Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Thus far, determining the relative contribution of Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and Ca²⁺-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca²⁺, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO₄/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO₄/mol RLC, respectively, within 2.5 min. Ca²⁺ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation     

Regulation of endothelial cell gap formation and barrier dysfunction: Role of myosin light chain phosphorylation

Endothelial cell (EC) contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. We tested the hypothesis that phosphorylation of regulatory myosin light chains (MLC) by Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) is critical to EC barrier dysfunction elicited by thrombin. Thrombin stimulated a rapid (<15 sec) increase in [Ca²⁺]_i which preceded maximal MLC phosphorylation (60 sec) with a 6 to 8-fold increase above constitutive levels of phosphorylated MLC. Dramatic cellular shape changes indicative of contraction and gap formation were observed at 5 min with maximal increases in albumin permeability occurring by 10 min. Neither the Ca²⁺ ionophore, A23187, nor phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), alone or in combination, produced MLC phosphorylation. The combination was synergistic, however, in stimulating EC contraction/gap formation and barrier dysfunction (3 to 4-fold increase). Down-regulation or inhibition of PKC activity attenuated thrombin-induced MLC phosphorylation (～40% inhibition) and both thrombin- and PMA-induced albumin clearance (～50% inhibition). Agents which augmented [cAMP]_i partially blocked thrombin-induced MLC phosphorylation (～50%) and completely inhibited both thrombin- and PMA-induced EC permeability (100% inhibition). Furthermore, cAMP produced significant reduction in the basal levels of constitutive MLC phosphorylation. Finally, MLCK inhibition (with either ML-7 or KT 5926) or Ca²⁺/calmodulin antagonism (with either trifluoperazine or W-7) attenuated thrombin-induced MLC phosphorylation and barrier dysfunction. These results suggest a model wherein EC contractile events, gap formation and barrier dysfunction occur via MLCK-dependent and independent mechanisms and are significantly modulated by both PKC and cAMP-dependent protein kinase A activities. © 1995 Wiley-Liss, Inc.     

Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K⁺, the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca²⁺ signal, and removal of external Ca²⁺ promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca²⁺ did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na⁺-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na⁺-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca²⁺ signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport. membrane potential; Na⁺-alanine cotransport; epithelium; phosphatidylinositol 3-kinase; LLC-PK₁ cells     

Neutrophil transepithelial migration: evidence for sequential,contact-dependent signaling events and enhanced paracellular permeability independent of transjunctional migration

Active migration of polymorphonuclear leukocytes (PMN) through the intestinal crypt epithelium is a hallmark of inflammatory bowel disease and correlates with patient symptoms. Previous in vitro studies have shown that PMN transepithelial migration results in increased epithelial permeability. In this study, we modeled PMN transepithelial migration across T84 monolayers and demonstrated that enhanced paracellular permeability to small solutes occurred in the absence of transepithelial migration but required both PMN contact with the epithelial cell basolateral membrane and a transepithelial chemotactic gradient. Early events that occurred before PMN entering the paracellular space included increased permeability to small solutes (<500 Da), enhanced phosphorylation of regulatory myosin L chain, and other as yet undefined proteins at the level of the tight junction. No redistribution or loss of tight junction proteins was detected in these monolayers. Late events, occurring during actual PMN transepithelial migration, included redistribution of epithelial serine-phosphorylated proteins from the cytoplasm to the nucleus in cells adjacent to migrating PMN. Changes in phosphorylation of multiple proteins were observed in whole cell lysates prepared from PMN-stimulated epithelial cells. We propose that regulation of PMN transepithelial migration is mediated, in part, by sequential signaling events between migrating PMN and the epithelium.     

Signaling through Myosin Light Chain Kinase in Smooth Muscles

Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca²⁺/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca²⁺ sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.     

Novel interaction of cortactin with endothelial cell myosin light chain kinase

Inflammatory mediators such as thrombin evoke increases in vascular permeability through activation of endothelial contractile mechanisms which involve increased levels of MLC phosphorylation catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK). We previously noted that the high molecular weight endothelial MLCK isoform (EC MLCK) is stably associated with a complex containing p60(src) and 80kDa cortactin, an actin-binding protein and known p60(src) target. In this study we have utilized in vitro binding assays to confirm specific interaction between EC MLCK and cortactin. Tyrosine phosphorylation of either EC MLCK (Y(464), Y(471)) or cortactin (Y(421), Y(466), and Y(482)) by p60(src) significantly increased this direct association. Site-specific antibody and peptide studies subsequently confirmed EC MLCK AA #972-979 and 1019-1025 as sites of cortactin interaction. EC MLCK-cortactin interaction in vitro failed to modulate MLCK enzymatic activity but appeared to inhibit EC MLCK binding to F-actin, while EC MLCK abolished cortactin-mediated augmentation of Arp2/3-stimulated actin polymerization. These data suggest that cortactin-EC MLCK interaction may be a novel determinant of endothelial cortical actin-based cytoskeletal rearrangement.     

Biochemistry of smooth muscle myosin light chain kinase

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca²⁺-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.     

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.     

Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability

Tumor necrosis factor-alpha (TNF-alpha) is known to induce changes in endothelial cell morphology and permeability, but the mechanisms have not been extensively characterized. TNF-alpha rapidly induced RhoA activation and myosin light chain phosphorylation, but caused only small changes to cortical F-actin, without significantly increasing paracellular permeability up to 30 min after stimulation. TNF-alpha subsequently caused a progressive increase in permeability and in stress fiber reorganization, cell elongation, and intercellular gap formation over 8-24 h. Consistent with the increased permeability, Occludin and JAM-A were removed from tight junctions and ZO-1 was partially redistributed. Rho/ROCK but not MLCK inhibition prevented the long-term TNF-alpha-induced changes in F-actin and cell morphology, but ROCK inhibition did not affect permeability. These results suggest that the gradual increase in permeability induced by TNF-alpha does not reflect contractile mechanisms mediated by Rho, ROCK, and MLCK, but involves long-term reorganization of tight junction proteins.     

Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.     

ROCK mediates thrombin's endothelial barrier dysfunction

Thrombin-induced endothelial monolayer hyperpermeability is thought toresult from increased F-actin stress fiber-related contractile tension,a process regulated by the small GTP-binding protein Rho. We testedwhether this process was dependent on the Rho-associated proteinkinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects ofY-27632 on thrombin-induced myosin light chain phosphorylation (MLCP)and tyrosine phosphorylation of p125 focal adhesion kinase(p125^FAK) and paxillin were measured by Western blotting.F-actin organization and content were analyzed by digital imaging, andendothelial monolayer permeability was measured in bovine pulmonaryartery endothelial cell (EC) monolayers using a size-selectivepermeability assay. Y-27632 enhanced EC monolayer barrier function dueto a decline in small-pore number that was associated with increased ECsurface area, reduced F-actin content, and reorganization of F-actin to-catenin-containing cell-cell adherens junctions. Although Y-27632prevented thrombin-induced MLCP, stress fiber formation, and theincreased phosphotyrosine content of paxillin and p125^FAK,it attenuated but did not prevent the thrombin-induced formation oflarge paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to bepartially ROCK dependent.

     

Regulation of arterial tone by smooth muscle myosin type II

Theinitiation of contractile force in arterial smooth muscle (SM) isbelieved to be regulated by the intracellular Ca²⁺concentration and SM myosin type II phosphorylation. We tested thehypothesis that SM myosin type II operates as a molecular motor proteinin electromechanical, but not in protein kinase C (PKC)-induced,contraction of small resistance-sized cerebral arteries. We utilized aSM type II myosin heavy chain (MHC) knockout mouse model and measuredarterial wall Ca²⁺ concentration([Ca²⁺]_i) and the diameter of pressurizedcerebral arteries (30-100 µm) by means of digital fluorescencevideo imaging. Intravasal pressure elevation caused a graded[Ca²⁺]_i increase and constricted cerebralarteries of neonatal wild-type mice by 20-30%. In contrast,intravasal pressure elevation caused a graded increase of[Ca²⁺]_i without constriction in (/)MHC-deficient arteries. KCl (60 mM) induced a further[Ca²⁺]_i increase but failed to inducevasoconstriction of (/) MHC-deficient cerebral arteries. Activationof PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM)induced a strong, sustained constriction of (/) MHC-deficientcerebral arteries without changing [Ca²⁺]_i.These results demonstrate a major role for SM type II myosin in thedevelopment of myogenic tone and Ca²⁺-dependentconstriction of resistance-sized cerebral arteries. In contrast, thesustained contractile response did not depend on myosin andintracellular Ca²⁺ but instead depended on PKC. We suggestthat SM myosin type II operates as a molecular motor protein in thedevelopment of myogenic tone but not in pharmacomechanical coupling byPKC in cerebral arteries. Thus PKC-dependent phosphorylation ofcytoskeletal proteins may be responsible for sustained contraction invascular SM.

     

Mutation analysis of the non-muscle myosin light chain kinase (MLCK) deletion constructs on CV1 fibroblast contractile activity and proliferation

Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(19) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated growth rate of mutant MLCK-transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility.     

Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125(FAK)), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.     

Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability

The actomyosincomplex is the major cytoskeletal component that controls cellcontraction. In this study, we investigated the effects of actomyosininteraction on endothelial barrier function and gap formation.Activated myosin light chain kinase (MLCK) protein was transferred intocoronary venular endothelial cell (CVEC) monolayers. Uptake of theactivated protein resulted in a significant shift in myosin light chain(MLC) from an unphosphorylated to a diphosphorylated form. In addition,MLCK induced a hyperpermeability response of the monolayer as measuredby albumin transendothelial flux. Microscopic examination ofMLCK-treated CVECs revealed widespread gap formation in the monolayer,loss of peripheral -catenin, and increases in actin stress fibers.Inhibition of all of the above responses by a specific MLCK inhibitorsuggests they are the direct result of exogenously added MLCK. Thesedata suggest that activation of MLCK in CVECs causes phosphorylation ofMLC and contraction of CVECs, resulting in gap formation andconcomitant increases in permeability. This study uses a noveltechnique to measure the effects of an activated kinase on both itssubstrate and cellular morphology and function through directtransference into endothelial cells.

     

A Calcium Signaling Cascade Essential for Myosin Thick Filament Assembly in Xenopus Myocytes

Spontaneous calcium release from intracellular stores occurs during myofibrillogenesis, the process of sarcomeric protein assembly in striated muscle. Preventing these Ca²⁺ transients disrupts sarcomere formation, but the signal transduction cascade has not been identified. Here we report that specific blockade of Ca²⁺ release from the ryanodine receptor (RyR) activated Ca²⁺ store blocks transients and disrupts myosin thick filament (A band) assembly. Inhibition of an embryonic Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) by blocking the ATP-binding site, by allosteric phosphorylation, or by intracellular delivery of a pseudosubstrate peptide, also disrupts sarcomeric organization. The results indicate that both RyRs and MLCK, which have well-described calcium signaling roles in mature muscle contraction, have essential developmental roles during construction of the contractile apparatus.     

Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation

Fibroblasts form fibers when grown inculture medium containing native type 1 collagen. The contractileforces generated can be precisely quantified and used to analyze thesignal transduction pathways regulating fibroblast contraction. Calfserum (30%) induces a sustained contraction that is accompanied by atransient increase in intracellular calcium([Ca²⁺]_i). W-7, a calmodulin inhibitor,KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, andML-7, a myosin light-chain kinase inhibitor, had no effects on eitherthe contraction or the [Ca²⁺]_i responses.Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, aprotein kinase C inhibitor, had major effects on force or[Ca²⁺]_i. In contrast, the Rho kinaseinhibitors(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca²⁺]_iresponse. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did notalter mono- or diphosphorylation of the myosin regulatory light chain(MRLC) compared with control untreated fibers. These results suggestthat the sustained contraction of NIH 3T3 fibroblast fibers induced bycalf serum is mediated by Rho kinase but is independent of a sustainedincrease in [Ca²⁺]_i, calcium/calmodulin- orprotein kinase C-dependent pathways, or increases in MRLC phosphorylation.

     

Length-dependent modulation of smooth muscle activation: effects of agonist, cytochalasin, and temperature

We tested thehypothesis that mechanical strain modulates agonist sensitivity ofsmooth muscle by measuring myosin phosphorylation and contractile forcein bovine tracheal smooth muscle activated by various concentrations ofthe muscarinic receptor agonist carbachol and at various musclelengths. Increasing carbachol concentration by 10,000-fold did notrestore myosin phosphorylation levels at shorter muscle lengths to thelevel at optimal length(L_o). Maximum levels of myosin phosphorylation induced by carbachol at 0.6, 0.8, and1.0 L_o weresimilar but became lower at <0.6L_o. Cytochalasin D significantly attenuated carbachol-induced contraction by 54%. Inaddition, cytochalasin D treatment induced a parallel downward shift inthe length-myosin phosphorylation relation. Lowering temperature from37 to 23°C did not significantly change the length dependencies ofcarbachol-induced active force and myosin phosphorylation. Theseresults have led us to conclude that1) agonist sensitivity and maximumlevel of activation (as measured by myosin phosphorylation) are targetsof length-dependent modulation, 2)actin filaments involved in contraction and length-dependent modulationare distinct in sensitivity to cytochalasin D, and3) length-dependent modulation isrelatively temperature insensitive.

     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Rho activation in excitatory agonist-stimulated vascular smooth muscle

Small GTPase Rho and its downstream effector, Rho kinase, havebeen implicated in agonist-stimulated Ca²⁺ sensitization of20-kDa myosin light chain (MLC₂₀) phosphorylation andcontraction in smooth muscle. In the present study we demonstrated forthe first time that excitatory receptor agonists induce increases inamounts of an active GTP-bound form of RhoA, GTP-RhoA, in rabbit aorticsmooth muscle. Using a pull-down assay with a recombinant RhoA-bindingprotein, Rhotekin, we found that a thromboxane A₂ mimetic,U-46619, which induced a sustained contractile response, induced asustained rise in the amount of GTP-RhoA in a dose-dependent mannerwith an EC₅₀ value similar to that for the contractile response. U-46619-induced RhoA activation was thromboxaneA₂ receptor-mediated and reversible. Other agonistsincluding norepinephrine, serotonin, histamine, and endothelin-1 (ET-1)also stimulated RhoA, albeit to lesser extents than U-46619. Incontrast, ANG II and phorbol 12,13-dibutyrate failed to increaseGTP-RhoA. The tyrosine kinase inhibitor genistein substantiallyinhibited RhoA activation by these agonists, except for ET-1. Thusexcitatory agonists induce Rho activation in an agonist-specificmanner, which is thought to contribute to stimulation ofMLC₂₀ phosphorylation Ca²⁺ sensitivity.