A role for focal adhesion kinase in the contraction of murine gastric fundus smooth muscle evoked by cholinergic neurotransmission

Abstract

Cholinergic neurotransmission‐induced smooth muscle contraction involves the regulation of myosin light chain (LC20) phosphorylation and dephosphorylation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). C‐kinase potentiated protein phosphatase‐1 inhibitor protein of 17 kDa (CPI‐17), and myosin phosphatase target subunit 1 (MYPT1) are crucial for gastrointestinal smooth muscle contractile regulation via their inhibition of MLCP activity. Besides these mechanisms, integrin‐mediated cell‐matrix adhesions connect the cytoskeleton with the extracellular matrix to facilitate cohesion and to generate tension throughout the whole muscle in response cholinergic neurotransmission. Cell‐matrix adhesions and integrin signaling involve the dynamic recruitment and assembly of several proteins, primarily focal adhesion kinase (FAK), at the adhesive site. FAK is a tyrosine kinase whose phosphorylation state and activity is tightly linked with cell adhesion to the extracellular matrix through integrin receptors. FAK participates in transmitting the force generated by the activation of contractile proteins in smooth muscle cells (SMC), to the extracellular matrix filaments and throughout the smooth muscle tissue.Here, we address the hypothesis that FAK activation is involved in the contraction of gastric fundus smooth muscle in response to cholinergic stimulation. FAK (not Pyk2) was identified as a major tyrosine‐phosphorylated protein in response to electric field stimulation (EFS). EFS in the presence of L‐NNA and MRS2500 contracted gastric fundus smooth muscle strips and significantly increased FAK phosphorylation at Tyr397. In addition, EFS increased the association of FAK with Beta‐1 integrin. The FAK inhibitor PF‐431396 inhibited the EFS‐stimulated contractions, the increase in FAK Tyr397 phosphorylation, and the association of FAK with Beta‐1 integrin. PF‐431396 also blocked the EFS‐induced increase in MYPT1 Thr853, CPI‐17 Thr38, and LC20 Ser19 phosphorylation. The muscarinic acetylcholine receptor antagonist atropine, but not the L‐type Ca2+ channel blocker nicardipine, prevented the EFS‐induced increase in FAK Tyr397 phosphorylation. However, FAK Tyr397 phosphorylation was increased during high K+‐induced contraction, and both were inhibited by PF‐431396. Our findings demonstrate that FAK Tyr397 phosphorylation and association with integrin receptors in SMC plays a role in the contractile response of gastric fundus smooth muscles to cholinergic transmission.Support or Funding InformationThis project was supported by a Takeda Innovation Center Grant, Takeda Pharmaceuticals, Takeda California, San Diego, CA 92121