Muscarinic m2 receptor‐mediated actin polymerization via PI3 kinase γ and integrin‐linked kinase in gastric smooth muscle

Abstract

Background Actin polymerization plays an important role in smooth muscle contraction. Integrin‐linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle. Methods Phosphorylation of paxillin, neuronal Wiskott‐Aldrich syndrome protein (N‐WASp), and association of paxillin with GEF proteins (Cool2/αPix [Cool2/PAK‐interacting exchange factor alpha], Cool1/βPix [Cool1/PAK‐interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N‐WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F‐actin/G‐actin ratio. Results Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3Kγ inhibitor (AS‐605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N‐WASp and its association with Arp2/3 complex were inhibited by ILK (R211). Conclusion In gastric smooth muscle cells, activation of PI3Kγ by muscarinic m2 receptors causes ILK‐dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N‐WASp and its association with Arp2/3 complex leading to actin polymerization.

Molecular mechanisms of voiding dysfunction in a novel mouse model of acute urinary retention

“…In canine trachea exposed to high mechanical loads, expression of both FAK and paxillin were elevated and the force applied directly stimulated FAK activation 57 . FA proteins are also involved in responding to the muscarinic signaling cascade as shown in gastric smooth muscle where paxillin phosphorylation and actin polymerization were directly stimulated by contractile signaling from acetylcholine 58 . Muscarinic receptors and contractility were both impaired in our AUR model.…”

Epigenetic Modulation of GPER Expression in Gastric and Colonic Smooth Muscle of Male and Female Non-Obese Diabetic (NOD) Mice: Insights into H3K4me3 and H3K27ac Modifications

“…Then, the gastric and colonic mucosa layers were scraped to separate them from the muscle layers of their corresponding organs, resulting in smooth muscle strips. Smooth muscle strips from gastric and colonic smooth muscle layers were used for genomic DNA extraction for bisulfite and chromatin immunoprecipitation (ChIP) assays, nuclear and total extractions for the DNMT3A/B assay, histone K4 trimethylation (H3K4me3), histone K27 acetylation (H3K27AC), RNA isolation, qPCR, and Western blot analyses [36].…”

“…Following a one-hour incubation in PBS-T (phosphate-buffered saline with 0.1% Tween-20) containing 1% (w/v) non-fat dried milk with the corresponding horseradish peroxidase-conjugated secondary antibody (1:5000), immunoreactive proteins were visualized using the Pierce ECL Western blotting Substrate Kit (ThermoFisher Scientific, Waltham, MA, USA). Throughout the procedure, PBS-T was used for all washing steps, and the protein bands were identified using the enhanced chemiluminescence reagent within the GE Amersham 680 imaging system [36]. The quantitative analysis was conducted by measuring the band intensity using ImageJ software, ImageJ 1.54; Java 1.8.0_345.…”

Ouabain-induced activation of phospholipase C zeta and its contributions to bovine sperm capacitation

“…Actin remodelling involves actin polymerization, a process of formation of globular actin (G-actin; monomer unit) to produce F-actin, whereas depolymerization involves the reverse process. Actin polymerization, facilitated through mediator proteins involving a signaling pathway (Mahavadi et al 2019;Zhang et al 2018), increases in the head of human sperm during capacitation, creating a physical barrier between the outer acrosomal OAM and the overlying PM, thereby preventing spontaneous acrosomal exocytosis (Shabtay and Breitbart 2016). Gelsolin (actin severing protein) depolymerizes actin through its PLC-dependent dephosphorylation mediated by EGFR (Finkelstein et al 2010).…”