The effects are studied of the oral administration of RU38486, a potent selective glucocorticoid antagonist, on muscle weight, non-collagen protein content, and selected enzyme activities (choline acetyltransferase, glucose 6-phosphate dehydrogenase, and glutamine synthetase) following denervation of rat skeletal muscle. Neither decreases in muscle weight, protein content, and choline acetyltransferase activity, nor increases in the activities of glucose 6-phosphate dehydrogernase and glutamine synthetase were affected by RU38486. These data do not support the hypothesis that denervation atrophy results from enhanced sensitivity of muscle to endogenous glucocorticoids

Konagaya, Masaaki

Konagaya, Yoko

Max, Stephen R.

English

NASA Technical Reports Server

PAGSIS fiCc*^ W/t* #191/86
OE POOR QUALITY.
 0- Neurol
(Revised)
Evaluation of the Endogenous Glucocorticoid Hypothesis of Denervation Atrophy
Masaaki Konagaya , Yoko Konagaya* and Stephen R. Max
Department of Neurology
University of Maryland Jf(J "57 "~*
School of Medicine
Baltimore, MD 21201 /y^~72./
U.S.A.
,Key Words: denervation, atrophy, gl ucoco'rticoid, ar.ti :1 ucocortic
glutamine synthetase, g lucose 5-phosphate dehydrogenase, cho l i ne
acetyltransferase, muscle
Send proofs to: ^
—xj
Stephen R. Max, Ph.D. ^ ::
Department of Neurology ^ •
University of Maryland --> --V>[I!
School of Medicine '•" '"o>m
Baltimore, MD 21201 U.S.A. D=> ?-V' o
(301) 528-3436 °° "-<
,«.-,
*r, • , "^Permanent address:
Department of Neurology
!;ara nediccl U n ^ v i r s ' i j
840 Shijo-cho, Kashihara
, iKASi-ci-182818) IVAIOfiTICK OF THE N88-21157
EKDOGENOOS GiDCCCOE1ICOID E3POTBESJS OF
DEKEEVfi l lON ITBO'PBY (Earylano. Dni-v..) 13 p
CSC1 06B Onclas
G3/51 0146721
https://ntrs.nasa.gov/search.jsp?R=19880014773 2020-03-20T06:54:35+00:00Z
SUMMARY
We studied the effects of oral administration of RU38486, a potent and
selective glucocorticoid antagonist, on muscle weight, non-collagen protein
content, and selected enzyme activit ies (choline acetyltransferase, glucose 6-
phosphate dehydrogenase, and glutamine synthetase) following denervation of
rat skeletal muscle. Neither decreases in muscle weight, protein content, and
choline acetyl transferase activity, nor increases in the activit ies of glucose
6-phosphate dehydrogenase and glutamine synthetase were affected by RU38486.
These data do not support the hypothesis that denervation atrophy results from
enhanced sensitivity of muscle to endogenous glucocorticoids.
INTRODUCTION
The mechanism by wh ich denervat ion causes atrophy of skeletal muscle is
not known. DuBois and Almon (1980, 1981) proposed an i n t r i g u i n g hypothesis ,
according to w h i c h enhanced sens i t iv i ty of musc le to endogenous
glucocorticoids might play a role in the promotion of denervat ion atrophy.
This hypothesis was based upon the demons t ra t ion of s t r i k i n g l y increased
cytoplasmic gl ucocor t icoid receptor b i n d i n g after denerva t ion ( rev iewed in
Karpa t i , 1984). Indeed, D u B o i s and Almon noted enhanced cytoplasmic
g lucocor t i co id receptor b i n d i n g in a number of other causes of atrophy
i n c l u d i n g d isuse (1980), m u r i n e (1984b) and a v i a n (1982) dyst rophies , and
atrophy of the l eva to r ani m u s c l e f o l l o w i n g orchiec tomy ( 1 9 S 4 a ) ; they
suggested that a t rophy , i r r e s p e c t i v e of c a u s e , is r e l a t e d to gl u c o c o r t i c o i d
a c t i o n s . I t i s w e l l - e s t a b l i s h e d tha t exogenous g l u c o c o r t i c o i d s can cause
pronounced m u s c l e a trophy ( K o s k i et al ., 1974; K e l l y et al ., 1986; Kona.gaya et
al... 1986a). The hypothesis of DuBois and A l m o n is therefore of in terest and
warrants i n v e s t i g a t i o n , e s p e c i a l l y because of the p o s s i b i l i t y of t r e a t i n g
d e n e r v a t e d . m u s . c l e s w i th gl ucocorticoid an t agon i s t s^ We (Konagaya et a l . ,
. . - ; . . . . . , . 1986a.)..were, .able, to prevent , to a s i g n i f i c a n t extent, glucocorticoid-.mediated
musc le atrophy in the rat u s ing RU38486 ( P h i l i b e r t , 1984), a potent and
se lec t ive gl ucocor t i co id a n t a g o n i s t . Support for the hypothes i s of D u B o i s and
Almon was provided by the a b i l i t y of RU38486 to prevent, to a s i g n i f i c a n t
extent, gonadectomy-mediated a t rophy of the l eva tor ani m u s c l e (Konagaya and
M a x , 1986). We have now used RU38485 to assess whe ther ch ron ic b lockade of
gl ucocorticoid receptors can i n f l u e n c e the course of musc le atrophy f o l l o w i n g
^ e n e r v a t i o n . V.'e assessed c h o l i n e acetyl t r a n s f erase and g lucose 6 -phospha te
dehydrooenase a c t i v i t i e s because they a re ore- and pos t - synap t i c b i o c h e m i c a l
i n d i e - ' - : f ry.-scle d e n e r v a t i o n ( W a g n e r and M a x , 1979; Max e t a l . , 1931; Max 'e t
2 ORIGINAL PAGE IS
OE POOR QUALITY
al.. 1982). We studied glutamine synthetase because 1) its activity is
enhanced following denervation (Konagaya et al.. 1986b), 2) it is involved in
mobilization of ami no acids from muscle proteins (Goldberg and Chang, 1979),
and 3) it may be an important index of muscle wast ing (Max et al., 1986; Max
and Silbergeld, 1986). Further, glutamine synthetase is induced in muscle by
glucocorticoids (Smith et al., 1984; King et al., 1983; Max et al., 1986).
MATERIALS AND METHODS
Adult, male rats (Crl :CD (SD) BR Strain, Charles River Breeding
Laboratories, Wi lmington, MA) weighing 200 g were used in all experiments.
They were given Purina Rodent Laboratory Chow (#5001, Ralston-Purina, St.
Louis, MO) and water ad libitum, and they were maintained on a lighting
schedule of 12 h of right and 12 h of darkness. Denervation was accomplished
under ether anesthesia by uni lateral sect ion of the sciat ic nerve at the
hi p. RU38486 (113 -(4-dimethyl ami nophenyl )"l 73-hydroxy-l 7c-(prop-l-ynyl )estra-
4, 9-dien-3-one; Roussel - UCLAF - Paris, France) was given p.o. at 50 mg/kg
beginning the day before denervation and .every day thereafter until the
'•' termination -of the experiment; This regimen-has been shown to cause blockade.
of muscle .glucocorti.coid. receptors., (Konagaya et_aj_., 19.86), . A t . 2 and 7 days
following denervation, rats were decapitated and denervated and contra!ateral
control extensor digitorum longus and plantaris muscles were removed and
weighed. The extensor digitorum longus muscle was assayed for choline
acetyltransf erase (Max et al_., 1982; Fonnum, 1975) and glucose 5-phosphate
dehydrogenase activi t ies (Max _et al_. , 1931; Schaerf _et aj_., 1982) as
described. Plantaris muscles were used for determination of glutamine
synthetase act iv i ty as described (Rowe, 1985; Smith et al.. 1984), using 5 roM
-" - " - - r te -:s substrate. Both musc les were used to provide sufficient material
for the assays.
ORIGINAL PAGE IS
OE P.OOR QUALITY
Non-collagen protein was assayed as described (Rifenberick et al.,
1974). Protein in supernatant fractions was determined by the method of Lowry
et al. (1951). .
Statistical analyses were made with a paired t-test.
RESULTS
As expected, denervated muscles lost weight. Seven days after neurotomy,
denervated extensor digitorum longus muscles weighed 70% of the contra! ateral
control muscle, while denervated plantaris muscles weighed 75% of the
central ateral control muscle (Table 1). Losses of muscle wet weight were
reflected in corresponding losses of non'-coll agen protein, (Table 1). Glucose
5-phosphate dehydrogenase act ivi ty increased 2- fo ld in denervated extensor
digitoruni longus musc les , as expected (V.'agner and Max, 1979 } (Tab le 2).
Choline acetyl transferase activity in extensor digitorum longus muscles
declined to about 25% of control (Table 2). Glutamine synthetase activity in
plantaris muscles increased 5.5-fold 8 days after denervation (Table 1). None
of these changes was altered by administration'of RU38486 (Tables 1 and 2).
• DISCUSSION . , ' . . . ' . , . ' . . . . " . . ' . - ' . . . . . '
. . . . . . . . The results ..described above ..reveal no .effect, .of chronic blockade of
gl ucocorticoid receptors on four, denervation-mediated events. Choline
acetyl transf erase, a marker of presynaptic integrity (Max et al ., 1981)
decreased, glucose 6-phosphate dehydrogenase (Max et al., 1981) and glutamine
synthetase (Konagaya et aj_., 1986b), biochemical markers of post-synaptic
( i .e . , musc le ) integri ty increased, and muscle weight and protein content
decreased. Concurrent treatment with RU38485 had no effect on the progression
of these a l terat ions fo l lowing denervat ion. These data do not support the
hypothesis that denervat ion-mediated muscular atrophy results from receptor-
mediated gl ucocort icoid action (Karpat i , 1984). Tremblay et al. (1985)
ORIGINAL' PAGE IS.
OF POOR
compared denervation atrophy in adrenalectomized rats with controls; they
found no effect of adrenalectomy in denervation atrophy. Thus, we have
confirmed and extended the conclusions of Tremblay et al. (1986) by using a
potent glucocorticoid receptor blocker, RU38486, and by measuring the
biochemical indices noted above.
We consider glutamine synthetase to be a valuable marker in the present
work because its activity is enhanced in muscle following denervation
(Konagaya et al., 1986b) or administration of gl ucocorticoids (Smith et a! .,
1984; King et al.. 1983; Max et al.. 1986). That the denervation effect is
not blocked by RU38436 (Table 1), whereas that of gl ucocorticoids is blocked
by this compound (Max et aj_., 1986; Max et al. - 1987) suggests two different
itiechani sms for the increases. The present results suggest that endogenous
gl ucocorticoids do not cause the increase in glutamine synthetase act iv i ty
fol lowing denervation. Thus, glucocorticoid- and denervation-mediated muscle
atrophy may be subserved via distinct mechanisms. However, glucocorticoid
'hormones may be involved in enhanced glutamine synthetase activity in soleus
muscles from ..tail-c.asted, hind-limb suspended rats (Jaspers et al_., 1986),
.. ... . b e c a u s e , adrenal ectomy. abolished .differences, in. muscle, .glutamine, .synthetase..
between normal and suspended rats. Jaspers and Tischler (1986) concluded that
elevation of circulating gl ucocorti coi ds is unlikely to be solely responsible
for muscle atrophy secondary to reduced activity, but that catabolic levels of
gl ucocorticoids could alter the response of muscle to unloading. RU38486 has
been shown to be e f fec t i ve in t reat ing a patient with Cushing 's syndrome
(Nieman et al_., 1985). Unfortunately, it seems not to be effective in
preventing denarvat i on-n?edi ated ef fects on muscle. The s igni f icance of the
interesting increase in g lucocort ico id receptor binding in denervated and
disused muscles (DuBois and Almon, 1980, 1981) warrants further exploration.
ORIGINAL PAGE IS
5 OE POOR QUALITY
ACKNOWLEDGEMENTS
We thank Drs. B.H. Sohmer and A. Mattia for helpful comments, Ms. Marilyn
Wennes for technical assistance and Ms. 8. Pasko for preparation of the
typescript. RU38486 was the generous gift of Dr. R. Deraedt, Roussel-UCLAF
(Paris, France). This work was supported in part by grants from NASA (NAG 2-
100), NIH (HD 16956), and the Bressler and Chancel lor 's Funds of the
University of Maryland.
REFERENCES
DuBois, D.C. and R.R. Almon (1980) Disuse atrophy of skeletal muscle is
associated with an increase in number of glucocorticoid receptors.
Endocrinology 107:1649-1551.
DuBois, D.C. and R.R. Almon (1981) A possible role for glucocortico.ids in
denervation atrophy. Muscle Nerve 4:370-373.
DuBois, D.C. and R.R. Almon (1982) The chicken dystrophic model: Does
hypersensitivity to glucocorticoids cause atrophy? Exp. Neurol. 75 :555-
565. .
DuBois, D.C. and R.R. Almon (1984a) Perineal muscles: Possible androgen
regulation of glucocort icoid receptor sites in the rat. J. Endocr.
102:225-229.
DuBois, D.C. and R.R. Almon (19845) Increased content of glucocort icoid
receptors in mouse muscular dystrophy. Endocrine Res. 10:3-10.
Fonnum, F. (1975) A rapid radiochemical method for the determination of
choline acetyl transferase, j. Neurochem. 24:407-409.
Goldberg, A.I. and. T .W. Chang. (1979) Regulat ion .and s igni f icance of am-ino
. .. acid metabolism, in skeletal .rnus.cl e..,.. .Fed. ...P.roc.., 37.:2301.-.23Q7.... .. . • . . . . •
Jaspers, S.R., S. Jacob and M. Tischler (1986) Metabolism of ami no acids by
the atrophied soleus of tai l -casted, suspended rats. Hetabolism 35:215-
223.
Jaspers, S.R. and Tischler, M.E. (1986) Role of gl ucocorti coids in the
response of rat leg musc les to reduced activity. Muscle Merv 9 :554-561.
Karpati, G. (1984) Denervation and disuse atrophy of skeletal muscles -
Involvement of endogenous 3! ucocort ico id hormones? Trends Neurosci.
7:61-62.
ORIGINAL PAGE IS
DE EOOR QUALITY
Kelly, J., J.A. McGrath, D.F. Goldspink, and M.J. Cullen (1986) A
morphological/biochemical study on the actions of corticosteroids on rat
skeletal muscle. Muscle Nerve 9:1-10.
King, P.A., L. Goldstein and E.A. Newsholme (1983) Glutamine synthetase
activity of muscle in acidosis. Biochem. J. 216:523-525.
Konagaya, M., P.A. Bernard and S.R. Max (1986a) Blockade of gl ucocorticoid
receptor binding and inhibition of dexamethasone-induced muscle atrophy
in the rat by RU38486, a potent glucocorticoid antagonist. Endocrinology
119:375-380.
Konagaya, M., Y. Konagaya and S.R. Max (1986b) Neural control of glutamine
synthetase act iv i ty in rat skeletal muscle. Soc. Neurosci. Abstr.
12:1107.
Konagaya, M. and S.R. Max (1986) A poss ib le role for endogenous
glucocort icoids in orchiectomy-induced atrophy of the rat levator ani
muscle: Studies with RU38485, a potent and se lect ive
antigl ucocort icoid. J. Steroid Biochem., in press.
Koski, C.L. , D.H. Rifenberick, .and S.R. Max. (1974) Oxidat ive metabolism of
. skeletal, .muscle., in steroid .atrophy... .Arch... N.eurol. 31_:407-41 0... . . - • ' . . • • , , . - • •
Lowry, O.H., N.S. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein
determination with the folin phenol reagent. J. 8io1. Chem. 193:265-275.
Max, S.R. and E.K. Silbergeld (1986) Skeletal muscle gl ucocorticoid receptor
and glutamine synthetase activity in the wasting syndrome in rats treated
with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Tox ico l . Appl. Pharmacol. In
press.
Max, S .R . , S . S . Oeshpande, and E . X . Albuquerque (1982) Neural regulation of
muscle glucose 5-phosphate dehydrogenase: Effect of batrachotoxin and
tetrodotoxin. J. Neurochem. 38:385-391.
ORIGINAL PAGE IS.
OF POOR
X
Max, S .R. , M. Konagaya, Y. Konagaya, J.W. Thomas, C. Baumer, and L. Vitkovic
(1987) Dexamethasone regulates glutamine synthetase expression in rat
skeletal muscles. Fed. Proc., in press.
Max, S.R., J.W. Thomas, C. Banner, L. Vitkovic, M. Konagaya and Y. Konagaya
(1986) Glucocorti coid receptor-mediated induction of glutamine synthetase
in skeletal muscle cells in vitro. Endocrinology, submitted for
publication.
Max, S.R., J.L. Young and R.F. Mayer (1981) Neural regulation of glucose 6-
phosphate dehydrogenase in rat muscle: Effect of denervation and
reinnervation. Brain Res. 220:131-138.
Nieman, L .K. , G.P. Chrousos, C. Kellner, KM. Spitz, ' B . C . Nisula, G.B.
Cutlery, G.R. Merriam, • C . W . Barden, and D.L. Lon'aux (1985) Successful
treatment of Cush ing ' s syndrome with the glucocorticoid antagonist
RU485. J. Clin. Endocrinol Metab. 61:535-540.
Philibert, D. (1984) RU38485: An original, mult i faceted antihormone in
v i vo. In: Agarwal , M.K. (ed. ) Adrenal Steroid Antagonism, Walter de
Gruyter and Co. , Berlin, pp 1-25.
.-,. Hifen.berick,. D..H.., .,C.L.. .Koski . and,.-S ..R... Max ,0.97.4) Metabolic studies,.of muscle,
regeneration. , Exp. Neurol. 45 :527-540..
Rowe, W . B . (1985) Glutamine synthetase from muscle. Methods Enzymol . 113:199-
212.
Schaerf, F .W. , T. Patz and S .R . Max (1982) Estrogen modulates neural control
of muscle glucose 6-phosphate dehydrogenase. J. Neurochem. 33:1765-1767.
Smith, R.J., S. Larson, S.E. Stred and R.P. Durschlag (1984) Regulation of
glutamine synthetase and glutaminase act iv i t ies in cultured skeletal
musc le ce l ls . J. Cell Physio!. 120:197-203.
ORIGINAL PAGE IS
OS EOOR QUALITY
Tremblay, R.R., M.A. Ho-Kim, C. Champagne, J. Gagnor and J.Y. Dube'(1986)
Variat ions in gl ucocorticoid receptors in intact or denervated muscles:
Lack of cause-effect relationship with muscle atrophy in the rat. J.
Receptor Res. 6_:183-193.
Wagner, K.R. and S.R. Max (1979) Neurotrophic regulation of glucose 6-
phosphate dehydrogenase in rat skeletal muscle. Brain Res. 170:572-576.
10
TABLE 1
EFFECT OF DENERVATION AND RU38486 ON MUSCLE WEIGHT AND NON-COLLAGEN PROTEIN CONTENT.
Treatment Muscle Weight
(mg)
Extensor Digi'torum
Longus
Plantaris
Non-Collagen Protein
(mg/muscle)
•
 i : , i
Extensor Digitorum Plantaris
Longus
Control
Denervated
Control + RU38486
Denervated + RU38486 '
135.0
94.8
132.9
loo.'o
±
±
±
±
i i ' '' i i
31.5
:r /i*5.4
12.5
5.8**
230.0
172.9
242.9
181.4
± 25.
± 21.
± 19.
± 15.
8 %
4*
8
7**
24.41
16.26
22.71
. 15.54
± 2
± 2
± 2
± 1
.92
.10*
.05
. .** .
.44
47.61
29.63
43.97
27.00
± 4.35
± 2.45*
± 4.39
± 4 . 3 7 * *
Data are means ± SD, n = 7. Rats were decapitated and muscles removed 8 days post-denervation. RU38486 was given
orally at 50 mg/kg.
Significantly different from untreated contra!ateral control, p < 0.01. ;
**Significantly different from central ateral control + RU38486, p < 0.01.
There were no significant differences between untreated and RU38486-treated groups. Experimental procedures are
described in the text. ..
TABLE 2.
EFFECT OF DENERVATION AND RU38486 ON GLUCOSE 6-PHOSPHATE DEHYDROGENASE, GLUTAMINE SYNTHETASE, AND CHOLINE
ACETYLTRANSFERASE ACTIVITIES.
Denervated + RU38486
Glucose 6-Phosphate
Dehydrogenase
(nmol/min/mg protein)
Glutamine Choline
Synthetase Acetyltransferase
(nmol/h/mg protein) (pmol/min/mg non-collagen protein)
Control
Denervated
Control + RU38486
•' 1.50 ± 0.13
3.27 ± 0.50*
' .. 1.64 ± 0.22
5.4 ± 0.8
. 29.8 ± 11.5*
5.8 ± 1.6
11.99 ± 1.11
2.97 ± 1.25*
11.31 ± 1.25
3.30 ± 0.38** 29.0 ± 9.1** 2.99 ± 1.25**
Data are means ± $D,'
 n = 7. Rats were decapitated and muscles removed 8 days post-denervation. Glucose 6-phosphate
dehydrogenase and choline acetyltransferase activit ies were assayed in extensor digitorum muscles. Glutamine synthetase
activity was assayed in plantaris muscles. RU38486 was given orally at 50 mg/kg. ';:
i.
Significantly different from untreated contra! ateral control, p < 0.01; :
**Significantly different from contralateral control + RU38486, p < 0.01.
There were no significant differences between untreated and RU38486-treated groups.
Experimental procedures are described in the text.


Evaluation of the endogenous glucocorticoid hypothesis of denervation atrophy

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server