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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The thyroid hormones have direct effects on vascular smooth muscle and are potent vasorelaxants. In the present study, the effects of d- and l-thyroxine (d-T4 and l-T4), 3,5,3'-triiodo-d-thyronine (d-T3), and 3,5,3'-triiodo-l-thyronine (l-T3) on the isolated mesenteric artery of the rabbit and superprecipitation of actomyosin from bovine aorta were examined. These thyroid hormones dose dependently relaxed vascular strips previously contracted with 50 mM KCl in the presence of phentolamine (1 microM), propranolol (1 microM), and atropine (0.3 microM), and the order of the inhibitory potency was l-T4 greater than d-T4 greater than l-T3 greater than d-T3 for the contraction. Pretreatment with l-T4 (10 and 30 microM) inhibited the contractile response concomitant with the inhibition of the 20,000-Da myosin light chain phosphorylation, without significant suppression of the increase in La3+-resistant 45Ca influx and uptake (5 and 30 min) induced by 50 mM KCl, suggesting that the inhibitory effect of l-T4 may not be primarily related to Ca2+ entry through the voltage-dependent Ca2+ channel. The l-T4 (10 and 30 microM) showed noncompetitive antagonism against the Ca2+-induced contraction in the high K+-depolarized vascular strips. Superprecipitation of actomyosin was inhibited by the addition of l-T4, in a dose-dependent manner, and calmodulin (1 microgram/ml) partly reversed the inhibitory effect of l-T4. Thyroid hormones were found to inhibit Ca2+/calmodulin-dependent smooth muscle myosin light chain kinase, and the Ki value for l-T4 was 2.5 microM. Although the concentrations of l-T4 used in this study are high, relative to circulating physiological levels, thyroid hormones act directly at the blood vessel wall to cause inhibition of the contractile process in vascular smooth muscle in vitro. Modulation of the 20,000-Da myosin light chain phosphorylation via the inhibition of myosin light chain kinase activity may at least in part contribute to the inhibitory effect of l-T4.
Mol Pharmacol 1989 Jun
PMID:Thyroid hormones directly interact with vascular smooth muscle strips. 273 94

We have found that phosphorylation of the 18,000 mol. wt protein in rat basophilic leukemia cells (RBL-2H3 cells) is enhanced by stimulation by an antigen. This phenomenon was also observed when cells were treated with phorbol myristate (TPA) and a calcium ionophor, A23187. The phosphorylated 18,000 mol. wt protein was mainly located in the membrane fraction. It was identified as one of the myosin light chains as follows: (1) the mol. wt of one of the major myosin light chains of RBL-2H3 cells was 18,000; (2) more than half of the phosphorylated 18,000 mol. wt protein was recovered in an actomyosin fraction; (3) this phosphorylated 18,000 mol. wt protein was immunoprecipitated with anti-myosin antibody. Since the presence of Ca2+ in the cell culture medium was essential for the phosphorylation of the 18,000 mol. wt protein and, since trifluoperazine (a potent inhibitor of calmodulin as well as of the degranulation process of RBL-2H3 cells) inhibited the reaction, the phosphorylation may be catalyzed by a Ca2+-calmodulin-dependent process, most likely by myosin light chain kinase. These results, together with our previous observation [Teshima et al. Molec Immun. 23, 279-284 (1986)], suggest that simultaneous phosphorylation of the 18,000 mol. wt myosin light chain and a 36,000 mol. wt membranous protein is a prerequisite for the degranulation of RBL-2H3 cells.
Mol Immunol 1989 Jul
PMID:Enhancement of the phosphorylation of membrane bound myosin light chain by antigen stimulation in rat basophilic leukemia cells. 277 87

Myosin was purified from chicken brush border cells to greater than 95% homogeneity and in a predominantly non-phosphorylated state. The effects of light chain phosphorylation by a Ca2+-calmodulin-dependent myosin light chain kinase on the conformational, enzymatic and filament assembly properties of this myosin were investigated. The actin-activated MgATPase activity of the non-phosphorylated myosin was low, and upon light chain phosphorylation an eight- to ninefold increase in this activity was observed, which was further potentiated by tropomyosin. Light chain phosphorylation was shown to control the assembly and disassembly of brush border myosin filaments. For example, turbidity measurements and electron microscopy demonstrated that MgATP disassembled non-phosphorylated myosin filaments; the disassembled myosin could reassemble when the light chains were phosphorylated, and could be disassembled again by dephosphorylating the light chains with phosphatase. In the electron microscope, the disassembled non-phosphorylated myosin molecules appeared in a folded conformation, and they were extended when phosphorylated. Proteolytic digestion was used to probe further the conformation of these folded and extended molecules, and their subunit organizations were characterized by a gel overlay technique. Quantitative analysis further demonstrated that light chain phosphorylation alters dramatically the monomer/polymer equilibrium of brush border myosin, shifting it towards filament formation. Comparison of analogous data for myosin from gizzard and thymus shows that each myosin has distinct solubility properties.
J Mol Biol 1986 Apr 05
PMID:Regulation in vitro of brush border myosin by light chain phosphorylation. 294 99

Ca2+-dependent myosin phosphorylation by Ca2+/calmodulin-dependent myosin light chain kinase (MLC-kinase) and protein kinase C were studied using selective inhibitors, isoquinolinesulfonamide derivatives. Both protein kinases were potently inhibited by 1-(8-chloro-5-isoquinolinesulfonyl)piperazine (HA-156) and its derivatives. Kinetic analysis indicated that HA-156 inhibited both enzymes competitively with respect to ATP, and Ki values of HA-156 for MLC-kinase and protein kinase C were 7.3 and 7.2 microM, respectively. To clarify molecular mechanisms of the isoquinolinesulfonamides to inhibit the Ca2+-dependent protein kinases, we examined the structure-activity relationships of HA-156 and its derivatives. The dechlorinated analogues, HA-100 and HA-142, markedly decreased the affinity for MLC-kinase, suggesting that the inhibitory effect of isoquinolinesulfonamide derivatives depends upon hydrophobicity of the compounds. There is a good correlation between MLC-kinase inhibition and hydrophobicity determined by reverse phase chromatography. In contrast, HA-140 and HA-142 showed weak inhibition of protein kinase C, suggesting that the electron density of the nitrogen in the isoquinoline ring of the compounds correlates with the potency to inhibit protein kinase C activity. These pairs of isoquinolinesulfonamides will aid in elucidating the biological roles of Ca2+-dependent myosin phosphorylation in intact cells. HA-156 and HA-140 inhibited myosin light chain phosphorylation in platelets exposed to collagen, whereas HA-142 and HA-100 did not, significantly. These isoquinolinesulfonamide derivatives should prove to be useful tools for distinguishing between the biological functions of Ca2+-activated, phospholipid-dependent, and Ca2+/calmodulin-dependent myosin light chain phosphorylation, in vivo.
Mol Pharmacol 1987 Jul
PMID:Selective modulation of calcium-dependent myosin phosphorylation by novel protein kinase inhibitors, isoquinolinesulfonamide derivatives. 295 13

We investigated systolic blood pressure (BP), ventricular myosin isoenzyme (MI) pattern, and myosin P-light chain phosphorylation (MP) of male and female normotensive (WKY) and spontaneously hypertensive rats (SHRSP). BP increased in SHRSP of both sexes during maturation. Male SHRSP reached a significantly higher BP (262 mmHg at week 64) than female SHRSP (217 mmHg at week 64). WKY remained at approximately 114 mmHg throughout the life-span investigated (5 to 64 weeks). MI pattern (expressed as %V1/%V3) shifted age-dependent to the V3 form: In female SHRSP MI pattern was 41/25 at week 18, 34/35 and 40/38 within week 22 to 32, and shifted to 18/53 until week 64. In male SHRSP MI pattern was 25/44 at week 18 and shifted gradually to 13/60 until week 53. MI patterns of WKY of both sexes were 100% V1 within week 5 to 12, shifted gradually to 51/23 and then remained constant until week 64. MP of the ventricle of female WKY and SHRSP was approximately 41% until week 52. At week 64, however, MP of female SHRSP decreased to 18% whereas female WKY remained at approximately 41%. MP of the ventricle of male WKY and SHRSP was approximately 38% until week 38. At week 44, however, MP of male SHRSP decreased to 22% whereas male WKY remained constant. Isometric tension generation of chemically skinned rat ventricular fibres increased after MP by calcium-calmodulin-dependent myosin light chain kinase. Both the shift to the V3 form and the decreased MP level might contribute to the development of cardiac failure in old SHRSP of both sexes.
J Mol Cell Cardiol 1988 Oct
PMID:Chronic hypertension changes myosin isoenzyme pattern and decreases myosin phosphorylation in the rat heart. 297 39

The enzyme, myosin light chain kinase, has been purified to homogeneity from bovine aortic vascular smooth muscle. Approximately 10 mg of enzyme could be obtained from 1 kg of fresh aortas with an overall yield of 26% of the original activity. The vascular myosin light chain kinase has a molecular weight of 160 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antiserum raised to the aortic myosin light chain kinase in rabbits strongly inhibited phosphotransferase activity. In addition, the antiserum was used to identify myosin kinase in a crude homogenate of vascular smooth muscle by radioimmunoblotting. A single species of the enzyme (Mr = 160 000) was identified. The bovine aortic myosin kinase could be phosphorylated by both cyclic AMP- and GMP-dependent protein kinases. Approximately 2 mols PO4/mole of enzyme could be incorporated by the cyclic AMP-dependent protein kinase in the absence of calmodulin. If Ca2+ and calmodulin were included in the reaction mixture, phosphate incorporation by the cyclic AMP-dependent protein kinase was reduced to 1 mol and phosphorylation by cyclic GMP-dependent protein kinase was completely inhibited. These results were confirmed by tryptic peptide mapping. Two distinct phosphopeptides were identified: site-1 and site-2. Both could be phosphorylated by the cyclic AMP-dependent protein kinase but only site-1 was phosphorylated by the cyclic GMP-dependent enzyme. In the presence of Ca2+ and calmodulin, phosphorylation by cAMP-dependent protein kinase was restricted to site-1. The effect of phosphorylation on myosin light chain kinase activity was determined. Only phosphorylation by cyclic AMP-dependent protein kinase was found to alter the requirement of myosin kinase for calmodulin. The K0.5 (i.e. the concentration of calmodulin required for half-maximal enzyme activation) for calmodulin was 5 nM for the unphosphorylated myosin kinase. With 2 mol PO4/mol myosin kinase incorporated, the K0.5 for calmodulin was increased to 82 nM. When only 1 mol PO4/mol myosin kinase was incorporated, no effect on calmodulin requirement was observed. Moreover, single site phosphorylation had no effect on other activity parameters, including Km for ATP and for light chains. Our studies suggest that cyclic AMP-dependent protein kinase may play an important role in the regulation of vascular myosin kinase activity. Moreover, our results indicate that cyclic GMP-dependent protein kinase does not affect calmodulin-activation of myosin kinase or several other activity parameters.
J Mol Cell Cardiol 1985 Sep
PMID:Phosphorylation of myosin light chain kinase from vascular smooth muscle by cAMP- and cGMP-dependent protein kinases. 299 88

The relationship between the functions of calmodulin (CaM) and Ca2+-induced smooth muscle contraction was investigated using a newly synthesized CaM antagonist, 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4- -phenylpiperidinyl)propyl]benzenesulfonamide (HT-74). We noted a selectivity of HT-74 for CaM, compared to other calcium-binding proteins and target enzymes of CaM. As HT-74 had no significant effect on the intensity of 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence in the presence of the Ca2+-CaM complex, the HT-74-binding sites may differ from those of naphthalenesulfonamides and phenothiazines which decrease ANS fluorescence. The Ca2+ binding to CaM was inhibited significantly by 1.0 microM HT-74, in sharp contrast to phenothiazines and naphthalenesulfonamides which increase the extent of the Ca2+ binding to CaM. Increasing CaM concentrations reversed the HT-74-induced inhibition of CaM-dependent enzymes such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase, with Ki values of 0.5 microM and 0.4 microM, respectively. In the presence of 0.3 microM HT-74, potassium-depolarized rabbit aortic strips pre-contracted with 0.3 mM CaCl2 relaxed, and this relaxation was completely reversed by the addition of an excess amount of CaCl2 (10 mM). This compound shifted the dose-response curve for CaCl2 to the right, in a competitive manner. However, HT-74 inhibited the phenylephrine-induced contraction elicited in Ca2+-free solution and the calcium ionophore A23187-induced contraction in the presence of calcium ion. Therefore, this agent affects intracellular actions of Ca2+ rather than membrane receptors or the influx of Ca2+. HT-74 is a CaM antagonist which binds to CaM in a manner different from that heretofore reported. It inhibits Ca2+ binding to CaM and produces a competitive inhibition of Ca2+-induced contractions of depolarized vascular smooth muscle.
Mol Pharmacol 1986 Mar
PMID:Modulation of calmodulin function and of Ca2+-induced smooth muscle contraction by the calmodulin antagonist, HT-74. 300 34

Ro 22-4839, a new cerebral circulation improver, has shown to be a potent calmodulin antagonist toward myosin light chain kinase (MLCK). It inhibited in vitro activity of calmodulin-activated cyclic AMP phosphodiesterase isolated from either bovine heart or brain and ATP-induced superprecipitation of chicken gizzard actomyosin with respective IC50 values of 20 microM, 17 microM, and 2.0 microM. The inhibitory action of Ro 22-4839 on the contractile system of the smooth muscle was demonstrated directly by its inhibition of chicken gizzard MLCK. Ro 22-4839 was found to potently inhibit MLCK with an IC50 value of 3.1 microM but was unable to inhibit the activity of MLCK rendered Ca2+/calmodulin independent by limited tryptic digestion. The inhibition of MLCK induced by Ro 22-4839 was completely overcome by addition of excess calmodulin. In contrast, Ro 22-4839 hardly inhibited calmodulin-activated Ca2+, Mg2+-ATPase from rat erythrocyte membrane or adenylate cyclase from rat brain. Use of hydrophobic fluorescence probes showed that Ro 22-4839 binds to the hydrophobic region of calmodulin like other calmodulin antagonists, trifluoperazine and W-7. However, the precise binding site of Ro 22-4839 to calmodulin is different from those of trifluoperazine and W-7, as suggested from differing IC50 values of these compounds against the probes. We conclude that Ro 22-4839 inhibits calmodulin-activated enzymes, most significantly of MLCK, highly specific to smooth muscle contractile systems by binding to the hydrophobic domain of the calmodulin and inducing its conformational change in the presence of calcium.
Mol Pharmacol 1987 Jul
PMID:Selective calmodulin inhibition toward myosin light chain kinase by a new cerebral circulation improver, Ro 22-4839. 303 98

Controversial views have been reported regarding the role of myosin light chain phosphorylation in the regulation of cardiac contractility (for review see. In the past, adenosine 5'-(-thio)triphosphate) (ATP gamma S) instead of ATP has frequently been used to study mechanical and biochemical consequences of myosin P-light chain (P-LC, LC-2) phosphorylation since thiophosphorylated sites are not significantly attacked by phosphatases. Unlike thiophosphorylation phosphorylation of myosin by myosin light chain kinase did neither decrease maximal (unloaded) shortening velocity of cardiac skinned fibres nor ATPase activity of cardiac myofibrils. We have accordingly investigated the phosphorylation pattern of purified cardiac myosin light chains using radioactive labeled ATP gamma S and ATP. We found that both the 28 kDa myosin light chain (LC-1) and the 18 kDa myosin light chain (LC-2, P-LC) were phosphorylated when ATP gamma S was present. In the presence of ATP, however, only LC-2 was found to be phosphorylated.
J Mol Cell Cardiol 1988 Jul
PMID:Different phosphorylation patterns of cardiac myosin light chains using ATP and ATP gamma S as substrates. 317 47

We investigated the effects of a newly synthesized compound, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor of superprecipitation of actomyosin, isometric tension development, and phosphorylation of the 20,000-Da myosin light chain (LC20) in vascular smooth muscle. Superprecipitation of actomyosin from bovine aorta was inhibited by the addition of ML-9 in a dose-dependent manner. In chemically skinned smooth muscles of the rabbit mesenteric artery, ML-9 inhibited the Ca2+-independent contraction provoked by application of trypsin-treated MLCK. In the intact rabbit mesenteric artery, increases in LC20 phosphorylation reached a maximal value of 0.49 mol of Pi/mol of LC20 within 10 sec from a resting value of 0.15 mol of Pi/mol of LC20 and then declined to near the basal level during the maintained isometric force developed in response to 50 mM KCl. Preincubation with 10-30 microM ML-9 for 30 min significantly inhibited both the maximal rate and extent of KCl-induced contraction and the phosphorylation of LC20, in a dose-dependent manner. There was a linear relationship between the initial rate of tension development and the extent of LC20 phosphorylation at 10 sec after stimulation. ML-9 nonspecifically antagonized the contraction induced by various contractile agonists, such as CaCl2, norepinephrine, serotonin, histamine, and angiotensin II. ML-9 dose dependently produced a shift to the right and down, in the dose-response curves, to all the agonists tested. These results suggest that ML-9 inhibits the actin-myosin interaction through the modulation of LC20 phosphorylation via the inhibition of MLCK activity. Thus, ML-9 may be a useful compound for investigating the physiologic role of myosin light chain phosphorylation by MLCK in living cells and tissues as well as in vitro.
Mol Pharmacol 1988 Jun
PMID:ML-9 inhibits the vascular contraction via the inhibition of myosin light chain phosphorylation. 338 76


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