Function of Gastrointestinal Smooth Muscle: From
Signaling to Contractile Proteins
Khalil N. Bitar, PhD
The action of smooth muscle in the intestinal wall
produces tonic contractions that maintain organ dimension against an imposed load such as a bolus of
food, as well as forceful contractions that produce
muscle shortening to propel the bolus along the
gastrointestinal tract. These functions are regulated
by intrinsic electrical and mechanical properties of
smooth muscle. The complex signaling process
that underlies these functions is discussed in this
article. We propose a model that describes the facilitation of sustained contraction of smooth muscle
cells in the gut. Am J Med. 2003;115(3A):15S–23S.
© 2003 by Excerpta Medica, Inc.
S
mooth muscle is responsible for the contractility of
hollow organs such as blood vessels, the gastrointestinal (GI) tract, gall bladder, bladder, and uterus.
The structure of smooth muscle differs greatly from that
of skeletal muscle. Smooth muscle can develop isometric
force per cross-sectional area that is equal to that of skeletal muscle. However, smooth muscle contractions are
far slower, but are sustained longer, than skeletal muscle
contractions.
The main function of smooth muscle of the GI tract is
to mix and propel intraluminal contents to enable efficient digestion of food, progressive absorption of nutrients, and evacuation of residues. These functions are regulated by intrinsic electrical and mechanical properties of
smooth muscle, such as the ability to maintain tone or
undergo phasic contraction. Thus, the action of smooth
muscle in the gut wall is 2-fold: tonic contractions that
maintain organ dimension against an imposed load such
as a bolus of food, and development of force and muscle
shortening similar to skeletal muscle. Both functions
contribute to the role of smooth muscle in modulating
the “reservoir” capacity and “propulsion” of food along
the length of the GI tract. This article describes the role of
signaling to contractile proteins occurring in smooth
muscle contraction in the GI tract.
STRUCTURE OF SMOOTH MUSCLE
From the Department of Pediatrics, University of Michigan Medical
School, Ann Arbor, Michigan, USA.
Supported by Grant Nos. RO1-DK57020 and RO1-DK42876 from
the National Institutes of Health.
Requests for reprints should be addressed to Khalil N. Bitar, PhD,
1150 W. Medical Center Drive, A520 MSRBI, Ann Arbor, Michigan
48109-0656.
© 2003 by Excerpta Medica, Inc.
All rights reserved.
In general, smooth muscle cells are about 200 to 300 ␮m
in length and 5 to 15 ␮m wide. The most striking feature
of smooth muscle is the lack of visible cross-striations.
Dense bodies and contractile filaments occupy about
80% of the interior of the cell. It is postulated that dense
bodies function as Z-lines. Three types of filaments can be
distinguished in smooth muscle cells: thin actin filaments, thick myosin filaments, and intermediate filaments. Intermediate filaments link dense bodies in the
cytoplasm to dense bands on the plasma membrane.
In smooth muscle, the actin filaments are organized
through attachments to the dense bodies that contain
␣-actinin, a Z-band protein in skeletal muscle. Actin is a
ubiquitous 42-kDa globular protein (G-actin) that polymerizes to form 2-stranded helical filaments (F-actin).
Inserted in the grooves of the actin helix is another protein, tropomyosin. Thin filaments in smooth muscle have
a distinct polarity, they insert into and emerge from the
dense bodies, and they are arranged in bundles that run
parallel to the long axis of the cells.
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A Symposium: Function of Gastrointestinal Smooth Muscle/Bitar
Thick filaments are aggregates of myosin molecules
formed from the association of 6 different proteins: 1 pair
of myosin heavy chains and 2 pairs of myosin light chains
(MLCs). The heavy chains are coiled around each other
to form an ␣-helical core or tail. Each strand of the core
terminates in a globular head surrounded by 2 MLCs: a
20-kDa regulatory chain, and a 17-kDa chain. Each globular head contains a binding site for actin and an actinactivated magnesium-adenosine triphosphatase (MgATP). A hinge located at the junction of the globular head
and core enables the head to rotate about the core. Another hinge in the core enables the globular heads to
project laterally. The globular heads and tail segments of
the core between the 2 hinges are called cross-bridges
because they constitute a link or bridge between thick
myosin and thin actin filaments.
CONTRACTION OF SMOOTH MUSCLE
OF THE GUT
The hydrolysis of ATP is the fundamental reaction
whereby chemical energy is converted into mechanical
energy in smooth muscle. The reaction generates force or
induces shortening as a result of the sliding of overlapping, interdigitating thin and thick filaments. The force
generated by crossbridge cycling depends on the number
of crossbridges acting in parallel. The crossbridges do not
cycle in unison; thus, in smooth muscle, unlike striated
muscle, both the number and cycling rate of crossbridges
are regulated.1
A rise in the concentration of cytosolic free calcium
(Ca⫹⫹) is the major trigger for the contraction of smooth
muscle.2 Smooth muscle contraction is regulated by
pharmacologic coupling mechanisms, whereby agonists
induce contraction without depolarizing the membrane,
and by electromechanical coupling, which involves
membrane depolarization.
Agonist-induced contraction of GI smooth muscle results in activation of G-protein– coupled receptors and in
sequential signal transduction events mediated through
several enzymes. These include: phosphatidyl–inositolspecific phospholipase C-␤ 1 and 3, phosphatidylcholine-specific phospholipase D, and phospholipase A2.3
Two different contractile pathways have been identified
in GI smooth muscle cells: a transient contraction, which
is calmodulin (CaM)-dependent, and is mediated by inositol 1,4,5-trisphosphate-dependent Ca⫹⫹ release,4 and a
sustained contraction induced by contractile agonists like
bombesin and ceramide.5,6 The sustained contraction is
mediated by extracellular Ca⫹⫹ influx and by a protein
kinase C (PKC)– dependent, CaM-independent pathway.7–9 Preincubation of smooth muscle cells with calphostin C, a PKC inhibitor, results in inhibition of contraction.10 Thus, stimulation with different agonists leads
to elevated Ca⫹⫹ via Ca⫹⫹ influx from the extracellular
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spaces through Ca⫹⫹-permeable nonspecific cation
channels,11 or voltage-dependent L-type Ca⫹⫹ channels,
and via Ca⫹⫹ release from the sarcoplasmic reticulum.
The elevation of intracellular Ca⫹⫹ leads to CaM-dependent activation of MLC kinase (MLCK), and hence
phosphorylation of the 20-kDa light chains (LCs) of myosin (MLC20) II at Ser19.12,13 This phosphorylation reaction triggers cycling of myosin crossbridges along the
actin filaments, which induces force development or
shortening of the muscle. Thus, regulation by myosin
phosphorylation is of particular importance for smooth
muscle, and is a feature of the actomyosin contractile system. In striated muscle, LC phosphorylation plays a modulatory role in the crossbridge cycle, and the on/off switch
of the troponin system located in the intermediate filament modulates the response to stimulation. The consequence is that the rapid binding of Ca⫹⫹ to 1 molecule of
troponin C renders 7 actin molecules available to interact
with as many heads of myosin molecules as they can accommodate. In smooth muscle, however, binding of
Ca⫹⫹ to CaM merely activates the enzyme, which in turn
must activate each myosin head independently, resulting
in a much slower process.1
The Actomyosin Crossbridge Cycle
The crossbridge cycle is a series of coupled biochemical
and mechanical events (Figure 1). The efficient conversion of biochemical energy requires a very precise temporal coupling between the biochemical and mechanical
events, and the structure of the myosin head is presumably designed to achieve this end. The crossbridge cycle
for fast skeletal muscle is the most thoroughly studied
system, and the description of the cycle that follows refers
to this actomyosin. The basic cycle is believed to be the
same for all myosins, but the relative rates of individual
steps are altered by changes in the amino acid sequence of
myosin to tune each myosin for its particular physiologic
role. Comparisons of the properties of different myosins,
together with mutagenesis of specific amino acid residues
or of short sequences, are the most active areas of current
research.
The proposed sequence of events is as follows: ATP
binding to either a resting length myosin head, or to a
head bearing a load, results in a change in conformation
in the myosin head, causing a rapid dissociation of the
myosin head from actin. After detachment from actin,
the ATP is hydrolyzed to adenosine diphosphate (ADP)
and inorganic phosphate (Pi), both of which remain very
tightly bound to the myosin head. The hydrolysis is relatively rapid (⬃10 msec) and reversible. The free energy of
ATP hydrolysis is not released but remains within the
structure of the myosin–ADP–Pi complex. This suggests
that the hydrolysis is accompanied by a major conformational change of myosin, which represents the reversal or
a repriming of the power stroke. The new structures are
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Figure 1. (1) Adenosine triphosphate (ATP) binds to the myosin head in the thick filaments. (2) ATP is hydrolyzed by myosin; the
products ADP and inorganic phosphate (Pi) are bound to myosin. The energy released by the splitting of ATP is stored in the myosin
molecule. The myosin–ADP–Pi complex is a high-energy state; this is the predominant state at rest. (3) Upon muscle stimulation, the
inhibition of actin–myosin interaction is lifted, and, consequently, the myosin with bound ADP and Pi attaches to actin. It is believed
that the angle of crossbridge attachment is 90°. (4) The actin–myosin interaction triggers the sequential release of Pi and ADP from
the myosin head, resulting in the working stroke. It is thought that the energy stored in the myosin molecule brings about a
conformational change in the crossbridge, tilting the angle from 90° to 45°. This tilting pulls the actin filament, while the energy
stored in myosin is used.
quite stable, and ADP and Pi will remain bound to the
myosin head until the myosin binds to an actin site. The
affinity of myosin–ADP–Pi for actin is significantly
higher than that of myosin–ATP. If an actin site is within
reach of the myosin head, it will bind rapidly and reversibly to the actin site. In doing so, it can explore several
potential actin-binding sites.14
The binding of myosin to actin can promote a major
change in conformation (the power stroke), which is accompanied by the dissociation of Pi. Crystal structures
suggest that the power stroke consists of a reorientation
of part of the myosin head distal to the actin-binding site
and includes the “converter” region and the LC binding
domain (LCBD). This results in the displacement of the
tip of the LCBD by up to 10 nm. The structural changes in
the actin–myosin interface that produce the power stroke
remain undefined.
A different pattern of crossbridge cycling is observed
during sustained (tonic) contraction of smooth muscle.
This type of contraction is useful, as mentioned earlier,
for allowing smooth muscle to maintain the dimension of
hollow organs (arteries or GI tract) against imposed loads
with minimal consumption of energy. In these contractions, after the initial peak in cytosolic (Ca⫹⫹) there is a
decrease in cytosolic Ca⫹⫹, while muscle contraction attains a peak and maintains its near steady state. This state
has been called the “latch” state, and refers to a transition
from a state of rapidly cycling crossbridges to a population of attached noncycling or slowly cycling crossbridges. Latch bridges maintain force in sustained
contraction.
Role of Calcium
As in skeletal muscle, Ca⫹⫹ plays a central role in the
contractility of smooth muscle. The amount of intracellular free Ca⫹⫹ is the key to regulation of smooth muscle
tone. Smooth muscle (as well as skeletal and cardiac muscle) contains MLCK, activated by Ca⫹⫹–CaM, the en-
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zyme that transfers the phosphate from ATP to either
serine or threonine hydroxyl groups of the phosphorylatable LC.15 In the smooth muscle cell, Ca⫹⫹ binds to CaM,
in contrast to striated muscles, where [Ca⫹⫹] binds to the
thin filament-associated protein troponin.16 The Ca⫹⫹–
CaM complex activates MLCK by association with the
catalytic subunit of the enzyme. The active MLCK catalyzes the phosphorylation of the regulatory LC subunits
of myosin (MLC20). Phosphorylated MLC20 activates
myosin ATPase, thus triggering cycling of the myosin
heads (crossbridges) along the actin filaments, resulting
in contraction of the smooth muscle. A decrease in the
intracellular level of Ca⫹⫹ induces a dissociation of the
Ca⫹⫹–CaM MLCK complex, resulting in dephosphorylation of the MLC20 by MLC phosphatase (MLCP) and
relaxation of the smooth muscle.17 Due to the antagonism between MLCK and MLCP, inhibition of MLCP
results in an increase in the phosphorylation content of
the LC with concomitant increase in muscle contraction.
Under certain conditions, submaximal levels of Ca⫹⫹ are
sufficient for maximal contraction, referred to as increased Ca⫹⫹ sensitivity (or “Ca⫹⫹ sensitization”).2
Secondary Regulatory Pathways:
Role of Phosphatases
Although MLCK is a Ca⫹⫹–CaM-dependent MLC-specific protein kinase (PK), it is primarily responsible for
the phosphorylation of myosin in smooth muscle,17–20
because an increase in cytosolic Ca⫹⫹ concentration induces MLC phosphorylation in smooth muscle. On the
other hand, the force development of smooth muscle is
not simply determined by the concentration of Ca⫹⫹. In
smooth muscle, the force/Ca⫹⫹ ratio is variable, and depends partly on specific activation mechanisms. It was
found that an agonist could induce an increase in force
development in smooth muscle even when cytoplasmic
Ca⫹⫹ is clamped.21,22 This agonist-induced “Ca⫹⫹ sensitization” strongly suggests that there are additional
mechanisms that can regulate smooth muscle contraction. Ca⫹⫹ does not always parallel the extent of MLC20
phosphorylation and contraction. In the case of tonic
force development, the relation between force and
MLC20 phosphorylation can be modified. Thus, in
smooth muscle, secondary regulatory pathways are functionally important in the control of Ca⫹⫹-independent
levels of MLC20 phosphorylation and Ca⫹⫹-independent contraction.
The increase in force induced by an agonist at a given
Ca⫹⫹ concentration is due to inhibition of MLCP.21,23
The effects of Ca⫹⫹-sensitizing agonists are mediated
by guanosine triphosphate (GTP)-binding proteins that
generate PKC or arachidonic acid as second messengers.
Thus, the major mechanism of Ca⫹⫹ sensitization of
smooth muscle contraction is through inhibition of the
smooth muscle myosin phosphatase, thus increasing
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MLC20 phosphorylation through elevated basal levels of
MLCK activity.
PROTEIN KINASE C
Members of the PKC family are involved in transducing
signals for hormone receptors, growth factors, and neurotransmitters. Large numbers of studies have indicated
that PKC activity is related to its subcellular localization.24 Many investigators have described association of
PKC with the plasma membrane after agonist stimulation
in smooth muscle cells.8,25,26 Membrane association is
reflected in a shift in subcellular localization and translocation from cytosolic PKC to membrane compartments.
This process is controlled by protein–protein interactions
that play an important role in localization and function of
PKC isozymes. The interaction between PKCs and cytoskeletal proteins is isozyme selective. Upon stimulation
with contractile agonists, the ␣ isoform of PKC (PKC-␣)
translocates to the cell membrane in adult rabbit colon
smooth muscle cells27 and associates with translocated
RhoA and Hsp27.26,28
PKC plays a role in the regulation of myosin phosphorylation,29,30 via activation of the MLCP inhibitor,
CPI17,31 whose inhibitory activity requires the phosphorylation of Thr 38 by PKC.32 It has been recently
shown that the phosphorylation of CPI17 increases in
smooth muscle after agonist stimulation.33 The resulting
myosin phosphorylation and subsequent smooth muscle
contraction therefore occurs without a change in sarcoplasmic Ca⫹⫹ concentration.
RhoA-Dependent Kinase
In smooth muscle, a Rho-regulated system of MLCP
exists, and Rho-kinase is the major enzyme in this system.
Ca⫹⫹ sensitization by the RhoA/Rho-kinase pathway has
been shown to contribute to the tonic phase of the agonist-induced contraction in smooth muscle.34 MLCP
consists of 3 subunits: a myosin-binding large subunit
(MBS), a 20-kDa small subunit, and a catalytic subunit of
the type 1 protein serine/threonine phosphatase family.35–37 MBS can be phosphorylated by RhoA-dependent
kinase, resulting in a decrease in MLCP activity.38 Rhokinase phosphorylates MBS at 2 sites in vitro, Thr 641 and
Thr 799; Thr 641 has been described as responsible for the
inhibition of MLCP.39 Thus, RhoA-kinase phosphorylation of MBS could result in downregulation of MLCP and
thereby increase contraction.
Relaxation
Return of Ca⫹⫹ to resting levels results in dissociation of
Ca⫹⫹ from CaM, inactivation of MLCK, and dephosphorylation of myosin catalyzed by MLCP. Relaxation
generally occurs after return of intracellular [Ca⫹⫹] to
resting levels. The main relaxant peptides in the gut,
vasoactive intestinal peptide and pituitary adenylate
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cyclase-activating peptide, stimulate cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in smooth muscle and activate both
cAMP-dependent PK (PKA), and cGMP-dependent PK
(PKG). PKG can act on various targets to regulate Ca⫹⫹
mobilization and induce relaxation.40 – 42 PKG inhibits
the formation of inositol 1,4,5-triphosphate (IP3)-dependent Ca⫹⫹ release, and stimulates plasmalemmal and
sarcoplasmic Ca⫹⫹-ATPase pumps, thereby increasing
Ca⫹⫹ uptake by stimulating Ca⫹⫹ efflux from the cells. It
also inhibits Ca⫹⫹ channel activity and stimulates K⫹
channel activity, causing membrane hyperpolarization
and a reduction in Ca⫹⫹ influx into the cell via voltagedependent Ca⫹⫹ channels. Some of these properties are
shared by PKA.
The resulting effect is dissociation of Ca⫹⫹ from CaM,
inactivation of MLCK, and dephosphorylation of myosin
catalyzed by MLCP. Both PKA and PKG can also induce
relaxation by acting on targets downstream of Ca⫹⫹ mobilization. Both decrease MLC phosphorylation during
the initial phase of contraction by inhibiting Ca⫹⫹–
CaM-dependent activation of MLCK or by activating
MLCP via telokin.43– 45 During the sustained phase of
contraction, which in GI smooth muscle is Ca⫹⫹ independent, both kinases decrease MLC phosphorylation by
inhibiting the monomeric G protein RhoA.
Role of Low Molecular Weight Heat-Shock
Protein Hsp27
The special biochemistry of smooth muscle is complicated by the role of other contractile proteins, namely
caldesmon and calponin, as well as by the low molecular
weight heat-shock protein 27 (Hsp27), which has been
identified more recently. Both caldesmon and calponin
bind to actin and CaM, and both have the capacity in
vitro to inhibit the Mg-ATPase of smooth muscle in a
Ca⫹⫹-regulated manner.
In smooth muscle there is a mechanism for the regulation and modulation of contraction involving the thin
filaments.15 Smooth muscle cells, upon agonist stimulation and triggering of signaling pathways, adjust their
functional properties by modulating association of contractile proteins and reorganizing their cytoskeleton.
Hsp27 has significant effects on actin cytoskeletal reorganization46,47; it has been implicated in the regulation of
the contraction and relaxation of smooth muscle.48,49
Preincubation of smooth muscle cells from the rabbit
rectosigmoid with a monoclonal antibody to Hsp27 inhibits PKC-induced contraction.48 Upon stimulation of
freshly isolated intestinal smooth muscle cells with contractile agonists, Hsp27 colocalizes and coimmunoprecipitates with the contractile proteins, actin, tropomyosin, and caldesmon27; it also associates with translocated
PKC-␣ and with translocated RhoA in the particulate
fraction.26
Hsp27 is a member of the mammalian small heatshock protein family. It is expressed in a variety of tissues
including smooth muscles, in the presence or absence of
stress, and has been shown to exhibit chaperone activity
in vitro and modulate actin filament microdynamics.
Hsp27 is phosphorylated in response to heat shock and in
response to different stimuli such as cytokines, growth
factors, and peptide hormones.28,50 –52 Landry et al.51
have mapped the phosphorylation sites in human Hsp27,
and showed that mitogen-activated protein kinase–activated protein (MAPKAP) kinase-2 phosphorylates human Hsp27 protein on Ser15, Ser78, and Ser82. Ser82
appears to be the major site of in vivo phosphorylation.53
Phosphorylation of Hsp27 changes the actin cytoskeleton and modulates actin-associated events,54 including
actin–myosin interaction. Recently, we have reported
that agonist-induced contraction is associated with phosphorylation of Hsp27, and that transfecting smooth muscle cells with the nonphosphomimic mutant of Hsp27
results in inhibition of agonist-induced association of actin–myosin in colonic smooth muscle cells.28 Results
from our laboratory also have confirmed the association
and translocation of Hsp27 with contractile proteins27
and with signaling proteins.6,26 Thus, in smooth muscle
cells, Hsp27 appears to be the link between the signal
transduction cascade and the contractile machinery.26
Mechanisms by which Hsp27 affects agonist-induced
contraction are not clear. In intact smooth muscle, under
physiologic conditions, Hsp27 probably regulates actin
cytoskeleton structure and may modulate the interaction
of actin and myosin. Hsp27 has significant effects on the
actin cytoskeleton that are regulated by phosphorylation
and dephosphorylation.46 Recently, we have shown that
agonist-induced phosphorylation of Hsp27 modulates
actin–myosin interaction through thin-filament regulation of tropomyosin (Figure 2).28 Thus, control of in vivo
activity of Hsp27 can be attributed to its phosphorylation
state. Inhibition of Hsp27 phosphorylation substantially
inhibited angiotensin II–induced contraction but, interestingly, had no effect on phenylephrine-induced contraction.54 Anti-Hsp27 antibodies also partially inhibit
endothelin-1–induced Ca⫹⫹ sensitization of chemically
permeabilized canine pulmonary artery strips.55 There is
a body of evidence that suggest that phosphorylation of
Hsp27 plays a crucial role in the association of Hsp27
with translocated PKC-␣ and with RhoA during agonistinduced smooth muscle contraction.
Role of Calponin
Studies suggest that CaM-binding thin filament–associated proteins, such as caldesmon and calponin, play an
important role in smooth muscle contractility.56,57
Calponin, an actin-binding protein, inhibits actomyosin
ATPase and slows the detachment of myosin from actin.58 In its unphosphorylated state, calponin binds to
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Figure 2. A model for heat shock protein Hsp27 regulation of the actin–tropomyosin filament. Through its phosphorylation and
reorganization, Hsp27 regulates the state of the actin–tropomyosin filament. Hsp27 binds to both actin and tropomyosin in the “on”
and “off ” states. Upon agonist-induced phosphorylation, Hsp27 undergoes conformational changes and reorganization inside the
cell. Phosphorylated (P)Hsp27 modulates Hsp27–tropomyosin binding. The binding of phosphorylated Hsp27 to tropomyosin may
help uncover the myosin binding site on actin. As a result, actin molecules are more available to myosin heads and facilitate sustained
contraction.
actin and inhibits the Mg-ATPase of myosin. Upon phosphorylation by PKC, its inhibiting activity is lost.59
PKC-␣ blocks calponin-dependent interference with the
actin–myosin interaction and, hence, leads to muscle
contraction.60 PKC regulation of calponin phosphorylation is thought to be of physiologic importance.61– 63
Calponin was originally discovered in smooth muscle
as an F-actin-, CaM-, and tropomyosin-binding protein.64 Three types of calponin isoforms—acidic, neutral,
and basic calponin— have been classified on the basis of
their isoelectric point.64 – 67 Basic calponin is distributed
relatively specifically in smooth muscle tissues,62 and has
been well characterized in vitro.62,68,69 Birukov et al.69
performed histochemical studies that showed that calponin is distributed more toward the center rather than the
periphery in a resting cell. Reports indicate that PKC/
PKC-␣ interacts with calponin.70,71 Calponin is also
shown to form a substrate for Rho-kinase in vitro.72 In
addition, it was recently reported that calponin may facilitate extracellular-regulated kinase– dependent signaling, thus playing a significant role in regulation of vascular smooth muscle contraction.73
Because of its ability to bind to actin, calponin is hypothesized to be a cytoskeleton regulatory protein functioning as a bridge between actin and intermediate fila20S August 18, 2003 THE AMERICAN JOURNAL OF MEDICINE威
ment networks in smooth muscles.74 The smooth
muscle–specific variant of calponin was originally identified as actin-associated protein from chicken gizzard.75
Potential binding partners for calponin include CaM,
s100 proteins, tropomyosin, myosin, and caldesmon.76 A
direct interaction of calponin with phospholipids and
with Hsp90 has also been suggested.77,78 Calponin also
associates with PKC-⑀ in vascular smooth muscles.71
Association of Calponin with Hsp27
Stimulation of colonic smooth muscle cells with acetylcholine (ACh) induces an increase in the association of
calponin with Hsp27 in the particulate fraction of colonic
smooth muscle. This increased association is inhibited
when the cells are pretreated with dibutyryl-cAMP
(dbcAMP), indicating that the association is concomitant
with ACh-induced contraction, and inhibited upon inhibition of contraction by preincubating the cells with
dbcAMP. ACh-induced contraction is also associated
with a concomitant increase in the association of calponin with PKC-␣ in the particulate fraction. There is a
concomitant reduction of these proteins in the cytosolic
fraction upon induction of smooth muscle cells with
ACh. The interaction of calponin with PKC has also been
shown in vascular smooth muscle.71
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Figure 3. Model of maintenance of smooth muscle contraction. Agonist-induced contraction results in activation and translocation of both protein kinase C (PKC)–␣ and RhoA to the membrane. Translocated PKC-␣ and RhoA associate with phosphorylated
(P) Hsp27 in the membrane. Translocated PKC-␣ also associates with calponin, thus blocking the inhibitory effect of calponin on
actin and allowing “free” actin monomers to bind to myosin. This facilitates sliding and cross bridge cycling of myosin and actin and
facilitates sustained contraction.
Actin-binding proteins play a key role in shaping the
actin cytoskeleton. Calponin is a CaM-dependent small
protein that interacts with actin. Calponin is phosphorylated by PKC-␣.70 If calponin plays an important role in
cytoskeleton formation or contractility, one would hypothesize that it will also be able to interact not only with
the proteins of microfilaments, but also with different
proteins involved in signal transduction. We are proposing a model (Figure 3) in which the association of calponin with PKC-␣ is mediated by phosphorylation of
Hsp27. The translocation of PKC-␣, and its association
with calponin, results in release of inhibitory calponin
molecules from actin and thus opens a window of “empty” actin molecules that would allow further binding of
myosin to actin. This model would be predicted to facilitate sustained contraction of smooth muscle cells.
ACKNOWLEDGMENT
We thank Dana Thomas for her assistance with technical editing and figure development.
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