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The development of age-related deficits in several presynaptic processes associated with brain [3H]acetylcholine release
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The development of age-related deficits in several presynaptic processes associated with brain [3H]acetylcholine release Edwin M. Meyer*, Jennifer H. Judkins Department of Pharmacology and Therapeutics, University of Florida College of Medicine. PO Box 100267, Health Science Center, Gainesville, FL 32610-0267, USA
(Received 14 March 1993; revision received 23 July 1993; accepted 13 August 1993)
Abstract Isolated nerve terminals were prepared from the neocortices and striate cortices of Fischer 344 rats from 6 to 26 months of age and then assayed for release of newly synthesized [3Hlacetylcholine (ACh) triggered by secretagogues with different mechanisms of action: 35 mM K +, 10 #M veratridine and 5/~M A23187. Secretagogue-induced release of newly synthesized [3H]ACh decreased with age in both brain regions, with reductions in A23187induced release paralleling those seen with depolarizing agents. This observation was consistent with the hypothesis that aging attenuates the release-triggering ability of calcium ions coincident with or before it affects voltage-sensitive calcium influx. In neocortex, phorbolstimulated translocation of protein kinase C (PKC) activity was attenuated in isolated nerve terminals concomitantly with A23187-induced release deficits. These results suggest that one of the earliest deficits in the ACh-release process may involve intracellular calcium potency, which may be associated with the onset of functional PKC deficits. Both brain regions also displayed gradual, age-related reductions in [3H]ACh synthesis, but this effect was more pronounced in the striatum. Choline acetyltransferase (CAT) activity decreased only in the striatum with aging. Key words: ACh-release and aging; Protein kinase C; Acetylcholine; Aging
I. Introduction One o f the most consistent p r e s y n a p t i c actions o f aging on brain cholinergic transmission involves the c o m p r o m i s e d ability o f neurons to release acetylcholine ( A C h ) * Corresponding author. 0047-6374/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0047-6374(93)01395-0
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E.M. Meyer, J.H. Judkins/ Mech. Ageing Dev. 72 (1993) 119-128
in response to depolarization [1-111. Aging also has been shown to interfere with several presynaptic processes underlying neurotransmitter-release, including K ÷ depolarization-induced calcium influx [4,5], the potency of intracellular calcium ions for triggering release as probed with A23187 [2,3] and the phorbol ester activated translocation of protein kinase C (PKC) [12,13]. Another membrane component important for neurotransmission with a potential, yet unknown role, in the age-related release-deficit is the voltage-sensitive sodium channel, which drugs such as veratridine can hold open to induce depolarization [14,15]. It is unclear as to what extent each of the foregoing presynaptic processes, differentially affected by K ÷, A23187, veratridine, or phorbol ester, underlies the agerelated ACh release-deficit, at least in part because there has been no investigation comparing their respective ages-of-onset. [3H]ACh release triggered by K ÷, A23187 or veratridine was therefore measured in nerve terminals isolated from the neocortex and striate cortex at different ages, based on the hypothesis that a comparison of these agents might reveal at least one process involved in the early stages of the release-deficit. We also measured several other processes associated with cholinergic transmission in the isolated nerve terminal preparation to determine their potential role in release-deficits: phorbol ester-stimulated translocation of PKC, choline acetyltransferase activity (CAT) and [3H]ACh synthesis. 2. Materials and methods
2.1. Chemicals All enzymes and non-radioactive chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. [3H-Methyilcholine (80 Ci/mmol) and [3H-acetyl]acetyl coenzyme A (1.2 Ci/mmol) were purchased from New England Nuclear, Co. (Boston, MA). Unless otherwise specified, drugs were dissolved in Krebs Ringer (KR) buffer. KR buffer consisted of 139 mM NaC1, 5.5 mM KCI, 1 mM CaC12, 1 mM MgCi2, 10 mM glucose, 1 mM NaH2PO4 and 15 mM NaHCO3; the final pH was 7.4. A23187 was dissolved in ethanol and added to samples such that the final ethanol concentration was 0.5%, which we find to have no effect on [3H]ACh release. 2.2. Isolated nerve terminal preparation Rat neocortical and striate cortical isolated nerve terminals were prepared as described previously from male Fischer 344 albino rats of the specified ages purchased from the NIA [16]. Tissues were suspended (0.5-1 mg protein/ml) in ice-cold KR buffer and then perfused for 5 min with 95:5 O2/CO2. 2.3. [3I-I]ACh synthesis and release The release of newly synthesized [3H]ACh was measured essentially as described previously [3]. Tissues (5-10 ml) were preincubated for 20 min at 37°C with 1 /~M [3H]choline (final specific activity 15 Ci/mmol) in oxygenated KR buffer. The tissues were washed twice by centrifugation (5 min at 15 000 × g) with ice-cold KR or calcium-free KR buffer containing 50 ~tM eserine. They were either assayed immediately for [3H]ACh to determine synthesis (using the radioenzymatic procedure
E.M. Meyer, J.H. Judkins/Mech. Ageing Dev. 72 (1993) 119-128
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described below), or suspended in 10 ml of ice-cold KR or calcium-free buffer containing eserine; 300-#1 aliquots were transferred to centrifuge tubes containing 0.7 ml KR buffer plus eserine pre-warmed to 37°C. Some centrifuge tubes also contained specified concentrations of secretagogues. Following a 2.5-min incubation at 37°C, the tubes were placed in an ice bath for 5 min and then centrifuged at 15 000 x g for 5 min. The supernatants were removed and assayed for released [3H]ACh and [3H]choline radioenzymatically with a choline kinase incubation and subsequent ion-pair extraction as described previously [3]. The [3H]ACh released in calciumfree KR was subtracted from that in normal KR for each preparation; the resulting calcium-dependent release value was used for statistical analyses. 2.4. CA T activity CAT activity was measured in isolated nerve terminal preparations essentially according to Roskoski [17]. This procedure involved incubating butanol-treated isolated nerve terminals, resuspended in KR buffer immediately after their preparation, with excess [3H-acetyl]acetyl coenzyme A (200 /~M) and choline (2 mM) in the presence of 100/~M eserine, 0.1 mM EDTA, 50 mM KC1, 10 mM KH2PO4 and KR buffer diluted 3:1. This 15 min incubation at 37°C was followed by the extraction of [3H]ACh from labeled substrate by ion-pair extraction with tetraphenylboron into butyrylnitrile. The assay was linear with tissue concentrations in the range of tissue concentrations used, as well as with time up to the 15 min incubation used. 2.5. PKC activity Isolated nerve terminals were incubated for 60 s in the presence or absence of 10 nM beta-phorbol 12-myristearic 13-acetate (/3-PMA) in KR buffer at 37°C. At that time, they were washed by centrifugation three times and then assayed for bound and free PKC by modifications of a previously described method [12] based on Friedman and Wang [13] and Matthies at al. [18]. Tissues were homogenized in 20 mM TRIS buffer (pH 7.4), 1 mM dithiothreitol, 2 mM EGTA, 50 #M of leupeptin and 0.2 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 40 000 x g for 30 min to obtain membrane-bound (pellet) and unbound (supernatant) fractions. Pellets were resuspended in the same lysis buffer to which 0.5% Triton X-100 (v/v) was added. Aliquots (10/~1) of this solubilized membrane-bound fraction and the previous supernatant unbound fraction were assayed by addition to an assay buffer (300 #1) containing 20 mM Tris-HCl (pH 7.5), 5 mM MgC12, 0.1 mM CaCI2, 40 /~g/ml phosphatidylserine, 10 nM/3-PMA, 5 #g/ml calf thymus H1 histone and 10 ~M [gamma-32p]ATP (S.A. 10-800 dpm/pmol). After a 60 s incubation at 30°C, the reaction was stopped by adding aliquots to 2 × 2 cm strips of phosphocellulose followed by addition of 70 mM phosphoric acid (3 x 5 ml washes). Radioactivity was determined by liquid scintillation spectrophotometry using a Beckman 1800A scintillation counter (counting efficiency over 90%). Basal activity was first determined by replacing the calcium, phosphatidylserine and/3-PMA with 0.5 mM EGTA and then subtracted from each corresponding PKC-activated sample. 2.6. Statistics Statistical analyses were performed with the Statview program. Multiple corn-
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parisons of means were performed using one-way analysis of variance (ANOVA: Scheffe F-test). All values are expressed as the mean ~: S.E.M. and every assay was performed in duplicate, with at least three preparations. 2.7. Protein estimates
Protein concentrations of synaptosome preparations were estimated with the Biorad reagent, which uses the Coomassie blue staining of peptidergic bonds [19]. 3. Results
Neocortical [3H]ACh release was elevated by 35 mM KC1, 10 izM veratridine or 5 /~M A23187 in 6-month-old animals (Fig. 1). Although there appeared to be a gradual decrease in the basal release of transmitter, only the 26-month age group was significantly different from the 6-month value. Aging interfered with the [3H]ACh release triggered by each secretagogue in a gradual manner. The K+-induced
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Fig. l. Age-related changes in neocortical [3HJACh release. Isolated nerve terminals were prepared t~om rats of the specified ages and preloaded with [3HIACh by incubating them with 1 ~M [-~H]choline for 20 min at 37°C in oxygenated KR buffer as described in the text. They were incubated in KR buffer at 37°C for 2 rain in the presence of 10 #M eserine with the specified treatment (NaCI removed from the 35 mM KCI samples to maintain osmolarityt. The [3HIACh released into the medium was assayed as described in the text and expressed as the mean ~ S.E.M. of at least five animals per group. *P < 0.05 compared with the 6-month-old group, same treatment (one-way ANOVA).
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[3H]ACh release values at 20, 24 and 26 months were each less than the corresponding 6-month values. Veratridine-induced [3H]ACh release was similarly inhibited at 16 months compared with 6 months and again at 24 and 26 months of age. A23187-induced release was consistently inhibited in 16- to 26-month-old tissues compared with the corresponding 6-month-old value. In the striate cortex, basal [3H]ACh release was reduced at 24 and 26 months of age compared with that in 6-month-old tissues (Fig. 2). K÷-induced release was less in the 16- to 26-month-old animals, slightly before this decrease was observed in the neocortex. Similarly veratridine- and A23187-induced release were both reduced by 16 months of age. CAT activity was not affected by aging in the neocortex, but was significantly reduced in the striate cortex compared with 6-month-old values at 24 and 26 months of age (Fig. 3). Neocortical and striatal [3H]ACh synthesis were both reduced at 24 and 26 months of age (Fig. 4). The percentage inhibition in [3H]ACh synthesis at 24 months of age in the cerebrum and striatum was 22% and 38%, respectively. Bound levels of PKC decreased in the neocortex under basal conditions by 24 months of age (Fig. 5). The phorbol-induced translocation in activity to membranebound fractions also decreased in the brain region by 16 months of age.
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Fig, 4. Effects of age on neocortical and striatal [3H]ACh synthesis. Isolated nerve terminals were incubated with [3H]choline for 20 min as described in Fig. I above and then assayed for [3H]ACh synthesis as described in the text. Each value is the mean m S.E.M. of five animals per group. *P < 0.05 compared with the 6-month-old value (one-way ANOVA).
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Fig. 5. PKC activity in bound and free fractions in isolated nerve terminals at different ages. Isolated terminals were incubated with (stimulated) or without (basal) I0 nM phorbol 12-myristearic acid for 60 s at 37°C. Tissues were then washed by centrifugation, homogenized, separated into bound and unbound fractions and assayed for PKC activity as described in the text. Each value is the mean ± S,E.M of four separate preparations, each assayed in duplicate.
4. Discussion This study characterized long-term ontogenetic deficits in several components of the [3H]ACh-release process in the neocortex and the striate cortex. Each region is susceptible to at least one age-related neurodegenerative condition (e.g. Aizheimer's or Parkinson's disease) in which a cholinergic system plays a direct or modulatory role. Previous studies have found that ACh release is diminished with age in both regions [4,5,7,8,10]. We now report that aging interferes with the A23187-induced release of [3H]ACh at least as soon as it inhibits K ÷ or veratridine-induced release of transmitter in both the neocortex and striate cortex. While the present results do not rule out the possibility that age-related changes in calcium influx into cholinergic terminals also occur over this interval, they do indicate that the reduction in calcium potency for triggering release must be accounted for even during the early stages of this deficit. Age-related changes in other transmitter systems which may also modulate ACh release were minimized in the present study by using an isolated nerve terminal preparation instead of intact preparations such as slices. Our studies also focused on newly synthesized transmitter, which is preferentially released by nerve terminals. Newly synthesized as well as older, unlabelled pools of ACh have both been shown to be sensitive to age-related reductions in calcium-triggered release though not identically under all conditions [20].
126
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Age-related deficits in veratridine-induced release essentially paralleled those with K + depolarization, arguing against any role for sodium channels in these deficits, at least under these conditions. Alternatively, if these channels remain open for shorter than normal intervals during aging, veratridine could mask this difference by artificially holding them open. PKC is normally activated by calcium ions and diacylglycerol in a manner that involves the translocation of the enzyme to phosphatidylserine binding sites along the cytoplasmic surface of plasma membranes [18]. PKC activation appears to modulate the release of a number of transmitters, including ACh, though its precise role in the release process remains unknown [13,18,21,22]. Friedman and Wang [ 13] demonstrated that aging interfered with the ability of secretagogues to trigger the translocation of PKC to neocortical membranes. However, aging has not consistently been found to reduce basal PKC levels in brain (e.g. Ref. 25 vs. 26). The present results are consistent with an age-related decrement in the function of this enzyme in the neocortex; unfortunately, we were unable to assay this enzyme activity in the striatum because of the lack of tissue. Nonetheless, we found for the first time a temporal correlation between the deficit in PKC activation and that in calciuminduced ACh release, consistent with the hypothesis that both deficits may be functionally related. Our results also demonstrate a regional difference in the age sensitivity of cholinergic terminals with respect to CAT activity and ACh synthesis in the rat brain. In the striate cortex, the coincident loss in ACh synthesis and CAT activity probably reflects a generalized loss of functional cholinergic terminals. The mechanism underlying the reduction in neocortical ACh synthesis may involve changes in high affinity choline uptake or in the disposition of the transmitter. That the latter process may be involved in the altered release of transmitter was suggested by Weiler [9], who found that loading rat neostriatal slices with labeled choline for 3 h attenuated the release-deficit when compared with shorter loading intervals [9]. The reduction in basal release of [3H]ACh from nerve terminals in both neocortex and striate cortex may be related at least in part to the reduction in ACh synthesis. That the basal release decreased more quickly and to a greater extent in the striate cortex than in the cerebrum probably reflects the greater reduction in striatal [3H]ACh synthesis. However, it should be noted that basal release of [3H]ACh in synaptosomal preparations is about 30% calcium-dependent [23] and at least some component in the reduction in basal release may be due to a change in calcium potency or influx. Many studies of aging and brain cholinergic activity have been published, sometimes with disparate results that may be accountable in part by the various preparations used. We observed concomitant age-related differences in isolated nerve terminal ACh synthesis and basal transmitter release in both regions studied, with greater synthesis-deficits associated with greater basal release reductions. In another recent study using tissue slices, the potassium-evoked, but not the spontaneous, release of ACh was markedly depressed in the neocortices and striate cortices of 27-month-old rats [8]. Another study found no age-related deficit in CAT activity or in basal ACh release from neostriatal slices from Fischer 344 rats at 28 months of age [9]. Tissue slices presumably contain perikeryal CAT-activity that the isolated
E.M. Meyer, J.H. Judkins/Mech. Ageing Dev. 72 (1993) 119-128
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terminals do not. Further, basal release in the slice may be more sensitive to compensatory age-related changes in the release of other transmitters o n t o cholinergic terminals. These changes would be eliminated largely in the isolated nerve terminal preparation used in the present study. In summary, this investigation demonstrates that, while there are regional differences with respect to how aging affects several presynaptic cholinergic processes, an early deficit involves the ability of intracellular calcium ions to trigger [3H]ACh release, as d e m o n s t r a t e d using A23187. This change in calcium potency may be sufficient to account for at least some of the age-related deficits observed with more classical depolarizing agents such as potassium or veratridine; further, it appears to be correlated temporally with changes in the functional translocation of P K C activity, at least in the neocortex. These studies suggest that i n t r a n e u r o n a l calcium potency and calcium-sensitive t r a n s d u c t i o n processes should remain one of foci of research in this field.
5. Acknowledgements This work was supported by N I H grant AG06226, The authors t h a n k Judy A d a m s for secretarial assistance.
6. References 1 R.T. Bartus, R.L. Dean, B. Beer and A.S. Lippa, The cholinergic hypothesis of geriatric memory dysfunction. Science. 217 (1982)408-414. 2 F.T.Crews, E.M. Meyer, R.A. Gonzales, D.H. Otero and K. Larsen, Pre-synapticand postsynaptic approaches to enhance central cholinergic neurotransmission. In Treatment and Development Strategies for AIzheimer's Disease, Mark Powley Association, Madison, CT, 1992, pp. 385-420. 3 E.M. Meyer, F.T. Crews, D,H. Otero and K. Larsen, Aging decreases the sensitivityof rat cortical synaptosomes to calcium ionophore induced acetylcholine release. J. Neurochem., 47 (1986) 1244-1246. 4 F. Pedata, L. Giovannelli, G.G. Spignoli and G. Pepeu, Phosphatidyl-serineincreasesacetylcholine release from cortical slices in aged rats. Neurobiol. Aging, 6 (1985) 337-340. 5 C. Peterson and G.E. Gibson, Aging and 3,4 diaminopyridine alter synaptosomal calcium uptake. J. Biol. Chem., 258 (1983) 11482-11486. 6 G.E. Gibson and C. Peterson, Aging decreases oxidative metabolism and the release and synthesis of acetylcholine. J. Neurochem.. 37 ( 1981) 978-984. 7 K.A.Sherman and E. Friedman, Pre- and post-synapticcholinergicdysfunction in aged rodent brain regions: new findings and an interpretative review. Int. J. Dev. Neurosci.. 8 (1990) 689-708. 8 D.M. Araujo, P.A. Lapchak, M.J. Meaney, B. Collier and R. Quirion, Effects of aging on nicotinic and muscarinic autoreceptor function in the rat brain: relationship to presynaptic cholinergic markers and binding sites. J. Neurosci., 10 (1990) 3069-3078. 9 M.H. Weiler, Acetylcholine release from striatal slices of young adult and aged Fischer 344 rats. Neurobiol. Aging, 11 (1990) 401-407. 10 C.G. Wu, R. Bertorelli, M. Sacconi, G. Pepeu and S. Consolo, Decrease of brain acetylcholine release in aging freely-movingrats detected by microdialysis. Neurobiol. Aging, 9 (1988) 357-361. 11 L.R. Williams, R.J. Rylett, H.C. Moises and A.H. Tang, Exogenous NGF affects cholinergic transmitter function and Y-maze behavior in aged Fischer 344 male rats. Can. J. Neurol. Sci., 18 ( 1991) 403-407. 12 F.T. Crews, L.J. Chandler, G. Calderni and E.M. Meyer, Changes in membrane calcium mediated signals in senescence. In G. Valenti (ed.), Psychoneuroendocrinalogy ~[ Aging: Basic and Clinical Aspeets, Liviana Press, Padova, 1988, pp..7-15.
128 13 14 15 16
17 18 19 20 21
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E. Friedman, E, and H.-Y. Wang, Effect of age on brain cortical protein kinase C and its mediation of 5-hydroxytryptamine release, k Neurochem., 52 (1989) 187-192. W.A. Catterall, Activation of the action potential sodium ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin. J. Biol. Chem., 250 0975) 4053-4059. E.M. Meyer and J.R. Cooper, Cobalt ions dissociate between calcium uptake through voltage sensitive sodium and calcium channels in rat cortical synaptosomes. Brain Res., 265 (1983) 173-176. G.P. Sgaragli, I.L. Sen, A. Baba, R.A. Schulz and J.R. Cooper, The mechanism of action of collagenase on the inhibition of acetyl-choline release from brain slices and the acetylcholine content of subcellular fractions prepared from brain. J. Neurochem.. 27 (1977) 71-76. R. Roskoski, Choline acetyltransferase. Evidence for an acetyl-enzyme reaction intermediate. Biochemistry, 12 0973) 3709-3713. H.J.G. Matthies, H.C. Palfrey, L.D. Hirning and R.J. Miller, Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release. J. Neurosci., 7 (1987) 1198-1206. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248-254. M.G. Vannucchi, F. Casamenti and G. Pepeu, Decrease of acetyl-choline release from cortical slices in aged rats: investigations into its reversal by phosphatidylserine. J. Neurochem., 55 (1990) 819-825. T. Oda, M.S. Shearman and Y. Nishizuka, Synaptosomal protein kinase C subspecies: Down regulation promoted by phorbol ester and its effect on evoked norepinephrine release. J. Neurochem., 56 0991) 1263-1269. R.A. Nichols, J.W. Haycock, J.K.T. Wang and P. Greengard, Phorbol ester enhancement of neurotransmitter release from rat brain synaptosomes. J. Neurochem., 48 (1987) 615-621. E. St. Onge, D.H. Otero, D. Bottiglieri and E.M. Meyer, Effects of several membrane channels and intracellular messengers on the muscarinic modulation of acetylcholine release. Neurochem Res.. I 1 (1986) 1547-1556.
Mechanisms of Ageing and Development
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The development of age-related deficits in several presynaptic processes associated with brain [3H]acetylcholine release Edwin M. Meyer*, Jennifer H. Judkins Department of Pharmacology and Therapeutics, University of Florida College of Medicine. PO Box 100267, Health Science Center, Gainesville, FL 32610-0267, USA
(Received 14 March 1993; revision received 23 July 1993; accepted 13 August 1993)
Abstract Isolated nerve terminals were prepared from the neocortices and striate cortices of Fischer 344 rats from 6 to 26 months of age and then assayed for release of newly synthesized [3Hlacetylcholine (ACh) triggered by secretagogues with different mechanisms of action: 35 mM K +, 10 #M veratridine and 5/~M A23187. Secretagogue-induced release of newly synthesized [3H]ACh decreased with age in both brain regions, with reductions in A23187induced release paralleling those seen with depolarizing agents. This observation was consistent with the hypothesis that aging attenuates the release-triggering ability of calcium ions coincident with or before it affects voltage-sensitive calcium influx. In neocortex, phorbolstimulated translocation of protein kinase C (PKC) activity was attenuated in isolated nerve terminals concomitantly with A23187-induced release deficits. These results suggest that one of the earliest deficits in the ACh-release process may involve intracellular calcium potency, which may be associated with the onset of functional PKC deficits. Both brain regions also displayed gradual, age-related reductions in [3H]ACh synthesis, but this effect was more pronounced in the striatum. Choline acetyltransferase (CAT) activity decreased only in the striatum with aging. Key words: ACh-release and aging; Protein kinase C; Acetylcholine; Aging
I. Introduction One o f the most consistent p r e s y n a p t i c actions o f aging on brain cholinergic transmission involves the c o m p r o m i s e d ability o f neurons to release acetylcholine ( A C h ) * Corresponding author. 0047-6374/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0047-6374(93)01395-0
120
E.M. Meyer, J.H. Judkins/ Mech. Ageing Dev. 72 (1993) 119-128
in response to depolarization [1-111. Aging also has been shown to interfere with several presynaptic processes underlying neurotransmitter-release, including K ÷ depolarization-induced calcium influx [4,5], the potency of intracellular calcium ions for triggering release as probed with A23187 [2,3] and the phorbol ester activated translocation of protein kinase C (PKC) [12,13]. Another membrane component important for neurotransmission with a potential, yet unknown role, in the age-related release-deficit is the voltage-sensitive sodium channel, which drugs such as veratridine can hold open to induce depolarization [14,15]. It is unclear as to what extent each of the foregoing presynaptic processes, differentially affected by K ÷, A23187, veratridine, or phorbol ester, underlies the agerelated ACh release-deficit, at least in part because there has been no investigation comparing their respective ages-of-onset. [3H]ACh release triggered by K ÷, A23187 or veratridine was therefore measured in nerve terminals isolated from the neocortex and striate cortex at different ages, based on the hypothesis that a comparison of these agents might reveal at least one process involved in the early stages of the release-deficit. We also measured several other processes associated with cholinergic transmission in the isolated nerve terminal preparation to determine their potential role in release-deficits: phorbol ester-stimulated translocation of PKC, choline acetyltransferase activity (CAT) and [3H]ACh synthesis. 2. Materials and methods
2.1. Chemicals All enzymes and non-radioactive chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. [3H-Methyilcholine (80 Ci/mmol) and [3H-acetyl]acetyl coenzyme A (1.2 Ci/mmol) were purchased from New England Nuclear, Co. (Boston, MA). Unless otherwise specified, drugs were dissolved in Krebs Ringer (KR) buffer. KR buffer consisted of 139 mM NaC1, 5.5 mM KCI, 1 mM CaC12, 1 mM MgCi2, 10 mM glucose, 1 mM NaH2PO4 and 15 mM NaHCO3; the final pH was 7.4. A23187 was dissolved in ethanol and added to samples such that the final ethanol concentration was 0.5%, which we find to have no effect on [3H]ACh release. 2.2. Isolated nerve terminal preparation Rat neocortical and striate cortical isolated nerve terminals were prepared as described previously from male Fischer 344 albino rats of the specified ages purchased from the NIA [16]. Tissues were suspended (0.5-1 mg protein/ml) in ice-cold KR buffer and then perfused for 5 min with 95:5 O2/CO2. 2.3. [3I-I]ACh synthesis and release The release of newly synthesized [3H]ACh was measured essentially as described previously [3]. Tissues (5-10 ml) were preincubated for 20 min at 37°C with 1 /~M [3H]choline (final specific activity 15 Ci/mmol) in oxygenated KR buffer. The tissues were washed twice by centrifugation (5 min at 15 000 × g) with ice-cold KR or calcium-free KR buffer containing 50 ~tM eserine. They were either assayed immediately for [3H]ACh to determine synthesis (using the radioenzymatic procedure
E.M. Meyer, J.H. Judkins/Mech. Ageing Dev. 72 (1993) 119-128
121
described below), or suspended in 10 ml of ice-cold KR or calcium-free buffer containing eserine; 300-#1 aliquots were transferred to centrifuge tubes containing 0.7 ml KR buffer plus eserine pre-warmed to 37°C. Some centrifuge tubes also contained specified concentrations of secretagogues. Following a 2.5-min incubation at 37°C, the tubes were placed in an ice bath for 5 min and then centrifuged at 15 000 x g for 5 min. The supernatants were removed and assayed for released [3H]ACh and [3H]choline radioenzymatically with a choline kinase incubation and subsequent ion-pair extraction as described previously [3]. The [3H]ACh released in calciumfree KR was subtracted from that in normal KR for each preparation; the resulting calcium-dependent release value was used for statistical analyses. 2.4. CA T activity CAT activity was measured in isolated nerve terminal preparations essentially according to Roskoski [17]. This procedure involved incubating butanol-treated isolated nerve terminals, resuspended in KR buffer immediately after their preparation, with excess [3H-acetyl]acetyl coenzyme A (200 /~M) and choline (2 mM) in the presence of 100/~M eserine, 0.1 mM EDTA, 50 mM KC1, 10 mM KH2PO4 and KR buffer diluted 3:1. This 15 min incubation at 37°C was followed by the extraction of [3H]ACh from labeled substrate by ion-pair extraction with tetraphenylboron into butyrylnitrile. The assay was linear with tissue concentrations in the range of tissue concentrations used, as well as with time up to the 15 min incubation used. 2.5. PKC activity Isolated nerve terminals were incubated for 60 s in the presence or absence of 10 nM beta-phorbol 12-myristearic 13-acetate (/3-PMA) in KR buffer at 37°C. At that time, they were washed by centrifugation three times and then assayed for bound and free PKC by modifications of a previously described method [12] based on Friedman and Wang [13] and Matthies at al. [18]. Tissues were homogenized in 20 mM TRIS buffer (pH 7.4), 1 mM dithiothreitol, 2 mM EGTA, 50 #M of leupeptin and 0.2 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 40 000 x g for 30 min to obtain membrane-bound (pellet) and unbound (supernatant) fractions. Pellets were resuspended in the same lysis buffer to which 0.5% Triton X-100 (v/v) was added. Aliquots (10/~1) of this solubilized membrane-bound fraction and the previous supernatant unbound fraction were assayed by addition to an assay buffer (300 #1) containing 20 mM Tris-HCl (pH 7.5), 5 mM MgC12, 0.1 mM CaCI2, 40 /~g/ml phosphatidylserine, 10 nM/3-PMA, 5 #g/ml calf thymus H1 histone and 10 ~M [gamma-32p]ATP (S.A. 10-800 dpm/pmol). After a 60 s incubation at 30°C, the reaction was stopped by adding aliquots to 2 × 2 cm strips of phosphocellulose followed by addition of 70 mM phosphoric acid (3 x 5 ml washes). Radioactivity was determined by liquid scintillation spectrophotometry using a Beckman 1800A scintillation counter (counting efficiency over 90%). Basal activity was first determined by replacing the calcium, phosphatidylserine and/3-PMA with 0.5 mM EGTA and then subtracted from each corresponding PKC-activated sample. 2.6. Statistics Statistical analyses were performed with the Statview program. Multiple corn-
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Protein concentrations of synaptosome preparations were estimated with the Biorad reagent, which uses the Coomassie blue staining of peptidergic bonds [19]. 3. Results
Neocortical [3H]ACh release was elevated by 35 mM KC1, 10 izM veratridine or 5 /~M A23187 in 6-month-old animals (Fig. 1). Although there appeared to be a gradual decrease in the basal release of transmitter, only the 26-month age group was significantly different from the 6-month value. Aging interfered with the [3H]ACh release triggered by each secretagogue in a gradual manner. The K+-induced
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E.M. Meyer, J.H. Judkins/Mech. Ageing Dev. 72 (1993) 119-128
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[3H]ACh release values at 20, 24 and 26 months were each less than the corresponding 6-month values. Veratridine-induced [3H]ACh release was similarly inhibited at 16 months compared with 6 months and again at 24 and 26 months of age. A23187-induced release was consistently inhibited in 16- to 26-month-old tissues compared with the corresponding 6-month-old value. In the striate cortex, basal [3H]ACh release was reduced at 24 and 26 months of age compared with that in 6-month-old tissues (Fig. 2). K÷-induced release was less in the 16- to 26-month-old animals, slightly before this decrease was observed in the neocortex. Similarly veratridine- and A23187-induced release were both reduced by 16 months of age. CAT activity was not affected by aging in the neocortex, but was significantly reduced in the striate cortex compared with 6-month-old values at 24 and 26 months of age (Fig. 3). Neocortical and striatal [3H]ACh synthesis were both reduced at 24 and 26 months of age (Fig. 4). The percentage inhibition in [3H]ACh synthesis at 24 months of age in the cerebrum and striatum was 22% and 38%, respectively. Bound levels of PKC decreased in the neocortex under basal conditions by 24 months of age (Fig. 5). The phorbol-induced translocation in activity to membranebound fractions also decreased in the brain region by 16 months of age.
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Fig. 5. PKC activity in bound and free fractions in isolated nerve terminals at different ages. Isolated terminals were incubated with (stimulated) or without (basal) I0 nM phorbol 12-myristearic acid for 60 s at 37°C. Tissues were then washed by centrifugation, homogenized, separated into bound and unbound fractions and assayed for PKC activity as described in the text. Each value is the mean ± S,E.M of four separate preparations, each assayed in duplicate.
4. Discussion This study characterized long-term ontogenetic deficits in several components of the [3H]ACh-release process in the neocortex and the striate cortex. Each region is susceptible to at least one age-related neurodegenerative condition (e.g. Aizheimer's or Parkinson's disease) in which a cholinergic system plays a direct or modulatory role. Previous studies have found that ACh release is diminished with age in both regions [4,5,7,8,10]. We now report that aging interferes with the A23187-induced release of [3H]ACh at least as soon as it inhibits K ÷ or veratridine-induced release of transmitter in both the neocortex and striate cortex. While the present results do not rule out the possibility that age-related changes in calcium influx into cholinergic terminals also occur over this interval, they do indicate that the reduction in calcium potency for triggering release must be accounted for even during the early stages of this deficit. Age-related changes in other transmitter systems which may also modulate ACh release were minimized in the present study by using an isolated nerve terminal preparation instead of intact preparations such as slices. Our studies also focused on newly synthesized transmitter, which is preferentially released by nerve terminals. Newly synthesized as well as older, unlabelled pools of ACh have both been shown to be sensitive to age-related reductions in calcium-triggered release though not identically under all conditions [20].
126
E.M. Meyer, J.tl. Judkins/ Mech. A~ein~ l)cv 72 <1993~ IltJ-12~
Age-related deficits in veratridine-induced release essentially paralleled those with K + depolarization, arguing against any role for sodium channels in these deficits, at least under these conditions. Alternatively, if these channels remain open for shorter than normal intervals during aging, veratridine could mask this difference by artificially holding them open. PKC is normally activated by calcium ions and diacylglycerol in a manner that involves the translocation of the enzyme to phosphatidylserine binding sites along the cytoplasmic surface of plasma membranes [18]. PKC activation appears to modulate the release of a number of transmitters, including ACh, though its precise role in the release process remains unknown [13,18,21,22]. Friedman and Wang [ 13] demonstrated that aging interfered with the ability of secretagogues to trigger the translocation of PKC to neocortical membranes. However, aging has not consistently been found to reduce basal PKC levels in brain (e.g. Ref. 25 vs. 26). The present results are consistent with an age-related decrement in the function of this enzyme in the neocortex; unfortunately, we were unable to assay this enzyme activity in the striatum because of the lack of tissue. Nonetheless, we found for the first time a temporal correlation between the deficit in PKC activation and that in calciuminduced ACh release, consistent with the hypothesis that both deficits may be functionally related. Our results also demonstrate a regional difference in the age sensitivity of cholinergic terminals with respect to CAT activity and ACh synthesis in the rat brain. In the striate cortex, the coincident loss in ACh synthesis and CAT activity probably reflects a generalized loss of functional cholinergic terminals. The mechanism underlying the reduction in neocortical ACh synthesis may involve changes in high affinity choline uptake or in the disposition of the transmitter. That the latter process may be involved in the altered release of transmitter was suggested by Weiler [9], who found that loading rat neostriatal slices with labeled choline for 3 h attenuated the release-deficit when compared with shorter loading intervals [9]. The reduction in basal release of [3H]ACh from nerve terminals in both neocortex and striate cortex may be related at least in part to the reduction in ACh synthesis. That the basal release decreased more quickly and to a greater extent in the striate cortex than in the cerebrum probably reflects the greater reduction in striatal [3H]ACh synthesis. However, it should be noted that basal release of [3H]ACh in synaptosomal preparations is about 30% calcium-dependent [23] and at least some component in the reduction in basal release may be due to a change in calcium potency or influx. Many studies of aging and brain cholinergic activity have been published, sometimes with disparate results that may be accountable in part by the various preparations used. We observed concomitant age-related differences in isolated nerve terminal ACh synthesis and basal transmitter release in both regions studied, with greater synthesis-deficits associated with greater basal release reductions. In another recent study using tissue slices, the potassium-evoked, but not the spontaneous, release of ACh was markedly depressed in the neocortices and striate cortices of 27-month-old rats [8]. Another study found no age-related deficit in CAT activity or in basal ACh release from neostriatal slices from Fischer 344 rats at 28 months of age [9]. Tissue slices presumably contain perikeryal CAT-activity that the isolated
E.M. Meyer, J.H. Judkins/Mech. Ageing Dev. 72 (1993) 119-128
127
terminals do not. Further, basal release in the slice may be more sensitive to compensatory age-related changes in the release of other transmitters o n t o cholinergic terminals. These changes would be eliminated largely in the isolated nerve terminal preparation used in the present study. In summary, this investigation demonstrates that, while there are regional differences with respect to how aging affects several presynaptic cholinergic processes, an early deficit involves the ability of intracellular calcium ions to trigger [3H]ACh release, as d e m o n s t r a t e d using A23187. This change in calcium potency may be sufficient to account for at least some of the age-related deficits observed with more classical depolarizing agents such as potassium or veratridine; further, it appears to be correlated temporally with changes in the functional translocation of P K C activity, at least in the neocortex. These studies suggest that i n t r a n e u r o n a l calcium potency and calcium-sensitive t r a n s d u c t i o n processes should remain one of foci of research in this field.
5. Acknowledgements This work was supported by N I H grant AG06226, The authors t h a n k Judy A d a m s for secretarial assistance.
6. References 1 R.T. Bartus, R.L. Dean, B. Beer and A.S. Lippa, The cholinergic hypothesis of geriatric memory dysfunction. Science. 217 (1982)408-414. 2 F.T.Crews, E.M. Meyer, R.A. Gonzales, D.H. Otero and K. Larsen, Pre-synapticand postsynaptic approaches to enhance central cholinergic neurotransmission. In Treatment and Development Strategies for AIzheimer's Disease, Mark Powley Association, Madison, CT, 1992, pp. 385-420. 3 E.M. Meyer, F.T. Crews, D,H. Otero and K. Larsen, Aging decreases the sensitivityof rat cortical synaptosomes to calcium ionophore induced acetylcholine release. J. Neurochem., 47 (1986) 1244-1246. 4 F. Pedata, L. Giovannelli, G.G. Spignoli and G. Pepeu, Phosphatidyl-serineincreasesacetylcholine release from cortical slices in aged rats. Neurobiol. Aging, 6 (1985) 337-340. 5 C. Peterson and G.E. Gibson, Aging and 3,4 diaminopyridine alter synaptosomal calcium uptake. J. Biol. Chem., 258 (1983) 11482-11486. 6 G.E. Gibson and C. Peterson, Aging decreases oxidative metabolism and the release and synthesis of acetylcholine. J. Neurochem.. 37 ( 1981) 978-984. 7 K.A.Sherman and E. Friedman, Pre- and post-synapticcholinergicdysfunction in aged rodent brain regions: new findings and an interpretative review. Int. J. Dev. Neurosci.. 8 (1990) 689-708. 8 D.M. Araujo, P.A. Lapchak, M.J. Meaney, B. Collier and R. Quirion, Effects of aging on nicotinic and muscarinic autoreceptor function in the rat brain: relationship to presynaptic cholinergic markers and binding sites. J. Neurosci., 10 (1990) 3069-3078. 9 M.H. Weiler, Acetylcholine release from striatal slices of young adult and aged Fischer 344 rats. Neurobiol. Aging, 11 (1990) 401-407. 10 C.G. Wu, R. Bertorelli, M. Sacconi, G. Pepeu and S. Consolo, Decrease of brain acetylcholine release in aging freely-movingrats detected by microdialysis. Neurobiol. Aging, 9 (1988) 357-361. 11 L.R. Williams, R.J. Rylett, H.C. Moises and A.H. Tang, Exogenous NGF affects cholinergic transmitter function and Y-maze behavior in aged Fischer 344 male rats. Can. J. Neurol. Sci., 18 ( 1991) 403-407. 12 F.T. Crews, L.J. Chandler, G. Calderni and E.M. Meyer, Changes in membrane calcium mediated signals in senescence. In G. Valenti (ed.), Psychoneuroendocrinalogy ~[ Aging: Basic and Clinical Aspeets, Liviana Press, Padova, 1988, pp..7-15.
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E. Friedman, E, and H.-Y. Wang, Effect of age on brain cortical protein kinase C and its mediation of 5-hydroxytryptamine release, k Neurochem., 52 (1989) 187-192. W.A. Catterall, Activation of the action potential sodium ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin. J. Biol. Chem., 250 0975) 4053-4059. E.M. Meyer and J.R. Cooper, Cobalt ions dissociate between calcium uptake through voltage sensitive sodium and calcium channels in rat cortical synaptosomes. Brain Res., 265 (1983) 173-176. G.P. Sgaragli, I.L. Sen, A. Baba, R.A. Schulz and J.R. Cooper, The mechanism of action of collagenase on the inhibition of acetyl-choline release from brain slices and the acetylcholine content of subcellular fractions prepared from brain. J. Neurochem.. 27 (1977) 71-76. R. Roskoski, Choline acetyltransferase. Evidence for an acetyl-enzyme reaction intermediate. Biochemistry, 12 0973) 3709-3713. H.J.G. Matthies, H.C. Palfrey, L.D. Hirning and R.J. Miller, Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release. J. Neurosci., 7 (1987) 1198-1206. M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72 (1976) 248-254. M.G. Vannucchi, F. Casamenti and G. Pepeu, Decrease of acetyl-choline release from cortical slices in aged rats: investigations into its reversal by phosphatidylserine. J. Neurochem., 55 (1990) 819-825. T. Oda, M.S. Shearman and Y. Nishizuka, Synaptosomal protein kinase C subspecies: Down regulation promoted by phorbol ester and its effect on evoked norepinephrine release. J. Neurochem., 56 0991) 1263-1269. R.A. Nichols, J.W. Haycock, J.K.T. Wang and P. Greengard, Phorbol ester enhancement of neurotransmitter release from rat brain synaptosomes. J. Neurochem., 48 (1987) 615-621. E. St. Onge, D.H. Otero, D. Bottiglieri and E.M. Meyer, Effects of several membrane channels and intracellular messengers on the muscarinic modulation of acetylcholine release. Neurochem Res.. I 1 (1986) 1547-1556.