Abstract
In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.
Highlights
The current understanding of the molecular mechanisms that lead to smooth muscle contraction is based on the phosphorylation of the 20- kDa myosin II regulatory light chain (MLC20), an essential requirement for both initiating and sustaining contraction
Stadie-Riggs tissue slicer was purchased from Thomas Scientific (Swedesboro, NJ); RhoA, Cdc42 and filamentous actin (F-actin) to globular actin (G-actin) (F/G-actin) ratio assay kits were purchased from Cytoskeleton (Denver, CO); the Rho kinase inhibitor Y27632 and the actin related protein complex 2/3 (Arp2/3) complex inhibitor CK666 were purchased from EMD Millipore (Billerica, MA); FAK inhibitor FP 573228 and the actin polymerization inhibitors latrunculin A, and cytochalasin D were purchased from Tocris Bioscience (Bristol, UK); All PCR reagents, RNA isolation kit, TURBO DNase, SuperScript II, Lipofectamine 2000 Transfection Reagent as well as phosphoPyk2(Tyr402) and phospho-paxillin(Tyr118) antibodies were purchased from Thermo Fisher Scientific (Rockford, IL)
Anti-Arp2/3, N-WASP antibodies were purchased from Abcam (Cambridge, UK); Cdc42 antibody, phospho-FAK, and paxillin antibodies were purchased from BD Biosciences; and Cool1/β-Pix, Cool2/α-Pix, DOCK 180, and FAK antibodies were purchased from Cell Signaling Technology (Danvers, MA)
Summary
The current understanding of the molecular mechanisms that lead to smooth muscle contraction is based on the phosphorylation of the 20- kDa myosin II regulatory light chain (MLC20), an essential requirement for both initiating and sustaining contraction. The chemical energy derived from actin-activated actomyosin is converted into mechanical force that induces both cyclical sliding of overlapping actin and myosin filaments (cross-bridge cycles and cell shortening) [1,2,3,4]. It is increasingly evident that in order to transmit force, the sliding actomyosin filaments must be anchored to the opposing sides of a smooth muscle cell, as well as to other smooth muscle cells via the extra cellular matrix (ECM). This anchoring process occurs as part of a dynamic, stimulus-driven reorganization of cytoskeletal proteins at membrane adhesion junctions [5]
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