Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle

Abstract

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that ϳ40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca 2؉ -dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/ cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.

The effects of the small GTPase RhoA on the muscarinic contraction of airway smooth muscle result from its role in regulating actin polymerization

“…RhoA has also been shown to modulate the activity of integrinlinked kinase (28,31), which binds to both ␤-integrins and paxillin at cell adhesion junctions, and can regulate airway muscle contractility and actin polymerization through its scaffolding activity (65). There is also evidence that RhoA can regulate activity of the actin dynamizing protein cofilin (2,37,44), which is involved in regulating stimulus-induced actin filament polymerization and reorganization during the contraction of airway smooth muscle (67). RhoA activation may be involved in catalyzing multiple cytoskeletal processes that regulate actin dynamics and cytoskeletal organization during the contractile activation of airway smooth muscle.…”

“…Contractile agonists have been shown to stimulate actin polymerization in a number of smooth muscle cells and tissues, and the critical role of actin polymerization in regulating active tension development in smooth muscle is well documented (4,8,12,16,18,22,29,38,63,64,67). The inhibition of actin polymerization using either pharmacologic or molecular approaches has been shown to depress tension development in many smooth muscles with little or no effect on MLC phosphorylation or cross-bridge cycling (16, 38, 42, 43, 49, 51, 57-59, 64, 65).…”

Recruitment of β-Catenin to N-Cadherin Is Necessary for Smooth Muscle Contraction

“…We used reversible permeabilization (24,28,29) to introduce the constructs of WT or mutant ␤-catenin into human bronchi. Briefly, the contractile responses of human tissues were determined, after which they were placed in 0.5-ml tubes and incubated successively in each of the following solutions: Solution 1 (at 4°C for 120 min) containing 10 mM EGTA, 5 mM Na 2 ATP, 120 mM KCl, 2 mM MgCl 2 , and 20 mM TES; Solution 2 (at 4°C overnight) containing 0.1 mM EGTA, 5 mM Na 2 ATP, 120 mM KCl, 2 mM MgCl 2 , 20 mM TES, and 10 g/ml plasmids; Solution 3 (at 4°C for 30 min) containing 0.1 mM EGTA, 5 mM Na 2 ATP, 120 mM KCl, 10 mM MgCl 2 , and 20 mM TES; and Solution 4 ( at 22°C for 60 min) containing 110 mM NaCl, 3.4 mM KCl, 0.8 mM MgSO 4 , 25.8 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , and 5.6 mM dextrose.…”

“…The contractile response of human bronchial rings to ACh was evaluated. Plasmids encoding WT or mutant ␤-catenin were introduced into bronchial rings by reversible permeabilization (24,28,29). Tissues were then incubated in the medium for 3 days.…”

The Role of Actin Filament Dynamics in the Myogenic Response of Cerebral Resistance Arteries

“…136 Acetylcholineinduced contraction of canine tracheal smooth muscle correlates with dephosphorylation (activation) of cofilin, and expression of the dominant-negative phosphomimetic S3E mutant of cofilin in canine tracheal smooth muscle inhibited acetylcholine-induced cofilin dephosphorylation, actin polymerization and contraction. 128 Cofilin function appears to be restricted to a subcellular pool of actin that is distinct from the stable actin filaments involved in actomyosin complex formation and cross-bridge cycling. The latter pool is associated with tropomyosin, which inhibits cofilin binding, whereas the cortical actin pool involved in dynamic polymerization-depolymerization is free of tropomyosin and, therefore, accessible to cofilin.…”