1N P’WO AND I?4 P’VITROEFFECTS OF BROMOACETYLCHOLINE ON RAT BRAIN ACETYLCHOLINE LEVELS AND CHOLINE ACETYLTRANSFERASE ACTIVITY R. C. SPETH,D. E. SCHMIDT,*B. V. RAI\IASASTRYand D. M. BUXBALJM Tennessee Neuropsychiatric Institute and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN (Accepted 19 September1975)
Summary-Intraventricular injection of bromoacetylcholine to rats did not lower brain acetylcholine levels, nor did it inhibit choline a~tyItransferase activity. In uitro experiments indicated that bromoacetylcholine can penetrate intact neuronal tissue and inhibit choline acetyltransferase, but that in uiuo it is most likely hydrolyzed by cholinesterase before it can exert any inhibitory action. For this reason bromoacetylcholine is unsuitable for in viva inhibition of choline acetyltransferase.
in water immediately prior to use to minimize autohydrolysis. Animals in which ACh levels were measured were sacrificed by whole body microwave irradiation in a Litton 550 microwave oven as previously described (SCHMIDT,SPETH,WEJ_XX and S~~DT, 1972). Acetylchoiine was qu~titatively determined by the pyrelysis gas chromatographic method of SCHMIDTet al. (1972). Following intraventricular a~inistration of BrACh, rats were sacrificed by cervical dislocation, brains were removed (excluding cerebellum and brainstem), and homogenized in cold 0.32~ sucrose containing 2 x 10e4 M physostigmine to give a tissue con~ntration of I~m~rnl. Small portions of the homogenate were then assayed for ChA activity by the method of SCHRIERand SHUSTER(1967). In some experiments, to reduce in viva ChE activity, animals were pretreated with either a single dose of physostigmine (@75mg/kg, i.p.) 20 min prior to administration of BrACh or with Oq7mgJkg of paraoxon daily for 3 days prior to administration of BrACh. Bromoacetylcholine was a~inistered 1 hr following the final dose of paraoxon. In addition to the in vivo studies, the effect of various concentrations of BrACh on ChA activity was MATERIALS AND METHODS studied on three different in vitro systems. SynaptoIntraventricular injection carmulae were chronically somes were prepared by homogenizing brain tissue implanted in male, 20@225 g Holtzman rats by the in sufficient cold, oxygenated 0.32~ sucrose, using a method of DE BALBIANVERSTER,ROBINSON,HENGEL- teflon-glass homogenizer (5 up and down strokes), VELDand SANDERS-BUSH (1971). A minimum of 3 days to attain a final tissue concentration of l~m~ml. recovery foliowing ~pl~tation was allowed prior to Portions of the crude homogenate were then incudrug administration. Compounds were administered bated for 10 min with BrACh at 37”C, either in the intraventricularly to conscious rats in a volume of #absence or presence of physostigmine (2 x 10F4~). 10 ~1. Bromoacetylcholine perchlorate was dissolved Following incubation, physostigmine was added to the non-physosti~ine homogenates and ChA ac* Send communications and inquiries to: Dr. Dennis tivity measured as previously described. In some exSchmidt, Tennessee Neuropsychiatric Institute, Central periments, n-butanol was added to disrupt the synapState Hospital, Nashville, TN 37217.
The halogenoacetylcholines, first synthesized by CHIOU and SASTRY(1968), have proved to be potent, specific and essentially irreversible inhibitors of choline acetyltransferase (CM) activity in vitro, with an Iso (concentration required to produce 50% maximum inhibition) of 3 x lO^‘? M for brain ChA (SASTRYand HENDERSON, 1976). The availability of specific and irreversible ChA inhibitors, which are effective in vivo, would be a valuable tool in investigating the cholinergic system in the central nervous system. This investigation was therefore initiated to determine the effects of in vivo administration of bromoacetylcholine (BrACh) on ChA activity and acetylcholine (ACh) levels and to thereby ascertain whether the halogenoa~tylcholines might be useful compounds for the in vivo inhibition of ChA. Bromoacetylcholine was chosen by virtue of its stability to hydrolysis by cholinesterase (ChE) and its low mu~rinic agonist properties, relative to the other halogenoacetylcholines. It is siightly less potent than iodoacetylcholine, the most potent inhibitor of the halogenoacetylcholines, but is considerably more resistant to ChE hydrolysis than iodoa~tyl~holine (CHIOIJand SASTRY,1968).
287
288
R. C.
SPETH,
D. E.
SCHMIDT,
B. V.
tosomes (BULL and ODERFELD-NOWAK, 1971) prior to incubation with BrACh. Choline acetyitransfemse activity was also measured following incu~tion of both brain slices and hemi-brains with BrACh. Brain slices, 500~ thick, were prepared on a McIlwain tissue chopper. Slices were then incubated for 10min at 37°C in oxygenated 0.32 M sucrose containing BrACh, either in the absence or presence of physostigmine (2 x 10-4~). Following incubation, the slices were washed to remove exogenous BrACh and homogenized (100 mg/ml) in 032 M sucrose containing 2 x 10s4 M physostigmine, and analyzed for ChA activity as described above. Finally, hemi-brains were prepared by dividing the whole brain longitudinally. The resulting hemi-brains were treated in the same manner as brain slices. Apparent Iso values were determined at fixed concentrations of choline (56 x 1O-3 M) and acetyl coenzyme A (25 x 10m4M) by using probit transformation (LI’RXFIELD and WILCOXON, 1949). Bromoacetylcholin~ perchlorate was prepared by the method of CHIOU and SASTRY(1968). [i4C]-Acetyl coenzyme A (50 $Zi/~mol) was obtained from New England Nuclear.
RESULTS AND
RAMA
SA~TRYand D. M.
BUBALJM
Table 1. Whole brain acetylcholine levels after administration of various doses of bromoacetylcholine Amount of BrACh
ACh level*
Saline 25 pg 50 Irg lOO%I 250 I*g 500 iug
226 + 24 20.5 + 1.8 20.0 _t 1.7 21.0 + 3.2 19.9 & 3.1 19.2 i: I,7
* nmol ACh/g brain + S.D. of 6 animals sacrificed 5 min after BrACh administration. In vivo CkA activity
To determine whether the failure of BrACh to lower ACh levels was due to a lack of inhibition of ChA activity or to other factors, ChA activity in brain was measured 30 min following intraventricular administration of 1OOpg of BrACh. There was no inhibition of ChA activity (Table 3). Several explanations could account for these observations: (1) BrACh is hydrolyzed before it can inhibit ChA; (2) BrACh cannot enter the neurone and, therefore, never reaches the intracellular site of ChA; and (3) BrACh does not diffuse freely following intraventricular administration and its local effects are too
DISCUSSION
Table 2. Whole brain acetylcholine levels at of various times after administration bromoacetylcholine
Brain ACk Eevels No significant changes in whole brain ACh levels were observed 5 min after intraventricular administration of either 25, 50, 100, 250 or 5OOpg of BrACh (Table 1). Acetylcholine levels also were not changed at time intervals of l-30 min follow~g 1OOpg of BrACh (Table 2). In addition, control animals which received 065 ~01 (molar equivalent to 5OOpg BrACh) of either NaCl, sodium perchlorate, hydrolyzed bromoacetylcholine, sodium bromoacetate and choline chloride, also failed to show any changes in ACh levels 10 and 30 min after a~inistration.
ACh level*
Time (min)
22-6 * 2.4 23.9 + 3.2 23.5 i: 0.8 21.0 f 3.2 25.9 _t 24 25.7 i: 4.3
0 (saline) 1 2 5 10 30
* nmol ACh/g brain & SD. of 6 animals following a~inistration of 100 pig of BrACh.
Table 3. Effect of inhibition of cholinesterase on in vioo inhibition of choline acetyltransferase Pretreatment
Intraventricular
injection
by bromoacetylcholine
Choline acetyltransferase activity
Saline Bromoacetylcholine,
100 pg
3.41 * 0.49’ 3.53 f 0.45 3.35 2 0.37
Physostigmine 0.75 mg/kg 20 min prior to intraventricular injection
Saline Bromoacetylcholine,
100 pg.
3.50 It 044 3.48 + 0.33
Paraoxon 0.7 m&kg/per day for 3 days, last injection 60 min prior to intraventricular injection
Saline Bromoacetylcholine,
100 pg.
3.53 * 029 353 i_ 053
None
None
* Expressed as ~01 ACh synthesizedjg brain per hr & S.D. Activity was measured 30 min after the indicated intraventricuiar injection.
Bromoacetylcholine Table 4. lrz vitro effects of bromoa~t~lcholine
Tissue preparation Purified choline acetyltransferaset Intact synaptosomes Butanol-treated synaptosomes Brain slices Hemi-brain
and ChA
289
on choline a~tyltransfer~e
activity
Apparent &,* No 2 X 10-&M Physostigmine Physostigmine 3 X lo-‘M 4.6 x 10-7M 1.4 X 10-7M No inhibition at 10m4~ BrACh No inhibition at tO-4~ BrACh
1.5
X
10-‘M
3.3 X 10-6M 30% inhibition at 10-4~ BrACh
* Calculated using probit transformation (LITCHFIELD and WILCOXON,1949). t From SASIXYand HENDERSON (1976).
small to produce changes in whole brain ChA activity. To attempt to resolve this question, 1OOpg BrACh was injected intraventricularly to animals which had been previously treated with ChE inhibitors (Table 3). Again, there was no inhibition of ChA activity, even though 3 day administration of paraoxon has been shown to reduce ChE levels to only approx. 7% of control (CEHOVIC,DET~BARN and WELSCH,1972).
To further investigate this problem, in t&o studies were undertaken as outlined in the Methods (Table 4). In the preparation containing intact synaptosomes and in the presence of physostigmine, BrACh was a potent inhibitor of ChA, having an I,, of 1.5 x lo-*M. When physostigmine was not present, the ability of BrACh to inhibit ChA in intact synaptosomes was si~i~~ntly decreased, having an I,, of 46 x lo-’ M.When the s~aptosomes were disrupted by addition of butanol, ChA activity increased from 3.5 ,umol ACh synthesized/g per hr to 6.5 ,mnol ACh synthesized/g per hr. The ability of BrACh to inhibit ChA in disrupted synaptosomes in the absence of physostigmine (I,, = 1.4 x lo-’ M) was similar to the I,, observed using intact synaptosomes (Table 4). The s~i~rity of the ability of BrACh to inhibit ChA in an intact s~aptosomal preparation versus its ability to inhibit purified ChA (Table 4) and the fact that disruption of synaptosomal membranes did not increase inhibitory potency (Table 4), indicates that BrACh was able to enter synaptosomes and produce inhibition of ChA activity, and, in synaptosomes, this inhibition was attained even in the presence of active ChE. To more closely approximate the in viljo situation, in which BrACh would have to diffuse through multicellular layers and would therefore have increased exposure to ChE, brain slices and hemi-brains were investigated. In brain slices, in the presence of physo.&mine (2 x 10e4M), BrACh still had an I,, of 3.3 x 10m6M. In the absence of physostigmine, however, concentrations of BrACh as high as 10V4M did not produce inhibition of ChA activity. Finally, in the hemibrain preparation, 30 min incubation with
10e4~ BrACh produced a 35% inhibition of ChA activity in the presence of physosti~ine, and no inhibition in the absence of phy~sti~ine. These in vitro results using synaptosomeg brain slices and hemi-brains indicate that BrACh can indeed cross neuronal cell membranes, since significant inhibition of ChA was observed in all these preparations in the presence of physostigmme. Even when synaptosomal membranes were disrupted by butanol treatment, as indicated by the increase in observed enzyme activity, BrACh had no greater inhibitory potency than it did in non-disrupted tissue. Nowever, BrACh was considerably less potent in the absence of ChE inhibition. In fact, there was no inhibition of ChA activity in the absence of physostigmine in slices or hemi-brains using BrACh concentrations as high as 10V4M. The most likely conclusion for the in vivo results, therefore, is that BrACh is hydrolyzed by ChE before it can interact with ChA. The decreasing potency of BrACh in the presence of physostigmine obtained using butanol-treated synaptosomes versus intact synaptosomes, versus brain slices, versus hemibrains may result from either a slow diffusion of BrACh through multicellular layers or it may reflect a decrease in the inhibition of intracellular ChE by physostigmine with increasing structural integrity. Of particular interest was the observation that in animals in which 93% of their ChE had been inhibited, BrACh was still without effect on ChA activity. This indicates that there is a large excess of ChE in brain and that the small amount of ChE activity remaining following such treatment must occupy a strategic location in cholinergic neurones. That is, they are still able to function and there is apparently sufficient ChE activity to prevent the access of BrACh to the ~tra~llular site of ChA. In summary, BrACh is unsuitable for in vivo inhibition of ChA, due to rapid hydroxylysis by ChE. It may be possible to synthesize ChE resistant derivatives of the halogenoacetylcholines such as the carbamy1 or ketone analogue, and these might hopefully prove to be useful in uivo ChA inhibitors. Ack~Qwle~eme~ts-This work was supported by USPH Grants DA-000319, MH-11468; training Grant MH-08107 and USAMRDC Contract DADA 17-73-C-3130.
290
R. C. SPETH,D. E. SCHMIDT,B. V. RAMASASSY and D. M. BUXEIALJM REFERENCES
BULL, G. and ODERFELD-NOWAK, B. (1971). Standardization of a radiochemical assay of choline acetyltransferase and a study of the activation of the enzyme in rabbit brain. J. Neurochem. 18: 935-941. CEHOVIC,G., DETTBARN,W. D. and WEIXH, F. (1972). Paraoxon: effects on rat brain cholinesterase and on growth hormone and prolactin of pituitary. Science, N.Y.175: 12561258. CHIOU,C. Y. and SASTRY,B. V. R. (1968). Acetylcholinesterase hydrolysis of halogen substituted acetylcholines. Biochem. Pharmac. 17: 805-815.
LITCHFIELD, J. T., JR. and WILCOXON,F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther. 96: 99-113. SASTRY,B. V. R. and HENDERSON, G. I. (1976). Mechanisms of acetylcholine synthesis by rat brain choline acetyltransferase and its regulation by products and inhibitors, In: Drugs and Central Synaptic Transmission (BRADLEY, P. B. and DHAWAN,B. N., Eds.) Macmillan, London. (In press). SC&&T, D. E., SPETH,R. C., WELSCH,F. and SCHMIDT, M. J. (1972). The use of microwave radiation in the determination of acetylcholine in the rat brain. Brain Res. 38: 377-389.
DEBALBIANVERSTER,F., ROBINSON,C. A., HENGELVELD,,SCHRIER,B. K. and SHUSTER,L. A. (1967). A simplified C. A. and SANDERS-BUSH, E. (1971). Free hand cerebroradiochemical assav for choline acetvltransferase. J. ventricular injection technique for unanesthetized rats. Neurochem. 14: 977-985. L.$ Sci. 10 (I): 1395-1402.
Summary-Intraventricular injection of bromoacetylcholine to rats did not lower brain acetylcholine levels, nor did it inhibit choline a~tyItransferase activity. In uitro experiments indicated that bromoacetylcholine can penetrate intact neuronal tissue and inhibit choline acetyltransferase, but that in uiuo it is most likely hydrolyzed by cholinesterase before it can exert any inhibitory action. For this reason bromoacetylcholine is unsuitable for in viva inhibition of choline acetyltransferase.
in water immediately prior to use to minimize autohydrolysis. Animals in which ACh levels were measured were sacrificed by whole body microwave irradiation in a Litton 550 microwave oven as previously described (SCHMIDT,SPETH,WEJ_XX and S~~DT, 1972). Acetylchoiine was qu~titatively determined by the pyrelysis gas chromatographic method of SCHMIDTet al. (1972). Following intraventricular a~inistration of BrACh, rats were sacrificed by cervical dislocation, brains were removed (excluding cerebellum and brainstem), and homogenized in cold 0.32~ sucrose containing 2 x 10e4 M physostigmine to give a tissue con~ntration of I~m~rnl. Small portions of the homogenate were then assayed for ChA activity by the method of SCHRIERand SHUSTER(1967). In some experiments, to reduce in viva ChE activity, animals were pretreated with either a single dose of physostigmine (@75mg/kg, i.p.) 20 min prior to administration of BrACh or with Oq7mgJkg of paraoxon daily for 3 days prior to administration of BrACh. Bromoacetylcholine was a~inistered 1 hr following the final dose of paraoxon. In addition to the in vivo studies, the effect of various concentrations of BrACh on ChA activity was MATERIALS AND METHODS studied on three different in vitro systems. SynaptoIntraventricular injection carmulae were chronically somes were prepared by homogenizing brain tissue implanted in male, 20@225 g Holtzman rats by the in sufficient cold, oxygenated 0.32~ sucrose, using a method of DE BALBIANVERSTER,ROBINSON,HENGEL- teflon-glass homogenizer (5 up and down strokes), VELDand SANDERS-BUSH (1971). A minimum of 3 days to attain a final tissue concentration of l~m~ml. recovery foliowing ~pl~tation was allowed prior to Portions of the crude homogenate were then incudrug administration. Compounds were administered bated for 10 min with BrACh at 37”C, either in the intraventricularly to conscious rats in a volume of #absence or presence of physostigmine (2 x 10F4~). 10 ~1. Bromoacetylcholine perchlorate was dissolved Following incubation, physostigmine was added to the non-physosti~ine homogenates and ChA ac* Send communications and inquiries to: Dr. Dennis tivity measured as previously described. In some exSchmidt, Tennessee Neuropsychiatric Institute, Central periments, n-butanol was added to disrupt the synapState Hospital, Nashville, TN 37217.
The halogenoacetylcholines, first synthesized by CHIOU and SASTRY(1968), have proved to be potent, specific and essentially irreversible inhibitors of choline acetyltransferase (CM) activity in vitro, with an Iso (concentration required to produce 50% maximum inhibition) of 3 x lO^‘? M for brain ChA (SASTRYand HENDERSON, 1976). The availability of specific and irreversible ChA inhibitors, which are effective in vivo, would be a valuable tool in investigating the cholinergic system in the central nervous system. This investigation was therefore initiated to determine the effects of in vivo administration of bromoacetylcholine (BrACh) on ChA activity and acetylcholine (ACh) levels and to thereby ascertain whether the halogenoa~tylcholines might be useful compounds for the in vivo inhibition of ChA. Bromoacetylcholine was chosen by virtue of its stability to hydrolysis by cholinesterase (ChE) and its low mu~rinic agonist properties, relative to the other halogenoacetylcholines. It is siightly less potent than iodoacetylcholine, the most potent inhibitor of the halogenoacetylcholines, but is considerably more resistant to ChE hydrolysis than iodoa~tyl~holine (CHIOIJand SASTRY,1968).
287
288
R. C.
SPETH,
D. E.
SCHMIDT,
B. V.
tosomes (BULL and ODERFELD-NOWAK, 1971) prior to incubation with BrACh. Choline acetyitransfemse activity was also measured following incu~tion of both brain slices and hemi-brains with BrACh. Brain slices, 500~ thick, were prepared on a McIlwain tissue chopper. Slices were then incubated for 10min at 37°C in oxygenated 0.32 M sucrose containing BrACh, either in the absence or presence of physostigmine (2 x 10-4~). Following incubation, the slices were washed to remove exogenous BrACh and homogenized (100 mg/ml) in 032 M sucrose containing 2 x 10s4 M physostigmine, and analyzed for ChA activity as described above. Finally, hemi-brains were prepared by dividing the whole brain longitudinally. The resulting hemi-brains were treated in the same manner as brain slices. Apparent Iso values were determined at fixed concentrations of choline (56 x 1O-3 M) and acetyl coenzyme A (25 x 10m4M) by using probit transformation (LI’RXFIELD and WILCOXON, 1949). Bromoacetylcholin~ perchlorate was prepared by the method of CHIOU and SASTRY(1968). [i4C]-Acetyl coenzyme A (50 $Zi/~mol) was obtained from New England Nuclear.
RESULTS AND
RAMA
SA~TRYand D. M.
BUBALJM
Table 1. Whole brain acetylcholine levels after administration of various doses of bromoacetylcholine Amount of BrACh
ACh level*
Saline 25 pg 50 Irg lOO%I 250 I*g 500 iug
226 + 24 20.5 + 1.8 20.0 _t 1.7 21.0 + 3.2 19.9 & 3.1 19.2 i: I,7
* nmol ACh/g brain + S.D. of 6 animals sacrificed 5 min after BrACh administration. In vivo CkA activity
To determine whether the failure of BrACh to lower ACh levels was due to a lack of inhibition of ChA activity or to other factors, ChA activity in brain was measured 30 min following intraventricular administration of 1OOpg of BrACh. There was no inhibition of ChA activity (Table 3). Several explanations could account for these observations: (1) BrACh is hydrolyzed before it can inhibit ChA; (2) BrACh cannot enter the neurone and, therefore, never reaches the intracellular site of ChA; and (3) BrACh does not diffuse freely following intraventricular administration and its local effects are too
DISCUSSION
Table 2. Whole brain acetylcholine levels at of various times after administration bromoacetylcholine
Brain ACk Eevels No significant changes in whole brain ACh levels were observed 5 min after intraventricular administration of either 25, 50, 100, 250 or 5OOpg of BrACh (Table 1). Acetylcholine levels also were not changed at time intervals of l-30 min follow~g 1OOpg of BrACh (Table 2). In addition, control animals which received 065 ~01 (molar equivalent to 5OOpg BrACh) of either NaCl, sodium perchlorate, hydrolyzed bromoacetylcholine, sodium bromoacetate and choline chloride, also failed to show any changes in ACh levels 10 and 30 min after a~inistration.
ACh level*
Time (min)
22-6 * 2.4 23.9 + 3.2 23.5 i: 0.8 21.0 f 3.2 25.9 _t 24 25.7 i: 4.3
0 (saline) 1 2 5 10 30
* nmol ACh/g brain & SD. of 6 animals following a~inistration of 100 pig of BrACh.
Table 3. Effect of inhibition of cholinesterase on in vioo inhibition of choline acetyltransferase Pretreatment
Intraventricular
injection
by bromoacetylcholine
Choline acetyltransferase activity
Saline Bromoacetylcholine,
100 pg
3.41 * 0.49’ 3.53 f 0.45 3.35 2 0.37
Physostigmine 0.75 mg/kg 20 min prior to intraventricular injection
Saline Bromoacetylcholine,
100 pg.
3.50 It 044 3.48 + 0.33
Paraoxon 0.7 m&kg/per day for 3 days, last injection 60 min prior to intraventricular injection
Saline Bromoacetylcholine,
100 pg.
3.53 * 029 353 i_ 053
None
None
* Expressed as ~01 ACh synthesizedjg brain per hr & S.D. Activity was measured 30 min after the indicated intraventricuiar injection.
Bromoacetylcholine Table 4. lrz vitro effects of bromoa~t~lcholine
Tissue preparation Purified choline acetyltransferaset Intact synaptosomes Butanol-treated synaptosomes Brain slices Hemi-brain
and ChA
289
on choline a~tyltransfer~e
activity
Apparent &,* No 2 X 10-&M Physostigmine Physostigmine 3 X lo-‘M 4.6 x 10-7M 1.4 X 10-7M No inhibition at 10m4~ BrACh No inhibition at tO-4~ BrACh
1.5
X
10-‘M
3.3 X 10-6M 30% inhibition at 10-4~ BrACh
* Calculated using probit transformation (LITCHFIELD and WILCOXON,1949). t From SASIXYand HENDERSON (1976).
small to produce changes in whole brain ChA activity. To attempt to resolve this question, 1OOpg BrACh was injected intraventricularly to animals which had been previously treated with ChE inhibitors (Table 3). Again, there was no inhibition of ChA activity, even though 3 day administration of paraoxon has been shown to reduce ChE levels to only approx. 7% of control (CEHOVIC,DET~BARN and WELSCH,1972).
To further investigate this problem, in t&o studies were undertaken as outlined in the Methods (Table 4). In the preparation containing intact synaptosomes and in the presence of physostigmine, BrACh was a potent inhibitor of ChA, having an I,, of 1.5 x lo-*M. When physostigmine was not present, the ability of BrACh to inhibit ChA in intact synaptosomes was si~i~~ntly decreased, having an I,, of 46 x lo-’ M.When the s~aptosomes were disrupted by addition of butanol, ChA activity increased from 3.5 ,umol ACh synthesized/g per hr to 6.5 ,mnol ACh synthesized/g per hr. The ability of BrACh to inhibit ChA in disrupted synaptosomes in the absence of physostigmine (I,, = 1.4 x lo-’ M) was similar to the I,, observed using intact synaptosomes (Table 4). The s~i~rity of the ability of BrACh to inhibit ChA in an intact s~aptosomal preparation versus its ability to inhibit purified ChA (Table 4) and the fact that disruption of synaptosomal membranes did not increase inhibitory potency (Table 4), indicates that BrACh was able to enter synaptosomes and produce inhibition of ChA activity, and, in synaptosomes, this inhibition was attained even in the presence of active ChE. To more closely approximate the in viljo situation, in which BrACh would have to diffuse through multicellular layers and would therefore have increased exposure to ChE, brain slices and hemi-brains were investigated. In brain slices, in the presence of physo.&mine (2 x 10e4M), BrACh still had an I,, of 3.3 x 10m6M. In the absence of physostigmine, however, concentrations of BrACh as high as 10V4M did not produce inhibition of ChA activity. Finally, in the hemibrain preparation, 30 min incubation with
10e4~ BrACh produced a 35% inhibition of ChA activity in the presence of physosti~ine, and no inhibition in the absence of phy~sti~ine. These in vitro results using synaptosomeg brain slices and hemi-brains indicate that BrACh can indeed cross neuronal cell membranes, since significant inhibition of ChA was observed in all these preparations in the presence of physostigmme. Even when synaptosomal membranes were disrupted by butanol treatment, as indicated by the increase in observed enzyme activity, BrACh had no greater inhibitory potency than it did in non-disrupted tissue. Nowever, BrACh was considerably less potent in the absence of ChE inhibition. In fact, there was no inhibition of ChA activity in the absence of physostigmine in slices or hemi-brains using BrACh concentrations as high as 10V4M. The most likely conclusion for the in vivo results, therefore, is that BrACh is hydrolyzed by ChE before it can interact with ChA. The decreasing potency of BrACh in the presence of physostigmine obtained using butanol-treated synaptosomes versus intact synaptosomes, versus brain slices, versus hemibrains may result from either a slow diffusion of BrACh through multicellular layers or it may reflect a decrease in the inhibition of intracellular ChE by physostigmine with increasing structural integrity. Of particular interest was the observation that in animals in which 93% of their ChE had been inhibited, BrACh was still without effect on ChA activity. This indicates that there is a large excess of ChE in brain and that the small amount of ChE activity remaining following such treatment must occupy a strategic location in cholinergic neurones. That is, they are still able to function and there is apparently sufficient ChE activity to prevent the access of BrACh to the ~tra~llular site of ChA. In summary, BrACh is unsuitable for in vivo inhibition of ChA, due to rapid hydroxylysis by ChE. It may be possible to synthesize ChE resistant derivatives of the halogenoacetylcholines such as the carbamy1 or ketone analogue, and these might hopefully prove to be useful in uivo ChA inhibitors. Ack~Qwle~eme~ts-This work was supported by USPH Grants DA-000319, MH-11468; training Grant MH-08107 and USAMRDC Contract DADA 17-73-C-3130.
290
R. C. SPETH,D. E. SCHMIDT,B. V. RAMASASSY and D. M. BUXEIALJM REFERENCES
BULL, G. and ODERFELD-NOWAK, B. (1971). Standardization of a radiochemical assay of choline acetyltransferase and a study of the activation of the enzyme in rabbit brain. J. Neurochem. 18: 935-941. CEHOVIC,G., DETTBARN,W. D. and WEIXH, F. (1972). Paraoxon: effects on rat brain cholinesterase and on growth hormone and prolactin of pituitary. Science, N.Y.175: 12561258. CHIOU,C. Y. and SASTRY,B. V. R. (1968). Acetylcholinesterase hydrolysis of halogen substituted acetylcholines. Biochem. Pharmac. 17: 805-815.
LITCHFIELD, J. T., JR. and WILCOXON,F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmac. exp. Ther. 96: 99-113. SASTRY,B. V. R. and HENDERSON, G. I. (1976). Mechanisms of acetylcholine synthesis by rat brain choline acetyltransferase and its regulation by products and inhibitors, In: Drugs and Central Synaptic Transmission (BRADLEY, P. B. and DHAWAN,B. N., Eds.) Macmillan, London. (In press). SC&&T, D. E., SPETH,R. C., WELSCH,F. and SCHMIDT, M. J. (1972). The use of microwave radiation in the determination of acetylcholine in the rat brain. Brain Res. 38: 377-389.
DEBALBIANVERSTER,F., ROBINSON,C. A., HENGELVELD,,SCHRIER,B. K. and SHUSTER,L. A. (1967). A simplified C. A. and SANDERS-BUSH, E. (1971). Free hand cerebroradiochemical assav for choline acetvltransferase. J. ventricular injection technique for unanesthetized rats. Neurochem. 14: 977-985. L.$ Sci. 10 (I): 1395-1402.
