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Methadone inhibition of glycogen synthase and phosphorylase in rat skeletal muscle
Life Sciences Vol. 13, pp. 1353-1361, 1973 . Printed in Great Britain
Pergamon Press
METHADONE INHIBITION OF GLYCOGEN SYNTHASE AND PHOSPHORYLASE IN RAT SKELETAL MUSCLE Desmond R .H . Gourleyl and Josef D . Schwarzmeier 2 Department of Pharmacology, University of Virginia School
of Medicine, Charlottesville, Virginia 22903
(Received 29 June 1973 ; in final form 8 October 1973) SUMMARY The activities of glycogen synthase (I and total) and phosphorylase (a and total) in crude extracts of isolated extensor dTgitorum longus and soleus muscles of the rat incubated in vitro in the absence or presence .Addition of glycogen during of methadone were very-1-6whomogenization increased the activities of both enzymes in control muscles . Even at optimal concentrations of glycogen, however, the activities of both enzymes from methadone-treated muscles were significantly lower than their activities in control muscles . The activity of phosphoglucomutase was not altered by incubation with methadone or by homogenization with glycogen . It is suggested that the addition of optimal amounts of glycogen during extraction of the enzymes enhances the extractability of glycogen synthase and increases the activity of phosphorylase by some other mechanism and that these processes are interfered with when the muscles are pretreated with methadone .
When added to intact extensor digitorum longus and soleus (EDL + SOL) muscles of the rat in vitro , methadone has a rapid glycogenolytic action (1,2) .
Within 45 min, the glycogen content
of muscles exposed to 5 mM methadone is reduced to about 0 .4 mg/g wet weight, which is only approximately 35% of the glycogen content
1 Present address : Department of Pharmacology, Eastern Vir inia Medical School, 358 Mowbray Arch, Norfolk, Virginia 2357 . 2 Present address : I . Medizinische Universitätsklinik, A-1097 Vienna, Austria . 1353
1354
of the corresponding paired control whether this on
Vol . 13, No . 10
Methadone and Enzymes of Glycogen Metabolism muscles .
To investigate
loss of glycogen was a result of actions of the drug
the primary enzymes of glycogenesis and glycogenolysis,
activities of glycogen
synthase (UDP-glucose :
a-4-glucosyltransferase, (a-1,4-glucan :
EC 2 .4 .1 .11)
in cross-paired
phosphoglucomutase
also measured to compare primary enzymes
EC
had
The activity of
2 .7 .5 .1)
in
the
same muscles was
and glycogenolysis and on an
involved in glycogen metabolism .
synthase and phosphorylase are thought to
associated with glycogen particles Since the
as a functional
be
unit of the
glycogen content of the muscles
after
incubation was known to be extremely low, especially in presence of methadone routinely in
been
the possible effects of methadone on the
of glycogenesis
enzyme not primarily
(3-5) .
EC 2 .4 .1 .1)
(o(-D-glucose-1,6-diphosphate : oL-D-glucose-l-
phosphate phosphotransferase,
cell
and phosphorylase
EDL + SOL muscles which
incubated without or with methadone .
Glycogen
glycogen
orthophosphate glucosyltransferase,
were determined
the
varying
(2),
the
the muscle extracts were prepared
concentrations
of added glycogen .
MATERIALS AND METHODS EDL + SOL muscles of rats weighing 61-87 incubated without or with 5 mM methadone as (2) .
After incubation,
previously described
the muscles were frozen
powdered in a mortar chilled in the
g were prepared and
in liquid
liquid nitrogen,
powder were homogenized at Oo in 5 volumes
and samples of
of an extraction
solution in a glass homogenizer with a motor-driven The extraction solution contained 50 mM Tris, 50 mM potassium fluoride (pH rabbit
7 .8)
plus
0,
nitrogen,
teflon pestle .
5 mM EDTA,
and
0 .5, or 1 .0 mg/ml
liver glycogen which had been purified by passage through
a mixed ion exchange
resin
(Amberlite MB-3) .
The homogenate was
Vol. 13, No. 10
Methadone and Enzymes of Glycogen Metabolism
1355
cleared by centrifugation at 35,000 x 9. for 15 min and the supernatant was passed through Sephadex G-50 at 50 and monitored spectrophotometrically at 250 nm .
The first peak, which contained
the protein fraction, was retained for the enzyme assays . The activities of glycogen synthase I and total glycogen synthase were assayed by the method of Thomas et al .
(6) .
Phosphorylase activities were assayed by a modification of the method of Villar-Palasi and Gazquez-Martinez (7) . Phosphoglucomutase activity was assayed by a modification of the method of Bergmeyer and Klotzsch (8) for the determination of D-glucose-l-phosphate previously described (2) . Glycogen was isolated from a sample of the frozen muscle powder by the method of Walaas and Walaas (9) .
After hydrolysis
of the glycogen, glucose was determined by the Glucostat (Worthington Biochemical Corp .) procedure .
The total glycogen
concentration in each homogenate was calculated from the weight of the muscle powder it contained and the amount of glycogen added to the extraction solution .
The mean glycogen contents of
five homogenates without added glycogen were : control muscles, 0 .23 + 0 .03 mg/ml ; methadone-treated muscles, 0 .08 + 0 .04 mg/ml (+ S .E .M .) . et al .
Protein was determined by the method of Lowry
(10) . RESULTS
The activities of glycogen synthase I and total glycogen synthase in the supernatant fractions after homogenization in different concentrations of glycogen are shown in Fig . 1 . Glycogen synthase activity was significantly lower in the muscles incubated with methadone .
Synthase activity in the homogenates
of both control muscles and methadone-treated muscles, however, increased with increasing concentrations of glycogen present in
1356
Methadone and Enzymes of Glycogen Metabolism T
&rooax 11r1IiE 1
vol. 13, No . 10
,
TOflll GLTC 6El1
m
10
0.5
1.0 GLYCOGEN
a 0.5 m ExrWia FLUID (WPL )
1.0
FIG . 1 The activities of glycogen synthase (synthase I in left panel, total synthase in right panel) in crude extracts of EDL + S01: muscles of the rat which were homogenized in the presence of different concentrations of glycogen . The abscissa represents the total glycogen present in the extraction fluids ; that is, the glycogen present in the homogenized muscle plus added glycogen (if any) . The crosspaired muscles had been previously incubated in the absence (O) or presence of 5 mM methadone (0) . Each point represents the mean synthase activities of preparations from 5 different incubations . The vertical bars represent 2 S .E .M . The synthase activities of all three samples of the methadone-treated muscles were significantly lower than the activities of the corresponding control muscle samples (P<0 .05) . the extraction solution during homogenization .
Maximal activities
of glycogen synthase I and total glycogen synthase were attained when the glycogen concentration was about 1 .0 mg/ml . Similar results were obtained with phosphorylase, as is shown in Fig . 2 .
The activities of phosphorylase a and total
phosphorylase in the control muscles increased with increasing concentrations of glycogen in the homogenate and were approaching a maximum when the glycogen concentration was about 1 .0 mg/ml . Phosphorylase activities in homogenates of methadone-treated muscles, however, were not significantly increased by added
Vol . 13, No. 10
Methadone and Enzymes of Glycogen Metabolism
PIDSPI DRRM a
s
1357
TOTAL RWPIARnM
40
áI
0
0 .5
1 .0 0 0 .5 GLTCDGEII IN EXTRACTION FLUID Gr/O
1.0
FIG . 2 The activities of phosphorylase (phosphor lase a in left panel, total phosphorylase in right paned in crude extracts of EDL + SOL muscles of the rat which were homogenized in the presence of different concentrations of glycogen which are shown on the abscissa. as in Fig . l . (0) represents extracts from control muscles ; ($) represents extracts from muscles previously incubated Each point represents the mean with 5 mM methadone . phosphorylase activity of the same preparations in Fig . 1 . The vertical bars represent 2 S .E .M . The phosphorylase a and total phosphorylase activities of the control and methadone-treated muscle homogenates to which no glycogen was added (first two points in each panel) were not significantly different (P>0 .3) . The phosphorylase activities of all other paired samples were significantly different at a level of 5% or lower . glycogen .
Thus it was only at higher concentrations of glycogen
in the extraction fluid that the activities of phosphorylase a and total phosphorylase of methadone-treated muscles were significantly lower than those of the corresponding control muscles . The activity of phosphoglucomutase was not affected by the addition of methadone during incubation of the muscles or by the addition of glycogen during homogenization, as is shown in Fig . 3 .
1358
Methadone and Enzymes of Glycogen Metabolism
f-
Vol. 13, No . 10
f
4
50D 0
0
0 .5
1.0
GLYCOGEN IN EXTRACTION FLUID (ns/mL) FIG .
3
The activities of phosphoglucomutase in crude extracts of EDL + SOL muscles of the rat which were homogenized in the presence of different concentrations of glycogen which are shown on the abscissa as in Fig . 1 . (p) represents extracts from control muscles ; (0) represents extracts from muscles incubated with 5 mM methadone . Each point represents the mean phosphoglucomutase activity of 4 of the 5 preparations described in Fig . 1 . The vertical bars represent 2 S .E .M . There was no significant difference in the phosphoglucomutase activities of any paired samples of control and methadone-treated homogenates (P>0 .05) . DISCUSSION The significant decreases in the activities of glycogen synthase and phosphorylase in the glycogen-enriched extracts of muscles preincubated with methadone contrast strikingly with the lack of effect of methadone on the activities of phosphoglucomutase in the same extracts . include the following :
Possible explanations
(1) Both glycogen synthase and
phosphorylase require glycogen for full activation
(11,12) and
prior incubation with methadone may have prevented subsequent glycogen activation .
The lack of effect of methadone on
phosphoglucomutase, which is not activated by glycogen, is consistent with this suggestion .
(2) Both glycogen synthase
and phosphorylase appear to be associated with glycogen particles in the liver (3,4) and the phosphorylase-glycogen association
Vol. 13, No . 10
Methadone and Enzymes of Glycogen Metabolism
has been confirmed in skeletal muscle (5) .
1359
Depletion of glycogen,
particularly after incubation of the muscles with methadone, no doubt would alter this association .
Thus, in the relative
absence of glycogen, the enzymes may have become less soluble for extraction, perhaps as a result of aggregation .
(3) Added
glycogen may have prevented inactivation of the affected enzymes by still other mechanisms in the control extracts but not in the extracts of methadone-treated muscles . A comparison of the curves in Fig . 1 and 2 suggests that the same explanation may not apply to the actions of methadone on glycogen synthase and on phosphorylase .
In the case of
glycogen synthase, the proportional decrease in activity in the muscles pretreated with methadone was approximately the same over the entire range of glycogen concentrations present during extraction .
The most likely explanation of those suggested above
is that the addition of glycogen increased the extractability of glycogen syntha.s e from the muscles but that prior incubation with methadone significantly interfered with this process . The data for phosphorylase differ from the data for glycogen synthase in two ways .
First, without added glycogen,
the activities of phosphorylase in both control and methadone treated muscles were the same and hence the extractability of the enzyme from both control and methadone-treated muscles, at least at low glycogen concentrations, was the same .
Second, the
addition of glycogen significantly increased the activity of phosphorylase in the control muscle extracts but had no effect on the activity of phosphorylase in the extracts of methadonetreated muscles .
In the case of phosphorylase, therefore,
pretreatment of the muscles with methadone prevented the increase in enzyme activity attributable to glycogen addition .
It is not
1360
Methadone and Enzymes of Glycogen Metabolism
Vol . 13, No . 10
suggested that the increase in activity of the crude phosphorylase preparation from control muscles in the present experiments occurred by the glycogen activation mechanism proposed for phosphorylase by Wang et al . (12) because the conditions under which phosphorylase was extracted and assayed in the present experiments were quite different from the more rigorous conditions specified by Wang et al . A decision between these and other possible explanations of the depression of the activities of glycogen synthase and phosphorylase in muscle as a result of exposure to methadone must await further investigation .
Regardless of the mechanisms
involved, the results of the present experiments emphasize the importance, in assays of glycogen synthase and phosphorylase activities, of adding sufficient glycogen during extraction when there is reason to .believe that glycogen depletion has occurred, owing, for example . to the action of drugs . This investigation was undertaken to determine whether the -glycogenolytic action of methadone in vitro could be explai .ned'by an action on one or both of the primary enzymes of glycogen synthesis . and degradation .
From the results obta.ined, it seems
unlikely that the .glycogenolyt1c,.acti.on of methadone can be -.-directly attributed to its demonstrated effects on the activities of glycogen synthase and phosphorylase .
At optimal concentrations
of glycogen, the activity of the glycogenolytic enzyme, phosphorylase a, is inhibited to an even greater extent than the activity of the synthetic enzyme, glycogen synthase I . .
It is not suggested that the glycogenolytic effect of
methadone is related to its pharmacologic actions .
Further studies
of this interesting in vitro effect of methadone which bear on this point will be presented elsewhere .
Vol . 13, No . 10 .
Methadone and Enzymes of Glycogen Metabolism
1361
ACKNOWLEDGEMENTS This investigation was supported in part by NIH grant AM 11901 and by NSF grant GB 29558 . J .D .S . wa s a Visiting Research Scholar under the Fulbright-Hays program . The authors appreciate the conscientious technical assistance of Kathleen N . Badal and Suzanne K . Beckner, and the advice of Drs . L .C . Huang, H . Kimura, J . Larner and C . Villar-Palasi . REFERENCES 1 . D .R .H . GOURLEY, Pharmacologist 13 1 3, 263 (1971) . 2 . D .R .H . GOURLEY, Biochem . Pharmacol . i n press . 3 . D .J .L . LUCK, J . biophys . biochem . CCytol .
0, 195-209 (1961. ) .
4 . J .R . TATA, Biochem . J . 90, 284-292 (1964) . 5 . F . MEYER, L .M .G . HEILMEYER, Jr ., R .H . HASCHKE and E,H . FISCHER, J . biol . Chem . 245, 6642-6648 (1970) . 6 . J .A . THOMAS, K .K . SCHLENDER and J . LARNER, Anal . Biochem . Q 25, 486-499 (1968) .
7 . C . VILLAR-PALASI and I . GAZQUEZ-MARTINEZ, Biochim . Biophys . Acta 159, 479-489 (1968) . 8 . H .-U BERGMEYER and H . KLOTZSCH, in Methods of Enzymatic Analysis (Ed . H .-U BERGMEYER) pp . 131-133, Academic Press, New York (1963) . 9 . 0 . WALAAS and E . WALAAS, J . biol . Chem . li, 769-776 (1950) . 10 . O .H . LOWRY, N .J . ROSEBROUGH, A .L . FARR and R .J . RANDALL, J . biol . Chem . 193, 265-275 (1951) . 11 . N .E . BROWN and J . LARNER, Biochim . Biophys . Acta 242, 69-80 (1971) . 12 . J .H . WANG, M .L . SHONKA and D .J . GRAVES, Biochemistry 4, 2296-2301 (1965) .
0~4i
Pergamon Press
METHADONE INHIBITION OF GLYCOGEN SYNTHASE AND PHOSPHORYLASE IN RAT SKELETAL MUSCLE Desmond R .H . Gourleyl and Josef D . Schwarzmeier 2 Department of Pharmacology, University of Virginia School
of Medicine, Charlottesville, Virginia 22903
(Received 29 June 1973 ; in final form 8 October 1973) SUMMARY The activities of glycogen synthase (I and total) and phosphorylase (a and total) in crude extracts of isolated extensor dTgitorum longus and soleus muscles of the rat incubated in vitro in the absence or presence .Addition of glycogen during of methadone were very-1-6whomogenization increased the activities of both enzymes in control muscles . Even at optimal concentrations of glycogen, however, the activities of both enzymes from methadone-treated muscles were significantly lower than their activities in control muscles . The activity of phosphoglucomutase was not altered by incubation with methadone or by homogenization with glycogen . It is suggested that the addition of optimal amounts of glycogen during extraction of the enzymes enhances the extractability of glycogen synthase and increases the activity of phosphorylase by some other mechanism and that these processes are interfered with when the muscles are pretreated with methadone .
When added to intact extensor digitorum longus and soleus (EDL + SOL) muscles of the rat in vitro , methadone has a rapid glycogenolytic action (1,2) .
Within 45 min, the glycogen content
of muscles exposed to 5 mM methadone is reduced to about 0 .4 mg/g wet weight, which is only approximately 35% of the glycogen content
1 Present address : Department of Pharmacology, Eastern Vir inia Medical School, 358 Mowbray Arch, Norfolk, Virginia 2357 . 2 Present address : I . Medizinische Universitätsklinik, A-1097 Vienna, Austria . 1353
1354
of the corresponding paired control whether this on
Vol . 13, No . 10
Methadone and Enzymes of Glycogen Metabolism muscles .
To investigate
loss of glycogen was a result of actions of the drug
the primary enzymes of glycogenesis and glycogenolysis,
activities of glycogen
synthase (UDP-glucose :
a-4-glucosyltransferase, (a-1,4-glucan :
EC 2 .4 .1 .11)
in cross-paired
phosphoglucomutase
also measured to compare primary enzymes
EC
had
The activity of
2 .7 .5 .1)
in
the
same muscles was
and glycogenolysis and on an
involved in glycogen metabolism .
synthase and phosphorylase are thought to
associated with glycogen particles Since the
as a functional
be
unit of the
glycogen content of the muscles
after
incubation was known to be extremely low, especially in presence of methadone routinely in
been
the possible effects of methadone on the
of glycogenesis
enzyme not primarily
(3-5) .
EC 2 .4 .1 .1)
(o(-D-glucose-1,6-diphosphate : oL-D-glucose-l-
phosphate phosphotransferase,
cell
and phosphorylase
EDL + SOL muscles which
incubated without or with methadone .
Glycogen
glycogen
orthophosphate glucosyltransferase,
were determined
the
varying
(2),
the
the muscle extracts were prepared
concentrations
of added glycogen .
MATERIALS AND METHODS EDL + SOL muscles of rats weighing 61-87 incubated without or with 5 mM methadone as (2) .
After incubation,
previously described
the muscles were frozen
powdered in a mortar chilled in the
g were prepared and
in liquid
liquid nitrogen,
powder were homogenized at Oo in 5 volumes
and samples of
of an extraction
solution in a glass homogenizer with a motor-driven The extraction solution contained 50 mM Tris, 50 mM potassium fluoride (pH rabbit
7 .8)
plus
0,
nitrogen,
teflon pestle .
5 mM EDTA,
and
0 .5, or 1 .0 mg/ml
liver glycogen which had been purified by passage through
a mixed ion exchange
resin
(Amberlite MB-3) .
The homogenate was
Vol. 13, No. 10
Methadone and Enzymes of Glycogen Metabolism
1355
cleared by centrifugation at 35,000 x 9. for 15 min and the supernatant was passed through Sephadex G-50 at 50 and monitored spectrophotometrically at 250 nm .
The first peak, which contained
the protein fraction, was retained for the enzyme assays . The activities of glycogen synthase I and total glycogen synthase were assayed by the method of Thomas et al .
(6) .
Phosphorylase activities were assayed by a modification of the method of Villar-Palasi and Gazquez-Martinez (7) . Phosphoglucomutase activity was assayed by a modification of the method of Bergmeyer and Klotzsch (8) for the determination of D-glucose-l-phosphate previously described (2) . Glycogen was isolated from a sample of the frozen muscle powder by the method of Walaas and Walaas (9) .
After hydrolysis
of the glycogen, glucose was determined by the Glucostat (Worthington Biochemical Corp .) procedure .
The total glycogen
concentration in each homogenate was calculated from the weight of the muscle powder it contained and the amount of glycogen added to the extraction solution .
The mean glycogen contents of
five homogenates without added glycogen were : control muscles, 0 .23 + 0 .03 mg/ml ; methadone-treated muscles, 0 .08 + 0 .04 mg/ml (+ S .E .M .) . et al .
Protein was determined by the method of Lowry
(10) . RESULTS
The activities of glycogen synthase I and total glycogen synthase in the supernatant fractions after homogenization in different concentrations of glycogen are shown in Fig . 1 . Glycogen synthase activity was significantly lower in the muscles incubated with methadone .
Synthase activity in the homogenates
of both control muscles and methadone-treated muscles, however, increased with increasing concentrations of glycogen present in
1356
Methadone and Enzymes of Glycogen Metabolism T
&rooax 11r1IiE 1
vol. 13, No . 10
,
TOflll GLTC 6El1
m
10
0.5
1.0 GLYCOGEN
a 0.5 m ExrWia FLUID (WPL )
1.0
FIG . 1 The activities of glycogen synthase (synthase I in left panel, total synthase in right panel) in crude extracts of EDL + S01: muscles of the rat which were homogenized in the presence of different concentrations of glycogen . The abscissa represents the total glycogen present in the extraction fluids ; that is, the glycogen present in the homogenized muscle plus added glycogen (if any) . The crosspaired muscles had been previously incubated in the absence (O) or presence of 5 mM methadone (0) . Each point represents the mean synthase activities of preparations from 5 different incubations . The vertical bars represent 2 S .E .M . The synthase activities of all three samples of the methadone-treated muscles were significantly lower than the activities of the corresponding control muscle samples (P<0 .05) . the extraction solution during homogenization .
Maximal activities
of glycogen synthase I and total glycogen synthase were attained when the glycogen concentration was about 1 .0 mg/ml . Similar results were obtained with phosphorylase, as is shown in Fig . 2 .
The activities of phosphorylase a and total
phosphorylase in the control muscles increased with increasing concentrations of glycogen in the homogenate and were approaching a maximum when the glycogen concentration was about 1 .0 mg/ml . Phosphorylase activities in homogenates of methadone-treated muscles, however, were not significantly increased by added
Vol . 13, No. 10
Methadone and Enzymes of Glycogen Metabolism
PIDSPI DRRM a
s
1357
TOTAL RWPIARnM
40
áI
0
0 .5
1 .0 0 0 .5 GLTCDGEII IN EXTRACTION FLUID Gr/O
1.0
FIG . 2 The activities of phosphorylase (phosphor lase a in left panel, total phosphorylase in right paned in crude extracts of EDL + SOL muscles of the rat which were homogenized in the presence of different concentrations of glycogen which are shown on the abscissa. as in Fig . l . (0) represents extracts from control muscles ; ($) represents extracts from muscles previously incubated Each point represents the mean with 5 mM methadone . phosphorylase activity of the same preparations in Fig . 1 . The vertical bars represent 2 S .E .M . The phosphorylase a and total phosphorylase activities of the control and methadone-treated muscle homogenates to which no glycogen was added (first two points in each panel) were not significantly different (P>0 .3) . The phosphorylase activities of all other paired samples were significantly different at a level of 5% or lower . glycogen .
Thus it was only at higher concentrations of glycogen
in the extraction fluid that the activities of phosphorylase a and total phosphorylase of methadone-treated muscles were significantly lower than those of the corresponding control muscles . The activity of phosphoglucomutase was not affected by the addition of methadone during incubation of the muscles or by the addition of glycogen during homogenization, as is shown in Fig . 3 .
1358
Methadone and Enzymes of Glycogen Metabolism
f-
Vol. 13, No . 10
f
4
50D 0
0
0 .5
1.0
GLYCOGEN IN EXTRACTION FLUID (ns/mL) FIG .
3
The activities of phosphoglucomutase in crude extracts of EDL + SOL muscles of the rat which were homogenized in the presence of different concentrations of glycogen which are shown on the abscissa as in Fig . 1 . (p) represents extracts from control muscles ; (0) represents extracts from muscles incubated with 5 mM methadone . Each point represents the mean phosphoglucomutase activity of 4 of the 5 preparations described in Fig . 1 . The vertical bars represent 2 S .E .M . There was no significant difference in the phosphoglucomutase activities of any paired samples of control and methadone-treated homogenates (P>0 .05) . DISCUSSION The significant decreases in the activities of glycogen synthase and phosphorylase in the glycogen-enriched extracts of muscles preincubated with methadone contrast strikingly with the lack of effect of methadone on the activities of phosphoglucomutase in the same extracts . include the following :
Possible explanations
(1) Both glycogen synthase and
phosphorylase require glycogen for full activation
(11,12) and
prior incubation with methadone may have prevented subsequent glycogen activation .
The lack of effect of methadone on
phosphoglucomutase, which is not activated by glycogen, is consistent with this suggestion .
(2) Both glycogen synthase
and phosphorylase appear to be associated with glycogen particles in the liver (3,4) and the phosphorylase-glycogen association
Vol. 13, No . 10
Methadone and Enzymes of Glycogen Metabolism
has been confirmed in skeletal muscle (5) .
1359
Depletion of glycogen,
particularly after incubation of the muscles with methadone, no doubt would alter this association .
Thus, in the relative
absence of glycogen, the enzymes may have become less soluble for extraction, perhaps as a result of aggregation .
(3) Added
glycogen may have prevented inactivation of the affected enzymes by still other mechanisms in the control extracts but not in the extracts of methadone-treated muscles . A comparison of the curves in Fig . 1 and 2 suggests that the same explanation may not apply to the actions of methadone on glycogen synthase and on phosphorylase .
In the case of
glycogen synthase, the proportional decrease in activity in the muscles pretreated with methadone was approximately the same over the entire range of glycogen concentrations present during extraction .
The most likely explanation of those suggested above
is that the addition of glycogen increased the extractability of glycogen syntha.s e from the muscles but that prior incubation with methadone significantly interfered with this process . The data for phosphorylase differ from the data for glycogen synthase in two ways .
First, without added glycogen,
the activities of phosphorylase in both control and methadone treated muscles were the same and hence the extractability of the enzyme from both control and methadone-treated muscles, at least at low glycogen concentrations, was the same .
Second, the
addition of glycogen significantly increased the activity of phosphorylase in the control muscle extracts but had no effect on the activity of phosphorylase in the extracts of methadonetreated muscles .
In the case of phosphorylase, therefore,
pretreatment of the muscles with methadone prevented the increase in enzyme activity attributable to glycogen addition .
It is not
1360
Methadone and Enzymes of Glycogen Metabolism
Vol . 13, No . 10
suggested that the increase in activity of the crude phosphorylase preparation from control muscles in the present experiments occurred by the glycogen activation mechanism proposed for phosphorylase by Wang et al . (12) because the conditions under which phosphorylase was extracted and assayed in the present experiments were quite different from the more rigorous conditions specified by Wang et al . A decision between these and other possible explanations of the depression of the activities of glycogen synthase and phosphorylase in muscle as a result of exposure to methadone must await further investigation .
Regardless of the mechanisms
involved, the results of the present experiments emphasize the importance, in assays of glycogen synthase and phosphorylase activities, of adding sufficient glycogen during extraction when there is reason to .believe that glycogen depletion has occurred, owing, for example . to the action of drugs . This investigation was undertaken to determine whether the -glycogenolytic action of methadone in vitro could be explai .ned'by an action on one or both of the primary enzymes of glycogen synthesis . and degradation .
From the results obta.ined, it seems
unlikely that the .glycogenolyt1c,.acti.on of methadone can be -.-directly attributed to its demonstrated effects on the activities of glycogen synthase and phosphorylase .
At optimal concentrations
of glycogen, the activity of the glycogenolytic enzyme, phosphorylase a, is inhibited to an even greater extent than the activity of the synthetic enzyme, glycogen synthase I . .
It is not suggested that the glycogenolytic effect of
methadone is related to its pharmacologic actions .
Further studies
of this interesting in vitro effect of methadone which bear on this point will be presented elsewhere .
Vol . 13, No . 10 .
Methadone and Enzymes of Glycogen Metabolism
1361
ACKNOWLEDGEMENTS This investigation was supported in part by NIH grant AM 11901 and by NSF grant GB 29558 . J .D .S . wa s a Visiting Research Scholar under the Fulbright-Hays program . The authors appreciate the conscientious technical assistance of Kathleen N . Badal and Suzanne K . Beckner, and the advice of Drs . L .C . Huang, H . Kimura, J . Larner and C . Villar-Palasi . REFERENCES 1 . D .R .H . GOURLEY, Pharmacologist 13 1 3, 263 (1971) . 2 . D .R .H . GOURLEY, Biochem . Pharmacol . i n press . 3 . D .J .L . LUCK, J . biophys . biochem . CCytol .
0, 195-209 (1961. ) .
4 . J .R . TATA, Biochem . J . 90, 284-292 (1964) . 5 . F . MEYER, L .M .G . HEILMEYER, Jr ., R .H . HASCHKE and E,H . FISCHER, J . biol . Chem . 245, 6642-6648 (1970) . 6 . J .A . THOMAS, K .K . SCHLENDER and J . LARNER, Anal . Biochem . Q 25, 486-499 (1968) .
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