Abstract
Contractile stimulation induces actin polymerization in smooth muscle tissues and cells, and the inhibition of actin polymerization depresses smooth muscle force development. In the present study, the role of Cdc42 in the regulation of actin polymerization and tension development in smooth muscle was evaluated. Acetylcholine stimulation of tracheal smooth muscle tissues increased the activation of Cdc42. Plasmids encoding wild type Cdc42 or a dominant negative Cdc42 mutant, Asn-17 Cdc42, were introduced into tracheal smooth muscle strips by reversible permeabilization, and tissues were incubated for 2 days to allow for protein expression. Expression of recombinant proteins was confirmed by immunoblot analysis. The expression of the dominant negative Cdc42 mutant inhibited contractile force and the increase in actin polymerization in response to acetylcholine stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type Cdc42 had no significant effect on force, actin polymerization, or myosin light chain phosphorylation. Contractile stimulation increased the association of neuronal Wiskott-Aldrich syndrome protein with Cdc42 and the Arp2/3 (actin-related protein) complex in smooth muscle tissues expressing wild type Cdc42. The agonist-induced increase in these protein interactions was inhibited in tissues expressing the inactive Cdc42 mutant. We conclude that Cdc42 activation regulates active tension development and actin polymerization during contractile stimulation. Cdc42 may regulate the activation of neuronal Wiskott-Aldrich syndrome protein and the actin related protein complex, which in turn regulate actin filament polymerization initiated by the contractile stimulation of smooth muscle.
Highlights
Introduction of Plasmids Encoding RecombinantCdc42 into Tracheal Smooth Muscle Tissues—The constructs of human wild type Cdc42 and the Asn-17 Cdc42 dominant negative mutants have been previously described [11, 20]
The essential role of actin polymerization in the regulation of active tension development in response to stimulation with contractile agonists has been documented in a number of smooth muscle tissues [2, 4, 7,8,9,10, 26]; the signaling pathways by which contractile stimuli activate the polymerization of actin in smooth muscle remain to be determined
The results of the present study demonstrate that Cdc42 plays a critical role in the regulation of actin polymerization and active tension development in tracheal smooth muscle
Summary
Cdc into Tracheal Smooth Muscle Tissues—The constructs of human wild type Cdc and the Asn-17 Cdc dominant negative mutants have been previously described [11, 20]. The cDNAs encoding wild type Cdc and Asn-17 mutant were subcloned into the mammalian expression vector pcDNA 3.1 (Invitrogen). Plasmids carrying wild type or mutant Cdc were introduced into the smooth muscle strips by reversible permeabilization ( referred to as chemical loading) using methods we have described previously [4, 21]. Solution 1 (at 4 °C for 120 min) contained 10 mM EGTA, 5 mM Na2ATP, 120 mM KCl, 2 mM MgCl2, and 20 mM TES, and Solution 2 (at 4 °C overnight) contained 0.1 mM EGTA, 5 mM Na2ATP, 120 mM KCl, 2 mM MgCl2, 20 mM TES, and 10 g/ml plasmids. We have shown previously that transfection efficiencies of ϳ90% are achieved in smooth muscle tissues using this reversible permeabilization procedure [4, 21]
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