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The influence of chronic treatment with dexamethasone on the acetylcholinesterase activity in rat skeletal muscle
Chem.-Biol. Interactions, 87 (1993) 2 4 9 - 2 5 2
249
Elsevier Scientific Publishers Ireland Ltd.
THE INFLUENCE OF CHRONIC TREATMENT WITH DEXAMETHASONE ON THE ACETYLCHOLINESTERASE ACTIVITY IN RAT SKELETAL MUSCLE
MARTINA BRANK and ZORAN
GRUBI(~
Institute of Pathophysiology, School of Medicine, University of Ljubljana, 61105 Ljubljana (Slovenia)
SUMMARY
In the conditions of chronically elevated glucocorticoid agents in plasma, a drop in AChE activity of about 45% was reported. This data suggests the possibility that among other factors glucocorticoids also control AChE activity in the skeletal muscles. The question addressed in the present investigation was if AChE activity was reduced uniformly or selectively in the rat skeletal muscles after chronic application of dexamethasone? Selective effects of glucocorticoids on the AChE activity in different muscles and/or different types or regions of muscles would suggest the potential of these agents to regulate AChE metabolism in the skeletal muscle according to the environmental demands. Specific activity of skeletal muscle AChE was reduced in sternomastoideus (SM), extensor digitorum longus (EDL) and diaphragm (D) but not in soleus (SOL) after chronic dexamethasone treatment. Axial SM (white part) was more affected than distal white muscle EDL. AChE was better preserved in red rather than in white parts of muscles. The endplate-free region lost twice as much of specific AChE activity than the endplate-rich region. Our results suggest, but do not prove that glucocorticoid agents act in a selective way on the AChE metabolism of the skeletal muscles.
Key words: Dexamethasone -- Acetylcholinesterase -- Skeletal muscle
INTRODUCTION
Hormonal regulation of the metabolism of the cholinergic system components in the skeletal muscle is a rather neglected field, at least in comparison to the attention paid to the neural regulation of these components. However, a drop in specific acetylcholinesterase (ACHE) activity of about 45% was reported in the Correspondence to: Zoran Grubi~, Institute of Pathophysiology, School of Medicine, 61105 Ljubljana, Zalos"ka 4, Slovenia. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
250
quadriceps femoris muscle under conditions of glucocorticoid-induced muscle atrophy [1,2]. Such atrophy is observed after chronically elevated concentrations of glucocorticoids in plasma and is the result of reduced protein synthesis in the muscle [3]. Fast twitch fibers are more affected than slow twitch ones [4], which suggests the selective action of glucocorticoids on the protein synthesis in the skeletal muscle. The present data suggest that glucocorticoids, which are known as transcriptional controllers [5] might play a role in the regulation of the AChE activity in the skeletal muscle in vivo. At the beginning of our investigations of the glucocorticoid effects on the AChE metabolism in the skeletal muscle, the question of whether AChE activity reduced uniformly, or selectively in the skeletal muscles of the rat after chronic application of dexamethasone was addressed. Selective action of glucocorticoids on the AChE activity in different muscles and/or different types or regions of muscle would suggest the potential of these agents to regulate AChE metabolism in the skeletal muscle according to environmental demands. In order to elucidate this problem, AChE activity was determined after chronic dexamethasone application in rat sternomastoideus (SM), diaphragm (D), soleus (SOL) and extensor digitorum longus (EDL) muscle. Red and white parts of the SM were studied separately. Endplaterich and endplate-free regions of the D and of both red and white part of the SM were also studied separately. Relevant metabolic parameters like body weight, nitrogen balance, food and water consumption and urinary output were followed in order to follow the dexamethasone actions in rats. METHODS
Treatment of animals Female Wistar strain albino rats (about 180 g) were treated with dexamethasone (2.5 mg/kg i.p.) until they had lost about 15-20% of their body weight, which took from 10- 15 days. Controls were treated with saline. Daily food intake and urea excretion in urine at 24-h intervals were determined using Techniplast rat metabolic cages and urea nitrogen determination kit obtained from Sigma. Water consumption and urine excretion were also determined. Isolation of tissues Rats were anaesthetized with ether and perfused with saline through the inferior vena cava. SM, D, SOL and EDL muscles were isolated, weighed and frozen in liquid nitrogen until homogenized. The red and white parts of SM, which could be distinguished macroscopically, were divided and weighed separately. Endplate-rich and endplate-free regions of both white and red parts of the SM and of the D were also divided. AChE activity was determined in the homogenates prepared as described previously [6]. RESULTS AND DISCUSSION
Body weights of rats treated with dexamethasone for 10 - 15 days were reduced to about 15% of their initial body weights. In comparison to controls, rats lost
251
25% of their body weight. Increase in liver weight of 3%, loss in brain weight of 5% and loss of skeletal muscle weight from 13% (D) to 26% (EDL) were observed in comparison to control rats (Fig. 1). In accordance with previous reports, predominantly fast twitch white muscle EDL lost more of its weight than predominantly slow twitch red muscle SOL. An exception is a relatively low weight loss in the D, which is considered a fast twitch muscle. Food consumption and water balance did not differ significantly between the treated and the
O) sternomastoideus
c) soleus
Change (%) 15%
W-SM 5%
R-SM
wh.m. cp.f. e.p.
wh.m. ep.L c.p.
-5%
Chan ~(%) 15% 9%
5% -5%
-15%
-15%
-6% -14%
n=32 -25%
-35%
-25%
22; 7__;o_
-35%
.40%
-45 %
-45%
n=6
d) extensor dio_itorum lcngus Chan e (%) 15%
n=||
Chang,: (%) 15%
8%
5% -5%
M.wI. -13% n=22
-25%
',*h.m.
ep. S~A. J
-15%
5%
ep.t.
T.A.
-5% -15%
-17% n-~
-25% -26%
-35% -45%
n=35
-35% -45 %
-40%
Fig. I. Changes in muscle weights (M.W.), AChE specific activities (S.A.) and total activities (T.A.) m (a) sternomastoideus, (b) diaphragm, (c) soleus and (d) extensor digitorum longus muscles after treatment with dexamethasone (2.5 mg/kg i.p.) for about 14 days. The results are presented as differences from the corresponding mean values in control rats. W-SM, white part of the sternomastoideus; R-SM, red part of the sternomastoideus; wh.m., whole muscle; e.p.f., endplate-free region; e.p., endplate region. * Statistically non-significant difference (t-test, P < 0.05). Total activity was calculated by S.A. x M.W.
252
control group. Differences in body weights and weights of particular organs were therefore not the result of reduced appetite or disturbed water balance. Nitrogen balance was slightly negative in the treated group, which is the result of the glucocorticoid-mediated reduction of protein synthesis. Specific activity of skeletal muscle AChE was reduced in SM, EDL and D (endplate-free region) but not in soleus (SOL) and D (endplate-rich region) after chronic dexamethasone treatment (Fig. 1). Axial SM (white part) muscle was more affected than distal white muscle EDL. AChE was better preserved in red as opposed to white muscles. The endplate-free region lost twice as much of specific AChE activity than the endplate-rich region. Our results suggest, but do not prove that glucocorticoid agents act in a selective way on the AChE metabolism of various skeletal muscles, muscle types and muscle regions. The differences observed might also be as a result of differences in the AChE turnover in various muscles and their regions. If, for example dexamethasone reduces the rate of AChE synthesis equally in all muscle fibers, AChE activity will be more impaired in fibers with high AChE turnover and less in fibers with slow AChE turnover provided that AChE degradation rate remains the same. Preservation of AChE from the endplate-rich region might therefore reflect either the resistance of AChE synthesis against dexamethasone or slow turnover of AChE in this region or both. This problem will be investigated in our future studies in which AChE molecular forms and AChE mRNA will also be determined. REFERENCES 1
S. Chokroverty, J. Bernsohn, M.G. Reyes and M. Chokroverty, Effect of adrenocorticotrophic hormone on muscle acetylcholinesterase and nonspecific esterase, Acta. Neurol. Scand., 55 (1977) 226 - 230. 2 A. Gibson and D. Pollock, Reduction in the cholinesterase activity of the rat anococygeus muscle produced by corticosterone, Br. J. Pharmacol., 55 (1975) 66-72. 3 S. Shoji, Myofibrilar protein catabolism in rat steroid myopathy measured by 3-methylhistidine excretion in the urine, J. Neurol. Sci., 93 (1989) 333-339. 4 R.L. Ruff, D. Martyn and A.M. Gordon, Glucocorticoid-induced atrophy is not due to impaired excitability of rat muscle, Am. J Physiol., 243 (Endocrinol. Metab. 6) (1983) E512- E521. 5 M. Muller and R. Renkawitz, The glucocorticoid receptor, Biochim. Biophys. Acta, 1088 (1991) 171 - 182. 6 Z. Grubie, J. Sketelj, B. Klinar and M. Brzin, 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 (1981) 909-916.
249
Elsevier Scientific Publishers Ireland Ltd.
THE INFLUENCE OF CHRONIC TREATMENT WITH DEXAMETHASONE ON THE ACETYLCHOLINESTERASE ACTIVITY IN RAT SKELETAL MUSCLE
MARTINA BRANK and ZORAN
GRUBI(~
Institute of Pathophysiology, School of Medicine, University of Ljubljana, 61105 Ljubljana (Slovenia)
SUMMARY
In the conditions of chronically elevated glucocorticoid agents in plasma, a drop in AChE activity of about 45% was reported. This data suggests the possibility that among other factors glucocorticoids also control AChE activity in the skeletal muscles. The question addressed in the present investigation was if AChE activity was reduced uniformly or selectively in the rat skeletal muscles after chronic application of dexamethasone? Selective effects of glucocorticoids on the AChE activity in different muscles and/or different types or regions of muscles would suggest the potential of these agents to regulate AChE metabolism in the skeletal muscle according to the environmental demands. Specific activity of skeletal muscle AChE was reduced in sternomastoideus (SM), extensor digitorum longus (EDL) and diaphragm (D) but not in soleus (SOL) after chronic dexamethasone treatment. Axial SM (white part) was more affected than distal white muscle EDL. AChE was better preserved in red rather than in white parts of muscles. The endplate-free region lost twice as much of specific AChE activity than the endplate-rich region. Our results suggest, but do not prove that glucocorticoid agents act in a selective way on the AChE metabolism of the skeletal muscles.
Key words: Dexamethasone -- Acetylcholinesterase -- Skeletal muscle
INTRODUCTION
Hormonal regulation of the metabolism of the cholinergic system components in the skeletal muscle is a rather neglected field, at least in comparison to the attention paid to the neural regulation of these components. However, a drop in specific acetylcholinesterase (ACHE) activity of about 45% was reported in the Correspondence to: Zoran Grubi~, Institute of Pathophysiology, School of Medicine, 61105 Ljubljana, Zalos"ka 4, Slovenia. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
250
quadriceps femoris muscle under conditions of glucocorticoid-induced muscle atrophy [1,2]. Such atrophy is observed after chronically elevated concentrations of glucocorticoids in plasma and is the result of reduced protein synthesis in the muscle [3]. Fast twitch fibers are more affected than slow twitch ones [4], which suggests the selective action of glucocorticoids on the protein synthesis in the skeletal muscle. The present data suggest that glucocorticoids, which are known as transcriptional controllers [5] might play a role in the regulation of the AChE activity in the skeletal muscle in vivo. At the beginning of our investigations of the glucocorticoid effects on the AChE metabolism in the skeletal muscle, the question of whether AChE activity reduced uniformly, or selectively in the skeletal muscles of the rat after chronic application of dexamethasone was addressed. Selective action of glucocorticoids on the AChE activity in different muscles and/or different types or regions of muscle would suggest the potential of these agents to regulate AChE metabolism in the skeletal muscle according to environmental demands. In order to elucidate this problem, AChE activity was determined after chronic dexamethasone application in rat sternomastoideus (SM), diaphragm (D), soleus (SOL) and extensor digitorum longus (EDL) muscle. Red and white parts of the SM were studied separately. Endplaterich and endplate-free regions of the D and of both red and white part of the SM were also studied separately. Relevant metabolic parameters like body weight, nitrogen balance, food and water consumption and urinary output were followed in order to follow the dexamethasone actions in rats. METHODS
Treatment of animals Female Wistar strain albino rats (about 180 g) were treated with dexamethasone (2.5 mg/kg i.p.) until they had lost about 15-20% of their body weight, which took from 10- 15 days. Controls were treated with saline. Daily food intake and urea excretion in urine at 24-h intervals were determined using Techniplast rat metabolic cages and urea nitrogen determination kit obtained from Sigma. Water consumption and urine excretion were also determined. Isolation of tissues Rats were anaesthetized with ether and perfused with saline through the inferior vena cava. SM, D, SOL and EDL muscles were isolated, weighed and frozen in liquid nitrogen until homogenized. The red and white parts of SM, which could be distinguished macroscopically, were divided and weighed separately. Endplate-rich and endplate-free regions of both white and red parts of the SM and of the D were also divided. AChE activity was determined in the homogenates prepared as described previously [6]. RESULTS AND DISCUSSION
Body weights of rats treated with dexamethasone for 10 - 15 days were reduced to about 15% of their initial body weights. In comparison to controls, rats lost
251
25% of their body weight. Increase in liver weight of 3%, loss in brain weight of 5% and loss of skeletal muscle weight from 13% (D) to 26% (EDL) were observed in comparison to control rats (Fig. 1). In accordance with previous reports, predominantly fast twitch white muscle EDL lost more of its weight than predominantly slow twitch red muscle SOL. An exception is a relatively low weight loss in the D, which is considered a fast twitch muscle. Food consumption and water balance did not differ significantly between the treated and the
O) sternomastoideus
c) soleus
Change (%) 15%
W-SM 5%
R-SM
wh.m. cp.f. e.p.
wh.m. ep.L c.p.
-5%
Chan ~(%) 15% 9%
5% -5%
-15%
-15%
-6% -14%
n=32 -25%
-35%
-25%
22; 7__;o_
-35%
.40%
-45 %
-45%
n=6
d) extensor dio_itorum lcngus Chan e (%) 15%
n=||
Chang,: (%) 15%
8%
5% -5%
M.wI. -13% n=22
-25%
',*h.m.
ep. S~A. J
-15%
5%
ep.t.
T.A.
-5% -15%
-17% n-~
-25% -26%
-35% -45%
n=35
-35% -45 %
-40%
Fig. I. Changes in muscle weights (M.W.), AChE specific activities (S.A.) and total activities (T.A.) m (a) sternomastoideus, (b) diaphragm, (c) soleus and (d) extensor digitorum longus muscles after treatment with dexamethasone (2.5 mg/kg i.p.) for about 14 days. The results are presented as differences from the corresponding mean values in control rats. W-SM, white part of the sternomastoideus; R-SM, red part of the sternomastoideus; wh.m., whole muscle; e.p.f., endplate-free region; e.p., endplate region. * Statistically non-significant difference (t-test, P < 0.05). Total activity was calculated by S.A. x M.W.
252
control group. Differences in body weights and weights of particular organs were therefore not the result of reduced appetite or disturbed water balance. Nitrogen balance was slightly negative in the treated group, which is the result of the glucocorticoid-mediated reduction of protein synthesis. Specific activity of skeletal muscle AChE was reduced in SM, EDL and D (endplate-free region) but not in soleus (SOL) and D (endplate-rich region) after chronic dexamethasone treatment (Fig. 1). Axial SM (white part) muscle was more affected than distal white muscle EDL. AChE was better preserved in red as opposed to white muscles. The endplate-free region lost twice as much of specific AChE activity than the endplate-rich region. Our results suggest, but do not prove that glucocorticoid agents act in a selective way on the AChE metabolism of various skeletal muscles, muscle types and muscle regions. The differences observed might also be as a result of differences in the AChE turnover in various muscles and their regions. If, for example dexamethasone reduces the rate of AChE synthesis equally in all muscle fibers, AChE activity will be more impaired in fibers with high AChE turnover and less in fibers with slow AChE turnover provided that AChE degradation rate remains the same. Preservation of AChE from the endplate-rich region might therefore reflect either the resistance of AChE synthesis against dexamethasone or slow turnover of AChE in this region or both. This problem will be investigated in our future studies in which AChE molecular forms and AChE mRNA will also be determined. REFERENCES 1
S. Chokroverty, J. Bernsohn, M.G. Reyes and M. Chokroverty, Effect of adrenocorticotrophic hormone on muscle acetylcholinesterase and nonspecific esterase, Acta. Neurol. Scand., 55 (1977) 226 - 230. 2 A. Gibson and D. Pollock, Reduction in the cholinesterase activity of the rat anococygeus muscle produced by corticosterone, Br. J. Pharmacol., 55 (1975) 66-72. 3 S. Shoji, Myofibrilar protein catabolism in rat steroid myopathy measured by 3-methylhistidine excretion in the urine, J. Neurol. Sci., 93 (1989) 333-339. 4 R.L. Ruff, D. Martyn and A.M. Gordon, Glucocorticoid-induced atrophy is not due to impaired excitability of rat muscle, Am. J Physiol., 243 (Endocrinol. Metab. 6) (1983) E512- E521. 5 M. Muller and R. Renkawitz, The glucocorticoid receptor, Biochim. Biophys. Acta, 1088 (1991) 171 - 182. 6 Z. Grubie, J. Sketelj, B. Klinar and M. Brzin, 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 (1981) 909-916.