Inhibition Of Myosin Light Chain Phosphorylation Research Articles

Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction.

Activation of cyclic nucleotide-dependent signaling pathways leads to relaxation of bovine carotid artery smooth muscle contractions and is associated with increased phosphorylation of the small heat shock-related protein (HSP20). Previous reports have shown that human umbilical artery smooth muscle is uniquely resistant to cyclic nucleotide-dependent relaxation, and HSP20 is not phosphorylated. In this investigation, we determined the phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. In carotid artery, activation of cyclic nucleotide-dependent signaling pathways inhibited contractile responses to serotonin but did not inhibit myosin light chain phosphorylation or oxygen consumption. The inhibition of contraction was associated with increases in HSP20 phosphorylation. In umbilical artery, activation of cyclic nucleotide-dependent signaling pathways did not inhibit serotonin-induced contraction or myosin light chain phosphorylation. The lack of contractile inhibition in umbilical artery was not associated with significant increases in HSP20 phosphorylation. In conclusion, cyclic nucleotide-dependent contractile inhibition is independent of the inhibition of myosin light chain phosphorylation or oxygen consumption but does correlate with increased HSP20 phosphorylation.

Stellettamide-A, a novel inhibitor of calmodulin, isolated from a marine sponge.

1. Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K+(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC50 of 88 microM. 2. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca2+ (3 and 10 microM)-induced contractions with an IC50 of 46 microM for 3 microM Ca2+ and 105 microM for 10 microM Ca2+. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca2+. ST-A had no effect on this contraction. 3. ST-A inhibited Mg2+-ATPase activity in native actomyosin prepared from chicken gizzard with an IC50 of 25 microM. 4. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC50 of 152 microM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin. 5. ST-A inhibited calmodulin activity, assessed by Ca2+/calmodulin-dependent enzymes, (Ca2+-Mg2+)-ATPase of erythrocyte membrane, with an IC50 of 100 microM and phosphodiesterase prepared from bovine cardiac muscle with an IC50 of 52 microM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration. 6. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3-300 microM). Similar results were obtained in the presence or absence of Ca2+ suggesting that ST-A binds to calmodulin and that Ca2+ is not essential for the binding of ST-A to calmodulin. 7. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.

Myosin light chain phosphorylation: modulation of basal and agonist-stimulated venular permeability.

We have previously demonstrated that agonists increase microvascular permeability through a phospholipase C-nitric oxide synthase-guanylate cyclase cascade. The aim of this study was to further investigate the downstream end of the signaling pathway with a focus on myosin light chain (MLC) phosphorylation. The apparent permeability coefficient to albumin was measured in isolated coronary venules. Under control conditions, the nitric oxide donor sodium nitroprusside, as well as the guanosine 3',5'-cyclic monophosphate-dependent protein kinase (PKG) activator 8-bromoguanosine 3',5'-cyclic monophosphate, increased venular permeability two- to threefold. Similarly, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly elevated permeability. Inhibition of MLC phosphorylation with ML-7 significantly attenuated the hyperpermeability responses to the agonists. Furthermore, ML-7 dose dependently reduced basal venular permeability. Consistently, inhibition of dephosphorylation with the protein phosphatase inhibitor calyculin dramatically increased basal permeability. These results suggest that 1) PKG and PKC play an important signaling role in the regulation of endothelial barrier function and 2) MLC phosphorylation contributes to basal and agonist-stimulated microvascular permeability.

Effects of a myosin light chain kinase inhibitor, wortmannin, on cytoplasmic Ca2+ levels, myosin light chain phosphorylation and force in vascular smooth muscle.

Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 2157-2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 microM) decreased MLC phosphorylation and the amplitude of contractions induced by high K+ (72.7 mM) to a level seen at rest. This occurred without a change in cytosolic Ca2+ levels ([Ca2+]i). In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 microM) and prostaglandin F2 alpha (PGF2 alpha, 10 microM) without a change in the [Ca2+]i. On the other hand, wortmannin (1 or 10 microM) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF2 alpha to a level seen at rest. In the absence of external Ca2+, caffeine (20 mM) induced a transient increase in [Ca2+]i and force with an increase in MLC phosphorylation. Wortmannin completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [Ca2+]i. In the absence of external Ca2+, phenylephrine induced a small transient increase in [Ca2+]i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca2+]i and MLC phosphorylation. Wortmannin (1 microM) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca2+]i nor the sustained contraction. Wortmannin inhibited the Ca2(+)-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the Ca(2+)-independent, ATP-induced contraction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entry of Ca2+ or through the release of Ca2+ from the sarcoplasmic reticulum. The results also suggest that the activation of receptors by norepinephrine and PGF2 alpha induces a contraction via a MLC phosphorylation-independent pathway or through a pathway which is dependent on the resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.

Inhibitory effect of forskolin on myosin phosphorylation-dependent and independent contractions in bovine tracheal smooth muscle.

In bovine tracheal smooth muscle, carbachol (CCh, 1 microM) and high K+ (72.7 mM) induced sustained increases in cytosolic Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction. Forskolin (FK, 1-10 microM) inhibited the CCh-induced increase in [Ca2+]i, MLC phosphorylation and force in parallel. In contrast, FK inhibited the high K(+)-induced contraction and MLC phosphorylation without changing [Ca2+]i. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), CCh (10 microM) and caffeine (20 mM) induced transient increase in [Ca2+]i and contractile force by releasing Ca2+ from cellular store. FK strongly inhibited the CCh-induced Ca2+ transient, but failed to inhibit the caffeine-induced Ca2+ transient. In the absence of external Ca2+, 12-deoxyphorbol 13-isobutylate (DPB, 1 microM) induced sustained contraction without increase in [Ca2+]i and MLC phosphorylation. FK inhibited this contraction without changing [Ca2+]i. In permeabilized muscle, Ca2+ induced contraction in a concentration-dependent manner. FK (10 microM) and cAMP (1-100 microM) shifted the Ca(2+)-force curve to the higher Ca2+ levels. CCh with GTP, GTP gamma S or DPB enhanced contraction in the presence of constant level of Ca2+. Forskolin and cAMP also inhibited the enhanced contractions in the permeabilized muscle. In the permeabilized, thiophosphorylated muscle, ATP induced contraction in the absence of Ca2+. cAMP (300 microM) had no effect on this contraction. These results suggest that forskolin inhibits agonist-induced contraction in tracheal smooth muscle by multiple mechanisms of action; 1) inhibition of MLC phosphorylation by reducing Ca2+ influx and Ca2+ release, 2) inhibition of MLC phosphorylation by changing the MLC kinase/phosphatase balance, and 3) inhibition of regulatory mechanism which is not dependent on MLC phosphorylation.

Staurosporine inhibits STA2-induced platelet aggregation by inhibition of myosin light-chain phosphorylation in rabbit washed platelets.

Annals of the New York Academy of SciencesVolume 714, Issue 1 p. 315-317 Staurosporine Inhibits STA2-Induced Platelet Aggregation by Inhibition of Myosin Light-Chain Phosphorylation in Rabbit Washed Platelets SHIZUKO TAKANO, SHIZUKO TAKANO Department of Pharmacology Fukushima Medical College Fukushima 960–12, JapanSearch for more papers by this author SHIZUKO TAKANO, SHIZUKO TAKANO Department of Pharmacology Fukushima Medical College Fukushima 960–12, JapanSearch for more papers by this author First published: April 1994 https://doi.org/10.1111/j.1749-6632.1994.tb12066.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume714, Issue1Platelet–dependent Vascular OcclusionApril 1994Pages 315-317 RelatedInformation

Effects of 2,3-butanedione monoxime on activation of contraction and crossbridge kinetics in intact and chemically skinned smooth muscle fibres from guinea pig taenia coli.

The effects of 2,3-butanedione monoxime (BDM) were studied in smooth muscle fibres from guinea pig taenia coli. In intact muscle, active force during contractions induced by high-K+ was inhibited by about 10% in 1 mM BDM and by approximately 70% in 10 mM BDM. Intracellular [Ca2+] during contraction, measured with the fura-2 technique, was reduced in the presence of BDM. The reduction in force and [Ca2+] in the presence of 1 and 10 mM BDM could be reproduced by reduction in extracellular Ca2+, suggesting that BDM influences the Ca2+ entry or release. In skinned muscle preparations, BDM decreased the Ca2+ sensitivity of active force. This change could be explained by a decreased level of myosin light chain phosphorylation. In fibres maximally activated by thiophosphorylation, the effect of BDM on force occurred at higher concentrations; 10 mM gave no reduction of force and 60 mM 15% reduction. The maximal shortening velocity (Vmax) and force were unaffected by 30 mM BDM in thiophosphorylated muscle and decreased almost in parallel in Ca(2+)-activated contractions. The present results suggest that BDM inhibits myosin light chain phosphorylation, directly decreases force generation at the crossbridge level and inhibits the Ca2+ translocation in smooth muscle. The effect on force in skinned fibres is observed at higher BDM concentrations than those reported to be required for inhibition of force in striated muscle. The inhibition of force in intact smooth muscle could be explained by an influence on Ca2+ translocation.

Inhibition of calmodulin and protein kinase C by amiodarone and other class III antiarrhythmic agents

Class III antiarrhythmic agents may prolong refractoriness via modulation of ion channels, which may be sensitive to Ca2+ regulatory proteins or enzymes. Accordingly, the purpose of this study was to quantitate the effects of several structurally diverse class III antiarrhythmic agents on calmodulin-regulated enzymes and protein kinase C activity, and to evaluate the ability of these agents and known calmodulin antagonists to prolong cardiac refractoriness in vivo. The rank order of potency (IC50;microM) of selected class III antiarrhythmic agents and reference calmodulin antagonists as inhibitors of calmodulin-regulated phosphodiesterase activity were: calmidazolium (0.12 microM) greater than amiodarone (0.62 microM) greater than desethylamiodarone (1.5 microM) greater than trifluoperazine (4.3 microM), bepridil (5 microM) greater than W-7 (7.5 microM), clofilium (13 microM). Similar concentration-related inhibition was evident in a second calmodulin-regulated system, inhibition of myosin light-chain phosphorylation and superprecipitation of arterial actomyosin. Sotalol and tetraethylammonium were inactive at 100 microM. Protein kinase C activity was also inhibited by some of these agents; desethylamiodarone (IC50 = 11 microM) was more potent than the reference agent, H-7 (IC50 = 79 microM), or amiodarone (38% inhibition at 100 microM) and clofilium (32% inhibition at 100 microM). In vivo, the minimally effective doses required to increase ventricular effective refractory periods in paced guinea pigs were (in mg/kg) bepridil, sotalol [1] greater than clofilium [3] greater than amiodarone [10] greater than W-7, desethylamiodarone [20]. No changes in refractory period were noted with maximum testable doses of calmidazolium or trifluoperazine. These studies show that some, but not all, class III antiarrhythmic agents are effective and potent calmodulin antagonists or protein kinase C inhibitors. Moreover, some calmodulin antagonists are effective at prolonging refractoriness in vivo. However, a lack of correlation between these agents suggests that these mechanisms are not solely responsible for the prolongation of refractoriness of all class III agents.

Direct inhibition of contractile apparatus by analogues of amiloride in the smooth muscle of guinea-pig taenia caecum and chicken gizzard

The relaxant effects of amiloride and its analogues, benzamil, 5-( N', N'-diethyl)-amiloride (DEAM) and 5-( N-ethyl- N-isopropyl)-amiloride (EIAM), were investigated using smooth muscle of guinea-pig taenia caeci and chicken gizzard. High K +-induced contractions of intact taenia and gizzard were inhibited by these compounds (1–100 μM) with the order of potency; benzamil ≥ EIAM > DEAM > amiloride. Contractions of permealized taenia and gizzard were also inhibited by these compounds at concentrations 8–35 times higher than those needed to inhibit the contractions of intact tissues. These compounds inhibited 20 K myosin light chain (MLC) phosphorylation at the concentrations needed to inhibit the contraction in the permealized muscles. Calmodulin (CaM) activity, as monitored by erythrocyte membrane (Ca 2+ + Mg 2+)-ATPase and phosphodiesterase activities, was inhibited by DEAM and EIAM at similar concentrations as those to inhibit the MLC phosphorylation. Benzamil also inhibited CaM activity at concentrations 4–8 times higher than those required to inhibit MLC phosphorylation. However, amiloride failed to inhibit CaM activity. Among these compounds, amiloride and benzamil inhibited Ca 2+/CaM-independent MLC phosphorylation due to trypsin-treated MLC kinase. Taenia tissue gradually accumulated these compounds and the tissue/medium ratio exceeded 3.5–17 after a 3-hr incubation period. These results indicate that amiloride and its analogues inhibit smooth muscle contraction mainly by the direct inhibition of MLC phosphorylation. The inhibitory effect of amiloride may be attributable to the inhibition of MLC kinase, whereas the inhibitory effect of DEAM and EIAM may largely be attributable to the inhibition of CaM. Benzamil may inhibit contraction by the inhibition of both MLC kinase and CaM. Differences in the drug-sensitivity between intact and permealized tissues may be attributable to the difference in drug accumulation by the cell.

Structural Variants of Verapamil and W-7 with Combined Ca2+ Entry Blockade/Myosin Phosphorylation Inhibitory Mechanisms

Arylalkylsulfonamides, identified as Wy-46,622 and Wy-47,324, combine pharmacological properties of the Ca2+ entry blocker verapamil and the calmodulin antagonist W-7. These agents directly inhibit arterial actin-myosin interactions via inhibition of myosin light chain (MLC) phosphorylation in actomyosin. Potencies of both Wy-46,622 (IC50 = 26 microM) and Wy-47,324 (IC50 = 18 microM) are greater than W-7 (IC50 = 35 microM); verapamil is inactive at 100 microM. Both Wy-46,622 and Wy-47,324 are less potent than verapamil, but more potent than W-7, at inhibiting K+-depolarized force development in paced rabbit atria. At higher concentrations (30 microM) which inhibit MLC phosphorylation, Wy-46,622 and Wy-47,324 are either equal to or more efficacious than verapamil in inhibiting receptor-mediated contractions in intact porcine coronary (histamine, serotonin, prostaglandin F2 alpha, carbocyclic thromboxane A2) or guinea pig aortic (leukotriene C4) smooth muscle. Both Wy-47,324 (IC50 = 16 microM) and Wy-46,622 (IC50 = 23 microM) are inhibitors of the second phase of epinephrine-induced human platelet aggregation; Wy-47,324 is more potent than verapamil or W-7 (IC50 for each = 29 microM). These results suggest that these agents possess combined Ca2+ entry blocker/MLC phosphorylation inhibitory mechanisms. Furthermore, they are more vascular specific than verapamil, and are effective inhibitors of intracellular Ca2+-mediated events in vascular smooth muscle and platelets.

Chemical modification of lysine and arginine residues in the myosin regulatory light chain inhibits phosphorylation

The contribution of lysine and arginine residues to the substrate specificity of the myosin light-chain kinase has been studied using chemically modified myosin light chains. Succinylation of maleylation of the myosin light chains caused complete inhibition of their phosphorylation. Modification of 50% of the lysine residues resulted in 90% inhibition of phosphorylation and this was accompanied by a 25-fold increase in the apparent K m. In contrast, phosphorylation of the myosin light chains by the cAMP-dependent protein kinase was relatively insensitive to lysine modification, with only a 15% reduction in phosphorylation following succinylation of 50% of the lysine residues. Treatment with either cyclohexane-1,2-dione or camphorquinone-10-sulfonic acid resulted in between 90 and 98% inhibition of myosin light-chain phosphorylation. These reagents caused modification of both lysine and arginine residues, and accordingly only part of the inhibition can be attributed to arginine modification. Modification of all of the cysteine and methionine residues caused only a 40% inhibition of phosphorylation. The results of this study support the concept that lysine and arginine residues act as essential specificity determinants for the myosin light-chain kinase in protein substrates.

Smooth muscle myosin kinase requires residues on the COOH-terminal side of the phosphorylation site. Peptide inhibitors.

The COOH-terminal residue in peptide analogs of the phosphorylation site sequence in smooth muscle myosin light chains, Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -(P)Asn20-Val21-Phe22-Ala23, were shown to have a strong influence on the kinetics of peptide phosphorylation. The peptides 11-19, 11-20, 11-21, 11-22, and 11-23 were all phosphorylated by the myosin light chain kinase with similar apparent Km values in the range 11-17 microM. The Vmax varied 40-fold, with the peptides 11-19, 11-20, 11-21, 11-22, and 11-23 having Vmax values of 0.035, 0.045, 0.32, 1.74, and 1.43 mumol X min-1 X mg-1 respectively. These results indicated that Ala23 was not essential whereas Phe22 and Val21 had a strong influence on the Vmax of peptide phosphorylation. This series of peptides competitively inhibited myosin light chain phosphorylation with Ki values similar to their respective Km values. Peptide 11-19 had a Ki value of approximately 10 microM and a Vmax less than 0.1% of the value with myosin light chains and is therefore an effective inhibitor of the smooth muscle myosin kinase.

Mechanism of Smooth Muscle Contraction

The phosphorylation hypothesis of smooth muscle contraction relates formation of cyclic AMP, resulting from beta-adrenergic stimulation, to inhibition of myosin light-chain phosphorylation and hence to bronchial relaxation. The hypothesis can therefore explain why beta-adrenergic blockade promotes bronchospasm in susceptible individuals. In the light of this molecular schema, various novel approaches to the use of beta-adrenergic blocking therapy for hypertension in the presence of asthma are discussed.

Role of phosphorylation in mediating the association of myosin with the cytoskeletal structures of human platelets.

The effect of myosin light chain phosphorylation on the association of myosin with the cytoskeletal structures of platelets was quantitated. In unstimulated platelets, little myosin light chain was phosphorylated and myosin remained in solution when cytoskeletons from Triton X-100 lysates of platelets were sedimented by centrifugation. In platelets activated by thrombin, the calcium ionophore A23187, or collagen, the rate and extent of myosin light chain phosphorylation paralleled the association of myosin with platelet cytoskeletal structures. Dephosphorylation of myosin light chain and myosin dissociation from the cytoskeleton occurred at comparable rates at longer times after addition of the stimulating agents to platelets. Quantitation of radioactive phosphate in the cytoskeleton-associated myosin and in the soluble myosin showed that the phosphorylated myosin light chain was selectively isolated with the Triton-insoluble cytoskeletons, whereas nonphosphorylated myosin was not associated. Inhibition of the light chain kinase with the calmodulin antagonist trifluoperazine inhibited myosin light chain phosphorylation and incorporation of myosin into the platelet cytoskeletons. Inhibition of light chain phosphorylation by prostaglandin E1 and prostacyclin produced similar effects. Thus, phosphorylation of the myosin light chain stabilizes the association of myosin with the contractile structures within platelets.