Am J Physiol Cell Physiol
279: C1722–C1732, 2000.
Determinants of the contractile properties
in the embryonic chicken gizzard and aorta
OZGUR OGUT1 AND FRANK V. BROZOVICH1,2
Departments of 1Physiology and Biophysics and 2Medicine (Cardiology),
Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970
Received 20 January 2000; accepted in final form 4 August 2000
controls the tone of many
involuntary muscles, such as those lining arteries and
the walls of the stomach. On the basis of their contraction profiles in response to K⫹ depolarization (43),
smooth muscles are divided into two general contractile phenotypes: tonic and phasic. Tonic smooth muscles demonstrate slow rates of force activation and relaxation, lower maximum speeds of shortening (Vmax)
as well as force maintenance, whereas phasic smooth
muscles display rapid rates of force activation and
relaxation and have a high Vmax but poor force maintenance (reviewed in Ref. 15). In both smooth muscle
types, phosphorylation of the 20-kDa regulatory myosin light chains (MLC20) by Ca2⫹-calmodulin-activated
myosin light chain kinase (MLCK) promotes actomyosin cycling to produce force. Although previous studies
have suggested that isoform expression of myosin and
actin filament-associated proteins are molecular determinants of the contractile properties of smooth muscle
(46), the molecular determinants of the unique contractile properties of phasic versus tonic smooth muscle
phenotypes remain unclear.
Proteins controlling the activation of the myosin
ATPase activity, namely MLCK and myosin light chain
phosphatase (MLCP), have been suggested as candidates that define contractile properties in phasic and
tonic smooth muscles (12, 23). These proteins control
MLC20 phosphorylation and therefore the level of activation. However, the unique rates of force activation in
phasic and tonic smooth muscles are not simply explained by differences in Ca2⫹ transients or rates of
MLC20 phosphorylation because phasic tissues maintain higher rates of force activation when skinned or
when the MLC20 is thiophosphorylated to dissociate
force activation from MLCK and MLCP kinetics (22).
Nonetheless, maximum shortening velocity (Vmax) in
smooth muscle is related to the level of activation as
defined by MLC20 phosphorylation (1, 10). Furthermore, it has been proposed that MLCP may play a role
in the regulation of relaxation because protein kinase
C activation and subsequent MLCP inhibition decreases the rate of MLC20 dephosphorylation and force
relaxation (25, 34), parameters thought to reflect a
decrease in MLCP catalytic activity. Moreover, the
MLCP holoenzyme may exist as isoforms, which differ
in the expression of two unique myosin binding subunits (M130/M133; Ref. 41). Therefore, in addition to
Address for reprint requests and other correspondence: F. V.
Brozovich, Dept. of Physiology and Biophysics, Case Western Reserve Univ. School of Medicine, 10900 Euclid Ave., Cleveland, OH
44106-4970 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
force; 17-kilodalton myosin light chain; phasic contractile
properties; tonic smooth muscle; development
SMOOTH MUSCLE CONTRACTION
C1722
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
Ogut, Ozgur, and Frank V. Brozovich. Determinants of
the contractile properties in the embryonic chicken gizzard
and aorta. Am J Physiol Cell Physiol 279: C1722–C1732,
2000.—Smooth muscle is generally grouped into two classes
of differing contractile properties. Tonic smooth muscles
show slow rates of force activation and relaxation and slow
speeds of shortening (Vmax) but force maintenance, whereas
phasic smooth muscles show poor force maintenance but
have fast Vmax and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular
determinants that define phasic (gizzard) and tonic (aortic)
contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid
rates of force activation and relaxation compared with the
aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin
light chain (MLC17a). Previous data from in vitro motility
assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res
Cell Motil 18: 103–110, 1997) have postulated that myosin
heavy chain (MHC) isoform expression is a determinant for
Vmax in intact tissues. In the current study, differences in
Vmax did not correlate with previously published differences
in MHC or MLC17a isoforms. Rather, Vmax was increased
with thiophosphorylation of the 20-kDa regulatory myosin
light chain (MLC20) in the gizzard, suggesting that a significant internal load exists. Furthermore, Vmax in the gizzard
increased during postnatal development without changes in
MHC or MLC17 isoforms. Although the rate of MLC20 phosphorylation was similar at ED 20, the rate of MLC20 dephosphorylation was significantly higher in the gizzard versus the
aorta, correlating with expression of the M130 isoform of the
myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique
MLCP and MLC17 isoform expression marks the phasic contractile phenotype.
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
MATERIALS AND METHODS
Identification of regulatory protein isoforms in the embryonic chicken. Fertilized chicken eggs were incubated at 37°C
in humidified air and collected at embryonic days (ED) 10, 12,
14, 16, 18, and 20. The gizzard and thoracic aortas from five
or more embryos were dissected in Ca2⫹-free physiological
saline solution (in mM: 140 NaCl, 4.7 KCl, 0.3 Na2HPO4, 2
MOPS, 0.5 EGTA, 0.2 EDTA, 1.2 MgCl2, 5.6 glucose, final pH
adjusted to 7.4 with 1 N KOH). The tissues were immediately
homogenized in SDS-PAGE buffer, heated to 80°C for 5 min,
and clarified by centrifugation at 14,000 g in a microcentrifuge. The samples were resolved by either 12% SDS-PAGE
with an acrylamide-to-bisacrylamide ratio of 29:1 (calponin,
MLC20) or 14% SDS-PAGE with an acrylamide-to-bisacryl-
amide ratio of 180:1 (MLCK, myosin binding subunit of
MLCP). To ensure comparable sample loading, the samples
were normalized according to the G-actin band in SDSPAGE. Once sample loadings were comparable, the SDSPAGE-resolved proteins were transferred to 0.22-␮m nitrocellulose membrane in a transfer buffer (100 mM glycine, 10
mM Tris, pH 8.6, 20% methanol) and blocked at room temperature for 3 h using Tris-buffered saline (TBS; 150 mM
NaCl, 50 mM Tris 䡠 HCl, pH 7.4) containing 1% BSA. Blocked
nitrocellulose membranes were incubated overnight at 4°C in
TBS-0.1% BSA with monoclonal antibodies (MAbs) against
calponin (CP1; 26), MLC20 (My-21; Sigma Immunochemicals), MLCK (LKH3 and LKH18; Ref. 6), or MLCP myosin
binding subunit (Covance). For MLCK identification, a purified smooth muscle MLCK protein sample was used as a
control. The membranes were washed three times for 10 min
in TBS containing either 0.05% Tween 20 or 0.5% Triton
X-100–0.05% SDS and then rinsed with TBS. The membranes were subsequently incubated with TBS-0.1% BSA
containing alkaline phosphatase-conjugated goat anti-mouse
IgG or IgM second antibody for 50 min at room temperature.
After membranes were washed after second antibody incubation, the color reaction was developed in alkaline phosphatase substrate buffer (in mM: 110 NaCl, 5 MgCl2, 100 Tris,
final pH 9.5) containing 0.04% (wt/vol) nitro blue tetrazolium
and 0.02% (wt/vol) 5-bromo-4-chloro-3-indolylphosphate.
Tissue strips from ED 12, 16, and 20 aorta and gizzard
were also freshly dissected and fixed in buffered Formalin
solution for paraffin embedding. The tissues were sectioned
at 7 ␮m and stained with hematoxylin and eosin according to
standard protocols.
Force of contraction of developing chicken gizzard and
aorta. Embryonic chicken gizzard and aorta strips were excised from embryos at various stages of development,
skinned, and allowed to contract in high Ca2⫹ (0.1 mM free
Ca2⫹) and relax in low Ca2⫹ (1 nM free Ca2⫹) solutions. The
solutions were mixed according to a computer program that
calculated the amount of stock solutions required for a given
set of free ion concentrations (3). The binding constants for
the ionic species present were corrected for temperature and
ionic strength. The strips were clipped between two aluminum foil T clips (11) and skinned at room temperature for 15
(gizzard) or 30 min (aorta) in a pCa9 solution [in mM: 5
Na2ATP, 5 EGTA, 25 potassium methane sulfonate, pH 7, 6.9
MgCl2 (1 mM free Mg2⫹), 25 creatine phosphate, 2 glutathione, and 400 ␮M ␤-escin, final pH to 7 with 1 N KOH]. The
times used for skinning provided optimal Ca2⫹ activated
force per cross section for the preparations; longer times
compromised the force response. After skinning, the tissues
were transferred to pCa9 relaxing solution [in mM: 5.4
Na2ATP, 5 EGTA, 71 potassium methane sulfonate, 7.1
MgCl2 (1 free Mg2⫹), 25 creatine phosphate, 25 N,N-bis(2hydroxyethyl)-2-aminoethanesulfonic acid (BES), and 12 ␮M
CaCl2, final pH to 7 with 1 N KOH] and mounted on the stage
of a computer-controlled mechanics workstation. One end of
the tissue strip was hooked to a length driver (Physik Instrumente, Waldbron, Germany) and the other to a force transducer (Akers AE 801 Sensonor, Horten, Norway). The strips
were stretched to 1.3 times their resting length (or to the
length for maximal force development) while in relaxing
solution. Contractions were initiated by transferring the
strips to a well containing pCa4 activating solution [in mM:
60 potassium methane sulfonate, pH 7, 5 EGTA, 5.3 CaCl2,
6.98 MgCl2 (1 free Mg2⫹), 5.56 Na2ATP, 25 creatine phosphate, 25 BES, final pH to 7 with 1 N KOH]. Previous studies
(7) have demonstrated that tissue from the developing aorta
and gizzard is predominantly smooth muscle cells. Force
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
contractile protein isoform diversity, the control of actomyosin kinetics in smooth muscle may also rely on
factors modulating MLC20 phosphorylation.
The smooth muscle myosin heavy chain (MHC) is a
single gene with two pairs of alternatively spliced exons. Two isoforms, named SM1 and SM2, differ by
expression of a 43- or 9-amino acid COOH-terminus,
respectively (5). Their contribution to contractility remains controversial (2, 27, 36). In addition, alternative
splicing of an exon near the NH2-terminal 25- to 50kDa junction of the myosin head accounts for a 7-amino
acid insert in visceral SM1 and SM2 MHCs (28). MHC
isoform diversity at the NH2-terminus has been hypothesized as a determinant of the tonic and phasic
contractile properties in smooth muscle (28, 30, 46).
Additionally, the 17-kDa essential myosin light chains
(MLC17) associated with the neck region of MHCs also
show isoform diversity. Two splice variants of MLC17
have been found in smooth muscle, and they differ by
inclusion (MLC17b) or exclusion (MLC17a) of a 39-bp
exon (38). Studies have shown a correlation between
increased MLC17b expression and decreased actin activated Mg2⫹-ATPase activity of myosin (18) as well as
decreased Vmax in intact tissues (31).
We assumed that the major factors controlling the
contractile properties of developing gizzard and aortic
smooth muscle would be the myosin isoforms (MHC
and MLC17) or proteins controlling myosin activation
through MLC20 phosphorylation (MLCK and MLCP).
Because MHC and MLC17 isoforms switch during development of the embryonic chicken aorta and gizzard
(7), we hypothesized that changes in protein isoform
expression throughout development would be reflected
by changes in the contractile phenotype. Therefore, we
investigated MHC, MLC20, MLC17, MLCK, MLCP, and
thin filament-associated proteins to determine the
markers for phasic and tonic contractile properties. In
the aorta and gizzard, the tonic phenotype was the
default during early embryonic development. Approximately 1 wk before hatching, the phasic phenotype
developed in the gizzard. We demonstrate that the
MLC17 and MLCP isoforms are molecular markers for
the phasic contractile phenotype. Expression of
MLC17a defined the rapid rate of force activation,
whereas expression of a gizzard-specific MLCP isoform
was a determinant for the rapid rate of relaxation, both
hallmarks of phasic smooth muscles.
C1723
C1724
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
acetone. The tissues were frozen in liquid nitrogen for 30
min. After denaturation, the tissue strips were washed twice
with acetone, dried using a Speed-Vac evaporator and homogenized in PAGE sample buffer containing 6 M urea, 20
mM glycine, 22 mM Tris 䡠 HCl, pH 8.6, and 1 mM EDTA.
Glycerol (5% vol/vol) and bromophenol blue (0.1% wt/vol)
were added to the samples before loading. The samples were
resolved by a 19:1 (acrylamide-bisacrylamide) 10% polyacrylamide gel containing 40% (vol/vol) glycerol and 4 M urea and
polymerized in a gel buffer containing 20 mM glycine and 22
mM Tris 䡠 HCl, pH 8.6. The gel was prerun in the electrophoresis buffer (20 mM glycine, 22 mM Tris 䡠 HCl, pH 8.6, 1
mM thioglycolic acid) for 1 h at 200 V before loading of the
samples. The samples were resolved overnight at 200 V or
roughly 2 h after the bromophenol blue dye front had exited
the gel. Resolved proteins were transferred to nitrocellulose
membrane, and the phosphorylated and unphosphorylated
MLC20 were identified by immunoblotting as described earlier. The percentage of MLC20 phosphorylated was calculated
by determining the ratio of the phosphorylated MLC20 band
versus the total MLC20 (phosphorylated ⫹ unphosphorylated
forms) identified by the mAb.
The rate of dephosphorylation of MLC20 was measured in
a fashion similar to phosphorylation. After the tissue strips
were skinned, all were activated for 300 s in the activation
buffer. After activation, the strips were allowed to relax in a
Ca2⫹-free rigor solution without ATP or creatine phosphate
(173 mM potassium methane sulfonate, pH 7, 5 mM EGTA,
12 ␮M CaCl2, 1.16 mM MgCl2, 25 mM BES, final pH to 7 with
1 N KOH) for 0, 30, 60, 120, and 300 s. The dephosphorylation was terminated by denaturing the tissue strips in 15%
trichloroacetic acid in acetone. The tissues were prepared for
glycerol-urea-PAGE as before. For phosphorylation and dephosphorylation experiments, results are expressed as the
average ⫾ SE of three paired experiments. The experimental
data were well fit to a single exponential.
Tissue strips were also activated with ATP-␥-S as described
before, and the extent of thiophosphorylation was determined
by glycerol-urea-PAGE. Thiophosphorylation of MLC20 was
near 100% in samples of aorta (86.9 ⫾ 6.6%, n ⫽ 3) and gizzard
(89.6 ⫾ 9.3%, n ⫽ 3) incubated for 300 s in thiophosphorylation
solution. To ensure that tissues were thiophosphorylated as
intended, glycerol-urea-PAGE was also performed on samples
that were incubated in the dephosphorylation buffer for 300 s
after thiophosphorylation. Because thiophosphorylated MLC20
is not a substrate for MLCP, the high percentage of thiophosphorylated MLC20 should remain. This was confirmed by the
persistence of the phosphorylated form of MLC20 in glycerolurea-PAGE analysis of both the aorta (90.2 ⫾ 5.0%; n ⫽ 3) and
the gizzard (89.7 ⫾ 9.5%, n ⫽ 3) after 300 s incubation in the
Ca2⫹-free rigor solution.
RESULTS
Rapid rate of force activation in the developing gizzard. Representative contraction profiles for embryonic
aorta and gizzard at various stages demonstrate the
development of the contractile phenotypes of these
tissues (Fig. 1). Both smooth muscle tissues show a
slow rate of force activation early during embryonic
development (ED 8–12). The faster contractile properties of the gizzard strips become apparent by ED 16,
with clearly increased rates of force activation and
relaxation compared with the aorta (Table 1 and see
Table 6). The rates of activation between ED 12 gizzard
and aorta were not different (P ⬎ 0.05). Although the
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
generated was normalized to force per cross-sectional area
(mN/cm2) by measuring the dimensions of the tissues and
assuming that the tissues were rectangular. Tissue strips
were of comparable sizes on given days of development and
were typically 250 and 1,000 ␮m long, 80–150 ␮m wide, and
80–150 ␮m thick. Results are reported as the average ⫾ SE.
All experiments were done at room temperature.
To measure the rate of force activation or the rate of
relaxation, tissue strips were skinned and allowed to contract
in the pCa4 activating solution until force reached a steady
plateau and then allowed to relax by transferring the tissues
to pCa9 relaxing solution. The contraction profiles were
stored on a digital oscilloscope (Nicolet 310). To determine a
rate, the force activation and relaxation profiles were fit to a
single exponential. Results are reported as the average ⫾ SE.
Differences were deemed significant if P ⬍ 0.05 by Student’s
t-test.
Experiments were also done wherein MLC20 was thiophosphorylated to assure maximal activation of myosin heads
during contraction (20). To thiophosphorylate MLC20,
skinned tissues were incubated for 5 min in an activating
solution wherein 5.56 mM (5 mM free) ATP-␥-S (Calbiochem)
was substituted for ATP and creatine phosphate. The tissues
were then transferred into pCa9 relaxing solution containing
ATP and force developed. The rates of force activation for
thiophosphorylated tissues were determined as described
above.
Vmax in developing chicken gizzard and aorta. The unloaded speed of shortening of the skinned tissues was determined from the force versus velocity relationship (19). A
computer program based on Smith and Barsotti (42) was
written in LabView (National Instruments, Dallas, TX) to
control length for constant loads. Force and length signals
were sampled and controlled using an analog-to-digital-digital-to-analog board (National Instruments PCI-MIO-16XE10, 100 ⫻ 103 samples/s, 16 bit). After activation of the
gizzard and aorta strips, the load on the tissues was decreased to any set percentage of the isometric force. The
tissues were maintained at a fraction of the initial isometric
load (1–90%) by a program monitoring tension (200–300
samples/s) and rapidly adjusting (shortening) length to provide constant load. A force clamp from an initial tone to any
desired new load was achieved within 10 (300 samples/s) to
30 ms (200 samples/s). The shortening profiles of the tissues
were saved, and the speed of shortening at various loads was
calculated from the tangent to the length versus time trace at
150 ms after the step change in load. The unloaded speed of
shortening was calculated by fitting the force versus velocity
relationship to the Hill equation (19) or taken as the speed of
shortening at 1% of initial isometric load (42). The speeds of
shortening were normalized to muscle lengths per second
(ML/s). For each tissue, at least three separate measurements were taken at each load tested; n represents the
number of tissues tested. However, the Vmax for smooth
muscles before ED 12 could not be reliably determined because of the low forces of Ca2⫹-activated contractions. Results are presented as the average ⫾ SE.
Analysis of MLC20 phosphorylation in developing chicken
gizzard and aorta. To determine the extent of phosphorylation of MLC20, a glycerol-urea-PAGE procedure (17) was
adapted to resolve the phosphorylated and unphosphorylated
forms of MLC20. Strips of chicken gizzard or aorta were
skinned as before, washed, and resuspended in the relaxing
buffer. To measure the rate of phosphorylation of MLC20,
tissue strips were incubated for 0, 30, 60, 120, and 300 s in
pCa4 activating solution. Activation of the strips was terminated by adding excess 15% (vol/vol) trichloroacetic acid in
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
C1725
rates of force activation were not significantly different
between ED 16 and 20 gizzard, they were always
significantly faster than either ED 16 or 20 aorta (P ⬍
0.05). The rates of force activation increased with development in the gizzard and were significantly faster
at ED 20 versus ED 12 (P ⬍ 0.05). Thiophosphorylation
of MLC20 significantly increased the rates of force
activation in both ED 20 aorta (0.018 ⫾ 0.003 s⫺1) and
gizzard (0.060 ⫾ 0.009 s⫺1) versus Ca2⫹ activation (P ⬍
0.05). However, the rate of force activation for ED 20
gizzard remained threefold faster than for ED 20 aorta
with or without thiophosphorylation.
Orientation of cells in the developing aorta and gizzard. To determine whether changes in the orientation
of the cells were occurring with development, ED 12,
16, and 20 aorta and gizzard sections were stained with
hematoxylin and eosin (Fig. 2). With development in
both tissues, an increase in cell density was apparent.
In the aorta, there was no change in the orientation of
the cells with development. In the gizzard, however,
the orientation of cells changed by ⬃20° between ED 12
and 16, with cells changing from parallel to the axis of
the strip at ED 12 to 20° off the axis at ED 16. No
subsequent change in cell orientation was observed
from ED 16 to 20 gizzard.
Table 1. The rates of force activation in embryonic
chicken aorta and gizzard
Stage
ED
ED
ED
ED
12
16
20 (ATP)
20 (ATP-␥-S)
Aorta
n
Gizzard
n
0.003 ⫾ 0.002
0.003 ⫾ 0.001
0.005 ⫾ 0.001
0.018 ⫾ 0.003
3
5
7
4
0.007 ⫾ 0.004
0.012 ⫾ 0.001
0.017 ⫾ 0.005
0.060 ⫾ 0.009
3
8
8
4
Vales are means ⫾ SE in s⫺1. The rates of force activation were
defined from single exponential fits to the data. ED, embryonic day.
Protein isoform expression in the developing aorta
and gizzard. The expression of multiple regulatory
protein isoforms is a common theme in developing
muscle. Representative smooth muscle-specific markers were chosen, and their isoform expression throughout development of the embryonic chicken gizzard and
aorta was monitored (Fig. 3). Calponin, an actin filament-associated marker of smooth muscle, was present
throughout development of the gizzard and aorta as a
single isoform. Using MAb MY-21 (Sigma), we identified a single isoform of MLC20 expressed throughout
development of the aorta and gizzard. Comparison of
the expression of the M130/M133 isoforms of the myosin binding subunit of MLCP (41) indicated that only
M133 was expressed throughout development of the
aorta. On the other hand, a developmental isoform
switch of M130 for M133 was observed in the developing gizzard, with M130 expression beginning at ED 20.
This is consistent with upregulation of M130 expression to become the dominant myosin binding subunit
isoform expressed in adult chicken gizzard (41). The
smooth muscle MLCK isoform was the abundant
MLCK isoform expressed in both the gizzard and aorta
during embryonic development. Because the Western
blots only monitored isoform expression, we could not
make any conclusions with respect to changes in protein content with development.
Force of contraction of the developing gizzard and
aorta. We measured the force per cross-sectional area
in developing aorta and gizzard to determine whether
a correlation could be made with myosin isoform expression. Although the force per cross-sectional area
increased with development for both embryonic aorta
and gizzard (Table 2), ED 20 gizzard was significantly
higher (P ⬍ 0.05) than ED 20 aorta, with or without
thiophosphorylation of the myosin regulatory light
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 1. Contraction profiles of developing embryonic chick aorta and gizzard
strips. Representative tracings of
skinned embryonic days (ED) 8, 12, 16,
and 20 chicken aorta and gizzard
strips after Ca2⫹ activation. Early in
development, both the aorta and gizzard show tonic contractile properties
marked by a slow rate of force activation. The rapid rate of force activation
in the gizzard emerges by ED 16, implying that the determinants of tonic
and phasic contractile properties are
established. A similar trend is seen for
the rates of force relaxation, as ED 16
gizzard shows faster rates of relaxation
compared with the aorta (Table 5).
C1726
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
chain. The increases in force per cross section may be
achieved by differences in actin/myosin content or tissue content of smooth muscle.
Vmax in embryonic chicken gizzard and aorta. To
monitor changes in Vmax with development of the
smooth muscle contractile phenotypes, the Vmax for
embryonic aorta and gizzard strips was determined at
Table 2. Force per cross-sectional area in embryonic
chicken aorta and gizzard
Fig. 3. The expression of regulatory protein isoforms during development. With the use of specific monoclonal antibodies, the expression pattern of the myosin binding subunit of myosin light chain
phosphatase (MLCP), myosin light chain kinase (MLCK), h1-calponin, and 20-kDa myosin light chain (MLC20) were determined in the
developing gizzard and aorta. All proteins showed expression of a
single protein isoform throughout development except for MLCP.
The gizzard showed expression of the M130 myosin binding subunit
beginning between ED 18 and 20, with M130 being the dominant
isoform expressed at ED 20.
Aorta
Stage
ED
ED
ED
ED
12
16
20 (ATP)
20 (ATP-␥-S)
Gizzard
Force per
cross section
n
Force per
cross section
n
17.6 ⫾ 1.8
31.3 ⫾ 9.7
119.8 ⫾ 12.7
90.0 ⫾ 11.6
4
5
3
10
74.2 ⫾ 24.6
57.8 ⫾ 30.5
403.5 ⫾ 91.9
518.5 ⫾ 76.9
4
4
3
11
Values are means ⫾ SE in mN/cm2.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
Fig. 2. The orientation of cells in embryonic
aorta and gizzard tissues. Tissue samples
from ED 12, 16, and 20 aorta and gizzard
were sectioned and stained with hematoxylin
and eosin to determine the orientation of the
cells. During development of the aorta, no
change in the orientation of the cells was
observed. During development of the gizzard,
a change of ⬃20° in the orientation of the cells
is evident from ED 12 to 16. No change in
orientation was seen between ED 16 and 20
gizzard. Bars denote 10 ␮m.
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
Table 3. The unloaded speed of shortening
in embryonic chicken aorta and gizzard
Aorta
Stage
ED
ED
ED
ED
12
16
20 (ATP)
20 (ATP-␥-S)
Fig. 4. The force versus velocity relationship in thiophosphorylated
ED 20 aorta and gizzard. After thiophosphorylation of skinned ED 20
aorta and gizzard strips, the force versus velocity relationship was
determined by decreasing the load on the strips to various levels and
measuring the speed of shortening. For a given tissue, either the
absolute speeds of shortening [muscle length (ML)/s] at various loads
were reported (A) or speeds of shortening were normalized by dividing by the maximum speed of shortening (Vmax) of the tissue and
expressing the speed as a fraction of the Vmax (B). A: the force versus
velocity relationship from single ED 20 aorta and gizzard fibers.
Data points are the average ⫾ SE for 3 measurements at each load.
B: pooled data for ED 20 aorta (n ⫽ 8) and ED 20 gizzard (n ⫽ 11)
strips. The pooled data were fit to the Hill equation. Note that the
gizzard displays a flatter relationship, indicating that a higher absolute Vmax is achieved at loads ⱖ40% of maximum (A) and that a
greater percentage of maximum speed is achieved for isotonic contractions above zero load (B).
Gizzard
Speed, ML/s
n
Speed, ML/s
n
0.039 ⫾ 0.013
0.054 ⫾ 0.003
0.144 ⫾ 0.068
0.164 ⫾ 0.028
3
4
4
8
0.032 ⫾ 0.001
0.028 ⫾ 0.006
0.038 ⫾ 0.004
0.084 ⫾ 0.018
4
7
5
11
Values are means ⫾ SE in muscle lengths (ML/s). All unloaded
speeds of shortening were determined from shortening speeds of
tissues maintained at 1% of isometric load, except ED 20 aorta and
ED 20 aorta treated with ATP-␥-S, where the unloaded speed of
shortening of 3 and 4 of the tissues, respectively, were calculated by
extrapolation of the Hill equation.
Therefore, calculation of the time for half-maximal
phosphorylation of MLC20 would very likely be an
underestimate of the actual time course and may mask
differences in the rates of MLC20 phosphorylation.
Nonetheless, maximum force in the skinned smooth
muscles was achieved well after 30 s (Fig. 1), indicating
that maximum MLC20 phosphorylation preceded maximum force. Therefore, the time course of MLC20 phosphorylation would not be rate limiting for the rate of
force activation in ED 20 aorta and gizzard.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
three stages of development. The Vmax for the developing aorta was higher than for the developing gizzard at
all stages (Table 3). This was surprising given that
adult phasic tissues have faster speeds of shortening
than tonic tissues (15). Thiophosphorylation of MLC20
with ATP-␥-S resulted in no significant change of Vmax
for ED 20 aorta but a doubling in the gizzard (0.038 ⫾
0.004 vs. 0.084 ⫾ 0.018 ML/s). Additional experiments
with Ca2⫹-activated skinned gizzard strips from 1-wkold chicks indicated that Vmax increased after hatching
(0.115 ⫾ 0.013 ML/s, n ⫽ 3). Therefore, Vmax continues
to increase significantly after birth, given that adult
chicken gizzard strips have a Vmax of ⬃0.3 ML/s (1).
Force-velocity curves of thiophosphorylated fibers
showed that the shortening velocity as a percentage of
Vmax (Fig. 4B) achieved by the gizzard was higher at
most loads compared with the aorta. The extent of
MLC20 thiophosphorylation in the preparations was
comparable (Table 4).
Time course of MLC20 phosphorylation and dephosphorylation. To assess whether phosphorylation-dephosphorylation of MLC20 played a significant role in
the rates of force activation and relaxation, we determined the time to reach half-maximal phosphorylation
and dephosphorylation in skinned ED 16 and 20 aorta
and gizzard. We chose ED 16 and 20 because phasic
and tonic contractile phenotypes appeared to be well
established at these days of development. The levels of
MLC20 phosphorylation in the gizzard and aorta after
0 or 300 s of activation were not significantly different
(Fig. 5). Both ED 20 aorta and gizzard showed similar
levels of maximum MLC20 phosphorylation (aorta,
47.3 ⫾ 4.0%, n ⫽ 7; gizzard, 44.2 ⫾ 3.3%, n ⫽ 7), which
were not different from ED 16 aorta (42.6 ⫾ 5.0%, n ⫽
8) or gizzard (42.7 ⫾ 4.6%, n ⫽ 7), indicating that the
maximum level of activation does not change between
ED 16 and 20. Likewise, for dephosphorylation experiments, we found no difference between the level of
MLC20 phosphorylation in the gizzard and aorta after
0 or 300 s of dephosphorylation (Fig. 6).
Our experiments characterizing the time course of
MLC20 phosphorylation in ED 20 aorta and gizzard
showed no significant difference between the two tissues. However, MLC20 phosphorylation was near maximal at 30 s, which was the first time point measured.
C1727
C1728
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
Table 4. Maximum levels of MLC20 phosphorylation
in embryonic chicken aorta and gizzard
Stage
Aorta
n
Gizzard
n
ED 16
ED 20 (ATP)
ED 20 (ATP-␥-S)
42.6 ⫾ 5.0
47.3 ⫾ 4.0
86.9 ⫾ 6.6
8
7
3
42.7 ⫾ 4.6
44.2 ⫾ 3.3
89.6 ⫾ 9.3
7
7
3
Values are means ⫾ SE. The extent of 20-kDa myosin light chain
(MLC20) phosphorylation is presented as a percentage of the total
MLC20.
Fig. 6. Dephosphorylation of MLC20 in ED 20 gizzard is faster than
ED 20 aorta. After activation of the skinned tissues for 5 min, the
time course of dephosphorylation of MLC20 was followed by incubating the tissue strips in a Ca2⫹-free rigor solution. Dephosphorylation
of MLC20 was significantly faster in ED 20 gizzard compared with
ED 20 aorta (P ⬍ 0.05). The solid line shows a fit to the data by a
single exponential with a rate of 0.051 ⫾ 0.016 s⫺1 in the gizzard and
0.021 ⫾ 0.003 s⫺1 in the aorta.
relaxation in ED 20 gizzard was significantly faster
(P ⬍ 0.05) than in ED 16 gizzard.
DISCUSSION
Regulatory protein isoform heterogeneity during development. Through development, regulatory proteins
may be expressed as multiple isoforms. During chicken
development, MHC and MLC17 expression both show
default tonic phenotypes at early embryonic stages,
with expression of MLC17b and noninsert containing
MHC (7). However, in the gizzard, polymerase chain
reaction and Western blot analyses have shown that
expression of the 7-amino acid insert containing MHCs
dominates expression in SM1 and SM2 MHC isoforms
from ED 10 (⬎70%) to adult (⬃100%; 7, 28), whereas
the aorta did not express the 7-amino acid insert in
SM1 and SM2 myosins. Furthermore, MLC17a expression is upregulated with development to hatching in
the gizzard (⬎50% at ED 12, ⬎70% at ED 16, and
⬃100% for adult; 7, 28), whereas MLC17b is predominant throughout aorta development (⬎60% from ED 12
to adult; 7, 28). Therefore, in the gizzard, MHC and
MLC17 isoform maturation correlates with development of the phasic phenotype.
We detected expression of only the smooth musclespecific MLCK isoform in both the developing gizzard
and aorta (Fig. 3), suggesting that isoform switching of
Table 5. Rates of MLC20 dephosphorylation
in embryonic chicken aorta and gizzard
Fig. 5. The phosphorylation of MLC20 in ED 20 aorta and gizzard.
With the use of skinned ED 20 aorta and gizzard strips, the time
course of MLC20 phosphorylation was followed by separation of the
phosphorylated and unphosphorylated MLC20 by glycerol-ureaPAGE. There was no difference in the time course of phosphorylation
or maximal level of MLC20 phosphorylation in the tonic and phasic
tissues.
Stage
Aorta
n
Gizzard
n
ED 16
ED 20
0.016 ⫾ 0.004
0.021 ⫾ 0.003
3
3
0.022 ⫾ 0.008
0.051 ⫾ 0.016
3
3
Values are means ⫾ SE in s⫺1. The rates of MLC20 dephosphorylation were defined from single exponential fits to the data.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
For the dephosphorylation of MLC20, no differences
were observed for the rates of dephosphorylation between ED 16 aorta (0.016 ⫾ 0.004 s⫺1) and gizzard
(0.022 ⫾ 0.008 s⫺1). For ED 20 aorta, the rate of
dephosphorylation of MLC20 (0.021 ⫾ 0.003 s⫺1) was
not significantly different from ED 16 aorta or gizzard.
On the other hand, the rate of MLC20 dephosphorylation for ED 20 gizzard (0.051 ⫾ 0.016 s⫺1) increased
significantly versus ED 16 gizzard (P ⬍ 0.05) and was
faster than the rate of MLC20 dephosphorylation measured in ED 16 or 20 aorta (Table 5).
Rate of force relaxation in embryonic chicken smooth
muscles. We measured the rates of force relaxation for
skinned embryonic gizzard and aorta strips at ED 12,
16, and 20 (Table 6) to determine whether relaxation
could be correlated with differences in the rate of
dephosphorylation of MLC20. For ED 12, 16, and 20
aorta, rates of relaxation were 0.001 ⫾ 0.001 (n ⫽ 3),
0.004 ⫾ 0.001 (n ⫽ 5), and 0.005 ⫾ 0.002 s⫺1 (n ⫽ 7),
respectively. These were not different from the rate of
relaxation of ED 12 gizzard (0.003 ⫾ 0.002 s⫺1, n ⫽ 3).
However, the rates of relaxation for ED 16 gizzard
(0.019 ⫾ 0.002 s⫺1, n ⫽ 8) and ED 20 gizzard (0.030 ⫾
0.004 s⫺1, n ⫽ 8) were significantly faster compared
with the aorta (P ⬍ 0.05). Furthermore, the rate of
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
Table 6. Rates of relaxation in embryonic
chicken aorta and gizzard
Stage
Aorta
n
Gizzard
n
ED 12
ED 16
ED 20
0.001 ⫾ 0.001
0.004 ⫾ 0.001
0.005 ⫾ 0.002
3
5
7
0.003 ⫾ 0.002
0.019 ⫾ 0.002
0.030 ⫾ 0.004
3
8
8
Values are means ⫾ SE in s⫺1. The rates of relaxation were defined
from single exponential fits to the data.
steady-state MLCK activity may be higher in the gizzard, its contribution to the differences in the rates of
Ca2⫹-activated force appears to be small. Furthermore,
differences in the rates of force activation between
aorta and gizzard are unlikely to be due to diffusional
effects. Activation of thiophosphorylated gizzard fibers
(wherein ATP diffusion is limiting) showed that diffusion can sustain rates ⬍0.060 s⫺1. Therefore, the differences in the rates of force activation in Ca2⫹-activated gizzard and aorta fibers (wherein rates were
⬍0.02 s⫺1) would not be due to diffusion. In fact,
thiophosphorylated phasic and tonic tissues activated
by photolysis of caged ATP still show significant differences in the rates of force activation (21). With flash
photolysis, diffusion is not a factor and differences in
the rates of force activation point to unique kinetics of
the actomyosin interaction in these tissues. In addition, the differences in the rates of force activation for
Ca2⫹-activated smooth muscle strips was not due to
differences in calmodulin content (or loss) after skinning, because thiophosphorylation achieved near complete levels of MLC20 phosphorylation in both ED 20
aorta and gizzard (Table 4) while preserving the differences in the rates of force activation (Table 1). We
also considered the possibility that changes in maximum force could be due to variations in the orientation
of sarcomere equivalents. However, because force is
related to the cosine of the angle between the axis of
tissue preparation and the orientation of the cells,
large changes in angle (⬎60°) would be required to
affect maximum force by twofold. The data in Fig. 2
demonstrate that changes in cell orientation were not
an issue with the developing aorta. In the gizzard, the
⬃20° change in orientation between ED 12 and 16
would result in the underestimation of the measured
force at ED 16 and 20 by ⬃6%. This suggests that the
increase in force with development is due to more
smooth muscle cells per cross section; this is supported
by data that the force per stiffness is not significantly
different with development (data not shown). However,
the orientation of cells is a factor only for the force
measured, not the rate of force activation. The data
indicate that the primary determinant for the rapid
rate of force activation in the gizzard is at the level of
the actin and myosin filaments. Because the MLC20
phosphorylation measurements (Fig. 5) were done on
skinned preparations activated directly with Ca2⫹, we
excluded possible modulation of MLCK activity in the
aorta and gizzard through second messenger pathways
(44), but not through Ca2⫹-activated kinases. However,
thiophosphorylated fibers maintained differences in
the rates of force activation, downplaying the importance of Ca2⫹-activated kinases (other than MLCK),
unless a kinase was specifically coupled to the contractile apparatus in only gizzard or only aortic smooth
muscle. Therefore, differences in the rates of force
activation between gizzard and aortic smooth muscle
remain in the absence of tissue-specific agonist-dependent pathways that differentially modulate MLCK/
MLCP activity through phosphorylation (9).
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
MLCK does not impart phasic or tonic contractile properties. It has been demonstrated that two splice variants of the myosin binding subunit of MLCP, designated M130 and M133 (41), are expressed in the
chicken gizzard. Monoclonal antibody detection of the
myosin binding subunits indicated that M130 expression begins between ED 18 and 20 in the gizzard (Fig.
3), consistent with M130 being the dominant isoform
expressed in the adult (41). On the other hand, the
M133 subunit was constitutively expressed in the developing aorta. Therefore, expression of a gizzard-specific MLCP holoenzyme may contribute to the differences in phasic versus tonic contractile properties.
Kinetics of MLC20 phosphorylation in embryonic gizzard and aorta. Although MLC20 phosphorylation controls smooth muscle force production, the increased
rate of force activation in the gizzard is not well explained by MLCK activity. Phosphorylation of MLC20
in both aorta and gizzard preceded maximal force activation (see Figs. 1 and 5). In addition, both tissues
expressed identical MLCK isoforms (Fig. 3) and
reached the same maximum level of MLC20 phosphorylation (Fig. 5). However, between ED 20 gizzard and
aorta, we detected a significant difference in the time
course of MLC20 dephosphorylation (Fig. 6). If MLCP
activity is constitutive in the presence and absence of
Ca2⫹, this implies that steady-state MLCK activity
was higher in ED 20 gizzard than in the aorta. This
difference was not resolved on the time scale of our in
vitro phosphorylation experiments. However, the difference between MLCK activity for the aorta and gizzard can be determined assuming that the steady-state
level of MLC20 phosphorylation is defined by MLCK
and MLCP activities as described by MLCK/(MLCK ⫹
MLCP). Because steady-state MLC20 phosphorylation
levels were not significantly different between ED 20
aorta and gizzard (⬃45%) and assuming that the rates
of dephosphorylation are accurate, the MLCK activity
in the aorta would calculate to 41% of that in the
gizzard. This implies that the rates of phosphorylation
were underestimated. Although thiophosphorylation of
MLC20 in ED 20 aorta and gizzard increased the rates
of force activation compared with phosphorylation of
MLC20, an approximately threefold difference remained (Table 1) in the rates of force activation in
these tissues. Therefore, MLC20 phosphorylation was
not a limiting factor in the slow rate of force activation
in the aorta, indicating that with phasic muscle the
rapid rate of force activation is not explained by increased MLCK activity alone. Thus although the
C1729
C1730
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
tissues. It remains a possibility that changes in content
or isoform of calponin and caldesmon (37) may contribute to the mechanical parameters measured in these
experiments.
Interestingly, after thiophosphorylation of MLC20,
there was a significant increase in the speed of shortening in the gizzard, without a significant change in
the aorta. This implies that there is a higher internal
load on ED 20 gizzard cells, which is overcome by
maximal (⬃90%) phosphorylation of MLC20 or by increasing the level of activation of the preparation. This
result is similar to cardiac muscle wherein shortening
velocity is dependent on the level of Ca2⫹ activation
(33). In fact, the Vmax of Ca2⫹-activated gizzard strips
increases 1 wk after hatching, approaching the Vmax of
⬃0.3 ML/s measured from adult chicken gizzard strips
(1). The postnatal increases in Vmax occur independent
of changes in the level of MLC20 phosphorylation from
ED 20 (this study) to adult (1) or differences in MHC/
MLC17 isoform expression, because myosin expression
has already matured to the adult patterns by ED 20.
Experiments characterizing isolated myosins in in
vitro motility assays (28, 40) have indicated that intestinal myosins propel actin filaments faster, along with
having a higher actin-activated Mg2⫹-ATPase activity,
although care must be taken when interpreting data
from in vitro motility assays (13). With the optical trap,
the MHC determined the kinetics of cross-bridge attachment (30). Nonetheless, our data indicate that
because of a significant internal load, the level of activation is a major factor in determining Vmax (1, 10).
The presence of an internal load in single smooth
muscle cells has been shown previously to affect the
extent of shortening (47). In the case of ED 20 gizzard,
the internal load was overcome by increasing the level
of MLC20 phosphorylation from 45 to 90% in thiophosphorylated fibers, presumably increasing the number
of actively cycling, force-generating myosin heads. In
fact, the increase in Vmax of the gizzard with development from hatching to birth and after birth takes place
in the absence of changes in myosin isoforms or MLC20
phosphorylation. One possible explanation for the increase is a decrease in the internal load on gizzard
myosins with maturation, either defined by changes in
the intracellular or extracellular matrix properties.
This is also supported by the significant increase of
Vmax with development of the aorta wherein there is a
modest change in MLC17 isoform expression but no
changes in MHC isoform expression or MLC20 phosphorylation. Thus changes in internal load and thus
the level of activation or MLC20 phosphorylation appears to be the major determinant of Vmax in this
model.
Role of MLC17 isoforms in determining tonic and
phasic contractile properties. It is well known that the
20-kDa regulatory myosin light chains play a defining
role in controlling actomyosin interaction in smooth
muscle. However, the function of the 17-kDa essential
myosin light chain has not been as well characterized.
The essential myosin light chains line the neck region
of the MHC dimers, possibly acting as a scaffold for
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
Rates of MLCP dephosphorylation in developing gizzard and aorta. There was a significant difference in
the rates of MLC20 dephosphorylation between ED 20
gizzard and ED 16 gizzard or ED 16 and 20 aorta
(Table 5; Fig. 6). The increased rate of MLC20 dephosphorylation in ED 20 gizzard correlated with expression of a unique isoform of the myosin binding subunit
of MLCP (M130; 16, 41) that was not expressed in the
aorta (Fig. 3). There was no difference in the rates of
dephosphorylation in ED 16 aorta and gizzard versus
ED 20 aorta (Table 4), wherein all smooth muscle
tissues expressed M133. Therefore, our results suggest
that the MLCP holoenzyme containing the M130 myosin binding subunit correlates with the increased rate
of MLC20 dephosphorylation in ED 20 gizzard, either
through increased activity of this MLCP holoenzyme or
an increase in the MLCP-to-myosin ratio. Coupled with
data characterizing the rates of force relaxation, two
conclusions may be drawn. First, we demonstrated
that the rate of relaxation in the gizzard is faster than
in the aorta at both ED 16 and 20. At ED 16, both
tissues express the MLCP holoenzyme with the M133
subunit and show similar time courses for MLC20 dephosphorylation. Therefore, the differences in the rates
of force relaxation between ED 16 aorta and gizzard
(Table 6) are best attributed to changes in the kinetics
of the myosin isoforms in the cross-bridge cycle, likely
due to inclusion (gizzard) or exclusion (aorta) of the
7-amino acid insert in the MHC or different MLC17
isoform expression in the two tissues. With laser trap
experiments, it has been demonstrated that heavy
meromyosin composed of the noninsert (aortic) form of
MHC has an attachment time on actin roughly twice as
long as the insert (gizzard) form (30). This would agree
with our results that relaxation in ED 16 gizzard is
faster than in ED 16 aorta, although the tissues show
similar rates of MLC20 dephosphorylation. Nonetheless, the increase in the rate of relaxation of ED 20
versus 16 gizzard indicates that expression of MLCP
holoenzymes containing the M130 myosin binding subunit correlates with increases in the rate of MLC20
dephosphorylation and thus the rate of force relaxation, whether by increased MLCP isoform activity or
increased MLCP-to-myosin ratio.
Unloaded speed of shortening in developing chicken
gizzard and aorta. Previous reports have demonstrated that increased MLC17a content may account for
faster speeds of shortening in smooth muscle tissues
(31). Increasing the proportion of MLC17a expression
by exchange of MLC17 isoforms in trifluoperazinetreated smooth muscle was also shown to increase the
shortening velocity (35), although contraction in these
tissues was independent of Ca2⫹ or MLC20 phosphorylation. On the other hand, in in vitro motility assays,
exchange of MLC17 isoforms had no effect on the speed
of actin filament movement by myosins (28, 40). Although the embryonic gizzard expressed a higher proportion of MLC17a than the aorta (7), in our study, the
gizzard had slower speeds of shortening under all conditions tested (Table 3). Therefore, MLC17a content
was not a factor in defining a faster Vmax in these
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
We thank Dr. Guay-haur Shue for writing the LabView Force
Clamp program and Albert Rhee and Carie Trbovich for assistance.
We thank Dr. Jian-Ping Jin for the anti-calponin mAb and Drs.
Mitsuo Ikebe and Steven Fisher for the anti-MLCK mAbs. We also
thank Drs. Michiko Watanabe and Midori Hitomi for histochemical
staining of tissue sections.
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-44181 and an American Heart Association grantin-aid.
REFERENCES
1. Barsotti RJ, Ikebe M, and Hartshorne DJ. Effect of Ca2⫹,
Mg2⫹, and myosin phosphorylation on skinned smooth muscle
fibers. Am J Physiol Cell Physiol 252: C543–C554, 1987.
2. Bhatti S, Pyne GJ, Manaves V, Franks R, Strauch AR, Paul
RJ, and Martin AF. Expression of a transgene encoding smooth
muscle SMaSM2 myosin heavy chain isoform in mice alters
contractility of the aorta. Biophys J 78: 23A, 2000.
3. Brozovich FV and Yamakawa M. Thin filament regulation of
force activation is not essential in single a-toxin permeabilized
vascular smooth muscle. Am J Physiol Cell Physiol 268: C237–
C242, 1995.
4. Dillon PF, Aksoy MO, Driska SP, and Murphy RA. Myosin
phosphorylation and the cross-bridge cycle in arterial smooth
muscle. Science 211: 495–497, 1981.
5. Eddinger TJ and Murphy RA. Two smooth muscle myosin
heavy chains in cultured aorta smooth muscle cells. A comparative study. J Biol Chem 2262: 7282–7288, 1987.
6. Fisher SA and Ikebe M. Developmental and tissue distribution
of expression of nonmuscle and smooth muscle isoforms of myosin light chain kinase. Biochem Biophys Res Commun 217: 696–
703, 1995.
7. Fisher SA, Ikebe M, and Brozovich F. Endothelin-1 alters the
contractile phenotype of cultured embryonic smooth muscle cells.
Circ Res 80: 885–893, 1997.
8. Ford LE, Huxley AF, and Simmons RM. The relation between stiffness and filament overlap in stimulated frog muscle
fibres. J Physiol (Lond) 311: 219–249, 1981.
9. Gallagher PJ, Herring BP, and Stull JT. Myosin light chain
kinases. J Muscle Res Cell Motil 18: 1–16, 1997.
10. Gerthoffer WT and Murphy RA. Myosin phosphorylation and
regulation of cross-bridge cycle in tracheal smooth muscle. Am J
Physiol Cell Physiol 244: C182–C187, 1983.
11. Goldman YE, Hibberd MG, and Trentham DR. Relaxation of
rabbit psoas muscle fibres from rigor by photochemical generation of adenosine-5⬘-triphosphate. J Physiol (Lond) 354: 577–
604, 1984.
12. Gong MC, Cohen P, Kitazawa T, Ikebe M, Masuo M, Somlyo AP, and Somlyo AV. Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and
phasic smooth muscle. J Biol Chem 267: 14662–14668, 1992.
13. Gordon AM, Homsher E, and Regnier M. Regulation of
contraction in striated muscle. Physiol Rev 80: 853–924, 2000.
14. Hai CM and Murphy RA. Cross-bridge phosphorylation and
regulation of latch state in smooth muscle. Am J Physiol Cell
Physiol 254: C99–C106, 1988.
15. Hartshorne DJ. Biochemistry of the contractile process in
smooth muscles. In: Physiology of the Gastrointestinal Tract (2nd
ed.), edited by Johnson LR. New York: Raven, 1987, p. 423–482.
16. Hartshorne DJ, Ito M, and Erdodi F. Myosin light chain
phosphatase: subunit composition, interactions and regulation.
J Muscle Res Cell Motil 18: 325–341, 1997.
17. Hathaway DR and Haeberle JR. A radioimmunoblotting
method for measuring myosin light chain phosphorylation levels
in smooth muscle. Am J Physiol Cell Physiol 249: C345–C351,
1985.
18. Helper DJ, Lash JA, and Hathaway DR. Distribution of
isoelectric variants of the 17,000-dalton myosin light chain in
mammalian smooth muscle. J Biol Chem 263: 15748–15753,
1988.
19. Hill AV. The heat of shortening and the dynamic constants of
muscle. Proc R Soc Lond B Biol Sci 126: 136–195, 1938.
20. Hoar PE, Kerrick WG, and Cassidy PS. Chicken gizzard:
relation between calcium-activated phosphorylation and contraction. Science 204: 503–506, 1979.
21. Homsher E, Wang F, and Sellers J. Factors affecting filament
velocity in in vitro motility assays and their relation to unloaded
shortening velocity in muscle fibers. Adv Exp Med Biol 332:
279–289, 1993.
22. Horiuti K, Somlyo AV, Goldman YE, and Somlyo AP. Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscle. J Gen
Physiol 94: 769–781, 1989.
23. Horowitz A, Menice CB, Laporte R, and Morgan KG. Mechanisms of smooth muscle contraction. Physiol Rev 76: 967–1003,
1996.
24. Huang QQ, Fisher SA, and Brozovich FV. Forced expression
of essential myosin light chain isoforms demonstrates their role
in smooth muscle force production. J Biol Chem 274: 35095–
35098, 1999.
25. Ikebe M and Brozovich FV. Protein kinase C increases force
and slows relaxation in smooth muscle: evidence for regulation of
the myosin light chain phosphatase. Biochem Biophys Res Commun 225: 370–376, 1996.
26. Jin JP, Walsh MP, Resek ME, and McMartin GA. Expression and epitopic conservation of calponin in different smooth
muscles and during development. Biochem Cell Biol 74: 187–
196, 1996.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
this extended helix (39). In this respect, the interaction
of different MLC17 isoforms with the neck region of
MHC dimers may result in changes in the stiffness of
this lever arm. The rapid rate of force activation in the
gizzard due to MLC17a expression may be explained if
this isoform provided added stiffness to the myosin
lever arm.
If MLC17 is a determinant in regulating the actomyosin cross bridge’s ability to bear a load, then we would
expect that speed of shortening would be higher with
MLC17a than MLC17b at or near physiological loads, as
shown in Fig. 4. In fact, the gizzard, which expresses
predominantly MLC17a, is able to develop a higher
relative percentage of its Vmax at all loads compared
with the aorta, which expresses predominantly
MLC17b. This is coupled by higher absolute isotonic
shortening velocities at loads ⱖ40% of isometric level,
supporting the conclusion that MLC17 is a major determinant of the speed of contraction under physiological loads. In addition, MHC isoform contribution to
the rate of force activation is not significant. This is
supported by previous experiments demonstrating
that endothelin-1 treatment of gizzard smooth muscle
cells increased MLC17b isoform expression, did not
change MHC isoform expression, but decreased the
rate of force activation (7). Furthermore, transfection
of single aortic smooth muscle cells with eukaryotic
plasmids expressing MLC17a increases the rate of force
activation, whereas transfection of gizzard smooth
muscle cells with MLC17b decreases the rates of force
activation, both in the absence of MHC isoform
changes (24).
In summary, we show that MLC17 and MLCP are
molecular markers for the phasic contractile phenotype of smooth muscle. The MLC17 isoforms regulate
the rate of force activation and expression of myosin
binding subunit isoforms of the MLCP holoenzyme
modulate the rate of force relaxation, two hallmarks
for tonic and phasic contractile properties.
C1731
C1732
CONTRACTILE PROPERTIES OF SMOOTH MUSCLE
38. Nabeshima Y, Nonomura Y, and Fujii-Kuriyama Y. Nonmuscle and smooth muscle myosin light chain mRNAs are generated from a single gene by the tissue-specific alternative RNA
splicing. J Biol Chem 262: 10608–10612, 1987.
39. Rayment I, Rypniewski WR, Schmidyt-Base K, Smith R,
Tomchick DR, Benning MM, Winkelmann DA, Wesenberg
G, and Holden HM. Three-dimensional structure of myosin
subfragment-1: a molecular motor. Science 261: 50–58, 1993.
40. Rovner AS, Freyzon Y, and Trybus KM. An insert in the
motor domain determines the functional properties of expressed
smooth muscle myosin isoforms. J Muscle Res Cell Motil 18:
103–110, 1997.
41. Shimizu H, Ito M, Miyahara M, Ichikawa K, Okubo S,
Konishi T, Naka M, Tanaka T, Hirano K, Hartshorne DJ,
and Nakano T. Characterization of the myosin-binding subunit
of smooth muscle myosin phosphatase. J Biol Chem 269: 30407–
30411, 1994.
42. Smith JP and Barsotti RJ. A computer-based servo system for
controlling isotonic contractions of muscle. Am J Physiol Cell
Physiol 265: C1424–C1432, 1993.
43. Somlyo AP and Somlyo AV. Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J Pharmacol
Exp Ther 159: 129–145, 1968.
44. Somlyo AP and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231–236, 1994.
45. Somlyo AV, Goldman YE, Fujimori T, Bond M, Trentham
DR, and Somlyo AP. Cross-bridge kinetics, cooperativity, and
negatively strained cross-bridges in vertebrate smooth muscle. A
laser-flash photolysis study. J Gen Physiol 91: 165–192, 1988.
46. Szymanski PT, Chacko TK, Rovner AS, and Goyal RJ.
Differences in contractile protein content and isoforms in phasic
and tonic smooth muscles. Am J Physiol Cell Physiol 275: C684–
C692, 1998.
47. Warshaw DM, McBride WJ, and Work SS. Corkscrew-like
shortening in single smooth muscle cells. Science 236: 1457–
1459, 1987.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
27. Kelley CA, Sellers JR, Goldsmith PK, and Adelstein RS.
Smooth muscle myosin is composed of homodimeric heavy
chains. J Biol Chem 267: 2127–2130, 1992.
28. Kelley CA, Takahashi M, Yu JH, and Adelstein RS. An
insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. J Biol Chem 268: 12848–12854, 1993.
29. Khromov A, Somlyo AV, Trentham DR, Zimmermann B,
and Somlyo AP. The role of MgADP in force maintenance by
dephosphorylated cross-bridges in smooth muscle: a flash photolysis study. Biophys J 69: 2611–2622, 1995.
30. Lauzon AM, Tyska MJ, Rovner AS, Freyzon Y, Warshaw
DM, and Trybus KM. A 7-amino-acid insert in the heavy chain
nucleotide binding loop alters the kinetics of smooth muscle
myosin in the laser trap. J Muscle Res Cell Motil 19: 825–837,
1998.
31. Malmqvist U and Arner A. Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening in smooth muscle. Pflügers Arch 418: 523–530, 1991.
32. Marston SB and Huber PAJ. Caldesmon. In: Biochemistry of
Smooth Muscle Contraction, edited by Barany M. San Diego, CA:
Academic, 1996, p. 77–90.
33. Martyn DA, Rondinone JF, and Huntsman LL. Myocardial
segment velocity at a low load: time, length, and calcium dependence. Am J Physiol Heart Circ Physiol 244: H708–H714, 1983.
34. Masuo M, Reardon S, Ikebe M, and Kitazawa T. A novel
mechanism for the Ca2⫹-sensitizing effect of protein kinase C on
vascular smooth muscle: inhibition of myosin light chain phosphatase. J Gen Physiol 104: 265–286, 1994.
35. Matthew JD, Khromov AS, Trybus KM, Somlyo AP, and
Somlyo AV. Myosin essential light chain isoforms modulate the
velocity of shortening propelled by nonphosphorylated crossbridges. J Biol Chem 273: 31289–31296, 1998.
36. Meer DP and Eddinger TJ. Expression of smooth muscle
myosin heavy chains and unloaded shortening in single smooth
muscle cells. Am J Physiol Cell Physiol 273: C1259–C1266, 1997.
37. Menon C and Chacko S. Expression of smooth muscle caldesmon
in developing chicken gizzard. Tissue Cell 30: 118–126, 1998.