Phosphorylation In Smooth Muscle Research Articles

Propofol-induced changes in myosin light chain phosphorylation in smooth muscle are reversed by Okadaic Acid

Propofol-induced changes in myosin light chain phosphorylation in smooth muscle are reversed by Okadaic Acid

Signal Transduction in Lung Cells

Part 1 General concepts: molecular diversity and functional correlates in transmembrane signalling by G proteins, Lutz Birnbaumer et al regulation of G protein-linked receptors - analysis from the genome to the cell membrane, Craig C. Malbon and John R. Hadcock G proteins in pulmonary and cardiovascular signal transduction systems, Vsevolod A. Tkachuk et al small GTPases, Burton F. Dickey and Jeffrey B. Rubins phosphoinositides and intracellular calcium, Frank S. Menniti et al protein-tyrosine kinases, Timothy O'Hare et al protein tyrosine phosphatases - a multigene family of diverse structure and function, Deborah E. Cool purinergic signal transduction - extracellular ATP and adenosine as agonists for receptor-mediated signalling, George R. Dubyak. Part 2 Epithelial cells: epithelial cells Na+ and Cl- channels, Sadis Matalon et al protein phosphorylation and dephosphorylation systems in alveolar epithelial cells, David Warburton et al growth factors and pulmonary epithelia, Rita M. Ryan and Jacob N. Finkelstein expression and regulation of chemotactic cytokine genes by nonimmune pulmonary cells, Steven L. Kunkel et al cellular and biochemical mechanisms of airway epithelial mucin release, K. Chul Kim dysregulation of cellular genes by latent viral genes, Lois J. Geist and Gary W. Hunninghake. Part 3 Smooth muscle cells: role of calcium in the regulation of smooth muscle tone, Katheleen G. Morgan the role of myosin phosphorylation in smooth muscle and macrophage signalling, Primal de Lanerolle et al growth factors and signal transduction in smooth muscle systems, Morley D. Hollenberg. Part 4 Endothelial cells: ionic currents in the membranes of cultured endothelial cells, P. Bregestovski et al receptor-dependent regulation of [Ca2+] and phospholipase C in vascular endothelial cells, Pavel Avdonin et al secretion profiles of vasoactive amine-induced leukocyte chemoattractants - evidence for endothelial cell heterogeneity, Dennis J. Beer and Harrison W. Farber high-altitude pulmonary hypertension and signal transduction in the cardiovascular system, A.A. Aldashev. Part 5 Cells of the immune system: alveolar macrophage growth-factor signalling systems, James C. Bonner and Arnold R. Brody the role of the GTPase superfamily in T-lymphocyte signal transduction, Thomas C. Ryan and William W. Cruikshank mechanisms of mast cell signalling, Stephen I. Wasserman et al beta-adrenergic receptors on alveolar macrophages and lymphocytes - studies with cells from asthmatics, Sergey A. Rudchenko et al.

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

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

Expression of myosin regulatory light-chain isoforms and regulation of phosphorylation in smooth muscle

Our objectives were to 1) determine how growth state and cell density affect the expression of the smooth muscle (SM) and nonmuscle (NM) isoforms of the 20-kDa myosin regulatory light chains (MLC20) in cultured rat aortic smooth muscle cells (SMC) and 2) to determine whether angiotensin II stimulates differential phosphorylation of SM and NM MLC20 isoforms in an effort to assess whether the SM and NM isoforms may subserve different cellular functions. The results demonstrated that changes in the SM MLC20 isoform content were inversely correlated with cell growth but independent of cell density. MLC20 phosphorylation levels were 20.8 +/- 2.9 and 19.4 +/- 3.7% for SM and NM isoforms, respectively, in unstimulated, substrate-attached SMC. Angiotensin II transiently elevated phosphorylation levels of both the SM and NM MLC20 isoforms to 60-70%. No differences in either the magnitude or the kinetics of phosphorylation were observed for the SM vs. NM isoforms. Forskolin, 3-isobutyl-1-methylxanthine, or isoproterenol treatment led to parallel dephosphorylation of the SM- and NM-specific isoforms followed by depolymerization of stress fibers and cell arborization. The studies provide evidence that growth arrest of cultured SMC enhances expression of cell-specific/-selective proteins characteristic of differentiated SM. However, there was no evidence for differential phosphorylation changes of SM and NM MLC20 isoforms in response to activating or relaxing agents as expected if these isoforms subserve different cellular functions.

O-303 - Str and its derivatives inhibited MLC20 phosphorylation in smooth muscle.

O-303 - Str and its derivatives inhibited MLC20 phosphorylation in smooth muscle.

Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle.

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.

Protein phosphorylation in smooth muscle

Protein phosphorylation in smooth muscle

Regulation of myosin light chain phosphorylation in smooth muscle: James Stull, Daniel Fitzsimons, Patricia Gallagher, Paul Herring, Kristine Kamm, Da-Chun Tang, Malu Tansey, Ann Word. Department of Physiology, UT Southwestern, Dallas, TX 75235 USA

Regulation of myosin light chain phosphorylation in smooth muscle: James Stull, Daniel Fitzsimons, Patricia Gallagher, Paul Herring, Kristine Kamm, Da-Chun Tang, Malu Tansey, Ann Word. Department of Physiology, UT Southwestern, Dallas, TX 75235 USA

Modulation of the [Ca2+] sensitivity of myosin phosphorylation in intact swine arterial smooth muscle.

1. The [Ca2+] sensitivity of myosin light chain phosphorylation in vascular smooth muscle is dependent on the form of stimulation. Contractile agonist stimulation, when compared to high-KCl depolarization, is associated with an increase in [Ca2+] sensitivity of phosphorylation. I evaluated potential mechanisms for this stimulus-specific response by measuring aequorin-estimated myoplasmic [Ca2+], myosin phosphorylation, and isometric stress in swine carotid media. 2. The relative [Ca2+] sensitivity of phosphorylation depended on the type of stimulus (ranked high to low sensitivity): contractile agonists (histamine, phenylephrine) = endothelin (sustained contraction) = combination of histamine and NaF greater than NaF alone = endothelin (initial contraction) = combination of histamine and depolarization = combination of NaF and depolarization greater than depolarization = Bay K 8644 = combination of depolarization and low-dose phorbol diester. 3. Activation of L-type Ca2+ channels with Bay K 8644 induced a [Ca2+] sensitivity of phosphorylation similar to depolarization, suggesting that any other effects of high KCl (such as cellular swelling) were not responsible for the low [Ca2+] sensitivity of phosphorylation. 4. The addition of either histamine or NaF (an activator of G proteins) to depolarized tissues produced similar increases in the [Ca2+] sensitivity of phosphorylation, suggesting that NaF (possibly by activation of a G protein) can mimic contractile agonist-induced increases in the [Ca2+] sensitivity of phosphorylation. 5. Phorbol dibutyrate enhanced the contractile effect of depolarization, and this enhancement was primarily caused by increases in [Ca2+] rather than an alteration in the [Ca2+] sensitivity of phosphorylation. 6. These data suggest that the [Ca2+] sensitivity of phosphorylation in smooth muscle may be regulated by agonists (possible by G protein activation); however, the role of protein kinase C activation or depolarization induced Ca2+ compartmentalization requires further study.

O-061 - Effects of HA1077 on tension development and myosin light chain phosphorylation in smooth muscle

O-061 - Effects of HA1077 on tension development and myosin light chain phosphorylation in smooth muscle

Stimulus-specific patterns of myosin light chain phosphorylation in smooth muscle of rabbit thoracic artery

When the rabbit thoracic artery was stimulated with submaximal concentrations of agonist [40 mM K+, 30 microM prostaglandin F2 alpha (PGF2 alpha) or 7 microM histamine], about 90% of a maximal contraction occurred. Each agonist induced a rapid development of contraction followed by a sustained response. The maximal rate of force generation stimulated with PGF2 alpha was twice that seen with K+ or histamine. Stimulation with 40 mM K+ increased the extent of monophosphorylated 20 kDa myosin light chain (MLC-P) for up to 1 min to a maximal value of 38.8 +/- 1.0%, there was a subsequent rapid decrease and the MLC-P level remained just above the basal value for 40 min (6.8 +/- 3.0%). In the case of stimulation with 7 microM histamine, MLC-P level increased rapidly and was sustained for up to 40 min (28.0 +/- 4.9%). In contrast to the stimulation with K+ or histamine, PGF2 alpha induced both mono- and diphosphorylated MLC20 (MLC-P and MLC-P2 respectively) at a low concentration (3 microM). The monophosphorylation of MLC20 induced by 30 microM PGF2 alpha reached the maximal value of 32.8 +/- 5.2%, and was sustained for up to 40 min (15.2 +/- 5.4%). The diphosphorylation of MLC20 increased rapidly (7.4 +/- 4.0% at 5 min), then decreased to the basal value within 40 min. These results suggest that different modes of stimulation of smooth muscle contraction produce different profiles of MLC20 phosphorylation. The implications of these observations are that the diphosphorylated form, specifically induced by certain agents, may modify the mode of contraction of the aortic artery.

Phosphatase inhibition with okadaic acid does not alter the relationship between force and myosin light chain phosphorylation in permeabilized smooth muscle

The phosphatase inhibitor, okadaic acid, has been used to test the hypothesis that myosin light chain phosphatase activity plays a central role in latchbridge formation in smooth muscle. In the permeabilized rabbit portal vein there is a non-linear relationship between myosin light chain phosphorylation and force production such that maximum force output occurs with about 50% phosphorylation. Treatment of the muscle with okadaic acid does not change this relationship even though there is a profound inhibition of phosphatase activity. The data suggest that dephosphorylation of the myosin light chain while the myosin is in the force producing state does not account for the high force output with low levels of light chain phosphorylation in smooth muscle.

Myosin light chain and heavy chain phosphorylation in smooth muscle: potential regulatory roles for calcium, phospholipids and cyclic nucleotides.

Myosin light chain and heavy chain phosphorylation in smooth muscle: potential regulatory roles for calcium, phospholipids and cyclic nucleotides.

Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle.

We have proposed a model that incorporates a dephosphorylated "latch bridge" to explain the mechanics and energetics of smooth muscle. Cross-bridge phosphorylation is proposed as a prerequisite for cross-bridge attachment and rapid cycling. Features of the model are 1) myosin light chain kinase and phosphatase can act on both free and attached cross bridges, 2) dephosphorylation of an attached phosphorylated cross bridge produces a noncycling "latch bridge," and 3) latch bridges have a slow detachment rate. This model quantitatively predicts the latch state: stress maintenance with reduced phosphorylation, cross-bridge cycling rates, and ATP consumption. In this study, we adapted A. F. Huxley's formulation of crossbridge cycling (A. F. Huxley, Progr. Biophys. Mol. Biol. 7: 255-318, 1957) to the latch-bridge model to predict the relationship between isotonic shortening velocity and phosphorylation. The model successfully predicted the linear dependence of maximum shortening velocity at zero external load (V0) on phosphorylation, as well as the family of stress-velocity curves determined at different times during a contraction when phosphorylation values varied. The model implies that it is unnecessary to invoke an internal load or multiple regulatory mechanisms to explain regulation of V0 in smooth muscle.

Effect of dinitrophenol and anoxia on isometric tension in rabbit colon smooth muscle.

In a previous study, it was suggested that inhibition of oxidative phosphorylation in smooth muscle is accompanied by a release of Ca2+ from mitochondria (Pettersson 1983); in this investigation, the effects of 2,4-dinitrophenol (DNP) and anoxia on isometric tension in pieces of smooth muscle from rabbit colon were studied. Addition of DNP to the bathing medium elicited a transient and dose-dependent contraction with an ED50 value of 2 X 10(-4) M. The contraction was not inhibited by pretreatment with atropine (1 X 10(-6) M). The removal of external Ca2+ did not reduce the contracting action of DNP. Addition of the local anaesthetic D-mepivacaine (1 X 10(-3) M), which reduces the Ca2+ efflux through the plasma membrane of the smooth muscle cells, augmented the response to DNP. Gassing the bathing medium with 95% N2 + 5% CO2 (anoxia) for 60 min. did not affect the basal tension, but markedly reduced the contractile response to DNP. The results indicate that DNP increased cytoplasmic Ca2+ significantly enough to induce a contraction of rabbit colon smooth muscle by a large release from an intracellular store, probably from the mitochondria. Since anoxia did not mimic the contracting action of DNP, it is suggested that a high concentration of DNP would be a more effective stimulus for mitochondrial Ca2+ release than anoxia.

The function of myosin and myosin light chain kinase phosphorylation in smooth muscle.

Due to the massive data sets available for drug candidates, modern drug discovery has advanced to the big data era. Central to this shift is the development of artificial intelligence approaches to implementing innovative modeling based on the dynamic, ...Read More

Adenosine and 5‘-chloro-5‘-deoxyadenosine inhibit the phosphorylation of phosphatidylinositol and myosin light chain in calf aorta smooth muscle.

Smooth muscle from calf aorta is homogenized and centrifuged. The insoluble material is subjected to sucrose density gradient centrifugation. When the heaviest fraction so obtained is incubated with radioactive ATP, two components incorporate most of the acid-insoluble radioactivity. One is a phosphoprotein with a molecular weight of 21,000. It has been identified as myosin light chain by its molecular weight, isoelectric point, and precipitation by antibody to calf aorta myosin. Its phosphorylation is strongly inhibited by EGTA, in agreement with published reports that myosin light chain kinase of smooth muscle is Ca2+ dependent. The other product is of low molecular weight, is extracted into acidic chloroform-methanol, and has been identified as phosphatidylinositol 4-phosphate. Adenosine and 5'-chloro-5'-deoxyadenosine, which are vasodilators, inhibit the phosphorylation of both substrates. Phosphorylation of phosphatidylinositol is inhibited at lower concentrations of the nucleosides than is the phosphorylation of myosin light chain. The inhibitory effects of the two nucleosides are not associated with changes in the concentration of cyclic AMP. The precise function of phosphatidylinositol phosphorylation in smooth muscle is not known, but correlations between smooth muscle contraction and increased turnover of phosphatidylinositol and its mono- and diphosphates have been reported. Myosin light chain is phosphorylated under conditions which favor smooth muscle contraction. We conclude that the inhibitory effects of adenosine described here are consistent with their physiological action as vasodilators.

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase.

In smooth muscle the Mr 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of Mr 17,000 that binds 4 moles of Ca2+; and a larger protein of Mr circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca2+. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of Mr 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of Mr 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca2+-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.

Studies on the effect of phosphorylation of the 20,000 Mr light chain of vertebrate smooth muscle myosin

Myosin was rapidly prepared from turkey gizzard muscle to a high level of purity, in high yield and in a non-phosphorylated state. It was consistently observed that the actin-activated Mg2+ ATPase activity of this myosin was dependent on the level of phosphorylation of the 20,000 Mr light chain, for example, in the non-phosphorylated state, the myosin Mg2+ ATPase activity was not activated by actin whereas, when the light chains were phosphorylated, the Mg2+ ATPase activity of the myosin was activated approximately ninefold by actin. Using the "desensitized" scallop myosin test system (Kendrick-Jones et al., 1976; Sellers et al., 1980) it was further demonstrated that phosphorylation of the 20,000 Mr gizzard light chain has a regulatory role. These results also suggest that the regulatory mechanisms mediated by smooth muscle myosin light chains and molluscan myosin regulatory light chains are similar, i.e. in the absence of Ca2+, both types of light chain inhibit myosin interaction with actin and this inhibition is relieved by either phosphorylation in smooth muscle or by direct calcium binding in molluscan myosins. The basis of regulation exerted by these light chains is therefore repression derepression. Using a variety of techniques, i.e. turbidity measurements, quantitative high speed centrifugation, electron microscopy and dark field light microscopy, it was observed that the stability of gizzard myosin filaments at approximately physiological conditions (0.15 M-NaCl, 1 mM-MgATP, pH 7.0) was dependent on the level of light chain phosphorylation. Using purified calmodulin-dependent light chain kinase and phosphatase, it was further shown that these gizzard myosin filaments can be reversibly assembled and disassembled as a result of phosphorylation-dephosphorylation of the 20,000 Mr light chain.