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Properties of binding sites for [3H]cyclohexyladenosine in the hippocampus and other regions of rat brain: A quantitative autoradiographic study
] [6
Neuroscience Letter~, 64 ( 19,~61 I 16 [ 2i~ P;Isevier Scientific Publisher~ Ireland fad
NSL 03764
PROPERTIES OF BINDING SITES FOR [3H]CYCLOHEXYLADENOSINE IN TI-~E HIPPOCAMPUS AND OTHER REGIONS OF RAT BRAIN: A QUANTITATIVE AUTORADIOGRAPHIC STUDY
A N D R E A S E R F U R T H and MARTIN R E D D I N G T O N * Department ~1 Neuromorphology, Max-Planck-lnstitute for Psychiatry, Am Klol~fer,spitz 18a. D-8033 Martinsried (F. R.G. ) (Received October 10lh, 1985; Revised version received November l lth, 1985: Accepted November 13th. 1985)
Key words:
adenosine - receptor
hippoeampus
autoradiography
guanyl nucleotide
rat
The properties of binding sites for the adenosine receptor ligand, [3H]cyclohexyladenosine (pH]CHA), were investigated in rat brain using quantitative autoradiography. Scalchard analysis of the binding data showed that there were no significant differences between Ka values for [3H]CHA in any of the regions investigated. The highest concentrations of[3H]CHA binding sites were found in the cerebellum (molecular layer) and the stratum oriens and stratum radiatum of the hippocampus (CAI region). Displacemenl curves obtained using N-ethylcarboxamidoadenosine (NECA) and the R- and S-diastereoisomers of phenylisopropyl adenosine (PIA) showed the [3H]CHA binding sites to have the pharmacological properties of At-adenosine receptors, i.e. the order of potency for these derivatives was R-PIA > NECA > S-PIA, in all regions tested. Further. pH]CHA binding was in all cases attenuated by the guanosine triphosphate derivative,/L)'-imidoguanosine triphosphate. These results indicate that [3H]CHA binding sites throughout the central nervous system have the properties of A~-adenosine receptors and that these are in all regions associated with guanine nucleotide regulatory proteins.
The importance of adenosine as a neuromodulator in the mammalian hippocampus has been well documented in recent years. The modulatory action of adenosine on nerve cell activity in the central nervous system (CNS) is mediated by extracellular receptors having pharmacological properties similar to those which attenuate adenylate cyclase [3, 12]. These receptors, referred to as Awadenosine receptors [9, 16], can be characterized biochemically using the radioactively labelled agonist, pH]cyclohexyladenosine ([3H]CHA) [1]. Autoradiographic studies using this ligand have shown Awreceptors to be specifically localized in certain brain regions, being highly concentrated in the cerebellum and in certain hippocampal subregions [6, 8]. Such qualitative studies do not, however, enable a possible heterogeneity of binding sites to be distinguished. We have therefore used quantitative autoradiographic techniques to examine the properties of [3H]CHA binding sites in rat brain with specific reference to the hippocampus, cerebellum and striatum. In particular we have examined the .
_
m
*Author for correspondence. 0304-3940/86/'$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
117
atlinities of these regions for p H ] C H A and some other adenosine receptor agonists, as well as the ability of guanine nucleotides to regulate agonist binding. Cryostat sections cut from rat brain were used. Sections (20 Ira0 were allowed to dry at room temperature before being incubated for 90 min at room temperature in 50 mM Tris-HC1, pH 7.4, containing 2.5 units/ml adenosine deaminase (Sigma Chenile, Miinchen). For the construction of displacement curves with non-radioactive ligands the concentration of p H ] C H A (New England Nuclear Chemicals, Drcieich) used was 10 nM. Saturation curves for p H ] C H A were constructed using concentrations of labelled ligand in the range 0.5 to 50 nM. Autoradiography was performed using ['H]Ultrofihn (LKB) with exposure times of 4- 6 weeks. Extinction wtlues w,ere measured using a Leitz Texture Analysis System and converted to dpm/mg protein by comparison with standards constructed from a rat cerebral cortical (grey matter) brain mash containing w~rious amounts of [3H]adenosine (Amersham) [15]. Saturation curves using dilfercnt concentrations of [~H]CHA yielded Scatchard plots as show.n for the stratum radiatum of the CA I region of hippocampus in Fig. la. Thc B...... and Kj values obtained for various brain regions are sho~n in Table 1. Although no significant differences were found between the apparent altinities for [~H]('HA in various brain regions, the actual K,; values obtained were somcwhat higher than those found in membrane preparations with this ligand [10. 13]. In addition, B...... values were somewhat higher than published values for membrane preparations, a phenomenon also reported for the binding of ligands to other receptor svs0.4-
100.
a
b
A
"5-
•
•
--
-~0.3
E "7 E
80
t_ 4,-
g LJ
o~ 60
O
E
g g
~- 0.2
g
z,O
c_
~g g 01
kg
20
,
,
i
0.5 1 1.5 2.0 bound(pmoLmg protein -1)
2.5
-9
-8
-'7
-6
[og[tigand]
Fig. 1. Properties of [3H]CttA binding sites in rat brain, a: Scatchard plot of p t t ] C I t A binding (0.5 50 nM) to the stralum radiatum of the hippocampus (CAI region), b: displacemenl of 10 nM I~HI('IIA from slriatum by R-PIA (circles), N E C A (squares) and S-PIA (triangles). All data were obtained by quantitative dcnsitometry of autoradiographs as described m the text.
1i8
terns measured by quantitative autoradiography [1 l]. The highest concentrations of [~HJCHA binding sites were found in the cerebellum (molecular layer) and in the stratum radiatum and stratum oriens of the hippocampal CA1 region. The numbers of pH]CHA binding sites in the hippocampus lay between 1.21 and 2.34 pmol/mg protein in the various subregions. An example of displacement curves obtained using quantitative autoradiography is shown in Fig. lb. In the rat striatum the ligands R-phenylisopropyladenosine (RPIA), N-ethylcarboxamidoadenosine (NECA) and S-phenylisopropyladenosine (SPIA) displaced pH]CHA from cryostat sections in a dose-dependent manner such that the rank order of their affinities was R-PIA > NECA > S-PIA. As has also been found in binding studies with isolated membrane fractions, the [3H]CHA binding sites measured in cryostat sections therefore have the characteristics of adenosine receptors of the A~-type [1, 9, 16]. Table I summarizes the equilibrium binding proTABLE 1 E Q U I L I B R I U M B I N D I N G C H A R A C T E R I S T I C S OF [3H]CHA B I N D I N G SITES IN V A R IO U S RL;G I O N S OF RAT BRAIN Scatchard parameters were derived from saturation curves performed using a concentration range of 11.5 to 51) nM [3H]CHA and are given in fmol/mg protein (Bin,x) and nM (K,~). The K, values for unlabelled ligands were derived from ICso values obtained with 10 nM [3H]CHA and various concentrations of RPIA, S-PIA or NECA, using the measured Kj values for the appropriate region obtained by Scatchard analysis. The effect of the guanine nucleotide derivative, G p p N t t p , was tested at a concentration of 100 ItM. All nqeasurcments were made using quantitativc autoradiography as described in the text.
B ......
Hippocampal subregions: Stratum oricns, CAI Stratum radiatum, CA1 Stratum lacunarc Molecular layer, dentate gyrus Granular layer, dentate gyrus Infragranular layer, internal leaf CA4 cell zone lnfragranular layer, external leaf Stratum radiatum, CA3 Stratum oriens, CA3 Cerebral cortex, area 41, layer 4 Cerebral cortex, area 41, layer 6 Cerebral cortex, area 2a~ layer 4 Cerebral cortex, area 2a, layer 6 Cerebellum. molecular layer Cerebellum, granular laycr Striatum Lateral posterior thalamic nucleus Lateral seplal nucleus (dorsal aspect)
2.31 2.34 1.49 2. l0 1.21 2.08 1.28 1.96 1.78 1.75 1.90 1.27 1.44 1.07 2.59 0.83 1.113 1.70 I. 15
K,~
6.12 5.88 6.32 7.01 7.23 6.18 7.25 6.22 7.60 7.77 6.22 6,21 7.65 6.45 5.19 5.90 7.23 8.02 5.62
K, values (nM)
±GppNttp (% control)
R-PIA
S-HA
N EC A
3.11 3.07 3.38 3.99 3.66 3.97 2.62 2.88 4.211 4.19 3.46 3.04 4.59 -2.58 4.26 -
294 226
13.9 16.7 16./)
314 . . . .
254 275 250
237 260 . . . . .
16.4 16.0 14.9
11.9 16.2 -
41.6 35.(i 41,0 30.6 45.7 36.6 45.tt 35.3 41.8 44.8 36.3 40.9 19.0 21.3 22.9
119 perties o f [3H]CHA b i n d i n g sites d e t e r m i n e d in v a r i o u s rat b r a i n regions using quantitative d e n s i t o m e t r y o f a u t o r a d i o g r a p h s . T h e respective a p p a r e n t K, values for RP I A , S - P I A and N E C A were similar in all regions, a n d in all cases R - P I A was m o r e effective than N E C A in displacing [3H]CHA. The binding o f a d e n o s i n e r e c e p t o r agonists to A~-adenosine receptors has been shown to be a t t e n u a t e d by g u a n i n e nucleotides such that a shift is observed from a high to a low aftinity state [1, 4, 17]. As f o u n d in m e m b r a n e p r e p a r a t i o n s , a d d i t i o n o f the p o o r l y h y d r o l y s a b l e g u a n o s i n e t r i p h o s p h a t e derivative, /L',,-imido g u a n o s i n e t r i p h o s p h a t e ( G p p N H p ) , to cryostat sections from rat brain decreased the binding o f p H ] C H A in all regions tested (Table l). These d a t a indicate the a s s o c i a t i o n o f guanine nucleotide r e g u l a t o r y m e c h a n i s m s with A~-receptors t h r o u g h o u t the central nervous system. In s u m m a r y , all regions o f the rat C N S e x a m i n e d here which b o u n d p H ] C H A showed p r o p e r t i e s consistent with the presence o f a d e n o s i n e receptors o f the A~-type. N o evidence was o b t a i n e d from studies o f e q u i l i b r i u m binding p r o p e r t i e s for a heterogeneity o f high-attinity b i n d i n g sites with discrete localizations. In a d d i t i o n , all regions tested showed the a s s o c i a t i o n o f A ] - r e c e p t o r s with r e g u l a t o r y guanine nucleotide b i n d i n g c o m p o n e n t s . These d a t a suggest that all A r r e c e p t o r - m e d i a t e d events in the C N S , whether p r e s y n a p t i c [2, 5] or p o s t s y n a p t i c [7, 14] involve similar types o f ligand recognition sites. It remains to be established whether these receptors are associated with the same or different effector mechanisms. The a u t h o r s w o u l d like to t h a n k Professor G . W . K r e u t z b e r g for his active s u p p o r t a n d e n c o u r a g e m e n t t h r o u g h o u t this work. O u r t h a n k s are also due to Dr. K.S. Lee l\~r much helpful discussion, Dr. W o l f r a m Tetzlaff for setting up the q u a n t i t a t i v e d e n s i t o m e t r y and Petra Grfimmel for help in m e a s u r i n g the a u t o r a d i o g r a p h s . This study is part o f the w o r k to be s u b m i t t e d by A.E. in partial fulfilhnent o f the degree o f l ) o c t o r o f Medicine at the University o f Munich. The financial s u p p o r t o f the Deutsche F o r s c h u n g s g e m e i n s c h a f t is gratefully a c k n o w l e d g e d .
1 Bruns, R.F., Daly, J.W. and Snyder, S.1t., Adenosine receptors in brain membranes: binding of N"cyclohexyl [3H]adenosine and 1,3-diethyl-8-pH]-phenylxanthine, Proc. Natl. Acad. Sci. USA, 77 (1980) 5547 5551. 2 Dolphin. A.C. and Archer, E.R., An adenosine agonisl inhibits and a cyclic AMP analogne enhances the releasc of glutamate but not GABA from slices of rat dentate gyrus, Neurosci. kerr., 43 (1983) 49 54. 3 Dunwiddie, T.V. and Fredholm, B.B., Adenosine receptors mediating inhibitory elcctrophysiological rcsponscs in rat hippocampus are different from receptors mediating cyclic AMP accumulation. Naunyn-Schmiedebergs Arch. Pharmacol., 326 (1984) 294 301. 4 Goodman, R.R., Cooper, M.J., Gavish, M. and Snyder, S.tt., Guanine nucleotide and cation regulation of the binding of [~H]cyclohexyladenosineand [3H]diethylphenylxanthine to adenosine A~ receptors in brain membranes, Mol. Pharmacol., 21 (1982) 329 335. 5 Goodman, R.R., Kuhar, M.J., Hester, L. and Snyder, S.H., Adenosine receptors: autoradiographic evidence for their location on axon terminals of excitatory neurons, Science, 220 (1983) 967 969. 6 Goodman, R.R. and Snyder, S.H., Autoradiographic localisation of adenosine receptors in rat brain using [~H]cyclohexyladenosine,J. Neurosci., 2 (1982) 1230 1241.
120 7 Lee, K.S., Schubert, P. and Heinemann, U., The anticonvulsive action of adenosine: a postsynaptic, dendritic action by a possible endogenous anticonvulsant, Brain Res., 321 (1984) 160-104. 8 Lewis, M.E., Patel, J., Edley, S.M. and Marangos, P.J., Autoradiographic visualisation of rat brain adenosine receptors using N6-cyclohexyl-[3H]adenosine, Eur. J. Pharmacol., 73 ( 1981 ) 109 110. 9 Londos, (,., Cooper, D.M.F. and Wolff, J., Subclasses of external adenosine receptors. Proc. Nall Acad. Sci. USA 77 (1980) 2551 2554. 10 Marangos, P.J.. PateL J., Martino, A.M., Dilli, M. and Boulenger, J.P., Differential binding properties of adenosine receptor agonists and antagonists in brain, J. Neurochem., 41 (1983) 367 374. I I Rainbow, T.C., Bicgon, A. and Berck, D.J., Quantitative receptor autoradiography with tritium-labeled ligands: comparison of biochemical and densitometric measurements, J. Neurosci. Meth.. 11 (1984) 231 241. 12 Reddington, M., Lee, K.S. and Schubert, P., An A~-adenosine receptor, characterised by [~H]cyclohexyladenosine binding, mediates the depression of evoked potentials in a rat hippocampal slice preparation, Neurosci. Left., 28 (1982) 275 279. 13 Reddington, M., Lee, K.S., Schubert, P. and Kreutzberg, G.W., Biochemical and electrophysiological characterisation of adenosine receptors in rat brain. In F. DeFeudis and P. Mandel (Eds.), CNS Receptors: From Molecular Pharmacology to Behaviour, Adv. Biochem. Psychopharm., Vol. 37, Raven Press, New York, 1983, pp. 465 476. 14 Siggins, G. and Schubert, P., Adenosine depression of hippocampal neurons in x,itro: an intracellulai study of dose-dependent actions on synaptic and membrane potentials, Neurosci. Lett.. 13 (1981) 55 61). 15 Unnerstall, J R . , Niehoff, D.L., Kuhar, M.J. and Palacios, J.M., Quantitative receptor autoradiography using [3H]Ultrofilm: application to multiple benzodiazepine receptors, J. Neurosci. Melh., 6 (1982) 59 73. 16 VanCalker, D., Mfiller, M. and tlamprechl, B., Adenosine regulates via two different types of receptors the accumulation of cyclic AMP in cultured brain cells, J. Neurochem., 33 (1979) 999 1005. 17 Yeung~ S.M. and Green, R.D., Agonist and antagonist affinities for inhibitory adenosine receptors are reciprocally affected by 5'-guanylylimidophosphate or N-ethylmaleimide, J. Biol. Chem, 258 (19831 2334 2339.
Neuroscience Letter~, 64 ( 19,~61 I 16 [ 2i~ P;Isevier Scientific Publisher~ Ireland fad
NSL 03764
PROPERTIES OF BINDING SITES FOR [3H]CYCLOHEXYLADENOSINE IN TI-~E HIPPOCAMPUS AND OTHER REGIONS OF RAT BRAIN: A QUANTITATIVE AUTORADIOGRAPHIC STUDY
A N D R E A S E R F U R T H and MARTIN R E D D I N G T O N * Department ~1 Neuromorphology, Max-Planck-lnstitute for Psychiatry, Am Klol~fer,spitz 18a. D-8033 Martinsried (F. R.G. ) (Received October 10lh, 1985; Revised version received November l lth, 1985: Accepted November 13th. 1985)
Key words:
adenosine - receptor
hippoeampus
autoradiography
guanyl nucleotide
rat
The properties of binding sites for the adenosine receptor ligand, [3H]cyclohexyladenosine (pH]CHA), were investigated in rat brain using quantitative autoradiography. Scalchard analysis of the binding data showed that there were no significant differences between Ka values for [3H]CHA in any of the regions investigated. The highest concentrations of[3H]CHA binding sites were found in the cerebellum (molecular layer) and the stratum oriens and stratum radiatum of the hippocampus (CAI region). Displacemenl curves obtained using N-ethylcarboxamidoadenosine (NECA) and the R- and S-diastereoisomers of phenylisopropyl adenosine (PIA) showed the [3H]CHA binding sites to have the pharmacological properties of At-adenosine receptors, i.e. the order of potency for these derivatives was R-PIA > NECA > S-PIA, in all regions tested. Further. pH]CHA binding was in all cases attenuated by the guanosine triphosphate derivative,/L)'-imidoguanosine triphosphate. These results indicate that [3H]CHA binding sites throughout the central nervous system have the properties of A~-adenosine receptors and that these are in all regions associated with guanine nucleotide regulatory proteins.
The importance of adenosine as a neuromodulator in the mammalian hippocampus has been well documented in recent years. The modulatory action of adenosine on nerve cell activity in the central nervous system (CNS) is mediated by extracellular receptors having pharmacological properties similar to those which attenuate adenylate cyclase [3, 12]. These receptors, referred to as Awadenosine receptors [9, 16], can be characterized biochemically using the radioactively labelled agonist, pH]cyclohexyladenosine ([3H]CHA) [1]. Autoradiographic studies using this ligand have shown Awreceptors to be specifically localized in certain brain regions, being highly concentrated in the cerebellum and in certain hippocampal subregions [6, 8]. Such qualitative studies do not, however, enable a possible heterogeneity of binding sites to be distinguished. We have therefore used quantitative autoradiographic techniques to examine the properties of [3H]CHA binding sites in rat brain with specific reference to the hippocampus, cerebellum and striatum. In particular we have examined the .
_
m
*Author for correspondence. 0304-3940/86/'$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
117
atlinities of these regions for p H ] C H A and some other adenosine receptor agonists, as well as the ability of guanine nucleotides to regulate agonist binding. Cryostat sections cut from rat brain were used. Sections (20 Ira0 were allowed to dry at room temperature before being incubated for 90 min at room temperature in 50 mM Tris-HC1, pH 7.4, containing 2.5 units/ml adenosine deaminase (Sigma Chenile, Miinchen). For the construction of displacement curves with non-radioactive ligands the concentration of p H ] C H A (New England Nuclear Chemicals, Drcieich) used was 10 nM. Saturation curves for p H ] C H A were constructed using concentrations of labelled ligand in the range 0.5 to 50 nM. Autoradiography was performed using ['H]Ultrofihn (LKB) with exposure times of 4- 6 weeks. Extinction wtlues w,ere measured using a Leitz Texture Analysis System and converted to dpm/mg protein by comparison with standards constructed from a rat cerebral cortical (grey matter) brain mash containing w~rious amounts of [3H]adenosine (Amersham) [15]. Saturation curves using dilfercnt concentrations of [~H]CHA yielded Scatchard plots as show.n for the stratum radiatum of the CA I region of hippocampus in Fig. la. Thc B...... and Kj values obtained for various brain regions are sho~n in Table 1. Although no significant differences were found between the apparent altinities for [~H]('HA in various brain regions, the actual K,; values obtained were somcwhat higher than those found in membrane preparations with this ligand [10. 13]. In addition, B...... values were somewhat higher than published values for membrane preparations, a phenomenon also reported for the binding of ligands to other receptor svs0.4-
100.
a
b
A
"5-
•
•
--
-~0.3
E "7 E
80
t_ 4,-
g LJ
o~ 60
O
E
g g
~- 0.2
g
z,O
c_
~g g 01
kg
20
,
,
i
0.5 1 1.5 2.0 bound(pmoLmg protein -1)
2.5
-9
-8
-'7
-6
[og[tigand]
Fig. 1. Properties of [3H]CttA binding sites in rat brain, a: Scatchard plot of p t t ] C I t A binding (0.5 50 nM) to the stralum radiatum of the hippocampus (CAI region), b: displacemenl of 10 nM I~HI('IIA from slriatum by R-PIA (circles), N E C A (squares) and S-PIA (triangles). All data were obtained by quantitative dcnsitometry of autoradiographs as described m the text.
1i8
terns measured by quantitative autoradiography [1 l]. The highest concentrations of [~HJCHA binding sites were found in the cerebellum (molecular layer) and in the stratum radiatum and stratum oriens of the hippocampal CA1 region. The numbers of pH]CHA binding sites in the hippocampus lay between 1.21 and 2.34 pmol/mg protein in the various subregions. An example of displacement curves obtained using quantitative autoradiography is shown in Fig. lb. In the rat striatum the ligands R-phenylisopropyladenosine (RPIA), N-ethylcarboxamidoadenosine (NECA) and S-phenylisopropyladenosine (SPIA) displaced pH]CHA from cryostat sections in a dose-dependent manner such that the rank order of their affinities was R-PIA > NECA > S-PIA. As has also been found in binding studies with isolated membrane fractions, the [3H]CHA binding sites measured in cryostat sections therefore have the characteristics of adenosine receptors of the A~-type [1, 9, 16]. Table I summarizes the equilibrium binding proTABLE 1 E Q U I L I B R I U M B I N D I N G C H A R A C T E R I S T I C S OF [3H]CHA B I N D I N G SITES IN V A R IO U S RL;G I O N S OF RAT BRAIN Scatchard parameters were derived from saturation curves performed using a concentration range of 11.5 to 51) nM [3H]CHA and are given in fmol/mg protein (Bin,x) and nM (K,~). The K, values for unlabelled ligands were derived from ICso values obtained with 10 nM [3H]CHA and various concentrations of RPIA, S-PIA or NECA, using the measured Kj values for the appropriate region obtained by Scatchard analysis. The effect of the guanine nucleotide derivative, G p p N t t p , was tested at a concentration of 100 ItM. All nqeasurcments were made using quantitativc autoradiography as described in the text.
B ......
Hippocampal subregions: Stratum oricns, CAI Stratum radiatum, CA1 Stratum lacunarc Molecular layer, dentate gyrus Granular layer, dentate gyrus Infragranular layer, internal leaf CA4 cell zone lnfragranular layer, external leaf Stratum radiatum, CA3 Stratum oriens, CA3 Cerebral cortex, area 41, layer 4 Cerebral cortex, area 41, layer 6 Cerebral cortex, area 2a~ layer 4 Cerebral cortex, area 2a, layer 6 Cerebellum. molecular layer Cerebellum, granular laycr Striatum Lateral posterior thalamic nucleus Lateral seplal nucleus (dorsal aspect)
2.31 2.34 1.49 2. l0 1.21 2.08 1.28 1.96 1.78 1.75 1.90 1.27 1.44 1.07 2.59 0.83 1.113 1.70 I. 15
K,~
6.12 5.88 6.32 7.01 7.23 6.18 7.25 6.22 7.60 7.77 6.22 6,21 7.65 6.45 5.19 5.90 7.23 8.02 5.62
K, values (nM)
±GppNttp (% control)
R-PIA
S-HA
N EC A
3.11 3.07 3.38 3.99 3.66 3.97 2.62 2.88 4.211 4.19 3.46 3.04 4.59 -2.58 4.26 -
294 226
13.9 16.7 16./)
314 . . . .
254 275 250
237 260 . . . . .
16.4 16.0 14.9
11.9 16.2 -
41.6 35.(i 41,0 30.6 45.7 36.6 45.tt 35.3 41.8 44.8 36.3 40.9 19.0 21.3 22.9
119 perties o f [3H]CHA b i n d i n g sites d e t e r m i n e d in v a r i o u s rat b r a i n regions using quantitative d e n s i t o m e t r y o f a u t o r a d i o g r a p h s . T h e respective a p p a r e n t K, values for RP I A , S - P I A and N E C A were similar in all regions, a n d in all cases R - P I A was m o r e effective than N E C A in displacing [3H]CHA. The binding o f a d e n o s i n e r e c e p t o r agonists to A~-adenosine receptors has been shown to be a t t e n u a t e d by g u a n i n e nucleotides such that a shift is observed from a high to a low aftinity state [1, 4, 17]. As f o u n d in m e m b r a n e p r e p a r a t i o n s , a d d i t i o n o f the p o o r l y h y d r o l y s a b l e g u a n o s i n e t r i p h o s p h a t e derivative, /L',,-imido g u a n o s i n e t r i p h o s p h a t e ( G p p N H p ) , to cryostat sections from rat brain decreased the binding o f p H ] C H A in all regions tested (Table l). These d a t a indicate the a s s o c i a t i o n o f guanine nucleotide r e g u l a t o r y m e c h a n i s m s with A~-receptors t h r o u g h o u t the central nervous system. In s u m m a r y , all regions o f the rat C N S e x a m i n e d here which b o u n d p H ] C H A showed p r o p e r t i e s consistent with the presence o f a d e n o s i n e receptors o f the A~-type. N o evidence was o b t a i n e d from studies o f e q u i l i b r i u m binding p r o p e r t i e s for a heterogeneity o f high-attinity b i n d i n g sites with discrete localizations. In a d d i t i o n , all regions tested showed the a s s o c i a t i o n o f A ] - r e c e p t o r s with r e g u l a t o r y guanine nucleotide b i n d i n g c o m p o n e n t s . These d a t a suggest that all A r r e c e p t o r - m e d i a t e d events in the C N S , whether p r e s y n a p t i c [2, 5] or p o s t s y n a p t i c [7, 14] involve similar types o f ligand recognition sites. It remains to be established whether these receptors are associated with the same or different effector mechanisms. The a u t h o r s w o u l d like to t h a n k Professor G . W . K r e u t z b e r g for his active s u p p o r t a n d e n c o u r a g e m e n t t h r o u g h o u t this work. O u r t h a n k s are also due to Dr. K.S. Lee l\~r much helpful discussion, Dr. W o l f r a m Tetzlaff for setting up the q u a n t i t a t i v e d e n s i t o m e t r y and Petra Grfimmel for help in m e a s u r i n g the a u t o r a d i o g r a p h s . This study is part o f the w o r k to be s u b m i t t e d by A.E. in partial fulfilhnent o f the degree o f l ) o c t o r o f Medicine at the University o f Munich. The financial s u p p o r t o f the Deutsche F o r s c h u n g s g e m e i n s c h a f t is gratefully a c k n o w l e d g e d .
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