Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.     

Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors

Signaling mechanisms forstretch-dependent growth and differentiation of vascular smooth musclewere investigated in mechanically loaded rat portal veins in organculture. Stretch-dependent protein synthesis was found to depend onendogenous release of angiotensin II. Autoradiography after[³⁵S]methionine incorporation revealed stretch-dependentsynthesis of several proteins, of which SM22 and actin wereparticularly prominent. Inhibition of RhoA activity by cell-permeant C3toxin increased tissue mechanical compliance and reducedstretch-dependent extracellular signal-regulated kinase (ERK)1/2activation, growth, and synthesis of actin and SM22, suggesting a roleof the actin cytoskeleton. In contrast, inhibition of Rho-associatedkinase by Y-27632 did not reduce ERK1/2 phosphorylation or actin and SM22 synthesis and did not affect tissue mechanical compliance butstill inhibited overall growth. The actin polymerization inhibitors latrunculin B and cytochalasin D both inhibited growth and caused increased tissue compliance. Whereas latrunculin Bconcentration-dependently reduced actin and SM22 synthesis,cytochalasin D did so at low (10⁸ M) but not at high(10⁶ M) concentration. The results show that stretchstabilizes the contractile smooth muscle phenotype. Stretch-dependentdifferentiation marker expression requires an intact cytoskeleton forstretch sensing, control of protein expression via the level ofunpolymerized G-actin, or both.

     

Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization

Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins gamma-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, >15 min; cofilin-P, >1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.     

Myosin and actin from ascidian smooth muscle and their interaction

Myosin and actin were purified from ascidian smooth muscle. Ascidian myosin contained two classes of light chains and the pH dependence of Ca2+-activated ATPase and the KCl dependence of actin-activated ATPase of ascidian myosin differed from those of vertebrate skeletal myosin. Troponin-tropomyosin complex from ascidian increased the ATPase activity of ascidian reconstituted actomyosin in a Ca2+-dependent manner. Ascidian myosin provided the reconstituted actomyosin with the responsiveness to calcium ions. Two actin isoforms were present in ascidian, which were distinguished by isoelectric points.     

Electron microscopic study of actin polymerization in airway smooth muscle

Actin polymerization as part of the normal smooth muscle response to various stimuli has been reported. The actin dynamics are believed to be necessary for cytoskeletal remodeling in smooth muscle in its adaptation to external stress and strain and for maintenance of optimal contractility. We have shown in our previous studies in airway smooth muscle that myosins polymerized in response to contractile activation as well as to adaptation at longer cell lengths. We postulated that the same response could be elicited from actins under the same conditions. In the present study, actin filament formation was quantified electron microscopically in cell cross sections. Nanometer resolution allowed us to examine regional distribution of filaments in a cell cross section. Airway smooth muscle bundles were fixed in relaxed and activated states at two lengths; muscle preparations were also fixed after a period of oscillatory strain, a condition known to cause depolymerization of myosin filaments. The results indicate that contractile activation and increased cell length nonsynergistically enhanced actin polymerization; the extent of actin polymerization was substantially less than that of myosin polymerization. Oscillatory strain increased thin filament formation. Although thin filament density was found higher in cytoplasmic areas near dense bodies, contractile activation did not preferentially enhance actin polymerization in these areas. It is concluded that actin thin filaments are dynamic structures whose length and number are regulated by the cell in response to changes in extracellular environment and that polymerization and depolymerization of thin filaments occur uniformly across the whole cell cross section.     

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.     

Myosin heavy-chain isoforms in adult and developing rabbit vascular smooth muscle

Two monoclonal antibodies specific for smooth muscle myosin (designated SM-E7 and SM-A9) and one monoclonal anti-(human platelet myosin) antibody (designated NM-G2) have been used to study myosin heavy chain composition of smooth muscle cells in adult and in developing rabbit aorta. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting experiments revealed that adult aortic muscle consisted of two myosin heavy chains (MCH) of smooth muscle type, named MHC-1 (205 kDa), and MHC-2 (200 kDa). In the fetal/neonatal stage of development, vascular smooth muscle was found to contain only MHC-1 but not MHC-2. Non-muscle myosin heavy chain, which showed the same electrophoretic mobility as the slower migrating MHC, was expressed in an inverse manner with respect to MHC-2, i.e. it was detectable only in the early stages of development. The distinct pattern of smooth and non-muscle myosin isoform expression during development may be related to the different functional properties of smooth muscle cells during vascular myogenesis.     

Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells

In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105, 82, 79, 77, 73, 45 and 40 kDa in time- and dose-dependent manners. Other vasoconstrictors such as [Arg]vasopressin, 5-hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C-activating phorbol ester, phorbol 12 myristate 13-acetate, and the Ca2+ ionophore, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.     

Permanently proliferating rat vascular smooth muscle cell with maintained expression of smooth muscle characteristics, including actin of the vascular smooth muscle type

Cells of an established clonal line (RVF-SMC) derived from rat vena cava are described by light and electron microscope methods and biochemical analysis of the major proteins. The cells are flat, and they moderately elongate and form monolayers. They are characterized by prominent cables of microfilaments bundles decoratable with antibodies to actin and alpha-actinin. These bundles contain numerous densely stained bodies and are often flanked by typical rows of surface caveolae and vesicles. The cells are rich in intermediate-sized filaments of the vimentin type but do not show detectable amounts of desmin and cytokeratin filaments. Isoelectric focusing and protein chemical studies have revealed actin heterogeneity. In addition to the two cytoplasmic actins, beta and gamma, common to proliferating cells, two smooth muscle-type actins (an acidic alpha-like and a gamma-like) are found. The major (alpha-type) vascular smooth muscle actin accounts for 28% of the total cellular actin. No skeletal muscle or cardiac muscle actin has been detected. The synthesis of large amounts of actin and vimentin and the presence of at least three actins, including alpha- like actin, have also been demonstrated by in vitro translation of isolated poly(A)+ mRNAs. This is, to our knowledge, the first case of expression of smooth muscle-type actin in a permanently growing cell. We conclude that permanent cell growth and proliferation is compatible with the maintained expression of several characteristic cell features of the differentiated vascular smooth muscle cell including the formation of smooth muscle-type actin.     

Caveolin regulates microtubule polymerization in the vascular smooth muscle cells

Microtubule and caveolin have common properties in intracellular trafficking and the regulation of cellular growth. Overexpression of caveolin in vascular smooth muscle cells increased the polymer form of microtubule without changing in the total amount of tubulin, and downregulation of caveolin decreased the polymer form of microtubule. Fractionation of cellular proteins followed by immunodetection as well as immunostaining of caveolin and microtubule revealed that caveolin and a portion of microtubule were co-localized in caveolar fractions. A caveolin scaffolding domain peptide, which mimics caveolin function, did not alter the polymerization of microtubule in vitro, but dramatically inhibited the depolymerization of microtubule induced by stathmin, a microtubule destabilizing protein, which was also found in caveolar fractions. Accordingly, it is most likely that caveolin increased the polymer form of microtubule through the inhibition of a microtubule destabilizer, stathmin, suggesting a novel role of caveolin in regulating cellular network and trafficking.     

Integrin-linked kinase regulates N-WASp-mediated actin polymerization and tension development in tracheal smooth muscle

The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a beta-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and alpha-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and alpha-parvin form a stable cytosolic complex and that the ILK.PINCH.alpha-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with beta-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK.PINCH.alpha-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle.     

Myosin filament structure in vertebrate smooth muscle

The in vivo structure of the myosin filaments in vertebrate smooth muscle is unknown. Evidence from purified smooth muscle myosin and from some studies of intact smooth muscle suggests that they may have a nonhelical, side-polar arrangement of crossbridges. However, the bipolar, helical structure characteristic of myosin filaments in striated muscle has not been disproved for smooth muscle. We have used EM to investigate this question in a functionally diverse group of smooth muscles (from the vascular, gastrointestinal, reproductive, and visual systems) from mammalian, amphibian, and avian species. Intact muscle under physiological conditions, rapidly frozen and then freeze substituted, shows many myosin filaments with a square backbone in transverse profile. Transverse sections of fixed, chemically skinned muscles also show square backbones and, in addition, reveal projections (crossbridges) on only two opposite sides of the square. Filaments gently isolated from skinned smooth muscles and observed by negative staining show crossbridges with a 14.5-nm repeat projecting in opposite directions on opposite sides of the filament. Such filaments subjected to low ionic strength conditions show bare filament ends and an antiparallel arrangement of myosin tails along the length of the filament. All of these observations are consistent with a side-polar structure and argue against a bipolar, helical crossbridge arrangement. We conclude that myosin filaments in all smooth muscles, regardless of function, are likely to be side-polar. Such a structure could be an important factor in the ability of smooth muscles to contract by large amounts.     

Nonmuscle Myosin motor of smooth muscle

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371-375). Intracellular calcium was measured in urinary bladders from SM-MHC-deficient and SM-MHC-expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC-deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC-deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC-deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC-expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.     

Myosin heavy-chain isoforms in human smooth muscle

The myosin heavy-chain composition of human smooth muscle has been investigated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, enzyme immunoassay, and enzyme-immunoblotting procedures. A polyclonal and a monoclonal antibody specific for smooth muscle myosin heavy chains were used in this study. The two antibodies were unreactive with sarcomeric myosin heavy chains and with platelet myosin heavy chain on enzyme immunoassay and immunoblots, and stained smooth muscle cells but not non-muscle cells in cryosections and cultures processed for indirect immunofluorescence. Two myosin heavy-chain isoforms, designated MHC-1 and MHC-2 (205 kDa and 200 kDa, respectively) were reactive with both antibodies on immunoblots of pyrophosphate extracts from different smooth muscles (arteries, veins, intestinal wall, myometrium) electrophoresed in 4% polyacrylamide gels. In the pulmonary artery, a third myosin heavy-chain isoform (MHC-3, 190 kDa) electrophoretically and antigenically distinguishable from human platelet myosin heavy chain, was specifically recognized by the monoclonal antibody. Analysis of muscle samples, directly solubilized in a sodium dodecyl sulfate solution, and degradation experiments performed on pyrophosphate extracts ruled out the possibility that MHC-3 is a proteolytic artefact. Polypeptides of identical electrophoretic mobility were also present in the other smooth muscle preparations, but were unreactive with this antibody. The presence of three myosin heavy-chain isoforms in the pulmonary artery may be related to the unique physiological properties displayed by the smooth muscle of this artery.