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Biophys J, September 2000, p. 1511-1523, Vol. 79, No. 3
*Department of Physiology and Biophysics and
Department of Medicine, Case Western Reserve University
School of Medicine, Cleveland, Ohio 44106-4970 USA
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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The mechanical characteristics of smooth muscle can be
broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (Somlyo and Somlyo, 1968
, Pharmacol.
Rev. 20:197-272). To determine if differences in the
cross-bridge cycle and/or distribution of the cross-bridge states could
contribute to differences in the mechanical properties of smooth
muscle, we determined force and stiffness as a function of frequency in
Triton-permeabilized strips of rabbit portal vein (phasic) and aorta
(tonic). Permeabilized muscle strips were mounted between a
piezoelectric length driver and a piezoresistive force transducer.
Muscle length was oscillated from 1 to 100 Hz, and the stiffness was
determined as a function of frequency from the resulting force
response. During calcium activation (pCa 4, 5 mM MgATP), force and
stiffness increased to steady-state levels consistent with the
attachment of actively cycling cross-bridges. In smooth muscle, because
the cross-bridge states involved in force production have yet to be
elucidated, the effects of elevation of inorganic phosphate
(Pi) and MgADP on steady-state force and stiffness were
examined. When portal vein strips were transferred from activating
solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM
Pi, force and stiffness decreased proportionally,
suggesting that cross-bridge attachment is associated with
Pi release. For the aorta, elevating Pi
decreased force more than stiffness, suggesting the existence of an
attached, low-force actin-myosin-ADP- Pi state. When portal
vein strips were transferred from activating solution (pCa 4, 5 mM
MgATP) to activating solution with 5 mM MgADP, force remained
relatively constant, while stiffness decreased ~50%. For the aorta,
elevating MgADP decreased force and stiffness proportionally,
suggesting for tonic smooth muscle that a significant portion of force
production is associated with ADP release. These data suggest that in
the portal vein, force is produced either concurrently with or after Pi release but before MgADP release, whereas in aorta,
MgADP release is associated with a portion of the cross-bridge
powerstroke. These differences in cross-bridge properties could
contribute to the mechanical differences in properties of phasic and
tonic smooth muscle.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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In the 1970s it was recognized that smooth muscle
mechanics could be modeled using the same sliding filament/cross-bridge population paradigms as those for striated muscle (Huxley and Simmons,
1971). Although smooth muscle and skeletal muscle have similar basic
mechanisms, there are crucial differences between the two that define
their individual characteristics. Mechanically, smooth muscle is
broadly categorized as either phasic or tonic (Somlyo and Somlyo,
1968). Phasic smooth muscle is characterized by relatively rapid rates
of force activation and relaxation, high actomyosin ATPase activity,
and a fast maximum velocity of muscle shortening
(vmax). On the other hand, tonic
smooth muscle has relatively slow rates of force activation and
relaxation, a slow actomyosin ATPase, and a slow
vmax. There are also differences in
the splice variant expression of the myosin heavy chain (Kelley et al.,
1993) and essential light chain (MLC17) isoforms
(Nabeshima et al., 1987) between phasic and tonic smooth muscle
(Szymanski et al., 1998). These structural differences are thought to
lead to differences in distribution and kinetics of the cross-bridge cycle (Morano et al., 1997) and the ADP affinity of the cross-bridge (Fuglsang et al., 1993; Nishiye et al., 1993; Khromov et al., 1995).
Traditionally, the quick release methods of force clamps (Hill, 1938)
and length steps (Huxley and Simmons, 1971) have been used to
investigate cross-bridge properties. In 1980, Kawai and Brandt
developed a method that expanded this basic concept to the detection of
shifts in the cross-bridge population, using separate, fixed-frequency
sine wave measurements or fixed-frequency sinusoidal length
perturbations. The fixed-frequency sinusoidal length perturbation
protocol has a major advantage over traditional methods in that it is
able to detect shifts in the cross-bridge population (Kawai and Zhao,
1993). However, this method requires multiple contraction and
relaxation cycles, resulting in limited temporal resolution because the
stiffness is determined separately at each frequency. To increase the
accuracy, frequency, and temporal resolution of the fixed-frequency
sinusoidal length perturbation protocol, a novel length perturbation
protocol was recently developed in our laboratory that combined and
linked linearly increasing multiplet sine waves or increasing
multi-sine-wave length perturbation protocols (Shue and Brozovich,
1999). In tissue strip preparations, the increasing multi-sine-wave
length perturbation protocol yields a linear frequency response of
1-100 Hz with a frequency resolution of 1 Hz and a temporal resolution
of 14 s.
Energy transduction in muscle is accomplished by a cyclic
interaction between the globular head of myosin interacting with actin
in the thin filaments. Relaxed muscle contains predominantly detached
or weakly attached cross-bridges with bound products of ATP hydrolysis
(refer to Fig. 1, state a).
Upon activation, the cross-bridge attaches to the actin filament and
tilts, to produce the sliding force between the thick and thin
filaments. Force generation has been associated primarily with the
release of Pi from the active site in smooth
muscle (Fig. 1, step 4; Osterman and Arner, 1995; He et al.,
1998). X-ray diffraction has predicted that the release of
Pi from the active site results in local
rearrangements of the protein structure near the active site,
translating into a 5-20-nm movement of the light chain region at the
head-rod junction (Rayment et al., 1993). However, in smooth muscle,
force generation may also be associated with ADP release (Fig. 1,
step 6; Whittaker et al., 1995; Gollub et al., 1996). The
release of ADP from the S1 nucleotide binding
cleft results in the ~23° movement of the light chain binding
helix, resulting in a ~35-Å movement of the last heavy chain
residue. After ADP release (Fig. 1, step 6) in the presence
of MgATP, there is rapid ATP binding (Fig. 1, step 1) that
induces cross-bridge detachment (Fig. 1, step 2). Hydrolysis of ATP by the S1 head reforms actin+myosin-ADP-
Pi (A+M-ADP-Pi) (Fig. 1,
step 3). Evidence suggests that two
A-M-ADP-Pi states are present in skeletal muscle,
and force is thought to occur during a rapid isomerization that
immediately precedes Pi release (Geeves, 1991;
Kawai and Halvorson, 1991; Dantzig et al., 1992). However, we have not
included this state (A-M-ADP- Pi) in the scheme
because there is no experimental evidence that it exists in smooth
muscle (Fuglsang et al., 1993).
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We chose to investigate the cross-bridge cycle in smooth muscle to determine if differences exist between tonic and phasic smooth muscle. In skinned smooth muscle, we reasoned that if an elevation of Pi changes the distribution of the cross-bridge states and increases the relative population of the A+M-ADP-Pi state (Fig. 1, state a), then an increase in MgADP should similarly result in an increase in the relative population of the A-M-ADP state(s) (Fig. 1, states b and c). Furthermore, if force generation is coincident with the release of Pi, elevation of Pi should result in a similar fall in both force and stiffness. However, if ADP release is associated with force production, an elevation of MgADP should produce a decrease in force with little to no change in muscle stiffness. To determine if there are differences in the distribution of the cross-bridge states that could contribute to the mechanical differences between phasic and tonic smooth muscle, we characterized phasic and tonic smooth muscles by using increasing multi-sine-wave length perturbations of muscle length during the steady state for relaxed, activated, and rigor states of permeabilized smooth muscle strips. We then examined the changes in force and stiffness that occur when the relative distributions of cross-bridge states are perturbed with the elevation of Pi or MgADP.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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Muscle preparation
The portal vein and aorta were removed from 4-6- kg adult New
Zealand white rabbits. The smooth muscles were quickly removed, rinsed
in physiological saline solution several times, and stored in
oxygenated, 4°C physiological saline solution for up to 8 h. Fat
and connective tissue were removed, and small smooth muscle strips
(~0.75 × 1.0 mm) were dissected from the portal vein and aorta
and attached to aluminum T-clips (Goldman and Simmons, 1984). The
portal vein and aortic membranes were chemically permeabilized in
relaxing solution containing 1% Triton (Sigma UltraPure; Table 1) at 25°C for 45 and 105 min,
respectively. The permeabilized tissue was removed from the detergent
and rinsed with relaxing solution before it was mounted on the
experimental apparatus.
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The experimental apparatus
Measurements of fiber mechanics were made on an experimental
apparatus similar to that described by Smith and Barsotti (1993). The
muscle was mounted between a solid-state force transducer (AE801;
Sensonor, Horten, Norway) and a piezoelectric stack length driver
(Physiks Instruments, Walbronn, Germany). Two small hooks were
fashioned from 50-µm-diameter stainless steel rods and connected to
both the force transducer and the piezoelectric length driver. The
length driver was controlled through an amplifier interfaced with a
computer, using a National Instrument D/A and A/D data acquisition
board (PCI-MIO-16XE-10; 100 kS/S, 16-bit; National Instruments). The
frequency response of the force transducer, hooks, and attached muscle
was 2 kHz. The tissue was bathed in 200-µl wells of a rotating Teflon
platform, and the solutions were changed using a stepped, optical
feedback-looped motor. The length driver, force transducer, and
rotating stage were mounted on an aluminum platform on a vibration
isolation table (Micro-G; TMC, Peabody, MA) inside an
electrically grounded cage.
Increasing frequency sinusoidal sequence
The length perturbation sequence combined 4 cycles/frequency of
a 5.3-µm (0.7% total tissue length,
Lo) peak-to-peak sine wave ramping
from 1 to 100 Hz in 1-Hz increments. The end of each series was padded
with 0.36 s of zeroes to create a total sequence length of 14 s. After Fourier transformation, the sequence yielded a frequency
response with a 100-Hz bandwidth at 1-Hz resolution (Shue and
Brozovich, 1999). The sine waves were generated at 4 kHz, and the
resultant force and length of the tissue during length perturbation
were sampled at 4 kHz. Data were collected during the steady state of
the force response after solution changes. The length perturbation
sequence was continuously applied for 14 s and averaged over five
consecutive trials to reduce the influence of noise on the frequency
response of muscle stiffness measurements.
For both preparations (portal vein and aorta),
Lo averaged 130% of the resting
length (data not shown), and thus in these experiments, the
preparations were stretched to Lo. We
have shown that stiffness is constant for length changes between 0.5%
and 2.0% of Lo in tissue strips, and
at length changes larger than 2% Lo,
force and stiffness fell, suggesting that the perturbations detached
cross-bridges (data not shown). Similar experiments in single smooth
muscle cells show that stiffness is constant for length changes smaller
than 1.3% of cell length (Shue and Brozovich, 1999). In addition, the
intercept of stiffness determined from quick release experiments in
single smooth muscle cells has been reported to be ~1.5% (Warshaw
and Fay, 1983), further suggesting that a 0.7% oscillation would not
result in the detachment of attached cross-bridges. For each
experiment, force and stiffness in relaxing solution were set at zero
and used as the reference for the frequency response of all other states.
Chemicals and solutions
Solutions and calcium buffers were mixed according to a computer
program that calculates a given set of free ion concentrations for the
amount of stock solutions to be mixed (Brozovich and Yamakawa, 1995).
The binding constants for the ionic species were corrected for both
temperature and ionic strength (Andrews et al., 1991); the composition
of the solutions is listed in Table 1. The solutions were adjusted to
pH 7.1, with a final ionic strength of 200 mM. All experiments were
performed at room temperature (22°C).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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Relaxed state
The relaxed state was obtained after stretching the tissue to
Lo, changing the well solution three
times with fresh relaxing solution, and allowing the preparation to
equilibrate. After steady state was reached, force and stiffness were
determined as a function of frequency (see Materials and Methods). The
relaxed state was characterized by low force and low stiffness for both
the portal vein and aorta (Fig. 2)
and is consistent with previously published reports (Martin and
Barsotti, 1994; Khromov et al., 1996). It has been suggested that
attached cross-bridges are present in relaxed smooth muscle. To
investigate this possibility we used ATP-
-S, a nonhydrolyzable ATP
analog that shifts the population of cross-bridges to the detached
A+M-ATP--S state (Goody et al., 1980). The ATPase/ADPase apyrase
(Barsotti and Ferenczi, 1988; Nishiye et al., 1993) was also added to
the ATP--S relaxing solution to remove contaminating endogenous ATP
and ADP from permeabilized fibers. Using thin-layer chromatography, we
demonstrated that a 5-min treatment of ATP--S relaxing solution with
apyrase eliminated contaminating ADP but did not hydrolyze ATP--S
(data not shown). Upon transfer from relaxing solution to ATP--S
relaxing solution with apyrase, the portal vein did not exhibit a
significant change in stiffness (
1 ± 1%, n = 4, Fig. 3 a). However, in the
aorta, the transfer from relaxing solution to ATP--S relaxing
solution with apyrase decreased stiffness (33 ± 4%,
n = 3, Fig. 3 b), suggesting that for aortic
smooth muscle in relaxing solution, there exists a population of
attached cross-bridges.
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Rigor state
The rigor (A-M) state was achieved by incubating the portal vein
or aorta in rigor solution for at least 1 h while changing to
fresh rigor solution every 15 min. After this protocol in rigor solution, stiffness was determined for the portal vein and aorta. As
reported by others (Kawai and Brandt, 1980), we expected the rigor
(A-M) state to be characterized by a constant
stiffness-versus-frequency profile over the measured frequencies. The
portal vein demonstrated a flat and constant stiffness profile. On the
other hand, the aorta did not display these "rigor"
characteristics; but instead, stiffness was not constant for all
frequencies of oscillation. This suggests that for the aorta in rigor
solution, not all cross-bridges populate the rigor (A-M) state. To test
for the presence of a cross-bridge population other than A-M in rigor
solution for both the portal vein and aorta, we utilized apyrase.
Apyrase catalyzes the hydrolysis of any endogenous ATP and/or ADP in
the permeabilized muscle strips when bound to cross-bridges. In the
portal vein, there was no significant differences in stiffness
(3 ± 1%, n = 4, Fig.
4 a) between rigor solution
with apyrase and rigor solution. On the other hand, in the aorta, the
addition of apyrase to the rigor solution increased stiffness (+22 ± 3%, n = 4, Fig. 4 b) compared to rigor
solution. These results support the idea that for the aorta, in rigor
solution, the cross-bridge population is mixed, consisting of both
A-M-ADP and A-M states.
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Active state
Compared to the relaxed baseline, calcium activation of portal
vein and aorta significantly increased stiffness from a resting level
of 17.5 ± 2.4 N/m (n = 8) to 66.3 ± 4.1 N/m
(n = 10) in the portal vein and from 47.5 ± 4.2 N/m (n = 7) to 96.1 ± 6.7 N/m (n = 9) in the aorta (Fig. 5). This is
consistent with an increase in the number of actively cycling,
force-producing cross-bridges and is similar to reports by others for
skeletal (Goldman and Simmons, 1984), cardiac (Barsotti and Ferenczi,
1988), and smooth (Somlyo et al., 1988) muscle.
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Phosphate release step and its association with force production
The effect of increasing the relative population of the
prephosphate release state (Fig. 1, state a) in portal vein
and aorta was analyzed by measuring the change in force and stiffness
when the tissue preparation was transferred from activating solution to
activating solution containing 12 mM inorganic
Pi. The increase in inorganic
Pi resulted in a decrease in force for both the
portal vein (42 ± 1%, n = 4, Fig.
6 a) and aorta (30 ± 1%, n = 3, Fig. 7
a). In addition for the portal vein, stiffness also
decreased (40 ± 4%, n = 4, Fig. 6
b), while in the aorta stiffness similarly dropped
(18 ± 1%, n = 4, Fig. 7 b). These
results are consistent with several published reports, which
demonstrate a decrease in force with the elevation of inorganic
Pi (Osterman and Arner, 1995; He et al., 1998).
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ADP release step and force production
To investigate the role of ADP release in the cross-bridge cycle
and force production for portal vein and aorta, the relative population
of pre-ADP release cross-bridge states (Fig. 1, states b and
c) was increased using 5 mM MgADP. In portal vein, when the
preparation was moved from activating solution to activating solution
with 5 mM MgADP, steady-state force decreased slightly (2 ± 1%, n = 6, Fig. 8
a). Surprisingly, there was a large decrease (50 ± 3%, n = 6, Fig. 8 b) in stiffness,
suggesting the detachment of actin-bound, low-force-producing
cross-bridges. However, in the aorta, transfer from activating solution
to activating solution with 5 mM MgADP decreased force (30 ± 3%, n = 3, Fig. 9
a) and stiffness (28 ± 3%, n = 3, Fig. 9 b), suggesting that the ADP release step is
associated with force production.
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To determine whether the affect of 5 mM MgADP on force and stiffness in
the aorta was due to only the elevation of MgADP and not to other
factors, we performed two different control experiments. First, we
assessed the possibility that the MgADP effect is reversible. When the
aorta was transferred from activating solution with 5 mM MgADP to
activating solution (Fig. 10
a), the aorta redeveloped force back to the original maximum
Ca2+ activated level (n = 4),
which demonstrates that the MgADP effect is reversible. Second, to show
that this effect was not due to a change in MLC20
phosphorylation, the skinned aortic strips were first
thiophosphorylated with ATP--S. After thiophosphorylation, the
preparations were activated with MgATP, and force rapidly increased
(Fig. 10 b). After force reached a steady state, the tissue
strip was transferred to activating solution with 5 mM MgADP and force
fell (26 ± 1%, n = 4), an effect similar to
that obtained with nonthiophosphorylated preparations. Again, this MgADP effect was reversible with force redevelopment when the preparation was transferred from activating solution with 5 mM MgADP to
activating solution.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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Relaxed state
When the tissue strips were transferred from the relaxing
solution to ATP--S relaxing solution with apyrase, stiffness and force did not change for the portal vein but decreased for the aorta.
These data suggest that in the aorta, a population of attached cross-bridges exists in relaxing solution. Because ATP--S is a
nonhydrolyzable analog of ATP (Goody et al., 1980), substitution of
ATP--S for ATP would detach any attached cross-bridges present in
relaxing solution and result in an increase in the population of
detached, A+M-ATP--S cross-bridges. Detachment of cross-bridges by
ATP--S in relaxing solution would decrease overall stiffness, with
force decreasing if these cross-bridges were also in a force-producing state(s).
The existence of a population of attached cross-bridges in the relaxed
state has been proposed by previous work from this laboratory (Shue and
Brozovich, 1999). This previous investigation demonstrated that in
single portal vein cells, the phase angle was consistent with the
presence of an energy-generating process in the relaxed state. However,
in portal vein tissue strips, we did not detect a change in the
stiffness when the preparation was moved from relaxing to ATP--S
relaxing solution (Fig. 3 a). Nevertheless, in aortic tissue
we were still able to detect changes in the stiffness when the skinned
aortic strip was transferred from relaxing solution to ATP--S
relaxing solution (Fig. 3 b). Therefore in the aorta, the
data suggest that there is a larger population of attached
cross-bridges in comparison to the portal vein in relaxing solution.
Because previous work from this laboratory has demonstrated that
attached cross-bridges are present in relaxing solution for single
portal vein cells, we suggest that for ATP--S relaxing solution, the
lack of change in stiffness for the portal vein compared to the aorta
could be attributable to a smaller population of attached cross-bridges
in relaxing solution.
It has been shown previously that MLC20
phosphorylation is required for detached cross-bridges to attach and
produce force (Brozovich and Yamakawa, 1995). However, if a population
of attached cross-bridges already exists in relaxing solution, these
cross-bridges can produce force by moving through a force-generating
step and consequently increasing force without increasing
MLC20 phosphorylation (Brozovich and Yamakawa,
1995; Shue and Brozovich, 1999). The existence of a population of
attached cross-bridges could be the mechanism for those contractions
that occur without changes in MLC20
phosphorylation, such as stimulation by phorbol esters (Jiang and
Morgan, 1987) and sodium hydrosulfite (Yu et al., 1998).
Rigor state
Several other investigators have shown the existence of a
population of A-M-ADP cross-bridges (either the A-M-ADP or A-M-ADP* state) in rigor solution for both cardiac (Martin and Barsotti, 1994)
and smooth (Nishiye et al., 1993) muscle. When the portal vein is
transferred from rigor solution to rigor solution with apyrase, the
lack of change in stiffness (Fig. 4 a) suggests that the
portal vein in rigor solution has a large population of cross-bridges in the rigor (A-M) state. However, in the aorta, the change from rigor
solution to rigor solution with apyrase resulted in an increase in
stiffness (Fig. 4 b). These data suggest that for the aorta, a population of A-M-ADP and/or A-M-ADP* cross-bridges (Fig. 1) exists
in rigor solution. Apyrase hydrolyzes ADP and shifts the ADP-bound
cross-bridges toward the rigor (A-M) state, resulting in an increase in
force as A-M-ADP moves through the force-producing isomerization to
A-M-ADP* before entering the A-M cross-bridge state (Fig. 1,
steps 5 and 6). Furthermore, the overall increase in stiffness suggests that the apyrase treatment increases the total
number of attached cross-bridges.
These apyrase results demonstrate that in rigor solution for the aorta,
there exists a significant percentage of cross-bridges in the ADP-bound
states (A-M-ADP and/or A-M-ADP*). The existence of a significant
population of ADP-bound cross-bridges in rigor solution for aorta
compared to portal vein could be due to the difference in ADP affinity
and disassociation between phasic and tonic tissue (Arner et al., 1987;
Somlyo et al., 1988; Nishiye et al., 1993). The isoforms of
MLC17 have been correlated with the relative
sensitivities of the different smooth muscles types to MgADP (Fuglsang
et al., 1993). The concentration of the more basic
MLC17 isoform was highest in tonic tissue, which
also has the highest affinity for MgADP. In contrast, phasic tissue
predominantly expresses the acidic MLC17 isoform,
which has a lower affinity for MgADP (Fuglsang et al., 1993), faster
shortening velocities (Malmqvist and Arner, 1991), faster rates of
force development (Horiuti et al., 1989), and higher ATPase activity
(Hasegawa and Morita, 1992). In addition, the differences in the
7-amino acid insert near the ATP binding site, which differs in phasic
and tonic smooth muscle myosin heavy chains (Kelley et al., 1993; White
et al., 1993), may also affect MgADP affinity in phasic and tonic
smooth muscle. Therefore, in tonic tissue, a lower ADP Kd would result in a larger population
of MgADP-bound cross-bridges (A-M-ADP and/or A-M-ADP*). ADP
disassociation is higher in the portal vein compared to the aorta
(Khromov et al., 1996), which may produce a nondetectable population of
ADP-bound cross-bridges in rigor solution. Others (Sweeney et
al., 1998) have also suggested that for tonic smooth muscle, a
lower Kd for ADP would result in a
slower cycling rate compared to phasic smooth muscle. This could
partially explain the slower force generation and lower ATPase
activity of tonic compared to phasic smooth muscle.
Active state
The large increase in force and stiffness (Fig. 5) for both the
portal vein and aorta is consistent with an increase in the number of
cycling, force-producing cross-bridges during activation. As expected,
force production was faster in the portal vein than in the aorta,
consistent with their phasic and tonic properties, respectively.
Similar increases in force and stiffness have been reported by others
(Somlyo et al., 1988).
Phosphate release
Phosphate release has been shown to be regulated by
MLC20 phosphorylation (Sellers, 1985) and, in
smooth muscle, is thought to occur concurrently with or just before
cross-bridge attachment and force generation (Fig. 1, step
4). Thus inhibition of Pi release should
inhibit cross-bridge attachment and increase the relative population of
the A+M-ADP-Pi state (Fig. 1, state
a). If inhibition of Pi release occurs with
the elevation of Pi, one would predict that force
and stiffness should fall proportionally. Similar to studies in
skeletal (Kawai et al., 1987; Pate et al., 1998), cardiac (Barsotti and
Ferenczi, 1988), and smooth (Itoh et al., 1986; Osterman and Arner,
1995) muscle, we have demonstrated that elevating Pi decreases force and stiffness. Our results for
both the portal vein (Fig. 6) and aorta (Fig. 7) suggest that elevation
of Pi decreases steady-state force and stiffness
by increasing the relative population of the weakly bound or detached
A+M-ADP-Pi state.
Others (Brozovich et al., 1988) have suggested that in skeletal muscle
there exists an attached but low-force A-M-ADP-Pi
state in addition to the detached, A+M-ADP-Pi
state. If this state exists in smooth muscle, an increase in
Pi should shift the population of cross-bridge
states and lead to a relative increase in the attached,
low-force-producing A-M-ADP-Pi state. An increase
in the relative population of an attached but low-force-producing A-M-ADP-Pi state should produce a smaller fall in stiffness than in
force. In the portal vein, elevation of Pi
produced a proportionate fall in stiffness and force. These results
suggest that a low-force A-M-ADP-Pi does not
exist for the portal vein (Fig. 1). However, in the aorta, stiffness
decreased by 18% while force fell by 30%, a fall in stiffness that is
40% less than the fall in force. This suggests that an attached,
low-force-producing A-M-ADP-Pi cross-bridge state
may exist in the aorta. Consequently, for tonic smooth muscle, the
cross-bridge model in Fig. 1 requires modifications to include this
potential state (Fig. 11, state
a.2).
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ADP release
Cooke and Pate (1985) have demonstrated in skeletal muscle fibers
that force increases when the fiber is transferred from activating
solution to activating solution with elevated MgADP, which suggests
that force is produced before ADP release from the cross-bridge.
However, recent literature (Whittaker et al., 1995; Gollub et al.,
1996; Jontes and Milligan, 1997) suggests that in smooth muscle the
binding of MgADP to the cross-bridge is associated with a
conformational change in the light-chain domain, resulting in a
significant movement of the head-rod junction. This change represents
an extra movement of smooth muscle S1 beyond that
of skeletal muscle S1 (Whittaker et al., 1995;
Gollub et al., 1996). If there is force production associated with the
extra movement of the S1 head by ADP release,
then elevating MgADP to increase the relative population of the A-M-ADP
states should decrease force.
Portal vein
When portal vein tissue was transferred from activating solution to activating solution containing 5 mM MgADP, there was a small decrease in force but a large, ~50% decrease in stiffness (Fig. 8 b). Therefore, for the portal vein, our data suggest that ADP release is not a force-producing step (Fig. 1, step 6). This correlates with similar reports for other phasic smooth muscle (Khromov et al., 1996) and fits well with the original cross-bridge model scheme presented by Fuglsang et al. (1993). This cross-bridge scheme (Fig. 1) suggests that the force-generating step is an isomerization from the low-force to the high-force A-M-ADP state (Fig.
1, step 5). Similar to our results, Dantzig et al. (1999) reported a decrease in stiffness when rigor tissue was exposed to
elevated MgADP. The large decrease (~50%) in stiffness we observed when transferring the portal vein tissue from activating solution to
activating solution containing 5 mM MgADP suggests that elevated MgADP
preferentially affects low-force-producing cross-bridges (Fig. 1,
state b), leading to their detachment (Fig. 1, state a). Detachment of low-force-producing cross-bridges (Fig. 1,
state b) can be explained by the cross-bridges exiting
backward out of the cross-bridge cycle with subsequent detachment (Fig.
1, reversal of step 4), or, alternatively, by the existence
of a separate, detached A+M-ADP state (not shown), as suggested by the
model of Piazzesi et al. (1993). According to Piazzesi et al. (1993),
an intermediate, detached state (A+M-ADP) exists that would allow the
attached, ADP-bound cross-bridges (Fig. 1, states b and
c) to detach without moving backward to the
A+M-ADP-Pi state (Fig. 1, state a) and
reattach to actin without ADP dissociation. This type of mechanism can
be used to explain the multiple steps of the myosin head per hydrolysis
of a single ATP (Lombardi et al., 1992; Kitamura et al., 1999).
Aorta
In the aortic preparations, transferring the tissue from activating solution to activating solution with 5 mM MgADP decreased force and stiffness proportionally (Fig. 9), suggesting that the elevation of MgADP decreases force and detaches cross-bridges. These results were surprising, particularly because the release of ADP has not generally been thought to be coupled to a major portion of the powerstroke. However, if the extra 28° angle shift in the smooth muscle S1 resulting from MgADP release is associated with a significant portion of the force-producing step(s) (Whittaker et al., 1995; Gollub et al., 1996;
Jontes and Milligan, 1997), elevation of ADP concentrations should
prevent ADP release and thus result in a fall in force. The explanation for the decrease in stiffness with the transfer from activating solution to activating solution with 5 mM MgADP could be similar to
that above for the portal vein: by the cross-bridge exiting backward
out the cycle (reversal of step 4, Fig. 1) or through a
detached A+M-ADP state (Piazzesi et al., 1993).
In the aorta, given that our data suggest that a significant portion of
the force-producing step is associated with ADP release, the subsequent
rigor (A-M) state has to be a force-producing state (Fig. 1,
state e). However, ATP-induced detachment from the rigor (A-M) state has been shown to occur at a rate of
~105 M
1
s1 (Nishiye et al., 1993; Khromov et al.,
1996). At 5 mM ATP, the rate of cross-bridge detachment would occur at
500 s1. Thus the rigor (A-M) state would be
very short lived and produce little or no force. To maintain force,
these data suggest that a separate, force-producing rigor state (A-M*;
Fig. 11, state d) would have to exist that is similar to
that suggested by measurements of force and ATPase activity of single
myosin S1 heads (Ishijima et al., 1998).
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CONCLUSIONS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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The data suggest that for tonic smooth muscle, the ADP affinity
for the cross-bridge is higher than that for phasic smooth muscle. Our
data also suggest that the force-generating mechanism, specifically the
state involved, is not the same for the portal vein and aorta. For
portal vein, our data are consistent with force production occurring
concurrently with and/or after Pi release, but
before ADP release, which fits well with the cross-bridge model
presented in Fig. 1 (Fuglsang et al., 1993). For the aorta, our data
suggest that an attached, low-force A-M-ADP- Pi
state exists and that a significant portion of the cross-bridge power stroke is linked with the ADP release. Thus in the aorta, the cross-bridge model in Fig. 1 must include the attached, low-force A-M-ADP-Pi and the attached, high-force A-M*
states (Fig. 11, states a.2 and d, respectively).
These differences in cross-bridge properties could account for the
mechanical differences between phasic and tonic smooth muscle.
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ACKNOWLEDGMENTS |
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We thank Robert Barsotti for his critical reading of the manuscript, Jose Whittenbury for the bioinstrumentation, Guayhaur Shue for the program/software implementation, and Ozgur Ogut for his technical assistance.
This work was supported by National Institutes of Health grants HL44181 (FVB) and T32HL07653 (AYR).
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FOOTNOTES |
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Received for publication 20 December 1999 and in final form 30 May 2000.
Address reprint requests to Dr. Frank V. Brozovich, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4970. Tel.: 216-844-8955; Fax: 216-368-5586; E-mail: fxb9{at}po.cwru.edu.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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Biophys J, September 2000, p. 1511-1523, Vol. 79, No. 3
© 2000 by the Biophysical Society 0006-3495/00/09/1511/13 $2.00
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