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A specific effect of glucocorticoids in decreasing neutral ribonuclease II in skeletal muscle
Vol. 70, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A SPECIFIC EFFECTOF GLUCOCORTICOIDS IN DECREASING NEUTRALRIBONUCLEASEII IN SKELETALMUSCLE' Robert M. Kershner2 and William
L. Meyer
Department of Biochemistry University of Vermont College of Medicine Burlington, Vermont 05401
Received
March
25,1976
Summa=. Neutral ribonuclease II (EC 3.1.4.22) activity decreases to as little as 20% of its normal level in skeletal muscle of mice within several hours to several days after administration of any of a variety of glucocorticoids. The magnitude of the change varies directly with dose and duration of treatment. Nonglucocorticoid steroids fail to lower ribonuclease activity. The decrease in neutral ribonuclease II levels specifically induced by glucocorticoids may play an important role in the substantial elevation of intracellular RNA concentration known to be an early effect of these hormones. Neutral
ribonuclease
has a specificity
tissues,
bovine pancreatic
of the latter
of this
pattern
similar
In certain
tissues,
inhibitor in a free,
where it
acid and protein variety
corticoids activity
turnover
of physiologic
of increasing
active
substantially
in skeletal
this
muscle and liver,
effect
with
of RNase activ-
of inhibitor
in the altered
of muscle have generally Here we report
in decreasing
is
an alteration
of the diseased muscle.
(1, 3, 5, and 6).
and specific
there
and patients
is an increase
may play a key role
perturbations
have a potent
mice (l-3)
exceeds the level
characteristic
RNase II levels
including
We have described
form.
(4) in which there
We have suggested that
to those of the well-known
of the enzyme occurs such that
in the muscle of dystrophic
ity to the point
RNases commonly found in
and properties
Duchenne muscular dystrophy
ent.
one of two "bulk"
RNase (1).
an excess of a protein little
II,
presnucleic
A wide
had the effect that
neutral
glucoRNase II
muscle of mice.
'This work was supported by grant number NSlOlOS from the National Institute of Neurological and Communicative Disorders and Stroke. 2Recipient of a Summer Scholarship from the Muscular Dystrophy Association. Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
513
Vol. 70, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
=I [
IQ
100
6 w
\
---
----w
4 100
1.0 DOSE
:
:
:
:
:
:
:
:
(mg/kg)
DAYS OF TREATNEWT
Figure 1. Effect of glucocorticoid dose on depression of RNase II activity. Experimental protocol was exactly as described in Table 1 and Methods. Dashed line, triamcinolone acetonide; solid line, dexamethasone sodium phosphate. The curves are from separate studies. Figure ministration as described 5 mg/kg body one control activity is of f 15 units.
2. Time course of depression of RNase II activity with adof triamcinolone acetonide. Experimental protocol was exactly in Table 1 and Methods. Triamcinolone acetonide daily dose was Each point is for one experimental animal compared to weight. killed the same day. The mean of the seven control values for 97 units per gm wet weight of muscle with a standard deviation The RNase unit is defined in references 5 and 7.
Methods. Albino male mice 35-40 days old were given daily intraperitoneamtions (0.1 cc) of either 0.9% NaCl (controls) or preparations of various steroids. Animals were allowed free access to food and water and periodically were ktlled by concussion 24 hours following the last inj ect ion. Gastrocnemius muscle was excised and homogenized with 8-10 volumes of 300 mM KCl-1 mM Na,+EDTA. Centrifugation at 37,000 g for 15 min at O” yielded a supernate in which RNase II was assayed at 45’ using an RNA preparation and buffered cadmium precipitating procedure developed in our laboratory was assayed by our general method (5) modified by (7) - RNase II inhibitor Two other the use of free RNase II prepared from mouse skeletal muscle. neutral hydrolases were measured: RNase I (7) and N-acetyl-L-phenylalanine ethyl esterase (8). Results istration
The data
and Discussion. of
glucocorticoids
results
activity
in skeletal
muscle
of mice.
effect
was seen with
every
glucocorticoid
without
effect.
creased
dose
rapid
The depression of glucocorticoids
and progressive
over
of Table
1 indicate
that
in decreased
levels
of total
In this
and other
tested.
of RNase activity as illustrated
a period
of days
514
becomes in Figure
(Figure
2).
the
admin-
RNase II
experiments,
Injection
the
vehicles greater 1.
:
’ 1C
5
0
were
with
The response
RNase activity
inis has
BIOCHEMICAL
Vol. 70, No. 2, 1976
Table
1.
Steroid
effects
AND BIOPHYSICAL
on muscle
RESEARCH COMMUNICATIONS
RNase II
activity
Dose CWW
Steroid Glucocorticoids: Hydrocortisone acetate Triamcinolone acetonide Cortisone acetate Dexamethasone sodium phosphate Prednisolone sodium succinate Mineralocorticoids : Fludrocortisone acetate Deoxycorticosterone acetate Others : Estrone Testosterone 17-a-methyl-testosterone Progesterone Stigmasterol Cholesterol
RNase units/gm (% of control)
10 2 10 0.4 2.5
3s 39 56 60 83
0.3 12.5
69 139
7.5 10 10 10 10 10
82 82 97 103 110 96
Results are for total RNase II activity/gm wet weight of muscle expressed as % of activity in control muscle. Measurements were made 24 hours following the last of two daily injections with an amount of steroid per kg of body weight as indicated. Data are for single animals collected from separate studies run on three different days and in each case the value is compared to one or two control animals An indication of the absolute values for enzyme activfor that day. ity and of the variation among controls is presented in the legend to Figure 2.
never
leveled
corticoid, effect
off
at
dosage
less
than
schedule,
on RNase II
or length
is relatively
or N-acetyl-L-phenylalanine weight
ratio
occur
Depression Other
steroids
tude
comparable
fludrocortisone response
values
in short
of RNase II (Table
esterase
term
experiments
1) do not
to that
elicited
experiments
given
like do not in Table
such as those effect
glucocorticoids.
known glucocorticoid
those
of Figure
1 show that,
cause
progressive
decreases
1.
515
changes or muscle
any consistent
by all
what
gluco-
has been employed.
activities
is a specific
cause
no matter
no appreciable
ethyl
activity
values
of treatment
specific;
may be due to its
nonglucocorticoids the
20% of control
The
in RNase I to body
described
above.
of glucocorticoids.
depression
of
a magni-
The decrease activity unlike
(9).
with Dose-
glucocorticoids,
in RNase activity
below
BIOCHEMICAL
Vol. 70, No. 2, 1976
Free total,
RNase II
also
inhibitor
activity,
decreases
In view istic
of the
of murine
glucocorticoids results
coids.
and perhaps It
well
effect
agents
to results
would
of the
tion
(ll),
changes
of
assay
mechanism increase
are
character-
investigation
diseases
is
of
indicated
relevant
by the
these
of
usually
cause
if
action.
a prominent
precedes
the
induction
in
actually
total
and other
variable
were
liver,
and other
activities
bases
for
some of the
increase
the
general
glucocorticoid
516
With
Because reported tissue
(13),
one excep-
of
large
simply
(11).
glucocorticoids or modest
to
may
has been re-
treatment that
the
mass declined
RNase activity
composition.
in RNase activity
comparison,
has been reported
(12-18).
indicated
given
RNase II,
treatment
have
measured;
of
tissue
after
is very
combinations
lymphoid
liver
on
varying
as follows.
or tumor
RNase
report
assayed
activities
of RNase while
on tissue
No others
may be summarized
cell-type
any change
is
effects
RNase II,
weight,
with
and which
preparation,
glucocorticoid
also
specific,
hormone which
difficult.
of several
and tissue
in the
few studies,
is
mixtures
tissue
to increase
in RNA levels
RNase activities
been due to persistence
or due to change
in liver
by glucocorti-
of glucocorticoid
of the
studies
following
levels
may be a widespread,
glucocorticoid
literature
With
RNase II depressed
tissues
for
RNase I and free
in lymphoid
little
RNase
(10).
RNase activity
be elevated
ported
therein
in the
be measured.
bulk
(l-4),
above,
levels
in certain
Frequently,
RNase,
in these
RNase II
in the
Identification
“acid”
have
of
of our results
conditions
of total
of RNase II which
to be substantially
to the
enzymes
difficult.
of the
levels
dystrophies
reported
and found
depression
Comparison
the
experiments
may contribute
muscle.
fraction
here.
of glucocorticoids
levels
levels
as therapeutic
key element
of specific
a small
treatment;
high muscular
measured
Thus,
is
RESEARCH COMMUNICATIONS
unaffected.
and Duchenne
described
were
normally
glucocorticoid
abnormally
In some of the also
which
with
are relatively
AND BIOPHYSICAL
decreases
A may in
BIOCHEMICAL
Vol. 70, No. 2,1976
either
RNase activity Our results
identity
aware of no evidence that
with regard
with a role The authors
in the uterus.
of RNase II inhibitor
of RNase inhibitor
interest
evidence consistent
of RNase activity
RESEARCH COMMUNICATIONS
(13, 19-21) or ribonucleolysis
are of particular
(23) which presents lation
levels
AND BIOPHYSICAL
and 8-estradiol
(22). to a recent report
for estrogen
suggest the possible
receptor
protein.
is more than tenuous with respect
and any steroid
receptor
in the regu-
We are
to the identity
protein.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Meyer, W. L., Little, B. W., Feussner, J. R. and Meyer, D. H. (1972) in Research in Muscle Development and the Muscle Spindle, B. Banker, R. Przybylski, J. Van Der Meulen, and M. Victor, Ed., pp. 195-217, Excerpta Medica, Amsterdam. Little, B. W., and Meyer, W. L. (1970) Science, 170, 747-749. Little, B. W., Pittman, D. G., and Meyer, W. L. (1973) in Basic Research in Myology, B. A. Kakulas, Ed., pp. 235-241, Excerpta Medica, Amsterdam. Kakulas, B. A., and Meyer, W. L. (1976) in Recent Advances in Myology, W. G. Bradley, D. Gardner-Medwin, and J. N. Walton, Ed., pp. 277-282, Excerpta Medica, Amsterdam. Meyer, D. H., Meyer, W. L., and Kakulas, B. A. (1976) in Recent Advances in Myology, W. G. Bradley, D. Gardner-Medwin, and J. N. Walton, Ed., pp. 283-288, Excerpta Medica, Amsterdam. Nwizu, C. S., Meyer, D. H., and Meyer, W. L. (1974) Abstracts, Third International Congress on Muscle Diseases, p. 7, Excerpta Medica, Amsterdam. Meyer, D. H., and Meyer, W. L. (1975) J. Insect Biochem., 5, 747-758. B. W. (1973) in Meyer, W. L., Smith, D. B., Stearns, K., and Little, Basic Research in Myology, B. A. Kakulas, Ed., pp. 242-247, Excerpta Medica, Amsterdam. Travis, R. H., and Sayers, G. (1965) in The Pharmacological Basis of Therapeutics, 3rd Ed., L. S. Goodman and A. Gilman, Ed., p. 1633, Macmillan, New York. Interactions, King, R. J. B., and Mainwaring, W. I. P. (1974) Steroid-Cell pp. 353-354, University Park Press, Baltimore. Pena, A., Dvorkin, B., and White, A. (1966) J. Biol. Chem. 241, 2144-2150. Maor, D., Eylan, E., and Alexander, P. (1973) Acta Endocrinol. (Copenhagen), 74, 201-208. Maor, D., Niviy, S., and Eylan, E. (1973) Biomedicine, 19, 374-378. Ambellan, E., and Hollander, V. P. (1966) Anal. Biochem., 17, 474-484. Ambellan, E., and Hollander, V. P. (1966) Cancer Res., 26, 903-909. Ambellan, E., and Roth, J. S. (1967) Biochem. Biophys. Res. Commun., 28, 244-248. MacLeod, R. M., King, C. E., and Hollander, V. P. (1963) Cancer Res., 23, 1045-1050. (Copenhagen), Wiernik, P. H., and MacLeod, R. M. (1965) Acta Endocrinol. 49, 138-144. Terner, J. Y., Goodman, R. M., and Spiro, D., (1967) Exp. Cell Res., 45, 550-558.
517
Vol. 70, No. 2, 1976
20. 21. 22. 23.
BIOCHEMICAL
AND BIOPHYSICAL
de Lamirande, G., and Allard, C. (1959) Ann. 570-584. Allard, C., de Lamirande, G., Weber, G., and Biochem. Physiol., 34, 170-179. Barnabei, O., and Ottolenghi, C. (1968) Adv. Zan-Kowalczewska, M., and Roth, J. S. (1975) commun. ) 65, 833-837.
518
RESEARCH COMMUNICATIONS
N.Y. Acad. Sci., Cantero,
A.
81,
(1956) Can. J.
Enzyme Regul., 6, 189-209. Biochem. Biophys. Res.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A SPECIFIC EFFECTOF GLUCOCORTICOIDS IN DECREASING NEUTRALRIBONUCLEASEII IN SKELETALMUSCLE' Robert M. Kershner2 and William
L. Meyer
Department of Biochemistry University of Vermont College of Medicine Burlington, Vermont 05401
Received
March
25,1976
Summa=. Neutral ribonuclease II (EC 3.1.4.22) activity decreases to as little as 20% of its normal level in skeletal muscle of mice within several hours to several days after administration of any of a variety of glucocorticoids. The magnitude of the change varies directly with dose and duration of treatment. Nonglucocorticoid steroids fail to lower ribonuclease activity. The decrease in neutral ribonuclease II levels specifically induced by glucocorticoids may play an important role in the substantial elevation of intracellular RNA concentration known to be an early effect of these hormones. Neutral
ribonuclease
has a specificity
tissues,
bovine pancreatic
of the latter
of this
pattern
similar
In certain
tissues,
inhibitor in a free,
where it
acid and protein variety
corticoids activity
turnover
of physiologic
of increasing
active
substantially
in skeletal
this
muscle and liver,
effect
with
of RNase activ-
of inhibitor
in the altered
of muscle have generally Here we report
in decreasing
is
an alteration
of the diseased muscle.
(1, 3, 5, and 6).
and specific
there
and patients
is an increase
may play a key role
perturbations
have a potent
mice (l-3)
exceeds the level
characteristic
RNase II levels
including
We have described
form.
(4) in which there
We have suggested that
to those of the well-known
of the enzyme occurs such that
in the muscle of dystrophic
ity to the point
RNases commonly found in
and properties
Duchenne muscular dystrophy
ent.
one of two "bulk"
RNase (1).
an excess of a protein little
II,
presnucleic
A wide
had the effect that
neutral
glucoRNase II
muscle of mice.
'This work was supported by grant number NSlOlOS from the National Institute of Neurological and Communicative Disorders and Stroke. 2Recipient of a Summer Scholarship from the Muscular Dystrophy Association. Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
513
Vol. 70, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
=I [
IQ
100
6 w
\
---
----w
4 100
1.0 DOSE
:
:
:
:
:
:
:
:
(mg/kg)
DAYS OF TREATNEWT
Figure 1. Effect of glucocorticoid dose on depression of RNase II activity. Experimental protocol was exactly as described in Table 1 and Methods. Dashed line, triamcinolone acetonide; solid line, dexamethasone sodium phosphate. The curves are from separate studies. Figure ministration as described 5 mg/kg body one control activity is of f 15 units.
2. Time course of depression of RNase II activity with adof triamcinolone acetonide. Experimental protocol was exactly in Table 1 and Methods. Triamcinolone acetonide daily dose was Each point is for one experimental animal compared to weight. killed the same day. The mean of the seven control values for 97 units per gm wet weight of muscle with a standard deviation The RNase unit is defined in references 5 and 7.
Methods. Albino male mice 35-40 days old were given daily intraperitoneamtions (0.1 cc) of either 0.9% NaCl (controls) or preparations of various steroids. Animals were allowed free access to food and water and periodically were ktlled by concussion 24 hours following the last inj ect ion. Gastrocnemius muscle was excised and homogenized with 8-10 volumes of 300 mM KCl-1 mM Na,+EDTA. Centrifugation at 37,000 g for 15 min at O” yielded a supernate in which RNase II was assayed at 45’ using an RNA preparation and buffered cadmium precipitating procedure developed in our laboratory was assayed by our general method (5) modified by (7) - RNase II inhibitor Two other the use of free RNase II prepared from mouse skeletal muscle. neutral hydrolases were measured: RNase I (7) and N-acetyl-L-phenylalanine ethyl esterase (8). Results istration
The data
and Discussion. of
glucocorticoids
results
activity
in skeletal
muscle
of mice.
effect
was seen with
every
glucocorticoid
without
effect.
creased
dose
rapid
The depression of glucocorticoids
and progressive
over
of Table
1 indicate
that
in decreased
levels
of total
In this
and other
tested.
of RNase activity as illustrated
a period
of days
514
becomes in Figure
(Figure
2).
the
admin-
RNase II
experiments,
Injection
the
vehicles greater 1.
:
’ 1C
5
0
were
with
The response
RNase activity
inis has
BIOCHEMICAL
Vol. 70, No. 2, 1976
Table
1.
Steroid
effects
AND BIOPHYSICAL
on muscle
RESEARCH COMMUNICATIONS
RNase II
activity
Dose CWW
Steroid Glucocorticoids: Hydrocortisone acetate Triamcinolone acetonide Cortisone acetate Dexamethasone sodium phosphate Prednisolone sodium succinate Mineralocorticoids : Fludrocortisone acetate Deoxycorticosterone acetate Others : Estrone Testosterone 17-a-methyl-testosterone Progesterone Stigmasterol Cholesterol
RNase units/gm (% of control)
10 2 10 0.4 2.5
3s 39 56 60 83
0.3 12.5
69 139
7.5 10 10 10 10 10
82 82 97 103 110 96
Results are for total RNase II activity/gm wet weight of muscle expressed as % of activity in control muscle. Measurements were made 24 hours following the last of two daily injections with an amount of steroid per kg of body weight as indicated. Data are for single animals collected from separate studies run on three different days and in each case the value is compared to one or two control animals An indication of the absolute values for enzyme activfor that day. ity and of the variation among controls is presented in the legend to Figure 2.
never
leveled
corticoid, effect
off
at
dosage
less
than
schedule,
on RNase II
or length
is relatively
or N-acetyl-L-phenylalanine weight
ratio
occur
Depression Other
steroids
tude
comparable
fludrocortisone response
values
in short
of RNase II (Table
esterase
term
experiments
1) do not
to that
elicited
experiments
given
like do not in Table
such as those effect
glucocorticoids.
known glucocorticoid
those
of Figure
1 show that,
cause
progressive
decreases
1.
515
changes or muscle
any consistent
by all
what
gluco-
has been employed.
activities
is a specific
cause
no matter
no appreciable
ethyl
activity
values
of treatment
specific;
may be due to its
nonglucocorticoids the
20% of control
The
in RNase I to body
described
above.
of glucocorticoids.
depression
of
a magni-
The decrease activity unlike
(9).
with Dose-
glucocorticoids,
in RNase activity
below
BIOCHEMICAL
Vol. 70, No. 2, 1976
Free total,
RNase II
also
inhibitor
activity,
decreases
In view istic
of the
of murine
glucocorticoids results
coids.
and perhaps It
well
effect
agents
to results
would
of the
tion
(ll),
changes
of
assay
mechanism increase
are
character-
investigation
diseases
is
of
indicated
relevant
by the
these
of
usually
cause
if
action.
a prominent
precedes
the
induction
in
actually
total
and other
variable
were
liver,
and other
activities
bases
for
some of the
increase
the
general
glucocorticoid
516
With
Because reported tissue
(13),
one excep-
of
large
simply
(11).
glucocorticoids or modest
to
may
has been re-
treatment that
the
mass declined
RNase activity
composition.
in RNase activity
comparison,
has been reported
(12-18).
indicated
given
RNase II,
treatment
have
measured;
of
tissue
after
is very
combinations
lymphoid
liver
on
varying
as follows.
or tumor
RNase
report
assayed
activities
of RNase while
on tissue
No others
may be summarized
cell-type
any change
is
effects
RNase II,
weight,
with
and which
preparation,
glucocorticoid
also
specific,
hormone which
difficult.
of several
and tissue
in the
few studies,
is
mixtures
tissue
to increase
in RNA levels
RNase activities
been due to persistence
or due to change
in liver
by glucocorti-
of glucocorticoid
of the
studies
following
levels
may be a widespread,
glucocorticoid
literature
With
RNase II depressed
tissues
for
RNase I and free
in lymphoid
little
RNase
(10).
RNase activity
be elevated
ported
therein
in the
be measured.
bulk
(l-4),
above,
levels
in certain
Frequently,
RNase,
in these
RNase II
in the
Identification
“acid”
have
of
of our results
conditions
of total
of RNase II which
to be substantially
to the
enzymes
difficult.
of the
levels
dystrophies
reported
and found
depression
Comparison
the
experiments
may contribute
muscle.
fraction
here.
of glucocorticoids
levels
levels
as therapeutic
key element
of specific
a small
treatment;
high muscular
measured
Thus,
is
RESEARCH COMMUNICATIONS
unaffected.
and Duchenne
described
were
normally
glucocorticoid
abnormally
In some of the also
which
with
are relatively
AND BIOPHYSICAL
decreases
A may in
BIOCHEMICAL
Vol. 70, No. 2,1976
either
RNase activity Our results
identity
aware of no evidence that
with regard
with a role The authors
in the uterus.
of RNase II inhibitor
of RNase inhibitor
interest
evidence consistent
of RNase activity
RESEARCH COMMUNICATIONS
(13, 19-21) or ribonucleolysis
are of particular
(23) which presents lation
levels
AND BIOPHYSICAL
and 8-estradiol
(22). to a recent report
for estrogen
suggest the possible
receptor
protein.
is more than tenuous with respect
and any steroid
receptor
in the regu-
We are
to the identity
protein.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
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