Vol. 281, Issue 6, R1769-R1777, December 2001
Cross-bridge regulation by Ca2+-dependent
phosphorylation in amphibian smooth muscle
C. J.
Wingard1,2,
J. M.
Nowocin1, and
R. A.
Murphy1
1 Department of Physiology, Medical College of Georgia,
Augusta, Georgia 30912; and 2 Department of Molecular
Physiology and Biological Physics, University of Virginia Health
Sciences Center, Charlottesville, Virginia 22908
 |
ABSTRACT |
A covalent
regulatory mechanism involving Ca2+-dependent cross-bridge
phosphorylation determines both the number of cycling cross bridges and
cycling kinetics in mammalian smooth muscle. Our objective was to
determine whether a similar regulatory mechanism governed smooth muscle
contraction from a poikilothermic amphibian in a test of the hypothesis
that myosin regulatory light chain (MRLC) phosphorylation could
modulate shortening velocity. We measured MRLC phosphorylation of
Rana catesbiana urinary bladder strips at 25°C in tonic
contractions in response to K+ depolarization, field
stimulation, or carbachol stimulation. The force-length relationship
was characterized by a steep ascending limb and a shallow descending
limb. There was a rapid rise in unloaded shortening velocity early in a
contraction, which then fell and was maintained at low rates while high
force was maintained. In support of the hypothesis, we found a positive
correlation of the level of myosin phosphorylation and an estimate of
tissue shortening velocity. These results suggest that MRLC
phosphorylation in amphibian smooth muscle modulates both the number of
attached cross bridges (force) and the cross-bridge cycling kinetics
(shortening velocity) as in mammalian smooth muscle.
cross-bridge cycling; excitation-contraction coupling; muscle
mechanics; myosin regulatory light chains
| |
INTRODUCTION |
MAMMALIAN SMOOTH
MUSCLE differs from skeletal muscle because both the extent of
cross-bridge recruitment (determining steady-state force) and the
kinetics of cross-bridge cycling (manifested by unloaded shortening
velocities or power output) vary with the level of activation
(18). This difference is not due to the myosin motors,
although the smooth muscle isoforms have a low ATPase activity, but is
conferred by differences in the regulatory mechanisms. In skeletal
muscle, Ca2+ acts allosterically by binding to a
thin-filament regulatory protein, troponin. By contrast, the primary
regulatory mechanism in mammalian smooth muscle is covalent.
Ca2+ binds to calmodulin, and this complex activates myosin
light chain kinase (MLCK). Phosphorylation of Ser19 of the
myosin regulatory light chain (MRLC) by MLCK allows the cross bridge to
bind to the thin filaments and cycle. Thus Ca2+-dependent
phosphorylation determines cross-bridge recruitment.
While myosin light chain phosphatase (MLCP) activity can be regulated,
it is considered constitutively active in mammalian smooth muscle in
the simplest regulatory paradigm for steady-state activation. When
myoplasmic Ca2+ concentrations are elevated, the kinetics
of cross-bridge phosphorylation and dephosphorylation are comparable to
those of cross-bridge cycling. This leads to a situation in which both
phosphorylated and dephosphorylated cross bridges contribute to force
development (18). However, the kinetics of detachment
differ between the phosphorylated and dephosphorylated cross bridges,
leading to differences in cycling rate (5). Thus
phosphorylation also determines shortening velocities. Physiologically,
this system is advantageous because it allows comparatively rapid
phasic contractions and also the slowing of cycling rates and ATP
consumption during sustained tonic contractions when muscle in the
walls of hollow organs serves a structural role in stabilizing organ
dimensions against imposed loads.
There is limited information on the mechanics and contractile behavior
of isolated frog stomach cells (2, 23, 27, 28) and the
pharmacology of teleost and reptile vascular smooth muscle (17,
30) and frog bladder smooth muscle (3, 12, 31). However, we found no measurements of cross-bridge phosphorylation or
information on whether ectothermic vertebrate smooth muscle exhibits
"latch" (i.e., maintained force in the presence of low MRLC
phosphorylation and cross-bridge cycling).
Our objectives were to see whether Ca2+-dependent MRLC
phosphorylation triggers contraction in an amphibian smooth muscle and whether cross-bridge cycling rates are regulated by this mechanism, in
a test of the hypothesis that MRLC phosphorylation regulates shortening velocity.
| |
MATERIALS AND METHODS |
Tissue preparation.
All animal care and use was conducted in accordance with National
Institutes of Health guidelines and a protocol approved by the Medical
College of Georgia and the University of Virginia Animal Care and Use
Committees. Urinary bladders were removed from Rana catesbiana
after anesthesia with MS-222 and euthanasia by double pithing. Two
3- to 4-mm-wide strips were dissected from each hemisphere of the
bladder. Each strip was folded over on itself, and the ends were
secured in an aluminum foil clip with cyanoacrylate to form a ring.
All bladders were bathed either in a bicarbonate-based Harris amphibian
Ringer solution (ARS) at 25°C, bubbled with a 20% oxygen-75%
nitrogen-5% CO2 mix, and adjusted to pH 7.4, or in a
MOPS-based physiological saline solution (PSS) also adjusted to a pH of
7.4 at 25°C and bubbled with air. The ARS contained (in mM) 90.4 NaCl, 2.9 KCl, 0.48 MgSO4, 30.0 NaHCO3, 1.2 Na2HPO4, 3.0 D-glucose, and 1.5 CaCl2. The MOPS-based buffer contained (in mM) 140.0 NaCl,
5.0 KCl, 1.6 CaCl2, 1.2 MgCl2, 1.2 Na2HPO4, 5.6 D-glucose, 2.03 MOPS,
and 0.02 EDTA (to chelate trace heavy metals). The use of both buffers
resulted in similar force-generating capacities of the bladder strips
in response to K+ depolarization or carbachol stimulation.
The MOPS-based buffer was used for all mechanics and myosin
phosphorylation experiments for its ability to allow storage of tissues
overnight in the cold without continued gassing to maintain the pH.
Spontaneous contractions were seen in <20% of all tissues examined.
When spontaneous contractions occurred, the CaCl2
concentration in either solution was lowered to 0.8 mM and then
returned to 1.5 or 1.6 mM in the potassium-containing solutions
(K+-ARS or K+-PSS). Lowering of
Ca2+ concentration had previously been shown to reduce or
eliminate spontaneous contraction (21). The
K+-ARS or K+-PSS solutions contained
stoichiometrically substituted KCl for NaCl. If preparations displayed
oscillations during agonist stimulation, the steady-state force
was estimated by fitting a straight line through the trace.
During field stimulation, 0.5 mM ascorbic acid was added to all
solutions to protect against free radical formation. A Protech (JDR
Microdevices, San Jose, CA) sweep function generator, model B-801,
provided 2- to 20-Hz direct current square-wave pulses delivered
through two platinum foil electrodes with 10- to 120-mA current. The
electrodes had a surface area of 0.8 cm2 and were separated
by a distance of 8 mm. Chemicals and drugs were purchased from either
Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
Determination of reference length, L0.
Urinary bladder rings were suspended between two stainless
steel support posts in a 25-ml water-jacketed organ bath. The upper post was attached to a Grass FT 03 force transducer (Astro-Med/Grass Instrument Division, West Warwick, RI) containing springs allowing force measurements up to 200 g. The lower post was attached to a
Unislide drum micrometer (Velmax, East Bloomfield, NY) (0.01-mm resolution). The compliance of the system was primarily due to transducer displacement, and a correction factor was applied to all
reported data. Force was recorded on a Gould eight-channel recorder
(model 8800, Gould Instruments Systems, Valley View, OH). All force
measurements were converted into stress values (force/cross-sectional
area) (29). Cross-sectional area of tissues at
L0 was calculated from the tissue measurements
of length, mass, and density [area = (wet weight/length at
L0)/(1.055 g/cm3)].
Mounted rings were equilibrated for 30 min in ARS or PSS at a length at
which there was <2 g of resting force. The rings were then stretched
using several protocols to determine the force-length relationship.
Initially, rings were stretched using three 0.5-mm increments with
intervening stress relaxation for 8-15 min. After the third
stretch, a 0.5-mm quick release was imposed (Fig.
1A). The passive force
(Fp) was estimated as the minimal force after the release before any tone redevelopment. Variations in the stretching protocol included larger 0.75-mm incremental stretches with a smaller
0.25-mm release, or a single 1.0-mm stretch followed by three 0.5-mm
stretches with a 0.5-mm quick release. Only after the release and force
stabilization at the new length were the preparations stimulated. The
tissues were depolarized (73 mM K+) for a minimum of 3 min
and relaxed in ARS or PSS for a period of 15 min. The next increment of
stretches was performed only when resting force in PSS equaled the
prestimulus resting force. The protocol was repeated until forces fell
to 0.5 peak values or the tissues became too long to remain immersed in
the bath.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Determination of stress-length relationship in Rana
catesbiana bladder strips. A: representative force
trace used in the generation of a force-length profile. B:
representative steady-state force-length profile. Filled symbols
represent data collected during tissue lengthening protocol. Open
symbols and line are the arithmetic determination of active force
(Fa = Ft  Fp, where Ft is the total
force in stimulated tissue and Fp is passive
force) and the third-order polynomial fit to these data. The vertical
dashed line identifies the theoretical length at which maximal force
was generated in response to 73 mM K+ depolarization.
C: summary stress (S)-length (L)
relationship. Individual force values were converted to stress values,
and then data were normalized to the reference length
(L0) and force (F0)
values as determined by individual polynomial fits. Normalized values
were grouped in 0.2-length unit bins, and means ± SE
were determined for each bin. Sample number for each
bin ranged from 3 to 65 individual strips from a total of 20 animals.
Lines are linear regressions of ascending (dashed) and descending
(solid) limbs of the stress-length relationship.
S0, reference stress.
|
|
Fp and total force (Ft)
at each length were measured. Active force
(Fa = Ft Fp) was calculated after correction for
apparatus compliance. The active force-length data for individual
preparations were fitted by a third-order polynomial (29).
The choice of this function was based on the best statistical fit
(typically r2 > 0.9) and has no
theoretical significance. The equation allowed a reference length
(L0) to be identified for each preparation (where L0 = L when
dF/dL = 0). Normalized force or stress
values are reported as the measured value (F or
S, respectively) divided by the calculated value of
F0 or S0, respectively,
where F0 = F and
S0 = S at
L0.
Microscopy.
Several rings were fixed for histological examination in 1.5%
glutaraldehyde/0.05 M sodium cacodylate buffer at
L0 for 2 h to determine the orientation and
fractional content of smooth muscle cells. The tissues were then washed
in 0.05 M sodium cacodylate buffer and rinsed in veronal acetate. The
tissues were stained en bloc with 3% uranyl acetate in veronal acetate
for 2 h. After washing in veronal acetate, the tissues were
dehydrated in increasing percentages of 70-100% ethanol, followed
by two washes in 100% acetone. The tissues were then infiltrated with
an acetone-Poly/Bed 812 mix (Polysciences, Warrington, PA). After two
additional changes of pure Poly/Bed 812 resin, the tissues were
embedded in the resin at 60°C for 2 days. Thick sections were cut
with a LKB III ultramicrotome (Leica, Deerfield, IL). The sections were
dried on glass slides and stained with toluidine blue.
Shortening velocity.
Shortening velocities were calculated from the estimated time to force
redevelopment after releases of 10, 12.5, and 15% of tissue length
during K+ depolarization at L0. The
times for force redevelopment for a set of releases at 5, 10, 20, 30, 60, and 90 s into a contraction were fitted by linear regression.
The slope of the regression represents the unloaded shortening
velocity, and the Y-intercept provides an estimate of the
series elasticity. Fits were done on individual tissues, and a mean
value was calculated for all preparations. Only those fits that
returned r2 values of >0.85 were included in
the subsequent analysis.
Cross-bridge phosphorylation.
Phosphorylation determinations were made in separate tissues under
identical treatment conditions because of the destructive nature of the
assay. A dry ice-acetone slurry (78°C) was used to freeze rings
after treatments at selected time intervals. The rings were slowly
thawed in acetone over a 2-h period, air dried, weighed, and
homogenized in a cold aqueous solution containing 1% (wt/vol) SDS,
10% glycerol (vol/vol), and 20 mM dithiothreitol (7).
Segments of the tissue secured and glued with cyanoacrylate were
discarded before homogenization.
Phosphorylation of the smooth muscle-specific 20-kDa MRLC isoform was
determined by two-dimensional isoelectric focusing (IEF) and SDS-PAGE
(7). Dephosphorylated and phosphorylated myosin light
chains were separated in the first dimension by IEF using an ampholyte
gradient of pH 4-6.5 (Pharmacia LKB Technology, Piscataway, NJ).
Myosin light chains were then separated from other proteins by
molecular weight in the second dimension using SDS-PAGE. The gels were
stained with colloidal Coomassie blue (ICN Biomedicals, Aurora,
OH). Phosphorylation values were determined densitometrically using a
BioImage 2000 digital image system (Bio Image, Ann Arbor, MI) and a Sun
Sparc 10 computer.
Alternatively, MRLC phosphorylation was determined using a
one-dimensional IEF slab gel protocol (4) with an
ampholyte gradient of pH 4-6.5. The separated proteins in the slab
gels were then transferred to either 0.22-µm nitrocellulose or
polyvinylidene difluoride (PVDF) membranes (Micro Separations,
Westborough, MA) via a tank blotter (Hoffer Scientific, San Francisco,
CA). Immunolabeling of the smooth muscle-specific MRLC isoform was
accomplished using a monoclonal anti-MRLC raised against turkey gizzard
MRLC (20 kDa) clone MY-21 (Sigma, St. Louis, MO), 1:1,000 dilution in
0.1% Tween-Tris-buffered saline. Detection used a 1:2,000 dilution of
a goat anti-mouse IgM-specific peroxidase-conjugated secondary antibody. Protein was detected using a
3,3'-diaminobenzidine-based colorimetric substrate, digitally
scanned for density and analyzed using ImageQuant software. All
phosphorylation values are reported as the density of the
monophosphorylated MRLC relative to the density of the total amount of
MRLC isoform detected, from the following equation:
%MRLC-Pi = (phosphorylated band
density)/(unphosphorylated band density + phosphorylated band
density) × 100.
Statistical analysis.
Data were analyzed using ANOVA for repeated measures with post hoc
comparisons made by Student-Newman-Keuls test. Student's t
analysis was used where appropriate. Statistical significance was set
at P < 0.05.
| |
RESULTS |
Preparations of urinary bladder from R. catesbiana
contracted in response to electrical field stimulation, carbachol
stimulation, and K+ depolarization. These contractions were
sustainable for more than 3 min and displayed a fast rising peak force
component and a slower sustained force component.
Physical properties of urinary bladder strips.
Force in the urinary bladder rings depends on tissue length (Fig. 1).
In response to 73 mM K+ depolarization, the peak
Fa length-tension relationship of individual preparations could be approximated by a third-order polynomial, and a
theoretical maximal force and optimal length for force generation (L0) could be identified (29) (Fig.
1B). The average force-length relationship for the urinary
ring preparation had a flat plateau (0.9-1.1
L0) and a very shallow descending limb
(1.1-1.7 L0) (Fig. 1C)
compared with mammalian smooth muscle (29) (Table
1).
To ensure that the bladder preparations were oriented in their
proper direction for monitoring force generation, representative strips
were fixed at L0 and sectioned for light
microscopy. Cross-sectional and longitudinal sections revealed
extensive connective tissue and nonmuscle cells. Smooth muscle occurred
in discrete bundles in these sections (Fig.
2A). The higher magnification
of longitudinal sections revealed smooth muscle cells aligned at a
small angle to the vector of force measurements. (Fig. 2B).
The fractional content of the smooth muscle cells of these preparations
was small, composing only 10.1 ± 8.5% (n = 4) of
cross-sectional area. The basic physical characteristics of the urinary
ring preparations, including wet weight, cross-sectional area at
L0, and contribution of
Fp to K+-dependent force, are
reported in Table 2.
View larger version (122K):
[in this window]
[in a new window]
|
Fig. 2.
Photomicrographs of R. catesbiana bladder
strips. A: cross-sectional view (0.75-µm-thick section).
Image depicts segment of bladder strip preparation with the mucosal
surface on right and the serosal side toward the
left. The section was stained with toluidine blue.
Arrowheads indicate smooth muscle bundles. Magnification, ×80.
B: longitudinal section (0.75-µm-thick section) of bladder
preparation. Arrowheads indicate individual smooth muscle cells in
plane of the section, stained with toluidine blue. Magnification,
×160.
|
|
Stimulus-response behavior.
Unstimulated bladder preparations occasionally showed spontaneous
contractile activity. This was abolished or diminished by reducing the
bathing Ca2+ concentration from 1.6 to 0.8 mM (data not
shown). Reduced CaCl2 did not affect the subsequent
stress-generating ability of the tissue in response to K+
depolarization, carbachol, or field stimulation, provided that 1.6 mM
Ca2+ was present in the stimulation solution and that time
was given to restore Ca2+ stores between stimulations.
The phasic component of the contractile response to K+
depolarization had an EC50 of 17.5 mM K+ and
was maximal above 30 mM K+. The same behavior was seen for
the steady-state stress response (Fig.
3A). The presence of 0.5 µM
tetrodotoxin to block neural effects before and during stimulation
resulted in a small increase in peak and steady-state stress generation
with K+ depolarization (Fig. 3, B and
C). However, this small increase in stress was not
statistically significant in paired comparisons (P > 0.125).
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Stress generation of Rana bladder rings during
graded K+ depolarization. A: representative
force trace from a bladder ring preparation submaximally stimulated
with KCl without TTX. B: peak active stress to graded
K+ concentration in presence and absence of 0.5 µM TTX.
C: steady-state active stress in bladder rings stimulated
with K+ in presence and absence of 0.5 µM TTX. Values are
presented as means ± SE (n = 6 for peak value
determinations and 8 for steady-state determinations).
* Statistically significant difference from value reported at 73 mM
K+ (P < 0.025).
|
|
The contractile responses of the urinary bladder to carbachol were
biphasic with increasing levels of peak and steady-state stress
generation occurring over the concentration range of 0.01-1.0 µM
(Fig. 4A). Concentrations
greater than 100 µM significantly reduced both the peak and
steady-state stress responses (Fig. 4B). The
EC50 for carbachol was 6.4 nM.
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Active stress generation of Rana bladder rings
stimulated with carbachol. A: representative force traces
for a cumulative carbachol dose response of a bladder ring preparation.
B: peak and steady-state stress responses are reported for
graded carbachol stimulation. Values are presented as means ± SE
(n = 4). * Statistically significant difference from
value reported at 1 µM carbachol (P < 0.001).
|
|
The response of Rana bladder to field stimulation was
strongly biphasic (Fig. 5). When we used
a fixed 20-Hz stimulation frequency, the peak and steady-state stress
response increased steeply with increased current. Peak stress occurred
with 60 mA of current with a threshold of 30 mA (Fig. 5B).
The dependence of stress on stimulus frequency was biphasic, having a
significantly larger peak stress value in the first 30 s of a
stimulus. Subsequently, stress declined to the lower steady-state
values (Fig. 5A). The peak stress values were constant over
a 2- to 20-Hz range and somewhat lower at the highest frequencies (Fig.
5C). However, the steady-state stress levels fell with
increasing frequency of stimulation.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Stress-generating capacity of Rana bladder
rings in response to electrical field stimulation. A:
representative force trace field stimulation with increasing frequency
at 45-mA constant current. B: peak and steady-state active
stress generation in response to graded stimulus current at a constant
20-Hz stimulation frequency. C: peak and steady-state active
stress generation with changing stimulation frequency during constant
60-mA current stimulation. Values are presented as means ± SE
(n = 6). * Statistically significant difference from
value reported at 60 mA or 20 Hz (P < 0.02).
|
|
MRLC phosphorylation and cross-bridge cycling.
MRLC phosphorylation was initially determined using the two-dimensional
gel technique for separation of protein based on their IEF point and
then by their molecular weight (Fig.
6A). The Rana bladder and the swine carotid media exhibited similar major protein compositions in gels stained with Coomassie blue. However, proteins with mobilities characteristic of the nonmuscle MRLC isoforms were
absent in the frog bladder, which is also true for mammalian visceral
and urogenital smooth muscle (1, 7).
View larger version (102K):
[in this window]
[in a new window]
|
Fig. 6.
Gel electrophoretic separation and immunodetection of
myosin regulatory light chains (MRLCs). A: isoelectric
focusing (IEF)/SDS 2-dimensional gel separation of 73 mM
K+-depolarized R. catesbiana bladder
(left) and swine carotid homogenates (right)
stained with a colloidal Coomassie blue. Identified are actin, the
phosphorylated (SM-MLCPi) and nonphosphorylated forms of
the smooth muscle MRLC (SM-MLC), and the phosphorylated
(NM-MLCPi) and nonphosphorylated forms of nonmuscle MRLC
(NM-MLC). B: immunodetection of MRLC from bladder
homogenates subjected to different periods of K+
depolarization. Proteins were detected using a monoclonal anti-MRLC and
detected with 3,3'-diaminobenzidine colorimetric substrate.
|
|
Detection of MRLC was also accomplished by Western blots of two
dimensions using a monoclonal anti-mouse IgG antibody labeling the
smooth muscle myosin light chain spots, identified as SM-MLC. We also
used a simplified gel preparation where homogenates of the urinary
bladder were loaded in lanes of an IEF slab gel and run overnight under
identical conditions used to run the first dimension of the
two-dimensional system. The gel was then transferred to either
nitrocellulose paper or PVDF membrane and exposed to the MRLC antibody
(Fig. 6B). The one-dimensional IEF and transfer system for
MRLC detection provided reproducible results and similar quantitative
results to that reported by the two-dimensional system. We report here
quantitative changes in light chain phosphorylation by the
two-dimensional Coomassie blue detection because of limitations in the
detection linearity of Western reaction products. MRLC phosphorylation
levels rose from an unstimulated value of 11.6 ± 1.5% to a peak
of 50.9 ± 1.1% in 20 s of stimulation with 73 mM
K+ (Table 3). Phosphorylation
then fell to 26.7 ± 1.2% by 60 s and remained at ~25%
for the remainder of a stimulation (up to 3 min, data not shown).
View this table:
[in this window]
[in a new window]
|
Table 3.
Time-dependent changes in MRLC phosphorylation, stress, unloaded
shortening velocity, and series elastic compliance estimate of the Rana
bladder during 73 mM K+ depolarization
|
|
Unloaded shortening velocities.
Cross-bridge cycling kinetics as estimated from unloaded shortening
velocity (Vus) measurements were dependent on
MRLC phosphorylation. In individual bladder preparations the slack
times after 10, 12.5, and 15% length releases were fitted with a
linear regression. The slope of this regression-estimated shortening
velocity and the intercept provided an estimate of the series elastic
shortening in a K+-induced contraction. The slack times for
10, 12.5, and 15% length releases during a K+
depolarization at 5, 30, and 90 s are shown in Fig.
7B. The fits revealed an
initial slope at 5 s, which progressively steepened, peaking at
30 s, and then fell to a slope shallower than the initial 5-s
determination at 90 s. The calculated mean
Vus normalized for tissue lengths is reported in
Table 3. The mean Vus exhibited a linear
dependence on the level of MRLC phosphorylation with a slope of
~0.008 L0/s per %MRLC phosphorylation (Fig.
7C). These results also provided an estimate of the mean
series elasticity of 5.9 ± 0.8% at the maximal force generated
(Table 3).
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Determination of unloaded shortening velocities and their
dependence on MRLC phosphorylation. A: representative force
trace from a protocol determining slack times during a maximal
K+ depolarization with 15% length releases occurring at
10, 20, 30, and 60 s into a 73 mM K+ depolarization.
B: time dependence of slack time on percentage of length
release. All tissues were subjected to releases of 10, 12.5, and 15%
at different times during a K+ depolarization. Solid lines
are regressions fitted to the mean data plotted at 5, 30, and 90 s
into a K+ depolarization. * Statistically significant
difference from peak values at 30 s. Data are reported as
means ± SE (n = 7). C: dependence of
unloaded shortening velocity on suprabasal MRLC phosphorylation. Slopes
of individual regressions were determined and normalized for tissue
length and averaged and are reported as means ± SE
(n = 5-15). Suprabasal MRLC phosphorylations are
reported as means ± SE for time points in a K+
depolarization. Values were calculated from the difference between
resting and stimulated phosphorylation levels in tissues from the same
bladder (n = 3-10). Solid line represents a linear
regression of the means.
|
|
The time-dependent changes in stress generation, shortening velocity,
and MRLC phosphorylation are shown in Fig.
8. Stress rose monotonically, achieving a
plateau in 30 s. The averaged data did not show the typical
initial phasic peak (Fig. 8) due to variability reflected by the large
SE (dashed lines). Statistical analysis of mean data showed no
differences in stress values between the 20-, 30-, 60-, and 90-s time
points (see Table 3). Increases in MRLC phosphorylation were correlated
with increases in stress and Vus. However,
stress was sustained while MRLC phosphorylation and
Vus fell. This phenomenon of force maintenance
with reduced cross-bridge cycling rates as manifested by
Vus is behavior termed "latch" in
mammalian smooth muscle.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8.
Time course of stress generation, MRLC phosphorylation
(MRLC-Pi), and unloaded shortening velocity
(Vus) measurements during a maximal 73 mM
K+ depolarization in R. catesbiana bladder. Data
are reported as means ± SE [n = 4-8
(Vus), n = 8 (active stress),
and n = 4-7 (MRLC-Pi)]. Dashed line
represents ±SE of the mean stress.
|
|
| |
DISCUSSION |
The frog urinary bladder exhibited contractile properties similar
to those of mammalian smooth muscle tissues, including latch at 25°C.
This mechanical behavior was correlated with changes seen in MRLC
phosphorylation responsible for the activation of the smooth muscle
cross bridge. Both observations support the hypothesis that
cross-bridge phosphorylation can regulate tissue shortening and
contractile behavior in Rana bladder.
The micrographs of the bladder strips confirmed previously described
cellular arrangements in the urinary bladder (20). There
was a relatively thick stromal layer containing collagen, blood
vessels, and smooth muscle bundles bounded by mucosal and serosal
epithelial layers. The alignment of the smooth muscle cells in the
preparations was reasonable, if not optimal, for mechanical measurements.
The dependence of force on length was measured using a protocol
developed for the swine carotid media (29). Our results are consistent with described force-length relationships for mammalian bladder preparations (6, 16, 26). The bladder strips
display a broad range of lengths around L0 where
there is little change in the active stress (Fig. 1 and Ref.
8). This behavior may be attributed to anatomic
arrangements of the smooth muscle bundles resulting in different
bundles becoming optimized for force generation at different lengths.
The descending limb of the force-length relationship was relatively
shallow compared with skeletal and mammalian smooth muscles. This
observation may also reflect the anatomic arrangement of the muscle
bundles. The biphasic stress-length relationship is consistent with a
sliding filament/cross-bridge paradigm (9) although
correlation with filament overlap is lacking in any vertebrate smooth muscle.
The maximal force response to most stimuli was usually biphasic with a
peak generated within 60 s followed by a slow decline to a
steady-state level. Such a response is typical for most mammalian urinary bladder preparations (13-15). The sustained
contractile response to K+ depolarization with or without
an initial transient (Figs. 3 and 8) is characteristic of a tonic
smooth muscle (24).
The series elastic component (SEC) was estimated to be ~6% in
K+-depolarized tissues, generating around 3.0 × 104 N/m2 (Table 3). This value is not
significantly different from that reported for the rabbit bladder
(26). This SEC value does not reflect the SEC of the cross
bridge itself but is a refection of the ensemble SEC of the tissue
preparation, and its accuracy is limited by the method used to fit
slack times. The maximal shortening velocity of 0.30 L0/s was also comparable to a variety of
mammalian bladder preparations (26). The mechanical
performance of the Rana bladder strips at 25°C is
remarkably similar to the reported behavior of mammalian bladder at
37°C.
Phosphorylation of the MRLC is accomplished by the
Ca2+-dependent action of MLCK and is reversed by MLCP.
Recent evidence suggests that equivalent changes in MLCK and MLCP
activities seen at different temperatures should have a minimal impact
on cross-bridge recruitment or phosphorylation kinetics
(22). Thus we anticipated a minimal impact on the
relationship between myosin phosphorylation, isometric force
generation, and shortening velocity for the frog bladder at room
temperature compared with a mammalian preparation at 37°C. The
maximal force generation in mammalian smooth muscle is only slightly
affected by change in temperatures from 22 to 37°C and only
moderately affected over the temperature range of 10-22°C (8, 11, 19). However, the shortening velocities and the rates of force generation and relaxation are more highly temperature dependent. The conclusions drawn from these studies were that the
temperature dependence of the ADP release step, as well as the
temperature dependence of the regulatory system, are likely responsible
for the differences (11). If the in situ temperature dependency of MLCK and MLCP in amphibian smooth muscle were the same,
one would predict that lowering temperature would not increase net MRLC
phosphorylation in response to a specific stimulus-induced elevation in
myoplasmic Ca2+. Several studies on the effect of moderate
cooling on contractile response in mammalian smooth muscle have
demonstrated that the hypersensitivity is related to an increased
release of intracellular Ca2+ and alteration in the
electrogenic Na+/K+ exchanger, resulting in a
general increase in intracellular Ca2+ concentration
(25). Such a rise in Ca2+ would lend itself to
an increased MLCK activity and MRLC phosphorylation. Because
Vus is directly proportional to MRLC
phosphorylation (Fig. 7), this would offset any slowing due to the
temperature dependence of cross-bridge cycling.
The behavior of the amphibian bladder at 25°C in response to a
muscarinic agonist, depolarization, and field stimulation was quantitatively similar to mammalian bladder at 37°C. Our data support
the hypothesis that Ca2+-dependent cross-bridge
phosphorylation is responsible for activation in response to excitatory
stimuli. As in mammals, high forces can be sustained with moderate
elevations in MRLC phosphorylation and with reduced cross-bridge
cycling rates as estimated from Vus in the frog
bladder preparation at room temperature.
| |
ACKNOWLEDGEMENTS |
We thank Drs. J. Strauss and R. Wardle for helpful discussions
during the data collection and analysis. Thanks are extended to A. Browne-Teage for invaluable technical assistance.
| |
FOOTNOTES |
This study was supported by American Heart Association (AHA)
Virginia Affiliate Grant VA-95-G-15 and AHA Southeast Region Affiliate
Grant 9960075V to C. J. Wingard and by National Institutes of
Health Grants PO1-HL-19242 and R01-DK-56034 to R. A. Murphy.
This paper includes work performed in the completion of an
undergraduate honors study course for J. M. Nowocin.
Address for reprint requests and other correspondence:
C. J. Wingard, Dept. of Physiology, Medical College of
Georgia, 1120 15th St., Augusta, GA 30912-3000 (E-mail:
Cwingard{at}mail.mcg.edu).
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.
Received 29 September 2000; accepted in final form 30 July 2001.
| |
REFERENCES |
1.
Bárány, K,
and
Bárány M.
Myosin light chains.
In: Biochemistry of Smooth Muscle Contraction, edited by Bárány M.. San Diego, CA: Academic, 1996, p. 21-35.
2.
Becker, PL,
Singer JJ,
Walsh JV,
and
Fay FS.
Regulation of calcium concentration in voltage-clamped smooth muscle cells.
Science
244:
211-214,
1989[Abstract/Free Full Text].
3.
Bowers, CW,
and
Kolton L.
The efferent role of sensory axons in nerve-evoked contractions of bullfrog bladder.
Neuroscience
23:
1157-1168,
1987[ISI][Medline].
4.
De Lanerolle, P,
Strauss JD,
Felsen R,
Doerman GE,
and
Paul RJ.
Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle.
Circ Res
68:
457-465,
1991[Abstract/Free Full Text].
5.
Fuglsang, A,
Khromov A,
Torok K,
Somlyo AV,
and
Somlyo AP.
Flash photolysis studies of relaxation and cross-bridge detachment: higher sensitivity of tonic than phasic smooth muscle to MgADP.
J Muscle Res Cell Motil
14:
666-677,
1993[ISI][Medline].
6.
Gabella, G,
and
Uvelius B.
Urinary bladder of rat: fine structure of normal and hypertrophic musculature.
Cell Tissue Res
262:
67-79,
1990[ISI][Medline].
7.
Gaylinn, BD,
Eddinger TJ,
Martino PA,
Monical PL,
Hunt DF,
and
Murphy RA.
Expression of nonmuscle myosin heavy and light chains in smooth muscle.
Am J Physiol Cell Physiol
257:
C997-C1004,
1989[Abstract/Free Full Text].
8.
Gibbs, CL,
and
Loiselle DS.
Effect of temperature on mechanical and myothermic properties of rabbit smooth muscle.
Am J Physiol Cell Physiol
238:
C49-C55,
1980[Abstract/Free Full Text].
9.
Gordon, AM,
Huxley AF,
and
Julian FJ.
The variation in isometric tension with sarcomere length in vertebrate muscle fibres.
J Physiol (Lond)
184:
170-192,
1966[Abstract/Free Full Text].
10.
Herlihy, JT,
and
Murphy RA.
Length-tension relationship of smooth muscle of the hog carotid artery.
Circ Res
33:
275-283,
1973[Abstract/Free Full Text].
11.
Jaworowski, A,
and
Arner A.
Temperature sensitivity of force and shortening velocity in maximally activated skinned smooth muscle.
J Muscle Res Cell Motil
19:
247-255,
1998[ISI][Medline].
12.
Kurihara, S.
The effect of procaine on the urinary bladder smooth muscle of bullfrogs.
Jpn J Physiol
23:
309-324,
1973[ISI][Medline].
13.
Levin, RM,
Brendler K,
and
Wein AJ.
Comparative pharmacological response of an in vitro whole bladder preparation (rabbit) with response of isolated smooth muscle strips.
J Urol
130:
377-381,
1983[ISI][Medline].
14.
Levin, RM,
Longhurst PA,
Kato K,
McGuire EJ,
Elbadawi A,
and
Wein AJ.
Comparative physiology and pharmacology of the cat and rabbit urinary bladder.
J Urol
143:
848-852,
1990[ISI][Medline].
15.
Levin, RM,
Ruggieri MR,
Gill HS,
Haugaard N,
and
Wein AJ.
Studies on the biphasic nature of urinary bladder contraction and function.
Neurourol Urodyn
6:
339-350,
1987.
16.
Longhurst, PA,
Kang JS,
Wein AJ,
and
Levin RM.
Comparative length-tension relationship of urinary bladder strips from hamsters, rats, guinea-pigs, rabbits, and cats.
Comp Biochem Physiol A Physiol
96:
221-225,
1990.
17.
Miller, VM,
and
Vanhoutte PM.
Endothelium-dependent responses in isolated blood vessels of lower vertebrates.
Blood Vessels
23:
225-235,
1986[ISI][Medline].
18.
Murphy, RA.
What is special about smooth muscle? The significance of covalent crossbridge regulation.
FASEB J
8:
311-318,
1994[Abstract].
19.
Paul, RJ,
Doerman G,
Zeugner C,
and
Rüegg JC.
The dependence of unloaded shortening velocity on Ca2+, calmodulin, and duration of contraction in "chemically skinned" smooth muscle.
Circ Res
53:
342-351,
1983[Abstract/Free Full Text].
20.
Peachey, LD,
and
Rasmussen H.
Structure of the toad's urinary bladder as related to its physiology.
J Biophys Biochem Cytol
10:
529-553,
1961[Abstract/Free Full Text].
21.
Prosser, CL.
Rhythmic electrical and mechanical activity in stomach of toad and frog.
Am J Physiol Gastrointest Liver Physiol
269:
G386-G395,
1995[Abstract/Free Full Text].
22.
Sato, O,
and
Ogawa Y.
The modulatory effect of MgATP on heterotrimeric smooth muscle myosin phosphatase activity.
J Biochem (Tokyo)
126:
787-797,
1999[Abstract/Free Full Text].
23.
Shonnard, PY,
and
Sanders KM.
Effects of acetylcholine and substance P on electrical activity of intact toad gastric muscles. Influence of prostaglandins on electrical and mechanical activities of gastric muscles of Bufo marinus.
Am J Physiol Gastrointest Liver Physiol
258:
G12-G15,
1990[Abstract/Free Full Text].
24.
Somlyo, AP,
and
Somlyo AV.
Vascular smooth muscle. I. Normal structure, pathology, biochemistry, and biophysics.
Pharmacol Rev
20:
197-272,
1968[Free Full Text].
25.
Souilem, O,
Bidon JC,
Gogny M,
Blin M,
Vu AT,
and
Jondet A.
Effect of moderate cooling on contractile responses in mouse vas deferens and its relation to calcium.
Naunyn Schmiedebergs Arch Pharmacol
352:
337-455,
1995[ISI][Medline].
26.
Uvelius, B.
Influence of muscle length on the force-velocity relation of K+-contractures in smooth muscle from rabbit urinary bladder.
Acta Physiol Scand
101:
270-277,
1977[ISI][Medline].
27.
Walsh, JV,
and
Singer JJ.
Calcium action potentials in single freshly isolated smooth muscle cells.
Am J Physiol Cell Physiol
239:
C162-C174,
1980[Abstract/Free Full Text].
28.
Warshaw, DM,
and
Fay FS.
Tension transients in single isolated smooth muscle cells.
Adv Exp Med Biol
170:
617-622,
1984[ISI][Medline].
29.
Wingard, CJ,
Browne AK,
and
Murphy RA.
Dependence of force on length at constant cross-bridge phosphorylation in the swine carotid media.
J Physiol (Lond)
488:
729-739,
1995[ISI][Medline].
30.
Wright, GL,
and
Hurn E.
Vascular smooth muscle contractile properties in the turtle Pseudemys scripta elegans.
Comp Biochem Physiol C Pharmacol Toxicol Endocrinol
106:
155-163,
1993.
31.
Yano, K,
Vaudry H,
and
Conlon JM.
Spasmogenic actions of frog urotensin II on the bladder and ileum of the frog, Rana catesbeiana.
Gen Comp Endocrinol
96:
412-419,
1994[ISI][Medline].
Am J Physiol Regul Integr Comp Physiol 281(6):R1769-R1777
0363-6119/01 $5.00
Copyright © 2001 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
