Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain

The contraction of smooth muscle is regulated primarily by intracellular Ca²⁺ signal. It is well established that the elevation of the cytosolic Ca²⁺ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca²⁺ concentration and force development revealed that the alteration of the Ca²⁺-sensitivity of the contractile apparatus as well as the Ca²⁺ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca²⁺ concentration is considered to contribute to the major mechanisms regulating the Ca²⁺-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca²⁺ elevation is increased either by Ca²⁺-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca²⁺-dependent manner but also by multiple kinases in a Ca²⁺-independent manner, thus adding a novel mechanism to the regulation of the Ca²⁺-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.     

Thin filament flexibility and its role in muscle contraction

Contemporary experimental methods do not allow unequivocal determination of molecular structural events during muscle contraction. To analyze existing contradictions, an original computer program has been developed. This program reconstructs the hexagonal lattice of a sarcomere for different states of muscle and finds the most realistic structure by comparing the calculated Fourier spectrum with the actual diffraction pattern. Previously, the new approach allowed reconstructing the actual structure of a myosin filament from mammalian striated muscle (http://zope.ibib.waw.pl/pspk). In this work, the thin filament is reconstructed for three states: relaxed, activated, and contracting. The good fit between the calculated Fourier spectra and the actual diffraction patterns taken from the literature suggests that the thin filament owing to its flexibility may play an active role in muscle contraction, as myosin cross-bridges do.     

The importance of Rho-associated kinase-induced Ca2+ sensitization as a component of electromechanical and pharmacomechanical coupling in rat ureteric smooth muscle

Ureteric peristalsis, which occurs via alternating contraction and relaxation of ureteric smooth muscle, ensures the unidirectional flow of urine from the kidney to the bladder. Understanding of the molecular mechanisms underlying ureteric excitation–contraction coupling, however, is limited. To address these knowledge deficits, and in particular to test the hypothesis that Ca²⁺ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway plays an important role in ureteric smooth muscle contraction, we carried out a thorough characterization of the electrical activity, Ca²⁺ signaling, MYPT1 (myosin targeting subunit of myosin light chain phosphatase, MLCP) and myosin regulatory light chain (LC₂₀) phosphorylation, and force responses to membrane depolarization induced by KCl (electromechanical coupling) and carbachol (CCh) (pharmacomechanical coupling). The effects of ROK inhibition on these parameters were investigated. We conclude that the tonic, but not the phasic component of KCl- or CCh-induced ureteric smooth muscle contraction is highly dependent on ROK-catalyzed phosphorylation of MYPT1 at T855, leading to inhibition of MLCP and increased LC₂₀ phosphorylation.     

Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

Myosin head mechanical performance under different conformational change mechanisms

The present paper puts forward a mathematical approach to model the conformational changes of the myosin head due to ATP hydrolysis, which determine the head swinging and consequent sliding of the actin filament. Our aim is to provide a simple but effective model simulating myosin head performance to be integrated into the overall model of sarcomere mechanics under development at our Laboratory (J. Biomech. 34 (2001) 1607). We began by exploring myosin head mechanics in recent findings about myosin ultrastructure, morphology and energetics in order to calculate the working stroke distance (WS) and the force transmitted to the actin filament during muscle contraction. Two different working stroke mechanisms were investigated, assuming that the swinging of the myosin head occurs either as a consequence of purely conformational changes (Science 261 (1993a) 58) or by thermally driven motion (ratchet mechanism) followed by conformational changes (Cell 99 (1999) 421). Our results show that force and WS values vary markedly between the two models. The maximum force generated is about 10 pN for the first model and 31 pN for the second model, and the WSs are about 13 and 4 nm, respectively. These results are then discussed and compared with published data. The experimental data used for comparison are scarce and non-homogeneous; hence, the final remarks do not lead to definite conclusions. In any event, relatively speaking, the first model is more coherent with experimental findings.     

A mathematical model of airway and pulmonary arteriole smooth muscle

Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca²⁺), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca²⁺ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca²⁺ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca²⁺ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca²⁺ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca²⁺-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration.     

Fine structure and contraction of isolated muscle actomyosin

Summary A modified thread model of isolated cross-striated muscle actomyosin was produced, which a priori consisted of both actin and myosin filaments forming a random network. This modified model contracts to the same extent as the normal model which lacks myosin filaments prior to contraction.The striking difference in the contraction behavior of the two models indicates 1) that in the normal model myosin filament formation occurs during contraction and 2) that the pre-existence of myosin filaments in the modified model increases the speed of contraction. Hence, the sliding mechanism involving myosin filaments is able to operate at a higher speed than the sliding mechanism which utilizes oligomeric myosin.     

Myosin filament sliding through the Z-disc relates striated muscle fibre structure to function

Striated muscle contraction requires intricate interactions of microstructures. The classic textbook assumption that myosin filaments are compressed at the meshed Z-disc during striated muscle fibre contraction conflicts with experimental evidence. For example, myosin filaments are too stiff to be compressed sufficiently by the muscular force, and, unlike compressed springs, the muscle fibres do not restore their resting length after contractions to short lengths. Further, the dependence of a fibre''s maximum contraction velocity on sarcomere length is unexplained to date. In this paper, we present a structurally consistent model of sarcomere contraction that reconciles these findings with the well-accepted sliding filament and crossbridge theories. The few required model parameters are taken from the literature or obtained from reasoning based on structural arguments. In our model, the transition from hexagonal to tetragonal actin filament arrangement near the Z-disc together with a thoughtful titin arrangement enables myosin filament sliding through the Z-disc. This sliding leads to swivelled crossbridges in the adjacent half-sarcomere that dampen contraction. With no fitting of parameters required, the model predicts straightforwardly the fibre''s entire force–length behaviour and the dependence of the maximum contraction velocity on sarcomere length. Our model enables a structurally and functionally consistent view of the contractile machinery of the striated fibre with possible implications for muscle diseases and evolution.     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.     

Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption

Lymphatic absorption is a highly regulated process driven by both an extrinsic mechanism (external force) and an intrinsic mechanism (lymphatic vessel contractility). The lymphatic muscle is a specialized smooth muscle with unique mechanical properties. To understand the molecular mechanism and relative contribution of smooth muscle contraction in lymphatic absorption, we analyzed mice with a smooth muscle-specific deletion of Mylk, a critical gene for smooth muscle contraction. Interestingly, the knockout mice were significantly resistant to anesthesia reagents. Upon injection in the feet with FITC-dextran, the mutant mice displayed a 2-fold delay of the absorption peak in the peripheral circulation. Examining the ear lymphatic vessels of the mutant mice revealed a reduction in the amount of fluid in the lumens of the lymphangions, suggesting an impairment of lymph formation. The Mylk-deficient lymphatic muscle exhibited a significant reduction of peristalsis and of myosin light chain phosphorylation in response to depolarization. We thus concluded that MLCK and myosin light chain phosphorylation are required for lymphatic vessel contraction. Lymphatic contractility is not an exclusive requirement for lymphatic absorption, and external force appears to be necessary for absorption.     

Peroxynitrite-mediated oxidative modifications of myosin and implications on structure and function

Abstract

The peroxynitrite-induced functional impairment of myosin was studied in different reaction conditions, known to alter the oxidative chemistry of peroxynitrite, to better understand the molecular mechanisms of this interaction. It is shown that peroxynitrite is able to enhance the basal MgATPase activity up to 2-fold while inhibiting the actin-stimulated ATPase activity of myosin and that the extent of these functional alterations is dependent on the reaction medium. The observed changes in the stimulation of the MgATPase activity correlate with the extent of carbonyl formation in myosin. The enzyme inhibition is more potent in conditions where the efficiency of tyrosine nitration and peroxynitrite reactivity towards sulphydryls are lower. Together with the observation that reversion of sulphydryl oxidation did not lead to the recovery of myosin functional and structural impairments, these results point out to the importance of protein carbonylation as a post-translational modification in the peroxynitrite-induced myosin functional impairment.     

An ionic–chemical–mechanical model for muscle contraction

The dynamic process underlying muscle contraction is the parallel sliding of thin actin filaments along an immobile thick myosin fiber powered by oar‐like movements of protruding myosin cross bridges (myosin heads). The free energy for functioning of the myosin nanomotor comes from the hydrolysis of ATP bound to the myosin heads. The unit step of translational movement is based on a mechanical‐chemical cycle involving ATP binding to myosin, hydrolysis of the bound ATP with ultimate release of the hydrolysis products, stress‐generating conformational changes in the myosin cross bridge, and relief of built‐up stress in the myosin power stroke. The cycle is regulated by a transition between weak and strong actin–myosin binding affinities. The dissociation of the weakly bound complex by addition of salt indicates the electrostatic basis for the weak affinity, while structural studies demonstrate that electrostatic interactions among negatively charged amino acid residues of actin and positively charged residues of myosin are involved in the strong binding interface. We therefore conjecture that intermediate states of increasing actin–myosin engagement during the weak‐to‐strong binding transition also involve electrostatic interactions. Methods of polymer solution physics have shown that the thin actin filament can be regarded in some of its aspects as a net negatively charged polyelectrolyte. Here we employ polyelectrolyte theory to suggest how actin–myosin electrostatic interactions might be of significance in the intermediate stages of binding, ensuring an engaged power stroke of the myosin motor that transmits force to the actin filament, and preventing the motor from getting stuck in a metastable pre‐power stroke state. We provide electrostatic force estimates that are in the pN range known to operate in the cycle.     

A model of muscle contraction based on the Langevin equation with actomyosin potentials

We propose a muscle contraction model that is essentially a model of the motion of myosin motors as described by a Langevin equation. This model involves one-dimensional numerical calculations wherein the total force is the sum of a viscous force proportional to the myosin head velocity, a white Gaussian noise produced by random forces and other potential forces originating from the actomyosin structure and intra-molecular charges. We calculate the velocity of a single myosin on an actin filament to be 4.9–49 μm/s, depending on the viscosity between the actomyosin molecules. A myosin filament with a hundred myosin heads is used to simulate the contractions of a half-sarcomere within the skeletal muscle. The force response due to a quick release in the isometric contraction is simulated using a process wherein crossbridges are changed forcibly from one state to another. In contrast, the force response to a quick stretch is simulated using purely mechanical characteristics. We simulate the force–velocity relation and energy efficiency in the isotonic contraction and adenosine triphosphate consumption. The simulation results are in good agreement with the experimental results. We show that the Langevin equation for the actomyosin potentials can be modified statistically to become an existing muscle model that uses Maxwell elements.     

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca²⁺ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca²⁺ saturation. A simple theory was developed to account for this fact.     

Ca2+ causes release of myosin heads from the thick filament surface on the milliseconds time scale

We have used electron microscopy to study the structural changes induced when myosin filaments are activated by Ca2+. Negative staining reveals that when Ca2+ binds to the heads of relaxed Ca2+ -regulated myosin filaments, the helically ordered myosin heads become disordered and project further from the filament surface. Cryo-electron microscopy of unstained, frozen-hydrated specimens supports this finding, and shows that disordering is reversible on removal of Ca2+. The structural change is thus a result of Ca2+ binding alone and not an artifact of staining. Comparison of the two techniques suggests that negative staining preserves the structure induced by Ca2+ -binding. We therefore used a time-resolved negative staining technique to determine the time scale of the structural change. Full disordering was observed within 30 ms of Ca2+ addition, and had started to occur within 10 ms, showing that the change occurs on the physiological time scale. Comparison with studies of single heavy meromyosin molecules suggests that an increased mobility of myosin heads induced by Ca2+ binding underlies the changes in filament structure that we observe. We conclude that the loosening of the array of myosin heads that occurs on activation is real and physiological; it may function to make activated myosin heads freer to contact actin filaments during muscle contraction.     

Cardiovascular adaptations to mechanical overload

The cardiac changes resulting from mechanical overload of the left ventricle have been well documented and a variety of compensatory mechanisms described. These include a decrease in maximum velocity (V₀) of shortening in the absence of reduction in active tension (P₀), and a reversible decrease in myofibrillar adenosine triphosphatase activity resulting from isoenzymic shift from, predominantly, a form of myosin with high ATPase activity (V₁) to another with low (V₃). The thermodynamic advantage of the transition is the hypertrophied muscle possesses a more energy-efficient form of contraction. These reversible transitions resulted from altered gene expression of isoenzymic forms of myosin heavy chain. It must be borne in mind that the adaptational modifications just described appear to occur only in smaller animals such as the rat, that possesses several myosin isozymes. In large mammals it is mainly the V₃ form of myosin that is present, which does not change with altered contractile state. Responses of the large arteries to hypertension have been poorly studied. This is surprising when one recalls that degenerative disease of such vessels, that include the aorta, carotids and ileo-femoral arteries is almost an obligatory concomitant of hypertension. Such studies as have been carried out indicate that hyperplasia is specific for abdominal aortic stenosis while hypertrophy is found in aortic smooth muscle in rats with systemic hypertension. Mechanically, an increase in V₀ with no change in P₀ have been reported; an increase in myofibrillar ATPase activity was also reported. Though two myosin heavy chain isozymes have been found in aortic smooth muscle densitometry did not reveal any difference in distribution between tissues from control and hypertensive rats. The cause of the increased ATPase activity must be in increased phosphorylation of the muscles' 20,000 dalton light chain.     

p116Rip promotes myosin phosphatase activity in airway smooth muscle cells

Myosin phosphatase-Rho interacting protein (p116^Rip) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116^Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116^Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116^Rip in cellular contractile processes, we suppressed the expression of p116^Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116^Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116^Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116^Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116^Rip expression in asthma pathogenesis.     

肌肉收缩的理论研究

基于肌球蛋白工作循环模型,从物理学的角度出发,利用化学动力学方法,给出肌动蛋白丝的动力学方程,讨论肌球蛋白的运力学行为,发现肌动蛋白运动呈锯齿状,并得到振动周期约为3.0s,与实验结果基本吻合。结论是宏观的肌肉运动是单分子运动的集体协同行为,为肌肉的运动训练和治疗提供理论参考。     

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction

Different interacting signaling modules involving Ca²⁺/calmodulin-dependent myosin light chain kinase, Ca²⁺-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K⁺-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.