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Function of gastrointestinal smooth muscle: from signaling to contractile proteins
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.
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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. 0002-9343/03/$22.00 15S doi:10.1016/S0002-9343(03)00189-X
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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 16S August 18, 2003 THE AMERICAN JOURNAL OF MEDICINE威
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 18S August 18, 2003 THE AMERICAN JOURNAL OF MEDICINE威
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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protein kinase C. Isolation from porcine aorta media and characterization. J Biochem. 1995;118:1104 –1107. Kitazawa T, Eto M, Woodsome TP, Brautigan DL. Agonists trigger G protein-mediated activation of the CPI17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem. 2000;275:9897–9900. Somlyo AP, Somlyo AV. Signal transduction by G-proteins, Rho-kinase, and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol. 2000;522:177–185. Shirazi H, Iizuka K, Fadden P, et al. Purification and characterization of the mammalian myosin light chain phosphatase holoenzyme: the differential effects of the holoenzyme and its subunits on smooth muscle. J Biol Chem. 1994;269: 31598 –31606. Shimizu H, Ito M, Miyahara M, et al. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J Biol Chem. 1994;269:30407–30411. Alessi D, MacDougall LK, Sola MM, Ikebe M, Cohen P. The control of protein phosphatase-1 by targeting subunits: the major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem. 1992; 210:1023–1035. Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rhokinase). Science. 1996;273:245–248. Feng J, Ito M, Ichikawa K, et al. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem. 1999;274:37385–37390. Lincoln TM, Cornwell TL. Intracellular cyclic GMP receptor proteins. FASEB J. 1993;7:328 –338. Francis SH, Corbin JD. Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci. 1999;36:275–328. Carvajal JA, Germain AM, Huidobro-Toro JP, Weiner CP. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol. 2000;184:409 –420. Surks HK, Mochizuki N, Kasai Y, et al. Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase I alpha. Science. 1999;286:1583–1587. MacDonald JA, Walker LA, Nakamoto RK, et al. Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle. FEBS Lett. 2000;479:83–88. Lee MR, Li L, Kitazawa T. Cyclic GMP causes Ca2⫹ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase. J Biol Chem. 1997;272: 5063–5068. Gerthoffer WT, Gunst SJ. Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle. J Appl Physiol. 2001;91:963–972. Landry J, Huot J. Regulation of actin dynamics by stressactivated protein kinase 2 (SAPK2)-dependent phosphorylation of heat-shock protein of 27 kDa (Hsp27). Biochem Soc Symp. 1999;64:79 –89. Bitar KN, Kaminski MS, Hailat N, Cease KB, Strahler JR. Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin. Biochem Biophys Res Commun. 1991;181:1192–1200. Brophy CM, Molinaro JR, Dickinson M. The macromolecular associations of heat shock protein-27 in vascular smooth muscle. Surgery. 2000;128:320 –326. Huot J, Lambert H, Lavoie JN, Guimond A, Houle F, Landry J. Characterization of 45-kDa/54-kDa Hsp27 kinase, a
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stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein Hsp27. Eur J Biochem. 1995;227:416 – 427. Landry J, Lambert H, Zhou M, et al. Human Hsp27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 Kinase II. J Biol Chem. 1992;267:794 –803. Rouse J, Cohen P, Trigon S, et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell. 1994;78:1027–1037. Stokoe D, Engel K, Campbell DG, Cohen P, Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 1992;313:307–313. Meloche S, Landry J, Huot J, Houle F, Marceau F, Giasson E. p38 MAP kinase pathway regulates angiotensin II-induced contraction of rat vascular smooth muscle. Am J Physiol Heart Circ Physiol. 2000;279:H741–H751. Yamboliev I, Wiesmann K, Singer C, Hedges J, Gerthoffer W. Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle. Am J Physiol. 2000;279:C352–C360. Chalovich JM. Actin mediated regulation of muscle contraction [review]. Pharmacol Ther. 1992;55:95–148. Winder SJ, Walsh MP. Calponin: thin filament–linked regulation of smooth muscle contraction. Cell Signal. 1993;5: 677–686. Haeberle JR. Calponin decreases the rate of cross-bridge cycling and increases maximum force production by smooth muscle myosin in an in vitro motility assay. J Biol Chem. 1994;269:12424 –12431. Winder S, Walsh M. Inhibition of the actomyosin MgATPase by chicken gizzard calponin. Prog Clin Biol Res. 1990;327: 141–148. Malmqvist U, Arner A. Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle. Pflugers Arch. 1991; 418:523–530. Pfitzer G, Sonntag-Bensch D, Brkic-Koric D. Thiophosphorylation-induced Ca2⫹ sensitization of guinea pig ileum contractility is not mediated by Rho-associated kinase. J Physiol. 2001;533(Pt 3):651–664. Parker CA, Takahashi K, Tao T, Morgan KG. Agonist-induced redistribution of calponin in contractile vascular smooth muscle cells. Am J Physiol. 1994;267:C1262– C1270. Parker CA, Takahashi K, Tang JX, Tao T, Morgan KG. Cytoskeletal targeting of calponin in differentiated, contrac-
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tile smooth muscle cells of the ferret. J Physiol. 1998;508(Pt 1):187–198. Winder SJ, Walsh MP. Calponin. Curr Top Cell Regul. 1996; 34:33–61. Takahashi K, Nadal-Ginard B. Molecular cloning and sequence analysis of smooth muscle calponin. J Biol Chem. 1991;266:13284 –13288. Strasser P, Gimona M, Moessler H, Herzog M, Small JV. Mammalian calponin: identification and expression of genetic variants. FEBS Lett. 1993;330:13–18. Applegate D, Feng W, Green RS, Taubman MB. Cloning and expression of a novel acidic calponin isoform from rat aortic vascular smooth muscle. J Biol Chem. 1994;269: 10683–10690. Kasuga M. Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation. Mol Cell Biol. 1994;14:6674 –6682. Birukov KG, Stepanova OV, Nanaev AK, Shirinsky VP. Expression of calponin in rabbit and human aortic smooth muscle cells. Cell Tissue Res. 1991;266:579 –584. Nakamura F, Mino T, Yamamoto J, Naka M, Tanaka T. Identification of the regulatory site in smooth muscle calponin that is phosphorylated by protein kinase C. J Biol Chem. 1993;268:6194 –6201. Leinweber B, Parissenti AM, Gallant C, et al. Regulation of protein kinase C by the cytoskeletal protein calponin. J Biol Chem. 2000;275:40329 –40336. Kaneko T, Amano M, Maeda A, et al. Identification of calponin as a novel substrate of Rho-kinase. Biochem Biophys Res Commun. 2000;273:110 –116. Leinweber BD, Leavis PC, Grabarek Z, Wang CL, Morgan KG. Extracellular regulated kinase (ERK) interaction with actin and the calponin homology (CH) domain of actinbinding proteins. Biochem J. 1999;344(Pt 1):117–123. Mabuchi K, Li B, Ip W, Tao T. Association of calponin with desmin intermediate filaments. J Biol Chem. 1997;272: 22662–22666. Takahashi K, Hiwada K, Kokubu T. Isolation and characterization of a 34,000-dalton calmodulin- and F-actin-binding protein from chicken gizzard smooth muscle. Biochem Biophys Res Commun. 1986;141:20 –26. Small JV, Gimona M. The cytoskeleton of the vertebrate smooth muscle cell. Acta Physiol Scand. 1998;164:341– 348. Bogatcheva NV, Ma Y, Urosev D, Gusev NB. Localization of calponin binding sites in the structure of 90 kDa heat shock protein (Hsp90). FEBS Lett. 1999;457:369 –374. Fujii T, Yamana K, Ogoma Y, Kondo Y. Interaction of calponin with phospholipids. J Biochem. 1995;117:999 –1003.
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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.
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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. 0002-9343/03/$22.00 15S doi:10.1016/S0002-9343(03)00189-X
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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 16S August 18, 2003 THE AMERICAN JOURNAL OF MEDICINE威
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 18S August 18, 2003 THE AMERICAN JOURNAL OF MEDICINE威
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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