Jourriul oj Nrurochrmofry. I975 Vol. 25. pp. 853-860. Pergamon Press. Prlnted in Great Britain.
ACETYLCHOLINESTERASE TURNOVER IN BRAIN, CEREBROSPINAL FLUID AND PLASMA T. L. YAKSH,’M. G. FILWRT,L. W. HARRIS and H. I. YAMAMVRA~ Mcdical Research Division, Biomedical Laboratory, Edgewood Arsenal, APG, M D 21010, U.S.A. (Received 25 April 1975. Accepted 14 M a y 1975) Abstract-By assay of acetylcholine hydrolysis to measure total cholinesterase activity and acetyl-Pmcthylcholine hydrolysis to measure acetylcholinesterase (E.C 3.1.1.7) activity, patterns of regeneration of enzyme activity were measured in seven areas of brain, cerebrospinal fluid and plasma of cats after administration of an irreversible inhibitor. Halftimes o f recovery of total cholinesterase in the brain tissues ranged from 0-9 to 3.8 days (av = 2.5 days) and acetylcholinesterase recovery halftimes ranged from 1.2 to 5.3 days (av = 3.6 days). Regeneration of total cholincsterase was also followed in subcellular fractions of guinea-pig and rat brains aftcr similar inhibition. In both species, the fastest recovery occurred in the soluble fraction with halftimes of 1.8 and 1.6 days, while the synaptosomal fractions exhibited the slowest recoveries with halftimes of 8.3 and 4.1 days. Regeneration of activity in plasma and CSF most nearly resembled that of the soluble brain fraction.
ACETYLCHOLINESTERASE (EC 3.1.1-7) plays an important role in the control of normal cerebral activity by hydrolyzing and, thereby, terminating the action of the neurotransmitter, acetylcholine. In brain, AChE occurs in both soluble and membrane-bound forms (HOLLUNGER & NIKLASSON, 1973; WENTHOLD et a/., 1974~).AChE has also been found in cerebrospinal fluid (CSF) (KALSBEEK et al., 1950; SVENSMARK, 1961; JOHNSON & DOMINO,1971; YAKSH et a!., 1974). Neither the origin nor the physiological function of the enzyme in the CSF is known. The CSF, itself, is thought to be elaborated principly from blood plasma through the choroid plexus (DAVSON,1967). It is also known that protein can enter the CSF directly from brain through the extracellular spaces (WELCH,1963; CUTLER et nl., 1970; HOCHWALD, 1970) and it has been suggested that the CSF may serve as a ‘sink‘ to aid in the removal of brain extracellular protein (DAVSON & OLDENDOKF, 1967). It was tempting to speculate that AChE in the CSF might be derived from the soluble enzyme in the brain which had entered the ventricular system via the immediately adjacent extracellular spaces of the brain. A comparison of the turnover rates of the AChE in CSF with that of various brain fractions as well as with plasma might shed some light on the In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care” as promulgated by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research Council. Present address: School of Pharmacy, Pharmacology Section, University of Wisconsin. Madison, WI 53706, U.S.A. Present address: Department of Pharmacology, College of Medicine, University of Arizona, Tucson, A Z 721 85, U SA .
’
contribution of these sources to the AChE in the CSF. The turnover rate of AChE was studied by following recovery of enzyme activity after inhibition by soman (pinacolyl methylphosphonofluoridate). Inhibition by soman i s considered to be irreversible, since significant dephosphorylation of the soman-inhibited enzyme has not been observed (FLEISHER & HARRIS, 1965; COULT& MARSH,1966; HOVANEC e l al., 1974). Because the return of enzyme activity, after inhibition by soman, is thought to be by de nouo synthesis (DOMSCHKE et a/., 1970; HARRIS et a/., 1971; FILBERT et al., 1972a,b),measuring the reappearance of enzyme activity should give an approximation of the turnover rate of AChE in tissue. The return of enzyme activity in these studies was followed by measuring the rate of hydrolysis of acetylcholine (ACh) as well as that of acetyl-P-methylcholine (MeCh). The activity measured by the hydrolysis of ACh has been designated total cholinesterase activity and that activity observed with MeCh designated A C E . MATERIALS AND METHODS Materials All reagents used were of analytical or spectral grade. Special chemicals: Soman of 95% purity was obtained from the Chemical Process Labs, Edgewood Arsenal, MD; [t -L4C]acetylcholine iodide (2.4 Ci/m-mol) and [I -14C] acetyl-fi-methylcholine iodide (4.2 mCi/m-mol) were purchased from New England Nuclcar Corp., Boston, MA.; Amberlite CG-120 resin, sodium form, 200-400 mesh was a product of Mallinckrodt Co., St. Louis, MO; naphthalene was obtained from Eastman Organic Chemicals, Rochester, NY; 2,5-diphenyloxazole (PPO) and 1,4 bis-2-(5 pheny1oxazolyl)-benzene (POPOP) were purchased from Packard Instruments Co., Downers Grove, IL; Lubrol WX was obtained from I.C.I. America, Inc., Charlotte, NC; and BW 284C-51 was a gift from Burroughs Wellcome Company, Tuckahoe, New York. 853
854
L. W. HARRIS and H. I. T. L. YAKSIX,M. G. FILBERT,
Collection of' CSF and plasma To collect CSF, cats of both sexes weighing from 2 . 4 4 5 kg were implanted permanently, while under sodium pentobarbital anesthesia (40 mg/kg), with a cannula so that the tip of the cannula rested just dorsal to the atlantooccipital membrane of the cisterna magna (YAKSHet al., 1973, 1975). After a 10-14-day period of recovery, CSF samples (1.2-1.4ml) were taken at intervals of 24 h by inserting a sterile needle into the cannula and piercing the atlanto-occipital membrane. To reduce variation in enzyme levels due to possible diurnal fluctuation, all sampling was performed between 0900 and loo0 h. Cerebrospinal fluid was then obtained by outflow into a length of PE-90 tubing, previously filled with saline. Blood was also collected at this time from the brachial vein, using a sterile heparinized syringe. All samples were then centrifuged at lo00 g for 10min at 4°C. CSF samples having a noticeable collection of red cells following centrifugation were rare, but were discarded when found. Repeated appearance of blood in the CSF was cause to terminate the experiments in the animal. The plasma was separated from the red blood cells. Assays were performed on aliquots of pure CSF while plasma was diluted 1 :3 with distilled water for enzyme assays and 1 : 100 for protein measurements. Preparation of brain homogenates and subcellular >actions
Cats were killed with sodium pentobarbital and the brains were perfused in situ before death with 0.9% sodium chloride for 3-5 min or until clear of blood, after which the brain was removed and rinsed in cold saline. After bisecting the brain in a sagittal plane, tissue samples were taken from the hypothalamus, thalamus, caudate nucleus, cerebral cortex, cerebellar cortex, pons and lower brainstem. Homogenates of these brain regions were prepared immediately after dissection and weighing. Samples were ground in a glass homogenizer with a Teflon pestle in 10 vol of an ice-cold buffered solution consisting of 0.1 Msodium phosphate buffer at ph 7.8, 0.3 M-NaCl and 1% Lubrol-WX. These homogenates were then diluted to 1% with additional volumes of the same buffer. Protein and enzyme measurements were performed with the 1% homogenates. Subcellular fractions of guinea-pig brainstem and rat whole brain were prepared by differential and sucrosedensity gradient centrifugation according .to the procedure described by WHITTAKER (1969). All procedures were carried out at O"4"C or in a refrigerated centrifuge at 2°C. Ten per cent homogenates (w/v) were prepared in 0.4 M-sucrose with a tightly fitting all-glass tissue grinder. The homogenates were separated into crude nuclear (P,), crude mitochondrial (P2).microsomal (P,) and supernatant (S,) fractions. Fraction Pzwas resuspended in 3 ml of 0 4 M-SUCTOSe and layered on a gradient of 4 ml of 0.8 M-SUCTOSe over 10 ml of 1.2 M-sucrose. The preparation was centrifuged in the SW-25.3 rotor in the Beckman L-2 ultracentrifuge at 78,700 g for 60 min. The layer forming at the interface between 0.8 M and 1.2 M-SUCTOSe corresponding to the nerve ending particle (NEP) fraction of GRAY& WHITTAKER (1962) was aspirated, diluted with 20 vol of 0.25 M-sucrose, and recentrifuged at 150,000 g for 60 min. The supernatant above fraction Pz was centrifuged at 105,o(Io g for 120 min to yield P,, the microsomal pellet, and S,, the supernatant fraction. P, and the NEP's were resus-
'
Biomedical Laboratory, Edgewood Arsenal, Aberdeen Proving Ground, Maryland.
YAh4AMURA
pended in 0.25 M-SucrOSe. Aliquots of the original whole homogenate, of P, and NEP resuspensions and of the SOL uble fraction (S,) were assayed for AChE activity and for protein content. Electron micrographs of the subcellular fractions were prepared and examined by Dr. John P. Petrali.' Synaptosomes were the major component In the NEP fraction with occasional synaptosomal ghosts, membrane fragments and small mitochondria. Enzyme assays The radiometric assay described by SWKOTOS ef al. (1969) was used to measure enzyme activity. [l-'4C]acetylcholine was used as substrate for measuring total cholinesterase activity and [l-'4C]acetyl-~-methylcholinefor acetylcholinesterase activity. A 0.1 ml aliquot of 1% brain homogenate and 0.1 ml of 0.1 M-sodium phosphate buffer, pH 7.8, containing 0.3 M-NaCI and 1% Lubrol WX were put into a centrifuge tube and equilibrated at 37°C. One hundred p1 of 3 x M labelled substrate was added and the mixture was incubated for the required period of time at 37°C. Nonenzymatic hydrolysis of the substrate was measured with solution containing 0 1 ml of water instead of tissue. The reaction was stopped by adding 5 ml of a dioxane-resin suspension (SO g Amberlite CG-I 20 in 250 ml dioxane) to each tube. This was diluted to 10 ml with dioxane, mixed thoroughly and centrifuged at 900 g for 1 min. Five ml of the clear supernatant was transferred to a scintillation vial and 10 ml of a modified 'cocktail' (BRAY,1960) was added. The [14C]hydrolysis product was counted at 80% efficiency in a Packard scintillation spectrometer (Model 3375). Standard curves were prepared and c.p.m. were converted to pmol of substrate hydrolysed. Protein concentration was measured using the method of ~ W R etY a[. (1951) with crystalline bovine serum albumin as the standard. All assays for protein and for enzyme activity were performed in duplicate and the results reported here are expressed as the mean of each pair. Inhibition of enzyme activiry
Soman was prepared immediately before use each day at a concentration of 27 pg/ml in ice-cold saline. All cats, including control animals, were pretreated with the peripheral, antimuscarinic compound atropine methylnitrate (0.5 mg/kg, i.m.) to reduce bronchial secretions. One hour later, soman (27 p@g) or an equal volume of saline (control) was injected subcutaneously into a shaved region on the back between the scapulae. When atropine methylnitrate is given 10 min or more before soman, this dose of agent is non-lethal. In other experiments designed to examine the rate of recovery following soman inhibition in subcellular fractions of brain from guinea-pigs and rats, atropine sulfate (16 mg/kg) was injected i.m. and followed 5 min later by soman (32 pg/kg, s.c.) in guinea-pigs and (120 pg/kg, s.c.) in rats. These doses are non-lethal when the animals are pretreated with atropine sulfate. The animals were killed by decapitation at varying time intervals. The brains were removed, quickly weighed, rinsed and prepared as described above. RESULTS
Distribution of enzyme activity The specific activities of AChE and total ChE (pmol/h per mg) in CSF and plasma obtained from
AChE turnover in brain, CSF, and plasma
TABLE 1.
SPECIFIC ACTIVITY* OF
ChE
AND
AChE
FOR
CSF,
855
PLASMA AND VARIOIJS BRAIN PARTS OF THE CAT
Brain parts1 Enzyme
Caudate CSFt Plasmat nucleus
Thalamus
Cerebellar cortex
Cerebral cortex
ChE
8.81 0.69 I 1 . 2 k0.08
AChE
1.11 007 + 0 0 7 ,0.01
Hypothalamus Pons
Brainstem
Whole brain
5.73 (0.41)
1.59 (0.32)
4-89 (022)
0.63 (0.11)
1.20 (013)
1.80 (018)
1.60 (0.21)
2.78 (0.31)
2.01 (0.68)
0.39 (003)
1.1I (0.10)
015 (005)
0.27 . 0.33 (009) (0.08)
0.26 (0.06)
0.64 (0.05)
035
025
023
024
0.23
0.16
023
AChE ChE
0.13
0.10
0.18
* Specific activity expressed as pmol/h per mg protein. t Data for CSF and plasma levels obtained from samples taken concurrently in 5 cats. Variation expressed as S.E.M. 1Brain parts were obtained from 2 cats. The mean of both values is shown with the range in parenthesis. 5 cats as well as enzyme levels of various parts of the brain obtained from 2 cats are given in Table 1. Total ChE activity was highest in the caudate nucleus (5-73pmol/h per mg) and the lowest in the cerebral cortex (063 pmol/h per mg). Examination of AChE activity in the brain tissues indicated that the highest levels also were in the caudate nucleus (2.01 pmollh per mg) followed by the cerebellar cortex (1.11 pmol/h per mg) and the lowest activity was in the cerebral cortex (015 pmol/h per mg). The ratio of hydrolysis of [ l-l4C]MeCh to [1-14C]ACh. reflecting the relative concentration of true to total activity (AChE:ChE), ranged from 0.35 in the caudate nucleus to 0.16 in the brain stem. In spite of these extremes, most tissue ratios. including that for the whole brain homogenale. fell between 023 and 0.25. Since AChE TABL~: 2. SPECIFIC ACTIVITY* IN CSF AND PLASMA OF AChE AND ChE EXPREFSED IN TFRMS OF VOLUMt OF FLUID? Enzyme AChE ChE
CSF
Plasma
0.40 k 0.03 3.51 i 023
3.53 t- 0.32 34.55 f 3.11
* Specific activity expressed as pmol/h
t Data represent TABLE 3. CONTROL
mean values LEVELS OF
S.E.M.
per ml. from 5 cats
hydrolyzes ACh at 10-3Mthree tinics faster than it , or true to total hydrolyzes MeCh at 1 0 - 3 ~the (AChE:ChE) will be 0.3 when only ~lictrue enzyme is present and will decrease as the proportion of pseudo or butyryl cholinesterase increases (SwKoros et al., 1969). The level of total ChE in CSF was 13 times higher than that found in plasma (8.81 vs 069 p o l / h per mg; P < 0.01) while the activity of AChE in CSF was 16 times that found in plasma (1.1 1 vs 007 pmol/h per mg; P < 010). Further, when all of the ratios (AChE:ChE) are arranged according to magnitude, it is apparent that the ratios in brain uniformly exceed those found in both plasma and CSF, indicating that the levels of AChE as compared to total ChE in brain tissue are higher than those 'in the 2 fluids. The values for specific activity are expressed in terms of mg of protein to allow comparisons with the brain homogenates. It must be remembered, however, that the plasma contains about 125 times as much protein (50 mg/ml) (YAKSHef al., 1973) as the CSF (04 mg/ml) (YAKSHet al., 1974). The absolute levels of cholinesterase activity are thus not reflected. Table 2 presents CSF and plasma specific activities for total ChE and AChE expressed in terms of pmol/h per ml.
AChE
AND ChE ACTIVITItS* AND PROTEIN CONCENTRATION? OBSERVED DALLY FOR IN ClSTERNAL CEREBROSPiNAL FLUIDS FROM THE CAT
14 DAYS
-
Day CSF Component
1
2
3
4
5
6
7
8
9 1 0 1 1 1 2 1 3 1 4
AChE
0.41 0.44 0.39 041 0.39 042 0.42 0.38 0.41 0.47 0-40 0.39 042 040 (004) (0.05) (OW) (0.04) (0.03) (0.05) (0.03) (0.06) (0.05) (008) (004) (005) (OW) (008)
ChE
3.6 (0.6)
Protein
0.32 0.38 0.36 033 038 031 0.39 0.38 0.33 0.33 0.32 032 034 035 (0.06) (0.05) (004) (008) (0.06) (0.08) (0.02) (0.03) (0.03) (0.04) (0.04) (006) (0.05) (004)
* AChE
3.5 (04)
3.5 3.2 (0.4) (0.2)
3.1 3.7 3.1 3.6 (0.3) (0.2) (0.3) (0.6)
3.1 3.2 3.6 (0.6) (0.4) (08)
33 3.3 (0.3) (03)
3.4 (03)
and ChE specific activities expressed as pmol/h/ml i- S.E.M. expressed as mg/ml. ml) were taken between 0900 and I000 hs each day from 4 cats. The values shown are
t Protein concentration 1 CSF samples (1-2-1.4 the mean k S.E.M.
~ A R R I Sand
T. L. YAKSH,M. G . FILBERT, L. W
856
0
2
H. I. YAMAMURA
A
6
8
1
0
1
2
DAYS AFTER INHIBITION
FIG. 1. Recovery of enzyme activity given as the log-% inhibition of AChE and ChE activity in CSF and plasma as a function of time in days after the administration of soman (27pg/kg, s.c.). Values represent the mean enzyme activity at each time period of the same 4 cats. Sequential CSF samples were taken as described in text.
Control levels of AChE, total ChE activity and protein for the CSF are presented in Table 3. These values were found in 4 cats over a period of 14 consecutive days. As can be seen, despite the daily withdrawal of samples of CSF, the enzyme levels remained stable. The mean and standard error for AChE and total ChE activity was 041 k 0.05 and 3.4 0.4 ,umol/h per ml, respectively. Likewise, the protein levels showed no significant fluctuation.
Enzyme recovery in CSF and plasma following soman inhibition After the injection of soman (27 & k g ) CSF-AChE specific activity fell to 5% of preinjection levels during the first hour from 1.11 k 0.07 to 0.064 0.01 pmol/ h per mg. Similarly, total ChE activity was reduced 96.4%,from 8.81 k 1-2 to 029 k 0.05 pnol/h per mg. CSF samples from control and poisoned animals were taken at 0.5 h, and then on 1,2,3,4,5,7 and 13 days after drug administration. The results of the assays are given in Fig. 1. The best fit linear regression lines were calculated using the least squares method for each set of data and the time required for the recovery of 50% of the original specific activity (t1,2)r was obtained using the derived regression equations TABLE 4.
CALCULATED
rliZ DAYS OF
RECOVERY OF
(Table 4). Statistical comparisons of the slopes of the regression lines were made employing the F ratio values derived from the regression data (WINER, 1962). As seen in Fig. 1 and Table 4, the tljz for AChE recovery in CSF was 1.4 days as compared to 2.1 days for total ChE activity. Comparison of the two slopes reveals this difference not to be statistically different ( P > 0.10). As reported elsewhere by YAKSHet al. (1974), mean CSF protein levels do not change significantly following soman treatment. In the present experiments, statistical comparisons of the protein levels before and after administration of soman, using a one-way analysis of variance for repeated measures, showed no significant alteration in cisternal CSF protein concentrations (P > 0.10). Venous blood samples were taken concurrently with the cisternal drains and AChE and total ChE specific activities were measured. As with CSF, AChE and total ChE activity in the plasma was significantly inhibited during the first hour after soman (94.9 and 95.6% respectively). The recovery of enzyme activity in plasma also is shown in Fig. 1. Like CSF, there was no significant difference in the slopes of the AChE and total ChE recovery curves (P > 010 with the tl,2 being 1.8 and 2.2 days, respectively (Table
AChE AND
TOTAL IN BRAIN,
CSF
AND PLASMA IN THE CAT*
Brain parts Enzyme
Caudate Hypothalamus ~~~
AChE ChE
2.54 1.18
Thalamus
Cerebral cortex
Cerebellar cortex
Brainstem
Pons
CSF
Plasma
2.09 1 -03
5.34 3 85
4-49 2-85
4.68 3.79
4.63 3.85
1.37 209
1.78 2.18
~
1.15 0.92
* The t I j zvalues were derived from the least square equations used to fit the data presented in Figs. 1-4. Consult text for further details.
AChE turnover in brain, CSF, and plasma
857
4). When CSF and blood samples obtained from soman treated animals were mixed with aliquots of control tissue, no inhibition of enzyme activity occurred in the control sample. This was taken to indicate the absence of free soman in these fluids at 1 h after treatment. Enzyme recovery in brain following soman inhibition To examine the rate of recovery of brain AChE > t and total ChE activities after inhibition by soman, U cats were given 27 pgkg and 1 each was killed at 1 h and then at 1,2,3, or 5 days later. In general, b Z Broinrlem Pons the maximum inhibition observed in brain tissue was somewhat less than that observed in CSF or plasma. The activity in whole brain homogenate, for example, fell 92%, from 2.78 to 0.22 pmol/h per mg. To be certain that the 1-h sample did, indeed, represent the maximum level of inhibition, 18 guinea-pigs were given soman (27 ,ug/kg, s.c.) and killed at 15, 30 or 60 min after the injection and the brains assayed for enzyme activity. The greatest inhibition occurred within the first 30 min and no change from this level was noted during the first hour. Thus, the specific activity of the enzyme at 1 h was taken to represent 0 2 4 6 0 2 A 6 thc maximum degree of inhibition. Further, at 1 h DAYS AFTER INHIBITION no free soman remained in brain homogenates as FIG. 3. Recovery of enzyme activity given as the log-”/, judged by the absence of inhibition when samples inhibition of AChE and ChE activity in cerebral cortex, from soman-treated animals were incubated with cerebellar cortex, brainstem and pons at several time perequal volumes of homogenates obtained from control iods following the administration of soman (27 &kg, s.c.). animals. Figure 2 shows the log percentage of inhibi- Each value represents the data obtained from one animal killed at the time. tion in the caudate nucleus, hypothalamus and thalamus at various times after soman injection. Figure 3 presents the recovery of enzyme activities in the respectively, whereas the longest period of recovery cerebral cortex and cerebellar cortex as well as the was observed in the cerebral cortex, brainstem, pons recovery in the pons and brainstem. The most rapid and cerebellar cortex, with tIi,’s between 4.5 and 5.3 recovery of AChE activity was observed in the days. The t1,2 for the total ChE recovery varied in hypothalamus (1.2 days) followed by the thalamus a similar manner from 0.9 days in the hypothalamus and caudate, which had t,,, rates of 2.1 and 2 3 days to 3.9 days in the pons and cerebral cortex. No strict correlation was observed between the recovery rate Hypoiholamur Ilrolonur -Coudole - - - - -N-------of enzyme specific activity or AChE to ChE ratio.
z
_______-^__
Recovery of total cholinesterase activity in subcellular fraction of brain tissue Figure 4 shows the log percentage of inhibition of AChE in guinea-pig brainstem subcellular fractions after soman intoxication. Table 5 presents the specific activities of the various fractions. As can he seen, the highest specific activity was observed in the microsoma1 and synaptosomal fractions (I 1.76 and 4.04 pmol/ h per mg, respectively) with the lowest in the soluble fraction (2.93 ,umol/h per mg). Corresponding half times of recovery were calculated using the equations 0 7 1 b 0 7 d 6 derived from the least squares analysis used to plot D A Y S AFTER INHIB’TION the data in Fig. 4. The shortest times were observed FIG. 2. Recovery of enzyme activity given as the log-% in the soluble fraction (1.8 days), the longest in the inhibition of AChE and ChE activity in caudate nucleus, synaptosomes (8.3 days) and intermediately in the thalamus, and hypothalamus at several time periods folmicrosomes (3.4 days). Total ChE levels in the rat lowing the administration of soman (27 pg/kg, s.c.). Each value represents the data obtained from 1 animal killed brain fractions are presented in Table 6. Distribution at that time. Samplcs were prepared and enzyme activity of enzyme activity in the subcellular fractions is identical with that observed in guinea-pig with the highest determined as described in the text.
T. L. YAKSH, M. G. FILBERT, L. W. HARRIS and H. I. YAMAMURA
858
DISCUSSION
D A Y S AFTER INHIBITION
FIG. 4. Recovery of enzyme activity given as the log-% inhibition of total ChE activity in the subcellular fractions of guinea-pig brainstem observed at several time periods following the administration of soman (32 pg/kg, s.c.). Each data point represents the results obtained from one animal after soman intoxication. Fractions were prepared as described in the text.
activity found in the microsomes, less in the synaptosoma1 fraction and least in the soluble fraction (15.18, 744 and 3.01 pmol/h per mg respectively). Again the fastest return of enzyme activity was found in the soluble fraction with a tl,, (1.6 days) similar to that found in the guinea-pig, and the slowest recovery also occurred in the synaptosomal fraction (flj2 = 4.1 days). TABLE 5. ChE ACTIVITY IN GUINEA-PIG BRAIN SUBCELLULAR FRACTIONS AND CALCULATED HALFTIME OF ChE RECOVERY FOLLOWING SOMAN ChE Specific activityt
Fraction Whole brain homogenate Soluble fraction Microsomes Synaptosomes
4.84 2.93 I 1.76
4.04
(Days)
t,/z*
4.7 14 3.4 8.3
* The tIiz derived from regression equations by the method of least squares. Consult text for further details. t Specific activity expressed as pmol/h per mg protein. TABLE 6. ChE
ACTIVITY IN RAT BRAIN SUBCELLULAR FRACTIONS AND CALCULATED SOMAN
Fraction Whole homogenate Soluble fraction Microsomes Synaptosomes
* Specific activity,
Control ChE activity* 5.57 (4.58-6.50) 3.01 (2.70-3.3 I ) 15.18 (1 3.38-16.88) 7.44 (489-9.99)
HALF-TIMF
OF RFCOVERY FOLLOWING
"/, Inhibitiont 0.5 h after soman 83.3 (91.1-75-4) 82.9 (90475.3) 82.6 (89.2-76.1) 81-8 (92671.0)
mean and (95% confidence limits), expressed as pmollh per mg.
t % inhibition, mean and (95% confidence limits). Days.
Whether soman can be used as an inhibitor for studying the rate of synthesis of AChE or total ChE in animals depends upon three assumptions: (1) that the inhibition is irreversible; ( 2 ) that excess, free inhibitor is not present to combine with newly formed enzyme; (3) soman has no direct effect on protein synthesis. We believe that these assumptions are justified for the following reasons. (1) Inhibition of cholinesterases by soman is due to phosphonylation of the active site serine (SCHAFFERet at., 1973). The soman+nzyme complex then ages, i.e. it loses the pinacolyl group to form a product that is not subject to reactivation with oximes (BERRY& DAVIS,1966). The f,,, for aging of soman-AChE complex in monkey brain is 1 min (FILBERTet al., 1972) and is 2.4 min in bovine erythrocytes (FLEISHER & HARRIS, 1965). In addition, FLEISHER & HARRIS(1965) have shown that there was no loss of the residual methylphosphonate from the aged enzyme in vivo up to 72 h. (2) The absence of free soman in tissues was verified by incubation of aliquots of tissues from poisoned animals with aliquots taken from tissues of normal animals. The enzyme in tissues from normal animals was not inhibited under these conditions. (3) Whether soman has no direct influence on protein synthesis is more difficult to assess. HARRIS et d.(1974) examined the effects of soman on the rate of synthesis of AChE as well as the incorporation of ~-[U-'~C]leucine into protein and the uptake of C3H]methylthymidine by DNA in rabbit bone marrow cells which were in an active state of erythropoiesis. They found no differences between normal and soman treated cells even though the AChE had been reduced to less than 5% of the control levels. In another study by HARRIS(1975) there was also no difference between control and soman poisoned animals in the ~-[U-'~C]leucineincorporation into protein by the guinea-pig retina. On the basis of these findings, those investigators concluded that soman has no effect on the overall protein synthesis. The specific activity of AChE in the present experiments for normal cat whole brain (0.64 pmol/h per et a!. (1969) mg) is lower than that found by SIAKOTOS in the mouse, rat, guinea-pig and rabbit (1.38-2.20 pmol/h per mg) but is similar to that found by them in the bovine (0.47 pmol/h per mg). Examination of
24 h after soman 6 6 9 (71.1-62.7) 60.4 (63.9-57'6) 65.9 (69.2-62.2) 73.5 (80.1-66.8)
t,,,:
2.3 1.6 2.2 4.1
AChE turnover in brain, CSF, and plasma
859
the specific activity of AChE in different brain areas the values obtained for the other species reported here indicates that as in cats, rats and dogs (CLOUET & and this uniformity of recovery rates across species, WAELSCH, 1961; KLINGet al., 1969; QUAYet al., 1971; we feel, substantiates the existence of a rapid rate of THOMASet al., 1971) the highest levels are to be found synthesis for this essential enzyme by brain tissue. In in the caudate nucleus while the lowest are generally general, protein turnover in brain has been thought observed in the cerebral cortex. Despite the fact that to be somewhat longer (1C-20 days) (VON HUNGEN species variation exists with respect to specific acti- e t p l . , 1968; LAJTHAet al., 1971). AUSTINet al. (1972) vity, there is similarity in the ratios of AChE to total found an apparent mean halftime of 3.2 days for proChE (i.e. hydrolysis of MeCh: ACh). For example, tein turnover in rat whole brain as well as the existvalues ranging from 0.2 in mouse whole brain homo- ence of a protein fraction with a turnovcr time of genates to 0.3 for rabbit have been found (SIAKOTOSless than 2 days. The present data suggest that AChE et al., 1969). Similarly, ratios of AChE to total ChE would fall within this rapidly changing protein fracfor different topographical regions generally fall in tion. this range although the caudate nucleus typically has The source of AChE in the CSF has not been identhe highest ratio. tified. Since the recovery of AChE after inhibition by AChE activity in CSF fluid obtained from the cis- soman is presumed to be by de novo synthesis and terna magna of normal cats is 6 times that found there is no synthetic capability within the CSF, the in human lumbar CSF by JOHNSON& DOMiNO (1970). return of the enzyme must be associated with moveProtein concentrations in the cat cisternal CSF ment of newly synthesized protein into the CSF. It reported here were also slightly higher than values has been established that while the brain enzyme is reported by CUTLEK (1970), but are identical to values mostly bound to membranes, a significant amount reported by HOCHWALD (1970) and previously found exists in a soluble form (HOLLUNGER et al., 1973; by these laboratories (YAKSHet al., 1973, 1974). Since WENTHOLD et ul., 1974~)and this soluble enzyme there was no change in either protein concentration could be the source of AChE in the CSF. It is signifior AChE when fluid was removed daily for 14 con- cant, therefore, that while the recovery rates of the secutive days, we have concluded that repeated with- AChE in the subcellular fractions differ markedly, drawal of CSF does not lead to the formation of a without exception, the most rapid recovery was more dilute CSF nor to excess protein from extra observed in the soluble fraction (AUSTIN & JAMES, et al. (1972a,b; present study). FurtherCSF sources. This is not unexpected. in as much as 1970; FILBERT the rate of formation of CSF in the cat is about 30 more, this more rapid rate of recovery in the soluble fraction coincides with thc rate of recovery in the ml/day (HOCHWALD, 1970). In the present studies, we observed that the t1,2 CSF. In spite of the strong correlation which exists for regeneration of AChE in brain tissues after irreversible inhibition by soman ranged from 1.2 days between the turnover rates of the brain soluble in the hypothalamus to 5.3 days in the cerebral cortex enzyme and CSF enzyme, the possibility that some with a mean halftime for the seven areas examined AChE in the CSF derives from plasma cannot be of 3-6 days. The halftime of recovery in the rat whole excluded. Small amounts of plasma protein may pass brain was 2.3 days and in the guinea-pig brainstem, through capillary walls and, thereby, gain access to 4.7 days. These regeneration rates are comparable to the brain extracellular space which is in direct com& DAVSON, the t1,2 for recovery of AChE in monkey brain after munication with the CSF (OLDENDORF 1970). Human and horse plasmas soman poisoning (2.5 days) observed by FILBERT et 1967; HOCHWALD, 1948). al. (1972b). From the data of GLOWet ul. (1966) we contain essentially no AChE (ALJGUSTINSSON, have calculated halftimes of 7-11 days for recovery However, cat and rat plasma do show significant of AChE in various parts of rat brain after inhibition AChE activity, a fact that has been established by by diisopropylphosphorofluoridate(DFP) and a t1,2 the use of specific substrates and specific inhibitors. et al. (1969) have shown that rat plasma of 16.5 days from the data of Ausrw & JAMES(1970) SIAKOTOS who also used DFP to measure regeneration rates still hydrolyzes MeCh at about 80% of the control of AChE in rat cerebral cortex. The reason for the rate in the presence of IO-jM-iso-OMPA, a specific longer recovery times after DFP inhibition is not inhibitor of pseudocholinesterase. Similarly, FILBERT, MOORE& YAKSH(manuscript in preparation) clear at this time, especially in the light of the findings H~RKIS, et al. (19746) who determined AChE have shown that the hydrolysis of ACh by cat plasma of WENTHOLD turnover in rat cerebral cortex by measuring the time is reduced 80% by 1 0 - 5 ~ - B W284C-51 a specific course of disappearance of radiolabel introduced inhibitor for AChE. Whether a significant quantity of AChE can enter into the enzyme by intraventricular injection of ~-[1-' 4C]leucine. Assuming steady-state conditions the CSF from the plasma via the capillary wall is where the rate of degradation of protein equals the not known. It should be pointed out, however,, that & MARKS,1971), they deter- there appears to be a substantial delay in passage rate of synthesis (LAJTHA (1970), mined the tL,2to be 2.8 days. This value for AChE of protein from plasma to CSF. HOCHWALD turnover agrees well with the tl,2 for resynthesis studying the movement of 1311-labelledalbumin and found by us in the rat. It also correlates well with ' 'I-labelled y-globulin, observed that it took between
860
L. W. HARRISand H. I. YAMAMURA T. L. YAKSH,M. G. FILBERT,
HARRISL. W. (1975) M.S. Thesis, Towson State College, Towson, Maryland. HOCHWALD G. M. (1970) J. neurol. SC~S10. 269-278. HoLLUNGER E. G . & NlKLASSON B. H. (1973) J . Neurochem. 20. 821-826. HOVANEC J. H., LIESKEC. N., ROWLAND S. L. & LANKS K. (1974) Abstracts, Priestly Bicentennial Middle Atlantic Regional Meeting ofthe American Chemical Society, Wilkes-Barre, Pennsylvania. D. 41. JOHNSONS. & D o ~ m bG. F.*(1971) Cfinica chim. Acta AckriowlidgementsThe authors wish to thank L. A. 35. 421428. FEDELE for his excellent assistance and Dr. C. A. BROOMKALSBEEK F., &HEN J. A. & BOVENS B. R. (1950) Biochim. FIELD, Mr. K. M. WILSON and Capt. J. R. LOWEfor helpful biophys. Acia 5. 548-560. commentary on the manuscript. KLINGA,, FINERS. & GILMOUR J. (1969) I n t . 1. Neuropharmac. 8. 2531. REFERENCES LAJTHAA. & MARKSN. (1971) in Handhook of Neurochemistry (LAITHAA., ed.) Vol. 5, pp. 551-629. Plenum AUGUSTINSWNK. B. (1948) Acta physiol. scand. 15. Suppl. Press, New York. 52. 1-182. 0. N., ROSEBROUGH N. J., FARR A. L. & RANDALL AUSTINL. & JAMESK. A. C. (1970) J. Neurochem. 17, 705- LOWRY 707. R. J. (1951) 1.biol. Chem. 193. 265-267. AUSTIN L., LOWRY0. H., BROWNJ. G. & CARTERJ. G. OLDENWRP W. H. & DAVSONH. (1967) Archs Neurol. Psy(1972) Biochern. J. 126. 351-359. chiat. Chicago 17. 196-205. QUAYW. B., BENNETE. L., MORIMOTO BERRYW. K. & DAVISD. R. (1966) Biochem. J. 100. 572H. & HERBERT 576. M. (1971) Comp. gen. Pharmac. 2. 402410. BRAYG. A. (1960) Analyt. Biochem. 1. 279-285. SCHAFFER N. K., MICHELH. 0. & BRIDGES A. F. (1973) CLOUET D. H. & WAELSCH H. (1961) J . Neurochem. 8. 201Biochem., Easton 12. 29462950. 215. SIAKOTOS A. N., FILBERT M. G. & HESTERR. (1969) BioCOULTD. B. & MARSHD. J. (1966) Biochem. J. 98. 869chem. M e d . 3. 1-12. 873. SVENSMARK 0. (1961) Actaphysiol. scand. 52. 372-37s. J. E. & CORNICK L. R. (1970) THOMAS CUTLERR. W. P., MURRAY N., FLEISHER J. H. & HARRIS L. W. (1971) Biochim. E x p f . Neurol. 28. 257-265. biophys. Acta 235. 542-547. DAVSQNH. (1967) Physiology of the Cerebrospinal Fluid VON HUNGENK., MAHLERH. R. & MOOREW. J. (1968) pp. 132-1 33. Churchill, London. J . biol. Chem. 243. 1415-1423 DAVSON H. & OLDENWRF W. H. (1967) Proc. R. SOC.Med. WELCHK. (1963) Am. J . Physiol. 205. 617-624. 60. 32C329. WENTHOLD R. .I. MAHLER , H. R. & MOORE W. J. (1974a) DoMsCHKE w., DOMAGK G. F., DOMSCHKE s. & ERDMANN J. Neurochem. 22. 945-949. W. D. (1970) Arch. Tox 26. 142-148. WENTHOLD R. J., MAHLERH. R. & MOORE W. J. (1974b) FILBERT M. G., LOCHNERM. A., YAMAMLJRA H. I., HARRIS J. Neurochem. 22. 941-943. L. W. & FLEISHER J. H. (1972n) Frdn Proc. Fedn Am. WHIITAKER V. P. (1969) in Handbook of Neurochemistry Socs. rxp. Biol. 31. 515. (LAJTHAA., ed.) Vol. 2, pp. 329-364. Plenum Press, New FILBERT M. G., LQCHNER M. A. & FLEISHER J. H. (1972b) York. Biochim. biophys. Acta 264. 164-174. WINERB. J. (1962) Statistical Principles in Experimental FLEISHER J. H. & HARRISL. W. (1965) Biochem. Pharmac. Design. McGraw-Hill, New York. 14. 641-650. YAKSHT. L., FEDELE L. A. & YAMAMURA H. I. (1973) PhyGLOWP. I., ROSE S . & RICHARDSONA. (1966) Aust. J . swl. Behau. 10. 149-151. exp. Biol. med. Sci. 44. 73-86. YAKSH T. L., FEDELE L. A. & YAMAMURAH. I. (1974) G R A Y E. J. & WHIlTAKER V. P. (1962) J. Anat. 96. 79-88. Experientia 30. 38-39. HARRISL. W., YAMAMURAH. I. & FLEISHER J. H. (1971) YAKSHT. L., YAMAMURA H. I. & RUDYT. A. (1975) EdgeBiochem. Pharmac 20. 2927-2930. wood Arsenal Special Publication EB-SP-74031. Aberdeen V. F. & MOORER. D. (1974) BioHARRISL. W., GARRY Proving Ground, Maryland. chem. Pharmac. 23. 21552163.
8 and 16 h for the brain to plasma ratio of radioactivity to approach unity. If plasma protein were contributjng sjmjfiantly to the enzyme levels observed in CSF during recovery, we would expat the r1,2 in CSF t o exceed the t I i z in plasma, reflecting the time required for movement between the The i.e. the t l / 2 appears to be the case, for the CSF is less than the tl,* for plasma.
ACETYLCHOLINESTERASE TURNOVER IN BRAIN, CEREBROSPINAL FLUID AND PLASMA T. L. YAKSH,’M. G. FILWRT,L. W. HARRIS and H. I. YAMAMVRA~ Mcdical Research Division, Biomedical Laboratory, Edgewood Arsenal, APG, M D 21010, U.S.A. (Received 25 April 1975. Accepted 14 M a y 1975) Abstract-By assay of acetylcholine hydrolysis to measure total cholinesterase activity and acetyl-Pmcthylcholine hydrolysis to measure acetylcholinesterase (E.C 3.1.1.7) activity, patterns of regeneration of enzyme activity were measured in seven areas of brain, cerebrospinal fluid and plasma of cats after administration of an irreversible inhibitor. Halftimes o f recovery of total cholinesterase in the brain tissues ranged from 0-9 to 3.8 days (av = 2.5 days) and acetylcholinesterase recovery halftimes ranged from 1.2 to 5.3 days (av = 3.6 days). Regeneration of total cholincsterase was also followed in subcellular fractions of guinea-pig and rat brains aftcr similar inhibition. In both species, the fastest recovery occurred in the soluble fraction with halftimes of 1.8 and 1.6 days, while the synaptosomal fractions exhibited the slowest recoveries with halftimes of 8.3 and 4.1 days. Regeneration of activity in plasma and CSF most nearly resembled that of the soluble brain fraction.
ACETYLCHOLINESTERASE (EC 3.1.1-7) plays an important role in the control of normal cerebral activity by hydrolyzing and, thereby, terminating the action of the neurotransmitter, acetylcholine. In brain, AChE occurs in both soluble and membrane-bound forms (HOLLUNGER & NIKLASSON, 1973; WENTHOLD et a/., 1974~).AChE has also been found in cerebrospinal fluid (CSF) (KALSBEEK et al., 1950; SVENSMARK, 1961; JOHNSON & DOMINO,1971; YAKSH et a!., 1974). Neither the origin nor the physiological function of the enzyme in the CSF is known. The CSF, itself, is thought to be elaborated principly from blood plasma through the choroid plexus (DAVSON,1967). It is also known that protein can enter the CSF directly from brain through the extracellular spaces (WELCH,1963; CUTLER et nl., 1970; HOCHWALD, 1970) and it has been suggested that the CSF may serve as a ‘sink‘ to aid in the removal of brain extracellular protein (DAVSON & OLDENDOKF, 1967). It was tempting to speculate that AChE in the CSF might be derived from the soluble enzyme in the brain which had entered the ventricular system via the immediately adjacent extracellular spaces of the brain. A comparison of the turnover rates of the AChE in CSF with that of various brain fractions as well as with plasma might shed some light on the In conducting the research described in this report, the investigators adhered to the “Guide for Laboratory Animal Facilities and Care” as promulgated by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research Council. Present address: School of Pharmacy, Pharmacology Section, University of Wisconsin. Madison, WI 53706, U.S.A. Present address: Department of Pharmacology, College of Medicine, University of Arizona, Tucson, A Z 721 85, U SA .
’
contribution of these sources to the AChE in the CSF. The turnover rate of AChE was studied by following recovery of enzyme activity after inhibition by soman (pinacolyl methylphosphonofluoridate). Inhibition by soman i s considered to be irreversible, since significant dephosphorylation of the soman-inhibited enzyme has not been observed (FLEISHER & HARRIS, 1965; COULT& MARSH,1966; HOVANEC e l al., 1974). Because the return of enzyme activity, after inhibition by soman, is thought to be by de nouo synthesis (DOMSCHKE et a/., 1970; HARRIS et a/., 1971; FILBERT et al., 1972a,b),measuring the reappearance of enzyme activity should give an approximation of the turnover rate of AChE in tissue. The return of enzyme activity in these studies was followed by measuring the rate of hydrolysis of acetylcholine (ACh) as well as that of acetyl-P-methylcholine (MeCh). The activity measured by the hydrolysis of ACh has been designated total cholinesterase activity and that activity observed with MeCh designated A C E . MATERIALS AND METHODS Materials All reagents used were of analytical or spectral grade. Special chemicals: Soman of 95% purity was obtained from the Chemical Process Labs, Edgewood Arsenal, MD; [t -L4C]acetylcholine iodide (2.4 Ci/m-mol) and [I -14C] acetyl-fi-methylcholine iodide (4.2 mCi/m-mol) were purchased from New England Nuclcar Corp., Boston, MA.; Amberlite CG-120 resin, sodium form, 200-400 mesh was a product of Mallinckrodt Co., St. Louis, MO; naphthalene was obtained from Eastman Organic Chemicals, Rochester, NY; 2,5-diphenyloxazole (PPO) and 1,4 bis-2-(5 pheny1oxazolyl)-benzene (POPOP) were purchased from Packard Instruments Co., Downers Grove, IL; Lubrol WX was obtained from I.C.I. America, Inc., Charlotte, NC; and BW 284C-51 was a gift from Burroughs Wellcome Company, Tuckahoe, New York. 853
854
L. W. HARRIS and H. I. T. L. YAKSIX,M. G. FILBERT,
Collection of' CSF and plasma To collect CSF, cats of both sexes weighing from 2 . 4 4 5 kg were implanted permanently, while under sodium pentobarbital anesthesia (40 mg/kg), with a cannula so that the tip of the cannula rested just dorsal to the atlantooccipital membrane of the cisterna magna (YAKSHet al., 1973, 1975). After a 10-14-day period of recovery, CSF samples (1.2-1.4ml) were taken at intervals of 24 h by inserting a sterile needle into the cannula and piercing the atlanto-occipital membrane. To reduce variation in enzyme levels due to possible diurnal fluctuation, all sampling was performed between 0900 and loo0 h. Cerebrospinal fluid was then obtained by outflow into a length of PE-90 tubing, previously filled with saline. Blood was also collected at this time from the brachial vein, using a sterile heparinized syringe. All samples were then centrifuged at lo00 g for 10min at 4°C. CSF samples having a noticeable collection of red cells following centrifugation were rare, but were discarded when found. Repeated appearance of blood in the CSF was cause to terminate the experiments in the animal. The plasma was separated from the red blood cells. Assays were performed on aliquots of pure CSF while plasma was diluted 1 :3 with distilled water for enzyme assays and 1 : 100 for protein measurements. Preparation of brain homogenates and subcellular >actions
Cats were killed with sodium pentobarbital and the brains were perfused in situ before death with 0.9% sodium chloride for 3-5 min or until clear of blood, after which the brain was removed and rinsed in cold saline. After bisecting the brain in a sagittal plane, tissue samples were taken from the hypothalamus, thalamus, caudate nucleus, cerebral cortex, cerebellar cortex, pons and lower brainstem. Homogenates of these brain regions were prepared immediately after dissection and weighing. Samples were ground in a glass homogenizer with a Teflon pestle in 10 vol of an ice-cold buffered solution consisting of 0.1 Msodium phosphate buffer at ph 7.8, 0.3 M-NaCl and 1% Lubrol-WX. These homogenates were then diluted to 1% with additional volumes of the same buffer. Protein and enzyme measurements were performed with the 1% homogenates. Subcellular fractions of guinea-pig brainstem and rat whole brain were prepared by differential and sucrosedensity gradient centrifugation according .to the procedure described by WHITTAKER (1969). All procedures were carried out at O"4"C or in a refrigerated centrifuge at 2°C. Ten per cent homogenates (w/v) were prepared in 0.4 M-sucrose with a tightly fitting all-glass tissue grinder. The homogenates were separated into crude nuclear (P,), crude mitochondrial (P2).microsomal (P,) and supernatant (S,) fractions. Fraction Pzwas resuspended in 3 ml of 0 4 M-SUCTOSe and layered on a gradient of 4 ml of 0.8 M-SUCTOSe over 10 ml of 1.2 M-sucrose. The preparation was centrifuged in the SW-25.3 rotor in the Beckman L-2 ultracentrifuge at 78,700 g for 60 min. The layer forming at the interface between 0.8 M and 1.2 M-SUCTOSe corresponding to the nerve ending particle (NEP) fraction of GRAY& WHITTAKER (1962) was aspirated, diluted with 20 vol of 0.25 M-sucrose, and recentrifuged at 150,000 g for 60 min. The supernatant above fraction Pz was centrifuged at 105,o(Io g for 120 min to yield P,, the microsomal pellet, and S,, the supernatant fraction. P, and the NEP's were resus-
'
Biomedical Laboratory, Edgewood Arsenal, Aberdeen Proving Ground, Maryland.
YAh4AMURA
pended in 0.25 M-SucrOSe. Aliquots of the original whole homogenate, of P, and NEP resuspensions and of the SOL uble fraction (S,) were assayed for AChE activity and for protein content. Electron micrographs of the subcellular fractions were prepared and examined by Dr. John P. Petrali.' Synaptosomes were the major component In the NEP fraction with occasional synaptosomal ghosts, membrane fragments and small mitochondria. Enzyme assays The radiometric assay described by SWKOTOS ef al. (1969) was used to measure enzyme activity. [l-'4C]acetylcholine was used as substrate for measuring total cholinesterase activity and [l-'4C]acetyl-~-methylcholinefor acetylcholinesterase activity. A 0.1 ml aliquot of 1% brain homogenate and 0.1 ml of 0.1 M-sodium phosphate buffer, pH 7.8, containing 0.3 M-NaCI and 1% Lubrol WX were put into a centrifuge tube and equilibrated at 37°C. One hundred p1 of 3 x M labelled substrate was added and the mixture was incubated for the required period of time at 37°C. Nonenzymatic hydrolysis of the substrate was measured with solution containing 0 1 ml of water instead of tissue. The reaction was stopped by adding 5 ml of a dioxane-resin suspension (SO g Amberlite CG-I 20 in 250 ml dioxane) to each tube. This was diluted to 10 ml with dioxane, mixed thoroughly and centrifuged at 900 g for 1 min. Five ml of the clear supernatant was transferred to a scintillation vial and 10 ml of a modified 'cocktail' (BRAY,1960) was added. The [14C]hydrolysis product was counted at 80% efficiency in a Packard scintillation spectrometer (Model 3375). Standard curves were prepared and c.p.m. were converted to pmol of substrate hydrolysed. Protein concentration was measured using the method of ~ W R etY a[. (1951) with crystalline bovine serum albumin as the standard. All assays for protein and for enzyme activity were performed in duplicate and the results reported here are expressed as the mean of each pair. Inhibition of enzyme activiry
Soman was prepared immediately before use each day at a concentration of 27 pg/ml in ice-cold saline. All cats, including control animals, were pretreated with the peripheral, antimuscarinic compound atropine methylnitrate (0.5 mg/kg, i.m.) to reduce bronchial secretions. One hour later, soman (27 p@g) or an equal volume of saline (control) was injected subcutaneously into a shaved region on the back between the scapulae. When atropine methylnitrate is given 10 min or more before soman, this dose of agent is non-lethal. In other experiments designed to examine the rate of recovery following soman inhibition in subcellular fractions of brain from guinea-pigs and rats, atropine sulfate (16 mg/kg) was injected i.m. and followed 5 min later by soman (32 pg/kg, s.c.) in guinea-pigs and (120 pg/kg, s.c.) in rats. These doses are non-lethal when the animals are pretreated with atropine sulfate. The animals were killed by decapitation at varying time intervals. The brains were removed, quickly weighed, rinsed and prepared as described above. RESULTS
Distribution of enzyme activity The specific activities of AChE and total ChE (pmol/h per mg) in CSF and plasma obtained from
AChE turnover in brain, CSF, and plasma
TABLE 1.
SPECIFIC ACTIVITY* OF
ChE
AND
AChE
FOR
CSF,
855
PLASMA AND VARIOIJS BRAIN PARTS OF THE CAT
Brain parts1 Enzyme
Caudate CSFt Plasmat nucleus
Thalamus
Cerebellar cortex
Cerebral cortex
ChE
8.81 0.69 I 1 . 2 k0.08
AChE
1.11 007 + 0 0 7 ,0.01
Hypothalamus Pons
Brainstem
Whole brain
5.73 (0.41)
1.59 (0.32)
4-89 (022)
0.63 (0.11)
1.20 (013)
1.80 (018)
1.60 (0.21)
2.78 (0.31)
2.01 (0.68)
0.39 (003)
1.1I (0.10)
015 (005)
0.27 . 0.33 (009) (0.08)
0.26 (0.06)
0.64 (0.05)
035
025
023
024
0.23
0.16
023
AChE ChE
0.13
0.10
0.18
* Specific activity expressed as pmol/h per mg protein. t Data for CSF and plasma levels obtained from samples taken concurrently in 5 cats. Variation expressed as S.E.M. 1Brain parts were obtained from 2 cats. The mean of both values is shown with the range in parenthesis. 5 cats as well as enzyme levels of various parts of the brain obtained from 2 cats are given in Table 1. Total ChE activity was highest in the caudate nucleus (5-73pmol/h per mg) and the lowest in the cerebral cortex (063 pmol/h per mg). Examination of AChE activity in the brain tissues indicated that the highest levels also were in the caudate nucleus (2.01 pmollh per mg) followed by the cerebellar cortex (1.11 pmol/h per mg) and the lowest activity was in the cerebral cortex (015 pmol/h per mg). The ratio of hydrolysis of [ l-l4C]MeCh to [1-14C]ACh. reflecting the relative concentration of true to total activity (AChE:ChE), ranged from 0.35 in the caudate nucleus to 0.16 in the brain stem. In spite of these extremes, most tissue ratios. including that for the whole brain homogenale. fell between 023 and 0.25. Since AChE TABL~: 2. SPECIFIC ACTIVITY* IN CSF AND PLASMA OF AChE AND ChE EXPREFSED IN TFRMS OF VOLUMt OF FLUID? Enzyme AChE ChE
CSF
Plasma
0.40 k 0.03 3.51 i 023
3.53 t- 0.32 34.55 f 3.11
* Specific activity expressed as pmol/h
t Data represent TABLE 3. CONTROL
mean values LEVELS OF
S.E.M.
per ml. from 5 cats
hydrolyzes ACh at 10-3Mthree tinics faster than it , or true to total hydrolyzes MeCh at 1 0 - 3 ~the (AChE:ChE) will be 0.3 when only ~lictrue enzyme is present and will decrease as the proportion of pseudo or butyryl cholinesterase increases (SwKoros et al., 1969). The level of total ChE in CSF was 13 times higher than that found in plasma (8.81 vs 069 p o l / h per mg; P < 0.01) while the activity of AChE in CSF was 16 times that found in plasma (1.1 1 vs 007 pmol/h per mg; P < 010). Further, when all of the ratios (AChE:ChE) are arranged according to magnitude, it is apparent that the ratios in brain uniformly exceed those found in both plasma and CSF, indicating that the levels of AChE as compared to total ChE in brain tissue are higher than those 'in the 2 fluids. The values for specific activity are expressed in terms of mg of protein to allow comparisons with the brain homogenates. It must be remembered, however, that the plasma contains about 125 times as much protein (50 mg/ml) (YAKSHef al., 1973) as the CSF (04 mg/ml) (YAKSHet al., 1974). The absolute levels of cholinesterase activity are thus not reflected. Table 2 presents CSF and plasma specific activities for total ChE and AChE expressed in terms of pmol/h per ml.
AChE
AND ChE ACTIVITItS* AND PROTEIN CONCENTRATION? OBSERVED DALLY FOR IN ClSTERNAL CEREBROSPiNAL FLUIDS FROM THE CAT
14 DAYS
-
Day CSF Component
1
2
3
4
5
6
7
8
9 1 0 1 1 1 2 1 3 1 4
AChE
0.41 0.44 0.39 041 0.39 042 0.42 0.38 0.41 0.47 0-40 0.39 042 040 (004) (0.05) (OW) (0.04) (0.03) (0.05) (0.03) (0.06) (0.05) (008) (004) (005) (OW) (008)
ChE
3.6 (0.6)
Protein
0.32 0.38 0.36 033 038 031 0.39 0.38 0.33 0.33 0.32 032 034 035 (0.06) (0.05) (004) (008) (0.06) (0.08) (0.02) (0.03) (0.03) (0.04) (0.04) (006) (0.05) (004)
* AChE
3.5 (04)
3.5 3.2 (0.4) (0.2)
3.1 3.7 3.1 3.6 (0.3) (0.2) (0.3) (0.6)
3.1 3.2 3.6 (0.6) (0.4) (08)
33 3.3 (0.3) (03)
3.4 (03)
and ChE specific activities expressed as pmol/h/ml i- S.E.M. expressed as mg/ml. ml) were taken between 0900 and I000 hs each day from 4 cats. The values shown are
t Protein concentration 1 CSF samples (1-2-1.4 the mean k S.E.M.
~ A R R I Sand
T. L. YAKSH,M. G . FILBERT, L. W
856
0
2
H. I. YAMAMURA
A
6
8
1
0
1
2
DAYS AFTER INHIBITION
FIG. 1. Recovery of enzyme activity given as the log-% inhibition of AChE and ChE activity in CSF and plasma as a function of time in days after the administration of soman (27pg/kg, s.c.). Values represent the mean enzyme activity at each time period of the same 4 cats. Sequential CSF samples were taken as described in text.
Control levels of AChE, total ChE activity and protein for the CSF are presented in Table 3. These values were found in 4 cats over a period of 14 consecutive days. As can be seen, despite the daily withdrawal of samples of CSF, the enzyme levels remained stable. The mean and standard error for AChE and total ChE activity was 041 k 0.05 and 3.4 0.4 ,umol/h per ml, respectively. Likewise, the protein levels showed no significant fluctuation.
Enzyme recovery in CSF and plasma following soman inhibition After the injection of soman (27 & k g ) CSF-AChE specific activity fell to 5% of preinjection levels during the first hour from 1.11 k 0.07 to 0.064 0.01 pmol/ h per mg. Similarly, total ChE activity was reduced 96.4%,from 8.81 k 1-2 to 029 k 0.05 pnol/h per mg. CSF samples from control and poisoned animals were taken at 0.5 h, and then on 1,2,3,4,5,7 and 13 days after drug administration. The results of the assays are given in Fig. 1. The best fit linear regression lines were calculated using the least squares method for each set of data and the time required for the recovery of 50% of the original specific activity (t1,2)r was obtained using the derived regression equations TABLE 4.
CALCULATED
rliZ DAYS OF
RECOVERY OF
(Table 4). Statistical comparisons of the slopes of the regression lines were made employing the F ratio values derived from the regression data (WINER, 1962). As seen in Fig. 1 and Table 4, the tljz for AChE recovery in CSF was 1.4 days as compared to 2.1 days for total ChE activity. Comparison of the two slopes reveals this difference not to be statistically different ( P > 0.10). As reported elsewhere by YAKSHet al. (1974), mean CSF protein levels do not change significantly following soman treatment. In the present experiments, statistical comparisons of the protein levels before and after administration of soman, using a one-way analysis of variance for repeated measures, showed no significant alteration in cisternal CSF protein concentrations (P > 0.10). Venous blood samples were taken concurrently with the cisternal drains and AChE and total ChE specific activities were measured. As with CSF, AChE and total ChE activity in the plasma was significantly inhibited during the first hour after soman (94.9 and 95.6% respectively). The recovery of enzyme activity in plasma also is shown in Fig. 1. Like CSF, there was no significant difference in the slopes of the AChE and total ChE recovery curves (P > 010 with the tl,2 being 1.8 and 2.2 days, respectively (Table
AChE AND
TOTAL IN BRAIN,
CSF
AND PLASMA IN THE CAT*
Brain parts Enzyme
Caudate Hypothalamus ~~~
AChE ChE
2.54 1.18
Thalamus
Cerebral cortex
Cerebellar cortex
Brainstem
Pons
CSF
Plasma
2.09 1 -03
5.34 3 85
4-49 2-85
4.68 3.79
4.63 3.85
1.37 209
1.78 2.18
~
1.15 0.92
* The t I j zvalues were derived from the least square equations used to fit the data presented in Figs. 1-4. Consult text for further details.
AChE turnover in brain, CSF, and plasma
857
4). When CSF and blood samples obtained from soman treated animals were mixed with aliquots of control tissue, no inhibition of enzyme activity occurred in the control sample. This was taken to indicate the absence of free soman in these fluids at 1 h after treatment. Enzyme recovery in brain following soman inhibition To examine the rate of recovery of brain AChE > t and total ChE activities after inhibition by soman, U cats were given 27 pgkg and 1 each was killed at 1 h and then at 1,2,3, or 5 days later. In general, b Z Broinrlem Pons the maximum inhibition observed in brain tissue was somewhat less than that observed in CSF or plasma. The activity in whole brain homogenate, for example, fell 92%, from 2.78 to 0.22 pmol/h per mg. To be certain that the 1-h sample did, indeed, represent the maximum level of inhibition, 18 guinea-pigs were given soman (27 ,ug/kg, s.c.) and killed at 15, 30 or 60 min after the injection and the brains assayed for enzyme activity. The greatest inhibition occurred within the first 30 min and no change from this level was noted during the first hour. Thus, the specific activity of the enzyme at 1 h was taken to represent 0 2 4 6 0 2 A 6 thc maximum degree of inhibition. Further, at 1 h DAYS AFTER INHIBITION no free soman remained in brain homogenates as FIG. 3. Recovery of enzyme activity given as the log-”/, judged by the absence of inhibition when samples inhibition of AChE and ChE activity in cerebral cortex, from soman-treated animals were incubated with cerebellar cortex, brainstem and pons at several time perequal volumes of homogenates obtained from control iods following the administration of soman (27 &kg, s.c.). animals. Figure 2 shows the log percentage of inhibi- Each value represents the data obtained from one animal killed at the time. tion in the caudate nucleus, hypothalamus and thalamus at various times after soman injection. Figure 3 presents the recovery of enzyme activities in the respectively, whereas the longest period of recovery cerebral cortex and cerebellar cortex as well as the was observed in the cerebral cortex, brainstem, pons recovery in the pons and brainstem. The most rapid and cerebellar cortex, with tIi,’s between 4.5 and 5.3 recovery of AChE activity was observed in the days. The t1,2 for the total ChE recovery varied in hypothalamus (1.2 days) followed by the thalamus a similar manner from 0.9 days in the hypothalamus and caudate, which had t,,, rates of 2.1 and 2 3 days to 3.9 days in the pons and cerebral cortex. No strict correlation was observed between the recovery rate Hypoiholamur Ilrolonur -Coudole - - - - -N-------of enzyme specific activity or AChE to ChE ratio.
z
_______-^__
Recovery of total cholinesterase activity in subcellular fraction of brain tissue Figure 4 shows the log percentage of inhibition of AChE in guinea-pig brainstem subcellular fractions after soman intoxication. Table 5 presents the specific activities of the various fractions. As can he seen, the highest specific activity was observed in the microsoma1 and synaptosomal fractions (I 1.76 and 4.04 pmol/ h per mg, respectively) with the lowest in the soluble fraction (2.93 ,umol/h per mg). Corresponding half times of recovery were calculated using the equations 0 7 1 b 0 7 d 6 derived from the least squares analysis used to plot D A Y S AFTER INHIB’TION the data in Fig. 4. The shortest times were observed FIG. 2. Recovery of enzyme activity given as the log-% in the soluble fraction (1.8 days), the longest in the inhibition of AChE and ChE activity in caudate nucleus, synaptosomes (8.3 days) and intermediately in the thalamus, and hypothalamus at several time periods folmicrosomes (3.4 days). Total ChE levels in the rat lowing the administration of soman (27 pg/kg, s.c.). Each value represents the data obtained from 1 animal killed brain fractions are presented in Table 6. Distribution at that time. Samplcs were prepared and enzyme activity of enzyme activity in the subcellular fractions is identical with that observed in guinea-pig with the highest determined as described in the text.
T. L. YAKSH, M. G. FILBERT, L. W. HARRIS and H. I. YAMAMURA
858
DISCUSSION
D A Y S AFTER INHIBITION
FIG. 4. Recovery of enzyme activity given as the log-% inhibition of total ChE activity in the subcellular fractions of guinea-pig brainstem observed at several time periods following the administration of soman (32 pg/kg, s.c.). Each data point represents the results obtained from one animal after soman intoxication. Fractions were prepared as described in the text.
activity found in the microsomes, less in the synaptosoma1 fraction and least in the soluble fraction (15.18, 744 and 3.01 pmol/h per mg respectively). Again the fastest return of enzyme activity was found in the soluble fraction with a tl,, (1.6 days) similar to that found in the guinea-pig, and the slowest recovery also occurred in the synaptosomal fraction (flj2 = 4.1 days). TABLE 5. ChE ACTIVITY IN GUINEA-PIG BRAIN SUBCELLULAR FRACTIONS AND CALCULATED HALFTIME OF ChE RECOVERY FOLLOWING SOMAN ChE Specific activityt
Fraction Whole brain homogenate Soluble fraction Microsomes Synaptosomes
4.84 2.93 I 1.76
4.04
(Days)
t,/z*
4.7 14 3.4 8.3
* The tIiz derived from regression equations by the method of least squares. Consult text for further details. t Specific activity expressed as pmol/h per mg protein. TABLE 6. ChE
ACTIVITY IN RAT BRAIN SUBCELLULAR FRACTIONS AND CALCULATED SOMAN
Fraction Whole homogenate Soluble fraction Microsomes Synaptosomes
* Specific activity,
Control ChE activity* 5.57 (4.58-6.50) 3.01 (2.70-3.3 I ) 15.18 (1 3.38-16.88) 7.44 (489-9.99)
HALF-TIMF
OF RFCOVERY FOLLOWING
"/, Inhibitiont 0.5 h after soman 83.3 (91.1-75-4) 82.9 (90475.3) 82.6 (89.2-76.1) 81-8 (92671.0)
mean and (95% confidence limits), expressed as pmollh per mg.
t % inhibition, mean and (95% confidence limits). Days.
Whether soman can be used as an inhibitor for studying the rate of synthesis of AChE or total ChE in animals depends upon three assumptions: (1) that the inhibition is irreversible; ( 2 ) that excess, free inhibitor is not present to combine with newly formed enzyme; (3) soman has no direct effect on protein synthesis. We believe that these assumptions are justified for the following reasons. (1) Inhibition of cholinesterases by soman is due to phosphonylation of the active site serine (SCHAFFERet at., 1973). The soman+nzyme complex then ages, i.e. it loses the pinacolyl group to form a product that is not subject to reactivation with oximes (BERRY& DAVIS,1966). The f,,, for aging of soman-AChE complex in monkey brain is 1 min (FILBERTet al., 1972) and is 2.4 min in bovine erythrocytes (FLEISHER & HARRIS, 1965). In addition, FLEISHER & HARRIS(1965) have shown that there was no loss of the residual methylphosphonate from the aged enzyme in vivo up to 72 h. (2) The absence of free soman in tissues was verified by incubation of aliquots of tissues from poisoned animals with aliquots taken from tissues of normal animals. The enzyme in tissues from normal animals was not inhibited under these conditions. (3) Whether soman has no direct influence on protein synthesis is more difficult to assess. HARRIS et d.(1974) examined the effects of soman on the rate of synthesis of AChE as well as the incorporation of ~-[U-'~C]leucine into protein and the uptake of C3H]methylthymidine by DNA in rabbit bone marrow cells which were in an active state of erythropoiesis. They found no differences between normal and soman treated cells even though the AChE had been reduced to less than 5% of the control levels. In another study by HARRIS(1975) there was also no difference between control and soman poisoned animals in the ~-[U-'~C]leucineincorporation into protein by the guinea-pig retina. On the basis of these findings, those investigators concluded that soman has no effect on the overall protein synthesis. The specific activity of AChE in the present experiments for normal cat whole brain (0.64 pmol/h per et a!. (1969) mg) is lower than that found by SIAKOTOS in the mouse, rat, guinea-pig and rabbit (1.38-2.20 pmol/h per mg) but is similar to that found by them in the bovine (0.47 pmol/h per mg). Examination of
24 h after soman 6 6 9 (71.1-62.7) 60.4 (63.9-57'6) 65.9 (69.2-62.2) 73.5 (80.1-66.8)
t,,,:
2.3 1.6 2.2 4.1
AChE turnover in brain, CSF, and plasma
859
the specific activity of AChE in different brain areas the values obtained for the other species reported here indicates that as in cats, rats and dogs (CLOUET & and this uniformity of recovery rates across species, WAELSCH, 1961; KLINGet al., 1969; QUAYet al., 1971; we feel, substantiates the existence of a rapid rate of THOMASet al., 1971) the highest levels are to be found synthesis for this essential enzyme by brain tissue. In in the caudate nucleus while the lowest are generally general, protein turnover in brain has been thought observed in the cerebral cortex. Despite the fact that to be somewhat longer (1C-20 days) (VON HUNGEN species variation exists with respect to specific acti- e t p l . , 1968; LAJTHAet al., 1971). AUSTINet al. (1972) vity, there is similarity in the ratios of AChE to total found an apparent mean halftime of 3.2 days for proChE (i.e. hydrolysis of MeCh: ACh). For example, tein turnover in rat whole brain as well as the existvalues ranging from 0.2 in mouse whole brain homo- ence of a protein fraction with a turnovcr time of genates to 0.3 for rabbit have been found (SIAKOTOSless than 2 days. The present data suggest that AChE et al., 1969). Similarly, ratios of AChE to total ChE would fall within this rapidly changing protein fracfor different topographical regions generally fall in tion. this range although the caudate nucleus typically has The source of AChE in the CSF has not been identhe highest ratio. tified. Since the recovery of AChE after inhibition by AChE activity in CSF fluid obtained from the cis- soman is presumed to be by de novo synthesis and terna magna of normal cats is 6 times that found there is no synthetic capability within the CSF, the in human lumbar CSF by JOHNSON& DOMiNO (1970). return of the enzyme must be associated with moveProtein concentrations in the cat cisternal CSF ment of newly synthesized protein into the CSF. It reported here were also slightly higher than values has been established that while the brain enzyme is reported by CUTLEK (1970), but are identical to values mostly bound to membranes, a significant amount reported by HOCHWALD (1970) and previously found exists in a soluble form (HOLLUNGER et al., 1973; by these laboratories (YAKSHet al., 1973, 1974). Since WENTHOLD et ul., 1974~)and this soluble enzyme there was no change in either protein concentration could be the source of AChE in the CSF. It is signifior AChE when fluid was removed daily for 14 con- cant, therefore, that while the recovery rates of the secutive days, we have concluded that repeated with- AChE in the subcellular fractions differ markedly, drawal of CSF does not lead to the formation of a without exception, the most rapid recovery was more dilute CSF nor to excess protein from extra observed in the soluble fraction (AUSTIN & JAMES, et al. (1972a,b; present study). FurtherCSF sources. This is not unexpected. in as much as 1970; FILBERT the rate of formation of CSF in the cat is about 30 more, this more rapid rate of recovery in the soluble fraction coincides with thc rate of recovery in the ml/day (HOCHWALD, 1970). In the present studies, we observed that the t1,2 CSF. In spite of the strong correlation which exists for regeneration of AChE in brain tissues after irreversible inhibition by soman ranged from 1.2 days between the turnover rates of the brain soluble in the hypothalamus to 5.3 days in the cerebral cortex enzyme and CSF enzyme, the possibility that some with a mean halftime for the seven areas examined AChE in the CSF derives from plasma cannot be of 3-6 days. The halftime of recovery in the rat whole excluded. Small amounts of plasma protein may pass brain was 2.3 days and in the guinea-pig brainstem, through capillary walls and, thereby, gain access to 4.7 days. These regeneration rates are comparable to the brain extracellular space which is in direct com& DAVSON, the t1,2 for recovery of AChE in monkey brain after munication with the CSF (OLDENDORF 1970). Human and horse plasmas soman poisoning (2.5 days) observed by FILBERT et 1967; HOCHWALD, 1948). al. (1972b). From the data of GLOWet ul. (1966) we contain essentially no AChE (ALJGUSTINSSON, have calculated halftimes of 7-11 days for recovery However, cat and rat plasma do show significant of AChE in various parts of rat brain after inhibition AChE activity, a fact that has been established by by diisopropylphosphorofluoridate(DFP) and a t1,2 the use of specific substrates and specific inhibitors. et al. (1969) have shown that rat plasma of 16.5 days from the data of Ausrw & JAMES(1970) SIAKOTOS who also used DFP to measure regeneration rates still hydrolyzes MeCh at about 80% of the control of AChE in rat cerebral cortex. The reason for the rate in the presence of IO-jM-iso-OMPA, a specific longer recovery times after DFP inhibition is not inhibitor of pseudocholinesterase. Similarly, FILBERT, MOORE& YAKSH(manuscript in preparation) clear at this time, especially in the light of the findings H~RKIS, et al. (19746) who determined AChE have shown that the hydrolysis of ACh by cat plasma of WENTHOLD turnover in rat cerebral cortex by measuring the time is reduced 80% by 1 0 - 5 ~ - B W284C-51 a specific course of disappearance of radiolabel introduced inhibitor for AChE. Whether a significant quantity of AChE can enter into the enzyme by intraventricular injection of ~-[1-' 4C]leucine. Assuming steady-state conditions the CSF from the plasma via the capillary wall is where the rate of degradation of protein equals the not known. It should be pointed out, however,, that & MARKS,1971), they deter- there appears to be a substantial delay in passage rate of synthesis (LAJTHA (1970), mined the tL,2to be 2.8 days. This value for AChE of protein from plasma to CSF. HOCHWALD turnover agrees well with the tl,2 for resynthesis studying the movement of 1311-labelledalbumin and found by us in the rat. It also correlates well with ' 'I-labelled y-globulin, observed that it took between
860
L. W. HARRISand H. I. YAMAMURA T. L. YAKSH,M. G. FILBERT,
HARRISL. W. (1975) M.S. Thesis, Towson State College, Towson, Maryland. HOCHWALD G. M. (1970) J. neurol. SC~S10. 269-278. HoLLUNGER E. G . & NlKLASSON B. H. (1973) J . Neurochem. 20. 821-826. HOVANEC J. H., LIESKEC. N., ROWLAND S. L. & LANKS K. (1974) Abstracts, Priestly Bicentennial Middle Atlantic Regional Meeting ofthe American Chemical Society, Wilkes-Barre, Pennsylvania. D. 41. JOHNSONS. & D o ~ m bG. F.*(1971) Cfinica chim. Acta AckriowlidgementsThe authors wish to thank L. A. 35. 421428. FEDELE for his excellent assistance and Dr. C. A. BROOMKALSBEEK F., &HEN J. A. & BOVENS B. R. (1950) Biochim. FIELD, Mr. K. M. WILSON and Capt. J. R. LOWEfor helpful biophys. Acia 5. 548-560. commentary on the manuscript. KLINGA,, FINERS. & GILMOUR J. (1969) I n t . 1. Neuropharmac. 8. 2531. REFERENCES LAJTHAA. & MARKSN. (1971) in Handhook of Neurochemistry (LAITHAA., ed.) Vol. 5, pp. 551-629. Plenum AUGUSTINSWNK. B. (1948) Acta physiol. scand. 15. Suppl. Press, New York. 52. 1-182. 0. N., ROSEBROUGH N. J., FARR A. L. & RANDALL AUSTINL. & JAMESK. A. C. (1970) J. Neurochem. 17, 705- LOWRY 707. R. J. (1951) 1.biol. Chem. 193. 265-267. AUSTIN L., LOWRY0. H., BROWNJ. G. & CARTERJ. G. OLDENWRP W. H. & DAVSONH. (1967) Archs Neurol. Psy(1972) Biochern. J. 126. 351-359. chiat. Chicago 17. 196-205. QUAYW. B., BENNETE. L., MORIMOTO BERRYW. K. & DAVISD. R. (1966) Biochem. J. 100. 572H. & HERBERT 576. M. (1971) Comp. gen. Pharmac. 2. 402410. BRAYG. A. (1960) Analyt. Biochem. 1. 279-285. SCHAFFER N. K., MICHELH. 0. & BRIDGES A. F. (1973) CLOUET D. H. & WAELSCH H. (1961) J . Neurochem. 8. 201Biochem., Easton 12. 29462950. 215. SIAKOTOS A. N., FILBERT M. G. & HESTERR. (1969) BioCOULTD. B. & MARSHD. J. (1966) Biochem. J. 98. 869chem. M e d . 3. 1-12. 873. SVENSMARK 0. (1961) Actaphysiol. scand. 52. 372-37s. J. E. & CORNICK L. R. (1970) THOMAS CUTLERR. W. P., MURRAY N., FLEISHER J. H. & HARRIS L. W. (1971) Biochim. E x p f . Neurol. 28. 257-265. biophys. Acta 235. 542-547. DAVSQNH. (1967) Physiology of the Cerebrospinal Fluid VON HUNGENK., MAHLERH. R. & MOOREW. J. (1968) pp. 132-1 33. Churchill, London. J . biol. Chem. 243. 1415-1423 DAVSON H. & OLDENWRF W. H. (1967) Proc. R. SOC.Med. WELCHK. (1963) Am. J . Physiol. 205. 617-624. 60. 32C329. WENTHOLD R. .I. MAHLER , H. R. & MOORE W. J. (1974a) DoMsCHKE w., DOMAGK G. F., DOMSCHKE s. & ERDMANN J. Neurochem. 22. 945-949. W. D. (1970) Arch. Tox 26. 142-148. WENTHOLD R. J., MAHLERH. R. & MOORE W. J. (1974b) FILBERT M. G., LOCHNERM. A., YAMAMLJRA H. I., HARRIS J. Neurochem. 22. 941-943. L. W. & FLEISHER J. H. (1972n) Frdn Proc. Fedn Am. WHIITAKER V. P. (1969) in Handbook of Neurochemistry Socs. rxp. Biol. 31. 515. (LAJTHAA., ed.) Vol. 2, pp. 329-364. Plenum Press, New FILBERT M. G., LQCHNER M. A. & FLEISHER J. H. (1972b) York. Biochim. biophys. Acta 264. 164-174. WINERB. J. (1962) Statistical Principles in Experimental FLEISHER J. H. & HARRISL. W. (1965) Biochem. Pharmac. Design. McGraw-Hill, New York. 14. 641-650. YAKSHT. L., FEDELE L. A. & YAMAMURA H. I. (1973) PhyGLOWP. I., ROSE S . & RICHARDSONA. (1966) Aust. J . swl. Behau. 10. 149-151. exp. Biol. med. Sci. 44. 73-86. YAKSH T. L., FEDELE L. A. & YAMAMURAH. I. (1974) G R A Y E. J. & WHIlTAKER V. P. (1962) J. Anat. 96. 79-88. Experientia 30. 38-39. HARRISL. W., YAMAMURAH. I. & FLEISHER J. H. (1971) YAKSHT. L., YAMAMURA H. I. & RUDYT. A. (1975) EdgeBiochem. Pharmac 20. 2927-2930. wood Arsenal Special Publication EB-SP-74031. Aberdeen V. F. & MOORER. D. (1974) BioHARRISL. W., GARRY Proving Ground, Maryland. chem. Pharmac. 23. 21552163.
8 and 16 h for the brain to plasma ratio of radioactivity to approach unity. If plasma protein were contributjng sjmjfiantly to the enzyme levels observed in CSF during recovery, we would expat the r1,2 in CSF t o exceed the t I i z in plasma, reflecting the time required for movement between the The i.e. the t l / 2 appears to be the case, for the CSF is less than the tl,* for plasma.
