- Email: [email protected]
Contribution of de novo synthesis of acetylcholinesterase to spontaneous recovery of neuromuscular transmission following soman intoxication
European Journal 01 Pharmacology,
381
149 (1988) 381-384
Elsevier EIP 20127
Short communication
Contribution of de novo synthesis of acetylcholinesterase to spontaneous recovery of neuromuscular transmission following soman intoxication Cornelia
J. Van Dongen
*, P. Willy Valkenburg
and Herman
Medical Biological L.&oratory TNO, P.O. Box 45, 2280 AA Rvswijk,
P.M. Van Helden
The Netherlands
Received 15 December 1987, accepted 15 March 1988
The role of the de nova synthesis of acetylcholinesterase in the spontaneous recovery of neuromuscular transmission was studied in diaphragms isolated from soman-intoxicated rats. Ten minutes after soman (3 X LDso i.v.), the transmission appeared to be completely blocked. acetylcholinesterase activity and the neuromuscular Acetylcholinesterase activity in endplate and endplate-free regions recovered linearly during a 3 h experiment (1.5 and 2.9%h, respectively); and neuromuscular transmission was also improved. Since both inhibition of the de novo synthesis of acetylcholinesterase by cycloheximide and the re-inhibition of acetylcholinesterase in vitro by soman did not affect the improvement of neuromuscular transmission, it was concluded that this recovery of neuromuscular transmission can not be attributed to synthesis of new acetylcholinesterase. Acetylcholinesterase
activity; Neuromuscular
1. Introduction Increasing doses of acetylcholinesterase (AChE) inhibiting organophosphates produce a progressive failure of neuromuscular transmission (NMT), i.e. muscles lose their ability to sustain a tetanic contraction upon indirect stimulation with higher frequencies. When kept alive with artificial ventilation, partial recovery of NMT was found in the hours that followed (Meeter and Wolthuis, 1968). Although these authors did not measure AChE activity, they assumed that de novo synthesis of this enzyme did not play a role in this recovery of NMT, since the data available at that time suggested that such a de novo synthesis would take days or weeks. Moreover, in experiments with diisopropyl phosphorofluoridate (DFP), a second dose of this organophosphate did not cause complete NMT again.
* To whom all correspondence should be addressed. 0014-2999/88/$03.50
0 1988 Elsevier Science Publishers
transmission;
(De novo synthesis)
However, since then it has been shown by several authors (Grubic et al., 1981; Fernandez and Stiles, 1984) that newly synthesized AChE-’ activity starts to return in the synaptic regions within several hours after intoxication. In addition, it is becoming increasingly clear that the AChE inhibitor, soman, differs in many ways from DFP, a compound that is mostly used in this type of experiments and that also inhibits serinesterases other than AChE (Churchill et al., 1987). Because the above-mentioned authors did not investigate NMT in direct relation to AChE inhibition, it was decided to study this relationship after soman intoxication. However, it is not excluded that other forms of AChE are synthesized that do contribute to the recovery of NMT but which may be less susceptible to soman. The lack of an effect of a second dose of soman on both enzyme activity and NMT might then erroneously suggest that recovery of NMT is caused by the return of AChE activity. Therefore, it was at-
B.V. (Biomedical
Division)
382
tempted enzymes inhibitor,
to eliminate de novo synthesis of such by administering the protein synthesis cycloheximide.
2. Materials and methods Soman (O-pinacolyl-methylphosphonylfluoridate) was synthesized at the Prins Maurits Laboratory TNO, Rijswijk, The Netherlands. All other compounds were of analytical grade and were obtained commercially. Male Small Wistar (WAG/RIJ) rats which a body weight of 170-180 g were anaesthetized with sodium hexobarbital (175 mg/kg i.p.) and sodium barbital (215 mg/kg i.p.). Atropine sulphate (50 mg/kg i.p.) was given 5 min before the animals were intoxicated with 3 x LD,, soman (248 pg/kg i.v.). The animals were kept alive with artificial ventilation. Some animals received cycloheximide in buffer (200 pg/200 ~1 per h in 154 mM NaCl in 10 mM Na/K phosphate buffer, pH 7.4.), delivered i.p. by osmotic minipumps during 240 min, starting 60 min before soman intoxication. Immediately before, and 10, 90 or 180 min after soman, the animals were killed and diaphragm strips weighing approximately 50 mg each were dissected. The diaphragm strips used to determine NMT were tested in vitro by field stimulation as described by Wolthuis et al. (1981). Each test consisted of three 3 s periods of indirect stimulation at frequencies of 25 (a), 50 (b) and 100 (c) Hz respectively, followed by a 3 s period at 50 Hz with pulses of 30 ps, resulting in a contraction curve (d) by direct muscle stimulation, used as a reference curve ‘within a preparation’. After a first test, soman (0.55 PM) was added to the bath and 10 min later the preparation was tested again. Finally, tubocurarine was added to the bath to ascertain whether stimulation to obtain curves a, b and c had been indirect. NMT was calculated by the empirical formula: NMT = (2a + b + 2/3c)/ 3d. This value was expressed as a percentage of the NMT of control preparations. To assess AChE activity, diaphragm strips were weighed and homogenized with a glass-glass potter in 50 mM Tris/HCl (pH 7.4), 1 M NaCl, 5 mM
EDTA and 1% Triton X-100 (1 : 10, w/v) (Fernandez and Stiles, 1984) at 0-4°C. In some experiments the endplate (e.p.) regions were obtained by isolating an approximately 3 mm wide piece of muscle (1.5 mm on both sides) around the branches of the phrenic nerve. The remainder was considered to be the non-endplate (n.e.p.) region. AChE activity was determined radiometrically according to Johnson and Russell (1975) and protein concentrations according to Bradford (1976). The AChE activity was expressed as a percentage of that in control rats. In some experiments the homogenates were incubated with 1,5-bis-(4-allyldimethylammoniumphenyl)penton-3-one dibromide (BW284c51) or ethopropazine (both lo-’ M) for 60 min at 0-4°C to distinguish between true and pseudocholinesterase.
3. Results Figure 1 shows the AChE activities in e.p. and n.e.p. diaphragm regions. Enzyme activity was not detected 10 min after soman, whereas a clearcut and apparently linear recovery of enzyme activity was found 90 and 180 min after soman. This recovery was slower in the e.p. (1.5%/h) than in the n.e.p. (2.9%/h) regions. Pooling of both regions resulted in an overall recovery of 7.4% in 180 min (table 1). By using BW284c51 and
10
% -a
1
x
z .>
zCl
5_
w
5 a
, 0 10
90
180
time imInI Fig. 1. Recovery of AChE activity in endplate (0) and endplate-free (0) regions of diaphragms from rats intoxicated with soman (3 x LD,, iv.). Values are expressed as percentages of AChE activities found in untreated rats and each point represents the mean k SD. of three determinations.
383 TABLE
1
The effect of cycloheximide (200 pg/h i.p., 240 min) on the recovery of AChE activity and neuromuscular transmission (NMT) in diaphragms from rats isolated either 10 or 180 mm following intoxication with 3 X LD,, soman. Diaphragms used for testing NMT in vitro were again exposed to soman (0.55 PM) for 10 min. AChE activity and NMT are expressed as percentages of the values found in untreated control animals. The values given represent the means k SE.; the number of experiments is given in parentheses
Control
Soman (10min) Soman (180)
Cycleheximide
‘%AChE
+
100 106
k7
% NMT after second dose of soman in vitro
% NMT
(7) 100 (4) (7) 100*2(5)
-
0.5 f 0.3 (3)
+
7.4*0.2(6) 1.3*0.3(7)
8*1
(5)
21+2(6) 21*2(6)
3+1(4) 7*2(5)
9+3
(2)
16+2(3) 17+2(6)
ethopropazine, inhibitors of true and pseudocholinesterase, respectively, we could show that at least 90% of the enzyme activity measured was attributable to true cholinesterase. NMT in these diaphragms was reduced to 8% of that in controls 10 min after soman but recovered to 21% at 180 min after soman. When the animals were treated with cycloheximide, the return of enzyme activity was blocked by 82% at 180 min after soman AChE activity was also completely absent 10 min after a second dose of soman (0.55 PM). However, in both cases a significant reduction of NMT did not take place. By itself, this dose of cycloheximide did not affect NMT (table 1).
4. Discussion The literature on the return of AChE activity following inhibition by organophosphates is slightly confusing. Using DFP, Austin and James (1970) reported that AChE activity in rat brain slowly returned over a period of two days. With the same inhibitor, Fernandez and Stiles (1984) found a return of AChE activity (1.5%/h) in the e.p. regions of the anterior gracilis muscle of the rat. With soman, Grubic et al. (1981) first found a
return of biochemically detectable low molecular AChE activity in the rat diaphragm after two days, whereas with cytochemical methods newly synthesized enzyme was detected within 5-12 h. These findings indicated that newly synthesized AChE activity may reappear much faster than previously expected. Since AChE activity was not measured in the earlier experiments in our laboratory (Meeter and Wolthuis, 1968) and certain assumptions were made with respect to de novo synthesis of AChE and its possible role in the recovery of NMT, a re-assessment of this problem was necessary. In the present experiments with soman, a clearcut and approximately linear return of AChE activity was already detectable within 180 min; the rate of recovery in the e.p. region (1.5%/h) was the same as found by Fernandez and Stiles (1984). The present results also indicate that the return of AChE activity in the n.e.p. regions was higher (2.9%/h) than in the e.p. regions. When e.p. and n.e.p. regions were pooled, 7.4% of the enzyme activity was found in 180 min. This enzyme activity seems small, but a comparison between the minimal amount of NMT required for adequate respiration and the amount of AChE activity in muscle tissue (Van Der Meer and Wolthuis, 1965), indicates that a 5-10s return of enzyme activity should be enough for adequate breathing. Since we used homogenates of muscle in the present study, it was not possible to distinguish between functional (extracellular) and non-functional (intracellular) molecular forms of AChE. The finding that the recovery of NMT was not related to the return of enzyme activity, suggests that the latter does not belong to a ‘functional’ molecular form of AChE. In agreement with this, Fernandez and Stiles (1984) found that the activity of extracellular AChE returned more slowly than that of intracellular AChE in muscle from rats. The finding that the protein synthesis inhibitor, cycloheximide, prevented this return of AChE activity indicates that this enzyme activity is newly formed. Abolishing the AChE activity by a second dose of soman or by preventing the synthesis of these enzymes with cycloheximide hardly affected the recovery of NMT.
384
In conclusion, the recovery of NMT in the first hours after inhibition by soman is independent of AChE activity.
Acknowledgement The authors tance.
thank
G.N.
Fokkema
for his technical
assis-
References Austin, L. and K.A.C. James, 1970, Rates of regeneration of acetyl-cholinesterase in rat brain subcellular fractions following DFP inhibition, J. Neurochem. 17, 705. Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. B&hem. 72. 248. Churchill, L., T.L. Pazdernik, R.S. Cross, M.P. Giesler, S.R. Nelson and F.E. Samson, 1987, Cholinergic systems influence local cerebral glucose use in specific anatomical
areas: diisopropyl phosphorofluoridate versus soman, Neuroscience 20, 329. Femandez, H.L. and J.R. Stiles, 1984, Intra versus extracellular recovery of 16s acetylcholinesterase following organophosphate inactivation in the rat, Neurosci. Lett. 49, 117. Grubic, Z., J. Sketelj, B. Klinar and M. Brzin, 1981, Recovery of acetylcholinesterase in the diaphragm, brain and plasma of the rat after irreversible inhibition by soman: a study of cytochemical localization and molecular forms of the enzyme in the motor end plate, J. Neurochem. 37, 909. Johnson, CD. and R.L. Russell, 1975, A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations, Anal. Biochem. 64, 229. Meeter, E. and O.L. Wolthuis, 1968, The spontaneous recovery of respiration and neuromuscular transmission in the rat after anticholinesterase poisoning, European J. Pharmacol. 2, 377. Van Der Meer, C. and O.L. Wolthuis, 1965, The effect of oximes on isolated organs intoxicated with organophosphorens antichlinesterases, Biochem. Pharmacol. 14, 1299. Wolthuis, O.L., F. Berends and E. Meeter 1981, Problems in the therapy of soman poisoning, Fundam. Appl. Toxicol. 1, 183.
381
149 (1988) 381-384
Elsevier EIP 20127
Short communication
Contribution of de novo synthesis of acetylcholinesterase to spontaneous recovery of neuromuscular transmission following soman intoxication Cornelia
J. Van Dongen
*, P. Willy Valkenburg
and Herman
Medical Biological L.&oratory TNO, P.O. Box 45, 2280 AA Rvswijk,
P.M. Van Helden
The Netherlands
Received 15 December 1987, accepted 15 March 1988
The role of the de nova synthesis of acetylcholinesterase in the spontaneous recovery of neuromuscular transmission was studied in diaphragms isolated from soman-intoxicated rats. Ten minutes after soman (3 X LDso i.v.), the transmission appeared to be completely blocked. acetylcholinesterase activity and the neuromuscular Acetylcholinesterase activity in endplate and endplate-free regions recovered linearly during a 3 h experiment (1.5 and 2.9%h, respectively); and neuromuscular transmission was also improved. Since both inhibition of the de novo synthesis of acetylcholinesterase by cycloheximide and the re-inhibition of acetylcholinesterase in vitro by soman did not affect the improvement of neuromuscular transmission, it was concluded that this recovery of neuromuscular transmission can not be attributed to synthesis of new acetylcholinesterase. Acetylcholinesterase
activity; Neuromuscular
1. Introduction Increasing doses of acetylcholinesterase (AChE) inhibiting organophosphates produce a progressive failure of neuromuscular transmission (NMT), i.e. muscles lose their ability to sustain a tetanic contraction upon indirect stimulation with higher frequencies. When kept alive with artificial ventilation, partial recovery of NMT was found in the hours that followed (Meeter and Wolthuis, 1968). Although these authors did not measure AChE activity, they assumed that de novo synthesis of this enzyme did not play a role in this recovery of NMT, since the data available at that time suggested that such a de novo synthesis would take days or weeks. Moreover, in experiments with diisopropyl phosphorofluoridate (DFP), a second dose of this organophosphate did not cause complete NMT again.
* To whom all correspondence should be addressed. 0014-2999/88/$03.50
0 1988 Elsevier Science Publishers
transmission;
(De novo synthesis)
However, since then it has been shown by several authors (Grubic et al., 1981; Fernandez and Stiles, 1984) that newly synthesized AChE-’ activity starts to return in the synaptic regions within several hours after intoxication. In addition, it is becoming increasingly clear that the AChE inhibitor, soman, differs in many ways from DFP, a compound that is mostly used in this type of experiments and that also inhibits serinesterases other than AChE (Churchill et al., 1987). Because the above-mentioned authors did not investigate NMT in direct relation to AChE inhibition, it was decided to study this relationship after soman intoxication. However, it is not excluded that other forms of AChE are synthesized that do contribute to the recovery of NMT but which may be less susceptible to soman. The lack of an effect of a second dose of soman on both enzyme activity and NMT might then erroneously suggest that recovery of NMT is caused by the return of AChE activity. Therefore, it was at-
B.V. (Biomedical
Division)
382
tempted enzymes inhibitor,
to eliminate de novo synthesis of such by administering the protein synthesis cycloheximide.
2. Materials and methods Soman (O-pinacolyl-methylphosphonylfluoridate) was synthesized at the Prins Maurits Laboratory TNO, Rijswijk, The Netherlands. All other compounds were of analytical grade and were obtained commercially. Male Small Wistar (WAG/RIJ) rats which a body weight of 170-180 g were anaesthetized with sodium hexobarbital (175 mg/kg i.p.) and sodium barbital (215 mg/kg i.p.). Atropine sulphate (50 mg/kg i.p.) was given 5 min before the animals were intoxicated with 3 x LD,, soman (248 pg/kg i.v.). The animals were kept alive with artificial ventilation. Some animals received cycloheximide in buffer (200 pg/200 ~1 per h in 154 mM NaCl in 10 mM Na/K phosphate buffer, pH 7.4.), delivered i.p. by osmotic minipumps during 240 min, starting 60 min before soman intoxication. Immediately before, and 10, 90 or 180 min after soman, the animals were killed and diaphragm strips weighing approximately 50 mg each were dissected. The diaphragm strips used to determine NMT were tested in vitro by field stimulation as described by Wolthuis et al. (1981). Each test consisted of three 3 s periods of indirect stimulation at frequencies of 25 (a), 50 (b) and 100 (c) Hz respectively, followed by a 3 s period at 50 Hz with pulses of 30 ps, resulting in a contraction curve (d) by direct muscle stimulation, used as a reference curve ‘within a preparation’. After a first test, soman (0.55 PM) was added to the bath and 10 min later the preparation was tested again. Finally, tubocurarine was added to the bath to ascertain whether stimulation to obtain curves a, b and c had been indirect. NMT was calculated by the empirical formula: NMT = (2a + b + 2/3c)/ 3d. This value was expressed as a percentage of the NMT of control preparations. To assess AChE activity, diaphragm strips were weighed and homogenized with a glass-glass potter in 50 mM Tris/HCl (pH 7.4), 1 M NaCl, 5 mM
EDTA and 1% Triton X-100 (1 : 10, w/v) (Fernandez and Stiles, 1984) at 0-4°C. In some experiments the endplate (e.p.) regions were obtained by isolating an approximately 3 mm wide piece of muscle (1.5 mm on both sides) around the branches of the phrenic nerve. The remainder was considered to be the non-endplate (n.e.p.) region. AChE activity was determined radiometrically according to Johnson and Russell (1975) and protein concentrations according to Bradford (1976). The AChE activity was expressed as a percentage of that in control rats. In some experiments the homogenates were incubated with 1,5-bis-(4-allyldimethylammoniumphenyl)penton-3-one dibromide (BW284c51) or ethopropazine (both lo-’ M) for 60 min at 0-4°C to distinguish between true and pseudocholinesterase.
3. Results Figure 1 shows the AChE activities in e.p. and n.e.p. diaphragm regions. Enzyme activity was not detected 10 min after soman, whereas a clearcut and apparently linear recovery of enzyme activity was found 90 and 180 min after soman. This recovery was slower in the e.p. (1.5%/h) than in the n.e.p. (2.9%/h) regions. Pooling of both regions resulted in an overall recovery of 7.4% in 180 min (table 1). By using BW284c51 and
10
% -a
1
x
z .>
zCl
5_
w
5 a
, 0 10
90
180
time imInI Fig. 1. Recovery of AChE activity in endplate (0) and endplate-free (0) regions of diaphragms from rats intoxicated with soman (3 x LD,, iv.). Values are expressed as percentages of AChE activities found in untreated rats and each point represents the mean k SD. of three determinations.
383 TABLE
1
The effect of cycloheximide (200 pg/h i.p., 240 min) on the recovery of AChE activity and neuromuscular transmission (NMT) in diaphragms from rats isolated either 10 or 180 mm following intoxication with 3 X LD,, soman. Diaphragms used for testing NMT in vitro were again exposed to soman (0.55 PM) for 10 min. AChE activity and NMT are expressed as percentages of the values found in untreated control animals. The values given represent the means k SE.; the number of experiments is given in parentheses
Control
Soman (10min) Soman (180)
Cycleheximide
‘%AChE
+
100 106
k7
% NMT after second dose of soman in vitro
% NMT
(7) 100 (4) (7) 100*2(5)
-
0.5 f 0.3 (3)
+
7.4*0.2(6) 1.3*0.3(7)
8*1
(5)
21+2(6) 21*2(6)
3+1(4) 7*2(5)
9+3
(2)
16+2(3) 17+2(6)
ethopropazine, inhibitors of true and pseudocholinesterase, respectively, we could show that at least 90% of the enzyme activity measured was attributable to true cholinesterase. NMT in these diaphragms was reduced to 8% of that in controls 10 min after soman but recovered to 21% at 180 min after soman. When the animals were treated with cycloheximide, the return of enzyme activity was blocked by 82% at 180 min after soman AChE activity was also completely absent 10 min after a second dose of soman (0.55 PM). However, in both cases a significant reduction of NMT did not take place. By itself, this dose of cycloheximide did not affect NMT (table 1).
4. Discussion The literature on the return of AChE activity following inhibition by organophosphates is slightly confusing. Using DFP, Austin and James (1970) reported that AChE activity in rat brain slowly returned over a period of two days. With the same inhibitor, Fernandez and Stiles (1984) found a return of AChE activity (1.5%/h) in the e.p. regions of the anterior gracilis muscle of the rat. With soman, Grubic et al. (1981) first found a
return of biochemically detectable low molecular AChE activity in the rat diaphragm after two days, whereas with cytochemical methods newly synthesized enzyme was detected within 5-12 h. These findings indicated that newly synthesized AChE activity may reappear much faster than previously expected. Since AChE activity was not measured in the earlier experiments in our laboratory (Meeter and Wolthuis, 1968) and certain assumptions were made with respect to de novo synthesis of AChE and its possible role in the recovery of NMT, a re-assessment of this problem was necessary. In the present experiments with soman, a clearcut and approximately linear return of AChE activity was already detectable within 180 min; the rate of recovery in the e.p. region (1.5%/h) was the same as found by Fernandez and Stiles (1984). The present results also indicate that the return of AChE activity in the n.e.p. regions was higher (2.9%/h) than in the e.p. regions. When e.p. and n.e.p. regions were pooled, 7.4% of the enzyme activity was found in 180 min. This enzyme activity seems small, but a comparison between the minimal amount of NMT required for adequate respiration and the amount of AChE activity in muscle tissue (Van Der Meer and Wolthuis, 1965), indicates that a 5-10s return of enzyme activity should be enough for adequate breathing. Since we used homogenates of muscle in the present study, it was not possible to distinguish between functional (extracellular) and non-functional (intracellular) molecular forms of AChE. The finding that the recovery of NMT was not related to the return of enzyme activity, suggests that the latter does not belong to a ‘functional’ molecular form of AChE. In agreement with this, Fernandez and Stiles (1984) found that the activity of extracellular AChE returned more slowly than that of intracellular AChE in muscle from rats. The finding that the protein synthesis inhibitor, cycloheximide, prevented this return of AChE activity indicates that this enzyme activity is newly formed. Abolishing the AChE activity by a second dose of soman or by preventing the synthesis of these enzymes with cycloheximide hardly affected the recovery of NMT.
384
In conclusion, the recovery of NMT in the first hours after inhibition by soman is independent of AChE activity.
Acknowledgement The authors tance.
thank
G.N.
Fokkema
for his technical
assis-
References Austin, L. and K.A.C. James, 1970, Rates of regeneration of acetyl-cholinesterase in rat brain subcellular fractions following DFP inhibition, J. Neurochem. 17, 705. Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. B&hem. 72. 248. Churchill, L., T.L. Pazdernik, R.S. Cross, M.P. Giesler, S.R. Nelson and F.E. Samson, 1987, Cholinergic systems influence local cerebral glucose use in specific anatomical
areas: diisopropyl phosphorofluoridate versus soman, Neuroscience 20, 329. Femandez, H.L. and J.R. Stiles, 1984, Intra versus extracellular recovery of 16s acetylcholinesterase following organophosphate inactivation in the rat, Neurosci. Lett. 49, 117. Grubic, Z., J. Sketelj, B. Klinar and M. Brzin, 1981, Recovery of acetylcholinesterase in the diaphragm, brain and plasma of the rat after irreversible inhibition by soman: a study of cytochemical localization and molecular forms of the enzyme in the motor end plate, J. Neurochem. 37, 909. Johnson, CD. and R.L. Russell, 1975, A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations, Anal. Biochem. 64, 229. Meeter, E. and O.L. Wolthuis, 1968, The spontaneous recovery of respiration and neuromuscular transmission in the rat after anticholinesterase poisoning, European J. Pharmacol. 2, 377. Van Der Meer, C. and O.L. Wolthuis, 1965, The effect of oximes on isolated organs intoxicated with organophosphorens antichlinesterases, Biochem. Pharmacol. 14, 1299. Wolthuis, O.L., F. Berends and E. Meeter 1981, Problems in the therapy of soman poisoning, Fundam. Appl. Toxicol. 1, 183.