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Changes in activities of calmodulin-mediated enzymes in rat brain during aging
Mechanisms of Ageing and Development, 26 (1984) 231 -239
231
Elsevier Scientific Publishers Ireland Ltd.
CHANGES IN ACTIVITIES OF CALMODULIN-MEDIATED ENZYMES IN RAT BRAIN DURING AGING
BETH HOSKINS* and JENNIFER M. SCOTT Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216 (U.S.A.)
(Received September 28th, 1983) (Revision received February 7th, 1984) SUMMARY Components of the calmodulin system, (i.e. calmodulin levels and activities of the following calmodulin-dependent enzymes: (Ca 2÷ + Mg2*)-ATPase, adenylate and guanylate cyclases, cyclic AMP and cyclic GMP phosphodiesterases, and Ca2+-dependent protein kinase) were studied in brains from rats of three different ages: 3 weeks old, 3 months old and 1 year old. With the exception of adenylate cyclase activity, all components measured were found to significantly decrease with increasing age. Adenylate cyclase activity was significantly higher "in brains from the 3-month-old rats than in those from 3-week-old rats. Brains from year-old rats had adenylate cyclase activity that was intermediate between the two younger ages and was significantly different from both groups. The study provides evidence for important changes in the activity of this fundamental cell regulatory system in the central nervous system during the aging process.
Key words: Calmodulin, (Ca 2+ + Mg2+)-ATPase; Phosphodiesterase; Adenylate cyclase;
Guanylate cyclase; Protein kinase. INTRODUCTION The calcium ion has a premier position among the physiologic cations. Its metabolism is intimately linked to the normal functioning of all cells [1]. As essential as the calcium ion is, it is inactive; its activity being modulated by calcium-binding proteins. Calmodulin stands out as the ubiquitous calcium-binding protein throughout eukaryotes, lacking both tissue- and species-specificity. Calmodulin was discovered in the late 1960s as the protein activator of cyclic nucleotide phosphodiesterase [2]. It has been studied exi
*To whom correspondence should be addressed. 0047-6374/84/$03.00 Printed and Published in Ireland.
© 1984 ElsevierScientific Publishers Ireland Ltd.
232 tensively over the past decade by many investigators and has been established as an important mediator of calcium in cell functions. In a recent review of calmodulin-regulated processes, authored by its discoverer [3], it was pointed out that the cellular effects of calcium ions and of cyclic AMP are interrelated and that these second messenger systems can now be integrated on a molecular basis via the common denominator, calmodulin. Calmodulin has been isolated from several vertebrate and invertebrate organisms as well as from plants [4]. The remarkable similarity between the various calmodulins and the known ubiquity of the protein reflects its ability to mediate the control of the activity of a large number of cellular processes in which Ca 2+ acts as a second messenger [5]. Figure 1 depicts some of the several enzymes and cellular processes regulated by calmodulin (asterisks denote components of the present study). According to Fig. 2, upon phosphorylation of membrane proteins (A) which are substrates for both cAMP-dependent (B) and calmodulin-regulated Ca2*-dependent (C) protein kinases, calmodulin is released from synaptosomal membranes (D) [16]. The binding of Ca2+ to calmodulin brings about a conformation change (E) which allows the Ca2*--calmodulin complex to interact with the apoenzyme [e.g. protein kinase (C), adenylate cyclase (F), guanylate cyclase (G), phosphodiesterase (H), Ca2*-ATPase (1)] to form a ternary complex which is the active species [3]. Adenylate cyclase (F) has been shown to be a dopamine receptor in the central nervous system (CNS) [17]. On the other hand, guanylate cyclase (G)has been shown to be a muscarinic receptor for acetylcholine in the brain [ 18]. The protein kinases (B) and (C) regulated by both calmodulin and cAMP, are responsible for many biochemical functions of the cell. In terms of neuronal activity, phos-
Adenylate* cyclase [6] Myosinlight chain kinase[14]
F //~
-
Phosphor~lase kinase [13] PhospholipaseA2 [12]
CaM*
~Ca2
Ca2+-dependenterotein. kinase [11]
Guanyl te* cyclasea[7] ~_ Cyclicnucleotide* r phosphodiesterase[8] + + Mg2+-ATPase*[9] Neurotransmitter release [15]
NADkinase[I0] Fig. 1. Examples of'enzymes and cellular processes that are calmodulin- (CaM)-dependent. Asterisks indicate those components that were measured in the present study.
233 Presynaptlc Terminal
_
®
ACh
Ca**
"
GMP
pro¢
5-Hr
"cAMP
procemms
'
Postsynaptlc Terminal
Fig. 2. Schematic model of the central neuronal calmodulin (CAM) system. Abbreviations: proteinPO, = phosphorylated protein(s); PK = protein kinase; AC = adenylate cyclase; GC = guanylate cyclase; PDE = cyclic nucleotide phosphodiesterase; TH = tyrosine hydroxylase; T-5-M = tryptophan-5-monooxygenase; DA = dopamine; NE = norepinephrine; 5-HT = serotonin. Asterisks indicate active forms. phorylation o f a synaptic membrane protein might be expected to alter membrane permeability. Thus, cellular flux o f ions as well as presynaptic release o f neurotransmitters [15] could be altered. Protein kinase is also involved in activation o f tyrosine hydroxylase (J) [15], the enzyme that controls the rate o f biosynthesis o f the catecholamines, dopamine and norepinephrine.
234 Calmodulin regulates the activity of Ca2÷-ATPase (I) of synaptic membranes [9]. As intracellular Ca 2÷ increases, calmodulin activates the membrane Ca2÷-ATPase, which extrudes Ca 2÷ into the extracellular space lowering intracellular Ca 2÷ concentration to a steady-state level - an excellent example of feedback control. Thus, calmodulin acts not only as a mediator of Ca2÷ action but also as a modulator of its intracellular level. Since calmodulin is a ubiquitous, calcium-binding protein which regulates the activity of so many calcium-dependent enzymes and processes, modification of the amount and/or activity of calmodulin would be expected to have profound biological consequences. With this in mind, therefore, we undertook the present study in an effort to ascertain if the aging process itself results in alterations of components of this regulatory system. This study represents a first step towards a better understanding of the molecular mechanisms that are altered during aging; thus, a first step in the possible prevention or reversal of age-induced debilitating changes in the CNS. METHODS
Animals Male Sprague-Dawley rats were used throughout tile study. Three age groups of ten rats each were used. Their ages were: Group I, 3 weeks old; Group II, 3 months old; and Group III, 1 year old.
Chemicals Kits for radioimmunoassays (RIA) of calmodulin cAMP and cGMP and Aquasol were obtained from New England Nuclear Corp., Boston, MA, as were [aH]ATP (50 mCi/ mmol), [SH]GTP (40 mCi/mmol) and ['r-a2P]ATP (2900 Ci/mmol). [8)H]Adenosine 3',5'-phosphate (20 Ci/mmol) and [8)H]guanosine 3',5'.phosphate (8 Ci/mmol) were obtained from Schwarz/Mann. cAMP, cGMP, ATP, GTP, snake venom (Crotalus atrox), Fiske-Subbarow reducer, dithiothreitol, phosphoenol pyruvate and pyruvate kinase were obtained from Sigma, St. Louis, MO. All other chemicals of analytical grade were obtained from commercial suppliers.
Experimental procedures Rats were decapitated and their brains were quickly removed. Each brain was divided in half and each half was quickly weighed. One half of each brain was homogenized in 5 ml of ice-cold 30 mM imidazole buffer, pH 7.0. The homogenate was divided into five 1.0 ml aliquots which were then stored at --4°C for subsequent use in enzyme assays (performed within one week). The other half was immediately homogenized in two volumes of buffer containing 50 mM Tris-HC1, pH 7.8, 3 mM MgSO4, 1 mM dithiothreitel, and 1 mM EGTA. Then calmodulin was extracted according to the procedure described by Wallace and Cheung [19]. The calmodulin extract so obtained was used to assay calmodulin levels using a calmodulin RIA kit.
235 For the assays of (Ca 2+ + Mg2+)-ATPase activities, 0.1 ml of thawed brain homogenate was added to 0.3 ml of assay medium consisting of 6 mM MgC12, 8 × 10 -4 M CaC12, 100 mM NaC1, 20 mM KC1, 1 × 10 -4 M ouabain, and 30 mM imidazole hydrochloride (pH 7.0). The reaction was started by addition of 0.1 ml of NaATP (1.65 mg/ml)to give a final concentration of 6 nM ATP, then the tubes were transferred to a 37°C shaking water bath. After 15 min of incubation, the reaction was terminated by the addition of 0.7 ml of a solution containing 0.5 M H2SO4, 0.5% (w/v) ammonium molybdate and 2% (w/v) sodium dodecyl sulfate. Fiske-Subbarow reducer (20 tzl) was then added to each tube and the color formation exactly 30 min later was measured at 650 nm. Appropriate phosphate standards demonstrated that the absorbance was linear at 650 nm with inorganic phosphate concentrations of 0-0.5/amol. Cyclic nucleotide phosphodiesterases were assayed using the two-stage enzymatic procedure of Thompson and Appleman [20] at substrate concentrations of 10 -4 M cAMP and 1.3 X 10 -s M cGMP. To 1 ml of the thawed brain homogenate described above, 1 ml of 21.8% sucrose was added to give the sucrose concentration of 10.9% needed. Adenylate cyclase and guanylate cyclase were assayed according to the procedure described by Wallace et at [21] using 500/aM ATP as substrate for adenylate cyclase and 500 taM GTP as substrate for guanylate cyclase. After 10 min incubation at 37°C, the reaction was terminated by the addition of 1.0 ml of ice-cold 6% trichloroacetic acid. The amounts of cyclic nucleotides formed during the reactions were determined by radioimmunoassay of the contents of each reaction vessel using RIA kits. The procedures yielded 99% recovery of marker [3H]cAMP or [3I-I]cGMP. Ca2+-Dependent protein kinase activities of the brain homogenates were measured using slight modifications of the procedure described by Forn and Greengard [22] for cerebral cortex slices. Aliquots (0.1 ml) of thawed brain homogenates were added to 0.4 ml of Krebs-Ringer bicarbonate buffer containing (in mmol/1): NaC1, 124; KC1, 5.0; NaHCO3, 25; CaC12, 1.0; Na2HPO4, 1.5; MgSO4, 1.5; glucose, 10; cAMP, 0.01; and ATP, 1.0 (approximately 6 × 104 cpm/mmol). After 10 min incubation at 37°C, the reaction was stopped by immersing the vessels in a boiling water bath for 10 min. The contents of each vessel were then centrifuged at 4000 g for 20 min. The precipitates were washed three times with 1.0 ml portions of distilled water. The precipitates were dissolved in 1.0 ml 0.4 N NaOH and transferred to counting vials. Aquasol was then added and radioactivity due to 32p transferred to protein was determined by liquid scintillation spectrometry. Determinations of protein contents of homogenates were performed using Bradford's method [23]. Data were analyzed using analysis of variance. Differences between means were considered significant at P < 0.05. RESULTS Results of all studies are summarized in Table 1. With the exception of adenylate cyclase activity, levels of calmodulin and activities of all calmodulin-stirnulated enzymes
236 TABLE I EFFECTS OF AGING ON SOME COMPONENTS OF THE CALMODULIN SYSTEM IN WHOLE BRAINS OF RATS
Body weights (g) mgprotein/gbrain Calmodulinlevel ~g/g b rain) cAMP-PDE (nmol cAMP hydrolyzed per g per 10 min) cGMP-PDE nmol cGMP hydrolyzed per g per 10 min) Adenylate cyclase (nmol cAMP formed per g per 10 min) Guanylate cyclase (nmol cGMP formed per g per 10 min) Proteinkinase ~ M PO 4 transferred to protein per g brain per 10 min (Ca 2÷ + Mg2+)-ATP-ase (mmol Pi released per mg per 15 min) 8
.
Group I (3 weeks)
Group H (3 months)
Group 111 (1 year)
92.2 + 2.9 121.4~ 6.6 13.2-+ 0.4
246.5 -+ 3.0 a'c 118.3-+ 1.6 11.9-+ 0.3 a'c
461.5 -+ 10.8 a'b 116.8 -+ 3.3 9.0 -+ 0.3 a'b
68.5 -+ 3.4
58.0 -+ 2.2 a
52.14 ± 2.3 a
133.8 -+ 5.1
106.7 -+ 2.6 a'c
16.5 -+ 0.4
34.3 -+ 2.7 a'c
27.1
-+ 2.0 a'b
283.3 -+ 19.7
195.2 -+ 10.7 a'c
110.9
+- 10.0 a'b
3.6 -+ 0.1
76.5 -+ 4.7
2.8 -+ 0.01 a'c
49.0 -+ 2.5 a'c
83.06 -+ 4.6 a'b
2.3 -+ 0.1 a'b
35.4 -+ 4.2 a'b
.
bSlgmficantly different (P < 0.05) from group I. Significantly different from Group II. CSignificantly different from Group III.
decreased w i t h increasing ages o f rats. The activity o f adenylate cyclase in brains s h o w e d a biphasic pattern in which activities apparently increased f r o m birth to m a t u r i t y , t h e n decreased during the latter stages o f life. Ratios o f guanylate cyclase to adenylate cyclase activities were 17.2 for G r o u p I, 5.7 for G r o u p II and 4.1 for G r o u p III. On the o t h e r hand, ratios o f cGMP p h o s p h o d i e s t e r a s e / c A M P phosphodiesterase activities were 1.95 for G r o u p I, 1.84 for G r o u p II and 1.59 for G r o u p III. DISCUSSION Since e n d o g e n o u s brain c a l m o d u l i n was n o t r e m o v e d f r o m the h o m o g e n a t e s used for the different e n z y m e assays, we m a y appropriately assume that the e n z y m e activities m e a s u r e d represent activities in the presence o f activator (calmodulin).
237 The decreased levels of calmodulin in brain of the mature (Group II) and old (Group III) rats, compared to levels in the young (Group I) rats, in the presence of relatively higher activities of adenylate cyclases might mean that, when calmodulin levels are low, the protein preferentially activates adenylate cyclase such that cAMP-dependent processes are favored during the first part of life. Later, however, there appears to be a lessened activity of adenylate cyclase. It has been demonstrated that an imbalance of dopaminergic (inhibitory) and cholinergic (excitatory) systems in the basal ganglia is associated with motor dysfunction, particularly Parkinsonism [24]. The altered ratios of activities of two nucleotide cyclases during aging may have some relationship to age-induced Parkinsonism. Our data suggest that, during the normal aging process, the ratio of activities of these enzymes shifts towards more activity of the adenylate cyclase (i.e. greater dopaminergic reserve), to produce a new balance with guanylate cyclase (cholinergic reserve). Thus, if dopamine levels do not increase to maintain the n e w balance between dopaminerglc (inhibitory) and cholinergic (excitatory) activity in the basal ganglia, Parkinson-like tremors would be apparent. Our finding slight but significantly decreased activity of protein kinase during aging might result in dopamine deficiency in the basal ganglia - certainly deficient if, indeed, increases of dopamine are needed later in life to induce a new balance of dopaminergic/cholinergic activity. Both pre- and postsynaptically, phosphorylated proteins serve as effectors or modulators of various cellular reactions or physiological responses [17,18]. The present study only shows a general decreased activity of whole brain protein kinase. We cannot yet say which proteins were not phosphorylated during the aging process. Future studies on this aspect of aging are certainly indicated and should be of great value in understanding molecular aspects of aging. The decreased activity of the divalent cation ATPase during aging may be in response to decreased levels of calmodulin. It might also reflect age-induced decreased permeability of neurons to passive influx of Ca 2÷ such that intraceUular, free calcium concentrations are low. The results of this study clearly demonstrate a general decrease in components of the calmodulin system in the brain as aging takes place. It should be noted that the oldest animals in our studies were one year old, Le. at the middle of their normal life span. Thus, the changes we report may be in part due to development and maturity and not to senescence. Future studies in our laboratory will be designed to determine how these changes in the caimodulin system may relate to differences in effects and efficacy of drugs when given to subjects of different ages. ACKNOWLEDGEMENTS This work was supported by BRSG Grant 2 S07 RR05386 awarded by the Biomedical Support Grant Program, Division of Research Resources, National Institutes of Health, a grant from the Mississippi Heart Association and a MARC Undergraduate Research
238 Grant (5-T34-GM7651-06) awarded to Tougaloo College. The authors are grateful for the technical assistance o f Martha A. Robertson and for the secretarial assistance of Annette McGrigg in the preparation of this manuscript. REFERENCES 1 H. Rasmussen and D.B.P. Goodman, Relationships between calcium and cyclic nucleotides in cell activation. Physiol. Rev., 57 (1977)421-509. 2 W.Y. Cheung, Activation of a partially inactive cyclic 3',5'-nucleotide phosphodiesterase. Fed. Proc., 27 (1968)783 (abstract). 3 W.Y. Cheung, Calmodulin plays a pivotal role in cellular regulation. Science, 207 (1980) 19-27. 4 T.C. Vanaman, Structure, function and evolution of calmodulin. In W.Y. Cheung (ed.) Calcium and Cell Function, Vol. 1, Academic Press, New York, 1980, pp. 41-58. 5 H. Rasmussen, Cell communication, calcium ion and cyclic adenosine monophosphate. Science, 170 (1970) 404-412. 6 W.Y. Cheung, L.S. Bradham, T.J. Lynch, Y.M. Lin and E.A. Tallant, Protein activator of cyclic 3'-St-nucleotide phosphodiesterases of bovine or rat brain also activates its adenylate cyclase. Biochem. Biophys. Res. Commun., 65 (1975) 1055-1062. 7 S. Nago, Y. Suzuki, Y. Watanabe and Y. Nozawa, Activation by a calcium-binding protein of guanylate cyclase in Tetrahymena pyriformis~ Biochem. Biophys. Res. Commun., 90 (1979) 261-268, 8 W.Y. Cheung, Cyclic 3',5'-nucleotide phosphodiesterase. Demonstration of an activator. Biochem. Biophy. Res. Commun., 38 (1970) 533-538. 9 S. Kenji, S. Ichida, H. Yoshida, R. Yamazaki and S. Kakiuchi, Occurrence of a Ca2÷- and modulator protein-activatable ATPase in the synaptic plasma membranes of brain. FEBS Lett., 99 (1979) 199-202. 10 D. Epel, C. Patton, R.W. Wallace and W.Y. Cheung, Calmodulin activates NAD kinase of sea urchin eggs: an early event of f e r ~ a t i o n . Cell, 23 (1981) 543-549. 11 H. Schulman and P. Greengard, Stimulation of brain membrane protein phosphorylation by calcium and an endogenous heat-stable protein. Nature, 271 (1978) 478-479. 12 P.Y.-K. Wong and W.Y. Cheung, Calmodulin stimulates human platelet phospholipase A 2. Biochem. Biophys. Res. Commun., 90 (1979) 473-480. 13 P. Cohn, A. Burchell, J.G. Foulkes, P.T.W. Cohen, T.C. Vanaman and A.C. Nakn, Identification of the Caa+-dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase. FEBS Lett., 92 (1978) 287-293. 14 R. Dabrowska, J.M.F. Sherry, D.K. Aromatorio and D.J. Hartshorne, Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry, 1 7 (1978) 253-258. 15 R.J. DeLorenzo, S.D. Freedman, W.B. Yoke and S.C. Maurer, Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from syna~tic vesicles. Proc. Natl. Acad. Sci. USA, 76 (1979) 1838-1842. 16 W. Seighart, J. Forn and P. Greengard, Ca2+ and cyclic AMP regulate phosphorylation of same two membrane-associated proteins specific to nerve tissue. Proc. Natl. Acad. Sci. USA, 76 (1979) 2475-2479. 17 P. Greengard, Cyclic Nucleotides, Phosphorylated Protein and Neuronal Function. Raven Press. New York, 1978. 18 E.G. Krebs and J.A. Beavo, Phosphorylation-dephosphory 'lation of enzymes. Annu. Rev. Biochem., 48 (1979) 923-959. 19 R.W. Wallace and W.Y. Cheung, Calmodulin: production of an antibody in rabbit and development of a radioimmunoassay. J. Biol. Chem., 254 (1979) 6564-6571. 20 W.J. Thompson and M.M. Appleman, Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry, I0 (1971) 311-316.
239 21 R.W. Wallace, T.J. Lynch, E.A. Tallant and W.Y. Cheung, An endogenous inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. Arch. Biochem. Biophys., 187 (1978) 328-334. 22 J. Forn and P. Greengard, Depolarizing agents and cyclic nncleotides regulate the phosphorylation of specific neuronal proteins in rat cerebral cortex slices. Proc. Natl. Acad. Sci. USA, 75 (1979) 5195-5199. 23 M.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. 24 O. Hornykiewicz, Parkinson's disease and its chemotherapy. Biochem. Pharmacol., 24 (1975) 1061-1065.
231
Elsevier Scientific Publishers Ireland Ltd.
CHANGES IN ACTIVITIES OF CALMODULIN-MEDIATED ENZYMES IN RAT BRAIN DURING AGING
BETH HOSKINS* and JENNIFER M. SCOTT Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216 (U.S.A.)
(Received September 28th, 1983) (Revision received February 7th, 1984) SUMMARY Components of the calmodulin system, (i.e. calmodulin levels and activities of the following calmodulin-dependent enzymes: (Ca 2÷ + Mg2*)-ATPase, adenylate and guanylate cyclases, cyclic AMP and cyclic GMP phosphodiesterases, and Ca2+-dependent protein kinase) were studied in brains from rats of three different ages: 3 weeks old, 3 months old and 1 year old. With the exception of adenylate cyclase activity, all components measured were found to significantly decrease with increasing age. Adenylate cyclase activity was significantly higher "in brains from the 3-month-old rats than in those from 3-week-old rats. Brains from year-old rats had adenylate cyclase activity that was intermediate between the two younger ages and was significantly different from both groups. The study provides evidence for important changes in the activity of this fundamental cell regulatory system in the central nervous system during the aging process.
Key words: Calmodulin, (Ca 2+ + Mg2+)-ATPase; Phosphodiesterase; Adenylate cyclase;
Guanylate cyclase; Protein kinase. INTRODUCTION The calcium ion has a premier position among the physiologic cations. Its metabolism is intimately linked to the normal functioning of all cells [1]. As essential as the calcium ion is, it is inactive; its activity being modulated by calcium-binding proteins. Calmodulin stands out as the ubiquitous calcium-binding protein throughout eukaryotes, lacking both tissue- and species-specificity. Calmodulin was discovered in the late 1960s as the protein activator of cyclic nucleotide phosphodiesterase [2]. It has been studied exi
*To whom correspondence should be addressed. 0047-6374/84/$03.00 Printed and Published in Ireland.
© 1984 ElsevierScientific Publishers Ireland Ltd.
232 tensively over the past decade by many investigators and has been established as an important mediator of calcium in cell functions. In a recent review of calmodulin-regulated processes, authored by its discoverer [3], it was pointed out that the cellular effects of calcium ions and of cyclic AMP are interrelated and that these second messenger systems can now be integrated on a molecular basis via the common denominator, calmodulin. Calmodulin has been isolated from several vertebrate and invertebrate organisms as well as from plants [4]. The remarkable similarity between the various calmodulins and the known ubiquity of the protein reflects its ability to mediate the control of the activity of a large number of cellular processes in which Ca 2+ acts as a second messenger [5]. Figure 1 depicts some of the several enzymes and cellular processes regulated by calmodulin (asterisks denote components of the present study). According to Fig. 2, upon phosphorylation of membrane proteins (A) which are substrates for both cAMP-dependent (B) and calmodulin-regulated Ca2*-dependent (C) protein kinases, calmodulin is released from synaptosomal membranes (D) [16]. The binding of Ca2+ to calmodulin brings about a conformation change (E) which allows the Ca2*--calmodulin complex to interact with the apoenzyme [e.g. protein kinase (C), adenylate cyclase (F), guanylate cyclase (G), phosphodiesterase (H), Ca2*-ATPase (1)] to form a ternary complex which is the active species [3]. Adenylate cyclase (F) has been shown to be a dopamine receptor in the central nervous system (CNS) [17]. On the other hand, guanylate cyclase (G)has been shown to be a muscarinic receptor for acetylcholine in the brain [ 18]. The protein kinases (B) and (C) regulated by both calmodulin and cAMP, are responsible for many biochemical functions of the cell. In terms of neuronal activity, phos-
Adenylate* cyclase [6] Myosinlight chain kinase[14]
F //~
-
Phosphor~lase kinase [13] PhospholipaseA2 [12]
CaM*
~Ca2
Ca2+-dependenterotein. kinase [11]
Guanyl te* cyclasea[7] ~_ Cyclicnucleotide* r phosphodiesterase[8] + + Mg2+-ATPase*[9] Neurotransmitter release [15]
NADkinase[I0] Fig. 1. Examples of'enzymes and cellular processes that are calmodulin- (CaM)-dependent. Asterisks indicate those components that were measured in the present study.
233 Presynaptlc Terminal
_
®
ACh
Ca**
"
GMP
pro¢
5-Hr
"cAMP
procemms
'
Postsynaptlc Terminal
Fig. 2. Schematic model of the central neuronal calmodulin (CAM) system. Abbreviations: proteinPO, = phosphorylated protein(s); PK = protein kinase; AC = adenylate cyclase; GC = guanylate cyclase; PDE = cyclic nucleotide phosphodiesterase; TH = tyrosine hydroxylase; T-5-M = tryptophan-5-monooxygenase; DA = dopamine; NE = norepinephrine; 5-HT = serotonin. Asterisks indicate active forms. phorylation o f a synaptic membrane protein might be expected to alter membrane permeability. Thus, cellular flux o f ions as well as presynaptic release o f neurotransmitters [15] could be altered. Protein kinase is also involved in activation o f tyrosine hydroxylase (J) [15], the enzyme that controls the rate o f biosynthesis o f the catecholamines, dopamine and norepinephrine.
234 Calmodulin regulates the activity of Ca2÷-ATPase (I) of synaptic membranes [9]. As intracellular Ca 2÷ increases, calmodulin activates the membrane Ca2÷-ATPase, which extrudes Ca 2÷ into the extracellular space lowering intracellular Ca 2÷ concentration to a steady-state level - an excellent example of feedback control. Thus, calmodulin acts not only as a mediator of Ca2÷ action but also as a modulator of its intracellular level. Since calmodulin is a ubiquitous, calcium-binding protein which regulates the activity of so many calcium-dependent enzymes and processes, modification of the amount and/or activity of calmodulin would be expected to have profound biological consequences. With this in mind, therefore, we undertook the present study in an effort to ascertain if the aging process itself results in alterations of components of this regulatory system. This study represents a first step towards a better understanding of the molecular mechanisms that are altered during aging; thus, a first step in the possible prevention or reversal of age-induced debilitating changes in the CNS. METHODS
Animals Male Sprague-Dawley rats were used throughout tile study. Three age groups of ten rats each were used. Their ages were: Group I, 3 weeks old; Group II, 3 months old; and Group III, 1 year old.
Chemicals Kits for radioimmunoassays (RIA) of calmodulin cAMP and cGMP and Aquasol were obtained from New England Nuclear Corp., Boston, MA, as were [aH]ATP (50 mCi/ mmol), [SH]GTP (40 mCi/mmol) and ['r-a2P]ATP (2900 Ci/mmol). [8)H]Adenosine 3',5'-phosphate (20 Ci/mmol) and [8)H]guanosine 3',5'.phosphate (8 Ci/mmol) were obtained from Schwarz/Mann. cAMP, cGMP, ATP, GTP, snake venom (Crotalus atrox), Fiske-Subbarow reducer, dithiothreitol, phosphoenol pyruvate and pyruvate kinase were obtained from Sigma, St. Louis, MO. All other chemicals of analytical grade were obtained from commercial suppliers.
Experimental procedures Rats were decapitated and their brains were quickly removed. Each brain was divided in half and each half was quickly weighed. One half of each brain was homogenized in 5 ml of ice-cold 30 mM imidazole buffer, pH 7.0. The homogenate was divided into five 1.0 ml aliquots which were then stored at --4°C for subsequent use in enzyme assays (performed within one week). The other half was immediately homogenized in two volumes of buffer containing 50 mM Tris-HC1, pH 7.8, 3 mM MgSO4, 1 mM dithiothreitel, and 1 mM EGTA. Then calmodulin was extracted according to the procedure described by Wallace and Cheung [19]. The calmodulin extract so obtained was used to assay calmodulin levels using a calmodulin RIA kit.
235 For the assays of (Ca 2+ + Mg2+)-ATPase activities, 0.1 ml of thawed brain homogenate was added to 0.3 ml of assay medium consisting of 6 mM MgC12, 8 × 10 -4 M CaC12, 100 mM NaC1, 20 mM KC1, 1 × 10 -4 M ouabain, and 30 mM imidazole hydrochloride (pH 7.0). The reaction was started by addition of 0.1 ml of NaATP (1.65 mg/ml)to give a final concentration of 6 nM ATP, then the tubes were transferred to a 37°C shaking water bath. After 15 min of incubation, the reaction was terminated by the addition of 0.7 ml of a solution containing 0.5 M H2SO4, 0.5% (w/v) ammonium molybdate and 2% (w/v) sodium dodecyl sulfate. Fiske-Subbarow reducer (20 tzl) was then added to each tube and the color formation exactly 30 min later was measured at 650 nm. Appropriate phosphate standards demonstrated that the absorbance was linear at 650 nm with inorganic phosphate concentrations of 0-0.5/amol. Cyclic nucleotide phosphodiesterases were assayed using the two-stage enzymatic procedure of Thompson and Appleman [20] at substrate concentrations of 10 -4 M cAMP and 1.3 X 10 -s M cGMP. To 1 ml of the thawed brain homogenate described above, 1 ml of 21.8% sucrose was added to give the sucrose concentration of 10.9% needed. Adenylate cyclase and guanylate cyclase were assayed according to the procedure described by Wallace et at [21] using 500/aM ATP as substrate for adenylate cyclase and 500 taM GTP as substrate for guanylate cyclase. After 10 min incubation at 37°C, the reaction was terminated by the addition of 1.0 ml of ice-cold 6% trichloroacetic acid. The amounts of cyclic nucleotides formed during the reactions were determined by radioimmunoassay of the contents of each reaction vessel using RIA kits. The procedures yielded 99% recovery of marker [3H]cAMP or [3I-I]cGMP. Ca2+-Dependent protein kinase activities of the brain homogenates were measured using slight modifications of the procedure described by Forn and Greengard [22] for cerebral cortex slices. Aliquots (0.1 ml) of thawed brain homogenates were added to 0.4 ml of Krebs-Ringer bicarbonate buffer containing (in mmol/1): NaC1, 124; KC1, 5.0; NaHCO3, 25; CaC12, 1.0; Na2HPO4, 1.5; MgSO4, 1.5; glucose, 10; cAMP, 0.01; and ATP, 1.0 (approximately 6 × 104 cpm/mmol). After 10 min incubation at 37°C, the reaction was stopped by immersing the vessels in a boiling water bath for 10 min. The contents of each vessel were then centrifuged at 4000 g for 20 min. The precipitates were washed three times with 1.0 ml portions of distilled water. The precipitates were dissolved in 1.0 ml 0.4 N NaOH and transferred to counting vials. Aquasol was then added and radioactivity due to 32p transferred to protein was determined by liquid scintillation spectrometry. Determinations of protein contents of homogenates were performed using Bradford's method [23]. Data were analyzed using analysis of variance. Differences between means were considered significant at P < 0.05. RESULTS Results of all studies are summarized in Table 1. With the exception of adenylate cyclase activity, levels of calmodulin and activities of all calmodulin-stirnulated enzymes
236 TABLE I EFFECTS OF AGING ON SOME COMPONENTS OF THE CALMODULIN SYSTEM IN WHOLE BRAINS OF RATS
Body weights (g) mgprotein/gbrain Calmodulinlevel ~g/g b rain) cAMP-PDE (nmol cAMP hydrolyzed per g per 10 min) cGMP-PDE nmol cGMP hydrolyzed per g per 10 min) Adenylate cyclase (nmol cAMP formed per g per 10 min) Guanylate cyclase (nmol cGMP formed per g per 10 min) Proteinkinase ~ M PO 4 transferred to protein per g brain per 10 min (Ca 2÷ + Mg2+)-ATP-ase (mmol Pi released per mg per 15 min) 8
.
Group I (3 weeks)
Group H (3 months)
Group 111 (1 year)
92.2 + 2.9 121.4~ 6.6 13.2-+ 0.4
246.5 -+ 3.0 a'c 118.3-+ 1.6 11.9-+ 0.3 a'c
461.5 -+ 10.8 a'b 116.8 -+ 3.3 9.0 -+ 0.3 a'b
68.5 -+ 3.4
58.0 -+ 2.2 a
52.14 ± 2.3 a
133.8 -+ 5.1
106.7 -+ 2.6 a'c
16.5 -+ 0.4
34.3 -+ 2.7 a'c
27.1
-+ 2.0 a'b
283.3 -+ 19.7
195.2 -+ 10.7 a'c
110.9
+- 10.0 a'b
3.6 -+ 0.1
76.5 -+ 4.7
2.8 -+ 0.01 a'c
49.0 -+ 2.5 a'c
83.06 -+ 4.6 a'b
2.3 -+ 0.1 a'b
35.4 -+ 4.2 a'b
.
bSlgmficantly different (P < 0.05) from group I. Significantly different from Group II. CSignificantly different from Group III.
decreased w i t h increasing ages o f rats. The activity o f adenylate cyclase in brains s h o w e d a biphasic pattern in which activities apparently increased f r o m birth to m a t u r i t y , t h e n decreased during the latter stages o f life. Ratios o f guanylate cyclase to adenylate cyclase activities were 17.2 for G r o u p I, 5.7 for G r o u p II and 4.1 for G r o u p III. On the o t h e r hand, ratios o f cGMP p h o s p h o d i e s t e r a s e / c A M P phosphodiesterase activities were 1.95 for G r o u p I, 1.84 for G r o u p II and 1.59 for G r o u p III. DISCUSSION Since e n d o g e n o u s brain c a l m o d u l i n was n o t r e m o v e d f r o m the h o m o g e n a t e s used for the different e n z y m e assays, we m a y appropriately assume that the e n z y m e activities m e a s u r e d represent activities in the presence o f activator (calmodulin).
237 The decreased levels of calmodulin in brain of the mature (Group II) and old (Group III) rats, compared to levels in the young (Group I) rats, in the presence of relatively higher activities of adenylate cyclases might mean that, when calmodulin levels are low, the protein preferentially activates adenylate cyclase such that cAMP-dependent processes are favored during the first part of life. Later, however, there appears to be a lessened activity of adenylate cyclase. It has been demonstrated that an imbalance of dopaminergic (inhibitory) and cholinergic (excitatory) systems in the basal ganglia is associated with motor dysfunction, particularly Parkinsonism [24]. The altered ratios of activities of two nucleotide cyclases during aging may have some relationship to age-induced Parkinsonism. Our data suggest that, during the normal aging process, the ratio of activities of these enzymes shifts towards more activity of the adenylate cyclase (i.e. greater dopaminergic reserve), to produce a new balance with guanylate cyclase (cholinergic reserve). Thus, if dopamine levels do not increase to maintain the n e w balance between dopaminerglc (inhibitory) and cholinergic (excitatory) activity in the basal ganglia, Parkinson-like tremors would be apparent. Our finding slight but significantly decreased activity of protein kinase during aging might result in dopamine deficiency in the basal ganglia - certainly deficient if, indeed, increases of dopamine are needed later in life to induce a new balance of dopaminergic/cholinergic activity. Both pre- and postsynaptically, phosphorylated proteins serve as effectors or modulators of various cellular reactions or physiological responses [17,18]. The present study only shows a general decreased activity of whole brain protein kinase. We cannot yet say which proteins were not phosphorylated during the aging process. Future studies on this aspect of aging are certainly indicated and should be of great value in understanding molecular aspects of aging. The decreased activity of the divalent cation ATPase during aging may be in response to decreased levels of calmodulin. It might also reflect age-induced decreased permeability of neurons to passive influx of Ca 2÷ such that intraceUular, free calcium concentrations are low. The results of this study clearly demonstrate a general decrease in components of the calmodulin system in the brain as aging takes place. It should be noted that the oldest animals in our studies were one year old, Le. at the middle of their normal life span. Thus, the changes we report may be in part due to development and maturity and not to senescence. Future studies in our laboratory will be designed to determine how these changes in the caimodulin system may relate to differences in effects and efficacy of drugs when given to subjects of different ages. ACKNOWLEDGEMENTS This work was supported by BRSG Grant 2 S07 RR05386 awarded by the Biomedical Support Grant Program, Division of Research Resources, National Institutes of Health, a grant from the Mississippi Heart Association and a MARC Undergraduate Research
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