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Effect of pyridostigmine administration in mice on the expression of motoneuron β-endorphin and muscle acetylcholinesterase
Xth International Symposium on Cholinergic Mechanisms
There is considerable evidence that B-endorphin and its C-terminal dipeptide derivative, glycylglutamine, can control the expression of acetylcholinesterase (AChE) in cholinergic tissues, including skeletal muscle (Haynes et al.. 1984; Haynes and Smith, 1985). heart (Nyquist-Battie and Millington, 1993), and sympathetic ganglion (Koelle, 1983). In previous work we have shown that repeated administration in mice of a low dose of pyridostigmine causes increases in the levels of AChE in skeletal muscle, after the acute inhibitory phase has worn off (Lintern et al, 1997; Lintern et al., 1997). We have :also shown that this drug increases the expression of POMC-derived peptides, including B-endorphin, in motoneurons (Amos et al.. 1996). Here we have compared the time course of the changes in AChE activity in the diaphragm muscle, and the expression of P-endorphin in motoneurons in the cervical spinal cord, following repeated administration of pytidostigmine, in the same animals. In another study we showed that repeated treatment in mice with pyridostigmine sensitised the muscle to a subsequent low dose of the drug given 2 weeks after the repeated treatment was
0
-
C
0 Time
1 Weeks
Figure 1. Time course of the changes in A, the proportion phin immunoreactive motoneurone in cervical spinal cord. tional A12 AChE activity in diaphragm muscle, at 24 h (0 and 2 weeks, after repeated treatment with pyridostigmine. compared to controls (C).
2
of B-endorand B, funcweeks) and I *Significant
495
discontinued (Lintern et al., 1997). Therefore the effect of a booster dose of pyridostigmine on both muscle AChE and motoneuron p-endorphin was also studied. Pyridostigmine hromide (0.4 pmol/kg) was administered to male BKW (6 month old) mice. twice a day for 3 weeks. In one group a further single low dose (0.4 pmol/kg) of the drug wa\ administered at 2 weeks after cessation of the repeated treatment, and the effects on the levels of AChE in the diaphragm muscle and the incidence of P-endorphin-immunoreactive motoneurons were compared with the effect of a single dose in animals which had had no previous treatment with the drug. The molecular forms of AChE were separated on a sucrose density gradient as described before (Haynes et al., 1984) and the enzyme activity was determined using the method of Johnson and Russell (1975). Immunoreactivity for B-endorphin was detected in 20 pm histological sections of cervical spinal cord using the immunoperoxidase method a\ described before (Hughes and Smith, 1994). At 24 h after the end of the 3-week repeated treatment there was a down-regulation of total AChE activity and the activity of the major component molecular forms, including the functional A12 form, in the diaphragm. followed 1 week later by an upregulation to levels significantly higher than normal (figure I). The incidence of motoneurons in the cervical spinal cord which were immunoreactive for B-endorphin was extremely low in untreated mice but there was a significant increase in the incidence after the pyridostigmine treatment. However, the increase in B-endorphin immunoreactivity preceded the increase in AChE activity. In the animals which had been given a further low dose of the drug at 2 weeks after the treatment the AChE activity had initially returned to normal, although the B-endorphin immunoreactivity was still significantly (over four-fold) elevated (&urr 1). The effect of the ‘booster’ dose of the drug in these animals wa, compared with administration of the same dose of the drug to previously untreated animals. In the pretreated animals the booster dose caused a significant (approximately 1304) increase in the levels of the enzyme in the muscle at 3 h, but when the same single low dose of the drug was administered to untreated mice in which the levels of B-endorphin in the motoneurons was initially extremely low figure I, controls), the enzyme activity wa\ unchanged at 3 h (although there was a three-fold increase in immunoreactive motoneurons at this ttme). Thus after treatment with pyridostigmine when an increase in AChE levels in the muscle w’as seen. the increases were preceded by an increase in P-endorphin expression in the motoneurons. These findings are consistent with the increased expression of P-endorphin in the motoneuron being a prerequisite for the upregulation of AChE in skeletal muscle. Thi? work has been carried out with the support of CBD Porton Down. (c) British Crown copyright 1998/DERA. Published with permission of the Controller of Her Britannic Majesty’s Stationary Office. References Amos ML, Smith ME, Ferry Cl3 (1996). the effect of pyridostigmine bromide administration in mice on the expression of POMC-derived peptidec: in motoneurones. J Pbyaiol 491P. l46P.
ABSTRACTS
496
Haynes LW, Smith ME (1985) Induction of endplate-specific acetylcholinesterase by b-endorphin C-terminal dipeptide in rat and chick muscle cells. Biochem Sot Transac 13, 174-175.
Koelle GB. Ruth GA (1983) Demonstration of a neurotrophic factor for the maintenance of AChE and BuChE in the preganglionically denervated superior cervical ganglion of the cat. Proc Nat Acad Sci 80, 3106-31
Haynes LW, Smith ME, Smyth DG (1984) Evidence for the neurotrophic regulation of acetylcholinesterase in immature skeletal muscle by b-endorphin. J Neurochem 42. 1542-1551. Hughes S and Smith ME (1994). Upregulation of the pro-opiomelanocortin gene in motoneurones after nerve section in mice. Mol Brain Res 25. 4149. Johnson CD, Russell RL (1975) A rapid and simple radiometric assay for cholinesterase, suitable for multiple determinations. Analyt Biothem 64, 220-238.
Metrifonate
affects the peak interval L. Spowart,
t
performance
in the rat
B.H. Schmidt, F.J. van der Staay
Metrifonate is a member of the second generation of cholinesterase inhibitors (Giacobini, 1991) that has been found to be a promising therapeutic agent to combat Alzheimer’s disease (Becker et al., 1990). In a number of studies metrifonate has been shown to improve the cognitive performance in various behavioural models (Schmidt et al., 1997). In this study the effect of metrifonate on timing behaviour was assessed. The rats were trained on a discrete-trial 20 s peak interval (PI) timing procedure in ten identical Skinnerboxes. The PI procedure is a modification of the standard fixed interval procedure of reinforcement (Catania, 1970; Roberts, 1981). On a random half of the trials a lever is presented and the animal is free to respond at any time, but only the first lever-press res-
ui
IO.
Lmtem ML, Smith ME, Ferry CB (1997) Effects of pyridostigmine on acetylcholinesterase in different muscles of the mouse. Human C Exp Toxicol, 16. 18-24. Lintern ML, Smith ME, Ferry CB (1997) Effect of repeated treatment with pyridostigmine on acetylcbolinesterase in mouse muscles. Human Exp Toxicol. 16. 158-165. Nyquist-Battie EC, Hagler KE, Millington WR (1993) Glycylglutamine regulates the expression of asymmetric acetylcholinesterase molecular forms in cultured cardiac postnatal myocytes J 5101 Cell Cardiol 25, I I1 1-I 128.
ponse after 20 s is reinforced with a 45 mg food pellet. On the remaining trials. the peak trials, no food is given and the lever remains available until 50 s have elapsed. Then the lever is retracted for IO seconds before the next trial starts. On the peak trials, response rate of trained animals increases to a peak, then the response rate decreases. The effects of a single administration of metrifonate (10 mg/kg and 30 mg/kg) on responding on a 20 h peak procedure were assecsed in Harlan Wistar rats. The animals had received six daily sessions on this schedule prior to drug administration. Metrifonate was admimstered orally 30 min before the hcventh session started. Data were collected for each rat only on peak trials and sessions lasted 30 min.
Session 20
t
Vehicle
--
Metrtfonate
--
Metrifonate
I On&Kg-’ 3Omg*Kg -1
Z-Second Bins
1. The effects of metrifonate in doses of IO mg/kg. 30 mg/kg and vehicle (sodium citrate buffer) on response frequency during the 20 s peak procedure. Results are f S.E.M., n = 9-10 per group.
Figure
2. Mean respome frequency peak procedure at sessmn 20
Figure
+ S.E.M.
(II = 30) during the 20 s
There is considerable evidence that B-endorphin and its C-terminal dipeptide derivative, glycylglutamine, can control the expression of acetylcholinesterase (AChE) in cholinergic tissues, including skeletal muscle (Haynes et al.. 1984; Haynes and Smith, 1985). heart (Nyquist-Battie and Millington, 1993), and sympathetic ganglion (Koelle, 1983). In previous work we have shown that repeated administration in mice of a low dose of pyridostigmine causes increases in the levels of AChE in skeletal muscle, after the acute inhibitory phase has worn off (Lintern et al, 1997; Lintern et al., 1997). We have :also shown that this drug increases the expression of POMC-derived peptides, including B-endorphin, in motoneurons (Amos et al.. 1996). Here we have compared the time course of the changes in AChE activity in the diaphragm muscle, and the expression of P-endorphin in motoneurons in the cervical spinal cord, following repeated administration of pytidostigmine, in the same animals. In another study we showed that repeated treatment in mice with pyridostigmine sensitised the muscle to a subsequent low dose of the drug given 2 weeks after the repeated treatment was
0
-
C
0 Time
1 Weeks
Figure 1. Time course of the changes in A, the proportion phin immunoreactive motoneurone in cervical spinal cord. tional A12 AChE activity in diaphragm muscle, at 24 h (0 and 2 weeks, after repeated treatment with pyridostigmine. compared to controls (C).
2
of B-endorand B, funcweeks) and I *Significant
495
discontinued (Lintern et al., 1997). Therefore the effect of a booster dose of pyridostigmine on both muscle AChE and motoneuron p-endorphin was also studied. Pyridostigmine hromide (0.4 pmol/kg) was administered to male BKW (6 month old) mice. twice a day for 3 weeks. In one group a further single low dose (0.4 pmol/kg) of the drug wa\ administered at 2 weeks after cessation of the repeated treatment, and the effects on the levels of AChE in the diaphragm muscle and the incidence of P-endorphin-immunoreactive motoneurons were compared with the effect of a single dose in animals which had had no previous treatment with the drug. The molecular forms of AChE were separated on a sucrose density gradient as described before (Haynes et al., 1984) and the enzyme activity was determined using the method of Johnson and Russell (1975). Immunoreactivity for B-endorphin was detected in 20 pm histological sections of cervical spinal cord using the immunoperoxidase method a\ described before (Hughes and Smith, 1994). At 24 h after the end of the 3-week repeated treatment there was a down-regulation of total AChE activity and the activity of the major component molecular forms, including the functional A12 form, in the diaphragm. followed 1 week later by an upregulation to levels significantly higher than normal (figure I). The incidence of motoneurons in the cervical spinal cord which were immunoreactive for B-endorphin was extremely low in untreated mice but there was a significant increase in the incidence after the pyridostigmine treatment. However, the increase in B-endorphin immunoreactivity preceded the increase in AChE activity. In the animals which had been given a further low dose of the drug at 2 weeks after the treatment the AChE activity had initially returned to normal, although the B-endorphin immunoreactivity was still significantly (over four-fold) elevated (&urr 1). The effect of the ‘booster’ dose of the drug in these animals wa, compared with administration of the same dose of the drug to previously untreated animals. In the pretreated animals the booster dose caused a significant (approximately 1304) increase in the levels of the enzyme in the muscle at 3 h, but when the same single low dose of the drug was administered to untreated mice in which the levels of B-endorphin in the motoneurons was initially extremely low figure I, controls), the enzyme activity wa\ unchanged at 3 h (although there was a three-fold increase in immunoreactive motoneurons at this ttme). Thus after treatment with pyridostigmine when an increase in AChE levels in the muscle w’as seen. the increases were preceded by an increase in P-endorphin expression in the motoneurons. These findings are consistent with the increased expression of P-endorphin in the motoneuron being a prerequisite for the upregulation of AChE in skeletal muscle. Thi? work has been carried out with the support of CBD Porton Down. (c) British Crown copyright 1998/DERA. Published with permission of the Controller of Her Britannic Majesty’s Stationary Office. References Amos ML, Smith ME, Ferry Cl3 (1996). the effect of pyridostigmine bromide administration in mice on the expression of POMC-derived peptidec: in motoneurones. J Pbyaiol 491P. l46P.
ABSTRACTS
496
Haynes LW, Smith ME (1985) Induction of endplate-specific acetylcholinesterase by b-endorphin C-terminal dipeptide in rat and chick muscle cells. Biochem Sot Transac 13, 174-175.
Koelle GB. Ruth GA (1983) Demonstration of a neurotrophic factor for the maintenance of AChE and BuChE in the preganglionically denervated superior cervical ganglion of the cat. Proc Nat Acad Sci 80, 3106-31
Haynes LW, Smith ME, Smyth DG (1984) Evidence for the neurotrophic regulation of acetylcholinesterase in immature skeletal muscle by b-endorphin. J Neurochem 42. 1542-1551. Hughes S and Smith ME (1994). Upregulation of the pro-opiomelanocortin gene in motoneurones after nerve section in mice. Mol Brain Res 25. 4149. Johnson CD, Russell RL (1975) A rapid and simple radiometric assay for cholinesterase, suitable for multiple determinations. Analyt Biothem 64, 220-238.
Metrifonate
affects the peak interval L. Spowart,
t
performance
in the rat
B.H. Schmidt, F.J. van der Staay
Metrifonate is a member of the second generation of cholinesterase inhibitors (Giacobini, 1991) that has been found to be a promising therapeutic agent to combat Alzheimer’s disease (Becker et al., 1990). In a number of studies metrifonate has been shown to improve the cognitive performance in various behavioural models (Schmidt et al., 1997). In this study the effect of metrifonate on timing behaviour was assessed. The rats were trained on a discrete-trial 20 s peak interval (PI) timing procedure in ten identical Skinnerboxes. The PI procedure is a modification of the standard fixed interval procedure of reinforcement (Catania, 1970; Roberts, 1981). On a random half of the trials a lever is presented and the animal is free to respond at any time, but only the first lever-press res-
ui
IO.
Lmtem ML, Smith ME, Ferry CB (1997) Effects of pyridostigmine on acetylcholinesterase in different muscles of the mouse. Human C Exp Toxicol, 16. 18-24. Lintern ML, Smith ME, Ferry CB (1997) Effect of repeated treatment with pyridostigmine on acetylcbolinesterase in mouse muscles. Human Exp Toxicol. 16. 158-165. Nyquist-Battie EC, Hagler KE, Millington WR (1993) Glycylglutamine regulates the expression of asymmetric acetylcholinesterase molecular forms in cultured cardiac postnatal myocytes J 5101 Cell Cardiol 25, I I1 1-I 128.
ponse after 20 s is reinforced with a 45 mg food pellet. On the remaining trials. the peak trials, no food is given and the lever remains available until 50 s have elapsed. Then the lever is retracted for IO seconds before the next trial starts. On the peak trials, response rate of trained animals increases to a peak, then the response rate decreases. The effects of a single administration of metrifonate (10 mg/kg and 30 mg/kg) on responding on a 20 h peak procedure were assecsed in Harlan Wistar rats. The animals had received six daily sessions on this schedule prior to drug administration. Metrifonate was admimstered orally 30 min before the hcventh session started. Data were collected for each rat only on peak trials and sessions lasted 30 min.
Session 20
t
Vehicle
--
Metrtfonate
--
Metrifonate
I On&Kg-’ 3Omg*Kg -1
Z-Second Bins
1. The effects of metrifonate in doses of IO mg/kg. 30 mg/kg and vehicle (sodium citrate buffer) on response frequency during the 20 s peak procedure. Results are f S.E.M., n = 9-10 per group.
Figure
2. Mean respome frequency peak procedure at sessmn 20
Figure
+ S.E.M.
(II = 30) during the 20 s