Smooth muscle activation and regulation

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Smooth muscle activation and regulation

• Actin polymerization and depolymerization

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Organization of smooth muscle cells

• Contractile apparatus actin and myosin
  filaments
• The ratio of thin to thick filaments is much
  higher in smooth muscle (151) than in skeletal
  muscle (61).
• Cytoskeleton compartment actin filaments and
  intermediate filaments
• Desmin and vimentin two major intermediate
  filament proteins
• The sarcomeric structure is poorly understood due
  to the lack of visible cross striations within
  the cells in the light or electron microscope.

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Small and Gimona model
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Components of contractile apparatus and
cytoskeleton
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The model of malleable sarcomeric structure of
contractile filament
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Smooth muscle contraction

• Sharing the mechanism of the sliding-filament,
  cross-bridge cycling as proposed in striated
  muscle.
• Optimize the contractile filament overlap at any
  cell length within its physiological range and to
  maintain the ability to generate maximal force
  over that length range.
• Contractile activation involves cytoskeleton
  rearrangements
• Dynamic remodeling of the actin filament lattice
  within cellular microdomains in response to local
  mechanical and pharmacological events enables
  the cell to maintain its external environment.
• As the contraction occurs, the cytoskeletal
  lattice stabilizes, solidifies, and forms a rigid
  structure for transmission of tension generated
  by the interaction of myosin and actin. The
  integrated molecular transitions take place
  within the contractile cycle.

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Thick filament regulation of smooth muscle
contraction

• The Ca2-dependent phosphorylation/dephosphorylati
  on of MRLC by myosin light chain kinase (MLCK)/
  myosin light chain phosphatase (MLCP) is thought
  to control the contraction-relaxation cycle of
  smooth muscle.
• Smooth muscle myosin similar to striated muscle
  counterpart contains two heavy chains (MHC) and
  two pairs of light chains, one 20-kD regulatory
  light chains (MRLC) and the other 17-kD essential
  light chains (MELC).
• Activated by Ca2-calmodulin, MLCK causes
  phosphorylation of serine-19 and/or threonine-18
  at MRLC, and MRLC phosphorylation increases
  myosins actin-activated ATPase activity at least
  100-fold
• Once phosphorylated, the myosin cross-bridge can
  bind to actin, generating force by cross-bridge
  cycling.
• On the other hand, dephosphorylation is brought
  about by MLCP.

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Thin filament regulation of smooth muscle
contraction

• The demonstration of Ca2 sensitivity of myosin
  ATPase activity in preparations containing
  skeletal muscle myosin and smooth muscle thin
  filament
• Reports that cross-bridge cycling rates can vary
  without detectable changes in MRLC
  phosphorylation
• Reports of dissociations between MRLC
  phosphorylation and tension
• Under some conditions, unphosphorylated
  cross-bridges are not completely turned off,
  which indicated that force is also regulated by
  thin filament associated proteins

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Actins

• At least six tissue-specific isoforms
  a-skeletal, a-cardiac, and a-vascular g-enteric
  and g-cytoplasmic and b-cytoplasmic actins
• Highly homologous in their primary structure
  with most sequence differences in the N-terminal
  region of the molecule. This part of the molecule
  is not functionally involved in filament assembly
  but is the site for interaction with various
  actin binding proteins
• The a-, b- and g- isoforms can be resolved by
  isoelectric focusing, proceeding from the most
  acidic to the least acidic
• The smooth muscle actins have been associated
  with the contractile filaments, whereas the
  cytoplasmic actins have been associated with the
  non- contractile cytoskeleton and sub-
  plasmalemma cortex.

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Tropomyosin (TM)

• From four highly conserved genes a-, b-, g-,
  d-TM , via alternative splicing, give rise to
  more than 40 isoforms
• a-TM is a major isoform in smooth muscle with
  minor b-TM to form a homo- or hetero-dimer that
  binds along the major groove of actin filament in
  a head-to-tail manner
• In vitro studies have implicated TMs in the
  stabilization of the actin cytoskeleton by
  protecting actin filaments from the severing
  action of gelsolin and the depolymerizing action
  of ADF/cofilin
• X-ray studies have suggested that the activation
  of smooth muscle leads to the movement of TM in a
  manner similar to that in striated muscle
• The movement of smooth muscle TM by myosin
  binding is more easily facilitated by
  phosphorylated myosin than unphosphorylated
  myosin, providing for possible cross talk between
  thick and thin filament regulation.
• TM is necessary for full inhibition of actomyosin
  ATPase activity by caldesmon

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Caldesmon (CaD)

• CaD is an actin, Tm, myosin, and calmodulin (CaM)
  binding protein.
• Two isoforms are produced by a single gene
  through alternative splicing to generate a smooth
  muscle form, h-CaD with high molecular weight of
  130140 kD and a non-muscle l-CaD with low
  molecular weight of 6090 kD
• The difference between h- and l-CaD is a highly
  charged repeating sequence, corresponding to a 35
  nm-long single helical region that separates the
  N-terminal domain from the C-terminal domain of
  h-CaD.

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CaD

• An elongated protein containing two relatively
  compact domains at the N- and the C-terminal ends
  
• The C-terminal domains are responsible for actin
  binding and inhibition of myosin ATPase activity
  in vitro.
• Binding of CaM or phosphorylation of sites
  between the two C-terminal actin binding domains
  can reverse some of the inhibitory actions of CaD
  in vitro.
• The N-terminal half of the molecule has been
  shown to bind myosin and, in vitro, tether myosin
  to actin in conjunction with C-terminal actin
  binding domains of CaD

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Domain structure of CaD
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Calponin (CaP)

• CaP is an actin binding protein that also binds
  to CaM, myosin, desmin, and phospholipids
• There are three different isoforms encoded by
  separate genes and expressed in smooth muscle (h1
  basic CaP), cardiac muscle (h2 neutral CaP), and
  non-muscle cells (acidic CaP), respectively.
• In smooth muscle, CaP interacts with F-actin and
  inhibits the actomyosin Mg-ATPase activity in
  vitro. When phosphorylated in vitro by either
  CaMKII or PKC, the inhibitory action of CaP on
  the actin-myosin interaction is reversed
• Smooth muscle CaP is an elongated molecule (MW
  32 kD) of 18 nm long
• The domain structure containing a CaP homology
  domain (CH), followed by a TnI-like domain, and
  three C-terminal repeat sequence. The CH domain
  in smooth muscle CaP is thought to localize the
  signaling molecules to the actin cytoskeleton
  since this domain can bind to ERK.
• Both TnI-like domain and C-terminal repeat can
  bind to actin.

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Domain structure of CaP
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Actin polymerization and depolymerization

• Actin exists in two forms, globular (G) monomer
  and filamentous (F) polymer.
• G-actin contains bound ATP, which, on
  polymerization, is transformed into
  F-actin-bound-ADP and Pi
• At intermediate concentration of free G-actin
  subunits, the filaments loose actin-ADP at the
  minusend (pointed end) and assemble actin-ATP at
  the plus-end (barbed end). This phenomenon is
  called treadmilling of actin filament
  polymerization/depolymerization.
• The treadmilling of actin filament is the basic
  mechanism for motility in nonmuscle cells

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Actin associated proteins in actin filament
polymerization and deplymerization

• Proteins that bind to G-actin
• -profilin, thymosin
• Proteins that nucleate F-actin
• -formin, Arp2/3 (also branching effect)
• Proteins that bind to F-actin ends and control
  polymerization
• - cofilin, gelsolin severing, and capping at
  the plus end
• - gCAP39, capZ capping at the plus end
• - tropomodulin capping at the minus end
• - severin severing, and capping
• - villin crosslinking, severing, and capping
• Proteins that attach actin to the plasma
  membrane Ezrin/Radixin/Moesin (ERM) protein
  family
• Proteins that crosslink/branch F-actin filamin,
  fimbrin, a-actinin, fascin, spectrin

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Arp2/3 complex downstream target of multiple
signaling pathways leading to actin assembly

• Seven conserved subunits including Arp2 and
  Arp3 (Machesky et al., 1994).
• Generates new actin filaments in a
  stimulus-responsive and spatially controlled
  fashion.

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Arp 2/3 complex mediates branching of actin
filaments
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Physiological functions

• Using the methods of DNase inhibition and actin
  affecting drugs (phalloidin, cytochalasin, and
  latrunculin), Mehta and Gunst first found that
  the G-actin content was 30 lower in extracts of
  muscle strips activated with acetylcholine than
  in extracts from unstimulated muscle strips. The
  decrease in G-actin in response to contractile
  stimulation was prevented by inhibitors of actin
  polymerization with no effect on MRLC
  phosphorylation.
• Actin polymerization in smooth muscle is
  functionally related to contractile activity
  under physiological conditions
• Bárány and his colleagues found a rapid exchange
  of the G-actin bound-ATP in intact arterial
  smooth muscle, indicating that dynamic
  rearrangement of actin filament occurs in intact
  smooth muscle.
• All these studies have suggested that
  polymerization of G-actin monomer into
  filamentous F-actin may play an important role in
  contractile activation in smooth muscle
  independent of activation of myosin ATPase
  activity in response to MRLC phosphorylation.

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Contractile stimulation inducing the formation of
linkages between the cytoskeleton and integrin
proteins

• Receptor coupled G-protein (Rho) activation by
  extracellular stimulation of carbachol may
  involve in actin reorganization in human airway
  smooth muscle.
• Signaling cascade of integrin activation to
  remodeling of cytoskeleton in regulation of
  smooth muscle contraction
• Cholinergic receptor stimulation recruits actin-
  and integrin- binding proteins (e.g. a-actinin,
  vinculin, talin, focal adhesion kinase, and
  paxillin) from the cytoplasm to the membrane
  cytoskeleton.
• Depletion of paxillin and focal adhesion kinase
  led to loss of force in tracheal smooth muscle
  tissues

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Linkages between the cytoskeleton and integrin
proteins

• Nonphosphorylatable paxillin mutants in tracheal
  muscle inhibits tension development and actin
  polymerization, without affecting MRLC
  phosphorylation
• Down-regulation of profilin, an actin regulatory
  protein, was found to attenuate the force
  development of arterial smooth muscle as well
• Membrane cytoskeletal linker proteins (e.g.
  a-actinin, vinculin, talin etc.) and integrin
  associated proteins (e.g. FAK, and paxillin)
  plays an important role in reorganization of
  actin filament polymerization and
  depolymerization in the membrane/cytoskeletal
  compartment during smooth muscle contraction.
• Contractile stimulation could induce formation of
  the structural linkage between the cytoskeleton
  and integrin proteins that mediate tension
  transmission between the contractile apparatus
  and the extracellular matrix in smooth muscle.

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Actin binding protein regulation of actin filament

• Formin and Arp 2/3 complexes (actin related
  proteins 2 and 3, seven strongly associated
  subunits) are the two nucleating proteins that
  facilitate the polymerization of actin filament.
• While the formin protein induces a linear form of
  actin filament, the Arp 2/3 protein complex
  branches the actin filaments to form a network
  within the cells.
• Profilin is an actin binding protein to promote
  the nucleotide exchange of G-actin and to
  increase the polymerization rate of actin
  filament formation.
• Cofilin associated with actin depolymerization
  factor (ADF/cofilin), gelsolin, severin, are
  known to cut off the actin filament and involved
  in depolymerization of actin filament.

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Wiskott-Aldrich syndrome protein (WASp)

• Originally described in fibroblasts could induce
  actin polymerization in connection with the small
  GTPase cdc42
• WASp family proteins bind to Arp2/3 complex and
  induce Arp2/3 complex activation and the
  nucleation of actin filaments
• WASp contains a family of homologous proteins
  found in different cells.
• All families of WASp contain a conserved region
  at the C-terminus with a proline-rich (PRO)
  region followed by one or two WH2 (also called V
  for verproline homology) domain, a central (C)
  region and an acidic (A) domain. The PRO region
  binds profilin and SH3-containing adaptor
  proteins, WH2 domain for binding G-actin, and A
  domain for Arp 2/3 binding. On the other hand,
  two domains at the the N-terminus, the GBD domain
  binds small G-protein Cdc42, and WH1 for F-actin
  binding.

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Domain structures of WASp family protein

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Mechanism

• In the unstimulated conditions, the WASp molecule
  has the inhibitory interaction between the N- and
  C-terminus and burries the binding site for Arp
  2/3. Upon stimulation, Cdc42 binding to GBD
  domain activates WASp, and unfold the N-and
  C-terminal domains of the molecule, and expose
  the binding site for Arp 2/3, finally initiate
  the actin filament polymerization .

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Signaling pathway by WASp regulation in smooth
muscle

• The signaling cascades by profilin and its
  upstream regulator, p130 Crk-associated substrate
  are necessary for regulation of actin filament
  polymerization and force generation in arterial
  smooth muscle in response to contractile
  stimulation.
• Small GTPase Cdc42 regulates actin polymerization
  and also tension development during contractile
  stimulation of tracheal smooth muscle.
• Crk II regulation of N-WASp-mediated activation
  of the Arp2/3 complex is also necessary for actin
  polymerization and tension development in
  response to muscarinic stimulation in tracheal
  smooth muscle.
• These studies provide evidence showing that the
  signaling pathway by the regulation of WASp
  activation upon contractile stimulation cause the
  reorganization of the actin filament network and
  smooth muscle contraction.
• Newly formed actin filaments may localize
  beneath the cell membrane (dense bodies) and may
  be critical for the transmission of tension from
  the contractile apparatus to adhesion sites on
  the membrane during contractile stimulation.