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Disappearance of low affinity adenosine binding sites in aging rat cerebral cortex and hippocampus
Neuroscience Letters, 49 (1984) 143-146
143
Elsevier Scientific Publishers Ireland Ltd. NSL 02858
D I S A P P E A R A N C E OF LOW AFFINITY ADENOSINE BINDING SITES IN AGING RAT CEREBRAL CORTEX A N D H I P P O C A M P U S
RENATO CORRADETTI, PEPEU***
LECH
KIEDROWSKI*,
OIE N O R D S T R O M * *
and G I A N C A R L O
Department of Pharmacology, Florence University, Viale G.B. Morgagni 65, 50134 Firenze (Italy) (Received February 16th, 1984; Revised version received May 18th, 1984; Accepted May 22nd, 1984)
Key words: adenosine - receptor - aging brain - cerebral cortex - hippocampus - rat
Al adenosine receptor binding was investigated, using the selective agonist, [3H]cyclohexyladenosine, on m e m b r a n e s prepared from the cerebral cortex and hippocampus of 3- and 24-month-old rats. The Scatchard analysis of the binding results obtained in the cerebral cortex of y o u n g animals showed two distinct binding sites with apparent Kd of 2 and 24 nM and Bmax o f 259 and 675 f m o l / m g protein, respectively. Conversely, in the old rats only one population of high affinity binding sites with a Kd of 2.2 nM and a B ~ of 450 f m o l / m g protein was found. Displacement curves of labelled ligand carried out on hippocampal membranes also demonstrate the disappearance of a low affinity subpopulation of A1 receptors in the old rat brain.
Evidence has been gathered recently that adenosine behaves as a neuromodulator or neurotransmitter, inhibiting neuronal firing and synaptic transmission, by decreasing neurotransmitter release and altering cAMP concentration in brain tissue (see refs. in Stone [12] and in Phillis and Wu [11]). These effects occur through the activation of specific receptors which have been classified in various ways by different authors [12]. However it is commonly accepted [3, 4] that AI receptors are responsible for the decrease in neurotransmitter release which adenosine induces. An example of this effect is the marked dose-dependent decrease in acetylcholine release from electrically stimulated brain slices [9] caused by adding adenosine to the superfusion fluid. This effect, which is antagonized by aminophylline, was detectable in cortical slices taken from newborn and 3-month-old rats but not in slices prepared from 24-month-old rats [10]. The latter observation prompted us to investigate whether the decrease in adenosine effect depends upon a decrease in the number of adenosine binding sites. * Present address: Nencki Institute for Experimental Biology, Warszawa, Poland. ** Present address: Department of Biochemistry, University of Stockholm, S- 10691 Stockholm, Sweden. *** A u t h o r for correspondence.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
144
Adenosine binding sites have been characterized using cyclohexyl[3H]adenosine ([3H]CHA), as described by Patel et al. [8]. Cyclohexyladenosine (CHA) offers many advantages as a ligand for adenosine receptors. It is neither inactivated by adenosine deaminase nor taken up by cells [3]. It is a more potent agonist than adenosine, and its affinity for A1 is 10,000-fold higher than for A: receptors [3]. Recently it has also been shown that C H A binds to two populations of A1 receptors with different affinity constants [8]. [3H]CHA binding to brain membranes was investigated as follows. After decapitation, the skull was opened and the cerebral cortex and hippocampus from 3- and 24-month-old Sprague-Dawley rats were rapidly dissected out and homogenized in 15 vols. sucrose (0.32 M) in teflon glass homogenizers (Thomas Elvejner-Potter). Following 1000 g (10 min at 0°C) centrifugation, the supernatant was further centrifuged at 30,000 g for 30 min at 0°C and resuspended in 25 ml TrisHC1 buffer, pH 7.4. This procedure was repeated twice in order to remove all endogenous adenosine from the membranes. After resuspension in 10 vols. Tris-HC1 buffer and incubation for 30 min with adenosine deaminase (2 units/ml; Boehringer-Mannheim) at room temperature, the homogenates were centrifuged (30,000 g, 30 min at 0°C), resuspended in 20 vols. Tris-HCl buffer and stored at - 7 0 ° C never longer than a month. Saturation curves were carried out in cortical membranes prepared from young and old rats by adding increasing concentrations (0.2-60 nM) of [3H]CHA (13.5 Ci/mmol; New England Nuclear) to a constant amount of membranes (about 300 mg protein/0.6 ml final volume). All experiments were performed in triplicate. Non-specific binding was quantified by adding 10/~M of unlabelled C H A (Boehringer-Mannheim). Samples filtered under vacuum through G F / B filters (Whatman) were washed three times with 3 ml ice cold TrisHCI buffer. Bound radioactivity was counted in 10 ml Instagel (Packard) scintillation fluid by means of a Tricarb 460C counter with 40°70 efficiency. Displacement curves were carried out on hippocampal membranes by adding increasing concentrations of unlabelled C H A in the presence of [3H]CHA 12 nM. Binding parameters were calculated according to the criteria indicated by Munson and Rodbard [7]. Protein content was measured according to the method described by Lowry et al. [6]. As shown in Fig. 1, the Scatchard analysis of the binding results showed two distinct binding sites for [3H]CHA in adult rats (n = 9) with apparent Ka of 2.0 and 24 nM, and Bmax of 259 and 675 fmol/mg protein, respectively. The Hill plot was linear (r= 0.99) and had a slope of 0.69. The semi-logarithmic plot [5] was found to confirm, reliably, total Bmax obtained by extrapolation from the Scatchard plot. Scatchard plots obtained from old rat membranes (n = 5) showed a straight line (r = 0.99) which lead to an apparent Ka of 2.2 nM and a Bmax of 450 fmol/mg protein. The Hill plot consistently showed a line (r = 0.98) with a slope of 0.96 which is not significantly different from unity. Displacement curves of [3H]CHA on hippocampal membranes also showed dif-
145
200 :
:
C O N T R O L (3 months)
% 150
'
o 0 f-
X LL
m
\
~
~
,oo",,,:\ 0
OLD (28 months)
\ 200
400
BOUND
600
fmol/mg
800
1000
protein
Fig. 1. [3H]CHA binding to cerebral cortex of adult and young rats. Data from saturation isotherms were subjected to Scatchard analysis. Each point represents the mean of 9 determinations in triplicate for 3-month-old rats (e) or 5 determinations for 24-month-old rats ( + ) . Binding was measured over a radioligand concentration range from 0.2 to 60 nM. Non-specific binding was determinated in the presence of l0 #M CHA. Dashed lines represent the two components of 3-month-old rats binding as calculated according to Munson [7]; site h Kd = 2 nM and Bmax = 259 f m o l / m g of membrane protein; site 2: Kd = 24 nM and Bmax = 675 f m o l / m g of membrane protein.
ferences between the young and old rats. When analyzed by means of Hill plot the experimental points gave straight lines with a slope of 0.60 for the young (r = 0.98; n = 5) and 0.98 (r-- 0.99; n = 4) for old rats. Consistent with these findings, the ICs0 calculated from the Hill plots showed a decrease from 7.4 to 5.4 nM. These results indicate through direct evidence from the cerebral cortex and through the changes in ICs0 in the hippocampus, that a low affinity subpopulation o f A1 receptors labelled by [3H]CHA in young rat brain disappears in old rat brain, and it is partially replaced by high affinity binding sites. It appears therefore that the lack of inhibitory effect of adenosine on ACh release from electrically stimulated cortical slices observed in old rats [10] is paralleled by the disappearance of low affinity C H A binding sites. It is conceivable that the low affinity adenosine receptors play an essential role in modulating acetylcholine release. Furthermore, the finding reported by Pedata et al. [10] helps to rule out the possibility, which is
146
difficult to dismiss on the basis of pure binding studies, that the curvature of the Scatchard plot observed in young rat cortical membranes may be due to negative cooperativity. In fact, given the disappearance of the negative cooperativity in old animals, one would not expect a decrease in inhibitory activity of adenosine but rather the opposite effect. Finally the changes in affinity and function of A~ receptors may offer a biochemical explanation for the electrophysiological compensatory modifications following the granular cell loss observed in the aging hippocampus [1]. The compensation could result from a reduction of the inhibitory action of adenosine on excitatory neurotransmitter release. This work was supported by CNR Grant 82.002266.04. We thank the Italian Group for Brain Aging for the gift of the old rats. 1 Barnes, C.A. and McNaughton, B.L., Physiological compensation for loss of afferent synapses in rat hippocampal granule cells during senescence, J. Physiol. (Lond.), 309 (1980) 473-485. 2 Bruns, R.F., Daly, J.W. and Snyder, S.H., Adenosine receptors in brain membranes: binding of N6-cyclohexyl[3H]adenosine and 1,3-diethyl-8-[3H]phenylxanthine, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 5547-5551. 3 Daly, J.W., Adenosine receptors: targets for future drugs, J. Med. Chem., 25 (1982) 197-207. 4 Fredholm, B.B., Jonzon, B. and Lindgren, E., Inhibition of noradrenaline release from hippocampal slices by a stable adenosine analogue, Acta physiol, scand., Suppl. 515 (1983) 7-10. 5 Klotz, I.M., Numbers of receptor sites from Scatchard graphs: facts and fantasies, Science, 217 (1982) 1247-1248. 6 Lowry, D.H., Rosebrough, N.J., Farr, L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 7 Munson, P.J. and Rodbard, D., L1GAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem., 107 (1980) 220-239. 8 Patel, J., Marangos, P.J., Stivers, J. and Goodwin, F.K., Characterization of adenosine receptors in brain using N6-cyclohexyl[3H]adenosine, Brain Res., 237 (1982) 203-214. 9 Pedata, F., Antonelli, T., Lambertini, L., Beani, L. and Pepeu, G., Effect of adenosine, adenosine triphosphate, adenosine deaminase, dipyridamole and aminophylline on acetylcholine release from electrically-stimulated brain slices, Neuropharmacology, 22 (1983) 609-614. 10 Pedata, F., Slavikova, J., Kotas, A. and Pepeu, G., Acetylcholine release from rat cortical slices during postnatal development and aging, Neurobiol. Aging, 4 (1983) 31-35. 11 Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Progr. Neurobiol., 16 (1981) 187-239. 12 Stone, T.W., Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system, Neuroscience, 6 (1981) 523-555.
143
Elsevier Scientific Publishers Ireland Ltd. NSL 02858
D I S A P P E A R A N C E OF LOW AFFINITY ADENOSINE BINDING SITES IN AGING RAT CEREBRAL CORTEX A N D H I P P O C A M P U S
RENATO CORRADETTI, PEPEU***
LECH
KIEDROWSKI*,
OIE N O R D S T R O M * *
and G I A N C A R L O
Department of Pharmacology, Florence University, Viale G.B. Morgagni 65, 50134 Firenze (Italy) (Received February 16th, 1984; Revised version received May 18th, 1984; Accepted May 22nd, 1984)
Key words: adenosine - receptor - aging brain - cerebral cortex - hippocampus - rat
Al adenosine receptor binding was investigated, using the selective agonist, [3H]cyclohexyladenosine, on m e m b r a n e s prepared from the cerebral cortex and hippocampus of 3- and 24-month-old rats. The Scatchard analysis of the binding results obtained in the cerebral cortex of y o u n g animals showed two distinct binding sites with apparent Kd of 2 and 24 nM and Bmax o f 259 and 675 f m o l / m g protein, respectively. Conversely, in the old rats only one population of high affinity binding sites with a Kd of 2.2 nM and a B ~ of 450 f m o l / m g protein was found. Displacement curves of labelled ligand carried out on hippocampal membranes also demonstrate the disappearance of a low affinity subpopulation of A1 receptors in the old rat brain.
Evidence has been gathered recently that adenosine behaves as a neuromodulator or neurotransmitter, inhibiting neuronal firing and synaptic transmission, by decreasing neurotransmitter release and altering cAMP concentration in brain tissue (see refs. in Stone [12] and in Phillis and Wu [11]). These effects occur through the activation of specific receptors which have been classified in various ways by different authors [12]. However it is commonly accepted [3, 4] that AI receptors are responsible for the decrease in neurotransmitter release which adenosine induces. An example of this effect is the marked dose-dependent decrease in acetylcholine release from electrically stimulated brain slices [9] caused by adding adenosine to the superfusion fluid. This effect, which is antagonized by aminophylline, was detectable in cortical slices taken from newborn and 3-month-old rats but not in slices prepared from 24-month-old rats [10]. The latter observation prompted us to investigate whether the decrease in adenosine effect depends upon a decrease in the number of adenosine binding sites. * Present address: Nencki Institute for Experimental Biology, Warszawa, Poland. ** Present address: Department of Biochemistry, University of Stockholm, S- 10691 Stockholm, Sweden. *** A u t h o r for correspondence.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
144
Adenosine binding sites have been characterized using cyclohexyl[3H]adenosine ([3H]CHA), as described by Patel et al. [8]. Cyclohexyladenosine (CHA) offers many advantages as a ligand for adenosine receptors. It is neither inactivated by adenosine deaminase nor taken up by cells [3]. It is a more potent agonist than adenosine, and its affinity for A1 is 10,000-fold higher than for A: receptors [3]. Recently it has also been shown that C H A binds to two populations of A1 receptors with different affinity constants [8]. [3H]CHA binding to brain membranes was investigated as follows. After decapitation, the skull was opened and the cerebral cortex and hippocampus from 3- and 24-month-old Sprague-Dawley rats were rapidly dissected out and homogenized in 15 vols. sucrose (0.32 M) in teflon glass homogenizers (Thomas Elvejner-Potter). Following 1000 g (10 min at 0°C) centrifugation, the supernatant was further centrifuged at 30,000 g for 30 min at 0°C and resuspended in 25 ml TrisHC1 buffer, pH 7.4. This procedure was repeated twice in order to remove all endogenous adenosine from the membranes. After resuspension in 10 vols. Tris-HC1 buffer and incubation for 30 min with adenosine deaminase (2 units/ml; Boehringer-Mannheim) at room temperature, the homogenates were centrifuged (30,000 g, 30 min at 0°C), resuspended in 20 vols. Tris-HCl buffer and stored at - 7 0 ° C never longer than a month. Saturation curves were carried out in cortical membranes prepared from young and old rats by adding increasing concentrations (0.2-60 nM) of [3H]CHA (13.5 Ci/mmol; New England Nuclear) to a constant amount of membranes (about 300 mg protein/0.6 ml final volume). All experiments were performed in triplicate. Non-specific binding was quantified by adding 10/~M of unlabelled C H A (Boehringer-Mannheim). Samples filtered under vacuum through G F / B filters (Whatman) were washed three times with 3 ml ice cold TrisHCI buffer. Bound radioactivity was counted in 10 ml Instagel (Packard) scintillation fluid by means of a Tricarb 460C counter with 40°70 efficiency. Displacement curves were carried out on hippocampal membranes by adding increasing concentrations of unlabelled C H A in the presence of [3H]CHA 12 nM. Binding parameters were calculated according to the criteria indicated by Munson and Rodbard [7]. Protein content was measured according to the method described by Lowry et al. [6]. As shown in Fig. 1, the Scatchard analysis of the binding results showed two distinct binding sites for [3H]CHA in adult rats (n = 9) with apparent Ka of 2.0 and 24 nM, and Bmax of 259 and 675 fmol/mg protein, respectively. The Hill plot was linear (r= 0.99) and had a slope of 0.69. The semi-logarithmic plot [5] was found to confirm, reliably, total Bmax obtained by extrapolation from the Scatchard plot. Scatchard plots obtained from old rat membranes (n = 5) showed a straight line (r = 0.99) which lead to an apparent Ka of 2.2 nM and a Bmax of 450 fmol/mg protein. The Hill plot consistently showed a line (r = 0.98) with a slope of 0.96 which is not significantly different from unity. Displacement curves of [3H]CHA on hippocampal membranes also showed dif-
145
200 :
:
C O N T R O L (3 months)
% 150
'
o 0 f-
X LL
m
\
~
~
,oo",,,:\ 0
OLD (28 months)
\ 200
400
BOUND
600
fmol/mg
800
1000
protein
Fig. 1. [3H]CHA binding to cerebral cortex of adult and young rats. Data from saturation isotherms were subjected to Scatchard analysis. Each point represents the mean of 9 determinations in triplicate for 3-month-old rats (e) or 5 determinations for 24-month-old rats ( + ) . Binding was measured over a radioligand concentration range from 0.2 to 60 nM. Non-specific binding was determinated in the presence of l0 #M CHA. Dashed lines represent the two components of 3-month-old rats binding as calculated according to Munson [7]; site h Kd = 2 nM and Bmax = 259 f m o l / m g of membrane protein; site 2: Kd = 24 nM and Bmax = 675 f m o l / m g of membrane protein.
ferences between the young and old rats. When analyzed by means of Hill plot the experimental points gave straight lines with a slope of 0.60 for the young (r = 0.98; n = 5) and 0.98 (r-- 0.99; n = 4) for old rats. Consistent with these findings, the ICs0 calculated from the Hill plots showed a decrease from 7.4 to 5.4 nM. These results indicate through direct evidence from the cerebral cortex and through the changes in ICs0 in the hippocampus, that a low affinity subpopulation o f A1 receptors labelled by [3H]CHA in young rat brain disappears in old rat brain, and it is partially replaced by high affinity binding sites. It appears therefore that the lack of inhibitory effect of adenosine on ACh release from electrically stimulated cortical slices observed in old rats [10] is paralleled by the disappearance of low affinity C H A binding sites. It is conceivable that the low affinity adenosine receptors play an essential role in modulating acetylcholine release. Furthermore, the finding reported by Pedata et al. [10] helps to rule out the possibility, which is
146
difficult to dismiss on the basis of pure binding studies, that the curvature of the Scatchard plot observed in young rat cortical membranes may be due to negative cooperativity. In fact, given the disappearance of the negative cooperativity in old animals, one would not expect a decrease in inhibitory activity of adenosine but rather the opposite effect. Finally the changes in affinity and function of A~ receptors may offer a biochemical explanation for the electrophysiological compensatory modifications following the granular cell loss observed in the aging hippocampus [1]. The compensation could result from a reduction of the inhibitory action of adenosine on excitatory neurotransmitter release. This work was supported by CNR Grant 82.002266.04. We thank the Italian Group for Brain Aging for the gift of the old rats. 1 Barnes, C.A. and McNaughton, B.L., Physiological compensation for loss of afferent synapses in rat hippocampal granule cells during senescence, J. Physiol. (Lond.), 309 (1980) 473-485. 2 Bruns, R.F., Daly, J.W. and Snyder, S.H., Adenosine receptors in brain membranes: binding of N6-cyclohexyl[3H]adenosine and 1,3-diethyl-8-[3H]phenylxanthine, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 5547-5551. 3 Daly, J.W., Adenosine receptors: targets for future drugs, J. Med. Chem., 25 (1982) 197-207. 4 Fredholm, B.B., Jonzon, B. and Lindgren, E., Inhibition of noradrenaline release from hippocampal slices by a stable adenosine analogue, Acta physiol, scand., Suppl. 515 (1983) 7-10. 5 Klotz, I.M., Numbers of receptor sites from Scatchard graphs: facts and fantasies, Science, 217 (1982) 1247-1248. 6 Lowry, D.H., Rosebrough, N.J., Farr, L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 7 Munson, P.J. and Rodbard, D., L1GAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem., 107 (1980) 220-239. 8 Patel, J., Marangos, P.J., Stivers, J. and Goodwin, F.K., Characterization of adenosine receptors in brain using N6-cyclohexyl[3H]adenosine, Brain Res., 237 (1982) 203-214. 9 Pedata, F., Antonelli, T., Lambertini, L., Beani, L. and Pepeu, G., Effect of adenosine, adenosine triphosphate, adenosine deaminase, dipyridamole and aminophylline on acetylcholine release from electrically-stimulated brain slices, Neuropharmacology, 22 (1983) 609-614. 10 Pedata, F., Slavikova, J., Kotas, A. and Pepeu, G., Acetylcholine release from rat cortical slices during postnatal development and aging, Neurobiol. Aging, 4 (1983) 31-35. 11 Phillis, J.W. and Wu, P.H., The role of adenosine and its nucleotides in central synaptic transmission, Progr. Neurobiol., 16 (1981) 187-239. 12 Stone, T.W., Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system, Neuroscience, 6 (1981) 523-555.