Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca²⁺ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions     

Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Phosphorylation of myosin II regulatory light chains (RLC) by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Post-translational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPase-dependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 +/- 0. 21 mol of PO(4)/mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.     

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.     

Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca(2+) concentration ([Ca(2+)](i)). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca(2+)](i) with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.     

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.     

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus

KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca²⁺ channels that leads to increases in cytosolic free Ca²⁺ ([Ca²⁺]_i), Ca²⁺-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca²⁺ sensitization, the relationship between [Ca²⁺]_i and force produced by GPCR agonists. Studies done in the late 1980s demonstrated that a unique relationship between stimulus-induced [Ca²⁺]_i and force does not exist: for a given increase in [Ca²⁺]_i, GPCR activation can produce greater force than KCl, and relaxant agents can produce the opposite effect to cause Ca²⁺ desensitization. Such changes in Ca²⁺ sensitivity are now known to involve multiple cell signaling strategies, including translocation of proteins from cytosol to plasma membrane, and activation of enzymes, including RhoA kinase and protein kinase C. However, recent studies show that KCl can also cause Ca²⁺ sensitization involving translocation and activation of RhoA kinase. Rather than complicating the Ca²⁺ sensitivity story, this surprising finding is already providing novel insights into mechanisms regulating Ca²⁺ sensitivity of smooth muscle contraction. KCl as a "simple" stimulus promises to remain a standard tool for smooth muscle cell physiologists, whose focus is to understand mechanisms regulating Ca²⁺ sensitivity. K⁺ depolarization; cell signaling; signal transduction; contraction     

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.

     

Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca²⁺-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent K_i value of 3.4 µM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues. inhibitory peptide; calcium sensitization     

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     

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.     

Constitutive Phosphorylation of Cardiac Myosin Regulatory Light Chain in Vivo

In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca²⁺ sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.     

Selenoprotein N Was Required for the Regulation of Selenium on the Uterine Smooth Muscle Contraction in Mice

Selenium (Se) is an essential micronutrient affecting various aspects of health. The balance of the Se concentration has an important protective and promoter effect on physiological function in inducing muscular disorders in smooth muscle. Selenoprotein N (SelN) is closely related to Ca²⁺ release. The present study aimed to determine the effects and mechanism of action of dietary Se on uterine smooth muscle contraction via SelN using a mouse model. Quantitative polymerase chain reaction (qPCR) analysis was performed to detect mRNA levels. Western blotting was performed to detect protein levels. The results of the immunohistochemical analysis showed that Se had an effect on the uterine smooth muscle. The Se-supplement increased the release of Ca²⁺, Ca²⁺-calmodulin (CaM) expression, myosin light chain kinase (MLCK) expression, and myosin light chain (MLC) phosphorylation but did not affect ROCK and RhoA in uterine smooth muscle. Furthermore, the lack of Se showed an opposite impact. The effects of Se regulation were closely related to SelN. The interference of mouse SelN was performed on the uterine smooth muscle cell. Additionally, the results displayed the regulation of Se on the release of Ca²⁺, CaM expression, MLCK expression, and MLC phosphorylation were significant inhibited, and there was no effect on ROCK and RhoA. In conclusion, Se played an important role in regulating the process of contraction in uterine smooth muscle with SelN.     

Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation

This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 microm forskolin, 40 microm 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.     

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.

     

Signaling Processes for Initiating Smooth Muscle Contraction upon Neural Stimulation

Relationships among biochemical signaling processes involved in Ca²⁺/calmodulin (CaM)-dependent phosphorylation of smooth muscle myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) were determined. A genetically-encoded biosensor MLCK for measuring Ca²⁺-dependent CaM binding and activation was expressed in smooth muscles of transgenic mice. We performed real-time evaluations of the relationships among [Ca²⁺]_i, MLCK activation, and contraction in urinary bladder smooth muscle strips neurally stimulated for 3 s. Latencies for the onset of [Ca²⁺]_i and kinase activation were 55 ± 8 and 65 ± 6 ms, respectively. Both increased with RLC phosphorylation at 100 ms, whereas force latency was 109 ± 3 ms. [Ca²⁺]_i, kinase activation, and RLC phosphorylation responses were maximal by 1.2 s, whereas force increased more slowly to a maximal value at 3 s. A delayed temporal response between RLC phosphorylation and force is probably due to mechanical effects associated with elastic elements in the tissue. MLCK activation partially declined at 3 s of stimulation with no change in [Ca²⁺]_i and also declined more rapidly than [Ca²⁺]_i during relaxation. The apparent desensitization of MLCK to Ca²⁺ activation appears to be due to phosphorylation in its calmodulin binding segment. Phosphorylation of two myosin light chain phosphatase regulatory proteins (MYPT1 and CPI-17) or a protein implicated in strengthening membrane adhesion complexes for force transmission (paxillin) did not change during force development. Thus, neural stimulation leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation in phasic smooth muscle, showing a tightly coupled Ca²⁺ signaling complex as an elementary mechanism initiating contraction.Increases in [Ca²⁺]_i3 in smooth muscle cells lead to Ca²⁺/CaM-dependent MLCK activation and RLC phosphorylation. Phosphorylation of RLC increases actin-activated myosin MgATPase activity leading to myosin cross-bridge cycling with force development (1–3).The activation of smooth muscle contraction may be affected by multiple cellular processes. Previous investigations show that free Ca²⁺/CaM is limiting for kinase activation despite the abundance of total CaM (4–6). The extent of RLC phosphorylation is balanced by the actions of MLCK and myosin light chain phosphatase, which is composed of three distinct protein subunits (7). The myosin phosphatase targeting subunit, MYPT1, in smooth muscle binds to myosin filaments, thus targeting the 37-kDa catalytic subunit (type 1 serine/threonine phosphatase, PP1c) to phosphorylated RLC. RLC phosphorylation and muscle force may be regulated by additional signaling pathways involving phosphorylation of RLC by Ca²⁺-independent kinase(s) and inhibition of myosin light chain phosphatase, processes that increase the contraction response at fixed [Ca²⁺]_i (Ca²⁺-sensitization) (8–14). Many studies indicate that agonist-mediated Ca²⁺-sensitization most often reflects decreased myosin light chain phosphatase activity involving two major pathways including MYPT1 phosphorylation by a Rho kinase pathway and phosphorylation of CPI-17 by PKC (8, 14–16). Additionally, phosphorylation of MLCK in its calmodulin-binding sequence by a Ca²⁺/calmodulin-dependent kinase pathway has been implicated in Ca²⁺ desensitization of RLC phosphorylation (17–19). How these signaling pathways intersect the responses of the primary Ca²⁺/CaM pathway during physiological neural stimulation is not known.There is also evidence that smooth muscle contraction requires the polymerization of submembranous cytoskeletal actin filaments to strengthen membrane adhesion complexes involved in transmitting force between actin-myosin filaments and external force-transmitting structures (20–23). In tracheal smooth muscle, paxillin at membrane adhesions undergoes tyrosine phosphorylation in response to contractile stimulation by an agonist, and this phosphorylation increases concurrently with force development in response to agonist. Expression of nonphosphorylatable paxillin mutants in tracheal muscle suppresses acetylcholine-induced tyrosine phosphorylation of paxillin, tension development, and actin polymerization without affecting RLC phosphorylation (24, 25). Thus, paxillin phosphorylation may play an important role in tension development in smooth muscle independently of RLC phosphorylation and cross-bridge cycling.Specific models relating signaling mechanisms in the smooth muscle cell to contraction dynamics are limited when cells in tissues are stimulated slowly and asynchronously by agonist diffusing into the preparation. Field stimulation leading to the rapid release of neurotransmitters from nerves embedded in the tissue avoids these problems associated with agonist diffusion (26, 27). In urinary bladder smooth muscle, phasic contractions are brought about by the parasympathetic nervous system. Upon activation, parasympathetic nerve varicosities release the two neurotransmitters, acetylcholine and ATP, that bind to muscarinic and purinergic receptors, respectively. They cause smooth muscle contraction by inducing Ca²⁺ transients as elementary signals in the process of nerve-smooth muscle communication (28–30). We recently reported the development of a genetically encoded, CaM-sensor for activation of MLCK. The CaM-sensor MLCK contains short smooth muscle MLCK fused to two fluorophores, enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, linked by the MLCK calmodulin binding sequence (6, 14, 31). Upon dimerization, there is significant FRET from the donor enhanced cyan fluorescent protein to the acceptor enhanced yellow fluorescent protein. Ca²⁺/CaM binding dissociates the dimer resulting in a decrease in FRET intensity coincident with activation of kinase activity (31). Thus, CaM-sensor MLCK is capable of directly monitoring Ca²⁺/CaM binding and activation of the kinase in smooth muscle tissues where it is expressed specifically in smooth muscle cells of transgenic mice. We therefore combined neural stimulation with real-time measurements of [Ca²⁺]_i, MLCK activation, and force development in smooth muscle tissue from these mice. Additionally, RLC phosphorylation was measured precisely at specific times following neural stimulation in tissues frozen by a rapid-release electronic freezing device (26, 27). Results from these studies reveal that physiological stimulation of smooth muscle cells by neurotransmitter release leads to rapid increases in [Ca²⁺]_i, MLCK activation, and RLC phosphorylation at similar rates without the apparent activities of Ca²⁺-independent kinases, inhibition of myosin light chain phosphatase, or paxillin phosphorylation. Thus, the elemental processes for phasic smooth muscle contraction are represented by this tightly coupled Ca²⁺ signaling complex.     

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.

     

G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.     

Lysophosphatidic acid enhances collagen gel contraction by hepatic stellate cells: association with rho-kinase

We studied the effect of lysophosphatidic acid (LPA) on collagen gel contraction by cultured rat hepatic stellate cells (HSCs) in association with the function of Rho-kinase, one of the target molecules of small GTPase Rho. Binding studies showed a single class-binding site of LPA on HSCs. LPA enhanced the contraction of a collagen lattice seeded with HSCs. LPA increased the number of HSCs with polygonal morphology that contained actin stress fibers, and enhanced the phosphorylation of myosin light chain and the assembly of focal adhesion kinase and RhoA around fibronectin-coated beads seeded on HSCs. The electric cell-substrate impedance sensor system showed that LPA enhanced adhesion of HSC to extracellular substrate. All the effects of LPA were suppressed by Y-27632, Rho-kinase inhibitor. These data support the notion that LPA is involved in modulating HSC morphology, its attachment to surrounding extracellular matrix and its contraction by a mechanism involving Rho-kinase.     

Mathematical model of excitation-contraction in a uterine smooth muscle cell

Uterine contractility is generated by contractions of myometrial smooth muscle cells (SMCs) that compose most of the myometrial layer of the uterine wall. Calcium ion (Ca²⁺) entry into the cell can be initiated by depolarization of the cell membrane. The increase in the free Ca²⁺ concentration within the cell initiates a chain of reactions, which lead to formation of cross bridges between actin and myosin filaments, and thereby the cell contracts. During contraction the SMC shortens while it exerts forces on neighboring cells. A mathematical model of myometrial SMC contraction has been developed to study this process of excitation and contraction. The model can be used to describe the intracellular Ca²⁺ concentration and stress produced by the cell in response to depolarization of the cell membrane. The model accounts for the operation of three Ca²⁺ control mechanisms: voltage-operated Ca²⁺ channels, Ca²⁺ pumps, and Na⁺/Ca²⁺ exchangers. The processes of myosin light chain (MLC) phosphorylation and stress production are accounted for using the cross-bridge model of Hai and Murphy (Am J Physiol Cell Physiol 254: C99–C106, 1988) and are coupled to the Ca²⁺ concentration through the rate constant of myosin phosphorylation. Measurements of Ca²⁺, MLC phosphorylation, and force in contracting cells were used to set the model parameters and test its ability to predict the cell response to stimulation. The model has been used to reproduce results of voltage-clamp experiments performed in myometrial cells of pregnant rats as well as the results of simultaneous measurements of MLC phosphorylation and force production in human nonpregnant myometrial cells. cellular calcium control mechanisms; myometrial contractions; myosin light chain phosphorylation     

Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation

The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F- actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.