Vol. 176, No. 1, 1991 April 15, 1991
OXYTOCIN
BIOCHEMICAL
CONTRACTS WITHOUT
Kazuhiko
AND BIOPHYSICAL
RAT UTERINE
ANY
SMOOTH
PHOSPHORYLATION
RESEARCH COMMUNICATIONS Pages 122-l 28
MUSCLE
IN Ca*+-FREE
OF MYOSIN
LIGHT
CHAIN
+, Naoe Minakawa-Matsuo, Osamu Hiromi Takano-Ohmuro Karibe, Kazuhiro Kohama’, and Masaatsu K. Uchida
Oishil,
Department of Molecular Pharmacology, l-35-23 Nozawa, Setagaya-ku, +Tokyo Metropolitan Institute Honkomagome, Bunkyo-ku,
MEDIUM
Suga, Hideji
Meiji College of Pharmacy, Tokyo 154, Japan of Medical Sciences, Tokyo 113, Japan
*Department of Pharmacology, The University of Gunma, Shouwa-machi, Maebashi, Gunma 371, Japan Received
February
19,
1991
SUMMARY: Contraction of rat uterine smooth muscle related to phosphorylation state of myosin light chain under various conditions was investigated. In the Ca2+-containing medium, both high K+ and oxytocin induced marked contraction of the muscle accompanied by pronounced phosphorylation of myosin light chain. In the Ca2+-free medium, although both vanadate and oxytocin induced slight contraction, phosphorylation of myosin light chain was only evident for vanadate but not for oxytocin. It was suggested that another mechanism distinct from myosin light chain phosphorylation might be involved in Ca2+-independent contraction of uterine smooth muscle elicted by oxytocin. B 1991 AcademicPress, Inc.
Oxytocin Ca2+ This
induces
after the muscle caused
reported
quin-2 indicates
contraction
with 3 mM EGTA
for
1 h (Ca-free
in Ca 2+-free medium gives in the presence of Ca 2+ (normal
antagonists
such
as D-600
no effect on Ca-free
that cytosolic
and nicardipine
contraction
(2,3).
free Ca 2+ level ([Ca*+]i)
in the solution 515%
accepted
mechanism KT-5926
that phosphorylation
to initiate
smooth
(5) effectively
of myosin
muscle inhibit
contraction
normal
of the
uterine
. ’ To whom correspondence
should be addressed.
Abbreviations used are: EGTA, ethylene glycol bis(R-aminoethyl ether) N,N,N’,N’tetraacetic acid; MLCK, myosin light chain kinase; PPi, pyrophosphate; PAGE, polyacrylamide gel electrophoresis; TPA, 12-0-tetradecanoylphorbol-t3-acetate. @306-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. AN rights of reproduction in any form reserved.
122
Ca2+-
is the most
In fact, MLCK of rat
Ca2+
this Ca-free
is rather
light chain by MLCK
contraction
We have
the study with
does not rise during
(4).
(1,2). maximal
and intracellular
Furthermore,
without
contraction)
contraction).
Therefore, it is predicted that this contraction that is, it is not triggered by the usual increase in [Ca2f]i.
It is generally and
smooth muscle
(3).
independent, probable
have
of rat uterine
contraction by oxytocin
that calcium
chelator fura-
contraction
is incubated
oxytocin-induced
contraction
ML-9
sustained
inhibitors, smooth
Vol.
BIOCHEMICAL
176, No. 1, 1991
muscle. myosin
But they have phosphorylation
no effect by MLCK
AND BIOPHYSICAL
on Ca-free
contraction
is not involved
study was performed
to clarify the phosphorylation
sustained
by means
contraction
(7,8) and urea-glycerol associated
of two different
PAGE
(9).
with any phosphorylation
(6), indicating
in this process.
a possibility
Therefore,
that
the present
state of myosin in an early stage of this electrophoresis
We reported of myosin
RESEARCH COMMUNICATIONS
systems,
here that Ca-free
namely,
PPi PAGE
contraction
was not
light chain.
EXPERIMENTAL PROCEDURES The rat uterine longitudinal smooth muscle was obtained as described by Matsuo and Uchfda (10). The muscle strips were mounted in a 10 ml organ bath filled with normal LockeRinger solution aerated with 5% carbon dioxide in oxygen at 30°C. After equilibration in normal Locke-Ringer solution for 1 h at a resting tension of 0.59, the stripes were incubated in Ca2+-free solution containing 3 mM EGTA for 1 h at a resting tension of 0.29 (EGTA treatment). The medium was then replaced by Ca 2+-free solution containing 0.2 mM EGTA. After further incubation for 10 min. contraction was induced by the addition of 10-2 unit/ml oxytocin (Ca-free contraction). Isometric tension was monitored with a force displacement transducer. At indicated times, the organ bath was removed without influencing tonic tension and strips were rapidly frozen by submersion in dry ice-acetone. Normal Locke-Ringer solution had the following composition (mM): NaCI,154; KCl,5.63; CaCl2,2.16; MgCl2,2.10; NaHC03,5.95; and glucose,5.55. Ca2+-free solution had the same composition except that CaC12 was substituted by 3 or 0.2 mM EGTA. The frozen strips were crushed in a frozen stainless steel mill at -17OOC. The frozen powder was extracted in 60 volumes of PPi sample buffer containing 100 mM Na4P207, 5 mM EGTA, 2.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 0.6 M KI and 10% glycerol (pH 8.8), which was slightly modified from the original composition of Persechini et al. (11). After centrifugation of the extract, glycerol and sucrose were added to give final concentration of 30 and 20%, respectively. The samples were subjected to PPi PAGE as described previously by Takano-Ohmuro and Kohama (7,8) for 16 h to examine the phosphorylation state of myosin by separating phosphorylated myosin from unphosphorylated one. The pattern of protein on PPi PAGE disk gel was visualized by staining with 0.1% Coomassie brilliant blue R-250. In order to analyze phosphorylation state of myosin light chain by urea-glycerol PAGE, PPi PAGE of the muscle extract was carried out for 3 h After staining briefly with Coomassie brilliant blue R-250, a major protein band which contained unseparated myosin and filamine was excised from the gel and treated with the urea solution containing 9 M urea, 100 mM Tris-HCI (pH 6.8) and 5% 2mercaptoethanol for 10 min. Urea-glycerol PAGE of the excised gel was then performed as described previously (9) to examine the phosphorylation state of myosin light chain by separating phosphorylated myosin light chain from unphosphorylated one in order of increased mobility. The pattern of protein on urea-glycerol PAGE slab gel was visualized by means of a slightly modified procedure (12) of the highly sensitive silver stain of Oakley et a/. (13), which is approximately 20-fold sensitive than the Coomassie brilliant blue staining. Identification of filamin on PPi PAGE gel was carried out as described previously (7). RESULTS Exposure sustained in Fig.1.
of the muscle strips to 45.6 mM K+ medium contraction
which gave a sub-maximal
At time zero,
unphosphorylated
gizzard
one myosin
tension
(designated
myosin was observed
15 set after the muscle was exposed migrated
band
in the presence by M) which
123
comigrated
with the
just under the filamin band on PPi PAGE.
myosin
a
of about 2g after 60 set, as shown
to the high Kf medium,
faster than the unphosphorylated
of Ca2+ caused
additional
band appeared
At
two bands which were
on PPi PAGE.
The upper
Vol.
176,
BIOCHEMICAL
No. 1, 1991
0
15
60
AND BIOPHYSICAL
0
set
RESEARCH COMMUNICATIONS
5
15
6Osec
2 2
k&i 2
c CL
01
456mM
Kf
I
02
-4
1 Omin
F -M
1CJ2unit/ml
oxytoc\n
I
-
I
1 Omin
&J. Analysis of the phosphorylation state of myosin by PPi PAGE during 45.6 mM K+induced contraction in the medium containing Ca 2+. After equilibration of uterine smooth muscle in normal Locke-Ringer solution for 1 h at a resting tension of 0.59, contraction was induced by changing the medium to K + rich solution containing 45.6 mM K+. Isometric tension was monitored with a force displacement transducer. Myosin phosphorylation was determined for individual strips frozen after indicated times of stimulation (indicated with the arrows). Unphosphorylated myosin (M), the myosin with one mono-phosphorylated 20 kDa light chain (MPl) and the myosin witQ two mono-phosphorylated 20 kDa light chains (MP2) were separated in the order of increasing mobility on PPi PAGE. The pattern of protein on the PPi PAGE disk gel was visualized by staining with 0.1% Coomassie brilliant blue R-250. The band represented by “F” was filamin. To the right of M, MPl and MP2 is schematically shown how myosin is phosphorylated at 20 kDa light chain, which was deduced from the results of urea-glycerol PAGE analysis of each myosin band separated on the PPi PAGE (figures not shown). Fia.2. Analysis of the phosphorylation state of myosin by PPi PAGE during oxytocintension of 0.59, induced contraction in the medium containing Ca 2+. After 1 h at a resting contraction was induced by the addition of 10W2 unit/ml oxytocin. See”Fig.1 legend” for details.
band (MPl)
was the myosin
with one unphosphorylated
band (MP2)
was the myosin
with two mono-phosphorylated
from the results of subsequent glycerol
analysis
PAGE (data not shown).
increased.
Approximately
expressed
by the percent
In the presence stage followed detected
40%
of
Ca2+,
oxytocin
were
(10s2
contraction
and MP2 bands was observed
by PPi PAGE. was about
myosin
unit/ml)
deduced
disk gel by urea-
state of these two bands
phosphorylated
induced
light chain
induced
one-tenth
induced
of oxytocin
form
at
a tonic
this
time
contraction
Phosphorylation
at
as
and a time-dependent
agreement
increase
sustained
of normal
that the initiation
phosphorylation contraction
contraction 124
early
of myosin
by high K+ (Fig.1) and oxytocin
of myosin during oxytocin-induced Oxytocin
the
as shown in Fig.2.
of Ca2+ and also confirm the general requires
band from PPi PAGE
up to 60 sec. These results indicate the involvement
in the contractions
Phosphorylation which
myosin
as early as 5 set after the addition
contraction
20 kDa light chains,
After 60 set the phosphorylation of
and the lower
of total myosin.
by a pendular
phosphorylation presence
of excised
20 kDa light chain
was
of MPI
of myosin
(Fig.2)
of smooth
in the muscle
(4). Ca-free with
(Fig.3).
contraction a maximal
was then examined tension
At any indicated
of about
0.29
times of 5, 15,
Vol.
BIOCHEMICAL
176, No. 1, 1991
s E .. 2
AND BIOPHYSICAL
F M-
.
vanadate &g.J. induced induced
Analysis of the phosphorylation contraction in the Ca2+-free by the addition of 10M2 unit/ml
Fjg&.
Analysis
induced induced
of the
states medium. oxytocin.
phosphorylation
light
3~
indicate
that Ca-free
state
contraction
I0-h
t
4
1Omin
of myosin by PPi PAGE during oxytocinAfter “EGTA treatment” contraction was See “Fig.1 legend” for details. of myosin
by PPi
contraction in the Ca 2+-free medium. After “EGTA by the addition of 10e5 M vanadate. See “Fig.1 legend”
and 60 set, we failed to detect phosphorylated results
PAGE
during
treatment” for details.
vanadate-
contraction
was
myosin bands such as MPI and MP2.
occured
without
any phosphorylation
These
of
myosin
chain.
We next examined Ca*+-free
the phosphorylation
medium.
Vanadate
manner
(Fig.4).
muscle
is still obscure,
Although
phosphorylation
the mechanism
of myosin
the
were
contraction
the
observed
urea-glycerol
presence
manner of Ca*+
phosphorylated contraction
causing
MLCK,
during
in a similar uterine
smooth of the
inhibitor
KT-5926
in Ca*+-free
contraction,
(5)
medium).*
small but significant M vanadate-induced
3~10~~
in
to be a result
a MLCK
M vanadate
was employed
in an
early The
(Fig.5B, PAGE
b, c, and
analysis
and
phosphorylation
in
the
light chain
d). also
of 20
of contractions
phosphorylated
contraction
20 kDa myosin
kDa
and
Uchida,M.K.,
myosin of
was observed
These
results that
oxytosin
light
chain.
myosin
light chain
induced
absence
indicated
unpublished
phosphorylation
state of the myosin
stage
(Fig.SA).
to analyze
confirmed
2 Karibe,H.
seems
because
Ca-free
contraction
in contracting
contraction
by 3~10~~
The phosphorylation
vanadate-induced
PPi
by
on PPi PAGE
PAGE
light chain.
time-dependent
during
induced
sustained
contraction
MPl
In and
sustained
(Fig.4).
Finally, myosin
contraction
vanadate-induced
of action of vanadate
light chains
to the case of oxytocin-induced
during
M) induced
the vanadate-induced
inhibits
MP2 bands
of myosin
(3~10~~
contrast
by
RESEARCH COMMUNICATIONS
observations.
125
light
chain
and was
(Fig.5B.
during the
was increased
by oxytocin
Ca*+
induced
state of 20 kDa
f).
also
Ca-free
K+
observations contraction
in
observed
However,
oxytocin-induced
above
high
in a
no
Ca-free obtained without
Vol.
BIOCHEMICAL
176, No. 1, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
. VlLCp
a
b
d
c
e
f
9
B
abcdef &&. Analysis of the phosphorylation state of 20 kDa myosin light chain by ureaThe PPi PAGE samples prepared as described in glycerol PAGE during contractions. “EXPERIMENTAL PROCEDURES” were subjected to PPi PAGE for 3 h to separate and recover whole myosin in the sample. The myosin excised from the PPi PAGE gel was treated with the urea solution. Unphosphorylated (RLC) and monophosphorylated (RLCp) forms of the light chain were separated as a function of increasing mobility in urea-glycerol PAGE. The pattern of urea-glycerol PAGE gel was visualized by the silver staining. (A) Urea-glycerol PAGE showing the state of 20 kDa myosin light chain phosphorylation during contractions in the presence of Ca2+. Muscles were frozen at 0 set (lane a), 15 set (lane b), and 60 set (lane c) after changing to KC rich (45.6 mM) solution and at 0 set (lane d), 5 set (lane e), 15 set (lane f), and 60 set (lane g) after the addition of 10m2 unit/ml oxytocin. (B) Ureaglycerol PAGE showing the state of 20 kDa myosin light chain phosphorylation during were frozen at 0 set (lane a), 5 set (lane b), contractions in the absence of Ca 2c. Muscles 15 set (lane c), and 60 set (lane d) after the addition of 10s2 unit/ml oxytocin and at 0 set (lane e) and 15 set (lane f) after the addition of 10m5 M vanadate.
DISCUSStON PPi
and urea-glycerol
PAGE
uterine
smooth
muscle
were
Kg) of myosin molecule purification.
(78).
myosin
light
smooth
muscle
MLCK
phosphorylated
occurs
whereas
increased
bands
contraction
by oxytocin
in the absence
and
charge (MPl
high
Ca-free
PAGE
systems
showing
that oxytocin
small
And
amount
mobility
of myosin were
the
(Figs.1,
of
is phosphorylated
by
on urea-glycerol
of Ca2+
Phosphorylation
however,
and
was not detected without
chain.
126
causing
during
the
vanadate-induced the involvement
of myosin
To our best knowledge,
contraction
In this study, the
on PPi PAGE
2, and 4), indicating
without any mobility
light chain
evident
(l-2
of regulatory
lastly,
as a result of phosphorylation.
K+ in the presence
and 5B).
can induce
(7,8).
only when the myosin
and MP2)
of Ca 2+
contraction, (Fig.3
Firstly,
nor dephosphorylation
analysis
by MLCK in these processes.
glycerol light
PAGE
the increased
contraction
oxytocin-induced
phosphorylation
PPi
negative
reasons.
states in contracting
by PPi PAGE from small tissue samples
on PPi PAGE increases
myosin
phosphorylation
neither during
myosin phosphorylation
by following
can be analyzed
myosin
(7,8,14,15),
employed
Secondly,
chain
PAGE reflects
PAGE to analyze
light chain
in both
of
during
PPi and urea-
this is the first report
phosphorylation
of myosin
Vol.
BIOCHEMICAL
176, No. 1, 1991
In Ca*+-induced increased chain.
contraction
[Ca*+]i
induces
systems
activation
As a result, the formation
initiates
muscle
case because previous
contraction. no myosin
observation
is independent
contraction
by agonist
phenomenon. smooth
This
muscles
of the rise stimulation
phosphorylation,
although
of
its usual
oxytocin-induced
than
Ca-free
small,
to initiate
since
can
contraction However,
no phosphorylation
by the
contraction
of
of Ca*+-induced
account
for
the
protein
Such mechanism
(18).
myosin
is also supported
by GTP-binding
muscle.3
increase
contraction
that
light
this is not the
that Ca-free
Sensitization
mediated
smooth
increase
fura-
(3).
is a possibility
and uterine
contraction,
This finding
indicator
that
of myosin
actin and phosphorylated
Ca-free
in [Ca*+]i
is probably
in myosin
[Ca*+]i
Ca *+
2, and 5, it is obvious
by phosphorylation
between
is observed.
fluoresent
RESEARCH COMMUNICATIONS
in Figs.1,
followed
of crossbridges
phosphorylation
mechanism
(16,17)
as shown
of MLCK
But in oxytocin-induced
by using
rat uterus
AND BIOPHYSICAL
above
in various
is likely to result
even
with much smaller
this
is also
of myosin
unlikely
light
in
chain
is
observed. Besides
receptor
agonist,
a phorbol
C, clearly
caused
Ca-free
contraction
others
that TPA contracts
(20.21). protein
Although kinase
cytoskeletal
ester TPA, at a concentration in rat thoracic
some vascular
no phosphorylation C phosphorylates
proteins
(22).
and cytoskeletal
inhibitors
might be actually
involved
(6).
smooth
muscles
of myos.
light
various
Indeed, in Ca-free
functional
Ca-free
Protein
aorta
contraction
kinases
contraction.
(19).
to activate
protein
even under Ca*+-free chain
was observed
proteins
including
was inhibited
(but not MLCK)
kinase
It has been reported
by
conditions in this study,
contractile
by protein
and cytoskeletal
and
kinase
C
elements
Further study is need to be done.
ACKNOWLEDGMENTS: We thank Dr. J.F. Kuo, Department of Pharmacology, for helpful advice and critical reading of the manuscript. This work was supported by Grant-in-Aid (No.2640575 and 60571062) for Scientific Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. Sakai,K. and Uchida,M. (1980) Jpn. J. Pharmacol. a, 394-396. 2. Uchida,M.K., Sakai,K., and Matsuo,K. (1989) Gen. Pharmacol. a 117-121. 3. Matsuo,K., Gokita,T., Karibe,H., and Uchida, M.K. (1989) Biochem. Biophys. Res. Commun. m, 722-727. 4. Kamm,K.E. and Stul1,J.T. (1985) Annu.Rev. Pharmacol. Toxicol. a, 593-620. 5. Nakanishi,S., Yamada,K., Iwahashi,K., Kuroda,K., and Kase,H. (1990) Mol. Pharmacol. 37, 482-488. 6. Karibe,H., Matsuo,K., Gokita,T., and Uchida,M.K. (1990) Eur. J. Pharmacol. 188. 407. 410. 7. Takano-Ohmuro,H. and Kohama,K. (1986) J. Biochem. .jQQ, 1681-1684. 8. Takano-Ohmuro,H. and Kohama,K. (1986) J. Biochem. I.QQ, 259-268. 9. Perrie,W.T. and Perry,S.V. (1970) Biochem. J. 119, 31-38. 10. Matsuo,K. and Uchida,M.K. (1987) Eur. J. Pharmacol. j/&Q, 294-301.
3
Suga,O.,
Oishi,K.,
Karibe,H.,
and Uchida,M.K., 127
Eur. J. Pharmacol.,
submitted
for publication.
Vol.
176, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
11. Persechini,A., Kamm,K.E., and Stul1,J.T. (1986) J. Biol. Chem. m, 6293-6299. 12. Mikawa,T., Takeda,S., Shimizu,T., and Kitaura,T. (1981) J. Biochem. 89, 1951-1962. 13. Oakley,B.R., Kirsch,D.R., and Morris,N.R. (1980) Anal. Biochem. m, 361-363 14. Takano-Ohmuro,H. and Kohama,K. (1987) J. Biochem. m, 971-974. 15. Takano-Ohmuro,H., Kohama,K., Kathuria,S., and Fujita-Yamaguchi,Y. (1989) Proc. Japan Acad f& 200-202. 16. Nishimura,J., Kolber,M., and van Breemen,C. (1988) Biochem. Biophys. Res. Commun. m, 677-683. 17. Kizawa,T., Kobayashi,S., Horiuchi,K., Somlyo,A.V., and Somlyo,A.P. (1989) J. Biol. Chem. u 53395342. 18. Karaki,H. (1989) Trends Pharmacol. Sci. u, 320-325. 19. Karibe,H. and Uchida,M.K. (1990) Gen. Pharmacol. 2, 191-197. 20. Geason,M.E. and Flaim,S.F. (1986) Biochem. Biophys. Res. Commun. 138, 1362.1369. 21. Ito,H. and Lederis,K. (1987) Am. J. Physiol. 252, C244-247. 22. Park,S. and Rasmussen,H. (1986) J. Biol. Chem. a, 15734-15739.
128
OXYTOCIN
BIOCHEMICAL
CONTRACTS WITHOUT
Kazuhiko
AND BIOPHYSICAL
RAT UTERINE
ANY
SMOOTH
PHOSPHORYLATION
RESEARCH COMMUNICATIONS Pages 122-l 28
MUSCLE
IN Ca*+-FREE
OF MYOSIN
LIGHT
CHAIN
+, Naoe Minakawa-Matsuo, Osamu Hiromi Takano-Ohmuro Karibe, Kazuhiro Kohama’, and Masaatsu K. Uchida
Oishil,
Department of Molecular Pharmacology, l-35-23 Nozawa, Setagaya-ku, +Tokyo Metropolitan Institute Honkomagome, Bunkyo-ku,
MEDIUM
Suga, Hideji
Meiji College of Pharmacy, Tokyo 154, Japan of Medical Sciences, Tokyo 113, Japan
*Department of Pharmacology, The University of Gunma, Shouwa-machi, Maebashi, Gunma 371, Japan Received
February
19,
1991
SUMMARY: Contraction of rat uterine smooth muscle related to phosphorylation state of myosin light chain under various conditions was investigated. In the Ca2+-containing medium, both high K+ and oxytocin induced marked contraction of the muscle accompanied by pronounced phosphorylation of myosin light chain. In the Ca2+-free medium, although both vanadate and oxytocin induced slight contraction, phosphorylation of myosin light chain was only evident for vanadate but not for oxytocin. It was suggested that another mechanism distinct from myosin light chain phosphorylation might be involved in Ca2+-independent contraction of uterine smooth muscle elicted by oxytocin. B 1991 AcademicPress, Inc.
Oxytocin Ca2+ This
induces
after the muscle caused
reported
quin-2 indicates
contraction
with 3 mM EGTA
for
1 h (Ca-free
in Ca 2+-free medium gives in the presence of Ca 2+ (normal
antagonists
such
as D-600
no effect on Ca-free
that cytosolic
and nicardipine
contraction
(2,3).
free Ca 2+ level ([Ca*+]i)
in the solution 515%
accepted
mechanism KT-5926
that phosphorylation
to initiate
smooth
(5) effectively
of myosin
muscle inhibit
contraction
normal
of the
uterine
. ’ To whom correspondence
should be addressed.
Abbreviations used are: EGTA, ethylene glycol bis(R-aminoethyl ether) N,N,N’,N’tetraacetic acid; MLCK, myosin light chain kinase; PPi, pyrophosphate; PAGE, polyacrylamide gel electrophoresis; TPA, 12-0-tetradecanoylphorbol-t3-acetate. @306-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. AN rights of reproduction in any form reserved.
122
Ca2+-
is the most
In fact, MLCK of rat
Ca2+
this Ca-free
is rather
light chain by MLCK
contraction
We have
the study with
does not rise during
(4).
(1,2). maximal
and intracellular
Furthermore,
without
contraction)
contraction).
Therefore, it is predicted that this contraction that is, it is not triggered by the usual increase in [Ca2f]i.
It is generally and
smooth muscle
(3).
independent, probable
have
of rat uterine
contraction by oxytocin
that calcium
chelator fura-
contraction
is incubated
oxytocin-induced
contraction
ML-9
sustained
inhibitors, smooth
Vol.
BIOCHEMICAL
176, No. 1, 1991
muscle. myosin
But they have phosphorylation
no effect by MLCK
AND BIOPHYSICAL
on Ca-free
contraction
is not involved
study was performed
to clarify the phosphorylation
sustained
by means
contraction
(7,8) and urea-glycerol associated
of two different
PAGE
(9).
with any phosphorylation
(6), indicating
in this process.
a possibility
Therefore,
that
the present
state of myosin in an early stage of this electrophoresis
We reported of myosin
RESEARCH COMMUNICATIONS
systems,
here that Ca-free
namely,
PPi PAGE
contraction
was not
light chain.
EXPERIMENTAL PROCEDURES The rat uterine longitudinal smooth muscle was obtained as described by Matsuo and Uchfda (10). The muscle strips were mounted in a 10 ml organ bath filled with normal LockeRinger solution aerated with 5% carbon dioxide in oxygen at 30°C. After equilibration in normal Locke-Ringer solution for 1 h at a resting tension of 0.59, the stripes were incubated in Ca2+-free solution containing 3 mM EGTA for 1 h at a resting tension of 0.29 (EGTA treatment). The medium was then replaced by Ca 2+-free solution containing 0.2 mM EGTA. After further incubation for 10 min. contraction was induced by the addition of 10-2 unit/ml oxytocin (Ca-free contraction). Isometric tension was monitored with a force displacement transducer. At indicated times, the organ bath was removed without influencing tonic tension and strips were rapidly frozen by submersion in dry ice-acetone. Normal Locke-Ringer solution had the following composition (mM): NaCI,154; KCl,5.63; CaCl2,2.16; MgCl2,2.10; NaHC03,5.95; and glucose,5.55. Ca2+-free solution had the same composition except that CaC12 was substituted by 3 or 0.2 mM EGTA. The frozen strips were crushed in a frozen stainless steel mill at -17OOC. The frozen powder was extracted in 60 volumes of PPi sample buffer containing 100 mM Na4P207, 5 mM EGTA, 2.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 0.6 M KI and 10% glycerol (pH 8.8), which was slightly modified from the original composition of Persechini et al. (11). After centrifugation of the extract, glycerol and sucrose were added to give final concentration of 30 and 20%, respectively. The samples were subjected to PPi PAGE as described previously by Takano-Ohmuro and Kohama (7,8) for 16 h to examine the phosphorylation state of myosin by separating phosphorylated myosin from unphosphorylated one. The pattern of protein on PPi PAGE disk gel was visualized by staining with 0.1% Coomassie brilliant blue R-250. In order to analyze phosphorylation state of myosin light chain by urea-glycerol PAGE, PPi PAGE of the muscle extract was carried out for 3 h After staining briefly with Coomassie brilliant blue R-250, a major protein band which contained unseparated myosin and filamine was excised from the gel and treated with the urea solution containing 9 M urea, 100 mM Tris-HCI (pH 6.8) and 5% 2mercaptoethanol for 10 min. Urea-glycerol PAGE of the excised gel was then performed as described previously (9) to examine the phosphorylation state of myosin light chain by separating phosphorylated myosin light chain from unphosphorylated one in order of increased mobility. The pattern of protein on urea-glycerol PAGE slab gel was visualized by means of a slightly modified procedure (12) of the highly sensitive silver stain of Oakley et a/. (13), which is approximately 20-fold sensitive than the Coomassie brilliant blue staining. Identification of filamin on PPi PAGE gel was carried out as described previously (7). RESULTS Exposure sustained in Fig.1.
of the muscle strips to 45.6 mM K+ medium contraction
which gave a sub-maximal
At time zero,
unphosphorylated
gizzard
one myosin
tension
(designated
myosin was observed
15 set after the muscle was exposed migrated
band
in the presence by M) which
123
comigrated
with the
just under the filamin band on PPi PAGE.
myosin
a
of about 2g after 60 set, as shown
to the high Kf medium,
faster than the unphosphorylated
of Ca2+ caused
additional
band appeared
At
two bands which were
on PPi PAGE.
The upper
Vol.
176,
BIOCHEMICAL
No. 1, 1991
0
15
60
AND BIOPHYSICAL
0
set
RESEARCH COMMUNICATIONS
5
15
6Osec
2 2
k&i 2
c CL
01
456mM
Kf
I
02
-4
1 Omin
F -M
1CJ2unit/ml
oxytoc\n
I
-
I
1 Omin
&J. Analysis of the phosphorylation state of myosin by PPi PAGE during 45.6 mM K+induced contraction in the medium containing Ca 2+. After equilibration of uterine smooth muscle in normal Locke-Ringer solution for 1 h at a resting tension of 0.59, contraction was induced by changing the medium to K + rich solution containing 45.6 mM K+. Isometric tension was monitored with a force displacement transducer. Myosin phosphorylation was determined for individual strips frozen after indicated times of stimulation (indicated with the arrows). Unphosphorylated myosin (M), the myosin with one mono-phosphorylated 20 kDa light chain (MPl) and the myosin witQ two mono-phosphorylated 20 kDa light chains (MP2) were separated in the order of increasing mobility on PPi PAGE. The pattern of protein on the PPi PAGE disk gel was visualized by staining with 0.1% Coomassie brilliant blue R-250. The band represented by “F” was filamin. To the right of M, MPl and MP2 is schematically shown how myosin is phosphorylated at 20 kDa light chain, which was deduced from the results of urea-glycerol PAGE analysis of each myosin band separated on the PPi PAGE (figures not shown). Fia.2. Analysis of the phosphorylation state of myosin by PPi PAGE during oxytocintension of 0.59, induced contraction in the medium containing Ca 2+. After 1 h at a resting contraction was induced by the addition of 10W2 unit/ml oxytocin. See”Fig.1 legend” for details.
band (MPl)
was the myosin
with one unphosphorylated
band (MP2)
was the myosin
with two mono-phosphorylated
from the results of subsequent glycerol
analysis
PAGE (data not shown).
increased.
Approximately
expressed
by the percent
In the presence stage followed detected
40%
of
Ca2+,
oxytocin
were
(10s2
contraction
and MP2 bands was observed
by PPi PAGE. was about
myosin
unit/ml)
deduced
disk gel by urea-
state of these two bands
phosphorylated
induced
light chain
induced
one-tenth
induced
of oxytocin
form
at
a tonic
this
time
contraction
Phosphorylation
at
as
and a time-dependent
agreement
increase
sustained
of normal
that the initiation
phosphorylation contraction
contraction 124
early
of myosin
by high K+ (Fig.1) and oxytocin
of myosin during oxytocin-induced Oxytocin
the
as shown in Fig.2.
of Ca2+ and also confirm the general requires
band from PPi PAGE
up to 60 sec. These results indicate the involvement
in the contractions
Phosphorylation which
myosin
as early as 5 set after the addition
contraction
20 kDa light chains,
After 60 set the phosphorylation of
and the lower
of total myosin.
by a pendular
phosphorylation presence
of excised
20 kDa light chain
was
of MPI
of myosin
(Fig.2)
of smooth
in the muscle
(4). Ca-free with
(Fig.3).
contraction a maximal
was then examined tension
At any indicated
of about
0.29
times of 5, 15,
Vol.
BIOCHEMICAL
176, No. 1, 1991
s E .. 2
AND BIOPHYSICAL
F M-
.
vanadate &g.J. induced induced
Analysis of the phosphorylation contraction in the Ca2+-free by the addition of 10M2 unit/ml
Fjg&.
Analysis
induced induced
of the
states medium. oxytocin.
phosphorylation
light
3~
indicate
that Ca-free
state
contraction
I0-h
t
4
1Omin
of myosin by PPi PAGE during oxytocinAfter “EGTA treatment” contraction was See “Fig.1 legend” for details. of myosin
by PPi
contraction in the Ca 2+-free medium. After “EGTA by the addition of 10e5 M vanadate. See “Fig.1 legend”
and 60 set, we failed to detect phosphorylated results
PAGE
during
treatment” for details.
vanadate-
contraction
was
myosin bands such as MPI and MP2.
occured
without
any phosphorylation
These
of
myosin
chain.
We next examined Ca*+-free
the phosphorylation
medium.
Vanadate
manner
(Fig.4).
muscle
is still obscure,
Although
phosphorylation
the mechanism
of myosin
the
were
contraction
the
observed
urea-glycerol
presence
manner of Ca*+
phosphorylated contraction
causing
MLCK,
during
in a similar uterine
smooth of the
inhibitor
KT-5926
in Ca*+-free
contraction,
(5)
medium).*
small but significant M vanadate-induced
3~10~~
in
to be a result
a MLCK
M vanadate
was employed
in an
early The
(Fig.5B, PAGE
b, c, and
analysis
and
phosphorylation
in
the
light chain
d). also
of 20
of contractions
phosphorylated
contraction
20 kDa myosin
kDa
and
Uchida,M.K.,
myosin of
was observed
These
results that
oxytosin
light
chain.
myosin
light chain
induced
absence
indicated
unpublished
phosphorylation
state of the myosin
stage
(Fig.SA).
to analyze
confirmed
2 Karibe,H.
seems
because
Ca-free
contraction
in contracting
contraction
by 3~10~~
The phosphorylation
vanadate-induced
PPi
by
on PPi PAGE
PAGE
light chain.
time-dependent
during
induced
sustained
contraction
MPl
In and
sustained
(Fig.4).
Finally, myosin
contraction
vanadate-induced
of action of vanadate
light chains
to the case of oxytocin-induced
during
M) induced
the vanadate-induced
inhibits
MP2 bands
of myosin
(3~10~~
contrast
by
RESEARCH COMMUNICATIONS
observations.
125
light
chain
and was
(Fig.5B.
during the
was increased
by oxytocin
Ca*+
induced
state of 20 kDa
f).
also
Ca-free
K+
observations contraction
in
observed
However,
oxytocin-induced
above
high
in a
no
Ca-free obtained without
Vol.
BIOCHEMICAL
176, No. 1, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
. VlLCp
a
b
d
c
e
f
9
B
abcdef &&. Analysis of the phosphorylation state of 20 kDa myosin light chain by ureaThe PPi PAGE samples prepared as described in glycerol PAGE during contractions. “EXPERIMENTAL PROCEDURES” were subjected to PPi PAGE for 3 h to separate and recover whole myosin in the sample. The myosin excised from the PPi PAGE gel was treated with the urea solution. Unphosphorylated (RLC) and monophosphorylated (RLCp) forms of the light chain were separated as a function of increasing mobility in urea-glycerol PAGE. The pattern of urea-glycerol PAGE gel was visualized by the silver staining. (A) Urea-glycerol PAGE showing the state of 20 kDa myosin light chain phosphorylation during contractions in the presence of Ca2+. Muscles were frozen at 0 set (lane a), 15 set (lane b), and 60 set (lane c) after changing to KC rich (45.6 mM) solution and at 0 set (lane d), 5 set (lane e), 15 set (lane f), and 60 set (lane g) after the addition of 10m2 unit/ml oxytocin. (B) Ureaglycerol PAGE showing the state of 20 kDa myosin light chain phosphorylation during were frozen at 0 set (lane a), 5 set (lane b), contractions in the absence of Ca 2c. Muscles 15 set (lane c), and 60 set (lane d) after the addition of 10s2 unit/ml oxytocin and at 0 set (lane e) and 15 set (lane f) after the addition of 10m5 M vanadate.
DISCUSStON PPi
and urea-glycerol
PAGE
uterine
smooth
muscle
were
Kg) of myosin molecule purification.
(78).
myosin
light
smooth
muscle
MLCK
phosphorylated
occurs
whereas
increased
bands
contraction
by oxytocin
in the absence
and
charge (MPl
high
Ca-free
PAGE
systems
showing
that oxytocin
small
And
amount
mobility
of myosin were
the
(Figs.1,
of
is phosphorylated
by
on urea-glycerol
of Ca2+
Phosphorylation
however,
and
was not detected without
chain.
126
causing
during
the
vanadate-induced the involvement
of myosin
To our best knowledge,
contraction
In this study, the
on PPi PAGE
2, and 4), indicating
without any mobility
light chain
evident
(l-2
of regulatory
lastly,
as a result of phosphorylation.
K+ in the presence
and 5B).
can induce
(7,8).
only when the myosin
and MP2)
of Ca 2+
contraction, (Fig.3
Firstly,
nor dephosphorylation
analysis
by MLCK in these processes.
glycerol light
PAGE
the increased
contraction
oxytocin-induced
phosphorylation
PPi
negative
reasons.
states in contracting
by PPi PAGE from small tissue samples
on PPi PAGE increases
myosin
phosphorylation
neither during
myosin phosphorylation
by following
can be analyzed
myosin
(7,8,14,15),
employed
Secondly,
chain
PAGE reflects
PAGE to analyze
light chain
in both
of
during
PPi and urea-
this is the first report
phosphorylation
of myosin
Vol.
BIOCHEMICAL
176, No. 1, 1991
In Ca*+-induced increased chain.
contraction
[Ca*+]i
induces
systems
activation
As a result, the formation
initiates
muscle
case because previous
contraction. no myosin
observation
is independent
contraction
by agonist
phenomenon. smooth
This
muscles
of the rise stimulation
phosphorylation,
although
of
its usual
oxytocin-induced
than
Ca-free
small,
to initiate
since
can
contraction However,
no phosphorylation
by the
contraction
of
of Ca*+-induced
account
for
the
protein
Such mechanism
(18).
myosin
is also supported
by GTP-binding
muscle.3
increase
contraction
that
light
this is not the
that Ca-free
Sensitization
mediated
smooth
increase
fura-
(3).
is a possibility
and uterine
contraction,
This finding
indicator
that
of myosin
actin and phosphorylated
Ca-free
in [Ca*+]i
is probably
in myosin
[Ca*+]i
Ca *+
2, and 5, it is obvious
by phosphorylation
between
is observed.
fluoresent
RESEARCH COMMUNICATIONS
in Figs.1,
followed
of crossbridges
phosphorylation
mechanism
(16,17)
as shown
of MLCK
But in oxytocin-induced
by using
rat uterus
AND BIOPHYSICAL
above
in various
is likely to result
even
with much smaller
this
is also
of myosin
unlikely
light
in
chain
is
observed. Besides
receptor
agonist,
a phorbol
C, clearly
caused
Ca-free
contraction
others
that TPA contracts
(20.21). protein
Although kinase
cytoskeletal
ester TPA, at a concentration in rat thoracic
some vascular
no phosphorylation C phosphorylates
proteins
(22).
and cytoskeletal
inhibitors
might be actually
involved
(6).
smooth
muscles
of myos.
light
various
Indeed, in Ca-free
functional
Ca-free
Protein
aorta
contraction
kinases
contraction.
(19).
to activate
protein
even under Ca*+-free chain
was observed
proteins
including
was inhibited
(but not MLCK)
kinase
It has been reported
by
conditions in this study,
contractile
by protein
and cytoskeletal
and
kinase
C
elements
Further study is need to be done.
ACKNOWLEDGMENTS: We thank Dr. J.F. Kuo, Department of Pharmacology, for helpful advice and critical reading of the manuscript. This work was supported by Grant-in-Aid (No.2640575 and 60571062) for Scientific Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. Sakai,K. and Uchida,M. (1980) Jpn. J. Pharmacol. a, 394-396. 2. Uchida,M.K., Sakai,K., and Matsuo,K. (1989) Gen. Pharmacol. a 117-121. 3. Matsuo,K., Gokita,T., Karibe,H., and Uchida, M.K. (1989) Biochem. Biophys. Res. Commun. m, 722-727. 4. Kamm,K.E. and Stul1,J.T. (1985) Annu.Rev. Pharmacol. Toxicol. a, 593-620. 5. Nakanishi,S., Yamada,K., Iwahashi,K., Kuroda,K., and Kase,H. (1990) Mol. Pharmacol. 37, 482-488. 6. Karibe,H., Matsuo,K., Gokita,T., and Uchida,M.K. (1990) Eur. J. Pharmacol. 188. 407. 410. 7. Takano-Ohmuro,H. and Kohama,K. (1986) J. Biochem. .jQQ, 1681-1684. 8. Takano-Ohmuro,H. and Kohama,K. (1986) J. Biochem. I.QQ, 259-268. 9. Perrie,W.T. and Perry,S.V. (1970) Biochem. J. 119, 31-38. 10. Matsuo,K. and Uchida,M.K. (1987) Eur. J. Pharmacol. j/&Q, 294-301.
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
11. Persechini,A., Kamm,K.E., and Stul1,J.T. (1986) J. Biol. Chem. m, 6293-6299. 12. Mikawa,T., Takeda,S., Shimizu,T., and Kitaura,T. (1981) J. Biochem. 89, 1951-1962. 13. Oakley,B.R., Kirsch,D.R., and Morris,N.R. (1980) Anal. Biochem. m, 361-363 14. Takano-Ohmuro,H. and Kohama,K. (1987) J. Biochem. m, 971-974. 15. Takano-Ohmuro,H., Kohama,K., Kathuria,S., and Fujita-Yamaguchi,Y. (1989) Proc. Japan Acad f& 200-202. 16. Nishimura,J., Kolber,M., and van Breemen,C. (1988) Biochem. Biophys. Res. Commun. m, 677-683. 17. Kizawa,T., Kobayashi,S., Horiuchi,K., Somlyo,A.V., and Somlyo,A.P. (1989) J. Biol. Chem. u 53395342. 18. Karaki,H. (1989) Trends Pharmacol. Sci. u, 320-325. 19. Karibe,H. and Uchida,M.K. (1990) Gen. Pharmacol. 2, 191-197. 20. Geason,M.E. and Flaim,S.F. (1986) Biochem. Biophys. Res. Commun. 138, 1362.1369. 21. Ito,H. and Lederis,K. (1987) Am. J. Physiol. 252, C244-247. 22. Park,S. and Rasmussen,H. (1986) J. Biol. Chem. a, 15734-15739.
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