RU38486, a potent and selective antiglucocorticoid, was employed to study a possible role for endogenous glucocorticoids in atrophy of the levator ani muscle secondary to castration of male rats. RU38486 was shown to block (3H) triamcinolone acetonide binding to cytosol from levator ani muscle. Daily oral administration of RU38486 to castrated rats partially prevented atrophy of the levator ani muscle, as well as a decrease in RNA concentration. In a control group receiving RU38486 alone, the levator ani underwent significant (20%) hypertrophy. Administration of exogenous dexamethasone also caused pronounced atrophy of the levator ani muscle. This atrophy was prevented, to a significant degree, by simultaneous oral administration of RU38486. It is concluded that endogenous glucocorticoids, the actions of which are blocked by RU38486, may be involved in regulation of the mass of the levator ani muscle in intact rats

Konagaya, M.

Max, S. R.

English

NASA Technical Reports Server

ORIGINAL
OF POOR QUAUTt
A POSSIBLE ROLE FOR ENDOGENOUS GLUCOCORTICOIDS
IN ORCHIECTOMY-INDUCED ATROPHY OF THE RAT LE\'ATOR ANI MUSCLE:
STUDIES WITH RU3-31)36, A POTENT AND SELECTIVE ANTIGLUCOCORTICOID
MASAAKI KONAGAYA and STEPHEN R. ,MAX
Department of Neurology
University of Maryland
School of Medicine
Baltimore, MD 21201 (U.S.A.)
R u n n i n g Tit le: Glucocort icoids and Muscle Atrophy
L-CB-177029) > ft POSSIBLE BOIE POR N86-30343
E N D O G E N O U S GLUCOCOBTICOIDS IN
OBCHIECTOMY-INDUCED ftTBOPHY, OF THE BAT
LEV&TOB SNI HUSCLE: STUDIES WITH £038486,, A
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POTENT AND SELECTIVE aNTIGLOCC-CGBTICOID -^/55 43232
Send proofs to:
Stephen R. Max , P h . D .
Department of Neurology
Univers i ty of Mary l and
School of M e d i c i n e
Bal t imore , MD 21201
U . S . A .
sPer;n<inont address :
Depar tment of 'Jrjrol o^y
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https://ntrs.nasa.gov/search.jsp?R=19860020871 2020-03-20T14:37:25+00:00Z
ORIGINAL PASS 8S
OF POOR QUALITY
SUMMARY
We employed RU3S^86, a potent and selective antiglucocorticoid, to s tudy
a possible role for endogenous glucocorticoids in atrophy of the levator ani
r jusc le secondary to castration of male rats. RU33^86 was shown to block [ H]
triaraeinolone acetonide b inding to cytosol f rom levator ani muscle . Dai ly
oral adminis t ra t ion of EUSS^SS to castrated rats par t ia l ly prevented atrophy
of the levator ani muscle, as well as a decrease in BNA concentrat ion. In a
control group receiving RU38^86 alone, the levator ani unde rwen t s ignif icant
(20%) hyper t rophy. Adminis t ra t ion of exogenous dexaraethasone also caused
pronounced atrophy of the levator ani muscle. This atrophy was p reven ted , to
a significant degree, by s imultaneous oral adminis t ra t ion of RU33 ; J86. It is
concluded that endogenous glucocorticoids, the actions of wh ich are blocked by
?i'J38^86, inay be involved in regulation of the mass of the levator ani muscle
in intact rats.
ORIGINAL PAGE SS
OF. POOR QUALITY
INTRODUCTION
The leva tor ani muscle of the rat is hormone dependen t ; it undergoes
atrophy following orchiectomy, and it hypertrophies when male rats are given
testosterone [1 ], The muscle is present in female rats at bi r th . Shortly
thereaf ter , unless the animals are given andrcgens, the muscle disappears L2,
3]. An obvious hormonal regulator of the size of this muscle is
testosterone. Recent ly , however , DuBois and Almon reported that castration of
male rats caused a two-fold increase in the cytosolic glucocorticoid receptor
in this rauscle (together with the bulbocavernosus muscle) [4] . DuBois and
Almon also demonstrated increases in cytoplasraic glucocorticoid receptor
binding in skeletal muscle folio-wing denervat ion [5], disuse [6], and in
nrurine [7] or avian muscular dystrophy [8]. Based on these resul ts , it was
hypothesized that endogenous glucocorticoids may be involved in a fundaiTiental
manner in muscle atrophy in these diverse condit ions [9]. Thus , maintenance
of the mass of the leva tor ani muscle , a n d , possibly, "ordinary" rVele ta l
musc le , may involve a balance between the anabolic actions of androgens and
the catabolic actions of glucocorticoids. We tested this hypothesis by
assessing the effects of a potent and selective antiglucocorticoid, RU38 ' l86 ,
on dexamethasone- and orchiectoray-induced atrophy of the rat levator ani
rauscle.
EXPERIMENTAL
Adul t male rats of the Cr l :CD(SD)BR strain (Charles River Breeding
Laboratories, Boston, M A ) , weighing 131) ± 9 grams (orchiectomy s tudy) or 157 ±
6 grams (dexarnethasone s tudy) were used in all exper iments . Yhsy were
maintained in a. 12h on:12h off light cycle, and they were fed Pur ina Lab Chow
#5001 and water ad_ lib_itu_ra. Bilateral orchiectomy was carried out by the
abdominal route under ether anesthesia. Dexaroethasone was i n ; < ; j l : d
K
ORIGINAL PAGE IS
Of. POOR QUALITY
subcutaneously at 5 rag/kg. RUBS^SS was administered orally at 50 mg/kg in an
aqueous vehicle comprising carboxyraethylcellulose (0.25?) and polysorbate 80
(0.20?) (10).
Glucocorticoid receptor binding in gastrocnemius muscle was assessed
using [^H] t r iamcinolone acetonide (9a- f luoro-11B- l6a , 17a,21 te t rahydroxy-
pregn-l , i )-diene-3, 20-dione-l6, 17-acetonide, TA, specific act ivi ty 30
Ci/rnrool) . Minced muscle was homogenized in a polytron horaogenizer in ice-cold
buf fe r (5 ral/g tissue) comprising 25 raM Na2 HP0 4 , 1.5 raM EDTA, 10? ( v / v ) ,
glycerol, 2 mM dithiothreitol, 10 mM sodium raolybdate and 1 raM
phenylraethylsulfonyl f luor ide , pH 7.2. The homogenate was centrifuged for 60
rnin at 110,000 x g in a Sorvall OTD-50 preparat ive u l t racen t r i fuge ; the
resulting supernate was designated the cytosol. Receptor binding to the
cytosol f ract ion was assessed at a single saturat ing concentrat ion (15 nM) of
TA. Portions ( 0 . ^ ml) of the cytosol were incubated in duplicate with 0.1 ml
of [ H] TA in buf fe r in either the presence or absence of a 200-fold mo la r
excess of unlabelled TA. Incubat ion was carried out for 20 h at 0-^°C. These
conditions permit optimal exchange in rat skeletal muscle cytosol [11]-
Hydroxylapa t i t e was employed to separate bound and unbound ligand [12-14].
Et'nanol-extracted hydroxylapat i te (2 ml) was added to 16 ml Liquiscint aqueous
fluor (National Diagnostics, Soraerville, N J ) , and radioactivity was determined
at 32? ef f ic iency in a Beckiaan LS-235 l iquid scinti l lat ion spectrometer.
Specific binding was def ined as the d i f fe rence in the value of b ind ing in the
absence and in the presence of excess unlabelled steroid.
Non-collagen protein was determined as described [15] using the protein
assay of Lowry et aU [l6].
SNA and ONA were de termined according to Fleck and Munro D?].
ORIGINAL PAGE 33
OF POOR QUALITY
Statistical analyses were rcade by analysis of variance and Dunnet t ' s
mult iple comparisons test p8].
RESDLTS
As reported for skeletal muscle [l9], RU38^86 blocks [^H]-TA specific
binding to cytosolic glucocorticoid receptors in levator ani muscle in vivo
(Fig. 1). Receptor blockade remained complete for 12 h a f te r oral
adminis t ra t ion of RU33486. By 2}4 h after RU38486 adminis t ra t ion receptor
binding returned to the control value (Fig. 1). The regiraen described above
for BU3SJJ86 was effect ive to a s ignif icant ' extent' in preventing muscular
atrophy secondary to daily dexaraetbasone injections or to castration. Fig. 2
shows levator ani Tuscle wet weights f rom control, dexaroethasone-treated ,
dexaraethasone + RU38'486-treated, and RU38 !l86-treated rats. These were
expressed as % body weight [20], because body weight changes f o l l o w i n g
dexamethasone adminis t ra t ion [19J. Dexamethasone caused about a 50? less of
levator ani rauscle wet weight in 8 days of daily injections. Tn dexaraethasone
+ RU38M86-treated rats, the loss of rauscle wet weight was s ignif icant ly
less. Surprisingly, in the group treated with RU38')86 a lone, the levator ani
muscle wet weight was s ignif icant ly (20£) larger than muscle f rom the
unt rea ted group (Fig. 2). Similar changes were noted in the RNA content of
levator ani muscles. In Table I, we see that neither non-collagen protein
concentration nor DNA concentration changed s ignif icant ly in the
dexamethasone-treated rats. There was, however , a significant loss of RHA in
the dexamethasone- t reated group. This effect was part ly amel iora ted by
RU38486 (Fig. 2) .
Hypertrophy caused by HU33^36 suggested that endogenous g l u o c c - r t i - j : I s
night inf luence l-ivaoor -i.ii auscle weight, since RU3o^85 is a : '<
ORIGINAL PAGE flS
0E POOR QUALITY
antiandrogen [id]. Indeed, RU33^86 was able to prevent , to a significant
degree, atrophy of the leva tor ani muscle secondary to castration (Fig. 3)-
Eight days after orchiectomy , levator ani wet weight was about ^0* of the
control value. .Adminis t ra t ion of RU38M86 , beginning the day before castration
and continuing dai ly thereaf ter , prevented this atrophy to a marked extent
(Fig. 3). Table II shows R N A , D N A , and non-collagen protein data for
castrated rats. Again the DNA and non-collagen protein concentrat ion of the
muscles did not decrease. However , there was RNA loss, which was part ia l ly
prevented by RU3SH86 (Table II).
DISCUSSION
The data presented above indicate that B'JSSMSG can prevent , to a.
s ignificant degree, atrophy of the levator ani .r.usele secondary to exogenous
glucocorticoid adminis t ra t ion or following gonadectorny. In the f irst
ins tance , the data confi rm our results on h ind- l imb muscles (gast rocnernius ,
plantaris, extensor digitorum longus) [19J. Since the levator ani muscle is
composed homogeneously of type II muscle f ibers , it was expected that it would
be sensitive to glucocorticoid adminis t ra t ion , and that the resulting atrophy
would be retarded by RU38^86. The surprising result of this experiment was
that RU38M85 alone caused a 20* increase in the wet weight of this muscle.
Because RUSS^SS is a weak antiandrogen [lO], and because there is no evidence
for the presence of a progestin receptor in muscle [ s . R . K a x , unpubl ished
observations; 21, 22], it is l ikely that the an t ig lucocor t i co id act ivi ty of
31333^86 was responsible for this e f fec t . Thus, main tenance of the mass of the
Isvator ani muscle nay not be dependent exc lus ive ly on the presence of
androgens; endogenous glucocorticoids also might be involved. This hypothesis
is supported by the cat?, of ?is. 3 and Table 1. In this -.-xperi.r.-'Vc 7(1)38-'! 36 -.-as
able to p reven t , to ~. s ign i f i can t ex ton t , atrophy of the levator ani muscle
secondary to gonadec tomy. If the sole action of RU38^86 is to block
glucocorticoid receptors, as appears to be the case, then regulation of the
mass of the levator ani muscle depends upon a balance between anabolic and
catabolic hormones. if the balance is upset by relative androgen excess, the
muscle hypertrophies. If the balance is upset by relative glucocorticoid
excess, the muscle atrophies.
As noted in the tables, the RNA content of the atrophying levator ani
muscles decreased. This result is in contrast to our f inding with skeletal
muscle [19], in which dexaraethasone administrat ion caused no significant
decrease in RNA concentrat ion. Others [23, 21) ] have reported that
glucocorticoid-induced muscle atrophy is accompanied by decreased RNA
content . The loss of RNA in rat levator ani muscle was prevented by n U S S M S S .
These data provide a .novel perspective on muscular a t rophy, and they
suggest possible cl inical applications of glucocorticoid antagonists.
•£• US
ACKNOWLEDGEMENTS
Vie thank Dr. B.H. Sohmer for helpful comments, Ms. M. Wennes for
technical assistance, and Ms. B. Pasko for preparation of the typescript.
This work was supported in part by NASA grant NAG 2-100.
? POOR QUALITY
ORIGINAL PAGE m
OF POOR QUALITY
REFERENCES
1. Venab le , J . H . Morphology of the cells of normal , testosterone-deprived
and testosterone-stimulated levator ani muscle. J. Anat.119 (1966) 271-
302.
2. Cihak, R. , G u t m a n n , E. and Hanz l ikova , V.: Involution and hormone-induced
persistence of the M. sphincter ( levator) ani in female rats. J. Ana t .
106 (1970) 93-110.
3- Hanzl ikova, V., Schiaff ino, S. and Settembrini, P. Histoc'nemical fiber
type characteristics in the normal and the persistent levator ani muscle
of the rat . Histochemie 22 (1970) i)5-50.
4 . DuBois , D . C . and A l m o n , R . R . Ferineal muscles: possible andrcgen
regulation of glucocorticoid receptor sites in the rat . J. Sndocrinol.
102 (198*0 225-229.
5. DuBois, D . C . and A l m o n , R . R . A possible role for glucocorticoids in
denerva t ion a t rophy . Muscle Nerve 4 (1931) 370-373.
6. DuBois, D . C . and Alraon, R . R . : Disuse atrophy of skeletal muscle is
associated w i t h an increase in the number , of glucocorticoid receptors.
Endocrinology K)_7 (1981) 16*19-1651.
7. DuBois , D . C . and Al raon , R . R . Increased content of glucocorticoid
receptors in incuse muscular dyst rophy. Endocrine Res. 10 (1934) 3-10.
8. DuBois, D . C . and Al raon , R . R . The chicken dystroohic model : Docs
hypersensi t iv i ty to glucocorticoids cause atrophy? gxp. J;!51H1O-:L- ^2.:
(1984) 555-565.
9. Karpati, G. Denervation and disuse atrophy of skeletal muscles
involvement of endogenous glucocorticoid hormones. Trends J*eurosci_. 7_
(19S'O 61-62.
ORIGINAL .PAGE IS
OF POOR QUALITY
10. Philibert , D. RU3S186: An original roultifaceted antihormone j.n vivo.
In: Adrena l Steroid Antagonism (Edited by M . K . Agarwal) Walter de Gruyter
and Co . , Berlin (1981) p. 1-25.
11. Ho-Kira, M . A . , Trerablay, . R . R . and Dube' J . Y . Determination of occupied
' cytoplasroic glucocorticoid receptor sites by an exchange assay in rat
muscle. J. Steroid Biochem. IB (1983) 179-181.
12. Liao, S., Wi t t e , D. , ' Schilling, K. and Chang , C. The use of
hydroxylapat i te filter steroid receptor assay method in the study of the
modulation of androgen receptor interaction. J. Steroid Bioc'nero. 2Q_
(1981)- 11-17.
13. Sakai, D. and Gorski, J. Estrogen receptor t ransformat ion to a high
a f f i n i t y site wi thout subuni t -subuni t interactions. j?ipj^he_T!_istr_y_ 31
(1931) 3511-3-517.
ll. Williams, D. and Gorski, J. Equilibrium binding of estradiol by uterine
cell suspensions and whole uteri in vitro. giocneroistry 13 (1961) 5537-
5512.
15. Ri fenber ick , D . H . , Koski , C . L . and Max , S .R . Metabolic studies of
skeletal muscle regeneration. Sxp. iieurol. _15_ (1971) 527-510.
16. Lovjry, O . H . , Rosebrough, N . S . , Farr , A . L . , and R a n d a l l , R . J . Protein
measurement with the Folin phenol reagent. J. 3iol. Chen). 193 (1951)
265- 275.
17. M u n r o , H . N . and Fleck, A. Recent developments in the measurement of
nucleic acids in biological mater ia l s . Ana lys t , 7^ (1935) 79-88.
18. Winer , 3.J. Principles of Exper imental Design, pp. 302-313, M c G r a w - H i l l ,
New York, 1962.
19. Kon?-s=ya, M. , Bernard , P. A. and '/.ax, S . R . Glucocort icoid receptor Vndi r .g
and inh ib i t ion of dexansethasons - induced .:-uscle atrophy in the ."-1 ":y a
10
ORIGINAL PAGE US
POOR QUALITY
potent glucocort icoid antagonist. Endocrinology Submitted for
publication.
20. Flynn , D. and Max , S .R. Effects of suspension hypokinesia/hypodynaroia on
rat skeletal muscle. A v i a t . Space Environ. Med . , in press.
21. Scbreiber, J . R . and Hsueh , . 4 . J . W . Progesterone "receptor" in rat ovary.
Endocrinology, 105, (1979) 915-919-
22. Perro t -Applanat , M. , nogeat, P., Groyer-Picard , M . T . , and Mil gron), E.
Imraunocy toc'neraical study of n-.arciLalian progesterone receptor using
monoclonal antibodies. Endocrinology 116:1^73-1^89.
23. Rannels , S . R . , Rannels , . D . G . , Pegg, • A . E . and Jefferson, L.S.
Glucocorticoid effects on pept ide-cbain initiation in skeletal muscle and
heart. .-^.^^PjTy^sj^oJU 235, (19-32) S13J-!-El39.
2^J . Odedra , B . R . and Mi l l ia rd , D . J . Ef fec t of corticosterone treatment on
muscle protein turnover in adrenalec tomized rats and diabetic rats
mainta ined on insul in . Biochem. J. 20^ (1982) 663-572.
11
FIGURE LEGENDS
Figure 1 - Tims-course of blockade of [ H] triamcinolone acetonide
binding to the cytosolic glucocorticoid receptor from rat levator ani
muscle. Experimental procedures are described in the text.
. Figure 2 - Effec t of dexamethasone (5 ing/kg/cay, subcutaneous) and
RU38^86 (50 mg/kg/day, oral) on levator ani nroscle wet weight as * body
weight. V = vehicle, dex = dexamethasone . Exper imental procedures are
described in the text. Data are means + SD. ^Significantly d i f f e ren t from V,
p < 0.001.
Figure 3_ - Effec t of RU33^86 on wet weight of rat levator ani muscle
following castration ( G D X ) . V, vehicle. Experimental procedures a re
described in the text. -Signif icantly d i f f e ren t f rom V, p < 0.01; 6,
significantly d i f f e r e n t from G D X , p < 0.001.
OF POOR QUALJTY
Table I
Ef fec t of Oexarnethasone
'•Jon-collagen SNA* DNA R N A / D N A
protein
(mg/g wet w.) (mg/g wet w.) ( rog/g wet w.)
Control 175 + 20 1.57 ± 0.18 2 . 4 3 ± 0.3^ 0 .66 ± 0.17
Dexarncthasone 191) ± 2}-i 1.20 + 0.15 2'. 52 ± 0.55 O . b O t 0.1^4
Dexaniethasone
+ F033'486 191 ± 23 1.35 + 0.09 2.09 ± 0.58 0.69 ± 0.21
X Jt
RU33*186 172 ± 17 1.69 ± 0 .20" 2.61) ± 0 .60 0.65 ± O. l i}
Significantly d i f f e r e n t f rom control, p < 0.0025.
Significantly d i f f e ren t f rom dexaroetbasone, dexarnethasone + R U 3 3 U 8 6 ,
D < 0 .025.
OF POOR QUALmf
Table II
Effec t of Orchiectoiiiy and RU38436 on Non-Collagen Protein,
RNA and DNA of Rat Levator Ani Muscle
Non-collagen RNA DNA R N A / D N A
protein " dng/g wet w.) (njg/g wet w.)
(rcg/g wet w. )
Control
Crehiec toray
171 ± 1H
173 ± 22
1.41 + 0.07 :
1.17 ± 0.15* -
1.90 + 0.25
2.14 ± 0 .45
0.75 ± 0.9
0.57 ± 0.15
Orchiec toray
+ RU33436 134 ± 12 1.36 + 0.13** 2,26 + 0.53 0.63 ± 0.16
*
Signi f icant ly d i f f e r e n t f rom control and orcniectomy + RU33486 , p < 0.00?5.
*«
Signi f icant ly less than orchiectomy, p < 0.01
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A possible role for endogenous glucocorticoids in orchiectomy-induced atrophy of the rat levator ani muscle: Studies with RU38486, a potent and selective antiglucocorticoid

A possible role for endogenous glucocorticoids in orchiectomy-induced atrophy of the rat levator ani muscle: Studies with RU38486, a potent and selective antiglucocorticoid

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