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Age-dependent change in the inhibitory effect of an adenosine agonist on hippocampal acetylcholine release in rats
0361-9230/93 $6.00 + .OO Copyright 0 1992 Pergamon Press Ltd.
Brain ResearchBulletin.Vol. 30, pp. 149-152, 1993 Printed in the USA. All rights reserved.
Age-Dependent Change in the Inhibitory Effect of an Adenosine Agonist on Hippocampal Acetylcholine Release in Rats Z. L. JIN,’ T. F. LEE, S. J. ZHOU AND L. C. H. WANG* Department
ofZoology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada Received 30 March 1992; Accepted 9 June 1992
JIN, Z. L., T. F. LEE, S. J. ZHOU AND L. C. H. WANG. Age-dependent change in the inhibitory effect of an adenosine agonist on hippocampal acetylcholine release in rats. BRAIN RES BULL 30( l/2) 149-152, 1993.-To investigate the possibility that age-dependent deficits in acetylcholine (ACh) release are precipitated by the alteration of endogenous purinergic activities, the effects of (-)N’-phenylisopropyladenosine (PIA), an adenosine agonist, in modulating K+ (25 mM)-induced [-‘H]ACh release from the hippocampal slices of young (3-6 months old) and old rats (26-30 months old) were examined. In young rats, PIA (0.1-10 PM) caused a dose-related inhibition of [3H]ACh release from the hippocampal slices and a significant reduction in [3H]ACh release was observed in the presence of 1 rM PIG. In old rats, a similar pattern of PIA suppression of K+-induced [3H]ACh release was observed; however, a IO-fold higher concentration of PIA (10 PM) was required to elicit a significant inhibition. This age-dependent reduction in responsiveness to PIA may be due to an enhanced endogenous adenosine activity in aged rats leading to downregulation of the adenosine receptors. This notion is supported by the finding that both the adenosine concentration and activity of S-nucleotidase, an enzyme partially governing adenosine synthesis, were increased in the hippocampus of old rats as compared to their younger counterparts. Adenosine
Adenosine receptor
Acetylcholine
Hippocampus
TO correlate age-dependent changes in many physiological and behavioral functions, numerous studies have examined the alteration with aging of activities of various neurotransmitters in the CNS. Much attention has been focused on the brain cholinergic systems because their deterioration has been suggested to underlie geriatric memory disorders (for review, see refs. I and 2). Dramatic reductions in cholinergic enzymes, muscarinic receptors, and acetylcholine (ACh) release have been reported in specific brain regions, including the hippocampus, cortex, and striatum, during aging (for review, see refs. 14 and 20). Other than the possibility that the impairment of ACh release is due to a loss of choline& terminals with age, the exact mechanism(s) that causes the age-dependent deficit in ACh release remains obscure. Adenosine is a dephosphorylation product of ATP known to be released from stimulated CNS neurons. It acts as a neuromodulator in inhibiting neuronal firing and synaptic transmission (for review, see refs. 5 and 16). In the hippocampus, a high
Aging
density of adenosine receptors has been demonstrated (6,1 I), and adenosine and its analogs can suppress the release of ACh through presynaptic inhibition (for review, see refs. 5 and 9). Although the exact mechanism of this effect is still unknown, it has been suggested that activation of adenosine A, receptors is responsible for the inhibitory effect of adenosine on neurotransmitter release (8,lS). Recently, an age-related increase in purinergic activity has been demonstrated in in vitro human fibroblast cultures (7) and rat neck muscle following cold stimulation (2 1). Even though no direct measurement ofhippocampal adenosine activity has been carried out in aged rats, a subpopulation of low-affinity A, receptors in the hippocampus has been observed to disappear in old rats (4). These age-related changes in adenosine concentration and ligand-receptor interaction raise the question whether the age-dependent change in CNS cholinergic function could be related to adenosine metabolism. To answer this, we investigated the modulatory role of an adenosine analog (-)N6-phenylisopropyladenosine (PIA), on ACh release
’ Current address: Department of Biology, Peking University Branch, Beijing 100083, China. ’ To whom requests for reprints should be addressed.
149
JIN ET AL.
150
in the hippocampus. To evaluate further the age-dependent changes in endogenous purinergic metabolism, the hippocampal adenosine concentration and its metabolic enzyme activities were also measured in rats of different ages. METHOD
All experimental protocols used in the present study received prior approval of the University of Alberta Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Two groups of adult, male Sprague-Dawley rats, 36 and 26-30 months old, were used. They were housed individually in polycarbonate cages with wood shaving bedding at 22 + 1“C in a walk-in environmental chamber under a 12L: 12D photoperiod. Food (Rodent Blox, consisting of 24% protein, 4% fat, 65% carbohydrate, 4.5% fiber and vitamins; Wayne Laboratory Animal Diets, Chicago, IL) and water were made available at all times. Animals were sacrificed by decapitation and brains were rapidly removed. Both hippocampi were dissected out and sliced to a thickness of 0.3 mm using a McIlwain tissue chopper. Slices were incubated for 30 min at 37°C in 1 ml oxygenated Krebs medium (pH 7.4) containing paraoxon (1 PM) and 0.1 PM [3H]choline Cl (specific activity 79.3 Ci/mmol, Amersham Corp., Arlington Heights, IL). After labeling, aliquots of 100 ~1 of tissue suspension were transferred to each of four superlusion chambers (about 80 mg of wet tissue per chamber) and superfused with Krebs medium with a flow rate of 1 ml/min at 37°C. Tissues within each chamber were stimulated twice at 46 min (S,) and 76 min (S,) after the onset of superfusion by exposure to a medium containing 25 mM KCI for 6 min. The Si was used as self-control and various concentrations of PIA were added to the superfusion medium immediately after S, and remained present throughout the rest of the experiment. Samples of the superfusate were collected at 2-min intervals 30 min after onset of superfusion. At the end of the experiment, the slices were solubilized with 1.0 ml 1 N NaOH and the radioactivity in the slices and superfusate determined by liquid scintillation spectrometry. [3H]Choline was separated from [3H]ACh according to the procedure described by Briggs and Cooper (3). A 125~~1 aliquot of a solution containing choline kinase (1 mu), adenosine triphosphate (50 mM), and MgClz (6.25 mM) in glycylglycine buffer (250 mM, pH 8.5) was added to 0.5 ml of each fraction of perfusate to convert choline to phosphorylcholine. The remaining [3H]ACh was then extracted with 750 ~1 3-heptanone containing 10 mg/ml sodium tetraphenylborate. The amount of [‘H]ACh in 500~~1 aliquots of organic supernatant was determined by liquid scintillation spectrometry. [3H]ACh makes up 46.6 1 f 1.75% (n = 8) of the total ‘H released. The amount of ‘H released in a 2-min sample was expressed as a fraction of the total tissue 3H content within the same chamber at the onset of the respective collection period. The percentage of radioactivity released above the basal level by the two pulses of K+ was expressed as the ratio of S2/S, for both the control and drug-treated slices. To quantify the effects of drugs on the stimulation-evoked outflow, the S2/S, ratios of the drug-treated slices were compared with the ratios calculated under the respective control conditions. To measure the adenosine concentration within the hippocampus, the tissue samples were homogenized in 1 N perchloric acid and the homogenate was then centrifuged to remove the precipitated protein. The amount of adenosine in the supernate was assayed by high-performance liquid chromatography (HPLC) as described by Jackson and Ohnishi ( 13). Owing to the
short half-life of adenosine ( 15) the absolute level of adenosine may not be positively correlated with the physiological responses. To examine the enzyme activity governing adenosine synthesis, the hippocampal 5’-nucleotidase (ND, EC 3.1.3.5) activity from animals of different ages was compared. After removing it from the rat, the hippocampus was homogenized with 25 times (w/ v) 0.1 M Tris-buffer (pH 7.4) and the activity of 5’-ND in the homogenate was determined by a conventional enzymatic method (Sigma Kit 265, Sigma Chemical Co., St. Louis, MO). Statistical analysis was by the unpaired t test and the significance was set at p < 0.05 unless otherwise stated. RESULTS
Ejkts
ofPIA
on hippocampal [“H]ACh release
Even though the fractional release of [3]ACh induced by 25 mM KCI was about the same between young and old rats (the ratios of S2:SI were 0.75 f 0.05 and 0.79 f 0.05 for young and old rats, respectively; Fig. I), the total ACh outflow elicited in the hippocampal slices of young rats (9.11 + 0.17%, n = 10) during the first period of K+-induced stimulation (S,) was significantly higher than that of old rats (8.60 -t 0.14%, n = 12). In experimental slices, addition of PIA (0.1-10 PM). immediately after S,, inhibited the K’-evoked release of [3H]ACh from the hippocampal slices of both young and old rats in a dose-related manner (Fig. 1). In young rats, a significant suppression (about 31%) of ACh outflow was observed at 1 PM PIA. In old rats, however, 10 PM PIA was required to elicit a similar inhibition on [3H]ACh release (Fig. 1).
Changc~ in hippocampal adenosine concentrati0n.s and S-nucleotia’asr activity with aging To examine whether the reduction in responsiveness to exogenous PIA was due to enhanced endogenous purinergic activity, the hippocampal adenosine concentration was measured in both young and old rats and the results are shown in Table 1. The hippocampal adenosine concentration of old rats was significantly higher (about 6 1%) than that found in their younger counterparts. In parallel with that observed with adenosine concentration, the hippocampal 5’-ND activity was also significantly higher (about 57%) in old than in young rats (Table 1).
Control
*** I vii 0.7
1.0
PIA
10.0,
f,uMT
FIG. 1. Effect of (-)N6-phenylisopropyladenosine (PIA) on K+-evoked [‘Hlacetylcholine (ACh) release from hippocampal slices of either young (open columns) or old (closed columns) rats. Each column represents the mean + SEM from 6-10 and 8-12 experiments for young and old rats, respectively. ‘Significantly different from respective control value,
p < 0.05.
ADENOSINE
AND
HIPPOCAMPAL
TABLE
I51
ACH RELEASE
1
HIPPOCAMPAL ADENOSINE CONCENTRATION AND S-ND ACTIVITY IN YOUNG AND OLD RATS
Adenosine concentration (nmol/mg protein) 5’-ND activity (mU/mg
protein)
Young Rats
Old Rats
161.60 + 14.1 (8)
260.8 I!Z33.4* (8)
75.57 f
2.46 (6)
118.50 f
2.99* (6)
Each value represents the mean k SE from number of animals shown in parentheses. * Significantly paired t test).
different
from the young rat, p < 0.01 (two-tailed
un-
DISCUSSION
It is well documented that endogenous adenosine can modulate central neuronal transmission by inhibiting presynaptic release of various neurotransmitters (for review, see refs. 5 and 9). In the hippocampus, activation of A, adenosine receptors has been reported to suppress the electrically stimulated ACh release (8). To investigate the possibility that the enhanced endogenous adenosine activity may be involved in blunting stimulated ACh release in older animals, the effect of PIA, a selective A, receptor agonist. on K+-stimulated ACh release from the hippocampal slices of both young and old rats was examined. Addition of PIA to the perfusion medium caused a dose-related suppression of K+-evoked ACh outflow from the hippocampal slices of the young rat. Even though a different stimulation method was used in our study, the concentration of PIA ( 1 PM) that caused a significant inhibition of ACh release was comparable to the concentration used previously in inhibiting electricallystimulated ACh release from the hippocampal slices of rats (8). Similar to earlier reports (for review, see ref. 20), the hippocampal ACh release evoked by 25 mM K+ was reduced with increasing age. The most interesting finding of the present study is that there was a decrease in responsiveness of the hippocampal ACh release to PIA in old rats. Ten times higher concentration of PIA than that used for young rats was required to elicit a significant suppression of K+-evoked ACh outflow in old rats. Similar reduction in responsiveness to exogenously applied
adenosine has also been observed previously in electrically stimulated ACh release from the cortical slices of old rats (10.17). Because age-related increase in adenosine release has been demonstrated in rat neck muscle following cold stimulation (2 1) and human fibroblast cultures (7) it is possible that the reduced responsiveness of hippocampal ACh release to PIA seen in old rats is due to a reduction in adenosine receptor efficacy consequent to an age-dependent increase in endogenous purinergic outflow. This suggestion is supported by the present finding that the hippocampal adenosine concentration was about 6 1% higher in old than in young rats. Coinciding with the increase in adenosine concentration, the activity of hippocampal S-ND, which has been proposed to partially govern adenosine synthesis (I 2). was also significantly higher in old than in young rats. Based on these observations, the age-dependent decrease in the inhibitory effect of PIA on hippocampal ACh release could be due to an enhanced endogenous purinergic activity during senescence. Alteration in the activity of an endogenous neurotransmitter is known to cause changes in the efficacy of its receptor(s). The net result of an increase in endogenous purinergic activity may possibly lead to conformational changes in adenosine receptor(s). This speculation is supported by the finding that the disappearance of a subpopulation of the low-affinity A, receptors in the hippocampus of 24-month-old rats is partly substituted by the high-affinity A, receptors (4). The decrease in responsiveness of the old rat hippocampal slices to PIA is, however, counter to the observed increase in receptor binding affinity. As suggested previously (4) this apparent paradox may possibly indicate that the low-affinity adenosine A, receptors, rather than the highaffinity A, receptors. are functionally important in modulating ACh release. If this interpretation is correct, then the observed age-related decrease in sensitivity to the inhibitory effect of an adenosine agonist is consistent with an increased endogenous purinergic activity with aging. In view of the recent finding that the cognitive deficit can be improved by pretreatment with a selective A, receptor antagonist ( 19). it is worthwhile to carry out further investigations on central adenosine metabolism and cholinergic activity during senescence. ACKNOWLEDGEMENT
The present study was supported by a Medical Research Council of Canada Operating Grant to L.W. We are indebted to SM. Paproski for excellent technical assistance.
REFERENCES I. Barnes, C. A. Spatial learning and memory process: The search for their neurobiological mechanisms in the rats. Trends Neurosci. 5: 163-169: 1988. 2. Bartus, R. T.; Dean, R. L., III; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 2 17:408-4 17; 1982. 3. Briggs, C. A.: Cooper. J. R. A synaptosomal preparation from the guinea pig ileum myenteric plexus. J. Neurochem. 36: 1097-I 108; 1981. 4. Corradetti, R.; Kiedrowski, L.; Nordstrom, 0.; Pepeu, G. Disappearance of low affinity adenosine binding sites in aging rat cerebral cortex and hippocampus. Neurosci. Lett. 49: 143-146; 1984. 5. Dunwiddle, T. V. The physiological role of adenosine in the central nervous system. Int. Rev. Neurobiol. 27:63-l 39; 1985. 6. Erfurth, A.; Reddington, M. Properties of binding sites for [‘Hlcyclohexyladenosine in the hippocampus and other regions of rat brain: A quantitative autoradiographic study. Neurosci. Lett. 64: 116-120; 1986.
7. Ethier, M. F.; Hickler, R. B.; Dobson. J. G. Aging increases adenosine and inosine release by human fibroblast cultures. Mech. Ageing Dev. 50:159-168; 1989. 8. Fredholm, B. B. Adenosine A,-receptor-mediated inhibition of evoked acetylcholine release in the rat hippocampus does not depend on protein kinase C. Acta Physiol. Stand. 140:245-255; 1990. 9. Fredholm. B. B.; Dunwiddie, T. V. How does adenosine inhibit transmitter release? Trends Pharmacol. Sci. 9: 130- 134: 1988. 10. Giovannelli, L.; Giovannini, M. G.; Pedata. F.; Pepeu, G. Purinergjc modulation of cortical acetylcholine release is decreased in aging rats. Exp. Gerontol. 23:175-181; 1988. Il. Goodman. R. R.: Snyder, S. H. Autoradiographic localization of adenosine receptors in rat brain using (‘H]cyclohexyladenosine. J. Neurosci. 2:1230-1241; 1982. 12. Henderson, J. F. The study of adenosine metabolism in isolated cells and tissues. In: Paton, D. M., ed. Methods in pharmacology. vol. 6. Methods used in adenosine research. New York: Plenum Press; 1985:67-82.
152
13. Jackson, E. K.; Ohnishi, A. Development and application of a simple microassay for adenosine in rat plasma. Hypertension 10:189- 197; 1987. 14. McGeer, P. L.; McGeer, E. G.; Suzuki, J.; Dolman, C. E.; Nagai, T. Aging, Alzheimer’s disease, and the cholinergic system of the basal forebrain. Neurology 34:741-745; 1984. 15. Moser, G. H.: Schmder, J.; Deussen, A. Turnover of adenosine in plasma of human and dog blood. Am. J. Physiol. 256:C799<806; 1989. 16. Ohisalo. J. J. Regulatory functions of adenosine. Med. Biol. 65: I8 l191; 1987. 17. Pedata, F.; Antonelli. T.; Lambertini, L.; Beani, L.; Pepeu, G. Effect of adenosine, adenosine triphosphate, adenosine deaminase, dipyridamole and aminophylline on acetylcholine release from electricallystimulated brain slices. Neuropharmacology 22:609-614: 1983.
JIN ET AL.
IS. Phillis, J. W.; Barraco, R. A. Adenosine, adenylate cyclase, and transmitter release. Adv. Cyclic Nucleotide Prot. Phosphoryl. Res. 191243-257; 1985. 19. Schingnitz, G.; Kufner-Muhl, U.; Ensinger, H.; Lehr, E.; Kuhn, F. J. Selective A, for treatment of cognitive deficits. Nucleosides Nucleotides lO:lO67-1076; 1991. 20. Sherman, K. A.; Friedman, E. Pre- and postsynaptic cholinergic dysfunction in aged rodent brain regions: New findings and an interpretive review. Int. J. Dev. Neurosci. 8:689-708; 1990. 21. Wang, L. C. H.; Jin, Z. L.; Lee, T. F. Decrease in cold tolerance of aged rats caused by the enhanced endogenous adenosine activity. Pharmacol. Biochem. Behav. 43:117-123: 1992.
Brain ResearchBulletin.Vol. 30, pp. 149-152, 1993 Printed in the USA. All rights reserved.
Age-Dependent Change in the Inhibitory Effect of an Adenosine Agonist on Hippocampal Acetylcholine Release in Rats Z. L. JIN,’ T. F. LEE, S. J. ZHOU AND L. C. H. WANG* Department
ofZoology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada Received 30 March 1992; Accepted 9 June 1992
JIN, Z. L., T. F. LEE, S. J. ZHOU AND L. C. H. WANG. Age-dependent change in the inhibitory effect of an adenosine agonist on hippocampal acetylcholine release in rats. BRAIN RES BULL 30( l/2) 149-152, 1993.-To investigate the possibility that age-dependent deficits in acetylcholine (ACh) release are precipitated by the alteration of endogenous purinergic activities, the effects of (-)N’-phenylisopropyladenosine (PIA), an adenosine agonist, in modulating K+ (25 mM)-induced [-‘H]ACh release from the hippocampal slices of young (3-6 months old) and old rats (26-30 months old) were examined. In young rats, PIA (0.1-10 PM) caused a dose-related inhibition of [3H]ACh release from the hippocampal slices and a significant reduction in [3H]ACh release was observed in the presence of 1 rM PIG. In old rats, a similar pattern of PIA suppression of K+-induced [3H]ACh release was observed; however, a IO-fold higher concentration of PIA (10 PM) was required to elicit a significant inhibition. This age-dependent reduction in responsiveness to PIA may be due to an enhanced endogenous adenosine activity in aged rats leading to downregulation of the adenosine receptors. This notion is supported by the finding that both the adenosine concentration and activity of S-nucleotidase, an enzyme partially governing adenosine synthesis, were increased in the hippocampus of old rats as compared to their younger counterparts. Adenosine
Adenosine receptor
Acetylcholine
Hippocampus
TO correlate age-dependent changes in many physiological and behavioral functions, numerous studies have examined the alteration with aging of activities of various neurotransmitters in the CNS. Much attention has been focused on the brain cholinergic systems because their deterioration has been suggested to underlie geriatric memory disorders (for review, see refs. I and 2). Dramatic reductions in cholinergic enzymes, muscarinic receptors, and acetylcholine (ACh) release have been reported in specific brain regions, including the hippocampus, cortex, and striatum, during aging (for review, see refs. 14 and 20). Other than the possibility that the impairment of ACh release is due to a loss of choline& terminals with age, the exact mechanism(s) that causes the age-dependent deficit in ACh release remains obscure. Adenosine is a dephosphorylation product of ATP known to be released from stimulated CNS neurons. It acts as a neuromodulator in inhibiting neuronal firing and synaptic transmission (for review, see refs. 5 and 16). In the hippocampus, a high
Aging
density of adenosine receptors has been demonstrated (6,1 I), and adenosine and its analogs can suppress the release of ACh through presynaptic inhibition (for review, see refs. 5 and 9). Although the exact mechanism of this effect is still unknown, it has been suggested that activation of adenosine A, receptors is responsible for the inhibitory effect of adenosine on neurotransmitter release (8,lS). Recently, an age-related increase in purinergic activity has been demonstrated in in vitro human fibroblast cultures (7) and rat neck muscle following cold stimulation (2 1). Even though no direct measurement ofhippocampal adenosine activity has been carried out in aged rats, a subpopulation of low-affinity A, receptors in the hippocampus has been observed to disappear in old rats (4). These age-related changes in adenosine concentration and ligand-receptor interaction raise the question whether the age-dependent change in CNS cholinergic function could be related to adenosine metabolism. To answer this, we investigated the modulatory role of an adenosine analog (-)N6-phenylisopropyladenosine (PIA), on ACh release
’ Current address: Department of Biology, Peking University Branch, Beijing 100083, China. ’ To whom requests for reprints should be addressed.
149
JIN ET AL.
150
in the hippocampus. To evaluate further the age-dependent changes in endogenous purinergic metabolism, the hippocampal adenosine concentration and its metabolic enzyme activities were also measured in rats of different ages. METHOD
All experimental protocols used in the present study received prior approval of the University of Alberta Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Two groups of adult, male Sprague-Dawley rats, 36 and 26-30 months old, were used. They were housed individually in polycarbonate cages with wood shaving bedding at 22 + 1“C in a walk-in environmental chamber under a 12L: 12D photoperiod. Food (Rodent Blox, consisting of 24% protein, 4% fat, 65% carbohydrate, 4.5% fiber and vitamins; Wayne Laboratory Animal Diets, Chicago, IL) and water were made available at all times. Animals were sacrificed by decapitation and brains were rapidly removed. Both hippocampi were dissected out and sliced to a thickness of 0.3 mm using a McIlwain tissue chopper. Slices were incubated for 30 min at 37°C in 1 ml oxygenated Krebs medium (pH 7.4) containing paraoxon (1 PM) and 0.1 PM [3H]choline Cl (specific activity 79.3 Ci/mmol, Amersham Corp., Arlington Heights, IL). After labeling, aliquots of 100 ~1 of tissue suspension were transferred to each of four superlusion chambers (about 80 mg of wet tissue per chamber) and superfused with Krebs medium with a flow rate of 1 ml/min at 37°C. Tissues within each chamber were stimulated twice at 46 min (S,) and 76 min (S,) after the onset of superfusion by exposure to a medium containing 25 mM KCI for 6 min. The Si was used as self-control and various concentrations of PIA were added to the superfusion medium immediately after S, and remained present throughout the rest of the experiment. Samples of the superfusate were collected at 2-min intervals 30 min after onset of superfusion. At the end of the experiment, the slices were solubilized with 1.0 ml 1 N NaOH and the radioactivity in the slices and superfusate determined by liquid scintillation spectrometry. [3H]Choline was separated from [3H]ACh according to the procedure described by Briggs and Cooper (3). A 125~~1 aliquot of a solution containing choline kinase (1 mu), adenosine triphosphate (50 mM), and MgClz (6.25 mM) in glycylglycine buffer (250 mM, pH 8.5) was added to 0.5 ml of each fraction of perfusate to convert choline to phosphorylcholine. The remaining [3H]ACh was then extracted with 750 ~1 3-heptanone containing 10 mg/ml sodium tetraphenylborate. The amount of [‘H]ACh in 500~~1 aliquots of organic supernatant was determined by liquid scintillation spectrometry. [3H]ACh makes up 46.6 1 f 1.75% (n = 8) of the total ‘H released. The amount of ‘H released in a 2-min sample was expressed as a fraction of the total tissue 3H content within the same chamber at the onset of the respective collection period. The percentage of radioactivity released above the basal level by the two pulses of K+ was expressed as the ratio of S2/S, for both the control and drug-treated slices. To quantify the effects of drugs on the stimulation-evoked outflow, the S2/S, ratios of the drug-treated slices were compared with the ratios calculated under the respective control conditions. To measure the adenosine concentration within the hippocampus, the tissue samples were homogenized in 1 N perchloric acid and the homogenate was then centrifuged to remove the precipitated protein. The amount of adenosine in the supernate was assayed by high-performance liquid chromatography (HPLC) as described by Jackson and Ohnishi ( 13). Owing to the
short half-life of adenosine ( 15) the absolute level of adenosine may not be positively correlated with the physiological responses. To examine the enzyme activity governing adenosine synthesis, the hippocampal 5’-nucleotidase (ND, EC 3.1.3.5) activity from animals of different ages was compared. After removing it from the rat, the hippocampus was homogenized with 25 times (w/ v) 0.1 M Tris-buffer (pH 7.4) and the activity of 5’-ND in the homogenate was determined by a conventional enzymatic method (Sigma Kit 265, Sigma Chemical Co., St. Louis, MO). Statistical analysis was by the unpaired t test and the significance was set at p < 0.05 unless otherwise stated. RESULTS
Ejkts
ofPIA
on hippocampal [“H]ACh release
Even though the fractional release of [3]ACh induced by 25 mM KCI was about the same between young and old rats (the ratios of S2:SI were 0.75 f 0.05 and 0.79 f 0.05 for young and old rats, respectively; Fig. I), the total ACh outflow elicited in the hippocampal slices of young rats (9.11 + 0.17%, n = 10) during the first period of K+-induced stimulation (S,) was significantly higher than that of old rats (8.60 -t 0.14%, n = 12). In experimental slices, addition of PIA (0.1-10 PM). immediately after S,, inhibited the K’-evoked release of [3H]ACh from the hippocampal slices of both young and old rats in a dose-related manner (Fig. 1). In young rats, a significant suppression (about 31%) of ACh outflow was observed at 1 PM PIA. In old rats, however, 10 PM PIA was required to elicit a similar inhibition on [3H]ACh release (Fig. 1).
Changc~ in hippocampal adenosine concentrati0n.s and S-nucleotia’asr activity with aging To examine whether the reduction in responsiveness to exogenous PIA was due to enhanced endogenous purinergic activity, the hippocampal adenosine concentration was measured in both young and old rats and the results are shown in Table 1. The hippocampal adenosine concentration of old rats was significantly higher (about 6 1%) than that found in their younger counterparts. In parallel with that observed with adenosine concentration, the hippocampal 5’-ND activity was also significantly higher (about 57%) in old than in young rats (Table 1).
Control
*** I vii 0.7
1.0
PIA
10.0,
f,uMT
FIG. 1. Effect of (-)N6-phenylisopropyladenosine (PIA) on K+-evoked [‘Hlacetylcholine (ACh) release from hippocampal slices of either young (open columns) or old (closed columns) rats. Each column represents the mean + SEM from 6-10 and 8-12 experiments for young and old rats, respectively. ‘Significantly different from respective control value,
p < 0.05.
ADENOSINE
AND
HIPPOCAMPAL
TABLE
I51
ACH RELEASE
1
HIPPOCAMPAL ADENOSINE CONCENTRATION AND S-ND ACTIVITY IN YOUNG AND OLD RATS
Adenosine concentration (nmol/mg protein) 5’-ND activity (mU/mg
protein)
Young Rats
Old Rats
161.60 + 14.1 (8)
260.8 I!Z33.4* (8)
75.57 f
2.46 (6)
118.50 f
2.99* (6)
Each value represents the mean k SE from number of animals shown in parentheses. * Significantly paired t test).
different
from the young rat, p < 0.01 (two-tailed
un-
DISCUSSION
It is well documented that endogenous adenosine can modulate central neuronal transmission by inhibiting presynaptic release of various neurotransmitters (for review, see refs. 5 and 9). In the hippocampus, activation of A, adenosine receptors has been reported to suppress the electrically stimulated ACh release (8). To investigate the possibility that the enhanced endogenous adenosine activity may be involved in blunting stimulated ACh release in older animals, the effect of PIA, a selective A, receptor agonist. on K+-stimulated ACh release from the hippocampal slices of both young and old rats was examined. Addition of PIA to the perfusion medium caused a dose-related suppression of K+-evoked ACh outflow from the hippocampal slices of the young rat. Even though a different stimulation method was used in our study, the concentration of PIA ( 1 PM) that caused a significant inhibition of ACh release was comparable to the concentration used previously in inhibiting electricallystimulated ACh release from the hippocampal slices of rats (8). Similar to earlier reports (for review, see ref. 20), the hippocampal ACh release evoked by 25 mM K+ was reduced with increasing age. The most interesting finding of the present study is that there was a decrease in responsiveness of the hippocampal ACh release to PIA in old rats. Ten times higher concentration of PIA than that used for young rats was required to elicit a significant suppression of K+-evoked ACh outflow in old rats. Similar reduction in responsiveness to exogenously applied
adenosine has also been observed previously in electrically stimulated ACh release from the cortical slices of old rats (10.17). Because age-related increase in adenosine release has been demonstrated in rat neck muscle following cold stimulation (2 1) and human fibroblast cultures (7) it is possible that the reduced responsiveness of hippocampal ACh release to PIA seen in old rats is due to a reduction in adenosine receptor efficacy consequent to an age-dependent increase in endogenous purinergic outflow. This suggestion is supported by the present finding that the hippocampal adenosine concentration was about 6 1% higher in old than in young rats. Coinciding with the increase in adenosine concentration, the activity of hippocampal S-ND, which has been proposed to partially govern adenosine synthesis (I 2). was also significantly higher in old than in young rats. Based on these observations, the age-dependent decrease in the inhibitory effect of PIA on hippocampal ACh release could be due to an enhanced endogenous purinergic activity during senescence. Alteration in the activity of an endogenous neurotransmitter is known to cause changes in the efficacy of its receptor(s). The net result of an increase in endogenous purinergic activity may possibly lead to conformational changes in adenosine receptor(s). This speculation is supported by the finding that the disappearance of a subpopulation of the low-affinity A, receptors in the hippocampus of 24-month-old rats is partly substituted by the high-affinity A, receptors (4). The decrease in responsiveness of the old rat hippocampal slices to PIA is, however, counter to the observed increase in receptor binding affinity. As suggested previously (4) this apparent paradox may possibly indicate that the low-affinity adenosine A, receptors, rather than the highaffinity A, receptors. are functionally important in modulating ACh release. If this interpretation is correct, then the observed age-related decrease in sensitivity to the inhibitory effect of an adenosine agonist is consistent with an increased endogenous purinergic activity with aging. In view of the recent finding that the cognitive deficit can be improved by pretreatment with a selective A, receptor antagonist ( 19). it is worthwhile to carry out further investigations on central adenosine metabolism and cholinergic activity during senescence. ACKNOWLEDGEMENT
The present study was supported by a Medical Research Council of Canada Operating Grant to L.W. We are indebted to SM. Paproski for excellent technical assistance.
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