Date Published: December 20, 2018
Publisher: Public Library of Science
Author(s): Sunila Mahavadi, Ancy D. Nalli, Hongxia Wang, Derek M. Kendig, Molly S. Crowe, Vijay Lyall, John R. Grider, Karnam S. Murthy, Ed Manser.
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.
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 phosphorylation of MLC20 enhances the ability of actin to activate myosin-Mg2+-ATPase and stimulate hydrolysis of ATP on the myosin head [1–3]. 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) [1–4]. Although cross-bridge cycling is essential for generating force, it does not provide a mechanism for transmitting force across the cell or between cells. 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 .
Others and we have demonstrated that the contraction of gastrointestinal smooth muscle is mediated via the phosphorylation of MLC20 [1, 3, 4]. The phosphorylation levels of MLC20 are regulated by two enzymes: MLCK and MLCP. MLCK is a Ca2+/CaM-dependent enzyme. In gastrointestinal smooth muscle, activation of the muscarinic M3 receptor by ACh increases PLC-β1 activity, IP3 formation, Ca2+ release, Ca2+/CaM-dependent MLCK activity, and phosphorylation of MLC20 at Ser19 to cause initial muscle contraction. Sustained muscle contraction is a Ca2+ independent mechanism mediated by two RhoA-dependent pathways. One pathway involves the activation of RhoA, which phosphorylates MYPT1 at Thr696 by Rho kinase, and another pathway involves phosphorylation of CPI-17 at Thr38 by PKC. The phosphorylation of both MYPT1 and CPI-17 leads to the inhibition of MLCP’s catalytic activity to augment MLC20 phosphorylation and cause sustained muscle contraction [1, 3, 4, 11].