.5 nM; 60-80 Ciimmol; DuPont-NEN) and the ligand for the study of A1A receptors was [“HI-CGS 2 I680 (2-[p-(2-carboxyethyl)-phenethylamino]-5’-N-ethylcarboxamidoadenosine; 2 nM, 48.1 Ci/mmol DuPont-NEN, Stockholm, Sweden). Non-specific binding was defined by 20 pM R-PIA (for A, receptors) or 20 pM 2-chloroadenosine (2-CADO; for A2~ receptors) (11). Sections were then washed twice for 5 min each in ice-cold Tris-HCI, 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 Hyperfdm-3H (Amersham) for 5 weeks. The autoradiograms were analysed with a Microcomputer Imaging Device (Imaging Research). Optical densities were converted to binding
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density (fmol/mg grey matter) using the plastic standards and the specific activity of the radioligands. In some experiments a high concentration of GTP was added to the incubation mixture. It is well known that GTP shifts virtually all agonist binding to G protein coupled receptors to the low affinity state (see 12). This facilitates the dissociation of agonist ligands, including endogenous adenosine from the A1 receptor (13, 14). It has been shown that such treatment with GTP (or a GTP analogue) can significantly increase antagonist binding and that this is due to the removal of endogenous adenosine formed in the slices and cryptically bound to the receptors (14). In order to measure binding, without having to take into account possible changes in the amount of endogenous agonist, GTP is added. The results from the quantitative receptor autoradiography were analysed by ANOVA procedures in the SYSTAT program. Statistical hypotheses were considered significant if pcO.05.
Statistics.
Results In agreement with previous results the Ai receptor ligand was enriched in hippocampus (CAl, CA2, CA3), cerebellum and some cortical layers (15, 16). Since the autoradiograms looked virtually identical to those already published they are not shown. The mentioned regions as well as the medial and lateral geniculate nucleus, globus pallidus and the dorsolateral part of the caudate-putamen were used to quantitate Ai receptor binding. Binding was considerably higher in sections treated with 100 pM GTP (Fig I), probably indicating the removal of endogenous adenosine cryptically bound to the receptors (14, 16). Whereas neither R-PIA nor 8 PST given alone produced any significant changes in Ai ligand binding, their combination produced a significant decrease in binding sites in all regions studied except the cortex in the absence of GTP (Fig la). The magnitude of the change was similar in all regions and quite modest (IO-20% decrease). In the presence of GTP significant effects were observed only in the CA3 region of the hippocampus, in the medial geniculate nucleus and in the caudate-putamen. The magnitude of the change was at least as great as in the absence of GTP and the apparent difference between results in the two assay conditions is probably mainly due to the fact that twelve measurements were made in the absence of GTP and only six in its presence. Since GTP dramatically decreases the affinity of agonists to the receptor, the results show that the effect of systemic R-PIA-treatment on the number of DPCPX binding sites is not due to the exogenous agonist competing for the binding sites. Whereas the binding of DPCPX was no significant changes in the amount lc). These results can be compared receptor antagonist, caffeine, which receptors without any accompanying
reduced, indicating a reduced number of receptors, there were of Ai receptor mRNA as judged by in situ hybridisation (Fig with the effects of long-term treatment with an adenosine demonstrated a significant up-regulation of the number of changes at the mRNA level (3).
R-PIA is an agonist not only at Ai adenosine receptors but it can also weakly activate adenosine Azn receptors (see 7) which are enriched in the dopamine-rich regions of the brain such as the striatum (17). However, R-PIA treatment was unable to alter the binding of the selective agonist ligand CGS 21680 or the amount of A 2~ receptor mRNA (Fig 2). Under the incubation conditions used (2 nM ligand and development of film for 5 weeks) there is detectable binding only in the caudate-putamen, nucleus accumbens and tuberculum olfactorium. The detection of binding sites in other regions requires either higher ligand concentrations or much longer exposure times (18).
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Fig. 1. Histograms showing the effect of R-PIA (0.3 mgkg). S-PST (10 mgkg ip) and their combination on A, receptors in selected brain regions. First bar (untilled) untreated controls: Second bar (striped) animals treated with R-PIA: Third bar (cross-hatched) animals treated with g-PST; Fourth bar (filled) animals treated with both drugs. The following regions were analysed: Cere - cerebellum; CAl, CA2 and CA3 regions of the hippocampus: Cort - cortex (all three panels); GP - globus pallidus; CP - caudate putamen (only for binding). In addition the medial and lateral geniculate nuclei were analysed and statistically evaluated, but the results from these regions are not shown. Panel a. shows the binding of the A, receptor antagonist DPCPX in the absence of added GTP. Mean f s.e.m. of 11 to 12 sections from 6 animals. There were highly significant differences (p
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0
Control
I
Both
Fig. 2. Histograms showing the effect of R-PIA (0.3 mgkg), S-PST (10 mg/kg i.p.) and their combination on A?* receptor binding. measured as CGS 21680 binding, and AlrZ receptor mRNA in the caudateputamen. The former is given as fmol bound/mg tissue, the latter in arbitrary density units scaled similarly. Mean i s.c.m. of 10 to 11 sections from 6 animals. There were no significant differences between treatments.
Discusssion
The present results demonstrate that systemic long-term administration of the somewhat adenosine A1 receptor selective agonist R-PIA can cause a down-regulation of adenosine A, receptor binding sites in several regions of the brain. This effect cannot be ascribed to changes in the expression of the corresponding mRNA, and is therefore mediated at the protein level. The effect also cannot be ascribed to peripheral actions of the agonist since the magnitude of the changes was larger if these peripheral effects were blocked by the peripheral receptor antagonist S-PST (19) than if they were not. There are several possible explanations for the finding that R-PIA treatment alone had no significant effect whereas the drug given together with S-PST was effective. The most likely explanation is that this dose of R-PIA causes a major decrease in blood pressure and heart rate and thus in cardiac output (19, 20). Therefore it is expected that a much smaller amount of the drug reaches the brain. In the same doses, R-PIA, given without a peripheral antagonist, also causes a marked reduction of body temperature, while the shivering activity is suppressed (I 9, 20). It is easy to imagine that these profound peripheral effects will also have central counterparts. When all the results from all the examined brain regions were pooled, nificant change in the number of A1 receptors was found in the group
a small and statistically sigof animals treated only with
R-PIA Down-regulates Brain At Receptors
775
the peripherally acting antagonist S-PST. It is important that this was seen examined in the absence of GTP. Under these circumstances changes in the agonist bound to the receptor can significantly affect the apparent receptor no significant effect of S-PST alone was seen in the slices treated with GTP does not alter receptor number.
only in the brain slices amount of endogenous number. The fact that suggests that the drug
Vol. 58, No. 9, 1996
The absence of any effect of R-PIA on ATAreceptors could be ascribed either to its being a weak agonist at these receptors or to the fact that down-regulation of A2r, receptor mediated signalling following agonist treatment is due not to changes at the receptor level but to subsequent steps in the signalling cascade (21). In favour of the latter possibility speaks the fact that long-term treatment with antagonists that are as active on A 2Aas on Ar receptors only affect the latter receptors (3). There are functional adaptations in adenosine A, but not in adenosine Axn receptor mediated responses (22). Furthermore, in brain slices treated with adenosine analogues adenosine Ai, but not adenosine A*n, receptor responses are down-regulated (23). Earlier studies have shown that the amount of R-PIA that reaches the brain immediately after injection is very low and has insignificant functional consequences (24) The present results suggest that after repeated administration the compound can reach central adenosine Ai receptors in sufficient concentrations to affect the receptors. This conclusion is also substantiated by recent findings that long-term treatment with another adenosine Ai receptor agonist can lead to adaptive changes in centrally evoked epileptic seizures (25). The results also suggest that long-term treatment with adenosine receptor agonists could lead to a tolerance to the central actions just as it has been reported to do for the peripheral actions (see 8).
Acknowlednments These studies were supported by grants from the Swedish Medical Research Council (proj. no 2553) by Karolinska Institutet, by Ostermans Foundation and by the 1987 Fund for Stroke Research. References 1. B.B. FREDHOLM, Acta Physiol. Stand. 115 283-286 (1982). 2. V. RAMKUMAR, J.R. BUMGARNER, K.A. JACOBSON and G.L. STILES, J. Clin. Invest. g 242-247 (1988). 3. B. JOHANSSON, S. AHLBERG, I. VAN DER PLOEG, S. BRENE, N. LINDEFORS, H. PERSSON and B.B. FREDHOLM, Naunyn-Schmiedeberg’s Arch. Pharmacol. 347 407-414 (1993). 4. R.C. SANDERS and T.F. MURRAY, Neurosci. Lett. 101325-330 (1989). 5. V. GEORGIEV, B. JOHANSSON and B.B. FREDHOLM, Brain Res. 612 271-277 (1993). 6. N.M. PORTER, M. RADULOVACKI and R.D. GREEN, J. Pharmacol. Exp. Ther. 244 218-225 (1988). 7. B.B. FREDHOLM, M.P. ABBRACCHIO, G. BURNSTOCK, J.W. DALY, T.K. HARDEN, K.A. JACOBSON, P. LEFF and M. WILLIAMS, Pharmacol. Rev. 46 143-156 (1994). 8. C. CASATI, S. MONOMOLI, S. DIONISOTTI, C. ZOCCHI, E. BONIZZONI and E. ONGINI, J. Pharmacol. Exp. Ther. 268 1506-1511 (1994). 9. L.C. MAHAN, L.D. MCVITTIE, E.M. SMYK-RANDALL, H. NAKATA, F.J. MONSMA JR, C.R. GERFEN and D.R. SIBLEY, Mol. Pharmacol. 40 l-7 (1991). 10. S.N. SCHIFFMANN, F. LIBERT, G. VASSART, J.E. DUMONT and J-J. VANDERHAEGHEN, Brain Res. 519 333-337 (1990).
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11. F.E. PARKINSON and B..B. FREDHOLM, Mol. Neuropharmacol. 1 179- 186 (1992). 12. A.G. GILMAN, Ann. Rev. Biochem. 56 615-649 (I 987). 13. I. VAN DER PLOEG, F.E. PARKINSON and B.B. FREDHOLM, J. Neurochem. 58 12211229 (1992). 14. F.E. PARKINSON and B.B. FREDHOLM, J. Neurochem. r 94 I-950 (I 992). 15. J. FASTBOM, A. PAZOS and J.M. PALACIOS, Neuroscience 22 8 13-826 (1987). 16. J. FASTBOM and B.B. FREDHOLM, Neuroscience 34 759-769 (1990). 17. F.E. PARKINSON and B.B. FREDHOLM, Naunyn-Schmiedeberg’s Arch Pharmacol. 342 8589 (1990). 18. B. JOHANSSON, V. GEORGIEV, F.E PARKINSON and B.B. FREDHOLM, Eur. J. Pharmacol., Mol. Pharmacol. Sect. 247 103-I 10 (1993). 19. B. JONZON, A. BERGQUIST, Y-O. LI and B.B. FREDHOLM, Acta Physiol Stand. 126 491-498 (1986). 20. H. VAPAATALO, D. ONKEN, P.J. NEUVONEN and E. WESTERMANN, Arzneimittelforschung 25 407-410 (1975). 21. Y. CHERN, H-L. LAI, J.C. FONG and Y LIANG, Mol. Pharmacol. 44 950-958 (1993). 22. B.B. FREDHOLM, B. JONZON and E LINDGREN, Acta Physiol. Stand. 122 55-59 (1984). 23. M.P. ABBRACCHIO, G. FOGLIATTO, A.M. PAOLETTI, G.E ROVATI and F CATTABENI, Eur. J. Pharmacol., Mol. Pharmacol. Sect 258 3 17-324 (1992) 24. M.S. BRODIE, K. LEE, B.B. FREDHOLM, L. STkILE and T.V. DUNWIDDIE, Brain Res. 415 323-330 (1987). 25. D.K.J.E. VON LUBITZ, I.A. PAUL, X-D. JI, M. CARTER and K.A. JACOBSON, Eur. J. Pharmacol. 253 95-99 (1994).