We hypothesized that IP3-induced Ca2+ release during the initial phase would activate Pyk2
We hypothesized that IP3-induced Ca2+ release during the initial phase would activate Pyk2. 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 , ( and PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome GSK-2881078 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 GSK-2881078 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. Introduction The current understanding of the molecular mechanisms that lead to smooth muscle contraction is based on the phosphorylation of the 20- kDa myosin GSK-2881078 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 [1C3]. The chemical energy GSK-2881078 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) [1C4]. 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 . Upon stimulation, contraction of smooth muscle occurs in two phases: an initial Ca2+-dependent contraction followed by a sustained Ca2+-independent contraction, with minimal overlap. Initial contraction involves sequential activation of Gq and PLC-1 by G protein-coupled receptors, resulting in inositol 1,4,5-trisphosphate (IP3) formation, IP3-dependent Ca2+ release via IP3R-1/Ca2+ channels, increase in cytosolic Ca2+ ([Ca2+]for 10 min to eliminate broken cells and organelles. Dispersed muscle cells isolated from the stomach were re-suspended in DMEM containing 200 U/ml penicillin, 200 g/ml streptomycin, 100 g/ml gentamycin, 2.5 g/ml amphotericin B and 10% fetal bovine serum (DMEM-10). The muscle cells were plated at a concentration of 5 105 cells/ml and incubated at 37 C in a CO2 incubator. The DMEM-10 medium was replaced every 3 days for 2C3 weeks until confluence was attained. All experiments were done on cells in the first passage [24C27]. Measurement of mRNA expression for FAK, paxillin, -Pix, -Pix and DOCK-180 by RT-PCR Total RNA was extracted from cultured gastric smooth muscle cells by treatment with RNAqueous reagent, and contaminant genomic DNA was removed by treatment with TURBO DNase as described previously [25, 27]. RNA from each preparation was reversely transcribed using the SuperScript II system containing 50 mM TrisCHCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 0.5 mM deoxynucleoside triphosphates (dNTP), 2.5 M random hexamers and 200 units of reverse transcriptase (RT) in a 20 l reaction volume. The reactions were carried out at room temperature for 10 min, then at 42C for 50 min, and terminated by heating to 70C for 15 min. Three l of the reverse transcription complementary DNA (RT cDNA) were amplified in a final GSK-2881078 volume of 50 l by PCR in standard conditions (2 mM MgCl2, 200 M dNTP, and 2.5 units Taq polymerase) with specific primers for Jag1 FAK, Cool2/PAK interacting exchange factor alpha (Cool2/Pix), Cool2/PAK interacting exchange factor beta (Cool1/Pix), paxillin, and DOCK 180. The.